Establishment of isogenic induced pluripotent stem cells with or without pathogenic mutation for understanding the pathogenesis of myeloproliferative neoplasms.

Identification and functional characterization of disease-associated genetic traits are crucial for understanding the pathogenesis of hematologic malignancies. Various in vitro and in vivo models, including cell lines, primary cells, and animal models, have been established to examine these genetic alterations. However, their nonphysiologic conditions, diverse genetic backgrounds, and species-specific differences often limit data interpretation. To evaluate somatic mutations in myeloproliferative neoplasms (MPNs), we used CRISPR/Cas9 combined with the piggyBac transposon system to establish isogenic induced pluripotent stem (iPS) cell lines with or without JAK2V617F mutation, a driver mutation of MPNs. We induced hematopoietic stem/progenitor cells (HSPCs) from these iPS cells and observed phenotypic differences during hematopoiesis using fluorescence-activated cell sorting analysis. HSPCs with pathogenic mutations exhibited cell-autonomous erythropoiesis and megakaryopoiesis, which are hallmarks in the bone marrow of patients with MPNs. Furthermore, we used these HSPCs as a model to validate therapeutic compounds and showed that interferon alpha selectively inhibited erythropoiesis and megakaryopoiesis in mutant HSPCs. These results demonstrate that genome editing is feasible for establishing isogenic iPS cells, studying genetic elements to understand the pathogenesis of MPNs, and evaluating therapeutic compounds against MPNs.

Mutant calreticulin interacts with MPL in the secretion pathway for activation on the cell surface.

Studies have shown that mutant calreticulin (CALR) constitutively activates the thrombopoietin (TPO) receptor MPL and thus plays a causal role in the development of myeloproliferative neoplasms (MPNs). To further elucidate the molecular mechanism by which mutant CALR promotes MPN development, we studied the subcellular localization of mutant CALR and its importance for the oncogenic properties of mutant CALR. Here, mutant CALR accumulated in the Golgi apparatus, and its entrance into the secretion pathway and capacity to interact with N-glycan were required for its oncogenic capacity via the constitutive activation of MPL. Mutant CALR-dependent MPL activation was resistant to blockade of intracellular protein trafficking, suggesting that MPL is activated before reaching the cell surface. However, removal of MPL from the cell surface with trypsin shut down downstream activation, implying that the surface localization of MPL is required for mutant CALR-dependent activation. Furthermore, we found that mutant CALR and MPL interact on the cell surface. Based on these findings, we propose a model in which mutant CALR induces MPL activation on the cell surface to promote MPN development.

Skewed megakaryopoiesis in human induced pluripotent stem cell-derived haematopoietic progenitor cells harbouring calreticulin mutations.

Somatic mutations in the calreticulin (CALR) gene have been found in most patients with JAK2- and MPL-unmutated Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). It has recently been shown that mutant CALR constitutively activates the thrombopoietin receptor MPL and, thus, plays a causal role in the development of MPNs. However, the roles of mutant CALR in human haematopoietic cell differentiation remain predominantly elusive. To examine the impact of the 5-base insertion mutant CALR gene (Ins5) on haematopoietic cell differentiation, we generated induced pluripotent stem cells from an essential thrombocythaemia (ET) patient harbouring a CALR-Ins5 mutation and from a healthy individual (WT). Megakaryopoiesis was more prominent in Ins5-haematopoietic progenitor cells (Ins5-HPCs) than in WT-HPCs, implying that the system recapitulates megakaryocytosis observed in the bone marrow of CALR-mutant ET patients. Ins5-HPCs exhibited elevated expression levels of GATA1 and GATA2, suggesting a premature commitment to megakaryocytic differentiation in progenitor cells. We also demonstrated that 3-hydroxy anagrelide markedly perturbed megakaryopoiesis, but not erythropoiesis. Collectively, we established an in vitro model system that recapitulates megakaryopoiesis caused by mutant CALR. This system can be used to validate therapeutic compounds for MPN patients harbouring CALR mutations and in detailed studies on mutant CALR in human haematological cell differentiation.

Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.

Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway.

Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.

A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.