Gurpreet S. Kapoor

Birinapant (TL32711), a Bivalent SMAC Mimetic, Targets TRAF2-Associated cIAPs, Abrogates TNF-Induced NF-κB Activation, and Is Active in Patient-Derived Xenograft Models

The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient–derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion. Mol Cancer Ther; 13(4); 867–79. ©2014 AACR.

Distinct Domains in the SHP-2 Phosphatase Differentially Regulate Epidermal Growth Factor Receptor/NF-κB Activation through Gab1 in Glioblastoma Cells

ABSTRACT The transcription factor nuclear factor κB (NF-κB) plays an important role in inflammation and cancer, is activated by a variety of stimuli including tumor necrosis factor alpha, interleukin-1, UV irradiation, and viruses, as well as receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). Although previous studies suggest that EGFR can induce NF-κB, the mechanism of this activation remains unknown. In this study, we identify the components of the EGFR-induced signalosome in human glioblastoma cells required to regulate NF-κB activation. Immunoprecipitation analyses with ErbB-modulated cells indicate that association between SHP-2 and Grb2-associated binder 1 (Gab1) is the critical step in the formation of the signalosome linking EGFR to NF-κB activation. We also show that EGFR-induced NF-κB activation is mediated by the PI3-kinase/Akt activation loop. Overexpression of SHP-2, Gab1, and myristoylated Akt significantly upregulated NF-κB transcriptional activity and DNA binding activity in glioblastoma cells. Interestingly, overexpression of either one of the two SH2 domain mutants of SHP-2, R32E or R138E, slightly reduced NF-κB activity relative to that of wild-type SHP-2, indicating that the SH2 domains of SHP-2 are required for EGFR-induced NF-κB activation. On the other hand, ectopic overexpression of either a Gab1 mutant incapable of binding to SHP-2 (Y627F) or a phosphatase-inactive SHP-2 mutant (C459S) caused a significant increase in NF-κB activity. Moreover, SHP-2 C459S-expressing cells displayed higher Gab1 phosphotyrosine content, suggesting that SHP-2 regulates Gab1 phosphorylation through its phosphatase domain, which confers a negative regulatory effect on NF-κB activity. These results indicate that SHP-2/Gab1 association is critical for linking EGFR to NF-κB transcriptional activity via the PI3-kinase/Akt signaling axis in glioblastoma cells and that SHP-2 acts as a dual regulator of NF-κB activation.

Critical Role of Diacylglycerol- and Phospholipid-Regulated Protein Kinase Cε in Induction of Low-Density Lipoprotein Receptor Transcription in Response to Depletion of Cholesterol

ABSTRACT Induction of low-density lipoprotein (LDL) receptor transcription in response to depletion of cellular sterols in animal cells is well established. The intracellular signal or signals involved in regulating this process, however, remain unknown. Using a specific inhibitor of protein kinase C (PKC), calphostin C, we show the requirement of this kinase in the induction process in human hepatoma HepG2 cells. Overexpression of PKCε, but not PKCα, -γ, -δ, or -ζ was found to dramatically induce (approximately 18-fold) LDL receptor promoter activity. Interestingly, PKCε-mediated induction was found to be sterol resistant. To further establish that PKCε is involved in the sterol regulation of LDL receptor gene transcription, endogenous PKCε was specifically inhibited by transfection with antisense PKCε phosphorothionate oligonucleotides. Antisense treatment decreased endogenous PKCε protein levels and completely blocked induction of LDL receptor transcription following sterol depletion. PKCε-induced LDL receptor transcription is independent of the extracellular signal-regulated kinase 1 and 2 (p42/44MAPK) cascade, because the MEK-1/2 inhibitor, PD98059 did not inhibit, even though it blocked p42/44MAPK activation. Finally, photoaffinity labeling studies showed an isoform-specific interaction between PKCε and sterols, suggesting that sterols may directly modulate its function by hampering binding of activators. This was confirmed by PKC activity assays. Altogether, these results define a novel signaling pathway leading to induction of LDL receptor transcription following sterol depletion, and a model is proposed to account for a new function for PKCε as part of a sterol-sensitive signal transduction pathway in hepatic cells.

Activation of Raf-1/MEK-1/2/p42/44(MAPK) cascade alone is sufficient to uncouple LDL receptor expression from cell growth.

Our previous observation that induction of low density lipoprotein (LDL) receptor expression by a variety of extracellular signals is blocked by PD98059, a specific mitogen-activated protein kinase kinase inhibitor, led to the suggestion that the growth-responsive p42/44MAPK cascade plays a critical role in regulating LDL receptor transcription. To analyze the specific contribution of the p42/44MAPK cascade in regulating cell growth and LDL receptor induction, we established a HepG2-derived cell line that stably expresses an inducible form of oncogenic human Raf-1 kinase. Using this system, we provide direct evidence that specific activation of this cascade alone is not only required but is sufficient to fully induce LDL receptor expression. Interestingly, degree of p42/44MAPK activation determines the extent of LDL receptor induction. However, activation of p42/44MAPK in the above cells led to the inhibition of DNA synthesis, caused growth arrest, decrease in cyclin A and upregulation of p21Cip expression in a time-dependent manner. These results suggest that each of these two processes can be regulated independently of each other in response to p42/44MAPK activation. Thus, extent of p42/44MAPK activation may be important in transducing divergent cellular responses in human cells with implications for altered signaling resulting in hypercholesterolemia.

Abstract 2278: The SMAC-mimetic birinapant regulates autocrine TNF production by caspase-8:RIPK1 complex via p38MAPK pathway

Birinapant (TL32711), a SMAC-mimetic currently in clinical trials, antagonizes multiple members of the inhibitor of apoptosis (IAP) protein family to promote apoptosis in cancer cells through the formation of a caspase-8:RIPK1 complex and induction of autocrine tumor necrosis factor (TNF). Evidently, birinapant-resistant cells do not produce TNF upon drug treatment. However, the mechanism by which birinapant-sensitive cells produce TNF is not clear. Owing to the fact that mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes, we explored the role of MAPKs in birinapant-induced TNF production. Western blot analyses on a panel of birinapant-sensitive (SK-OV-3, EVSA-T, TOV-21G: all with an IC50 10μM) cell lines showed that birinapant induced rapid and robust phosphorylation of p38MAPK in sensitive cells, which corresponded to increased TNF production as assessed by ELISA. However, resistant cells were not affected at multiple time points. The p38MAPK inhibitor, LY2228820, reduced the levels of birinapant-induced TNF (2-fold with 5nM birinapant after 24h) in birinapant-sensitive cancer cells (SK-OV-3), whereas inhibitors of Erk1/2 (PD98059) and JNK (SP600125) had no effect on TNF production. Interestingly, RNAi-mediated inhibition of p38α (MAPK14) isoform significantly inhibited birinapant-induced TNF production (∼3-fold with 5nM birinapant after 24h) in SK-OV-3 cells while inhibition of p38β (MAPK11) isoform upregulated TNF production (∼3-fold), suggesting that there may be a balance between p38MAPK isoforms to regulate TNF production. Pharmacological and RNAi-mediated inhibition of RIPK1 and caspase-8 activities in SK-OV-3 cells resulted in reduced birinapant-induced p38MAPK phosphorylation and TNF production. Furthermore, commercially available pooled siRNAs targeting different isoforms of cFLIP, a catalytically-inactive homologue of caspase-8, upregulated p38MAPK phosphorylation (∼4-fold) and TNF production (∼8 to 26-fold) in birinapant-resistant cancer cell lines (T24, HT1376 & IGROV-1), and conferred differential sensitivity to the drug treatment (T24-IC50: 7nM; HT1376-IC50: >10nM; IGROV-1-IC50: >1μM). Experiments involving cFLIP long (cFLIP-L) and short (cFLIP-S) isoform-specific siRNAs showed that inhibition of cFLIP-L, but not cFLIP-S, in T24 & HT1376 cells induced p38MAPK phosphorylation and TNF production, and conferred sensitivity to birinapant. However, the combined inhibition of cFLIP-L and cFLIP-S in IGROV-1 cells was required to induce p38MAPK phosphorylation, TNF production and apoptosis. Collectively, these observations indicate that birinapant-induced autocrine TNF production is triggered by the activation of the caspase-8/RIPK1/p38MAPK axis, and that cFLIP isoforms may confer a context-dependent negative regulatory effect. Citation Format: Gurpreet Singh Kapoor, Christopher A. Benetatos, Yasuhiro Mitsuuchi, Eric M. Neiman, Guangyao Yu, Mark A. Mckinlay, Jennifer Burns, John Silke, Stephen M. Condon, Srinivas K. Chunduru. The SMAC-mimetic birinapant regulates autocrine TNF production by caspase-8:RIPK1 complex via p38MAPK pathway. [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 2278. doi:10.1158/1538-7445.AM2014-2278

Abstract 1806: Birinapant, a bivalent SMAC-mimetic, promotes efficient cellular IAP E3 ligase activity and formation of a pro-apoptotic RIPK1:caspase-8 complex while monovalent IAP inhibitors are less efficient - implications for therapeutic utility

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The second mitochondria-derived activator of caspases (SMAC) is thought to exert its pro-apoptotic activity as a homodimeric protein. Both monovalent and bivalent peptidomimetics of the SMAC tetrapeptide are being developed for cancer therapy. Birinapant/TL32711 is a bivalent SMAC-mimetic that targets the inhibitor of apoptosis (IAP) proteins whose gene abnormalities have been implicated in various cancers. Owing to structural differences between bivalent SMAC-mimetics and monovalent IAP-inhibitors, we sought to compare and contrast the biochemical activity of birinapant with several monovalent IAP-inhibitors including a monovalent- version of birinapant/TL32711, MV711. Previous studies have shown that both bivalent and monovalent agents promote auto-ubiquitylation and subsequent degradation of cIAP1 and cIAP2, which triggers tumor necrosis factor receptor (TNFR)-mediated cell death in certain tumor cell lines. However, birinapant showed substantial differences from IAP-inhibitors in degrading TRAF2-associated cIAP1 and cIAP2. Here we show that MV711 was less efficient at degrading cIAP1 by a factor of 7-fold (16 vs. 118 nM, birinapant vs. MV711, respectively) and inhibiting TNF-mediated NF-κB activation by 220-fold (9 vs. 1985 nM, respectively). In addition, a linker-lengthened variant of birinapant was less able to inhibit NF-κB activation by 71-fold (9 vs. 642 nM, respectively). We also studied the effect of birinapant or IAP-inhibitor treatment on SKOV-3, MDA-MB-231 and EVSA-T cancer cell lines in vitro. Comparable cIAP1 BIR3 domain binding constants and IC50 values for the degradation of cIAP1 and cIAP2 (ΔcIAP1/2) were observed for these two classes of agents, and both birinapant and IAP-inhibitors showed dose-dependent induction of cell death. However, despite such comparable potencies, the IAP-inhibitors did not completely kill SKOV-3 or MDA-MB-231 cells even with concentrations >100-times their ΔcIAP1/2 IC50 values. Birinapant revealed the highest suppression of cancer cell growth in the cell lines tested, even after the agent was removed, whereas the cell lines treated with the IAP-inhibitors showed rapid restoration of cell proliferation within 24 h following removal of the agents. These results suggested that monovalent IAP-inhibitors require maintenance of high steady state levels of drug to effectively suppress tumor growth in vivo. In agreement with their inability to induce cell death, IAP-inhibitors were less efficient in stimulating the formation of a RIPK1:caspase-8 complex when compared to birinapant in EVSA-T or SKOV-3 cells. These observations may be partly attributed to the reduced ability of IAP-inhibitors to degrade TRAF2-associated cIAP1 which serves a central role in the activation of NF-κB via TNFR. Citation Format: Yasuhiro Mitsuuchi, Christopher A. Benetatos, Thomas Haimowitz, Yijun Deng, Angeline C. Mufalli, Martin E. Seipel, Jennifer M. Burns, Gurpreet S. Kapoor, C. Glenn Begley, Stephen M. Condon. Birinapant, a bivalent SMAC-mimetic, promotes efficient cellular IAP E3 ligase activity and formation of a pro-apoptotic RIPK1:caspase-8 complex while monovalent IAP inhibitors are less efficient - implications for therapeutic utility. [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 1806. doi:10.1158/1538-7445.AM2014-1806

Abstract 3336: The Smac Mimetic Birinapant Synergistically Induces Apoptosis in Combination with Type I Interferons and GM-CSF.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The Inhibitor of Apoptosis (IAP) protein family is able to inhibit cell death and promote survival signaling triggered by a wide variety of stimuli. Two members of this family in particular, cIAP1 and cIAP2, play key roles in enabling tumor necrosis factor alpha (TNFα)-induced nuclear factor-kappa beta (NF-kB) activation and inflammatory responses. Birinapant (TL32711) is a bivalent Smac Mimetic that antagonizes multiple IAP family members resulting in the conversion of TNFα-induced survival signaling into an apoptotic response, an effect which is more prominent in tumor cells than their normal counterparts. Combining birinapant with agents capable of increasing the levels of TNFα in the tumor microenvironment represents a novel approach to cancer treatment. We have tested the ability of interferon-alpha/-beta (IFN-α, IFN-β) and granulocyte macrophage colony stimulating factor (GM-CSF), both known to be immunomodulatory agents, for their ability to stimulate TNFα production by peripheral blood mononuclear cells (PBMC). Treatment with each cytokine induced the production of TNFα by cultured PBMC as measured by ELISA. Supernatants of IFN-α/-β and GM-CSF-treated PBMC cultures were able to sensitize resistant tumor cells to Smac Mimetics in a TNFα-dependent manner. Furthermore, the combination of birinapant and either IFN-β or GM-CSF synergistically inhibited the development of RENCA tumors in a syngeneic mouse prevention model. Due to the highly aggressive nature of the syngeneic tumors, this model is performed by simultaneous implantation of tumor cells and initiation of dosing of test agents. Activity in this model is measured by prevention of tumor outgrowth. Birinapant in combination with IFN-β resulted in only 5 of 18 measurable tumors, versus 19 of 20 tumors in vehicle-treated mice, 16 of 20 tumors in IFN-β alone-treated mice and 13 of 20 tumors in birinapant alone-treated mice. In addition, when tumor growth occurred it was delayed in mice treated with the combination. Similarly, the birinapant and GM-CSF combination treatment resulted in 9 of 20 tumors, versus 20 of 20 tumors in the vehicle-treated mice, 20 of 20 tumors in the GM-CSF alone-treated mice, and 16 of 20 tumors in the birinapant alone-treated mice. In addition when tumors did develop in the combination-treated mice, their growth was substantially delayed: 4 of 10 mice treated with the combination of birinapant and GM-CSF were still on study on day 85 post initiation - approximately 40 days post dosing - with no measureable tumor. The combination of birinapant with immunomodulatory agents that induce TNF represents a potential novel therapeutic strategy. Citation Format: Christopher A. Benetatos, Jennifer M. Burns, Ernest C. Borden, Daniel Lindner, Yasuhiro Mitsuuchi, Mark A. Mckinlay, Gurpreet Singh Kapoor, Eric M. Neiman, Martin E. Seipel, Guangyao Yu, Martin Graham, David Weng, Stephen M. Condon, C. Glenn Begley, Srinivas Chunduru. The Smac Mimetic Birinapant Synergistically Induces Apoptosis in Combination with Type I Interferons and GM-CSF. [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 3336. doi:10.1158/1538-7445.AM2013-3336

Abstract 5302: Characterization of tumor cell lines resistant to birinapant, a novel bivalent smac mimetic.: Table 1 -

Acquired resistance to cancer therapies remains a major therapeutic challenge. Identification of resistance mechanisms and biomarkers of acquired resistance are important for management of patients with drug refractory tumors. The present study was aimed to understand the potential mechanism(s) of acquired resistance to birinapant (TL32711), a bivalent Smac Mimetic (SM), currently in clinical trials for the treatment of solid and hematological malignancies. We generated SM resistant cell lines from four tumor cell lines (SKOV-3, EFM192A, EVSA-T and MDA-MB-231) which were initially sensitive to birinapant. Resistant variants of each cell line were resistant to 10μM concentration of birinapant, equivalent to 1000-10,000x IC50 values. Variants were also cross-resistant to other, structurally diverse, SM compounds suggesting common mechanisms of resistance. Sensitivity to birinapant in combination with TRAIL and TNF was also measured. These experiments revealed a range of sensitivity among the resistant variants. In order to identify changes which occurred during development of resistance, total mRNAs were extracted from SM sensitive cell lines and their resistant variants and were subjected to gene expression analyses using commercially available apoptosis pathway real-time PCR arrays. Preliminary data analysis identified several candidate genes that were down- or up-regulated in the SM resistant cell line relative to their sensitive counterparts. Three of four SM-resistant subclones exhibited decreased expression of genes including NAIP, BCL2A1, DAPK1, TNF, cFLIP, Smac, and TNFSF10 or increased expression of BCL2L2, or BAK1 (Table 1). Identification of genes modulated during acquired resistance provides a starting point for the discovery of novel biomarkers that may explain mechanism(s) of acquired resistance, and could potentially be exploited to achieve maximum anti-tumor response with birinapant as a single agent or in combination with other treatment regimens. Citation Format: Eric M. Neiman, Christopher A. Benetatos, Gurpreet S. Kapoor, Yasuhiro Mitsuuchi, Mark A. McKinlay, Martin E. Seipel, Guangyao Yu, Stephen M. Condon, Srinivas K. Chunduru. Characterization of tumor cell lines resistant to birinapant, a novel bivalent smac mimetic. [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 5302. doi:10.1158/1538-7445.AM2013-5302

Abstract 3333: Birinapant, a novel bivalent Smac mimetic drug, is superior to monovalent Smac mimetics in inhibition of NF-kB by targeting TRAF2-bound cIAP1 and cIAP2.

A variety of Smac-mimetic drugs have been developed to target the inhibitor of apoptosis (IAP) proteins including X chromosome-linked IAP (XIAP), cellular IAP proteins (cIAP1 and cIAP2) and ML-IAP whose gene mutations, amplifications and chromosomal translocations have been implicated in various malignancies. The IAPs also play an important role in multiple signaling pathways including the TNFα-mediated NF-kB and MAPK pathways (inflammatory responses) and pattern recognition receptor signaling pathways (innate immunity). Smac-mimetics have been designed to mimic the IAP-binding motif of the second mitochondria-derived activator of caspase (Smac). The IAP-binding motif consists of four amino acids (AVPI) that serve as an endogenous IAP antagonist. Structurally different Smac-mimetic compounds have been published and showed different binding affinity to IAP proteins. These differences in structure and binding have important consequences in terms of biological activity. Here, we present evidence that bivalent Smac-mimetics are superior to monovalent Smac-mimetics in their ability to inhibit NF-kB in cells stimulated with TNFα. Over 300 monovalent and 300 bivalent Smac-mimetics, including compounds whose structures have been published, were tested for ability to degrade cIAP1 and cIAP2, and inhibit NF-kB in cell-based assays. Inhibition of NF-kB by bivalent Smac-mimetics correlated (R²=0.78) with the degradation of cIAP1 over a range of four logs, while monovalent Smac-mimetics did not (R²=0.20). Furthermore, monovalent Smac-mimetics degraded TRAF2-bound cIAP1 and cIAP2 less effectively (1/10-1/100 fold) compared to bivalent Smac-mimetics. While bivalent Smac-mimetics effectively degraded cIAP1 and cIAP2, birinapant (TL32711), a bivalent Smac-mimetic currently in Phase 2 clinical trials, was unique in its ability to preferentially degrade TRAF2-bound cIAP1 and cIAP2. Inhibition of NF-kB by birinapant was further characterized by the ImageStream cytometry, which showed that the nuclear translocation of p65 in response to TNFα stimulation was blocked in both HeLa and HL-60 cells. These data demonstrate that bivalent Smac-mimetics are superior to monovalent Smac-mimetics in degrading TRAF2-bound cIAPs, in inhibition of NF-kB and efficiently mediating cell death. Furthermore, the unique profile of birinapant versus other bivalent Smac-mimetics may explain its preclinical and clinical safety profile. Citation Format: Yasuhiro Mitsuuchi, Stephen M. Condon, Eric M. Neiman, Christopher A. Benetatos, Martin E. Seipel, Gurpreet Singh Kapoor, Angeline C. Mufalli, Guangyao Yu, Orla Maguire, Hans Minderman, Mark A. Mckinlay, Martin Graham, David Weng, Srinivas Chunduru. Birinapant, a novel bivalent Smac mimetic drug, is superior to monovalent Smac mimetics in inhibition of NF-kB by targeting TRAF2-bound cIAP1 and cIAP2. [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 3333. doi:10.1158/1538-7445.AM2013-3333

Abstract LB-11: PTEN modulates EGFRvIII-induced SHP2 phosphorylation and translocation to affect glioblastoma sensitivity to tyrosine kinase inhibitors (TKIs)

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Co-expression of EGFRvIII and PTEN in a small subset of recurrent glioblastoma tumors has been shown to increase sensitivity to tyrosine kinase inhibitors (TKIs). However, the exact mechanism of EGFRvIII and PTEN interaction in response to TKIs is still unresolved. Our recent work has shown that SHP2 PTPase is required for EGFRvIII-mediated transformation. The present work was aimed to study the effect of PTEN on EGFRvIII-induced SHP2 phosphorylation and translocation and modulate glioblastoma sensitivity to tarceva treatment. We show that tarceva treatment (10μM/ml) abolished EGFRvIII, EGFR, Gab1, SHP2 and Erk1/2 phosphorylation in LN229.EGFRvIII cells at all time intervals. On the contrary, phosphorylation of EGFRvIII and Erk1/2 in U87MG.EGFRvIII cells was inhibited at early time points, but was restored in 2 to 6 hours. Interestingly, phosphorylation of Akt, Gab1 and SHP2 (Tyr580) was unaffected in U87MG.EGFRvIII cells, but EGFR and SHP2 (Tyr542) phosphorylation were inhibited in a time-dependent manner. MTT proliferation and soft agar transformation assays demonstrated that U87MG.EGFRvIII cells were resistant to tarceva treatment when compared to LN229.EGFRvIII cells. Immunofluorescent labeling of U87MG.EGFRvIII cells with an anti-phospho-SHP2 (Tyr542) antibody showed membrane, cytoplasmic, perinuclear and nuclear localization of SHP2, whereas LN229.EGFRvIII cells exhibited membrane staining of phosphorylated SHP2. However, both the cell lines exhibited strong nuclear staining of phospho-SHP2 (Tyr580). Interestingly, tarceva treatment inhibited membrane localization of phospho-SHP2 (Tyr542) and nuclear localization of phospho-SHP2 (Tyr580) in LN229.EGFRvIII cells after 1 hr, but did not affect SHP2 phosphorylation and localization in U87MG.EGFRvIII cells at all time intervals. Furthermore, stable expression of PTEN in U87MG.EGFRvIII cells conferred relocalization of phospho-SHP2 (Tyr542) to the membrane. Notably, U87MG.EGFRvIII/PTEN clones showed a partially untransformed phenotype. Furthermore, phosphorylation of SHP2 (Tyr580) and Erk1/2 was totally abolished in U87MG.EGFRvIII/PTEN subclones. Our data indicate that the expression of PTEN in U87MG.EGFRvIII cells conferred a phenotype similar to LN229.EGFRvIII cells. Collectively, these observations infer that SHP2 is a downstream effecter of PTEN and that PTEN deficiency may lead to SHP2 activation and nuclear localization by EGFRvIII, which may result in increased resistance to TKIs. 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 LB-11. doi:10.1158/1538-7445.AM2011-LB-11