Effects of the treatment with pegIFNa, Azacytidine or pegIFNa+Azacytidine in LT-HSCs from JAK2-V617F MPN mice or JAK2-V617F;Dnmt3a-KO MPN mice.
Pegylated interferon alpha (pegIFNa) can induce molecular remissions in JAK2-V617F-positive myeloproliferative neoplasms (MPN) patients by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFNa. We investigated if DNMT3A loss leads to alterations in JAK2-V617F LT-HSCs functions conferring resistance to pegIFNa treatment in a mouse model of MPN and in hematopoietic progenitors from MPN patients. Long-term treatment with pegIFNa normalized blood parameters, reduced splenomegaly and JAK2-V617F-chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFNa in VF;Dnmt3a?/? (VF;Dm?/?) mice worsened splenomegaly and failed to reduce JAK2-V617F-chimerism. Furthermore, LT-HSCs from VF;Dm?/? mice compared to VF were less prone to accumulate DNA damage and exit dormancy upon pegIFNa treatment. RNA-sequencing showed that IFNa induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;Dm?/? compared to VF mice, indicating that the resistance of VF;Dm?/? LT-HSC was not due to failure in IFNa signaling. Transplantations of bone marrow from pegIFNa treated VF;Dm?/? mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from MPN patients with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F-positive colonies upon IFNa exposure, whereas in patients with JAK2-V617F alone the percentages of JAK2-V617F-positive colonies decreased or remained unchanged. PegIFNa combined with 5-azacytidine only partially overcame resistance in VF;Dm?/? mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone. Overall design: A cohort of MPN mice was generated by transplanting bone marrow (BM) cells from either JAK2-V617F transgenic mice (VF) or JAK2-V617F;Dnmt3a-KO mice (VFDM) into WT mice in competition with 10-fold excess BM from WT donor mice. Once the mice developed MPN phenotype (4 weeks after transplantation), they were treated with vehicle, pegIFNa (IFN), 5-azacytidine (Aza) or the combination (COMBO) of both drugs (n=4). After 8 weeks of treatment, LT-HSCs were sorted (lin- ckit+ Sca1+ CD150+ CD48-) and RNA was extracted and sequenced.