Pharmacological Inhibition Of PTEN Research Articles

PTEN Regulates Myofibroblast Activation in Valvular Interstitials Cells based on Subcellular Localization.

Aortic valve stenosis (AVS) is characterized by altered mechanics of the valve leaflets, which disrupts blood flow through the aorta and can cause left ventricle hypotrophy. These changes in the valve tissue result in activation of resident valvular interstitial cells (VICs) into myofibroblasts, which have increased levels of αSMA in their stress fibers. The persistence of VIC myofibroblast activation is a hallmark of AVS. In recent years, the tumor suppressor gene phosphatase and tensin homolog (PTEN) has emerged as an important player in the regulation of fibrosis in various tissues (e.g., lung, skin), which motivated us to investigate PTEN as a potential protective factor against matrix-induced myofibroblast activation in VICs. In aortic valve samples from humans, we found high levels of PTEN in healthy tissue and low levels of PTEN in diseased tissue. Then, using pharmacological inducers to treat VIC cultures, we observed PTEN overexpression prevented stiffness-induced myofibroblast activation, whereas genetic and pharmacological inhibition of PTEN further activated myofibroblasts. We also observed increased nuclear PTEN localization in VICs cultured on stiff matrices, and nuclear PTEN also correlated with smaller nuclei, altered expression of histones and a quiescent fibroblast phenotype. Together, these results suggest that PTEN not only suppresses VIC activation, but functions to promote quiescence, and could serve as a potential pharmacological target for the treatment of AVS.

Deletion of PTEN in microglia ameliorates chronic neuroinflammation following repetitive mTBI

Traumatic brain injury is a leading cause of morbidity and mortality in adults and children in developed nations. Following the primary injury, microglia, the resident innate immune cells of the CNS, initiate several inflammatory signaling cascades and pathophysiological responses that may persist chronically; chronic neuroinflammation following TBI has been closely linked to the development of neurodegeneration and neurological dysfunction. Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been shown to regulate several key mechanisms in the inflammatory response to TBI. Increasing evidence has shown that the modulation of the PI3K/AKT signaling pathway has the potential to influence the cellular response to inflammatory stimuli. However, directly targeting PI3K signaling poses several challenges due to its regulatory role in several cell survival pathways. We have previously identified that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN), the major negative regulator of PI3K/AKT signaling, is dysregulated following exposure to repetitive mild traumatic brain injury (r-mTBI). Moreover, this dysregulated PI3K/AKT signaling was correlated with chronic microglial-mediated neuroinflammation. Therefore, we interrogated microglial-specific PTEN as a therapeutic target in TBI by generating a microglial-specific, Tamoxifen inducible conditional PTEN knockout model using a CX3CR1 Cre recombinase mouse line PTENfl/fl/CX3CR1+/CreERT2 (mcg-PTENcKO), and exposed them to our 20-hit r-mTBI paradigm. Animals were treated with tamoxifen at 76 days post-last injury, and the effects of microglia PTEN deletion on immune-inflammatory responses were assessed at 90-days post last injury. We observed that the deletion of microglial PTEN ameliorated the proinflammatory response to repetitive brain trauma, not only reducing chronic microglial activation and proinflammatory cytokine production but also rescuing TBI-induced reactive astrogliosis, demonstrating that these effects extended beyond microglia alone. Additionally, we observed that the pharmacological inhibition of PTEN with BpV(HOpic) ameliorated the LPS-induced activation of microglial NFκB signaling in vitro. Together, these data provide support for the role of PTEN as a regulator of chronic neuroinflammation following repetitive mild TBI.

Primary human monocytes are stimulated in a pro-atherosclerotic manner in hyperhomocysteine conditions by attenuated PTEN phosphatase function

Abstract Purpose Monocytes are essential for atherosclerosis and hereby crucial for the detrimental consequences of cardiovascular diseases like coronary heart disease. Interestingly, homocysteine (HCY) was identified as an idependent risk factor for atherosclerosis development. However, the specific implication of HCY on monocytes has not yet been clarified and was therefore investigated in the present study. Methods Primary human monocytes, isolated from peripheral blood through the immuno-magnetic method, were treated with a clinically relevant dose of 400 μM HCY for 24 hours. Monocyte migration was investigated with transwell migration assays. Adhesion to inflamed endothelium (HUVECs) was studied under static and physiological flow conditions. Expression of relevant molecules was quantified with Western Blot, RT-qPCR and FACS. 5-azacytidine (AZA) was used to inhibit DNA methyltransferase 1 (DNMT1) and SF1670 to inhibit PTEN. Results First, we studied monocyte migration phenotype. Under hyperhomocysteine conditons monocytes revealed elevated chemokinesis and enhance chemotaxis towards MCP-1. In addition, monocytes show a pro-adhesive phenotype under HCY treatment which was indicated by heightened adhesion to inflamed endothelial cells, both in static and physiological flow conditions. These data could be sustained by observation of HCY-induced augmented expression of adhesion molecule CD11a on primary human monocytes. Interestingly, we observed decreased PTEN phosphatase function and activity in HCY-treated monocytes, which was reflected downstream in increased activation of AKT at serine 473. Based on these data, it was also possible to induce the pro-migratory phenotype in monocytes by pharmacological inhibition of PTEN alone. Since HCY can modify the methylation status of PTEN and hereby regulates its expression, we inhibited DNMT1 with AZA which could prevent downregulation of PTEN expression by HCY treatment. In addition, through inhibition of DNMT1 HCY-induced aberrant migration and adhesion was rescued. In line with that, HCY-induced pro-migratory and pro-adhesive phenotype was also rescued by co-treatment with the cofactors (30 μM Vitamin B12 and 3 μM folic acid) responsible for homocysteine to methionine catabolism due to normalized PTEN expression. Conclusions The present work deciphers a previously unknown mechanism how an increased concentration of HCY induces a pro-atherosclerotic activation of monocytes. Accumulation of HCY leads to a methylation-dependent inactivation of PTEN phosphatase. This subsequently causes increased phosphorylation of AKT at serine 473, which results in an augmented migration behaviour of monocytes. At the same time, we were able to reduce HCY-induced aberrant monocyte activation by inhibiting DNTM1 or by interfering with HCY metabolism by adding vitamin B12 or folic acid. In summary, these findings provide a new approach to reset pro-atherosclerotic monocytes in hyperhomocysteinemic conditions. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): IZKF SEED Project 14/20

Pharmacological inhibition of the lipid phosphatase PTEN ameliorates heart damage and adipose tissue inflammation in stressed rats with metabolic syndrome.

Phosphatidylinositol 3‐kinase (PI3K) signaling promotes the differentiation and proliferation of regulatory B (Breg) cells, and the lipid phosphatase phosphatase and tensin homolog deleted on chromosome 10 (PTEN) antagonizes the PI3K–Akt signaling pathway. We previously demonstrated that cardiac Akt activity is increased and that restraint stress exacerbates hypertension and both heart and adipose tissue (AT) inflammation in DS/obese rats, an animal model of metabolic syndrome (MetS). We here examined the effects of restraint stress and pharmacological inhibition of PTEN on heart and AT pathology in such rats. Nine‐week‐old animals were treated with the PTEN inhibitor bisperoxovanadium‐pic [bpV(pic)] or vehicle in the absence or presence of restraint stress for 4 weeks. BpV(pic) treatment had no effect on body weight or fat mass but attenuated hypertension in DS/obese rats subjected to restraint stress. BpV(pic) ameliorated left ventricular (LV) inflammation, fibrosis, and diastolic dysfunction as well as AT inflammation in the stressed rats. Restraint stress reduced myocardial capillary density, and this effect was prevented by bpV(pic). In addition, bpV(pic) increased the proportions of Breg and B‐1 cells as well as reduced those of CD8+ T and B‐2 cells in AT of stressed rats. Our results indicate that inhibition of PTEN by bpV(pic) alleviated heart and AT inflammation in stressed rats with MetS. These positive effects of bpV(pic) are likely due, at least in part, to a reduction in blood pressure, an increase in myocardial capillary formation, and an altered distribution of immune cells in fat tissue that result from the activation of PI3K–Akt signaling.

Elevated miR-29a Contributes to Axonal Outgrowth and Neurological Recovery After Intracerebral Hemorrhage via Targeting PTEN/PI3K/Akt Pathway.

Spontaneous intracerebral hemorrhage (ICH) is a clinical challenge with high disability and lacks an effective treatment. miR-29a strongly expressed in the brain has been implicated in various neurological disorders. In this study, we investigated the biological roles of miR-29a in axonal outgrowth and neurological outcomes after ICH and relevant molecular mechanism. The rat model of ICH was established by injection of autologous whole blood into the right basal ganglia. First, a significant decrease in miR-29a level was found in perihematomal brain tissues and cerebrospinal fluid (CSF) after ICH in vivo and hemin-treated neurons in vitro. Further study documented that lentivirus-mediated miR-29a overexpression could remarkably attenuate hemorrhagic brain injury, promoted regenerative outgrowth of injured axons and improved neurobehavioral and cognitive impairments after ICH in rats. In addition, we also identified that overexpression of miR-29a obviously alleviated neuronal damage and mitochondrial dysfunctions, and facilitated neurite outgrowth in cultured neurons exposed to hemin in vitro. Furthermore, luciferase reporter assay showed that miR-29a directly targeted the 3'-UTR region of phosphatase and tensin homolog (PTEN) mRNA and negatively regulated its expression. More importantly, pharmacological inhibition of PTEN has similar neuroprotective effects as miR-29a overexpression involving activation of the PI3K/Akt pathway after hemorrhagic stroke. Collectively, these results suggested that elevated miR-29a could contribute to axonal outgrowth and neurological recovery through targeting PTEN/PI3K/Akt pathway after ICH, thereby providing a potential therapeutic target for patients with ICH.

PTEN activation contributes to neuronal and synaptic engulfment by microglia in tauopathy

Phosphatase and tensin homolog (PTEN) regulates synaptic density in development; however, whether PTEN also regulates synapse loss in a neurodegenerative disorder such as frontotemporal lobar degeneration with Tau deposition (FTLD-Tau) has not been explored. Here, we found that pathological Tau promotes early activation of PTEN, which precedes apoptotic caspase-3 cleavage in the rTg4510 mouse model of FTLD-Tau. We further demonstrate increased synaptic and neuronal exposure of the apoptotic signal phosphatidylserine that tags neuronal structures for microglial uptake, thereby linking PTEN activation to synaptic and neuronal structure elimination. By applying pharmacological inhibition of PTEN's protein phosphatase activity, we observed that microglial uptake can be decreased in Tau transgenic mice. Finally, we reveal a dichotomous relationship between PTEN activation and age in FTLD-Tau patients and healthy controls. Together, our findings suggest that in tauopathy, PTEN has a role in the synaptotoxicity of pathological Tau and promotes microglial removal of affected neuronal structures.

Autoimmunity Checkpoints As Therapeutic Targets in B- and T-Cell Malignancies

▪Background. Chronic active BCR or TCR signaling or its oncogenic mimics result in nuclear accumulation of NF-kB. Oncogenic mimicry of BCR- and TCR signaling can be induced by viral oncoproteins (e.g. LMP2A (EBV), K1 (KSHV) and Tax (HTLV1), activating mutations in the CD79A, CD79B and CD3Z signaling chains or genetic lesions driving tyrosine kinase (e.g. BCR-ABL1), ERK (RAS, BRAF, PLCG1) or PI3K (PIK3CA, PIK3R1) signaling. While BCR and TCR signaling induces positive selection, survival and proliferation in normal lymphocytes, the majority of B cell and T cell malignancies are driven by oncogenic activation of this pathway.Concept & central hypothesis. Oncogenic drivers in B- and T-lymphoid malignancies function as mimics of B- and T-cell receptor (BCR or TCR) signaling. Oncogenic activation of BCR- or TCR-signaling represents the functional equivalent of positive selection during normal lymphocyte development. Addiction to survival and proliferation signals (or the equivalent of positive selection) is a common feature in many types of cancer. However, B- and T-lymphoid malignancies are unique in that they are also subject to an active negative selection process. B- and T-cells expressing autoreactive antibodies and TCRs, respectively, can cause systemic autoimmunity. As a safeguard against autoimmune diseases, lymphocyte development evolved autoimmunity checkpoints (AIC) to eliminate autoreactive clones . Owing to negative selection of autoreactive B- and T-cells through AIC activation, lymphoid cells fundamentally differ in their signaling requirements from other cell types. Four recent studies from our group showed that despite malignant transformation, B cell- and T cell-lineage leukemia and lymphoma cells are fully sensitive to negative selection and AIC-activation resulting (Chen et al., Nature 2015; Shojaee et al., Cancer Cell 2015; Shojaee et al., Nature Med 2016; Chan et al., Nature 2017).Results: AIC-activation in various lymphoid malignancies is achievable by pharmacological hyperactivation of BCR- and TCR-signaling above a maximum threshold, e.g. pharmacological inhibition of PTEN, SHIP1 or DUSP6 phosphatases (Figure). Studying genetic models of B cell malignancies, we observed that Cre-mediated deletion of Pten, Ship1 and constitutive deletion of Dusp6 caused hyperactivation of SYK, PI3K and ERK-pathways, leading to AIC-activation. Unlike other types of cancer, B- and T-cell malignancies are uniquely susceptible to clonal deletion induced by hyperactive signaling from an autoreactive BCR or TCR. Hence, targeted AIC-activation can be leveraged for eradication of drug-resistant leukemia and lymphoma clones. Studying engineered B cells for inducible activation of autoreactive BCR-signaling, we discovered that targeted hyperactivation of SYK, PI3K and ERK in B cell malignancies represents the functional equivalent of an autoimmunity checkpoint (AIC) for elimination of autoreactive clones. Likewise, transformed B cell tumors are uniquely vulnerable to AIC activation, suggesting that targeted activation of this checkpoint represents a novel strategy to induce cell death in otherwise drug-resistant B cell malignancies.Conclusion: Normal B- and T-cells are positively selected for BCR or TCR signaling of intermediate strength (moderate activation of SYK, PI3K and ERK). In the absence of a functional BCR/TCR, SYK, PI3K and ERK activity fall below a minimum threshold, resulting in death by neglect. Hyperactivation above maximum thresholds (e.g. autoreactive BCR/TCR) triggers negative selection and cell death via AIC-activation. Targeted therapy of cancer typically focuses onagents that suppress oncogenic signaling below a minimum threshold . Our results support a novel strategy to overcome drug-resistance in B- and T-cell malignancies based on targeted activation of autoimmunity checkpoints (AIC) for removal of autoreactive cells. [Display omitted] DisclosuresMuschen:Pfizer: Research Funding; AbbVie: Research Funding.

Inhibition of Phosphatase and Tensin Homologue (PTEN) in Human Ovary In Vitro Results in Increased Activation of Primordial Follicles But Compromises Development of Growing Follicles

The overwhelming majority of human ovarian follicles exist as a quiescent population from fetal life until puberty. Only a small number of follicles from this dormant population are recruited, undergo development, go on to complete growth, and release a mature fertilizable oocyte. The phosphoinositide 3-kinase (PI3K)–protein kinase B (Akt) signaling pathway has been identified as a key mechanism involved in the maintenance of follicle growth and loss. A number of growth factors stimulate PI3K to promote phosphorylation of Akt and downstream transcription factors including the forkhead winged helix box O1 (FOXO1); this results in follicle survival and activation of growth. These events are reversed by the action of phosphatase and tensin homolog (PTEN). In murine models, the effect of PTEN can be inhibited by the vanadate derivative, dipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate (V) (bpV(HOpic), and thereby promote the downstream phosphorylation of Akt. Previous studies suggest that PTEN inhibitors could be used to generate mature oocytes in women whose oocyte reserve is impaired because of illness or treatment. There are few or no data on the response of human follicles to bpV(HOpic) in vitro. It is not known whether selective promotion of the PI3K pathway by pharmacological inhibition of PTEN in vitro would promote initiation of growth in quiescent human ovarian follicles within tissue fragments and could provide a population of biopsy-derived growing secondary follicles of sufficient quality for in vitro maturation. The aim of this study was to investigate the effect of bpV(HOpic), a reversible inhibitor of PTEN, on the activation, survival, and development of human ovarian follicles in vitro. Ovarian cortical tissue was obtained by biopsy from a total of 17 adult women aged 23 to 46 years. Fragments of the biopsied tissue were cultured for 24 hours in control medium (n = 128) or medium supplemented with 1 μM bpV(HOpic (n = 146). Media in both cultures were then replaced with control medium, and tissues were incubated for a further 5 days. After the initial 24-hour culture, half of the tissue fragments from both groups were fixed, and phosphorylated Akt was quantified by Western blot analysis. Following 6 days of incubation, tissue fragments were examined under light microscopy, and any secondary follicles 100 μm in diameter or greater were isolated. Then, follicles isolated from tissue fragments were cultured individually in control medium supplemented with 100 ng/mL recombinant human activin A. Tissue fragments without detectable follicles or with only unilaminar follicles were fixed and processed for histological and immunohistochemical analysis. Microscopic examination showed that after 6 days incubation, initiation of follicle growth had occurred in both treatment groups, but significantly more follicles progressed to the secondary stage of development in the presence of 1 bpV(HOpic) than in the control (31% vs 16%; P < 0.05). Increased activation of the PI3K pathway in tissue exposed to bpV(HOpic) was confirmed by an increase in Akt phosphorylation and nuclear export of FOXO3. However, isolated and cultured secondary follicles treated with bpV(HOpic) exhibited limited growth and reduced survival compared with follicles from control fragments (P < 0.05). These data demonstrate that inhibition of PTEN with bpV(HOpic) promotes activation and development of isolated follicles to the secondary stage, as in rodents, but severely compromises their continued growth and survival.

MP29-04 THE LOSS OF PTEN CONSEQUENT TO URETERAL OBSTRUCTION CONTRIBUTES TO RENAL FIBROSIS

INTRODUCTION AND OBJECTIVES: Phosphate Tensin Homologue on Chromosome 10 (PTEN), the principle negative regulator of PI3K pathway, is a known tumor suppressor that is mutated or lost in many cancer types. Recent studies highlight the emerging role of tumor suppressor PTEN in organ fibrosis including lung and skin where loss of PTEN expression is linked to progression of fibrosis via promoting epithelial plasticity in the lung and induction of myofibroblasts in the skin. However, potential involvement of this molecule in obstructive uropathies is not defined. METHODS: Obstruction induced renal fibrosis was modeled in vivo by unilateral ureteral obstruction (UUO) in mice and in vitro by gene silencing and pharmacological inhibition of PTEN in HK-2 human renal tubular epithelial cells and NRK-49F rat renal fibroblasts. RESULTS: PTEN expression is dramatically decreased in the obstructed (ligated) kidney, while profibrotic TGF-b1/SMAD3 and p53 signal transduction is increased compared to contralateral control or sham kidney. PTEN silencing induced phenotypic modifications from typical cuboidal to an elongated fibroblast-like shape (4-fold increase) and a 40% decrease in the relative cell number compared to control shRNA expressing HK-2 cultures (p<0.01). Stable gene depletion of PTEN in HK-2 cells long term (3-5 days) resulted in increased expression of the profibrotic markers CTGF, PAI-1 and fibronectin compared to control shRNA expressing cultures. Loss of PTEN resulted in activation of SMAD2/3 transcription factor. SMAD3 knockdown or pharmacological inhibition of SMAD3 in PTEN depleted HK-2 cells reversed growth inhibition and suppressed PAI-1 induction, confirming a critical role of SMAD3 downstream of PTEN in orchestrating fibrotic responses. CONCLUSIONS: Unilateral ureteral obstruction induces loss of PTEN expression, resulting in the increased expression of profibrotic cytokines in renal tubular epithelial cells that is dependent on the SMAD3 signaling pathway. PTEN is a potential therapeutic target in the treatment of ureteral obstruction and renal fibrosis. This model paves the way for further clinical studies in humans.

PTEN inhibition enhances neurite outgrowth in human embryonic stem cell–derived neuronal progenitor cells

We investigated the role of PTEN (phosphatase and tensin homolog deleted on chromosome 10) during neurite outgrowth of human embryonic stem cell (hESC)-derived neuronal progenitors. PTEN inhibits phosphoinositide 3-kinase (PI3K)/Akt signaling, a common and central outgrowth and survival pathway downstream of neuronal growth factors. It is known that PTEN inhibition, by either polymorphic mutation or gene deletion, can lead to the development of tumorigenesis (Stambolic et al., ; Tamura et al., ). However, temporary inhibition of PTEN, through pharmacological manipulation, could regulate signaling events such as the PI3K/Akt signaling pathway, leading to enhanced recovery of central nervous system (CNS) injury and disease. We demonstrate that pharmacological inhibition of PTEN in hESC-derived neuronal progenitors significantly increased neurite outgrowth in vitro in a dose- and time-dependent manner. Our results indicate that inhibition of PTEN augments neurite outgrowth beyond that of traditional methods such as elevation of intracellular cyclic adenosine monophosphate (cAMP) levels, and depends on upregulation of the PI3K/Akt signaling pathway and its downstream effectors, such as mammalian target of rapamycin (mTOR). PTEN inhibition also rescued neurite outgrowth over an inhibitory substrate in vitro. These findings indicate a remarkable impact on hESC-derived neuronal progenitor plasticity through PTEN inhibition. Overall, these findings identify a novel therapeutic strategy for neurite outgrowth in CNS injury and disease.

PTEN signaling modulates lung epithelial cells migration and wound repair through changes in cellular mechanics

The PI3K/Akt pathway is a vital component of survival in lung epithelia. Previous work by our group indicates that pharmacological inhibition of PTEN, a major suppressor of this pathway, results in enhanced wound repair and cell migration following lung injury. However, the precise biomechanical mechanisms responsible for enhanced wound repair are not known. In this study, we used atomic force microscopy (AFM) and traction force microscopy (TFM) to characterize the biomechanical effects of PTEN inhibition in a bronchial epithelial cell line (BEAS2B). PTEN inhibition was found to induce a significant decrease in both cell stiffness and the power-law exponent which indicates that PTEN-inhibited cells behave more like a soft-elastic-solid than a viscous-fluid. TFM measurements revealed that PTEN inhibited cells exhibited decreased contractility, consistent with weaker adhesion and increased migration. Concurrent inhibition of both PTEN activity and Akt phosphorylation resulted in viscoelasticity profiles similar to those obtained in control samples. These results indicate that enhanced cell migration and wound repair during PTEN inhibition is due to changes in the cell's biomechanical properties. In addition, these changes in cell mechanics appear to be mediated by Akt signaling pathways. Thus, Akt pathways may represent a novel target for wound repair following lung injury. Supported by NSF 0852417.

Phosphatase and tensin homologue deleted on chromosome ten (PTEN) as a molecular target in lung epithelial wound repair

Epithelial injury contributes to lung pathogenesis. Our work and that of others have identified the phosphoinositide-3 kinase (PI3K)/Akt pathway as a vital component of survival in lung epithelia. Therefore, we hypothesized that pharmacological inhibition of PTEN, a major suppressor of this pathway, would enhance wound closure and restore lung epithelial monolayer integrity following injury. We evaluated the ability of two bisperoxovanadium derivatives, bpV(phen) and bpV(pic), in differentiated primary human airway epithelia and BEAS2B cultures for their ability to inhibit PTEN, activate the PI3K/Akt pathway and restore epithelial monolayer integrity following mechanical injury. BpV(phen) and bpV(pic) induced Akt phosphorylation in primary and BEAS2B cells in a dose and time dependent manner. Minimal toxicity was observed as measured by lactate dehydrogenase (LDH) release. To verify that Akt phosphorylation is specifically induced by PTEN inhibition, the PTEN positive cell line, DU145, and two PTEN negative cell lines, LNCaP and PC3, were examined. PTEN positive cells demonstrated a dose responsive increase in Akt phosphorylation whereas PTEN negative cells showed no response indicating that bpV(phen) directly suppresses PTEN without affecting auxiliary pathways. Next, we observed that exposure to either compound resulted in accelerated wound closure following mechanical injury. Similar effects were observed after transfection with a dominant negative isoform of PTEN and PTEN specific siRNA. From these studies, we conclude that PTEN is a valid target for future studies directed at restoring epithelial barrier function after lung injury.

Unlike Inactivating PTEN Mutations, Pharmacological Inhibition of PTEN Does Not Increase the Sensitivity of Multiple Myeloma Cells to Inhibition of the PI3K/Akt/mTOR Pathway.

PTEN-deficient multiple myeloma (MM) and other tumor cell lines have been shown to be remarkably sensitive to inhibitors of the PI3K/Akt/mTOR pathway (Shi Y, et al. Cancer Res 2002; Simth Y, et al. Proc Am Assoc Cancer Res 2002; Neshat MS, et al. Proc. Natl. Acad. Sci. USA 2001.). Inactivating mutations of PTEN, however, are very rare in human MM cells. We investigated whether pharmacological inhibition of wild type PTEN might also sensitize MM cells to inhibitors of PI3K and mTOR, and therefore provide the rationale for novel therapeutically useful combinations. We tested the effect of the PTEN antagonist, SF1740 (Semafore Inc, Indianapolis, IN), in combination with the mTOR inhibitor, rapamycin, or the PI3K inhibitor, SF1101, (also known as LY294002, from Semafore Inc, Indianapolis, IN) in U266, MM1R, MM1S myeloma cells. Using the Malachite Green assay, the IC50 for PTEN inhibition by SF1740 was 0.4 μM, and maximal inhibition was achieved at a concentration of 1μM. We treated MM cells with rapamycin (1, 5, 10 nM) or SF1101 (10, 25, 50 μM) alone or in combination with SF1740 for 24, 48, 72 and 96 hours. Cellular toxicity was assessed using the MTT assay, apoptosis by annexin V/PI staining, and the effect on intracellular signaling was monitored by Western blotting. SF1740 (1 μM) alone increased cell growth, and failed to sensitize cells to the inhibitory effects of rapamycin (1–10 nM) at all time points. There were no significant differences in cell survival between the combination of the two drugs and rapamycin alone when treated from 48–96hr. Cytotoxicity was unaffected by the sequence of drug treatment; concurrent or 24-hour pre-treatment with SF1740. Similar results were obtained using the combination of SF1740 (1 μM) and SF1101 used at concentrations of 10, 25, and 50 μM, with no difference in MM cell inhibition and survival between the SF1101 and the combination with SF1740. We conclude that in MM cells, pharmacological inhibition of wild type PTEN does not appear to replicate the effect of inactivating mutations of PTEN in increasing cell sensitivity to inihibitors of the PI3K/Akt/mTOR pathway. The results do not support the clinical use of the PTEN inhibitor SF1740 in combination with either PI3K or mTOR inhibitors. Xiaojing Wang and Shuhong Zhang contributed equally to this work.