Stathmin 1 Silencing Amplifies Ruxolitinib-Induced Apoptosis in JAK2^V617F Cells

Introduction: The JAK/STAT pathway is constitutively activated in the myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). JAK/STAT pathway has been targeted by Ruxolitinib, a selective JAK1/2 inhibitor approved by FDA for intermediate and high-risk PMF treatment. Results from phase III clinical trials demonstrated that Ruxolitinib is well-tolerated, reduces inflammatory cytokines and splenomegaly, ameliorated constitutional symptoms; however, Ruxolitinib did not reverse bone marrow fibrosis, suggesting that the development of better approaches are necessary. In addition to the STAT3 transcriptional activity in the nucleus, activated STAT3 binds to and inhibits Stathmin 1 in the cytoplasm, resulting in microtubule stability. Stathmin 1 is an important cytoplasmic microtubule-destabilizing protein that plays critical roles in proliferation and accurate chromosome segregation through regulation of microtubule dynamics. Paclitaxel, an anti-neoplastic compound, is a microtubule-targeted drug that induces microtubule polymerization, cell-cycle block at the metaphase-anaphase transition and cell death, and inhibits Stathmin 1 activity. We hypothesized that an undesirable effect of Ruxolitinib treatment could be Stathmin 1 activation as a consequence of STAT3 inhibition; hence targeting Stathmin 1 in addition to JAK2 might be a reasonable approach in MPN.

Aims: To investigate Stathmin 1 expression in CD34⁺ cells from MPN patients and the effects of Stathmin 1 silencing or Paclitaxel-induced microtubule stability on Ruxolitinib JAK2^V617F-treated cells.

Materials and Methods: CD34⁺ cells were obtained from a total of 104 peripheral blood (PB) samples collected from healthy donors (n=22) and patients with MPN (n=82; ET=34, PV=23, PMF=25; JAK2^V617F=52, JAK2^wt=30, CALR^mut=19 and CALR^wt=54). Gene and protein expressions were evaluated by qPCR and Western blot. In JAK2^V617F-positive HEL cells, Stathmin 1 was stably knocked down with specific shRNA-expressing lentiviral vector, and cell growth, clonogenicity and apoptosis were evaluated by MTT assay, colony formation, and AnnexinV/PI and Caspase 3 activation by flow cytrometry, respectively. Alternatively, HEL cells were submitted or not to Paclitaxel treatment (5 and 10 nM). All conditions were evaluated in the presence or not of Ruxolitinib (100 and 300 nM). Statistical analyses were performed by Student’s t-test or Mann-Whitney test, as appropriate.

Results: Stathmin 1 transcripts were significantly increased in PB CD34⁺ cells from PMF patients (2.73 [0.49-8.32]; p=.005) compared with healthy donors (1.08 [0.08-7.17]). No difference in Stathmin 1 expression was observed between healthy donors, ET and PV PB CD34⁺ cells. Stathmin 1 levels did not significantly differ between JAK2^wtvs. JAK2^V617F (p=.20); and CALR^wtvs. CALR^mut patients (p=.63). In HEL cells, Stathmin 1 silencing significantly reduced cell proliferation (p<.05) and clonal growth (p<.01). Although Stathmin 1 silencing itself did not modulate apoptosis, it increased apoptosis induced by Ruxolitinib treatment (p<.01). Stathmin 1 silencing resulted in microtubule stability (increased alpha-tubulin acetylation), whereas Ruxolitinib treatment induced microtubule instability (decreased alpha-tubulin acetylation) and inhibited JAK2 and STAT3 activity. Stathmin 1 silencing in association with Ruxolitinib prevented microtubule instability. In the attempt to pharmacologically inhibit Stathmin 1 activity, cells were treated with Paclitaxel, in association or not Ruxolitinib. Combined treatment resulted in a significantly increased apoptosis compared to monotherapy (p<.01). The ability of Paclitaxel of inhibiting Stathmin 1 was confirmed by increased phosphorylation of Stathmin 1 at serine 16 (an inhibitory site) in either Paclitaxel-treated cells and in Paclitaxel plus Ruxolitinib-treated cells, which was not observed in Ruxolitinib-treated cells.

Conclusions:Stathmin 1 is highly expressed in PMF. Our study adds new insights for Stathmin 1 in hematological malignancies and indicates a possible role of Stathmin 1 in the aberrant JAK2^V617F signaling pathway. Targeting Stathmin 1 or microtubule instability may improve Ruxolitinib response in JAK2^V617F positive MPN patients.