Transcriptional regulation of the ABCC6 gene and the background of impaired function of missense disease-causing mutations.

The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein expressed primarily in the liver and to a lesser extent in the kidneys and the intestines. We review here the mechanisms of this restricted tissue-specific expression and the role of hepatocyte nuclear factor 4alpha which is responsible for the expression pattern. Detailed analyses uncovered further regulators of the expression of the gene pointing to an intronic primate-specific regulator region, an activator of the expression of the gene by binding CCAAT/enhancer-binding protein beta, which interacts with other proteins acting in the proximal promoter. This regulatory network is affected by various environmental stimuli including oxidative stress and the extracellular signal-regulated protein kinases 1 and 2 pathway. We also review here the structural and functional consequences of disease-causing missense mutations of ABCC6. A significant clustering of the missense disease-causing mutations was found at the domain-domain interfaces. This clustering means that the domain contacts are much less permissive to amino acid replacements than the rest of the protein. We summarize the experimental methods resulting in the identification of mutants with preserved transport activity but failure in intracellular targeting. These mutants are candidates for functional rescue by chemical chaperons. The results of such research can provide the basis of future allele-specific therapy of ABCC6-mediated disorders like pseudoxanthoma elasticum or the generalized arterial calcification in infancy

Combined inhibition of Wee1 and Chk1 as a therapeutic strategy in multiple myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative stress. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways. In this study, we reported that high CHK1 and WEE1 expression is associated with poor outcome in independent cohorts of MM patients treated with high dose melphalan chemotherapy or anti-CD38 immunotherapy. Combined targeting of Chk1 and Wee1 demonstrates synergistic toxicities on MM cells and was associated with higher DNA double-strand break induction, as evidenced by an increased percentage of γH2AX positive cells subsequently leading to apoptosis. The therapeutic interest of Chk1/Wee1 inhibitors’ combination was validated on primary MM cells of patients. The toxicity was specific of MM cells since normal bone marrow cells were not significantly affected. Using deconvolution approach, MM patients with high CHK1 expression exhibited a significant lower percentage of NK cells whereas patients with high WEE1 expression displayed a significant higher percentage of regulatory T cells in the bone marrow. These data emphasize that MM cell adaptation to replicative stress through Wee1 and Chk1 upregulation may decrease the activation of the cell-intrinsic innate immune response. Our study suggests that association of Chk1 and Wee1 inhibitors may represent a promising therapeutic approach in high-risk MM patients characterized by high CHK1 and WEE1 expression

The BLM helicase is a new therapeutic target in multiple myeloma involved in replication stress survival and drug resistance

Multiple myeloma (MM) is a hematologic cancer characterized by accumulation of malignant plasma cells in the bone marrow. To date, no definitive cure exists for MM and resistance to current treatments is one of the major challenges of this disease. The DNA helicase BLM, whose depletion or mutation causes the cancer-prone Bloom’s syndrome (BS), is a central factor of DNA damage repair by homologous recombination (HR) and genomic stability maintenance. Using independent cohorts of MM patients, we identified that high expression of BLM is associated with a poor outcome with a significant enrichment in replication stress signature. We provide evidence that chemical inhibition of BLM by the small molecule ML216 in HMCLs (human myeloma cell lines) leads to cell cycle arrest and increases apoptosis, likely by accumulation of DNA damage. BLM inhibition synergizes with the alkylating agent melphalan to efficiently inhibit growth and promote cell death in HMCLs. Moreover, ML216 treatment re-sensitizes melphalan-resistant cell lines to this conventional therapeutic agent. Altogether, these data suggest that inhibition of BLM in combination with DNA damaging agents could be of therapeutic interest in the treatment of MM, especially in those patients with high BLM expression and/or resistance to melphalan

Enolase is regulated by Liver X Receptors.

International audienceEnolase is a glycolytic enzyme known to inhibit cholesteryl ester hydrolases (CEHs). Cholesteryl ester loading of macrophages, as occurs during atherosclerosis, is accompanied by increased Enolase protein and activity. Here, we describe that J774 macrophages treated with LXR agonists exhibit reduced Enolase transcript and protein abundance. Moreover, we show that this reduction is further potentiated by activation of the LXR/RXR heterodimer with the RXR ligand 9-cis retinoic acid. Enolase levels are also reduced in vivo following activation of LXRs in the intestine, but not in the liver. This effect is lost in Lxralphabeta-/- mice. In aggregate, our study identified Enolase as a new target of LXRs in vivo, which may promote cholesterol mobilization for subsequent efflux

ABCC6 Expression Is Regulated by CCAAT/Enhancer-Binding Protein Activating a Primate-Specific Sequence Located in the First Intron of the Gene

Pseudoxanthoma elasticum (PXE), a rare recessive genetic disease causing skin, eye, and cardiovascular lesions, is characterized by the calcification of elastic fibers. The disorder is due to loss-of-function mutations of the ABCC6 gene, but the pathophysiology of the disease is still not understood. Here we investigated the transcriptional regulation of the gene, using DNase I hypersensitivity assay followed by luciferase reporter gene assay. We identified three DNase I hypersensitive sites (HSs) specific to cell lines expressing ABCC6. These HSs are located in the proximal promoter and in the first intron of the gene. We further characterized the role of the HSs by luciferase assay and demonstrated the transcriptional activity of the intronic HS. We identified the CCAAT/enhancer-binding protein β (C/EBPβ) as a factor binding the second intronic HS by chromatin immunoprecipitation and corroborated this finding by luciferase assays. We also showed that C/EBPβ interacts with the proximal promoter of the gene. We propose that C/EBPβ forms a complex with other regulatory proteins including the previously identified regulatory factor hepatocyte nuclear factor 4α (HNF4α). This complex would account for the tissue-specific expression of the gene and might serve as a metabolic sensor. Our results point toward a better understanding of the physiological role of ABCC6

Lack of liver X receptors leads to cell proliferation in a model of mouse dorsal prostate epithelial cell.

Recent studies underline the implication of Liver X Receptors (LXRs) in several prostate diseases such as benign prostatic hyperplasia (BPH) and prostate cancer. In order to understand the molecular mechanisms involved, we derived epithelial cells from dorsal prostate (MPECs) of wild type (WT) or Lxrαβ-/- mice. In the WT MPECs, our results show that LXR activation reduces proliferation and correlates with the modification of the AKT-survival pathway. Moreover, LXRs regulate lipid homeostasis with the regulation of Abca1, Abcg1 and Idol, and, in a lesser extent, Srebp1, Fas and Acc. Conversely cells derived from Lxrαβ-/- mice show a higher basal phosphorylation and consequently activation of the survival/proliferation transduction pathways AKT and MAPK. Altogether, our data point out that the cell model we developed allows deciphering the molecular mechanisms inducing the cell cycle arrest. Besides, we show that activated LXRs regulate AKT and MAPK transduction pathways and demonstrate that LXRs could be good pharmacological targets in prostate disease such as cancer