Unveiling CXCR2 as a promising therapeutic target in renal cell carcinoma: exploring the immunotherapeutic paradigm shift through its inhibition by RCT001.

BACKGROUND: In clear cell renal cell carcinoma (ccRCC), first-line treatment combines nivolumab (anti-PD-1) and ipilimumab (anti-CTLA4), yielding long-term remissions but with only a 40% success rate. Our study explored the potential of enhancing ccRCC treatment by concurrently using CXCR2 inhibitors alongside immunotherapies. METHODS: We analyzed ELR + CXCL levels and their correlation with patient survival during immunotherapy. RCT001, a unique CXCR2 inhibitor, was examined for its mechanism of action, particularly its effects on human primary macrophages. We tested the synergistic impact of RCT001 in combination with immunotherapies in both mouse models of ccRCC and human ccRCC in the presence of human PBMC. RESUTS: Elevated ELR + CXCL cytokine levels were found to correlate with reduced overall survival during immunotherapy. RCT001, our optimized compound, acted as an inverse agonist, effectively inhibiting angiogenesis and reducing viability of primary ccRCC cells. It redirected M2-like macrophages without affecting M1-like macrophage polarization directed against the tumor. In mouse models, RCT001 enhanced the efficacy of anti-CTLA4 + anti-PD1 by inhibiting tumor-associated M2 macrophages and tumor-associated neutrophils. It also impacted the activation of CD4 T lymphocytes, reducing immune-tolerant lymphocytes while increasing activated natural killer and dendritic cells. Similar effectiveness was observed in human RCC tumors when RCT001 was combined with anti-PD-1 treatment. CONCLUSIONS: RCT001, by inhibiting CXCR2 through its unique mechanism, effectively suppresses ccRCC cell proliferation, angiogenesis, and M2 macrophage polarization. This optimization potentiates the efficacy of immunotherapy and holds promise for significantly improving the survival prospects of metastatic ccRCC patients.

Single-cell RNA sequencing reveals intratumoral heterogeneity in primary uveal melanomas and identifies HES6 as a driver of the metastatic disease.

Intratumor heterogeneity has been recognized in numerous cancers as a major source of metastatic dissemination. In uveal melanomas, the existence and identity of specific subpopulations, their biological function and their contribution to metastasis remain unknown. Here, in multiscale analyses using single-cell RNA sequencing of six different primary uveal melanomas, we uncover an intratumoral heterogeneity at the genomic and transcriptomic level. We identify distinct transcriptional cell states and diverse tumor-associated populations in a subset of the samples. We also decipher a gene regulatory network underlying an invasive and poor prognosis state driven in part by the transcription factor HES6. HES6 heterogenous expression has been validated by RNAscope assays within primary human uveal melanomas, which further unveils the existence of these cells conveying a dismal prognosis in tumors diagnosed with a favorable outcome using bulk analyses. Depletion of HES6 impairs proliferation, migration and metastatic dissemination in vitro and in vivo using the chick chorioallantoic membrane assay, demonstrating the essential role of HES6 in uveal melanomas. Thus, single-cell analysis offers an unprecedented view of primary uveal melanoma heterogeneity, identifies bona fide biomarkers for metastatic cells in the primary tumor, and reveals targetable modules driving growth and metastasis formation. Significantly, our findings demonstrate that HES6 is a valid target to stop uveal melanoma progression.

Endoplasmic reticulum stress mediates resistance to BCL-2 inhibitor in uveal melanoma cells.

To address unmet clinical need for uveal melanomas, we assessed the effects of BH3-mimetic molecules, the ABT family, known to exert pro-apoptotic activities in cancer cells. Our results uncovered that ABT-263 (Navitoclax), a potent and orally bioavailable BCL-2 family inhibitor, induced antiproliferative effects in metastatic human uveal melanoma cells through cell cycle arrest at the G0/G1 phase, loss of mitochondrial membrane potential, and subsequently apoptotic cell death monitored by caspase activation and poly-ADP ribose polymerase cleavage. ABT-263-mediated reduction in tumor growth was also observed in vivo. We observed in some cells that ABT-263 treatment mounted a pro-survival response through activation of the ER stress signaling pathway. Blocking the PERK signaling pathway increased the pro-apoptotic ABT-263 effect. We thus uncovered a resistance mechanism in uveal melanoma cells mediated by activation of endoplasmic reticulum stress pathway. Therefore, our study identifies ABT-263 as a valid therapeutic option for patients suffering from uveal melanoma.

Lysosomal acid ceramidase ASAH1 controls the transition between invasive and proliferative phenotype in melanoma cells.

Phenotypic plasticity and subsequent generation of intratumoral heterogeneity underly key traits in malignant melanoma such as drug resistance and metastasis. Melanoma plasticity promotes a switch between proliferative and invasive phenotypes characterized by different transcriptional programs of which MITF is a critical regulator. Here, we show that the acid ceramidase ASAH1, which controls sphingolipid metabolism, acted as a rheostat of the phenotypic switch in melanoma cells. Low ASAH1 expression was associated with an invasive behavior mediated by activation of the integrin alphavbeta5-FAK signaling cascade. In line with that, human melanoma biopsies revealed heterogeneous staining of ASAH1 and low ASAH1 expression at the melanoma invasive front. We also identified ASAH1 as a new target of MITF, thereby involving MITF in the regulation of sphingolipid metabolism. Together, our findings provide new cues to the mechanisms underlying the phenotypic plasticity of melanoma cells and identify new anti-metastatic targets.

Focus on cutaneous and uveal melanoma specificities.

Cutaneous melanoma (CM) and uveal melanoma (UM) derive from cutaneous and uveal melanocytes that share the same embryonic origin and display the same cellular function. However, the etiopathogenesis and biological behaviors of these melanomas are very different. CM and UM display distinct landscapes of genetic alterations and show different metastatic routes and tropisms. Hence, therapeutic improvements achieved in the last few years for the treatment of CM have failed to ameliorate the clinical outcomes of patients with UM. The scope of this review is to discuss the differences in tumorigenic processes (etiologic factors and genetic alterations) and tumor biology (gene expression and signaling pathways) between CM and UM. We develop hypotheses to explain these differences, which might provide important clues for research avenues and the identification of actionable vulnerabilities suitable for the development of new therapeutic strategies for metastatic UM.

FANCD2 functions as a critical factor downstream of MiTF to maintain the proliferation and survival of melanoma cells.

Proteins involved in genetic stability maintenance and safeguarding DNA replication act not only against cancer initiation but could also play a major role in sustaining cancer progression. Here, we report that the FANC pathway is highly expressed in metastatic melanoma harboring the oncogenic microphthalmia-associated transcription factor (MiTF). We show that MiTF downregulation in melanoma cells lowers the expression of several FANC genes and proteins. Moreover, we observe that, similarly to the consequence of MiTF downregulation, FANC pathway silencing alters proliferation, migration and senescence of human melanoma cells. We demonstrate that the FANC pathway acts downstream MiTF and establish the existence of an epistatic relationship between MiTF and the FANC pathway. Our findings point to a central role of the FANC pathway in cellular and chromosomal resistance to both DNA damage and targeted therapies in melanoma cells. Thus, the FANC pathway is a promising new therapeutic target in melanoma treatment.