The JAK1/2 Inhibitor Ruxolitinib Kills Malignant Plasma Cells and Is Synergistic with PI3K and mToR Inhibitors

Abstract 1844

Malignant plasma cell growth and survival in multiple myeloma (MM) is regulated by cytokines produced in the tumor environment. Specifically, IL-6 plays a key role by activating important signaling pathways through its gp130 receptor associated Janus kinases (JAK). Ruxolitinib (INC424/INCB018424; Novartis/Incyte) is the first small molecule JAK inhibitor approved for the treatment of patients with myelofibrosis. It is a potent inhibitor of both JAK1 and JAK2 and has an approx. 6-fold selectivity against Tyk2 and marked selectivity against JAK3 (more than 130-fold) and additional kinases. The aim of our study was to evaluate the effects of ruxolitinib on malignant plasma cells as well as its activity in combination with other pathway inhibitors.

Ruxolitinib activity was evaluated in MTS-based colorimetric cell growth assays or by [3]H-thymidine uptake. IC50 concentrations and combination index (CI) were calculated with CalcuSyn (Biosoft). Evaluating seven human plasma cell lines, ruxolitinib showed a strong cytotoxic activity on the only IL-6 dependent line INA-6 (IC50 0.23 μM). Complete growth inhibition was achieved at 1 μM, even in the presence of bone marrow stromal cells, whereas stromal cell viability and IL-6 production, as measured by specific ELISA, were maintained. Consistent with the dose-dependent inhibition of IL-6 induced STAT3 phosphorylation, apoptosis was induced, resulting in 39% and 63% annexin V-positive cells in the presence of 1 μM ruxolitinib after 48 or 72 hours, respectively. Likewise, significant growth inhibition was seen in purified tumor cells from a patient with plasma cell leukemia that were stimulated with IL-6 (IC50 0.16 μM), and in a LIF-responsive tumor subline of INA-6 (IC50 0.12 μM). In contrast, autonomously growing MM cell lines were not directly inhibited by ruxolitinib, pointing to the kinase specificity of the drug. However, IL-6 mediated drug resistance can be reversed as shown in dexamethasone-sensitive MM1.S cells.

Simultaneous inhibition of JAKs with additional signaling pathways that may be activated in myeloma cells by mutations and/or cytokines such as insulin-like growth factor-1, is hypothesized to result in increased cytotoxicity. In INA-6 cells, p44/p42 MAPK activation due to mutated N-Ras and phosphorylation of S6 protein, a downstream target of the PI3K/AKT/mToR pathway, were not abrogated by ruxolitinib. Using combinations of ruxolitinib with inhibitors of PI3K (Ly294002, NVP-BKM120) and mToR (rapamycin), synergistic effects were achieved with a combination index (CI) <1 at the effective dose levels ED50, ED75 and ED90. Other combinations are currently under evaluation.

In conclusion, ruxolitinib has strong direct cytotoxic activity against malignant plasma cells that are dependent on JAK/STAT pathway activation. The rationale exists to combine it with inhibitors of complementary pathways and other drugs to potentiate its activity and overcome cytokine or stromal cell mediated drug resistance. Thus, ruxolitinib, as a generally well tolerated drug, may offer therapeutic options for patients with MM. Clearly, the identification of molecular markers may be helpful to assess its precise use alone or in combination, and to select for patients who will benefit from such a treatment.