- Email: [email protected]
Conserved transcriptional programmes regulating blood cell development
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
S101
3201 - CYCLIN D1/CDK4-, BUT NOT CYCLIN E1/CDK2-MEDIATED ACCELERATION OF G1 CELL CYCLE PHASE TRANSIT TIME PROVIDES A COMPETITIVE ADVANTAGE FOR HUMAN HEMATOPOIETIC STEM CELLS IN VIVO Nicole Mende and Claudia Waskow TU Dresden, Dresden, Germany
3203 - CONSERVED TRANSCRIPTIONAL PROGRAMMES REGULATING BLOOD CELL DEVELOPMENT Adam C. Wilkinson1, Yosuke Tanaka1, Wei Wang2, David J. Ryan2, Pentao Liu2, and Bertie Gottgens1 1 Cambridge University, Cambridge, United Kingdom; 2Wellcome Trust Sanger Institute, Cambridge, United Kingdom
Hematopoietic stem cells (HSC) give rise to all mature blood cell types and are crucial for blood homeostasis in steady-state and under stress conditions. To maintain this function in the long-term HSC cell fate choices are well balanced between exhaustive differentiation and the preservation of stem cell qualities by quiescence or self-renewing divisions. It has been proposed that stem cell fate is influenced by the tight regulation of G1 cell cycle phase progression but it remains unknown whether the transit time through early or late G1 itself influences HSC fate. We aimed at testing whether a ‘shortened’ G1 phase effects fate choice of human HSCs, hypothesizing that a ‘short’ G1 phase may provide insufficient opportunities for the delivery of differentiation-inducing mitogenic signals compared to a ‘long’ G1 phase. To shorten the G1 phase experimentally, we transduced human cord blood CD34+ HSPCs with the early G1-phase regulators cyclin D1 / cyclin-dependent kinase 4 (CYC D1/CDK4, 4D) resulting in a reduced time span HSCs spend in the G1 cycle phase. Serial transplantation revealed an increased human leukocyte engraftment in primary and secondary recipient mice. HSPC pool sizes between 4D and controls were comparable in primary recipients but engraftment in secondary recipients was much improved by 4D, suggesting that an accelerated G1 transit improves the maintenance and function of human HSCs in vivo. 4D-mediated protection from differentiation-inducing signals was confirmed by LPS stimulation in vitro. In contrast, overexpression of late G1 cell cycle phase regulator CYC E1/CDK2 (2E) results in rapid loss of functional HSPCs in vivo. Taken together our data suggests that a balanced transit time through the G1 cell cycle phase is a key regulator for human HSC function and provides a new regulatory platform to ensure lifelong hematopoiesis.
Transcription factors (TFs) act within wider regulatory networks to control cell identity and fate. Numerous TFs are known to regulate developmental and adult haematopoiesis, but how they act within the wider TF network is still poorly understood. Developmental blood cell formation by the endothelial-to-haematopoietic transition (EHT) provides a tractable system to ask fundamental questions about the molecular identity, stabilisation and transitions of TF network states. Additionally, further understanding of this process has potential translational impact for efforts to generate blood cells in vitro for clinical transfusion. Using Transcription Activator-Like Effectors (TALEs) to perturb expression of key haematopoietic TFs during in vitro mouse embryonic stem cell differentiations, we have previously uncovered novel roles for the TF PU.1 during early haematopoietic specification and molecular details of its activity within the wider TF network (Wilkinson et al., DEVELOPMENT 2014). Follow-on work using this system has further implicated PU.1 as an important regulator of megakaryocyte generation from embryonic stem cells. To understand the evolutionarily conservation of the regulatory network that underlies the EHT in mammals and explore the translational potential of our findings, we have set up a comparable in vitro differentiation system from human na€ıve state pluripotent stem cells. Efficient CRISPR/Cas9 gene targeting in these cells has allowed generation of human TF gene knock-in reporters and perturbations for investigating the human EHT. We will present our cellular characterisation and molecular dissection of the EHT using these systems, and the conserved TF network components uncovered.
3202 - CD97 EXPRESSION INFLUENCES MIGRATION AND ADHESION OF FLT3-ITD POSITIVE ACUTE MYELOID LEUKEMIA CELLS AND MODULATES THE BONE MARROW STROMAL MICROENVIRONMENT Manja Wobus1, Uta Oelschl€agel1, Claudia Ortlepp1, Angela Jacobi2, and Martin Bornh€ auser1 1 Department of Medicine I, University Hospital Dresden, Dresden, Germany; 2 Biotechnology Center, TU Dresden, Dresden, Germany
3204 - DECLINED PRESENTATION Vb17 T CELL CLONES MAY RESPOND TO ABL-T315I MUTATION PROTEIN IN CHRONIC MYELOID LEUKEMIA IN BLASTIC CRISIS Ling Xu1, Jie Chen2, Tao Zhang2, Xianfeng Zha2, Shaohua Chen1, Lijian Yang1, Yuhong Lu2, Kanger Zhu2, and Yangqiu Li1 1 Institute of Hematology, Jinan University, Guangzhou, Guangdong, China; 2The First Affiliated Hospital of Jinan Universitiy, Guangzhou, Guangdong, China
Bone marrow niches are specialized microenvironments that facilitate homing and survival of normal hematopoietic stem and progenitor cells (HSPCs) as well as leukemic stem cells (LCSs). Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. However, defined mechanisms mediating interactions of acute myeloid leukemia (AML) cells with the microenvironment remain largely unexplored. We recently demonstrated that expression of the adhesion GPCR CD97 is elevated in blasts of FLT3-ITD positive AML patients. Therefore, we aim to investigate how CD97 expression influences malignant cell characteristics and the impact on mesenchymal stromal cells (MSCs) as a main cellular component of the bone marrow niche. Real-time PCR and FACS analysis of AML cell lines with or without FLT3-ITD or NPM1 mutation revealed 20- and 10-fold higher CD97 expression levels in cells carrying these mutations, as MV4-11 and OCI-AML3. CD97 knock-down in MV4-11 cells was achieved by lentiviral transduction of plko1.6/shRNACD97 and inhibited trans-well migration towards fetal calf serum (FCS) and lysophosphatidic acid (LPA) being at least in part Rho-A dependent. Moreover, knock-down of CD97 led to a reduced adhesion of AML cells to a stromal layer. To test the impact of leukemic cells and their CD97 expression on the MSC phenotype, FACS analysis was performed after 3 days of MSC incubation with tumor cell conditioned medium. Interestingly, CD90 and CD146 expression levels were increased by about 50% by MV4-11 wildtype cell medium but declined to the basic level after incubation with conditioned medium of CD97 knock-down cells. In contrast, the expression levels of CD73 were increased by MV4-11 medium and even further elevated by CD97 knock-down cell medium. In summary, our data suggest a modulation of the bone marrow microenvironment by leukemic cells expressing CD97 and mutated FLT3 rendering it a potential new theragnostic target.
The clonally expanded T cells identified in cancer patients that specific respond to tumor-associated antigen (TAA) have definite. We previously identified a high frequency of oligoclonal expansion of the Vb21 T cell subfamily in the peripheral blood (PB) from BCR-ABL positive chronic myeloid leukemia (CML) and B cell acute lymphocytic leukemia (B-ALL) patients, and the TCR Va13/Vb21 gene modified T cell maintained anti-CML cytotoxicity. However, little is known whether there are any TCR Vb specific for the mutant BCR-ABL protein. In this study, we analyzed the TCR b repertoire in PB from two cases with CML in blastic crisis (CML-BC) with ABL gene mutation, case 1 with B-cell resembling lymphoid blast crisis (LBC) contained single kinase domain mutation (T315I), and case 2 with AML resembling myeloid blast crisis (MBC) contained compound-mutant BCR-ABL1 (L387M and T315I). Using RT-PCR and genescan analysis, clonally expanded Vb17 was identified in samples from both cases. The size of clonal peak in PCR products which indicate the Vb17 CDR3 length, was all in 181 bp, this indicated that both samples contained Vb17 clones with similar CDR3 rearrangement. Interesting, the same size clonally expanded Vb17 T cells was also identified in case 2 at 51 days after HLA-matched sibling hematopoietic stem cell transplantation (HSCT), which is thought from donor origin rather than recipient origin. This result indicated the possibility that the expanded Vb17 clones may respond to CML associated antigen, particularly for mutant T315I-ABL. Moreover, clonally expanded Vb18 T cells was identified in case 1, while clonally expanded Vb15 T cells was found in case 2, this is thought to be individual response to CML associated antigen. In conclusion, our findings showed a identical clonal TCR Vb17 expansion in CMLBC cases with T315I-ABL mutation. Further investigation will be performed to characterize the function of Vb17+T cell clone in T315I-ABL mutant CML-BC patients. This study was supported by grants from the NSFC (81270604 and 81400109)
S101
3201 - CYCLIN D1/CDK4-, BUT NOT CYCLIN E1/CDK2-MEDIATED ACCELERATION OF G1 CELL CYCLE PHASE TRANSIT TIME PROVIDES A COMPETITIVE ADVANTAGE FOR HUMAN HEMATOPOIETIC STEM CELLS IN VIVO Nicole Mende and Claudia Waskow TU Dresden, Dresden, Germany
3203 - CONSERVED TRANSCRIPTIONAL PROGRAMMES REGULATING BLOOD CELL DEVELOPMENT Adam C. Wilkinson1, Yosuke Tanaka1, Wei Wang2, David J. Ryan2, Pentao Liu2, and Bertie Gottgens1 1 Cambridge University, Cambridge, United Kingdom; 2Wellcome Trust Sanger Institute, Cambridge, United Kingdom
Hematopoietic stem cells (HSC) give rise to all mature blood cell types and are crucial for blood homeostasis in steady-state and under stress conditions. To maintain this function in the long-term HSC cell fate choices are well balanced between exhaustive differentiation and the preservation of stem cell qualities by quiescence or self-renewing divisions. It has been proposed that stem cell fate is influenced by the tight regulation of G1 cell cycle phase progression but it remains unknown whether the transit time through early or late G1 itself influences HSC fate. We aimed at testing whether a ‘shortened’ G1 phase effects fate choice of human HSCs, hypothesizing that a ‘short’ G1 phase may provide insufficient opportunities for the delivery of differentiation-inducing mitogenic signals compared to a ‘long’ G1 phase. To shorten the G1 phase experimentally, we transduced human cord blood CD34+ HSPCs with the early G1-phase regulators cyclin D1 / cyclin-dependent kinase 4 (CYC D1/CDK4, 4D) resulting in a reduced time span HSCs spend in the G1 cycle phase. Serial transplantation revealed an increased human leukocyte engraftment in primary and secondary recipient mice. HSPC pool sizes between 4D and controls were comparable in primary recipients but engraftment in secondary recipients was much improved by 4D, suggesting that an accelerated G1 transit improves the maintenance and function of human HSCs in vivo. 4D-mediated protection from differentiation-inducing signals was confirmed by LPS stimulation in vitro. In contrast, overexpression of late G1 cell cycle phase regulator CYC E1/CDK2 (2E) results in rapid loss of functional HSPCs in vivo. Taken together our data suggests that a balanced transit time through the G1 cell cycle phase is a key regulator for human HSC function and provides a new regulatory platform to ensure lifelong hematopoiesis.
Transcription factors (TFs) act within wider regulatory networks to control cell identity and fate. Numerous TFs are known to regulate developmental and adult haematopoiesis, but how they act within the wider TF network is still poorly understood. Developmental blood cell formation by the endothelial-to-haematopoietic transition (EHT) provides a tractable system to ask fundamental questions about the molecular identity, stabilisation and transitions of TF network states. Additionally, further understanding of this process has potential translational impact for efforts to generate blood cells in vitro for clinical transfusion. Using Transcription Activator-Like Effectors (TALEs) to perturb expression of key haematopoietic TFs during in vitro mouse embryonic stem cell differentiations, we have previously uncovered novel roles for the TF PU.1 during early haematopoietic specification and molecular details of its activity within the wider TF network (Wilkinson et al., DEVELOPMENT 2014). Follow-on work using this system has further implicated PU.1 as an important regulator of megakaryocyte generation from embryonic stem cells. To understand the evolutionarily conservation of the regulatory network that underlies the EHT in mammals and explore the translational potential of our findings, we have set up a comparable in vitro differentiation system from human na€ıve state pluripotent stem cells. Efficient CRISPR/Cas9 gene targeting in these cells has allowed generation of human TF gene knock-in reporters and perturbations for investigating the human EHT. We will present our cellular characterisation and molecular dissection of the EHT using these systems, and the conserved TF network components uncovered.
3202 - CD97 EXPRESSION INFLUENCES MIGRATION AND ADHESION OF FLT3-ITD POSITIVE ACUTE MYELOID LEUKEMIA CELLS AND MODULATES THE BONE MARROW STROMAL MICROENVIRONMENT Manja Wobus1, Uta Oelschl€agel1, Claudia Ortlepp1, Angela Jacobi2, and Martin Bornh€ auser1 1 Department of Medicine I, University Hospital Dresden, Dresden, Germany; 2 Biotechnology Center, TU Dresden, Dresden, Germany
3204 - DECLINED PRESENTATION Vb17 T CELL CLONES MAY RESPOND TO ABL-T315I MUTATION PROTEIN IN CHRONIC MYELOID LEUKEMIA IN BLASTIC CRISIS Ling Xu1, Jie Chen2, Tao Zhang2, Xianfeng Zha2, Shaohua Chen1, Lijian Yang1, Yuhong Lu2, Kanger Zhu2, and Yangqiu Li1 1 Institute of Hematology, Jinan University, Guangzhou, Guangdong, China; 2The First Affiliated Hospital of Jinan Universitiy, Guangzhou, Guangdong, China
Bone marrow niches are specialized microenvironments that facilitate homing and survival of normal hematopoietic stem and progenitor cells (HSPCs) as well as leukemic stem cells (LCSs). Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. However, defined mechanisms mediating interactions of acute myeloid leukemia (AML) cells with the microenvironment remain largely unexplored. We recently demonstrated that expression of the adhesion GPCR CD97 is elevated in blasts of FLT3-ITD positive AML patients. Therefore, we aim to investigate how CD97 expression influences malignant cell characteristics and the impact on mesenchymal stromal cells (MSCs) as a main cellular component of the bone marrow niche. Real-time PCR and FACS analysis of AML cell lines with or without FLT3-ITD or NPM1 mutation revealed 20- and 10-fold higher CD97 expression levels in cells carrying these mutations, as MV4-11 and OCI-AML3. CD97 knock-down in MV4-11 cells was achieved by lentiviral transduction of plko1.6/shRNACD97 and inhibited trans-well migration towards fetal calf serum (FCS) and lysophosphatidic acid (LPA) being at least in part Rho-A dependent. Moreover, knock-down of CD97 led to a reduced adhesion of AML cells to a stromal layer. To test the impact of leukemic cells and their CD97 expression on the MSC phenotype, FACS analysis was performed after 3 days of MSC incubation with tumor cell conditioned medium. Interestingly, CD90 and CD146 expression levels were increased by about 50% by MV4-11 wildtype cell medium but declined to the basic level after incubation with conditioned medium of CD97 knock-down cells. In contrast, the expression levels of CD73 were increased by MV4-11 medium and even further elevated by CD97 knock-down cell medium. In summary, our data suggest a modulation of the bone marrow microenvironment by leukemic cells expressing CD97 and mutated FLT3 rendering it a potential new theragnostic target.
The clonally expanded T cells identified in cancer patients that specific respond to tumor-associated antigen (TAA) have definite. We previously identified a high frequency of oligoclonal expansion of the Vb21 T cell subfamily in the peripheral blood (PB) from BCR-ABL positive chronic myeloid leukemia (CML) and B cell acute lymphocytic leukemia (B-ALL) patients, and the TCR Va13/Vb21 gene modified T cell maintained anti-CML cytotoxicity. However, little is known whether there are any TCR Vb specific for the mutant BCR-ABL protein. In this study, we analyzed the TCR b repertoire in PB from two cases with CML in blastic crisis (CML-BC) with ABL gene mutation, case 1 with B-cell resembling lymphoid blast crisis (LBC) contained single kinase domain mutation (T315I), and case 2 with AML resembling myeloid blast crisis (MBC) contained compound-mutant BCR-ABL1 (L387M and T315I). Using RT-PCR and genescan analysis, clonally expanded Vb17 was identified in samples from both cases. The size of clonal peak in PCR products which indicate the Vb17 CDR3 length, was all in 181 bp, this indicated that both samples contained Vb17 clones with similar CDR3 rearrangement. Interesting, the same size clonally expanded Vb17 T cells was also identified in case 2 at 51 days after HLA-matched sibling hematopoietic stem cell transplantation (HSCT), which is thought from donor origin rather than recipient origin. This result indicated the possibility that the expanded Vb17 clones may respond to CML associated antigen, particularly for mutant T315I-ABL. Moreover, clonally expanded Vb18 T cells was identified in case 1, while clonally expanded Vb15 T cells was found in case 2, this is thought to be individual response to CML associated antigen. In conclusion, our findings showed a identical clonal TCR Vb17 expansion in CMLBC cases with T315I-ABL mutation. Further investigation will be performed to characterize the function of Vb17+T cell clone in T315I-ABL mutant CML-BC patients. This study was supported by grants from the NSFC (81270604 and 81400109)