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

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 <50 nM. Interestingly, Pacritinib also inhibited IRAK1 (IC₅₀ =13.6 nM). Pacritinib was comparable to quizartinib and ruxolitinib in inhibiting the growth of FLT3- and JAK2/3-dependent cells, respectively, but also reduced the viability and survival of AML cell lines and primary samples harboring a wide variety of genetic abnormalities, including RUNX translocation and RAS mutations (median IC­₅₀ = 300 nM; range = 70-600 nM). We observed a similar trend of pacritinib sensitivity in primary AML samples (median IC₅₀= 130 nm; range = 2.0-1000 nM), with a higher response rate (18/23, or 78%) than that to ruxolitinib (26%) or quizartinib (56%). Interestingly, treatment of primary AML samples with IL-1 sensitized primary AML cells to pacritinib showing a 91% response. The IL-1 dependent growth inhibition was more evident with pacritinib than with either ruxolitinib or quizartinib, suggesting that pacritinib specifically targets this inflammatory pathway. Pacritinib-treated AML cells revealed inhibition of the IL-1 pathway, including IRAK1 and p38MAPK. Consistent with the inhibitory effects of pacritinib on FLT3 and JAK2, we observed inhibition of FLT3 and ERK1/2, and of JAK2/JAK3/STAT5 in FLT3- or JAK-dependent AML cells, respectively. Computational modeling indicated that pacritinib binds to the ATP binding-pocket of IRAK1 making a critical interaction with S295. Homology alignments of IRAK1 with JAK2, FLT3, and CDK2 guided the creation of S295D, DG294, and D298K mutations in IRAK1. These mutations, which cause IRAK1 to resemble the pacritinib-insensitive CDK2, abrogated the inhibitory effect of pacritinib.

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.