Dr. Largaespada's laboratory is working to exploit insertional mutagenesis for cancer gene discovery and functional genomics in the mouse. The Largaespada lab has heavily invested in the use of a vertebrate-active transposon system, called Sleeping Beauty (SB), for insertional mutagenesis in mouse somatic and germline cells, and for gene therapy.
The identity of the mutations and other changes that drive the development of cancer must be determined for developing molecularly targeted therapeutics. Studies on human cancer exon re-sequencing suggest that a large number of mutations are present in breast and colorectal tumors (Sjoblom et al., Science, 2006). But, the identification of those changes that are selected for is going to be difficult because the number of “passenger” alterations not selected for during tumorigenesis is very large.
The human cancer genome project promises to help reveal the typical landscape of genomic changes in human cancer, but must be supplemented with complementary large-scale approaches for functional validation of targets and genetic screens that can identify cancer gene candidates. The Largaespada lab has developed approaches, using the SB transposon system, which can meet these needs. They have shown that SB transposon vectors can be mobilized in the soma of transgenic mice allowing forward genetic screens for cancer genes involved in sarcoma and lymphoma/leukemia to be performed in living mice (Collier et al., Nature, 2005; Dupuy et al., Nature, 2005). The system requires creating mice that harbor both a transposon array of the insertionally mutagenic SB vector, T2/Onc, and express the transposase enzyme in the target somatic tissue. If transposition can induce cancer, then tumor DNA is studied by cloning insertion sites. These insertion sites are analyzed and one looks for T2/Onc insertions at reproducibly mutated genes, called common insertion sites (CIS). The system has now been altered so that tissue-specific transposon mutagenesis for cancer gene discovery in various organs can be accomplished.
In one illustrative project mice harboring mutagenic (SB) transposons were crossed to mice expressing SB transposase in gastrointestinal tract epithelium (Starr et al., Science, 2009). All mice developed intestinal lesions including intraepithelial neoplasia, adenomas, and adenocarcinomas. Analysis of over 95,000 transposon insertions from these tumors identified 77 candidate gastrointestinal tract cancer genes. These genes were then compared to those mutated in human cancer, including colorectal cancer (CRC), or amplified, deleted or misexpressed in CRC, which allowed us to generate an 18 gene list that is highly likely to contain driver mutations for CRC. These genes include many of the most commonly known genes mutated in human CRC, such as APC, BMPR1A,
SMAD4 PTEN, FBXW7, DCC, MCC, in addition to several novel CRC candidate genes that function in pathways widely expected to participate in CRC such as the proliferation, adhesiveness and motility of epithelial cells. Similar work has revealed drivers for hepatocellular carcinoma development (Keng et al, Nature Biotech, 2009). These studies demonstrate the power of transposon-based mutagenesis when combined with human studies for identifying the driver mutations that cause cancer. Similar results have been obtained for hepatocellular carcinoma, brain tumors (including glioma and medulloblastoma), sarcomas and several other types of cancer.
Dr. Largaespada is also using mouse models of murine leukemia virus induced acute myeloid leukemia (AML) to identify and characterize genes that have a role in leukemia progression after disease is initiated by mutations relevant to human AML. This work also includes genetic studies of myeloid leukemia chemotherapy resistance and relapse. AML is the most common adult leukemia. It is clear that genetically defined subsets of AML have varying prognoses. AML frequently harbor chromosomal translocations that create fusion oncoproteins that act as transcription factors or constitutively active kinases. These fusion genes are thought to be insufficient, by themselves, for AML induction. Instead, secondary mutations cooperate with them to produce AML. The full set of cooperating mutations and their usefulness as therapeutic targets are important unknown quantities. The lab is exploring these questions by using MuLV mutagenesis in mice carrying specific human translocation fusion oncogenes known to play a role in human AML. The lab has developed MuLV-accelerated models of AML initiated by expression of the MLL-AF9 and AML1-ETO fusion oncoproteins (Bergerson et al., Blood 2012). We have cloned
4,731 unique proviral insertions from 89 MuLV accelerated Mll-AF9/+ leukemia and 79 control MuLV-induced leukemia. Preliminary analysis reveals ~90 common insertion sites with many showing strong bias for Mll-AF9+ leukemias.
Comparisons to expression microarray data on human AML with MLL gene translocations are in progress. These data may help to distinguish between genes that are direct targets of MLL-AF9, those that are a cause of AML development and those that cooperate with MLL-AF9 to induce AML
In another area of AML research, we have sought to address the role of the activated NRAS oncogene in AML maintenance. We therefore developed Vav-tTA (expressed inhematopoietic cells) and TRE-NRASG12V transgenic lines in FVB/n mice. Interestingly, the doubly transgenic Vav-tTA plus TRE-NRASG12V mice developed a myeloproliferative disease very similar to human aggressive systemic mastocytosis (ASM) without other detectable hematopoietic tumors (Wiesner et al., Blood, 2005). To determine the ability of NRASG12D to cooperate with a fusion oncogene encoding an altered transcription factor we created triple transgenic Vav-tTA; TRE-NRASG12V; Mll-AF9 lines in C57BL/6J X FVB/n F1 mice. AML were obtained in triple transgenic mice. When we transplanted triple transgenic Vav-tTA; TRE-NRASG12V; Mll-AF9 AML into SCID mice we found that doxycycline (DOX) treatment via the drinking water could prevent AML engraftment or eliminate AML cells after letting them grow to full-blown leukemia in recipients. However, at least some of these mice develop DOX-resistant AML, which do not re-express the NRASG12V (Kim et al., Blood, 2009). This suggests that RAS oncoproteins may be good therapeutic targets, even in complex tumors induced in cooperation with another strong oncogene. The mechanisms for oncogene addiction are not clearly understood. We are currently exploring the mechanism of AML cell death after NRAS oncogene suppression, the mechanism by which rare AML cells escape death in this context, and interactions between RAS targeted therapies and conventional chemotherapy.
(For a comprehensive list of recent publications, refer to PubMed, a service provided by the National Library of Medicine.)
- Tschida BR, Temiz NA, Kuka TP, Lee LA, Riordan JD, Tierrablanca CA, Hullsiek R, Wagner S, Hudson WA, Linden MA, Amin K, Beckmann PJ, Heuer RA, Sarver AL, Yang JD, Roberts LR, Nadeau JH, Dupuy AJ, Keng VW, Largaespada DA. Sleeping beauty insertional mutagenesis in mice identifies drivers of steatosis-associated hepatic tumors.Cancer Res. 2017;77:6576-6588.
- Kurata M, Yamamoto K, Moriarity BS, Kitagawa M, Largaespada DA. CRISPR/Cas9 library screening for drug target discovery. J Hum Genet. 2017 Nov 20. doi: 10.1038/s10038-017-0376-9.
- Patel AV, Chaney KE, Choi K, Largaespada DA, Kumar AR, Ratner N. An ShRNA screen identifies MEIS1 as a driver of malignant peripheral nerve sheath tumors. EBioMedicine. 2016;9:110-119.
- Becker CM, Oberoi RK, McFarren SJ, Muldoon DM, Pafundi DH, Pokorny JL, Brinkmann DH, Ohlfest JR, Sarkaria JN, Largaespada DA, Elmquist WF. Decreased affinity for efflux transporters increases brain penetrance and molecular targeting of a PI3K/mTOR inhibitor in a mouse model of glioblastoma. Neuro Oncol. 2015;17(9):1210-9.
- Kempema AM, Widen JC, Hexum JK, Andrews TE, Wang D, Rathe SK, Meece FA, Noble KE, Sachs Z, Largaespada DA, Harki DA. Synthesis and antileukemic activities of C1-C10-modified parthenolide analogues. Bioorg Med Chem. 2015;23(15):4737-45.
- Dorr C, Janik C, Weg M, Been RA, Bader J, Kang R, Ng B, Foran L, Landman SR, O'Sullivan MG, Steinbach M, Sarver AL, Silverstein KA, Largaespada DA, Starr TK. Transposon Mutagenesis Screen Identifies Potential Lung Cancer Drivers and CUL3 as a Tumor Suppressor. Mol Cancer Res. 2015;13(8):1238-47.
- Mirabello L, Koster R, Moriarity BS, Spector LG, Meltzer PS, Gary J, Machiela MJ, Pankratz N, Panagiotou OA, Largaespada D, Wang Z, Gastier-Foster JM, Gorlick R, Khanna C, de Toledo SR, Petrilli AS, Patiño-Garcia A, Sierrasesúmaga L, Lecanda F, Andrulis IL, Wunder JS, Gokgoz N, Serra M, Hattinger C, Picci P, Scotlandi K, Flanagan AM, Tirabosco R, Amary MF, Halai D, Ballinger ML, Thomas DM, Davis S, Barkauskas DA, Marina N, Helman L, Otto GM, Becklin KL, Wolf NK, Weg MT, Tucker M, Wacholder S, Fraumeni JF Jr, Caporaso NE, Boland JF, Hicks BD, Vogt A, Burdett L, Yeager M, Hoover RN, Chanock SJ, Savage SA A Genome-Wide Scan Identifies Variants in NFIB Associated with Metastasis in Patients with Osteosarcoma. Cancer Discov. 2015;5(9):920-31.
- Zuckermann M, Hovestadt V, Knobbe-Thomsen CB, Zapatka M, Northcott PA, Schramm K, Belic J, Jones DT, Tschida B, Moriarity B, Largaespada D, Roussel MF, Korshunov A, Reifenberger G, Pfister SM, Lichter P, Kawauchi D, Gronych J. Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling. Nat Commun. 2015 Jun 11;6:7391.
- Hexum JK, Becker CM, Kempema AM, Ohlfest JR, Largaespada DA, Harki DA. Parthenolide prodrug LC-1 slows growth of intracranial glioma. Bioorg Med Chem Lett. 2015;25(12):2493-5.
- Moriarity BS, Otto GM, Rahrmann EP, Rathe SK, Wolf NK, Weg MT, Manlove LA, LaRue RS, Temiz NA, Molyneux SD, Choi K, Holly KJ, Sarver AL, Scott MC, Forster CL, Modiano JF, Khanna C, Hewitt SM, Khokha R, Yang Y, Gorlick R, Dyer MA, Largaespada DA. A Sleeping Beauty forward genetic screen identifies new genes and pathways driving osteosarcoma development and metastasis. Nat Genet. 2015;47(6):615-24.
- Moriarity BS, Largaespada DA. Sleeping Beauty transposon insertional mutagenesis based mouse models for cancer gene discovery. Curr Opin Genet Dev. 2015 Feb;30:66-72.
- Sachs Z, LaRue RS, Nguyen HT, Sachs K, Noble KE, Mohd Hassan NA, Diaz-Flores E, Rathe SK, Sarver AL, Bendall SC, Ha NA, Diers MD, Nolan GP, Shannon KM, Largaespada DA. NRASG12V oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia. Blood. 2014;124(22):3274-83.
- Litterman AJ, Zellmer DM, LaRue RS, Jameson SC, Largaespada DA. Antigen-specific culture of memory-like CD8 T cells for adoptive immunotherapy. Cancer Immunol Res. 2014 Sep;2(9):839-45.
- Rathe SK, Moriarity BS, Stoltenberg CB, Kurata M, Aumann NK, Rahrmann EP, Bailey NJ, Melrose EG, Beckmann DA, Liska CR, Largaespada DA. Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia. Sci Rep. 2014 Aug 13;4:6048.
- Tseng YY, Moriarity BS, Gong W, Akiyama R, Tiwari A, Kawakami H, Ronning P, Reuland B, Guenther K, Beadnell TC, Essig J, Otto GM, O'Sullivan MG, Largaespada DA, Schwertfeger KL, Marahrens Y, Kawakami Y, Bagchi A. PVT1 dependence in cancer with MYC copy-number increase. Nature. 2014;512(7512):82-6.
- Rahrmann EP, Moriarity BS, Otto GM, Watson AL, Choi K, Collins MH, Wallace M, Webber BR, Forster CL, Rizzardi AE, Schmechel SC, Ratner N, Largaespada DA. Trp53 haploinsufficiency modifies EGFR-driven peripheral nerve sheath tumorigenesis. Am J Pathol. 2014;184:2082-98.
- Li MV, Shukla D, Rhodes BH, Lall A, Shu J, Moriarity BS, Largaespada DA. HomeRun Vector Assembly System: a flexible and standardized cloning system for assembly of multi-modular DNA constructs. PLoS One. 2014 Jun 24;9(6):e100948.
- Been RA, Linden MA, Hager CJ, DeCoursin KJ, Abrahante JE, Landman SR, Steinbach M, Sarver AL, Largaespada DA, Starr TK. Genetic signature of histiocytic sarcoma revealed by a sleeping beauty transposon genetic screen in mice. PLoS One. 2014 May 14;9(5):e97280.
- Moriarity BS, Rahrmann EP, Beckmann DA, Conboy CB, Watson AL, Carlson DF, Olson ER, Hyland KA, Fahrenkrug SC, McIvor RS, Largaespada DA. Simple and efficient methods for enrichment and isolation of endonuclease modified cells. PLoS One. 2014 May 5;9(5):e96114.
- Watson AL, Anderson LK, Greeley AD, Keng VW, Rahrmann EP, Halfond AL, Powell NM, Collins MH, Rizvi T, Moertel CL, Ratner N, Largaespada DA. Co-targeting the MAPK and PI3K/AKT/mTOR pathways in two genetically engineered mouse models of schwann cell tumors reduces tumor grade and multiplicity. Oncotarget. 2014;5:1502-14.
- Tschida BR, Largaespada DA, Keng VW. Mouse models of cancer: Sleeping Beauty transposons for insertional mutagenesis screens and reverse genetic studies. Semin Cell Dev Biol. 2014;27:86-95.
- Keng VW, Largaespada DA, Villanueva A. Why men are at higher risk for hepatocellular carcinoma? J Hepatol. 2012;57:453-454.
- Keng VW, Rahrmann EP, Watson AL, Tschida BR, Moertel CL, Jessen WJ, Rizvi TA, Collins MH, Ratner N, Largaespada DA. PTEN and NF1 inactivation in Schwann cells produces a severe phenotype in the peripheral nervous system that promotes the development and malignant progression of peripheral nerve sheath tumors. Cancer Res. 2012;72:3405-3413
- Bergemann TL, Starr TK, Yu H, Steinbach M, Erdmann J, Chen Y, Cormier RT, Largaespada DA, Silverstein KA. New methods for finding common insertion sites and co-occurring common insertion sites in transposon- and virus-based genetic screens. Nucleic Acids Res. 2012;40:3822-3833.
- Bergerson RJ, Collier LS, Sarver AL, Been RA, Lugthart S, Diers MD, Zuber J, Rappaport AR, Nixon MJ, Silverstein KA, Fan D, Lamblin AF, Wolff L, Kersey JH, Delwel R, Lowe SW, O'Sullivan MG, Kogan SC, Adams DJ, Largaespada DA. An insertional mutagenesis screen identifies genes that cooperate with Mll-AF9 in a murine leukemogenesis model. Blood. 2012;119:4512-4523.
- Pérez-Mancera PA, Rust AG, van der Weyden L, Kristiansen G, Li A, Sarver AL, Silverstein KA, Grützmann R, Aust D, Rümmele P, Knösel T, Herd C, Stemple DL, Kettleborough R, Brosnan JA, Li A, Morgan R, Knight S, Yu J, Stegeman S, Collier LS, ten Hoeve JJ, de Ridder J, Klein AP, Goggins M, Hruban RH, Chang DK, Biankin AV, Grimmond SM; Australian Pancreatic Cancer Genome Initiative, Wessels LF, Wood SA, Iacobuzio-Donahue CA, Pilarsky C, Largaespada DA, Adams DJ, Tuveson DA. The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma. Nature. 2012;486(7402):266-70.
- Seshagiri S, Stawiski EW, Durinck S, Modrusan Z, Storm EE, Conboy CB, Chaudhuri S, Guan Y, Janakiraman V, Jaiswal BS, Guillory J, Ha C, Dijkgraaf GJ, Stinson J, Gnad F, Huntley MA, Degenhardt JD, Haverty PM, Bourgon R, Wang W, Koeppen H, Gentleman R, Starr TK, Zhang Z, Largaespada DA, Wu TD, de Sauvage FJ. Recurrent R-spondin fusions in colon cancer. Nature. 2012;488:660-664.
- Wu X, Northcott PA, Dubuc A, Dupuy AJ, Shih DJ, Witt H, Croul S, Bouffet E, Fults DW, Eberhart CG, Garzia L, Van Meter T, Zagzag D, Jabado N, Schwartzentruber J, Majewski J, Scheetz TE, Pfister SM, Korshunov A, Li XN, Scherer SW, Cho YJ, Akagi K, MacDonald TJ, Koster J, McCabe MG, Sarver AL, Collins VP, Weiss WA, Largaespada DA, Collier LS, Taylor MD. Clonal selection drives genetic divergence of metastatic medulloblastoma. Nature. 2012;482(7386):529-533.
- Bergemann TL, Starr TK, Yu H, Steinbach M, Erdmann J, Chen Y, Cormier RT, Largaespada DA, Silverstein KA. New methods for finding common insertion sites and co-occurring common insertion sites in transposon- and virus-based genetic screens. Nucleic Acids Res. 2012;40:3822-33.
- Wilber A, Ulloa Montoya F, Hammer L, Moriarity BS, Geurts AM, Largaespada DA, Verfaillie CM, McIvor RS, Lakshmipathy U. Efficient non-viral integration and stable gene expression in multipotent adult progenitor cells. Stem Cells Int. 2011;2011:717069.
- Wiesner SM, Geurts JL, Diers MD, Bergerson RJ, Hasz DE, Morgan KJ, Largaespada DA. Nf1 mutant mice with p19ARF gene loss develop accelerated hematopoietic disease resembling acute leukemia with a variable phenotype. Am J Hematol. 2011;86:579-85.
- Starr TK, Scott PM, Marsh BM, Zhao L, Than BL, O'Sullivan MG, Sarver AL, Dupuy AJ, Largaespada DA, Cormier RT. A Sleeping Beauty transposon-mediated screen identifies murine susceptibility genes for adenomatous polyposis coli (Apc)-dependent intestinal tumorigenesis. Proc Natl Acad Sci U S A. 2011;108:5765-5770.
- Keng VW, Tschida BR, Bell JB, Largaespada DA. Modeling hepatitis B virus X-induced hepatocellular carcinoma in mice with the Sleeping Beauty transposon system. Hepatology. 2011;53:781-90.
Former Graduate Student(s):
Chani Becker (Ph.D. 2016, Neuroscience, University of Minnesota).