Abstract RF4-04: Mechanisms of resistance to capivasertib in combination with CDK4/6 inhibitor (CDK4/6i) plus fulvestrant in patients with hormone receptor-positive/HER2-negative (HR+/HER2−) advanced breast cancer (ABC): exploratory analysis from the

Abstract Background: PI3K/AKT pathway activation is implicated in resistance to endocrine therapy (ET) + CDK4/6i. Simultaneous inhibition of PI3K/AKT and CDK4/6 pathways may delay ET and/or CDK4/6i resistance, and/or re-sensitize tumors to ET plus CDK4/6i. Results from Phase 1b of the CAPItello-292 Phase 1b/3 trial (NCT04862663) have shown that triplet therapy with pan-AKT inhibitor capivasertib + CDK4/6i (palbociclib or ribociclib) + fulvestrant is tolerable in patients with HR+/HER2− ABC, with preliminary evidence of clinical activity. Most Phase 1b patients were heavily pre-treated and had received prior CDK4/6i treatment. This exploratory analysis from Phase 1b of CAPItello-292 examined matched pre- and post-treatment circulating tumor DNA (ctDNA) to identify potential genomic mechanisms associated with resistance to the triplet treatment. Methods: Baseline and end-of-treatment (EOT) ctDNA were tested using the Guardant360 Liquid assay on the Guardant Infinity platform. Somatic alterations of known/likely significance with variant allele fraction ≥0.3% (limit of quantification) were evaluated. Copy number changes, single nucleotide variants, fusions, and indels in EOT ctDNA were considered to be acquired only if absent in baseline ctDNA and archival tumor tissue. Separately, genome-wide CRISPR-Cas9 knockout screens were performed in estrogen receptor-positive (ER+) breast cancer cell lines (MCF7, CAMA-1, and T47D) to discover genomic modifiers of response/resistance following 3 weeks of treatment with combination treatments: capivasertib + fulvestrant and fulvestrant + CDK4/6i. Moreover, a stringent genome-wide CRISPR knockout screen (10 days treatment) was conducted in ER+ breast cancer cells to elucidate mechanisms of resistance to capivasertib + fulvestrant + palbociclib. Results: Across the CAPItello-292 Phase 1b population, paired ctDNA samples from 48 patients were analyzed; 42/48 patients discontinued treatment due to disease progression. 259 genetic alterations were detected at baseline and 304 alterations at the EOT. 30 patients had ≥1 newly detected alteration at EOT (30/48, 63%). Alterations in targets downstream of AKT were observed in 15% (7/48) at EOT, compared with 8% (4/48) at baseline, particularly loss-of-function mutations in TSC1, a tumor suppressor that controls mTORC1 signaling, and amplifications in RICTOR, which encodes the catalytic subunit of the mTORC2 complex. RAS pathway alterations were present in 25% (12/48) of patients at EOT versus 17% (8/48) at baseline. Cell cycle gene alterations were detected at EOT in 46% (22/48) versus 42% (20/48) at baseline. Newly detected PIK3CA/PTEN alterations were found at EOT in 6% (3/48) of patients. The specific PIK3CA/PTEN alterations identified at EOT included PIK3CA E545K, PIK3CA H1047R, and PTEN rearrangement, known to be sensitive to capivasertib. CRISPR resistance screen identified loss of TSC1, TSC2 and STK11 (AMPK pathway regulator) as key genes limiting response to capivasertib + fulvestrant. Resistance to the fulvestrant + palbociclib combination was associated with loss of cell cycle control in particular loss of RB. Identified mechanisms of resistance to capivasertib + fulvestrant + palbociclib combination further confirmed that loss of TSC1, TSC2 and STK11 attenuates the effectiveness of the triplet therapy. Conclusions: Consistent with AKT being a pivotal node in the PI3K/AKT signaling pathway, these data show that treatment with capivasertib + fulvestrant + CDK4/6i resulted in emergence of alterations associated with activation of alternate mechanisms, such as mTORC1, RAS, and AMPK. The CRISPR data provides additional support to clinical observations. Citation Format: D. R. Sudhan, C. Salinas-Souza, V. Cutano, I. De Toma, S. Dunn, J. Bradley, P. Neven, M. Beeram, E. Hamilton, B. Pistilli, A. Raskov Kodahl, P. Lau, V. F. Borges, M. Campone, T. Foukakis, E. Lim, I. Ługowska, P. Wysocki, I. Oshiomogho, V. Bhagawati Prasad, C. Gresty, U. McDermott, S. Barry, E. de Bruin, J. Armenia, H. S. Rugo. Mechanisms of resistance to capivasertib in combination with CDK4/6 inhibitor (CDK4/6i) plus fulvestrant in patients with hormone receptor-positive/HER2-negative (HR+/HER2−) advanced breast cancer (ABC): exploratory analysis from the Phase 1b CAPItello-292 study [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr RF4-04.