- Email: [email protected]
securin as a quantitative trait locus candidate gene controlling progenitor cell survival and organismal lifespan
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Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3049 - PTTG1/SECURIN AS A QUANTITATIVE TRAIT LOCUS CANDIDATE GENE CONTROLLING PROGENITOR CELL SURVIVAL AND ORGANISMAL LIFESPAN Desiree Sch€ utz1, Andreas Brown1, Hartmut Geiger2, and Kalpana Nattamai3
ing this, TLR2 surface expression is often (38% of cases) lost upon transformation. Ongoing experiments are aimed at determining the mechanism by which TLR2 signaling promotes premalignant cell death.
1 Institute of Molecular Medicine, Ulm University, Ulm, Germany; 2EHCB, CCHMC, Cincinnati, USA, Ulm, Germany; 3EHCB, CCHMC, Cincinnati, USA, Cincinnati, United States
Prior work identified a positive correlation between the percentage of hematopoietic progenitor cells (HPCs), defined as cobblestone-area forming cells (CAFC) day 7, killed by the genotoxic agent hydroxyurea (HU) and median lifespan in eight inbred mouse strains. These data suggest a common underlying molecular mechanism for both traits (response of HPCs to HU and lifespan). Given that HU kills proliferating cells, this finding was interpreted to mean that the fraction of proliferating HSC/ HPCs was significantly higher in long-lived C57BL/6 (B6) versus short-lived DBA/2 mice. Using recombinant inbred mice derived from the B6 and DBA/2 mouse strains we mapped a quantitative trait locus (QTL) on chromosome 11 that is responsible for the HU-killing phenotype. The locus was found to overlap with a locus that is linked to lifespan, further emphasizing a likely common mechanism. With this knowledge, congenic mice were generated that have a B6 background and the specific locus on chromosome 11 replaced by the corresponding segment of DBA/2 mice, named line A, or vice versa for DBA/2 mice, named line K. Transfer of the loci was verified by single-nucleotide polymorphism (SNP) analyses. Using the CAFC approach, we could show that HPCs from DBA/2 and line A mice exhibit higher sensitivity to HU, which confirms that the locus on chromosome 11 indeed determines the sensitivity of HPCs to HU. We also analyzed the cell cycle dynamics of HPCs from B6, DBA/2 as well as the congenic strains. Surprisingly the HPCs of the four mouse strains do not differ in cell cycle dynamics, which reveals that the distinct response of HPCs to HU is not correlated to HPCs proliferation. Pituitary tumor-transforming gene 1 (PTTG1, also known as securin) has been identified as a candidate gene within the locus on chromosome 11, which is primarily involved in the regulation of sister chromatid separation during cell division. PTTG1 shows high expression in progenitor cells from DBA/2 and line A mice. Experiments are underway to verify PTTG1 as the quantitative trait gene in this locus and to reveal the underlying molecular mechanisms that determine the distinct response of HPCs to HU treatment.
3050 - TOLL LIKE RECEPTOR 2 (TLR2) SIGNALING PROMOTES APOPTOSIS OF PREMALIGNANT HEMATOPOIETIC STEM AND PROGENITOR CELLS IN A MOUSE MODEL OF MYELODYSPLASTIC SYNDROME Laura Schuettpelz1, Darlene Monlish2, Sima Bhatt2, John Keller2, and Molly Romine2
3051 - DECIPHERING THE ONCOGENIC NETWORK OF PRC2-LOSS GUIDED LEUKEMOGENESIS Dorit Schneider1, Adrian Schwarzer2, Sabine Knoess2, Jan-Henning Klusmann2, and Dirk Heckl2 1 Hannover Medical School, Pediatric Hematology and Oncology, Hannover, Germany; 2Hannover Medical School, Hannover, Germany
1
Washington University School of Medicine, Webster Groves, United States; Washington University, St Louis, United States
2
The myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis and a high risk of leukemic transformation. The only curative treatment is stem cell transplantation; thus new therapies are needed. Multiple studies have shown a correlation between MDS and enhanced toll like receptor 2 (TLR2) signaling, suggesting this might contribute to disease pathogenesis. In fact, an inhibitory TLR2 antibody is currently in clinical trial for use in MDS (OPN-305, Opsona Therapeutics). Despite enthusiasm for TLR2 as a potential drug target, the role of TLR2 in MDS remains unclear. Moreover, high TLR2 expression predicts higher, rather than lower, survival rates, suggesting TLR2 may actually have a protective role in MDS. Thus, we are exploring how TLR2 signaling regulates cell fate in MDS. To investigate the contribution of TLR2 signaling to MDS pathogenesis, we determined the effects of TLR2 loss in a mouse model of MDS (expressing the NUP98-HOXD13 fusion). These ‘‘NHD13’’ mice recapitulate many features of human MDS, and die of leukemia or cytopenias (Lin et al, Blood 2005). Similar to humans, we find increased TLR2 on their HSCs compared to controls. NHD13 mice were crossed to Tlr2-/- mice to generate NHD13+;Tlr2-/-, NHD13+;TLR2+/+, NHD13-;Tlr2-/and NHD13-;Tlr2+/+ groups. Interestingly, loss of TLR2 was associated with accelerated leukemogenesis and worse survival (p50.019). Assessment of the hematopoietic stem and progenitor cells (HSPCs) of young adult mice revealed that TLR2 deficiency led to enhanced cell survival (decreased Annexin V+ HSPCs; p ! .01). Furthermore, the accumulated HSPCs in the NHD13;Tlr2-/- mice showed elevated levels of H2AX, suggesting that TLR2 loss promotes the survival of damaged HSPCs. As apoptosis of HSPCs is characteristic of low-risk MDS, these data suggest that TLR2 signaling may be protective against transformation via promotion of apoptosis of damaged premalignant HSCs. Further support-
Acute myeloid leukemia is elicited by the sequential acquisition of cooperating mutations (mostly three to five) in hematopoietic stem and progenitor cells, which strongly indicates that a single mutation is not sufficient to induce AML. As mutations leading to the loss of PRC2 activity - including -7/-7q deletions – frequently occur in human AML, we aimed to decipher the oncogenic network guided by loss of PRC2. Via a subsequent molecular profiling of these leukemias we sought to unravel novel therapeutic targets in EZH2 loss AML. To this end, a newly established 96-well based CRISPR-Cas9 in vitro cooperation screening enabled us to efficiently screen for the transforming capacity of different combinations of sgRNAs in addition to the Ezh2 sgRNA. Four out of six pools (5 genes each), representing 148 possible sgRNA combinations, reproducibly transformed LSK cells with distinct clonal output. The central role of the Ezh2-loss during transformation in this setting was highlighted by Ezh2 rescue experiments. The same four pools also induced leukemia in recipient mice after bone marrow transplants. Leukemic phenotypes were confirmed in secondary transplants. Sequencing for induced mutations in the leukemic cells highlighted loss of Runx1, Dnmt3a and Nf1 as potential cooperating events, whereas loss of Cohesin complex subunits seemed to be dispensable. To define oncogenic dependencies global gene expression spectra were analyzed in the in vitro transformed clones revealing the repression of differentiation-associated genes and a distinct expression pattern for each sgRNA combination. However, we could identify overlaps of differentially regulated genes for different sgRNA combinations, which may represent a core Ezh2 signature in AML demonstrating the existence of oncogenic dependencies and potentially containing novel therapeutic targets. CRISPR-Cas9 cooperation screenings in vivo and newly established high throughput in vitro screenings proved to have the power to probe oncogenic interaction. Both assays unraveled cooperating mutations for the Ezh2-loss and provided valuable cellular resources to study molecular mechanisms of oncogenic synergies and dependencies.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3049 - PTTG1/SECURIN AS A QUANTITATIVE TRAIT LOCUS CANDIDATE GENE CONTROLLING PROGENITOR CELL SURVIVAL AND ORGANISMAL LIFESPAN Desiree Sch€ utz1, Andreas Brown1, Hartmut Geiger2, and Kalpana Nattamai3
ing this, TLR2 surface expression is often (38% of cases) lost upon transformation. Ongoing experiments are aimed at determining the mechanism by which TLR2 signaling promotes premalignant cell death.
1 Institute of Molecular Medicine, Ulm University, Ulm, Germany; 2EHCB, CCHMC, Cincinnati, USA, Ulm, Germany; 3EHCB, CCHMC, Cincinnati, USA, Cincinnati, United States
Prior work identified a positive correlation between the percentage of hematopoietic progenitor cells (HPCs), defined as cobblestone-area forming cells (CAFC) day 7, killed by the genotoxic agent hydroxyurea (HU) and median lifespan in eight inbred mouse strains. These data suggest a common underlying molecular mechanism for both traits (response of HPCs to HU and lifespan). Given that HU kills proliferating cells, this finding was interpreted to mean that the fraction of proliferating HSC/ HPCs was significantly higher in long-lived C57BL/6 (B6) versus short-lived DBA/2 mice. Using recombinant inbred mice derived from the B6 and DBA/2 mouse strains we mapped a quantitative trait locus (QTL) on chromosome 11 that is responsible for the HU-killing phenotype. The locus was found to overlap with a locus that is linked to lifespan, further emphasizing a likely common mechanism. With this knowledge, congenic mice were generated that have a B6 background and the specific locus on chromosome 11 replaced by the corresponding segment of DBA/2 mice, named line A, or vice versa for DBA/2 mice, named line K. Transfer of the loci was verified by single-nucleotide polymorphism (SNP) analyses. Using the CAFC approach, we could show that HPCs from DBA/2 and line A mice exhibit higher sensitivity to HU, which confirms that the locus on chromosome 11 indeed determines the sensitivity of HPCs to HU. We also analyzed the cell cycle dynamics of HPCs from B6, DBA/2 as well as the congenic strains. Surprisingly the HPCs of the four mouse strains do not differ in cell cycle dynamics, which reveals that the distinct response of HPCs to HU is not correlated to HPCs proliferation. Pituitary tumor-transforming gene 1 (PTTG1, also known as securin) has been identified as a candidate gene within the locus on chromosome 11, which is primarily involved in the regulation of sister chromatid separation during cell division. PTTG1 shows high expression in progenitor cells from DBA/2 and line A mice. Experiments are underway to verify PTTG1 as the quantitative trait gene in this locus and to reveal the underlying molecular mechanisms that determine the distinct response of HPCs to HU treatment.
3050 - TOLL LIKE RECEPTOR 2 (TLR2) SIGNALING PROMOTES APOPTOSIS OF PREMALIGNANT HEMATOPOIETIC STEM AND PROGENITOR CELLS IN A MOUSE MODEL OF MYELODYSPLASTIC SYNDROME Laura Schuettpelz1, Darlene Monlish2, Sima Bhatt2, John Keller2, and Molly Romine2
3051 - DECIPHERING THE ONCOGENIC NETWORK OF PRC2-LOSS GUIDED LEUKEMOGENESIS Dorit Schneider1, Adrian Schwarzer2, Sabine Knoess2, Jan-Henning Klusmann2, and Dirk Heckl2 1 Hannover Medical School, Pediatric Hematology and Oncology, Hannover, Germany; 2Hannover Medical School, Hannover, Germany
1
Washington University School of Medicine, Webster Groves, United States; Washington University, St Louis, United States
2
The myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis and a high risk of leukemic transformation. The only curative treatment is stem cell transplantation; thus new therapies are needed. Multiple studies have shown a correlation between MDS and enhanced toll like receptor 2 (TLR2) signaling, suggesting this might contribute to disease pathogenesis. In fact, an inhibitory TLR2 antibody is currently in clinical trial for use in MDS (OPN-305, Opsona Therapeutics). Despite enthusiasm for TLR2 as a potential drug target, the role of TLR2 in MDS remains unclear. Moreover, high TLR2 expression predicts higher, rather than lower, survival rates, suggesting TLR2 may actually have a protective role in MDS. Thus, we are exploring how TLR2 signaling regulates cell fate in MDS. To investigate the contribution of TLR2 signaling to MDS pathogenesis, we determined the effects of TLR2 loss in a mouse model of MDS (expressing the NUP98-HOXD13 fusion). These ‘‘NHD13’’ mice recapitulate many features of human MDS, and die of leukemia or cytopenias (Lin et al, Blood 2005). Similar to humans, we find increased TLR2 on their HSCs compared to controls. NHD13 mice were crossed to Tlr2-/- mice to generate NHD13+;Tlr2-/-, NHD13+;TLR2+/+, NHD13-;Tlr2-/and NHD13-;Tlr2+/+ groups. Interestingly, loss of TLR2 was associated with accelerated leukemogenesis and worse survival (p50.019). Assessment of the hematopoietic stem and progenitor cells (HSPCs) of young adult mice revealed that TLR2 deficiency led to enhanced cell survival (decreased Annexin V+ HSPCs; p ! .01). Furthermore, the accumulated HSPCs in the NHD13;Tlr2-/- mice showed elevated levels of H2AX, suggesting that TLR2 loss promotes the survival of damaged HSPCs. As apoptosis of HSPCs is characteristic of low-risk MDS, these data suggest that TLR2 signaling may be protective against transformation via promotion of apoptosis of damaged premalignant HSCs. Further support-
Acute myeloid leukemia is elicited by the sequential acquisition of cooperating mutations (mostly three to five) in hematopoietic stem and progenitor cells, which strongly indicates that a single mutation is not sufficient to induce AML. As mutations leading to the loss of PRC2 activity - including -7/-7q deletions – frequently occur in human AML, we aimed to decipher the oncogenic network guided by loss of PRC2. Via a subsequent molecular profiling of these leukemias we sought to unravel novel therapeutic targets in EZH2 loss AML. To this end, a newly established 96-well based CRISPR-Cas9 in vitro cooperation screening enabled us to efficiently screen for the transforming capacity of different combinations of sgRNAs in addition to the Ezh2 sgRNA. Four out of six pools (5 genes each), representing 148 possible sgRNA combinations, reproducibly transformed LSK cells with distinct clonal output. The central role of the Ezh2-loss during transformation in this setting was highlighted by Ezh2 rescue experiments. The same four pools also induced leukemia in recipient mice after bone marrow transplants. Leukemic phenotypes were confirmed in secondary transplants. Sequencing for induced mutations in the leukemic cells highlighted loss of Runx1, Dnmt3a and Nf1 as potential cooperating events, whereas loss of Cohesin complex subunits seemed to be dispensable. To define oncogenic dependencies global gene expression spectra were analyzed in the in vitro transformed clones revealing the repression of differentiation-associated genes and a distinct expression pattern for each sgRNA combination. However, we could identify overlaps of differentially regulated genes for different sgRNA combinations, which may represent a core Ezh2 signature in AML demonstrating the existence of oncogenic dependencies and potentially containing novel therapeutic targets. CRISPR-Cas9 cooperation screenings in vivo and newly established high throughput in vitro screenings proved to have the power to probe oncogenic interaction. Both assays unraveled cooperating mutations for the Ezh2-loss and provided valuable cellular resources to study molecular mechanisms of oncogenic synergies and dependencies.