Momelotinib and Citarinostat: Co-Targeting JAK2/STAT3 and HDAC6 in Lymphoid Malignancies, a New Potential Therapeutic Combination

Background . The JAK2/STAT3 signaling pathway is abnormally activated in hematological malignancies. HDAC6 has been reported to be overexpressed in primary and cultured multiple myeloma cells and B and T-cell lymphoma. Momelotinib (CYT-387) is a potent inhibitor of JAK1 and JAK2 that demonstrated efficacy in patients with primary and secondary myelofibrosis. Citarinostat (ACY-241), a second generation HDAC6 selective inhibitor, has the potential for a substantially reduced side effect profile versus current nonselective HDAC inhibitor drugs candidates due to reduced potency against Class I HDACs while retaining the potential for anticancer effectiveness. Both drugs are currently under investigation in clinical trials, they show a good toxicity profile and are both orally available.

Methods. Screening: momelotinib and citarinostat alone and in combination were tested in 12 lymphoid malignant cell lines: B-cell lymphomas (WSU-NHL, RL, Karpas-422), mantle cell lymphoma (Granta-519, Jeko-1), cutaneous T cell lymphoma (Hut-78), anaplastic large cell lymphoma (Karpas-299), Hodgkin lymphoma (L-1236, L-540), multiple myeloma (U266, RPMI8266) and chronic lymphocytic leukemia (MEC-1). IC₅₀ values of each drug were calculated based on momelotinib concentrations (1 - 10 μM), and citarinostat (1- 100 μM) after 24 - 48 h. Serial dilutions of the two agents were assessed using low concentrations and the interaction was evaluated using the Chou-Talalay method. Cell viability was measured by MTT assay. Cell cycle was determined by flow cytometry. Cell apoptosis was detected by Annexin V/PI staining and apoptosis-related proteins (Bcl₂, Bcl-xL, Bax, Bim, PARP and caspase 8) were detected by western blotting. Enzymatic activity of caspases 3, -9 was measured using colorimetric assay. JAK2/STAT3 and the related proteins were analysis by western blotting.

Results . Momelotinib alone exhibited anti-proliferation potency in the cell lines examined with IC₅₀ values ranging from 1.8 to 9.6 μM. Exposure for 24 - 48 h with citarinostat alone resulted in time- and dose-dependent inhibition of cell growth with IC₅₀ values ranging from 1.51 to 60 μM. After 24 h, low concentrations of the two drugs, momelotinib (1 μM) and citarinostat (4 μM) had a synergistic effect in WSU-NHL, RL, Karpas-422, Jeko-1, Hut-78, Karpas-299, L-540, RPMI8226 and U266 cells with CI (combination index) values < 1 and antagonist effect in L-1236, Granta-519 and Mec-1 cells with CI > 1. From the panel, we selected three lymphoid cell lines (WSU-NHL, RL, L-540) which were particularly sensitive to the drug combination and two cell lines (L-1236, Granta-519) that showed an IC > 1. While in these cell lines the combination treatment had minimal or no cytotoxic effect, WSU-NHL, RL and L-540 cells showed an increase of cells in sub-G0/G1 phase and a reduction in S phase after 24 h. Compared with single agents, the percentage of apoptotic cells ranged from 35% to 42%. Apoptosis induced by the drug combination was exerted mainly via the mitochondrial apoptotic pathway (intrinsic apoptotic pathway) as demonstrated by upregulation of caspase -9 that was especially evident in WSU-NHL and L-540 with a fold induction of 3.2 and 3.3 compared to control. The apoptosis was associated with activation of caspase-3 and PARP which were prevented by the ZVAD-fmk broad caspase inhibitor. No caspase-8 activation (extrinsic apoptotic pathway) was induced within 24 h of combination treatment. The effect was mediated by the increased expression of the pro-apoptotic proteins Bax and Bim and downregulation of key anti-apoptotic regulators, Bcl₂ and Bcl-xL. In addition, momelotinib/citarinostat combination inhibited the phosphorylation of JAK2 and its downstream mediators, including STAT3 and STAT5.

Conclusions. These preliminary results indicate that momelotinib can be potentially combined with the HDAC6 inhibitor citarinostat in lymphoid malignancies. Due to the good toxicity profile and the oral administration the combination has the potential for a completely orally therapeutic approach. The study is still ongoing and further investigation is required to better define the underlying molecular mechanisms.