A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas
Authors: Munshi Manit , Liu Xia , Kofides Amanda , Tsakmaklis Nickolas , Demos Maria G , Guerrera Maria Luisa , Hunter Zachary R , Palomba M. Lia , Argyropoulos Kimon V. , Patterson Christopher J , Meid Kirsten , Gustine Joshua , Castillo Jorge J. , Sarosiek Shayna , Flynn Catherine A , Wang Jinhua , Buhrlage Sara J , Gray Nathanael S , Munshi Nikhil C. , Anderson Kenneth C. , Yang Guang , Treon Steven P 
Published: November 2021

Abstract
Activating mutations in MYD88 (MYD88 Mut) are common in B-cell malignancies including Waldenstrom Macroglobulinemia (WM) and ABC subtype of diffuse B-cell lymphoma (ABC DLBCL). MYD88 is a component of the Toll-like receptor (TLR) pathway. We and others previously showed that MYD88 Mut triggers assembly of a "Myddosome" complex that leads to downstream pro-survival signaling that includes IRAK4/IRAK1 and BTK triggered NF-κB (Ngo et al, Nature 2011; Treon et al, NEJM 2012; Yang et al, Blood 2013) and HCK mediated BTK/NF-κB, PI3K/AKT, and MAPK/ERK signaling (Yang et al, Blood 2016; Liu et al Blood Adv. 2020). The activation of the B-cell receptor (BCR) signaling component SYK has also been observed in MYD88 Mut WM (Argyropoulos et al, Leukemia 2016). In ABC DLBCL, chronic active BCR signaling underlies SYK activation that is triggered by the SRC family member LYN (Davis et al, Nature 2010). These observations led us to explore potential drivers of BCR/SYK activation in WM. We previously reported that MYD88 Mut triggered activation of SYK in WM and ABC DLBCL cells (Munshi et al, BCJ 2020). Herein, we investigated if HCK, a SRC family member that is transcriptionally upregulated and activated by MYD88 Mut could trigger the BCR pathway through SYK activation. Since LYN is an integral part of BCR signaling, we first examined its expression and activation state in MYD88 Mut WM and ABC DLBCL cells. While MYD88 Mut TMD8, HBL-1 and OCI-Ly3 ABC DLBCL cells showed strong expression of p-LYN, such expression was absent or low in MYD88 Mut BCWM.1 and MWCL-1 cells, as well as CD19-selected bone marrow derived primary lymphoplasmacytic cells (LPCs) from WM patients. In view of the above findings, we next interrogated a direct role for HCK in mediating SYK activation. We over-expressed wild-type HCK (HCK WT) or gatekeeper mutated HCK (HCK T333M) in MYD88 Mut BCWM.1 and MWCL-1 WM cell lines, and TMD8 ABC DLBCL cells. In all these cell lines, over-expression of HCK WT or HCK T333M triggered a robust increase in phosphorylation of SYK Y525/Y526 in comparison to vector only transduced cells. Moreover, using an inducible vector system, knockdown of HCK showed a marked reduction in phosphorylation of SYK Y525/Y526 in MYD88 Mut BCWM.1 WM and TMD8 ABC DLBCL cells. We next sought to clarify if HCK and activated SYK were present in the same signaling complex. We performed co-immunoprecipitation experiments using an HCK antibody in MYD88 Mut BCWM.1, TMD8 and wild-type MYD88 (MYD88 WT) Ramos cells. The HCK antibody effectively pulled down p-SYK in MYD88 Mut BCWM.1 and TMD8 cells, but not in MYD88 WT Ramos cells. To confirm whether SYK activation was a result of HCK kinase activity, we next performed rescue experiments with the HCK inhibitors A419259 and KIN-8194 in MYD88 Mut BCWM.1 and MWCL-1 WM and TMD8 ABC DLBCL cells expressing either HCK WT or the HCK T333M protein that abrogated the activity of these inhibitors against HCK. Expression of the HCK T333M protein produced marked resistance to A419259 as well as KIN-8194 versus vector or HCK WT transduced BCWM.1 and MWCL-1 cells. By PhosFlow analysis, we observed that expression of HCK T333M but not HCK WT led to persistent activation of HCK and SYK in the presence of A419259 or KIN-8194 in BCWM.1 and MWCL-1 WM cells, and TMD8 ABC DLBCL cells. Consistent with these observations, treatment of primary MYD88 mutated WM LPCs cells with either A419259 or KIN-8194 also showed marked reduction in both p-HCK and p-SYK expression by PhosFlow analysis. Taken together, our findings show that SYK is activated by HCK in MYD88 Mut B-cell lymphomas cells; broaden the pro-survival signaling generated by aberrant HCK expression in response to MYD88 Mut; and help further establish HCK as an important therapeutic target in MYD88 Mut B-cell lymphomas.

Disclosures
Palomba: Juno: Patents & Royalties; Rheos: Honoraria; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Notch: Honoraria, Other: Stock; Kite: Consultancy; Novartis: Consultancy; BeiGene: Consultancy; Priothera: Honoraria; Nektar: Honoraria; PCYC: Consultancy; Wolters Kluwer: Patents & Royalties; WindMIL: Honoraria; Magenta: Honoraria; Pluto: Honoraria; Lygenesis: Honoraria; Ceramedix: Honoraria. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Gray: Syros, C4, Allorion, Jengu, B2S, Inception, EoCys, Larkspur (board member) and Soltego (board member: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield and Sanofi: Research Funding. Munshi: Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy. Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.