ALBERT

Description: Background:Acute promyelocytic leukemia (APL) is a favorable-risk subgroup of AML characterized by the t(15;17) translocation. The leading cause of early death in APL is uncontrolled bleeding mostly attributed to aberrant expression of tissue factor (F3) and annexin A2 (ANXA2) on leukemic promyelocytes leading to disseminated intravascular coagulation and hyperfibrinolysis, respectively. To prevent or treat such complications, early suspicion of APL and rapid initiation of therapy and supportive measures are critical. Podoplaninor PDPN is a surface glycoprotein expressed in most cell types (http://www.gtexportal.org), but not in blood cells. CLEC-2, the PDPN receptor, is expressed on normal platelets and was found to be necessary for the separation of blood and lymphatic vessels during embryogenesis. PDPN expression (whether endogenous or ectopic) in cell lines induces platelet aggregation, which can be inhibited by chemical tool compounds or by monoclonal antibodies (Chang et al, Oncotarget, 2015, Kato et al, Biochem. Biophys. Res. Commun., 2006). Aims and Methods:We analyzed the transcriptome of 30 APL comprised in the Leucegene 430 AML cohort, aiming to identify clinically useful markers and to better understand the hemostasis-related transcriptomic landscape of this subgroup. Patient cohorts and sorted normal hematopoietic cell populations (n=63) were previously reported (Lavallée et al, Nature Genetics, 2015 and Lavallée et al, Blood, 2016). Comparative analysis of gene expression and mutations were performed as previously described. Results:Our analytical pipeline identified several mutated genes in this cohort, most of which are non-specific and previously identified. CEBPE mutations were the only exception and were specific to APL specimens in this cohort (2/30 vs 0/400, p= 0.005). Most interestingly, we identified PDPN as the single most differentially overexpressed gene in APL (median 2.6 vs 0 RPKM, q = 7 x 10-29, Fig A-B). We also found that PDPN is not expressed in whole blood, bone marrow and in any sorted cell subpopulations from these normal tissues, including promyelocytes (median PDPN expression = 0 RPKM, range 0-0.018). This indicates that platelets are never exposed to PDPN in the adult vasculature and reveals that this gene is ectopically expressed in APL promyelocytes. Accordingly, our hypothesis is that aberrant PDPN expression on leukemic promyelocytes contributes to abnormal platelet aggregation in APL patients. We found that high PDPN expression is associated with lower platelet counts at presentation (18 vs 34 x 1012/L, median PDPN expression ≥ 10 vs 〈 10 RPKM, p = 0.016, Fig C). Furthermore, a strong inverse correlation was observed between the number of estimated circulating PDPN+ promyelocytes and platelet counts (leukocyte count x [% PDPN+ cells] 〉 1 x 109/L, Fig C, p=0.007). By incorporating anti-PDPN antibody (clone NC-08, Biolegend) in the EuroFlow protocol, we observed that PDPN expression test was 90% sensitive and 100% specific for APL (n= 48 and 50 APL and non-APL primary AML, respectively). Of note, 5 APL cases considered positive expressed low levels of PDPN. Interestingly, by comparing expression of all coagulation and fibrinolysis genes in APL (n=30) to that of non-APL specimens (n=400), we found that PDPN was the most discriminatory transcript (Fig. A). This result stands in sharp contrast with that found with F3 and ANXA2 which largely overlap in these APL versus non-APL human AML (Fig. A-B). This reinforces the hypothesis that aberrant PDPN expression may be a strong and unappreciated contributor to platelet consumption in APL. Conclusion:I) CEPBE is recurrently mutated in a small subset of APL patients. II) PDPN is the most specific aberrant transcript in this disease and is a new biomarker for APL. Systematic assessment of podoplanin by flow cytometry in newly diagnosed AML could lead to an earlier detection of unsuspected APL cases. III) PDPN expression by leukemic promyelocytes likely contributes to defective primary hemostasis, representing a new mechanism of APL-related bleeding. This provides a strong rationale for evaluating PDPN-CLEC-2 axis inhibitors in this setting. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Description: RUNX1 is an essential transcription factor for definite hematopoiesis and plays important roles in immune function. Mutations in RUNX1 occur in 5-13% of Acute Myeloid Leukemia (AML) patients (RUNX1mut ) and are associated with adverse outcome, highlighting the need for better genetic characterization of this AML subgroup and for the design of efficient therapeutic strategies for patients with RUNX1mut AML. Toward this goal, we performed RNA-sequencing of a cohort of RUNX1mut primary AML specimens and used a chemogenomic approach combining mutational and gene expression analysis of these specimens with assessment of their drug sensitivity profile through chemical screening. Sequencing data analysis demonstrated that samples with no remaining RUNX1 wild-type allele are clinically and genetically distinct and display a more homogeneous gene expression profile. Interestingly, these studies also unveiled the increased susceptibility of RUNX1-mutated specimens to Glucocorticoids (GCs) and revealed that RUNX1 allele dosage dictates sensitivity to these compounds in AML patient cells, unravelling a new role for RUNX1 in the Glucocorticoid Receptor (GR) pathway. GR is a nuclear receptor that modulates the expression of thousands of genes involved in several biological processes such as metabolism, immune function, skeletal growth, etc. GCs are commonly used to treat cancers of the lymphoid system, however their potential benefits for AML treatment have never been assessed formally and the mechanism of action of these drugs is not fully understood. Transcriptome analyses identified NR3C1 (encoding the GR) as one of the genes whose expression is determined by RUNX1 allele dosage, with increased expression in RUNX1mut specimens, indicating that RUNX1 inactivation could lead to GR upregulation, which might explain the increased sensitivity to GCs. We previously showed that RUNX1 silencing sensitizes human AML cell lines to GCs and that this acquired sensitivity is accompanied by the upregulation of the GR both at the transcript and protein levels. However, basal levels of GR could not explain GC sensitivity in all cases, indicating that other mechanisms are involved in the GC response. By performing co-immunoprecipitations (co-IP), we demonstrated that RUNX1 and GR physically interact in AML cells. Overexpression of FLAG-tagged RUNX1 mutants in HEK293 cells followed by co-IP identified the C-terminal inhibitory domain of RUNX1 as essential for the interaction with GR. Our results suggest that RUNX1 could be part of the GR transcriptional complex and could modulate the transcription of genes involved in the response to GCs. To identify regulators of the GC response, we are currently performing genome-wide CRISPR-Cas9-based genetic screens in AML cell lines. We generated RUNX1-deficient (GCs sensitive) AML cell lines that uniformly express Cas9 under a doxycycline-inducible promoter. These cell lines, along with parental cell lines (GC resistant) were used to conduct screens in GCs-supplemented media for the identification of genes that confer sensitivity or resistance to GCs. Mechanistic insights gained from these experiments will allow the design of additional therapeutic strategies to potentiate the effect of GCs on poor outcome AML. Disclosures No relevant conflicts of interest to declare.

Description: Key Points CSF3R was the most frequently mutated gene identified in this CEBPAbi AML cohort analyzed by next-generation sequencing. CEBPA bi AML that have a characteristic transcriptomic profile are more sensitive to JAK inhibitors than CEBPAwt AML.

Description: Acute myeloid leukemia (AML) is associated with poor overall survival and the development of more effective therapies is urgently needed. G Protein-Coupled Receptors (GPCRs) represent the largest family of membrane receptors, with an estimated 800 members in humans, and are attractive therapeutic targets, accounting for approximately 30% of targets of marketed drugs. These receptors are key transducers that bind a vast diversity of ligands (e.g. glycoproteins, peptides, amino acids, nucleotides, nucleosides, ions) allowing the cells to adapt to their environment by regulating a wide variety of physiological processes including the control of blood pressure, heart rate, digestive processes, hormone secretion, cell growth and migration, as well as vision and olfaction. Binding to their ligands leads to conformational rearrangements promoting the engagement and modulation of many distinct downstream signaling effectors that are both G protein-dependent and independent. Several GPCRs are critical for cell proliferation and survival, and can be aberrantly expressed in cancer cells. For example, the chemokine receptor CXCR4 plays an important role in metastasis and angiogenesis in breast cancer and many other types of tumors. In AML, CXCR4 overexpression has been associated with poor outcome. Moreover, in vivo mouse studies have shown that the use of a small molecule antagonist of CXCR4 increases the mobilization of AML cells into the peripheral blood and improves the apoptotic effects of chemotherapy. This activity has been explored in a phase I/II clinical study showing that the addition of CXCR4 antagonists to chemotherapy in AML might improve the remission rate. The role of GPCRs in mouse leukemogenesis has also been suggested in a transcriptome analysis of two related leukemia clones that differ in their stem cell frequency. In this study, GPCRs were the most differentially expressed class of genes between the two clones. Currently, an extensive assessment of GPCR expression in human AML is lacking. To address this issue, we studied the expression of GPCRs in a large cohort of AML samples, as well as in normal blood cells, bone marrow cell populations, and cord blood-derived CD34+ cells as controls.The 772 GPCRs analyzed in this study consist of all the GPCR members included in the International Union of Basic and Clinical Pharmacology (IUPHAR) database, as well as 370 olfactory, 24 taste and 4 vomeronasal receptors. RNA sequencing data analysis was performed as previously described (Lavallée et al, Blood 2015 Jan 1;125(1):140-3) and revealed that 240 GPCRs are expressed in cells from this AML cohort. Among these receptors, 30 are upregulated and 19 are downregulated in AML samples compared to CD34+ normal cells. Upregulated GPCRs are enriched in chemokine (CCR1, CXCR4, CCR2, CX3CR1, CCR7, and CCRL2), adhesion (CD97, EMR1, EMR2, and GPR114) and purine (including P2RY2 and P2RY13) receptor sub-families. The downregulated members include adhesion GPCRs such as LPHN1, GPR125, GPR56, CELSR3, and GPR126, protease-activated receptors (F2R and F2RL1), and the Frizzled family receptors SMO and FZD6. Interestingly, specific deregulation was observed in genetically distinct subgroups of AML, a subset of GPCRs being differentially expressed in normal karyotype AML with NPM1 or FLT3 -ITD mutations, and in specimens with Core Binding Factor and MLL rearrangements, thereby representing promising therapeutic targets or diagnostic markers. In conclusion, our results demonstrate that several GPCR members are deregulated in AML with a clear enrichment in distinct classes, providing the rationale for functional assays using available agonists or antagonists to leukemia-enriched GPCRs. Since these receptors are the targets of several US Federal and Drug Administration approved drugs, our results pave the way to explore selected GPCRs as novel AML therapeutic targets. Disclosures Bouvier: American Society of Nephrology: Other: speaker; Domain Therapeutics: Other: Company SAB member, Research Funding; Vertex Pharmaceutical: Research Funding; Ontario Genomic Institute: Other: SAB meeting; BMS: Research Funding; DalCor Pharmaceutics: Other: Company SAB member; Pfizer: Other: Speaker, Research Funding; Novo-Nordisc: Research Funding.

Description: Insights into the complex clonal architecture of acute myeloid leukemia (AML) unravelled by deep sequencing technologies have challenged the concept of AML as a hierarchically organised disease initiated and driven by rare self-renewing leukemic stem cells (LSCs). In contrast to normal human hematopoietic stem cells (HSCs), which are highly enriched in the CD34+ CD38- population, LSCs have also been found in the CD34- and the CD38+ fractions questioning the existence of a consistent LSC surface marker profile for AML. Besides, low LSC frequencies in primary samples, rapid onset of differentiation upon ex vivo culture, and genetic inter-specimen heterogeneity hamper the dissection of the molecular machinery that drives LSC self-renewal. We performed RNA-Sequencing of primary human AML samples and assessed LSC frequencies by limiting dilution analyses for 56 of these in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. By comparing gene expression profiles between high vs low LSC frequency leukemias, we identified the G-protein coupled receptor 56 (GPR56) has significantly more expressed in high LSC frequency leukemias. We validated the RNA-seq data with protein expression by FACS and found an excellent correlation. To determine whether GPR56 positive cells overlapped with the known LSC-associated phenotype CD34+ CD38-, we stained 45 AML samples with CD34, CD38, GPR56, and antibodies against other described LSC markers. Although CD34+ GPR56+ and CD34+ CD38- compartments identified the same population in some samples, we found in the majority of samples that GPR56 further subdivided the CD34+ CD38- compartment. Accordingly, not only the proportions of total GPR56+ and CD34+ GPR56+ cells were significantly higher in LSChigh versus LSClow samples, but also the proportion of GPR56+ cells within the CD34+ CD38- compartment was significantly different between the groups indicating that GPR56 might be of additional value to what is currently considered the best described LSC phenotype. The percentage of total CD34 positive cells did not correlate with LSC frequency clearly distinguishing GPR56 from CD34 or CD38, which are only suitable LSC markers when used in combination. We analysed other potential LSC markers (TIM3, CD96, CD44, CD123, CLL1 and CD47) in our RNA-Seq dataset and by FACS analysis in combination with CD34 as we did for GPR56 and none of them correlated with LSC frequency in our sample collection. To determine whether GPR56 discriminates engrafting LSCs from non-LSCs, we sorted GPR56+ and GPR56- cells within the CD34-positive and -negative compartments from selected specimens with known engraftment potential. We found that GPR56 identified the engrafting fraction in CD34positive AML samples, with a 〉50 fold enrichment in LSC in the CD34+GRP56+ fraction vs the CD34+GPR56- fraction within the same sample, demonstrating that GPR56 is a good LSC marker. Specimens with high molecular or cytogenetic risk such as chromosome 5 or 7 anomalies and EVI1- rearrangementexpressed high levels of both, GPR56 and CD34, while samples with coexistent FLT3 -ITD, DNMT3A, and NPM1 mutations displayed a unique CD34low GPR56high profile. Moreover, we found a divergent distribution of variant allele frequencies in GPR56+ versus GPR56- fractions identifying GPR56 as a discriminator of leukemic sub-clones with high and low NSG engrafting capacity. Analysis of engrafted cells re-sorted based on GPR56 after being harvested from mouse bone marrow revealed reduced complexity of the clonal composition. Most importantly, GPR56 positive cells differentiated to GPR56 negative cells in mice, which did not happen in the human niche, in which GPR56 positive and negative fractions represented two independently evolved subclones. In summary our work identifies GPR56 as a novel LSC marker in AML and also shows that GPR56 readily identifies a functionally distinct LSC-rich subclone in the majority of human AML patients and reveals hitherto unforeseen complexity in the interaction between human LSCs and the NSG mouse environment. Disclosures No relevant conflicts of interest to declare.

Description: Background: Acute myeloid leukemia (AML) with complex karyotype (CK) is associated with adverse prognosis with current therapies, in particular in the presence of TP53 mutations. Identification of novel therapeutic strategies is therefore urgently needed for this subgroup of patients. Methods and aims: As part of the Leucegene project, we RNA sequenced a cohort of 415 clinically annotated primary human AML specimens. Comparative transcriptomic analysis of the 68 CK AML specimens with the non-CK samples of the cohort was performed to identify features associated with the CK AML subgroup. Based on these results, we performed a chemical screen aimed at identifying active agents toward CK AML. Gene expression analysis, primary AML specimen culture and chemical screening were performed as previously described by our group (Lavallée et al., Nature Genetics, 2015; Pabst et al., Nature Methods, 2014; Simon et al., Clinical cancer research, 2017). Results: CK AML is a heterogeneous disease by definition and accordingly, Multidimensional Scaling (MDS) analysis fails to identify a distinct transcriptional signature. Nonetheless, comparative transcriptome analysis of CK and non-CK AML specimens revealed highly deregulated genes between these subgroups (Figure 1A). Among them, EDA2R, a known target to p53, is specifically downregulated in CK AML. This reflects the frequent TP53 alterations observed in CK AML. Most importantly, we found that the recently identified AML prognostic gene HMGA2 is overexpressed in the majority of these patients, independently of the TP53 status. HMGA2 is an oncofetal gene expressed in hematopoietic stem cell (HSC) -enriched populations while its expression decreases in progenitors and is nearly absent in mature cells. Interestingly, Gene Set Enrichment Analysis (GSEA) revealed an enrichment for HSC genes in AML specimens with high HMGA2 expression levels, which could reflect a stem-cell origin or a more immature state for these leukemias. Global transcriptomic analysis of CK AML also identified a G2/M checkpoint signature for HMGA2-high specimens (Figure 1B). Accordingly, we observed that chemical inhibitors of the G2/M regulators ATR, CHK1 and WEE1 are preferentially active on HMGA2-high CK specimens and that engineered overexpression of HMGA2 in different leukemia cell lines enhanced sensitivity to G2/M inhibition (Figure 1C). This specific inhibition was independent of the TP53 status. Conclusions: Here we report a comprehensive transcriptomic analysis of the CK AML specimens of the Leucegene cohort and identified aberrant expression of the HMGA2 oncogene in close to 80% of these samples. Chemical interrogation of primary AML specimens revealed a TP53-independent but HMGA2-mediated sensitization of CK AML to G2/M checkpoint inhibition. Thus, our results reveal a novel vulnerability for AML expressing high levels of HMGA2 and identify potential biological targets for these high-risk patients. Since several CHK1 and PLK1 inhibitors have been or are currently under evaluation in clinical trials, our findings suggest that HMGA2-high AML patients could benefit from this therapeutic approach. Figure 1: A/ Volcano plot representing the genes differentially expressed in CK versus non-CK AML. Samples with low group median expression (2; n=39) and HMGA2 null (RPKM=0; n=83) AML specimens. C/ Dose-response curves and IC50 values for representative compounds in K562 cells infected with control YFP vector (blue) or HMGA2-YFP expressing vector (red). Disclosures Sauvageau: ExCellThera: Employment, Equity Ownership.

Description: Cord blood (CB) transplants have fallen into disfavor in large part due to low cell dose leading to prolonged hospitalizations and high transplant related mortality (TRM). UM171, a novel and potent agonist of hematopoietic stem cell (HSC) self-renewal could solve this major limitation, allowing for CB's important qualities of lower risk of chronic GVHD and relapse to prevail. In addition, UM171 could permit transplantation of smaller, better HLA matched cords, associated with lower TRM. Hence, we initiated a clinical trial to test the safety and efficacy of UM171 expanded CB (eCB). Our goal was to design a clinically viable eCB transplant with a TRM as low or lower than other HSC sources all the while maintaining CB's low relapse rate. Patients (pts) received a myeloablative conditioning regimen. On day (D)-7 of transplant, CB was thawed and CD34+ selected. The CD34- lymphocyte containing fraction was cryopreserved and infused on D+1. The CD34+ component was placed in a closed culture system with UM171 and media was injected once a day until D0, when cells were washed and infused. This fed-batch culture system allowed for small culture volumes, saving cost and labor. Between 7/16-6/18, 21 adult pts (median age 44 years) were transplanted with an eCB. Median final culture volume and net viable CD34 fold expansion were 670 mL and 35, respectively. Median 1st day of 100 and 500 neutrophils were D+10 and D+18, respectively. Achieving 100 neutrophils was 5 days faster than seen with our pts receiving peripheral blood (PB) or marrow (BM) and appeared cell dose independent, suggesting that clinically meaningful expansion of an early repopulating myeloid progenitor is at saturation even with smaller CBs. In contrast, attaining 500 neutrophils was accelerated but dependent on cell dose. More importantly, pts appeared to derive clinical benefit beyond neutrophil engraftment (defined as 500 neutrophils). Pts' median last day of fever prior to 500 neutrophils was D+8, much earlier than engraftment and 4 days earlier than seen with our PB-BM pts. We offer 2 hypotheses as explanation: i) 100 neutrophils, which are attained early, provide significant defense against infection, ii) the graft contains a significant proportion of dendritic cell precursors (30-40%) which offer mucosal protection during severe neutropenia. Duration of hospitalization was shorter by 12 days and longer by 2 days compared to our non eCB and PB-BM transplants, respectively. In addition, because cell dose requirements were lower, 12/21 pts received a better HLA matched CB, thus 〉80% of patients were transplanted with a ≥6/8 HLA matched eCB. As a result of lower minimal cell dose criteria, we can now use ∼half the CBs in the banks instead of only 5% for a 70 kg patient. Platelet engraftment occurred at a median of 42 days. With a median follow up of 14 months, there has been no CMV disease, no PTLD, 2 adenovirus cystitis, 2 (10%) grade 3-4 acute GVHD, no moderate/severe chronic GVHD and 1 TRM (5%) despite a median comorbidity index of 2 (0-5). Full donor chimerism was achieved in all cell subsets. Immune recovery was faster than seen in our unrelated donor transplants who routinely receive ATG prophylaxis with 196, 300 and 413 CD4+/µL at 3, 6 and 12 months, respectively. Interestingly, transcriptome analysis of UM171-eCB cells shows an enhanced lymphoid progenitor-associated gene signature when compared to DMSO exposed cells. Animals transplanted with UM171-eCB cells showed a 20 to 35-fold increase in thymic cellularity at 8 weeks post-transplant. Despite some very high risk pts in our trial, only 3 relapsed. Overall, progression free, and GVHD/relapse free survival (GRFS) are excellent at 95, 77 and 67%, respectively, at 12 months. A 7 day UM171 single eCB protocol is feasible and provides clinical benefits beyond faster engraftment with fewer infectious complications, better HLA matching and very low TRM, all the while saving production and hospitalization costs. Nevertheless, longer follow up will be required to better assess relapse howbeit encouraging preliminary results. Furthermore, patients' quality of life is paramount and best evaluated by GRFS which is excellent thanks to a very low rate of significant chronic GVHD all the while maintaining a low risk of relapse. In conclusion, this 1st trial documents the potency of UM171 and positions UM171-eCB as a promising HSC source which could compete with the current standard of care. Figure. Figure. Disclosures Cohen: ExCellThera: Patents & Royalties: Royalities from sales of UM171. Roy:ExCellThera: Patents & Royalties: Royalities from sales of UM171. Lachance:ExCellThera: Patents & Royalties: Royalities from sales of UM171. Roy:Hopital Maisonneuve Rosemont: Patents & Royalties: Author on patent; Kiadis Pharma: Other: Travel support; University of Montreal: Patents & Royalties: Author on patent. Busque:BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Paladin: Consultancy. Kiss:Alexion: Membership on an entity's Board of Directors or advisory committees, Research Funding; Otsuka: Membership on an entity's Board of Directors or advisory committees, Research Funding. Caudrelier:ExCellThera: Employment. Zandstra:ExCellThera: Equity Ownership. Sauvageau:ExCellThera: Employment, Equity Ownership.

Description: Acute myeloid leukemias (AML) are aggressive blood cancers characterized by an overall survival of 27% at 5 years. The main challenge in AML treatment originates from the genetic heterogeneity of the disease that contributes to the wide range of clinical outcomes observed. A large proportion (~35%) of AML patients exhibit no distinguishable chromosomal abnormalities that can be used to guide treatment selection and are therefore classified in the poorly characterized intermediate risk category. Approximately 50-60% of intermediate risk AML patients carry mutations in the NPM1 gene. These mutations are associated with a favorable outcome unless a concomitant mutation in the FLT3 gene is detected, which accounts for 39% of cases. The survival rate is further worsened when a third mutation is detected in the DNMT3A gene, dropping from 40% to 20% 5 years post-treatment for double and triple mutants, respectively. This study aimed to identify drugs selectively affecting the viability of leukemic cells from AML patients with NPM1 mutations. To achieve this, we took advantage of culture conditions developed by our group that prevent differentiation of leukemic cells and preserve leukemia stem cell activity from primary AML specimens (Pabst et al., Nature methods, 2014), enabling chemical screening of primary AML specimens. We conducted a chemical screen using a collection of ~300 clinical grade drugs on a cohort of 38 primary human AML specimens containing NPM1 mutated (NPM1c+) and NPM1 wild-type (NPM1wt) samples. These specimens belonged to the Leucegene collection of sequenced and clinically annotated samples. The screen identified ABT-199 as the compound with the most discriminatory effect toward NPM1c+ AML. ABT-199 is a specific BH3-mimetic that prevents anti-apoptotic BCL2 from binding pro-apoptotic BAX and BAK1 proteins, leading to apoptosis. ABT-199 demonstrated encouraging results for AML treatment, but the determinants of drug sensitivity have not been well defined. Analysis of the enrichment of clinical variables in ABT-199 sensitive and resistant groups of AML specimens to define characteristics/biomarkers associated with ABT-199 sensitivity in AML revealed that mutations in NPM1, RAD21, IDH1, IDH2, DNMT3A and FLT3 (ITD), as well as normal karyotype and the FAB M1 class all significantly associate with increased ABT-199 sensitivity. At the other side of the spectrum, mutations in TP53 and FAB class M5B were significantly enriched in the resistant group. Additional analyses revealed that NPM1c+/DNMT3Amut/FLT3-ITD specimens are sensitive to ABT-199, which may provide a rationale to prioritize patients from this adverse risk AML subgroup for explorative ABT-199 based regimens. Specimens with RAD21 mutations were the most sensitive to ABT-199 treatment and further analyses demonstrated a clear association between mutation of cohesin genes (RAD21, SMC1A, SMC3, STAG2) and increased ABT-199 sensitivity. In line with this, we demonstrated that RAD21 knockdown alone is able to sensitise AML cell lines to BCL2 inhibition. Comparative transcriptome analysis of ABT-199 sensitive and resistant specimens also revealed an apoptotic gene signature linked to ABT-199 resistance with BCL2A1, an anti-apoptotic BCL2 homolog, being the most differentially expressed apoptotic gene between these response groups and showing increased expression in the resistant subset. Expression correlation analysis over the 415 specimens of the Leucegene cohort showed that BCL2A1 is one of the top genes anti-correlated to BCL2, and accordingly, high BCL2 and BCL2A1 expressors were enriched among ABT-199 sensitive and resistant specimens, respectively. In conclusion, using an unbiased pharmacogenomic approach, we identified ABT-199, a compound with the potential to eradicate NPM1c+ AML, which has already been tested in a phase 2 clinical trial for AML. Our results shed light on determinants of ABT-199 sensitivity which could readily impact AML therapy by providing a rationale for prioritizing patients with NPM1, RAD21, IDH1 and/or IDH2 mutations for ABT-199 AML trials. Our results also uncover potential mechanisms of resistance to ABT-199, providing grounds to design combination therapies to overcome ABT-199 chemoresistance. Disclosures Sauvageau: ExCellThera: Employment, Equity Ownership.

Description: Key Points EVI1-r AMLs have recurrent mutations in RAS and other signaling genes, splicing factors, and at a lower frequency, IKZF1 and TP53. EVI1-r AMLs show a characteristic transcriptome profile marked by high expression of MECOM, PREX2, MYCT1, PAWR, and VIP.