Abstract PR07: Novel oncogenic mutations in the beta subunit of heteromeric G-proteins identified by functional cDNA library screening.
Authors: Yoda Akinori , Adelmant Guillaume , Shindoh Nobuaki , Chapuy Bjoern , Yoda Yuka , Weigert Oliver , Kopp Nadja , Wu Shuo-Chieh , Kim Sunhee S. , Liu Huiyun , Tivey Trevor , Tyner Jeffrey W. , Gotlib Jason , Deininger Michael W. , Turley Shannon , Marto Jarrod A. , Lane Andrew A. , Weinstock David M. 
Published: August 2014

Abstract
Although next-generation sequencing can delineate the genetic alterations within a primary tumor specimen, it can be difficult to distinguish the small number of driver mutations from the large number of passenger mutations. To overcome this issue, we developed a system for identifying oncogenic alterations directly from tumor cells. In this system, retroviral cDNA libraries built from cancer cell lines and directly from primary cancer samples are transduced into BaF3 cells, an IL3-dependent B cell line. Transformation by oncogenes promotes IL3-independent survival, allowing for the isolation of individual transformed clones and sequencing of the integrated cDNA. In the past, we identified CRLF2 as a novel oncogene in acute lymphoblastic leukemia (Yoda et al. PNAS 2010). We have improved the method and demonstrated 100% sensitivity for isolating well-characterized oncogenes, including EGFR, HER2, RAS and ALK (Shindoh et al. PLoS One 2012). Recently, we isolated a mutated GNB1 K89E allele from a cDNA library generated from a primary blastic plasmacytoid dendritic cell neoplasm (BPDCN). BPDCN is a rare and aggressive leukemia with a dismal prognosis. GNB1 encodes the beta subunit of the heterotrimeric G-protein, a binding complex that transduces signals from G-protein coupled receptors to multiple downstream pathways. Gain-of-function mutations have been reported in alpha subunits of the G-protein, however, the contributions of beta subunits to cancer remains undefined. To investigate downstream signaling from GNB1 K89E, we performed gene expression profiling and mass spectrometry (MS)-based phosphoproteomics and found significant activation of RAS/MAPK and PI3K/AKT pathways in GNB1 K89E-expressing cells compared to isogenic cells expressing wild-type GNB1. To target GNB1 K89E signaling, we screened kinase inhibitors using a multiplex panel of small molecules and found selective sensitivity of GNB1 K89E cells to MEK and pan-PI3-kinase inhibitors. Next, we transduced GNB1 alleles into bone marrow cells from Cdkn2a-deficient mice and transplanted into wild-type recipient mice. Within 4 months after transplantation, all mice (n=10) that received bone marrow transduced with GNB1 K89E developed a lethal dendritic cell malignancy, confirming the transforming effects of GNB1 K89E in vivo. A search of published cancer mutations identified four cases with GNB1 I80T/N in chronic lymphocytic leukemia or B-cell lymphomas, five cases with GNB1 K57E/T in myeloid malignancies, one case of GNB1 K89E in acute lymphoblastic leukemia, and two cases with GNB2 M101T/V in ovarian cancer. All of these alleles promoted GM-CSF-independent growth in human TF1 cells. Interestingly, the mutated codons are all located on the GNB1 molecular surface that is critical for interactions between GNB1 and both alpha subunits and downstream effectors. Immunoprecipitation followed by MS demonstrated that GNB1 K89E and I80T mutants failed to bind inhibitory G alpha subunits GNAI2 and GNAI3 as well as GNA11 that are bound by wild-type GNB1. Thus, gain-of-function mutations in G-protein beta subunits occur across a broad range of malignancies, can drive in vivo transformation, and activate targetable downstream kinases by modifying essential interactions with partner proteins.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR07.
Citation Format: Akinori Yoda, Guillaume Adelmant, Nobuaki Shindoh, Bjoern Chapuy, Yuka Yoda, Oliver Weigert, Nadja Kopp, Shuo-Chieh Wu, Sunhee S. Kim, Huiyun Liu, Trevor Tivey, Jeffrey W. Tyner, Jason Gotlib, Michael W. Deininger, Shannon Turley, Jarrod A. Marto, Andrew A. Lane, David M. Weinstock. Novel oncogenic mutations in the beta subunit of heteromeric G-proteins identified by functional cDNA library screening. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR07.