Oncogenic chimeric transcription factors drive tumor-specific transcription, processing, and translation of silent genomic regions.

Many cancers are characterized by gene fusions encoding oncogenic chimeric transcription factors (TFs) such as EWS::FLI1 in Ewing sarcoma (EwS). Here, we find that EWS::FLI1 induces the robust expression of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome. These neogenes (NGs) are virtually undetectable in large collections of normal tissues or non-EwS tumors and can be silenced by CRISPR interference at regulatory EWS::FLI1-bound microsatellites. Ribosome profiling and proteomics further show that some NGs are translated into highly EwS-specific peptides. More generally, we show that hundreds of NGs can be detected in diverse cancers characterized by chimeric TFs. Altogether, this study identifies the transcription, processing, and translation of novel, specific, highly expressed multi-exonic transcripts from otherwise silent regions of the genome as a new activity of aberrant TFs in cancer.

MDM4 amplification in atypical lipomatous tumors/well-differentiated liposarcoma: Private event or alternative oncogenic mechanism?

Adipocytic tumors are the most common mesenchymal tumors in soft tissues. Among them, a diagnostic challenge relies in the distinction between lipoma and atypical lipomatous tumor (ALT)/well differentiated liposarcoma (WDLPS), as both entities are often undistinguishable not only from a radiological point of view, but also at the microscopic level and particularly when dealing with small tumor specimen. Thus, detection of recurrent MDM2 amplifications may be the only criteria to discriminate malignant tumors from lipomas. In this study, we report the case of a patient diagnosed with a well differentiated, adipocytic tumor located in the inferior limb and lacking MDM2 amplification, whose diagnosis was reclassified for ALT/WDLPS after identification of an alternative MDM4 amplification by comparative genomic hybridization profiling, whole exome sequencing and fluorescence in situ hybridization (FISH). Screening of a cohort of 37 large, deep-seated, well-differentiated adipocytic tumors previously classified as lipomas using RT-qPCR and FISH failed to detect other cases of MDM4-amplified ALT/WDLPS. This report shows that MDM4 amplification is an exceptional molecular event alternative to MDM2 amplification in ALT/WDLPS. This alteration should be considered and looked for in suspicious adipocytic tumors to optimize their surgical management.

Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis.

Alterations of chromatin modifiers are frequent in cancer, but their functional consequences often remain unclear. Focusing on the Polycomb protein EZH2 that deposits the H3K27me3 (trimethylation of Lys27 of histone H3) mark, we showed that its high expression in solid tumors is a consequence, not a cause, of tumorigenesis. In mouse and human models, EZH2 is dispensable for prostate cancer development and restrains breast tumorigenesis. High EZH2 expression in tumors results from a tight coupling to proliferation to ensure H3K27me3 homeostasis. However, this process malfunctions in breast cancer. Low EZH2 expression relative to proliferation and mutations in Polycomb genes actually indicate poor prognosis and occur in metastases. We show that while altered EZH2 activity consistently modulates a subset of its target genes, it promotes a wider transcriptional instability. Importantly, transcriptional changes that are consequences of EZH2 loss are predominantly irreversible. Our study provides an unexpected understanding of EZH2's contribution to solid tumors with important therapeutic implications.

Neoadjuvant Everolimus for Adult Giant Mesenteric Cystic Lymphangioma with mTOR Pathway Activation.

Cystic lymphangioma are rare benign vascular or lymphatic tumors, diagnosed mostly in newborns or children, that may become life-threatening because of local invasiveness. Surgical "en-bloc" resection with negative margins is the only curative treatment, but some patients are diagnosed with unresectable tumors. We describe the case of a young adult with giant unresectable mesenteric lymphangioma. Extensive pathological characterization as well as whole exome and transcriptome sequencing enabled us to identify mTOR pathway activation within endothelial tumor cells. The patient was treated with everolimus and experienced major partial response, leading to the surgical resection of the residual lesions. This case highlights the importance of molecular characterization of adult cystic lymphangioma for mTOR pathway activation because multidisciplinary therapeutic approaches, including neoadjuvant everolimus and secondary surgery, can lead to complete cure of this rare condition. KEY POINTS: The case of an adult patient diagnosed with giant unresectable mesenteric cystic lymphangioma, in which activation of the mTOR pathway was documented at both the pathological and transcriptomic levels, is reported. This patient showed major partial response to the mTOR inhibitor everolimus, which led to the successful resection of residual tumor lesions after 9 months of treatment. This report shows that mTOR targeting should be considered as neoadjuvant treatment in adult large cystic lymphangioma, as it can lead to complete surgery and cure of this rare condition.

Medullary Breast Carcinoma, a Triple-Negative Breast Cancer Associated with BCLG Overexpression.

Medullary breast carcinoma (MBC) is a rare subtype of triple-negative breast cancer with specific genomic features within the spectrum of basal-like carcinoma (BLC). In this study of 19 MBCs and 36 non-MBC BLCs, we refined the transcriptomic and genomic knowledge about this entity. Unsupervised and supervised analysis of transcriptomic profiles confirmed that MBC clearly differs from non-MBC BLC, with 92 genes overexpressed and 154 genes underexpressed in MBC compared with non-MBC BLC. Immunity-related pathways are the most differentially represented pathways in MBC compared with non-MBC BLC. The proapoptotic gene BCLG (official name BCL2L14) is by far the most intensely overexpressed gene in MBC. A quantitative RT-PCR validation study conducted in 526 breast tumors corresponding to all molecular subtypes documented the specificity of BCLG overexpression in MBC, which was confirmed at the protein level by immunohistochemistry. We also found that most MBCs belong to the immunomodulatory triple-negative breast cancer subtype. Using pan-genomic analysis, it was found that MBC harbors more losses of heterozygosity than non-MBC BLC. These observations corroborate the notion that MBC remains a distinct entity that could benefit from specific treatment strategies (such as deescalation or targeted therapy) adapted to this rare tumor type.

LIN7A is a major determinant of cell-polarity defects in breast carcinomas.

BACKGROUND: Polarity defects are a hallmark of most carcinomas. Cells from invasive micropapillary carcinomas (IMPCs) of the breast are characterized by a striking cell polarity inversion and represent an interesting model for the analysis of polarity abnormalities. METHODS: In-depth investigation of polarity proteins in 24 IMPCs and a gene expression profiling, comparing IMPC (n = 73) with invasive carcinomas of no special type (ICNST) (n = 51) have been performed. RESULTS: IMPCs showed a profound disorganization of the investigated polarity proteins and revealed major abnormalities in their subcellular localization. Gene expression profiling experiments highlighted a number of deregulated genes in the IMPCs that have a role in apico-basal polarity, adhesion and migration. LIN7A, a Crumbs-complex polarity gene, was one of the most differentially over-expressed genes in the IMPCs. Upon LIN7A over-expression, we observed hyperproliferation, invasion and a complete absence of lumen formation, revealing strong polarity defects. CONCLUSION: This study therefore shows that LIN7A has a crucial role in the polarity abnormalities associated with breast carcinogenesis.

JUN oncogene amplification and overexpression block adipocytic differentiation in highly aggressive sarcomas.

The human oncogene JUN encodes a component of the AP-1 complex and is consequently involved in a wide range of pivotal cellular processes, including cell proliferation, transformation, and apoptosis. Nevertheless, despite extensive analyses of its functions, it has never been directly involved in a human cancer. We demonstrate here that it is highly amplified and overexpressed in undifferentiated and aggressive human sarcomas, which are blocked at an early step of adipocyte differentiation. We confirm by cellular and xenograft mouse models recapitulating these sarcoma genetics that the failure to differentiate is dependent upon JUN amplification/overexpression.

A siRNA screen identifies RAD21, EIF3H, CHRAC1 and TANC2 as driver genes within the 8q23, 8q24.3 and 17q23 amplicons in breast cancer with effects on cell growth, survival and transformation.

RNA interference has boosted the field of functional genomics, by making it possible to carry out 'loss-of-function' screens in cultured cells. Here, we performed a small interfering RNA screening, in three breast cancer cell lines, for 101 candidate driver genes overexpressed in amplified breast tumors and belonging to eight amplicons on chromosomes 8q and 17q, investigating their role in cell survival/proliferation. This screening identified eight driver genes that were amplified, overexpressed and critical for breast tumor cell proliferation or survival. They included the well-described oncogenic driver genes for the 17q12 amplicon, ERBB2 and GRB7. Four of six other candidate driver genes-RAD21 and EIF3H, both on chromosome 8q23, CHRAC1 on chromosome 8q24.3 and TANC2 on chromosome 17q23-were confirmed to be driver genes regulating the proliferation/survival of clonogenic breast cancer cells presenting an amplification of the corresponding region. Indeed, knockdown of the expression of these genes decreased cell viability, through both cell cycle arrest and apoptosis induction, and inhibited the formation of colonies in anchorage-independent conditions, in soft agar. Strategies for inhibiting the expression of these genes or the function of the proteins they encode are therefore of potential value for the treatment of breast cancers presenting amplifications of the corresponding genomic region.

Polarity gene alterations in pure invasive micropapillary carcinomas of the breast.

INTRODUCTION: Pure invasive micropapillary carcinoma (IMPC) is a special type of breast carcinoma characterised by clusters of cells presenting polarity abnormalities. The biological alterations underlying this pattern remain unknown. METHODS: Pangenomic analysis (n=39), TP53 (n=43) and PIK3CA (n=41) sequencing in a series of IMPCs were performed. A subset of cases was also analysed with whole-exome sequencing (n=4) and RNA sequencing (n=6). Copy number variation profiles were compared with those of oestrogen receptors and grade-matched invasive ductal carcinomas (IDCs) of no special type. RESULTS: Unsupervised analysis of genomic data distinguished two IMPC subsets: one (Sawtooth/8/16) exhibited a significant increase in 16p gains (71%), and the other (Firestorm/Amplifier) was characterised by a high frequency of 8q (35%), 17q (20% to 46%) and 20q (23% to 30%) amplifications and 17p loss (74%). TP53 mutations (10%) were more frequently identified in the amplifier subset, and PIK3CA mutations (4%) were detected in both subsets. Compared to IDC, IMPC exhibited specific loss of the 6q16-q22 region (45%), which is associated with downregulation of FOXO3 and SEC63 gene expression. SEC63 and FOXO3 missense mutations were identified in one case each (2%). Whole-exome sequencing combined with RNA sequencing of IMPC allowed us to identify somatic mutations in genes involved in polarity, DNAH9 and FMN2 (8% and 2%, respectively) or ciliogenesis, BBS12 and BBS9 (2% each) or genes coding for endoplasmic reticulum protein, HSP90B1 and SPTLC3 (2% each) and cytoskeleton, UBR4 and PTPN21 (2% each), regardless of the genomic subset. The intracellular biological function of the mutated genes identified by gene ontology analysis suggests a driving role in the clinicopathological characteristics of IMPC. CONCLUSION: In our comprehensive molecular analysis of IMPC, we identified numerous genomic alterations without any recurrent fusion genes. Recurrent somatic mutations of genes participating in cellular polarity and shape suggest that they, together with other biological alterations (such as epigenetic modifications and stromal alterations), could contribute to the morphological pattern of IMPC. Though none of the individual abnormalities demonstrated specificity for IMPC, whether their combination in IMPC may have a cumulative effect that drives the abnormal polarity of IMPC needs to be examined further with in vitro experiments.

PPAPDC1B and WHSC1L1 are common drivers of the 8p11-12 amplicon, not only in breast tumors but also in pancreatic adenocarcinomas and lung tumors.

Amplification of the 8p11-12 chromosomal region is a common genetic event in many epithelial cancers. In breast cancer, several genes within this region have been shown to display oncogenic activity. Among these genes, the enzyme-encoding genes, PPAPDC1B and WHSC1L1, have been identified as potential therapeutic targets. We investigated whether PPAPDC1B and WHSC1L1 acted as general driver genes, thereby serving as therapeutic targets in other tumors with 8p11-12 amplification. By using publicly available genomic data from a panel of 883 cell lines derived from different cancers, we identified the cell lines presenting amplification of both WHSC1L1 and PPAPDC1B. In particular, we focused on cell lines derived from lung cancer and pancreatic adenocarcinoma and found a correlation between the amplification of PPAPDC1B and WHSC1L1 with their overexpression. Loss-of-function studies based on the use of siRNA and shRNA demonstrated that PPAPDC1B and WHSC1L1 played a major role in regulating the survival of pancreatic adenocarcinoma and small-cell lung cancer-derived cell lines, both in anchorage-dependent and anchorage-independent conditions, displaying amplification and overexpression of these genes. We also demonstrated that PPAPDC1B and WHSC1L1 regulated xenograft growth in these cell lines. Finally, quantitative RT-PCR experiments after PPAPDC1B and WHSC1L1 knockdown revealed exclusive PPAPDC1B and WHSC1L1 gene targets in small-cell lung cancer and pancreatic adenocarcinoma-derived cell lines compared with breast cancer.

A metabolic crosstalk between liposarcoma and muscle sustains tumor growth.

Dedifferentiated and Well-differentiated liposarcoma are characterized by a systematic amplification of the Murine Double Minute 2 (MDM2) oncogene. We demonstrate that p53-independent metabolic functions of chromatin-bound MDM2 are exacerbated in liposarcoma and mediate an addiction to serine metabolism to sustain tumor growth. However, the origin of exogenous serine remains unclear. Here, we show that elevated serine levels in mice harboring liposarcoma-patient derived xenograft, released by distant muscle is essential for liposarcoma cell survival. Repressing interleukine-6 expression, or treating liposarcoma cells with Food and Drugs Administration (FDA) approved anti-interleukine-6 monoclonal antibody, decreases de novo serine synthesis in muscle, impairs proliferation, and increases cell death in vitro and in vivo. This work reveals a metabolic crosstalk between muscle and liposarcoma tumor and identifies anti-interleukine-6 as a plausible treatment for liposarcoma patients.

Metastatic renal cell carcinoma with occult primary: a multicenter prospective cohort.

Metastatic carcinoma of presumed renal origin (rCUP) has recently emerged as a new entity within the heterogeneous entity of Cancers of Unknown Primary (CUP) but their biological features and optimal therapeutic management remain unknown. We report the molecular characteristics and clinical outcome of a series of 25 rCUP prospectively identified within the French National Multidisciplinary Tumor Board for CUP. This cohort strongly suggests that rCUP share similarities with common RCC subtypes and benefit from renal-tailored systemic treatment. This study highlights the importance of integrating clinical and molecular data for optimal diagnosis and management of CUP.

Cytokine profile of anti-spike CD4+T cells predicts humoral and CD8+T cell responses after anti-SARS-CoV-2 mRNA vaccination.

Coordinating immune responses - humoral and cellular - is vital for protection against severe Covid-19. Our study evaluates a multicytokine CD4+T cell signature's predictive for post-vaccinal serological and CD8+T cell responses. A cytokine signature composed of four cytokines (IL-2, TNF-α, IP10, IL-9) excluding IFN-γ, and generated through machine learning, effectively predicted the CD8+T cell response following mRNA-1273 or BNT162b2 vaccine administration. Its applicability extends to murine vaccination models, encompassing diverse immunization routes (such as intranasal) and vaccine platforms (including adjuvanted proteins). Notably, we found correlation between CD4+T lymphocyte-produced IL-21 and the humoral response. Consequently, we propose a test that offers a rapid overview of integrated immune responses. This approach holds particular relevance for scenarios involving immunocompromised patients because they often have low cell counts (lymphopenia) or pandemics. This study also underscores the pivotal role of CD4+T cells during a vaccine response and highlights their value in vaccine immunomonitoring.

Single-cell multiomics profiling reveals heterogeneous transcriptional programs and microenvironment in DSRCTs.

Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive sarcoma driven by the EWSR1::WT1 chimeric transcription factor. Despite this unique oncogenic driver, DSRCT displays a polyphenotypic differentiation of unknown causality. Using single-cell multi-omics on 12 samples from five patients, we find that DSRCT tumor cells cluster into consistent subpopulations with partially overlapping lineage- and metabolism-related transcriptional programs. In vitro modeling shows that high EWSR1::WT1 DNA-binding activity associates with most lineage-related states, in contrast to glycolytic and profibrotic states. Single-cell chromatin accessibility analysis suggests that EWSR1::WT1 binding site variability may drive distinct lineage-related transcriptional programs, supporting some level of cell-intrinsic plasticity. Spatial transcriptomics reveals that glycolytic and profibrotic states specifically localize within hypoxic niches at the periphery of tumor cell islets, suggesting an additional role of tumor cell-extrinsic microenvironmental cues. We finally identify a single-cell transcriptomics-derived epithelial signature associated with improved patient survival, highlighting the clinical relevance of our findings.

Cellular origin and clonal evolution of human dedifferentiated liposarcoma.

Dedifferentiated liposarcoma (DDLPS) is the most frequent high-grade soft tissue sarcoma subtype. It is characterized by a component of undifferentiated tumor cells coexisting with a component of well-differentiated adipocytic tumor cells. Both dedifferentiated (DD) and well-differentiated (WD) components exhibit MDM2 amplification, however their cellular origin remains elusive. Using single-cell RNA sequencing, DNA sequencing, in situ multiplex immunofluorescence and functional assays in paired WD and DD components from primary DDLPS tumors, we characterize the cellular heterogeneity of DDLPS tumor and micro-environment. We identify a population of tumor adipocyte stem cells (ASC) showing striking similarities with adipocyte stromal progenitors found in white adipose tissue. We show that tumor ASC harbor the ancestral genomic alterations of WD and DD components, suggesting that both derive from these progenitors following clonal evolution. Last, we show that DD tumor cells keep important biological properties of ASC including pluripotency and that their adipogenic properties are inhibited by a TGF-ß-high immunosuppressive tumor micro-environment.

New developments in the pathology and molecular biology of retroperitoneal sarcomas.

Retroperitoneal sarcomas (RPS) refer to a heterogeneous group of malignancies of mesenchymal origin developing from retroperitoneal tissues and vessels. The most frequent RPS are well differentiated/dedifferentiated liposarcomas and leiomyosarcomas, but other rare histological subtypes can be observed. Over the last decade, significant advances have been made in the pathological and molecular characterization of sarcomas. These advances have led to major changes in their diagnostic management as well as in the development of new therapeutic strategies based on tumor biology and microenvironment. This review describes the current knowledge and recent findings in the pathology and molecular biology of the most frequent RPS subtypes.

Reversible transitions between noradrenergic and mesenchymal tumor identities define cell plasticity in neuroblastoma.

Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity.

CXCR6 expressing T cells: Functions and role in the control of tumors.

CXCR6 is a receptor for the chemokine CXCL16, which exists as a membrane or soluble form. CXCR6 is a marker for resident memory T (TRM) cells that plays a role in immunosurveillance through their interaction with epithelial cells. The interaction of CXCR6 with CXCL16 expressed at the membrane of certain subpopulations of intratumor dendritic cells (DC) called DC3, ideally positions these CXCR6+ T cells to receive a proliferation signal from IL-15 also presented by DC3. Mice deficient in cxcr6 or blocking the interaction of CXCR6 with its ligand, experience a poorer control of tumor proliferation by CD8+ T cells, but also by NKT cells especially in the liver. Intranasal vaccination induces CXCL16 production in the lungs and is associated with infiltration by TRM expressing CXCR6, which are then required for the efficacy of anti-tumor vaccination. Therapeutically, the addition of CXCR6 to specific CAR-T cells enhances their intratumoral accumulation and prolongs survival in animal models of pancreatic, ovarian and lung cancer. Finally, CXCR6 is part of immunological signatures that predict response to immunotherapy based on anti-PD-(L)1 in various cancers. In contrast, a protumoral role of CXCR6+T cells has also been reported mainly in Non-alcoholic steatohepatitis (NASH) due to a non-antigen specific mechanism. The targeting and amplification of antigen-specific TRM expressing CXCR6 and its potential use as a biomarker of response to immunotherapy opens new perspectives in cancer treatment.

Single-cell transcriptomics reveals shared immunosuppressive landscapes of mouse and human neuroblastoma.

BACKGROUND: High-risk neuroblastoma is a pediatric cancer with still a dismal prognosis, despite multimodal and intensive therapies. Tumor microenvironment represents a key component of the tumor ecosystem the complexity of which has to be accurately understood to define selective targeting opportunities, including immune-based therapies. METHODS: We combined various approaches including single-cell transcriptomics to dissect the tumor microenvironment of both a transgenic mouse neuroblastoma model and a cohort of 10 biopsies from neuroblastoma patients, either at diagnosis or at relapse. Features of related cells were validated by multicolor flow cytometry and functional assays. RESULTS: We show that the immune microenvironment of MYCN-driven mouse neuroblastoma is characterized by a low content of T cells, several phenotypes of macrophages and a population of cells expressing signatures of myeloid-derived suppressor cells (MDSCs) that are molecularly distinct from the various macrophage subsets. We document two cancer-associated fibroblasts (CAFs) subsets, one of which corresponding to CAF-S1, known to have immunosuppressive functions. Our data unravel a complex content in myeloid cells in patient tumors and further document a striking correspondence of the microenvironment populations between both mouse and human tumors. We show that mouse intratumor T cells exhibit increased expression of inhibitory receptors at the protein level. Consistently, T cells from patients are characterized by features of exhaustion, expressing inhibitory receptors and showing low expression of effector cytokines. We further functionally demonstrate that MDSCs isolated from mouse neuroblastoma have immunosuppressive properties, impairing the proliferation of T lymphocytes. CONCLUSIONS: Our study demonstrates that neuroblastoma tumors have an immunocompromised microenvironment characterized by dysfunctional T cells and accumulation of immunosuppressive cells. Our work provides a new and precious data resource to better understand the neuroblastoma ecosystem and suggest novel therapeutic strategies, targeting both tumor cells and components of the microenvironment.

Mild dyslipidemia accelerates tumorigenesis through expansion of Ly6Chi monocytes and differentiation to pro-angiogenic myeloid cells.

Cancer and cardiovascular disease (CVD) share common risk factors such as dyslipidemia, obesity and inflammation. However, the role of pro-atherogenic environment and its associated low-grade inflammation in tumor progression remains underexplored. Here we show that feeding C57BL/6J mice with a non-obesogenic high fat high cholesterol diet (HFHCD) for two weeks to induce mild dyslipidemia, increases the pool of circulating Ly6Chi monocytes available for initial melanoma development, in an IL-1ß-dependent manner. Descendants of circulating myeloid cells, which accumulate in the tumor microenvironment of mice under HFHCD, heighten pro-angiogenic and immunosuppressive activities locally. Limiting myeloid cell accumulation or targeting VEGF-A production by myeloid cells decrease HFHCD-induced tumor growth acceleration. Reverting the HFHCD to a chow diet at the time of tumor implantation protects against tumor growth. Together, these data shed light on cross-disease communication between cardiovascular pathologies and cancer.