Development and Characterization of Novel Dual HCK/BTK Bifunctional Protacs That Potently Inhibit Kinase Function and Demonstrate Robust in Vitro and In Vivo Degradation of Targets for the Treatment of MYD88-Mutated B-Cell Malignancies

Background: Activating mutations in MYD88 are prevalent in many B-cell malignancies, including Waldenström Macroglobulinemia (95-97%), primary CNS lymphoma (70-80%), ABC DLBCL (40%), marginal zone lymphoma (5-10%), and CLL (5-15%). Mutated MYD88 upregulates the SRC family member HCK, which triggers multiple pro-survival cascades including BTK and SYK. Our previous work led to the development of KIN-8194, a novel HCK/BTK kinase inhibitor that was highly active in vitro and in MYD88-mutated xenografts, including BTK^C481S ibrutinib-resistant disease models. Proteolysis targeting chimeras (PROTACs) offer greater selectivity and sustained target inhibition over traditional kinase inhibitors. We therefore sought to develop novel HCK/BTK-targeting bifunctional PROTACs that abrogate kinase function and potently degrade their targets for treating MYD88-driven lymphomas.

Methods: Structural activity relationship studies were performed on an extensive series of candidate scaffolds that demonstrated selective targeting of HCK and BTK. Both cereblon and VHL targeting PROTACs were synthesized of the most promising scaffolds and evaluated for kinase inhibition by KINOMEscan assay against 468 kinases; flow cytometic and western blot analyses. Selective degradation of target was characterized by western blot analyses and/or proteomic screening in MYD88 mutated lymphoma cells expressing wild-type as well as Cys481 mutated BTK. Cytotoxicity and apoptosis assays were performed on MYD88 mutated and wild-type lymphoma cell lines; BTK wild-type and mutated Cys481 expressing lymphoma cells; as well as primary MYD88 mutated WM cells. In vitro ADME profiling and extensive in vivo mouse pharmacokinetic studies, and pharmacodynamic studies in a MYD88 mutated TMD8 xenograft murine model were performed for the most promising candidates.

Results: Two novel bifunctional PROTACs DFCI-002-05 and DFCI-002-06 were identified that exhibited potent kinase inhibition and degradation of HCK and BTK, including in lymphoma cells expressing mutated BTK Cys481. Both compounds exhibited low IC50 values (1-100 nM) in proliferation assays of MYD88-mutated WM and ABC DLBCL cells, as well as high levels of apoptosis in MYD88-mutated WM and ABC DLBCL cell lines, including cell lines expressing mutated BTK Cys 481; and primary WM tumor cells. Importantly both DFCI-002-05 and DFCI-002-06 demonstrated high levels of bioavailability (F~40%) and CMax of 700-2700 nM at single oral doses of 10-60 mg/kg, well in excess of cellular IC50s for both compounds. Significant degradation of both HCK and BTK occurred in TMD8 xenografted tumors following oral administration at dose ranges of 5-30 mg/kg. Robust dose dependent tumor growth suppression was also observed following treatment of TMD8 xenografted mice with both DFCI-002-05 and DFCI-002-06.

Conclusions: Our studies detail the development of two highly potent and selective bifunctional HCK/BTK PROTACs that show potent target kinase inhibition and protein degradation, as well as anti-proliferative and/or apoptotic activity including in lymphoma cells expressing mutated BTK Cys481. Both PROTACs showed robust bioavailability and degradation of HCK and BTK in murine TMD8 xenograft models as well as potent tumor suppression. These studies provide a framework for advancing bifunctional HCK/BTK PROTACs for treating MYD88-mutated lymphomas, including covalent BTK-inhibitor resistant disease carrying BTKCys481 mutations.