Combination Therapy In Mast Cell Neoplasms: Co-Targeting KIT and NFAT Signaling Pathways

Activating KIT mutations are a hallmark of human systemic mast cell disease and have also been identified in gastrointestinal stromal tumors, AML, melanoma, and seminoma. Traditionally, constitutively active KIT mutations have been targeted by tyrosine kinase inhibitors; however, currently available inhibitors have limited activity against resistant mutationsand sustainable responses are often not achieved. Our goal has been to identify a synergistic drug combination that increases the effectiveness of KIT inhibitors. A recent study in chronic myelogenous leukemiareported enhanced anti-proliferative/pro-apoptotic effects when combining a BCR-ABL inhibitor with inhibitors targeting a novel Wnt/Ca++/ Calcineurin/ NFATpathway. Based on shared downstream signaling mechanisms between BCR-ABL and KIT kinases we hypothesized that a similar combination would work to target KIT mutant kinases. We found that combining a KIT inhibitor with a calcineurin phosphatase inhibitorled to a synergistic decrease (average combination index (CI) values less than 1.0) in cell viability as well as an increase in apoptosis in KIT-mutant cell lines including six mastocytosis cell lines. For example, we found that 1uM of cyclosporine A (CSA) plus 20nM of dasatinibin the p815 cell line consistently reduced cell viability by 50% compared to dasatinibalone. This decrease in cell viability corresponded to a 40 fold increase in capsase 3/7 activity ([Figure 1][1]). Similar results were obtained using the other five mastocytosis cell lines. ![Figure 1][2] Figure 1 Cell viability and caspase 3/7activity following 48hrs of treatment in murine P815 mastocytomacell line The activity of NFATtranscription factors is known to be regulated by the phosphatase activity of calcineurin. To confirm our results were calcineurin- and NFAT-dependent, we repeated the previous combination experiments with the NFATspecific inhibitors, rocaglamideand tributylhexadecylphosphonium bromide (THPB). We observed a synergistic decrease (average CI value 0.81 for rocaglamideand 0.43 for THPB) in cell viability and increase in apoptosis when combining these NFAT-specific and KIT kinase inhibitors, indicating NFATas an important mediator of the observed synergy. Next we characterized NFATin KIT mutant mast cells and found constitutive NFATactivation in each cell line, as indicated by nuclear localization and dephosphorylation under unstimulated conditions. NFAT-dependent transcriptional activity, as measured by a luciferase reporter system, decreased following treatment with either calcineurinor NFATinhibitors. Unexpectedly, NFAT-dependent transcriptional activity also decreased following treatment with a number of KIT inhibitors. However, only calcineurininhibitors and not KIT inhibitors, completely blocked NFATactivation following calcium influx, suggesting a unique mechanism by which KIT inhibition affects NFAT-dependent transcriptional activity. To determine downstream effectors of the observed synergy, we measured changes in RNA expression following mono- and combination therapy. RNA-Seq revealed synergistic decreases in a number of genes including members of the JAK-STAT and MAPK pathways, which are druggabletargets downstream of this transcriptional program. Further validation studies of these targets are currently underway and will be reported later. The constitutive activation of NFAThas not been previously reported in KIT mutant cell lines. Further, our data suggest a point of convergence between KIT and NFATsignaling pathways, possibly through the interaction of NFATwith a DNA binding partner. Importantly, our results indicate that combination therapy using an NFATinhibitor and a KIT kinase inhibitor results in synergistic killing of KIT mutant mast cells. However, existing calcineurin phosphatase inhibitorssuch as CSA have known chronic long-term toxicities, including immunosuppression. To identify alternative drug targets we used RNA-Seq and found that simultaneous calcineurinand KIT inhibition modulated members of the JAK-STAT and MAPK pathways. Preliminary data indicates that targeting these pathways in combination with dasatinibachieves the same synergistic effect on KIT mutant cancer cells. We are currently testing the potential of these alternative combination therapies for the treatment of advanced mast cell disease. Disclosures: Heinrich : Onyx: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; MolecularMD: Consultancy, Equity Ownership; AROG: Research Funding. [1]: #F1 [2]: pending:yes