Combined PD-1 and CTLA-4 Blockade in an International Cohort of Patients with Acral Lentiginous Melanoma.

BACKGROUND: Combination immune checkpoint blockade targeting PD-1 and CTLA-4 leads to high response rates and improved survival in advanced cutaneous melanoma (CM). Less is known about the efficacy of this combination in acral lentiginous melanoma (ALM). OBJECTIVES: To determine the efficacy of combination immune checkpoint blockade targeting PD-1 and CTLA-4 in a real-world, diverse population of ALM. METHODS: This multi-institutional retrospective study analyzed patients with histologically confirmed ALM treated with the combination of PD-1 and CTLA-4 inhibitors between 2010-2022. The primary objective of the study was objective response rate (ORR) per RECIST criteria. The secondary objectives were progression-free survival (PFS) and overall survival (OS). RESULTS: In total, 109 patients with advanced ALM treated with combined PD-1 and CTLA-4 blockade in any line of treatment were included. The majority of patients had stage IV disease (n=81, 74.2%). The ORR for the entire cohort was 18.3% (95% CI 11.6-26.9%), with 9 (8.3%) complete responses (CR) and 11 (10.1%) partial responses (PR). An additional 22 patients (20.2%) had stable disease (SD), and the disease control rate (DCR) was 38.5%. The median PFS was 4.2 months [95% CI 3.25-5.62], while the median OS was 17 months [95% CI 12.4%-23.1%]. A total of 95 patients (87.2%) had a treatment-related adverse event, with 40.4% (n=44/109) experiencing at least one grade 3 or 4 toxicity. Elevated LDH (p=.04), 2+ lines of prior therapy (p=.03), and Asian race/ethnicity (p=.04) were associated with worse OS, while Hispanic/Latino race/ethnicity was associated with better OS (p=.02). CONCLUSIONS: Combination of PD-1 and CTLA-4 blockade is less effective for ALM, as compared to CM, despite similar toxicity. Asian patients, in particular, appear to derive lower benefit from this regimen. Novel treatment approaches are needed for this rare melanoma subtype.

Mutually exclusive genetic interactions and gene essentiality shape the genomic landscape of primary melanoma.

Melanoma is a heterogenous malignancy with an unpredictable clinical course. Most patients who present in the clinic are diagnosed with primary melanoma, yet large-scale sequencing efforts have focused primarily on metastatic disease. In this study we sequence-profiled 524 American Joint Committee on Cancer Stage I-III primary tumours. Our analysis of these data reveals recurrent driver mutations, mutually exclusive genetic interactions, where two genes were never or rarely co-mutated, and an absence of co-occurring genetic events. Further, we intersected copy number calls from our primary melanoma data with whole-genome CRISPR screening data to identify the transcription factor interferon regulatory factor 4 (IRF4) as a melanoma-associated dependency. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Pro-Apoptotic Antitumoral Effect of Novel Acridine-Core Naphthoquinone Compounds against Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a global public health problem with high incidence and mortality. The chemotherapeutic agents used in the clinic, alone or in combination, usually lead to important side effects. Thus, the discovery and development of new antineoplastic drugs are essential to improve disease prognosis and reduce toxicity. In the present study, acridine-core naphthoquinone compounds were synthesized and evaluated for their antitumor activity in OSCC cells. The mechanism of action, pharmacokinetics, and toxicity parameters of the most promising compound was further analyzed using in silico, in vitro, and in vivo methods. Among the derivatives, compound 4e was highly cytotoxic (29.99 µM) and selective (SI 2.9) at levels comparable and generally superior to chemotherapeutic controls. Besides, compound 4e proved to be non-hemolytic, stable, and well tolerated in animals at all doses tested. Mechanistically, compound 4e promoted cell death by apoptosis in the OSCC cell, and molecular docking studies suggested this compound possibly targets enzymes important for tumor progression, such as RSK2, PKM2, and topoisomerase IIα. Importantly, compound 4e presented a pharmacological profile within desirable parameters for drug development, showing promise for future preclinical trials.

TOP1 modulation during melanoma progression and in adaptative resistance to BRAF and MEK inhibitors.

In melanomas, therapy resistance can arise due to a combination of genetic, epigenetic and phenotypic mechanisms. Due to its crucial role in DNA supercoil relaxation, TOP1 is often considered an essential chemotherapeutic target in cancer. However, how TOP1 expression and activity might differ in therapy sensitive versus resistant cell types is unknown. Here we show that TOP1 expression is increased in metastatic melanoma and correlates with an invasive gene expression signature. More specifically, TOP1 expression is highest in cells with the lowest expression of MITF, a key regulator of melanoma biology. Notably, TOP1 and DNA Single-Strand Break Repair genes are downregulated in BRAFi- and BRAFi/MEKi-resistant cells and TOP1 inhibition decreases invasion markers only in BRAFi/MEKi-resistant cells. Thus, we show three different phenotypes related to TOP1 levels: i) non-malignant cells with low TOP1 levels; ii) metastatic cells with high TOP1 levels and high invasiveness; and iii) BRAFi- and BRAFi/MEKi-resistant cells with low TOP1 levels and high invasiveness. Together, these results highlight the potential role of TOP1 in melanoma progression and resistance.

Poor clinical outcome in metastatic melanoma is associated with a microRNA-modulated immunosuppressive tumor microenvironment.

BACKGROUND: Interaction between malignant cells and immune cells that reside within the tumor microenvironment (TME) modulate different aspects of tumor development and progression. Recent works showed the importance of miRNA-containing extracellular vesicles in this crosstalk. METHODS: Interested in understanding the interplay between melanoma and immune-related TME cells, we characterized the TCGA's metastatic melanoma samples according to their tumor microenvironment profiles, HLA-I neoepitopes, transcriptome profile and classified them into three groups. Moreover, we combined our results with melanoma single-cell gene expression and public miRNA data to better characterize the regulatory network of circulating miRNAs and their targets related to immune evasion and microenvironment response. RESULTS: The group associated with a worse prognosis showed phenotypic characteristics that favor immune evasion, including a strong signature of suppressor cells and less stable neoantigen:HLA-I complexes. Conversely, the group with better prognosis was marked by enrichment in lymphocyte and MHC signatures. By analyzing publicly available melanoma single-cell RNA and microvesicle microRNAs sequencing data we identified circulating microRNAs potentially involved in the crosstalk between tumor and TME cells. Candidate miRNA/target gene pairs with previously reported roles in tumor progression and immune escape mechanisms were further investigated and demonstrated to impact patient's overall survival not only in melanoma but across different tumor types. CONCLUSION: Our results underscore the impact of tumor-microenvironment interactions on disease outcomes and reveal potential non-invasive biomarkers of prognosis and treatment response.

Abrogation of BRAFV600E-induced senescence by PI3K pathway activation contributes to melanomagenesis.

Human melanocytic nevi (moles) are benign lesions harboring activated oncogenes, including BRAF. Although this oncogene initially acts mitogenically, eventually, oncogene-induced senescence (OIS) ensues. Nevi can infrequently progress to melanomas, but the mechanistic relationship with OIS is unclear. We show here that PTEN depletion abrogates BRAF(V600E)-induced senescence in human fibroblasts and melanocytes. Correspondingly, in established murine BRAF(V600E)-driven nevi, acute shRNA-mediated depletion of PTEN prompted tumor progression. Furthermore, genetic analysis of laser-guided microdissected human contiguous nevus-melanoma specimens recurrently revealed identical mutations in BRAF or NRAS in adjacent benign and malignant melanocytes. The PI3K pathway was often activated through either decreased PTEN or increased AKT3 expression in melanomas relative to their adjacent nevi. Pharmacologic PI3K inhibition in melanoma cells suppressed proliferation and induced the senescence-associated tumor suppressor p15(INK4B). This treatment also eliminated subpopulations resistant to targeted BRAF(V600E) inhibition. Our findings suggest that a significant proportion of melanomas arise from nevi. Furthermore, these results demonstrate that PI3K pathway activation serves as a rate-limiting event in this setting, acting at least in part by abrogating OIS. The reactivation of senescence features and elimination of cells refractory to BRAF(V600E) inhibition by PI3K inhibition warrants further investigation into the therapeutic potential of simultaneously targeting these pathways in melanoma.

Protease-activated receptor 2 (PAR2) upregulates granulocyte colony stimulating factor (G-CSF) expression in breast cancer cells.

Protease-activated receptor 2 (PAR2) is a G-protein coupled receptor which is activated upon cleavage of its N-terminal region. PAR2 has been associated with many aspects regarding tumor progression, such as the production of pro-tumoral cytokines. Granulocyte colony-stimulating factor (G-CSF) is a cytokine essential to neutrophil production and maturation, and it is often overexpressed in tumors. In this study, we evaluated the ability of PAR2 to modulate G-CSF expression. PAR2 and G-CSF were significantly more expressed in metastatic (4T1 and MDA-MB-231) as compared to non-metastatic (67NR and MCF7) breast cancer cell lines. In addition, PAR2 stimulation by a synthetic agonist peptide significantly increased G-CSF gene expression in the metastatic cell lines. Knockdown of PAR2 in 4T1 cells decreased G-CSF expression and secretion. In addition, treatment of 4T1 with the commercial PAR2 antagonist, ENMD-1068, significantly decreased G-CSF expression. cBioPortal analyses of the TCGA database showed a significant co-occurrence of G-CSF and PAR2 gene overexpression in breast cancer samples. In conclusion, our data suggest that PAR2 contributes to G-CSF expression in breast cancer cells, possibly favoring tumor progression.

PhosphoPath: Visualization of Phosphosite-centric Dynamics in Temporal Molecular Networks.

Protein phosphorylation is an essential post-translational modification (PTM) regulating many biological processes at the cellular and multicellular level. Continuous improvements in phosphoproteomics technology allow the analysis of this PTM in an expanding biological content, yet up until now proteome data visualization tools are still very gene centric, hampering the ability to comprehensively map and study PTM dynamics. Here we present PhosphoPath, a Cytoscape app designed for the visualization and analysis of quantitative proteome and phosphoproteome data sets. PhosphoPath brings knowledge into the biological network by importing publically available data and enables PTM site-specific visualization of information from quantitative time series. To showcase PhosphoPath performance we use a quantitative proteomics data set comparing patient-derived melanoma cell lines grown in either conventional cell culture or xenografts.

ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma.

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down-regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.

Ancestry and somatic profile predict acral melanoma origin and prognosis.

Acral melanoma, which is not ultraviolet (UV)-associated, is the most common type of melanoma in several low- and middle-income countries including Mexico. Latin American samples are significantly underrepresented in global cancer genomics studies, which directly affects patients in these regions as it is known that cancer risk and incidence may be influenced by ancestry and environmental exposures. To address this, here we characterise the genome and transcriptome of 128 acral melanoma tumours from 96 Mexican patients, a population notable because of its genetic admixture. Compared with other studies of melanoma, we found fewer frequent mutations in classical driver genes such as BRAF, NRAS or NF1. While most patients had predominantly Amerindian genetic ancestry, those with higher European ancestry had increased frequency of BRAF mutations and a lower number of structural variants. These BRAF-mutated tumours have a transcriptional profile similar to cutaneous non-volar melanocytes, suggesting that acral melanomas in these patients may arise from a distinct cell of origin compared to other tumours arising in these locations. KIT mutations were found in a subset of these tumours, and transcriptional profiling defined three expression clusters; these characteristics were associated with overall survival. We highlight novel low-frequency drivers, such as SPHKAP, which correlate with a distinct genomic profile and clinical characteristics. Our study enhances knowledge of this understudied disease and underscores the importance of including samples from diverse ancestries in cancer genomics studies.

[Have you eaten any DNA today? Science communication during Science and Technology Week in Brazil]. / Você já comeu DNA hoje? Divulgação científica durante a Semana da Ciência e Tecnologia no Brasil.

During the first National Science and Technology Week held in 2004, science centers and museums, universities and schools engaged in activities with the idea of divulging science to the people. Demonstrations of the extraction of DNA from fruits were conducted in supermarkets in 11 Brazilian cities by two institutions, DNA Vai à Escola and Conselho de Informação e Biotecnologia. This article describes the formation of a national network of people interested in communicating information about genetics to the lay public and the implementation of a low-cost science communication activity in different parts of the country simultaneously. It also analyzes the impact caused by this initiative and the perceptions of those involved in its organization.

Acral melanoma: new insights into the immune and genomic landscape.

Acral melanoma is a rare subtype of melanoma that arises on the non-hair bearing skin of the nail bed, palms of the hand and soles of the feet. It is unique among melanomas in not being linked to ultraviolet radiation (UVR) exposure from the sun, and, as such, its incidence is similar across populations who are of Asian, Hispanic, African and European origin. Although research into acral melanoma has lagged behind that of sun-exposed cutaneous melanoma, recent studies have begun to address the unique genetics and immune features of acral melanoma. In this review we will discuss the latest progress in understanding the biology of acral melanoma across different ethnic populations and will outline how these new discoveries can help to guide the therapeutic management of this rare tumor.

Impact of clinical and histopathological characteristics on the disease-free survival of stage I-II acral melanoma patients.

BACKGROUND: Acral melanoma is rare and associated with a worse prognosis compared to cutaneous melanoma in other locations. Despite this, few studies have focused on the prognosis of acral melanoma, particularly in patients with initial clinical stage. The aim of this study was to assess the impact of clinical and histopathological characteristics on the disease-free survival (DFS) of stage I-II patients. METHODS: We analyzed 154 stage I-II acral melanoma cases, all of whom underwent a review of the histopathological and clinical parameters. Patients were divided into groups based on the presence or absence of disease recurrence within 5 years. We used Cox proportional regression to analyze independent risk factors and computed DFS curves using the Kaplan-Meier method. RESULTS: Within 5 years, 27.9% of patients experienced disease recurrence, with 90.4% occurring during the first 3 years. Univariate and multivariate analyses did not identify any clinical parameters with a significant influence on DFS. The DFS rate at 5 years was 72.7%. The median duration of disease recurrence after the initial diagnosis was 21 months. However, Breslow thickness, presence of ulceration, >3 mitosis/mm2 , presence of tumor-infiltrating lymphocytes (TIL), and perineural invasion were significantly associated with a decrease in time to first recurrence. CONCLUSIONS: Despite the favorable prognosis of stage I-II acral melanoma compared with advance stage, clinical and histopathological characteristics can impact prognosis. In addition to Breslow thickness and ulceration, attention should be paid to mitotic rate, presence of TIL, and perineural invasion to optimize follow-up of acral melanoma patients diagnosed in the initial clinical stage.

More than just acral melanoma: the controversies of defining the disease.

Acral melanoma (AM) is a malignant cutaneous melanocytic tumour specifically located on the palms, soles, and nail apparatus, which are areas of glabrous (hairless) skin. Acral lentiginous melanoma, a subtype of AM, represents a histopathological subtype diagnosis of cutaneous melanoma with unique morphological and structural features. Despite clear definitions, the misuse of these terms and the inconsistency in reporting the histopathological features of AM cases have become a major obstacle to the study of the disease. In this review, we discuss the epidemiology, histopathological features, prognosis, and genetic profile of AM, highlighting the differences observed when histopathological subtypes are considered. The increasing global effort to characterise AM cases from ethnically diverse populations would benefit greatly from a more consistent classification of the disease.

Acral lentiginous melanoma: Basic facts, biological characteristics and research perspectives of an understudied disease.

Acral lentiginous melanoma is a histological subtype of cutaneous melanoma that occurs in the glabrous skin of the palms, soles and the nail unit. Although in some countries, particularly in Latin America, Africa and Asia, it represents the most frequently diagnosed subtype of the disease, it only represents a small proportion of melanoma cases in European-descent populations, which is partially why it has not been studied to the same extent as other forms of melanoma. As a result, its unique genomic drivers remain comparatively poorly explored, as well as its causes, with current evidence supporting a UV-independent path to tumorigenesis. In this review, we discuss current knowledge of the aetiology and diagnostic criteria of acral lentiginous melanoma, as well as its epidemiological and histopathological characteristics. We also describe what is known about the genomic landscape of this disease and review the available biological models to explore potential therapeutic targets.

The role of BRAF mutation and p53 inactivation during transformation of a subpopulation of primary human melanocytes.

Melanocytic nevi frequently harbor oncogenic BRAF mutations, but only a minority progress to melanoma. In human melanocytes, persistent BRAF(V600E) expression triggers oncogene-induced senescence, which implies that bypass of oncogene-induced senescence is necessary for malignant transformation of melanocytes. We show that a subpopulation of primary human melanocytes with persistent expression of BRAF(V600E) do not enter oncogene-induced senescence, but instead survive despite heightened MAPK activity. Disruption of the p53 pathway using short-hairpin RNA initiated rapid growth of these V600E(+) melanocytes in vitro. The resultant V600E(+)/p53(sh) melanocytes grew anchorage-independently in soft agar, formed pigmented lesions reminiscent of in situ melanoma in artificial skin reconstructs, and were weakly tumorigenic in vivo. Array comparative genomic hybridization analysis demonstrated that the transformed melanocytes acquired a substantial deletion in chromosome 13, which encodes the Rb1 tumor suppressor gene. Gene expression profiling study of nevi and melanomas showed that p53 target genes were differentially expressed in melanomas compared with nevi, suggesting a dysfunctional p53 pathway in melanoma in vivo. In summary, these data demonstrate that a subpopulation of melanocytes possesses the ability to survive BRAF(V600E)-induced senescence, and suggest that p53 inactivation may promote malignant transformation of these cells.

Extracellular vesicle-mediated crosstalk between melanoma and the immune system: Impact on tumor progression and therapy response.

Melanoma is a very lethal tumor type that easily spreads and colonizes regional and distant tissues. Crucial phenotypic changes that favor melanoma metastasis are interposed by the tumor microenvironment (TME), representing a complex network in which malignant cells communicate not only with each other but also with stromal and immune cells. This cell-cell communication can be mediated by extracellular vesicles (EVs), which are lipid bilayer-delimited particles capable of carrying a wide variety of bioactive compounds. Both melanoma-derived or TME-derived EVs deliver important pro- and antitumor signals implicated in various stages of tumor progression, such as proliferation, metastasis, and treatment response. In this review, we highlight the recent advances in EV-mediated crosstalk between melanoma and immune cells and other important cells of the TME, and address different aspects of this bidirectional interaction as well as how this may hinder or trigger the development and progression of melanoma. We also discuss the potential of using EVs as biomarkers and therapeutic strategies for melanoma.

Integrated in vivo genetic and pharmacologic screening identifies co-inhibition of EGRF and ROCK as a potential treatment regimen for triple-negative breast cancer.

Breast cancer is the second most common cause of cancer-related deaths worldwide among women. Despite several therapeutic options, 15% of breast cancer patients succumb to the disease owing to tumor relapse and acquired therapy resistance. Particularly in triple-negative breast cancer (TNBC), developing effective treatments remains challenging owing to the lack of a common vulnerability that can be exploited by targeted approaches. We have previously shown that tumor cells have different requirements for growth in vivo than in vitro. Therefore, to discover novel drug targets for TNBC, we performed parallel in vivo and in vitro genetic shRNA dropout screens. We identified several potential drug targets that were required for tumor growth in vivo to a greater extent than in vitro. By combining pharmacologic inhibitors acting on a subset of these candidates, we identified a synergistic interaction between EGFR and ROCK inhibitors. This combination effectively reduced TNBC cell growth by inducing cell cycle arrest. These results illustrate the power of in vivo genetic screens and warrant further validation of EGFR and ROCK as combined pharmacologic targets for breast cancer.

Cooperative induction of apoptosis in NRAS mutant melanoma by inhibition of MEK and ROCK.

No effective targeted therapy is currently available for NRAS mutant melanoma. Experimental MEK inhibition is rather toxic and has only limited efficacy in clinical trials. At least in part, this is caused by the emergence of drug resistance, which is commonly seen for single agent treatment and shortens clinical responses. Therefore, there is a dire need to identify effective companion drug targets for NRAS mutant melanoma. Here, we show that at concentrations where single drugs had little effect, ROCK inhibitors GSK269962A or Fasudil, in combination with either MEK inhibitor GSK1120212 (Trametinib) or ERK inhibitor SCH772984 cooperatively caused proliferation inhibition and cell death in vitro. Simultaneous inhibition of MEK and ROCK caused induction of BimEL , PARP, and Puma, and hence apoptosis. In vivo, MEK and ROCK inhibition suppressed growth of established tumors. Our findings warrant clinical investigation of the effectiveness of combinatorial targeting of MAPK/ERK and ROCK in NRAS mutant melanoma.