Pacritinib, a Dual FLT3/JAK2 Inhibitor, Reduces Irak-1 Signaling in Acute Myeloid Leukemia

Abstract

Background: A momentous challenge in the treatment of acute myeloid leukemia (AML) is the significant molecular heterogeneity in driving abnormalities and rapid emergence of resistance. Successful clinical translation of novel targeted agents has been impeded by an incomplete understanding of the genetic drivers and the role of the bone marrow microenvironment that modulates response to therapy. There is thus a need for further investigation of the signaling mechanisms contributing to disease pathogenesis. We previously identified growth dependency on interleukin-1 (IL-1) signaling in >50% of the 90 primary AML samples tested. IL-1 signals through IL-1 receptor-associated kinase (IRAK1), a major mediator of innate immunity and inflammatory responses and a potentially critical therapeutic target in hematopoietic malignancies. These results suggest that targeting the commonly dysregulated IL-1 signaling pathway would be therapeutically beneficial to AML patients. However, there is no clinically viable molecule available to selectively inhibit IL-1 signaling. Here we uncovered that pacritinib, a dual FLT3/JAK2 inhibitor, has high specificity and sensitivity to target IRAK1 in various hematopoietic malignancies, including AML. Methods: We used a combination of biochemical, structural biology, and functional approaches to elucidate the mechanism of action of pacritinib and establish its sensitivity and specificity for target inhibition. Kinome screening analysis against 429 recombinant kinases in the presence of 100 nM pacritinib (approximately 50% of the steady state concentration of free pacritinib at the phase 3 dose of 400 mg QD) was followed by titration (1-100 nM) against those kinases that were >50% inhibited at 100 nM. Because pacritinib is an established dual FLT3/JAK2 inhibitor, we then compared the efficacy of pacritinib to the FLT3 inhibitor quizartinib and the JAK2 inhibitor ruxolitinib in 16 AML cell lines and 25 primary AML samples harboring various genetic lesions. The effects of drug treatments were evaluated on cell viability, survival and downstream signaling. Patient samples having IC50below 1000 nM were considered responsive. To validate the selectivity of pacritinib for binding IRAK1, we identified key interacting residues by molecular docking simulation and performed targeted mutagenesis studies. Results: Pacritinib inhibited the activity of FLT3 and all JAK family members except JAK1 at IC50 values of Conclusions: Pacritinib is a specific inhibitor of IRAK1 that blocks IL-1 signaling in AML-derived cell lines and primary patient samples. To our knowledge, pacritinib is the first clinically viable molecule to robustly and selectively inhibit IRAK1. These findings may be extended to a variety of cancer types with a dependence on IRAK1 kinase. Disclosures Druker:Novartis Pharmaceuticals: Research Funding; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; ARIAD: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Oncotide Pharmaceuticals: Research Funding; Oregon Health & Science University: Patents & Royalties; Roche TCRC, Inc.: Consultancy, Membership on an entity9s Board of Directors or advisory committees; AstraZeneca: Consultancy; McGraw Hill: Patents & Royalties; Millipore: Patents & Royalties; Sage Bionetworks: Research Funding; Leukemia & Lymphoma Society: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Gilead Sciences: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Bristol-Myers Squibb: Research Funding; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees. Singer:CTI BioPharma, Corp: Employment, Equity Ownership. Agarwal:CTI BioPharma: Research Funding.