RhoF GTPase Transcription Is Upregulated in Germinal Center B-Cells, Shows Altered Expression in Malignant B-Cells, and Interacts with the ATM Pathway.
Authors: Gouw Launce G. , Reading N. Scott , Crockett David K. , Szankasi Philippe , Lim Megan S. , Elenitoba-Johnson Kojo S.J. 
Published: April 2020

Abstract
Follicular lymphoma (FL) is the most common low-grade B-cell non Hodgkin lymphoma in the Western hemisphere. A significant proportion of FL undergo histologic transformation to diffuse large B-cell lymphoma (DLBCL). Using cDNA microarray analysis, we identified an expressed sequence tag GI#10952525 consistently differentially expressed in transformed follicular lymphomas (tFL). This was characterized as RhoF, a novel member of the Rho family. Rho GTPases play central roles in cytoskeletal dynamics, cell-cell interactions, and intracellular signaling pathways involved in migration, proliferation and survival. Dysregulation of Rho proteins are key events implicated in tumorigenesis.
To define the role of RhoF in lymphocyte physiology and lymphoma transformation, we assessed its expression across phenotypically defined lymphocyte subpopulations, using quantitative real-time PCR. We determined relative RhoF levels in immunomagnetic bead purified normal lymphoid subpopulations [naïve B-cells, memory B-cells, germinal center B-cells and T-cells], reactive lymphoid tissues (n=5), cell lines [derived from t(14;18) tFL (n =3), de novo DLBCL (n=7), and T-cell malignancy (n=3)] and tissue from primary human lymphoid neoplasms [FL (n=5), de novo DLBCL (n=5), tFL (n=5), CLL/SLL (n=4), anaplastic large cell lymphoma (n=8), mantle cell lymphoma (n=5), and T-cell acute lymphoblastic leukemia (n=5)]. RhoF was expressed at significantly higher levels in B-cells relative to T-cells. We saw this pattern in purified lymphocyte subpopulations, in cell lines, and in primary lymphoma tissue samples. Notably, we detected elevated levels of RhoF transcript in B-cells of germinal center (GC) origin, both in the reactive and neoplastic samples of GC-derived B-cells. The highest transcriptional levels of RhoF were in malignant B-cells of GC origin; both in heterogeneous primary tissue samples and in homogeneous tissue culture preparations.
To investigate its functional role, we cloned RhoF into a vector coding for a C-terminal polyhistidine- and V5 epitope-tag. We expressed the constructs in HEK 293T cells, and purified the RhoF-containing complexes using a tandem affinity purification approach. We ran cell lysates through a nickel column; non-interacting proteins were washed off under native conditions and the bound RhoF complexes eluted with imidazole. Eluate was immunoprecipitated with sepharose-bound anti-V5 antibody. Immunoprecipitated complexes were denatured and resolved by 1D-PAGE. Unique bands representing RhoF interacting proteins were isolated and enzymatically cleaved with trypsin. Resultant peptides underwent liquid chromatography and tandem mass spectrometry. Data were searched against the NCBI nr.FASTA nonredundant protein database using the SEQUEST algorithm and false positive rates determined with INTERACT and ProteinProphet. Among several putative RhoF interactors, we identified ATM as an important RhoF binding partner.
In conclusion, our demonstration of the differential expression of RhoF in GC-derived cells and its upregulation in tFL provide evidence for a connection between the role of this novel protein in B-cell development and malignancy. In addition, evidence of an association between RhoF and ATM may provide a link between DNA repair, cell cycle control and morphological dynamics.