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Introduction: TP53 gene mutations (m TP53), found in up to 20% of MDS or AML pts and 30-40% of therapy-related (TR) MDS/AML cases, represent a distinct molecular cohort with poor outcomes. Hypomethylating agents (HMA) are the frontline standard of care, with CR rates of ~20% and median OS of < 12 months. APR-246 is a novel, first-in-class small molecule that reactivates the mutant p53 protein and targets cellular redox balance, ultimately inducing apoptosis and ferroptosis in m TP53 cancer cells. We previously reported the Phase 2 results of 2 parallel trials of APR-246+AZA (Sallman et al and Cluzeau et al., JCO 2021). We now report analyses of the combined Phase 2 cohorts and long-term follow-up. Methods: This is a multicenter, international collaboration of the US MDS clinical research consortium and the GFM of HMA-naïve m TP53 higher-risk MDS, MDS/MPN and oligoblastic AML (≤ 30% blasts) pts (NCT03072043/NCT03588078). Pts received APR-246 4500mg IV (days 1-4) + AZA 75 mg/m 2 SC/IV x 7 days (days 4-10 or 4-5 and 8-12) in 28-day cycles. Primary objective was CR rate by International Working Group (IWG) 2006 criteria. Secondary objectives included ORR, OS, outcome following allogeneic hematopoietic stem cell transplant (allo-HSCT), with serial high depth next generation sequencing (NGS, 0.1% cutoff) for evaluation of measurable residual disease (MRD). Results: As of July 15, 2021, 100 pts had been enrolled with a median age of 68 years (range, 34-87; 47% male). By WHO, 74 pts had MDS, 22 AML-MRC and 4 CMML/MDS-MPN; 83% complex karyotype (CK) and 88% were CK and/or biallelic for TP53 mutations; 92% had a TP53 missense mutation in the DNA binding domain. In 63 pts, TP53 was the only mutation detected (i.e. isolated m TP53). Median time on treatment was 6 cycles (1-25+) with 5 pts ongoing and 23 pts who proceeded to allo-HSCT. Non-hematologic treatment (Tx)-related adverse events (AEs) in ≥20% of pts included nausea/vomiting (58%), ataxia (26%), and dizziness (23%). Neurologic AEs were reversible in 100% of cases. Febrile neutropenia occurred in 37% of pts. Thirty and 60-day mortality was 1% (n=1) and 7% (n=7), respectively. Dose reductions of APR-246 and AZA occurred in 16% and 1% of pts, respectively, with only 1 treatment discontinuation due to a treatment-related AE. By intention-to-treat (ITT) analysis, ORR by IWG was 69% with 43 CR, 1 PR, 10 marrow CR (mCR)+HI, 9 HI alone, and 6 with mCR. Of non-responders, 6 had stable disease and 7 pts had progressive disease. The median duration of CR/PR was 10.6 months (95% CI 8.8-12.3, 23+ months ongoing). CR/PR rate for MDS was 49% (36/74), 36% for AML (8/22) and 0% for MDS/MPN (0/4) with an ORR rate of 70%/64%/75%, respectively. Isolated m TP53 was predictive for a higher CR rate (52% vs 30%; P=.04). Patients who had biallelic TP53 or CK had a significantly higher CR rate vs pts without (49% vs 8%; P=0.01). On serial TP53 NGS using a VAF cutoff of 5%, 40 pts achieved NGS negativity with 6 pts MRD negative (VAF < 0.1%). Of NGS negative pts (TP53 VAF At data-cutoff by ITT analysis with a median follow up of 27.8 months, median OS was 11.8 months (95% CI 9.4-14.3). Pts undergoing allo-HSCT had a median OS of 16.1 months (95% CI 14-18.1). Impact of response and NGS clearance was evaluated by landmark analysis at 6 months. Pts achieving CR/PR or NGS negativity had improved OS (15.8 vs 10.1 months; P=0.002; Fig 1A). Additionally, pts who became MRD negative had a 2-year OS of 50% vs 23% (P=0.21). Although allo-HSCT was not predictive of OS in the overall cohort by landmark analysis (14.7 vs 14.4 months; P=0.15; Fig 1B), significant OS improvement was noted in allo-HSCT pts based on CR/PR or NGS negativity (P=0.002; Fig 1C). Notably, pts who achieved CR/PR/NGS negativity and were bridged to allo-HSCT had a median OS that was not reached (95% CI 10.4-NR) vs 9.1 months (95% CI 7.4-NR) in allo-HSCT pts who did not achieve this response (P=0.01). Conclusions: In this international, combined analysis of P2 APR-246+AZA pts, the combination was well-tolerated with high response rates in m TP53 MDS/AML. Quality of response and NGS negativity strongly predicted OS, particularly in the setting of allo-HSCT, validating NGS clearance as a critical biomarker of allo-HSCT outcomes in m TP53 pts. Figure 1 Figure 1. Disclosures Sallman: Intellia: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite: Membership on an entity's Board of Directors or advisory committees; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Magenta: Consultancy; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Agios: Membership on an entity's Board of Directors or advisory committees. Komrokji: BMS: Honoraria, Speakers Bureau; JAZZ: Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau; Novartis: Honoraria; Geron: Honoraria; Acceleron: Honoraria; Agios: Honoraria, Speakers Bureau. DeZern: Taiho: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sebert: BMS: Consultancy; Abbvie: Consultancy. Steensma: Novartis: Current Employment. Roboz: Astellas: Consultancy; Otsuka: Consultancy; Blueprint Medicines: Consultancy; AbbVie: Consultancy; Bayer: Consultancy; Glaxo SmithKline: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Helsinn: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Jasper Therapeutics: Consultancy; MEI Pharma - IDMC Chair: Consultancy; Janssen: Research Funding; AstraZeneca: Consultancy; Jazz: Consultancy; Amgen: Consultancy; Daiichi Sankyo: Consultancy; Mesoblast: Consultancy; Agios: Consultancy; Pfizer: Consultancy; Astex: Consultancy; Actinium: Consultancy; Roche/Genentech: Consultancy. Sekeres: BMS: Membership on an entity's Board of Directors or advisory committees; Takeda/Millenium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Sweet: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kuykendall: Incyte: Consultancy; Novartis: Honoraria, Speakers Bureau; Prelude: Research Funding; PharmaEssentia: Honoraria; CTI Biopharma: Honoraria; Celgene/BMS: Honoraria, Speakers Bureau; BluePrint Medicines: Honoraria, Speakers Bureau; Abbvie: Honoraria; Protagonist: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Recher: Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria; Janssen: Honoraria; Jazz: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MaatPharma: Research Funding; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Raffoux: ASTELLAS: Consultancy; ABBVIE: Consultancy; PFIZER: Consultancy; CELGENE/BMS: Consultancy. Padron: Incyte: Research Funding; BMS: Research Funding; Blueprint: Honoraria; Kura: Research Funding; Taiho: Honoraria; Stemline: Honoraria. Attar: Aprea Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kantarjian: Amgen: Honoraria, Research Funding; KAHR Medical Ltd: Honoraria; BMS: Research Funding; AbbVie: Honoraria, Research Funding; Ascentage: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Astellas Health: Honoraria; Jazz: Research Funding; Aptitude Health: Honoraria; Astra Zeneca: Honoraria; Ipsen Pharmaceuticals: Honoraria; NOVA Research: Honoraria; Precision Biosciences: Honoraria; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Taiho Pharmaceutical Canada: Honoraria. List: Precision BioSciences: Current Employment, Current equity holder in publicly-traded company; Aileron Therapeutics: Consultancy; CTI Biosciences: Consultancy; Halia Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company. Ades: Celgene: Honoraria, Research Funding; Abbvie: Honoraria; Takeda: Honoraria; Novartis: Honoraria; JAZZ: Honoraria. Lancet: BerGenBio: Consultancy; Celgene/BMS: Consultancy; Millenium Pharma/Takeda: Consultancy; Jazz: Consultancy; Astellas: Consultancy; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Agios: Consultancy; AbbVie: Consultancy. Fenaux: Janssen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; JAZZ: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Syros Pharmaceuticals: Honoraria.

Introduction : TP53 mutated (TP53m) MDS and AML have very poor outcome irrespective of the treatment received, including 40% responses (20% CR) with azacitidine (AZA) with short response duration and a median overall survival (OS) of about 8 months (Bejar, Blood 2014). APR-246 is a prodrug spontaneously converted to methylene quinuclidinone (MQ), a Michael acceptor that binds covalently to cysteines in mutant p53, leading to protein reconformation that reactivates its pro apoptotic and cell cycle arrest functions. The combination of AZA and APR 246 showed promising results in a phase Ib study in TP53m MDS (Sallman, ASH 2018). We report interim results of a GFM phase 2 study of AZA+ APR-246 in TP53m MDS and AML, conducted in parallel with a similar US MDS consortium study. Patients and Methods : The study included hypomethylating agent (HMA) naïve and not previously allografted intermediate, high or very high IPSS-R TP53m MDS and AML adult patients. Patients received APR-246 4500 mg IV /d (6 hour infusions) (days 1-4) followed by AZA 75 mg/m²/d (days 4-10) in 28 day cycles. Response (primary endpoint, assessed by IWG 2006 for MDS and ELN criteria for AML) was evaluated after 3 and 6 cycles in the intent to treat (ITT) population, ie all patients who had received any protocol treatment, and in patients who had at least a blood and bone marrow evaluation after cycle 3 (evaluable population). Allo-SCT, when possible, was proposed after 3 to 6 cycles, and treatment with reduced APR 246 and AZA doses could be continued after allo-SCT. Results : 53 patients were enrolled between Sept 2018 and July 2019 in 7 GFM centers, with a median age of 73 years (range 44-87), and M/F: 28/25. 34 patients had MDS (including 74% very high IPSS-R) and 19 had AML. IPSS-R cytogenetic risk was very poor in 30/34 MDS, and unfavorable in 18/19 AML, complex in 89% of the patients. Median baseline mutated TP53 VAF was 21% (range 3-76). Nineteen of the 53 patients had been included at least 7 months before date of analysis (25 July 2019), had received protocol treatment and were thus potentially evaluable for response after 6 treatment cycles (ITT population). One of them died after only one cycle from an unrelated cause (cerebral ischemic stroke), and 2 during the third cycle (from bleeding and sepsis, respectively). In the remaining 16 patients (evaluable population per protocol), the response rate was 75% including 9 (56%) CR, 3 (19%) marrow CR or stable disease with hematological improvement (HI), and 4 treatment resistance. In the ITT population, the response rate was 63%, including 47% CR, and 16% stable or marrow CR+ HI. Among CR patients, complete cytogenetic CR and negative NGS for TP53 mutation (VAF cutoff of 2%) were achieved in 7/9 (78%) and 8/8 (100%), respectively. So far, 1 patient has undergone allo-SCT. All 53 patients had received at least one treatment cycle, and no increased myelosuppression, compared with AZA alone, was apparent. Treatment related AEs observed in ≥ 20% of patients were febrile neutropenia in 19 (36%) and neurological AEs in 21 (40%) of the patients. The latter, reviewed with a neurological team, were mainly grade 1 or 2 and consisted of ataxia (n=13), sometimes associated with cognitive impairment (n=4), suggesting a cerebellar origin. Other patients experienced acute confusion (n=4), isolated dizziness (n=3) and facial paresthesia (n=1). Neurological AEs reached grade III in 3 cases (1 acute confusion, 2 ataxia). Occurrence of neurological AEs was correlated with lower glomerular filtration rate at treatment onset (p Conclusion : In this very high-risk elderly population of TP53m MDS and AML, generally with complex karyotype, a promising 56% CR rate at 6 cycles was reached in the evaluable population with AZA+ APR 246 combination, with deep molecular remission in all CR patients. We observed manageable neurologic AEs, mainly in elderly patients with reduced renal function, who therefore require close monitoring and dose reduction if necessary. An update regarding safety and efficacy in the 53 patients, including survival data, will be available at the meeting. A phase III international trial comparing AZA alone and AZA+ APR 246 in TP53m MDS is ongoing. Disclosures Cluzeau: Abbvie: Consultancy; Jazz Pharma: Consultancy; Menarini: Consultancy. Peterlin:AbbVie Inc: Consultancy; Astellas: Consultancy; Jazz Pharma: Consultancy; Daiichi-Sankyo: Consultancy. Recher:Daiichi-Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; chugai: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Stamatoullas:Celgene: Honoraria; Takeda: Consultancy. Berthon:JAZZPHARMACEUTICAL: Other: DISCLOSURE BOARD; CELGEN: Other: DISCLOSURE BOARD; PFIZER: Other: DISCLOSURE BOARD. Sallman:Celyad: Membership on an entity's Board of Directors or advisory committees. Ades:Amgen: Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Helsinn Healthcare: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Fenaux:Celgene Corporation: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Aprea: Research Funding.

Introduction Myelodysplastic syndromes (MDS) are clinically diverse malignant disorders of aging with a propensity to evolve to acute myeloid leukemia (AML) or bone marrow failure. In early MDS, whole genome sequencing identified mutations distributed in few clones. Evidence have been provided for the existence of a MDS-initiating cell in cases harboring a 5q deletion. However, the clonal heterogeneity and its consequences on the phenotypic diversity of non-del(5q) MDS is little documented. Here we focused on studying the hierarchical organization and the functionality of clones defined by molecular profiling. The clonal architecture of the CD34+CD38- hematopoietic stem/progenitor cell (HSPC) compartment was investigated and dominant clones were examined as MDS-initiating cells. Material and methods Bone marrow (BM) samples were obtained from 20 patients with non-del(5q) MDS enrolled in the national Programme Hospitalier de Recherche Clinique MDS-04 after informed consent in accordance with ethics committee guidelines. BM samples, cytaphereses from age-matched healthy individuals and cord bloods were used as controls. Targeted NGS of a selected panel of 39 genes was used to define the mutational landscape on BM mononuclear cells (MNC). To study the clonal architecture at the HSPC level, single CD34+CD38- cells were seeded in 96-well plates coated with MS-5 stromal cells and cultured in H5100 MyeloCult medium (StemCell Technologies, Vancouver, Canada) with cytokines for six weeks. For long-term culture-initiating cell (LTC-IC) assays, CD34+ progenitors were cultured for six weeks on MS-5-coated plates without cytokines and then tested for colony-forming cells. For clonogenic assays, CD34+CD38- cells were seeded in methylcellulose for two weeks. All animal experimentations were performed in NSG mice. Results In the 20 cases of non-del(5q) MDS, genomic lesions were traced down to single CD34+CD38- HSPC-derived colonies. Clonal organization was mostly linear in 13/17 patients and branched in 4 cases with retention of a dominant subclone. The clone detected in LTC-IC compartment and that reconstituted short-term human hematopoiesis in xenotransplantation models was usually the dominant clone, which gave rise to the myeloid and to a lesser extent to the lymphoid lineage. Other mutations not detected in LTC-IC can appear in CD34+CD38- compartment or at the level of lineage-committed progenitors. The pattern of mutations may differ between common myeloid (CMP), granulo-monocytic (GMP) and megakaryocytic-erythroid (MEP) progenitors. For instance, a major truncating BCOR gene mutation affecting HSPC and CMP was beneath the threshold of detection in GMP or MEP. Consistently, BCOR knockdown by shRNA in normal CD34+ progenitors impaired their granulocytic and erythroid differentiation. By contrast, a STAG2 gene mutation, not detected in CMP or MEP, amplified in a GMP, which drove the transformation to AML. Conclusion In the present study, we characterized the first genetic hits that initiate disease in a dominant clone of the CD34+CD38- HSPC compartment, which exhibits LTC-IC activity and reconstitutes human short-term hematopoiesis in NSG mice. The genetic heterogeneity in non-del(5q) MDS arises within the HSPC compartment and in lineage-committed progenitors which ultimately support the transformation into AML. The clonal architecture of HSPC compartment and mutations selection along differentiation contribute to the phenotype of MDS. Defining the hierarchy of driver mutations provides insights into the process of transformation, and may guide the search for novel therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

Background: Myelofibrosis (MF) is the less frequent Philadelphia-negative myeloproliferative neoplasms (MPN). We have included in a nationwide database primary (P), post-polycythemia (PPV) and post-essential thrombocythemia (PET) MF diagnosed in France since 2005. Methods: Inclusion criteria were: diagnostic of MF after 2005; according strictly to WHO (bone marrow biopsy mandatory); informed consent. The registry was launched in Oct. 2013, and 26 hematology centers included patients (pts). Summary statistics were reported, namely median [Interquartile range, IQR] or percentage. Baseline characteristics were compared across IPSS groups using chi-square test or Mann and Whitney test. Kaplan-Meier survival curves were plotted and compared by the log-rank test. Results: At time of analysis (June 2016) a total of 527 pts were included in the registry, complete baseline data were available in 499 (95%), and follow-up (FU) data in 433 (87%). Median [IQR] age and M/F sex ratio were 71 [63-78] years and 315/184 (1.7), respectively (resp). 301 (60%) pts had PMF, 182 (36%) had secondary MF (including 64 PPVMF and 118 PETMF) and 16 had pre-fibrotic MF. Five percent of pts had a familial history of hematologic malignancy, and 22% a history of thrombosis or hemorrhage. Splenomegaly was present in 386 (77%) with a median [IQR] spleen size of 4 cm [1-8] below costal margin, and was symptomatic in 11% of pts. Constitutional symptoms were present in 107 (21%) (weight loss in 56, night sweats in 61, fever in 14), and ECOG score was 0, 1, 2, and 3 in 53%, 36%, 11% and 0.3% of pts, resp. Median [IQR] Hemoglobin, WBC and platelet counts were 109 g/L [94-122], 9.3 G/L [5.7-16.0] and 257 G/L [138-430], resp. Circulating blast cells were present in 41%, LDH was above normal value in 95% of pts, median EPO level was 54 [11 - 57] U/L. Grade of fibrosis (WHO) was 1, 2, and 3 in 2%, 66% and 32% of pts, resp. Karyotype was done in 321 pts, normal in 173 (54%), abnormal with favorable prognostic value in 89 (30%) and unfavorable in 30 (10%) (29 failures). A total of 461 (92%) pts had molecular testing: 60% were JAK2V617F positive, 4% had MPL and 7% had CALR mutations, and 99 (28%) over the 352 pts with triple testing were triple negative. IPSS risk categories were low, int-1, int-2 and high in 68 (14%), 168 (34%), 158 (32%), and 105 (21%) pts, resp. In addition to constitutional symptoms, there was a significant increase in the prevalence of clinical signs across IPSS categories (from low to high risk): symptomatic spleen (p=0.016) -though no difference in spleen size was found (p=0.18)-, early satiety (p=0.013), ECOG score (p= 0.0001), bone pain (p=0.002). Moreover, among biological parameters, there was an increase across IPSS groups in WBC (p=0.029), LDH (p=0.0007), ferritin (p=0.003), circulating CD34+ cells (p=0.020), EPO level (p=0.035). In contrast, a decrease was seen for hemoglobin and platelets (p=0.0001 for both). Lastly, frequency of grade 3 fibrosis increased with IPSS (p=0.043), while no evidence of difference was found regarding abnormal karyotype and mutational pattern. Median FU was 33 months [9-63 months]. Among those 433 pts with FU data, median FU was 38 months [19-68], and 124 (29 %) pts had died at the time of analysis, including 13%, 17%, 31%, and 40% of pts from the Low, Int-1, Int-2, and High risk groups, resp (p= 0.0001, figure 1). In the 450 pts with treatment data, treatments received during FU included cytoreductive drug (41%), Jak-inhibitors (35%), Interferon alpha (18%), IMIDs (4%). Splenectomy was performed in only 14 (3%) pts. Forty percent of pts received packed red blood cells, and 12% platelets transfusions. 49 (11%) patients participated in a clinical trial, and 27 (6%) were allografted. Conclusion: This is the first analysis of the French MF observatory after inclusion of more than 500 pts diagnosed and treated during the past 10 years. Complete baseline data and follow-up information available for the majority of pts should allow for new studies of outcomes and influence of clinical and biological parameters, as well as reassessment of prognostic models in the era of new targeted therapies. Figure 1 Comparison of overall survival according to IPPS Figure 1. Comparison of overall survival according to IPPS Figure 2 Figure 2. Disclosures Etienne: Pfizer: Speakers Bureau; BMS: Speakers Bureau; ARIAD: Speakers Bureau; novartis: Consultancy, Speakers Bureau. Tavitian:Novartis: Membership on an entity's Board of Directors or advisory committees. Ugo:Novartis: Membership on an entity's Board of Directors or advisory committees. Kiladjian:Novartis: Honoraria, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 4786 Background Chronic myelomonocytic leukemia (CMML) is a rare clonal hematopoietic stem cell disorder whom biology remains unclear.CMML is associated with many different somatic mutations in genes involved in key cellular processes including signaling (N/K-Ras, CBL, JAK2); differentiation (RUNX1, NPM1, CEBPa); epigenetic regulation (TET2, ASXL1, IDH1/2, EZH2, DNMT3A); and RNA splicing (SRSF2, U2AF1, SF3B1 and ZRSR2). FLT3 mutations are very rare but provide the rationale for FLT3 tyrosine kinase inhibitor use to treat this disease. We report the case of a 60-years-old patient diagnosed with a hepatocarcinoma metastasis which legitimized the introduction of anti-angiogenic therapy using the VEGF-R2 inhibitor, sorafenib. The patient was addressed to the hematology department with a myeloproliferative-like CMML in transformation. We show here that the molecular analyses of this hematological disorders allow us to use sorafenib as a targeted therapy to inhibit the consequences of a FLT3-ITD mutation. Methods Cytogenetic analysis and genome wide array-based comparative genomic hybridization (aCGH) were performed at diagnosis. The reference standard used for aCGH was matched genomic constitutional DNA (CD3+ T cells sorted from a blood sample). Serum samples collected from the patient before or under treatment with sorafenib were assessed for their plasma inhibitory activity by western blotting analyses of signaling molecules downstream the FLT3-ITD mutation. Genomic DNA samples extracted from BMMCs and peripheral blood (PB) cells at diagnosis were screened for mutations in 18 classical genes. To monitor the FLT3-ITD mutation, the exon 15 of FLT3 was amplified by a specific PCR using a 6FAM-labeled forward primer. Results The patient developed hyperleucocytosis (48.2 G/L) with neutrophilia (30.4 G/L), monocytosis (11.6 G/L) and basophilia (0.5 G/L) in January 2011. The BM was hypercellular with granulocytic and monocytic proliferation, dysgranulopoiesis and dysmegacaryopoiesis. Blast cells plus promonocytes accounted for 30% of the nucleated BM cells, leading to a diagnosis of AML secondary to CMML in the WHO classification BM karyotype identified no clonal abnormalities and aCGH analysis of BMMCs produced normal findings. BM and PB cells were screened for mutations in 18 CMML-associated genes. Only two abnormalities were identified: a 27 base pair (bp) insertion FLT3-ITD mutation (exon 15) detected in BM cells with near complete disappearance of the wild type (WT) FLT3 allele (FLT3-ITD/FLT3-WT ratio at 9.62) and a classical heterozygous mutation (dupG) was found in the exon 12 of ASXL1. ASXL1 and FLT3-ITD mutations were not detected in purified CD3+ T lymphocytes. Five months after sorafenib introduction, PB was strictly normal and BM examination demonstrated normal richness, blast cells and promonocytes accounting for 2% of the nucleated BM cells but persistent moderate dysgranulopoiesis and dysmegakaryopoiesis, indicative of complete remission. At this time, the FLT3-ITD/FLT3-WT ratio was 1.66 and 0.58 in the BM and PB, respectively. In January 2012, the WBC profile was still normal and a BM smear only showed moderate dysgranulopoiesis. On the molecular side, FLT3-ITD mutation was undetectable, indicative of complete molecular response. But ASXL1 mutation was evident at all time points. The serum of the patient, obtained before and under sorafenib was tested on cell line harboring FLT3-ITD mutation. Constitutive FLT3 Y591, Akt S473, STAT5 Y694 and ERK1/2 T202/Y204 phosphorylations were fully inhibited in the presence of the serum extracted under sorafenib treatment. Conclusion Our patient clearly had a myeloproliferative-CMML driven by an homozygous FLT3-ITD mutation. This is the first report of such a CMML patient achieving sustained CR and CMR after treatment with an FLT3-ITD tyrosine kinase inhibitor. In this case, the ASXL1 mutation remained detectable upon sorafenib treatment after the suppression of FLT3-ITD-driven malignant hematopoiesis, suggesting that it arose from a FLT3-WT subclone that contributed to the CMML phenotype with some dysplastic feature. In conclusion, we propose that mutations in the FLT3 gene should be examinated in all CMML cases, even their low frequency because FLT3 TKI may induce dramatic and sustained responses without significant toxicity and eventually allow for allogenic transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 1024 Poster Board I-46 The treatment of acute myeloid leukemia (AML) still represents a challenge. As the growth and survival of AML cells are enhanced by the deregulation of signaling pathways such as PI3K, ERK/MAPK or mTOR, major efforts are made to develop targeted molecules against these activated pathways. The mTOR serine/threonine kinase belongs to two separate complexes, mTORC1 and mTORC2. The mTORC1 complex, defined by the interaction between mTOR and raptor, is inhibited by rapamycin in most models. It controls mRNA translation through the phosphorylation of the physiological translation repressor 4E-BP1. A constitutive activation of mTORC1 is usually detected in primary AML cells, indicating this pathway as a major target for AML therapy. However, inhibition of mTORC1 with rapamycin has only modest effects in this disease (Recher, Beyne-Rauzy et al., Blood 2005; Xu, Thompson et al.,Blood 2005). Accordingly, we recently showed that the translation process is deregulated and rapamycin resistant in AML, although specifically targeting translation with the 4E-BP1 mimetic 4EGI-1 markedly impairs AML cells survival (Tamburini, Green et al., Blood 2009). The mTORC2 complex is defined by the interaction between rictor and mTOR and is generally reported as resistant to rapamycin. An emerging role for mTORC2 has recently been rose in cancer biology, including in AML, in part through its ability to activate the oncogenic kinase Akt by phosphorylation on S473 (Zeng, Sarbassov dos et al., Blood 2007). In AML, the PI3K and mTOR activities are closely connected: inhibition of mTORC1 activates an IGF-1/IGF-1R autocrine loop that overactivates PI3K but mTORC1 activity is PI3K-independent (Tamburini, Chapuis et al., Blood 2008). These results strongly suggest a rational for simultaneous inhibition of PI3K and mTOR to enhance the killing of AML cells (Park, Chapuis et al., Leukemia 2008; Tamburini, Chapuis et al., Blood 2008). The NVP-BEZ235 compound (Novartis®) is an ATP-competitive inhibitor specific of PI3K and mTOR kinases, actually in phase I/II trial in oncology. We tested for the first time NVP-BEZ235 in 3 human AML cell lines (MV4-11, MOLM-14 and OCI-AML3) and in primary AML samples (n=10). This compound inhibited PI3K and mTOR signaling without off-target effects against other kinases usually activated in AML cells (ERK1/2 and STAT5). In contrast to rapamycin, NVP-BEZ235 dose-dependently inhibited the catalytic activity of mTOR as attested by inhibition of the mTOR autophosphorylation site on S2481. Consequently, NVP-BEZ235 fully inhibited the rapamycin-resistant phosphorylation of the translation repressor 4E-BP1 on T37/46, T70 and S65 residues. This resulted in a marked inhibition of mRNA translation in AML cells, attested by (i) an inhibition of the assembly of the translation initiating complex eIF4F, (ii) a shift from large to small polysomes in NVP-BEZ235-treated AML cells and (iii) a decreased expression of the oncogenic cap-dependant proteins Bcl-XL, c-myc and cyclinD1. Moreover, to study the effects of NVP-BEZ235 on mTORC2 activity independently of Akt, we investigated the regulation of the mTORC2 substrate paxillin (Jacinto, Loewith et al., Nat Cell biol 2004). We first confirmed that paxillin Y118 residue was a substrate for mTORC2 activity in primary AML cells as the siRNA-mediated rictor knockdown dramatically inhibited this phosphorylation event. In primary AML samples, NVP-BEZ235 inhibited paxillin Y118 phosphorylation in a dose-dependent way and this closely correlated to the inhibition of Akt S473 phosphorylation. Targeting the catalytic activity of mTOR represses therefore both mTORC1 and mTORC2 activity. Accordingly, the NVP-BEZ235 compound had marked anti-leukemic activity in AML. Indeed, it strongly reduced the proliferation of AML cells and blocked the cell-cycle as attested by a decrease of AML cells in S-phase and an accumulation in G1-phase. Finally, NVP-BEZ235 induced an important apoptotic response in AML blast cells but its effects on normal hematopoiesis were limited ex vivo. Overall, NVP-BEZ235 strongly inhibited protein synthesis and induced the killing of AML cells through the catalytic inhibition of the oncogenic PI3K and mTOR kinases. Given these results, NVP-BEZ235 may be tested urgently in clinical trials in AML. Disclosures: No relevant conflicts of interest to declare.

In most cancer models, the PI3K/Akt pathway controls the activity of the mammalian Target of Rapamycin Complex 1 (mTORC1) that regulates cell growth through the phosphorylation of P70S6K and 4E-BP1. In Acute Myeloid Leukemia (AML), both constitutive activation of PI3K (PI3K+) and of mTORC1 are detected. We previously reported in AML that PI3K activity is due to the expression of the p110d isoform of class Ia PI3K and is constitutive in 50% of samples at diagnosis. We show here in primary AML samples that the constitutive activity of mTORC1, detected by the phosphorylation of P70S6K on Thr389, is not due to PI3K/Akt activation. In PI3K+ samples, the specific inhibition of p110d with IC87114 does not inhibit mTORC1. Furthermore, a constitutive activity of mTORC1 is detected in almost all AML samples regardless of their PI3K/ Akt activation status. Similarly, the inhibition of ERK/MAPK activity with UO126 does not result in mTORC1 inhibition. Interestingly, in primary AML cells, we shows that mTORC1 is fully inhibited following leucine starvation as observed in non-transformed cells, suggesting that the kinase activity of mTOR is not deregulated and remains accessible to target inhibition. We thus focused on the links between the AMP kinase (AMPK) and mTORC1. AMPK is a physiological cellular energy sensor and becomes phosphorylated at Thr172 downstream of LKB1 in response to a decrease in cellular ATP levels. AMPK then controls multiple metabolic processes such as fatty acid oxidation, glucose production and protein synthesis. Interestingly, AMPK is usually unphosphorylated in cancer cells including AML and thereby represents a potential therapeutic target. We thus investigated the effects of metformin, a potent AMPK activator developed as an oral anti-diabetic drug, in primary AML cells. This compound induces a dose-dependent activation of AMPK that correlates with a decrease of mTORC1 activity. Primary AML blast cells cultured with metformin show a significant inhibition of proliferation, of clonogenic growth and apoptosis suggesting that AMPK may be an attractive anticancer therapeutic target in AML. However, as metformin may exerts AMPK-independent effects, we are producing a lentiviral construct expressing a constitutively activated AMPK mutant to more specifically determine the consequences of AMPK activation in mTORC1 signaling and blast cells survival.

Background: The PI3K/AKT signaling pathway is frequently activated in blast cells from AML patients, and also detected in the leukemic stem cell (LSCs). The p110δ catalytic subunit of class IA PI3K is constantly expressed in leukemic cells, and IC87114, a specific p110δ inhibitor, inhibits Akt phosphorylation, reduces blast cell proliferation, but does not induce apoptosis in AML cells. The mTORC1 pathway is also activated in 50 to 70% of AML samples. Rapamycin and its derivative RAD001, inhibit mTORC1 activity, decrease the clonogenicity of leukemic progenitors, but induce low rates of apoptosis. LY294002 inhibits all p110 isoforms of PI3K as well as most kinases of the PIKK family but can not be used in clinical practice because of its toxicity. Pharmaceutical industry effort identified more selective PI3K inhibitors, and among them, PI-103, a small synthetic molecule of the pyridofuropyrimidine class, is potentially interesting as a selective PI3K inhibitor and also as an mTOR inhibitor. Materials and methods: Bone marrow (BM) samples were obtained from 20 newly diagnosed patients with de novo AML, treated in the AML2001 trial of chemotherapy, initiated by the GOELAMS. The MOLM-14 and OCI-AML-3 leukemic cell lines were obtained from ATCC. CD34+ cells from 5 normal allogeneic donors were purified after cytapheresis. Results: We studied the effect of PI-103 in MOLM-14 and OCI-AML-3. We observed a dose-dependent inhibition of AKT (Ser 473 and Thr 308), FOXO3a (Thr 32), and p70S6-kinase (Thr 389) phosphorylation with 0.1, 1 or 5 μM PI-103. 1μM PI-103 was as effective as 25 μM LY294002 or 10 nM RAD001 at inhibiting PI3K/AKT or mTORC1 activity, respectively. 1μM PI-103 dramatically inhibited the proliferation of both cell lines. However, PI-103 was essentially cytostatic and did not induce significant apoptosis in these cells. For subsequent experiments on primary AML blasts, the dose of 1 μM PI-103 was chosen as it was sufficient to inhibit AKT and p70S6K phosphorylations. The effect of 1μM PI-103 on PI3K/AKT and mTORC1 activation was evaluated in 5 AML samples (3 with constitutive PI3K activation (PI3K+) and 2 without constitutive PI3K activation (PI3K-)). 1 μM PI-103 inhibited completely basal and SCF or Flt3-L-stimulated AKT, FOXO3A phosphorylation and P70S6K phosphorylation in these AML samples. PI-103 dramatically inhibited both basal and growth factor-stimulated proliferation of blast cells. Interestingly, PI-103 induced significant apoptosis in 6 AML samples at 1μM and a significant decrease of CFU-L colonies in 6 other AML samples. In contrast, specific inhibition of the catalytic activity of the p110δ isoform and of the mTORC1 complex, with IC87114 and RAD001 respectively, did not induce apoptosis. Finally, excess of apoptosis was also obtained in the CD34+ CD38Low/Neg CD123+ LSCs from 4 patients in presence of PI-103 alone. Moreover, the functional effects of PI-103 on normal CD34+ cells were low, indicating that this compound may have a favourable therapeutic index. Conclusions: Our results suggest that multi-targeted therapy toward PI3K/AKT and mTOR may be efficient in AML and particularly useful to target LSCs.