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Key Points DNMT3A R882, TET2, ASXL1, and SRSF2 mutations identified at the time of diagnosis are associated with delayed count recovery. Persistence of preleukemic mutations in remission at high variant allele frequency is associated with delayed count recovery.

Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Novel therapies that target LSCs have the potential to improve clinical outcomes. An important step towards achieving this goal is identifying the fundamental processes that regulate cell fate decisions in LSCs. Perturbation of these processes may impair LSC activity and form the basis of novel therapies. Here, we investigated the role of mitochondrial quality control in the regulation of LSCs by inhibiting PTEN-induced kinase 1 (PINK1). PINK1 is a serine/threonine kinase that serves as a critical sensor of mitochondrial damage and is at the apex of multiple quality control pathways that maintain mitochondrial homeostasis. Damage to mitochondria results in activation of PINK1 on the outer mitochondrial membrane, which in turn triggers mitochondrial fission and mitophagy. We first determined if the overall quality of the mitochondrial pool differs between LSCs and their more differentiated progenies (henceforth termed "non-LSCs") in two human AML cultures known as OCI-AML-8227 and OCI-AML-21. In both cultures, LSCs assayed by xenotransplantation are found only in the CD34 +CD38 -fraction and not in the CD34 +CD38 + and CD34 - fractions. We monitored the turnover rate of the mitochondrial pool in the different fractions by expressing MitoTimer, a mitochondria-targeted fluorescent protein that matures from green to red fluorescence over 48 hours. A high ratio of green to red fluorescence is indicative of active mitochondrial protein turnover and high overall mitochondrial quality. In both cell lines, the fluorescence ratio was highest in the CD34 +CD38 - fraction compared with the other fractions, suggesting that LSCs maintain a higher quality pool of mitochondria than non-LSCs. Based on the above findings, we hypothesized that PINK1-dependent mitochondrial quality control mechanisms are involved in the regulation of LSC fate. To test this hypothesis, we silenced the expression of PINK1 in OCI-AML-8227 cells using lentiviral vectors expressing validated shRNAs under a doxycycline-inducible promoter. Depletion of PINK1 shifted the MitoTimer fluorescence from green to red, reduced oxygen consumption rate, and disrupted mitochondrial ultrastructure in all cell fractions, consistent with a reduction in mitochondrial quality. Unexpectedly, PINK1 downregulation resulted in a block in differentiation and cell cycle arrest at G1/G0 phase in CD34 +CD38 - cells. Re-expression of PINK1 was unable to reverse the cell cycle arrest, suggestive of a state of cellular senescence. Indeed, we observed other hallmarks of senescence including an increase in p21 WAF1 and p16 INK4a expression and SA-β-gal activity. To determine the mechanism by which PINK1 depletion causes senescence, we performed RNA sequencing analysis of sorted CD34 +CD38 - cells expressing PINK1 shRNAs. This analysis revealed a marked decrease in the expression of MYC target genes, with TERT (Telomerase Reverse Transcriptase) being the most downregulated gene. Consistent with the role of TERT in telomere maintenance and telomere shortening as a trigger of senescence, we found that PINK1 knockdown decreased telomere length in CD34 +CD38 - cells, and overexpression of TERT effectively rescued the senescence phenotype. These findings collectively indicate that inhibition of PINK1-dependent mitochondrial quality control pathways induces senescence of LSCs through telomere shortening. To determine whether these changes translated to a reduction in functional LSC activity, we performed colony forming unit assays and serial xenotransplantation assays in immunodeficient NSG mice. Depletion of PINK1reduced the colony forming capacity and engraftment potential of 3 primary AML samples in primary and secondary recipients. Importantly, PINK1 depletion had minimal impact on the colony forming capacity and engraftment potential of normal CD34 + hematopoietic stem and progenitor cells (HSPCs) derived from cord blood, suggestive of a therapeutic window in vivo. In summary, our results demonstrate that mitochondrial quality control pathways regulate cell fate decision in LSCs. Inhibition of PINK1 activity impairs LSC activity by inducing senescence, while sparing normal HSPCs. Our findings provide the basis for exploring PINK1 as a therapeutic target against LSCs in AML. Disclosures Chan: AbbVie: Research Funding; BMS: Research Funding.

BACKGROUND: Isocitrate dehydrogenase 2 (IDH2) mutations are found in about 10-15% of acute myeloid leukemia (AML) cases. Enasidenib (ENA) is a first-in-class mutant IDH2 inhibitor that induces differentiation of IDH2-mutated leukemic cells. However, the clinical efficacy of single agent ENA therapy in relapsed or refractory (R/R) AML is limited, underscoring the need for combination therapy. Preclinical studies have shown that IDH-mutated leukemic cells are particularly sensitive to BCL2 inhibition with venetoclax (VEN), a finding supported by clinical studies of VEN combination therapies. We previously demonstrated that the combination of ENA and VEN can be more effective than each drug alone, in reducing leukemic burden in patient-derived xenograft models of IDH2-mutated AML. Here, we report preliminary safety and efficacy results of an ongoing open-label, single-arm, phase Ib/II trial (NCT04092179) of ENA in combination with VEN in patients with IDH2-mutated myeloid malignancies. METHODS: Patients 18 years of age or above with IDH2-mutated R/R AML or high-risk MDS/MPN and an ECOG performance status of 0-2 were eligible. Patients previously treated with an IDH2 or BCL2 inhibitor were excluded. Participants received VEN continuously starting on cycle 1 day 1 with a 3-day ramp-up to a target dose of 400 mg daily (dose level 0) in cohorts 1 and 2. ENA was administered at 100 mg daily continuously starting on cycle 1 day 15. Each cycle was 28 days. Concurrent use of a moderate or strong CYP3A4 inhibitor was allowed with 50% dose reduction of VEN after completion of the first 2 cycles at 100% target dose. Interruptions of VEN and/or ENA were permitted for management of adverse events (AEs). Primary endpoints were 1) safety and tolerability and 2) overall response rate (ORR) defined as complete remission (CR) + CR with incomplete blood count recovery (CRi) + morphologic leukemia-free state (MLFS) + partial remission (PR) by revised IWG criteria. Secondary endpoints include pharmacokinetic (PK) profiles of VEN, duration of response, overall survival (OS), and IDH2 mutant allele burden in bone marrow by ddPCR. RESULTS: The study opened to recruitment in November of 2020. As of July 28, 2021 (data cutoff), 11 patients were enrolled on study; 10 with R/R AML and 1 with very-high risk MDS by IPSS-R. Six patients had a R140Q mutation, and 5 had a R172K mutation. Median age was 72 years (range: 32 - 80); 6 patients were male. Participants had received a median of 2 prior lines of therapies (range: 1 - 4). Six of 10 AML patients had primary refractory disease. The MDS patient experienced secondary azacitidine failure. Key treatment emergent grade ≥ 3 AEs regardless of attribution were: febrile neutropenia (n=3), intracranial hemorrhage (n=3), lung infection (n=2), other infection (n=2), elevated AST/ALT (n=2), sepsis (n=1), leukocytosis (n=1), TRALI (n=1), and small bowel obstruction (n=1). No cases of differentiation or tumor lysis syndrome were observed. No patients discontinued the study due to AEs. The addition of ENA, a known inhibitor of CYP3A4, did not significantly affect the PK profiles of VEN. Nine of the AML patients completed at least 1 cycle of treatment and are considered evaluable for efficacy. One AML patient died from intracranial hemorrhage prior to completion of cycle 1. Median duration of observation is 3.5 months. Of the evaluable patients, CR was achieved in 2 patients (22%) and CRi in 3 patients (33%) for an ORR of 55% (Fig. 1). Median number of cycles to response was 3. All responders remain in remission and on study with a median of 6 cycles received to date (range: 3 - 8). Of the remaining 4 patients, 2 patients (22%) remain on study with stable disease, and 2 patients (22%) experienced progressive disease and died (one after 7 cycles and the other after 1 cycle). Median OS for the entire cohort has not been reached. A sustained reduction in mutant IDH2 allele frequency in bone marrow correlated with response (Fig. 2). For the MDS patient, no response was observed after 1 cycle of treatment. CONCLUSIONS: VEN in combination with ENA is a well-tolerated regimen with no dose-limiting toxicities observed at the current dose level. The preliminary efficacy of this combination is encouraging with an ORR of 55% in evaluable R/R AML patients, with some responders achieving deep molecular remissions. Patient enrollment in dose-escalation cohorts is ongoing. Figure 1 Figure 1. Disclosures Chan: AbbVie: Research Funding; BMS: Research Funding. Gupta: Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Constellation Pharma: Consultancy, Honoraria; Incyte: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Roche: Consultancy; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy. Maze: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene-BMS: Honoraria; Takeda: Research Funding; PharmaEssentia: Research Funding; Kronos Bio: Research Funding. Minden: Astellas: Consultancy. Schimmer: Takeda Pharmaceuticals: Consultancy, Research Funding; Medivir AB: Research Funding; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Otsuka Pharmaceuticals: Consultancy, Honoraria; UHN: Patents & Royalties. Schuh: AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlycoMimetics: Research Funding; Kite/Gilead: Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees; Teva: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yee: Pfizer: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Research Funding; F. Hoffmann La Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Onconova: Research Funding; Bristol-Myers Squibb/Celgene: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees; Paladin: Membership on an entity's Board of Directors or advisory committees; Geron: Research Funding; MedImmune: Research Funding; Jazz: Research Funding; TaiHo: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria; Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding. DiNardo: Agios/Servier: Consultancy, Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; AbbVie: Consultancy, Research Funding; Novartis: Honoraria; Foghorn: Honoraria, Research Funding; Forma: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. OffLabel Disclosure: Enasidenib is approved for the treatment of relapsed or refractory AML as single agent. Venetoclax, in combination with azacitidine or low-dose cytarabine, is approved for the treatment of newly diagnosed AML patients who are 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy.

Mutations in isocitrate dehydrogenase 1 (IDH1) promote leukemic transformation through the production of an oncometabolite, 2-hydroxyglutarate (2-HG). Ivosidenib is an inhibitor of mutant IDH1 approved for the treatment of IDH1-mutated AML. Treatment with ivosidenib can induce terminal differentiation of leukemic blasts via suppression of 2-HG. However, ivosidenib as a single agent has limited efficacy, highlighting the need to better understand the mechanisms of drug resistance. The study of drug resistance mechanisms has been hindered by the lack of IDH1/2-mutated AML cell lines models. To address this issue, we derived an Idh1-mutated AML cell line from bone marrow cells of a murine AML model generated by crossing mice expressing mutant Idh1R132H with mice expressing mutant Npm1 (Npm1c). This cell line (henceforth termed "OCI-mIDH1/N") undergoes partial myeloid differentiation in response to ivosidenib treatment in vitro. The establishment of OCI-mIDH1/N enabled us to perform a genome-wide CRISPR knockout screen to identify genes that upon inactivation, increased the differentiation response to ivosidenib. Through this screen, we identified C-type lectin member 5a (Clec5a) as one of the top hits. Clec5a encodes a cell surface receptor that signals through the intracellular spleen tyrosine kinase (SYK). To confirm this hit, we generated Clec5a knockout clones of OCI-mIDH1/N cells. Consistent with results of the screen, ivosidenib treatment induced higher levels of Gr-1, a myeloid differentiation marker, on Clec5a-/- cells than on parental Clec5a+/+ cells. Next, we investigated the role in SYK. Clec5a-/- cells had lower levels of pSYK compared with Clec5a+/+ cells, and overexpression of Syk in Clec5a-/- cells reversed their sensitization to ivosidenib. Furthermore, direct inhibition of SYK with fostamatinib was sufficient to sensitize Clec5a+/+ cells to ivosidenib. Our findings show that CLEC5A-SYK signaling promotes resistance to ivosidenib-induced differentiation. Mechanistically, we found that CLEC5A-SYK signaling drives ivosidenib resistance through STAT5 dependent expression of self-renewal genes in the HOX family. Clec5a-/- and Syk-/- OCI-mIDH1/N cells exhibited lower levels of STAT5 activation compared with wildtype cells. Furthermore, SYK inhibitor treatment downregulated pSTAT5 and decreased STAT5 occupancy at HOX gene clusters. Functionally, overexpression of constitutively active STAT5 in Clec5a-/- and Syk-/- cells reversed their heightened sensitivity to ivosidenib and increased the expression of key HOX self-renewal genes. To determine the clinical relevance of these findings, we analyzed two independent RNA-seq datasets from patients treated with IDH inhibitors (Quek et al., 2018; Wang et al., 2021). Gene set enrichment analysis revealed that poor responders expressed significantly higher levels of STAT5 target genes compared with responders in both datasets. Mutations in the receptor tyrosine kinase (RTK) pathway have previously been shown to be associated with resistance to IDH inhibitors. Given that these pathways signal through STAT5, we hypothesized that the mechanism by the RTK mutations confer ivosidenib resistance is through STAT5 activation. To test this hypothesis, we ectopically expressed KRASG12D or PTPN11E76G in OCI-mIDH1/N cells. Expression of the oncogenes increased pSTAT5 and conferred resistance to ivosidenib-induced differentiation. Importantly, knockdown of Stat5 completely restored their sensitivity to ivosidenib, in support for our hypothesis. Next, we tested if the STAT5 inhibitor pimozide synergizes with ivosidenib to treat patient-derived xenograft (PDX) models of human IDH1-mutated AML. In two PDX models where ivosidenib alone induced moderate differentiation, the addition of pimozide to ivosidenib significantly increased the expression of myeloid differentiation markers on AML cells. In two PDX models where ivosidenib alone induced no differentiation, pimozide alone was sufficient to induce profound differentiation of AML cells. Collectively, these results suggest that ivosidenib resistant cells shift their dependence from 2-HG to STAT5 to maintain their undifferentiated state. In summary, our findings demonstrate that STAT5 is a critical mediator of resistance to ivosidenib, and combination therapy with pimozide and ivosidenib is a promising therapeutic approach for IDH1-mutated AML. Disclosures Chan: BMS: Research Funding; AbbVie: Research Funding.

Clonal Hematopoiesis of Indeterminate Potential (CHIP) refers to the clonal expansion of hematopoietic stem and progenitor cells (HSPCs) carrying mutations associated with hematologic malignancies in individuals without evidence of a blood disorder. CHIP carriers are at a 10-fold higher risk of developing blood cancers relative to non-carriers. The time interval from acquisition of CHIP to overt neoplasia can span many years. This finding suggests a potential window of opportunity to intervene to prevent or delay malignant transformation; however, this goal is not yet possible due to the lack of known effective interventions. One of the most commonly mutated genes in CHIP is TET2, which encodes an epigenetic modifier involved in DNA demethylation. Tet2 knockout mouse models have an expansion of HSPCs and are at increased risk of development of myeloid malignancies, thus establishing TET2 mutation as a bona fide driver of pre-leukemia. To identify drugs that selectively target Tet2-deficient HSPCs, we generated isogenic murine cell lines by overexpressing HOXB4 in Sca-1 + HSPCs derived from a Tet2 knockout (Tet2-/-) mouse and a Tet2 wildtype (Tet2 +/+) littermate. HOXB4 overexpression has previously been shown to expand and immortalize HSPCs indefinitely without causing leukemogenesis. Using this isogenic cell line system termed HPC HOXB4, we conducted a drug screen of 37 small-molecule inhibitors of epigenetic signaling to find compounds that selectively reduced the proportion of Tet2-/- to Tet2+/+ HPC HOXB4 cells in competition assays. Through this screen, we discovered that DOT1L inhibitors, including SGC0946 and pinometostat, preferentially reduced the competitive advantage of Tet2 -/- HPC HOXB4 cells. Importantly, SGC0946 treatment also reduced the competitive advantage of unmodified Tet2 -/- murine HSPCs over wildtype cells in short-term competition assays. Mechanistic studies revealed that DOT1L inhibition induced higher levels of apoptosis in Tet2 -/- than in Tet2+/+ HPC HOXB4 cells and promoted the differentiation of Tet2-/- HPC HOXB4 cells. Genetic knockdown of Dot1l expression using shRNAs phenocopied the effects of pharmacologic DOT1L inhibition. DOT1L is a histone methyltransferase that catalyzes histone H3K79 methylation. Unexpectedly, we found that global H3K79me2 and H3K79me3 levels were higher in Tet2 -/- than in Tet2+/+ HPC HOXB4 cells. SGC0946 treatment effectively removed the H3K79me2/3 mark in Tet2 -/- cells. Next, we conducted RNA-seq analysis to gain insights into the transcriptomic changes in Tet2 -/- HPC HOXB4 after DOT1L inhibition. We found that expression of Mpl, which encodes the thrombopoietin receptor (TPOR), was over 20-fold higher in Tet2 -/- HPC HOXB4 than in wildtype cells at baseline and was strongly decreased after SGC0946 treatment. In contrast, SGC0946 had no effect on Mpl expression in Tet2 +/+ HPC HOXB4 cells. Mpl expression was also elevated in unmodified Tet2 -/- HSPCs compared with Tet2+/+ cells. Given that TPO signaling promotes HSC self-renewal and proliferation, we hypothesized that the effects of Dot1L inhibition are mediated through suppression of Mpl. Consistent with our hypothesis, enforced expression of Mpl in Tet2 -/- HPC HOXB4 cells completely rescued the effects of DOT1L inhibition, and downregulation of Mpl expression using shRNAs phenocopied the effects of DOT1L inhibition on Tet2 -/- HPC HOXB4 cells. Based on the findings above, we hypothesized that inhibition of TPOR signaling would selectively target Tet2 -/- over Tet2+/+ HSPCs. Given that Janus Kinase 2 (JAK2) is required to transduce signals downstream of the TPOR, we studied the effects of two potent JAK2 inhibitors, fedratinib and AZD1480, along with a JAK1/2 inhibitor, ruxolitinib, on Tet2 -/- and Tet2+/+ HPC HOXB4 cells in competition assays. In line with our hypothesis, all three compounds reduced the competitive advantage of Tet2 -/- HPC HOXB4 cells. Ruxolitinib also reduced the competitive advantage of unmodified Tet2 -/- HPSCs cells over wildtype cells in a dose-dependent manner. In summary, our data demonstrate that 1) the TPOR signaling pathway is upregulated in TET2-mutated HSPCs through epigenetic dysregulation by DOT1L, and 2) inhibition of DOT1L or the MPL/JAK2 signaling axis selectively targets TET2-mutated HSPCs. Our findings have important implications in the development of pharmacologic interventions against TET2-mutation driven CHIP. Disclosures Chan: AbbVie: Research Funding; BMS: Research Funding.

The main barrier to curing acute myeloid leukemia (AML) is disease relapse, which occurs due to therapy resistance and persistence of leukemic stem cells (LSCs) after conventional induction chemotherapy. Thus drug discovery efforts must focus on identifying agents that effectively target LSCs and not just bulk blasts. To this end, we employed a multi-parametric stemness screen of 1200 bioactive small molecules to identify drugs targeting LSCs, based on reduction of the stem cell compartment of a functionally-characterized hierarchical AML model (OCI-AML-8227) assessed by flow cytometry. In this cell line, self-renewing LSCs are restricted to the CD34+CD38- fraction. The screen identified a number of compound classes with the potential to antagonize LSC properties, including those already in clinical use for AML as well as classes of compounds whose effects in AML have not been previously reported (Figure 1A). Top hits were further validated based on treatment-induced alteration of the expression profile of 104 LSC genes (LSC104) differentially expressed between LSC+ and LSC- fractions of primary AML, which captures stemness properties. The LSC17 score, which is strongly associated with survival and response to standard therapy in AML, was derived from the LSC104 gene set. Notably, all Polo-like kinase 1 (PLK1) inhibitors in the library were identified as top hits in the screen. In vitro treatment of OCI-AML-8227 cells with PLK1 inhibitors over 3 days selectively inhibited the CD34+CD38- fraction enriched in LSCs (Figure 1B), and decreased correlation of gene expression to the LSC104 signature in bulk cells (Figure 1C). Together, these data support a role for PLK1 in regulating leukemic stemness, and we prioritized this class of compounds for validation studies. PLK1 is an important regulator of cell cycle and its best studied role is in the regulation of mitotic entry. However, PLK1 is expressed in and likely plays an important role in all phases of the cell cycle. For instance, PLK1 has been described to regulate cilia disassembly at G0/1. The PLK1 inhibitor volasertib was previously tested in a Phase III trial against AML in combination with low-dose cytarabine (LDC) for elderly patients not eligible for induction chemotherapy. In this trial, although efficacy was observed, significant toxicity in the volasertib+LDC treatment arm resulted in poor survival outcomes for this group of patients. We evaluated the toxicity of volasertib treatment in vitro against two hierarchical AML cell lines (OCI-AML-8227 and OCI-AML-21) as well as normal cord blood (CB). Similar to CB, self-renewing stem cells for these two AML cell lines are restricted to the CD34+CD38- fraction. Treatment with volasertib at 20nM over three days resulted in significantly more cytotoxicity to the AML cell lines compared to CB (Figure 1D), especially in the CD34+CD38- compartment, suggesting that a therapeutic window exists. To evaluate the effects of PDK1 inhibitors against LSCs in vivo, we treated mice bearing AML patient xenografts with single-agent volasertib at a low dose (10mg/kg twice weekly for 4 weeks by oral gavage) starting 4 weeks post-transplant. The gene expression profile for 2 of 4 samples tested showed decreased correlation to the LSC104 signature after volasertib treatment, supporting an effect on stemness (Figure 1E). Volasertib treatment significantly reduced AML engraftment in 4 of 7 samples (Figure 1F). To evaluate the effect of volasertib on LSCs in primary treated mice, we performed secondary transplantation at limiting doses. Volasertib treatment significantly reduced LSC frequency in 2 of 4 samples tested (Figure 1G). Notably, sample AML5 showed a 31.8-fold reduction in LSC frequency compared to controls (p = 0.039) despite no significant reduction in bulk engraftment in primary treated mice, suggesting that volasertib may selectively target LSCs in this sample. In conclusion, our data indicate that the PLK1 inhibitor volasertib, identified as a top hit in a stemness-based drug screen, can target LSCs and decrease stemness properties in some primary AML samples. These findings support further studies of the potential of PLK1 inhibitors for the treatment of AML. Figure Disclosures Wang: Trilium Therapeutics: Patents & Royalties.

Current chemotherapeutic regimens for acute myeloid leukemia (AML) often fail to eliminate leukemic stem cells (LSCs) which contribute to disease relapse. A key step towards the development of more effective therapies is the identification of vulnerabilities that are unique to LSCs. Here, we sought to identify LSC-specific metabolic dependencies by performing a flow cytometry-based screen of 110 metabolically-focused drugs against a primary human AML sample. This sample harbored distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation was restricted to the CD34+CD38- fraction. Through this screen, we found that inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in the NAD+ salvage pathway, preferentially depleted CD34+CD38- cells, implicating NAMPT inhibitors as potential anti-LSC agents. To evaluate the therapeutic potential of NAMPT inhibitors, we focused on KPT-9274, a small-molecule NAMPT inhibitor currently under clinical development for other cancer types. Treatment with KPT-9274 depleted the CD34+CD38- fraction across multiple primary human AML samples through induction of apoptosis. The preferential sensitivity of CD34+CD38- cells to NAMPT inhibition correlated with a lower basal level of intracellular NAD+ and greater dependency on NAMPT activity for NAD+ generation relative to the other fractions. In contrast, normal CD34+ HSPCs were largely resistant to the cytotoxic effects of KPT-9274 due to their capacity to utilize the Preiss-Handler pathway for NAD+ generation. Consistent with the in vitro findings, KPT-9274 treatment significantly reduced LSC activity as determined by secondary engraftment potential in 2 of 3 patient-derived xenograft (PDX) models of human AML and had minimal impact on normal HSC activity in mice engrafted cord blood cells. To gain mechanistic insights into how NAMPT inhibition induces cell death, we performed transcriptomic analysis of sorted CD34+CD38- cells treated with KPT-9274. This analysis revealed a striking upregulation of genes involved in cholesterol and lipid synthesis including the stearoyl-CoA desaturase (SCD) gene. The upregulated genes were highly enriched for known targets of the sterol regulatory element binding protein (SREBP) transcription factors. Functional studies demonstrated that this transcriptional response was protective against the cytotoxic effect of NAMPT inhibition in AML cells. To uncover the metabolic basis of this protective effect, we performed global metabolomic profiling of AML cells treated with KPT-9274 and observed a decrease in the ratio of monounsaturated fatty acids (MUFAs) to saturated fatty acids (SFAs) upon drug treatment. This drop in MUFA:SFA ratio reflected a reduction in SCD activity which catalyzes the desaturation of SFAs to MUFAs in a NADPH-dependent reaction. Since depletion of intracellular MUFAs could trigger apoptosis, we hypothesized that the SREBP response might protect against cell death through upregulation of SCD activity and consequent increase in MUFA synthesis. In line with this hypothesis, we found that exogenous oleic acid, a MUFA, completely rescued cell death induced by KPT-9274, while treatment with SCD inhibitors sensitized AML cells to the cytotoxic effects of NAMPT inhibition. To explore the translational application of our findings, we tested whether dipyridamole (DP), a clinically approved anti-platelet agent with inhibitory activity against SREBP signaling, can be repurposed to enhance the anti-leukemic effects of KPT-9274. We showed that treatment with DP, at non-toxic concentrations, potentiated the cytotoxicity of KPT-9274 against AML cells in vitro. Importantly, in vivo combination treatment with KPT-9274 and DP effectively targeted LSC activity in a PDX model that was refractory to KPT-9274 as single agent. In summary, our findings demonstrate that LSCs are preferentially dependent on NAMPT activity for survival over non-LSCs and normal HSCs. We further uncovered that NAMPT inhibition results in dysregulation of lipid homeostasis and induces a lipogenic response coordinated by SREBPs that protects AML cells against NAD+ depletion. These findings offer insights into drug combination strategies to enhance the efficacy of NAMPT inhibitors and provide the rationale for testing NAMPT inhibitors in the treatment of AML in clinical trials. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium therapeutics: Patents & Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .

Introduction: CFI-400945 is a first-in-class, potent, selective, orally active inhibitor of Polo-like kinase 4 (PLK4) (Ki=0.26nM), a master regulator of centriole duplication, necessary for genomic integrity (Mason et al. Cancer Cell 2014; 26:163-76). CFI-400945 has activity in leukemia cell lines and primary leukemia samples including those with complex karyotype, inversion 3 and monosomy 7 (Minden. personal communications). This suggests that CFI-400945 may provide an effective treatment of patients with AML. The objectives of this phase 1 trial was to establish the safety, tolerability, and recommend phase II dose (RP2D) of CFI-400945 in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Methods: Patients with relapsed/refractory AML or MDS and patients with untreated AML who refused induction chemotherapy or who are not candidates for intensive chemotherapy were eligible. Dose escalation followed a standard 3+3 design with a starting dose of 64 mg orally once daily. Plasma levels of CFI-400945 free base were measured on Days 1, 2, & 29 of Cycle 1 and Day 15 on all subsequent cycles. Peripheral blood and/or bone marrow were obtained at baseline, Day 8 of Cycle 1 and Day 1 of each subsequent cycle prior to dosing for pharmacodynamic monitoring. Safety assessments using the NCI CTCAE version 4.03 were performed. Results: From May 2018 to June 2019, nine patients have been enrolled on study across three pre-defined dose levels (64 mg [n=3], 96 mg [n=4], and 128 mg [n=2]). Three patients had untreated AML, five patients had relapsed/refractory AML and one patient had myelodysplastic syndrome/myeloproliferative disorder (MDS/MPN). Patient characteristics at diagnosis are outlined in Table 1. Six (67%) patients had baseline high throughput sequencing; the most frequent mutations were TP53 (33%), TET2 (33%), KRAS (33%) and DNMT3A (33%). A total of 20 cycles were administered with a median of 1 cycle (range, 0 to 7 cycles). The most common non-hematological drug related toxicities of any grade, which occurred in over 20%, were diarrhea (44%), headache (44%), colitis (33%), vomiting (33%), bilirubin increase (22%), dizziness (22%), fatigue (22%), and nausea (22%). One patient on the 96 mg dose level was not evaluable for DLT and hence, replaced. Both patients treated at the 128 mg/day dose level developed DLTs, consisting of grade 3 colitis and grade 5 sepsis and colitis. Pharmacokinetic profile indicated low interpatient variability between patients. Maximum exposure did not correlate with toxicity Six patients were evaluable for disease response. Two (33%) achieved complete remission (CR), 3 pts (50%) had stable disease (with one patient having a 78% reduction in marrow blast count). The patient with MDS/MPN who did not complete 1 cycle of therapy progressed to AML (Figure 1). Both patients who obtained a CR had an early response within 2 cycles. One CR has been durable for 218 days with no measurable residual disease (MRD) by flow cytometry. The additional patient, who obtained a CR with incomplete platelets recovery, with subsequent best response of CR, had a sustained response for 91 days before relapse was confirmed by bone marrow examination (Figure 1). Conclusion: Single agent CFI-400945 has activity in patients with poor risk AML. The RP2D in this population is 96 mg once daily. Dose expansion is occurring at the RP2D level. A phase 2 study with CFI-400945 single agent or in combination study with azacitidine or decitabine is planned. Disclosures Leber: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Lundbeck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda/Palladin: Honoraria, Membership on an entity's Board of Directors or advisory committees; Treadwell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bray:Treadwell Therapeutics: Current Employment; TIO Discovery: Current Employment. Gupta:Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding. Maze:Novartis: Honoraria; Pfizer: Consultancy; Takeda: Research Funding. McNamara:Novartis: Honoraria. Schimmer:Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock ; Takeda: Honoraria, Research Funding; Novartis: Honoraria.

BACKGROUND: Multiparameter flow cytometry (MFC) has increasingly been used for measurable residual disease (MRD) assessment in patients with acute myeloid leukemia (AML), while next-generation sequencing (NGS)-based MRD monitoring tool is in clinical development for its application. Clonal hematopoiesis (CH), in which leukemia-associated somatic mutations gene are present in individuals with no apparent hematologic disease, adds a challenge in the detection of MRD. In patients with AML, CH could be potentially pre-leukemic, while persistent mutations in DNMT3A, TET2 orASXL1 (DTA) in remission marrow are usually removed from the analysis of residual leukemic cells. However, reports suggest that persistent DTA mutations in remission may be correlated with an increased relapse risk. In the patients with DTA mutations, the use of NGS for MRD monitoring is limited or modified due to the presence of CH clone in the remission marrow. We evaluated whether MFC-MRD can be adjunctive to predict the risk of AML relapse in this population of 221 patients with DTA mutation (DNMT3A (n=123), ASXL1 (n=56) or TET2 (n=100). METHODS: The present study evaluated long-term outcomes in AML patients who achieved first complete remission (CR1) and compared outcomes according to MFC-based MRD status (was defined as negative if patients achieved 0.1 or less) assessed at the time of CR1. A total of 435 patients diagnosed with AML and treated with induction chemotherapy between 2015 and 2018 were included. MFC-MRD was assessed in 336 patients in CR1 (77%). NGS was performed using samples obtained at the time of initial diagnosis and used for mutational subgroup classification. Overall survival (OS) was calculated as the date of CR1 to the date of death and censored on the date of the last follow-up. Relapse-free survival (RFS) was defined as the time from the date of CR1 to the date of relapse or death from any cause. Cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) were calculated considering competing risk. The Kaplan-Meier method using a log-rank test and a multivariate Cox proportional hazard model was used for analyses of time-to-event endpoints. For CIR and NRM, Gray test was performed for the risk factors and the Fine-Gray model was adopted for the multivariate model. RESULTS: According to the MFC-MRD status, i.e., the group with positive MRD (MRDpos; n=118, 35%) vs. those with negative MRD (MRDneg; n=218, 65%), we evaluated OS, RFS, and CIR. The MFC-MRDneg group showed better OS at 2 years 67.0% than the MFC-MRDpos group 40.7% (p We divided the groups according to the number of induction treatment courses, AML type, cytogenetics risk, and age ( Also, we evaluated MFC-MRD status at CR by mutational profile subgroup. Long-term outcomes such as OS, RFS, CIR or NRM were compared by the mutational subgroup. It consistently showed a trend of superior OS, RFS and lower risk of CIR in patients with MFC-MRDneg compared to MFC-MRDposTab1. Of interest, in the subgroup of patients carrying any DTA mutations (n=221), those with MFC-MRDneg (n=103) showed better OS (HR 1.61 [1.01-2.55%]; p=0.042), RFS (HR 1.66 [1.06-2.61%]; p=0.026) and CIR (HR 1.99[1.03-3.83%]; p=0.04) compared to those MFC-MRDpos (n=64; Fig 1). Multivariate analysis confirmed that the MFC-MRDneg is an independent prognostic factor in patients with DTAmutwith respect to OS: MFC-MRDpos (HR 1.63, p=0.04) and age (≥60; HR 2.04, p=0.008) for OS; for RFS, MFC-MRDpos (HR 1.71, p=0.02) and age (≥60; HR 2.32, p= 0.001); for CIR, MFC-MRDpos (HR 2.31, p=0.01) and HCT (HR 0.14, p= Conclusion: These findings suggest that in AML patients with DTAmut, MFC-MRD status at the time of remission assessment can be a tool for MRD assessment when NGS-based MRD assessment is limited. Further study is strongly warranted to reach a clearer conclusion with multiple cohorts. Disclosures Schimmer: Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock . Tierens:Amgen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees. McNamara:Novartis: Honoraria. Maze:Pfizer: Consultancy; Novartis: Honoraria; Takeda: Research Funding. Gupta:Pfizer: Consultancy; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding.

Introduction: Acute myeloid leukemia (AML) is driven by a subpopulation of leukemia stem cells (LSCs), which possess properties such as quiescence and self-renewal that are linked to therapy resistance and relapse. The LSC17 score was derived from genes differentially expressed between functionally validated LSC+ and LSC- fractions from 78 AML patients and is strongly associated with survival and response to standard therapy. A critical advantage of the LSC17 test over cytogenetic and molecular analysis is its rapid turnaround time (24-48h on a NanoString platform), providing clinicians with a rapid and powerful tool for upfront risk stratification. We have developed a clinical assay for the LSC17 score validated in a CAP/CLIA-lab setting. Methods: We conducted a prospective, multicenter validation and feasibility study to test the prognostic value of the LSC17 assay under real-world conditions in AML patients treated with curative intent. Patients with a possible new diagnosis of AML were eligible. Patients with a confirmed diagnosis of acute promyelocytic leukemia were excluded from analysis. Standard prognostic markers including cytogenetics, molecular studies and targeted sequencing using a standard AML panel were performed in parallel to the LSC17 score. Treatment was administered according to physician preference, based on patient history and results of standard prognostic assays, when available. Survival data was censored on June 14th, 2020. Results: 381 patients were recruited to the study between June 2016 and March 2020. 4 patients were excluded for quality control reasons (one sample had insufficient RNA and three samples failed quality control checks). 103 were excluded as they had alternative diagnoses. 84 patients were excluded because they did not receive intensive chemotherapy. LSC17 scores ranged from 0 to 1.25, and were classified as high or low according to the median score of 0.51 from a previously validated reference cohort (Ng et al, Nature 2016). Of the 190 patients included in this analysis, 84 had a low LSC17 score and 106 had a high LSC17 score. The median age was 61 years (range 18-79); 86 (45%) were female. When stratified according to ELN 2017 criteria, 48 (27%), 51 (29%), and 77 (44%) patients had favorable, intermediate, and adverse risk disease, respectively. Low LSC17 score was associated with normal cytogenetics (high vs low, 33% vs 58%; P We first considered response to induction chemotherapy (Table 1). 141 patients had standard induction chemotherapy with 3+7, 40 had Flag-IDA and 9 had CPX-351. High score patients had inferior responses to 3+7 with only 59% achieving complete remission (CR) after 1 cycle of chemotherapy compared to 96% of low score patients; responses for LSC17 high score patients were better in the Flag-IDA group with 80% achieving CR after 1 cycle. When considering overall CR rates after 2 cycles of induction, patients with a high LSC17 score were less likely to achieve CR (high vs low, 87% vs 98%; P=0.02). However, this difference was predominantly observed in patients treated with 3+7 (87% vs 99% CR rate in high vs low score patients, respectively); response rates to Flag-IDA were not significantly different between the 2 groups. Measurable residual disease (MRD) monitoring by flow cytometry was performed at the time of CR in 135 (71%) patients enrolled at Princess Margaret Cancer Centre. Patients with a high LSC17 score were significantly more likely to have MRD compared to low score patients (46% vs 10% respectively, P Conclusion: AML patients with a high LSC17 score have inferior outcomes following 3+7 induction chemotherapy. The LSC17 score should be considered as a tool to identify and stratify high-risk patients to alternative upfront therapies such as Flag-IDA. A risk adapted study is planned to validate these results. Disclosures Gupta: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding. Maze:Novartis: Honoraria; Takeda: Research Funding; Pfizer: Consultancy. McNamara:Novartis: Honoraria. Schimmer:Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock ; Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria. Leber:Takeda/Palladin: Honoraria, Membership on an entity's Board of Directors or advisory committees; Treadwell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees; Lundbeck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Tierens:Amgen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees. Wang:Trilium therapeutics: Patents & Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .

Background and objectives: CPX-351 is a liposomal formulation of daunorubicin and cytarabine in a fixed synergistic ratio of 5:1. It has been approved by both the FDA and EMA for use in high risk AML including therapy related AML (t-AML) and AML with myelodysplastic related change (AML-MRC). When compared with '3+7', CPX-351 resulted in superior OS (9.56 vs 5.96 months) and overall response rate (47.7% v 33.3%; P = .016; Lancet JCO 2018). However, this is little data of CPX-351 in a real world setting. The primary objective of our multi-centre study was to assess outcomes and associated toxicities of CPX-351 in these high risk AML patients. Methods: Patients aged 18 and over who were treated with CPX-351 and who met the WHO criteria for t-AML and AML-MRC were included in the study. Retrospective data was collected from 6 centres throughout Canada. Targeted sequencing was performed on DNA samples using the TruSight Myeloid Sequencing Panel isolated from peripheral blood or bone marrow samples at diagnosis. Overall survival (OS) and progression free survival (PFS) rates were calculated using the Kaplan-Meier method. Results: Fifty patients treated with CPX-351 were identified from six centres. Baseline characteristics are seen in Table 1. Cytogenetics was available in 45 (90%) patients and an abnormal karyotype was seen in 30 (60%) patients. Monosomal (33%) and complex (28%) karyotype were the most common chromosomal abnormalities seen. Targeted sequencing was performed on 64% patients (32/50) and the average number of mutations was 2(0-7). RUNX1 was the most commonly mutated gene found in 22% (7/32), followed by SRSF2 in 19% (6/32) with ASXL1 and NRAS both at 16% (5/32). Other commonly mutated genes were NPM1, IDH2, DNMT3a and TP53 all occurred at a frequency of 12% (4/32). Of the 10 patients (20%) that were FLT3 mutated (80% ITD and 20% TKD), 5 patients received the FLT3 inhibitor midostaurin in combination with CPX-351. Assessing treatment responses, 50% (25/50) of patients achieved a complete remission (CR) or a complete remission with incomplete recovery (CRi). Median neutrophil recovery (≥500/μl) was 32 days (range 18-68) and median platelet recovery (≥50 000/μl) was 34 days (range 19-70). Proven or probable fungal infection was seen in 10 (20%) patients. 30 day mortality was 4% (2/50) and 60-day mortality was 10% (5/50). The median CR duration was short at 7.17 months. Of the 25 patients who did not achieve a CR, 6 (24%) had prior azacitidine use for MDS suggesting CPX-351 may not have activity in this group of patients. Median follow up was 7.43 months (range 0.2 to 18 months). OS was 44% at 12 months (0.28-0.58 95% CI) and 29% at 18 months (Fig 1; 0.13-0.46 95% CI). PFS was 28% at 12 months (0.15-0.43 95% CI) and 18% at 18 months (0.06-0.39 95% CI). There were no differences in OS when stratified by ELN risk (p=0.25), adverse risk cytogenetics (p=0.1485), or poor risk mutations such as FLT3-ITD (p=0.29), RUNX1 (p=0.123) or ASXL1 (p=0.06). This is consistent with previous studies, which suggest that CPX-351 overcomes the poor prognosis associated with these mutations. 18 (36%) patients received an allogeneic stem cell transplant (ASCT) in CR1. Patients who received a allogeneic transplant had significant improvement of OS of 62% at 18 months compared to those who did not receive a transplant of 14.5% at 18 months (Fig 2; p=0.0008). Conclusion Our study reveals that CPX-351 produces high remission rates in patients with AML with a similar efficacy and toxicity profile seen in the Phase 3 trial data (Lancet JCO 2018). However, response rates are short and therefore CPX-351 is most effective when used as a bridge for allogeneic stem cell transplantation in this high risk population. Disclosures Assouline: AbbVie: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Takeda: Research Funding; BeiGene: Consultancy, Honoraria, Research Funding. Brandwein:Celgene: Honoraria; Astellas: Honoraria; Taiho: Honoraria; Roche: Honoraria; Jazz Pharmaceuticals: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Gupta:Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Maze:Pfizer: Consultancy; Takeda: Research Funding; Novartis: Honoraria. McNamara:Novartis: Honoraria. Sanford:Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Schimmer:Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock ; Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria.

Upfront therapy for newly diagnosed patients with acute myeloid leukemia (AML) includes intensive induction chemotherapy with curative intent, low dose chemotherapy, best supportive care, and clinical trials. The choice between these therapies is influenced by multiple factors including age, cytogenetic and molecular mutations, and performance status. In our single payer provincial health care system, induction chemotherapy and clinical trials are only offered at a small number of specialized quaternary care centers with geographically large catchment areas. As a result, some patients are required to travel long distances for their appointments, which may constitute a barrier to care, especially among elderly patients. We therefore asked whether distance from the quaternary center influences the choice of care for AML. We reviewed the records of patients ≥18 years of age diagnosed with AML from 2015-2017 and assessed at our quaternary care center in Toronto, Canada. We compared upfront therapy choice and survival between patients living close versus distant from the cancer center (empirically defined as 50km) and stratified by age. A total of 675 patients were assessed by our quaternary center for a new diagnosis of AML during the timeframe studied. Of those patients, 477 (71%) patients lived ≤50km, and 198 (29%) patients lived >50km from the quaternary center. The overall median distance from patient residence to the quaternary center was 33.2km (range: 1-1791km), and the median distance of patients in the >50km group was 93km (range: 50.2-1791km). Age, sex, baseline Eastern Cooperative Oncology Group Performance Status (ECOG), and cytogenetic risk were not significantly different between the two groups. There were no differences in the proportion of patients receiving induction chemotherapy or clinical trial as upfront therapy between patients living close versus distant from the quaternary center, even when stratified for age ≥70 years. There was no difference in overall survival between patients living ≤50km versus >50km from the quaternary center either overall, or when stratified by age. In conclusion, geographic distance from treatment center does not appear to impact choice of upfront therapy, access to clinical trials, or clinical outcomes in this study of newly diagnosed patients with AML treated in a single payer environment. Disclosures Gupta: Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding; Pfizer: Consultancy; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maze:Novartis: Honoraria; Pfizer: Consultancy; Takeda: Research Funding. McNamara:Novartis: Honoraria. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .

Introduction: Clinical outcomes of acute myeloid leukemia (AML) in adolescents and young adults (AYA) are rarely reported as an isolated subgroup. Treatments vary little across age groups, and treatment intensity depends upon comorbid conditions and performance status. Optimal treatment strategies focused on disease behavior, biological factors, and the distinct needs of this subset of AML patients remain elusive. The purpose of this retrospective analysis is to determine the characteristics and outcomes of AYA AML patients treated at a specialized adult leukemia cancer center in comparison to older adults with AML (40-60 years). Methods: A retrospective analysis was performed on all patients treated at Princess Margaret Cancer Center from 2008-2018. Patients with acute promyelocytic leukemia were excluded. Clinical characteristics, treatment strategies, and survival outcomes were recorded for all patients. Overall survival (OS) and disease-free survival (DFS) rates were calculated using the Kaplan-Meier product-limit method and the impact of covariates were assessed using the Log-rank test. Finally, we compared the outcomes of AYA patients treated at our centre between 2015-2018 with older patients. Results: A total of 175 patients aged 18-39 were identified. Patient characteristics are shown in (Table 1). Cytogenetic were available in 163 patients. Based on MRC criteria, 27 (16%) were favorable risk, intermediate in 95 (54%), adverse in 39 (22%), and missing/failed in 14(8%). NPM1 status was available in 110 patients of whom 38 (35%) were positive. FLT3-ITD was available in 67 patients with 24 (36%) positive. Both mutations were present in 13 (54%) patients. There were no significant differences in terms of risk stratification based on cytogenetic and molecular markers based on age (18-29 vs.30-39) (P= 0.98). Most patients 172 (98%) received induction, 157 (91%) with 3+7, and 15 (9%) with FLAG-IDA. Complete remission (CR) was achieved in 133 (77%) after first induction [120 (76%) after 3+7 and 11 (73%) after FLAG-IDA]. Induction related mortality was low (2%). Of the 39 who did not achieve CR, thirty-four patients received re-induction (13 FLAG-IDA, 16 NOVE-HiDAC, 5 others) with CR in 21 (62%). Overall, 154 (89.5%) achieved CR1. Sixty-four (42%) proceeded to hematopoietic stem cell transplantation (HSCT) in CR1. 59 (38%) patients relapsed in CR1 with 8 (12%) relapsing post HSCT. Fifty-five (5 post HSCT) patients received reinduction with 30 (51%) (2 after HSCT) achieving CR2. Fifteen patients received HSCT in CR2. OS and DFS at 2 years were 62% (95% CI 0.53-0.69) and 50% (95% CI 0.41-0.57), respectively. Stratified by cytogenetic risk, OS was 81% for favorable risk, 61% for intermediate, and 50% for adverse risk (P=0.0001), respectively. DFS in these groups was 85%, 57%, and 46 % (P=0.0025), respectively. We further compared outcomes in the 18-29y and 30-39y age groups. The OS was 61.9% compared to 62.5% (P=0.91) and DFS of 52.1% compared to 47% (P=0.65) respectively. On univariate analysis for OS and DFS, cytogenetic risk stratification was the only significant variable (P=0.0004 and P=0.0042). We then compared the outcomes 67 sequential patients aged I8-39 treated from 2014-2018, with those of 176 sequential patients aged 40-60 treated during the same period (table 2). OS at 2 years was not statistically higher in the younger group compared to the older group (66.7% vs. 61.2%, P=0.372). While relapse rate was lower in older patients (15.5% vs. 22.6%, P=0.093), NRM was higher in older patients (29.7% vs. 18.8%,P=0.094). Conclusion: AYA pts. occupy a unique niche amongst AML as a whole. While treatment responses have improved in general, there may be potential for further gains in these patients. Increased tolerance for more intense treatment strategies as well as the incorporation of novel agents into standard treatment protocols may provide a means to optimize care in AYA patients. Finally, research is needed to elucidate biological mechanisms and predictors of disease behavior instead of arbitrary, age-stratified treatment schema. Disclosures McNamara: Novartis Pharmaceutical Canada Inc.: Consultancy. Schimmer:Jazz Pharmaceuticals: Consultancy; Medivir Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy. Schuh:Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Teva Canada Innovation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees. Maze:Pfizer Inc: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yee:Astellas: Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MedImmune: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Hoffman La Roche: Research Funding. Minden:Trillium Therapetuics: Other: licensing agreement. Gupta:Incyte: Honoraria, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction: A 17-gene stemness score (LSC17 score) had been reported to determine the risk of therapy resistance in acute myeloid leukemia (Nature 2016), and this was replicated successfully in 5 independent cohorts (n=908). When the patients were stratified according to the median value of the LSC17 score, allogeneic hematopoietic stem cell transplantation (HCT) did not affect overall survival (OS) from initial diagnosis for either high- or low-score patients (p=0.2 for high and p=0.06 for low LSC17 score groups). In the present study, we aimed to further perform a subgroup analysis confined to the patients receiving allogeneic HCT and determine whether the LSC17 score at leukemia diagnosis was associated with treatment outcomes including OS, leukemia-free survival (LFS), non-relapse mortality (NRM), relapse incidence (RI), and acute/chronic GVHD following allogeneic HCT. Methods and patients: Out of 452 patients with available LSC17 scores, 123 patients were included into the final analysis who received allogeneic HCT using matched (n=104, 84.6%) or mismatched/haploidentical donors (n=19, 15.4%). 80 patients were from the previous study (Nature 2016), while 43 patients were a prospectively accrued cohort during 2016-2018. Patients and transplant characteristics were: male/female (n=61/62); median age, 51 (17-73); CR status prior to HCT, CR1 (n=93, 75.6%), CR2 (n=30, 24.4%); Conditioning regimen, reduced intensity/myeloablative conditioning (n=59, 48.0% vs n=64, 52.0%); GVHD prophylaxis using post-transplant cyclophosphamide (PTCy; n=45, 36.6%) or T cell depletion (n=62, 50.4%); Cytogenetic risk, favorable (n=10, 8.1%), intermediate (n=70, 56.9%), adverse (n=26, 21.1%), inconclusive or not done (n=17, 13.8%). The LSC17 score for each patient was measured in a diagnostic sample using a NanoString assay and compared to the high/low threshold of a reference AML cohort (Ng et al, Nature 2016 and unpublished data). Transplant outcomes were compared according to the LSC17 risk group for OS, LFS, NRM and RI. Univariate and multivariate analyses were conducted for OS and LFS using Cox's proportional hazard model or for NRM and RI using Fine-Gray model, respectively. The following variables were included in the model: the LSC17 score group (high vs low LSC17 score), chronic GVHD, CR status (CR2 vs CR1), Cytogenetic risk (adverse vs favorable/intermediate/inconclusive), GVHD prophylaxis (PTCy vs others, T-cell depletion vs others), Age (above 60 vs others), donor type (mismatched/haploidentical vs matched donors). Results: With a median follow-up duration of 22 months among survivors after HCT, 23 patients experienced relapse (n=23, 18.7%) while 63 deaths (51.2%) were noted. Out of 123 patients, 58 (47.1%) had a low LSC17 score and 65 (52.9%) had a high LSC17 score. There was no difference in the distribution of LSC17 scores between the group who received HCT (n=123; 0.479±0.026) vs not (n=229; 0.456±0.019; p=0.491). LFS survival was significantly better in the low LSC17 score group (51.5 vs 32.4% for 2-year LFS rate, p=0.0219), and there was a trend to higher OS rate in the low LSC17 group (48.1%) compared to the high LSC17 group at 2 years (34.2%, p=0.09). Furthermore, patients with a low LSC17 score had a significantly lower RI (14.9% vs 27.3% for 2-year relapse incidence, p=0.028). There is no difference of NRM between the groups (37.2% vs 38.2% at 2 years, p=0.647). Multivariate analysis confirmed that the high LSC17 score group was associated with worse LFS (HR 1.874 [1.080-3.249], p=0.025). However, it was not confirmed with respect to OS or relapse incidence. As expected, it was not associated with NRM. Conclusion: A low 17-gene stemness score is associated with better leukemia-free survival and lower relapse incidence after allogeneic HCT, and is suggested to be associated with OS. The high LSC17 score group may be considered for novel therapeutic strategies to reduce the risk of relapse after allogeneic HCT. Figure Disclosures Chan: Celgene: Honoraria, Research Funding; AbbVie Pharmaceuticals: Research Funding; Agios: Honoraria. Minden:Trillium Therapetuics: Other: licensing agreement. Michelis:CSL Behring: Other: Financial Support. Mattsson:Gilead: Honoraria; Celgene: Honoraria; Therakos: Honoraria. Wang:Pfizer AG Switzerland: Honoraria, Other: Travel and accommodation; Pfizer International: Honoraria, Other: Travel and accommodation; Trilium therapeutics: Other: licensing agreement, Research Funding; NanoString: Other: Travel and accommodation.

Background: AML is driven by a small subpopulation of leukemia stem cells (LSCs), which possess stem-cell properties such as quiescence and self-renewal that are linked to therapy resistance and relapse. The LSC17 score was derived from genes differentially expressed between functionally validated LSC+ and LSC- cell fractions from 78 AML patients. The LSC17 score was strongly associated with survival in 4 independent cohorts of AML patients treated with curative intent (n = 908), and accurately predicted initial response. Patients with high LSC17 scores had poor outcomes with standard treatment strategies. The LSC17 score remained highly significant in multivariate analyses, independent of commonly used prognostic factors. A critical advantage of the LSC17 test over cytogenetic analysis is its rapid turnaround time (24-48h on a NanoString platform), providing clinicians with a powerful tool for upfront risk stratification. To date, no RNA-based, stem cell-derived score has been transitioned into a Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Study design and methods: The study consists of 2 phases. Phase 1 aims to validate the assay in a CLIA certified laboratory setting. Phase 2 aims to determine prospectively the feasibility and prognostic power of LSC17 score testing in newly diagnosed AML patients in the real-world setting. Clinical endpoints include primary induction failure rate, relapse free survival and overall survival. All patients with a suspected diagnosis of de novo or secondary AML, who are deemed fit and appropriate by their treating physician to undergo intensive induction chemotherapy, are considered for this study. Patients who received any prior anti-leukemia treatments (except hydroxyurea) and patients with a confirmed diagnosis of acute promyelocytic leukemia are excluded. Current participating centres include Princess Margaret Cancer Centre (Toronto), Juravinski Cancer Centre (Hamilton), and The Ottawa Hospital Cancer Centre (Ottawa). Pre-study sample size analysis suggests that 150 patients will be required to demonstrate a hazard ratio for death of 2.3 between patients with a high and low LSC17 score (α = 0.05, power = 0.8). The survival for the high and low LSC17 score groups will be compared using the Cox proportional hazards model. Traditional risk stratification will also be tested within a Cox proportional hazards model. Phase 1 of the study has been completed and several key quality control measures have been created. Initial derivation and validation of the LSC17 score was performed using standard chemistry on the NanoString platform; for CLIA lab validation, the assay was transitioned to Elements© chemistry, which does not require custom codeset manufacture by NanoString. The original AML reference cohort was retested using Elements© chemistry to derive an absolute median threshold for prospective LSC17 score determination in individual patients. The lab validation process compared and found no difference in LSC17 scores between samples processed by Ficoll or collected in Paxgene for ease of processing. A standardised quality assurance (QA) process was completed to identify optimal sample requirements as well as specimen storage conditions, score stability during sample storage and turnaround time for testing. An algorithm has been created using the laboratory information system to allow standardised and rapid calculation of the LSC17 score from NanoString nCounter output data. The LSC17 score can be tested on peripheral blood or bone marrow, although bone marrow samples are preferred for patients with very low peripheral blast counts. Samples are ideally stored in RNA Paxgene tubes for RNA stability and to maximize RNA yield. The prospective phase of the study (Phase 2) opened in April 2018 and as of June 2019, 233 patients have been enrolled, of which 120 received induction chemotherapy. 54 patients were excluded due to an alternative diagnosis or failed QA. The remaining patients had non-intensive therapy based on patient choice. Standard prognostic markers including cytogenetics, molecular studies and targeted sequencing using a 49-gene AML panel are performed in parallel to the LSC17 score. Treatment was administered according to physician preference, based on patient history and results of standard prognostic assays, when available. The study continues to recruit and is open to collaborations in other centres. Disclosures Ng: Celgene: Research Funding. Leber:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sabloff:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; ASTX: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; Actinium Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi Canada: Research Funding. Minden:Trillium Therapetuics: Other: licensing agreement. Wang:NanoString: Other: Travel and accommodation; Trilium therapeutics: Other: licensing agreement, Research Funding; Pfizer AG Switzerland: Honoraria, Other: Travel and accommodation; Pfizer International: Honoraria, Other: Travel and accommodation.

Background: Venous thromboembolism (VTE) is a well-known complication in adults with acute lymphoblastic leukemia (ALL) receiving asparaginase (ASNase) based therapy. The MD Anderson Cancer Centre reported an overall VTE incidence of 29% (14/49) in patients with Philadelphia positive ALL (Ph+ ALL) treated with Hyper-CVAD and in the absence of ASNase (Vu et al. Cancer Medicine. 2015;4(1):27-35. doi:10.1002/cam4.332). The VTE incidence in adult Ph+ ALL patients treated with tyrosine kinase inhibitor plus the pediatric-inspired regimen modified from the Dana Farber Cancer Institute (DFCI) ALL protocol remains unclear. We have previously compared the incidence of VTE in adults with Ph+ ALL treated with or without ASNase during the intensification phase of the modified DFCI treatment. This study suggested that patients with Ph+ve ALL experience high incidence of VTE, even in the absence of ASNase, and that this risk is not restricted to the intensification phase. As ASNase was omitted from the protocol due to toxicity since 2008, in this report, we provide further insight into the risk of VTE in adults with Ph+ ALL during the entire ASNase-free DFCI treatment course. Methods: A single-institution retrospective review of sequential Ph+ ALL patients treated at Princess Margaret Cancer Center (PMCC) from 2008-2018 with an imatinib-containing DFCI protocol-based regimen that omitted ASNase. VTE events were recorded from the time of diagnosis to the end of modified DFCI treatment, including at the time of allogenic bone marrow transplantation. Co-variants of age, sex, weight, BMI, Padua score, white blood cell count, and platelet count were also collected. Statistical analyses were performed using the 2-tailed t test. Results: Overall, 130 patients were included for analysis; none received prophylactic anticoagulation. Among the 130 Ph+ ALL patients treated with the modified DFCI protocol, 28 (21.5%) patients had at least 1 VTE events from diagnosis to the end of treatment. The mean age of patients with and without VTE were 56.8 and 49.0 years, respectively (p=0.014) (Table 1). The majority of the VTE events occurred during active treatment, with 9 (7.0%), 11 (8.5%), and 4 (3.1%) occurring during induction, intensification, and maintenance, respectively (Figure 1). Only four patients (3.1%) presented with VTE at the time of diagnosis. Of all VTE events, 14 (50%) were DVT and/or PE, 12 (43%) were line-related, and 2 (7%) involved a cardiac thrombus (Table 2). The average Padua score for these patients was 5.4 with a range of 3-10. All VTE events were treated with low weight molecular heparin (LMWH), with 2 patients experiencing a recurrent episode within a year while on LMWH treatment. Bleeding following anticoagulation was rare. One patient experienced a major bleeding into the psoas muscle while on LMWH plus platelet transfusion for a right atrial clot during the induction phase in ICU. No other significant bleeding event was observed. Conclusion: VTE was observed in more than 20% of adults with Ph+ ALL patients treated with imatinib plus a modified DFCI pediatric ALL protocol. The majority of VTE events occurred during active treatment (induction and intensification), and DVT/PE incidence was similar to that of line-related thrombosis. The high observed VTE incidence suggests that prophylactic anticoagulation should be considered for adult patients with Ph+ ALL even in ASNase-free regimens, especially if additional thromboembolic risk factors are present. Disclosures Maze: Pfizer Inc: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Chan:Celgene: Honoraria, Research Funding; Agios: Honoraria; AbbVie Pharmaceuticals: Research Funding. Gupta:Sierra Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding. Yee:Novartis, Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas, Celgene, Otsuka, Shire, Takeda: Membership on an entity's Board of Directors or advisory committees; Agensys, Astex, Hoffman La Roche, MedImmune, Merck, Millenium, Roche/Genentech: Research Funding. Minden:Trillium Therapetuics: Other: licensing agreement. Schimmer:Novartis Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy; Medivir Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Schuh:Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Teva Canada Innovation: Honoraria, Membership on an entity's Board of Directors or advisory committees. McNamara:Novartis Pharmaceutical Canada Inc.: Consultancy.

Acute myeloid leukemia (AML) cells and stem cells have unique mitochondrial characteristics with an increased reliance on oxidative phosphorylation (OXPHOS). To identify new biological vulnerabilities in the mitochondrial proteome of AML cells, we conducted an shRNA screen and identified neurolysin (NLN), a zinc metalloprotease whose mitochondrial function is not well understood and whose role in AML has not been previously reported. To begin our investigation into the role of NLN in AML, we analyzed NLN gene expression in a database of 536 AML and 73 normal bone marrow samples. NLN was overexpressed in 41% of AML samples. Overexpression of NLN in primary AML cells compared to normal hematopoietic cells was confirmed by immunoblotting. To validate the results of the screen and to determine whether NLN is required for AML growth and viability, we knocked down NLN in the leukemia cell lines OCI-AML2, MV4-11, NB4, and TEX with shRNA. NLN knockdown reduced leukemia growth and viability by 50-70%. Moreover, knockdown of NLN in AML cells reduced the clonogenic growth of leukemic cells in vitro and the engraftment of AML cells into mouse marrow after five weeks by up to 80% and 85%, respectively. The mitochondrial function of NLN is largely unknown, so we identified NLN's mitochondrial protein interactors in T-REx HEK293 cells using proximity-dependent biotin labeling (BioID) coupled with mass spectrometry (MS). This screen identified 73 mitochondrial proteins that preferentially interacted with NLN and were enriched for functions including respiratory chain complex assembly, respiratory electron transport, and mitochondrion organization. Therefore, we assessed the effects of NLN knockdown on OXPHOS. NLN knockdown reduced basal and maximal oxygen consumption, but there were no changes in the levels of individual respiratory chain complex subunits. To understand how NLN influences OXPHOS, we examined the formation of respiratory chain supercomplexes (RCS). Respiratory chain complexes I, III, and IV assemble into higher order quaternary structures called RCS, which promote efficient oxidative metabolism. NLN knockdown significantly impaired RCS formation in T-REx HEK293, OCI-AML2, and NB4 cells, which was rescued by overexpressing wild-type shRNA-resistant NLN. RCS have not been previously studied in leukemia. Therefore, we analyzed their levels in primary AML patient samples and normal hematopoietic cells. RCS assembly was increased in a subset of AML patient samples and positively correlated with NLN protein expression (R2 = 0.83, p < 0.05), suggesting that NLN mediates RCS assembly in AML. To investigate how NLN may be regulating RCS assembly, we analyzed our BioID results to identify NLN interactors that are known regulators of supercomplex formation. Among the top interactors was the known RCS regulator, LETM1. Knockdown of NLN in AML cells impaired LETM1 assembly. Of note, knockdown of LETM1 also reduced growth and oxygen consumption of AML cells. As a chemical approach to evaluate the effects of NLN inhibition on AML cells, we used the allosteric NLN inhibitor R2, (3-[(2S)-1-[(3R)-3-(2-Chlorophenyl)-2-(2-fluorophenyl)pyrazolidin-1-yl]-1-oxopropan-2-yl]-1-(adamantan-2-yl)urea), whose anti-cancer effects have not been previously reported. R2 reduced viability of AML cells, as well as two primary AML culture models, 8227 and 130578. R2 impaired RCS formation in OCI-AML2, NB4, 8227, and primary AML cells. Moreover, R2 reduced the CD34+CD38- stem cell enriched population in 8227 cells, reduced LETM1 complex assembly, and impaired OXPHOS in OCI-AML2 and 8227 cells. Finally, we assessed the effects of inhibiting NLN in mice engrafted with primary AML and normal hematopoietic cells in vivo. Treatment of mice with R2 reduced the leukemic burden in these mice without toxicity. Moreover, inhibiting NLN targeted the AML stem cells as evidenced by reduced engraftment in secondary experiments. In contrast, inhibiting NLN did not reduce the engraftment of normal hematopoietic cells. Collectively, these results demonstrate that inhibition of NLN preferentially targets AML cells and stem cells as compared to normal hematopoietic cells. In summary, we defined a novel role for NLN in RCS formation. We show that RCS are necessary for oxidative metabolism in AML and highlight NLN inhibition as a potential therapeutic strategy. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Chan:Agios: Honoraria; AbbVie Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding. Schimmer:Medivir Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy.

Mixed phenotype acute leukemia (MPAL) is a heterogeneous disease consisting of acute leukemia expressing markers of both myeloid and lymphoid lineage. The role of hematopoietic stem cell transplantation (alloHSCT) remains uncertain due to lack of prospective trials . Several studies have retrospectively examined its role in pediatric and younger adult subgroups. Myeloablative conditioning (MAC) can result in an overall survival (OS) of 56% at 3 years, but is not suitable for older and frail patients. With expanding transplant availability and alternative donors, we sought to evaluate the outcomes of our cohort of MPAL patients, including older adults unfit for MAC, in a heterogeneous, real-world scenario compared with the outcomes of patients who were consolidated with chemotherapy only. Methods: Seventy four patients, aged 18 years or older, at the Princess Margaret Cancer Center between January 1, 2000 and December 31, 2018 were evaluated. All patients included in analysis met the WHO 2016 classification criteria for MPAL. Overall survival and relapse free survival rates were calculated using the Kaplan-Meier product-limit method. The log-rank test was used to compare the two groups with respect to OS and relapse free survival. Results: Baseline characteristics of the patient cohorts are shown in Table 1. Among the 74 MPAL patients included in this study, 59 (79%) received induction chemotherapy. Complete remission (CR) was achieved in 47 (79%) treated patients. Twenty-five of 36(80%) achieved a CR using ALL protocols (DFCI Protocol, Hyper-CVAD), while 9 of 23 (39%) achieved a CR using AML protocols (3+7, FLAG-Ida). Consolidation treatment post CR was evenly split, with 24(51%) receiving chemotherapy followed by an alloHSCT and 23 (49%) receiving chemotherapy only. In the alloHSCT group, RIC was used in 20 (81%) patients while MAC was used in four younger patients with a median age of 51 and 30 respectively. Donor types included sixteen matched unrelated donors, five sibling donors, two haploidentical donors, and one double umbilical cord. The median age of the transplant cohort was 50 years with a median OS of 21 months. In the chemotherapy only group, the median age was 57 with a median OS of 18 months. ALL-type treatment was used for consolidation in 74% of patients with 83% using the asparaginase-containing DFCI protocol. Univariate analysis demonstrated no difference in outcomes with several parameters including age, induction chemotherapy, donor source, and conditioning intensity. The sole predictor of poor OS on univariate analysis was acute graft versus host disease (aGVHD) with a hazard ratio of 3.3 (95% CI 0.9-11). In total, 12 patients (50%) had aGVHD with a median OS of 16 months. However, when Grade 1/2 and Grade 3/4 aGVHD were compared, median OS was not reached in patients with G1/2, compared with an OS of 11 months in those patients with G3/4 aGVHD. Chronic GVHD was present in 6 patients and did not impact OS (HR 0.9892 CI 95% 0.2-2.9). Post-transplant relapses occurred solely in the RIC group and were early, occurring within a median of 124 days (range 87-188). Although, there was no relapses in the MAC group, the non-relapse mortality (NRM) was 75%, with patients dying from either aGVHD or infection. The NRM in the RIC group was 17.65%. The 3 year OS and relapse free survival (RFS) for the entire cohort were 30% and 32%, respectively. When comparing those patients that had undergone alloSCT and those that who received chemo alone, survival in the two groups were similar with a 1 year OS of 75% vs. 68% and a 3 year OS of 29% vs 32%, respectively (Figure 1). The 3 year RFS was also similar at 29% and 34%, respectively. Subgroup analysis between the two groups was performed for patients with specific poor prognostic factors (older age, complex cytogenetics, higher WCC, conditioning regimen used), but results showed no significant survival differences. Conclusions Older patients with MPAL have an inferior prognosis and worse outcomes with alloHSCT compared with those of younger adults or children. Despite the increase in access to alloHSCT and an expanding pool of alternative donors, outcomes with RIC remain similar to consolidation with chemotherapy alone. There may be some evidence to suggest that the graft versus leukemia effect can be harnessed to improve outcomes in selected patients. Further research towards optimizing patient outcomes is clearly required to address the needs of older patients unfit for MAC. Disclosures Gupta: Sierra Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding. Mattsson:Gilead: Honoraria; Celgene: Honoraria; Therakos: Honoraria. Maze:Pfizer Inc: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Michelis:CSL Behring: Other: Financial Support. McNamara:Novartis Pharmaceutical Canada Inc.: Consultancy. Schimmer:Jazz Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy; Medivir Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Consultancy. Schuh:Teva Canada Innovation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Teva Canada Innovation: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yee:Astex: Research Funding; MedImmune: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffman La Roche: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding. Minden:Trillium Therapetuics: Other: licensing agreement.

Introduction: The introduction of next-generation sequencing (NGS) has expedited the discovery of novel genetic lesions in acute myeloid leukemia (AML), thereby allowing better risk stratification with respect to overall survival (OS). We have previously reported that AML patients with PTPN11 and NPM1 mutations had longer OS following chemotherapy, while those carrying mutations in ASXL1, JAK2, RUNX1, TP53 and SRSF2 had a shorter OS (Daher-Reyes,ASH 2018). Little is known, however, regarding the impact of genetic profiles (somatic mutations and cytogenetic abnormalities) at initial AML diagnosis on the treatment outcomes following allogeneic hematopoietic stem cell transplantation (HCT). Methods & Patients: We enrolled AML patients who had available NGS data at time of initial diagnosis as part of the AGILE project between February 2015 and December 2018, and who subsequently underwent allogeneic HCT. NGS was performed on DNA samples isolated from peripheral blood or bone marrow samples at diagnosis. Analysis was performed using the TruSight Myeloid Sequencing Panel on the MiSeq sequencer (Illumina; San Diego, CA). Transplant outcomes (overall survival (OS), relapse-free survival (RFS), relapse incidence (RI), and non-relapse mortality (NRM)) after HCT were compared according to genetic profiles defined at diagnosis. Survival analysis for OS and RFS was performed using Cox's proportional hazard model, while the Fine-Gray model was used for RI and NRM analyses. Variables considered in the model included CR status prior to HCT (CR1 vs. beyond CR1), de novo AML (vs. secondary/therapy-related AML), induction chemotherapy used (3+7 vs. others), conditioning regimen (myeloablative vs. reduced intensity), WBC, age, donor type, mutation status of commonly mutated genes, and the composite adverse genetic profile (defined as having at least one of monosomal karyotype (MK), TP53 mutation, del(5), complex karyotype (CK), and monosomy 7), given that these 5 features were highly co-occurring, adverse prognostic factors (Figure 1A). Results: We identified 435 patients in whom frontline NGS was performed, of whom a total of 178 patients (40.9%) received HCT and were included in the final analysis. A total of 598 (median 4, IQR 2-5) mutations were identified in 165 patients (n=165/178, 92.7%). Among 54 genes in the panel, 12 genes were mutated in more than 10% of the cohort, with the most commonly mutated genes being DNMT3A (30.3%), TET2 (25.3%), NPM1 (22.5%), RUNX1 (18.5%), IDH2 (16.9%), FLT3 (15.7%), ASXL1 (12.4%), BCOR (12.4%), CEBPA (11.2%), NRAS (11.2%), IDH1 (10.1%), and SRSF2 (10.1%). In univariate analysis, the groups with a composite adverse genetic profile (n=30/178, 16.9%) showed decreased OS (HR 2.19 [1.30-3.67]; p=0.003), while patients harbouring spliceosome gene (SF3B1, SRSF2, U2AF1, and ZRSR2) mutations (n=37/178, 20.8%) had longer OS (HR 0.39 [0.18-0.85]; p=0.018), with 2-year OS rates of 24.9% and 57.9%, respectively (p=0.002)) (Figure 1B). The composite adverse genetic profile was also associated with shorter RFS (HR 2.23 [1.34-3.69]; p=0.002), while spliceosome gene mutations were associated with longer RFS (HR 0.42 [0.20-0.88]; p=0.022), with 2-year RFS rates of 23.7% vs. 57.9%, respectively (p=0.001)). The composite adverse genetic profile was also associated with higher RI (HR 2.94 [1.52-5.66]; p=0.001), with 2-year RI rates of 47.2% vs. 17.2%, respectively, for patients with and without adverse genetic features (p=0.002) (Figure 1C). Neither the composite adverse genetic profile, nor spliceosome gene mutations, were associated with NRM, with HR of 1.21 [0.55-2.65], p=0.64) and 0.45 [0.16-1.31], p=0.15, respectively (Figure 1D). Multivariate analyses confirmed that the composite adverse genetic profile and spliceosome gene mutations were independent prognostic factors for OS, RFS, and RI (p=0.004, p=0.002, and p=0.001, respectively) and for OS and RFS (p=0.020 and p=0.022, respectively). Conclusion: In our cohort, the composite adverse genetic profile (i.e. having at least one of MK, TP53 mutation, del(5), CK and monosomy 7 remained as a poor prognostic factor even after allogeneic HCT. To clarify the role of genetic risk stratification in HCT, further analysis using a larger cohort is warranted. In addition, a comparative analysis between HCT vs no-HCT groups according to the genetic profile, is ongoing in a in a larger patient cohort. Figure 1 Disclosures Michelis: CSL Behring: Other: Financial Support. Mattsson:Gilead: Honoraria; Celgene: Honoraria; Therakos: Honoraria. Schimmer:Novartis Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Medivir Pharmaceuticals: Research Funding. McNamara:Novartis Pharmaceutical Canada Inc.: Consultancy. Maze:Pfizer Inc: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Gupta:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Yee:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astex: Research Funding; Hoffman La Roche: Research Funding; MedImmune: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Millennium: Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Minden:Trillium Therapetuics: Other: licensing agreement. Schuh:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria; Teva Canada Innovation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction: Although induction chemotherapy results in a complete remission (CR) in ~70% of newly diagnosed AML patients, post-remission therapies are needed to eliminate minimal residual disease (MRD) and prevent relapse. Consolidation chemotherapy, either as definitive therapy or bridge to bone marrow transplantation (BMT), is currently the most common form of post-remission therapy. Yet, our understanding of its impact on MRD remains limited. In this study, we investigated the effects of consolidation chemotherapy on molecular MRD (mMRD) burden using ultra-deep next generation sequencing (NGS) and correlated treatment response with disease characteristics and survival outcomes in AML patients. Patients and Methods: 91 newly diagnosed AML patients who achieved CR following standard induction chemotherapy were evaluated. Targeted conventional NGS using a 54-gene panel was performed on whole blood (PB) or bone marrow samples collected at diagnosis. PB samples were collected during remission at two consecutive time points (T1 and T2), before and after 1 (n=79) or 2 (n=12) cycles of consolidation chemotherapy, for each patient. To detect mMRD, we used a custom 37-gene hybrid-capture panel and error-corrected NGS based on the duplex sequencing approach with a variant allele frequency (VAF) detection limit of ~1x10-4. For 10 patients, we also performed duplex sequencing analysis on their relapsed samples. Results: NGS of the diagnostic samples identified a total of 298 putative oncogenic mutations in 92% (n=84) of the 91 patients. Ninety percent of these mutations (n=267) were trackable by the custom hybrid-capture panel. Duplex sequencing detected persistence of 56% (n=149) of the trackable mutations in T1 samples; 34% (n=50) of which were clonal hematopoiesis-associated DTA mutations (those involving DNMT3A, TET2, or ASXL1), and the remaining 66% (n=99) were non-DTA mutations. Analysis of T2 samples showed that consolidation chemotherapy reduced the VAF of non-DTA mutations by a median of 73% and cleared 27% (n=27) of them at T2. In contrast, the burden of DTA mutations increased by 0.5% (P < 0.0001 by Mann-Whitney test), and only 2% (n=1) of the mutations was cleared (P = 0.0001 by Fisher's exact test). These findings are consistent with prior studies demonstrating that non-DTA mutations are more reliable markers of leukemic burden than DTA mutations. To study the impact of consolidation chemotherapy at the level of individual patients, the mean VAF of all persistent non-DTA mutations was calculated for each sample and used as a composite measure of mMRD burden (henceforth referred to as "cmMRD"). Analysis of the 10 patients with relapsed samples showed that cmMRD levels tracked well with achievement of remission and disease progression (Fig. 1). In the subset of patients with persistent non-DTA mutations at T1 (n=61), consolidation chemotherapy decreased cmMRD levels by a median of 36% at T2. However, we observed high interpatient variability (Fig. 2); 36% (n=22) of the patients experienced an increase in cmMRD burden after consolidation chemotherapy, and 36% (n=22) had less than a 1 log reduction. Only 28% (n=17) of the patients achieved a log reduction of greater than 1. The likelihood and magnitude of cmMRD response were significantly associated with cytogenetic risk (P = 0.026 by 3x3 Chi-square test; Fig. 3). The proportion of patients with favorable, intermediate, and poor-risk cytogenetics who experienced cmMRD expansion was 17%, 27%, and 71%, respectively. Consistent with these findings, a suboptimal response (defined as cmMRD ratio [T2/T1] > 0.4) was associated with inferior overall survival (HR = 3.29, P = 0.007 by log-rank test; Fig. 4). Conclusions: Our analysis showed that mMRD response to consolidation chemotherapy was highly variable among patients. Although consolidation chemotherapy was effective in deepening the remission for a subset of patients, it failed to lower MRD levels for a substantial proportion of patients, especially those with poor risk cytogenetics. These findings challenge the practice of using consolidation chemotherapy to achieve a deeper remission prior to BMT for high-risk patients and indicate that the opposite outcome may occur instead. NGS-based monitoring of mMRD can potentially be used to distinguish between patients who can remain on consolidation chemotherapy as definitive therapy and those who require a switch in post-remission therapy. Disclosures Gupta: Sierra Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding. Maze:Pfizer Inc: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. McNamara:Novartis Pharmaceutical Canada Inc.: Consultancy. Minden:Trillium Therapetuics: Other: licensing agreement. Schimmer:Medivir Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy. Schuh:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Teva Canada Innovation: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yee:Novartis, Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas, Celgene, Otsuka, Shire, Takeda: Membership on an entity's Board of Directors or advisory committees; Agensys, Astex, Hoffman La Roche, MedImmune, Merck, Millenium, Roche/Genentech: Research Funding. Bratman:SVB: Other: is co-inventor of a patent relating to circulating tumor DNA detection technology, which has been licensed to Roche Molecular Diagnostics.. Chan:Agios: Honoraria; AbbVie Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding.

Introduction Core-binding factor (CBF) acute myeloid leukemias (AML) are characterized by the chromosomal abnormalities t(8;21) or inv(16)/t(16;16) and their fusion proteins RUNX1/RUNX1T1 and CBFB-MYH11, respectively. Despite their relatively favorable prognosis, it has been reported that up to 45% of patients with CBF-AML relapse. Current guidelines recommend monitoring the peripheral blood (PB) or bone marrow (BM) for molecular evidence of residual disease (RD) every 3 months for 2 years following remission. By monitoring patients for rising molecular transcripts, those at risk of impending relapse can be identified and treated prior to the emergence of overt disease. However, the relapse kinetics of CBF-AML are poorly-understood, and it is unknown if serial RD monitoring can detect molecular relapses with sufficient lead-time to intervene and prevent morphologic relapse. Methods After local REB approval, we identified patients with CBF-AML treated at the Princess Margaret Cancer Centre in Toronto, Canada between 2000 and 2017. Patients underwent induction and consolidation chemotherapy according to standard protocols followed by RD monitoring with polymerase chain reaction (qPCR) of RUNX1/RUNX1T1 or CBFB-MYH11 transcripts in a CAP/CLIA certified lab every 3 months for a median of 1.2 years (range 0-5.3). RD assessment was performed on BM aspirates, but PB could be tested in patients who could not tolerate repeat BM aspirates. Morphologic relapse was defined as emergence of >5% blasts in the PB or BM, and molecular relapse as a positive transcript PCR (if previous PCR measurements were undetectable) or an increase by 1 log (if previous PCR measurements were detectable) on 2 successive samples without morphologic relapse. Rapid relapse was defined as Results We included 114 patients with CBF-AML. Median age was 46.5 years (range 18-79). t(8;21) was present in 59% and inv(16)/t(16;16) in 41% of patients. All patients achieved remission with 7+3 induction chemotherapy. Over a median follow-up time of 3.7 years (range 0.2-14.3), RD measurements were performed a mean of 5 times per patient with mean sampling interval of 103±54 days. Remission was maintained in 71 (62%) patients but 43 (38%) developed morphological (n=34) or isolated molecular relapse (n=9), with median time to relapse of 4.4 months (range 1.4-31.4). Patients with relapsed disease were significantly less likely to have achieved ≥3 log reduction in RUNX1/RUNX1T1 or CBFB-MYH11 BM transcripts at the end of consolidation chemotherapy compared to patients who remained in remission (75% vs 90%, p=0.046). Of the 43 patients who relapsed, the majority (74.4%, n=32) had rapid relapse kinetics. Of these 43 patients, 25 received reinduction chemotherapy with achievement of CR2, 6 had refractory disease or death, 1 was lost to follow-up, and 17 went on to receive allotransplantation. RD monitoring enabled timely detection of impending relapse and permitted intervention prior to morphologic relapse in only 11 patients (25.6%). Among these 11 patients with slower relapse kinetics, 6 received reinduction chemotherapy with achievement of CR2, 2 received reinduction chemotherapy with refractory disease, and 8 went on to receive allotransplantation. Median overall survival was 20 months (range 3-128) for patients with rapid relapse kinetics and 28 months (range 14-166) for patients with slow relapse kinetics (p=0.02). Clinical features, BM vs PB RD measurements, additional molecular mutations, and cytogenetic abnormalities did not distinguish patients with rapid vs slow relapse kinetics. Conclusions Current guidelines recommend molecular RD monitoring every 3 months for CBF-AML. However, in the majority of patients who relapsed at our institution, RD monitoring every 3 months provided insufficient lead-time to identify molecular relapses prior to morphologic relapse. Further research is warranted to identify the patients with CBF at the highest risk of relapse and the best strategies to monitor these patients over time. Disclosures Gupta: Novartis: Consultancy, Honoraria, Research Funding; Incyte: Research Funding. Maze:Novartis: Consultancy, Honoraria. Schuh:Celgene: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Jazz: Consultancy; Amgen Inc.: Consultancy; Shire: Consultancy; Teva: Consultancy; Otsuka: Consultancy. Yee:Celgene, Novartis, Otsuka: Membership on an entity's Board of Directors or advisory committees; Agensys, Astex, GSK, Onconova, Genentech/Roche: Research Funding. Schimmer:Otsuka Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow associated with poor clinical outcomes. Conventional chemotherapies are effective in debulking the leukemic burden in most AML patients. However, a small population of disease-sustaining leukemic stem cells (LSCs) frequently persists and contributes to relapsed disease. Novel therapies that eradicate LSCs have the potential to improve clinical outcomes in AML. To discover novel anti-LSC agents, we performed a high-throughput flow cytometry-based drug screen of 1,220 compounds against a primary AML sample (8227). This sample harbors distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation is restricted to the CD34+CD38- fraction. Through this screen, we identified compounds that selectively depleted the CD34+CD38- fraction including four structurally-unrelated NAMPT inhibitors (FK866, STF-118804, GMX1778, and KPT-9274). These inhibitors also depleted the LSC-enriched CD34+CD38- fraction in two other primary AML samples, indicating that the effect was not unique to 8227 cells. To further evaluate their impact on LSCs in 8227, we measured the expression of 104 genes that were previously found to be differentially expressed between LSC+ and LSC- cell fractions isolated from patient samples. Treatment with NAMPT inhibitors reduced the correlation between the measured LSC gene signature and the LSC+ reference profile, providing additional evidence for their anti-LSC activity. To determine whether the selective loss of CD34+CD38- cells was due to cell death or differentiation, we sorted subsets of 8227 cells based on CD34 and CD38 expression and treated each fraction with FK866. NAMPT inhibition preferentially triggered apoptosis as measured by Annexin V staining in the CD34+CD38- and CD34+CD38+ fractions over the CD34- fraction. We did not observe significant changes in the expression of CD34, CD38, or other myeloid differentiation markers (CD14 and CD15) in the remaining viable cells. Our subsequent mechanistic studies focused on KPT-9274 because it is the furthest along in clinical development. NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway that converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), a direct NAD+ precursor. To confirm a decrease in intracellular NAD+ with KPT-9274 treatment, we introduced expression of genetically-encoded biosensors for measuring NAD+ levels in different cellular compartments in an AML cell line. KPT-9274 treatment for 15 hours lowered the free NAD+ pool in the cytosol and mitochondria but not in nucleus. To determine whether the drop in NAD+ was necessary for the effects of KPT-9274 on LSCs, we supplemented the primary AML samples with nicotinamide riboside (NR) which can be directly converted to NMN, thereby bypassing the requirement for NAMPT activity to generate NAD+. The addition of NR completely rescued the effects of KPT-9274 on the CD34+CD38- fraction. Niacin can also generate NAD+ through an alternative pathway that depends on nicotinic acid phosphoribosyltransferase (NAPRT). However, niacin supplementation failed to rescue the effects of NAMPT inhibition which correlated with the lack of NAPRT expression in LSC-enriched CD34+CD38- cells. Next, we studied the effects of KPT-9274 on normal CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from human cord blood. Although HSPCs were sensitive to the pro-apoptotic effects of KPT-9274, their survival was fully rescued by both NR and niacin. The rescue by niacin correlated with a higher expression of NAPRT in HSPCs. As the blood concentration of niacin is ~1,000 fold higher than that of NR, KPT-9274 is predicted to have a favorable therapeutic window in vivo. To demonstrate its in vivo activity, we treated immunodeficient NOD/SCID/IL2Rγ-null (NSG) mice engrafted with a luciferase-tagged AML cell line (OCI-AML3) with KPT-9274 at a dose of 150 or 250 mg/kg/day or vehicle control for 50 consecutive days by oral administration. KPT-9274 treatment significantly lowered leukemia burden and prolonged survival in both dosing cohorts. In summary, our results indicate that NAMPT inhibition represents an effective approach to target human LSCs through reduction in intracellular NAD+ levels and induction of apoptosis. Our data provide the preclinical rationale for investigating the use of KPT-9274 in AML clinical trials. Disclosures Chan: Genentech: Research Funding; Celgene: Research Funding; AbbVie: Research Funding.

Acute myeloid leukemia (AML) is a clinically and biologically heterogeneous disease. Traditionally, cytogenetic analysis has been the backbone for prognostication and treatment decisions. Outcomes vary between age groups with older adults generally having a poorer prognosis. Next Generation Sequencing (NGS) has expedited the discovery of novel genetic lesions in AML to better predict response to intensive chemotherapy and overall survival (OS). The aims of our study were to describe the genetic profile of older adults with AML and to determine its impact on treatment response and survival. We included all new patients with a diagnosis of AML (≥20% blasts in peripheral blood or bone marrow; acute promyelocytic leukemia and myeloid sarcoma were excluded), treated at Princess Margaret Cancer Centre between February 2015 and August 2017. NGS was performed on DNA isolated from peripheral blood or bone marrow samples at diagnosis. Analysis was performed using the TruSight Myeloid Sequencing Panel (Illumina; San Diego, CA) on the MiSeq benchtop genome sequencer (Illumina). Of the 54 genes included in the panel, the complete coding region was sequenced in 15, with hotspot region coverage provided for 39. Demographic and clinical data were obtained from the Princess Margaret Cancer Centre Registry. We identified 454 patients with a new diagnosis of AML in whom frontline NGS was performed. Of these, 300 were 60 years (range 60-92) or older. Demographic and clinical data are shown in Table 1. The median age overall at presentation was 67 yrs (range 18-92) and 57% were male. de novo AML was diagnosed in 329 patients (72%), secondary AML in 17%, and therapy related myeloid neoplasm in 11%. Secondary AML (sAML) was more frequently seen in the ≥60 vs the In total 283 patients (62%) received intensive chemotherapy (95% in the younger group and 45% in the older cohort). 216 patients (76%) achieved a response (CR/CRi/morphologic leukemia free state). No statistically significant difference was seen between age groups (P=0.73) NGS detected 1655 variants. Of these, 1353 classified as oncogenic and 302 as variants of unknown significance (the latter were excluded from subsequent analysis). 14 patients had no mutations identified by NGS. Forty three genes were recurrently mutated in the study cohort: 36 were mutated in >1% of patients, and 11 genes were mutated in >10% of patients. The most commonly mutated genes included DNMT3A (25%), NPM1 (21%), TET2 (19%), RUNX1 (16%), TP53 (16%), ASXL1 (15%), IDH2 (15%), SRSF2 (15%), NRAS (12%), and IDH1 (11%). Older patients had a greater number of mutations overall compared to younger adults (median: 3 vs 2; P = 0.001). In total 986 oncogenic variants were identified in the older group (median 3, range 1-12), while 367 oncogenic variants (median 0, range 0-2) were present in patients < 60 years old (P < 0.001). The older group more commonly harbored mutations in TET2 (25 vs 10%), ASXL1 (21 vs 4%), RUNX1 (20 vs 8 %), SRFS2 (20 vs 6 %), STAG2 (11 vs 6%) and U2AF1 (8 vs 2%). Patients with PTPN11 and NPM1 mutations had longer OS, while patients carrying mutations in ASXL1, JAK2, RUNX1, TP53 and SRSF2 had a shorter OS (Table 2). No differences in OS between age groups (P= 0.2). Multivariate Cox regression analysis showed that male sex (P= 0.0057), sAML (P We then analyzed the correlation between mutations and response rate in patients receiving intensive treatment. PTPN11 and NPM1 mutations were associated with a greater likelihood of achieving a response, while patients with mutations in BCOR, TP53, SF3B1 and U2AF1 had a lower chance of response. Multivariate Cox regression analysis of mutations known to affect response did not show any differences by age, gender, or diagnosis. Mutations in BCOR (P= 0.045), TP53 (P= 0.032) and U2FA1 (P=0.019) were significantly associated with primary induction failure. Overall, older AML patients harbored a greater number of mutations than did their younger counterparts. No age-related differences were observed in mutations known to affect response and/or OS. TP53 mutations, adverse cytogenetics and sAML, were poor prognostic factors regardless of age. Disclosures Schimmer: Medivir AB: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy. Yee:Agensys, Astex, GSK, Onconova, Genentech/Roche: Research Funding; Celgene, Novartis, Otsuka: Membership on an entity's Board of Directors or advisory committees. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte: Research Funding. Maze:Novartis: Consultancy, Honoraria. Schuh:Novartis: Consultancy; Otsuka: Consultancy; Pfizer: Consultancy; Teva: Consultancy; Celgene: Consultancy; Amgen Inc.: Consultancy; Shire: Consultancy; Jazz: Consultancy.

The majority of mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and imported into the mitochondria. A subset of cysteine-rich proteins destined for the mitochondrial intermembrane space are oxidized and folded by the Mitochondrial IMS Assembly (MIA) pathway. We found that genes encoding substrates of the MIA pathway are over-expressed in leukemic stem cells compared to bulk AML cells. Therefore, we assessed the effects of inhibiting the MIA pathway in AML by targeting the FAD linked sulfhydryl oxidase ALR, an integral part of the MIA machinery. Knockdown of ALR with shRNA reduced the growth and viability of OCI-AML2, TEX and NB4 leukemia cells. In addition, ALR knockdown reduced engraftment of TEX cells into mouse marrow, demonstrating an effect on the leukemia initiating cells. The small molecule selective ALR inhibitor, MitoBloCK-6 killed AML cells (IC50 of 5-10 mM) and preferentially reduced the clonogenic growth of primary AML cells over normal hematopoietic cells. MitoBloCK-6 treatment (80 mg/kg i.p. 5 of 7 days x 2 weeks) of mice engrafted with primary AML cells strongly reduced the leukemic burden without changing mouse body weight, serum chemistries, or organ histology. In contrast, MitoBloCK-6 did not change engraftment of normal cord blood in similar experiments. As evidenced by secondary transplants, MitoBloCK-6 also targeted leukemic stem cells. As expression levels of ALR substrates are increased in AML stem cells we assessed the effects of ALR inhibition on differentiation in AML. Genetic or chemical inhibition of ALR induced the differentiation of AML cells as evidenced by changes in gene expression, increased differentiation associated CD surface marker expression and increased non-specific esterase. Interrogation of the effects of ALR inhibition on its substrates identified the mitochondrial copper chaperone, Cox17 as the primary downstream target in leukemic cells. Genetic or chemical inhibition of ALR selectively reduced levels of Cox17 protein. Validating the functional importance of these findings, knockdown of Cox17 phenocopied ALR inhibition and reduced AML proliferation and induced AML differentiation. Cox17 is a copper metallochaperone that promotes respiratory chain complex IV assembly by loading copper into the respiratory complex. COX17 knockdown slightly reduced the complex IV enzymatic activity, but did not change basal oxygen consumption or ROS production. However, COX17 knockdown increased intracellular levels of free copper 16-fold as measured by atomic mass spectrometry. Copper is a known inhibitor of adenosylhomocysteinase, a key enzyme involved in the preservation of S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio in cells. SAM is a global methyl donor and is critical for DNA methylation. Knockdown of COX17 or ALR inhibition with MitoBloCK-6 decreased levels of SAM and reduced DNA methylation in AML cells. Likewise, the enzymatic activity of adenosylhomocysteinase was reduced in OCI-AML2 cells after MitoBloCK-6 treatment. Importantly, co-treatment with the copper chelator, penicillamine, rescued reductions in SAM, DNA methylation, and cell viability after COX17 knockdown or MitoBloCK-6 treatment. Thus, we have discovered a novel copper-dependent mechanism by which mitochondrial pathways regulate epigenetics and stemness in AML. Moreover, inhibitors of ALR or COX17 may be a novel therapeutic strategy to promote the differentiation of AML cells and stem cells. Disclosures Schimmer: Otsuka Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medivir AB: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Background Venous thromboembolism (VTE) is a well-known complication in adults receiving asparaginase (ASNase)-based intensification chemotherapy for acute lymphoblastic leukemia (ALL). The optimal preventative strategy is unclear. We previously reported high thrombosis rates during intensification without VTE prophylaxis. Our objective is to determine the effects of weight-adjusted enoxaparin as primary VTE prophylaxis in ambulatory adults receiving ASNase-based intensification chemotherapy for ALL. Methods Patients who achieved complete remission following induction from 2011-2017 went on to receive ASNase-based intensification on the Dana Farber Cancer Institute (DFCI) 91-01 protocol. Patients already on therapeutic anticoagulation for prior VTE were excluded. VTE prophylaxis commenced on day 1, cycle 1 of intensification until the completion of the entire 21-30 week. From 2011 to 2014 patients received enoxaparin subcutaneously once daily, at a dose of 40 mg for patients weighing less than 80 kg, and 60 mg for those 80 kg and over for the first 3 years. Due to continuing occurrence of VTE from 2014-2017 patients received an escalated dose aiming at 1mg/kg daily (rounded to the nearest 20 mg). Results were compared to an historical patient cohort that received the same regimen without VTE prophylaxis prior to 2011. Result: In our historical cohort of adult ALL that did not receive prophylactic anticoagulation (n=99), the rate of VTE was 27%. 124 patients received one of the above prophylactic anticoagulation schedules. The treated and control groups did not differ with respect to median age, gender, weight and number of ASNase treatment cycles per patient. 16 of 124 patients (12.9 %) in the prophylaxis groups developed symptomatic VTE. Sites of VTE in the prophylaxis group included lower extremity (10), sagittal sinus (2), subclavian line related (3), and pulmonary embolism (4). There were no major bleeding complications observed in the prophylaxis group. In the first patient cohort (n=42), receiving fixed enoxaparin doses of 40 or 60 mg, dosing ranged from 0.39-0.69 mg/kg. The mean enoxaparin dose administered was 0.57 mg/kg. Seven (16.6 %) developed a symptomatic VTE, which was not significantly different from the historical non-prophylaxis cohort. In the second patient cohort (n=82), all patients received weight-adjusted enoxaparin > 0.7 mg/kg of enoxaparin. The mean enoxaparin dose administered was 0.88 mg/kg. Nine of 82 patients (10.9 %)) in this cohort had a symptomatic VTE, which was lower than the historical cohort (OR = 0.33 {95% CI, 0.15-0.77} and P< 0.01). Conclusions: Weight-adjusted enoxaparin prophylaxis targeting 1 mg/kg/day reduced the incidence of symptomatic VTE in adult ALL patients receiving intensification chemotherapy with ASNase. This treatment is a viable VTE prevention option with no documented major bleeding. Disclosures Schimmer: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy; Medivir AB: Research Funding; Jazz Pharmaceuticals: Consultancy. Schuh:Teva: Consultancy; Jazz: Consultancy; Pfizer: Consultancy; Amgen Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Shire: Consultancy; Otsuka: Consultancy. Maze:Novartis: Consultancy, Honoraria. Yee:Celgene, Novartis, Otsuka: Membership on an entity's Board of Directors or advisory committees; Agensys, Astex, GSK, Onconova, Genentech/Roche: Research Funding. Gupta:Incyte: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Brandwein:Pfizer: Consultancy; Celgene: Consultancy; Lundbeck: Consultancy; Novartis: Consultancy; Boehringer Ingelheim: Consultancy, Research Funding.

Introduction:Induction chemotherapy debulks the leukemic burden in AML patients. Blood count recovery usually occurs during the fourth week of starting chemotherapy in patients who achieve a morphologic remission in bone marrow. However, a subset of patients experience significantly delayed recovery. The relevance of delayed recovery on long-term clinical outcomes and its contributing factors have not been well studied. Specifically, the association between recurrent mutations in AML and hematologic recovery is unknown. Methods:We studied a total of 262 newly diagnosed adult AML patients treated between September 2014 and December 2017 at Princess Margaret Cancer Centre who achieved a complete remission (CR) or CR with incomplete count recovery (CRi) after one cycle of induction chemotherapy. The regimens consisted of 3+7 (N=194) and FLAG-IDA (N=68). We collected information on disease characteristics and blood count results at baseline and during chemotherapy. Mutation profiling of diagnostic samples was performed using a 54-gene next generation sequencing panel (TruSight Myeloid Sequencing Panel, Illumina). Detection of persistent mutations in remission samples was performed using a custom 37-gene duplex sequencing platform with a lower detection limit of ~0.05% variant allele frequency (VAF). Results:Of the cohort of 262 patients, 256 patients (97.7%) achieved neutrophil recovery (defined as > 1x109/L), with time to recovery ranging from 17 to 84 days. Two hundred forty-four (93.1%) patients achieved platelet recovery (defined as > 100x109/L); time to recovery ranged from 17 to 117 days. The percentage of patients who achieved neutrophil and platelet count recovery before day 35 was 82.4% and 84.0% respectively (Fig. 1). To evaluate the prognostic significance of delayed recovery, we categorized patients who achieved CR into two groups, "normal" or "delayed" recovery, according to whether they achieved recovery before or after day 35, respectively. Relapse-free survival (RFS) of patients with delayed recovery was significantly worse than those with normal recovery and only marginally better than those with CRi (P=0.02; Fig. 2). Analysis restricted to 3+7 treated patients showed the same trend (P=0.02), excluding the possibility that the inferior outcome was due to treatment of higher risk patients with more intensive regimens. To study the factors associated with delayed recovery, we performed multivariable Cox regression analysis that included clinical factors and mutations identified at the time of diagnosis as covariates. Four factors were found to be independently correlated with delayed recovery: treatment with FLAG-IDA, truncating ASXL1mutations, SRSF2mutations, and DNMT3AR882 mutations (Table 1). Because FLAG-IDA is the preferred frontline regimen for higher risk patients at our institution, we performed a secondary analysis restricted to patients treated with 3+7 to exclude chemotherapy regimen as a potential confounding variable. This analysis identified six independent factors: AML with myelodysplasia-related changes, lower hemoglobin levels at presentation, truncating ASXL1mutations, TET2mutations, CEBPAmutations, and DNMT3AR882 mutations (Table 1). Somatic mutations in DNMT3A, TET2, ASXL1, and SRSF2(DTAS) mutations are associated with preleukemic conditions, such as myelodysplastic syndrome and age-related clonal hematopoiesis, and frequently persist in remission. These mutations are acquired in hematopoietic stem cells resulting in their propagation to progenitors and terminally differentiated blood cells. We hypothesized that the persistence of DTAS mutations in progenitors might compromise their capacity for reconstitution of normal hematopoiesis resulting in delayed recovery. To test this hypothesis, we performed duplex sequencing on peripheral blood DNA samples collected from a random subset of 43 patients during remission. The detection of DTAS mutations in remission above a VAF of 2% was strongly associated with delayed recovery (P=0.0004; Fig. 3). Conclusion:Delayed hematologic recovery in AML patients after induction chemotherapy is associated with inferior RFS and persistence of preleukemic mutations (i.e., DTAS mutations). Our results support a model in which progenitors harboring DTAS mutations have reduced repopulation capacity leading to delayed hematologic recovery after induction chemotherapy. Disclosures Gupta: Incyte: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Schimmer:Otsuka Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medivir AB: Research Funding. Yee:Agensys, Astex, GSK, Onconova, Genentech/Roche: Research Funding; Celgene, Novartis, Otsuka: Membership on an entity's Board of Directors or advisory committees. Maze:Novartis: Consultancy, Honoraria. Bratman:Roche: Other: SVB is a co-inventor on a patent describing methods for circulating tumor DNA analysis, which has been licensed to Roche Molecular Diagnostics.. Schuh:Shire: Consultancy; Jazz: Consultancy; Novartis: Consultancy; Otsuka: Consultancy; Teva: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen Inc.: Consultancy.

Mutations in isocitrate dehydrogenase 2 (IDH2) promote AML pathogenesis through production of 2-hydroxyglutarate (2-HG). Enasidenib is an inhibitor of mutant IDH2 activity and induces the differentiation of IDH2-mutated leukemic blasts. In a phase I/II clinical trial, enasidenib monotherapy resulted in an overall response rate of 40% and median duration of response of 6 months in relapsed/refractory AML (Stein et. al. Blood 2017). We previously discovered that mutant IDH activity sensitizes AML cells to BCL-2 inhibition through accumulation of 2-HG (Chan et. al. Nature Medicine 2015). Pharmacologic inhibition of BCL-2 activity with venetoclax, a highly specific BH3 mimetic, preferentially targets IDH-mutated human AML cells. In a phase II clinical trial of venetoclax monotherapy, IDH-mutated relapsed/refractory AML patients had a response rate of 33% compared to 10% in IDH-wildtype patients (Konopleva et. al. Cancer Discovery 2016). Based on the above findings, we hypothesized that combination therapy with enasidenib and venetoclax may demonstrate superior anti-leukemic activity in comparison to single agents in the treatment of IDH2-mutant AML. However, given that the mechanism by which IDH mutations increase BCL-2 dependence is via 2-HG, reduction of 2-HG with enasidenib may antagonize venetoclax activity. As such, we further hypothesized that a sequential dosing schedule may be superior to concurrent dosing. To test our hypotheses, we conducted a preclinical study to evaluate the efficacy of monotherapy versus combination therapy in reducing the leukemic burden in three patient-derived xenograft (PDX) models of human IDH2-mutant AML. All three PDX models were derived from samples with co-occurring IDH2R140Q and NPM1c mutations. Engrafted animals were randomly assigned to one of six treatment arms (N=5 per arm): vehicle (arm 1), enasidenib alone (arm 2), venetoclax alone (arm 3), concurrent combination (arm 4), and sequential combinations (arms 5 and 6; see Figure 1 for details). Enasidenib and venetoclax were administered by oral gavage at a dose of 40 mg/kg twice a day and 100 mg/kg daily, respectively. Tumor burden was measured in bone marrow samples collected immediately prior to treatment and every 2 weeks during the 12-week treatment period. Concurrent combination treatment (arm 4) resulted in the greatest reduction in leukemia engraftment compared to all other treatment arms, including the sequential dosing arms, in two of the three models (#1 and #2, henceforth termed "responders"; Figure 2A). Although venetoclax monotherapy reduced engraftment in all three models, persistent disease above a threshold of 0.1% was detectable in 12 of 13 animals by flow cytometry (9 of 13 by ddPCR) after 12 weeks of treatment (Figure 2B). In contrast, disease was detectable in only 2 of 9 animals (0 of 9 by ddPCR) treated with concurrent therapy in responders. In the remaining model (#3, henceforth termed "non-responder"), combination therapy was not superior to venetoclax monotherapy but importantly, co-treatment with enasidenib did not antagonize venetoclax activity. Interestingly, enasidenib monotherapy increased expression of myeloid differentiation markers, CD15 and CD11b, only in responders, indicating that differentiation might be a precondition for responsiveness to concurrent therapy. We confirmed that the lack of response in non-responders was not due to selection of an IDH2 wildtype clone or failure to block 2-HG production by enasidenib. To gain insights into the mechanism by which enasidenib might enhance venetoclax sensitivity, we performed quantitative intracellular flow cytometry staining for the anti-apoptotic proteins BCL-2, BCL-xL and MCL-1 in leukemic cells collected after 12 weeks of treatment. Enasidenib monotherapy resulted in a significant decrease in BCL-2 expression in responders. The reduction in anti-apoptotic protein expression could potentiate mitochondrial priming and sensitization to venetoclax. In summary, our findings demonstrate that concurrent combination therapy with enasidenib and venetoclax is a promising therapeutic approach for IDH2-mutated AML. Responsiveness to combination therapy is associated with enasidenib-induced differentiation and reduction in anti-apoptotic protein expression. Our findings support ongoing and future clinical investigations in combination therapies with mutant IDH and BCL-2 inhibitors. Disclosures Cojocari: AbbVie Inc: Employment. Phillips:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. Leverson:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. MacBeth:Celgene Corporation: Employment, Equity Ownership. Nicolay:Agios: Employment. Narayanaswamy:Agios: Employment. Ronseaux:Agios: Employment. Liu:Agios: Employment, Equity Ownership. Chan:AbbVie: Research Funding; Genentech: Research Funding; Celgene: Research Funding.

The vast majority of mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and then imported into the mitochondria. A subset of these imported proteins are folded into their mature and functional forms in the mitochondrial inter-membrane space (IMS) by the Mitochondrial IMS Assembly (MIA) pathway. We found that genes encoding substrates of the MIA pathway are over-expressed in leukemic stem cells compared to bulk AML cells. Therefore, we assessed the effects of inhibiting the MIA pathway in AML. We knocked down the mitochondrial sulfhydryl oxidase ALR, a key regulator of the MIA pathway. Knockdown of ALR with shRNA reduced the growth and viability of OCI-AML2, TEX and NB4 leukemia cells. In addition, knockdown of ALR reduced the engraftment of TEX cells into mouse marrow, demonstrating an effect on the leukemia initiating cells. The small molecule selective ALR inhibitor, MitoBloCK-6, mimicked the effects of ALR knockdown and killed AML cells with an IC50 of 5-10 μM. MitoBloCK-6 preferentially reduced the clonogenic growth of primary AML cells (n=4/5) over normal hematopoietic cells (n=4). However, only 3/10 bulk AML cells were sensitive to MitoBloCK-6 induced cell death by Annexin V/PI staining. Next, we evaluated the efficacy and toxicity of ALR inhibition in vivo . We injected primary AML cells or normal cord blood into the femurs of mice and then treated mice with MitoBloCK-6 (80 mg/kg i.p. 5 of 7 days x 2 weeks). MitoBloCK-6 strongly reduced the engraftment of primary AML samples but did not affect engraftment of cord blood. In secondary transplants, MitoBloCK-6 also targeted leukemic stem cells. No change in mouse body weight, serum chemistries, or organ histology was seen. As expression levels of ALR substrates are increased in AML stem cells, we assessed the effects of ALR inhibition on differentiation in AML. Genetic or chemical inhibition of ALR induced the differentiation of AML cells as evidenced by increased CD surface marker expression and increased non-specific esterase. In addition, ALR inhibition was preferentially cytotoxic towards undifferentiated cells and stem cells over differentiated bulk AML cells. Interrogation of the effects of ALR inhibition on its substrates identified the mitochondrial copper chaperone, Cox17 as the primary downstream target in leukemic cells. Inhibition of ALR selectively reduced levels of Cox17 protein and altered mitochondrial cristae structure. Validating the functional importance of these findings, knockdown of Cox17 phenocopied ALR inhibition and reduced AML proliferation, induced differentiation of AML cells, and altered mitochondrial cristae structure, without changing respiratory chain activity or oxygen consumption. Of note, cristae remodelling independent of respiratory chain function has been recently implicated in cellular differentiation and in yeast, Cox17 regulates the cristae organizing machinery. Thus, we have identified novel mechanisms by which mitochondrial pathways regulate the fate and differentiation of AML cells and stem cells Moreover, inhibition of ALR may be a novel therapeutic strategy to promote the differentiation of AML cells and stem cells. Disclosures Schimmer: Takeda Pharmaceuticals: Research Funding; Medivir: Research Funding; Novartis Pharmaceuticals: Honoraria.

The prognosis of Philadelphia positive acute lymphoblastic leukemia (Ph+ ALL) has improved markedly after the introduction of BCR ABL1 targeted tyrosine kinase inhibitors (TKI). With the addition of upfront TKI, ~90 % of patient will achieve complete remission (CR). Allogenic stem cell transplant (alloSCT) remains the cornerstone of post remission therapy based on large trials that predate the introduction of TKIs. This approach has since been challenged in more recent studies in which similar outcomes were observed with lifetime TKI therapy. Several reports have suggested a correlation between achieving a molecular negativity with chemotherapy + TKI and long term outcome, in patients who did not receive alloSCT, suggesting that a monitoring strategy might spare some patients with excellent response to chemotherapy and TKI the toxicity of (alloSCT), and with comparable outcome. We previously reported good overall survival and event free survival in patients treated with a TKI-modified version of the Dana Farber Cancer Institute (DFCI) pediatric ALL protocol. In this retrospective analysis we compared the long term outcome of Ph+ ALL patients who were transplanted and those who were not, mainly due to the unavailability of a donor. In addition, we reviewed the effect of residual disease monitoring (RDM) at post induction, 3, 6, 9 and 12 months, and correlated the achievement of complete and major molecular responses with survival. In the present study, 167 patients diagnosed with Ph+ ALL in our center were evaluated from year 2001-2015. One hundred and thirty three (133) received DFCI chemotherapy + imatinib. One hundred and nineteen (119; 89%) patients achieved CR following the induction phase. Fifty nine (59; 44.3 %) subsequently received an allogenic stem cell transplant from a sibling or matched unrelated donor, leaving 74 patients who were not transplanted and were evaluated for RDM. While there was no significant difference between the overall survival (OS) for the transplanted and not transplanted cohorts (median survival 3.3 years vs 5.3 years, and 5 year- OS 40% vs 50 %, respectively; p value = 0.40), there was an improved relapse free survival (RFS) in the transplanted group (5- year RFS, 55% vs 75%, respectively; p value= 0.033). Achieving CMR with un-detectable transcripts post-induction, and at 3 and 6 months, showed superior OS compared to those not achieving CMR (p values of 0.029, 0.0086 and 0.028, respectively), whereas superior RFS was associated with molecular negativity at 3, 6 and 9 months ( p values of 0.0076,0.0023 and 0.03, respectively). Molecular negativity at 12 months had no impact on survival. In conclusion, our data confirm that while the RFS of patients with Ph+ ALL who did not undergo alloSCT as post remission therapy was inferior to that of those who did, OS was similar in the two groups. Monitoring of residual disease at early time points during therapy, and especially at 3 and 6 months, appears to identify patients with a lower chance of relapse who can likely be spared the toxicity of alloSCT, and receive chemotherapy + TKI, followed by ongoing TKI maintenance. Further RDM studies are needed to better stratify such patients and to facilitate alloSCT decision making. Figure Figure. Disclosures Gupta: Incyte Corporation: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Schimmer:Novartis: Honoraria. Schuh:Amgen: Membership on an entity's Board of Directors or advisory committees.

Introduction: Mutations substituting arginine 132 of isocitrate dehydrogenase 1 (IDH1) are recurrent in acute myeloid leukemia (AML) and several other cancers, resulting in the aberrant production of the onco-metabolite, R-2-hydroxyglutarate (2-HG), as well as an inability to convert cytoplasmic alpha-ketoglutarate to isocitrate via reductive carboxylation. Currently, small molecules that effectively inhibit the neomorphic enzyme and abrogate the production of 2-HG, such as AG-120, are in clinical trials with promising results. However, these inhibitors have not proven to be curative in most AML cases, indicating a need for additional targeted therapies. We have previously investigated synthetic lethal vulnerabilities in IDH1-mutated AML and identified an interaction with BCL2 leading to increased susceptibility to ABT-199 (Chan et al, 2015). Synthetic lethal approaches targeting 2-HG independent metabolic vulnerabilities conferred by mutant IDH1 may complement IDH1 mutant inhibitors. Using a novel computational method (MiSL) based on Boolean implication (if-then rules) mining of pan-cancer data, we identified acetyl CoA carboxylase (ACACA) as a potential druggable target in IDH1-mutated AML. ACACA is the rate-limiting step in the de novo synthesis of fatty acids, and mutant IDH1 leads to a reduction in malonyl-CoA, a key building block for fatty acids, in a 2-HG independent manner. This finding led us to investigate a potential synthetic lethal interaction between mutant IDH1 and ACACA based on the hypothesis that the combination causes marked inhibition of fatty acid synthesis required for cell growth. Methods: Boolean implications (MiSL) were used to identify candidate synthetic lethal interactions with mutant IDH1 by isolating genes deleted only in the absence of the mutation and with differential gene expression within pan-cancer TCGA data. Validation was performed using THP-1 cells transduced with doxycycline-inducible wildtype and R132H mutant IDH1 lentiviral vectors, and primary patient IDH1-mutant and wildtype AML samples, using both shRNAs and a targeted pharmacologic inhibitor of ACACA. Metabolomics was performed using semi-targeted mass spectrometry and liquid chromatography. Finally, primary AML samples and IDH1-mutant and wildtype cancer cell lines (HT-1080, U118, U87) were transduced with validated shRNA and engrafted into NSG mice. Results: Our computational method found that IDH1 mutation and ACACA deletions were mutually exclusive in pan-cancer TCGA data, ACACA deletions resulted in lowered expression of ACACA, and ACACA was differentially over-expressed in IDH1-mutant AML compared to IDH1-wildtypeAML. Pharmacologic inhibition of ACACA with 2 uM TOFA caused a marked reduction in cell growth in the presence of IDH1 R132H (+ dox), but not in its absence (- dox; p = 0.0001). Similarly, knockdown of ACACA with independent shRNAs caused a defect in viable cell growth in the presence of IDH1 R132H (+ dox), but not in its absence (- dox) or with scrambled shRNA (p=0.009, shRNA #1 vs. scrambled; p=0.01, shRNA #2 vs. scrambled). Primary IDH1 R132 mutated purified AML blasts were selectively sensitive to TOFA treatment compared to IDH1 wildtype normal karyotype blasts (IC50 0.6 uM vs 6 uM, p=0.009) in viable growth assays. Furthermore, when transduced with lentivirus encoding shRNA to ACACA, primary IDH1-mutant AML cells exhibited markedly reduced engraftment of RFP-positive human CD45+CD33+ leukemic cells compared to scrambled non-targeting shRNA (p < 0.05, Mann-Whitney U). As predicted, IDH1-mutant AML blasts pre-treated with 10uM AG-120 (sufficient to inhibit production of detectable 2-HG) remained susceptible to ACACA inhibition in vitro. Strikingly, in vivo models of IDH1 R132C mutated, but not wildtype, sarcoma cell lines exhibited a dramatic decrease in cell growth after ACACA inhibition that was not reversible by treatment with AG-120. Finally, metabolomic profiling revealed a major perturbation in multiple phospholipid fatty acid species and decreased malonyl-CoA conferred by IDH1 R132H, consistent with our proposed mechanism. Conclusion: We have identified de novo lipogenesis through ACACA as a critical metabolic vulnerability linked to IDH1 mutation in AML and provide evidence that therapeutic inhibition of ACACA with small molecules may be beneficial in AML, as well as in other cancers with IDH1 mutations. Disclosures Majeti: Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Background The role of thromboprophylaxis in solid organ malignancies is well established. Hematologic malignancies can also be associated with a considerable risk of thromboembolic complications. The incidence of these events is variable and is influenced by multiple factors. Limited data are available regarding the incidence of venous thromboembolism (VTE) in hospitalized acute leukemia (AL) patients. The management of symptomatic VTE in patients with AL can be challenging due to the increased risk of thrombocytopenia-related bleeding. Methods The Discharge summary Database (DAD) was used to extract post-admitted PE and DVT volumes in AL patients admitted to Princess Margaret Cancer Centre from 2007-2016. ICD-10-CA diagnosis codes for acute leukemia, and both PE and DVT events, were used. Only patients diagnosed with VTE at least 48 hours post-admission were included to restrict the cohort to patients that developed VTE during their admission. Results We analyzed a total of 10,041 patients that were admitted to Princess Margaret Cancer Centre during the 2007-2016 period. (Table 1) Of these, 7759 had a solid tumor diagnosis (271 VTE events, 3.4%) and 2282 patients had AL (1675 AML, 464 ALL, 144 APL). The AL patients (AML, ALL, APL) admitted to our Centre for chemotherapy or for the management of complications were further evaluated to determine VTE incidence and to evaluate its management in this setting. As of September 2012, patients with solid tumors treated at our Centre received standard thromboprophylaxis as part of an institutional in-patient (VTE) prophylaxis policy (IPP) that reduced the incidence of VTE from 4.8% (219/4520) to 1.6% (3239/52) before and after the policy was initiated, respectively. AL patients are not given prophylactic anticoagulation. 37 AL patients (22 AML, 10 ALL, and 5 APL; overall incidence 1.6%) developed symptomatic VTE (DVT only 23, PE only 8, DVT + PE 6). VTE was reported as central venous catheter related in 12/37 patients (32.4%). PE was detected in all cases by CT-PE. Median age of VTE patients was 53 years (range 32-77), with a median hospital stay of 35 days (range 2-144). Chemotherapy was given to 26 of the 37 patients that developed VTE, with 20 receiving initial induction chemotherapy. 17/37 (46%) patients had PLT Conclusions In our experience, the incidence of VTE in AL patients during prolonged hospital stay is relatively low, raising questions about the need for routine VTE prophylaxis in this group. The relatively increased risk of VTE in AL patients receiving chemotherapy (particularly, induction chemotherapy), should prompt particular scrutiny in symptomatic patients. Disclosures Schuh: Amgen: Membership on an entity's Board of Directors or advisory committees. Yee:Novartis Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding. Gupta:Incyte Corporation: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Schimmer:Novartis: Honoraria.

Background: Venous thromboembolism (VTE) is a well-known complication in adults receiving asparaginase (ASNase) based intensification chemotherapy for acute lymphoblastic leukemia (ALL). We previously reported a high VTE rate in patients receiving a modified Dana Farber Cancer Institute (DFCI) intensification phase, which included weekly ASNase. We report thrombosis rates during induction and the results using two different dosing schedules of enoxaparin as primary VTE prophylaxis in adults treated with the same protocol. Methods: We reviewed pts who received induction chemotherapy, without VTE prophylaxis, using DFCI protocol (containing one dose of ASNase) between 2012-2016. Pts achieving complete remission (CR) subsequently received weekly ASNase-based modified DFCI intensification phase for at least 7 cycles (21 weeks) with VTE prophylaxis, using two dosing schedules in consecutive cohorts: A. Low-dose group received enoxaparin 40 mg subcutaneously (SC) daily for patients weighing < 80 kg and 60 mg daily for those ≥ 80 kg; B. Dose-escalated group received enoxaparin 1 mg/kg SC daily (rounded to the nearest 20 mg). VTE rates were calculated for pts during induction and intensification phase (low-dose and escalated dose prophylaxis). Results were compared to a similar group of 99 pts previously treated with the same DFCI protocol who did not receive VTE prophylaxis during intensification. Patients not achieving CR, relapsing, undergoing alloSCT after induction, not completing at least 21 weeks of intensification phase or developing VTE before induction were not included. Results: The VTE rate during induction (n-=144) was 2.8%. Of 111 pts who received intensification prophylaxis, the overall VTE rate was 19.8% (p The actual mean dose of enoxaparin in the low-dose prophylaxis group was 0.62 mg/kg, as compared to 0.90 mg/kg in the dose-escalated group. There were no major bleeding complications observed in the prophylaxis groups. The minor bleeding rate in the entire prophylaxis cohort was 4.5% (5/111), and was similar between the low-dose and escalated dose groups. Sites of VTE in the prophylaxis groups included lower extremity (11 cases), sagittal sinus (3), subclavian line related (5), pulmonary embolism (9), and cardiac thrombus (1); some patients had more than one site involved. Conclusions: Our data confirmed a high VTE rate during intensification, even with prophylaxis. Dose-escalation of enoxaparin to 1 mg/kg was safe with a trend toward reduction in VTE rates, particularly in patients weighing > 80 kg; however, a larger cohort would be needed to determine if this difference is significant. The use of novel anticoagulants in this setting could be considered. Disclosures Schuh: Amgen: Membership on an entity's Board of Directors or advisory committees. Yee:Novartis Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding. Schimmer:Novartis: Honoraria.

The outcome of adult patients with AML who are primary non-responders to two courses of induction chemotherapy is poor. However, the utility of a 3rd induction for a select subgroup of these patients is uncertain. Here, we evaluated the rates of response and survival after a 3rd course of induction chemotherapy for primary non-responders with AML. We identified 98 patients from the Princess Margaret Cancer Centre between May 1999 and March 2015 who were non-responders to induction and reinduction chemotherapy. No-response to re-induction chemotherapy was defined according to the Revised Recommendations of the International Working Group for AML (JCO, 2003) as patients who survived > 7 days post re-induction and had persistent AML in blood or bone marrow (>5%). Median age was 58.3 years [range: 20-76.6]. 50 (51%) were male. 2% had favorable, 18% normal, 18% intermediate, and 48% adverse cytogenetics. 50% had de novo AML, 23% had AML secondary to MDS or MPN, and 17% had therapy-related AML. Induction chemotherapy consisted of "7+3" (n =88), Nove-HiDAC (n=1), Flag-Ida (n= 2), or similar variants (n=7). Reinduction chemotherapy consisted of Nove-HiDAC (n=70), Flag-Ida (n=7), "7+3" (n=1) or other similar variants (n =20). No patients received the same regimen for both induction and reinduction. Of the 98 primary non-responders, 15 received a 3rd induction regimen, while the others received supportive/palliative care ± low-dose chemotherapy (57 pts), or a non-induction clinical trial (26 pts). Average age was 56.4 (sd: 12.9) for patients who received supportive/palliative care and 47.0 (sd: 17.5) for patients who received a 3rd induction (p=0.008). Other baseline characteristics including gender, cytogenetic risk, marrow blast count post 2nd induction, and time between 1st and 2nd induction, did not differ between patients who did and did not receive a 3rd induction. Time to 3rd induction was a median of 54 days [range:36-126] from the start of the 2nd induction. Of the 15 third inductions, 7 were clinical trials evaluating novel agents in combination with induction chemotherapy, while the other 8 were combinations of standard chemotherapeutics (Flag-Ida n=1), AMSA+HiDAC (n=2), Daunorubicin+ HiDAC (n=1), Nove-HiDAC (n=4). Of the 15 patients who received a 3rd induction, 3 (20%) achieved a CR following Nove-HiDAC and Flag-Ida or AMSA+HiDAC chemotherapy, where the Ara-C was given as continuous infusion. 1 patient underwent allogeneic stem cell transplant (SCT) approximately 3.7 months after 3rd induction and remains alive 4.6 years post CR. 2 patients relapsed 2.3 and 4.7 months post CR without having received alloSCT. None of the 12 other patients responded to the 3rd induction and none had prolonged aplasia. 2 of 15 (13%) died during 3rd induction. Among the 83 patients who did not receive a 3rdinduction, 1 achieved a CR after a phase 1 clinical trial (MDM2 inhibitor) and remains in CR 3.6 years following an alloSCT. For patients who survived the immediate post induction period and were discharged from hospital median overall survival from the start of the 2nd induction did not differ between patients who did and did not receive a 3rd induction (276 days [range: 78-1304] vs 181.5 days [range: 47-1855] respectively p= 0.14). Median duration of hospital stay (including subsequent admissions) was longer for patients receiving a 3rd induction compared to those who did not (94 days following start of the 2nd induction [range: 47-169] vs 57 days [range: 51-181], respectively;(p= 0.003)). In summary, remissions after 3rd inductions for primary non-responders are uncommon, and short-lived, suggesting that 3rd inductions should be considered with caution and only when an SCT strategy is in place. Disclosures Gupta: Incyte Corporation: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Schuh:Amgen: Membership on an entity's Board of Directors or advisory committees. Yee:Novartis Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding. Schimmer:Novartis: Honoraria.

Background: The prognostication of cytogenetically normal AML (CN-AML) continues to evolve with the use of NGS-based risk stratification. Emerging data indicate that the presence of additional mutations in good- and intermediate-risk patients (as defined by conventional cytogenetic and molecular analyses) changes the behavior of their disease, suggesting that more personalized treatment approaches are needed. Methods: We analyzed the mutational profile of newly diagnosed AML patients with DNMT3A mutations (N = 48) seen at our center and described their clinical characteristics and associated mutations. These patients were identified as part of the Advanced Genomics in Leukemia (AGILE) clinical project currently underway at the Princess Margaret Cancer Centre, Toronto. NGS molecular profiling was performed using the TruSight Myeloid Sequencing Panel (TMSP; Illumina) on the MiSeq benchtop genome sequencer (Illumina). This process permitted profiling of 54 genes (39 in the hotspot region; 15 complete coding region coverage) using amplicon-based library preparation and sequencing by synthesis. 160 patients with newly diagnosed AML were evaluated for this project from February 2015 to February 2016. All were analyzed in parallel by the standard cytogenetic and molecular (NPM1, FLT3-ITD/TKD) diagnostic algorithm. Results: 48 unique patients were identified bearing mutated DNMT3A. Of these, 31 patients had a normal karyotype. Their clinical characteristics are depicted in Table 1. The R882X DNMT3A variant was detected in 12 patients. A total of 100 additional mutations were identified on sequencing (Figure1). The most common associated mutations were in NPM1 (38.7%) followed by IDH1 (29%), RUNX1 (25.8%) and TET2 (22.6%). PTPN11 mutations were identified in 6 patients, 75% of which also had mutated NPM1. Two patients were found to have biallelic CEBPA mutations. Potential gene-gene interactions were also examined and led to the identification of subgroups such as NPM1-FLT3-DNMT3A mutated (n=4), DNMT3A-IDH2R140 (n=3) and DNMT3A-IDH2R172 (n=3) based on recent data by Papaemmanuil, et al (New England Journal of Medicine, 2016) defining these subgroups as being prognostically relevant.( Patients >60 years of age had more frequent mutations in NRAS, BCOR, BCORL1 and TET2. NRAS and BCOR mutations were mutually exclusive with NPM1. RUNX1, IDH2, SRSF2 and U2AF1 mutations were also seen exclusively in the NPM1 negative group. Eligible patients (n=25) received induction therapy with daunorubicin and cytarabine leading to CR1 in 18 patients (72%). Primary induction failure occurred in 6 cases (24%). All 6 cases were NPM1 negative and had missense mutations in DNMT3A including 3 R882X variants. (Figure 2) Additional mutations were identified in IDH1, RUNX1 and/or TET2 in all 6 patients. During the median follow up of 8 months (range 1 - 15 months), 4 patients relapsed, 2 of which had mutated NPM1 with wild type FLT3-ITD. Sixteen patients are alive at this point and 12 are in CR, six having received allogeneic stem cell transplants. One patient with relapsed disease entered a clinical trial of an IDH1 inhibitor Conclusion: Genomic analysis is increasingly recognized as a vital adjunct to conventional diagnostic and prognostic approaches. With ongoing advancements in technology leading to increasing cost effectiveness and decreased turnaround times, the use of NGS is likely to become an up-front investigation resulting in a more personalized approach to therapy. Also, unique patient subgroups defined by gene-gene interactions can be identified to further predict clinical behavior and potentially identify druggable targets for therapy. Disclosures Gupta: Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding. Schimmer:Novartis: Honoraria. Yee:Novartis Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kamel-Reid:BMS: Research Funding. Schuh:Amgen: Membership on an entity's Board of Directors or advisory committees.

Recurrent mutations in the components of the cohesin complex (RAD21, SMC1A, SMC3, and STAG2) have been identified in human AML and other myeloid malignancies, and have been shown to occur as pre-leukemic mutations in HSC. Cohesin functions to hold chromatin strands within a ring-like structure composed of the four core components, and although its best-established role is to maintain the polarity of sister chromatids during mitosis, cohesin is also involved in double-stranded DNA damage repair and regulation of transcription. As little is known about their contributions to leukemogenesis, we sought to investigate the effects of cohesin mutants on human hematopoiesis, particularly hematopoietic stem and progenitor cells (HSPC). Introduction of mutant cohesin into AML cell lines and primary human HSPC resulted in a differentiation block with an increased frequency of CD34+ progenitor cells. A similar phenotype was observed with knockdown of core component RAD21 both in vitro and in vivo, indicating that mutant cohesin can act either through haploinsufficiency or dominant-negative mechanisms. Mutant cohesin increased the serial replating ability of HSPC in vitro and showed enrichment for HSC and leukemia stem cell gene expression programs, indicating an effect to enforce stem cell functions. Furthermore, we observed a skewing toward the myeloid lineage in cohesin mutant colonies cultured in methylcellulose, which was recapitulated by a strong myeloid skewing of human engrafted cells in vivo. Thus, mutant cohesin enforces stem cell programs and impairs human hematopoietic progenitor differentiation. Since cohesin complex mutations were identified in pre-leukemic HSC in many of the cases we investigated, we hypothesized that they may impart their phenotype in a cell context-dependent manner. To investigate this hypothesis, six human HSPC subpopulations (HSC, MPP, LMPP, CMP, GMP, and MEP) were isolated from cord blood, and these cells were transduced with cohesin WT, cohesin mutant, RAD21 shRNA, or control lentivirus. Transduced cells were then cultured in either myeloid differentiation or erythroid differentiation-promoting conditions. Strikingly, a strong myeloid differentiation block was only observed with cohesin mutant-transduced HSC and MPP, but not GMP. Similarly, a strong erythroid differentiation block was also observed in HSC and MPP, but not MEP. These results indicate that the effect of mutant cohesin is context dependent and restricted to the most immature HSPC. We next sought to elucidate the mechanism by which cohesin mutants exert their effects on human HSPC. Since the cohesin complex functions to establish and maintain DNA accessibility, and knockdown of cohesin can led to a decrease in chromatin accessibility at transcription factor (TF) clustered regions (Yan et al., 2013), we hypothesized that cohesin mutants impart their phenotypic effects through modulation of chromatin accessibility. To investigate this hypothesis, we used a newly developed method known as ATAC-Seq (Buenrostro et al., 2013) to assess genome-wide accessibility in cohesin WT and mutant HSPC. As expected, we found that cohesin mutants exhibited globally reduced chromatin accessibility at transcriptional regulatory elements. However, we detected increased chromatin accessibility at motifs for transcription factors known to be highly expressed in and critical regulators of HSPC including ERG, GATA2 and RUNX1. Further footprinting analysis, a proxy for ChIP-Seq experiments, showed a strong enrichment of binding of these factors in the mutant cells compared to WT cells. Based on these results, we developed a model in which the functional effects of mutant cohesin on human HSPC are mediated by transcription factors exhibiting increased chromatin accessibility such as ERG, GATA2 and RUNX1. From this model, we hypothesized that knockdown of these transcription factors would prevent the enforcement of stem cell programs and increase in CD34-expressing cells observed with cohesin mutants. As predicted, knockdown of ERG, GATA2, or RUNX1, but not GATA1 or PU.1, in the presence of cohesin mutants completely prevented the increase in CD34-expressing cells. These results strongly support our proposed model that mutant cohesin impairs hematopoietic differentiation and enforces stem cell programs through the modulation of ERG, GATA2, and RUNX1 chromatin accessibility, expression, and activity. Disclosures Majeti: Forty Seven, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Introduction: Somatic mutations in cancer can directly or indirectly perturb signalling and metabolic pathways that can render a cancer cell susceptible to synthetic lethality. We have developed a novel computational method to accelerate identification of synthetic lethal partners for recurrent mutations in acute myeloid leukemia. Our method is based on the hypothesis that, across multiple cancers, synthetic lethal partners of a mutation will be amplified more frequently or deleted less frequently, with concordant changes in expression, in primary tumor samples harboring the mutation of interest.It uses Boolean implication (if-then rules) mining (Sinha et al, Blood 2015) to efficiently identify candidate synthetic lethal partners of a given mutation. The method is distinct from existing work in that it is not reliant on data collected from cell-lines, which are not biologically equivalent to primary tissue and do not always share the composition of mutations found in vivo, but instead utilizes large pan-cancer primary patient datasets. Pan-cancer analysis discovers robust relationships that are more likely to be independent of cancer subtypes, as well as increases statistical power. Methods: We utilized TCGA data of 12 non-AML cancer data-sets (TCGA Research Network et al, Nat. Gen. 2013) for which recurrent AML mutations were present with a frequency of at least 2.5%. These mutations include Cohesin, IDH1, WT1, KRAS, and RUNX1. Boolean implications (FDR < 0.05) were used to identify genes that have more copies in the presence of a mutation as determined by (i) preferred amplification in the presence of the mutation - if gene B is amplified, then mutation A is present, (ii) deletion only in the absence of the mutation - if mutation A is present, then gene B is not deleted. Next, we remove genes that are passengers in large chromosomal alterations using gene expression filtering. Finally, the resulting gene set is filtered by differential gene expression in AML to yield the set of candidate synthetic lethal (SL) partners for a given mutation in AML. Results: To validate our novel method, we compared our putative SL partners to an independent shRNA library screen (DECIPHER) performed in our laboratory for the IDH1 R132 mutation (mut) expressed in THP-1 cells using a doxycycline-inducible promoter (Chan et al, Nat. Med. 2015). We found 6 out of 29 predicted genes showed synthetic lethality when knocked down in the presence of the mutation (Fisher's exact test, p=0.002) indicating our method could find experimentally confirmed interactions. Interestingly, our method predicted Bcl-w to be a SL partner of IDH1 mut, consistent with the SL interaction we previously described between Bcl-2 family members and IDH1 mut in primary AML. Importantly, we found that acetyl-CoA carboxylase alpha (ACACA), the rate-limiting enzyme that controls lipid biosynthesis, was predicted to be a strong SL partner for IDH1 mut. Selective inhibition of ACACA with independently validated shRNA or the small molecule inhibitors, 5-(tetradecyloxy)-2-furoic acid (TOFA) and Soraphen A, prevented cell proliferation in the presence of IDH1 mut but not with IDH1 wildtype. (R)-2-hydroxyglutarate inhibited oxidative phosphorylation and sensitised cells to ACACA inhibitors suggesting the interaction was mediated through the oncometabolite. Gene expression profiling of IDH1 mut cells indicated upregulation of lipid biogenesis pathways (PHOSPHOLIPID METABOLISM, p=0.001). Furthermore, gene expression of ACACA is higher in primary IDH1 mut samples compared to IDH1 wildtype (p=0.008, fold change = 1.2), and cultured primary IDH1mut blasts show selective sensitisation to ACACA inhibition in vitro (n=5/6 IDH1mut/IDH1 wt, p=0.04). Conclusion: We have developed a computational tool that can predict SL interactions for recurrent mutations in AML, with applicability to other cancers. Our method identified de novo lipogenesis as a critical metabolic pathway linked to a specific mutation and suggests therapeutic inhibition of ACACA with small molecules may be beneficial in IDH1 mut AML. This is consistent with recent understanding of the Warburg effect, which postulates that certain oncogenic mutations may indirectly stimulate macromolecule biosynthesis pathways to promote unrestrained cell growth. Our results indicate that a function of the IDH1 mutation is to inhibit oxidative phosphorylation and stimulate de-novo lipid synthesis. Disclosures Majeti: Forty Seven, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Inhibition of glutaminase (GLS), the principal enzyme in the glutamine utilization pathway that coverts glutamine (Gln) to glutamate (Glu), is an attractive therapeutic approach in many cancers. Gln plays a unique role in the metabolism of proliferating cancer cells, providing building blocks to sustain cell proliferation and regulating redox homeostasis and signal transduction pathways. Recent findings indicate that leukemic cells depend on Gln as a major carbon source for growth and survival [Willems et al., Blood, 2013]. We previously reported that a subset of acute myeloid leukemia (AML) cell lines are sensitive to Gln deprivation as well as inhibition of glutaminase by the small molecule BPTES [Matre et al. ASH 2013 #606]. Here we report the efficacy of CB-839, a novel, potent, orally bioavailable GLS inhibitor currently under clinical investigation, in the Gln-dependent subset of AML. First, expression of GLS gene splice variants glutaminase C (GAC) and kidney glutaminase (KGA) and of the GLS2 gene was determined through analysis of RNA sequencing data from 173 newly diagnosed AML patients in the TCGA dataset. Of the GLS gene splice variants, the expression levels of GAC were much higher than those of KGA; GLS2 was expressed at low levels. Levels of both GAC and KGA mRNA were significantly higher (two-sample Wilcoxon test) in AML patients with complex cytogenetics and monosomal karyotype (n=31) than in those with diploid AML (n=88, p=0.019 and p=0.01); GAC levels were higher in core-binding factor AML (n=14) than in diploid AML (p=0.018). These findings indicate high expression of the GLSGAC splice variant in specific AML subsets. Analysis of a panel of AML cell lines showed that, in a subset of leukemia cells, CB-839 treatment decreased viable cell number and induced apoptosis. In sensitive cell lines (Molm14, OCI-AML3, MV4;11), CB-839 decreased viable cell number with IC50s between 10nM and 100nM and induced significant apoptosis. HL60, MOLM13, KG1α, and OCI-AML2 cells were less sensitive (IC50 100-1000nM) and responded with minor induction of cell death. CB-839 decreased viability by >40% in blasts from 9 of 20 (45%) primary AML samples. GC- or LC-MS metabolic profiling of OCI-AML3 and THP1 cell lines as well as primary patient samples revealed that GLS inhibition by BPTES or CB-839 was accompanied by concomitant decrease in concentration of downstream GLS metabolites such as glutamate, α-ketoglutarate (a-KG), aspartate, fumarate, and malate. Investigation of the effects of CB-839 on mitochondrial OXPHOS by the Seahorse Bioscience XF96 Analyzer showed that CB-839 exposure for 16 h caused a dose-dependent decrease in maximal respiratory capacity in OCI-AML3 cells, indicating reduced availability of the substrates for OXPHOS. Similar results were obtained upon treatment with BPTES and in AML cells stably transduced with GLS shRNA. Gln, through Glu, is a precursor for cellular α-KG, which can undergo further metabolism through the Krebs cycle or be further metabolized to 2-hydroxyglutarate (2-HG) by mutant isocitrate dehydrogenase (IDH). In THP1 cell lines stably transduced with doxycycline-inducible mutant IDH1-R132H or IDH2-R140Q construct, CB-839 exposure for 4 days reduced intracellular 2-HG oncometabolite levels by >50%. This was associated with induction of differentiation marker CD11b and morphological signs of differentiation in CB-839–treated IDH2-R140Q cells [30%±2% increase in CD11b mean fluorescent intensity (p In summary, these results indicate that GLS is a relevant therapeutic target in AML, warranting future inclusion of GLS inhibitors in the armamentarium of multi-agent therapeutic approaches. In particular, reduction of production of the oncometabolite 2-HG in conjunction with therapeutic blockade of Gln metabolism may serve as a tailored therapeutic strategy in IDH-mutated AML cells. Disclosures Konopleva: Calithera Biosciences: Research Funding.

IDH1 and IDH2 are two of the most frequently mutated genes in acute myeloid leukemia at an overall frequency of about 15-20%. The genes encode enzymes in the citric acid cycle that normally catalyze the oxidative decarboxylation of isocitrate, producing α-ketoglutarate (α-KG). The mutant enzymes gain a neomorphic activity that catalyzes the conversion of α-KG to (R)-2-hydroxyglutarate (2-HG). The intracellular concentration of (R)-2-HG is over 100-fold higher in IDH-mutated cells than in wildtype cells. (R)-2-HG has been shown to be a competitive inhibitor of multiple α-KG dependent dioxygenases including TET2, and the Jumonji-C domain containing histone demethylases. These enzymes are thought to be the main targets through which (R)-2-HG exerts its effects on leukemogenesis. We previously reported that inhibition of the anti-apoptotic BCL-2 protein is synthetic lethal against mutant IDH which we discovered through a large-scale pooled lentiviral RNA interference screen. We confirmed that expression of mutant IDH1 or IDH2 strikingly sensitized AML cells to shRNA-mediated BCL-2 knockdown and pharmacologic BCL-2 inhibition with ABT-199, a highly specific BH3 mimetic. Importantly, we found that primary human AML blasts harboring IDH mutations were significantly more sensitive to ABT-199 than blasts with wildtype IDH ex vivo and in xenograft transplant models. Furthermore, we showed that ABT-199 was able to target the leukemic stem cell compartment in IDH-mutated samples. Here, we present our work to uncover the synthetic lethal mechanism. An important clue to the mechanism surfaced with the finding that treatment with a cell-permeable precursor of (R)-2-HG sensitized AML cells to BCL-2 inhibition, indicating that the intracellular accumulation of (R)-2-HG found in IDH-mutated cells is sufficient to mediate the phenotype. In addition, we found that (R)-2-HG was able to sensitize isolated mitochondria to ABT-199 with collapse of the mitochondrial transmembrane potential as a surrogate marker for commitment to apoptosis. This finding indicates that 1) the target mediating the synthetic lethal phenotype is localized to the mitochondria, and 2) changes in the epigenome and expression of nuclear-encoded genes are not required for the synthetic lethal phenotype. To identify the potential mitochondrial molecular target, we focused our analysis on the effect of (R)-2-HG on the enzymatic activity of individual complexes in the electron transport chain (ETC), given that ETC dysfunction can potentially alter the threshold for apoptosis. We found that (R)-2-HG at concentrations found in IDH mutated cells inhibited the in vitro enzymatic activity of complex IV (cytochrome C oxidase (COX)) in a dose-dependent manner, but had no effect on the remaining ETC complexes. This finding has in vivo significance as COX activity in intact IDH-mutated primary AML cells was found to be significantly decreased compared with non-mutated AML cells. Next, we investigated the possibility that COX inhibition is sufficient to induce BCL-2 dependence. We found that suppression of COX activity with chemical inhibitors or genetically through shRNA-mediated knockdown of a COX subunit (COX-IV) was sufficient to sensitize AML cells to ABT-199. Furthermore, treatment with tigecycline, a FDA-approved antibiotic that has previously been shown to disrupt ETC function through inhibition of mitochondrial translation resulting in decreased expression of mitochondrial-encoded proteins including the catalytic subunits of the COX complex, reproduced the sensitization effect in non-IDH mutated AML cells. Based on the above findings, we propose a mechanistic model in which (R)-2-HG accumulation in IDH mutant cells directly inhibits COX, thereby lowering the mitochondrial threshold for triggering apoptosis upon BCL-2 inhibition. In summary, we discovered that in addition to the previously described inhibition of α-KG dependent dioxygenases through (R)-2-HG production, IDH mutations also affect mitochondrial bioenergetics. This finding opens up the intriguing possibility that IDH mutations contribute to leukemogenesis not only through epigenetic changes but also through metabolic dysregulation. Lastly, our findings form the rational basis for combining agents that disrupt ETC function such as tigecycline with ABT-199 to target resistant cancer cells and maximize the clinical utility of this promising drug. Disclosures Medeiros: Agios: Consulting - Ad board Other.

PI3Kinase/Akt signaling is frequently activated in acute myelogenous leukemia (AML) and inhibitors of this pathway have shown some promise in both pre-clinical and early clinical studies. In particular, much attention has been focused on components of the mTOR complex as downstream effectors of Akt activation that promote cell proliferation and survival in AML and other malignancies. We have discovered a direct effect of activated and phosphorylated Akt in increasing ribosomal RNA (rRNA) synthesis in both leukemic cell lines and primary AML cells by a novel pathway that is independent of mTOR. We have defined TIF-90 as a splice variant of TIF-1A, a molecule that tethers RNA Polymerase I to the ribosomal DNA promoter and is required for rRNA synthesis. TIF-90 is localized in the nucleolus and directly co-locates with Pol I in imaging, pull down, and rDNA promoter occupancy studies. Activation and phosphorylation of Akt correlates with high levels of TIF-90 expression and increased TIF-90 occupancy of the rDNA promoter in primary AML cells, while overexpression of constitutively activated Akt (Akt-Myr) increases rRNA synthesis in a TIF-90-dependent fashion in cell lines. Activation of Akt increases the expression of TIF-90 by inhibiting its ubiquitination and proteasomal degradation by the p53 ubiquitin ligase, Mdm2. Furthermore, activated Akt directly inhibits the cleavage of the actin-binding protein, Filamin A, into a 90 kDa fragment that interacts with TIF-90 and prevents it from occupying the rDNA promoter. Expression of the 90 kDa Filamin A cleavage fragment alone is sufficient to inhibit rRNA synthesis by binding to TIF-90. Thus, inhibition of Filamin A cleavage by Akt is associated with increased rRNA synthesis. The Akt inhibitor AZD8055, but not the mTORC1 inhibitor Rapamycin, promotes the cleavage of filamin A, decreasing both TIF-90 occupancy of the rDNA promoter and rRNA synthesis. Examination of 21 primary AML samples reveals an inverse correlation between the phosphorylation of Akt and the cleavage of Filamin A; conversely, AZD8055 inhibits both Akt phosphorylation and rRNA synthesis in these samples while promoting the cleavage of Filamin A. These data demonstrate a novel pathway by which Akt directly enhances rRNA synthesis. This pathway is active in primary AML cells and in cell lines that have high levels of phosphorylated Akt. Inhibition of rRNA synthesis has been shown to result in nucleolar disruption, cell cycle arrest, and ultimately cell death. Our data support an mTOR-independent effect of Akt activation on cell proliferation that would require direct targeting of PI3Kinase or activated Akt to have a major therapeutic effect in AML. Disclosures: No relevant conflicts of interest to declare.