Abstract 32: Hypercholesterolemia-induced Priming of Hematopoietic Stem and Progenitor Cells Aggravates Atherosclerosis

During homeostasis hematopoietic stem and progenitor stem cells (HSPCs) give rise to lymphoid and myeloid cells as well as platelets and erythrocytes. However, during chronic inflammatory conditions hematopoiesis is often skewed towards the myeloid lineage, thereby potentially aggravating the ongoing inflammation. Here we investigated the effects of hypercholesterolemia on HSPCs during atherogenesis. Hypercholesterolemia increased HSPCs, defined as Lin - Sca1 + cKit - , in the bone marrow (BM) of LDLr -/- mice by 253.1%. The number of granulocyte-monocyte progenitors, BM granulocytes and BM monocytes was increased by 18.1%, 34.8% and 13.2%, respectively. In accordance, the myeloid colony forming potential of hypercholesterolemic BM was increased by 25.8%. Peripheral blood monocytes and granulocytes were increased by 203.0% and 161.1%, respectively. Competitive bone marrow transplantations (cBMT) in which we compared the effects of normo- vs. hypercholesterolemia primed HSPCs confirmed that the hypercholesterolemic microenvironment activates HSPCs, as reflected by a 26.5% increased reconstitution of peripheral blood leukocytes 10 weeks after the cBMT. Moreover, hypercholesterolemia-primed, and not normocholesterolemia-primed HSPCs acquired an enhanced propensity to generate myeloid cells, especially granulocytes and Ly6C high monocytes, even under long-term normocholesterolemic conditions in the recipient animals. cBMT demonstrated that hypercholesterolemia-induced activation of HSPCs increased atherosclerosis in LDLr -/- mice by 122.1% and increased CD45.1 + plaque leukocytes by 76.1%. Macrophages differentiated from hypercholesterolemia-primed BM produced increased levels of TNFα (+21.3%), IL6 (+17.4%) and MCP1 (+10.5%) compared to their normocholesterolemic counterparts, demonstrating that hypercholesterolemia-induced priming of HSPCs increased the inflammatory phenotype of their mature offspring. These results demonstrate that hypercholesterolemia-induced priming of HSPCs aggravates atherosclerosis by skewing hematopoiesis towards the pro-inflammatory myeloid lineages. Inhibition of this pro-inflammatory differentiation pathway on HSPC level has the potential to reduce atherosclerosis.

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Mobilization of long-term reconstituting hematopoietic stem cells in mice by recombinant human interleukin 7.

Journal of Experimental Medicine ◽

10.1084/jem.181.1.369 ◽

1995 ◽

Vol 181 (1) ◽

pp. 369-374 ◽

Cited By ~ 26

Author(s):

K J Grzegorzewski ◽

K L Komschlies ◽

S E Jacobsen ◽

F W Ruscetti ◽

J R Keller ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Peripheral Blood ◽

Peripheral Blood Leukocytes ◽

Gene Modification ◽

Blood Leukocytes ◽

Hematopoietic Stem ◽

Interleukin 7 ◽

Cell Repopulation

Administration of recombinant human interleukin 7 (rh)IL-7 to mice has been reported by our group to increase the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakarocyte macrophage) from the bone marrow to peripheral organs (blood, spleen[s], and liver). We now report that IL-7 also stimulates a sixfold increase in the number of more primitive CFU-S day 8 (CFU-S8) and day 12 (CFU-S12) in the peripheral blood leukocytes (PBL) of mice treated with rhIL-7 for 7 d. Moreover, > 90% of lethally irradiated recipient mice that received PBL from rhIL-7-treated donor mice have survived for > 6 mo whereas none of the recipient mice that received an equal number of PBL from diluent-treated donors survived. Flow cytometry analysis at 3 and 6 mo after transplantation revealed complete trilineage (T, B, and myelomonocytic cell) repopulation of bone marrow, thymus, and spleen by blood-borne stem/progenitor cells obtained from rhIL-7-treated donor mice. Thus, IL-7 may prove valuable for mobilizing pluripotent stem cells with long-term repopulating activity from the bone marrow to the peripheral blood for the purpose of gene modification and/or autologous or allogeneic stem cell transplantation.

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Long-Term Clinical Follow-Up and Safety/Toxicity Analysis in 3 X-CGD Patients Treated by Gene Therapy and Non-Myeloablative Conditioning.

Blood ◽

10.1182/blood.v108.11.199.199 ◽

2006 ◽

Vol 108 (11) ◽

pp. 199-199 ◽

Cited By ~ 1

Author(s):

Marion G. Ott ◽

Manfred Schmidt ◽

Stefan Stein ◽

Kerstin Schwarzwaelder ◽

Ulrich Siler ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Myeloid Cells ◽

Adult Patients ◽

Hematopoietic Stem

Abstract Gene transfer into hematopoietic stem cells has been successfully used to correct immunodeficiencies affecting the lymphoid compartment. However, similar results have not been reported for diseases affecting myeloid cells, mainly due to low engraftment levels of gene-modified cells observed in unconditioned patients. Here we report on two adult patients (P1 and P2, follow up >24 months) and one child (P3, 6 years, follow up 15 months) who received gene-transduced hematopoietic stem cells in combination with nonmyeloablative bone marrow conditioning for the treatment of X-linked Chronic Granulomatous Disease (X-CGD), a primary immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes. Therapeutically significant gene marking was detected in neutrophils of both adult patients (P1 and P2) leading to large numbers (up to 60%) of functionally corrected phagocytes 24 months after gene therapy. This high correction resulted from an unexpected but temporarily restricted expansion of gene transduced myeloid cells in vivo. In contrast gene marking and functionally reconstitution levels in P3 have been low (1–2%). Both adult patients suffered from active infections prior to gene therapy (P1 of bacterial liver abscesses and P2 of lung aspergillosis) and were free of severe bacterial and fungal infections until 24 months after transplantation. P3 suffered from an Aspergillus infection of the spinal cord with paraparesis before transplantation and recovered after gene therapy despite low numbers of functionally corrected cells in the peripheral blood. Large-scale mapping of retroviral integration site distribution revealed that activating insertions in the zinc finger transcription factor homologs MDS1/EVI1, PRDM16, or in SETBP1 have expanded gene-corrected long term myelopoiesis 3- to 4-fold in both adults, providing direct evidence in humans that these genes may influence regulation of normal long-term hematopoiesis. The hematopoietic repopulation in P1 was polyclonal until 18 months after therapy. P1 died of a severe bacterial sepsis after colon perforation 27 months after gene therapy. No evidence of malignant transformation was found in peripheral blood or bone marrow aspirates from this patient. Gene marking at death was still 60%; however the function of gene transduced cells, the number of corrected cell clones and the activity of a predominant clone was greatly decreased. P2 has been free of infections since transplantation (last monitoring: month 26). Hematopoietic repopulation was polyclonal in P2 until day 560. In conclusion, gene therapy in combination with bone marrow conditioning has provided a transitory therapeutic benefit for all 3 patients. Further improvements in vector design and conditioning regimes are under investigation to provide a stable and long term correction of the disease.

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E-Cigarette Exposure Decreases Bone Marrow Hematopoietic Progenitor Cells

Cancers ◽

10.3390/cancers12082292 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2292 ◽

Cited By ~ 1

Author(s):

Gajalakshmi Ramanathan ◽

Brianna Craver-Hoover ◽

Rebecca J. Arechavala ◽

David A. Herman ◽

Jane H. Chen ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Electronic Cigarettes ◽

Selective Advantage ◽

Tobacco Product ◽

Long Term Effects ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Electronic cigarettes (E-cigs) generate nicotine containing aerosols for inhalation and have emerged as a popular tobacco product among adolescents and young adults, yet little is known about their health effects due to their relatively recent introduction. Few studies have assessed the long-term effects of inhaling E-cigarette smoke or vapor. Here, we show that two months of E-cigarette exposure causes suppression of bone marrow hematopoietic stem and progenitor cells (HSPCs). Specifically, the common myeloid progenitors and granulocyte-macrophage progenitors were decreased in E-cig exposed animals compared to air exposed mice. Competitive reconstitution in bone marrow transplants was not affected by two months of E-cig exposure. When air and E-cig exposed mice were challenged with an inflammatory stimulus using lipopolysaccharide (LPS), competitive fitness between the two groups was not significantly different. However, mice transplanted with bone marrow from E-cigarette plus LPS exposed mice had elevated monocytes in their peripheral blood at five months post-transplant indicating a myeloid bias similar to responses of aged hematopoietic stem cells (HSC) to an acute inflammatory challenge. We also investigated whether E-cigarette exposure enhances the selective advantage of hematopoietic cells with myeloid malignancy associated mutations. E-cigarette exposure for one month slightly increased JAK2V617F mutant cells in peripheral blood but did not have an impact on TET2−/− cells. Altogether, our findings reveal that chronic E-cigarette exposure for two months alters the bone marrow HSPC populations but does not affect HSC reconstitution in primary transplants.

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CFU-GM content of bone marrow graft correlates with time to hematologic reconstitution following autologous bone marrow transplantation with 4- hydroperoxycyclophosphamide-purged bone marrow

Blood ◽

10.1182/blood.v70.1.271.bloodjournal701271 ◽

1987 ◽

Vol 70 (1) ◽

pp. 271-275

Author(s):

SD Rowley ◽

M Zuehlsdorf ◽

HG Braine ◽

OM Colvin ◽

J Davis ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Autologous Bone Marrow Transplantation ◽

Autologous Bone Marrow ◽

Marrow Graft ◽

Blood Leukocytes ◽

Cell Content ◽

Hematopoietic Stem ◽

Bone Marrow Graft ◽

Autologous Bone

Autologous bone marrow transplants (BMTs) can repopulate the hematologic system of patients treated with marrow-ablative chemotherapy and/or radiotherapy. However, treatment of the bone marrow graft to eliminate residual tumor cells prior to reinfusion can delay the return of peripheral blood elements, presumably from damage to or loss of hematopoietic stem cells responsible for hematologic recovery. To develop a model predictive of hematologic recovery, we studied the progenitor cell contents of 4-hydroperoxycyclophosphamide (100 micrograms/mL)-purged bone marrow grafts of 40 consecutive patients undergoing autologous BMT at this center. Granulocyte-macrophage colonies (CFU-GM) were grown from all grafts after treatment with this chemotherapeutic agent, but erythroid (BFU-E) and mixed (CFU-GEMM) colonies were grown from only 44% and 33% of the grafts respectively. The recovery of CFU-GM after purging ranged from 0.07% to 23%. The logarithm of CFU-GM content of the treated grafts was linearly correlated with the time to recovery of peripheral blood leukocytes (r = -0.80), neutrophils (r = -0.79), reticulocytes (r = -0.60), and platelets (r = -0.66). The CFU-GM content of purged autologous bone marrow grafts may reflect the hematopoietic stem cell content of the grafts and thus predict the rate of hematologic recovery in patients undergoing autologous BMT.

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ALL Cells Mobilized by AMD3100 Are More Proliferative, and Remain In the Circulation Longer Than Normal HSC, Enhancing the Action of Vincristine

Blood ◽

10.1182/blood.v116.21.2137.2137 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2137-2137 ◽

Cited By ~ 1

Author(s):

Linda J. Bendall ◽

Robert Welschinger ◽

Florian Liedtke ◽

Carole Ford ◽

Aileen Dela Pena ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Bone Marrow Microenvironment ◽

Hematopoietic Progenitors ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Cell Mobilization ◽

Cycle Dependent

Abstract Abstract 2137 The chemokine CXCL12, and its receptor CXCR4, play an essential role in homing and engraftment of normal hematopoietic cells in the bone marrow, with the CXCR4 antagonist AMD3100 inducing the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. We have previously demonstrated that AMD3100 similarly induces the mobilization of acute lymphoblastic leukemia (ALL) cells into the peripheral blood. The bone marrow microenvironment is thought to provide a protective niche for ALL cells, contributing to chemo-resistance. As a result, compounds that disrupt leukemic cell interactions with the bone marrow microenvironment are of interest as chemo-sensitizing agents. However, the mobilization of normal hematopoietic stem and progenitor cells may also increase bone marrow toxicity. To better evaluate how such mobilizing agents affect normal hematopoietic progenitors and ALL cells, the temporal response of ALL cells to the CXCR4 antagonist AMD3100 was compared to that of normal hematopoietic progenitor cells using a NOD/SCID xenograft model of ALL and BALB/c mice respectively. ALL cells from all 7 pre-B ALL xenografts were mobilized into the peripheral blood by AMD3100. Mobilization was apparent 1 hour and maximal 3 hours after drug administration, similar to that observed for normal hematopoietic progenitors. However, ALL cells remained in the circulation for longer than normal hematopoietic progenitors. The number of ALL cells in the circulation remained significantly elevated in 6 of 7 xenografts examined, 6 hours post AMD3100 administration, a time point by which circulating normal hematopoietic progenitor levels had returned to baseline. No correlation between the expression of the chemokine receptor CXCR4 or the adhesion molecules VLA-4, VLA-5 or CD44, and the extent or duration of ALL cell mobilization was detected. In contrast, the overall motility of the ALL cells in chemotaxis assays was predictive of the extent of ALL cell mobilization. This was not due to CXCL12-specific chemotaxis because the association was lost when correction for background motility was undertaken. In addition, AMD3100 increased the proportion of actively cells ALL cells in the peripheral blood. This did not appear to be due to selective mobilization of cycling cells but reflected the more proliferative nature of bone marrow as compared to peripheral blood ALL cells. This is in contrast to the selective mobilization of quiescent normal hematopoietic stem and progenitor cells by AMD3100. Consistent with these findings, the addition of AMD3100 to the cell cycle dependent drug vincristine, increased the efficacy of this agent in NOD/SCID mice engrafted with ALL. Overall, this suggests that ALL cells will be more sensitive to effects of agents that disrupt interactions with the bone marrow microenvironment than normal progenitors, and that combining agents that disrupt ALL retention in the bone marrow may increase the therapeutic effect of cell cycle dependent chemotherapeutic agents. Disclosures: Bendall: Genzyme: Honoraria.

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Gabp Transcription Factor Is Required for Self-Renew and Proliferation of Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v118.21.1284.1284 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1284-1284

Author(s):

Zhongfa Yang ◽

Karen Drumea ◽

James Cormier ◽

Junling Wang ◽

Xuejun Zhu ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Transcription Factor ◽

Progenitor Cells ◽

Myeloid Cells ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Significant Difference ◽

Stem And Progenitor Cells ◽

Ets Domain

Abstract Abstract 1284 GABP is an ets transcription factor that regulates genes which are required for normal hematopoietic development. In myeloid cells, GABP is an essential component of a retinoic acid-inducible enhanceosome that mediates granulocytic gene expression and, in lymphoid cells, GABP regulates expression of IL7-R and the essential transcription factor, Pax5. GABP is a tetrameric complex that includes GABPa, which binds DNA via its ets domain, and GABPb, which contains the transcription activation domain. Genetic disruption of mouse Gabpa caused early embryonic lethality. We created mice in which loxP recombination sites flank exons that encode the Gabpa ets domain, and bred them to mice that bear the Mx1Cre recombinase; injection with pIC induced Cre expression and efficiently deleted Gabpa in hematopoietic cells. One half of the Gabpa knock-out (KO) mice died within two weeks of pIC injection in association with widespread visceral hemorrhage. Gabpa KO mice exhibited a rapid loss of mature granulocytes, and residual myeloid cells exhibited myelodysplasia due, in part, to regulation by Gabp of the transcriptional repressor, Gfi-1. We used bone marrow transplantation to demonstrate that the defect in Gabpa null myeloid cells is cell intrinsic. Although hematopoietic progenitor cells in Gabpa KO bone marrow were decreased more than 100-fold compared to pIC treated control mice, there was not a statistically significant difference in the numbers of Lin−c-kit+Sca-1− hematopoietic stem cells (HSCs) between KO and control mice. Genetic disruption of Gfi-1 disruption in HSCs caused increased cell cycle activity – an effect that is diametrically opposite of the effect of Gabpa KO; this suggests that the effect of Gabpa on HSCs is not due to its control of Gfi-1. In contrast, Gabpa KO HSCs exhibited a marked decrease in cell cycle activity, but did not demonstrate increased apoptosis. The defects in S phase entry of Gabpa null HSCs are reminiscent of the cell cycle defects in Gabpa null fibroblasts, in which expression of Skp2 E3 ubiquitin ligase, which controls degradation of the cyclin dependent kinase inhibitors (CDKIs) p21 and p27, was markedly reduced following Gabpa disruption. We showed that Gabpa KO cells express reduced levels of Skp2. We propose that GABP controls self-renewal and proliferation of mouse bone marrow stem and progenitor cells, in part, through its regulation of Skp2. Thus, Gabpa is a key regulator of myeloid differentiation through its control of Gfi-1, but it is required for cell cycle activity of HSCs, by a distinct effect that may be due to its control of Skp2 and CDKIs. Disclosures: No relevant conflicts of interest to declare.

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The Disease-Related Bone Marrow Microenvironment Alters Hematopoietic Stem and Progenitor Function in Multiple Myeloma Patients

Blood ◽

10.1182/blood.v118.21.2898.2898 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2898-2898

Author(s):

Ingmar Bruns ◽

Ron-Patrick Cadeddu ◽

Ines Brückmann ◽

Sebastian Buest ◽

Julia Fröbel ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Progenitor Cells ◽

Bone Marrow Microenvironment ◽

Tgf Beta ◽

Hematopoietic Stem ◽

Healthy Donors ◽

Stem And Progenitor Cells ◽

Related Bone Marrow

Abstract Abstract 2898 Multiple myeloma (MM) patients often suffer from hematopoietic impairment already at the time of diagnosis with anemia as the prevailing symptom. Given the overt affection of the bone marrow in MM patients by the invasion of malignant plasma cells, we hypothesized that hematopoietic insufficiency in these patients may originate from a functional impairment of hematopoietic stem and progenitor cells. Quantitative analysis of BM CD34+ HSPC cell subsets from MM patients and age-matched healthy donors showed a significant decline of all HSPC subsets including hematopoietic stem cells, common myeloid and lymphoid progenitors, granulocyte-macrophage progenitors and megakaryocyte-erythrocyte progenitors in MM patients. The greatest diminution was observed in megakaryocyte-erythrocyte progenitors (MEP) which were 4.9-fold reduced in comparison to healthy donors. Transcriptional analyses of CD34+ HSPC subsets revealed a significant deregulation of signaling pathways that was particularly striking for TGF beta signaling and suggested increased activation of this signaling pathway. Immunhistochemical staining of phosphorylated smad2, the downstream mediator of TGF receptor I kinase activation, in bone marrow sections and immunoblotting of purified CD34+ HSPC of MM patients confirmed the overactivation of TGF beta signaling. On a functional level, we observed significantly reduced long-term self-renewal and clonogenic growth, particularly of the erythroid precursors BFU-E and CFU-E, in CD34+ HSPC of MM patients which could be restored by inhibition of TGF beta signaling. Proliferation and cell cycle analyses revealed a significantly decreased proliferation activity in CD34+ HSPC and, particularly, MEP. Again, this was reversible after inhibition of TGF beta signaling. In addition, the transcriptional analyses showed disturbance of pathways involved in the adhesion and migration of HSPC and the gene encoding for the principal hyaluronan receptor CD44 throughout the HSPC subsets. This was corroborated by immunofluorescence imaging of CD44 on HSPC subsets showing a marked downregulation in the patients' cells. In line, the adhesion of CD34+ HSPC subsets to hyaluronan and their migration towards SDF-1 was significantly inhibited. Subsequent xenotransplantation of CD34+ HSPC from MM patients and healthy donors into myeloma-free recipients revealed even increased long-term engraftment of CD34+ HSPC obtained from MM patients and normal differentiation capacities suggesting that the observed functional alterations in fact depend on the MM-related bone marrow microenvironment. Our data show that hematopoietic impairment in patients with multiple myeloma originates, at least in part, from functional alterations of hematopoietic stem and progenitor cells. These alterations seem to depend on the disease-related changes of the bone marrow microenvironment. Currently, experiments are underway to elucidate in more detail the role of the microenvironment and the responsible structures for the impairment of HSPC in MM patients. These data will be presented. Disclosures: Kobbe: Celgene: Consultancy, Research Funding; Ortho Biotec: Consultancy.

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Prostaglandin E2 Inhibits Apoptosis in Hematopoietic Stem and Progenitor Cells and Enhances Their Survival Following Sub-Lethal Radiation Injury,

Blood ◽

10.1182/blood.v118.21.3401.3401 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3401-3401

Author(s):

Rebecca L Porter ◽

Mary A Georger ◽

Laura M Calvi

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Radiation Injury ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Cox 2 ◽

Apoptotic Genes

Abstract Abstract 3401 Hematopoietic stem and progenitor cells (HSPCs) are responsible for the continual production of all mature blood cells during homeostasis and times of stress. These cells are known to be regulated in part by the bone marrow microenvironment in which they reside. We have previously reported that the microenvironmentally-produced factor Prostaglandin E2 (PGE2) expands HSPCs when administered systemically in naïve mice (Porter, Frisch et. al., Blood, 2009). However, the mechanism mediating this expansion remains unclear. Here, we demonstrate that in vivo PGE2 treatment inhibits apoptosis of HSPCs in naïve mice, as measured by Annexin V staining (p=0.0083, n=6–7 mice/group) and detection of active-Caspase 3 (p=0.01, n=6–7 mice/group). These data suggest that inhibition of apoptosis is at least one mechanism by which PGE2 expands HSPCs. Since PGE2 is a local mediator of injury and is known to play a protective role in other cell types, we hypothesized that it could be an important microenvironmental regulator of HSPCs during times of injury. Thus, these studies explored the role of PGE2 signaling in the bone marrow following myelosuppressive injury using a radiation injury model. Endogenous PGE2 levels in the bone marrow increased 2.9-fold in response to a sub-lethal dose of 6.5 Gy total body irradiation (TBI)(p=0.0004, n=3–11 mice/group). This increase in PGE2 correlated with up-regulation of microenvironmental Cyclooxygenase-2 (Cox-2) mRNA (p=0.0048) and protein levels at 24 and 72 hr post-TBI, respectively. Further augmentation of prostaglandin signaling following 6.5 Gy TBI by administration of exogenous 16,16-dimethyl-PGE2 (dmPGE2) enhanced the survival of functional HSPCs acutely after injury. At 24 hr post-TBI, the bone marrow of dmPGE2-treated animals contained significantly more LSK cells (p=0.0037, n=13 mice/group) and colony forming unit-spleen cells (p=0.037, n=5 mice/group). Competitive transplantation assays at 72 hr post-TBI demonstrated that bone marrow cells from irradiated dmPGE2-treated mice exhibited increased repopulating activity compared with cells from vehicle-treated mice. Taken together, these results indicate that dmPGE2 treatment post-TBI increases survival of functional HSPCs. Since PGE2 can inhibit apoptosis of HSPCs in naïve mice, the effect of dmPGE2 post-TBI on apoptosis was also investigated. HSPCs isolated from mice 24 hr post-TBI demonstrated statistically significant down-regulation of several pro-apoptotic genes and up-regulation of anti-apoptotic genes in dmPGE2-treated animals (3 separate experiments with n=4–8 mice/group in each), suggesting that dmPGE2 initiates an anti-apoptotic program in HSPCs following injury. Notably, there was no significant change in expression of the anti-apoptotic gene Survivin, which has previously been reported to increase in response to ex vivo dmPGE2 treatment of bone marrow cells (Hoggatt et. al., Blood, 2009), suggesting differential effects of dmPGE2 in vivo and/or in an injury setting. Additionally, to ensure that this inhibition of apoptosis was not merely increasing survival of damaged and non-functional HSPCs, the effect of early treatment with dmPGE2 post-TBI on hematopoietic recovery was assayed by monitoring peripheral blood counts. Interestingly, dmPGE2 treatment in the first 72 hr post-TBI significantly accelerated recovery of platelet levels and hematocrit compared with injured vehicle-treated mice (n=12 mice/group). Immunohistochemical analysis of the bone marrow of dmPGE2-treated mice also exhibited a dramatic activation of Cox-2 in the bone marrow microenvironment. This suggests that the beneficial effect of dmPGE2 treatment following injury may occur, both through direct stimulation of hematopoietic cells and also via activation of the HSC niche. In summary, these data indicate that PGE2 is a critical microenvironmental regulator of hematopoietic cells in response to injury. Exploitation of the dmPGE2-induced initiation of an anti-apoptotic program in HSPCs may represent a useful method to increase survival of these cells after sub-lethal radiation injury. Further, amplification of prostaglandin signaling by treatment with PGE2 agonists may also represent a novel approach to meaningfully accelerate recovery of peripheral blood counts in patients with hematopoietic system injury during a vulnerable time when few therapeutic options are currently available. Disclosures: No relevant conflicts of interest to declare.

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Aberrant Hedgehog Pathway Activity Marks Clinical MDS Progression and Accelerates Leukemic Transformation in Vivo

Blood ◽

10.1182/blood.v124.21.528.528 ◽

2014 ◽

Vol 124 (21) ◽

pp. 528-528 ◽

Cited By ~ 2

Author(s):

Lukasz P Gondek ◽

Yiting Lim ◽

Hideki Makishima ◽

Qiuju Wang ◽

Jaroslaw P. Maciejewski ◽

...

Keyword(s):

Bone Marrow ◽

Disease Progression ◽

Peripheral Blood ◽

Myeloid Cells ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Pathway Activity ◽

Pathway Activation ◽

Hh Signaling

Abstract Introduction:Myelodysplastic Syndromes (MDS) represent a heterogeneous group of hematopoietic stem cell (HSC) disorders with varying clinical outcomes, but prognosis uniformly worsens with transformation to secondary acute myeloid leukemia (AML). Despite recent progress in genomics, the mechanisms responsible for disease progression are not fully understood as most of the somatic mutations defined thus far can be found at early stages of the disease. Previous studies have identified aberrant activation of the Hedgehog (Hh) signaling pathway in a subset of AML patients and the expression of the Hh-regulated transcription factor GLI2 correlated with inferior overall and progression free survival. In solid tumors, Hh pathway activation has been associated with metastatic disease progression, and we examined its role in MDS progression and transformation to AML. Methods/Results: We initially quantified changes in Hh pathway activity in CD34+ cells isolated from serial bone marrow samples collected from MDS patients at the time of diagnosis and following progression to AML. We found that the expression of the Hh target genes GLI1 and PTCH1 was increased in 67% of patients (4/6) suggesting that pathway activation was involved in the development of secondary AML. We also analyzed gene expression in 135 MDS patients and found significantly higher GLI2 expression in high-risk MDS (N=80) compared to low-risk MDS (N=55) (p=0.036). In addition, bone marrow blast percentage was significantly higher in the MDS cohort with higher GLI2 expression (mean±SEM=7.1±0.7%) than with lower expression (5.4±0.5%, p=0.039). In order to mechanistically study the effects of Hh pathway activation on MDS progression, we studied mice expressing the Nup98-HoxD13 (NHD13) fusion gene under the control of the vav promoter that generates progressive cytopenia and, in some animals, progression to AML. We crossed NHD13 mice with mice conditionally expressing the constitutively active mutant of the Hh signal transduction regulator Smoothened (SmoM2) in hematopoietic cells expressing Mx1-Cre. The survival of double transgenic animals (NHD13/SmoM2) was significantly shorter compared to mice expressing NHD13 (median survival of 3 months vs. 12 months, p<0.001, Fig. 1). Similar to previous reports we found that the expression of SmoM2 alone had no significant effect on survival or hematologic phenotype. Compared to NHD13 mice, NHD13/SmoM2 mice had significantly higher peripheral blood WBC (93.9k vs. 5.2k, p<0.001) and splenomegaly (633mg vs. 462mg, p=0.006) at the time of disease progression. Furthermore, an immature population of myeloid cells (Mac1+/Gr1 dim) was widely present in peripheral blood, bone marrow and spleen of NHD13/SmoM2 mice. We also examined mice prior to the onset of disease (4-8 weeks post Smo M2 induction) and found an increased frequency of myeloid cells (Mac1+/Gr1+) within the peripheral blood compared to wild type and NHD13/Smo M2 mice (wild type – 17.2%, NHD13 - 40.5%, NHD13/SmoM2 - 68%, p<0.01). Within the bone marrow, hematopoietic stem/progenitor cells (Lin-Sca1+cKit+) were depleted within both NHD13 and NHD13/SmoM2 mice. To examine the impact of Hh activation on clonogenic growth and self-renewal potential, we plated bone marrow cells in methylcellulose and found that NHD13/SmoM2 cells contained a significantly higher number of CFU compared to NHD13 cells (37 vs. 2, p=0.002) that was sustained with subsequent passages (400 vs. 30, p<0.001). In order to define the compartment enriched for leukemia initiating potential (LIC) we transplanted Lin- and Lin+ leukemic fractions from NHD13/SmoM2 mice into naive recipients and found that Lin+ cells led to death within 3 months suggesting that the Lin+ compartment has acquired self- renewal potential. Conclusions:We found that the Hh signaling pathway may be aberrantly activated in a subset of secondary AML patients progressing from MDS. We also demonstrated that activation of Hh signaling induces fatal AML in a mouse model of MDS characterized by inferior survival and widespread expansion of immature myeloid cells. This disease progression is driven by the acquisition of self-renewal and tumor initiating potential in differentiated Lin+ hematopoietic cells. Our findings suggest that Hh pathway inhibition may be a promising approach for AML arising from MDS. Figure 1 Figure 1. Disclosures Maciejewski: Alexion: Speakers Bureau; Celgene: Speakers Bureau.

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PTPN11D61Y/+; Vavcre+ Animals Commonly Succumb to Leukemia Relapse Despite Robust Engraftment of Transplanted Wild-Type Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v128.22.496.496 ◽

2016 ◽

Vol 128 (22) ◽

pp. 496-496

Author(s):

Stefan P. Tarnawsky ◽

Mervin C. Yoder ◽

Rebecca J. Chan

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Wild Type ◽

Hematopoietic Stem ◽

Donor Cells ◽

Stem And Progenitor Cells ◽

Leukemia Relapse ◽

Conditioning Regimens

Juvenile Myelomonocytic Leukemia (JMML) is a rare childhood myelodysplastic / myeloproliferative overlap disorder. JMML exhibits myeloid populations with mutations in Ras-Erk signaling genes, most commonly PTPN11, which confer growth hypersensitivity to GM-CSF. While allogeneic hematopoietic stem cell transplant (HSCT) is the treatment of choice for children with JMML, 50% of children succumb to leukemia relapse; however, the mechanism leading to this high relapse rate is unknown. We hypothesized that the hyperinflammatory nature of JMML may damage the bone marrow microenvironment, leading to poor engraftment of normal donor cells following transplant, permitting residual leukemia cells to outcompete the normal graft, and thus promoting leukemia relapse. Using Vav1 promoter-directed Cre, we generated a mouse model of JMML that conditionally expresses gain-of-function PTPN11D61Yin utero during development. While PTPN11D61Y/+; VavCre+embryos did not demonstrate in utero lethality, we observed a modest reduction of PTPN11D61Y/+; VavCre+ mice at the time of weaning compared to predicted Mendelian frequencies. Further, surviving PTPN11D61Y/+; VavCre+ mice developed elevated peripheral blood leukocytosis and monocytosis as early as 4 weeks of age compared to PTPN11+/+; VavCre+ controls. To address the hypothesis that an aberrant bone marrow microenvironment in the PTPN11D61Y/+ mice leads to poor engraftment of wild-type donor cells following transplant, we examined engraftment of wild-type hematopoietic stem and progenitor cells (HSPCs) in the PTPN11D61Y/+; VavCre+ mice and monitored animals for disease relapse. 16-24 week-old diseased PTPN11D61Y/+; VavCre+ and control PTPN11+/+; VavCre+ mice were lethally irradiated (11 Gy split dose) and transplanted with 5 x 105 CD45.1+ wild-type bone marrow low density mononuclear cells (LDMNCs), which simulates a limiting stem cell dose commonly available in a human HSCT setting. 6 weeks post-HSCT, PTPN11D61Y/+; VavCre+recipients demonstrated an unexpected elevated CD45.1+ donor cell contribution in peripheral blood compared to the control PTPN11+/+; VavCre+ recipients. However, despite superior engraftment in the PTPN11D61Y/+; VavCre+ recipients, these mice had a significantly shorter median survival post-HSCT due to a resurgence of recipient CD45.2-derived leukemic cells. We repeated the experiment using a high dose of CD45.1+ LDMNCs (10 x 106 cells) to determine if providing a saturating dose wild-type cells could prevent the relapse of recipient-derived leukemogenesis and normalize the survival of the PTPN11D61Y/+; VavCre+recipients. While this saturating dose of wild-type cells resulted in high peripheral blood chimerism in both the PTPN11D61Y/+; VavCre+ and PTPN11+/+; VavCre+ recipients, the PTPN11D61Y/+; VavCre+ animals nevertheless demonstrated significantly reduced overall survival. When we examined the cause of mortality in the HSCT-treated PTPN11D61Y/+; VavCre+mice, we found enlarged spleens, hypercellular bone marrow, and enlarged thymuses. Flow cytometry revealed that the majority of cells in the peripheral blood, bone marrow, and spleen were recipient-derived CD45.2+ CD4+ CD8+ T cells. To verify that the disease was neoplastic in origin, secondary transplants into CD45.1/.2 recipients were performed from two independent primary PTPN11D61Y/+; VavCre+and two independent primary PTPN11+/+; VavCre+ controls. Secondary recipients of bone marrow from PTPN11D61Y/+; VavCre+ animals rapidly succumbed to a CD45.2-derived T-cell acute lymphoid leukemia (T-ALL). Previous studies demonstrated that wild-type PTPN11 is needed to protect the integrity of the genome by regulating Polo-like kinase 1 (Plk1) during the mitosis of the cell cycle (Liu et al., PNAS, 2016). We now demonstrate that even when PTPN11 mutant animals are provided with saturating doses of wild-type HSCs, dysregulated residual recipient cells are able to produce relapsed disease. Collectively, these studies highlight the propensity of residual mutant PTPN11 cells to transform after being subjected to mutagenic agents that are commonly used for conditioning regimens prior to allogeneic HSCT. These findings suggest that modified pre-HSCT conditioning regimens bearing reduced mutagenicity while maintaining adequate cytoreductive efficacy may yield lower post-HSCT leukemia relapse in children with PTPN11mutations. Disclosures No relevant conflicts of interest to declare.

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