Reversine exhibits antineoplastic activity in JAK2V617F-positive myeloproliferative neoplasms

JAK2/STAT signaling participates in the Ph-negative myeloproliferative neoplasms (MPN) pathophysiology and has been targeted by ruxolitinib, a JAK1/2 inhibitor. In the present study, the impact of ruxolitinib treatment on cytoskeleton-related genes expression was explored. In SET2 cells, AURKA and AURKB expression/activity were downregulated in a dose- and time-dependent manner by ruxolitinib. Reversine, a multikinase inhibitor selective for aurora kinases, reduced cell viability in a dose- and/or time-dependent manner in JAK2V617F cells. Reversine significantly increased apoptosis and mitotic catastrophe, and reduced cell proliferation and clonogenic capacity in SET2 and HEL cells. In the molecular scenario, reversine induced DNA damage and apoptosis markers, as well as, reduced AURKA and AURKB expression/activity. In SET2 cells, reversine modulated the expression of 32 out of 84 apoptosis-related genes investigated, including downregulation of antiapoptotic (BCL2, BCL2L1, and BIRC5) and upregulation of proapoptotic (BIK, BINP3, and BNIP3L) genes. Synergism experiments indicated that low dose of reversine had a potentiating effect under ruxolitinib treatment at low doses in SET2 cells. In summary, our exploratory study establishes new targets, related to the regulation of the cellular cytoskeleton, for potential pharmacological intervention in MPN. These findings indicate that AURKA and AURKB participate in the JAK2/STAT signaling pathway and contribute to the MPN phenotype.


Supplementary
. Primer sequences and concentrations.
Supplementary Figure 1. Raw data of the synergism test in SET2 and HEL cells. Dose-response cytotoxicity for combined treatment were analyzed by methylthiazoletetrazolium (MTT) assay for SET2 and HEL cells treated with graded concentrations of reversine (1, 2.5, 5, 10, 25, and 50 μM) and ruxolitinib (3, 10, 30, 100, 300, and 1000 nM) alone or in combination with each other for 48 hours. Values are expressed as the percentage of viable cells for each condition relative to untreated controls. Results are shown as the mean±SD of four independent experiments.
Supplementary Figure 2. Long-term reversine exposure inhibits autonomous clonal growth in JAK2 V617F cell lines. Colonies containing viable cells were detected by MTT after 10 days of culture of SET2 and HEL cells exposed to reversine (1, 2.5, 5, 10 μM) and normalized to the corresponding DMSO-treated controls (Ø). Colony images are shown for one experiment and the bar graphs show the mean±SD of at least four independent experiments. ***p<0.0001; ANOVA test and Bonferroni post-test.
Supplementary Figure 3. Effects of reversine treatment on AURKA and AURKB mRNA expression in SET2 and HEL cells. qPCR analysis of AURKA and AURKB mRNA expression in SET2 (A) and HEL (B) cells treated with graded concentrations of reversine (vehicle, 1, 2.5, 5, or 10 µM) for 48 hours. Bar graphs represent the mean±SD of at least four independent experiments. The p values are indicated in the graphs; *p<0.05, **p<0.001, ***p<0.0001; ANOVA test and Bonferroni post-test.
Supplementary Figure 5. Effects of ruxolitinib treatment on AURKA and AURKB expression/activation in HEL cells. qPCR analysis of AURKA (A) and AURKB (B) mRNA expression in HEL cells treated with graded concentrations of ruxolitinib (vehicle, 100, 300, or 1000 nM) for 48 hours. Bar graphs represent the mean±SD of at least four independent experiments. The p values are indicated in the graphs; *p<0.05, **p<0.001; ANOVA test and Bonferroni post-test. (C) Western blot analysis for p-STAT3 Y705 , p-STAT5 Y694 , AURKA, AURKB, and p-histone H3 S10 , in total cell extracts from HEL cells treated with graded concentrations of ruxolitinib (vehicle, 100, 300, or 1000 nM) for 48 hours; membranes were reprobed with the antibody for the detection of the respective total protein or α-tubulin, and developed with the SuperSignal™ West Dura Extended Duration Substrate system using a Gel Doc XR+ imaging system. Figure 6. Cell viability upon treatment with AURKA, AURKB, MPS1, and JNK selective inhibitors in SET2 and HEL cells. Dose-response cytotoxicity was analyzed by methylthiazoletetrazolium (MTT) assay for SET2 and HEL cells treated with graded concentrations of Aurora-A Inhibitor I (A), AZD1152-HQPA (B), NMS-P715 (C) and SP600125 (D) for 48 hours. Values are expressed as the percentage of viable cells for each condition relative to untreated controls. Results are shown as the mean±SD of at least four independent experiments. The p values and cell lines are indicated in the graphs; ***p<0.0001; ANOVA test and Bonferroni post-test. Figure 7. Whole gel images of western blotting analysis. Western blot analysis for protein phosphorylation and expression in total cell extracts from SET2 and HEL cells treated, or not, with ruxolitinib or reversine, as indicated; membranes were reprobed with the antibody for the detection of the respective total protein or αtubulin. The molecular weight of the ladder, antibodies, merged and unmerged images are indicated.