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 Dnmt3a^fl/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 Dnmt3a^fl/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 Dnmt3a^fl/fl animals. Compared to WT B1a cells, Dnmt3a^fl/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.