Amandine Alard

DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity

Radiotherapy is under investigation for its ability to enhance responses to immunotherapy. However, the mechanisms by which radiation induces anti-tumour T cells remain unclear. We show that the DNA exonuclease Trex1 is induced by radiation doses above 12–18 Gy in different cancer cells, and attenuates their immunogenicity by degrading DNA that accumulates in the cytosol upon radiation. Cytosolic DNA stimulates secretion of interferon-β by cancer cells following activation of the DNA sensor cGAS and its downstream effector STING. Repeated irradiation at doses that do not induce Trex1 amplifies interferon-β production, resulting in recruitment and activation of Batf3-dependent dendritic cells. This effect is essential for priming of CD8+ T cells that mediate systemic tumour rejection (abscopal effect) in the context of immune checkpoint blockade. Thus, Trex1 is an upstream regulator of radiation-driven anti-tumour immunity. Trex1 induction may guide the selection of radiation dose and fractionation in patients treated with immunotherapy.

NAD(P)H Quinone-Oxydoreductase 1 Protects Eukaryotic Translation Initiation Factor 4GI from Degradation by the Proteasome

ABSTRACT The eukaryotic translation initiation factor 4GI (eIF4GI) serves as a central adapter in cap-binding complex assembly. Although eIF4GI has been shown to be sensitive to proteasomal degradation, how the eIF4GI steady-state level is controlled remains unknown. Here, we show that eIF4GI exists in a complex with NAD(P)H quinone-oxydoreductase 1 (NQO1) in cell extracts. Treatment of cells with dicumarol (dicoumarol), a pharmacological inhibitor of NQO1 known to preclude NQO1 binding to its protein partners, provokes eIF4GI degradation by the proteasome. Consistently, the eIF4GI steady-state level also diminishes upon the silencing of NQO1 (by transfection with small interfering RNA), while eIF4GI accumulates upon the overexpression of NQO1 (by transfection with cDNA). We further reveal that treatment of cells with dicumarol frees eIF4GI from mRNA translation initiation complexes due to strong activation of its natural competitor, the translational repressor 4E-BP1. As a consequence of cap-binding complex dissociation and eIF4GI degradation, protein synthesis is dramatically inhibited. Finally, we show that the regulation of eIF4GI stability by the proteasome may be prominent under oxidative stress. Our findings assign NQO1 an original role in the regulation of mRNA translation via the control of eIF4GI stability by the proteasome.

4E-BP restrains eIF4E phosphorylation.

In eukaryotes, mRNA translation is dependent on the cap-binding protein eIF4E. Through its simultaneous interaction with the mRNA cap structure and with the ribosome-associated eIF4G adaptor protein, eIF4E physically posits the ribosome at the 5′ extremity of capped mRNA. eIF4E activity is regulated by phosphorylation on a unique site by the eIF4G-associated kinase MNK. eIF4E assembly with the eIF4G-MNK sub-complex can be however antagonized by the hypophosphorylated forms of eIF4E-binding protein (4E-BP). We show here that eIF4E phosphorylation is dramatically affected by disruption of eIF4E-eIF4G interaction, independently of changes in MNK expression. eIF4E phosphorylation is actually strongly downregulated upon eIF4G shutdown or upon sequestration by hypophosphorylated 4E-BP, consequent to mTOR inhibition. Downregulation of 4E-BP renders eIF4E phosphorylation insensitive to mTOR inhibition. These data highlight the important role of 4E-BP in regulating eIF4E phosphorylation independently of changes in MNK expression.

Dual mTORC1/2 Inhibition as a Novel Strategy for the Resensitization and Treatment of Platinum-Resistant Ovarian Cancer.

There is considerable interest in the clinical development of inhibitors of mTOR complexes mTORC1 and 2. Because mTORC1 and its downstream mRNA translation effectors may protect against genotoxic DNA damage, we investigated the inhibition of mTORC1 and mTORC1/2 in the ability to reverse platinum resistance in tissue culture and in animal tumor models of serous ovarian cancer. Cell survival, tumor growth, PI3K–AKT–mTOR pathway signaling, DNA damage and repair response (DDR) gene expression, and translational control were all investigated. We show that platinum-resistant OVCAR-3 ovarian cancer cells are resensitized to low levels of carboplatin in culture by mTOR inhibition, demonstrating reduced survival after treatment with either mTORC1 inhibitor everolimus or mTORC1/2 inhibitor PP242. Platinum resistance is shown to be associated with activating phosphorylation of AKT and CHK1, inactivating phosphorylation of 4E-BP1, the negative regulator of eIF4E, which promotes increased cap-dependent mRNA translation and increased levels of CHK1 and BRCA1 proteins. Animals with platinum-resistant OVCAR-3 tumors treated with carboplatin plus mTORC1/2 inhibition had significantly longer median survival and strikingly reduced metastasis compared with animals treated with carboplatin plus everolimus, which inhibits only mTORC1. Reduced tumor growth, metastasis, and increased survival by mTORC1/2 inhibition with carboplatin treatment was associated with reduced AKT-activating phosphorylation and increased 4E-BP1 hypophosphorylation (activation). We conclude that mTORC1/2 inhibition is superior to mTORC1 inhibition in reversing platinum resistance in tumors and strongly impairs AKT activation, DNA repair responses, and translation, promoting improved survival in the background of platinum resistance. Mol Cancer Ther; 15(7); 1557–67. ©2016 AACR.

Hyperactive mTOR and MNK1 phosphorylation of eIF4E confer tamoxifen resistance and estrogen independence through selective mRNA translation reprogramming

The majority of breast cancers expresses the estrogen receptor (ER+) and is treated with anti-estrogen therapies, particularly tamoxifen in premenopausal women. However, tamoxifen resistance is responsible for a large proportion of breast cancer deaths. Using small molecule inhibitors, phospho-mimetic proteins, tamoxifen-sensitive and tamoxifen-resistant breast cancer cells, a tamoxifen-resistant patient-derived xenograft model, patient tumor tissues, and genome-wide transcription and translation studies, we show that tamoxifen resistance involves selective mRNA translational reprogramming to an anti-estrogen state by Runx2 and other mRNAs. Tamoxifen-resistant translational reprogramming is shown to be mediated by increased expression of eIF4E and its increased availability by hyperactive mTOR and to require phosphorylation of eIF4E at Ser209 by increased MNK activity. Resensitization to tamoxifen is restored only by reducing eIF4E expression or mTOR activity and also blocking MNK1 phosphorylation of eIF4E. mRNAs specifically translationally up-regulated with tamoxifen resistance include Runx2, which inhibits ER signaling and estrogen responses and promotes breast cancer metastasis. Silencing Runx2 significantly restores tamoxifen sensitivity. Tamoxifen-resistant but not tamoxifen-sensitive patient ER+ breast cancer specimens also demonstrate strongly increased MNK phosphorylation of eIF4E. eIF4E levels, availability, and phosphorylation therefore promote tamoxifen resistance in ER+ breast cancer through selective mRNA translational reprogramming.

Abstract 4525: mTOR complex inhibition as a novel therapeutic strategy in high-grade papillary serous ovarian cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Objectives: To compare mTOR complex 1 inhibition (RAD001) vs. mTOR complex 1/2 inhibition (PP242) as single agents and with Carboplatin (CPP) in a preclinical model of serous ovarian cancer (OVCA). Methods: In vitro: OVCAR3 and SKOV3 cell lines were exposed to the following treatment conditions: RAD001 or PP242 as single agents, vehicle, CPP alone, and RAD001 or PP242 followed by CPP. Colony forming assays (CFAs) were performed and quantified. mRNA levels of AKT, downstream targets of mTOR and the DNA repair response (ATR, ATM, BRCA1/2) were quantified by qRTPCR. Immunoblots characterized the protein expression of key components of mTOR and DNA repair pathways. Non-parametric analyses were used to compare results across groups on SPSS. In vivo: OVCAR3 cells expressing F-Luciferase were injected IP into SCID-BG mice. 15 wks post-injection, mice were exposed to the treatment conditions described above for 4 weeks. Tumor growth and response to treatment were assessed using bioluminescence imaging (IVIS). Results were analyzed on Living Image and Prism6. Results: In vitro: OVCAR3 and SKOV3 cells are highly sensitive to mTOR inhibition. CFAs showed significantly decreased colony counts and diameter in groups exposed to either PP242 or RAD001 vs. control, an effect that was potentiated by CPP (Fig 1). qRTPCR revealed a significant decrease in 4EBP1 mRNA with mTOR inhibition (p<0.0001), but no change in other biomarkers. Although treatment with both mTOR inhibitors resulted in decreased expression of p-S6 by immunoblots, treatment with the dual inhibitor (PP242) caused a decrease in p-AKT, p-4EBP1, total and p-CHK1, and total and p-BRCA1. The levels of these proteins were not changed by addition of CPP despite the significant effect observed in the functional assay (CFA). In vivo: Treatment with CPP + PP242 was associated with a longer median survival than other treatment groups (Table 1). A decrease in tumor burden was seen on IVIS and tumor flux (photons/sec) at the end of treatment was significantly lower in mice treated with CPP+PP242 compared to other groups (p<0.0001). Conclusions: Our preclinical model supports the concurrent use of dual mTOR inhibitors and platinum chemotherapy in the treatment of OVCA. mTOR complex 1/2 inhibition impairs the DNA repair response and correlates with improved survival in a murine model. Citation Format: Fernanda Musa, Amandine Alard, Gizelka David-West, Iulia Giuroiu, Stephanie Blank, Bhavana Pothuri, John P. Curtin, Robert Schneider. mTOR complex inhibition as a novel therapeutic strategy in high-grade papillary serous ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4525. doi:10.1158/1538-7445.AM2014-4525

A widespread alternate form of cap-dependent mRNA translation initiation

Translation initiation of most mammalian mRNAs is mediated by a 5′ cap structure that binds eukaryotic initiation factor 4E (eIF4E). However, inactivation of eIF4E does not impair translation of many capped mRNAs, suggesting an unknown alternate mechanism may exist for cap-dependent but eIF4E-independent translation. We show that DAP5, an eIF4GI homolog that lacks eIF4E binding, utilizes eIF3d to facilitate cap-dependent translation of approximately 20% of mRNAs. Genome-wide transcriptomic and translatomic analyses indicate that DAP5 is required for translation of many transcription factors and receptor capped mRNAs and their mRNA targets involved in cell survival, motility, DNA repair and translation initiation, among other mRNAs. Mass spectrometry and crosslinking studies demonstrate that eIF3d is a direct binding partner of DAP5. In vitro translation and ribosome complex studies demonstrate that DAP5 and eIF3d are both essential for eIF4E-independent capped-mRNA translation. These studies disclose a widespread and previously unknown mechanism for cap-dependent mRNA translation by DAP5-eIF3d complexes.Binding of eIF4E to the 5′ cap of mRNAs is a key early step in canonical translation initiation, but the requirement for eIF4E is not universal. Here the authors show that the eIF4G homolog DAP5 interacts with eIF3 to promote cap-dependent translation of a significant number of mRNA in an eIF4E-independent manner.

Abstract MIP-051: EFFICACY OF MTORC1/2 INHIBITION ON OVARIAN CANCER STEM CELLS

INTRODUCTION: Ovarian cancer is the most lethal gynecologic malignancy. Initial treatment with platinum and taxane drugs are effective, however inevitably these patients will recur and become resistant to cytotoxic therapies. Thus, new alternatives for durable treatments need to be identified. One approach is to explore the heterogeneity of epithelial ovarian cancer and identify the subpopulation of cells that are resistant to treatment. It has been hypothesized, that cancer stem cells (CSCs) can survive chemotherapy and have enhanced tumor-initiating capabilities. The AKT-PI3K-mTOR pathway is well studied and has been shown to be critical for tumor cell survival. This pathway closely regulates both mTOR complexes (mTORC1 and mTORC2). mTORC1 regulates protein synthesis and autophagy whereas mTORC2 regulates cell motility. mTORC1 and 2 ultimately affect cell proliferation, survival and angiogenesis, all inherently important in tumorgenesis. OBJECTIVES: Rapalogs are specific and potent inhibitors of mTORC1 but have no effect of mTORC2. We investigate the efficacy of dual mTORC1/2 inhibitors and its effect on the CSC population. METHODS: Carboplatin resistant ovarian cancer cell line OVCAR3 were cultured and treated with dimethyl sulfoxide (DMSO), carboplatin, or INK128, a potent dual inhibitor of mTORC1/2. Using cell surface markers CD133 and CD44, CSCs were quantified by flow cytometry. OVCAR3 spheroids were cultured as a surrogate for CSCs. Colony formation-survival assays were performed with spheroids treated with the above mentioned conditions. Analysis was performed using ArrayScan technology. Doxycyline inducible silencing cell lines derived from OVCAR3 were generated; Non-silenced (Nsi), Raptor (Sh-mTORC1) and Rictor (Sh-mTORC2). Colony formation assays using spheroids from silenced lineages were performed to evaluate growth and survival. Western blot analysis was performed to confirm silencing of targets, and to evaluate protein expression in CSCs. RESULTS: 4.87% of the untreated OVCAR3 population were CSCs. INK128 treatment of the cells confered a 2 fold increase of CD44+/CD133+ cells, presumably CSCs, P CONCLUSIONS: The PI3K/AKT/mTOR pathway appears to have a role in the biogenesis of platinum resistant ovarian cancer cells. The inhibition of mTORC1/2 delays growth of OVCAR3 cells, and may have a role in activating in stem like CSCs. However, it does not appear to selectively inhibit CD133+/CD44+ cells. These findings suggest that targeting CSCs may lead to advances in ovarian cancer treatment. Citation Format: Jing-Yi Chern MD, Melissa Frey MD, Fernanda Musa MD, Amandine Alard PhD, Stephanie V. Blank, MD, Robert Schneider, PhD. EFFICACY OF MTORC1/2 INHIBITION ON OVARIAN CANCER STEM CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-051.

Abstract 2262: Evidence that the translational initiation factor DAP5 plays a critical role in breast cancer metastasis

The metastatic process is highly inefficient. Among the thousands cells that escape daily from the primary tumor only a few survive and posses the ability of developing overt metastases in a distant organ. Yet, metastasis is the cause of 90% of cancer-related deaths. Despite its clinical importance, little is known about genetic and biochemical determinants of metastasis and new therapeutic targets are desperately needed. During their journey to colonize a distant organ, cancer cells undergo a wide variety of stresses (hypoxia, anoikis or colonization) that down-regulate global cap-dependent protein synthesis. In order to produce the proteins needed to overcome these stresses cancer cells rely on alternative mechanisms for translation initiation, typically through non-cap-dependent means such as internal ribosome entry (IRES). DAP5 is a poorly studied translation initiation factor known to mediate IRES-driven translation of cellular mRNAs that include anti-apoptotic factors and others factors likely involved in stress responses. Therefore, we hypothesized that DAP5 is crucial for the expression of the disseminated cancer cell proteome and cell survival during metastasis. We engineered highly metastatic murine 4T1 cancer cells to express a doxycycline (dox) inducible system shRNA silencing of DAP5 (4T1 shDAP5). These cells or the non silencing (NS) shRNA control cells were grown s.c. in immunocompetent syngeneic BALB-c mice. Dox induction was started by drinking water addition 12 days post cell injection and tumors measured twice weekly. Animals were sacrificed 18 days later, lungs collected and fixed in 4% PFA. While no significant effect was observed on cell proliferation in vitro, silencing DAP5 significantly decreased primary tumor growth and lung metastatic colonization. Importantly, when the same tumor study was performed in immunodeficient animals (NOD SCID γ) the inhibition of tumor growth with DAP5 silencing was completely abolished. These data suggest that DAP5 likely enables cancer cells to elude the immune response. However in this model, DAP5 silencing impaired the metastatic process and significantly decreased the number of lung metastases observed, implying that at least one of the mechanisms by which DAP5 prevents metastasis is immune system-independent. These results have been preliminarily confirmed in human metastatic breast cancer cells that efficiently disseminate to lungs. We engineered them with the same dox inducible system. When tested on NOD SCID mice, a significant decrease in the metastatic burden was also observed upon DAP5 silencing. Our results suggest that the translation initiation factor DAP5 likely plays a critical role in breast cancer metastasis and provides new concepts for therapeutic strategies involving translational regulation and immunotherapeutics. Indeed, inhibiting DAP5 might be a powerful approach, as it impacts the whole proteome of the disseminated cancer cell. Citation Format: Amandine Alard, Fernanda Musa, Robert Schneider. Evidence that the translational initiation factor DAP5 plays a critical role in breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2262. doi:10.1158/1538-7445.AM2015-2262