Maf Proteins Research Articles

Epidermal structure and differentiation.

In this Primer, Moreci and Lechler follow the lifetime of an epidermal cell from its birth to its ultimate death, and detail how this journey is necessary for epidermal function.

The Critical Role of Bach2 in Shaping the Balance between CD4+ T Cell Subsets in Immune-Mediated Diseases.

The transcription factor Bach2 which is predominantly expressed in B and T lymphocytes represses the expression of genes by forming heterodimers with small Maf and Batf proteins and binding to the corresponding sequence on the DNA. In this way, Bach2 serves as a highly conserved repressor which controls the terminal differentiation and maturation of both B and T lymphocytes. It is required for class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes in activated B cells, and its function in B cell differentiation has been well-described. Furthermore, emerging data show that Bach2 regulates transcriptional activity in T cells at super enhancers or regions of high transcriptional activity, thus stabilizing immunoregulatory capacity and maintaining T cell homeostasis. Bach2 is also critical for the formation and function of CD4+ T cell lineages (Th1, Th2, Th9, Th17, T follicular helper (Tfh), and regulatory T (Treg) cells). Genetic variations within Bach2 locus are associated with numerous immune-mediated diseases including multiple sclerosis (MS), rheumatoid arthritis (RA), chronic pancreatitis (CP), type 2 chronic airway inflammation, inflammatory bowel disease (IBD), and type 1 diabetes. Here, we reveal a critical role of Bach2 in regulating T cell biology and the correlation with these immune-mediated diseases.

Cis-element architecture of Nrf2-sMaf heterodimer binding sites and its relation to diseases.

Cellular detoxication is essential for health because it provides protection against various chemicals and xenobiotics. The KEAP1-NRF2 system is important for cellular defense against oxidative and electrophilic stresses as NRF2 activates the transcription of an array of cytoprotective genes, including drug-metabolizing and antioxidant enzymes, in a stress-dependent manner. The CNC family of transcription factors, including NRF2, form heterodimers with small Maf (sMaf) proteins and bind to consensus DNA sequences that have been referred to as antioxidant response element, electrophile response element, or NF-E2-binding element. These sequences are now collectively called CNC-sMaf binding element (CsMBE). In addition to forming a heterodimer with CNC proteins, sMaf proteins can form homodimers and recognize regulatory motifs called Maf recognition element (MARE). Although the CsMBE sequence substantially overlaps with that of MARE, the sequences differ. NRF2 selectively recognizes CsMBE, which is critical for cytoprotection. Recent advances in high-throughput sequencing and population-scale genome analysis provide new insights into the transcriptional regulation involved in the stress response. The integration of a genome-wide map of NRF2 occupancy with disease-susceptibility loci reveals the associations between polymorphisms in CsMBE and disease risk, information useful for the personalized medicine of the future.

Expression and function of FRA1 protein in tumors.

AP-1 is a dimeric complex that is composed of JUN, FOS, ATF and MAF protein families. FOS-related antigen 1 (FRA1) which encoded by FOSL1 gene, belongs to the FOS protein family, and mainly forms an AP-1 complex with the protein of the JUN family to exert an effect. Regulation of FRA1 occurs at levels of transcription and post-translational modification, and phosphorylation is the major post-translational modification. FRA1 is mainly regulated by the mitogen-activated protein kinases signaling pathway and is degraded by ubiquitin-independent proteasomes. FRA1 can affect biological functions, such as tumor proliferation, differentiation, invasion and apoptosis. Studies have demonstrated that FRA1 is abnormally expressed in many tumors and plays a relevant role, but the specific condition varies from the target organs. FRA1 is overexpressed in breast cancer, lung cancer, colorectal cancer, prostate cancer, nasopharyngeal cancer, thyroid cancer and other tumors. However, the expression of FRA1 is decreased in cervical cancer, and the expression of FRA1 in ovarian cancer and oral squamous cell carcinoma is still controversial. In this review, we present a detailed description of the regulatory factors and functions of FRA1, also, the expression of FRA1 in various tumors and its function in relative tumor.

Direct and Specific Functional Evaluation of the Nrf2 and MafG Heterodimer by Introducing a Tethered Dimer into Small Maf-Deficient Cells.

A group of cytoprotective genes is regulated by heterodimers composed of the cap'n'collar (CNC) family member Nrf2 and one of the small Maf (sMaf) proteins (MafF, MafG, or MafK) through the antioxidant response element (ARE, also referred to as the CNC-sMaf binding element [CsMBE]). Many lines of evidence support this model; however, a direct and specific evaluation of the Nrf2-sMaf heterodimer remains to be executed. To address this issue, we constructed a tethered Nrf2-MafG (T-N2G) heterodimer using a flexible linker peptide. We then introduced the T-N2G construct into cells lacking all three sMaf proteins to specifically evaluate the function of the tethered heterodimer without interference from other endogenous CNC-sMaf heterodimers or sMaf homodimers. In response to an Nrf2 activator, diethyl maleate, the T-N2G protein can widely activate the target genes of Nrf2 but not those of Nrf1, such as proteasome subunit genes. Genome-wide binding analysis showed that the T-N2G protein preferentially bound to the CsMBE motifs in the regulatory regions of the Nrf2 target genes. These results provide direct evidence that the Nrf2-MafG heterodimer acts as a transcriptional activator of Nrf2-dependent genes and show that this assay system will be a powerful tool to specifically examine the function of other CNC-sMaf heterodimers.

The Oncogenic Action of NRF2 Depends on De-glycation by Fructosamine-3-Kinase.

The NRF2 transcription factor controls a cell stress program that is implicated in cancer and there is great interest in targeting NRF2 for therapy. We show that NRF2 activity depends on Fructosamine-3-kinase (FN3K)-a kinase that triggers protein de-glycation. In its absence, NRF2 is extensively glycated, unstable, and defective at binding to small MAF proteins and transcriptional activation. Moreover, the development of hepatocellular carcinoma triggered by MYC and Keap1 inactivation depends on FN3K invivo. N-acetyl cysteine treatment partially rescues the effects of FN3K loss on NRF2 driven tumor phenotypes indicating a key role for NRF2-mediated redox balance. Mass spectrometry reveals that other proteins undergo FN3K-sensitive glycation, including translation factors, heat shock proteins, and histones. How glycation affects their functions remains to be defined. In summary, our study reveals a surprising role for the glycation of cellular proteins and implicates FN3K as targetable modulator of NRF2 activity in cancer.

MAF proteins: a family of regulating and regulated molecules

The proteins of MAF family (the cellular counterpart of viral oncogene MAF, isolated from avian musculoaponeurotic fibrosarcoma) are transcription factors regulating gene expression. On the bases of the size, they are sub-grouped into two families: the “large” (L-MAFs: MAFA, MAFB, c-MAF, and Nrl) and the “small” (S-MAFs: MAFF, MAFK, and MAFG) MAF proteins (1).

PO-380 Epigenetically regulated MAF is a new potential tumour suppressor gene in LSCC

IntroductionLaryngeal cancer (LSCC) is one of the most common tumours among head and neck squamous cell carcinomas. Recently, analysis of the meaning of epigenetic alterations in LSCC development has gained momentum. In this field we have shown recurrent overexpression of the miR-1290 in LSCC. Therefore, in this study we aim at clarifying the oncomiR functionality of miR-1290 by analysing its interaction with the transcriptional modulator MAF – a putative suppressor gene.Material and methodsThe identification of miR-1290 overexpression in LSCC was based on global miRNA expression profiles (Agilent) of 16 LSCC cell lines and 3 non-tumour controls which were validated on 50 tumour samples by real-time qPCR (RT-qPCR) with LNA modified primers. Target genes of miR-1290 were selected by in silico analysis using miRDB (TS >90) and miRWALK (gene listed in at least 2 in 4 databases). The influence of miR-1290 on its putative target gene MAF was analysed by transient transfection of 2 LSCC cell lines using miRNA inhibitor and successive expression analysis of MAF in the transfected cells by RT-qPCR and Western blot. To confirm the downregulation of MAF in primary LSCC we conducted RT-qPCR on 22 tumour samples and 5 non-tumour controls. In addition, the nuclear MAF protein abundance was evaluated by immunohistochemistry on tissue microarray containing 128 LSCC tumour samples and 19 tumour free resection margins.Results and discussionsBased on the in sillico analysis MAF has been selected as a the putative miR-1290 target gene with high suppressor potential. Importantly, MAF is a transcription factor, involved in regulation of apoptosis and TP53 expression, processes deregulated in LSCC. Functional analyses, showed that inhibition of miR-1290 resulted in 3.47 fold increase in MAF mRNA expression and 50% increase of MAF protein abundance. Moreover, by using RT-qPCR we have shown recurrent MAF downregulation in LSCC tumour samples compared to non-tumour controls. In line with these findings we have indicated by immunohistochemical staining that 74/128 LSCC tumour samples were characterised by lack of nuclear expression of MAF in opposite to nuclear expression observed in 19/19 margins.ConclusionOur results demonstrate MAF as a new potential tumour suppressor gene epigenetically downregulated in LSCC by the overexpression of miR-1290. Further studies are required for fully understanding the role of MAF in LSCC pathogenesis.

Maf links Neuregulin1 signaling to cholesterol synthesis in myelinating Schwann cells.

Cholesterol is a major constituent of myelin membranes, which insulate axons and allow saltatory conduction. Therefore, Schwann cells, the myelinating glia of the peripheral nervous system, need to produce large amounts of cholesterol. Here, we define a crucial role of the transcription factor Maf in myelination and cholesterol biosynthesis and show that Maf acts downstream from Neuregulin1 (Nrg1). Maf expression is induced when Schwann cells begin myelination. Genetic ablation of Maf resulted in hypomyelination that resembled mice with defective Nrg1 signaling. Importantly, loss of Maf or Nrg1 signaling resulted in a down-regulation of the cholesterol synthesis program, and Maf directly binds to enhancers of cholesterol synthesis genes. Furthermore, we identified the molecular mechanisms by which Nrg1 signaling regulates Maf levels. Transcription of Maf depends on calmodulin-dependent kinases downstream from Nrg1, whereas Nrg1-MAPK signaling stabilizes Maf protein. Our results delineate a novel signaling cascade regulating cholesterol synthesis in myelinating Schwann cells.

Microcystin induction small Maf protein involve in transcriptional regulation of GST from freshwater mussel Cristaria plicata

The small Mafs, MafF, MafG and MafK play critical roles in morphogenesis and homeostasis through associating with Cap “n” Collar family of transcription factors. In this study, we tried to identify a small Maf protein in the freshwater mussel Cristaria plicata. The MafK cDNA of C. plicata, designated as CpMafK, was cloned from the hemocytes using degenerate primers by the rapid amplification of cDNA ends PCR. The full length cDNA of CpMafK is 2170 bp, which includes an open reading frame of 570 bp, encoding 189 amino acids. CpMafK possesses four conserved domains and shows a low level (54–63%) of sequence similarity to small Mafs from other species. The results of Real-time quantitative PCR revealed that CpMafK mRNA was constitutively expressed in tissues, and the highest expression level was in hepatopancreas. After microcystin challenge, the expression levels of CpMafK mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpMafK was cloned into the plasmid pET-32, and the recombinant protein was expressed in Escherichia coli BL21(DE3). CpMafK could combine to the promoters of CpGST1 and CpGST2 with high-affinity in vitro. Therefore, CpMafK could regulate the expression of detoxification.

Bach1: Function, Regulation, and Involvement in Disease.

The transcription factor BTB and CNC homology 1 (Bach1) is widely expressed in most mammalian tissues and functions primarily as a transcriptional suppressor by heterodimerizing with small Maf proteins and binding to Maf recognition elements in the promoters of targeted genes. It has a key regulatory role in the production of reactive oxygen species, cell cycle, heme homeostasis, hematopoiesis, and immunity and has been shown to suppress ischemic angiogenesis and promote breast cancer metastasis. This review summarizes how Bach1 controls these and other cellular and physiological and pathological processes. Bach1 expression and function differ between different cell types. Thus, therapies designed to manipulate Bach1 expression will need to be tightly controlled and tailored for each specific disease state or cell type.

A new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe

YhdE is a Maf (multicopy associated filamentation) proteins from Escherichia coli which exhibits pyrophosphatase activity towards selected nucleotides, although its catalytic mechanism remains unclear. Herein we used a novel fluorescence probe (4-isoACBA–Zn(II) complex) to characterize the enzymatic properties of YhdE and its mutant, establishing a new method for assaying pyrophosphatase catalytic function. Our results reveal for the first time that the new fluorescence sensor confers high sensitivity and specificity and pyrophosphate (PPi) is the direct catalytic product of YhdE. Crystal structures of a mutant in the active-site loop (YhdE_E33A) show conformational flexibility implicated in the catalytic mechanism of YhdE. ITC experiments and computational docking further reveal that Asp70 and substrate dTTP coordinate Mn2+. Quantum mechanics calculations indicate that YhdE hydrolysis appears to follow a stepwise pathway in which a water molecule first attacks the α-phosphorus atom in the substrate, followed by the release of PPi from the pentavalent intermediate.

The ubiquitin-conjugating enzyme UBE2O modulates c-Maf stability and induces myeloma cell apoptosis

BackgroundUBE2O is proposed as a ubiquitin-conjugating enzyme, but its function was largely unknown.MethodsMass spectrometry was applied to identify c-Maf ubiquitination-associated proteins. Immunoprecipitation was applied for c-Maf and UBE2O interaction. Immunoblotting was used for Maf protein stability. Luciferase assay was used for c-Maf transcriptional activity. Lentiviral infections were applied for UBE2O function in multiple myeloma (MM) cells. Flow cytometry and nude mice xenografts were applied for MM cell apoptosis and tumor growth assay, respectively.ResultsUBE2O was found to interact with c-Maf, a critical transcription factor in MM, by the affinity purification/tandem mass spectrometry assay and co-immunoprecipitation assays. Subsequent studies showed that UBE2O mediated c-Maf polyubiquitination and degradation. Moreover, UBE2O downregulated the transcriptional activity of c-Maf and the expression of cyclin D2, a typical gene modulated by c-Maf. DNA microarray revealed that UBE2O was expressed in normal bone marrow cells but downregulated in MGUS, smoldering MM and MM cells, which was confirmed by RT-PCR in primary MM cells, suggesting its potential role in myeloma pathophysiology. When UBE2O was restored, c-Maf protein in MM cells was significantly decreased and MM cells underwent apoptosis. Furthermore, the human MM xenograft in nude mice showed that re-expression of UBE2O delayed the growth of myeloma xenografts in nude mice in association with c-Maf downregulation and activation of the apoptotic pathway.ConclusionsUBE2O mediates c-Maf polyubiquitination and degradation, induces MM cell apoptosis, and suppresses myeloma tumor growth, which provides a novel insight in understanding myelomagenesis and UBE2O biology.

MOLECULAR MECHANISMS OF β-AMYLOID-INDUCED INHIBITION OF GLUTATHIONE SYNTHESIS IN RATS

Oxidative stress is one of the main pathogenic mechanisms of Alzheimer's disease (AD). Glutathione (GSH) is an endogenous antioxidant protecting cells from oxidative injury. However, it is reported that the content of GSH is lower both in the brain of AD patients and the AD animal models. The reason why GSH content is lower in such an oxidative environment of AD brain is not clear. Glutamatecysteine ligase (GCL), composed of a catalytic (GCLC) and a modifier (GCLM) subunit, is the rate-limiting enzyme in GSH synthesis. The transcription of both GCLC and GCLM are regulated by Keap1-Nrf2-ARE signal pathway. Meanwhile, as a chaperone protein of Nrf2, small Maf proteins form heterodimers with Nrf2 leading to activation or repression of ARE-mediated gene expression. The current study aimed at investigating the molecular mechanisms of regulation on GCL transcription by amyloid-β (Aβ) involving Keap1-Nrf2-ARE signal pathway. Aβ intrahippocampal injection rat model and human neuroblastoma SH-SY5Y cells treated with Aβ were used. The expressions of Nrf2, GCLC, GCLM and small Maf proteins as well as GSH level under Aβ insult at different time point were tested. Overexpression and knockdown of Maf proteins were used to explore the roles of Maf proteins on GCL expression. The banding of Keap1 and Nrf2 was examined by CoIP. The expressions of GCLC, GCLM and GSH level were increased in acute stage of Aβ treatment (within 6h), while the expressions of GCLC, GCLM and GSH level were decreased accompanying with activation of Keap1-Nrf2-ARE signal pathway after Aβ treatment for 6–24h. The nuclear level of Nrf2 restored to normal level accompanying with down regulation of GCL and low GSH in the late stage of Aβ treatment. The nuclear expressions of MafG and MafK were largely induced and displacement of Nrf2 nuclear binding to ARE in the late stage of Aβ treatment, while, MafG or MafK knockdown restored GCLC expression and GSH level. Meanwhile, the banding of Keap1 and Nrf2 were enhanced with decreased expression of p-Nrf2 Ser40. Aβ-mediated inhibition of Keap1-Nrf2-ARE signal pathway and induction of nuclear Maf proteins contributed to lowering GSH content due to inhibition of GCL expression.

Genetic polymorphisms of MAFK, encoding a small Maf protein, are associated with susceptibility to ulcerative colitis in Japan.

AIMTo investigate whether single nucleotide polymorphisms in maf protein K (MAFK), which encodes the MAFK, lead to increased susceptibility to ulcerative colitis in the Japanese population.METHODSThis case control study examined the associations between MAFK single nucleotide polymorphisms (rs4268033 G>A, rs3735656 T>C and rs10226620 C>T) and ulcerative colitis susceptibility in 174 patients with ulcerative colitis (UC) cases, and 748 subjects without no lower abdominal symptoms, diarrhea or hematochezia (controls). In addition, as the second controls, we set 360 subjects, who have an irregular bowel movement without abnormal lower endoscopic findings (IBM controls).RESULTSThe genotype frequency of rs4268033 AA and allelic frequency of the rs4268033A allele were significantly higher in the UC cases than in both controls (P = 0.0005 and < 0.0001, P = 0.015 and 0.0027 vs controls and IBM controls, respectively). Logistic regression analysis after adjustment for age and gender showed that the rs4268033 AA and rs3735656 CC genotypes were significantly associated with susceptibility to UC development (OR = 2.63, 95%CI: 1.61-4.30, P = 0.0001 and OR = 1.81; 95%CI: 1.12-2.94, P = 0.015, respectively). Similar findings were observed by the comparison with IBM controls. In addition, the rs4268033 AA genotype was significantly associated with all phenotypes of UC except early onset. There was no significant association between rs10226620 and ulcerative colitis.CONCLUSIONOur results provide the first evidence that MAFK genetic polymorphisms are significantly associated with susceptibility to UC development. In particular, rs4268033 is closely associated with an increased risk for the development of UC.

MiR-128 Is Implicated in Stress Responses by Targeting MAFG in Skeletal Muscle Cells.

MAFG (v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog G) is a bZIP-type transcriptional regulator that belongs to the small MAF (sMAFs) protein family. By interacting with other bZIP transcription factors, sMAFs can form homo- and heterodimers governing either repressive or activating transcriptional functions. As heterodimeric partner of Nrf2, MAFG positively influences the ARE-dependent antioxidant/xenobiotic pathways, at least in condition of a correct MAFG:Nrf2 balance. MicroRNAs (miRs) participate to different regulatory networks being involved as fine-tuning regulators of gene expression. However, the connections between cellular surveillance to stresses mediated by MAFG:Nrf2 and miR regulations are not well understood. Here, we explored the impact of miR-128 in expression of genes related to stress response. Bioinformatic predictions coupled with functional analysis revealed the presence of miR-128 binding site in the 3′UTR of MAFG. Ectopic miR-128 expression correlated with reduced expression of endogenous MAFG-dependent genes and negatively affected ARE-mediated molecular phenotype based on Nrf2 activity. Indeed, miR-128 impairs redox-dependent pathways induced in response to oxidative stress. Moreover, in condition of hypoxia, MAFG induction correlated with reduced levels of miR-128. This lead to increased mRNA levels of HMOX-1 and x-CT for blunting stress. Overall, these findings identify MAFG as novel direct target of miR-128.

MAF protein mediates innate resistance to proteasome inhibition therapy in multiple myeloma

AbstractMultiple myeloma (MM) patients with the t(14;16) translocation have a poor prognosis, and unlike other molecular subgroups, their outcome has not improved with the introduction of bortezomib (Bzb). The mechanism underlying innate resistance of MM to Bzb is unknown. In the present study, we have investigated how MAF overexpression impacts resistance to proteasome inhibitor (PI) therapy (Bzb and carfilzomib). High levels of MAF protein were found in t(14;16) cell lines; cell lines from the t(4;14) subgroup had intermediate levels, whereas cell lines from the other subgroups had low levels. High expression of MAF protein in t(14;16) was associated with significantly higher PI half-maximum inhibitory concentration values compared with other molecular subgroups. PI exposure abrogated glycogen synthase kinase 3β (GSK3β)-mediated degradation of MAF protein, resulting in increased MAF protein stability and PI resistance. Subsequent studies using loss-of-function and gain-of-function models showed that silencing MAF led to increased sensitivity to PIs, enhanced apoptosis, and activation of caspase-3, -7, -8, -9, poly (ADP-ribose) polymerase, and lamin A/C. In contrast, overexpression of MAF resulted in increased resistance to PIs and reduced apoptosis. These results define the role of MAF and GSK3 in the resistance of t(14;16) MM to PIs and identifies a novel mechanism by which MAF protein levels are regulated by PIs, which in turn confers resistance to PIs.

The Co-Occurrence of MAF Translocations in RAS Mutated Multiple Myeloma Confers Resistance to MEK Inhibition

IntroductionMutant NRAS and KRAS lead to the activation of the RAS/RAF/MEK/ERK pathway in approximately 50% of multiple myeloma (MM). Blocking this pathway with MEK1/2 inhibitors (MEKi) such as trametinib (Tram) is a therapeutic option but the response rate in MM varies between 30-50% (Heuck et al, Leukemia 2015). In MM it is unknown whether RAS mutation status correlates with sensitivity to Tram. The purpose of this study was to characterize factors which predict response to Tram and to identify mechanism mediating resistance.MethodsWe established the IC50 of Tram using MTT assays in 32 MM cell lines (HMCL) including 16 RAS mutant positive (RASm+), and 15 wildtype RAS (RASm-), and 1 BRAF mutant (BRAFm+) line which acted as a positive control. HMCLs were classified according to the IC50 value as sensitive (<0.05μM); intermediate sensitive (IMS) (0.05 and 10μM); and resistant (>10μM). All lines underwent immunoblotting for pERK at baseline and following treatment with serial concentrations of Tram to identify correlation of activation with sensitivity. BrdU incorporation analyzed by FACS was performed to determine the molecular action of Tram. A lentiviral mediated expression system was used to engineer a MAF overexpressing cell line in a RASm+ HMCL lacking MAF (MMRASm+MAF) and silencing MAF in two lines with co-occurring MAF and RASm+ (MMRASm+shMAF). The clinical characteristics of 84 relapsed RASm+ patients who received Tram either as a single agent or in combination with other anti-MM therapies were also examined.Results6/16 (37.5%) of RASm+ HMCLs were sensitive to Tram, 5/16 (31.1%) were IMS and 5/16 (31.1%) resistant. There was no difference in sensitivity to Tram between KRASm+ (IC50 = 9.5μM, n = 11) and NRASm+ (IC50 =12.5μM, n=4, p=0.65). In contrast, 13/15 (87%) RASm- HMCLs were resistant to Tram. Mechanistically, Tram blocked cell cycle progression in Tram-sensitive RASm+ cells with an increase in G0/G1 phase (22.25%) and a decrease in S phase (16.76%) compared with untreated controls (p<0.01). These in-vitro findings correlated with our clinical experience where there was no difference in progression free or overall survival (OS) between KRASm+ (n=46) or NRASm+ (n=38) patients treated with Tram.Given that RASm+ lines exhibit a range of sensitivity (IC50 0.005-30μM), we further defined additional molecular modifiers that influence sensitivity by comparing KRASm+ alone with KRASm+ co-occurring with t(14;16) (n=4) or t(4;14) (n=3). The mean IC50 value of Tram (0.025μM) in 5 RASm+ was significantly lower than those with a co-occurring t(14;16) (IC50 =16μM; P<0.001) or a t(4;14) (IC50 =11.6μM, P<0.05). Among 15 RASm- HMCLs, the IC50 (37.5μM, n=9) of Tram was much higher in t(14;16) HMCL than in t(4;14) (IC50=9.16μM, n=3, P<0.01). A similar pattern was seen in patients, with a shorter OS when RASm+ co-occurred with t(14;16) or t(4;14) (n=17 OS=0.99yrs) than those with RASm+ alone (n= 67, OS=1.85yrs, P=0.01). These findings provide evidence that co-occurring MAF expression renders RAS+ MM less sensitive to MAKP inhibition.To determine if activation of MAPK pathway caused by RAS mutation was associated with sensitivity to MEKi, we assess baseline and post Tram treatment levels of p-ERK1/2, a downstream substrate of MEK1/2 activation. MM HMCLs displayed variable basal levels of pERK. There was no correlation between basal levels of pERK and RASm+, with the highest baseline levels of pERK being seen in HMCLs with a t(14;16) or t(14;20) and wildtype RAS, intermediate levels in RASm+ with a t(14;16) and lowest in RASm+ alone. The concentrations of Tram for complete inhibition of pERK were higher in HMCLs with co-occurring t(14;16) ( >or = 500 nM) than for RASm+ alone.To further confirm the ability of MAF to confer resistance of RASm+ cells to MEKi, we examined the sensitivity of MMRASm+/MAF to Tram.MTT assay showed that the IC50 of MMRASm+/MAF cells was 50% higher than that of MMEV cells (P <0.001). Similar results were observed in two other HMCLs with transient ectopic expression of MAF indicating that increase in MAF protein in RAS mutant cells reduces sensitivity to MEKi. In contrast, silencing of MAF in two RASm+ HMCLs significantly increased sensitivity (75% decrease in IC50) to Tram, compared with MMRASm+shCont (P<0.001).ConclusionOur results demonstrate that RASm+ MM is sensitive to Tram but the co-occurrence of t(14;16) leading to MAF overexpression confers resistance both in-vitro and in patients. DisclosuresMorgan:Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding. Davies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

83 - Nuclear Factor (Erythroid-Derived 2)-Like-1 (NFE2L1): At the Crossroads of Stress Responses

The Antioxidant Response Element (ARE, also known as the Electrophile Response Element (EpRE)) activates gene expression in response to various stresses including oxidative stress, xenobiotic stress and inflammation. The factors that bind to AREs/EpREs include the Nuclear Factor (Erythroid-derived 2)-Like proteins (NFE2L or Nrf) and the small Maf proteins. Nrf is a member of the Cap’n’Collar (CNC) family of basic-region leucine zipper (bZIP) transcription factors (Nrf1, Nrf2 and Nrf3) and activate gene expression from AREs/EpREs found in the promoter and enhancer regions of the genes for antioxidant enzymes, enzymes of xenobiotic metabolism and inflammatory factors. The Nrf proteins also regulate expression of subunits of the proteasome, and in this manner, may autoregulate their own levels. In comparison to Nrf2, there are relatively fewer studies on Nrf1. This may be due to a) their very different mechanisms by which they are regulated, b) the knockout mouse for Nrf2 is completely viable, whereas the knockout mouse for Nrf1 is embryonic lethal and c) their different locations within the cell (Nrf2 is cytosolic, whereas Nrf1 is bound to the endoplasmic reticulum (ER)). Our research focuses on Nrf1 and its responses to various stresses (oxidative stress, hypoxia, ER stress and toxin stresses). Here we show evidence of “cross-talk” between these stresses through effects upon Nrf1 function. Specifically, ER stress impinges upon the response of Nrf1 to other types of stresses. The functional units of Nrf1, and the interacting proteins, that may be involved in the regulation of the response of Nrf1 to multiple stresses will be outlined. The implications of such stress cross-talk, in the response to the individual stresses, will be discussed.

Small Maf deficiency recapitulates the liver phenotypes of Nrf1- and Nrf2-deficient mice.

Nrf1 and Nrf2 (NF-E2-related factors 1 and 2, respectively) are transcription factors that belong to the Cap'n'collar (CNC) family and play critical roles in various tissues, including the liver. Liver-specific Nrf1 knockout mice show hepatic steatosis, accompanied by dysregulation of various metabolic genes. Nrf2 knockout mice show impairment in the induction of antioxidant and xenobiotic-metabolizing enzyme genes. Although it has been shown that small Maf (sMaf) proteins act as obligatory partners of CNC proteins, their precise contributions to the function of CNC proteins remain unclear especially in the context of adult liver functions. To address this issue, we generated mice that conditionally lack expression of all sMaf proteins in the liver. The liver-specific sMaf-deficient mice develop hepatic steatosis and dysregulation of genes involved in lipid and amino acid metabolism and proteasomal subunit expression. Importantly, the gene expression profiles in the sMaf-deficient livers share a strong similarity with those in Nrf1-deficient livers. In addition, the basal expression levels of a number of Nrf2 target genes were diminished in the sMaf-deficient livers. These results provide the first genetic evidence that sMaf proteins are indispensable for liver functions as heterodimeric partners for Nrf1 and Nrf2.