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Item The autotaxin-LPA2 GPCR axis is modulated by γ-irradiation and facilitates DNA damage repair(Elsevier, 2015-09) Balogh, Andrea; Shimizu, Yoshibumi; Lee, Sue Chin; Norman, Derek D.; Gangwar, Ruchika; Bavaria, Mitul; Moon, ChangSuk; Shukla, Pradeep; Rao, Radakrishna; Ray, Ramesh; Naren, Anjaparavanda P.; Banerje, Souvik; Miller, Duane D.; Balazs, Louisa; Pelus, Louis; Tigyi, Gabor; Department of Microbiology and Immunology, IU School of MedicineIn this study we characterized the effects of radiation injury on the expression and function of the autotaxin (ATX)-LPA2 GPCR axis. In IEC-6 crypt cells and jejunum enteroids quantitative RT-PCR showed a time- and dose-dependent upregulation of lpa2 in response to γ-irradiation that was abolished by mutation of the NF-κB site in the lpa2 promoter or by inhibition of ATM/ATR kinases with CGK-733, suggesting that lpa2 is a DNA damage response gene upregulated by ATM via NF-κB. The resolution kinetics of the DNA damage marker γ-H2AX in LPA-treated IEC-6 cells exposed to γ-irradiation was accelerated compared to vehicle, whereas pharmacological inhibition of LPA2 delayed the resolution of γ-H2AX. In LPA2-reconstituted MEF cells lacking LPA1&3 the levels of γ-H2AX decreased rapidly, whereas in Vector MEF were high and remained sustained. Inhibition of ERK1&2 or PI3K/AKT signaling axis by pertussis toxin or the C311A/C314A/L351A mutation in the C-terminus of LPA2 abrogated the effect of LPA on DNA repair. LPA2 transcripts in Lin(-)Sca-1(+)c-Kit(+) enriched for bone marrow stem cells were 27- and 5-fold higher than in common myeloid or lymphoid progenitors, respectively. Furthermore, after irradiation higher residual γ-H2AX levels were detected in the bone marrow or jejunum of irradiated LPA2-KO mice compared to WT mice. We found that γ-irradiation increases plasma ATX activity and LPA level that is in part due to the previously established radiation-induced upregulation of TNFα. These findings identify ATX and LPA2 as radiation-regulated genes that appear to play a physiological role in DNA repair.Item CELLULAR THERAPY AND HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR CANCER(Office of the Vice Chancellor for Research, 2010-04-09) Farag, Sherif S.; Srivastava, Shivani; Schwartz, Jennifer; Nelson, Robert; Homsi, Yasser; Zhang, Shuhong; Dinauer, Mary; Cornetta, Kenneth; March, Kieth; Pelus, Louis; Broxmeyer, HalThe Center for Cellular Therapy and Hematopoietic Stem Cell Transplantation for Cancer was established in July 2007 to promote translational and clinical research in cellular therapy for cancer. The primary goal of the Center is translate discoveries from bench-to-clinic through phase I and early phase II cellular therapy clinical trials. To achieve this objective, the Center has brought together the unique expertise in hematopoiesis, immunology, gene therapy, graft engineering, and clinical hematopoietic stem cell transplantation (HCT) available at IUPUI. Since its establishment, we have completed two phase I clinical trials developing novel preparative regimens for allogeneic and autologous stem cell transplantation for patients with refractory leukemia and lymphoma, respectively. In addition, we have also initiated 5 additional early phase clinical trials that directly translate IUPUI laboratory discoveries to patients with hematological cancers. The Center has successfully competed for external funding through peerreviewed grants and pharmaceutical contracts. In this presentation, we highlight some important examples of the Center’s ongoing and completed research. An important clinical research focus of our Center is the ability to extend the curative potential of allogeneic HCT to patients without suitably HLA-matched donors. We are currently exploring ways to improve the outcomes of umbilical cord blood (UCB) and haplotype-mismatched stem cell transplantation for patients with hematological cancers. The discovery in Dr. Broxmeyer’s Laboratory, Indiana University, Indianapolis, that inhibition of the enzyme CD26 promotes homing and engraftment of limiting numbers of UCB stem cells has been translated to the first clinical trial in vivo CD26 inhibition using sitagliptin in adult leukemia patients undergoing UCB transplantation. Our preliminary data indicates that high-dose sitagliptin is well tolerated and appears to shorten the time of engraftment. As our data is further confirmed in this pilot study, we plan to investigate this potentially paradigm changing approach in a larger national study. As an extension of this research, Dr. Pelus’ Laboratory, Indiana University, Indianapolis, has shown that short-term ex vivo treatment of hematopoietic progenitors using PGE2 will also promote engraftment. We are currently investigating the potential synergy of PGE2 treatment with CD26 inhibition to further enhance engraftment, which if results appear promising will also be translated to a phase I clinical trial. In haplotypemismatched allogeneic HCT, mismatching of donor KIR receptors on natural killer (NK) cells with recipient KIR ligands expressed on the patient’s tumor cells exerts a NK cell-mediated antileukemia effect that contributes to reduced relapse after transplantation. We (Dr. Farag’s Laboratory, Indiana University, Indianapolis) have shown that in vivo donor derived NK cells developing from donor stem cells have an “inhibitory” receptor phenotype that may suboptimally function against leukemia. This has resulted in a phase I trial of purified NK cell infusion following mismatched HCT to investigate the feasibility and safety of this approach, as a prelude to a larger study to investigate its efficacy. Although the highest dose level of NK cells has not yet been investigated, the preliminary data indicates that such a novel approach is feasible. In additional studies based on our laboratory findings, we are exploring the harnessing of NK cells in the therapy of cancer through the monoclonal antibodies that block KIR receptors in combination with immuno-modulatory agents (e.g., lenalidomide) and antibodies that promote antibody-dependent cellular cytotoxicity (e.g., rituximab, anti-CS1). We have initiated patents for these discoveries, and are currently planning to transplant these into phase I clinical trials. Other ongoing research includes enhancing immune function against cancer through STAT3 inhibition to overcome tumor-mediated impairment of dendritic cell maturation, ex vivo specific expansion of cytotoxic of NK cell subsets for clinical use, and enhancing immune cell function following transplantation. The continued success of our Center will depend on a continuing pipeline of novel laboratory discoveries and their translation to early phase clinical trials to assess feasibility and safety as a prelude to larger trials assessing efficacy. Initial funding of the Center by IUPUI has allowed the Center’s conception, and the bringing together of basic and clinical researchers to the “research table” to make this translational/clinical research endeavor a reality, and has allowed us to be competitive for external funding. An important developing outcome of this initiative is the preparation for a Program Project grant in Mobilization and Engraftment of Stem Cells.Item Effects of Altering Cell Proliferation on Hematopoietic Stem and Progenitor Cell Function(2011-06-14) Rohrabaugh, Sara L.; Broxmeyer, Hal E.; Pelus, Louis; Roman, Ann; Yoder, Mervin C.Cell cycle checkpoints guarantee movement through the cell cycle in an appropriate manner. The spindle assembly checkpoint (SAC) ensures the proper segregation of chromosomes into daughter cells during mitosis. Mitotic arrest deficiency 2 (Mad2), a member of the mitotic checkpoint proteins, appears to be crucial for generating the wait anaphase signal to prevent onset of anaphase. We first studied the SAC in hematopoietic stem cells (HSC) to ensure that it was functional. Our previous studies found that prolonged SAC activation was uncoupled from apoptosis initiation in mouse and human embryonic stem cells (ESC). We found that upon treatment with a microtubule-destabilizing agent, HSC arrested in M-phase and subsequently initiated apoptosis. Thus unlike ESC, HSC exhibit coupling of prolonged SAC activation with apoptosis. We studied the effects of Mad2+/- on in vivo recovery of bone marrow HPC from cytotoxic effects and also effects of cytostatic agents on HPC growth in vitro using Mad2-haploinsufficient (Mad2+/-) mice. We found that Mad2+/- HPCs were protected from the cytotoxic effects of cytarabine (Ara-C), a cycle specific agent, consistent with Mad2+/- HPCs being in a slow or non-cycling state. Mad2 haploinsufficiency did not affect recovery of functional HPC after treatment with cyclophosphamide or high sub-lethal dose irradiation, both non-cycle specific agents. There were no differences in immunophenotype defined HSCs in Mad2+/- and Mad2+/+ mice, data confirmed by functional HSC competitive repopulation assays. To better understand the role of Mad2 in HPC, E3330, a cytostatic agent, was used to assess the redox function of Ape1/Ref-1, and colony formation in vitro was examined under normoxic and lowered O2 tension. Mad2+/- HPCs were less responsive to E3330 than Mad2+/+ HPCs, and E3330 was more effective under lowered O2 tension. Mad2+/- HPCs did not exhibit enhanced growth in lowered oxygen tension, in contrast to Mad2+/+ HPCs. Our studies have unexpectedly found that Mad2 haploinsufficiency is protective from the cytotoxic effects of a cycle specific DNA synthesis agent in vivo, and Ape1/Ref-1 inhibitor in vitro.Item Neurexophilin1 suppresses the proliferation of hematopoietic progenitor cells(2012-03-16) Kinzfogl, John M; Broxmeyer, Hal E.; Pelus, Louis; Harrington, Maureen A.; Nakshatri, HarikrishnaNeurexin I alpha (NRXN1α) and Dystroglycan (DAG1) are membrane receptors which serve as mutual ligands in the neuronal system. Neurexophilins (NXPHs) bind NRXN1α. Both NRXN1α and DAG1 were expressed in primitive populations in human cord blood (huCB) and murine bone marrow (muBM), with high concentrations of NXPHs in huCB plasma. We evaluated effects of these molecules on huCB and muBM hematopoietic progenitor (HPC) and stem (HSC) cells. At both a single and population level in vitro, we found that NXPH1 is a potent inhibitor of HPC proliferation acting through NRXN1α, an effect antagonized by DAG1. Injection of recombinant NXPH1 in vivo resulted in myelo- and lymphosuppression, with absolute numbers and cycling status of functional and phenotypically defined HPCs dose- and time-dependently decreased, and absolute numbers and cycling status of phenotypically defined longer-term repopulation HSCs increased. Competitive transplants showed an initial decrease in engraftment of NXPH1-treated cells, with an intermediate stage increase in engraftment. The increase in HSCs is at least partially mediated by the mTOR pathway and is thought to be homeostatic in nature. These results demonstrate the presence and function of a regulated signaling axis in hematopoiesis centered on NRXN1α and its modulation by DAG1 and NXPH1.Item Prostaglandin E₂ promotes recovery of hematopoietic stem and progenitor cells after radiation exposure(2014-07-11) Stilger, Kayla N.; Broxmeyer, Hal E.; Kacena, Melissa A.; Srour, Edward F.; Pelus, LouisThe hematopoietic system is highly proliferative, making hematopoietic stem and progenitor cells (HSPC) sensitive to radiation damage. Total body irradiation and chemotherapy, as well as the risk of radiation accident, create a need for countermeasures that promote recovery of hematopoiesis. Substantive damage to the bone marrow from radiation exposure results in the hematopoietic syndrome of the acute radiation syndrome (HS-ARS), which includes life-threatening neutropenia, lymphocytopenia, thrombocytopenia, and possible death due to infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow HSPC may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E2 (PGE2) is known to have pleiotropic effects on hematopoiesis, inhibiting apoptosis and promoting self-renewal of hematopoietic stem cells (HSC), while inhibiting hematopoietic progenitor cell (HPC) proliferation. We assessed the radiomitigation potential of modulating PGE2 signaling in a mouse model of HS-ARS. Treatment with the PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) at 24 hours post-irradiation resulted in increased survival of irradiated mice compared to vehicle control, with greater recovery in HPC number and colony-forming potential measured at 30 days post-irradiation. In a sublethal mouse model of irradiation, dmPGE2-treatment at 24 hours post-irradiation is associated with enhanced recovery of HSPC populations compared to vehicle-treated mice. Furthermore, dmPGE2-treatment may also act to promote recovery of the HSC niche through enhancement of osteoblast-supporting megakaryocyte (MK) migration to the endosteal surface of bone. A 2-fold increase in MKs within 40 um of the endosteum of cortical bone was seen at 48 hours post-irradiation in mice treated with dmPGE2 compared to mice treated with vehicle control. Treatment with the non-steroidal anti-inflammatory drug (NSAID) meloxicam abrogated this effect, suggesting an important role for PGE2 signaling in MK migration. In vitro assays support this data, showing that treatment with dmPGE2 increases MK expression of the chemokine receptor CXCR4 and enhances migration to its ligand SDF-1, which is produced by osteoblasts. Our results demonstrate the ability of dmPGE2 to act as an effective radiomitigative agent, promoting recovery of HSPC number and enhancing migration of MKs to the endosteum where they play a valuable role in niche restoration.Item Role of STAT3 and SDF-1/CXCL 12 in mitochondrial function in hematopoietic stem and progenitor cells(2016-08-10) Messina-Graham, Steven V.; Broxmeyer, Hal E.; Pelus, Louis; Harrington, Maureen; Srour, EdwardMitochondria are the major ATP producing source within cells. There is increasing data supporting a direct involvement of mitochondria and mitochondrial function in regulating stem cell pluripotency. Mitochondria have also been shown to be important for hematopoietic stem and progenitor cell function. Hematopoietic stem cells have lower numbers of mitochondria (mass), lower mitochondrial membrane potential, and lower ATP levels as compared to other blood cell types. Mitochondria play an important role in hematopoietic stem and progenitor cells, thus we investigated the role of the chemokine, SDF-1/CXCL12, in mitochondrial function in hematopoietic stem and progenitor cells using an SDF-1/CXCL12 transgenic mouse model. We found increased mitochondrial mass is linked to CD34 surface expression in hematopoietic stem and progenitor cells, suggesting that mitochondrial biogenesis is linked to loss of pluripotency. Interestingly these hematopoietic progenitor cells have low mitochondrial membrane potential and these mitochondrial become active prior to leaving the progenitor cell compartment. We also tested the ability of SDF-1/CXL12 to modulate mitochondrial function in vitro by treating the human leukemia cell line, HL-60, and primary mouse lineage- bone marrow cells with SDF-1/CXCL12. We found significantly reduced mitochondrial function at two hours while mitochondrial function was significantly increased at 24 hours. This suggests that SDF1/CXCL12 regulates mitochondrial function in a biphasic manner in a model of hematopoietic progenitors and immature blood cells. This suggests SDF1/CXCL12 may play a role in regulating mitochondrial function in hematopoiesis. We also investigated STAT3 in hematopoietic stem and progenitor cells. Mitochondrial STAT3 plays an essential role in regulating mitochondrial function. By using a knockout (Stat3-/-) mouse model we found that Stat3-/- hematopoietic progenitor cells had reduced colony forming ability, slower cell cycling status, and loss of proliferation in response to multi-cytokine synergy. We also found mitochondrial dysfunction in Stat3-/- hematopoietic stem and progenitor cells. Our results suggest an essential role for mitochondria in HSC function and a novel role for SDF-1/CXCL12 and STAT3 in regulating mitochondrial function in hematopoietic stem and progenitor cells.Item SIRT1 DEFICIENCY COMPROMISES MOUSE EMBRYONIC STEM CELL DIFFERENTIATION, AND EMBRYONIC AND ADULT HEMATOPOIESIS IN THE MOUSE(2011-03-16) Ou, Xuan; Broxmeyer, Hal E.; Pelus, Louis; Roman, Ann; Yoder, Mervin C.SIRT1 (Sirtuin 1) is a founding member of a family of seven proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1-/- mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1-/- mouse embryonic stem (mES) cells in vitro, and hematopoietic progenitors in SIRT1+/+, SIRT1+/-, and SIRT1-/- mice. SIRT1-/- ES cells exhibited markedly delayed/immature formation of blast colony-forming cells (BL-CFCs). When individual blast colonies were analyzed for hematopoietic and endothelial potential, replated SIRT1-/- BL-CFC possessed limited hematopoietic potential, whereas endothelial potential was essentially unaltered. The ability of SIRT1-/- ES cells to form primitive erythroid progenitors was not only delayed but greatly decreased. Moreover, after differentiation of SIRT1-/- mES cells, there were also significant decreases in granulocyte-macrophage (CFU-GM) and multipotential (CFU-GEMM) progenitor cells. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5, decreased β-H1 globin, β-major globin, and Scl gene expression and reduced activation of the Erk1/2 pathway upon SIRT1-/- ES cell commitment. Reintroduction of WT SIRT1 into SIRT1-/- cells partially rescued the primitive erythroid progenitor formation of SIRT1-/- cells and the expression of hemoglobin genes, Hbb-bh1 and Hbb-b1, suggesting that the defect of hematopoietic commitment is due to deletion of SIRT1, and not to genetic drifting of SIRT1-/- cells. To confirm the requirement for SIRT1 for normal development of hematopoietic progenitor cells, we assessed embryonic and adult hematopoiesis in SIRT1+/+, SIRT1+/- and SIRT1-/- mice. Yolk sacs from SIRT1 mutant embryos generated fewer primitive erythroid precursors compared to wild-type (WT) and heterozygous mice. Moreover, knockout of SIRT1 decreased primary bone marrow hematopoietic progenitor cells (HPCs) in 5 week and 12 month old mice, which was especially notable at lower (5%) O2 tension. In addition these progenitors survived less well in vitro under conditions of delayed growth factor addition. Taken together, these results demonstrate that SIRT1 plays a role in ES cell hematopoietic differentiation and mouse hematopoiesis.Item Transfer of intracellular HIV Nef to endothelium causes endothelial dysfunction(2014) Wang, Ting; Pelus, Louis; Clauss, Matthias A.; Yu, Andy; Dent, Alexander L.; Wek, Ronald C.With effective antiretroviral therapy (ART), cardiovascular diseases (CVD), are emerging as a major cause of morbidity and death in the aging population with HIV infection. Although this increase in CVD could be partially explained by the toxic effects of combined anti-retroviral therapy (ART), more recently, HIV infection has emerged as an independent risk factor for CVD. However, it is unclear how HIV can contribute to CVD in patients on ART, when viral titers are low or non-detectable. Here, we provide several lines of evidence that HIV-Nef, produced in infected cells even when virus production is halted by ART, can lead to endothelial activation and dysfunction, and thus may be involved in CVD. We demonstrate that HIV-infected T cell-induced endothelial cell activation requires direct contact as well as functional HIV-Nef. Nef protein from either HIV-infected or Nef-transfected T cells rapidly transfers to endothelial cells while inducing nanotube-like conduits connecting T cells to endothelial cells. This transfer or transfection of endothelial cells results in endothelial apoptosis, ROS generation and release of monocyte attractant protein-1 (MCP-1). A Nef SH3 binding site mutant abolishes Nef-induced apoptosis and ROS formation and reduces MCP-1 production in endothelial cells, suggesting that the Nef SH3 binding site is critical for Nef effects on endothelial cells. Nef induces apoptosis of endothelial cells through both NADPH oxidase- and ROS-dependent mechanisms, while Nef-induced MCP-1 production is NF-kB dependent. Importantly, Nef can be found in CD4 positive and bystander circulating blood cells in patients receiving virally suppressive ART, and in the endothelium of chimeric SIV-infected macaques. Together, these data indicate that Nef could exert pro-atherogenic effects on the endothelium even when HIV infection is controlled and that inhibition of Nef-associated pathways may be promising new therapeutic targets for reducing the risk for cardiovascular disease in the HIV-infected population.