De-Hua Yu

Preclinical Pharmacology of AZD5363, an Inhibitor of AKT: Pharmacodynamics, Antitumor Activity, and Correlation of Monotherapy Activity with Genetic Background

AKT is a key node in the most frequently deregulated signaling network in human cancer. AZD5363, a novel pyrrolopyrimidine-derived compound, inhibited all AKT isoforms with a potency of 10 nmol/L or less and inhibited phosphorylation of AKT substrates in cells with a potency of approximately 0.3 to 0.8 μmol/L. AZD5363 monotherapy inhibited the proliferation of 41 of 182 solid and hematologic tumor cell lines with a potency of 3 μmol/L or less. Cell lines derived from breast cancers showed the highest frequency of sensitivity. There was a significant relationship between the presence of PIK3CA and/or PTEN mutations and sensitivity to AZD5363 and between RAS mutations and resistance. Oral dosing of AZD5363 to nude mice caused dose- and time-dependent reduction of PRAS40, GSK3β, and S6 phosphorylation in BT474c xenografts (PRAS40 phosphorylation EC50 ∼ 0.1 μmol/L total plasma exposure), reversible increases in blood glucose concentrations, and dose-dependent decreases in 2[18F]fluoro-2-deoxy-d-glucose (18F-FDG) uptake in U87-MG xenografts. Chronic oral dosing of AZD5363 caused dose-dependent growth inhibition of xenografts derived from various tumor types, including HER2+ breast cancer models that are resistant to trastuzumab. AZD5363 also significantly enhanced the antitumor activity of docetaxel, lapatinib, and trastuzumab in breast cancer xenografts. It is concluded that AZD5363 is a potent inhibitor of AKT with pharmacodynamic activity in vivo, has potential to treat a range of solid and hematologic tumors as monotherapy or a combinatorial agent, and has potential for personalized medicine based on the genetic status of PIK3CA, PTEN, and RAS. AZD5363 is currently in phase I clinical trials. Mol Cancer Ther; 11(4); 873–87. ©2012 AACR.

The AKT inhibitor AZD5363 is selectively active in PI3KCA mutant gastric cancer, and sensitizes a patient-derived gastric cancer xenograft model with PTEN loss to Taxotere.

IntroductionActivation of the PI3K/AKT pathway is a common phenomenon in cancer due to multiple mechanisms, including mutation of PI3KCA, loss or mutation of PTEN, or over-expression of receptor tyrosine kinases. We recently developed a novel AKT kinase inhibitor, AZD5363, and demonstrated that HGC27, a cell line harboring both PI3KCA mutation and PTEN loss, displayed the greatest sensitivity to this AKT inhibitor in vitro and in vivo.Case preparationTo further elucidate the correlation between AZD5363 response and genetic alterations in gastric cancer (GC) and identify GC patients with both PI3KCA mutations and PTEN loss, we investigated the effects of pharmacological inhibition of AKT on a panel of 20 GC cell lines and genetic aberrations in tumor samples from a cohort of Chinese GC patients. We demonstrated that GC cells with PI3KCA mutations were selectively sensitive to AZD5363. Disease linkage studies showed that PI3KCA activating mutations or PTEN loss were found in 2.7% (4/150) and 23% (14/61) of Chinese GC patients respectively. To further dissect the role of PI3KCA mutation and PTEN loss in response to AKT inhibition, we tested the antitumor activity of AZD5363 in two patient-derived GC xenograft (PDGCX) models harboring either PI3KCA mutation or PTEN loss. Our data indicated that AZD5363 monotherapy treatment led to a moderate response in the PI3KCA mutant PDGCX model. Whilst monotherapy AZD5363 or Taxotere were ineffective in the PTEN negative PDGCX model, significant anti-tumor activity was observed when AZD5363 was combined with Taxotere.ConclusionOur results indicated that PI3KCA mutation is an important determinant of response to AKT inhibition in GC and combination with AZD5363 can overcome innate resistance to Taxotere in a PTEN loss PDGCX model. It is suggested that AKT inhibitor is an attractive option for treatment of a new segment of GC patients with aberrant PI3K/AKT signaling.

Whole genome gene copy number profiling of gastric cancer identifies PAK1 and KRAS gene amplification as therapy targets.

Gastric cancer is the second leading cause of death from cancer worldwide, with an approximately 20% 5‐year survival rate. To identify molecular subtypes associated with the clinical prognosis, in addition to genetic aberrations for potential targeted therapeutics, we conducted a comprehensive whole‐genome analysis of 131 Chinese gastric cancer tissue specimens using whole‐genome array comparative genomic hybridization. The analyses revealed gene focal amplifications, including CTSB, PRKCI, PAK1, STARD13, KRAS, and ABCC4, in addition to ERBB2, FGFR2, and MET. The growth of PAK1‐amplified gastric cancer cells in vitro and in vivo was inhibited when the corresponding mRNA was knocked down. Furthermore, both KRAS amplification and KRAS mutation were identified in the gastric cancer specimens. KRAS amplification was associated with worse clinical outcomes, and the KRAS gene mutation predicted sensitivity to the MEK1/2 inhibitor AZD6244 in gastric cancer cell lines. In summary, amplified PAK1, as well as KRAS amplification/mutation, may represent unique opportunities for developing targeted therapeutics for the treatment of gastric cancer.

Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target.

Protein phosphatase methylesterase 1 (PPME1) is a protein phosphatase 2A (PP2A)-specific methyl esterase that negatively regulates PP2A through demethylation at its carboxy terminal leucine 309 residue. Emerging evidence shows that the upregulation of PPME1 is associated with poor prognosis in glioblastoma patients. By performing an array comparative genomic hybridization analysis to detect copy number changes, we have been the first to identify PPME1 gene amplification in 3.8% (5/131) of Chinese gastric cancer (GC) samples and 3.1% (4/124) of Chinese lung cancer (LC) samples. This PPME1 gene amplification was confirmed by fluorescence in situ hybridization analysis and is correlated with elevated protein expression, as determined by immunohistochemistry analysis. To further investigate the role of PPME1 amplification in tumor growth, short-hairpin RNA-mediated gene silencing was employed. A knockdown of PPME1 expression resulted in a significant inhibition of cell proliferation and induction of cell apoptosis in PPME1-amplified human cancer cell lines SNU668 (GC) and Oka-C1 (LC), but not in nonamplified MKN1 (GC) and HCC95 (LC) cells. The PPME1 gene knockdown also led to a consistent decrease in PP2A demethylation at leucine 309, which was correlated with the downregulation of cellular Erk and AKT phosphorylation. Our data indicate that PPME1 could be an attractive therapeutic target for a subset of GCs and LCs.

The oncogenic role of PKCiota gene amplification and overexpression in Chinese non-small cell lung cancer

BACKGROUND The atypical protein kinase C isozyme iota (PKCiota) has been proposed as an oncogene based on its transformation property and amplification identified in Caucasian non-small cell lung cancer (NSCLC) patients. Because the geography difference of some genetic aberrance such as EGFR mutations between Caucasian and Asian NSCLC patients has been identified previously, it is important to know whether the PKCiota amplification also occurs in Asian NSCLC patients. METHODS The PKCiota gene copy number changes and protein expression in Chinese patients samples were detected by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. Logistic regression was used to assess the association of PKCiota expression with clinicopathological parameters. siRNA-mediated gene silencing was applied to demonstrate the role of PKCiota in promoting cell growth in PKCiota gene amplified and protein overexpressed cancer cells. RESULTS The result showed that PKCiota gene was amplified in 20.2% (24/119) of the tested primary tumor samples from Chinese NSCLC patients. Interestingly this gene amplification was highly enriched in squamous NSCLC patients (37.1%, 23/62). Further IHC analysis indicated that PKCiota protein was highly expressed (IHC score 2+ and 3+) in 91.6% (109/119) of Chinese NSCLC tumors. Moreover, the PKCiota gene amplification was also correlated with gender, subtype and distant metastasis. Knockdown of PKCiota gene in the PKCiota gene amplified and protein overexpressed cells led to significant growth inhibition. CONCLUSION Taken together, our data demonstrate that PKCiota is a potential oncogene and therapeutic target in Chinese NSCLC.

Abstract 3224: Pre-clinical targeting of the metabolic phenotype of lymphoma by AZD3965, a selective inhibitor of monocarboxylate transporter 1 (MCT1)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction - The metabolic phenotype of tumours (relative to most normal tissues) is shifted from oxidative phosphorylation to aerobic (Warburg Effect) or anaerobic glycolysis. This enables tumours to meet their energetic and biosynthetic demands even under conditions of low nutrient and O2. The end-product of glycolysis is lactate, a metabolic dead end, which if allowed to accumulate in the tumour cell may cause feedback inhibition of glycolysis and intra-cellular acidification resulting in inhibition of cell growth or survival. Experimental data - We have studied the effects of a potent, selective and orally available inhibitor of MCT1 in lymphoma cell lines and in vivo models. AZD3965 is a small molecule inhibitor of MCT1 with a binding affinity of 1.6 nM, is 6 fold selective over MCT2 and does not inhibit MCT4 at 10 μM. Both lactate transport and cell growth are potently inhibited by AZD3965 in lymphoma cell lines that preferentially express MCT1. Lactate transport inhibition in some cell lines also induces a cytotoxic effect. In vitro combination studies show that lactate transport inhibition can enhance the induction of cell death by doxorubicin. Blocking lactate transport in vitro also leads to a rapid inhibition of glucose uptake in the Raji Burkitts lymphoma cell line. In vivo, AZD3965 is well tolerated and induces a dose and time dependent accumulation of lactate in the tumours, suppresses tumour growth and in the Raji model potentiates the effects of Rituxan, doxorubicin and bendamustine. Conclusions - Here we demonstrate that selective inhibition of lactate transport by the MCT1 inhibitor AZD3965 offers an novel mechanism for targeting the metabolic phenotype in tumours that preferentially express MCT1. 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 3224. doi:1538-7445.AM2012-3224

Abstract 2923: Identification and validation of dysregulated MAPK7 (ERK5) as a novel oncogenic target in squamous cell lung and esophageal carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA MAPK7/ERK5 (extracellular-signal-regulated kinase 5) is the effector kinase of a canonical three-tiered MAPK (mitogen activated protein kinase) signalling cascade comprising MEK (MAPK/ERK kinase) 5, MEKK(MEK kinase) 2/3 and ERK5 itself(1). Under normal physiological conditions, MEK5 and ERK5 are ubiquitously expressed and are activated by growth factors and cellular stresses to perform roles in blood vessel, cardiac and muscle development(2). Although the MAPK7-axis is the least well studied of the MAPK-signaling pathways, evidence suggests that it plays a role in the pathology of cancer, in particular; breast cancer progression, prostate cancer bone metastases and hepatocellular carcinoma(3-5). Array-based comparative genomic hybridisation profiling (aCGH) of non-small cell lung cancer (NSCLC) patient tumor samples initially identified MAPK7 gene amplification as a novel genetic event. To further explore the role of MAPK7 in human cancer, we assessed the true incidence of MAPK7 gene amplification using fluorescence in situ hybridization (FISH) analysis in a cohort of 80 NSCLC and 95 squamous esophageal cancers (sqEC). High level MAPK7 gene amplification was detected in 4% (3/80) of NSCLC (enriched to 6% (3/49) in squamous cell carcinoma) and 2% (2/95) of sqEC. Immunohistochemical (IHC) analysis of the same tissue samples revealed a good correlation of MAPK7 gene amplification and high level protein expression but importantly, also identified a high prevalence of IHC ‘3+’ MAPK7 protein expression (20% - 15/74 NSCLC) in the absence of gene amplification. To validate MAPK7 as an oncogenic driver of tumor cell proliferation, we performed in vitro siRNA knockdown of MAPK7 in amplified and non-amplified tumor cell lines. Potent anti-proliferative effects were only observed when MAPK7 protein expression was ablated in MAPK7-amplified lines (eg. KYSE30). To support drug discovery efforts, a novel MEK5/MAPK7 co-transfected HEK293 cell line was developed and used for routine cell-based pharmacodynamic screening. In parallel, reverse-phase protein arrays (RPPA) were used to identify novel candidate downstream pharmacodynamic biomarkers of MAPK7 kinase inhibition in tumor cells (pMEF2A and pMEF2C). Finally, a novel MAPK7-amplified esophageal xenograft model was established and used to demonstrate direct pharmacodynamic modulation of pMAPK7 using a commercially available tool compound (XMD8-92). Taken together, these data highlight a broader role for dysregulated MAPK7 in driving tumorigenesis within niche populations of highly prevalent tumor types, and describe current efforts in establishing a robust drug discovery screening cascade. 1. Zhou, G. (1995) J. Biol. Chem. 270, 12665-12669 2. Lochhead PA. (2012) Biochem Soc Trans. 40(1):251-6. 3. Song, H. (2004) Oncogene 23, 8301-8309 4. Mehta, P.B. (2003) Oncogene 22, 1381-1389 5. Zen, K. (2009) Genes Chromosomes Cancer 48,109-120 Citation Format: Paul R. Gavine, Mei Wang, Dehua Yu, Eva Hu, Chunlei Huang, Jenny Xia, Xinying Su, Joan Fan, Tianwei Zhang, Qingqing Ye, Li Zheng, Liang Xie, Luna Han, Guanshan Zhu, Ziliang Qian, Qingquan Luo, Yingyong Hou, Qunsheng Ji. Identification and validation of dysregulated MAPK7 (ERK5) as a novel oncogenic target in squamous cell lung and esophageal carcinoma. [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 2923. doi:10.1158/1538-7445.AM2014-2923

Abstract 5186: PPME1 is a potential novel molecular target in gastric and lung cancer.

Protein phosphatase 2A (PP2A) is a human tumor suppressor, and it accounts for the majority of serine/threonine phosphatase activity which is tightly regulated via the methylation of carboxy-terminal of PP2A. Methylation at the carboxyterminal Leu 309 residue by S-adenosylmethionine-dependent leucine carboxyl methytransferase-1 (LCMT1) leads to the activation of PP2A, whilst demethylation of Leu 309 by specific protein phosphatase methylesterase-1 (PPME1) suppresses the PP2A activity. Recent evidence showed that PPME1 is up-regulated in glioblastoma patients and associated with a poor prognosis. In this study we profiled 131 Chinese gastric cancer (GC) samples and 124 Chinese non-small cell lung carcinoma (NSCLC) samples by Affy and aCGH to identify potential drug targets, and found PPME1 gene amplification in 3.8% of the GC samples and 3.1% of the NSCLC samples. The PPME1amplification correlated with its mRNA expression. In addition, we confirmed that PPME1 protein expressions were also elevated in the PPME1-amplified tumor samples indicated by immunohistochemistry as compared with corresponding adjacent non-tumor samples. To further investigate the role of PPME1 amplification in tumor growth, we utilized siRNA-mediated gene silencing approaches to specifically knock down PPME1 expression in the PPME1 amplified human cancer cell lines SNU668 (GC) and Oka-C1 (NSCLC). We found that suppression of the PPME1 expression resulted in significant inhibition of cell proliferation and induction of cell death in those cells. In contrast, little effects were observed on the cell growth and survival of non PPME1-amplified human cancer cells, MKN1 (GC) and HCC95 (NSCLC). Moreover, we showed that the PPME-1 knock-down also resulted in a decrease of PP2A demethylation at Leu309, which correlated with down-regulation of phosphorylation of MAPK and AKT in the cells. Taken together, our data indicate that PPME-1 could be an attractive therapeutic target for subsets of gastric and lung cancers. Citation Format: Jing Li, Sufang Han, Ziliang Qian, Xinying Su, Shuqiong Fan, Jiangang Fu, Charles Liu, Lucy Yin, Zeren Gao, Guanshan Zhu, Jingchuan Zhang, De-Hua Yu, Qunsheng Ji. PPME1 is a potential novel molecular target in gastric and lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5186. doi:10.1158/1538-7445.AM2013-5186

Abstract 4368: Cathepsin B is a potential therapeutic target in pancreatic and gastric cancer .

Proteases are critical in tumorigenesis by facilitating rapid cell cycling, mediating local invasion, fueling angiogenesis, and promoting metastasis. Cathepsin B (CTSB), a member of papain subfamily of lysosomal cysteine proteases, is involved in tumor cell invasion, metastasis, and angiogenesis. Emerging evidence indicated that CTSB is up-regulated in many cancer nodes and metastatic lesions and functions to protect tumor cells from apoptosis. Hererin, we have performed microarray-based comparative hybridization (aGCH) to screen gene copy number variations in primary tumor samples and found CTSB gene is amplified in 13% (14/107) of Chinese gastric cancer and 2% (1/54) of Chinese pancreatic samples. Theses amplifications were confirmed by fluorescence in situ hybridization (FISH) analysis. By using siRNA-mediated gene silencing, we demonstrated for the first time a role of CTSB in cancer cell proliferation. Down-regulation of CTSB by siRNA interference resulted in significantly reduced cell proliferation and increased cell death in HupT4, a CTSB amplified human pancreatic cancer cell line, but not in the non-amplified cell lines. Together, our results suggest that CTSB is a potential therapeutic target for pancreatic and gastric cancer with CTSB amplification. Citation Format: Mei Wang, Chunlei Huang, Harvey Dong, Ziliang Qian, Shuqiong Fan, Xinying Su, Li Zheng, Qunsheng Ji, De-Hua Yu. Cathepsin B is a potential therapeutic target in pancreatic and gastric cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4368. doi:10.1158/1538-7445.AM2013-4368