F. Michael Yakes

PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization.

We have shown a novel mechanism of Akt-mediated regulation of the CDK inhibitor p27kip1. Blockade of HER2/neu in tumor cells inhibits Akt kinase activity and upregulates nuclear levels of the CDK inhibitor p27Kip1. Recombinant Akt and Akt precipitated from tumor cells phosphorylated wild-type p27 in vitro. p27 contains an Akt consensus RXRXXT157D within its nuclear localization motif. Active (myristoylated) Akt phosphorylated wild-type p27 in vivo but was unable to phosphorylate a T157A-p27 mutant. Wild-type p27 localized in the cytosol and nucleus, whereas T157A-p27 localized exclusively in the nucleus and was resistant to nuclear exclusion by Akt. T157A-p27 was more effective than wild-type p27 in inhibiting cyclin E/CDK2 activity and cell proliferation; these effects were not rescued by active Akt. Expression of Ser473 phospho Akt in primary human breast cancers statistically correlated with expression of p27 in tumor cytosol. These data indicate that Akt may contribute to tumor-cell proliferation by phosphorylation and cytosolic retention of p27, thus relieving CDK2 from p27-induced inhibition.

Cabozantinib (XL184), a novel MET and VEGFR2 inhibitor, simultaneously suppresses metastasis, angiogenesis, and tumor growth

The signaling pathway of the receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF) is important for cell growth, survival, and motility and is functionally linked to the signaling pathway of VEGF, which is widely recognized as a key effector in angiogenesis and cancer progression. Dysregulation of the MET/VEGF axis is found in a number of human malignancies and has been associated with tumorigenesis. Cabozantinib (XL184) is a small-molecule kinase inhibitor with potent activity toward MET and VEGF receptor 2 (VEGFR2), as well as a number of other receptor tyrosine kinases that have also been implicated in tumor pathobiology, including RET, KIT, AXL, and FLT3. Treatment with cabozantinib inhibited MET and VEGFR2 phosphorylation in vitro and in tumor models in vivo and led to significant reductions in cell invasion in vitro. In mouse models, cabozantinib dramatically altered tumor pathology, resulting in decreased tumor and endothelial cell proliferation coupled with increased apoptosis and dose-dependent inhibition of tumor growth in breast, lung, and glioma tumor models. Importantly, treatment with cabozantinib did not increase lung tumor burden in an experimental model of metastasis, which has been observed with inhibitors of VEGF signaling that do not target MET. Collectively, these data suggest that cabozantinib is a promising agent for inhibiting tumor angiogenesis and metastasis in cancers with dysregulated MET and VEGFR signaling. Mol Cancer Ther; 10(12); 2298–308. ©2011 AACR.

Loss of PTEN/MMAC1/TEP in EGF receptor-expressing tumor cells counteracts the antitumor action of EGFR tyrosine kinase inhibitors

We have examined the possible mechanisms of resistance to the epidermal growth factor receptor (EGFR) inhibitors in tumor cells with variable levels of EGFR. ZD1839 (Iressa) is a small-molecular-weight, ATP-mimetic that specifically inhibits the EGFR tyrosine kinase. A431 cell growth was markedly inhibited by ZD1839 (IC50⩽0.1 μM) whereas the MDA-468 cells were relatively resistant (IC502 μM). Low doses of ZD1839 delayed cell cycle progression and induced apoptosis in A431 cells but not in MDA-468 cells. In both cell lines, 0.1 μM ZD1839 eliminated EGFR phosphorylation. However, the basal activity of the phosphatidylinositol-3 kinase (PI3 K) target Akt was eliminated in A431 but not in MDA-468 cells, implying that their Akt activity is independent of EGFR signals. A431 cells express PTEN/MMAC1/TEP, a phosphatase that can dephosphorylate position D3 of phosphatidylinositol-3,4,5 trisphosphate, the site that recruits the plecstrin-homology domain of Akt to the cell membrane. On the contrary, MDA-468 cells lack the phosphatase and tensin homolog (PTEN), potentially setting Akt activity at a high threshold that is unresponsive to EGFR inhibition alone. Therefore, we reintroduced (PTEN) by retroviral infection in MDA-468 cells. In MDA-468/PTEN but not in vector controls, treatment with ZD1839 inhibited P-Akt levels, induced relocalization of the Forkhead factor FKHRL1 to the cell nucleus, and increased FKHRL1-dependent transcriptional activity. ZD1839 induced a greater degree of apoptosis and cell cycle delay in PTEN-reconstituted than in control cells. These data suggest that loss of PTEN, by permitting a high level of Akt activity independent of receptor tyrosine kinase inputs, can temporally dissociate the inhibition of the EGFR with that of Akt induced by EGFR inhibitors. Thus, in EGFR-expressing tumor cells with concomitant amplification(s) of PI3K-Akt signaling, combined blockade of the EGFR tyrosine kinase and Akt should be considered as a therapeutic approach.

Hydrogen Peroxide– and Peroxynitrite-Induced Mitochondrial DNA Damage and Dysfunction in Vascular Endothelial and Smooth Muscle Cells

The mechanisms by which reactive species (RS) participate in the development of atherosclerosis remain incompletely understood. The present study was designed to test the hypothesis that RS produced in the vascular environment cause mitochondrial damage and dysfunction in vitro and, thus, may contribute to the initiating events of atherogenesis. DNA damage was assessed in vascular cells exposed to superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite. In both vascular endothelial and smooth muscle cells, the mitochondrial DNA (mtDNA) was preferentially damaged relative to the transcriptionally inactive nuclear beta-globin gene. Similarly, a dose-dependent decrease in mtDNA-encoded mRNA transcripts was associated with RS treatment. Mitochondrial protein synthesis was also inhibited in a dose-dependent manner by ONOO(-), resulting in decreased cellular ATP levels and mitochondrial redox function. Overall, endothelial cells were more sensitive to RS-mediated damage than were smooth muscle cells. Together, these data link RS-mediated mtDNA damage, altered gene expression, and mitochondrial dysfunction in cell culture and reveal how RS may mediate vascular cell dysfunction in the setting of atherogenesis.

HER (erbB) tyrosine kinase inhibitors in the treatment of breast cancer.

Protein tyrosine kinases are tightly regulated enzymes that play an important role in the control of most fundamental cellular processes, including cell proliferation, differentiation, metabolism, migration, and survival. These signaling proteins are the frequent target of oncogenic mutations or other genetic alterations leading to dysregulated tyrosine kinase activity, cellular transformation, and subsequent tumor progression. Many of the known dominant oncogenes encode aberrant protein tyrosine kinases and are causally associated with a significant fraction of human neoplasms, including breast carcinoma. The epidermal growth factor receptor and HER2/neu are two transmembrane tyrosine kinases that are members of the HER (erbB) signaling network. Aberrant signaling by this network is present in a cohort of breast carcinomas. Structure/function studies of these kinases have led to the identification of molecular approaches aimed at disabling signaling by this transforming network. Trastuzumab, a monoclonal antibody that binds the ectodomain of HER2, was recently shown to induce regression of HER2-overexpressing breast cancers, confirming the role of HER2 in tumor maintenance and progression. A rational therapeutic approach that builds on these results with trastuzumab and expands the targeting of the HER network will be presented.

Inhibition of the T790M gatekeeper mutant of the epidermal growth factor receptor by EXEL-7647.

Purpose: Agents inhibiting the epidermal growth factor receptor (EGFR) have shown clinical benefit in a subset of non–small cell lung cancer patients expressing amplified or mutationally activated EGFR. However, responsive patients can relapse as a result of selection for EGFR gene mutations that confer resistance to ATP competitive EGFR inhibitors, such as erlotinib and gefitinib. We describe here the activity of EXEL-7647 (XL647), a novel spectrum-selective kinase inhibitor with potent activity against the EGF and vascular endothelial growth factor receptor tyrosine kinase families, against both wild-type (WT) and mutant EGFR in vitro and in vivo. Experimental Design: The activity of EGFR inhibitors against WT and mutant EGFRs and their effect on downstream signal transduction was examined in cellular assays and in vivo using A431 and MDA-MB-231 (WT EGFR) and H1975 (L858R and T790M mutant EGFR) xenograft tumors. Results: EXEL-7647 shows potent and long-lived inhibition of the WT EGFR in vivo. In addition, EXEL-7647 inhibits cellular proliferation and EGFR pathway activation in the erlotinib-resistant H1975 cell line that harbors a double mutation (L858R and T790M) in the EGFR gene. In vivo efficacy studies show that EXEL-7647 substantially inhibited the growth of H1975 xenograft tumors and reduced both tumor EGFR signaling and tumor vessel density. Additionally, EXEL-7647, in contrast to erlotinib, substantially inhibited the growth and vascularization of MDA-MB-231 xenografts, a model which is more reliant on signaling through vascular endothelial growth factor receptors. Conclusions: These studies provide a preclinical basis for clinical trials of XL647 in solid tumors and in patients bearing tumors that are resistant to existing EGFR-targeted therapies.

Tyrosine kinase inhibitors: Rationale, mechanisms of action, and implications for drug resistance

Tyrosine kinases play a role in normal cellular regulatory processes. However, aberrant tyrosine kinase activity can lead to cellular transformation and can be causally associated with tumor maintenance and progression. In the last few years, high-throughput screening and the use of combinatorial, computational, and medicinal chemistry have led to the identification of small molecules that compete with the adenosine triphosphate binding site of the catalytic domain of several oncogenic tyrosine kinases. Some of these compounds are highly specific to a single tyrosine kinase, while others can inhibit several homologous kinase pockets simultaneously. At a practical level, the relative promiscuity of these inhibitors against more than one oncogenic tyrosine kinase may have clinical merit as well as implications for host tissue toxicity. Many of these small molecules are in different stages of preclinical and clinical development against several solid tumors and will be discussed.

Discovery of a First-in-Class, Potent, Selective, and Orally Bioavailable Inhibitor of the p97 AAA ATPase (CB-5083)

The AAA-ATPase p97 plays vital roles in mechanisms of protein homeostasis, including ubiquitin-proteasome system (UPS) mediated protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. Herein we describe our lead optimization efforts focused on in vitro potency, ADME, and pharmaceutical properties that led to the discovery of a potent, ATP-competitive, D2-selective, and orally bioavailable p97 inhibitor 71, CB-5083. Treatment of tumor cells with 71 leads to significant accumulation of markers associated with inhibition of UPS and ERAD functions, which induces irresolvable proteotoxic stress and cell death. In tumor bearing mice, oral administration of 71 causes rapid accumulation of markers of the unfolded protein response (UPR) and subsequently induces apoptosis leading to sustained antitumor activity in in vivo xenograft models of both solid and hematological tumors. 71 has been taken into phase 1 clinical trials in patients with multiple myeloma and solid tumors.

Abstract 951: CB-5083 is a novel first in class p97 inhibitor that disrupts cellular protein homeostasis and demonstrates anti-tumor activity in solid and hematological models

Background: The AAA-ATPase p97/VCP facilitates the extraction and degradation of ubiquitinated proteins by converting chemical energy into mechanical force. p97 is closely involved in several facets of protein homeostasis, including ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD) and autophagy. p97 has been increasingly linked to cancer: it showed elevated protein expression in tumors, it can mediate the degradation of proteins in cancer-relevant pathways and is required for orchestrating the ubiquitin-governed DNA-damage response. In this context, p97 inhibitors may have an advantage versus other protein homeostasis inhibitors and may be active in solid tumors where 26S proteasome inhibitors, bortezomib and carfilzomib, have shown poor efficacy. We report here p97 inhibition as a novel approach to exploit cancer cell addiction to protein homeostatic mechanisms. Results: We have discovered novel small molecule inhibitors of p97 ATPase activity with nanomolar enzymatic and cellular potency. In cellular models, treatment of cancer cells with our lead compound CB-5083 causes disruptions in specific p97 functions, including ubiquitin-dependent protein degradation, ERAD, endocytosis and autophagy. In mouse models, CB-5083 is orally bio-available and causes rapid and sustained accumulation of poly-ubiquitin in tumor xenografts after a single administration. Concurrent with increases in polyubiquitin levels, activation of ER stress response pathways and induction of apoptosis markers are also observed. Sub-chronic oral administration of CB-5083 is generally well-tolerated with 50% reduction in M-spike. Additional efforts are focused on the development of translational assays to monitor p97 target engagement and antitumor efficacy in upcoming clinical trials of CB-5083. Conclusion: These data demonstrate that CB-5083 is a potent inhibitor of p97 that translates to tumor growth inhibition in multiple rodent models of human cancer. Furthermore, CB-5083 appears to exhibit greater potency over current proteasome inhibitors that further validate targeting p97 and protein homeostasis in the treatment of cancer. Citation Format: Ronan Le Moigne, Steve Wong, Ferdie Soriano, Eduardo Valle, Daniel J. Anderson, Stevan Djakovic, Mary-Kamala Menon, Bing Yao, Julie Rice, Jinhai Wang, Szerenke Kiss Von Soly, Brajesh Kumar, Marta Chesi, P. Leif Bergsagel, Han-Jie Zhou, David Wustrow, Mark Rolfe, F. Michael Yakes. CB-5083 is a novel first in class p97 inhibitor that disrupts cellular protein homeostasis and demonstrates anti-tumor activity in solid and hematological models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 951. doi:10.1158/1538-7445.AM2014-951

Abstract C188: Novel small molecule inhibitors of p97 disrupt cellular protein homeostasis and demonstrate anti-tumor activity in solid and hematological models.

Background: The ubiquitin-proteasome system (UPS) sustains cancer cell viability by alleviating proteotoxic stress caused by an imbalance of protein synthesis and degradation. Bortezomib and carfilzomib are 26S proteasome inhibitors approved for the treatment of multiple myeloma. Unfortunately, these agents have poor efficacy in solid tumors, prompting the need for discovery of novel drugs targeting other enzymes within the UPS. The AAA-ATPase p97/VCP functions by converting chemical energy into mechanical force and is closely involved in several facets of protein homeostasis. These include ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. p97 inhibition will provide a novel approach to exploit cancer cell addiction to protein homeostatic mechanisms. Results: Through a targeted medicinal chemistry effort, we have discovered novel small molecule inhibitors of p97 ATPase activity with nanomolar enzymatic and cellular potency. Sequencing of human cancer cell lines that have developed resistance to our p97 inhibitors has revealed mutations in p97, strongly suggesting on-target cellular activity. Treatment of normal and cancer cells with this class of p97 inhibitors causes a dramatic increase in poly-ubiquitinated proteins and an accumulation of substrates of the UPS and ERAD. These inhibitors also disrupt other specific p97 cellular functions including macroautophagy and receptor endocytosis. Inhibitor treatment ultimately leads to a blockade of NF-kB signaling and a decrease in downstream survival factors followed by the induction of caspase cleavage and apoptosis. In animal models, our p97 inhibitors are orally bio-available and cause rapid accumulation of poly-ubiquitin in tumor xenografts at levels that exceed the accumulation seen with bortezomib. Furthermore, significant tumor growth inhibition was observed in a number of solid tumor and hematological models. Conclusion: Together, these data provide novel insights into the role of p97 in cancer cell growth and the mechanism of death due to p97 inhibition. Animal model data suggests promise for our inhibitors as therapeutic agents for patients with hematological and solid tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C188. Citation Format: Stevan N. Djakovic, Daniel J. Anderson, Szerenke Kiss von Soly, Ronan Le Moigne, Julie Rice, Mark Rolfe, Ferdie Soriano, Eduardo Valle, Jinhai Wang, Steve Wong, David Wustrow, F. Michael Yakes, Bing Yao, Han-Jie Zhou. Novel small molecule inhibitors of p97 disrupt cellular protein homeostasis and demonstrate anti-tumor activity in solid and hematological models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C188.