Abstract A077: Multi-pathway inhibition and synergistic anticancer activity of novel biguanides in triple negative breast cancer models

Abstract

Triple-negative breast cancer (TNBC) is an aggressive subtype defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and HER2 amplification and is associated with early recurrence, metastasis, and poor overall survival. Increasing evidence implicates metabolic dysregulation as a major promoter of TNBC progression, particularly hyperinsulinemia and activation of the PI3K/AKT/mTOR signaling cascade. Novel therapies such as cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have transformed the management of ER-positive breast cancer (BC), leading to significantly improved clinical outcomes, however lack efficacy in TNBC. The PI3K/AKT/mTOR signaling cascade is the most frequently activated pathway in metastatic BC and provides a bypass route that enables continued cell proliferation despite CDK4/6 blockade, thereby driving resistance to CDK4/6 inhibitors and justifying a combined treatment targeting cell cycle and mTOR signaling. We have developed a novel treatment for TNBC based on the biguanide metformin structure, termed JD006, which significantly inhibits TNBC cell growth in vitro and in vivo in a dose-dependent manner and at lower doses than its parental metformin (p<0.01). In syngeneic mouse TNBC models, JD006 reduced TNBC tumor growth and lung metastasis. Transcriptome analysis revealed a decrease in expression of oxidative phosphorylation genes in the lungs of treated mice, and oral administration of JD006 did not produce significant alteration of blood glucose or lactate levels. Mechanistically, JD006 treatment induces AMPK phosphorylation and attenuates mTORC1 signaling, evidenced by reduced phosphorylation of S6 ribosomal protein and 4E-BP1. Seahorse assays reveal marked inhibition of oxidative phosphorylation and ATP synthase along with a compensatory increase in glycolytic ATP production. Furthermore, JD006 induces apoptosis through the intrinsic pathway by reducing levels of Bcl-2, Bcl-xL, and Bax. In addition, JD006 decreases cyclin D1 expression, thereby functionally intersecting with CDK4/6-mediated control of Rb phosphorylation and the G1–S cell cycle transition. When combined with selective CDK4/6 inhibitors, JD006 produces additive and synergistic antiproliferative effects in vitro, accompanied by enhanced suppression of Rb phosphorylation compared with either agent alone. In vivo, combination therapy significantly reduced tumor growth relative to single-agent alone in orthotopic TNBC mouse xenografts (p<0.01). Collectively, these findings support a therapeutic paradigm in which simultaneous disruption of mitochondrial bioenergetics, apoptosis promotion and cyclin D–CDK4/6–Rb signaling exploits a key metabolic–cell cycle dependency in TNBC. This work not only elucidates previously underappreciated mechanisms of biguanide-mediated metabolic reprogramming but also provides a strong preclinical rationale for combining next-generation biguanides with CDK4/6 inhibitors as a novel strategy for this high-risk breast cancer subtype. Julie Liu, Mario Morales Martinez, Eduardo Mauricio Gonzalez, Linsey Stiles, Nalo Hamilton, Michael E. Jung, Richard J. Pietras, Diana Marquez-Garban. Multi-pathway inhibition and synergistic anticancer activity of novel biguanides in triple negative breast cancer models [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr A077.