Project 2 Josefowicz

PROJECT 2: SUMMARY/ABSTRACT Activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL) and Epstein Barr virus associated viral lymphomas (EBV+ lymphoma) have in common that they are among the most aggressive and difficult to treat lymphomas and both hijack potent signaling cascades to drive aggressive growth, survival, and resistance to

treatment. These hijacked signaling programs flip a potent epigenetic switch that results in activation of positive- selection and differentiation programs that are characteristic of aged autoimmune B cells (AAB) (P3). This chromatin program involves loss of PRC2/H3K27me3 and stimulation of CREBBP (P4), KMT2D (P1) and is

highly active at ABC-DLBCL and EBV+ lymphoma signature genes. The mechanisms by which immune synapse signaling, viral oncogenes, or ABC-DLBCL mutations activate these epigenetic programs have not been characterized. Our preliminary data show that histone H3 phosphorylation (H3ph) at both residue S28 (H3S28ph)

and residue 31 (H3.3S31ph), downstream of immune synapse signaling, can potently disrupt and reshape chromatin states, acting as a sensitive signaling scaffold. Normally absent in resting B cells, we find constitutive high-level H3ph at lymphoma signature genes in ABC-DLBCL and EBV+ lymphoma. Further, in human

lymphomas we found that H3S28ph high tumors are associated with decreased survival and that both ABC- DLBCL and EBV+ lymphoma cells are selectively sensitive to histone kinase inhibition. Based on these preliminary data, we hypothesize that ABC-DLBCL signaling mutations and EBV oncoproteins signal through

histone kinases and H3ph to constitutively switch on chromatin at lymphoma driver genes. To pursue this hypothesis we will first use mechanistic, biochemical, and epigenetic studies of murine and human models of ABC-DLBCL and EBV+ lymphoma to determine the sequence of epigenetic events that activate gene expression

downstream from Myd88 and Cd79b mutations or EBV oncoproteins, respectively, expecting that H3ph is an initiating and dominant epigenetic event (Aim 1). Next, we will determine the direct contribution of H3ph to malignant transformation of GC B cells by employing first-of-their-kind histone functional mutagenesis

approaches that confer constitutive activation or suppression of H3ph in mouse and human lymphoma (Aim 2). Finally, we will determine whether chromatin kinases and histone phosphorylation are required for maintenance of ABC-DLBCL and viral lymphomas using small-molecule inhibitors and inducible knock-down of MSK and

IKKa, and CRISPR editing of histone genes in well-characterized models of ABC-DLBCL and EBV+ lymphoma. We expect to find that ABC-DLBCL and EBV+ lymphoma require histone kinases and H3ph for lymphomagenesis and are addicted to their constitutive activity for maintenance. We anticipate that these studies

will establish histone kinases as novel dependencies for these aggressive lymphomas for which there are currently few treatment options.