ABSTRACT
PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms â³11 and â³11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood.We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials.Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations.
Subject(s)
BRCA1 Protein , Drug Resistance, Neoplasm , Exons , Ovarian Neoplasms , Poly(ADP-ribose) Polymerase Inhibitors , RNA Splice Sites , Humans , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Drug Resistance, Neoplasm/genetics , BRCA1 Protein/genetics , Female , Animals , Mice , Ovarian Neoplasms/genetics , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Mutation , Breast Neoplasms/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Xenograft Model Antitumor Assays , Cell Line, TumorABSTRACT
Investigating how transcription factors control complex cellular processes requires tools that enable responses to be visualised at the single-cell level and their cell fate to be followed over time. For example, the tumour suppressor p53 (also called TP53 in humans and TRP53 in mice) can initiate diverse cellular responses by transcriptional activation of its target genes: Puma to induce apoptotic cell death and p21 to induce cell cycle arrest/cell senescence. However, it is not known how these processes are regulated and initiated in different cell types. Also, the context-dependent interaction partners and binding loci of p53 remain largely elusive. To be able to examine these questions, we here developed knock-in mice expressing triple-FLAG-tagged p53 to facilitate p53 pull-down and two p53 response reporter mice, knocking tdTomato and GFP into the Puma/Bbc3 and p21 gene loci, respectively. By crossing these reporter mice into a p53-deficient background, we show that the new reporters reliably inform on p53-dependent and p53-independent initiation of both apoptotic or cell cycle arrest/senescence programs, respectively, in vitro and in vivo.
ABSTRACT
Mutations in the tumor suppressor TP53 cause cancer and impart poor chemotherapeutic responses, reportedly through loss-of-function, dominant-negative effects and gain-of-function (GOF) activities. The relative contributions of these attributes is unknown. We found that removal of 12 different TP53 mutants with reported GOFs by CRISPR/Cas9 did not impact proliferation and response to chemotherapeutics of 15 human cancer cell lines and colon cancer-derived organoids in culture. Moreover, removal of mutant TP53/TRP53 did not impair growth or metastasis of human cancers in immune-deficient mice or growth of murine cancers in immune-competent mice. DepMap mining revealed that removal of 158 different TP53 mutants had no impact on the growth of 391 human cancer cell lines. In contrast, CRISPR-mediated restoration of wild-type TP53 extinguished the growth of human cancer cells in vitro. These findings demonstrate that LOF but not GOF effects of mutant TP53/TRP53 are critical to sustain expansion of many tumor types. SIGNIFICANCE: This study provides evidence that removal of mutant TP53, thereby deleting its reported GOF activities, does not impact the survival, proliferation, metastasis, or chemotherapy responses of cancer cells. Thus, approaches that abrogate expression of mutant TP53 or target its reported GOF activities are unlikely to exert therapeutic impact in cancer. See related commentary by Lane, p. 211 . This article is featured in Selected Articles from This Issue, p. 201.
Subject(s)
Colonic Neoplasms , Tumor Suppressor Protein p53 , Humans , Mice , Animals , Cell Line, Tumor , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Mutation , Colonic Neoplasms/genetics , Cell ProliferationABSTRACT
Transcriptional activation of target genes is essential for TP53-mediated tumour suppression, though the roles of the diverse TP53-activated target genes in tumour suppression remains poorly understood. Knockdown of ZMAT3, an RNA-binding zinc-finger protein involved in regulating alternative splicing, in haematopoietic cells by shRNA caused leukaemia only with the concomitant absence of the PUMA and p21, the critical effectors of TRP53-mediated apoptosis and cell cycle arrest respectively. We were interested to further investigate the role of ZMAT3 in tumour suppression beyond the haematopoietic system. Therefore, we generated Zmat3 knockout and compound gene knockout mice, lacking Zmat3 and p21, Zmat3 and Puma or all three genes. Puma-/-p21-/-Zmat3-/- triple knockout mice developed tumours at a significantly higher frequency compared to wild-type, Puma-/-Zmat3-/- or p21-/-Zmat3-/-deficient mice. Interestingly, we observed that the triple knockout and Puma-/-Zmat3-/- double deficient animals succumbed to lymphoma, while p21-/-Zmat3-/- animals developed mainly solid cancers. This analysis suggests that in addition to ZMAT3 loss, additional TRP53-regulated processes must be disabled simultaneously for TRP53-mediated tumour suppression to fail. Our findings reveal that the absence of different TRP53 regulated tumour suppressive processes changes the tumour spectrum, indicating that different TRP53 tumour suppressive pathways are more critical in different tissues.
Subject(s)
Neoplasms , Tumor Suppressor Protein p53 , Animals , Mice , Apoptosis/genetics , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Incidence , Mice, Knockout , Neoplasms/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Background: Despite initial response to platinum-based chemotherapy and PARP inhibitor therapy (PARPi), nearly all recurrent high-grade serous ovarian cancer (HGSC) will acquire lethal drug resistance; indeed, ~15% of individuals have de novo platinum-refractory disease. Objectives: To determine the potential of anti-microtubule agent (AMA) therapy (paclitaxel, vinorelbine and eribulin) in platinum-resistant or refractory (PRR) HGSC by assessing response in patient-derived xenograft (PDX) models of HGSC. Design and methods: Of 13 PRR HGSC PDX, six were primary PRR, derived from chemotherapy-naïve samples (one was BRCA2 mutant) and seven were from samples obtained following chemotherapy treatment in the clinic (five were mutant for either BRCA1 or BRCA2 (BRCA1/2), four with prior PARPi exposure), recapitulating the population of individuals with aggressive treatment-resistant HGSC in the clinic. Molecular analyses and in vivo treatment studies were undertaken. Results: Seven out of thirteen PRR PDX (54%) were sensitive to treatment with the AMA, eribulin (time to progressive disease (PD) ⩾100 days from the start of treatment) and 11 out of 13 PDX (85%) derived significant benefit from eribulin [time to harvest (TTH) for each PDX with p < 0.002]. In 5 out of 10 platinum-refractory HGSC PDX (50%) and one out of three platinum-resistant PDX (33%), eribulin was more efficacious than was cisplatin, with longer time to PD and significantly extended TTH (each PDX p < 0.02). Furthermore, four of these models were extremely sensitive to all three AMA tested, maintaining response until the end of the experiment (120d post-treatment start). Despite harbouring secondary BRCA2 mutations, two BRCA2-mutant PDX models derived from heavily pre-treated individuals were sensitive to AMA. PRR HGSC PDX models showing greater sensitivity to AMA had high proliferative indices and oncogene expression. Two PDX models, both with prior chemotherapy and/or PARPi exposure, were refractory to all AMA, one of which harboured the SLC25A40-ABCB1 fusion, known to upregulate drug efflux via MDR1. Conclusion: The efficacy observed for eribulin in PRR HGSC PDX was similar to that observed for paclitaxel, which transformed ovarian cancer clinical practice. Eribulin is therefore worthy of further consideration in clinical trials, particularly in ovarian carcinoma with early failure of carboplatin/paclitaxel chemotherapy.
ABSTRACT
BRCA1 splice isoforms Δ11 and Δ11q can contribute to PARP inhibitor (PARPi) resistance by splicing-out the mutation-containing exon, producing truncated, partially-functional proteins. However, the clinical impact and underlying drivers of BRCA1 exon skipping remain undetermined. We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs), predicted in silico to drive exon skipping. Predictions were confirmed using qRT-PCR, RNA sequencing, western blots and BRCA1 minigene modelling. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials. We demonstrate that SSMs drive BRCA1 exon 11 skipping and PARPi resistance, and should be clinically monitored, along with frame-restoring secondary mutations.
ABSTRACT
Mutant TP53 proteins are thought to drive the development and sustained expansion of cancers at least in part through the loss of the wild-type (wt) TP53 tumour suppressive functions. Therefore, compounds that can restore wt TP53 functions in mutant TP53 proteins are expected to inhibit the expansion of tumours expressing mutant TP53. APR-246 has been reported to exert such effects in malignant cells and is currently undergoing clinical trials in several cancer types. However, there is evidence that APR-246 may also kill malignant cells that do not express mutant TP53. To support the clinical development of APR-246 it is important to understand its mechanism(s) of action. By establishing isogenic background tumour cell lines with different TP53/TRP53 states, we found that APR-246 can kill malignant cells irrespective of their TP53/TRP53 status. Accordingly, RNAseq analysis revealed that treatment with APR-246 induces expression of the same gene set in Eµ-Myc mouse lymphoma cells of all four possible TRP53 states, wt, wt alongside mutant, knockout and knockout alongside mutant. We found that depending on the type of cancer cell and the concentration of APR-246 used, this compound can kill malignant cells through induction of various programmed cell death pathways, including apoptosis, necroptosis and ferroptosis. The sensitivity of non-transformed cells to APR-246 also depended on the cell type. These findings reveal that the clinical testing of APR-246 should not be limited to cancers expressing mutant TP53 but expanded to cancers that express wt TP53 or are TP53-deficient.
Subject(s)
Genes, p53 , Tumor Suppressor Protein p53 , Animals , Mice , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Apoptosis , Cell Line, Tumor , MutationABSTRACT
Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes. SIGNIFICANCE: Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.
Subject(s)
Antineoplastic Agents , Carcinoma , Carcinosarcoma , Ovarian Neoplasms , Humans , Female , Epithelial-Mesenchymal Transition/genetics , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Cell Transformation, Neoplastic , Antineoplastic Agents/pharmacology , Microtubules , Carcinosarcoma/genetics , Carcinosarcoma/pathologyABSTRACT
Cell death, cell cycle arrest and cellular senescence are three distinct cellular responses that can be induced by oncogene activation and diverse anti-cancer agents, and this often requires the action of the tumour suppressor TP53. Within a cell population, or even within an individual cell, these processes are not necessarily mutually exclusive. It is therefore important to measure all these processes simultaneously. However, current assays generally visualise only one or at best two responses, often only detecting the dominant one. Here, we present a novel flow cytometric assay that allows simultaneous assessment of cell viability and cell cycling through measurement of DNA content and DNA synthesis, and markers of cell senescence at the single cell level. We demonstrate that this assay can be performed on both human and murine cells, that are either cancerous or non-transformed, and can help to dissect complex cell fate decisions. We believe that this experimental tool will be useful for the study of diverse biological processes.
Subject(s)
Cellular Senescence , DNA , Animals , Cell Cycle , Cell Cycle Checkpoints , Cell Death , Cellular Senescence/genetics , Humans , Mice , Tumor Suppressor Protein p53/metabolismABSTRACT
In high-grade serous ovarian carcinoma (HGSC), deleterious mutations in DNA repair gene RAD51C are established drivers of defective homologous recombination and are emerging biomarkers of PARP inhibitor (PARPi) sensitivity. RAD51C promoter methylation (meRAD51C) is detected at similar frequencies to mutations, yet its effects on PARPi responses remain unresolved.In this study, three HGSC patient-derived xenograft (PDX) models with methylation at most or all examined CpG sites in the RAD51C promoter show responses to PARPi. Both complete and heterogeneous methylation patterns were associated with RAD51C gene silencing and homologous recombination deficiency (HRD). PDX models lost meRAD51C following treatment with PARPi rucaparib or niraparib, where a single unmethylated copy of RAD51C was sufficient to drive PARPi resistance. Genomic copy number profiling of one of the PDX models using SNP arrays revealed that this resistance was acquired independently in two genetically distinct lineages.In a cohort of 12 patients with RAD51C-methylated HGSC, various patterns of meRAD51C were associated with genomic "scarring," indicative of HRD history, but exhibited no clear correlations with clinical outcome. Differences in methylation stability under treatment pressure were also observed between patients, where one HGSC was found to maintain meRAD51C after six lines of therapy (four platinum-based), whereas another HGSC sample was found to have heterozygous meRAD51C and elevated RAD51C gene expression (relative to homozygous meRAD51C controls) after only neoadjuvant chemotherapy.As meRAD51C loss in a single gene copy was sufficient to cause PARPi resistance in PDX, methylation zygosity should be carefully assessed in previously treated patients when considering PARPi therapy. SIGNIFICANCE: Homozygous RAD51C methylation is a positive predictive biomarker for sensitivity to PARP inhibitors, whereas a single unmethylated gene copy is sufficient to confer resistance.
Subject(s)
Cystadenocarcinoma, Serous/genetics , DNA Methylation , DNA-Binding Proteins/genetics , Drug Resistance, Neoplasm/genetics , Ovarian Neoplasms/genetics , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Promoter Regions, Genetic , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Computational Biology , Cystadenocarcinoma, Serous/drug therapy , Cystadenocarcinoma, Serous/metabolism , Cystadenocarcinoma, Serous/pathology , DNA-Binding Proteins/metabolism , Disease Models, Animal , Female , Gene Expression Profiling , Gene Silencing , Homozygote , Humans , Mice , Neoplasm Grading , Neoplasm Staging , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Prognosis , Xenograft Model Antitumor AssaysABSTRACT
Patients with cancer treated with PARP inhibitors (PARPi) experience various side effects, with hematologic toxicity being most common. Short-term treatment of mice with olaparib resulted in depletion of reticulocytes, B-cell progenitors, and immature thymocytes, whereas longer treatment induced broader myelosuppression. We performed a CRISPR/Cas9 screen that targeted DNA repair genes in Eµ-Myc pre-B lymphoma cell lines as a way to identify strategies to suppress hematologic toxicity from PARPi. The screen revealed that single-guide RNAs targeting the serine/threonine kinase checkpoint kinase 2 (CHK2) were enriched following olaparib treatment. Genetic or pharmacologic inhibition of CHK2-blunted PARPi response in lymphoid and myeloid cell lines, and in primary murine pre-B/pro-B cells. Using a Cas9 base editor, we found that blocking CHK2-mediated phosphorylation of p53 also impaired olaparib response. Our results identify the p53 pathway as a major determinant of the acute response to PARPi in normal blood cells and demonstrate that targeting CHK2 can short circuit this response. Cotreatment with a CHK2 inhibitor did not antagonize olaparib response in ovarian cancer cell lines. Selective inhibition of CHK2 may spare blood cells from the toxic influence of PARPi and broaden the utility of these drugs. IMPLICATIONS: We reveal that genetic or pharmacologic inhibition of CHK2 may offer a way to alleviate the toxic influence of PARPi in the hematologic system.
Subject(s)
Checkpoint Kinase 2/antagonists & inhibitors , Ovarian Neoplasms/drug therapy , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Animals , Cell Line, Tumor , DNA Repair/drug effects , Female , Humans , Male , Mice , Mice, Inbred C57BL , Ovarian Neoplasms/metabolism , Phthalazines/pharmacology , Piperazines/pharmacology , Signal Transduction/drug effectsABSTRACT
Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease.
Subject(s)
Benzothiazoles/pharmacology , Cystadenocarcinoma, Serous/drug therapy , DNA Damage , Naphthyridines/pharmacology , Ovarian Neoplasms/drug therapy , Animals , Cell Line, Tumor , Cystadenocarcinoma, Serous/genetics , Cystadenocarcinoma, Serous/metabolism , DNA Replication/drug effects , Drug Resistance, Neoplasm , Enzyme Inhibitors/pharmacology , Female , Heterografts , Homologous Recombination , Humans , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Models, Biological , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , RNA Polymerase I/antagonists & inhibitors , TranscriptomeABSTRACT
BACKGROUND: We conducted a Phase 1 study to evaluate safety and activity of olaparib tablets and oral cyclophosphamide. METHODS: Patients had metastatic breast cancer (BC) or recurrent high-grade serous ovarian cancer (HGSOC), performance status 0-2, and ≤3 lines of prior therapy. Patients were treated using a dose escalation strategy with cohort expansion once maximal tolerated dose (MTD) was determined. Dose level 1 (DL1): olaparib 300 mg bid, cyclophosphamide 50 mg on days 1, 3, and 5, weekly. DL2: olaparib 300 mg bid, cyclophosphamide 50 mg, days 1-5 weekly. RESULTS: Of 32 patients, 23 had HGSOC (germline BRCA mutation [gBRCAm] 70%) and 9 had BC (gBRCAm 67%). Four were treated at DL1 and 28 at DL2, the MTD. Haematological adverse events (AEs) were most common: grade 3/4 AEs: lymphopenia 75%, anaemia 31%, neutropenia 37%, thrombocytopenia 47%. Two permanently discontinued treatment due to haematological AEs. In BC, no objective response was reported. Unconfirmed objective response was 48% and 64% for all HGSOC and gBRCAm subset, respectively. CA125 responses were 70% (all HGSOC) and 92% (gBRCAm). CONCLUSIONS: In HGSOC and BC, olaparib 300 mg bid and cyclophosphamide 50 mg on days 1-5 weekly were tolerable and active, particularly in gBRCAm, and is worthy of further investigation.
Subject(s)
Cyclophosphamide/administration & dosage , Ovarian Neoplasms/drug therapy , Phthalazines/administration & dosage , Piperazines/administration & dosage , Triple Negative Breast Neoplasms/drug therapy , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Cyclophosphamide/adverse effects , Disease-Free Survival , Dose-Response Relationship, Drug , Drug-Related Side Effects and Adverse Reactions/classification , Drug-Related Side Effects and Adverse Reactions/pathology , Female , Germ-Line Mutation/genetics , Humans , Middle Aged , Neoplasm Metastasis , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Phthalazines/adverse effects , Piperazines/adverse effects , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathologyABSTRACT
Activation of the tumour suppressor p53 upon cellular stress can induce a number of different cellular processes. The diverse actions of these processes are critical for the protective function of p53 in preventing the development of cancer. However, it is still not fully understood which process(es) activated by p53 is/are critical for tumour suppression and how this might differ depending on the type of cells undergoing neoplastic transformation and the nature of the drivers of oncogenesis. Moreover, it is not clear why upon activation of p53 some cells undergo cell cycle arrest and senescence whereas others die by apoptosis. Here we discuss some of the cellular processes that are crucial for p53-mediated tumour suppression and the factors that could impact cell fate upon p53 activation. Finally, we describe therapies aimed either at activating wild-type p53 or at changing the behaviour of mutant p53 to unleash tumour growth suppressive processes for therapeutic benefit in malignant disease.
Subject(s)
Tumor Suppressor Protein p53/metabolism , Animals , Apoptosis/genetics , Apoptosis/physiology , Cell Cycle Checkpoints/genetics , Cell Cycle Checkpoints/physiology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cellular Senescence/genetics , Cellular Senescence/physiology , Humans , Tumor Suppressor Protein p53/geneticsABSTRACT
Mutations in Trp53, prevalent in human cancer, are reported to drive tumorigenesis through dominant-negative effects (DNEs) over wild-type TRP53 function as well as neomorphic gain-of-function (GOF) activity. We show that five TRP53 mutants do not accelerate lymphomagenesis on a TRP53-deficient background but strongly synergize with c-MYC overexpression in a manner that distinguishes the hot spot Trp53 mutations. RNA sequencing revealed that the mutant TRP53 DNE does not globally repress wild-type TRP53 function but disproportionately impacts a subset of wild-type TRP53 target genes. Accordingly, TRP53 mutant proteins impair pathways for DNA repair, proliferation, and metabolism in premalignant cells. This reveals that, in our studies of lymphomagenesis, mutant TRP53 drives tumorigenesis primarily through the DNE, which modulates wild-type TRP53 function in a manner advantageous for neoplastic transformation.
Subject(s)
Carcinogenesis/genetics , Mutation , Tumor Suppressor Protein p53/genetics , Animals , Lymphoma/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Tumor Suppressor Protein p53/metabolismABSTRACT
Accurately identifying patients with high-grade serous ovarian carcinoma (HGSOC) who respond to poly(ADP-ribose) polymerase inhibitor (PARPi) therapy is of great clinical importance. Here we show that quantitative BRCA1 methylation analysis provides new insight into PARPi response in preclinical models and ovarian cancer patients. The response of 12 HGSOC patient-derived xenografts (PDX) to the PARPi rucaparib was assessed, with variable dose-dependent responses observed in chemo-naive BRCA1/2-mutated PDX, and no responses in PDX lacking DNA repair pathway defects. Among BRCA1-methylated PDX, silencing of all BRCA1 copies predicts rucaparib response, whilst heterozygous methylation is associated with resistance. Analysis of 21 BRCA1-methylated platinum-sensitive recurrent HGSOC (ARIEL2 Part 1 trial) confirmed that homozygous or hemizygous BRCA1 methylation predicts rucaparib clinical response, and that methylation loss can occur after exposure to chemotherapy. Accordingly, quantitative BRCA1 methylation analysis in a pre-treatment biopsy could allow identification of patients most likely to benefit, and facilitate tailoring of PARPi therapy.