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Significance Standard of care for metastatic castration-resistant prostate cancer (mCRPC) mainly relies on suppression of androgen receptor (AR) signaling. This approach has no lasting benefit due to the emergence of resistance mechanisms, such as ligand-independent splicing variant AR-V7. A metabolic feature of mCRPC is the upregulation of de novo lipogenesis to provide substrates and fuel for metastatic spread. Whether increased levels of fats affect AR signaling to promote an aggressive disease remains to be determined. Using a selective and potent inhibitor of fatty acid synthase we demonstrate that suppression of this key enzyme inhibits AR, most importantly AR-V7, and reduces mCRPC growth. Our findings offer a therapeutic opportunity for mCRPC and a potential mechanism to overcome resistance to AR inhibitors., A hallmark of prostate cancer progression is dysregulation of lipid metabolism via overexpression of fatty acid synthase (FASN), a key enzyme in de novo fatty acid synthesis. Metastatic castration-resistant prostate cancer (mCRPC) develops resistance to inhibitors of androgen receptor (AR) signaling through a variety of mechanisms, including the emergence of the constitutively active AR variant V7 (AR-V7). Here, we developed an FASN inhibitor (IPI-9119) and demonstrated that selective FASN inhibition antagonizes CRPC growth through metabolic reprogramming and results in reduced protein expression and transcriptional activity of both full-length AR (AR-FL) and AR-V7. Activation of the reticulum endoplasmic stress response resulting in reduced protein synthesis was involved in IPI-9119–mediated inhibition of the AR pathway. In vivo, IPI-9119 reduced growth of AR-V7–driven CRPC xenografts and human mCRPC-derived organoids and enhanced the efficacy of enzalutamide in CRPC cells. In human mCRPC, both FASN and AR-FL were detected in 87% of metastases. AR-V7 was found in 39% of bone metastases and consistently coexpressed with FASN. In patients treated with enzalutamide and/or abiraterone FASN/AR-V7 double-positive metastases were found in 77% of cases. These findings provide a compelling rationale for the use of FASN inhibitors in mCRPCs, including those overexpressing AR-V7.

STAT3 (signal transducers and activators of transcription 3) is a transcription factor and a member of the STAT protein family that is activated through a variety of different cytokine and growth factor receptors via JAKs, as well as through oncogenic fusion proteins and gain-of-function (GoF) mutations in STAT3 itself. STAT3 hyperactivation and GoF mutations are found in numerous cancers, including clinically aggressive hematologic malignancies with high unmet medical need, such as peripheral T cell lymphomas (PTCLs) (Andersson et al., 2020). We have previously shown that a potent and selective STAT3 heterobifunctional degrader, KTX-201, strongly represses cell growth in models of STAT3-dependent heme malignancies (Csibi et al., 2019). Herein, we report on the cellular mechanisms underlying the anti-tumor effect of STAT3 degradation in PTCL and provide a model for the relationship between pharmacokinetics/ pharmacodynamics (PK/PD) and activity of KTX-201 in vivo. The relationship between STAT3 degradation by KTX-201, anti-tumor mechanism of action and in vivo activity were investigated in anaplastic large T cell lymphoma (ALCL) models, a subset of PTCLs. In vitro, a decrease of STAT3 by 90% for 48hr was required for ALCL cells to commit to death. To identify anti-tumor mechanism(s) of KTX-201 at the systems level, we performed a time-resolved analysis of the proteomic changes of SU-DHL-1 cells undergoing growth inhibition mediated by KTX-201 at GI95. We measured the abundance of 10,000 proteins and confirmed selective degradation of STAT3 by KTX-201 after 8h of treatment. Significant changes in several marker proteins known to be involved in STAT3-mediated proximal signaling in ALCL including SOCS3, Myc and Granzyme B were observed after 16h. Functional annotation analysis of proteins identified pathways that were significantly enriched in at least one time point. Using unsupervised hierarchical clustering of annotations, we found that proteins that increased in abundance over 48h of exposure to KTX-201 were associated with markers of apoptosis and those that decreased in abundance by 24h and 48h were associated with cytokine signaling and cell cycle, respectively. Based on these data, this study identifies inhibition of cytokine signaling, G1 cell cycle arrest and induction of apoptosis as key anti-tumor mechanisms associated with KTX-201 consistent with observed cell phenotypes. STAT3 degradation in tumor was characterized in mice bearing SU-DHL-1 tumors following single dose IV administration. The STAT3 PD response in tumor was correlated with exposures in tumor. At the dose of 25 mg/kg weekly where complete tumor regression was achieved, KTX-201 achieves >90% STAT3 degradation at 24h post dosing in SUDHL1 xenografts. STAT3 degradation was maintained at 90% at 4 days post dosing. The results from the PK/PD study suggests that STAT3 degradation in tumor of >90% is necessary for anti-tumor efficacy in vivo of KTX-201, but only for a limited duration, such as 4 days out of a weekly dosing cycle. Collectively, our data demonstrate that significant STAT3 degradation for a limited time during dosing interval with KTX-201 in ALCL promotes early changes in key signaling nodes involved with proliferation and cytokine stimulation, followed by profound changes in apoptotic proteins. By integrating mechanistic biology with a deep understanding of PK/PD and efficacy, this study provides a foundation for the clinical development of STAT3 degraders using intermittent dosing regimen for treatment of PTCL and other STAT3-dependent heme malignancies. Disclosures Rong: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Csibi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company, Ended employment in the past 24 months. Yang:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rusin:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Shi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayo:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yuan:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chutake:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonald:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhu:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ji:Kymera Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Liu:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Li:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Walker:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollob:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mainolfi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Desavi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Signal transducer and activator of transcription 3 (STAT3) has been implicated in multiple aspects of tumorigenesis. In addition to increasing cancer cell proliferation and survival, constitutively activated STAT3 is proposed to regulate cross-talk between tumor, stroma and immune cells to promote immune-evasion. STAT3 activity in tumors promotes the production of immune-suppressive factors that activate STAT3 in diverse immune-cell subsets. Mechanistically, genetic studies support a direct role of activated STAT3 in regulating myeloid cell differentiation to contribute to an immune-suppressed tumor microenvironment (TME) (Kortylewski et al.; Nat. Med. 2005 and Curr. Opin. Immunol. 2008) Therefore, STAT3 is a highly attractive target for immune-oncology. Here, we demonstrate that the degradation of STAT3 with a potent and selective STAT3 degrader reverses immune suppression in preclinical models. KYM-003 is a heterobifunctional molecule that hijacks the ubiquitin-proteasome system (UPS) for rapid STAT3 degradation. KYM-003 robustly degraded STAT3 in both human peripheral blood mononuclear cells (hPBMC)-derived monocytes and lymphocytes with DC50 < 100 nM. Degradation in hPBMCs was highly selective for STAT3 vs >10,000 other detected proteins (including all other STAT family members) as evaluated by deep tandem mass tag proteomics. IL-6 treatment of hPBMCs upregulates STAT3 phosphorylation, resulting in transcription of genes involved in myeloid cell-mediated immune suppression, such as IL-10 and CD163, a marker of M2 macrophage differentiation. Treatment of cells with KYM-003 at DC90 for 6 hours abrogated the IL-6 induced up-regulation of immune-suppressive gene signatures consistent with a role of STAT3 in mediating an immune suppressive environment by regulating macrophages and other myeloid cells. In several tumor cell lines, degradation of STAT3 by KYM-003 led to strong downregulation of PD-L1. Importantly, in the BioMap StroNSCLC (DiscoverRx), a co-culture system composed of NCI-H1299 lung cancer cells, hPBMCs and primary human fibroblasts that models immune-suppressed TME biology, KYM-003 treatment decreased angiogenic and immune-suppressive cytokines, including VEGF and IL-10, and promoted the pro-inflammatory anti-tumor cytokines IL-2, IFNg and TNFa. Lastly, administration of KYM-003 to mice bearing syngeneic tumors exhibited anti-tumor activity as monotherapy in the CT26 syngeneic model. We have shown that targeting STAT3 for degradation may have a role in restoring an immune-permissive environment in tumors by both rescuing the suppressed immunologic microenvironment and directly downregulating immune checkpoint signals in tumor cells. These data support STAT3 degraders as a promising new therapeutic modality as immune-oncology agents.Citations: Kortylewski et al (2005) Nat. Med, 11(12):1314-21. Kortylewski et al (2008) Curr. Opin. Immunol, 20(2):228-33 Citation Format: Alfredo Csibi, Bin Yang, Yogesh Chutake, Karen Yuan, Michele Mayo, Veronica Campbell, Alice McDonald, Scott Rusin, Kirti Sharma, Hari Kamadurai, Henry Li, Mike Sintchak, Sean Zhu, Sharon Townson, Anthony Slavin, Haojing Rong, Phillip Liu, Chris De Savi, Jared Gollob, Duncan Walker, Nan Ji, Nello Mainolfi. A STAT3 selective targeted protein degrader decreases the immunesuppressive tumor microenvironment and drives antitumor activity in preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-088.

A kornyezet es a sajat enkep altal tamasztott egyre novekedő elvarasok, a szemelyisegbeli es motivacios rendszer sajatossagai, valamint az előzetes tapasztalatok alapjan rogzult valaszadasi modok egyarant befolyasoljak az agressziv viselkedes sajatossagait es az alkalmazott megkuzdesi stilust a serdulők koreben. Jelen tanulmanyban celunk a serdulők agressziv megnyilvanulasainak jobb megertese, valamint a megkuzdes sajatossagainak felterkepezese volt. Vizsgalatunk az agresszio kifejezesenek nemek es eletkor szerinti kulonbozősegeit, valamint az agresszio es a megkuzdes kapcsolatat elemzi. Eszkozeink a magyarorszagi populaciora adaptalt Spielberger-fele Harag es Duh Kifejezesi Mod Skala, valamint a megkuzdesi stilust vizsgalo Lazarus es Folkman-fele Konfliktusmegoldo Kerdőiv, Rozsa es mtsai adaptacioja utan. A vizsgalt minta 148 serdulőt tartalmaz a 9–12. evfolyamos korcsoportbol, atlageletkoruk 17 ev volt. Eredmenyeink szerint a fiuk es a lanyok agresszioja hasonlo mertekűnek bizonyult, ugyanakkor a ket ne...

Purpose: Targeted protein degradation is a novel therapeutic modality that holds the promise to target previously “undruggable” proteins by harnessing the innate ubiquitin proteasome system in cells. In this study we describe the discovery and characterization of KYM-003, a novel, selective degrader of the oncogenic transcription factor STAT3 that shows potent antitumor activity in vitro and in vivo in STAT3-dependent models. Introduction: STAT3 is a transcription factor downstream of several signaling events including the IL-6-JAK pathway. Activating mutations and aberrant STAT3 activation are found in numerous cancers and STAT3 activation has been directly linked to the promotion of cancer cell survival, proliferation and immune evasion, making it a highly attractive target for oncology. Potent and selective agents specifically and directly targeting STAT3 have remained elusive, however. Herein we report the discovery of a potent and selective STAT3 degrader, KYM-003, which displays strong anti-tumor activity in models of heme malignancies. Methods: The binary binding of KYM-003 to STAT3 and the E3 ligase were characterized in fluorescence polarization assays. An AlphaLISA immunoassay was used to measure the formation of the E3-KYM-003-STAT3 ternary complex. Ubiquitination of endogenous STAT3 was evaluated in human cell lysates in the presence of KYM-003. STAT3 levels in multiple primary cells, cell lines, and xenograft tumor tissues were quantified by immunoassays. STAT3 signaling was monitored through its target gene levels by RT-qPCR. Cell viability was monitored by CellTiter-Glo. Tumor xenograft studies were conducted by implanting human cancer cell lines into immune-compromised mice and assessment of tumor volume changes. Key data: KYM-003 degrades STAT3 via an E3 ligase-dependent mechanism. KYM-003 potently binds to and demonstrated strong cooperativity in promoting the formation of the STAT3-KYM-003-E3 ligase ternary complex. In a biochemical assay with A549 cell lysates, KYM-003 potently induced ubiquitination of endogenous STAT3. KYM-003 robustly degraded STAT3 in numerous primary cells or cell lines with DC50 < 100 nM. Degradation was highly selective for STAT3 vs >10,000 other detected proteins (including all other STAT family members) in A549 and hPBMCs. Degradation of STAT3 by KYM-003 led to strong downregulation of STAT3 targets gene expression, such as SOCS3, MYC, and PIM1 Importantly, total STAT3 and pSTAT3 levels in tumors were reduced by >90% for at least 24 hours after a single dose of KYM-003 and repeated dosing of KYM-003 showed dose-dependent antitumor activity in xenograft models of heme maliganancies. Conclusion: KYM-003 is a potent and selective STAT3 degrader that demonstrated strong anti-tumor activity in heme cancer models. These data support STAT3 degraders as a promising new therapeutic opportunity. Citation Format: Fred Csibi, Nan Ji, Bin Yang, Karen Yuan, Michele Mayo, Haojing Rong, Scott Rusin, Kirti Sharma, Christine Loh, Henry Li, Sharon Townson, Hari Kamadurai, Jesse Chen, Duncan Walker, Jared Gollob, Nello Mainolfi. Discovery of KYM-003, a potent and selective STAT3 degrader with antitumor activity in heme malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C054. doi:10.1158/1535-7163.TARG-19-C054

Targeted protein degradation mediated by small molecule degraders represents a new and exciting therapeutic modality to target difficult-to-drug oncogenic proteins including transcription factors. These molecules bind to both the target protein and an E3 ligase, enabling the formation of a ternary complex that leads to ubiquitination and subsequent degradation of the target protein by the proteasome. STAT3 (signal transducers and activators of transcription 3) is a transcription factor and a member of the STAT protein family. In response to cytokines and growth factors, STAT3 is phosphorylated by receptor-associated serine/threonine kinases, and phosphylated STAT3 (pSTAT3) then forms dimers that translocate into the nucleus, binds to DNA, and regulate transcription. STAT3 is frequently mutated and activated in numerous cancers including clinically aggressive hematologic malignancies with high unmet medical need. Mechanistically, aberrant activation of STAT3 has been directly linked to the promotion of cancer cell survival, proliferation and immune evasion, thus making it a highly attractive target for oncology. Potent and selective agents specifically and directly targeting STAT3 have remained elusive, however. Herein we report the discovery of a potent and selective STAT3 heterobifunctional degrader, KYM-003, which displays strong anti-tumor activity in models of STAT3-dependent heme malignancies. KYM-003 degrades STAT3 via an E3 ligase-dependent mechanism. It strongly binds to STAT3 and a E3 ligase, leading to the formation of the a productive ternary complex, which leads to ubiquitination of STAT3 and subsequent proteasomal degradation. KYM-003 robustly degraded STAT3 in a number of primary cells or cell lines with DC50 < 100 nM. Degradation was highly selective for STAT3 vs >10,000 other detected proteins (including all other STAT family members) in cell lines and human PBMCs. Degradation of STAT3 by KYM-003 led to significant downregulation of STAT3 target genes, such as SOCS3, MYC, and PIM1. Importantly, total STAT3 and pSTAT3 levels in tumors were reduced by >90% for at least 24 hours after a single dose of KYM-003 and repeated dosing of KYM-003 showed dose-dependent antitumor activity in xenograft models of heme malignancies. Collectively, our data demonstrates that KYM-003 is a potent and selective STAT3 degrader that exhibited strong anti-tumor activity in vitro and in vivo. These data support STAT3 degraders as a new and exciting therapeutic opportunity in heme malignancies. Disclosures Csibi: kymera Therapeutics: Employment, Equity Ownership. Ji:Kymera Therapeutics: Employment, Equity Ownership. Yang:Kymera Therapeutics: Employment, Equity Ownership. Yuan:Kymera Therapeutics: Employment, Equity Ownership. Mayo:kymera Therapeutics: Employment, Equity Ownership. Rong:Kymera Therapeutics: Employment, Equity Ownership. Rusin:Kymera Therapeutics: Employment, Equity Ownership. Sharma:kymera Therapeutics: Employment, Equity Ownership. Loh:Kymera Therapeutics: Employment, Equity Ownership. Li:Kymera Therapeutics: Employment, Equity Ownership. Townson:Kymera Therapeutics: Employment, Equity Ownership. Chen:kymera therapeutics: Employment, Equity Ownership. Kamadurai:Kymera Therapeutics: Employment, Equity Ownership. Walker:Kymera Therapeutics: Employment, Equity Ownership. Gollob:Kymera Therapeutics: Employment, Equity Ownership. Mainolfi:Kymera Therapeutics: Employment, Equity Ownership.

Estradiol enhances COX-2 expression and prostaglandin biosynthesis in TSC2-deficient cells via a rapamycin-insensitive, mTORC2-dependent mechanism., Lymphangioleiomyomatosis (LAM) is a progressive neoplastic disorder that leads to lung destruction and respiratory failure primarily in women. LAM is typically caused by tuberous sclerosis complex 2 (TSC2) mutations resulting in mTORC1 activation in proliferative smooth muscle–like cells in the lung. The female predominance of LAM suggests that estradiol contributes to disease development. Metabolomic profiling identified an estradiol-enhanced prostaglandin biosynthesis signature in Tsc2-deficient (TSC−) cells, both in vitro and in vivo. Estradiol increased the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin biosynthesis, which was also increased at baseline in TSC-deficient cells and was not affected by rapamycin treatment. However, both Torin 1 treatment and Rictor knockdown led to reduced COX-2 expression and phospho-Akt-S473. Prostaglandin production was also increased in TSC-deficient cells. In preclinical models, both Celecoxib and aspirin reduced tumor development. LAM patients had significantly higher serum prostaglandin levels than healthy women. 15-epi-lipoxin-A4 was identified in exhaled breath condensate from LAM subjects and was increased by aspirin treatment, indicative of functional COX-2 expression in the LAM airway. In vitro, 15-epi-lipoxin-A4 reduced the proliferation of LAM patient–derived cells in a dose-dependent manner. Targeting COX-2 and prostaglandin pathways may have therapeutic value in LAM and TSC-related diseases, and possibly in other conditions associated with mTOR hyperactivation.

International audience; The AMPactivated protein kinase (AMPK) is a serine/threonine protein kinase that acts as a sensor of cellular energy status switch regulating several systems including glucose and lipid metabolism. Recently, AMPK has been implicated in the control of skeletal muscle mass by decreasing mTORC1 activity and increasing protein degradation through regulation of ubiquitin-proteasome and autophagy pathways. In this review, we give an overview of the central role of AMPK in the control of skeletal muscle plasticity. We detail particularly its implication in the control of the hypertrophic and atrophic signaling pathways. In the light of these cumulative and attractive results, AMPK appears as a key player in regulating muscle homeostasis and the modulation of its activity may constitute a therapeutic potential in treating muscle wasting syndromes in humans.

The control of muscle cell size is a physiological process balanced by a fine tuning between protein synthesis and protein degradation. MAFbx/Atrogin-1 is a muscle specific E3 ubiquitin ligase upregulated during disuse, immobilization and fasting or systemic diseases such as diabetes, cancer, AIDS and renal failure. This response is necessary to induce a rapid and functional atrophy. To date, the targets of MAFbx/Atrogin-1 in skeletal muscle remain to be identified. We have recently presented evidence that eIF3-f, a regulatory subunit of the eukaryotic translation factor eIF3 is a key target that accounts for MAFbx/Atrogin-1 function in muscle atrophy. More importantly, we showed that eIF3-f acts as a "translational enhancer" that increases the efficiency of the structural muscle proteins synthesis leading to both in vitro and in vivo muscle hypertrophy. We propose that eIF3-f subunit, a mTOR/S6K1 scaffolding protein in the IGF-1/Akt/mTOR dependent control of protein translation, is a positive actor essential to the translation of specific mRNAs probably implicated in muscle hypertrophy. The central role of eIF3-f in both the atrophic and hypertrophic pathways will be discussed in the light of its promising potential in muscle wasting therapy.

International audience; In response to cancer, AIDS, sepsis and other systemic diseases inducing muscle atrophy, the E3 ubiquitin ligase Atrogin1/MAFbx (MAFbx) is dramatically upregulated and this response is necessary for rapid atrophy. However, the precise function of MAFbx in muscle wasting has been questioned. Here, we present evidence that during muscle atrophy MAFbx targets the eukaryotic initiation factor 3 subunit 5 (eIF3-f) for ubiquitination and degradation by the proteasome. Ectopic expression of MAFbx in myotubes induces atrophy and degradation of eIF3-f. Conversely, blockade of MAFbx expression by small hairpin RNA interference prevents eIF3-f degradation in myotubes undergoing atrophy. Furthermore, genetic activation of eIF3-f is sufficient to cause hypertrophy and to block atrophy in myotubes, whereas genetic blockade of eIF3-f expression induces atrophy in myotubes. Finally, eIF3-f induces increasing expression of muscle structural proteins and hypertrophy in both myotubes and mouse skeletal muscle. We conclude that eIF3-f is a key target that accounts for MAFbx function during muscle atrophy and has a major role in skeletal muscle hypertrophy. Thus, eIF3-f seems to be an attractive therapeutic target

It is well known that the large caliber vessels, the nutrition of the wall differs in the internal part in comparison to the external one. In the present study we assessed the presence and distribution of vascularisation of aortic media in 3 species: lamb, rabbit and chinchilla. Samples were harvested from a group of 3 animals for each species. After histological processing, we stained the sections and assessed the presence and distribution of vasa vasorum. In lamb, blood vessels were present in the media of the first aortic segments (with a larger caliber), and starting with abdominal descending aorta they were absent. In rabbit and chinchilla, blood vessels were not present in tunica media. The present study shows that vasa vasorum are present only in tunica media of arteries exceeding a certain caliber.

Genomic characterization of hematologic and solid cancers has revealed recurrent somatic mutations affecting genes encoding the RNA splicing factors SF3B1, U2AF1, SRSF2 and ZRSR2. Recent data reveal that these mutations confer an alteration of function inducing aberrant splicing and rendering spliceosome mutant cells preferentially sensitive to splicing modulation compared with wildtype (WT) cells. Here we describe a novel orally bioavailable small molecule SF3B1 modulator identified through a medicinal chemistry effort aimed at optimizing compounds for preferential lethality in spliceosome mutant cells. H3B-8800 potently binds to WT or mutant SF3b complexes and modulates splicing in in vitro biochemical splicing assays and cellular pharmacodynamic assays. The selectivity of H3B-8800 was confirmed by observing lack of activity in cells expressing SF3B1R1074H, the SF3B1 mutation previously shown to confer resistance to other splicing modulators. Although H3B-8800 binds both WT and mutant SF3B1, it results in preferential lethality of cancer cells expressing SF3B1K700E, SRSF2P95H, or U2AF1S34F mutations compared to WT cells. In animals xenografted with SF3B1K700E knock-in leukemia K562 cells or mice transplanted with Srsf2P95H/MLL-AF9 mouse AML cells, oral H3B-8800 treatment demonstrated splicing modulation and inhibited tumor growth, while no therapeutic impact was seen in WT controls. These data were also evident in patient-derived xenografts (PDX) from patients with CMML where H3B-8800 resulted in a substantial reduction of leukemic burden only in SRSF2-mutant but not in WT CMML PDX models. Additionally, due to the high frequency of U2AF1 mutations in non-small cell lung cancer, H3B-8800 was tested in U2AF1S34F-mutant H441 lung cancer cells. Similar to the results from leukemia models, H3B-8800 demonstrated preferential lethality of U2AF1-mutant cells in vitro and in in vivo orthotopic xenografts at well tolerated doses. RNA-seq of isogenic K562 cells treated with H3B-8800 revealed dose-dependent inhibition of splicing. Although global inhibition of RNA splicing was not observed; H3B-8800 treatment led to preferential intron retention of transcripts with shorter and more GC-rich regions compared to those unaffected by drug. Interestingly, H3B-8800-retained introns commonly disrupted the expression of spliceosomal genes, suggesting that the preferential effect of H3B-8800 on spliceosome mutant cells is due to the dependency of these cells on expression of WT spliceosomal genes. These data identify a novel therapeutic approach with selective lethality in leukemias and lung cancers bearing a spliceosome mutation. Despite the essential nature of splicing, cancer cells without a spliceosome mutation were less sensitive to H3B-8800 compared with potent eradication of mutant counterparts. H3B-8800 is currently undergoing clinical evaluation in patients with MDS, AML, and CMML. Citation Format: Silvia Buonamici, Akihide Yoshimi, Michael Thomas, Michael Seiler, Betty Chan, Benjamin Caleb, Fred Csibi, Rachel Darman, Peter Fekkes, Craig Karr, Gregg Keaney, Amy Kim, Virginia Klimek, Pavan Kumar, Kaiko Kunii, Stanley Chun-Wei Lee, Xiang Liu, Crystal MacKenzie, Carol Meeske, Yoshiharu Mizui, Eric Padron, Eunice Park, Ermira Pazolli, Sudeep Prajapati, Nathalie Rioux, Justin Taylor, John Wang, Markus Warmuth, Huilan Yao, Lihua Yu, Ping Zhu, Omar Abdel-Wahab, Peter Smith. H3B-8800, a novel orally available SF3b modulator, shows preclinical efficacy across spliceosome 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 1185. doi:10.1158/1538-7445.AM2017-1185

Growth-promoting signaling molecules, including the mammalian target of rapamycin complex 1 (mTORC1), drive the metabolic reprogramming of cancer cells required to support their biosynthetic needs for rapid growth and proliferation [1]. Glutamine is catabolyzed to α-ketoglutarate (αKG), a tricarboxylic acid (TCA) cycle intermediate, through two deamination reactions, the first requiring glutaminase (GLS) to generate glutamate and the second occurring via glutamate dehydrogenase (GDH) or transaminases [2]. Activation of the mTORC1 pathway has been shown previously to promote the anaplerotic entry of glutamine to the TCA cycle via GDH. Moreover, mTORC1 activation also stimulates the uptake of glutamine, but the mechanism is unknown [3]. It is generally thought that rates of glutamine utilization are limited by mitochondrial uptake via GLS, suggesting that, in addition to GDH, mTORC1 could regulate GLS. Here we demonstrate that mTORC1 positively regulates GLS and glutamine flux through this enzyme. We show that mTORC1 controls GLS levels through the S6K1-dependent regulation of c-Myc (Myc). Molecularly, S6K1 enhances Myc translation efficiency by modulating the phosphorylation of eukaryotic initiation factor eIF4B, which is critical to unwind its structured 5' untranslated region (5'UTR). Finally, our data show that the pharmacological inhibition of GLS is a promising target in pancreatic cancers expressing low levels of PTEN. ispartof: Current biology : CB vol:24 issue:19 pages:2274-80 ispartof: location:England status: published

Proliferating mammalian cells use glutamine as a source of nitrogen and as a key anaplerotic source to provide metabolites to the tricarboxylic acid cycle (TCA) for biosynthesis. Recently, mammalian target of rapamycin complex 1 (mTORC1) activation has been correlated with increased nutrient uptake and metabolism, but no molecular connection to glutaminolysis has been reported. Here, we show that mTORC1 promotes glutamine anaplerosis by activating glutamate dehydrogenase (GDH). This regulation requires transcriptional repression of SIRT4, the mitochondrial-localized sirtuin that inhibits GDH. Mechanistically, mTORC1 represses SIRT4 by promoting the proteasome-mediated destabilization of cAMP-responsive element binding 2 (CREB2). Thus, a relationship between mTORC1, SIRT4, and cancer is suggested by our findings. Indeed, SIRT4 expression is reduced in human cancer, and its overexpression reduces cell proliferation, transformation, and tumor development. Finally, our data indicate that targeting nutrient metabolism in energy-addicted cancers with high mTORC1 signaling may be an effective therapeutic approach. ispartof: Cell vol:153 issue:4 pages:840-54 ispartof: location:United States status: published

SummaryDNA damage elicits a cellular signaling response that initiates cell cycle arrest and DNA repair. Here, we find that DNA damage triggers a critical block in glutamine metabolism, which is required for proper DNA damage responses. This block requires the mitochondrial SIRT4, which is induced by numerous genotoxic agents and represses the metabolism of glutamine into tricarboxylic acid cycle. SIRT4 loss leads to both increased glutamine-dependent proliferation and stress-induced genomic instability, resulting in tumorigenic phenotypes. Moreover, SIRT4 knockout mice spontaneously develop lung tumors. Our data uncover SIRT4 as an important component of the DNA damage response pathway that orchestrates a metabolic block in glutamine metabolism, cell cycle arrest, and tumor suppression.

Metformin inhibits cancer cell proliferation, and epidemiology studies suggest an association with increased survival in patients with cancer taking metformin; however, the mechanism by which metformin improves cancer outcomes remains controversial. To explore how metformin might directly affect cancer cells, we analyzed how metformin altered the metabolism of prostate cancer cells and tumors. We found that metformin decreased glucose oxidation and increased dependency on reductive glutamine metabolism in both cancer cell lines and in a mouse model of prostate cancer. Inhibition of glutamine anaplerosis in the presence of metformin further attenuated proliferation, whereas increasing glutamine metabolism rescued the proliferative defect induced by metformin. These data suggest that interfering with glutamine may synergize with metformin to improve outcomes in patients with prostate cancer., German Science Foundation (Grant FE1185), National Institutes of Health (U.S.), Glenn Foundation for Medical Research, National Institutes of Health (U.S.) (Grant 5-P50-090381-09), National Institutes of Health (U.S.) (Grant 5-P30-CA14051-39), Burroughs Wellcome Fund, Smith Family Foundation, Damon Runyon Cancer Research Foundation, National Institutes of Health (U.S.) (Grant 1R01DK075850-01), National Institutes of Health (U.S.) (Grant 1R01CA160458-01A1)

The eukaryotic initiation factor 3 subunit f (eIF3f) is one of the 13 subunits of the translation initiation factor complex eIF3 required for several steps in the initiation of mRNA translation. In skeletal muscle, recent studies have demonstrated that eIF3f plays a central role in skeletal muscle size maintenance. Accordingly, eIF3f overexpression results in hypertrophy through modulation of protein synthesis via the mTORC1 pathway. Importantly, eIF3f Was described as a target of the E3 ubiquitin ligase MAFbx/atrogin-1 for proteasome-mediated breakdown under atrophic conditions. The biological importance of the MAFbx/atrogin-1-dependent targeting of eFI3f is highlighted by the finding that expression of ad eIF3f mutant insensitive to MAFbx/atrogin-1 polyubiquitination is associated with enhanced protection against starvation-induced muscle atrophy. A better understanding of the precise role of this subunit should lead to the development of new therapeutic approaches to prevent or limit muscle wasting that prevails in numerous physiological and pathological states such as immobilization, aging, denervated conditions, neuromuscular diseases, AIDS, cancer, diabetes. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting. (C) 2013 Elsevier Ltd. All rights reserved.

Aims In polycystic ovary syndrome (PCOS), metabolic and cardiovascular dysfunction is related to hyperandrogenic status and insulin resistance, however, Vitamin D3 has a beneficial effect partly due to its anti-oxidant capacity. Nitrative stress is a major factor in the development of cardiovascular dysfunction and insulin resistance in various diseases. Our aim was to determine the effects of vitamin D3 in a rat model of PCOS, particularly the pathogenic role of nitrative stress. Main methods Female Wistar rats weighing 100–140 g were administered vehicle (C), dihydrotestosterone (DHT) or dihydrotestosterone plus vitamin D3 (DHT + D) (n = 10 per group). On the 10th week, acetylcholine (Ach) induced relaxation ability of the isolated thoracic aorta rings was determined. In order to examine the possible role of endothelial nitric oxide synthase (eNOS) and cyclooxygenase-2 (COX-2) pathways in the impaired endothelial function, immunohistochemical labeling of aortas with anti-eNOS and anti-COX-2 antibodies was performed. Leukocyte smears, aorta and ovary tissue sections were also immunostained with anti-nitrotyrosine antibody to determine nitrative stress. Key findings Relaxation ability of aorta was reduced in group DHT, and vitamin D3 partly restored Ach induced relaxation. eNOS labeling was significantly lower in DHT rats compared to the other two groups, however COX-2 staining showed an increment. Nitrative stress showed a significant increase in response to dihydrotestosterone, while vitamin D3 treatment, in case of the ovaries, was able to reverse this effect. Significance Nitrative stress may play a role in the pathogenesis of PCOS and in the development of the therapeutic effect of vitamin D3.

The elevated metabolic requirements of cancer cells reflect their rapid growth and proliferation and are met through mutations in oncogenes and tumor suppressor genes that reprogram cellular processes. For example, in tuberous sclerosis complex (TSC)-related tumors, the loss of TSC1/2 function causes constitutive mTORC1 activity, which stimulates glycolysis, resulting in glucose addiction in vitro. In research published in Cell and Bioscience, Jiang and colleagues show that pharmacological restriction of glucose metabolism decreases tumor progression in a TSC xenograft model. See research article: http://www.cellandbioscience.com/content/1/1/34

Angiotensin II (Ang II) plays a major role in the pathogenesis of insulin resistance and diabetes by inhibiting insulin's metabolic and potentiating its trophic effects. Whereas the precise mechanisms involved remain ill-defined, they appear to be associated with and dependent upon increased oxidative stress. We found Ang II to block insulin-dependent GLUT4 translocation in L6 myotubes in an NO- and O(2)(*-)-dependent fashion suggesting the involvement of peroxynitrite. This hypothesis was confirmed by the ability of Ang II to induce tyrosine nitration of the MAP kinases ERK1/2 and of protein kinase B/Akt (Akt). Tyrosine nitration of ERK1/2 was required for their phosphorylation on Thr and Tyr and their subsequent activation, whereas it completely inhibited Akt phosphorylation on Ser(473) and Thr(308) as well as its activity. The inhibitory effect of nitration on Akt activity was confirmed by the ability of SIN-1 to completely block GSK3alpha phosphorylation in vitro. Inhibition of nitric oxide synthase and NAD(P)Hoxidase and scavenging of free radicals with myricetin restored insulin-stimulated Akt phosphorylation and GLUT4 translocation in the presence of Ang II. Similar restoration was obtained by inhibiting the ERK activating kinase MEK, indicating that these kinases regulate Akt activation. We found a conserved nitration site of ERK1/2 to be located in their kinase domain on Tyr(156/139), close to their active site Asp(166/149), in agreement with a permissive function of nitration for their activation. Taken together, our data show that Ang II inhibits insulin-mediated GLUT4 translocation in this skeletal muscle model through at least two pathways: first through the transient activation of ERK1/2 which inhibit IRS-1/2 and second through a direct inhibitory nitration of Akt. These observations indicate that not only oxidative but also nitrative stress play a key role in the pathogenesis of insulin resistance. They underline the role of protein nitration as a major mechanism in the regulation of Ang II and insulin signaling pathways and more particularly as a key regulator of protein kinase activity., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

International audience; Ubiquitin ligase Atrogin1/Muscle Atrophy F-box (MAFbx) up-regulation is required for skeletal muscle atrophy but substrates and function during the atrophic process are poorly known. The transcription factor MyoD controls myogenic stem cell function and differentiation, and seems necessary to maintain the differentiated phenotype of adult fast skeletal muscle fibres. We previously showed that MAFbx mediates MyoD proteolysis in vitro. Here we present evidence that MAFbx targets MyoD for degradation in several models of skeletal muscle atrophy. In cultured myotubes undergoing atrophy, MAFbx expression increases, leading to a cytoplasmic-nuclear shuttling of MAFbx and a selective suppression of MyoD. Conversely, transfection of myotubes with sh-RNA-mediated MAFbx gene silencing (shRNAi) inhibited MyoD proteolysis linked to atrophy. Furthermore, overexpression of a mutant MyoDK133R lacking MAFbx-mediated ubiquitination prevents atrophy of mouse primary myotubes and skeletal muscle fibres in vivo. Regarding the complex role of MyoD in adult skeletal muscle plasticity and homeostasis, its rapid suppression by MAFbx seems to be a major event leading to skeletal muscle wasting. Our results point out MyoD as the second MAFbx skeletal muscle target by which powerful therapies could be developed

Lymphangioleiomyomatosis LAM is a female predominant and devastating pulmonary disease, characterized by diffusely infiltrated smooth muscle like cells that carry mutations in the tuberous sclerosis complex (TSC) genes. TSC1, TSC2 and TBC1D7 interact and inhibit the mammalian target of rapamycin complex 1 (mTORC1). The reasons that LAM exclusively affects women and how TSC1 or TSC2 deficiency contributes to the pathogenesis of LAM are not yet fully understood. We previously discovered that estrogen promotes the survival and lung metastases of tuberin-deficient rat uterine leiomyoma-derived ELT3 cells in a xenograft tumor model (PNAS 2009). Recently, we reported that estrogen and mTORC2 coordinate to enhance prostaglandin biosynthesis and tumorigenesis in LAM (J. Expt. Med. 2013). Prostaglandins are lipid mediators that participate in tumor survival, growth, invasion, and inflammation. Phospholipase A2 (PLA2), Cyclooxygenase-2 (COX-2) and prostacyclin synthase (PTGIS) are critical enzymes responsible for the production of prostaglandins. Prostaglandin receptors (EPs) mediate the biological function of prostaglandins. This study is to determine whether suppression of prostaglandin biosynthesis pathway potentially leads to tumor regression LAM in both cell culture and preclinical models of LAM. To identify additional pathways activated by TSC loss, we performed bioinformatics analysis of public expression arrays and found a rapamycin-insensitive upregulation of prostaglandin biosynthesis genes including (PLA2), cyclooxygenase-2 (COX-2), prostacyclin synthase (PTGIS), and prostaglandin E2 (PGE2) receptor 3 (EP3), in TSC2-deficient LAM patient-derived cells compared to TSC2-addback cells. Real-time RT-PCR assays validated the enhanced expression of PLA2, COX-2, PTGIS and EP3 in TSC2-deficient cells. Immunoblotting analysis showed the increased levels of PLA2, COX-2, PTGIS and EP3 in TSC2-deficient cells compared to TSC2-addback cells. Immunohistochemistry demonstrated abundant accumulation of PLA2, COX-2 and EP3 in LAM lung lesions compared to adjacent normal tissues. Interestingly, PGE2 specifically stimulated the growth of TSC2-deficient LAM patient-derived cells compared to TSC2-addback cells. Importantly, treatment of TSC2-deficient LAM patient-derived cells with PLA2 inhibitor or EP3 inhibitor more potently reduced cell proliferation in dose-dependent manner compared to TSC2-addback cells. Our data documents that loss of TSC2 leads to the aberrant expression and accumulation of prostaglandin biosynthesis regulators PLA2, COX-2, PTGIS and EP3, thereby enhancing prostaglandin production and promoting TSC-related cell growth and tumor development. Our data supports the potential application of prostaglandin metabolites as biomarkers of disease severity and the development of prostaglandin biosynthesis inhibitors as alternative therapeutic options for lesions occurring in LAM patients and in other gender-specific neoplasm. Citation Format: Chenggang Li, Po-Shun Lee, Yang Sun, Erik Zhang, Xiaoxiao Gu, Jing Li, Kai-Feng Xu, Alfredo Csibi, John Blenis, Elizabeth Petri Henske, Bruce Levy, David Kwiatkowski, Jane J. Yu. Estradiol and mTORC2 cooperate to enhance prostaglandin biosynthesis and tumorigenesis in tuberous sclerosis complex. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B10.

Regulation of organ size is achieved through the action of the mTOR and Hippo signalling pathways, which control cell proliferation and cell growth in response to extracellular cues. A link between these pathways is revealed by the finding that YAP downregulates PTEN to promote cell growth and tissue hyperplasia.

The mTOR complex 1 (mTORC1) is a ser/thr protein kinase complex and critical regulator of cellular metabolism, protein synthesis, autophagy and cell growth. This signaling system is normally carefully controlled by the integration of inputs from growth factors, nutrients, cellular energy status, oxygen availability and stress. A variety of loss-of-function or gain-of-function alterations in key mTORC1 regulatory proteins, very often results in inappropriate regulation of this signaling system. Uncontrolled mTORC1 activation contributes on its own or in combination with other pathways to metabolic diseases ranging from diabetes and obesity, cardiac hypertrophy, neurodegeneration, aging, benign tumor syndromes to highly malignant cancers. Mechanistically, uncontrolled mTORC1 activation leads to a constant positive input into anabolic processes and suppression of many catabolic processes. As a result, these cells require increased energy and carbon sources to meet their high metabolic needs for cell growth. To meet this demand, we have found in cells that are PTEN or TSC1/2 null, that activated mTORC1 uses distinct mechanisms to increase glutamine consumption (glutaminolysis) by elevating the expression of glutaminase and the activity of glutamate dehydrogenase. Furthermore, under nutrient-rich conditions, these tumor cells sense the increased energy production and in a positive feedback loop, promote more mTORC1 assembly via an AMP kinase (AMPK)-dependent, and a novel AMPK-independent mechanism. This acquired addiction to glutamine provides a novel therapeutic strategy for treating patients with activated mTORC1. Indeed, by acutely blocking the ability of energetically stressed cells with hyperactive mTORC1 to metabolize glutamine, we can selectively kill some tumor cells without damaging normal cells. We anticipate that these studies will lead to the development of “synthetic-lethal” drugs useful in the treatment of patients with diseases linked to inappropriate activation of mTORC1. Research supported by NIH RO1-GM51405 and the LAM Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY29-01. doi:1538-7445.AM2012-SY29-01

Anne Poupon: Biologie et Bioinformatique des Systèmes de Signalisation, UMR Physiologie de la Reproduction et des Comportements, INRA, Nouzilly, France. Sanchez A.M.J. : Equipe Remodelage Musculaire et Signalisation Contact: leibovs@supagro.inra.fr; The mTORC1 pathway is required for both the terminal muscle differentiation and hypertrophy by controlling the mammalian translational machinery via phosphorylation of S6K1 and 4E-BP1. mTOR and S6K1 are connected by interacting with the eIF3 initiation complex. The regulatory subunit eIF3f plays a major role in muscle hypertrophy and is a key target that accounts for MAFbx function during atrophy. Here we present evidence that in MAFbx-induced atrophy the degradation of eIF3f suppresses S6K1 activation by mTOR, whereas an eIF3f mutant insensitive to MAFbx polyubiquitination maintained persistent phosphorylation of S6K1 and rpS6. During terminal muscle differentiation a conserved TOS motif in eIF3f connects mTOR/raptor complex, which phosphorylates S6K1 and regulates downstream effectors of mTOR and Cap-dependent translation initiation. Thus eIF3f plays a major role for proper activity of mTORC1 to regulate skeletal muscle size.

International audience; Myostatin deficiency leads in skeletal muscle overgrowth but the precise molecular mechanisms underlying this hypertrophy are not well understood. In this study, to gain insight into the role of endogenous myostatin in the translational regulation, we used an in vitro model of cultured satellite cells derived from myostatin knock-out mice. Our results show that myostatin knock-out myotubes are larger than control myotubes and that this phenotype is associated with an increased activation of the Akt/mTOR signaling pathway, a known regulator of muscle hypertrophy. These results demonstrate that hypertrophy due to myostatin deficiency is preserved in vitro and suggest that myostatin deletion results in an increased protein synthesis. Accordingly, the rates of global RNA content, polysome formation and protein synthesis are all increased in myostatin-deficient myotubes while they are counteracted by the addition of recombinant myostatin. We furthermore demonstrated that genetic deletion of myostatin stimulates cap-dependent translation by positively regulating assembly of the translation preinitiation complex. Together the data indicate that myostatin controls muscle hypertrophy in part by regulating protein synthesis initiation rates, that is, translational efficiency.