Rictor is required for early B cell development in bone marrow

Rictor is required for early B cell development in bone marrow

Poster Presentations/ Experimental Hematology 42 (2014) S23–S68 S39 P1064 - RICTOR IS REQUIRED FOR EARLY B CELL DEVELOPMENT IN BONE MARROW Chunlan H...

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Poster Presentations/ Experimental Hematology 42 (2014) S23–S68


P1064 - RICTOR IS REQUIRED FOR EARLY B CELL DEVELOPMENT IN BONE MARROW Chunlan Hua, Tianyuan Hu, Yingchi Zhang, Tao Cheng, and Weiping Yuan State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin, China

P1066 - DECLINED PRESENTATION PERIPHERAL BLOOD SMEAR FINDINGS IN BURN PATIENTS Jungwon Hyun and Hyun Soo Kim Department of Laboratory Medicine, Hallym University Dongtan Sacred Heart Hospital, Gyeonggi-do, Republic of Korea

Mammalian target of rapamycin (mTOR) receives signals such as growth factors, nutrients and stress, and regulates cell growth and proliferation. mTOR forms mTORC1 and mTORC2 complexes and have been shown to have distinct functions in hematopoietic stem cells (HSCs). The early B cells are developed from HSC through a series of well-characterized stages in bone marrow (BM) and are regulated by PI3K/Akt/ mTOR signaling. However, the role of mTORC2 in the development of early B cells is still not fully understood. In this study, by using the Rictor conditional knockout mice model and Mx-1 cre mice, we demonstrated here that Rictor deletion significantly increased the Pro-B, Pre-B and immature B cells and decreased mature B cells in BM in adult Rictor-deleted mice. We then transplanted Rictor deficient BM cells on to WT mice and found that the percentages of pro-B, pre-B, and immature B cells were significantly lower in the BM of WT mice receiving Rictor-deficient BM cells than those in the control. This indicates that the defect of B cell development in Rictor deficient mice was hematopoietic environment independent and indicates that Rictor regulates the early B cell development in a cell intrinsic manner. Since FoxO1 and Rag-1 were implicated as the key regulators of B cell development, we studied their role in Rictor-deleted mice. We found that Rictor deletion caused the aberrant increase of FoxO1 and Rag-1 proteins in BM B cells. Knocking-down of FoxO1 in Rictor-deleted HSCs and hematopoietic progenitor cells (HPCs) rescued the defect of early B cell development with down-regulation of Rag-1 and IL-7R expression in B cells in recipient mice. It indicates that Rictor regulates early B cell development in BM via suppressing FoxO1 and Rag-1. Furthermore, we found that inhibition of mTORC1 with rapamycin in recipient mice transplanted with Rictor-deleted BMMNCs aggravated the defect of B cell development that was induced by mTORC2 depletion. It suggests that both mTORC1 and mTORC2 regulate the development of early B cells.

Background: Microscopic examination of peripheral blood films plays an important role to supplement the information by automated hematology analyzers. Various hematologic changes were reported after burn injury. In this study, we examined the peripheral blood smears and investigated the characteristics in burn patients. Methods: Sixty-two blood films received within 48 hours after admission from patients with burns were included. Leukocyte count, manual differential counts, absolute neutrophils count (ANC), and the existences of nucleated RBCs (nRBCs), toxic granulations, D€ohle bodies, vacuolations and left-shift were compared according to the total body surface area (TBSA) burned and survival. Results: Mean leukocyte count and ANC were 19,471/mL and 18,593/mL, respectively, and showed no significant differences between patients with burns !60% TBSA and $60% TBSA (16,752/mL vs. 21,307/mL, P 5 0.116; 14,522/mL vs. 19,291/mL, P 5 0.086) and between survivors and nonsurvivors (17,432/mL vs. 21,044/mL, P 5 0.246; 15,411/mL vs. 18,878/mL, P 5 0.247). Manual differential counts also showed no differences between those groups. However, nRBCs were observed more frequently in nonsurvivors than survivors and showed statistically significance (7/35, 20.0% vs. 0/27, 0%; P 5 0.014). Vacuolations were observed less frequently (3/37, 8.1% vs. 9/25, 36.0%; P 5 0.006) and left-shift was observed more frequently (37/37, 100% vs. 20/25, 80.0%; P 5 0.005) in patients with burns $60% TBSA. Toxic granulations and D4hle bodies did not show any difference between those groups. Conclusions: In burn patients, leukocyte counts and ANC were increased and left-shift was frequently observed. The existence of nRBCs has prognostic power with regard to mortality and vacuolation and left-shift shows association with the extent of burned area.

P1065 - RICTOR/MTORC2 REGULATES MOUSE T-ALL DEVELOPMENT VIA FOXO3A Chunlan Hua1, Huidong Guo1, Jiachen Bu2, Qianfei Wang2, Tao Cheng1, and Weiping Yuan1 1 State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Tianjin, China; 2Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Beijing, China

P1067 - INHIBITION OF TELOMERASE IMPAIRS NORMAL MEGAKARYOPOIESIS Goar Mosoyan1, Kevin Eng2, Craig Parker2, Ronald Hoffman1, and Camelia Iancu-Rubin1 1 Hematology/Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, New York, USA; 2Geron Corporation, Menlo Park, California, USA

T-cell acute lymphoblastic leukemia (T-ALL) is accounted for about 25% of adult ALL and 10-15% of childhood ALL. Hyper-activation of the PI3K/Akt/mTOR pathway sustains Notch1 mutation induced T-ALL viability. Mammalian TOR (mTOR) participates in two distinct biochemical complexes formation, namely, mTORC1 and mTORC2. mTORC2 mediates cell proliferation and survival by phosphorylating AKT at its Ser473 site to facilitate its full activation. Deletion of Rictor, an essential component of mTORC2, inactivated mTORC2 and affected leukemia progression in PTEN deficient mice model. However, how mTORC2 affects leukemogenesis is still not fully understood. Here we investigated the role of mTORC2 in T-ALL in a Rictor deficient mouse model driven by NOTCH1. We found that loss of Rictor led to a prolonged life span in T-ALL mice, accompanied with a slower growth of leukemia cells in the peripheral blood (PB), less splenomegaly, and less infiltration in liver, lung and kidney. We then transplanted Rictor deficient and control T-ALL cells on to WT mice, and found that Rictor deficiency significantly arrested more leukemia cells at G0 phase. Interestingly, the absence of Rictor led to the appearance of two new CD4+CD8- and CD4-CD8- subgroups. However, the transplantation studies revealed that their leukemogenesis abilities are similar to the CD4+CD8+ and CD4-CD8+ subgroups in WT mice and have no enhanced leukemia induction ability. Since the FoxO transcription factors are involved in multiple signaling pathways and is a downstream target of AKT, we studied the role of FoxO3a in our T-ALL model. We found Rictor deletion caused the overexpression of FoxO3a and FoxO3a activated genes. Furthermore, knocking-down of FoxO3a in Rictor-deleted and control T-ALL cells accelerated the leukemia progression. Our study indicates that PI3K/Akt/mTOR/FoxO3a axis regulates T-ALL development and progression and FoxO3a could be a potential drug target for the treatment of T-ALL leukemia.

Imetelstat (GRN163L) is a telomerase inhibitor which has shown preliminary activity for the treatment of hematological malignancies, including myelofibrosis. In clinical trials, the primary dose-limiting toxicity is thrombocytopenia. We utilized GRN163L in order to explore the possible role of telomerase in human megakaryocytes (MKs) and the mechanisms underlying the drug’s inhibitory effects on platelet production. MKs were generated from normal primary CD34+ cells in an ex vivo system of megakaryopoiesis. We first showed that both telomerase activity (TA) and the expression of its catalytic unit hTERT were higher in proliferating CD34+ cells and declined gradually during MK differentiation. GRN163L treatment during the first 7 days of culture did not interfere with the ability of CD34+ cells to commit to MKs since the proportion of CD34+/CD41+ cells was similar in its absence and presence. However, the absolute number of CD34+/CD41+ MK in GRN163L-treated cultures was 50% lower than that found in control. To further evaluate the effects of GRN163L on MK maturation, MK precursors were allowed to mature for 7 additional days. Although the absolute number of immature CD34+/CD41+ MK in was similar in control and GRN163L-treated cultures, the proportion of CD34+/CD41+ cells in drugtreated cultures was twice that observed in control cultures. Moreover, the cultures treated with GRN163L had 70% fewer mature CD41+/CD42b+ MK as compared to control. These inhibitory effects on terminal MK maturation were also supported by preliminary observations showing that drug-treated cultures inhibited the ability of MK to become polyploid. Ongoing studies are now being pursued to determine if telomerase plays a direct role in MK endomitosis. We conclude that GRN163L-mediated inhibition of telomerase affects megakaryopoiesis not only by impairing the ability of CD34+/CD41+ MK precursors to proliferate but also by blocking their further maturation. These findings provide a possible explanation for GRN163L’s propensity to induce thrombocytopenia and support a previously unrecognized role for telomerase in the regulation of normal MK development.