Patient-Derived Induced Pluripotent Stem Cells Identified SLITRK4 As a Causative Gene of Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML), the most frequent disease entity of myelodysplastic syndrome/myeloproliferative neoplasm is a clonal hematopoietic malignancy that is characterized by persistent monocytosis, morphologic myeloid dysplasia, and progression to acute myeloid leukemia. The pathogenesis of CMML remains entirely elusive because of the lack of suitable mouse models and the difficulties in the establishment of CMML cell lines.

We have previously reported that we established induced pluripotent stem cells (iPSC) from CMML CD34 positive leukemic cells (CMML-iPSC) as a new disease model. Co-cultured with C3H10T1/2 stromal cells in the presence of vascular endothelial growth factor, CMML-iPSC generated CD34 CD43 double-positive hematopoietic progenitor cells (CMML-HPC). CMML-HPC have recapitulated important disease features of parental CMML cells in terms of genetic abnormalities, acceleration of cell proliferation, and aberrant surface markers expression. In addition, a novel human CMML xenograft mouse model has been established through secondary transplantation of human HPCs from CMML-iPSC-derived teratomas. This model produced HPCs that mimicked the properties of CMML in vivo.

To identify key molecular abnormalities that contribute to the pathophysiology of CMML, we conducted comprehensive gene expression and DNA methylation profiling analyses of normal and CMML parental CD34 positive cells, iPSC, and their hematopoietic progenies, respectively. Correlation analysis revealed that gene expression and DNA methylation status between normal and CMML iPSC-derived HPC exhibited similar pattern (R² = 0.92 and 0.96, respectively), although normal and CMML parental CD34 positive cells were quite different (R² = 0.72 and 0.90, respectively), indicating that reprogramming followed by re-differentiation may enable to obtain more homogenous population of normal and CMML cells that reside in almost the same differentiation stage. These results allowed us to determine the difference in the genetic and epigenetic status between normal and CMML iPSC-derived HPC, which remained through reprogramming and re-differentiation, in order to find out causative genes in the pathogenesis of CMML.

Using these combined omics platforms, we identified SLIT and NTRK like family member 4 (SLITRK4) as a candidate gene involving in pathogenesis of CMML, whose expression was enhanced and whose promoters were hypo-methylated in CMML-HPC. In other CMML patients' CD34 positive leukemic cells, the expression of SLITRK4 was up-regulated compared to healthy CD34 positive bone marrow cells and other leukemia cells. In addition, we revealed SLITRK4 had pro-proliferative activity as the knockdown of SLITRK4 inhibited proliferation of leukemic cell lines OCI-AML3. To elucidate whether SLITRK4 exert any biological functions in CMML, we established CMML-iPSC clones harboring hetero-knockout (wt/-) or homo-knockout (-/-) of SLITRK4 gene by CRISPR/Cas9 system. Although SLITRK4 (wt/-) and (-/-) clones did not exhibit any morphological and proliferative difference in CMML-iPSC, the production of HPC from CMML-iPSC was dramatically attenuated in SLITRK4-dependent manner. Therefore, while little has been known about the roles of SLITRK molecules in tumorigenesis, we demonstrated SLITRK4 was indispensable for generation of CMML leukemic cells and suggested the possibility of novel molecular therapy targeting SLITRK4, based on the findings obtained from our combined omics platforms.

In summary, we identified SLITRK4 as a novel candidate gene responsible for the pathogenesis of CMML through our combined omics platform using patient-derived iPSC. This platform may provide a potential to trace causative genes in a variety of diseases.