ABSTRACT
The blood system serves as a key model for cell differentiation and cancer. It is orchestrated by precise spatiotemporal expression of crucial transcription factors. One of the key master regulators in the hematopoietic systems is PU.1. Reduced levels of PU.1 are characteristic for human acute myeloid leukemia (AML) and are known to induce AML in mouse models. Here, we show that transcriptional downregulation of PU.1 is an active process involving an alternative promoter in intron 3 that is induced by RUNX transcription factors driving noncoding antisense transcription. Core-binding factor (CBF) fusions RUNX1-ETO and CBFß-MYH11 in t(8;21) and inv(16) AML, respectively, activate the PU.1 antisense promoter that results in a shift from sense toward antisense transcription and myeloid differentiation blockade. In patients with CBF-AML, we found that an elevated antisense/sense transcript and promoter accessibility ratio represents a hallmark compared with normal karyotype AML or healthy CD34+ cells. Competitive interaction of an enhancer with the proximal or the antisense promoter forms a binary on/off switch for either myeloid or T-cell development. Leukemic CBF fusions thus use a physiological mechanism used by T cells to decrease sense transcription. Our study is the first example of a sense/antisense promoter competition as a crucial functional switch for gene expression perturbation by oncogenes. Hence, this disease mechanism reveals a previously unknown Achilles heel for future precise therapeutic targeting of oncogene-induced chromatin remodeling.
Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor beta Subunit/genetics , Gene Expression Regulation, Leukemic , Leukemia, Myeloid, Acute/genetics , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Antisense Elements (Genetics)/genetics , Cell Line, Tumor , Gene Fusion , Humans , Oncogene Proteins, Fusion/genetics , Promoter Regions, Genetic , RUNX1 Translocation Partner 1 Protein/genetics , Tumor Cells, CulturedABSTRACT
The BCR/ABL1 inhibitor Nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). Although otherwise well-tolerated, Nilotinib has been associated with the occurrence of progressive arterial occlusive disease (AOD). Our objective was to determine the exact frequency of AOD and examine in vitro and in vivo effects of Nilotinib and Imatinib on endothelial cells to explain AOD-development. In contrast to Imatinib, Nilotinib was found to upregulate pro-atherogenic adhesion-proteins (ICAM-1, E-selectin, VCAM-1) on human endothelial cells. Nilotinib also suppressed endothelial cell proliferation, migration and tube-formation and bound to a distinct set of target-kinases, relevant to angiogenesis and atherosclerosis, including angiopoietin receptor-1 TEK, ABL-2, JAK1 and MAP-kinases. Nilotinib and siRNA against ABL-2 also suppressed KDR expression. In addition, Nilotinib augmented atherosclerosis in ApoE-/- mice and blocked reperfusion and angiogenesis in a hindlimb-ischemia model of arterial occlusion, whereas Imatinib showed no comparable effects. Clinically overt AOD-events were found to accumulate over time in Nilotinib-treated patients. After a median observation-time of 2.0 years, the AOD-frequency was higher in these patients (29.4%) compared to risk factor- and age-matched controls (<5%). Together, Nilotinib exerts direct pro-atherogenic and anti-angiogenic effects on vascular endothelial cells, which may contribute to development of AOD in patients with CML.
Subject(s)
Endothelium, Vascular/drug effects , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Protein Kinase Inhibitors/adverse effects , Pyrimidines/adverse effects , Vascular Diseases/chemically induced , Adult , Aged , Aged, 80 and over , Animals , Apolipoproteins E/genetics , Atherosclerosis/chemically induced , Endothelium, Vascular/cytology , Female , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle AgedABSTRACT
The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.
