Novel ratio-metric features enable the identification of new driver genes across cancer types

An emergent area of cancer genomics is the identification of driver genes. Driver genes confer a selective growth advantage to the cell. While several driver genes have been discovered, many remain undiscovered, especially those mutated at a low frequency across samples. This study defines new features and builds a pan-cancer model, cTaG, to identify new driver genes. The features capture the functional impact of the mutations as well as their recurrence across samples, which helps build a model unbiased to genes with low frequency. The model classifies genes into the functional categories of driver genes, tumour suppressor genes (TSGs) and oncogenes (OGs), having distinct mutation type profiles. We overcome overfitting and show that certain mutation types, such as nonsense mutations, are more important for classification. Further, cTaG was employed to identify tissue-specific driver genes. Some known cancer driver genes predicted by cTaG as TSGs with high probability are ARID1A, TP53, and RB1. In addition to these known genes, potential driver genes predicted are CD36, ZNF750 and ARHGAP35 as TSGs and TAB3 as an oncogene. Overall, our approach surmounts the issue of low recall and bias towards genes with high mutation rates and predicts potential new driver genes for further experimental screening. cTaG is available at https://github.com/RamanLab/cTaG.

Genomic stability of mouse spermatogonial stem cells in vitro

Germline mutations underlie genetic diversity and species evolution. Previous studies have assessed the theoretical mutation rates and spectra in germ cells mostly by analyzing genetic markers and reporter genes in populations and pedigrees. This study reported the direct measurement of germline mutations by whole-genome sequencing of cultured spermatogonial stem cells in mice, namely germline stem (GS) cells, together with multipotent GS (mGS) cells that spontaneously dedifferentiated from GS cells. GS cells produce functional sperm that can generate offspring by transplantation into seminiferous tubules, whereas mGS cells contribute to germline chimeras by microinjection into blastocysts in a manner similar to embryonic stem cells. The estimated mutation rate of GS and mGS cells was approximately 0.22 × 10−9 and 1.0 × 10−9 per base per cell population doubling, respectively, indicating that GS cells have a lower mutation rate compared to mGS cells. GS and mGS cells also showed distinct mutation patterns, with C-to-T transition as the most frequent in GS cells and C-to-A transversion as the most predominant in mGS cells. By karyotype analysis, GS cells showed recurrent trisomy of chromosomes 15 and 16, whereas mGS cells frequently exhibited chromosomes 1, 6, 8, and 11 amplifications, suggesting that distinct chromosomal abnormalities confer a selective growth advantage for each cell type in vitro. These data provide the basis for studying germline mutations and a foundation for the future utilization of GS cells for reproductive technology and clinical applications.

Insulin Receptor Isoforms Differently Regulate Cell Proliferation and Apoptosis in the Ligand-Occupied and Unoccupied State

The insulin receptor (IR) presents two isoforms (IR-A and IR-B) that differ for the α-subunit C-terminal. Both isoforms are expressed in all human cells albeit in different proportions, yet their functional properties-when bound or unbound to insulin-are not well characterized. From a cell model deprived of the Insulin-like Growth Factor 1 Receptor (IGF1-R) we therefore generated cells exhibiting no IR (R-shIR cells), or only human IR-A (R-shIR-A), or exclusively human IR-B (R-shIR-B) and we studied the specific effect of the two isoforms on cell proliferation and cell apoptosis. In the absence of insulin both IR-A and IR-B similarly inhibited proliferation but IR-B was 2–3 fold more effective than IR-A in reducing resistance to etoposide-induced DNA damage. In the presence of insulin, IR-A and IR-B promoted proliferation with the former significantly more effective than the latter at increasing insulin concentrations. Moreover, only insulin-bound IR-A, but not IR-B, protected cells from etoposide-induced cytotoxicity. In conclusion, IR isoforms have different effects on cell proliferation and survival. When unoccupied, IR-A, which is predominantly expressed in undifferentiated and neoplastic cells, is less effective than IR-B in protecting cells from DNA damage. In the presence of insulin, particularly when present at high levels, IR-A provides a selective growth advantage.

Enhanced Vasculogenic Capacity Induced by 5-Fluorouracil Chemoresistance in a Gastric Cancer Cell Line

Chemotherapy is still widely used as a coadjutant in gastric cancer when surgery is not possible or in presence of metastasis. During tumor evolution, gatekeeper mutations provide a selective growth advantage to a subpopulation of cancer cells that become resistant to chemotherapy. When this phenomenon happens, patients experience tumor recurrence and treatment failure. Even if many chemoresistance mechanisms are known, such as expression of ATP-binding cassette (ABC) transporters, aldehyde dehydrogenase (ALDH1) activity and activation of peculiar intracellular signaling pathways, a common and universal marker for chemoresistant cancer cells has not been identified yet. In this study we subjected the gastric cancer cell line AGS to chronic exposure of 5-fluorouracil, cisplatin or paclitaxel, thus selecting cell subpopulations showing resistance to the different drugs. Such cells showed biological changes; among them, we observed that the acquired chemoresistance to 5-fluorouracil induced an endothelial-like phenotype and increased the capacity to form vessel-like structures. We identified the upregulation of thymidine phosphorylase (TYMP), which is one of the most commonly reported mutated genes leading to 5-fluorouracil resistance, as the cause of such enhanced vasculogenic ability.

From the (Epi)Genome to Metabolism and Vice Versa; Examples from Hematologic Malignancy

Hematologic malignancies comprise a heterogeneous group of neoplasms arising from hematopoietic cells or their precursors and most commonly presenting as leukemias, lymphomas, and myelomas. Genetic analyses have uncovered recurrent mutations which initiate or accumulate in the course of malignant transformation, as they provide selective growth advantage to the cell. These include mutations in genes encoding transcription factors and epigenetic regulators of metabolic genes, as well as genes encoding key metabolic enzymes. The resulting alterations contribute to the extensive metabolic reprogramming characterizing the transformed cell, supporting its increased biosynthetic needs and allowing it to withstand the metabolic stress that arises as a consequence of increased metabolic rates and changes in its microenvironment. Interestingly, this cross-talk is bidirectional, as metabolites also signal back to the nucleus and, via their widespread effects on modulating epigenetic modifications, shape the chromatin landscape and the transcriptional programs of the cell. In this article, we provide an overview of the main metabolic changes and relevant genetic alterations that characterize malignant hematopoiesis and discuss how, in turn, metabolites regulate epigenetic events during this process. The aim is to illustrate the intricate interrelationship between the genome (and epigenome) and metabolism and its relevance to hematologic malignancy.

Vaccinia virus gene acquisition through non-homologous recombination

Many of the genes encoded by poxviruses are orthologs of cellular genes. These virus genes serve different purposes, but perhaps of most interest is the way some have been repurposed to inhibit the antiviral pathways that their cellular homologs still regulate. What is unclear is how these virus genes were acquired although it is presumed to have been catalyzed by some form(s) of non-homologous recombination (NHR). We used transfection assays and substrates encoding a fluorescent and drug selectable marker to examine the NHR frequency in vaccinia virus (VAC) infected cells. These studies showed that when cells were transfected with linear duplex DNAs bearing VAC N2L gene homology it yielded a recombinant frequency (RF) of 6.7×10−4. In contrast, DNA lacking any VAC homology reduced the yield of recombinants ∼400-fold (RF = 1.6×10−6). DNA·RNA hybrids were also substrates, although homologous molecules yielded fewer recombinants (RF = 2.1×10−5) and non-homologous substrates yielded only rare recombinants (RF ≤ 3×10−8). NHR was associated with genome rearrangements ranging from simple insertions with flanking sequence duplications to large-scale indels that produced helper-dependent viruses. The insert was often also partially duplicated and would rapidly rearrange through homologous recombination. Most of the virus-insert junctions exhibited little or no pre-exiting microhomology, although a few encoded VAC topoisomerase recognition sites (C/T·CCTT). These studies show that VAC can catalyze NHR through a process that may reflect a form of aberrant replication fork repair. Although it is less efficient than classical homologous recombination, the rates of NHR may still be high enough to drive virus evolution. IMPORTANCE Large DNA viruses sometimes interfere in antiviral defenses using repurposed and mutant forms of the cellular proteins that mediate these same reactions. Such virus orthologs of cellular genes were presumably captured through non-homologous recombination, perhaps in the distant past, but nothing is known about the processes that might promote “gene capture” or even how often these events occur over the course of an infectious cycle. This study shows that non-homologous recombination in vaccinia virus infected cells is frequent enough to seed a small but still significant portion of novel recombinants into large populations of newly replicated virus particles. This offers a route by which a pool of virus might survey the host genome for sequences that offer a selective growth advantage and potentially drive discontinuous virus evolution (saltation) through the acquisition of adventitious traits.

Increased Pathway Complexity Is a Prognostic Biomarker in Metastatic Castration-Resistant Prostate Cancer

Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease, characterized by common and rare driver gene alterations that provide a selective growth advantage for progressing tumour cells. We hypothesized that the number of distinct gene driver alteration-affected pathways or gene classes was associated with poor prognosis in patients initiating androgen receptor signalling inhibitors (ARSi). We performed a post hoc analysis of an amalgamated baseline circulating tumour DNA (ctDNA) mutational landscape dataset of ARSi-treated men with mCRPC (n = 342). We associated the detected hotspot, pathogenic, and/or high impact protein function-affecting perturbations in 39 genes into 13 pathways. Progression-free (PFS) and overall survival (OS) were analysed using Kaplan–Meier curves and multivariate Cox regression models. Driver gene alterations were detected in 192/342 (56.1%) evaluable patients. An increased number of affected pathways, coined pathway complexity index (PCI), resulted in a decremental PFS and OS, and was independently associated with prognosis once ≥3 pathway or gene classes were affected (PFS HR (95%CI): 1.7 (1.02–2.84), p = 0.04, and OS HR (95%CI): 2.5 (1.06–5.71), p = 0.04). Additionally, visceral disease and baseline PSA and plasma ctDNA levels were independently associated with poor prognosis. Elevated PCI is associated with poor ARSi outcome and supports comprehensive genomic profiling to better infer mCRPC prognosis.

Loss-of-function mutations in the histone methyltransferase EZH2 promote chemotherapy resistance in AML

Chemotherapy resistance is the main impediment in the treatment of acute myeloid leukaemia (AML). Despite rapid advances, the various mechanisms inducing resistance development remain to be defined in detail. Here we report that loss-of-function mutations (LOF) in the histone methyltransferase EZH2 have the potential to confer resistance against the chemotherapeutic agent cytarabine. We identify seven distinct EZH2 mutations leading to loss of H3K27 trimethylation via multiple mechanisms. Analysis of matched diagnosis and relapse samples reveal a heterogenous regulation of EZH2 and a loss of EZH2 in 50% of patients. We confirm that loss of EZH2 induces resistance against cytarabine in the cell lines HEK293T and K562 as well as in a patient-derived xenograft model. Proteomics and transcriptomics analysis reveal that resistance is conferred by upregulation of multiple direct and indirect EZH2 target genes that are involved in apoptosis evasion, augmentation of proliferation and alteration of transmembrane transporter function. Our data indicate that loss of EZH2 results in upregulation of its target genes, providing the cell with a selective growth advantage, which mediates chemotherapy resistance.

The NRF2, Thioredoxin, and Glutathione System in Tumorigenesis and Anticancer Therapies

Cancer remains an elusive, highly complex disease and a global burden. Constant change by acquired mutations and metabolic reprogramming contribute to the high inter- and intratumor heterogeneity of malignant cells, their selective growth advantage, and their resistance to anticancer therapies. In the modern era of integrative biomedicine, realizing that a personalized approach could benefit therapy treatments and patients’ prognosis, we should focus on cancer-driving advantageous modifications. Namely, reactive oxygen species (ROS), known to act as regulators of cellular metabolism and growth, exhibit both negative and positive activities, as do antioxidants with potential anticancer effects. Such complexity of oxidative homeostasis is sometimes overseen in the case of studies evaluating the effects of potential anticancer antioxidants. While cancer cells often produce more ROS due to their increased growth-favoring demands, numerous conventional anticancer therapies exploit this feature to ensure selective cancer cell death triggered by excessive ROS levels, also causing serious side effects. The activation of the cellular NRF2 (nuclear factor erythroid 2 like 2) pathway and induction of cytoprotective genes accompanies an increase in ROS levels. A plethora of specific targets, including those involved in thioredoxin (TRX) and glutathione (GSH) systems, are activated by NRF2. In this paper, we briefly review preclinical research findings on the interrelated roles of the NRF2 pathway and TRX and GSH systems, with focus given to clinical findings and their relevance in carcinogenesis and anticancer treatments.

Nfkbiz 3′ UTR Mutations Confer Selective Growth Advantage and Affect Drug Response in Diffuse Large B-Cell Lymphoma

Introduction: The activated B-cell-like (ABC) molecular subgroup of diffuse large B-cell lymphoma (DLBCL) is characterized by activation of NF-κB signaling and increased mortality. Although recurrent mutations affecting genes such as MYD88, CD79A/B and TNFAIP3 contribute to this phenomenon in some cases, there remain tumors with no known genetic basis for NF-κB-activation. Previously, amplification (AMP) of the NFKBIZ locus has been reported in 10% of ABC DLBCLs and it was demonstrated that this contributes to activation of NF-κB signaling. We recently described a novel pattern of non-coding mutations affecting the 3′ untranslated region (UTR) of NFKBIZ resulting in an overall mutation rate of ~30% (UTR or AMP) in ABC DLBCL. NFKBIZ mutations are mutually exclusive with MYD88 mutations, suggesting they may also act as driver mutations. The NFKBIZ protein interacts with NF-κB transcription factors and is thought to regulate canonical NF-κB signaling. We hypothesized that NFKBIZ 3′ UTR mutations affect post-transcriptional regulation of the mRNA by disrupting the conserved secondary structure of the UTR. The binding of regulatory proteins is abrogated by the structural changes induced by mutations, which consequently lead to stabilization of the mRNA. This causes an accumulation of protein and may be a novel mechanism to promote cell growth and survival in ABC DLBCL. Methods: NFKBIZ 3′ UTR mutations were introduced into the WSU-DLCL2 DLBCL cell line using the CRISPR-Cas9 system, producing eight different CRISPR-mutant lines. Custom droplet digital PCR assays and western blotting were used to asses mRNA and protein levels, respectively. Competitive growth assays with wild-type (WT) and CRISPR-mutant lines were performed to assess whether UTR mutations provide a growth advantage in vitro. A similar study was performed in vivo by engrafting a mix of WT and mutant cells into NSG mice. We separately compared gene expression profiles (generated by RNA-Seq) of the parental cell line and a subset of CRISPR-mutant lines. Genes up- and down-regulated by NFKBIZ 3' UTR mutations were identified and analyzed for pathway enrichment. Finally, the IC50 of drugs relevant to DLBCL was determined by WST-1 assays after drug treatment on WT and mutant lines. Results: Introduction of NFKBIZ mutations into DLBCL cells confirmed that UTR mutations lead to varying degrees of increased NFKBIZ mRNA and protein levels. NFKBIZ UTR deletions afforded DLBCL cells a selective growth advantage over WT both in vitro and in vivo. In an assay containing all mutants and WT, mutants with the highest NFKBIZ expression had the largest advantage, suggesting NFKBIZ expression drives this growth advantage. In assays comparing individual mutants to WT, each mutant out-competed WT over time despite varying degrees of NFKBIZ expression, suggesting that all of these mutations act as drivers. Analysis of differentially expressed genes revealed some known NF-κB targets as well as overlap with multiple targets of MYD88, which supports our hypothesis that NFKBIZ and MYD88 regulate a common set of genes. We also discovered potential novel targets of NFKBIZ, including CD274 (PD-L1) and the src kinase HCK. Western blot confirmed that HCK protein is highly expressed in NFKBIZ CRISPR-mutant lines. HCK is a potentially relevant therapeutic target as it has been shown to be overexpressed in multiple cancer types and has been associated with poor overall survival. Expression of PD-L1 in NFKBIZ mutant cases could suggest that immunotherapies may be useful in patients with these mutations, as immunotherapies have had limited success in DLBCL, this may be a way to select patients likely to respond. Mutant cell lines had significantly higher IC50 compared to WT for the drugs Ibrutinib, Idelalisib and Masitinib, but not Bortezomib, suggesting that NKFBIZ UTR mutations confer resistance to drugs specifically targeting the NF-κB pathway. Conclusions: This work directly establishes a role for NFKBIZ amplifications and 3′ UTR mutations in driving ABC DLBCL through NF-κB signaling. We demonstrate that these mutations can cause over-expression of NFKBIZ and provide a selective growth advantage to tumor cells. We also identified novel targets of NFKBIZ including HCK and PD-L1, both of which have implications as therapeutic targets in this subset of DLBCLs. In addition, we found that these mutant lines were more resistant to some targeted lymphoma drugs. Disclosures Scott: NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding. Steidl:Seattle Genetics: Consultancy; AbbVie: Consultancy; Bayer: Consultancy; Curis Inc: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy. Morin:Celgene: Consultancy.