Jane Guan

Predictive Biomarkers of Sensitivity to the Phosphatidylinositol 3′ Kinase Inhibitor GDC-0941 in Breast Cancer Preclinical Models

Purpose: The class I phosphatidylinositol 3′ kinase (PI3K) plays a major role in proliferation and survival in a wide variety of human cancers. A key factor in successful development of drugs targeting this pathway is likely to be the identification of responsive patient populations with predictive diagnostic biomarkers. This study sought to identify candidate biomarkers of response to the selective PI3K inhibitor GDC-0941. Experimental Design: We used a large panel of breast cancer cell lines and in vivo xenograft models to identify candidate predictive biomarkers for a selective inhibitor of class I PI3K that is currently in clinical development. The approach involved pharmacogenomic profiling as well as analysis of gene expression data sets from cells profiled at baseline or after GDC-0941 treatment. Results: We found that models harboring mutations in PIK3CA, amplification of human epidermal growth factor receptor 2, or dual alterations in two pathway components were exquisitely sensitive to the antitumor effects of GDC-0941. We found that several models that do not harbor these alterations also showed sensitivity, suggesting a need for additional diagnostic markers. Gene expression studies identified a collection of genes whose expression was associated with in vitro sensitivity to GDC-0941, and expression of a subset of these genes was found to be intimately linked to signaling through the pathway. Conclusion: Pathway focused biomarkers and the gene expression signature described in this study may have utility in the identification of patients likely to benefit from therapy with a selective PI3K inhibitor. Clin Cancer Res; 16(14); 3670–83. ©2010 AACR.

GDC-0980 Is a Novel Class I PI3K/mTOR Kinase Inhibitor with Robust Activity in Cancer Models Driven by the PI3K Pathway

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene, loss or mutation of phosphatase and tensin homolog (PTEN), and deregulation of mammalian target of rapamycin (mTOR) complexes. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K and mTOR are promising therapeutic targets for cancer. We discovered GDC-0980, a selective, potent, orally bioavailable inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2) with excellent pharmacokinetic and pharmaceutical properties. GDC-0980 potently inhibits signal transduction downstream of both PI3K and mTOR, as measured by pharmacodynamic (PD) biomarkers, thereby acting upon two key pathway nodes to produce the strongest attainable inhibition of signaling in the pathway. Correspondingly, GDC-0980 was potent across a broad panel of cancer cell lines, with the greatest potency in breast, prostate, and lung cancers and less activity in melanoma and pancreatic cancers, consistent with KRAS and BRAF acting as resistance markers. Treatment of cancer cell lines with GDC-0980 resulted in G1 cell-cycle arrest, and in contrast to mTOR inhibitors, GDC-0980 induced apoptosis in certain cancer cell lines, including those with direct pathway activation via PI3K and PTEN. Low doses of GDC-0980 potently inhibited tumor growth in xenograft models including those with activated PI3K, loss of LKB1 or PTEN, and elicited an exposure-related decrease in PD biomarkers. These preclinical data show that GDC-0980 is a potent and effective dual PI3K/mTOR inhibitor with promise for the clinic. Mol Cancer Ther; 10(12); 2426–36. ©2011 AACR.

GDC-0941, a Novel Class I Selective PI3K Inhibitor, Enhances the Efficacy of Docetaxel in Human Breast Cancer Models by Increasing Cell Death In Vitro and In Vivo

Purpose: Docetaxel is a front-line standard-of-care chemotherapeutic drug for the treatment of breast cancer. Phosphoinositide 3-kinases (PI3K) are lipid kinases that regulate breast tumor cell growth, migration, and survival. The current study was intended to determine whether GDC-0941, an orally bioavailable class I selective PI3K inhibitor, enhances the antitumor activity of docetaxel in human breast cancer models in vitro and in vivo. Experimental Design: A panel of 25 breast tumor cell lines representing HER2+, luminal, and basal subtypes were treated with GDC-0941, docetaxel, or the combination of both drugs and assayed for cellular viability, modulation of PI3K pathway markers, and apoptosis induction. Drug combination effects on cellular viability were also assessed in nontransformed MCF10A human mammary epithelial cells. Human xenografts of breast cancer cell lines and patient-derived tumors were used to assess efficacy of GDC-0941 and docetaxel in vivo. Results: Combination of GDC-0941 and docetaxel decreased the cellular viability of breast tumor cell lines in vitro but to variable degrees of drug synergy. Compared with nontransformed MCF10A cells, the addition of both drugs resulted in stronger synergistic effects in a subset of tumor cell lines that were not predicted by breast cancer subtype. In xenograft models, GDC-0941 enhanced the antitumor activity of docetaxel with maximum combination efficacy observed within 1 hour of administering both drugs. GDC-0941 increased the rate of apoptosis in cells arrested in mitosis upon cotreatment with docetaxel. Conclusion: GDC-0941 augments the efficacy of docetaxel by increasing drug-induced apoptosis in breast cancer models. Clin Cancer Res; 18(14); 3901–11. ©2012 AACR.

Nuclear Phospho-Akt Increase Predicts Synergy of PI3K Inhibition and Doxorubicin in Breast and Ovarian Cancer

Akt phosphorylation in response to DNA damage predicts the sensitivity of cancer cells to combinations of a PI3K inhibitor and the replication inhibitor doxorubicin. Timing the Delivery of a One-Two Punch How to effectively target and eradicate lingering cancer cells that evade surgical, radiological, or even chemotherapeutic treatments remains one of the most confounding questions in cancer biology. Although complex mechanisms underlie the etiology and inherent heterogeneity of the disease, the manipulation of growth signaling pathways offers several avenues for efficient intervention. In many cancers, a key growth pathway—the phosphatidylinositol 3-kinase (PI3K) pathway—can be activated at one or several points, encouraging efforts to inhibit critical proteins. Yet, cancer cells are smart; they reach out and evolve new ways of resisting such setbacks, turning on new pathways or alternate effector proteins to achieve the same result: incessant growth. Now, Wallin and colleagues have taken aim at this pathway in a large panel of breast and ovarian cancer cell lines by investigating the combinatorial effects of PI3K pathway inhibitors and DNA damage inflicted with a common chemotherapeutic agent, doxorubicin. When the authors probed the basis of the cell lines’ susceptibility to growth inhibition in the presence of one or both drugs, they found that activation of a master serine-threonine kinase, Akt, by phosphorylation was higher in cancer lines that benefited from the drugs in combination, irrespective of PI3K pathway activation status or p53 mutational status. Similar experiments in mice carrying tumors recapitulated these findings and, importantly, the drug combination did not elevate Akt phosphorylation in nontumor tissues. Together, these findings suggest that the activation of Akt in tumor tissues may mark the vulnerability of a cancer to combination therapy with a PI3K inhibitor and a DNA-damaging agent. This proposal will need to be explored further in the clinic, but the utility of tandem drug treatments shown here offers hope for curbing this class of cancers. The phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway is frequently disrupted in cancer and implicated in multiple aspects of tumor growth and survival. In addition, increased activity of this pathway in cancer is associated with resistance to chemotherapeutic agents. Therefore, it has been hypothesized that PI3K inhibitors could help to overcome resistance to chemotherapies. We used preclinical cancer models to determine the effects of combining the DNA-damaging drug doxorubicin with GDC-0941, a class I PI3K inhibitor that is currently being tested in early-stage clinical trials. We found that PI3K inhibition significantly increased apoptosis and enhanced the antitumor effects of doxorubicin in a defined set of breast and ovarian cancer models. Doxorubicin treatment caused an increase in the amount of nuclear phospho-AktSer473 in cancer cells that rely on the PI3K pathway for survival. This increased phospho-AktSer473 response to doxorubicin correlates with the strength of GDC-0941’s effect to augment doxorubicin action. These studies predict that clinical use of combination therapies with GDC-0941 in addition to DNA-damaging agents will be effective in tumors that rely on the PI3K pathway for survival.

Active PI3K pathway causes an invasive phenotype which can be reversed or promoted by blocking the pathway at divergent nodes.

The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents an attractive target for therapeutic intervention. To investigate the primary phenotypes mediated by increased pathway signaling in a clean, patient-relevant context, an activating PIK3CA mutation (H1047R) was knocked-in to an endogenous allele of the MCF10A non-tumorigenic human breast epithelial cell line. Introduction of an endogenously mutated PIK3CA allele resulted in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype, compared to isogenic wild-type cells. The invasive phenotype was linked to enhanced PIP3 production via a S6K-IRS positive feedback mechanism. Moreover, potent and selective inhibitors of PI3K were highly effective in reversing this phenotype, which is optimally revealed in 3-dimensional cell culture. In contrast, inhibition of Akt or mTOR exacerbated the invasive phenotype. Our results suggest that invasion is a core phenotype mediated by increased PTEN/PI3K pathway activity and that therapeutic agents targeting different nodes of the PI3K pathway may have dramatic differences in their ability to reverse or promote cancer metastasis.

The PI3K inhibitor taselisib overcomes letrozole resistance in a breast cancer model expressing aromatase.

Letrozole is a commonly used treatment option for metastatic hormone receptor-positive (HR+) breast cancer, but many patients ultimately relapse. Due to the importance of phosphoinositide-3 kinase (PI3K) in breast cancer, PI3K inhibitors such as taselisib are attractive for combination with endocrine therapies such as letrozole. Taselisib was evaluated as a single agent and in combination with letrozole in a breast cancer cell line engineered to express aromatase. The combination of taselisib and letrozole decreased cellular viability and increased apoptosis relative to either single agent. Signaling cross-talk between the PI3K and ER pathways was associated with efficacy for the combination. In a secreted factor screen, multiple soluble factors, including members of the epidermal and fibroblast growth factor families, rendered breast cancer cells non-responsive to letrozole. It was discovered that many of these factors signal through the PI3K pathway and cells remained sensitive to taselisib in the presence of the soluble factors. We also found that letrozole resistant lines have elevated PI3K pathway signaling due to an increased level of p110α, but are still sensitive to taselisib. These data provide rationale for clinical evaluation of PI3K inhibitors to overcome resistance to endocrine therapies in ER+ breast cancer.

Abstract P2-17-01: The PI3K inhibitor GDC-0032 is selectively potent against PIK3CA mutant breast cancer cell lines and tumors

Mutations in the phosphoinositide-3 kinase alpha isoform (PIK3CA) are frequent in breast cancer and activate the PI3K signaling pathway. We discovered GDC-0032, a selective, potent, orally bioavailable inhibitor of PI3Ka with a Ki = 0.2nM, and with reduced inhibitory activity against PI3Kβ. This selectivity profile, and excellent pharmacokinetic and pharmaceutical properties, allowed for greater efficacy in vivo at the maximum tolerated dose relative to a pan Class I PI3K inhibitor in PIK3CA mutant xenografts. Notably, GDC-0032 preferentially inhibited PIK3CA mutant cells relative to cells with wild-type PI3K. GDC-0032 potently inhibits signal transduction downstream of PI3K and induces apoptosis at low concentrations in breast cancer cell lines and xenograft models that harbor PIK3CA mutations. The mutant-bias of GDC-0032 is linked to unique properties of GDC-0032, including cellular potency against the mutant isoform and reduction of receptor tyrosine kinase (RTK) signaling. Endocrine therapies such as letrozole are commonly used treatment options for metastatic Hormone Receptor positive (HR+) breast cancer but many patients ultimately relapse. Due to the importance of PI3K in breast cancer, PI3K inhibitors such as GDC-0032 are attractive for combination with endocrine therapies. GDC-0032 was evaluated in breast cancer lines and models in combination with letrozole, and assayed for cellular viability, modulation of PI3K pathway, modulation of ER pathway markers, and apoptosis induction. The combination of GDC-0032 and letrozole decreased cellular viability and increased apoptosis relative to either single agent. We observed cross-talk between the PI3K and ER pathways that suggest a mechanism of action for the combination. In a secreted factor screen we found that multiple soluble factors render breast cancer cells non-responsive to letrozole. It was discovered that many of these factors signal through the PI3K pathway and GDC-0032 in combination with letrozole was able to overcome the growth inhibition caused by the soluble factor. We also established letrozole resistant cell lines that grow independently of any estrogen source. These letrozole resistant lines have elevated PI3K pathway signaling and are still sensitive to GDC-0032. Taken together, these data provide rationale for evaluating GDC-0032 in combination with endocrine therapies for ER+ breast cancer treatment in the clinic. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-17-01.

Abstract DDT02-01: Discovery of GDC-0032: A beta-sparing PI3K inhibitor active against PIK3CA mutant tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Modifications of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway are frequent in cancer due to multiple mechanisms, including activating mutations of the alpha isoform of PI3K. The dysregulation of this pathway has been implicated in many processes involved in oncogenesis. Thus, PI3K is a promising therapeutic target for cancer. Previously we have disclosed GDC-0941, a class 1 selective PI3K inhibitor and our class 1 PI3K/mTOR kinase inhibitor, GDC-0980. In this presentation we describe the design and discovery of a new class of PI3K inhibitors, which selectively inhibit the activated PI3Kα isoform relative to the PI3Kβ isoform. A lead was identified from a high throughput screen (HTS) that resulted in a novel chemical series of kinase inhibitors. Through a structure-based approach, this lead was optimized to provide very potent inhibitors of PI3K. In addition, this chemical series allowed for designing molecules that have different selectivity patterns with respect to the class 1 PI3K isoforms. In particular, a series of inhibitors were designed that could preferentially inhibit PI3Kα relative to PI3Kβ (“beta-sparing”). Further modification of the physicochemical properties led to the discovery of GDC-0032. GDC-0032 is a potent inhibitor of PI3Kα (PIK3CA) isoform with a Ki =0.2 nM, and with reduced inhibitory activity against PI3Kβ. This selectivity profile allowed for greater efficacy in vivo at the maximum tolerated dose relative to a pan inhibitor in representative PI3Kα (PIK3CA) mutant xenografts. It is notable that GDC-0032 preferentially inhibited PI3Kα (PIK3CA) mutant cells relative to cells with wild-type PI3K. Taken together, GDC-0032 is a potent and effective beta-sparing PI3K inhibitor, which currently is in clinical trials. Citation Format: Alan G. Olivero, Timothy P. Heffron, Matthew Baumgardner, Marcia Belvin, Leanne Berry Ross, Nicole Blaquiere, Erin Bradley, Georgette Castanedo, Mika Derynck, Steven Do, Jennafer Dotson, Danette Dudley, Kyle Edgar, Adrian Folkes, Ross Francis, Tony Gianetti, Richard Goldsmith, Paul Goldsmith, Jane Guan, Trevor Harrison, Robert Heald, Jerry Hsu, Phillip Jackson, Graham Jones, Amy Kim, Aleks Kolesnikov, Mark Lackner, Leslie Lee, John Lesnick, Cristina Lewis, Michael Mamounas, Neville McLean, Jeremy Murray, Chudi Ndubaku, Jim Nonomiya, Jodie Pang, Neil Pegg, Wei Wei Prior, Laurent Salphati, Deepack Sampath, Stephen Sideris, Michael Siu, Steven Staben, Daniel Sutherlin, Mark Ultsch, Jeff Wallin, Lan Wang, Christian Wiesmann, Xiaolin Zhang, Lori S. Friedman. Discovery of GDC-0032: A beta-sparing PI3K inhibitor active against PIK3CA mutant tumors. [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 DDT02-01. doi:10.1158/1538-7445.AM2013-DDT02-01

Abstract P5-19-02: Selective PI3K and dual PI3K/mTOR inhibitors enhance the efficacy of endocrine therapies in breast cancer models

Purpose: Phosphoinositide 3-kinases (PI3K) are lipid kinases that can regulate breast tumor cell growth, migration and survival. Standard of care drugs such as estrogen receptor (ER) antagonists including fulvestrant and tamoxifen, and aromatase inhibitors such as letrozole are indicated for the treatment of hormone receptor positive breast cancer. The current study is focused on investigating preclinical activity in breast cancer models, for GDC-0941, a class I PI3K inhibitor, GDC-0032, a PI3K inhibitor, and GDC-0980, a dual mTOR kinase and class I PI3K inhibitor. Investigation into PI3K inhibitor efficacy in combination with endocrine therapies is also explored. Experimental Design: A panel of ER+ breast cancer cell lines were treated with GDC-0941, GDC-0032 and GDC-0980 either as single agents or in combination with fulvestrant or tamoxifen and assayed for cellular effects. MCF-7 cells ectopically expressing aromatase were utilized to test the efficacy of aromatase inhibitors in combination with PI3K inhibitors in vitro. In addition, human xenografts of breast cancer cell lines were employed to assess combination efficacy of PI3K inhibitors with fulvestrant and tamoxifen in vivo. Results: Combination of GDC-0941, GDC-0032 or GDC-0980 with endocrine therapies resulted in a decrease in cellular viability and an increase in cell death. Synergy of PI3K inhibitor combinations with fulvestrant or tamoxifen was assessed using Combination Index (C.I.), and C.I. values as low as 0.1 indicated strong synergy in some contexts. Combination activity of PI3K inhibitors and letrozole was also observed in MCF7 cells expressing aromatase. In MCF-7 xenografts, the combination of GDC-0980, GDC-0032 and GDC-0941 enhanced activity of fulvestrant resulting in tumor regressions and tumor growth delay (116% tumor growth inhibition (TGI) for GDC-0980 and 91% TGI for GDC-0941 and GDC-0032). In addition, the combination of GDC-0941 or GDC-0032 with tamoxifen enhanced the efficacy of tamoxifen in vivo (83%TGI for GDC-0941 and 102%TGI for GDC-0032). Mechanism of action and biomarker studies are underway. Conclusion: Collectively, the non-clinical efficacy data provide a strong rationale to evaluate the combination of PI3K inhibitors with anti-estrogen therapy in hormone receptor positive breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-19-02.

Abstract 298: Mutant PIK3CA increases the expression of individual tubulin isoforms and promotes resistance to anti-mitotic chemotherapy drugs

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Resistance to many anti-cancer agents has been attributed to increased PI3K/PTEN pathway signaling. Hence, inhibitors of the pathway have potential utility in combination with these therapies. To investigate chemotherapy drug sensitivities downstream of mutant PIK3CA in a controlled and patient-relevant context we utilized human non-tumorigenic MCF10A parental and isogenic knock-in cell lines that harbor a common activating PIK3CA kinase domain mutation (H1047R). We found the mutant cells to be least sensitive to anti-mitotic drugs, but the resistance could be overcome in combination with GDC-0941, a Class I PI3K inhibitor currently being evaluated in clinical trials. To understand the mechanism of resistance we analyzed microarray data for expression of known anti-mitotic resistance genes. We found that a subset of tubulin isoforms were increased with PI3K pathway signaling, but were reduced with GDC-0941 treatment. The changes in mRNA expression were confirmed at the protein level and were extended to additional cell lines. RNAi knockdown of TUBB2, the isoform we found to be most influenced by PI3K pathway signaling, increased the sensitivity of both parental and H1047R clones to anti-mitotic drugs, but did not influence GDC-0941 potency. Thus, reduced expression of the target increases the potency of the anti-mitotic drug. Overall, these findings suggest a mechanism of action for anti-mitotic drugs in combination with inhibitors of PI3K. 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 298.