Abstract LB_C07: Utilizing a BCL-2 inhibitor (venetoclax) to overcome doxorubicin resistance in triple-negative breast cancer
Authors: Dus Evan D , Smoots Stephen G , Schreiber Anna R , Jackson Marilyn M , Bagby Stacey M , Dominguez Adrian T A , Binns Cameron A , Pitts Todd M , Diamond Jennifer R 
Published: December 2023

Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of the progesterone receptor, estrogen receptor, and human epidermal growth factor-2 over-expression.  TNBC comprises 10-15% of all breast cancer cases and compared to other subtypes of breast cancer, TNBC is associated with a higher risk for metastatic recurrence and the chemotherapy agent doxorubicin (dox) remains a mainstay of treatment.  Senescence is a cellular phenomenon where cells are committed to an arrested state, however, senescent cells can secrete pro-tumorigenic factors which can help to promote tumor progression and invasion. Senescence is one potential mechanism of resistance to doxorubicin in breast cancer. Avoiding senescence-mediated resistance and promoting apoptosis is one strategy to improve response to dox in TNBC. Venetoclax is an orally bioavailable FDA approved BCL-2 inhibitor with activity in preclinical models of TNBC. The purpose of this study was to investigate venetoclax in combination with dox to overcome senescence-mediated resistance and induce apoptosis.

Experimental Procedures: TNBC cell lines were exposed to a no drug, dox, venetoclax or combination at increasing doses for 72 hours and cell viability was assessed via CellTiter-Glo. Synergy scores were calculated from three independent replicates using Synergy Finder+.  Apoptosis at 48 hours was analyzed by flow cytometry using Annexin V on cells treated in combination and with single agents.  In vitro cell survival following drug treatment for 24 hours was assessed via clonogenic assay.  In vitro senescence-associated β-gal was performed after 6 days drug treatment.  Athymic nude mice were subcutaneously injected with 5 million MDA-MB-231 cells for in vivo studies.  Mice were treated with either no drug, 60 mg/kg venetoclax PO QD, 1.5 mg/kg dox IP QW, or the combination.

Results: The combination of venetoclax and dox resulted in synergy based on CellTiter-Glo data, which was used to calculate Bliss scores using Synergy Finder+ in CAL-51, MDA-MB-231, and CAL-120 TNBC cell lines. The dox and venetoclax combination caused a statistically significant increase in apoptosis measured by Annexin V staining when compared to single agents and control in all cell lines.  The combination resulted in a decrease in cellular survival in all cell lines tested as measured by the clonogenic assay.  Senescence-associated beta-galactosidase showed an increase in senescent cells for both dox and venetoclax treated cells, which was eradicated in combination. Dox and venetoclax were further tested in vivo, where significant tumor growth inhibition was observed in combination compared to doxorubicin (p = 0.0031) and venetoclax alone (p = 0.0038) after 48 days of treatment.

Conclusion: The addition of venetoclax to doxorubicin resulted in decreased cellular proliferation, increased apoptosis, and decreased senescence.  As a result, venetoclax is a promising combination partner for doxorubicin to overcome doxorubicin resistance and promote apoptosis in TNBC.

Citation Format: Evan D Dus, Stephen G Smoots, Anna R Schreiber, Marilyn M Jackson, Stacey M Bagby, Adrian T A Dominguez, Cameron A Binns, Todd M Pitts, Jennifer R Diamond. Utilizing a BCL-2 inhibitor (venetoclax) to overcome doxorubicin resistance in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C07.