NFl LOSS IS A FUNCTIONAL GENOMIC EVENT IN MELANOMA by Moriah Heller Nissan A Dissertation Presented to the Faculty of the Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy New York, NY December, 2013 ]2~<;~ David B. Solit, MD Date Dissertation Mentor Copyright Moriah Heller Nissan 2013 Dedication “No man can reveal to you aught but that which already lies half asleep in the dawning of your knowledge. The teacher who walks in the shadow of the temple, among his followers, gives not of his wisdom but rather of his faith and his lovingness. If he is indeed wise he does not bid you enter the house of his wisdom, but rather leads you to the threshold of your own mind. The astronomer may speak to you of his understanding of space, but he cannot give you his understanding. The musician may sing to you of the rhythm which is in all space, but he cannot give you the ear which arrests the rhythm nor the voice that echoes it. And he who is versed in the science of numbers can tell of the regions of weight and measure, but he cannot conduct you thither. For the vision of one man lends not its wings to another man. And even as each one of you stands alone in God's knowledge, so must each one of you be alone in his knowledge of God and in his understanding of the earth.” -Kahlil Gibran, The Prophet For all of my teachers, in gratitude for their unfathomable patience. For my parents: my first teachers. For my husband, whose patience conveying the rhythm of space onto occasionally deaf ears deserves distinct laud and recognition. iii Abstract Melanoma is a disease characterized by lesions that activate ERK. Though 70% of cutaneous melanomas harbor activating mutations in the BRAF and NRAS genes, the alterations that drive tumor progression in the remaining 30% are largely undefined. Vemurafenib, a selective inhibitor of RAF kinases, has clinical utility restricted to BRAF mutant tumors. MEK inhibitors, which have shown clinical activity in NRAS mutant melanoma, may be effective in other ERK pathway-dependent settings. We investigated a panel of melanoma cell lines wild-type for BRAF and NRAS to determine the genetic alteration driving their transformation and their dependence on ERK signaling in order to elucidate a candidate set for MEK inhibitor treatment. From 191 melanoma cell lines, we collected a set of 66 BRAFWT/NRASWT cell lines for our study. We screened these cell lines for functional alterations that activated RAS, and found a subset of cell lines with elevated RAS-GTP. NF1 negatively regulates RAS and is found to be somatically altered in a growing number of cancers. We examined the NF1 status of these cell lines and identified a cohort of 6 cell lines with high RAS-GTP and loss of NF1 protein expression. Deep sequencing via the IMPACT assay revealed a genomic mechanism for NF1 loss in all of the NF1- null cell lines. Data from the melanoma Cancer Genome Atlas (TCGA) revealed alteration of NF1 via missense mutation, nonsense mutation or deletion in 14% of melanoma tumors, confirming that NF1 loss occurs in human melanomas. In contrast to prior studies in other tissue contexts in which NF1-null cells were shown to be mTOR dependent, we find that NF1-null melanoma cell lines are not dependent on TORC1 but rather on the MAPK pathway for proliferation and cell cycle progression. Inhibition of ERK signaling by some MEK inhibitors was short lived in NF1-null melanoma cell lines due to loss of negative feedback and reactivation of pERK. Enhanced antitumor effects were observed with trametinib, a compound that blocked phosphorylation of MEK by RAF and thus prevented pathway reactivation. NF1 loss in the context of BRAF(V600E) mutation was sufficient to raise RAS-GTP levels and confer resistance iv to the RAF inhibitor vemurafenib. However, these cells remained sensitive to the MEK inhibitor trametinib, suggesting a potential therapeutic strategy for patients with NF1 loss and BRAF(V600E) mutation. We questioned whether NF1 loss was sufficient to induce melanoma formation, and modeled somatic NF1 loss in melanocytes using a Nf1flox/flox; Tyr::CreER mouse model. Nf1 loss as a single alteration was not sufficient for melanoma formation, but caused hyperpigmentation and mild hyperproliferation of melanocytes. In summary, NF1 loss occurs in a subset of cutaneous melanomas and results in RAS activation, MEK dependence, and RAF inhibitor resistance. v Vitae Moriah Nissan (née Heller) was raised in Andover, Massachusetts. She attended the University of Rochester in Rochester, NY with a Bausch and Lomb Honorary Science Scholarship from 2004- 2008. There, she was active in the Department of Chemistry as a general chemistry workshop leader under the mentorship of Professor Todd Krauss, and the Department of Biochemistry and Biophysics as a laboratory teaching assistant under the mentorship of Dr. Harold Smith. She joined the laboratory of Dr. Joseph Wedekind in the Department of Biochemistry and Biophysics in 2006 and worked for two years alongside Robert Spitale, a graduate student studying the structure and function of the hairpin ribozyme. Her project was synthesizing modified nucleotides for incorporation into the active site of the hairpin ribozyme to study the cleavage mechanism of this class of ribozymes. Her work yielded two publications with Spitale and Wedekind. Moriah graduated from the University of Rochester in 2008 magna cum laude with a BS in biochemistry. Upon graduation in 2008, Moriah matriculated at the Gerstner Sloan-Kettering Graduate School. There she joined the laboratory of Dr. David Solit in the Human Oncology and Pathogenesis Program to study occult MAPK pathway mutations in melanoma. In addition to this project, Moriah spearheaded an effort to genotype, document and organize a melanoma cell line and matched tumor database with the Wolchok laboratory in order to facilitate use of MSKCC- derived melanoma cell lines. Moriah also worked under the clinical mentorship of Dr. Paul Chapman, initiating an IRB-approved clinical survey of neurofibromatosis patients with melanoma in order to further understand the relationship between the two diseases. She first- authored a review article, a commentary, and a book chapter with Dr. Solit in addition to the first- author peer reviewed article she submitted prior to her defense. Additionally, Moriah presented her work at the 2013 AACR-NCI-EORTC Molecular Targets and Cancer Therapeutics vi Conference where she was the recipient of a Scholar-in-Training award for “outstanding abstract”. In addition to her laboratory work, Moriah spent three years actively involved in recruitment for the Gerstner Sloan-Kettering Graduate School, traveling to the 2009, 2010 and 2011 SACNAS and 2011 ABRCMS conferences to meet with potential applicants to the school. She was also co- founder of the GSK Women in Science (GWIS) group, and remained actively involved in the group until her graduation. vii Acknowledgements In kindergarten my parents brought me to Plimouth Plantation, which featured actors dedicated to reenacting life in the year 1627 in the famous harbor-side village. Their commitment to the historical accuracy of the time was, to say the least, convincing. At school the following day I told my teacher I had met the pilgrims, to which she informed me that, actually, the pilgrims are dead. Naturally, what she said conflicted with my experiences and beliefs, so, naturally, I informed her that she was wrong. Since then, I have come to interpret my stubbornness more as a form of inertia (which, I will have you know, is a property of ALL matter. It’s not just me). It is for this reason, among many others, that I am so grateful for the mentorship provided me by David Solit. David spent 5 years nurturing my abilities and interests whilst putting up with my “inertia” on a whole range of topics. In addition to his unwavering patience, he has been a wonderful role model to me in his responsibilities as both scientist and parent. Some of my favorite meetings involved him invoking analogies between lab work and Harry Potter, or between the peer-review process and Legally Blonde. Football analogies, I am sorry to say, were lost on me. However, I will take away from my experience the balance between “getting it done” and “needing to finish early so I can pick up the kids from [insert activity]”. David is a living example that success can come to people who work hard but who have lives outside of their careers. For this, I will be eternally grateful. I have also been extremely fortunate to have the co-mentorship of Neal Rosen. Neal has given me wonderful guidance, input, and, most importantly, criticism over the years; I would worry if Neal didn’t have something critical to say, as it meant either that he wasn’t paying attention or that he didn’t care. I am grateful that the criticism was constructive and frequent. He has made me a better scientist, and continues to do so. I also had the pleasure of having the clinical mentorship of Paul Chapman. Dr. Chapman met with me on a monthly basis to discuss the state of the clinic and the current clinical trials in addition to reviewing data for my project, viii and I am extremely appreciative for the time and effort he put into this mentorship. My project was also the result of an outstanding and productive collaboration with Taha Merghoub and the Wolchok lab. Taha was my animal mentor, and helped me plan, execute and analyze my mouse experiments.