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The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial-mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein., Competing Interests: The authors declare no conflicts of interest. The funding organization had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abstract: Trichilemmoma is a benign cutaneous neoplasm that recapitulates the outer root sheath of the hair follicle. Trichilemmomas may occur sporadically or in association with Cowden syndrome, which is characterized by germline mutations in the lipid phosphatase PTEN (phosphatase and tensin homolog on chromosome 10). Interestingly, most sporadic trichilemmomas do not show PTEN aberrations, but rather activating mutations in HRAS. Despite these important advances, a comprehensive genetic analysis of trichilemmoma has not been reported. Here, we used a next-generation DNA sequencing platform to study 9 sporadic trichilemmoma cases. Seven cases (7/9; 78%) harbored activating mutations in HRAS, consistent with previous findings. Unexpectedly, we identified recurrent mutations in the tyrosine phosphatase PTPN14 (protein tyrosine phosphatase nonreceptor type 14) in 4 cases (4/9; 44%). Three of these cases also harbored HRAS mutations, whereas one case occurred in the absence of a HRAS mutation and showed evidence of biallelic inactivation of PTPN14. Finally, one case (1/9; 11%) showed biallelic inactivation of PTEN in the absence of a HRAS (or PTPN14) mutation. These data suggest at least 3 distinct pathways of molecular pathogenesis in sporadic trichilemmoma and identify PTPN14 as a potentially important contributor to trichilemmoma biology., Competing Interests: J. Hanna has disclosed that he is a recipient of research funding from the Bertarelli Rare Cancers Fund. Lippincott CME Institute has identified and resolved all conflicts of interest concerning this educational activity., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

A common limitation of cancer treatments is chemotherapy resistance. We have previously identified that endothelial cell (EC)-specific deletion of focal adhesion kinase (FAK) sensitises tumour cells to DNA-damaging therapies, reducing tumour growth in mice. The present study addressed the kinase activity dependency of EC FAK sensitisation to the DNA-damaging chemotherapeutic drug, doxorubicin. FAK is recognised as a therapeutic target in tumour cells, leading to the development of a range of inhibitors, the majority being ATP competitive kinase inhibitors. We demonstrate that inactivation of EC FAK kinase domain (kinase dead; EC FAK-KD) in established subcutaneous B16F0 tumours improves melanoma cell sensitisation to doxorubicin. Doxorubicin treatment in EC FAK-KD mice reduced the percentage change in exponential B16F0 tumour growth further than in wild-type mice. There was no difference in tumour blood vessel numbers, vessel perfusion or doxorubicin delivery between genotypes, suggesting a possible angiocrine effect on the regulation of tumour growth. Doxorubicin reduced perivascular malignant cell proliferation, while enhancing perivascular tumour cell apoptosis and DNA damage in tumours grown in EC FAK-KD mice 48 h after doxorubicin injection. Human pulmonary microvascular ECs treated with the pharmacological FAK kinase inhibitors defactinib, PF-562,271 or PF-573,228 in combination with doxorubicin also reduced cytokine expression levels. Together, these data suggest that targeting EC FAK kinase activity may alter angiocrine signals that correlate with improved acute tumour cell chemosensitisation. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland., (© 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.)

Superficial angiomyxomas (SAMs) are benign cutaneous tumors that arise de novo and in the setting of the Carney complex (CC), an autosomal dominant disease with several cutaneous manifestations including lentigines and pigmented epithelioid melanocytomas. Although most SAM do not pose a diagnostic challenge, a subset can demonstrate histopathologic overlap with other myxoid tumors that arise in the skin and subcutis. Traditional immunohistochemical markers are of limited utility when discriminating SAM from histopathologic mimics. Since protein kinase A regulatory subunit 1 alpha (PRKAR1A) genetic alterations underlie most CC cases, we investigated whether SAM demonstrate loss of PRKAR1A protein expression by immunohistochemistry. In our series, 29 SAM, 26 myxofibrosarcoma, 5 myxoid dermatofibrosarcoma protuberans, 11 superficial acral fibromyxomas, and 18 digital mucous cysts were characterized. Of the 29 SAM examined in this study, 1 was associated with documented CC in a 5-year-old girl. SAM tended to arise in adults (mean 49.7 y; range: 5 to 87 y). Loss of PRKAR1A was seen in 55.2% of cases (16/29) and had a male predilection (87.5%, 12/16). PRKAR1A-inactivated SAM demonstrated significant nuclear enlargement (100%, 16/16 vs. 23.1%, 3/13), multinucleation (81.3%, 13/16 vs. 23.1%, 3/13), and presence of neutrophils (43.8%, 7/16 vs. 0%, 0/13). In contrast, PRKAR1A was retained in all cases of myxofibrosarcoma (100%, 26/26), myxoid dermatofibrosarcoma protuberans (100%, 5/5), superficial acral fibromyxomas (100%, 11/11), and digital mucous cyst (100%, 18/18). Taken together, PRKAR1A loss by immunohistochemistry can be used as an adjunctive assay to support the diagnosis of SAM given the high specificity of this staining pattern compared with histopathologic mimics., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Melanoma cells expressing mutant B-RAF V600E are susceptible to treatment with the combination of a B-RAF inhibitor and a MEK1/2 inhibitor. We investigated the impact of the ERBB family and MAP4K inhibitor neratinib on the biology of PDX isolates of cutaneous melanoma expressing B-RAF V600E. Neratinib synergized with HDAC inhibitors to kill melanoma cells at their physiologic concentrations. Neratinib activated ATM, AMPK, ULK1, and PERK and inactivated mTORC1/2, ERK1/2, eIF2 alpha, and STAT3. Neratinib increased expression of Beclin1, ATG5, CD95, and FAS-L and decreased levels of multiple toxic BH3 domain proteins, MCL1, BCL-XL, FLIP-s, and ERBB1/2/4. ATG13 S318 phosphorylation and autophagosome formation was dependent upon ATM, and activation of ATM was dependent on reactive oxygen species. Reduced expression of ERBB1/2/4 required autophagosome formation and reduced MCL1/BCL-XL levels required eIF2 alpha phosphorylation. Maximal levels of eIF2 alpha phosphorylation required signaling by ATM-AMPK and autophagosome formation. Knock down of eIF2 alpha, CD95, FAS-L, Beclin1, and ATG5 or over-expression of FLIP-s significantly reduced killing. Combined knock down of Beclin1 and CD95 abolished cell death. Our data demonstrate that PDX melanoma cells expressing B-RAF V600E are susceptible to being killed by neratinib and more so when combined with HDACi., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The aim of this study is to explore the role of mammalian target of rapamycin (mTOR) in cutaneous squamous cell carcinoma (CSCC), Bowen's disease (BD), and actinic keratosis (AK) with squamous cell differentiation abnormality and its relationship with the degree of tumor proliferation. Thirty cases of clinical paraffin specimens of CSCC, BD, and AK were each collected from Jinhua Fifth Hospital, while 30 cases of normal skin specimens surgically resected in Department of Plastic Surgery were selected as controls. The expressions of mTOR and Ki-67 in tissues were detected by immunohistochemical staining. The positive expression rate of mTOR in the CSCC group was higher than those in the BD group and AK group ( P < 0.05), while it was higher in the BD group and AK group than in the normal skin group ( P < 0.05). The CSCC group had a higher positive expression rate of Ki-67 than the AK group ( P < 0.01). The results of logistic regression analysis showed that the pathogenic site [odds ratio (OR) = 1.189, 95% confidence interval (95%CI): 1.028-1.381, P = 0.021], course of disease (OR = 2.059, 95%CI: 1.036-4.087, P = 0.043), and differentiation degree (OR = 1.325, 95%CI: 1.169-1.512, P = 0.001) were independent factors for the positive expression of mTOR. OR>1, indicating that the factor is a risk factor. The expression levels of mTOR in CSCC, BD, and AK were positively correlated with the expression level of Ki-67 ( r = 0.827, P < 0.01, r = 0.608, P < 0.01, r = 0.368, P = 0.045). These results suggest that mTOR may be involved in the pathogenesis of CSCC, and related to the proliferation degree of CSCC, as an index reflecting the proliferation status of CSCC.

Spindle and kinetochore-associated complex subunit 3 (SKA3) is reportedly a key contributor to the progression of various cancers. The present work aimed to evaluate the possible role of SKA3 in cutaneous melanoma (CM). A high SKA3 level was found in CM tissues and predicted a poor prognosis. SKA3 silencing markedly repressed the proliferation, invasion, and epithelial-mesenchymal transition and induced the apoptosis of CM cells. SKA3 silencing decreased the phosphorylation of PI3K and Akt. Akt inhibition markedly reversed SKA3 overexpression-induced oncogenic effects on CM cells. SKA3 silencing significantly prohibited the formation and growth of CM-derived xenograft tumors in nude mice in vivo. Our findings demonstrated SKA3 inhibition repressed the progression of CM by downregulating the PI3K/Akt pathway. This study indicates that SKA3 has potential as an anticancer candidate for CM., (© 2021 Wiley Periodicals LLC.)

Cell death provides host defense and maintains homeostasis. Zα-containing molecules are essential for these processes. Z-DNA binding protein 1 (ZBP1) activates inflammatory cell death, PANoptosis, whereas adenosine deaminase acting on RNA 1 (ADAR1) serves as an RNA editor to maintain homeostasis. Here, we identify and characterize ADAR1's interaction with ZBP1, defining its role in cell death regulation and tumorigenesis. Combining interferons (IFNs) and nuclear export inhibitors (NEIs) activates ZBP1-dependent PANoptosis. ADAR1 suppresses this PANoptosis by interacting with the Zα2 domain of ZBP1 to limit ZBP1 and RIPK3 interactions. Adar1 fl/fl LysM cre mice are resistant to development of colorectal cancer and melanoma, but deletion of the ZBP1 Zα2 domain restores tumorigenesis in these mice. In addition, treating wild-type mice with IFN-γ and the NEI KPT-330 regresses melanoma in a ZBP1-dependent manner. Our findings suggest that ADAR1 suppresses ZBP1-mediated PANoptosis, promoting tumorigenesis. Defining the functions of ADAR1 and ZBP1 in cell death is fundamental to informing therapeutic strategies for cancer and other diseases., Competing Interests: Declaration of interests St. Jude Children’s Research hospital filed a provisional patent application on the IFN and NEI treatment strategy described in this study, listing R.K. and T.-D.K. as inventors (serial no. 63/196,986)., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Melanoma brain metastases (MBM) have a poor prognosis. Systemic treatments that have improved outcomes in advanced melanoma have been shown to have an intracranial (IC) effect. We studied the efficacy and outcomes of combined immune checkpoint inhibitor ipilimumab/nivolumab (Combi-ICI) or targeted therapy (Combi-TT) as first-line treatment in MBM., Methods: MBM patients treated with Combi-ICI or Combi-TT within 3 months after MBM diagnosis. Endpoints were progression-free survival (PFS) and overall survival (OS)., Results: 53 patients received Combi-ICI, 32% had symptomatic MBM and 33.9% elevated LDH. 71.7% required local treatment. The disease control rate was 60.3%. IC response rate (RR) was 43.8% at 3-months with durable responses at 6- (46.5%) and 12-months (53.1%). Extracranial (EC) RR was 44.7% at 3-months and 50% at 12-months. Median PFS was 9.6 months (95% CI 3.6-NR) and median overall survival (mOS) 44.8 months (95% CI; 26.2-NR). 63 patients received Combi-TT, 55.6% of patients had symptomatic MBM, 57.2% of patients had elevated LDH and 68.3% of patients required local treatment. The disease control rate was 60.4%. ICRR was 50% at 3-months, but dropped at 6-months (20.9%). ECRR was 69.2% at 3-months and 17.6% at 12-months. Median PFS was 5.8 months (95% CI 4.2-7.6) and mOS 14.2 months (95% CI 8.99-26.8). In BRAFV600 patients, 26.7% of patients received Combi-ICI and 73.3% Combi-TT with OS (p = 0.0053) and mPFS (p = 0.03) in favour to Combi-ICI., Conclusion: Combi-ICI showed prolonged mOS with sustainable IC and EC responses. Despite the initially increased efficacy, Combi-TT responses at 12 months were low. Combi-ICI appeared superior to Combi-TT for OS and PFS in BRAFV600 patients. Other clinical factors are determinants for first-line treatment choice., Competing Interests: Conflict of interest statement RD has intermittent, project focused consulting and/or advisory relationships with Novartis, Merck Sharp & Dhome (MSD), Bristol-Myers Squibb (BMS), Roche, Amgen, Takeda, Pierre Fabre outside the submitted work. JM has intermittent project focused consultant or advisory relationships with Merck/Pfizer, Merck Sharp & Dohme, Amgen, Novartis and Pierre Fabre and has received travel support from Ultrasun, L’ oreal, Merck Sharp & Dohme, Bristol Myers and Squibb und Pierre Fabre outside of the submitted work. PL has received honoraria from Bristol-Myers Squibb (BMS) and Pfizer. PB receives travel support from MSD and honoraria from Novartis. GAM has been a consultant or advisor for Provectus; received research funding from Pfizer, Celgene, and Ventana; and has had travel accommodation and expenses paid for by Roche and Novartis. OM reports grants and personal fees from Bristol Myers Squibb (BMS), grants and personal fees from MSD, personal fees from Roche, outside the submitted work. LZ reports honoraria from Roche, BMS, MSD, Novartis, Pierre Fabre; Consultant or Advisory Role: BMS, Novartis, Pierre Fabre, Sunpharma, Sanofi, MSD; Research funding to institution: Novartis; Travel support: BMS, Pierre Fabre, Sanofi, Amgen, Novartis, Sunpharma. MPL receives project specific research funding, unrelated to the work presented here, from Roche, Novartis, Molecular Partners, and Oncobit AG. FD, CLG, MLH, KK and PC have declared no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Fumarate hydratase (FH), encoded by the FH gene, is an enzyme which catalyses the conversion of fumarate to L-malate as part of the tricarboxylic acid cycle. Biallelic germline mutations in FH result in fumaric aciduria, a metabolic disorder resulting in severe neurological and developmental abnormalities. Heterozygous germline mutations in FH result in hereditary leiomyomatosis and renal cell carcinoma, a cancer predisposition syndrome. FH deficiency has multiple oncogenic mechanisms including through promotion of aerobic glycolysis, induction of pseudohypoxia, post-translational protein modification and impairment of DNA damage repair by homologous recombination. FH-deficient neoplasms can present with characteristic morphological features that raise suspicion for FH alterations and also frequently demonstrate loss of FH immunoreactivity and intracellular accumulation of 2-succinocysteine, also detected by immunohistochemistry., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

The 1858C>T allele of the tyrosine phosphatase PTPN22 (causing amino acid substitution R620W in encoded protein lymphoid tyrosine phosphatase) is present in 5-10% of the North American population and is strongly associated with numerous autoimmune diseases. Although much research has been done to define how this allele potentiates autoimmunity, the influence PTPN22 and its proautoimmune allele have in tumor immunity is poorly defined. To interrogate the role this allele may have in the antitumor immune response, we used CRISPR/Cas9 to generate mice in which the ortholog of lymphoid tyrosine phosphatase, PEST domain-enriched protein (PEP), is mutated at position 619 to produce the relevant proautoimmune mutation (R619W). Results of this study show that mice homozygous for this alteration (PEP-619WW) resist tumor growth as compared with wild-type mice. Consistent with these results, tumors from PEP-619WW mice have more CD45 infiltrates containing more activated CD8 T cells and CD4 T cells. In addition, there are more conventional dendritic cell type 1 (cDC1) cells and fewer myeloid-derived suppressor cells in tumors from PEP-619WW mice. Interestingly, the tumor-infiltrating PEP-619WW cDC1 cells have decreased PD-L1 expression compared with cDC1 cells from PEP-wild-type mice. Taken together, our data show that the proautoimmune allele of Ptpn22 drives a strong antitumor response in innate and adaptive immune cells resulting in superior control of tumors., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Immune escape is an early phenomenon in cancer development/progression. Indoleamine 2,3-dioxygenase 1 (IDO1) is a normal endogenous mechanism of acquired peripheral immune tolerance and may therefore be tumor-promoting. This study investigated the clinical relevance of IDO1 expression by immune cells in the lymph nodes and blood and of the serum kynurenine/tryptophan (Kyn/Trp) ratio in 65 systemic treatment naïve stage I-III melanoma patients. Blood samples were collected within the first year of diagnosis. Patients had a median follow-up of 61 months. High basal IDO1 expression in peripheral monocytes and low IFNγ-induced IDO1 upregulation correlated with worse outcome independent from disease stage. Interestingly studied factors were not interrelated. During follow-up, the risk of relapse was 9% (2/22) in the subgroup with high IFNγ-induced IDO1 upregulation in monocytes. In contrast, if IDO1 upregulation was low, relapse occurred in 30% (3/10) of patients with low basal IDO1 expression in monocytes and in 61.5% (8/13) in the subgroup with high basal IDO1 expression in monocytes (Log-Rank test, p=0.008). This study reveals some immune features in the blood of early stage melanoma that may be of relevance for disease outcome. These may offer a target for sub-stratification and early intervention., Competing Interests: NS reported travel grants from Merck Sharpe & Dohme, Astellas, Bayer and Bristol-Myers Squibb. PO received a research grant from Ferring, Merck, Varian, Bayer, consultancy fees from Ferring, Bayer, Janssen, Sandoz, Sanofi. MH was employed by Dermpat. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Meireson, Ferdinande, Haspeslagh, Hennart, Allorge, Ost, Sundahl, Spaas, Demeyer and Brochez.)

Genetic alterations affecting RAS proteins are commonly found in human cancers. Roughly a fourth of melanoma patients carry activating NRAS mutations, rendering this malignancy particularly challenging to treat. Although the development of targeted as well as immunotherapies led to a substantial improvement in the overall survival of non-NRAS mut melanoma patients (e.g. BRAF mut ), patients with NRAS mut melanomas have an overall poorer prognosis due to the high aggressiveness of RAS mut tumors, lack of efficient targeted therapies or rapidly emerging resistance to existing treatments. Understanding how NRAS-driven melanomas develop therapy resistance by maintaining cell cycle progression and survival is crucial to develop more effective and specific treatments for this group of melanoma patients. In this review, we provide an updated summary of currently available therapeutic options for NRAS mut melanoma patients with a focus on combined inhibition of MAPK signaling and CDK4/6-driven cell cycle progression and mechanisms of the inevitably developing resistance to these treatments. We conclude with an outlook on the most promising novel therapeutic approaches for melanoma patients with constitutively active NRAS. STATEMENT OF SIGNIFICANCE: An estimated 75000 patients are affected by NRAS mut melanoma each year and these patients still have a shorter progression-free survival than BRAF mut melanomas. Both intrinsic and acquired resistance occur in NRAS-driven melanomas once treated with single or combined targeted therapies involving MAPK and CDK4/6 inhibitors and/or checkpoint inhibiting immunotherapy. Oncolytic viruses, mRNA-based vaccinations, as well as targeted triple-agent therapy are promising alternatives, which could soon contribute to improved progression-free survival of the NRAS mut melanoma patient group., (Copyright © 2021. Published by Elsevier Ltd.)

In the last decade, immunotherapy and target therapy have revolutionized the prognosis of patients with BRAF-V600 mutation-positive metastatic melanoma. To date, three different combinations of BRAF/MEK inhibitors have been approved for this population, showing comparable efficacy and unique toxicity profiles. Several immune-checkpoint inhibitors, including pembrolizumab, nivolumab and the combination of nivolumab plus ipilimumab, are also available options for untreated metastatic melanoma patients. A novel approach has emerged by combining immune-checkpoint inhibitors and targeted agents, based on preclinical hints of synergy, prompting clinical results from large randomized trials. Specifically, the triplet of atezolizumab, vemurafenib and cobimetinib has been recently approved by FDA for patients with untreated BRAF-mutant metastatic melanoma. With a wide variety of available treatment options in this setting, it is paramount to establish criteria to select the most effective and safe frontline tailored approaches, for each patient. Results from ongoing studies are awaited, to maximise the benefits in survival outcomes and quality of life for patients, balancing adverse events and clinical benefit. The purpose of this review is to summarize the current landscape of standard and experimental treatment strategies for the first line treatment of patients with BRAF-mutated advanced melanoma and discuss the best patient-centered tailored strategies in the first-line setting., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The incidence of melanoma and nonmelanoma skin cancer has increased tremendously in recent years. Although novel treatment options have significantly improved patient outcomes, the prognosis for most patients with an advanced disease remains dismal. It is, thus, imperative to understand the molecular mechanisms involved in skin carcinogenesis in order to develop new targeted treatment strategies. Receptor tyrosine kinases (RTK) like the ERBB receptor family, including EGFR/ERBB1, ERBB2/NEU, ERBB3, and ERBB4, are important regulators of skin homeostasis and their dysregulation often results in cancer, which makes them attractive therapeutic targets. Members of the leucine-rich repeats and immunoglobulin-like domains protein family (LRIG1-3) are ERBB regulators and thus potential therapeutic targets to manipulate ERBB receptors. Here, we analyzed the function of LRIG1 during chemically induced skin carcinogenesis in transgenic mice expressing LRIG1 in the skin under the control of the keratin 5 promoter (LRIG1-TG mice). We observed a significant induction of melanocytic tumor formation in LRIG1-TG mice and no difference in papilloma incidence between LRIG1-TG and control mice. Our findings also revealed that LRIG1 affects ERBB signaling via decreased phosphorylation of EGFR and increased activation of the oncoprotein ERBB2 during skin carcinogenesis. The epidermal proliferation rate was significantly decreased during epidermal tumorigenesis under LRIG1 overexpression, and the apoptosis marker cleaved caspase 3 was significantly activated in the epidermis of transgenic LRIG1 mice. Additionally, we detected LRIG1 expression in human cutaneous squamous cell carcinoma and melanoma samples. Therefore, we depleted LRIG1 in human melanoma cells (A375) by CRISPR/Cas9 technology and found that this caused EGFR and ERBB3 downregulation in A375 LRIG1 knockout cells 6 h following stimulation with EGF. In conclusion, our study demonstrated that LRIG1-TG mice develop melanocytic skin tumors during chemical skin carcinogenesis and a deletion of LRIG1 in human melanoma cells reduces EGFR and ERBB3 expression after EGF stimulation., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Overcoming acquired drug resistance is a primary challenge in cancer treatment. Notably, more than 50% of patients with BRAF V600E cutaneous metastatic melanoma (CMM) eventually develop resistance to BRAF inhibitors. Resistant cells undergo metabolic reprogramming that profoundly influences therapeutic response and promotes tumor progression. Uncovering metabolic vulnerabilities could help suppress CMM tumor growth and overcome drug resistance. Here we identified a drug, HA344, that concomitantly targets two distinct metabolic hubs in cancer cells. HA344 inhibited the final and rate-limiting step of glycolysis through its covalent binding to the pyruvate kinase M2 (PKM2) enzyme, and it concurrently blocked the activity of inosine monophosphate dehydrogenase, the rate-limiting enzyme of de novo guanylate synthesis. As a consequence, HA344 efficiently targeted vemurafenib-sensitive and vemurafenib-resistant CMM cells and impaired CMM xenograft tumor growth in mice. In addition, HA344 acted synergistically with BRAF inhibitors on CMM cell lines in vitro . Thus, the mechanism of action of HA344 provides potential therapeutic avenues for patients with CMM and a broad range of different cancers. SIGNIFICANCE: Glycolytic and purine synthesis pathways are often deregulated in therapy-resistant tumors and can be targeted by the covalent inhibitor described in this study, suggesting its broad application for overcoming resistance in cancer., (©2021 American Association for Cancer Research.)

This investigation addressed the impact of integrin-initiated signaling pathways on senescence of tumor cells. In a model of human SK-Mel-147 melanoma cells, the silencing of integrin α2β1 strongly reduced cell proliferation and enhanced the percentage of SA-β-Gal-positive cells, a phenotypic feature of cellular senescence. These changes were accompanied by a significant increase in the activity of Akt and mTOR protein kinases and also in the expression of p53 and p21 oncosuppressors. Pharmacological inhibition of Akt and mTORC1 and genetic inhibition of p53 and p21 reduced the senescence of α2β1-depleted SK-Mel-147 cells to the level of control cells. Based on our earlier data on the non-canonical functions of Akt isomers in the invasion and anoikis of SK-Mel-147 cells, we investigated the role of Akt isomers in senescence induced by α2β1 suppression. The inhibition of Akt1 strongly reduced the percentage of SA-β-Gal-positive cells in the α2β1-depleted cell population, while the inhibition of Akt2 did not have a noticeable effect. Our data demonstrated for the first time that α2β1 is involved in the protection of tumor cells against senescence and that senescence, which is induced by the downregulation of α2β, is based on a signaling mechanism in which Akt1 performs a non-canonical function.

Context.—: Molecularly distinct from cutaneous melanomas arising from sun-exposed sites, acral lentiginous melanomas (ALMs) typically lack ultraviolet-signature mutations, such as telomerase reverse transcriptase (TERT) promoter mutations. Instead, ALMs show a high degree of copy number alterations, often with multiple amplifications of TERT, which are associated with adverse prognosis. The prognostic value of TERT protein expression in acral melanomas, however, is not established., Objective.—: To evaluate the frequency and pattern of TERT immunoreactivity and assess the potential utility of TERT expression as a prognostic indicator in ALMs., Design.—: TERT expression by immunohistochemistry was analyzed in a series of 57 acral and nonacral melanocytic lesions, including 24 primary and 6 metastatic ALMs. Clinical outcome in patients with ALMs by TERT expression was assessed., Results.—: TERT expression was more frequent in ALMs than in nonlentiginous acral melanomas and nonacral cutaneous melanomas, and was absent in acral nevi (P = .01). When present, TERT expression in ALMs was cytoplasmic and more intense than TERT expression in other melanocytic lesions (P = .05) with a higher H-score (P = .01). There was a trend toward decreased overall survival in patients with ALMs with TERT immunoreactivity, but it did not reach statistical significance. Furthermore, no correlation was found between TERT expression and disease-specific survival in patients with ALMs., Conclusions.—: Although TERT protein expression was frequently detected in both primary and metastatic ALMs, TERT immunoreactivity in ALMs did not correlate with survival in our study. Further studies with larger cohorts are needed to elucidate the prognostic value of TERT expression in ALMs.

The molecular background of a significant proportion of spitzoid neoplasms is still unknown. Recently, activating mutations in MAP2K1 have been described in a few spitzoid lesions, but not in benign Spitz nevi. We report four cases of melanocytic tumors with spitzoid features in which a MAP2K1 mutation was detected. The lesions did not show a single distinct phenotype and ranged from benign to malignant. Two cases resembled desmoplastic Spitz nevi. Based on the combination of morphological, immunohistochemical, and molecular findings, one case was classified as benign, one as probably benign, possibly intermediate low-grade (MELTUMP-melanocytic tumor of unknown malignant potential), one case was classified as intermediate (MELTUMP), and one case was considered a superficial spreading melanoma with spitzoid features. Based on this, we conclude that MAP2K1 mutations can indicate a spitzoid genetic signature and can be found in both benign and malignant spitzoid neoplasms., (© 2020. The Author(s).)

Basal cell carcinoma (BCC) is the most common skin malignancy, which rarely metastasizes but has a great ability to infiltrate and invade the surrounding tissues. One of the molecular players involved in the metastatic process are matrix metalloproteinases (MMPs). MMPs are enzymes that can degrade various components of the extracellular matrix. In the skin, the expression of MMPs is increased in response to various stimuli, including ultraviolet (UV) radiation, one of the main factors involved in the development of BCC. By modulating various processes that are linked to tumor growth, such as invasion and angiogenesis, MMPs have been associated with UV-related carcinogenesis. The sources of MMPs are multiple, as they can be released by both neoplastic and tumor microenvironment cells. Inhibiting the action of MMPs could be a useful therapeutic option in BCC management. In this review that reunites the latest advances in this domain, we discuss the role of MMPs in the pathogenesis and evolution of BCC, as molecules involved in tumor aggressiveness and risk of recurrence, in order to offer a fresh and updated perspective on this field.

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieve a durable remission. Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that regulates metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we showed that SIRT5 was required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 was required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we found that SIRT5 was required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably included MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a druggable genotype-independent addiction in melanoma.

Indoleamine 2-3 dioxygenase 1 (IDO1) expression may contribute to immunologic escape by melanoma metastases. However, a recent clinical trial failed to identify any clinical benefits of IDO1 inhibition in patients with unresectable metastatic melanoma, and prior characterizations of IDO1 expression have predominately studied primary lesions and local metastases, generating uncertainty regarding IDO1 expression in distant metastases. We hypothesized that IDO1 expression in such lesions would be low and correlated with decreased overall survival (OS). Metastases from patients (n=96) with stage IIIb to IV melanoma underwent tissue microarray construction and immunohistochemical staining for IDO1. Th1-related gene expression was determined quantitatively. Associations between OS and IDO1 expression were assessed with multivariate models. Of 96 metastatic lesions, 28% were IDOpos, and 85% exhibited IDO1 expression in <10% of tumor cells. IDOpos lesions were associated with improved OS (28.9 vs. 10.5 mo, P=0.02) and expression of Th1-related genes. OS was not associated with IDO1 expression in a multivariate analysis of all patients; however, IDO1 expression (hazard ratio=0.25, P=0.01) and intratumoral CD8+ T-cell density (hazard ratio=0.99, P<0.01) were correlated with OS in patients who underwent metastasectomy with curative-intent. IDOpos metastases were less likely to recur after metastasectomy (54% vs. 16%, P=0.01). IDO1 expression was low in melanoma metastases and correlated with OS after metastasectomy with curative-intent. Intratumoral CD8+ T cells and Th1-related genes were correlated with IDO1 expression, as was tumor recurrence. These suggest that IDO1 expression may be a marker of immunologic tumor control, and may inform participant selection in future trials of IDO1 inhibitors., Competing Interests: Conflicts of Interest and Source of Funding: Support provided by the United States Public Health Services Training Grants T32HL007849 (K.T.L.), T32CA009109-41 (M.K.), and T32CA163177 (M.K., M.O.M.), as well as the Farrow Fellowship and Rebecca Clary Harris Memorial Fellowship, both from the University of Virginia (M.K.). Additional support was received from the Biostatistics Shared Resource, University of Virginia Cancer Center (N.A.W.) and the Cancer Center Support Grant P30CA044579 (C.L.S.). The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Cutaneous squamous cell carcinomas (cSCC) are among the most commonly diagnosed malignancies, causing significant morbidity and mortality. Tumor-associated macrophage (TAM) expression of arginase is implicated in tumor progression, and therapeutic use of arginase inhibitors has been studied in various cancers. However, investigating potential cSCC immunotherapies including arginase inhibition in pre-clinical models is hampered by the lack of appropriate tumor models in immunocompetent mice. PDV is a cSCC cell line derived from chemical carcinogenesis of mouse keratinocytes. PDVC57 cells were derived from a PDV tumor in C57BL/6 (B6) mice. Unlike PDV, PDVC57 tumors grow consistently in B6 mice, and have increased TAMs, decreased dendritic and T cell intra-tumor infiltration. Arginase inhibition in cSCC tumors using Nω-hydroxy-nor-arginine (nor-NOHA) reduced tumor growth in B6 mice but not immunodeficient Rag1-deficient mice. nor-NOHA administration increased dendritic and T cell tumor-infiltration and PD-1 expression. The combination of nor-NOHA and anti-PD-1 therapy with nivolumab enhanced anti-PD-1 therapeutic efficacy. This study demonstrates the therapeutic potential of transcutaneous arginase inhibition in cSCC. A competent immune microenvironment is required for tumor growth inhibition using this arginase inhibitor. Synergistic co-inhibition of tumor growth in these results, supports further examination of transcutaneous arginase inhibition as a therapeutic modality for cSCC.

The clinical use of cisplatin are limited due to its drug resistance. Thus, it is urgent to find effective combination therapy that sensitizes tumor cells to this drug. The combined chemo-photodynamic therapy could increase anti-tumor efficacy while also reduce the side effects of cisplatin. Berberine is an isoquinoline alkaloid, which has been reported to show high photosensitizing activity. In this study, we have examined the effect of a combination of cisplatin and berberine-PDT in cisplatin-resistant melanoma cells. The cytotoxic effects of berberine-PDT alone or in combination with cisplatin were tested by MTT assays. We then examined the subcellular localization of berberine with confocal fluorescence microscopy. The percentage of apoptotic cells, the mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) generation assessed using flow cytometry analysis. Western blotting used in this study to determine the expression levels of MAPK signaling pathways and apoptosis-related proteins. Experimental data revealed that the mode of cell death is the caspase-dependent mitochondrial apoptotic pathways. Excessive accumulation of ROS played a key role in this process, which is confirmed by alleviation of cytotoxicity upon pretreatment with NAC. Furthermore, we found that the combined treatment activated MAPK signaling pathway. The inhibition of p38 MAPK by pretreating with SB203580 block the combined treatment-induced apoptotic cell death. In conclusion, berberine-PDT could be used as a chemo-sensitizer by promoting cell death through activation of a ROS/p38/caspase cascade., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mycosis fungoides (MF) represents the most common type of cutaneous lymphoma. In the majority of patients, the disease has a slow evolution and a protracted course; however, a subset of patients shows poor oncologic outcomes. Unfortunately, there are no reliable prognostic markers for MF, and the currently available treatments are only effective in a minority of patients. This study aimed to evaluate the expression and clinical significance of PARP-1 and CAF-1/p60 in MF. Sixty-four MF representatives of the different stages of disease were assessed by immunohistochemistry for PARP-1 and CAF-1/p60. The association of PARP-1 and CAF-1/p60 with the MF stage and outcome was assessed by using Fisher's exact test and Kaplan-Meier survival analysis with the Log-rank test; a p value < 0.05 was considered significant. PARP-1 was overexpressed in 57.9% of MF and was significantly associated with a MF stage > II (p = 0.034) but not with the risk of death (p = 0.237). CAF-1/p60 was overexpressed in 26.8% of MF and was significantly associated with decreased overall survival (p < 0.001) but not with the MF stage (p = 1). A significant association was found between PARP-1 overexpression and CAF-1/p60 overexpression (p = 0.0025). Simultaneous overexpression of PARP-1 and CAF-1/p60 was significantly associated with decreased overall survival (p < 0.001), although less strongly than CAF-1/p60 alone (χ 2  = 14.916 vs 21.729, respectively). In MF, PARP-1 is overexpressed in advanced stages, while CAF-1/p60 is overexpressed in the cases with shorter overall survival, appearing as a significant prognostic marker. A role for PARP-1 inhibitors and anti-CAF-1/p60 targeted therapy may be reasonably hypothesized in MF.

Dendritic cells (DCs) play an important role in linking innate and adaptive immunity. DCs can sense endogenous and exogenous antigens and present those antigens to T cells to induce an immune response or immune tolerance. During activation, alternative splicing (AS) in DCs is dramatically changed to induce cytokine secretion and upregulation of surface marker expression. PTBP1, an RNA-binding protein, is essential in alternative splicing, but the function of PTBP1 in DCs is unknown. Here, we found that a specific deficiency of Ptbp1 in DCs could increase MHC II expression and perturb T-cell homeostasis without affecting DC development. Functionally, Ptbp1 deletion in DCs could enhance antitumour immunity and asthma exacerbation. Mechanistically, we found that Pkm alternative splicing and a subset of Ifn response genes could be regulated by PTBP1. These findings revealed the function of PTBP1 in DCs and indicated that PTBP1 might be a novel therapeutic target for antitumour treatment., (© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.)

Background: In the tumor microenvironment, tumor cells are able to suppress antitumor immunity by competing for essential nutrients, including amino acids. However, whether amino acid depletion modulates the activity of CD8 + tumor-infiltrating lymphocytes (TILs) is unclear., Method: In this study, we evaluated the roles of amino acids and the Rag complex in regulating mammalian target of rapamycin complex 1 (mTORC1) signaling in CD8 + TILs., Results: We discovered that the Rag complex, particularly RagD, was crucial for CD8 + T-cell antitumor immunity. RagD expression was positively correlated with the antitumor response of CD8 + TILs in both murine syngeneic tumor xenografts and clinical human colon cancer samples. On RagD deficiency, CD8 + T cells were rendered more dysfunctional, as demonstrated by attenuation of mTORC1 signaling and reductions in proliferation and cytokine secretion. Amino acids maintained RagD-mediated mTORC1 translocation to the lysosome, thereby achieving maximal mTORC1 activity in CD8 + T cells. Moreover, the limited T-cell access to leucine (LEU), overshadowed by tumor cell amino acid consumption, led to impaired RagD-dependent mTORC1 activity. Finally, combined with antiprogrammed cell death protein 1 antibody, LEU supplementation improved T-cell immunity in MC38 tumor-bearing mice in vivo., Conclusion: Our results revealed that robust signaling of amino acids by RagD and downstream mTORC1 signaling were crucial for T-cell receptor-initiated antitumor immunity. The characterization the role of RagD and LEU in nutrient mTORC1 signaling in TILs might suggest potential therapeutic strategies based on the manipulation of RagD and its upstream pathway., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

A subset of Spitz tumors harbor fusions of NTRK3 with ETV6, MYO5A, and MYH9. We evaluated a series of 22 melanocytic tumors in which an NTRK3 fusion was identified as part of the diagnostic workup. Tumors in which NTRK3 was fused to ETV6 occurred in younger patients were predominantly composed of epithelioid melanocytes and were classified by their histopathologic features as Spitz tumors. In contrast, those in which NTRK3 was fused to MYO5A were predominantly composed of spindled melanocytes arrayed in fascicles with neuroid features such as pseudo-Verocay bodies. To further investigate the effects of the fusion kinases ETV6-NTRK3 and MYO5A-NTRK3 in melanocytes, we expressed them in immortalized melanocytes and determined their subcellular localization by immunofluorescence. ETV6-NTRK3 was localized to the nucleus and diffusely within the cytoplasm and caused melanocytes to adopt an epithelioid cytomorphology. In contrast, MYO5A-NTRK3, appeared excluded from the nucleus of melanocytes, was localized to dendrites, and resulted in a highly dendritic cytomorphology. Our findings indicate that ETV6-NTRK3 and MYO5A-NTRK3 have distinct subcellular localizations and effects on cellular morphology.

Cutaneous squamous cell carcinomas (cSCCs) account for about 20% of keratinocyte carcinomas, the most common cancer in the UK. Therapeutic options for cSCC patients who develop metastasis are limited and a better understanding of the biochemical pathways involved in cSCC development/progression is crucial to identify novel therapeutic targets. Evidence indicates that the phosphoinositide 3-kinases (PI3Ks)/Akt pathway plays an important role, in particular in advanced cSCC. Questions remain of whether all four PI3K isoforms able to activate Akt are involved and whether selective inhibition of specific isoform(s) might represent a more targeted strategy. Here we determined the sensitivity of four patient-derived cSCC cell lines to isoform-specific PI3K inhibitors to start investigating their potential therapeutic value in cSCC. Parallel experiments were performed in immortalized keratinocyte cell lines. We observed that pan PI3Ks inhibition reduced the growth/viability of all tested cell lines, confirming the crucial role of this pathway. Selective inhibition of the PI3K isoform p110α reduced growth/viability of keratinocytes and of two cSCC cell lines while affecting the other two only slightly. Importantly, p110α inhibition reduced Akt phosphorylation in all cSCC cell lines. These data indicate that growth and viability of the investigated cSCC cells display differential sensitivity to isoform-specific PI3K inhibitors.

Ethnopharmacological Relevance: 22β-hydroxytingenone (22-HTG) is a quinonemethide triterpene isolated from Salacia impressifolia (Miers) A. C. Smith (family Celastraceae), which has been used in traditional medicine to treat a variety of diseases, including dengue, renal infections, rheumatism and cancer. However, the anticancer effects of 22-HTG and the underlying molecular mechanisms in melanoma cells have not yet been elucidated., Aim of the Study: The present study investigated apoptosis induction and antimetastatic potencial of 22-HTG in SK-MEL-28 human melanoma cells., Materials and Methods: First, the in vitro cytotoxic activity of 22-HTG in cultured cancer cells was evaluated. Then, cell viability was determined using the trypan blue assay in melanoma cells (SK-MEL-28), which was followed by cell cycle, annexin V-FITC/propidium iodide assays (Annexin/PI), as well as assays to evaluate mitochondrial membrane potential, production of reactive oxygen species (ROS) using flow cytometry. Fluorescence microscopy using acridine orange/ethidium bromide (AO/BE) staining was also performed. RT-qPCR was carried out to evaluate the expression of BRAF, NRAS, and KRAS genes. The anti-invasiveness potential of 22-HTG was evaluated in a three-dimensional (3D) model of reconstructed human skin., Results: 22-HTG reduced viability of SK-MEL-28 cells and caused morphological changes, as cell shrinkage, chromatin condensation, and nuclear fragmentation. Furthermore, 22-HTG caused apoptosis, which was demonstrated by increased staining with AO/BE and Annexin/PI. The apoptosis may have been caused by mitochondrial instability without the involvement of ROS production. The expression of BRAF, NRAS, and KRAS, which are important biomarkers in melanoma development, was reduced by the 22-HTG treatment. In the reconstructed skin model, 22-HTG was able to decrease the invasion capacity of melanoma cells in the dermis., Conclusions: Our data indicate that 22-HTG has anti-tumorigenic properties against melanoma cells through the induction of cell cycle arrest, apoptosis and inhibition of invasiveness potential, as observed in the 3D model. As such, the results provide new insights for future work on the utilization of 22-HTG in malignant melanoma treatment., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ponatinib is a broad-spectrum tyrosine kinase inhibitor that targets numerous receptor tyrosine kinases (RTKs), including but not limited to fibroblast growth factor receptor (FGFR)-1, platelet derived growth factor receptor (PDGFR)-α, and vascular endothelial growth factor receptor (VEGFR)-2. This study evaluated the expression of FGFR-1, PDGFR-α, and VEGFR-2 in three canine mast cell tumor (MCT) cell lines (CM-MC1, VI-MC1, CoMS) and the effects of ponatinib on these MCT cell lines. Quantitative RT-PCR confirmed the expression of FGFR-1, PDGFR-α, and VEGFR-2 in the three MCT cell lines. Ponatinib exhibited dose- and time-dependent cytotoxicity in MCT cell lines via MTT assay. The IC 50 for 24, 48, and 72 h across the three cell lines ranged from 38.47 nM to 103.3 nM, which is clinically comparable to dose ranges established for humans. Significantly increased apoptosis in each cell line was seen between 12 and 18 h after treatment with IC 50 of ponatinib via Annexin-V and Caspase-3/7 assays. These data suggest that ponatinib could be a possible therapeutic agent for canine MCTs. Further studies are needed to investigate the prognostic value of FGFR-1, PDGFR-α, and VEGFR-2 in canine MCTs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose of Review: BRAF/MEK inhibitor has changed the treatment landscape in patients with advanced and metastatic melanoma with prolonged overall survival and progression-free survival. Since three treatment combinations exist with similar efficacy therapy decisions are often made based on the side effect profile. Additionally, on-target side effects or class effects have to be properly managed to ensure treatment adherence., Recent Findings: Sequential treatment with BRAF/MEK inhibition and immunotherapy might increase toxicity with a sepsis-like syndrome and triple therapy with concomitant BRAF/MEK inhibition and anti-PD1/PD-L1 antibody therapy induces severe side effects in the vast majority of patients., Summary: Toxicity of combination therapy with BRAF/MEK inhibitors is generally manageable, reversible and infrequently associated with treatment discontinuation. In case of persisting off-target effects the change to another combination therapy can resolve side effects., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Functional evaluation of genetic lesions can discover a role in cancer initiation and progression and help develop novel therapeutic strategies. We previously identified the negative MAPK regulator SPRED1 as a novel tumor suppressor in KIT-driven melanoma. Here, we show that SPRED1 is also frequently deleted in human melanoma driven by mutant BRAF. We found that SPRED1 inactivation in human melanoma cell lines and primary zebrafish melanoma conferred resistance to BRAFV600E inhibition in vitro and in vivo. Mechanistically, SPRED1 loss promoted melanoma cell proliferation under mutant BRAF inhibition by reactivating MAPK activity. Consistently, biallelic deletion of SPRED1 was observed in a patient whose melanoma acquired resistance to MAPK-targeted therapy. These studies combining work in human cells and in vivo modeling in zebrafish demonstrate a new mechanism of resistance to BRAFV600E inhibition in melanoma., Competing Interests: Disclosures:   R.S. Lo reported grants from Pfizer, Merck, OncoSec, and BMS outside the submitted work. L.I. Zon reported personal fees from Scholar Rock Inc., Fate Therapeutics, CAMP4 Therapeutics, Amagma Therapeutics, Celularity, and Cellarity outside the submitted work. No other disclosures were reported., (© 2020 Ablain et al.)

Background: As most clinical trials evaluating BRAF and MEK inhibitor combination therapy (B + Minh) have been conducted in Western countries, little is known about the effect of B + Minh among East Asian populations., Material and Methods: Data from patients with advanced melanoma treated using B + Minh (either dabrafenib + trametinib or encorafenib + binimetinib) were retrospectively collected from 16 institutes in Japan. Response rates, adverse events, patterns of failure and survival were analysed., Results: We analysed 112 of 144 collected patient records and, of these, 14 had acral/mucosal melanoma. The response rate for the entire cohort was 75.0%. There were no statistical differences in response rates between acral/mucosal and cutaneous melanomas (64.3% versus 76.5%), whereas previous treatment using immune checkpoint inhibitors (ICIs) did not affect response (72.7% versus 73.9%) to B + Minh, response to ICI after B + Minh was only 20%. Patients who achieved complete response had the best overall survival rates at 24 months (94.7%). Elevated serum lactate dehydrogenase levels and 3 or more metastatic sites were independently associated with survival. The most common relapse site was the brain (17.9%). More than half of the patients (58.8%) experienced grade III/IV pyrexia., Conclusion: B + Minh was effective among Japanese patients with melanoma, including those with acral/mucosal melanoma. Factors associated with survival were similar to previous Western studies. B + Minh response was not affected by the previous use of ICI; however, vigilance against brain metastasis during B + Minh therapy is required as the brain was our most commonly encountered relapse site., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose of Review: Although immune checkpoint inhibitors and small molecule inhibitors targeting the MAPK pathway have revolutionized the management of metastatic melanoma, long-term disease control occurs only for a minority of patients because of multiple resistance mechanisms. One way to tackle resistance is to develop the next-generation of RAF, MEK and ERK inhibitors using our understanding of the molecular mechanisms that fine-tune the MAPK pathway., Recent Findings: Studies on the regulation of the MAPK pathway have revealed a dominant role for homo-dimerization and hetero-dimerization of RAF, MEK and ERK. Allosteric inhibitors that break these dimers are, therefore, undergoing various stages of preclinical and clinical evaluation. Novel MEK inhibitors are less susceptible to differences in MEK's activation state and do not drive the compensatory activation of MEK that could limit efficacy. Innovations in targeting ERK originate from dual inhibitors that block MEK-catalyzed ERK phosphorylation, thereby limiting the extent of ERK reactivation following feedback relief., Summary: The primary goal in RAF, MEK and ERK inhibitors' development is to produce molecules with less inhibitor paradox and off-target effects, giving robust and sustained MAPK pathway inhibition., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Dipeptidyl peptidase-4 (DPP4) is a multifunctional, transmembrane glycoprotein present on the cell surface of various tissues. It is present in multiple molecular forms including cell surface and soluble. The role of DPP4 and its inhibition in cutaneous dermatoses have been a recent point of investigation. DPP4 exerts a notable influence on T-cell biology, the induction of skin-specific lymphocytes, and the homeostasis between regulatory and effector T cells. Moreover, DPP4 interacts with a broad range of molecules, including adenosine deaminase, caveolin-1, CXCR4 receptor, M6P/insulin-like growth factor II-receptor and fibroblast activation protein-α, triggering downstream effects that modulate the immune response, cell adhesion and chemokine activity. DPP4 expression on melanocytes, keratinocytes and fibroblasts further alters cell function and, thus, has crucial implications in cutaneous pathology. As a result, DPP4 plays a significant role in bullous pemphigoid, T helper type 1-like reactions, cutaneous lymphoma, melanoma, wound healing and fibrotic disorders. This review illustrates the multifactorial role of DPP4 expression, regulation, and inhibition in cutaneous diseases., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose of Review: Patients with advanced or metastatic v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutated melanoma can be treated with a BRAF inhibitor in combination with a MAPK/ERK kinase (MEK) inhibitor, achieving high but short-lived response rates. Immune checkpoint inhibitors (ICIs), in contrast, give lower response rates but more durable responses. Preclinical and translational data indicate that combining BRAF and MEK inhibitors with ICI could exceed the limitations of each class and potentially lead to longer lasting responses., Recent Findings: Vemurafenib, dabrafenib and encorafenib are designed to block mutated forms of BRAF, which cause abnormal signalling inside cancer cells leading to tumour growth. Trametinib, binimetinib and cobimetinib are designed to target and inhibit MEK1/2, proteins in a cell signalling pathway that help cell growth and survival. Pembrolizumab, nivolumab, durvalumab and atezolizumab are ICIs which can inhibit the pathway of programmed death-1/ programmed death-ligand-1 proteins, allowing tumours to avoid detection by the immune system., Summary: Treating patients with targeted therapy would allow the release of antigens from tumour cells, which could be more easily acknowledged by the immune system. Efficacy can also be increased by combining ICIs with the aim of maintaining a longer response. The possibility to administer three drugs in combination, would allow to induce tumour regression and produce an immune response with a synergistic effect., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Melanoma is the main cause of skin cancer deaths, with special emphasis in those cases carrying BRAF mutations that trigger the mitogen-activated protein kinases (MAPK) signaling and unrestrained cell proliferation in the absence of mitogens. Current therapies targeting MAPK are hindered by drug resistance and relapse that rely on metabolic rewiring and Akt activation. To identify new drug candidates against melanoma, we investigated the molecular mechanism of action of the Octopus Kaurna -derived peptide, Octpep-1, in human BRAF(V600E) melanoma cells using proteomics and RNAseq coupled with metabolic analysis. Fluorescence microscopy verified that Octpep-1 tagged with fluorescein enters MM96L and NFF cells and distributes preferentially in the perinuclear area of MM96L cells. Proteomics and RNAseq revealed that Octpep-1 targets PI3K/AKT/mTOR signaling in MM96L cells. In addition, Octpep-1 combined with rapamycin (mTORC1 inhibitor) or LY3214996 (ERK1/2 inhibitor) augmented the cytotoxicity against BRAF(V600E) melanoma cells in comparison with the inhibitors or Octpep-1 alone. Octpep-1-treated MM96L cells displayed reduced glycolysis and mitochondrial respiration when combined with LY3214996. Altogether these data support Octpep-1 as an optimal candidate in combination therapies for melanoma BRAF(V600E) mutations.

Background: Angiolipoma (AL) is considered as a lipoma variant that is characterized by the combination of mature adipocytes and capillary blood vessels diffusely distributed within the tumor. With the exception of recurrent PRKD2 mutations of uncertain pathogenetic significance, the genetic abnormalities of ALs are unknown, in the absence of any of the specific chromosomal aberrations described in other lipoma variants., Methods: Formalin-fixed and paraffin-embedded blocks of 13 conventional ALs and 5 cellular ALs from 17 individuals were retrieved and analyzed for mutations in exons 9 and 20 of PIK3CA by polymerase chain reaction and Sanger sequencing., Results: Activating PIK3CA mutations were identified in 14 tumors (78%). All PIK3CA-mutated samples carried the same exon 9 mutation, c.1634A>C (p.E545A). No mutation was detected in exon 20 of PIK3CA. No significant difference between PIK3CA-mutated and wild-type samples appeared to exist based on age, gender, and location of the tumor. All 5 cellular ALs carried the p.E545A PIK3CA mutation., Conclusion: The high frequency of the p.E545A PIK3CA mutation in both conventional and cellular ALs suggests that activation of the PI3K/AKT pathway plays a key role in AL pathogenesis and reinforces the concept that cellular AL should be regarded as a variant of AL., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Understanding the mechanisms of the Warburg shift to aerobic glycolysis is critical to defining the metabolic basis of cancer. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an aggressive cancer characterized by biallelic inactivation of the gene encoding the Krebs cycle enzyme fumarate hydratase, an early shift to aerobic glycolysis, and rapid metastasis. We observed impairment of the mitochondrial respiratory chain in tumors from patients with HLRCC. Biochemical and transcriptomic analyses revealed that respiratory chain dysfunction in the tumors was due to loss of expression of mitochondrial DNA (mtDNA)-encoded subunits of respiratory chain complexes, caused by a marked decrease in mtDNA content and increased mtDNA mutations. We demonstrated that accumulation of fumarate in HLRCC tumors inactivated the core factors responsible for replication and proofreading of mtDNA, leading to loss of respiratory chain components, thereby promoting the shift to aerobic glycolysis and disease progression in this prototypic model of glucose-dependent human cancer., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Melanoma is an aggressive form of cancer with poor prognosis therefore, identification of associated pathophysiological mechanisms is imperative towards the development of new therapeutic strategies. The KLK6 is a serine protease normally expressed in the epidermis. Recently, we found that elimination of Klk6 in mice results in enhanced resistance to chemically induced non-melanoma skin cancer. To delineate putative roles of KLK6 in melanoma, the invasive KLK6-non-expressing MDA-MB-435 melanoma cell line was stably transfected with the full-length KLK6 cDNA and expression of the corresponding RNA and protein were confirmed. Interestingly, restoration of KLK6 expression resulted in markedly suppressed growth of primary tumors when orthotopically implanted in SCID mice. Analysis of data retrieved from the human protein atlas revealed that melanomas with high KLK6 expression have a trend for longer survival. Collectively, we suggest that KLK6 inhibits growth of melanomas., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

The incidence of cutaneous keratinocyte-derived cancers is increasing globally. Basal cell carcinoma (BCC) is the most common malignancy worldwide, and cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. BCC can be classified into subtypes based on the histology, and these subtypes are classified further into low- and high-risk tumors. There is an increasing need to identify new therapeutic strategies for the treatment of unresectable and metastatic cSCC, and for aggressive BCC variants such as infiltrating, basosquamous or morpheaform BCCs. The most important risk factor for BCC and cSCC is solar UV radiation, which causes genetic and epigenetic alterations in keratinocytes. Similar gene mutations are noted already in sun-exposed normal skin emphasizing the role of the alterations in the tumor microenvironment in the progression of cSCC. Early events in cSCC progression are alterations in the composition of basement membrane and dermal extracellular matrix induced by influx of microbes, inflammatory cells and activated stromal fibroblasts. Activated fibroblasts promote inflammation and produce growth factors and proteolytic enzymes, including matrix metalloproteinases (MMPs). Transforming growth factor-β produced by tumor cells and fibroblasts induces the expression of MMPs by cSCC cells and promotes their invasion. Fibroblast-derived keratinocyte growth factor suppresses the malignant phenotype of cSCC cells by inhibiting the expression of several MMPs. These findings emphasize the importance of interplay of tumor and stromal cells in the progression of cSCC and BCC and suggest tumor microenvironment as a therapeutic target in cSCC and aggressive subtypes of BCC., (© 2020 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

The crosstalk between Notch and MAPK pathway plays a role in MEK inhibitor resistance in BRAF V600E metastatic melanoma (MM) and promotes migration in GNAQ Q209L uveal melanoma (UM) cells. We determined the cytotoxicity of combinatorial inhibition of MEK and Notch by cobimetinib and γ-secretase inhibitor (GSI) nirogacestat, in BRAF V600E and BRAF wt MM and GNAQ Q209L UM cells displaying different Erk1/2 and Notch activation status, with the aim to elucidate the impact of Notch signaling in the response to MEK inhibitor. Overall the combination was synergic in BRAF V600E MM and GNAQ Q209L UM cells and antagonistic in BRAF wt one. Focusing on UM cells, we found that cobimetinib resulted in G0/G1 phase arrest and apoptosis induction, whereas the combination with GSI increased treatment efficacy by inducing a senescent-like state of cells and by blocking migration towards liver cancer cells. Mechanistically, this was reflected in a strong reduction of cyclin D1, in the inactivation of retinoblastoma protein and in the increase of p27 KIP1 expression levels. Of note, each drug alone prevented Notch signaling activation resulting in inhibition of c-jun(Ser63) and Hes-1 expression. The combination achieved the strongest inhibition on Notch signaling and on both c-jun(Ser63) and Erk1/2 activation level. In conclusion we unveiled a coordinate action of MAPK and Notch signaling in promoting proliferation of BRAF V600E MM and GNAQ Q209L UM cells. Remarkably, the simultaneous inhibition of MEK and Notch signaling highlighted a role for the second pathway in protecting cells against senescence in GNAQ Q209L UM cells treated with the MEK inhibitor., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

With the advent of better molecular characterization of Spitz melanocytic neoplasms, there has been increasing effort to better understand and describe the relationships between specific driver fusion and/or mutations with the clinical and histomorphological characteristics of the lesions. Structural rearrangements in mitogen activated protein kinase genes have recently been noted to be important in Spitz neoplasms. Only very few reports, however, have described in detail melanocytic tumors with in frame deletions in MAP2K1. Cases in the literature with this aberration have been described as having a diagnosis of Spitz, deep penetrating nevi, or pigmented epithelioid melanocytoma. In this study, we describe a cohort of 6 cases with MAP2K1 activating in frame deletions. The morphologic spectrum of the cases was broad. Common features of these cases include Spitzoid cytomorphology (5/6) cases, prominent melanin pigmentation (4/6) cases, and deep penetrating nevi-like plexiform architecture (3/6) cases. The diagnoses at the time of clinical care of these cases included nevus of Reed (1/6), desmoplastic Spitz tumor (1/6), BAPoma (1/6), deep penetrating melanocytic nevus (2/6), and melanoma (1/6). Clinical follow-up was available in 3 of the 6 cases. None of the patients had a tumor recurrence. This builds on the growing literature to help expand the spectrum of changes associated with Spitzoid melanocytic neoplasms.

While the genomics of BRAF, NRAS, and other key genes influencing MAP kinase (MAPK) activity have been thoroughly characterized in melanoma, mutations in MAP2K1 (MEK1) have received significantly less attention and have consisted almost entirely of missense mutations considered secondary oncogenic drivers of melanoma. Here, we investigated melanomas with in-frame deletions of MAP2K1, alterations characterized as MAPK-activating in recent experimental models. Our case archive of clinical melanoma samples with comprehensive genomic profiling by a hybrid capture-based DNA sequencing platform was searched for MAP2K1 genetic alterations. Clinical data, pathology reports, and histopathology were reviewed for each case. From a cohort of 7119 advanced melanomas, 37 unique cases (0.5%) featured small in-frame deletions in MAP2K1. These included E102&#95;I103del (n = 11 cases), P105&#95;A106del (n = 8), Q58&#95;E62del (n = 6), I103&#95;K104del (n = 5), I99&#95;K104del (n = 3), L98&#95;I103del (n = 3), and E41&#95;F53del (n = 1). All 37 were wild type for BRAF, NRAS, and NF1 genomic alterations ("triple wild-type"), representing 2.0% of triple wild-type melanomas overall (37/1882). Median age was 66 years and 49% were male. The majority arose from primary cutaneous sites (35/37; 95%) and demonstrated a UV signature when available (21/25; 84%). Tumor mutational burden was typical for cutaneous melanoma (median = 9.6 mut/Mb, range 0-35.7), and frequently mutated genes included TERTp (63%), CDKN2A (46%), TP53 (11%), PTEN (8%), APC (8%), and CTNNB1 (5%). Histopathology revealed a spectrum of appearances typical of melanoma. For comparison, we evaluated 221 cases with pathogenic missense single nucleotide variants in MAP2K1. The vast majority of melanomas with missense SNVs in MAP2K1 showed co-mutations in BRAF (58%), NF1 (23%), or NRAS (18%). In-frame deletions in MAP2K1, previously shown in experimental models to be strongly MAPK-activating, characterized a significant subset of triple wild-type melanoma (2.0%), suggesting a primary oncogenic role for these mutations. Comprehensive genomic profiling of melanomas enables detection of this alteration, which may have implications for potential therapeutic options.

Downregulation of integrins α3β1 and α5β1 strongly decreased cell colony formation and in vitro invasion and markedly enhanced anoikis in SK-Mel-147 human melanoma cells. These modifications were accompanied by a marked increase in the levels of active Akt protein kinase, which indicated it played a non-canonical function in the melanoma cells. Pharmacological inhibition of Akt1, an Akt isozyme, in cells depleted of α3β1 or α5β1 restored their invasive activity, while inhibition of the Akt 2 isoform did not cause a visible effect. Similar to our previous results with the α2β1 integrin, this finding suggested that in signaling pathways initiated by α3β1 and α5β1, the Akt1 isoform performs a non-canonical function in regulating invasive phenotype of melanoma cells. In contrast, when the effects of Akt inhibitors on anoikis of the melanoma cells were compared, the Akt2 isoform demonstrated a non-canonical activity in which Akt2 suppression led to a significant attenuation of apoptosis in cells with downregulated α3β1 or α5β1. Our results were the first evidence that, in the same tumor cells, different integrins can control various manifestations of tumor progression through distinct signaling pathways that are both common to various integrins and specific to a particular receptor.