A Phase I Expansion Cohort Study Evaluating the Safety and Efficacy of the CHK1 Inhibitor LY2880070 with Low-dose Gemcitabine in Patients with Metastatic Pancreatic Adenocarcinoma.

PURPOSE: Combining gemcitabine with CHK1 inhibition has shown promise in preclinical models of pancreatic ductal adenocarcinoma (PDAC). Here, we report the findings from a phase I expansion cohort study (NCT02632448) investigating low-dose gemcitabine combined with the CHK1 inhibitor LY2880070 in patients with previously treated advanced PDAC. PATIENTS AND METHODS: Patients with metastatic PDAC were treated with gemcitabine intravenously at 100 mg/m2 on days 1, 8, and 15, and LY2880070 50 mg orally twice daily on days 2-6, 9-13, and 16-20 of each 21-day cycle. Pretreatment tumor biopsies were obtained from each patient for correlative studies and generation of organoid cultures for drug sensitivity testing and biomarker analyses. RESULTS: Eleven patients with PDAC were enrolled in the expansion cohort between August 27, 2020 and July 30, 2021. Four patients (36%) experienced drug-related grade 3 adverse events. No objective radiologic responses were observed, and all patients discontinued the trial by 3.2 months. In contrast to the lack of efficacy observed in patients, organoid cultures derived from biopsies procured from two patients demonstrated strong sensitivity to the gemcitabine/LY2880070 combination and showed treatment-induced upregulation of replication stress and DNA damage biomarkers, including pKAP1, pRPA32, and γH2AX, as well as induction of replication fork instability. CONCLUSIONS: No evidence of clinical activity was observed for combined low-dose gemcitabine and LY2880070 in this treatment-refractory PDAC cohort. However, the gemcitabine/LY2880070 combination showed in vitro efficacy, suggesting that drug sensitivity for this combination in organoid cultures may not predict clinical benefit in patients.

TGFß pathway is required for viable gestation of Fanconi anemia embryos.

Overexpression of the TGFß pathway impairs the proliferation of the hematopoietic stem and progenitor cells (HSPCs) pool in Fanconi anemia (FA). TGFß promotes the expression of NHEJ genes, known to function in a low-fidelity DNA repair pathway, and pharmacological inhibition of TGFß signaling rescues FA HSPCs. Here, we demonstrate that genetic disruption of Smad3, a transducer of the canonical TGFß pathway, modifies the phenotype of FA mouse models deficient for Fancd2. We observed that the TGFß and NHEJ pathway genes are overexpressed during the embryogenesis of Fancd2-/- mice and that the Fancd2-/-Smad3-/- double knockout (DKO) mice undergo high levels of embryonic lethality due to loss of the TGFß-NHEJ axis. Fancd2-deficient embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. Strikingly, the few DKO survivors have activated the non-canonical TGFß-ERK pathway, ensuring expression of NHEJ genes during embryogenesis and improved survival. Activation of the TGFß-NHEJ axis was critical for the survival of the few Fancd2-/-Smad3-/- DKO newborn mice but had detrimental consequences for these surviving mice, such as enhanced genomic instability and ineffective hematopoiesis.

CCAR2 functions downstream of the Shieldin complex to promote double-strand break end-joining.

The 53BP1-RIF1 pathway restricts the resection of DNA double-strand breaks (DSBs) and promotes blunt end-ligation by non-homologous end joining (NHEJ) repair. The Shieldin complex is a downstream effector of the 53BP1-RIF1 pathway. Here, we identify a component of this pathway, CCAR2/DBC1, which is also required for restriction of DNA end-resection. CCAR2 co-immunoprecipitates with the Shieldin complex, and knockout of CCAR2 in a BRCA1-deficient cell line results in elevated DSB end-resection, RAD51 loading, and PARP inhibitor (PARPi) resistance. Knockout of CCAR2 is epistatic with knockout of other Shieldin proteins. The S1-like RNA-binding domain of CCAR2 is required for its interaction with the Shieldin complex and for suppression of DSB end-resection. CCAR2 functions downstream of the Shieldin complex, and CCAR2 knockout cells have delayed resolution of Shieldin complex foci. Forkhead-associated (FHA)-dependent targeting of CCAR2 to DSB sites re-sensitized BRCA1-/-SHLD2-/- cells to PARPi. Taken together, CCAR2 is a functional component of the 53BP1-RIF1 pathway, promotes the refill of resected DSBs, and suppresses homologous recombination.