Differential Impact of Interferon Alpha on JAK2V617F and Calr Mutated Hematopoietic Stem and Progenitor Cells in Classical MPN

Abstract

Introduction: Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are acquired clonal disorders of hematopoietic stem cells (HSC) leading to the hyperplasia of one or several myeloid lineages. They are due to three main recurrent mutations affecting the JAK/STAT signaling pathway: JAK2V617F and mutations in the calreticulin (CALR) and thrombopoietin receptor (MPL). Interferon alpha (IFNα) is the only drug that not only induces a hematological response in ET, PV and early MF, but also a significant molecular response on both JAK2V617F or CALR-mutated cells. Our broad aim was to understand the mechanism of action of IFNα. Previously, our group and others have shown that IFNα specifically targets JAK2V617F HSC in a chimeric JAK2V617F knock-in mouse model. In this study, we wanted to know how and how fast IFNα impacts the different mutated human hematopoietic compartments. Methods: A prospective study was performed with a cohort of 47 patients treated by IFNα for 3-5 years. The MPN disease distribution was 40% ET, 49% PV and 11% MF. This cohort included 33 JAK2V617F-mutated patients, 11 CALR-mutated patients (7 type 1/type 1-like and 4 type 2/type 2-like), 2 both JAK2V617F- and CALR-mutated patients and 1 MPLW515K-mutated patient. At 4-month intervals, the JAK2V617F or/and CALR mutation allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, the clonal architecture was also determined by studying the presence of the JAK2V617F or CALR mutations in colonies derived from the different hematopoietic stem and progenitor cell (HSPC) populations (CD90+CD34+CD38- HSC-enriched progenitors, CD90-CD34+CD38- immature progenitors and CD90- CD34+CD38+ committed progenitors). Results: After a median follow-up of 33 months, IFNα targets more efficiently and rapidly the HSPC particularly in HSC-enriched progenitors, than the mature blood cells in JAK2V617F patients (p<.05). Moreover, homozygous JAK2V617F clones responded more rapidly than heterozygous clones in all hematopoietic cell compartments showing that the intensity of JAK2V617F signaling is correlated with the efficacy of IFNα. This efficacy was slightly increased after a median follow-up of 51 months. In contrast, during a median follow-up of 33 months for CALR-mutated patients, IFNα targeted similarly the HSPC and the mature cells. Moreover, IFNα induced a slower response in targeting CALR-mutated HSPC than the JAK2V617F HSPC (p<.05) (see Figure). The role of associated mutations at diagnosis was also investigated in the IFNα-mediated HSPC molecular responses using a NGS targeted myeloid panel. In JAK2V617F-mutated patients, the number of associated mutations did not impact the HSPC molecular response. In contrast, in CALR-mutated patients, the only molecular responders were not associated with other mutations, although the lower number of cases should be expanded. Using Ba/F3-MPL cellular models and primary cells, we observed that JAK2V617F was more prone to sensitize to IFNα signaling (increased Phospho-STAT1 and IFN-stimulating genes (ISGs)) compared to controls or CALRdel52 mutated cells. Conclusion: Altogether, our results show that IFNα targets more efficiently the human JAK2V617F-HSPCthan the mature cells. Moreover, IFNα has a greater efficacy on JAK2V617F HSPC thanCALR-mutated HSPC. This former result was associated with a greater priming of the IFNα signaling by JAK2V617F than by CALRdel52. The molecular response was dependent not only on mutational status, but also on the presence of other associated mutations for the CALR-mutated HSPC. Patient data are currently incorporated into a mathematical model taking into account clonal architecture and associated mutations to develop an algorythm able to predict patient response. Figure. Figure. Disclosures Kiladjian: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Emerging translational science discoveries, clonal approaches, and treatment trends in chronic myeloproliferative neoplasms

“…The results are in keeping with data from patients with MF that were treated with pegIFNa, describing a decrease in JAK2 V617F but not CALR allele burden . In addition, these results were recently confirmed in an independent study presented at ASH 2018, reporting greater efficacy of IFNa in patients with JAK2 V617F vs CALR ‐mutant MPN as well as priming of JAK2 V617F ‐positive cells toward IFN‐α response . Together, these results suggest that JAK2 V617F , but not CALR mutants, sensitize cells to IFN‐α and that higher doses of IFN‐α may be needed in patients with CALR mutations (Figure ).…”

“…Myeloid cell lines expressing CALR mutants require at least 5-fold higher doses IFNa doses compared with JAK2 V617F positive cells. 50 This was not due to a decrease of IFNa receptors or increase in inhibitors of IFNa signaling, but rather to a genuine IFN-α sensitization by mutant JAK2 V617F protein: IFN-α target genes were upregulated in both PBMCs and CD34+ immature hematopoietic cells from patients with JAK2 V617Fbut not CALRmutant MPN patients. These effects were dependent on the presence of JAK2V617F, but also STAT1, suggesting that mutant JAK2 V617F increases JAK1 and STAT1 activity through a yet undefined mechanism, possibly involving the IFN-gamma receptor.…”

“…48 In addition, these results were recently confirmed in an independent study presented at ASH 2018, reporting greater efficacy of IFNa in patients with JAK2 V617F vs CALR-mutant MPN as well as priming of JAK2 V617F -positive cells toward IFN-α response. 50 Together, these results suggest that 4 JAK2V617F but not calreticulin (CALR)-mutants sensitize hematopoietic cells toward interferon alpha (IFNa). JAK2 V617F and CALR (mut CALR) mutants both associate with the MPL (thrombopoietin) receptor, thus activating the JAK2-STAT5 signaling axis and leading to transcription of expression of proliferative genes.…”

“…Koschmieder et al analyzed the mechanism for this discrepancy. Myeloid cell lines expressing CALR mutants require at least 5‐fold higher doses IFNa doses compared with JAK2 V617F positive cells . This was not due to a decrease of IFNa receptors or increase in inhibitors of IFNa signaling, but rather to a genuine IFN‐α sensitization by mutant JAK2 V617F protein: IFN‐α target genes were upregulated in both PBMCs and CD34+ immature hematopoietic cells from patients with JAK2 V617F ‐ but not CALR‐mutant MPN patients.…”

Targeting Abnormal Hematopoietic Stem Cells in Chronic Myeloid Leukemia and Philadelphia Chromosome-Negative Classical Myeloproliferative Neoplasms

“…Sustained molecular, haematological response and regression of BM fibrosis were seen in some patients after discontinuation of Peg-IFNα-2a, indicating the eradication of MPN stem cells [ 65 , 69 ] ( Table 2 ). Interestingly, the effect of Peg-IFNα-2a on JAK2 V617F+ stem cells was greater than that on CALR-mutated stem cells, with no difference in hematological response [ 70 , 71 ]. This is due to the phosphorylation and activation of JAK1-STAT1 pathway in JAK2 V617F cells, but not in CALR -mutated cells, resulting in JAK2 V617F-positive cells priming towards Peg-IFNα-2a [ 70 ].…”