Phase I crossover study of DNA-protein kinase inhibitor peposertib in healthy volunteers: Effect of food and pharmacokinetics of an oral suspension.

Peposertib is an orally administered inhibitor of DNA-dependent protein kinase. We evaluated the effect of food on its pharmacokinetics, and examined the pharmacokinetics of an oral suspension (OS) of disintegrated tablets, in a phase I, open-label, crossover three-period study (NCT04702698). Twelve healthy volunteers were randomized to one of six treatment sequences. They received a single dose of peposertib 100 mg as film-coated tablets under fasted or fed conditions ("tablet fasted" or "tablet fed") or as an OS under fasted conditions ("OS fasted"), with washout between treatments. Using healthy volunteers was possible because, despite its mechanism of action being suppression of DNA repair, peposertib has shown no genotoxic effect in animals. A mild food effect was observed with peposertib tablets. Fed-to-fasted ratios were: area under the curve from time 0 to time t (AUC0-t ), 123.81% (90% confidence interval [CI]: 108.04, 141.87%); AUC from zero to infinity (AUC0-∞ ), 110.28% (90% CI 100.71, 120.77%); and maximum concentration (Cmax ) 104.47% (90% CI: 79.15, 137.90%). Cmax was delayed under fed conditions (median time to maximum concentration [Tmax ] was 3.5 h [tablet fed] vs. 1 h [tablet fasted]). OS-to-tablet (fasted) ratios were: AUC0-t , 124.83% (90% CI: 111.50%, 139.76%); AUC0-∞ , 119.05% (90% CI: 104.47, 135.67%); and Cmax 173.29% (90% CI: 135.78, 221.16%). Median Tmax was 0.5 h (OS fasted) versus 1 h (tablet). All treatments were well-tolerated in healthy volunteers. Peposertib tablets can be taken with or without food; if combined with chemotherapy or radiotherapy, the delay in Cmax must be considered to optimize the chemo- or radiosensitizing effect. The peposertib OS form represents an alternative route of administration in patients with specific cancers causing dysphagia. However, the OS form should be part of future dose optimization strategies in relevant settings.

Fanconi anemia proteins are required to prevent accumulation of replication-associated DNA double-strand breaks.

Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.

Yeast two-hybrid screens imply involvement of Fanconi anemia proteins in transcription regulation, cell signaling, oxidative metabolism, and cellular transport.

Mutations in one of at least eight different genes cause bone marrow failure, chromosome instability, and predisposition to cancer associated with the rare genetic syndrome Fanconi anemia (FA). The cloning of seven genes has provided the tools to study the molecular pathway disrupted in Fanconi anemia patients. The structure of the genes and their gene products provided few clues to their functional role. We report here the use of 3 FA proteins, FANCA, FANCC, and FANCG, as "baits" in the hunt for interactors to obtain clues for FA protein functions. Using five different human cDNA libraries we screened 36.5x10(6) clones with the technique of the yeast two-hybrid system. We identified 69 proteins which have not previously been linked to the FA pathway as direct interactors of FANCA, FANCC, or FANCG. Most of these proteins are associated with four functional classes including transcription regulation (21 proteins), signaling (13 proteins), oxidative metabolism (10 proteins), and intracellular transport (11 proteins). Interaction with 6 proteins, DAXX, Ran, IkappaBgamma, USP14, and the previously reported SNX5 and FAZF, was additionally confirmed by coimmunoprecipitation and/or colocalization studies. Taken together, our data strongly support the hypothesis that FA proteins are functionally involved in several complex cellular pathways including transcription regulation, cell signaling, oxidative metabolism, and cellular transport.

The effects of the Fanconi anemia zinc finger (FAZF) on cell cycle, apoptosis, and proliferation are differentiation stage-specific.

FAZF, a member of the BTB/POZ family of transcriptional repressor proteins, has been shown to bind to FANCC, the protein defective in patients with the bone marrow failure syndrome Fanconi anemia complementation group C. Because bone marrow failure in Fanconi anemia has been attributed to a failure of the hematopoietic stem cell population to produce sufficient progeny, we documented the expression of FAZF in human CD34(+) hematopoietic progenitor cells. FAZF was expressed at high levels in early stages of differentiation but declined during subsequent differentiation into erythroid and myeloid lineages. Consistent with its presumed role as a transcriptional repressor, FAZF was found in the nuclear compartment, where it resides in distinct nuclear speckles at or near sites of DNA replication. Using a FAZF-inducible myeloid cell line, we found that enforced expression of FAZF was accompanied by accumulation in the G(1) phase of the cell cycle followed later by apoptosis. These results suggest an essential role for FAZF during the proliferative stages of primitive hematopoietic progenitors, possibly acting in concert with (a subset of) the Fanconi anemia proteins.