Stem Cell Biology, Cancer Mechanisms, and Novel Cellular and Molecular Therapeutics
Fitness of Smoothened Variants in Drug Resistant Tumors
Advanced basal cell carcinomas (BCCs) frequently acquire resistance to Smoothened (SMO) inhibitors through unknown mechanisms, providing a unique opportunity to study human tumor evolution in patients through whole exome, RNA, and targeted sequencing. We find SMO mutations in 50% of resistant BCCs compared with 6% of untreated BCCs. Alterations both ligand binding pocket mutations that define sites of inhibitor binding constitutively active SMO mutants define pivotal residues of SMO that ensure receptor autoinhibition. Both classes of SMO variants respond to the aPKC-i/l inhibitor PSI and GLI2 antagonist ATO that operate downstream of SMO, setting the stage for the clinical use of GLI antagonists we are developing in our lab.
Highly Recombinogenic AAV for Genome Editing of Somatic and ES Cells
The efficient targeting of human somatic and ES cells by AAV-DJ together with the observed low frequency of off-target events, makes AAV-DJ-based homologous recombination a promising strategy for ex-vivo therapy for human genetic disorders.
Atypical Protein Kinase C in Gli Activation
Our studies demonstrate aPKC is critical for Hh-dependent processes, acts directly on the Gli transcription factor and implicates the kinase as a new, tumor-selective therapeutic target for the treatment of Smo-inhibitor resistant cancers.
Therapeutic Reprogramming for Epidermolysis Bullosa
Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. Our lab has developed a scalable and cGMP compatible protocol to generate patient-derived COL7A1 gene-corrected epithelial keratinocyte sheets for autologous grafting. iPSC-derived keratinocytes are produced with minimal heterogeneity and secreted wild-type type VII collagen, resulting in stratified epidermis in vitro and in vivo in mice. Sequencing of corrected cell lines prior to tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1 corrected banks with minimal mutational burden for downstream epidermis production.