Overexpression Of Receptor Tyrosine Kinases Research Articles

Design and synthesis of improved active-site SHP2 inhibitors with anti-breast cancer cell effects

The Src homology containing phosphotyrosyl phosphatase 2 (SHP2) is a bona fide oncogene particularly in cancers driven by overexpression of receptor tyrosine kinases (RTKs). As such, there is a growing interest to target SHP2 in cancer. Based on these premises, several active site (type I) and allosteric site (type II) inhibitors have been developed, but no SHP2 targeting therapies have reached the clinic yet. In an effort to fill these gaps, we embarked on producing optimized versions of our parent active-site SHP2 inhibitor CNBDA. The objectives were to produce derivatives with increased inhibitory potential and improved selectivity. Accordingly, we designed derivatives around the CNBDA scaffold and predicted their binding property by in silico molecular modeling. Based on comparative differences in free energy of binding to the SHP2 versus the SHP1 active sites, ten were selected, chemically synthesized, and evaluated by NMR and mass spectroscopy for structural integrity. Among the ten derivatives, BPDA2 was found to be the most potent and highly selective compound, inhibiting the SHP2 enzyme activity with an IC50 of 92 nM when DiFMUP was used as a substrate and with an IC50 of 47 nM when pNPP was used as a substrate. Furthermore, enzyme kinetic analyses showed that BPDA2 is a competitive SHP2 inhibitor. Selectivity comparisons in a PTPase assay using DiFMUP as a substrate demonstrated that BPDA2 is more selective to SHP2 than to SHP1 and PTP1B by more than 369-fold and 442-fold, respectively. Evaluation with a cellular thermal shift assay (CETSA) confirmed that BPDA2 binds to wild-type SHP2 in a cellular context, and stabilizes it in solution. Treatment of cells with DBDA2 downregulates mitogenic and cell survival signaling and RTK expression in a concentration dependent manner. Furthermore, treatment of cells with BPDA2 suppresses anchorage independent growth and cancer stem cell properties of breast cancer cells. Overall, data described in this report show that BPDA2 is a more potent derivative of CNBDA with a highly improved selectivity for SHP2.

Abstract 3749: MER receptor tyrosine kinase overexpression potentiates survival signal in chronic lymphocytic leukemia cells

Abstract Chronic lymphocytic leukemia (CLL) is the most common form of adult B-cell leukemia in the United States. It is predominantly characterized by the accumulation of CD19+/CD5+/CD23+ monoclonal B-cells in peripheral blood, bone marrow and lymphoid tissues. BCR signaling pathway has emerged as a key driver for the expansion of neoplastic B-cell clones and pathogenesis in several B-cell malignancies including CLL. Stimulation of BCR induces activation of a cascade of downstream signal mediators including Bruton's tyrosine kinase (BTK), PI3K/AKT and ERK1/2. While the use of BCR-targeted agents, ibrutinib (BTK-inhibitor) or idelalisib (PI3Kδ-inhibitor), has been shown to be effective in relapsed/refractory CLL patients, the responses are limited to partial remissions. Therefore, insights into drugs with unique mechanism(s) of action that are nontoxic to the bone marrow/immune system and feasible in the older patient population (average age: 65 - 70 years) are urgently needed. To achieve such a goal it is critical to identify other targets that are constitutively active and may crosstalk with the BCR signal or other receptor tyrosine kinases (RTKs). MER RTK of the TAM (Tyro3, AXL, MER) family offers such an alternative approach. Previously, we detected that CLL B-cells express highly active AXL that acts as a docking site of multiple kinases/lipase including LYN, SYK/ZAP70, PI3K and PLCγ2 that, when subject to specific inhibition induces apoptosis. Most recently, we found that CLL B-cells from 50 - 60% of previously untreated CLL patients (n=30) also overexpressed MER at variable levels while, normal B-cells from health subjects did not show any such expression. Interestingly, MER in CLL B-cells remains constitutively phosphorylated (active). In addition, we also detected overexpression of its ligand, growth arrest specific gene 6 (Gas6) in CLL B-cells as compared to normal B-cells. Of note, leukemic B-cells from the Eμ-TCL1 transgenic mice which recapitulate aggressive human CLL also showed overexpression of MER. To this end, we found that exogenous addition of recombinant Gas6 to primary CLL B-cell culture or MEC1 (a CLL cell line) cells activated MER in a time-dependent manner. Interestingly, we also detected co-activation of BTK of the BCR pathway, following MER activation in CLL cells while, in vitro stimulation of BCR did not activate MER. Together, these findings suggest that MER activation may regulate the BCR signal in CLL B-cells in order to potentiate the survival signal. In conclusion, we detected overexpression of constitutively activated MER in CLL B-cells from some CLL patients which may represent high-risk CLL clones for clinical progression. Importantly, activation of MER may positively regulate the BCR signal inducing increased survival and apoptotic resistance of the leukemic B-cells to BCR-targeted agents. Further studies are in progress to elucidate the impact of targeting MER in CLL B-cell survival and resistance to therapy. Citation Format: Mariana T. Mendez, Hasan Mahmud, Sara K. Vesely, Jennifer Holter-Chakrabarty, Asish K. Ghosh. MER receptor tyrosine kinase overexpression potentiates survival signal in chronic lymphocytic leukemia cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3749.

Genomic Profiling of Biliary Tract Cancer Cell Lines Reveals Molecular Subtypes and Actionable Drug Targets.

SummaryBiliary tract cancers (BTCs) currently have no approved targeted therapies. Although genomic profiling of primary BTCs has identified multiple potential drug targets, accurate models are needed for their evaluation. Genomic profiling of 22 BTC cell lines revealed they harbor similar mutational signatures, recurrently mutated genes, and genomic alterations to primary tumors. Transcriptomic profiling identified two major subtypes, enriched for epithelial and mesenchymal genes, which were also evident in patient-derived organoids and primary tumors. Interrogating these models revealed multiple mechanisms of MAPK signaling activation in BTC, including co-occurrence of low-activity BRAF and MEK mutations with receptor tyrosine kinase overexpression. Finally, BTC cell lines with altered ERBB2 or FGFRs were exquisitely sensitive to specific targeted agents, whereas surprisingly, IDH1-mutant lines did not respond to IDH1 inhibitors in vitro. These findings establish BTC cell lines as robust models of primary disease, reveal specific molecular disease subsets, and highlight specific molecular vulnerabilities in these cancers.

Drak/STK17A Drives Neoplastic Glial Proliferation through Modulation of MRLC Signaling.

Glioblastoma (GBM) and lower grade gliomas (LGG) are the most common primary malignant brain tumors and are resistant to current therapies. Genomic analyses reveal that signature genetic lesions in GBM and LGG include copy gain and amplification of chromosome 7, amplification, mutation, and overexpression of receptor tyrosine kinases (RTK) such as EGFR, and activating mutations in components of the PI3K pathway. In Drosophila melanogaster, constitutive co-activation of RTK and PI3K signaling in glial progenitor cells recapitulates key features of human gliomas. Here we use this Drosophila glioma model to identify death-associated protein kinase (Drak), a cytoplasmic serine/threonine kinase orthologous to the human kinase STK17A, as a downstream effector of EGFR and PI3K signaling pathways. Drak was necessary for glial neoplasia, but not for normal glial proliferation and development, and Drak cooperated with EGFR to promote glial cell transformation. Drak phosphorylated Sqh, the Drosophila ortholog of nonmuscle myosin regulatory light chain (MRLC), which was necessary for transformation. Moreover, Anillin, which is a binding partner of phosphorylated Sqh, was upregulated in a Drak-dependent manner in mitotic cells and colocalized with phosphorylated Sqh in neoplastic cells undergoing mitosis and cytokinesis, consistent with their known roles in nonmuscle myosin-dependent cytokinesis. These functional relationships were conserved in human GBM. Our results indicate that Drak/STK17A, its substrate Sqh/MRLC, and the effector Anillin/ANLN regulate mitosis and cytokinesis in gliomas. This pathway may provide a new therapeutic target for gliomas.Significance: These findings reveal new insights into differential regulation of cell proliferation in malignant brain tumors, which will have a broader impact on research regarding mechanisms of oncogene cooperation and dependencies in cancer.See related commentary by Lathia, p. 1036.

Drosophila melanogaster as a Model System for Human Glioblastomas.

Glioblastoma multiforme (GBM) is the most common primary malignant adult brain tumor. Genomic amplifications, activating mutations, and overexpression of receptor tyrosine kinases (RTKs) such as EGFR, and genes in core RTK signaling transduction pathways such as PI3K are common in GBM. However, efforts to target these pathways have been largely unsuccessful in the clinic, and the median survival of GBM patients remains poor at 14-15months. Therefore, to improve patient outcomes, there must be a concerted effort to elucidate the underlying biology involved in GBM tumorigenesis. Drosophila melanogaster has been a highly effective model for furthering our understanding of GBM tumorigenesis due to a number of experimental advantages it has over traditional mouse models. For example, there exists extensive cellular and genetic homology between humans and Drosophila, and 75% of genes associated with human disease have functional fly orthologs. To take advantage of these traits, we developed a Drosophila GBM model with constitutively active variants of EGFR and PI3K that effectively recapitulated key aspects of GBM disease. Researchers have utilized this model in forward genetic screens and have expanded on its functionality to make a number of important discoveries regarding requirements for key components in GBM tumorigenesis, including genes and pathways involved in extracellular matrix signaling, glycolytic metabolism, invasion/migration, stem cell fate and differentiation, and asymmetric cell division. Drosophila will continue to reveal novel biological pathways and mechanisms involved in gliomagenesis, and this knowledge may contribute to the development of effective treatment strategies to improve patient outcomes.

CSIG-26. MET FUSION, AMPLIFICATION, AND/OR OVEREXPRESSION DEFINES DIFFUSELY INVASIVE TUMOR CELLS IN PEDIATRIC AND ADULT GLIOBLASTOMA

Glioblastomas (GBMs) are WHO Grade IV neoplasms of the central nervous system (CNS) that are the most common and most aggressive glioma. Genomics show that the most frequent changes in GBMs include amplification, mutation, and/or overexpression of receptor tyrosine kinases (RTKs) as well as mutations that constitutively activate the PI-3 kinase (PI3K) pathway. Current data indicates that RTK-PI3K signaling cooperates with other mutations that drive GBM pathology and confer therapeutic resistance. To best understand the factors that drive tumor growth and therapeutic resistance, we have developed a research program that utilizes primary human GBM neurosphere cultures created from surgical specimens. These GBM neurosphere cultures retain many of the defining features and mutations found in primary tumors, and, in mouse xenograft assays, they yield highly malignant invasive tumors. We currently have more than 30 GBM neurosphere lines created from tumors that have been genomically profiled for copy number abnormalities and for common mutations. In addition, neurosphere lines are subject to RNA sequencing to determine their over-all transcriptome and to identify novel and recurrent mutations in tumor cells. Through these analyses we have identified a number of copy number changes, point mutations, and gene fusions in several loci. We identified alterations in the MET gene, and found that diffusely invasive GBM cells express MET. Our sequencing data showed that MET is co-overexpressed with Class 3 Semaphorins and their receptors, genes that augment MET activity in migrating neural cells in the developing embryonic brain; these genes may cooperate with MET alterations to worsen GBM. Therefore, we propose to 1) test drugs that inhibit MET and in our models of MET-mutant GBMs, and 2) determine if MET mutations and alterations cooperate with Semaphorins to drive GBM tumor cell invasion. Our results could lead to new clinical trials of MET targeted therapies in GBM.

HER3 Expression Is a Marker of Tumor Progression in Premalignant Lesions of the Gastroesophageal Junction.

Overexpression of receptor tyrosine kinases (RTK), including members of the HER family, has prognostic and therapeutic significance in invasive esophagogastric carcinoma. RTK expression in premalignant gastroesophageal lesions has not been extensively explored. Formalin-fixed paraffin-embedded tissue samples of esophageal biopsy specimens from 73 patients with Barrett’s esophagus with either low-grade dysplasia (LGD) (n = 32) or high-grade dysplasia (HGD) (n = 59) were analyzed for HER1, HER2, HER3 and CMET expression by immunohistochemistry (IHC). Immunophenotype was correlated with histologic and clinical features. High-grade dysplasia (HGD) was associated with overexpression of HER1 (20.7% vs. 3.1%, p = 0.023), HER2 (5.3% vs. 0.0%, p = 0.187) and HER3 (47.4% vs. 9.4%, p<0.001) compared to low-grade dysplasia (LGD). There was a significant association of HER2 (20.0% vs. 2.1%, p = 0.022) and HER3 (80.0% vs. 40.4%, p = 0.023) overexpression in HGD lesions associated with foci of invasive carcinoma compared to those without invasive foci. Overexpression of CMET was observed in 42.9% of specimens, was increasingly observed with HGD compared to LGD (58.3% vs. 36.7%, p = 0.200), and was most often co-expressed with HER3 (62.5% of HER3-positive specimens vs. 38.2% of HER3-negative specimens, p = 0.212). In summary, HER3 is frequently overexpressed in high-grade dysplastic lesions of the gastroesophageal junction and may be a marker of invasive progression. These data provide rationale for targeting HER2 and HER3 pathways in an early disease setting to prevent disease progression.

A Nexus Consisting of Beta-Catenin and Stat3 Attenuates BRAF Inhibitor Efficacy and Mediates Acquired Resistance to Vemurafenib

Acquired resistance to second generation BRAF inhibitors (BRAFis), like vemurafenib is limiting the benefits of long term targeted therapy for patients with malignant melanomas that harbor BRAF V600 mutations. Since many resistance mechanisms have been described, most of them causing a hyperactivation of the MAPK- or PI3K/AKT signaling pathways, one potential strategy to overcome BRAFi resistance in melanoma cells would be to target important common signaling nodes. Known factors that cause secondary resistance include the overexpression of receptor tyrosine kinases (RTKs), alternative splicing of BRAF or the occurrence of novel mutations in MEK1 or NRAS.In this study we show that β-catenin is stabilized and translocated to the nucleus in approximately half of the melanomas that were analyzed and which developed secondary resistance towards BRAFi. We further demonstrate that β-catenin is involved in the mediation of resistance towards vemurafenib in vitro and in vivo. Unexpectedly, β-catenin acts mainly independent of the TCF/LEF dependent canonical Wnt-signaling pathway in resistance development, which partly explains previous contradictory results about the role of β-catenin in melanoma progression and therapy resistance. We further demonstrate that β-catenin interacts with Stat3 after chronic vemurafenib treatment and both together cooperate in the acquisition and maintenance of resistance towards BRAFi.

Abstract 4696: Combining AZD8186, an inhibitor of PI3Kbeta, with inhibitors of PI3Kalpha and mTORC1/2 gives comprehensive pathway suppression and enhanced antitumor activity in PTEN null tumors

Abstract The PI3K pathway drives the progression of many human tumours via mutation of PI3KCA or AKT, loss of tumour suppressors PTEN or TSC1/2, or over-expression of receptor tyrosine kinases, such as EGFR, Erb2, or IGFR. Targeting the PI3K pathway has been challenging due to toxicities with the first generation compounds. Despite this, positive clinical activity has been achieved, particularly in combination. Maximum benefit with inhibitors of the PI3K pathway is likely to be in combination, with agents that target other pathways, or PI3K signalling at different nodes. In cells, AZD8186 inhibits PI3Kbeta- and PI3Kdelta-dependent phosphorylation of AKT with IC50s of 3nM and 17nM respectively. Tumour cells can become dependent on PI3Kbeta when they lose PTEN protein function. PI3Kbeta inhibitors are differentiated from PI3Kalpha, mTOR and AKT inhibitors as they do not modulate peripheral glucose and other aspects of the canonical PI3Kalpha signalling pathway. Therefore in tumours where PI3Kbeta is activated it may be possible to combine AZD8186 with other inhibitors of the PI3K pathway to achieve comprehensive pathway suppression. Delivering comprehensive pathway suppression has become critically important with the realisation that resistance to the PI3K inhibitors can occur through feedback-mediated or mutation-mediated reactivation of the pathway through alternate PI3K isoforms. We have explored intra-pathway combinations of AZD8186 with inhibitors of PI3Kalpha (AZD8835 and BYL719) and mTOR (AZD2014 and everolimus) in PTEN protein deficient models. In particular, combining AZD8186 with inhibitors of mTOR increased efficacy across a range of tumour models. In TNBC (HCC-70) or renal (786-0) xenograft models, combined inhibition resulted in tumour regression. The degree of tumour regression was influenced by dose and schedule. For example, intermittent dosing of AZD8186 with continuous AZD2014 regressed 786-0 tumours by between 38% and 82%, while HCC70 showed a 21% regression. Tumour regressions were also observed when AZD8186 and AZD2014 were dosed intermittently, a 5 day on AZD8186 with 2 day on AZD2014 concurrent dosing schedule in 786-0 gave 41% and 22% regression respectively in HCC70. The anti-tumour effects were associated with increased depth and duration of inhibition of AKT pathway biomarkers, but significantly also modification of biomarkers associated with cellular metabolic function and survival pathways. The ability of intermittent doses of the combination regimen to deliver tumour regression establishes the principle that it is possible to move away from chronic pathway suppression, offering the flexibility to mitigate potential tolerability challenges, potential acute resistance mechanisms, but also drive increased phenotypic effects in the tumour. Citation Format: Urs Hancox, Urszula Polanska, James T. Lynch, Carol Lenaghan, Cath Trigwell, Oona Depuelch, Phillippa Dudley, Juliana Maynard, Lara Ward, Kevin Hudson, Jon Curwen, Francisco Cruzalegui, Stephen Green, Klinowska Teresa, Simon T. Barry. Combining AZD8186, an inhibitor of PI3Kbeta, with inhibitors of PI3Kalpha and mTORC1/2 gives comprehensive pathway suppression and enhanced antitumor activity in PTEN null tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4696. doi:10.1158/1538-7445.AM2015-4696

Prognostic relevance of receptor tyrosine kinase expression in breast cancer: a meta-analysis.

Receptor tyrosine kinases (RTKs) may facilitate tumor progression if activated aberrantly. The prognostic impact of human epidermal growth factor receptor 2 (HER2) overexpression and effectiveness of its therapeutic targeting is well established, but the effects on prognosis of overexpression of other RTKs is unknown. Here we evaluate the association of RTK expression and survival in breast cancer. PubMed was searched to identify studies evaluating the association between expression of RTKs other than HER2 and survival of women with breast cancer. Published data were extracted and computed into odds ratios (OR) for death at 5 years with 95% confidence intervals (CI). Data were pooled in a meta-analysis using the Mantel-Haenszel random-effect model. For studies reporting data for more than one RTK the lowest and highest OR were used for separate analyses. Sixteen studies comprising 11,056 patients were included in the analysis. There was an association between overexpression of RTKs and decreased 5-year OS and this was highly significant when using highest ORs from studies reporting more than one RTK (OR=2.42; 95% CI=1.92-3.06, P<0.001). Similar results were observed for 5-year BCSS. Worse OS was seen with overexpression of fibroblast growth factor receptor 2/3 (FGFR) (OR=3.81; 95% CI=1.79-8.11) and epidermal growth factor receptor (EGFR)/HER1 (OR=2.45; 95% CI=1.90-3.15). Overexpression of various RTKs is associated with poor outcomes. This data suggests the clinical evaluation of combination of agents against RTKs or relevant oncogenic nodes.

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.

Phosphotyrosine Signaling Proteins that Drive Oncogenesis Tend to be Highly Interconnected

Mutation and overexpression of receptor tyrosine kinases or the proteins they regulate serve as oncogenic drivers in diverse cancers. To better understand receptor tyrosine kinase signaling and its link to oncogenesis, we used protein microarrays to systematically and quantitatively measure interactions between virtually every SH2 or PTB domain encoded in the human genome and all known sites of tyrosine phosphorylation on 40 receptor tyrosine kinases and on most of the SH2 and PTB domain-containing adaptor proteins. We found that adaptor proteins, like RTKs, have many high affinity bindings sites for other adaptor proteins. In addition, proteins that drive cancer, including both receptors and adaptor proteins, tend to be much more highly interconnected via networks of SH2 and PTB domain-mediated interactions than nononcogenic proteins. Our results suggest that network topological properties such as connectivity can be used to prioritize new drug targets in this well-studied family of signaling proteins.

Abstract 525: Induction of apoptosis in ovarian clear cell carcinomas with wild-type TP53 by inhibiting PI3K/mTOR signaling pathway.

Abstract Background:Epithelial ovarian cancer is a leading cause of death from gynecological malignancies.Ovarian clear cell adenocarcinoma (OCCA) is the second most common cause of death from ovarian cancer with a higher incidence in Asia, especially Japan (&amp;gt;25%). Due to low response rates to conventional platinum-based chemotherapy, the clinical outcome is generally poor. PIK3CA mutations are frequent (&amp;gt;40%) in OCCA compared with other histology types and overexpression of receptor tyrosine kinases (RTKs), including HER2 and cMET, is also frequently observed in OCCA. Thus, the RTK-PI3K (phosphatidylinositol 3-kinase)/mTOR (mammalian target of rapamycin) signaling axis is broadly activated in OCCA. This study evaluated the anti-tumor efficacy of DS-7423, a novel PI3K/mTOR dual inhibitor, in a panel of OCCA cell lines. Material and methods: We evaluated the phosphorylation status of RTK-PI3K/mTOR signaling pathway in 9 OCCA cell lines. Four of those cell lines possessed PIK3CA mutations; 3 of the 5 remaining cell lines overexpressed HER2, HER3 and/or cMET. We determined the effect of DS-7423 by MTT assay, FACS analysis, and Western blotting. Apoptosis induction was detected by annexin-V and propidium iodide (PI) staining. Induction of p53 target genes, including p53AIP1, was evaluated by RT-PCR. Results: IC50 values for DS-7423 were &amp;lt;100 nM for all 9 OCCA cell lines. Efficacy did not significantly differ between PIK3CA mutant and wild type OCCA cells. AKT and its downstream targets (GSK3β, FOXO1/3a, MDM2, 4EBP1, and S6) were phosphorylated in OCCA cell lines regardless of PIK3CA mutational status. DS-7423 suppressed the phosphorylation levels of AKT and its target proteins in all these OCCA cell lines. FACS analysis revealed an increase in sub-G1 cell populations by treatment at ≥156 nM DS-7423 in 6 out of 9 OCCA cell lines. In these 6 cell lines, the number of apoptotic cells, as determined by annexin-V and PI staining, increased in a dose-dependent manner and DS-7423 induced apoptosis more effectively in TP53 wild type OCCA cell lines than in TP53 mutant OCCA cell lines. In accordance with decreased phospho-MDM2 levels, phosphorylation levels of p53 (Ser46) were increased and expression of p53AIP1 and PUMA, genes regulating p53-dependent apoptosis, were induced by DS-7423 in these p53 wild type cells. We also evaluated the expression status of bcl-2 family proteins in the OCCA cell line OVISE, and observed that DS-7423 treatment induced up-regulation of proapoptotic Bim and down-regulation of anti-apoptotic Mcl-1. Conclusions: Dual PI3K/mTOR inhibitors such as DS-7423 may constitute a promising molecular targeted therapy for OCCA, regardless of PIK3CA status. DS-7423 may induce apoptosis via activation of p53 and bcl-2 family members. As p53 mutations ar generally rare in OCCA, DS-7423 might induce p53-dependent apoptosis in this cancer type. Citation Format: Tomoko Kashiyama, Katsutoshi Oda, Yuji Ikeda, Yoshinobu Shiose, Yasuhide Hirora, Aki Miyasaka, Takahiro Koso, Kanako Inaba, Tomohiko Fukuda, Keiko Shoji, Michihiro Tanikawa, Kazunori Nagasaka, Osamu Hiraike-Wada, Kei Kawana, Hiroyuki Aburatani, Shunsuke Nakagawa, Tomoyuki Fujii, Tetsu Yano, Shiro Kozuma. Induction of apoptosis in ovarian clear cell carcinomas with wild-type TP53 by inhibiting PI3K/mTOR signaling pathway. [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 525. doi:10.1158/1538-7445.AM2013-525

Abstract 1207: Development of a quantitative gastroesophageal cancer selected reaction monitoring mass Spectrometric Multiplex Assay for use in FFPE tumor tissues.

Abstract Aberrant over-expression of receptor tyrosine kinases, (e.g. MET, HER, FGFR, and IGFR) as well as other oncogenic mediators (e.g. KRAS, PI3 Kinase and SRC) are known drivers of gastroesophageal adenocarcinoma (GEC), subdividing the disease into distinct molecular subsets. Inter/intrapatient tumor heterogeneity suggests that an expedient, reliable, medium throughput oncogene protein expression profiling will provide vital information to better personalize cancer care. To date, clinical quantification of protein in formalin fixed paraffin embedded (FFPE) tissues is limited to immunohistochemistry (IHC), which is semi-quantitative at best. Moreover, IHC of multiple proteins of interest is laborious, time consuming, wasteful of scarce tissue, and costly. We present a quantitative mass spectrometric (MS) assay for FFPE GEC utilizing Liquid Tissue - Selected Reaction Monitoring (SRM), with subsequent multiplex quantification of relevant oncoproteins in a panel of gastroesophageal cancer (GEC) cell lines and tissues. Using trypsin digestion mapping of recombinant oncoproteins, we identified unique peptide sequences, and built quantitative MS assays which could be multiplexed into a single SRM analysis of 1μg of tumor protein. Assays were preclinically validated on 10 different formalin fixed (FF) cell lines. We then tested the GEC-plex assay on a panel of FFPE GEC cell lines characterized by immunoblot (IB), IHC, and gene copy number by FISH. In addition to RON, we multiplexed SRM quantification of Met, EGFR, HER2, HER3, IGF1R, FGFR2, KRAS and cSRC. We evaluated 17 GEC lines including AGS wild type, scrambled shRNA (AGS-SC) and RON shRNA knockdown (AGS-KD) to assess ‘post-treatment’ changes in oncogene expression. We then evaluated 100 GEC human cancer tissues with paired peritoneal metastases when available and select paraneoplastic normal tissues using laser microdissection of tumor tissue from a single unstained 10μm thick section. Validation of the GEC-plex SRM assay on GEC cell lines revealed very high concordance when compared to IB and IHC measurement. The AGS-WT/SC cells showed comparable levels of RON (284/323 amol/μg cell protein), while RON was not detected in AGS-KD cells, as expected. Measurement of oncoproteins in GEC cell lines and tissues correlated well with IHC and FISH data. Multiplex oncogene quantification of all cell lines and tissues, along with expression profile changes in the AGS RON KD line compared to AGS-WT/SC will be presented. Taken together, these data demonstrate a sensitive, accurate, and quantitative assay to measure relevant actionable oncoproteins in FF cells. The GEC-plex multiplexed oncogene expression of these tumors was feasible and expedient using limited tissue from clinical samples, and is a novel clinically applicable approach for tumor characterization for baseline and post-treatment assessment. Citation Format: Daniel V. Catenacci, Peng Xu, Les Henderson, Wei-Li Liao, Sheeno Thyparambil, Jon Burrows, Todd Hembrough. Development of a quantitative gastroesophageal cancer selected reaction monitoring mass Spectrometric Multiplex Assay for use in FFPE tumor tissues. [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 1207. doi:10.1158/1538-7445.AM2013-1207

Abstract 41: Development of a quantitative colorectal cancer SRM assay for use in FFPE tumor tissues.

Abstract Introduction: Aberrant over-expression of receptor tyrosine kinases, including the MET, HER, FGFR, and IGFR families along with other critical downstream oncogenic mediators including KRAS, BRAF, PI3 Kinase and SRC are known drivers of colorectal cancer (CRC), subdividing the disease into distinct molecular subsets. Inter-patient tumor heterogeneity suggests that an expedient, reliable, medium throughput oncogene protein expression profiling will provide vital information to better personalize cancer care. Moreover, intra-patient tumor heterogeneity from primary tumor to metastatic disease is likely to influence biomarker prediction of response to specific targeted agents. To date, clinical quantification of protein in formalin fixed paraffin embedded (FFPE) tissues is limited to immunohistochemistry (IHC), which is semi-quantitative at best. Moreover, IHC of multiple proteins of interest is laborious, time consuming, wasteful of scarce tissue, and costly. Other protein quantification methods (ELISA, ECL) would require non-standard tissue processing for analysis. We present a quantitative mass spectrometric (MS) assay for CRC utilizing Liquid Tissue - Selected Reaction Monitoring (SRM), with subsequent multiplex quantification of relevant oncoproteins in a cohort of CRC paired primary and metastatic tumor tissues. Methods: Using trypsin digestion mapping of recombinant oncoproteins, we identified unique peptide sequences, and built quantitative MS assays which could be multiplexed into a single SRM analysis of 1μg of tumor protein. Assays were preclinically validated on 10 different formalin fixed (FF) cell lines. We then tested the ‘CRC-plex’ MS assay with multiplexed SRM quantification of Met, RON, EGFR, HER2, HER3, IGF1R, FGFR2, KRAS and cSRC on 42 primary human CRC cancer tissues, with paired metastases when available obtained from core biopsy or metastatectomy, using laser capture microdissection of the target material from a single unstained 10μm thick section per sample. Results: Validation of the CRC-plex SRM assay on cell lines and FFPE tissues revealed very high concordance when compared to IB and IHC. Multiplex oncogene quantification of all tissues, to the attomole/microgram level, will be presented, highlighting inter-patient and intra-patient (from primary to metastasis) heterogeneity of samples. Conclusions: Taken together, these data demonstrate a sensitive, accurate, and quantitative assay to measure relevant actionable oncoproteins in FFPE clinical samples. The CRC-plex multiplexed oncogene expression of these tumors was feasible and expedient using limited tissue from clinical samples, and is a novel clinically applicable approach for tumor characterization for baseline and/or post-treatment assessment. Citation Format: Todd Hembrough, Wei-Li Liao, Les Henderson, Peng Xu, Sheeno Thyparambil, Jon Burrows, Daniel V. Catenacci. Development of a quantitative colorectal cancer SRM assay for use in FFPE tumor tissues. [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 41. doi:10.1158/1538-7445.AM2013-41

Expression of receptor tyrosine kinases in esophageal carcinosarcoma

Esophageal carcinosarcoma (ECS) is a rare malignant neoplasm associated with a poor patient prognosis. It is characterized by the presence of both malignant epithelial and mesenchymal components. Molecular-targeted therapy of several receptor tyrosine kinases (RTKs) has been reported to be effective in the treatment of various malignant tumors, including carcinosarcoma of several organs. This study aimed to assess the therapeutic potential of targeting RTKs in ECS. Overexpression of RTKs was assessed in 21ECS cases by immunohistochemistry (IHC). Positively stained cases were further examined for RTK gene mutations and amplifications by direct sequencing analysis and fluorescence insitu hybridization. In epithelial components, KIT, platelet-derived growth factor receptor (PDGFR)A, PDGFRB, MET, epidermal growth factor receptor (EGFR) and HER-2 were overexpressed in 1 (4.8%), 1 (4.8%), 0 (0%), 11 (52.4%), 13 (61.9%) and 2 (9.5%) cases, respectively. In the mesenchymal components the corresponding numbers of cases were 2 (9.5%), 2 (9.5%), 0 (0%), 12 (57.1%), 11 (52.4%) and 0 (0%). No mutations in the c-kit, PDGFRA and c-met genes were found. Among 19EGFR-positive tumors, 2 had EGFR missense mutations (T790A, exon 20) only in the mesenchymal component. Gene amplification or high polysomy of c-kit, PDGFRA, c-met and EGFR was observed in 1 (33.3%), 0 (0%), 3 (18.8%) and 10 (52.6%) cases, respectively. In conclusion, various RTKs, particularly MET and EGFR were overexpressed in ECSs suggesting that molecular-targeted therapies directed to MET, EGFR or other RTKs may be effective in inhibiting the growth or progression of the epithelial and/or mesenchymal component of ECS.

P04.12 - Development of Quantitative Gastrointestinal Carcinoma (GEC and CRC) SRM Assays for Use in FFPE Tumor Tissues

Aberrant over-expression of receptor tyrosine kinases, (e.g. MET, HER, FGFR, and IGFR) as well as other oncogenic mediators (e.g. KRAS, PI3 Kinase and SRC) are known drivers of cancer, subdividing the disease into distinct molecular subsets. Inter/intrapatient tumor heterogeneity suggests that an expedient, reliable, medium throughput oncogene protein expression profiling will provide vital information to better personalize cancer care. To date, clinical quantification of protein in formalin fixed paraffin embedded (FFPE) tissues is limited to immunohistochemistry (IHC), which is semi-quantitative at best.

350P - Antitumor Activity of Dual PI3K and ER Blockade in ER Positive Breast Cancer Models

ABSTRACT Background Activation of the phosphoinositide 3-kinase (PI3K) pathway, either by receptor tyrosine kinase overexpression or PI3K/Akt/mTOR axis dysregulation, has been implicated in endocrine therapy resistance, prompting combination of PI3K inhibitors and antiestrogen therapy in the clinical setting such as in the recent BOLERO-2 study. We hypothesize that dissecting the molecular crosstalk between the PI3K and estrogen receptor (ER) pathways will help define the subset of patients most responsive to combined PI3K/antiestrogen therapy. Methods ER+ cell lines MCF7, MCF7-long term estrogen deprived (LTED), MCF7-fulvestrant resistant clones Fslx64 and Fslx70, T47D, ZR75-1, CAMA1, MDA361, KPL-1, BT474, EFM19, HCC1428, UACC812, were treated with the ER degrader, fulvestrant, and the p110α-specific PI3K inhibitor BYL719 in vitro. Cell viability was measured by CellTiter-Glo and Crystal Violet. MCF7 and ER+ patient-derived xenografts were treated with fulvestrant, BYL719 or the combination in vivo. Protein expression was measured by Western blot and immunohistochemistry. Results Fulvestrant or BYL719 treatment resulted in variable inhibition of cell viability in all ER+ cell lines. Combination treatment was significantly superior to monotherapy in MCF7, MCF7-LTED, MCF7-Fslx64 and MCF7-Fslx70. While MCF7 clones Fslx64 and Fslx70 were resistant to >1 µM of fulvestrant, they were exquisitely sensitive to BYL719. Moreover, PI3K inhibition led to ER upregulation in ER+ cell lines, including those most sensitive to combination treatment. Total ER levels also increased in MCF7 xenografts treated with therapeutic doses of BYL719. Conclusions Combined treatment with BYL719 and fulvestrant in vitro was superior to single-agent treatment in 4 of 13 ER+ cell lines. Importantly, dual PI3K/ER blockade was effective in cells resistant to ER deprivation and/or degradation. Induced ER levels following PI3K suppression may represent a feedback mechanism by which ER+ cells escape PI3K inhibition. We are currently studying whether this phenomenon predicts sensitivity to dual PI3K/ER blockade in ER+ breast cancer models. Disclosure J. Baselga: J. Baselga is a consultant/advisory board member for Aragon, AstraZeneca, Sanofi, Bayer, Onyx, Chugai, Constellation, Exelixis, Intellikine, Merck, Novartis, and Roche Genentech. All other authors have declared no conflicts of interest.

Glioblastoma—a moving target

The slow development of effective treatment of glioblastoma is contrasted by the rapidly advancing research on the molecular mechanisms underlying the disease. Amplification and overexpression of receptor tyrosine kinases, particularly EGFR and PDGFRA, are complemented by mutations in the PI3K, RB1, and p53 signaling pathways. In addition to finding effective means to target these pathways, we may take advantage of the recent understanding of the hierarchical structure of tumor cell populations, where the progressive expansion of the tumor relies on a minor subpopulation of glioma stem cells, or glioma-initiating cells. Finding ways to reprogram these cells and block their self-renewal is one of the most important topics for future research.

Abstract 4835: Genetic determinants of mTOR inhibitor response in breast and endometrial cancer

Abstract Background: Breast and endometrial cancers frequently harbour genetic aberrations that result in increased activation of the PI3K-AKT-mTOR signalling pathway including amplification and overexpression of receptor tyrosine kinases, activating PIK3CA mutations, and inactivating PTEN mutations or loss of PTEN function. Given the pivotal role of the PI3K-AKT-mTOR pathway in cancer cell growth and survival, inhibitors of several of its components are currently being tested in clinical trials. The aim of this study was to define the genetic determinants of mTOR inhibitor response in breast and endometrial cancer cells. Methods: A panel of 31 breast and 26 endometrial cancer cell lines was treated with serial dilutions of ‘rapalogs’ (i.e. allosteric inhibitors of mTOR complex 1 (mTORC1)) and mTOR kinase inhibitors targeting mTORC1 and mTORC2. Cell viability was determined using CellTiter Blue. PTEN protein levels and mutations in PTEN transcripts were assessed in all cell lines using western blotting and Sanger sequencing, respectively. PIK3CA mutations were assessed using Sanger sequencing in the breast cancer cells, and hot-spot mutations in PIK3CA and 18 additional oncogenes using the Sequenom OncoCarta Panel v1.0 in the endometrial cancer cells. Results: Response of breast cancer cells to both the rapalog everolimus and the mTOR kinase inhibitor PP242 was determined by the presence of activating PIK3CA mutations but not PTEN loss of function. In addition, PP242 treatment induced a more efficient response in HER2-amplified breast cancer cell lines, irrespective of the PIK3CA mutation status. By contrast, endometrial cancer cell lines not only harbouring mutations in PIK3CA but also in PTEN were sensitive to treatment with the rapalog temsirolimus. The mTOR kinase inhibitor AZD8055 induced a strong reduction in cell viability in all endometrial cancer cell lines tested. Conclusions: Our results suggest that response to mTOR inhibitors in breast and endometrial cancer cells may be determined by the presence of alterations affecting PIK3CA and PTEN. Our data further indicate, however, that the epistatic interactions between different components of the PI3K-AKT-mTOR pathway are distinct in cancers of the breast and endometrium. 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 4835. doi:1538-7445.AM2012-4835