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Supplementary Fig. S1. The chemical structure of VS-4718.

Supplemental Figure S1. Changes in protein expression levels induced by MCL-1 inhibitor. Supplemental Figure S2. Impact of MCL-1 inhibitors on total Mcl-1, T163 and S159 phosphorylation.Supplemental Figure S3. MCL-1 inhibitors induce a transient decrease in Mule.Supplemental Figure S4. In vitro De-ubiquitinating activity assay of USP9x with MCL-1 inhibitors.

Supplementary Methods

Supplemental Table S1- CLL Patient CharacteristicsSupplemental Table S2. Name and source of antibodiesSupplemental Table 3 - RT-PCR Primers

Purpose:Several MCL-1 inhibitors (MCL-1i), including AMG-176 and AZD5991, have shown promise in preclinical studies and are being tested for the treatment of hematologic malignancies. A unique feature of these agents is induction and stability of Mcl-1 protein; however, the precise mechanism is unknown. We aim to study the mechanism of MCL-1i–induced Mcl-1 protein stability.Experimental Design:Using several B-cell leukemia and lymphoma cell lines and primary chronic lymphocytic leukemia (CLL) lymphocytes, we evaluated molecular events associated with Mcl-1 protein stability including protein half-life, reverse-phase protein array, protein–protein interaction, phosphorylation, ubiquitination, and de-ubiquitination, followed by molecular simulation and modeling.Results:Using both in vivo and in vitro analysis, we demonstrate that MCL-1i–induced Mcl-1 protein stability is predominantly associated with defective Mcl-1 ubiquitination and concurrent apoptosis induction in both cell lines and primary CLL subjects. These MCL1i also induced ERK-mediated Mcl-1Thr163 phosphorylation, which partially contributed to Mcl-1 stability. Disruption of Mcl-1:Noxa interaction followed by Noxa degradation, enhanced Mcl-1 de-ubiquitination by USP9x, and Mule destabilization are the major effects of these inhibitors. However, unlike other BH3 proteins, Mule:Mcl-1 interaction was unaffected by MCL-1i. WP1130, a global deubiquitinase (DUB) inhibitor, abrogated Mcl-1 induction reaffirming a critical role of DUBs in the observed Mcl-1 protein stability. Further, in vitro ubiquitination studies of Mcl-1 showed distinct difference among these inhibitors.Conclusions:We conclude that MCL-1i blocked Mcl-1 ubiquitination via enhanced de-ubiquitination and dissociation of Mcl-1 from Noxa, Bak and Bax, and Mule de-stabilization. These are critical events associated with increased Mcl-1 protein stability with AMG-176 and AZD5991.

Supplementary Table 1 from Effect of Long-term Storage in TRIzol on Microarray-Based Gene Expression Profiling

Background: Although TRIzol is widely used for preservation and isolation of RNA, there is suspicion that prolonged sample storage in TRIzol may affect array-based gene expression profiling (GEP) through premature termination during reverse transcription.Methods: GEP on Illumina arrays compared paired aliquots (cryopreserved or stored in TRIzol) of primary samples of multiple myeloma (MM) and acute myeloid leukemia (AML). Data were analyzed at the “probe level” (a single consensus value) or “bead level” (multiple measurements provided by individual beads).Results: TRIzol storage does not affect standard probe-level comparisons between sample groups: different preservation methods did not generate differentially expressed probes (DEP) within MM or AML sample groups, or substantially affect the many DEPs distinguishing between these groups. Differences were found by gene set enrichment analysis, but these were dismissible because of instability with permutation of sample labels, unbalanced restriction to TRIzol aliquots, inconsistency between MM and AML groups, and lack of biological plausibility. Bead-level comparisons found many DEPs within sample pairs, but most (73%) were Conclusions: TRIzol preserves RNA quality well, without a deleterious effect on GEP. Samples stored frozen with and without TRIzol may be compared by GEP with only minor concern for systematic artifacts.Impact: The standard practice of prolonged sample storage in TRIzol is suitable for GEP. Cancer Epidemiol Biomarkers Prev; 19(10); 2445–52. ©2010 AACR.

Supplementary Figures and Tables

Supplementary Table 2 from Effect of Long-term Storage in TRIzol on Microarray-Based Gene Expression Profiling

Supplementary Table 3 from Effect of Long-term Storage in TRIzol on Microarray-Based Gene Expression Profiling

Purpose: Wnt/β-catenin signaling is required for leukemic stem cell function. FLT3 mutations are frequently observed in acute myeloid leukemia (AML). Anomalous FLT3 signaling increases β-catenin nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKI) are used clinically to treat FLT3-mutated AML patients, but with limited efficacy. We investigated the antileukemia activity of combined Wnt/β-catenin and FLT3 inhibition in FLT3-mutant AML.Experimental Design: Wnt/β-catenin signaling was inhibited by the β-catenin/CBP antagonist C-82/PRI-724 or siRNAs, and FLT3 signaling by sorafenib or quizartinib. Treatments on apoptosis, cell growth, and cell signaling were assessed in cell lines, patient samples, and in vivo in immunodeficient mice by flow cytometry, Western blot, RT-PCR, and CyTOF.Results: We found significantly higher β-catenin expression in cytogenetically unfavorable and relapsed AML patient samples and in the bone marrow–resident leukemic cells compared with circulating blasts. Disrupting Wnt/β-catenin signaling suppressed AML cell growth, induced apoptosis, abrogated stromal protection, and synergized with TKIs in FLT3-mutated AML cells and stem/progenitor cells in vitro. The aforementioned combinatorial treatment improved survival of AML-xenografted mice in two in vivo models and impaired leukemia cell engraftment. Mechanistically, the combined inhibition of Wnt/β-catenin and FLT3 cooperatively decreased nuclear β-catenin and the levels of c-Myc and other Wnt/β-catenin and FLT3 signaling proteins. Importantly, β-catenin inhibition abrogated the microenvironmental protection afforded the leukemic stem/progenitor cells.Conclusions: Disrupting Wnt/β-catenin signaling exerts potent activities against AML stem/progenitor cells and synergizes with FLT3 inhibition in FLT3-mutant AML. These findings provide a rationale for clinical development of this strategy for treating FLT3-mutated AML patients. Clin Cancer Res; 24(10); 2417–29. ©2018 AACR.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor (NR) family of transcription factors with important regulatory roles in cellular growth, differentiation, and apoptosis. Using proteomic analysis, we showed expression of PPARγ protein in a series of 260 newly diagnosed primary acute myelogenous leukemia (AML) samples. Forced expression of PPARγ enhanced the sensitivity of myeloid leukemic cells to apoptosis induced by PPARγ agonists 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and 15-deoxy-12,14-15DPGJ2, through preferential cleavage of caspase-8. No effects on cell cycle distribution or differentiation were noted, despite prominent induction of p21 in PPARγ-transfected cells. In turn, antagonizing PPARγ function by small interfering RNA or pharmacologic PPARγ inhibitor significantly diminished apoptosis induction by CDDO. Overexpression of coactivator protein DRIP205 resulted in enhanced differentiation induction by CDDO in AML cells through PPARγ activation. Studies with DRIP205 deletion constructs showed that the NR boxes of DRIP205 are not required for this coactivation. In a phase I clinical trial of CDDO (RTA-401) in leukemia, CDDO induced an increase in PPARγ mRNA expression in six of nine patient samples; of those, induction of differentiation was documented in four patients and that of p21 in three patients, all expressing DRIP205 protein. In summary, these findings suggest that cellular levels of PPARγ regulate induction of apoptosis via caspase-8 activation, whereas the coactivator DRIP205 is a determinant of induction of differentiation, in response to PPARγ agonists in leukemic cells. Cancer Res; 70(12); 4949–60. ©2010 AACR.

Supplementary Figures 1-4 from Role of Peroxisome Proliferator-Activated Receptor-γ and Its Coactivator DRIP205 in Cellular Responses to CDDO (RTA-401) in Acute Myelogenous Leukemia

Supplementary Figure Legends 1-6, Methods, Table 2 legend from Role of Peroxisome Proliferator-Activated Receptor-γ and Its Coactivator DRIP205 in Cellular Responses to CDDO (RTA-401) in Acute Myelogenous Leukemia

Supplementary Figure 5 from Role of Peroxisome Proliferator-Activated Receptor-γ and Its Coactivator DRIP205 in Cellular Responses to CDDO (RTA-401) in Acute Myelogenous Leukemia

Supplementary Table 1 from Role of Peroxisome Proliferator-Activated Receptor-γ and Its Coactivator DRIP205 in Cellular Responses to CDDO (RTA-401) in Acute Myelogenous Leukemia

Malignancies can become reliant on glutamine as an alternative energy source and as a facilitator of aberrant DNA methylation, thus implicating glutaminase (GLS) as a potential therapeutic target. We demonstrate preclinical synergy of telaglenastat (CB-839), a selective GLS inhibitor, when combined with azacytidine (AZA), in vitro and in vivo, followed by a phase Ib/II study of the combination in patients with advanced MDS. Treatment with telaglenastat/AZA led to an ORR of 70% with CR/mCRs in 53% patients and a median overall survival of 11.6 months. scRNAseq and flow cytometry demonstrated a myeloid differentiation program at the stem cell level in clinical responders. Expression of non-canonical glutamine transporter, SLC38A1, was found to be overexpressed in MDS stem cells; was associated with clinical responses to telaglenastat/AZA and predictive of worse prognosis in a large MDS cohort. These data demonstrate the safety and efficacy of a combined metabolic and epigenetic approach in MDS.

Background: Multiagent induction chemotherapy consisting of fludarabine, cytarabine, G-CSF, and idarubicin (FLAG-IDA) results in complete remission in ~ 85% of newly diagnosed patients with acute myeloid leukemia (ND-AML), yet 30-40% experience relapse. Combining the BCL-2 inhibitor venetoclax (VEN) with chemotherapy results in synergistic leukemic cell death, inferring FLAG-IDA+VEN may improve outcomes in ND-AML. Design: A phase 1b/2 study evaluating FLAG-IDA+VEN (Fig. 1A) in ND and relapsed/refractory (R/R) AML patients 18 years of age. Phase 1b results were previously reported (DiNardo, JCO 2021). The Phase 2 (P2) portion enrolled patients into two cohorts: ND and R/R-AML. ND-AML outcomes are reported herein. Objectives: The primary P2 objective is determination of overall activity of FLAG-IDA+VEN including overall response rate (ORR: CR+CRi+CRh+MLFS+PR). Secondary objectives include determination of overall survival (OS), event-free survival (EFS), duration of response (DOR), and biomarkers predictive of VEN sensitivity. Results: Forty-one patients with ND-AML (de novo AML: n=29, secondary AML [sAML]: n=7, therapy-related AML [tAML]: n=5) enrolled. Median age was 44 years (range 20-65). ELN risk was favorable, intermediate, and adverse in 20%, 37%, and 44% of patients. Common mutations included NRAS (29%; n=12), IDH2 (17%; n=7), RUNX1 (15%, n=6), and NPM1 (15%, n=6). TP53 mutations and KMT2A-rearrangements were identified in 10% (n=4) and 12% (n=5) of patients at diagnosis, respectively. The median number of treatment cycles received was 2 (range 1-6). The ORR was 98% (CR: n=30 [73%], CRh: n=5 [12%], CRi: n=1 [2%], MLFS: n=4 [10%]) and CRc (CR+CRh+CRi) rate was 88% (n=36). 92% of CRc patients attained MRD-negative status by flow cytometry. Median time to best response was 29 days (range 22-94). Sixty-six percent (n=27) of patients transitioned to allogeneic transplantation (HSCT) after a median of 3.8 (range 3.2-4.9) months. Median DOR was NR (95% CI: 17-NR). Median cycle length for cycles 1 and 2 were 31 and 41 days. Median time to count recovery (ANC 500 cells/mm 3 and platelet count 50,000 cells/mm 3) was longer following cycle 2 compared to cycle 1 (47 vs. 32 days, p-value < 0.001). Common adverse events included febrile neutropenia (39%; n=16), pneumonia (24%; n=10), and bacteremia (19%; n=8). No 30 or 60-day mortality occurred. Nine patients relapsed (ELN intermediate: N=3, adverse: N=6) including 100% of patients with baseline TP53 mutations (N= 4), and the one patient with inv(3). TP53 mutations were identifiable in 67% (n=6) of patients at relapse, including two patients without TP53 mutations at diagnosis. Four deaths have occurred, all in the setting of relapsed disease. With median follow up of 16 months, median OS and EFS are both NR, with 1-year OS and EFS rates of 96% and 77%, respectively (Fig. 1B). Patients with KMT2A rearrangements (n=5) and NPM1, IDH1, and/or IDH2 mutations (n=13) have experienced an 18-month survival rate of 100%. Patients with TP53 mutations at diagnosis have significantly inferior OS (24 months vs. NR, p-value: 0.03) and EFS (median EFS 8 months vs. NR, p-value A propensity score matched analysis compared to a historical cohort of patients receiving frontline FIA (fludarabine, idarubicin, cytarabine) induction (N=74) at our institution indicated FLAG-IDA+VEN was associated with improved median OS (NR vs. 47 months, p-value: 0.022), and a trend towards improved OS and EFS in patients not undergoing HSCT (FLAG-IDA+VEN vs. FAI median OS: 24 vs. 19 months, p-value: 0.09; median EFS: NR vs. 8 months, p-value: 0.064). On multivariate analysis, increased age and ELN risk group correlated with increased risk of death (age [HR 1.05, 95% CI: 1.02-1.09, p-value: 0.001]; ELN risk group [HR 1.92; 95% CI: 1.185-3.11, p-value: 0.008]), while decreased risk was observed based on treatment with FLAG-IDA+VEN vs. FIA (HR 0.28, 95% CI: 0.096-0.84, p-value: 0.02). Conclusions: FLAG-IDA+VEN induction and consolidation results in high MRD-negative CRc rates in ND-AML with an expected safety profile. Durable responses were observed at 12-months, with favorable outcomes compared to historical cohorts of intensive therapy. Patients with TP53 mutations treated with FLAG-IDA+VEN experience inferior outcomes compared to patients with wild-type TP53. Prospective randomized trials are warranted to confirm these results. Figure 1 Figure 1. Disclosures DiNardo: AbbVie: Consultancy, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Foghorn: Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Forma: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Takahashi: GSK: Consultancy; Celgene/BMS: Consultancy; Novartis: Consultancy; Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Loghavi: Abbvie: Current equity holder in publicly-traded company; Curio Sciences: Honoraria; Gerson Lehrman Group: Consultancy; Guidepoint: Consultancy; Peerview: Honoraria; Qualworld: Consultancy. Kadia: Amgen: Other: Grant/research support; Liberum: Consultancy; Jazz: Consultancy; Ascentage: Other; Novartis: Consultancy; Cellonkos: Other; Sanofi-Aventis: Consultancy; Cure: Speakers Bureau; Dalichi Sankyo: Consultancy; Aglos: Consultancy; AstraZeneca: Other; AbbVie: Consultancy, Other: Grant/research support; BMS: Other: Grant/research support; Genentech: Consultancy, Other: Grant/research support; Pfizer: Consultancy, Other; Pulmotech: Other; Genfleet: Other; Astellas: Other. Daver: ImmunoGen: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Abbvie: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; Hanmi: Research Funding; Novimmune: Research Funding; Novartis: Consultancy; Daiichi Sankyo: Consultancy, Research Funding; Glycomimetics: Research Funding; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Short: Jazz Pharmaceuticals: Consultancy; AstraZeneca: Consultancy; Astellas: Research Funding; NGMBio: Consultancy; Takeda Oncology: Consultancy, Research Funding; Novartis: Honoraria; Amgen: Consultancy, Honoraria. Sasaki: Novartis: Consultancy, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees. Wang: Stemline Therapeutics: Honoraria. Borthakur: Astex: Research Funding; Ryvu: Research Funding; ArgenX: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; University of Texas MD Anderson Cancer Center: Current Employment; Protagonist: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy. Issa: Novartis: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Kura Oncology: Consultancy, Research Funding. Alvarado: Jazz Pharmaceuticals: Research Funding; BerGenBio: Research Funding; CytomX Therapeutics: Consultancy; Sun Pharma: Consultancy, Research Funding; FibroGen: Research Funding; Daiichi-Sankyo: Research Funding; Astex Pharmaceuticals: Research Funding; MEI Pharma: Research Funding. Pemmaraju: Springer Science + Business Media: Other; Roche Diagnostics: Consultancy; Cellectis S.A. ADR: Other, Research Funding; Plexxicon: Other, Research Funding; Daiichi Sankyo, Inc.: Other, Research Funding; CareDx, Inc.: Consultancy; MustangBio: Consultancy, Other; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; Sager Strong Foundation: Other; DAVA Oncology: Consultancy; Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Samus: Other, Research Funding; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; Aptitude Health: Consultancy; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; LFB Biotechnologies: Consultancy; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Celgene Corporation: Consultancy; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; Incyte: Consultancy; Affymetrix: Consultancy, Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Clearview Healthcare Partners: Consultancy; Blueprint Medicines: Consultancy; Bristol-Myers Squibb Co.: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy. Yilmaz: Daiichi-Sankyo: Research Funding; Pfizer: Research Funding. Jain: Genentech: Honoraria, Research Funding; Servier: Honoraria, Research Funding; TG Therapeutics: Honoraria; Adaptive Biotechnologies: Honoraria, Research Funding; ADC Therapeutics: Honoraria, Research Funding; Beigene: Honoraria; Fate Therapeutics: Research Funding; Incyte: Research Funding; AstraZeneca: Honoraria, Research Funding; Pfizer: Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Cellectis: Honoraria, Research Funding; Janssen: Honoraria; Precision Biosciences: Honoraria, Research Funding; Aprea Therapeutics: Research Funding; AbbVie: Honoraria, Research Funding; Pharmacyclics: Research Funding. Andreeff: Medicxi: Consultancy; AstraZeneca: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Senti-Bio: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; ONO Pharmaceuticals: Research Funding; Oxford Biomedica UK: Research Funding; Amgen: Research Funding; Syndax: Consultancy; Glycomimetics: Consultancy; Aptose: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Karyopharm: Research Funding; Breast Cancer Research Foundation: Research Funding. Ravandi: Xencor: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astex: Honoraria, Research Funding; Taiho: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Agios: Honoraria, Research Funding; AstraZeneca: Honoraria; AbbVie: Honoraria, Research Funding; Novartis: Honoraria; Prelude: Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding. Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Konopleva: Calithera: Other: grant support, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; AstraZeneca: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Ascentage: Other: grant support, Research Funding; Cellectis: Other: grant support; Agios: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Ablynx: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Stemline Therapeutics: Research Funding; KisoJi: Research Funding. Kantarjian: Amgen: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Immunogen: Research Funding; BMS: Research Funding; Ascentage: Research Funding; Astellas Health: Honoraria; Precision Biosciences: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Jazz: Research Funding; Aptitude Health: Honoraria; Ipsen Pharmaceuticals: Honoraria; KAHR Medical Ltd: Honoraria; NOVA Research: Honoraria; Astra Zeneca: Honoraria; Taiho Pharmaceutical Canada: Honoraria. OffLabel Disclosure: Will describe use of venetoclax in combination with FLAG-IDA induction and consolidation therapy in AML.

Microsatellite instability (MSI) is caused by defects in DNA mismatch repair (MMR) components. Inactivation of any MMR gene(s), including hMLH1, hMSH2, hMSH6, and hPMS2, can result in MSI. Immunohistochemistry (IHC) is a sensitive and specific screening tool for MSI that can detect loss of expression of one or more MMR components. Of the four MMR markers, hMLH1 and hMSH2 are considered most informative of MSI status. There has been renewed interest in MSI status in view of its favorable association with response to immune checkpoint inhibitors in some cancers. MMR expression patterns in acute myeloid leukemia (AML) have not been evaluated systematically.We used clinically-validated IHC assays to assess the expression of hMLH1, hMSH2, hMSH6, and/or hPMS2 in formalin-fixed paraffin-embedded tissue sections of bone marrow core biopsies from patients diagnosed with AML. Mutation profiling was performed using next-generation sequencing to assess for mutations in MMR genes.The study group included 236 patients with AML, including a cohort treated on a clinical trial of azacitidine and nivolumab (NCT02397720). In addition, hMSH6, and/or hPMS2 expression was assessed in 99 AML patients with diploid karyotype. All patients, except two, had retained expression of all MMR markers assessed: One patient from the azacytidine+nivolumab group had zonal patchy loss of staining of hMLH1 and, to a lesser extent, a similar staining pattern of hMSH2; and one patient from the AML with diploid karyotype group had loss of hMSH2 but retained expression of hMLH1, hMSH6 and hPMS2. In addition, a retrospective analysis on a separate cohort of 139 patients with primary AML, on which next generation sequencing profiling was performed, identified 14 cases with alterations in MMR genes.MMR loss is a rare event in AML, thus does not appear to underlie response patterns to anti-PD1 therapy.

BACKGROUND—Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear. METHODS—We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis. RESULTS—AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcriptionfactor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumorsuppressor genes (16%), DNA-methylation–related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories. CONCLUSIONS—We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.) The molecular pathogenesis of acute myeloid leukemia (AML) has been studied with the use of cytogenetic analysis for more than three decades. Recurrent chromosomal structural variations are well established as diagnostic and prognostic markers, suggesting that acquired genetic abnormalities (i.e., somatic mutations) have an essential role in pathogenesis. 1,2 However, nearly 50% of AML samples have a normal karyotype, and many of these genomes lack structural abnormalities, even when assessed with high-density comparative genomic hybridization or single-nucleotide polymorphism (SNP) arrays 3-5 (see Glossary). Targeted sequencing has identified recurrent mutations in FLT3, NPM1, KIT, CEBPA, and TET2. 6-8 Massively parallel sequencing enabled the discovery of recurrent mutations in DNMT3A 9,10 and IDH1. 11 Recent studies have shown that many patients with

B cell Acute Lymphoblastic Leukemia (B-ALL) arises from the transformation of progenitor B cells. The transcription factor STAT5 is required for transformation but how STAT5 mediates this effect is unclear. Previous studies suggested that STAT5 only acts to promote survival of progenitor B cells. However, other roles for STAT5 in B cell development and B-ALL have not been explored. Here we show that STAT5 activation drives leukemia in cooperation with defects in a linear signaling pathway emanating from the pre-BCR, including Blnk, Btk, Prkcb, Nfkb1, and Ikzf1. Using microarray analysis and ChIP-Seq we demonstrate that STAT5 disrupts the function of superenhancer binding transcription factor networks that normally promote B cell development. STAT5 versus NFkB or IKAROS binding to these enhancers largely had opposing effects on target gene expression. The antagonism between STAT5 and IKAROS or NFκB has direct clinical relevance as the balance between these transcription factors affects patient outcome; patients with high ratios of active STAT5 to NFκB or IKAROS had more aggressive disease characterized by shorter remission and decreased survival. Thus, our studies illustrate how modest perturbations in two opposing transcriptional programs can have dramatic consequences for B cell development and transformation and how the degree of antagonism between these transcriptional programs ultimately predicts patient outcome to therapy.

We recently found that STAT5 activation correlates with poor response to therapy in B-ALL. To identify genetic defects that cooperate with STAT5 activation to initiate transformation we carried out a forward genetic screen involving the mutagenic transposon Sleeping Beauty. This screen identified a number of cooperating partners including gain-of-function mutations in Sos1 and loss-of-function mutations in Ebf1 and Ikzf1 (i.e., Ikaros). Haploinsufficiency of Ebf1 or the related transcription factor Pax5 synergized with STAT5 to rapidly induce progenitor B-ALL in 100% of mice. The leukemic cells displayed reduced expression of Ebf1 and Pax5, which affected a small subset of EBF1 or PAX5 target genes, including tumor suppressor genes and oncogenes. To test whether compound haploinsufficiency of Ebf1 and Pax5 alone is sufficient to cause transformation, we generated Ebf1+/- x Pax5+/- mice. Over 80% of these mice developed progenitor B-ALL. We observed similar results in Ebf1+/- x Ikzf1+/-, Pax5+/- x Ikzf1+/-, and Pax5+/- x Ebf1+/- x Ikzf1+/- mice; interestingly, although most leukemias were B-ALL we also observed T-ALL suggesting that T-All may arise from a B cell progenitor. Our findings suggest a model in which STAT5 activation cooperates with small perturbations in a self-reinforcing network of transcription factors critical for B cell development to initiate ALL and that loss of any two alleles in the network results in a loss of network tumor suppressor function.

STAT5 is activated in a subset of patients with ALL and high levels of STAT5 activation prior to treatment predicted poor response to therapy. In mice, we identified synergy between constitutive activation of STAT5 (STAT5b-CA) and Ebf1 or Pax5 haploinsufficiency that rapidly induced progenitor B-ALL in 100% of the mice. The leukemic cells displayed 2-fold reduced expression of Ebf1 and Pax5 that affected a small subset of EBF1 or PAX5 target genes, including tumor suppressor genes and oncogenes. To test whether compound haploinsufficiency of Ebf1 and Pax5 is sufficient to cause transformation we generated Ebf1+/- x Pax5+/- mice. Over 80% of these mice developed progenitor B-ALL. Although these mice lack the Stat5b-CA transgene, the leukemic cells exhibit increased STAT5 activation. In preliminary studies we observed similar results in Ebf1+/- x Ikzf1+/- and Pax5+/- x Ikzf1+/- mice. Our findings suggest a model in which small perturbations in a self-reinforcing network of transcription factors critical for B cell development cooperate with STAT5 to initiate ALL and that EBF1, PAX5 and IKZF1 form an interrelated network of transcription such that loss of any two alleles in the network results in a loss of tumor suppressor function. This hypothesis represents a departure from the conventional idea that transformation is a result of deletion at both alleles of one tumor suppressor gene, and may explain why mutations in Pax5, Ebf1 and Ikzf1 are so common in human ALL.

In Ph+ ALL, complete remission (CR) rates with intensive chemotherapy such hyper-CVAD is 90%, but most patients (pts) relapse within a median time of 16 months [Kantarjian et al, JCO18:547, 2000; Kantarjian et al, Cancer101:2788, 2004]. Single agent therapy with the tyrosine kinase inhibitor imatinib mesylate in relapsed or refractory Ph+ ALL or chronic myelogenous leukemia in lymphoid blast phase yielded CR rates of 20% with rapid disease recurrence. A phase II clinical trial of concurrent hyper-CVAD and imatinib was designed to improve these results, with the initial regimen of imatinib 400 mg orally daily days 1–14 of each course (fractionated cyclophosphamide, vincristine [VCR], doxorubicin and dexamethasone alternating with high dose methotrexate and cytarabine) followed by imatinib, VCR and prednisone maintenance with intensifications months 6 and 13. Allogeneic stem cell transplant (SCT) was performed in CR if feasible. Preliminary results of first 20 pts treated were encouraging [Thomas et al, Blood103:4396, 2004]. Recent modifications included increasing dosing of imatinib to 600 mg daily days 1–14 of course 1, then daily if tolerated with courses 2–8. Maintenance was extended to 24 months with imatinib indefinitely. To date, 43 pts with Ph+ ALL have been treated from April 2004 to July 2005. Thirty-six pts had active disease, either untreated (n=31) or refractory (n=1) to one induction course without imatinib; 7 pts were in CR after one induction course without imatinib. Of 35 evaluable pts, 33 (94%) achieved CR (1 induction death, 1 failed to meet platelet criteria for CR). Median days to response was 21 days. 13 pts underwent allogeneic SCT within a median of 3 months from start of therapy (range, 1–12). After a median follow-up of 3 yrs (range 1–48 months), 1 primary refractory pt relapsed at 12 months, 1 de novo pt had isolated CNS relapse, 2 pts relapsed after allogeneic SCT (no post SCT imatinib) and 2 pts changed therapy for persistent Ph+ metaphases (1 relapsing). Deaths in CR included 5 older pts without allogeneic SCT (1 osteomyelitis, 1 mucormycosis, 1 C. difficile colitis, 1 sudden death, 1 GNR sepsis) and 4 pts after allogeneic SCT (3 graft-versus-host disease, 1 GNR sepsis). Outcome with the hyper-CVAD and imatinib regimen continues to demonstrate favorable disease-free survival rates compared with hyper-CVAD alone, particularly for the de novo group. Use of higher dose imatinib concurrently appears to be feasible. Molecular response rates appear to be improved with the higher dose imatinib; additional accrual will be required to assess impact of modifications, including role of allogeneic SCT.