Abstract
Myelodysplastic syndromes (MDS) are disorders originated from hematopoietic stem cells (HSCs), which are characterized by ineffective hematopoiesis, dysplasia mainly in the myeloid lineage, and high progression ratio to acute myeloid leukemia (AML). Recently, we identified mutations of the c-CBL (Casitas B cell lymphoma, a cellular homologue of v-CBL) gene in patients with MDS and myeloproliferative neoplasms (MPN). The mutations are detected in about 8% of the patients with the highest frequency in chronic myelomonocytic leukemia (CMML) cases with acquired uniparental disomy (aUPD) at 11q. c-CBL encodes a RING finger-based E3 ubiquitin ligase that negatively regulates receptor-mediated intracellular signaling. c-CBL is highly expressed in HSCs, strongly suggesting that it functions as a fine regulator of hematopoietic homeostasis. In fact, c-CBL knockout (KO) mice showed a myeloproliferative phenotype, owing to the hyper-responsiveness of HSCs to cytokine stimulation and subsequent augmented hematopoietic progenitor pool. In addition, c-CBL knockin (KI) mice harboring a mutation in the RING finger domain in one allele and a null mutation in the other allele exhibit an MPD-like disease and eventually progress to AML. These findings indicate that dysfunction of c-CBL perturbs normal hematopoietic development and contributes to hematopoietic abnormalities, but the precise leukemogenic mechanism(s) remains elusive. To gain insights into this issue and to create a novel animal model for mutated c-CBL-harboring leukemia, we generated conditional knock-in (cKI) mice that express wild-type c-CBL at steady state and inducibly express c-CBLQ367P, which was identified in patients with chronic myelomonocytic leukemia (CMML). After induced expression of c-CBLQ367P, the cKI mice exhibited a rapid and sustained increase in myelomonocytic cells with dysplasia in the peripheral blood and splenic enlargement with proliferation of myeloid cells, which closely resemble to the phenotype of human CMML. The bone marrow (BM) was hypercellular with predominance of myeloid cells, and increased number of HSC subpopulations and early myeloid progenitors were observed. In addition, phosphorylation of AKT, STAT3 and STAT5 was detected in long-term hematopoietic stem cells (LT-HSCs) of c-CBLQ367P cKI mice, indicating that PI3K/AKT and JAK/STAT signaling pathways are activated in c-CBLQ367P LT-HSCs. Moreover, competitive repopulation assays revealed that mice transplanted with c-CBLQ367P LT-HSCs showed significantly higher donor-derived chimerism than those transplanted with control LT-HSCs and displayed expansion of myelomonocytic cells as observed in c-CBLQ367P cKI mice, indicating that c-CBLQ367P conferred a proliferative advantage to LT-HSCs and that the phenotypes observed in c-CBLQ367P cKI mice were hematopoietic cell-intrinsic. CMML is known to progress to AML, possibly with additional genetic aberrations. To investigate the mechanism(s) underlying the disease evolution, we performed retroviral insertional mutagenesis using MOL4070A, a derivative of Moloney murine leukemia virus capable of inducing myeloid diseases. Almost all MOL4070A-infected c-CBLQ367P cKI mice developed AML, while no disease was observed in virus-injected control mice. Inverse PCR method identified Evi1 gene as a common integration site in the diseased mice and high Evi1 expression was detected in Evi1-integrated tumors. Mice transplanted with Evi1-transduced c-CBLQ367P cKI c-kit-positive BM cells developed AML at a high frequency and in a shortened period as compared to those transplanted with Evi1-transduced control cells. Taken together, we demonstrated that acquired expression of c-CBLQ367P plays a causative role in the development of CMML by activating PI3K/AKT and JAK/STAT pathways in HSCs and found that Evi1 overexpression cooperates with c-CBLQ367P to develop AML. Our mouse model provides a powerful tool for understanding of the pathogenesis of CMML and for developing novel therapeutic strategies.
Disclosures
No relevant conflicts of interest to declare.
Generation and Analysis of a Novel Mouse Model for Chronic Myelomonocytic Leukemia (CMML) with Acquired Expression of c-CBLQ367P