The treatment of melanoma with checkpoint inhibitor (CPI) therapy has produced rapid and substantial gains in survival, and as a result, the indications for its use have steadily increased. As CPIs are used in more patients, and particularly as part of a neoadjuvant therapeutic strategy, the significance of the challenge presented by toxicities has also increased. Immune-related adverse events (irAEs), always a meaningful concern in the use of CPIs, can present with increased severity in the neoadjuvant setting, and can delay timely progression to surgery in addition to their impacts on morbidity, mortality, and the ability to maintain an uninterrupted course of CPI treatment. irAE in the skin, the most frequently affected organ, can present with a variety of morphologies that mimic a collection of naturally-occurring inflammatory dermatoses. The management strategies of these irAE is built on the treatment of their naturally-occurring namesakes. Much about the immunologic mechanisms of the irAE remains poorly understood, and as a result, management decisions are made empirically, to mixed results. Here, we propose to address this challenge by leveraging the distinct strengths of our team’s institutions and laboratories to compile a thorough characterization of the immune infiltrates across morphologies of cutaneous irAE, and compare these to the corresponding naturally-occurring dermatoses. Beginning with a substantial existing repository of patient samples, we will use sequential-slice tissue sections to generate paired single-cell and spatial transcriptomic analyses of both irAE and morphologically-matched dermatoses occurring outside the context of CPIs. This strategy will allow us to combine the power of single-cell resolution to generate information about population shifts and transcriptional program activation with the tissue-level context and cell-cell interaction mapping that spatial sequencing makes possible, generating in the process a first-of-its-kind library of transcriptional data across a class of immune-mediated skin disease. We will then interrogate this library to reveal features that connect irAE to (or distinguish them from) their naturally-occurring counterparts, along with those features that define cutaneous irAE as a class of toxicities to melanoma therapeutics. Building on these results, we will utilize multiplex RNA in situ hybridization, quantitative RT-PCR, and protein-level cytokine arrays to validate significant driver genes, while assessing the relationship between in-skin inflammatory response and patient-level shifts in circulating cytokine levels. Collectively, this work will represent a foundational step toward the understanding of the breakdown of selftolerance that is commonly a feature of the treatment of melanoma patients with CPI, and will provide mechanistic support for what are currently empiric treatment decisions that can determine the success of multiple CPI delivery strategies.

Uveal melanoma (UM) is a type of aggressive eye cancer that often spreads to the liver and has a poor prognosis. We don’t fully understand how UM spreads, which makes it difficult to develop targeted treatments. In this study, we created a new model of UM by injecting human cancer cells into the eyes of mice. This model consistently led to large liver metastasis, whereas other organs were not as strongly affected, mimicking how UM behaves in humans.

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Traditional cancer therapy involves surgery, radiotherapy, and chemotherapy. These modalities aim to ablate the tumor, yet generally the disease relapses due to the inefficiencies of the therapy and the ensuing growth of distant and/or resistant micrometastases. Immunotherapy is a new paradigm referring to the reactivation of the immune system by monoclonal antibodies against inhibitory molecules that are expressed on immune cells in the tumor microenvironment (TME). Monoclonal antibodies against CTLA-4, PD-1, and PD-L1 have been approved for clinical use in multiple cancers and can induce long-term, durable immune responses that result in the clearance of tumors. The most important advantage of immunotherapy is that, thanks to the evolving nature of the immune system, a responding patient is often cured of cancer.

Patients with lymph-node positive melanoma have a high risk of recurrence despite curative surgery. While adjuvant therapy given after surgery improves RFS and OS, ~25% of patients particularly those with bulky lymph-node disease, progress prior to commencing adjuvant therapy.

Melanoma is the most aggressive form of skin cancer. Although progress has been made for advanced melanoma patients with 13 new FDA-approved therapies since 2011, resistance arises in most cases. Our focus is on melanomas that harbor activating BRAF mutations (~50% of patients). Most of these patients respond dramatically to combination therapy with a BRAF and MEK inhibitor (BRAFi/MEKi). However, four out of every five patients relapse within two years due to the persistence of therapy-resistant subpopulations of melanoma cells. This expanding BRAFi/MEKi-resistant patient cohort is the greatest challenge of the field; few experience durable benefit from immune therapy and no alternative effective therapies exist. Therefore, there is an unmet need to develop more effective strategies. We have characterized therapy-resistant subpopulations and identified common features; 1) existence prior to therapy, 2) a slow-growing state, 3) high metastatic potential and 4) stem cell-like molecular and biological properties that allow for high adaptability in stressful conditions including therapy. Shared gene signatures by stem cells and melanoma cells are poorly understood. In our initial studies, we identified a developmental receptor, LPAR1, as key for the survival of melanoma and stem cells. LPAR1 increases the proliferation of neuronal stem cells and aggressiveness of breast and lung cancer. We show LPAR1 expression increases with progression in melanoma patient tumor tissue relative to benign nevi. Further, a) LPAR1 expression is higher in BRAFi/MEKi resistant melanoma cells, b) hyperactivation of a down-stream LPAR1 effector, YAP1, increases the presence of resistant stem cell-like melanoma cells, and c) genetic or pharmacological targeting of LPAR1 kills BRAFi/MEKi resistant melanoma cells. This provides strong scientific rationale for investigating LPAR1 as a novel target to overcome BRAFi/MEKi resistance. We propose to validate LPAR1 as a clinically relevant target by using models that closely mimic the in vivo biology of melanoma. This includes 3D human skin-, spheroid-, and a collection of >500 patient-derived xenograft (PDX)-models where patient tumor material is inoculated directly into mice, including >200 patients that relapsed on BRAFi/MEKi. Towards this goal, we will define the molecular consequences of inhibiting LPAR1 on the survival and growth of stem cell-like melanoma cells and in BRAFi/MEKi resistance. We will identify the most potent LPAR1 inhibitor that can synergize with BRAFi/MEKi to eliminate all tumor cells without causing toxicity. As LPAR1 inhibitors are currently being clinically investigated, we expect our proposed studies will provide the scientific rationale to clinically test new therapeutic strategies that will increase the curative potential of BRAFi/MEKi and facilitate the development of future clinical trials. 

The Hippo pathway in cancer, mediated by YAP1, has been implicated in therapy resistance and aggressive tumor behavior in melanomas treated with targeted therapies. However, the role of this pathway in response and resistance to immunotherapy has not yet been characterized. There is existing data suggesting that activation of this pathway may lead to immune evasion by tumors. Therefore, we hypothesize that concurrent inhibition of the Hippo pathway in combination with immunotherapy may be complementary. We will use a unique resource of longitudinal tumors from patients treated with immunotherapy in whom analysis of molecular data is already underway to assess if there is regulation of the Hippo pathway during treatment with immunotherapy in melanoma and if this correlates with response or resistance to therapy (Aim 1). We will then use cell lines to establish the mechanism of Hippo pathway activity in melanoma (Aim 2), and finally will examine the impact of activating/inactivating the Hippo pathway in combination with immunotherapy in a mouse model of melanoma. This proposal draws from our respective strengths in translational medicine (Dr. Boland), computational biology (Dr. Liu) and mechanisms of gene regulation (Dr. Saladi).

Immune therapies termed “immune checkpoint inhibitors” remove key “brakes” on immune cells. These treatments lead to long-term responses and cures in some patients with melanoma, but may also cause severe side effects. These toxicities result from abnormal immune cell activation, and are highly unpredictable. This presents several important obstacles to improving melanoma treatment: 1) We are unable to predict which patients will develop severe toxicities; 2) More aggressive treatment combinations cause more toxicity, limiting our ability to develop more active combinations; and 3) Patients who have long-term benefit may still develop devastating side effects. To address these problems, we propose to perform an in-depth study of the most common severe toxicity: colitis (inflammation of the colon). To do this, we plan the following aims. First, we will look at genetic and immune factors that may predispose patients to colitis. Many autoimmune conditions, that have similarities with immune colitis, have such genetic and immune system links. Second, we will take colon biopsies from patients who have colitis, and perform in-depth analysis of these tissues. We will compare the specific immune cells between the colon, blood, and tumor to better understand how the immune system is working in all three areas, and how to “turn off” abnormal activation while maintaining activity against the tumor. Third, we will collect cases from large melanoma centers to determine exactly when and how colitis occurs, and how best to treat it. Ultimately, we propose that in-depth study of this common and severe toxicity will allow us to understand it, prevent it, and treat it more effectively.

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