Lysin (K)-specific demethylase 1 inhibition enhances proteasome inhibitor response and overcomes drug resistance in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is an incurable plasma cell malignancy, accounting for approximately 1% of all cancers. Despite recent advances in the treatment of MM, due to the introduction of proteasome inhibitors (PIs) such as bortezomib (BTZ) and carfilzomib (CFZ), relapses and disease progression remain common. Therefore, a major challenge is the development of novel therapeutic approaches to overcome drug resistance, improve patient outcomes, and broaden PIs applicability to other pathologies. METHODS: We performed genetic and drug screens to identify new synthetic lethal partners to PIs, and validated candidates in PI-sensitive and -resistant MM cells. We also tested best synthetic lethal interactions in other B-cell malignancies, such as mantle cell, Burkitt's and diffuse large B-cell lymphomas. We evaluated the toxicity of combination treatments in normal peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs). We confirmed the combo treatment' synergistic effects ex vivo in primary CD138+ cells from MM patients, and in different MM xenograft models. We exploited RNA-sequencing and Reverse-Phase Protein Arrays (RPPA) to investigate the molecular mechanisms of the synergy. RESULTS: We identified lysine (K)-specific demethylase 1 (LSD1) as a top candidate whose inhibition can synergize with CFZ treatment. LSD1 silencing enhanced CFZ sensitivity in both PI-resistant and -sensitive MM cells, resulting in increased tumor cell death. Several LSD1 inhibitors (SP2509, SP2577, and CC-90011) triggered synergistic cytotoxicity in combination with different PIs in MM and other B-cell neoplasms. CFZ/SP2509 treatment exhibited a favorable cytotoxicity profile toward PBMCs and BMSCs. We confirmed the clinical potential of LSD1-proteasome inhibition in primary CD138+ cells of MM patients, and in MM xenograft models, leading to the inhibition of tumor progression. DNA damage response (DDR) and proliferation machinery were the most affected pathways by CFZ/SP2509 combo treatment, responsible for the anti-tumoral effects. CONCLUSIONS: The present study preclinically demonstrated that LSD1 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients and that this combination might be exploited for the treatment of other B-cell malignancies, thus extending the therapeutic impact of the project.

Euchromatic Histone Lysine Methyltransferase 2 Inhibition Enhances Carfilzomib Sensitivity and Overcomes Drug Resistance in Multiple Myeloma Cell Lines.

Proteasome inhibitors (PIs) are extensively used for the therapy of multiple myeloma. However, patients continuously relapse or are intrinsically resistant to this class of drugs. In addition, adverse toxic effects such as peripheral neuropathy and cardiotoxicity could arise. Here, to identify compounds that can increase the efficacy of PIs, we performed a functional screening using a library of small-molecule inhibitors covering key signaling pathways. Among the best synthetic lethal interactions, the euchromatic histone-lysine N-methyltransferase 2 (EHMT2) inhibitor UNC0642 displayed a cooperative effect with carfilzomib (CFZ) in numerous multiple myeloma (MM) cell lines, including drug-resistant models. In MM patients, EHMT2 expression correlated to worse overall and progression-free survival. Moreover, EHMT2 levels were significantly increased in bortezomib-resistant patients. We demonstrated that CFZ/UNC0642 combination exhibited a favorable cytotoxicity profile toward peripheral blood mononuclear cells and bone-marrow-derived stromal cells. To exclude off-target effects, we proved that UNC0642 treatment reduces EHMT2-related molecular markers and that an alternative EHMT2 inhibitor recapitulated the synergistic activity with CFZ. Finally, we showed that the combinatorial treatment significantly perturbs autophagy and the DNA damage repair pathways, suggesting a multi-layered mechanism of action. Overall, the present study demonstrates that EHMT2 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients.

Extraction Bottleneck in the Diagnosis of SARS-CoV-2: Evaluation of an Alternative Protocol Derived from Veterinary Use.

The COVID-19 pandemic represented a challenge for health-care systems, and a major bottleneck in SARS-CoV-2 diagnosis was the unavailability of extraction reagents. To overcome this limitation, we performed a comparative analysis to evaluate the performance of an alternative extraction protocol derived from veterinary use adapted to an open robotic platform (Testing method). A total of 73 nasopharyngeal swabs collected for diagnosis of SARS-CoV-2 infection were simultaneously extracted with the Testing protocol and the laboratory Standard of Care in order to assess the performance of the first one. The Cohen's coefficient between both procedures was excellent (K Value = 0.955). Analysis of cycle threshold and linear regression showed a significant correlation between the two methods for each tested genetic target. Although validated for veterinary applications, the Testing method showed excellent performances in RNA extraction, with several advantages: lower sample input volume, the possibility to overcome the lack of deep-well plates and adaptability to robotic liquid handlers.

Activation of long non-coding RNA NEAT1 leads to survival advantage of multiple myeloma cells by supporting a positive regulatory loop with DNA repair proteins.

Long non-coding RNA NEAT1 is the core structural component of the nuclear paraspeckle (PS) organelles and it has been found to be deregulated in multiple myeloma (MM) patients. Experimental evidence indicated that NEAT1 silencing negatively impacts proliferation and viability of MM cells, both in vitro and in vivo, suggesting a role in DNA damage repair (DDR). In order to elucidate the biological and molecular relevance of NEAT1 upregulation in MM disease we exploited the CRISPR/Cas9 synergistic activation mediator genome editing system to engineer the AMO-1 MM cell line and generate two clones that para-physiologically transactivate NEAT1 at different levels. NEAT1 overexpression is associated with oncogenic and prosurvival advantages in MM cells exposed to nutrient starvation or a hypoxic microenvironment, which are stressful conditions often associated with more aggressive disease phases. Furthermore, we highlighted the NEAT1 involvement in virtually all DDR processes through, at least, two different mechanisms. On one side NEAT1 positively regulates the posttranslational stabilization of essential PS proteins, which are involved in almost all DDR systems, thus increasing their availability within cells. On the other hand, NEAT1 plays a crucial role as a major regulator of a molecular axis that includes ATM and the catalytic subunit of DNA-PK kinase proteins, and their direct targets pRPA32 and pCHK2. Overall, we provided novel important insightsthe role of NEAT1 in supporting MM cells adaptation to stressful conditions by improving the maintenance of DNA integrity. Taken together, our results suggest that NEAT1, and probably PS organelles, could represent a potential therapeutic target for MM treatment.

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies.

Gene expression is controlled by epigenetic deregulation, a hallmark of cancer. The DNA methylome of canine diffuse large B-cell lymphoma (cDLBCL), the most frequent malignancy of B-lymphocytes in dog, has recently been investigated, suggesting that aberrant hypermethylation of CpG loci is associated with gene silencing. Here, we used a multi-omics approach (DNA methylome, transcriptome and copy number variations) combined with functional in vitro assays, to identify putative tumour suppressor genes subjected to DNA methylation in cDLBCL. Using four cDLBCL primary cell cultures and CLBL-1 cells, we found that CiDEA, MAL and PCDH17, which were significantly suppressed in DLBCL samples, were hypermethylated and also responsive (at the DNA, mRNA and protein level) to pharmacological unmasking with hypomethylating drugs and histone deacetylase inhibitors. The regulatory mechanism underneath the methylation-dependent inhibition of those target genes expression was then investigated through luciferase and in vitro methylation assays. In the most responsive CpG-rich regions, an in silico analysis allowed the prediction of putative transcription factor binding sites influenced by DNA methylation. Interestingly, regulatory elements for AP2, MZF1, NF-kB, PAX5 and SP1 were commonly identified in all three genes. This study provides a foundation for characterisation and experimental validation of novel epigenetically-dysregulated pathways in cDLBCL.

The Landscape of Signaling Pathways and Proteasome Inhibitors Combinations in Multiple Myeloma.

Multiple myeloma is a malignancy of terminally differentiated plasma cells, characterized by an extreme genetic heterogeneity that poses great challenges for its successful treatment. Due to antibody overproduction, MM cells depend on the precise regulation of the protein degradation systems. Despite the success of PIs in MM treatment, resistance and adverse toxic effects such as peripheral neuropathy and cardiotoxicity could arise. To this end, the use of rational combinatorial treatments might allow lowering the dose of inhibitors and therefore, minimize their side-effects. Even though the suppression of different cellular pathways in combination with proteasome inhibitors have shown remarkable anti-myeloma activities in preclinical models, many of these promising combinations often failed in clinical trials. Substantial progress has been made by the simultaneous targeting of proteasome and different aspects of MM-associated immune dysfunctions. Moreover, targeting deranged metabolic hubs could represent a new avenue to identify effective therapeutic combinations with PIs. Finally, epigenetic drugs targeting either DNA methylation, histone modifiers/readers, or chromatin remodelers are showing pleiotropic anti-myeloma effects alone and in combination with PIs. We envisage that the positive outcome of patients will probably depend on the availability of more effective drug combinations and treatment of early MM stages. Therefore, the identification of sensitive targets and aberrant signaling pathways is instrumental for the development of new personalized therapies for MM patients.

Genomic Instability in Multiple Myeloma: A "Non-Coding RNA" Perspective.

Multiple myeloma (MM) is a complex hematological malignancy characterized by abnormal proliferation of malignant plasma cells (PCs) within a permissive bone marrow microenvironment. The pathogenesis of MM is unequivocally linked to the acquisition of genomic instability (GI), which indicates the tendency of tumor cells to accumulate a wide repertoire of genetic alterations. Such alterations can even be detected at the premalignant stages of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) and, overall, contribute to the acquisition of the malignant traits underlying disease progression. The molecular basis of GI remains unclear, with replication stress and deregulation of DNA damage repair pathways representing the most documented mechanisms. The discovery that non-coding RNA molecules are deeply dysregulated in MM and can target pivotal components of GI pathways has introduced a further layer of complexity to the GI scenario in this disease. In this review, we will summarize available information on the molecular determinants of GI in MM, focusing on the role of non-coding RNAs as novel means to tackle GI for therapeutic intervention.

Author Correction: Therapeutic potential of KLF2-induced exosomal microRNAs in pulmonary hypertension.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Next Generation Sequencing (NGS) Strategies for Genetic Testing of Cerebral Cavernous Malformation (CCM) Disease.

The application of next generation sequencing (NGS) technique has a great impact on complex disease studies. Indeed, genetic heterogeneity, phenotypic variability, and disease rarity are all factors that make the traditional diagnostic approach to genetic disorders, whereby a specific gene is selected for sequencing based on the clinical phenotype, very challenging and obsolete.Exome sequencing, which sequences the protein-coding region of the genome, has been rapidly applied to variant discovery in research settings. Recent coverage and accuracy improvements have accelerated the development of clinical exome sequencing (CES) platforms targeting disease-related genes and enabling variant identification in patients with suspected genetic diseases. Nowadays, CES is rapidly becoming the diagnostic test of choice in patients with suspected Mendelian diseases, especially for those with heterogeneous etiology and clinical presentation. Reporting large CES series can improve guidelines on best practices for test utilization, and a better variant interpretation through clinically oriented data sharing.Herein, we suggest a feasible CES procedure for the genetic testing of Cerebral Cavernous Malformation (CCM) disease, including proband identification, library preparation, data analysis, and variant interpretation.

Therapeutic potential of KLF2-induced exosomal microRNAs in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a severe disorder of lung vasculature that causes right heart failure. Homoeostatic effects of flow-activated transcription factor Krüppel-like factor 2 (KLF2) are compromised in PAH. Here, we show that KLF2-induced exosomal microRNAs, miR-181a-5p and miR-324-5p act together to attenuate pulmonary vascular remodelling and that their actions are mediated by Notch4 and ETS1 and other key regulators of vascular homoeostasis. Expressions of KLF2, miR-181a-5p and miR-324-5p are reduced, while levels of their target genes are elevated in pre-clinical PAH, idiopathic PAH and heritable PAH with missense p.H288Y KLF2 mutation. Therapeutic supplementation of miR-181a-5p and miR-324-5p reduces proliferative and angiogenic responses in patient-derived cells and attenuates disease progression in PAH mice. This study shows that reduced KLF2 signalling is a common feature of human PAH and highlights the potential therapeutic role of KLF2-regulated exosomal miRNAs in PAH and other diseases associated with vascular remodelling.

NAMPT Over-Expression Recapitulates the BRAF Inhibitor Resistant Phenotype Plasticity in Melanoma.

Serine-threonine protein kinase B-RAF (BRAF)-mutated metastatic melanoma (MM) is a highly aggressive type of skin cancer. Treatment of MM patients using BRAF/MEK inhibitors (BRAFi/MEKi) eventually leads to drug resistance, limiting any clinical benefit. Herein, we demonstrated that the nicotinamide adenine dinucleotide (NAD)-biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) is a driving factor in BRAFi resistance development. Using stable and inducible NAMPT over-expression systems, we showed that forced NAMPT expression in MM BRAF-mutated cell lines led to increased energy production, MAPK activation, colony-formation capacity, and enhance tumorigenicity in vivo. Moreover, NAMPT over-expressing cells switched toward an invasive/mesenchymal phenotype, up-regulating expression of ZEB1 and TWIST, two transcription factors driving the epithelial to mesenchymal transition (EMT) process. Consistently, within the NAMPT-overexpressing cell line variants, we observed an increased percentage of a rare, drug-effluxing stem cell-like side population (SP) of cells, paralleled by up-regulation of ABCC1/MRP1 expression and CD133-positive cells. The direct correlation between NAMPT expression and gene set enrichments involving metastasis, invasiveness and mesenchymal/stemness properties were verified also in melanoma patients by analyzing The Cancer Genome Atlas (TCGA) datasets. On the other hand, CRISPR/Cas9 full knock-out NAMPT BRAFi-resistant MM cells are not viable, while inducible partial silencing drastically reduces tumor growth and aggressiveness. Overall, this work revealed that NAMPT over-expression is both necessary and sufficient to recapitulate the BRAFi-resistant phenotype plasticity.

Wild-Type IDH Enzymes as Actionable Targets for Cancer Therapy.

Isocitrate dehydrogenases (IDHs) are enzymes that catalyze the oxidative decarboxylation of isocitrate, producing α-ketoglutarate (αKG) and CO2. The discovery of IDH1 and IDH2 mutations in several malignancies has brought to the approval of drugs targeting IDH1/2 mutants in cancers. Here, we summarized findings addressing the impact of IDH mutants in rare pathologies and focused on the relevance of non-mutated IDH enzymes in tumors. Several pieces of evidence suggest that the enzymatic inhibition of IDHs may have therapeutic potentials also in wild-type IDH cancers. Moreover, IDHs inhibition could enhance the efficacy of canonical cancer therapies, such as chemotherapy, target therapy, and radiotherapy. However, further studies are required to elucidate whether IDH proteins are diagnostic/prognostic markers, instrumental for tumor initiation and maintenance, and could be exploited as targets for anticancer therapy. The development of wild-type IDH inhibitors is expected to improve our understanding of a potential non-oncogenic addition to IDH1/2 activities and to fully address their applicability in combination with other therapies.

Integration of transcriptional and mutational data simplifies the stratification of peripheral T-cell lymphoma.

The histological diagnosis of peripheral T-cell lymphoma (PTCL) can represent a challenge, particularly in the case of closely related entities such as angioimmunoblastic T-lymphoma (AITL), PTCL-not otherwise specified (PTCL-NOS), and ALK-negative anaplastic large-cell lymphoma (ALCL). Although gene expression profiling and next generations sequencing have been proven to define specific features recurrently associated with distinct entities, genomic-based stratifications have not yet led to definitive diagnostic criteria and/or entered into the routine clinical practice. Herein, to improve the current molecular classification between AITL and PTCL-NOS, we analyzed the transcriptional profiles from 503 PTCLs stratified according to their molecular configuration and integrated them with genomic data of recurrently mutated genes (RHOA G17V , TET2, IDH2 R172 , and DNMT3A) in 53 cases (39 AITLs and 14 PTCL-NOSs) included in the series. Our analysis unraveled that the mutational status of RHOA G17V , TET2, and DNMT3A poorly correlated, individually, with peculiar transcriptional fingerprints. Conversely, in IDH2 R172 samples a strong transcriptional signature was identified that could act as a surrogate for mutational status. The integrated analysis of clinical, mutational, and molecular data led to a simplified 19-gene signature that retains high accuracy in differentiating the main nodal PTCL entities. The expression levels of those genes were confirmed in an independent cohort profiled by RNA-sequencing.

IDH2 inhibition enhances proteasome inhibitor responsiveness in hematological malignancies.

Proteasome inhibitors (PI) are extensively used for the therapy of multiple myeloma (MM) and mantle cell lymphoma. However, patients continuously relapse or are intrinsically resistant to this class of drugs. Here, to identify targets that synergize with PI, we carried out a functional screening in MM cell lines using a short hairpin RNA library against cancer driver genes. Isocitrate dehydrogenase 2 (IDH2) was identified as a top candidate, showing a synthetic lethal activity with the PI carfilzomib (CFZ). Combinations of US Food and Drug Administration-approved PI with a pharmacological IDH2 inhibitor (AGI-6780) triggered synergistic cytotoxicity in MM, mantle cell lymphoma, and Burkitt lymphoma cell lines. CFZ/AGI-6780 treatment increased death of primary CD138+ cells from MM patients and exhibited a favorable cytotoxicity profile toward peripheral blood mononuclear cells and bone marrow-derived stromal cells. Mechanistically, the CFZ/AGI-6780 combination significantly decreased tricarboxylic acid cycle activity and adenosine triphosphate levels as a consequence of enhanced IDH2 enzymatic inhibition. Specifically, CFZ treatment reduced the expression of nicotinamide phosphoribosyltransferase (NAMPT), thus limiting IDH2 activation through the NAD+-dependent deacetylase SIRT3. Consistently, combination of CFZ with either NAMPT or SIRT3 inhibitors impaired IDH2 activity and increased MM cell death. Finally, inducible IDH2 knockdown enhanced the therapeutic efficacy of CFZ in a subcutaneous xenograft model of MM, resulting in inhibition of tumor progression and extended survival. Taken together, these findings indicate that NAMPT/SIRT3/IDH2 pathway inhibition enhances the therapeutic efficacy of PI, thus providing compelling evidence for treatments with lower and less toxic doses and broadening the application of PI to other malignancies.

Wiskott-Aldrich syndrome protein (WASP) is a tumor suppressor in T cell lymphoma.

In T lymphocytes, the Wiskott-Aldrich Syndrome protein (WASP) and WASP-interacting-protein (WIP) regulate T cell antigen receptor (TCR) signaling, but their role in lymphoma is largely unknown. Here we show that the expression of WASP and WIP is frequently low or absent in anaplastic large cell lymphoma (ALCL) compared to other T cell lymphomas. In anaplastic lymphoma kinase-positive (ALK+) ALCL, WASP and WIP expression is regulated by ALK oncogenic activity via its downstream mediators STAT3 and C/EBP-ß. ALK+ lymphomas were accelerated in WASP- and WIP-deficient mice. In the absence of WASP, active GTP-bound CDC42 was increased and the genetic deletion of one CDC42 allele was sufficient to impair lymphoma growth. WASP-deficient lymphoma showed increased mitogen-activated protein kinase (MAPK) pathway activation that could be exploited as a therapeutic vulnerability. Our findings demonstrate that WASP and WIP are tumor suppressors in T cell lymphoma and suggest that MAP-kinase kinase (MEK) inhibitors combined with ALK inhibitors could achieve a more potent therapeutic effect in ALK+ ALCL.

ALK expression favorably impacts the prognosis of NRAS-mutated metastatic melanomas.

Recent studies reported the expression of anaplastic lymphoma kinase (ALK) in malignant melanomas. The aim of this study was to investigate whether ALK expression is associated with specific clinical and molecular characteristics of melanoma metastases, and to evaluate its correlation with survival outcomes. Seventy-one patients with metastatic melanoma were investigated. Clinical features and survival outcomes were analyzed and correlated to ALK expression, as detected by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction, and to the mutational status of BRAF, KRAS, NRAS, and PIK3CA. No translocations or ALK alternative isoforms were identified. ALK expression was mainly detected in NRAS mutated metastatic lesions. Interestingly, among NRAS-mutated patients, ALK positive samples displayed a significantly more favorable outcome in terms of disease specific survival, as compared to ALK negative ones. In conclusion, we suggest that ALK positive/NRAS mutated metastases represent a specific subset of metastatic melanomas, associated with a better prognosis. Validation of these observations in larger cohorts could contribute to understand the molecular events cooperating to melanoma progression, in addition to open new perspectives in the clinical and therapeutic management of this subgroup of patients.

The AP-1-BATF and -BATF3 module is essential for growth, survival and TH17/ILC3 skewing of anaplastic large cell lymphoma.

Transcription factor AP-1 is constitutively activated and IRF4 drives growth and survival in ALK+ and ALK- anaplastic large cell lymphoma (ALCL). Here we demonstrate high-level BATF and BATF3 expression in ALCL. Both BATFs bind classical AP-1 motifs and interact with in ALCL deregulated AP-1 factors. Together with IRF4, they co-occupy AP-1-IRF composite elements, differentiating ALCL from non-ALCL. Gene-specific inactivation of BATFs, or global AP-1 inhibition results in ALCL growth retardation and/or cell death in vitro and in vivo. Furthermore, the AP-1-BATF module establishes TH17/group 3 innate lymphoid cells (ILC3)-associated gene expression in ALCL cells, including marker genes such as AHR, IL17F, IL22, IL26, IL23R and RORγt. Elevated IL-17A and IL-17F levels were detected in a subset of children and adolescents with ALK+ ALCL. Furthermore, a comprehensive analysis of primary lymphoma data confirms TH17-, and in particular ILC3-skewing in ALCL compared with PTCL. Finally, pharmacological inhibition of RORC as single treatment leads to cell death in ALCL cell lines and, in combination with the ALK inhibitor crizotinib, enforces death induction in ALK+ ALCL. Our data highlight the crucial role of AP-1/BATFs in ALCL and lead to the concept that some ALCL might originate from ILC3.

IRF4 Mediates the Oncogenic Effects of STAT3 in Anaplastic Large Cell Lymphomas.

Systemic anaplastic large cell lymphomas (ALCL) are a category of T-cell non-Hodgkin's lymphomas which can be divided into anaplastic lymphoma kinase (ALK) positive and ALK negative subgroups, based on ALK gene rearrangements. Among several pathways aberrantly activated in ALCL, the constitutive activation of signal transducer and activator of transcription 3 (STAT3) is shared by all ALK positive ALCL and has been detected in a subgroup of ALK negative ALCL. To discover essential mediators of STAT3 oncogenic activity that may represent feasible targets for ALCL therapies, we combined gene expression profiling analysis and RNA interference functional approaches. A shRNA screening of STAT3-modulated genes identified interferon regulatory factor 4 (IRF4) as a key driver of ALCL cell survival. Accordingly, ectopic IRF4 expression partially rescued STAT3 knock-down effects. Treatment with immunomodulatory drugs (IMiDs) induced IRF4 down regulation and resulted in cell death, a phenotype rescued by IRF4 overexpression. However, the majority of ALCL cell lines were poorly responsive to IMiDs treatment. Combination with JQ1, a bromodomain and extra-terminal (BET) family antagonist known to inhibit MYC and IRF4, increased sensitivity to IMiDs. Overall, these results show that IRF4 is involved in STAT3-oncogenic signaling and its inhibition provides alternative avenues for the design of novel/combination therapies of ALCL.

PD-L1 up-regulation in melanoma increases disease aggressiveness and is mediated through miR-17-5p.

PD-L1 is expressed by a subset of patients with metastatic melanoma (MM) with an unfavorable outcome. Its expression is increased in cells resistant to BRAF or MEK inhibitors (BRAFi or MEKi). However, the function and regulation of expression of PD-L1 remain incompletely understood.After generating BRAFi- and MEKi-resistant cell lines, we observed marked up-regulation of PD-L1 expression. These cells were characterized by a common gene expression profile with up-regulation of genes involved in cell movement. Consistently, in vitro they showed significantly increased invasive properties. This phenotype was controlled in part by PD-L1, as determined after silencing the molecule. Up-regulation of PD-L1 was due to post-transcriptional events controlled by miR-17-5p, which showed an inverse correlation with PD-L1 mRNA. Direct binding between miR-17-5p and the 3'-UTR of PD-L1 mRNA was demonstrated using luciferase reporter assays.In a cohort of 80 BRAF-mutated MM patients treated with BRAFi or MEKi, constitutive expression of PD-L1 in the absence of immune infiltrate, defined the patient subset with the worst prognosis. Furthermore, PD-L1 expression increased in tissue biopsies after the metastatic lesions became resistant to BRAFi or MEKi. Lastly, plasmatic miR-17-5p levels were higher in patients with PD-L1+ than PD-L1- lesions.In conclusion, our findings indicate that PD-L1 expression induces a more aggressive behavior in melanoma cells. We also show that PD-L1 up-regulation in BRAFi or MEKi-resistant cells is partly due to post-transcriptional mechanisms that involve miR-17-5p, suggesting that miR-17-5p may be used as a marker of PD-L1 expression by metastatic lesions and ultimately a predictor of responses to BRAFi or MEKi.

The RNA-binding protein ESRP1 promotes human colorectal cancer progression.

Epithelial splicing regulatory protein 1 (ESRP1) is an epithelial cell-specific RNA binding protein that controls several key cellular processes, like alternative splicing and translation. Previous studies have demonstrated a tumor suppressor role for this protein. Recently, however, a pro-metastatic function of ESRP1 has been reported. We thus aimed at clarifying the role of ESRP1 in Colorectal Cancer (CRC) by performing loss- and gain-of-function studies, and evaluating tumorigenesis and malignancy with in vitro and in vivo approaches. We found that ESRP1 plays a role in anchorage-independent growth of CRC cells. ESRP1-overexpressing cells grown in suspension showed enhanced fibroblast growth factor receptor (FGFR1/2) signalling, Akt activation, and Snail upregulation. Moreover, ESRP1 promoted the ability of CRC cells to generate macrometastases in mice livers. High ESRP1 expression may thus stimulate growth of cancer epithelial cells and promote colorectal cancer progression. Our findings provide mechanistic insights into a previously unreported, pro-oncogenic role for ESRP1 in CRC, and suggest that fine-tuning the level of this RNA-binding protein could be relevant in modulating tumor growth in a subset of CRC patients.