Functional Genomics Initiative Research Awards fund projects that address functional and mechanistic implications of genetic information.
Effects of cancer-associated SOX17 mutations on cell lineage specification and tissue morphogenesis
Description: SOX17, an evolutionarily conserved transcription factor involved in the development of embryos, has also been identified as a tumor suppressor. It is mutated in cancers of internal organs, including endometrial cancer, colon cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, and lung cancer. Using a combination of sophisticated genetic manipulations and state-of-the-art single-cell genomics approaches, combined with functional studies in vivo in embryos and in organoids, we will investigate the function of tumor-associated mutations in SOX17.
Precise modeling of cancer-associated single nucleotide variants using CRISPR base editing
Description: Cancer arises as a consequence of mutations in genes that promote cell proliferation, survival, and metastasis. DNA-sequencing efforts have cataloged the mutations that can occur in individual human cancers, and indeed, most people treated at Memorial Sloan Kettering are now tested for the gene mutations that are present in their tumors. These cataloging efforts have identified hundreds of genes that can be altered in human tumors, often through different types of mutations in the same gene and in a range of different gene combinations. These efforts have helped better classify tumors and, in some cases, suggested therapeutic strategies that can be tailored to individuals with cancer. Still, to achieve the full benefit of this information, it is important to understand the functional consequence of specific mutations, such as how each alteration alters normal cell physiology and how mutant gene combinations produce distinct cancer behaviors. Our project aims to develop a suite of tools that will facilitate engineering the range of cancer-associated mutations observed in MSK’s patients for investigators to study. Eventually, we plan to develop strategies to target these mutations. This approach takes advantage of new genome-editing technology using CRISPR that has been optimized by the team to efficiently produce cancer-associated mutations in cultured cells and animal models. To illustrate the potential use of these tools, we will generate and study a series of mutations in the p53 tumor suppressor, which is the most frequently mutated gene observed in human tumors. These efforts will produce tools that will enable functional cancer research throughout the MSK community and will yield biological insights into a fundamentally important cancer gene.
Deciphering the role of ADAM proteases in Notch and EGFR pathways using the genetic data from the CMO and TCGA
Description: ADAM metalloproteases regulate normal and tumorigenic signaling by cleaving cell-surface substrates, including Notch, EGFR, and their ligands. Our proposed studies are aimed at understanding the molecular mechanisms of ADAM protease regulation and specifically the role of conformational ADAM ectodomain rearrangements in substrate recognition and cleavage. We will utilize the large amount of available CMO sequencing data from patients’ tumors with mutations in the substrate-binding domains of ADAM10 and ADAM17. We will study how ADAMs switch between “opened” and “closed” conformations and how this affects the downstream Notch and EGFR signaling.
The roles of BLM and FANCM homologs in attenuating replication stress
(Co-PI: Xiaolan Zhao)
Description: Mutations in the human FANCM and BLM DNA helicases underlie severe cancer predisposition syndromes. Consistent with their role in protecting the genome from cancer-causing mutations, previous studies have demonstrated an evolutionarily conserved role for FANCM and BLM helicases in the repair of DNA lesions. Here, exploiting the genetic and biochemical tractability of their budding yeast orthologues, we will investigate a recently emerged novel role for these helicases in promoting DNA replication during replication stress. This condition is often observed in cancer cells and is characterized by the impairment of replication forks. These studies will enhance our understanding of the mechanisms that link genome instability and cancer.
Immunomodulatory function of E-cadherin in cancer
Description: With immunotherapy being actively adopted for cancer care, a better understanding of tumor-elicited immune responses and tumor immune escape mechanisms is crucial for the development of new cancer treatments and for stratifying people for effective therapy. Our recent studies have revealed a novel tumor-resident cytotoxic lymphocyte response that’s broadly induced in human and murine malignancies. The current proposal will investigate how tumor-expressed E-cadherin supports the cancer immunosurveillance response and whether the E-cadherin mutations seen in patients’ tumors promote tumor immune escape.
Integrated approaches annotate functions of cancer-associated H3K36 methyltransferases
(Co-PI: John Chodera)
Description: Among frequent cancer mutations on the MSK-IMPACT™ list are those associated with H3K36 protein methyltransferases (PMTs). This project will implement the integrated computational-experimental approach to elucidate the molecular mechanisms through which point mutations of H3K36 PMTs impact downstream functions. Our work will be essential to annotate gain/loss-of-function variants for PMTs.
Defining the oncogenic impact of ATP6AP1- and ATP6AP2-inactivating somatic mutations in granular cell tumors
(Co-PIs: Britta Weigelt and Fresia Pareja)
Description: Granular cell tumors are rare lesions composed of cells that contain numerous granules whose nature is unknown. We have sequenced these tumors and found that the vast majority of them have mutations in the genes ATP6AP1 or ATP6AP2, which render them inactive. These genes, albeit not previously linked to cancer, play pivotal roles in the trafficking of materials inside cells. Our preliminary analyses have shown that inactivation of ATP6AP1 or ATP6AP2 in normal cells grown in petri dishes results in the formation of granules similar to those found in granular cell tumors. In this project, we will be seeking to develop models to study granular cell tumors and to understand how mutations in ATP6AP1 and ATP6AP2 result in the development of these tumors and in the production of the characteristic granules.
The determinants of immunogenicity of mutationally derived neoantigens of human cancers
(Co-PI: Andrea Schietinger)
Description: There are hundreds to thousands of genetic mutations detected in cancers, yet few of them can be treated with existing drugs. In spite of this, tumors with the highest number of mutations respond best to immunotherapies, even though we generally do not know which of the mutations are the target of the immune system. In this proposal, we seek to discover the rules for predicting which tumor mutations are immunogenic. If successful, we can apply these rules to the mutations found by the Marie-Josée and Henry R. Kravis Center for Molecular Oncology (CMO). We have developed a high-throughput, pooled-screening method (called PresentER) to discover which major histocompatibility complex (MHC)-presented protein sequences can be effectively recognized by human killer T cells and possibly cause tumor rejection. Longer term, this method may allow the CMO to vastly increase the number of therapeutically actionable mutations that are discovered, as well as to design new immunotherapies based on vaccines, engineered T cells, and T cell receptor mimic antibodies.
Functional requirement for DSS1 and the DBD domain of BRCA2 in maintenance of genome integrity
Description: The tumor suppressor gene BRCA2 is known to be mutated in families with a history of breast and ovarian cancer. However, more recently BRCA2 has been found to be mutated in prostate and other tumors in individuals with no family history of the mutations. This grant focuses on understanding the role of the DNA-binding domain of the large BRCA2 protein to clarify its role in tumor suppression, as well as its sensitivity to therapy and acquired resistance.
Understanding and exploiting replication stress in cohesin-deficient cancers
Description: Our project investigates the impact of patient-derived mutations in cohesin, a ring-shaped complex that traps and tethers pairs of DNA molecules. While most famously involved in mitotic cell division, cohesin is now appreciated to have a direct role in DNA replication itself, mainly through work from our laboratory. In this project, we take advantage of our new discovery that two different cohesin subunits (STAG2 and PDS5B) control the speed and stability of replication forks (sites of DNA synthesis) and also affect cellular sensitivity to drugs that induce replication stress, a state in which replication fork integrity is partially compromised. This situation is commonly seen in cancer. Our studies will illuminate the link between cancer-derived cohesin mutations and replication stress, possibly suggesting routes for selectively targeting such cancers therapeutically.
The role of the oncoprotein TRIM37 in the surveillance of S phase and mitosis
Description: Our preliminary studies found that two genes involved in promoting cell fitness are frequently amplified in human tumors, as indicated in the Center for Molecular Oncology’s data. This novel surveillance pathway is likely essential for the long-term fitness and robustness of cancer cells. Our proposal aims to understand the molecular mechanism by which cells maintain their efficiency and fitness in both normal and disease conditions.
ATM as a molecular rheostat during class switch recombination
Description: DNA breaks, especially DNA double strand breaks, are one of the most toxic lesions that can occur in a cell. Double strand breaks occur in a cell following genotoxic stress such as exposure to ionizing radiation or as mistakes during replication. If unrepaired, double strand breaks either lead to cell death or participate in aberrant DNA transactions leading to cancer. Despite the toxicity, for development of a robust immune system, B cells go through a process called class switch recombination in which DNA double strand breaks are deliberately introduced into the genome. A failure to generate such breaks lead to immunodeficiency syndromes while impaired repair leads to B cell lymphomas. Thus, mechanisms that regulate class switch recombination is highly relevant to both immunity and preservation of genomic integrity. We have discovered that a DNA repair protein, ATM, participates in both the generation and the repair of DNA breaks during class switching. However, the molecular mechanism by which ATM executes this dual function is completely unknown. This proposal uses data available from the MSKCC Functional Genomic database to elucidate the role of ATM in promoting immunity through the precise and controlled generation of DNA breaks and maintaining genomic integrity during this otherwise dangerous process. Successful completion of the project will illuminate novel pathways that will have major impact in our understanding of the ontogeny and intervention of B cell lymphomagenesis.
Quantifying and Correlating Tissue Pathology with the MSK-IMPACT Genotype
Computational Biology Program
(Co-PI Hikmat Al-Ahmadie, Nikolaus Schultz, Sahussapont Joseph Sirintrapun)
Description: The histological slides of samples that have undergone MSK-IMPACT testing are a prime example of “dark data” — stored, but unleveraged. Tapping into this terabyte-scale resource will provide unparalleled possibilities for novel, quantitative research for MSK scientists and clinicians. We will apply and develop machine learning algorithms based on ensemble classifiers and deep learning to statistically describe and quantify tumor morphology from image data. This allows us to automatically correlate the resulting phenotype descriptors with genes or alleles from MSK-IMPACT. With this framework we can not only automatically screen for correlations but answer questions like: “Can we predict a genotype from a specific tissue morphology?”
Dysregulation of the Nutrient-Sensing Pathway in Cancer
Description: Uncontrolled cell growth is the most fundamental feature of cancer. In this proposal, we will define whether and how tumor cells acquire growth advantage by mutating key molecules involved in nutrient sensing. These studies can help to stratify cancer patients for treatment with drugs targeting the nutrient signaling pathway.
Delineating the impact of 12-lipoxygenase deletion on epithelial maintenance, inflammation and transformation in live zebrafish
Description: 12-lipoxygenase is a scarcely studied member of an enzyme cascade that produces powerful bioactive lipids involved in inflammation, epithelial barrier maintenance, and tumorigenesis. Cancer genome database analysis (cbioportal.org) shows that 12-lipoxygenase is absent in various tumors due to a chromosome deletion. Using genomic and live imaging approaches in zebrafish, we will test causal connections between 12-lipoxygenase deletion, loss of epithelial barrier integrity, epithelial inflammation, and tumorigenesis to identify potential tumor suppressive functions for this enzyme.
The mechanism of eukaryotic initiation factor 4E (eIF4E) mediated translational control
Description: Eukaryotic initiation factor 4E (eIF4E) overexpression is oncogenic and it is commonly identified in cancers including several studies in CBioPortal. Interestingly, despite its characterization as a general translation initiation factor, eIF4E overexpression was found to selectively stimulate the translation of a subset of mRNAs that are essential for tumorigenesis. In this proposal, we will use a novel approach that combines our newly developed single-molecule translation assay with genomic RNA sequencing to better understand the molecular mechanism of eIF4E-mediated translational control of gene expression.
Understanding and Targeting Mutations in MEK1/2 in Cancer
Description: Genetic alterations which result in activation of the “Mitogen Activate Protein Kinase” (“MAPK”) pathway are amongst the most common alterations in cancer. Normally, the MAPK pathway communicates signals from receptors on the surface of a cell to the DNA in the nucleus of the cell. In cancer, however, this pathway is often altered in a manner which results in hyperactivation of this pathway. The MAPK is also sometimes referred to as the “Ras-Raf-MEK-ERK pathway” after the names of each of the proteins in the chain of proteins that communicate signals in this pathway. While activating mutations in the first 2 members of this chain (RAS and RAF) are very well studied in cancer, mutations affecting the 3rd member of this chain, MEK, are far less understood. This is in part due to the fact that, until very recently, MEK proteins were thought to be rarely mutated at a high frequency in any one common form of cancer. However, the increased understanding of the genetics of many forms of cancer have highlighted that MEK mutations are seen consistently at a low frequency across many forms of cancer and are also common in a few rare forms of cancer. Thus, MEK-mutant cancers appear to represent an important subtype of cancer in aggregate. Moreover, at least a proportion of MEK mutations appear to sensitize cancer patients to specific drugs which inhibit MEK. MEK mutations may therefore be extremely important to understand clinically. Through generous funding from the Functional Genomics Initiative of MSKCC, we now aim to systematically understand the consequences of MEK mutations across a wide variety of cancers. We are studying the effects of these mutations on activation of the MAP kinase pathway, biological effects in cancer formation models, and response to inhibitors of MEK. We believe this work may be very important in promoting clinical efforts to treat cancer patients with MEK inhibitors and help promote the use of more selective and efficacious therapies for cancer patients.
Molecular genetic analysis of cancer hotspot mutations in core miRNA machinery
Description: microRNAs (miRNAs) are ~22 nucleotide (nt) RNAs that mediate broad networks of gene regulation. Notably, cancer genome sequencing efforts (such as the TCGA and MSKCC-IMPACT projects) reveals somatic hotspot mutations that affect core factors in the miRNA pathway. We will conduct an integrated set of biochemical, genetic, and computational studies to understand what effects these mutations have on miRNA production and cellular behavior. Our goal is to use this information to understand why these mutations in miRNA machinery are beneficial to particular tumors, as this could provide a molecular framework for treating patients with these cancer signatures.
Functional analysis of cancer-associated PPP2R1A mis-sense mutations during cell division
Description: While most human cells have 46 chromosomes, tumor cells often have more or fewer chromosomes, a condition known as aneuploidy. Tumor sequencing data indicate that mutations and deletions in one protein family, called protein phosphatase 2A, positively correlate with aneuploidy. We aim to define the molecular mechanisms linking these cancer-associated aberrations to aneuploidy, which may provide insight into tumor development and growth.
The functional characterization of RhoA mutations found in diffuse gastric cancer
Cell Biology Program
Description: Diffuse gastric cancer is a highly malignant adenocarcinoma of the stomach wall. Recent genomic sequencing efforts have revealed a significant incidence of somatic mutations in RhoA (23 percent in one study). Our goal is to characterize the functional significance of RhoA mutations, using both epithelial cell lines as well as patient-derived gastric tumor cells.
Mechanisms of mTOR activating mutations in cancer
Description: mTOR inhibitors (mTORi) have been approved for the use to treat kidney cancer and ER+ breast cancer. However, sporadic cases of exceptional benefits drawn from mTORi for patients of other cancer types have been reported, indicating the need to select genomics-defined patients for precision medicine. We have discovered a series of mTOR activation mutations in kidney cancer patients and characterized their activities. Importantly, these mutations remain sensitive to mTORi. In this proposal, we mined the CMO dataset, which reports 388 mTOR missense mutations. There are 192 (~50 percent) mutations residing at the FAT or kinase domains. Importantly, most mutations either appear in clusters or focus at evolutionarily conserved positions. The remaining 180 are dispersed throughout the HEAT domain.
Hence, we propose to systemically interrogate these cancer-derived mTOR mutations. We hypothesize that a number of the mutations in FAT and kinase domains and certain mutations that were observed at evolutionary conversed positions in the HEAT domain are gain-of-function cancer driver mutations. With clear functional annotation of these mutants, oncologists could soon prescribe mTORi for tumors carrying activation mTOR mutations.
Mechanisms of genomic instability and epigenetic dysregulation in rare cancers with cohesion complex mutations
Description: The ring-shaped cohesin complex plays vital roles in shaping, expressing, transmitting, and repairing the genome. As such, recent sequencing efforts at MSK and elsewhere have unearthed frequent alterations in cohesin subunits in brain, bladder, soft tissue, and hematologic malignancies. Nonetheless, the precise function(s) of cohesin that are disrupted by these mutations, and their impact on the response to cytotoxic chemotherapy, remain poorly understood. In this project we will use genome editing to model and correct cancer-associated cohesin mutations in cultured cells. Using these systems, we will explore pathogenetic mechanisms and test strategies for enhancing therapeutic potency.
Deciphering the molecular mechanisms of Eph signaling using the genetic data from the CMO
Description: Our studies take advantage of the availability of large amounts of CMO sequencing data from tumors to study the molecular mechanisms of Eph receptor signaling and to understand how its misregulation causes cancer. The focus of our studies are three receptors, EphA3, EphA5, and EphB1, which are often mutated in multiple myeloma, lung adenocarcinoma, and melanoma, and which will be investigated using a combination of genomic, cell-biological, biochemical, and structural approaches.
Understanding the role FOXA1 in prostate cancer growth, transdifferentiation, metastasis and drug resistance using a novel prostate organoid system
Description: Recent DNA sequencing studies of hundreds of prostate cancer samples obtained from men with localized or metastatic disease have identified recurrent mutations in a gene called FOXA1. FOXA1 collaborates with another important gene encoding the androgen receptor, which is the target of hormonal therapies in prostate cancer. We hypothesize that mutations in FOXA1 perturb the normal function of the androgen receptor, setting the stage for prostate cancer to develop and develop resistance to hormone therapy. We will test this hypothesis using a novel prostate organoid system that allows us to grow prostate cells obtained from patients in the lab. The results may also have implications in breast and lung cancer, where alterations in FOXA1 are also frequently observed.
Functional consequences of BLM cancer mutations to genome maintenance
Description: Mutations in the DNA helicase BLM underlie the pathology of Bloom syndrome, one of the most penetrant cancer predisposition syndromes demonstrating genome instability. A better understanding of the role of BLM could provide vital insight into the link between genome instability and tumorigenesis, as well as inform the development of potential diagnostic and treatment strategies. We propose to examine BLM cancer mutations at the highly conserved residues identified in the CMO genomics dataset in gastric and ovarian cancers, in both yeast and mammalian model systems, to elucidate molecular mechanisms of BLM for genome maintenance.