The Role of Cathepsin K in Lung Destruction in Lymphangioleiomyomatosis (LAM)

Lymphangioleiomyomatosis (LAM) is a rare disease of women which causes lung damage leading to breathlessness, lung collapse and sometimes respiratory failure. How this occurs is not well understood, although it is thought that LAM cells which build up in the lungs produce proteases, powerful enzymes that, when activated in the wrong place, can damage lung tissue. Cathepsin K is a protease which is present in LAM cells, but not normal lung tissue. We have shown that cathepsin K is produced by cells present in LAM lungs, and that LAM cells create an acid environment necessary to activate cathepsin K. We will examine the production and activation of cathepsin K in LAM derived cells and tissue. We will see which proteins contributing to the acidic environment are present in LAM, why they become activated and how this affects cathepsin K. We will test how cathepsin K contributes to lung damage in models of LAM and if current drugs that block the action of cathepsin K can reduce lung damage. Finally, we will determine if the amount of cathepsin K in the lungs of women with LAM is related to the amount of lung damage and how this changes over time. Together this work will determine whether blocking the action of cathepsin K should be investigated in a trial for women with LAM.

Genome-Wide Vulnerability Mapping in a TSC Mutant Cell-line Using CRISPR Knockout Screening

This proposal uses an unbiased approach called synthetic lethality screening to discover new drug targets in the treatment of TSC and LAM, in which the causative genes TSC1 and TSC2 are mutated or non-functional. In this approach, we systematically perturb hundreds of genes to identify vulnerabilities that specifically affect cells with mutated TSC1 or TSC2 but leave normal cells unaffected. The TSC1 and TSC2 genes are highly evolutionarily conserved. Our previous results showed that cells from the simple fruit fly can be used to identify vulnerabilities. Moreover, three of these vulnerabilities were conserved in mouse and human cells lacking TSC2. This rapidly led us to the identification of a drug that selectively inhibits TSC2-mutated mouse and human cells while leaving normal cells unaffected. Our previous study used a small screening library that represents one twenty-fifth of the fly genome. We hypothesize that increasing the scale of the screening approach will lead to the identification of more vulnerabilities and thus more potential therapeutics, but this is difficult using our current methods. Here, we adapt a newly discovered technology called CRISPR to the identification of conserved vulnerabilities in TSC and LAM to reveal all remaining vulnerabilities in the fly cells, and then additionally propose to test selected hits in mouse and human cells. Extending our evolutionary approach to identifying TSC and LAM vulnerabilities is likely to lead to new drug therapies for the treatment of TSC and LAM.

PD-L1 as a Potential Therapeutic Target for Patients with Advanced or Refractory-to-Rapamycin LAM

Tumor manipulation of host immunity is important for tumor survival and invasion. Little is known, however, about the immunobiology of LAM and specific role of the immune inhibitory molecule PD-L1. Our proposal will elucidate a relationship between loss of TSC2 and PD-L1 upregulation and will shed new light on the immunobiology of LAM. Because blockage of immune checkpoint protein PD-L1 is among the most promising approaches in cancer immunotherapy, our findings could provide a new opportunity for therapeutic targeting for LAM patients unresponsive to rapamycin treatment and with advanced LAM disease.

Assessing mTOR dependent and independent pathways in LAM

A major obstacle for LAM research and the development of new LAM treatments has been the difficulty in isolation of LAM cells from patients to study LAM or to perform drug screens. When LAM cells have been isolated, these cells don’t behave like LAM in the culture dish or they stop growing. To solve these problems we have made LAM-like cells from both female and male human stem cells and we have developed a gel that has many of the properties of a human lung so that we can grow LAM or LAM-like cells in a 3D lung-like environment. We found that TSC deficient, LAM-like cells behave differently in 2D (normal) and 3D culture. In this proposal we will use our LAM-like cell lines and our 3D lung tissue-like culture system to tease apart why rapamycin only slows down LAM but does not stop it, and to explore why mutations in TSC2 predispose a woman LAM more than mutations in TSC1. In addition, we will perform a drug screen of nearly 1,300 FDA-approved drugs to identify drugs that can work alone or in concert with rapamycin to preferentially kill LAM cells, or to stop their production of MMPs which destroy the lungs of LAM patients. In addition, since we are using both male and female cells, we may learn why LAM only strikes women.

T Cell Receptor Transduced T Cells Supported by Anti-PD-1 to Treat LAM

LAM cells share some features with the cells that make pigment in the skin. The molecules that are responsible for pigmentation can be misread as foreign particles by the immune system in patients that develop the autoimmune disease vitiligo. We thus isolated immune cells from vitiligo patient skin and identified the recognition molecule on the surface of T cells that allows these immune cells to recognize gp100. Expression of gp100 is also a hallmark of LAM, and HMB45 staining can be used to detect it and confirm a LAM diagnosis in patients. We now have the gp100-reactive T cell receptor in hand so that it can be introduced into patient T cells to be redirected towards LAM tissue. The gp100-reactive T cells may become exhausted before the diseased tissue is effectively eliminated by the redirected T cells. In fact, we have seen that our LAM cell line is equipped with the tools to exhaust reactive T cells. We thus want to test the anti-PD1 reagent, recently approved for use in melanoma and in lung cancer, for its value in the treatment of LAM, to prevent exhaustion in our redirected T cells. To put this combination therapy to the test, we will make use of a preclinical model of LAM using a human LAM cell line and redirected human T cells. This live model allows for the outgrowth of traceable human LAM cells and their interaction with T cells in presence of the anti-PD-1 reagent. The outcome of these studies may serve as the basis for clinical studies in (wo)man.

Exploring Pathobiology of LAM Through Isolation of Cellular Components of LAM Lesions, Their Characterizations and the Reconstruction of LAM Lesions by Patient-Derived LAM Cells and Lymphatic Endothelial Cells

The prevailing LAM models at present are -knockout mice or a mouse model explanting -null cell line or human -null angiomyolipoma cells. However, these have some faults since they unfortunately lack lymphatic endothelial cells (LEC), an important component of LAM lesions. In addition, no human LAM cells with mutations have been isolated from LAM patients. Therefore, the pathobiology of LAM would have not been fully disclosed by the existing models. We are going to establish a flow cytometry (FACS)-based method by which that HMB45-positive and mutation-harboring LAM cells can be isolated from human LAM lung tissues. In addition, other cellular components of LAM lesions, e.g. LEC and mesenchymal cells, are simultaneously obtained. We would like to establish the culture technique to expand these cellular components , and then reconstitute LAM lesions in a 3-dimedional gel. We expect that this reconstitute would work as a new “ LAM model” to explore what kind of cell-cell interactions work there and what the key mechanism for LAM cells survive.

NKG2D Receptor-Ligand Interactions in LAM Pathogenesis

Lymphangioleiomyomatosis (LAM) is a rare lung disease of women in which smooth muscle cells from an unknown source proliferate and infiltrate the lung. Proliferating smooth muscle cells lead to destruction of lung tissue, leading to respiratory failure. Typically, abnormally proliferating cells are recognized by immune cells through expression of surface markers that target them for elimination. It is important to understand how LAM cells evade this immune detection. Recognition of abnormal cells by cytotoxic lymphocytes is mediated in part through the NKG2D receptor that recognizes ligands on abnormal cells. NKG2D ligand expression is highly restricted and absent on healthy cells, but abundant on the surface of tumor cells. The importance of NKG2D–mediated control of abnormal cell proliferation is established. However, recent studies demonstrate that the elaboration of soluble NKG2D ligands suppress tumor immunity by acting as a sink and downregulating NKG2D expression and function. This proposal will test the possibility that soluble ligands for the NKG2D receptor are useful biomarkers for disease progression. Further, we will examine the function of the NKG2D receptor in LAM patients to gain insight into disease progression. Several therapeutics aimed at modulating NK cell receptors are in development for chronic diseases, autoimmune diseases, and cancer. Our hope is that these novel agents will provide exciting therapeutic options for LAM patients in the near future.

Use of 18F-Glutamine PET in the Diagnosis of LAM and Other Pulmonary Diseases

Diagnosing many lung diseases, including LAM, can be challenging. Though sometimes a disease can be identified by a combination of physical exam findings, imaging findings (like X-rays or CT scans), and blood tests, often a definitive diagnosis requires a lung biopsy. This can be difficult to accomplish safely, and even under the best of circumstance, there is always some degree of risk and inconvenience involved. This project hopes to improve the use of special imaging techniques so that LAM and other lung diseases can be more confidently diagnosed without lung biopsies, and so that responses to existing and new treatments can be monitored more easily.

The Safety of Simvastatin in Patients with Sporadic Pulmonary Lymphangioleiomyomatosis (LAM) and LAM Associated with Tuberous Sclerosis Complex (The SOS Trial)

Pulmonary LAM, which can be sporadic (LAM-S) or associated with tuberous sclerosis (LAM-TS), is a rare lung disease affecting predominantly women of childbearing age that manifests by neoplastic growth of atypical smooth muscle-like LAM cells, lung cyst formation, obstruction of lymphatics, and spontaneous pneumothoraces (1). Current treatment with rapamycin analogs, which are not FDA-approved for treatment of LAM-S and LAM-TS, but are commonly used based upon published reports, stabilize lung function but does not prevent further disease progression (1, 2). The rapalogs are cytostatic agents and cessation of treatment resumes decline in lung function. Because of this limitation new combinational therapy targeting LAM cell death is needed. Preclinical evidence demonstrates that simvastatin acts as a pro-apoptotic agent in in vitro and in vivo studies in cells and tumor lesions deficient for TSC2, inactivation mutation of which cause LAM and TS (3, 4). HMG-CoA reductase inhibitors (statins) are very safe, highly effective therapies used by millions of people (5, 6). Simvastatin decreases cholesterol, reduces cancer incidence, stabilizes the endothelial cell layer and decreases inflammation. In preclinical studies the combination of rapamycin and simvastatin inhibited xenograft tumor growth and completely blocked the recurrence of xenograft tumors after treatment withdrawal (4). In mouse model of LAM, treatment with rapamycin and simvastatin prevented both growth of TSC2-null lesions and lung destruction (3). These findings have fundamental translational significance for combinatorial therapeutic strategies to induce the death of LAM cells, potentially obviating the need for continuous, life-long suppressive therapies. We have designed a Phase II, open label, safety trial to initiate the study of this potentially useful intervention. We propose to conduct a safety trial of simvastatin treatment of patients with LAM-S and LAM-TS. We aim to determine the safety of simvastatin in combination with sirolimus or everolimus over 4 months.

Genomewide Association Studies (GWAS)

Genome wide association studies (GWAS) are a type of study commonly used by medical genetic researchers to try to identify genes and their variants that are associated with, or in some way predispose to, human disease. They have been performed for almost every common disease you can think of, including diabetes, cancer, and heart disease. A GWAS has not been performed in LAM previously. We suspect that genetic risk may be significant in LAM given the strong association of LAM development with female sex and the consistent occurrence of TSC2 gene mutations in LAM cells. One great thing about GWAS is that they are unbiased. This means that the entire genome is scrutinized, so that any gene anywhere that has a role in LAM development may be discovered. It is well-known that many genes have been identified in this manner in other diseases by GWAS which were completely unexpected. Therefore, there is the possibility that something completely different from what we know about LAM will be discovered from GWAS. Therefore, we propose to perform a GWAS of LAM with appropriate follow-up studies.

Mechanism of Tumorigenesis by mTORC1-Medicated mRNA Alternative Splicing

Cells can make different types of proteins from a single gene by cutting, removing, and rejoining part of the gene. This process, called alternative splicing, normally occurs to make specific protein types required for each organ and developmental time. Cancer research has mostly focused on investigating gene expression or protein quality control. However, recent large-scale screen technologies have found that alternative splicing is remarkably changed in tumors. For example, cancer cells express an embryonic type of a certain metabolic protein to provide growth advantage. In breast cancer patients resistant to chemotherapy, aberrantly spliced oncogene was identified to confer drug resistance. I discovered that alternative splicing of specific genes is changed in LAM cells. Moreover, I found that the activity of splicing regulating factors is also dysregulated in LAM cells. I will perform in-depth mechanistic studies to identify signaling processes controlling alternative splicing and investigate the effect of altered splicing in LAM pathogenesis. Successful completion of the proposed work will demonstrate that the alternative splicing process is likely to be a rich source of novel drug targets and diagnostic markers, which will lead to development of more selective and effective therapeutic interventions for LAM.

Estradiol and mTORC2 Orchestrate to Enhance Prostaglandin Biosynthesis and Tumorigenesis in LAM

Lymphangioleiomyomatosis (LAM), the pulmonary manifestation of tuberous sclerosis complex (TSC), is a devastating disease affecting women, often leading to end-stage lung disease during or soon after pregnancy. The pathogenesis of LAM is very unusual: LAM cells are histologically-benign smooth muscle cells carrying TSC2 mutations that are believed to transfer or metastasize to the lungs, where they cause lung degeneration. The only proven treatment for LAM is lung transplantation, which carries significant one-year mortality and after which LAM can recur in the transplanted lungs. The reasons that LAM affects women almost exclusively are not yet clearly defined, and animal models that mimic the metastatic behavior of LAM cells have not been previously developed. Our animal model provides in vivo evidence that estrogen promotes the survival and metastasis of Tsc2-null cells and increased expression of COX-2, which allows us to investigate the possibility that targeting COX-2 pathways may have a major role in the treatment of TSC and LAM. In addition, this model will allow other novel agents and/or combinations of existing agents to be tested in preclinical stage. This will further facilitate the eventual development of effective therapies for TSC and LAM, which are urgently needed.

MicroRNA in LAM: Therapeutic Targets and Biomarkers

Lymphagioleiomyomatosis (LAM) is a devastating disease of women that is associated with lung destruction and progressive lung function decline. LAM is caused by mutations in TSC1 and 2, resulting in hyperactive mammalian Target of Rapamycin (mTORC1), which leads to excessive cell growth and proliferation. Rapamycin and related “rapalogs” inhibit mTORC1 and promote tumor regression; however, tumors regrow upon treatment cessation suggesting that rapalogs prevent LAM cell proliferation, but do not induce specific LAM cell death. To identify novel therapies that promote cell death in TSC-deficient cells, we sought to determine whether dysregulated expression of microRNA (miRNA or miR) contributes to TSC pathogenesis and rapamycin-resistance. miRNA are small molecules that can target multiple mRNA transcripts, negatively regulating the formation of proteins from genes. In recently published experiments using TSC2-deficient cells we identified rapamycindependent miRNA “Rapa-miRs”. Amongst the most highly expressed Rapa-miRs we identified pro-tumorigenic miRNA, “onco-miRs,” including miR-21, miR-221 and miR-29b. We hypothesize that targeting these pro-survival miRNAs will enhance rapamycin effectiveness. To explore this hypothesis we will investigate the regulation of miRNA by rapamycin, identify target genes regulated by rapa-miRs, determine the in vitro and in vivo effects of rapa-miR knockdown, and measure TSCLAM miRNA biomarkers in patient plasma versus healthy patient controls. These studies will identify novel therapeutics and potential biomarkers, which may have a profound impact on the clinical diagnosis and treatment of LAM patients.