Aberrant HCK Survival Signaling By Mutated MYD88 Requires PAX5 in Waldenstrom's Macroglobulinemia and ABC DLBCL Cells

Activating mutations in MYD88 are present in 95% and 40% of patients with WM and ABC DLBCL, respectively. MYD88 mutations are known to trigger growth and survival of WM and ABC DLBCL cells through BTK/IRAK1/IRAK4 directed NF-kB signaling (Ngo et al, Nature 2011; Yang et al, Blood 2013). Recently, we identified that mutated MYD88 can trigger the SRC family member HCK, a kinase that is downregulated in later stages of B-cell ontogeny (Yang et al, Blood 2016). HCK promotes malignant cell survival through activation of BTK, as well as AKT and ERK1/2, whereas knockdown of HCK leads to decreased survival in WM and ABC DLBCL cells. The regulatory mechanisms that promote mutated MYD88 directed HCK signaling are unknown, and may permit therapeutic advances for MYD88 mutated diseases. To clarify whether HCK expression is regulated by TLRs / MYD88 or BCR signaling, we performed activation studies using triggers of TLR9 (ODN-2006) and TLR4 (LPS-EP) signaling, and BCR (anti-IgM/IgG) signaling in MYD88 wild-type or mutated B-cell lymphoma cells. Stimulation of TLRs but not BCR increased HCK transcription, particularly in wild-type MYD88 expressing B-cell lymphoma cells thereby demonstrating the importance of TLR/MYD88 signaling in driving HCK expression. To better understand the mechanism for the aberrant upregulation of HCK transcription in MYD88 mutated B-cell lymphoma cells, we performed promoter-binding TFs profiling, and observed that among the TFs, PAX5 signal was reduced the most by the addition of HCK promoter into BCWM.1 cell nuclear extracts. The signals of many other TFs that can be regulated by NF-kB (AP1, C\EBP, Myb, NF-kB), MAPK (AP1, C\EBP, CREB, HNF4, NF-1) and STAT3 signaling were also reduced. These results indicate that PAX5 and mutated MYD88 directed TFs including NF-kB, AP-1 and STAT3 bind to endogenous HCK promoter sequence. HCK promoter binding consensus sequence analysis and ChIP studies confirmed the presence of PAX5, as well as AP-1, NF-kB and STAT3 binding sites on the HCK promoter. Knockdown of PAX5 reduced HCK mRNA level in BCWM.1 and TMD-8 cells indicating an activating function of PAX5 in the regulation of HCK expression. The deletion of AP-1, NF-kB and STAT3 binding sites on HCK promoter reduced HCK promoter activity, thereby supporting a role for AP-1, NF-kB and STAT3 in HCK gene expression in MYD88 mutated cells. Treatment of BCWM.1 and TMD-8 cells with inhibitors of AP-1, NF-kB and STAT3 decreased HCK promoter activities by luciferase reporter assay, as well as endogenous HCK mRNA transcription levels in MYD88 mutated cells. Our data indicate PAX5 along with mutated MYD88 triggered transcription factors NF-kB, AP-1 and STAT3 orchestrate aberrant HCK expression. The findings provide a framework for efforts directed at blocking MYD88 triggered HCK survival signaling in MYD88 mutated B-cell lymphomas.