In high-grade serous ovarian carcinoma (HGSC), deleterious mutations in DNA repair gene
are established drivers of defective homologous recombination and are emerging biomarkers of PARP inhibitor ...(PARPi) sensitivity.
promoter methylation (me
) 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
promoter show responses to PARPi. Both complete and heterogeneous methylation patterns were associated with
gene silencing and homologous recombination deficiency (HRD). PDX models lost me
following treatment with PARPi rucaparib or niraparib, where a single unmethylated copy of
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
-methylated HGSC, various patterns of me
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 me
after six lines of therapy (four platinum-based), whereas another HGSC sample was found to have heterozygous me
and elevated
gene expression (relative to homozygous me
controls) after only neoadjuvant chemotherapy.As me
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
methylation is a positive predictive biomarker for sensitivity to PARP inhibitors, whereas a single unmethylated gene copy is sufficient to confer resistance.
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.
Mutations in
, 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
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.
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.
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.
Mutations in p53 occur in ∼50% of cancers. Mutant p53 usually accumulates in cells and differs from wildtype (wt) p53 by only one amino acid within its DNA binding domain. It is thought to exhibit ...loss of function (an inability to regulate genes that wt p53 is able to), dominant negative effects (suppressing the activity of wt p53 in cells expressing both wt and mutant p53) and gain of function effects (the ability to regulate proteins not regulated by wt p53). It is unclear what impact these functions have on cells undergoing neoplastic transformation, if these functions are required for sustained tumour growth, or response to anti-cancer therapies.
Given the high mutation rate and the increased difficulty in treating these cancers, the thought of targeting mutant p53 in cancer therapy is an exciting prospect. However, for effective therapies to be designed, answers to the above questions are required.
To answer this, we have generated two switchable mutant p53 mouse models using CRISPR-based genome editing. From conception, these mice encode wt p53 in all cells. After FLP recombination, mutant p53 protein is expressed in specifically targeted cells. In the first strain, CRE recombination leads to restoration of wt p53 expression, and in the second strain it leads to a p53 null state. We are currently investigating the role of this p53 mutation in the development of lymphomas by crossing them onto an Eµ-Myc background, where mice spontaneously develop lymphomas that are accelerated by loss or mutation of p53.
These switchable mutant p53 mouse models have the potential to answer some of the most important open questions in cancers, such as whether deleting mutant p53 protein or restoring wt p53 function will have an effect on tumour growth. The findings from such studies will inform on the development of novel, improved therapies for cancers with defects in p53.
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.
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
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.
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.
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.
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