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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.

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.

Introduction: Multiple myeloma (MM) is a genetically complex disease with extensive clonal heterogeneity. Substantial genomic instability in MM is illustrated by extensive copy number variations (CNVs) that can be detected in almost every MM patient. The molecular basis of this genomic instability in MM is not clear. Linker histones are dynamic components of chromatin and mutations in these molecules are present in ~6% of MM patients. Two of the linker histone super-family, HIST1H1Eand HIST1H1Care the most frequently mutated members of the family in MM and their mutations mostly occur in a clonal fashion. Interestingly, it has been reported in human cell lines and in other species that linker histone loss affects DNA damage/repair pathways and leads to transcription-replication conflicts. Based on these data we hypothesized that mutation or genomic loss of linker histones affects the genome stability of MM cells. To test this hypothesis, we developed an experimental system using CRISPR/Cas9 genome editing to generate MM linker histone-deficient cells. Low-pass whole genome sequencing (LPWGS), immunoblotting and immunofluorescent experiments were performed for genomic, molecular and functional characterization. We found that HIST1H1E,HIST1H1Cand H1FXwere the most abundantly expressed members of the linker histone family in primary myeloma cells and that myeloma cells have the highest dependency on HIST1H1Eand HIST1H1Cwhen compared to all other cancer cell lines derived from other tissues. We used OPM2 and U266 myeloma cell lines and generated knock-out variants of HIST1H1E, HIST1H1Cand H1FXlinker histones by inserting a biallelic stop codon, followed by generation of individual single-cell clones that were used as replicates. We first asked if linker histone deficient cells preserve genome stability. To address this question, we performed low pass whole genome sequencing and found more copy number abnormalities in linker histone deficient myeloma cells, when compared to wild-type cells. Moreover, linker histone deficient cells showed increased DNA damage as indicated by higher frequency of nuclear foci that were positive for damage dependent phosphorylation of the histone variant H2AX ( γH2AX). This was associated with an increased frequency of micronuclei in linker histones deficient cells, suggesting defects in mitotic fidelity and in genome stability. These micronuclei were positive for γH2AX by microscopic staining, indicative of DNA damage. We then asked if the DNA damage in micronuclei is due to defective and asynchronous DNA replication when the myeloma cells are exposed to etoposide, a topoisomerase inhibitor that induces DNA replication stress and double-strand DNA breaks (DSBs). Etoposide treatment of myeloma cells caused DNA replication stress, as measured by immunofluorescent staining of micronuclei for Replication Protein A (RPA). Conclusions: Our results demonstrate that loss of linker histones is associated with increased copy number abnormalities, extensive DNA damage and increased frequency of micronuclei, most likely as a consequence of replication stress. These data provide a potential mechanism of how chromatin structure dynamics preserve genome stability in myeloma cells. Disclosures Lohr: Celgene: Research Funding; T2 Biosystems: Honoraria.

Introduction: Despite recent advances in the treatment of multiple myeloma, responses may be short-lived and therapeutic resistance develops almost invariably. Non-genetic cellular plasticity and dedifferentiation have recently emerged as a basis for therapeutic resistance in cancer as cells acquire transcriptional states which no longer depend on the drug target. Therefore, a better understanding of plasticity and adaptive state changes in myeloma cells is critical to develop effective therapeutic approaches that can overcome drug resistance. Here we show that cellular plasticity, though frequently invoked as a basis for therapeutic resistance in cancer, can also lead to new therapeutic opportunities. Methods: To define transcriptional states in myeloma at a single cell level, we performed fluorescence activated cell sorting and full-length single-cell RNA sequencing. We assayed a total 6000 CD38+CD138+ plasma cells and CD45+ immune cells from the bone marrow of 8 patients with relapsed and refractory multiple myeloma (RRMM) before and after immuno-modulatory treatment on a clinical trial with elotuzumab, pomalidomide, bortezomib and dexamethasone (Elo-PVD; NCT02718833) and 2 healthy donors. Surface expression of selected markers was validated by flow cytometry. Results: Assessing pre-treatment samples, we discovered that the transcriptional states of single myeloma cells are highly distinct between individual patients, despite the presence of the same established genomic classifiers, such as t(11;14). Furthermore, distinct transcriptional states co-exist within individual patients, indicating there is substantial inter- and intra-individual heterogeneity. Transcriptional states diverge from normal plasma cells towards more immature cells, of the B lymphoid lineage, suggesting a substantial cellular plasticity. Notably, we detected co-expression of myeloid and lymphoid developmental programs in the same single cells. Interestingly, these altered differentiation states were associated with up-regulation of potential immunotherapeutic targets, such as CD20, CD19, and CD33, indicating that this plasticity may result in novel therapeutic vulnerabilities. To define gene-regulatory relationships, we identified a shared core regulatory network present in malignant and normal plasma cells with the active transcription factors XBP1, ATF4, and CREB3, suggesting that myeloma cells retain lineage-specific regulons. However, we further identified patient-specific regulons not detected in any of the mature immune cell populations assayed, such as TEAD4, ELF3 and SNAI1, illustrating an aberrant and promiscuous activation of transcriptional regulators in myeloma cells. Consistent with this finding, we observed an increased number of expressed genes in myeloma cells compared to normal plasma cells as well as an increase in single cell transcriptional entropy, measures that have been linked to cell potency in normal development and cancer. Comparison of pre- and post-treatment samples interestingly revealed a further increase in transcriptional diversity and signatures associated with stemness and developmental potential following treatment. Conclusions: In conclusion, we find that higher transcriptional diversity and activation of alternate gene regulatory programs facilitate the emergence of altered transcriptional states. Interestingly, these altered states are associated with up-regulation of putative immune-therapeutic targets in myeloma cells, thus providing novel therapeutic vulnerabilities. Disclosures Lipe: amgen: Research Funding; Celgene: Consultancy; amgen: Consultancy. O'Donnell:Celgene: Consultancy; Takeda: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Amgen: Consultancy. Munshi:Celgene: Consultancy; Amgen: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Takeda: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Anderson:Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder . Lohr:T2 Biosystems: Honoraria; Celgene: Research Funding. OffLabel Disclosure: Samples for ancillary research were obtained in the context of a phase II clinical trial evaluating Elotuzumab, pomalidomide, bortezomib, dexamethasone The combination of elo-PVD is off label.

Introduction: Treatment of multiple myeloma (MM) has improved over the last decade. Although long-term survival is noted in some patients, emergence of resistant disease still prevents cures. The objective of this study was to define "liquid biopsy" parameters that identify patients who do not benefit from a particular treatment before relapse becomes evident by serological markers. Having such parameters at hand will potentially inform changes in treatment. Methods: Here, we apply low-pass whole genome sequencing to profile a uniform cohort of 45 relapsed and refractory MM (RRMM) patients who have been treated in a multicenter phase II trial evaluating the combination of elotuzumab, pomalidomide, bortezomib and dexamethasone (elo-PVd; NCT02718833). Peripheral blood plasma samples were acquired for circulating tumor DNA (ctDNA) evaluation at four different timepoints (screening, cycle 3 day 1 (C3D1), cycle 5 day 1 and end of treatment). The concentration, relative fraction and copy number profile of myeloma-derived ctDNA were determined across all timepoints. Results: At the time of this preliminary analysis, 17 patients (35%) continue on treatment whereas 28 patients (58%) have developed progressive disease (PD). Our data suggest that ctDNA levels at screening and C3D1 strongly correlate with progression-free survival (PFS). Patients with available follow-up samples (n=40) were stratified according to ctDNA levels at C3D1 of treatment. Patients with a residual ctDNA level Conclusions: These data indicate that "liquid biopsy" evaluation of ctDNA may refine prognostication and provide added predictive value over serological markers alone. While in the large majority of cases ctDNA has excellent concordance with M protein and SFLC for monitoring of MM disease progression, ctDNA may identify patients where relapse is imminent before it can be detected by serological parameters. This approach may therefore complement our framework for treatment decisions. Notably, this approach is highly scalable, cost-efficient and provides information about the clonal evolution of MM without the need for a bone marrow biopsy. Disclosures Yee: Adaptive: Consultancy; Amgen: Consultancy, Honoraria; Takeda: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding. Lipe:amgen: Research Funding; Celgene: Consultancy; amgen: Consultancy. O'Donnell:Sanofi: Consultancy; Amgen: Consultancy; Celgene: Consultancy; BMS: Consultancy; Takeda: Consultancy. Munshi:Abbvie: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Takeda: Consultancy; Oncopep: Consultancy. Richardson:Bristol-Myers Squibb: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Anderson:OncoPep: Other: Scientific founder ; Sanofi-Aventis: Other: Advisory Board; Janssen: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder ; Gilead Sciences: Other: Advisory Board. Raje:Celgene Corporation: Consultancy; Amgen Inc.: Consultancy; Merck: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy. Lohr:T2 Biosystems: Honoraria; Celgene: Research Funding. OffLabel Disclosure: This abstract reports on the quadruple regimen elotuzumab, pomalidomide, bortezomib and dexamethasone which is not yet approved for the treatment of multiple myeloma in the United States.

Introduction: Constant clonal evolution and outgrowth of clones that harbor resistance mutations are likely explanations for the emergence of drug-resistant disease in multiple myeloma (MM). Activating mutations in BRAF, KRAS and NRAS have been suggested as potential therapeutic targets. In this study, we investigate resistance to BRAF inhibition in the context of BRAF-mutated MM which accounts for about 5-12% of all patients with relapsed/ refractory MM. Methods: Resistance to dabrafenib was modeled in vitro in the BRAF-mutated MM cell lines (MMCL) U266 (K601Nmut) and DP6 (BRAFV600Emut). Low-pass whole genome sequencing (LPWGS), RNA sequencing, ChIP sequencing and immunoblotting were performed for genomic, transcriptomic, epigenomic and molecular characterization. Functional validation was performed by genome editing using CRISPR/Cas9 technology. Results: Modeling of dabrafenib resistance in vitro revealed an initial decline of cell numbers, followed by a plateau phase and a gradual outgrowth of resistant cells after ~80 days of treatment. As expected, exposure of BRAFmut MMCL to dabrafenib led to initial downregulation of pERK and pMEK. At later timepoints, upregulation of pERK and pMEK was observed, suggesting that re-activation of the ERK/MEK pathway ultimately overcomes BRAF inhibition. This outgrowth was associated with highly distinct copy number profiles in each resistant clone, implying clonal selection with outgrowth of genetically resistant clones as one mechanism of drug resistance in MM. Additionally, we found that BRAF inhibition of BRAFmut MMCL promotes changes of the transcriptional circuitry that appears independent of clonal outgrowth of genetically resistant clones. These transcriptional changes were highly homogenous, occurred as early as 7-14 days after starting treatment and were associated with de-differentiation of MMCL into a more immature B lymphocytic phenotype. This phenotype was associated with greater mRNA expression of CD19 and CD81, as well as upregulation of the B-lymphocyte activation antigen B7-2 (CD86) and PI3K pathway genes. We next investigated if targeting the PI3K pathway and B7.2 can be exploited for effective killing of dabrafenib-resistant BRAFmut MM cells. Studies for the PI3Kδ inhibitor idelalisib in dabrafenib-persistent MMCL revealed higher sensitivity as compared to dabrafenib-naïve controls. Genome editing suggests a survival advantage for CD86WT as compared to CD86KO MMCL. Conclusions: Our data suggest that resistance to BRAF inhibition in vitro is mediated by two distinct mechanisms: 1) clonal outgrowth of genetically distinct resistant clones, and 2) transcriptional rewiring that leads to activation of alternative signaling pathways. The latter is characterized by changes in cellular differentiation and upregulation of PI3K and CD28/CD86 signaling. These concepts may provide a framework for revealing therapeutic vulnerabilities and to overcome drug resistance mediated by genetic heterogeneity in MM. Disclosures Munshi: Oncopep: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy; Amgen: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Yee:Karyopharm: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Adaptive: Consultancy. Raje:Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Merck: Consultancy. Lohr:Celgene: Research Funding; T2 Biosystems: Honoraria. OffLabel Disclosure: Dabrafenib is a potent inhibitor of BRAF mutated at codon 600 (BRAFV600). Here we explored the efficacy of dabrafenib a preclinical model of multiple myeloma cell lines with BRAFV600E and BRAFK601N mutations.