Novel Role of LMO2 in DNA Repair Control in Diffuse Large B Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common and aggressive type of Non-Hodgkin Lymphoma (NHL). However, about 40% of patients still do not respond to the current therapy and die. Therefore, there is an urgent need for developing new strategies to improve their outcome. We have shown that LIM domain Only 2 (LMO2) expression is increased in germinal center (GC) B-lymphocytes and GC-derived lymphomas. LMO2 protein is the best single prognostic biomarker which high expression is associated with longer survival of DLBCL patients treated with R-CHOP therapy, irrespective of the cell of origin of these tumors. LMO2 is indirectly involved in gene regulation by forming multipartite DNA-binding complexes with other transcription factors. LMO2 plays an important role in the normal development of hematopoietic and endothelial system, in erythropoiesis, and functions as an oncogene in T-cell acute lymphoblastic leukemia. However, its role in B cell biology as well as B-cell lymphoma is still unclear. We have recently shown that LMO2 over-expression in DLBCL results in genomic instability (Cubedo et al Blood 2012). These studies suggested that LMO2 expression could compromise proper DNA repair.

To address whether LMO2 has a role in DNA repair, we evaluated the capacity of DLBCL cells with high expression of LMO2 (DLBCL^LMO2+) to repair DNA double-strand breaks (DSBs). DNA DSBs are repaired by two major pathways: Non-homologous end joining (NHEJ) and Homologous recombination (HR). Therefore, next we evaluated whether LMO2 affects the activity of these DSBs repair pathways. To this end, we analyzed the core factors: 53BP1 for NHEJ and Rad51 for HR. We exposed DLBCL^LMO2+ and controlcells to genotoxic agents used in DLBCL therapy to produce DSBs and evaluated 53BP1 and Rad51 recruitment to DSBs site via immunofluorescence (IF) studies. Phosphorylated form of H2AX, known as gammaH2AX was used as a DSB molecular marker. In these studies we found a similar frequency of 53BP1 foci in both DLBCL^LMO2+ and control cells. These results suggested that NHEJ is active irrespective of LMO2 protein levels. However, DLBCL^LMO2+ cells showed a decreased frequency of Rad51 foci after DNA damage. Importantly, DLBCL^LMO2+ cells showed similar Rad51 protein levels and cell-cycle distribution when compared to control cells. These results showed a deficient HR pathway in DLBCL^LMO2+ cells and suggested a role for LMO2 in the control of HR. In support of this, overexpression of LMO2 in control cells produced a similar HR-deficiency as observed in DLBCL^LMO2+ cells. In addition, knockdown of LMO2 in DLBCL^LMO2+ cells restored Rad51 foci formation to the levels observed in control cells. These results showed that HR activity in DLBCL cells depends on LMO2 protein levels. It is well known that deficiency in HR can be rescued by deleting 53BP1, which is a HR inhibitor. Hence, in order to address the question whether deletion of 53BP1 in DLBCL^LMO2+ cells can rescue HR pathway, we knockdown 53BP1 in DLBCL^LMO2+ cells via shRNA and examined the frequency of Rad51 foci after exposure to different genotoxic agents. Interestingly, we found an increase in Rad51 foci formation in DLBCL^LMO2+sh53BP1 cells to near control cells levels. This result shows that LMO2-dependent inhibition of HR depends on 53BP1 and suggests that LMO2 and 53BP1 form a complex at the DSB site. Two observations support this hypothesis: 1) LMO2 co-localize with 53BP1 at the DSB site; and 2) LMO2 expression increases the frequency of 53BP1-Rif1 complex at the DSB site. Since Rif1 (Rap1-interacting factor 1) is one of the 53BP1 effector proteins that inhibits HR, here we propose that LMO2 inhibition of HR is through the stabilization of the 53BP1-Rif1 complex.

Our study reveals a novel role for LMO2 in the control of genome stability by controlling homologous recombination repair activity. Defect in HR pathway in LMO2 expressing cells could explains why DLBCL patients with higher level of LMO2 are more sensitive to chemotherapy, and its significance as the best prognostic marker for survival. Our studies can potentially give new insight for the treatment of DLBCL patient and improve their outcome.