Inducing Synthetic Lethality in LAM Cells Using Novel Antagomir Biologics

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. We have exploited these models of LAM to perform a genetic screen called synthetic lethality to identify genes required by TSC2 deficient cells to live. The rationale for this screen is that recently several RNA-based drugs have been approved by the FDA, demonstrating that drug development is no longer confined to inhibitors of enzymes. Surprisingly, many of the top mutations resulting in synthetic lethality were found to be in miRNAs, small RNAs that target protein coding mRNAs for degradation. In this proposal we will test the ability of inhibitors of 4 different miRNAs known as antagomirs to induce cell death in various types of TSC deficient cells. Furthermore, we will use next generation sequencing based technologies to identify the mRNAs targeted for degradation by these 4 miRNAs. These experiments have the potential to identify a new class of drugs to target LAM.

Therapeutic Targeting of Macropinocytosis-Mediated Nutrient Uptake in LAM

Lymphangioleiomyomatosis (LAM) is a rare disease that causes lung destruction mostly in women. LAM is caused by a mutation in the TSC1 or TSC2 gene, leading to activation of the mammalian target of rapamycin complex 1 (mTORC1). In the clinic, treatment with mTORC1 inhibitors (rapamycin, everolimus) stabilize lung function, but the lung cysts are not completely removed. Therapies for LAM that eliminate LAM cells are not currently available, and identification of such treatments would have a major clinical impact. The overall objective of this project is to understand how LAM can be treated by understanding their ability to take up nutrients and exosomes from their environment. Many studies have established that LAM cells are highly dependent on nutrients such as sugars (for example, glucose) and amino acids (for example, glutamine). I have found that TSC2-deficient cells, which I use as a model for studying LAM, uptake nutrients via a mechanism called “macropinocytosis.” Basically, macropinocytosis means to drink liquid into the cell. This “drinking” allows cells to take up proteins (which contain amino acids), fats (lipids, including cholesterol), and sugars.
I found that macropinocytosis is strongly increased (more than 3-fold) in TSC2-deficient cells (Figure 1-3), allowing cells to take up nutrients. Pharmacologic inhibitors of this process can target either the later stages of “drinking” (for example, inhibiting an enzyme called VPS34) or earliest stages (for example, inhibiting the engulfment of the nutrients using a drug called Amiloride, also called EIPA). I found that TSC2-deficient cells can be selectively killed using the combination of Amiloride and downregulation of genes that initiate autophagy, the cellular nutrient recycling process). Importantly, SAR405 induces the death of TSC2-deficient cells (75% reduction, Figure 6), with no effect on normal cells that have TSC2.
My objectives will be addressed in three Aims: 1) to better understand how TSC2 loss increases the movement of nutrients inside the cell within specific compartments (termed “endocytic trafficking”), 2) to better understand how macropinocytosis affects the metabolism of TSC2-deficient cells and what the consequences are for tumor cells in LAM, and 3) to use the increased macropinocytosis and treat TSC2-deficient cells with exosomes (small vesicles that exist in the human body) to restore normal mTORC1 activity in preclinical models of LAM.

LAM cells reprogram the epithelial-mesenchymal crosstalk in the alveolar niche

Lymphangioleiomyomatosis (LAM) is a fatal lung disease occurring exclusively in young women. LAM cells grow in the lungs in an uncontrolled manner, causing tissue breakdown and manifesting clinically as respiratory failure. One of the biggest challenges in improving treatment for LAM patients is the lack of understanding of where LAM cells originate and the specific factors causing LAM cells to have uncontrolled growth, movement and metastasis. The first objective of this application is to address the fundamental question, what are LAM cells and what key regulators and signaling pathways in addition to m-TOR regulate LAM cells’ destructive behavior. LAM lesions consist of two major cell types, small, spindle shaped smooth muscle positive cells and cuboidal epithelioid cells that express a premelanosomal protein (PMEL) also found in cancer. Cancer cells are in crosstalk with the tumor microenvironment and ‘activate’ normal resident cells to become Cancer Associated Fibroblasts (CAF). The second objective of this application is to interrogate the cellular crosstalk between LAM cells, epithelial cells and stromal cells, to understand how the LAM cells reprogram the alveolar niche. To validate our bioinformatics predictions and to identify the sender and receiver cells locations in the LAM lung we will perform molecular staining of LAM tissue samples using signatures identified using bioinformatics approach. This way we will connect computational data analyses and the underlying histopathology, possible identifying new therapeutic targets.

Defining the mTORC1-dependent Signaling Mechanisms Mediating Epigenetic Modifications

LAM, or lymphangioleiomyomatosis, is a rare lung disease. In LAM, abnormal, muscle-like cells begin to grow out of control in certain organs or tissues, especially the lungs, lymph nodes, and kidneys. Over time, these LAM cells can grow throughout the lungs and destroy the normal lung tissue inducing a respiratory failure. This disease is mainly due to a loss of protein called TSC (Tuberous Sclerosis Complex) which lead to a hyperactivation of an oncogene called mTOR (mechanistic Target Of Rapamycin). Currently, there is no efficient treatment against LAM disease. Epigenetics is the science that aims to understand exactly how our DNA is packaged. Changes in DNA packaging can occur through regulation of epigenetic modulators and metabolism that can directly modify DNA and proteins contributing to the progression of LAM. We hypothesize that mTOR could drive changes in DNA packaging in LAM cells leading therefore to abnormal cell growth. We will quantify the mTOR-dependent alteration of metabolic conditions in LAM cells, to identify metabolic and epigenetic states that are sufficient to drive LAM. Subsequently, we will map these changes in vivo in tumor mouse model, with the final goal to unravel metabolic and epigenetic routes responsible of LAM progression. Elucidating the interface between metabolic state and DNA packaging will provide a better understanding of tumor initiating events occurring in LAM and ultimately will lead to new therapeutic strategies to fight LAM.

Natural Killer Cell Phenotype and Function in Lymphangioleiomyomatosis

The proposed research is relevant to The LAM Foundation because it represents novel line of investigation in the discovery of immune surveillance in the rare but deadly disease lymphangioleiomyomatosis (LAM). This project will address the function and significance of the NK cells and the NKG2D receptor, as well as the role of soluble NKG2D ligands in the regulation of these pathways in LAM. The successful completion of this project is expected to lead to novel therapeutic strategies to modulate the development and progression of disease in LAM and potentially identify novel prognostic biomarkers of disease progression. This contribution would decrease the morbidity and mortality, and offer new therapeutic approaches for LAM patients.

Home Spirometry to Evaluate Disease Progression and Treatment Response in Patients with LAM

Lymphangioleiomyomatosis (LAM) is a rare, progressive lung disease primarily seen in women of childbearing age. The current standard of care in LAM to assess both the natural history as well as response to treatment involves periodic assessments of lung function conducted in a physician’s office, and it takes multiple measurements (usually 3 or more) of lung function conducted over a prolonged period (usually 12 months or longer) to make a reliable judgment about the trajectory of lung function. We propose to add home spirometry evaluation to a recently funded clinical trial in LAM - the Multicenter Interventional LAM Early Disease (MILED) trial, in order to determine the feasibility and reliability of home spirometry in patients with LAM, and to test if more frequent monitoring of lung function at home can help reduce the time needed to establish the disease trajectory, and ultimate help save time and resources.

Dysregulated Phosphatidylcholine Metabolism in TSC and LAM Pathogenesis and Therapy

Lymphangioleiomyomatosis (LAM) is a rare disease of young women characterized by progressive destruction of the lung associated with diffuse growth of so called “LAM cells”, renal angiomyolipomas, and lymphangiomyomas. LAM occurs in ~30% of women with Tuberous Sclerosis Complex (TSC). Rapamycin, the FDA-approved treatment for LAM, stabilizes lung function in LAM patients; however, lung function worsens when therapy is discontinued. More efficient therapeutic strategies remain needed in LAM.

We have found that autotaxin is a secreted protein involved in the metabolism and survival mechanisms of TSC2-deficient cells derived from a LAM patient.

We propose to test the effect of autotaxin inhibition on the biology of TSC2-deficient cells. We will investigate mechanisms of tumor growth and tumor-associated lymphatic vessel formation. Importantly, the drug targeting autotaxin (GLPG1690) that we are currently testing has been shown to be safe in clinical trials of patients with pulmonary fibrosis.

Our goal is to identify therapies that can selectively remove LAM cells. This project will potentially lead to better understanding of LAM pathogenesis and novel therapeutic opportunities for LAM.

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 11C-Glutamine PET in the Diagnosis and Monitoring of LAM

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 can be more confidently diagnosed without lung biopsies, and so that responses to existing and new treatments can be monitored more easily.

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.