Metabolism-driven in vitro/in vivo disconnect of an oral ERɑ VHL-PROTAC.

Targeting the estrogen receptor alpha (ERα) pathway is validated in the clinic as an effective means to treat ER+ breast cancers. Here we present the development of a VHL-targeting and orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of ERα. In vitro studies with this PROTAC demonstrate excellent ERα degradation and ER antagonism in ER+ breast cancer cell lines. However, upon dosing the compound in vivo we observe an in vitro-in vivo disconnect. ERα degradation is lower in vivo than expected based on the in vitro data. Investigation into potential causes for the reduced maximal degradation reveals that metabolic instability of the PROTAC linker generates metabolites that compete for binding to ERα with the full PROTAC, limiting degradation. This observation highlights the requirement for metabolically stable PROTACs to ensure maximal efficacy and thus optimisation of the linker should be a key consideration when designing PROTACs.

The Next-Generation Oral Selective Estrogen Receptor Degrader Camizestrant (AZD9833) Suppresses ER+ Breast Cancer Growth and Overcomes Endocrine and CDK4/6 Inhibitor Resistance.

Oral selective estrogen receptor degraders (SERD) could become the backbone of endocrine therapy (ET) for estrogen receptor-positive (ER+) breast cancer, as they achieve greater inhibition of ER-driven cancers than current ETs and overcome key resistance mechanisms. In this study, we evaluated the preclinical pharmacology and efficacy of the next-generation oral SERD camizestrant (AZD9833) and assessed ER-co-targeting strategies by combining camizestrant with CDK4/6 inhibitors (CDK4/6i) and PI3K/AKT/mTOR-targeted therapy in models of progression on CDK4/6i and/or ET. Camizestrant demonstrated robust and selective ER degradation, modulated ER-regulated gene expression, and induced complete ER antagonism and significant antiproliferation activity in ESR1 wild-type (ESR1wt) and mutant (ESR1m) breast cancer cell lines and patient-derived xenograft (PDX) models. Camizestrant also delivered strong antitumor activity in fulvestrant-resistant ESR1wt and ESR1m PDX models. Evaluation of camizestrant in combination with CDK4/6i (palbociclib or abemaciclib) in CDK4/6-naive and -resistant models, as well as in combination with PI3Kαi (alpelisib), mTORi (everolimus), or AKTi (capivasertib), indicated that camizestrant was active with CDK4/6i or PI3K/AKT/mTORi and that antitumor activity was further increased by the triple combination. The response was observed independently of PI3K pathway mutation status. Overall, camizestrant shows strong and broad antitumor activity in ER+ breast cancer as a monotherapy and when combined with CDK4/6i and PI3K/AKT/mTORi. SIGNIFICANCE: Camizestrant, a next-generation oral SERD, shows promise in preclinical models of ER+ breast cancer alone and in combination with CDK4/6 and PI3K/AKT/mTOR inhibitors to address endocrine resistance, a current barrier to treatment.

Discovery of a Potent and Orally Bioavailable Zwitterionic Series of Selective Estrogen Receptor Degrader-Antagonists.

Herein, we report the optimization of a meta-substituted series of selective estrogen receptor degrader (SERD) antagonists for the treatment of ER+ breast cancer. Structure-based design together with the use of modeling and NMR to favor the bioactive conformation led to a highly potent series of basic SERDs with promising physicochemical properties. Issues with hERG activity resulted in a strategy of zwitterion formation and ultimately in the identification of 38. This compound was shown to be a highly potent SERD capable of effectively degrading ERα in both MCF-7 and CAMA-1 cell lines. The low lipophilicity and zwitterionic nature led to a SERD with a clean secondary pharmacology profile and no hERG activity. Favorable physicochemical properties resulted in good oral bioavailability in preclinical species and potent in vivo activity in a mouse xenograft model.

A Phase I Study Investigating AZD8186, a Potent and Selective Inhibitor of PI3Kß/δ, in Patients with Advanced Solid Tumors.

PURPOSE: To characterize safety and tolerability of the selective PI3Kß inhibitor AZD8186, identify a recommended phase II dose (RP2D), and assess preliminary efficacy in combination with abiraterone acetate or vistusertib. PATIENTS AND METHODS: This phase I open-label study included patients with advanced solid tumors, particularly prostate cancer, triple-negative breast cancer, and squamous non-small cell lung cancer. The study comprised four arms: (i) AZD8186 monotherapy dose finding; (ii) monotherapy dose expansion; (iii) AZD8186/abiraterone acetate (with prednisone); and (iv) AZD8186/vistusertib. The primary endpoints were safety, tolerability, and identification of the RP2D of AZD8186 monotherapy and in combination. Secondary endpoints included pharmacokinetics (PK), pharmacodynamics, and tumor and prostate-specific antigen (PSA) responses. RESULTS: In total, 161 patients were enrolled. AZD8186 was well tolerated across all study arms, the most common adverse events being gastrointestinal symptoms. In the monotherapy dose-finding arm, four patients experienced dose-limiting toxicities (mainly rash). AZD8186 doses of 60-mg twice daily [BID; 5 days on, 2 days off (5:2)] and 120-mg BID (continuous and 5:2 dosing) were taken into subsequent arms. The PKs of AZD8186 were dose proportional, without interactions with abiraterone acetate or vistusertib, and target inhibition was observed in plasma and tumor tissue. Monotherapy and combination therapy showed preliminary evidence of limited antitumor activity by imaging and, in prostate cancer, PSA reduction. CONCLUSIONS: AZD8186 monotherapy had an acceptable safety and tolerability profile, and combination with abiraterone acetate/prednisone or vistusertib was also tolerated. There was preliminary evidence of antitumor activity, meriting further exploration of AZD8186 in subsequent studies in PI3Kß pathway-dependent cancers.

Serial monitoring of genomic alterations in circulating tumor cells of ER-positive/HER2-negative advanced breast cancer: feasibility of precision oncology biomarker detection.

Nearly all estrogen receptor (ER)-positive (POS) metastatic breast cancers become refractory to endocrine (ET) and other therapies, leading to lethal disease presumably due to evolving genomic alterations. Timely monitoring of the molecular events associated with response/progression by serial tissue biopsies is logistically difficult. Use of liquid biopsies, including circulating tumor cells (CTC) and circulating tumor DNA (ctDNA), might provide highly informative, yet easily obtainable, evidence for better precision oncology care. Although ctDNA profiling has been well investigated, the CTC precision oncology genomic landscape and the advantages it may offer over ctDNA in ER-POS breast cancer remain largely unexplored. Whole-blood (WB) specimens were collected at serial time points from patients with advanced ER-POS/HER2-negative (NEG) advanced breast cancer in a phase I trial of AZD9496, an oral selective ER degrader (SERD) ET. Individual CTC were isolated from WB using tandem CellSearch® /DEPArray™ technologies and genomically profiled by targeted single-cell DNA next-generation sequencing (scNGS). High-quality CTC (n = 123) from 12 patients profiled by scNGS showed 100% concordance with ctDNA detection of driver estrogen receptor α (ESR1) mutations. We developed a novel CTC-based framework for precision medicine actionability reporting (MI-CTCseq) that incorporates novel features, such as clonal predominance and zygosity of targetable alterations, both unambiguously identifiable in CTC compared to ctDNA. Thus, we nominated opportunities for targeted therapies in 73% of patients, directed at alterations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), fibroblast growth factor receptor 2 (FGFR2), and KIT proto-oncogene, receptor tyrosine kinase (KIT). Intrapatient, inter-CTC genomic heterogeneity was observed, at times between time points, in subclonal alterations. Our analysis suggests that serial monitoring of the CTC genome is feasible and should enable real-time tracking of tumor evolution during progression, permitting more combination precision medicine interventions.

Macrophage Activation Status Rather than Repolarization Is Associated with Enhanced Checkpoint Activity in Combination with PI3Kγ Inhibition.

Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1-blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell-mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.

Discovery of AZD9833, a Potent and Orally Bioavailable Selective Estrogen Receptor Degrader and Antagonist.

Herein we report the optimization of a series of tricyclic indazoles as selective estrogen receptor degraders (SERD) and antagonists for the treatment of ER+ breast cancer. Structure based design together with systematic investigation of each region of the molecular architecture led to the identification of N-[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl]pyridin-3-amine (28). This compound was demonstrated to be a highly potent SERD that showed a pharmacological profile comparable to fulvestrant in its ability to degrade ERα in both MCF-7 and CAMA-1 cell lines. A stringent control of lipophilicity ensured that 28 had favorable physicochemical and preclinical pharmacokinetic properties for oral administration. This, combined with demonstration of potent in vivo activity in mouse xenograft models, resulted in progression of this compound, also known as AZD9833, into clinical trials.

A Randomized, Open-label, Presurgical, Window-of-Opportunity Study Comparing the Pharmacodynamic Effects of the Novel Oral SERD AZD9496 with Fulvestrant in Patients with Newly Diagnosed ER+ HER2- Primary Breast Cancer.

PURPOSE: Fulvestrant, the first-in-class selective estrogen receptor (ER) degrader (SERD), is clinically effective in patients with ER+ breast cancer, but it has administration and pharmacokinetic limitations. Pharmacodynamic data suggest complete ER degradation is not achieved at fulvestrant's clinically feasible dose. This presurgical study (NCT03236974) compared the pharmacodynamic effects of fulvestrant with AZD9496, a novel, orally bioavailable, nonsteroidal, potent SERD, in treatment-naïve patients with ER+ HER2- primary breast cancer awaiting curative intent surgery. PATIENTS AND METHODS: Patients were randomized 1:1 to receive AZD9496 250 mg twice daily from day 1 for 5-14 days, or fulvestrant 500 mg on day 1. On-treatment imaging-guided core tumor biopsies were taken between day 5 and 14 and compared with pretreatment diagnostic biopsies. The primary objective was to compare the effects of AZD9496 and fulvestrant on ER expression. Secondary objectives included changes in progesterone receptor (PR) and Ki-67 pharmacokinetic/pharmacodynamic relationships and safety. RESULTS: Forty-six women received treatment (AZD9496 n = 22; fulvestrant n = 24); 35 paired biopsies were evaluable (AZD9496 n = 15; fulvestrant n = 20). The least square mean estimate for ER H-score reduction was 24% after AZD9496 versus 36% after fulvestrant treatment (P = 0.86). AZD9496 also reduced PR H-scores (-33.3%) and Ki-67 levels (-39.9%) from baseline, but was also not superior to fulvestrant (PR: -68.7%, P = 0.97; Ki-67: -75.4%, P = 0.98). No new safety findings were identified. CONCLUSIONS: This was the first presurgical study to demonstrate that an oral SERD affects its key biological targets. However, AZD9496 was not superior to fulvestrant at the dose tested.

HO-1 drives autophagy as a mechanism of resistance against HER2-targeted therapies.

PURPOSE: Targeted therapies have resulted in major advances in the treatment of HER2-positive breast cancers. Despite this, up to 70% of patients will develop resistance to treatment within 2 years and new strategies for targeting resistant disease are needed. METHODS: To identify potential resistance mechanisms, we used the mouse MMTV-NIC-PTEN+/- spontaneous model of HER2-positive breast cancer and the pan-HER family kinase inhibitor sapatinib. Vehicle and sapatinib-treated tumors were evaluated by immunohistochemistry and proteomic analysis. In vitro studies were carried out to define the role of heme oxygenase 1 (HO-1) and autophagy in resistance to sapatinib and lapatinib, another pan-HER family kinase inhibitor. RESULTS: Treatment of tumor-bearing MMTV-NIC-PTEN+/- mice with sapatinib resulted in delayed tumor progression and increased survival. However, tumors eventually progressed on treatment. Proteomic analysis identified proteins associated with cellular iron homeostasis as being upregulated in the sapatinib-treated tumors. This included HO-1 whose overexpression was confirmed by immunohistochemistry. Overexpression of HO-1 in HER2-expressing SKBR3 breast cancer cells resulted in reduced sensitivity to both pan-HER family kinase inhibitors sapatinib and lapatinib. This was associated with increased autophagy in the HO-1 over-expressing cells. Furthermore, increased autophagy was also seen in the sapatinib-treated tumors. Treatment with autophagy inhibitors was able to increase the sensitivity of the HO-1 over-expressing cells to both lapatinib and sapatinib. CONCLUSION: Together these data indicate a role for HO-1-induced autophagy in resistance to pan-HER family kinase inhibitors.

Building Bridges in a Series of Estrogen Receptor Degraders: An Application of Metathesis in Medicinal Chemistry.

Herein we report the use of metathesis to construct a novel tetracyclic core in a series of estrogen receptor degraders. This improved the chemical stability, as assessed using an NMR-MS based assay, and gave a molecule with excellent physicochemical properties and pharmacokinetics in rat. X-ray crystallography established minimal perturbation of the bridged compounds relative to the unbridged analogues in the receptor binding pocket. Unfortunately, despite retaining excellent binding to ERα, this adversely affected the ability of the compounds to degrade the receptor.

Circulating Biomarkers and Resistance to Endocrine Therapy in Metastatic Breast Cancers: Correlative Results from AZD9496 Oral SERD Phase I Trial.

PURPOSE: Common resistance mechanisms to endocrine therapy (ET) in estrogen receptor (ER)-positive metastatic breast cancers include, among others, ER loss and acquired activating mutations in the ligand-binding domain of the ER gene (ESR1LBDm). ESR1 mutational mediated resistance may be overcome by selective ER degraders (SERD). During the first-in-human study of oral SERD AZD9496, early changes in circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) were explored as potential noninvasive tools, alongside paired tumor biopsies, to assess pharmacodynamics and early efficacy. EXPERIMENTAL DESIGN: CTC were enumerated/phenotyped for ER and Ki67 using CellSearch in serial blood draws. ctDNA was assessed for the most common ESR1LBDm by droplet digital PCR (BioRad). RESULTS: Before starting AZD9496, 11 of 43 (25%) patients had ≥5 CTC/7.5 mL whole blood (WB), none of whom underwent reduction to <5 CTC/7.5 mL WB on C1D15. Five of 11 patients had baseline CTC-ER+, two of whom had CTC-ER+ reduction. CTC-Ki67 status did not change appreciably. Patients with ≥5 CTC/7.5 mL WB before treatment had worse progression-free survival (PFS) than patients with <5 CTC (P = 0.0003). Fourteen of 45 (31%) patients had ESR1LBDm + ctDNA at baseline, five of whom had ≥2 unique mutations. Baseline ESR1LBDm status was not prognostic. Patients with persistently elevated CTC and/or ESR1LBDm + ctDNA at C1D15 had worse PFS than patients who did not (P = 0.0007). CONCLUSIONS: Elevated CTC at baseline was a strong prognostic factor in this cohort. Early on-treatment changes were observed in CTC-ER+ and ESR1LBDm + ctDNA, but not in overall CTC number. Integrating multiple biomarkers in prospective trials may improve outcome prediction and ET resistance mechanisms' identification over a single biomarker.

Combined Inhibition of PI3Kß and mTOR Inhibits Growth of PTEN-null Tumors.

Loss of the tumor suppressor PTEN confers a tumor cell dependency on the PI3Kß isoform. Achieving maximal inhibition of tumor growth through PI3K pathway inhibition requires sustained inhibition of PI3K signaling; however, efficacy is often limited by suboptimal inhibition or reactivation of the pathway. To select combinations that deliver comprehensive suppression of PI3K signaling in PTEN-null tumors, the PI3Kß inhibitor AZD8186 was combined with inhibitors of kinases implicated in pathway reactivation in an extended cell proliferation assay. Inhibiting PI3Kß and mTOR gave the most effective antiproliferative effects across a panel of PTEN-null tumor cell lines. The combination of AZD8186 and the mTOR inhibitor vistusertib was also effective in vivo controlling growth of PTEN-null tumor models of TNBC, prostate, and renal cancers. In vitro, the combination resulted in increased suppression of pNDRG1, p4EBP1, as well as HMGCS1 with reduced pNDRG1 and p4EBP1 more closely associated with effective suppression of proliferation. In vivo biomarker analysis revealed that the monotherapy and combination treatment consistently reduced similar biomarkers, while combination increased nuclear translocation of the transcription factor FOXO3 and reduction in glucose uptake. These data suggest that combining the PI3Kß inhibitor AZD8186 and vistusertib has potential to be an effective combination treatment for PTEN-null tumors. Mol Cancer Ther; 17(11); 2309-19. ©2018 AACR.

A First-in-Human Study of the New Oral Selective Estrogen Receptor Degrader AZD9496 for ER+/HER2- Advanced Breast Cancer.

Purpose: AZD9496 is an oral nonsteroidal, small-molecule inhibitor of estrogen receptor alpha (ERα) and a potent and selective antagonist and degrader of ERα. This first-in-human phase I study determined the safety and tolerability of ascending doses of oral AZD9496 in women with estrogen receptor (ER)+/HER2- advanced breast cancer, characterized its pharmacokinetic (PK) profile, and made preliminary assessment of antitumor activity.Patients and Methods: Forty-five patients received AZD9496 [20 mg once daily (QD) to 600 mg twice daily (BID)] in a dose-escalation, dose-expansion "rolling 6" design. Safety, tolerability, and PK activity in each cohort were reviewed before escalating to the next dose. PK was determined by mass spectrometry. Adverse events (AEs) were graded according to the Common Terminology Criteria for Adverse Events (CTCAE) v4.0. Objective tumor response was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1.Results: Most common causally related AEs were diarrhea (35.6%), fatigue (31.1%), and nausea (22.2%), and seven patients had grade ≥3 AEs. Three patients experienced a dose-limiting toxicity: one each at 150 mg BID (abnormal hepatic function), 400 mg BID (diarrhea and elevated liver function tests), and 600 mg BID (diarrhea), and all were reversible. The maximum tolerated dose was not reached. Partial response was confirmed in one patient, who also had decreased tumor marker Ca15.3. Four patients had stable disease at 12 months' follow-up.Conclusions: AZD9496 is well tolerated with an acceptable safety profile, showing evidence of prolonged disease stabilization in heavily pretreated patients with ER+/HER2- advanced breast cancer. Clin Cancer Res; 24(15); 3510-8. ©2018 AACRSee related commentary by Jordan, p. 3480.

Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor.

Precision medicines exert selective pressure on tumour cells that leads to the preferential growth of resistant subpopulations, necessitating the development of next-generation therapies to treat the evolving cancer. The PIK3CA-AKT-mTOR pathway is one of the most commonly activated pathways in human cancers, which has led to the development of small-molecule inhibitors that target various nodes in the pathway. Among these agents, first-generation mTOR inhibitors (rapalogs) have caused responses in 'N-of-1' cases, and second-generation mTOR kinase inhibitors (TORKi) are currently in clinical trials. Here we sought to delineate the likely resistance mechanisms to existing mTOR inhibitors in human cell lines, as a guide for next-generation therapies. The mechanism of resistance to the TORKi was unusual in that intrinsic kinase activity of mTOR was increased, rather than a direct active-site mutation interfering with drug binding. Indeed, identical drug-resistant mutations have been also identified in drug-naive patients, suggesting that tumours with activating MTOR mutations will be intrinsically resistant to second-generation mTOR inhibitors. We report the development of a new class of mTOR inhibitors that overcomes resistance to existing first- and second-generation inhibitors. The third-generation mTOR inhibitor exploits the unique juxtaposition of two drug-binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants.

Utilization of Structure-Based Design to Identify Novel, Irreversible Inhibitors of EGFR Harboring the T790M Mutation.

A novel series of covalent inhibitors of EGFR (epidermal growth factor receptor) kinase was discovered through a combination of subset screening and structure-based design. These compounds preferentially inhibit mutant forms of EGFR (activating mutant and T790M mutant) over wild-type EGFR in cellular assays measuring EGFR autophosphorylation and proliferation, suggesting an improved therapeutic index in non-small cell lung cancer patients would be achievable relative to established EGFR inhibitors. We describe our design approaches, resulting in the identification of the lead compound 5, and our efforts to develop an understanding of the structure-activity relationships within this series. In addition, strategies to overcome challenges around metabolic stability and aqueous solubility are discussed. Despite limitations in its physical properties, 5 is orally bioavailable in mice and demonstrates pronounced antitumor activity in in vivo models of mutant EGFR-driven cancers.

Identification of novel pathways linking epithelial-to-mesenchymal transition with resistance to HER2-targeted therapy.

Resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapies in the treatment of HER2-positive breast cancer is a major clinical problem. To identify pathways linked to resistance, we generated HER2-positive breast cancer cell lines which are resistant to either lapatinib or AZD8931, two pan-HER family kinase inhibitors. Resistance was HER2 independent and was associated with epithelial-to-mesenchymal transition (EMT), resulting in increased proliferation and migration of the resistant cells. Using a global proteomics approach, we identified a novel set of EMT-associated proteins linked to HER2-independent resistance. We demonstrate that a subset of these EMT-associated genes is predictive of prognosis within the ERBB2 subtype of human breast cancers. Furthermore, targeting the EMT-associated kinases Src and Axl potently inhibited proliferation of the resistant cells, and inhibitors to these kinases may provide additional options for the treatment of HER2-independent resistance in tumors.

Use of a genetically engineered mouse model as a preclinical tool for HER2 breast cancer.

Resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapies presents a major clinical problem. Although preclinical studies have identified a number of possible mechanisms, clinical validation has been difficult. This is most likely to reflect the reliance on cell-line models that do not recapitulate the complexity and heterogeneity seen in human tumours. Here, we show the utility of a genetically engineered mouse model of HER2-driven breast cancer (MMTV-NIC) to define mechanisms of resistance to the pan-HER family inhibitor AZD8931. Genetic manipulation of MMTV-NIC mice demonstrated that loss of phosphatase and tensin homologue (PTEN) conferred de novo resistance to AZD8931, and a tumour fragment transplantation model was established to assess mechanisms of acquired resistance. Using this approach, 50% of tumours developed resistance to AZD8931. Analysis of the resistant tumours showed two distinct patterns of resistance: tumours in which reduced membranous HER2 expression was associated with an epithelial-to-mesenchymal transition (EMT) and resistant tumours that retained HER2 expression and an epithelial morphology. The plasticity of the EMT phenotype was demonstrated upon re-implantation of resistant tumours that then showed a mixed epithelial and mesenchymal phenotype. Further AZD8931 treatment resulted in the generation of secondary resistant tumours that again had either undergone EMT or retained their original epithelial morphology. The data provide a strong rationale for basing therapeutic decisions on the biology of the individual resistant tumour, which can be very different from that of the primary tumour and will be specific to individual patients.

AZD2014, an Inhibitor of mTORC1 and mTORC2, Is Highly Effective in ER+ Breast Cancer When Administered Using Intermittent or Continuous Schedules.

mTOR is an atypical serine threonine kinase involved in regulating major cellular functions, such as nutrients sensing, growth, and proliferation. mTOR is part of the multiprotein complexes mTORC1 and mTORC2, which have been shown to play critical yet functionally distinct roles in the regulation of cellular processes. Current clinical mTOR inhibitors only inhibit the mTORC1 complex and are derivatives of the macrolide rapamycin (rapalogs). Encouraging effects have been observed with rapalogs in estrogen receptor-positive (ER(+)) breast cancer patients in combination with endocrine therapy, such as aromatase inhibitors. AZD2014 is a small-molecule ATP competitive inhibitor of mTOR that inhibits both mTORC1 and mTORC2 complexes and has a greater inhibitory function against mTORC1 than the clinically approved rapalogs. Here, we demonstrate that AZD2014 has broad antiproliferative effects across multiple cell lines, including ER(+) breast models with acquired resistance to hormonal therapy and cell lines with acquired resistance to rapalogs. In vivo, AZD2014 induces dose-dependent tumor growth inhibition in several xenograft and primary explant models. The antitumor activity of AZD2014 is associated with modulation of both mTORC1 and mTORC2 substrates, consistent with its mechanism of action. In combination with fulvestrant, AZD2014 induces tumor regressions when dosed continuously or using intermittent dosing schedules. The ability to dose AZD2014 intermittently, together with its ability to block signaling from both mTORC1 and mTORC2 complexes, makes this compound an ideal candidate for combining with endocrine therapies in the clinic. AZD2014 is currently in phase II clinical trials.

Combining AZD8931, a novel EGFR/HER2/HER3 signalling inhibitor, with AZD5363 limits AKT inhibitor induced feedback and enhances antitumour efficacy in HER2-amplified breast cancer models.

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signalling network is frequently de-regulated in breast cancer and has been shown to mediate resistance to anti-HER2 agents. Whilst constitutive activation of this pathway is emerging as a marker of sensitivity to various PI3K pathway inhibitors, activity of these agents in the clinic may be limited by the presence of feedback loops, leading to reactivation of receptor tyrosine kinases, such as HER2/HER3. To determine whether inhibition of HER2 could increase the efficacy of AZD5363, a novel AKT inhibitor, a panel of breast cancer cells was dosed with AZD5363 in combination with AZD8931, an inhibitor of EGFR/HER2/HER3 signalling. We show that the combined treatment resulted in synergistic growth inhibition and enhanced cell death, specifically in the HER2-amplified cell lines. Investigation of the mechanism by western blot analysis revealed that the addition of AZD8931 prevented the induction of HER2/HER3 phosphorylation induced by AZD5363 and resulted in concomitant inhibition of both the PI3K/AKT/mTOR and ERK signalling pathways and induction of apoptosis. Using the HCC1954 xenograft model, which is resistant to trastuzumab, we show that the combination of AZD5363 and AZD8931 is more efficacious than either agent alone, resulting in profound tumour regressions. We conclude that the activity of AZD5363 in HER2-amplified breast cancer cells is enhanced by the addition of AZD8931 and that dual targeting of AKT and EGFR/HER2/HER3 signalling is an attractive treatment option to be explored in the clinic.

Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor.

Epidermal growth factor receptor (EGFR) inhibitors have been used clinically in the treatment of non-small-cell lung cancer (NSCLC) patients harboring sensitizing (or activating) mutations for a number of years. Despite encouraging clinical efficacy with these agents, in many patients resistance develops leading to disease progression. In most cases, this resistance is in the form of the T790M mutation. In addition, EGFR wild type receptor inhibition inherent with these agents can lead to dose limiting toxicities of rash and diarrhea. We describe herein the evolution of an early, mutant selective lead to the clinical candidate AZD9291, an irreversible inhibitor of both EGFR sensitizing (EGFRm+) and T790M resistance mutations with selectivity over the wild type form of the receptor. Following observations of significant tumor inhibition in preclinical models, the clinical candidate was administered clinically to patients with T790M positive EGFR-TKI resistant NSCLC and early efficacy has been observed, accompanied by an encouraging safety profile.