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

• Interferon alpha (IFNa) induced apoptosis in polycythemia vera immature progenitors while erythroid differentiation was not inhibited

• IFNa reduced ribosome genes in apoptotic cells and high ribosomal activity clones were most targeted hinting at vital ribosome-altering role

Interferon alpha (IFNa) is approved for the therapy of patients (pts) with polycythemia vera (PV), a subtype of myeloproliferative neoplasms (MPN). Some pts achieve molecular responses (MR), but clonal factors sensitizing for MR remain elusive. We integrated colony formation and differentiation assays with single-cell RNA seq and genotyping in PV-derived cells vs. healthy controls (HC) to dissect how IFNa targets diseased clones during erythroid differentiation. IFNa significantly decreased colony growth in MPN and HC, with variable transcriptional responses observed in individual colonies. scRNAseq of colonies demonstrated more mature erythroid PV-derived colonies compared with HC. JAK2V617F-mutant cells exhibited upregulated STAT5A, heme, and G2M checkpoint pathways compared to JAK2WT cells from the same pts. 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. CD71-/CD235a+ cells from HC (p<0.05) but not from PV pts were inhibited by IFNa, and number of reticulocytes was less affected in PV. Robust IFNa responses persisted throughout differentiation, leading to significant apoptosis in PV. Apoptotic cells displayed 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, characterized by elevated expression of ribosomal genes. Our findings indicate that PV-derived clones either undergo apoptosis or pass through differentiation, overall reducing cycling mutant cells over long-term treatment. Further, the significance of ribosomal genes and clonal prerequisites in IFNa's therapeutic mechanism is underscored, shedding light on the intricate dynamics of IFNa treatment in PV.