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Acute myeloid leukemia (AML) is a high remission, high relapse fatal blood cancer. Although mTORC1 is a master regulator of cell proliferation and survival, its inhibitors have not performed well as AML treatments. To uncover the dynamics of mTORC1 activity in vivo, fluorescent probes are developed to track single cell proliferation, apoptosis and mTORC1 activity of AML cells in the bone marrow of live animals and to quantify these activities in the context of microanatomical localization and intra-tumoral heterogeneity. When chemotherapy drugs commonly used clinically are given to mice with AML, apoptosis is rapid, diffuse and not preferentially restricted to anatomic sites. Dynamic measurement of mTORC1 activity indicated a decline in mTORC1 activity with AML progression. However, at the time of maximal chemotherapy response, mTORC1 signaling is high and positively correlated with a leukemia stemness transcriptional profile. Cell barcoding reveals the induction of mTORC1 activity rather than selection of mTORC1 high cells and timed inhibition of mTORC1 improved the killing of AML cells. These data define the real-time dynamics of AML and the mTORC1 pathway in association with AML growth, response to and relapse after chemotherapy. They provide guidance for timed intervention with pathway-specific inhibitors. The role of mTORC1 in AML has not yet been proven due to the mixed results of its inhibitors in clinical trials. Here the authors show the real-time dynamics of the mTORC1 pathway in association with AML growth and response to chemotherapy with fluorescent markers, providing guidance for timed intervention with pathway-specific inhibitors. [ABSTRACT FROM AUTHOR]

Activating mutations of c-Kit are frequently found in acute myeloid leukemia (AML) patients harboring t(8;21) chromosomal translocation generating a fusion protein AML1-ETO. Here we show that an active mutant of c-Kit cooperates with AML1-ETO to induce AML in mouse bone marrow transplantation models. Leukemic cells expressing AML1-ETO with c-Kit were serially transplantable. Transplantation experiments indicated that lineagec-KitSca-1 (KSL) leukemic cells, but not lineage leukemic cells, were enriched for leukemia stem cells (LSCs). Comparison of gene expression profiles between KSL leukemic and normal cells delineated that CD200R1 was highly expressed in KSL leukemic cells as compared with KSL normal cells. Upregulation of CD200R1 was verified in lineage leukemic cells, but not in lineage leukemic cells. CD200R1 expression in the lineage leukemic cells was not correlated with the frequency of LSCs, indicating that CD200R1 is not a useful marker for LSCs in these models. Interestingly, CD200R1 was upregulated in KSL cells transduced with AML1-ETO, but not with other leukemogenic mutants, including c-Kit, AML1, and AML1. Consistently, upregulation of CD200R1 in lineage leukemic cells was observed only in the BM of mice suffering from AML1-ETO-positive leukemia. In conclusion, AML1-ETO upregulated CD200R1 in lineage cells, which was characteristic of AML1-ETO-positive leukemia in mice. [ABSTRACT FROM AUTHOR]

The Bcr-Abl oncogene causes human Philadelphia chromosome-positive (Ph) leukemias, including B-cell acute lymphoblastic leukemia (B-ALL) and chronic myeloid leukemia (CML) with chronic phase (CML-CP) to blast crisis (CML-BC). Previous studies have demonstrated that Src family kinases are required for the induction of B-ALL, but not for CML, which is induced by Bcr-Abl in mice. In contrast, it has been reported that Fyn is up-regulated in human CML-BC compared with CML-CP, implicating Fyn in the blast crisis transition. Here, we aimed to delineate the exact role of Fyn in the induction/progression of Ph leukemias. We found that Fyn is expressed in mouse hematopoietic cells at varying stages of development, including c-kitSca-1Lin cells. Notably, Fyn is highly expressed in some of human lymphomas, but not in human Ph leukemias including CML-BC. In mouse bone marrow transplantation models, mice transplanted with wild-type or Fyn-deficient bone marrow cells transduced with Bcr-Abl showed no differences in the development of B-ALL or CML-like diseases. Similarly, Fyn deficiency failed to impact the development of myeloid CML-BC induced by Bcr-Abl and Hes1. Elevated expression of Fyn was not found in mouse samples of Bcr-Abl-mediated CML- and CML-BC-like diseases. Thus, Fyn is not required for the pathogenesis of Bcr-Abl-mediated leukemias. [ABSTRACT FROM AUTHOR]

In this report we review the concept of standardization and propose an exhaustive framework for the proper management of technology on pluripotent stem cells based on studies of global and regional initiatives. We demonstrated detailed observational analysis on global initiatives for the standardization of related technologies as well as regional attempts with particular interest in the cases of the UK, the US and Japan. Consequently, we came up two fundamental issues: first, these initiatives and attempts tend to be limited to each of currently existing categories of pluripotent stem cells, whereas the technological opportunity to enable clinical/commercial application is equally open to all stem cell types. Second, the subject to be examined for standardization is set to a quite narrow range compared to precedent practices in other industrial sectors. To address these issues, we propose a strategic framework for standardization with an emphasis on comprehensiveness covering various technological opportunities and consistency to learning in the management science. By utilizing this framework development of intellectual property rights not only through patents but through taking leadership in standardization can be considered as means for improving research and development competence. Of particular concerns is the formation of quality standards for final products/services and core elemental technology, especially specific pluripotent stem cell lines. Furthermore, we inferred two stages of the standardization process, individualization where a particular product/service is qualified by the market, then standardization where the elemental technology is subsequently established as compatibility standards. [ABSTRACT FROM AUTHOR]

It is now conceivable that leukemogenesis requires two types of mutations, class I and class II mutations. We previously established a mouse bone marrow-derived HF6, an IL-3-dependent cell line, that was immortalized by a class II mutation MLL/SEPT6 and can be fully transformed by class I mutations such as FLT3 mutants. To understand the molecular mechanism of leukemogenesis, particularly progression of myelodysplastic syndrome (MDS) to acute leukemia, we made cDNA libraries from the samples of patients and screened them by expression-cloning to detect class I mutations that render HF6 cells factor-independent. We identified RasGRP4, an activator of Ras, as a candidate for class I mutation from three of six patients (MDS/MPD = 1, MDS-RA = 1, MDS/AML = 2, CMMoL/AML = 1 and AML-M2 = 1). To investigate the potential roles of RasGRP4 in leukemogenesis, we tested its in vivo effect in a mouse bone marrow transplantation (BMT) model. C57BL/6J mice transplanted with RasGRP4-transduced primary bone marrow cells died of T cell leukemia, myeloid leukemia, or myeloid leukemia with T cell leukemia. To further examine if the combination of class I and class II mutations accelerated leukemic transformation, we performed a mouse BMT model in which both AML1 mutant (S291fsX300) and RasGRP4 were transduced into bone marrow cells. The double transduction led to early onset of T cell leukemia but not of AML in the transplanted mice when compared to transduction of RasGRP4 alone. Thus, we have identified RasGRP4 as a gene potentially involved in leukemogenesis and suggest that RasGRP4 cooperates with AML1 mutations in T cell leukemogenesis as a class I mutation. [ABSTRACT FROM AUTHOR]