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Next-generation oral selective estrogen receptor degraders (ngSERDs) are aiming to become the backbone endocrine therapy (ET) for patients with estrogen receptor positive (ER+) breast cancer (BC) by achieving greater ER signaling blockade than current therapies and tackling key mechanisms of resistance. Camizestrant (AZD9833) is an ngSERD for the treatment of ER+ BC which has demonstrated selective ERα degradation, pure ER antagonism and significant anti-tumor activity in ESR1 wild-type (ESR1wt) and mutant (ESR1m) tumors, as well as encouraging clinical activity in early phase trials. ER+ BC is responsive to therapies targeting ER and CDK4/6 signaling in the adjuvant and metastatic settings. To explore camizestrant combination potential as a backbone ET, camizestrant was partnered with either palbociclib or abemaciclib in CDK4/6 inhibitor (CDK4/6i) naïve and resistant in vitro and in vivo models. Combination benefit was observed in vitro in 3 parental ER+ BC cell lines, moreover camizestrant plus abemaciclib showed activity in palbociclib-resistant cell lines including lines harboring CCNE1amp and RB1 loss. In vivo, combination of camizestrant and either CDK4/6i was well tolerated with the combinations promoting improved efficacy in ESR1wt and ESR1m PDX tumor models compared to monotherapy arms. ER+ BC has a high prevalence of alterations in the PI3K/AKT/PTEN pathway which provides opportunities for treatment with PI3K/AKT pathway inhibitors. Camizestrant delivered enhanced efficacy when combined with mTORC1 inhibitor everolimus and AKT inhibitor capivasertib in CTC-174, an ESR1m and PI3KCAm tumor model. Additionally, combination with capivasertib was more efficacious than single treatments, at clinically relevant dose and schedules, in both PI3Kwt and mutated pathway in PDX models. These data demonstrate the interplay between PI3K/AKT/PTEN pathway inhibition and camizestrant mechanism of action. Finally, we explored a potential triple combination of camizestrant, capivasertib and palbociclib in Palbociclib-resistant models representative of PI3KCA/AKT/PTEN wt and altered tumors. This strategy delivered robust efficacy across these models comparing to single treatments and double combinations. The triplet led to durable regressions at clinically achievable doses of all compounds, irrespective of genetic background and aligned to biomarker modulation of the three signaling axis. These preclinical data demonstrate the potential of camizestrant to become the backbone ET, with high combinability in vivo with inhibitors of CDK4/6, mTOR and AKT. These combinations demonstrate the opportunity to impact care of patients with early and metastatic ER+BC, delivering benefit to broad patient populations including those with ESR1wt or ESR1m tumors, independent of PI3K/AKT/PTEN pathway mutation status, and in patients with both CDK4/6i sensitive and resistant tumors. Citation Format: Larissa Carnevalli, Susana Ros, Jelena Urosevic, Natalie Cureton, Sophie Darcy, Mandy Lawson, Stuart Williamson, Pablo Morentin Gutierrez, Azadeh Bashi, Jennifer Moss, Christopher Morrow, Barry Simon, Teresa Klinowska. PD10-04 Combination of the next generation oral SERD camizestrant (AZD9833) with CDK4/6 and mTOR/AKT inhibitors delivers robust efficacy in a broad range of ER+ breast tumors. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-04.

PDF file - 235KB, Activity of AZD9291 and metabolites against rare EGFR mutations using cellular phosphorylation assays.

PDF file - 294KB, Phenotype and phosphorylation assay data for AZD9291 against lung cancer associated HER2 mutation.

Supp Fig 1 Tumour efficacy Supp Fig 2 In vitro gene expression modulation Supp Fig 3 In vivo biomarker analysis Supp Fig 4 HCC70 In vivo FDG uptake imaging Supp Fig 5 U87MG in vivo FDG uptake imaging

PDF file - 107KB, Mass Spectrometry study showing covalent binding of AZD9291 to cysteine 797 in EGFR T790M.

Pharmacokinetics modelling of AZD2014 intermittent dosing schedule.

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.

Supplementary Figure 1. Pharmacokinetic (PK) properties of AZD3458 in mouse. Supplementary Figure 2. In vivo pharmacodynamic properties of AZD3458 in mouse neutrophils. Supp Fig 3. PI3K-�/mTOR pathway regulates expression of IL-10/IL-12 via LPS-induced glycolytic burst. Supplementary Figure 4. AZD3458 improves checkpoint activity in CT-26 tumor models. Supplementary Figure 5. AZD3458 improves checkpoint activity in 4T1 tumor models. Supplementary Figure 6. Top canonical IPA pathways across tissues in AZD3458+PD-1 combination group. Supplementary Figure 7. Graphical abstract.

HCC1428-eveR and HCC1428-LTED-eveR cell lines resistant to everolimus remain sensitive to dual mTORC1/2 inhibitor AZD2014.

PDF file - 312KB, Efficacy of AZD9291 in H3255, PC-9VanR and A431 xenograft models. Weight loss data for chronically dosed AZD9291 in PC-9 and H1975 xenograft models.

Supplementary Materials and Methods. Suplementary Table 1. Tumor Immuno-phenotyping. Suplementary Table 2. Flow cytometry panels.

Loss of the tumor suppressor PTEN confers a tumor cell dependency on the PI3Kβ 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 PI3Kβ inhibitor AZD8186 was combined with inhibitors of kinases implicated in pathway reactivation in an extended cell proliferation assay. Inhibiting PI3Kβ 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 PI3Kβ 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.

Supplementary Table 1: Cell lines; Supplementary Table 2: Antibodies; Supplementary Table 3: Gene lists

Supplementary data on cell lines

Pharmacokinetics and PK/PD relationships for AZD2014

PDF file - 151KB, Activity of AZD9291 and metabolites against cellular phospho-HER2 assays.

PDF file - 470KB, Effect of AZD9291 and AZ5104 in transgenic models of EGFR-TKI sensitizing (C/L858R and C/ex19del) and T790M resistant (C/L+T) lung cancer.

Supplementary table 1: Biochemical and cellular activity of AZD2014. Supplementary table 2: Biochemical activity of AZD2014. Supplementary table 3: Anti proliferative activity of AZD2014 in a panel of ER+, endocrine resistant and LTED cell lines. Supplementary table 4: Activity of AZD2014 in a number of ER+ breast cancer in vivo models.

PDF file - 182KB, Pharmacokinetic analysis of AZD9291, AZ5104 and AZ7550 total plasma concentrations vs time following single oral dose of 25mg/kg AZD9291 in mice.

PDF file - 89KB, Additional biochemical and cellular profiling data for AZD9291 and metabolites.

Supplementary Figure from A Phase I Study Investigating AZD8186, a Potent and Selective Inhibitor of PI3Kβ/δ, in Patients with Advanced Solid Tumors

Supplementary Figures: Supplementary Figure 1. Summary of duration on AZD9496 therapy, previous therapies, and circulating biomarkers status at baseline for each patient ranked by ascending dose; Supplementary Figure 2. Tumor pharmacodynamics. Supplementary Tables: Supplementary Table 1. Study Plan showing time-points of collection of biosamples for exploratory research; Supplementary Table 2. ddPCR primers and probes;Supplementary Table 3. Antibodies and conditions for IHC analysis Antibodies for IHC analysis; Supplementary Table 4. Patients with {greater than or equal to}1 CTC/7.5 mL WB within 120 days of fulvestrant wash-out period prior C1D1.

Purpose:To characterize safety and tolerability of the selective PI3Kβ 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 PI3Kβ pathway–dependent cancers.

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 +, 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 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.

Supplementary Data from AZD8931, an Equipotent, Reversible Inhibitor of Signaling by Epidermal Growth Factor Receptor, ERBB2 (HER2), and ERBB3: A Unique Agent for Simultaneous ERBB Receptor Blockade in Cancer

PK parameters for M5 following a single dose (Day 1) and multiple doses (Day 15)

GAZD9496 metabolite, M3 (Panel A) and M5 (Panel C), geometric mean plasma concentration following a single dose of AZD9496 on Day 1 (semi-log scale). AZD9496 metabolite, M3 (Panel B) and M5 (Panel D), geometric mean plasma concentration following multiple doses of AZD9496 on Day 15 (semi-log scale). On Day 15 at all doses except 20 mg, the 24 h time point is the 12 h trough concentration following the evening dose of AZD9496 and therefore not shown. aPooled data from dose escalation and expansion groups. BID=twice daily; QD=once daily; EXP=expansion cohort.

Supplementary Data from A Phase I Study Investigating AZD8186, a Potent and Selective Inhibitor of PI3Kβ/δ, in Patients with Advanced Solid Tumors

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 AACR.See related commentary by Jordan, p. 3480

A total of 45 patients were recruited and four to six patients were allocated to cohorts to receive escalating doses of AZD9496. In the BID cohorts, patients received QD dosing on Day 1 and BID dosing thereafter. Six patients entered the expansion cohort, receiving AZD9496 250 mg BID. BID=twice daily, DLT=dose-limiting toxicity; n=number of patients included; LFT=liver function tests; QD=once daily.

Purpose: To test the hypothesis that simultaneous, equipotent inhibition of epidermal growth factor receptor (EGFR; erbB1), erbB2 (human epidermal growth factor receptor 2), and erbB3 receptor signaling, using the novel small-molecule inhibitor AZD8931, will deliver broad antitumor activity in vitro and in vivo.Experimental Design: A range of assays was used to model erbB family receptor signaling in homodimers and heterodimers, including in vitro evaluation of erbB kinase activity, erbB receptor phosphorylation, proliferation in cells, and in vivo testing in a human tumor xenograft panel, with ex vivo evaluation of erbB phosphorylation and downstream biomarkers. Gefitinib and lapatinib were used to compare the pharmacological profile of AZD8931 with other erbB family inhibitors.Results: In vitro, AZD8931 showed equipotent, reversible inhibition of EGFR (IC50, 4 nmol/L), erbB2 (IC50, 3 nmol/L), and erbB3 (IC50, 4 nmol/L) phosphorylation in cells. In proliferation assays, AZD8931 was significantly more potent than gefitinib or lapatinib in specific squamous cell carcinoma of the head and neck and non–small cell lung carcinoma cell lines. In vivo, AZD8931 inhibited xenograft growth in a range of models while significantly affecting EGFR, erbB2, and erbB3 phosphorylation and downstream signaling pathways, apoptosis, and proliferation.Conclusions: AZD8931 has a unique pharmacologic profile providing equipotent inhibition of EGFR, erbB2, and erbB3 signaling and showing greater antitumor activity than agents with a narrower spectrum of erbB receptor inhibition in specific preclinical models. AZD8931 provides the opportunity to investigate whether simultaneous inhibition of erbB receptor signaling could be of utility in the clinic, particularly in the majority of solid tumors that do not overexpress erbB2. Clin Cancer Res; 16(4); 1159–69

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Background Camizestrant (C), a next-generation oral selective estrogen receptor (ER) antagonist and degrader (ngSERD) has shown promising clinical activity in ER+ breast cancer (BC) in the Phase 1 SERENA-1 study1,2 with a dose-dependent safety profile. The Phase 2 randomized SERENA-2 study (NCT04214288) initially assessed three doses of C vs fulvestrant (F) in post-menopausal women with ER+ HER2˗ BC with disease recurrence or progression after ≤1 endocrine therapy (ET) in the advanced setting. Methods SERENA-2 evaluated efficacy and safety of C 75, 150 or 300 mg monotherapy QD vs F (per label). Eligible patients were randomized 1:1:1:1. The Primary objective was to determine clinical efficacy of C vs F by investigator-assessed progression-free survival (PFS). Secondary endpoints included objective response rate, response duration, clinical benefit rate at 24 weeks, overall survival and safety. Patients had no prior F or oral SERD and ≤1 ET and ≤1 chemotherapy (CTX) in the advanced setting. To assess the impact of prior CDK4/6 inhibitor (CDK4/6i) treatment, randomization was stratified so that 50% of patients had prior CDK4/6i. Planned enrolment of 288 patients began in April 2020. The C 300 arm was closed after 20 patients were enrolled, changing target enrolment to 236. By August 2021, 240 patients had been randomized. Primary analysis was triggered when 108 progression events (75% maturity) had occurred in the best performing pair (C vs F) in August 2022. Efficacy analyses compared C 75 and 150 mg doses with F, with no formal analyses of C 300 vs F. 108 events for pairwise comparison vs F gave 86% power at the 2-sided 10% significance level. Primary analyses used a Cox proportional hazards model to compare PFS, adjusting for prior CDK4/6i and lung/liver metastases. ESR1 mutations (ESR1m) were detected in plasma samples using next-generation sequencing. Results 119/240 (49.6%) patients had had prior CDK4/6i therapy. At baseline, 88 (36.7%) patients had detectable ESR1m and 140 (58.3%) had lung/liver metastases. Prior CTX or ET rates in the advanced setting were 19.2 and 65.4%. Treatment-emergent adverse events (AEs) (grade ≥3) occurred in 77.0 (12.2), 90.4 (21.9) and 68.5 (13.7) % of patients in the C 75, C 150 and F arms. AEs leading to treatment discontinuation occurred in 2.7, 0 and 0% of patients in the C 75, C 150 and F arms. The most common AEs considered by the investigator to be causally related to study drug were photopsia (18.4%) and (sinus) bradycardia (13.6%) – all were grade 1 or 2. Hot flush (2.7%) and myalgia (2.7%) were the most common AEs related to F. Conclusions SERENA-2 is the first Phase 2 trial investigating multiple dose levels of an ngSERD vs F in post-menopausal women with advanced ER+ HER2˗ BC with disease recurrence or progression after ≤1 ET in the advanced setting. C at both 75 and 150 mg dose levels showed a statistically significant and clinically meaningful benefit in PFS vs F in the overall study population, and was well tolerated. The results of SERENA-2 support the further development of C in ER+ HER2- BC. Acknowledgements AstraZeneca sponsored this trial and funded medical writing support from Helen Heffron (InterComm International). References 1. Baird R, Oliveira M, Ciruelos Gil EM, et al. SABCS 2020 Virtual Meeting. Abstract PS11-05; 2. Oliveira M, Hamilton EP, Incorvati J et al. ASCO 2022 Annual Meeting, Chicago, IL, USA. Abstract 1032. Citation Format: Mafalda Oliveira, Denys Pominchuck, Zbigniew Nowecki, Erika Hamilton, Yaroslav Kulyaba, Timur Andabekov, Yevhen Hotko, Tamar Melkadze, Gia Nemsadze, Patrick Neven, Yuriy Semegen, Vladmir Vladmirov, Claudio Zamagni, Hannelore Denys, Frederic Forget, Zsolt Horvath, Alfiya Nesterova, Maxine Bennett, Bistra Kirova, Teresa Klinowska, Justin Lindemann, Delphine Lissa, Alastair Mathewson, Christopher Morrow, Zuzana Traugottova, Ruaan Van Zyl, Ekaterine Arkania. GS3-02 Camizestrant, a next generation oral SERD vs fulvestrant in post-menopausal women with advanced ER-positive HER2-negative breast cancer: Results of the randomized, multi-dose Phase 2 SERENA-2 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS3-02.

Background: AZD9833 is an oral selective estrogen receptor (ER) antagonist and degrader (SERD) in Phase 2 clinical development for the treatment of ER+ HER2− breast cancer. Here we report data from Parts C and D of the ongoing Phase 1 study (SERENA-1) examining AZD9833 in combination with palbociclib, together with updated data from Parts A and B examining AZD9833 monotherapy. Methods: SERENA-1 (NCT03616587) is an ongoing open-label Phase 1 study of AZD9833 in pre- and post-menopausal women with ER+, HER2− advanced breast cancer who have previously received ≥1 endocrine therapy and ≤2 prior chemotherapies. Prior treatment with fulvestrant and/or CDK4/6 inhibitors was permitted. The primary objective is to determine the safety and tolerability of once daily (QD) AZD9833, with dose-limiting toxicities (DLTs) in the first 28 days defining the maximum tolerated dose. Secondary objectives include anti-tumor response (including circulating tumor [ct] DNA response) and pharmacokinetics. Parts A (escalation) and B (expansion) assess AZD9833 as a monotherapy, and Parts C (escalation) and D (expansion) assess AZD9833 in combination with palbociclib. Results: At a data cut-off of March 24 2020, 17 patients had received either 150 mg or 300 mg AZD9833 in combination with palbociclib, given according to its product labeling. Eighty patients had received AZD9833 monotherapy at doses of 25, 75, 150, 300, and 450 mg QD. In patients treated with AZD9833 and palbociclib, treatment-related adverse events (AEs; experienced by ≥10% of patients) included visual disturbances*, bradycardia*, asthenia, anemia, QTcF prolongation, nausea, neutropenia, decreased white blood cell count, and vomiting (*combined terms). All instances of AZD9833-related bradycardia were Grade 1. One DLT was observed in the 150 mg cohort: CTCAE Grade 2 visual disturbances, which began on Cycle 1 Day 8 and resolved by Cycle 1 Day 9 following dose interruption. The patient restarted treatment on Cycle 1 Day 15 at 75 mg and continued this dose until data cut-off. No causally related AEs led to discontinuation of AZD9833. The tolerability of AZD9833 with palbociclib was consistent with the observed tolerability profile of AZD9833 monotherapy, and the known tolerability profile of palbociclib. Pharmacokinetic analysis showed similar AZD9833 exposure for monotherapy and palbociclib combination therapy. Similarly, palbociclib exposure was comparable with simulations using a published population pharmacokinetic model. In Part A, ESR1 hotspot mutations were detected in ctDNA at baseline in 26/56 (46%) patients; 13/26 (50%) of these patients achieved a partial response or stable disease at 24 weeks, including 5/10 (50%) with a Y537S ESR1 mutation. Further, in patients with ESR1 mutations and samples available for longitudinal ctDNA analysis, 17/20 (85%) exhibited a reduction or loss of mutant ESR1 on treatment with AZD9833. Efficacy data to be presented include objective response rate and clinical benefit rate at 24 weeks. Of note, unconfirmed partial responses have been observed in Part C after the data cut-off for this abstract. Conclusions: AZD9833 continues to show an encouraging efficacy and dose-dependent safety profile as a monotherapy or in combination with palbociclib. A Phase 2 study comparing the efficacy and safety of three doses of AZD9833 versus fulvestrant (NCT04214288), and a Phase 2 pre-surgical ‘window of opportunity’ study (EUDRA-CT; 2019-003706-2) are ongoing. Citation Format: Richard Baird, Mafalda Oliveira, Eva Maria Ciruelos Gil, Manish R Patel, Begoña Bermejo de las Heras, Manuel Ruiz-Borrego, Javier García-Corbacho, Anne Armstrong, Udai Banerji, Chris Twelves, Valentina Boni, Jason Incorvati, Peter Kabos, Adam L Cohen, Bruno de Paula, Marta Capelán Rodríguez, Judy S Wang, Christina Hernando, Alejandro Falcón Gonzalez, Ivan Victoria Ruiz, Julia Lai-Kwon, Anosheh Afghani, Christos Vaklavas, Tim Brier, Steven Fox, Bistra Kirova, Teresa Klinowska, Chris Leach, Justin PO Lindemann, Richard Mather, Rhiannon Maudsley, Christopher J Morrow, Nitharsan Sathiyayogan, Andy Sykes, Li Zhang, Erika Hamilton. Updated data from SERENA-1: A Phase 1 dose escalation and expansion study of the next generation oral SERD AZD9833 as a monotherapy and in combination with palbociclib, in women with ER-positive, HER2-negative advanced breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-05.

[Image: see text] 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.

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.

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.

Loss of the tumor suppressor PTEN confers a tumor cell dependency on the PI3Kβ 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 PI3Kβ inhibitor AZD8186 was combined with inhibitors of kinases implicated in pathway reactivation in an extended cell proliferation assay. Inhibiting PI3Kβ 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 PI3Kβ 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.

Fulvestrant, the first-in-class selective estrogen receptor (ER) degrader (SERD), is clinically effective in patients with ERPatients 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.Forty-six women received treatment (AZD9496This 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.

Background AZD9496 is a potent orally bioavailable ER antagonist and degrader that has shown antitumor efficacy in a range of preclinical xenograft models including ESR1wild-type tamoxifen-resistant and long term estrogen deprived models and an ESR1 mutant model. Methods This is a phase I, open label global multicenter study in women with ER+ HER2–ve BC either metastatic or locoregionally recurrent, not amenable to treatment with curative intent. Patients are post-menopausal, or pre-menopausal women receiving LHRH agonist therapy, with disease progression after ≥6 months endocrine therapy for ER+ BC (no limit on number of prior endocrine therapies; ≤2 prior chemotherapies in advanced setting). The primary objective is to determine the safety and tolerability of AZD9496. Cohorts of 3-6 patients received daily oral therapy and dose limiting toxicities (DLTs) occurring in cycle 1 (28 days) were assessed. Patients are dosed until MTD (defined as ≤1/6 patients with a DLT) or maximum feasible dose (MFD) is reached. Key secondary objectives include determination of single and multiple dose pharmacokinetics (PK), and preliminary antitumor efficacy. ER target modulation by protein and gene expression is evaluated in circulating tumor cells and paired tumor biopsies. In addition to the dose escalation phase, expansion cohort(s) in patients with or without ESR1 mutations can be enrolled to examine the safety, tolerability, PK and biological activity of AZD9496 further. Results Preliminary data as of 30th April 2016: 45 patients (median age 62 (range 41-83); 38 post-menopausal, 7 pre/perimenopausal; visceral metastases 76%, prior fulvestrant 25/45) received AZD9496 in 7 dose escalation cohorts: 20mg QD n=4, 40mg BID n=6, 80mg BID n=5, 150mg BID n=6, 250mg BID n=6, 400mg BID n=6, 600mg BID n=6 and also a 250mg BID expansion cohort n=6. The majority of adverse events (AEs) were grade 1 or 2; the most common treatment-related AEs (≥10%) have been diarrhoea (33%), fatigue (27%), nausea (22%), upper abdominal pain (13%) and increased liver function tests (13%). Six patients had treatment-related grade 3 AEs, 5 of which were manageable with dose interruption +/- dose reduction. Specifically, three had DLTs: grade 3 increased AST/ALT/GGT-150mg BID, serious adverse reaction (SAR) leading to withdrawal; grade 3 diarrhoea and grade 3 increased AST/ALT/GGT-400mg BID, SAR, manageable with dose reductions; grade 3 diarrhoea 600mg BID, manageable with dose reduction. The MTD/MFD has not been reached. Following the first dose up to 400mg the AZD9496 exposure increased in reasonable proportion to increasing dose. At 600mg a more than dose-proportional increase in exposure was observed. Evidence of reduced ER and Ki67 has been observed in on-study biopsies at 150mg BID and above. 10 subjects received treatment for >3-6- Conclusions AZD9496 has a tolerable safety profile, evidence of PD biomarker modulation and prolonged stabilisation of disease in women with heavily pre-treated ER+ve ABC. Citation Format: Hamilton E, Patel M, Armstrong A, Baird R, Jhaveri K, Hoch M, Morgan S, Dowdall T, Schiavon G, Klinowska T, Weir H, Bujac S, Nash T, Im S-A. A phase I study of AZD9496, a novel oral, selective estrogen receptor degrader (SERD) in women with estrogen receptor positive, HER-2 negative advanced breast cancer (ABC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-03.

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.

Background AZD9833 is an orally bioavailable selective estrogen receptor (ER) antagonist and degrader (SERD) that has shown anti-tumor efficacy in a range of preclinical breast cancer models. SERENA-1, an ongoing first-in-human study assessing AZD9833 as a monotherapy and in combination with palbociclib, established dose-dependent tolerability in pre- and post-menopausal women at doses of 25-450 mg once daily (QD), with clinical benefit and target engagement at all dose levels. Two randomized, open-label Phase 2 trials are also ongoing in women with ER+ HER2- breast cancer. SERENA-2 compares the efficacy of AZD9833 with fulvestrant in post-menopausal women with advanced breast cancer following treatment with ≤1 endocrine therapy. SERENA-3 will examine the biological effects of different doses of AZD9833 in treatment-naïve women with primary breast cancer. Methods SERENA-3 is a randomized, open-label, parallel-group, pre-surgical study to investigate the biological effects of different doses of AZD9833 in ER+, HER2- primary breast cancer. Eligible patients will be post-menopausal (and potentially pre-menopausal) women awaiting curative-intent surgery for newly diagnosed, ER+ HER2- primary breast cancer. The study is designed in two stages. In Stage 1, 24 post-menopausal women will be randomized 1:1 to receive either 75 mg or 150 mg oral AZD9833 QD for 5-7 days, followed by a minimum 5-day pre-surgery washout period; Stage 2 gives provision for additional groups depending on emerging data from Stage 1. The primary objective of this study is to explore the effect of AZD9833 on ER expression in pre- and on-treatment tumor samples from women with primary breast cancer, as assessed by immunohistochemistry and H-score. Safety and tolerability will be assessed as secondary endpoints, along with the pharmacokinetic and pharmacodynamic effects of AZD9833 on other biomarkers. Blood will be collected at screening, on the day of biopsy and the day of surgery to assess circulating tumor DNA and exploratory biomarkers. Primary endpoint analysis will be performed on the pharmacodynamic analysis set. The study will be conducted in 20 centers across 3 countries. For more information please contact Professor John Robertson at: john.robertson@nottingham.ac.uk. Citation Format: John FR Robertson, Iain Moppett, Juan Enrique Bargalló Rocha, Giorgi Dzagnidze, Joanna Harding, Teresa Klinowska, Richard Mather, Alastair Mathewson, Rhiannon Maudsley, Christopher J Morrow, Andrew Saunders, Andy Sykes, Li Zhang, Justin PO Lindemann. A randomized, pre-surgical study to investigate the biological effects of AZD9833 doses in women with ER-positive HER2-negative primary breast cancer (SERENA-3) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-09-05.

Background AZD9833 is a next-generation oral selective estrogen receptor (ER) antagonist and degrader (SERD) that has shown anti-tumor efficacy in a range of pre-clinical xenograft models of breast cancer. The first-in-human study, assessing AZD9833 as a monotherapy and in combination with palbociclib (SERENA-1; NCT03616587), established a dose-dependent safety profile with clinical benefit and target engagement in pre- and post-menopausal women at all dose levels. Here, we describe the design of SERENA-2, a Phase 2 randomized, open-label trial of three different doses of AZD9833 versus fulvestrant. Methods SERENA-2 is a global comparative study of three different doses of AZD9833 versus fulvestrant in post-menopausal women with advanced ER+, HER2− breast cancer with disease recurrence or progression after ≥1 endocrine therapy. The study will evaluate the efficacy and safety of AZD9833 monotherapy once daily at three dose levels, versus fulvestrant monotherapy administered according to its label. Eligible patients will have received no prior fulvestrant or other oral SERD, and no more than one endocrine therapy and one chemotherapy in the advanced setting. Prior treatment with CDK4/6 inhibitors is permitted. Patients will be randomized 1:1:1:1 to one of four treatment groups: AZD9833 75 mg, 150 mg, 300 mg, or fulvestrant. The primary objective of the study is to determine the clinical efficacy of AZD9833 as assessed by progression-free survival, compared with fulvestrant. Secondary objectives include objective response rate, duration of response, percentage change in tumor size at 16 weeks, clinical benefit rate at 24 weeks, and overall survival. Pharmacokinetics, pharmacodynamic biomarker changes from baseline, and effects of AZD9833 and fulvestrant on patients’ health-related quality of life will also be assessed. Exploratory endpoints include predictive markers of response and/or acquired resistance to AZD9833 and fulvestrant, including circulating tumor DNA mutation profiling and dynamics, circulating tumor cell enumeration, and analysis of tumor samples. Patient enrollment commenced in Q2 2020, with a target enrollment of 288 patients across approximately 100 sites in up to 17 countries. Efficacy analyses will compare each dose of AZD9833 with fulvestrant. Sample size was calculated to provide 80% power for the primary endpoint. The primary analysis will use a Cox proportional hazards model stratified by prior use of CDK4/6 inhibitors and presence of lung and/or liver metastases to compare progression-free survival in each dose of AZD9833 versus fulvestrant. Another randomized, open-label, parallel-group, pre-surgical study investigating the biological effects of AZD9833 in ER+, HER2− primary breast cancer (SERENA-3) is also ongoing. For more information please contact Dr Mafalda Oliveira at: moliveira@vhio.net. Citation Format: Mafalda Oliveira, Maxine Bennett, Ali Khalil, Richard Mather, Rhiannon Maudsley, Sam McGuinness, Christopher J Morrow, Andy Sykes, Li Zhang, Teresa Klinowska, Justin PO Lindemann, Danielle Carroll. A randomized, open-label, parallel-group, multicenter phase 2 study comparing the efficacy and safety of oral AZD9833 versus fulvestrant in women with advanced ER-positive HER2-negative breast cancer (SERENA-2) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-09-02.

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.