Katherine Rendahl

CHIR-124, a Novel Potent Inhibitor of Chk1, Potentiates the Cytotoxicity of Topoisomerase I Poisons In vitro and In vivo

Purpose: Chk1 kinase is a critical regulator of both S and G2-M phase cell cycle checkpoints in response to DNA damage. This study aimed to evaluate the biochemical, cellular, and antitumor effects of a novel Chk1 inhibitor, CHIR124. Experimental Design: CHIR-124 was evaluated for its ability to abrogate cell cycle checkpoints, to potentiate cytotoxicity, and to inhibit Chk1-mediated signaling induced by topoisomerase I poisons in human tumor cell line and xenograft models. Results: CHIR-124 is a quinolone-based small molecule that is structurally unrelated to other known inhibitors of Chk1. It potently and selectively inhibits Chk1 in vitro (IC50 = 0.0003 μmol/L). CHIR-124 interacts synergistically with topoisomerase poisons (e.g., camptothecin or SN-38) in causing growth inhibition in several p53-mutant solid tumor cell lines as determined by isobologram or response surface analysis. CHIR-124 abrogates the SN-38–induced S and G2-M checkpoints and potentiates apoptosis in MDA-MD-435 breast cancer cells. The abrogation of the G2-M checkpoint and induction of apoptosis by CHIR-124 are enhanced by the loss of p53. We have also shown that CHIR-124 treatment can restore the level of cdc25A protein, which is normally targeted by Chk1 for degradation following DNA damage, indicating that Chk1 signaling is suppressed in the presence of CHIR-124. Finally, in an orthotopic breast cancer xenograft model, CHIR-124 potentiates the growth inhibitory effects of irinotecan by abrogating the G2-M checkpoint and increasing tumor apoptosis. Conclusions: CHIR-124 is a novel and potent Chk1 inhibitor with promising antitumor activities when used in combination with topoisomerase I poisons.

Fibroblast growth factor-2 gene delivery stimulates axon growth by adult retinal ganglion cells after acute optic nerve injury.

Basic fibroblast growth factor (or FGF-2) has been shown to be a potent stimulator of retinal ganglion cell (RGC) axonal growth during development. Here we investigated if FGF-2 upregulation in adult RGCs promoted axon regrowth in vivo after acute optic nerve injury. Recombinant adeno-associated virus (AAV) was used to deliver the FGF-2 gene to adult RGCs providing a sustained source of this neurotrophic factor. FGF-2 gene transfer led to a 10-fold increase in the number of axons that extended past 0.5 mm from the lesion site compared to control nerves. Detection of AAV-mediated FGF-2 protein in injured RGC axons correlated with growth into the distal optic nerve. The response to FGF-2 upregulation was supported by our finding that FGF receptor-1 (FGFR-1) and heparan sulfate (HS), known to be essential for FGF-2 signaling, were expressed by adult rat RGCs. FGF-2 transgene expression led to only transient protection of injured RGCs. Thus the effect of this neurotrophic factor on axon extension could not be solely attributed to an increase in neuronal survival. Our data indicate that selective upregulation of FGF-2 in adult RGCs stimulates axon regrowth within the optic nerve, an environment that is highly inhibitory for regeneration. These results support the hypothesis that key factors involved in axon outgrowth during neural development may promote regeneration of adult injured neurons.

Tightly Regulated Long-Term Erythropoietin Expression In Vivo Using Tet-Inducible Recombinant Adeno-Associated Viral Vectors

Recombinant adeno-associated viral (rAAV) vectors containing an improved tetracycline (tet) system of transcriptional regulation are an efficient strategy for the control of long-term therapeutic gene expression. In vivo studies with the original tet-off and tet-on vectors, while promising, have failed to demonstrate complete repression in the uninduced state. To address this issue, we incorporated the tTS(kid) fusion of the tet repressor and a KRAB-derived transcriptional silencer into the tet-on system in the context of rAAV vectors. The tTS(kid) repressor and rtTA activator were expressed constituitively from a regulator vector, and the repressor and an erythropoietin (Epo) transgene were expressed inducibly via a second vector. Following intramuscular co-injection of these vectors, we observed repeated induction of serum Epo protein following drug administration and undetectable levels after its withdrawal. Four cycles of regulation were achieved over a 32-week period. Thus, the tet-on system plus the tTS(kid) repressor delivered via nonpathogenic rAAV vectors is a powerful tool for controlling the in vivo expression of therapeutic transgenes. In a clinical setting, the repressor could provide a mechanism for abolishing transgene expression if it were no longer needed or if the safety of a patient became compromised.

Development of a Screening Assay for Surrogate Markers of Chk1 Inhibitor-Induced Cell Cycle Release

Chk1 is a key regulator of the S and G2/M checkpoints and is activated following DNA damage by agents such as the topoisomerase I inhibitor camptothecin (CPT). It has been proposed that Chk1 inhibitors used in combination with such a DNA damaging agent to treat tumors would potentiate cytotoxicity and increase the therapeutic index, particularly in tumors lacking functional p53. The aim of this study was to determine whether gene expression analysis could be used to inform lead optimization of a novel series of Chk1 inhibitors. The candidate small-molecule Chk1 inhibitors were used in combination with CPT to identify potential markers of functional Chk1 inhibition, as well as resulting cell cycle progression, using cDNA-based microarrays. Differential expression of several of these putative marker genes was further validated by RT-PCR for use as a medium-throughput assay. In the presence of DNA damage, Chk1 inhibitors altered CPT-dependent effects on the expression of cell cycle and DNA repair genes in a manner consistent with a Chk1-specific mechanism of action. Furthermore, differential expression of selected marker genes, cyclin E2, EGR1, and DDIT3, was dose dependent for Chk1 inhibition. RT-PCR results for these genes following treatment with a panel of Chk1 inhibitors showed a strong correlation between marker gene response and the ability of each compound to abrogate cell cycle arrest in situ following CPT-induced DNA damage. These results demonstrate the utility of global expression analysis to identify surrogate markers, providing an alternative method for rapid compound characterization to support advancement decisions in early drug discovery.