Dissecting Richter's Syndrome in a Multiplexed CRISPR-Based Mouse Model Reveals Key Changes in MYC, Interferon and BCR Signaling Underlying Transformation

Richter's syndrome (RS) represents one of the foremost challenges in CLL management, and its pathogenesis remains largely undefined. We recently leveraged CRISPR-Cas9 in vivo gene editing to develop mouse models of RS by engineering multiplexed loss-of-function lesions typical of CLL (Atm, Trp53, Chd2, Birc3, Mga, Samhd1) in early stem and progenitor cells [Lineage - Sca-1 + c-kit + (LSK)] from MDR-Cd19Cas9 donor mice. These animals express Cas9-GFP in a B-cell restricted fashion and the leukemogenic MDR lesion, which mimics del(13q) when the sgRNA-transduced LSK cells are transplanted in CD45.1 immunocompetent recipients. Through these methods, we observed not only development of CLL, but also transformation into RS, and even captured a stage where CLL and RS were co-existing in the same animal (CLL/RS). We hypothesized that the molecular events underlying RS development would be markedly distinct from those of CLL and performed transcriptome analysis of FACS-sorted CLL and/or RS cells (5 CLL, 4 CLL/RS, 10 RS) and normal B cell controls from 4 age-matched wild type MDR-Cd19Cas9 mice. We identified a unique transcriptional profile of RS (ANOVA, FDR<0.1), characterized by upregulation of pathways involved in cell survival and proliferation (E2F/MYC targets, G2-M checkpoint, mitotic spindle). In contrast, genes involved in interferon gamma response, JAK-STAT and BCR signaling were predominantly downregulated. We asked whether these oncogenic circuitries would be recapitulated in human RS. By correlating the differentially expressed genes in murine RS with those of 7 human RS cases (compared to matched CLL), we identified similar pathway dysregulations with >100 commonly altered genes including upregulated cell cycle regulators (CDK1, CCNA2) and downregulated signaling adapters (ITPKB, MAP3K9). To further dissect gene regulatory networks driving transformation in the mouse, we profiled one CLL and one RS case by single cell ATAC sequencing (scATAC-seq). Consistent with the RNA-seq profiles, we detected increased chromatin accessibility of MYC-family associated transcription factor motifs (MAX, MYCN), and reduced accessibility of the pro-inflammatory STAT2 motif in RS (-log10adjP>50). Functionally, decreased interferon gamma responses were confirmed by the reduced ability of RS cells to phosphorylate STAT1 and STAT3 at 5' and 15' after IFN-gamma stimulation, compared to CLL and normal B cells (Western Blot). To define the genetic landscape underlying these changes, we performed whole genome sequencing analysis, and identified loss of chr12 and chr16 as recurrent events in RS (6/8 cases) and CLL/RS (2/2), but not in CLL cases (0/5). Among the genes encoded by these chromosomes, we identified several epigenetic drivers (Dnmt3a, Crebbp, Setd3/4), MAP kinase family members (Map4k5, Mapk1), cytoskeletal regulators (Hcls1, Rhoj), and interferon family receptors (Ifnar1/2, Ifngr2), suggesting that broad epigenetic modifications together with loss of BCR and interferon signaling molecules represent key events of transforming disease. RS cases were also characterized by a significantly higher number of full chromosome amplifications or deletions (median=6; range: 2-9), as compared to CLL or CLL/RS (1; 0-5, P=0.0008), consistent with the high degree of genomic instability observed in human disease. Finally, we asked whether the observed changes would impact RS therapeutic vulnerabilities, and exposed 15 primary murine RS splenocyte samples to 20 drugs in vitro for 24 hours, followed by CellTiter-Glo assessment of cellular viability. We observed strong sensitivity to the BRD4 inhibitor JQ1 and the mTOR inhibitor everolimus (both reported to interfere with MYC signaling, P<0.0001), and to CDK inhibitors (e.g. the CDK4/6 inhibitor palbociclib, P=0.0007), modest activity of the JAK1/2 inhibitor ruxolitinib (P=0.05), and minimal, if any, response to ibrutinib, venetoclax and fludarabine. In conclusion, we define the evolutionary trajectories and therapeutic vulnerabilities of RS in a mouse model, with unique transcriptional, genetic, and epigenetic features, indicative of broad changes in MYC, IFN and BCR signaling pathways and remarkable similarities with human disease. In-depth analyses of BCR signaling and in vivo treatment studies are underway and will refine mechanistic insights into the biology of RS. Disclosures Davids: Surface Oncology: Research Funding; Eli Lilly and Company: Consultancy; Genentech: Consultancy, Research Funding; Takeda: Consultancy; MEI Pharma: Consultancy; Janssen: Consultancy; Verastem: Consultancy, Research Funding; Ascentage Pharma: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Merck: Consultancy; Adaptive Biotechnologies: Consultancy; Research to Practice: Consultancy; AbbVie: Consultancy; MEI Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Celgene: Consultancy; BeiGene: Consultancy. Letai: Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board; Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Wu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company.

Maturity State and MCL-1 Dependence Predetermines Response to NOTCH1 Inhibition in T-ALL

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.

Identification of Novel Targets Based on Splicing Alterations for Undruggable RAS/CDK Signaling Cascade in Multiple Myeloma

Background: RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis. Targeting these pathways represents a novel therapeutic strategy in MM. Our ongoing studies (>420 patients) demonstrate that aberrantly spliced transcript expressions can predict MM patient survival outcomes better than gene expression alone, indicating a significant role of splicing mechanism in MM pathophysiology. These studies also identified intron retentions as the predominant recurrent alterations (~32% of spliced genes were retained introns) in MM. We evaluated splicing alterations associated with pathway-level responses after RAS/CDK inhibition in order to identify and validate novel molecular targets. Methods/results: MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to inhibit RAS and CDK pathways. Our studies demonstrated strong synergistic (IC<0.5) MM cytotoxicity triggered by this combination treatment, which triggered dose-dependent manner G0/G1 phase growth arrest. We assessed early death cascade in MM cells after Ei+Ci treatment, and demonstrated significant priming to selective peptides BIM, BAD, and MS1 or HRK, suggesting dependency on BCL2 and MCL1 or on BCL-XL proteins. Our studies showed that Ei+Ci treatment induced inhibition of key target molecules in Erk1/2 and CDK4/6 signaling including c-myc, p-RSK, p-S6, p-RB, and E2F1, suggesting on-target activity of Ei and Ci. Patient MM cells co-cultured with or without autologous BM stromal cells remain equally sensitive to Ei+Ci, suggesting that this combination can overcome the protective effects of the MM BM milieu. Moreover, our in vivo study demonstrated a significant (P=0.0004) MM burden decrease in Ei+Ci-treated mice. We evaluated the effect of Ei+Ci treatment on target gene expression in BM cells isolated from flushed femurs of treated animals with Ei, Ci or Ei+Ci, and observed downregulation of Erk1/2-CDK4/6-dependent gene signature. Therefore, we suggest that these inhibitors selectively target Erk1/2, CDK4/6 and their downstream substrates both in vitro/vivo. We next evaluated aberrantly spliced transcript expression in MM cells, with/without Erk1/2 knockdown (KD) or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. This observation was further supported by principal component analyses: upregulation in response to Erk1/2 KD and downregulation due to treatment with Ei+Ci were considered spliced gene-signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complex and are involved in intron retention processes, a marker of malignant transformation. We compared signature-gene expressions from 558 MM patient samples to the signature-genes in plasma cells from normal donors and observed significant (p<2e-11) upregulation of genes with progression from MGUS to sMM, and, also to overt MM . SNRPB overexpression is associated with shorter overall patient survival (p<0.01), while RAVER1 is linked with poor outcomes. SNRPB proteins are also overexpressed in MM cells. Our studies evaluating SNRPB effects on RNA splicing showed both upregulation of transcripts with full intron retention and transcripts with cryptic stop codons utilizing intronic sequences causing their partial retention. We evaluated RAVER1 and SNRPB expression in BM cells from animals treated with Ei and Ci alone or in combination. We observed significant downregulation of RAVER1/SNRPB (p=0.001) in BM samples obtained from animals treated with Ei+Ci. We observed decreased intron retention events in genes in treated samples, consistent with our in vitro analyses in MM cell lines and patient samples. Thus, RAVER1/SNRPB overexpression contributes to the aberrant transcriptome splicing associated with RAS/CDK cascade in MM. Conclusions: Our studies 1) show an association between RNA processing and RAS-CDK pathways in MM, 2) identify a core splicing protein, SNRPB/RAVER1, as a novel target for modulating this cascade, and 3) suggest that targeting spliceosome complexes represents a promising therapy in MM. 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. Anderson: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: 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; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

B Cell-Restricted Depletion of Dnmt3a Activates Notch Signaling and Causes Chronic Lymphocytic Leukemia

Previous studies have revealed a critical role of methylation deregulation in the onset and progression of chronic lymphocytic leukemia (CLL). In mammalian cells, DNA methylation is dynamically established by the DNA methyltransferase 3 (DNMT3) family of de novo methyltransferases DNMT3A. Although mutations of DNMT3A are rarely observed in CLL, our RNA-sequencing (RNA-seq) analysis of 107 human CLLs show that low DNMT3A expression is associated with more aggressive disease, and supports a driving role of DNMT3A loss in CLL. To test this hypothesis, we generated a conditional knock-out mouse model with B cell-restricted deletion of Dnmt3a. Homozygous Dnmt3a depletion in B cells resulted in the development of CD5+ B cell leukemia mimicking human CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yielded a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haplo-insufficient tumor suppressor. Given the known role of Dnmt3a as a de novo methyltransferase, we first evaluated the impact of Dnmt3a depletion on global DNA methylation in non-leukemic CD5+ B cells isolated from the peritoneal cavity by cell sorting (i.e. B1a cells) using reduced representation bisulfite sequencing (RRBS). We identified a set of differentially methylated regions (DMRs) (difference>0.2), mostly hypomethylated, in Dnmt3afl/fl versus WT B1a cells (473 hypomethylated, 19 hypermethylated). Genes with dysregulated methylation were highly enriched in pathways involved in immune response (e.g., Interferon-α signaling, JAK/STAT3 signaling) and proliferation (Wnt Signaling and Notch signaling). Given the prominent hypomethylation changes observed in Dnmt3a depleted B1a cells, we investigated whether these would lead to altered gene transcript expression. Using RNA-seq, we detected 460 downregulated and 168 upregulated genes in the Dnmt3afl/fl B1a cells compared to WT B1a cells (FDR<0.05, fold change >2). Consistent with the methylation data, differentially expressed genes were likewise enriched for JAK/STAT3 signaling, Wnt Signaling and Notch signaling, supporting a direct influence of dysregulated methylation on downstream signaling cascades. We investigated the changes in methylomes of the CLL cells arising from the Dnmt3afl/fl animals. Compared to WT B1a cells, Dnmt3afl/fl CLL cells generated 1335 hypomethylated and 2369 hypermethylated DMRs in. Focusing on genes that were hypomethylated in CLL compared to WT B1a cells, we found that these were highly enriched for several oncogenic signaling pathways including Notch signaling and Wnt Signaling, consistent with the pre-leukemia findings. RNA-seq analysis identified more upregulated (n=2801) than downregulated (n=1244) genes in CLL cells compared to WT B1a cells (FDR<0.05, FC>2), supporting a role of Dnmt3a depletion in transcriptional activation. We observed a general upregulation of Notch signaling genes and the downstream Notch targets, implicating Notch activation in this CLL mouse model. Of note, we showed Dnmt3a-depleted CLL cells to be highly sensitive to Notch inhibitor DAPT both in vitro and in a transplantable mouse model. Consistently, primary human CLL cells with low DNMT3A expression were more sensitive to DAPT than those with higher DNMT3A expression (P=0.005, Spearman correlation), despite similar sensitivity to ibrutinib and venetoclax. Together, our results have confirmed the causal role of Dnmt3a downregulation in CLL generation. We provide evidence in support of the interaction between Dnmt3a-dependent methylation changes and hyperactivation of Notch signaling in transcriptional reprogramming and transformation of B1a cells into CLL. Furthermore, we demonstrate differential sensitivity of DNMT3A high and low expressing primary CLLs to Notch inhibition, indicative of consistent dependencies of human and murine CLLs. Thus, the Dnmt3a models provides a unique opportunity for the study of non-mutational Notch activation, and a useful platform for the study of Notch-signaling targeted therapeutics. Disclosures Kipps: Abbott Laboratories: Consultancy, Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Pharmacyclics LLC, an Abbvie Company: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Genentech, Inc.: Honoraria, Research Funding, Speakers Bureau; Gilead Sciences, Inc.: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; MedImmune Inc: Research Funding; Moores Cancer Center: Current Employment; Oncternal Therapeutics, Inc.: Current holder of stock options in a privately-held company, Other: Stock or other ownership, Patents & Royalties: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory., Research Funding; AbbVie: Consultancy, Honoraria, Other, Speakers Bureau; DAVAOncology: Consultancy, Honoraria, Other; DAVA Pharmaceuticals: Speakers Bureau; Bionest Partner: Other; Celgene: Consultancy, Honoraria, Other, Research Funding; Genetech: Honoraria, Other; Genentech-Roche: Consultancy; Gilead Sciences: Consultancy, Honoraria, Other, Speakers Bureau; Janssen: Consultancy, Honoraria, Other, Research Funding, Speakers Bureau; Roche: Honoraria, Other; MD Anderson Cancer Center: Research Funding; Velos: Research Funding; CRIM: Research Funding; Indy Hematology Review: Other; TG Therapeutics: Other; Verstem: Other, Speakers Bureau; University of California, San Diego: Current Employment; Pharmacyclics/AbbVie: Honoraria, Research Funding; Breast Cancer Research Foundation: Research Funding; SCOR - The Leukemia and Lymphoma Society: Research Funding; National Cancer Institute/NIH: Honoraria, Research Funding; Genentech/Roche: Honoraria; European Research Initiative on CLL (ERIC): Honoraria. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Letai: Flash Therapeutics: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board. Wu: BioNTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

Effect of Director’s Tunneling on Assets Utilization: Evidence from Corporate Organizations in Nigeria

This study evaluates the effect of director’s tunnelling on asset utilization of companies in consumer goods sector in Nigeria using a panel data collected from annual financial report of thirty listed consumer goods firm in Nigeria between 2011 and 2016. The study was based on ex-post-facto research design and the data collected were analysed using descriptive statistics, correlation analysis and multiple regression. The study finds that the director’s pay and equity holding varies widely among consumer goods firms. Chairman’s pay and director’s equity holding have a statistically significant effect on asset utilization at 5% level. While the director’s pay policy has no statistically significant effect on asset utilization. The finding shows pay, chairman’s pay and director’s equity holding are three major avenues used for tunnelling as they have a significant effect on tunnelling. The study recommends that policymaker should formulate a policy that will reduce the tunnelling tendency of directors and board chairman.