Search

Therapeutic targeting of dysregulated transcription has emerged as a promising strategy for the treatment of cancers, such as leukaemias. The therapeutic response to small molecule inhibitors of Bromodomain-Containing Proteins (BRD), such as BRD2 and BRD4, P300/cAMP-response element binding protein (CBP) and Cyclin Dependent Kinases (CDKs), is generally attributed to the selective disruption of oncogenic gene expression driven by enhancers, super-enhancers (SEs) and lineage-specific transcription factors (TFs), including the c-MYC oncogene. The selectivity of compounds targeting the transcriptional machinery may be further shaped by post-transcriptional processes. To quantitatively assess the contribution of post-transcriptional regulation in responses to transcription inhibition, we performed multi-omics analyses to accurately measure mRNA production and decay kinetics. We demonstrate that it is not only the selective disruption of mRNA production, but rather mRNA decay rates that largely influence the selectivity associated with transcriptional inhibition. Accordingly, genes down-regulated with transcriptional inhibitors are largely characterized by extremely rapid mRNA production and turnover. In line with this notion, stabilization of the c-MYC transcript through swapping of its 3' untranslated region (UTR) rendered c-MYC insensitive to transcriptional targeting. This failed to negate the impact on c-MYC downstream targets and did not abrogate therapeutic responses. Finally, we provide evidence that modulating post-transcriptional pathways, such as through ELAVL1 targeting, can sensitize long-lived mRNAs to transcriptional inhibition and be considered as a combination therapy approach in leukaemia. Taken together, these data demonstrate that mRNA kinetics influence the therapeutic response to transcriptional perturbation and can be modulated for novel therapeutic outcomes using transcriptional agents in leukaemia., (© The Author(s) 2024. Published by Oxford University Press on behalf of NAR Cancer.)

Hereditary SDHB -mutant pheochromocytomas (PC) and paragangliomas (PG) are rare tumours with a high propensity to metastasize although their clinical behaviour is unpredictable. To characterize the genomic landscape of these tumours and identify metastasis biomarkers, we performed multi-omic analysis on 94 tumours from 79 patients using seven molecular methods. Sympathetic (chromaffin cell) and parasympathetic (non-chromaffin cell) PCPG had distinct molecular profiles reflecting their cell-of-origin and biochemical profile. TERT and ATRX -alterations were associated with metastatic PCPG and these tumours had an increased mutation load, and distinct transcriptional and telomeric features. Most PCPG had quiet genomes with some rare co-operative driver events observed, including EPAS1 /HIF-2α mutations. Two mechanisms of acquired resistance to DNA alkylating chemotherapies were also detected - MGMT overexpression and mismatch repair-deficiency causing hypermutation. Our comprehensive multi-omic analysis of SDHB -mutant PCPG therefore identified features of metastatic disease and treatment response, expanding our understanding of these rare neuroendocrine tumours., Competing Interests: Competing interests The authors declare no competing interests.

Background: Metazoans inherited genes from unicellular ancestors that perform essential biological processes such as cell division, metabolism, and protein translation. Multicellularity requires careful control and coordination of these unicellular genes to maintain tissue integrity and homeostasis. Gene regulatory networks (GRNs) that arose during metazoan evolution are frequently altered in cancer, resulting in over-expression of unicellular genes. We propose that an imbalance in co-expression of unicellular (UC) and multicellular (MC) genes is a driving force in cancer., Results: We combine gene co-expression analysis to infer changes to GRNs in cancer with protein sequence conservation data to distinguish genes with UC and MC origins. Co-expression networks created using RNA sequencing data from 31 tumor types and normal tissue samples are divided into modules enriched for UC genes, MC genes, or mixed UC-MC modules. The greatest differences between tumor and normal tissue co-expression networks occur within mixed UC-MC modules. MC and UC genes not commonly co-expressed in normal tissues form distinct co-expression modules seen only in tumors. The degree of rewiring of genes within mixed UC-MC modules increases with tumor grade and stage. Mixed UC-MC modules are enriched for somatic mutations in cancer genes, particularly amplifications, suggesting an important driver of the rewiring observed in tumors is copy number changes., Conclusions: Our study shows the greatest changes to gene co-expression patterns during tumor progression occur between genes of MC and UC origins, implicating the breakdown and rewiring of metazoan gene regulatory networks in cancer development and progression., (© 2024. The Author(s).)

Purpose: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT)., Experimental Design: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1-CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case-control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls., Results: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)-like (N = 226). We then examined their association with breast cancer risk in the case-control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35-3.41), 1.57 (95% CI, 1.41-1.75), and 1.19 (95% CI, 1.08-1.31), respectively. The meta-analysis of population-specific datasets showed similar results., Conclusions: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers., (©2023 The Authors; Published by the American Association for Cancer Research.)

Background: Triple negative BCa (TNBC) is defined by a lack of expression of estrogen (ERα), progesterone (PgR) receptors and human epidermal growth factor receptor 2 (HER2) as assessed by protein expression and/or gene amplification. It makes up ~ 15% of all BCa and often has a poor prognosis. TNBC is not treated with endocrine therapies as ERα and PR negative tumors in general do not show benefit. However, a small fraction of the true TNBC tumors do show tamoxifen sensitivity, with those expressing the most common isoform of ERβ1 having the most benefit. Recently, the antibodies commonly used to assess ERβ1 in TNBC have been found to lack specificity, which calls into question available data regarding the proportion of TNBC that express ERβ1 and any relationship to clinical outcome., Methods: To confirm the true frequency of ERβ1 in TNBC we performed robust ERβ1 immunohistochemistry using the specific antibody CWK-F12 ERβ1 on 156 primary TNBC cancers from patients with a median of 78 months (range 0.2-155 months) follow up., Results: We found that high expression of ERβ1 was not associated with increased recurrence or survival when assessed as percentage of ERβ1 positive tumor cells or as Allred > 5. In contrast, the non-specific PPG5-10 antibody did show an association with recurrence and survival., Conclusions: Our data indicate that ERβ1 expression in TNBC tumours does not associate with prognosis., (© 2023. The Author(s).)

Objective: To determine the feasibility of universal genetic testing of women with newly diagnosed breast cancer, to estimate the incidence of pathogenic gene variants and their impact on patient management, and to evaluate patient and clinician acceptance of universal testing., Design, Setting, Participants: Prospective study of women with invasive or high grade in situ breast cancer and unknown germline status discussed at the Parkville Breast Service (Melbourne) multidisciplinary team meeting. Women were recruited to the pilot (12 June 2020 - 22 March 2021) and expansion phases (17 October 2021 - 8 November 2022) of the Mutational Assessment of newly diagnosed breast cancer using Germline and tumour genomICs (MAGIC) study., Main Outcome Measures: Germline testing by DNA sequencing, filtered for nineteen hereditary breast and ovarian cancer genes that could be classified as actionable; only pathogenic variants were reported. Surveys before and after genetic testing assessed pilot phase participants' perceptions of genetic testing, and psychological distress and cancer-specific worry. A separate survey assessed clinicians' views on universal testing., Results: Pathogenic germline variants were identified in 31 of 474 expanded study phase participants (6.5%), including 28 of 429 women with invasive breast cancer (6.5%). Eighteen of the 31 did not meet current genetic testing eligibility guidelines (probability of a germline pathogenic variant ≥ 10%, based on CanRisk, or Manchester score ≥ 15). Clinical management was changed for 24 of 31 women after identification of a pathogenic variant. Including 68 further women who underwent genetic testing outside the study, 44 of 542 women carried pathogenic variants (8.1%). Acceptance of universal testing was high among both patients (90 of 103, 87%) and clinicians; no decision regret or adverse impact on psychological distress or cancer-specific worry were reported., Conclusion: Universal genetic testing following the diagnosis of breast cancer detects clinically significant germline pathogenic variants that might otherwise be missed because of testing guidelines. Routine testing and reporting of pathogenic variants is feasible and acceptable for both patients and clinicians., (© 2023 The Authors. Medical Journal of Australia published by John Wiley & Sons Australia, Ltd on behalf of AMPCo Pty Ltd.)

Background: Breast cancers (BCs) that arise in individuals heterozygous for a germline pathogenic variant in a susceptibility gene, such as BRCA1 and BRCA2, PALB2, and RAD51C, have been shown to exhibit biallelic loss in the respective genes and be associated with triple-negative breast cancer (TNBC) and distinctive somatic mutational signatures. Tumor sequencing thus presents an orthogonal approach to assess the role of candidate genes in BC development., Methods: Exome sequencing was performed on paired normal-breast tumor DNA from 124 carriers of germline loss-of-function (LoF) or missense variant carriers in 15 known and candidate BC predisposition genes identified in the BEACCON case-control study. Biallelic inactivation and association with tumor genome features including mutational signatures and homologous recombination deficiency (HRD) score were investigated., Results: BARD1-carrying TNBC (4 of 5) displayed biallelic loss and associated high HRD scores and mutational signature 3, as did a RAD51D-carrying TNBC and ovarian cancer. Biallelic loss was less frequent in BRIP1 BCs (4 of 13) and had low HRD scores. In contrast to other established BC genes, BCs from carriers of CHEK2 LoF (6 of 17) or missense (2 of 20) variant had low rates of biallelic loss. Exploratory analysis of BC from carriers of LoF variants in candidate genes such as BLM, FANCM, PARP2, and RAD50 found little evidence of biallelic inactivation., Conclusions: BARD1 and RAD51D behave as classic BRCA-like predisposition genes with biallelic inactivation, but this was not observed for any of the candidate genes. However, as demonstrated for CHEK2, the absence of biallelic inactivation does not provide definitive evidence against the gene's involvement in BC predisposition., (© The Author(s) 2022. Published by Oxford University Press.)

Background: PALB2 is the most important contributor to familial breast cancer after BRCA1 and BRCA2 . Large genomic rearrangements (LGRs) in BRCA1 and BRCA2 are routinely assessed in clinical testing and are a significant contributor to the yield of actionable findings. In contrast, the contribution of LGRs in PALB2 has not been systematically studied., Methods: We performed targeted sequencing and real-time qPCR validation to identify LGRs in PALB2 in 5770 unrelated patients with familial breast cancer and 5741 cancer-free control women from the same Australian population., Results: Seven large deletions ranging in size from 0.96 kbp to 18.07 kbp involving PALB2 were identified in seven cases, while no LGRs were identified in any of the controls. Six LGRs were considered pathogenic as they included one or more exons of PALB2 and disrupted the WD40 domain at the C terminal end of the PALB2 protein while one LGR only involved a partial region of intron 10 and was considered a variant of unknown significance. Altogether, pathogenic LGRs identified in this study accounted for 10.3% (6 of 58) of the pathogenic PALB2 variants detected among the 5770 families with familial breast cancer., Conclusions: Our data show that a clinically important proportion of PALB2 pathogenic mutations in Australian patients with familial breast cancer are LGRs. Such observations have provided strong support for inclusion of PALB2 LGRs in routine clinical genetic testing., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Pheochromocytomas (PC) and paragangliomas (PG) are rare neuroendocrine tumors associated with autonomic nerves. Here we use single-nuclei RNA-seq and bulk-tissue gene-expression data to characterize the cellular composition of PCPG and normal adrenal tissues, refine tumor gene-expression subtypes and make clinical and genotypic associations. We confirm seven PCPG gene-expression subtypes with significant genotype and clinical associations. Tumors with mutations in VHL, SDH-encoding genes (SDHx) or MAML3-fusions are characterized by hypoxia-inducible factor signaling and neoangiogenesis. PCPG have few infiltrating lymphocytes but abundant macrophages. While neoplastic cells transcriptionally resemble mature chromaffin cells, early chromaffin and neuroblast markers are also features of some PCPG subtypes. The gene-expression profile of metastatic SDHx-related PCPG indicates these tumors have elevated cellular proliferation and a lower number of non-neoplastic Schwann-cell-like cells, while GPR139 is a potential theranostic target. Our findings therefore clarify the diverse transcriptional programs and cellular composition of PCPG and identify biomarkers of potential clinical significance., (© 2022. The Author(s).)

Background: Interferon gamma (IFNγ) is a pro-inflammatory cytokine that directly activates the JAK/STAT pathway. However, the temporal dynamics of chromatin remodeling and transcriptional activation initiated by IFNγ have not been systematically profiled in an unbiased manner. Herein, we integrated transcriptomic and epigenomic profiling to characterize the acute epigenetic changes induced by IFNγ stimulation in a murine breast cancer model., Results: We identified de novo activation of cis-regulatory elements bound by Irf1 that were characterized by increased chromatin accessibility, differential usage of pro-inflammatory enhancers, and downstream recruitment of BET proteins and RNA polymerase II. To functionally validate this hierarchical model of IFNγ-driven transcription, we applied selective antagonists of histone acetyltransferases P300/CBP or acetyl-lysine readers of the BET family. This highlighted that histone acetylation is an antecedent event in IFNγ-driven transcription, whereby targeting of P300/CBP acetyltransferase activity but not BET inhibition could curtail the epigenetic remodeling induced by IFNγ through suppression of Irf1 transactivation., Conclusions: These data highlight the ability for epigenetic therapies to reprogram pro-inflammatory gene expression, which may have therapeutic implications for anti-tumor immunity and inflammatory diseases., (© 2022. The Author(s).)

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies., Significance: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397., (©2022 The Authors; Published by the American Association for Cancer Research.)

Clonal analysis of tumour sequencing data enables the evaluation of the relationship of histologically distinct synchronous lesions, such as co-existing benign areas, and temporally distinct tumours, such as primary-recurrence comparisons. In this review, we summarise statistical approaches that are commonly employed to define tumour clonal relatedness using data from bulk DNA technologies. We discuss approaches using total copy number, allele-specific copy number and mutation data, and the relative genomic resolution required for analysis and summarise some of the current tools for inferring clonal relationships. We argue that the impact of the biological context is critical in selecting any particular approach, such as the relative genomic complexity of the lesions being compared, and we recommend considering this context before employing any method to a new dataset., (© 2022. The Author(s).)

While protein-truncating variants in RAD51C have been shown to predispose to triple-negative (TN) breast cancer (BC) and ovarian cancer, little is known about the pathogenicity of missense (MS) variants. The frequency of rare RAD51C MS variants was assessed in the BEACCON study of 5734 familial BC cases and 14,382 population controls, and findings were integrated with tumour sequencing data from 21 cases carrying a candidate variant. Collectively, a significant enrichment of rare MS variants was detected in cases (MAF < 0.001, OR 1.57, 95% CI 1.00-2.44, p = 0.05), particularly for variants with a REVEL score >0.5 (OR 3.95, 95% CI 1.40-12.01, p = 0.006). Sequencing of 21 tumours from 20 heterozygous and 1 homozygous carriers of nine candidate MS variants identified four cases with biallelic inactivation through loss of the wild-type allele, while six lost the variant allele and ten that remained heterozygous. Biallelic loss of the wild-type alleles corresponded strongly with ER- and TN breast tumours, high homologous recombination deficiency scores and mutational signature 3. Using this approach, the p.Gly264Ser variant, which was previously suspected to be pathogenic based on small case-control analyses and loss of activity in in vitro functional assays, was shown to be benign with similar prevalence in cases and controls and seven out of eight tumours showing no biallelic inactivation or characteristic mutational signature. Conversely, evaluation of case-control findings and tumour sequencing data identified p.Ile144Thr, p.Arg212His, p.Gln143Arg and p.Gly114Arg as variants warranting further investigation., (© 2022. The Author(s).)

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) are an approved treatment for hormone receptor-positive breast cancer and are currently under evaluation across hundreds of clinical trials for other cancer types. The clinical success of these inhibitors is largely attributed to well-defined tumor-intrinsic cytostatic mechanisms, whereas their emerging role as immunomodulatory agents is less understood. Using integrated epigenomic, transcriptomic, and proteomic analyses, we demonstrated a novel action of CDK4/6 inhibitors in promoting the phenotypic and functional acquisition of immunologic T-cell memory. Short-term priming with a CDK4/6 inhibitor promoted long-term endogenous antitumor T-cell immunity in mice, enhanced the persistence and therapeutic efficacy of chimeric antigen receptor T cells, and induced a retinoblastoma-dependent T-cell phenotype supportive of favorable responses to immune checkpoint blockade in patients with melanoma. Together, these mechanistic insights significantly broaden the prospective utility of CDK4/6 inhibitors as clinical tools to boost antitumor T-cell immunity. SIGNIFICANCE: Immunologic memory is critical for sustained antitumor immunity. Our discovery that CDK4/6 inhibitors drive T-cell memory fate commitment sheds new light on their clinical activity, which is essential for the design of clinical trial protocols incorporating these agents, particularly in combination with immunotherapy, for the treatment of cancer. This article is highlighted in the In This Issue feature, p. 2355 ., (©2021 American Association for Cancer Research.)

Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis and limited treatment strategies. Determining the role of cell-extrinsic regulators of leukemic cells is vital to gain clinical insights into the biology of AML. Iron is a key extrinsic regulator of cancer, but its systemic regulation remains poorly explored in AML. To address this question, we studied iron metabolism in patients with AML at diagnosis and explored the mechanisms involved using the syngeneic MLL-AF9-induced AML mouse model. We found that AML is a disorder with a unique iron profile, not associated with inflammation or transfusion, characterized by high ferritin, low transferrin, high transferrin saturation (TSAT), and high hepcidin. The increased TSAT in particular, contrasts with observations in other cancer types and in anemia of inflammation. Using the MLL-AF9 mouse model of AML, we demonstrated that the AML-induced loss of erythroblasts is responsible for iron redistribution and increased TSAT. We also show that AML progression is delayed in mouse models of systemic iron overload and that elevated TSAT at diagnosis is independently associated with increased overall survival in AML. We suggest that TSAT may be a relevant prognostic marker in AML., (© 2021 by The American Society of Hematology.)

The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment., (© 2021. Crown.)

Breast cancer (BC) has a significant heritable component but the genetic contribution remains unresolved in the majority of high-risk BC families. This study aims to investigate the monogenic causes underlying the familial aggregation of BC beyond BRCA1 and BRCA2, including the identification of new predisposing genes. A total of 11,511 non-BRCA familial BC cases and population-matched cancer-free female controls in the BEACCON study were investigated in two sequencing phases: 1303 candidate genes in up to 3892 cases and controls, followed by validation of 145 shortlisted genes in an additional 7619 subjects. The coding regions and exon-intron boundaries of all candidate genes and 14 previously proposed BC genes were sequenced using custom designed sequencing panels. Pedigree and pathology data were analysed to identify genotype-specific associations. The contribution of ATM, PALB2 and CHEK2 to BC predisposition was confirmed, but not RAD50 and NBN. An overall excess of loss-of-function (LoF) (OR 1.27, p = 9.05 × 10 -9 ) and missense (OR 1.27, p = 3.96 × 10 -73 ) variants was observed in the cases for the 145 candidate genes. Leading candidates harbored LoF variants with observed ORs of 2-4 and individually accounted for no more than 0.79% of the cases. New genes proposed by this study include NTHL1, WRN, PARP2, CTH and CDK9. The new candidate BC predisposition genes identified in BEACCON indicate that much of the remaining genetic causes of high-risk BC families are due to genes in which pathogenic variants are both very rare and convey only low to moderate risk.

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically., Competing Interests: Declaration of interests The Johnstone laboratory receives funding support from Roche, BMS, Astra Zeneca, and MecRx. R.W.J. is a shareholder in MecRx and receives consultancy payments., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-IHC/immunofluorescence (m-IHC/IF). CD4 + or CD8 + T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2 - γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T-cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions. See related Spotlight on p. 600 ., (©2021 American Association for Cancer Research.)

Bi-allelic loss-of-function (LoF) variants in the base excision repair (BER) gene NTHL1 cause a high-risk hereditary multi-tumor syndrome that includes breast cancer, but the contribution of heterozygous variants to hereditary breast cancer is unknown. An analysis of 4985 women with breast cancer, enriched for familial features, and 4786 cancer-free women revealed significant enrichment for NTHL1 LoF variants. Immunohistochemistry confirmed reduced NTHL1 expression in tumors from heterozygous carriers but the NTHL1 bi-allelic loss characteristic mutational signature (SBS 30) was not present. The analysis was extended to 27,421 breast cancer cases and 19,759 controls from 10 international studies revealing 138 cases and 93 controls with a heterozygous LoF variant (OR 1.06, 95% CI: 0.82-1.39) and 316 cases and 179 controls with a missense variant (OR 1.31, 95% CI: 1.09-1.57). Missense variants selected for deleterious features by a number of in silico bioinformatic prediction tools or located within the endonuclease III functional domain showed a stronger association with breast cancer. Somatic sequencing of breast cancers from carriers indicated that the risk associated with NTHL1 appears to operate through haploinsufficiency, consistent with other described low-penetrance breast cancer genes. Data from this very large international multicenter study suggests that heterozygous pathogenic germline coding variants in NTHL1 may be associated with low- to moderate- increased risk of breast cancer.

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAF V600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic Braf V600E Cdkn2a -/- Pten -/- melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103 + dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma., (©2020 American Association for Cancer Research.)

Low-grade serous ovarian carcinoma (LGSOC) is associated with a poor response to existing chemotherapy, highlighting the need to perform comprehensive genomic analysis and identify new therapeutic vulnerabilities. The data presented here represent the largest genetic study of LGSOCs to date (n = 71), analysing 127 candidate genes derived from whole exome sequencing cohorts to generate mutation and copy-number variation data. Additionally, immunohistochemistry was performed on our LGSOC cohort assessing oestrogen receptor, progesterone receptor, TP53, and CDKN2A status. Targeted sequencing identified 47% of cases with mutations in key RAS/RAF pathway genes (KRAS, BRAF, and NRAS), as well as mutations in putative novel driver genes including USP9X (27%), MACF1 (11%), ARID1A (9%), NF2 (4%), DOT1L (6%), and ASH1L (4%). Immunohistochemistry evaluation revealed frequent oestrogen/progesterone receptor positivity (85%), along with CDKN2A protein loss (10%) and CDKN2A protein overexpression (6%), which were linked to shorter disease outcomes. Indeed, 90% of LGSOC samples harboured at least one potentially actionable alteration, which in 19/71 (27%) cases were predictive of clinical benefit from a standard treatment, either in another cancer's indication or in LGSOC specifically. In addition, we validated ubiquitin-specific protease 9X (USP9X), which is a chromosome X-linked substrate-specific deubiquitinase and tumour suppressor, as a relevant therapeutic target for LGSOC. Our comprehensive genomic study highlighted that there is an addiction to a limited number of unique 'driver' aberrations that could be translated into improved therapeutic paths. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

High-grade serous ovarian carcinoma (HGSOC) has a significant hereditary component, approximately half of which cannot be explained by known genes. To discover genes, we analyse germline exome sequencing data from 516 BRCA1/2-negative women with HGSOC, focusing on genes enriched with rare, protein-coding loss-of-function (LoF) variants. Overall, there is a significant enrichment of rare protein-coding LoF variants in the cases (p < 0.0001, chi-squared test). Only thirty-four (6.6%) have a pathogenic variant in a known or proposed predisposition gene. Few genes have LoF mutations in more than four individuals and the majority are detected in one individual only. Forty-three highly-ranked genes are identified with three or more LoF variants that are enriched by three-fold or more compared to GnomAD. These genes represent diverse functional pathways with relatively few involved in DNA repair, suggesting that much of the remaining heritability is explained by previously under-explored genes and pathways.