The number of diagnoses of melanoma increased 3% per year between 2005 and 2014. Nearly 10,000 Americans die from the disease every year. Most deaths are associated with the metastatic form of the disease, spreading to the liver, lungs, bone, and brain1. Since 2011, three immune checkpoint inhibitors have been approved for metastatic melanoma and have quickly been adopted as standard of care. These monoclonal antibodies block signaling through the tolerance-associated receptors CTLA-4 (ipilimumab) and PD-1 (pembrolizumab and nivolumab), increasing the antitumor immune response. Despite a major improvement in remission rates, more than half of patients do not respond to immune checkpoint inhibitors2. The identification of molecular events leading to resistance to immune checkpoint inhibitors and approaches to overcome this resistance are crucially needed.
My data shows that at levels that maintain body weight, a methionine restricted diet dramatically reduces the number of metastases in an in vivo immunocompetent murine melanoma model3. This phenomenon may be associated with two mechanisms: methionine dependence and epigenetic perturbations. Methionine dependence describes the complete reliance of most cancer cells on exogenous sources of the essential amino acid methionine, contrarily to normal cells that can thrive off the remethylation of homocysteine4. The molecular bases for methionine dependence in cancer cells are not known. In addition to its role in protein synthesis, methionine is the direct precursor for S-adenosylmethionine, the universal methyl donor required for both DNA and histone methylation. These two epigenetic marks regulate gene expression, and undergo important changes during carcinogenesis. Inhibitors of DNA and histone methyltransferases increase the responsiveness to immune checkpoint blockers in preclinical and clinical studies5–8. In addition, the Human Protein Atlas (www.proteinatlas.org) reports seven methylation-related proteins associated with an unfavorable prognosis in melanoma. These are strong indications that methylation plays a key role in the progression and treatment of metastatic melanoma. The combination of restricted methyl group supply and immune checkpoint inhibitor responsiveness has never been tested.
According to my data and supporting scientific literature, the scientific premise for the proposed work is that the methylation status of the patient influences tumor metabolism and gene expression, in turn influencing immune checkpoint inhibitor responsiveness. My hypothesis is that restricting the availability of methyl groups provides a significant benefit when used as an adjuvant therapy for metastatic melanoma. To test this hypothesis, I propose to study the links between melanoma, methylation status, methyl donors, and responsiveness to immune checkpoints blockers.
Aim 1: Investigate the links between methylation status and response to immune checkpoint blockade in samples from patients with metastatic melanoma. I will use formalin-fixed paraffin-embedded (FFPE) samples for melanoma patients that present at the oncology clinic to correlate a common single nucleotide polymorphism in the rate-limiting enzyme MTHFR to outcome of therapy in patients with melanoma. I will also create MTHFR constructs to measure the impact of the variants on metabolite levels in the methylation cycle and on PD-L1 promoter methylation.
Aim 2: Identify the molecular bases of methionine dependence in cancer. In order to target the mechanism of methionine dependence in cancer, I will perform a proteomic screen on two human melanoma cell lines; the methionine independent MeWo and the methionine dependent A101D. In conjunction, I will analyze gene expression with RNA-Seq. Comparison under conditions of methionine stress is expected to uncover the metabolic pathway(s) underlying this phenomenon and provide therapeutic targets. Validation will use additional dependent and independent cell lines and patient samples.
Aim 3: Use a preclinical metastatic melanoma model to screen for methylation-altering interventions that potentiate response to immune checkpoint inhibitors. I will test the effect of dietary methionine restriction on tumor growth, DNA and histone methylation, and specifically on CTLA-4 and PD-1 expression and promoter methylation in a preclinical immunocompetent tumor model. As an alternative approach, I will also assess the administration of methionine-degrading methionine gamma-lyase enzyme.
Impact: My study investigates a novel approach to allow more metastatic melanoma patients to benefit from the life-saving effects of immunotherapy. I also will provide a clearer understanding of how methyl donor availability alters progression in this disease. The overarching goal of this project is to improve survival rates and quality of life in patients with metastatic melanoma.

1KL2TR003108-01

2019-08-01

2021-03-31