Abstract: Background Deferasirox (DFX) is widely employed as iron chelation therapy (ICT) in the current clinical practice in patients with myelodysplastic syndromes (MDS) and chronic transfusion need. The efficacy of DFX in reducing median ferritin levels in different cohorts of these patients has been reported in many trials, but the lack of worldwide accepted criteria of individual response to ICT makes it difficult to appreciate its clinical relevance for any single patient. Aim To highlight the clinical impact of ICT with DFX in a large real-life cohort of MDS patients, based on different individual ferritin variation during treatment. Methods A retrospective cohort of 301 consecutive MDS patients [M/F 187/114 (62.1%/37.9%)] of any age followed in 20 hematological Centers in Italy was analyzed: the main features at diagnosis are reported in the Table 1. Individual response to ICT was categorized as complete response (CR) (ferritin levels 〈 500 ng/ml), partial response (PR) (ferritin levels 〈 1,000 ng/ml), ferritin improvement (FI) (ferritin reduction 〉 50% of baseline value but with levels 〉 1,000 ng/ml), ferritin stability (FS) (ferritin levels without changes from baseline during ICT) or no ferritin response (NR) (ferritin levels increasing during ICT). Results ICT was started after a median period from diagnosis and from transfusion start of 21.0 months [interquartile range (IQR) 8.9 - 44.3] and 11.3 months (IQR 7.1 - 21.7), respectively, with a median burden of red cell transfusions at baseline of 22 units (IQR 14 - 35). The main features of patients at baseline of ICT are reported in the Table 1. Starting DFX dose was 〈 10 mg/Kg in 38 patients (12.7%), 10 - 14 mg/Kg in 110 patients (36.6%), 15 - 19 mg/Kg in 57 patients (18.9%) and ≥ 20 mg/Kg in 96 patients (31.9%). As to individual response, 4 patients (1.3%) were too early for evaluation ( 〈 6 months of DFX treatment): in addition, 16 patients (5.4%) discontinued ICT behind 6 months from start, due to early toxicity (10 patients, 7 for gastro-intestinal toxicity and 3 for skin toxicity) or other reasons (unrelated death, AML evolution, transplant procedure). Among the remaining 281 patients, 37 (12.3%) achieved a CR, 65 (21.6%) a PR, 23 (7.6%) a FI, 112 (37.2%) a FS and 44 (14.6%) a NR. Five-year overall survival (OS) of the whole cohort from ICT start was 43.9% (95%CI 37.1 - 50.7). Five-year OS according to ICT response was 74.8% (95%CI 57.9 - 91.7) in patients with CR, 51.7% (95%CI 37.6 - 65.8) in patients with PR, 50.6% (95%CI 28.2 - 73.0) in patients with FI, 38.6% (95%CI 27.0 - 50.2) in patients with FS and 21.1% (95%CI 5.2 - 37.0) in patients with NR (p=0.002) (Figure 1). Five-year cumulative incidence of AML evolution (CIE) of the whole cohort from ICT start was 27.1% (95%CI 20.3 - 33.9). Five-year CIE according to ICT response was 7.6% (95%CI 0 - 18.0) in patients with CR, 27.0% (95%CI 13.0 - 40.5) in patients with PR, 38.3% (95%CI 15.5 - 61.7) in patients with FI, 20.8% (95%CI 10.4 - 31.2) in patients with FS and 57.7% (95%CI 31.9 - 83.5) in patients with NR (p=0.003) (Figure 2). Notably, no statistical difference was observed for both OS and CIE among patients achieving PR, FI or FS. Conclusions Present data highlight the clinical relevance of individual response in MDS patients receiving ICT with DFX. In particular, achievement of CR seemed related to a better OS and a lower CIE, while patients with NR had a significant worst OS and CIE: furthermore, the achievement of stable ferritin levels was associated with similar OS and CIE than PR and FI and thus should be considered as a response. Disclosures Latagliata: Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Oliva:Novartis: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Honoraria, Speakers Bureau; Apellis: Consultancy. Pilo:Novartis: Other: Advisory board. Molteni:Celgene: Membership on an entity's Board of Directors or advisory committees. Balleari:Celgene: Membership on an entity's Board of Directors or advisory committees. Breccia:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; Celgene: Honoraria. Foà:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Finelli:Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding, Speakers Bureau.

Abstract: The FDA-approval of potent targeted therapies has led to great changes in the therapeutic landscape of chronic lymphocytic leukemia (CLL). As a key example, venetoclax, a first-in-class BCL-2 inhibitor, leads to response in about 80% of patients with relapsed/refractory (R/R) CLL. Disease progression on venetoclax, however, has been increasingly observed, and better biologic understanding of resistance mechanisms to this agent is needed. To systematically discover the potential mechanisms of resistance to venetoclax, we performed both genome-scale loss- (LOF) and gain-of-function (GOF) genetic modifier screens in the BCL-2-driven OCI-Ly1 lymphoma cell line using CRISPR-Cas9 sgRNA and ORF libraries, respectively. Significant hits from both screens included the BCL-2 family: the LOF screen with pro-apoptotic genes (PMAIP1, BAX, BAK1, BCL-2L11) and the GOF screen with anti-apoptotic genes (BCL2L1, BCL2L2, BCL2, MCL1). In addition, the LOF screen uncovered genes in pathways relevant to lymphoid biology (i.e, NFKBIA) and lymphoid transcription factors and modulators (IKZF5, ID3, EP300, NFIA). The GOF screen also uncovered components of the energy-stress sensor PKA/AMPK signaling pathways (ADIPOQ, PRKAR2B, PRKAA2) and regulators of mitochondrial metabolism. In parallel, we performed an integrated transcriptome, whole proteome and functional characterization of an OCI-Ly1 cell line rendered resistant to venetoclax (OCI-Ly1-R) from the parental cell line (OCI-Ly1-S). RNA-seq and spectrometry-based proteomics revealed coordinated dysregulation of transcripts and proteins in the resistant line originating from genes critical to cellular metabolism, cell cycle, B-cell biology and autophagy. Of the transcripts and proteins significantly associated with the resistant cell line, only MCL-1 overlapped with the gene hits from the genome-scale screens. Treatment of the OCI-Ly-R cells with the MCL-1 inhibitor S63845 synergized with venetoclax. Given the dysregulation of proteins critical to metabolism in both the GOF screen and in OCI-Ly1-R cells, we also evaluated the role of metabolic reprogramming in venetoclax resistance. We first assessed mitochondrial respiration by measuring the oxygen consumption rate. Compared to OCI-Ly-S cells, OCI-Ly1-R cells demonstrated markedly higher respiration levels, suggesting a state of higher oxidative phosphorylation (OXPHOS). More directly, we measured oxygen consumption following venetoclax exposure. Consistent with impairment of OXPHOS by venetoclax, we observed both an immediate decrease in oxygen consumption and an immediate burst of glycolysis following venetoclax in the OCI-Ly1-S cells, but not in the OCI-Ly1-R cells. In line with these findings, the AMPK inhibitor dorsomorphin and mitochondrial electron transport chain (mETC) inhibitors synergized with venetoclax in OCI-Ly1-S cells. Transcriptome related to ID3 (identified as one of the LOF screen targets) was characterized in isogenic ID3-knockout OCI-Ly1 lines. It revealed PRKAR2B overexpression as a key effect, suggesting a role for ID3, and perhaps of other lymphoid transcription factors in regulating metabolic reprogramming associated with resistance. Indeed, exposure of ID3 knockout lines to mETC inhibitors overcame resistance to venetoclax. To determine if there is a genetic basis for the drug resistance seen in OCI-Ly1-R cells, we compared whole-exome sequencing (WES) results of DNA isolated from the OCI-Ly1-R and OCI-Ly1-S cell lines. A clear region was amplified on chromosome 1q23, which includes MCL1 and PRKAB2 (the regulatory subunit of AMPK). Similarly, a WES-based analysis of paired CLL DNA samples isolated from 6 R/R CLL patients just prior to venetoclax initiation and at time of progression on venetoclax was performed. We did not identify any non-silent somatic single nucleotide in BCL2 or its family members at baseline or at progression, despite marked clonal shifts in all patients. We confirmed the presence of the amp(1q23) as acquired at relapse after venetoclax in 3 out of 6 patients. Our study reveals that venetoclax resistance implicates changes not only for outer mitochondrial membrane (MCL-1 expression) but also for inner membrane (oxydative metabolism). Such mitochondrial reprogramming represents a new vulnerability that can potentially be exploited through combinatorial therapy with metabolic modulators to overcome resistance. Disclosures Guieze: abbvie: Honoraria; janssen: Honoraria; gilead: Honoraria. Thompson:Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Adaptive Biotechnologies: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Davids:Merck: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; MEI Pharma: Consultancy, Research Funding; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Surface Oncology: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Brown:Sun Pharmaceutical Industries: Research Funding; Abbvie: Consultancy; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy; Janssen: Consultancy; Sunesis: Consultancy; Roche/Genentech: Consultancy; Verastem: Consultancy, Research Funding; Boehringer: Consultancy; Loxo: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees; Invectys: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead: Consultancy, Research Funding; Pharmacyclics: Consultancy; Genentech: Consultancy. Wierda:AbbVie, Inc: Research Funding; Genentech: Research Funding. Letai:AstraZeneca: Consultancy, Other: Lab research report; Novartis: Consultancy, Other: Lab research report; AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Vivid Biosciences: Equity Ownership. Wu:Neon Therapeutics: Equity Ownership.

Abstract: Alterations in signaling pathways are critical to the pathogenesis of acute myeloid leukemia (AML) and are often driven by aberrant kinases. We previously identified spleen tyrosine kinase (SYK), a non-receptor, cytoplasmic tyrosine kinase, as a druggable target in AML and a critical regulator of FLT3, the most commonly mutated receptor tyrosine kinase in this disease. SYK was initially described as having an important role in B-cell development. More recent studies showed a broader relevance of SYK in hematopoietic signaling. Multiple orally bioavailable SYK inhibitors, including entospletinib (GS-9973) and TAK-659, are currently in Phase I/II clinical trials in patients with AML, with promising results. Targeted therapy, however, is typically associated with the emergence of resistance, and combination therapy is almost always needed for a durable therapeutic response. The aim of our study, therefore, was to identify resistance mechanisms to SYK inhibition in AML and to identify new synergistic drug combinations to overcome them. We conducted a genome-scale, pooled open reading frame (ORF) library screen to investigate the mechanisms of drug resistance to SYK inhibition. We screened the AML cell lines MV4;11 and MOLM-14, which are both highly sensitive to SYK inhibitors, with entospletinib after transduction with the ORF library. Expression of KRAS and HRAS ORFs, as well as other RAS signaling pathway-related proteins, emerged as conferring the strongest resistance to entospletinib in both cell lines. Upon overexpression of NRAS and KRAS in MV4;11 and MOLM-14 cells, both wildtype for RAS, we observed i) upregulation of phospho ERK 1/2, indicating activated RAS signaling, and ii) we confirmed markedly reduced sensitivity to entospletinib. We next screened 12 AML cell lines for sensitivity to entospletinib and found that the presence of activating mutations of the RAS signaling pathway was associated with innate resistance to entospletinib. To further study the mechanism of resistance to SYK inhibition, we generated entospletinib resistant cell lines by exposing MV4;11 cells to increasing concentrations of the drug for five months. In these cells, the entospletinib IC50 shifted from 500 nM to 5 μM, and these cells were also cross resistant to the SYK inhibitor PRT062607. RNA-sequencing showed increased expression of NRAS, KRAS and related pathway genes in entospletinib resistant compared to sensitive parental cells. Accordingly, gene set enrichment analysis revealed significant upregulation of RAS signaling-related gene sets. These findings were confirmed by qPCR and western blot analysis, supporting that the activation of the RAS/MAPK signaling pathway also confers acquired resistance to SYK inhibition in AML. Because RAS mutant AML cells have been reported to be sensitive to MEK inhibition, we next tested whether MEK inhibition using PD0325901 would recapture response to SYK inhibition in RAS/MAPK activated AML cells. Indeed, entospletinib was synergistic with PD0325901 in the MV4;11 entospletinib resistant cells. Moreover, entospletinib was synergistic with PD0325901 in five different AML cell lines, including AML cell lines with RAS pathway mutations. Additionally, this drug combination was synergistic when tested in a patient sample with NRAS G12D, KRAS G12D and PTPN11 G60V mutations. In conclusion, we found that RAS pathway activation results in resistance to entospletinib in AML that may be overcome with the combination of a SYK inhibitor with a MEK inhibitor. Disclosures No relevant conflicts of interest to declare.

Abstract: Metabolic reprogramming contributes to tumor development and sustains cancer cell proliferation. Like other cancers, acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival, has altered metabolic features, providing new possibilities for AML treatment. Since the niche can reshape the metabolic properties of cancer cells, it is critical to validate AML metabolic vulnerabilities in a proper microenvironment. To this end, we optimized a protocol for CRISPR screening in orthotopic xenograft AML models, including patient-derived-xenograft (PDX) models tractable for CRISPR-editing, to enable the systematic evaluation of the physiological relevance of top AML dependencies. We performed in vivo screens in MV4-11 and U937 cell lines and a PDX model, which converged to reveal the sodium/myo-inositol cotransporter SLC5A3 as a top-ranked in vivo gene target. We have validated the SLC5A3 dependency in additional AML cell line and PDX models; SLC5A3 deletion consistently induced apoptotic cell death in all AML models tested. We also observed cell-context dependent alterations of cell cycle and differentiation. In addition, using a PDX model with a doxycycline-inducible CRISPR system, we have confirmed that induction of SLC5A3 knockout post transplantation significantly represses AML progression and prolonged mouse survival. Next, we investigated whether the growth defect caused by SLC5A3 knockout results from the myo-inositol deficiency. Augmentation of myo-inositol concentration in the standard culture medium completely rescued the proliferation of SLC5A3-knockout cells. In accordance, depletion of myo-inositol from the culture medium largely impeded the growth of parental AML cells, causing similar phenotypes as SLC5A3-deletion, with cell-context dependent alterations of cell cycle and induction of apoptotic cell death. Together, these data reveal that myo-inositol is a critical metabolite for AML. Since a subset of AML cell lines were not dependent on SLC5A3 based on a genome-scale CRISPR screen dataset (DepMap), we explored the potential biomarkers associated with SLC5A3 essentiality in AML. Intriguingly, the low expression of Inositol-3-phosphate synthase 1 (ISYNA1) predicted a strong SLC5A3 dependency in AML cell lines. In addition to importing myo-inositol from the extracellular fluid, cells can also synthesize myo-inositol de novo from glucose 6-phosphate, and ISYNA1 encodes the rate-limiting enzyme in this myo-inositol biosynthesis pathway. We confirmed low expression of ISYNA1 protein in AML cells sensitive to SLC5A3 deletion, and importantly, overexpression of ISYNA1 can completely relieve the SLC5A3 dependency. Moreover, knockout of ISYNA1 in an ISYNA1-high cell line M07e exacerbated the growth defect associated with SLC5A3 deletion. Altogether, these results strongly demonstrate that SLC5A3 becomes essential in AML cells with insufficient myo-inositol biosynthesis capacity to support AML proliferation. Finally, we have investigated the clinical features associated with low ISYNA1 expression in genomic datasets of primary AML samples, including TCGA and Beat AML, to postulate the patient population that can benefit from a SLC5A3-directed therapy. Interestingly, low ISYNA1 expression is associated with FAB M4 and M5 AML subtypes. In accordance, a monocyte lineage gene signature is enriched in ISYNA1-low samples. In addition, AML samples with low ISYNA1 expression tend to have IDH2 or DNMT3 mutations. Collectively, our study demonstrated that SLC5A3 is a strong metabolic dependency in AML, and targeting SLC5A3 can provide a therapeutic opportunity for a subset of monocytic AML. Disclosures Dharia: Genentech: Current Employment. Piccioni: Merck Research Laboratories: Current Employment. Stegmaier: AstraZeneca: Consultancy; KronosBio: Consultancy; Bristol Myers Squibb: Consultancy; Auron Therapeutics: Consultancy, Current equity holder in publicly-traded company; Novartis: Research Funding.

Abstract: First-generation, large-scale functional genomic screens have revealed hundreds of potential genetic vulnerabilities in acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because these large-scale genetic screens were primarily performed in vitro in established AML cell lines, their translational relevance has been debated. Therefore, we established a protocol for CRISPR screening in orthotopic xenograft models of human AML, including patient-derived-xenograft (PDX) models that are tractable for CRISPR-editing. We first defined experimental conditions necessary for an optimal in vivo screen via barcoding experiments. We determined that sub-lethal irradiation was necessary for improved barcode representation in bone marrow and to reduce mouse-to-mouse variation. Moreover, it was critical to combine samples from multiple mice to achieve complete in vivo library representation. Next, using the Broad DepMap and other publicly available functional genomic screen data, we identified 200 genes that were stronger dependencies in AML cell lines compared to all other cancer types screened. Using this list, we created a secondary library and performed parallel in vivo and in vitro screens using the MV4-11 and U937 cell lines and a PDX model. In vitro and in vivo hits were surprisingly well correlated, although a modest number of targets did not score well in vivo. Notably, dependencies identified across AML cell line models were strongly recapitulated in the PDX model, validating the application of AML cell lines for dependency discovery. Our in vivo screens nominated the mitochondria-localized RING-type ubiquitin E3 ligase MARCH5 as a potential therapeutic target in AML. Using CRISPR, we first validated this in vitro dependency on MARCH5 and determined that MARCH5 is a critical guardian to prevent apoptosis in AML. MARCH5 depletion activates the canonical mitochondrial apoptosis pathway in a BAX/BAK1-dependent manner. Multiple genome-wide screens revealed that a dependency on MARCH5 is strongly correlated with a dependency on MCL1, but not other anti-apoptotic BCL2-family members, across the AML cell lines in the screen. As observed with MCL1 inhibition, MARCH5 depletion sensitized AML cells to venetoclax, a BCL2-specific inhibitor FDA-approved in combination with a hypomethylating agent for the treatment of older adults with AML. Importantly, MARCH5 depletion diminished the venetoclax resistance induced by MCL1 overexpression but not that caused by BCLXL overexpression. Altogether, these results suggest that MARCH5 is required for maintaining MCL1 activity specifically. Since there are no small molecule inhibitors directed against MARCH5, we deployed a dTAG system as an approximation of pharmacological inhibition. This approach uses a hetero-bifunctional small molecule that binds the FKBP12 F36V-fused MARCH5 and the E3 ligase VHL, leading to the ubiquitination and proteasome-mediated degradation of the fusion protein. dTAG-MARCH5 cells were established via deleting endogenous MARCH5 by CRISPR and expressing exogenous FKBP-tagged MARCH5 protein. MARCH5 degradation with the dTAG molecule dTAG V-1 markedly impaired cell growth in vitro. Additionally, we demonstrated the utility of dTAG system in vivo using a PDX model. The combination treatment of dTAG V-1 and venetoclax elicited a much stronger anti-leukemic effect compared to the treatment with only venetoclax or dTAG V-1, further highlighting MARCH5 as a promising synergistic target for enhancing the efficacy of venetoclax in AML. Taken together, our in vivo screening approach, coupled with CRISPR-competent PDX models and dTAG-directed protein degradation, constitute a useful platform for prioritizing AML targets emerging from in vitro screens to serve as the starting point for therapy development. Disclosures Dharia: Genentech: Current Employment. Piccioni: Merck Research Laboratories: Current Employment. Stegmaier: Bristol Myers Squibb: Consultancy; KronosBio: Consultancy; AstraZeneca: Consultancy; Auron Therapeutics: Consultancy, Current equity holder in publicly-traded company; Novartis: Research Funding.