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Our previous studies on melanoma antigens identified two new polypeptides, named MELOE-1 and MELOE-2, that are involved in immunosurveillance. Intriguingly, these antigens are coded by distinct open reading frames (ORF) of the meloe mRNA which is significantly expressed only in the melanocytic lineage. In addition, MELOE-1 and -2 specific T cell clones recognized melanoma cells but very poorly normal melanocytes suggesting differential translation of meloe in normal vs tumor cells. This prompted us to elucidate the mechanisms of translation of these antigens in melanoma cells. We first demonstrated that no splicing event or cryptic promoter could generate shorter meloe transcripts containing only one of the two ORFs. Triggering meloe RNA degradation with a siRNA close to the ORF coding for MELOE-2 abrogated expression of both MELOE-1 and MELOE-2, thus confirming that the two ORFs are always associated. Next we showed, in a bicistronic reporter system, that IRES activities could be detected upstream of MELOE-1 and MELOE-2 and finally confirmed their translation from full length meloe cDNA in melanoma cells with eGFP constructs. In conclusion, meloe is a polycistronic mRNA that generates both MELOE-1 and MELOE-2 antigens through IRES-dependent translation in melanoma cells and that may explain their tumor specificity.

Abstract In multiple myeloma, as in B‐cell malignancies, mono‐ and especially bi‐allelic TP53 gene inactivation is a high‐risk factor for treatment resistance, and there are currently no therapies specifically targeting p53 deficiency. In this study, we evaluated if the loss of cell cycle control in p53‐deficient myeloma cells would confer a metabolically actionable vulnerability. We show that CTP synthase 1 (CTPS1), which encodes a CTP synthesis rate‐limiting enzyme essential for DNA and RNA synthesis in lymphoid cells, is overexpressed in samples from myeloma patients displaying a high proliferation rate (high MKI67 expression) or a low p53 score (synonymous with TP53 deletion and/or mutation). This overexpression of CTPS1 was associated with reduced survival in two cohorts. Using scRNA‐seq analysis in 24 patient samples, we further demonstrate that myeloma cells in the S or G2/M phase display high CTPS1 expression. Pharmacological inhibition of CTPS1 by STP‐B induced cell cycle arrest in early S phase in isogenic NCI‐H929 or XG7 TP53+/+, TP53−/−, and TP53R175H/R175H cells and in a TP53−/R123STOP patient sample. The functional annotation of transcriptional changes in 10 STP‐B‐treated myeloma cell lines revealed a decrease in protein translation and confirmed the blockade of cells into the S phase. The pharmacological inhibition of ATR, which governs the intrinsic S/G2 checkpoint, in STP‐B‐induced S‐phase arrested cells synergistically induced cell death in TP53+/+, TP53−/−, and TP53R175H/R175H isogenic cell lines (Bliss score >15). This combination induced replicative stress and caspase‐mediated cell death and was highly effective in resistant/refractory patient samples with TP53 deletion and/or mutation and in TP53−/− NCI‐H929 xenografted NOD‐scid IL2Rgamma mice. Our in vitro, ex vivo, and in vivo data provide the rationale for combined CTPS1 and ATR inhibition for the treatment of p53‐deficient patients.

Aggressive B-cell malignancies, such as mantle cell lymphoma (MCL), are microenvironment-dependent tumors and a better understanding of the dialogs occurring in lymphoma-protective ecosystems will provide new perspectives to increase treatment efficiency. To identify novel molecular regulations, we performed a transcriptomic analysis based on the comparison of circulating MCL cells (n=77) versus MCL lymph nodes (n=107) together with RNA sequencing of malignant (n=8) versus normal B-cell (n=6) samples. This integrated analysis led to the discovery of microenvironment-dependent and tumor-specific secretion of interleukin-32 beta (IL32β), whose expression was confirmed in situ within MCL lymph nodes by multiplex immunohistochemistry. Using ex vivo models of primary MCL cells (n=23), we demonstrated that, through the secretion of IL32β, the tumor was able to polarize monocytes into specific MCL-associated macrophages, which in turn favor tumor survival. We highlighted that while IL32β-stimulated macrophages secreted several protumoral factors, they supported tumor survival through a soluble dialog, mostly driven by BAFF. Finally, we demonstrated the efficacy of selective NIK/alternative-NFkB inhibition to counteract microenvironment-dependent induction of IL32β and BAFF-dependent survival of MCL cells. These data uncovered the IL32β/BAFF axis as a previously undescribed pathway involved in lymphoma-associated macrophage polarization and tumor survival, which could be counteracted through selective NIK inhibition.

Antimitotic compounds, such as paclitaxel, induce cell death in cycling cancer cells only. Here, the authors show that paclitaxel-targeted breast cancer cells prime neighboring cells to apoptosis through a STING-mediated paracrine signaling pathway.

Abstract Background Human myeloma cell lines (HMCLs) are widely used for their representation of primary myeloma cells because they cover patient diversity, although not fully. Their genetic background is mostly undiscovered, and no comprehensive study has ever been conducted in order to reveal those details. Methods We performed whole-exon sequencing of 33 HMCLs, which were established over the last 50 years in 12 laboratories. Gene expression profiling and drug testing for the 33 HMCLs are also provided and correlated to exon-sequencing findings. Results Missense mutations were the most frequent hits in genes (92%). HMCLs harbored between 307 and 916 mutations per sample, with TP53 being the most mutated gene (67%). Recurrent bi-allelic losses were found in genes involved in cell cycle regulation (RB1, CDKN2C), the NFκB pathway (TRAF3, BIRC2), and the p53 pathway (TP53, CDKN2A). Frequency of mutations/deletions in HMCLs were either similar to that of patients (e.g., DIS3, PRDM1, KRAS) or highly increased (e.g., TP53, CDKN2C, NRAS, PRKD2). MAPK was the most altered pathway (82% of HMCLs), mainly by RAS mutants. Surprisingly, HMCLs displayed alterations in epigenetic (73%) and Fanconi anemia (54%) and few alterations in apoptotic machinery. We further identified mutually exclusive and associated mutations/deletions in genes involved in the MAPK and p53 pathways as well as in chromatin regulator/modifier genes. Finally, by combining the gene expression profile, gene mutation, gene deletion, and drug response, we demonstrated that several targeted drugs overcome or bypass some mutations. Conclusions With this work, we retrieved genomic alterations of HMCLs, highlighting that they display numerous and unprecedented abnormalities, especially in DNA regulation and repair pathways. Furthermore, we demonstrate that HMCLs are a reliable model for drug screening for refractory patients at diagnosis or at relapse.

The melanoma antigens MELOE-1 and MELOE-2 are encoded by a messenger, called meloe, overexpressed in melanomas compared with other tumour cell types and healthy tissues. They are both able to elicit melanoma-specific T cell responses in melanoma patients, and MELOE-1-specific CD8 T cells have been involved in melanoma immunosurveillance. With the aim to develop immunotherapies targeting this antigen, we investigated the transcriptional mechanisms leading to the preferential expression of meloe messenger in the melanocytic lineage. We defined the minimal promoter region of meloe gene and identified binding motifs for a set of transcription factors. Using mutagenesis, co-transfection experiments and chromatin immunoprecipitation, we showed that transcription factors involved in meloe promoter activity in melanomas were the melanocytic specific SOX9 and SOX10 proteins together with the activated P-CREB protein. Furthermore, we showed that meloe promoter was hypomethylated in melanomas and melanocytes, and hypermethylated in colon cancer cell lines and mesotheliomas, thus explaining the absence of P-CREB binding in these cell lines. This was a second key to explain the overerexpression of meloe messenger in the melanocytic lineage. To our knowledge, such a dual transcriptional control conferring tissue-specificity has never been described for the expression of tumour antigens.

BACKGROUND: Double positive (DP) CD4CD8 Talphabeta cells have been reported in normal individuals as well as in different pathological conditions including inflammatory diseases, viral infections and cancer, but their function remains to be elucidated. We recently reported the increased frequency of DP Talphabeta cells in human breast pleural effusions. This manuscript addresses the question of the existence and above all the role of this non-conventional DP sub-population among tumor associated lymphocytes in melanomas. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the intratumoral cell infiltrate in solid metastasis (n = 6) and tumor invaded lymph nodes (n = 26) samples from melanomas patients by multiparametric cytometry. Here we documented for the first time significant increased frequency of DP T cells in about 60% of melanoma tumors compared to blood samples. Interestingly, a high proportion of these cells produced TNF-alpha in response to autologous melanoma cell lines. Besides, they are characterized by a unique cytokine profile corresponding to higher secretion of IL-13, IL-4 and IL-5 than simple positive T cells. In deep analysis, we derived a representative tumor-reactive DP T cell clone from a melanoma patient's invaded lymph node. This clone was restricted by HLA-A*2402 and recognized both autologous and allogeneic tumor cells of various origins as well as normal cells, suggesting that the target antigen was a ubiquitous self antigen. However, this DP T cell clone failed to kill HLA-A*2402 EBV-transformed B cells, probably due to the constitutive expression of immunoproteasome by these cells. CONCLUSIONS/SIGNIFICANCE: In conclusion, we can postulate that, according to their broad tumor reactivity and to their original cytokine profile, the tumor associated DP T cells could participate in immune responses to tumors in vivo. Therefore, the presence of these cells and their role will be crucial to address in cancer patients, especially in the context of immunotherapies.