靶向 cyclin K-CDK12 通过限制 RPA 染色质加载与三阴性乳腺癌中的 ATR 抑制产生协同作用

Targeting cyclin K-CDK12 is a promising therapeutic due to its ability to induce homologous recombination (HR) repair deficiency through transcriptional regulation. Reduced CDK12 activity sensitizes HR repair-proficient tumor cells to PARP inhibitors (PARPi) and DNA-damaging agents. However, recent studies show that CDK12 -mutant cancers lack the genomic signatures associated with HR repair deficiency and derive limited therapeutic benefit from PARPi. It is therefore essential to understand additional mechanisms by which cyclin K-CDK12 depletion promotes genomic instability beyond impaired HR repair. Here, we demonstrate that cyclin K-CDK12 depletion drives a replication stress (RS)-associated vulnerability and synergizes with ATR inhibition, using CT7311 (Carrick Therapeutics), a protein degrader of cyclin K. Triple-negative breast cancer (TNBC) cells, especially those with high MYC expression, were the most sensitive to CT7311 compared with estrogen receptor-positive (ER+) BC and non-transformed breast cells. Notably, CT7311 sensitivity did not correlate with HR repair status. In a DNA damage response-focused CRISPR screen with CT7311, DNA replication-related genes were highly enriched among the sensitizers, with effects stronger than those of HR repair-related factors. We validated three sensitizers, TOPBP1 , PRIM2 , and PRPF19 , which are closely linked to ATR activation, a central RS regulator. Consistent with the screen, cyclin K-CDK12 depletion yielded synergistic cytotoxicity with ATR inhibition only in TNBC, not in ER+ BC cells. Mechanistically, CT7311 induces transcription-replication conflicts (TRCs), a known RS source, but fails to robustly activate ATR. Rather, it limits ATR activation by reducing the chromatin loading of Replication Protein A (RPA). This impaired ATR activation is further exacerbated when combined with tuvusertib (ATRi), leading to marked DNA damage specifically in replicating cells. Strikingly, RPA overexpression conferred resistance to the combination, indicating that the synergy arises from impaired availability of RPA. We further show that cyclin K-CDK12 regulates RPA chromatin loading via the PRPF19 E3-ligase complex. AlphaFold-Multimer predictions revealed interactions between cyclin K-CDK12 and the PRPF19 complex. CT7311 disrupted PRPF19 complex assembly, compromising its activity and impairing RPA ubiquitination. Notably, CDK12-mediated phosphorylation of CDC5L, a PRPF19 complex subunit, was required for RPA chromatin loading. Collectively, our work identifies cyclin K-CDK12 as a critical regulator of RPA dynamics and uncovers ATR dependency driven by cyclin K-CDK12 depletion. These insights extend the rationale for cyclin K-CDK12 targeted therapy beyond HR repair-focused applications, supporting its use in RS-high tumors including TNBC, as monotherapy or in combination with ATR inhibition.