Combining interferon-α and arsenic for the treatment of JAKV617F mutant MPN

Myeloproliferative neoplasms (MPN) are a group of stem cell diseases that are typified by an increased production of differentiated blood cells. The JAK^V617F driver mutation is common in polycythemia vera (PV), essential thrombocytopenia (ET), and primary myelofibrosis (MF). Treatment of MPN with interferon (IFN)-α, which targets JAK2^V617F clones, can bring about a complete molecular response, but only in 8–20% of patients.¹ The promyelocytic leukemia (PML) protein, a sensor of oxidative stress and inducer of senescence, has been shown to be transcriptionally activated by IFNs. In addition, arsenic trioxide (ATO), a curative treatment used for acute promyelocytic leukemia, binds to PML protein and enhances its activity.¹

Tracy Dagher and team recently published their work on the use of an IFN-α/arsenic combination therapy for the eradication of MPN cells harboring the JAK^V617F mutation.¹ The study, published in the Journal of Experimental Medicine, utilized mouse models and human cell lines to study the involvement of the PML protein in combination treatment with IFN-α and ATO.

Methods

The mouse models used conditional flexed Jak2^V617F knock-in mice, crossed with VavCre transgenic mice, and the resultant Jak2^V617F mutation-expressing mice were then also crossed with UbiGFP transgenic mice to enable the assessment of allelic burden. To assess PML involvement in IFN response, Pml^f/f mice were crossed with VavCre transgenic mice to produce mice with a germinal deletion of Pml exon 3.

The human cells used included the following:

• The human megakaryoblastic UT-7 11oc1 cell line, transduced with either wild-type JAK2 or JAK2^V617F vectors.
• Human CD34⁺ cells that had been purified from the peripheral blood of patients with PV or ET and healthy volunteers.

Results

• PML nuclear bodies (NB) were significantly greater in number and size in CD34⁺ cells from patients with JAK2^V612F MF compared with CD34⁺ cells from healthy volunteers. Treatment with IFN-α, ATO, or the combination of IFN-α and ATO further increased the size of PML NB both in patient cells and cells derived from mice Jak2^V612F, while control cells did not show significant changes. The increase in PML NB numbers after IFN-α plus ATO treatment was abrogated in Jak2^V617F mouse cells that were Pml^−/−.
• Growth inhibition of UT-7 cells with IFN-α was greater in cells expressing JAK2^V612F than in those not expressing JAK2^V612F, and it was further enhanced by the combination treatment of IFN-α and ATO. Furthermore, compared with IFN-α or ATO alone, IFN-α and ATO combination treatment significantly decreased erythroid colony formation and size in JAK2^V612F progenitor cells from patients with MF or PV. By contrast, little effect was seen on cells from healthy individuals or the JAK2^WT progenitor cells from patients with MF or PV. Results could be reproduced in bone marrow (BM) cells from Jak2^V612F and Jak2^WT mice ex vivo.
• Assessments of treatment in a Jak2^V612F mouse model demonstrated that the addition of ATO to IFN-α treatment enhanced the effect of IFN-α. ATO alone had only moderate effects on white blood cell counts and spleen size, however, when combined with IFN-α it significantly improved the IFN-α-induced hematological response, as seen by the decrease in white blood cells, hematocrit, and splenomegaly.
• In total, 53% of Jak2^V612 mice treated with ATO and IFN-α remained disease-free after 9 weeks, whereas none of the untreated mice were recurrence-free, and only 27% of the mice treated with IFNα alone remained recurrence-free. Using BM cells from these Jak2^V612F mice and transferring them into secondary recipient mice resulted in MPN development in 88% of the secondary recipient mice transplanted with cells from non-treated mice, in 100% of those transplanted from ATO-treated mice, and in 60% of those receiving BM cells from IFN-α-treated mice. By contrast, none of the mice who received a transplant of BM cells from mice who had been treated with a combination of ATO and IFN-α became ill.
• Using CD34⁺ cells from patients with JAK2^V612F MF, the team were able to demonstrate that silencing of the PML gene severely decreased the effect of ATO and IFN-α on colony formation. By treating Jak2^V612F mice that were chimeric for PML^-/- and PML^wt, the group further demonstrated that both IFN-α and ATO plus IFN-α were able to improve hematological parameters. However, only the combination was able to reduce the percentage of PML^wt cells, demonstrating that PML is involved in the removal of JAK2^V612F progenitors/stem cells in response to combination treatment.
• In UT-7 cells, the expression levels of genes associated with senescence were shown to be enhanced in JAK2^V612F expressing cells. Under culture conditions favoring megakaryocyte differentiation, progenitor cells from healthy individuals showed strongly enhanced senescence, whereas this was not seen in progenitor cells from patients with MF. However, treatment with ATO plus IFN-α restored senescence in those cells from patients with MF, which was abrogated when lentiviral-introduced short hairpin RNA targeting PML was expressed in these cells. Similarly, induction of senescence-associated genes was observed in Jak2^V617F mouse cells following ATO plus IFN-α combination treatment, but an absence of PML significantly reduced senescence gene expression.

Conclusion

IFN-α has been shown to target JAK2^V612F progenitors; however, long term efficacy has been lacking in most patients. In this study, the authors demonstrated that ATO plus IFN-α combination treatment improves IFN-α long-term efficacy by targeting JAK2^V612F disease initiating cells, with a favourable safety profile, and that this improvement is primarily through inhibition of PML. They also provided evidence that the ATO plus IFN-α combination reverts the pathological senescence response in JAK2^V612F expressing cells. The team highlights the parallels with acute promyelocytic leukemia, suggesting that PML may be a common feature of various cancer types. Based on the presented data, the authors predict that administration of ATO will enhance responses to IFN-α therapy in patients with MPN.