Induced Pluripotent Stem Cells Enable Disease Modeling and Drug Screening in Calreticulin del52 and ins5 Myeloproliferative Neoplasms

Abstract

Mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and primary myelofibrosis patients. To address the contribution of the human CALR mutants to the pathogenesis of myeloproliferative neoplasms (MPNs) in an endogenous context, we modeled the CALRdel52 and CALRins5 mutants by induced pluripotent stem cell (iPSC) technology using CD34+ progenitors from 4 patients. We describe here the generation of several clones of iPSC carrying heterozygous CALRdel52 or CALRins5 mutations. We showed that CALRdel52 induces a stronger increase in progenitors than CALRins5 and that both CALRdel52 and CALRins5 mutants favor an expansion of the megakaryocytic lineage. Moreover, we found that both CALRdel52 and CALRins5 mutants rendered colony forming unit–megakaryocyte (CFU-MK) independent from thrombopoietin (TPO), and promoted a mild constitutive activation level of signal transducer and activator of transcription 3 in megakaryocytes. Unexpectedly, a mild increase in the sensitivity of colony forming unit-granulocyte (CFU-G) to granulocyte-colony stimulating factor was also observed in iPSC CALRdel52 and CALRins5 compared with control iPSC. Moreover, CALRdel52-induced megakaryocytic spontaneous growth is more dependent on Janus kinase 2/phosphoinositide 3-kinase/extracellular signal-regulated kinase than TPO-mediated growth and opens a therapeutic window for treatments in CALR-mutated MPN. The iPSC models described here represent an interesting platform for testing newly developed inhibitors. Altogether, this study shows that CALR-mutated iPSC recapitulate MPN phenotypes in vitro and may be used for drug screening.

CALR frameshift mutations in MPN patient-derived iPSCs accelerate maturation of megakaryocytes

“…This might be due to the reported oncogenic interaction of the TPO receptor and mutant CALR before reaching the cell surface ( Masubuchi et al., 2020 ; Pecquet et al., 2019 ), already sufficiently occupying the TPO binding site. However, Secardin et al. (2021) observed an increase of CFU-MKs, generated from iPSC-derived CALR-mutated CD34 + CD43 + progenitors, in the presence of TPO.…”

“…In the present study, we generated patient-specific iPSC lines harboring CALR del52, CALR ins5, or CALR del31 mutations, and the iPSC-based model was applied to study the impact of CALR mutant zygosity on hematopoietic and more precisely on MK differentiation at the clonal level. Patient-derived iPSCs carrying het CALR mutations have been recently described, but detailed comparisons of type 1 and type 2 CALR mutations with focus on their zygosity and detailed characterization of derived disease-driving MKs are missing ( Gomez Limia et al., 2017 , 2018 ; Secardin et al., 2021 ; Takei et al., 2018 ). To obtain isogenic CALR -repaired clones for CALR ins5 and CALR del52-mutated iPSCs, we used CRISPR/Cas9 technology to correct the mutations.…”

ANKRD26 is a new regulator of type I cytokine receptor signaling in normal and pathological hematopoiesis

“…Three different clones were studied for each patient-derived iPSC. As a control, we used three different iPSC lines derived from healthy individuals 28-30 .…”

“…The iPSC control cell lines C1, C2 and C3 were already established and previously characterized. 28-30…”

Induced Pluripotent Stem Cells as a Tool for Modeling Hematologic Disorders and as a Potential Source for Cell-Based Therapies

“…The utilization of different types of JAK2 mutations in patient-derived iPSCs or generated mutant iPSCs for the discovery of pathogenesis and candidate therapy for MPNs has been demonstrated [149,150]. Additionally, hiPSCs derived from patients with the calreticulin (CALR) gene mutation, which is usually found in ET and primary myelofibrosis patients, could reflect disease phenotypes by representing megakaryopoiesis and prominent colony-forming unit megakaryocytes (CFU-MK) [151,152].…”