A P53-Independent DNA Damage Response Suppresses Oncogenic Proliferation and Genome Instability

The Mre11-Rad50-Nbs1 complex is a DNA double-strand break sensor that mediates a tumor-suppressive DNA damage response (DDR) in cells undergoing oncogenic stress, yet the mechanisms underlying this effect are poorly understood. Using a genetically inducible primary mammary epithelial cell model, we demonstrate that Mre11 suppresses proliferation and DNA damage induced by diverse oncogenic drivers through a p53-independent mechanism. Breast tumorigenesis models engineered to express a hypomorphic Mre11 allele exhibit increased levels of oncogene-induced DNA damage, R-loop accumulation, and chromosomal instability with a characteristic copy number loss phenotype. Mre11 complex dysfunction is identified in a subset of human triple-negative breast cancers and is associated with increased sensitivity to DNA-damaging therapy and inhibitors of ataxia telangiectasia and Rad3 related (ATR) and poly (ADP-ribose) polymerase (PARP). Thus, deficiencies in the Mre11-dependent DDR drive proliferation and genome instability patterns in p53-deficient breast cancers and represent an opportunity for therapeutic exploitation

MED12 somatic mutations in fibroadenomas and phyllodes tumours of the breast

Aims: Somatic mutations in exon 2 of the mediator complex subunit 12 (MED12) gene have been identified in 60% of breast fibroadenomas (FAs). The aim of this study was to define whether phyllodes tumours (PTs) would harbour MED12 somatic mutations in a way akin to FAs. Methods and results: A collection of 73 fibroepithelial tumours (including 26 FAs, 25 benign PTs, nine borderline PTs and 13 malignant PTs) from 64 patients was retrieved from the authors' institution. Sections from formalin-fixed paraffin-embedded (FFPE) blocks were microdissected to ensure an enrichment in neoplastic stromal elements of >70%. DNA samples extracted from tumour and matched normal tissues were subjected to Sanger sequencing of exon 2 of the MED12 gene. MED12 exon 2 somatic mutations, including 28 somatic single nucleotide variants and 19 insertions and deletions, were found in 65%, 88%, 78% and 8% of FAs, benign PTs, borderline PTs and malignant PTs, respectively. Malignant PTs harboured MED12 exon 2 somatic mutations significantly less frequently than FAs, benign and borderline PTs. Conclusions: Although MED12 exon 2 somatic mutations probably constitute the driver genetic event of most FAs, benign and borderline PTs, our results suggest that the majority of malignant PTs may be driven by other genetic/epigenetic alterations

HAMSAB diet ameliorates dysfunctional signaling in pancreatic islets in autoimmune diabetes

An altered gut microbiota is associated with type 1 diabetes (T1D), affecting the production of short-chain fatty acids (SCFA) and glucose homeostasis. We previously demonstrated that enhancing serum acetate and butyrate using a dietary supplement (HAMSAB) improved glycemia in non-obese diabetic (NOD) mice and patients with established T1D. The effects of SCFA on immune-infiltrated islet cells remain to be clarified. Here, we performed single-cell RNA sequencing on islet cells from NOD mice fed an HAMSAB or control diet. HAMSAB induced a regulatory gene expression profile in pancreas-infiltrated immune cells. Moreover, HAMSAB maintained the expression of b-cell functional genes and decreased cellular stress. HAMSAB-fed mice showed preserved pancreatic endocrine cell identity, evaluated by decreased numbers of poly-hormonal cells. Finally, SCFA increased insulin levels in human b-like cells and improved transplantation outcome in NOD/SCID mice. Our findings support the use of metabolite-based diet as attractive approach to improve glucose control in T1D.Valerie Vandenbempt, Sema Elif Eski, Manoja K. Brahma, Ao Li, Javier Negueruela, Ylke Bruggeman, Stephane Demine, Peng Xiao, Alessandra K. Cardozo, Nicolas Baeyens, Luciano G. Martelotto, Sumeet Pal Singh, Eliana Marin, o, Conny Gysemans, and Esteban N. Gurzo

Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease

Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (>99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMO-dependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness.Candice Alexandra Grzelak, Nicholas David Sigglekow, Janina Elke Eleonore Tirnitz-Parker, Elizabeth Jane Hamson, Alessandra Warren, Bharvi Maneck, Jinbiao Chen, Bramilla Patkunanathan, Jade Boland, Robert Cheng, Nicholas Adam Shackel, Devanshi Seth, David Geoffrey Bowen, Luciano Gaston Martelotto, D. Neil Watkins, Geoffrey William McCaugha

Combined BRAF, MEK, and CDK4/6 inhibition depletes intratumoral immune-potentiating myeloid populations in melanoma

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.Emily J. Lelliott, Stefano Mangiola, Kelly M. Ramsbottom, Magnus Zethoven, Lydia Lim, Peter K.H. Lau, Amanda J. Oliver, Luciano G. Martelotto, Laura Kirby, Claire Martin, Riyaben P. Patel, Alison Slater, Carleen Cullinane, Anthony T. Papenfuss, Nicole M. Haynes, Grant A. McArthur, Jane Oliaro, and Karen E. Sheppar

Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.Charles C. Bell, Katie A. Fennell, Yih-Chih Chan, Florian Rambow, Miriam M. Yeung, Dane Vassiliadis, Luis Lara, Paul Yeh, Luciano G. Martelotto, Aljosja Rogiers, Brandon E. Kremer, Olena Barbash, Helai P. Mohammad, Timothy M. Johanson, Marian L. Burr, Arindam Dhar, Natalie Karpinich, Luyi Tian, Dean S. Tyler, Laura MacPherson, Junwei Shi, Nathan Pinnawala, Chun Yew Fong, Anthony T. Papenfuss, Sean M. Grimmond, Sarah-Jane Dawson, Rhys S. Allan, Ryan G. Kruger, Christopher R. Vakoc, David L. Goode, Shalin H. Naik, Omer Gilan, Enid Y.N. Lam, Jean-Christophe Marine, Rab K. Prinjha, Mark A. Dawso

CDK4/6 inhibition promotes antitumor immunity through the induction of T-cell memory

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.Emily J. Lelliott, Isabella Y. Kong, Magnus Zethoven, Kelly M. Ramsbottom, Luciano G. Martelotto, Deborah Meyran, Joe Jiang Zhu, Matteo Costacurta, Laura Kirby, Jarrod J. Sandow, Lydia Lim, Pilar M. Dominguez, Izabela Todorovski, Nicole M. Haynes, Paul A. Beavis, Paul J. Neeson, Edwin D. Hawkins, Grant A. McArthur, Ian A. Parish, Ricky W. Johnstone, Jane Oliaro, Karen E. Sheppard, Conor J. Kearney, and Stephin J. Vervoor

gamma delta T cells in merkel cell carcinomas have a proinflammatory profile prognostic of patient survival

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. 600Nicholas A. Gherardin, Kelly Waldeck, Alex Caneborg, Luciano G. Martelotto, Shiva Balachander, Magnus Zethoven ... et al

Antigen-driven EGR2 expression is required for exhausted CD8(+) T cell stability and maintenance

Chronic stimulation of CD8⁺ T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.Mayura V. Wagle, Stephin J. Vervoort, Madison J. Kelly, Willem Van Der Byl, Timothy J. Peters, Ben P. Martin ... et al

The repertoire of somatic genetic alterations of acinic cell carcinomas of the breast : an exploratory, hypothesis-generating study

Acinic cell carcinoma (ACC) of the breast is a rare form of triple-negative (that is, oestrogen receptor-negative, progesterone receptor-negative, HER2-negative) salivary gland-type tumour displaying serous acinar differentiation. Despite its triple-negative phenotype, breast ACCs are reported to have an indolent clinical behaviour. Here, we sought to define whether ACCs have a mutational repertoire distinct from that of other triple-negative breast cancers (TNBCs). DNA was extracted from microdissected formalin-fixed, paraffin-embedded sections of tumour and normal tissue from two pure and six mixed breast ACCs. Each tumour component of the mixed cases was microdissected separately. Tumour and normal samples were subjected to targeted capture massively parallel sequencing targeting all exons of 254 genes, including genes most frequently mutated in breast cancer and related to DNA repair. Selected somatic mutations were validated by targeted amplicon resequencing and Sanger sequencing. Akin to other forms of TNBC, the most frequently mutated gene found in breast ACCs was TP53 (one pure and six mixed cases). Additional somatic mutations affecting breast cancer-related genes found in ACCs included PIK3CA, MTOR, CTNNB1, BRCA1, ERBB4, ERBB3, INPP4B, and FGFR2. Copy number alteration analysis revealed complex patterns of gains and losses similar to those of common forms of TNBCs. Of the mixed cases analysed, identical somatic mutations were found in the acinic and the high-grade non-acinic components in two out of four cases analysed, providing evidence of their clonal relatedness. In conclusion, breast ACCs display the hallmark somatic genetic alterations found in high-grade forms of TNBC, including complex patterns of gene copy number alterations and recurrent TP53 mutations. Furthermore, we provide circumstantial genetic evidence to suggest that ACCs may constitute the substrate for the development of more aggressive forms of triple-negative disease