Resistance to therapy remains a critical challenge in cancer. This is exemplified by TGF-β pathway inhibitors which, despite broad clinical testing, have failed to deliver survival benefit. More precise SMAD3 targeting has shown preclinical promise, yet resistance mechanisms to SMAD3 inhibition remain entirely unknown. Using complementary genome-wide CRISPR activation and knockout screens, we discover that cancer cells escape SMAD3 inhibition through metabolic reprogramming. Strikingly, this escape is driven not by transcriptional bypass but by the lactate transporter MCT1, with the effect preserved across BRAF-mutated melanoma, KRAS-mutated lung adenocarcinoma, and mouse melanoma. Mechanistically, SMAD3 inhibition creates energetic stress that cancer cells exploit through MCT1. By conferring metabolic flexibility and switching toward glycolysis under drug pressure, MCT1 increases anabolic activity to drive lipid and cholesterol synthesis. Pharmacological and genetic MCT1 inhibition synergizes strongly with SMAD3 blockade to impair tumor viability in BRAF-mutated melanoma, while suppressing drug-tolerant cell emergence in BRAF inhibitor-resistant cells. Critically, this synergy extends to preclinical models, where co-inhibition of SMAD3 and MCT1 potently reduces tumor growth in vivo. Given the additional and well-established role of MCT1 in shaping the tumor immune microenvironment via lactate transport, these results position MCT1 as both a metabolic and immunological target and provide a strong rationale for the repurposing of TGF-β/SMAD3 pathway inhibitors in combination.

Antisense oligonucleotides (ASOs) belong to promising therapeutics for the treatment of neurological, muscular, and metabolic disorders. Several ASOs have been approved so far and more than 100 clinical trials are currently underway covering a dozen therapeutic areas. Yet, the mechanisms of internalization and cell trafficking of these molecules remain poorly understood. Moreover, with only a small fraction of ASOs reaching the correct cellular compartment after systemic delivery, the majority of targeted diseases require recurrent injections of ASOs. A deeper understanding of these mechanisms would guide the improvement of their potency and, thus, reduce the amount of delivered ASOs and their potential side effects. Here, using a CRISPR screen, we investigated intracellular proteins involved in ASOs efficiency using a whole genome approach and identified several potential regulators that could significantly impact ASOs potency in melanoma cells. We validated WD repeat domain 91, a regulator of endosomal maturation, as a modulator whose depletion significantly inhibits ASO productive activity. This study provides a list of ASO modulators using a biologically relevant assay to estimate the role of these proteins. In conclusion, these data could lead to a better understanding of the mechanisms favoring productive uptake or improved endosomal escape of ASOs.

Cholangiocarcinoma is a devastating liver cancer characterized by high aggressiveness and therapy resistance, resulting in poor prognosis. Long non-coding RNAs and signals imposed by oncogenic pathways, such as transforming growth factor β (TGFβ), frequently contribute to cholangiocarcinogenesis. Here, we explore novel effectors of TGFβ signalling in cholangiocarcinoma. LINC00313 is identified as a novel TGFβ target gene. Gene expression and genome-wide chromatin accessibility profiling reveal that nuclear LINC00313 transcriptionally regulates genes involved in Wnt signalling, such as the transcriptional activator TCF7. LINC00313 gain-of-function enhances TCF/LEF-dependent transcription, promotes colony formation in vitro and accelerates tumour growth in vivo. Genes affected by LINC00313 over-expression in CCA tumours are associated with KRAS and TP53 mutations and reduce overall patient survival. Mechanistically, ACTL6A and BRG1, subunits of the SWI/SNF chromatin remodelling complex, interact with LINC00313 and affect TCF7 and SULF2 transcription. We propose a model whereby TGFβ induces LINC00313 in order to regulate the expression of hallmark Wnt pathway genes, in co-operation with SWI/SNF. By modulating key genes of the Wnt pathway, LINC00313 fine-tunes Wnt/TCF/LEF-dependent transcriptional responses and promotes cholangiocarcinogenesis.

Purpose: Uveal melanoma (UM) is a deadly cancer with limited therapeutic options. At advanced stages, UM cells metastasize almost exclusively into the liver, where targeting metastatic UM cells remain a clinical challenge. Transforming growth factor beta (TGF-I3) exhibits a functional duality in cancer, with one arm limiting tumor growth at an early stage and the second arm promoting metastasis at an advanced stage, notably by inducing an epithelial-to-mesenchymal transition. Thus, we hypothesized that targeting the TGF-I3 pathway could be relevant in the treatment of metastatic UM. Methods: In this study, we first characterized the pseudoepithelial/mesenchymal phenotype of UM cell lines Mel270 and 92.1. We then treated the cell lines with TGF-I3 to evaluate their responsiveness to the cytokine and to characterize the functional impact of TGF-I3 on their cell viability. Results: The results demonstrated, first, that the UM cell lines exhibited a mesenchymal phenotype and responded to TGF-I3 treatment in vitro and, second, that TGF-I3 promoted a cytostatic effect on the UM cell lines. Conclusions: Our findings indicate that UM cells are sensitive to the two arms of TGF-I3 signaling, which suggests that targeting the TGF-I3 pathway could be challenging in UM and would require a precise selection of patients in which only the prometastatic arm of TGF-I3 is activated.

Characterization of the canine anaplastic oligodendroglioma.

Characterization of the canine DLBCL.

Sexual development is a complex process relying on numerous genes. Disruptions in some of these genes are known to cause differences of sexual development (DSDs). Advances in genome sequencing allowed the discovery of new genes implicated in sexual development, such as PBX1. We present here a fetus with a new PBX1 NM_002585.3: c.320G>A,p.(Arg107Gln) variant, presenting with severe DSD along with renal and lung malformations. Using CRISPR-Cas9 gene editing on HEK293T cells, we generated a KD cell line for PBX1. The KD cell line showed reduced proliferation and adhesion properties compared with HEK293T cells. HEK293T and KD cells were then transfected plasmids coding either PBX1 WT or PBX1-320G>A (mutant). WT or mutant PBX1 overexpression rescued cell proliferation in both cell lines. RNA-seq analyses showed less than 30 differentially expressed genes, in ectopic mutant-PBX1-expressing cells compared with WT-PBX1. Among them, U2AF1, encoding a splicing factor subunit, is an interesting candidate. Overall, mutant PBX1 seems to have modest effects compared with WT PBX1 in our model. However, the recurrence of PBX1 Arg107 substitution in patients with closely related phenotypes calls for its impact in human diseases. Further functional studies are needed to explore its effects on cellular metabolism.

Primers used for validation of the canine gene fusions.

Characterization of the canine dermatofibrosarcoma protuberans-like.

Abstract Canine cancers represent a tremendous natural resource due to their incidence and striking similarities to human cancers, sharing similar clinical and pathologic features as well as oncogenic events, including identical somatic mutations. Considering the importance of gene fusions as driver alterations, we explored their relevance in canine cancers. We focused on three distinct human-comparable canine cancers representing different tissues and embryonic origins. Through RNA-Seq, we discovered similar gene fusions as those found in their human counterparts: IGK-CCND3 in B-cell lymphoma, MPB-BRAF in glioma, and COL3A1-PDGFB in dermatofibrosarcoma protuberans-like. We showed not only similar partner genes but also identical breakpoints leading to oncogene overexpression. This study demonstrates similar gene fusion partners and mechanisms in human–dog corresponding tumors and allows for selection of targeted therapies in preclinical and clinical trials with pet dogs prior to human trials, within the framework of personalized medicine. Cancer Res; 77(21); 5721–7. ©2017 AACR.

Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced β-galactosidase (β-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim to deliver a functional copy of the GLB1 gene to stop disease progression. In this study, we show that 41% of GLB1 pathogenic SNVs can be replaced by adenine base editors (ABEs). Our results demonstrate that ABE efficiently corrects the pathogenic allele in patient-derived fibroblasts, restoring therapeutic levels of β-gal activity. Off-target DNA analysis did not detect off-target editing activity in treated patient's cells, except a bystander edit without consequences on β-gal activity based on 3D structure bioinformatics predictions. Altogether, our results suggest that gene editing might be an alternative strategy to cure GM1 gangliosidosis.

Alignment of the human and canine BRAF proteins.

Fusions identified by RNA-Seq in the analyzed canine tumors.

Alignment of the human and canine PDGFB proteins.

Mucosal melanoma (MM) is a rare, aggressive clinical cancer. Despite recent advances in genetics and treatment, the prognosis of MM remains poor. Canine MM offers a relevant spontaneous and immunocompetent model to decipher the genetic bases and explore treatments for MM. We performed an integrative genomic and transcriptomic analysis of 32 canine MM samples, which identified two molecular subgroups with a different microenvironment and structural variant (SV) content. The overexpression of genes related to the microenvironment and T-cell response was associated with tumors harboring a lower content of SVs, whereas the overexpression of pigmentation-related pathways and oncogenes, such as TERT, was associated with a high SV burden. Using whole-genome sequencing, we showed that focal amplifications characterized complex chromosomal rearrangements targeting oncogenes, such as MDM2 or CDK4, and a recurrently amplified region on canine chromosome 30. We also demonstrated that the genes TRPM7, GABPB1, and SPPL2A, located in this CFA30 region, play a role in cell proliferation, and thus, may be considered as new candidate oncogenes for human MM. Our findings suggest the existence of two MM molecular subgroups that may benefit from dedicated therapies, such as immune checkpoint inhibitors or targeted therapies, for both human and veterinary medicine.

PBX1 is a highly conserved atypical homeodomain transcription factor (TF) belonging to the TALE (three amino acid loop extension) family. Dimerized with other TALE proteins, it can interact with numerous partners and reach dozens of regulating sequences, suggesting its role as a pioneer factor. PBX1 is expressed throughout the embryonic stages (as early as the blastula stage) in vertebrates. In human, PBX1 germline variations are linked to syndromic renal anomalies (CAKUTHED). In this review, we summarized available data on PBX1 functions, PBX1-deficient animal models, and PBX1 germline variations in humans. Two types of genetic alterations were identified in PBX1 gene. PBX1 missense variations generate a severe phenotype including lung hypoplasia, cardiac malformations, and sexual development defects (DSDs). Conversely, truncating variants generate milder phenotypes (mainly cryptorchidism and deafness). We suggest that defects in PBX1 interactions with various partners, including proteins from the HOX (HOXA7, HOXA10, etc.), WNT (WNT9B, WNT3), and Polycomb (BMI1, EED) families are responsible for abnormal proliferation and differentiation of the embryonic mesenchyme. These alterations could explain most of the defects observed in humans. However, some phenotype variability (especially DSDs) remains poorly understood. Further studies are needed to explore the TALE family in greater depth.

Background: The essential amino acid, tryptophan, is predominantly metabolised through the kynurenine pathway (KP) to generate kynurenine, an aryl-hydrocarbon receptor (AhR) pro-ligand that exerts its effects in a ligand-dependent manner. Interaction between kynurenine and the AhR is an effector mechanism of immunosuppression. We previously found that the KP is involved in multiple sclerosis (MS) disease progression. We postulated that AhR activation by kynurenine might be neuroprotective by encouraging differentiation of Tregs. In this study, we assess both the prophylactic and therapeutic efficiency of kynurenine on disease severity and progression in mice with experimental autoimmune encephalomyelitis (EAE), an MS model. Methods: Myelin oligodendrocyte glycoprotein induced EAE mice (n = 6 per group) were treated with 200 mg/kg L-kynurenine once daily for 10 days beginning on either day 1 of EAE induction (prophylactic) or once they demonstrated motor weakness (therapeutic). Clinical disease severity measured by disease score, time on rotarod, and body weight. Results: The prophylactic kynurenine treatment significantly ( P < .0001) prevented the development of a more severe disease course with mice demonstrating diminished relapse rate and improved clinical and behavioural outcomes. However, therapeutic kynurenine did not significantly ( P = .4463) decrease the clinical signs until 36 days following induction of disease; after 36 days, it also significantly ( P = .0479) reduced disease relapse. Mean body weight measurements only correlated with time on rotarod ( r = −.6410; P = .0007) but not clinical scores ( r = .1925; P = .3674). Conclusions: Kynurenine ameliorates EAE disease progression prophylactically and reduces relapses therapeutically. Further investigations are needed to elucidate the molecular mechanism explaining the therapeutic role of kynurenine for MS.

Uveal melanoma (UM), the most common primary intraocular tumor in adults, has been extensively characterized by omics technologies during the last 5 yr. Despite the discovery of gene signatures, the molecular actors driving cancer aggressiveness are not fully understood, and UM is still associated with very poor overall survival (OS) at the metastatic stage. By defining the miR-16 interactome, we revealed that miR-16 mainly interacts via non-canonical base-pairing to a subset of RNAs, promoting their expression levels. Consequently, the canonical miR-16 activity, involved in the RNA decay of oncogenes, such as cyclin D3, is impaired. This non-canonical base-pairing can explain both the derepression of miR-16 targets and the promotion of oncogene expression observed in patients with poor OS in two cohorts. miR-16 activity, assessment using our RNA signature, discriminates the patient's OS as effectively as current methods. To the best of our knowledge, this is the first time that a predictive signature has been composed of genes belonging to the same mechanism (miR-16) in UM. Altogether, our results strongly suggest that UM is a miR-16 disease.