Novel Protacs Demonstrate Dual Kinase Inhibition and Degradation of HCK and BTK, and Are Highly Active in MYD88 Mutated WM and ABC DLBCL Cells

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 transcriptionally upregulates the SRC family member HCK, which in turn serves as a master signal for triggering multiple pro-survival cascades in mutated MYD88 lymphoma cells including BTK/NFKB, SYK, and ERK (Blood 127:3237-52; Blood Adv 4:141-153; Blood Adv 6:3332-38). In previous work, we developed and characterized a novel HCK kinase inhibitor, KIN-8194 (Blood 138:1966-79). KIN-8194 blocked both HCK and BTK and was active both in vitro and in vivo in MYD88 mutated xenograft lymphoma models, including BTK ^Cys481Ser ibrutinib resistant models. Proteolysis targeting chimeras (PROTACs) represent a novel approach for blocking kinase based signaling and may provide an advantage over kinase inhibitors with greater selectivity and sustained target inhibition. We therefore initiated a medicinal chemistry campaign to develop highly potent, selective, and bio-available HCK/BTK targeting PROTACS using KIN-8194 scaffold for the treatment of MYD88 driven lymphomas.

Methods: We developed and characterized a large series of PROTACs using KIN-8194 as a scaffold. We rationally designed linkers to facilitate E3 ubiquitin ligase binding and improve physical properties leading to degraders with ideal oral pharmacokinetic properties and bioavailability. We characterized their kinome profiling by performing a KINOMEscan assay against a panel of 468 kinases at 1 uM ( Fig. 1). Subsequently, we conducted rigorous biological efficacy screenings using the ATP-based Cell Titer-Glo Luminescent Cell Viability Assay to identify the most potent compounds. Compounds with IC ₅₀ scores lower than KIN-8194 were selected for further validation through flow cytometry based quantitative assays that verified apoptotic and cytotoxic effects on WM and MYD88-mutated lymphoma cell lines, as well as primary human peripheral blood mononuclear cells (PBMCs), utilizing Annexin V and Propidium iodide (PI) staining.

Results: We identified and characterized several novel PROTACs from the KIN-8194 scaffold that exhibited potent dual inhibition of HCK and BTK kinase activity at 1 nM, as well as marked degradation of HCK and BTK by western blot analysis at dose levels as low as 100 nM in MYD88 mutated WM and ABC DLBCL cell lines. KINOMEscan profiling showed greater selectivity for novel PROTACs as exemplified in Fig. 1. Enhanced cell killing due to degradation of HCK and BTK versus inhibition alone was confirmed using respective PROTAC analogues with modified IMID warheads that do not bind cereblon. We observed suppression of downstream pro-survival NFKB and ERK signaling in response to the dual HCK/BTK PROTACs. Moreover, several HCK/BTK PROTACs (DFCI-004, -005, -006) demonstrated remarkable low IC ₅₀ values (1-100 nM) in proliferation assays of MYD88 mutated BCWM.1 WM and TMD-8 ABC DLBCL cells. Importantly, the dual HCK/BTK PROTACs elicited higher levels of apoptosis in BCWM.1 WM and TMD-8 ABC DLBCL cells in comparison to the native KIN-8194 kinase inhibitor and showed sparing of healthy donor B- and T-cells akin to KIN-8194. Finally, murine PK studies showed that the HCK/BTK PROTACs DFCI-005 and -006 were highly bioavailable with F=40-42%, and C _Max of 700-2700 nM at single oral dose ranges of 10-60 mg/kg.

Conclusions: In these studies, we demonstrated the development and characterization of novel, dual HCK/BTK PROTACs that demonstrate potent and selective kinase inhibition and protein degradation of HCK and BTK. Importantly, the novel PROTACs showed enhanced apoptosis of MYD88 mutated WM and ABC DLBCL cells over the native kinase inhibitor KIN-8194 and sparing of healthy donor B- and T-cells. Lastly, novel HCK/BTK PROTACs exhibited high levels of bioavailability. Our studies therefore provide a framework for the advancement of dual HCK/BTK PROTACs for the treatment of MYD88 mutated lymphomas.