The Second Generation Type II JAK2 Inhibitor, AJ1-10502, Demonstrates Enhanced Selectivity, Improved Therapeutic Efficacy and Reduced Mutant Cell Fraction Compared to Type I JAK2 Inhibitors in Models of Myeloproliferative Neoplasms (MPNs)

Abstract

Type I JAK inhibition (JAKi) represents a mainstay therapy for myelofibrosis and hydroxyurea-resistant high-risk polycythemia vera. Current type I JAKi, including ruxolitinib (RUX) and fedratinib, improve symptoms and outcomes in MPNs; however, JAK2VF allele burden remains essentially unchanged, and efficacy wanes over time. Sustained JAK/STAT signaling plays a critical role in MPN cell persistence in the setting of type I inhibition. CHZ868, a type II JAKi that binds the inactive conformation of the kinase domain, overcomes RUX persistence in vitro and reduces Jak2VF allele fraction in vivo suggesting improved JAK2 targeting might enhance clinical efficacy. However, current type II JAKi, including CHZ868, are limited by lack of kinome specificity and off-target toxicity. We therefore sought to develop novel type II JAK2i with improved potency and selectivity. Computational free energy perturbation and structure-activity relationship-based methods were used to identify lead type II inhibitor compounds. This work, followed by Absorption, Distribution, Metabolism and Excretion modeling, led to the development of AJ1-10502. Kinome selectivity profiling of AJ1-10502 revealed potent JAK2 selectivity with minimal kinase cross-reactivity compared to CHZ868, including among other JAK family tyrosine kinases (Figure 1A). Assessment of in vitro efficacy of AJ1-10502 in SET2 inhibitor naïve and SET2 RUX persistence (RUXper) cells revealed concentration-dependent inhibition of proliferation of RUXper cells comparable to that of CHZ868. The IC50 for AJ1-10502 in RUXper cells was similar to the IC50 observed in inhibitor naïve cells. We next evaluated AJ1-10502 in vivo using a novel dual Dre/Cre-recombinase Jak2VF knock-in/knock-out model allowing for the comparison of JAKi to Jak2VF genetic deletion. AJ1-10502 demonstrated reductions in leukocytosis comparable to that of RUX (K/uL: VEH 18.7 vs. RUX 10.1 vs. AJ1-10502 9.3) but dose-dependent improvements in hematocrit, platelet levels, and spleen weights superior to RUX, with reductions in spleen weights on par to that of Jak2VF deletion (mg: VEH 480 vs. RUX 255 vs. AJ1-10502 145 vs. del 107, p<0.05). A greater degree of restoration of splenic architecture was also observed with AJ1-10502 compared to type I JAKi. Critically, we observed reductions in peripheral blood (PB) and bone marrow (BM) mutant allele fraction with AJ1-10502 not observed with RUX, including within Mac1+Gr1+ myeloid cell fractions (VEH 94% vs. RUX 92% vs. AJ1-10502 78.5%, p<0.05), consistent with a mutant-biased reduction in myeloid output. In a separate series of competitive transplants using a Cre-inducible human JAK2VF transgenic mouse line, we validated the phenotypic changes in regard to leukocytosis, hematocrit, and spleen weight reduction seen with the DreCre model. Most importantly, we confirmed significant reductions in mutant cell fraction within the hematopoietic stem (HSC) compartment of BM and spleen, including among granulocytic-monocytic progenitor (GMP) and long-term HSC (LT-HSC) populations (Figure 1B). Finally, a separate study comparing AJ1-10502 to CHZ868 revealed no significant weight loss with AJ1-10502 (-0.5g) compared to CHZ868 (-2.2g, p<0.05) despite similar reductions in Jak2VF allele fraction suggesting comparable efficacy without systemic toxicity. In sum, AJ1-10502 is a potent, selective type II JAK2i with improved efficacy compared to RUX and an enhanced safety profile compared to previous non-selective type II JAKi. Most importantly, AJ1-10502 results in superior reductions in PB and BM mutant cell fraction in vivo not observed with type I JAKi. These data demonstrate the preclinical utility of type II JAKi with AJ1-10502 and inform a path to clinical development of type II JAKi for MPN patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal