Genetic dissection of the 11q13 amplicon in squamous cell carcinoma

Abstract

Loss of normal keratinocyte function is the cause of a significant number of epidermal or oral diseases and conditions, as detailed in Chapter 1. Development of representative genetic models is crucial to gain a better understanding of these disorders, such as epidermolysis bullosa and squamous cell carcinoma. Additionally, engineered keratinocyte-based cell therapies are a promising option to treat a variety of dermal conditions, such as burn wounds. However, the lack of efficient genome engineering strategies currently limits the generation of engineered keratinocytes for such purposes. In Chapter 2 we present a novel electroporation-based cell engineering workflow for efficient transfection of primary keratinocytes with CRISPR RNPs and demonstrate its use in both cancer modeling and the development of novel allogeneic cell therapies. Our main results include: 1. Delivery of mRNA and CRISPR-RNPs to primary adult keratinocytes derived from four distinct anatomical sites with efficiencies between 97% and 100%, without compromising cell viability or cell morphology 2. Genome editing efficiencies upon CRISPR RNP transfection in primary adult keratinocytes between 85% and 100%, in a fast, robust, scalable, and GMP- compatible manner 3. A proof of concept for the generation of hypoimmune universal donor primary adult keratinocytes for the use in cell therapies Overall, we present a highly efficient and fast workflow for the generation of engineered keratinocytes, facilitating future developments for disease modeling and the field of keratinocyte-based cell therapies.

Over 25% of Head and Neck Squamous Cell Carcinomas (HNSCC) display amplification of genes in the 11q13 region, yet it is unclear which genetic elements of the amplicon are the key driver events in these tumors. In Chapter 3 we describe a comparative analysis on the contribution of each 11q13 gene to HNSCC tumorigenesis and the identification of three critical drivers of the amplicon. In Chapter 4 and Chapter 5, we further analyze the mechanism behind these critical drivers and explore potential therapeutic strategies. Our main findings include: 1. CCND1, ORAOV1, and MIR548K are the critical drivers of the 11q13 amplicon in HNSCC. These genes have distinct effects on tumorigenesis. 2. MIR548K contributes to the epithelial-mesenchymal transition. 3. Primary keratinocytes are exclusively dependent on CCND2, whereas CCND1 amplification induces cyclin D1 oncogene addiction in cancer cells. 4. CCND1 amplification drives the cell cycle in a CDK4/6/RB1-independent fashion. 5. CCND1 amplification induces RRM2 expression, conferring a dependency on RRM2. 6. ORAOV1 is a potent oncogene that is capable of initiating in vivo tumor formation at a level similar to CCND1. 7. ORAOV1 regulates reactive oxygen species through activation of the thioredoxin pathway. Thus, 11q13 amplification drives tumorigenesis through a combination of at least three independent oncogenic events. Through better understanding of the individual contributions, we find dependencies on these genes and their downstream mechanisms that are unique to the cancer cells. Exploiting these weaknesses could be a novel therapeutic approach for 11q13-amplified HNSCC.