Selective PI3K Inhibitor Research Articles

Up-regulation of BSEP and MRP2 by Calculus Bovis administration in 17α-ethynylestradiol-induced cholestasis: Involvement of PI3K/Akt signaling pathway

Ethnopharmacology relevanceCalculus Bovis, also known as Niuhuang, is a rare traditional Chinese medicine that has been widely used in China for 2000 years in pharmacology for sedation, anti-spasm, relieving fever, diminishing inflammation and recovering gallbladder functions. Aim of the studyThis study aimed to investigate the choleretic potential and molecular responses in rats to Calculus Bovis (CB) administration after 17α-ethynylestradiol (EE)-induced cholestasis. Material and methodsCB (50 and 100mg/kg per day) was intragastrically (i. g.) given to experimental rats for five consecutive days in coadministration with EE (5mg/kg daily for five days, s.c.). The levels of serum biomarkers were determined biochemically. The histopathology of the liver tissue was evaluated. Expression of bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2) were studied by western blot and immunohistochemical assay. The expression of Akt and phospho-Akt (pAkt) were also measured by western blot. ResultsIn response to EE, CB treatment significantly prevented an increase in serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyltransferase (GGT) and total bilirubin (TBIL). CB treatment also repaired tissue lesions caused by EE. Western blots showed that EE significantly decreased the protein expression of BSEP and MRP2. EE also dramatically increased levels of pAkt and decreased levels of Akt. Compared to the EE group, CB treatment increased levels of hepatic BSEP and MRP2 while pAkt levels decreased and Akt levels increased. Immunohistochemistry also indicated that EE decreased the expression of BSEP and MRP2. LY294002 is a selective PI3K inhibitor and showed similar beneficial effects as CB. Decreased expression of BSEP and MRP2 caused by EE were also prevented by LY294002 treatment. ConclusionCalculus Bovis administration can alleviate liver injury and up-regulate the expression of BSEP and MRP2 in 17α-ethynylestradiol-induced cholestasis by a mechanism that may involve inhibiting the activated PI3K/Akt signaling pathway.

A phase II study of the PI3K inhibitor taselisib (GDC-0032) combined with fulvestrant (F) in patients (pts) with HER2-negative (HER2-), hormone receptor-positive (HR+) advanced breast cancer (BC).

520Background: The PI3K pathway is activated in HR+ BC, often via gain-of-function mutations in PIK3CA that occur in ~40% of HR+ BC. Taselisib is a potent and selective PI3K inhibitor, with greater selectivity against mutant PI3Kα isoforms than wild type (WT) PI3Kα. Phase Ib data demonstrated good tolerability and preliminary efficacy for taselisib + F in HR+ BC. Methods: This Phase II, open-label, single-arm study enrolled post-menopausal pts with HER2-, HR+ locally advanced or metastatic BC (mBC) who had progression or non-response to ≥ 1 prior endocrine therapy in adjuvant or mBC settings. Pts received taselisib (6 mg capsule PO qd) plus F (500 mg IM on Cycle 1 Day 1, Cycle 1 Day 15, then q4w Day 1 of each cycle) until progressive disease or unacceptable toxicity. PIK3CA mutation status was centrally confirmed retrospectively on archival tumor tissue by 'cobas' PIK3CA Mutation Test. Primary endpoints were objective response rate (RECIST version 1.1) and clinical benefit rate (CBR; confirmed CR and PR, ...

POSEIDON trial phase 1b results: Safety and preliminary efficacy of the isoform selective PI3K inhibitor taselisib (GDC-0032) combined with tamoxifen in hormone receptor (HR) positive, HER2-negative metastatic breast cancer (MBC) patients (pts) - including response monitoring by plasma circulating tumor (ct) DNA.

2520Background: Endocrine therapy resistance remains a major challenge in the treatment of HR-positive breast cancer. The strategy of combining PI3K-AKT-mTOR pathway inhibitors with endocrine therapy can improve clinical outcomes (eg. BOLERO-2, BELLE-2) but at the cost of increased toxicity. Taselisib is a potent, selective, beta-isoform sparing inhibitor of PI3K designed to have a better therapeutic index than first-generation pan-PI3K inhibitors. Methods: Pts with prior exposure to endocrine therapy were enrolled using a 3+3 design, testing taselisib in combination with tamoxifen 20mg qd. A (21 day on / 7 day off) intermittent schedule was also compared with daily continuous dosing (28/28). The primary objective was to determine the recommended phase II dose and schedule. Other objectives included: pharmacokinetics (PK), pharmacodynamics (PD) and serial monitoring of plasma ctDNA using digital PCR / tagged amplicon deep-sequencing. Results: 30 pts were enrolled in 3 cohorts: taselisib 2mg (21/7), 4mg (2...

Novel anti-cancer agent myrtucommulone-A and thymoquinone abrogate epithelial-mesenchymal transition in cancer cells mainly through the inhibition of PI3K/AKT signalling axis.

Epithelial-mesenchymal transition (EMT) plays a prominent role in cancer progression and metastasis. Inhibition of EMT-associated regulators may hold a huge promise for cancer therapy. Although TGF-β signalling has a pivotal role in the induction of EMT, alterations during the EMT process are usually initiated and controlled by the cross-talk of multiple signalling pathways, and in most cases this is context-dependent. In the present study, we aimed at identifying the molecular mechanisms during the inhibition of EMT by novel anti-cancer agent myrtucommulone-A (MC-A) and thymoquinone (TQ). We used epithelial cancer cells to study the effects of MC-A and TQ on EMT. We first showed the functional inhibition of EMT by MC-A or TQ using migration assays and confirmed the EMT inhibition by analysing the expression of EMT markers with RT-PCR, immunocytochemistry and Western blotting. We evaluated the changes in intracellular dynamics by Western blotting and compared the effects of MC-A and TQ with the effects of selective inhibitors of PI3K (LY294002), ERK 1/2 (U0126) and TGF-βR (SB431542). We demonstrate that both MC-A and TQ treatment negatively regulate the EMT process through modulation of signalling pathways in cancer cells. MC-A and TQ treatment inhibited phosphorylation of multiple proteins in a context-dependent manner. Novel anti-cancer agent MC-A and TQ regulate distinct signalling pathways for the repression of EMT which emphasises the significance of combinational therapies in cancer treatment. MC-A and TQ could be considered as candidate molecules for combinational therapies with their ability to interfere signalling pathways regulating cancer cell behaviour.

Activating Mutations in PIK3CB Confer Resistance to PI3K Inhibition and Define a Novel Oncogenic Role for p110β.

Activation of the PI3K pathway occurs commonly in a wide variety of cancers. Experience with other successful targeted agents suggests that clinical resistance is likely to arise and may reduce the durability of clinical benefit. Here, we sought to understand mechanisms underlying resistance to PI3K inhibition in PTEN-deficient cancers. We generated cell lines resistant to the pan-PI3K inhibitor GDC-0941 from parental PTEN-null breast cancer cell lines and identified a novel PIK3CB D1067Y mutation in both cell lines that was recurrent in cancer patients. Stable expression of mutant PIK3CB variants conferred resistance to PI3K inhibition that could be overcome by downstream AKT or mTORC1/2 inhibitors. Furthermore, we show that the p110β D1067Y mutant was highly activated and induced PIP3 levels at the cell membrane, subsequently promoting the localization and activation of AKT and PDK1 at the membrane and driving PI3K signaling to a level that could withstand treatment with proximal inhibitors. Finally, we demonstrate that the PIK3CB D1067Y mutant behaved as an oncogene and transformed normal cells, an activity that was enhanced by PTEN depletion. Collectively, these novel preclinical and clinical findings implicate the acquisition of activating PIK3CB D1067 mutations as an important event underlying the resistance of cancer cells to selective PI3K inhibitors.

Abstract B23: Mutant NRAS regulates glycolysis in melanoma through ERK, mTOR and the MYC/HIF1α/MondoA network of transcriptional regulators

Abstract Background/Aims: After BRAF, NRAS is the most commonly mutated oncogene in melanoma. However therapies targeting NRAS remain elusive. Recent investigations have demonstrated that mutated RAS can increase glycolytic metabolism in cancer cells, and our previous work has elucidated a role for glycolysis in the sensitivity of BRAF-mutant melanoma cells to inactivation of BRAF(1). This study aims to identify the molecular pathways controlling glycolysis in NRAS-mutant melanoma in the context of therapeutic targeting of critical oncogenic pathways. Methods: A panel of NRAS and BRAF-mutant melanoma cell lines were screened for changes in viability and glycolysis following treatment with a panel of small molecule inhibitors targeting the MAPK and PI3K pathways. Glycolytic responses of melanoma cells, following MEK, ERK, selective-PI3K, AKT, mTOR or combined PI3K/mTOR inhibition (alone or in combination), were investigated by assessing cell proliferation, glucose uptake, lactate production, and known transcriptional regulators of glycolysis. Results: In NRAS-mutant melanoma, MEK and ERK inhibition resulted in a reduction in lactate production, and similar responses were observed following treatment with mTORC1/2 and PI3K/mTOR inhibitors but not AKT or selective PI3K inhibitors. Treatment with MEK or ERK inhibitors resulted in a consistent reduction in glucose uptake, with a more modest effect being observed following combined PI3K/mTOR inhibition. Treatment with a combination of MEK or ERK inhibitor with either an mTORC1/2 or PI3K/mTOR inhibitor, resulted in synergy with respect to a reduction in cell proliferation. However, synergy was not demonstrated when lactate production and glucose uptake were assessed indicating that inhibition of MEK or ERK was sufficient to inhibit glycolysis in NRAS-mutant cells. Notably, treatment with these inhibitors also resulted in altered expression of transcriptional regulators of glycolysis (reduced MYC and HIF1α, increased TXNIP protein expression via MondoA), similar to that seen in BRAF-mutant melanoma cells. The role of each individual transcriptional regulator of glycolysis in the response of NRAS-mutant cells to MEK/ERK inhibition is under investigation. Conclusion: NRAS-mutant melanoma cells demonstrate a similar glycolytic phenotype to BRAF-mutant melanoma cells, with MAPK pathway or mTOR inhibition resulting in decreased lactate production, glucose uptake and modulation of the MYC/HIF1α/MondoA network of transcriptional regulators of glycolysis. These findings identify the mechanism of oncogene-driven glycolysis in NRAS-mutant melanoma, and open new possibilities for targeting glycolysis as a therapeutic target in the treatment of patients with NRAS-mutant melanoma.

Effect of pioglitazone on the abrogated cardioprotective effect of ischemic preconditioning in hyperlipidemic rat heart.

Objectives:The signaling pathways upstream of glycogen synthase kinase-3β (GSK-3β) get reduced during ischemic preconditioning (IPC) in hyperlipidemic rat heart. Pioglitazone, an insulin sensitizer, exerts cardioprotection through GSK-3β. The objective of the study is to investigate the role of pioglitazone on the attenuated cardioprotective effect of IPC in hyperlipidemic rat heart.Materials and Methods:The rats were administered high-fat diet for 8 weeks to induce experimental hyperlipidemia (HL). After mounting on a Langendorff apparatus, isolated perfused hearts were given four cycles of IPC; each consists of 5 min of both ischemia and reperfusion followed by 30 min of ischemia and 120 min of reperfusion. Insulin (50 mU/ml) was perfused alone and in combination with pioglitazone (2 μM), while in other groups, this combination was repeated with wortmannin (100 nM), a selective PI3K inhibitor and rapamycin (1 nM), a selective mammalian target of rapamycin (mTOR) inhibitor, separately, and in combination. Myocardial injury was assessed by measuring infarct size and the levels of creatinine kinase-myocardial band (CK-MB) and lactate dehydrogenase (LDH) in the coronary effluent.Results:IPC significantly decreased the infarct size and levels of LDH and CK-MB in normal but not in HL rat heart. Perfusion of insulin along with pioglitazone significantly reduced the infarct size and release of CK-MB and LDH in IPC-treated HL rat hearts. Perfusion of wortmannin or rapamycin alone significantly and in combination almost completely abolished the pioglitazone-induced restored cardioprotection (P < 0.05).Conclusion:Cardioprotective effect of IPC gets lost in hyperlipidemic rat heart. The results suggest that perfusion of pioglitazone restored the cardioprotective effect of IPC in hyperlipidemic rat heart, an effect that may be via PI3K and mTOR.

Functional mapping of multiple myeloma kinome with library of small molecule inhibitors

The functional role of individual kinases in multiple myeloma (MM) cells has been primarily interrogated using RNAi approaches, which are mechanistically dissimilar to the pharmacological action of small molecule inhibitors used in the clinic; and in stroma-free cultures, which don’t account for stroma-induced signaling events. To address these dissimilarities, we performed functional mapping of the kinome in 16 MM cell lines, cultured in the presence vs. absence of stromal cells, using a panel of 273 kinase inhibitors (100nM, 24-72 h exposure) targeting a total of 43 known primary oncogenic targets. The cell-autonomous activity of the various inhibitors was broadly potent for Aurora, PLK, and mTORC1/2 inhibitors (consistent with high proliferative rates of MM cell lines in vitro); modest to minimal for selective PDK1, PI3K (excluding those that also inhibit mTOR), and Akt inhibitors; virtually none for selective inhibitors of c-met, ALK, EGFR, HER2, c-kit, PDGFR, VEGFR, Flt3, FAK, Syk, Src, and BTK (even in cell lines with detectable transcripts for the respective kinases). We also observed modest activity for FGFR3 inhibitors against FGFR3expressing cell lines; and none for BRAF inhibitors in our BRAF wild-type cell lines (some of which had increased proliferation upon treatment; consistent with known adaptive activation of signaling through C-RAF). Testing 8 different MEK1/2 inhibitors identified lines with low, intermediate vs. high response; the latter being KRAS-mutant and BRAF-wild-type cells with high baseline p-ERK levels (e.g. due to MEK2-activating mutation in one cell line). Importantly, comparison of results obtained in the presence vs. absence of stroma, indicate heterogeneous effect of bone marrow stromal cells on MM cell response to inhibitors across different targets and cell lines, including stroma-induced resistance to the same kinase inhibitor for some cell lines vs. sensitization for others. Our studies demonstrate the feasibility of functional annotations of the kinome in MM using libraries of small-molecule kinase inhibitors in phenotypic assays against panels of cell lines. Our results provide insight into the relative role of different classes of kinases in MM, and how the genetic (e.g. MEK2 or BRAF mutation status) and microenvironmental context can influence the role of these kinases and possible future individualized administration of inhibitors against these kinases in MM. BP-032

RICTOR involvement in the PI3K/AKT pathway regulation in melanocytes and melanoma.

Several studies have highlighted the importance of the PI3K pathway in melanocytes and its frequent over-activation in melanoma. However, little is known about regulation of the PI3K pathway in melanocytic cells. We showed that normal human melanocytes are less sensitive to selective PI3K or mTOR inhibitors than to dual PI3K/mTOR inhibitors. The resistance to PI3K inhibitor was due to a rapid AKT reactivation limiting the inhibitor effect on proliferation. Reactivation of AKT was linked to a feedback mechanism involving the mTORC2 complex and in particular its scaffold protein RICTOR. RICTOR overexpression in melanocytes disrupted the negative feedback, activated the AKT pathway and stimulated clonogenicity highlighting the importance of this feedback to restrict melanocyte proliferation. We found that the RICTOR locus is frequently amplified and overexpressed in melanoma and that RICTOR over-expression in NRAS-transformed melanocytes stimulates their clonogenicity, demonstrating that RICTOR amplification can cooperate with NRAS mutation to stimulate melanoma proliferation. These results show that RICTOR plays a central role in PI3K pathway negative feedback in melanocytes and that its deregulation could be involved in melanoma development.

Abstract 2399: Circulating tumor DNA (ctDNA) analysis of PIK3CA and AKT1 mutations in patients enrolled onto the Phase 1b study of the PI3K inhibitor taselisib (GDC-0032) in solid malignancies

Abstract The PI3K signaling pathway is one of the most frequently activated pathways in cancer, and controls critical proliferative and survival processes. Taselisib (GDC-0032) is a novel, oral, selective inhibitor of PI3K, sparing inhibition of PI3Kβ. (Ndubaku CO et al. J Med Chem 2013). Preclinically, taselisib demonstrates enhanced potency in PIK3CA mutant cells, with greater signaling and growth/survival effects in mutant cells vs. wild-type cells. In the phase Ia study with single agent taselisib, partial responses (PRs) were observed in 6/34 enrolled patients. All 6 responses were observed in patients with PIK3CA mutant tumors (Juric D. et al. AACR 2013), indicating the need to determine PIK3CA mutation status from patients treated with taselisib. Circulating tumor DNA (ctDNA) provides an up-to-date liquid biopsy that can be used to detect somatic mutation in oncogenes especially when tumor tissue is not available. Analysis was undertaken on five cohorts of patients enrolled onto the taselisib Phase 1b study comprised of 106 patients. In total, 87 patients had tumor tissue available for PIK3CA mutation testing. Plasma was collected from 91 patients prior to treatment and 10 patients had matched plasma collected at the study discontinuation visit. Tissue samples were assessed using the Roche cobas PIK3CA mutation test that detects 17 PIK3CA hotspot mutations, of which 47 (54%) tissue samples were positive for PIK3CA mutations. Analysis of PIK3CA and AKT1 mutations from plasma was performed using an Inostics BEAMing digital PCR Oncobeam panel, which detects 8 PIK3CA hotspot mutations and 1 AKT1 mutation. ctDNA was detected in all 91 patients sampled, and ranged from 1.3ng to 1ug per milliliter of plasma. PIK3CA mutations were identified in the plasma of 51 patients (56%), and an AKT1 mutation was identified in one patient. For 80 patients that had both tissue and plasma available for PIK3CA mutation testing, a sensitivity of 81%, a specificity of 74% and an overall accuracy of 78% was observed. Allele frequency ranged from 0.02% to 75.58% with a median of 2.72%. In two cohorts that enrolled hormone receptor positive, HER2 negative breast cancer patients, a sensitivity of 84%, a specificity of 87% and an overall accuracy of 88% was observed between 42 matched tissue and plasma samples. In a patient who experienced a sustained clinical partial response to taselisib in combination with fulvestrant, a decrease in plasma PIK3CA allele frequency was observed from 3.05% in the pretreatment sample to 0.14% in the study discontinuation sample. In summary, analysis of PIK3CA mutations from plasma in patients enrolled onto trials testing the efficacy of taselisib may be a useful surrogate for patients when tissue is unavailable, an observation that is being assessed in ongoing trials. Citation Format: Timothy R. Wilson, Heidi Savage, Junko Aimi, Jessica Jin, Hema Parmar, Jerry Hsu, Ian Krop, Cristina Saura, Andres Cervantes, Jasgit Sachdev, Manish Patel, Juan Cejalvo, Mafalda Oliveira, Eric Winer, Daniel Von Hoff, Jose Baselga, Dejan Juric. Circulating tumor DNA (ctDNA) analysis of PIK3CA and AKT1 mutations in patients enrolled onto the Phase 1b study of the PI3K inhibitor taselisib (GDC-0032) in solid malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2399. doi:10.1158/1538-7445.AM2015-2399

Abstract A45: In depth molecular characterization of novel PI3K-mTOR inhibitor resistant NSCLC cell lines

Abstract Introduction: Non-small cell lung cancer (NSCLC) is the leading cause of cancer morbidity and mortality globally in males, with rates increasing in females in recent years. New therapeutic intervention strategies will be crucial in improving the current survival rates for patients with this disease, which currently stand at less than 15% at 5 years. The PI3K-Akt- mTOR pathway regulates cell growth and proliferation and is often dysregulated in cancers including NSCLC, making it an attractive new therapeutic target in this setting. GDC-0980 is a selective dual inhibitor of PI3K and mTOR, which is currently in Phase II clinical trials for solid tumours. As with all targeted therapies, acquired resistance to GDC-0980 is anticipated to be a major hurdle in the success of this drug. The aim of this project is to develop and characterise four cell line models of resistance to GDC-0980, each representing a different molecular subtype of NSCLC, in order to identify biomarkers of response/resistance to the drug that may dictate beneficial treatment strategies. Methods: H460, A549, H1975 and SKMES-1 cells were cultured in GDC-0980 at IC50 concentrations over a period of several months, along with matched ‘parent’ cell lines. Development of resistance was assessed by monthly BrdU proliferation assays. Cell growth patterns were compared across the sensitive and resistant cell lines in real time using the xCELLigence platform. Cell lines were then interrogated for alterations in DNA (Sequenom sequencing of 547 mutations in 46 genes), mRNA (SABiosciences arrays profiling expression of &amp;gt;150 genes), miRNA (Exiqon expression profiling of 2100 miRNAs) and protein (R&amp;D Phospho Kinase array expression profiling of 43 kinases and 2 associated total proteins, bottoms-up label-free mass spectrometry and Western blot analysis). Results: H1975 cells (PIK3CA mutant and activated pAkt (Ser473/Thr308), pmTOR, pS6R) were most sensitive to GDC-0980, however they were the first to develop resistance to the drug. Results obtained from xCELLigence studies identified H1975 resistant cells as having the highest cell index out of all parent and resistant cell lines after 100 hours of cell growth, suggesting that these are the most aggressive cells. A 33 miRNA signature was identified contrasting H1975 parent and resistant cells. 1,200 proteins were found to be differentially expressed between H1975 parent and resistant cells (p &amp;lt; 0.05, fold change &amp;gt;2) by bottoms-up label-free mass spectrometry. Preliminary results based on these screens have identified dysregulated metabolism and IGF-1 pathway signalling as potential mechanisms of resistance to GDC-0980 in H1975 cells. Comparison of Nanog, Sox2, Klf4, Oct4 and c-Myc expression highlighted a putative stem-like signature in all resistant cell lines. Several dysregulated genes and proteins will be presented including a kinase which plays a key role in cell proliferation and cancer progression and is targetable by dual and triple kinase inhibitors. Discussion and Conclusion: While the panel of four NSCLC cell lines all responded well to GDC-0980 treatment initially, resistance to the drug developed rapidly in 3 of the cell lines, within 4-6 months. Initial data highlights IGF-1 signalling pathway proteins and the emergence of a stem-like signature as potential mediators of resistance to the drug. Of particular interest is the identification of a specific kinase which is targetable by third generation small molecule inhibitors. Further elucidation of these bypass strategies are under investigation and are crucial to the design of optimal treatment protocols for patients. Citation Format: Susan Heavey, Paul Dowling, Sinéad Toomey, Aoife Carr, Bryan Hennessy, Michael O'Neill, Martin P Barr, Stephen Finn, Sinéad Cuffe, Kenneth J. O'Byrne, Kathy Gately. In depth molecular characterization of novel PI3K-mTOR inhibitor resistant NSCLC cell lines. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A45.

Abstract A46: Inhibition of PIK3CA with BYL719 can overcome resistance to cetuximab in squamous cell carcinoma of the head and neck (SCCHN)

Abstract Background: Preclinical data suggest squamous cell tumors, such as SCCHN, are driven by epidermal growth factor receptor (EGFR) overexpression. However, many patients are refractory to anti-EGFR treatment due to intrinsic and acquired resistance. PI3K/AKT/mTOR pathway activation is a potential mechanism of resistance to EGFR-directed therapy. BYL719, a selective α-isoform PI3K inhibitor, enhances cetuximab (EGFR inhibitor) activity in SCCHN cell lines. A Phase Ib/II study combining BYL719 with cetuximab in patients with recurrent/metastatic SCCHN is testing this hypothesis (Razak et al, ASCO 2014, abst 6044; NCT01602315). Here, we outline the preclinical rationale for combining BYL719 with cetuximab in the setting of cetuximab-resistant squamous cell carcinoma, highlight Phase Ib safety and efficacy data in patients with SCCHN who have received prior cetuximab, and describe the design of the Phase II portion of the study, which is assessing both cetuximab-naive and -pretreated patients. Methods: The combination of BYL719 and cetuximab was tested in vivo in two xenograft models of esophageal squamous cell carcinoma: KYSE180 (cetuximab-sensitive model) and KYSE180_CR (cetuximab-resistant, induced by long-term in vivo treatment). In the Phase Ib study, BYL719 was administered once daily (QD) in 28-day cycles with standard weekly (QW) cetuximab (400 mg/m2 on Cycle 1 Day 1; 250 mg/m2 QW thereafter) to adults with histologically/cytologically confirmed recurrent/metastatic SCCHN resistant/intolerant to platinum-based chemotherapy (prior cetuximab therapy was allowed). Results: In the KYSE180 model, the addition of BYL719 to cetuximab demonstrated an additive effect leading to tumor regression; furthermore, in the KYSE180_CR model, adding BYL719 to cetuximab completely restored sensitivity and led to similar activity as seen in the cetuximab-sensitive model, suggesting that inhibiting PIK3CA signaling may overcome resistance to cetuximab. In the Phase Ib study, as of March 10, 2014, 37 patients were treated with BYL719 300 mg QD (n=32) or 400 mg QD (n=5) and cetuximab. Frequent adverse events (&amp;gt;30%; all-grade/Grade 3/4) were hyperglycemia (54/24), stomatitis (38/5), and dermatitis acneiform (35/3). Best overall response (BOR) per RECIST v1.1 in the full population (n=37) was 4 partial responses (PRs), 16 stable disease (SD; of which 5 were unconfirmed PRs), 10 unknown (UNK) responses, and 1 non-complete response/non-progressive disease (PD) at 300 mg or 400 mg. Six patients had PD as BOR. The overall response rate (ORR) in the full population was 4/37 (11%) and the disease control rate (DCR) was 20/37 (54%). Within this population, 7 patients had received prior cetuximab therapy (6 in the metastatic/recurrent setting and 1 curative; BOR: 4 SD, 1 PR, 1 PD, and 1 UNK). Of these 7 patients, 1 had a confirmed PR, 2 had unconfirmed PRs, 2 had SD, and 1 had PD with BYL719 and cetuximab. One patient with prior cetuximab therapy had an UNK response due to death from tumor lysis syndrome after 1 week in the study. The ORR among patients with prior cetuximab was 1/7 (14%) and DCR was 5/7 (71%). Phase II tests the combination of BYL719 at the recommended Phase II dose of 300 mg QD in two second-line SCCHN patient populations. Patients who are cetuximab-naive are randomized to cetuximab alone or to BYL719 in combination with cetuximab, while patients who have received cetuximab and platinum therapy in the first-line setting are enrolled in a single arm to receive BYL719 with cetuximab. Conclusion: Combined inhibition of PI3Kα and EGFR by BYL719 and cetuximab, respectively, overcame cetuximab resistance in the preclinical setting, was well tolerated, and demonstrated encouraging antitumor activity in both cetuximab-naive and -pretreated patients with SCCHN. The Phase II part of the study in both patient populations is ongoing. Citation Format: Pamela Munster, Moshe Elkabets, Jill Gilbert, Albiruni R Abdul Razak, Myung-Ju Ahn, Chia-Jui Yen, Se-Hoon Lee, Hung-Ming Wang, Carla van Herpen, Wan-Teck Lim, David Demanse, Rupam Ranjan Pal, Alan Huang, Qing Sheng, Chiara Lambertini, Malte Peters, Christina Coughlin, Maurizio Scaltriti, José Baselga, George Blumenschein. Inhibition of PIK3CA with BYL719 can overcome resistance to cetuximab in squamous cell carcinoma of the head and neck (SCCHN). [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A46.

Abstract P5-19-24: Phase Ib/II study of LEE011 and BYL719 and letrozole in ER+, HER2– breast cancer: Safety, preliminary efficacy and molecular analysis

Abstract Background: Activation of the PI3K/AKT/mTOR and cyclin D–CDK4/6–INK4–Rb pathways, including through alteration of PIK3CA and CCND1, has been implicated in resistance to aromatase inhibitors. BYL719 (BYL), an α-isoform selective PI3K inhibitor, demonstrated clinical activity as a single agent and in combination with hormone therapy in pts with advanced HR+ breast cancer (BC), while the CDK4/6 inhibitor LEE011 (LEE) showed evidence of clinical activity as a single agent in pts with advanced solid tumors and in combination with letrozole (LET) in pts with heavily pretreated advanced ER+ BC. In ER+ BC models, the triple combination of LEE, BYL, and LET had enhanced activity vs each agent alone. A Ph Ib/II, 3-arm study is currently investigating the combination of LEE, BYL, and LET in pts with ER+ BC. Here we report on safety, preliminary efficacy, and molecular analysis from Arm (A)1 (LEE + LET) and A2 (BYL + LET) of the Ph Ib part of the study. Methods: Postmenopausal pts with advanced ER+, HER2– BC receive daily oral LEE (3-wks-on/1-wk-off; A1) or BYL (continuous; A2), plus fixed, daily LET (2.5 mg, continuous) in 28-day cycles. Primary objective: determine the MTD and/or RP2D of each combination. A Bayesian Logistic Regression Model using the escalation with overdose control principle and real-time PK guide dose escalation. Secondary objectives: safety, PK, and preliminary efficacy. Potential biomarkers that are predictive of response are also being assessed by next-generation sequencing of tumor samples. Results: As of March 28, 2014, 10 pts received 600 mg LEE plus LET (A1), and 7 pts 300 mg BYL plus LET (A2). At study entry, all pts had stage IV disease; number of prior endocrine regimens for advanced disease was: 0–1 (47%); 2–3 (35%); 4–5 (18%); 35% of pts had previously received PI3K/AKT/mTOR pathway inhibitors for advanced disease. Of the 15 pts evaluable for dose determination (10 in A1 and 5 in A2), 1 dose-limiting toxicity was observed (A1: Grade [G]4 neutropenia; data cut-off: May 15). Most common (all grade &amp;gt;30%) study drug-related AEs (all grade/G3–4) were: A1: neutropenia (90%/50%) and nausea (40%/0%); A2: hyperglycemia (57%/14%), decreased appetite, diarrhea, and nausea (43%/0% each). PK of LEE and BYL on Days 1 and 21, and LET on Day 1, are comparable with historic single-agent data. PK of LET at steady state is being evaluated. In A1, of 6 pts with known response, 1 pt had a PR, 2 pts had SD, 1 pt without measurable disease had NCRNPD, and 2 pts had PD. In A2, of 5 pts with known response, 2 pts had SD, and 3 pts had NCRNPD. Biomarker analysis showed that 2 pts in A2 with SD who are still on study (1 with 25% tumor shrinkage) had PIK3CA mutations. Conclusion: LET plus LEE or BYL had an acceptable safety profile and preliminary clinical activity in pts with ER+/HER2– advanced BC. Dose escalation continues, and upon determination of the MTD/RP2D in A1 and A2, enrollment into A3 (LEE + BYL + LET) will begin to determine the MTD/RP2D. Genetic alterations in PIK3CA were observed in A2. Analysis of baseline aberrations in the cyclin D–CDK4/6–INK4–Rb pathway in A1 and A3 are ongoing and will be updated. The Ph II part of the trial will compare LET plus LEE or BYL with the triple combination. Citation Format: Dejan Juric, Erika Hamilton, Laura Garcia Estévez, Richard H De Boer, Ingrid Mayer, Mario Campone, Shizuka Asano, Suraj Bhansali, Vickie Zhang, Becker Hewes, Pamela Munster. Phase Ib/II study of LEE011 and BYL719 and letrozole in ER+, HER2– breast cancer: Safety, preliminary efficacy and molecular analysis [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 P5-19-24.

Targeting PI3K/AKT/mTOR network for treatment of leukemia.

Increased activity of PI3K/AKT/mTOR pathway has been observed in a huge number of malignancies. This pathway can function as a prosurvival factor in leukemia stem cells and early committed leukemic precursors and its inhibition is regarded as a therapeutic approach. Accordingly, the aim of this review is to evaluate the PI3K/Akt/mTOR inhibitors used in leukemia models. Inhibition of the PI3K/AKT/mTOR pathway has been reported to have beneficial therapeutic effects in leukemias, both in vitro in leukemia cell lines and in vivo in animal models. Overall, the use of dual PI3K/mTOR inhibitor, dual Akt/RTK inhibitor, Akt inhibitor, selective inhibitor of PI3K, mTOR inhibitor and dual PI3K/PDK1 inhibitor in CML, AML, APL, CLL, B-ALL and T-ALL has a better therapeutic effect than conventional treatments. Targeting the PI3K/Akt/mTOR pathway may have pro-apoptotic and antiproliferative effects on hematological malignancies. Furthermore, modulation of miRNA can be used as a novel therapeutic approach to regulate the PI3K/Akt/mTOR pathway. However, both aspects require further clinical studies.

Neuroprotection of Sevoflurane Against Ischemia/Reperfusion-Induced Brain Injury Through Inhibiting JNK3/Caspase-3 by Enhancing Akt Signaling Pathway.

In this study, we investigated the neuroprotective effect of sevoflurane against ischemic brain injury and its underlying molecular mechanisms. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with sevoflurane alone or sevoflurane combined with LY294002/wortmannin (selective inhibitor of PI3K) before ischemia. Cresyl violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. Immunoblotting and immunoprecipitation were performed to measure the phosphorylation of Akt1, PRAS40, ASK1, and JNK3 and the expression of cleaved-caspase-3. The results demonstrated that a moderate dose of sevoflurane inhalation of 2% for 2 h had significant neuroprotective effects against ischemia/reperfusion induced hippocampal neuron death. Sevoflurane significantly increased Akt and PRAS40 phosphorylation and decreased the phosphorylation of ASK1 at 6 h after reperfusion and the phosphorylation of JNK3 at 3 days after reperfusion following 15 min of transient global brain ischemia. Conversely, LY294002 and wortmannin significantly inhibited the effects of sevoflurane. Taken together, the results suggest that sevoflurane could suppress ischemic brain injury by downregulating the activation of the ASK1/JNK3 cascade via increasing the phosphorylation of Akt1 during ischemia/reperfusion.

Neuroprotection induced by sevoflurane-delayed post-conditioning is attributable to increased phosphorylation of mitochondrial GSK-3β through the PI3K/Akt survival pathway

Background and purposePost-conditioning with volatile anesthetics can create ischemic tolerance against cerebral ischemia–reperfusion injury. The present study was designed to determine whether delayed exposure to sevoflurane could induce ischemic tolerance and if this effect was dependent on increasing phosphorylated Akt-Ser473 and GSK-3β-Ser9 expression in the mitochondria, via a mechanism involving the PI3K/Akt pathway. MethodsAdult male Sprague–Dawley rats were subjected to focal cerebral ischemia. Sevoflurane post-conditioning was achieved by administration of 2.5% sevoflurane for 60min, 15min after reperfusion. Phosphorylated Akt-Ser473 and GSK-3β-Ser9 in the cytosol and mitochondria of the ischemic penumbra were evaluated 4, 12, 24, and 72h after reperfusion. Neurological deficit score and activity of caspase-3 and -9 were evaluated 24 and 72h after reperfusion. Apoptosis, as measured by TUNEL staining and cerebral infarct size,was determined 24h after reperfusion. ResultsSevoflurane-delayed post-conditioning significantly increased levels of phosphorylated Akt-Ser473 and GSK-3β-Ser9 in the mitochondria and inhibited the activities of caspase-3 and -9, showing an improved neurological deficit score and a decreased infarct size. However, LY294002, a selective PI3K inhibitor, not only eliminated the neuroprotection of sevoflurane, as indicated by an increased infarct size and a larger number of TUNEL-positive cells, but also reversed the elevation of p-Akt and p-GSK-3β expression in the mitochondria induced by sevoflurane post-conditioning. ConclusionsOur data suggested that delayed application of sevoflurane after reperfusion provides neuroprotection by activating phosphorylated Akt-Ser473 and GSK-3β-Ser9 in the mitochondria via the PI3K/Akt pathway.

Functional Mapping of Multiple Myeloma Kinome Using a Small Molecule Inhibitor Library

In multiple myeloma (MM) and other neoplasias, several kinases have been extensively evaluated as potential therapeutic targets using RNAi-based approaches or pharmacological inhibitors. Attempts to map the functional dependence of MM cells on individual kinases have primarily utilized RNAi, a mechanistic approach inherently dissimilar to small molecule inhibitors that are applied in the clinic. For many of these oncogenic kinases, large numbers of such inhibitors have been designed: these inhibitors often exhibit very similar effect on their primary designated target(s), but also perturb other secondary kinases, which may vary for different inhibitors within the same class. Using large sets of such inhibitors can enable comparative analyses to reveal the functional roles of both the respective primary target(s), as well as non-overlapping secondary targets. We therefore pursued the functional mapping of the kinome dependencies of 16 MM cell lines, using a panel of 273 kinase inhibitors (100nM, 24-72 h exposure), which target a total of 43 known primary oncogenic targets. In this study, we observed universally potent activity of Aurora (n=18 compounds), PLK (n=5), and mTORC1/2 (n=20) inhibitors; this observation is consistent with the high proliferative rate of MM cell lines in vitro. In contrast, we observed modest to minimal cell-autonomous susceptibility of MM cells to selective inhibitors of PDK1, PI3K (excluding those that also inhibit mTOR), and Akt: this suggests that PDK1- and Akt-independent mechanisms mediate the effect of PI3K signaling on the survival of most of these cell lines. In addition, we observed lack of response in virtually all tested cell lines to inhibitors of c-met (n=17 inhibitors), ALK (n=2), EGFR superfamily members (EGFR, HER2; n=25 inhibitors), c-kit (n=3), PDGFR(n=5), VEGFR (n=21), Flt3 (n=7), FAK (n=2), Syk (n=5), Src (n=5) and BTK: this result was observed even in those cell lines with detectable transcript against the respective kinases. Notable exceptions to this pattern were inhibitors that, in addition to their primary target, also possess activity to other kinases with known roles in MM (e.g. potent activity of FAK or ALK inhibitors that also target IGF1R, such as TAE226 and GSK1838705A, respectively). Consistent with prior experience, several FGFR3 inhibitors showed modest activity against FGFR3- expressing cell lines (e.g. KMS11, KMS18, OPM2, KMS34). Our screen also revealed several previously underappreciated classes of inhibitors with "non-consensus", heterogeneous, activity across the tested MM cell lines. For instance, we identified 3 clusters of cell lines with high (e.g. AMO1, Karpas-620); intermediate (e.g. KMS20, MM1S), and low responsiveness, to 8 different MEK1/2 inhibitors. Notably, both Karpas-620 and AMO1 cells are KRAS-mutant, BRAF-wild-type and have inherently high levels of p-ERK; while AMO1 cells also harbor a MEK2-Q60P mutation, previously reported to positively regulate the kinase domain activity of MEK2 and induce resistance of BRAF-V600E mutant melanoma cells to MEK1/2 inhibitors. These results raise the possibility that the response to MEK1/2 inhibitors and the role of specific mutations, such as MEK2-Q60P, are tumor-type dependent and/or influenced by concurrent BRAF mutation status. Notably, BRAF inhibitors (n=7) were inactive as cytoreductive agents against our cell line panel of BRAF wild-type cells; while several MM cell lines exhibited significantly increased proliferation upon treatment with these inhibitors. This stimulation has been previously noted in melanoma and has been attributed to activation and signaling through C-RAF; it also suggests that treatment of MM patients harboring both V600E-BRAF mutant and wild-type clones with BRAF inhibitor may decrease the burden of the former clone(s), but select for outgrowth of the latter. In summary, our studies establish the value of using large libraries of small-molecule kinase inhibitors in phenotypic assays against panels of tumor cell lines, as an approach to functionally annotate the kinome dependencies across a given neoplasia, such as MM. Furthermore, our studies provide insight into the possible clinical implications that specific molecular lesions (e.g. mutation status of MEK2 or BRAF) can have on the individualized administration of kinase inhibitors targeting the respective pathway. DisclosuresMitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Research Funding; Johnson & Johnson: Research Funding.

Design, synthesis and biological evaluation of acylhydrazone derivatives as PI3K inhibitors

Abstract Since the PI3K signaling pathway is the most commonly activated in human cancers, inhibition of PI3K is a promising approach to cancer therapy. In this study, a series of 2-methyl-5-nitrobenzeneacylhydrazones were designed and synthesized. All the new derivatives were tested by p110 α enzymatic and Rh30 cellular assays. Further enzyme selectivity profiling proved that 6e and 7 were potential selective PI3K inhibitors.

Abstract 4022: Combination of PI3K inhibitor BAY 1082439 with radium-223 is a promising treatment of cancer with bone metastases

Abstract About 80% of advanced prostate, breast and lung cancer patients develop bone metastases, which cause significant morbidity including chronic pain and pathologic fractures. Patients with bone metastases have a poor prognosis and currently there is no curative treatment. This is largely due to the unique biological microenvironments of bone where a wide range of growth factors promote tumor cell survival and tumor cells stimulate bone remodeling. Activation of PI3K plays important role in prostate and breast cancer cell proliferation, survival and metastasis. PI3K also mediates critical signaling pathways involving tumor and stromal cell interaction, including cancer-induced bone turnover and osteolysis. BAY 1082439 is a highly selective and potent PI3K inhibitor with balanced activity against PI3kα and PI3Kβ isoforms and currently being evaluated in a phase I clinical trial (N. Liu, et al. AACR 2012 Abstract #2799). Radium-223 dichloride (radium-223), a novel therapy recently approved for treatment of castration-resistant prostate cancer (CRPC) with bone metastases, selectively targets bone and kills metastatic cancer cells by the emission of alpha particles (Parker et al. N Engl J Med. 2013; 369:213). To identify more effective therapy to treat bone metastases, we evaluated the combination of PI3K inhibitor BAY 1082439 and radium-223 in vitro and in the syngeneic 4T1 metastatic breast cancer model in mice. BAY 1082439 and radium-223 showed synergistic anti-proliferative effects in vitro in both hormone receptor positive (HR+, MCF7 and LNCaP) and negative (4T1 and PC3) breast and prostate tumor cell lines. Pronounced synergistic effects on apoptosis induction were observed in MCF7 and LNCaP cell lines. In vivo, combination of BAY 1082439 at the maximum tolerable dose and radium-223 at the efficacious dose was well tolerated. BAY 1082439 significantly decreased whole body tumor burden as single agent (67% reduction, p=0.00156) and more effectively in combination with radium-223 (81% tumor reduction, p=0.0012), while the effect of radium-223 alone was not statistically significant. Furthermore, BAY 1082439 strongly inhibited tumor-induced osteolysis measured by radiography and led to 53.2% (p=0.16) and 84% (p=0.00698) reduction in total osteolytic area as single agent and in combination with radium-223, respectively. The frequency of soft tissue metastases was also decreased by treatment with BAY 1082439 alone (e.g. 73% reduction in kidney, p=0.012) and more pronounced with radium-223 combination treatment (e.g. complete inhibition of kidney metastasis, p=0.0003). In summary, our data indicate synergistic effects of the PI3K inhibitor BAY 1082439 and radium-223 in inhibiting tumor cell proliferation, survival, total and bone marrow tumor burden, and tumor-induced osteolysis, and warrant further clinical evaluation of this promising combination therapy for the treatment of cancer with bone metastases. Citation Format: Mari I. Suominen, Katja Fagerlund, Enrico Stasik, Andrea Haegebarth, Arne Scholz, Jukka P. Rissanen, Martin Kornacker, Dominik Mumberg, Jussi M. Halleen, Karl Ziegelbauer, Ningshu Liu. Combination of PI3K inhibitor BAY 1082439 with radium-223 is a promising treatment of cancer with bone metastases. [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 4022. doi:10.1158/1538-7445.AM2014-4022

Abstract 4774: The antitumor effects of PI3K beta inhibitors in PTEN negative prostate cancer are enhanced by inhibition of reactivated PI3K alpha signaling

Abstract The PI3K pathway is dysregulated in many cancers via selective activation of class 1 isoforms. In tumors with deficient PTEN function, signaling is driven by PI3K beta. We show here that a selective inhibitor of PI3K beta inhibits the AKT/mTOR pathway in tumors with defective PTEN function, but is ineffective in those where the pathway is driven by receptor tyrosine kinases. However, inhibition of PI3K signaling by PI3K beta inhibitors is limited by relief of AKT/mTOR dependent feedback and reactivation of IGF1R and other receptors. This results in activation of PI3K alpha and a rebound of PI3K-AKT signaling. This rebound is suppressed and tumor cell inhibition is enhanced with combined inhibition of PI3K alpha and beta. Combined administration of isoform selective PI3K inhibitors may more effectively inhibit the pathway than pan-PI3K inhibitors because of the greater selectivity and decreased off-target toxicity of the former. In PTEN deficient models of prostate cancer, triple therapy with PI3K alpha and beta selective inhibitors combined with a potent androgen receptor inhibitor suppresses the reciprocal feedback activation of both pathways and results in marked (complete) eradication of tumors in vivo. Citation Format: Sarit Schwartz, Brett S. Carver, John Wongvipat, Vanessa Rodrik-Outmezguine, Elisa De Stanchina, Cath Trigwell, Simon Barry, Jose Baselga, Sarat Chandarlapaty, Howard I. Scher, Charles L. Sawyers, Neal Rosen. The antitumor effects of PI3K beta inhibitors in PTEN negative prostate cancer are enhanced by inhibition of reactivated PI3K alpha signaling. [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 4774. doi:10.1158/1538-7445.AM2014-4774