Lacking PTEN Expression Research Articles

Targeting mTOR Pathway in PTEN Deleted Newly Isolated Chordoma Cell Line.

Chordomas are rare primary malignant tumours of notochordal origin usually arising along the axial skeleton with particular predilection of the skull base and sacrococcygeal region. Albeit usually slow-growing, chordomas can be aggressive mostly depending on their invasive behaviour and according to different histotypes and molecular alterations, including TBXT duplication and SMARCB1 homozygous deletion. Partial or complete PTEN deficiency has also been observed. PTEN is a negative regulator of the Akt/mTOR pathway and hyperactivation of Akt/mTOR in cells lacking PTEN expression contributes to cell proliferation and invasiveness. This pathway is targeted by mTOR inhibitors and the availability of in vitro models of chordoma cells will aid in further investigating this issue. However, isolation and maintenance of chordoma cell lines are challenging and PTEN-deleted chordoma cell lines are exceedingly rare. Hereby, we established and characterized a novel human PTEN-deleted chordoma cell line (CH3) from a primary skull base chordoma. Cells exhibited morphological and molecular features of the parent tumour, including PTEN loss and expression of Brachyury and EMA. Moreover, we investigated the activation of the mTOR pathway and cell response to mTOR inhibitors. CH3 cells were sensitive to Rapamycin treatment suggesting that mTOR inhibitors may represent a valuable option for patients suffering from PTEN-deleted chordomas.

Combined inhibition of topoisomerase I and poly(ADP-ribose) polymerase: A synergistic therapeutic strategy for glioblastoma with phosphatase and tensin homolog deficiency.

Deletions or loss-of-function mutations in phosphatase and tensin homolog (PTEN) are common in glioblastoma (GBM) and have been associated with defective DNA damage repair. Here we investigated whether PTEN deficiency presents a vulnerability to a simultaneous induction of DNA damage and suppression of repair mechanisms by combining topoisomerase I (TOP1) and PARP inhibitors. Patient-derived GBM cells and isogenic PTEN-null and PTEN-WT glioma cells were treated with LMP400 (Indotecan), a novel non-camptothecin TOP1 inhibitor alone and in combination with a PARP inhibitor, Olaparib or Niraparib. RNAseq analysis was performed to identify treatment-induced dysregulated pathways. We found that GBM cells lacking PTEN expression are highly sensitive to LMP400; however, rescue of the PTEN expression reduces sensitivity to the treatment. Combining LMP400 with Niraparib leads to synergistic cytotoxicity by inducing G2/M arrest, DNA damage, suppression of homologous recombination-related proteins, and activation of caspase 3/7 activity significantly more in PTEN-null cells compared to PTEN-WT cells. LMP400 and Niraparib are not affected by ABCB1 and ABCG2, the major ATP-Binding Cassette (ABC) drug efflux transporters expressed at the blood-brain barrier (BBB), thus suggesting BBB penetration which is a prerequisite for potential brain tumor treatment. Animal studies confirmed both an anti-glioma effect and sufficient BBB penetration to prolong survival of mice treated with the drug combination. Our findings provide a proof of concept for the combined treatment with LMP400 and Niraparib in a subset of GBM patients with PTEN deficiency.

EXTH-03. COMBINED TOP1 AND PARP INHIBITION ENFORCES GENOMIC INSTABILITY AND CELL DEATH IN PTEN-DEFICIENT GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma is an aggressive brain tumor with high mortality. The development of new therapies is critical for improving patient outcomes. LMP400, a novel topoisomerase I (TOP1) inhibitor, traps TOP1 cleavage complexes, thereby generating DNA damage. Poly(ADP-ribose) polymerase (PARP) is involved in DNA repair responses triggered by TOP1 inhibition. Niraparib is a potent PARP inhibitor that can cross the blood-brain barrier. Loss of phosphatase and tensin homolog (PTEN) occurs in 40% of GBM patients and is known to promote DNA damage repair deficiency. Here, we hypothesize that PTEN loss presents a vulnerability to a combined induction of DNA damage and inhibition of repair mechanisms. METHODS Human glioblastoma cells (U251, SNB-75, SF-295, LN18) and patient-derived glioblastoma stem cells (GSC923 and GSC827) were treated with LMP400 and/or Niraparib. Cell viability and apoptosis were examined using Celigo image cytometer and Annexin V/PI assay at 72h after treatment. Single clones after PTEN knockdown using shRNA were isolated after puromycin selection. For planned studies of PTEN knockout, sgRNA plasmids targeting PTEN will be transiently transfected and GFP-positive single KO clones will be isolated. PTEN will be restored in PTEN-null cells using lentiviral transduction. RESULTS CRISPR-Cas9 KO screening in GSC923 cells suggests that LMP400 is unlikely a substrate for ABC transporters. LMP400 and Niraparib synergistically induced cytotoxic effects in U251, SF-295, GSC923, GSC827 cells lacking PTEN expression. Combined LMP400/Niraparib led to increased expression of gamma-H2AX, cleaved caspase 3 and PARP, indicative of enhanced DNA damage and cell death. CONCLUSION LMP400 and Niraparib act synergistically to target PTEN-deficient glioblastoma by inducing DNA damage and cell death. These results will be further verified in isogenic cells in vitro as well as in vivo in a mouse model driven by PTEN deletion which would strongly support a novel therapeutic strategy in a subset of glioblastoma with PTEN loss.

Lack of PTEN in osteocytes increases circulating phosphate concentrations by decreasing intact fibroblast growth factor 23 levels

Fibroblast growth factor 23 (FGF23) has been centric to the regulation of phosphate (Pi) metabolism; however, the regulatory network of FGF23 in osteocytes has not yet been defined in detail. We herein investigated the role of PTEN (phosphatase and tensin homolog deleted from chromosome 10) in this regulation. We created mice lacking PTEN expression mainly in osteocytes by crossing Pten-flox mice with Dmp1-Cre mice. The lack of PTEN in the osteocytes of these mice was associated with decreased skeletal and serum intact FGF23 levels, which, in turn, resulted in reductions of urinary Pi excretion and elevations of serum Pi levels. Mechanistically, the knockdown of PTEN expression in osteoblastic UMR106 cells activated the AKT/mTORC1 (mechanistic target of rapamycin complex 1) pathway and this was associated with reductions in Fgf23 expression. Furthermore, the suppression of Fgf23 expression by PTEN knockdown or insulin simulation in UMR106 cells was partially restored by the treatment with the mTORC1 inhibitor, rapamycin. These results suggest that FGF23 expression in osteoblastic cells is in part regulated through the AKT/mTORC1 pathway and provide new insights into our understanding of the regulatory network of Pi metabolism.

Role of androgen receptor splice variant-7 (AR-V7) in prostate cancer resistance to 2nd-generation androgen receptor signaling inhibitors.

The role of truncated androgen receptor splice variant-7 (AR-V7) in prostate cancer biology is an unresolved question. Is it simply a marker of resistance to 2nd generation androgen receptor signaling inhibitors (ARSi) like Abiraterone Acetate (Abi) and Enzalutamide (Enza) or a functional driver of lethal resistance via its ligand-independent transcriptional activity? To resolve this question, the correlation between resistance to ARSi and genetic chances and expression of full length AR (AR-FL) vs. AR-V7 were evaluated in a series of independent patient-derived xenografts (PDXs). While all PDXs lack PTEN expression, there is no consistent requirement for mutation in TP53, RB1, BRCA2, PIK3CA, or MSH2, or expression of SOX2 or ERG and ARSi-resistance. Elevated expression of AR-FL alone is sufficient for Abi- but not Enza-resistance, even if AR-FL is gain-of-function (GOF) mutated. Enza-resistance is consistently correlated with enhanced AR-V7 expression. In vitro and in vivo growth responses of Abi-/Enza-resistant LNCaP-95 cells in which CRISPR-Cas9 was used to knockout AR-FL or AR-V7 alone or in combination were evaluated. Combining these growth responses with RNAseq analysis demonstrates that both AR-FL and AR-V7 dependent transcriptional complementation are needed for Abi/Enza resistance.

Modulation of PI3K/PTEN Pathway Does Not Affect Catalytic Activity of PDK1 in Jurkat Cells.

Unopposed phosphoinositide 3-kinase (PI3K) activity and 3-phosphoinositide production in Jurkat cells, due to a mutation in the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor-suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB. In Jurkat cells, PKB is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3-Phosphoinositide-dependent protein kinase-1 (PDK1), an enzyme that also contains a PH domain, catalyses Thr308 phosphorylation of PKB in addition to other kinase families such as PKC isoforms. It is unknown, however, whether the loss of PTEN in Jurkat cells also results in unregulated PDK1 activity and whether such loss has an impact on activation-loop phosphorylation of other PDK1 substrates e.g. PKC. In this study, we addressed whether loss of PTEN in Jurkat cells affects PDK1 catalytic activity and intracellular localization. We demonstrated that reducing the level of 3-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3K or expression of PTEN does not affect PDK1 activity or its intracellular localization. We conclude, therefore, that although Jurkat cells lack PTEN expression, only a subset of pathways downstream of PDK1 are perturbed as a consequence of PTEN loss.

Immunohistochemical analysis of PTEN, HER2/neu, and ki67 expression in patients with gastric cancer and their association with survival

BackgroundConsidering the poor prognosis of patients with gastric cancer, molecular diagnostic and prognostic markers for this cancer should be established. The aims of our study were to assess the correlations between PTEN, HER2/neu, and Ki67 expressions and clinicopathological factors of gastric cancer patients in upper Egypt, as well as their influence on OS and DFS. Patients and methodsIn this descriptive-analytic study, 42 patients with gastric carcinoma treated by postioerative chemoradiation between 2004 and 2014. Pathological review was done. Immunohistochemical staining and evaluation were performed. ResultsAll the studied markers were significantly correlated with increased TNM stage. Her2/neu overexpression and positive Ki67 expression were significantly associated with histological grade. High percentage of positive Her2/neu and Ki67expression was found in gastric carcinoma tissue samples which lack PTEN expression. The one-year OS rate for the entire group (n=42) was 77.4%, whereas the DFS rate was 45%. Pathological T stage PTEN status significantly affected both OS (p=0.029 and 0.027 respectively) and DFS (p=0.006 and 0.012 respectively) rates. Multivariate Cox analyses showed that only pathological T stage was an independent prognostic factor affecting OS (P=0. 007, HR: 2.02; 95% CI: 1.2–3.38)and DFS(P<0.0001, HR: 2.69; 95% CI: 1.54–4.69). ConclusionAll the studied molecular markers, was significantly correlated with pathological T stage that significantly affected both OS and DFS rates. These findings indicate that these markers have an important role in gastric cancer growth and dissemination so these markers can be used as a prognostic biomarker. In addition, therapies targeting Her2 and PTEN may help develop novel therapeutics for gastric cancer.

Abstract 2618: PTEN loss as a putative biomarker of synergistic growth inhibitory activity of combined MEK/ERK and PI3K/mTOR pathway blockade

Abstract Background. Complex feedback loops and crosstalk between the MEK/ERK and PI3K/mTOR pathways, thus molecular predictors of sensitivity/resistance and synergistic/antagonistic interactions are urgently needed for the effective clinical development of combination strategies. Here we hypothesize that PTEN status critically modulates the growth inhibitory activity of single and combined MEK and PI3K/mTOR inhibition. Methods. Molecular and functional effects of single and combined MEK (trametinib, T) and mTOR (everolimus, E) blockade were assessed in a panel of 29 cancer cell lines with different molecular ‘drivers’. Pharmacologic interactions were analyzed by conservative isobologram analysis. PTEN role was mechanistically assessed by either silencing PTEN expression by shRNA or overexpressing a functional PTEN protein by stable transfection. Specific proteins and their phosphorylation states were analyzed using Kinexus Antibody Microarrays. Results. In the cell line panel analyzed, the presence of a wt-PTEN was the only significant predictor of sensitivity to T (p=0.001), while BRAF mutations were significantly related to E resistance (p=0.015). PTEN silencing slightly increased resistance to T in the wt-PTEN melanoma cell line M14; conversely, wt-PTEN overexpression in the PTEN-del WM115 cell line, dramatically increased sensitivity to T. E-induced growth inhibition was not significantly affected. Combined MEK and mTOR inhibition resulted in a striking growth inhibitory synergism in cells lacking PTEN expression (CI: 0.4-0.0005), but not in those with an intact PTEN (CI: 1.2-107; p=0.001). However, PTEN silencing restored growth inhibitory synergism of combined T and E in M14 cells (CI:0.36); similarly, the slope of the CI/fraction affected curve was dramatically altered in PTEN-overexpressing WM115 cells, as compared to their control-transfected counterpart, again indicating that PTEN expression/function causally influences functional response to combined MEK and mTOR inhibition. Similar results in terms of synergistic/antagonistic pharmacologic interactions were obtained when either double PI3K/mTOR kinase inhibitors (PF-5212384) or AKT allosteric inhibitors (MK-2206) were used to block the PI3K/mTOR pathway in combination with T. Proteomic analysis indicated that a greater modification of protein expression/phosphorylation profiles in response to MEK or combined MEK/mTOR inhibition occurs in cells lacking a functional PTEN, as compared to the wt-PTEN-expressing ones; preliminary analysis suggests that AKT phosphorylation and NF-κB activation maybe be crucial mediators of synergistic growth-inhibitory interactions occurring with combined treatment. Conclusions. PTEN loss may constitute a suitable genetic/molecular marker of synergistic activity interactions between MEK/ERK and PI3K/mTOR pathway inhibitors. Citation Format: Ludovica Ciuffreda, Italia Falcone, Silvia Matteoni, Andrea Sacconi, Federico Malusa, Teresa De Luca, Ursula Cesta Incani, Anais Del Curatolo, Marina Konopleva, Michael Andreeff, Adriana Eramo, Ruggero De Maria, Donatella Del Bufalo, Francesco Cognetti, Michele Milella. PTEN loss as a putative biomarker of synergistic growth inhibitory activity of combined MEK/ERK and PI3K/mTOR pathway blockade. [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 2618. doi:10.1158/1538-7445.AM2014-2618

Synergistic Growth Inhibitory Activity of Combined Mek/Mtor Pathway Blockade in Pten-Null Cancers

ABSTRACT Aim: We have recently shown that induction of PTEN expression plays an integral role in MEK inhibitors' antitumor activity. Here we hypothesize that PTEN status critically influences the functional outcome of combined MEK and PI3K/mTOR inhibition. Methods: Single and combined MEK (trametinib, T) and mTOR (everolimus, E) blockade were assessed in a panel of cancer cell lines and in patient-derived lung and colon cancer stem cells (L- or C-CSC). Pharmacologic interactions were analyzed by conservative isobologram analysis. PTEN role was assessed by shRNA-mediated silencing or overexpression by stable transfection. Treatment-induced changes in the phosphoproteome were analyzed by antibody microarrays. Results: The presence of a wt-PTEN was the only significant predictor of sensitivity to T (p=0.001) in cancer cell lines. Combined MEK and mTOR inhibition resulted in a striking growth inhibitory synergism only in cells lacking PTEN expression (CI: 0.4-0.0005). PTEN silencing or its overexpression dramatically altered both sensitivity to T and the nature of its interaction with E (synergism vs antagonism), indicating PTEN as a fundamental determinant of functional response to combined MEK/mTOR inhibition. Similar results were obtained when either the double PI3K/mTOR kinase inhibitor PF-5212384 or the AKT allosteric inhibitors MK-2206 were substituted for E in combination with T. Concomitant T+E treatment was also highly synergistic in 3/5 L-CSC in vitro and, most importantly, in a C-CSC-derived model in vivo. Proteomic analysis indicated that a greater modification of protein expression/phosphorylation profiles in response to MEK or combined MEK/mTOR inhibition occurs in cells lacking a functional PTEN; preliminary analysis suggests that AXL, JAK1, and MAPK14 maybe be crucial mediators of synergistic growth-inhibitory interactions occurring with combined treatment. Conclusions: PTEN loss may constitute a suitable genetic/molecular marker of synergistic activity interactions between MEK/ERK and PI3K/mTOR pathway inhibitors and could be proposed as a potential patient selection biomarker for trials exploring such horizontal combinations. Disclosure: All authors have declared no conflicts of interest.

PTEN expression and function in adult cancer stem cells and prospects for therapeutic targeting.

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a non-redundant lipid phosphatase that restrains and fine tunes the phosphatidylinositol-3-kinase (PI3K) signaling pathway. PTEN is involved in inherited syndromes, which predispose to different types of cancers and is among the most frequently inactivated tumor suppressor genes in sporadic cancers. Indeed, loss of PTEN function occurs in a wide spectrum of human cancers through a variety of mechanisms, including mutations, deletions, transcriptional silencing, or protein instability. PTEN prevents tumorigenesis through multiple mechanisms and regulates a plethora of cellular processes, including survival, proliferation, energy metabolism and cellular architecture. Moreover, recent studies have demonstrated that PTEN is able to exit, exist, and function outside the cell, allowing for inhibition of the PI3K pathway in neighboring cells in a paracrine fashion. Most recently, studies have shown that PTEN is also critical for stem cell maintenance and that PTEN loss can lead to the emergence and proliferation of cancer stem cell (CSC) clones. Depending on the cellular and tissue context of origin, PTEN deletion may result in increased self-renewal capacity or normal stem cell exhaustion and PTEN-defìcient stem and progenitor cells have been reported in prostate, lung, intestinal, and pancreatic tissues before tumor formation; moreover, reversible or irreversible PTEN loss is frequently observed in CSC from a variety of solid and hematologic malignancies, where it may contribute to the functional phenotype of CSC. In this review, we will focus on the role of PTEN expression and function and downstream pathway activation in cancer stem cell biology and regulation of the tumorigenic potential; the emerging role of PTEN in mediating the crosstalk between the PI3K and MAPK pathways will also be discussed, together with prospects for the therapeutic targeting of tumors lacking PTEN expression.

Vandetanib is effective in EGFR-mutant lung cancer cells with PTEN deficiency

The effectiveness of vandetanib, an agent that targets RET, VEGFR and EGFR signaling, against EGFR-mutant lung cancer cells with PTEN loss was investigated. Two EGFR mutant non-small cell lung cancer (NSCLC) cell lines, PC-9 (PTEN wild type) and NCI-H1650 (PTEN null), were used. We transfected an intact PTEN gene into H1650 cells and knocked down PTEN expression in PC-9 cells using shRNA. The effectiveness of gefitinib and vandetanib was assessed using a xenograft model. While PC-9 cells were more resistant to vandetanib than gefitinib, H1650 cells were more sensitive to vandetanib than gefitinib. Both gefitinib and vandetanib suppressed the activation of EGFR and MAPK in H1650 cells, although phosphorylated AKT levels were not affected. In an H1650 cell xenograft model, vandetanib was also more effective than gefitinib. Although PTEN-transfected H1650 cells did not show restoration of sensitivity to gefitinib in vitro, the xenograft tumors responded to gefitinib and vandetanib. Knockdown of PTEN in PC-9 cells caused resistance to gefitinib. In conclusion, vandetanib might be effective in NSCLC with EGFR mutations that lack PTEN expression. The contribution of PTEN absence to vandetanib activity in NSCLC cells harboring EGFR mutations should be further examined.

Abstract 5222: A novel mechanism of microenvironment-mediated drug resistance in BRAF V600E melanoma with loss of PTEN

Abstract Despite recent success of the BRAF kinase inhibitor vemurafenib in the clinic, nearly all of those treated follow a course in which initial tumor regression was followed by eventual relapse, which constitutes a major barrier to long-term therapeutic management of melanoma. We have identified a novel mechanism of microenvironment-mediated drug resistance that arises from the release of TGFβ from melanomas that lack PTEN expression leading to the recruitment of host fibroblasts and the generation of a protective microenvironment through autocrine and paracrine fibronectin secretion facilitating therapeutic escape. Preliminary studies show that BRAF V600E mutant melanoma cell lines that lacked PTEN expression dramatically increased their secretion of TGF-β following vemurafenib treatment, as detected by western blot, immunofluorescence and ELISA assays. Furthermore, autocrine TGFβ secretion initiated with vemurafinib treatment stimulated the upregulation and secretion of fibronectin in PTEN- cells. The vemurafenib-mediated ECM remodeling in this case was dependent upon autocrine TGFβ signaling and could be attenuated following pre-treatment with the TGFβ receptor kinase inhibitor SB505124 or by siRNA knockdown of TGFβ receptor I. A mass spectrometry based phospho-proteomic screen in conjunction with innovative network modeling approaches suggested that increased integrin α5β1 signaling was a key adaptive response of PTEN- melanoma cells to BRAF inhibitor therapy. The importance of increased fibronectin expression and integrin α5β1 signaling in the therapeutic escape of PTEN- melanoma cells was confirmed by the significant increases in vemurafenib-induced apoptosis observed following siRNA knockdown of either integrin α5, β1, or fibronectin expression. A mass spectrometry screen (LC-MRM) of 18 BH3 proteins demonstrated Mcl-1 and Bak to be critical apoptosis regulators in the microenvironment-mediated drug resistance response. The importance of PTEN loss and Mcl-1 downregulation in therapeutic escape was validated in PTEN- melanoma cell lines expressing doxycycline inducible forms of PTEN, lipid phosphatase mutant PTEN and Mcl-1. Novel organotypic co-culture models were developed to demonstrate that TGF-β released from PTEN- melanoma cell lines activated host fibroblasts leading to the attenuation of drug response in the tumor cells. Our overall hypothesis is that BRAF inhibition remodels both the melanoma and host microenvironments to provide a protective “sanctuary” for the minor populations of melanoma cells that escape therapy. We postulate that tumor eradication can only be achieved by a dual therapeutic strategy that targets both the bulk tumor and the microenvironment. 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 5222. doi:1538-7445.AM2012-5222

Abstract 1752: A phase I/IIa, first time in human, open-label dose-escalation study of GSK2636771 in subjects with advanced solid tumors with PTEN deficiency

Abstract Background: The phosphoinositide 3-kinase (PI3K) pathway is among the most commonly mutated pathways in human cancer. The majority of first-generation PI3K inhibitors currently in clinical trials inhibit multiple PI3K isoforms. The PIK3CB gene encodes p110α isoform, which has been reported to be required for pathway activation in phosphatase and tensin homolog (PTEN) deficient cancer cells [Wee, 2008; Jia 2008]. The observed dependence of PTEN-deficient tumors on p110α provides an opportunity for identification of a novel isoform-selective therapeutic. In addition, as PTEN is commonly mutated, deleted, or epigenetically silenced in a wide range of solid tumors, measurement of PTEN protein expression would provide an identifiable target patient population using a clinically-available biomarker. Methods: GSK2636771 (‘771) is a potent, orally bioavailable, beta isoform-selective inhibitor of PI3K. Study P3B115717 is a Phase I/IIa first-time-in-human dose-escalation study of ‘771. The objectives of the study include the characterization of the safety, tolerability, and pharmacokinetic (PK) profile of ‘771 following single- and repeat-dose administration, evaluation of the pharmacodynamic (PD) effects of ‘771 in tumor and surrogate tissue, and evaluation of the anti-tumor effects of ‘771 in advanced solid tumors with PTEN deficiency. A standard 3+3 dose escalation scheme will be used, however this study incorporates multiple elements designed to accelerate clinical development. These include a single-dose phase for rapid determination of a pharmacokinetically acceptable starting dose, a “backfill” strategy for acquiring parallel samples for PD analyses, mandatory tumor biopsies for select patient cohorts, inclusion of subjects with glioblastoma multiformae, and, importantly, only patients whose tumors lack PTEN expression are eligible for this study, beginning with dose escalation. PD and anti-tumor response will be assessed in-stream to determine biologically effective doses. Anti-tumor efficacy in three tumor types with high prevalence of PTEN loss will be assessed using volumetric imaging in addition to RECIST 1.1, as well as circulating tumor cell enumeration for prostate and breast tumors. Exploratory analyses include evaluation of alterations in tumor growth rates following treatment with ‘771 compared to patient's prior lines of therapy, correlation of serum markers (e.g., IGFBP-2) with tumor PTEN expression, comparison of anti-tumor response to DNA point mutations detected in circulating tumor DNA, and PD assessment in a surrogate tissue (change in pAKT, pGSK3β, and pS6 in platelets). This study initiated in November 2011 and is currently accruing patients (GSK Study P3B115717 - NCT01458067). 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 1752. doi:1538-7445.AM2012-1752

939 poster EFFECTS OF RADIATION-INDUCED ALPHAVBETA3 INTEGRIN MODULATION ON CILENGITIDE EFFICIENCY IN PEDIATRIC GLIOMA CELLS

The efficacy of PARP inhibition under treatment conditions of RT plus temozolomide (TMZ) for glioblastoma (GBM) has not been previously reported, therefore we sought to determine the effects of PARP inhibition as well as the molecular determinants of response to PARP inhibitors in combination with standard treatment of GBM. Materials: In the present study, PJ-34, a highly potent PARP inhibitor, was used to investigate the effects of PTEN status on PARP inhibition in combination with RT and TMZ using in vitro and in vivo models of GBM. First, two primary GBM cell lines that lack PTEN expression and two primary GBM cells lines that express PTEN were utilized to determine if PTEN status affects the efficacy of PARP inhibition in combination with RT and TMZ. Next, DNA damage and ATP levels were measured after the addition of a PARP inhibitor to RT and TMZ with respect to PTEN status. The role of glucose metabolism and PI3K/ AKT signaling in the response to PARP inhibition was then characterized in correlation to PTEN status. Finally, a U87-MG xenograft model was used to validate our in vitro findings. Results: Cell lines lacking PTEN were sensitized to PJ-34 in the setting of RT plus TMZ, whereas PTEN expression was associated with resistance. AKT activation was higher in resistant cell lines and was associated with increased ATP levels. Additionally, forced expression of PTEN conferred resistance to PJ-34. Treatment with the glycolytic inhibitor 2-deoxyglucose (2-DG) or the phosphatidylinosotide-3 kinase (PI3K) inhibitor LY294002 sensitized resistant PTEN-expressing cell lines to PJ-34 suggesting PI3K/AKT signaling is likely responsible for resistance in PTEN positive cells. The therapeutic benefits of PARP inhibition observed in PTEN-null cell lines may be dependent on RT, as treatment with TMZ alone prevented sensitization. Conclusions: In summary, PTEN status may be a promising marker to identify tumors that will respond to PARP inhibition when administered with TMZ and RT. Our findings suggest that the ability of PARP inhibitors, such as PJ34, to enhance the anti-tumor effects of RT and TMZ is highly dependent on the equilibrium between PARP’s pro-survival (increased PI3K/AKT signaling) and pro-death (increased DNA damage) functions. To this end, we are the first to report that the ability of PARP inhibition to enhance TMZ and RT response depends on the proteomic profile of GBM tumors, especially PTEN status.

Abstract 5370: PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression

Abstract The current study has addressed the role of PTEN loss in intrinsic resistance to the BRAF inhibitor PLX4720. Immunohistochemical staining of a tissue array covering all stages of melanocytic neoplasia (n=147) revealed PTEN expression to be lost in &amp;gt;10% of all melanoma cases. Although PTEN status did not predict for sensitivity to the growth inhibitory effects of PLX4720, it was predictive for apoptosis, with little cell death observed in melanomas lacking PTEN expression (PTEN-). Mechanistically, PLX4720 was found to stimulate AKT signaling in the PTEN- but not the PTEN+ cell lines. Liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) was performed to identify differences in apoptosis signaling between the two cell line groups. PLX4720 treatment significantly increased BIM expression in the PTEN+ (&amp;gt;14-fold) compared to the PTEN- cell lines (4-fold). A role for PTEN in the regulation of PLX4720-mediated BIM expression was confirmed by siRNA knockdown of PTEN and through re-introduction of PTEN into cells that were PTEN-. Further studies showed that siRNA knockdown of BIM significantly blunted the apoptotic response in PTEN+ melanoma cells. Dual treatment of PTEN- cells with PLX4720 and a PI3K inhibitor enhanced both BIM expression and the level of apoptosis. The importance of rebound PI3K/AKT signaling in BRAF inhibitor resistance was confirmed in 3D organotypic cell culture and long-term colony formation assays. In conclusion, we have shown for the first time that loss of PTEN contributes to intrinsic BRAF inhibitor resistance via the suppression of BIM-mediated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5370. doi:10.1158/1538-7445.AM2011-5370

PTEN Loss Confers BRAF Inhibitor Resistance to Melanoma Cells through the Suppression of BIM Expression

This study addresses the role of PTEN loss in intrinsic resistance to the BRAF inhibitor PLX4720. Immunohistochemical staining of a tissue array covering all stages of melanocytic neoplasia (n = 192) revealed PTEN expression to be lost in >10% of all melanoma cases. Although PTEN expression status did not predict for sensitivity to the growth inhibitory effects of PLX4720, it was predictive for apoptosis, with only limited cell death observed in melanomas lacking PTEN expression (PTEN-). Mechanistically, PLX4720 was found to stimulate AKT signaling in the PTEN- but not the PTEN+ cell lines. Liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) was performed to identify differences in apoptosis signaling between the two cell line groups. PLX4720 treatment significantly increased BIM expression in the PTEN+ (>14-fold) compared with the PTEN- cell lines (four-fold). A role for PTEN in the regulation of PLX4720-mediated BIM expression was confirmed by siRNA knockdown of PTEN and through reintroduction of PTEN into cells that were PTEN-. Further studies showed that siRNA knockdown of BIM significantly blunted the apoptotic response in PTEN+ melanoma cells. Dual treatment of PTEN- cells with PLX4720 and a PI3K inhibitor enhanced BIM expression at both the mRNA and protein level and increased the level of apoptosis through a mechanism involving AKT3 and the activation of FOXO3a. In conclusion, we have shown for the first time that loss of PTEN contributes to intrinsic BRAF inhibitor resistance via the suppression of BIM-mediated apoptosis.

Preclinical evaluation of PARP inhibition as a treatment for endometrioid endometrial carcinomas.

5065 Background: Loss of function of PTEN is the most common genetic defect in endometroid endometrial carcinomas (EEC). PTEN plays a pivotal role in the regulation of the AKT and mTOR signalling pathways and is reported to be inactivated in up to 80% of EECs. PTEN loss of function has recently been shown to cause genetic instability through defects in homologous recombination (HR) DNA repair of double strand breaks. Cancer cells with defective HR have an exquisite sensitivity to inhibitors of the poly(ADP) ribose polymerase (PARP) inhibitors. Recent data suggest PTEN deficiency may also lead to sensitivity to PARP inhibition. Methods: PTEN expression was tested in a panel of eight EEC cell lines by western blotting. Cells were tested for their ability to elicit phospho-γ-H2AX foci (a marker of induction of DNA damage) and RAD51 foci (a marker of competent HR DNA repair) when challenged with ionising radiation. The sensitivity of these cell lines to the PARP inhibitor KU0058948 was tested in vitro. PTEN was silenced and re-expressed in cell lines by generating stably transfected cell lines with either two short hairpin-RNAs against PTEN or with a pBABE retroviral PTEN expression vector, respectively. Results: Out of the 8 EEC cell lines, two cell lines expressed PTEN (Hec-1b, EFE-184). ECC cells lacking PTEN expression (Nou-1, Ishikawa, RL95-2, EN, AN3-CA, Hec-59) were unable to elicit HR DNA repair. PTEN deficient EEC cells showed a significantly greater sensitivity to KU0058948 than PTEN wild-type EEC cells (IC50 10−8M vs 10−4M, respectively). PTEN silencing in Hec-1b cells resulted in a reduced RAD51 foci formation and in increased sensitivity to PARP inhibition. PTEN re-expression in Ishikawa and Nou-1 cells resulted in enhanced RAD51 foci formation and in a lower sensitivity to KU0058948. Conclusions: Given that most EECs lack PTEN expression and that EEC cell lines lacking PTEN expression show an exquisite sensitivity to KU0058948, our results suggest that PARP inhibitors may constitute a novel therapeutic approach for the majority of EECs. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Merck Merck AstraZeneca, Pfizer AstraZeneca

Dual Inhibition of Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin with NVP-BEZ235 as a New Therapeutic Option for T-Cell Acute Lymphoblastic Leukemia.

Abstract 2025Poster Board II-2 Introduction:Recent findings have highlighted that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL) where it strongly influences cell proliferation and survival. Pathway activation could be due to several reasons which include Notch1 activation leading to HES1-mediated transcriptional suppression of PTEN gene, PTEN phosphorylation or oxidation, and inactivation of SHIP1 phosphatase. These findings lend compelling weight for the application of PI3K/Akt/mTOR inhibitors in T-ALL. Rapamycin and its analogues have shown some promising effects in pre-clinical models of T-ALL. However, mTOR inhibitors are mainly cytostatic and could hyperactivate Akt due to the existence of feedback loops between mTOR, p70 S6 kinase, PI3K, and Akt. Recently, dual PI3K/mTOR inhibitors have been synthesized. Here, we have analyzed the therapeutic potential of the novel, dual PI3K/mTOR inhibitor, NVP-BEZ235, an orally bioavailable imidazoquinoline derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. Methods and Patients:We employed a panel of cell lines with up-regulated PI3K/Akt/mTOR signaling, including CEM-R cells [which overexpress high levels of the membrane transporter, 170-kDa P-glycoprotein (P-gp)], MOLT-4 and CEM-S cells (which lack PTEN expression), Jurkat cells (which do not express both PTEN and SHIP1), and RPMI-8402 and BE-13 cells. MOLT-4, CEM, and Jurkat cells have a non-functional p53 pathway. Moreover, both Jurkat and MOLT-4 cells have aberrant Notch1 signaling. Patients samples displayed pathway activation as documented by increased levels of p-Akt, p-4E-BP1, and p-S6 ribosomal protein, as well as low/absent PTEN expression. Results:NVP-BEZ235 was cytotoxic to the panel of cell lines as documented by MTT assays. NVP-BEZ235 IC50 ranged from 80 to 280 nM at 24 h. A comparison between NVP-BEZ235 and the dual PI3K/mTOR inhibitor PI-103, a small synthetic molecule of the pyridofuropyrimidine class with the same targets, demonstrated that NVP-BEZ235 was more effective than PI-103 when employed at equimolar concentrations. NVP-BEZ235 did not significantly affect the proliferation of peripheral blood T-lymphocytes from healthy donors stimulated with phytohemagglutinin and interleukin-2, whereas it blocked leukemic cells in the G1 phase of the cell cycle, and this was accompanied by decreased levels of phosphorylated Retinoblastoma protein. NVP-BEZ235 treatment also resulted in apoptotic cell death (about 20-30% at 6 h of exposure, when employed at 200 nM), as documented by Annexin V/propidium iodide staining and cytofluorimetric analysis. Moreover, NVP-BEZ235 activated caspase-8 and caspase-3, as demonstrated by western blot. Western blot documented a dose- and time-dependent dephosphorylation of Akt and its downstream target, GSK-3β, in response to NVP-BEZ235. mTOR downstream targets were also efficiently dephosphorylated, including p70S6 kinase, S6 ribosomal protein, and 4E-BP1. Remarkably, NVP-BEZ235 targeted the side population (SP, identified by Hoechst 33342 staining and ABCG2 expression) of T-ALL cell lines, which might correspond to leukemia initiating cells, and synergized with several chemotherapeutic agents (dexamethasone, vincristine, cyclophosphamide, Ara-C) currently employed for treating T-ALL patients. NVP-BEZ235 reduced chemoresistance to vincristine induced in Jurkat cells by co-culturing with MS-5 stromal cells which mimic the bone marrow microenvironment. NVP-BEZ235 was cytotoxic (IC50: 10-15 nM at 96 h) to primary lymphoblasts from patients with T-ALL, where the drug dephosphorylated 4E-BP1, at variance with rapamycin. Of note, NVP-BEZ235 targeted the SP also in T-ALL patient samples. Conclusions:NVP-BEZ235 was cytotoxic to T-ALL cell lines and patient lymphoblasts (including SP cells) at concentrations that have been previously reported to be achievable in vivo. Taken together, our findings indicate that longitudinal inhibition at two nodes of the PI3K/Akt/mTOR network with NVP-BEZ235, either alone or in combination with other drugs, may serve as an efficient treatment towards T-ALL cells (including those overexpressing P-gp and independently from p53 status) which require upregulation of this signaling pathway for their survival and growth. Disclosures:No relevant conflicts of interest to declare.

PI-103, a Dual Inhibitor of Class IA Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin, Has Cytotoxic Activity in T-Cell Acute Lymphoblastic Leukemia Cells: A New Therapeutic Strategy in T-Cell Acute Lymphoblastic Leukemia.

Constitutively activated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukaemia (T-ALL). Recently, it was demonstrated that activated Notch-1 leads to constitutive activation of the PI3K/Akt/mTOR pathway by HES1-mediated transcriptional suppression of the PTEN gene. In addition, PTEN is mutated in about 20% of T-ALL patients, and virtually all T-ALL cell lines that are resistant to Notch-1 inhibition with γ-secretase inhibitors, contain mutations leading to either no or low PTEN expression. These findings lend compelling weight for the application of PI3K/Akt/mTOR inhibitors in T-ALL. However, our knowledge of PI3K/Akt/mTOR signalling in T-ALL is still limited and it is not clear whether it could be an effective target for innovative therapeutic strategies. Here, we have characterized PI3K/Akt/mTOR signalling in T-ALL cell lines (Jurkat, MOLT-4, CEM) lacking PTEN expression, including one (CEM-R) which over expresses high levels of the membrane transporter, 170-kDa P-glycoprotein (P-gp), one of the main determinants of multidrug-resistance. While MOLT-4 cells display wild-type p53, both Jurkat and CEM have a non-functional p53 pathway. Moreover, we have analyzed the therapeutic potential of the dual PI3K/mTOR inhibitor, PI-103, a small synthetic molecule of the pyridofuropyrimidine class, on both T-ALL cell lines and patient samples. T-ALL cell lines expressed p110α, p110β, p110γ, and p110α PI3K. Moreover, they expressed Akt1 and Akt2, both of which were found to be constitutively phosphorylated on Ser 473 and Ser 474, respectively, by immunoprecipitation experiments. Treatment of T-ALL cell lines with selective pharmacological inhibitors of p110 PI3K isoforms, demonstrated that only a p110α PI3K inhibitor (PIK75) was cytotoxic, resulting in a 40–50% reduction of cell growth when used at 2 μM. Consistently, only PIK75 induced Akt1 and Akt2 dephosphorylation on Ser 473 or Ser 474, respectively, hinting that p110α is the most important isoform for the activation of downstream signalling events. PI-103 was cytotoxic to all T-ALL cell lines including P-gp overexpressing cells, as it reduced cell growth by approximately 70% when employed at 2 μM for 24 h. PI-103 IC50 ranged from 0.5 to 1.0 μM at 24 h. PI-103 treatment resulted in apoptotic cell death (about 30% at 6 h of exposure, when employed at 0.75 μM), as demonstrated by Annexin V/propidium iodide staining and cytofluorimetric analysis. PI-103 caused both Akt1 and Akt2 dephosphorylation, accompanied by dephosphorylation of the Akt downstream target, glycogen synthase kinase (GSK) -3β. Also mTOR downstream targets were dephosphorylated in response to PI-103, including p70S6 kinase, ribosomal S6 protein, and 4E-BP1. Moreover, PI-103 resulted in lower levels of c-Myc expression. PI-103 activated caspase-3, -8, and -9. In contrast, an mTOR inhibitor (rapamycin) was less cytotoxic than PI-103 (25–30% reduction of cell growth at 100 nM after 24 h), blocked cells in the G1 phase of the cell cycle, and was much less effective in inducing apoptosis (about 5% at 6 h of treatment). Remarkably, rapamycin was almost completely ineffective against CEM-R cells. A combination consisting of PIK75 and rapamycin was less cytotoxic to T-ALL cell lines than PI-103 alone. Furthermore, rapamycin treatment, at variance with PI-103, resulted in an overactivation of the Akt/ GSK-3β axis, as documented by increased phosphorylation levels of both Akt and GSK- 3β. PI-103 was also cytotoxic to primary lymphoblasts from patients with T-ALL (IC50: 0.15 nM at 96 h), displaying constitutive phosphorylation of Akt and 4E-BP1, as well as low/absent PTEN expression. These data indicate that multi-targeted therapy towards PI3K and mTOR, may serve as an efficient treatment towards T-ALL cells (including those over expressing P-gp and independently from p53 state) which require upregulation of PI3K/Akt/mTOR signaling for their survival and growth.

Activation of Nonreceptor Tyrosine Kinase Bmx/Etk Mediated by Phosphoinositide 3-Kinase, Epidermal Growth Factor Receptor, and ErbB3 in Prostate Cancer Cells

Pathways activated downstream of constitutively active phosphatidylinositol (PI) 3-kinase in PTEN-deficient prostate cancer (PCa) cells are possible therapeutic targets. We found that the nonreceptor Tec family tyrosine kinase Bmx/Etk was activated by tyrosine phosphorylation downstream of Src and PI 3-kinase in PTEN-deficient LNCaP and PC3 PCa cells and that Bmx down-regulation by short interfering RNA markedly inhibited LNCaP cell growth. Bmx also associated with ErbB3 in LNCaP cells, and heregulin-beta1 enhanced this interaction and further stimulated Bmx activity. Epidermal growth factor (EGF) similarly stimulated an interaction between Bmx and EGF receptor and rapidly increased Bmx kinase activity. Bmx stimulation in response to heregulin-beta1 and EGF was Src-dependent, and heregulin-beta1 stimulation of Bmx was also PI 3-kinase-dependent. In contrast, the rapid tyrosine phosphorylation and activation of Bmx in response to EGF was PI 3-kinase-independent. Taken together, these results demonstrate that Bmx is a critical downstream target of the constitutively active PI 3-kinase in PTEN-deficient PCa cells and further show that Bmx is recruited by the EGF receptor and ErbB3 and activated in response to their respective ligands. Therefore, Bmx may be a valuable therapeutic target in PCa and other epithelial malignancies in which PI 3-kinase or EGF receptor family pathways are activated.