SHP2 Inhibition Suppresses RAS-ERK Signaling, the Development of JAK2 Inhibitor Persistence, and MPN Development in Pre-Clinical Models of Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are neoplastic myeloid diseases driven by aberrant JAK2 signaling due to mutations in JAK2, MPL and CALR. There are three FDA approved JAK2 inhibitors for MPN patients: ruxolitinib, fedratinib and pacritinib. These inhibitors reduce MPN patients’ constitutional symptoms and hence improve quality of life. However, JAK2 inhibitors have limited ability to reduce mutant JAK2 allele burden and do not readily alter the natural history of disease, seemingly due to MPN-driving cells’ ability to persistently survive chronic JAK2 inhibitor therapy. However, preclinical studies continue to demonstrate a requirement for aberrant JAK2 activity to maintain disease phenotypes, thus indicating MPN patients may benefit from better JAK2 inhibitors or combination therapies.

In order to identify potential mechanisms of JAK2 inhibitor persistent survival, we carried out phosphoproteomic analyses in two MPN model cell lines (UKE-1 and SET2) in ruxolitinib naïve and persistent states. These analyses revealed altered levels of phosphorylation in persistent cells, including peptides belonging to SHP2, an oncogenic phosphatase known to regulate JAK-STAT signaling as well as RAS-ERK activation and which has been shown to contribute to adaptive resistance to targeted cancer therapies. One reported mechanism of JAK2 inhibitor persistence is via JAK2-independent ERK activation. Therefore, we hypothesized that SHP2 may contribute to persistent survival of MPN cells during JAK2 inhibitor treatment. To assess the role of SHP2 in JAK2 inhibitor persistence we used allosteric SHP2 inhibitors, including RMC-4550, and observed that ruxolitinib persistent cells were more sensitive to SHP2 inhibition compared to drug naïve cells. In addition, inhibition of SHP2 synergized with ruxolitinib, antagonizing the development of ruxolitinib persistence in part through enhanced induction of apoptosis. Importantly, SHP2 and JAK2 inhibition also synergistically suppressed the neoplastic growth of cells isolated from JAK2-V617F-positive MPN patients.

Interestingly, while we observed phospho-ERK return within 24 hours of ruxolitinib treatment, this was suppressed when SHP2 activity was inhibited. Since SHP2 signals to activate the RAS-ERK pathway and inhibition of SHP2 suppressed ERK reactivation in ruxolitinib treated cells, we next assessed the levels of active RAS in these cells. We found significantly reduced RAS-GTP levels in UKE-1 and SET2 cells treated with the combination of JAK2 and SHP2 inhibitors compared to the treatment with each drug alone. Moreover, co-immunoprecipitation studies suggested that SHP2 complexes with SOS1 and GAB1 and this interaction is inhibited by combined JAK2 and SHP2 inhibitor treatment. Interestingly, we observed that a SHP2-GAB1 complex is lost in JAK2 inhibitor persistent cells, suggesting a potential signaling rewiring that allows for continued cell signaling that is associated with ruxolitinib persistent cell growth and survival (e.g., phospho-ERK).

We next used the MPL-W515L bone marrow transplant MPN mouse model to test the therapeutic potential of SHP2 inhibition. While our initial experiments indicated RMC-4550 displayed impressive efficacy to antagonize MPN phenotypes, we also assessed RMC-4550 (20 mg/kg, q.o.d.) in combination with ruxolitinib (30 mg/kg, q.d.) in this therapeutic model. While we observed a significant reduction in white blood cell counts after a week of treatment with RMC-4550 (~4-fold reduction) and ruxolitinib (~2.5-fold reduction) alone compared to vehicle treated mice, the combination had the most pronounced effect (~8-fold reduction). After another week of treatment, we observed while white blood cell counts started going up in monotherapy cohorts, but the combination of ruxolitinib and RMC-4550 continued to suppress cell counts lower than those at the initiation of treatment, and not surprisingly mice treated with the combination therapy ultimately survived significantly longer. These ongoing experiments suggest SHP2 inhibition may enhance the efficacy of ruxolitinib and antagonize the development of JAK2 inhibitor persistence, possibly through greater suppression of RAS-ERK signaling.

In summary, these results suggest that inhibition of SHP2 as a monotherapy or in combination with JAK2 inhibition may provide a promising therapeutic approach to improve targeted therapies for MPN patients.

Amin:Iovance Biotherapeutics, Inc.: Current Employment. Zhang:Sensei Biotherapeutics: Current Employment; mProbe Inc.: Ended employment in the past 24 months. Akuffo:AstraZeneca: Current Employment. Haura:Ellipses Pharma: Consultancy; ORI Capital II, Inc.: Consultancy; Revolution Medicines, Inc.: Research Funding; Amgen Inc.: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Kanaph Therapeutics, Inc.: Consultancy. Epling-Burnette:AbbVie Inc.: Current Employment, Current holder of stock options in a privately-held company, Other: Travel, Accommodations, and Expenses; Ibis Therapeutics: Ended employment in the past 24 months; Merck: Current holder of stock options in a privately-held company; TEVA: Current holder of stock options in a privately-held company; AstraZeneca: Current holder of stock options in a privately-held company. Reuther:Revolution Medicines, Inc.: Research Funding; Incyte Corporation: Research Funding.