Abstract 599: Combination of CDK4/6 inhibitors with biguanides for treatment of triple negative breast cancer
Authors: Martinez Mario Morales , Gonzalez Eduardo Mauricio , Deng Gang , Ma Gaoyuan , Kim Hyunsoo , Stiles Linsey , Hamilton Nalo , Jung Michael E. , Pietras Richard J. , Marquez-Garban Diana C. 
Published: March 2024

Abstract
Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC) and is associated with early relapse and high frequency of metastasis. This has been attributed in part to the lack of targeted therapies. TNBC occurs more frequently in younger women of African and Hispanic ancestry and with a higher risk of mortality than women of European ancestry. Emerging data shows activation of the AKT/mTOR pathway by insulin may occur in aggressive TNBC and inhibition of mTOR signaling by rapamycin, a potent mTOR inhibitor, induces cell cycle arrest and downregulation of cyclin D1 in TNBC cells. CDK4/6 inhibitors have demonstrated to be an effective therapeutic for estrogen receptor positive BC and although their use in TNBC patients remains uncertain their use in combination with other targeted therapies may be promising. We have developed a set of newly designed biguanides with potent anticancer action and safety doses in vivo that in combination with CDK4/6 inhibitors block TNBC cell growth in vitro and in vivo. Using TNBC cell proliferation in vitro assays, new biguanides show dose-dependent inhibition of cell proliferation (P&lt;0.01). Further, combination of biguanides with ribocliclib, an orally bioavailable CDK4/6 inhibitor, produced additive and synergistic effects in TNBC cell lines in vitro (P&lt;0.01). Biguanides are known to activate de LKB1-AMPK pathway. Western immunoblots showed our biguanide analogues also induce AMPK phosphorylation and significantly reduce phosphorylation of downstream mTOR signaling pathway components including S6 ribosomal protein and 4E-BP1, an effect that is potentiated by combination treatment with ribociblib. Further, cyclin D1 is known to be overexpressed in about 50% of BCs and binding to CDK 4/6 induces phosphorylation of Rb and cell cycle progression. Treatment with biguanides downregulated cyclin D1 expression in TNBC cells and combination treatment of ribociclib with biguanide JD006 was more effective at decreasing Rb phosphorylation than each drug alone. In addition, seahorse analysis evaluated mitochondrial respiratory function. Treatment with biguanides inhibited oxidative phosphorylation in a dose dependent manner, an important metabolic pathway that can drive drug resistance to therapy in cancer cells. In vivo, combination treatment of ribociclib with biguanide JD006 was more effective than each agent alone in stopping human TNBC xenograft progression in nude mouse models (P&lt;0.05). Our preliminary data offer evidence that human TNBC are highly vulnerable to metabolic reprogramming by new biguanides. This work can yield insight on unanticipated mechanisms of biguanide action and rationale for new combination therapies with CDK 4/6 inhibitors as a new option for treatment of TNBC. Funding: CBCRP B27IB3869, JCCC Breast Cancer Award, Hickey Family Foundation, NCI U54 CA143930, Team Research Grant, UCLA TDG.
Citation Format: Mario Morales Martinez, Eduardo Mauricio Gonzalez, Gang Deng, Gaoyuan Ma, Hyunsoo Kim, Linsey Stiles, Nalo Hamilton, Michael E. Jung, Richard J. Pietras, Diana C. Marquez-Garban. Combination of CDK4/6 inhibitors with biguanides for treatment of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 599.