No major predisposition gene for familial myeloproliferative neoplasms (MPN) has been identified. Here we demonstrate that the autosomal dominant transmission of a 700-kb duplication in four genetically related families predisposes to myeloid malignancies, including MPN, frequently progressing to leukemia. Using induced pluripotent stem cells and primary cells, we demonstrate that overexpression of ATG2B and GSKIP enhances hematopoietic progenitor differentiation, including of megakaryocytes, by increasing progenitor sensitivity to thrombopoietin (TPO). ATG2B and GSKIP cooperate with acquired JAK2, MPL and CALR mutations during MPN development. Thus, the germline duplication may change the fitness of cells harboring signaling pathway mutations and increases the probability of disease development.
JAK2V617F is the predominant mutation in myeloproliferative neoplasms (MPN). Modeling MPN in a human context might be helpful for the screening of molecules targeting JAK2 and its intracellular signaling. We describe here the derivation of induced pluripotent stem (iPS) cell lines from 2 polycythemia vera patients carrying a heterozygous and a homozygous mutated JAK2V617F, respectively. In the patient with homozygous JAK2V617F, additional ASXL1 mutation and chromosome 20 allowed partial delineation of the clonal architecture and assignation of the cellular origin of the derived iPS cell lines. The marked difference in the response to erythropoietin (EPO) between homozygous and heterozygous cell lines correlated with the constitutive activation level of signaling pathways. Strikingly, heterozygous iPS cells showed thrombopoietin (TPO)-independent formation of megakaryocytic colonies, but not EPO-independent erythroid colony formation. JAK2, PI3K and HSP90 inhibitors were able to block spontaneous and EPO-induced growth of erythroid colonies from GPA+CD41+ cells derived from iPS cells. Altogether, this study brings the proof of concept that iPS can be used for studying MPN pathogenesis, clonal architecture, and drug efficacy.
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.
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During the last several years, iPS cells have been described as a new powerful tool for disease modeling and drug screening. It became possible to reprogram adult patients cells into specific pluripotent cell lines harboring inherited and/or acquired genetic abnormalities. We have focused our work on modeling Jak2V617F positive MPN patients in iPS cells.
By retroviral infection (OCT4, SOX2, KLF4, c-Myc), several iPS cell lines were generated from CD34+ cells of a healthy donor (control), a heterozygous JAK2 V617F patient and a homozygous JAK2 V617F patient. All cell lines expressed pluripotent surface markers (TRA1–81, SSEA4), pluripotent genes (NANOG, OCT4, SOX2) and were capable to induce teratomas in immunodeficient mice. Moreover, they were silenced for the expression of the 4 exogenous transgenes and showed no new clonal karyotypic abnormalities. All the iPS cell lines derived from the JAK2V617F heterozygous and homozygous patients were also heterozygous and homozygous for JAK2V617F. No JAK2 wild type iPS could be obtained from the patients CD34+ cells. Interestingly all the homozygous JAK2V617Fhad no 20q deletion by cytogenetic and CGH analysis demonstrating that this deletion was a secondary event in this MPN.
JAK2V617F mutation did not elicit a significant increase in hematopoiesis in comparison to the control iPS. However, marked differences in cytokine sensitivity were found. Homozygous iPS-derived erythroid progenitors had spontaneous growth, hypersensitivity to EPO and gave rise to slightly larger erythroid colonies in methylcellulose assays as compared to the control, whereas erythroid progenitors from JAK2V617F heterozygous iPS has similar Epo-response than those from the control. Interestingly, Megakaryocytes (MK) progenitors from JAK2V617F heterozygous iPS had hypersensitivity to TPO whereas those from homozygous iPS showed a complete TPO independence. Those cytokine-response profiles recapitulate the disease's primary features and validate the iPS as a good tool for JAK2V617F MPN modeling. MK differentiation showed a maturation defect with an excess of immature MKs only in the JAK2 V617Fhomozygous lines, who was derived with a post-PV myelofibrosis, suggesting the existence of intrinsic abnormalities of the MK lineage.
Finally, we tested several JAK2, PI3K and ERK inhibitors on the JAK2V617F-mediated erythroid growth and showed that only JAK2 and PI3K inhibitors were able to block the erythroid spontaneous growth.
iPS technology recapitulates the cytokine hypersensitivity of JAK2V617F MPNs with marked differences between heterozygous and homozygous mutations, enabling us to screen various pharmaceutical inhibitors. In an addition it can also be used to study the clonal hierarchy in a MPN and in the future to study the synergistic effects of different mutations.
Disclosures:
No relevant conflicts of interest to declare.
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