Deciphering the complex clonal heterogeneity of polycythemia vera and the response to interferon alpha

Abstract Background: Interferon alpha (IFNa) is approved for the therapy of patients (pts) with polycythemia vera (PV), a subtype of myeloproliferative neoplasms (MPN), achieving high hematologic response (HR) rates. A fraction of pts also achieve molecular responses (MR), but clonal factors sensitizing for MR remain elusive. Methods: We integrated colony formation and differentiation assays with single-cell RNA sequencing (scRNAseq) and cell genotyping in PV patient-derived cells vs. healthy controls (HC) to dissect how IFNa targets diseased clones during erythroid differentiation. Results: IFNa significantly decreased colony growth in MPN and HC. Differences in MR were observed, with a higher degree of MR in pts with high variant allele frequency (VAF). While lower STAT1 but higher IFIT2 inducibility of PV vs. HC was observed, responses in individual colonies were much more variable. scRNAseq of colonies demonstrated more mature erythroid PV-derived colonies compared with HC and showed differential activation of JAK-STAT and hypoxia signaling. JAK2V617F-mutant cells showed significantly upregulated STAT5A-, heme- and G2M checkpoint-associated pathways relative to JAK2WT cells from the same pts. IFNa induced viral response genes in mutated cells from PV pts and antagonized the upregulation of metabolism genes. Subgroup analysis revealed that IFNa significantly decreased immature erythrocytic cells in PV (basophilic erythroblasts p <0.05; polychromatic erythroblasts p <0.05) but not in HC. CD71neg‑CD235a+ cells from HC ( p <0.05) but not from PV pts were inhibited by IFNa, and the number of reticulocytes was less affected in PV than in HC. Robust IFNa responses persisted throughout differentiation, with PV showing more pronounced effects than HC. Apoptosis was significantly upregulated by IFNa in PV but not HC, and the most apoptotic cells were characterized by downregulation of ribosomal genes. This link between apoptosis and ribosomal genes was corroborated through analysis of mitochondrial variants, demonstrating IFNa-induced eradication of specific clones, which harbored significantly elevated expression of ribosomal genes. Conclusion: Our findings indicate that PV-derived clones either undergo apoptosis or pass through their typical differentiation cycle, contributing to the long-term exhaustion of mutant cells. The pivotal roles of ribosomal genes and clonal prerequisites in the therapeutic mechanism of IFNa are underscored by our study.