An Information Retrieval System for Multiomics Data Integration and LAM Biomarker Discovery

Lymphangioleiomyomatosis (LAM) is a rare lung disease occurring almost exclusively in women, causing tissue remodeling and progressive respiratory failure. Biomarkers are useful in LAM patient care and clinical studies: diagnostic markers can assist in non-invasive LAM diagnosis; prognostic markers enable prediction of an individual's likely course of the disease; and predictive biomarkers provide information on the likelihood of responses to specific treatments. Pivotal clinical trials and research demonstrated that serum VEGF-D is a clinically useful diagnostic, prognostic, and predictive biomarker of LAM. However, 30~40% of LAM patients have normal serum VEGF-D, highlighting an urgent unmet need for novel biomarkers, either used alone or in combination with VEGF-D, to improve LAM patient care. To address this, we collected a wealth of LAM multiomics and clinical data, which provides large-scale complementary information on RNA expression and chromatin states in individual lung cells in LAM and protein concentrations in blood samples of LAM. We reasoned that the integration of these data will enable robust and novel LAM biomarker discovery at single cell resolution. We will develop GLAMOR, a novel computational system that employs theories of search engines (e.g., Google), to map the complex data and rank molecular candidates for LAM biomarkers. We anticipate that GLAMOR will discover new biomarkers that can contribute to diagnostic, predictive and prognostic tools for LAM.

The TSC-mTORC1 Network Controls Bicarbonate Uptake to Support Cell Growth

In LAM, abnormal muscle-like cells in specific organs or tissues grow out of control, especially the lungs, lymph nodes, and kidneys. Over time, these LAM cells can grow throughout the lungs and destroy the normal lung tissue inducing respiratory failure. This disease is mainly due to a loss of a protein called TSC (Tuberous Sclerosis Complex), leading to hyperactivation of a protein called mTOR (mammalian Target Of Rapamycin). Currently, there are no effective treatments against LAM. mTOR is widely activated in most cancers, leading to increased energy intake in cancer and LAM cells. The alteration of small molecules called metabolites in TSC-deficient cells could explain the abnormal proliferation of cells in LAM and cancer disease. In our research project, we observed that the molecule bicarbonate was stimulated by mTOR and increased in LAM cells. Bicarbonate is metabolically consumed to support the cellular biomass required for cell proliferation. Therefore, we hypothesized that targeting the metabolic enzyme under the control of mTOR that enables the accumulation of bicarbonate would eliminate the growth of LAM cells. Firstly, we will determine the global change in cellular metabolism in LAM cells by performing metabolomics experiments using the mass spectrometry instrument. Then, by using specific inhibitors targeting bicarbonate metabolism, we will determine whether bicarbonate could be exploited to target the pathophysiology of LAM.

Nanomedicine for Eliminating Rapamycin Tolerant Persister Cells

Rapalogs are the only effective treatment lymphangioleiomyomatosis (LAM) but induce a cytostatic effect with stabilized lung function in LAM. Lung function continues to decline upon treatment cessation. Our single cell analysis of LAM specimens identified a subset of LAM cells with elevated stemness and dormancy programs, two typical features of drug tolerance/resistance. These cells are characterized with a unique transcriptional profile enriched with cytokines and transcriptional/epigenetic factors previously reported to be expressed in drug tolerant tumor cells. Our preliminary data showed that targeting on some of these factors re-sensitized TSC-deficient cells and significantly enhanced rapamycin efficacy in terms of decreasing tumor burden. However, many of these cytokines and transcriptional/epigenetic factors are undruggable due to cellular location or lack of small molecule binding pocket. We have developed a tumor cell-targeting nanocarrier that can deliver siRNA therapeutics targeting these factors specifically into tumor cells. This approach greatly expands the pool of druggable targets because siRNA molecules can theoretically target any gene, including transcription factors. The development of tumor cell-targeting nanoparticle-assisted RNA medicine will open a new therapeutic paradigm for LAM.

Monitoring Risk of Rapid Lung Function Decline: The LAM Prediction (LAMP) Early Translation Study

Lymphangioleiomyomatosis, or LAM, is a rare lung disease primarily affecting women. LAM can cause cells to grow in organs and tissues, including lungs, which can damage healthy tissue and lead to poor lung function. Physicians and researchers measure lung function as forced expiratory volume in one second, or FEV1. Maintaining FEV1 is essential to survival in LAM, but many patients experience precipitous drops in FEV1 and these drops are highly variable between patients and within a single patient over time. These bouts can be clinically termed rapid decline, and they can lead to irreversible lung damage and failure. It is imperative to have monitoring tools that can detect, and possibly prevent, rapidly declining FEV1. We performed a prognostic study of registry data suggesting that rapid FEV1 decline can be predicted with a high degree of accuracy. The proposed pilot project seeks to translate these prognostic data into a clinical decision aid for providers treating patients with LAM. This real-time LAM prediction (LAMP) will be developed and tested in a single center for point-of-care assessments on usability and acceptability of the physicians and patients. Retrospective data will be used to compare LAMP detection to the current detection approach used in clinic today. The findings will form the basis to design and execute large-scale LAMP intervention testing across the LAM Clinic Network, thereby increasing the quality of life and longevity of individuals with LAM.

Interaction of Lymphatic Endothelial and LAM Cells Driving Lymphangioleiomyomatosis Pathogenesis

Lymphangioleiomyomatosis (LAM) is a rare disease affecting multiple organs, especially the lung, leading to a chronic decline in lung function with the need for oxygen supplementation and even lung transplantation. The disease predominantly affects women and is caused by abnormal growth of smooth muscle-like cells in the lung and lymphatic system. Currently, the only therapeutic option is rapamycin, which slows the progression of the disease, there is not cure. Studying LAM pathogenesis using relevant cells has been challenging and progress to identify new therapeutic options limited. In this study we will further develop co-culture models where complex interactions between the LAM-smooth muscle-like cells and the lymphatic system can be investigated. Such studies are likely to identify new intercellular signaling that augments LAM progression and may be suitable for therapeutic intervention.

LAM Cell Atlas (LCA): An Intuitive Web Portal for Integrative Analysis and Visualization of LAM Single Cell Multiomics Data

Lymphangioleiomyomatosis (LAM) is a rare lung disease of women, with unclear cell origin, tumor microenvironment, and cellular interactions in the LAM lesion. The recent application of single-cell transcriptomics in LAM has shed light on LAM cell identity and likely origin, brought new insights into pathways and gene networks related to LAM disease pathogenesis; but the technical challenges of handling and analyzing a large amount of single-cell multi-omics datasets; and the lack of intuitive and easy-to-use tools for data analysis and visualization, have limited experimental biologists and LAM research experts from directly exploring and utilizing the rich single-cell data resources. We propose to integrate a compendium of LAM single-cell multi-omics datasets and construct an intuitive, interactive, multidimensional LAM cell atlas of LAM lesions and their surrounding microenvironment to expedite data sharing, data reanalyzing, and to facilitate hypothesis generation and collaborative LAM research. Considering the rarity of LAM disease and the limited availability of fresh tissue samples, our long-term goal is to establish a LAM Cell Atlas as a living resource that will iteratively expand to include new single-cell omics data from LAM researchers around the world.

Using Artificial Intelligence to Predict Need for Therapy in LAM

Women with LAM lose lung function at different rates, meaning some can develop respiratory failure in a decade, whereas others remain well for many years. This variation in the progression of LAM means that advising on prognosis and identifying women who will benefit from treatment with Sirolimus can be difficult in early disease. In this work we will use ‘artificial intelligence’ or ‘machine learning’ and other methods to identify patterns of features present in early LAM which are associated with loss of lung function and the need for Sirolimus therapy. Using data from carefully studied women with LAM at the UK LAM Centre we will generate, test and refine a prediction tool to identify those at risk of progressive loss of lung function. Through collaborations with LAM clinicians internationally we will examine the performance of the tool in other populations to determine its wider applicability with the eventual aim of generating a predictive tool that can used to target treatment at diagnosis to those at risk of progressive disease and give better prognostic information.

Targeting Immune Suppression in Lymphangioleiomyomatosis Using Adjuvant Immunotherapy

Pulmonary Iymphangioleiomyomatosis (LAM) is a slow progressing, metastasizing neoplasm primarily affecting women of reproductive age. Recent evidence has shown that LAM creates an immunosuppressive microenvironment, through expression of lymphocyte inhibitory molecules, including PD-L1, and checkpoint immunotherapy blocking these molecules enhances survival in murine metastatic LAM. Here, we will determine the effects of LAM cells on lymphatic transport and immunomodulation, and therapeutic efficacy of adjuvant-based vaccines against LAM. This work will bring novel insights into the effects of LAM on lymphatic functions and may provide new avenues for LAM treatments. Adjuvant based therapy is another promising immunotherapeutic approach used to also treat immunosuppressive cancers. Adjuvants function by effectively ‘turning on’ the immune response through activation of antigen presenting cells that subsequently activate lymphocyte activity. We have exciting preliminary data suggesting that treatment with a specific adjuvant, PolyI:C, enhances survival in murine metastatic LAM. In this proposal we explore the use of PolyI:C alone and in combination with anti-PD-1 checkpoint inhibitors as new LAM therapy, and also to explore how PolyI:C treatment affects the immune response in LAM. Finding that PolyI:C and its combination with checkpoint inhibition effectively treats LAM, would not only lead to adjuvant treatments for LAM, but also open the door to designing anti-LAM vaccinations as new LAM therapeutics. Furthermore, the proposed work will shed new light into LAM immunity that could identify new treatment targets in LAM, all crucial to finding a cure for this devastating disease.

Targeting the Immune Checkpoint Molecule B7-H3 in the Therapy of LAM

One of the most exciting recent discoveries in LAM research is that the immune cells in the LAM niche play a critical role in disease progression and potentially a pivotal role in LAM therapy. Recent work by two groups, including ours, have shed a light on the potential utilization of immune checkpoint inhibitors for LAM. The power of immunotherapy to eliminate LAM cells could represent the next therapeutic breakthrough in LAM.

B7-H3 is an immune checkpoint molecule (and a homolog of PD-L1), that is highly expressed by LAM cells; however, its role in LAM disease progression and therapy is unknown. This project aims to identify the mechanisms by which B7-H3 remodels the immune microenvironment in LAM and define the impact of targeting B7-H3 in preclinical models of LAM. Importantly, B7-H3 neutralizing monoclonal antibodies and chimeric antigen receptor (CAR) T cells have shown efficacy in mouse models, and human clinical trials of anti-B7-H3 antibody therapy have revealed no dose-limiting toxicities. This highly favorable toxicity profile highlights the potential of this project to have transformative benefits for women with LAM. The immune-mediated strategies represent a completely new approach for LAM, with the potential for a profound and even paradigm-shifting clinical impact.

Applying 68Ga-citrate PET/CT and PET/MR to Measure Lymphangioleiomyomatosis Disease Burden

The clinical progression of LAM is highly unpredictable, with some patients experiencing stable disease for years, while others endure rapidly declining health. This heterogeneity underscores the urgent need for non-invasive biomarkers that clearly measure disease burden and/or activity in all regions of the body, and that can be conveniently applied many times over a patient’s lifetime to study changes in disease state. To this end, we have shown that LAM cells to take up more iron than normal cells, and this biological effect can be detected in living subjects with a positron emission tomography scan that we developed. The goal of this project is to test if the iron imaging assay can detect diseased tissue in patients with LAM. A secondary goal of this project is to test if the PET imaging assay can be combined with anatomical imaging using CT to improve the detection of LAM cells in diagnostically challenging regions of the body like the lungs. Finally, we will conduct exploratory studies to determine if advanced MR technologies we have developed can be applied to image LAM cells in the lungs, which could represent a first step toward developing a radiation free imaging platform to visualize LAM. In summary, this project aims to establish a new imaging-based biomarker platform to better monitor LAM progression and response to approved or experimental therapies. Because iron can also be targeted therapeutically to treat diseases, the imaging data we collect during this project could also stimulate new drug trials toward curing LAM.

Deciphering the Role of Estrogen in LAM Cell Stemness and Dormancy

LAM almost exclusively affects adult women, with worsening symptoms often observed during the premenopausal years. About 40% of sporadic LAM patients also have AML. The reasons for the female predominance of LAM are not yet entirely understood. Our lab previously reported that estrogen promotes the survival and pulmonary metastasis of tuberin-null cells.

My preliminary single cell data of ten LAM and AML samples identified a subpopulation of LAM and AML cells showing a strong stem cell-like transcriptional signature and upregulated markers of dormancy. In the cancer field, “dormant” cells are known to resist therapy, and can re-emerge after long periods of time to regrow and metastasize. Interestingly, this subpopulation also showed higher expression of estrogen receptor alpha (ESR1). This suggest that this dormant subpopulation, induced by estrogen, could be critical to both the cause and the treatment of LAM. Remarkably, dramatic co-expression of tumor lymphangiogenic growth factor midkine (MDK) was observed in the dormant subpopulation in both LAM and AML. The potential role of MDK in lymphangiogenesis and metastasis in LAM is entirely unknown.

This project aims to reveal how estrogen-related pathways lead to “dormancy” in LAM. This dormancy may affect the response to rapamycin treatment and the effects of estrogen on LAM progression. I will also provide proof-of-concept that targeting on key nodes in the estrogen pathway can suppress the proliferation and metastasis of LAM cells.

Role of Homeobox Genes in Hormonal Regulation of Transcriptional Activation in LAM Progression

LAM is a fatal lung disease occurring exclusively in young women in their reproductive years. LAM cells travel to the lungs and, once established, start to grow uncontrollably causing tissue breakdown and respiratory failure. Currently, there is no cure for LAM patients. Treatment with the drug, sirolimus, can prevent lung lesions from growing, stabilize lung function, and improve symptoms in LAM patients. Unfortunately, sirolimus does not make the tumors go away, and tumors enlarge further as soon as treatment is stopped. In addition, not only can long-term treatment with sirolimus lead to side effects, but also some patients do not respond. Therefore, it is important to develop new therapies that both eliminate tumors and do not have major side effects.

Multiple observations implicate female hormones in LAM progression, including gender specificity, expression of hormone receptors, and worsening of symptoms during pregnancy and the menstrual cycle. Understanding the female predominance has long thought to be a strategy to development of novel therapies. This application proposes to provide such increased understanding.

What are objectives of our research? The main objective is to understand why LAM develops primarily in women, explain how female hormone estrogen affects the behavior of LAM cells, and develop new treatment method to kill LAM cells without affecting normal cells. We have preliminary data that has identified specific pathways accounting for estrogen’s actions. Our goal for this project is to show more definitively the role of these pathways and to demonstrate the potential of a cure by using this new understanding to design a treatment that will kill abnormal LAM cells without affecting normal cells.

What are the likely contributions of this proposed research project to advancing the field of LAM research and/or patient care? Our proposal will study a previously unknown mechanism controlling LAM progression in the lung. This establishes a new potential therapeutic target for treatment of LAM. Importantly, by identifying the new pathways used by LAM cells only, new therapies distinct from sirolimus may be discovered that, unlike sirolimus, are curative. In summary, these pre-clinical studies described in our proposal provide the possibility of a major long-term breakthrough in treatment of LAM.

Targeting Immune Suppression in Lymphangioleiomyomatosis Using Adjuvant Immunotherapy

This proposal has been partially funded with a Seed Grant to support gathering of preliminary data for a future grant submission.

LAM is a devastating disease and patients have little treatment options other than lifestyle changes, dietary changes, oxygen, and rapamycin, none of which cause disease regression and often only slow progression. Recent evidence has demonstrated that LAM has many similarities to cancer, including inducing an immune suppressive microenvironment in the lungs. Several immunotherapy treatments have been tested, but so far adjuvants remain untapped as LAM therapies. Here, we seek to address this gap in knowledge. If we find that adjuvants and/or their combination with checkpoint inhibition (and rapamycin) effectively treat LAM, it would not only lead to adjuvant treatments for LAM, but also open the door to designing anti-LAM vaccinations as new LAM therapeutics. Furthermore, the proposed work will shed new light into LAM immunity that could identify new treatment targets in LAM, all crucial to finding a cure for this devastating disease.

Use of Gallium-68 DOTA PET/CT to Determine Disease Activity in LAM

This proposal has been partially funded with a Seed Grant to support gathering of preliminary data for a future grant submission.

Whole body Gallium-68 DOTA PET/CT scans will be performed on 5 patients with known sporadic or TSC-LAM, with anatomical coverage from the vertex to mid-thighs. Image interpretation will be performed by an expert PET radiologist with experience in clinical Ga-DOTA PET/CT interpretation. The PET images will be reviewed on a hybrid nuclear medicine display workstation. A qualitative visual assessment will be performed for any increased tracer uptake in the lungs, using the low-dose attenuation correction CT for anatomical localization of tracer activity to any areas with cystic lung change. Assessment for any focal extra-pulmonary (renal, osseous and cerebral) uptake will be performed. Semi-quantitative measurements of any visually increased area of tracer uptake will also be performed in the form of maximum and mean standardized uptake values (SUVs).

Study of Monoamine Metabolism in Lymphangioleiomyomatosis: Novel Biomarkers and Therapeutic Opportunities

This proposal has been partially funded with a Seed Grant to support gathering of preliminary data for a future grant submission.

Lymphangioleiomyomatosis (LAM) is a rare lung disease that presents important challenges to patient care and clinical management. Inhibition of mTOR with sirolimus (rapamycin) is the current standard of care, but this treatment is not sufficiently tolerated in all patients and, critically, a substantial portion of the cases suffer continuous decline in their lung function despite receiving therapy. The inhibition of mTOR by rapamycin does not completely eradicate diseased cells. In parallel, high plasma levels of VEGFD are a biomarker of the disease’s diagnosis and progression; however, measures of this factor show a degree of variation whose origin is unknown, and low levels of it do not necessarily mean there is no pathology present. In this project, we address both fundamental limitations to improving LAM care by studying novel blood plasma biomarkers and assessing novel therapies.

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.