Abstract
Abstract 1961
Poster Board I-984
LRF (Leukemia/Lymphoma Related Factor) is a transcriptional repressor originally identified as an interaction partner of the oncoprotein BCL6 (B cell Lymphoma 6). We previously found that LRF acts as a proto-oncogene by repressing tumor suppressor ARF (Alternative Reading Frame, also known as p19 in mice and p14 in humans) and is highly expressed in 60-80% of human Non-Hodgkin Lymphoma (NHL) cases (Maeda et al., Nature 2005). LRF was also found to be indispensable for hematopoietic stem cells (HSCs) to commit to the B cell lineage by opposing Notch function (Maeda et al., Science 2007). Considering that: 1) LRF is normally expressed in Germinal Center B cells (GCB) and overexpressed in NHL tissues and 2) LRF opposes Notch function to maintain normal B cell fate at HSC/progenitor levels, we explored the role of LRF in B cell development and its functional interaction with the Notch pathway in vivo. Upon T cell dependent (TD) immunization, GC formation was severely impaired in secondary lymphoid organs of B cell specific LRF conditional knockout mice (LRFflox/flox mb1-Cre+). While a GC reaction was robustly induced in control mice upon immunization, only few GCB cells were noted in secondary lymphoid organs of LRFflox/flox mb1-Cre+ mice. To assess functional significance of LRF loss in antigen response in vivo, titers of class-switched immunoglobulin (Ig) were measured in the serum; baseline serum titers of IgG1, IgG2b and IgG3 were perturbed, and the primary and secondary antibody response against the TD antigen was impaired in LRFflox/flox mb1-Cre+ mice. Absolute numbers of memory B cells and long-lived BM plasma cells were reduced in LRFflox/flox mb1-Cre+ mice 20 wk after immunization. To determine the cause of defective GC formation, apoptosis and proliferation of GCB cells were examined by FACS. While proportions of apoptotic (AnnexinV positive) GCB cells were similar, regardless of genotypes, LRF deficient GCB cells failed to proliferate upon antigen stimuli. Short-term kinetic analysis demonstrated 5-ethynyl-2'-deoxyuridine (EdU) incorporation was markedly decreased in LRF deficient GCB cells and that the proportion of GCB cells in S phase was reduced in LRFflox/flox mb1-Cre+ mice. In agreement with these findings, quantitative RT-PCR analysis in FACS-sorted GCB cells demonstrated up-regulation of p19Arf and p21, but not p53, mRNA levels in LRF deficient GCB cells. Up-regulation of p19Arf protein levels was also observed in Western Blots. Furthermore, microarray analysis and subsequent Gene Set Enrichment Analysis in FACS-sorted GCB cells showed signatures of defective proliferation, further implicating a critical role for LRF in GCB cell proliferation. Signals mediated by Notch2 are necessary for transitional B cells to commit to the marginal zone B cells (MZB). Inactivation of a component of the Notch pathway in mice resulted in no MZB development and increased follicular B cells (FOB). On the contrary, deletion of the MINT/SHARP gene, a suppressor of Notch signaling, lead to increase of MZB cells and concomitant reduction of FOB cells, indicating that Notch induces MZB cell fate at the transitional B cell stage. While B cell development in the BM was grossly normal, a reduction of FOB cells and a concomitant increase of MZB cells were observed in LRFflox/flox mb1-Cre+ mice. Since the phenotype was reminiscent of that seen in MINT/SHARP knockout mice and opposite to that observed in Notch2 knockout mice, we hypothesized that LRF antagonizes Notch2 mediated signal during the FOB vs. MZB fate determination process. To test this, LRF/Notch2 double knockout mice (LRFflox/flox Notch2flox/flox mb1-Cre+) were established and their mature B cell compartments analyzed. As expected, loss of the Notch2 gene led to an increase of FOB cells and decrease of MZB in LRFflox/flox mb1-Cre+ mice, suggesting that LRF regulates FOB vs. MZB fate in a Notch2 dependent manner. However, Notch2 deficiency did not restore GC formation in LRFflox/flox mb1-Cre+ mice. In summary, our genetic studies strongly indicate that the proto-oncogene LRF is required for normal mature B cell development and function via distinct mechanisms. We propose that LRF is necessary for mature B cell fate by blocking Notch2-mediated signals and plays a critical role in GCB cell proliferation via suppressing p19Arf mediated cell cycle arrests. Our findings provide a further rational for targeting LRF for the treatment of B cell malignancies as well as autoimmune diseases.
Disclosures:
No relevant conflicts of interest to declare.
Density of the Notch ligand Delta1 determines generation of B and T cell precursors from hematopoietic stem cells
