Abstract PS1-13-27: A Non-invasive Approach to Assess the Response to CDK4/6 Inhibitors

Abstract Background: Dysregulation of cell cycle is one of the most important hallmarks of cancer. Cell cycle progression is driven by the phasic expression of cyclins and activation of cyclin-dependent kinases. Given that the CDK4/6-Rb axis is essential for the transition from G1 to S phase in the cell cycle, it is a popular drug target in several types of cancer. Although CDK4/6 inhibitors can effectively improve the prognosis of HR+/HER2- breast cancer, there are differences in the sensitivity to CDK4/6 inhibitors among individuals. Some patients show de novo resistance, while others acquire resistance to CDK4/6 inhibitors after a few cycles of therapy. There is thus an urgent need for a precise method to assess the therapeutic response to CDK4/6 inhibitors and to differentiate who is likely to be sensitive to those drugs. Methods: HSA-ICi were obtained by conjugated CDK4/6 inhibitor (Palbociclib or Ribociclib) with ICG-NHS, and blended them with human serum albumin (HSA) through self-assembly. To study the ability of HSA-ICi to assess the CDK4/6 kinase activity in living cells, luminal A breast cancer cells (including MCF-7 and T-47D) were pre-treated with different siRNA or Palbociclib and detected the change of the pRb protien, cell cycle distribution and mean fluorescence intensity (MFI) of HSA-ICi. The targeting ability of HSA-ICi in vivo was investigated by injecting the luminal A breast cancer cells-bearing ectopic and orthotopic tumor mice (hereafter called PalS-mice) with HSA-ICi or HSA-ICG and imaging them in NIR-II imaging system. To study whether HSA-ICi could assess the response to CDK4/6 inhibitor, we subjected the PalS-mice, or Palbociclib-resistant luminal A breast cancer cells-bearing tumor mice (hereafter called PalR-mice) to daily treatment with 150 mg/kg Palbociclib for 7 days. NIR-II imaging of mice before and after treatment were conducted. Results: We treated the MCF-7 with siRNA targeting CDK4 or CDK6 or CyclinD1 or CDK4/6-CyclinD1 complex, leading to a partial reduction of pRb level and G1 phase block in varying degrees in different groups. The MFI of the above cells with HSA-ICi showed a corresponding decrease. Palbociclib caused a dose-dependent decrease in pRb level, and as the concentration of Palbociclib increased, cells were almost arrested in G1 phase. When we applied HSA-ICi to visualize the inhibition of CDK4/6 kinase activity, we found that the does-dependent reduction of MFI was coincided with the decrease of pRb. The fluorescence signal of PalS-mice injected with HSA-ICi intensified primarily in the tumor, and the intensity could be significantly reduced by blocking with CDK4/6 inhibitor (P 0.01). Meanwhile, the tumor MFI of PalR-mice was significantly lower than that of PalS-mice (P 0.01). Subsequently, after a 7-day treatment with Palbociclib to PalS-mice and PalR-mice, we found there were clear reductions in the tumor MFI and cells positive for Ki67 and pRb in tumor tissue between baseline and day 7 of PalS-mice, even the volume of tumors did not decrease, yet there were no significant change in the tumor MFI of PalR-mice, demonstrating that HSA-ICi can non-invasively provide information of CDK4/6 kinase activity through in vivo NIR-II fluorescence imaging, allowing for the evaluation of early treatment efficacy of CDK4/6 inhibitors. And, HSA-ICi exhibited good biosafety. Conclusions: In this study, we developed a novel NIR-II fluorescence probe using clinically accessible materials. We successfully utilized it to detect CDK4/6 kinase activity and assess the efficacy of CDK4/6 inhibitors. This technology could offer an alternative method for non-invasive clinical assessment of CDK4/6 inhibitors efficacy and for distinguishing patients who are sensitive to CDK4/6 inhibitors. Citation Format: Y. Gao, J. Bai, G. Zhang. A Non-invasive Approach to Assess the Response to CDK4/6 Inhibitors 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 PS1-13-27.