Combination Of GDC-0941 Research Articles

Comprehensive analysis of T cell leukemia signals reveals heterogeneity in the PI3 kinase-Akt pathway and limitations of PI3 kinase inhibitors as monotherapy.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K γ-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kγ-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner.

Sensitizing Triple-Negative Breast Cancer to PI3K Inhibition by Cotargeting IGF1R.

Targeted therapies have proven invaluable in the treatment of breast cancer, as exemplified by tamoxifen treatment for hormone receptor-positive tumors and trastuzumab treatment for HER2-positive tumors. In contrast, a subset of breast cancer negative for these markers, triple-negative breast cancer (TNBC), has met limited success with pathway-targeted therapies. A large fraction of TNBCs depend on the PI3K pathway for proliferation and survival, but inhibition of PI3K alone generally has limited clinical benefit. We performed an RNAi-based genetic screen in a human TNBC cell line to identify kinases whose knockdown synergizes with the PI3K inhibitor GDC-0941 (pictilisib). We discovered that knockdown of insulin-like growth factor-1 receptor (IGF1R) expression potently increased sensitivity of these cells to GDC-0941. Pharmacologic inhibition of IGF1R using OSI-906 (linsitinib) showed a strong synergy with PI3K inhibition. Furthermore, we found that the combination of GDC-0941 and OSI-906 is synergistic in 8 lines from a panel of 18 TNBC cell lines. In these cell lines, inhibition of IGF1R further decreases the activity of downstream PI3K pathway components when PI3K is inhibited. Expression analysis of the panel of TNBC cell lines indicates that the expression levels of IGF2BP3 can be used as a potential predictor for sensitivity to the PI3K/IGF1R inhibitor combination. Our data show that combination therapy consisting of PI3K and IGF1R inhibitors could be beneficial in a subset of TNBCs. Mol Cancer Ther; 15(7); 1545-56. ©2016 AACR.

Abstract P6-07-01: Anti-tumor efficacy of PI3K-mTOR pathway inhibitors in combination with PARP inhibitor plus carboplatin in BRCA1-competent TNBC, beyond PTEN: A proof of concept study

Abstract Background: Recently we reported that PI3K-mTOR inhibition potentiated anti-tumor effects in BRCA-competent TNBC cells when combined with ABT888 (A) and carboplatin (C) (De et al., Neoplasia, 2014). Pro-proliferative and anti-apoptotic actions of this pathway constitute one of the main effector signals of RAS. Here, we tested the anti-tumor effect of either single or dual node blockade of PI3K-mTOR pathway when combined with A and C on BRCA-competent TNBC cells with WT-PTEN background and activated RAS-RAF pathway. Materials & Methods: Athymic mice bearing TNBC xenograft tumors were treated with pan PI3K inhibitor, GDC-0941 or PI3K-mTOR dual inhibitor, GDC-0980 alone or in combination with A and C. Mechanism-based in vitro studies were performed using a panel of BRCA-wt/mutants TNBC cells with varying genetic backgrounds. Results: Blocking a single nodal point of PI3K by GDC-0941 failed to significantly inhibit growth of pre-established tumors (> 20%) even in combination with A and C in MDA-MB231 xenografts, while GDC-0980 potentiated an anti-tumor effect by inhibiting tumor growth by 90%. In vitro treatment (1) decreased proliferation signals (pAKT, pP70S6K, p4EBP1, pS6RP), cell cycle progression, vascular mimicry & 2D clonogenic growth, and (2) increased apoptosis markers (cl-caspase3, 9, BIM, cl-PARP, & annexinV positivity). GDC-0980 in combination with A plus C concomitantly decreased Ki67, cl-caspse3, pVEGFR, CD31, p4EBP1, and pS6RP in vivo (IHC). In contrast, combination of GDC-0941 with A plus C failed to affect the cell cycle, apoptosis or 2D clonogenic growth in MDA-MB231 cells. Alternatively, Treatment with RAD001 increased pAKT while it perturbed the activation of S6RP/4EBP1. Conclusion: Our data indicate that following dual inhibition of PI3K-mTOR, S6RP/4EBP1 de-phosphorylation tracks more consistently with the drug’s tumor-growth inhibitory response rather than the upstream state of AKT-activation. Unlike mTORC1 inhibitor RAD001, GDC-0980 potently eliminates (in vitro & in vivo) feed-back re-activation of the pathway as, (1) it targets PI3K, reactivation of AKTT308 is blocked and (2) inhibition of the mTORC2 complex blocks the reactivation of AKTS473. TNBC tumors with PTEN independent RAS/RAF mutation-mediated activation of PI3K-mTOR pathway can be controlled by dual node blockade of the pathway when combined with a PARP inhibitor and carboplatin. Citation Format: Nandini Dey, Yuliang Sun, Jennifer Carlson, Lori Friedman, Pradip De, Brian Leyland-Jones. Anti-tumor efficacy of PI3K-mTOR pathway inhibitors in combination with PARP inhibitor plus carboplatin in BRCA1-competent TNBC, beyond PTEN: A proof of concept study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-01.

Abstract 2925: Mechanism-based combinatorial treatment of KRAS mutant colorectal cancer with MEK and PI3K pathway inhibitors

Abstract Treatment of metastatic colorectal cancer remains a considerable hurdle as typically <20% of patients respond to available targeted drugs. The most advanced treatment options for these patients are anti-EGFR therapies. However, it is now accepted that tumors with KRAS mutations will not respond to these therapies. Genomic aberrations, including KRAS mutation, frequently activate the MAPK and PI3K pathways in cancer, and combined inhibition of these pathways are now being trialed widely in the clinic. We used a panel of 28 colorectal cancer cell lines to explore combinations of PI3K (PI3Ki) and MEK inhibitors (MEKi). As expected BRAF mutant tumors were sensitive to allosteric MEKi. KRAS/BRAF mutant tumors were resistant to PI3K pathway inhibitors compared to wild-type cells, while tumors with PIK3CA and KRAS/BRAF mutations had intermediate sensitivity. PI3Ki induced a robust G1 cell cycle arrest in sensitive cells and a potent autophagic response in less sensitive lines. MEKi induced a cytostatic response. Combination of MEKi with PI3K pathway inhibitors of different chemotype and selectivity demonstrated that class I PI3Ki were potent inducers of synergy with MEK inhibition. Combination of the MEKi with mTOR inhibitors was at best additive, while class I PI3K/mTOR inhibitors were borderline synergistic/additive with MEKi. Normal non-transformed colon cell lines did not show synergy, which was in part due to a lack of response to the MEKi. We selected a combination of GDC0941 (class I PI3Ki) with GDC0973 (MEKi) to explore the mechanism behind the synergistic interaction. A potent combinatorial effect was observed at the level of FOXO-regulated transcription. Regulators of apoptosis were affected both at the transcriptional and post-translational level by the combination, and this resulted in switching from cytostasis/autophagy to an apoptotic response. Gene knockout and a siRNA screen targeting apoptotic regulators identified a dual requirement for BIM and BBC3 BH3-proteins that acted through activation of BAX and abrogation of MCL1/BCL-XL to drive apoptosis. Continuous exposure to the combination resulted in acquired resistance, through resistance to the MEKi. This resistant line retained sensitivity to an ERK inhibitor and a synergistic interaction when combined with GDC0941. Overall, our data suggest that combination of MEK and class I PI3Ki may be a treatment option for the particularly hard to treat KRAS mutant colorectal cancers. Resistance to targeted agents is major challenge in clinic and may be overcome by combinatorial treatments. Intriguingly, our observations indicate that, as with single agent treatments, cancer cells can also evolve resistance to combinatorial regimes. This suggests that combined treatment with two targeted agents may not be sufficient to overcome resistance and that combinations of multiple (>2) agents or intermittent/alternating treatment strategies will be required. Citation Format: Paul A. Clarke, Toby Roe, Paul Workman. Mechanism-based combinatorial treatment of KRAS mutant colorectal cancer with MEK and PI3K pathway inhibitors. [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 2925. doi:10.1158/1538-7445.AM2014-2925

Abstract P5-06-01: The PI3K inhibitor GDC-0941 combines with trastuzumab for superior anti-tumor efficacy in HER2+ breast cancer models

Abstract Background: PI3K-AKT-mTOR pathway signaling is important for the oncogenic function of HER2. Activating alterations of this pathway are frequently observed in HER2-enriched breast cancer and generally herald a poor response and resistance to trastuzumab (T). Purpose: Targeting the PI3K-AKT-mTOR pathway is an attractive strategy in HER2+ breast cancer that is refractory to trastuzumab. The hypothesis is that the suppression of this pathway by pan-PI3K inhibitor, GDC-0941 may lead to overcome trastuzumab-resistance. Experimental Design: The antiproliferative and HER2-mediated cellular signaling (pAKT, pP70S6K, pS6RP, p4EBP1 and p-ERK) effects of GDC-0941 alone and in combination with T were evaluated in HER2 amplified T-sensitive (BT474), T-resistant (BT474HR), and HER2 amplified/PIK3CA mutated (HCC1954, UACC893) BT cell lines by MTT assay and Western blots. Clonogenic growth was tested by 3D ON-TOP assay and apoptosis markers were also tested. Athymic mice bearing BT474 and BT474HR xenograft tumors were treated with GDC-0941 and T (alone and in combination). Results: (1) GDC-0941 exhibited in vitro cell killing activity in MTT assay with IC50's ranging from 0.35 μM to 1 μm and potency was augmented by the addition of T, (2) inhibition of phosphorylation of AKT(S473, T308), P70S6K, S6RP, and 4EBP1(T37/46, T70) was observed following GDC-0941 treatment, and the combination of GDC-0941 and T more effectively blocked the PI3K-AKT-mTOR pathway, (3) GDC-0941 treatment increased apoptosis markers (CL-CASPASE3 and annexinV positivity), (4) GDC-0941 dose-dependently blocked 3D-ON-TOP clonogenic growth of HER2+ cells. This effect was potentiated in the presence of T and (5) in vivo, the combination of GDC-0941 and T significantly reduced established tumor growth in both sensitive (82%) and resistant (79%) models with concomitant decrease of different PD markers. Conclusions: Our data suggest that 1) therapeutic targeting of the PI3K-AKT-mTOR signaling should be effective in abrogating resistance to T therapy in HER2+ BT, and 2) targeting both the HER2 and the PI3K signaling pathways is an attractive strategy to enhance the clinical efficacy of T therapy, as well as to prevent or delay the development of resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-06-01.

PI3K inhibitor GDC-0941 enhances apoptotic effects of BH-3 mimetic ABT-737 in AML cells in the hypoxic bone marrow microenvironment

Both phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling and antiapoptotic Bcl-2 family members are critical for survival of acute myeloid leukemia (AML) cells. Here, we demonstrate the antileukemic effects of simultaneous inhibition of PI3K by the selective class I PI3K inhibitor GDC-0941 and of Bcl-2 family members by the BH3 mimetic ABT-737 in the context of the bone marrow microenvironment, where hypoxia and interactions with bone marrow stromal cells promote AML cell survival and chemoresistance. The combination of GDC-0941 and ABT-737 profoundly downregulated antiapoptotic Mcl-1 expression levels, activated BAX, and induced mitochondrial apoptosis in AML cells co-cultured with bone marrow stromal cells under hypoxic conditions. Hypoxia caused degradation of Mcl-1 and rendered Mcl-1-overexpressing OCI-AML3 cells sensitive to ABT-737. Our findings suggest that pharmacologic PI3K inhibition by GDC-0941 enhances ABT-737-induced leukemia cell death even under the protective conditions afforded by the bone marrow microenvironment. Combined blockade of PI3K and Bcl-2 pathways down-regulates anti-apoptotic Mcl-1 expression PI3K and Bcl-2 induced Mcl-1 down-regulation activates BAX PI3K and Bcl-2 blockage induces apoptosis in AML under hypoxic BM microenvironment.

Effect of pan-PI3K and HER2 blockade on antitumor activity in preclinical models of breast cancer resistance to trastuzumab therapy.

e13570 Background: PI3K-AKT-mTOR pathway signaling is important for the oncogenic function of HER2. Activating alterations of this pathway are frequently observed in HER2-enriched breast cancer and generally herald a poor response and resistance to trastuzumab (T). Targeting the PI3K-AKT-mTOR pathway is an attractive strategy in HER2+ breast cancer that is refractory to trastuzumab. The hypothesis is that the suppression of this pathway by pan-PI3K inhibitor, GDC-0941 may lead to overcome trastuzumab-resistance. Methods: The antiproliferative and HER2-mediated cellular signaling (pAKT, pP70S6K, pS6RP, p4EBP1 and p-ERK) effects of GDC-0941 alone and in combination with T were evaluated in HER2 amplified T-sensitive (BT474), T-resistant (BT474HR), and HER2 amplified/PIK3CA mutated (HCC1954, UACC893) BT cell lines by MTT assay and Western blots. Athymic mice bearing BT474 and BT474HR xenograft tumors were treated with GDC-0941 and T (alone and in combination). Results: (1) GDC-0941 exhibited in vitro cell killing activity in MTT assay with IC50’s ranging from 0.35 µM to 1 µm and potency was augmented by the addition of T, (2) inhibition of phosphorylation of AKT(S473, T308), P70S6K, S6RP, and 4EBP1(T37/46, T70) was observed following GDC-0941 treatment, and the combination of GDC-0941 and T more effectively blocked the PI3K-AKT-mTOR pathway, (4) GDC-0941 dose-dependently blocked 3D-ON-TOP clonogenic growth of HER2+ cells. This effect was potentiated in the presence of T and (5) xenograft data show that the combination of GDC-0941 and T has strongly enhanced anti-tumor effect in both sensitive (82%) and resistant models (79%), something that cannot be achieved by either monotherapy. Conclusions: Our data suggest that 1) therapeutic targeting of the PI3K-AKT-mTOR signaling should be effective in abrogating resistance to T therapy in HER2+ BT, and 2) targeting both the HER2 and the PI3K signaling pathways is an attractive strategy to enhance the clinical activity of T therapy, as well as to prevent or delay the development of resistance.

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

Abstract 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 P4-07-01: The Class I Selective PI3K Inhibitor GDC-0941 Enhances the Efficacy of Docetaxel in Human Breast Cancer Models by Increasing the Rate of Apoptosis

Abstract Docetaxel (DTX) is commonly used as a front-line treatment option for breast cancer but many patients ultimately relapse and succumb to disease progression. Phosphatidylinositol 3-kinases (PI3K) are lipid kinases that regulate breast tumor cell growth, migration and survival. Given that phosphatidylinositol 3-kinase (PI3K) is frequently activated in breast cancer and induces chemo-resistance, it is an attractive target for combination therapy with standard of care drugs such as DTX. The current study was intended to determine if GDC-0941, an orally bioavailable class I selective PI3K inhibitor, enhances the anti-tumor activity of DTX in human breast cancer models in vitro and in vivo. A panel of 25 breast tumor cell lines representing luminal, HER2+ and basal sub-types were treated with GDC-0941, DTX or the combination of both drugs and assayed for cellular viability, modulation of PI3K pathway markers and apoptosis induction. The combination of GDC-0941 and DTX decreased the cellular viability of breast tumor cell lines in vitro but with variable drug synergy. The addition of both drugs resulted in stronger synergistic effects in a sub-set of tumor cell lines that was not predicted by breast cancer sub-type or mutations within the PI3K pathway. Human xenografts of breast cancer cell lines and patient-derived tumors were utilized to assess efficacy of GDC-0941 and DTX in vivo. In these models, the best combination efficacy was detected when the two drugs were dosed within 1 hour of each other. We also observed that GDC-0941 increased the rate of apoptosis in cells arrested in mitosis upon co-treatment with DTX. Our data provides a preclinical rationale for evaluating GDC-0941 in combination with DTX for breast cancer treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-07-01.

Phosphoinositide 3-Kinase (PI3K) Pathway Alterations Are Associated with Histologic Subtypes and Are Predictive of Sensitivity to PI3K Inhibitors in Lung Cancer Preclinical Models

Class 1 phosphatidylinositol 3-kinase (PI3K) plays a major role in cell proliferation and survival in a wide variety of human cancers. Here, we investigated biomarker strategies for PI3K pathway inhibitors in non-small-cell lung cancer (NSCLC). Molecular profiling for candidate PI3K predictive biomarkers was conducted on a collection of NSCLC tumor samples. Assays included comparative genomic hybridization, reverse-transcription polymerase chain reaction gene expression, mutation detection for PIK3CA and other oncogenes, PTEN immunohistochemistry, and FISH for PIK3CA copy number. In addition, a panel of NSCLC cell lines characterized for alterations in the PI3K pathway was screened with PI3K and dual PI3K/mTOR inhibitors to assess the preclinical predictive value of candidate biomarkers. PIK3CA amplification was detected in 37% of squamous tumors and 5% of adenocarcinomas, whereas PIK3CA mutations were found in 9% of squamous and 0% of adenocarcinomas. Total loss of PTEN immunostaining was found in 21% of squamous tumors and 4% of adenocarcinomas. Cell lines harboring pathway alterations (receptor tyrosine kinase activation, PI3K mutation or amplification, and PTEN loss) were exquisitely sensitive to the PI3K inhibitor GDC-0941. A dual PI3K/mTOR inhibitor had broader activity across the cell line panel and in tumor xenografts. The combination of GDC-0941 with paclitaxel, erlotinib, or a mitogen-activated protein-extracellular signal-regulated kinase inhibitor had greater effects on cell viability than PI3K inhibition alone. Candidate biomarkers for PI3K inhibitors have predictive value in preclinical models and show histology-specific alterations in primary tumors, suggesting that distinct biomarker strategies may be required in squamous compared with nonsquamous NSCLC patient populations.

The Antitumor Effect of GDC-0941 Alone and in Combination with Rapamycin in Breast Cancer Cells

Background: The phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is a key potential target in breast cancer therapy. Because some cancer cell lines are resistant to mTOR inhibition, we combined the mTOR inhibitor with the PI3K inhibitor and assayed the inhibitory effect of this combination versus that of a single inhibitor. Methods: The proliferation of MCF7, SK-BR-3, T-47D, and MDA-MB-231 cells was measured by MTT assay in the presence of GDC-0941 and/or rapamycin. Afterwards, we determined the visible changes in signaling in the PI3K/AKT/mTOR pathway by Western blotting. Results: GDC-0941 exhibited excellent inhibition on MCF7, T-47D and SK-BR-3 cells with different characteristics. In addition, GDC-0941 blocked the feedback of PI3K/Akt through S6K1, resulting in decreased Akt activity by rapamycin activation. The combination of GDC-0941 and rapamycin downregulated the key components of the cell cycle machinery, such as cyclin D1 and upregulated the apoptotic markers. Conclusion: Our findings suggest that GDC-0941, either alone or in combination with rapamycin, may serve as a new, promising approach for breast cancer treatment.

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

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. 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. 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. GDC-0941 augments the efficacy of docetaxel by increasing drug-induced apoptosis in breast cancer models.

GDC-0941 sensitizes breast cancer to ABT-737 in vitro and in vivo through promoting the degradation of Mcl-1

The present study showed that GDC-0941 potently sensitized breast cancer to ABT-737 in vitro and in vivo. ABT-737 exhibited limited lethality in breast cancer cells; however, when combined with GDC-0941, it displayed strong synergistic cytotoxicity and enhanced caspase-mediated apoptosis. GDC-0941 promoted proteasomal degradation of Mcl-1, of which the overexpression has been validated to confer ABT-737 resistance, thereby enhanced the anticancer efficacy of ABT-737. Furthermore, the combination of GDC-0941 and ABT-737 exerted increased anti-tumor efficacy on MDA-MB-231 xenograft models. Overall, our data described unprecedentedly the promising therapeutic potential and underlying mechanisms of combining GDC-0941 with ABT-737 in treating breast cancer.

A phase Ib study to evaluate the PI3-kinase inhibitor GDC-0941 with paclitaxel (P) and carboplatin (C), with and without bevacizumab (BEV), in patients with advanced non-small cell lung cancer (NSCLC).

3044 Background: Genetic alterations affecting the PI3K pathway are frequently observed in NSCLC (eg. PTEN loss, PIK3CA gene amplification). In preclinical tumor models GDC-0941 is synergistic with chemotherapy and increases the efficacy of anti-VEGF when dosed concurrently. This study sought to determine whether GDC-0941 can be safely combined with P + C +/- BEV. METHODS: In Arm A, BEV-ineligible patients (pts) with advanced NSCLC and 0 or 1 prior chemotherapy regimen received increasing GDC-0941 doses (3 + 3 design) with P (200 mg/m(2)) and C (AUC 6 mg/mL·min) every 3 weeks. BEV-eligible pts in Arm B received BEV (15 mg/kg) in addition to P and C. In both arms, GDC-0941 was given once daily on Days 1-14 of a 21-day cycle. P and C were given up to 6 cycles, while GDC-0941 (with or without BEV) was given to progression or toxicity. Study objectives were to evaluate safety and pharmacokinetics (PK), and to determine the maximum tolerated dose (MTD) of GDC-0941 in both arms. RESULTS: As of 9 January 2011, 18 pts were enrolled in GDC-0941 cohorts of 60, 100, and 165 mg (Arm A) and GDC-0941 cohorts of 100, 165 and 250 mg (Arm B). Eleven of 18 pts were active. Treatment-related adverse events (AEs) in ≥20% of pts were alopecia, asthenia, nausea, stomatitis, neutropenia, decreased appetite, paresthesia, epistaxis, arthralgia, peripheral neuropathy and rash. All treatment-related AEs were Grade 1 and 2 except for neutropenia. Grade 3 (16%) and 4 (11%) neutropenia events were not dose-limiting. PK characteristics of GDC-0941, P, 6-OH-P and C were similar to historical single-agent profiles. Based on preclinical efficacy, target exposures were achieved in the 250 mg cohort. Dose escalation will continue to a maximum of 400 mg. Confirmed partial responses (PR) were seen in 8 of 18 (44%) patients, including 1 squamous NSCLC pt with a pathologic complete response upon resection of the primary lung lesion (Arm A, 165 mg GDC-0941). Median follow up at the time of data cut-off was 5.4 months (range 0.6-10.3 m). CONCLUSIONS: The combination of GDC-0941, P and C (+/- BEV) was well tolerated. The combination MTD has not yet been reached. Antitumor activity was observed.

Abstract 1: Mcl-1 downregulation plays a crucial role in the synergistic anticancer activities between PI-3 kinase inhibitors and histone deacetylase inhibitors in primary NSCLC tumors in vitro and in vivo

Abstract Interactions between the PI-3 kinase inhibitors and histone deacetylase inhibitors (HDACi) were examined in vivo using SCID mice bearing patient-derived primary NSCLC xenografts which contain the “hot spot” mutation PIK3CA [E545K (G1633A)] (K-Ras and EGFR wild-type), and in vitro using the primary cell cultures derived directly from the in vivo xenografts. Co-treatment of primary cell cultures to marginally toxic concentrations of GDC-0941 (1.0 uM) and HDACi LBH589 (2.5 nM) induced a striking increase of apoptosis, as evidenced by Annex V positive staining, cytochrome c release and caspase 3 cleavage. Similar effects were observed using other HDACis (e.g. SAHA or MS275). The combination was associated with profound Akt inactivation, GSK-3β activation and Mcl-1 attenuation without affecting other Bcl-2 family members. Although LBH589 induced p21CIP1/WAF1 expression, GDC-0941 blocked LBH589-mediated p21CIP1/WAF1 accumulation. Ectopic expression of the constitutively active (myristolated) Akt, dominant negative GSK-3β (K85A), Mcl-1, or p21CIP1/WAF1 significantly blocked apoptosis induced by this combination. Inactivation of GSK-3β by either shRNA knockdown or its pharmacological inhibitor markedly diminished GDC-0941/LBH589-induced cell death, and abrogated the Mcl-1 and p21CIP1/WAF1 downregulation seen with the combination, indicating that GSK-3β modulation is the process upstream of Mcl-1 and p21. However, overexpression of either Mcl-1 or p21CIP1/WAF1 did not demonstrate an interaction with each other under the co-treatment, suggesting an independent functional cascade for each Mcl-1 and p21CIP1/WAF1. The downregulation of Mcl-1 and p21CIP1/WAF1 was reversed by proteasome inhibitor, indicating a proteasome-dependent degradation of Mcl-1 and p21CIP1/WAF1. Lastly, the combination of GDC-0941/LBH589 in vivo inhibited the growth of primary tumor xenografts more efficiently than either agent alone. In vivo downregulation of Mcl-1 and p21CIP1/WAF1 was also observed, consistent with the modulation of molecular events presented in vitro. Taken together, our results demonstrate that GDC-0941 potentiates HDACi-mediated apoptosis through PI3K/Akt/GSK-3β pathway, and subsequently attenuates Mcl-1 and abrogates p21CIP1/WAF1 induction in primary NSCLC xenografts in vivo and the primary cell cultures in vitro, thereby suggesting a novel therapeutic approach for NSCLC. 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 1. doi:10.1158/1538-7445.AM2011-1

Activity of GDC-0941, an inhibitor of phosphoinositol 3 kinase (PI3K), in gastrointestinal stromal tumor (GIST) xenograft and duration of response after discontinuation of treatment in combination with imatinib.

10020 Background: Oncogenic signaling in GISTs is sustained mainly via PI3K/AKT rather than RAS/MAPK pathway. We tested the efficacy of GDC-0941 (GDC) alone and in combination with imatinib (IM) in IM-sensitive GIST xenograft. Methods: Nude mice were grafted bilaterally with human GIST bearing KIT exon 11 mutation. They were orally dosed over 19 days in 4 groups: A (control); B (GDC-0941 75mg/kg/d); C (IM 50mg/kg/bid); D (GDC+IM, doses/schedule=B+C). In addition, xenografts re-growth after treatment withdrawal was evaluated. Micro-FDG-PET assessed standardized uptake values (SUV) and tumor/lesion glycolysis (TLG) over 8 weeks. Histopathology included H&E, KIT and Ki-67 staining. KIT signaling status was assessed by Western blot. Results: IM and GDC+IM reduced tumor volume by 86% and 95%, respectively, while GDC had no significant effect. IM and GDC treatments showed only grade 1 or grade 1&2 histologic response (HR), while grade 3 HR was observed in 75% of tumors under GDC+IM. Compared to control, mitotic activity decreased 2-fold under GDC, while it was not detectable in the other arms. Apoptotic activity was respectively 3- and 15-fold higher than control under IM and GDC+IM, being almost unaffected under GDC alone. After 24 hrs, SUV decreased in all treated groups, and remained low particularly under IM. The highest reduction in TLG was observed under GDC+IM. In GDC and IM groups, inhibition of AKT was visible but incomplete. Only GDC+IM induced complete PI3K/AKT pathway knockdown. After treatment discontinuation, immediate tumor re-growth was observed in IM group, whereas GDC+IM treatment yielded long-lasting volumetric effect (98% and 15% of baseline, respectively, after 4 weeks of treatment end). SUV and TLG increased constantly under IM, whereas same parameters remained low in GDC+IM. Mitotic index in GDC+IM was still 2-fold lower than in controls. Conclusions: The combination of GDC-0941 and imatinib has extensive antitumor efficacy in GIST xenograft, inducing more substantial apoptosis and durable effects than imatinib alone. This effect was sustained even after treatment withdrawal. No significant financial relationships to disclose.

Abstract 5399: Growth inhibition, cytotoxicity and signaling studies of PI3K & MEK inhibitor combinations in colorectal carcinoma cell lines

Abstract MAPK and PI3K signalling pathways are frequently activated in human cancers, and represent promising therapeutic targets. Previous studies suggest that combined targeting of these pathways may be necessary for optimal therapeutic activity, hence the aim of this study was to evaluate the MEK inhibitors, ARRY-142886 and PD 0325901, alone and in combination with the dual mTOR/PI3K inhibitor, NVP-BEZ235, or the pan class I PI3K inhibitor, GDC-0941, in colorectal cancer cell lines. Growth inhibition, survival and signal transduction were measured using the Sulforhodamine B assay, clonogenicity and western blotting, respectively. Median effect analysis revealed that all MEK/PI3K inhibitor combinations exhibited marked synergistic growth inhibition in both HCT116 and HT29 cell lines. GDC-0941 displayed the greatest synergy in combination with either MEK inhibitor in the HCT116 cell line, and preliminary results suggest that this is also the case in the HT29 cell line. At concentrations up to 10 µM only NVP-BEZ235 was cytotoxic after 72 hours exposure in either colorectal cancer cell line with an LC50 of 0.51 µM in the HCT116 cell line and an LC50 of 0.44 µM in the HT29 cell line. However, no increase in cytotoxicity was observed with MEK/PI3K inhibitor combinations in the HCT116 cell line, and studies in the HT29 cell line are ongoing. Western blotting using HCT116 cell lysates revealed that NVP-BEZ235 exhibits stronger inhibition of AKT and 4EBP1 phosphorylation, and similar inhibition of S6 phosphorylation, compared to GDC-0941. Both PD 0325901 and ARRY-142886 inhibited ERK phosphorylation. However, no clear or consistent additional effect on S6, ERK or 4EBP1 phosphorylation was observed when the MEK and PI3K inhibitors were combined. These studies confirm that MEK and PI3K inhibitors are predominantly cytostatic, as opposed to cytotoxic, when used alone or in combination. The dual mTOR/PI3K inhibitory action of NVP-BEZ235 may increase its ability to inhibit 4EBP1 phosphorylation, and thereby protein translation. Furthermore, the lower level of synergy exhibited by NVP-BEZ235 in combination with MEK inhibitors, compared to GDC-0941, may be due to the inhibition of mTOR and thereby 4EBP1 phosphorylation. Hence studies have commenced using the mTOR inhibitor, rapamycin, in combination with GDC-0941. In the HCT116 cell line, preliminary results suggest that the combination of GDC-0941 and rapamycin is considerably more growth inhibitory than either compound alone. These studies confirm that dual targeting of PI3K and MEK can induce synergistic growth inhibition; however, the detailed effects of specific inhibitors should be investigated to identify optimal combinations. The research was funded by grants from the Medical Research Council UK and UCB Celltech Ltd. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 5399.