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2016 - GENE EDITED STEM CELLS COMBINED WITH TARGETED IMMUNOTHERAPY: A NOVEL APPROACH TO TREAT MYELOID MALIGNANCIES
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Short Talk Presentations / Experimental Hematology 2019;76 (Suppl): S42−S50
2016 - GENE EDITED STEM CELLS COMBINED WITH TARGETED IMMUNOTHERAPY: A NOVEL APPROACH TO TREAT MYELOID MALIGNANCIES Florence Borot1, Hui Wang2, Abdullah Ali3, Siddhartha Mukherjee3 1 Columbia University Medical Center, New York, United States; 2 Humanized Mouse Core, Columbia University Medical Center, New York, United States; 3Myelodysplastic Syndromes Center, Columbia University Medical Center, New York, United States Despite improvements in supportive care and bone marrow transplantation, only 25% of patients diagnosed with Acute Myeloid Leukemia will survive beyond 5 years. While the emergence of targeted immunotherapy has opened new possibilities, clinical studies with chimeric antigen T-cells (CAR-T) cells have shown limited success beyond CD19-targeted immunotherapy, due to lack of unique targetable cell surface antigens and the possible management of B cell aplasia with immunoglobulin supplements. To be an ideal candidate for immunotherapy, an antigen should be unique to cancer cells, indispensable for their survival and not expressed on normal cells. Frequent expression of CD33 on AML blasts and leukemic stem cells have made it an attractive target for AML therapy. The recently reapproved anti-CD33 drug conjugate gemtuzumab ozogamicin (GO) shows promising results but leads to neutropenia and low platelets because of its off-tumor effects targeting CD33 expressed on normal cells. We have designed a novel approach combining immunotherapy targeting a lineage specific antigen expressed by cancer cells with the transplantation of hematopoietic stem cells (HSCs) lacking that lineage specific antigen. As a proof of concept, using CRISPR/Cas 9 technology, we have deleted all CD33 isoforms in human CD34+ stem cells, generating CD34+33Del human stem cells. After confirming in vivo, that the deletion of CD33 in HSCs doesn’t impair their ability to engraft and to repopulate a fully functional hematopoietic system overtime, we demonstrate that leukemic humanized mice transplanted with CD34 +33Del stem cells and treated with CD33-targeted immunotherapy (anti-CD33CART cells and/or GO) show full remission and full engraftment of CD34+33stem cells overtime. With the present study, we have demonstrated a pioneering new approach to improve targeted immunotherapies to treat blood disorders
2017 - MICROBIAL SIGNAL INSTRUCTS EARLY HEMATOPOIESIS UPON INTESTINAL TISSUE DAMAGE Yoshikazu Hayashi1 , Maiko Sezaki1, Sumit Sheoran1, Tatsuya Morishima1, Gaku Nakato2, Shinji Fukuda3, Hitoshi Takizawa1 1 Kumamoto University, Kumamoto, Japan; 2Kanagawa Institute of Industrial Science and Technology, Kawasaki, Japan; 3Keio University, Yamagata, Japan Hematopoietic stem cells (HSCs) slowly self-renew and differentiate in adult bone marrow (BM) to sustain lifelong hematopoiesis, and can be activated to self-renew or differentiate when hematopoietic need increases. However, it remains unclear how HSC and progenitor cells (HSPCs) integrate the peripheral organ-derived demand signal to hematopoietic production and what the biological consequence of HSPCs activation is on their cell fate decision. We have previously shown that systemic challenge of gram negative bacteria directly activates dormant HSCs to proliferation and impairs their competitive fitness via Toll like receptor (TLR)-4 signaling (Cell Stem Cell 2017). These findings let us hypothesize that commensal bacteria, often referred to as microbiota, might also impact on early hematopoiesis upon intestinal barrier damage. Here, we employed inflammatory bowel disease (IBD) model to induce gut inflammation. Acute IBD induced expansion of HSPCs such as multipotent progenitors (MPPs) in primary BM followed by their localization in mesenteric lymph node (MLN), a local inflammatory site. Myeloid compartment, especially neutrophils and monocytes dominantly increased and suppressed IBD-induced colitis as shown by antibody-based cell depletion study, indicating possible contribution of MPP in tissue damage and repair. Genetic and pharmacological studies revealed that the HSPC expansion and their directed migration depend on a TLR/IL-1 receptor signaling and specific type of microbiota, suggesting that microbial signal generated in the distal organs regulates early hematopoietic cell proliferation and localization. Uncovering the underlying mechanism for gut associated inflammation will help to understand inflammatory feedback signals through cross-organ communications that orchestrate hematopoiesis, and might be relevant to ageingassociated chronic disorders.
2018 - TRANSPOSABLE ELEMENTS ENHANCE HEMATOPOIETIC REGENERATION VIA ACTIVATION OF INNATE IMMUNE SIGNALLING Eirini Trompouki1, Thomas Clapes1, Aikaterini Polyzou1, Pia Prater1, Sagar Sagar1, Barbara Hummel1, Daniel Mariczka2, Stylianos Lefkopoulos1, Ibrahim Ilik1, Lheanna Klaeyle1, Rolf Backofen2, Asifa Akhtar1, Ritwick Sawarkar1, Dominic Gr€ un1 1 Max Planck Institute of Immunobiolgy and Epigenetics, Freiburg, Germany; 2 Albert-Ludwigs-Universitaet, Freiburg, Germany Transposable elements (TEs) are transcriptionally upregulated upon stress signals. In the hematopoietic system, stress, like myeloablation, leads to a regenerative response, where hematopoietic stem cells (HSCs) exit quiescence and differentiate. A potential role of TEs in this process is currently unknown. Here we used myeloablation by 5fluorouracil injection as a hematopoietic regeneration system. A time course of chromatin accessibility and expression analysis revealed that during regeneration, robust chromatin rearrangement takes place in HSCs concomitant with transcriptional upregulation of TEs and activation of inflammatory signalling. We reasoned that TE upregulation could be responsible for the inflammatory signalling activation by acting as ligands for the innate immune receptor MDA5. Indeed, by sequencing the RNA that is crosslinked to MDA5 upon stress we found several TE transcripts bound to MDA5. Loss of MDA5 in HSCs did not affect TE upregulation during regeneration but led to impaired inflammatory signalling activation. Since inflammatory signalling is crucial for the quiescent status of HSCs, we then examined the ability of Mda5-/- animals to activate their HSCs. Mda5-/- HSCs were more quiescent both at steady state and during regeneration. This quiescent phenotype was validated by metabolic assays and single-cell RNA-seq analysis. Finally, overexpression of a single TE element in HSCs led to their activation in a wild type but not an Mda5-/- background. Our step-by-step model proposes that during regeneration, TE upregulation leads to binding and activation of the innate immune receptor MDA5 that in turn stimulates the inflammatory signalling necessary for HSCs to exit quiescence and regenerate the hematopoietic system. Thus, using hematopoiesis as an exemplary system, our work uncovers a novel function of TEs in tissue regeneration.
2019 - WITHDRAWN
Short Talk Presentations / Experimental Hematology 2019;76 (Suppl): S42−S50
2016 - GENE EDITED STEM CELLS COMBINED WITH TARGETED IMMUNOTHERAPY: A NOVEL APPROACH TO TREAT MYELOID MALIGNANCIES Florence Borot1, Hui Wang2, Abdullah Ali3, Siddhartha Mukherjee3 1 Columbia University Medical Center, New York, United States; 2 Humanized Mouse Core, Columbia University Medical Center, New York, United States; 3Myelodysplastic Syndromes Center, Columbia University Medical Center, New York, United States Despite improvements in supportive care and bone marrow transplantation, only 25% of patients diagnosed with Acute Myeloid Leukemia will survive beyond 5 years. While the emergence of targeted immunotherapy has opened new possibilities, clinical studies with chimeric antigen T-cells (CAR-T) cells have shown limited success beyond CD19-targeted immunotherapy, due to lack of unique targetable cell surface antigens and the possible management of B cell aplasia with immunoglobulin supplements. To be an ideal candidate for immunotherapy, an antigen should be unique to cancer cells, indispensable for their survival and not expressed on normal cells. Frequent expression of CD33 on AML blasts and leukemic stem cells have made it an attractive target for AML therapy. The recently reapproved anti-CD33 drug conjugate gemtuzumab ozogamicin (GO) shows promising results but leads to neutropenia and low platelets because of its off-tumor effects targeting CD33 expressed on normal cells. We have designed a novel approach combining immunotherapy targeting a lineage specific antigen expressed by cancer cells with the transplantation of hematopoietic stem cells (HSCs) lacking that lineage specific antigen. As a proof of concept, using CRISPR/Cas 9 technology, we have deleted all CD33 isoforms in human CD34+ stem cells, generating CD34+33Del human stem cells. After confirming in vivo, that the deletion of CD33 in HSCs doesn’t impair their ability to engraft and to repopulate a fully functional hematopoietic system overtime, we demonstrate that leukemic humanized mice transplanted with CD34 +33Del stem cells and treated with CD33-targeted immunotherapy (anti-CD33CART cells and/or GO) show full remission and full engraftment of CD34+33stem cells overtime. With the present study, we have demonstrated a pioneering new approach to improve targeted immunotherapies to treat blood disorders
2017 - MICROBIAL SIGNAL INSTRUCTS EARLY HEMATOPOIESIS UPON INTESTINAL TISSUE DAMAGE Yoshikazu Hayashi1 , Maiko Sezaki1, Sumit Sheoran1, Tatsuya Morishima1, Gaku Nakato2, Shinji Fukuda3, Hitoshi Takizawa1 1 Kumamoto University, Kumamoto, Japan; 2Kanagawa Institute of Industrial Science and Technology, Kawasaki, Japan; 3Keio University, Yamagata, Japan Hematopoietic stem cells (HSCs) slowly self-renew and differentiate in adult bone marrow (BM) to sustain lifelong hematopoiesis, and can be activated to self-renew or differentiate when hematopoietic need increases. However, it remains unclear how HSC and progenitor cells (HSPCs) integrate the peripheral organ-derived demand signal to hematopoietic production and what the biological consequence of HSPCs activation is on their cell fate decision. We have previously shown that systemic challenge of gram negative bacteria directly activates dormant HSCs to proliferation and impairs their competitive fitness via Toll like receptor (TLR)-4 signaling (Cell Stem Cell 2017). These findings let us hypothesize that commensal bacteria, often referred to as microbiota, might also impact on early hematopoiesis upon intestinal barrier damage. Here, we employed inflammatory bowel disease (IBD) model to induce gut inflammation. Acute IBD induced expansion of HSPCs such as multipotent progenitors (MPPs) in primary BM followed by their localization in mesenteric lymph node (MLN), a local inflammatory site. Myeloid compartment, especially neutrophils and monocytes dominantly increased and suppressed IBD-induced colitis as shown by antibody-based cell depletion study, indicating possible contribution of MPP in tissue damage and repair. Genetic and pharmacological studies revealed that the HSPC expansion and their directed migration depend on a TLR/IL-1 receptor signaling and specific type of microbiota, suggesting that microbial signal generated in the distal organs regulates early hematopoietic cell proliferation and localization. Uncovering the underlying mechanism for gut associated inflammation will help to understand inflammatory feedback signals through cross-organ communications that orchestrate hematopoiesis, and might be relevant to ageingassociated chronic disorders.
2018 - TRANSPOSABLE ELEMENTS ENHANCE HEMATOPOIETIC REGENERATION VIA ACTIVATION OF INNATE IMMUNE SIGNALLING Eirini Trompouki1, Thomas Clapes1, Aikaterini Polyzou1, Pia Prater1, Sagar Sagar1, Barbara Hummel1, Daniel Mariczka2, Stylianos Lefkopoulos1, Ibrahim Ilik1, Lheanna Klaeyle1, Rolf Backofen2, Asifa Akhtar1, Ritwick Sawarkar1, Dominic Gr€ un1 1 Max Planck Institute of Immunobiolgy and Epigenetics, Freiburg, Germany; 2 Albert-Ludwigs-Universitaet, Freiburg, Germany Transposable elements (TEs) are transcriptionally upregulated upon stress signals. In the hematopoietic system, stress, like myeloablation, leads to a regenerative response, where hematopoietic stem cells (HSCs) exit quiescence and differentiate. A potential role of TEs in this process is currently unknown. Here we used myeloablation by 5fluorouracil injection as a hematopoietic regeneration system. A time course of chromatin accessibility and expression analysis revealed that during regeneration, robust chromatin rearrangement takes place in HSCs concomitant with transcriptional upregulation of TEs and activation of inflammatory signalling. We reasoned that TE upregulation could be responsible for the inflammatory signalling activation by acting as ligands for the innate immune receptor MDA5. Indeed, by sequencing the RNA that is crosslinked to MDA5 upon stress we found several TE transcripts bound to MDA5. Loss of MDA5 in HSCs did not affect TE upregulation during regeneration but led to impaired inflammatory signalling activation. Since inflammatory signalling is crucial for the quiescent status of HSCs, we then examined the ability of Mda5-/- animals to activate their HSCs. Mda5-/- HSCs were more quiescent both at steady state and during regeneration. This quiescent phenotype was validated by metabolic assays and single-cell RNA-seq analysis. Finally, overexpression of a single TE element in HSCs led to their activation in a wild type but not an Mda5-/- background. Our step-by-step model proposes that during regeneration, TE upregulation leads to binding and activation of the innate immune receptor MDA5 that in turn stimulates the inflammatory signalling necessary for HSCs to exit quiescence and regenerate the hematopoietic system. Thus, using hematopoiesis as an exemplary system, our work uncovers a novel function of TEs in tissue regeneration.
2019 - WITHDRAWN