Oct-2: The Achilles Heel of Germinal Center derived Lymphomas.

RANUNCOLO, SM; NGO, VN; XIAO, W; WRIGHT G; STAUDT, LM

Saxton River, Vermont

Congreso; Hematological Malignancies; 2009

Federation of American Societies for Experimental Biology (FASEB)

To identify genes requiered for the proliferation and survival of Diffuse Large B Cell Lymphomas we conducted an ?Achilles Heel? RNA interference screen in DLBCL cell lines model of ABC (Activated B cell-like) and GCB (Germinal Center B-cell like) DLBCL subtypes. One of the most toxic shRNAs in this screen targeted Oct-2, encoding a primarily lymphoid restricted transcriptional activator. Oct2 was identified based on its ability to bind the highly conserved DNA octamer motif (ATGCAAAT) within immunoglobulin (Ig) genes promoters. Oct2 and the ubiquitously expressed Oct1 belong to a group of homeodomain transcription factors that contain the bipartite DNA binding POU domain. The B cell specific co-activator OCA-B interacts with the octamer binding proteins-POU domain enhancing their transactivation potential. Due to the B cell restricted expression pattern, Oct2 and OCA-B were the main candidates to explain the B cell specific Ig promoter activity by virtue of the oct binding proteins interaction with the ?believed? functionally essential octamer element. However, individual targetting of these two genes, failed to demonstrate a compelling role of either Oct2 or OCA-B in Ig transcription. The outstanding feature of the Oct2 and OCA-B knock out mice is the lack of germinal center (GC) development. Twenty years after these first studies we now elucidated the Oct2 controlled genomic network, both in normal and malignant B cells by profiling gene expression changes upon Oct-2 knoced down and merging this data with genome wide assessment of Oct2 binding sites. Surprisingly, these techniques reveled that Oct2 does not always behave in the canonical expected way and that the perfect octamer DNA binding motif does not fully determine Oct2 controled genes. A loss of function screen for genes required for Diffuse Large B Cell Lymphoma (DLBCL) cell proliferation and survival using an RNA interference library, revaled Oct2 as an essential factor for the survival of ABC (Activated B cell-like) and GCB (Germinal Center B-cell like) DLBCL cell lines but not for other B cell malignancies. Cell death was specifically due to Oct2 depletion since the expression of an Oct2 cDNA was able to rescue lymphoma cells from the Oct2 shRNA induced toxicity. Coupling Chromatin immunoprecipitation assay (ChIP) with high-throughput sequencing technologies (ChIP-Seq) uncovered an extensive network of Oct2 target genes in DLBCL cells. More than 60% of the Oct2 target genes also showed OCA-B biding. This Oct2/OCA-B overlaping set of targets was enriched for genes selectively expressed in pan-B cells and GC B cells. We found that Oct2/OCA-B lie upstream many of the main transcription factors known to play an essential role in inducing and mantaining the GC stage of B cell development such as BCL6, MTA3, PU.1, IRF8, SpiB and OCA-B, among others. All these genes are bonafide targets since they are down-regulated following shOct-2 induction at the mRNA as well as the protein level, shown by expression arrays and western blot, both in normal and transformed B cells. Strickingly, among Oct-2 downstream effectors, BCL6 cDNA was enough to rescue DLBCL cells from the Oct2 shRNA lethal effect. The DNA binding of Oct2/OCA-B on BCL6 promoter was confirmed in vivo by single locus ChIP in different GCB-DLBCL cell lines as well as in primary centroblasts isolated from human tonsils. Gel shifts experiments showed Oct2 binding to more than one ?non-canonical? octamer motif within the BCL6 promoter. ChIP-Seq findings opened an entire and exciting new chapter in the Oct2 biology field. Pou transcription factors were suppossed to regulate the activity of octamer containing promoters. Interestingly, Oct2 binds and control the expression of many GC specific genes that do not harbor a canonical octamer motif. Even when Oct2 is expressed through out the different stages of B cell maturation, both mRNA and protein levels are enhanced in centroblasts as compared to pre-germinal center B cells. We found Oct2 capable of inducing its own expression as well as Oct-1 and OCA-B. This autoregulatory circuit might partially account for the Oct2 predominant role in GC specific genes expression control. Genetic aberrations of Oct2 are rare in non Hodgkin lymphoma patients. Nonetheless, lymphoma cells become addicted to the Oct2 controlled network that sustain cell survival, which makes Oct2 an attractive therapeutic target. We showed that BCL6 is one of the critical Oct2 downstream effectors and that all GC and Post-GC B cells tested requiered Oct-2 to survive, indicating that Oct2-directed therapy might have a broder activity spectrum than the BCL6-directed therapy. The Oct2/OCA-B binding interface would be amenable to attack with potential manageable toxicity, since this interaction is exclusively required in GC B cells.