Abstract
Expression of DAPK1, a critical regulator of autophagy and apoptosis, is lost in a wide variety of tumors, although the mechanisms are unclear. A transcription factor complex consisting of ATF6 (an endoplasmic reticulum-resident factor) and C/EBP-β is required for the IFN-γ-induced expression of DAPK1 IFN-γ-induced proteolytic processing of ATF6 and phosphorylation of C/EBP-β are obligatory for the formation of this transcriptional complex. We report that defects in this pathway fail to control growth of chronic lymphocytic leukemia (CLL). Consistent with these observations, IFN-γ and chemotherapeutics failed to activate autophagy in CLL patient samples lacking ATF6 and/or C/EBP-β. Together, these results identify a molecular basis for the loss of DAPK1 expression in CLL.
Highlights
Multiple genes are inactivated during the progression of a normal cell into malignant one
IFN-␥-stimulated Autophagy Correlates with death-associated protein kinase-1 (DAPK1) Expression Levels in B Cells—We first investigated whether DAPK1 expression was induced by IFN-␥ in B cell tumor line MEC1 and compared it with a control B cell line
DAPK1 Expression in B Cell Lines Requires activating transcription factor 6 (ATF6) and C/EBP-—Because our recent studies using mouse embryonic fibroblasts (MEFs) identified a major role for ATF6 and C/EBP- in promoting DAPK1 expression, we investigated the relevance of these transcription factors (Fig. 2A) to DAPK1 expression in B cells, using RNAi
Summary
IFN-␥-stimulated Autophagy Correlates with DAPK1 Expression Levels in B Cells—We first investigated whether DAPK1 expression was induced by IFN-␥ in B cell tumor line MEC1 (derived from a case of CLL) and compared it with a control B cell line. Immunofluorescence analyses showed that only B cells with scrambled shRNA control yielded maximum numbers of LC3 puncta upon IFN-␥ treatment, when compared with either ATF6 or C/EBP- depletion, signifying their importance in IFN-induced autophagy (Fig. 2C). QPCR analyses of the steady state levels of DAPK1, ATF6, BECN1, and CEBPB mRNAs in the patient samples showed a loss of their expression in the majority of CLLs (Fig. 3A). We investigated whether there were any differences in mRNA levels of IFN-induced tumor suppressor genes IRF1 and IRF8 [24, 25] These transcripts were abundantly expressed in CLL samples, and their levels were comparable with those of healthy controls (Fig. 3B). To address whether the ATF61⁄7C/EBP- complex is recruited to the DAPK1 promoter upon IFN-␥ treatment in the patient samples, we performed ChIP assays with specific antibodies.
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