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CDX2 promotes leukemogenesis by modulating leukemic cell - bm niche interactions
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3066 - CDX2 PROMOTES LEUKEMOGENESIS BY MODULATING LEUKEMIC CELL - BM NICHE INTERACTIONS Anna Paczulla1, Marcelle Ndoh Mbarga1, Leticia Quintanilla-Martinez2, and Claudia Lengerke1
S73
3068 - DECLINED PRESENTATION SHIKONIN INDUCES REACTIVE OXIGEN SPECIES MEDIATED APOPTOSIS ON PRIMARY EFFUSION LYMPHOMA CELLS Seiji Okada1,2, Alam Md Masud3, and Ryusho Kariya3
1 University of Basel and University Hospital Basel, Department for Biomedicine, Basel, Switzerland; 2University of Tuebingen, Department of Pathology, Tuebingen, Germany
1 Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, Kumamoto, Japan; 2Kumamoto, Japan; 3Center for AIDS Research, Kumamoto University, Kumamoto, Japan
Objectives: The caudal-type homeobox (CDX) gene family regulates embryonic hematopoiesis via downstream HOX genes and interactions with the WNT signaling pathway. CDX2 expression is not detected in healthy bone marrow (BM) cells but present in O80% of human acute myeloid (AML) and lymphoid leukemia (ALL). Ectopic activation in murine BM cells induces myeloid leukemia. Here, we explore the functional role and molecular targets of CDX2 in human leukemia. Methods: CDX2 expression was genetically modulated (via lentiviral or siRNA treatment) in healthy human cord blood-derived CD34+ cells (for CDX2 overexpression) and in human AML cell lines (SKM1, EOL-1) and primary patient-derived cells (both for CDX2 knockdown). CDX2 modified and control cells were subjected to growth, colony forming (CFU), cell cycle, flow cytometry, adhesion and qRT-PCR assays and analyzed in vivo upon xenotransplantation in NOD/SCID/IL2Rgnull (NSG) mice for bone marrow (BM) homing and leukemogenesis. Results: CDX2 knockdown in AML cells strongly reduced clonogenicity while leaving proliferation, apoptosis and cell cycle unaltered. Importantly, CDX2 knockdown profoundly suppressed in vivo leukemogenic properties. Gene set enrichment analyses (GSEA) of microarray data collected on CDX2 overexpressing versus control leukemic cells revealed cell adhesion genes as one of the pathways most prominently regulated by CDX2. Furthermore in vitro adhesion assays of human AML cell lines or respectively primary patient-derived AML cells on murine MS5 stromal cells revealed CDX2 to positively regulate leukemic cell adhesion to stroma. These data suggest that CDX2 expression might promote leukemogenicity by enhancing AML cell adherence to protective BM niches. Consistently, high CDX2 expression levels associated with enhanced homing of AML cells to the BM in in vivo assays. Conclusion: Our data suggest that CDX2 plays important roles in human AML cells during in vivo leukemogenesis by inducing clonogenicity and BM niche adherence.
Primary effusion lymphoma (PEL) is an incurable hematological malignancy and novel biological-based treatments are urgently necessitated in the clinical settings. Shikonin (SHK), a natural naphthoquinone, has been reported to trigger cell death and overcome drug resistance in anti-tumour therapy. In the present study, we investigated the effectiveness and molecular mechanism of SHK in treatment with PEL both in vitro and in animal model. Treatment of PEL cells with SHK resulted in profound induction of apoptosis in a dose dependent manner accompanied by rapid generation of reactive oxygen species (ROS), striking activation of cJun-N-terminal kinase (JNK) and p38, marked decrease the mitochondrial membrane potential (Djm), activation of caspase-9, -8 and -3. Scavenging of ROS in the presence of N-acetylcysteine (NAC) almost blocked the JNK activation, caspase-3 cleavage, the loss of Djm as well as the induction of apoptosis in the SHK treated PEL cells. SP600125, a specific inhibitor of JNK rescued the cells from the apoptosis of SHK, indicated that JNK pathway plays main role for SHK dependent apoptosis. In a PEL xenograft Nude-Rag2/Jak3 double deficient mice model, SHK treatment reduced the amount of ascites without showing significant systemic toxicity. These findings demonstrated that SHK would be a potential anti-tumor agent for the therapeutic treatment of PEL.
3067 - DECLINED PRESENTATION THE STANDARD TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) IS BASED ON POLYCHEMOTHERAPY 1 € , Sarah Ebinger2, Christoph Ziegenhain3, Erbey Ziya Ozdemir Wolfgang Enard3, and Irmela Jeremias2
3069 - A NOVEL STRATEGY FOR ABL TYROSINE KINASE INHIBITOR RESISTANT PH-CHROMOSOME POSITIVE LEUKEMIA CELLS Seiichi Okabe, Tetsuzo Tauchi, Yuko Tanaka, and Kazuma Ohyashiki
1
Department of Gene Vectors, Helmholtz Zentrum M€unchen, Munich, Germany; Department of Gene Vectors, Helmholtz Zentrum M€unchen, Germany, Munich, Germany; 3 Department Biology II, Ludwig-Maximilians-Universit€at M€unchen, Munich, Germany 2
The standard treatment of acute lymphoblastic leukemia (ALL) is based on polychemotherapy. Often high tumor burdens can be reduced and patients reach remission. However, potentially remaining leukemic cells after therapy, e.g., in the disease stage of minimal residual disease (MRD), indicate a high risk for relapse resulting in a bad prognosis. Novel treatment strategies are urgently needed and have to target the clinically challenging MRD cells. However, so far no preclinical model was available to develop novel approaches for targeting infinitesimal amounts of leukemic cells surviving chemotherapy. In this study we established two distinct patient-derived xenograft (PDX) mouse models for the identification and analysis of minor ALL populations associated with challenging features of dormancy and chemotherapy resistance. Genetic engineering of the PDX cells facilitated the detection and isolation of these cells from the murine bone marrow by expression of reporter genes and a luciferase. In the first PDX model, a long-term dormant subpopulation of ALL cells growing in mice was identified by CFSE stainings. In the second PDX model, a therapy-resistant ALL population was generated by application of a combination chemotherapy over several weeks in order to reduce high leukemia burden to MRD levels of less than 0.1% of PDX cells in the murine bone marrow. Functional studies showed high plasticity and similar features between the both critical ALL populations in the two PDX models. The identified dormant ALL cells were chemotherapy resistant in vivo and the generated chemotherapy resistant ALL cells displayed a dormant phenotype. Furthermore, no of the both critical ALL populations were enriched for stemness and chemotherapy resistance compared to the untreated dividing controls. Gene expression profiles (GEP), obtained by bulk and single cell RNA-seq, revealed high similarities between dormant and chemotherapy resistant PDX populations. The clinical relevance of these cells was shown by high correlation of their GEP with primary GEP, obtained from patients in MRD. This two established PDX models provide an unique platform to develop novel treatment strategies against MRD cells.
Tokyo Medical University, Tokyo, Japan Background: Although ABL tyrosine kinase inhibitors (TKIs) such as imatinib have demonstrated the potency against Philadelphia chromosome (Ph)-positive leukemia patients, resistance to ABL TKI can develop in chronic myeloid leukemia (CML) patients. Aims: We hypothesized that ABL TKI resistance may often happen due to additional somatic mutations in the oncogene. Methods: We established several TKI-resistant in vitro cell line models. We also investigated model to evaluate the next-generation sequencing (NGS) panel. Results: We established ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinibR, K562 ponatinib-R, Ba/F3 T315I and Ba/F3 ponatinib-R) in this study. BCR-ABL expression levels were not increased. We could not detect the BCR-ABL point mutation. However, the exon 4 deletion in the BCR-ABL gene was found in K562 ponatinib-R cells. In contrast, compound mutations in BCR-ABL were found in Ba/F3 ponatinib-R cells. We next evaluated the NGS panel (GeneRead DNAseq Targeted Panels V2) to investigate the mutation. We found that several somatic mutations in TET2, FLT3, RB1, TP53, SETBP1, ASXL1, and BCORL1 in parental K562 cells. We also found that additional somatic mutations in K562 imatinib-R (IDH1 and KRAS), K562 dasatinib-R (IDH1) and K562 ponatinib-R (SF3A1). We could not detect additional mutation in K562 nilotinib-R cells. Combined treatment of ABLTKI resistant K562 with imatinib or dasatinib and MEK inhibitor or IDH1 inhibitor caused more cytotoxicity. Because aberrant activation of PI3K signaling pathway and deregulation of HDAC activity may be a cause of malignant disease in humans, we examined the PI3K and HDAC inhibitor in ABL TKI resistant cells. The inhibitor of class I PI3K as well as class I and II HDAC enzymes, CUDC-907 exhibits cell growth inhibition. Summary: Our study indicated that leukemia cells have acquired resistance through somatic mutation or exon 4 deletion in the BCR-ABL gene, suggested that individual based investigations may be important to evaluate the ABL TKI resistance. We also provide the promising clinical relevance as a candidate drug for treatment of ABL TKI resistant leukemia patients.
3066 - CDX2 PROMOTES LEUKEMOGENESIS BY MODULATING LEUKEMIC CELL - BM NICHE INTERACTIONS Anna Paczulla1, Marcelle Ndoh Mbarga1, Leticia Quintanilla-Martinez2, and Claudia Lengerke1
S73
3068 - DECLINED PRESENTATION SHIKONIN INDUCES REACTIVE OXIGEN SPECIES MEDIATED APOPTOSIS ON PRIMARY EFFUSION LYMPHOMA CELLS Seiji Okada1,2, Alam Md Masud3, and Ryusho Kariya3
1 University of Basel and University Hospital Basel, Department for Biomedicine, Basel, Switzerland; 2University of Tuebingen, Department of Pathology, Tuebingen, Germany
1 Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, Kumamoto, Japan; 2Kumamoto, Japan; 3Center for AIDS Research, Kumamoto University, Kumamoto, Japan
Objectives: The caudal-type homeobox (CDX) gene family regulates embryonic hematopoiesis via downstream HOX genes and interactions with the WNT signaling pathway. CDX2 expression is not detected in healthy bone marrow (BM) cells but present in O80% of human acute myeloid (AML) and lymphoid leukemia (ALL). Ectopic activation in murine BM cells induces myeloid leukemia. Here, we explore the functional role and molecular targets of CDX2 in human leukemia. Methods: CDX2 expression was genetically modulated (via lentiviral or siRNA treatment) in healthy human cord blood-derived CD34+ cells (for CDX2 overexpression) and in human AML cell lines (SKM1, EOL-1) and primary patient-derived cells (both for CDX2 knockdown). CDX2 modified and control cells were subjected to growth, colony forming (CFU), cell cycle, flow cytometry, adhesion and qRT-PCR assays and analyzed in vivo upon xenotransplantation in NOD/SCID/IL2Rgnull (NSG) mice for bone marrow (BM) homing and leukemogenesis. Results: CDX2 knockdown in AML cells strongly reduced clonogenicity while leaving proliferation, apoptosis and cell cycle unaltered. Importantly, CDX2 knockdown profoundly suppressed in vivo leukemogenic properties. Gene set enrichment analyses (GSEA) of microarray data collected on CDX2 overexpressing versus control leukemic cells revealed cell adhesion genes as one of the pathways most prominently regulated by CDX2. Furthermore in vitro adhesion assays of human AML cell lines or respectively primary patient-derived AML cells on murine MS5 stromal cells revealed CDX2 to positively regulate leukemic cell adhesion to stroma. These data suggest that CDX2 expression might promote leukemogenicity by enhancing AML cell adherence to protective BM niches. Consistently, high CDX2 expression levels associated with enhanced homing of AML cells to the BM in in vivo assays. Conclusion: Our data suggest that CDX2 plays important roles in human AML cells during in vivo leukemogenesis by inducing clonogenicity and BM niche adherence.
Primary effusion lymphoma (PEL) is an incurable hematological malignancy and novel biological-based treatments are urgently necessitated in the clinical settings. Shikonin (SHK), a natural naphthoquinone, has been reported to trigger cell death and overcome drug resistance in anti-tumour therapy. In the present study, we investigated the effectiveness and molecular mechanism of SHK in treatment with PEL both in vitro and in animal model. Treatment of PEL cells with SHK resulted in profound induction of apoptosis in a dose dependent manner accompanied by rapid generation of reactive oxygen species (ROS), striking activation of cJun-N-terminal kinase (JNK) and p38, marked decrease the mitochondrial membrane potential (Djm), activation of caspase-9, -8 and -3. Scavenging of ROS in the presence of N-acetylcysteine (NAC) almost blocked the JNK activation, caspase-3 cleavage, the loss of Djm as well as the induction of apoptosis in the SHK treated PEL cells. SP600125, a specific inhibitor of JNK rescued the cells from the apoptosis of SHK, indicated that JNK pathway plays main role for SHK dependent apoptosis. In a PEL xenograft Nude-Rag2/Jak3 double deficient mice model, SHK treatment reduced the amount of ascites without showing significant systemic toxicity. These findings demonstrated that SHK would be a potential anti-tumor agent for the therapeutic treatment of PEL.
3067 - DECLINED PRESENTATION THE STANDARD TREATMENT OF ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) IS BASED ON POLYCHEMOTHERAPY 1 € , Sarah Ebinger2, Christoph Ziegenhain3, Erbey Ziya Ozdemir Wolfgang Enard3, and Irmela Jeremias2
3069 - A NOVEL STRATEGY FOR ABL TYROSINE KINASE INHIBITOR RESISTANT PH-CHROMOSOME POSITIVE LEUKEMIA CELLS Seiichi Okabe, Tetsuzo Tauchi, Yuko Tanaka, and Kazuma Ohyashiki
1
Department of Gene Vectors, Helmholtz Zentrum M€unchen, Munich, Germany; Department of Gene Vectors, Helmholtz Zentrum M€unchen, Germany, Munich, Germany; 3 Department Biology II, Ludwig-Maximilians-Universit€at M€unchen, Munich, Germany 2
The standard treatment of acute lymphoblastic leukemia (ALL) is based on polychemotherapy. Often high tumor burdens can be reduced and patients reach remission. However, potentially remaining leukemic cells after therapy, e.g., in the disease stage of minimal residual disease (MRD), indicate a high risk for relapse resulting in a bad prognosis. Novel treatment strategies are urgently needed and have to target the clinically challenging MRD cells. However, so far no preclinical model was available to develop novel approaches for targeting infinitesimal amounts of leukemic cells surviving chemotherapy. In this study we established two distinct patient-derived xenograft (PDX) mouse models for the identification and analysis of minor ALL populations associated with challenging features of dormancy and chemotherapy resistance. Genetic engineering of the PDX cells facilitated the detection and isolation of these cells from the murine bone marrow by expression of reporter genes and a luciferase. In the first PDX model, a long-term dormant subpopulation of ALL cells growing in mice was identified by CFSE stainings. In the second PDX model, a therapy-resistant ALL population was generated by application of a combination chemotherapy over several weeks in order to reduce high leukemia burden to MRD levels of less than 0.1% of PDX cells in the murine bone marrow. Functional studies showed high plasticity and similar features between the both critical ALL populations in the two PDX models. The identified dormant ALL cells were chemotherapy resistant in vivo and the generated chemotherapy resistant ALL cells displayed a dormant phenotype. Furthermore, no of the both critical ALL populations were enriched for stemness and chemotherapy resistance compared to the untreated dividing controls. Gene expression profiles (GEP), obtained by bulk and single cell RNA-seq, revealed high similarities between dormant and chemotherapy resistant PDX populations. The clinical relevance of these cells was shown by high correlation of their GEP with primary GEP, obtained from patients in MRD. This two established PDX models provide an unique platform to develop novel treatment strategies against MRD cells.
Tokyo Medical University, Tokyo, Japan Background: Although ABL tyrosine kinase inhibitors (TKIs) such as imatinib have demonstrated the potency against Philadelphia chromosome (Ph)-positive leukemia patients, resistance to ABL TKI can develop in chronic myeloid leukemia (CML) patients. Aims: We hypothesized that ABL TKI resistance may often happen due to additional somatic mutations in the oncogene. Methods: We established several TKI-resistant in vitro cell line models. We also investigated model to evaluate the next-generation sequencing (NGS) panel. Results: We established ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinibR, K562 ponatinib-R, Ba/F3 T315I and Ba/F3 ponatinib-R) in this study. BCR-ABL expression levels were not increased. We could not detect the BCR-ABL point mutation. However, the exon 4 deletion in the BCR-ABL gene was found in K562 ponatinib-R cells. In contrast, compound mutations in BCR-ABL were found in Ba/F3 ponatinib-R cells. We next evaluated the NGS panel (GeneRead DNAseq Targeted Panels V2) to investigate the mutation. We found that several somatic mutations in TET2, FLT3, RB1, TP53, SETBP1, ASXL1, and BCORL1 in parental K562 cells. We also found that additional somatic mutations in K562 imatinib-R (IDH1 and KRAS), K562 dasatinib-R (IDH1) and K562 ponatinib-R (SF3A1). We could not detect additional mutation in K562 nilotinib-R cells. Combined treatment of ABLTKI resistant K562 with imatinib or dasatinib and MEK inhibitor or IDH1 inhibitor caused more cytotoxicity. Because aberrant activation of PI3K signaling pathway and deregulation of HDAC activity may be a cause of malignant disease in humans, we examined the PI3K and HDAC inhibitor in ABL TKI resistant cells. The inhibitor of class I PI3K as well as class I and II HDAC enzymes, CUDC-907 exhibits cell growth inhibition. Summary: Our study indicated that leukemia cells have acquired resistance through somatic mutation or exon 4 deletion in the BCR-ABL gene, suggested that individual based investigations may be important to evaluate the ABL TKI resistance. We also provide the promising clinical relevance as a candidate drug for treatment of ABL TKI resistant leukemia patients.