Gouri Nanjangud

Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas

Genomic instability promotes tumorigenesis and can occur through various mechanisms, including defective segregation of chromosomes or inactivation of DNA mismatch repair. Although B-cell lymphomas are associated with chromosomal translocations that deregulate oncogene expression, a mechanism for genome-wide instability during lymphomagenesis has not been described. During B-cell development, the immunoglobulin variable (V) region genes are subject to somatic hypermutation in germinal-centre B cells. Here we report that an aberrant hypermutation activity targets multiple loci, including the proto-oncogenes PIM1, MYC, RhoH/TTF (ARHH) and PAX5, in more than 50% of diffuse large-cell lymphomas (DLCLs), which are tumours derived from germinal centres. Mutations are distributed in the 5′ untranslated or coding sequences, are independent of chromosomal translocations, and share features typical of V-region-associated somatic hypermutation. In contrast to mutations in V regions, however, these mutations are not detectable in normal germinal-centre B cells or in other germinal-centre-derived lymphomas, suggesting a DLCL-associated malfunction of somatic hypermutation. Intriguingly, the four hypermutable genes are susceptible to chromosomal translocations in the same region, consistent with a role for hypermutation in generating translocations by DNA double-strand breaks. By mutating multiple genes, and possibly by favouring chromosomal translocations, aberrant hypermutation may represent the major contributor to lymphomagenesis.

The Eph-receptor A7 is a soluble tumor suppressor for follicular lymphoma

Insights into cancer genetics can lead to therapeutic opportunities. By cross-referencing chromosomal changes with an unbiased genetic screen we identify the ephrin receptor A7 (EPHA7) as a tumor suppressor in follicular lymphoma (FL). EPHA7 is a target of 6q deletions and inactivated in 72% of FLs. Knockdown of EPHA7 drives lymphoma development in a murine FL model. In analogy to its physiological function in brain development, a soluble splice variant of EPHA7 (EPHA7(TR)) interferes with another Eph-receptor and blocks oncogenic signals in lymphoma cells. Consistent with this drug-like activity, administration of the purified EPHA7(TR) protein produces antitumor effects against xenografted human lymphomas. Further, by fusing EPHA7(TR) to the anti-CD20 antibody (rituximab) we can directly target this tumor suppressor to lymphomas in vivo. Our study attests to the power of combining descriptive tumor genomics with functional screens and reveals EPHA7(TR) as tumor suppressor with immediate therapeutic potential.

Recurring chromosomal abnormalities in non-hodgkin's lymphoma: Biologic and clinical significance

Non-Hodgkins lymphomas (NHLs) are a group of clinically important neoplasms with a complex biology that makes their classification and treatment difficult. Their incidence is increasing and they cause significant morbidity and mortality. NHLs result from transformation of B and T/natural killer (NK) cells. Their genetic hallmark is chromosomal translocations resulting from aberrant rearrangements of IG and TCR genes, which lead to inappropriate expression of genes at reciprocal breakpoints that regulate a variety of cellular functions, including gene transcription, cell cycle, apoptosis, and tumor progression. Cytogenetics followed by molecular genetic analysis of some of the recurring translocations continues to provide new insights into lymphomagenesis and cell biology. More recently, chromosomal and gene amplification and gene deletion have been recognized as frequent genetic changes that may play a role in lymphoma progression and clinical behavior. In this review, cytogenetic data pertaining to recurring chromosomal changes on lymphomas are reviewed and examined in relation to their relevance to lymphoma development, classification, and clinical behavior.

Chromosomal instability drives metastasis through a cytosolic DNA response

Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS–STING (cyclic GMP-AMP synthase–stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

An approach to suppress the evolution of resistance in BRAFV600E-mutant cancer

The principles that govern the evolution of tumors exposed to targeted therapy are poorly understood. Here we modeled the selection and propagation of an amplification in the BRAF oncogene (BRAFamp) in patient-derived tumor xenografts (PDXs) that were treated with a direct inhibitor of the kinase ERK, either alone or in combination with other ERK signaling inhibitors. Single-cell sequencing and multiplex fluorescence in situ hybridization analyses mapped the emergence of extra-chromosomal amplification in parallel evolutionary trajectories that arose in the same tumor shortly after treatment. The evolutionary selection of BRAFamp was determined by the fitness threshold, the barrier that subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for inhibitors of ERK signaling, suggesting that sequential monotherapy is ineffective and selects for a progressively higher BRAF copy number. Concurrent targeting of the RAF, MEK and ERK kinases, however, imposed a sufficiently high fitness threshold to prevent the propagation of subclones with high-level BRAFamp. When administered on an intermittent schedule, this treatment inhibited tumor growth in 11/11 PDXs of lung cancer or melanoma without apparent toxicity in mice. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose the highest fitness threshold will likely prevent the evolution of resistance-causing alterations and, thus, merit testing in patients.

Clonal Selection in Malignant Transformation of Human Fibroblasts Transduced with Defined Cellular Oncogenes

Recent evidence has implied that disruption of a limited number of defined cellular pathways is necessary and sufficient for neoplastic conversion of a variety of normal human cell types in tissue culture. We show instead that malignancy in such models results from an iterative process of clonal selection in vitro and/or in vivo. Normal human fibroblasts underwent malignant transformation after transduction with telomerase, cyclin-dependent kinase 4, dominant-negative p53, and activated Ras or MEK. Furthermore, culture conditions favoring overgrowth resulted in clonal selection, which with added Ras or MEK oncogenes led to the emergence of tumorigenic clones. Such tumors showed variable degrees of malignancy with some even exhibiting metastasis. SV40 small t antigen (ST) has been reported to be necessary and sufficient to convert human fibroblasts with these pathway aberrations to a polyclonal tumor. However, we observed that clonal tumors emerged even with ST addition. Genomic instability was markedly increased by p53 and Rb pathway abrogation. Under the same conditions, fibroblasts with these alterations failed to induce tumors, implying that genomic instability may be necessary but not sufficient for malignant transformation. These findings indicate that the minimum number of events required for malignant transformation of human fibroblasts is greater than has been enumerated by such oncogene addition strategies and support a stochastic cancer progression model initiated by four defined cellular alterations.

Whole-genome single-cell copy number profiling from formalin-fixed paraffin-embedded samples

A substantial proportion of tumors consist of genotypically distinct subpopulations of cancer cells. This intratumor genetic heterogeneity poses a substantial challenge for the implementation of precision medicine. Single-cell genomics constitutes a powerful approach to resolve complex mixtures of cancer cells by tracing cell lineages and discovering cryptic genetic variations that would otherwise be obscured in tumor bulk analyses. Because of the chemical alterations that result from formalin fixation, single-cell genomic approaches have largely remained limited to fresh or rapidly frozen specimens. Here we describe the development and validation of a robust and accurate methodology to perform whole-genome copy-number profiling of single nuclei obtained from formalin-fixed paraffin-embedded clinical tumor samples. We applied the single-cell sequencing approach described here to study the progression from in situ to invasive breast cancer, which revealed that ductal carcinomas in situ show intratumor genetic heterogeneity at diagnosis and that these lesions may progress to invasive breast cancer through a variety of evolutionary processes.

Molecular analysis of aggressive renal cell carcinoma with unclassified histology reveals distinct subsets.

Renal cell carcinomas with unclassified histology (uRCC) constitute a significant portion of aggressive non-clear cell renal cell carcinomas that have no standard therapy. The oncogenic drivers in these tumours are unknown. Here we perform a molecular analysis of 62 high-grade primary uRCC, incorporating targeted cancer gene sequencing, RNA sequencing, single-nucleotide polymorphism array, fluorescence in situ hybridization, immunohistochemistry and cell-based assays. We identify recurrent somatic mutations in 29 genes, including NF2 (18%), SETD2 (18%), BAP1 (13%), KMT2C (10%) and MTOR (8%). Integrated analysis reveals a subset of 26% uRCC characterized by NF2 loss, dysregulated Hippo–YAP pathway and worse survival, whereas 21% uRCC with mutations of MTOR, TSC1, TSC2 or PTEN and hyperactive mTORC1 signalling are associated with better clinical outcome. FH deficiency (6%), chromatin/DNA damage regulator mutations (21%) and ALK translocation (2%) distinguish additional cases. Altogether, this study reveals distinct molecular subsets for 76% of our uRCC cohort, which could have diagnostic and therapeutic implications.

Identifying actionable targets through integrative analyses of GEM model and human prostate cancer genomic profiling

Copy-number alterations (CNA) are among the most common molecular events in human prostate cancer genomes and are associated with worse prognosis. Identification of the oncogenic drivers within these CNAs is challenging due to the broad nature of these genomic gains or losses which can include large numbers of genes within a given region. Here, we profiled the genomes of four genetically engineered mouse prostate cancer models that reflect oncogenic events common in human prostate tumors, with the goal of integrating these data with human prostate cancer datasets to identify shared molecular events. Met was amplified in 67% of prostate tumors from Pten p53 prostate conditional null mice and in approximately 30% of metastatic human prostate cancer specimens, often in association with loss of PTEN and TP53. In murine tumors with Met amplification, Met copy-number gain and expression was present in some cells but not others, revealing intratumoral heterogeneity. Forced MET overexpression in non–MET-amplified prostate tumor cells activated PI3K and MAPK signaling and promoted cell proliferation and tumor growth, whereas MET kinase inhibition selectively impaired the growth of tumors with Met amplification. However, the impact of MET inhibitor therapy was compromised by the persistent growth of non–Met-amplified cells within Met-amplified tumors. These findings establish the importance of MET in prostate cancer progression but reveal potential limitations in the clinical use of MET inhibitors in late-stage prostate cancer. Mol Cancer Ther; 14(1); 278–88. ©2014 AACR.

Deregulation of the carbohydrate (chondroitin 4) sulfotransferase 11 (CHST11) gene in a B-cell chronic lymphocytic leukemia with a t(12;14)(q23;q32).

The t(12;14)(q23;q32) breakpoints in a case of B-cell chronic lymphocytic leukemia (B-CLL) were mapped by fluorescence in situ hybridization (FISH) and Southern blot analysis and cloned using an IGH switch-γ probe. The translocation affected a productively rearranged IGH allele and the carbohydrate (chondroitin 4) sulfotransferase 11 (CHST11) locus at 12q23, with a reciprocal break in intron 2 of the CHST11 gene. CHST11 belongs to the HNK1 family of Golgi-associated sulfotransferases, a group of glycosaminoglycan-modifying enzymes, and is expressed mainly in the hematopoietic lineage. Northern Blot analysis of tumor RNA using CHST11-specific probes showed expression of two CHST11 forms of abnormal size. 5′- and 3′-Rapid Amplification of cDNA Ends (RACE) revealed IGH/CHST11 as well as CHST11/IGH fusion RNAs expressed from the der(14) and der(12) chromosomes. Both fusion species contained open reading frames making possible the translation of two truncated forms of CHST11 protein. The biological consequence of t(12;14)(q23;q32) in this case presumably is a disturbance of the cellular distribution of CHST11 leading to deregulation of a chondroitin-sulfate-dependent pathway specific to the hematopoietic lineage.