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A pooled lentiviral mirna overexpression screen in hematopoietic stem and progenitor cells reveals miR-10a and miR-335 as regulators of lymphopoiesis
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Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
as day 3. Surprisingly, the similar effect of TGF-b1 was also detected when TGF-b1 was present only on days 5-7, indicating that TGF-b1 similarly suppresses the function of cycling HSCs throughout culture. Single cell transplantation (6 months) showed that both total and myeloid reconstitution levels were dramatically reduced by 10 and 100 pg/ml TGF-b1. Single-cell RNA-sequencing showed that SCF+TPO drove HSCs into cycling, while TGF-b1 inhibited their cycling in about half of the cells. Conclusion: TGF-b1 can let cycling HSCs go back into the quiescent state, but in these quiescent HSCs, the self-renewal potential is reduced and the differentiation potential is biased towards lymphoid lineage.
3186 - A POOLED LENTIVIRAL MIRNA OVEREXPRESSION SCREEN IN HEMATOPOIETIC STEM AND PROGENITOR CELLS REVEALS MIR-10A AND MIR-335 AS REGULATORS OF LYMPHOPOIESIS unsche2, Nina Hofmann2, Tim Kindinger2, Elias Eckert1, Peer W€ Manfred Schmidt2, Claudia Ball2, Friederike Herbst2, and Hanno Glimm2 1
National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany; 2National Center for Tumor Diseases (NCT) Heidelberg, Germany We used a global viral integration site (IS) data set from clinical gene therapy to identify novel hematopoietic regulators. Within the 130,699 unique IS we found clusters of integration sites significantly overrepresented at loci of known hematopoietic regulatory genes as well as in the vicinity of 8 miRNA genes. To subsequently investigate the influence of the selected miRNA genes on hematopoiesis, we developed a lentiviral based pooled miR-overexpression (OE) approach using uniquely barcoded (BC) vectors. This miR-OE library was used to transduce stem and progenitor cells to perform both CFU assays as well as serial transplantation (TP) experiments. We detected an increase in colony forming potential of miRNA library OE cells compared to GFP control cells as well as a significant overrepresentation of barcode counts from 2/8 candidates. BC vectors encoding for two other miRNAs were significantly reduced. We confirmed these results in single candidate OE experiments (miR-10a: 1.8460.19 fold, miR-335: 0.4560.02 fold vs GFP, n54). In line with our results in vitro, we observed a 1.2960.13 (p50.044) fold enrichment of barcodes of miR10a and a depletion of the two other miRNA genes (miR-335: 0.6860.13 fold; p50.033) in PB samples eight weeks after TP. Single candidate overexpression confirmed that miR-335 leads to a reduction of B-cell output and spleen size after 20 weeks. Interestingly, BC sequencing of lineage sorted fractions suggests that miR-10a has a specific influence on T-cell differentiation (fc51.5060.20, p50.029, 20 weeks post TP). Finally, we could show that miR-10a leads to increased T-cell output in PB (1.5660.14) and a 3.760.85 fold increase of the CLP fraction in single candidate OE experiments 20 weeks post TP. In summary, our systematic stepwise approach combining the comprehensive analysis of the integration site repertoire in a clinical gene therapy study with subsequent functional validation is a useful strategy to identify and characterize novel hematopoietic regulators.
3187 - IMMUNOSURVEILLANCE DURING THE FORMATION AND PROGRESSION OF AML Monika Dudenh€offer-Pfeifer, Amol Ugale, and David Bryder Lund University, Lund, Sweden Despite progress in the treatment of acute leukemia, specific subtypes still have a very bad prognosis, highlighting the need for increased knowledge that can be harnessed to develop novel therapeutic strategies. While experimental data from the last decade have conclusively established that the immune system can critically influence on the development of cancer, with a range of immunomodulatory strategies reaching clinical use, most studies have been conducted using solid cancer models of non-hematological origin. Such cancers evidently have requirements very distinct from hematological cancers that pertain to initiation, maintenance and spreading (metastasis). Also, constraints of experimental models precluding spatiotemporal control have to a large extent hindered investigations of immunosurveillance and immunoediting mechanisms during the multistep progression of acute leukemia. Here, we make use of a recently described mouse model of acute myeloid leukemia AML (Ugale et al, 2014) in which physiologically relevant expression levels of the fusion oncogene MLL-ENL can be conditionally activated in vivo as a leukemic ‘‘first-hit’’. This model associates with a phase of ‘‘pre-leukemic’’ contraction after which overt clonal transformation commences, highlighting the critical influence of secondary but less defined events during the course of disease. By investigating aspects of the immune system during the formation and progression of AML, we show that transformation follows similar kinetics regardless of developing in wild type (WT) or immune-deficient (Rag2-/- or Rag2-/gc-/-) environments. By contrast, immunity critically influences on the propagation of established leukemia, but with little evidence of primary immune-editing. Rather, intrinsic properties of individual leukemic clones, regardless of their origin, underlie the aggressiveness of arising disease. Antibody-mediated in vivo depletion experiments revealed a critical role for CD8+ cells in this process. Ongoing investigations focus on analyzing and characterizing CD8+ T lymphocyte subsets during the disease course of AML.
3188 - LEUKEMIC MESENCHYMAL STROMAL CELL COX2-PG SIGNALING REGULATES DONOR ANTI-LEUKEMIA IMMUNITY Wei Du, Limei Wu, Jian Xu, Sara MacLaughlin, and Xue Li WVU, Morgantown, United States Hematopoietic stem cell transplantation (HSCT) is considered the gold standard for treatment of hematologic malignancies, including Fanconi anemia (FA), a cancer-prone disease with extremely high incidence of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, eradication of residual leukemia stem cells (LSCs), which often contributes to relapse, remains the challenge for HSCT. Here we investigate the interaction between leukemic mesenchymal niche and donor hematopoietic stem progenitor cells (HSPCs) by modeling FA HSCT. We found that healthy donor CD34+ HSPCs cocultured on mesenchymal stromal cells (MSCs) derived from patients with AML exhibit high human engraftment characteristic of HSPC and myeloid expansion in NOD/SCID/IL-2gamma-/-/SGM3 (NSGS) mice. LC/MS-based untargeted metabolomics analysis revealed that prostaglandins (PGs), an inflammatory component of the mesenchymal secretome, are the only metabolites that are progressively elevated in MDS and AML MSCs compared to the MSCs from healthy donors or patients with cytopenias but without cancer. Inhibition of the inflammatory cyclooxygenase 2 (COX2) in the AML MSCs ex vivo ameliorates HSPC and myeloid expansion in transplanted recipients of the cocultured CD34+ cells. In addition, transcriptome analysis demonstrated dysregulation of genes involved in the NR4A family of nuclear hormone transcription factors (TFs) and the WNT/-catenin signaling pathway in the CD34+ cells co-cultured on MSCs derived from AML patients. Consistently, reduced MSC secretion of PGs subsequent to inhibition of COX2 leads to a significant decrease in the expression of the NR4A TFs and the WNT signaling genes including Wnt ligand WNT5A, -catenin (CTNNB1) and the WNT effector LEF1 in cocultured CD34+ cells. Furthermore, knocking down the NR4A TFs or CTNNB1 abrogated the expansion of progeny of the AML MSC-cocultured HSPCs in recipient mice. Together, these findings suggest that specific interactions between leukemic mesenchymal niche and donor HSPCs orchestrate a novel COX2/PG/NR4A signaling axis, connecting inflammation, cellular metabolism and cancer immunity.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
as day 3. Surprisingly, the similar effect of TGF-b1 was also detected when TGF-b1 was present only on days 5-7, indicating that TGF-b1 similarly suppresses the function of cycling HSCs throughout culture. Single cell transplantation (6 months) showed that both total and myeloid reconstitution levels were dramatically reduced by 10 and 100 pg/ml TGF-b1. Single-cell RNA-sequencing showed that SCF+TPO drove HSCs into cycling, while TGF-b1 inhibited their cycling in about half of the cells. Conclusion: TGF-b1 can let cycling HSCs go back into the quiescent state, but in these quiescent HSCs, the self-renewal potential is reduced and the differentiation potential is biased towards lymphoid lineage.
3186 - A POOLED LENTIVIRAL MIRNA OVEREXPRESSION SCREEN IN HEMATOPOIETIC STEM AND PROGENITOR CELLS REVEALS MIR-10A AND MIR-335 AS REGULATORS OF LYMPHOPOIESIS unsche2, Nina Hofmann2, Tim Kindinger2, Elias Eckert1, Peer W€ Manfred Schmidt2, Claudia Ball2, Friederike Herbst2, and Hanno Glimm2 1
National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany; 2National Center for Tumor Diseases (NCT) Heidelberg, Germany We used a global viral integration site (IS) data set from clinical gene therapy to identify novel hematopoietic regulators. Within the 130,699 unique IS we found clusters of integration sites significantly overrepresented at loci of known hematopoietic regulatory genes as well as in the vicinity of 8 miRNA genes. To subsequently investigate the influence of the selected miRNA genes on hematopoiesis, we developed a lentiviral based pooled miR-overexpression (OE) approach using uniquely barcoded (BC) vectors. This miR-OE library was used to transduce stem and progenitor cells to perform both CFU assays as well as serial transplantation (TP) experiments. We detected an increase in colony forming potential of miRNA library OE cells compared to GFP control cells as well as a significant overrepresentation of barcode counts from 2/8 candidates. BC vectors encoding for two other miRNAs were significantly reduced. We confirmed these results in single candidate OE experiments (miR-10a: 1.8460.19 fold, miR-335: 0.4560.02 fold vs GFP, n54). In line with our results in vitro, we observed a 1.2960.13 (p50.044) fold enrichment of barcodes of miR10a and a depletion of the two other miRNA genes (miR-335: 0.6860.13 fold; p50.033) in PB samples eight weeks after TP. Single candidate overexpression confirmed that miR-335 leads to a reduction of B-cell output and spleen size after 20 weeks. Interestingly, BC sequencing of lineage sorted fractions suggests that miR-10a has a specific influence on T-cell differentiation (fc51.5060.20, p50.029, 20 weeks post TP). Finally, we could show that miR-10a leads to increased T-cell output in PB (1.5660.14) and a 3.760.85 fold increase of the CLP fraction in single candidate OE experiments 20 weeks post TP. In summary, our systematic stepwise approach combining the comprehensive analysis of the integration site repertoire in a clinical gene therapy study with subsequent functional validation is a useful strategy to identify and characterize novel hematopoietic regulators.
3187 - IMMUNOSURVEILLANCE DURING THE FORMATION AND PROGRESSION OF AML Monika Dudenh€offer-Pfeifer, Amol Ugale, and David Bryder Lund University, Lund, Sweden Despite progress in the treatment of acute leukemia, specific subtypes still have a very bad prognosis, highlighting the need for increased knowledge that can be harnessed to develop novel therapeutic strategies. While experimental data from the last decade have conclusively established that the immune system can critically influence on the development of cancer, with a range of immunomodulatory strategies reaching clinical use, most studies have been conducted using solid cancer models of non-hematological origin. Such cancers evidently have requirements very distinct from hematological cancers that pertain to initiation, maintenance and spreading (metastasis). Also, constraints of experimental models precluding spatiotemporal control have to a large extent hindered investigations of immunosurveillance and immunoediting mechanisms during the multistep progression of acute leukemia. Here, we make use of a recently described mouse model of acute myeloid leukemia AML (Ugale et al, 2014) in which physiologically relevant expression levels of the fusion oncogene MLL-ENL can be conditionally activated in vivo as a leukemic ‘‘first-hit’’. This model associates with a phase of ‘‘pre-leukemic’’ contraction after which overt clonal transformation commences, highlighting the critical influence of secondary but less defined events during the course of disease. By investigating aspects of the immune system during the formation and progression of AML, we show that transformation follows similar kinetics regardless of developing in wild type (WT) or immune-deficient (Rag2-/- or Rag2-/gc-/-) environments. By contrast, immunity critically influences on the propagation of established leukemia, but with little evidence of primary immune-editing. Rather, intrinsic properties of individual leukemic clones, regardless of their origin, underlie the aggressiveness of arising disease. Antibody-mediated in vivo depletion experiments revealed a critical role for CD8+ cells in this process. Ongoing investigations focus on analyzing and characterizing CD8+ T lymphocyte subsets during the disease course of AML.
3188 - LEUKEMIC MESENCHYMAL STROMAL CELL COX2-PG SIGNALING REGULATES DONOR ANTI-LEUKEMIA IMMUNITY Wei Du, Limei Wu, Jian Xu, Sara MacLaughlin, and Xue Li WVU, Morgantown, United States Hematopoietic stem cell transplantation (HSCT) is considered the gold standard for treatment of hematologic malignancies, including Fanconi anemia (FA), a cancer-prone disease with extremely high incidence of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, eradication of residual leukemia stem cells (LSCs), which often contributes to relapse, remains the challenge for HSCT. Here we investigate the interaction between leukemic mesenchymal niche and donor hematopoietic stem progenitor cells (HSPCs) by modeling FA HSCT. We found that healthy donor CD34+ HSPCs cocultured on mesenchymal stromal cells (MSCs) derived from patients with AML exhibit high human engraftment characteristic of HSPC and myeloid expansion in NOD/SCID/IL-2gamma-/-/SGM3 (NSGS) mice. LC/MS-based untargeted metabolomics analysis revealed that prostaglandins (PGs), an inflammatory component of the mesenchymal secretome, are the only metabolites that are progressively elevated in MDS and AML MSCs compared to the MSCs from healthy donors or patients with cytopenias but without cancer. Inhibition of the inflammatory cyclooxygenase 2 (COX2) in the AML MSCs ex vivo ameliorates HSPC and myeloid expansion in transplanted recipients of the cocultured CD34+ cells. In addition, transcriptome analysis demonstrated dysregulation of genes involved in the NR4A family of nuclear hormone transcription factors (TFs) and the WNT/-catenin signaling pathway in the CD34+ cells co-cultured on MSCs derived from AML patients. Consistently, reduced MSC secretion of PGs subsequent to inhibition of COX2 leads to a significant decrease in the expression of the NR4A TFs and the WNT signaling genes including Wnt ligand WNT5A, -catenin (CTNNB1) and the WNT effector LEF1 in cocultured CD34+ cells. Furthermore, knocking down the NR4A TFs or CTNNB1 abrogated the expansion of progeny of the AML MSC-cocultured HSPCs in recipient mice. Together, these findings suggest that specific interactions between leukemic mesenchymal niche and donor HSPCs orchestrate a novel COX2/PG/NR4A signaling axis, connecting inflammation, cellular metabolism and cancer immunity.