PTEN Wt Research Articles

Capivasertib combines with docetaxel to enhance anti-tumour activity through inhibition of AKT-mediated survival mechanisms in prostate cancer

Background/objectiveTo explore the anti-tumour activity of combining AKT inhibition and docetaxel in PTEN protein null and WT prostate tumours.MethodsMechanisms associated with docetaxel capivasertib treatment activity in prostate cancer were examined using a panel of in vivo tumour models and cell lines.ResultsCombining docetaxel and capivasertib had increased activity in PTEN null and WT prostate tumour models in vivo. In vitro short-term docetaxel treatment caused cell cycle arrest in the majority of cells. However, a sub-population of docetaxel-persister cells did not undergo G2/M arrest but upregulated phosphorylation of PI3K/AKT pathway effectors GSK3β, p70S6K, 4E-BP1, but to a lesser extent AKT. In vivo acute docetaxel treatment induced p70S6K and 4E-BP1 phosphorylation. Treating PTEN null and WT docetaxel-persister cells with capivasertib reduced PI3K/AKT pathway activation and cell cycle progression. In vitro and in vivo it reduced proliferation and increased apoptosis or DNA damage though effects were more marked in PTEN null cells. Docetaxel-persister cells were partly reliant on GSK3β as a GSK3β inhibitor AZD2858 reversed capivasertib-induced apoptosis and DNA damage.ConclusionCapivasertib can enhance anti-tumour effects of docetaxel by targeting residual docetaxel-persister cells, independent of PTEN status, to induce apoptosis and DNA damage in part through GSK3β.

Abstract 2884: Cancer-associated fibroblasts potentiate subcutaneous tumor growth in a novel bladder cancer mouse model with a genotype dependency

Abstract Introduction Interrogation of the relationship of genotype (mutation in relevant gene) and phenotype (sensitivity to therapy) in targeted therapy would be facilitated by the use of isogenic in vitro and in vivo models. The PI3K/AKT/mTOR pathway is widely activated but heretofore untargeted in bladder cancer. We sought to develop an immunocompetent CRISPR-edited bladder cancer mouse model that recapitulates common PI3K/AKT/mTOR mutations to study a genotype-phenotype relationship. Our in vitro CRISPR-edited organoids are immunogenic in vivo, as such, we explored utilizing Cancer-Associated Fibroblasts (CAFs) to mediate immunosuppression and promote a pro-tumorigenic environment. Methods and Results We isolated bladder urothelial cells from Rosa26 LSL-Cas9/GFP transgenic mice and grew them as organoids. Trp53 and Rb1 were co-edited to establish ‘base organoids’. As proof of concept, Pten was edited in base organoids in anticipation of developing a series of organoids with different mTOR pathway genotypes. We found that Pten-KO base organoids were 9x more sensitive to ipatasertib than Pten WT organoids. The edited urothelial cells were engrafted into mouse bladder to further test kinase inhibitor susceptibility as preclinical evidence to support human clinical trials. However, the cells successfully engrafted but disappeared after 3-4 weeks before tumors formed. Subcutaneous allografts were also rejected after 3-4 weeks and showed CD45+ immune infiltration during rejection. When cells were injected subcutaneously with CAFs, tumors persisted for 12 weeks, but only in organoids with both Trp53 loss and loss of murine chromosome 4qC4 (which is homologous to 9p21 loss in humans). 9p21 contains 3 genes which are co-deleted in ~40% of bladder cancers: MTAP, CDKN2A, and CDKN2B. Histopathological analysis of these masses confirmed their malignant nature and revealed an immune-excluded architecture. IHC staining of 9p21/Trp53 KO tumors for CD206 and CD11c revealed greater recruitment of M2 macrophages than M1 macrophages. Conclusions CAFs remodel the tumor-microenvironment in bladder cancer via the recruitment of pro-tumorigenic M2 macrophages. This may occur in a genotype-specific manner and 9p21 loss may be a requirement for some genotypes to be tumorigenic. Citation Format: Henkel Lee Valentine, Uttam Satyal, Alexander Metz, Laura Bukavina, Philip H. Abbosh. Cancer-associated fibroblasts potentiate subcutaneous tumor growth in a novel bladder cancer mouse model with a genotype dependency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2884.

Abstract PD3-02: Linking proliferative signal to DNA-damage signal in a tumor cell: A contextual synergy between the PI3K pathway and the DDR pathway in TNBC

Abstract The phosphoinositide 3-kinase (PI3K) pathway signals not only for cell survival and proliferation in tumor cells but also controls DNA damage repair (DDR) and maintains HR (homologous recombination) steady-state the translational significance of which has been reported [Juvekar A. et al., 2012]. In breast cancers (BC), alterations of the PI3K pathway genes are both subtype-specific as well as contextual, and alterations of DDR pathway genes are one such contextual event occurring with the upregulation of the PI3K pathway. We hypothesized that there is contextual cooperation between the cellular signals of the PI3K and DDR pathways. We sought to identify the co-alterations of the PI3K and DDR pathway genes in our triple-negative BC (TNBC) patient cohort to (A) interrogate the contextuality of such alterations and (B) understand the mechanism of the contextual cooperation using an algorithmically chosen combination of pathway targeted drugs. We analyzed the somatic alterations profile of genes (FoundationOne, USA) in 264 BC patients (Avera Cancer Institute, SD, USA). We experimentally validated the cooperation between the pathways using alteration-guided targeted inhibitors; isoform-specific PI3K and PARP inhibitor(s) in the TNBC model, to demonstrate the efficacy of a combination of a node-specific targeted inhibitor(s) of the two pathways. The predominant type of mutation of genes in the PI3K pathway in our patients with TNBC was found in PTEN (Y68C, Y180*, loss, loss exons 1-5, and deletion exon1) and PIK3CA (amplification, E545K, H1047R). The other common mutation found was in TP53 (>80%) and somatic BRCA1/2 (~15%) genes. The interaction between the two pathways was evaluated, applying STRING10 to test the association at the highest 0.900 confidence views. The highest level of efficiency of drugs is demonstrated following the treatment of p110beta inhibitor (AZD 6482) in combination with PARP inhibitor (Olaparib/Talazoparib) plus DNA damaging agent (carboplatin) in BRCA-incompetent PTEN-null TNBC cells (SUM149) as compared to the efficiency of a combination of a PI3K pathway targeted inhibitor (of p110alpha) with a PARP inhibitor in BRCA-competent PTEN WT, but PIK3CA altered TNBC cells (BT20). The context-dependent synergy between the PI3K and DDR pathways in the TNBC model is comprehended at the levels of (1) load of DNA damage, (2) PARP activity, (3) of BRAC1/2 competency or HRD scores, and (4) mode of upregulation of PI3K pathway. We present an algorithm for a rational combination of the PI3K and the DDR pathway targeted drugs in TNBC. Citation Format: Nandini Dey, Jennifer Carlson Aske, Pradip De. Linking proliferative signal to DNA-damage signal in a tumor cell: A contextual synergy between the PI3K pathway and the DDR pathway in TNBC [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-02.

A high throughput proliferation and cytotoxicity assay for co-cultured isogenic cell lines

PTEN is a well-known tumor suppressor that is inactivated or suppressed at a high frequency in cancer. We sought an assay to screen compounds for ones that differentially inhibited proliferation or induced cytotoxicity in PTEN mutated cancer cells. We employed the isogenic pair of cell lines MCF10-A breast cell line (wild type, WT) and the same cell line with PTEN knocked out (KO) by CRISPR. We sought an assay where these PTEN WT and KO isogenic cell lines were co-cultured in the same well for compound testing. The KO cell line, but not the WT, was tagged with the red fluorescent protein mKate2. We employed a real time microscopic imaging instrument to identify cell populations in co-culture based on red fluorescence to obtain a cell count for each cell line. To acquire cytotoxicity data for each population, the dye CellTox Green was added to the media. To assess the assay, we determined the concentration response of paclitaxel. In order to assess the potential for screening, we performed mock screening in 384-well plate format. Thus, we developed a high throughput co-culture cell cytotoxicity and proliferation assay method that could be employed for any pair of cell lines to identify selective compounds.

Combination Benefit of Capivasertib and Venetoclax in Preclinical Models of Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy among adults and despite approximately 65% of patients with DLBCL being cured with RCHOP therapy, nonresponsive and relapsed patients have inadequate treatment options, highlighting the importance for innovative treatment regimens. Blockade of B-cell receptor (BCR) downstream signaling components with various targeted agents is emerging as a clinically tractable treatment strategy across multiple B-cell malignancies. Protein Kinase B (AKT) signaling downstream of the BCR complex has been shown to be a central node in germinal center B-cell (GCB) DLBCL and the potent, selective inhibitor of AKT1, AKT2, AKT3, capivasertib, currently being evaluated in multiple clinical trials by targeting AKT-driven solid cancers, has been shown to induce apoptosis in a subset of GCB-DLBCL cell lines and cause tumor stasis in xenograft mouse models (Erdman et al., 2017). Since the monotherapy capivasertib responses in GCB DLBCL models are partial and lack durability, we hypothesized a combination approach could deliver even greater therapeutic benefit. To identify optimal partners, we conducted a capivasertib centric in vitro combination screen with specific with BH3 family members across a panel of 15 DLBCL cell lines, which revealed a synergistically active combination with the BCL2 inhibitor, venetoclax which is currently being evaluated in DLBCL. The activity was specifically enhanced in cell lines of the GCB subtype, with 4 PTEN del and 2 PTEN wt cell line models showing combination benefit. To determine the ability of this combination to drive stronger and durable responses, we assessed capivasertib and ventoclax activity in xenograft mouse models using two GCB-DLBCL cell line lines, SUDHL4 (PTEN wt) and WSU-DLCL2 (PTEN del). Oral administration of either monotherapy capivasertib (130 mg/kg BID, 4-day on/3-day off) or venetoclax (100 mg/kg QD) provided partial tumor growth inhibition (capivasertib TGI = 74% in SUDHL4 and 29% in WSU-DLCL2, and venetoclax TGI = 46% in SUDHL4 and 0% in WSU-DLCL2), whereas the combination of capivasertib and venetoclax both on a 4-day on/3-day off schedule produced complete tumor regression (100% regression) in both xenograft GCB cell line models during the dosing period. Notably, in both xenograft models all mice (5/5 per model) remained tumor free for at least 30 days following dosing cessation demonstrating high durability of response for the combination. Additionally, this combination is currently being evaluated in clinically relevant GCB and non-GCB PDX mouse models. Taken together, our results provide preclinical evidence for the rational combination of AKT and BCL-2 blockade with capivasertib and venetoclax respectively in patients with relapsed/refractory GCB-DLBCL. DisclosuresWillis: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Neveras: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Dry: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Mongeon: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Rosen: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Mettetal: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Barry: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options.

Developmental Pleiotropy of PTEN in Congenital Hydrocephalus and Autism Spectrum Disorder

Abstract INTRODUCTION A population-based study has found that approximately one quarter of individuals diagnosed with congenital hydrocephalus (CH) are also diagnosed with autism spectrum disorder (ASD); however, a common mechanism underlying these seemingly disparate disorders is unknown. We hypothesize that rare, damaging mutations in genes cause a pleiotropic effect responsible for the development of both CH and ASD. METHODS Using WES studies >250 CH patients, we selected 42 DNMs in genes with the greatest probability of loss-of-function (LoF) (pLI > .9), to determine whether there was an enrichment of ASD genes (pLI > .9) found in denovo-db, a national database of ASD DNMs A 2 × 2 contingency table revealed a 2.18-fold enrichment of ASD genes in sporadic CH genes (P-value = . 011). Gene Ontology analysis found >100-fold enrichment of TORC1 signaling (GO: 0031931) as a significantly enriched module (P-value = .000152). We recapitulated the CH phenotype in a genetic mouse model; wherein, Pten, a direct negative regulator of torc1 signaling, is conditionally knocked-out (Pten cKO) in a select subset of NSCs previously associated with an ASD phenotype in mice. Kaplan-Meier survival curves were constructed for Pten cKO and their WT, control littermates. Rapamycin was injected intraperitoneally in Pten cKO and WT mice daily from postnatal (P) days 10 to 22. RESULTS Pten cKO resulted in fully penetrant, fatal hydrocephalus with a median survival of P17.5. WT mice (n = 41) survived significantly longer than Pten cKO mice (n = 10) (P < .0001). Rapamycin rescued the fatal hydrocephalus phenotype (n = 8) by inhibition of mTOR (P < .0001). CONCLUSION PTEN mutations have previously been identified in ASD patients. Here, we present statistical analyses and a mouse model to support our hypothesis that ASD diagnoses in some CH patients may be a neurodevelopmental sequelae of PTEN LoF. These findings suggest that early ASD service interventions may benefit a subset of CH patients who would otherwise be managed solely with surgery.

PB1648 THE CLINICAL RELEVANCE OF INDIRECT MYC INHIBITORS FOR PERSONALIZED T‐ALL TREATMENT

Background:Therapy refractory and relapsed T cell lymphoblastic leukemia (T‐ALL) is still one of the most common reasons for cancer related death in children. The current therapy option is stem cell transplantation, which is associated with many complications and therefore, targeted therapies with low toxicity profiles are urgently needed. The oncogenic transcription factor MYC is excellently suited as a therapeutic target as it has proven to be a potent driver for T‐ALL development. So far, direct inhibition of MYC is not possible due to its undruggable structure. Hence, inhibition of MYC can be achieved indirectly or by targeting synergistic pathways.Aims:The aim of our study is to identify major key regulators in MYC driven T‐ALL. Targeting synergistic pathways of MYC like the PI3K/AKT signalling pathway seems to be a promising strategy to precisely eliminate tumour cells without affecting healthy cells.Methods:We profiled several T‐ALL and a T cell lymphoma cell line model with a specific interest for genes implicated in MYC regulation on mRNA and protein level including major MYC regulator pathways like Notch‐signaling, Raf‐MEK‐ERK cascade, BRD4, protein folding and PI3K/AKT/mTOR pathway. In parallel, we designed a leukemia specific compound library (n = 180) of diverse phase I ‐ III inhibitors including clinically available chemotherapeutics, diverse kinase‐, HSP90‐, proteasome‐, HDAC‐ and BET inhibitors. Synergy studies were performed with preselected compounds in an 11 × 11 matrix.Results:Protein and mRNA profiling displayed a varying distribution of MYC expression, including its associated downstream and upstream signaling pathways. PTEN loss of function mutants (PTEN mut) are described to have an increased pAKT level due to PI3K dysregulation which has a stabilising effect on MYC, and besides that it has also been associated with a poor outcome in T‐ALL patients. Interestingly, PTEN mut expressing T‐ALL cell lines (MOLT4, HPBALL, JURKAT and SUPT1) shown a simultaneous hyper AKT phosphorylation (pAKT), whereas, PTEN wildtype (wt) cell lines (TALL1, HSB2, DND41 and PEER) exhibit remarkably low pAKT. However MYC expression did not strongly correlate with the PTEN/pAKT status. Therefore, we stratified high and low MYC expressing cell lines according to their PTEN mutational status. Thus, we screened the T‐ALL cell lines on our compound library, which covered major MYC regulatory pathways. Remarkably, specific classes of drugs, including PI3K/AKT/mTOR and Aurora kinase A inhibitors exhibited a promising drug profile in PTEN mut models, while MEK and ERK1/2 inhibitors were particularly potent in PTEN wt. These results were corroborated in a (PTEN wt) low MYC expressing T‐ALL1 cell line model, which showed an increased sensitivity toward Aurora Kinase A inhibitors when MYC was overexpressed. Furthermore, combination studies of PI3K inhibitors with the established indirect MYC inhibitors, e.g. BRD4 or Aurora kinase A inhibitors revealed an synergistic effect in PTEN mut when compared to PTEN wt expressing cell lines.Summary/Conclusion:The source of MYC overexpression is very heterogeneous because its maintenance is highly dependent upon many regulatory pathways. Knocking down the oncogenic transcription factor MYC via synergistic pathways is a promising strategy for targeted therapies in the future.

Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities.

High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTOR-inhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell line-specific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.

Germline PARP4 mutations in patients with primary thyroid and breast cancers.

Germline mutations in the PTEN gene, which cause Cowden syndrome, are known to be one of the genetic factors for primary thyroid and breast cancers; however, PTEN mutations are found in only a small subset of research participants with non-syndrome breast and thyroid cancers. In this study, we aimed to identify germline variants that may be related to genetic risk of primary thyroid and breast cancers. Genomic DNAs extracted from peripheral blood of 14 PTEN WT female research participants with primary thyroid and breast cancers were analyzed by whole-exome sequencing. Gene-based case-control association analysis using the information of 406 Europeans obtained from the 1000 Genomes Project database identified 34 genes possibly associated with the phenotype with P < 1.0 × 10(-3). Among them, rare variants in the PARP4 gene were detected at significant high frequency (odds ratio = 5.2; P = 1.0 × 10(-5)). The variants, G496V and T1170I, were found in six of the 14 study participants (43%) while their frequencies were only 0.5% in controls. Functional analysis using HCC1143 cell line showed that knockdown of PARP4 with siRNA significantly enhanced the cell proliferation, compared with the cells transfected with siControl (P = 0.02). Kaplan-Meier analysis using Gene Expression Omnibus (GEO), European Genome-phenome Archive (EGA) and The Cancer Genome Atlas (TCGA) datasets showed poor relapse-free survival (P < 0.001, Hazard ratio 1.27) and overall survival (P = 0.006, Hazard ratio 1.41) in a PARP4 low-expression group, suggesting that PARP4 may function as a tumor suppressor. In conclusion, we identified PARP4 as a possible susceptibility gene of primary thyroid and breast cancer.

Abstract 2561: Contextual combination of PARP inhibitors with p110beta inhibitors: Functional logistics to tame PTEN null tumors

Abstract Background: Hyper-activation of the PI3K pathway due to a functional loss of PTEN is a characteristic feature of many tumors including TNBC and GBM. Although p110b rarely undergoes activating mutations in these cancers, loss of PTEN function makes these tumor cells dependent specifically on p110b. In a mechanism-based study we have recently established an integral role of the PI3K pathway in DNA damage repair (DDR)-mediated anti-tumor efficacy of PARP inhibitor wherein we have demonstrated that PTEN-nullness confers in vivo sensitivity to the combination as compared to tumors carrying PTEN WT (De et al., Neoplasia, 2014). Thus here we hypothesize that PTEN-null tumors will be more susceptible to a combination of p110b inhibitor (p110b i) plus PARP inhibitor (PARP i). Method: To test our hypothesis we used PTEN-null TNBC and GBM models. Isogenic cell lines as well as SiRNA-directed PTEN-knockdown cells were tested for the (a) IC50 (MTT) (b) real-time confluence assay (Incucyte) (c) cell cycle (flow cytometry) (d) clonogenecity (ON-TOP assay) (e) cytosolic proliferation and apoptotic signals and (f) nuclear HRD signals for DDR using 3 p110b i (TGX221, AZD6482, GSK2636771) and 5 PARPi (BMN673, Niraparib, Olaparib, Rucaparib, Veliparib) (procured from Selleckchem, USA). Results: Individually (1) AZD6482 was the most effective among p110b i in controlling the cell cycle when tested in PTEN null U87MG GBM cells and (2) AZD6482 had the lowest dose (1-10 uM) for achieving the minimum effective dose (MED) in the confluence studies in TNBC and GBM models as compared to other p110b i. The MED of 5 PARPi for TNBC and GBM cells were determined by clonogenic assay (7-10 days). The most potent among 5 PARPi was BMN673 with a dose range of 100 nM-500 nM. Based on the effectiveness of individual drugs, we used a combination dose range of 500 nM-10 uM of 5 PARPi with 10 uM fixed dose of p110b i, AZD6482, and tested the effect of this combination using (1) real time apoptosis assay (Incucyte) and (2) 3D clonogenic assay. The combination of each PARPi with AZD6482 was more effective in perturbing clonogenic growth of PTEN-null TNBC and GBM cells as compared to the individual agent(s) which can be corroborated with the increased apoptosis in the cells treated with the combination and their effect on cell cycle. Significance: Better understanding of the mechanistic involvement of the combination of p110b i and PARPi in BRCA-competent PTEN null tumors may provide a novel target for future therapeutic intervention and provide insight to PTEN-mediated carcinogenesis in these tumors which eventually will extend the realm of PARP i use in cancer therapy. Citation Format: Jennifer H. Carlson, Yuliang Sun, Xiaoqian Lin, Pradip De, Brian Leyland-Jones, Nandini Dey. Contextual combination of PARP inhibitors with p110beta inhibitors: Functional logistics to tame PTEN null tumors. [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 2561. doi:10.1158/1538-7445.AM2015-2561

Abstract 59: PDK1 in melanoma development and metastasis

Abstract Phosphoinositide-dependent kinase-1 (PDK-1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and PKC, and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. Selective inactivation of Pdk1 in the melanocytes of BrafV600E::Pten-/- or BrafV600E::Cdkn2a-/-::Pten-/- mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-HT. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in BrafV600E::Pten-/- mice. Pdk1-/- melanomas exhibit a marked decrease in the activity of AKT, p70S6K and PKC. Notably, genetic inactivation or pharmacological inhibition of PDK1 were even more effective in attenuating melanoma development and metastasis of human tumors with Pten WT as in genetic model of BrafV600E::Cdkn2a-/-::Pten+/+ mice, representing an additional fraction of human tumors harboring such mutation. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes and inhibition of FOXO3a restored proliferation and colony formation of Pdk1-/- melanoma cells. PDK1 effect on FOXO3a and melanoma development and progression appears to be AKT independent. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression. Citation Format: Marzia Scortegagna, Chelsea Ruller, Yongmei Feng, Rossitza Lazova, Harriet Kluger, Jian-Liang Li, Surya K. De, Robert Rickert, Maurizio Pellecchia, Marcus Bosenberg, Ze'ev Ronai. PDK1 in melanoma development and metastasis. [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 59. doi:10.1158/1538-7445.AM2014-59

Abstract 2254: Pro-angiogenic CXCL8 signalling underpins microenvironment-induced relapse to anti-androgen therapy of prostate cancer

Abstract Introduction and Objectives: Bicalutamide induces a rapid and sustained hypoxia in growing LNCaP prostatic tumors. This treatment-induced hypoxia (&amp;gt;14 days) increases synthesis of the CXC-chemokine Interleukin-8(CXCL8) co-incident with increasing oxygen tension and the restoration of tumor vascularisation1. Consequently, our current hypothesis is that hypoxia induced CXCL8 signalling underpins resistance to bicalutamide-treated tumors and facilitates the adoption of more aggressive cancer cells. Results: Culturing of LNCaP (PTEN null) and 22Rv1 (PTEN wt) cells under hypoxic conditions (mimicking a bicalutamide-treated tumor microenvironment) increased transcriptional activity of the androgen receptor (AR), Hypoxia-Inducible Factor (HIF-1) and NF-kB, inducing expression of downstream target genes associated with survival, metabolic adaptation and angiogenesis. The magnitude and duration of these responses was greater in PTEN-null LNCaP cells. Experiments were conducted to determine how bicalutamide-mediated inhibition of AR signalling would affect hypoxia-induced potentiation of AR, HIF and NF-kB transcription. Bicalutamide or an AR-targeted siRNA failed to reduce expression of pro-angiogenic, pro-survival and metabolism-associated genes. However, concurrent knockdown of both HIF-1 and NF-kB activity using gene-targeted siRNA's repressed expression of these hypoxia-induced disease-progressing genes. Amongst the genes up-regulated in hypoxic and PTEN-deficient LNCaP cells was expression of CXCL8 and its receptors, CXCR1 and CXCR2, which we have previously shown to be regulated by HIF-1 and NF-kB activity. Potentiation of CXCL8 signalling under hypoxia was found to sustain AR, HIF and NF-kB transcription. Combination of bicalutamide with the proteasome inhibitor bortezomib attenuated hypoxia-induced AR expression and transcriptional activity and decreased HIF-1 activity but failed to reduce NF-kB activity. In contrast, only direct inhibition of CXCL8 signalling was shown to repress activity of AR, HIF-1 and NF-kB in hypoxic LNCaP cells and increased LNCaP cells sensitivity to bicalutamide. Conclusion: Our data suggests that targeting CXCL8 signalling may therefore be a relevant strategy to enhance tumor response to anti-androgen therapies. Inhibiting signalling of this chemokine attenuates key transcriptional approaches induced in the hypoxic microenvironment of bicalutamide-treated tumors. Moreover, inhibition of CXCL8 signalling may prevent the re-vascularization previously observed in hypoxic LNCaP tumors. Citation Format: Melanie McKechnie, Pamela J. Maxwell, David J. J. Waugh. Pro-angiogenic CXCL8 signalling underpins microenvironment-induced relapse to anti-androgen therapy of prostate cancer. [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 2254. doi:10.1158/1538-7445.AM2014-2254

Abstract 976: BRAF inhibitor GSK2118436 - single agent and combination activity with other targeted agents in BRAF V600 mutant melanoma

Abstract Although BRAF inhibitors display promising clinical response in BRAFV600 mutant melanoma patients, not all patients respond to these agents and the durability of the response is limited. To improve BRAF targeted therapy, we characterized a panel of BRAFV600 mutant melanoma cell lines and determined their response to cell growth inhibition, apoptosis induction and cell signaling changes by GSK2118436 alone and in combination with the MEK inhibitor GSK1120212 or PI3K/mTOR inhibitor GSK2126458. Nine of 11 BRAFV600E, 4/5 BRAFV600K and 1/1 BRAFV600D mutant lines were sensitive to GSK2118436 (IC50&amp;lt;0.3µM). These sensitive lines had wild-type MEK1/2 with or without PTEN mutations. Two of the three lines insensitive to GSK2118436 had either MEK1P124S or MEK2K66_K68del mutations. GSK2118436 treatment decreased MEK and ERK phosphorylation and cyclin D1 in both the sensitive and insensitive lines. However, GSK2118436 decreased S6 ribosomal protein phosphorylation (pS6P) only in the sensitive lines. Cell lines sensitive to GSK2118436 were also sensitive to the allosteric MEK inhibitor GSK1120212 (IC50&amp;lt;0.01µM). The combination of GSK2118436 and GSK1120212 had a synergistic to additive effect on cell growth inhibition in the sensitive lines, but had minimal benefit in the three insensitive lines. Although 5/6 PTEN NULL lines were sensitive to GSK2118436 based on their IC50s, growth of these lines was inhibited less effectively by GSK2118436 compared to the PTEN WT lines evaluated by IC70. PTEN NULL cells displayed higher levels of basal AKT phosphorylation (pAKT) than PTEN WT lines. Treatment of PTEN NULL lines with GSK2118436 increased pAKT while similar treatment of PTEN WT lines decreased pAKT and increased PTEN expression. In combination with the PI3K/mTOR inhibitor GSK2126458, GSK2118436 or GSK1120212 effectively inhibited MEK/ERK and PI3K/mTOR signaling, enhanced cell growth inhibition, and induced apoptosis evaluated by caspase 3/7 activation and/or PARP cleavage in both PTEN WT and PTEN NULL lines. In PTEN deficient lines, these combinations were more effective than the combination of GSK2118436 and GSK1120212. These results indicate MEK1/2 mutations associate with lack of sensitivity to single agent BRAF and MEK inhibitors. In addition, PTEN deficiency may reduce the effectiveness of BRAF or MEK inhibitors. The combination of GSK2118436 with GSK1120212 or GSK2126458 may improve the effectiveness of BRAF targeted therapy in BRAFV600 mutant melanoma. Our results support the testing of GSK2118436 with these combinations in the clinic and warrant further investigation of MEK and PTEN mutation status and pS6P as a pharmacodynamic marker to improve treatment outcome in BRAFV600 mutant melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 976. doi:1538-7445.AM2012-976

Abstract LB-261: A novel notch-AKT-NF-KB axis in triple negative breast cancer cells

Abstract BACKGROUND: Basal-like and claudin-low are two major subgroups of triple-negative breast cancers (TNBC). The clinical subtype claudin-low is represented by MDA-MB 231 cells. The Notch and NF-κB signaling pathways are known to regulate proliferation, differentiation, and apoptosis by regulating the transcription of target genes. Both pathways are commonly active in TNBC. There is strong evidence for cross-talk between these pathways: We previously published that in cervical cancer cells, Notch-1 can activate NF-kB through nuclear IKKα. We published that Notch-1 can activate the estrogen receptor alpha (ERα), also through nuclear IKKα and the IKK signalosome in T-LL cells. However, the mechanism(s) of IKK downstream of Notch remain unknown. Notch1 is highly expressed in TNBC, and Notch ligands Jagged1 and 2 correlate with poor prognosis. IKKα (CHUK) is expressed in all subtypes of TNBC. We investigated whether Notch-1 can activate NF-kB in TNBC cells of claudin-low and basal like subtypes, whether this requires IKKα and how IKKα is activated. METHODS: We used Retroviral RNAi transduction of MDA-MB231 cells to establish Notch1 siRNA. ChIP (chromatin Immunoprecipitation), CoIP (co-immunoprecipitation), Western blot, luciferase reporter assays, co-culture MDA-MB 231 cells with LTK-JAG mouse fibroblasts, qPCR, xenograft tumors in nude mice. RESULTS: We have determined that phosphorylation of Akt is mediated through a non-canonical Notch signaling pathway and this is dependent on PTEN status. Unlike the canonical Notch pathway, Notch activation by Jagged1-expressing stroma cells leads to phosphorylation of Akt within minutes of contact between Jagged1-expressing stroma cells in PTEN wt, MDA-MB 231 cells through a non-transcriptional mechanism. Our data suggest that Notch complexes with PI3K subunits, p85 and p110, after ligand-mediated activation. Inhibition of Notch activity through GSIs (γ-secretase inhibitors) leads to decreased phosphorylation of Akt. Dominant-negative AKT abolishes IKKα phosphorylation. Additionally, the combination of the AKT inhibitor, perifosine and gamma-secretase inhibitor MRK-003 is synergistic in vitro and highly active in MDA-MB231 xenografts. CONCLUSIONS: Our findings suggest a novel non-canonical cross-talk mechanism including Jagged, Notch, AKT and IKKα connecting three major oncogenic pathways in TNBC: Notch, AKT and NF-kB. Moreover, our findings suggest that Akt inhibitors may be combined with GSI for therapeutic purposes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-261. doi:1538-7445.AM2012-LB-261

Protein Interactions of Phosphatase and Tensin Homologue (PTEN) and Its Cancer-associated G20E Mutant Compared by Using Stable Isotope Labeling by Amino Acids in Cell Culture-based Parallel Affinity Purification

The tumor suppressor PTEN (phosphatase and tensin homologue) negatively regulates the PI3K pathway through its lipid phosphatase activity and is one of the most commonly lost tumor suppressors in human cancers. Though the tumor suppressive function involves the lipid phosphatase-dependent and -independent activities of PTEN, the mechanism leading to the phosphatase-independent function of PTEN is understood poorly. Some PTEN mutants have lipid phosphatase activity but fail to suppress cell growth. Here, we use a cancer-associated mutant, G20E, to gain insight into the phosphatase-independent function of PTEN by investigating protein-protein interactions using MS-based stable isotope labeling by amino acids in cell culture (SILAC). A strategy named parallel affinity purification (PAP) and SILAC has been developed to prioritize interactors and to compare the interactions between wild-type and G20E PTEN. Clustering of the prioritized interactors acquired by the PAP-SILAC approach shows three distinct clusters: 1) wild-type-specific interactors, 2) interactors unique to the G20E mutant, and 3) proteins common to wild-type and mutant. These interactors are involved mainly in cell migration and apoptosis pathways. We further demonstrate that the wild-type-specific interactor, NUDTL16L1, is required for the regulatory function of wild-type PTEN in cell migration. These findings contribute to a better understanding of the mechanisms of the phosphatase-dependent and -independent functions of PTEN.

Targeted combinatorial therapy of androgen receptor in androgen-independent prostate cancer cells.

29 Background: Gain of function of androgen receptor (AR) and PI3K/Akt/mTOR pathway due to loss of PTEN function are interrelated determinants of AIPC progression and resistance to antiandrogens. We determined that histone deacetylase inhibitors (HDACi), known to regulate AR expression and transcriptional activity, restore sensitivity to bicalutamide (Bic) of AIPC cells and the combination is synergistic. We postulate that adding a dual inhibitor of PI3K/mTOR would potentiate growth inhibition and apoptotic response. Methods: Androgen-independent, PTEN null AI-LNCaP (AI-cells) and wt PTEN RV1-cell lines were treated for 72h with the HDACi Panobinostat (PAN), the dual PI3K/mTOR inhibitor BEZ235 (E) and Bic as single agents or in doublet or triplet combinations. Growth was measured by MTT assay, Akt/mTOR pathway target proteins p-Akt, p-4EBP1 and p-S6K by western blot. Activation of ligand-independent AR downstream targets: CDC20, IDI1, CDK1, UBE2C, PRDM4 by qRT-PCR. Apoptosis by oligonucleosome quantification by ELISA was compared to docetaxel. Results: In both cell lines, the IC50s were markedly reduced in response to the doublet combinations and further decreased by the triplet. The isobolograms showed synergy. The apoptosis response increased by two fold in AI and RV1 cells in response to the doublet PAN+E and by four fold with the addition of Bic in AI cells. Only E decreased the protein levels of p473-akt, p-4EBP1, p-S6K markedly while only PAN decreased AR and these levels were not affected by the combinations. However, the triplet but not the doublets decreased by 2-fold the expression of AR targets in AI-cells but not in RV1 cells. Conclusions: The combination of PAN+E is highly effective in AIPC cells regardless of PTEN status but the addition of Bic is essential to maximize the effect in PTEN null AI-cells, suggesting they are more dependent on aberrant activation of AR. [Table: see text] No significant financial relationships to disclose.

Invasiveness and anchorage independent growth ability augmented by PTEN inactivation through the PI3K/AKT/NFkB pathway in lung cancer cells

PTEN is inactivated in a subset of lung cancer; therefore, we investigated the involvement of PTEN inactivation in invasiveness of lung cancer cells. AKT at Ser473 was phosphorylated in several lung cancer cell lines with loss of PTEN expression. Therefore, we created a tetracycline inducible expression system of wild-type PTEN (PTEN-WT) as well as catalytically (PTEN-G129R) and lipid phosphatase (PTEN-G129E) inactive PTEN mutants using the PC14, PC9 and PC3 lung adenocarcinoma cell lines, in which endogenous PTEN expression was not detected and AKT at Ser473 was phosphorylated by Western blot analysis. Induction of PTEN-WT reduced phosphorylation of AKT and inhibited the transcriptional activity of NFkB, whereas PTEN mutants did not, suggesting that PTEN inactivation results in the activation of the AKT/NFkB pathway in PC14, PC9 and PC3 cells. Furthermore, overexpression of PTEN-WT suppressed anchorage independent growth in soft agar and reduced invasiveness in a trans-well chamber assay of PC14 cells. Neither PTEN-G129R nor PTEN-G129E had suppressive effects on anchorage independent growth and invasiveness. Augmentation of invasiveness by constitutively active AKT was also shown in mouse NIH3T3 cells. Therefore, it was strongly indicated that activation of the PI3K/AKT/NFkB pathway by PTEN inactivation results in augmented invasiveness in lung cancer cells and lipid phosphatase activity of PTEN plays a key role in this process.

Abstract SY28-04: Rational incorporation of novel agents into multimodality treatment of glioma and neuroblastoma

Abstract SY28-04: Rational incorporation of novel agents into multimodality treatment of glioma and neuroblastoma

Abstract 3125: EGFRvIII translocates to the nucleus and regulates gene transcription with STAT5

Abstract Dysregulation of receptor tyrosine kinases is a major contributor to cancer, including glioblastoma, where amplification and mutation of epidermal growth factor receptor (EGFR) is common. The most common mutation is in-frame deletion of exons 2-7, known as EGFRvIII, EGFR* or ΔEGFR. EGFRvIII confers enhanced tumorigenicity on glioma cells in vivo, reducing apoptosis and increasing proliferation. EGFRvIII signals constitutively and at approximately 5-to-10-fold lower intensity than wild-type. We are investigating the pathways that are preferentially activated by EGFRvIII, using a phosphotyrosine-directed, mass spectrometry-based shotgun phosphoproteomics approach in two different cell lines, LNZ308 (PTEN null) and LN428 (PTEN wt). We identified 433 proteins with 772 peptides out of which 249 proteins corresponding to 354 peptides showed tyrosine phosphorylation. Statistical analysis revealed several signals that were prevalent in EGFRvIII expressing glioma cells, including phosphorylation of STAT5, Gab1 and MIG6. Identification of STAT5 as a target of EGFRvIII prompted an investigation of whether EGFRvIII is active in the nucleus. We have identified EGFRvIII associated with phosphorylated STAT5 on chromatin, and capable of activating the expression of STAT5 target genes, including Aurora A kinase, which positively regulate glioblastoma malignancy. STAT5 promotes expression of the EGFRvIII target gene Bcl-XL, and knockdown of STAT5 reduces the transformation induced by EGFRvIII. Furthermore, we have mutated signals in EGFRvIII that regulate nuclear localization, and are examining the impact of these on its ability to promote tumor growth. In a related study we have used artificial receptor dimerization, mediated by technology from Ariad Pharmaceuticals, to enhance the EGFRvIII signal. A chimeric version of EGFRvIII was created, in which the receptor was fused N-terminally with two FKBP-F36V domains that could be activated by the chemical inducer of dimerization, AP20187. This has resulted in a more oncogenic form of EGFRvIII that emits a stronger signal, and we are using this to deepen our phosphoproteomics analysis to seek additional preferential targets of EGFRvIII action, which will be reported. 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 3125.

Ubiquitination of PTEN (Phosphatase and Tensin Homolog) Inhibits Phosphatase Activity and Is Enhanced by Membrane Targeting and Hyperosmotic Stress

The PTEN (phosphatase and tensin homolog) tumor suppressor is a phosphatase that inhibits phosphoinositide 3-kinase-dependent signaling by metabolizing the phosphoinositide lipid phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)) at the plasma membrane. PTEN can be mono- or polyubiquitinated, and this appears to control its nuclear localization and stability, respectively. Although PTEN phosphorylation at a cluster of C-terminal serine and threonine residues has been shown to stabilize the protein and inhibit polyubiquitination and plasma membrane localization, details of the regulation of ubiquitination are unclear. Here, we show that plasma membrane targeting of PTEN greatly enhances PTEN ubiquitination and that phosphorylation of PTEN in vitro does not affect subsequent ubiquitination. These data suggest that C-terminal phosphorylation indirectly regulates ubiquitination by controlling membrane localization. We also show that either mono- or polyubiquitination in vitro greatly reduces PTEN phosphatase activity. Finally, we show that hyperosmotic stress increases both PTEN ubiquitination and cellular PtdInsP(3) levels well before a reduction in PTEN protein levels is observed. Both PTEN ubiquitination and elevated PtdInsP(3) levels were reduced within 10 min after removal of the hyperosmotic stress. Our data indicate that ubiquitination may represent a regulated mechanism of direct reversible control over the PTEN enzyme.