PROJECT 2 ABSTRACT Melanoma treatment has had instrumental advances in two modern modalities: immunotherapy and targeted kinase inhibitor treatments. Nevertheless, there is a crucial clinical need to address dedifferentiation as a resistance mechanism to modern melanoma therapies. Notably, dedifferentiation is a documented cross- resistance mechanism that impacts both of the new expanding standards of care: BRAF and MEK kinase inhibitor therapies, and immunotherapies such as immune checkpoint blockade (e.g. anti-PD-1/PD-L1). Our discovery of a new dedifferentiation-associated sensitivity to iron-dependent oxidative stress (ferroptosis), provides a new angle from which to complement current standard of care therapies for melanoma. In our studies, sensitivity to ferroptosis is an Achilles heel for dedifferentiated cells independent of whether they were dedifferentiated at baseline, or induced to dedifferentiate by BRAF and MEK inhibitor therapy or by immunotherapy associated cytokines. Thus, using preclinical models, we will further pursue this orthogonal sensitivity to prevent the dedifferentiation escape route. Our discovery and proposed experiments brings the programmed cell death process of ferroptosis into the field of melanoma and melanocytes. We will investigate how the redox stress protection mechanisms specific to redox-regulated melanin production in melanocytes interact with iron metabolism, ferritinophagy and ferroptosis in melanoma cells. Studies in the melanoma and melanocyte context will help advance our knowledge of this relatively recently discovered cell death mechanism. We will test the efficacy of combining ferroptosis induction with either kinase inhibitor therapy, or checkpoint inhibitor immunotherapy using preclinical models. We will interrogate human samples from clinical trials to assess the immunotherapy contexts in which dedifferentiation presents the highest clinical challenge and opportunity. Our discovery of dedifferentiation-linked ferroptosis sensitivity was the result of an integrative genomics and pharmacogenomics approach. We will continue to expand our framework of the regulation of melanoma dedifferentiation using integrative genomics to delineate transcription and epigenetic regulatory programs. To expand upon the multiple links between metabolism, redox buffering, differentiation, and ferroptosis, we will incorporate metabolomics and lipidomics in our discovery and mechanistic delineation approaches. Our goal in these aims is to mechanistically understand and enhance the therapeutic approach of targeting the melanoma dedifferentiation resistance escape route.