Project description:The aim of this experiment was to study the effects of a kinase-dead mutant of the cell cycle kinase CDK6 (CDK6-K43M) on gene expression in a murine model of BCR-ABL driven leukemia.
Project description:The aim of this experiment was to study the chromatin accessibility in BCR-ABL(p185)-positive pre/pro B-cells expressing wildtype CDK6 or a kinase-dead mutant (CDK6 K43M)
Project description:Metabolic reprogramming and cell cycle deregulation are hallmarks of cancer cells. We here investigate the role of the cell cycle kinase CDK6 in the regulation of cellular energetics in BCR-ABL+ leukemia. Gene expression data and ChIP-Seq analysis from murine BCR-ABL+ cell lines expressing kinase-inactive CDK6 or no CDK6 highlight an activating role for the kinase in regulating the oxidative phosphorylation gene set by interacting with respective promoter regions. Our data imply a competition of CDK6 and Nrf-1, a master regulator of genes required for mitochondrial respiration, at the same sites. In line, cells expressing kinase- inactive CDK6 show signs of a defective electron transport chain and morphologically changed mitochondria. An enhanced cytoplasm/mitochondria ATP ratio together with high levels of pyruvate and lactate point towards a metabolic switch to glycolysis in those cells. Combinatorial treatment of BCR-ABL+ cell lines with the clinically used CDK4/6 inhibitor palbociclib and the glycolysis inhibitor 2-deoxyglocose (2-DG) reduced proliferation and led to enhanced apoptosis compared to single-agent treatments. Our data, suggest a new therapeutic avenue for hematologic malignancies with high CDK6 expression.
Project description:The aim of this experiment was to study the DNA interaction of a kinase-dead mutant of the cell cycle kinase CDK6 (CDK6-K43M) in a murine model of BCR-ABL driven leukemia.
Project description:The transcription factor c-JUN and its upstream kinase JNK1 have been implicated in BCR-ABL induced leukemogenesis. JNK1 has been shown to regulate BCL2 expression thereby altering leukemogenesis, but the impact of c-JUN remained unclear. In this study we show that JNK1 and c-JUN promote leukemogenesis via separate pathways, since lack of c-JUN impairs proliferation of p185BCR-ABL transformed cells without affecting viability. The decreased proliferation of c-JunD/D cells is associated with the loss of cyclin dependent kinase 6 (CDK6) expression. In c-JunD/D cells CDK6 expression becomes down-regulated upon BCR-ABL induced transformation which correlates with CpG island methylation within the 5´ region of Cdk6. We verified the impact of Cdk6 deficiency by using Cdk6-/- mice that developed BCR-ABL induced B-lymphoid leukemia with significantly increased latency and an attenuated disease phenotype. In addition we show that re-expression of CDK6 in BCR-ABL transformed c-JunD/D cells reconstitutes proliferation and tumor formation in Nu/Nu mice. In summary, our study reveals a novel function for the AP-1 transcription factor c-JUN in leukemogenesis by antagonizing promoter methylation. Moreover, we identify CDK6 as relevant and critical target of AP-1 regulated DNA methylation upon BCR-ABL induced transformation, thereby accelerating leukemogenesis.
Project description:The transcription factor c-JUN and its upstream kinase JNK1 have been implicated in BCR-ABL induced leukemogenesis. JNK1 has been shown to regulate BCL2 expression thereby altering leukemogenesis, but the impact of c-JUN remained unclear. In this study we show that JNK1 and c-JUN promote leukemogenesis via separate pathways, since lack of c-JUN impairs proliferation of p185BCR-ABL transformed cells without affecting viability. The decreased proliferation of c-JunD/D cells is associated with the loss of cyclin dependent kinase 6 (CDK6) expression. In c-JunD/D cells CDK6 expression becomes down-regulated upon BCR-ABL induced transformation which correlates with CpG island methylation within the 5´ region of Cdk6. We verified the impact of Cdk6 deficiency by using Cdk6-/- mice that developed BCR-ABL induced B-lymphoid leukemia with significantly increased latency and an attenuated disease phenotype. In addition we show that re-expression of CDK6 in BCR-ABL transformed c-JunD/D cells reconstitutes proliferation and tumor formation in Nu/Nu mice. In summary, our study reveals a novel function for the AP-1 transcription factor c-JUN in leukemogenesis by antagonizing promoter methylation. Moreover, we identify CDK6 as relevant and critical target of AP-1 regulated DNA methylation upon BCR-ABL induced transformation, thereby accelerating leukemogenesis. Overall, 8 samples consisting of 4 wild type and 4 c-jun knock out samples were hybridized to MoGene-1_0-st-v1 microarrays.
Project description:Tyrosine kinase activity is the crucial enzymatic activity driving all known functions of the BCR-ABL protein and is required for protection from apoptosis by BCR-ABL, therefore, targeting this enzyme is an effective approach for therapeutic strategies. Recently, a novel structural entity, imatinib (STI571; Novartis, Basel, Switzerland), a potent and selective inhibitor of the tyrosine kinase activity of BCR-ABL, has shown promise against Ph-positive leukemia in human clinical trials. However, the emergence of imatinib resistance in patients with acute forms of Ph-positive leukemia has highlighted the need for overriding chemotherapy to eradicate this disease. AMN107 and BMS-354825 are clinically active “next-generation” BCR-ABL inhibitors. One potentially powerful approach is to use these compounds in combination with imatinib. The rationale for such approaches is that an inhibitor cocktail may target the widest range of resistant clones and thereby delay the onset of acquired drug resistance. More potent BCR-ABL inhibitors would be to target residual leukemia that persists in patients in whom imatinib induces durable remission but failed to eradicate the disease. From these points, our studies are performed to determine (1) the differences of molecular signaling pathways between BMS-354825 and imatinib (2) the mechanisms by which drug resistance of BMS-354825 and imatinib occur except for point mutation of BCR-ABL kinase domain. Keywords: drug sensitivity
Project description:BCR-ABL was used to transform Cdk6+/+, Cdk6-K43M or Cdk6-/- bone marrow cells. RNA was isolated from individual colonies formed in methylcellulose at day 10 post-transduction