The Beta-Subunit Of Heterotrimeric G Proteins Harbors Gain-Of-Function Mutations In Multiple Hematologic Malignancies

To identify new oncogene alleles directly from primary tumor specimens, we generate and screen cDNA libraries from patient samples for gain-of-function alterations that can substitute for cytokine signaling in cytokine-dependent cells. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia of plasmacytoid dendritic cells with a dismal prognosis. No driver oncogenes have been identified in cases of BPDCN. Screening of a cDNA library generated from a BPDCN resulted in multiple cytokine-independent clones that expressed a full-length transcript of GNB1 with a K89E mutation. GNB1 encodes a 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, including GNAQ/GNA11 in uveal melanoma and GNAS in pituitary tumors, 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. ERK and AKT activation by GNB1 K89E were confirmed by western blotting. To target GNB1 K89E signaling, we screened kinase inhibitors using a multiplex assay of small molecules and found selective sensitivity of GNB1 K89E cells to MEK and pan-PI3-kinase inhibitors. To assay the transforming effects of GNB1 K89E in vivo, we transduced GNB1 (wild-type or K89E) into bone marrow from Cdkn2a-deficient donors after 5-FU treatment and transplanted into wild-type recipients. We opted to utilize Cdkn2a-deficient donors as the loss of CDKN2A is common in cases of BPDCN. Within 4 months after transplantation, all mice (n=10) that received bone marrow transduced with GNB1 K89E developed a lethal malignancy characterized by pancytopenia and massive hepatosplenomegaly. Spleens were infiltrated by large, spindly cells with extensive dendritic projections, as well as extensive fibrosis that completely effaced the normal splenic architecture. The cells were negative for T-cell (CD2, CD3) and B-cell (CD19, B220) markers but positive for the dendritic cell/macrophage markers MAC-2 and MAC-3. Further characterization by flow cytometry demonstrated that the cells infiltrating the spleen were CD8, CD103, MHC class II, CD26, FLT3 and CD11c positive, consistent with neoplastic dendritic cells. Serial transplantation of splenic cells from five different GNB1 K89E-transplanted mice into secondary wild-type recipients resulted in 100% fatality within 50 days. We searched published datasets from exome, transcriptome and whole genome sequencing of hematologic malignancies for GNB1 mutations. We identified one case of K89E in B-cell acute lymphoblastic leukemia (ALL), four cases with I80T/N in chronic lymphocytic leukemia or B-cell lymphomas, six cases with K57E/T in myeloid neoplasms, and D76G in T-cell ALL. Expression of any of these alleles but not wild-type GNB1 was sufficient to promote cytokine-independent growth of human TF1 cells. The published structure of GNB1 (Ford et al. Science 1998) reported a small number of residues, including K57, I80 and K89 that mediate interactions with both G-alpha subunits and effector proteins. In fact, affinity purification followed by MS using tagged GNB1 (wild-type, I80T and K89E) demonstrated that, unlike wild-type GNB1, the GNB1 mutants fail to bind distinct Gα subunits. The repertoire of protein interactors, which includes potential G protein effectors, also differed between different GNB1 alleles. Thus, gain-of-function mutations in GNB1 occur across a broad range of hematologic malignancies, modify essential interaction G-protein subunit interactions, can drive in vivo transformation, and activate targetable downstream kinases.