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2031 - DELINEATING MECHANISMS OF ACQUIRED RESISTANCE TO IDH INHIBITORS IN AML
Short Talk Presentations / Experimental Hematology 2019;76 (Suppl): S42−S50 2028 - PULSATION IN BLOOD VESSELS STIMULATES MECHANOSENSITIVE AND EPIGENETIC MECHANISMS TO PROMOTE THE BIRTH OF HEMATOPOIETIC STEM CELLS Giorgia Scapin, Dhvanit Shah Nationwide Children’s Hospital, Ohio State University College of Medicine, Columbus, United States During fetal development, a subset of endothelial cells changes their fate to become HSCs in the aorta-gonad-mesonephros (AGM) region. However, the mechanisms regulating the endothelial-to-hematopoietic transition (EHT) remain largely unknown. Our machine learning and confocal imaging demonstrate that the rhythmic contraction in ventral part of dorsal aorta is concurrent with EHT events. We also found that pulsation-mediated cyclical contraction stretches AGM in circumferential direction. Using serial transplant, limiting dilution, and replating assays, we found that the circumferential stretching of hemogenic endothelial cells or Piezo1 activation yields 3-times higher amounts of Long Term (LT)-HSC formation; which reconstitute to normal multi-lineage adult blood. Using vaccine-challenge, adult globin expression, MPO enzyme activity, immunoglobulins, and T-cell receptor rearrangement analyses, we found that circumferential stretching or Piezo1 activationderived HSCs reconstitute to functional T and B cells, adult erythrocytes, and myeloid cells. Our Piezo1fl/flxScl-Cre conditional knockout, gene-silencing, & confocal imaging further demonstrate that circumferential stretching of blood vessels activates Piezo1; which enhances epigenetic regulator Dnmt3b expression to stimulate the EHT. Our Cut&Run & MassArray analyses demonstrate that Dnmt3b suppresses endothelial genes during EHT. In conclusion, we found that pulsation-mediated circumferential stretching of AGM stimulates the EHT by Piezo1 activation and Dnmt3b over-expression. This leads to the formation of long-term self-renewing HSCs engrafting upon serial transplantations and reconstituting normal adult blood. To our knowledge, this is the first report demonstrating how biomechanical forces utilize epigenetic machinery to promote cell fate transition as well as a bioreactor to develop and expand LT-HSCs.
2029 - THE RELAPSED B-CELL ACUTE LYMPHOBLASTIC LEUKAEMIA IMMUNE MICROENVIRONMENT Matthew Witkowski1, Igor Dolgalev1, Nikki Evensen1, Kathryn Roberts2 , Sheetal Sreeram1, Yuling Dai1, Anastasia Tikhonova1, Cynthia Loomis1, Charles Mullighan2, Aristotelis Tsirigos1, William Carroll1 , Iannis Aifantis1 1 New York University School of Medicine, New York, United States; 2St. Jude Children’s Research Hospital, Memphis, United States As with most cancer types, there remains a subset of B-cell acute lymphoblastic leukaemia (B-ALL) patients who will relapse and succumb to therapyresistant disease. It is believed that tumour heterogeneity underpins therapy failure leading to a Darwinian model of clonal evolution, however, such studies do not account for the role of the bone marrow microenvironment in supporting leukaemia survival, progression and escape from treatment. Here, we perform single-cell RNA-Sequencing (scRNA-Seq) to generate a comprehensive map of the primary human B-ALL bone marrow immune microenvironment throughout three distinct stages of the human leukemic disease process: diagnosis, remission and relapse. These studies show extensive re-modelling of the immune microenvironment composition and cell-to-cell interactions throughout the course conventional chemotherapy, and uncover a role for inflammatory leukaemia-associated monocytes in promoting B-ALL pathogenesis in vivo. These monocytic subsets are predictive of Ph+ B-ALL patient event-free survival and when targeted in B-ALL animal models, lead to prolonged disease remission. Our profiling of the human B-ALL bone marrow immune microenvironment provides a greater understanding of the potential extrinsic regulators of B-ALL survival and may highlight previously unknown environmental factors influencing immune-based treatment approaches to high-risk B-ALL.
S49
2030 - RIBONUCLEASE INHIBITOR (RNH1) REGULATES HEMATOPOIETIC CELL SPECIFIC TRANSLATION Martina Stilinovic1,2, Nicola Andina3, Aubry Tardivel3, Irene Keller2, Ramanjaneyulu Allam3 1 Department of Hematology, Inselspital, Bern University Hospital, Bern, Switzerland; 2Department for BioMedical Research, University of Bern, Switzerland, Bern, Switzerland; 3Department of Hematology, Inselspital, Bern University Hospital. Department for BioMedical Research, University of Bern, Switzerland, Bern, Switzerland Regulation of gene expression is important for normal development and is mainly controlled at the level of transcription. However, recent studies show that ribosomal proteins (RPs) regulate specific gene expression by selectively facilitating translation of specific mRNAs. Indeed, in Diamond- Blackfan anemia (DBA) and 5q− syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. How mutations in RP genes leads to hematopoietic specific defects is largely unknown. Similar to transcription factors the existence of cell type specific translation regulators remain elusive. Here, we report that Ribonuclease inhibitor (RNH1) regulates hematopoietic cell specific translation. Recently, we published that RNH1 is a ribosomal associated protein and regulates erythropoiesis by regulating GATA1 mRNA translation. In this study, we found that RNH1-deficiency in human hematopoietic origin cells such as erythroid leukemia cells, monocytic cells and T lymphocytes decreased polysome formation but not in non-hematopoietic origin cells such as HEK293, HaCat, HeLa cells. Similarly, OP-Puro incorporation experiments in mice revealed that RNH1-deficency leads to translation defect in hematopoietic cells but not in non- hematopoietic cells. At molecular level, we found that RNH1 binds to ribosomes and regulates RPs gene expression at translation level independent of mTOR signaling. Interestingly, it has been shown that RNH1 expression is translationally down regulated in RPS19 knockdown cells, which is frequently mutated in DBA patients. Supporting RNH1 role in translation, over expression of RNH1 rescues erythroid and translation defects in RPS19 knockdown cells. Collectively, our result unravels the existence of cell type specific translation regulators and may partially explain cell type specific defects caused by mutations in RP genes.
2031 - DELINEATING MECHANISMS OF ACQUIRED RESISTANCE TO IDH INHIBITORS IN AML Lev Kats1, Emily Gruber1, Rohini Narayanaswamy2, Sebastien Ronseaux2, Brandon Nicolay2, Ricky Johnstone1 1 Peter MacCallum Cancer Centre, Parkville, Australia; 2Agios Pharmaceuticals, Cambridge, United States Approximately 20% of acute myeloid leukaemia (AML) patients carry mutations in genes that encode the metabolic enzymes isocitrate dehydrogenase (IDH) 1 and -2. Mutant IDH proteins possess a neomorphic enzymatic activity and produce the oncometabolite D-2-hydroxyglutarate (2-HG) that modulates numerous pathways implicated in cell fate decisions and cancer. We and others have shown that IDH mutations can contribute to leukaemia initiation and maintenance both in vitro and in vivo. Small molecule inhibitors that block production of 2-HG are starting to enter the clinic, however, the biomarkers of response and mechanisms of de novo and acquired resistance to mutant IDH inhibition remain poorly understood. We have engineered a series of murine AML models driven by inducible expression of mutant IDH proteins. To uncover the molecular mechanisms that underpin response and adaptation to mutant IDH inhibition we integrated bulk and single-cell RNA sequencing with chromatin immuno-precipitation sequencing. We demonstrate that genetic depletion and pharmacological inhibition of mutant IDH has striking effects, altering self-renewal and differentiation programs of leukaemic cells. Importantly, we identify a population of cells that persist during treatment and characterise genetic programs that circumvent effective IDH inhibition, ultimately leading to therapy resistance and disease relapse. Our studies provide detailed mechanistic insight into the oncogenic properties of mutant IDH proteins in maintaining the transformed state of AML cells.
2029 - THE RELAPSED B-CELL ACUTE LYMPHOBLASTIC LEUKAEMIA IMMUNE MICROENVIRONMENT Matthew Witkowski1, Igor Dolgalev1, Nikki Evensen1, Kathryn Roberts2 , Sheetal Sreeram1, Yuling Dai1, Anastasia Tikhonova1, Cynthia Loomis1, Charles Mullighan2, Aristotelis Tsirigos1, William Carroll1 , Iannis Aifantis1 1 New York University School of Medicine, New York, United States; 2St. Jude Children’s Research Hospital, Memphis, United States As with most cancer types, there remains a subset of B-cell acute lymphoblastic leukaemia (B-ALL) patients who will relapse and succumb to therapyresistant disease. It is believed that tumour heterogeneity underpins therapy failure leading to a Darwinian model of clonal evolution, however, such studies do not account for the role of the bone marrow microenvironment in supporting leukaemia survival, progression and escape from treatment. Here, we perform single-cell RNA-Sequencing (scRNA-Seq) to generate a comprehensive map of the primary human B-ALL bone marrow immune microenvironment throughout three distinct stages of the human leukemic disease process: diagnosis, remission and relapse. These studies show extensive re-modelling of the immune microenvironment composition and cell-to-cell interactions throughout the course conventional chemotherapy, and uncover a role for inflammatory leukaemia-associated monocytes in promoting B-ALL pathogenesis in vivo. These monocytic subsets are predictive of Ph+ B-ALL patient event-free survival and when targeted in B-ALL animal models, lead to prolonged disease remission. Our profiling of the human B-ALL bone marrow immune microenvironment provides a greater understanding of the potential extrinsic regulators of B-ALL survival and may highlight previously unknown environmental factors influencing immune-based treatment approaches to high-risk B-ALL.
S49
2030 - RIBONUCLEASE INHIBITOR (RNH1) REGULATES HEMATOPOIETIC CELL SPECIFIC TRANSLATION Martina Stilinovic1,2, Nicola Andina3, Aubry Tardivel3, Irene Keller2, Ramanjaneyulu Allam3 1 Department of Hematology, Inselspital, Bern University Hospital, Bern, Switzerland; 2Department for BioMedical Research, University of Bern, Switzerland, Bern, Switzerland; 3Department of Hematology, Inselspital, Bern University Hospital. Department for BioMedical Research, University of Bern, Switzerland, Bern, Switzerland Regulation of gene expression is important for normal development and is mainly controlled at the level of transcription. However, recent studies show that ribosomal proteins (RPs) regulate specific gene expression by selectively facilitating translation of specific mRNAs. Indeed, in Diamond- Blackfan anemia (DBA) and 5q− syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. How mutations in RP genes leads to hematopoietic specific defects is largely unknown. Similar to transcription factors the existence of cell type specific translation regulators remain elusive. Here, we report that Ribonuclease inhibitor (RNH1) regulates hematopoietic cell specific translation. Recently, we published that RNH1 is a ribosomal associated protein and regulates erythropoiesis by regulating GATA1 mRNA translation. In this study, we found that RNH1-deficiency in human hematopoietic origin cells such as erythroid leukemia cells, monocytic cells and T lymphocytes decreased polysome formation but not in non-hematopoietic origin cells such as HEK293, HaCat, HeLa cells. Similarly, OP-Puro incorporation experiments in mice revealed that RNH1-deficency leads to translation defect in hematopoietic cells but not in non- hematopoietic cells. At molecular level, we found that RNH1 binds to ribosomes and regulates RPs gene expression at translation level independent of mTOR signaling. Interestingly, it has been shown that RNH1 expression is translationally down regulated in RPS19 knockdown cells, which is frequently mutated in DBA patients. Supporting RNH1 role in translation, over expression of RNH1 rescues erythroid and translation defects in RPS19 knockdown cells. Collectively, our result unravels the existence of cell type specific translation regulators and may partially explain cell type specific defects caused by mutations in RP genes.
2031 - DELINEATING MECHANISMS OF ACQUIRED RESISTANCE TO IDH INHIBITORS IN AML Lev Kats1, Emily Gruber1, Rohini Narayanaswamy2, Sebastien Ronseaux2, Brandon Nicolay2, Ricky Johnstone1 1 Peter MacCallum Cancer Centre, Parkville, Australia; 2Agios Pharmaceuticals, Cambridge, United States Approximately 20% of acute myeloid leukaemia (AML) patients carry mutations in genes that encode the metabolic enzymes isocitrate dehydrogenase (IDH) 1 and -2. Mutant IDH proteins possess a neomorphic enzymatic activity and produce the oncometabolite D-2-hydroxyglutarate (2-HG) that modulates numerous pathways implicated in cell fate decisions and cancer. We and others have shown that IDH mutations can contribute to leukaemia initiation and maintenance both in vitro and in vivo. Small molecule inhibitors that block production of 2-HG are starting to enter the clinic, however, the biomarkers of response and mechanisms of de novo and acquired resistance to mutant IDH inhibition remain poorly understood. We have engineered a series of murine AML models driven by inducible expression of mutant IDH proteins. To uncover the molecular mechanisms that underpin response and adaptation to mutant IDH inhibition we integrated bulk and single-cell RNA sequencing with chromatin immuno-precipitation sequencing. We demonstrate that genetic depletion and pharmacological inhibition of mutant IDH has striking effects, altering self-renewal and differentiation programs of leukaemic cells. Importantly, we identify a population of cells that persist during treatment and characterise genetic programs that circumvent effective IDH inhibition, ultimately leading to therapy resistance and disease relapse. Our studies provide detailed mechanistic insight into the oncogenic properties of mutant IDH proteins in maintaining the transformed state of AML cells.