The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, with an IC 50 of 0.8 nmol/L. AZD8055 showed excellent selectivity (∼1,000-fold) against all class I phosphatidylinositol 3-kinase (PI3K) isoforms and other members of the PI3K-like kinase family. Furthermore, there was no significant activity against a panel of 260 kinases at concentrations up to 10 μmol/L. AZD8055 inhibits the phosphorylation of mTORC1 substrates p70S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins. The rapamycin-resistant T37/46 phosphorylation sites on 4E-BP1 were fully inhibited by AZD8055, resulting in significant inhibition of cap-dependent translation. In vitro, AZD8055 potently inhibits proliferation and induces autophagy in H838 and A549 cells. In vivo, AZD8055 induces a dose-dependent pharmacodynamic effect on phosphorylated S6 and phosphorylated AKT at plasma concentrations leading to tumor growth inhibition. Notably, AZD8055 results in significant growth inhibition and/or regression in xenografts, representing a broad range of human tumor types. AZD8055 is currently in phase I clinical trials. Cancer Res; 70(1); 288-98. ©2010 AACR.

Acquired resistance to MEK1/2 inhibitors (MEKi) arises through amplification of BRAF V600E or KRAS G13D to reinstate ERK1/2 signalling. Here we show that BRAF V600E amplification and MEKi resistance are reversible following drug withdrawal. Cells with BRAF V600E amplification are addicted to MEKi to maintain a precise level of ERK1/2 signalling that is optimal for cell proliferation and survival, and tumour growth in vivo. Robust ERK1/2 activation following MEKi withdrawal drives a p57 KIP2 -dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, selecting against those cells with amplified BRAF V600E . p57 KIP2 expression is required for loss of BRAF V600E amplification and reversal of MEKi resistance. Thus, BRAF V600E amplification confers a selective disadvantage during drug withdrawal, validating intermittent dosing to forestall resistance. In contrast, resistance driven by KRAS G13D amplification is not reversible; rather ERK1/2 hyperactivation drives ZEB1-dependent epithelial-to-mesenchymal transition and chemoresistance, arguing strongly against the use of drug holidays in cases of KRAS G13D amplification.

ATM inhibitors, such as 7, have demonstrated the antitumor potential of ATM inhibition when combined with DNA double-strand break-inducing agents in mouse xenograft models. However, the properties of 7 result in a relatively high predicted clinically efficacious dose. In an attempt to minimize attrition during clinical development, we sought to identify ATM inhibitors with a low predicted clinical dose (<50 mg) and focused on strategies to increase both ATM potency and predicted human pharmacokinetic half-life (predominantly through the increase of volume of distribution). These efforts resulted in the discovery of 64 (AZD0156), an exceptionally potent and selective inhibitor of ATM based on an imidazo[4,5- c]quinolin-2-one core. 64 has good preclinical phamacokinetics, a low predicted clinical dose, and a high maximum absorbable dose. 64 has been shown to potentiate the efficacy of the approved drugs irinotecan and olaparib in disease relevant mouse models and is currently undergoing clinical evaluation with these agents.

Deregulation of the cell cycle has long been recognized as an essential driver of tumorigenesis, and agents that selectively target key cell cycle components continue to hold promise as potential therapeutics. We have developed AZD5438, a 4-(1-isopropyl-2-methylimidazol-5-yl)-2-(4-methylsulphonylanilino) pyrimidine, as a potent inhibitor of cyclin-dependent kinase (cdk) 1, 2, and 9 (IC 50 , 16, 6, and 20 nmol/L, respectively). In vitro, AZD5438 showed significant antiproliferative activity in human tumor cell lines (IC 50 range, 0.2-1.7 μmol/L), causing inhibition of the phosphorylation of cdk substrates pRb, nucleolin, protein phosphatase 1a, and RNA polymerase II COOH-terminal domain and blocking cell cycling at G 2 -M, S, and G 1 phases. In vivo, when orally administered at either 50 mg/kg twice daily or 75 mg/kg once daily, AZD5438 inhibited human tumor xenograft growth (maximum percentage tumor growth inhibition, range, 38-153; P < 0.05). In vivo, AZD5438 reduced the proportion of actively cycling cells. Further pharmacodynamic analysis of AZD5438-treated SW620 xenografts showed that efficacious doses of AZD5438 (>40% tumor growth inhibition) maintained suppression of biomarkers, such as phospho-pRbSer 249 /Thr 252 , for up to 16 hours following a single oral dose. A comparison of different schedules indicated that chronic daily oral dosing provided optimal cover to ensure antitumor efficacy. These data indicate that broad cdk inhibition may provide an effective method to impair the dysregulated cell cycle that drives tumorigenesis and AZD5438 has the pharmacologic profile that provides an ideal probe to test this premise.

Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells AZ32, with enhanced blood-brain barrier (BBB) penetration, was highly efficient as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53., apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. .

Background AZD0156 and AZD6738 are potent and selective inhibitors of ataxia-telangiectasia-kinase (ATM) and ataxia-telangiectasia-mutated and Rad3-related (ATR), respectively, important sensors/signallers of DNA damage. Methods We used multiplexed targeted-mass-spectrometry to select pRAD50(Ser635) as a pharmacodynamic biomarker for AZD0156-mediated ATM inhibition from a panel of 45 peptides, then developed and tested a clinically applicable immunohistochemistry assay for pRAD50(Ser635) detection in FFPE tissue. Results We found moderate pRAD50 baseline levels across cancer indications. pRAD50 was detectable in 100% gastric cancers ( n = 23), 99% colorectal cancers ( n = 102), 95% triple-negative-breast cancers (TNBC) ( n = 40) and 87.5% glioblastoma-multiformes ( n = 16). We demonstrated AZD0156 target inhibition in TNBC patient-derived xenograft models; where AZD0156 monotherapy or post olaparib treatment, resulted in a 34–72% reduction in pRAD50. Similar inhibition of pRAD50 (68%) was observed following ATM inhibitor treatment post irinotecan in a colorectal cancer xenograft model. ATR inhibition, using AZD6738, increased pRAD50 in the ATM-proficient models whilst in ATM-deficient models the opposite was observed, suggesting pRAD50 pharmacodynamics post ATR inhibition may be ATM-dependent and could be useful to determine ATM functionality in patients treated with ATR inhibitors. Conclusion Together these data support clinical utilisation of pRAD50 as a biomarker of AZD0156 and AZD6738 pharmacology to elucidate clinical pharmacokinetic/pharmacodynamic relationships, thereby informing recommended Phase 2 dose/schedule.

Irinotecan, a standard of care therapy for CRC, elicits cytotoxic effects by generating double strand breaks resulting in DNA damage. The activation of the ATM pathway plays a fundamental role in regulating the cellular response and repair to DNA damage. The objective of this preclinical study was to determine whether ATM inhibition would enhance sensitivity to irinotecan treatment. Treatment effects of AZ31, irinotecan or AZ31 + irinotecan were investigated in CRC cell lines and CRC patient derived xenografts. Activation of ATM and downstream targets p-RAD50 and p-H2AX were evaluated by immunohistochemistry. Combinational effects were demonstrated in 4 out of 8 CRC explants. Interestingly, each of the combinational sensitive CRC PDX models were shown to be more resistant to irinotecan single agent therapy. Treatment with irinotecan significantly elevated the ATM pathway evident by an increase in the activation of H2AX and RAD50. Combinational therapy reduced the activation of H2AX and RAD50 when compared to irinotecan alone in the combination sensitive CRC098. AZ31 + irinotecan was effective at reducing tumor growth in tumors that exhibited resistance to irinotecan in our CRC PDX model. These findings support further investigation of this combinational therapy for the treatment of CRC patients.

Total number of figures and tables: 6 Translational RelevanceInhibiting the DNA damage response mechanism is a promising strategy for enhancing the efficacy of chemotherapy and radiotherapy in cancer treatment. DNAdependent protein kinase (DNA-PK) is a key mediator of nonhomologous end joining, and is involved in repairing double-strand breaks. It has been shown that AZD7648 is a potent and selective DNA-PK inhibitor and has radiosensitizing effects.Here, we show that AZD7648 with radiotherapy not only increases cancer cell death via increased DNA damage, but also that cell death was immunogenic, inducing CD8 + cytotoxic T cells and type I interferon dependent anti-tumor responses. As the result, significant tumor growth control was observed in pre-clinical models.Our research highlights the important role of tumor microenvironment and immunological memory in the mechanism of this combination, suggesting that efficacy may be greater than predicted by immunodeficient models. Phase I/II clinical trials for AZD7648 in combination with chemotherapy (NCT03907969) and radiotherapy (NCT04550104) are ongoing.Research.