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Metabolites significantly changed by p62 knockdown in Tsc2-/- MEFs

p62/sequestosome-1 (SQSTM1) is a multifunctional adaptor protein and autophagic substrate that accumulates in cells with hyperactive mTORC1, such as kidney cells with mutations in the tumor suppressor genes tuberous sclerosis complex (TSC)1 or TSC2. Here we report that p62 is a critical mediator of TSC2-driven tumorigenesis, as Tsc2+/− and Tsc2f/f CAGGCreERT2+ mice crossed to p62−/− mice were protected from renal tumor development. Metabolic profiling revealed that depletion of p62 in Tsc2-null cells decreased intracellular glutamine, glutamate, and glutathione (GSH). p62 positively regulated the glutamine transporter Slc1a5 and increased glutamine uptake in Tsc2-null cells. We also observed p62-dependent changes in Gcl, Gsr, Nqo1, and Srxn1, which were decreased by p62 attenuation and implicated in GSH production and utilization. p62 attenuation altered mitochondrial morphology, reduced mitochondrial membrane polarization and maximal respiration, and increased mitochondrial reactive oxygen species and mitophagy marker PINK1. These mitochondrial phenotypes were rescued by addition of exogenous GSH and overexpression of Sod2, which suppressed indices of mitochondrial damage and promoted growth of Tsc2-null cells. Finally, p62 depletion sensitized Tsc2-null cells to both oxidative stress and direct inhibition of GSH biosynthesis by buthionine sulfoximine. Our findings show how p62 helps maintain intracellular pools of GSH needed to limit mitochondrial dysfunction in tumor cells with elevated mTORC1, highlighting p62 and redox homeostasis as nodal vulnerabilities for therapeutic targeting in these tumors. Cancer Res; 77(12); 3255–67. ©2017 AACR.

Sod2 overexpression reduces the percentage of MitoSOX positive cells assessed by flow cytometry

Supplementary Materials and Methods

Raw data and bioinformatic analysis of metabolomics and ion torrent expression profiling

The PI3K-mTOR pathway is one of the most commonly dysregulated pathways in human tumors. Rapalogs have been used extensively in human clinical trials but exhibit modest clinical benefit for the most part, possibly due to their lack of effect on 4E-BP1, a key target downstream of mTORC1. ATP-competitive mTOR inhibitors can fully inhibit mTORC1 but are poorly tolerated possibly due to their inhibition of mTORC2. A new class of selective mTORC1 inhibitors has been developed and termed ‘bi-steric’, which comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. We report that bi-steric mTORC1-selective inhibitors (RMC-4627 and RMC-6272) have greater activity than rapalogs for tumors with mTORC1 hyperactivation. RMC-4627 and RMC-6272 showed more effective growth inhibition in multiple tumor cell lines with mTORC1 dysfunction compared to rapamycin. Both RMC-4627 and RMC-6272 at ~1 nM showed near complete inhibition of p4E-BP1T37/46, which was not seen with rapamycin treatment. Rapamycin and both bi-steric compounds markedly reduced kidney tumor burden and human bladder cancer PDX tumor size after four weeks of treatment. Tumor regrowth assessed after two-month treatment cessation was significantly reduced in the RMC-6272 group as compared to the rapamycin group. Finally, TUNEL staining showed RMC-6272 treatment led to a greater induction of apoptosis in kidney tumor cells and human bladder cancer PDX tumors relative to rapamycin after a single dose of each compound. This is the first time that clear evidence for cell death after mTORC1 inhibition has been seen. Integrative multi-omic analysis reveals differential global rewiring induced by RMC-6272 in comparison to rapamycin. Genes involved in cell cycle transition were downregulated by RMC-6272 vs. rapamycin. Proteins downregulated by RMC-6272 treatment relative to rapamycin were enriched for translation and ribosome-related proteins, consistent with effects of non-phosphorylated 4E-BP1 on synthesis of those proteins. Multiple purine metabolites were decreased to a greater extent in RMC-6272 treated cells as compared to rapamycin treatment, including deoxyadenosine, AICAR, AMP, IMP, and GMP. Notably, there was no significant change in R5P, suggesting that downregulation of the purine de novo synthesis pathway was the cause of these changes. Strong tumor cell apoptosis was induced by the suppression of de novo purine synthesis through mTORC1-PRPS1 axis. PRPS1, the rate-limit enzyme involving in purine synthesis was regulated by mTORC1 activity. In summary, RMC-4627 and RMC-6272 demonstrate improved in vitro and in vivo inhibition of mTORC1 in comparison to rapamycin, and induced more cell death in preclinical models, indicating the potential of bi-steric mTORC1-selective inhibitors as a novel therapeutic strategy to treat tumors with mTORC1 dysregulation. Citation Format: Heng Du, Yu Chi Yang, Heng-Jia Liu, Min Yuan, John Asara, Kwok-Kin Wong, Mallika Singh, David Kwiatkowski. Bi-steric mTORC1 inhibitors are superior to rapamycin and induce apoptotic cell death in tumor models with hyperactivated mTORC1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4859.

The Rac-GEF, P-Rex1, activates Rac1 signaling downstream of G protein-coupled receptors and PI3K. Increased P-Rex1 expression promotes melanoma progression; however, its role in breast cancer is complex, with differing reports of the effect of its expression on disease outcome. To address this we analyzed human databases, undertook gene array expression analysis, and generated unique murine models of P-Rex1 gain or loss of function. Analysis of PREX1 mRNA expression in breast cancer cDNA arrays and a METABRIC cohort revealed that higher PREX1 mRNA in ER(+ve)/luminal tumors was associated with poor outcome in luminal B cancers. Prex1 deletion in MMTV-neu or MMTV-PyMT mice reduced Rac1 activation in vivo and improved survival. High level MMTV-driven transgenic PREX1 expression resulted in apicobasal polarity defects and increased mammary epithelial cell proliferation associated with hyperplasia and development of de novo mammary tumors. MMTV-PREX1 expression in MMTV-neu mice increased tumor initiation and enhanced metastasis in vivo, but had no effect on primary tumor growth. Pharmacological inhibition of Rac1 or MEK1/2 reduced P-Rex1-driven tumoroid formation and cell invasion. Therefore, P-Rex1 can act as an oncogene and cooperate with HER2/neu to enhance breast cancer initiation and metastasis, despite having no effect on primary tumor growth.

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor syndrome, characterized by tumor development in multiple organs, including renal angiomyolipoma. Biallelic loss of TSC1 or TSC2 is a known genetic driver of angiomyolipoma development, however, whether an altered transcriptional repertoire contributes to TSC-associated tumorigenesis is unknown. RNA-seq analyses showed that MITF A isoform (MITF-A) was consistently highly expressed in angiomyolipoma, immunohistochemistry showed microphthalmia-associated transcription factor nuclear localization, and Chromatin immuno-Precipitation Sequencing analysis showed that the MITF-A transcriptional start site was highly enriched with H3K27ac marks. Using the angiomyolipoma cell line 621-101, MITF knockout (MITF.KO) and MITF-A overexpressing (MITF.OE) cell lines were generated. MITF.KO cells showed markedly reduced growth and invasion in vitro, and were unable to form xenografted tumors. In contrast, MITF.OE cells grew faster in vitro and as xenografted tumors compared to control cells. RNA-Seq analysis showed that both ID2 and Cysteine-rich angiogenic inducer 61 (CYR61) expression levels were increased in the MITF.OE cells and reduced in the MITF.KO cells, and luciferase assays showed this was due to transcriptional effects. Importantly, CYR61 overexpression rescued MITF.KO cell growth in vitro and tumor growth in vivo. These findings suggest that MITF-A is a transcriptional oncogenic driver of angiomyolipoma tumor development, acting through regulation of CYR61.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic multisystem disease characterized by the nodular proliferation of smooth muscle-like LAM cells, progressive cystic changes of the lung, lymphatic abnormalities, and renal angiomyolipomas (AMLs). LAM can arise sporadically or in women with the autosomal dominant disorder, tuberous sclerosis complex (TSC), in which hamartomatous tumors of brain, heart, skin, kidney, and lung are found. LAM and TSC are caused by mutations in the TSC1 or TSC2 tumor suppressor genes leading to elevated mechanistic/mammalian target of rapamycin complex activity. Recent data indicate that T cells within LAM nodules and renal AMLs exhibit features of T-cell exhaustion, with coinhibitory receptor programmed cell death protein 1 (PD-1) expression on tumor-infiltrating T cells. Treatment of animal models of TSC and LAM with anti-PD-1 antibodies or with the combination of anti-PD-1 and anti-CTLA4 antibodies has led to remarkable results, suppressing TSC2-null tumor growth and inducing tumor rejection. Here we review our current knowledge about the potential for immunotherapy for the treatment of LAM and TSC and highlight critical unknowns and key next steps.

Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are caused by aberrant mechanistic Target of Rapamycin Complex 1 (mTORC1) activation due to loss of either TSC1 or TSC2. Cytokine profiling of TSC2-deficient LAM patient-derived cells revealed striking upregulation of Interleukin-6 (IL-6). LAM patient plasma contained increased circulating IL-6 compared with healthy controls, and TSC2-deficient cells showed upregulation of IL-6 transcription and secretion compared to wildtype cells. IL-6 blockade repressed the proliferation and migration of TSC2-deficient cells and reduced oxygen consumption and extracellular acidification. U-13C glucose tracing revealed that IL-6 knockout reduced 3-phosphoserine and serine production in TSC2-deficient cells, implicating IL-6 in de novo serine metabolism. IL-6 knockout reduced expression of phosphoserine aminotransferase 1 (PSAT1), an essential enzyme in serine biosynthesis. Importantly, recombinant IL-6 treatment rescued PSAT1 expression in the TSC2-deficient, IL-6 knockout clones selectively and had no effect on wildtype cells. Treatment with anti-IL-6 (aIL-6) antibody similarly reduced cell proliferation and migration and reduced renal tumors in Tsc2+/- mice, while reducing PSAT1 expression. These data reveal a novel mechanism through which IL-6 regulates serine biosynthesis, with potential relevance to the therapy of tumors with mTORC1 hyperactivity.ClassificationMajor category: Biological Sciences Minor category: Cell Biology

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2+/− mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells., Tuberous sclerosis complex (TSC) is a multiorgan disease that can lead to hyperactive mTORC1 due to deficient TSC1 or TSC2 protein function. Here, the authors find that despite high mTORC1 activity, TFEB localizes to the nucleus and drives lysosomal gene expression via a non-canonical Rag-dependent mechanism.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by germline inactivating mutations of TSC1 or TSC2. In TSC-associated tumors of the brain, heart, skin, kidney and lung, inactivation of both alleles of TSC1 or TSC2 leads to hyperactivation of the mTORC1 pathway. The TSC/mTORC1 pathway is a key regulator of cellular processes related to growth, proliferation and autophagy. We and others have previously found that mTORC1 regulates microRNA biogenesis, but the mechanisms are not fully understood. Microprocessor, a multi-protein complex including the nuclease Drosha, processes the primary miR transcript. Using a dual-luciferase reporter, we found that inhibition of mTORC1 or downregulation of Raptor decreased Microprocessor activity, while loss of TSC2 led to a striking increase (∼5-fold) in Microprocessor activity. To determine the global impact of TSC2 on microRNAs we quantitatively analyzed 752 microRNAs in Tsc2-expressing and Tsc2-deficient cells. Out of 259 microRNAs expressed in both cell lines, 137 were significantly upregulated and 24 were significantly downregulated in Tsc2-deficient cells, consistent with the increased Microprocessor activity. Microprocessor activity is known to be regulated in part by GSK3β. We found that total GSK3β levels were higher in Tsc2-deficient cells, and the increase in Microprocessor activity associated with Tsc2 loss was reversed by three different GSK3β inhibitors. Furthermore, mTOR inhibition increased the levels of phospho-GSK3β (S9), which negatively affects Microprocessor activity. Taken together these data reveal that TSC2 regulates microRNA biogenesis and Microprocessor activity via GSK3β.

BackgroundBiomarkers of disease progression and treatment response are urgently needed for patients with lymphangioleiomyomatosis (LAM). Activity-based nanosensors, an emerging biosensor class, detect dysregulated proteases in vivo and release a reporter to provide a urinary readout of disease. Because proteases are dysregulated in LAM and may directly contribute to lung function decline, activity-based nanosensors may enable quantitative, real-time monitoring of LAM progression and treatment response. We aimed to assess the diagnostic utility of activity-based nanosensors in a pre-clinical model of pulmonary LAM.MethodsTsc2-null cells were injected intravenously into female nude mice to establish a mouse model of pulmonary LAM. A library of 14 activity-based nanosensors, designed to detect proteases across multiple catalytic classes, was administered into the lungs of LAM mice and healthy controls, urine was collected, and mass spectrometry was performed to measure nanosensor cleavage products. Mice were then treated with rapamycin and monitored with activity-based nanosensors. Machine learning was performed to distinguish diseased from healthy and treated from untreated mice.ResultsMultiple activity-based nanosensors (PP03 (cleaved by metallo, aspartic and cysteine proteases), padjustedadjusted=0.017)) were differentially cleaved in diseased and healthy lungs, enabling strong classification with a machine learning model (area under the curve (AUC) 0.95 from healthy). Within 2 days after rapamycin initiation, we observed normalisation of PP03 and PP10 cleavage, and machine learning enabled accurate classification of treatment response (AUC 0.94 from untreated).ConclusionsActivity-based nanosensors enable noninvasive, real-time monitoring of disease burden and treatment response in a pre-clinical model of LAM.

The PI3K-mTOR pathway is one of the most commonly dysregulated pathways in human tumors. Rapalogs have been used extensively in human clinical trials but exhibit modest clinical benefit for the most part, possibly due to their lack of effect on 4E-BP1, a key target downstream of mTORC1. ATP-competitive mTOR inhibitors can fully inhibit mTORC1 but are poorly tolerated possibly due to their inhibition of mTORC2. A new class of selective mTORC1 inhibitors has been developed and termed ‘bi-steric', which comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. RMC-5552 is the first clinical candidate of this class and clinical testing is planned in 2021. RM-001 and RM-006 (also known as RMC-6272) are representative bi-steric tool compounds that exhibit potent and selective (> 10-fold) inhibition of mTORC1 over mTORC2. We report that bi-steric mTORC1-selective inhibitors have greater activity than rapalogs for tumors with mTORC1 hyperactivation secondary to TSC1/2 loss. RM-001 and RM-006 showed more effective growth inhibition in multiple TSC1 or TSC2 mutant tumor cell lines compared to rapamycin. RM-001 and RM-006 caused a more profound growth inhibition in the TSC1 or TSC2 mutant cells than the wild type cells. Two-week low dilution clonogenic assays in TSC1 or TSC2 mutant cells showed clone numbers were significantly reduced by rapamycin, RM-001, and RM-006, whereas in the wild type cells, Rapamycin and RM compounds had less reduction in clone numbers, indicating a differential sensitivity in TSC-null cells. Both RM-001 and RM-006 at ~1 nM showed near complete inhibition of p4E-BP1T37/46, which was not seen with rapamycin treatment, while inhibition of pS6S240/244 levels was similar for rapamycin and RM compounds. Further, the RM compounds had prolonged anti-proliferative activity in TSC-null cells, as shown by washout studies. Rapamycin, MLN0128 and both RM compounds markedly reduced kidney tumor burden in Tsc2+/- A/J mice after four weeks of treatment. Tumor regrowth was assessed two months after treatment cessation, tumor burden was significantly reduced in the RM-006 group as compared to the rapamycin and MLN0128 groups. Finally, TUNEL staining showed RM-006 treatment led to a greater induction of apoptosis in kidney tumor cells relative to rapamycin and MLN0128 after a single dose of each compound in Tsc2+/- A/J mice. This is the first time that clear evidence for cell death after mTORC1 inhibition has been seen in TSC rodent models. In summary, RM-001 and RM-006 demonstrate improved in vitro and in vivo inhibition of mTORC1 in comparison to rapamycin, and induced more cell death in TSC2 null tumors in vivo, indicating the potential of bi-steric mTORC1-selective inhibitors as a novel therapeutic strategy to treat tumors with mTORC1 dysregulation. Citation Format: Heng Du, Yu Chi Yang, Mallika Singh, Heng-jia Liu, David Kwiatkowski. Bi-steric mTORC1-selective inhibitors demonstrate improved potency and efficacy in tumors with mTORC1 hyperactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1026.

// Hilaire C. Lam 1 , Heng-Jia Liu 1 , Christian V. Baglini 1 , Harilaos Filippakis 1 , Nicola Alesi 1 , Julie Nijmeh 1 , Heng Du 1 , Alicia Llorente Lope 1 , Katherine A. Cottrill 2 , Adam Handen 2 , John M. Asara 3 , David J. Kwiatkowski 1 , Issam Ben-Sahra 4 , William M. Oldham 1 , Stephen Y. Chan 2 and Elizabeth P. Henske 1 1 Department of Medicine, Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA 2 Department of Medicine, Division of Cardiology, Center for Pulmonary Vascular Biology and Medicine, Pittsburgh Heart, Lung, Blood, and Vascular Medicine Institute, University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center, Pittsburgh, PA, USA 3 Department of Medicine, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 4 Department of Biochemistry and Molecular Genetics, Northwestern University, Chicago, IL, USA Correspondence to: Elizabeth P. Henske, email: // Keywords : tuberous sclerosis complex, mTORC1, rapamycin, miR-21, mitochondria Received : June 20, 2017 Accepted : June 25, 2017 Published : August 04, 2017 Abstract mTORC1 hyperactivation drives the multi-organ hamartomatous disease tuberous sclerosis complex (TSC). Rapamycin inhibits mTORC1, inducing partial tumor responses; however, the tumors regrow following treatment cessation. We discovered that the oncogenic miRNA, miR-21, is increased in Tsc2-deficient cells and, surprisingly, further increased by rapamycin. To determine the impact of miR-21 in TSC, we inhibited miR-21 in vitro . miR-21 inhibition significantly repressed the tumorigenic potential of Tsc2-deficient cells and increased apoptosis sensitivity. Tsc2-deficient cells’ clonogenic and anchorage independent growth were reduced by ~50% ( p

p62/sequestosome-1 (SQSTM1) is a multifunctional adaptor protein and autophagic substrate that accumulates in cells with hyperactive mTORC1, such as kidney cells with mutations in the tumor suppressor genes tuberous sclerosis complex (TSC)1 or TSC2. Here we report that p62 is a critical mediator of TSC2-driven tumorigenesis, as Tsc2+/− and Tsc2f/f CAGGCreERT2+ mice crossed to p62−/− mice were protected from renal tumor development. Metabolic profiling revealed that depletion of p62 in Tsc2-null cells decreased intracellular glutamine, glutamate, and glutathione (GSH). p62 positively regulated the glutamine transporter Slc1a5 and increased glutamine uptake in Tsc2-null cells. We also observed p62-dependent changes in Gcl, Gsr, Nqo1, and Srxn1, which were decreased by p62 attenuation and implicated in GSH production and utilization. p62 attenuation altered mitochondrial morphology, reduced mitochondrial membrane polarization and maximal respiration, and increased mitochondrial reactive oxygen species and mitophagy marker PINK1. These mitochondrial phenotypes were rescued by addition of exogenous GSH and overexpression of Sod2, which suppressed indices of mitochondrial damage and promoted growth of Tsc2-null cells. Finally, p62 depletion sensitized Tsc2-null cells to both oxidative stress and direct inhibition of GSH biosynthesis by buthionine sulfoximine. Our findings show how p62 helps maintain intracellular pools of GSH needed to limit mitochondrial dysfunction in tumor cells with elevated mTORC1, highlighting p62 and redox homeostasis as nodal vulnerabilities for therapeutic targeting in these tumors. Cancer Res; 77(12); 3255–67. ©2017 AACR.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic multisystem disease characterized by the nodular proliferation of smooth muscle-like LAM cells, progressive cystic changes of the lung, lymphatic abnormalities, and renal angiomyolipomas (AMLs). LAM can arise sporadically or in women with the autosomal dominant disorder, tuberous sclerosis complex (TSC), in which hamartomatous tumors of brain, heart, skin, kidney, and lung are found. LAM and TSC are caused by mutations in the TSC1 or TSC2 tumor suppressor genes leading to elevated mechanistic/mammalian target of rapamycin complex activity. Recent data indicate that T cells within LAM nodules and renal AMLs exhibit features of T-cell exhaustion, with coinhibitory receptor programmed cell death protein 1 (PD-1) expression on tumor-infiltrating T cells. Treatment of animal models of TSC and LAM with anti-PD-1 antibodies or with the combination of anti-PD-1 and anti-CTLA4 antibodies has led to remarkable results, suppressing TSC2-null tumor growth and inducing tumor rejection. Here we review our current knowledge about the potential for immunotherapy for the treatment of LAM and TSC and highlight critical unknowns and key next steps.

miR-29b has been identified as a rapamycin-induced microRNA (miRNA) in Tsc2-deficient, mTORC1-hyperactive cells. The biological significance of this induction of miR-29b is unknown. We have found that miR-29b acts as an oncogenic miRNA in Tsc2-deficient cells: inhibition of miR-29b suppressed cell proliferation, anchorage-independent cell growth, cell migration, invasion, and the growth of Tsc2-deficient tumors in vivo. Importantly, the combination of miR-29b inhibition with rapamycin treatment further inhibited these tumor-associated cellular processes. To gain insight into the molecular mechanisms by which miR-29b promotes tumorigenesis, we used RNA sequencing to identify the tumor suppressor retinoid receptor beta (RARβ) as a target gene of miR-29b. We found that miR-29b directly targeted the 3′UTR of RARβ. Forced expression of RARβ reversed the effects of miR-29b overexpression in proliferation, migration, and invasion, indicating that it is a critical target. miR-29b expression correlated with low RARβ expression in renal clear cell carcinomas and bladder urothelial carcinomas, tumors associated with TSC gene mutations. We further identified growth family member 4 (ING4) as a novel interacting partner of RARβ. Overexpression of ING4 inhibited the migration and invasion of Tsc2-deficient cells while silencing of ING4 reversed the RARβ-mediated suppression of cell migration and invasion. Taken together, our findings reveal a novel miR-29b/RARβ/ING4 pathway that regulates tumorigenic properties of Tsc2-deficient cells, and that may serve as a potential therapeutic target for TSC, lymphangioleiomyomatosis (LAM), and other mTORC1-hyperactive tumors.

p62/sequestosome-1 (SQSTM1) is a multifunctional adaptor protein and autophagic substrate which accumulates in cells with hyperactive mTORC1, such as kidney cells with mutations in the tumor suppressor genes TSC1 or TSC2. Here we report that p62 is a critical mediator of TSC2-driven tumorigenesis, as Tsc2+/− and Tsc2f/f Ksp-CreERT2+ mice crossed to p62−/− mice were protected from renal tumor development. Metabolic profiling revealed that depletion of p62 in Tsc2-null cells decreased intracellular glutamine, glutamate, and glutathione (GSH). p62 positively regulated the glutamine transporter Slc1a5 and increased glutamine uptake in Tsc2-null cells. We also observed p62-dependent changes in Gcl, Gsr, Nqo1 and Srxn1 which were decreased by p62 attenuation and implicated in GSH production and utilization. p62 attenuation altered mitochondrial morphology, reduced mitochondrial membrane polarization and maximal respiration, and increased mitochondrial ROS and mitophagy marker PINK1. These mitochondrial phenotypes were rescued by addition of exogenous GSH and overexpression of Sod2, which suppressed indices of mitochondrial damage and promoted growth of Tsc2-null cells. Finally, p62 depletion sensitized Tsc2-null cells to both oxidative stress and direct inhibition of glutathione biosynthesis by buthionine sulfoximine (BSO). Our findings show how p62 helps maintain intracellular pools of glutathione needed to limit mitochondrial dysfunction in tumor cells with elevated mTORC1, highlighting p62 and redox homeostasis as nodal vulnerabilities for therapeutic targeting in these tumors.

Tuberous sclerosis complex (TSC) is an incurable multisystem disease associated with hamartomatous tumors of brain, heart, skin, kidney and lung. The TSC proteins (TSC1 and TSC2) inhibit the mechanistic/mammalian target of rapamycin complex 1 (mTORC1), an exquisitely sensitive molecular sensor that regulates cell growth, cell metabolism and autophagy. mTORC1 is hyperactive in many malignancies, including TSC. Treatment of TSC-associated tumors with mTOR inhibitors leads to partial responses, with prompt regrowth upon treatment cessation, highlighting the unmet clinical need for cytocidal therapies. We previously discovered that miR-29b expression is strongly upregulated following rapamycin treatment. However, the functional significance and therapeutic impact of miR-29b induction by rapamycin in the therapy of TSC is unknown. We found that miR-29b expression is induced by rapamycin in vitro in 4 different TSC2-deficient cells and in vivo TSC-2 deficient tumors. Inhibition of miR-29b significantly decreased anchorage-independent cell growth by 40% (p < 0.01), and combined rapamycin treatment and miR-29b inhibition further suppressed growth by 73% (p < 0.0001). In addition, miR-29b knockdown inhibited cell migration and invasion by 68% (p < 0.05) and 50% (p < 0.01), respectively. miR-29b inhibition combined with rapamycin further decreased cell migration and invasion by 77% (p < 0.001) and 74% (p < 0.001), respectively. These findings suggest that miR-29b acts as an “onco-miR” in this context. Importantly, miR-29b knockdown in TSC2-deficient cells significantly suppressed tumorigenesis in vivo by 52% (p < 0.0001). Furthermore, the combination of rapamycin and miR-29b inhibition resulted in a more robust inhibition of tumor volume compared with miR control (47% vs. 24% inhibition) (p < 0.01). To gain insight into the molecular mechanisms by which miR-29b promotes tumorigenesis, we performed RNA-sequencing on miR-29b knockdown and control cells treated with rapamycin. 236 genes were upregulated in miR-29b knockdown cells by at least 2.0-fold and p < 0.05. Retinoid receptor beta (RARβ), one of the candidates, was increased in miR-29b knockdown cells at the mRNA level by 2.25-fold (p < 0.01) and protein expression by 2-fold (p < 0.05). RARβ is a tumor suppressor in many types of tumors. Interestingly, RARβ expression is downregulated by rapamycin (67% (p < 0.001)) and upregulated 4-fold following miR-29b inhibition (p < 0.05). Lastly, we found the 3'UTR activity of RARβ was upregulated in miR-29b knockdown cells by 4-fold (p < 0.001) using a luciferase reporter assay, supporting a model in which RARβ is a direct target of miR-29b. These studies demonstrate that miR-29b induction by rapamycin promotes TSC2-deficient tumorigenesis. miR-29b is a potential therapeutic target for TSC-associated tumors and possibly other mTORC-1 hyperactive tumors. Citation Format: Heng-Jia Liu, Hilaire C. Lam, Christian V. Baglini, Alex K. Conttrill, Stephen Y. Chan, Elizabeth P. Henske. Rapamycin-upregulated miR-29b promotes mTORC1-hyperactivative cell growth by downregulating retinoic acid receptor β (RARβ) [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A28.

Renal angiomyolipoma (AML) and cysts are seen in 70-80% of patients with the human tumor suppressor gene syndrome tuberous sclerosis complex (TSC). Renal cell carcinoma (RCC) also is relatively common in TSC. Although two-hit loss of TSC1/2 is established in AML and TSC-RCC, the mechanisms and signaling pathways underlying TSC tumor development are not well understood. TSC-related tumor models were generated through i.p. tamoxifen (TM) injection (10mg/kg) at embryonic day 17.5 in Tsc2f/fROSA26-CreERT2 (n=24), Tsc2f/f CAG-CreERT2 mice (n=2) and Tsc1f/f CAG-CreERT2 mice (n=19). Extensive TSC-associated kidney papillary RCC (PRCC) and hybrid oncocytic/chromophobe tumors (HOCT) were seen in every kidney of these mice at age of 6 months, confirmed by IHC. A phospho-RTK immunoblot screen was performed using lysates from kidney tumors in comparison to normal kidneys. pAXL levels were ~2.8-fold higher versus normal kidney, whereas other pRTKs showed no apparent change. Immunoblotting confirmed the upregulated levels of both p-AXL (Tyr 702) and total AXL in kidney tumor lysates vs. normal kidneys. Q-RT-PCR of mRNA also showed 2-fold increase in AXL mRNA. Bioinformatic analysis suggested that AXL is a probable target of the transcription factor JUN. RNA-seq data showed that AMLs had the highest level of JUN expression in comparison to 28 different kinds of cancer (from TCGA). JUN was highly expressed in the mouse kidney tumors compared to normal kidneys in both protein and mRNA levels. JUN levels were also assessed using TSC-null cell lines (TSC2-/- angiomyolipoma cells (h), Tsc2-/- renal cell carcinoma cells (m), TSC1-/- bladder cancer cells (h), Tsc1-/- lung cancer cells (m)) versus their TSC add-back counterparts. All TSC-null cells showed a higher level of JUN and AXL compared to their TSC add-back counterparts. Both rapamycin (20nM) and Torin (500nM) treatment of these cell lines for 24h led to a reduction in JUN expression, with a stronger effect from Torin than rapamycin. To identify connections between JUN and AXL, JUN and AXL CRISPR KO were performed in 2 Tsc2-/- renal cell carcinoma cell lines. JUN KO led to a major decrease in AXL expression, and AXL KO led to a decrease of JNK, ERK and JUN. A positive feedback loop may exist between JUN and AXL through JNK and ERK pathway. R428, an inhibitor of AXL, was used to treat our Tsc2f/f ROSA26-CreERT2 mice and Tsc2+/- AJ mice (12.5mg/kg, twice daily, oral gavage, 3 weeks). Kidney tumor volume decreased dramatically compared with vehicle-treated mice in both cohorts (31.38 vs 4.11 mm³, p=0.001; 1.84 vs 0.05 mm³, p=0.02). These findings suggest that JUN and AXL are both upregulated in response to TSC1/TSC2 loss and mTORC1 activation and contribute to tumorigenesis, and that there is potential therapeutic value to targeting these events. Citation Format: Heng Du, Heng-jia Liu, Damir Khabibullin, Mahsa Zarei, John Dreier, Chin-Lee Wu, Elizabeth Henske, David Kwiatkowski. Targeting mTOR/JUN/AXL pathway in TSC-related tumors [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B13.

Tuberous sclerosis complex (TSC) is an incurable multisystem disease characterized by mTORC1-hyperactive tumors. TSC1/2 mutations also occur in other neoplastic disorders, including lymphangioleiomyomatosis (LAM) and bladder cancer. Whether TSC-associated tumors will respond to immunotherapy is unknown. We report here that the programmed death 1 coinhibitory receptor (PD-1) is upregulated on T cells in renal angiomyolipomas (AML) and pulmonary lymphangioleiomyomatosis (LAM). In C57BL/6J mice injected with syngeneic TSC2-deficient cells, anti-PD-1 alone decreased 105K tumor growth by 67% (P < 0.0001); the combination of PD-1 and CTLA-4 blockade was even more effective in suppressing tumor growth. Anti-PD-1 induced complete rejection of TSC2-deficient 105K tumors in 37% of mice (P < 0.05). Double blockade of PD-1 and CTLA-4 induced rejection in 62% of mice (P < 0.01). TSC2 reexpression in TSC2-deficient TMKOC cells enhanced antitumor immunity by increasing T cell infiltration and production of IFN-γ/TNF-α by T cells, suggesting that TSC2 and mTORC1 play specific roles in the induction of antitumor immunity. Finally, 1 month of anti-PD-1 blockade reduced renal tumor burden by 53% (P < 0.01) in genetically engineered Tsc2+/- mice. Taken together, these data demonstrate for the first time to our knowledge that checkpoint blockade may have clinical efficacy for TSC and LAM, and possibly other benign tumor syndromes, potentially yielding complete and durable clinical responses.

We demonstrate that two interferometric autocorrelation traces obtained by sampling two second-harmonic tones around the carrier would be sufficient to algebraically retrieve the spectral phase of a 3.5 fs pulse.

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP3 and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP3/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP3 and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.

PtdIns(3,4,5)P3-dependent Rac exchanger 1 (PREX1) is a Rac-guanine nucleotide exchange factor (GEF) overexpressed in a significant proportion of human breast cancers that integrates signals from upstream ErbB2/3 and CXCR4 membrane surface receptors. However, the PREX1 domains that facilitate its oncogenic activity and downstream signaling are not completely understood. We identify that ERK1/2 MAPK acts downstream of PREX1 and contributes to PREX1-mediated anchorage-independent cell growth. PREX1 overexpression increased but its shRNA knockdown decreased ERK1/2 phosphorylation in response to EGF/IGF-1 stimulation, resulting in induction of the cell cycle regulators cyclin D1 and p21(WAF1/CIP1) PREX1-mediated ERK1/2 phosphorylation, anchorage-independent cell growth, and cell migration were suppressed by inhibition of MEK1/2/ERK1/2 signaling. PREX1 overexpression reduced staurosporine-induced apoptosis whereas its shRNA knockdown promoted apoptosis in response to staurosporine or the anti-estrogen drug tamoxifen. Expression of wild-type but not GEF-inactive PREX1 increased anchorage-independent cell growth. In addition, mouse xenograft studies revealed that expression of wild-type but not GEF-dead PREX1 resulted in the formation of larger tumors that displayed increased phosphorylation of ERK1/2 but not AKT. The impaired anchorage-independent cell growth, apoptosis, and ERK1/2 signaling observed in stable PREX1 knockdown cells was restored by expression of wild-type but not GEF-dead-PREX1. Therefore, PREX1-Rac-GEF activity is critical for PREX1-dependent anchorage-independent cell growth and xenograft tumor growth and may represent a possible therapeutic target for breast cancers that exhibit PREX1 overexpression.

Renal cell carcinoma (RCC) occurs in up to 4% of tuberous sclerosis complex (TSC) patients, often affecting children and young adults. The mechanisms of TSC-associated RCC are not well understood. We explored the pathogenesis of TSC-associated RCC using three mouse models. First, loss of Tsc2 was induced by i.p. tamoxifen (TM) injection (10mg/kg) at embryonic day 17.5 (E17.5) in Tsc2flox/floxROSA26-CreER+ mice (n=24), examined at age 6 m. Second, loss of Tsc1 was induced by injecting Cre-encoding adenovirus directly into the left kidney of Tsc1flox/flox mice at P1 (n=19), examined at age 1 year. Third, Tsc2 was knock out through 5 consecutive times of i.p. tamoxifen injection (1mg/mouse/day) when the Tsc2flox/floxROSA26-CreER+ mice were 6 weeks old (n=7), examined at age 6 month. We found the following: In the E17.5 TM injection mouse model, each kidney had more than 200 tumor lesions, which almost occupied the whole kidney cortex and medulla. Extensive TSC-associated kidney papillary RCC (PRCC) and hybrid oncocytic/chromophobe tumors (HOCT) were seen in every kidney. The same types of tumor but fewer tumor lesions (~40/kidney) were found in the Cre-encoding adenovirus group. All the females in the P6 week group had the same phenotype as E17.5 TM injection group, but only single cyst was found in the male kidneys. So, the earlier those mice lost TSC1/2, the more severe their tumor burdens. IHC was performed to further validate the diagnosis. All the PRCC showed strong and diffuse expression of phospho-S6-S240/244 (pS6), CK7, and carbonic anhydrase-IX, with negative vimentin staining, while all the HOCT were positive for pS6, S-100 (a diagnostic marker used for human HOCT) and vimentin. The single cysts in the P6 week TM injection male kidneys only showed pS6 and CK7 staining. To further explore the mechanism of tumorigenesis, a phospho-RTK screen was done using lysates from kidney tumors (PRCC and HOCT) and normal kidneys. The pAXL level was 2.8-fold higher in comparison to normal kidney. Immunoblotting revealed high levels of p-AXL and total AXL in kidney tumor lysates compared to normal kidney. AXL mRNA, measured by real-time RT-PCR, was increased 2-fold (p=0.011). c-Jun, a known driver of AXL expression, was also highly expressed in those tumors but negative in normal kidneys as shown by immunoblot. Immunoblot analysis of a cell line established from the kidney tumors showed TSC-dependent c-Jun and AXL expression. These novel mouse models of TSC-related RCC closely resemble those found in TSC patients and elucidate distinct mechanisms of disease development and potential therapeutic targets. Citation Format: Heng Du, Heng-jia Liu, Damir Khabibullin, Mahsa Zarei, John Dreier, Chin-Lee Wu, Elizabeth Henske, David Kwiatkowski. Pre- and early postnatal loss of tsc1/2 induces kidney hybrid oncocytic/chromophobe tumors (HOCT) via c-JUN [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 5096.

Tuberous sclerosis complex (TSC) is an incurable multisystem genetic disease characterized by mTORC1-hyperactive hamartomatous tumors of brain, kidney, and lung. TSC is caused by mutations in the two TSC1 or TSC2 tumor-suppressor genes. mTORC1 inhibitors lead to partial antitumor responses in TSC, with tumor regrowth upon treatment cessation. Understanding the interaction between T cells and tumor cells has led to the development of successful checkpoint blockade immunotherapy targeting PD-1, programmed cell death-ligand 1 (PD-L1), and CTLA-4. These therapies have proven to be highly effective in treating several malignancies, including non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), melanoma, bladder cancer and Hodgkin's lymphoma. It is currently unknown whether benign tumors, such as those arising in TSC, will also respond to checkpoint blockade therapy. Using multiparametric flow cytometry and immunohistochemistry of human angiomyolipomas and pulmonary LAM, we discovered that PD-1 is highly expressed on tumor-infiltrating T cells. Using immunocompetent preclinical models of TSC, we found that dual blockade of PD-1 and CTLA-4 inhibited the growth of TSC2-deficient tumors by 77% (p < 0.0001). This was associated with increased tumor-infiltrating CD8+ and CD4+ T cells and decreased Tregs, G-MDSCs and regulatory CD11b+ DCs. Strikingly, we discovered that complete regression of established tumors can be achieved in 37% of the mice using anti-PD-1 monotherapy and in 62% of the mice using combined anti-PD-1 and anti-CTLA-4 therapy. We also assessed a treatment regimen of rapamycin followed by PD-1 blockade and found that this sequential treatment delays regrowth of TSC2-deficient tumors. Furthermore, TSC2 re-expression in TSC2-deficient tumors promoted antitumor responses via increasing T cells and decreasing CD11b+Ly6G+Ly6Cmed G-MDSCs and CD11b+ DCs infiltration into tumors. Importantly, we found that the potency of dual PD-1 and CTLA-4 blockade is significantly enhanced by TSC2 re-expression. Our data indicate that PD-1 is upregulated on T cells in TSC-associated tumors and that dual blockade of PD-1 and CTLA-4 pathways is effective in delaying tumor growth and improving long-term survival, with long-term complete tumor responses. Therefore, PD-1 and/or CTLA-4 blockade may represent a promising dual immunotherapy for patients with TSC-associated tumors and women with the sporadic form of LAM. Our data may have relevance for the many sporadic human tumors with mTORC1 hyperactivation (~50% of all human malignancies) and also for other hamartomatous tumor syndromes such as neurofibromatosis. Furthermore, the specific role of intratumoral TSC2 in antitumor T cell responses that we discovered may help to elucidate the response to immunotherapy in sporadic human malignancies with mutations in the TSC genes, which include a subset of bladder cancer and renal cell carcinoma. Citation Format: Heng-Jia Liu, Patrick Lizotte, Heng Du, Maria Speranza, Spencer Vaughan, Nicola Alesi, Kwok-Kin Wong, Gordon Freeman, Arlene Sharpe, Elizabeth Henske. TSC2 enhances antitumor immunity and potentiates PD-1 and CTLA-4 blockade [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 1686.