Mutated MYD88 Regulates HCK Pro-Survival Signaling through JunB in MYD88 Mutated B-Lymphoma Cells

Hematopoietic cell kinase (HCK) is a member of the SRC family tyrosine kinases (SFKs) that is down-regulated in later stages of B-cell ontogeny. In MYD88 mutated B-cell lymphomas, HCK is aberrantly over-expressed and is activated and triggers multiple growth and survival pathways including BTK, PI3Kδ/AKT and ERK1/2 which are essential to tumor cell survival. Ibrutinib, a pleiotropic inhibitor of BTK, that produces remarkable responses in MYD88 mutated WM, ABC-DLBCL, and Primary CNS Lymphoma was found also potently inhibits HCK. Mutations that abolish ibrutinib-HCK binding greatly diminish anti-tumor activity in MYD88 mutated lymphoma cells, highlighting the importance of HCK as an essential target in MYD88 driven diseases. To clarify the transcriptional regulation of HCK in MYD88 mutated malignancies, we performed promoter binding TF profiling, PROMO weighted transcription factor (TF) consensus binding analysis, and chromatin immuno-precipitation (ChIP) studies. We identified PAX5, and mutated MYD88 downstream signaling mediators, STAT3, AP-1 and NF-kB as important drivers of HCK transcription. Knockdown of PAX5, a crucial regulatory factor required for B-cell commitment and identity, abrogated HCK transcription in MYD88 mutated lymphoma cells. Deletion of STAT3, AP-1 or NF-kB binding sites greatly reduced corresponding TFs binding and HCK promoter activity, indicating the importance of MYD88 directed signaling in the regulation of HCK transcription. Among AP-1 complex components, JunB but not c-Jun showed greatest relevance to TLR/MYD88 signaling and regulation of HCK transcription. Since STAT3 and NF-kB are known downstream mediators of mutated MYD88 signaling, we focused on the function of JunB. JunB phosphorylation increased following MYD88 pathway activation through TLR4 (with LPS-EB) or TLR9 (with ODN-2006), as well as lentiviral mediated expression of mutated MYD88 (L265P) but not wild type MYD88 (MYD88-WT) at Thr102 and Thr104 in either MYD88 mutated BCWM.1 cells or MYD88-WT Ramos cells. Conversely, c-Jun phosphorylation was not impacted by either intervention. Knockdown of MYD88 in BCWM.1 WM cells also reduced the phosphorylation of JunB, while c-Jun phosphorylation showed modest increase. Moreover, knockdown of JunB reduced HCK protein levels in MYD88 mutated WM and ABC-DLBCL cells. These data demonstrate that JunB is an important mediator of mutated MYD88 signaling among AP1 complex members and is directly involved in the regulation of HCK expression in MYD88 mutated B-cell lymphoma. The findings provide new insights into the transcriptional regulation of HCK by MYD88 driven TFs, and opportunities for further advancing targeted therapeutics in MYD88 driven B-cell malignancies.