Notch pathway inhibitors

Regardless of the data implicating the Notch pathway in tumor suppression as well as onco-genesis, chemotherapeutic targeting of the Notch pathway employing y-secretase inhibitors (GSI) to block release of the NICD has generated much interest [92]. GSIs, which were designed primarily for Alzheimer's disease, developed by Merck, Novartis, Pfizer and Roche are currently in clinical trials for a number of malignancies including T-ALL, lymphoma, breast, colorectal, brain, pancreatic, and non-small cell lung carcinoma. However, targeting the Notch pathway through the use of GSIs is problematic. Preclinical studies examining GSI function in vitro are difficult because, with the notable exception of GSI-1, these drugs do not display strong inhibitory effects on cell growth or survival in vitro. Also, while these drugs do inhibit Notch signaling, they display poor specificity. As an example, the inhibition of survival of breast carcinoma cell lines by GSI-1 was associated with inhibition of the proteosome and not effects on Notch signaling [93]. In addition to off-target effects, Notch inhibition by GSI has adverse effects on the intestinal system and immune function [94]. Lastly, as discussed above, the cell context determines whether the Notch pathway is onco-genic or tumor suppressive even within cancers of the same organ [86]. Thus, the consequence of inhibition of Notch receptor activation by GSI or inhibitory antibodies to Notch receptors/ligands is difficult to predict.

Inhibition of Notch activation by GSIs, inhibitory antibodies that bind DSL ligands, or other inhibitors of receptor activation target only the Notch activated state and they are less than ideal. However, the Notch pathway is central to oncogenesis, and this idea fuels the search for novel ways to inhibit the Notch signaling pathway [11]. Recently, the Bradner laboratory developed a stabilized peptide that mimics MAML and binds to the NICD-CSL complex to block interaction with endogenous MAML [95]. SAHM1, a 16 amino acid peptide which blocks MAML binding to the NICD-CSL complex is cell-permeable and lowers NOTCH-target gene expression when added to cells in culture [95]. SAHM1 lowers proliferation of TALL cell lines suggesting that these small molecules will be useful as probes to dissect the requirement for MAML in Notch signaling and as building blocks for a new generation of Notch inhibitors.

Davis and co-workers tested the idea that direct inhibition of CSL would not only abrogate Notch pathways in the activated oncogenic state, but also disrupt the transcriptional regulation of Notch pathway genes that are repressed in the Notch quiescent state [85]. According to this argument, in cells or tissues where Notch activation is tumor suppressive, inhibition of CSL would release the strong transcriptional repressive complexes positioned on Notch targets. Removal of CSL-dependent repressive complexes could mimic the tumor suppres-sive activity of the Notch pathway. Indeed, Davis and co-workers addressed the role of CSL in Notch-dependent signaling in prostate cancer cell lines, using lentiviral mediated transfer of shRNA specific for CSL to knockdown expression of CSL. CSL knockdown was tracked by EMSA and expression of the Notch pathway genes was documented using RT-PCR array profiling. Knockdown of CSL expression produced gene expression changes distinct from those induced by GSI inhibition of Notch signaling [85]. For example, inhibition of Notch receptor activation by DAPT resulted in repression of Hes1, a well-characterized CSL target in prostate and breast cancer cell lines. In contrast, Hes1 mRNA levels were unaffected by CSL ablation in prostate cancer cell lines, indicating that Hes1 expression does not require the activating function of CSL [85]. Thus, Notch pathway-dependent transcriptional regulation of Hes1 is primarily through repression and ablation of CSL partially mimics Notch receptor activation. While HES1 expression was not significantly altered by CSL knockdown, the expression of other Notch pathway genes did change. One such gene, DTX1 is thought to regulate Notch signaling either by targeting the NICD for ubiquitination and degradation or by altering NICD transcriptional functions, possibly by competing for co-activators [96]. Davis and coworkers failed to generate stable cell lines after infection with the CSL-specific shRNA but not with the control non-target (NT) shRNA. CSL knockdown cells were poorly attached and growth inhibited as compared to the NT infected cells [96]. These data provide strong evidence that CSL, the major Notch pathway effector, is required for cell growth in prostate cancer cells lines, and suggest that CSL is an important candidate for small molecule therapies in AIPC.

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