HCK Is a Highly Relevant Target of Ibrutinib in MYD88 Mutated Waldenstrom's Macroglobulinemia and Diffuse Large B-Cell Lymphoma
Authors: Yang Guang , Buhrlage Sara , Tan Li , Liu Xia , Chen Jie , Hunter Zachary , Xu Lian , Tsakmaklis Nicholas , Chen Jiaji , Patterson Christopher J , Castillo Jorge J , Zhang Wei , Gray Nathanael , Treon Steven P 
Published: October 2019

Abstract
Background: Ibrutinib was recently approved by the U.S. FDA, and the European Medicines Agency for the treatment of Waldenström's macroglobulinemia (WM) given the findings of a prospective, multicenter study that showed high levels of response and durable progression-free survival in previously treated patients (NEJM 372(15):1430-40). Mutations in MYD88 are highly prevalent in patients with WM (NEJM 367(9):826-33) and ABC subtype of DLBCL (Nature 470(7332):115-9), and activate BTK, a target of ibrutinib (Blood 122(7):1222-32). Ibrutinib is also known to target other kinases, which may be functionally relevant to its activity in WM, and other MYD88 mutated diseases.
Methodology: Transcriptomic profiling of primary WM patient cells; MYD88, HCK and BTK transduction experiments; Kinome profiling; and co-immunoprecipitation studies using biotin-labeled ibrutinib were performed to identify relevant targets of ibrutinib in MYD88 mutated cells. Functional studies were also performed to validate target activity.
Results: Transcriptomic profiling and quantitative RT-PCR validation studies identified aberrant over-expression of HCK in bone marrow derived malignant cells (CD19+) from WM patients with MYD88 mutations versus memory (CD19+ CD27+) and non-memory (CD19+ CD27-) B-cells from healthy donors. HCK was highly expressed in MYD88 mutated WM and ABC-type DLBCL cell lines (BCWM.1, MWCL-1, TMD-8, HBL-1, OCI-Ly3) versus MYD88 wild type cell lines (OCI-Ly7, OCI-Ly19, Ramos, MM1.S and RPMI-8226). Over-expression of MYD88 L265P but not wild-type MYD88 resulted in significantly higher levels of HCK, while knockdown of HCK and/or use of a known HCK inhibitor (A419259) reduced survival of MYD88 mutated WM and DLBCL cell lines, and primary WM cells. Importantly, HCK (Tyr411) was highly phosphorylated in primary WM cells, as well as WM and DLBCL cell lines expressing the MYD88 L265P mutation, and its level of activation was enhanced by treatment with IL-6, a major growth and survival factor. Knockdown of the IL-6 co-receptor GP-130 abrogated IL-6 triggered activation of HCK. Kinome screening showed that HCK was a target of ibrutinib, while biotin labeled ibrutinib but not AVL-292 (CC-292) showed robust binding to HCK in BCWM.1, MWCL-1 and TMD-8 cells. Furthermore, by use of a kinase active-site inhibition assay, ibrutinib but not AVL-292 blocked ATP binding to HCK in a dose-dependent manner, while both compounds blocked ATP binding to BTK. To elaborate on the importance of HCK inhibition by ibrutinib in WM growth and survival, WM cells were transduced with vectors expressing a HCK gatekeeper mutant (HCK-T338M) or an ibrutinib BTK binding site mutant (BTK-C481S). Transduction with HCK-T338M significantly increased the EC50 for ibrutinib or A419259 in comparison to wild type HCK expressing BCWM.1 cells, while no rescue effect was observed for AVL-292 treated cells. Transduction with BTK-C481S showed rescue for ibrutinib and AVL-292, but not A419259. By phospho-flow analysis, ibrutinib and A419259, but not AVL-292 significantly reduced HCK Tyr411 phosphorylation in MYD88 L265P expressing WM and DLBCL cell lines.
Conclusions: HCK is regulated by MYD88 L265P, and is activated in primary WM cells, as well as MYD88 L265P expressing WM and DLBCL cell lines. IL-6 activates HCK though GP130, and inhibition of HCK abrogates WM cell survival. Ibrutinib shows robust binding to HCK, and transduction of WM cells with a mutated HCK gatekeeper blocks ibrutinib related tumor cell killing. These studies identify HCK as a novel, and highly relevant target of ibrutinib in MYD88 mutated WM and DLBCL cells.

Disclosures
No relevant conflicts of interest to declare.