Tumor immune microenvironment permissive to metastatic progression of ING4-deficient breast cancer.

Deficiencies in the ING4 tumor suppressor are associated with advanced stage tumors and poor patient survival in cancer. ING4 was shown to inhibit NF-kB in several cancers. As NF-kB is a key mediator of immune response, the ING4/NF-kB axis is likely to manifest in tumor-immune modulation but has not been investigated. To characterize the tumor immune microenvironment associated with ING4-deficient tumors, three approaches were employed in this study: First, tissue microarrays composed of 246 primary breast tumors including 97 ING4-deficient tumors were evaluated for the presence of selective immune markers, CD68, CD4, CD8, and PD-1, using immunohistochemical staining. Second, an immune-competent mouse model of ING4-deficient breast cancer was devised utilizing CRISPR-mediated deletion of Ing4 in a Tp53 deletion-derived mammary tumor cell line; mammary tumors were evaluated for immune markers using flow cytometry. Lastly, the METABRIC gene expression dataset was evaluated for patient survival related to the immune markers associated with Ing4-deleted tumors. The results showed that CD68, CD4, CD8, or PD-1, was not significantly associated with ING4-deficient breast tumors, indicating no enrichment of macrophages, T cells, or exhausted T cell types. In mice, Ing4-deleted mammary tumors had a growth rate comparable to Ing4-intact tumors but showed increased tumor penetrance and metastasis. Immune marker analyses of Ing4-deleted tumors revealed a significant increase in tumor-associated macrophages (Gr-1loCD11b+F4/80+) and a decrease in granzyme B-positive (GzmB+) CD4+ T cells, indicating a suppressive and/or less tumoricidal immune microenvironment. The METABRIC data analyses showed that low expression of GZMB was significantly associated with poor patient survival, as was ING4-low expression, in the basal subtype of breast cancer. Patients with GZMB-low/ING4-low tumors had the worst survival outcomes (HR = 2.80, 95% CI 1.36-5.75, p = 0.0004), supportive of the idea that the GZMB-low immune environment contributes to ING4-deficient tumor progression. Collectively, the study results demonstrate that ING4-deficient tumors harbor a microenvironment that contributes to immune evasion and metastasis.

Human translesion DNA polymerases ι and κ mediate tolerance to temozolomide in MGMT-deficient glioblastoma cells.

Glioblastoma (GBM) is a highly aggressive brain tumor associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing temozolomide (TMZ). Resistance to TMZ is, however, a major challenge. Previous work from our group has identified candidate genes linked to TMZ resistance, including genes encoding translesion synthesis (TLS) DNA polymerases iota (PolÉ©) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of PolÉ© and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding PolÉ© and Polκ, respectively, and assess their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant decrease in viability following TMZ treatment compared to parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed increased cell cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction of γH2AX foci and flow cytometry data. This implies that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (double-strand break repair pathway inhibitor) further reduced the cell viability and increased DNA damage and γH2AX positive cells in TLS KO cells, but not in parental cells. These findings underscore the crucial roles of PolÉ© and Polκ in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. Targeting these TLS enzymes, along with double-strand break DNA repair inhibition, could, therefore, provide a promising strategy to enhance TMZ's effectiveness in treating GBM.

Cilia-deficient renal tubule cells are primed for injury with mitochondrial defects and aberrant tryptophan metabolism.

The exocyst and Ift88 are necessary for primary ciliogenesis. Overexpression of Exoc5 (OE), a central exocyst component, resulted in longer cilia and enhanced injury recovery. Mitochondria are involved in acute kidney injury (AKI). To investigate cilia and mitochondria, basal respiration and mitochondrial maximal and spare respiratory capacity were measured in Exoc5 OE, Exoc5 knockdown (KD), Exoc5 ciliary targeting sequence mutant (CTS-mut), control Madin-Darby canine kidney (MDCK), Ift88 knockout (KO), and Ift88 rescue cells. In Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells, these parameters were decreased. In Exoc5 OE and Ift88 rescue cells they were increased. Reactive oxygen species were higher in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells compared with Exoc5 OE, control, and Ift88 rescue cells. By electron microscopy, mitochondria appeared abnormal in Exoc5 KD, Exoc5 CTS-mut, and Ift88 KO cells. A metabolomics screen of control, Exoc5 KD, Exoc5 CTS-mut, Exoc5 OE, Ift88 KO, and Ift88 rescue cells showed a marked increase in tryptophan levels in Exoc5 CTS-mut (113-fold) and Exoc5 KD (58-fold) compared with control cells. A 21% increase was seen in Ift88 KO compared with rescue cells. In Exoc5 OE compared with control cells, tryptophan was decreased 59%. To determine the effects of ciliary loss on AKI, we generated proximal tubule-specific Exoc5 and Ift88 KO mice. These mice had loss of primary cilia, decreased mitochondrial ATP synthase, and increased tryptophan in proximal tubules with greater injury following ischemia-reperfusion. These data indicate that cilia-deficient renal tubule cells are primed for injury with mitochondrial defects in tryptophan metabolism.NEW & NOTEWORTHY Mitochondria are centrally involved in acute kidney injury (AKI). Here, we show that cilia-deficient renal tubule cells both in vitro in cell culture and in vivo in mice are primed for injury with mitochondrial defects and aberrant tryptophan metabolism. These data suggest therapeutic strategies such as enhancing ciliogenesis or improving mitochondrial function to protect patients at risk for AKI.

Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF.

Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1-3. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.

Combination of EZH2 and ATM inhibition in BAP1-deficient mesothelioma.

BACKGROUND: More than half of mesothelioma tumours show alterations in the tumour suppressor gene BAP1. BAP1-deficient mesothelioma is shown to be sensitive to EZH2 inhibition in preclinical settings but only showed modest efficacy in clinical trial. Adding a second inhibitor could potentially elevate EZH2i treatment efficacy while preventing acquired resistance at the same time. METHODS: A focused drug synergy screen consisting of 20 drugs was performed by combining EZH2 inhibition with a panel of anti-cancer compounds in mesothelioma cell lines. The compounds used are under preclinical investigation or already used in the clinic. The synergistic potential of the combinations was assessed by using the Bliss model. To validate our findings, in vivo xenograft experiments were performed. RESULTS: Combining EZH2i with ATMi was found to have synergistic potential against BAP1-deficient mesothelioma in our drug screen, which was validated in clonogenicity assays. Tumour growth inhibition potential was significantly increased in BAP1-deficient xenografts. In addition, we observe lower ATM levels upon depletion of BAP1 and hypothesise that this might be mediated by E2F1. CONCLUSIONS: We demonstrated the efficacy of the combination of ATM and EZH2 inhibition against BAP1-deficient mesothelioma in preclinical models, indicating the potential of this combination as a novel treatment modality using BAP1 as a biomarker.

A de novo paradigm for male infertility.

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10-5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10-4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder.

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.

Cxcl10 Chemokine Induces Migration of ING4-Deficient Breast Cancer Cells via a Novel Cross Talk Mechanism between the Cxcr3 and Egfr Receptors.

The chemokine Cxcl10 has been associated with poor prognosis in breast cancer, but the mechanism is not well understood. Our previous study has shown that CXCL10 was repressed by the ING4 tumor suppressor, suggesting a potential inverse functional relationship. We thus investigated a role for Cxcl10 in the context of ING4 deficiencies in breast cancer. We first analyzed public gene expression data sets and found that patients with CXCL10-high/ING4-low expressing tumors had significantly reduced disease-free survival in breast cancer. In vitro, Cxcl10 induced migration of ING4-deleted breast cancer cells but not of ING4-intact cells. Using inhibitors, we found that Cxcl10-induced migration of ING4-deleted cells required Cxcr3, Egfr, and the Gßγ subunits downstream of Cxcr3 but not Gαi. Immunofluorescent imaging showed that Cxcl10 induced early transient colocalization between Cxcr3 and Egfr in both ING4-intact and ING4-deleted cells, which recurred only in ING4-deleted cells. A peptide agent that binds to the internal juxtamembrane domain of Egfr inhibited Cxcr3/Egfr colocalization and cell migration. Taken together, these results presented a novel mechanism of Cxcl10 that elicits migration of ING4-deleted cells, in part by inducing a physical or proximal association between Cxcr3 and Egfr and signaling downstream via Gßγ. These results further indicated that ING4 plays a critical role in the regulation of Cxcl10 signaling that enables breast cancer progression.

Multiomic identification of factors associated with progression to cystic kidney disease in mice with nephron Ift88 disruption.

Ift88 gene mutations cause primary cilia loss and polycystic kidney disease (PKD) in mice. Nephron intraflagellar transport protein 88 (Ift88) knockout (KO) at 2 mo postnatal does not affect renal histology at 4 mo postnatal and causes PKD only in males by 11 mo postnatal. To identify factors associated with PKD development, kidneys from 4-mo-old male and female control and Ift88 KO mice underwent transcriptomic, proteomic, Western blot, metabolomic, and lipidomic analyses. mRNAs involved in extracellular matrix (ECM) synthesis and degradation were selectively upregulated in male KO mice. Proteomic analysis was insufficiently sensitive to detect most ECM components, while Western blot analysis paradoxically revealed reduced fibronectin and collagen type I in male KO mice. Only male KO mice had upregulated mRNAs encoding fibrinogen subunits and receptors for vascular endothelial growth factor and platelet-derived growth factor; period 2, period 3, and nuclear receptor subfamily 1 group D member 1 clock mRNAs were selectively decreased in male KO mice. Proteomic, metabolomic, and lipidomic analyses detected a relative (vs. the same-sex control) decrease in factors involved in fatty acid ß-oxidation in female KO mice, while increased or unchanged levels in male KO mice, including medium-chain acyl-CoA dehydrogenase, 3-hydroxybutyrate, and acylcarnitine. Three putative mRNA biomarkers of cystogenesis in male Ift88 KO mice (similar control levels between sexes and uniquely altered by KO in males) were identified, including high levels (fibrinogen α-chain and stromal cell-derived factor 2-like 1) and low levels (BTG3-associated nuclear protein) in male KO mice. These findings suggest that relative alterations in renal ECM metabolism, fatty acid ß-oxidation, and other pathways precede cystogenesis in Ift88 KO mice. In addition, potential novel biomarkers of cystogenesis in Ift88 KO mice have been identified.NEW & NOTEWORTHY Male, but not female, mice with nephron intraflagellar transport protein 88 (Ift88) gene knockout (KO) develop polycystic kidneys by ∼1 yr postnatal. We performed multiomic analysis of precystic male and female Ift88 KO and control kidneys. Precystic male Ift88 KO mice exhibited differential alterations (vs. females) in mRNA, proteins, metabolites, and/or lipids associated with renal extracellular matrix metabolism, fatty acid ß-oxidation, circadian rhythm, and other pathways. These findings suggest targets for evaluation in the pathogenesis of Ift88 KO polycystic kidneys.

Loss of hepatic Flcn protects against fibrosis and inflammation by activating autophagy pathways.

Non-alcoholic fatty liver disease (NAFLD) is the most frequent liver disease worldwide and can progress to non-alcoholic steatohepatitis (NASH), which is characterized by triglyceride accumulation, inflammation, and fibrosis. No pharmacological agents are currently approved to treat these conditions, but it is clear now that modulation of lipid synthesis and autophagy are key biological mechanisms that could help reduce or prevent these liver diseases. The folliculin (FLCN) protein has been recently identified as a central regulatory node governing whole body energy homeostasis, and we hypothesized that FLCN regulates highly metabolic tissues like the liver. We thus generated a liver specific Flcn knockout mouse model to study its role in liver disease progression. Using the methionine- and choline-deficient diet to mimic liver fibrosis, we demonstrate that loss of Flcn reduced triglyceride accumulation, fibrosis, and inflammation in mice. In this aggressive liver disease setting, loss of Flcn led to activation of transcription factors TFEB and TFE3 to promote autophagy, promoting the degradation of intracellular lipid stores, ultimately resulting in reduced hepatocellular damage and inflammation. Hence, the activity of FLCN could be a promising target for small molecule drugs to treat liver fibrosis by specifically activating autophagy. Collectively, these results show an unexpected role for Flcn in fatty liver disease progression and highlight new potential treatment strategies.

PinX1 Depletion Improves Liver Injury in a Mouse Model of Nonalcoholic Fatty Liver Disease via Increasing Telomerase Activity and Inhibiting Apoptosis.

PIN2/TRF1-interacting telomerase inhibitor 1 (PinX1) can inhibit tumor growth by inhibiting telomerase activity. However, only few studies investigated the expression and function of PinX1 in nonalcoholic fatty liver disease (NAFLD). Thus, here we aimed to explore the roles of PinX1 in high-fat diet (HFD)-induced NAFLD in mice and in isolated hepatocytes. The mRNA expression of PinX1 and mTERT as well as telomere length were analyzed by RT-PCR. Pathological changes were detected by HE staining and oil red O staining. Triglyceride, cholesterol, alanine aminotransferase, aspartic aminotransferase, and telomerase activity were detected by ELISA. Hepatocyte apoptosis was determined by TUNEL and flow cytometry, and protein expression was analyzed by western blotting. We found that the expression of PinX1 was upregulated in the HFD group compared with the WT group. PinX1 knockout improved HFD-induced liver injury in mice and exhibited less lipid accumulation in hepatocytes. Moreover, telomere length, telomerase activity, and mTERT expression were significantly reduced in liver tissues of HFD-induced mice and palmitic acid-induced hepatocytes, while PinX1 knockout attenuated the effect. Furthermore, HFD-induced PinX1-/- mice exhibited less hepatocyte apoptosis than HFD-induced WT mice. Besides, PinX1 knockout inhibited the increase of cleaved caspase-3 and cleaved PARP expression in vivo and in vitro. Moreover, inhibition of mTERT reversed the effect of PinX1 knockout in hepatocytes. Taken together, our findings indicate that PinX1 promotes hepatocyte apoptosis and lipid accumulation by decreasing telomere length and telomerase activity in the development of NAFLD. PinX1 might be a target for the treatment of NAFLD.

Pyrimethamine Modulates Interplay between Apoptosis and Autophagy in Chronic Myelogenous Leukemia Cells.

Pyrimethamine (Pyri) is being used in combination with other medications to treat serious parasitic infections of the body, brain, or eye and to also reduce toxoplasmosis infection in the patients with HIV infection. Additionally, Pyri can display significant anti-cancer potential in different tumor models, but the possible mode of its actions remains unclear. Hence, in this study, the possible anti-tumoral impact of Pyri on human chronic myeloid leukemia (CML) was deciphered. Pyri inhibited cell growth in various types of tumor cells and exhibited a marked inhibitory action on CML cells. In addition to apoptosis, Pyri also triggered sustained autophagy. Targeted inhibition of autophagy sensitized the tumor cells to Pyri-induced apoptotic cell death. Moreover, the activation of signal transducer and activator of transcription 5 (STAT5) and its downstream target gene Bcl-2 was attenuated by Pyri. Accordingly, small interfering RNA (siRNA)-mediated STAT5 knockdown augmented Pyri-induced autophagy and apoptosis and promoted the suppressive action of Pyri on cell viability. Moreover, ectopic overexpression of Bcl-2 protected the cells from Pyri-mediated autophagy and apoptosis. Overall, the data indicated that the attenuation of STAT5-Bcl-2 cascade by Pyri can regulate its growth inhibitory properties by simultaneously targeting both apoptosis and autophagy cell death mechanism(s).

IFT88 deficiency in proximal tubular cells exaggerates cisplatin-induced injury by suppressing autophagy.

Primary cilia are widely regarded as specialized sensors in differentiated cells that have been implicated in the regulation of cell proliferation, differentiation, and viability. We have previously shown that shortening of primary cilia sensitizes cultured kidney tubular cells to cisplatin-induced apoptosis. Intraflagellar transport 88 (IFT88) is an essential component for ciliogenesis and maintenance. Here, we have further examined the effect of proximal tubule-specific IFT88 ablation on cisplatin-induced acute kidney injury (AKI). In this study, more severe AKI occurred in IFT88 knockout mice than age- and sex-matched wild-type mice. Mechanistically, cisplatin stimulated autophagy in kidney tubular cells as an intrinsic protective mechanism. However, renal autophagy was severely impaired in IFT88 knockout mice. In cultured HK-2 cells, cisplatin induced more apoptosis when IFT88 was knocked down. Tat-beclin 1 peptide, a specific autophagy activator, could partially prevent IFT88-associated cell death during cisplatin treatment, although cilium length was not improved significantly. Reexpression of IFT88 partially restored autophagy in IFT88 knockdown cells and suppressed apoptosis during cisplatin treatment. Taken together, these results indicate that defective autophagy in IFT88-deficient kidney cells and tissues contributes to the exaggerated AKI following cisplatin exposure.NEW & NOTEWORTHY Almost every cell has one hair-like, nonmotile antenna projecting from the cell surface, named the primary cilium. In kidney tubular cells, the primary cilium has a protective role, but the underlying mechanism is unclear. This study shows that a short cilium leads to the suppression of autophagy, which is responsible for the heightened injury sensitivity. These findings provide the clues of how to manipulate primary cilium and autophagy to save kidneys.

ZNF668 deficiency causes a recognizable disorder of DNA damage repair.

The purpose of this study is to describe a Mendelian disorder of DNA damage repair. Phenotypic delineation of two families, one new and one previously published, with overlapping dysmorphic and neurodevelopmental features was undertaken. Functional characterization of DNA damage repair in fibroblasts obtained from the index individuals in each of the two families was pursued. We present new evidence of a distinct disorder caused by biallelic truncating variants in ZNF668 comprising microcephaly, growth deficiency, severe global developmental delay, brain malformation, and distinct facial dysmorphism. DNA damage repair defect was observed in fibroblasts of affected individuals. ZNF668 deficiency in humans results in a recognizable autosomal recessive disorder, which we propose to name ZNF668-related ZMAND (ZNF668-related brain malformation, microcephaly, abnormal growth, neurodevelopmental delay, and dysmorphism). Our results add to the growing list of Mendelian disorders of the DNA damage repair machinery.

Role of BCLAF-1 in PD-L1 stabilization in response to ionizing irradiation.

Programmed cell death ligand 1 (PD-L1) is a major immunosuppressive checkpoint protein expressed by tumor cells to subvert anticancer immunity. Recent studies have shown that ionizing radiation (IR) upregulates the expression of PD-L1 in tumor cells. However, whether an IR-induced DNA damage response (DDR) directly regulates PD-L1 expression and the functional significance of its upregulation are not fully understood. Here, we show that IR-induced upregulation of PD-L1 expression proceeds through both transcriptional and post-translational mechanisms. Upregulated PD-L1 was predominantly present on the cell membrane, resulting in T-cell apoptosis in a co-culture system. Using mass spectrometry, we identified PD-L1 interacting proteins and found that BCLAF1 (Bcl2 associated transcription factor 1) is an important regulator of PD-L1 in response to IR. BCLAF1 depletion decreased PD-L1 expression by promoting the ubiquitination of PD-L1. In addition, we show that CMTM6 is upregulated in response to IR and participates in BCLAF1-dependent PD-L1 upregulation. Finally, we demonstrated that the ATM/BCLAF1/PD-L1 axis regulated PD-L1 stabilization in response to IR. Together, our findings reveal a novel regulatory mechanism of PD-L1 expression in the DDR.

Should temozolomide be used on the basis of O6-methylguanine DNA methyltransferase status in patients with advanced neuroendocrine tumors? A systematic review and meta-analysis.

BACKGROUND: Temozolomide (TEM) is an active treatment in metastatic neuroendocrine tumors (NETs). Patients affected by glioblastoma multiforme or advanced melanoma treated with TEM who have deficiency of O6-methylguanine DNA methyltransferase (MGMT) have a better responses and survival. However, the predictive role of MGMT in patients with NETs treated with TEM is still debated. METHODS: We conducted a systematic review of the literature and meta-analysis, based on PRISMA methodology, searching in the main databases (PubMed, Embase, Scopus, Web of Science, Cochrane Library and clinical trial.gov) and the proceedings of the main international congresses, until April 26, 2021. RESULTS: Twelve out of 616 articles were selected for our analysis, regarding a total of 858 NET patients treated with TEM-based chemotherapy. The status of MGMT had been tested in 513 (60%) patients, using various methods. The pooled overall response rate (ORR) was higher in MGMT-deficient compared with MGMT-proficient NETs, with a risk difference of 0.31 (95% confidence interval, CI: 0.13-0.50; p < 0.001; I2: 73%) and risk ratio of 2.29 (95% CI: 1.34-3.91; p < 0.001; I2: 55%). The pooled progression free survival (PFS) (hazard ratio, HR = 0.56; 95% CI: 0.43-0.74; p < 0.001) and overall survival (OS) (HR = 0.41; 95% CI: 0.20-0.62; p = 0.011) were longer in MGMT-deficient versus MGMT-proficient NETs. CONCLUSIONS: Our meta-analysis suggested that MGMT status may be predictive of TEM efficacy. However, due to the high heterogeneity of the evaluated studies the risk of biases should be considered. On this hypothesis future homogeneous prospective studies are warranted.

Resistance to Targeted Therapy and RASSF1A Loss in Melanoma: What Are We Missing?

Melanoma is one of the most aggressive forms of skin cancer and is therapeutically challenging, considering its high mutation rate. Following the development of therapies to target BRAF, the most frequently found mutation in melanoma, promising therapeutic responses were observed. While mono- and combination therapies to target the MAPK cascade did induce a therapeutic response in BRAF-mutated melanomas, the development of resistance to MAPK-targeted therapies remains a challenge for a high proportion of patients. Resistance mechanisms are varied and can be categorised as intrinsic, acquired, and adaptive. RASSF1A is a tumour suppressor that plays an integral role in the maintenance of cellular homeostasis as a central signalling hub. RASSF1A tumour suppressor activity is commonly lost in melanoma, mainly by aberrant promoter hypermethylation. RASSF1A loss could be associated with several mechanisms of resistance to MAPK inhibition considering that most of the signalling pathways that RASSF1A controls are found to be altered targeted therapy resistant melanomas. Herein, we discuss resistance mechanisms in detail and the potential role for RASSF1A reactivation to re-sensitise BRAF mutant melanomas to therapy.

Loss of family with sequence similarity 107, member A (FAM107A) induces browning in 3T3-L1 adipocytes.

Induction of white fat browning (beiging) and activation of brown fat has been considered a promising strategy to treat obesity and associated metabolic complications. However, the molecular mechanisms regulating brown and beige fat-mediated thermogenesis remains unclear. Our study aimed to identify genes with a hitherto unknown mechanism in the metabolic functions of adipocytes and identified family with sequence similarity 107, member A (FAM107A) as a factor that interferes with fat browning in white adipocytes. We explored physiological roles of FAM107A in cultured 3T3-L1 white adipocytes and HIB1B brown adipocytes by using FAM107A-deficient adipocytes. Significant loss in FAM107A gene functionality induced fat browning was evidenced by evaluating the gene and protein expression level of brown fat-associated markers through real-time qRT-PCR and immunoblot analysis, respectively. Deficiency of FAM107A promoted mitochondrial biogenesis and significantly upregulated core fat-browning marker proteins (PGC-1α, PRDM16, and UCP1) and beige-specific genes (Cd137, Cited1, Tbx1, and Tmem26). Furthermore, FAM107A increased adipogenesis and negatively regulated lipid metabolism in 3T3-L1 adipocytes. In addition, in-silico analysis revealed a strong interaction between FAM107A and ß3-AR based on their energy binding score. Next, mechanistic study revealed that specific knockdown of FAM107A induces browning in white adipocytes via activation of ß3-AR, AMPK and p38 MAPK-dependent signaling pathways. Our data unveiled a previously unknown mechanism of FAM107A in the regulation of lipid metabolism and identified its significant role in metabolic homeostasis. This highlighted the potential of FAM107A as a pharmacotherapeutic target in treating obesity and related metabolic disorders.