Charlotte Knights - Academia.edu (original) (raw)
Papers by Charlotte Knights
ACS Chemical Biology, Oct 31, 2016
Deubiquitinating enzymes play an important role in a plethora of therapeutically relevant process... more Deubiquitinating enzymes play an important role in a plethora of therapeutically relevant processes and are emerging as pioneering drug targets. Herein, we present a novel probe, Ubiquitin Specific Protease (USP) inhibitor, alongside an alkyne-tagged activity-based probe analogue. Activity-based proteome profiling identified 12 USPs, including USP4, USP16, and USP33, as inhibitor targets using submicromolar probe concentrations. This represents the first intact cell activitybased profiling of deubiquitinating enzymes. Further analysis demonstrated functional inhibition of USP33 and identified a synergistic relationship in combination with ATR inhibition, consistent with USP4 inhibition.
Background: Early results from Phase II trials have shown that treatment with the PARP inhibitor ... more Background: Early results from Phase II trials have shown that treatment with the PARP inhibitor olaparib (AZD2281, KU-0059436) can induce tumour specific synthetic lethality in patients with BRCA-mutated breast and ovarian cancer, with little effect on normal tissues. Pre-clinical studies have indicated that sensitivity can also result from other perturbations affecting homologous recombination (HR), suggesting broader patient populations may benefit from this drug. We aimed to generate pre-clinical hypotheses as to the key biological mechanisms regulating response to this drug; and identify associated biomarkers by which responding patient subsets could be stratified. Methods: A panel of 95 cell lines representing breast, ovarian, colorectal, lung, head & neck and pancreatic cancers was tested for sensitivity to olaparib using 2D-clonogenic survival assays. Baseline (untreated) gene expression profiles (Affymetrix U133A Plus2) were determined for each cell line, alongside protein expression and mutational status of core HR genes. Dynamic pathway gene expression signatures were collated from the literature, and additional signatures generated using RNAi against core HR components. Statistical and bioinformatic approaches were then applied to prioritise correlated networks of genes displaying consistent pathway overlay and predictive of response in cell line subsets. Results: 30 cell lines were highly sensitive to olaparib treatment (IC50 4 µM). Deleterious mutations in BRCA1 or BRCA2 genes were associated with only a small subset of highly sensitive cell lines, highlighting the presence of other factors able to modulate olaparib responsiveness. Transcriptome analysis revealed DNA repair and proliferation associated genes to be most consistently correlated with olaparib response. Based on these results, a candidate predictive baseline gene expression profile was established. Conclusions: By profiling a large panel of cell lines we have determined that factors in addition to BRCA mutation can be linked with olaparib sensitivity. A list of candidate gene transcripts predictive of in vitro sensitivity to olaparib were identified which may have utility as predictive biomarkers in the clinic. Although primarily linked to mechanisms expected to influence response (DNA repair, cell-cycle checkpoints and proliferation), novel roles were suggested for other pathways such as Aurora A kinase signalling. Analyses also highlighted that some classical HR pathway components may prove to be more robust mRNA markers of pathway activity than others. Studies are already underway to determine whether this baseline transcript tumour profile can be correlated with patient responses to olaparib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3497.
Cancer Research, 2009
Abstract #1038 Background: Deficiencies in DNA repair mechanisms have been associated with breast... more Abstract #1038 Background: Deficiencies in DNA repair mechanisms have been associated with breast cancer. AZD2281, a potent, oral, PARP inhibitor has been shown to have clinical activity in patients with BRCA mutant breast cancers. Laboratory studies have suggested that non-BRCA mutant breast cancers may also be sensitive to PARP inhibition in tumors as a consequence of deficiencies in other homologous recombination (HR) repair components. Using a large panel of human breast cancer cell lines we tested the hypotheses that (1) there may be a subset of non-BRCA mutant breast cancers that are sensitive to single-agent AZD2281 and (2) AZD2281 would potentiate the cytotoxic effects of the DNA damaging agent cisplatin. Methods: 43 human cell lines representing known molecular subgroups of breast cancer (i.e. ER+, HER2 amplified, “triple-negative”), and 3 immortalized breast lines were treated in duplicate in adherent plates with AZD2281 using two-fold dilutions over 6 concentrations for 6 days. Dose response curves were generated using a cell count assay to calculate the IC50 of AZD2281. In addition, a subset of cell lines that grow under anchorage independent conditions were grown in triplicate in the presence and absence of 1 µM AZD2281 in soft agar for at least 3 weeks and growth inhibition was calculated as per cent of untreated control. Cell lines (both sensitive and resistant to single agent AZD2281) were also evaluated in combination with cisplatin in a cell count assay to assess the interaction between the two agents. Results: The majority of breast cancer cell lines evaluated in the short term 2-D growth assay did not show significant growth inhibition (IC50 < 1 µM) following AZD2281 treatment, including a known BRCA mutant cell line, suggesting this assay may not be ideal for determining sensitivity to AZD2281. However, in the longer term anchorage independent clonogenic assay, approximately half of the cell lines evaluated demonstrated an IC50 < 1 µM. Of note, the majority of the cell lines representing a “triple-negative” phenotype appeared sensitive to AZD2281 in this assay. In addition, pre-treatment with AZD2281 prior to cisplatin, potentiated the growth inhibition seen with cisplatin in both AZD2281 sensitive and resistant cell lines. Additional studies evaluating predictive markers other than BRCA status are ongoing. Conclusion: The PARP inhibitor AZD2281 has significant pre-clinical activity in human breast cancer cell lines. In a clonogenic assay, cell lines representing the “triple negative” subtype were especially sensitive to AZD2281 supporting clinical development in this population, regardless of BRCA status. In addition, these pre-clinical data support the hypothesis that PARP inhibition may potentiate the effects of chemotherapy induced DNA damage and provide further rationale for clinical development. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1038.
Ejc Supplements, Oct 1, 2008
Background: The oral PARP inhibitor olaparib (AZD2281, KU-0059436) is in Phase II clinical trials... more Background: The oral PARP inhibitor olaparib (AZD2281, KU-0059436) is in Phase II clinical trials in patients with BRCA-mutated breast and ovarian cancer. Early results in these trials have provided positive clinical evidence for the targeted therapy concept of synthetic lethality using PARP inhibitors in molecularly-defined tumors with little effect on normal tissues. However, olaparib offers the potential to treat a broader range of patient tumors and pre-clinical studies have indicated that homologous recombination deficiency (HRD) due to loss of other DNA repair genes or through impaired cell-cycle checkpoints can also lead to sensitivity to PARP inhibitors. The ability to stratify patients whose tumors will respond to treatment will be critical to realise the potential of this targeted therapy approach. In order to identify predictive biomarkers of olaparib response and to provide further insights into mechanisms of olaparib sensitivity a study was undertaken using a panel of cell lines correlating response with molecular profiles. Methods: A cross tumor-type panel of 95 cell lines (KU-95 panel) representing breast, ovarian, colorectal, lung, head & neck and pancreatic cancers was tested for sensitivity to olaparib using 2D-clonogenic survival assays. Baseline (untreated) gene expression profiles using Affymetrix genome-wide U133A 2.0 arrays together with protein expression and mutational status of genes, such as BRCA1, known to modify of olaparib response were determined for each cell line. Novel statistical, bioinformatics and pathway analysis approaches were used to identify genes that are predictive for olaparib response. Results: Across the KU-95 cell-line panel, 30 cell lines were highly sensitive to olaparib (IC50 <1 M) treatment and 36 were resistant (IC50 >4 M). Deleterious mutations in BRCA1 or BRCA2 genes were associated with only a small subset of highly sensitive cell lines indicating the presence of other factors able to modulate olaparib responsiveness. Directed analysis of several other DNA repair genes showed correlations with response consistent with previous data. Genome-wide transcriptome and pathway mapping analysis revealed DNA repair and proliferation associated genes to be most consistently correlated with olaparib sensitivity. Based on results from all these approaches a candidate baseline gene expression profile predictive of olaparib response was established. Conclusions: By profiling a large panel of cell lines we have determined that factors in addition to BRCA mutation can be linked with olaparib sensitivity. A list of candidate gene transcripts was identified that predicts sensitivity to olaparib across a broad range of tumor types in vitro and may have utility as predictive biomarkers in the clinic. Studies are already underway to determine whether this baseline transcript tumor profile can be correlated with patient responses to olaparib. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C119.
Background Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)is the member of a family of... more Background Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)is the member of a family of enzymes that catalyze the addition of ADP-ribose units to proteins that mediate DNA repair pathways. Olaparib (KU-0059436; AZD2281) is a potent oral inhibitor of PARP currently in clinical development, which has selective antitumor activity in cancers, associated with BRCA1 and BRCA2 mutations. Gastric cancer is the most common cancer and the second leading cause of cancer deaths in Korea. New therapies including molecular targeting agents are eagerly awaited, and treatment strategies to overcome chemoresistance are also needed in the treatment of gastric cancer. Materials and Methods We studied the growth inhibitory effects of Olaparib on gastric cancer (GC) cells using clonogenic survival assays and studied the effect of combination with chemotherapeutic agents using MTS assays. Cell cycle analysis and molecular changes induced by olaparib were also performed. Results Olaparib showed marked growth inhibitory activity against the SNU 601 cell line, with IC50 value of 0.015 μM. In addition, 5 out of 11 GC cells (45%) were sensitive to olaparib with IC50s ≤ 2μM. Olaprib induced G2/M cell cycle arrest and apoptosis in sensitive GC cells. We identified synergistic growth inhibitory effects of Olaparib in combination with clinically relevant cytotoxic agents (5-FU, cisplatin, and oxaliplatin). Furthermore, Olaparib-induced apoptosis was associated with PARP cleavage and caspase-3 activation. Conclusion: Olaparib showed growth inhibitory activity against GC cells as a single agent and acted synergistically with cytotoxic agents. These results provide a rationale for the future clinical trials of olaparib combined with cytotoxic drugs in the treatment of gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1775.
Supplementary materials and methods. Suppl Table 1. MMS combination PF50 cell line assays. Concen... more Supplementary materials and methods. Suppl Table 1. MMS combination PF50 cell line assays. Concentration-dependent potentiation of MMS (PF50 ratios). Suppl Table 2. Permeability of AZD2461 and olaparib were assessed. Suppl Table 3. Activity of olaparib and AZD2461 alone or in combination with the P-gp inhibitor verapamil in the colorectal cancer cell line HCT-15, which expresses high levels of P-gp. Suppl Fig. 1. Immunofluorescence for poly(ADP-ribose) in human A549 cells. Suppl Fig. 2. Analysis of both in vivo pharmacokinetics (PK) and pharmacodynamics (PD) in tumors taken from SW620 (human colon carcinoma) xenograft-bearing mice. Suppl Fig. 3. Colony formation (clonogenic) assays in the intrinsically high P-gp-expressing MRE11-deficient HCT-15 human colorectal cell line. Suppl. Fig. 4. Combination efficacy studies. Suppl Fig. 5. Relative body weight changes in mice following treatment with 10 mg/kg AZD2461 and olaparib in combination with temozolomide. Suppl. Fig. 6. A serial dilu...
Molecular Cancer Therapeutics, Dec 1, 2009
Ataxia telangiectasia mutated (ATM) is a protein kinase that regulates cell-cycle checkpoints, DN... more Ataxia telangiectasia mutated (ATM) is a protein kinase that regulates cell-cycle checkpoints, DNA repair and recombination (1). It has been shown that ATM interacts with and regulates NBS1 and BRCA complex which is essential for the homologous recombination (HR) repair in response to the DNA double-strand break (DSB) damage (2). These observations suggest that tumor cells with defective ATM expression or activity subsequently leading to HR deficiency are likely more sensitive to the targeted therapeutic/chemotherapeutic agents that cause the accumulation of DNA DSBs during replication. To test this, we studied the growth inhibitory effects of olaparib (AZD2281; KU-0059436) and SN-38 (the active metabolite of Irinotecan) on gastric cancer (GC) cells with reduced ATM expression. Olaparib is a potent oral inhibitor of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP), which has selective antitumor activity in cancers associated with BRCA1 and BRCA2 mutations (3). Our study here showed that 6 out of 7 GC cell lines with low or no expression of ATM are sensitive to olaparib inhibition with IC50s ≤ 1uM as measured by clonogenic survival assays. In contrast, the majority of other GC cell lines positive for ATM expression (11 out of 13) are less sensitive (1uM < IC50 ≤ 1.5uM) or resistant (IC50 > 1.5uM) to olaparib with only 2 of 13 lines sensitive. Similar correlation was also observed in these GC cell lines between reduced ATM expression and sensitivity to SN-38 treatment. In addition, combination of olaparib and SN-38 had a synergistic and selective inhibitory effect on cell growth of GC cells with low/no expression of ATM. These data suggest that loss of ATM in GC cell can sensitize their cellular response to olaparib or/and SN-38. Next, we asked whether this finding has clinical relevance in GC therapy. The tumor and tumor adjacent tissue specimens from more than 100 patients with gastric cancer were analyzed for ATM expression by immunohistochemistry. 22% (24/111) of the gastric tumor specimens were found stained ATM negative while the negative rate in the adjacent tissues was only 4% (1/26). These results suggested that loss of ATM expression is frequently associated with gastric tumorigenesis. Loss of ATM function may serve as an important tumor biomarker in GC and a PARP inhibitor as monotherapy or in combination with chemotherapy provides a promising therapeutic in this disease segment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B42.
The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first... more The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, which suggests that AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared with rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-dam...
Cancer research, Oct 22, 2016
The PARP inhibitor AZD2461 was developed as a next generation agent following olaparib, the first... more The PARP inhibitor AZD2461 was developed as a next generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, suggesting AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared to rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-damaging agent...
Cancer Research, 2015
Ubiquitin proteasome pathways are emerging as a growing source for novel anti-cancer therapeutics... more Ubiquitin proteasome pathways are emerging as a growing source for novel anti-cancer therapeutics. In this respect, by depleting a number of deubiquitylating enzymes (DUBs) through a combination of synthetic lethality screens and isogenic cell line models, MISSION has identified several novel DUBs essential for a range of different tumor types, including platinum-resistant ovarian cancers, DNA damage response (DDR) pathway deficient tumors (e.g. ATM, ATR or BRCA2 defective) and haematological tumors such as multiple myeloma. Illustrating the diversity of DUBs as oncology targets we identified USP11 and UCHL1 using our target validation platform. USP11 was identified as essential for proliferation of platinum-resistant cells but not platinum-sensitive tumor cells in an isogenic model derived from patient tumors before and after development of resistance. In addition, USP11 depletion displayed synthetic lethality in isogenic models of BRCA2 or ATR loss, supporting USP119s previously identified association with homologous recombination proteins as well as the importance such processes in platinum resistance mechanisms. UCHL1 on the other hand is a prototypical oncogene target that selectively drives proliferation in many tumor types, such as multiple myeloma and lung cancer. UCHL1 depletion leads to selective killing of a number of tumors whose proliferation is driven by UCHL1. Validation of targets like USP11 or UCHL1 for the selective treatment of cancer bearing genetic deficiencies or resistant to standard-of-care supports the rationale of developing DUB inhibitors for cancers with unmet medical need. A broad drug discovery platform combining unique biochemical, cellular, biophysical and structural assays was designed by MISSION to identify and optimise potent and selective DUB inhibitors. Early selective DUB hits, developed for USP11 and UCHL1, are successfully recapitulating target validation biology, including synthetic lethality in matched isogenic backgrounds. Lead optimisation of chemical series has recently been translated into compounds with adequate properties for in vivo proof-of-concept studies. The challenges and advances in demonstrating in vivo DUB target engagement will be discussed. MISSION9s integrated drug discovery methodologies exemplify the tractability of DUBs to pharmacologic intervention and the potential scope for DUB inhibitors in a number of cancer types, including those characterised by DDR deficiencies. Citation Format: Xavier Jacq, Niall MB Martin, Lisa Smith, Jeanine Harrigan, Charlotte Knights, Helen Robinson, Yaara Ofir-Rosenfeld, Aaron Cranston, Mark I. Kemp, Stephen P. Jackson. Discovery of highly selective DUB inhibitors with in vivo pre-clinical anti-tumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1728. doi:10.1158/1538-7445.AM2015-1728
Journal of Medicinal Chemistry, 2008
Poly(ADP-ribose) polymerase activation is an immediate cellular response to metabolic-, chemical-... more Poly(ADP-ribose) polymerase activation is an immediate cellular response to metabolic-, chemical-, or ionizing radiation-induced DNA damage and represents a new target for cancer therapy. In this article, we disclose a novel series of substituted 4-benzyl-2 H-phthalazin-1-ones that possess high inhibitory enzyme and cellular potency for both PARP-1 and PARP-2. Optimized compounds from the series also demonstrate good pharmacokinetic profiles, oral bioavailability, and activity in vivo in an SW620 colorectal cancer xenograft model. 4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2 H-phthalazin-1-one (KU-0059436, AZD2281) 47 is a single digit nanomolar inhibitor of both PARP-1 and PARP-2 that shows standalone activity against BRCA1-deficient breast cancer cell lines. Compound 47 is currently undergoing clinical development for the treatment of BRCA1- and BRCA2-defective cancers.
Molecular Cancer Therapeutics, 2009
Background: The oral PARP inhibitor olaparib (AZD2281, KU‐0059436) has shown anti‐tumor activity ... more Background: The oral PARP inhibitor olaparib (AZD2281, KU‐0059436) has shown anti‐tumor activity in patients with hereditary BRCA mutations and provides positive evidence for synthetic lethal approaches for the treatment of cancers with DNA repair defects. The BRCA genes are regulators of homologous recombination (HR) DNA repair pathway but deficiency in other mediators of this pathway, such as the MRE11‐NBS1‐Rad50 (MRN) complex have also been demonstrated to be synthetically lethal with PARP inhibition in pre‐clinical models. Cells deficient in HR DNA repair or signalling pathways are potentially sensitive to PARP inhibition and have been termed homologous recombination deficient (HRD). Examples of HRD include inactivating MRE11 mutations which have been linked to mismatch repair (MMR) deficient, microsatellite instable (MSI) colorectal and endometrial cancers. Previous studies have indicated a high frequency (up to 87% of MSI colorectal cancers) of −1/−2 frameshifts within the poly(T)11 microsatellite repeat sequence and a corresponding reduction in expression. We have therefore undertaken a pre‐clinical study to determine whether MRE11‐deficient colorectal cancer (CRC) respond to olaparib treatment. Methods: CRC cell line panels with known MSI status as well as isogenic matched cell lines models of MRE11 and MMR‐deficiency were assessed for their response to olaparib by both MTS viability and clonogenic survival assays. Quantitative RT‐PCR mRNA and protein expression analysis of MRE11 and DNA damage response genes were obtained for each cell line and HRD status determined. Expression of Pgp (ABCB1) multi‐drug transporter, a pre‐clinical resistance factor for olaparib and whose over‐expression is associated with CRC, was also determined. Results: MTS viability assays showed a limited range of sensitivities between CRC cell lines and only modest correlation with molecular profiles. Analysis using sensitive long term clonogenic survival assays showed a wider range of sensitivities and revealed that HRD (MRE11 mutant) and MSI CRC cells were sensitive to olaparib treatment while microsatellite stable (MSS) and non‐HRD (MRE11 wild‐type) cells remained insensitive. Using isogenic cell line models we confirmed that loss of MRE11 expression can confer sensitivity to olaparib whereas MMR‐deficiency did not. CRC cells which were HRD but overexpressed Pgp were more resistant to olaparib but could be re‐sensitized to treatment by co‐administration of the Pgp inhibitors or through the use of alternative non‐Pgp substrate PARP inhibitors. Conclusions: Here we demonstrate that CRC cell lines with HRD due to MRE11 mutation and loss of protein expression are highly sensitive to the PARP inhibitor olaparib. These data provide evidence that patients with MSI and MRE11‐deficient colorectal tumors could be suitable for treatment with olaparib. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A114.
Olaparib (AZD2281; KU-0059436) is a potent and well tolerated oral inhibitor of PARP. Olaparib of... more Olaparib (AZD2281; KU-0059436) is a potent and well tolerated oral inhibitor of PARP. Olaparib offers a potential synthetic lethal strategy for treatment of cancers with homologous recombination deficiency (HRD). A first-in-human Phase I dose escalation trial of olaparib was conducted and pharmacokinetic (PK) and pharmacodynamic (PD) studies were performed (Fong et al ., 2008). The trial consisted of patients with enrichment for those with hereditary BRCA1 and BRCA2 germline mutations. Dose levels rose from 10 mg daily given for 2 out of 3 weeks to continuous bid dosing at 600mg. PK data indicated rapid absorption and elimination. PARP inhibition PD studies were conducted using immunoblotting and an electrochemiluminescent immunoassay which utilises a functional ex vivo activation assay for poly(ADP)-ribose (PAR) formation (Menear et al ., 2008) in surrogate peripheral blood mononuclear cell (PBMC) and tumour tissue cell lysates. Substantial PARP inhibition of over 90% could be obse...
Cancer Research, 2009
AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO The oral PARP inhibitor olaparib (AZD2281, KU-0... more AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO The oral PARP inhibitor olaparib (AZD2281, KU-0059436) has been shown to have single agent clinical activity in tumours from patients with hereditary BRCA mutations (Fong et al ., 2008). The BRCA genes play key roles in homologous recombination (HR) repair of DNA double strand breaks (DSBs) and BRCA dysfunction is associated with an increased incidence of breast and ovarian cancers. In addition to BRCA, RNAi-mediated gene expression knockdown of other components of the HR DNA damage response pathway such as ATM, ATR and CHK2 has been shown to cause sensitivity to PARP inhibition pre-clinically (McCabe et al ., 2006). These data suggest broader clinical potential of PARP inhibitors in other tumours that may be homologous recombination deficient (HRD). Triple negative (TN) breast cancers represent a distinct clinical subgroup and are defined as ER, PR and HER-2 receptor negative. TN tumours are associated with increased aggressiveness ...
Article (Accepted Version) http://sro.sussex.ac.uk Rulten, Stuart and Caldecott, Keith (2016) The... more Article (Accepted Version) http://sro.sussex.ac.uk Rulten, Stuart and Caldecott, Keith (2016) The PARP inhibitor AZD2461 provides insights into the role of PARP3 inhibition for both synthetic lethality and tolerability with chemotherapy in preclinical models. Cancer Research, 76 (20).
Molecular Cancer Therapeutics, 2009
Background: The oral PARP inhibitor olaparib (AZD2281, KU‐0059436) is in Phase II clinical trials... more Background: The oral PARP inhibitor olaparib (AZD2281, KU‐0059436) is in Phase II clinical trials in patients with BRCA‐mutated breast and ovarian cancer. Early results in these trials have provided positive clinical evidence for the targeted therapy concept of synthetic lethality using PARP inhibitors in molecularly‐defined tumors with little effect on normal tissues. However, olaparib offers the potential to treat a broader range of patient tumors and pre‐clinical studies have indicated that homologous recombination deficiency (HRD) due to loss of other DNA repair genes or through impaired cell‐cycle checkpoints can also lead to sensitivity to PARP inhibitors. The ability to stratify patients whose tumors will respond to treatment will be critical to realise the potential of this targeted therapy approach. In order to identify predictive biomarkers of olaparib response and to provide further insights into mechanisms of olaparib sensitivity a study was undertaken using a panel of cell lines correlating response with molecular profiles. Methods: A cross tumor‐type panel of 95 cell lines (KU‐95 panel) representing breast, ovarian, colorectal, lung, head & neck and pancreatic cancers was tested for sensitivity to olaparib using 2D‐clonogenic survival assays. Baseline (untreated) gene expression profiles using Affymetrix genome‐wide U133A 2.0 arrays together with protein expression and mutational status of genes, such as BRCA1, known to modify of olaparib response were determined for each cell line. Novel statistical, bioinformatics and pathway analysis approaches were used to identify genes that are predictive for olaparib response. Results: Across the KU‐95 cell‐line panel, 30 cell lines were highly sensitive to olaparib (IC50 4 M). Deleterious mutations in BRCA1 or BRCA2 genes were associated with only a small subset of highly sensitive cell lines indicating the presence of other factors able to modulate olaparib responsiveness. Directed analysis of several other DNA repair genes showed correlations with response consistent with previous data. Genome‐wide transcriptome and pathway mapping analysis revealed DNA repair and proliferation associated genes to be most consistently correlated with olaparib sensitivity. Based on results from all these approaches a candidate baseline gene expression profile predictive of olaparib response was established. Conclusions: By profiling a large panel of cell lines we have determined that factors in addition to BRCA mutation can be linked with olaparib sensitivity. A list of candidate gene transcripts was identified that predicts sensitivity to olaparib across a broad range of tumor types in vitro and may have utility as predictive biomarkers in the clinic. Studies are already underway to determine whether this baseline transcript tumor profile can be correlated with patient responses to olaparib. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C119.
Cancer Research, 2010
Background Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)is the member of a family of... more Background Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)is the member of a family of enzymes that catalyze the addition of ADP-ribose units to proteins that mediate DNA repair pathways. Olaparib (KU-0059436; AZD2281) is a potent oral inhibitor of PARP currently in clinical development, which has selective antitumor activity in cancers, associated with BRCA1 and BRCA2 mutations. Gastric cancer is the most common cancer and the second leading cause of cancer deaths in Korea. New therapies including molecular targeting agents are eagerly awaited, and treatment strategies to overcome chemoresistance are also needed in the treatment of gastric cancer. Materials and Methods We studied the growth inhibitory effects of Olaparib on gastric cancer (GC) cells using clonogenic survival assays and studied the effect of combination with chemotherapeutic agents using MTS assays. Cell cycle analysis and molecular changes induced by olaparib were also performed. Results Olaparib showed marked growth inhibitory activity against the SNU 601 cell line, with IC50 value of 0.015 μM. In addition, 5 out of 11 GC cells (45%) were sensitive to olaparib with IC50s ≤ 2μM. Olaprib induced G2/M cell cycle arrest and apoptosis in sensitive GC cells. We identified synergistic growth inhibitory effects of Olaparib in combination with clinically relevant cytotoxic agents (5-FU, cisplatin, and oxaliplatin). Furthermore, Olaparib-induced apoptosis was associated with PARP cleavage and caspase-3 activation. Conclusion: Olaparib showed growth inhibitory activity against GC cells as a single agent and acted synergistically with cytotoxic agents. These results provide a rationale for the future clinical trials of olaparib combined with cytotoxic drugs in the treatment of gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1775.
ACS Chemical Biology, Oct 31, 2016
Deubiquitinating enzymes play an important role in a plethora of therapeutically relevant process... more Deubiquitinating enzymes play an important role in a plethora of therapeutically relevant processes and are emerging as pioneering drug targets. Herein, we present a novel probe, Ubiquitin Specific Protease (USP) inhibitor, alongside an alkyne-tagged activity-based probe analogue. Activity-based proteome profiling identified 12 USPs, including USP4, USP16, and USP33, as inhibitor targets using submicromolar probe concentrations. This represents the first intact cell activitybased profiling of deubiquitinating enzymes. Further analysis demonstrated functional inhibition of USP33 and identified a synergistic relationship in combination with ATR inhibition, consistent with USP4 inhibition.
Background: Early results from Phase II trials have shown that treatment with the PARP inhibitor ... more Background: Early results from Phase II trials have shown that treatment with the PARP inhibitor olaparib (AZD2281, KU-0059436) can induce tumour specific synthetic lethality in patients with BRCA-mutated breast and ovarian cancer, with little effect on normal tissues. Pre-clinical studies have indicated that sensitivity can also result from other perturbations affecting homologous recombination (HR), suggesting broader patient populations may benefit from this drug. We aimed to generate pre-clinical hypotheses as to the key biological mechanisms regulating response to this drug; and identify associated biomarkers by which responding patient subsets could be stratified. Methods: A panel of 95 cell lines representing breast, ovarian, colorectal, lung, head & neck and pancreatic cancers was tested for sensitivity to olaparib using 2D-clonogenic survival assays. Baseline (untreated) gene expression profiles (Affymetrix U133A Plus2) were determined for each cell line, alongside protein expression and mutational status of core HR genes. Dynamic pathway gene expression signatures were collated from the literature, and additional signatures generated using RNAi against core HR components. Statistical and bioinformatic approaches were then applied to prioritise correlated networks of genes displaying consistent pathway overlay and predictive of response in cell line subsets. Results: 30 cell lines were highly sensitive to olaparib treatment (IC50 4 µM). Deleterious mutations in BRCA1 or BRCA2 genes were associated with only a small subset of highly sensitive cell lines, highlighting the presence of other factors able to modulate olaparib responsiveness. Transcriptome analysis revealed DNA repair and proliferation associated genes to be most consistently correlated with olaparib response. Based on these results, a candidate predictive baseline gene expression profile was established. Conclusions: By profiling a large panel of cell lines we have determined that factors in addition to BRCA mutation can be linked with olaparib sensitivity. A list of candidate gene transcripts predictive of in vitro sensitivity to olaparib were identified which may have utility as predictive biomarkers in the clinic. Although primarily linked to mechanisms expected to influence response (DNA repair, cell-cycle checkpoints and proliferation), novel roles were suggested for other pathways such as Aurora A kinase signalling. Analyses also highlighted that some classical HR pathway components may prove to be more robust mRNA markers of pathway activity than others. Studies are already underway to determine whether this baseline transcript tumour profile can be correlated with patient responses to olaparib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3497.
Cancer Research, 2009
Abstract #1038 Background: Deficiencies in DNA repair mechanisms have been associated with breast... more Abstract #1038 Background: Deficiencies in DNA repair mechanisms have been associated with breast cancer. AZD2281, a potent, oral, PARP inhibitor has been shown to have clinical activity in patients with BRCA mutant breast cancers. Laboratory studies have suggested that non-BRCA mutant breast cancers may also be sensitive to PARP inhibition in tumors as a consequence of deficiencies in other homologous recombination (HR) repair components. Using a large panel of human breast cancer cell lines we tested the hypotheses that (1) there may be a subset of non-BRCA mutant breast cancers that are sensitive to single-agent AZD2281 and (2) AZD2281 would potentiate the cytotoxic effects of the DNA damaging agent cisplatin. Methods: 43 human cell lines representing known molecular subgroups of breast cancer (i.e. ER+, HER2 amplified, “triple-negative”), and 3 immortalized breast lines were treated in duplicate in adherent plates with AZD2281 using two-fold dilutions over 6 concentrations for 6 days. Dose response curves were generated using a cell count assay to calculate the IC50 of AZD2281. In addition, a subset of cell lines that grow under anchorage independent conditions were grown in triplicate in the presence and absence of 1 µM AZD2281 in soft agar for at least 3 weeks and growth inhibition was calculated as per cent of untreated control. Cell lines (both sensitive and resistant to single agent AZD2281) were also evaluated in combination with cisplatin in a cell count assay to assess the interaction between the two agents. Results: The majority of breast cancer cell lines evaluated in the short term 2-D growth assay did not show significant growth inhibition (IC50 < 1 µM) following AZD2281 treatment, including a known BRCA mutant cell line, suggesting this assay may not be ideal for determining sensitivity to AZD2281. However, in the longer term anchorage independent clonogenic assay, approximately half of the cell lines evaluated demonstrated an IC50 < 1 µM. Of note, the majority of the cell lines representing a “triple-negative” phenotype appeared sensitive to AZD2281 in this assay. In addition, pre-treatment with AZD2281 prior to cisplatin, potentiated the growth inhibition seen with cisplatin in both AZD2281 sensitive and resistant cell lines. Additional studies evaluating predictive markers other than BRCA status are ongoing. Conclusion: The PARP inhibitor AZD2281 has significant pre-clinical activity in human breast cancer cell lines. In a clonogenic assay, cell lines representing the “triple negative” subtype were especially sensitive to AZD2281 supporting clinical development in this population, regardless of BRCA status. In addition, these pre-clinical data support the hypothesis that PARP inhibition may potentiate the effects of chemotherapy induced DNA damage and provide further rationale for clinical development. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1038.
Ejc Supplements, Oct 1, 2008
Background: The oral PARP inhibitor olaparib (AZD2281, KU-0059436) is in Phase II clinical trials... more Background: The oral PARP inhibitor olaparib (AZD2281, KU-0059436) is in Phase II clinical trials in patients with BRCA-mutated breast and ovarian cancer. Early results in these trials have provided positive clinical evidence for the targeted therapy concept of synthetic lethality using PARP inhibitors in molecularly-defined tumors with little effect on normal tissues. However, olaparib offers the potential to treat a broader range of patient tumors and pre-clinical studies have indicated that homologous recombination deficiency (HRD) due to loss of other DNA repair genes or through impaired cell-cycle checkpoints can also lead to sensitivity to PARP inhibitors. The ability to stratify patients whose tumors will respond to treatment will be critical to realise the potential of this targeted therapy approach. In order to identify predictive biomarkers of olaparib response and to provide further insights into mechanisms of olaparib sensitivity a study was undertaken using a panel of cell lines correlating response with molecular profiles. Methods: A cross tumor-type panel of 95 cell lines (KU-95 panel) representing breast, ovarian, colorectal, lung, head & neck and pancreatic cancers was tested for sensitivity to olaparib using 2D-clonogenic survival assays. Baseline (untreated) gene expression profiles using Affymetrix genome-wide U133A 2.0 arrays together with protein expression and mutational status of genes, such as BRCA1, known to modify of olaparib response were determined for each cell line. Novel statistical, bioinformatics and pathway analysis approaches were used to identify genes that are predictive for olaparib response. Results: Across the KU-95 cell-line panel, 30 cell lines were highly sensitive to olaparib (IC50 <1 M) treatment and 36 were resistant (IC50 >4 M). Deleterious mutations in BRCA1 or BRCA2 genes were associated with only a small subset of highly sensitive cell lines indicating the presence of other factors able to modulate olaparib responsiveness. Directed analysis of several other DNA repair genes showed correlations with response consistent with previous data. Genome-wide transcriptome and pathway mapping analysis revealed DNA repair and proliferation associated genes to be most consistently correlated with olaparib sensitivity. Based on results from all these approaches a candidate baseline gene expression profile predictive of olaparib response was established. Conclusions: By profiling a large panel of cell lines we have determined that factors in addition to BRCA mutation can be linked with olaparib sensitivity. A list of candidate gene transcripts was identified that predicts sensitivity to olaparib across a broad range of tumor types in vitro and may have utility as predictive biomarkers in the clinic. Studies are already underway to determine whether this baseline transcript tumor profile can be correlated with patient responses to olaparib. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C119.
Background Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)is the member of a family of... more Background Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)is the member of a family of enzymes that catalyze the addition of ADP-ribose units to proteins that mediate DNA repair pathways. Olaparib (KU-0059436; AZD2281) is a potent oral inhibitor of PARP currently in clinical development, which has selective antitumor activity in cancers, associated with BRCA1 and BRCA2 mutations. Gastric cancer is the most common cancer and the second leading cause of cancer deaths in Korea. New therapies including molecular targeting agents are eagerly awaited, and treatment strategies to overcome chemoresistance are also needed in the treatment of gastric cancer. Materials and Methods We studied the growth inhibitory effects of Olaparib on gastric cancer (GC) cells using clonogenic survival assays and studied the effect of combination with chemotherapeutic agents using MTS assays. Cell cycle analysis and molecular changes induced by olaparib were also performed. Results Olaparib showed marked growth inhibitory activity against the SNU 601 cell line, with IC50 value of 0.015 μM. In addition, 5 out of 11 GC cells (45%) were sensitive to olaparib with IC50s ≤ 2μM. Olaprib induced G2/M cell cycle arrest and apoptosis in sensitive GC cells. We identified synergistic growth inhibitory effects of Olaparib in combination with clinically relevant cytotoxic agents (5-FU, cisplatin, and oxaliplatin). Furthermore, Olaparib-induced apoptosis was associated with PARP cleavage and caspase-3 activation. Conclusion: Olaparib showed growth inhibitory activity against GC cells as a single agent and acted synergistically with cytotoxic agents. These results provide a rationale for the future clinical trials of olaparib combined with cytotoxic drugs in the treatment of gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1775.
Supplementary materials and methods. Suppl Table 1. MMS combination PF50 cell line assays. Concen... more Supplementary materials and methods. Suppl Table 1. MMS combination PF50 cell line assays. Concentration-dependent potentiation of MMS (PF50 ratios). Suppl Table 2. Permeability of AZD2461 and olaparib were assessed. Suppl Table 3. Activity of olaparib and AZD2461 alone or in combination with the P-gp inhibitor verapamil in the colorectal cancer cell line HCT-15, which expresses high levels of P-gp. Suppl Fig. 1. Immunofluorescence for poly(ADP-ribose) in human A549 cells. Suppl Fig. 2. Analysis of both in vivo pharmacokinetics (PK) and pharmacodynamics (PD) in tumors taken from SW620 (human colon carcinoma) xenograft-bearing mice. Suppl Fig. 3. Colony formation (clonogenic) assays in the intrinsically high P-gp-expressing MRE11-deficient HCT-15 human colorectal cell line. Suppl. Fig. 4. Combination efficacy studies. Suppl Fig. 5. Relative body weight changes in mice following treatment with 10 mg/kg AZD2461 and olaparib in combination with temozolomide. Suppl. Fig. 6. A serial dilu...
Molecular Cancer Therapeutics, Dec 1, 2009
Ataxia telangiectasia mutated (ATM) is a protein kinase that regulates cell-cycle checkpoints, DN... more Ataxia telangiectasia mutated (ATM) is a protein kinase that regulates cell-cycle checkpoints, DNA repair and recombination (1). It has been shown that ATM interacts with and regulates NBS1 and BRCA complex which is essential for the homologous recombination (HR) repair in response to the DNA double-strand break (DSB) damage (2). These observations suggest that tumor cells with defective ATM expression or activity subsequently leading to HR deficiency are likely more sensitive to the targeted therapeutic/chemotherapeutic agents that cause the accumulation of DNA DSBs during replication. To test this, we studied the growth inhibitory effects of olaparib (AZD2281; KU-0059436) and SN-38 (the active metabolite of Irinotecan) on gastric cancer (GC) cells with reduced ATM expression. Olaparib is a potent oral inhibitor of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP), which has selective antitumor activity in cancers associated with BRCA1 and BRCA2 mutations (3). Our study here showed that 6 out of 7 GC cell lines with low or no expression of ATM are sensitive to olaparib inhibition with IC50s ≤ 1uM as measured by clonogenic survival assays. In contrast, the majority of other GC cell lines positive for ATM expression (11 out of 13) are less sensitive (1uM < IC50 ≤ 1.5uM) or resistant (IC50 > 1.5uM) to olaparib with only 2 of 13 lines sensitive. Similar correlation was also observed in these GC cell lines between reduced ATM expression and sensitivity to SN-38 treatment. In addition, combination of olaparib and SN-38 had a synergistic and selective inhibitory effect on cell growth of GC cells with low/no expression of ATM. These data suggest that loss of ATM in GC cell can sensitize their cellular response to olaparib or/and SN-38. Next, we asked whether this finding has clinical relevance in GC therapy. The tumor and tumor adjacent tissue specimens from more than 100 patients with gastric cancer were analyzed for ATM expression by immunohistochemistry. 22% (24/111) of the gastric tumor specimens were found stained ATM negative while the negative rate in the adjacent tissues was only 4% (1/26). These results suggested that loss of ATM expression is frequently associated with gastric tumorigenesis. Loss of ATM function may serve as an important tumor biomarker in GC and a PARP inhibitor as monotherapy or in combination with chemotherapy provides a promising therapeutic in this disease segment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B42.
The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first... more The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, which suggests that AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared with rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-dam...
Cancer research, Oct 22, 2016
The PARP inhibitor AZD2461 was developed as a next generation agent following olaparib, the first... more The PARP inhibitor AZD2461 was developed as a next generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, suggesting AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared to rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-damaging agent...
Cancer Research, 2015
Ubiquitin proteasome pathways are emerging as a growing source for novel anti-cancer therapeutics... more Ubiquitin proteasome pathways are emerging as a growing source for novel anti-cancer therapeutics. In this respect, by depleting a number of deubiquitylating enzymes (DUBs) through a combination of synthetic lethality screens and isogenic cell line models, MISSION has identified several novel DUBs essential for a range of different tumor types, including platinum-resistant ovarian cancers, DNA damage response (DDR) pathway deficient tumors (e.g. ATM, ATR or BRCA2 defective) and haematological tumors such as multiple myeloma. Illustrating the diversity of DUBs as oncology targets we identified USP11 and UCHL1 using our target validation platform. USP11 was identified as essential for proliferation of platinum-resistant cells but not platinum-sensitive tumor cells in an isogenic model derived from patient tumors before and after development of resistance. In addition, USP11 depletion displayed synthetic lethality in isogenic models of BRCA2 or ATR loss, supporting USP119s previously identified association with homologous recombination proteins as well as the importance such processes in platinum resistance mechanisms. UCHL1 on the other hand is a prototypical oncogene target that selectively drives proliferation in many tumor types, such as multiple myeloma and lung cancer. UCHL1 depletion leads to selective killing of a number of tumors whose proliferation is driven by UCHL1. Validation of targets like USP11 or UCHL1 for the selective treatment of cancer bearing genetic deficiencies or resistant to standard-of-care supports the rationale of developing DUB inhibitors for cancers with unmet medical need. A broad drug discovery platform combining unique biochemical, cellular, biophysical and structural assays was designed by MISSION to identify and optimise potent and selective DUB inhibitors. Early selective DUB hits, developed for USP11 and UCHL1, are successfully recapitulating target validation biology, including synthetic lethality in matched isogenic backgrounds. Lead optimisation of chemical series has recently been translated into compounds with adequate properties for in vivo proof-of-concept studies. The challenges and advances in demonstrating in vivo DUB target engagement will be discussed. MISSION9s integrated drug discovery methodologies exemplify the tractability of DUBs to pharmacologic intervention and the potential scope for DUB inhibitors in a number of cancer types, including those characterised by DDR deficiencies. Citation Format: Xavier Jacq, Niall MB Martin, Lisa Smith, Jeanine Harrigan, Charlotte Knights, Helen Robinson, Yaara Ofir-Rosenfeld, Aaron Cranston, Mark I. Kemp, Stephen P. Jackson. Discovery of highly selective DUB inhibitors with in vivo pre-clinical anti-tumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1728. doi:10.1158/1538-7445.AM2015-1728
Journal of Medicinal Chemistry, 2008
Poly(ADP-ribose) polymerase activation is an immediate cellular response to metabolic-, chemical-... more Poly(ADP-ribose) polymerase activation is an immediate cellular response to metabolic-, chemical-, or ionizing radiation-induced DNA damage and represents a new target for cancer therapy. In this article, we disclose a novel series of substituted 4-benzyl-2 H-phthalazin-1-ones that possess high inhibitory enzyme and cellular potency for both PARP-1 and PARP-2. Optimized compounds from the series also demonstrate good pharmacokinetic profiles, oral bioavailability, and activity in vivo in an SW620 colorectal cancer xenograft model. 4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2 H-phthalazin-1-one (KU-0059436, AZD2281) 47 is a single digit nanomolar inhibitor of both PARP-1 and PARP-2 that shows standalone activity against BRCA1-deficient breast cancer cell lines. Compound 47 is currently undergoing clinical development for the treatment of BRCA1- and BRCA2-defective cancers.
Molecular Cancer Therapeutics, 2009
Background: The oral PARP inhibitor olaparib (AZD2281, KU‐0059436) has shown anti‐tumor activity ... more Background: The oral PARP inhibitor olaparib (AZD2281, KU‐0059436) has shown anti‐tumor activity in patients with hereditary BRCA mutations and provides positive evidence for synthetic lethal approaches for the treatment of cancers with DNA repair defects. The BRCA genes are regulators of homologous recombination (HR) DNA repair pathway but deficiency in other mediators of this pathway, such as the MRE11‐NBS1‐Rad50 (MRN) complex have also been demonstrated to be synthetically lethal with PARP inhibition in pre‐clinical models. Cells deficient in HR DNA repair or signalling pathways are potentially sensitive to PARP inhibition and have been termed homologous recombination deficient (HRD). Examples of HRD include inactivating MRE11 mutations which have been linked to mismatch repair (MMR) deficient, microsatellite instable (MSI) colorectal and endometrial cancers. Previous studies have indicated a high frequency (up to 87% of MSI colorectal cancers) of −1/−2 frameshifts within the poly(T)11 microsatellite repeat sequence and a corresponding reduction in expression. We have therefore undertaken a pre‐clinical study to determine whether MRE11‐deficient colorectal cancer (CRC) respond to olaparib treatment. Methods: CRC cell line panels with known MSI status as well as isogenic matched cell lines models of MRE11 and MMR‐deficiency were assessed for their response to olaparib by both MTS viability and clonogenic survival assays. Quantitative RT‐PCR mRNA and protein expression analysis of MRE11 and DNA damage response genes were obtained for each cell line and HRD status determined. Expression of Pgp (ABCB1) multi‐drug transporter, a pre‐clinical resistance factor for olaparib and whose over‐expression is associated with CRC, was also determined. Results: MTS viability assays showed a limited range of sensitivities between CRC cell lines and only modest correlation with molecular profiles. Analysis using sensitive long term clonogenic survival assays showed a wider range of sensitivities and revealed that HRD (MRE11 mutant) and MSI CRC cells were sensitive to olaparib treatment while microsatellite stable (MSS) and non‐HRD (MRE11 wild‐type) cells remained insensitive. Using isogenic cell line models we confirmed that loss of MRE11 expression can confer sensitivity to olaparib whereas MMR‐deficiency did not. CRC cells which were HRD but overexpressed Pgp were more resistant to olaparib but could be re‐sensitized to treatment by co‐administration of the Pgp inhibitors or through the use of alternative non‐Pgp substrate PARP inhibitors. Conclusions: Here we demonstrate that CRC cell lines with HRD due to MRE11 mutation and loss of protein expression are highly sensitive to the PARP inhibitor olaparib. These data provide evidence that patients with MSI and MRE11‐deficient colorectal tumors could be suitable for treatment with olaparib. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A114.
Olaparib (AZD2281; KU-0059436) is a potent and well tolerated oral inhibitor of PARP. Olaparib of... more Olaparib (AZD2281; KU-0059436) is a potent and well tolerated oral inhibitor of PARP. Olaparib offers a potential synthetic lethal strategy for treatment of cancers with homologous recombination deficiency (HRD). A first-in-human Phase I dose escalation trial of olaparib was conducted and pharmacokinetic (PK) and pharmacodynamic (PD) studies were performed (Fong et al ., 2008). The trial consisted of patients with enrichment for those with hereditary BRCA1 and BRCA2 germline mutations. Dose levels rose from 10 mg daily given for 2 out of 3 weeks to continuous bid dosing at 600mg. PK data indicated rapid absorption and elimination. PARP inhibition PD studies were conducted using immunoblotting and an electrochemiluminescent immunoassay which utilises a functional ex vivo activation assay for poly(ADP)-ribose (PAR) formation (Menear et al ., 2008) in surrogate peripheral blood mononuclear cell (PBMC) and tumour tissue cell lysates. Substantial PARP inhibition of over 90% could be obse...
Cancer Research, 2009
AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO The oral PARP inhibitor olaparib (AZD2281, KU-0... more AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO The oral PARP inhibitor olaparib (AZD2281, KU-0059436) has been shown to have single agent clinical activity in tumours from patients with hereditary BRCA mutations (Fong et al ., 2008). The BRCA genes play key roles in homologous recombination (HR) repair of DNA double strand breaks (DSBs) and BRCA dysfunction is associated with an increased incidence of breast and ovarian cancers. In addition to BRCA, RNAi-mediated gene expression knockdown of other components of the HR DNA damage response pathway such as ATM, ATR and CHK2 has been shown to cause sensitivity to PARP inhibition pre-clinically (McCabe et al ., 2006). These data suggest broader clinical potential of PARP inhibitors in other tumours that may be homologous recombination deficient (HRD). Triple negative (TN) breast cancers represent a distinct clinical subgroup and are defined as ER, PR and HER-2 receptor negative. TN tumours are associated with increased aggressiveness ...
Article (Accepted Version) http://sro.sussex.ac.uk Rulten, Stuart and Caldecott, Keith (2016) The... more Article (Accepted Version) http://sro.sussex.ac.uk Rulten, Stuart and Caldecott, Keith (2016) The PARP inhibitor AZD2461 provides insights into the role of PARP3 inhibition for both synthetic lethality and tolerability with chemotherapy in preclinical models. Cancer Research, 76 (20).
Molecular Cancer Therapeutics, 2009
Background: The oral PARP inhibitor olaparib (AZD2281, KU‐0059436) is in Phase II clinical trials... more Background: The oral PARP inhibitor olaparib (AZD2281, KU‐0059436) is in Phase II clinical trials in patients with BRCA‐mutated breast and ovarian cancer. Early results in these trials have provided positive clinical evidence for the targeted therapy concept of synthetic lethality using PARP inhibitors in molecularly‐defined tumors with little effect on normal tissues. However, olaparib offers the potential to treat a broader range of patient tumors and pre‐clinical studies have indicated that homologous recombination deficiency (HRD) due to loss of other DNA repair genes or through impaired cell‐cycle checkpoints can also lead to sensitivity to PARP inhibitors. The ability to stratify patients whose tumors will respond to treatment will be critical to realise the potential of this targeted therapy approach. In order to identify predictive biomarkers of olaparib response and to provide further insights into mechanisms of olaparib sensitivity a study was undertaken using a panel of cell lines correlating response with molecular profiles. Methods: A cross tumor‐type panel of 95 cell lines (KU‐95 panel) representing breast, ovarian, colorectal, lung, head & neck and pancreatic cancers was tested for sensitivity to olaparib using 2D‐clonogenic survival assays. Baseline (untreated) gene expression profiles using Affymetrix genome‐wide U133A 2.0 arrays together with protein expression and mutational status of genes, such as BRCA1, known to modify of olaparib response were determined for each cell line. Novel statistical, bioinformatics and pathway analysis approaches were used to identify genes that are predictive for olaparib response. Results: Across the KU‐95 cell‐line panel, 30 cell lines were highly sensitive to olaparib (IC50 4 M). Deleterious mutations in BRCA1 or BRCA2 genes were associated with only a small subset of highly sensitive cell lines indicating the presence of other factors able to modulate olaparib responsiveness. Directed analysis of several other DNA repair genes showed correlations with response consistent with previous data. Genome‐wide transcriptome and pathway mapping analysis revealed DNA repair and proliferation associated genes to be most consistently correlated with olaparib sensitivity. Based on results from all these approaches a candidate baseline gene expression profile predictive of olaparib response was established. Conclusions: By profiling a large panel of cell lines we have determined that factors in addition to BRCA mutation can be linked with olaparib sensitivity. A list of candidate gene transcripts was identified that predicts sensitivity to olaparib across a broad range of tumor types in vitro and may have utility as predictive biomarkers in the clinic. Studies are already underway to determine whether this baseline transcript tumor profile can be correlated with patient responses to olaparib. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C119.
Cancer Research, 2010
Background Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)is the member of a family of... more Background Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)is the member of a family of enzymes that catalyze the addition of ADP-ribose units to proteins that mediate DNA repair pathways. Olaparib (KU-0059436; AZD2281) is a potent oral inhibitor of PARP currently in clinical development, which has selective antitumor activity in cancers, associated with BRCA1 and BRCA2 mutations. Gastric cancer is the most common cancer and the second leading cause of cancer deaths in Korea. New therapies including molecular targeting agents are eagerly awaited, and treatment strategies to overcome chemoresistance are also needed in the treatment of gastric cancer. Materials and Methods We studied the growth inhibitory effects of Olaparib on gastric cancer (GC) cells using clonogenic survival assays and studied the effect of combination with chemotherapeutic agents using MTS assays. Cell cycle analysis and molecular changes induced by olaparib were also performed. Results Olaparib showed marked growth inhibitory activity against the SNU 601 cell line, with IC50 value of 0.015 μM. In addition, 5 out of 11 GC cells (45%) were sensitive to olaparib with IC50s ≤ 2μM. Olaprib induced G2/M cell cycle arrest and apoptosis in sensitive GC cells. We identified synergistic growth inhibitory effects of Olaparib in combination with clinically relevant cytotoxic agents (5-FU, cisplatin, and oxaliplatin). Furthermore, Olaparib-induced apoptosis was associated with PARP cleavage and caspase-3 activation. Conclusion: Olaparib showed growth inhibitory activity against GC cells as a single agent and acted synergistically with cytotoxic agents. These results provide a rationale for the future clinical trials of olaparib combined with cytotoxic drugs in the treatment of gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1775.