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Background Older patients (≥ 60 years) with acute myeloid leukemia (AML) often have multiple, sequentially acquired, somatic mutations that drive leukemogenesis and are associated with poor outcome. Beat AML is a Leukemia and Lymphoma Society-sponsored, multicenter umbrella study that algorithmically segregates AML patients based upon cytogenetic and dominant molecular abnormalities (variant allele frequencies (VAF) ≥ 0.2) into different cohorts to select for targeted therapies. During the conception of the Beat AML design, a historical dataset was needed to help in the design of the genomic algorithm for patient assignment and serve as the basis for the statistical design of individual genomic treatment substudies for the Beat AML study. Methods We classified 563 newly diagnosed older AML patients treated with standard intensive chemotherapy on trials conducted by Cancer and Leukemia Group B based on the same genomic algorithm and assessed clinical outcomes. Results Our classification identified core-binding factor and NPM1-mutated/FLT3-ITD-negative groups as having the best outcomes, with 30-day early death (ED) rates of 0 and 20%, respectively, and median overall survival (OS) of > 1 year and 3-year OS rates of ≥ 20%. All other genomic groups had ED rates of 17–42%, median OS ≤ 1 year and 3-year OS rates of ≤ 15%. Conclusions By classifying patients through this genomic algorithm, outcomes were poor and not unexpected from a non-algorithmic, non-dominant VAF approach. The exception is 30-day ED rate typically is not available for intensive induction for individual genomic groups and therefore difficult to compare outcomes with targeted therapeutics. This Alliance data supported the use of this algorithm for patient assignment at the initiation of the Beat AML study. This outcome data was also used for statistical design for Beat AML substudies for individual genomic groups to determine goals for improvement from intensive induction and hopefully lead to more rapid approval of new therapies. Trial registration ClinicalTrials.gov Identifiers: NCT00048958 (CALGB 8461), NCT00900224 (CALGB 20202), NCT00003190 (CALGB 9720), NCT00085124 (CALGB 10201), NCT00742625 (CALGB 10502), NCT01420926 (CALGB 11002), NCT00039377 (CALGB 10801), and NCT01253070 (CALGB 11001).

Purpose: Spleen tyrosine kinase (SYK) signaling is a proposed target in acute myeloid leukemia (AML). Sensitivity to SYK inhibition has been linked to HOXA9 and MEIS1 overexpression in preclinical studies. This trial evaluated the safety and efficacy of entospletinib, a selective inhibitor of SYK, in combination with chemotherapy in untreated AML. Patients and Methods: This was an international multicenter phase Ib/II study, entospletinib dose escalation (standard 3+3 design between 200 and 400 mg twice daily) + 7+3 (cytarabine + daunorubicin) in phase Ib and entospletinib dose expansion (400 mg twice daily) + 7+3 in phase II. Results: Fifty-three patients (n = 12, phase Ib and n = 41, phase II) with previously untreated de novo (n = 39) or secondary (n = 14) AML were enrolled (58% male; median age, 60 years) in this study. The composite complete response with entospletinib + 7+3 was 70%. Patients with baseline HOXA9 and MEIS1 expression higher than the median had improved overall survival compared with patients with below median HOXA9 and MEIS1 expression. Common adverse events were cytopenias, febrile neutropenia, and infection. There were no dose-limiting toxicities. Entospletinib-related skin rash and hyperbilirubinemia were also observed. Conclusions: Entospletinib with intensive chemotherapy was well-tolerated in patients with AML. Improved survival was observed in patients with HOXA9/MEIS1 overexpression, contrasting published data demonstrating poor survival in such patients. A randomized study will be necessary to determine whether entospletinib was a mediator this observation.

Prognostic factors associated with chemotherapy outcomes in patients with acute myeloid leukemia (AML) are extensively reported, and one gene whose mutation is recognized as conferring resistance to several newer targeted therapies is protein tyrosine phosphatase non-receptor type 11 (PTPN11). The broader clinical implications of PTPN11 mutations in AML are still not well understood. The objective of this study was to determine which cytogenetic abnormalities and gene mutations co-occur with PTPN11 mutations and how PTPN11 mutations affect outcomes of patients treated with intensive chemotherapy. We studied 1725 patients newly diagnosed with AML (excluding acute promyelocytic leukemia) enrolled onto the Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology trials. In 140 PTPN11-mutated patient samples, PTPN11 most commonly co-occurred with mutations in NPM1, DNMT3A, and TET2. PTPN11 mutations were relatively common in patients with an inv(3)(q21q26)/t(3;3)(q21;q26) and a normal karyotype but were very rare in patients with typical complex karyotype and core-binding factor AML. Mutations in the N-terminal SH2 domain of PTPN11 were associated with a higher early death rate than those in the phosphatase domain. PTPN11 mutations did not affect outcomes of NPM1-mutated patients, but these patients were less likely to have co-occurring kinase mutations (ie, FLT3-ITD), suggesting activation of overlapping signaling pathways. However, in AML patients with wild-type NPM1, PTPN11 mutations were associated with adverse patient outcomes, providing a rationale to study the biology and treatment approaches in this molecular group. This trial was registered at www.clinicaltrials.gov as #NCT00048958 (CALGB 8461), #NCT00899223 (CALGB 9665), and #NCT00900224 (CALGB 20202).

Background: AML is a clinically and molecularly heterogeneous disease associated with poor survival. Multiple disease-related factors including cytogenetic findings and gene mutations, as well as patient-related factors, such as demographics and African American (AA) heritage, have been identified that impact on pt outcomes. However, with recent improved survival it is unknown whether racial health disparities persist. Moreover, we are not aware of a large study that assessed possible race-associated molecular differences. Thus, the goals of our study were to 1) analyze the outcomes of adult AML pts in a nationwide population study, including possible impacts of sociodemographic, financial and racial disparities and 2) characterize molecular features of AA compared with those of Caucasian AML pts. Methods: For a nationwide population analysis, the Surveillance Epidemiology End Results (SEER) Program of the National Cancer Institute was used to identify 11,190 adults aged 18-60 years (y) diagnosed with AML (excluding acute promyelocytic leukemia) between 1986 and 2015. To characterize molecular features we performed targeted sequencing of 81 genes in 1,339 AML pts treated on frontline Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology (Alliance) protocols based on standard intensity cytarabine/anthracycline induction followed by consolidation between 1986 and 2016. No Alliance pt received an allogeneic stem cell transplant in 1st complete remission (CR). Results: The associations between demographic parameters and risk of death among SEER registry AML pts are shown in Table 1. While there was a slightly higher risk of death for men (HR 1.09) and a lower risk of death for pts with a higher median household income (>79.6k vs Conclusion: Self-reported AA race is the most important pt-associated factor associated with poor survival in AML pts < 60 y of age based on SEER. Survival analyses in Alliance pts identify AA race as independent poor survival prognosticator in AML pts besides established molecular markers. . This disparity must be urgently addressed to ensure improved outcomes for AA AML pts, and larger studies to establish molecular risk profiles are needed. Support: U10CA180821, U10CA180882 U24CA196171, https://acknowledgments.alliancefound.org; Clinicaltrials.gov Identifiers: NCT00048958, NCT00899223, NCT00900224 Disclosures Bhatnagar: KaryoPharm Therapuetics: Research Funding; Cell Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; KITE: Membership on an entity's Board of Directors or advisory committees. Blachly:AbbVie, AstraZeneca, KITE Pharma: Consultancy. Mims:Novartis: Speakers Bureau; Jazz Pharmaceuticals: Other: Data Safety Monitoring Board; Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Leukemia and Lymphoma Society: Other: Senior Medical Director for Beat AML Study; Agios: Consultancy. Walker:Vigeo Therapeutics: Consultancy; Karyopharm: Current Employment, Current equity holder in publicly-traded company. Powell:Genentech: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Pfizer: Research Funding; Rafael Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Novartis: Research Funding. Kolitz:Magellan: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Stone:Biolinerx: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Other; Aztra-Zeneca: Consultancy; Jazz: Consultancy; Argenix: Other; Janssen: Consultancy; Daiichi-Sankyo: Consultancy; Pfizer: Consultancy; Gemoab: Consultancy; Syndax: Consultancy, Research Funding; Takeda: Other: DSMB; Macrogenics: Consultancy; Trovagene: Consultancy; Syntrix: Other: DSMB; Abbvie: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy; Arog: Consultancy, Research Funding; Syros: Consultancy; Stemline: Consultancy. Byrd:Trillium: Research Funding; Leukemia and Lymphoma Society: Other; Acerta Pharma: Research Funding; Syndax: Research Funding; Pharmacyclics LLC, an AbbVie Company, Janssen, Novartis, Gilead, TG Therapeutics: Other; Janssen: Consultancy; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, Novartis, Janssen: Speakers Bureau; Kartos Therapeutics: Research Funding; Vincera: Research Funding; Novartis: Research Funding. Eisfeld:Karyopharm: Current Employment, Current equity holder in publicly-traded company; Vigeo Therapeutics: Consultancy.

Purpose:Uniparental disomy (UPD) is a way cancer cells duplicate a mutated gene, causing loss of heterozygosity (LOH). Patients with cytogenetically normal acute myeloid leukemia (CN-AML) do not have microscopically detectable chromosome abnormalities, but can harbor UPDs. We examined the prognostic significance of UPDs and frequency of LOH in patients with CN-AML.Experimental Design: We examined the frequency and prognostic significance of UPDs in a set of 425 adult patients with de novo CN-AML who were previously sequenced for 81 genes typically mutated in cancer. Associations of UPDs with outcome were analyzed in the 315 patients with CN-AML younger than 60 years.Results:We detected 127 UPDs in 109 patients. Most UPDs were large and typically encompassed all or most of the affected chromosome arm. The most common UPDs occurred on chromosome arms 13q (7.5% of patients), 6p (2.8%), and 11p (2.8%). Many UPDs significantly cooccurred with mutations in genes they encompassed, including 13q UPD with FLT3-internal tandem duplication (FLT3-ITD; P < 0.001), and 11p UPD with WT1 mutations (P = 0.02). Among patients younger than 60 years, UPD of 11p was associated with longer overall survival (OS) and 13q UPD with shorter disease-free survival (DFS) and OS. In multivariable models that accounted for known prognostic markers, including FLT3-ITD and WT1 mutations, UPD of 13q maintained association with shorter DFS, and UPD of 11p maintained association with longer OS.Conclusions:LOH mediated by UPD is a recurrent feature of CN-AML. Detection of UPDs of 13q and 11p might be useful for genetic risk stratification of patients with CN-AML.

Current treatment options for older and relapsed or refractory (R/R) acute myeloid leukemia (AML) patients are limited and represent an unmet need. Based on preclinical studies showing strong anti-leukemic effects in vivo, this phase I dose-escalation study assessed the safety and preliminary clinical activity of the oral exportin-1 inhibitor, selinexor, in combination with the hypomethylating agent, decitabine 20 mg/m(2), in adults with R/R AML and in older (age ≥60) untreated AML patients. There were no protocol-defined dose limiting toxicities. The recommended phase 2 dose of selinexor was 60mg (~35 mg/m(2)) given twice-weekly. Notable grade ≥3 toxicities included asymptomatic hyponatremia (68%), febrile neutropenia (44%), sepsis (44%), hypophosphatemia (36%), and pneumonia (28%). In 25 patients, the overall response rate was 40%. Modification of selinexor to a flat dose of 60mg, twice-weekly for two weeks after decitabine, improved tolerability of the regimen and demonstrated preliminary clinical activity in poor-risk patients with AML.

Sole trisomies of chromosomes 4, 8, 11, 13 and 21 account for 89–95% of all sole trisomies in adult AML patients. We analyzed clinical and molecular characteristics of 138 de novo AML patients with sole +4, +8, +11, +13 or +21, and compared them with AML patients with those trisomies occurring in addition to other chromosome abnormalities (non-sole trisomy) and with cytogenetically normal AML (CN-AML) patients. Mutations in methylation-related genes were most commonly observed within each sole trisomy group (+4, 55%; +8, 58%; +11, 71%; +13, 71%; +21, 75% of patients). Patients with sole trisomies, excluding +4, also had frequent mutations in spliceosome genes (+8, 43%; +11, 65%; +13, 65%; +21, 45% of patients). In contrast, +4 patients frequently had mutations in transcription factor genes (44%) and NPM1 (36%). While 48% of patients with sole trisomies harbored mutations in a spliceosome gene, spliceosome mutations were observed in only 24% of non-sole trisomy (n = 131, P < 0.001) and 19% of CN-AML patients (n = 716, P < 0.001). Our data suggest that mutations affecting methylation-related genes are a molecular hallmark of sole trisomies. Mutations in spliceosome genes were also commonly observed in many sole trisomy patients and represent a novel finding in this cytogenetic subgroup.

Improvement in survival has been achieved for children and adolescents with AML but is largely attributed to enhanced supportive care as opposed to the development of better treatment regimens. High risk subtypes continue to have poor outcomes with event free survival rates, Pediatric AML is traditionally treated with chemotherapy and stem cell transplant but some subsets of patients have a poor response to therapy. Here, the authors perform a high throughput screen and identify several FDA approved drugs that might be useful in treating this disease.

The treatment of acute myelogenous leukemia (AML) relies heavily on cytarabine (Ara-C) containing chemotherapy. Even newly approved multi-kinase inhibitors improved survival only when combined with...

Purpose: Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (NAMPTi) are currently in development, but may be limited as single-agent therapy due to compound-specific toxicity and cancer metabolic plasticity allowing resistance development. To potentially lower the doses of NAMPTis required for therapeutic benefit against acute myeloid leukemia (AML), we performed a genome-wide CRISPRi screen to identify rational disease-specific partners for a novel NAMPTi, KPT-9274. Experimental Design: Cell lines and primary cells were analyzed for cell viability, self-renewal, and responses at RNA and protein levels with loss-of-function approaches and pharmacologic treatments. In vivo efficacy of combination therapy was evaluated with a xenograft model. Results: We identified two histone deacetylases (HDAC), HDAC8 and SIRT6, whose knockout conferred synthetic lethality with KPT-9274 in AML. Furthermore, HDAC8-specific inhibitor, PCI-34051, or clinical class I HDAC inhibitor, AR-42, in combination with KPT-9274, synergistically decreased the survival of AML cells in a dose-dependent manner. AR-42/KPT-9274 cotreatment attenuated colony-forming potentials of patient cells while sparing healthy hematopoietic cells. Importantly, combined therapy demonstrated promising in vivo efficacy compared with KPT-9274 or AR-42 monotherapy. Mechanistically, genetic inhibition of SIRT6 potentiated the effect of KPT-9274 on PARP-1 suppression by abolishing mono-ADP ribosylation. AR-42/KPT-9274 cotreatment resulted in synergistic attenuation of homologous recombination and nonhomologous end joining pathways in cell lines and leukemia-initiating cells. Conclusions: Our findings provide evidence that HDAC8 inhibition- or shSIRT6-induced DNA repair deficiencies are potently synergistic with NAMPT targeting, with minimal toxicity toward normal cells, providing a rationale for a novel–novel combination-based treatment for AML.

Clinical outcome of patients with acute myeloid leukemia (AML) is associated with cytogenetic and molecular factors and patient demographics (e.g., age and race). We compared survival of 25,523 non-Hispanic Black and White adults with AML using Surveillance Epidemiology and End Results (SEER) Program data and performed mutational profiling of 1,339 patients with AML treated on frontline Alliance for Clinical Trials in Oncology (Alliance) protocols. Black patients had shorter survival than White patients, both in SEER and in the setting of Alliance clinical trials. The disparity was especially pronounced in Black patients Significance: We show that young Black patients have not benefited as much as White patients from recent progress in AML treatment in the United States. Our data suggest that both socioeconomic factors and differences in disease biology contribute to the survival disparity and need to be urgently addressed. See related commentary by Vyas, p. 540. This article is highlighted in the In This Issue feature, p. 521

Effective treatment for AML is challenging due to the presence of clonal heterogeneity and the evolution of polyclonal drug resistance. Here, we report that TP-0903 has potent activity against protein kinases related to STAT, AKT, and ERK signaling, as well as cell cycle regulators in biochemical and cellular assays. In vitro and in vivo, TP-0903 was active in multiple models of drug-resistant FLT3 mutant AML, including those involving the F691L gatekeeper mutation and bone marrow microenvironment–mediated factors. Furthermore, TP-0903 demonstrated preclinical activity in AML models with FLT3-ITD and common co-occurring mutations in IDH2 and NRAS genes. We also showed that TP-0903 had ex vivo activity in primary AML cells with recurrent mutations including MLL-PTD, ASXL1, SRSF2, and WT1, which are associated with poor prognosis or promote clinical resistance to AML-directed therapies. Our preclinical studies demonstrate that TP-0903 is a multikinase inhibitor with potent activity against multiple drug-resistant models of AML that will have an immediate clinical impact in a heterogeneous disease like AML., TP-0903, a multikinase inhibitor, demonstrates preclinical activity in models of drug-resistant AML, including those involving FLT3 mutations, bone marrow microenvironment-mediated factors and recurrent mutations.

Acute myeloid leukemia (AML) is a heterogeneous and complex disease, and treatments for this disease have not been curative for the majority of patients. In younger patients, internal tandem duplication of FLT3 (FLT3-ITD) is a common mutation for which two inhibitors (midostaurin and gilteritinib) with varied potency and specificity for FLT3 are clinically approved. However, the high rate of relapse or failed initial response of AML patients suggests that the addition of a second targeted therapy may be necessary to improve efficacy. Using an unbiased large-scale CRISPR screen, we genetically identified BCL2 knockout as having synergistic effects with an approved FLT3 inhibitor. Here, we provide supportive studies that validate the therapeutic potential of the combination of FLT3 inhibitors with venetoclax in vitro and in vivo against multiple models of FLT3-ITD-driven AML. Our unbiased approach provides genetic validation for co-targeting FLT3 and BCL2 and repurposes CRISPR screening data, utilizing the genome-wide scope toward mechanistic understanding.

Balanced rearrangements involving the KMT2A gene, located at 11q23, are among the most frequent chromosome aberrations in acute myeloid leukemia (AML). Because of numerous fusion partners, the mutational landscape and prognostic impact of specific 11q23/KMT2A rearrangements are not fully understood. We analyzed clinical features of 172 adults with AML and recurrent 11q23/KMT2A rearrangements, 141 of whom had outcome data available. We compared outcomes of these patients with outcomes of 1,097 patients without an 11q23/KMT2A rearrangement categorized according to the 2017 European LeukemiaNet (ELN) classification. Using targeted next-generation sequencing, we investigated the mutational status of 81 leukemia/cancer-associated genes in 96 patients with 11q23/KMT2A rearrangements with material for molecular studies available. Patients with 11q23/KMT2A rearrangements had a low number of additional gene mutations (median, 1; range 0 to 6), which involved the RAS pathway (KRAS, NRAS, and PTPN11) in 32% of patients. KRAS mutations occurred more often in patients with t(6;11)(q27;q23)/KMT2A-AFDN compared with patients with the other 11q23/KMT2A subsets. Specific gene mutations were too infrequent in patients with specific 11q23/KMT2A rearrangements to assess their associations with outcomes. We demonstrate that younger (age

Patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) have poor outcomes and hematopoietic cell transplantation (HCT) is the only curative treatment. New targeted therapies improved survival in select patients with specific mutations, however management of patients without these molecular alterations is an unmet need. We conducted a phase one study of lenalidomide in combination with cytarabine/idarubicin salvage chemotherapy in patients with R/R AML and high‐risk myelodysplastic syndromes. A total of 33 patients were enrolled in the study (30 AML, 3 MDS), and treated at three dose levels with 3 + 3 design. Dose‐limiting toxicity (DLT) was seen in eight patients, including four hematologic DLTs. The most commonly observed non‐hematologic serious adverse events were febrile neutropenia, rash, sepsis and renal injury. Dose level −1, consisting of 25 mg/d lenalidomide D1‐21, 1 g/m2 cytarabine D5‐8, and 8 mg/m2 idarubicin D5‐7 was determined to be the maximum tolerated dose. Note, 15/33 (45%) of patients were able to receive pre‐planned 21 days of lenalidomide. Overall, 18 patients achieved complete remission (CR) (n = 14) or CR with incomplete count recovery (CRi) (n = 4) with total CR/CRi rate of 56%. The 1‐year and 2‐year overall survival (OS) were 24% and 10%, respectively. Among responders, 10/18 underwent allogeneic HCT and had a 1‐year OS of 40%. There was no molecular pattern associated with response. These data demonstrate that the combination had clinical activity in R/R AML. This regimen should be further investigated for patients who relapsed after HCT, and as a bridge therapy to HCT. (ClinicalTrials.gov identifier: NCT01132586).

Targeted mutation assessment of 81 genes in 1021 adults with de novo acute myeloid leukemia (AML) identified recurrent mutations in the neurofibromin 1 (NF1) gene in 52 (5.1%) patients, including 36 (5.2%) younger and 16 (4.8%) older patients, which suggests NF1 belongs to the 20 most frequently mutated genes in adult AML. NF1 mutations were found throughout the gene, and comprised missense, frameshift, and nonsense mutations. One mutation hotspot, at amino acid threonine 676 (Thr676), was found in 27% of AML patients with NF1 mutations. NF1-mutated patients belonged more often to the adverse European LeukemiaNet (ELN) risk category than NF1 wild-type patients. Among patients aged

KMT2A partial tandem duplication occurs in approximately 5-10% of patients with acute myeloid leukemia and is associated with adverse prognosis. KMT2A wild type is epigenetically silenced in KMT2A partial tandem duplication; re-expression can be induced with DNA methyltransferase and/or histone deacetylase inhibitors in vitro, sensitizing myeloid blasts to chemotherapy. We hypothesized that epigenetic silencing of KMT2A wildtype contributes to KMT2A partial tandem duplication-associated leukemogenesis and pharmacologic re-expression activates apoptotic mechanisms important for chemoresponse. We developed a regimen for this unique molecular subset, but due to relatively low frequency of KMT2A partial tandem duplication, this dose finding study was conducted in relapsed/refractory disease regardless of molecular subtype. Seventeen adults (< age 60) with relapsed/refractory acute myeloid leukemia were treated on study. Patients received decitabine 20 milligrams/meter2 daily on days 1-10 and vorinostat 400 milligrams daily on days 5-10. Cytarabine was dose-escalated from 1.5 grams/meter2 every 12 hours to 3 grams/meter2 every 12 hours on days 12, 14 and 16. Two patients experienced dose limiting toxicities at dose level 1 due to prolonged myelosuppression. However, as both patients achieved complete remission after Day 42, the protocol was amended to adjust the definition of hematologic dose limiting toxicity. No further dose limiting toxicities were found. Six of 17 patients achieved complete remission including 2 of 4 patients with KMT2A partial tandem duplication. Combination therapy with decitabine, vorinostat and cytarabine was tolerated in younger relapsed/refractory acute myeloid leukemia and should be explored further focusing on the KMT2A partial tandem duplication subset. (clinicaltrials.gov identifier 01130506).

Thus far, only 5-15% of AML patients aged ≥60 years are cured with chemotherapy. Identification of patients who are less (more) likely to respond to standard chemotherapy might enable early risk stratification toward alternative treatment regimens. We used a next-generation sequencing panel of 80 cancer- and/or leukemia-associated genes to profile molecularly 423 older patients with de novo AML. Using variables identified in multivariable models and co-occurring mutations in NPM1-mutated AML, we classified the patients into good-, intermediate-, and poor-risk groups for complete remission (CR) attainment, disease-free (DFS), and overall survival (OS). Whereas 81% of good-risk patients (comprising NPM1-mutated patients harboring mutations in chromatin remodeling, cohesin complex, methylation-related, spliceosome, and/or RAS pathway genes, FLT3-TKD, and/or patients without FLT3-ITD) achieved a CR, only 32% of poor-risk patients (with U2AF1, WT1 mutations and/or complex karyotype) did. Intermediate-risk patients had a 50% CR rate. Similarly, using NPM1 co-mutation patterns and SF1 mutation status, we identified patients with favorable DFS and OS 3-year rates of 46% and 45%, respectively. Patients with adverse genetic features had DFS and OS rates of only 2% and 4%. We show that application of our proposed criteria may refine the 2017 European LeukemiaNet classification for older patients treated with chemotherapy.

Genetic profiling of leukemic cells forms the basis for risk stratification in acute myeloid leukemia (AML). Genetic markers in AML are used to make recommendations for distinct treatment approaches, such as remission consolidation with chemotherapy rather than stem cell transplant for patients with favorable risk genetics as defined by the current guidelines from the European LeukemiaNet (ELN). Yet, several limitations remain, such as overall rarity of many mutations, hierarchical complexity in cases with multiple mutations, conflicting reports of associations with outcomes for some genetic markers, and the absence of markers with prognostic significance in some patients. We have recently described genome-wide DNA methylation signatures that underlie biological features of AML cells and their utility to classify patients [Giacopelli et al. Genome Res. 2021;31:747]. The additional value of epigenetic information for risk assessment in AML in the context of current genetic and other clinical prognostic markers remains largely unexplored. In this study, we have first developed a targeted approach for assessment of DNA methylation-based signatures and employed it to classify 1,262 patients with de novo AML enrolled onto the Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology studies. We successfully classified 87.5% of patients into one of 13 DNA methylation subgroups, termed 'epitypes' (Figure 1A,B). We found that epitypes are composed of a majority of patients with a specific genetic alteration (or a unique combination of alterations) in 9 of 13 epitypes. However, we also identified subgroups of patients that lack these highly recurrent alterations, and, instead, represent an epigenetic phenocopy of the dominant genetic feature (epiphenocopy). Epiphenocopies within epitypes were often enriched in specific lower frequency mutations, suggesting convergence of biological function(s) for these rare mutations. Epiphenocopying was also exhibited by patients displaying a DNA methylation signature involving hypomethylation of STAT DNA sequence motifs (termed the STAT hypomethylation signature, SHS) that mimicked FLT3-ITD mutations. Epitype and SHS DNA methylation signatures affected clinical outcomes separately to ELN risk groups (P To broadly examine the prognostic power of DNA methylation signatures, we combined methylation-based classifications into a knowledge bank containing a compendium of other prognostic markers. Using a recently developed machine-learning approach [Gerstung et al. Nat Genet. 2017;49(3):332], we found that DNA methylation retained a high degree of importance for clinical outcomes, including overall survival (Figure 1F). Specifically, SHS and 6 epitypes were the most significant features negatively associated with overall survival along with age (P Our study demonstrates that DNA methylation signatures advance our understanding of the biology of AML and improve risk stratification through the identification of patients with epiphenocopies that mimic genetic mutations and other biological features. Use of DNA methylation signatures may lead to more effective assignment of patients to existing and novel therapeutic approaches. Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org; ClinicalTrials.gov Identifiers: NCT00048958 (8461), NCT00899223 (9665), and NCT00900224 (20202) Figure 1 Figure 1. Disclosures Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Blum: Abbvie: Honoraria; AmerisourceBergen: Honoraria; Celyad Oncology: Research Funding; Xencor: Research Funding; Nkarta: Research Funding; Forma Therapeutics: Research Funding; Leukemia and Lymphoma Society: Research Funding; Syndax: Honoraria. Stone: Agios Pharmaceuticals Inc, Novartis;: Research Funding; ACI Clinical, Syntrix Pharmaceuticals, Takeda Oncology: Other: Data Safety & Monitoring; AbbVie Inc, Actinium Pharmaceuticals Inc, Aprea Therapeutics, BerGenBio ASA, ElevateBio, Foghorn Therapeutics, GEMoaB, GlaxoSmithKline, Innate Pharma, Syndax Pharmaceuticals Inc, Syros Pharmaceuticals Inc, Takeda Oncology: Other: Advisory Committee. Eisfeld: Karyopharm (spouse): Current Employment. Byrd: Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees.

Background: WBC levels vary widely in AML pts at diagnosis. Together with various cytogenetic and molecular abnormalities, WBC is a main prognostic factor for AML pts. Treatment decisions like need for intrathecal chemotherapy, trial enrollment eligibility, and stem cell transplant (SCT) considerations are often influenced by degree of WBC elevation. Despite such high clinical relevance, there are no standardized WBC-associated groups that improve prognostication and treatment guidance for AML pts. Aims: (1) define clinically relevant WBC level groups associated with outcome, (2) determine if WBC level has an independent prognostic impact in addition to established prognostic features [i.e., 2017 European LeukemiaNet (ELN) genetic-risk classification] and (3) characterize WBC level-associated gene-expression profiles to provide biologic insights into factors influencing WBC levels. Methods: We analyzed clinical and molecular features of 1,121 younger de novo AML pts similarly treated on frontline Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology protocols. No pt received an allogeneic SCT in 1 st complete remission (CR). Targeted next generation sequencing of 81 cancer- and leukemia-associated genes was done using MiSeq platform. We defined 3 WBC groups: low ( Results: Pts in the high WBC group had higher extramedullary disease burden at diagnosis than pts in the intermediate and low groups (38% vs 29% and 12%, respectively; P Conclusion: The 3 WBC groups we propose offer additional prognostic information for younger AML pts. An intermediate WBC group was associated with better outcome among all pts and in pts included in the ELN Favorable group, especially those with non-CBF-AML. We also showed differences in the metabolic pathways among WBC groups. Our results suggest that the paradigm that all pts who present with a high WBC have a poor prognosis should be re-evaluated, and can help guide therapy decisions for younger AML pts. U10CA180821, U10CA180882, U24CA196171, https://acknowledgments.alliancefound.org; Clinicaltrials.gov Ids: NCT00048958, NCT00899223, NCT00900224 Figure 1 Figure 1. Disclosures Walker: Karyopharm Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Blachly: AstraZeneca: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Larson: Takeda: Research Funding; CVS/Caremark: Consultancy; Gilead: Research Funding; Astellas: Consultancy, Research Funding; Epizyme: Consultancy; Rafael Pharmaceuticals: Research Funding; Cellectis: Research Funding; Novartis: Research Funding. Stone: Amgen: Membership on an entity's Board of Directors or advisory committees; Syntrix/ACI: Membership on an entity's Board of Directors or advisory committees; Foghorn Therapeutics: Consultancy; Bristol Myers Squibb: Consultancy; Gemoab: Membership on an entity's Board of Directors or advisory committees; Elevate Bio: Membership on an entity's Board of Directors or advisory committees; Aprea: Consultancy; Arog: Consultancy, Research Funding; Actinium: Membership on an entity's Board of Directors or advisory committees; Boston Pharmaceuticals: Consultancy; AbbVie: Consultancy; BerGen Bio: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy; Innate: Consultancy; Janssen: Consultancy; Jazz: Consultancy; Novartis: Consultancy, Research Funding; Onconova: Consultancy; Takeda: Consultancy; Agios: Consultancy, Research Funding; Celgene: Consultancy; Macrogenics: Consultancy. Byrd: Newave: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria. Eisfeld: Karyopharm (spouse): Current Employment. Mims: Kura Oncology: Consultancy; BMS: Consultancy; Abbvie: Consultancy; Genentech: Consultancy; Xencor: Research Funding; Leukemia and Lymphoma Society's Beat AML clinical study: Consultancy, Research Funding; Syndax Pharmaceuticals: Consultancy; Aptevo: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Glycomemetics: Research Funding; Kartos Pharmaceuticals: Research Funding; BMS: Consultancy; Jazz Pharmaceuticals: Consultancy; Aptevo: Research Funding.

Background: Chronic Lymphocytic Leukemia (CLL) is a genetically heterogeneous disease characterized by clonal expansion of B-lymphocytes that induce secondary immune suppression. CLL is now treated with inhibitors of Bruton tyrosine kinase (BTK) and BCL2. Virtually all patients respond to therapy, however resistance to these therapies has been described justifying the need for novel CLL therapies. Broad inhibition of cyclin dependent kinases (CDK) and associated alternative target enzymes with agents such as flavopiridol or dinaciclib have demonstrated significant clinical activity in CLL but are hindered by a relatively narrow therapeutic window. VIP152 is a highly specific inhibitor of CDK9 - considered the most important CDK kinase member for CLL clinical activity. VIP152 has favorable pharmacokinetic properties and has demonstrated durable, preliminary single-agent clinical activity in double-hit diffuse large B-cell lymphoma. Herein, we report the efficacy of VIP152 preclinically in CLL. Methods: On-target activity of VIP152 was measured using a KinomeScan from DiscoveryRx at 100nM and 1000nM. Kinase profiling for VIP152 was performed on 6 kinases in a 10-dose assay at ReactionBio. Cell-based viability and proliferation assays (MTS, Annexin-V (AV), and propidium iodine (PI)) were performed in primary CLL cells and the CLL cell lines, HG3 and MEC1. Transcriptional activity after VIP152 exposure was measured via qPCR and limiting-cell RNA sequencing (lcRNAseq). Proteomic and immunoblot studies were performed to measure perturbations in CDK9 binding partners and on-target activity of VIP152. A genome-wide CRISPR/CAS9 knockout screen was performed to identify any synthetically lethal targets and pathways. Results: The KinomeScan identified CDK9 as the kinase with maximal inhibition upon VIP152 treatment and no other CDKs were identified at 100nM. Kinase profiling revealed the IC 50 of VIP152 was lowest for CDK9/Cyclin T1 and CDK9/Cyclin T2 with close similarity to dinaciclib and greater than 1 log superiority over KB-0742. Co-immunoprecipitation and proteomics experiments have identified a CDK9 specific mechanism of action relating to perturbations of CDK9 binding partners. Specifically, we showed that CDK9 nuclear immunoprecipitation resulted in decreased co-immunoprecipitation of 7SK RNA components (HEXIM1 & MEPCE) as well as decreased RNA Polymerase II (RNAP2). The decrease in RNAP2 CoIP was further seen via proteomics. A 2-hour exposure of VIP152 against HG3 and MEC1 demonstrated growth inhibition, with an IC 50 of 0.9814µM and 1.092µM respectively. Continuous exposure of the compound for 24 hours resulted in a statistically significant drop in relative viability of 30% across a 10-fold dose range (0.1µM to 1.0µM) as measured by AV/PI. Primary CLL cells (n=10) responded with similar dosing strategies with a 54% reduction in viability at 1µM; moreover, stromal cell co-culture experiments demonstrated VIP152's ability to induce cell death and overcome stromal protection with short exposure. Induction of apoptosis was observed with pro-caspase-3 and PARP cleavage on immunoblot. qPCR and immunoblot studies demonstrated a time dependence of phosphorylated serine 2 (pS2) RNAP2 decreases alongside diminishment of MYC and MCL1 mRNA and protein. pS2 was shown to decrease as early as 2 hours after VIP152 treatment with similar decreases in MCL1 and MYC at both the protein and mRNA levels. We identified several pathways which are disrupted following treatment via lcRNAseq, including TNFR1 and TNFR2 signaling as well as upregulation of autophagy signals. Finally, CRISPR screen identified several potentially synergistically lethal targets, including transcriptional co-activators, DNA binding proteins, and cell proliferation pathways. Validation of these is ongoing as is an in vivo study of VIP152 in a CLL mouse model. Conclusions: Our data demonstrate VIP152 to be a highly selective and potent CDK9 inhibitor that disrupts the CDK9 nuclear complex and mediates significant preclinical activity against CLL cell lines and primary CLL cells. VIP152 also demonstrates predictable and new pharmacodynamic markers to assess target engagement. Collectively, these data support the recently initiated CLL clinical trial (NCT04978779). Disclosures Johnson: Vincerx: Current Employment; Janssen: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Frigault: Vincerx Pharma Inc: Current Employment; AstraZeneca: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months, Patents & Royalties. Greer: Gilead: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Vincerx Pharma Inc: Current Employment. Hamdy: Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Acerta Pharma Inc: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months, Patents & Royalties. Izumi: Acerta Pharma Inc: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months, Patents & Royalties; Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Hwang: Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company. Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Byrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees.

Background: Outcome of AML pts is known to be impacted by genetic factors such as cytogenetic abnormalities and gene mutations and sociodemographic factors such as age and race. Neighborhood factors are increasingly recognized as important drivers of disparities in cancer outcomes. Neighborhood socioeconomic deprivation has been associated with worse survival of numerous cancers, but studies in AML are lacking. Understanding the role of social deprivation in AML outcomes, and its impact in the context of known prognostic factors could identify areas that may benefit from additional health care resources. Methods: We analyzed the clinical and molecular features of 1,242 younger AML (age range, 17-59 y) pts similarly treated with intensive cytarabine/daunorubicin-based chemotherapy on frontline Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology protocols between 2001 and 2013. A neighborhood social deprivation index (SDI) was assigned to all pts based on pt reported zip code of residence. SDI was population weighted with resulting scores (1-100) corresponding to US zip code percentiles of SDI (e.g., 100 = top 1% of US population in zip codes of highest deprivation). We defined 2 SDI levels, low (1-50 n=664) and high (51-100, n=578), that were analyzed for associations with clinical and molecular features and outcomes. Results: Demographic and baseline clinical features did not differ between high and low SDI pts, except for a higher percentage of self-reported non-Hispanic Black pts residing in high compared to low SDI areas (15% v 2%, p Concerning clinical outcomes, there were no significant differences in early death (7% v 6%) or CR (67% for both) rates between low and high SDI pts. However, pts residing in high SDI areas had shorter disease-free survival (DFS; median: 1.3 v 2.4 y, p=.001; Fig. 1A) and overall survival (OS; median: 1.9 v 2.9 y, p We next assessed whether differences in molecular features between high and low SDI groups may help explain the observed survival disparities (n=656 pts). While we found no statistically significant differences in the frequencies of known prognosis-associated gene mutations by SDI levels, there was a trend towards lower frequencies in genes belonging to the cohesion complex and methylation-associated genes (both p=.06), indicating possible differences in underlying disease biology. Percentages of high and low SDI pts did not differ significantly within any of the 3 genetic-risk groups in the 2017 European Leukemia Net (ELN) classification. However, when we analyzed outcomes of pts within the ELN groups, we found that among pts belonging to the ELN Favorable-risk group, high SDI score was associated with shorter OS (p=.03) than low SDI score, but this was not true for ELN Intermediate- or Adverse-risk pts. Lastly, in multivariable analysis for OS in younger AML pts, a high SDI score associated with shorter OS (HR: 1.28, CI: 1.10-1.48, p=.01; Table 1), after correction for ELN groups (p Conclusion: In younger AML pts, residence in areas with high socioeconomic deprivation was associated with poor survival, especially for pts classified in the ELN favorable-risk group. With the exception of patient racial-ethnic identity and receipt of allogeneic transplant in first CR, SDI did not associate with clinical or molecular characteristics. Thus, area social deprivation among younger AML pts should be further investigated to overcome potentially avoidable survival disparities. Support: U10CA180821, U10CA180882 U24CA196171; Clinicaltrials.gov Identifiers: NCT00048958, NCT00899223, NCT00900224; https://acknowledgments.alliancefound.org Figure 1 Figure 1. Disclosures Mims: Leukemia and Lymphoma Society's Beat AML clinical study: Consultancy, Research Funding; Aptevo: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Glycomemetics: Research Funding; Kartos Pharmaceuticals: Research Funding; Xencor: Research Funding; Genentech: Consultancy; Abbvie: Consultancy; BMS: Consultancy; Kura Oncology: Consultancy; Syndax Pharmaceuticals: Consultancy; BMS: Consultancy; Jazz Pharmaceuticals: Consultancy; Aptevo: Research Funding. Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Blum: Nkarta: Research Funding; Celyad Oncology: Research Funding; Abbvie: Honoraria; AmerisourceBergen: Honoraria; Xencor: Research Funding; Forma Therapeutics: Research Funding; Leukemia and Lymphoma Society: Research Funding; Syndax: Honoraria. Uy: Agios: Consultancy; AbbVie: Consultancy; Novartis: Consultancy; GlaxoSmithKline: Consultancy; Genentech: Consultancy; Macrogenics: Research Funding; Astellas: Honoraria, Speakers Bureau; Jazz: Consultancy. Stone: Abbvie: Consultancy; Actinium: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Aprea: Consultancy; Arog: Consultancy, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; BerGen Bio: Membership on an entity's Board of Directors or advisory committees; Boston Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Elevate Bio: Membership on an entity's Board of Directors or advisory committees; Foghorn Therapeutics: Consultancy; Gemoab: Membership on an entity's Board of Directors or advisory committees; Glaxo Smith Kline: Consultancy; Innate: Consultancy; Jannsen: Consultancy; Jazz: Consultancy; Macrogenics: Consultancy; Novartis: Consultancy, Research Funding; Onconova: Consultancy; Syndax: Membership on an entity's Board of Directors or advisory committees; Syntrix/ACI: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy. Byrd: Newave: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria. Paskett: Pfizer: Research Funding; Merck: Research Funding. Eisfeld: Karyopharm (spouse): Current Employment.

Acute myeloid leukemia (AML) is the most commonly diagnosed acute leukemia in adults. Despite newly approved treatment, AML still results in poor outcomes especially in older patients (pts). Cytogenetic abnormalities, gene mutations, and their combinations contribute to the pathogenesis and pt outcomes in AML. The PTPN11 gene encodes the phosphatase Shp2, which activates the RAS-MAPK pathway. Despite the relatively high frequency of PTPN11 mutations in AML, little is known about associations of PTPN11 mutations with other genomic features and their influence on outcomes of pts with standard 7+3 chemotherapy. In addition, primary resistance to targeted therapy, such as venetoclax and enasidenib, has been preliminarily noted in PTPN11+ pts. This study sought to determine the type and frequency of PTPN11 mutations as well as associations with clinical, cytogenetic, and genomic features and outcome in adult AML pts treated with 7+3 induction chemotherapy followed by consolidation chemotherapy on Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology trials. 1,725 newly diagnosed AML pts, defined by the European LeukemiaNet 2017 recommendations (excluding acute promyelocytic leukemia), were examined using targeted next generation sequencing analysis and centrally reviewed metaphase cytogenetics. Missense, nonsense, or frameshift variants not reported in the 1000 Genomes database, dbSNP137 or dbSNP142, were considered mutations. Fisher's exact test was used to determine mutation association and complete remission (CR) rates while continuous variables are from Wilcoxon rank sum test. The median follow-up was 9 years. We identified 140 pts (8.1%) with PTPN11 mutations with the majority (61%) located in the N-terminal SH2 domain (Figure 1). 98 younger ( 0.3. NPM1 (61% vs 31%, P < .001) and DNMT3A (R882 or other) mutations (39% vs 22%, P < .001) were more likely to co-occur with PTPN11 mutations than wild-type (WT) PTPN11 (Figure 2). PTPN11 mutations were less common in FLT3-ITD pts than in those without (17% vs 25%, P = .07). PTPN11 mutations were more common in inv(3)(q21q26)/t(3;3)(q21;q26) pts (26%, P = .004) and were rare in pts with core-binding factor AML, inv(16)/t(16;16) (3%, P = .03) and t(8;21) (0%, P =.005). Clinical features of PTPN11+ pts were similar to those of WT pts except for elevated platelet counts (P < .001) and more extramedullary involvement (P = .03). For all pts, there was no difference in CR rate, disease-free (DFS), overall (OS), and event-free (EFS) survival between PTPN11+ and PTPN11- pts. DFS of older PTPN11+ pts was shorter (3-y rates: 5% vs 15%, P = .04). Given that PTPN11 mutations often co-occur with NPM1 mutations, which are typically associated with favorable outcome (in the absence of a high FLT3-ITD ratio), we focused on the contribution of PTPN11 mutations to outcomes in the NPM1+/FLT3-WT subset. Compared with PTPN11-/NPM1+/FLT3-WT, PTPN11+ pts had a lower CR rate (38% vs 64%, P = .001) and shorter EFS (3-y rates: 10% vs 21%, P = .01), whereas there was no significant differences in OS (3-y rates: 23% vs 32%, P = .13) or DFS (3-y rates: 27% vs 33%, P = .75). When considering the role of PTPN11 mutations with WT NPM1, there was a reduction in survival in PTPN11+/NPM1- pts compared with PTPN11-/NPM1- pts. Younger PTPN11+/NPM1- pts had a lower CR rate (45% vs 71%, P = .002) and shorter OS (3-y rates: 30% vs 41%, P = .04), and EFS (3-y rates: 13% vs 27%, P = .008). Compared to older PTPN11-/NPM1-, older PTPN11+/NPM1- pts had a lower CR rate (18% vs 43%, P = .04) and shorter DFS (3-y rates: 0% vs 10%, P = .02) and EFS (3-y rates: 0% vs 4%, P = .02), whereas OS (3-y rates: 12% vs 10%, P = .58) had no significant difference. To our knowledge, this study is the largest cohort of PTPN11+ pts in adult AML and demonstrates specific mutational and cytogenetic associations. When considering PTPN11+ pts based on NPM1 mutation status, we showed that PTPN11 mutations associated with worse outcome in both NPM1+ and NPM1- AML pts when treated with intensive chemotherapy. Developing targeted treatments to this genomic group in AML represents a research priority. Support: U10CA180821, U10CA180882, U24CA196171, R35CA198183; https://acknowledgments.alliancefound.org; Clinicaltrials.gov Identifier: NCT00048958, NCT00899223, NCT00900224 Disclosures Mims: Leukemia and Lymphoma Society: Other: Senior Medical Director for Beat AML Study; Agios: Consultancy; Kura Oncology: Membership on an entity's Board of Directors or advisory committees; Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Jazz Pharmaceuticals: Other: Data Safety Monitoring Board. Blachly:AbbVie, AstraZeneca, KITE Pharma: Consultancy. Stone:Takeda: Consultancy; Trovagene: Consultancy; Pfizer: Consultancy; Gemoab: Consultancy; Janssen: Consultancy; AbbVie: Consultancy, Research Funding; Actinium: Consultancy; Agios: Consultancy, Research Funding; Argenx: Consultancy, Other: Data and safety monitoring board; Arog: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Biolinerx: Consultancy; Celgene: Consultancy, Other: Data and safety monitoring board; Jazz: Consultancy; Novartis: Consultancy, Research Funding; Otsuka: Consultancy; Syntrix: Consultancy; Syros: Consultancy; Elevate: Consultancy; Syndax: Consultancy; Daiichi-Sankyo: Consultancy; Stemline: Consultancy; Macrogenics: Consultancy; Hoffman LaRoche: Consultancy. Wang:Bristol Meyers Squibb (Celgene): Consultancy; PTC Therapeutics: Consultancy; Macrogenics: Consultancy; Astellas: Consultancy; Jazz Pharmaceuticals: Consultancy; Stemline: Speakers Bureau; Genentech: Consultancy; Pfizer: Speakers Bureau; Abbvie: Consultancy. Kolitz:Pfizer: Membership on an entity's Board of Directors or advisory committees; Magellan: Membership on an entity's Board of Directors or advisory committees. Powell:Rafael Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Genentech: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Advisor, Research Funding. Eisfeld:Vigeo Therapeutics: Consultancy; Karyopharm: Current Employment, Current equity holder in publicly-traded company. Byrd:Syndax: Research Funding; Vincera: Research Funding; Novartis: Research Funding; Kartos Therapeutics: Research Funding; Acerta Pharma: Research Funding; Trillium: Research Funding; Leukemia and Lymphoma Society: Other; Janssen: Consultancy; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, Novartis, Janssen: Speakers Bureau; Pharmacyclics LLC, an AbbVie Company, Janssen, Novartis, Gilead, TG Therapeutics: Other.

Introduction: AML is a heterogeneous disease of mainly older pts, with a median age of 68 y at diagnosis. Previous studies have shown that age is one of the most important prognosticators for overall survival (OS) of AML pts. In addition, several cytogenetic and molecular markers have been used for risk stratification of AML pts and to guide therapeutic decisions. However, the role of these established molecular marker (e.g. FLT3-ITD and NPM1 mutations) in pts aged ≥70 y is not fully understood. Aims: The goal of this study was to analyze the prognostic role of FLT3-ITD and other molecular markers in AML pts ≥70 y. We also compared this cohort with pts 60-69 y, who received a similar treatment, to identify potential differences between these age groups. Methods: We analyzed 269 de novo AML pts ≥70 y and 250 pts 60-69 y. All pts were treated on frontline Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology protocols that were based on standard intensity cytarabine/anthracycline induction followed by consolidation with varied chemotherapy regimens. None of the pts received an allogeneic stem cell transplant in first complete remission (CR). In pretreatment bone marrow or blood samples, the mutational status of 80 cancer- and leukemia-associated genes (Eisfeld et al., Leukemia 2017;31:2211) were determined using a targeted next-generation sequencing panel. All variants that occurred with variant allele fractions of .05). The pts' pretreatment karyotypes were determined locally and reviewed centrally. Results: There were no significant differences in frequencies of any clinical or specific cytogenetic markers between AML pts ≥70 y and pts 60-69 y. Pts 60-69 y presented more often with Favorable-risk disease based on the European LeukemiaNet classification (Döhner et al. Blood 2017;129:424; 30% vs 23%, P Conclusions: Although pts aged ≥70 y had similar pretreatment clinical and cytogenetic features as pts 60-69 y, we showed that distinct differences can be found regarding prognosticators. Interestingly, we found that presence of a FLT3-ITD had no impact on outcome of pts ≥70 y, regardless of allelic ratio or presence of an additional NPM1 mutation. Only NPM1 mutations and complex karyotype had a prognostic impact on both age groups. Support: U10CA180821, U10CA180882 U24CA196171; https://acknowledgments.alliancefound.org; Clinicaltrials.gov Identifiers: NCT00048958 (CALGB-8461), NCT00899223 (CALGB 9665), and NCT00900224 (CALGB 20202) Disclosures Walker: Vigeo Therapeutics: Consultancy; Karyopharm: Current Employment, Current equity holder in publicly-traded company. Blachly:AbbVie, AstraZeneca, KITE Pharma: Consultancy. Wang:Bristol Meyers Squibb (Celgene): Consultancy; Astellas: Consultancy; Jazz Pharmaceuticals: Consultancy; Macrogenics: Consultancy; PTC Therapeutics: Consultancy; Abbvie: Consultancy; Stemline: Speakers Bureau; Pfizer: Speakers Bureau; Genentech: Consultancy. Kolitz:Pfizer: Membership on an entity's Board of Directors or advisory committees; Magellan: Membership on an entity's Board of Directors or advisory committees. Powell:Rafael Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Pfizer: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Novartis: Research Funding; Genentech: Research Funding. Stone:Gemoab: Consultancy; Syros: Consultancy; Arog: Consultancy, Research Funding; Celgene: Consultancy, Other; Macrogenics: Consultancy; Agios: Consultancy, Research Funding; Janssen: Consultancy; Aztra-Zeneca: Consultancy; Biolinerx: Consultancy; Trovagene: Consultancy; Daiichi-Sankyo: Consultancy; Argenix: Other; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Other: DSMB; Abbvie: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Astellas: Consultancy; Syntrix: Other: DSMB; Jazz: Consultancy; Stemline: Consultancy; Pfizer: Consultancy; Syndax: Consultancy, Research Funding. Byrd:Novartis: Research Funding; Acerta Pharma: Research Funding; Syndax: Research Funding; Pharmacyclics LLC, an AbbVie Company, Janssen, Novartis, Gilead, TG Therapeutics: Other; Vincera: Research Funding; Kartos Therapeutics: Research Funding; Trillium: Research Funding; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company, Gilead, TG Therapeutics, Novartis, Janssen: Speakers Bureau; Leukemia and Lymphoma Society: Other; Janssen: Consultancy. Eisfeld:Vigeo Therapeutics: Consultancy; Karyopharm: Current Employment, Current equity holder in publicly-traded company.

Investigate antileukemic activity of artemisinins, artesunate (ART), and dihydroartemisinin (DHA), in combination with cytarabine, a key component of acute myeloid leukemia (AML) chemotherapy using in vitro and in vivo models. Using ten human AML cell lines, we conducted a high-throughput screen to identify antimalarial agents with antileukemic activity. We evaluated effects of ART and DHA on cell viability, cytotoxicity, apoptosis, lysosomal integrity, and combination effects with cytarabine in cell lines and primary patient blasts. In vivo pharmacokinetic studies and efficacy of single-agent ART or combination with cytarabine were evaluated in three xenograft models. ART and DHA had the most potent activity in a panel of AML cell lines, with selectivity toward samples harboring MLL rearrangements and FLT3-ITD mutations. Combination of ART or DHA was synergistic with cytarabine. Single-dose ART (120 mg/kg) produced human equivalent exposures, but multiple dose daily administration required for in vivo efficacy was not tolerated. Combination treatment produced initial regression, but did not prolong survival in vivo. The pharmacology of artemisinins is problematic and should be considered in designing AML treatment strategies with currently available agents. Artemisinins with improved pharmacokinetic properties may offer therapeutic benefit in combination with conventional therapeutic strategies in AML.

The use of multikinase inhibitors (MKI) in oncology, such as sorafenib, is associated with a cutaneous adverse event called hand–foot skin reaction (HFSR), in which sites of pressure or friction become inflamed and painful, thus significantly impacting quality of life. The pathogenesis of MKI-induced HFSR is unknown, and the only available treatment options involve dose reduction or discontinuation of therapy, which have negative effects on primary disease management. To investigate the underlying mechanisms by which sorafenib promotes keratinocyte cytotoxicity and subsequent HFSR induction, we performed a transporter-directed RNAi screen in human epidermal keratinocytes and identified SLC22A20 (OAT6) as an uptake carrier of sorafenib. Further investigations into the intracellular mechanism of sorafenib activity through in situ kinome profiling identified the mitogen-activated protein kinase MAP3K7 (TAK1) as a target of sorafenib that induces cell death. Finally, we demonstrate that sorafenib induced keratinocyte injury in vivo and that this effect could be reversed by cotreatment with the OAT6 inhibitor probenecid. Collectively, our findings reveal a novel pathway that regulates the entry of some MKIs into keratinocytes and explains the basis underlying sorafenib-induced skin toxicity, with important implications for the therapeutic management of HFSR. Cancer Res; 76(1); 117–26. ©2015 AACR.

Using a panel of human cell lines representing subtypes associated with high-risk of relapse we combined high-throughput screening (HTS), intracellular accumulation assays, and in vivo efficacy studies to identify novel therapeutic strategies for pediatric acute myeloid leukemia (AML). We identified multiple drug classes with broad activity across multiple subtypes, including: cytotoxics, epigenetic modifiers, and targeted agents; select compounds were further evaluated using primary patient samples ex vivo. FDA-approved drugs (e.g. gemcitabine, cabazitaxel) not currently used to treat AML were identified to have greater anti-leukemic activity relative to the standard of care, cytarabine, both in vitro and in vivo. Our comprehensive approach provides advances in pre-clinical development of novel treatment strategies with potential for rapid clinical translation for pediatric AML.

Introduction: NPM1 gene mutations are a common molecular aberration in acute myeloid leukemia (AML). In the absence of concurrent high FLT3-ITD ratio mutations (>0.5), NPM1 mutations typically associate with higher complete remission (CR) rates following intensive induction chemotherapy. NPM1 mutations have been shown to be stable markers of persistent disease or impending relapse during CR or complete remission with incomplete count recovery (CRi). Given the clinical implications that persistent NPM1 mutations can have during CR/CRi, we used Deep Amplicon sequencing on CR/CRi bone marrow (BM) samples collected from adult de novoNPM1-mutated AML patients to determine the ability of NPM1 mutations at both a high and lower sensitivity next generation sequencing methods and also the presence of additional clonal abnormalities on relapse risk. Methods: We performed targeted next generation sequencing (NGS) analysis in addition to NPM1 Deep Amplicon sequencing on paired BM or blood samples collected from 38 newly diagnosed NPM1-mutated AML patients during CR/CRi after successful induction (1-2 courses of 7 + 3) and, if available, at relapse. NPM1 mutated NGS libraries were prepared using a KAPA HyperPlus Kit (Roche, Pleasanton, CA) and xGen Lockdown Probes (IDT, Coralville, IA). Libraries were sequenced using the Illumina HiSeq 4000 (Illumina, San Diego, CA). GATK's MuTect2 was used to perform variant calling. Variant allele frequency (VAF) cut-off for the NGS panel was 0.05 (5%) with the exception of hotspot variants in IDH1 (R132) and IDH2 (R140) where variants detected to a level of 0.01 (1%) were included. The VAF cut off used for NPM1 Deep Amplicon sequencing was 0.00012 (0.012%). Results: Targeted NGS analysis and NPM1 Deep Amplicon sequencing had exceptional concordance at the level of detection of VAF= 0.05 (Figure 1). Of 38 patients, 23 patients had undetectable NPM1 mutations as analyzed through NPM1 Deep Amplicon sequencing of whom 9 (38.1%) relapsed. In contrast, 15 patients were positive by NPM1 Deep Amplicon sequencing and 9 (60%) relapsed. Only 4 patients had detectable persistent NPM1 mutations after induction according to both detection techniques and two of these relapsed. We next examined the potential impact of clearing both NPM1 mutation and co-occurring mutations together on relapses (Figure 2). A total of 15 patients cleared all of their clonal abnormalities and 5 (27%) relapsed. In contrast, of the 23 patients who did not clear the NPM1 mutation and/or another co-occurring mutation at remission, 14 (61%) have relapsed. Eleven of the relapsed patients had relapse samples available of whom all had persistent NPM1 mutation at this time. Paired CR/CRi and relapsed samples showed acquisition or recurrence of several other mutations, most notably FLT3-ITD, IDH1, and IDH2 which are all targetable with small molecule therapeutics. Conclusions: The use of Deep Amplicon sequencing to identify NPM1 mutations at a lower detection threshold compared to standard NGS techniques was more sensitive, but did not appear to fully inform relapse rates in NPM1-mutated AML patients after receipt of induction therapy. The appearance of other AML-associated mutations, identified together with NPM1 at time of remission, was more frequent among patients relapsing. These pilot data provide support for concurrent assessment of Deep Amplicon sequencing together with a broad standard NGS AML mutational assay to further enhance risk stratification of NPM1-mutated patients. Additionally, while NPM1 clones are present in all patients examined at the time of relapse, persistence or development of targetable clones justifies repeat broad NGS sequencing at this time. Figure Disclosures Bhatnagar: Novartis and Astellas: Consultancy, Honoraria; Cell Therapeutics, Inc.: Other: Research support; Karyopharm Therapeutics: Other: Research support. Mims:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees. Behbehani:Fluidigm corporation: Other: Travel funding. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S.

Somatic mutation of the DNMT3A gene at the arginine R882 site is common in acute myeloid leukaemia (AML). The prognostic significance of DNMT3A R882 mutation clearance, using traditional diagnostic next generation sequencing (NGS) methods, during complete remission (CR) in AML patients is controversial. We examined the impact of clearing DNMT3A R882 mutations at diagnosis to the detectable threshold of ˂3% during CR on outcome in 56 adult AML patients. Mutational remission, defined as clearance of pre-treatment DNMT3A R882 and all other AML-associated mutations to a variant allele frequency ˂3%, occurred in 14 patients whereas persistent DNMT3A R882 mutations were observed in 42 patients. There were no significant differences in disease-free or overall survival between patients with and without DNMT3A R882 mutation clearance. Patients with persistent DNMT3A R882 who cleared all other AML mutations and did not acquire new mutations (n = 30), trended towards longer disease-free survival (1·6 vs. 0·6 years, P = 0·06) than patients with persistence of DNMT3A R882, in addition to other mutations or acquisition of new AML-associated mutations, such as those in TET2, JAK2, ASXL1 and TP53 (n = 12). These data demonstrate that DNMT3A R882 mutations, as assessed by traditional NGS methods, persist in the majority of AML patients in CR.

Approximately 45% of adult AML pts do not have cytogenetic abnormalities visible by microscopic karyotype analysis and are classified as CN-AML. However, these pts can still have one or more UPD (the loss of a chromosome or its segment and duplication of the remaining chromosome or segment), since UPDs are not detectable by typical karyotyping methods. Because UPDs have been shown to contribute to malignant transformation and outgrowth in various cancers and leukemias, we sought to identify recurrent UPDs in CN-AML pts and explore their associations with disease presentation, gene mutations and outcome. We used Infinium OmniExpress SNP arrays to detect UPDs in a cohort of 425 pts, who were similarly treated with intensive chemotherapy on Alliance for Clinical Trials in Oncology therapeutic trials. Pts having early death ( UPDs of 10 different chromosome arms were detected in >1% of pts (Figure A). The most commonly occurring UPDs were UPD of 13q (present in 7.5% of pts), UPD of 6p (in 2.4% of pts) and UPD of 11p (in 2.4% of pts). We examined associations between UPDs of 13q, 6p and 11p with demographic and clinical diagnostic characteristics. UPD of 6p was associated with higher peripheral blood (PB) blast percentage (median 86% vs 70%, P=.02); UPD of 11p was associated with lower platelet counts (41 vs. 60 x 109/L, P=.03) and higher PB blast percentage (85% vs 60%, P=.002); and UPD of 13q was associated with higher white blood cell count (53 vs 29 x 109/L, P=.02), higher PB blast percentage (77% vs 58%, P=.004) and higher bone marrow blast percentage (80% vs 69%, P=.005). We also examined the associations between UPDs and gene mutations present in >2% of CN-AML pts in our sample series. 13q UPD significantly co-occurred with FLT3-internal tandem duplication (ITD) (P Assessment of the association between UPDs and outcome was performed. Because of differences in induction therapies between pts younger and older than 60 years (y), outcome analyses must be performed separately in these two age groups. Based on sample size limitations we were only able to assess the impact of 11p UPD and 13q UPD on achievement of complete remission (CR), disease-free survival (DFS) and overall survival (OS) in the 315 pts younger than 60 y. UPD of 11p was associated with longer OS (3 y rates, 83% vs 45%, P=.02), and UPD of 13q was associated with shorter DFS and OS (3 y rates, 0% vs 38%, P Finally, since FLT3-ITD and UPD of 13q often co-occurred and were both found to be associated with shorter DFS in the MVA of adult younger pts, we examined younger pts who harbored FLT3-ITD for differences in DFS associated with UPD of 13q. We found those pts with 13q UPD had significantly shorter DFS (P=.003) compared to those without (Figure C), which further demonstrates 13q UPD status is useful for prognostic stratification independent from its association with the known prognostic marker FLT3-ITD. These data show the incorporation of 13q UPD might improve current genetic risk stratification models. Follow-up studies investigating the mechanistic roles of these recurrent UPDs, especially the relationship between UPD of 11p and bi-allelic CEBPA mutation, could shed light on novel pathways underlying leukemic transformation and proliferation in CN-AML. Disclosures Powell: Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Kolitz:Magellan Health: Consultancy, Honoraria.

Introduction: Patients (pts) with AML who harbor TP53 mutations (TP53m) have a poor prognosis, with complete remission (CR) rates of 20-30% with standard intensive cytarabine/daunorubicin induction regimens (7+3) and median disease-free survival (DFS) of 4-6 months. There is a high risk of relapse after allogeneic stem cell transplantation (allo SCT) with 3-year overall survival (OS) of 10-12%. Another member of the p53 family is TP73, expressed as multiple protein isoforms that range in function from tumor suppressive activity to promoting oncogenic activities through control of cell survival and cell renewal. The TP73 gene resides in the short arm of chromosome 1; although this region is frequently deleted in a broad range of tumors, TP73 is rarely mutated in cancer. In the era of next-generation sequencing (NGS), TP73 mutations (TP73m) have not yet been described in AML to our knowledge. Herein we describe AML pts with TP73m and compare their outcomes with those of pts with TP53m and pts without TP73m or TP53m. Methods: Mutational assessment of 81 leukemia- and/or cancer-associated genes using a customized NGS approach was performed on samples from 1165 pts (age ≥18 years) with newly diagnosed AML, treated with standard 7+3 followed by cytarabine +/- anthracycline based consolidation (Eisfeld et al., Leukemia 2017;31:2211-8). We identified 3 groups of pts: wild-type (wt) for both TP53 and TP73,TP53m (including 2 pts with TP73m), and TP73m/TP53wt (TP73m). We made statistical comparisons between groups regarding co-mutations, mutational functional groupings, and outcomes using Fisher's exact test, Kruskal Wallis test, Kaplan-Meier curves, and log rank test. Results: We identified 106 pts with TP53m and 11 pts with TP73m; 1048 pts were confirmed to be wt for both. TP73m pts were all found to have missense mutations, with the majority of variant allele frequencies (VAF) >20%, suggesting these mutations likely represent early mutational events similar to TP53m. Evaluation of mutations in other genes in TP73m pts revealed that most frequent were FLT3-ITD (27%) and mutations in ASXL1 (18%), DNMT3A (18%), IDH2 (18%), NPM1 (36%), RUNX1 (18%), STAG2 (18%), TET2 (27%), and U2AF1 (18%) genes. Frequencies of these mutations were similar to those seen in wt pts, except for higher frequencies of STAG2 (P=.05) and U2AF1 (P=.05) mutations in TP73m pts. No mutation occurred in TP53m pts with a significantly higher frequency than that found in wt pts. However, FLT3-ITD (P Pretreatment characteristics of pts with TP73m were similar to those of wt pts, except for lower white blood cell counts (WBC) in the former (P=.05). No differences were seen between TP73m and TP53m pts. In contrast, compared with wt pts, TP53m pts were older (P There were no significant differences in CR, DFS or OS between TP73m and wt pts. However, compared to both TP73m and wt pts, TP53m pts had shorter DFS (P=.005 for TP73m and P Conclusion: TP73m are recurrently found in adult AML pts, albeit with a lower frequency than TP53m. TP73m does not appear to portend the poor prognosis seen in TP53m pts in regards to DFS, EFS and OS and have outcomes that appear to be similar to wt pts. Support: U10CA180821, U10CA180882, U10CA180861, U24CA196171; ClinicalTrials.gov Ids: NCT00048958, NCT00900224 Figure. Figure. Disclosures Mims: Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Powell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Kolitz:Magellan Health: Consultancy, Honoraria.

Introduction: In contrast to younger pts with AML, those ages 60 years (y) and above often have multiple, sequentially acquired, somatic mutations that drive leukemogenesis. Long-term 10-y event-free survival (EFS) of non-transplanted older AML pts receiving intensive cytarabine/daunorubicin induction (7 and 3) is 2% and the median overall survival (OS) is less than 1 y (Vasu et al., Blood Adv 2018;2:1645-50). Divergent pathogenesis based upon sequentially acquired mutations with targetable founder alterations and recent introduction of new targeted therapies have prompted interest in precision medicine-based treatment of AML. In order to determine if new therapeutic treatment strategies perform better than current standards of care, it is important to understand the historical outcomes for defined genomic groups in older AML pts. Methods: In anticipation of a precision medicine-based trial initiated by the Leukemia and Lymphoma Society (LLS AML Master Study), we designed a precision medicine-based stratification considering "assignment to curative therapy with 7 and 3" for known responsive groups [i.e., core-binding factor (CBF) AML and NPM1-mutated(+)/FLT3-ITD- pts] followed by pts with defined driving cytogenetic aberrations [11q23/MLL-rearranged and TP53 wild-type/complex karyotype (≥3 chromosome abnormalities)] and pts with mutation clone with VAF ≥0.3 by next generation sequencing. For pts not assigned to any genomic group during the initial stratification, a second run-through of the algorithm was performed assessing for a mutation clone with VAF ≥0.2 (Figure 1). The following priority (in order of highest to lowest) was used: CBF-AML, NPM1+/FLT3-ITD-, 11q23/MLL-rearranged, IDH2+, IDH1+, TP53+, TP53 wild-type/complex karyotype, FLT3-ITD+ (both high and low allelic ratios included) or FLT3-TKD+, WT1+ or TET2+, and marker negative (all other karyotypes and mutations not defined by previous grouping). A well-characterized data set of 589 pts aged ≥60 y (range, 60-92) from the Alliance was analyzed to show outcomes of pts within specific genomic groups (Table 1). Identification of a second multi-institutional pt data set is currently on-going to validate these findings. Results: In the Alliance cohort, 516 of 589 (88%) pts were assigned solely based upon cytogenetic group or dominant mutational clone with VAF ≥0.3 and 11 (1%) patients were assigned based off of VAF ≥0.2, thus providing support for this algorithm. The remaining 62 pts were assigned to the marker-negative group. As this algorithm is designed to assign therapy and assess outcome on an intent-to-treat basis, pts who suffered early death (ED), defined as death within 30 days of starting therapy irrespective of cause, were included. Baseline characteristics among groups were similar with the following exceptions: 1) CBF-AML and NPM1+/FLT3-ITD- pts having equal male and female pts with other groups having male predominance; 2) white blood cell counts being highest in NPM1+/FLT3-ITD-, 11q23/MLL-rearranged and FLT3-ITD+ or TKD+ groups; 3) percent of bone marrow blasts were lowest in the TP53+ group. Outcome endpoints are summarized in Table 1. Early death (ED) occurred in 20% of pts, most commonly in TP53+ (41%), IDH1+ (24%) and FLT3-ITD+ or TKD+ (23%) groups. Considering ED, the complete remission (CR) rates were above 50% in the CBF-AML (72%), and NPM1+/FLT3-ITD- (67%) groups, whereas in other groups they ranged between 30% and 50%, except for TP53+ at 17%. The 3-y disease-free survival (DFS) ranged from 0 to 13% for most groups, whereas only NPM1+/FLT3-ITD- (27%) and CBF-AML (30%) pts appeared to gain long-term benefit from 7 and 3. Three-y OS was 0-16% for most groups, except NPM1+/FLT3-ITD- pts at 27% and CBF-AML pts at 33%. Conclusion: Our analysis of a large older AML pt cohort provides evidence of the feasibility of classifying this disease based upon long-term benefit from 7 and 3. We also use cytogenetic/dominant mutation clone identification for precision medicine-based assignment to novel targeted therapies where 7 and 3 lacks impact on long-term outcome. These data can also serve as a historical control population for pts treated as part of the LLS AML Master Study and for other efforts brought forth to advance novel therapies in select genomic groups of older AML pts. Support: U10CA180821, U10CA180882, U10CA180861, U24CA196171 ClinicalTrials.gov Identifiers: NCT00048958 (8461) and NCT00900224 (20202) Disclosures Mims: Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Borate:Agios: Consultancy; Novartis: Consultancy. Stein:Celgene: Consultancy; Bayer: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Novartis: Consultancy; Daiichi Sankyo: Consultancy. Kolitz:Magellan Health: Consultancy, Honoraria. Wang:Jazz: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy. Powell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Levine:Epizyme: Patents & Royalties; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Prelude: Research Funding; Loxo: Consultancy, Equity Ownership; C4 Therapeutics: Equity Ownership; Isoplexis: Equity Ownership; Gilead: Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Research Funding; Novartis: Consultancy; Celgene: Consultancy, Research Funding; Imago: Equity Ownership. Druker:Celgene: Consultancy; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Henry Stewart Talks: Patents & Royalties; Millipore: Patents & Royalties; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Beta Cat: Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Monojul: Consultancy; Fred Hutchinson Cancer Research Center: Research Funding; Bristol-Meyers Squibb: Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; Oregon Health & Science University: Patents & Royalties; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Acute Myeloid Leukemia (AML) is a heterogeneous disease that is characterized by an accumulation of neoplastic myeloid precursor cells in the bone marrow. Recently, multiple agents targeting AML associated mutations in FLT3, IDH1 and IDH2 have been developed. However, a majority of AML patients lack these mutations. Therefore, development of a novel therapeutic approach broadly relevant to AML would be attractive. The clonal capacity of AML cells is maintained by leukemic initiating cells (LICs), which possess self-renewal capabilities and are resistant against cytotoxic combination chemotherapy. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in the generation of NAD+, which is an important enzymatic cofactor and serves as a metabolite required for cellular respiration. LICs show a higher energy turnover rate than normal cells and are heavily reliant on oxidative phosphorylation. This suggests that energy generation processes, such as NAD+ biosynthesis, are critically required in myeloid malignancies. Thus, targeting the regeneration of NAD+ offers an attractive alternative therapeutic strategy in AML. Unlike many targeted therapies that are limited to one genetic subtype of AML, targeting regeneration of NAD+ via NAMPT inhibition could be relevant to a much broader patient population based upon metabolic differences between tumor and normal cells. We show that inhibition of NAMPT using the agent KPT-9274 induces loss of glycolytic and mitochondrial activity, more specifically a depleted total spare reserve capacity (p Disclosures Mims: Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Baloglu:Karyopharm Therpeutics: Employment. Senapedis:Karyopharm Therapeutics: Employment.

Acute myeloid leukemia (AML) is the most prevalent adult leukemia characterized by genetic or mutational disruption of myeloid differentiation, growth arrest, and apoptosis. Standard “7 + 3” chemotherapy consisting of cytarabine in combination with an anthracycline such as daunorubicin has been used for more than four decades. However this course only results in 10 year disease free survival in 15% of patients age 60 years. Several new successful therapies in AML are shown to work by promoting differentiation, and/or inducing apoptosis of the terminally differentiated cells. However these therapies, while promising, have the limitation of working in only a subset of AML with specific genetic profiles. The discovery of novel agents applicable to a broader patient population still represents a critical unmet need in this disease. Intracellular calcium is a common signaling molecule used in a variety of cellular processes including cell cycle progression. One of the major pathways which regulates calcium entry is store operated calcium entry (SOCE) mediated through calcium release-activated Ca2+ channels (CRAC), which respond to depleted calcium stores in ER lumen. This primarily occurs via interaction of ORAI1 (expressed on the plasma membrane) and STIM1 (expressed on the endoplasmic reticulum). Leukemic cells in particular often rely on the influx of calcium through CRAC for proliferation, therefore targeting these channels are a promising therapeutic option. In the current study, we explore the preclinical use of RP4010, a novel inhibitor of ORAI1 developed by Rhizen Pharmaceuticals, in AML. RP4010 is very effective at inhibiting proliferation of both AML cell lines and primary AML patient samples (IC50 values ranging from 0.2 - 1 μM using a tetrazolium-based colorimetric assay (MTS) assay). Importantly, RP4010 efficacy is not altered in the presence of stromal cell support. RP4010 also decreased colony growth in CFU assays. Finally, we evaluated the in vivo efficacy using an MV4-11 xenograft model. RP4010 treatment at 50 mg/kg once daily by oral gavage significantly prolonged survival compared to the vehicle control (p = 0.019). Furthermore, at the time of sacrifice, splenic tumor burden (percentage of human CD45 positive cells) was reduced in the mice treated with RP4010 compared to the vehicle group (p = 0.019). In conclusion, we provide here compelling evidence that inhibition of ORAI1 with RP4010 is a promising therapeutic strategy in AML. A Phase 1/1b study in R/R Non-Hodgkin Lymphoma (NHL) patients is currently underway in the US (ClinicalTrials.gov Identifier: NCT03119467), and our data would support expansion to other hematological malignancies as well, particularly AML. Senior authors J. C. B and E. H contributed equally to this work. Citation Format: Bridget Carmichael, Amy Lehman, Ola A. Elgamal, Shelley J. Orwick, Jean Truxall, Larry Beaver, Kumar V. Penmetsa, Srikant Viswanadha, John C. Byrd, Erin Hertlein. Store-operated calcium signaling is an effective therapeutic target in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1938.

Acute Myeloid Leukemia (AML) is a rapidly progressing hematopoietic malignancy arising from bone marrow myeloid progenitor cells. Treatment with cytotoxic chemotherapy has not changed for over four decades resulting in poor survival. The dismal prognosis could be attributed to the heterogeneity of this disease, where multiple genetically aberrant clones exist within the same patient. Mutations in the FMS-like tyrosine kinase (FLT3) occurs in 30% of AML patients, typically as an internal tandem duplication (ITD) resulting in a constitutively active FLT3 survival pathway. This has prompted the generation of selective FLT3 inhibitors such as Quizartinib and Gilteritinib which are currently being pursued in clinical trials. Still, acquired resistance to these selective FLT3 inhibitors due to the acquisition of tyrosine kinase domain mutations (TKD) can occur. This suggests that the use of an agent with a broader kinome inhibition profile (such as the recently granted FDA approved Midostaurin) could achieve more durable clinical benefit. ARQ 531 is a novel potent BTK inhibitor currently being investigated in a Phase 1 trial in patients with relapsed/refractory hematological malignancies (ClinicalTrials.gov Identifier: NCT03162536). We have found that ARQ 531 also has inhibitory activity against members of the Src family of kinases (SFK; including downstream target SYK) as well as FLT3. SYK directly binds to and trans-activates FLT3 which is essential for FLT3-ITD tumorigenicity, suggesting that ARQ 531 has therapeutic potential in AML. Therefore we have investigated the in vitro and in vivo efficacy of ARQ 531 in AML. Our preliminary studies demonstrate cytotoxicity for ARQ 531 in patient-derived primary AML cells harboring FLT3 wild type and FLT3-ITD, as well as multiple AML cell lines. Importantly, ARQ 531 is effective in a MOLM-13 tyrosine kinase inhibitor (TKI) resistant cell line harboring a FLT3-ITD-TKD-D835Y mutation. Furthermore, we show that ARQ 531 can reduce the level of phosphorylated FLT3, but unlike selective FLT3 inhibitors, it can also inhibit Src family phosphorylation and SYK phosphorylation. Additionally, ARQ 531 exhibited an anti-clonogenic effect on primary patient blasts using Methocult colony forming unit assay. Finally, to investigate the in vivo effect of ARQ 531, we used an aggressive AML MOLM-13 disseminated xenograft mouse model. NSG mice were randomized one-week post engraftment to either vehicle or daily oral gavage of 50 mg/kg ARQ 531. The estimated median survival for the ARQ 531 group was 23 days compared to 21 days for the vehicle group (p = 0.002) suggesting in vivo efficacy for ARQ 531 in AML. Collectively, we provide for the first time promising preclinical efficacy for ARQ 531 in AML supporting further mechanistic investigation of this agent, and potentially, expansion of the ongoing clinical studies to include AML patients. E. H. and J.C. B. contributed equally as co-senior authors to this work Citation Format: Ola A. Elgamal, Bridget Carmichael, Amy Lehman, Shelley J. Orwick, Minh Tran, Virginia M. Goettl, Shaneice Mitchell, Rosa Lapalombella, Jae Yoon Jeon, Sharyn D. Baker, Sudharshan Eathiraj, Brian Schwartz, Erin Hertlein, John C. Byrd. Preclinical evaluation of the tyrosine kinase inhibitor ARQ 531 in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1882.

The purpose of this study was to evaluate the affinity of docetaxel for 14 transporter proteins and assess the functional significance of 17 variants in five genes involved in drug elimination. Among the transfected models investigated, OATP1B3 (SLCO1B3) was identified as the most efficient influx transporter for docetaxel. None of the observed genotypes (SLCO1B3, ABCB1, and ABCC2) was related with docetaxel clearance in 92 white patients (P > 0.17). However, the simultaneous presence of the CYP3A4*1B and CYP3A5*1A alleles was associated with a 64% increase in docetaxel clearance (P = 0.0015), independent of both sex and CYP3A activity (as determined using the erythromycin breath test). This haplotype was also associated with increased midazolam clearance in another population (P = 0.0198). An analysis of the CYP3A locus among CEPH-HapMap samples revealed that CYP3A4*1B is present exclusively among a subset of CYP3A5 expressors. Therefore, future studies should first stratify the population on the basis of CYP3A5 genotype and then compare CYP3A activity between individuals with and without the CYP3A4*1B allele.

Using broad interrogation of clinically relevant drug absorption, distribution, metabolism, and excretion (ADME) genes on the DMET platform, we identified a genetic variant in SLCO1B1 (rs2291075; c.597C>T), encoding the transporter OATP1B1, associated with event-free (P = 0.006, hazard ratio = 1.74) and overall survival (P = 0.012, hazard ratio = 1.85) in children with de novo acute myeloid leukemia (AML). Lack of SLCO1B1 expression in leukemic blasts suggested the association might be due to an inherited rather than a somatic effect. rs2291075 was in strong linkage with known functional variants rs2306283 (c.388A>G) and rs4149056 (c.521T>C). Functional studies in vitro determined that four AML-directed chemotherapeutics (cytarabine, daunorubicin, etoposide, and mitoxantrone) are substrates for OATP1B1 and the mouse ortholog Oatp1b2. In vivo pharmacokinetic studies using Oatp1b2-deficient mice further confirmed our results. Collectively, these findings demonstrate an important role for OATP1B1 in the systemic pharmacokinetics of multiple drugs used in the treatment of AML and suggest that inherited variability in host transporter function influences the effectiveness of therapy.

Introduction: Exportin-1 (XPO1), a nuclear transport protein critical for the export of tumor suppressor proteins (TSPs) and select mRNAs to the cytoplasm, is highly expressed in acute myeloid leukemia (AML) and correlates with poor survival. Selinexor, an oral, first-in-class, selective inhibitor of nuclear export, blocks XPO1 function. We previously reported that sequential treatment of AML blasts using the hypomethylating agent decitabine followed by selinexor exhibited strong anti-leukemic effects in vivo by inducing the expression of silenced TSPs that are kept in the nucleus by XPO1 inhibition (Ranganathan, Blood 2015). Methods: Based on these findings, a phase I dose-escalation study was initiated to evaluate the safety, feasibility, maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), and preliminary clinical activity of selinexor in combination with decitabine in poor-risk AML pts (NCT02093403). Adults with relapsed or refractory (R/R) AML and older (age ≥60) unfit pts with untreated AML were eligible. Pts received 10-day decitabine induction(s) at 20mg/m2 on days 1-10 for up to four 28-day cycles in combination with selinexor once daily, twice weekly beginning on day 11. Pts with Results: Twenty-four pts were enrolled (see Table 1 for baseline pt characteristics). Nineteen pts had R/R AML (median of 3 prior chemotherapy regimens, range 1-4); five pts had previously untreated AML. Seventeen pts were treated on the dose escalation phase receiving selinexor doses ranging from 23mg/m2 to 55mg/m2. After two patients in DL 1 declined continuation of study therapy beyond cycle 1 due to grade Conclusions: The combination of selinexor plus decitabine is an active regimen in poor-risk AML pts, with CR/CRi/mCR rate of 80% in older untreated and 21% in R/R AML pts. Modification of selinexor exposure (flat dose of 60mg given twice weekly for 2 weeks after decitabine) improved tolerability of the regimen. Correlative studies, including targeted mutation analysis to assess whether specific mutations are associated with response, are ongoing and will be presented. Disclosures Bhatnagar: Karyopharm: Research Funding.

While dramatic improvements in survival have been achieved for children and adolescents with acute myeloid leukemia (AML), some subtypes are associated with event free survival of 8000 compounds. Compounds with >50% activity (N = 285) in the primary screen and known clinical candidates were prioritized for a secondary HTS performed in a dose-response manner (10 point curve, 0-10μM). Screening was performed using a 348-well plate format and a Biomek automation workstation for drug delivery. At 72 h cell viability was determined using Cell Titer Glo and automated Envision plate reader. Multiple drug classes had broad activity including: anti-malarials, cytotoxics (antimicrotubules, purine/pyrimidine antimetabolites, floxuridine, trimetrexate, topoI/II inhibitors), epigenetic modifiers (BET, DNMT and HDAC inhibitors), and agents targeting Bcl-2, Chk1, Parp, Hsp90, NF-êB, NAMPT, p53, proteasome, and Wee1. We validated 13 compounds from select drug classes (artesunate, cytarabine, gemcitabine, cabazitaxel, depsipeptide, panobinostat, vorinostat, ABT-199, RG117, bortezomib, carfilizomab, BMN673, MK-1775) using primary blast samples representing 3 high-risk groups (FLT3-ITD-positive, MLL-rearranged, FAB M7). Given the broad activity of gemcitabine and cabazitaxel across AML subtypes and the ability for repurposing in childhood AML, we further evaluated these agents alone and in combination in vitro. After 72h exposure, cabazitaxel was a potent inhibitor of viability in AML cell lines (IC50, 3-11nM) and primary blast samples (IC50, 3.4nM to >10μM); gemcitabine had similar activity (IC50, 3-62nM and 102nM to >10μM, respectively). The combination was evaluated using continuous and sequential administration schedules for up to 72h of treatment in several cell lines; simultaneous treatments were synergistic (CI, 0.2-0.45) and sequential treatments were additive to synergistic (CI, 0.82-0.95). Tolerable combination regimens were identified in NSG mice (5mg/kg cabazitaxel + 50mg/kg gemcitabine q3d x 4 weeks or q4d x 3 weeks) and efficacy studies in AML xenografts and primagrafts are ongoing. Further studies will aim to determine the underlying mechanisms of AML sensitivity to gemcitabine, cabazitaxel, and the combination. Our comprehensive approach to identify treatment leads provide advances in understanding the biology of a heterogeneous disease and development of novel treatment strategies with potential clinical benefit for pediatric AML. Citation Format: Christina D. Drenberg, Anang Shelat, Shelley J. Orwick, Hiroto Inaba, Raul C. Ribeiro, Jeffrey E. Rubnitz, Tanja A. Gruber, R K. Guy, Sharyn D. Baker. Integrated high-throughput screen to identify treatment leads for pediatric AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4786.

Improvements in survival have been achieved for children and adolescents with acute myeloid leukemia (AML), with the 5-year survival rate increasing between 1975 and 2010 from T), a gene encoding the hepatocellular uptake transporter OATP1B1. This SNP was not correlated with gene expression in leukemic blasts (P = 0.58), suggesting the association might be due to a host rather than a leukemia effect. Since this SNP is in strong linkage disequilibrium (P Citation Format: Christina D. Drenberg, Stanley Pounds, Lei Shi, Shelley Orwick, Lie Li, Shuiying Hu, Alice Gibson, Raul Ribeiro, Jeffrey Rubnitz, Alex Sparreboom, Sharyn D. Baker. Host variation in OATP1B1 is associated with treatment outcome in pediatric AML. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5464. doi:10.1158/1538-7445.AM2015-5464

A common mechanism associated with clinical resistance to FLT3 inhibitors (e.g., sorafenib and quizartinib) in FLT3-ITD+ AML is the acquisition of secondary FLT3 TKD mutations. Recently, we demonstrated that crenolanib, a potent and selective FLT3 inhibitor, has preclinical activity against FLT3 inhibitor-resistant FLT3-ITD+/TKD mutants, and the combination of sorafenib and crenolanib was more efficacious than single-agent TKIs in a FLT3-ITD+ xenograft model (Zimmerman et al. Blood 2013). Here, we evaluated continuous and intermittent schedules of sorafenib (SOR) and crenolanib (CRE) and the impact on efficacy and drug resistant profiles in vivo. We hypothesized that varying selective pressure through modulation of FLT3 inhibitor exposure (e.g., single-agent vs combination, high- vslow-dose intensity) would alter drug resistance patterns. For in vivo evaluations, one million MOLM13-luciferase+ cells were administered by tail vein injection to NSG mice and treatment was started 10 days later. Mice (7-10 per treatment group) were treated with vehicle (median survival, 16 days), SOR 60 mg/kg once daily (qd) on Monday, Wednesday, and Friday (3/7); SOR 60mg/kg qd for 5 days per week (5/7); CRE 15mg/kg twice daily (bid) 5/7; CRE 15mg/kg qd 5/7 and SOR 60mg/kg qd 5/7 (combination A); or CRE 15mg/kg bid 5/7 and SOR 60mg/kg qd 3/7 (combination B). Mice were monitored daily for assessment of toxicity and serial pharmacokinetic sampling was performed on days 15 or 16 of therapy. Mice were treated continuously until leukemic progression, sacrificed and bone marrow was harvested for assessment of leukemic infiltration and FLT3 TKD mutations in exons 17 and 20 by deep amplicon sequencing. Combinations A & B were tolerated and more efficacious than single-agent TKIs: median survival combination A vs sorafenib qd 5/7 (59 days vs 45 days, p Emergence of clinically relevant FLT3 TKD mutations during TKI treatment was modeled in a MOLM-13 bone marrow xenograft model. Although both drug combination schedules were more efficacious than single-agent TKIs, the combination incorporating more dose intensive crenolanib therapy suppressed the emergence of FLT3 TKD mutations. These results may allow for optimal clinical trial design of TKI combinations and have provided the foundation for a protocol in development for FLT3-ITD+ pediatric AML. Disclosures Schuster: AROG Pharmaceuticals: Employment. Ramachandran:AROG Pharmaceuticals: Employment. Inaba:Bayer/Onyx: Research Funding.

Introduction: We previously identified genes associated with cytarabine (AraC) response in acute myeloid leukemia (AML) and demonstrated increased expression of PIK3C3 to be significantly associated with a detrimental pattern of association (p=0.016) and multiple phenotypes (increased DNA synthesis, increased blast AraC IC50, poor clinical response, and worse event free survival) (Lamba et al., 2011). Here we evaluate the anti-leukemic activity of a selective PI3K inhibitor, GDC-0941, alone and in combination with AraC. Methods and Materials: A panel of 6 AML cell lines were assessed for potential activity of GDC-0941 alone (0.0625-4µM) and in combination with AraC (72h continuous, 4h pulse sequential or simultaneous exposure). We assessed cell viability by MTT assay and implemented the median effect approach to determine if combination drug effects, assessed as the combination index (CI) value, were synergistic, additive, or antagonistic. Induction of apoptosis and alterations in cell cycle were detected by caspase activation and propidium iodide staining, respectively. Western blot analyses were performed to determine drug effects on expression of PI3K isoforms, pAkt, pS6, and pErk signaling. Ex vivo cytotoxicity assays were performed on leukemic blasts isolated from murine primary c-Myc recipients to determine sensitivity to GDC-0941, AraC, and the combination. To evaluate in vivo efficacy, survival studies were performed using a c-Myc induced murine model of AML. Sublethally irradiated (550cGy) syngeneic tertiary recipients were randomized to receive vehicle, GDC-0941 (50mg/kg daily x 5 days, po), AraC (100mg/kg daily x 5 days, ip) or combination (week 1: AraC100mg/kg daily x 5 days; week 2: GDC-0941 50mg/kg daily x 5,) (N=8/treatment arm). Pharmacodynamic assessment of pAkt levels in leukemic blasts among the different treatment arms were also evaluated. Results: GDC-0941 treatment reduced cell viability in a dose-dependent manner and IC50s ranged from 0.36-4µM in AML cell lines. Caspase activity was induced by GDC-0941 alone and enhanced with the combination. For combination treatment, CI values were synergistic in all but 1 cell line (range, 0.20-0.97). Western blot analyses of the U937 cell line revealed that AraC induced both PI3Kα and pAkt signaling which could be abrogated by cotreatment with GDC-0941. Ex vivo combination drug treatments of c-Myc leukemic blasts demonstrated synergistic CI values. Combination treatment significantly prolonged survival in the c-Myc AML model compared to either drug alone or controls (control, median survival=21; GDC-0941, median survival=22; AraC, median survival=26; combination, median survival=28 days; p=0.01 and p=0.03 for AraC vs control and combination vs AraC, respectively) Conclusions: Our results show that AraC mediated induction of PI3Kα and pAkt in AML cells can be augmented by selective targeting of the PI3K/Akt pathway with GDC-0941. From these lines of preclinical evidence, combination of AraC with PI3K inhibitors is a promising strategy to enhance AraC efficacy in AML. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C170. Citation Format: Christina D. Drenberg, Brian Fermanski, Steven Zatechka, Yiping Fan, Shelley Orwick, Laura Janke, Sharyn D. Baker. GDC-0941 inhibits cytarabine-induced PI3K/AKT signaling and promotes synergistic activity in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C170.

Dasatinib, an orally available multikinase inhibitor, has been recently approved for imatinib- resistant chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. As with other tyrosine kinase inhibitors, dasatinib exhibits extensive interindividual pharmacokinetic variability, the causes of which are presently unknown. Previously, a notable degree of variability was observed with dasatinib oral absorption (time to peak concentration ranging from 0.5 to 6 h) and overall systemic exposure (CV, ∼50%) in patients (Demetri et al, Clin Cancer Res 2009). Identifying the factors underlying this variability may help enable clinicians to better manage the balance between dasatinib efficacy and toxicity. Here, we evaluated the role of ABCC4 (MRP4), a member of the ATP-binding cassette transporter family, as a factor regulating the oral absorption of dasatinib. Results from ABCC4-overexpressing inside-out vesicles indicated that dasatinib, at clinically relevant concentrations, was actively transported by ABCC4. Saturation of ABCC4-related transport was not seen even at a dasatinib concentration of 65 µM, suggesting high capacity transport. Dasatinib (1 µM) accumulation was increased ∼3-fold after a 5-min incubation in ABCC4-overexpressing vesicles compared to empty vector controls. The encouraging in vitro results prompted us to assess the in vivo role of ABCC4 in dasatinib absorption using Abcc4 knockout mice on a C57BL/6 background (Abcc4(-/-) mice) (Leggas et al, Mol Cell Biol 2004). Oral administration of dasatinib (10 mg/kg, in 50 mM sodium acetate buffer pH 4.6) to both wildtype mice and Abcc4(-/-) mice resulted in significant differences (P = 0.015) in dasatinib plasma concentrations at 4 out of 6 samples collected at serial time points. The largest difference was observed at 15 min, where dasatinib plasma concentrations in the Abcc4(-/-) mice were 5-fold decreased compared with wildtype mice. Although later time points also showed a decrease in dasatinib concentrations in Abcc4(-/-) mice (3.2-fold at 30 min; 2.2-fold at 1 h; 1.5-fold at 2 h), there was a convergence of the plasma curves by 4 h, suggesting that absorption rather than elimination pathways of dasatinib were altered in the Abcc4(-/-) mice. Indeed, pharmacokinetic analyses revealed a nearly 12-fold (P = 0.014) decrease in the dasatinib absorption rate constant in Abcc4(-/-) mice compared with wildtype mice, whereas the terminal half-life was unchanged (P = 0.41). The reduced absorption of dasatinib in Abcc4(-/-) mice was associated with a 4-fold (P = 0.019) decrease in peak concentration and a 2-fold decrease in AUC (P = 0.041). Collectively, these findings demonstrate that ABCC4 plays an important role in the intestinal absorption of dasatinib and reveal a new host factor that may contribute to enhanced interindividual pharmacokinetic variability in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1528.

There is an increasing body of evidence indicating that the bone marrow microenvironment can generate drug resistance in acute leukemia. The mechanisms underlying this effect have not yet been elucidated; signals triggered by direct contact with extracellular matrix components and by mesenchymal cell (MSC)-secreted factors have been implicated. The protective effect of the microenvironment has been primarily observed for classical chemotherapeutic drugs. Recent reports, however, indicate that this can also occur with molecularly targeted therapies. Thus, it was shown that interleukin (IL)-7 desensitizes BCR-ABL+ leukemic cells to imatinib (Williams RT et al. Genes Dev 2007) and MSC-conditioned media protects BCR-ABL+ cells to imatinib and nilotinib (Weisberg E at al. Mol Cancer Ther, 2008). Several tyrosine kinase inhibitors are in clinical development for the treatment of acute myeloid leukemia (AML). The aim of this study was to determine whether bone marrow MSC affected the sensitivity of AML cells to 3 promising tyrosine kinase inhibitors (sorafenib, sunitinib, and midostaurin) and, if so, to begin to elucidate the underlying mechanisms. Using proliferation assays, we found that 3 AML cells lines (MV4-11, U937, and THP1) were significantly less sensitive to the tyrosine kinase inhibitors when cultured in the presence of bone marrow-derived MSC for 24h before exposure to drugs for 72h. In experiments with MV4-11, IC50 increased from 4.7 nM to 55 nM for sorafenib, from 10 nM to 110 nM for sunitinib, and from 28 nM to 135 nM for midostaurin; in experiments with U937, IC50 increases were 5.1 μM to 11 μM, 6.2 μM to > 10 μM, and 230 to > 1000 nM for each drug; and in experiments with THP1, they were 6.3 μM to 11 μM, 2.2 μM to > 10 μM, and 211 nM to 996 nM. Coculture with MSC also reduced sorafenib- and sunitinib-induced apoptosis by > 60%. Interestingly, drug resistance increased even further after coculturing the cell lines with MSC for 4 weeks or longer: sunitinib had virtually no effect on the proliferation of MV4-11 cells at concentrations of up to 100 nM, and on THP-1 cells at 10 μM. To determine whether the induction of drug resistance was dependent on the direct contact of AML cells with MSC, we tested sensitivity to sorafenib after separating MV4-11cells from MSC with transwell inserts. Under these conditions, the protective effect of MSC was lessened but not abrogated. These results indicated that direct contact with MSC was not an absolute requirement for induction of drug resistance and that MSC-secreted soluble factors might be, at least in part, involved. We therefore determined the soluble factors secreted by MSC using a multiplex assay and tested whether their secretion was augmented by contact with AML cells. MSC secreted IL-6 (230 pg/mL), IL-8 (1880 pg/mL), and VCAM-1 (30 pg/mL). When cocultured with MV4-11, U937 and THP-1 cells for 24h, IL-6 secretion increased 1.3 to 1.8-fold, IL-8 increased 1.5 to 2.6-fold, and VCAM-1 increased 2.2 to 5.6-fold; after 72 of coculture, dramatically elevated levels of IL-6 (2140–3869 pg/mL), IL-8 (4296–8068 pg/mL), and VCAM-1 (5109–6389 pg/mL) were observed. The effects of these and other MSC-derived factors on the sensitivity of AML cells lines and primary AML cells to tyrosine kinase inhibitors are being tested. These results indicate that the anti-AML effect of tyrosine kinase inhibitors is strongly inhibited by bone marrow MSC cells, and support the concept that the microenvironment is an important determinant of resistance to these agents in leukemia. We suggest that the development of agents that interfere with the interaction between AML cells and MSC, and with the molecular mechanisms underlying this protective effect of MSC is a crucial step to improve cure rates.