Interpretation of allele-specific chromatin accessibility using cell state-aware deep learning.

Genomic sequence variation within enhancers and promoters can have a significant impact on the cellular state and phenotype. However, sifting through the millions of candidate variants in a personal genome or a cancer genome, to identify those that impact cis-regulatory function, remains a major challenge. Interpretation of noncoding genome variation benefits from explainable artificial intelligence to predict and interpret the impact of a mutation on gene regulation. Here we generate phased whole genomes with matched chromatin accessibility, histone modifications, and gene expression for 10 melanoma cell lines. We find that training a specialized deep learning model, called DeepMEL2, on melanoma chromatin accessibility data can capture the various regulatory programs of the melanocytic and mesenchymal-like melanoma cell states. This model outperforms motif-based variant scoring, as well as more generic deep learning models. We detect hundreds to thousands of allele-specific chromatin accessibility variants (ASCAVs) in each melanoma genome, of which 15%-20% can be explained by gains or losses of transcription factor binding sites. A considerable fraction of ASCAVs are caused by changes in AP-1 binding, as confirmed by matched ChIP-seq data to identify allele-specific binding of JUN and FOSL1. Finally, by augmenting the DeepMEL2 model with ChIP-seq data for GABPA, the TERT promoter mutation, as well as additional ETS motif gains, can be identified with high confidence. In conclusion, we present a new integrative genomics approach and a deep learning model to identify and interpret functional enhancer mutations with allelic imbalance of chromatin accessibility and gene expression.

ATP1A1 is a promising new target for melanoma treatment and can be inhibited by its physiological ligand bufalin to restore targeted therapy efficacy.

Despite advancements in treating metastatic melanoma, many patients exhibit resistance to targeted therapies. Our study focuses on ATP1A1, a sodium pump subunit associated with cancer development. We aimed to assess ATP1A1 prognostic value in melanoma patients and examine the impact of its ligand, bufalin, on melanoma cell lines in vitro and in vivo. High ATP1A1 expression (IHC) correlated with reduced overall survival in melanoma patients. Resistance to BRAF inhibitor was linked to elevated ATP1A1 levels in patient biopsies (IHC, qPCR) and cell lines (Western blot, qPCR). Additionally, high ATP1A1 mRNA expression positively correlated with differentiation/pigmentation markers based on data from The Cancer Genome Atlas (TCGA) databases and Verfaillie proliferative gene signature analysis. Bufalin specifically targeted ATP1A1 in caveolae, (proximity ligation assay) and influenced Src phosphorylation (Western blot), thereby disrupting multiple signaling pathways (phosphokinase array). In vitro, bufalin induced apoptosis in melanoma cell lines by acting on ATP1A1 (siRNA experiments) and, in vivo, significantly impeded melanoma growth using a nude mouse xenograft model with continuous bufalin delivery via an osmotic pump. In conclusion, our study demonstrates that ATP1A1 could serve as a prognostic marker for patient survival and a predictive marker for response to BRAF inhibitor therapy. By targeting ATP1A1, bufalin inhibited cell proliferation, induced apoptosis in vitro, and effectively suppressed tumor development in mice. Thus, our findings strongly support ATP1A1 as a promising therapeutic target, with bufalin as a potential agent to disrupt its tumor-promoting activity.

Cross-species analysis of enhancer logic using deep learning.

Deciphering the genomic regulatory code of enhancers is a key challenge in biology because this code underlies cellular identity. A better understanding of how enhancers work will improve the interpretation of noncoding genome variation and empower the generation of cell type-specific drivers for gene therapy. Here, we explore the combination of deep learning and cross-species chromatin accessibility profiling to build explainable enhancer models. We apply this strategy to decipher the enhancer code in melanoma, a relevant case study owing to the presence of distinct melanoma cell states. We trained and validated a deep learning model, called DeepMEL, using chromatin accessibility data of 26 melanoma samples across six different species. We show the accuracy of DeepMEL predictions on the CAGI5 challenge, where it significantly outperforms existing models on the melanoma enhancer of IRF4 Next, we exploit DeepMEL to analyze enhancer architectures and identify accurate transcription factor binding sites for the core regulatory complexes in the two different melanoma states, with distinct roles for each transcription factor, in terms of nucleosome displacement or enhancer activation. Finally, DeepMEL identifies orthologous enhancers across distantly related species, where sequence alignment fails, and the model highlights specific nucleotide substitutions that underlie enhancer turnover. DeepMEL can be used from the Kipoi database to predict and optimize candidate enhancers and to prioritize enhancer mutations. In addition, our computational strategy can be applied to other cancer or normal cell types.

Recent advances in radiosensitivity determinants in melanoma.

PURPOSE OF REVIEW: Radiotherapy has been proven to be useful but insufficient in melanoma management due to the intrinsic radioresistance of melanoma cells. Elucidation of the molecular mechanisms and pathways related to resistance/sensitivity to radiotherapy in melanoma is of paramount importance. In this review, we will summarize and discuss the recent 'discoveries' and advances in radiosensitivity determinants in melanoma. RECENT FINDINGS: The different levels of radiosensitivity among the various melanoma tumors could be attributed to the DNA damage signaling and repair proteins, tumor microenvironment, hypoxia, cell metabolism, glutathione and redox balance, protein kinase signaling pathways as well as pigmentation and melanin content. SUMMARY: It is therapeutically important to elucidate the factors involved in radiation resistance/sensitivity of melanoma. More importantly, improving radiosensitivity may 'widen the clinical utility' in melanoma of this important therapeutic modality.

Bufalin for an innovative therapeutic approach against cancer.

Bufalin is an endogenous cardiotonic steroid, first discovered in toad venom but also found in the plasma of healthy humans, with anti-tumour activities in different cancer types. The current review is focused on its mechanisms of action and highlights its very large spectrum of effects both in vitro and in vivo. All leads to the conclusion that bufalin mediates its effects by affecting all the hallmarks of cancer and seems restricted to cancer cells avoiding side effects. Bufalin decreases cancer cell proliferation by acting on the cell cycle and inducing different mechanisms of cell death including apoptosis, necroptosis, autophagy and senescence. Bufalin also moderates metastasis formation by blocking migration and invasion as well as angiogenesis and by inducing a phenotype switch towards differentiation and decreasing cancer cell stemness. Regarding its various mechanisms of action in cancer cells, bufalin blocks overactivated signalling pathways and modifies cell metabolism. Moreover, bufalin gained lately a huge interest in the field of drug resistance by both reversing various drug resistance mechanisms and affecting the immune microenvironment. Together, these data support bufalin as a quite promising new anti-cancer drug candidate.

Understanding the Radiobiological Mechanisms Induced by 177Lu-DOTATATE in Comparison to External Beam Radiation Therapy.

Radionuclide Therapy (RNT) with 177Lu-DOTATATE targeting somatostatin receptors (SSTRs) in neuroendocrine tumours (NET) has been successfully used in routine clinical practice, mainly leading to stable disease. Radiobiology holds promise for RNT improvement but is often extrapolated from external beam radiation therapy (EBRT) studies despite differences in these two radiation-based treatment modalities. In a panel of six human cancer cell lines expressing SSTRs, common radiobiological endpoints (i.e., cell survival, cell cycle, cell death, oxidative stress and DNA damage) were evaluated over time in 177Lu-DOTATATE- and EBRT-treated cells, as well as the radiosensitizing potential of poly (ADP-ribose) polymerase inhibition (PARPi). Our study showed that common radiobiological mechanisms were induced by both 177Lu-DOTATATE and EBRT, but to a different extent and/or with variable kinetics, including in the DNA damage response. A higher radiosensitizing potential of PARPi was observed for EBRT compared to 177Lu-DOTATATE. Our data reinforce the need for dedicated RNT radiobiology studies, in order to derive its maximum therapeutic benefit.

Galectins and Carcinogenesis: Their Role in Head and Neck Carcinomas and Thyroid Carcinomas.

Head and neck cancers are among the most frequently occurring cancers worldwide. Of the molecular drivers described for these tumors, galectins play an important role via their interaction with several intracellular pathways. In this review, we will detail and discuss this role with specific reference to galectins-1, -3, and -7 in angiogenesis, cell proliferation, and invasion as well as in cell transformation and cancer progression. Furthermore, we will evaluate the prognostic value of galectin expression in head and neck cancers including those with oral cavity, salivary gland, and nasopharyngeal pathologies. In addition, we will discuss the involvement of these galectins in thyroid cancers where their altered expression is proposed as a new diagnostic biomarker.

Tumour targeting and radiation dose of radioimmunotherapy with (90)Y-rituximab in CD20+ B-cell lymphoma as predicted by (89)Zr-rituximab immuno-PET: impact of preloading with unlabelled rituximab.

PURPOSE: To compare using immuno-PET/CT the distribution of (89)Zr-labelled rituximab without and with a preload of unlabelled rituximab to assess the impact of preloading with unlabelled rituximab on tumour targeting and radiation dose of subsequent radioimmunotherapy with (90)Y-labelled rituximab in CD20+ B-cell lymphoma. METHODS: Five patients with CD20+ B-cell lymphoma and progressive disease were prospectively enrolled. All patients underwent three study phases: initial dosimetric phase with baseline (89)Zr-rituximab PET/CT imaging without a cold preload, followed 3 weeks later by a second dosimetric phase with administration of a standard preload (250 mg/m(2)) of unlabelled rituximab followed by injection of (89)Zr-rituximab, and a therapeutic phase 1 week later with administration of unlabelled rituximab followed by (90)Y-rituximab. PET/CT imaging and tracer uptake by organs and lesions were assessed. RESULTS: With a cold rituximab preload, the calculated whole-body dose of (90)Y-rituximab was similar (mean 0.87 mSv/MBq, range 0.82-0.99 mSv/MBq) in all patients. Without a preload, an increase in whole-body dose of 59% and 87% was noted in two patients with preserved circulating CD20+ B cells. This increase in radiation dose was primarily due to a 12.4-fold to 15-fold higher dose to the spleen without a preload. No significant change in whole-body dose was noted in the three other patients with B-cell depletion. Without a preload, consistently higher tumour uptake was noticed in patients with B-cell depletion. CONCLUSION: Administration of the standard preload of unlabelled rituximab impairs radioconjugate tumour targeting in the majority of patients eligible for radioimmunotherapy, that is patients previously treated with rituximab-containing therapeutic regimens. This common practice may need to be reconsidered and further evaluated as the rationale for this high preload has its origin in the "prerituximab era". Clinical Trial Application: CTA 2011-005474-38 TRIAL REGISTRY: EudraCT.

Has removal of excess cysteine led to the evolution of pheomelanin? Pheomelanogenesis as an excretory mechanism for cysteine.

Pheomelanogenesis may have evolved as an excretory mechanism to remove excess cysteine, and in humans this might potentially confer a greater ability to avoid disease such as Parkinson's and Alzheimer's disease, in which excess cysteine is a contributory cause.

Electron paramagnetic resonance spectrometry and imaging in melanomas: comparison between pigmented and nonpigmented human malignant melanomas.

It has been known for a long time that the melanin pigments present in normal skin, hair, and most of malignant melanomas can be detected by electron paramagnetic resonance (EPR) spectrometry. In this study, we used EPR imaging as a tool to map the concentration of melanin inside ex vivo human pigmented and nonpigmented melanomas and correlated this cartography with anatomopathology. We obtained accurate mappings of the melanin inside pigmented human melanoma samples. The signal intensity observed on the EPR images correlated with the concentration of melanin within the tumors, visible on the histologic sections. In contrast, no EPR signal coming from melanin was observed from nonpigmented melanomas, therefore demonstrating the absence of EPR-detectable pigments inside these particular cases of skin cancer and the importance of pigmentation for further EPR imaging studies on melanoma.

Targeting Prohibitins to Inhibit Melanoma Growth and Overcome Resistance to Targeted Therapies.

Despite important advances in the treatment of metastatic melanoma with the development of MAPK-targeted agents and immune checkpoint inhibitors, the majority of patients either do not respond to therapies or develop acquired resistance. Furthermore, there is no effective targeted therapy currently available for BRAF wild-type melanomas (approximately 50% of cutaneous melanoma). Thus, there is a compelling need for new efficient targeted therapies. Prohibitins (PHBs) are overexpressed in several types of cancers and implicated in the regulation of signaling networks that promote cell invasion and resistance to cell apoptosis. Herein, we show that PHBs are highly expressed in melanoma and are associated with not only poor survival but also with resistance to BRAFi/MEKi. We designed and identified novel specific PHB inhibitors that can inhibit melanoma cell growth in 3D spheroid models and a large panel of representative cell lines with different molecular subtypes, including those with intrinsic and acquired resistance to MAPKi, by significantly moderating both MAPK (CRAF-ERK axis) and PI3K/AKT pathways, and inducing apoptosis through the mitochondrial pathway and up-regulation of p53. In addition, autophagy inhibition enhances the antitumor efficacy of these PHB ligands. More important, these ligands can act in synergy with MAPKi to more efficiently inhibit cell growth and overcome drug resistance in both BRAF wild-type and mutant melanoma. In conclusion, targeting PHBs represents a very promising therapeutic strategy in melanoma, regardless of mutational status.

The benefit of co-targeting PARP-1 and c-Met on the efficacy of radiotherapy in wild type BRAF melanoma.

Melanoma is known to be a radioresistant cancer. Melanoma radioresistance can be due to several factors such as pigmentation, antioxidant defenses and high Deoxyribonucleic acid (DNA) repair efficacy. However, irradiation induces intracellular translocation of RTKs, including cMet, which regulates response to DNA damage activating proteins and promotes DNA repair. Accordingly, we hypothesized that co-targeting DNA repair (PARP-1) and relevant activated RTKs, c-Met in particular, may radiosensitize wild-type B-Raf Proto-Oncogene, Serine/Threonine Kinase (WTBRAF) melanomas where RTKs are often upregulated. Firstly, we found that PARP-1 is highly expressed in melanoma cell lines. PARP-1 inhibition by Olaparib or its KO mediates melanoma cell sensitivity to radiotherapy (RT). Similarly, specific inhibition of c-Met by Crizotinib or its KO radiosensitizes the melanoma cell lines. Mechanistically, we show that RT causes c-Met nuclear translocation to interact with PARP-1 promoting its activity. This can be reversed by c-Met inhibition. Accordingly, RT associated with the inhibition of both c-Met and PARP-1 resulted in a synergistic effect not only on tumor growth inhibition but also on tumor regrowth control in all animals following the stop of the treatment. We thus show that combining PARP and c-Met inhibition with RT appears a promising therapeutic approach in WTBRAF melanoma.

Correction: Krayem et al. The Benefit of Reactivating p53 under MAPK Inhibition on the Efficacy of Radiotherapy in Melanoma. Cancers 2019, 11, 1093.

In the original article [...].

Disturbing the Redox Balance Using Buthionine Sulfoximine Radiosensitized Somatostatin Receptor-2 Expressing Pre-Clinical Models to Peptide Receptor Radionuclide Therapy with 177Lu-DOTATATE.

Peptide receptor radionuclide therapy with 177Lu-DOTATATE improves the outcome of patients with somatostatin receptor (SSTR)-expressing neuroendocrine tumours. Nevertheless, stable disease has been the main response pattern observed, with some rare complete responses. Lu-177 exerts about two-thirds of its biological effects via the indirect effects of ionizing radiation that generate reactive oxygen species, eventually leading to oxidative damage and cell death. This provides a rationale for targeting the antioxidant defence system in combination with 177Lu-DOTATATE. In the present study, the radiosensitizing potential and the safety of depleting glutathione (GSH) levels using buthionine sulfoximine (BSO) during 177Lu-DOTATATE therapy were assessed in vitro and in vivo using a xenograft mouse model. In vitro, the combination resulted in a synergistic effect in cell lines exhibiting a BSO-mediated GSH decrease. In vivo, BSO neither influenced 177Lu-DOTATATE biodistribution nor induced liver, kidney or bone marrow toxicity. In terms of efficacy, the combination resulted in reduced tumour growth and metabolic activity. Our results showed that disturbing the cell redox balance using a GSH synthesis inhibitor increased 177Lu-DOTATATE efficacy without additional toxicity. Targeting the antioxidant defence system opens new safe treatment combination opportunities with 177Lu-DOTATATE.

Cytokine Landscape in Central Nervous System Metastases.

The central nervous system is the location of metastases in more than 40% of patients with lung cancer, breast cancer and melanoma. These metastases are associated with one of the poorest prognoses in advanced cancer patients, mainly due to the lack of effective treatments. In this review, we explore the involvement of cytokines, including interleukins and chemokines, during the development of brain and leptomeningeal metastases from the epithelial-to-mesenchymal cell transition and blood-brain barrier extravasation to the interaction between cancer cells and cells from the brain microenvironment, including astrocytes and microglia. Furthermore, the role of the gut-brain axis on cytokine release during this process will also be addressed.

Understanding Molecular Mechanisms of Phenotype Switching and Crosstalk with TME to Reveal New Vulnerabilities of Melanoma.

Melanoma cells are notorious for their high plasticity and ability to switch back and forth between various melanoma cell states, enabling the adaptation to sub-optimal conditions and therapeutics. This phenotypic plasticity, which has gained more attention in cancer research, is proposed as a new paradigm for melanoma progression. In this review, we provide a detailed and deep comprehensive recapitulation of the complex spectrum of phenotype switching in melanoma, the key regulator factors, the various and new melanoma states, and corresponding signatures. We also present an extensive description of the role of epigenetic modifications (chromatin remodeling, methylation, and activities of long non-coding RNAs/miRNAs) and metabolic rewiring in the dynamic switch. Furthermore, we elucidate the main role of the crosstalk between the tumor microenvironment (TME) and oxidative stress in the regulation of the phenotype switching. Finally, we discuss in detail several rational therapeutic approaches, such as exploiting phenotype-specific and metabolic vulnerabilities and targeting components and signals of the TME, to improve the response of melanoma patients to treatments.

Development of fluorizoline analogues as prohibitin ligands that modulate C-RAF signaling, p21 expression and melanogenesis.

Fluorizoline is a cytotoxic trifluorothiazoline that targets the scaffold proteins prohibitins-1 and -2 (PHB1/2) to inhibit the kinase C-RAF and promote the expression of the cyclin-dependent kinase inhibitor p21 to induce cancer cell death. In melanocytes, fluorizoline also induces the synthesis of melanin. Herein we report the first structural requirement of fluorizoline analogues for these activities. We identified in particular some compounds that display enhanced anti-C-RAF and anti-MEK activities, and a higher cytotoxicity in HeLa cells compared to fluorizoline. These results provide a foundation for further optimization of PHB ligands for the treatment of cancers. We also discovered an analogue of fluorizoline that displays pharmacological effects opposed to those of fluorizoline and that can be used as a chemical tool to explore PHB signaling in cancers and other diseases.

Regorafenib Induces Senescence and Epithelial-Mesenchymal Transition in Colorectal Cancer to Promote Drug Resistance.

Potential intrinsic resistance mechanisms to regorafenib were explored after short exposure (3 days) on five CRC cell lines (HCT-116, SW1116, LS-1034, SW480, Caco-2). The observation of senescence-like features led to the investigation of a drug-initiated phenotype switch. Following long-term exposure (12 months) of HCT-116 and SW480 cell lines to regorafenib, we developed resistant models to explore acquired resistance. SW480 cells demonstrated senescent-like properties, including a cell arrest in the late G2/prophase cell cycle stage and a statistically significant decrease in the expression of G1 Cyclin-Dependent Kinase inhibitors and key cell cycle regulators. A specific senescence-associated secretome was also observed. In contrast, HCT-116 treated cells presented early senescent features and developed acquired resistance triggering EMT and a more aggressive phenotype over time. The gained migration and invasion ability by long-exposed cells was associated with the increased expression level of key cellular and extracellular EMT-related factors. The PI3K/AKT pathway was a significant player in the acquired resistance of HCT-116 cells, possibly related to a PI3KCA mutation in this cell line. Our findings provide new insights into the phenotypic plasticity of CRC cells able, under treatment pressure, to acquire a stable TIS or to use an early senescence state to undergo EMT.

Investigating intrinsic radiosensitivity biomarkers to peptide receptor radionuclide therapy with [177Lu]Lu-DOTATATE in a panel of cancer cell lines.

INTRODUCTION: [177Lu]Lu-DOTATATE is an effective systemic targeted radionuclide therapy for somatostatin receptor (SSTR) positive metastatic or inoperable neuroendocrine tumours (NET). However, for a given injected activity, tumour responses are variable. Our aim was to investigate whether SSTR expression/functionality and known characteristics of intrinsic radiosensitivity, namely proliferation rate, glucose metabolism, cell cycle phase, DNA repair and antioxidant defences were predictors of sensitivity to [177Lu]Lu-DOTATATE in SSTR expressing human cancer cell lines. METHODS: In six human cancer cell lines and under basal condition, SSTR expression was assessed by qRT-PCR and immunocytochemistry. Its functionality was evaluated by binding/uptake assays with [68Ga]Ga- and [177Lu]Lu-DOTATATE. The radiosensitivity parameters were evaluated as follows: proliferation rate (cell counting), glucose metabolism ([18F]FDG uptake), antioxidant defences (qRT-PCR, colorimetric assay, flow cytometry), DNA repair (qRT-PCR) and cell cycle (flow cytometry). Effect of [177Lu]Lu-DOTATATE on cell viability was assessed 3, 7 and 10 days after 4 h incubation with [177Lu]Lu-DOTATATE using crystal violet. RESULTS: Based on cell survival at day 10, cell lines were classified into two groups of sensitivity to [177Lu]Lu-DOTATATE. One group with <20% of survival decrease (-14 to -1%) and one group with >20% of survival decrease (-22 to -33%) compared to the untreated control cell lines. The latter had significantly lower total antioxidant capacity, glutathione (GSH) levels and glucose metabolism (p < 0.05) compared to the first group. SSTR (p = 0.64), proliferation rate (p = 0.74), cell cycle phase (p = 0.55), DNA repair (p > 0.22), combined catalase and GSH peroxidase expression (p = 0.42) and superoxide dismutase (SOD) activity (p = 0.41) were not significantly different between the two groups. CONCLUSION: Antioxidant defences may be major determinants in [177Lu]Lu-DOTATATE radiosensitivity.

RTK Inhibitors in Melanoma: From Bench to Bedside.

MAPK (mitogen activated protein kinase) and PI3K/AKT (Phosphatidylinositol-3-Kinase and Protein Kinase B) pathways play a key role in melanoma progression and metastasis that are regulated by receptor tyrosine kinases (RTKs). Although RTKs are mutated in a small percentage of melanomas, several receptors were found up regulated/altered in various stages of melanoma initiation, progression, or metastasis. Targeting RTKs remains a significant challenge in melanoma, due to their variable expression across different melanoma stages of progression and among melanoma subtypes that consequently affect response to treatment and disease progression. In this review, we discuss in details the activation mechanism of several key RTKs: type III: c-KIT (mast/stem cell growth factor receptor); type I: EGFR (Epidermal growth factor receptor); type VIII: HGFR (hepatocyte growth factor receptor); type V: VEGFR (Vascular endothelial growth factor), structure variants, the function of their structural domains, and their alteration and its association with melanoma initiation and progression. Furthermore, several RTK inhibitors targeting the same receptor were tested alone or in combination with other therapies, yielding variable responses among different melanoma groups. Here, we classified RTK inhibitors by families and summarized all tested drugs in melanoma indicating the rationale behind the use of these drugs in each melanoma subgroups from preclinical studies to clinical trials with a specific focus on their purpose of treatment, resulted effect, and outcomes.