The combination of CDK4/6i and ET has reshaped treatment for HR+/HER2– breast cancer (Johnston et al., 2019; Finn et al., 2015; Finn et al., 2016; Turner et al., 2018; Dickler et al., 2017; Hortobagyi et al., 2022; Slamon et al., 2020; Im et al., 2019). However, resistance commonly emerges, and no consensus second-line standard is established. Our data show that continued CDK4/6i treatment in drug-resistant cells engages a noncanonical, proteolysis-driven route of Rb inactivation, yielding attenuated E2F output and a pronounced delay in G1 progression (Figure 7G). Concurrent ET further deepens this blockade by suppressing c-Myc-mediated E2F amplification, thereby prolonging G1 and slowing population growth. Importantly, CDK2 inhibition alone was insufficient to control resistant cells. Robust suppression of both CDK2 activity and resistant-cell growth required CDK2i in combination with CDK4/6i, consistent with prior reports supporting dual CDK targeting (Pandey et al., 2020; Freeman-Cook et al., 2021; Dietrich et al., 2024; Al-Qasem et al., 2022; Kudo et al., 2024; Arora et al., 2023; Kumarasamy et al., 2025; Dommer et al., 2025). Moreover, cyclin E blunted the efficacy of the CDK4/6i+CDK2i combination by reactivating CDK2. Together, these findings provide a mechanistic rationale for maintaining CDK4/6i beyond progression and support testing the combination of CDK4/6i and CDK2i, as evidenced by concordant in vitro and in vivo results.
Our data indicate that maintaining both CDK4/6i and ET synergistically decelerates cell-cycle progression in drug-resistant cells by further delaying CDK2 activation kinetics and the G1/S transition without affecting the S and G2 phases. This dual effect stems from CDK4/6i causing suboptimal Rb inactivation, while ET suppresses the global transcription amplifier c-Myc, collectively leading to diminished E2F transcriptional activity. As a result, this reduced E2F activity lowers the expression of critical cell-cycle genes, such as cyclin E and A, extending the time needed for CDK2 activation. Given that CDK2 plays an essential role in initiating and advancing DNA replication (Tanaka et al., 2007; Krude et al., 1997), its delayed activation significantly prolongs the G1/S transition. Moreover, CDK2 activation also contributes to Rb phosphorylation and inactivation. High CDK2 activity is required to phosphorylate Rb, and CDK2-mediated Rb phosphorylation is tightly coupled with DNA replication timing (Kim et al., 2022; Chung et al., 2019). Thus, upon Rb phosphorylation by CDK2 at the G1/S transition, drug-resistant cells may effectively proceed through the cell cycle even under continued CDK4/6i treatment.
Clinical trials evaluating the efficacy of sustained CDK4/6i therapy predominantly use PFS as the primary endpoint (Llombart-Cussac et al., 2025; Mayer et al., 2024; Kalinsky et al., 2025; Jhaveri et al., 2025; Kalinsky et al., 2023). However, our findings suggest that drug-resistant tumors continue to proliferate despite CDK4/6i maintenance. Consequently, maintaining CDK4/6i appears to slow tumor growth rather than completely arrest it. This underscores the need for clinical trials to consider overall survival and tumor progression rates as more appropriate endpoints for assessing the true benefits of sustained CDK4/6i therapy. Furthermore, the distinct polypharmacology profiles among CDK4/6i (Hafner et al., 2019), with ribociclib being the most specific and abemaciclib the least, may explain the varying therapeutic outcomes observed among these inhibitors (Kalinsky et al., 2023; Navarro-Yepes et al., 2023).
Maintaining CDK4/6i treatment beyond progression may be particularly beneficial for about 70% of patients who do not acquire new genetic mutations (O’Leary et al., 2018). However, it is important to recognize that resistance to CDK4/6i often arises from mutations in genes associated with mitogenic or hormone-signaling pathways (O’Leary et al., 2018; Mao et al., 2020; Wander et al., 2020; Formisano et al., 2017; Costa et al., 2020). These include mutations in PIK3CA, ESR1, FGFR1–3, and HER2, which have been linked to increased c-Myc expression (Shang et al., 2000; Zhu et al., 2008; Tsai et al., 2012). Additionally, previous studies have identified FAT1 mutations as a driver of CDK4/6i resistance (Li et al., 2022; Li et al., 2018). These resistance mutations may reduce the efficacy of maintaining CDK4/6i and ET therapy. Moreover, about 4.7% of HR+/HER2– breast cancer patients exhibit Rb mutations (O’Leary et al., 2018; Wander et al., 2020), making CDK4/6i treatment unlikely to be effective, thus making its continuation inadvisable in these cases.
In conclusion, our study provides mechanistic rationale for maintaining CDK4/6i together with ET after disease progression in HR+/HER2– breast cancers that retain an intact Rb/E2F pathway. The combination of CDK4/6i and CDK2i can further provide durable growth suppression, consistent with prior studies (Pandey et al., 2020; Freeman-Cook et al., 2021; Dietrich et al., 2024; Al-Qasem et al., 2022; Kudo et al., 2024; Arora et al., 2023; Kumarasamy et al., 2025; Dommer et al., 2025). However, it is essential to acknowledge that CDK2/4/6 inhibition may promote whole-genome duplication (Kim et al., 2025a), potentially fueling more aggressive tumor evolution. Finally, we identify cyclin E overexpression as a key driver of resistance to dual CDK4/6i and CDK2i therapy, providing a basis for biomarker-guided patient selection and the development of strategies to overcome therapeutic resistance.