Ken Sadahira

Tet2 disruption leads to enhanced self-renewal and altered differentiation of fetal liver hematopoietic stem cells

Somatic mutation of ten-eleven translocation 2 (TET2) gene is frequently found in human myeloid malignancies. Recent reports showed that loss of Tet2 led to pleiotropic hematopoietic abnormalities including increased competitive repopulating capacity of bone marrow (BM) HSCs and myeloid transformation. However, precise impact of Tet2 loss on the function of fetal liver (FL) HSCs has not been examined. Here we show that disruption of Tet2 results in the expansion of Lin−Sca-1+c-Kit+ (LSK) cells in FL. Furthermore, Tet2 loss led to enhanced self-renewal and long-term repopulating capacity of FL-HSCs in in vivo serial transplantation assay. Disruption of Tet2 in FL also led to altered differentiation of mature blood cells, expansion of common myeloid progenitors and increased resistance for hematopoietic progenitor cells (HPCs) to differentiation stimuli in vitro. These results demonstrate that Tet2 plays a critical role in homeostasis of HSCs and HPCs not only in the BM, but also in FL.

Impaired hematopoietic differentiation of RUNX1-mutated induced pluripotent stem cells derived from FPD/AML patients

Somatic mutation of RUNX1 is implicated in various hematological malignancies, including myelodysplastic syndrome and acute myeloid leukemia (AML), and previous studies using mouse models disclosed its critical roles in hematopoiesis. However, the role of RUNX1 in human hematopoiesis has never been tested in experimental settings. Familial platelet disorder (FPD)/AML is an autosomal dominant disorder caused by germline mutation of RUNX1, marked by thrombocytopenia and propensity to acute leukemia. To investigate the physiological function of RUNX1 in human hematopoiesis and pathophysiology of FPD/AML, we derived induced pluripotent stem cells (iPSCs) from three distinct FPD/AML pedigrees (FPD-iPSCs) and examined their defects in hematopoietic differentiation. By in vitro differentiation assays, FPD-iPSCs were clearly defective in the emergence of hematopoietic progenitors and differentiation of megakaryocytes, and overexpression of wild-type (WT)-RUNX1 reversed most of these phenotypes. We further demonstrated that overexpression of mutant-RUNX1 in WT-iPSCs did not recapitulate the phenotype of FPD-iPSCs, showing that the mutations were of loss-of-function type. Taken together, this study demonstrated that haploinsufficient RUNX1 allele imposed cell-intrinsic defects on hematopoietic differentiation in human experimental settings and revealed differential impacts of RUNX1 dosage on human and murine megakaryopoiesis. FPD-iPSCs will be a useful tool to investigate mutant RUNX1-mediated molecular processes in hematopoiesis and leukemogenesis.

Gossypol induces apoptosis in multiple myeloma cells by inhibition of interleukin-6 signaling and Bcl-2/Mcl-1 pathway

Multiple myeloma (MM) is a clonal plasma cell disorder affecting the immune system with various systemic symptoms. MM remains incurable even with high dose chemotherapy using conventional drugs, thus necessitating development of novel therapeutic strategies. Gossypol (Gos) is a natural polyphenolic compound extracted from cotton plants, and has been shown to possess anti-neoplastic activity against various tumors. Recent studies have shown that Gos is an inhibitor for Bcl-2 or Bcl-XL acting as BH3 mimetics that interfere interaction between pro-apoptotic BH3-only proteins and Bcl-2/Bcl-XL. Since most of the patients with MM overexpress Bcl-2 protein, we considered Gos might be a promising therapeutic agent for MM. We herein show that Gos efficiently induced apoptosis and inhibited proliferation of the OPM2 MM cell line, in a dose- and time-dependent manner. Gos induced activation of caspase-3 and cytochrome c release from mitochondria, showing mitochondrial dysfunction pathway is operational during apoptosis. Further investigation revealed that phosphorylation of Bcl-2 at serine-70 was attenuated by Gos treatment, while protein levels were not affected. In addition, Mcl-1 was downregulated by Gos. Interestingly, phosphorylation of JAK2, STAT3, ERK1/2 and p38MAPK was inhibited by Gos-treatment, indicating that Gos globally suppressed interleukin-6 (IL-6) signals. Moreover, JAK2 inhibition mimicked the effect of Gos in OPM2 cells including Bcl-2 dephosphorylation and Mcl-1 downregulation. These results demonstrated that Gos induces apoptosis in MM cells not only through displacing BH3-only proteins from Bcl-2, but also through inhibiting IL-6 signaling, which leads to Bcl-2 dephosphorylation and Mcl-1 downregulation.

Direct reprogramming of terminally differentiated B cells into erythroid lineage

Hematopoietic progenitors have been shown to retain plasticity and switch lineages by appropriate stimuli. However, mature blood cells hardly showed such differentiation plasticity. In this paper, we tried to reprogram mature B cells into erythroid lineage by expressing various hematopoietic transcription factors. Among various factors, GATA‐1, SCL together with CCAAT/enhancer binding protein (C/EBP) α turned out to be a minimal set of factors that efficiently reprogrammed terminally differentiated mature B cells into erythroid lineage, as evidenced by colony forming assays and erythroid‐specific gene expressions. This study sets an avenue to generate autologous erythrocytes from peripheral B cells.