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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>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 (FDR2). 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 (FDR2), 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.

BCR signaling is a central pathogenetic pathway in chronic lymphocytic leukemia (CLL). Most CLL cells express BCRs of IgM and IgD isotypes, but the contribution of these isotypes to functional responses remains incompletely defined. We therefore investigated differences between IgM and IgD signaling in freshly isolated peripheral blood CLL cells and in CLL cells cultured with nurselike cells, a model that mimics the lymph node microenvironment. IgM signaling induced prolonged activation of ERK kinases and promoted CLL cell survival, CCL3 and CCL4 chemokine secretion, and downregulation of BCL6, the transcriptional repressor of CCL3 In contrast, IgD signaling induced activation of the cytoskeletal protein HS1, along with F-actin polymerization, which resulted in rapid receptor internalization and failure to support downstream responses, including CLL cell survival and chemokine secretion. IgM and IgD receptor downmodulation, HS1 and ERK activation, chemokine secretion, and BCL6 downregulation were also observed when CLL cells were cocultured with nurselike cells. The Bruton's tyrosine kinase inhibitor ibrutinib effectively inhibited both IgM and IgD isotype signaling. In conclusion, through a variety of functional readouts, we demonstrate very distinct outcomes of IgM and IgD isotype activation in CLL cells, providing novel insight into the regulation of BCR signaling in CLL.