BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we ...conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.
Response and resistance to anticancer therapies vary due to intertumor and intratumor heterogeneity
. Here, we map differentially enriched G-quadruplex (G4) DNA structure-forming regions (∆G4Rs) in ...22 breast cancer patient-derived tumor xenograft (PDTX) models. ∆G4Rs are associated with the promoters of highly amplified genes showing high expression, and with somatic single-nucleotide variants. Differences in ΔG4R landscapes reveal seven transcription factor programs across PDTXs. ∆G4R abundance and locations stratify PDTXs into at least three G4-based subtypes. ∆G4Rs in most PDTXs (14 of 22) were found to associate with more than one breast cancer subtype, which we also call an integrative cluster (IC)
. This suggests the frequent coexistence of multiple breast cancer states within a PDTX model, the majority of which display aggressive triple-negative IC10 gene activity. Short-term cultures of PDTX models with increased ∆G4R levels are more sensitive to small molecules targeting G4 DNA. Thus, G4 landscapes reveal additional IC-related intratumor heterogeneity in PDTX biopsies, improving breast cancer stratification and potentially identifying new treatment strategies.
Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal ...populations and temporal statistical models
. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model
to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.
Abstract
Background Pharmacological inhibition of PARP results in the specific killing of BRCA1/2 deficient tumor cells due to a synthetic lethal interaction between the concomitant impairment in ...homologous recombination and the DNA damage response. Despite the success of this approach, resistance to PARP inhibition has been observed in majority of patients with advanced cancer. Novel ways of interrogating PARP resistance are necessary to further elucidate the mechanisms of drug resistance and help identify ways to overcome it. To probe potential mechanisms of resistance we applied data-independent acquisition (DIA) mass spectrometry for unbiased global protein quantification in patient derived xenografts with demonstrated resistance to PARP inhibitors.
Methods Patient-derived tumor xenografts (PDTXs) were generated from breast cancer patient tumor material implanted in severely immuno-compromised NOD-scid IL2rgnull (NSG) mice. PDTXs, developed as part of the CRUK Cambridge Institute biobank of PDTXs, have previously undergone extensive molecular profiling. PDTXs and short-term cultured PDTX cells (or PDTCs) capture most of originating patient's sample features, including heterogeneity, and consequently, the PDTX/PDTC platform is a robust intermediate in oncogenic drug development. Resistance to the PARP inhibitors AZD-2281 (olaparib) and BMN-673 (talazoparib) was tested ex vivo in PDTCs from 6 sensitive and 11 resistant PDTXs. Deep quantitative proteome profiling of PDTXs samples was conducted by applying an LC-MS/MS setup operated in DIA mode. Data was extracted using Spectronaut™ (Biognosys) with a sample specific spectral library. Pathway analysis was conducted to highlight dysregulated biological functions and pathways and the proteomics data was correlated with transcriptomics readouts.
Results LC-MS/MS profiling allowed the identification of more than 11'000 proteins with more than 8'700 proteins quantified across the samples. 448 human and 430 murine proteins were significantly changed between PARP inhibitor resistant and sensitive PDTX models. Resistant models were characterized by increased expression of DNA damage response proteins including ATR and FANCD2, downregulation of TP53, TP53BP1, POLB and H2AFX, and upregulated EGFR, BRAF, ERBB2, STAT3, MLLT4 and CDKN2A. Most interestingly, we observed upregulation of human CD47/SIRPa immunomodulatory signals and upregulation of mouse Ly6G, CSF1R and SHP-1 suggesting the infiltration of immunosuppressive neutrophils and monocytes in the tumor microenvironment in the models resistant to PARP inhibition.
Conclusions To our knowledge, this is the first report aiming at interrogating PARP inhibitor resistance with unbiased quantitative proteomics. The proteomics data confirmed prior observations of resistance mechanisms to PARP and elucidated potential novel mechanisms involving modulation of the immune response in resistant tumors.
Citation Format: Jakob Vowinckel, Yuehan Feng, Dimitra Georgopoulou, Alejandra Bruna, Tobias Treiber, Abigail Shea, Giulia Lerda, Martin O'Reilly, Kristina Beeler, Carlos Caldas. Quantitative proteomics reveals novel immunomodulatory pathways of resistance to PARP therapy abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4077.
Tumour heterogeneity is thought to be a major barrier to successful cancer treatment due to the presence of drug resistant clonal lineages. However, identifying the characteristics of such lineages ...that underpin resistance to therapy has remained challenging. Here, we utilise clonal transcriptomics with WILD-seq;
holistic
nterrogation of
ineage
ynamics by
uencing, in mouse models of triple-negative breast cancer (TNBC) to understand response and resistance to therapy, including BET bromodomain inhibition and taxane-based chemotherapy. These analyses revealed oxidative stress protection by NRF2 as a major mechanism of taxane resistance and led to the discovery that our tumour models are collaterally sensitive to asparagine deprivation therapy using the clinical stage drug L-asparaginase after frontline treatment with docetaxel. In summary, clonal transcriptomics with WILD-seq identifies mechanisms of resistance to chemotherapy that are also operative in patients and pin points asparagine bioavailability as a druggable vulnerability of taxane-resistant lineages.
Autistic people’s perceptions of their interactions with criminal justice professionals are predominantly negative; however, little is known about the state of interactions on a global scale. To ...further understanding, a comprehensive stakeholder questionnaire was created. Aspects of reliability and validity including evidence for test content and internal structure were gathered using expert reviews, cognitive interviewing, pilot data collection, and a larger data collection effort (
N
= 1618). Data was gathered from the autism community through perspectives of parents/caregivers as well as from self-reported autistic adults. Criminal justice professionals included law enforcement officers, corrections professionals, probation and parole officers, forensic psychologists and legal professionals. The scale development process was detailed in order to sufficiently document the initial psychometric evidence and share the steps taken to gain diverse stakeholder input. This study is a critical first step in generating further information to facilitate policy and program development with wide applicability.
The continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug-resistant (XDR) ...tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents.
We performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis collected in KwaZulu-Natal from 2008 to 2013, in addition to three historical isolates, collected from patients in the same province and including an isolate from the 2005 Tugela Ferry XDR outbreak, a multidrug-resistant (MDR) isolate from 1994, and a pansusceptible isolate from 1995. We utilized an array of whole genome comparative techniques to assess the relatedness among strains, to establish the order of acquisition of drug resistance mutations, including the timing of acquisitions leading to XDR-TB in the LAM4 spoligotype, and to calculate the number of independent evolutionary emergences of MDR and XDR. Our sequencing and analysis revealed a 50-member clone of XDR M. tuberculosis that was highly related to the Tugela Ferry XDR outbreak strain. We estimated that mutations conferring isoniazid and streptomycin resistance in this clone were acquired 50 y prior to the Tugela Ferry outbreak (katG S315T isoniazid; gidB 130 bp deletion streptomycin; 1957 95% highest posterior density (HPD): 1937-1971), with the subsequent emergence of MDR and XDR occurring 20 y (rpoB L452P rifampicin; pncA 1 bp insertion pyrazinamide; 1984 95% HPD: 1974-1992) and 10 y (rpoB D435G rifampicin; rrs 1400 kanamycin; gyrA A90V ofloxacin; 1995 95% HPD: 1988-1999) prior to the outbreak, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and nine independent evolutionary events, respectively. Isoniazid resistance evolved before rifampicin resistance 46 times, whereas rifampicin resistance evolved prior to isoniazid only twice. We identified additional putative compensatory mutations to rifampicin in this dataset. One major limitation of this study is that the conclusions with respect to ordering and timing of acquisition of mutations may not represent universal patterns of drug resistance emergence in other areas of the globe.
In the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first-line drugs at the beginning of the antibiotic era. Subsequent accumulation of stepwise resistance mutations, occurring over decades and prior to the explosion of HIV in this region, yielded MDR and XDR, permitting the emergence of compensatory mutations. Our results suggest that drug-resistant strains circulating today reflect not only vulnerabilities of current TB control efforts but also those that date back 50 y. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics employed in South Africa that assess only rifampicin resistance.
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