Combination Therapy Using JAK2 and HSP90 Inhibitors Increased Efficacy in Myelofibrosis in Vivo

Abstract ⁸⁰⁵

Following the discovery of JAK2V617F mutation in a majority of patients with Myeloproliferative Neoplasms (MPN), JAK kinase inhibitors have entered clinical trials. JAK kinase inhibitors, including the JAK1/JAK2 inhibitor INCB18424 (ruxolitinib™, Jakafi®) improve patient constitutional symptoms and reduce splenomegaly, but do not significantly reduce mutant allele burden in the majority of MPN patients. We recently showed that chronic exposure to JAK kinase inhibitors results in JAK inhibitor persistence in cell lines murine models, and patient samples. JAK inhibitor persistence is associated with heterodimerization between JAK2 and other JAK kinases; this results in JAK2-transactivation and persistent JAK-STAT signaling. ShRNA studies and pharmacologic studies with HSP90 inhibitors showed that JAK2 depletion retained efficacy in JAK inhibitor persistent cells, such that these cells remain dependent on JAK2 expression for proliferation and survival. These data suggest that pharmacologic approaches that reduce JAK2 protein expression in MPN cells may demonstrate efficacy and potentiate the effects of JAK kinase inhibition in vivo. However, combination studies with JAK inhibitors and HSP90 inhibitors in vivo have not been reported to date. In this study we tested the effects of the purine scaffold inhibitor, PU-H71, in combination with INCB18424 in a murine model of PMF. We investigated the effects of a series of different regimens in the MPLW515L murine bone marrow transplant model of PMF, including INCB18424 monotherapy, PU-H71 monotherapy, combined JAK/HSP90 inhibition at treatment onset, and INCB18424 therapy followed by the addition of PU-H71 therapy after initial response. Of note, all regimens, including combination therapy with PU-H71 and INCB18424 at maximal doses used in monotherapy trials, were well tolerated without additive hematologic or non-hematologic toxicity. Mice treated with combination therapy gained more weight compared to mice that received either monotherapy. We noted a significant reduction in WBC (89.9 K/ml vs. 8.5K/ml, p<0.0001, n=10), platelet counts (2583 K/ml vs. 737 K/ml, p<0.0005, n=10) and spleen weights (0.601g vs. 0.192g) for mice treated with PU-H71 + INCB18424 compared to mice receiving INCB18424 alone in the first 14 days after treatment onset. Consistent with the effects seen with 14 days of treatment, we noted a significant reduction in WBC (214 K/ml vs. 35.2 K/ml, p<0.03, n=4), platelet counts (2323 K/ml vs. 613 K/ml, p<0.03, n=4) and spleen weights (0.539g vs. 0.339g) for mice treated with PU-H71 + INCB18424 compared to INCB18424 after 29 days of treatment. We observed similar additive effects in mice who received PU-H71 + INCB18424 after initial treatment with INCB18424 monotherapy. We noted a decrease in total and phosphorylated JAK2 levels and in activation of downstream signaling effectors (STAT3/5, MAPK, AKT) in mice receiving INCB18424 + PU-H71; of note PU-H71 prevented (initial combination therapy) or reversed (INCB18424 followed by combination therapy) the increase in pJAK2/JAK2 seen with chronic INCB18424 treatment. Consistent with the effects on blood counts and signaling, combination therapy was associated with pathologic improvement, a reduction in bone marrow fibrosis, and a reduction in downstream signaling with immunohistochemistry. In summary, combination treatment of HSP90 and JAK inhibitors demonstrates increased efficacy without increased hematopoietic/non-hematopoietic toxicities, and combination studies with JAK and HSP90 inhibitors are warranted in the clinical setting to improve outcomes for MPN patients.