Critical Interferon-Response Genes Are Induced By JAK2^V617F but Repressed By BCR-ABL, and They Are Responsible for the Differential Effects of Interferon in PV Vs. CML

Interferon-alpha (IFNa) is successfully used as monotherapy in JAK2V617F positive myeloproliferative neoplasms (MPN) such as polycythemia vera (PV), but with only limited success in chronic myeloid leukemia (CML). Here, we investigated the mechanisms of IFNa in JAK2V617F vs. BCR-ABL positive cells.

In 32D cells stably transduced with JAK2V617F, IFNa alone (100 U/mL) significantly decreased the viability to less than 50% of control but had no significant effect on 32D-BCR-ABL cell viability, as assessed by MTT assays. Gene expression microarray data of 32D-JAK2V617F cells revealed strong upregulation of a number of interferon-stimulated genes compared to empty vector-transduced (EV) cells, and RT-PCR validation confirmed significantly increased expression of Stat1, Stat2 and Irf9 and reversal of these effects by ruxolitinib treatment. Conversely, BCR-ABL led to a decrease of these three genes, and this was reversed by imatinib. Similar responses were seen in human HEL and K562 cells. Histone (H3K9) acetylation of Stat1, Stat2 and Irf9 promoters was significantly higher in 32D-JAK2V617F vs. 32-BCR-ABL cells, correlating with gene expression. IFNa treatment increased Stat1, Stat2 and Irf9 mRNA in both 32D-JAK2V617F and 32D-BCR-ABL cells, and this effect was reduced by ruxolitinib but potentiated by imatinib. In line with these results, the phosphorylation of STAT1 (P-STAT1) was induced by IFNa in all cell lines. In addition, Stat1, Stat2 and Irf9 mRNA was significantly downregulated in peripheral blood samples from CML but not PV patients, as compared to healthy controls (all subjects provided written informed consent, as approved by the local ethics committee). Likewise, Stat1 and Stat2 expression was significantly downregulated in SCLtTA/BCR-ABL but not JAK2V617F transgenic mice.

We next analyzed whether ectopic expression of STAT1 or STAT2 or their dominant-negative Y/F mutants affected the IFNa sensitivity of 32D-JAK2V617F vs. 32D-BCR-ABL cells. While there was little apoptosis in empty-vector- or STAT1-overexpressing 32D-BCR-ABL cells, overexpression of the STAT1Y701F mutant in these cells showed a significantly increased basal level of apoptosis, which was further enhanced by IFNa (p<0.05). This was associated with IFNa-induced P-STAT1 but complete absence of P-STAT2. Conversely, EV- or STAT1-overexpressing 32D-JAK2V617F cells showed strong IFNa-induced apoptosis, while STAT1Y701F overexpression in these cells led to abrogation of IFNa-induced apoptosis. This was associated with decreased P-STAT1 but preserved P-STAT2. On the other hand, STAT2 overexpression decreased P-STAT1 and P-STAT2 and led to mild IFNa-induced apoptosis in 32D-BCR-ABL cells, but preserved the high P-STAT1 and P-STAT2 as well as potent IFNa responsiveness in 32D-JAK2V617F cells.

In conclusion, JAK2V617F and BCR-ABL induce opposite effects on IFNa target genes in vitro and in vivo, suggesting that JAK2V617F but not BCR-ABL sensitizes cells to interferon. Moreover, while the major factor of IFNa-induced apoptosis in JAK2V617F positive cells is an increase of P-STAT1, the major determinant in BCR-ABL positive cells is a decrease of P-STAT2. Together, these results may possibly explain the differences of clinical responses to IFNa monotherapy in PV vs. CML.