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Abstract Background The identification of cancer driver genes from sequencing data has been crucial in deepening our understanding of tumor biology and expanding targeted therapy options. However, apart from the most commonly altered genes, the mechanisms underlying the contribution of other mutations to cancer acquisition remain understudied. Leveraging on our whole-exome sequencing of the largest Asian lung adenocarcinoma (LUAD) cohort (n = 302), we now functionally assess the mechanistic role of a novel driver, PARP4. Methods In vitro and in vivo tumorigenicity assays were used to study the functional effects of PARP4 loss and mutation in multiple lung cancer cell lines. Interactomics analysis by quantitative mass spectrometry was conducted to identify PARP4’s interaction partners. Transcriptomic data from cell lines and patient tumors were used to investigate splicing alterations. Results PARP4 depletion or mutation (I1039T) promotes the tumorigenicity of KRAS- or EGFR-driven lung cancer cells. Disruption of the vault complex, with which PARP4 is commonly associated, did not alter tumorigenicity, indicating that PARP4’s tumor suppressive activity is mediated independently. The splicing regulator hnRNPM is a potentially novel PARP4 interaction partner, the loss of which likewise promotes tumor formation. hnRNPM loss results in splicing perturbations, with a propensity for dysregulated intronic splicing that was similarly observed in PARP4 knockdown cells and in LUAD cohort patients with PARP4 copy number loss. Conclusions PARP4 is a novel modulator of lung adenocarcinoma, where its tumor suppressive activity is mediated not through the vault complex—unlike conventionally thought, but in association with its novel interaction partner hnRNPM, thus suggesting a role for splicing dysregulation in LUAD tumorigenesis.

Molecularly targeted cancer therapy has improved outcomes for patients with cancer with targetable oncoproteins, such as mutant EGFR in lung cancer. Yet, the long-term survival of these patients remains limited, because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations of EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alterations of the mRNA splicing factor RNA-binding motif 10 (RBM10) that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR-mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of (proapoptotic) Bcl-xS to (antiapoptotic) Bcl-xL isoforms of Bcl-x. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Coinhibition of Bcl-xL and mutant EGFR overcame the resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations and on the effect of splicing factor deficiency on the modulation of sensitivity to targeted kinase inhibitor cancer therapy.

Abstract Background Cancer predisposition is most often studied in the context of single cancers. However, inherited cancer predispositions can also give rise to multiple primary cancers. Yet, there is a paucity of studies on genetic predisposition in multiple primary cancers, especially those outside of well-defined cancer predisposition syndromes. This study aimed to identify germline variants associated with dual primary cancers of the breast and lung. Methods Exome sequencing was performed on germline DNA from 55 Singapore patients (52 [95%] never-smokers) with dual primaries in the breast and lung, confirmed by histopathology. Using two large control cohorts: the local SG10K_Health (n = 9770) and gnomAD non-cancer East Asians (n = 9626); and two additional local case cohorts of early-onset or familial breast cancer (n = 290), and lung cancer (n = 209), variants were assessed for pathogenicity in accordance with ACMG/AMP guidelines. In particular, comparisons were made with known pathogenic or likely pathogenic variants in the ClinVar database, pathogenicity predictions were obtained from in silico prediction software, and case–control association analyses were performed. Results Altogether, we identified 19 pathogenic or likely pathogenic variants from 16 genes, detected in 17 of 55 (31%) patients. Six of the 19 variants were identified using ClinVar, while 13 variants were classified pathogenic or likely pathogenic using ACMG/AMP guidelines. The 16 genes include well-known cancer predisposition genes such as BRCA2, TP53, and RAD51D; but also lesser known cancer genes EXT2, WWOX, GATA2, and GPC3. Most of these genes are involved in DNA damage repair, reaffirming the role of impaired DNA repair mechanisms in the development of multiple malignancies. These variants warrant further investigations in additional populations. Conclusions We have identified both known and novel variants significantly enriched in patients with primary breast and lung malignancies, expanding the body of known cancer predisposition variants for both breast and lung cancer. These variants are mostly from genes involved in DNA repair, affirming the role of impaired DNA repair in the predisposition and development of multiple cancers.

Introduction: To compare the performance of droplet digital polymerase chain reaction (ddPCR) and plasma next-generation sequencing (NGS) in detecting clearance of plasma EGFR (pEGFR) mutations. Methods: Patients with treatment-naive advanced EGFR-mutated lung cancer treated with first-line tyrosine kinase inhibitors (TKIs) were included. pEGFR were measured at baseline and first response assessment using ddPCR and NGS. Clearance of pEGFR was defined as undetectable levels after a positive baseline result. Results were correlated with time-to-treatment failure (TTF). In exploratory analysis, corresponding change in carcinoembryonic antigen (CEA) levels was evaluated. Results: Between January 1, 2020, and December 31, 2021, 27 patients were recruited. Ex19del comprised 74% (20 of 27) and L858R 26% (seven of 27). Osimertinib was used in 59% (16 of 27), dacomitinib 4% (one of 27), and gefitinib/erlotinib 37% (10 of 27). Sensitivity of ddPCR and NGS in detecting pEGFR mutation at baseline was 70% (19 of 27) and 78% (21 of 27), respectively (p = 0.16). All patients with detectable pEGFR by ddPCR were detected by NGS.At a median of 8 (range 3–24) weeks post-TKI initiation, clearance of pEGFR was achieved in 68% (13 of 19) and 71% (15 of 21) using ddPCR and NGS, respectively. Concordance between ddPCR and NGS was 79% (kappa = 0.513, p = 0.013). Clearance of pEGFR was associated with longer median TTF (not reached versus 6 months, p = 0.03) and median decrease in CEA levels by 70% from baseline.In another cohort of 124 patients, decrease in CEA levels by greater than 70% within 90 days of TKI initiation was associated with doubling of both TTF and overall survival. Conclusions: Plasma NGS trended toward higher sensitivity than ddPCR in detecting pEGFR, although both had similar concordance in detecting pEGFR clearance. Our results support using NGS at diagnosis and interchangeability of NGS and ddPCR for monitoring, whereas CEA could be explored as a surrogate for pEGFR clearance.

Abstract Tumors are complex masses composed of malignant and non-malignant cells. Variation in tumor purity (proportion of cancer cells in a sample) can both confound integrative analysis and enable studies of tumor heterogeneity. Here we developed PUREE, which uses a weakly supervised learning approach to infer tumor purity from a tumor gene expression profile. PUREE was trained on gene expression data and genomic consensus purity estimates from 7864 solid tumor samples. PUREE predicted purity with high accuracy across distinct solid tumor types and generalized to tumor samples from unseen tumor types and cohorts. Gene features of PUREE were further validated using single-cell RNA-seq data from distinct tumor types. In a comprehensive benchmark, PUREE outperformed existing transcriptome-based purity estimation approaches. Overall, PUREE is a highly accurate and versatile method for estimating tumor purity and interrogating tumor heterogeneity from bulk tumor gene expression data, which can complement genomics-based approaches or be used in settings where genomic data is unavailable.

Deep learning could be applied to the challenge of somatic variant calling in cancer by making use of large-scale genomic data. Here, the authors develop VarNet, a weakly supervised deep learning model for somatic variant calling in cancer with robust performance across multiple cancer genomics datasets.

Introduction: Although immune checkpoint inhibitors (ICIs) have dramatically improved outcomes for nononcogene-addicted NSCLC, monotherapy with programmed cell death protein-1 (PD1) inhibition has been associated with low efficacy in the EGFR-mutant setting. Given the potential for synergism with combination checkpoint blockade, we designed a trial to test the activity of combination nivolumab (N)-ipilimumab (NI) in EGFR-mutant NSCLC. Methods: This is a randomized phase 2 study (NCT03091491) of N versus NI combination in EGFR tyrosine kinase inhibitor (TKI)–resistant NSCLC, with crossover permitted on disease progression. The primary end point was the objective response rate, and the secondary end points included progression-free survival, overall survival, and safety of ICI after EGFR TKI. Results: Recruitment ceased owing to futility after 31 of 184 planned patients were treated. A total of 15 patients received N and 16 received NI combination. There were 16 patients (51.6%) who had programmed death-ligand (PDL1) 1 greater than or equal to 1%, and 15 (45.2%) harbored EGFR T790M. Five patients derived clinical benefits from ICI with one objective response (objective response rate 3.2%), and median progression-free survival was 1.22 months (95% confidence interval: 1.15–1.35) for the overall cohort. None of the four patients who crossed over achieved salvage response by NI. PDL1 and tumor mutational burden (TMB) were not able to predict ICI response. Rates of all grade immune-related adverse events were similar (80% versus 75%), with only two grade 3 events. Conclusions: Immune checkpoint inhibition is ineffective in EGFR TKI–resistant NSCLC. Whereas a small subgroup of EGFR-mutant NSCLC may be immunogenic and responsive to ICI, better biomarkers are needed to select appropriate patients.

Abstract Background Enhancers are distal cis-regulatory elements required for cell-specific gene expression and cell fate determination. In cancer, enhancer variation has been proposed as a major cause of inter-patient heterogeneity—however, most predicted enhancer regions remain to be functionally tested. Methods We analyzed 132 epigenomic histone modification profiles of 18 primary gastric cancer (GC) samples, 18 normal gastric tissues, and 28 GC cell lines using Nano-ChIP-seq technology. We applied Capture-based Self-Transcribing Active Regulatory Region sequencing (CapSTARR-seq) to assess functional enhancer activity. An Activity-by-contact (ABC) model was employed to explore the effects of histone acetylation and CapSTARR-seq levels on enhancer-promoter interactions. Results We report a comprehensive catalog of 75,730 recurrent predicted enhancers, the majority of which are GC-associated in vivo (> 50,000) and associated with lower somatic mutation rates inferred by whole-genome sequencing. Applying CapSTARR-seq to the enhancer catalog, we observed significant correlations between CapSTARR-seq functional activity and H3K27ac/H3K4me1 levels. Super-enhancer regions exhibited increased CapSTARR-seq signals compared to regular enhancers, even when decoupled from native chromatin contexture. We show that combining histone modification and CapSTARR-seq functional enhancer data improves the prediction of enhancer-promoter interactions and pinpointing of germline single nucleotide polymorphisms (SNPs), somatic copy number alterations (SCNAs), and trans-acting TFs involved in GC expression. We identified cancer-relevant genes (ING1, ARL4C) whose expression between patients is influenced by enhancer differences in genomic copy number and germline SNPs, and HNF4α as a master trans-acting factor associated with GC enhancer heterogeneity. Conclusions Our results indicate that combining histone modification and functional assay data may provide a more accurate metric to assess enhancer activity than either platform individually, providing insights into the relative contribution of genetic (cis) and regulatory (trans) mechanisms to GC enhancer functional heterogeneity.

Molecularly targeted cancer therapy has improved outcomes for patients with cancer with targetable oncoproteins, such as mutant EGFR in lung cancer. Yet, the long-term survival of these patients remains limited, because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations of EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alterations of the mRNA splicing factor RNA-binding motif 10 (RBM10) that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR-mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor–mediated apoptosis by decreasing the ratio of (proapoptotic) Bcl-xS to (antiapoptotic) Bcl-xL isoforms of Bcl-x. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Coinhibition of Bcl-xL and mutant EGFR overcame the resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations and on the effect of splicing factor deficiency on the modulation of sensitivity to targeted kinase inhibitor cancer therapy.

Senescence-associated secretory phenotype (SASP) involves secretion of factors such as pro-inflammatory cytokines. Here the authors show that MIR31HG regulates the expression and secretion of a subset of SASP components that induce paracrine invasion, through interaction with YBX1 and induction of IL1A translation.

Circulating tumour DNA (ctDNA) represents a non-invasive option to monitor cancer progression. Here, the authors perform deep sequencing of plasma cell-free DNA, and find that nucleosome-dependent cfDNA degradation at 6 specific regulatory regions is predictive of ctDNA burden.

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF7-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1., Introduction Gastric cancer (GC) is a leading cause of global cancer mortality (1), with a particularly high prevalence in East Asia. With the exception of Japan and South Korea, where [...]

DICER is involved in the processing of miRNAs, where the RNase IIIa and IIIb domains are thought to cut the 3p and 5p hairpin arms, respectively. Here, in endometrial cancer, the authors identify an RNase IIIa mutation, which phenocopies mutations in the RNase IIIb domain.

Background: Overexpression of epidermal growth factor receptor (EGFR), and downstream pathway activation appears to be a common oncogenic driver in the majority of head and neck squamous cell cancers (HNSCCs); yet targeting EGFR for the treatment of HNSCC has met with limited success. Apart from the anti-EGFR antibody cetuximab, no small molecule EGFR/tyrosine kinase inhibitors (TKIs) have progressed to routine clinical use. The aim of this study was to determine factors contributing to the lack of response to TKIs and identify alternative therapeutic vulnerabilities. Methods: Genomic and transcriptomic sequencing, high-throughput compound screens, overexpression and siRNA knockdown, western blot, in vivo xenograft studies. Findings: We derived three pairs of isogenic gefitinib (TKI)-sensitive and resistant patient-derived HNSCC cell lines. Genomic sequencing of gefitinib-resistant cell lines identified a lack of activating and resistance-associated EGFR mutations. Instead, transcriptomic sequencing showed upregulated EMT gene signature in the gefitinib-resistant cells with a corresponding increase in their migratory phenotype. Additionally, the resistant cell displayed reduced growth rate. Surprisingly, while gefitinib-resistant cells were independent of EGFR for survival, they nonetheless displayed activation of downstream ERK and AKT signalling. High-throughput screening (HTS) of druggable, small molecule libraries revealed that the gefitinib-resistant cells were particularly sensitive to inhibitors of genes involved in cell cycle and mitosis, such as Aurora kinase inhibitors (AKIs), cyclin-dependent kinase (CDK) inhibitors, and microtubule inhibitors. Notably our results showed that in the EGFR inhibited state, Aurora kinases are essential for cell survival. Interpretation: Our study demonstrates that in the absence of activating EGFR mutations, HNSCCs may gain resistance to gefitinib through decreased cell proliferation, which makes them exceptionally vulnerable to cell-cycle inhibitors. Funding: Agency for Science, Technology, and Research (A*STAR), National Medical Research Council (NMRC), and the National Institutes of Health (NIH)/National Cancer Institute (NCI).

The impact of non-coding somatic mutations in gastric cancer is unknown. Here, using whole genome sequencing data from 212 gastric tumors, the authors identify recurring mutations at specific CTCF binding sites that are common across gastrointestinal cancers and associated with chromosomal instability.

Summary: The BCL-2 family proteins are central regulators of apoptosis. However, cells deficient for BAX and BAK or overexpressing BCL-2 still succumb to oxidative stress upon DNA damage or matrix detachment. Here, we show that ΔNp63α overexpression protects cells from oxidative stress induced by oxidants, DNA damage, anoikis, or ferroptosis-inducing agents. Conversely, ΔNp63α deficiency increases oxidative stress. Mechanistically, ΔNp63α orchestrates redox homeostasis through transcriptional control of glutathione biogenesis, utilization, and regeneration. Analysis of a lung squamous cell carcinoma dataset from The Cancer Genome Atlas (TCGA) reveals that TP63 amplification/overexpression upregulates the glutathione metabolism pathway in primary human tumors. Strikingly, overexpression of ΔNp63α promotes clonogenic survival of p53−/−Bax−/−Bak−/− cells against DNA damage. Furthermore, co-expression of BCL-2 and ΔNp63α confers clonogenic survival against matrix detachment, disrupts the luminal clearance of mammary acini, and promotes cancer metastasis. Our findings highlight the need for a simultaneous blockade of apoptosis and oxidative stress to promote long-term cellular well-being. : Apoptosis-defective cells remain vulnerable to oxidative stress, which limits long-term survival. Wang et al. identify ΔNp63α as a central regulator of redox homeostasis through transcriptional control of a tightly coupled glutathione metabolic circuit. ΔNp63α alleviates oxidative stress and cooperates with the BCL-2 family to promote both long-term cellular well-being and cancer metastasis. Keywords: oxidative stress, ROS, apoptosis, necrosis, programmed necrotic death, TP63, BCL-2, ferroptosis, redox, glutathione metabolism