Development and Characterization of a Novel Bifunctional Protac Jh-XIII-05-1 That Shows Potent IRAK1 and IRAK4 Kinase Inhibition and Target Degradation and Is Highly Active in MYD88 Mutated B-Cell Lymphoma Cells Alone and in Combination with BTK- and BCL2- Inhibitors

Background: Activating mutations in MYD88 are prevalent in many B-cell malignancies, including Waldenström Macroglobulinemia (WM) (95-97%), primary CNS lymphoma (70-80%), ABC DLBCL (40%), marginal zone lymphoma (5-10%), and CLL (5-15%). Mutated MYD88 triggers divergent pro-survival pathways that include BTK and IRAK1/IRAK4. BTK-inhibitors are highly active in many MYD88 mutated B-cell malignancies but fail to achieve complete responses. The unabated activation of IRAK1 and IRAK4 may contribute to ongoing pro-survival signaling in patients receiving BTK-inhibitors. Moreover, our ongoing studies suggest that both the kinase activity and scaffold of IRAKs may contribute to their pro-survival signaling. Therefore, targeting IRAK1 and IRAK4 alone and in combination with BTK-inhibitors is a rational therapeutic approach in MYD88-mutated malignancies.

Methods: Structural activity relationship studies were performed on an extensive series of candidate scaffolds that demonstrated selective targeting of IRAK1 and IRAK4. 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 cytometric and western blot analyses. Selective degradation of target was characterized by western blot analyses and/or proteomic screening in MYD88 mutated lymphoma cells. Cytotoxicity and apoptosis assays were performed on MYD88 mutated 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: We identified a novel bifunctional PROTAC JH-XIII-05 that exhibited potent and highly selective kinase inhibition and degradation of IRAK1 and IRAK4, including in lymphoma cells expressing mutated BTK Cys481. JH-XIII-05 exhibited low IC50 values (3-90 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, the activity of JH-XIII-05 was superior in these studies to its non-PROTAC containing scaffold JH-XI-82-01. Pharmacokinetic studies revealed CMax of 2000-3200 nM following subcutaneous single dose administration at 10-30 mg/kg that was well in excess of cellular IC50s for JH-XIII-05 in MYD88 mutated lymphoma cells. Combination treatment with the BTK-inhibitor ibrutinib and the BCL2 inhibitor venetoclax demonstrated synergistic activity at most doses.

Conclusion: JH-XIII-05-1 is a novel bifunctional IRAK1 and IRAK4 PROTAC that shows potent and selective target kinase inhibition and degradation, and demonstrates potent anti-tumor activity in MYD88-mutated lymphoma cells. Synergistic interactions with BTK and BCL2 inhibitors were observed with JH-XIII-05. Our studies provide a framework for the advancement of bifunctional PROTACs targeting IRAK1 and IRAK4 alone and in combination with BTK and BCL-2 inhibitors for the treatment of MYD88 mutated lymphomas.