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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

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.

Osteoclasts are multinucleated cells of the myeloid lineage that are highly specialized for bone resorption through acidification and exocytosis of bone-degrading enzymes into the space between the osteoclast and underlying bone, termed the resorption lacuna. Physiologic osteoclast activity is essential for normal skeletal development and remodeling to repair skeletal microdamage. Osteopetrosis, a collection of monogenic diseases caused by nonfunctional or absent osteoclasts, underscores the importance of adequate osteoclast function for skeletal health. Excessive osteoclastic bone resorption also results in bone disease, including osteoporosis. In this chapter, we provide information on the cellular origin of osteoclasts, discuss the receptors and signaling pathways that drive osteoclast differentiation, and detail the transcriptional machinery required to express the osteoclast program. We detail the cytoskeletal reorganization and formation of a specialized resorption apparatus required for mineral and matrix resorption. Lastly, we discuss the consequences of perturbation of osteoclast formation or function, both in the context of genetic diseases that map to the osteoclast and in the context of pathologic conditions. The goal of this chapter is to provide a detailed overview of osteoclast formation and function that can serve as a reference for clinician’s interested in the role of the osteoclast in osteoporosis.

// 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.

// Harilaos Filippakis 1 , Nicola Alesi 1 , Barbara Ogorek 1 , Julie Nijmeh 1 , Damir Khabibullin 1 , Catherine Gutierrez 1 , Alexander J. Valvezan 2 , James Cunningham 3 , Carmen Priolo 1 and Elizabeth P. Henske 1 1 Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA 2 Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, Massachusetts, USA 3 Department of Medicine, Division of Hematology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA Correspondence to: Elizabeth P. Henske, email: // Keywords : TSC, mTORC1, chloroquine, cholesterol, lysosome Received : February 25, 2017 Accepted : April 15, 2017 Published : April 27, 2017 Abstract Tuberous sclerosis complex (TSC) is a multisystem disease associated with hyperactive mTORC1. The impact of TSC1/2 deficiency on lysosome-mediated processes is not fully understood. We report here that inhibition of lysosomal function using chloroquine (CQ) upregulates cholesterol homeostasis genes in TSC2-deficient cells. This TSC2-dependent transcriptional signature is associated with increased accumulation and intracellular levels of both total cholesterol and cholesterol esters. Unexpectedly, engaging this CQ-induced cholesterol uptake pathway together with inhibition of de novo cholesterol synthesis allows survival of TSC2-deficient, but not TSC2-expressing cells. The underlying mechanism of TSC2-deficient cell survival is dependent on exogenous cholesterol uptake via LDL-R, and endosomal trafficking mediated by Vps34. Simultaneous inhibition of lysosomal and endosomal trafficking inhibits uptake of esterified cholesterol and cell growth in TSC2-deficient, but not TSC2-expressing cells, highlighting the TSC-dependent lysosome-mediated regulation of cholesterol homeostasis and pointing toward the translational potential of these pathways for the therapy of TSC.

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.

Purpose: The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) is constitutively active in many human cancers and in tuberous sclerosis complex (TSC). mTORC1 hyperactivation drives extensive metabolic reprogramming via several interconnected mechanisms, including glucose and glutamine utilization, nucleic acid synthesis, and lipid synthesis. We recently found that treatment with chloroquine (CQ), a lysosomal/autophagy inhibitor, upregulates the uptake and endosomal processing of cholesterol selectively in TSC2-deficient cells, creating a TSC2-specific dependence on exogenous cholesterol. This dependence on exogenous nutrients and the mechanisms of nutrient uptake may be therapeutically targetable. Our central hypothesis is that Tsc2-deficient cells depend on the uptake of exogenous nutrients via macropinocytosis to maintain cellular metabolic homeostasis and proliferation. Methods and Results: To investigate the role of macropinocytosis-mediated nutrient uptake in Tsc2-deficient cells, we measured the uptake of dextran, a polysaccharide taken up via macropinocytosis. Tsc2-deficient cells showed a striking increase in both dextran (3.6-fold, p Conclusions: These data suggest that macropinocytosis is upregulated in Tsc2-deficient cells via a Vps34-dependent mechanism to support tumorigenesis. Macropinocytosis-mediated nutrient uptake represents an unexplored opportunity for therapeutic intervention in TSC and may also be relevant to other diseases characterized by mTORC1 hyperactivation. Citation Format: Charilaos Filippakis, Amine Belaid, Brian Siroky, Constance Wu, Nicola Alesi, Thomas Alesi, Julie Nijmeh, Hilaire Colletta Lam, Ellizabeth Henske. Vps34 promotes macropinocytosis in Tsc2-deficient cells [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 A01.

Tuberous Sclerosis Complex (TSC), a rare genetic disorder with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivation, is characterized by multi-organ hamartomatous benign tumors including brain, skin, kidney, and lung (Lymphangioleiomyomatosis). mTORC1 hyperactivation drives metabolic reprogramming including glucose and glutamine utilization, protein, nucleic acid and lipid synthesis. To investigate the mechanisms of exogenous nutrients uptake in Tsc2-deficient cells, we measured dextran uptake, a polysaccharide internalized via macropinocytosis. Tsc2-deficient cells showed a striking increase in dextran uptake (3-fold, p −/− MEFs) sensitized cells with Tsc2 downregulation to the Vps34 inhibitor, SAR405, resulting in growth inhibition (75%, p in vivo xenograft model of TSC. Our findings show that macropinocytosis is upregulated with Tsc2-deficiency via a Vps34-dependent mechanism to support their anabolic state. The dependence of Tsc2-deficient cells on exogenous nutrients may provide novel approaches for the treatment of TSC.

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.

Renal angiomyolipoma is a kidney tumor in the perivascular epithelioid (PEComa) family that is common in patients with Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) but occurs rarely sporadically. Though histologically benign, renal angiomyolipoma can cause life-threatening hemorrhage and kidney failure. Both angiomyolipoma and LAM have mutations in TSC2 or TSC1. However, the frequency and contribution of other somatic events in tumor development is unknown. We performed whole exome sequencing in 32 resected tumor samples (n = 30 angiomyolipoma, n = 2 LAM) from 15 subjects, including three with TSC. Two germline and 22 somatic inactivating mutations in TSC2 were identified, and one germline TSC1 mutation. Twenty of 32 (62%) samples showed copy neutral LOH (CN-LOH) in TSC2 or TSC1 with at least 8 different LOH regions, and 30 of 32 (94%) had biallelic loss of either TSC2 or TSC1. Whole exome sequencing identified a median of 4 somatic non-synonymous coding region mutations (other than in TSC2/TSC1), a mutation rate lower than nearly all other cancer types. Three genes with mutations were known cancer associated genes (BAP1, ARHGAP35 and SPEN), but they were mutated in a single sample each, and were missense variants with uncertain functional effects. Analysis of sixteen angiomyolipomas from a TSC subject showed both second hit point mutations and CN-LOH in TSC2, many of which were distinct, indicating that they were of independent clonal origin. However, three tumors had two shared mutations in addition to private somatic mutations, suggesting a branching evolutionary pattern of tumor development following initiating loss of TSC2. Our results indicate that TSC2 and less commonly TSC1 alterations are the primary essential driver event in angiomyolipoma/LAM, whereas other somatic mutations are rare and likely do not contribute to tumor development., Author Summary We performed comprehensive genome analysis of a kidney tumor called angiomyolipoma. These tumors are known to develop in most individuals who have Tuberous Sclerosis Complex (TSC) and those who have sporadic lymphangioleiomyomatosis (LAM), and are seen rarely in the general population. In these angiomyolipomas, we found consistent involvement of the TSC2 and TSC1 genes that are known to cause TSC, but very few (

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.

Cardiac aging is associated with lengthening of the QT interval, a condition that enhances malignant ventricular arrhythmias and sudden death. The aim of this study was to establish whether ionic currents are altered in old myocytes contributing to the protracted electrical recovery of the senescent heart. Thus, mice at 3-30 months of age were studied by ECG and patch-clamp; these physiological determinations were complemented with molecular assays for the analysis of ion channel proteins. By surface ECG and telemetry system, PR, QRS and QT intervals were prolonged in mice at 25 months or older. These delays were maintained in ex-vivo Langendorff preparations. In comparison to young, epicardial monophasic action potential (AP) duration at 50% and 90% repolarization were 1.6- and 1.2-fold larger in old LV, respectively. Moreover, senescent hearts presented a 60% higher incidence of arrhythmias. In isolated myocytes, prolongation of the early (+47%), intermediate (+117%) and late (+75%) repolarization phases of the AP were identified in cells from old animals, whereas resting membrane potential, upstroke amplitude and +dV/dt were preserved. Voltage-clamp experiments were then performed to measure ionic current properties. The rapidly activating K+ current, which consists of the transient outward and ultrarapid delayed rectifier (Ito+Kur), is responsible for the early repolarization of the AP, and was significantly reduced in old myocytes. Molecular studies revealed low levels of transcripts and proteins for K+ channel subunits Kv1.4, Kv1.5 and KChiP2 in senescent cells. Also, the late Na+ current INaL, which presents slow inactivation kinetics and is operative during AP repolarization, was 1.5-fold larger in old cells. These changes were associated with alterations in gene and protein expression of Na+ channel subunits. Inhibition of INaL with mexiletine significantly shortened the intermediate and late repolarization phases of the AP in both myocytes and perfused myocardium from old mice. Importantly, INaL inhibition in vivo shortened the QT interval of senescent mice by 12%. Thus, defects in ionic current occur with aging resulting in prolongation of the AP and delays in electrical recovery which may lead to malignant ventricular arrhythmias.

Tuberous sclerosis complex (TSC) is a multisystem disease associated with tumors of the brain, skin and kidney as well as progressive cystic lung destruction characteristic of lymphangioleiomyomatosis (LAM). TSC and LAM are caused by loss-of-function mutations in TSC1 or TSC2, resulting in hyperactive mechanistic Target of Rapamycin complex 1 (mTORC1) signaling. We previously discovered that mTORC1 hyperactivation in TSC inhibits autophagy and promotes the accumulation of oncogenic p62/sequestosome 1 (PNAS, 2011). p62 exerts a pleiotropic role in tumor progression, including the regulation of NRF2 to combat oxidative stress, NFκB to promote cell survival and the degradation of ubiquitinated proteins to efficiently recycle organelles and proteins by autophagy. Knockdown of p62 in Tsc2-null cells inhibited tumorigenesis in xenografts in vivo, but did not impact proliferation in vitro, leading us to hypothesize that p62 exerts non-cell autonomous functions influencing the tumor microenvironment to promote Tsc2-/- cell tumorigenic potential. In unpublished work, we explored the molecular mechanisms by which p62 promotes tumorigenesis in TSC/LAM. First, we analyzed renal tumor burden in Tsc2+/-; p62-/- mice. Compared to littermate control Tsc2+/-; p62+/+ mice, Tsc2+/-; p62-/- mice have a 2-fold (p Our data point toward a model in which p62 and mTORC1 cooperatively regulate cellular IL6 in Tsc2-null cells to promote tumorigenesis in TSC. IL6 is correlated with poor prognosis, advanced disease and metastatic potential in numerous cancers. Rapamycin dramatically decreases p62 levels in TSC-deficient cells. Therefore, the induction of IL6 by rapamycin through decreased p62 expression may represent one of the first non-cell autonomous mechanisms by which mTORC1 inhibition impacts tumorigenesis. Since mTORC1 is hyperactivated in the majority of malignant tumors and more than 100 cancer clinical trials are ongoing with mTORC1 inhibitors, our findings may yield insights with broad therapeutic potential. Citation Format: Hilaire C. Lam, Andrey Parkhitko, Alicia Llorente Lope, Nicola Alesi, Damir Khabibullin, Harilaos Filippakis, Ana Pereira, Barbara Ogorek, Erik Zhang, Jane Yu, Carmen Priolo, Elizabeth P. Henske. Regulation of the tumor microenvironment by oncogenic p62 in mTORC1-hyperactive diseases: Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM). [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A35.

It is well known that interindividual variability can affect the response to many drugs in relation to age, gender, diet, and organ function. Pharmacogenomic studies have also documented that genetic polymorphisms can exert clinically significant effects in terms of drug resistance, efficacy and toxicity by modifying the expression of critical gene products (drug-metabolizing enzymes, transporters, and target molecules) as well as pharmacokinetic and pharmacodynamic parameters. A growing body of in vitro and clinical evidence suggests that common polymorphisms in the folate gene pathway are associated with an altered response to methotrexate (MTX) in patients with malignancy and autoimmune disease. Such polymorphisms may also induce significant MTX toxicity requiring expensive monitoring and treatment. Although the available data are not conclusive, they suggest that in the future MTX pharmacogenetics could play a key role in clinical practice by improving and tailoring treatment. This review describes the genetic polymorphisms that significantly influence MTX resistance, efficacy, and toxicity.