Higher Rates of Molecular Response to Peg-IFNa in JAK2V617F Vs. Calr Mutant MPN Patients Are Due to JAK1-Mediated STAT1 Activation and Autoregulation

Abstract

Introduction: Pegylated interferon alpha (Peg-IFNa) treatment has been described to induce molecular remission in patients (pts) with myeloproliferative neoplasms (MPN), with partial molecular remission (PMR) being achieved in approximately 30-40% of pts. However, it is unclear whether pts with CALR mutations exhibit the same response as pts with JAK2V617F mutations. Also, the precise mechanisms accounting for the responses are still unknown.Methods: We report on hematological and molecular responses, according to ELN guidelines, in 52 MPN pts (JAK2V617F n=26 vs. CALR mutation n=26; ET n=25, prePMF n=8, PMF n=19), treated at Roskilde hospital with Peg-IFNa. The mechanism of IFNa response was assessed in stably transduced 32D murine cell lines and in a cohort of MPN patients within the Czech MPN registry MIND. All pts provided written informed consent, as approved by the local ethics committees. Cell proliferation was assessed by cell counting and/or MTT assay, signaling molecules were quantified using RT-PCR, flow cytometry and/or Western blotting.Results: Pts harboring JAK2V617F mutations were more likely to achieve PMR than those harboring CALR mutations (60% vs. 23%, p<0.05 by Chi square testing), while there was no significant difference in allelic burden before Peg-IFNa treatment. Accordingly, the median allelic burden decrease from baseline in the IFNa responding pts was 66% (range 15-99%) in JAK2V617F- vs. 29% (range 5-82%) in CALR-mutant pts. These differences were present independently of the fractions of ET, prePMF, or PMF pts in the two groups. There was no significant difference in the rates of hematological response. When examining primary cells from the Czech MPN cohort, cells from JAK2V617F positive pts showed higher expression of IFNa response-related genes such as Irf1 (p=0.0350) and Stat1 (trend at p=0.0874). Moreover, IFNa receptor expression was higher in JAK2V617F positive pts (p=0.0471) and the expression levels were associated with hemoglobin (p=0.0139) and hematocrit levels (p=0.0251). This was reflected in 32D cell lines, ectopically expressing both the MPL receptor and either JAK2V617F or CALR del 52. Here, the expression of Irf7, Irf-9, Ifi-27, and Stat2 was significantly higher in JAK2VF vs. CALR mutants (all p<0.001). In addition, JAK2V617F positive cells showed higher STAT1 phosphorylation and Stat1 mRNA expression levels (p=0.0499). In particular, STAT1 showed constitutive phosphorylation in JAK2V617F but not CALR mutant cells, indicating priming of these cells towards an IFNa response. Overexpression of STAT1 but not of a dominant-negative STAT1YF mutant was able to rescue cellular IFNa-induced growth arrest in CALR positive cells to the same level of JAK2V617F positive cells. In contrast, manipulation of STAT2 expression did not alter IFNa signaling. IFNa-induced growth arrest was counteracted by selective JAK1 inhibition (using itacitinib or filgotinib) but not selective JAK2 inhibition (using BMS-911543).Conclusions: MPN pts harboring JAK2V617F mutations show superior Peg-IFNa sensitivity against the malignant clone, as detected by molecular responses, compared to pts harboring CALR mutations. The higher IFNa sensitivity of JAK2V617F mutant cells was reflected in mutant-specific cell lines and is at least in part due to JAK1-mediated increases in STAT1 phosphorylation and STAT1 transcription. These results are of considerable importance for future clinical trials of Peg-IFNa as monotherapy or in combination with JAK inhibitors. DisclosuresBrümmendorf:Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding. Knudsen:Novartis: Other: Payed for ASH2016 registration fee, travel expenses and accommodation. Hasselbalch:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOR Orphan: Membership on an entity's Board of Directors or advisory committees. Koschmieder:Roche: Other: Clinical Trial participation; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support.