Search

High-grade serous ovarian carcinoma (HGSOC) has a significant hereditary component, only half of which is explained. Previously, we performed germline exome sequencing on BRCA1 and BRCA2-negative HGSOC patients, revealing three proposed and 43 novel candidate genes enriched with rare loss-of-function variants. For validation, we undertook case-control analyses using genomic data from disease-free controls. This confirms enrichment for nearly all previously identified genes. Additionally, one-hundred-and-eleven HGSOC tumours from variant carriers were sequenced alongside other complementary studies, seeking evidence of biallelic inactivation as supportive evidence. PALB2 and ATM validate as HGSOC predisposition genes, with 6/8 germline carrier tumours exhibiting biallelic inactivation accompanied by characteristic mutational signatures. Among candidate genes, only LLGL2 consistently shows biallelic inactivation and protein expression loss, supporting it as a novel HGSOC susceptibility gene. The remaining candidate genes fail to validate. Integrating case-control analyses with tumour sequencing is thus crucial for accurate gene discovery in familial cancer studies., Competing Interests: Competing interests: The authors declare no competing interests. Ethical approval: This study protocol was approved by the Human Research Ethics Committees at each participating ViP study recruitment centre and the Peter MacCallum Cancer Centre (approval nos. 11/50 and 09/29). All participants provided informed consent for genetic analysis of their germline and tumour DNA., (© 2025. The Author(s).)

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.

Intraductal papillomas (IDP) are challenging breast findings because of their variable risk of progression to malignancy. The molecular events driving IDP development and genomic features of malignant progression are poorly understood. In this study, genome-wide CNA and/or targeted mutation analysis was performed on 44 cases of IDP, of which 20 cases had coexisting ductal carcinoma in situ (DCIS), papillary DCIS or invasive ductal carcinoma (IDC). CNA were rare in pure IDP, but 69% carried an activating PIK3CA mutation. Among the synchronous IDP cases, 55% (11/20) were clonally related to the synchronous DCIS and/or IDC, only one of which had papillary histology. In contrast to pure IDP, PIK3CA mutations were absent from clonal cases. CNAs in any of chromosomes 1, 16 or 11 were significantly enriched in clonal IDP lesions compared to pure and non-clonal IDP. The observation that 55% of IDP are clonal to DCIS/IDC indicates that IDP can be a direct precursor for breast carcinoma, not limited to the papillary type. The absence of PIK3CA mutations and presence of CNAs in IDP could be used clinically to identify patients at high risk of progression to carcinoma., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2020.)

Objective: Mucinous ovarian carcinoma (MOC) is an uncommon ovarian cancer histotype that responds poorly to conventional chemotherapy regimens. Although long overall survival outcomes can occur with early detection and optimal surgical resection, recurrent and advanced disease are associated with extremely poor survival. There are no current guidelines specifically for the systemic management of recurrent MOC. We analyzed data from a large cohort of women with MOC to evaluate the potential for clinical utility from a range of systemic agents., Methods: We analyzed gene copy number (n = 191) and DNA sequencing data (n = 184) from primary MOC to evaluate signatures of mismatch repair deficiency and homologous recombination deficiency, and other genetic events. Immunohistochemistry data were collated for ER, CK7, CK20, CDX2, HER2, PAX8 and p16 (n = 117-166)., Results: Molecular aberrations noted in MOC that suggest a match with current targeted therapies include amplification of ERBB2 (26.7%) and BRAF mutation (9%). Observed genetic events that suggest potential efficacy for agents currently in clinical trials include: KRAS/NRAS mutations (66%), TP53 missense mutation (49%), RNF43 mutation (11%), ARID1A mutation (10%), and PIK3CA/PTEN mutation (9%). Therapies exploiting homologous recombination deficiency (HRD) may not be effective in MOC, as only 1/191 had a high HRD score. Mismatch repair deficiency was similarly rare (1/184)., Conclusions: Although genetically diverse, MOC has several potential therapeutic targets. Importantly, the lack of response to platinum-based therapy observed clinically corresponds to the lack of a genomic signature associated with HRD, and MOC are thus also unlikely to respond to PARP inhibition., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest. ADF, NT and DDLB have received research grant funding from AstraZeneca, unrelated to the contents on this manuscript. DDLB also reports funding from Roche-Genentech and BeiGene, also unrelated. CG reports funding from AstraZeneca, Roche, Clovis, Tesaro, Foundation One, Nucana, Aprea, Novartis, Chugai, and MSD, all outside the submitted work. CLS reports non-financial support and/or other support from Clovis Oncology, Roche, Eisai Australia, Beigene, Sierra Oncology, and AstraZeneca, all outside the submitted work., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Loss-of-function variants in RAD51C are associated with familial ovarian cancer, but its role in hereditary breast cancer remains unclear. The aim of this study was to couple breast tumor sequencing with case-control data to clarify the contribution of RAD51C to hereditary breast cancer., Methods: RAD51C was sequenced in 3080 breast cancer index cases that were negative in BRCA1/2 clinical tests and 4840 population-matched cancer-free controls. Pedigree and pathology data were analyzed. Nine breast cancers and one ovarian cancer from RAD51C variant carriers were sequenced to identify biallelic inactivation of RAD51C, copy number variation, mutational signatures, and the spectrum of somatic mutations in breast cancer driver genes. The promoter of RAD51C was analyzed for DNA methylation., Results: A statistically significant excess of loss-of-function variants was identified in 3080 cases (0.4%) compared with 2 among 4840 controls (0.04%; odds ratio = 8.67, 95% confidence interval = 1.89 to 80.52, P< .001), with more than half of the carriers having no personal or family history of ovarian cancer. In addition, the association was highly statistically significant among cases with estrogen-negative (P <. 001) or triple-negative cancer (P < .001), but not in estrogen-positive cases. Tumor sequencing from carriers confirmed bi-allelic inactivation in all the triple-negative cases and was associated with high homologous recombination deficiency scores and mutational signature 3 indicating homologous recombination repair deficiency., Conclusions: This study provides evidence that germline loss-of-function variants of RAD51C are associated with hereditary breast cancer, particularly triple-negative type. RAD51C-null breast cancers possess similar genomic and clinical features to BRCA1-null cancers and may also be vulnerable to DNA double-strand break inducing chemotherapies and poly ADP-ribose polymerase inhibitors., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mucinous ovarian carcinoma (MOC) is a unique subtype of ovarian cancer with an uncertain etiology, including whether it genuinely arises at the ovary or is metastatic disease from other organs. In addition, the molecular drivers of invasive progression, high-grade and metastatic disease are poorly defined. We perform genetic analysis of MOC across all histological grades, including benign and borderline mucinous ovarian tumors, and compare these to tumors from other potential extra-ovarian sites of origin. Here we show that MOC is distinct from tumors from other sites and supports a progressive model of evolution from borderline precursors to high-grade invasive MOC. Key drivers of progression identified are TP53 mutation and copy number aberrations, including a notable amplicon on 9p13. High copy number aberration burden is associated with worse prognosis in MOC. Our data conclusively demonstrate that MOC arise from benign and borderline precursors at the ovary and are not extra-ovarian metastases.

Context: von Hippel-Lindau (VHL) disease, comprising renal cancer, hemangioblastoma, and/or pheochromocytoma (PHEO), is caused by missense or truncating variants of the VHL tumor-suppressor gene, which is involved in degradation of hypoxia-inducible factors (HIFs). However, the role of synonymous VHL variants in the disease is unclear., Objective: We evaluated a synonymous VHL variant in patients with familial PHEO or VHL disease without a detectable pathogenic VHL mutation., Design: We performed genetic and transcriptional analyses of leukocytes and/or tumors from affected and unaffected individuals and evaluated VHL splicing in existing cancer databases., Results: We identified a synonymous VHL variant (c.414A>G, p.Pro138Pro) as the driver event in five independent individuals/families with PHEOs or VHL syndrome. This variant promotes exon 2 skipping and hence, abolishes expression of the full-length VHL transcript. Exon 2 spans the HIF-binding domain required for HIF degradation by VHL. Accordingly, PHEOs carrying this variant display HIF hyperactivation typical of VHL loss. Moreover, other exon 2 VHL variants from the The Cancer Genome Atlas pan-cancer datasets are biased toward expression of a VHL transcript that excludes this exon, supporting a broader impact of this spliced variant., Conclusion: A recurrent synonymous VHL variant (c.414A>G, p.Pro138Pro) confers susceptibility to PHEO and VHL disease through splice disruption, leading to VHL dysfunction. This finding indicates that certain synonymous VHL variants may be clinically relevant and should be considered in genetic testing and surveillance settings. The observation that other coding VHL variants can exclude exon 2 suggests that dysregulated splicing may be an underappreciated mechanism in VHL-mediated tumorigenesis., (Copyright © 2019 Endocrine Society.)

The current model for breast cancer progression proposes independent 'low grade (LG)-like' and 'high grade (HG)-like' pathways but lacks a known precursor to HG cancer. We applied low-coverage whole-genome sequencing to atypical ductal hyperplasia (ADH) with and without carcinoma to shed light on breast cancer progression. Fourteen out of twenty isolated ADH cases harboured at least one copy number alteration (CNA), but had fewer aberrations than LG or HG ductal carcinoma in situ (DCIS). ADH carried more HG-like CNA than LG DCIS (e.g. 8q gain). Correspondingly, 64% (7/11) of ADH cases with synchronous HG carcinoma were clonally related, similar to LG carcinoma (67%, 6/9). This study represents a significant shift in our understanding of breast cancer progression, with ADH as a common precursor lesion to the independent 'low grade-like' and 'high grade-like' pathways. These data suggest that ADH can be a precursor of HG breast cancer and that LG and HG carcinomas can evolve from a similar ancestor lesion. We propose that although LG DCIS may be committed to a LG molecular pathway, ADH may remain multipotent, progressing to either LG or HG carcinoma. This multipotent nature suggests that some ADH cases could be more clinically significant than LG DCIS, requiring biomarkers for personalising management. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Breast cancer (BC) diagnosed after a negative mammogram but prior to the next screening episode is termed an 'interval BC' (IBC). Understanding the molecular differences between IBC and screen-detected BCs (SDBC) could improve mammographic screening and management options. Therefore, we assessed both germline and somatic genomic aberrations in a prospective cohort. Utilising the Lifepool cohort of >54 000 women attending mammographic screening programs, 930 BC cases with screening status were identified (726 SDBC and 204 IBC). Clinico-pathological and family history information were recorded. Germline and tumour DNA were collected where available and sequenced for BC predisposition and driver gene mutations. Compared to SDBC, IBCs were significantly associated with a younger age at diagnosis and tumour characteristics associated with worse prognosis. Germline DNA assessment of BC cases that developed post-enrolment (276 SDBCs and 77 IBCs) for pathogenic mutations in 12 hereditary BC predisposition genes identified 8 carriers (2.27%). The germline mutation frequency was higher in IBC versus SDBC, although not statistically significant (3.90% versus 1.81%, p = 0.174). Comparing somatic genetic features of IBC and SDBC matched for grade, histological subtype and hormone receptor revealed no significant differences, with the exception of higher homologous recombination deficiency scores in IBC, and copy number changes on chromosome Xq in triple negative SDBCs. Our data demonstrates that while IBCs are clinically more aggressive than SDBC, when matched for confounding clinico-pathological features they do not represent a unique molecular class of invasive BC, but could be a consequence of timing of tumour initiation and mammographic screening. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Purpose: The identification of carriers of hereditary breast and ovarian cancer (HBOC) gene variants through family cancer history alone is suboptimal, and most population-based genetic testing studies have been limited to founder mutations in high-risk populations. Here, we determine the clinical utility of identifying actionable variants in a healthy cohort of women., Methods: Germline DNA from a subset of healthy Australian women participating in the lifepool project was screened using an 11-gene custom sequencing panel. Women with clinically actionable results were invited to attend a familial cancer clinic (FCC) for post-test genetic counseling and confirmatory testing. Outcomes measured included the prevalence of pathogenic variants, and the uptake rate of genetic counseling, risk reduction surgery, and cascade testing., Results: Thirty-eight of 5908 women (0.64%) carried a clinically actionable pathogenic variant. Forty-two percent of pathogenic variant carriers did not have a first-degree relative with breast or ovarian cancer and 89% pursued referral to an FCC. Forty-six percent (6/13) of eligible women pursued risk reduction surgery, and the uptake rate of cascade testing averaged 3.3 family members per index case., Conclusion: Within our cohort, HBOC genetic testing was well accepted, and the majority of high-risk gene carriers identified would not meet eligibility criteria for genetic testing based on their existing family history.

The BCL6 Corepressor (BCOR) is a component of a variant Polycomb repressive complex 1 (PRC1) that is essential for normal development. Recurrent mutations in the BCOR gene have been identified in acute myeloid leukaemia and myelodysplastic syndrome among other cancers; however, its function remains poorly understood. Here we examine the role of BCOR in haematopoiesis in vivo using a conditional mouse model that mimics the mutations observed in haematological malignancies. Inactivation of Bcor in haematopoietic stem cells (HSCs) results in expansion of myeloid progenitors and co-operates with oncogenic Kras G12D in the initiation of an aggressive and fully transplantable acute leukaemia. Gene expression analysis and chromatin immunoprecipitation sequencing reveals differential regulation of a subset of PRC1-target genes including HSC-associated transcription factors such as Hoxa7/9. This study provides mechanistic understanding of how BCOR regulates cell fate decisions and how loss of function contributes to the development of leukaemia.

PALB2 is established as the most clinically important moderate to high penetrance breast cancer predisposition gene after BRCA1 and BRCA2. Mutations in classical familial cancer predisposition genes are presumed to be recessive at the cellular level and therefore a second inactivating somatic mutation is required in the tumour tissue. However, from the limited data that exist, PALB2 may be an example of a cancer predisposition gene that does not conform to Knudson's 'two hit' paradigm. We conducted genome-wide copy number analysis and targeted sequencing of PALB2 and other breast cancer driver genes in 15 invasive breast cancers from individuals carrying pathogenic germline mutations in PALB2. The majority of cancers showed clear evidence of bi-allelic inactivation of PALB2 (10/15) either as loss of heterozygosity involving the wild-type allele (six tumours) or as somatic point mutations (four tumours). All PALB2-null cancers had high homologous recombination deficiency (HRD) scores consistent with a homologous recombination repair deficiency. Interestingly, all but one of the PALB2 heterozygous cancers also had high HRD scores, suggesting that alternative mechanisms of PALB2 functional loss might be operating in these cancers. Our findings demonstrate that PALB2 does undergo bi-allelic inactivation in the majority of breast cancers from PALB2 germline mutation carriers. This feature has implications for the discovery of new moderate to high penetrance breast cancer predisposition genes as it supports using the existence of a 'second hit' and mutation signatures as important search criteria. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background: Genome-wide association studies (GWASs) have identified numerous single-nucleotide polymorphisms (SNPs) associated with small increases in breast cancer risk. Studies to date suggest that some SNPs alter the expression of the associated genes, which potentially mediates risk modification. On this basis, we hypothesised that some of these genes may be enriched for rare coding variants associated with a higher breast cancer risk., Methods: The coding regions and exon-intron boundaries of 56 genes that have either been proposed by GWASs to be the regulatory targets of the SNPs and/or located < 500 kb from the risk SNPs were sequenced in index cases from 1043 familial breast cancer families that previously had negative test results for BRCA1 and BRCA2 mutations and 944 population-matched cancer-free control participants from an Australian population. Rare (minor allele frequency ≤ 0.001 in the Exome Aggregation Consortium and Exome Variant Server databases) loss-of-function (LoF) and missense variants were studied., Results: LoF variants were rare in both the cases and control participants across all the candidate genes, with only 38 different LoF variants observed in a total of 39 carriers. For the majority of genes (n = 36), no LoF variants were detected in either the case or control cohorts. No individual gene showed a significant excess of LoF or missense variants in the cases compared with control participants. Among all candidate genes as a group, the total number of carriers with LoF variants was higher in the cases than in the control participants (26 cases and 13 control participants), as was the total number of carriers with missense variants (406 versus 353), but neither reached statistical significance (p = 0.077 and p = 0.512, respectively). The genes contributing most of the excess of LoF variants in the cases included TET2, NRIP1, RAD51B and SNX32 (12 cases versus 2 control participants), whereas ZNF283 and CASP8 contributed largely to the excess of missense variants (25 cases versus 8 control participants)., Conclusions: Our data suggest that rare LoF and missense variants in genes associated with low-penetrance breast cancer risk SNPs may contribute some additional risk, but as a group these genes are unlikely to be major contributors to breast cancer heritability.

New treatments for triple-negative breast cancer (TNBC) are urgently needed. Despite there being little evidence of clinical activity as single-agent therapies, we show that dual blockade of PI3Kα and CDK4/6 is synergistically effective against multiple RB1 -wild-type TNBC models. Combined PI3Kα and CDK4/6 inhibition significantly increased apoptosis, cell-cycle arrest, and tumor immunogenicity and generated immunogenic cell death in human TNBC cell lines. Combination treatment also significantly improved disease control in human xenograft models compared with either monotherapy. Combined PI3Kα and CDK4/6 inhibition significantly increased tumor-infiltrating T-cell activation and cytotoxicity and decreased the frequency of immunosuppressive myeloid-derived suppressor cells in a syngeneic TNBC mouse model. Notably, combined PI3Kα and CDK4/6 inhibition, along with inhibition of immune checkpoints PD-1 and CTLA-4, induced complete and durable regressions (>1 year) of established TNBC tumors in vivo Overall, our results illustrate convergent mechanisms of PI3Kα and CDK4/6 blockade on cell-cycle progression, DNA damage response, and immune-modulation and may provide a novel therapeutic approach for TNBC. Cancer Res; 77(22); 6340-52. ©2017 AACR ., (©2017 American Association for Cancer Research.)