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The 1,287 genes targeted by the shRNA of the leukemia library.

Standard hematologic parameters in SIRT5-/- mice and SIRT5+/+ littermates.

Plasmids.

Patient sample information.

Antibodies.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.Significance:Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML.See related commentary by Li and Melnick, p. 198.

All supplemental figures and legends.

Oligonucleotide sequences.

Ranked list of 1,287 genes from shRNA library screen in primary AML cells. The 1,287 genes assessed with an shRNA library screen were sorted by the second highest percentile fold change present in 2 shRNA and across 2 samples, with the 34 genes showing a fold change in the top 2 percent in more than 2 samples listed first.

Supplementary Table 1

Supplementary Figures 1-6 and Supplementary Methods

Supplementary Table 6

The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor:macrophage ratio, was recapitulated in spleen by partial macrophage depletion, and was overcome by multiple, high-dose alkylating agents. Cyclophosphamide induced endoplasmic reticulum (ER) stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGFA, massive macrophage infiltration, and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGFA blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a “super-phagocytic” subset that expressed CD36/FCGR4. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance.Significance:mAbs are effective against only a small subset of cancers. Herein, we recapitulate compartment-specific antibody resistance and define an ER stress–dependent mechanism induced by high-dose alkylating agents that promotes phagocytosis of opsonized tumor cells. This approach induces synergistic effects with mAbs and merits testing across additional tumor types.See related commentary by Duval and De Palma, p. 834.This article is highlighted in the In This Issue feature, p. 813

Supplementary Table 3

Supplementary Table 2

Supplementary Table 5

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML. Significance: Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML. See related commentary by Li and Melnick, p. 198.

Recent studies have implicated DNA polymerases θ (Pol θ) and β (Pol β) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol θ, Pol δ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol δ promotes Alt-NHEJ in human cells at DSBs, including translocations.

The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor:macrophage ratio, was recapitulated in spleen by partial macrophage depletion, and was overcome by multiple, high-dose alkylating agents. Cyclophosphamide induced endoplasmic reticulum (ER) stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGFA, massive macrophage infiltration, and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGFA blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a “super-phagocytic” subset that expressed CD36/FCGR4. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance. Significance: mAbs are effective against only a small subset of cancers. Herein, we recapitulate compartment-specific antibody resistance and define an ER stress–dependent mechanism induced by high-dose alkylating agents that promotes phagocytosis of opsonized tumor cells. This approach induces synergistic effects with mAbs and merits testing across additional tumor types. See related commentary by Duval and De Palma, p. 834. This article is highlighted in the In This Issue feature, p. 813

Chimeric antigen receptor (CAR) T cells have emerged as a novel form of treatment of patients with B-cell malignancies. In particular, anti-CD19 CAR T-cell therapy has effected impressive clinical responses in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, not all patients respond, and relapse with antigen loss has been observed in all patient subsets. Here, we report on the design and optimization of a novel CAR directed to the surface antigen CD37, which is expressed in B-cell non-Hodgkin lymphomas, in chronic lymphocytic leukemia, and in some cases of cutaneous and peripheral T-cell lymphomas. We found that CAR-37 T cells demonstrated antigen-specific activation, cytokine production, and cytotoxic activity in models of B- and T-cell lymphomas in vitro and in vivo, including patient-derived xenografts. Taken together, these results are the first showing that T cells expressing anti-CD37 CAR have substantial activity against 2 different lymphoid lineages, without evidence of significant T-cell fratricide. Furthermore, anti-CD37 CARs were readily combined with anti-CD19 CARs to generate dual-specific CAR T cells capable of recognizing CD19 and CD37 alone or in combination. Our findings indicate that CD37-CAR T cells represent a novel therapeutic agent for the treatment of patients with CD37-expressing lymphoid malignancies.

T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models., T- and NK-cell lymphomas (TCL) are a group of lymphoid malignancies characterized by poor prognosis, but the absence of appropriate pre-clinical models has hampered the development of effective therapies. Here the authors establish several pre-clinical models and identify vulnerabilities that could be further exploited to treat patients afflicted by these diseases.

The oncogenic BCR-ABL1 tyrosine kinase is the driver of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). Tyrosine kinase inhibitors (TKIs) targeting ABL kinase are generally effective, but subsets of patients treated with single-agent TKIs develop resistance due to mutations in BCR-ABL1 that impair TKI binding. We have previously reported that BCR-ABL1 compound mutants (exhibiting two mutations within the same BCR-ABL molecule) that include the T315I gatekeeper mutation confer a high degree of resistance to all clinical ABL TKIs used as single agents, including ponatinib and the allosteric inhibitor asciminib. However, combining asciminib with ponatinib provides an effective strategy for overcoming compound mutation-based resistance (Eide et al. Cancer Cell 2019). As the clinical utility of ponatinib is limited by cardiovascular toxicity, including arterial occlusive events (AOEs), we decided to search for alternative molecules for use in combination with asciminib. To identify functional ponatinib analogs, we performed Quantum Similarity Modeling (QSM) on the reported crystal structure of T315I mutant ABL1 kinase in complex with nilotinib and asciminib (5MO4) (Wylie et al. Nature 2017) to search for other molecules. Compared to conventional computational modeling, QSM identifies novel classes of structurally distinct compounds that are comparable on a quantum level by precisely defining their interaction with the target. Affinity inferred by close complementarity with the shared ligand-protein surface in the region of the surveyed binding site is mapped, using multiple weak local associations. Our in silico QSM platform combines quantum methods with machine learning to investigate extensive chemical spaces. We screened several million compounds against BCR-ABL1 and identified 51 potential candidates predicted effectively to block T315I mutant BCR-ABL1 when combined with asciminib. To prioritize potent and non-toxic drug combinations for further development against compound mutants, we initially profiled all 51 compounds for their efficacy against Ba/F3 BCR-ABL T315I cells, alone and in combination with asciminib (1 nM). Of 51 compounds, LY3009120, a pan-RAF inhibitor that is currently in phase I clinical development for advanced solid malignancies (Sullivan et al. Mol Cancer Ther 2020), showed strong activity against BCR-ABL T315I when combined with asciminib. These data provided proof of principle for the QSM approach. We next tested the efficacy of all 51 candidates ± asciminib against Ba/F3 cells harboring T315I-inclusive BCR-ABL1 compound mutants, including Y253H/T315I, E255V/T315I, H396R/T315I, G250E/T315I, and T315L as the most resistant mutants. Neither single agent showed any effect. However, LY3009120 strongly inhibited BCR-ABL1 compound mutants when combined with asciminib. No toxicity was observed against Ba/F3 parental cells, confirming that the effects of the combinations are mediated by inhibition of BCR-ABL1. Synergy quantification of the dose-response matrix for the LY3009120/asciminib combination using the Zero Interaction Potency model demonstrated highly synergistic interactions (Synergy score > 10) between the two inhibitors. To directly assess the binding affinity of LY3009120 to the ABL1 kinase domain, we used the cell-based NanoBRET intracellular ABL1 kinase assay on HEK-293 cells expressing luciferase-tagged ABL1. The NanoBRET assay uses energy transfer to quantify the affinity of test compounds by competitive displacement of a cell-permeable fluorescent tracer that is reversibly bound to an ABL1-luciferase fusion protein. We found that LY3009120 competes off the fluorescent tracer at a low micromolar range (EC 50 = 0.75 μM), confirming direct binding of LY3009120 to the kinase domain of ABL1. We hypothesize that the binding of LY3009120 to the ABL1 kinase domain induces a conformational change that re-establishes asciminib binding to the myristoyl binding pocket, allowing for synergy. Studies to quantify the binding affinity of LY3009120 and asciminib to BCR-ABL1 mutants are underway, and data will be presented. In summary, our findings validate QSM as a novel in silico approach to identify TKI combinations. Combining LY3009120 with asciminib may be an effective, low-risk strategy to target BCR-ABL1 compound mutants in patients with clinical TKI resistance. Disclosures Deininger: SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Research Funding; Fusion Pharma, Medscape, DisperSol: Consultancy; Novartis: Consultancy, Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding.

Introduction: Acute T cell lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and young adults that frequently becomes treatment-refractory and relapses. The Notch1 pathway is a key oncogenic driver in T-ALL and is aberrantly activated in more than 50% of the cases. Despite promising pre-clinical data using gamma secretase inhibitors such as DBZ to target NOTCH1, resistance is rapidly occurring in vivo. As molecular heterogeneity has been linked to treatment escape, we focused our study on defining transcriptional cell states driving resistance to NOTCH inhibition and understanding their relation to mitochondrial priming. Methods: 5 primary T-ALLs harboring NOTCH activating mutations were engrafted in NSG (NOD-scidIL2Rgnull) mice. Upon reaching ~ 10% of human CD45+ positive leukemic blasts in the peripheral blood, randomized groups of 8 mice per primary T-ALL were treated with DBZ (Dipenzazepine; 10 μM/kg every other day through tail vein) or vehicle (VEH). 3 mice per group were sacrificed after one week of treatment to assess short-term effect of DBZ, while the remaining 5 mice were weekly monitored for disease progression, leukemic blasts were collected from lymphoid organs and overall survival was determined. Full-length transcriptome analysis of 3188 blasts present in the blood of 20 sensitive and 22 refractory mice was performed by Smart-Seq2. Based on scRNA features, 'scVelo' and 'CytoTRACE' were used to identify developmental potential and differentiation trajectories. Cell fate and transcriptional regulatory networks were defined and reconstructed using 'SCENIC'. Assessment of mitochondrial priming as measured by BH3 profiling was used to identify anti-apoptotic vulnerabilities present in these PDX models. Results: Upon DBZ, short or long-term disease control was observed in two strains, while rapid resistance occurred in three strains, thus establishing two sensitive and three refractories to NOTCH inhibition PDX models. Immunohistochemical analysis showed decreased expression of active NOTCH1 in spleen biopsies of all strains, validating the efficacy of DBZ and suggesting a mechanism of resistance independent of ICN1. Single cell transcriptional profiling showed enrichment of immature hematopoietic signatures and co-expression of lymphoid and myeloid progenitor programs in refractory models. Interestingly, pre-existing cells harboring refractory-like transcriptional circuits within the untreated sensitive population were identified. Upon treatment, despite increased differentiation in all models, lineage promiscuity was maintained in refractory strains, suggesting that cellular plasticity mediates treatment escape. Next, we characterized cell states driving treatment refraction. RNA velocity projections identified two distinct immature states differing in cell cycle and oncogenic signaling. Clustering of untreated, sensitive leukemic cells in immature state imply that aberrant lineage commitment can predict response to NOTCH inhibition in vivo. These observations were further confirmed by differentiation state analysis, where prior to treatment, high developmental potential was correlated to treatment escape. Surprisingly, in addition to early lineage differentiation drivers such as BCL11A, state-specific regulons analysis associated immature states with BCLAF1 a transcriptional regulator of apoptosis. We postulated that these transcriptional circuits lead to differential apoptotic priming, therefore the dependence on individual anti-apoptotic proteins was evaluated. Mitochondrial priming at baseline revealed BCL-2 dependence in sensitive strains whereas MCL1-dependence was observed in refractory ones. Upon DBZ treatment, while dependency profiles in refractory strains remained unchanged, a functional switch from BCL-2 to MCL1-dependency occurred in sensitive models. Conclusion: Our results suggest that response to NOTCH inhibition is predetermined by cell maturity states and their associated transcriptional circuits responsible for differential sensitivity to apoptotic priming via BCL2 and MCL1. These data suggest that combining BH3 and lineage commitment profiling may predict drug responses in vivo. Moreover, our findings highlight the importance of targeting co-existing cell states to overcome transcriptional heterogeneity as a driver of treatment escape. Disclosures Letai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Weinstock: Daiichi Sankyo: Consultancy, Research Funding; Verastem: Research Funding; Abcuro: Research Funding; Bantam: Consultancy; ASELL: Consultancy; SecuraBio: Consultancy; AstraZeneca: Consultancy; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.

Standard of care for AML includes chemotherapy and stem cell transplant, with 5-year survival rates Citation Format: Dongqing Yan, Anca Franzini, Anthony D. Pomicter, Brayden J. Halverson, Orlando Antelope, Clinton C. Mason, Jonathan M. Ahmann, Anna V. Senina, Courtney L. L. Jones, Matthew S. Zabriskie, Hein Than, Michael J. Xiao, Alexandria van Scoyk, Ami B. Patel, William L. L. Heaton, Shawn C. Owen, Joshua L. Andersen, Christina M. Egbert, Julie A. Reisz, Angelo D'Alessandro, James E. Cox, Kevin C. Gantz, Hannah M. Redwine, Siddharth M. Iyer, Jamshid S. Khorashad, Nima Rajabi, Christian A. Olsen, Thomas O'Hare, Michael W. Deininger. A critical role for SIRT5 in acute myeloid leukemia metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB109.

There is a pressing need for more effective therapies to treat patients with T-cell lymphomas (TCLs), including first-line approaches that increase the response rate to cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP) chemotherapy. We characterized the mitochondrial apoptosis pathway in cell lines and patient-derived xenograft (PDX) models of TCL and assessed the in vitro efficacy of BH3 mimetics, including the BCL2 inhibitor venetoclax, the BCL2/BCL-xL inhibitor navitoclax, and the novel MCL1 inhibitor AZD5991. The abundance of antiapoptotic BCL2 family members based on immunoblotting or RNA transcript levels correlated poorly with the activity of BH3 mimetics. In contrast, the functional approach BH3 profiling reliably predicted sensitivity to BH3 mimetics in vitro and in vivo. We used BH3 profiling to select TCL PDX that were dependent on MCL1. Mice xenografted with these PDX and treated with AZD5991 had markedly improved survival. The combination of AZD5991 and CHOP achieved synergy based on survival improvement beyond a mathematical “sum of benefits” model. Thus, MCL1 inhibition is a promising strategy as both a single agent and in combination with chemotherapy for patients with TCL and functional dependence on MCL1.

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classical‐ and alternative-nonhomologous end joining factors (NHEJ). We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. In contrast to c-NHEJ factors, we show here that Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class switch recombination in primary B cells and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting Parp1 may suppress DSB processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

Compartment-specific resistance to cancer therapy remains poorly understood. We utilized orthotopic xenografts of human double-hit lymphoma (DHL) to interrogate responses across involved sites. We identified resistance to multiple chemotherapies and the anti-CD52 antibody Alemtuzumab within the bone marrow (BM) that depended on extensive lymphoma involvement and impaired antibody-dependent cellular phagocytosis. This resistance was overcome by high doses of alkylating agents, including cyclophosphamide (CTX), which exhibited >80-fold in vivo synergy with Alemtuzumab. CTX induced ER stress in BM DHL cells leading to ATF4- mediated paracrine secretion of VEGF-A and massive macrophage infiltration. Macrophages from DHL-engrafted, CTX-treated mice had increased phagocytic capacity for lymphoma cells that was reversed by VEGF-A blockade and required SYK phosphorylation. A subset of these macrophages, defined by surface CD36/FcgRIV and a distinct transcriptional state, were “superphagocytic.” Together, our findings define a unique mechanism through which high-dose alkylating agents can overcome therapy-resistant niches by ER stress-induced activation of phagocytosis.

Introduction: Acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and adolescents that is associated with high rates of treatment failure and early relapse. T-ALL patients frequently harbor NOTCH1 activating mutations as the driving oncogene in this disease. A multitude of strategies preventing NOTCH1 cleavage and activation, such as Gamma-secretase inhibitors (GSIs) have been developed. Despite promising pre-clinical data, the rapid development of Notch1 inhibitor resistance in early clinical trials, prevented the translation of these inhibitors into the clinical setting. In previous work, our group demonstrated that T-ALL resistant to NOTCH1 inhibition carry altered epigenetic states conferring unique dependency on epigenetic modifiers, such as BRD4. The goal of this study was to study enhancer rewiring in Notch1 inhibitor resistant T-ALL in vivo and its relationship to apoptotic priming. Methods: After reaching 5% of circulating leukemic blasts, five established T-ALL PDX models with aberrant NOTCH1 expression were divided into to two treatment groups each (8 mice per group). 1 group received the Notch inhibitor DBZ (Dibenzazepine; 10 μM/kg intraperitoneal every other day) and the other group was treated with vehicle. Short-term effect of DBZ in vivo was assessed after 1 week of treatment, when 3 mice per group were sacrificed and leukemic blasts were isolated from spleen and bone marrow. The remaining 5 mice were monitored for disease burden (by flow cytometry staining for human CD45+) and followed for survival. After reaching moribund state, animals were sacrificed, spleens and bone marrows were collected and prepared for further analyses. To assess DBZ efficacy in vivo, the presence of active NOTCH1 (ICN1) in spleen and bone marrow was analyzed by Immunohistochemistry analysis (IHC). Enhancer landscapes were identified by chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) for Histone 3 Lysine 27 acetylation (H3K27ac). A custom computational pipeline that incorporates algorithms for demultiplexing, alignment, normalization, peak calling, and computation of signal intensities within peaks was used to call differential peaks and intersect with RNA-sequencing results. BH3 profiling was performed on leukemic blasts isolated from spleen to measure overall mitochondrial priming and to identify anti-apoptotic dependencies. Results: In four out of five T-ALL PDX models, IHC analysis of spleen and bone marrow demonstrated a drastic downregulation of active NOTCH1 upon DBZ treatment, validating the efficacy of the used inhibitor. Weak ICN1 staining that remained unchanged upon DBZ treatment, was observed in 1 of the models, resulting in the exclusion of this strain from further functional analysis. Survival analysis of the four T-ALL PDX models expressing ICN1, revealed the presence of two Notch inhibitor sensitive and two refractory strains. The latter strains developed DBZ resistance rapidly after starting treatment (less than 10 days). One sensitive strain eventually developed resistance, while the second showed long-term disease control. Transcriptional profiling (bulk RNA-seq) of Notch inhibitor refractory strains versus sensitive identified the intrinsic apoptotic pathway as one of the most deferentially deregulated GSEA signatures. H3K27ac ChIPseq analysis at pretreatment (baseline), showed increased signal intensity of H3K27ac peaks at BCL2 and MCL1 enhancers in the refractory strains compared to sensitive. Upon DBZ treatment, while the enhancer state in refractory T-ALL remained unchanged, in the sensitive strains the signal intensity of H3K27ac peaks within the BCL2 and MCL1 loci decreased. Mitochondrial BH3 profiling at baseline demonstrated BCL-2 dependency (measured via BAD peptide) in sensitive strains and MCL-1 dependency (measured via MS1 peptide) in refractory strains. Upon DBZ treatment, sensitive strains showed a decrease in BCL-2 dependency and compensatory switch to MCL1-dependency, while dependency profile remained unchanged in refractory T-ALL. Conclusions: Our results suggest that enhancer rewiring near anti-apoptotic genes is critical for Notch inhibitor resistance. Combining BH3 profiling with enhancer profiling may allow to predict drug responses in vivo and may contribute to the identification of novel therapeutic targets for combination therapy in resistant disease. Disclosures Letai: Zeno Pharmaceuticals, Vivid Bioscience, Flash Therapeutics, Dialectic Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder or Advisory Board member; AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding. Weinstock:Celgene: Research Funding. Lohr:T2 Biosystems: Honoraria; Celgene: Research Funding.

BCR-ABL1 tyrosine kinase inhibitors (TKIs) are the cornerstone of treatment in chronic myeloid leukemia. Although there are now four TKIs approved for use in the front-line setting, acquired TKI resistance via secondary kinase domain mutations remains a problem for patients. K0706 is a novel BCR-ABL1 TKI currently under clinical investigation with structural elements similar to those of ponatinib and dasatinib. In this article, we functionally characterize the anti-leukemic activity of K0706 using cell proliferation assays in conjunction with drug resistance screening. We provide details from molecular modeling to support our in vitro findings and additionally describe our limited clinical experience with this drug in two patients treated on trial. We demonstrate that although K0706 retains efficacy against a large spectrum of clinically relevant mutations, it does not appear to have activity against BCR-ABL1T315I. Early trial experience suggests excellent tolerability, which may positively affect the place of K0706 within the ever-expanding chronic myeloid leukemia treatment paradigm.

Outcomes for patients with aggressive T-cell lymphomas (TCLs) remain dismal. Monoclonal antibodies (mAbs) that inhibit engagement of the "don't eat me" signal CD47 with SIRPα on myeloid cells may induce phagocytosis and have exhibited promise in multiple cancers. We dissected the role of CD47 blockade in opsonization and phagocytosis-induction through Fc-Fc gamma receptor (FcγR) interactions. CD47 was expressed at higher levels on 22 TCL cell lines by flow cytometry compared to quiescent T cells from 10 healthy donors (mean fluorescent intensity, 176.9 versus 69.85; Welch t-test p=0.002). We determined tumor cell-specific protein expression of CD47 by calculation of H scores in a tissue microarray (TMA) of 68 primary TCLs. The common subtypes (PTCL-NOS (n=16), AITL (n=19), and ALCL (n=12)) each had heterogeneous CD47 expression that was not associated with progression-free or overall survival. We utilized B6H12, an anti-CD47 mAb to assess apoptosis, antibody-dependent cell mediated-cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) of TCL cells (HuT-78, HH, Myla, SU-PM2, KIJK, MAC-2A, SMZ1 and K-299) and no increase was observed except for ADCC by murine FcγRIII-expressing engineered Jurkat cells. Upon addition of B6H12 or SRF231, a fully human IgG4 mAb against CD47 that is currently in a Phase I clinical trial (NCT03512340), phagocytosis of CFSE+ TCL cells with human monocyte-derived macrophages (hMDMs) and murine bone-marrow derived macrophages (mBMDMs) was increased. In contrast, induction of phagocytosis of resting T cells from healthy donors was tested using B6H12 and no increase was observed (22.7 and 19.5%). A flow cytometry-based receptor occupancy assay demonstrated a linear correlation between receptor binding and phagocytosis induction in Jurkat T-cell lymphoblastic leukemia cells. hMDMs failed to induce phagocytosis of TCL cells with F(ab')2 portion of B6H12 suggesting complete dependence on Fc receptors. In contrast, mBMDM-mediated phagocytosis of TCL cells with the F(ab')2 portion of B6H12 (mean, 21.5%) was increased compared to negative control but reduced compared to full length (mean, 33.4%), suggesting both Fc-dependent and Fc-independent mechanisms. mBMDMs from FcγR knockout mice demonstrated a statistically significant decrease in phagocytosis (compared to wild-type mBMDMs) of Jurkat, HuT-78, MAC2A and K-299 cells with B6H12, confirming a role for Fc-FcγR engagement. Phagocytosis of tumor cells during CD47 blockade may depend on the ability of SLAMF7 to interact with the integrin Mac-1 (Chen J, Nature 2017). Phagocytosis assays of TCL cells with mBMDMs from Mac-1 knockout mice with full length and F(ab')2 B6H12 demonstrated a reduction in phagocytosis using Mac-1 knockout macrophages (compared to wild-type) only with full-length B6H12. Thus, the Mac-1 contribution to phagocytosis induced by B6H12 in this context is Fc-dependent. We tested SRF231 in vivo using patient-derived xenograft (PDX) models of systemic hepatosplenic (HSTL), transformed cutaneous, and NKTCL. After 10 days, SRF231-treated mice had remarkable reductions in tumor burden compared to untreated mice, across multiple compartments. SRF231 treatment resulted in prolonged survival despite a short course of therapy (Figure 1). Bone marrow from xenografted mice, collected at day 11 post-treatment with SRF231, had significantly increased cytokines known to promote monocyte recruitment. Ex-vivo phagocytosis assays using mBMDMs and hMDMs, enhanced engulfment of HSTL, T-PLL and anaplastic large cell-lymphoma PDX harvested cells upon exposure to SRF231. Finally, we observed that in our HSTL PDX model, depletion of macrophages with clodronate completely abrogated the antitumor efficacy of SRF231 while depletion of neutrophils with anti-Ly6G Ab had no effect, highlighting macrophages as the essential effectors of phagocytosis in this context. In summary, monoclonal antibodies that disrupt CD47-SIRPα have striking in vitro and in vivo preclinical efficacy but require interactions with human FcγR on macrophages to maximize efficacy. Disclosures Peluso: Surface Oncology: Employment, Equity Ownership. O'Connor:Surface Oncology: Employment, Equity Ownership. Paterson:Surface Oncology: Employment, Equity Ownership. Holland:Surface Oncology: Employment, Equity Ownership. Weinstock:Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties; Travera: Equity Ownership; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Genentech/Roche, Monsanto: Consultancy; Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy.