Preclinical profile of a potent gamma-secretase inhibitor targeting notch signaling with in vivo efficacy and pharmacodynamic properties

Abstract

Notch signaling is an area of great interest in oncology. RO4929097 is a potent and selective inhibitor of gamma-secretase, producing inhibitory activity of Notch signaling in tumor cells. The RO4929097 IC50 in cell-free and cellular assays is in the low nanomolar range with >100-fold selectivity with respect to 75 other proteins of various types (receptors, ion channels, and enzymes). RO4929097 inhibits Notch processing in tumor cells as measured by the reduction of intracellular Notch expression by Western blot. This leads to reduced expression of the Notch transcriptional target gene Hes1. RO4929097 does not block tumor cell proliferation or induce apoptosis but instead produces a less transformed, flattened, slower-growing phenotype. RO4929097 is active following oral dosing. Antitumor activity was shown in 7 of 8 xenografts tested on an intermittent or daily schedule in the absence of body weight loss or Notch-related toxicities. Importantly, efficacy is maintained after dosing is terminated. Angiogenesis reverse transcription-PCR array data show reduced expression of several key angiogenic genes. In addition, comparative microarray analysis suggests tumor cell differentiation as an additional mode of action. These preclinical results support evaluation of RO4929097 in clinical studies using an intermittent dosing schedule. A multicenter phase I dose escalation study in oncology is under way.

Figure 1

RO4929097 chemical structure and in vitro Aβ40 and Notch potency. A, RO4929097 was derived from a dibenzazepinone lead. B, RO4929097 was assayed in a cell-free γ-secretase enzyme assay and two cellular assays using engineered cell lines to monitor Aβ40 and Notch processing.

Figure 2

RO4929097 inhibits the production of ICN, reducing the expression of the downstream Notch target Hes1, producing a less transformed morphology in A549 cells. A, RT-PCR analysis was used to monitor Hes1 mRNA expression. B, Western blot analysis was used to monitor the protein expression of ICN and Hes1. C, tissue culture pictures showing a less transformed morphology. D, RO4929097 reduces the size of MDA-MB-468 soft agar colonies. Pictures were taken 3 weeks after start of dosing. Compound was added twice weekly.

Figure 3

In vivo efficacy of RO4929097 in the A549 xenograft model. A, nude mice bearing A549 s.c. tumors were dosed orally following the indicated schedule. Arrow, length of treatment. B, animals from the 60 mg/kg group were monitored after cessation of treatment for 32 d and then redosed for 7 d. C, animals were dosed on a daily 21-day schedule at the indicated concentrations and then monitored after cessation of treatment. D, summary of breadth of in vivo efficacy. TGI, tumor growth inhibition; bid, twice daily; qd, once daily.

Figure 4

RO4929097-treated A549 tumors have increased areas of necrosis with relative increase in extracellular matrix (ECM). A, representative H&E-stained tumor tissue sections from vehicle and 10 mg/kg/d × 21 d RO4929097-treated groups. B, Western blot analysis of xenograft protein was done. C, angiogenesis-specific RT-PCR array showing down-regulation of several angiogenic genes in the sensitive A549 xenograft model compared with the insensitive H460a model.

Figure 5

Hes1 mRNA can serve as a pharmacodynamic marker for tracking Notch inhibition in hair follicles. A, Hes1 mRNA expression change in in vitro treated human hair follicles (2 µmol/L). B, c-myc mRNA expression. C, schematic representation of in vivo treatment of rats. D, rat Hes1, c-myc, and p21 in vivo mRNA expression changes.

Figure 6

Comparative microarray analysis. A, heat map of human gene expression changes induced by RO4929097 at 6 and 24 h in A549 and H460 NSCLC-derived cell lines as monitored (Affymetrix U133 Plus 2 arrays). Red and green, genes up-regulated and down-regulated at least 1.5-fold after RO4929097 treatment (P < 0.001, ANOVA, compared with untreated controls), respectively. Boxes list mRNAs that were altered at both 6 and 24 h. B, heat map of genes that are differentially expressed in both RO4929097-treated A549 cells (versus untreated) and well-differentiated non–small cell lung adenocarcinomas (versus poorly differentiated) identified by Gene Set Enrichment Analysis using the NextBio software.