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CRTC1/MAML2 gain-of-function interactions with MYC PNAS PLUS create a gene signature predictive of cancers with CREB–MYC involvement Antonio L. Amelioa,1, Mohammad Fallahib, Franz X. Schauba, Min Zhangc, Mariam B. Lawania, Adam S. Alpersteina, Mark R. Southernd, Brandon M. Younge, Lizi Wuf, Maria Zajac-Kayec,g, Frederic J. Kayec, John L. Clevelanda, and Michael D. Conkrighta,d,2 aDepartment of Cancer Biology, bIT Informatics, dTranslational Research Institute and Department of Molecular Therapeutics, and eGenomics Core, The Scripps Research Institute, Jupiter, FL 33458; and Departments of cMedicine, fMolecular Genetics and Microbiology, and gAnatomy and Cell Biology, Shands Cancer Center, University of Florida, Gainesville, FL 32610 Edited* by Marc Montminy, The Salk Institute for Biological Studies, La Jolla, CA, and approved July 8, 2014 (received for review October 11, 2013) Chimeric oncoproteins created by chromosomal translocations are histology. As such, these are a class of epithelial cell malignancies among the most common genetic mutations associated with tu- that originate from mucous/serous glands present at different morigenesis. Malignant mucoepidermoid salivary gland tumors, as locations in the body (6, 8–15). well as a growing number of solid epithelial-derived tumors, can The genesis of these adult solid tumors was thought to be due arise from a recurrent t (11, 19)(q21;p13.1) translocation that gen- to the direct activation of both CREB and NOTCH/RBPJ, the erates an unusual chimeric cAMP response element binding protein transcription factor targets of the CRTC1 and MAML2 coac- (CREB)-regulated transcriptional coactivator 1 (CRTC1)/mastermind- tivators involved in the t (11, 19) translocation, respectively like 2 (MAML2) (C1/M2) oncoprotein comprised of two transcrip- (6, 15). However, forced expression of both CRTC1 and MAML2 tional coactivators, the CRTC1 and the NOTCH/RBPJ coactivator is not sufficient to provoke transformation, whereas ectopic MAML2. Accordingly, the C1/M2 oncoprotein induces aberrant ex- and/or inducible expression of C1/M2 transforms epithelial cells. pression of CREB and NOTCH target genes. Surprisingly, here we Furthermore, the domain within MAML2 required for inter- BIOCHEMISTRY report a gain-of-function activity of the C1/M2 oncoprotein that acting with NOTCH is absent in the C1/M2 fusion protein, and directs its interactions with myelocytomatosis oncogene (MYC) pro- those NOTCH genes originally identified as aberrantly regulated teins and the activation of MYC transcription targets, including C1/M2 targets were found to also possess CREB-responsive those involved in cell growth and metabolism, survival, and tumor- promoters (16, 17). However, a C1/M2 deletion mutant (C1/ igenesis. These results were validated in human mucoepidermoid Δ – tumor cells that harbor the t (11, 19)(q21;p13.1) translocation and M2 48 222) that is fully capable of interacting with and acti- express the C1/M2 oncoprotein. Notably, the C1/M2–MYC interac- vating CREB cannot drive transformation (17). Collectively, tion is necessary for C1/M2-driven cell transformation, and the these observations point to added levels of regulation outside of C1/M2 transcriptional signature predicts other human malignancies simply activating CREB or NOTCH, which is further supported having combined involvement of MYC and CREB.Thesefindings by clinical patient data, where the C1/M2 translocation alone is suggest that such gain-of-function properties may also be manifest in other oncoprotein fusions found in human cancer and that agents Significance targeting the C1/M2–MYC interface represent an attractive strategy for the development of effective and safe anticancer therapeutics in The prevailing dogma since the identification of the t (11, 19) tumors harboring the t (11, 19) translocation. translocation gene product as a fusion of the cAMP response element binding protein (CREB)-regulated transcriptional coac- ene regulatory circuits are generally controlled by transcrip- tivator 1 (CRTC1) and the NOTCH coactivator mastermind-like 2 Gtional mechanisms tied to signal transduction pathways, and (MAML2) in malignant salivary gland tumors has been that ab- they allow cells to rapidly respond to environmental cues to con- errant activation of CREB and/or NOTCH transcription programs trol cell survival, growth, metabolism, and biological function. drives oncogenesis. However, combined expression of the pa- These controls are lost in cancers through various means (1–3), rental coactivator molecules CRTC1 and MAML2 is not sufficient including chromosomal translocations that can augment the ex- to induce transformation, suggesting an added level of com- pression of oncogenes or that generate chimeric oncoproteins that plexity. Here we describe gain-of-function interactions between are necessary and sufficient to provoke malignancy (4). the CRTC1/MAML2 (C1/M2) coactivator fusion and myelocyto- Chromosomal translocations found in epithelial tumors fre- matosis oncogene (MYC) oncoproteins that are necessary for C1/ quently involve the fusion of signaling molecules and regulators M2-driven transformation. Our findings suggest that targeting of transcriptional activity (5). The t (11, 19)(q21;p13.1) trans- the C1/M2–MYC interface represents an attractive strategy for location gene product creates a unique oncoprotein fusion that is the development of effective and safe anticancer therapeutics in comprised of two transcriptional coactivators, the cAMP re- tumors harboring the t (11, 19) translocation. sponse element binding protein (CREB)-regulated transcrip- tional coactivator 1 (CRTC1) and the NOTCH/RBPJ coactivator Author contributions: A.L.A. and M.D.C. designed research; A.L.A., M.F., F.X.S., M.Z., M.B.L., – A.S.A., M.R.S., B.M.Y., L.W., M.Z.-K., and M.D.C. performed research; A.L.A., F.X.S., and M.D.C. mastermind-like 2 (MAML2) (6 11). The ensuing CRTC1/ contributed new reagents/analytic tools; A.L.A., M.F., L.W., M.Z.-K., F.J.K., J.L.C., and M.D.C. MAML2 (C1/M2) chimeric oncoprotein is comprised of the analyzed data; and A.L.A., F.J.K., J.L.C., and M.D.C. wrote the paper. N-terminal 42 residues of CRTC1 encompassing a coiled-coil The authors declare no conflict of interest. domain involved in CREB binding followed by the C-terminal *This Direct Submission article had a prearranged editor. 981 residues of MAML2, which includes a transcriptional acti- 1Present address: Lineberger Comprehensive Cancer Center, University of North Carolina vation domain. This C1/M2 coactivator fusion was originally at Chapel Hill, Chapel Hill, NC 27599-7455. identified in mucoepidermoid carcinomas (MECs) of the salivary 2To whom correspondence may be addressed. Email: [email protected]. gland and lungs, but has now also been detected in primary thy- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. roid, breast, cervix, and skin tumors that present with MEC-like 1073/pnas.1319176111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1319176111 PNAS Early Edition | 1of9 Downloaded by guest on October 1, 2021 characterized by a benign phenotype, and where the full tu- morigenic potential of mucoepidermoid C1/M2-positive cells requires additional cooperating signals for a full-blown malig- nancy (18). Collectively, these observations hint at additional functional activities present in the C1/M2 coactivator fusion that contribute to its oncogenic potential. To define these functions, we used a mammalian cell-based screen and discovered a C1/M2 gain-of- function, where C1/M2 binds to and coopts the function of MYC oncoproteins, and show that C1/M2-driven transformation requires MYC. Furthermore, a C1/M2 gene signature identifies human tumors having combined activation of MYC and CREB pathways. These findings suggest that gain-of-function activities may be a common feature of oncoprotein fusions and that such activities represent new avenues for therapeutic intervention. Results C1/M2 Interacts with the MYC Oncoprotein Network. To assess if the C1/M2 coactivator fusion interacts with transcriptional regu- lators in addition to CREB or NOTCH/RBPJ, we used a pre- viously developed and validated cell-based functional screen coined the “Coactivator Trap” composed of a library of nearly all human transcription factors fused to the GAL4 DNA binding domain (DBD) (SI Appendix, Fig. S1) (19). Surprisingly, the C1/M2 coactivator fusion selectively activated several proteins within the MYC:MAX network including MYCN, MXI (a.k.a. MAD2), and MAX (Fig. 1A). In contrast, neither parental CRTC1 nor MAML2 coactivator proteins displayed this activity. The catalytic subunit of PKA served as a control for the assay and displayed, as expected (20, 21), highly specific activation of GAL4–ATF1 and repression of GAL4–ATF4 reporter activ- ity. Furthermore, as expected, expression of CRTC1 activated Fig. 1. The C1/M2 chimeric oncoprotein selectively coactivates the MYC GAL4–ATF1 and repressed GAL4–ATF4 reporter activity, network. (A) Heat map of screen data displaying luciferase reporter activa- whereas MAML2 activated GAL4–MEF2, which are known tion (red) and repression (green). GAL4–DBD library proteins are along the y binding partners for these coactivators (22–24). Finally, trans- axis, with coexpressed C1/M2 and controls along the x axis. (B) HEK293T cells × were transiently cotransfected with CRTC1, MAML2, C1/M2, or empty ex- fection experiments with a 5 GAL4::UAS-luciferase reporter – × – pression vectors, and GAL4 DBD library proteins
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    G C A T T A C G G C A T genes Article Emerging Role of ODC1 in Neurodevelopmental Disorders and Brain Development Jeremy W. Prokop 1,2,3,*, Caleb P. Bupp 1,4 , Austin Frisch 1, Stephanie M. Bilinovich 1 , Daniel B. Campbell 1,3,5, Daniel Vogt 1,3,5, Chad R. Schultz 1, Katie L. Uhl 1, Elizabeth VanSickle 4, Surender Rajasekaran 1,6,7 and André S. Bachmann 1,* 1 Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503, USA; [email protected] (C.P.B.); [email protected] (A.F.); [email protected] (S.M.B.); [email protected] (D.B.C.); [email protected] (D.V.); [email protected] (C.R.S.); [email protected] (K.L.U.); [email protected] (S.R.) 2 Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824, USA 3 Center for Research in Autism, Intellectual, and Other Neurodevelopmental Disabilities, Michigan State University, East Lansing, MI 48824, USA 4 Spectrum Health Medical Genetics, Grand Rapids, MI 49503, USA; [email protected] 5 Neuroscience Program, Michigan State University, East Lansing, MI 48824, USA 6 Pediatric Intensive Care Unit, Helen DeVos Children’s Hospital, Grand Rapids, MI 49503, USA 7 Office of Research, Spectrum Health, Grand Rapids, MI 49503, USA * Correspondence: [email protected] (J.W.P.); [email protected] (A.S.B.) Abstract: Ornithine decarboxylase 1 (ODC1 gene) has been linked through gain-of-function variants Citation: Prokop, J.W.; Bupp, C.P.; to a rare disease featuring developmental delay, alopecia, macrocephaly, and structural brain anoma- Frisch, A.; Bilinovich, S.M.; Campbell, lies.
  • Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation

    Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation

    BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in