The poly (ADP-ribose) polymerase (PARP) inhibitor olaparib is FDA approved for the treatment of BRCA-mutated breast, ovarian and pancreatic cancers. Olaparib inhibits PARP1/2 enzymatic activity and ...traps PARP1 on DNA at single-strand breaks, leading to replication-induced DNA damage that requires BRCA1/2-dependent homologous recombination repair. Moreover, DNA damage response pathways mediated by the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia mutated and Rad3-related (ATR) kinases are hypothesised to be important survival pathways in response to PARP-inhibitor treatment. Here, we show that olaparib combines synergistically with the ATR-inhibitor AZD6738 (ceralasertib), in vitro, leading to selective cell death in ATM-deficient cells. We observe that 24 h olaparib treatment causes cells to accumulate in G2-M of the cell cycle, however, co-administration with AZD6738 releases the olaparib-treated cells from G2 arrest. Selectively in ATM-knockout cells, we show that combined olaparib/AZD6738 treatment induces more chromosomal aberrations and achieves this at lower concentrations and earlier treatment time-points than either monotherapy. Furthermore, single-agent olaparib efficacy in vitro requires PARP inhibition throughout multiple rounds of replication. Here, we demonstrate in several ATM-deficient cell lines that the olaparib and AZD6738 combination induces cell death within 1-2 cell divisions, suggesting that combined treatment could circumvent the need for prolonged drug exposure. Finally, we demonstrate in vivo combination activity of olaparib and AZD6738 in xenograft and PDX mouse models with complete ATM loss. Collectively, these data provide a mechanistic understanding of combined PARP and ATR inhibition in ATM-deficient models, and support the clinical development of AZD6738 in combination with olaparib.
The repair of toxic double-strand breaks (DSB) is critical for the maintenance of genome integrity. The major mechanisms that cope with DSB are: homologous recombination (HR) and classical or ...alternative nonhomologous end joining (C-NHEJ versus A-EJ). Because these pathways compete for the repair of DSB, the choice of the appropriate repair pathway is pivotal. Among the mechanisms that influence this choice, deoxyribonucleic acid (DNA) end resection plays a critical role by driving cells to HR, while accurate C-NHEJ is suppressed. Furthermore, end resection promotes error-prone A-EJ. Increasing evidence define Poly(ADP-ribose) polymerase 3 (PARP3, also known as ARTD3) as an important player in cellular response to DSB. In this work, we reveal a specific feature of PARP3 that together with Ku80 limits DNA end resection and thereby helps in making the choice between HR and NHEJ pathways. PARP3 interacts with and PARylates Ku70/Ku80. The depletion of PARP3 impairs the recruitment of YFP-Ku80 to laser-induced DNA damage sites and induces an imbalance between BRCA1 and 53BP1. Both events result in compromised accurate C-NHEJ and a concomitant increase in DNA end resection. Nevertheless, HR is significantly reduced upon PARP3 silencing while the enhanced end resection causes mutagenic deletions during A-EJ. As a result, the absence of PARP3 confers hypersensitivity to anti-tumoral drugs generating DSB.
High grade serous ovarian cancer (HGSOC) is a major cause of female cancer mortality. The approval of poly (ADP-ribose) polymerase (PARP) inhibitors for clinical use has greatly improved treatment ...options for patients with homologous recombination repair (HRR)-deficient HGSOC, although the development of PARP inhibitor resistance in some patients is revealing limitations to outcome. A proportion of patients with HRR-proficient cancers also benefit from PARP inhibitor therapy. Our aim is to compare mechanisms of resistance to the PARP inhibitor olaparib in these two main molecular categories of HGSOC and investigate a way to overcome resistance that we considered particularly suited to a cancer like HGSOC, where there is a very high incidence of
gene mutation, making HGSOC cells heavily reliant on the G2 checkpoint for repair of DNA damage and survival. We identified alterations in multiple factors involved in resistance to PARP inhibition in both HRR-proficient and -deficient cancers. The most frequent change was a major reduction in levels of poly (ADP-ribose) glycohydrolase (PARG), which would be expected to preserve a residual PARP1-initiated DNA damage response to DNA single-strand breaks. Other changes seen would be expected to boost levels of HRR of DNA double-strand breaks. Growth of all olaparib-resistant clones isolated could be controlled by WEE1 kinase inhibitor AZD1775, which inactivates the G2 checkpoint. Our work suggests that use of the WEE1 kinase inhibitor could be a realistic therapeutic option for patients that develop resistance to olaparib.
The dihydroceramide, ceramide, sphingomyelin, lactosylceramide, and ganglioside species of A2780 human ovarian carcinoma cells treated with the synthetic retinoids N-(4-hydroxyphenyl)retinamide ...(fenretinide, 4-HPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) in culture were characterized by ESI-MS. We characterized 32 species of ceramide and dihydroceramide, 15 of sphingomyelin, 12 of lactosylceramide, 9 of ganglioside GM2, and 6 of ganglioside GM3 differing for the long-chain base and fatty acid structures. Our results indicated that treatment with both 4-HPR and 4-oxo-4-HPR led to a marked increase in dihydroceramide species, while only 4-oxo-4-HPR led to a minor increase of ceramide species. Dihydroceramides generated in A2780 cells in response to 4-HPR or 4-oxo-4-HPR differed for their fatty acid content, suggesting that the two drugs differentially affect the early steps of sphingolipid synthesis. Dihydroceramides produced upon treatments with the drugs were further used for the synthesis of complex dihydrosphingolipids, whose levels dramatically increased in drug-treated cells.
Abstract
AstraZeneca obtained positive results from POLO, the first Phase III randomised, double-blind study that evaluated the efficacy of the PARP inhibitor (PARPi) olaparib, for maintenance ...monotherapy in patients with gBRCA mutated metastatic pancreatic cancer whose disease has not progressed on first-line platinum-based chemotherapy.
To support the application of olaparib for the proposed indication in these patients, a series of in vitro pharmacological tests were performed and will be here presented.
1. In a panel of pancreatic cancer cell lines, a striking correlation was observed between the responses to platinum and to olaparib. This indicates that it is predictable that olaparib will show efficacy in treatment of platinum-sensitive pancreatic cancer, as it has been previously demonstrated in other cancer indications.
2. Tests in isogenic cell line pairs and non-tumour cell lines revealed that platinum treatments lead to cytotoxic effects that are only ~10-20-fold more selective for BRCAm versus BRCAwt and non-tumour models; in contrast, olaparib is able to selectively kill BRCAm cells, with ~500-1000-fold sensitivity versus the BRCAwt cells.
3. In the panel of pancreatic cancer cells, the sensitivity to other DNA damaging agents used as standard of care, such as irinotecan (and, to some extent, 5FU, other key components of FOLFIRINOX as well as gemcitabine), correlated with sensitivity to olaparib. In contrast, no correlation was observed with paclitaxel (mitotic inhibitor), or with other agents.
4. The antiproliferative effects to olaparib treatments in the cancer cell panel were compared to that of other 5 clinical PARPi. High correlations were observed in the responses to all the PARPi except for veliparib, which inhibits the catalytic activity of PARP but does not act as PARP-trapper, confirming that the efficacy of PARPi in monotherapy depends on their ability to stabilise the interaction between PARP and the DNA.
Citation Format: Elisabetta Leo, Giuditta Illuzzi, Sabrina Bentouati. Olaparib antiproliferative effect in pancreatic cancer and correlation with the response to other anticancer agents 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 1829.
Abstract
Treatments with PARPi in cancers with impaired DNA repair mechanisms (i.e. with Homologous Recombination Repair Deficiency, HRD) causes unsupportable genomic instability resulting in tumor ...cell death. PARPi act via a dual mechanism: 1) they block PARylation activity that normally occurs in response to DNA damage; 2) they trap PARP onto DNA lesions creating potentially cytotoxic PARP-DNA complexes. The longer PARP is inhibited and trapped onto the DNA, the greater the cytotoxic effect of PARPi in preclinical models. However, differences in preclinical PARP trapping potency have not translated into increased clinical efficacy with standard clinical doses used. Therefore, the ability to assess PARP-chromatin trapping in cancer models is critical for understanding the MoA of existing clinical PARPi. To date, this has been achieved by low throughput assays in non-HRD models using high PARPi concentrations and the addition of exogenous DNA damage.
Here, we describe the development of a novel assay, a high throughput in situ cell extraction platform, where PARP-chromatin trapping is monitored by immunofluorescence and can be multiplexed with the analysis of other relevant biomarkers. Our novel assay has several advantages compared to conventional methods: the overall experimental process is simpler and less time consuming with results being quantitative and less error prone. Most importantly, the higher throughput allows a thorough evaluation of PARP1-chromatin trapping kinetics and their effects with dose-response in a time-dependent manner for clinical PARPi following both continuous treatment or after a wash out of drug.
Using this new assay, we tested the kinetics in isogenic cell line pairs (BRCA2-/- and WT) and followed in parallel the appearance of biomarkers of DNA damage (e.g. γ-H2AX). The data have revealed important elements of differentiation between the MoA of clinical PARPi including the important insight that the strongest PARP trapper (talazoparib) has clearly reduced cytotoxic specificity for HRD cells, which likely explains the significantly reduced dose used in the clinic compared to other PARPi. These along with other data presented highlight how this next generation PARP trapping assays can provide important insights into PARPi MoA.
Citation Format: Giuditta Illuzzi, Mark J O'Connor, Elisabetta Leo. A novel assay for PARP-DNA trapping provides insights into the mechanism of action (MoA) of clinical PARP inhibitors (PARPi) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2077.
Abstract
PARP inhibitors (PARPi) have demonstrated clinical efficacy in cancers with defects in the homologous recombination repair (HRR) pathway. Recent advances in the understanding of the PARPi ...mechanism of action via stabilization of the PARP-DNA complex (trapping) and of the biological roles of the different PARP family members led to the development of AZD5305, a next generation, potent and selective PARP1 inhibitor and trapper. In this work, we disclose for the first time the profiling of AZD5305 in cellular models and how its activity differentiates from other PARPi. AZD5305 was able to potently inhibits overall PARylation in A549 cell line with IC50 of 3 nM, and to target PARP1 ~500 times more potently than PARP2 in A549 PARP1-KO, confirming its selectivity in cells. With our novel, sensitive and high-throughput immunofluorescence-based assay, we tested the ability of PARPi to trap PARP1 or PARP2 onto the chromatin of damaged or undamaged cells; olaparib, talazoparib and veliparib will be presented as comparative examples of non-selective PARPi. Unlike all the current clinical PARPi, AZD5305 was able to selectively induce PARP1 trapping upon treatments with concentrations as low as nanomolar (nM), whereas PARP2-trapping was not observed at any of the tested conditions. These optimal profiles of AZD5305 translated into greatly improved targeted anticancer effects in vitro compared to all other PARPi. In BRCA mutant (BRCAm) cells, treatments with AZD5305 led to antiproliferative IC50 in the single-digit nM, whilst there was no- or minimal effect in the isogenic paired BRCA wild type (BRCAwt) cells after treatments with double-digit µM concentrations. We further explored the effects of AZD5305 in genetic backgrounds “beyond BRCAm” and confirmed its superior antiproliferative and selective activity, particularly in cells isogenic for relevant genes in the HRR pathway, like PALB2 and RAD51. Screening of AZD5305 and other PARPi in larger cell lines panels revealed a differential clustering of AZD5305-treated cells between the sensitive versus the insensitive ones; this indicates that AZD5305 is also a unique instrumental tool to explore and refine selective PARP1-related activities in cancer cells, and the effects of targeting them, in different genetic backgrounds. With this goal, we are currently performing CRISPR/Cas9 screens to identify genes that, upon downregulation, cause sensitization to AZD5305. Preliminary results of these screens will be presented here. In summary, the optimal PARP1 inhibition and trapping profile of AZD5305 in cells demonstrated that AZD5305 is a next generation PARPi, with great potentials to become the best in class and deliver a markedly improved therapeutic index in the clinic.
Citation Format: Giuditta Illuzzi, Lisa McWilliams, Kunzah Jamal, Alessandro Galbiati, Sabrina Bentouati, Daniel Griffiths, Elisabetta Leo. In vitro cellular profiling of AZD5305, novel PARP1-selective inhibitor and trapper abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1272.
We hypothesized that inhibition and trapping of PARP1 alone would be sufficient to achieve antitumor activity. In particular, we aimed to achieve selectivity over PARP2, which has been shown to play ...a role in the survival of hematopoietic/stem progenitor cells in animal models. We developed AZD5305 with the aim of achieving improved clinical efficacy and wider therapeutic window. This next-generation PARP inhibitor (PARPi) could provide a paradigm shift in clinical outcomes achieved by first-generation PARPi, particularly in combination.
AZD5305 was tested in vitro for PARylation inhibition, PARP-DNA trapping, and antiproliferative abilities. In vivo efficacy was determined in mouse xenograft and PDX models. The potential for hematologic toxicity was evaluated in rat models, as monotherapy and combination.
AZD5305 is a highly potent and selective inhibitor of PARP1 with 500-fold selectivity for PARP1 over PARP2. AZD5305 inhibits growth in cells with deficiencies in DNA repair, with minimal/no effects in other cells. Unlike first-generation PARPi, AZD5305 has minimal effects on hematologic parameters in a rat pre-clinical model at predicted clinically efficacious exposures. Animal models treated with AZD5305 at doses ≥0.1 mg/kg once daily achieved greater depth of tumor regression compared to olaparib 100 mg/kg once daily, and longer duration of response.
AZD5305 potently and selectively inhibits PARP1 resulting in excellent antiproliferative activity and unprecedented selectivity for DNA repair deficient versus proficient cells. These data confirm the hypothesis that targeting only PARP1 can retain the therapeutic benefit of nonselective PARPi, while reducing potential for hematotoxicity. AZD5305 is currently in phase I trials (NCT04644068).
Poly-ADP-ribose-polymerase (PARP) inhibitors have achieved regulatory approval in oncology for homologous recombination repair deficient tumors including BRCA mutation. However, some have failed in ...combination with first-line chemotherapies, usually due to overlapping hematological toxicities. Currently approved PARP inhibitors lack selectivity for PARP1 over PARP2 and some other 16 PARP family members, and we hypothesized that this could contribute to toxicity. Recent literature has demonstrated that PARP1 inhibition and PARP1-DNA trapping are key for driving efficacy in a BRCA mutant background. Herein, we describe the structure- and property-based design of
(AZD5305), a potent and selective PARP1 inhibitor and PARP1-DNA trapper with excellent in vivo efficacy in a BRCA mutant HBCx-17 PDX model. Compound
is highly selective for PARP1 over other PARP family members, with good secondary pharmacology and physicochemical properties and excellent pharmacokinetics in preclinical species, with reduced effects on human bone marrow progenitor cells in vitro.
We evaluated the properties and activity of AZD9574, a blood-brain barrier (BBB) penetrant selective inhibitor of PARP1, and assessed its efficacy and safety alone and in combination with ...temozolomide (TMZ) in preclinical models.
AZD9574 was interrogated in vitro for selectivity, PARylation inhibition, PARP-DNA trapping, the ability to cross the BBB, and the potential to inhibit cancer cell proliferation. In vivo efficacy was determined using subcutaneous as well as intracranial mouse xenograft models. Mouse, rat, and monkey were used to assess AZD9574 BBB penetration and rat models were used to evaluate potential hematotoxicity for AZD9574 monotherapy and the TMZ combination.
AZD9574 demonstrated PARP1-selectivity in fluorescence anisotropy, PARylation, and PARP-DNA trapping assays and in vivo experiments demonstrated BBB penetration. AZD9574 showed potent single agent efficacy in preclinical models with homologous recombination repair deficiency in vitro and in vivo. In an O6-methylguanine-DNA methyltransferase (MGMT)-methylated orthotopic glioma model, AZD9574 in combination with TMZ was superior in extending the survival of tumor-bearing mice compared with TMZ alone.
The combination of three key features-PARP1 selectivity, PARP1 trapping profile, and high central nervous system penetration in a single molecule-supports the development of AZD9574 as the best-in-class PARP inhibitor for the treatment of primary and secondary brain tumors. As documented by in vitro and in vivo studies, AZD9574 shows robust anticancer efficacy as a single agent as well as in combination with TMZ. AZD9574 is currently in a phase I trial (NCT05417594). See related commentary by Lynce and Lin, p. 1217.
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