Cynthia Seidel-Dugan

SCH529074, a Small Molecule Activator of Mutant p53, Which Binds p53 DNA Binding Domain (DBD), Restores Growth-suppressive Function to Mutant p53 and Interrupts HDM2-mediated Ubiquitination of Wild Type p53

Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1–2 μm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

Evaluating TBK1 as a Therapeutic Target in Cancers with Activated IRF3

TBK1 (TANK-binding kinase 1) is a noncanonical IκB protein kinase that phosphorylates and activates downstream targets such as IRF3 and c-Rel and, mediates NF-κB activation in cancer. Previous reports demonstrated synthetic lethality of TBK1 with mutant KRAS in non–small cell lung cancer (NSCLC); thus, TBK1 could be a novel target for treatment of KRAS-mutant NSCLC. Here, the effect of TBK1 on proliferation in a panel of cancer cells by both genetic and pharmacologic approaches was evaluated. In KRAS-mutant cancer cells, reduction of TBK1 activity by knockdown or treatment with TBK1 inhibitors did not correlate with reduced proliferation in a two-dimensional viability assay. Verification of target engagement via reduced phosphorylation of S386 of IRF3 (pIRF3S386) was difficult to assess in NSCLC cells due to low protein expression. However, several cell lines were identified with high pIRF3S386 levels after screening a large panel of cell lines, many of which also harbor KRAS mutations. Specifically, a large subset of KRAS-mutant pancreatic cancer cell lines was uncovered with high constitutive pIRF3S386 levels, which correlated with high levels of phosphorylated S172 of TBK1 (pTBK1S172). Finally, TBK1 inhibitors dose-dependently inhibited pIRF3S386 in these cell lines, but this did not correlate with inhibition of cell growth. Taken together, these data demonstrate that the regulation of pathways important for cell proliferation in some NSCLC, pancreatic, and colorectal cell lines is not solely dependent on TBK1 activity. Implications: TBK1 has therapeutic potential under certain contexts and phosphorylation of its downstream target IRF3 is a biomarker of TBK1 activity. Visual Overview: http://mcr.aacrjournals.org/content/12/7/1055/F1.large.jpg. Mol Cancer Res; 12(7); 1055–66. ©2014 AACR. Visual Overview

Prognostic and Predictive Molecular Markers in Cutaneous Malignant Melanoma: The First Step Toward Personalized Medicine

Cutaneous malignant melanoma represents one of the most aggressive human cancers with high metastatic po- tential. Differences in the response and toxicity to current melanoma therapies among individuals are observed in nearly all-available treatment regimens. The first step toward personalized medicine is identifying a panel of biomarkers that al- low classification of melanoma patients for appropriate treatment and prediction of probable response to therapy. The tra- ditional approach to biomarker detection relied on studying a few candidate markers suspected of affecting clinical out- come. However, these studies have yielded contradictory results because of the small number of molecular determinants examined. This has been a major limitation of translational studies in malignant melanoma. Recent studies using high- throughput technologies, such as gene expression profiling and serum proteomic fingerprinting, have explored the utility of molecular markers to discriminate between clinical stages and predict disease progression in melanoma patients. This expert review highlights key approaches for the discovery and validation of biomarkers at the levels of DNA, RNA and protein. It also summarizes biomarker work performed by less invasive approaches, i.e., RT-PCR in detection of circulat- ing melanoma cells and serum markers that may be used to monitor early response to treatment and guide the therapeutic strategy. We anticipate that pharmacogenomics will play an integral role in disease assessment, patient selection and treatment response in melanoma clinical management with the ultimate goal of individualizing treatment and improving overall survival for patients.

Discovery of Novel 3,3-Disubstituted Piperidines as Orally Bioavailable, Potent, and Efficacious HDM2-p53 Inhibitors.

A new subseries of substituted piperidines as p53-HDM2 inhibitors exemplified by 21 has been developed from the initial lead 1. Research focused on optimization of a crucial HDM2 Trp23-ligand interaction led to the identification of 2-(trifluoromethyl)thiophene as the preferred moiety. Further investigation of the Leu26 pocket resulted in potent, novel substituted piperidine inhibitors of the HDM2-p53 interaction that demonstrated tumor regression in several human cancer xenograft models in mice. The structure of HDM2 in complex with inhibitors 3, 10, and 21 is described.

Abstract 2785: Discovery of a novel HDM2 inhibitor with potent in vivo anti-tumor activity

HDM2 is a major negative regulator of the p53 tumor suppressor pathway. Aberrant HDM2 overexpression and gene amplification contributed to accelerated cancer development and growth. Several small molecule inhibitors of HDM2-p53 protein-protein interaction have been reported in recent years with anti-tumor activities in tumor xenograft models. Here we describe a novel and potent small molecule inhibitor of HDM-p53 inhibitor that binds selectively to HDM2 with high affinity compared to HDM4. Treatment of cancer cells with this HDM2 inhibitor results in activation of p53 pathway as demonstrated by study of pharmacodynamic biomarkers both in cell culture and in tumor xenograft in vivo. More importantly, cancer cells response to this HDM2 inhibitor is mechanism based and dependent on the presence of functional p53 status as shown in a profiling of a broad-panel of cancer cell lines. This inhibitor is very potent against cancer cell growth with IC50 below 200 nM for most cell lines tested in vitro. It is orally bioavailable and has single agent activity that results tumor regression in SJSA-1 osteosarcoma model or growth inhibition in A549 NSCLC and A2780 ovarian cancer xenograft models. In addition, combination of this HDM2 inhibitor with various chemotherapy agents results in added or synergistic anti-tumor response both in vitro and in vivo in several human cancer xenograft models with limited bone marrow toxicity at the efficacious dose. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2785. doi:1538-7445.AM2012-2785

Abstract 1945: Inhibition of PTK6 kinase activity reduces proliferation and migration of tumor cells

Protein kinase 6 (PTK6) is a member of the Frk family of non-receptor tyrosine kinase that is overexpressed in several types of cancers with the highest overexpression observed in breast tumors. PTK6 shows sequence homology to the src tyrosine kinase family. Its functional domains, including a SH3, a SH2 and a kinase domain, are arranged similarly with src family kinases although PTK6 lacks a myristoylation domain. We have identified a potent small molecule PTK6 kinase inhibitor from kinase cross screens that inhibits PTK6 autophosphorylation and phosphorylation of its substrate Sam68, a member of the KH domain containing RNA binding proteins. In cell culture, the compound inhibited proliferation, soft agar growth and migration of tumor cells. The compound inhibited soft agar growth of breast tumor cells more potently than dasatinib. A specific PTK6 kinase inhibitor may provide a novel approach to inhibit the growth of selected tumors, sensitize the response of the tumor cells to other chemotherapeutics and prevent/inhibit metastasis of cancer in a wide range of cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1945. doi:10.1158/1538-7445.AM2011-1945

Abstract 599: TMPRSS2-ERG gene fusion sensitizes VCaP human prostate tumors to the fully human anti-IGF-IR monoclonal antibody SCH 717454

A gene fusion of the androgen-regulated gene transmembrane protease, serine 2, (TMPRSS2) to the v-ets erythroblastosis virus E26 oncogene homolog (avian), ERG, of the erythroblast transformation-specific (ETS) family of transcription factors is a recently discovered genetic alteration in prostate cancer. Expression of the TMPRSS2-ERG fusion in prostate cancer patients is associated with an aggressive clinical phenotype and an early recurrence of the disease. Interestingly, in TMPRSS2-ERG positive prostate cancer cells, expression of this gene fusion is regulated by both estrogens and androgens. However, while the hormonal regulation of this fusion has been established the downstream effects of TPRSS2-ERG fusion gene have not been determined. Quantitative PCR analyses of 134 cancer cell lines, including 10 prostate cancer models, for expression of the TMPRSS2-ERG gene fusion showed that it was only present in VCaP, DuCaP, and NCI-H660 prostate cancer cells. We report here that VCaP human prostate cancer cells and tumor xenografts are sensitive to treatment with the fully human anti insulin-like growth factor-I receptor (IGF-IR) monoclonal antibody SCH 717454. After 28 days of treatment, single-agent SCH 717454 (0.5 mg, ip, 2 × week) caused complete inhibition of VCaP tumor growth. MDA PCa-2b, 22Rv1, DU-145, and LNCaP human prostate cancer tumor xenografts (negative for expression of the TMPRSS2-ERG gene fusion) did not respond to treatment with SCH 717454. Further, PTEN null NCI-H660 prostate tumors that also express the TMPRSS2-ERG gene fusion also did not respond to treatment with SCH 717454. When male SCID mice bearing established VCaP prostate cancer tumor xenografts were treated with the combination of surgical castration and SCH 717454 we observed 100% tumor regressions in all of the animals so that after 28 days of treatment there were no detectable tumors. When SCH 717454 was combined with the antiandrogen bicalutamide (50 mpk, po, bid), VCaP tumors regressed by 42%. That the combination of SCH 717454 and castration produced superior anti-tumor activity than the combination of SCH 717454 and bicalutamide provides further evidence that expression of the TMPRSS2-ERG gene fusion in VCaP prostate cancer tumors is regulated by both estrogens and androgens. TMPRSS2-ERG gene fusion expressing prostate cancer represents a new sub-classification of human prostate cancer. The precise role that the TMPRSS2-ERG fusion plays in the biology of human prostate cancer remains to be fully determined. These data indicate that PTEN wild-type VCaP prostate tumors that express the TMPRSS2-ERG gene fusion are sensitive to anti-IGF-IR treatment with SCH 717454 and that combining SCH 717454 with an androgen ablation therapy results in impressive anti-tumor activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 599. doi:10.1158/1538-7445.AM2011-599

Abstract 4534: A novel and potent small molecule antagonist of HDM2, SCH 1450206, activates the p53 pathway with mechanism-based activity

p53 is an attractive therapeutic target in oncology since aberrant regulation of p53 expression is associated with cancer development and progression. HDM2 is a primary negative regulator of p53 function in cells. The blockade of the p53-HDM2 interaction presents an attractive approach for development of drugs against tumors expressing wild-type p53. Here we report a novel and potent small molecule antagonist of HDM2, SCH 1450206. It binds selectively to the HDM2 protein with high affinity, with weak affinity to HDMX protein. SCH 1450206 induced dose- and time-dependent stabilization of p53 protein and its downstream targets in several human cancer cell lines with wild-type p53, but not in cell with p53 gene deletion. SCH 1450206-induced stabilization of p53 led to distinct changes in expression of p53 target genes regulating apoptosis and cell cycle checkpoints. Inhibition of cell proliferation and induction of apoptosis were surveyed in a panel of human tumor cell lines following SCH 1450206 treatment. Inactivation of p53 by shRNA in cell lines expressing wt-p53 abolished the inhibition of cell proliferation and appearance of senescence in response to SCH 1450206, suggesting the cellular activity of SCH 1450206 is p53 specific and mechanism-based. Taken together, our results demonstrated the mechanism of action of SCH 1450206, a novel HDM2 antagonist that can activate the p53 pathway, and offers a potential anti-tumor agent targeting tumors expressing wild-type p53. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4534.

Abstract 1536: Characterization of tumor cell lines selected for resistance to the HDM2 antagonist Nutlin-3a

The tumor suppressor p53 is activated in response to various cellular stress signals including DNA damage and oncogene activation. Activated p53 regulates the expression of multiple downstream target genes involved in cell cycle control, cell death and senescence. Dysfunction of the p53 pathway is the most frequent alteration in human cancers. HDM2 protein binds directly to p53 and acts as a major negative regulator of the p53 function. Nutlin-3a is an antagonist of the HDM2-p53 protein-protein interaction and restores p53 pathway function in tumors with wild-type p53. Nutlin-3a demonstrates anti-tumor activity both in vitro and in vivo. Emergence of resistance is frequently observed with many anti-cancer therapeutics. The goal of the current studies is to understand the mechanism of resistance to Nutlin-3a. Cancer cell lines were treated with Nutlin-3a for an extended period and resistant clones were isolated. We found using molecular profiling studies that the emergence of Nutlin resistance is primarily due to the inactivation of the p53 signaling pathway through either mutation of the p53 gene or loss of p53 protein expression. These Nutlin-resistant cells are also resistant to others antagonists of the HDM2-p53 protein-protein interaction. These results suggest that, following chronic treatment, resistance to HDM2 antagonists may occur in the clinic. It will be important to combine HDM2 antagonists with other anti-cancer drugs early on during the treatment cycle in order to minimize the emergence of resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1536.

Abstract 3197: Differential senescent and apoptotic responses to HDM2 antagonism in p53 wild-type human solid cancer cells

The p53 protein is a primary regulator of the cellular response to DNA damage and is a critical tumor suppressor. Restoration of p53 function in soft tissue sarcomas and hepatocellular carcinomas in mice was reported to cause tumor regressions associated with cellular senescence. These findings led us to investigate the ability of HDM2 antagonism to induce cellular senescence as well as apoptosis in a broad panel of wild-type p53 cell lines derived from human solid tumors. Here we report that the small molecule HDM2 antagonist Nutlin-3a activates p53 and induces cellular senescence in 10 out of 12 human tumor cell lines tested. Only two of these tumor cell lines also demonstrated an apoptotic response to Nutlin-3a. The kinetics of senescence induction by Nutlin-3a was slower than that of the apoptotic response, suggesting that apoptosis may be the first-line response, while senescence may be triggered as a back-up program when the apoptotic response fails to execute. A distinct expression profile of p53 target genes associated with apoptosis, cell cycle and senescence was observed and corresponded with the distinct phenotypic responses in this panel of cell lines. Depletion of p53 protein expression using shRNA blocked cellular senescence induced by HDM2 antagonism demonstrating the critical role for p53 in this process. The pronounced effects of Nutlin-3a on senescence in multiple wild-type p53 tumor cell lines underscores the unique anti-tumor mechanisms of HDM2 antagonism. This approach may provide a novel therapeutic strategy for future cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3197.