Combination of Momelotinib and Citarinostat Show Strong Synergistic Effect in Lymphoid Cell Lines Co-Targeting JAK2/STAT3 and HDAC6

Introduction. Janus kinases (JAKs) are well described signaling kinases comprising four family members JAK1, JAK2, JAK3 and TYK2 that are essential in hematological malignancy, as JAK mutations have been shown to contribute to the pathogenesis of myeloproliferative disorders. Momelotinib is a potent inhibitor of JAK1/JAK2 that demonstrated efficacy in patients with primary and secondary myelofibrosis. HDAC6 has been reported to be overexpressed in lymphoid cells and its inhibition has demonstrated activity in preclinical and clinical study of lymphoproliferative disease. Citarinostat, a second generation HDAC6 selective inhibitor, is a well-tolerated compound compared with nonselective HDAC inhibitors, with reduced potency against Class I HDACs while retaining anticancer effectiveness. Both drugs are currently under investigation in clinical trials, they show a good toxicity profile and are both orally available.

Methods. Momelotinib and citarinostat alone and in combination were tested in 12 lymphoid cell lines: WSU-NHL, RL, Karpas422 (follicular lymphoma), Granta519, Jeko1 (mantle cell lymphoma), Hut78 (cutaneous T cell lymphoma), Karpas299 (anaplastic large cell lymphoma), L540, L1236 (Hodgkin's lymphoma), U266, RPMI8266 (multiple myeloma) and MEC1 (chronic lymphocytic leukemia).

Synergistic interaction was evaluated using the Chou-Talalay method. Annexin V staining, ROS generation, cell cycle and migration assay were determined by flow cytometry. ATP levels and Mitochondrial Membrane Potential (ΔΨm) were evaluated by fluorometric assay. Lactate levels and Cyt-C were evaluated by colorimetric assay. Activity of caspases-8,-9 and 3 was measured using colorimetric assay. ER stress and apoptosis-related proteins and JAK2/STAT3 were detected by western blotting. Clonogenic survival was studied with the methylcellulose clonogenic assay. Co-cultures with bone marrow stromal cells were also performed.

Results. The combination of momelotinib (1 μM) and citarinostat (4 μM) at 24 h showed a synergistic effect in WSU-NHL, RL, Karpas422, Jeko1, Hut78, Karpas299, L540, RPMI8226 and U266 cells with CI values < 1 and antagonist effect in L1236, Granta519 and Mec1 cells with CI > 1. We studied seven lymphoid cell lines (WSU-NHL, RL, Karpas422, Jeko1, L-540) which were particularly sensitive to the drug combination and two cell lines (L-1236, Granta-519) that showed an IC > 1. Drug combination exhibited a strong cytotoxicity, evidenced by reduction of mitochondrial depolarization, depletion of ATP and lactate levels and Cyt-C release from the mitochondria but also by increase in cellular apoptotic index and reactive oxygen species levels, leading to arrest in the sub-G0/G1 phase of the cell cycle. Apoptosis induced by the drug combination was exerted via the mitochondrial apoptotic pathway as demonstrated by upregulation of caspase-9 that was especially evident in WSU-NHL and Karpas422 with a fold increase of 3.2 and 3.8. The extrinsic apoptotic pathway was active in Karpas422 and Jeko1 cells as evaluated by upregulation of caspase-8 but not in WSU-NHL, RL and L540. The apoptosis was associated with activation of caspase-3, PARP and with increased hallmarks of ER stress and was mediated by the increased expression of the pro-apoptotic proteins Bad, Bax and Bim and downregulation of Bcl2, Bcl-xL and Mcl-1. Drug combination inhibited the migration induced by CXCL12 (chemoattractant known as stromal-cell derived factor-1, SDF-1α), reduced clonogenic survival and suppressed cell viability of lymphoid cells when co-cultured with bone marrow mesenchymal stromal cells targeting JAK2/STAT3 pathway and confirming acetylation of acetyl-α tubulin.

Conclusion. Due to the good toxicity profile and the oral administration, combined therapy with momelotinib and citarinostat may represent a promising novel therapeutic modality for hematological malignancies. The study is ongoing and further investigation is required.