Kevin M. Haigis, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): My primary goal is to understand how mutational activation of K-RAS induces high grade dysplasia early during the malignant progression of colorectal cancer. My working hypothesis is that mutant K-RAS signals through the B-RAF kinase to suppress the differentiation of benign colonic tumor cells. The proposed studies include targeted investigations aimed at gaining mechanistic insight into known RAS effector pathways and exploratory studies aimed at identifying novel effectors of K-RAS signaling. I propose three specific aims: [unreadable] [unreadable] Aim 1. Analyze the phenotypic effects on the intestinal epithelium of activating K-ras, H-ras, or N-ras. [unreadable] Aim 2. Determine the relevance of candidate effector pathways to K-ras-induced tumor progression. [unreadable] Aim 3. Identify novel signaling effectors of mutationally activated K-ras. [unreadable] [unreadable] My studies will utilize both in vitro and in vivo experimental systems, namely human colorectal cancer cell lines and genetically engineered mice. Parallel studies conducted in these complementary systems will yield the greatest amount of insight into the oncogenic properties of K-RAS signaling. To successfully complete the Specific Aims, I will use small molecule inhibitors, genome-wide phosphoproteomic analysis, and emerging shRNA technology to dissect the signaling pathways that are required for oncogenic K-RAS to promote tumor progression. In the end, I am confident that therapeutic targeting of the RAS signaling pathway will represent a powerful means of fighting the malignant progression of colorectal cancer. [unreadable] [unreadable] Relevance to public health: In January, 2005 the American Cancer Society announced that cancer has replaced heart disease as the number one cause of death in the United States. Colorectal cancer alone accounts for more than 50,000 deaths per year in this country. Despite all that we have learned about the molecular pathogenesis of colorectal cancer, mortality due to this disease has remained constant over the past twenty years. Through my functional studies of the K-RAS oncogene, I aim to identify molecular targets for novel therapeutics that will eradicate colon cancer. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): It remains generally accepted that colorectal cancer (CRC) results from the accumulation of genetic events within the epithelial compartment. Unresolved issues include (1) whether the order of these genetic events matter (e.g., APC vs KRAS), (2) whether cancers can initiate from multiple distinct cells of origin, (3) the relationship between the normal colonic stem cell and the cells that function to maintain the tumor and (4) the contribution of the local environment to the neoplastic process. We hypothesize that colonic tumor initiation and progression is highly dependent upon the origin of the cell that incurs the relevant mutagenic events, as well as the regulation of growth and clonal selection of particular progeny that emanate from tumor-initiating cells. We have demonstrated that there are distinct normal colonic progenitor populations marked by expression of two specific cell surface markers, Lrig1 and Lgr5. Although the precise relationship between cells expressing these two markers is unknown, these two bona fide stem cell markers exhibit overlapping, yet distinct, expression patterns in both normal colon and in ApcMin/+ tumors. Of note, Lrig1 is induced by EGFR signaling and acts to negatively regulate EGFR signaling, whereas Lgr5 is a canonical Wnt target of unknown function. We have generated inducible Lrig1-CreERT2 mice, and our collaborator Hans Clevers has provided us with inducible Lgr5-EGFP-IRES-CreERT2 animals that allow for the specific activation of Cre recombinase within distinct colonic stem cell populations. We will use these mice to determine the importance of cell-of-origin in colon cancer initiation and progression. In addition, we have identified a gene, Slc26a3, that is selectively expressed in the differentiated compartment of the colon, and we have generated Slc26a3-CreERT2 mice that will allow us to resolve whether colonic tumors arise from the "top down" or "bottom up." PUBLIC HEALTH RELEVANCE: The cell in the colon which gives rise to colorectal cancer is unknown. We have generated novel genetically engineered mice that will allow us to answer this question. These results may allow us to determine whether colon cancer stem cells exist and their relationship to normal colonic stem cells, leading to identification of the colon cancer cell-of-origin and the colon cancer stem cell and offering exciting new therapeutic options.

? DESCRIPTION (provided by applicant): More than 200,000 Americans die as a result of lung and colorectal cancer each year. Decreasing the frequency of deaths due to these cancers will undoubtedly require tailoring an individual's treatment to the specific mutations that have occurred in their cancer. Activating mutations in the K-Ras oncoprotein are common in lung and colorectal cancers and are associated with particularly poor response to both conventional and targeted therapies. Our overarching goal is to understand the mechanisms underlying the oncogenic properties of mutant K-Ras in order to develop targeted therapeutic strategies. This project includes three phases. In the first phase of our project, we will use CRISPR technology to generate K-Ras wild-type derivatives of lung and colorectal cancer cells expressing endogenous mutant K-Ras. We will then comprehensively characterize the cellular and molecular phenotypes associated with loss of mutant K-Ras, for example by combining multiplexed mass spectrometry with computational modeling to identify the signal transduction network utilized by mutant K-Ras to transform cells. This study will also include an analysis of radiation response in wild-type and mutant cells, as activated K-Ras is known to confer resistance to ionizing radiation. In the second phase of our project, we will perform a variety of genome-wide and targeted screens for genes that when knocked down or over-expressed cause lethality in the context of mutant KRas in vitro and in vivo. These studies will utilize stat-of-the-art high-throughput screening technologies, including doxycycline-inducible shRNAs and open reading frames, that we have perfected over the past decade. In the final phase of the project, we will identify K-Ras synthetic lethals that are therapeutically targetable and then perform preclinical studies in genetically engineered mouse models of lung and colon cancer. The utilization of genetically controlled mouse and human experimental systems will allow us to identify gene products that are truly selectively required in cancer cells expressing mutant K-Ras. In the end, this work will have a major impact for patients who develop K-Ras mutant cancer.

Project Summary/Abstract The promise of precision medicine is that a physician can tailor a therapeutic regimen to suit each individual patient. In the case of cancer, this means a personalized therapeutic strategy based on the molecular features of an individual's cancer. But while successes in precision medicine have garnered significant attention in recent years, precision medicine has not made an impact for the vast majority of cancer patients. Our overarching goal is to use proteomics and systems biology to understand the relationships between cancer genotype and therapeutic response, with the long-term goal of expanding the prospects of precision medicine. Our study focuses primary on cancers expressing mutant forms of K-Ras, the most commonly mutated oncoprotein in cancer and one of the best biomarkers for the failure of a cancer to respond to therapy. Using a variety of experimental and computational approaches, this project will address three key questions related to K-Ras and the promise of precision medicine. First, we will exploit a relatively rare circumstance in which colorectal cancers expressing a specific mutant form of K-Ras are uniquely sensitive to inhibition of the MEK kinase. We will use mass spectrometry and computational modeling to determine why cancers expressing K-RasG12D and K-RasA146T are differentially sensitive to inhibition of MEK. Next, we will address the limitation of univariate genetic prediction of therapeutic efficacy by determining how genetic and epigenetic factors interact to establish network signaling state. We will use mass cytometry and computational modeling to explore how signaling downstream of mutant K-Ras is affected by cellular lineage and by secondary mutations in oncogenes and tumor suppressor genes. Finally, we will move beyond genotype as a predictor of therapeutic efficacy by developing an algorithm to predict sensitivity to kinase inhibition based on phospho-proteomic measurements. We will validate the computational approach via preclinical therapeutics studies in patient-derived xenografts. Altogether these studies will utilize state-of-the-art experimental and computational approaches to make personalized medicine a realistic goal for patients suffering from K-Ras mutant cancer.

Project Summary/Abstract Colorectal cancer (CRC) kills more than 50,000 Americans each year. Fluorouracil-based therapy remains the standard of care and there have been no targeted therapies approved for use in CRC in the past half decade. Mutational activation of the KRAS oncogene ? which occurs more than in 40% of cases ? is a major source of intrinsic and acquired resistance to both conventional and targeted therapies in CRC. Since there are no effective therapies that directly or indirectly target K-Ras or its downstream effector pathways, KRAS mutation is the single greatest barrier to medical treatment for CRC. Large scale sequencing of cancer genomes has revealed that, among those 40% of CRCs that express mutant K-Ras, the diversity of KRAS alleles is greater than in any other type of cancer. Epidemiological studies demonstrate that survival and response to therapy varies depending on the KRAS genotype of the patient's cancer, suggesting that different mutant forms of the K-Ras oncoprotein could exhibit distinct oncogenic properties. Experimental validation of allele-specific behaviors has never been achieved, however. We will use primary human and mouse organoids and genetically engineered mouse models to address three key questions relating to K-Ras oncogenicity: (1) Are different mutant forms of K-Ras equivalent in their ability to promote colorectal cancer initiation and progression? (2) Are genetic interactions between KRAS and other genes allele-specific? (3) How do mutant forms of K-Ras influence the tumor microenvironment in a non-cell- autonomous manner to promote cancer progression? The ultimate goal of this work is to decipher the ?KRAS Allele Code? in order to identify therapeutic strategies that are effective for cancers expressing specific K-Ras mutants. Precision medicine, where a physician tailors a patient's therapy to the genes that are mutated in his/her cancer, requires this level of understanding, especially for mutant oncoprotein that, like K-Ras, cannot be targeted with direct inhibitors.