There has been renewed interest in pursuing cyclin-dependent kinase 2 (CDK2) as a therapeutic target for cancer treatment, given its essential role in driving the survival of CCNE1-amplified tumors and mediating resistance to CDK4/6 inhibitor treatment in estrogen receptor positive breast cancer. This has resulted in the identification of a next generation of orthosteric inhibitors which have increased selectivity for CDK2 kinase versus other CDK-family members. This study aimed to contrast genetic and chemical perturbation of CDK2 in human tumor cell lines with and without CCNE1-amplification. Methods: Cellular responses were assessed using assays for proliferation (CyQUANT), clonogenicity, cell cycle (flow cytometry), and western blotting. NanoBRET tracer displacement assays were used to examine orthosteric kinase inhibition in cells. Genetic perturbation of CDK2 used siRNA and CRISPR-Cas9 editing with sgRNA. Four CDK2 inhibitors currently undergoing clinical trials were profiled. Results: CDK2-targeting siRNA conferred a >75% decrease in the clonogenicity of CCNE1-amplified OVCAR-3, Kuramochi and FUOV1 cells, thus confirming dependence of the cells on CDK2. The non-CCNE1 amplified cells TYK-nu and PEA2 were not sensitive to CDK2-siRNA. The orthosteric CDK2 inhibitors inhibited cellular CDK2 with IC50 values in the range of 1-20 nM. As expected, the compounds inhibited the proliferation of CCNE1-amplified cell lines but they also demonstrated activity against non-CCNE1 amplified TYK-nu cells (one example had GI50 values of 89 ± 8 nM in OVCAR-3 cells, and GI50 values of 122 ± 13 nM in TYK-nu cells, respectively). Consistent with the role of CDK2 in controlling the G1 checkpoint, we observed G1 arrest in the CCNE1-amplified cell line OVCAR-3 following CDK2 CRISPR knock-out. As expected, CDK2 gene deletion did not significantly impact the cell cycle in the TYK-nu non-CCNE1 amplified cell line. In contrast, an orthosteric CDK2 inhibitor induced a G2 arrest in the TYK-nu cell line at doses 2 - 5-fold greater than the compound GI50. Evidence of cyclin E1 accumulation was observed in both CCNE1-amplified and non-amplified cells following orthosteric CDK2 kinase inhibitor treatment (48 - 72 hours). It was also observed following generation of an F80G CDK2 mutant TYK-nu cell line and treatment with the bulky purine analog 3MB-PP1. In contrast, gene-silencing, or gene-editing of CDK2, had no effect on cyclin E1 levels. Conclusion: We demonstrate that the profiled orthosteric CDK2 inhibitors do not reproduce genetic perturbation of CDK2 in CCNE1-amplified and non-amplified cell lines. We propose the need for more selective CDK2 inhibitors and those that inhibit CDK2 in a manner that more closely reflects the genetic perturbation of CDK2. Joanne M. Munck, Susan J. Tudhope, Kleopatra Papa, Alex Howard, Sam Hogan, Suzanne Kyle, Jessica Watt, Luke Gaughan, Maria Ahn, Stephen R. Wedge, John F. Lyons. Orthosteric CDK2 kinase inhibitors have a distinctive profile when compared to genetic perturbation of CDK2 in CCNE1-amplified and non-amplified tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5331.

Abstract Aim We aimed to design an MDM2-p53 antagonist with a differentiated tolerability profile that could be used to treat patients with wild-type TP53 malignancies. As part of an alliance between Newcastle University, Astex Pharmaceuticals, and Cancer Research Horizons, we discovered ASTX295, a potent inhibitor of the MDM2-p53 interaction that is currently under clinical investigation in patients with solid tumors (NCT03975387). We selected ASTX295 as a compound with a predicted short plasma half-life, which we hypothesised would help to mitigate the dose-limiting neutropenia and thrombocytopenia observed with earlier MDM2-p53 antagonists in clinical studies. To examine this hypothesis in vitro, we determined time- and concentration-dependent responses to ASTX295 treatment in healthy volunteer-derived human bone marrow cells, megakaryocytes, and in a panel of human tumor cell lines. Methods Samples containing bone marrow cells from healthy patients undergoing hip surgery were obtained under the ethical approval of the Newcastle Biobank (REC 12/NE/0395). Following Lymphoprep™ separation, cells were treated ex vivo with ASTX295 and seeded for Granulocyte-macrophage (GM) colony-forming assays in methylcellulose. Megakaryocytes were obtained from in vitro differentiation of CD34+ stem/progenitor cells. Human tumor cell lines (including MDM2-amplified SJSA-1) were treated with ASTX295 in vitro and seeded at low density for colony-forming assays. Exposures of 6, 12, or 24h were examined, and the data plotted to calculate LC50 values. Results The clonogenic survival of tumor cells was time-dependent, with LC50 values (mean ± SEM) in SJSA1 cells being 238 ± 46nM and 75 ± 7nM respectively (n = 3-4), following a 12h or 24h exposure to ASTX295. Time-dependent effects were also evident in five human bone marrow samples but with LC50 values of 1.9, >3, >10, >10, and >10uM being achieved at 12h, and 860 ± 268nM at 24h. Megakaryocytes showed similar time-dependent sensitivities in which daily treatment of 2 or 6h over three days did not induce apoptosis while significant cell death was observed when the treatment time was extended to 16-24h daily. In contrast, short, daily pulse treatment of 2-6h in cell lines (MV4-11, MOLM-13, SJSA-1) over three days was sufficient to induce cell death. Conclusions ASTX295 is a potent antagonist of the MDM2-p53 interaction. Collectively, our in vitro data suggest that a shorter exposure to ASTX295 (up to 12h), may help to spare healthy bone marrow cells whilst killing tumor cells. Hence, intermittent exposure to an MDM2-p53 antagonist could favourably modulate its therapeutic index. The predicted short plasma half-life of ASTX295 should provide flexibility in controlling the duration of exposure in vivo, potentially enabling a more bone-marrow sparing approach to MDM2-p53 antagonism to be utilised. Citation Format: Elaine Willmore, Maria Ahn, Suzanne Kyle, Yan Zhao, Huw Thomas, Kenneth S. Rankin, Luke Bevan, Lynsey Fazal, Keisha Hearn, Nicola Wilsher, Justyna Kucia-Tran, Nicola Ferrari, Nicola Wallis, Neil Thompson, John Lyons, Duncan Miller, Celine Cano, Martin E. Noble, Ian R. Hardcastle, Steven Howard, Gianni Chessari, John Lunec, David R. Newell, Steve R. Wedge. Targeting the MDM2-p53 interaction: Time- and concentration-dependent studies in tumor and normal human bone marrow cells reveal strategies for an enhanced therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3333.

Abstract In response to cellular stress, the tumor suppressor p53 is activated to modulate cell cycle progression, DNA repair, and apoptosis. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 prevents its degradation and can reactivate p53 to elicit an anti-cancer effect. Targeting the p53-MDM2 interaction therefore remains a promising strategy for cancer therapy. However, development of first generation MDM2 antagonists has been challenged by dose-limiting, on-target bone marrow toxicities. Understanding of differential effects of p53 pathway activation in normal hematopoietic versus cancer cells (to be presented in a separate abstract) together with our expertise in structure-based drug design have led to the discovery of ASTX295, a potent MDM2 antagonist with differentiated pharmacokinetic profile aimed at sparing bone marrow toxicities and increasing the therapeutic index. Here, we present the first disclosure of the structure and pre-clinical characterisation of ASTX295. ASTX295 exhibits potent activity (IC50<1 nM) against MDM2 in an ELISA-based in vitro assay and induces significant growth reduction in p53 wild-type, MDM2-amplified SJSA-1 cells (GI50=27 nM). Antiproliferative activity of ASTX295 was further demonstrated in a panel of 219 p53 wild-type cell lines, with 143 cell lines showing GI50 values less than 1 μM and 50 showing values less than 0.1 μM. Effects of ASTX295 are shown in cell lines carrying functional p53 as confirmed in three p53 wild-type and mutant cell line pairs (SJSA1 and SN40R2, A2780 and A2780CP, HCT116 and HCT116 p53−/−). In addition to inhibiting cell cycle progression and cell proliferation, ASTX295 also potently induces apoptosis following 24-48 hour treatment. Further in vitro analyses of ASTX295 demonstrated an increase in the levels of p53 (EC50=10 nM after 2 hours) and its transcriptional targets such as p21 and MDM2. In vivo, ASTX295 shows robust induction of p53 and its target genes at 3 and 6 hours after oral administration together with dose-dependent inhibition of tumour growth in the SJSA-1 xenograft model. Importantly, ASTX295 exhibits optimised pharmacokinetic and pharmacodynamic profiles with relatively short duration of pathway modulation and a desired predicted human half-life of 2-8 hours. Based on our pre-clinical hypothesis on differential time-dependent sensitivities of normal versus cancer cells to p53 activation, achieving such a profile while maintaining potency may increase the therapeutic index. These data highlight the therapeutic potential of ASTX295, which is currently being tested in a Phase 1/2 clinical trial in advanced solid tumors with wild-type p53 (NCT03975387). We plan to present preliminary clinical data in a separate abstract at this meeting. Citation Format: Maria Ahn, Luke Bevan, Ildiko Buck, Celine Cano, Juan Castro, Ben Cons, Jane Endicott, Lynsey Fazal, Nicola Ferrari, Ian Hardcastle, Keisha Hearn, Rhian Holvey, Steven Howard, Chris Johnson, Claire Jennings, Justyna Kucia-Tran, Suzanne Kyle, John Lunec, John Lyons, Duncan Miller, David Rees, Martin Noble, David R. Newell, Judith Reeks, Harpreet Saini, Jeffrey St. Denis, Emiliano Tamanini, Huw Thomas, Neil Thompson, Mladen Vinkovic, George Ward, Nicola Wallis, Hugh Walton, Stephen Wedge, Pamela Williams, Elaine Willmore, Nicola Wilshire, Yan Zhao, Gianni Chessari. Discovery of ASTX295, a potent, next-generation small molecule antagonist of MDM2 with differentiated pharmacokinetic profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6588.

Supplementary Figure 3 from 6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

Supplementary Tables 1-2 from 6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

Adobe PDF - MCT-06-0552--Suppl_Table_S2.pdf from Preclinical selection of a novel poly(ADP-ribose) polymerase inhibitor for clinical trial

Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.

Poly(adenosine diphosphate ribose) polymerases (PARPs) are multifunctional proteins which play a role in many cellular processes. Namely, PARP1 and PARP2 have been shown to be involved in DNA repair, and therefore are valid targets in cancer treatment with PARP inhibitors, such as rucaparib, currently in clinical trials. Proton magnetic resonance spectroscopy (1 H-MRS) was used to study the impact of rucaparib in vitro and ex vivo in liver tissue from mice, via quantitative analysis of nicotinamide adenosine diphosphate (NAD+ ) spectra, to assess the potential of MRS as a biomarker of the PARP inhibitor response. SW620 (colorectal) and A2780 (ovarian) cancer cell lines, and PARP1 wild-type (WT) and PARP1 knock-out (KO) mice, were treated with rucaparib, temozolomide (methylating agent) or a combination of both drugs. 1 H-MRS spectra were obtained from perchloric acid extracts of tumour cells and mouse liver. Both cell lines showed an increase in NAD+ levels following PARP inhibitor treatment in comparison with temozolomide treatment. Liver extracts from PARP1 WT mice showed a significant increase in NAD+ levels after rucaparib treatment compared with untreated mouse liver, and a significant decrease in NAD+ levels in the temozolomide-treated group. The combination of rucaparib and temozolomide did not prevent the NAD+ depletion caused by temozolomide treatment. The 1 H-MRS results show that NAD+ levels can be used as a biomarker of PARP inhibitor and methylating agent treatments, and suggest that in vivo measurement of NAD+ would be valuable.

Ataxia telangiectasia mutated (ATM) kinase signals DNA double-strand breaks (DSB) to cell-cycle arrest via p53 and DNA repair. ATM-defective cells are sensitive to DSB-inducing agents, making ATM an attractive target for anticancer chemo- and radiosensitization. KU59403 is an ATM inhibitor with the potency, selectivity, and solubility for advanced preclinical evaluation. KU59403 was not cytotoxic to human cancer cell lines (SW620, LoVo, HCT116, and MDA-MB-231) per se but significantly increased the cytotoxicity of topoisomerase I and II poisons: camptothecin, etoposide, and doxorubicin. Chemo- and radiosensitization by ATM inhibition was not p53-dependent. Following administration to mice, KU59403 distributed to tissues and concentrations exceeding those required for in vitro activity were maintained for at least 4 hours in tumor xenografts. KU59403 significantly enhanced the antitumor activity of topoisomerase poisons in mice bearing human colon cancer xenografts (SW620 and HCT116) at doses that were nontoxic alone and well-tolerated in combination. Chemosensitization was both dose- and schedule-dependent. KU59403 represents a major advance in ATM inhibitor development, being the first compound to show good tissue distribution and significant chemosensitization in in vivo models of human cancer, without major toxicity. KU59403 provides the first proof-of-principle preclinical data to support the future clinical development of ATM inhibitors. Mol Cancer Ther; 12(6); 959–67. ©2013 AACR.

Background: Preclinical studies show that both duration and extent of PARP inhibition is critical for synthetically lethality in tumors with defects in homologous recombination repair (HRR). Rucaparib is undergoing clinical evaluation in HRR-defective tumors. Our aim was to determine whether multiple daily doses or an intermittent schedule will give the required “coverage” for anticancer activity. Methods: The accumulation of 14C-rucaparib and duration of PARP inhibition was determined in SW620 and BRCA2 mutant Capan-1 cells after a 30 minute pulse. Rucaparib concentration in plasma brain and Capan-1 tumor xenografts and PARP inhibition in brain and tumor was determined at intervals up to 1 week after a single dose of rucaparib. The efficacy of continuous and discontinuous schedules of rucaparib was determined in mice bearing Capan-1 xenografts. Results: Rucaparib accumulates in cells via a carrier-mediated transporter (Km of 8.4 ± 1.2 μM and Vmax of 469 ± 22 pmol/106cells/10 min) PARP activity in Capan-1 cells was suppressed by 80% for 72 hr after a pulse of 50 or 400 nM rucaparib, and still 40% reduced 7 days after 400 nM. Rucaparib was cleared rapidly from the plasma but it was detectable for up to 72 hr and suppressed PARP activity in the tumors for 7 days, being 25% and 10% of control after 10 mg/kg and 150 mg/kg, respectively. Peak levels in the brain were 2-10% of those in the tumor and only modest, transient PARP inhibition was observed in the brain. Tumor growth was suppressed by rucaparib at 150 mg/kg po on a weekly schedule as effectively as 10 mg/kg po on a daily x5 every week for 6 weeks. Conclusion: Rucaparib accumulates in human tumor cells and PARP inhibition by rucaparib is durable after a 30 min pulse. PARP is inhibited in tumor xenografts for up to 1 week after a single dose and when the drug concentrations are no longer detectable. Weekly dosing with rucaparib inhibits tumor growth. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C75. Citation Format: James C. Murray, Huw D. Thomas, Philip Berry, Suzanne Kyle, Christopher Jones, Ruth Plummer, Alan V. Boddy, Nicola J. Curtin. Rucaparib (CO-338) accumulation and persistence of PARP inhibition in vitro and in vivo and efficacy of intermittent vs continuous schedules. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C75.

AbstractAG014699 was the first inhibitor of the DNA repair enzyme PARP-1 to enter clinical trial in cancer patients. In addition to enhancing the cytotoxic effect of DNA-damaging chemotherapies, we have previously shown that AG014699 is vasoactive, thereby having the potential to improve drug biodistribution. The effectiveness of the clinical agent doxorubicin is confounded both by poor tumor penetration and cardiotoxicity elicited via PARP hyperactivation. In this study, we analyzed the impact of AG014699 on doxorubicin tolerance and response in breast (MDA-MB-231) and colorectal (SW620, LoVo) tumor models in vitro and in vivo. As anticipated, AG014699 did not potentiate the response to doxorubicin in vitro. In vivo, AG014699 did not influence the pharmacokinetics of doxorubicin; however, it did ameliorate cardiotoxicity. Both toxicity and extent of amelioration were more pronounced in male than in female mice. AG014699 improved vessel perfusion in both MDA-MB-231 and SW620 tumors; however, this neither led to improved tumor-accumulation of doxorubicin nor enhanced therapeutic response. In contrast, when combined with radiotherapy, AG014699 significantly enhanced response both in vitro and in vivo. Real-time assessment of tumor vessel function and companion histologic studies indicate that doxorubicin causes a profound antivascular effect that counters the positive effect of AG014699 on perfusion. These data indicate that although AG014699 can enhance response to some chemotherapeutic drugs via improved delivery, this does not apply to doxorubicin. PARP inhibitors may still be of use to counter doxorubicin toxicity, and if the gender effect translates from rodents to humans, this would have greater effect in males. Mol Cancer Ther; 10(12); 2320–9. ©2011 AACR.

BACKGROUND Mutations in BRCA1 and BRCA2 (BRCA1/2), components of the homologous recombination DNA repair (HRR) pathway, are associated with hereditary breast and ovarian cancers. Poly(ADP-ribose) polymerase (PARP) inhibitors are selectively cytotoxic to animal cells with defective HRR, but results in human cancer cells have been contradictory. We undertook, to our knowledge, the first comprehensive in vitro and in vivo investigations of the antitumor activity of the PARP inhibitor AG014699 in human cancer cells carrying mutated or epigenetically silenced BRCA1/2. METHODS We used nine human cell lines, four with nonmutated BRCA1/2 (MCF7, MDA-MB-231, and HCC1937-BRCA1 [breast cancer] and OSEC-2 [ovarian surface epithelial]), two with mutated BRCA1 (MDA-MB-436 and HCC1937 [breast cancer]), one with mutated BRCA2 (CAPAN-1 [pancreatic cancer]), one that was heterozygous for BRCA2 (OSEC-1 [ovarian surface epithelial]), and one with epigenetically silenced BRCA1 (UACC3199 [breast cancer]), and two Chinese hamster ovary cell lines, parental AA8 and XRCC3 mutated IRS 1SF. We assessed cytotoxicity, DNA damage, and HRR function. Antitumor activity of AG014699 was determined by growth of xenograft tumors (five mice per treatment group). Long-term safety of AG014699 was assessed. RESULTS AG014699 (≤10 μM) was cytotoxic to cells with mutated BRCA1/2 or XRCC3 and to UACC3199 cells with epigenetically silenced BRCA1 but not to cells without BRCA1/2 or XRCC3 mutations or that were heterozygous for BRCA2 mutation. AG014699 induced DNA double-strand breaks in all nine cell lines studied. HRR was observed only in cells with functional BRCA1/2 proteins. Growth of xenograft tumors with BRCA1/2 mutations or with epigenetically silenced BRCA1 was reduced by AG014699 treatment, and combination treatment with AG014699 plus carboplatin was more effective than either drug alone. AG014699 was not toxic in mice with nonmutated or heterozygous BRCA2. CONCLUSION Human cancer cells or xenograft tumors with mutated or epigenetically silenced BRCA1/2 were sensitive to AG014699 monotherapy, indicating a potential role for PARP inhibitors in sporadic human cancers.

Abstract Purpose: Poly(ADP-ribose) polymerase (PARP) inhibitors selectively target homologous recombination (HR)–defective cells and show good clinical activity in hereditary breast and ovarian cancer associated with BRCA1 or BRCA2 mutations. A high proportion (up to 50%) of sporadic epithelial ovarian cancers (EOC) could be deficient in HR due to genetic or epigenetic inactivation of BRCA1/BRCA2 or other HR genes. Therefore, there is a potential for extending the use of PARP inhibitors to these patients if HR status can be identified. We developed a functional assay of HR status in primary cultures of EOCs based on Rad51 focus formation that correlates well with sensitivity to the potent PARP inhibitor AG014699. Experimental Design: Primary cultures were derived from ascitic fluid from patients with EOCs. HR status was investigated by γH2AX and Rad51 focus formation by immunofluorescence. Cytotoxicity to PARP inhibitors was tested by sulforhodamine B and survival assay. Results: Twenty-five cultures were evaluated for HR status and cytotoxicity to PARP inhibitor. Following exposure to AG014699, there was an increase in Rad51 foci (HR competent) in 9 of 24 (36%) but no increase (HR deficient) in 16 of 24 (64%) cultures. Cytotoxicity was observed in 15 of 16 (93%) HR-deficient samples but not in 9 of 9 HR-competent samples following 24-hour exposure to 10 μmol/L AG014699. Conclusion: HR status can be determined in primary cancer samples by Rad51 focus formation, and this correlates with in vitro response to PARP inhibition. Use of this assay as a biomarker now needs testing in the setting of a clinical trial. Clin Cancer Res; 16(8); 2344–51. ©2010 AACR.

Abstract Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor–resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor–resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair–independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair–independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy. Cancer Res; 70(15); 6268–76. ©2010 AACR.

Abstract MAPK and PI3K signalling pathways are frequently activated in human cancers, and represent promising therapeutic targets. Previous studies suggest that combined targeting of these pathways may be necessary for optimal therapeutic activity, hence the aim of this study was to evaluate the MEK inhibitors, ARRY-142886 and PD 0325901, alone and in combination with the dual mTOR/PI3K inhibitor, NVP-BEZ235, or the pan class I PI3K inhibitor, GDC-0941, in colorectal cancer cell lines. Growth inhibition, survival and signal transduction were measured using the Sulforhodamine B assay, clonogenicity and western blotting, respectively. Median effect analysis revealed that all MEK/PI3K inhibitor combinations exhibited marked synergistic growth inhibition in both HCT116 and HT29 cell lines. GDC-0941 displayed the greatest synergy in combination with either MEK inhibitor in the HCT116 cell line, and preliminary results suggest that this is also the case in the HT29 cell line. At concentrations up to 10 µM only NVP-BEZ235 was cytotoxic after 72 hours exposure in either colorectal cancer cell line with an LC50 of 0.51 µM in the HCT116 cell line and an LC50 of 0.44 µM in the HT29 cell line. However, no increase in cytotoxicity was observed with MEK/PI3K inhibitor combinations in the HCT116 cell line, and studies in the HT29 cell line are ongoing. Western blotting using HCT116 cell lysates revealed that NVP-BEZ235 exhibits stronger inhibition of AKT and 4EBP1 phosphorylation, and similar inhibition of S6 phosphorylation, compared to GDC-0941. Both PD 0325901 and ARRY-142886 inhibited ERK phosphorylation. However, no clear or consistent additional effect on S6, ERK or 4EBP1 phosphorylation was observed when the MEK and PI3K inhibitors were combined. These studies confirm that MEK and PI3K inhibitors are predominantly cytostatic, as opposed to cytotoxic, when used alone or in combination. The dual mTOR/PI3K inhibitory action of NVP-BEZ235 may increase its ability to inhibit 4EBP1 phosphorylation, and thereby protein translation. Furthermore, the lower level of synergy exhibited by NVP-BEZ235 in combination with MEK inhibitors, compared to GDC-0941, may be due to the inhibition of mTOR and thereby 4EBP1 phosphorylation. Hence studies have commenced using the mTOR inhibitor, rapamycin, in combination with GDC-0941. In the HCT116 cell line, preliminary results suggest that the combination of GDC-0941 and rapamycin is considerably more growth inhibitory than either compound alone. These studies confirm that dual targeting of PI3K and MEK can induce synergistic growth inhibition; however, the detailed effects of specific inhibitors should be investigated to identify optimal combinations. The research was funded by grants from the Medical Research Council UK and UCB Celltech Ltd. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 5399.

Room temperature ionic liquids (RTILs) are organic salts which are liquids at ambient temperature. Composed of relatively large asymmetric organic cations and inorganic or organic anions, they have generated interest as ‘green’ solvents. Here we report on the solvency of alkyl imidazolium salts (PF6−Br−Cl−) for poorly water-soluble model drugs, albendazole and danazol, indicating their potential application as pharmaceutical solvents/cosolvents. The solubility of albendazole, for example, is increased by more than 10,000 times by 1-butyl-3-methylimidazolium hexaflourophosphate ([bmim]PF6−). Ionic liquids can be water-miscible or water-immiscible. The aqueous miscibility of a poorly water-miscible RTIL such as of [bmim]PF6− can be improved by the inclusion of a second more miscible RTIL (e.g. 1-hexyl-3-methylimidazolium bromide ([hmim]Br−)). The extent of improvement in water miscibility was found to correlate with the hydrophilicity of the second RTIL. This ability to modulate RTILs’ aqueous miscibility increases their usefulness as pharmaceutical solvents.

Abstract Purpose: Poly(ADP-ribose) polymerase (PARP) plays an important role in DNA repair, and PARP inhibitors can enhance the activity of DNA-damaging agents in vitro and in vivo. AG014699 is a potent PARP inhibitor in phase II clinical development. However, the range of therapeutics with which AG014699 could interact via a DNA-repair based mechanism is limited. We aimed to investigate a novel, vascular-based activity of AG014699, underlying in vivo chemosensitization, which could widen its clinical application. Experimental Design: Temozolomide response was analyzed in vitro and in vivo. Vessel dynamics were monitored using “mismatch” following the administration of perfusion markers and real-time analysis of fluorescently labeled albumin uptake in to tumors established in dorsal window chambers. Further mechanistic investigations used ex vivo assays of vascular smooth muscle relaxation, gut motility, and myosin light chain kinase (MLCK) inhibition. Results: AG014699 failed to sensitize SW620 cells to temozolomide in vitro but induced pronounced enhancement in vivo. AG014699 (1 mg/kg) improved tumor perfusion comparably with the control agents nicotinamide (1 g/kg) and AG14361 (forerunner to AG014699; 10 mg/kg). AG014699 and AG14361 relaxed preconstricted vascular smooth muscle more potently than the standard agent, hydralazine, with no impact on gut motility. AG014699 inhibited MLCK at concentrations that relaxed isolated arteries, whereas AG14361 had no effect. Conclusion: Increased vessel perfusion elicited by AG014699 could increase tumor drug accumulation and therapeutic response. Vasoactive concentrations of AG014699 do not cause detrimental side effects to gut motility and may increase the range of therapeutics with which AG014699 could be combined with for clinical benefit. (Clin Cancer Res 2009;15(19):6106–12)