Abstract: Abstract 1520 Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here we analyzed the therapeutic effect of LBH589 or panobinostat, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced a significant dose-dependent increase in cell apoptosis and a markedly inhibition of cell proliferation, which were associated with increased H3 and H4 histone acetylation. While apoptosis of ALL cells was detected between 12 and 24 hours after treatment with LBH589, changes in acetylated H3 and H4 were detected as early as 2 hours. Phosphorylation of H2AX, as an early marker of DNA damaged, was detected 12 to 24 hours after in vitro treatment with LBH589. These results suggest that H3 and H4 acetylation precede DNA damaged and induction of apoptosis indicating that inhibition of HDAC is likely to be responsible at least in part for LBH589 induced apoptosis and inhibition of cell proliferation. The in vivo activity of LBH589 was initially examined in a subcutaneous ALL mouse model. The ALL cell lines TOM-1 and MOLT-4 were transplanted (1×106 cell per animal) subcutaneously into the left flanks of 6-week-old female BALB/cA-Rag2−/−γc−/−. These cell lines develop into a rapidly growing tumor. Treatment with 5mg/kg of LBH589 was initiated 24 hours after injection of the leukemic cells, included 3 cycles of 5 consecutive days of LBH589 with two days rest between cycles and animals were monitored for 24 days. A significant inhibition of tumor growth was demonstrated in animals treated with LBH589 compared with control animals (P 〈 0.01). Inhibition of leukemia cell growth was associated with an increase in the levels of acetylated H3 and H4 and an increase in phosphorylated H2AX in the leukemic cells obtained after sacrifice of mice. These results suggest that LBH589 has a powerful antileukemic effect not only in vitro but also in vivo. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2−/−γc−/− mice was established, allowing continuous passages of transplanted cells to several mouse generations. A total of 10 million cells from a patient with T-ALL (ALL-T1) and a patient with B-ALL (ALL-B1) were administered intravenously into the tail vein of 6-week-old immunodeficient female BALB/cA-Rag2−/−γc−/− mice. Kinetics of engraftment of leukemic cells was monitored in PB and BM by phenotyping while organ infiltration was analyzed by immunohistochemistry. There were no significant differences in the genome, methylome or transcriptome between the original sample and the samples obtained after multiple generations on mice. To determine the efficacy of LBH589 alone or in combination with drugs currently used for treatment of ALL, BALB/cA-RAG2−/−γc−/− mice engrafted with ALL-T1 and ALL-B1 cells were treated with LBH589, Vincristine and Dexamethasone or a combination of LBH589 with Vincristine and Dexamethasone. Treatment was initiated when disease could be detected in PB by FACS (24 hours after injection of cells for ALL-T1 and between day 17 and 21 after injection for ALL-B1). LBH589 was administered i.p. on days 1–5, 8–12 and 15–19, Vincristine i.v. on days 1, 8 and 21 and Dexamethasone daily until day 21 i.p. and survival was analyzed. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving Vincristine and Dexametasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with Vincristine and Dexametasone. Our results demonstrate the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract: INTRODUCTION: High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is the standard treatment for patients with relapsed or refractory aggressive B-cell lymphoma, and is frequently used as part of first-line therapy in patients with peripheral T-cell lymphoma (PTCL). However, long-term remission rates with this strategy are inferior to 50%, so novel approaches are required. We have designed a prospective multicenter phase II study to evaluate the safety and efficacy of bendamustine as part of conditioning regimen in patients with aggressive lymphomas undergoing ASCT. METHODS: Inclusion criteria were: histologic diagnosis of i) relapsed or refractory diffuse large B-cell lymphoma (DLBCL) or grade 3B follicular lymphoma (FL) in partial response (PR) or complete remission (CR) after salvage therapy, or ii) transformed DLBCL or peripheral T-cell lymphoma (PTCL) in first or subsequent PR or CR. Conditioning regimen consisted of bendamustine (200 mg/m2, days -7 and -6), etoposide (200 mg/m2, days -5 to -2), cytarabine (400 mg/m2, days -5 to -2), and melphalan (140 mg/m2, day -1) (BendaEAM regimen). Primary endpoint was progression-free survival (PFS) at 3 years. Secondary endpoints were toxicity, response to transplant at 3 months, and overall survival (OS). This trial was registered at EMEA (EUDRACT number 2010-020926-17). RESULTS: Sixty patients (median age 54 years, range 27-70) from 22 Spanish hospitals were included since May 2011 to November 2012. Histologies were: 40 DLBCL, 3 grade 3B FL, 13 transformed DLBCL, and 7 PTCL. 82% of patients have received ³2 lines of treatment prior to ASCT. 37 patients (62%) were in CR at the time of transplant and 23 (38%) in PR. A median number of 4.05 x 106/Kg (range: 1.69-19.80) CD34+ cells were reinfused. All patients (except one who died early) engrafted after a median of 11 (range: 9 to 72) and 14 (range: 4 to 53) days, respectively, to achieve 〉 0.5 x109/L neutrophils and 〉 20 x109/L platelets. 39 serious adverse events (SAEs) were reported before day +100, including 14 infectious episodes, 2 of them resulting in respiratory failure and death (3.3% of transplant related mortality). Another major SAE was renal toxicity developed by 5 patients (8.3%) after bendamustine administration, reversible in all cases (3 of these patients had developed mild renal failure during previous salvage therapy). Non-relapse mortality after day +100 was 3.3% (1 patient died because of Wernicke's encephalopathy, and 1 patient from infectious complications). Concerning response to transplant, 44 patients (73.3%) achieved CR, 7 (11.7%) PR, and 6 patients (10%) did not respond. Univariate analysis showed that patients who received more than 2 lines of treatment prior to transplant (1 line: 100% of CR post-transplant; 2 lines: 71%; 〉 2 lines: 50%; p=0.013), and those who were in PR at transplant (48% vs 89%, p 〈 0.001) had lower CR rates after ASCT (day +100), although only disease status at transplant retained the significant influence in the multivariate analysis (RR: 0.11, 95% CI: 0.03-0.42, p=0.001). Histological diagnosis had no significant influence on CR rates after ASCT (DLBCL: 73%; transformed DLBCL: 69%; PTCL: 86%; p 〉 0.1). At the time of analysis, 13 patients (22%) had disease progression and 8 patients (13%) have died (4 from lymphoma, and 4 from other causes). With a median follow-up of 18.9 (9.5 to 32.3) months, the estimated 2-year PFS and OS were 73% and 88%, respectively. CONCLUSIONS: The BendaEAM conditioning regimen is feasible and active in patients with aggressive lymphomas. Toxicity profile is similar to that commonly observed in the ASCT setting, but renal toxicity can occur and should be carefully monitored, especially in patients with prior history of renal failure. Longer follow-up is needed to assess the long-term toxicity and the efficacy of this regimen, although patients who are not in CR before transplant seem to have poorer outcomes. Disclosures No relevant conflicts of interest to declare.

Abstract: We conducted a phase 2 trial to evaluate the safety and efficacy of bendamustine instead of BCNU (carmustine) in the BEAM (BCNU, etoposide, cytarabine and melphalan) regimen (BendaEAM) as conditioning for autologous stem‐cell transplantation (ASCT) in patients with aggressive lymphomas. The primary endpoint was 3‐year progression‐free survival (PFS). Sixty patients (median age 55 [28–71] years) were included. All patients (except one who died early) engrafted after a median of 11 (9–72) and 14 (4–53) days to achieve neutrophil and platelet counts of 〉 0.5 × 10 9 /l and 〉 20 × 10 9 /l, respectively. Non‐relapse mortality at 100 days and 1 year were 3.3% and 6.7%, respectively. With a median follow‐up of 67 (40–77) months, the estimated 3‐year PFS and overall survival (OS) were 58% and 75%, respectively. Patients in partial response at study entry had significantly worse PFS and OS than patients who underwent ASCT in complete metabolic remission, and this was the only prognostic factor associated with both PFS (Relative risk [RR], 0.27 [95% confidence interval {CI} [0.12–0.56] ) and OS (RR, 0.40 [95% CI 0.17–0.97]) in the multivariate analysis. BendaEAM conditioning is therefore a feasible and effective regimen in patients with aggressive lymphomas. However, patients not in complete metabolic remission at the time of transplant had poorer survival and so should be considered for alternative treatment strategies.

Abstract: Introduction: Allogeneic haematopoietic stem cell transplantation (alloHCT) is a potentially curative approach for patients (pts) with multiple myeloma (MM). The high transplant related mortality (TRM) rate with myeloablative conditioning has resulted in a shift to reduced intensity conditioning regimens (RIC). However, most MM pts who receive an alloRIC ultimately relapse and their treatment remains a challenge. Since alloHCT can modify the biology of the disease, including the immune environment, responses after alloHCT to rescue therapies previously used before alloHCT could be improved. Our main objective was to evaluate the efficacy of regimens including new drugs in MM pts relapsing after alloHCT comparing the efficacy achieved before and after alloHCT. Material and Methods: We report a retrospective multicenter analysis of 126 consecutive pts that underwent alloHCT for MM from 2010 to 2013 at 8 Spanish centers. Results: Baseline pts and transplant characteristics are shown in Table I. The median of prior therapies prior to alloHCT was 3 (1-9), 83% had received previous autologous HCT and 26% had high risk cytogenetic. 71 (56%) and 48 pts (38%) had been treated with regimens containing proteasome inhibitors (PI) and immunomodulatory drugs (IMIDs) before transplantation, respectively. Disease status at transplant was complete remission (CR) in 16 (13%) pts, partial response (PR) or very good PR in 86 (68%) and 24 (18.5%) had relapsed/progressive disease. 35 pts (28%) had active extramedullary disease at transplant. The majority of pts received peripheral blood HCT (90%), RIC (90%) with fludarabine plus melphalan based conditioning regimen (61%) and calcineurin inhibitor plus MTX as GVHD prophylaxis (68%). 19% receiving allo-HCT from an unrelated donor (91% 10/10 HLA matched). All pts engrafted. Grade II-IV acute GVHD occurred in 54% pts (grade IV 8%) and chronic GVHD in 45% (moderate 15%, severe 12%). TRM within the first 100 days after transplant was 6% (overall TRM 28%). 60% pts improved their pre-transplant response, with an overall response rate of 74% (56% CR). After a median follow-up of 92 months for pts alive (22-197), the OS was 51 and 43% at 2 and 5 years respectively. 75 pts (59.5%) relapsed after alloHCT, 57 of them with extramedullary involvement. Median time to relapse was 8 months post-transplant (1-141). The cumulative incidence of relapse was 79% at 3 years. Median OS after relapse was 22 months (8-33). Seventeen out of 75 pts who relapsed received IP both in the pre-transplant and in the post-transplant period. Sixteen pts out of 17 who received IP achieved at least PR pre-transplant while 10 out of these 16 pts responded again to PI post-HCT. Moreover, 1 patient reached a deeper response (CR) post as compared to pre alloHCT (PR) and 1 patient who was refractory pre-alloHCT did respond post-alloHCT. In addition, 6 out of 7 pts who did not respond to IP post-transplant reached stable disease with time to progression (TTP) lasting 4 to 12 months. Twelve out of 75 pts who relapsed received IMIDs both pre and post-alloHCT. Ten pts out of 12 who received IMID pre-alloHCT achieved at least PR, and 8 out of these 10 pts responded again to IMIDs post-alloHCT. Moreover, 1 patient who had been refractory to IMIDs in the pre-transplant period reached CR after alloHCT. In addition, 2 out of 4 pts who were refractory to IMIDs in the post-transplant period reached stable disease with TTP of 8 to 13 months. Remarkably, among pts who respond both in the pre and the post-transplant period to IP or IMIDs, the time to response (TTR) and time to progression (TTP) was similar despite the regimens used in the pre-transplant setting were more aggressive (TTR 3 vs 3.5 and TTP 9 vs 7 months before and after alloHCT for IP, and TTR 4 both before and after alloHCT and TTP 10 vs 9.5 months before and after alloHCT for IMIDs). All but 2 pts received first generation IP pre and post-alloHCT (two pts received carfilzomib) and all but 5 received first generation IMIDs (5 pts were treated with pomalidomide). Conclusions: MM pts relapsing after alloHCT should be considered candidates to receive new drugs, as they can achieve response rates at least in a similar proportion and durability to those observed in the pre-transplant setting. This finding is in contrast to the usual course of the disease outside the alloHCT setting, where response rates and TTP decreases with consecutive lines of treatment. Disclosures Mateos: Janssen: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Amgen: Honoraria.

Abstract: Myeloid neoplasms (MN) are usually sporadic late-onset cancers; nevertheless, growing evidence suggests that ~5% of the cases could emerge as a consequence of inherited predisposition. Indeed, the 2016 revision of the World Health Organization (WHO) includes hereditary myeloid malignancies (HMMs) as a separate disease entity. Distinguishing somatic from germline variants is of vital importance, in order to establish an appropriate individualized management and counsel the patients and their relatives. Germline tissues are required to discriminate HMMs alterations, but they are not always available for testing at the time of diagnosis; therefore additional strategies should be put in place in order to identify variants potentially conferring genetic predisposition to MN. The aim of the present work is to develop a tool to identify HMMs cases from tumour-only sequencing data. To this end, we reviewed tumor-only NGS reports from 299 cases of patients with myeloid malignancies sequenced with a custom Pan Myeloid Panel (PMP) that targets 48 genes, of which 21 are described in literature as HMM-associated genes. All patients signed a written informed consent form for genetic testing, research and tissue banking provided by the Biobank of the University of Navarra (UN) and were approved by the Ethical and Scientific Committees of the UN. Variant calling files (VCF) of the 299 cases were run through an algorithm aiming at identifying those variants suggestive of being of germline nature (Figure 1). We considered as indicative of potential inherited origin, variants detected in bone marrow samples at a ~50% VAF classified as pathogenic, likely pathogenic or of unknown significance. This first filter yielded 90 "likely HMMs" patients harboring a total of 104 suspicious of being germline variants affecting at least one of the 21 HMM-associated genes. Our algorithm included three additional filters: mutational patterns suggestive of germline nature of the variants, analysis of variant allele frequency (VAF) on follow up data, and sequencing of non-myeloid tissues (Figure 1). Firstly, we found 10 "likely HMMs" patients presenting double hit mutations in one of three genes: DDX41 (n=7), CEBPA (n=2) and RUNX1 (n=1). Additionally, there was available follow up data for 8 cases included in our cohort, harboring 11 suspicious variants. From them, VAFs in 4 variants affecting ASXL1, CBL, IKZF1 and GATA2 genes, drastically changed over time, indicating that those variants were somatic, whereas the other 7 variants affecting ASXL1 (n=2), DDX41 (n=2), IKZF1 (n=1), and RUNX1 (n=1) genes suspected to be germline maintained VAF ~50% with evident change of the accompanying variants over time. Finally, we collected non-myeloid tissue samples from 8 patients, harboring a total of 9 variants in ASXL1 (n=2), DDX41 (n=1), GATA2 (n=1), IKZF1 (n=1), NF1 (n=3), and SH2B3 (n=1) genes. Sequencing data confirmed germline nature of 6 of the 9 tested variants; 3 of the 6 germline variants have previously been shown to be linked to MN (DDX41 p.Asp140Glyfs*2 in UPN1, ASXL1 p.Gly967del in UPN11, and NF1 p.Met992del in UPN19). Of note, two of these bona-fide pathogenic germline mutations had been previously highlighted as potential germline variants when applying our criteria of mutational patterns and/or follow up data indicative of germline nature, in so showing the effectiveness of our algorithm (DDX41 in UPN1, and ASXL1 in UPN11). Regarding the usefulness of the different tissues, we found that skin fibroblasts, hair follicles and CD3+ T cells helped discriminating the nature of the variants; on the contrary, DNA from hair follicles showed poor DIN values and scarce DNA concentration. Similarly, DNA isolated from buccal swab showed in general poor quality metrics, and high level of contamination with tumour DNA in one case, proving it unsuitable for discrimination of the nature of the variants in some instances. Our data supports the importance of considering variants found upon tumor-only sequencing as potentially of germline origin, and we offer a pipeline to define the nature of the variants. We hope to provide insights for genetic laboratories facing this relatively new challenge of discriminating somatic from germline variants in tumor sequencing data. Disclosures No relevant conflicts of interest to declare.

Abstract: Acute myeloid leukemia (AML) is a malignant disease characterized by uncontrolled proliferation, differentiation arrest and accumulation of immature myeloid progenitors. Despite recent developments and the approval of new therapeutic agents in the last few years, long term survival of AML, particularly in elderly patients remains an unmet medical need.The use of all-trans retinoic acid (ATRA) in Acute Promyelocytic Leukemia has proven that differentiation therapy may significantly change the survival of AML patients, however the success in APL has not been translated to other groups of AML. Therefore, the identification of new therapeutic agents that may induce the differentiation of AML blasts represents an attractive new target. Furthermore, it is well known that epigenetic alterations have an important role in the development and maintenance of cancer and AML in particular. Thus, our aim was to develop new small molecules targeting epigenetic modifying enzymes like DNA methyltransferases (DNMT), histone methyltransferases or histone deacetylase (HDAC) with the aim of inducing differentiation in AML. We performed a screening of over 50 small molecules synthesized by our group. The design was performed in-house using a knowledge and structure based strategy and the read out of the screening was based on changes in expression of CD11b (a well described marker of myeloid differentiation) after in vitro treatment of AML cells lines. Interestingly, we found several compounds with high capacity to promote the differentiation of leukemic cells in AML cells lines at low non-cytotoxic doses, selecting CM-444 and CM-1758 as our lead compounds (Figure 1a).A complete biochemical characterization showed that both compounds are specific pan-HDACs inhibitors (HDACi). CM-444 and CM-1758 induced in vitro cell differentiation in all subtypes of AML, independently of the AML genetic subgroups or the presence of mutations, which was significantly more pronounced that differentiation induced by reference compounds such as Panobinostat, Vorinostat, Entinostat, Tubastatin or Quisinostat, previously described HDACi. CM-444 and CM-1758 also induced in vivo differentiation in xenogeneic models of AML. AML differentiation was associated with induction of CD11b, downregulation of c-MYC, overexpression of transcription factors that govern the myeloid differentiation and morphologic changes. In addition, these compounds promoted in vitro differentiation of patient-derived AML blasts. The complete transcriptome analysis by RNA-Seq carried out in AML cell lines after CM-444, CM-1758, Panobinostat or Vorinostat treatment showed changes in genes implicated in differentiation, but without explaining the differences among the different HDACi. Analysis of the complete acetylome and proteome before and after treatment with CM-444 and CM-1758 in comparison with other HDACi showed differential acetylation of non-histone proteins included in the GO categories of Zn binding proteins and nucleic acid binding proteins (Figure 1b). Most of these proteins are epigenetic enzymes and have been related to AML and myeloid differentiation, such as MLL2, EP300 or BRD4. In summary, we have developed and characterized novel epigenetic small molecules with a high in vitro and in vivo capacity of differentiating AML cells. These compounds might be an effective differentiation-based therapy to be tested in AML. Besides, the mechanism of differentiation of these compounds is due, at least in part, to the acetylation of non-histone epigenetic proteins, which are key in the myeloid differentiation. Disclosures Paiva: Celgene, Janssen, Sanofi and Takeda: Consultancy; Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche and Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria.

Abstract: Background: Chimeric Antigen Receptor-modified T cell (CAR-T) therapies have revolutionized cancer immunotherapy, especially in hematological malignancies. Although great results have been achieved during the last years, long-term efficacy is still compromised in some cases and factors behind CAR-T cell disfunction are not fully understood. Recent studies have shown that the control of CAR expression influences CAR-T fitness and antitumoral efficacy 1. Therefore, we hypothesized that CAR density on the membrane of CAR-T cells could directly affect CAR-T cell function. In this study we perform a functional and genomic analysis of FACS-isolated subpopulations of CAR-T cells with different CAR densities (CAR High and CAR Low). Methodology: Second generation CAR-T cells with 4-1BB costimulatory domain targeting BCMA were generated by lentiviral transduction of αCD3/αCD28 activated T cells that were expanded for 12-14 days in the presence of IL-7/IL-15. Phenotypic analyses were performed by flow cytometry before and after coculture with MM cells. Cytotoxic activity and cytokine production were measured by standard procedures. In vivo antitumoral efficacy was evaluated in xenogeneic tumor models in NSG mice. Transcriptomic (RNA-seq) and epigenetic (ATAC-seq) analysis were performed following stablished protocols 2. Single cell analysis was performed using the Chromium Single Cell Immune Profiling solution from 10x Genomic that allows simultaneous analysis of gene expression and paired T-cell receptors from a single cell. Gene Regulatory Network (GRN) analysis was performed using SimiC, a novel computational method that infers regulatory dissimilarities 3. Results: RNA-seq and ATAC-seq analysis revealed completely different profiles between CAR High- and CAR Low-T cells in both CD4 +and CD8 + cell subsets, with & gt;3500 differentially expressed genes (2086 for CD4 + and 1553 for CD8 +) that were related with increased tonic signaling, T cell activation and proliferation in CAR High-T cells. Functional studies at resting state (before antigen encounter) corroborated that CAR High-T cells presented increased tonic signaling, that lead to a higher basal activation and a more differentiated phenotype with skewed presence of CCR7 +/CD45RA +/CXCR3 + T SCM cells. After antigen-driven activation, increased cytotoxicity and cytokine production was observed in CAR High-T cells, that also presented higher percentage of terminally differentiated effector cells (CCR7 -/CD45RA +), along with increased exhaustion (PD1 +/LAG3 +/TIGIT +). This effect was also observed in the infusion products of CARTBCMA-HCB-01 clinical trial for patients with R/R MM (NCT04309981), where products enriched in CAR High-T cells presented increased cytotoxic activity. Although no significant differences were observed in the antitumoral efficacy in vivo, CAR Low-T cells presented increased persistence, suggesting that higher CAR levels could reduce long-term efficacy. Further characterization of CAR-T cells at single cell level (scRNA-seq) showed enrichment of CAR High-T cells in activated CD4 + and exhausted CD8 + cell clusters. The analysis of regulatory dissimilarities driven by different CAR densities with SimiC revealed an increased activity of the regulon associated to NR4A1 transcription factor (a well-known TF driving T cell exhaustion 4) in CAR High-T cells, providing mechanistic insights of the regulatory networks behind differential functionality of CAR High-T cells. Finally, to evaluate the impact of CAR density in the clinical outcome of CAR-T therapies, we developed a gene signature associated to increased CAR density, that was applied to transcriptomic data available from public studies 5. We score the infusion products of several clinical trials testing CTL019 (NCT01029366, NCT01747486 and NCT02640209) and we observed an enrichment on CAR High signature in the products from non-responder patients. Conclusions: Our data demonstrate that CAR density on the membrane of engineered T cells plays important roles in CAR-T activity with a significant impact on clinical outcome. Moreover, the comprehension of regulatory mechanisms driven by CAR densities at the single cell level offer an important tool for the identification of key regulatory factors that could be modulated for the development of improved therapies. Figure 1 Figure 1. Disclosures Rodríguez-Otero: Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kite: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Regeneron: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel and other expenses. Paiva: Bristol-Myers Squibb-Celgene, Janssen, and Sanofi: Consultancy; Adaptive, Amgen, Bristol-Myers Squibb-Celgene, Janssen, Kite Pharma, Sanofi and Takeda: Honoraria; Celgene, EngMab, Roche, Sanofi, Takeda: Research Funding. San-Miguel: AbbVie, Amgen, Bristol-Myers Squibb, Celgene, GlaxoSmithKline, Janssen, Karyopharm, Merck Sharpe & Dohme, Novartis, Regeneron, Roche, Sanofi, SecuraBio, Takeda: Consultancy, Other: Advisory board. Prósper: Oryzon: Honoraria; Janssen: Honoraria; BMS-Celgene: Honoraria, Research Funding.

Abstract: Acute myeloid leukemia (AML) is a hematologic neoplasm characterized by a remarkable phenotypic and genomic heterogeneity. The recent characterization of genomic subtypes of AML based on large sequencing studies has provided the rationale for the development of targeted therapies based on the presence of specific genomic abnormalities. However, long term survival particularly in older patients remains a unmet medicalneed. Additionally, recent studies using RNA interference (RNAi) libraries have determined the existence of genes that are essential for the survival of multiple cancer cells. Understanding the effect of genomic alterations (mutations, deletions, translocations) on gene essentiality could favor the development of targeted therapies for specific subgroups of AML patients. However, current statistical methods such as the Benjamini-Hochberg (BH) procedure have shown limitations for controlling the false discovery rate (FDR) and have suboptimal sensitivity (recall of true positives) because the P-value correction does not include any prior information of individual tests. For this reason, in this study we developed a new large-scale statistical algorithm, which combine the RNAi libraries (more than 17.000 genes) data with mutational profiles, to identify gene essentialities associated with specific genomic mutations in order to explore this approach in AML. We adapted the Independent Hypothesis Weighting (IHW) procedure to the problem of identifying mutations as surrogate markers of gene essentiality, by using the gene mutation state in each cell line as prior information of a IHW problem. This approach was tested in 19 tumor subtypes, of the Cancer Cell Line Encyclopedia (CCLE) showing that it recalls new discoveries that cannot be identified with standard procedures in 17 out of 19 tumors, including the identification of up to 1,000 discoveries in tumor types in which BH recalls no discovery. These results demonstrated the accuracy of the IHW-based approach to identify gene mutations as surrogate markers of gene essentiality in the future. Once validated, we applied this computational model to the15 AMLcell lines of CCLE. The number of discoveries with an FDR of 20% increases from 2 (using the traditional BH correction), to 38 using our procedure, showing NRAS as the top mutation biomarker in the ranking. Interestingly, the algorithm identified one essential gene (NRAS) for NRAS mutated (NRAS-mut) and another essential gene (PTPN11) for NRAS wild type (NRAS-wt) AML cells, covering all samples of AMLs. To validate this hypothesis, we examined the effect of two different specific siRNAs for each gene (siPTPN11 and siNRAS) on cell proliferation of four AML cell lines: two lines with NRAS-mut (HL-60 and OCIAML3) and two with NRAS-wt (MV4-11 and HEL). Downregulation of NRAS expression significantly decreases the cell proliferation only in the 2 NRAS-mutated AML cell lines. Whereas the inhibition of PTPN11expression produced an equivalent effect, but specifically in the 2 NRAS-wt AML cell lines (Figure 1). These results confirmed our predictions and showed the essential role of NRAS or PTNPN11 in AML cell lines either with NRAS mutated or wild type, respectively. These results demonstrate that the application of our algorithm in the context of specific gene mutation not only may allow identification of directed therapies based on the mutation but can also define new gene essentialities amenable for targeted therapies providing new therapeutic strategies in patients with AML and potentially in other tumors. Disclosures Paiva: Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche and Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Janssen, Sanofi and Takeda: Consultancy. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria.