Autophagy Inhibition Potentiates Ruxolitinib-Induced Apoptosis in JAK2^V617F Cells

Introduction: Autophagy is a paradoxical and evolutionarily conserved cellular process that is fundamental to eliminating harmful intracellular components and allowing cells to adapt to nutrient starvation or stress conditions. Ruxolitinib, a selective JAK1/2 inhibitor approved by the FDA for treatment of intermediate and high-risk primary myelofibrosis and polycythemia vera patients, provides clinical benefits, but fail to eliminate the myeloproliferative neoplasm (MPN)-initiating cells. Frequency of mutations in autophagy-related genes has recently been reported in myeloid neoplasms (Visconte et al. Leukemia 2017). Considering that JAK2^V617F mimics constitutive growth factors signal and leads to mTOR/PI3K activation, we hypothesized that ruxolitinib induces autophagy as a mechanism of resistance. Aims: To investigate the effects of ruxolitinib treatment on autophagy-related genes and cellular processes, the potential benefit of autophagy inhibitors plus ruxolitinib in JAK2^V617F cells, and to verify frequency and clinical impact of autophagy-related genes mutations in MPN patients. Methods: SET2 cells (JAK2^V617F-positive) were treated with increasing doses or exposure to ruxolitinib and subjected to protein extraction or autophagy analysis. Ruxotilinib (300 nM) and autophagy inhibitors (3-methyladenine [1 mM], bafilomycin A1 [10 nM] and chloroquine [20 μM]) were used in monotherapy or in combination. The expression of 79 autophagy-related genes was investigated in SET2 cells upon ruxolitinib treatment by RNA-seq (data obtained from Meyer et al. Cancer Cell 2015) and validated by qPCR and Western blot. Autophagy was evaluated by acridine orange staining and LC3BI/II or SQSTM1/p62 consumption, apoptosis by annexin V/PI staining and caspase 3 cleavage, and mitochondrial damage by JC-1 staining. Mutations in 124 autophagy-related genes were investigated by whole genome sequencing in a cohort of 65 MPN patients from a single Institution. ANOVA and Bonferroni post-test and Mann-Whitney test were used as appropriated. Kaplan-Meyer curves, log-rank test, and cox regression analysis were applied for survival analysis. Results: Ruxolitinib treatment promoted an accumulation of acidic vesicular organelles and consumption of LC3BI/II and SQSTM1/p62 proteins, characterizing autophagy induction. Using a 1.3-fold cutoff in both directions, treatment with ruxolitinib modulated the expression of 26 out of 79 autophagy-related genes investigated. BCL2 and BCL2L1 downregulation, BAX upregulation and stable BECN1 expression were validated, which impacted BCL2/BAX and BCL2/BECN1 ratios. In SET2 cells, ruxolitinib treatment reduced important autophagy regulators in a dose-dependent manner, including STAT3^Y705, STAT5^Y694, mTOR^S2448, p70S6K^T421/S424 and 4EBP1^T70 phosphorytation, and BCL2 and BCL-XL expression. All autophagy inhibitors significantly suppressed the ruxolitinib-induced autophagy, being bafilomycin A1 (reduction of 89%) and chroloquine (reduction of 70%) more efficient than 3-methyladenine (reduction of 10%) (all p<0.05). Autophagy pharmacological inhibition significantly increased ruxolitinib-induced apoptosis (all p<0.05) and accumulation of cells containing damaged mitochondria (all p<0.05). Mutations in autophagy-related genes were found in 25% (16 out of 65) of MPN patients and was associated with increased age (p<0.05). TP53 mutations were found in 6 patients and ARSA, TGFB1, APP, PPP2R3A, BECN1, SQSTM1, HGS, TNFSF13B, ATG2A, STAT3, ATG9B, WAC or PRKACB mutations were found in one case each. Mutated patients presented JAK2 (n=14), CALR (n=1) or MPL (n=1) mutations. Median overall survival for autophagy-related genes mutated and wild-type patients were 20 months vs. not reached (p=0.066), respectively. In multivariate analysis using mutations in TP53 only, mutations in other autophagy-related genes, and DIPSS as confounders, only DIPSS independently predicted overall survival (p<0.001). Conclusions: Ruxolitinib induced autophagy in JAK2^V617F cells through modulation of mTOR/p70S6K/4EBP1 and STAT/BCL2 axis, which potentially represent a mechanism of resistance to apoptosis. Our results indicate that combination of ruxolitinib with autophagy pharmacological inhibitors, especially chloroquine, may be a promissory strategy to improving apoptosis induction in JAK2^V617F cells.