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Primary myelofibrosis (PMF) is a hematological malignancy characterized by activation of the JAK/STAT pathway and risk of leukemic transformation. In this study, we generated an induced Pluripotent Stem (iPS) cell line derived from a 65-year old male PMF patient carrying the 5-pb insertion in the CALR gene (CALRins5) and the c.437 G > A mutation in the TP53 gene (p.W146X). The newly derived PMF3.17 iPS cell line harbors the original mutations and was characterized as bona fide iPS.Resource tableUnlabelled TableUnique stem cell line identifierINCABRi002-AAlternative name(s) of stem cell linePMF3.17InstitutionBrazilian National Cancer Institute and D'Or Institute for Research and EducationContact information of distributorMartin Bonamino, PhD, mbonamino@inca.gov.br, Bárbara Monte Mór, PhD, barbara.montemor@inca.gov.br, Stevens Rehen, PhD, srehen@lance-ufrj.orgType of cell lineiPS cellOriginHumanAdditional origin infoAge: 65Sex: maleCell sourcePeripheral bloodClonalityClonalMethod of reprogrammingSendai VirusGenetic modificationNoType of modificationNot applicableAssociated diseasePrimary myelofibrosisGeneCALR gene: CALRins5, c.1154_1155insTTGTC, p.K385 fs*47TP53 gene: c.437 G > A, p.W146XMethod of modificationNot applicableName of transgene or resistanceNot applicableInducible/constitutive systemNot applicableDate archived/stock dateJuly 7th, 2015Cell line repository/bankNot applicableEthical approvalEthics Committee of the Brazilian National Cancer Institute (INCA) under the number 062/08. Ethics Review Board-competent authority obtained.

Peripheral blood sample was donated by a 61 years old female patient diagnosed with acute myeloid leukemia secondary to a primary myelofibrosis harboring the 52-bp deletion in the CALR gene (c.1092_1143del, p.L367fs*46) and the R693X mutation in the ASXL1 gene (c.2077C>T, p.R693X). CD34+ cells were isolated from the sample and subjected to the reprogramming procedure by using the Sendai virus carrying the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc. iPS colonies generated retained the original mutations and displayed all the features of bona fide iPS cells.

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.