Category: Preclinical Development
Purpose: The RON receptor tyrosine kinase plays a critical role in the pathogenesis of breast tumors including triple-negative breast cancer (TNBC). Recently, accumulated evidence has indicated that TNBC derived stem like-cells (SLCs) contribute to malignancy and chemoresistance. The objectives of this study are to determine the role of RON in TNBC-SLCs biology and the efficacy of RON targeted antibody-drug conjugate (ADC) Zt/g4-MMAE in killing TNBC SLCs and evaluation in preclinical TNBC xenograft models.
Methods: Monoclonal antibody Zt/g4 specific to human RON was conjugated with chemotherapeutic MMAE to generate anti-RON antibody-drug conjugate Zt/g4-MMAE. Two populations of TNBC SLCs with RONlow/CD44high/CD24low or RONhigh/CD44high/CD24low phenotypes were isolated from a panel of TNBC cells through FACS sorting. Various biochemical and biological studies were performed to determine the role of RON in TNBC SLCs spheroid formation, chemosensitivity, receptor endocytosis, and viability to Zt/g4-MMAE. Xenograft tumors were established mediated by TNBC SLCs for treatment with Zt/g4-MMAE.
Results: Analysis of TNBC SLCs with RONlow/CD44high/CD24low or CD44high/CD24low/RONhigh phenotype revealed that TNBC SLCs with RON expression displayed different biological behaviors. Expression of RON appeared to facilitate TNBC SLC spheroid development. These cells also were highly resistant to conventional chemotherapy agents in comparison to TNBC SLCs without RON expression. In addition, inhibition of RON signaling using specific small molecule inhibitor BMS-777607 was able to dramatically impaired TNBC SLC spheroid formation. Additional studies demonstrated that TNBC SLCs with RON expression displayed clonogenic potential and harbored long-term self-renewal capacity with increased expression of CSC self-renewal markers. These results suggest that RON expression not only contributes importantly for development and formation of TNBC SLCs but also acts as a pathogenic factor for the acquired chemoresistance of TNBC. Another interesting finding is that expression levels of RON were relatively higher in TSLCs compared to regular TNBC cells. We further show that sustained RON expression in TNBC SLCs is a suitable target for Zt/g4-MMAE-directed cell killing. We show that Zt/g4-MMAE, upon interaction with TNBC SLCs, is rapidly internalized with internalization efficacy (IE50) of ~12h, which facilitates the delivery of MMAE into the intracellular compartment for cytotoxicity. The IC50 values for the reduced TNBC SLC viability in vitro were in the range of 0.2 ~ 2.2 µg/ml. These indicate that TNBC SLCs with RON expression are highly sensitive to RON targeted ADC. Finally, we demonstrate in xenograft models that tumors initiated by TNBC SLCs are highly sensitive to Zt/g4-MMAE in a single dose treatment regimen with complete inhibition and eradication with long-lasting effect.
Conclusion: Our findings demonstrate that RON is a functional biomarker of TNBC SLCs, which plays a critical role in the stemness of TNBC. It is also a valuable indicator of biological behaviors of TNBC SLCs. We conclude from these studies that RON is a pathogenic biomarker contributing to TNBC SLC tumorigenesis. Zt/g4-MMAE directed therapy is highly effective in eradicating TNBC SLCs in tumor xenograft models, which lays the foundation for using RON targeted ADCs as a novel strategy for potential TNBC treatment.