Characterization of disease-initiating hematopoietic stem cells in calreticulin-mutated myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) arise following the acquisition of a mutation in a hematopoietic stem cell (HSC) that causes oncogenic cytokine receptor signaling. Mutations in the calreticulin (CALR) gene are the second most common key driver mutations in MPNs, yet the identity of the disease-initiating HSCs and the mechanisms underlying their clonal expansion remain elusive. Knock-in mice bearing CALRdel52 and CALRins5 mutations recapitulate disease phenotypes, with a greater HSC amplification in CALRdel52 mice. Here, we crossed these strains with transgenic reporter mice expressing GFP under the control of the Von willebrand factor (Vwf) promoter, enabling discrimination between different HSC subsets. We show that CALR-mutated MPNs are mainly initiated from the expansion of platelet-biased, Vwf-positive HSCs, without substantially altering their lineage bias. Notably, the selective amplification of Vwf-positive CALRdel52 compared to CALRins5 HSCs is associated with increased signaling of the thrombopoietin receptor MPL and activation of the integrated stress response as evidenced by eIF2a phosphorylation downstream of PERK kinase triggered by endoplasmic reticulum stress. This pathway is also transcriptionally upregulated in CALRdel52-like compared with CALRins5-like patient hematopoietic stem and progenitor cells (HSPCs), as supported by reanalysis of a previous dataset, and is associated with increased phosphorylation of eIF2a. Pharmacological inhibition of PERK markedly reduces the proliferation of mouse CALRdel52 HSCs and impairs the megakaryocytic differentiation of CALRdel52-like HSPCs from patients while sparing cells from healthy donors. These findings identify the PERK/eIF2a pathway as a mechanistic vulnerability in the MPN cell-of-origin and offer a rationale for exploring new treatment approaches.