Search

Evaluation of caspae 3 staining after treatment with Aβ(25-35) in mice

CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. Although the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short β-amyloid–derived peptide (Aβ(25–35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of eight melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aβ(25–35) in ex vivo tumors from immunotherapy- and targeted therapy–resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aβ(25–35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein–protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aβ(25–35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy.Significance:The discovery of a means to specifically activate the CD271 death domain reveals unknown pathways mediated by the receptor and highlights new treatment possibilities for melanoma.

Tumor Profiler Consortium author list

Table ST5: Table of the 15 most informative proteins upregulated in MEK inhibitor-resistant cell culture (ranked by log2 fold-change).

S13: Immunohistochemical analysis of CyTof biomarker panel.

Supplementary Figures

Supplementary Figure Legends

Concurrent MEK and CDK4/6 inhibition shows promise in clinical trials for patients with advanced-stage mutant BRAF/NRAS solid tumors. The effects of CDK4/6 inhibitor (CDK4/6i) in combination with BRAF/MEK-targeting agents on the tumor immune microenvironment are unclear, especially in melanoma, for which immune checkpoint inhibitors are effective in approximately 50% of patients. Here, we show that patients progressing on CDK4/6i/MEK pathway inhibitor combinations exhibit T-cell exclusion. We found that MEK and CDK4/6 targeting was more effective at delaying regrowth of mutant BRAF melanoma in immunocompetent versus immune-deficient mice. Although MEK inhibitor (MEKi) treatment increased tumor immunogenicity and intratumoral recruitment of CD8+ T cells, the main effect of CDK4/6i alone and in combination with MEKi was increased expression of CD137L, a T-cell costimulatory molecule on immune cells. Depletion of CD8+ T cells or blockade of the CD137 ligand–receptor interaction reduced time to regrowth of melanomas in the context of treatment with CDK4/6i plus MEKi treatment in vivo. Together, our data outline an antitumor immune-based mechanism and show the efficacy of targeting both the MEK pathway and CDK4/6.

S16: scRNA-sequencing analysis of xenograft tumors M130227

Figure Legends for Supplemental Figures 1-4

Figure legend for supplementary figures.

Conformation of knock-down for siRNA experiments.

Many metastatic melanoma patients experience durable responses to anti-PD1 and/or anti-CTLA4; however, a significant proportion (over 50%) do not benefit from the therapies. In this study, we sought to assess pretreatment liquid biopsies for biomarkers that may correlate with response to checkpoint blockade. We measured the combinatorial diversity evenness of the T-cell receptor (TCR) repertoire (the DE50, with low values corresponding to more clonality and lack of TCR diversity) in pretreatment peripheral blood mononuclear cells from melanoma patients treated with anti-CTLA4 (n = 42) or anti-PD1 (n = 38) using a multi-N-plex PCR assay on genomic DNA (gDNA). A receiver operating characteristic curve determined the optimal threshold for a dichotomized analysis according to objective responses as defined by RECIST1.1. Correlations between treatment outcome, clinical variables, and DE50 were assessed in multivariate regression models and confirmed with Fisher exact tests. In samples obtained prior to treatment initiation, we showed that low DE50 values were predictive of a longer progression-free survival and good responses to PD-1 blockade, but, on the other hand, predicted a poor response to CTLA4 inhibition. Multivariate logistic regression models identified DE50 as the only independent predictive factor for response to anti-CTLA4 therapy (P = 0.03) and anti-PD1 therapy (P = 0.001). Fisher exact tests confirmed the association of low DE50 with response in the anti-CTLA4 (P = 0.041) and the anti-PD1 cohort (P = 0.0016). Thus, the evaluation of basal TCR repertoire diversity in peripheral blood, using a PCR-based method, could help predict responses to anti-PD1 and anti-CTLA4 therapies.

Targeting cyclin-dependent kinases 4/6 (CDK4/6) represents a therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor (MEKi) in melanoma; however, continuous dosing elicits toxicities in patients. Using quantitative and temporal in vivo reporting, we show that continuous MEKi with intermittent CDK4/6 inhibitor (CDK4/6i) led to more complete tumor responses versus other combination schedules. Nevertheless, some tumors acquired resistance that was associated with enhanced phosphorylation of ribosomal S6 protein. These data were supported by phospho-S6 staining of melanoma biopsies from patients treated with CDK4/6i plus targeted inhibitors. Enhanced phospho-S6 in resistant tumors provided a therapeutic window for the mTORC1/2 inhibitor AZD2014. Mechanistically, upregulation or mutation of NRAS was associated with resistance in in vivo models and patient samples, respectively, and mutant NRAS was sufficient to enhance resistance. This study utilizes an in vivo reporter model to optimize schedules and supports targeting mTORC1/2 to overcome MEKi plus CDK4/6i resistance.Significance: Mutant BRAF and NRAS melanomas acquire resistance to combined MEK and CDK4/6 inhibition via upregulation of mTOR pathway signaling. This resistance mechanism provides the preclinical basis to utilize mTORC1/2 inhibitors to improve MEKi plus CDK4/6i drug regimens. Cancer Discov; 8(5); 568–81. ©2018 AACR.See related commentary by Sullivan, p. 532.See related article by Romano et al., p. 556.This article is highlighted in the In This Issue feature, p. 517

Time course experiment for gene expression analysis of IL13RA2 and IL1R2.

Effect of IM on fibroblasts and melanoma cells and effect of TPA on SCC13 cells.

Chemical structure of ingenol mebutate. Confirmation of inhibition by MEK and ERK inhibitor

Selection of further investigated targets.

ERK is activated after IM treatment in a PKC-dependent manner.

Lymphocyte to leukocyte ratio in high and low ANC/ALC groups for anti-PD1 treated patients at baseline. (Wilcoxon test)

Supplementary Figures

Materials and Methods

Figure S1: Negative control for immunofluorescence and immunohistochemistry (A) Negative control for Cilia, and Sox9 (B) Representative image of negative control for CD4, CD8, FoxP3, MHC class I and HLA-DR-class II staining using tonsil.

Epithelial-to-mesenchymal transition (EMT)-inducing transcription factors (TF) are well known for their ability to induce mesenchymal states associated with increased migratory and invasive properties. Unexpectedly, nuclear expression of the EMT-TF ZEB2 in human primary melanoma has been shown to correlate with reduced invasion. We report here that ZEB2 is required for outgrowth for primary melanomas and metastases at secondary sites. Ablation of Zeb2 hampered outgrowth of primary melanomas in vivo, whereas ectopic expression enhanced proliferation and growth at both primary and secondary sites. Gain of Zeb2 expression in pulmonary-residing melanoma cells promoted the development of macroscopic lesions. In vivo fate mapping made clear that melanoma cells undergo a conversion in state where ZEB2 expression is replaced by ZEB1 expression associated with gain of an invasive phenotype. These findings suggest that reversible switching of the ZEB2/ZEB1 ratio enhances melanoma metastatic dissemination.Significance:ZEB2 function exerts opposing behaviors in melanoma by promoting proliferation and expansion and conversely inhibiting invasiveness, which could be of future clinical relevance.

Figure S2: Higher magnification photos of MHC class I expression (A) MHC class I expression on tumor cells after 4 week treatment with Hh inhibitors. Scale bar; 400 µm (upper), 50 µm (lower)

Legends for S1 and S2

Clinical management of melanomas with NRAS mutations is challenging. Targeting MAPK signaling is only beneficial to a small subset of patients due to resistance that arises through genetic, transcriptional, and metabolic adaptation. Identification of targetable vulnerabilities in NRAS-mutated melanoma could help improve patient treatment. Here, we used multiomics analyses to reveal that NRAS-mutated melanoma cells adopt a mesenchymal phenotype with a quiescent metabolic program to resist cellular stress induced by MEK inhibition. The metabolic alterations elevated baseline reactive oxygen species (ROS) levels, leading these cells to become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intratumor heterogeneity necessitates the combination of a ROS inducer and a MEK inhibitor to inhibit both tumor growth and metastasis. Ex vivo pharmacoscopy of 62 human metastatic melanomas confirmed that MEK inhibitor–resistant tumors significantly benefited from the combination therapy. Finally, oxidative stress response and translational suppression corresponded with ROS-inducer sensitivity in 486 cancer cell lines, independent of cancer type. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS inducers in melanoma and other cancers.Significance:Metabolic reprogramming in drug-resistant NRAS-mutated melanoma cells confers sensitivity to ROS induction, which suppresses tumor growth and metastasis in combination with MAPK pathway inhibitors.

Purpose: Basal cell carcinomas (BCCs) are tumors ignored by immune surveillance. Activated Hedgehog (Hh) signaling within primary cilia is a key driver in the pathogenesis of BCCs. We examined immune alterations during treatment with systemic Hh inhibitors.Experimental Design: We investigated biopsies from patients with BCC before (23 patients) and after 4 weeks of treatment (5 patients) with Hh signaling inhibitor. Ber-Ep4, BCL-2, Ki-67, CD4, CD8, MHC class I, HLA-DR-class II, and SOX9 were analyzed by immunohistochemistry. Primary cilia were analyzed by double immunofluorescence of acetylated tubulin and SOX9. Differential gene expression for 84 cytokines and chemokines was analyzed in 3 patients.Results: After 4 weeks of treatment, we found reduction of Ki-67, SOX9, Ber-EP4, and BCL-2 expression in tumors associated with morphologic signs of squamous differentiation. In addition, the number of cilia-positive BCC cells was significantly decreased. An upregulation of MHC I expression on the cell membranes of residual tumor cells and an influx of CD4+, HLA-DR-class II+, and CD8+ cells with invasion into the tumor cell nests were found. Finally, qPCR arrays showed the differential expression of genes involved in modulating immune responses.Conclusions: We show that Hh pathway inhibitor–induced tumor regression is accompanied by a dynamic change of the microenvironment with a disruption of immune privilege involving an influx of cytotoxic T cells, activation of the adaptive immune functions, and a profound alteration of the local chemokine/cytokine network. Clin Cancer Res; 21(6); 1289–97. ©2015 AACR.

Supplementary Figures. Supplementary Figure S1: Zeb2-wildtype cells outcompete Zeb2-negative cells in terms of expansion and differentiation. Supplementary Figure S2: Zeb2 does not accelerate melanoma development on a p53-wildtype background. Supplementary Figure S3: ZEB2 affects the phenotypical switch between proliferation and invasion in mouse and human melanoma cells. Supplementary Figure S4: ZEB2 affects the phenotypical switch between proliferation and invasion in human melanoma. Supplementary Figure S5: TGF-ß treatment mediates the switch from ZEB2 to ZEB1 and induces an invasive gene signature.

Background: The COVID-19 lockdown had a dramatic impact on primary care access and resulted in postponed skin cancer screenings. This raises concerns for a diagnostic delay on primary cutaneous melanomas, which can subsequently increase morbidity and mortality. Objectives: The aim of the study was to investigate the impact of the COVID-19-related restrictions on the melanoma diagnosis in five European skin cancer reference centres in Switzerland, Germany, Austria and Italy. Methods: A total of 7865 cutaneous melanoma cases were collected between 01 September 2018 and 31 August 2021. The time period was stratified into pre-COVID (pre-lockdown) and post-COVID (lockdown and post-lockdown) according to the established restrictions in each country. The data collection included demographic, clinical and histopathological data from histologically confirmed cutaneous melanomas. Personal and family history of melanoma, and presence of immunosuppression were used to assess the diagnosis delay in high-risk individuals. Results: There was an overall increase of the Breslow tumour thickness (mean 1.25 mm vs. 1.02 mm) during the post-COVID period, as well as an increase in the proportion of T3-T4 melanomas, rates of ulceration and the number of mitotic rates ≥2 (all, p < 0.001). Patients with immunosuppression and personal history of melanoma showed a decrease in the mean log10-transformed Breslow during lockdown and post-COVID. In the multivariate analysis, age at melanoma diagnosis (p < 0.01) and personal history of melanoma (p < 0.01) showed significant differences in the mean Breslow thickness. Conclusions: The study confirms the diagnostic delay in cutaneous melanomas due to the COVID-19 lockdown. High-risk individuals, such as patients with personal history of melanoma and elderly individuals, were more hesitant to restart their regular skin cancer screenings post-COVID. Further studies with longer follow-up are required to evaluate the consequences of this diagnostic delay in long-term outcomes. in press

The Covid-19 pandemic created new uncertainties in the management of metastatic melanoma patients. In particular, the impact of immunotherapy, targeted therapy, or chemotherapy on the risk of Sars-CoV-2 infection and severity was debated. In this study, we analyzed all patients with metastatic melanoma receiving therapy who developed Covid-19 between February 2020 and February 2022. We retrospectively collected demographic data, cancer-specific parameters, melanoma treatment regimen, comorbidities and Covid-19-specific parameters in these patients. Of the 350 patients with metastatic melanoma, 25 had Covid-19. The median age at the time of Covid-19 diagnosis was 66 years (range 36-86), 10 patients were female, and 15 patients were male. The treatment regimen during infection was immunotherapy in 12 cases, followed by targeted therapy (

CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. Although the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short β-amyloid–derived peptide (Aβ(25–35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of eight melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aβ(25–35) in ex vivo tumors from immunotherapy- and targeted therapy–resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aβ(25–35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein–protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aβ(25–35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy. Significance: The discovery of a means to specifically activate the CD271 death domain reveals unknown pathways mediated by the receptor and highlights new treatment possibilities for melanoma.

Reliable transportation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patient samples from a swabbing station to a diagnostics facility is essential for the generation of accurate results. Therefore, cooling or freezing the samples is recommended in case of longer transportation times. In this study, the impact on SARS-CoV-2 detectability by reverse transcriptase PCR was assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. SARS-CoV-2–positive patient swabs stored in viral transport medium were exposed to different temperatures (4°C, 25°C, and 35°C) and to repetitive freeze-thawing, to assess the effect of storage conditions on reverse transcriptase PCR detection. SARS-CoV-2 RNA was still reliably detected by reverse transcriptase PCR after 21 days of storage in viral transport medium, even when the samples had been stored at 35°C. The change of Ct value per day was 0.023 to 0.046 (±0.018 to 0.019). In addition, viral RNA was still detected after 15 freeze-thaw cycles, with a change of Ct value of 0.106 to 0.197 (±0.009 to 0.061) per freeze-thaw cycle. However, compared with storage at 4°C, RNA was significantly less detectable when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate that viral RNA levels, as measured by reverse transcriptase PCR assays, are significantly, but not substantially, altered by prolonged unrefrigerated storage of up to 21 days at temperatures ranging up to 35°C or after repeated freeze-thaw cycles.

The survival of patients with melanoma of unknown primary (MUP) is proposed to be more favourable than that for melanoma of known primary (MKP). This may be due to an enhanced initial immune response in patients with MUP, which could also affect the efficacy of immunotherapy in advanced disease. The present study compared therapeutic outcome and survival in Stage III and IV MUP and MKP. Medical records of 67 MUP and 536 MKP patients were reviewed. Median overall survival (OS) in Stage III patients was 77 months versus 54 months in patients with MUP and MKP, respectively (p = 0.11). Median OS was prolonged in MUP patients receiving adjuvant first-line ipilimumab (p = 0.14). In contrast, OS tended to be more favourable in patients with MKP after palliative first-line ipilimumab treatment (p = 0.16). Yet, no statistically significant differences in OS were detected between the groups. Moreover, survival after anti-PD-1-antibody treatment was similar in patients with MUP and MKP. Overall, we observed similar survival outcomes after immunotherapy in patients with MUP and MKP. These findings provide no evidence of difference in responsiveness to immunotherapy between patients with MUP and MKP.