Giovanni Ciriello

Multilevel Genomics-Based Taxonomy of Renal Cell Carcinoma

On the basis of multidimensional and comprehensive molecular characterization (including DNA methalylation and copy number, RNA, and protein expression), we classified 894 renal cell carcinomas (RCCs) of various histologic types into nine major genomic subtypes. Site of origin within the nephron was one major determinant in the classification, reflecting differences among clear cell, chromophobe, and papillary RCC. Widespread molecular changes associated with TFE3 gene fusion or chromatin modifier genes were present within a specific subtype and spanned multiple subtypes. Differences in patient survival and in alteration of specific pathways (including hypoxia, metabolism, MAP kinase, NRF2-ARE, Hippo, immune checkpoint, and PI3K/AKT/mTOR) could further distinguish the subtypes. Immune checkpoint markers and molecular signatures of T cell infiltrates were both highest in the subtype associated with aggressive clear cell RCC. Differences between the genomic subtypes suggest that therapeutic strategies could be tailored to each RCC disease subset.

Loss of the HVEM Tumor Suppressor in Lymphoma and Restoration by Modified CAR-T Cells

The HVEM (TNFRSF14) receptor gene is among thexa0most frequently mutated genes in germinal center lymphomas. We report that loss of HVEM leads toxa0cell-autonomous activation of B cell proliferationxa0and drives the development of GC lymphomasxa0inxa0vivo. HVEM-deficient lymphoma B cells also induce a tumor-supportive microenvironment marked by exacerbated lymphoid stroma activation and increased recruitment of T follicular helper (TFH) cells. These changes result from the disruption of inhibitory cell-cell interactions between the HVEM and BTLA (B and T lymphocyte attenuator) receptors. Accordingly, administration of the HVEM ectodomain protein (solHVEM(P37-V202)) binds BTLA and restores tumor suppression. To deliver solHVEM to lymphomas inxa0vivo, we engineered CD19-targeted chimeric antigen receptor (CAR) Txa0cells that produce solHVEM locally and continuously. These modified CAR-T cells show enhanced therapeutic activity against xenografted lymphomas. Hence, the HVEM-BTLA axis opposes lymphoma development, and our study illustrates the use of CAR-T cells as micro-pharmacies able to deliver an anti-cancer protein.

Comprehensive Genetic Landscape of Uveal Melanoma by Whole-Genome Sequencing

Uveal melanoma (UM) is a rare intraocular tumor that, similar to cutaneous melanoma, originates from melanocytes. To gain insights into its genetics, we performed whole-genome sequencing at very deep coverage of tumor-control pairs in 33 samples (24 primary and 9 metastases). Genome-wide, the number of coding mutations was rather low (only 17 variants per tumor on average; range 7-28), thus radically different from cutaneous melanoma, where hundreds of exonic DNA insults are usually detected. Furthermore, no UV light-induced mutational signature was identified. Recurrent coding mutations were found in the known UM drivers GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. Other genes, i.e., TP53BP1, CSMD1, TTC28, DLK2, and KTN1, were also found to harbor somatic mutations in more than one individual, possibly indicating a previously undescribed association with UM pathogenesis. De novo assembly of unmatched reads from non-coding DNA revealed peculiar copy-number variations defining specific UM subtypes, which in turn could be associated with metastatic transformation. Mutational-driven comparison with other tumor types showed that UM is very similar to pediatric tumors, characterized by very few somatic insults and, possibly, important epigenetic changes. Through the analysis of whole-genome sequencing data, our findings shed new light on the molecular genetics of uveal melanoma, delineating it as an atypical tumor of the adult for which somatic events other than mutations in exonic DNA shape its genetic landscape and define its metastatic potential.

Conditional Selection of Genomic Alterations Dictates Cancer Evolution and Oncogenic Dependencies

Cancer evolves through the emergence and selection of molecular alterations. Cancer genome profiling has revealed that specific events are more or less likely to be co-selected, suggesting that the selection of one event depends on the others. However, the nature of these evolutionary dependencies and their impact remain unclear. Here, we designed SELECT, an algorithmic approach to systematically identify evolutionary dependencies from alteration patterns. By analyzing 6,456 genomes from multiple tumor types, we constructed a map of oncogenic dependencies associated with cellular pathways, transcriptional readouts, and therapeutic response. Finally, modeling of cancer evolution shows that alteration dependencies emerge only under conditional selection. These results provide a framework for the design of strategies to predict cancer progression and therapeutic response.

The molecular basis of breast cancer pathological phenotypes.

The histopathological evaluation of morphological features in breast tumours provides prognostic information to guide therapy. Adjunct molecular analyses provide further diagnostic, prognostic and predictive information. However, there is limited knowledge of the molecular basis of morphological phenotypes in invasive breast cancer. This study integrated genomic, transcriptomic and protein data to provide a comprehensive molecular profiling of morphological features in breast cancer. Fifteen pathologists assessed 850 invasive breast cancer cases from The Cancer Genome Atlas (TCGA). Morphological features were significantly associated with genomic alteration, DNA methylation subtype, PAM50 and microRNA subtypes, proliferation scores, gene expression and/or reverse‐phase protein assay subtype. Marked nuclear pleomorphism, necrosis, inflammation and a high mitotic count were associated with the basal‐like subtype, and had a similar molecular basis. Omics‐based signatures were constructed to predict morphological features. The association of morphology transcriptome signatures with overall survival in oestrogen receptor (ER)‐positive and ER‐negative breast cancer was first assessed by use of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset; signatures that remained prognostic in the METABRIC multivariate analysis were further evaluated in five additional datasets. The transcriptomic signature of poorly differentiated epithelial tubules was prognostic in ER‐positive breast cancer. No signature was prognostic in ER‐negative breast cancer. This study provided new insights into the molecular basis of breast cancer morphological phenotypes. The integration of morphological with molecular data has the potential to refine breast cancer classification, predict response to therapy, enhance our understanding of breast cancer biology, and improve clinical management. This work is publicly accessible at www.dx.ai/tcga_breast. Copyright

Genetic and epigenetic inactivation of SESTRIN1 controls mTORC1 and response to EZH2 inhibition in follicular lymphoma

SESTRIN1 is a tumor suppressor deleted or epigenetically silenced by mutated EZH2. Lymphoma’s loss is a therapeutic gain Follicular lymphoma is a relatively common and difficult-to-treat hematologic malignancy, for which no specific targeted therapy is available. Knowing that deletions of chromosome 6q are common in this tumor type, Oricchio et al. examined the genes on this chromosome and identified SESTRIN1 as a likely tumor suppressor. The authors examined the mechanism by which the loss of SESTRIN1 contributes to tumorigenesis and identified a mechanistic connection between SESTRIN1 and EZH2, an epigenetic modifier that plays a role in multiple cancer types. The authors demonstrated that the effectiveness of targeting EZH2 depends on SESTRIN1 genetic and epigenetic status and also reported that mutations in EZH2 itself can sensitize cancer cells to additional targeted therapies. Follicular lymphoma (FL) is an incurable form of B cell lymphoma. Genomic studies have cataloged common genetic lesions in FL such as translocation t(14;18), frequent losses of chromosome 6q, and mutations in epigenetic regulators such as EZH2. Using a focused genetic screen, we identified SESTRIN1 as a relevant target of the 6q deletion and demonstrate tumor suppression by SESTRIN1 in vivo. Moreover, SESTRIN1 is a direct target of the lymphoma-specific EZH2 gain-of-function mutation (EZH2Y641X). SESTRIN1 inactivation disrupts p53-mediated control of mammalian target of rapamycin complex 1 (mTORC1) and enables mRNA translation under genotoxic stress. SESTRIN1 loss represents an alternative to RRAGC mutations that maintain mTORC1 activity under nutrient starvation. The antitumor efficacy of pharmacological EZH2 inhibition depends on SESTRIN1, indicating that mTORC1 control is a critical function of EZH2 in lymphoma. Conversely, EZH2Y641X mutant lymphomas show increased sensitivity to RapaLink-1, a bifunctional mTOR inhibitor. Hence, SESTRIN1 contributes to the genetic and epigenetic control of mTORC1 in lymphoma and influences responses to targeted therapies.

Pan-cancer inference of intra-tumor heterogeneity reveals associations with different forms of genomic instability

Genomic instability is a major driver of intra-tumor heterogeneity. However, unstable genomes often exhibit different molecular and clinical phenotypes, which are associated with distinct mutational processes. Here, we algorithmically inferred the clonal phylogenies of ~6,000 human tumors from 32 tumor types to explore how intra-tumor heterogeneity depends on different implementations of genomic instability. We found that extremely unstable tumors associated with DNA repair deficiencies or high chromosomal instability are not the most intrinsically heterogeneous. Conversely, intra-tumor heterogeneity is greatest in tumors exhibiting relatively high numbers of both mutations and copy number alterations, a feature often observed in cancers associated with exogenous mutagens. Independently of the type of instability, tumors with high number of clones invariably evolved through branching phylogenies that could be stratified based on the extent of clonal (early) and subclonal (late) instability. Interestingly, tumors with high number of subclonal mutations frequently exhibited chromosomal instability, TP53 mutations, and APOBEC-related mutational signatures. Vice versa, mutations of chromatin remodeling genes often characterized tumors with few subclonal but multiple clonal mutations. Understanding how intra-tumor heterogeneity depends on genomic instability is critical to identify markers predictive of the tumor complexity and envision therapeutic strategies able to exploit this association.

Pan-Cancer Landscape of Aberrant DNA Methylation across Human Tumors

The discovery of cancer-associated alterations has primarily focused on genetic variants. Nonetheless, altered epigenomes contribute to deregulate transcription and promote oncogenic pathways. Here, we designed an algorithmic approach (RESET) to identify aberrant DNA methylation and associated cis-transcriptional changes across >6,000 human tumors. Tumors exhibiting mutations of chromatin remodeling factors and Wnt signaling displayed DNA methylation instability, characterized by numerous hyper- and hypo-methylated loci. Most silenced and enhanced genes coalesced in specific pathways including apoptosis, DNA repair, and cell metabolism. Cancer-germline antigens (CG) were frequently epigenomically enhanced and their expression correlated with response to anti-PD-1, but not anti-CTLA4, in skin melanoma. Finally, we demonstrated the potential of our approach to explore DNA methylation changes in pediatric tumors, which frequently lack genetic drivers and exhibit epigenomic modifications. Our results provide a pan-cancer map of aberrant DNA methylation to inform functional and therapeutic studies.

Pan-SRC kinase inhibition blocks B-cell receptor oncogenic signaling in non-Hodgkin lymphoma.

In diffuse large B-cell lymphoma (DLBCL), activation of the B-cell receptor (BCR) promotes multiple oncogenic signals, which are essential for tumor proliferation. Inhibition of the Brutons tyrosine kinase (BTK), a BCR downstream target, is therapeutically effective only in a subgroup of patients with DLBCL. Here, we used lymphoma cells isolated from patients with DLBCL to measure the effects of targeted therapies on BCR signaling and to anticipate response. In lymphomas resistant to BTK inhibition, we show that blocking BTK activity enhanced tumor dependencies from alternative oncogenic signals downstream of the BCR, converging on MYC upregulation. To completely ablate the activity of the BCR, we genetically and pharmacologically repressed the activity of the SRC kinases LYN, FYN, and BLK, which are responsible for the propagation of the BCR signal. Inhibition of these kinases strongly reduced tumor growth in xenografts and cell lines derived from patients with DLBCL independent of their molecular subtype, advancing the possibility to be relevant therapeutic targets in broad and diverse groups of DLBCL patients.

Evolutionary dynamics and molecular features of intra-tumor heterogeneity

The systematic assessment of intra-tumor heterogeneity is still limited and often unfeasible. In silico investigations of large tumor cohorts can be used to decipher how multiple clones emerge and organize into complex architectures. Here, we addressed this challenge by integrating mathematical modeling of cancer evolution with algorithmic inference of clonal phylogenies in 2,600 human tumors from 15 tumor types. Through numerical simulations, we could discriminate between observable and hidden intra-tumor heterogeneity, the latter characterized by clones that are missed by DNA sequencing of human samples. To overcome this limitation in human tumors, we show that population frequencies of detectable clones can be used to estimate the extent of hidden heterogeneity. Overall, simulated and human clonal architectures were highly concordant and showed that high numbers of clones invariably emerge through branching lineages. Interestingly, high numbers of alterations were not necessarily associated with high intra-tumor heterogeneity. Indeed, tumors with alterations in proliferation-associated genes exhibited high numbers of clonal mutations, but few clones. Instead, mutations of chromatin remodeling genes characterized tumors with high numbers of subclonal alterations and multiple clones. Our results identify evolutionary and genetic determinants of tumor clonal architectures to guide functional investigations of intra-tumor heterogeneity.