ΔNp63-Senataxin circuit controls keratinocyte differentiation by promoting the transcriptional termination of epidermal genes.

SignificanceΔNp63 is a master regulator of skin homeostasis since it finely controls keratinocyte differentiation and proliferation. Here, we provide cellular and molecular evidence demonstrating the functional role of a ΔNp63 interactor, the R-loop-resolving enzyme Senataxin (SETX), in fine-tuning keratinocyte differentiation. We found that SETX physically binds the p63 DNA-binding motif present in two early epidermal differentiation genes, Keratin 1 (KRT1) and ZNF750, facilitating R-loop removal over their 3' ends and thus allowing efficient transcriptional termination and gene expression. These molecular events translate into the inability of SETX-depleted keratinocytes to undergo the correct epidermal differentiation program. Remarkably, SETX is dysregulated in cutaneous squamous cell carcinoma, suggesting its potential involvement in the pathogenesis of skin disorders.

Role of Hippo pathway dysregulation from gastrointestinal premalignant lesions to cancer.

BACKGROUND: First identified in Drosophila melanogaster, the Hippo pathway is considered a major regulatory cascade controlling tissue homeostasis and organ development. Hippo signaling components include kinases whose activity regulates YAP and TAZ final effectors. In response to upstream stimuli, YAP and TAZ control transcriptional programs involved in cell proliferation, cytoskeletal reorganization and stemness. MAIN TEXT: While fine tuning of Hippo cascade components is essential for maintaining the balance between proliferative and non-proliferative signals, pathway signaling is frequently dysregulated in gastrointestinal cancers. Also, YAP/TAZ aberrant activation has been described in conditions characterized by chronic inflammation that precede cancer development, suggesting a role of Hippo effectors in triggering carcinogenesis. In this review, we summarize the architecture of the Hippo pathway and discuss the involvement of signaling cascade unbalances in premalignant lesions of the gastrointestinal tract, providing a focus on the underlying molecular mechanisms. CONCLUSIONS: The biology of premalignant Hippo signaling dysregulation needs further investigation in order to elucidate the evolutionary trajectories triggering cancer inititation and develop effective early therapeutic strategies targeting the Hippo/YAP pathway.

Bcl-2 dependent modulation of Hippo pathway in cancer cells.

INTRODUCTION: Bcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2. METHODS: We performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts. RESULTS: We demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation. CONCLUSIONS: We discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.

ABCC1 Is a ΔNp63 Target Gene Overexpressed in Squamous Cell Carcinoma.

The transcription factor ΔNp63 plays a pivotal role in maintaining the integrity of stratified epithelial tissues by regulating the expression of distinct target genes involved in lineage specification, cell stemness, cell proliferation and differentiation. Here, we identified the ABC transporter subfamily member ABCC1 as a novel ΔNp63 target gene. We found that in immortalized human keratinocytes and in squamous cell carcinoma (SCC) cells, ∆Np63 induces the expression of ABCC1 by physically occupying a p63-binding site (p63 BS) located in the first intron of the ABCC1 gene locus. In cutaneous SCC and during the activation of the keratinocyte differentiation program, ∆Np63 and ABCC1 levels are positively correlated raising the possibility that ABCC1 might be involved in the regulation of the proliferative/differentiative capabilities of squamous tissue. However, we did not find any gross alteration in the structure and morphology of the epidermis in humanized hABCC1 knock-out mice. Conversely, we found that the genetic ablation of ABCC1 led to a marked reduction in inflammation-mediated proliferation of keratinocytes, suggesting that ABCC1 might be involved in the regulation of keratinocyte proliferation upon inflammatory/proliferative signals. In line with these observations, we found a significant increase in ABCC1 expression in squamous cell carcinomas (SCCs), a tumor type characterized by keratinocyte hyper-proliferation and a pro-inflammatory tumor microenvironment. Collectively, these data uncover ABCC1 as an additional ∆Np63 target gene potentially involved in those skin diseases characterized by dysregulation of proliferation/differentiation balance.

The Molecular Tumor Board of the Regina Elena National Cancer Institute: from accrual to treatment in real-world.

BACKGROUND: Molecular Tumor Boards (MTB) operating in real-world have generated limited consensus on good practices for accrual, actionable alteration mapping, and outcome metrics. These topics are addressed herein in 124 MTB patients, all real-world accrued at progression, and lacking approved therapy options. METHODS: Actionable genomic alterations identified by tumor DNA (tDNA) and circulating tumor DNA (ctDNA) profiling were mapped by customized OncoKB criteria to reflect diagnostic/therapeutic indications as approved in Europe. Alterations were considered non-SoC when mapped at either OncoKB level 3, regardless of tDNA/ctDNA origin, or at OncoKB levels 1/2, provided they were undetectable in matched tDNA, and had not been exploited in previous therapy lines. RESULTS: Altogether, actionable alterations were detected in 54/124 (43.5%) MTB patients, but only in 39 cases (31%) were these alterations (25 from tDNA, 14 from ctDNA) actionable/unexploited, e.g. they had not resulted in the assignment of pre-MTB treatments. Interestingly, actionable and actionable/unexploited alterations both decreased (37.5% and 22.7% respectively) in a subset of 88 MTB patients profiled by tDNA-only, but increased considerably (77.7% and 66.7%) in 18 distinct patients undergoing combined tDNA/ctDNA testing, approaching the potential treatment opportunities (76.9%) in 147 treatment-naïve patients undergoing routine tDNA profiling for the first time. Non-SoC therapy was MTB-recommended to all 39 patients with actionable/unexploited alterations, but only 22 (56%) accessed the applicable drug, mainly due to clinical deterioration, lengthy drug-gathering procedures, and geographical distance from recruiting clinical trials. Partial response and stable disease were recorded in 8 and 7 of 19 evaluable patients, respectively. The time to progression (TTP) ratio (MTB-recommended treatment vs last pre-MTB treatment) exceeded the conventional Von Hoff 1.3 cut-off in 9/19 cases, high absolute TTP and Von Hoff values coinciding in 3 cases. Retrospectively, 8 patients receiving post-MTB treatment(s) as per physician's choice were noted to have a much longer overall survival from MTB accrual than 11 patients who had received no further treatment (35.09 vs 6.67 months, p = 0.006). CONCLUSIONS: MTB-recommended/non-SoC treatments are effective, including those assigned by ctDNA-only alterations. However, real-world MTBs may inadvertently recruit patients electively susceptible to diverse and/or multiple treatments.

Che-1/AATF binds to RNA polymerase I machinery and sustains ribosomal RNA gene transcription.

Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.

The lncRNAMALAT1-WTAP axis: a novel layer of EMT regulation in hypoxic triple-negative breast cancer.

Early metastatic disease development is one characteristic that defines triple-negative breast cancer (TNBC) as the most aggressive breast cancer (BC) subtype. Numerous studies have identified long non-coding RNAs (lncRNA) as critical players in regulating tumor progression and metastasis formation. Here, we show that MALAT1, a long non-coding RNA known to promote various features of BC malignancy, such as migration and neo angiogenesis, regulates TNBC cell response to hypoxia. By profiling MALAT1-associated transcripts, we discovered that lncRNA MALAT1 interacts with the mRNA encoding WTAP protein, previously reported as a component of the N6-methyladenosine (m6A) modification writer complex. In hypoxic conditions, MALAT1 positively regulates WTAP protein expression, which influences the response to hypoxia by favoring the transcription of the master regulators HIF1α and HIF1ß. Furthermore, WTAP stimulates BC cell migratory ability and the expression of N-Cadherin and Vimentin, hallmarks of epithelial-to-mesenchymal transition (EMT). In conclusion, this study highlights the functional axis comprising MALAT1 and WTAP as a novel prognostic marker of TNBC progression and as a potential target for the development of therapeutic approaches for TNBC treatment.

Evolution of transcriptomic profiles in relapsed inv(16) acute myeloid leukemia.

Acute myeloid leukemia (AML) with inv(16) is typically associated with a favourable prognosis. However, up to 40 % of patients will eventually experience disease relapse. Herein, we dissected the genomic and transcriptomic profile of inv(16) AML to identify potential prognostic markers and therapeutic vulnerabilities. Sequencing data from 222 diagnostic samples, including 44 relapse/refractory patients, revealed a median of 1 concomitant additional mutation, cooperating with inv(16) in leukemogenesis. Notably, the mutational landscape at diagnosis did not differ significantly between patients experiencing primary induction failure or relapse when compared to the rest of the cohort, except for an increase in the mutational burden in the relapse/refractory group. RNA-Seq of unpaired diagnostic(n=7) and relapse(n=6) samples allowed the identification of oxidative phosphorylation (OXPHOS) as one of the most significantly downregulated pathways at relapse. Considering that OXPHOS could be targeted by Venetoclax/Azacitidine combination, we explored its biological effects on an inv(16) cell-line ME-1, but there was no additional advantage in terms of cell death over Azacitidine alone. To enhance Venetoclax efficacy, we tested in vitro effects of Metformin as a potential drug able to enhance chemosensitivity of AML cells by inhibiting the mitochondrial transfer. By challenging ME-1 with this combination, we observed a significant synergistic interaction at least similar to that of Venetoclax/Azacitidine. In conclusions, we identified a downregulated expression of oxidative phosphorylation (OXPHOS) at relapse in AML with inv(16), and explored the in vitro effects of metformin as a potential drug to enhance chemosensitivity in this setting.

Transcriptional Phenocopies of Deleterious KEAP1 Mutations Correlate with Survival Outcomes in Lung Cancer Treated with Immunotherapy.

PURPOSE: Co-occurring mutations in KEAP1 and STK11/KRAS have emerged as determinants of survival outcomes in patients with non-small cell lung cancer (NSCLC) treated with immunotherapy. However, these mutational contexts identify a fraction of nonresponders to immune checkpoint inhibitors. We hypothesized that KEAP1 wild-type tumors recapitulate the transcriptional footprint of KEAP1 mutations and that this KEAPness phenotype can determine immune responsiveness with higher precision compared to mutation-based models. EXPERIMENTAL DESIGN: The Cancer Genome Atlas was used to infer the KEAPness phenotype and explore its immunological correlates at the pan-cancer level. The association between KEAPness and survival outcomes was tested in two independent cohorts of patients with advanced NSCLC treated with immunotherapy and profiled by RNA sequencing (SU2C n = 153; OAK/POPLAR n = 439). The NSCLC TRACERx421 multiregion sequencing study (tumor regions, n = 947) was used to investigate evolutionary trajectories. RESULTS: KEAPness-dominant tumors represented 50% of all NSCLCs and were associated with shorter progression-free survival (PFS) and overall survival (OS) compared to KEAPness-free cases in independent cohorts of patients with NSCLC treated with immunotherapy (SU2C PFS P = 0.042, OS P = 0.008; OAK/POPLAR PFS P = 0.0014, OS P < 0.001). Patients with KEAPness tumors had survival outcomes comparable to those with KEAP1-mutant tumors. In the TRACERx421, KEAPness exhibited limited transcriptional intratumoral heterogeneity and immune exclusion, resembling the KEAP1-mutant disease. This phenotypic state occurred across genetically divergent tumors, exhibiting shared and private cancer genes under positive selection when compared to KEAP1-mutant tumors. CONCLUSIONS: We identified a KEAPness phenotype across evolutionary divergent tumors. KEAPness outperforms mutation-based classifiers as a biomarker of inferior survival outcomes in patients with NSCLC treated with immunotherapy.

Hippo pathway dysregulation in gastric cancer: from Helicobacter pylori infection to tumor promotion and progression.

The Hippo pathway plays a critical role for balancing proliferation and differentiation, thus regulating tissue homeostasis. The pathway acts through a kinase cascade whose final effectors are the Yes-associated protein (YAP) and its paralog transcriptional co­activator with PDZ­binding motif (TAZ). In response to a variety of upstream signals, YAP and TAZ activate a transcriptional program that modulates cellular proliferation, tissue repair after injury, stem cell fate decision, and cytoskeletal reorganization. Hippo pathway signaling is often dysregulated in gastric cancer and in Helicobacter pylori-induced infection, suggesting a putative role of its deregulation since the early stages of the disease. In this review, we summarize the architecture and regulation of the Hippo pathway and discuss how its dysregulation fuels the onset and progression of gastric cancer. In this setting, we also focus on the crosstalk between Hippo and other established oncogenic signaling pathways. Lastly, we provide insights into the therapeutic approaches targeting aberrant YAP/TAZ activation and discuss the related clinical perspectives and challenges.

Charting the transcriptomic landscape of primary and metastatic cancers in relation to their origin and target normal tissues.

Metastasis is a leading cause of cancer-related deaths, yet understanding how metastatic tumors adapt from their origin to target tissues is challenging. To address this, we assessed whether primary and metastatic tumors resemble their tissue of origin or target tissue in terms of gene expression. We analyzed gene expression profiles from various cancer types, including single-cell and bulk RNA-seq data, in both paired and unpaired primary and metastatic patient cohorts. We quantified the transcriptomic distances between tumor samples and their normal tissues, revealing that primary tumors are more similar to their tissue of origin, while metastases shift towards the target tissue. Pathway-level analysis highlighted critical transcriptomic changes during metastasis. Notably, primary cancers exhibited higher activity in cancer hallmarks, including Activating Invasion and Metastasis , compared to metastatic cancers. This comprehensive landscape analysis provides insight into how cancer tumors adapt to their metastatic environments, providing a transcriptome-wide view of the processes involved.

The long non-coding RNA NEAT1 is a ΔNp63 target gene modulating epidermal differentiation.

The transcription factor ΔNp63 regulates epithelial stem cell function and maintains the integrity of stratified epithelial tissues by acting as transcriptional repressor or activator towards a distinct subset of protein-coding genes and microRNAs. However, our knowledge of the functional link between ∆Np63 transcriptional activity and long non-coding RNAs (lncRNAs) expression is quite limited. Here, we show that in proliferating human keratinocytes ∆Np63 represses the expression of the lncRNA NEAT1 by recruiting the histone deacetylase HDAC1 to the proximal promoter of NEAT1 genomic locus. Upon induction of differentiation, ∆Np63 down-regulation is associated by a marked increase of NEAT1 RNA levels, resulting in an increased assembly of paraspeckles foci both in vitro and in human skin tissues. RNA-seq analysis associated with global DNA binding profile (ChIRP-seq) revealed that NEAT1 associates with the promoter of key epithelial transcription factors sustaining their expression during epidermal differentiation. These molecular events might explain the inability of NEAT1-depleted keratinocytes to undergo the proper formation of epidermal layers. Collectively, these data uncover the lncRNA NEAT1 as an additional player of the intricate network orchestrating epidermal morphogenesis.

DARPP-32 and t-DARPP in the development of resistance to anti-HER2 agents. Pre-clinical evidence from the STEP study.

The therapeutic scenario of Human Epidermal Growth Factor Receptor 2 positive advanced breast cancer (ABC) has been recently enriched by a number of innovative agents, which are reshaping treatment sequence. While randomized trials have documented an advantage in terms of efficacy, for the newly available agents we lack effectiveness and tolerability evidence from the real-world setting. Similarly, the identification of predictive biomarkers might improve clinical decision. We herein describe the outline of a prospective/retrospective study which aims to explore the optimal sequence of treatment in HER2+, pertuzumab pre-treated ABC patients treated in II line with anti-HER2 agents in clinical practice. As part of the pre-clinical tasks envisioned by the STEP study, in vitro cell models of resistance were exploited to investigate molecular features associated with reduced efficacy of HER2 targeting agents at the transcript level. The aggressive behavior of resistant cell populations was measured by growth assessment in mouse models. This approach led to the identification of DARPP-32 and t-DARPP proteins as possible predictive biomarkers of efficacy of anti-HER2 agents. Biomarkers validation and the clinical goals will be reached through patients' inclusion into two independent cohorts, i.e., the prospective and retrospective cohorts, whose setup is currently ongoing.

HSF-1/miR-145-5p transcriptional axis enhances hyperthermic intraperitoneal chemotherapy efficacy on peritoneal ovarian carcinosis.

Hyperthermic intraperitoneal administration of chemotherapy (HIPEC) increases local drug concentrations and reduces systemic side effects associated with prolonged adjuvant intraperitoneal exposure in patients affected by either peritoneal malignancies or metastatic diseases originating from gastric, colon, kidney, and ovarian primary tumors. Mechanistically, the anticancer effects of HIPEC have been poorly explored. Herein we documented that HIPEC treatment promoted miR-145-5p expression paired with a significant downregulation of its oncogenic target genes c-MYC, EGFR, OCT4, and MUC1 in a pilot cohort of patients with ovarian peritoneal metastatic lesions. RNA sequencing analyses of ovarian peritoneal metastatic nodules from HIPEC treated patients unveils HSF-1 as a transcriptional regulator factor of miR-145-5p expression. Notably, either depletion of HSF-1 expression or chemical inhibition of its transcriptional activity impaired miR-145-5p tumor suppressor activity and the response to cisplatin in ovarian cancer cell lines incubated at 42 °C. In aggregate, our findings highlight a novel transcriptional network involving HSF-1, miR145-5p, MYC, EGFR, MUC1, and OCT4 whose proper activity contributes to HIPEC anticancer efficacy in the treatment of ovarian metastatic peritoneal lesions.

A diagnostic circulating miRNA signature as orchestrator of cell invasion via TKS4/TKS5/EFHD2 modulation in human gliomas.

BACKGROUND: Altered microRNA profiles have been observed not only in tumour tissues but also in biofluids, where they circulate in a stable form thus representing interesting biomarker candidates. This study aimed to identify a microRNA signature as a non-invasive biomarker and to investigate its impact on glioma biology. METHODS: MicroRNAs were selected using a global expression profile in preoperative serum samples from 37 glioma patients. Comparison between serum samples from age and gender-matched controls was performed by using the droplet digital PCR. The ROC curve and Kaplan-Meier survival analyses were used to evaluate the diagnostic/prognostic values. The functional role of the identified signature was assessed by gain/loss of function strategies in glioma cells. RESULTS: A three-microRNA signature (miR-1-3p/-26a-1-3p/-487b-3p) was differentially expressed in the serum of patients according to the isocitrate dehydrogenase (IDH) genes mutation status and correlated with both patient Overall and Progression Free Survival. The identified signature was also downregulated in the serum of patients compared to controls. Consistent with these results, the signature expression and release in the conditioned medium of glioma cells was lower in IDH-wild type cells compared to the mutated counterpart. Furthermore, in silico analysis of glioma datasets showed a consistent deregulation of the signature according to the IDH mutation status in glioma tumour tissues. Ectopic expression of the signature negatively affects several glioma functions. Notably, it impacts the glioma invasive phenotype by directly targeting the invadopodia-related proteins TKS4, TKS5 and EFHD2. CONCLUSIONS: We identified a three microRNA signature as a promising complementary or even an independent non-invasive diagnostic/prognostic biomarker. The signature displays oncosuppressive functions in glioma cells and impacts on proteins crucial for migration and invasion, providing potential targets for therapeutic intervention.

KEAP1-Mutant NSCLC: The Catastrophic Failure of a Cell-Protecting Hub.

Mutations in the KEAP1-NRF2 pathway are common in NSCLC, albeit with a prevalence of KEAP1 mutations in lung adenocarcinoma and an equal representation of KEAP1 and NFE2L2 (the gene encoding for NRF2) alterations in lung squamous cell carcinoma. The KEAP1-NRF2 axis is a crucial modulator of cellular homeostasis, enabling cells to tolerate oxidative and metabolic stresses, and xenobiotics. The complex cytoprotective response orchestrated by NRF2-mediated gene transcription embraces detoxification mechanisms, ferroptosis protection, and metabolic reprogramming. Given that the KEAP1-NRF2 pathway controls core cellular functions, it is not surprising that a number of clinical studies connected KEAP1 mutations to increased resistance to chemotherapy, radiotherapy, and targeted agents. More recently, an immune-cold tumor microenvironment was described as a typical feature of KEAP1-mutant lung adenocarcinoma. Consistently, a reduced efficacy of immunotherapy was reported in the KEAP1-mutant background. Nevertheless, the connection between KEAP1 and immune resistance seems more complex and dependent on coexisting genomic alterations. Given the clinical implications of deregulated KEAP1-NRF2 pathway in lung cancer, the development of pathway-directed anticancer treatments should be considered a priority in the domain of thoracic oncology.

SEMA6A/RhoA/YAP axis mediates tumor-stroma interactions and prevents response to dual BRAF/MEK inhibition in BRAF-mutant melanoma.

BACKGROUND: Despite the promise of dual BRAF/MEK inhibition as a therapy for BRAF-mutant (BRAF-mut) melanoma, heterogeneous responses have been observed in patients, thus predictors of benefit from therapy are needed. We have previously identified semaphorin 6A (SEMA6A) as a BRAF-mut-associated protein involved in actin cytoskeleton remodeling. The purpose of the present study is to dissect the role of SEMA6A in the biology of BRAF-mut melanoma, and to explore its predictive potential towards dual BRAF/MEK inhibition. METHODS: SEMA6A expression was assessed by immunohistochemistry in melanoma cohort RECI1 (N = 112) and its prognostic potential was investigated in BRAF-mut melanoma patients from DFCI and TCGA datasets (N = 258). The molecular mechanisms regulated by SEMA6A to sustain tumor aggressiveness and targeted therapy resistance were investigated in vitro by using BRAF-mut and BRAF-wt melanoma cell lines, an inducible SEMA6A silencing cell model and a microenvironment-mimicking fibroblasts-coculturing model. Finally, SEMA6A prediction of benefit from dual BRAF/MEK inhibition was investigated in melanoma cohort RECI2 (N = 14). RESULTS: Our results indicate higher protein expression of SEMA6A in BRAF-mut compared with BRAF-wt melanoma patients and show that SEMA6A is a prognostic indicator in BRAF-mut melanoma from TCGA and DFCI patients cohorts. In BRAF-mut melanoma cells, SEMA6A coordinates actin cytoskeleton remodeling by the RhoA-dependent activation of YAP and dual BRAF/MEK inhibition by dabrafenib+trametinib induces SEMA6A/RhoA/YAP axis. In microenvironment-mimicking co-culture condition, fibroblasts confer to melanoma cells a proliferative stimulus and protect them from targeted therapies, whereas SEMA6A depletion rescues the efficacy of dual BRAF/MEK inhibition. Finally, in BRAF-mut melanoma patients treated with dabrafenib+trametinib, high SEMA6A predicts shorter recurrence-free interval. CONCLUSIONS: Overall, our results indicate that SEMA6A contributes to microenvironment-coordinated evasion of melanoma cells from dual BRAF/MEK inhibition and it might be a good candidate predictor of short-term benefit from dual BRAF/MEK inhibition.

KEAP1 and TP53 Frame Genomic, Evolutionary, and Immunologic Subtypes of Lung Adenocarcinoma With Different Sensitivity to Immunotherapy.

INTRODUCTION: The connection between driver mutations and the efficacy of immune checkpoint inhibitors is the focus of intense investigations. In lung adenocarcinoma (LUAD), KEAP1/STK11 alterations have been tied to immunoresistance. Nevertheless, the heterogeneity characterizing immunotherapy efficacy suggests the contribution of still unappreciated events. METHODS: Somatic interaction analysis of top-ranking mutant genes in LUAD was carried out in the American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia Information Exchange (GENIE) (N = 6208). Mutational processes, intratumor heterogeneity, evolutionary trajectories, immunologic features, and cancer-associated signatures were investigated, exploiting multiple data sets (AACR GENIE, The Cancer Genome Atlas [TCGA], TRAcking Cancer Evolution through therapy [Rx]). The impact of the proposed subtyping on survival outcomes was assessed in two independent cohorts of immune checkpoint inhibitor-treated patients: the tissue-based sequencing cohort (Rome/Memorial Sloan Kettering Cancer Center/Dana-Farber Cancer Institute, tissue-based next-generation sequencing [NGS] cohort, N = 343) and the blood-based sequencing cohort (OAK/POPLAR trials, blood-based NGS cohort, N = 304). RESULTS: Observing the neutral interaction between KEAP1 and TP53, KEAP1/TP53-based subtypes were dissected at the molecular and clinical levels. KEAP1 single-mutant (KEAP1 SM) and KEAP1/TP53 double-mutant (KEAP1/TP53 DM) LUAD share a transcriptomic profile characterized by the overexpression of AKR genes, which are under the control of a productive superenhancer with NEF2L2-binding signals. Nevertheless, KEAP1 SM and KEAP1/TP53 DM tumors differ by mutational repertoire, degree of intratumor heterogeneity, evolutionary trajectories, pathway-level signatures, and immune microenvironment composition. In both cohorts (blood-based NGS and tissue-based NGS), KEAP1 SM tumors had the shortest survival; the KEAP1/TP53 DM subgroup had an intermediate prognosis matching that of pure TP53 LUAD, whereas the longest survival was noticed in the double wild-type group. CONCLUSIONS: Our data provide a framework for genomically-informed immunotherapy, highlighting the importance of multimodal data integration to achieve a clinically exploitable taxonomy.

Control of replication stress and mitosis in colorectal cancer stem cells through the interplay of PARP1, MRE11 and RAD51.

Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. Here, we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. First, we demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Second, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Also, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations.