- Email: [email protected]
Regulation of hematopoietic stem cells by toll like receptor 2
Poster Presentations/ Experimental Hematology 44 (2016) S56–S110
3135 - REGULATION OF HEMATOPOIETIC STEM CELLS BY TOLL LIKE RECEPTOR 2 Laura Schuettpelz1, Darlene Monlish1, Angela Herman1, Sima Beat1, Molly Romine1, and Sabrina Zipper2 1 Washington University, St. Louis, USA; 2Washington University, Seattle, USA TLR2 is a member of the TLR family of pattern-recognition receptors that play a central role in innate immunity. Studies of TLRs have historically focused on mature immune cells, however TLRs are also expressed on HSCs and recent reports demonstrate that TLR signaling may influence the immune response from the level of the HSC. Furthermore, deregulated TLR signaling is associated with myelodysplastic syndromes (MDS), a group of HSC disorders characterized by ineffective hematopoiesis and a high risk of transformation to acute leukemia, suggesting that aberrant signaling through these receptors has detrimental effects on HSCs. While accumulating data support a role for TLR signaling in regulating HSCs, the mechanisms are not clear, and the contributions of cell-autonomous versus non-autonomous TLR signaling to the effects on HSCs are not well known. Our current studies aim to elucidate the role of TLR2 in HSCs, focusing on this particular TLR because it is expressed in HSCs, has a large cadre of known natural ligands, and enhanced TLR2 signaling is associated with MDS. We previously reported that TLR2 is not required for normal HSC function (in fact, loss of TLR2 leads to enhanced bone marrow repopulating activity). To model the enhanced TLR2 signaling seen in MDS, we treated mice with PAM3CSK4, a synthetic TLR1/2 ligand. Using flow cytometry, we find that in vivo exposure to this TLR2 ligand (100 ug IP every other day x3 doses) leads to increased HSC cycling, expansion of bone marrow and spleen HSCs, and a reduction in bone marrow megakaryocyte-erythroid progenitors (MEPs). In spite of increased HSC numbers, competitive repopulation transplants showed that TLR2 agonist treatment leads to a loss of HSC self-renewal. Treatment of chimeric animals revealed that these effects are largely cell non-autonomous (mediated by both radio-resistant and radio-sensitive populations), with a minor contribution from cell-autonomous TLR2 signaling. Cytokine arrays demonstrated an increase in G-CSF and TNFa following TLR2 agonist treatment. To elucidate the contribution of these cytokines to the TLR2 agonist-mediated effects on HSCs, respective receptor knockout models were employed. Inhibition of G-CSF enhanced the expansion of bone marrow HSCs in response to PAM3CSK4, but minimally reduced the mobilization of HSCs to the spleen. Loss of TNFa, a,however, significantly reduced the expansion of spleen HSCs in response to PAM3CSK4, and abrogated the PAM3CSK4-induced spleen HSC cycling. Taken together, these data suggest that TLR2 signaling affects HSCs via both cell cell-autonomous and non-autonomous mechanisms, with G-CSF and TNFa acontributing to TLR2 agonist-mediated effects on HSC cycling, mobilization, and function. Ongoing studies aim to determine the cell types that are crucial for mediating the effects of TLR2 signaling on HSCs and elucidate the role of this pathway in the pathogenesis of MDS.
3136 - UNDERSTANDING THE DEVELOPMENT OF MLL REARRANGED LEUKEMIAS Roshani Sinha1,2,3, Cristina Porcheri4, Teresa d’Altri5, Lluis Espinosa4, and Anna Bigas Salvans4 1 Institut Hospital del Mar d’Investigacions Mediques; 2Kingston University; 3Jawaharlal Nehru Technological University; 4Institut Hospital del Mar d’Investigacions Mediques, Barcelona, Spain; 5DanStem, Copenhagen, Denmark Leukemic initiating cells (LIC) share critical similarities with endogenous stem cells, and common elements of regulation might exist to preserve self-renewal and prevent differentiation. As hematopoietic cells progress through their development, we expect the early appearing progenitors to be differently primed to acquire an aberrant phenotype by conditions existing intrinsically in the cell or in its developing niche, resulting in a higher probability of transforming into cancer initiating cells, as is seen in the aggressive infant leukemias as opposed to other childhood or adult leukemias. Rearrangements involving the MLL gene 11q23 are found in majority of human infant leukemias (O80% of ALL and 35-50% AML cases) and have been associated with extremely poor prognosis of these patients. While far less frequent (total of 10% cases), they are found in leukemias affecting older children and adults as well, making MLL-rearranged leukemia models ideal for our study. We performed induction of the leukemic MLL/ENL chromosomal translocation in hematopoietic cells from distinct hematopoietic tissues at different developmental stages i.e., Aorta-gonads-mesonephric tissue (AGMs, E10.5), fetal liver (FL,
S99
E12.5) and bone marrow (BM, P60) to develop and recapitulate the human infant, childhood and adult leukemias respectively and to compare transformation capacities of these distinct hematopoietic tissues. After evaluation of several models to induce MLL-ENL recombination, we have reproducibly obtained MLL-ENL leukemia from adult animals with the interferon inducible Mx-1 Cre line. Moreover, we can induce this translocation in embryonic tissues with the same line and pIpC injection. We are currently evaluating the effect of embryonic induction in FL and AGM in vivo, which will be presented at the meeting. In conclusion, we are aiming to characterize a novel model of embryo-derived leukemia which could be of help to study the ontogeny of infant leukemias.
3137 - THE CHROMATIN REGULATOR CHD8 IS A CONTEXT-DEPENDENT MEDIATOR OF CELL SURVIVAL IN MURINE HEMATOPOIETIC MALIGNANCIES Jennifer Shingleton1 and Michael Hemann2 1 Duke University, Durham, USA; 2MIT, Cambridge, USA Aberrant chromatin regulation is a frequent driver of leukemogenesis. Mutations in chromatin regulators often result in more stem-like cells that seed a bulk leukemic population. Inhibitors targeting these proteins represent an emerging class of therapeutics, and identifying further chromatin regulators that promote disease progression may result in additional drug targets. We identified the chromatin-modifying protein CHD8 as necessary for cell survival through an RNAi screen previously carried out in a mouse model of BCR-Abl+ B-cell acute lymphoblastic leukemia. This disease has a poor prognosis despite treatment with kinase inhibitors targeting BCR-Abl. Although implicated as a risk factor in autism spectrum disorder, the mechanism of CHD8’s activity is still unclear and has never been studied in the context of hematopoietic malignancies. Here we demonstrate that depletion of CHD8 in B-ALL cells leads to cell death. While multiple B cell malignancies were dependent on CHD8 expression for survival, T cell malignancies displayed milder phenotypes upon CHD8 knockdown. In addition, ectopic expression of the Notch1 intracellular domain in a T cell malignancy partially alleviated the detrimental effect of CHD8 depletion. Our results demonstrate that CHD8 has a context-dependent role in cell survival, and its inhibition may be an effective treatment for B lymphoid malignancies.
3135 - REGULATION OF HEMATOPOIETIC STEM CELLS BY TOLL LIKE RECEPTOR 2 Laura Schuettpelz1, Darlene Monlish1, Angela Herman1, Sima Beat1, Molly Romine1, and Sabrina Zipper2 1 Washington University, St. Louis, USA; 2Washington University, Seattle, USA TLR2 is a member of the TLR family of pattern-recognition receptors that play a central role in innate immunity. Studies of TLRs have historically focused on mature immune cells, however TLRs are also expressed on HSCs and recent reports demonstrate that TLR signaling may influence the immune response from the level of the HSC. Furthermore, deregulated TLR signaling is associated with myelodysplastic syndromes (MDS), a group of HSC disorders characterized by ineffective hematopoiesis and a high risk of transformation to acute leukemia, suggesting that aberrant signaling through these receptors has detrimental effects on HSCs. While accumulating data support a role for TLR signaling in regulating HSCs, the mechanisms are not clear, and the contributions of cell-autonomous versus non-autonomous TLR signaling to the effects on HSCs are not well known. Our current studies aim to elucidate the role of TLR2 in HSCs, focusing on this particular TLR because it is expressed in HSCs, has a large cadre of known natural ligands, and enhanced TLR2 signaling is associated with MDS. We previously reported that TLR2 is not required for normal HSC function (in fact, loss of TLR2 leads to enhanced bone marrow repopulating activity). To model the enhanced TLR2 signaling seen in MDS, we treated mice with PAM3CSK4, a synthetic TLR1/2 ligand. Using flow cytometry, we find that in vivo exposure to this TLR2 ligand (100 ug IP every other day x3 doses) leads to increased HSC cycling, expansion of bone marrow and spleen HSCs, and a reduction in bone marrow megakaryocyte-erythroid progenitors (MEPs). In spite of increased HSC numbers, competitive repopulation transplants showed that TLR2 agonist treatment leads to a loss of HSC self-renewal. Treatment of chimeric animals revealed that these effects are largely cell non-autonomous (mediated by both radio-resistant and radio-sensitive populations), with a minor contribution from cell-autonomous TLR2 signaling. Cytokine arrays demonstrated an increase in G-CSF and TNFa following TLR2 agonist treatment. To elucidate the contribution of these cytokines to the TLR2 agonist-mediated effects on HSCs, respective receptor knockout models were employed. Inhibition of G-CSF enhanced the expansion of bone marrow HSCs in response to PAM3CSK4, but minimally reduced the mobilization of HSCs to the spleen. Loss of TNFa, a,however, significantly reduced the expansion of spleen HSCs in response to PAM3CSK4, and abrogated the PAM3CSK4-induced spleen HSC cycling. Taken together, these data suggest that TLR2 signaling affects HSCs via both cell cell-autonomous and non-autonomous mechanisms, with G-CSF and TNFa acontributing to TLR2 agonist-mediated effects on HSC cycling, mobilization, and function. Ongoing studies aim to determine the cell types that are crucial for mediating the effects of TLR2 signaling on HSCs and elucidate the role of this pathway in the pathogenesis of MDS.
3136 - UNDERSTANDING THE DEVELOPMENT OF MLL REARRANGED LEUKEMIAS Roshani Sinha1,2,3, Cristina Porcheri4, Teresa d’Altri5, Lluis Espinosa4, and Anna Bigas Salvans4 1 Institut Hospital del Mar d’Investigacions Mediques; 2Kingston University; 3Jawaharlal Nehru Technological University; 4Institut Hospital del Mar d’Investigacions Mediques, Barcelona, Spain; 5DanStem, Copenhagen, Denmark Leukemic initiating cells (LIC) share critical similarities with endogenous stem cells, and common elements of regulation might exist to preserve self-renewal and prevent differentiation. As hematopoietic cells progress through their development, we expect the early appearing progenitors to be differently primed to acquire an aberrant phenotype by conditions existing intrinsically in the cell or in its developing niche, resulting in a higher probability of transforming into cancer initiating cells, as is seen in the aggressive infant leukemias as opposed to other childhood or adult leukemias. Rearrangements involving the MLL gene 11q23 are found in majority of human infant leukemias (O80% of ALL and 35-50% AML cases) and have been associated with extremely poor prognosis of these patients. While far less frequent (total of 10% cases), they are found in leukemias affecting older children and adults as well, making MLL-rearranged leukemia models ideal for our study. We performed induction of the leukemic MLL/ENL chromosomal translocation in hematopoietic cells from distinct hematopoietic tissues at different developmental stages i.e., Aorta-gonads-mesonephric tissue (AGMs, E10.5), fetal liver (FL,
S99
E12.5) and bone marrow (BM, P60) to develop and recapitulate the human infant, childhood and adult leukemias respectively and to compare transformation capacities of these distinct hematopoietic tissues. After evaluation of several models to induce MLL-ENL recombination, we have reproducibly obtained MLL-ENL leukemia from adult animals with the interferon inducible Mx-1 Cre line. Moreover, we can induce this translocation in embryonic tissues with the same line and pIpC injection. We are currently evaluating the effect of embryonic induction in FL and AGM in vivo, which will be presented at the meeting. In conclusion, we are aiming to characterize a novel model of embryo-derived leukemia which could be of help to study the ontogeny of infant leukemias.
3137 - THE CHROMATIN REGULATOR CHD8 IS A CONTEXT-DEPENDENT MEDIATOR OF CELL SURVIVAL IN MURINE HEMATOPOIETIC MALIGNANCIES Jennifer Shingleton1 and Michael Hemann2 1 Duke University, Durham, USA; 2MIT, Cambridge, USA Aberrant chromatin regulation is a frequent driver of leukemogenesis. Mutations in chromatin regulators often result in more stem-like cells that seed a bulk leukemic population. Inhibitors targeting these proteins represent an emerging class of therapeutics, and identifying further chromatin regulators that promote disease progression may result in additional drug targets. We identified the chromatin-modifying protein CHD8 as necessary for cell survival through an RNAi screen previously carried out in a mouse model of BCR-Abl+ B-cell acute lymphoblastic leukemia. This disease has a poor prognosis despite treatment with kinase inhibitors targeting BCR-Abl. Although implicated as a risk factor in autism spectrum disorder, the mechanism of CHD8’s activity is still unclear and has never been studied in the context of hematopoietic malignancies. Here we demonstrate that depletion of CHD8 in B-ALL cells leads to cell death. While multiple B cell malignancies were dependent on CHD8 expression for survival, T cell malignancies displayed milder phenotypes upon CHD8 knockdown. In addition, ectopic expression of the Notch1 intracellular domain in a T cell malignancy partially alleviated the detrimental effect of CHD8 depletion. Our results demonstrate that CHD8 has a context-dependent role in cell survival, and its inhibition may be an effective treatment for B lymphoid malignancies.