Notch-Regulated Enhancers in B-Cell Lymphoma Activate MYC and Potentiate B-Cell Receptor Signaling

Gain-of-function mutations in Notch receptor genes occur in 10-15% of cases of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), and are associated with inferior clinical outcomes. Nearly all Notch mutations reported in B cell tumors lead to loss of the C-terminal negative regulatory PEST domain and result in stabilization of the activated form of Notch (intracellular Notch [ICN]), whereas mutations that lead to ligand-independent Notch activation (which are common in T cell acute lymphoblastic leukemia [T-ALL]) are rare. ICN can be detected in tumor cells within lymph nodes of >80% of patients with CLL, suggesting that Notch may have a broader oncogenic role than the incidence of Notch mutations would suggest. However, the downstream targets of Notch in B-cell tumors have not been identified.

We used a gamma-secretase inhibitor (GSI) washout strategy to determine the immediate, direct effects of Notch activation in three MCL cell lines with Notch gain-of-function mutations, including two cell lines with unusual Notch gene rearrangements that lead to ligand-independent Notch activation, as well as a third line with a Notch PEST domain mutation in which signaling was activated with recombinant Notch ligand. Using these models, we identified likely direct target genes and their associated genomic Notch response elements using RNA-seq and ChIP-Seq in the Notch-on and Notch-off states. Most of these response elements corresponded to long-range enhancers that showed Notch-dependent changes in H3K27 acetylation, and were bound by components of the Notch transcription complex (NTC) in both cell lines. We confirmed these associations by performing ChIP-Seq on primary CLL and MCL biopsies, and by identifying specific looping interactions with Notch target gene promoters in public genome-wide proximity ligation datasets (RNA Pol2 ChIA-PET) from a lymphoblastoid cell line expressing the EBV-encoded Notch surrogate protein EBNA2.

MYC was among the most strongly Notch-activated genes in Notch-dependent MCL cell lines and was associated with NTC binding at two B cell-specific 5' enhancers distinct from the Notch-dependent MYC enhancer previously identified in T-ALL. MCL cell line proliferation was blocked by Cas9 nuclease or epigenetic repressors targeting the 5' MYC enhancers, whereas cells were rescued from Notch inhibition by GSI via transduction with MYC. Gene set enrichment analysis of other direct Notch target genes identified in MCL models showed enrichment for regulators of B cell receptor (BCR) signaling, including the Src family kinase genes FYN, LYN, and BLK, and the signaling complex adaptor BLNK, as well as regulators of CD40 and cytokine signaling.

RNA-seq analysis of primary CLL lymph node biopsies revealed significantly higher expression of many Notch target genes in biopsies with high levels of ICN. To functionally validate Notch target genes in primary tumors, we co-cultured CLL and MCL cells obtained from peripheral blood with Notch ligand-expressing stromal cells in the presence ("notch off") or absence ("notch on") of GSI, and demonstrated increased expression of Notch target genes, including MYC, in the "notch-on" cells. Furthermore, "notch-on" CLL cells showed increased phosphorylation of the BCR signaling intermediates SYK and PLCg2 upon BCR crosslinking compared to GSI-treated cells. Finally, we validated Notch-dependent regulation of target genes in vivo in a patient-derived xenograft model of NOTCH1-mutant MCL. Notch target gene expression was significantly higher in MCL cells within the spleen versus bone marrow or blood, but was markedly reduced in animals treated for five days with GSI. Additional xenograft studies are ongoing, and will be described at the meeting.

Our data link active Notch signaling to two well-characterized oncogenic drivers in B cell lymphoma, MYC and BCR signaling, and may have important implications for the development of treatment strategies involving Notch antagonists and other targeted therapeutics, such as BCR targeting agents.