At the MONARCH 3 interim analysis, abemaciclib plus a nonsteroidal aromatase inhibitor (AI) significantly improved progression-free survival (PFS) and objective response rate (ORR) with a tolerable safety profile as initial treatment for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2−) advanced breast cancer (ABC). MONARCH 3 is a randomized, phase III, double-blind study of abemaciclib/placebo (150 mg twice daily, continuous) plus nonsteroidal AI (1 mg anastrozole or 2.5 mg letrozole, daily). A total of 493 postmenopausal women with HR+, HER2− ABC with no prior systemic therapy in this setting were enrolled. The primary endpoint was investigator-assessed PFS (final analysis after 240 events); other endpoints included response and safety evaluations. Here we analyze the final PFS data and update secondary endpoints. The abemaciclib arm had a significantly longer median PFS than the placebo arm (28.18 versus 14.76 months; hazard ratio [95% confidence interval], 0.540 [0.418–0.698]; p = .000002). The ORR was 61.0% in the abemaciclib arm versus 45.5% in the placebo arm (measurable disease, p = .003). The median duration of response was longer in the abemaciclib arm (27.39 months) compared to the placebo arm (17.46 months). The safety profile was consistent with previous reports. The most frequent grade ≥ 3 adverse events in the abemaciclib versus placebo arms were neutropenia (23.9% versus 1.2%), diarrhea (9.5% versus 1.2%), and leukopenia (8.6% versus 0.6%). Abemaciclib plus a nonsteroidal AI was an effective initial treatment with an acceptable safety profile for HR+, HER2− ABC.

Purpose Inhibitors of the DNA damage response (DDR) have great potential for radiosensitization of numerous cancers including glioblastomas (GBM), which are extremely radio- and chemo-resistant brain tumors. Currently, there are no DNA double-strand break (DSB) repair inhibitors that have been successful in treating GBM. Our lab has previously demonstrated that the dual PI3K/mTOR inhibitor NVP-BEZ235 can potently inhibit the two central DDR kinases, DNA-PKcs and ATM, in vitro. Here, we tested whether NVP-BEZ235 could also inhibit ATM and DNA-PKcs in tumors in vivo and assessed its potential as a radio- and chemo-sensitizer in pre-clinical mouse GBM models. Experimental design The radiosensitizing effect of NVP-BEZ235 was tested by following tumor growth in subcutaneous and orthotopic GBM models. Tumors were generated using the radioresistant U87-vIII glioma cell line and GBM9 neurospheres in nude mice. These tumors were then treated with ionizing radiation (IR) and/or NVP-BEZ235 and analyzed for DNA-PKcs and ATM activation, DSB repair inhibition, and attenuation of growth. Results NVP-BEZ235 potently inhibited both DNA-PKcs and ATM kinases and attenuated the repair of IR-induced DNA damage in tumors. This resulted in striking tumor radiosensitization, which extended the survival of brain tumor-bearing mice. Notably, tumors displayed a higher DSB-load when compared to normal brain tissue. NVP-BEZ235 also sensitized a subset of subcutaneous tumors to temozolomide, a drug routinely used concurrently with IR for the treatment of GBM. Conclusions These results demonstrate that it may be possible to significantly improve GBM therapy by combining IR with potent and bioavailable DNA repair inhibitors like NVP-BEZ235.

Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. While CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival, and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favor HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In sum, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice.

End resection of DNA double-strand breaks (DSBs) to generate 3'-single-stranded DNA facilitates DSB repair via error-free homologous recombination (HR) while stymieing repair by the error-prone non-homologous end joining (NHEJ) pathway. Activation of DNA end resection involves phosphorylation of the 5' to 3' exonuclease EXO1 by the phosphoinositide 3-kinase-like kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related) and by the cyclin-dependent kinases 1 and 2. After activation, EXO1 must also be restrained to prevent over-resection that is known to hamper optimal HR and trigger global genomic instability. However, mechanisms by which EXO1 is restrained are still unclear. Here, we report that EXO1 is rapidly degraded by the ubiquitin-proteasome system soon after DSB induction in human cells. ATR inhibition attenuated DNA-damage-induced EXO1 degradation, indicating that ATR-mediated phosphorylation of EXO1 targets it for degradation. In accord with these results, EXO1 became resistant to degradation when its SQ motifs required for ATR-mediated phosphorylation were mutated. We show that upon the induction of DNA damage, EXO1 is ubiquitinated by a member of the Skp1-Cullin1-F-box (SCF) family of ubiquitin ligases in a phosphorylation-dependent manner. Importantly, expression of degradation-resistant EXO1 resulted in hyper-resection, which attenuated both NHEJ and HR and severely compromised DSB repair resulting in chromosomal instability. These findings indicate that the coupling of EXO1 activation with its eventual degradation is a timing mechanism that limits the extent of DNA end resection for accurate DNA repair.