Abstract: Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy. Significance: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101

Abstract: The novel multiple myeloma (MM) cell line LNT1 was established from peripheral blood of a 66-year-old female patient with recurrent plasma cell leukemia of IgG/kappa type. Peripheral blood mononuclear cells from the heparinized peripheral blood separated by Ficoll-Hypaque density gradient centrifugation were cultured in RPMI 1640 medium/20% FBS, 100 U/ml penicillin and 50 μg/ml streptomycin, without additional growth factors. Cells were cultured by replacing ½ of the volume of the culture with fresh medium every 3-4 days for 3 months. In the 4th month, a slow yet sustained growth of cultured cells was noted, and designated LNT1. These cells grow as single cell suspension; IL-6-induced proliferation in a dose-dependent manner with 2 ng/ml of IL-6 inducing maximal DNA synthesis (by [3H]thymidine uptake) and survival (by MTT). In contrast, IGF-1 had no effect. Subsequently, 2 ng/ml of IL-6 was added to cultures and growth now sustained at 7 months. LNT1 was negative for Epstein-Barr virus, as evidenced by PCR using primers specific for EBNA-1. May-Grunwald-Giemsa staining showed that LNT1 cells have a high nucleocytoplasmic ratio with excentrically located nuclei, prominent nucleoli, and a deeply basophilic and vacuolated cytoplasm. Some cells were bi- or multi-nucleated. Surface antigen analysis showed that LNT1 cells express CD138, CD38, HM1.24, CS1, and HLA-A2, but did not express CD19, CD20, CD3, CD16, and CD14. Chromosome analysis at 7 months revealed the karyotype: 43, X, -X, del(1)(p13p22), psu dic(6;1)(q13;p11), del(12)(p11.2), −13, add(19)(q13.4), −22[2] /43, idem, del(2)(q11.2q2?3), der(6)ins(6;?)(q21;?)[7]. DNA fingerprinting was performed by simultaneously amplifying eight short tandem repeat (STR) loci and the amelogenin gene in a multiplex PCR reaction using specific primers to confirm the authentication of cell line. The established LNT1 cell line and the primary tumor showed identical band patterns of these 9 markers and the same immunoglobulin H rearrangement. An unique and identical VH3/IgG fragment was amplified using the appropriate VH family-specific framework region primer in conjunction with CH isotype specific (IgG) primers in DNA samples of both LNT1 cell line and the original tumor. Moreover, identical DNA sequence of VH3/IgG PCR from cell line and primary tumor further confirms clonality. LNT1 cell line also bears a N-RAS G12D mutation, as in the original tumor. Finally, LNT1 cell line is responsive to conventional (Dex, Malphalan) and novel or emerging (bortezomib, lenalidomide, perifosine, AS703026) anti-MM therapies. Therefore, the novel LNT1 cell line will provide a useful model system to study genetics and biology of MM, as well as to evaluate novel therapeutic strategies. Since LNT1 cells highly express HLA-A2, this line also serves a useful model to validate novel tumor-associated antigens for immunotherapy in MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 400.

Abstract: 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 & gt;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 & lt;0.05, fold change & gt;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 & lt;0.05, FC & gt;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.

Abstract: Mouse models represent invaluable tools for the systematic evaluation of cancer drivers, yet models that address the impact of putative genetic drivers of chronic lymphocytic leukemia (CLL) on B cell development and function are largely lacking. To study recurrent loss-of-function (LOF) mutations observed in human CLL, we established a transplant model that can rapidly evaluate genetic lesions. First, we crossed mice carrying B-cell restricted Cre expression (Cd19-cre) with mice carrying conditional Cas9-GFP, to generate a strain expressing B cell-restricted Cas9 (Cd19-Cas9). Next, we optimized methods for in vitro engineering of early stem and progenitor cells (Lin- Sca-1+ c-kit+ [LSK]) from Cd19-Cas9 mice using lentivirus expressing sgRNAs (mCherry+)targeting Atm, Trp53, Chd2, Birc3, Mga, or Samhd1. We chose LSKs because of their high transducibility and long-term repopulating potential. Last, we transplanted the single sgRNA-expressing LSKs into sub-lethally irradiated CD45.1 recipient mice, and then confirmed presence of ~45-85% gene-edited sequences ( & gt;70% carrying frameshift mutations) in edited B cells (GFP+mCherry+) at 2 months post-transplant, by PCR-based targeted deep sequencing and CRISPResso software analysis. We also verified presence of gene alterations (and putative off-target lesions) at the single cell DNA level (targeted sequencing by Tapestri, Mission Bio). We first asked whether presence of the 6 LOFs could impact B cell developmental trajectories in marrow, spleen and peritoneum at 4 months post-transplant, a time point by which B cells are considered to achieve optimal host reconstitution (n=5/group, including a non-targeting control group). No marked changes were observed in mice with Atmindel, Trp53indel, Chd2indel, Birc3indel or Samhd1indel, as analyzed by flow cytometry. Of interest, however, Mgaindel mice were detected to have increased germinal center (B220+CD95+CD38-) and marginal zone (B220+CD21highCD23-) splenic B cells, and also showed increased B1a (CD5+ B220low CD23- CD43+) and decreased B1b (CD5- B220low CD23- CD43+) cells in the peritoneum (p & lt;0.05, ANOVA). These results indicate that the likely negative regulatory role that Mga exerts on MYC networks may directly impact germinal center formation and cell fate determination in B cells. The overall abundance of edited B cells in spleen and blood of each group was higher (overall median: 17.0%; 90%CI 6.7-58.8%) than the non-targeting control (8.4%; 90%CI 1.6-14.2%) at 4 months post-transplant (n=8/group, p & lt;0.05, ANOVA), and abundance of edited cells increased in peripheral bleeds at 4 vs. 2 months (n=8/group, p & lt;0.05, Wilcoxon signed rank test). This suggests that presence of individual alterations can alter pro-survival pathways in mature B cells, through mechanisms that may, at least partly, be shared across LOFs. To address this question, we analyzed the transcriptional profiles of edited B cell splenocytes (n=3/group), and compared them to their non-edited counterparts (GFP+mCherry- splenocytes from the same animal), identifying a total of ~3900 differentially expressed genes among the 6 groups (p & lt;0.05, paired Student's t test). Notably, changes in gene expression were highly concordant across 5 of the 6 groups (Spearman r & gt;0.37 for each of the 10 pairs of 5 groups), with the exception of Mgaindel, consistent with its unique phenotype, observed in developmental studies. Gene ontology analyses using Enrichr confirmed commonalities in pathway dysregulations across the 5 similar groups of mice (p & lt;0.05), such as modulation of Notch signaling in Chd2indel, Samhd1indel, and Birc3indel, serine/glycine metabolism in Atmindel, Trp53indel, and Chd2indel, and oxidative phosphorylation in Atmindel and Samhd1indel. Unique to Mgaindel, we saw enrichment of the GOs for transcriptional mis-regulation in cancer and cellular senescence, both relevant for tumorigenesis and B cell development. In conclusion, we demonstrate that common LOFs typical of patients with CLL lead to increased cellular fitness in B-cell restricted mouse models, while dysregulating pro-survival pathways relevant to B cell development, CLL pathogenesis and more broadly to tumorigenesis. We are currently exploring phenotypic similarities and differences through tailored functional assays, while addressing the relative contribution of each alteration to CLL development in multiplexed edited mouse lines. Disclosures Wang: Mission Bio Inc.: Employment. Jacob:Mission Bio Inc.: Employment. Flynn:Mission Bio Inc.: Employment. Ruff:Mission Bio Inc.: Employment. Jones:Mission Bio Inc.: Employment. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

Abstract: Large-scale DNA methylation analysis of chronic lymphocytic leukemia (CLL) has identified a pervasive genome-wide level of discordance in local methylation state in leukemic cells compared to normal B cells. This is associated with variation in gene expression, increased clonal evolution and poorer clinical outcomes. We hypothesized that locally disordered methylation could lead to dysregulation of gene expression and hence contribute to cancer development and progression. To test this, we have engineered mouse lines with B-cell restricted homozygous or heterozygous knock-out of Dnmt3a by crossing Dnmt3a-floxed mice with CD19-Cre mice. Dnmt3a is a DNA methyltransferase, catalyzing the addition of a methyl group to CpG sequences in the DNA and thereby regulating gene expression. Although DNMT3A mutations are only rarely identified in CLL, RNA sequencing and protein expression analysis reveal dysregulation of DNMT3A. We confirmed partial or complete reduction in Dnmt3a protein levels in B cells from CD19-Cre;Dnmt3a heterozygous (Dnmt3a-het) and CD19-Cre;Dnmt3a homozygous mice (Dnmt3a-hom), respectively. These mice therefore provide a unique opportunity to study B cell restricted changes in locally discordant methylation over time. We first assessed the impact of Dnmt3a deletion on normal B cell development, prior to CLL development, by characterizing splenic B cell of CD19-Cre (control) or Dnmt3a-hom mice. Flow cytometry data using B220, CD21 and CD23 markers to identify B220+CD23+CD21- follicular B cells and B220+CD23+CD21high marginal zone B cells revealed elevated levels of follicular B cells (83.1% vs 87.6%, p=0.008) and reduced levels of marginal zone B cells (9.6% vs 4.1%, p=0.001) in Dnmt3a-hom mice in comparison to control mice (n=3 mice per group). These results indicate that mice with Dnmt3a deletion present with massive changes in their B cells, even prior to overt CLL development. We next monitored both Dnmt3a-het and Dnmt3a-hom cohorts over time for CLL development. We observed that 100% Dnmt3a-hom mice developed CLL-like disease by 7 months (n=23), characterized by CD5+B220+;Igk+ expression and evident within the blood, bone marrow (BM), spleen and peritoneum, suggesting a fundamental role of altered DNMT3A expression in generation of CLL. In comparison, 75% of Dnmt3a-het mice developed CLL-like disease by 18 months (n=12), with similar expansion of CD5+C220+ expansion in the BM and spleen. By RNA-sequencing analysis of normal splenic B cells from CD19-Cre and Dnmt3a-hom mice (n=3 mice, 10 weeks old), we detected substantial changes in gene expression, including 113 upregulated genes and 39 downregulated (p 〈 0.05, FC 〉 2). To explore the development of locally disordered methylation following transformation, CLL cells from Dnmt3a-hom mice (n=3) were subjected to reduced representation bisulfite sequencing (RRBS), a high-throughput technique to analyze genome wide methylation patterns. We found that murine CLL-like cells display locally disordered methylation, which was detected in all genomic features covered by this assay, indicating that disordered methylation is broadly affecting the murine CLL cells' epigenome. Additionally, we identified a set of differentially methylated regions (DMRs) between B cells from CD19-Cre vs CLL cells from Dnmt3a-hom (n = 2,839 DMRs), with a minimum difference of 0.2 and a minimum of 10 CpGs per DMR. Interestingly, gene ontology analysis demonstrated strong association with genes hypermethylated in TCL1 mouse model, linking this model with alternative murine models for CLL. In conclusion, we have studied B cell specific deletion of Dntm3a and showed the development of CLL in 100% of the case in Dnmt3a-hom mice. Our data suggest a fundamental role for Dnmt3a in CLL development through increased locally disordered methylation and changes in associated transcriptional signatures. This mouse model provides an exciting experimental model to undertake functional in vivo studies in order to elucidate the contribution of epigenetic changes on CLL development. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

Abstract: Collective large-scale sequencing efforts have unexpectedly revealed the high frequency of mutations in the splicing factor genes (SF3B1, U2AF1, SRSF2, ZRSR2) in various solid and hematological cancers, suggesting the association of splicing dysregulation with tumorigenesis. Mutations in SF3B1 occur in 5-20% of patients with chronic lymphocytic leukemia (CLL) and are associated with poorer overall survival and chemotherapy resistance. These mutations are restricted to hotspots ( 〉 50% at K700E site) and strongly co-occur with ATM mutations (loss-of-function) and deletion of 11q (ATM minimal deleted region). Numerous studies including ours have demonstrated that somatic alterations in this gene cause RNA splicing dysregulation, however, how this splicing factor mutation alone and in combination with ATM deletion impacts cellular processes and contributes to CLL remains to be fully defined. To this end, we modeled the effects of these combined alterations by crossing mice with conditional knockout of Atm and mice with a conditional knock-in allele of SF3B1 mutation (Sf3b1-K700E). We achieved B cell-restricted expression of heterozygous Sf3b1 mutation and Atm deletion by breeding these mice with CD19-Cre homozygous transgenic mice. Conditional expression of heterozygous Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters B-cell development, and induces a state of cellular senescence. Combined with Atm deletion in B cells led to the overcoming of cellular senescence and the development of clonal CLL cells in elderly mice at low penetrance (6%). These malignant cells could be propagated by in vivo passaging, with detectable disease within 4 weeks following transfer, thus making this mouse line amenable to further drug discovery and biologic investigations. To fully understand the underlying mechanisms of how the combined alterations led to CLL, we performed integrated genome, transcriptome, and proteome analysis using mouse CLL (DM-CLL) cells and B cells with either Sf3b1 mutation or Atm deletion, or with double genetic lesions (DM). Whole-genome sequencing of paired DNA from B cells (or DM-CLL) and non-B cell tissue (kidney) revealed the somatic mutation rate in the CLL cells to be ~0.5 mutations/Mb. Few recurrent mutations were identified among the samples. However, copy number variation analysis of DM-CLL cells revealed recurrent amplifications of chromosomes 15 and 17. RNA-seq analysis revealed that these amplifications were associated with overexpression of 835 of 987 Chr15 and Chr17 genes detected in DM-CLL vs. DM cells. Of note, 146 genes were overexpressed in human CLLs with SF3B1 mutations (DFCI cohort), compared to normal B-cells (p 〈 0.05). Integrated transcriptome and proteome analysis of the DM-CLL cells showed coordinated dysregulation of multiple CLL-associated cellular processes with B-cell receptor (BCR) signaling as the most dramatically downregulated compared to DM cells. Since BCR signaling is a therapeutic target in CLL and has critical roles in B cell biology, we asked how SF3B1 mutation contributes to gene expression of BCR signaling. Through RNA-seq data analysis derived from two independent patient cohorts (DFCI and ICGC), we identified downregulation of BCR gene expression in SF3B1 mutant CLL cells. In line with this, human CLLs harboring SF3B1 mutations exhibit greater sensitivity to in vitro treatment with ibrutinib, and altered response kinetics in vivo to ibrutinib, per analysis of patients with SF3B1 mutations treated with ibrutinib. These studies together highlight a role of SF3B1 mutation in BCR signaling. In summary, we have generated a genetically-engineered murine model that recapitulates human CLL genetics, and presents an informative model to functionally dissect the effects of mutant SF3B1 in a B cell context. Starting from computation-based identification of recurrent co-occurring events in CLL, our study employs murine lines that express genetic alterations in an lineage-specific fashion, utilizes integrated genomics and proteomics approaches to dissect pathways that are fundamental to CLL phenotype, and more importantly, links the dysregulated pathways back to human CLL gene expression data and clinical trials to reveal novel mechanisms underlying therapeutic response. Disclosures Wiestner: Pharmacyclics LLC, an AbbVie Company: Research Funding. Wu:Neon Therapeutics: Equity Ownership.

Abstract: Large-scale cancer sequencing of primary chronic lymphocytic leukemia (CLL) has identified SF3B1, an RNA splicing factor, as one of the most frequently mutated CLL genes. SF3B1 mutations localize to a hotspot ( & gt;50% at K700E site) and highly co-occur with mutations in ATM or deletion of chromosome 11q (minimally deleted region contains ATM). How this splicing factor mutation alone or in cooperation with ATM deletion contributes to CLL remains elusive. Genetically engineered mice are powerful tools in understanding genetic lesions and cancer phenotypes. We therefore generated a mouse line that conditionally expresses heterozygous Sf3b1-K700E mutation. We modeled the effects of the combined alterations by crossing mice with conditional knockout of Atm and mice with Sf3b1-K700E. By breeding these mice with homozygous CD19-Cre transgenic mice, we achieved B cell-restricted expression of heterozygous Sf3b1 mutation and Atm deletion. B cell co-expression of these two mutations in vivo led to clonal expansion of CD19+CD5+ B cells in blood, marrow and spleen in aged mice (18 to 24-month old) at low penetrance. No leukemia cells were found in the Sf3b1-K700E mice (up to 24-month old). The CLL cells from the double mutant mice could be engrafted in both immunocompetent and immunodeficient mice, with detectable disease within 2-4 weeks following transfer, thus making this mouse line amenable to drug discovery and biologic investigations. To investigate how Sf3b1 mutation and Atm deletion synergistically contribute to CLL, we asked if there are RNA and DNA level changes in the double mutant mice with CLL. First, we performed transcriptome sequencing of splenic B cell RNA collected from age-matched mice that either express wild-type, singly mutant alleles of Sf3b1 or Atm, or doubly mutant alleles with or without CLL-like disease. Using the tool JuncBASE, we classified and quantified splice variants associated with the different genetic alterations. Consistent with prior findings in human CLL, we observed that splice variants in Sf3b1-mutant mice alone were highly enriched at 3' splice sites. Sf3b1 and Atm doubly mutant B cells displayed a splicing pattern similar to that in Sf3b1 singly mutant cells. Moreover, we identified unique CLL splice variants in genes (Setdb2, Phf11c) previously shown to be associated with CLL. Next, we examined the mutation rate in DNA derived from splenic B cells collected from mice with a singly mutated allele of Sf3b1 or Atm, or with doubly mutated alleles with and without CLL-like disease through comparison against matched germline DNA from kidney by whole-genome sequencing. We have observed that co-expression of Sf3b1 mutation and deletion of Atm results in a higher mutation rate compared to singly mutant cells. Our analysis has revealed that altered RNA splicing and genomic instability all contribute to CLL leukemogenesis. We will further dissect how the two lesions contribute to CLL functionally using this model. Citation Format: Lili Wang, Jing Sun, Amaro Taylor-Weiner, Jaegil Kim, Zachary J. Cartun, Angela N. Brooks, Donna Neuberg, Mark D. Fleming, Benjamin L. Ebert, Gad Getz, Ruben Carrasco, Catherine J. Wu. Transcriptome and whole-genome sequencing analysis of a novel murine model of chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2449. doi:10.1158/1538-7445.AM2017-2449

Abstract: Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc–expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested (Sf3b1-Atm, Ikzf3, and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling. Significance: Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition.

Abstract: Large-scale DNA sequencing efforts in chronic lymphocytic leukemia (CLL) have identified a broad array of putative cancer drivers arising from somatic mutations in this disease, but functional understanding of the impact of these genetic events on CLL onset and progression remains to be elucidated. One such example is mutation in the IKZF3 gene, encoding the zinc finger protein AIOLOS, mutated in ~2% of CLLs and associated with fludarabine-refractory disease. AIOLOS is a lymphoid-restricted transcription factor and a chromatin remodeler that plays an essential role in B cell development and maturation. In CLL, the IKZF3 mutation, also reported in few cases of diffuse large B cell lymphoma and mantle cell lymphoma,targets a highly conserved hotspot (L162R, homologous to murine L161R) that is localized in the 2nd zinc finger of the DNA-binding domain, required for DNA sequence recognition. Given the localization of this hotspot mutation, we hypothesized that it impacts the function of AIOLOS to drive CLL. To characterize the effects of the IKZF3-L162R mutation, we generated a knock-in mouse line that conditionally expresses the point mutation in a B cell lineage context through crossing with Cd19-cre mice, generating mouse lines carrying Ikzf3-L161R as either a heterozygous mutation (Ikzf3-L161RHet), homozygous mutation (Ikzf3-L161RHomo) or wild-type Ikzf3(Ikzf3WT). Given the established role of Aiolos in lymphoid differentiation, we first asked how the mutation impacts B cell development. By flow cytometry, using established markers to detect marrow pro-B, pre-B, transitional and mature B cell populations, or peritoneal B1a and B1b cell populations, no differences in the proportion of cells were observed between Ikzf3WTor Ikzf3-L161RHet. In the spleen, however, the average proportion of marginal zone B cells (B220+CD23+CD21high) was markedly reduced in heterozygousmice compared to wild type mice (6 mice/group: 4.9% vs. 11.5%, p=0.006), while the average proportion of follicular B cells (B220+CD23+CD21-) was increased (76% vs. 63%; p=0.003). Immunohistochemical staining of spleen sections confirmed that the marginal zone area was significantly reduced in Ikzf3-L161RHetmice (p=0.01). In addition, we noted a higher proliferation rate of B cells from Ikzf3-L161RHetmice when stimulated with LPS and IL-4 for 3 days (p=0.01), suggesting that the mutation confers a survival advantage to B cells. Similar analyses in Ikzf3-L161RHomomice are ongoing. By immunofluorescence and immunoprecipitation, neither Aiolos binding with its partners CHD4, SIN3 or HDAC1, nor its cellular distribution were impacted by the mutation. Of note, the total protein level of Aiolos was increased in Ikzf3-L161RHetmice (9 mice/group; p 〈 0.05). Since the mutation localizes to a DNA binding domain, we hypothesized that it modifies the ability of Aiolos to control expression of its target genes. We therefore performed CHIP-seq in Ikzf3WTsplenic B cells, and identified Aiolos-associated high confidence peaks (fold change (FC) enrichment compared to input 〉 20) corresponding to DNA binding sites in the promoters of genes such as Rps19, Ogg1, Dusp2, Phf23 or Brfp1 and confident peaks (FC 〉 10) in the anti-apoptotic gene Mcl1 and in genes involved in BCR signaling (i.e.Syk, Pi3kr1, Nfkbid), suggesting that their expression is under the control of Aiolos. Comparison of the expression by qPCR of these 8 genes in splenic B cells from the 3 mouse lines revealed Dusp2, Mcl1, Syk, Nfkbid and Phf23 to be upregulated in Ikzf3-L161RHomoB cells (p 〈 0.05) but not in Ikzf3-L161RHetB cells. These findings suggest that the mutation directly impacts the expression level of Aiolos target genes. The upregulation of Mcl1 expression is particularly relevant in the context of CLL as dysregulation of anti-apoptotic signaling is characteristic of the disease. In conclusion, these data show that Aiolos mutation affects B cell subpopulation ontogeny, inducing a disproportionate abundance of follicular B cells endowed with high proliferative capacity. The mutation impacts Aiolos transcription capacity leading to upregulation of genes belonging to pathways cardinal to CLL development, including BCR signaling and apoptosis. Ongoing studies focus combining RNA-seq and CHIP-seq in mutant B cells, with the aim of identifying the breadth of differential expressed genes and dysregulated cellular pathways in mutant B cells in an unbiased manner. Disclosures Wu: Neon Therapeutics: Equity Ownership.