PTEN-mutant Cells Research Articles

AKT activation because of PTEN loss upregulates xCT via GSK3β/NRF2, leading to inhibition of ferroptosis in PTEN-mutant tumor cells

Here, we show that the tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) sensitizes cells to ferroptosis, an iron-dependent form of cell death, by restraining the expression and activity of the cystine/glutamate antiporter system Xc- (xCT). Loss of PTEN activates AKT kinase to inhibit GSK3β, increasing NF-E2 p45-related factor 2 (NRF2) along with transcription of one of its known target genes encoding xCT. Elevated xCT in Pten-null mouse embryonic fibroblasts increases the flux of cystine transport and synthesis of glutathione, which enhances the steady-state levels of these metabolites. A pan-cancer analysis finds that loss of PTEN shows evidence of increased xCT, and PTEN-mutant cells are resistant to ferroptosis as a consequence of elevated xCT. These findings suggest that selection of PTEN mutation during tumor development may be due to its ability to confer resistance to ferroptosis in the setting of metabolic and oxidative stress that occurs during tumor initiation and progression.

GH Action in Prostate Cancer Cells Promotes Proliferation, Limits Apoptosis, and Regulates Cancer-related Gene Expression.

Previous studies investigating the effects of blocking the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis in prostate cancer found no effects of the growth hormone receptor (GHR) antagonist, pegvisomant, on the growth of grafted human prostate cancer cells in vivo. However, human GHR is not activated by mouse GH, so direct actions of GH on prostate cancer cells were not evaluated in this context. The present study addresses the species specificity of GH-GHR activity by investigating GH actions in prostate cancer cell lines derived from a mouse Pten-deletion model. In vitro cell growth was stimulated by GH and reduced by pegvisomant. These in vitro GH effects were mediated at least in part by the activation of JAK2 and STAT5. When Pten-mutant cells were grown as xenografts in mice, pegvisomant treatment dramatically reduced xenograft size, and this was accompanied by decreased proliferation and increased apoptosis. RNA sequencing of xenografts identified 1765 genes upregulated and 953 genes downregulated in response to pegvisomant, including many genes previously implicated as cancer drivers. Further evaluation of a selected subset of these genes via quantitative reverse transcription-polymerase chain reaction determined that some genes exhibited similar regulation by pegvisomant in prostate cancer cells whether treatment was in vivo or in vitro, indicating direct regulation by GH via GHR activation in prostate cancer cells, whereas other genes responded to pegvisomant only in vivo, suggesting indirect regulation by pegvisomant effects on the host endocrine environment. Similar results were observed for a prostate cancer cell line derived from the mouse transgenic adenocarcinoma of the mouse prostate (TRAMP) model.

The Splicing Factor SF2 Is Critical for Hyperproliferation and Survival in a TORC1-Dependent Model of Early Tumorigenesis in Drosophila.

The Target of Rapamycin complex 1 (TORC1) is an evolutionarily conserved kinase complex coordinating cellular growth with nutritional conditions and growth factor signaling, and its activity is elevated in many cancer types. The use of TORC1 inhibitors as anticancer drugs is, however, limited by unwanted side-effects and development of resistance. We therefore attempted to identify limiting modulators or downstream effectors of TORC1 that could serve as therapeutic targets. Drosophila epithelial tissues that lack the tumor suppressor Pten hyperproliferate upon nutrient restriction in a TORC1-dependent manner. We probed candidates of the TORC1 signaling network for factors limiting the overgrowth of Pten mutant tissues. The serine/arginine-rich splicing factor 2 (SF2) was identified as the most limiting factor: SF2 knockdown drives Pten mutant cells into apoptosis, while not affecting control tissue. SF2 acts downstream of or in parallel to TORC1 but is not required for the activation of the TORC1 target S6K. Transcriptomics analysis revealed transcripts with alternatively used exons regulated by SF2 in the tumor context, including p53. SF2 may therefore represent a highly specific therapeutic target for tumors with hyperactive TORC1 signaling.

Therapeutic Stratification of PTEN-MYC Axis Appears to be Promising for Molecular Targeted Therapies in Refractory and Relapsed T-Cell Acute Lymphoblastic Leukemia

Therapy refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL) is still one of the most common causes for cancer related death in children. T-ALL is a highly heterogeneous disease with several established biomarkers, which promote its progression. These biomarkers are promising tools for patient stratification and can be used for targeted therapeutic approaches. For instance, PTEN is one of the most displayed mutations in pediatric T-ALL and plays an important role in proteolysis of the oncoprotein MYC. Therefore, it is not surprising that PTEN activity in T-ALL patients correlates with higher MYC protein levels. Importantly, PTEN mutations have recently been associated with poor overall survival in T-ALL patients, underscoring its importance for future treatment options using molecular targeted therapies. In this study, we validated the importance of PTEN as a MYC regulator in T-ALL and elucidated PTEN mutational status as a stratification marker for T-ALL patients. Comparative quantification of PTEN, phosphorylated AKT and MYC in leukemic T-cell lines (n=10) was analyzed at protein level, relative to healthy T-cells. PTEN loss or deceased expression was observed in half of the screened leukemic T-cell lines with concomitant high MYC expression and increased AKT phosphorylation. To identify the drug sensitivity of PTEN mutant (PTENm) and PTEN wild type (PTENwt) cell lines, high-throughput drug screening was performed with 180 inhibitors, including among others kinase-, BET- and gamma secretase inhibitors, as well as conventional chemotherapeutics. PTENm cell lines showed remarkable sensitivity in nanomolar ranges against PI3K inhibitors compared to PTENwt (P=0.001). Anticipating that tumor cells eventually escape the killing effect of single pathway targeted drugs, the need for molecular identification and characterization of synergistic drug combinations is evident. Therefore, diverse compound combinations were tested in an 11 by 11 matrix of different concentrations in PTENm and PTENwt cell lines. Synergism was predicted with Combenefit software using Loewe model to assess putative additive versus synergistic effects. We observed that simultaneous use of copanlisib (PI3K inhibitor) with JQ1 (BET inhibitor) had a strong killing effect in PTENm cell lines. To assess target downregulation after 24 hours of drug treatment, lysates were taken and visualized using western blot. A strong decrease of MYC expression was observed already with a combination of low concentrations of copanlisib and JQ1 with subsequent higher apoptosis induction as measured by Annexin V/PI staining. A second drug combination that showed synergy in PTENm was observed with copanlisib and alisertib, an Aurora kinase-A (AURKA) inhibitor, expecting changes in cell cycle and reduced MYC stability. Indeed, results of cell cycle analysis have revealed an inhibition during mitosis and cellular MYC levels were decreased, suggesting that this combination affects highly proliferative MYC-dependent tumor cells. Contrary to PTENm, in PTENwt cell lines, PI3K inhibition alone showed no cytotoxic effect, but synergy was detected in combination with the MEK inhibitor cobimetinib. Moreover, in this synergistic combination MYC was effectively downregulated on protein level accompanied with changes in regulatory pathways including AKT and MEK. Interestingly, phosphorylation of MYC changed from the stabilizing site (S62) to the degradative site (T58), proposing the progress of MYC degradation. In summary, we observed the importance of PTEN as a regulator for MYC proteolysis and related pathways including AKT and MEK. The loss of PTEN is accompanied with increased MYC. Synergy studies have observed effective drug combinations for PTENm and PTENwt with the aim to downregulate MYC and induce apoptosis in tumor cells. Based on these findings, PTEN-MYC axis seems suitable as a potential stratification marker for future therapy options in refractory and relapsed T-ALL. In the next step, promising drug combinations will be tested in a patient derived xenograft mouse model to validate these findings. Figure Disclosures No relevant conflicts of interest to declare.

The effect of miR-579 on the PI3K/AKT pathway in human glioblastoma PTEN mutant cell lines.

Glioblastoma multiform (GBM) is a type of aggressive brain cancer with limited success in standard treatment. MicroRNAs are one of the most beneficial tools for diagnosis, prognosis, and treatment of cancer. This study aimed to investigate the effect of miR-579 on cellular behaviors and expression of PI3K/AKT signaling pathway in GBM cell lines. In the present study, miR-579 was overexpressed in U251 and A-172 cell lines by using lentil vector, and its effect on cellular behavior such as proliferation and migration was investigated by the cell cycle, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Annexin V, colony formation, Transwell and wound healing assays. MiR-579 predicted target genes (AKT1, Rheb, PDK1, and a few others) were also evaluated by real-time polymerase chain reaction or luciferase assay and Western blot analysis. Our results represented that overexpression of miR-579 could inhibit proliferation, migration, cell cycle and also promoted the apoptosis of GBM cell lines. The luciferase reporter assay showed miR-579 directly targets the 3 UTR of mTOR, Rheb, and PDK1 and repressed their expressions. Furthermore, the Western blot analysis showed that miR-579 could downregulate the AKT1 and Rheb protein expression. Overall, our findings propose that miR-579 functions as a novel tumor suppressor gene in GBM by regulating the PI3K/AKT signaling pathway and may serve as a therapeutic target for clinical therapy of glioblastoma multiform.

The PHLPP2 phosphatase is a druggable driver of prostate cancer progression.

Metastatic prostate cancer commonly presents with targeted, bi-allelic mutations of the PTEN and TP53 tumor suppressor genes. In contrast, however, most candidate tumor suppressors are part of large recurrent hemizygous deletions, such as the common chromosome 16q deletion, which involves the AKT-suppressing phosphatase PHLPP2. Using RapidCaP, a genetically engineered mouse model of Pten/Trp53 mutant metastatic prostate cancer, we found that complete loss of Phlpp2 paradoxically blocks prostate tumor growth and disease progression. Surprisingly, we find that Phlpp2 is essential for supporting Myc, a key driver of lethal prostate cancer. Phlpp2 dephosphorylates threonine-58 of Myc, which renders it a limiting positive regulator of Myc stability. Furthermore, we show that small-molecule inhibitors of PHLPP2 can suppress MYC and kill PTEN mutant cells. Our findings reveal that the frequent hemizygous deletions on chromosome 16q present a druggable vulnerability for targeting MYC protein through PHLPP2 phosphatase inhibitors.

Abstract 5499: A potent BH3 mimetic targeting BCL-XL induces apoptosis regulated by PTEN loss and integrin alpha 5 in prostate cancer cells

Abstract Background: PTEN-loss is a highly prevalent genomic event associated with higher risk of metastatic progression and resistance to hormonal therapy in prostate cancer. Akt inhibitor therapy in combination with abiraterone demonstrates early promise in the clinical control of PTEN-deficient prostate cancers. We have previously implicated BCL-XL in the survival of PTEN-null PC-3 prostate cancer cells in vitro, regulated by integrin alpha 5 (ITGA5) (Ren, MCR 2016). Toward advancing therapeutic strategies in PTEN-deficient prostate cancer we further assessed the link between PTEN and BCL-XL mediated prostate cancer cell survival. Methods: Isogenic PTEN knockdown stable cell lines (VCAP and DU145) were generated by lentiviral transduction of pLKO-control-shRNA and two pLKO-PTEN-shRNAs, respectively. PTEN was exogenously expressed in PTEN-null prostate cancer cell lines (PC-3, LNCaP and C4-2B) by 1) transfecting cells with pCMV-Flag-vector or pCMV-Flag-PTEN 2) transfecting cells with pcDNA3-GFP-PTEN and then sorting GFP-PTEN-negative and GFP-PTEN-positive cells by flow cytometry. Cells were then treated with a single agent BCL-XL inhibitor (A1331852 or navitoclax (ABT263); AbbVie), PI3K (buparlisib; Selleck Chem) or Akt (ipatasertib, Selleck Chem) inhibitors or combined PI3K and BCL-2 family inhibitors. Cell survival was assessed by cell viability assay. Expression of pAkt, ITGA5 and apoptotic markers was assayed by Western blot analysis and/or flow cytometry. Results: Notwithstanding the reduction of phospho-bad expression with PI3K inhibition, the potent BCL-XL-targeting BH3 mimetic, A1331852, compared to single agent PI3K or Akt inhibitors in titrated doses was alone sufficient to induce apoptosis and loss of cell viability in all PTEN-null prostate cancer lines tested. PTEN expression reversed the apoptotic response to BCL-XL targeting and blocked the anti-proliferative effect of PI3K or Akt inhibition in PTEN-mutant cells. PTEN knockdown induced membrane expression of ITGA5 whereas PTEN overexpression diminished ITGA5 expression. Conclusions: PTEN regulates the apoptotic threshold in prostate cancer cells via BCL-XL independent of the PI3K/Akt pathway. The induction of ITGA5 membrane localization by PTEN loss defines a potential upstream signaling mechanism for the BCL-XL regulated apoptotic threshold in these cells. Highly potent BCL-XL inhibition requires further investigation as a therapeutic strategy in PTEN-deficient prostate cancer. Citation Format: Wenying Ren, Raghav Joshi, Paul Mathew. A potent BH3 mimetic targeting BCL-XL induces apoptosis regulated by PTEN loss and integrin alpha 5 in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5499.

Abstract SY22-02: Metabolic vulnerability of PTEN mutant cancer

Abstract Signaling pathways that act as drivers of cancer cell growth and proliferation activate metabolic pathways required to generate the building blocks required for cell doubling. Loss of PTEN activates PI3K, AKT and mTOR to stimulate glucose and glutamine flux to allow DNA replication. One of the metabolic pathways that is activated after deletion of both alleles of PTEN is the de novo pyrimidine synthesis pathway. PTEN null cells show evidence of increased DNA replication and low levels of damage at replication forks that can be rescued by providing supplementary uridine. Interruption of de novo pyrimidine synthesis with an inhibitor of dihydroorotate dehydrogenase (DHODH) increased DNA damage at replication forks of PTEN null cells that ultimately led to chromosome breaks detected in mitosis and cell death. This cellular vulnerability to inhibition of DHODH was rescued with uridine, demonstrating the specificity of the effect. Exploration of the mechanism that led to vulnerability of PTEN null cells demonstrated that it was due to defective ATR signaling at replication forks that was a consequence of activated AKT, which phosphorylate and attenuate TOPBP1 and CHK1. Complementation of PTEN null cells with mutants of TOPBP1 and CHK1 that cannot be phosphorylated by AKT rescued the cells from DNA damage and cell death due to inhibition of DHODH. Tumor cell lines lacking PTEN tended to be more sensitive to DHODH inhibition than PTEN wild type cells were. In conclusion, the attenuated ATR signaling and increased flux of nucleotides to newly synthesized DNA at replication forks makes PTEN mutant cells vulnerable to disruption of de novo pyrimidine synthesis. Citation Format: Ramon E. Parsons. Metabolic vulnerability of PTEN mutant cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY22-02.

Abstract 5802: Efficacy of PI3Kβ inhibitor AZD8186 in PTEN-deficient triple-negative breast cancer

Abstract Background: Phosphatase and tensin homologue (PTEN) loss is a frequently disrupted tumor suppressor in cancer. PTEN serves as a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway to promote balanced cell proliferation, survival and differentiation. Consequently, loss of PTEN function increases cell proliferation, decreases cell death and contributes to tumor initiation. PTEN loss occurs across a variety of cancer subtypes, and these PTEN deficient tumors are dependent on PI3Kβ activity. As a result, therapeutic approaches that focus on the inhibition of PI3Kβ isoform in PTEN-null cells are of interest. We evaluate the efficacy of PI3Kβ inhibitors focused on triple negative breast cancers with loss of PTEN function. Methods: We studied the effects of p110β inhibitor AZD8186 in a panel of ten TNBC cell lines, which included four PTEN-mutant cell lines. Three of four cell lines were confirmed to have PTEN loss on western blot. In vitro, cell proliferation assays were performed to determine the half maximal inhibitory concentration (IC50) after 3 days of treatment and to test the effects in combination with chemotherapy. We confirmed the sensitivity of the PTEN-null cell lines to AZD8186 with colony formation assays. Western blot analysis was performed to assess PTEN expression and PI3K pathway activation in MDA-MB-436 and MDA-MB-468 cell lines, and to evaluate effects of AZD8186 on PI3K signaling. Results: Cell lines with PTEN loss were significantly more sensitive to AZD8186 in vitro (p= 0.008). AZD8186 inhibited PI3K signaling. Western blot revealed decreased activation of pAKT, pGSK3β, pPRAS40 and pS6. Activation of other pathways were evaluated with no activation of the MAPK/ERK or MEK pathways appreciated. AZD8186 treatment resulted in increased apoptosis but did not have a significant effect on cell-cycle progression in PTEN-deficient cell lines. AZD8186 was not synergistic with eribulin, paclitaxel or carboplatin; combinations with novel targeted therapies in vitro and in vivo are ongoing. Conclusion: AZD8186 had single agent efficacy in PTEN-deficient triple negative breast cancer cell lines in vitro. Further study is needed to identify rationale combinations and in vivo efficacy. Citation Format: Nicci Owusu-Brackett, Ming Zhao, Argun Akcakanat, Kurt W. Evans, Erkan Yuca, Funda Meric-Bernstam. Efficacy of PI3Kβ inhibitor AZD8186 in PTEN-deficient triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5802.

Akt1 enhances the chemoradiotherapy resistance of gliomas through phosphorylation of ubiquitin-conjugating enzyme 2S: An experimental study

Objective To explore the mechanism of Akt1-associated enhancement of the chemoradiotherapy resistance of gliomas. Methods The immunofluorescence (IF) experiment was conducted to test the expression of ubiquitin-conjugating enzyme 2S (UBE2S) in the normal astrocytes and glioma cells (U87, U251, SF767 and U373). The Western blot was used to assess the differences of UBE2S expression in PTEN mutant and wild type glioma cell lines. To explore the role of PTEN/Akt signal pathway in UBE2S functions and the effect of UBE2S on the non-homologous end joining (NHEJ) repair, a constitutively activated Akt1 (Myr-HA-Akt1) and the UBE2S wild type or UBE2S T152A mutant were coexpressed. The flow cytometry (FCM) was used to test in the chemoradiotherapy sensitivity of the stable UBE2S knockdown glioma cells. Results Compared with the normal astrocytes, UBE2S was overexpressed in the malignant glioma cells. The expression of UBE2S was more stable in the PTEN mutant glioma cell line. Akt1 phosphorylated UBE2S at T152, which promoted the stabilization of UBE2S. UBE2S interacted with Ku70 and Ku80, which increased the expression of Ku70 (P=0.009). The results of Western blot showed that the expression of Ku70, Ku80 and DNA-PKcs decreased significantly after knockdown of UBE2S expression (P<0.001). The results of FCM showed that the knockdown of UBE2S could enhance the chemoradiotherapy sensitivity of gliomas(P<0.001). Conclusions UBE2S is highly expressed in the malignant glioma. Akt1 physically interacts with UBE2S and phosphorylates UBE2S at T152, which is critical for the stabilization of UBE2S. The knockdown of UBE2S expression could retard NHEJ-mediated DNA repair and thus increase the sensitivity of chemoradiotherapy. Key words: Glioma; Chemoradiotherapy; Ubiquitin-conjugating enzyme 2S; PTEN/Akt signal pathway

Mutation in the FGFR1 tyrosine kinase domain or inactivation of PTEN is associated with acquired resistance to FGFR inhibitors in FGFR1‐driven leukemia/lymphomas

Stem cell leukemia/lymphoma syndrome (SCLL) is driven by constitutive activation of chimeric FGFR1 kinases generated by chromosome translocations. We have shown that FGFR inhibitors significantly suppress leukemia and lymphoma development in vivo, and cell viability in vitro. Since resistance to targeted therapies is a major reason for relapse, we developed FGFR1-overexpressing mouse and human cell lines that are resistant to the specific FGFR inhibitors AZD4547 and BGJ398, as well as non-specific inhibitors, such as ponatinib, TKI258 and E3810. Two mutually exclusive mechanisms for resistance were demonstrated; an activating V561M mutation in the FGFR1 kinase domain and mutational inactivation of PTEN resulting in increased PI3K/AKT activity. Ectopic expression of PTEN in the PTEN-mutant cells resensitizes them to FGFR inhibitors. Treatment of resistant cells with BGJ398, in combination with the BEZ235 PI3K inhibitor, shows an additive effect on growth in vitro and prolongs survival in xenograft models in vivo. These studies provide the first direct evidence for both the involvement of the FGFR1 V561M mutation and PTEN inactivation in the development of resistance in leukemias overexpressing chimeric FGFR1. These studies also provide a potential strategy to treat leukemias and lymphomas driven by FGFR1 activation that become resistant to FGFR1 inhibitors.

Abstract 2162: MEK signaling marks resistance to synthetic lethality induced by PI3K/Akt and BCL-XL inhibition in PTEN-mutant cancers

Abstract Background: Loss of PTEN represents one of the most common disruptions of tumor suppressor activity in the progression of human neoplasia. There are no established precision medicine approaches defined in PTEN-deficient neoplasms which include prostate, breast, glioblastoma (GBM) and uterine cancers. In PTEN-mutant prostate cancer cells, the PI3K/Akt pathway collaborates with BCL-XL specifically, to regulate the apoptotic threshold (Ren, MCR 2016). Whereas single agent PI3K/Akt inhibitors are ineffective in inducing cell death, combined inhibition of PI3K/Akt and BCL-XL in PTEN-mutant prostate cancer cells results in synergistic apoptosis suggesting a novel therapeutic strategy with translational potential in this important subset of disease. Hypothesis: We hypothesized that synthetic lethality following combinatorial PI3K/BCL-XL inhibition is conserved across the range of PTEN-mutant cancers. Methods: PTEN-mutant cell lines (ATCC), validated for PTEN-loss and p-Akt expression, were assessed in apoptosis (cleaved Parp) and cell-viability assays following combined PI3K (Buparlisib, SelleckChem) and BCL-XL (A-1331852, AbbVie) inhibition (1µM each, 24h). Since MEK signaling has been associated with resistance to PI3K pathway inhibition, p-Erk was profiled as a candidate marker of resistance. The contribution of MEK signaling to the apoptotic threshold in p-Erk expressing PTEN-mutant cells was tested with pharmacological MEK inhibition (Trametinib, SelleckChem; 1,3,5 µM 48h) in combination. Results: MEK signaling and PI3K/BCL-XL inhibition in PTEN-mutant cancersSourceCell linePTENp-Aktp-ErkSynergistic Apoptosis with PI3K / BCL-XL inhibitionLoss of cell viabilityp-Erk suppression with MEK inhibitionApoptosis with concomitant MEK inhibitionProstatePC3AbsentPresentAbsentYesYesN/AN/AProstateLNCaPAbsentPresentAbsentYesYesN/AN/AProstateC4-2BAbsentPresentAbsentYesYesN/AN/ABreastZR-75-1AbsentPresentWeakYesYesN/AN/AUterineSK-UT-1AbsentPresentPresentNoNoYesYesGBMA-172AbsentPresentPresentNoNoYesNoCervixC33-AAbsentPresentPresentNoNo-- Conclusions: Synthetic lethality in PTEN-mutant tumor cells with PI3K/BCL-XL inhibition was confined to tumor cells without active MEK signaling. MEK inhibition inconsistently overcomes resistance to therapy suggesting heterogeneity of survival pathways in p-erk expressing PTEN-deficient tumors. These data have implications for precision medicine approaches in PTEN-deficient tumors as absence of p-Erk expression may predict for a responsive phenotype. Citation Format: Raghav Joshi, Wenying Ren, Paul Mathew. MEK signaling marks resistance to synthetic lethality induced by PI3K/Akt and BCL-XL inhibition in PTEN-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2162. doi:10.1158/1538-7445.AM2017-2162

PTEN Regulates Glutamine Flux to Pyrimidine Synthesis and Sensitivity to Dihydroorotate Dehydrogenase Inhibition

Metabolic changes induced by oncogenic drivers of cancer contribute to tumor growth and are attractive targets for cancer treatment. Here, we found that increased growth of PTEN-mutant cells was dependent on glutamine flux through the de novo pyrimidine synthesis pathway, which created sensitivity to the inhibition of dihydroorotate dehydrogenase, a rate-limiting enzyme for pyrimidine ring synthesis. S-phase PTEN-mutant cells showed increased numbers of replication forks, and inhibitors of dihydroorotate dehydrogenase led to chromosome breaks and cell death due to inadequate ATR activation and DNA damage at replication forks. Our findings indicate that enhanced glutamine flux generates vulnerability to dihydroorotate dehydrogenase inhibition, which then causes synthetic lethality in PTEN-deficient cells due to inherent defects in ATR activation. Inhibition of dihydroorotate dehydrogenase could thus be a promising therapy for patients with PTEN-mutant cancers.Significance: We have found a prospective targeted therapy for PTEN-deficient tumors, with efficacy in vitro and in vivo in tumors derived from different tissues. This is based upon the changes in glutamine metabolism, DNA replication, and DNA damage response which are consequences of inactivation of PTENCancer Discov; 7(4); 380-90. ©2017 AACR.See related article by Brown et al., p. 391This article is highlighted in the In This Issue feature, p. 339.

Abstract 1126: Importance of the REM (Ras exchange) motif for membrane interactions by RasGRP3

Abstract RasGRP comprises a family of guanine nucleotide exchange factors that activate small GTPases, regulating the dissociation of GDP from Ras GTPases to enhance the formation of the active GTP-bound form. RasGRP3 is a complex protein with REM (Ras exchange motif), GEF (catalytic domain), EF-hand, C1, SuPT, and PT (plasma membrane-targeting) domains, each of which can affect the cellular membranes to which RasGRP1/3 localizes. The C1 domain was the first membrane localizing domain identified in RasGRP1/3. It binds to the lipid second messenger diacylglycerol (DAG) and has the potential to mediate translocation of RasGRP1/3. The catalytic GEF domain and REM domain are both required for membrane localization of RasGRP1, affecting both endomembrane and plasma membrane targeting of RasGRP1. The objective of this study was to explore the importance of these two domains in translocation of RasGRP3. For these studies, we prepared full length RasGRP1/3, RasGRP1/3 C1 domains, and truncated RasGRP1/3 green fluorescent protein labeled constructs to transfect human prostate adenocarcinoma (LNCaP) cells. In the LNCaP cells, which are a PTEN mutant cell line with markedly elevated phosphoinositide levels, RasGRP1 translocates to the plasma membrane in response to PMA. In contrast, the full length RasGRP3 does not translocate to the plasma membrane even though it has a PT (plasma membrane-targeting) domain. In parallel, we examined the translocation of the C1 domain of RasGRP1/3 and of the truncated RasGRP1/3 constructs. The C1 domain of RasGRP3 was insufficient for targeting RasGRP3 to the plasma membrane. However, truncated RasGRP3s, which contain the PT domain and are missing the REM motif, showed stronger translocation. To better understand differences in the translocation pattern of these two proteins we compared RasGRP1 (“template”) and RasGRP3 (“target”) domains using Swiss-Model (homology or comparative modelling methods) and amino acid sequence alignment (NCBI/ BLAST). The percentage of identities showed remarkable differences in the structure of the REM and SuPT-PT domains. Based on these findings we prepared chimeras where we exchanged either the N or C terminus of RasGRP1/3. Exchange of the REM motif between RasGRP1 and RasGRP3 enhanced the translocation of RasGRP3, while it does not affect the translocation of RasGRP1. Exchange of C termini (SuPT-PT domain) of both proteins did not cause any changes in plasma membrane translocation. Our results clearly show that the N terminus (REM motif) of RasGRP3 is a critical element contributing to the difference between RasGRP1 and RasGRP3 in their ability to translocate to the plasma membrane. This difference in membrane translocation, in turn, should imply important differences in their regulation and, potentially, in their ligand selectivity in the living cell. Citation Format: Agnes Czikora, Noemi Kedei, Peter M. Blumberg. Importance of the REM (Ras exchange) motif for membrane interactions by RasGRP3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1126.

Abstract 4635: Comparative cancer cell line profiling differentiates the mechanism of action of different PI3K/mTOR, Aurora kinase and EZH2 inhibitors

Abstract [purpose] Profiling of drug candidates in cell line panels is an important tool to compare the selectivity and targeting of new anti-cancer agents. In addition, comparative profiling may be used to repurpose established therapeutics by identifying new mechanisms of action or cross-selectivities. [experimental procedures] A collection of more than 120 anti-cancer agents, targeting all important oncogenic signaling pathways, including classic cytotoxic agents as well as many targeted kinase inhibitors and epigenetic modulators, was profiled on a panel of 44 or 66 parallel cell line proliferation assays (Oncolines™) [1,2]. The Oncolines™ profiles of the compounds were compared by Pearson correlations of their inhibitor responses. Inhibitor sets were clustered using hierarchical trees. Response profiles were correlated to the genetic background of the cell lines by Analysis of Variance (Anova). [results] Reproducibility of the NTRC Oncolines™ cell panel was validated by monitoring cell growth rate and the variation in IC50s of replicate profiles over a period of three years. The Pearson correlation between replicates ranged between 0.60 and 0.99 for 16 different inhibitors, depending on dose-response curve shape. Correlation analyses of the &amp;gt; 120 profiled anti-cancer agents revealed separate clusters of, a.o., taxanes, platins, topo-isomerase inhibitors, and EGFR, ABL, MEK and BRAF inhibitors. This demonstrates that the Oncolines™ profiles are an unbiased representation of the compound's mechanisms. The profile of the BTK inhibitor ibrutinib correlated with EGFR inhibitors. In biochemical experiments we showed that this due to its cross-reactivity with EGFR. The six Aurora kinase inhibitors profiled fall into two separate clusters, which are related to their biochemical selectivity. Thus, Aurora A-selective inhibitors are relatively more active in cell lines with mutations in cell cycle checkpoint-related genes such as TP53 and RB1; whereas pan-Aurora inhibitors are more active in cell lines with mutations in growth factor signaling pathways, such as NRAS. Profiling of eleven PI3 kinase and mTOR inhibitors revealed four distinct clusters. PI3Kalpha and PI3Kdelta isoform selective inhibitors each target genetically distinct subgroups of cell lines. Rapamycin-analogs, such as everolimus, specifically target PTEN-mutant cell lines. Finally, profiling of EZH2 inhibitors indicates that there are essentially two classes and that these have a cellular profile that is distinct from HDAC, DOT1L or BET inhibitors. [conclusions] Comparative cancer cell line profiling is a powerful tool to rapidly explore the pharmacogenomics of drug action in cancer cells and to identify new or previously unnoted activities of compounds. [references]: [1] Uitdehaag et al. (2014) PLOS ONE 9(3) e92146; [2] Uitdehaag et al. (2015) PLOS ONE 10(6) e0132230. Citation Format: Joost C.M. Uitdehaag, Jeroen A.D.M. de Roos, Martine B.W. Prinsen, Judith R.F. de Vetter, Jelle Dylus, Antoon M. van Doornmalen, Suzanne J.C. van Gerwen, Jos de Man, Rogier C. Buijsman, Guido J.R. Zaman. Comparative cancer cell line profiling differentiates the mechanism of action of different PI3K/mTOR, Aurora kinase and EZH2 inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4635.

Highlights of This Issue

Deregulation of the transcription factor c-Myb has been implicated in the development of leukemia and several other types of cancer, including breast and colon cancer, adenoid cystic carcinoma, and certain types of glioma. By searching for compounds that disrupt the interaction of Myb with the coactivator p300, Uttarkar and colleagues have identified Naphthol AS-E phosphate as the first small molecule inhibitor of c-Myb. This work highlights a strategy for the generation of therapeutic Myb inhibitors, which may have potential for the treatment of leukemia and possibly other tumors driven by deregulated Myb.The helix-loop-helix transcription factor c-Myc is a powerful oncoprotein widely expressed in human cancer and has long been considered “undruggable.” The best available small molecule inhibitors inhibit dimerization with c-Myc's binding partner Max, but suffer from poor bioavailability that limits their use in vivo. Here, Soodgupta, Pan, and colleagues demonstrate that a lipid-conjugated prodrug based on a small molecule Myc inhibitor is effective and can be incorporated into nanoparticles targeted to integrin surface proteins expressed on multiple myeloma cells. Anti-Myc nanoparticles prolong the survival of mice with disseminated myeloma, suggesting that this approach may be clinically applicable.Focal adhesion kinase (FAK) overexpression portends a poor prognosis and decreased overall survival in many malignancies. PTEN, frequently mutated in uterine cancer, dephosphorylates FAK. Here, Thanapprapasr, Previs, and colleagues demonstrated that PTEN-mutated cells were more sensitive to FAK inhibition and combination chemotherapy than wild-type PTEN cells in vitro and in vivo. In a cohort of patients, FAK and pFAKY397 expression was correlated with poor overall survival, and PTEN levels were inversely related to pFAKY397 expression. These findings demonstrate that PTEN-mutated uterine cancers are more sensitive to FAK inhibition, and PTEN represents a potential biomarker for further clinical development.Quiescent cancer cells, residing in hypoxic and nutrient-deprived parenchyma of solid tumors, pose a major obstacle for successful drug treatment. In order to identify compounds targeting this cell population, Senkowski and colleagues performed a high-throughput screening for drugs selectively toxic to quiescent cancer spheroids. Out of 1,600 compounds, the five most 3D selective were all inhibitors of mitochondrial respiration. Of these, a FDA-approved anthelmintic drug, nitazoxanide, produced strong in vivo tumor growth inhibition when combined with the standard chemotherapeutic agent irinotecan. This study identified nitazoxanide, a drug with minor side effects and good oral bioavailability, as a promising candidate for drug repurposing against colorectal cancer.

PARP targeting counteracts gliomagenesis through induction of mitotic catastrophe and aggravation of deficiency in homologous recombination in PTEN-mutant glioma.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults and one of the most aggressive cancers. PARP-1 is a nuclear protein involved in multiple facets of DNA repair and transcriptional regulation. In this study we dissected the action of PARP inhibition in different GBM cell lines with either functional or mutated PTEN that confers resistance to diverse therapies. In PTEN mutant cells, PARP inhibition induced a severe genomic instability, exacerbated homologous recombination repair (HR) deficiency and down-regulated the Spindle Assembly Checkpoint (SAC) factor BUBR1, leading to mitotic catastrophe (MC). EGFR gene amplification also represents a signature of genetic abnormality in GBM. To more effectively target GBM cells, co-treatment with a PARP inhibitor and an EGFR blocker, erlotinib, resulted in a strong suppression of ERK1/2 activation and in vivo the combined effect elicited a robust reduction in tumour development. In conclusion, PARP inhibition targets PTEN-deficient GBM cells through accentuation of SAC repression and aggravation of HR deficiency, leading to the induction of genomic instability and eventually deriving to mitotic catastrophe (MC); the inhibition of PARP and co-treatment with an inhibitor of pro-survival pathways strongly retarded in vivo gliomagenesis.

Abstract 2788: Antiproliferative schweinfurthins reveal a role of the trans-Golgi-network in mTOR-AKT signaling and cancer cell survival

Abstract The schweinfurthin class of natural compounds showed selective and potent anti-proliferative activities in the NCI-60 cancer cell panel1,2). Recent work by Burgett et al3) suggested that schweinfurthins may bind to oxysterol-binding protein-related proteins (ORPs) for their anti-proliferative activity. However, the mechanism of the selectivity and potency is still unknown. Here we show that schweinfurthins preferentially inhibited cell proliferation of PTEN-deficient cancer cells across different cancer types, and specifically inhibited AKT phosphorylation in both PTEN wild-type and mutant cancer cells. Intracellularly, a fluorescent schweinfurthin probe localized within the endosome/lysosome structure. Treatment of cells with active schweinfurthins caused fragmentation of the trans-Golgi-network (TGN), a vesicular trafficking system, in a dose- and time-dependent manner, without impacting the cis, med or trans-Golgi matrix structures. An inactive trimethylated analog of schweinfurthins did not display this activity. Using biotinylated probes, we found that schweinfurthins bind to the sterol-binding domain of ORPs; the capacity of binding to ORPs was positively correlated with both TGN fragmentation activity and anti-proliferative activity. These results are consistent with the recent observation that knockdown of specific ORP proteins leads to TGN trafficking disruption4). The fragmentation of TGN by schweinfurthins resulted in specific inhibition of mTOR-AKT pathway signaling in two ways: 1) disruption of lipid raft organization at the plasma membrane, evidenced by diminished trafficking of ceramide, GM1 and sphingomyelin, which inhibited PDK1 phosphorylation; and 2) dislocation of mTOR from its active sites at endosomes and/or lysosomes. Furthermore, we found that PTEN-deficient DLBCL cancer cells were highly sensitive to schweinfurthin treatment in vitro (IC50 values &amp;lt; 10 nM). Normal human fibroblasts were 1000 times less sensitive than the DLBCL cells. Taken together, our results disclosed an important role of the TGN, which was specifically targeted by schweinfurthins, in mTOR-AKT signaling and in cancer cell survival, suggesting that analogs of schweinfurthin might offer promising novel therapeutics for treating mTOR-AKT signaling-addicted cancer cells such as PTEN-deficient DLBCL. 1. Beutler JA, et al. Cytotoxic geranyl stilbenes from Macaranga schweinfurthii. J Nat Prod., 1998, 61:1509-1512. 2. Yoder BJ, et al. Antiproliferative prenylated stilbenes and flavonoids from Macaranga alnifolia from the Madagascar rainforest. J Nat Prod., 2007, 70:342-346. 3. Burgett AW, et al. Natural products reveal cancer cell dependence on oxysterol-binding proteins. Nat Chem Biol., 2011, 7:639-647. 4. Du X, et al. A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking. J Cell Biol., 2011, 192:121-135. Citation Format: Xingfeng Bao, Naoko Hata, Kishan Agarwala, Zichun Wang, Winnie Lee, Lana Parent, Hongsheng Cheng, Dayong Qiu, Yongchun Shen, Bingfan Du, Wanjun Zheng, Nanding Zhao, Maarten Postema, Mary Woodall Jappe, Yasutaka Takase, Toshimitsu Uenaka, Kenichi Nomoto. Antiproliferative schweinfurthins reveal a role of the trans-Golgi-network in mTOR-AKT signaling and cancer cell survival. [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 2788. doi:10.1158/1538-7445.AM2014-2788

Abstract 4450: Comparison of structural elements in RasGRP3 and RasGRP1 controlling membrane interaction

Abstract RasGRP comprises a family of guanine nucleotide exchange factors that activate small GTPases, regulating the dissociation of GDP from Ras GTPases to enhance the formation of the active GTP-bound form. RasGRP3 has a region homologous to the phosphoinositide binding region of RasGRP1. The objective of this study was to explore the hypothesis that this region of RasGRP3, like that of RasGRP1, indeed contributes to phosphoinositide binding. For these studies, we prepared full length RasGRP1/3, RasGRP1/3 C1 domain, and truncated (C1-SuPT-PT) RasGRP1 and RasGRP3 green fluorescent protein labeled constructs to transfect human prostate adenocarcinoma (LNCaP) and human cervical carcinoma (HeLa) cells. In HeLa cells, both RasGRP1 and RasGRP3 translocated to the endonuclear membranes, most probably to the Gogli in response to phorbol ester (PMA) and other agents. In the LNCaP cells, which are a PTEN mutant cell line with markedly elevated phosphoinositide levels, RasGRP1 translocates to the plasma membrane. In contrast, the full length RasGRP3 does not translocate to the plasma membrane even though it has a PT (plasma membrane-targeting) domain with a basic/hydrophobic cluster of amino acids (BHC) similar to that of RasGRP1. In parallel, we examined the translocation of the C1 domains of RasGRP1/3 and of the truncated RasGRP1/3 constructs, as well as the corresponding constructs with amino acid substitutions in the predicted phosphoinositide binding domain of RasGRP3. The RasGRP1 translocated to the plasma membrane in response to PMA. The C1 domain of RasGRP3 was insufficient for targeting RasGRP3 to the plasma membrane. However, truncated RasGRP3, which contains the PT domain, showed stronger translocation. Substituting amino acids (Asp647, Lys648 and Ala651) in the PT domain of full length RasGRP3 triggered the plasma membrane translocation in the LNCaP cells. We conclude that the differences in the BHC section of the PT domain of RasGRP3 play an important role in the membrane translocation of RasGRP3 and will reduce its sensitivity to membrane phosphoinositide levels. Citation Format: Agnes Czikora, Noemi Kedei, Peter M. Blumberg. Comparison of structural elements in RasGRP3 and RasGRP1 controlling membrane interaction. [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 4450. doi:10.1158/1538-7445.AM2014-4450

The Relationship Between Estrogen and Genes in the Molecular Pathogenesis of Endometrial Carcinoma

Endometrial carcinoma is a common cancer of the female reproductive system. It comprises a common estrogen-dependent endometrioid carcinoma (EC) in peri- and postmenopausal women and an uncommon estrogen-independent serous carcinoma in older women. ECs exhibit specific mutations (e.g., in PTEN gene), but the link between hormones and genetic changes remains unclear. A subset of ECs is well known to possess an altered DNA mismatch repair (MMR) system, causing microsatellite instability. Recently, the catalytic subunit of DNA polymerase e (POLE), which functions in the DNA base excision repair (BER) system against mutations (e.g., by estrogen metabolite), was shown to be mutated in a small subset of ECs, exhibiting microsatellite stability. This suggests that a defect in either MMR or BER system provides alternative mechanisms to achieve genomic instability, resulting in acquisition of specific gene mutations. A model mouse for addressing the effect of estrogen in PTEN-mutated cells showed that estrogen clonally proliferates PTEN-mutated cells together with surrounding cells in its endometrium, and depletion of estrogen induces predominant growth of PTEN-mutated cells with estrogen-independent capabilities, resulting in neoplasia. Taken together, those mechanisms may explain why the incidence of ECs increases with peri- or post-menopausal status.