Functional Genomic Interrogation of Disease Progression
We develop and apply next generation genomic tools to elucidate the molecular mechanisms behind disease progression. We are primarily interested in drug resistance, though we also focus some efforts on studying the unperturbed metastatic cascade. The lab is situated at the Cedars-Sinai Medical Center in sunny Los Angeles, California. We are always searching for bright and motivated members and collaborators, so if you like what you see here please feel free to contact us at any time.
Dr. Knott’s research program is largely focused on the application of functional tools that he developed during his postdoctoral work to design combinatorial therapeutics for metastatic breast cancer. Function studies aimed at identifying cancer targets are driven by the premise that oncogenic changes alter the dependencies of cells, making them vulnerable to the loss of driving oncogenes and to addictions that the transformed state creates. There are cases where this paradigm have proven successful. However, inevitability some patients fail to respond or, more commonly, initially respond but later acquire resistance to single-target therapies. The reasons for outright resistance are several-fold, with heterogeneity in cancer cell populations being chief among them. The mechanisms behind delayed and/or acquired resistance are more complex. Cellular pathways are dense, highly connected and adaptable. Following the exposure to cancer cells to a targeted therapy, the pathway involved is typically altered as anticipated. However, cells can overcome this initial therapeutic onslaught by taking advantage of pathway plasticity. To overcome these obstacles, it is necessary to turn towards combinatorial agents that target multiple pathways. My goal is to implement a research program that develops strategies for the discovery of multi-target metastatic cancer therapies. Below I describe three aims that I will focus on over the next 5 years in order to achieve these goals.
1. Develop clinically relevant models of human breast cancer metastasis. A key to understanding breast cancer metastasis is having available a set of clinically relevant models that collectively capture the molecular heterogeneity that exists across the spectrum of disease subtypes. Patient derived xenograph (PDX) models have emerged as a powerful technology, capable of maintaining the molecular characteristics of their founding tumors. I wish to apply viral barcoding to develop models of intra- and inter-tumor heterogeneity from currently available PDX models of metastatic breast cancer. We are in the process of optimizing the viral “tagging” method, to allow cells harboring specific barcodes to be isolated from heterogeneous populations. This capability will essentially transform any heterogeneous population into a virtual library of thousands of cell lines.
2. Identify therapeutics for use in combination with anti-angiogenics. The strategy of starving cancer cells by targeting the tumor neovasculature has failed to provide the broad therapeutic benefit that was initially anticipated. This is likely due to the existence of alternative, non-endothelial based mechanisms for tumor perfusion. Here, I aim to identify the drivers of these alternative pathways and to assess the utility of therapies that target all modes of perfusion in combination.
3. Identify translatable combinatorial targets. The goal of this aim is to perform a broad search for combinatorial targets that can be rapidly translated into therapeutics. For this reason, it focuses on genes for which a targeting agent exists (drugged-genes) or for which, based on protein structure and localization, an agent is theoretically feasible (druggable-genes). Initially, this aim will focus on identifying co-targets for the drugged-genes with the most robust clinical data supporting their relevance to disease progression. In a second arm, I will develop a screening method, based on CRISPR/Cas9 technology, for the ab initio identification of synergistic drug targets.
AREAS OF EXPERTISE:
During his postdoctoral work, Simon designed molecular tools for interrogating gene function in mammalian cells. These were the basis for the biotechnology company TransOmic Technologies, which has commercially distributed these reagents to over 300 academic and industrial labs worldwide. In addition, he developed strategies to study tumor heterogeneity and its impact on disease progression. By combining these resources he was able to identify a key regulator of breast cancer metastasis. This target is now being developed in collaboration with the pharmaceutical company AstraZeneca, with the goal of identifying a small molecule inhibitor that can move into pre-clinical trials in the next year.
WHERE WE ARE NOW
In August 2016, our lab (The Knott lab) was formed at Cedars-Sinai Medical Center which is located in Los Angeles, California. Here, we are implementing a platform for the development of combination therapies that target both malignant and nonmalignant tumor cells to combat drug-resistance and metastasis.
The “War on Cancer” was founded upon the premise that an understanding of cancer biology would lead to effective cancer treatments. A modern reinterpretation of this philosophy is embodied by The Cancer Genome Atlas, which is predicated on the notion that the discovery of oncogenic drivers will afford therapeutic opportunities that can be both tumor specific and rationally guided. Drugs such as Gleevec, Tarceva and Erlotinib are cited as examples of the benefit gained from inhibiting driver oncogenes. While it is true that these drugs have minimal side-effects and can produce stunning responses, their impact is invariably short-lived. Due to their plasticity and anti-apoptotic tendencies, tumor cells are able to take advantage of parallel or related molecular pathways to evade single-target therapies, resulting in drug resistance and patient relapse. Thus, while the underlying philosophy of targeting cancer cell addictions is valid, we must clearly move away from a single target view and toward a paradigm in which we consider entire molecular networks.
It has also been argued that curative responses will only be achieved by simultaneously targeting malignant and non-malignant cells in the tumor, to eliminate interactions that drive resistance. For example, we have recently identified vascular mimicry as a driver of vasculature intravasation. Here, tumor cells form extravascular networks that deliver nutrients to cells, while simultaneously providing a route for their escape to the blood. This alternative mode of perfusion is a likely reason why the anti-angiogenic drug Avastin, which targets the endothelial cells of the tumor neovasculature, has failed to provide broad therapeutic benefit.
The goal of the Knott Lab is to cultivate a research program that develops multi-target cancer therapies that focus on interacting pathways in malignant and non-malignant tumor cells. This work entails developing models of resistance to established anti angiogenic inhibitors and immune-therapies. This work also involves applying novel computational tools to interpret the output from these animal studies, as well as relevant patient data. The ultimate goal of this research is to identify and characterize novel targets that can be moved into the drug-development pipeline.
Simon Knott, PhD. - Assistant Professor - Center for Bioinformatics and Functional Genomics at Cedar-Sinai Medical Center (firstname.lastname@example.org)
I combine computational biology and functional genomics to elucidate the molecular mechanisms that drive cancer progression and drug resistance. My multidisciplinary approach to science began during my second year as a PhD student in the Computational Biology PhD program at the University of Southern California (USC). After successfully completing the computational qualifying exam, I arranged to be co-supervised by a molecular and a computational biologist (Drs. Oscar Aparico and Simon Tavaré, respectively). For the remainder of my PhD I performed “wet lab” experiments in the Aparicio lab and developed algorithms to analyze the resultant data with Dr. Tavaré. This relationship resulted in several high-profile publications in the area of DNA replication.
After graduating, I joined Dr. Gregory Hannon’s lab at Cold Spring Harbor Laboratory (CSHL) for my postdoctoral work. My initial focus at CSHL was to elucidate the sequence determinants of short hairpin RNA (shRNA) efficacy and to develop a computational algorithm for predicting strong shRNAs for any target gene. After developing the algorithm, I lead a team of MSc students and technicians through a set of large-scale functional screens aimed at validating its predictive capacity. This work lead to both a high-profile publication and the commercialization of human and mouse shRNA libraries designed with the algorithm. Since the design of these libraries others have harnessed the prokaryotic CRISPR/Cas9 system for functional studies in mammalian cells. I have recently developed algorithms and vectors that together maximize the efficacy of CRISPR targeting molecules. I have also lead second team of PhD students and laboratory technicians through a set of experiments that demonstrate the superior efficacy of reagents designed with these principals.
While supervising these studies, I also became interested in tumor heterogeneity and metastasis, and I developed a method to “barcode” individual cells within complex populations so that they could be tracked throughout the various stages of disease progression. In an initial study, I lead a team of PhD and MSc students as well as technicians to apply this method to study tumor cell intravastation into the vascular. I have subsequently led a team of similar make-up through a set of in vivo shRNA screens to elucidate drivers of tumor cell extravasation from the vasculature. All together, my experience with these technologies makes me a suitable collaborator for a project that involves multiplexed CRISPR screening technologies.
Dustin Rollins - Research Associate I - Center for Bioinformatics and Functional Genomics at Cedar-Sinai Medical Center (Dustin.Rollins@cshs.org)
Originally from northern California, Dustin received his Bachelor of Science in Biological Sciences from California State University Chico. He went on to earn his Master of Science in Cancer Biology from Drexel University College of Medicine where he worked with Dr. Edna Cukierman at the Fox Chase Cancer Center. His research background is based in cellular and molecular biology, patient derived tissue culture and 3-Dimensional cell culture techniques. Dustin joined the Knott-Lab in early January 2017 .
Florian Raths - Graduate Student - Center for Bioinformatics and Functional Genomics at Cedar-Sinai Medical Center (Florian.Raths@cshs.org)
Florian was born in Germany and holds a B.Sc in Biology from the University of Marburg and a M.Sc. in Biotechnology from the University of Münster. He gained experience at the Max Planck Institute for Terrestrial Microbiology (Marburg), the Karolinska Institutet (Stockholm) and the Institute for Plant Biology and Biotechnology (Münster). He worked on projects related to molecular genetics, polymer science, microbiology and plant-pathogen systems.
Florian joined the Knott-Lab in Oct. 2016
- Knott SRV, Erard N, Khan S, Hannon GJ. An optimized CRISPR/Cas9 library for functional screens in mammalian cells. In preparation.
- Knott SRV, Wagenblast E, Kim SY, Soto M, Khan S, Gable AL, Maceli AR, Dickopf S, Erard N, Harrell C, Perou CM, Wilkinson JE, Hannon GJ. Asparagine availability governs metastasis in a model of breast cancer. In review.
- Wagenblast E, Soto M, Gutiérrez-Ángel S, Hartl CA, Gable AL, Maceli AR, Erard N, Williams AM, Kim SY, Dickopf S, Harrell CJ, Smith AD, Perou CM, Wilkinson JE, Hannon GJ & Knott SRV. A model of breast cancer heterogeneity reveals vascular mimicry as a driver of metastasis. Nature. 2015, 520:358-362.
- Knott SRV, Maceli A, Erard N, Chang K, Marran K, Zhou X, Gordon A, El Demerdash O, Wagenblast E, Kim S, Fellmann C, Hannon GJ. A computational algorithm to predict shRNA potency. Mol Cell. 2014, 56(6):796-807.
- Knott SRV, Peace JM, Ostrow AZ, Gan Y, Rex AE, Viggiani CJ, Tavaré S, Aparicio OM. Forkhead transcription factors establish origin timing and long-range clustering in S. cerevisiae. Cell. 2012, 148(1-2):99-111.
- Knott SRV, Viggiani CV, Aparicio OM, Tavaré S. Strategies for analyzing highly enriched IP-chip datasets. BMC Bioinformatics. 2009a, 10:305.
- Knott SRV, Viggiani CV, Tavaré S, Aparicio OM. Genome-wide replication profiles indicate an expansive role for Rpd3L in regulating replication initiation timing or efficiency, and reveal genomic loci of Rpd3 function in Saccharomyces cerevisiae. Genes Dev. 2009b, 23(9):1077-1090.