Examining Ras/MAP Kinase Signaling in Primary Myelofibrosis May Yield Insight into the Mechanisms of JAK Inhibitor Resistance and Provide New Therapeutic Targets

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) arising from a clonal expansion in myeloid-committed hematopoietic stem and progenitor cells (HSPCs). Aberrant cytokine signaling and the acquisition of additional mutations then collectively induce a recruitment of fibroblasts to the bone marrow, leading to collagen fibrosis. Hematopoietic stem cell transplantation remains the only curative therapy. In non-transplant candidates, JAK2-inhibitors are most commonly utilized for symptomatic relief and reduction in splenomegaly but are in general not considered to be disease-modifying therapies and may lead to worsening cytopenias themselves. Recent studies, accordingly, have begun exploring alternative signaling mechanisms in PMF beyond JAK/STAT. Amongst these, mutations activating Ras signaling are frequently detected in PMF and have been associated with shortened overall survival, progression to AML and advanced disease features.

Ras-activating mutations are common in myelofibrosis and many other pathways, including those involved in cytokine signaling, also activate Ras. In order to directly evaluate this process, we examined the levels of Ras activity, measured by the binding of effector Ras binding domain (RBD) via ELISA, in peripheral blood mononuclear cells from 27 PMF patients and compared it to 13 healthy donors and 12 patients with pre-MF conditions, including polycythemia vera (PV) or essential thrombocytopenia (ET). This revealed that Ras activity was significantly higher in patients with PMF as compared to healthy donors (1.74 fold, p=0.0304) and to these other MPNs (1.64 fold, 0.0462). In addition to Ras activating mutations (NRAS, KRAS, CBL, NF1), 8 of 27 samples with no detectable Ras activating mutations also demonstrated higher Ras activity as compared to patients with PV, ET and healthy donors. Interestingly, we also observed a significant increase in Ras activity from 1.594 to 5.105 (absolute difference of 3.511, p=0.0115) in patients not on treatment (5) compared to those on JAK inhibitors (11).

To directly evaluate the relationship between Jak inhibition and Ras activation, we treated a JAK2 homozygous human erythroleukemia (HEL) cell line with ruxolitinib at 0.5 µM over a 7-day time course alongside a vehicle (DMSO) control. Here, we observed a significant increase in Ras activity, measured by RBD binding, at nearly all timepoints from 4 hours to 7 days, with a range of percentage increase from 28.4 to 146.6 (p value range 0.0002 to 0.003). Activation of downstream MAP Kinase pathway signaling, measured by pERK levels, in cells exposed to ruxolitinib was also elevated relative to vehicle. Ras activity notably increased with longer periods of exposure to ruxolitinib, suggesting that the induction of Ras/MAP Kinase by JAK inhibition occurs in a time-dependent manner.

Our preliminary data, which requires additional confirmation, suggests that JAK2 inhibition leads to activation of Ras in JAK2 mutant cells, raising the possibility that Ras activation conveys resistance to JAK inhibitors. Targeting Ras in PMF, therefore, may improve the efficacy of JAK inhibitors. To begin testing this theory, we examined the effects of combining JAK and Ras pathway inhibitors in MF cells. Here, whole bone marrow from 4 different patients with PMF was plated in methylcellulose-enriched media formulated for the growth of CD34+ cells under 4 therapeutic conditions in colony forming assays: Ruxolitinib, cobimetinib, ruxolitinib + cobimetinib and an unstimulated control. While all four patients were variably sensitive to individual therapies, the combination of ruxolitinib and cobimetinib yielded a more significant reduction in colony formation. Specifically, treatment with ruxolitinib monotherapy resulted in a reduction in colony number to 73.93% of that of untreated marrow (p=0.0025) and cobimetinib monotherapy to 79.3% (p=0.0092). Combination therapy, however, resulted in an even more substantial reduction to 45.8% (p<0.0001). While expansion is required in a larger cohort, our findings suggest that MAP Kinase pathway modulation may serve as a therapeutic strategy to overcome JAK inhibitor resistance in PMF.

Talpaz:Arcus: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Imago: Membership on an entity's Board of Directors or advisory committees; KyowaKirin: Membership on an entity's Board of Directors or advisory committees; Sumitomo: Membership on an entity's Board of Directors or advisory committees; SierraOncology: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.