- Email: [email protected]
progenitor cells to tyrosine kinase inhibitors
S62
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3028 - SMO AND GLI2 ARE KEY REGULATORS MEDIATING RESISTANCE OF CML STEM/PROGENITOR CELLS TO TYROSINE KINASE INHIBITORS Kelly Turner1,2, Katharina Rothe1, Adrian Woolfson3, and Xiaoyan Jiang1 1
British Columbia Cancer Agency, Vancouver, Canada; 2University of British Columbia, Vancouver, Canada; 3Pfizer, Inc., New York, United States
3030 - CYTOKINE-MEDIATED ENDOCYTOSIS IS CRUCIAL FOR THE THERAPEUTIC RESISTANCE OF LEUKEMIAPROPAGATING CELLS Cedric Tremblay1, Sung Kai Chiu1, Jesslyn Saw1, Stefan Sonderegger1, Phillip Robinson2, and David Curtis1 1
Monash University, Melbourne, Australia; 2University of Sydney, Sydney, Australia
Growing evidence suggests that the Hedgehog (HH) pathway plays a key role in the survival of primitive chronic myeloid leukemia (CML) cells. However, it is not known whether the expression of key regulators of the pathway in leukemic stem cells (LSCs) and progenitor cells differ between ABL tyrosine kinase inhibitor (TKI) imatinib (IM) responders and non-responders. We performed RNA sequencing analysis of 6 CD34+ chronic phase (CP)-CML samples obtained at diagnosis. 3 patients were retrospectively classed as IM responders, and 3 as IM non-responders. We compared global gene expression changes between these samples with 3 healthy bone marrow (HBM) controls. We identified 27 differentially-expressed (O1.5 fold) HH pathway genes between CML patients and healthy controls. In particular, Smoothened (SMO) and GLI2 were highly upregulated in IM non-responders as compared with responders. We confirmed these results in an additional 18 CP-CML samples and 8 HBM samples using quantitative real-time PCR (p ! 0.05). SMO and GLI2 were most highly expressed in the stem-enriched subpopulation (lin-CD34+38-) as compared with progenitor (lin-CD34+38+) and the more mature (CD34-) subpopulations, especially in IM non-responders compared with IM responders (O50-fold for GLI2). We then tested the effects of a highly selective SMO inhibitor PF-04449913 in CD34+ cells from IM responders and IM non-responders. In accordance with the gene expression results, IM non-responders were more sensitive to SMO inhibition as compared with IM responders with regard to viability, induction of apoptosis, re-plating potential, and colony-forming ability following long-term (O6-weeks) culture. We also tested a treatment strategy comprising the second generation TKI bosutinib in combination with PF-04449913 in CD34+ IM non-responder cells. This was significantly better at reducing colony-forming ability (p ! 0.01) and re-plating potential (p ! 0.01) compared with either agent alone. The results suggest that dual inhibition of the BCR-ABL and HH pathways may provide a compelling strategy for targeting drug-insensitive LSCs.
Current therapeutic regimen transiently reduce the tumour burden of patients with Tcell acute lymphoblastic leukemia (T-ALL), but fail to eliminate refractory leukemiapropagating cells (LPCs), which are responsible for relapse. Hence, the eradication of LPCs that evade from chemotherapy, will be determinant for achieving long-term remission. These resistant LPCs are dependent on the growth factors produced by the thymic niche to develop and survive following chemotherapy treatment. Using the Lmo2 transgenic mouse model, we have shown previously that these LPCs have long-term self-renewal potential and resistance to chemotherapy. We have also shown that growth factors produced by the thymic niche, like IL-7 and Notch1, are crucial for the development and maintenance of these resistant LPCs during the early stage of the disease. In thymocytes, the activation of these signalling pathways is tightly controlled by endocytosis, which is dependent on the GTPase Dynamin 2 (DNM2). We found that treatment of Lmo2 transgenic DN3 T-cell progenitors with the specific DNM2 inhibitor Dynole 34-2 blocked IL-7R internalization and downstream activation of Stat5, confirming that DNM2 activity is essential for cytokine-mediated response in thymocytes. Inhibition of endocytosis with Dynole 34-2 significantly reduced the frequency and the engraftment of LPCs in transplantation assays. We then treated 2 month-old mice to address the importance of niche-mediated signalling in therapeutic resistance. Accordingly, we found that treatment with Dynole 34-2 sensitized LPCs to the current induction regimen used in the clinic for T-ALL. Finally, inhibition of DNM2 activity significantly delayed leukemia onset in mice treated with standard therapeutic agents, suggesting that endocytosis is required for the maintenance of treatment-resistant T-ALL. These results provide the most convincing in vivo evidence that endocytosis is crucial for the maintenance and therapeutic resistance of LPCs. Targeting endocytosis may represent an attractive therapeutic strategy for improving cure rates in refractory leukemias.
3029 - THE ROLE OF THE H3K4-DEMETHYLASE, KDM5C, IN MALIGNANT HEMATOPOIESIS Mette Louise Trempenau, Mikkel Schuster, Nicolas Rapin, and Bo Porse
3031 - A STANDARDIZED QUANTIFICATION TOOL FOR BONE MARROW COMPONENTS IN HISTOLOGICAL SECTIONS Josefine Tratwal1, Chiheb Boussema1, Olivier Burri2, Tereza Koliqi1, Vasco Campos1, Valentina Nardi3, Carmen Barcena4, Rossella Sarro5, Bettina Bisig5, Laurence De Leval5, and Olaia Naveiras1
Finsen Laboratory, Rigshospitalet, UCH, Copenhagen N, Denmark Background: Acute Myeloid Leukemia (AML) is an aggressive blood cancer, characterized by a rapid accumulation of immature myeloid blasts at the expense of hematopoiesis. Unfortunately, only limited improvements in standard treatment and in overall patient survival have been achieved over the last three to four decades. This emphasizes a need for better understanding of the tumor biology. Epigenetic factors have been implicated as crucial regulators of tumor biology, which prompted us to carry out in vivo shRNA screens to identify novel factors involved in AML progression. This led to the identification of Lysine Demethylase 5C (KDM5C) as a tumor suppressor in C/EBPa mutated AML. Aim: This project aims to elucidate the role of the epigenetic factor KDM5C in AML. Methods: Using murine AML models and flow cytometry assays we investigate the effect of KDM5C in leukemic initiation and progression. To elucidate the mechanisms underlying the effect of KDM5C, we will identify target genes by a combination of microarray and ChIP-seq and test the effects of these genes on tumor maintenance. Result: Knockdown of KDM5C leads to a tumor progressive advantage in C/EBPa mutated AML and presents a less differentiated leukemia. Furthermore, microarray analysis showed downregulation of AP1 transcription factor genes, known to have a role in myeloid differentiation. We further show that modulation of c-Jun expression, an AP1 factor, can partially rescue the phenotype of KDM5C knockdown in our AML model. Conclusion: We suggest that KDM5C functions as a tumor suppressor in AML in part by guiding differentiation through regulation of AP1 gene expression.
1
Laboratory of Regenerative Hematopoiesis, EPFL, Lausanne, Switzerland; Bioimaging and Optics Core Facility, EPFL, Lausanne, Switzerland; 3Massachusetts General Hospital, Harvard Medical School, Boston, United States; 4Department of Pathology, University Hospital Madrid, Spain; 5University Institute of Pathology, CHUV, Lausanne, Switzerland 2
The bone marrow (BM) is the seedbed of our blood, existing as hematopoietic or adipocytic marrow heterogeneously distributed with skeletal location, age, and physiological condition. Patients who undergo chemotherapy or suffer acute BM failure have a rapid adipocytic conversion of the marrow. After recovery such as upon hematopoietic stem cell transplantation (HSCT) following intensive chemotherapy, functional hematopoiesis is restored, for which the Gold Standard of quantification is pathologist assessment of BM cellularity in H&E sections. This expertise is not accessible to all laboratories, and multi-site standardization of BM cellularity quantification is often critical in the clinical and fundamental research setting. In our effort to systematically quantify BM components in histological sections in an unbiased manner, we developed and optimized a semi-automated image analysis tool for ImageJ, MarrowQuant. Area of hematopoietic cells, red blood cells, bone, and adipocyte ghosts are identified based on color and texture variations of H&E stains. We find that BM cellularity quantifications correlate directly with scoring by four independent clinical pathologists from different countries, while quantification of bone and adipocytes compare with microCT volumetric measurements. We have established a consistent map of cellularity in BM sections of homeostatic C57BL6 mice, and observe highly predictable hematopoietic-to-adipocytic marrow transitions in the tibia and caudal tail. With age, BM adiposity increases in the tibia and appears in the femur. Following HSCT, adipogenesis inversely correlates with kinetics of hematopoietic recovery. BM adipocytes reach maximum expansion 17 days post-transplant and hematopoiesis recovers after 25 days, consistent with the exit of severe neutropenia and recovery of pre-transplant blood counts. MarrowQuant has been
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3028 - SMO AND GLI2 ARE KEY REGULATORS MEDIATING RESISTANCE OF CML STEM/PROGENITOR CELLS TO TYROSINE KINASE INHIBITORS Kelly Turner1,2, Katharina Rothe1, Adrian Woolfson3, and Xiaoyan Jiang1 1
British Columbia Cancer Agency, Vancouver, Canada; 2University of British Columbia, Vancouver, Canada; 3Pfizer, Inc., New York, United States
3030 - CYTOKINE-MEDIATED ENDOCYTOSIS IS CRUCIAL FOR THE THERAPEUTIC RESISTANCE OF LEUKEMIAPROPAGATING CELLS Cedric Tremblay1, Sung Kai Chiu1, Jesslyn Saw1, Stefan Sonderegger1, Phillip Robinson2, and David Curtis1 1
Monash University, Melbourne, Australia; 2University of Sydney, Sydney, Australia
Growing evidence suggests that the Hedgehog (HH) pathway plays a key role in the survival of primitive chronic myeloid leukemia (CML) cells. However, it is not known whether the expression of key regulators of the pathway in leukemic stem cells (LSCs) and progenitor cells differ between ABL tyrosine kinase inhibitor (TKI) imatinib (IM) responders and non-responders. We performed RNA sequencing analysis of 6 CD34+ chronic phase (CP)-CML samples obtained at diagnosis. 3 patients were retrospectively classed as IM responders, and 3 as IM non-responders. We compared global gene expression changes between these samples with 3 healthy bone marrow (HBM) controls. We identified 27 differentially-expressed (O1.5 fold) HH pathway genes between CML patients and healthy controls. In particular, Smoothened (SMO) and GLI2 were highly upregulated in IM non-responders as compared with responders. We confirmed these results in an additional 18 CP-CML samples and 8 HBM samples using quantitative real-time PCR (p ! 0.05). SMO and GLI2 were most highly expressed in the stem-enriched subpopulation (lin-CD34+38-) as compared with progenitor (lin-CD34+38+) and the more mature (CD34-) subpopulations, especially in IM non-responders compared with IM responders (O50-fold for GLI2). We then tested the effects of a highly selective SMO inhibitor PF-04449913 in CD34+ cells from IM responders and IM non-responders. In accordance with the gene expression results, IM non-responders were more sensitive to SMO inhibition as compared with IM responders with regard to viability, induction of apoptosis, re-plating potential, and colony-forming ability following long-term (O6-weeks) culture. We also tested a treatment strategy comprising the second generation TKI bosutinib in combination with PF-04449913 in CD34+ IM non-responder cells. This was significantly better at reducing colony-forming ability (p ! 0.01) and re-plating potential (p ! 0.01) compared with either agent alone. The results suggest that dual inhibition of the BCR-ABL and HH pathways may provide a compelling strategy for targeting drug-insensitive LSCs.
Current therapeutic regimen transiently reduce the tumour burden of patients with Tcell acute lymphoblastic leukemia (T-ALL), but fail to eliminate refractory leukemiapropagating cells (LPCs), which are responsible for relapse. Hence, the eradication of LPCs that evade from chemotherapy, will be determinant for achieving long-term remission. These resistant LPCs are dependent on the growth factors produced by the thymic niche to develop and survive following chemotherapy treatment. Using the Lmo2 transgenic mouse model, we have shown previously that these LPCs have long-term self-renewal potential and resistance to chemotherapy. We have also shown that growth factors produced by the thymic niche, like IL-7 and Notch1, are crucial for the development and maintenance of these resistant LPCs during the early stage of the disease. In thymocytes, the activation of these signalling pathways is tightly controlled by endocytosis, which is dependent on the GTPase Dynamin 2 (DNM2). We found that treatment of Lmo2 transgenic DN3 T-cell progenitors with the specific DNM2 inhibitor Dynole 34-2 blocked IL-7R internalization and downstream activation of Stat5, confirming that DNM2 activity is essential for cytokine-mediated response in thymocytes. Inhibition of endocytosis with Dynole 34-2 significantly reduced the frequency and the engraftment of LPCs in transplantation assays. We then treated 2 month-old mice to address the importance of niche-mediated signalling in therapeutic resistance. Accordingly, we found that treatment with Dynole 34-2 sensitized LPCs to the current induction regimen used in the clinic for T-ALL. Finally, inhibition of DNM2 activity significantly delayed leukemia onset in mice treated with standard therapeutic agents, suggesting that endocytosis is required for the maintenance of treatment-resistant T-ALL. These results provide the most convincing in vivo evidence that endocytosis is crucial for the maintenance and therapeutic resistance of LPCs. Targeting endocytosis may represent an attractive therapeutic strategy for improving cure rates in refractory leukemias.
3029 - THE ROLE OF THE H3K4-DEMETHYLASE, KDM5C, IN MALIGNANT HEMATOPOIESIS Mette Louise Trempenau, Mikkel Schuster, Nicolas Rapin, and Bo Porse
3031 - A STANDARDIZED QUANTIFICATION TOOL FOR BONE MARROW COMPONENTS IN HISTOLOGICAL SECTIONS Josefine Tratwal1, Chiheb Boussema1, Olivier Burri2, Tereza Koliqi1, Vasco Campos1, Valentina Nardi3, Carmen Barcena4, Rossella Sarro5, Bettina Bisig5, Laurence De Leval5, and Olaia Naveiras1
Finsen Laboratory, Rigshospitalet, UCH, Copenhagen N, Denmark Background: Acute Myeloid Leukemia (AML) is an aggressive blood cancer, characterized by a rapid accumulation of immature myeloid blasts at the expense of hematopoiesis. Unfortunately, only limited improvements in standard treatment and in overall patient survival have been achieved over the last three to four decades. This emphasizes a need for better understanding of the tumor biology. Epigenetic factors have been implicated as crucial regulators of tumor biology, which prompted us to carry out in vivo shRNA screens to identify novel factors involved in AML progression. This led to the identification of Lysine Demethylase 5C (KDM5C) as a tumor suppressor in C/EBPa mutated AML. Aim: This project aims to elucidate the role of the epigenetic factor KDM5C in AML. Methods: Using murine AML models and flow cytometry assays we investigate the effect of KDM5C in leukemic initiation and progression. To elucidate the mechanisms underlying the effect of KDM5C, we will identify target genes by a combination of microarray and ChIP-seq and test the effects of these genes on tumor maintenance. Result: Knockdown of KDM5C leads to a tumor progressive advantage in C/EBPa mutated AML and presents a less differentiated leukemia. Furthermore, microarray analysis showed downregulation of AP1 transcription factor genes, known to have a role in myeloid differentiation. We further show that modulation of c-Jun expression, an AP1 factor, can partially rescue the phenotype of KDM5C knockdown in our AML model. Conclusion: We suggest that KDM5C functions as a tumor suppressor in AML in part by guiding differentiation through regulation of AP1 gene expression.
1
Laboratory of Regenerative Hematopoiesis, EPFL, Lausanne, Switzerland; Bioimaging and Optics Core Facility, EPFL, Lausanne, Switzerland; 3Massachusetts General Hospital, Harvard Medical School, Boston, United States; 4Department of Pathology, University Hospital Madrid, Spain; 5University Institute of Pathology, CHUV, Lausanne, Switzerland 2
The bone marrow (BM) is the seedbed of our blood, existing as hematopoietic or adipocytic marrow heterogeneously distributed with skeletal location, age, and physiological condition. Patients who undergo chemotherapy or suffer acute BM failure have a rapid adipocytic conversion of the marrow. After recovery such as upon hematopoietic stem cell transplantation (HSCT) following intensive chemotherapy, functional hematopoiesis is restored, for which the Gold Standard of quantification is pathologist assessment of BM cellularity in H&E sections. This expertise is not accessible to all laboratories, and multi-site standardization of BM cellularity quantification is often critical in the clinical and fundamental research setting. In our effort to systematically quantify BM components in histological sections in an unbiased manner, we developed and optimized a semi-automated image analysis tool for ImageJ, MarrowQuant. Area of hematopoietic cells, red blood cells, bone, and adipocyte ghosts are identified based on color and texture variations of H&E stains. We find that BM cellularity quantifications correlate directly with scoring by four independent clinical pathologists from different countries, while quantification of bone and adipocytes compare with microCT volumetric measurements. We have established a consistent map of cellularity in BM sections of homeostatic C57BL6 mice, and observe highly predictable hematopoietic-to-adipocytic marrow transitions in the tibia and caudal tail. With age, BM adiposity increases in the tibia and appears in the femur. Following HSCT, adipogenesis inversely correlates with kinetics of hematopoietic recovery. BM adipocytes reach maximum expansion 17 days post-transplant and hematopoiesis recovers after 25 days, consistent with the exit of severe neutropenia and recovery of pre-transplant blood counts. MarrowQuant has been