Marie-Claire Wagle

Evaluation and Clinical Analyses of Downstream Targets of the Akt Inhibitor GDC-0068

Purpose: The oncogenic PI3K/Akt/mTOR pathway is an attractive therapeutic target in cancer. However, it is unknown whether the pathway blockade required for tumor growth inhibition is clinically achievable. Therefore, we conducted pharmacodynamic studies with GDC-0068, an ATP competitive, selective Akt1/2/3 inhibitor, in preclinical models and in patients treated with this compound. Experimental Design: We used a reverse phase protein array (RPPA) platform to identify a biomarker set indicative of Akt inhibition in cell lines and human-tumor xenografts, and correlated the degree of pathway inhibition with antitumor activity. Akt pathway activity was measured using this biomarker set in pre- and post-dose tumor biopsies from patients treated with GDC-0068 in the dose escalation clinical trial. Results: The set of biomarkers of Akt inhibition is composed of 10 phosphoproteins, including Akt and PRAS40, and is modulated in a dose-dependent fashion, both in vitro and in vivo. In human-tumor xenografts, this dose dependency significantly correlated with tumor growth inhibition. Tumor biopsies from patients treated with GDC-0068 at clinically achievable doses attained a degree of biomarker inhibition that correlated with tumor growth inhibition in preclinical models. In these clinical samples, compensatory feedback activation of ERK and HER3 was observed, consistent with preclinical observations. Conclusion: This study identified a set of biomarkers of Akt inhibition that can be used in the clinical setting to assess target engagement. Here, it was used to show that robust Akt inhibition in tumors from patients treated with GDC-0068 is achievable, supporting the clinical development of this compound in defined patient populations. Clin Cancer Res; 19(24); 6976–86. ©2013 AACR.

Abstract B154: A first-in-human trial of GDC-0068: A novel, oral, ATP-competitive Akt inhibitor, demonstrates robust suppression of the Akt pathway in surrogate and tumor tissues.

GDC-0068 is a highly selective ATP-competitive small molecule that inhibits all three isoforms of Akt with IC50 values of 5 to 30 nM. GDC-0068 selectively inhibits cancer cells with activated Akt signaling (e.g. via PTEN loss or PIK3CA mutations). Patients with advanced solid tumors were treated with GDC-0068 using a 3+3 escalation design. GDC-0068 was dosed PO QD on a 21-day on, 7-day off schedule; endpoints included safety, pharmacokinetics (PK) and determination of pathway knockdown. Pharmacodynamics (PD) endpoints in surrogate tissue [platelet rich plasma (PRP)] were evaluated in all patients. In addition, at least two patients per cohort had pre- and on-treatment (day 15) tumor biopsies at doses ≥100mg QD. The tumor tissue samples were evaluated using reverse phase protein array (RPPA). Thirty patients were enrolled across 7 cohorts (25, 50, 100, 200, 400, 600 and 800 mg QD). GDC-0068 was generally well-tolerated at doses ≤ 600 mg. Preliminary PK analyses show a dose proportional increase in exposure over the dose range tested. In addition, clinical exposures at doses ≥ 200 mg QD met or exceeded GDC-0068 exposures associated with tumor stasis in multiple PTEN null preclinical xenograft models. PK/PD evaluation showed dose-dependent Akt pathway inhibition in the PRP assay, with ≥70% inhibition of pGSK3 in all patients at doses ≥ 200 mg QD. Pre- and on-treatment tumor biopsies showed ≥50% decrease in pPRAS40 and cyclin D1 in multiple patients at doses of 200 mg QD and higher. In addition, evidence of feedback activation of the MAP kinase pathway after treatment with GDC-0068 was observed in biopsy samples. One patient with PTEN low/ PIK3CA H1047R/ KRAS wt CRC had prolonged stable disease and showed Akt pathway suppression by multiple downstream markers. GDC-0068 is well tolerated at doses ≤ 600 mg with a favorable safety profile and dose proportional pharmacokinetics. Treatment with GDC-0068 results in substantial pathway knockdown in both surrogate and tumor tissues at doses ≥ 200 mg QD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B154.

Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors

Mitogen-activated protein kinase (MAPK) pathway dysregulation is implicated in >30% of all cancers, rationalizing the development of RAF, MEK and ERK inhibitors. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, these inhibitors had limited success in other MAPK dysregulated tumors, with insufficient pathway suppression and likely pathway reactivation. In this study we show that inhibition of either MEK or ERK alone only transiently inhibits the MAPK pathway due to feedback reactivation. Simultaneous targeting of both MEK and ERK nodes results in deeper and more durable suppression of MAPK signaling that is not achievable with any dose of single agent, in tumors where feedback reactivation occurs. Strikingly, combined MEK and ERK inhibition is synergistic in RAS mutant models but only additive in BRAF mutant models where the RAF complex is dissociated from RAS and thus feedback productivity is disabled. We discovered that pathway reactivation in RAS mutant models occurs at the level of CRAF with combination treatment resulting in a markedly more active pool of CRAF. However, distinct from single node targeting, combining MEK and ERK inhibitor treatment effectively blocks the downstream signaling as assessed by transcriptional signatures and phospho-p90RSK. Importantly, these data reveal that MAPK pathway inhibitors whose activity is attenuated due to feedback reactivation can be rescued with sufficient inhibition by using a combination of MEK and ERK inhibitors. The MEK and ERK combination significantly suppresses MAPK pathway output and tumor growth in vivo to a greater extent than the maximum tolerated doses of single agents, and results in improved anti-tumor activity in multiple xenografts as well as in two Kras mutant genetically engineered mouse (GEM) models. Collectively, these data demonstrate that combined MEK and ERK inhibition is functionally unique, yielding greater than additive anti-tumor effects and elucidates a highly effective combination strategy in MAPK-dependent cancer, such as KRAS mutant tumors.

A transcriptional MAPK Pathway Activity Score (MPAS) is a clinically relevant biomarker in multiple cancer types

KRAS- and BRAF-mutant tumors are often dependent on MAPK signaling for proliferation and survival and thus sensitive to MAPK pathway inhibitors. However, clinical studies have shown that MEK inhibitors are not uniformly effective in these cancers indicating that mutational status of these oncogenes does not accurately capture MAPK pathway activity. A number of transcripts are regulated by this pathway and are recurrently identified in genome-based MAPK transcriptional signatures. To test whether the transcriptional output of only 10 of these targets could quantify MAPK pathway activity with potential predictive or prognostic clinical utility, we created a MAPK Pathway Activity Score (MPAS) derived from aggregated gene expression. In vitro, MPAS predicted sensitivity to MAPK inhibitors in multiple cell lines, comparable to or better than larger genome-based statistical models. Bridging in vitro studies and clinical samples, median MPAS from a given tumor type correlated with cobimetinib (MEK inhibitor) sensitivity of cancer cell lines originating from the same tissue type. Retrospective analyses of clinical datasets showed that MPAS was associated with the sensitivity of melanomas to vemurafenib (HR: 0.596) and negatively prognostic of overall or progression-free survival in both adjuvant and metastatic CRC (HR: 1.5 and 1.4), adrenal cancer (HR: 1.7), and HER2+ breast cancer (HR: 1.6). MPAS thus demonstrates potential clinical utility that warrants further exploration.Biomarker: Gene signature predicts drug responses and patient outcomesA clinical score based on the activity of genes that regulate cell signaling can predict drug sensitivity and patient outcomes across a range of cancer types. Marie-Claire Wagle, Daniel Kirouac and their colleagues at Genentech in South San Francisco, California, USA developed an index that aggregates expression levels of 10 genes involved in modulating the mitogen-activated protein kinase (MAPK) pathway. This “MAPK Pathway Activity Score”, or MPAS, performed as well or better than other more complicated, genome-based tools at predicting whether drugs that inhibit MAPK-related enzymes were active against tumor cell lines. Retrospective analyses of clinical datasets also showed that MPAS correlated with survival outcomes in patients with melanoma, colon cancer, and breast cancer. The authors suggest that MPAS should be evaluated more broadly and perhaps implemented as a clinically informative biomarker.

Abstract 4470: Mechanisms of acquired resistance to the PI3K inhibitors in colorectal cancer cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene or loss of the tumor suppressor PTEN. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K is a promising therapeutic target for cancer. There are multiple PI3K inhibitors in clinical trials and the current study investigates preclinical mechanisms of resistance to two clinical PI3K inhibitors, GDC-0941 and GDC 0032. Acquired resistance to PI3K inhibitors was generated in vitro, in SW48 parental and SW48 knock-in clones harboring hotspot PIK3CA mutations. We found that GDC-0941 resistant clones and GDC-0032 resistant clones demonstrated a marked signaling increase in the PI3K/Akt/mTOR pathway when released from drug inhibition, and different nodes in the pathway were activated. GDC-0941 resistant clones have elevated pAKT levels due to loss of PTEN, and the pAKT is further increased when drug is removed. GDC-0032 resistant clones retained functional PTEN protein, but had increased pS6 levels in the absence of GDC-0032. The result of increased PI3K pathway signaling in cells with acquired resistance to PI3K inhibitors resulted in a decreased sensitivity to inhibitors of the MAPK pathway upon removal of drug. Resistant cells maintained on PI3K inhibitors gained sensitivity to MAPK pathway inhibitors. These preclinical data may provide rationale for combination therapy in the clinic. Citation Format: Kyle A. Edgar, Marie-Claire Wagle, Yibing Yan, Marcia Belvin, Lori Friedman, Jeffrey Wallin. Mechanisms of acquired resistance to the PI3K inhibitors in colorectal cancer cell lines. [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 4470. doi:10.1158/1538-7445.AM2013-4470

Abstract 3479: Sensitivity of endometrial cancer cells to inhibitors targeting different nodes of the PI3K pathway and their combination with a MEK inhibitor.

Activation of the PI3K pathway has been reported in endometrial cancers, and there are a number of inhibitors targeting different nodes of the PI3K pathway currently in clinical trials, including the PI3K/mTOR dual inhibitor GDC-0980 that is in a phase II trial for endometrial cancer (NCT01455493). In this study, we set to examine the prevalence of overlapping mutational activation of the PI3K pathway with other pathways, such as MAPK. We also tested whether targeting different nodes of PI3K pathway, such as PI3K, AKT, or PI3K/mTOR could exert differential effects on endometrial cancer cells, and whether combination with a MEK inhibitor could provide an additional benefit in inhibiting endometrial cancer cell growth. Alteration of oncogenes and tumor suppressor genes were profiled in 80 endometrial cancer samples by qRT-PCR, targeted deep sequencing, and IHC staining. Multiple PI3K inhibitors, including GDC-0941 (PI3K), GDC-0068 (AKT), GDC-0980 (PI3K/mTOR), and the MEK inhibitor GDC-0973 were tested in a panel of 27 endometrial cancer cell lines for their effects on cell growth. Cell signaling status at baseline and on-treatment was profiled by reverse phase protein array. Mutations and copy number variations of relevant oncogenes and tumor suppressor genes were examined in these cells by targeted deep sequencing. In the 80 endometrial tumor tissues we profiled, there were multiple instances of PI3K pathway alteration including 31% PIK3CA mutations, 9% AKT1 mutations, 16% MET mutations, and 38% PTEN null. In addition, KRAS mutations significantly overlapped with PTEN null or PIK3CA mutation in the same specimen. Endometrial cancer cell lines had distinct patterns of sensitivity to inhibitors targeting different nodes of the PI3K pathway. Preliminary results showed that GDC-0068 was more effective in cell lines with PTEN mutations, while GDC-0941 had a greater effect on those with PIK3CA mutations, and the cell lines were broadly sensitive to GDC-0980. In spite of the widespread activation of the PI3K pathway in endometrial cancer cells, synergistic inhibition of cell growth was observed when GDC-0973 was combined with either GDC-0068 or GDC-0941 in most of the cell lines tested. We will seek to identify genomic and/or proteomic features that will allow better selection as to which node of the PI3K pathway to target, and when to combine with a MEK inhibitor. Citation Format: Matthew J. Wongchenko, Yinghui Guan, Marie-Claire Wagle, Lisa Ryner, Shan Lu, Hartmut Koeppen, Garret Hampton, Mark Lackner, Yulei Wang, Yibing Yan. Sensitivity of endometrial cancer cells to inhibitors targeting different nodes of the PI3K pathway and their combination with a MEK inhibitor. [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 3479. doi:10.1158/1538-7445.AM2013-3479

Abstract B62: Multiple genomic aberrations in BCR-ABL inhibitor-resistant cells lead to sensitivity towards MEK inhibition

Purpose: Chronic myeloid leukemia (CML) can be effectively treated with BCR-ABL inhibitors such as imatinib, dasatinib, and nilotinib; however, resistance to these inhibitors develops over time causing patients to relapse. Recently, MEK inhibition was shown to synergize with BCR-ABL inhibitors to inhibit resistant CML cells harboring the T315I mutation (1). In this study we characterized mechanisms of resistance to BCR-ABL inhibitor in acquired resistant cells without BCR-ABL mutation by genomic and phosphoprotein profiling. We further evaluated the sensitivity of these resistant clones to MEK inhibitor, GDC0973, PI3K inhibitor, GDC0941, alone or in combination. Methods: The CML cell K562 was grown in an increasing concentration of imatinib or/and dasatinib to induce acquired resistance. Clones were isolated from the resistant pool and the GI50 of each clone was measured using the Cell-Titer Glo® viability assay. Genomic DNA from selected clones was tested using Oncoscan® (Affymetrix), which surveys whole genome for copy number variation (CNV) and 400 oncogenic mutations. Cell lysates were subjected to reverse phase protein array (RPPA) analysis of phosphorylation levels of ∼100 proteins. Genomic mutation and CNV profiles, as well as protein phosphorylation patterns of the clones were compared to the parental cells to identify potential pathways involved in resistance. Results: Four imatinib-resistant clones with varying GI50s (4–30 times higher than parental cells) were fully characterized with both Oncoscan and RPPA. None contained the T315I mutation. A genomic mutation scan showed that all the clones had multiple mutations affecting various signaling pathways including TGFβ (Smad-2), Wnt (APC), PI3K (PIK3CA) and EGFR. Likewise, whole genome copy number scan showed that CNV occurred in multiple genes including copy number gains in ADAM29, BMP2, Rspondin4 (Wnt) and PKA in multiple clones, and copy number losses in ADAMTS14, LIM-kinase and FzD9 (Wnt) in multiple clones. The protein phosphorylation patterns also showed multiple changes in the resistant clones. Three of the resistant clones (K14, 15 and 25) had 6-10-fold increased pMEK and pERK, relative to the parental cells. MEk inhibition resulted in a cytostatic response in the parental cells (GI50: 1900 nM), whereas resistant clones 14, 15, and 25 were highly sensitive to GDC0973-mediated apoptosis, with GI50s ranging from 60–75 nM. Clone 6 had lower pMEK and pERK levels than the others, but was still sensitive to GDC0973 (GI50: 600 nM). Phosphorylation of proteins in the PI3K pathway such as pS6, p70S6K and p-mTOR were elevated in all of the clones as compared to the parental cells (7–11 fold); however, none of these clones were sensitive to GDC0941 alone. Our data suggest that either GDC0973 or GDC0973 alone or in combination with GDC0941 could be effective at inducing cell death in resistant clones despite their various genetic backgrounds. Conclusion: The MEK inhibitor GDC0973 caused robust cellular apoptosis in all of the resistant clones in contrast to the cytostatic effect in parental cells. All of the clones had elevated PI3K signaling as shown by enhanced PI3K substrate phosphorylation. Despite the many genetic aberrations that occurred as cells became resistant; most noticeably changes in Wnt, PKA, ADAMs and BMP2 in all of the clones, resistant cells appear to channel escape/survival signals through the MAPK and/or PI3K pathway rendering them sensitive to GDC0973 alone or in combination with GDC0941.

Abstract A29: Different PIK3CA activation mutations can lead to distinct signaling mechanisms and EGFR inhibitor sensitivities in colorectal cancer cells

Introduction: The PI3K/AKT pathway is activated in a wide variety of human cancers, and it has previously been shown that its dysregulation can lead to EGFR inhibitor resistance. In colorectal cancer, activating mutations of the PIK3CA gene have been identified in 13–17% of patients, and typically occur in either the helical domain (E542K or E545K) or the kinase domain (H1047R). Here, we used the colorectal cancer cell SW48, which has an EGFR G719S activating mutation, as well as the isogenic lines with knock-in of either PIK3CA E545K or H1047R mutation to study the differential effects of these two mutations on cell signaling, and the impact that each mutation has on the activity of the EGFR inhibitors erlotinib (Tarceva®), and GDC-0068, an AKT inhibitor. Results: The effect on cell growth of the erlotinib was measured in parental, PIK3CA E545K, and PIK3CA H1047R SW48 cells. Compared to the parental SW48 cells, knock-in of E545K led to erlotinib resistance, while knock-in of H1047R significantly sensitized cells to erlotinib. The GI50 of cells exposed to erlotinib was 2,400, 21,000, and 20 nM, respectively. The EGFR/ErB2 inhibitor lapatinib (Tykerb®) had GI50 values of >30,000, >30,000, and 18 nM, respectively. To explore the mechanism of resistance and sensitivity rendered by the two PIK3CA mutations, we measured the phosphorylation levels of 80 proteins that represent several key signaling pathways in these isogenic cells. Compared to parental SW48 cells, H1047R cells were found to have increased activation of EGFR and ErbB2, as well as PDGFRβ and Ron, receptor tyrosine kinases that have been shown to heterodimerize with members of the ErbB family. Furthermore, these cells showed increased activity of the MEK/ERK pathway. Treatment with erlotinib led to attenuation of EGFR, ErbB2, PDGFRβ, Ron, and MEK/ERK pathway signaling, providing a mechanistic basis for the increased effect of erlotinib in H1047R cells. While cells with both the E545K and H1047R mutations had increased activity of the PI3K/AKT pathway, a greater increase was seen in E545K cells. To determine the impact of each mutation on the cells dependence on the PI3K/AKT pathway, we measured the effect of GDC-0068 on cell proliferation. SW48 parental, E545K, and H1047R cells had GI50 values of 2,500, 520, and 1,800 nM, respectively, highlighting the differential dependence on the PI3K/AKT pathway in cells with the E545K. While GDC-0068 inhibited downstream targets of PI3K/AKT in all three lines, increased suppression was seen in the E545K mutant. In addition, more substantial inhibition of targets that converge downstream of both PI3K/AKT and MEK/ERK was seen in E545K cells. Conclusion: Previous studies have shown that activating mutations of the PIK3CA gene lead to independence from receptor tyrosine kinase signaling, and insensitivity to EGFR inhibition. However, we have demonstrated that two distinct types of activating mutation of PIK3CA lead to differential sensitivity to EGFR inhibitors in CRC cells. PI3K with the H1047R mutation led to increased receptor tyrosine kinase activities and increased sensitivity to EGFR inhibitors, whereas PI3K with the E545K mutation caused hyperactivity of the PI3K/AKT pathway, which led to resistance to EGFR inhibitors and increased sensitivity to an Akt inhibitor. Our results, which added to potential explanations for the variability of clinical outcomes of EGFR inhibitors in patients with colorectal cancer, demonstrate the importance of informed patient selection.