Phosphatidylinositol 3-kinase Inhibitor GDC-0941 Research Articles

PCC0208018 exerts antitumor effects by activating effector T cells.

Poor prognosis is associated with melanoma due to immunosuppression profiles, suggesting that immune alterations have an important role in the occurrence, growth, and metastasis of melanoma. Here, we found that PCC0208018, a small-molecule compound, enhanced T cell proliferation and activation to release interferon gamma (IFN-γ) and interleukin-2 (IL-2) without blocking the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) binding and did not directly affect tumor cell viability in vitro. Furthermore, PCC0208018 increased the phosphorylation of protein kinase B (PKB/AKT) as well as extracellular regulated protein kinases (ERK) in human peripheral blood mononuclear cells (PBMCs) in vitro. The secretion of cytokines induced by PCC0208018 was significantly suppressed by the PI3K inhibitor GDC-0941. In B16-F10 melanoma-harboring mice, PCC0208018 significantly inhibited tumor growth as well as increasing CD3+, CD3+CD4+, and CD3+CD8+ T cell abundance in tumors without affecting PD-L1 expression. This study showed that PCC0208018 potentially increased PBMCs proliferation and function by activating the phosphatidylinositol 3 kinase (PI3K)/AKT and mitogen-activated protein kinase (MEK)/ERK pathways to exert antitumor effects.

Abstract 3149: Identification of genes associated with the cisplatin resistance in cervical cancer cells expressing E545K mutation

Abstract The phosphatidylinositol-3 kinase (PI3K)/AKT/ mTOR signaling pathway is activated in several human cancers and activation is frequently mediated by “hotspot” mutations including E542K, E545K and H1047R in the PIK3CA gene. Approximately 30% of cervical cancer patients have the PIK3CA-E545K mutation. Cisplatin with radiotherapy (RT) is the standard treatment of cervical cancer world-wide, used in both the radical and post-operative adjuvant settings. However, details of the molecular mechanisms responsible for cisplatin resistance remain unclear. We previously reported PIK3CA mutation in patients with earlier stage (IB/II) cervical cancer was associated with poor survival (McIntyre et al. Gynecol Oncol. 2013, PMID:23266353), and in our recent study we observed that PIK3CA-E545K mutation renders cervical cancer cells more resistant to cisplatin or cisplatin plus RT and results in a more migratory phenotype than isogenic cell lines with wild type-PIK3CA. Moreover, these phenotypes are reversed by the PI3K inhibitor GDC-0941/Pictilisib (Wani et al. Oncotarget. 2016, PMID:27489350). The aim of the present study is to identify the expression of genes related to cisplatin resistance in cervical cancer cells engineered to express PIK3CA-E545K. Microarray analysis identified 161 genes that were up-regulated and 189 that were down-regulated in the cervical cancer cells stably expressing PIK3CA-E545K, some of which are involved in well-characterized mechanisms that could be relevant to cisplatin resistance. We are currently validating some of those genes by Real-time PCR that will help us to determine the mechanism of PIK3CA-E545K induced cisplatin resistance and enhanced migration in cervical cancer cells expressing PIK3CA-E545K and extending our in vitro findings to animal models. Together, our data will provide a useful basis for screening candidate targets for risk stratification and provide valuable information for potential targeted intervention in patients whose tumors harbor cisplatin-resistant molecular characteristics. Citation Format: Wani Arjumand, Nicholas Jette, Jb McIntyre, Prafull Ghatage, Corinne M. Doll, Susan P. Lees-Miller. Identification of genes associated with the cisplatin resistance in cervical cancer cells expressing E545K mutation [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 3149. doi:10.1158/1538-7445.AM2017-3149

Phosphatidyl inositol-3 kinase (PIK3CA) E545K mutation confers cisplatin resistance and a migratory phenotype in cervical cancer cells.

The phosphatidylinositol-3 kinase (PI3K)/Akt/mTOR signaling pathway is activated in many human cancers. Previously, we reported that patients with early stage cervical cancer whose tumours harbour PIK3CA exon 9 or 20 mutations have worse overall survival in response to treatment with radiation and cisplatin than patients with wild-type PIK3CA. The purpose of this study was to determine whether PIK3CA-E545K mutation renders cervical cancer cells more resistant to cisplatin and/or radiation, and whether PI3K inhibition reverses the phenotype. We found that CaSki cells that are heterozygous for the PIK3CA-E545K mutation are more resistant to cisplatin or cisplatin plus radiation than either HeLa or SiHa cells that express only wild-type PIK3CA. Similarly, HeLa cells engineered to stably express PIK3CA-E545K were more resistant to cisplatin or cisplatin plus radiation than cells expressing only wild-type PIK3CA or with PIK3CA depleted. Cells expressing the PIK3CA-E545K mutation also had constitutive PI3K pathway activation and increased cellular migration and each of these phenotypes was reversed by treatment with the PI3K inhibitor GDC-0941/Pictilisib. Our results suggests that cervical cancer patients whose tumours are positive for the PIK3CA-E545K mutation may benefit from PI3K inhibitor therapy in concert with standard cisplatin and radiation therapy.

Strategically Timing Inhibition of Phosphatidylinositol 3-Kinase to Maximize Therapeutic Index in Estrogen Receptor Alpha-Positive, PIK3CA-Mutant Breast Cancer.

Phosphatidylinositol 3-kinase (PI3K) inhibitors are being developed for the treatment of estrogen receptor α (ER)-positive breast cancer in combination with antiestrogens. Understanding the temporal response and pharmacodynamic effects of PI3K inhibition in ER(+) breast cancer will provide a rationale for treatment scheduling to maximize therapeutic index. Antiestrogen-sensitive and antiestrogen-resistant ER(+) human breast cancer cell lines and mice bearing PIK3CA-mutant xenografts were treated with the antiestrogen fulvestrant, the PI3K inhibitor GDC-0941 (pictilisib; varied doses/schedules that provided similar amounts of drug each week), or combinations. Cell viability, signaling pathway inhibition, proliferation, apoptosis, tumor volume, and GDC-0941 concentrations in plasma and tumors were temporally measured. Treatment with the combination of fulvestrant and GDC-0941, regardless of dose/schedule, was significantly more effective than that with single-agent treatments in fulvestrant-resistant tumors. Short-term, complete PI3K inhibition blocked cell growth in vitro more effectively than chronic, incomplete inhibition. Longer-term PI3K inhibition hypersensitized cells to growth factor signaling upon drug withdrawal. Different schedules of GDC-0941 elicited similar tumor responses. While weekly high-dose GDC-0941 with fulvestrant continuously suppressed PI3K signaling for 72 hours, inducing a bolus of apoptosis and inhibiting proliferation, PI3K reactivation upon GDC-0941 washout induced a proliferative burst. Fulvestrant with daily low-dose GDC-0941 metronomically suppressed PI3K for 6 to 9 hours/day, repeatedly inducing small amounts of apoptosis and temporarily inhibiting proliferation, followed by proliferative rebound compared with fulvestrant alone. Continuous and metronomic PI3K inhibition elicits robust anticancer effects in ER(+), PIK3CA-mutant breast cancer. Clinical exploration of alternate treatment schedules of PI3K inhibitors with antiestrogens is warranted. Clin Cancer Res; 22(9); 2250-60. ©2016 AACRSee related commentary by Toska and Baselga, p. 2099.

Abstract A119: Investigating PIK3CA mutational status on cisplatin and radiation sensitivity in cervical cancer cell lines: Implications for PI3K inhibitor therapy

Abstract The phosphatidylinositol-3 kinase (PI3K)/AKT/ mTOR signaling pathway is activated in many different human cancers. Activation is frequently mediated by “hotspot” mutations including E542K, E545K and H1047R in the PIK3CA gene, which encodes the catalytic subunit of PI3Kα. We previously reported PIK3CA mutation in patients with early stage (IB/II) cervical cancer was associated with poor survival (McIntyre et al. Gynecol Oncol. 2013, 128(3): 409-14). The purpose of this study was to determine whether PIK3CA mutation renders cervical cancer cells more resistant to conventional therapy (cisplatin chemotherapy and/or radiation), and whether PI3K inhibition enhances cell kill and/or reverse this phenotype. Here, we report that the cervical cancer cell line CaSki, which expresses the PIK3CA-E545K mutation, is more resistant to cisplatin than cervical cancer cells with wild-type PIK3CA. To validate our findings, we depleted endogenous PIK3CA from HeLa cells using shRNA and stably expressed either shRNA resistant wild-type PIK3CA or PIK3CA-E545K. Cells expressing PIK3CA-E545K were more resistant to cisplatin and cisplatin plus ionizing radiation than cells expressing either wild-type PIK3CA or lacking PIK3CA. Preliminary results also suggest that cells expressing PIK3CA-E545K have an enhanced migratory phenotype. We are currently testing whether treatment with the PI3K inhibitor GDC-0941 restores sensitivity to cisplatin and reverts this aggressive phenotype. Together, these experiments will determine whether inhibition of the PI3K pathway in cervical cancers with PIK3CA activating mutation has potential for therapeutic benefit. Citation Format: ARJUMAND WANI, COLE MERRY, CHEN WANG, SHUJUAN FANG, ELIZABETH KORNAGA, Jb MCINTYRE, PRAFULL GHATAGE, MARTIN KöBEL, CORINNE M. DOLL, SUSAN P. LEES-MILLER. Investigating PIK3CA mutational status on cisplatin and radiation sensitivity in cervical cancer cell lines: Implications for PI3K inhibitor therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A119.

Ellagic Acid Enhances the Efficacy of PI3K Inhibitor GDC-0941 in Breast Cancer Cells.

The fact that the phosphatidylinositol 3 kinase (PI3K) signaling pathway is one of the most frequently deregulated signaling networks has triggered intensive efforts in the development of PI3K pathway inhibitors. However, recent clinical trial data have shown only limited activity of PI3K inhibitors at tolerated doses. Thus, there is an urgent need to identify rational combination therapy to improve the efficacy of PI3K-targeted cancer treatment. In this study, we investigated if dietary compound ellagic acid (EA) could improve the therapeutic efficacy of PI3K inhibitor GDC-0941 in breast cancer. Specifically, using a panel of breast cancer cell lines, we showed that combined use of EA and GDC-0941 significantly inhibited cell growth under attached and detached conditions, blocked migration and invasion in vitro as well as tumor initiation and metastasis in vivo. Furthermore, we found that EA promoted apoptosis and further reduced AKT/mTOR activation in GDC-0941- treated breast cancer cells. Together, our data suggest that EA may be a safe and effective agent to boost the efficacy of PI3K-directed breast cancer therapy and that such drug combination may merit further clinical investigation.

Preclinical efficacy of MEK inhibition in Nras-mutant AML

AbstractOncogenic NRAS mutations are highly prevalent in acute myeloid leukemia (AML). Genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis, but have limited independent prognostic significance. Using short hairpin RNA–mediated knockdown in human cell lines and primary mouse leukemias, we show that AML cells with NRAS/Nras mutations are dependent on continued oncogene expression in vitro and in vivo. Using the Mx1-Cre transgene to inactivate a conditional mutant Nras allele, we analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPCs) under normal and stressed conditions and found that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Treating recipient mice transplanted with primary NrasG12D AMLs with 2 potent allosteric mitogen-activated protein kinase kinase (MEK) inhibitors (PD0325901 or trametinib/GlaxoSmithKline 1120212) significantly prolonged survival and reduced proliferation but did not induce apoptosis, promote differentiation, or drive clonal evolution. The phosphatidylinositol 3-kinase inhibitor GDC-0941 was ineffective as a single agent and did not augment the activity of PD0325901. All mice ultimately succumbed to progressive leukemia. Together, these data validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors.

Preclinical Efficacy of MEK Inhibition in Nras Mutant Acute Myeloid Leukemia

Oncogenic NRAS mutations are highly prevalent in hematologic malignancies. In acute myeloid leukemia (AML), genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis. Furthermore, NRAS mutations identified at diagnosis may disappear at relapse, raising questions regarding the potential clinical benefits of inhibiting oncogenic N-Ras in AML. To directly investigate the consequences of Nras inactivation in normal hematopoiesis, we used the Mx1-Cre transgene to inactivate a conditional mutant Nras allele and analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPC) under normal and stressed conditions. We show that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Importantly, shRNA-mediated knockdown in human AML cell lines and primary mouse leukemias with oncogenic NRAS/Nras mutations revealed dependence on continued oncogene expression in vitro and in vivo. Next, we interrogated the functional consequences of pharmacologic inhibition of the canonical Ras effector pathways, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways,alone and in combination. Recipient mice transplanted with five independent primary mouse AMLs generated by infecting NrasG12D “knock in” mice with the MOL4070LTR retrovirus (Li et al, Blood 2011; 117:2022) were treated with the allosteric MEK inhibitors PD0325901 (PD901) or trametinib or the PI3K inhibitor GDC-0941. Both MEK inhibitors significantly prolonged survival and reduced proliferation and blast colony formation, but did not induce apoptosis, differentiation, or promote clonal evolution. PI3K inhibition alone was ineffective in vivo and combinations of MEK and PI3K inhibitors were no better than MEK inhibition alone. All mice ultimately succumbed from progressive leukemia. These data, along with observations that Nras is dispensable for normal hematopoiesis, validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors in leukemias harboring NRAS mutations. DisclosuresNo relevant conflicts of interest to declare.

A Genetic Mouse Model of Invasive Endometrial Cancer Driven by Concurrent Loss of Pten and Lkb1 Is Highly Responsive to mTOR Inhibition

Signals from the tumor suppressors PTEN and LKB1 converge on mTOR to negatively regulate its function in cancer cells. Notably, both of these suppressors are attenuated in a significant fraction of human endometrial tumors. In this study, we generated a genetic mouse model of endometrial cancer driven by concomitant loss of these suppressors to gain pathophysiological insight into this disease. Dual loss of Pten and Lkb1 in the endometrial epithelium led to rapid development of advanced endometrioid endometrial tumors with 100% penetrance and short host survival. The tumors displayed dysregulated phosphatidylinositol 3-kinase (PI3K)/Akt and Lkb1/Ampk signaling with hyperactivation of mTOR signaling. Treatment with a dual PI3K/mTOR inhibitor, BEZ235, extended the time before tumor onset and prolonged overall survival. The PI3K inhibitor GDC-0941 used as a single agent reduced the growth rate of primary tumor implants in Pten/Lkb1-deficient mice, and the mTOR inhibitor RAD001 was unexpectedly as effective as BEZ235 in triggering tumor regression. In parallel, we also found that ectopic expression of LKB1 in PTEN/LKB1-deficient human endometrial cancer cells increased their sensitivity to PI3K inhibition. Together, our results demonstrated that Pten/Lkb1-deficient endometrial tumors rely strongly on deregulated mTOR signaling, and they provided evidence that LKB1 status may modulate the response of PTEN-deficient tumors to PI3K or mTOR inhibitors.

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

BackgroundThe phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients.MethodsWe conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application.ResultsThe phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells.ConclusionsWe developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

The PI3K inhibitor GDC-0941 combines with existing clinical regimens for superior activity in multiple myeloma

The phosphatidylinositol 3'-kinase (PI3K) pathway is dysregulated in multiple myeloma (MM); we therefore tested a highly selective class I PI3K inhibitor, GDC-0941, for anti-myeloma activity. Functional and mechanistic studies were first performed in MM cell lines, then extended to primary MM patient samples cultured in vitro. GDC-0941 was then assessed as a single agent and in various combinations in myeloma tumor xenograft models. We show p110 α and β are the predominant PI3K catalytic subunits in MM and that a highly selective class I PI3K inhibitor, GDC-0941, has robust activity as a single agent to induce cell cycle arrest and apoptosis of both MM cell lines and patient myeloma cells. Mechanistic studies revealed an induction of cell cycle arrest at G0/G1, with decreased phospho-FoxO1/3a levels, decreased cyclin D1 and c-myc expression, and an increase in the cell cycle inhibitor, p27kip. Induction of apoptosis correlated with increased expression of the pro-apoptotic BH3-only protein BIM, cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP). In vitro, GDC-0941 synergized with dexamethasone (Dex) and lenalidomide (combination index values of 0.3-0.4 and 0.4-0.8, respectively); in vivo GDC-0941 has anti-myeloma activity and significantly increases the activity of the standard of care agents in several murine xenograft tumor models (additional tumor growth inhibition of 37-53% (Dex) and 22-72% (lenalidomide)). These data provide a clear therapeutic hypothesis for the inhibition of PI3K and provide a rationale for clinical development of GDC-0941 in myeloma.

GDC-0941 enhances the lysosomal compartment via TFEB and primes glioblastoma cells to lysosomal membrane permeabilization and cell death

Since phosphatidylinositol-3-kinase (PI3K) inhibitors are primarily cytostatic against glioblastoma, we searched for new drug combinations. Here, we discover that the PI3K inhibitor GDC-0941 acts in concert with the natural compound B10, a glycosylated derivative of betulinic acid, to induce cell death in glioblastoma cells. Importantly, parallel experiments in primary glioblastoma cultures similarly show that GDC-0941 and B10 cooperate to trigger cell death, underscoring the clinical relevance of this finding. Molecular studies revealed that treatment with GDC-0941 stimulates the expression and nuclear translocation of Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, the lysosomal membrane marker LAMP-1 and the mature form of cathepsin B. Also, GDC-0941 triggers a time-dependent increase of the lysosomal compartment in a TFEB-dependent manner, since knockdown of TFEB significantly reduces this GDC-0941-stimulated lysosomal enhancement. Importantly, GDC-0941 cooperates with B10 to trigger lysosomal membrane permeabilization, leading to increased activation of Bax, loss of mitochondrial membrane potential (MMP), caspase-3 activation and cell death. Addition of the cathepsin B inhibitor CA-074me reduces Bax activation, loss of MMP, caspase-3 activation and cell death upon treatment with GDC-0941/B10. By comparison, knockdown of caspase-3 or the broad-range caspase inhibitor zVAD.fmk inhibits GDC-0941/B10-induced DNA fragmentation, but does not prevent cell death, thus pointing to both caspase-dependent and -independent pathways. By identifying the combination of GDC-0941 and B10 as a new, potent strategy to trigger cell death in glioblastoma cells, our findings have important implications for the development of novel treatment approaches for glioblastoma.

Growth-Inhibitory Effects of a Class I PI3K Inhibitor GDC-0941 Are Modulated by Hypoxia-Induced mTOR Pathway Activation in Mantle Cell Lymphoma

Abstract 1424Hypoxia confers pro-survival signals to tumor cells via multiple mechanisms including activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. Constitutive activation of PI3K/Akt signaling pathway is documented in aggressive blastoid variants of mantle cell lymphoma (MCL) and is implicated in the pathogenesis of MCL. mTOR signaling, one of the important downstream targets of Akt, positively regulates protein translation by phosphorylating p70 ribosomal S6 kinase (S6K) or eukaryotic initiation factor 4E-binding protein-1 (4E-BP1). We have reported that the Class IA PI3K isoforms, mainly p110α and partially p110δ, are responsible for PI3K/Akt/mTOR signaling activation in blastoid MCL cells (Zhou et al., ASH abstract 2010). In this study, the activity and molecular mechanisms of action of a selective pan-isoform class I PI3K inhibitor GDC-0941 (Genentech) were investigated in the context of hypoxic microenvironment in MCL cells. For hypoxia experiments, MCL cells were cultured under 1% O2 for at least 14 days to assure their sustained proliferation and survival. Under hypoxia, more cells accumulated in G0/G1 phase, indicating that hypoxic conditions promote cell cycle quiescence in MCL cells. GDC-0941 treatment in normoxia resulted in a reduction of cell proliferation in a dose-dependent manner (IC50 at 48 hrs; 0.7 μM for Granta519, 0.5 μM for JVM2, 1.0 μM for MINO, 0.7 μM for Jeko-1, MTT test). In hypoxia, higher doses of GDC-0941 were required to cause cell growth inhibition compared to normoxic conditions in Granta519 (IC50; 1.7 μM), JVM2 (3.5 μM) and MINO (1.2 μM). Further, GDC-0941 failed to inhibit growth of Jeko-1 cells under hypoxia. GDC-0941 (0.5 μM) induced G0/1 cell cycle arrest under both normoxic and hypoxic conditions in Granta519, JVM2 and MINO cells. No significant induction of apoptosis by GDC-0941 (0.5 μM) was observed in any of the tested MCL cells. GDC-0941 completely inhibited phosphorylated (p-Ser473) Akt in all cell lines analyzed under both normoxic and hypoxic conditions, which resulted in decreased expression of p-GSK-3β and its downstream target cyclin D1. GDC-0941 further downregulated the mTOR downstream targets p-S6K and p-4EBP1 in Granta519, JVM2 and MINO cells under both, normoxia and hypoxia. On the contrary, GDC-0941 failed to affect hypoxia-induced p-S6K and p-4EBP1 expression levels in Jeko-1 cells, whereas potently suppressed these proteins at ambient oxygen level. These results suggest that in Jeko-1 cells hypoxia activates mTOR through PI3K-independent pathways. To test this, we examined consequences of mTOR blockade in Jeko-1 cells. mTOR inhibitor rapamycin (100 nM) moderately diminished cell growth under normoxic conditions causing accumulation of cells in G0/1 cell cycle phase but no apoptosis. In turn, under hypoxia rapamycin profoundly inhibited cell growth, inducing >80% of MCL cells to undergo cell death.These findings indicate that in certain MCL cells hypoxia activates mTOR through alternative, PI3K-independent pathways, which facilitate cell survival under hypoxia. Hence, blockade of class I PI3K may eliminate circulating MCL cells but not tissue-resident cells in hypoxic areas of the bone marrow or lymph nodes. In turn, simultaneous therapeutic ablation of PI3K/Akt and mTOR pathway may represent a promising strategy to target the aggressive blastoid variants of MCL cells in the hypoxic microenvironments. Disclosures:No relevant conflicts of interest to declare.

Preclinical pharmacokinetics of the novel PI3K inhibitor GDC-0941 and prediction of its pharmacokinetics and efficacy in human

The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregulation is associated with the development of many cancers.GDC-0941, a potent and selective inhibitor of PI3K, was characterised preclinically in in vitro and in vivo studies.Plasma protein binding was extensive, with free fraction less than 7%, and blood-to-plasma ratio ranged from 0.6 to 1.2 among the species tested. GDC-0941 human hepatic clearance was predicted to be moderate by liver microsomal incubations. GDC-0941 had high permeability in Madin-Darby canine kidney cells.The clearance of GDC-0941 was high in mouse (63.7 mL/min/kg), rat (49.3 mL/min/kg) and cynomolgus monkey (58.6 mL/min/kg), and moderate in dog (11.9 mL/min/kg). The volume of distribution ranged from 2.52 L/kg in rat to 2.94 L/kg in monkey. Oral bioavailability ranged from 18.6% in monkey to 77.9% in mouse.Predicted human clearance and volume of distribution using allometry were 6 mL/min/kg and 2.9 L/kg, respectively. The human efficacious doses were predicted based on results from preclinical pharmacokinetic studies and xenograft models.GDC-0941 preclinical characterisation and predictions of its properties in human supported its progression towards clinical development. GDC-0941 is currently in phase II clinical trials.

Abstract 3140: Analysis of candidate phosphatidylinositol 3’ kinase inhibitor biomarkers in matched synchronous primary and metastatic breast cancer tumors

Abstract The class I phosphatidylinositol 3’kinase (PI3K) is activated in a wide variety of human malignancies and inhibitors targeting the PI3K pathway hold great promise in the treatment and management of cancer. Successful development of such inhibitors will be enhanced by identification of responsive patients through the use of predictive biomarkers. Using cell lines and xenograft tumor models to evaluate a collection of putative biomarkers predictive of response to the selective inhibitor GDC-0941 in breast cancer, we have found excellent activity of the PI3K inhibitor GDC-0941 in luminal and HER2 amplified models, and that PIK3CA mutations and HER2 amplification are highly specific biomarkers of response to this agent. PTEN was predictive in some but not all cases, suggesting a need for further biomarkers of response in PTEN-driven disease. Here we report expanded analysis of human breast tumor samples, including matched primary tumor and lymph node metastases from 59 patients, suggesting primary and metastatic sites were generally concordant in terms of status of key biomarkers such as PTEN and PIK3CA, though several notable exceptions were discovered. We also compared PTEN status determined by immunohistochemistry (IHC) with a chromosomal fluorescence in situ hybridization (FISH) assay in a subset of tumors and found evidence for chromosomal deletion of the PTEN locus in several ER+ breast cancers, suggesting that some but not all of the PTEN loss observed in breast occurs at the chromosomal level and that both FISH and IHC may have utility as diagnostic assays. While we report that PIK3CA mutations and PTEN loss have reasonable prevalence to be considered as biomarkers for GDC-0941 in ER+ breast cancer, we have found a number of breast cancer models with no apparent pathway alterations but sensitivity to GDC-0941, indicating the need for additional predictive biomarkers. We identified a collection of genes for which expression levels indicate PI3K pathway activation and correlate with in vitro sensitivity to GDC-0941 as well as apparent prognostic significance in retrospective analysis of breast tumors. We report here expression analysis of these genes across matched primary breast tumors and lymph node metastases. In summary, our data provide strong rationale for developing a selective PI3K inhibitor in the ER+ breast cancer setting, and a framework for clinical implementation of predictive biomarker assays with potential utility in patient selection. 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 3140. doi:10.1158/1538-7445.AM2011-3140

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

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. 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. 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. 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.

Role of P-Glycoprotein and Breast Cancer Resistance Protein-1 in the Brain Penetration and Brain Pharmacodynamic Activity of the Novel Phosphatidylinositol 3-Kinase Inhibitor GDC-0941

2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) is a novel small molecule inhibitor of the phosphatidylinositol 3-kinase (PI3K) pathway currently evaluated in the clinic as an anticancer agent. The objectives of this study were to determine in vitro whether GDC-0941 was a substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp1) and to investigate the impact of these transporters on the pharmacokinetics, brain penetration, and activity of GDC-0941 in FVBn mice (wild-type) and Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)/Bcrp1(-/-) knockout mice. Studies with Madin-Darby canine kidney cells transfected with P-gp or Bcrp1 established that this compound was a substrate of both transporters. After administrations to mice, GDC-0941 brain-to-plasma ratio ranged from 0.02 to 0.06 in the wild-type and Bcrp1(-/-) mice and was modestly higher in the Mdr1a/b(-/-) mice, ranging from 0.08 to 0.11. In contrast, GDC-0941 brain-to-plasma ratio in Mdr1a/b(-/-)/Bcrp1(-/-) triple knockout mice was 30-fold higher than in the wild-type mice. The plasma clearance of GDC-0941 was similar in wild-type and all knockout mice, ranging from 15 to 25 ml/(min . kg) in the wild-type mice and from 18 to 35 ml/(min . kg) in the knockout mice. Exposure after oral administration was comparable in the four strains of mice. The PI3K pathway was markedly inhibited in the brain of Mdr1a/b(-/-)/Bcrp1(-/-) mice for up to 6 h postdose, as evidenced by a 60% suppression of the phosphorylated Akt signal, whereas no inhibition was detected in the brain of wild-type mice. The concerted effects of P-gp and Bcrp1 in restricting GDC-0941 access and pathway modulation in mouse brain may have implications for the treatment of patients with brain tumors.

The PI3K Inhibitor GDC-0941 Induces Growth Arrest and Apoptosis of Acute Myeloid Leukemia Cells.

Abstract 3787Poster Board III-723Acute myeloid leukemia (AML) is a biologically and molecularly heterogeneous malignancy characterized by an accumulation of myeloid-lineage cells in the blood and bone marrow. Phosphatidylinositol 3' kinase (PI3K) -mediated signaling is frequently dysregulated in cancer and controls fundamental cellular functions such as cell migration, growth, survival and development of drug resistance in many cancers, including AML, and therefore represents an attractive therapeutic target, even against the backdrop of AML disease heterogeneity. In AML, for example, activating mutations in Ras and Flt3 are common and a number of autocrine and paracrine signaling mechanisms have been proposed as important disease mediators; all converge on PI3K as an important signal transduction node to provide growth- and survival-promoting effects. Here, we demonstrate that PI3K p110-a,b,d catalytic subunits are all prevalent across a panel of ∼30 cell lines and that the majority of cell lines as well as primary patient samples show evidence of PI3K pathway activation. We demonstrate in vitro with both cell lines and patient cell isolates, that a potent and selective pan-isoform PI3K inhibitor, GDC-0941, modulates pharmacodynamic markers such as p-Akt, p-4E-BP1, and p-FOXO-3a, and that treated cells show a G0/G1 cell-cycle arrest. A strong induction of apoptosis is observed by annexin/PI staining with upregulation of cleaved caspases and PARP. Preliminary observations suggest significant induction of p27 and Bim as direct transcriptional targets of FOXO-3a, as well as downregulation of c-Myc levels; these targets may represent key mediators of observed cellular effects and are the subject of ongoing work. Additional studies are currently underway to identify clinical standard-of-care agents and new molecular entities (pipeline NMEs) that combine favorably with PI3k-inhibition in vitro, and the results of several in-vivo xenograft studies will also be presented. Disclosures:Sun:Genentech Inc.: Employment. Berry:Genentech: Employment, Patents & Royalties. Du:Genentech, Inc.: Employment, Equity Ownership. Ma:Genentech: Employment, Patents & Royalties. Shi:Genentech: Employment, Patents & Royalties. Medeiros:Genentech: Research Funding. Ebens:Genentech, Inc.: Employment, Equity Ownership, Patents & Royalties.

The PI3K Inhibitor GDC-0941 Synergizes with Standard of Care Therapies to Induce Growth Inhibition and Apoptosis of Multiple Myeloma Cells.

Abstract 3788Poster Board III-724Multiple myeloma (MM) is a malignancy characterized by clonal expansion and accumulation of long-lived plasma cells within the bone marrow. Phosphatidylinositol 3' kinase (PI3K) -mediated signaling is frequently dysregulated in cancer and controls fundamental cellular functions such as cell migration, growth, survival and development of drug resistance in many cancers, including MM, and therefore represents an attractive therapeutic target. Here, we demonstrate in vitro, that a potent and selective pan-isoform PI3Kinhibitor, GDC-0941, modulates the expected pharmacodynamic markers, inhibits cell-cycle progression and induces apoptosis; overcomes resistance to apoptosis in MM cells conferred by IGF-1 and IL-6; and is additive or synergistic with current standard of care drugs including dexamethasone, melphalan, lenolidamide and bortezomib. In cell lines we find sensitivity to GDC-0941 is positively correlated with pathway activation as determined by phospho-AKT-specific flow-cytometry and Western-blot analysis. Preliminary results indicate apoptosis of MM cells is correlated with increased expression of the proapoptotic BH3-only protein BIM; mechanisms of increased apoptosis in MM will be further explored and an update presented. We further extend these in vitro findings to show that GDC-0941 has activity as a single agent in vivo and combines well with standard of care agents in several murine xenograft models to delay tumor progression and prolong survival. Our results suggest that GDC-0941 may combine well with existing therapies, providing a framework for the clinical use of this agent, and a rational approach to improving the efficacy of myeloma treatment. Disclosures:Munugalavadla:Genentech: Employment, Patents & Royalties. Berry:Genentech: Employment, Patents & Royalties. Du:Genentech, Inc.: Employment, Equity Ownership. Mariathasan:Genentech: Employment, Patents & Royalties. Slaga:Genentech: Employment, Patents & Royalties. Sun:Genentech Inc.: Employment. Chesi:Genentech, Inc.: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Bergsagel:Genentech: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Ebens:Genentech, Inc.: Employment, Equity Ownership, Patents & Royalties.

High-Throughput Screening Compatible Cell-Based Assay for Interrogating Activated Notch Signaling

Aberrant Notch pathway function is associated with a wide array of developmental disorders, cancers, and neurodegenerative diseases. Thus, strategies to modulate Notch signaling may facilitate therapeutic intervention. Ligand binding to Notch receptors at the cell surface results in a series of cleavage events that release Notch intracellular domain (NICD) fragments that translocate to the nucleus where they function as transcriptional activators of downstream transcriptional programs. We have developed a cell-based assay that can be used to screen for modulators of NICD signaling by engineering HeLa cervical cancer cells with a Notch response element driving beta-lactamase (BLA) reporter gene expression along with a tetracycline-inducible NICD expression system. Induction of NICD expression leads to increased BLA reporter activity that can be knocked down using NICD-specific RNA interference as well as RNA interference against endogenous components of the NICD transactivation complex. Profiling of 19 known compounds in this assay identified several previously undescribed modulators of NICD signaling. The Wnt pathway inhibitor ICG-001 antagonized NICD signaling, whereas the histone deacetylase inhibitor suberoylanilide hydroxamic acid, the heat shock protein 90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin, and the dual phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin inhibitor PI-103 each further activated the NICD-driven reporter activity. The AKT inhibitor triciribine and the PI3K inhibitor GDC-0941 also resulted in the enhanced reporter activity, strongly implicating a role for the PI3K/AKT pathway in regulating NICD signaling. Together this cell-based assay system provides a sensitive, quantitative readout for NICD signaling that is amenable to high-throughput screening for NICD pathway modulators.