Our lab and the EB iPS Consortium, composed of Stanford's Marius Wernig, Univ Colorado Dennis Roop, and Columbia's Angela Christiano, will work with the newly formed Stanford Center for Definitive and Curative Medicine and the Laboratory for Cell and Gene Medicine to develop a clinically robust and widely available treatment.  We hope our efforts will also facilitate other normal and genetically corrected tissue replacement therapies that the center aims to develop at Stanford.

Stanford cGMP Cell Therapy Facility opens September 2016

Epidermolysis bullosa patients suffer profound skin fragility, ineffective wound healing and persistent erosions leading to chronic wounding and longer-term complications of scarring and increased risk of malignancy. Traditional medical approaches have focused on either a surgical approach, where the diseased tissue is removed, or a medical approach, where a pill is provided to the patient.  EB and diseases like EB fall outside the realm of traditional medical approach and require a third type of approach that focuses on tissue regeneration through the correction of the diseased protein.  The Stanford EB Consortium represents a campus-wide collaboration of clinicians and scientists focused on developing this third tissue approach using novel molecular and cellular therapies to alleviate patient suffering. Accelerating this effort is the construction of a cGMP Cell Therapy Facilty on the Stanford campus through a joint effort of Stanford Hospital, Packard Hospital and Stanford medical school.  This facility will allow the development of novel cell-based treatments and teach a new generation of physicians and scientists about definitive genetic therapy.  

Highly Recombinogenic AAV for Genome Editing of Somatic and ES Cells

An explosion in the ability to edit the human genome, in conjunction with the identification of human tissue and embryonic stem cells allows for the first time in history the ability for “definitive genetic therapy” through the correction of genetic mutations in stem cells.  We investigated the efficiency of genome editing tools with an eye for scalability and cGMP compatibility.  We have explored conventional targeting, zinc finger nucleases, transcriptional activator-like effector nucleases (TALENs), and CRISPR/Cas9-based editing. In addition, in collaboration with Mark Kay at Stanford, we have found a novel adeno-associated viral AAV variant (AAV-DJ) that has high recombinogenic activity. AAV-DJ–mediated targeting (AT) has the advantage that AAVs have previously been used in human gene therapy trials, have low off-target rates, and are easily detectable. In our initial paper, we demonstrated the ability of AAV-DJ to directly correct the LamA3 mutation in Junctional Epidermolysis Bullosa patient keratinocytes. This provided proof-of-principle that direct correction, without the use of selectable markers, could be used clinically. Moreover, in a followup paper, we showed that an AAV-DJ variant could correct the COL7A1 locus in Dystrophic Epidermolysis bullosa induced –pleuripotent cells, demonstrating its usefulness in manufacturing protocols for the Therapeutic Reprogramming of skin. Because AAV-DJ–mediated homologous recombination rates were comparable to those of CRISPR/Cas9, and potentially safer and easier to deliver, we concluded that AAV-DJ–mediated genome editing would provide the ideal genome-editing system for correction during therapeutic iPSC manufacturing.

In the course of developing the Therapeutic Reprogramming protocol for skin, we developed a reliable in vitro differentiation protocol for skin. This has allowed us to take a intensive genomics and bioinformatic / network analysis approach to understand how human skin progresses through pluripotent, simple and stratified epithelium in response to developmental morphogen retinoic acid and BMP.  Our studies using cohesin Hi-ChIP technology to analyze chromatin conformation have shown that developmental morphogens work with major lineage selectors like p63 to properly fold local chromatin and connect key regulatory elements to their neighboring genes. 

Loss of Primary Cilia Drives Switching from Hedgehog to Ras/MAPK Pathway Dependence in Resistant BCCs

We previously observed epigenetic pathway switching from BCCs to a different subtype of skin cancer, SCC as a mechanism of tumor evolution and resistance. In this work, we identify the primary cilium as a critical determinant controlling tumor pathway switching. Smoothened inhibitor-resistant BCCs have an increased mutational load in ciliome genes, resulting in reduced primary cilia and HH pathway activation compared with naive or Gorlin syndrome patient BCCs. Gene set enrichment analysis of resistant BCCs with a low HH pathway signature showed increased Ras/MAPK pathway activation. Our results provide insights into BCC treatment and identify the primary cilium as an important lineage gatekeeper, preventing HH-to-Ras/MAPK pathway switching.

Cytoskeletal/Fibrotic signaling through SRF-MRTF confers Drug resistance 

Using multi-dimensional genomic analysis of animal models and our patients with drug-resistant BCCs, we have found that Serum Response Factor/Myocardin Related Transcription Factor (SRF/MRTF) activation is a common and powerful mechanism of drug resistance.  SRF/MRTF have previosly been associated with fibrosis and inflammation, and MRTF inhibitors significantly slowed the growth of drug-resistant basal cell carcinomas in mice and signaling in primary patient tumors, highlighting the therapeutic potential of MRTF-class of drugs.

Pathway Switching from BCC to SCC during tumor resistance

Our group found a novel mechanism that advanced BCCs frequently acquire resistance to Smoothened (SMO) inhibitors:  through tumor type / signaling pathway switching from BCCs to squamous cell carcinomas (SCC).  Both BCC and SCC derive from a common cancer stem cell, with hedgehog pathway driving BCCs and RAS/MAPK signaling driving SCC. We had previously observed SCCs arising out of the same location where a drug-sensitive BCC had been treated. Our group showed that the SCC was related by lineage to the BCC but had shut off the hedgehog pathway and turned on RAS/MAPK. This work provides the molecular underpinnings for tumor type pathway switching.  Please see related work from our Stanford colleagues Ransohoff et al. NEJM 2015 373:1079-82. 

Fitness of Smoothened Variants in Drug Resistant human BCCs

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.

Efficacy of First Hedgehog Pathway Inhibitor Targeting Smoothened

In this multicenter, international, two-cohort, nonrandomized study funded by Genentech, Stanford patients with metastatic basal-cell carcinoma or those with locally advanced basal cell carcinoma who had inoperable disease, received the first hedgehog pathway inhibitor. The study demonstrated that Vismodegib is effective in 30-45% of patients, and validated Smoothened as a therapeutic target.  In the same issue of the New England Journal of Medicine, we describe a novel patient in the trial with advanced BCC from a (7;Y) translocation driving Sonic Hedgehog in the skin.

Actin Polymerization  controls cilia-mediated signaling

Primary cilia are polarized organelles that allow detection of extracellular signals such as Hedgehog (Hh). How the cytoskeleton supporting the cilium generates and maintains a structure that finely tunes cellular response remains unclear. Here, we find that regulation of actin polymerization controls primary cilia and Hh signaling. Disrupting actin polymerization, or knockdown of N-WASp/Arp3, increases ciliation frequency, axoneme length, and Hh signaling. Cdc42, a potent actin regulator, recruits both atypical protein pinase C iota/lambda (aPKC) and Missing-in-Metastasis (MIM) to the basal body to maintain actin polymerization and restrict axoneme length. Transcriptome analysis implicates the Src pathway as a major aPKC effector. aPKC promotes whereas MIM antagonizes Src activity to maintain proper levels of primary cilia, actin polymerization, and Hh signaling. Hh pathway activation requires Smoothened-, Gli-, and Gli1-specific activation by aPKC. Surprisingly, longer axonemes can amplify Hh signaling, except when aPKC is disrupted, reinforcing the importance of the Cdc42–aPKC–Gli axis in actin-dependent regulation of primary cilia signaling.

Primary cilium and the regulation of hedgehog signaling

Intense focus has been centered around how the primary cilia transduces the hedgehog signal from smoothened to the Gli transcription factors. New data indicate that ligand and signaling lipids help regulate small GTPase-dependent accumulation and activity of signaling components. These component regulate vesicle transport, regulatory post-translational modifications, and signaling intensity.

Mtss1, a founding member of the I-BAR family of cytoskeletal regulators, prevents human metastasis

The actin cytoskeleton is essential for cell viability and plays critical roles in cell signaling, proliferation, motility, and survival. Normal physiological functions are associated with a diversity of actin cytoskeletal regulatory proteins and their functions. However, it is becoming clear that local, rather than global, actin cytoskeletal regulation plays critical roles in homeostatic signaling and cancer progression. A major challenge in cell biology is to understand key mechanisms of actin regulation and the range of functions that they control in order to develop therapeutic targets and better understand the clinical context in which to use them. One family of context-dependent cytoskeletal regulators is the I-BAR (Bin/amphiphysin/Rvs)  family of cytoskeletal regulators. Our lab focuses on the in vitro and in vivo functions of one of the founding members Missing-in- Metastasis (MIM/Mtss1) in homeostasis and neoplasia. Mtss1 has been identified as a central inhibitor of metastasis in cohorts of human melanoma, breast cancer, and urothelial cancers. Our lab studies how Mtss1 contributes to the metastatic phenotype and how we can develop novel therapeutics for patients with metastatic tumors.