PF219 THE LEDGF/P75 HOMOLOG HRP2 IS NOT IMPORTANT FOR THE INITIATION OF MLL‐REARRANGED LEUKEMIA
Authors: S. Van Belle, S. El Ashkar, K. Cermáková, F. Matthijssens, S. Goossens, A. Canella, F. Christ, P. Van Vlierberghe, V. Veverka, J. De Rijck, Z. Debyser
Journal: HemaSphere (2019)

Background: HDGF‐related protein 2 or HRP2 is a member of the Hepatoma‐Derived Growth Factor‐related protein family. Through its N‐terminal PWWP domain it associates with methylated histone tails and functions as such as a chromatin reader. HRP2 also features an integrase binding domain (IBD), known for its interaction with cellular and viral proteins. Little is known about the cellular function of HRP2, yet it has been linked to the malignant hepatocellular carcinoma. HRP2 has mainly been studied in comparison with its paralog, Lens Epithelium Derived Growth Factor (LEDGF/p75), suggesting similar but not identical physiological roles for both proteins. Previous publications showed an important role for LEDGF/p75 in the development of MLL‐rearranged leukemia (MLL‐r). Interaction between the IBD domain of LEDGF/p75 and the N‐terminal part of an oncogenic MLL fusion protein results in aberrant expression of MLL target genes, which in turn are important for normal hematopoiesis. Aims: Since HRP2 is functionally related to LEDGF/p75 but its potential role in MLL‐r remains unknown, we aimed to investigate a potential role for HRP2 in MLL‐r leukemia. Methods: Protein‐protein interactions were investigated with immunoprecipitation and detected by western blot. Interactions were confirmed using recombinant proteins in Alpha Screen and NMR experiments. The role of HRP2 in normal hematopoiesis was investigated using a systemic HRP2 knockout mouse model. The role of HRP2 in mixed lineage leukemia was determined using lentiviral‐mediated knock down and rescue experiments in, and colony forming assay with human cell lines. In addition, we performed colony forming assay and bone marrow transplantation with primary mouse bone marrow cells, transduced with an MLL‐fusion containing vector. Results: We show for the first time that HRP2 directly interacts with MLL with similar affinities compared to LEDGF/p75. In addition, NMR analysis indicates a similar interface between MLL and either IBD domain. However, the HRP2‐MLL interaction occurs independently of MENIN, the stabilizing adaptor of the MLL‐LEDGF/p75 interaction. The systemic HRP2 knockout mice survived with wild type blood counts. Only upon serial plating of hematopoietic stem cells, a decrease in proliferation‐potential of HRP2 knockout cells was observed. Knocking down HRP2 in leukemic cell lines and primary murine MLL‐transformed cells resulted in a decreased transformation‐potential. In contrast, Hrp2 knockout cells from the systemic knock out mice were efficiently transformed by MLL fusions, suggesting that Hrp2 might be dispensable for the initiation of MLL‐rearranged leukemia. Summary/Conclusion: In summary, we report for the first time that HRP2 is able to interact with MLL, yet in a MENIN‐independent manner. However, our results suggest that HRP2 might not be important for the initiation of mixed lineage leukemia, pointing towards a different role for HRP2 and its homolog LEDGF/p75 in MLL‐r.