Abstract P4-01-10: A kinase inhibitor library screen reveals novel candidates that reverse CDK4/6 inhibitor resistance in CDK6 amplified HR(+) breast cancer
Authors: Lai Jiun-I , Zhuang Yong-Ji , Lin Ting-Yi , Chao Ta-Chung , Liu Chun-Yu , Tseng Ling-Ming 
Published: March 2022

Background and rationale: Metastatic HR (+) breast cancer currently still remains an incurable disease. CDK4/6 inhibitors in combination with endocrine therapy have emerged as standard 1st and 2nd lines in metastatic HR (+) breast cancer. Resistance to CDK4/6 inhibitors eventually occurs, and currently the optimal subsequent treatment for these patients remains unsettled. Based on the excellent tolerability and efficacy of CDK4/6 inhibitors, it remains a clinical interest to identify mechanisms of resistance towards CDK4/6 inhibitors in breast cancer. This is currently an area of intense research, with many resistance models being proposed, including loss of Rb, upregulation of cell cycle components (CDK2, cyclin E, AURKA), activation of growth factors (HER2, FGFGR), and the Hippo pathway. The activation of the Hippo pathway downstream gene YAP resulted in CDK6 overexpression that further led to CDK4/6 inhibitor resistance. This was confirmed in preclinical models and in patients developing resistance towards CDK4/6 inhibitors. The mechanism for how increased CDK6 activity restricts efficacy towards CDK4/6 inhibitors is unknown. We sought to discover kinase inhibitors that could downregulate CDK6 expression and CDK6 overexpression mediated resistance. Our goal is to develop therapeutics that could be added on CDK4/6 inhibitors upon progression to reverse the resistance, and to uncover pathways that modulate the relevant interactions. Results: We individually overexpressed YAP and CDK6 in the HR(+) breast cancer cell line MCF-7 and T47D and confirmed that each could increase the IC50 of CDK4/6 inhibitors. We screened a small library of 160 kinase inhibitors for candidates that could decrease CDK6 expression in a luciferase reporter assay and identified several PI3K inhibitors as well as the PKC inhibitor staurosporine that could significantly decrease CDK6 expression. Interestingly, we demonstrated that staurosporine could modulate the Hippo-YAP signaling pathway and thus modulate CDK6 expression. We further confirmed that when combined to CDK4/6 inhibitors, the addition of these candidates reversed the resistance in YAP or CDK6 overexpressing cells. Incidentally, we discovered that a mutant (D224Y) in CDK6 abolished the resistance in CDK6 overexpressing cells. CDK6-D224Y overexpressing MCF7 cells lost their resistance towards CDK4/6 inhibitors, despite that CDK6-D224Y was indistinguishable from CDK6 in kinase activity and function. D224Y resides in an alpha helix far apart from the activation loop of the CDK6 active site, therefore suggesting that mutations in CDK6 that leads to possible conformation changes could modulate the resistance to CDK4/6 inhibitors. We performed BRET (bioluminescence resonance energy transfer) and confirmed that the D224Y changed the BRET values for a palbociclib luminescent tracer, suggesting that this mutant could modulate the binding affinity of CDK4/6 inhibitors towards CDK6. In summary, we identified several novel candidates as well as a CDK6 mutant (D224Y) that could therapeutically reverse CDK4/6 inhibitor resistance conferred by CDK6 overexpression. Conclusions: We identified the PI3K pathway and the Hippo pathway as potential pathways that could modulate the CDK4/6 inhibitors resistance from CDK6 amplification. We further identified the D224Y mutant as a point mutation that abolished resistance. Our findings highlight the role of CDK6 and conformational variants in modulating CDK4/6 inhibitor resistance and uncover potential pathways that could be exploited for therapeutic development for subsequent treatment.

Citation Format: Jiun-I Lai, Yong-Ji Zhuang, Ting-Yi Lin, Ta-Chung Chao, Chun-Yu Liu, Ling-Ming Tseng. A kinase inhibitor library screen reveals novel candidates that reverse CDK4/6 inhibitor resistance in CDK6 amplified HR(+) breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-10.