ABSTRACT The PI3K–mTOR–AKT pathway regulates tumour proliferation, gene expression and metabolism, but pathway inhibition induces heterogeneous feedback reactivation, limiting anti-tumour responses. Measuring heterogeneity of pathway inhibition in tissues using protein biomarker phosphorylation or location is challenging. An integrated multi-modal imaging workflow was developed to assess the heterogeneity of AZD2014 (mTORC1/2 inhibitor) response in a PTEN-null renal cancer model. Spatial responses of metabolite biomarkers were analysed by mass spectrometry imaging (MSI). Control and treated tumours were classified according to metabolite-defined regions enriched in control versus AZD2014-treated tumours, respectively. Noticeably, AZD2014-treated tumours retained regions similar to regions dominant in untreated tumours. Imaging mass cytometry analysis of protein biomarkers in ‘control-like’ regions following AZD2014 treatment showed reduced phospho-S6, indicating suppression, but retained high expression of the glucose transporter GLUT1. Increasing PI3K–AKT inhibition by combining with AZD8186 (PI3Kβ inhibitor) further decreased the control-like metabolic signature, showing PI3K-dependent resistance. This demonstrates that MSI-based workflows yield novel insights into the pharmacodynamic effects of mTORC1/2 inhibition in tumours, which classical biomarkers do not resolve. Coupling these workflows with spatial-omics approaches can deliver greater insights into heterogeneity of treatment response.

Supplementary Figure 2 shows polyploidy and apoptosis are inhibited by high BCL2 levels

Supplementary table shows AZD2811 sensitivity, transcriptomic and proteomic profiles of SCLC cell lines

Supplementary Figure 3 shows the combination of AZD2811 and BCL2 inhibitor enhances apoptosis and cell death.

Supplementary Figure 1 shows BCL2 is a strong biomarker of resistance to AURK inhibition in SCLC.

Supplementary Figure 4 shows the combination of AZD2811NP with venetoclax is well tolerated in mice and less efficacious in SCLC models with low BCL2 expression.

Purpose/Objective(s) Glioblastoma (GBM), the most common primary brain malignancy in adults, is an aggressive cancer with limited life expectancy. The standard of care backbone for newly diagnosed GBM is intensity-modulated radiation therapy (IMRT) with concomitant and adjuvant temozolomide. AZD1390, an oral, highly potent, and selective ataxia telangiectasia mutated kinase inhibitor, is designed to augment the efficacy of IMRT without exacerbating neurotoxicity. AZD1390 is optimized for blood brain barrier penetration, confirmed in a human healthy volunteer PET study (NCT03215381) and a Phase 0 study in patients (pts) with GBM (NCT05182905). This global, Phase 1, open-label study (NCT03423628) evaluates the safety and preliminary efficacy of escalating doses of AZD1390 and radiation therapy (RT) in pts with GBM and brain metastases. Materials/Methods Eligible adult pts received escalating once-daily AZD1390 doses following a Bayesian continual reassessment method, with IMRT 35 Gy in 10 fractions over 2 weeks (Arm A, recurrent GBM) or IMRT 60 Gy in 30 fractions over 6 weeks (Arm C, newly diagnosed, O-6-methylguanine-DNA methyltransferase unmethylated GBM). Pts in both Arms received 2 additional weeks of adjuvant AZD1390 post-IMRT. Arm B included pts with brain metastases but was closed due to low recruitment and is not reported. Primary objective was safety; secondary objectives included clinical efficacy and pharmacokinetics (PK). Results As of Feb 6, 2024, 115 pts have received AZD1390 (75 in Arm A; 40 in Arm C) at doses of 10–900 mg/day. PK was linear with a slightly more than dose-proportional increase and a mean terminal elimination half-life around 9–11 hours. Most patients had ≥1 treatment-emergent adverse event (AE); the most common were fatigue (51.3%), nausea (39.1%) and headache (38.3%). Grade ≥3 AZD1390-related AEs occurred in 18/115 pts (15.7%). The maximum tolerated dose was identified as 400 mg in Arm A and 300 mg in Arm C. Dose limiting toxicities included, but were not limited to, creatinine kinase elevation in Arm A and radiation skin injury in Arm C. Treatment was well tolerated, with the majority of reported AEs being low-grade. AEs led to discontinuation of AZD1390 in 13.0% of pts. The safety profile was consistent across Arms despite the different RT schedules; the most notable difference was in radiation skin injury, a reversible event, with higher frequency and severity in Arm C. At doses demonstrating target engagement in the Phase 0 study, median overall survival was 12.7 months (95% CI = 10.7, 18.9, n = 21) for Arm A and is still maturing for Arm C. Conclusion Concurrent AZD1390 and IMRT administration is tolerated with a manageable safety profile, at doses shown to achieve clear target engagement in the Phase 0 study. Preliminary efficacy is encouraging in Arm A. These data suggest the potential for AZD1390 to act as a radiosensitizer for the treatment of GBM. Clinical investigation is ongoing.

Abstract BACKGROUND Glioblastoma is an aggressive cancer; intensity-modulated radiation therapy (IMRT) with concomitant and adjuvant temozolomide is the first-line standard of care. AZD1390, an ataxia telangiectasia mutated kinase inhibitor, aims to augment the efficacy of IMRT without exacerbating neurotoxicity. AZD1390 is optimized for blood–brain barrier penetration, confirmed in healthy volunteers (NCT03215381) and patients with glioblastoma (Phase 0; NCT05182905). This Phase 1, open-label study (NCT03423628) evaluated safety and efficacy of AZD1390 and IMRT in patients with glioblastoma. METHODS Adult patients received escalating once-daily AZD1390 doses with IMRT 35 Gy in 10 fractions over 2 weeks (Arm A, recurrent glioblastoma) or IMRT 60 Gy in 30 fractions over 6 weeks (Arm C, newly-diagnosed O-6-methylguanine-DNA methyltransferase unmethylated glioblastoma). Patients received 2 additional weeks of adjuvant AZD1390 post-IMRT. Primary objective was safety; secondary objectives included efficacy and pharmacokinetics. RESULTS As of Feb 6, 2024, 115 patients (Arm A=75; Arm C=40) received AZD1390 10–900 mg/day. Pharmacokinetics were linear, with a slightly more-than dose-proportional increase and mean elimination half-life ~9–11 hours. Most patients had ≥1 treatment-emergent adverse event (AE), most commonly fatigue (51.3%), nausea (39.1%) and headache (38.3%). Grade ≥3 AZD1390-related AEs occurred in 18/115 patients. The maximum tolerated dose was 400 mg (Arm A) and 300 mg (Arm C). Dose-limiting toxicities included creatinine kinase elevation (Arm A) and radiation skin injury (Arm C). AEs led to AZD1390 discontinuation in 15/115 patients. Safety profiles were mostly consistent across Arms despite different radiation schedules. At target-engaging doses, median overall survival was 12.7 months (95% CI: 10.7–18.9; Arm A) and still maturing (Arm C). CONCLUSIONS Concurrent AZD1390 and IMRT demonstrated manageable safety at doses with known target engagement. Preliminary efficacy is encouraging (Arm A). AZD1390 can potentially act as a radiosensitizer for glioblastoma treatment. Clinical investigation is ongoing.

Design and outcome details for each AZD8835 in vivo efficacy study.

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

MCF7 and MCF7-LTED cells were treated {plus minus} AZD5363 for 24-hours and RNA was submitted to microarray analysis.

Cell lines were treated as described with GI50 concentrations of each drug alone or in combination, as indicated in the graphs. For Combination Index Tables shown, each graph cells were treated with AZD5363 and a combination of (A) 4-OHT, (B) fulvestrant and (C) anastrozole over a 6-day period. CIs were derived using Calcusyn software and significance was calculated, as detailed in the Methods.

Combination of AZD8835 with mTOR kinase inhibitor AZD2014 (+/- fulvestrant) increases anti-tumor efficacy in breast xenografts

Gene expression changes in response to AZD2014, fulvestrant and the combination.

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

Body Weight time-courses for AZD8835 combination efficacy studies.

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.

Tumor cell line panel composition and growth inhibition sensitivity (GI50) to AZD8835.

Supplementary methods for in vivo studies

Cells were treated in absence or presence of exogenous E2 (0.01nM), 1�S or androstenedione and increasing concentrations of AZD5363. Treatments were performed at day 1 and day 3 after seeding. After 6 days of treatment, cell viability was analyzed by using a cell titre-glo assay. Data are expressed as fold-change relative to vehicle control. Error bars represent {plus minus} SEM.