PIK3CA Mutant Models Research Articles

Abstract 51: Potential and challenges in co-targeting mTORC and EGFR signaling as a therapeutic strategy in HNSCC

Abstract Background: Head and neck squamous cell carcinomas (HNSCCs) have high rates of mutation and other alterations along the PI3K/AKT/mTORC signaling axis. This has led to interest in the use of therapeutics targeting this pathway; however, identifying reliable predictive biomarkers to guide patient selection remains challenging. Despite excellent preclinical data, the use of these compounds as monotherapy has been underwhelming in initial clinical trials. The EGFR monoclonal antibody cetuximab remains the only approved targeted agent for HNSCC and with reasonable toxicity profiles, has potential use in combination therapy. Methods: Both catalytic mTORC (AZD8055) and PI3K/mTORC(NVP-BEZ-235) inhibitors were tested +/- cetuximab in several in vitro and in vivo pre-clinical models. A panel of HNSCC cell lines and patient derived xenografts (PDX) were evaluated for PI3K/AKT/mTORC pathway mutation by sequencing and potential protein biomarker by immunoblot and IHC. Cell lines were assayed for sensitivity to all three agents by growth inhibition and clonogenic survival assay. DNA replication (BrdU uptake) and apoptosis (Capase 3/7 activity) were investigated to assess the mechanism of inhibition. The specificity of the molecular targeted effects was confirmed by siRNA knockdown. Five unique PDX models that presented PIK3CA mutation or intrinsic cetuximab resistance were treated with a combination of cetuximab and the dual mTORC inhibitor AZD8055 in a nude mouse model. Matched PDX derived cell strains were generated to investigate differences in response observed in in vitro and in vivo settings. Results: Assessment of the panel of HNSCC cell lines by mutational hotspot sequencing did not reveal any obvious sensitizing mutations, whereas putative protein biomarkers (e.g. PIK3CA, pAKT) were elevated in some cell lines. All cell lines showed modest response to both PI3K/mTORC and dual mTORC inhibition. The addition of cetuximab to either agent produced modest additive effect. Mechanistic studies revealed that growth inhibition rather than death induction was the major anticancer effect. SiRNA knockdown showed similar molecular signaling and functional effects to drug inhibition. Using the PDX models, in vivo single agent mTORC inhibition inhibited growth of a PIK3CA mutant cancer, but had no effect on any PIK3CAWT or a second PIK3CA mutant model. In all models the combination therapy showed greater growth delay than monotherapy. In matched PDX derived cell strains, in vitro responses were similar when grown in 3D culture but cells displayed greater sensitivity when grown in 2D culture, suggesting that tumor microenvironment contributes to response. Conclusions: The uniform ability of PI3K/mTORC and mTORC inhibition to suppress the growth of HNSCC cells highlights the role of this signaling pathway to drive the proliferation. In vivo, despite some PDX models meeting likely selection criteria, the single agent therapy was largely ineffective. Conversely, the combination treatment produced growth delay and suggests the potential for adding a catalytic mTORC inhibitor to cetuximab therapy for HNSCC patients. Overall, these results add to a growing body of evidence suggesting approaches that attempt to match genetic alternation or other biomarker to the optimal therapy in HNSCC remain complex and challenging. Citation Format: Adam D. Swick, Prashanth J. Prabakaran, Margot C. Miller, Amal M. Javaid, Michael M. Fisher, Emmanuel Sampene, Irene M. Ong, Mari Iida, Deric L. Wheeler, Kwangok P. Nickel, Justine Y. Bruce, Randall J. Kimple. Potential and challenges in co-targeting mTORC and EGFR signaling as a therapeutic strategy in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 51.

Abstract 146: The PI3K inhibitor, taselisib, has a unique mechanism of action that leads to enhanced potency in PIK3CA mutant models

Abstract Activating mutations in PIK3CA are commonly found in a wide variety of human cancers, and the dysregulation of the phosphoinositide-3 kinase (PI3K) signaling pathway has been implicated in tumor cell growth and survival. Taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha, sparing inhibition of p110beta, is more potent in cancer cells bearing PIK3CA mutants than those with wildtype PIK3CA. Preclinical studies demonstrate that taselisib induces more apoptotic cell death in PIK3CA mutant cancer cells than other PI3K inhibitors. We have discovered that taselisib has a dual mechanism of action, both blocking PI3K signaling, and inducing a decrease in p110a protein levels. Mass spec analysis reveals that taselisib treatment leads to the specific depletion of mutant p110alpha without significant change in wildtype p110alpha protein levels. This drug-induced p110a protein depletion is rescued by E1 inhibitors and by proteasome inhibitors. Other clinical PI3K inhibitors, including p110alpha selective and pan-PI3K inhibitors, are unable to induce the depletion of mutant p110 alpha protein. Furthermore, we have discovered that taselisib more effectively maintains pathway suppression in PIK3CA mutant cells at 24 hrs in response to feedback. In comparison to other clinical-stage PI3K inhibitors administered at a maximum tolerated dose, taselisib has superior efficacy with increased tumor regressions in PIK3CA mutant xenograft models. In summary, these preclinical studies indicate that PI3K inhibitors, which have the ability to trigger degradation of mutant p110a protein, can more effectively suppress the signaling pathway, which may result in greater anti-tumor activity and improved therapeutic index in PIK3CA mutant tumors. Citation Format: Kyung W. Song, Kyle A. Edgar, Donald S. Kirkpatrick, Lilian Phu, Stephen Schmidt, Michelle Nannini, Rebecca Hong, Eric Cheng, Lisa Crocker, Amy Young, Deepak Sampath, Lori Friedman. The PI3K inhibitor, taselisib, has a unique mechanism of action that leads to enhanced potency in PIK3CA mutant models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 146. doi:10.1158/1538-7445.AM2017-146

Abstract 149: Co-targeting mTORC and EGFR signaling as a potential therapeutic strategy in HNSCC

Abstract Background - Head and neck squamous cell carcinomas (HNSCCs) have high rates of mutation and other alterations along the PI3K/AKT/mTORC signaling axis. This has led to interest in the use of therapeutics targeting this pathway, however identifying reliable predictive biomarkers to guide patient selection remains challenging. Despite excellent preclinical data, the use of these compounds as monotherapy has been underwhelming in initial clinical trials. The EGFR monoclonal antibody cetuximab remains the only approved targeted agent for HNSCC and with reasonable toxicity profiles, has potential use in combination therapy. Methods - Both catalytic mTORC (AZD8055) and PI3K/mTORC(NVP-BEZ-235) inhibitors were tested +/- cetuximab in several in vitro and in vivo pre-clinical models. A panel of HNSCC cell lines and patient derived xenografts (PDX) were evaluated for PI3K/AKT/mTORC pathway mutation by sequencing and potential protein biomarker by immunoblot and IHC. Cell lines were assayed for sensitivity to all three agents by growth inhibition and clonogenic survival assay. DNA replication(BrdU uptake) and apoptosis (Capase 3/7 activity) were investigated to assess the mechanism of inhibition. The specificity of the molecular targeted effects was confirmed by siRNA knockdown. Five unique PDX models that presented PIK3CA mutation or intrinsic cetuximab resistance were treated with a combination of cetuximab and the dual mTORC inhibitor AZD8055 in a nude mouse model. Results - Assessment of the panel of HNSCC cell lines by mutational hotspot sequencing did not reveal any obvious sensitizing mutations, whereas putative protein biomarkers (e.g. PIK3CA, pAKT) were elevated in some cell lines. All cell lines showed modest response to both PI3K/mTORC and dual mTORC inhibition. The addition of cetuximab to either agent produced modest additive effect. Mechanistic studies revealed that growth inhibition rather than death induction was the major anti-cancer effect. SiRNA knockdown showed similar molecular signaling and functional effects to drug inhibition. Using the PDX models, in vivo single agent mTORC inhibition inhibited growth of a PIK3CA mutant cancer, but had no effect on any PIK3CAWT or a second PIK3CA mutant model. In all models the combination therapy showed greater growth delay than monotherapy. Conclusions -The uniform ability of PI3K/mTORC and mTORC inhibition to suppress the growth of HNSCC cells highlights the role of this signaling pathway to drive the proliferation. In vivo, despite some PDX models meeting likely selection criteria, the single agent therapy was largely ineffective. Conversely the combination treatment produced growth delay and suggests the potential for adding a catalytic mTORC inhibitor to cetuximab therapy for HNSCC patients. Overall, these results add to a growing body of evidence suggesting that attempts to match genetic alternation or other biomarker to the optimal therapy in HNSCC remains complex and challenging. Citation Format: Adam D. Swick, Prashanth J. Prabakaran, Amal Javaid, Margot Miller, Michael Fisher, Emmanuel Sampene, Irene M. Ong, Kwangok Nickel, Randall J. Kimple. Co-targeting mTORC and EGFR signaling as a potential therapeutic strategy in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 149. doi:10.1158/1538-7445.AM2017-149

Cotargeting mTORC and EGFR Signaling as a Therapeutic Strategy in HNSCC.

Head and neck squamous cell carcinomas (HNSCC) are frequently altered along the PI3K/AKT/mTORC signaling axis. Despite excellent preclinical data, the use of compounds targeting this pathway as monotherapy has been underwhelming in initial clinical trials, and identification of predictive biomarkers remains challenging. To investigate mTORC-specific inhibition, we tested catalytic mTORC (AZD8055) and PI3K/mTORC (NVP-BEZ-235) inhibitors ± cetuximab in a panel of HNSCC cell lines and patient-derived xenografts (PDX). Cell lines were assayed for response to all agents and siRNA knockdown of targets by multiple approaches. All cell lines showed similar response to both drug and siRNA inhibition of both PI3K and mTORC pathways, with anti-EGFR combination producing modest additive effect. Five PDX models that presented PIK3CA mutation or intrinsic cetuximab resistance were treated with a combination of cetuximab and AZD8055. In vivo single-agent mTORC inhibition inhibited growth of one PIK3CA-mutant cancer, but had little effect on any PIK3CAWT or a second PIK3CA-mutant model. In all models, the combination therapy showed greater growth delay than monotherapy. The uniform ability of PI3K and mTORC inhibition to suppress the growth of HNSCC cells highlights the pathway's role in driving proliferation. Although single-agent therapy was largely ineffective in vivo, improved response of combination treatment in an array of PDXs suggests the potential for adding a catalytic mTORC inhibitor to cetuximab therapy. Overall, these results add to a growing body of evidence, suggesting that approaches that attempt to match biomarkers to the optimal therapy in HNSCC remain complex and challenging. Mol Cancer Ther; 16(7); 1257-68. ©2017 AACR.

Co-targeting mTORC and EGFR signaling as a therapeutic strategy in HNSCC.

e17509 Background: Head and neck squamous cell carcinomas (HNSCCs) are frequently altered along the PI3K/AKT/mTORC signaling axis leading to development of therapeutics targeting this pathway. Despite excellent preclinical data, the use of these compounds as monotherapy has been underwhelming in initial clinical trials and identification of predictive biomarkers remains challenging. The EGFR monoclonal antibody cetuximab remains the only approved targeted agent for HNSCC and has potential use in combination therapy. Methods: Both catalytic mTORC (AZD8055) and PI3K/mTORC(NVP-BEZ-235) inhibitors were tested +/- cetuximab in a panel of HNSCC cell lines and patient derived xenografts (PDX) evaluated for potential biomarkers of PI3K/AKT/mTORC pathway. Cell lines were assayed for response to all three agents by multiple approaches, and confirmed by siRNA knockdown. Five PDX models that presented PIK3CA mutation or intrinsic cetuximab resistance were treated with a combination of cetuximab and AZD8055. Results: In HNSCC cells no likely sensitizing mutations were identified, whereas putative protein biomarkers were elevated in some lines. All lines showed similar response to both PI3K/mTORC and dual mTORC inhibition with cetuximab combination producing modest additive effect with similar results for siRNA knockdown. In PDX models, in vivo single agent mTORC inhibition inhibited growth of a PIK3CA mutant cancer, but had little effect on any PIK3CAWT or a second PIK3CA mutant model. In all models the combination therapy showed greater growth delay than monotherapy. Conclusions: The uniform ability of PI3K and mTORC inhibition to suppress the growth of HNSCC cells highlights the pathway’s role in driving proliferation. In vivo, despite some PDX models meeting likely selection criteria, the single agent therapy was largely ineffective. Conversely improved response of combination treatment suggests the potential for adding a catalytic mTORC inhibitor to cetuximab therapy. Overall, these results add to a growing body of evidence suggesting approaches that attempt to match biomarkers to the optimal therapy in HNSCC remains complex and challenging.

Abstract S6-04: The PI3K inhibitor, taselisib, has enhanced potency in PIK3CA mutant models through a unique mechanism of action

Abstract Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutational activation of the PIK3CA gene. The dysregulation of this pathway has been implicated in tumor cell growth and survival, thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. Taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha, sparing inhibition of p110beta, is more potent against cancer cells bearing mutations in the PIK3CA gene than those with wildtype PIK3CA. The mechanism leading to this enhanced mutant selectivity is revealed in these preclinical studies. Uniquely among PI3K inhibitors, taselisib has a gain of potency in PIK3CA mutant SW48 isogenic cells compared to wildtype SW48 parental cells. Pathway inhibition and increased apoptosis are associated with the enhanced activity observed in PIK3CA mutant cells. In PIK3CA mutant cell culture-derived and patient-derived xenograft (PDX) models taselisib induces tumor regressions. In comparison to other clinical-stage PI3K inhibitors, taselisib confers superior anti-tumor activity in PIK3CA mutant xenografts when treated at a Maximum Tolerated Dose (MTD) in vivo. We have discovered that taselisib has a dual mechanism of action, both blocking kinase signaling and inducing down-regulation of the mutant p110alpha protein level in a dose-dependent and time-dependent manner. Taselisib treatment leads to the specific degradation of mutant p110alpha without significant change in wildtype p110alpha protein in cultured cells and in mutant xenograft models including PDX. Other clinical PI3K inhibitors, including PI3Kalpha selective and pan-PI3K inhibitors are unable to induce degradation of mutant p110 alpha. The taselisib-induced degradation of mutant p110a protein is ubiquitin-mediated and proteasome-dependent. These unique mechanistic effects of taselisib are most pronounced when comparing signaling suppression and p110a protein levels at 24 hours vs. 1 hour of drug exposure in PIK3CA mutant cell lines. This discovery indicates that PI3K inhibitors which trigger degradation of mutant p110a protein can more effectively suppress the signaling pathway in response to feedback, and may result in greater activity and improved therapeutic index. Citation Format: Friedman LS, Edgar KA, Song K, Schmidt S, Kirkpatrick DS, Phu L, Nannini MA, Hong R, Cheng E, Crocker L, Young A, Sampath D. The PI3K inhibitor, taselisib, has enhanced potency in PIK3CA mutant models through a unique mechanism of action [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S6-04.

Abstract 370: The PI3K inhibitor, taselisib (GDC-0032), has enhanced potency in PIK3CA mutant models through a unique mechanism of action

Abstract Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutational activation of the PIK3CA gene. The dysregulation of this pathway has been implicated in tumor cell growth and survival, thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. The mechanism of action of taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha sparing inhibition of p110beta, is investigated in these preclinical studies. Taselisib demonstrates greater potency in cancer cell lines harboring activating mutations in PIK3CA vs. wild-type lines, and induces regressions at tolerated doses in both PIK3CA mutant xenograft and patient-derived xenograft (PDX) models. When comparing taselisib to other clinical-stage PI3K inhibitors at Maximum Tolerated Dose (MTD) in vivo, taselisib confers greater activity in PIK3CA mutant models, which may indicate a larger therapeutic index. Unlike other PI3K inhibitors, taselisib has a gain of potency in PI3K alpha mutant SW48 isogenic cells compared to wildtype SW48 parental cells. Pathway inhibition and increased apoptosis are associated with the enhanced activity observed in PI3K alpha mutant cells. Other clinical PI3K inhibitors, including PI3K alpha selective and pan-PI3K inhibitors, do not have improved potency in PI3K alpha mutant cells due to their inability to maintain pathway suppression after alleviation of negative feedback. The unique mechanism of action of taselisib is most notable when comparing signaling suppression at 24 hours vs. 1 hour of drug exposure. In mutant cells, Taselisib displays greater pathway suppression at 24 hours and is more effective at maintaining pathway suppression upon re-activation of growth factor RTK signaling. This mechanism is specific to PIK3CA mutant cells and not observed in wildtype cells. Ongoing studies to further elucidate this mechanism of action will be presented. Citation Format: Kyle A. Edgar, Kyung Song, Stephen Schmidt, Don Kirkpatrick, Lilian Phu, Michelle Nannini, Rebecca Hong, Eric Cheng, Amy Young, Deepak Sampath, Lori S. Friedman. The PI3K inhibitor, taselisib (GDC-0032), has enhanced potency in PIK3CA mutant models through a unique mechanism of action. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 370.

Abstract 2619: Meausuring PIP3 reveals the unexpected role of p110B in luminal breast cancers

Abstract The phosphatidylinositol 3-kinase (PI3K) family of lipid kinases plays a prominent role in the growth and survival of several types of cancer, including breast cancers where the PI3K pathway is altered in up to 70% of cases. Due to the technical challenges of measuring the PI3K product, PIP3 directly, the phosphorylation of the downstream effector AKT has historically been used to indirectly assess the activity of class I PI3K. While this approach has proven to be rather useful, it is based on the assumption that this readout is proportional to the levels of PIP3, which is likely to apply only to a limited range of activation levels and within specific contexts. Indeed, evaluation of the efficacy of PI3K inhibitors with such indirect measurements may approximate and oversimplify the complex transduction mechanism of PI3K. During time course experiments using the p110A selective inhibitor, BYL719, we observed that both PIP3 and p-AKT levels were similarly restored after 24 hours in HER2 amplified breast cancer cells, while the rebound of p-AKT was minimal and did not correlate with increased PIP3 levels in PIK3CA mutant breast cancer models. Both p110B inhibition and p110B depletion were effective in blocking the PIP3 rebound observed. Consistent with this, the combination of p110A and p110B inhibitors decreased cell growth and induced a better apoptotic response compared to BYL719 alone. We further demonstrated that only the combination of p110A and p110B inhibitors induced tumor regression. Importantly, PIP3 levels correlated with efficacy more reliably than p-AKT in a PIK3CA mutant model in vivo. These data suggest that measurement of PIP3 levels following treatment may serve as a more effective biomarker than p-AKT levels to predict response to PI3K inhibitors within this subset of breast cancers. Citation Format: Carlotta Costa, Hiromichi Ebi, Anthony C. Faber, Madhuri Nishtala, Youngchul Song, Cyril H. Benes, Jeffrey A. Engelman. Meausuring PIP3 reveals the unexpected role of p110B in luminal breast cancers. [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 2619. doi:10.1158/1538-7445.AM2014-2619

Abstract A169: Assessment of genotype-correlated sensitivity to the investigational PI3Kα selective inhibitor MLN1117 in preclinical models.

Abstract The PI3K-Akt-mTOR signaling pathway is the most dysregulated pathway in cancer. Specifically, helical and kinase domain mutations in the p110α subunit (E542K, E545K, H1047R) account for greater than 15% mutations in many cancers. In addition, copy number gain of the PIK3CA locus (>2.5 copies) also occur at a high frequency suggesting that these events are also associated with the oncogenic process. MLN1117 is an investigational PI3Kα selective inhibitor that is currently in Phase I dose escalation in subjects with advanced cancer (NCT01449370). It is widely hypothesized that PI3Kα isoform selective inhibitors will demonstrate considerable therapeutic benefit in PI3K “addicted” tumors; the work presented here is focused on identifying the association of genetic alterations with sensitivity to MLN1117. We have assessed the sensitivity profile both in vitro (n=47) and in vivo (n=29) using human tumor cell lines and primary human tumor xenografts, representing 9 different tumor types. PIK3CA gene point mutations are associated with sensitivity to MLN1117, in both in vitro and in vivo settings. 14 of 17 (82%) PIK3CA mutant cell lines demonstrate an in vitro IC50 of ≤ 5.0 μM and all non mutant models demonstrate an IC50 of > 5.0 μM. In vivo 12 of 15 PIK3CA mutant models including two non-hotspot alterations (R88Q and G106_R108del) demonstrate a TGI value of > 50%. Although copy number gain of PIK3CA is a frequent genetic event and could also be considered as a potential marker of sensitivity to PI3Kα inhibitors, in our assessment this was not apparent. We assessed 25 in vitro models and 16 in vivo models with a gain in the PIK3CA copy number greater than 2.5. Using a cut-off of IC50 of <5μM and a TGI value of >50% in both settings we did not identify a significant association between PIK3CA amplification and MLN1117 sensitivity. Thus, PIK3CA mutation status is a significant predictor in classifying sensitive vs. non-sensitive models to MLN1117 whereas PIK3CA copy number status is not (compare p=0.00002 vs p = 0.7668). Our assessment of models with PTEN alterations found all models to be insensitive to MLN1117 using our established in vitro criteria whereas several of these models were found to have borderline sensitivity (range 41-50% TGI) in vivo. These results suggest the need for additional exploration of the PTEN role and MLN1117 sensitivity. Additional trends observed in this data set are 1) PIK3CA co-mutated with KRAS is insensitive to MLN1117 in both in vitro and in vivo systems; and 2) complex mutations involving PIK3CA and ARID1A or EIF4B demonstrate a distribution of sensitivities. We present data that support the use of PIK3CA mutation as a patient selection strategy for clinical studies of MLN1117 while the utility of identifying patients with increases in PIK3CA copy number for clinical studies of MLN1117 may be limited. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A169. Citation Format: Natasha Iartchouk, Erik Koenig, Pooja Shah, Jeff Szwaya, Michael Fitzgerald, Dong Mei Zhang, Yueying Cao, Keli Song, Rachael Brake. Assessment of genotype-correlated sensitivity to the investigational PI3Kα selective inhibitor MLN1117 in preclinical 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 A169.

Abstract 2781: Champions TumorGraft™ models possess PIK3CA mutations - A great tool to explore the PI3K/AKT pathway.

Abstract Phosphatidylinositol 3-kinase (PI3K) is an important intracellular signal transducer that is involved in many cellular functions such as cell growth, proliferation, and differentiation. PI3K is a heterodimeric molecule composed of a regulatory and a catalytic subunit. PIK3CA, which encodes the p110α catalytic subunit, is often mutated in a range of human cancers, such as colorectal and non-small-cell lung cancers. Increasing our understanding of the biological implications of these mutations and the role of PI3K pathway in cancer proliferation or survival may lead to potential new therapeutic opportunities targeting PI3K signaling. Champions Oncology focuses on the development of TumorGraft models derived from the direct implantation of patient tumors into immunocompromised mice. Compared to traditional cell line-based xenograft models, the patient derived Champions TumorGraft™ models maintain stable gene-expression patterns and mutational status and correlate to clinical predictability. Through extensively characterization, Champions has identified 19 TumorGraft models with PIK3CA mutations. Out of these 19 models, there are 11 colorectal cancers, 3 non-small cell lung cancers, 3 pancreatic cancers, 1 gastric cancer and 1 melanoma. Genomics analyses using the Ampliseq platform identified the majority of PIK3CA mutations in exon 9 and 20. Mutations in these regions of PIK3CA are known to confer resistance to some anti-cancer agents, such as HER2 antibodies. Additionally, it has been reported that PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers. Upon further analysis, we identified the same pattern in Champions TumorGraft models where∼70% (13 out of 19) of the PIK3CA mutant models possess either concurrent RAS or BRAF mutations. Notably, PI3K activates the downstream kinase AKT which ultimately triggers the mTOR pathway. Mutations in cancer often result in constitutive activation of this pathway. Given the role of the PI3K/AKT signaling in cancer and the presence of these specific mutations in the Champions TumorGraft bank, utilization of Champions’ TumorGraft models in oncology drug development allows for greater insight into new therapeutic opportunities targeting the inhibition of PI3K/AKT signaling and offers new promise for patients with these activated pathways. Citation Format: Tin Oo Khor, Dhanrajan Tiruchinapalli, Jay Friedman, Nathan Anderson, David Sidransky, Elizabeth M. Bruckheimer. Champions TumorGraft™ models possess PIK3CA mutations - A great tool to explore the PI3K/AKT 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 2781. doi:10.1158/1538-7445.AM2013-2781

Abstract 4504: Genetic determinants of AKT-dependence in epithelial ovarian cancer

Abstract Ovarian cancer is a genetically complex disease. Recent data compiled by The Cancer Genome Atlas Project highlights the heterogeneity within this disease, though distinct genetic signatures, such as PI3K pathway aberration, KRas alteration and Rb loss, are emerging that may condition treatment response. The high frequency of AKT activation in ovarian cancer, via PTEN mutation/deletion, PIK3CA/AKT mutation/amplification, RAS mutation, or mutation/amplification of upstream receptor tyrosine kinases (RTK), suggests that inhibitors of this pathway may be particularly effective treatments for this disease. We utilized a selective, allosteric inhibitor of AKT to determine the AKT-dependence of a panel of ovarian cancer cell lines. This AKT1/2 inhibitor (Merck AKTi-1/2) has been extensively tested against panels of kinases and PH-domain containing proteins and based upon these studies appears to be highly selective for AKT1 and AKT2. The drug effectively inhibited AKT activity as measured by a decrease in AKT phosphorylation at serine 473 in all cell lines. Of the 17 cell lines tested to date, 3 were hypersensitive (IC50 = 0.1um) to AKT inhibition, while 1 demonstrated intermediate sensitivity (IC50 = 2uM) and 13 were resistant (IC50 >=5uM). Levels of AKT phosphorylation at serine 473 were inversely correlated with Merck AKTi-1/2 IC50 (p = 0.036) but AKT activation alone was insufficient to confer sensitivity. Activation of the MAPK pathway, via KRas/BRaf/MEK1 mutation or KRas amplification, and Rb loss were predictive of resistance to AKT inhibition. In sensitive cell lines, AKT inhibition resulted in hypophosphorylation of RB and accumulation of cells in G1. Significant induction of apoptosis was not observed even in PTEN null, AKT2 amplified and PIK3CA mutant models. Treatment of a PTEN-mutant xenograft model of ovarian cancer with the AKTi-1/2 was also effective in inhibiting AKT phosphorylation and downstream signaling and resulted in a cytostatic tumor response. Ongoing studies are focused on the identification of rational combination strategies that result in induction of apoptosis in the cell lines in which the AKTi-1/2 alone induces growth arrest. The long-term goal will be to identify genetic predictors of AKT-dependence and AKT-inhibitor resistance to guide patient selection for future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4504.