LR11 Is a Novel Surface Marker for Normal Leukocytes and Leukemia Cells
Authors: Takeuchi Masahiro , Ohwada Chikako , Sakai Shio , Takeda Yusuke , Abe Daijiro , Shimizu Naomi , Sakaida Emiko , Kawaguchi Takeharu , Takubo Kohei , Ebinuma Hiroyuki , Fukamachi Isamu , Yokote Koutaro , Iwama Atsushi , Schneider Wolfgang J , Bujo Hideaki , Nakaseko Chiaki 
Published: October 2019

Abstract
Abstract 4834

Introduction:
LR11 (also called SorLA or SORL1) is a type I membrane protein, from which a large extracellular part, sLR11, is released by proteolytic shedding. LR11 plays a key role in the migration of undifferentiated vascular smooth muscle cells, and circulating sLR11 is a biomarker of carotid intima-media thickness. In accordance with sLR11 levels correlating with the fraction of immature vascular cells, human CD34+CD38- immature hematopoietic precursors display high levels of LR11 mRNA. We investigated the expression of LR11 in normal leukocytes, leukemia cell lines and acute leukemia cells.

Methods:
A2-2-3 anti LR11 monoclonal antibody was used for immunoblotting. Biotinylated or FITC-conjugated anti LR11 monoclonal antibodies, M3 and R15 were used for flow cytometric analysis and immunohistochemistry. Normal mononuclear cells were obtained from healthy volunteer donors. Leukemia cells were obtained from patients' bone marrow or peripheral blood. LR11 protein levels and sLR11 in the culture supernatant of human leukemic cell lines were examined by Western blotting and ELISA using specific monoclonal antibody against LR11. The expression of LR11 mRNA of the cells was examined by Real-Time PCR. Flow cytometric analysis of cell surface LR11 was performed with desktop cell sorter JSAN (Bay Bioscience).

Results:
Most human leukemia cell lines expressed high level of LR11 mRNA and protein. sLR11 was also detected in the culture supernatant. The levels of LR11 mRNA, the amount of cellular LR11 protein, and the amount of released sLR11 protein were significantly correlated with each other. Flow cytometric analysis of peripheral leukocytes using the anti-LR11 mAb M3, showed expression of LR11 in most CD14+ monocytes. LR11 was not significantly expressed on most T cells (CD4+, CD8+), B cells (CD19+), or granulocytes. However, the leukemia cell lines HL-60 (acute promyelocytic), CCRF-SB (lymphoblastic), and U937 (monocytic), but not K562 (chronic myelogenous) expressed LR11. Since LR11 is expressed by leukemia cells of different origins, we explored the expression of LR11 on the surface of patients' leukemia cells. We have examined 7 AML cases (M0, M1, M2, M3, M4, M5 and M6) and 3 ALL cases. Although expression level of LR11 differs among these cases, LR11 was detected in every case except one ALL case. The most dramatic M3-stained population was the clonally expanded CD19+ mononuclear fraction in MLL-AF4 positive early precursor B acute lymphoblastic leukemia (ALL). In addition, more than 50% blastic cells were positive for LR11 in a Philadelphia chromosome positive ALL patient. Over 50% of CD34+ mononuclear cells in AML (M0) were LR11-positive, whereas LR11-positive blasts predominated in the CD38- fraction. The majority of mononuclear cells in AML (M4) with high CD11b-expression were also LR11-positive. Thus, LR11 is specifically expressed on the surface of leukemic blasts in both ALL and AML. Furthermore, immunohistochemistry of bone marrow clot sections of AML and ALL revealed that cytoplasm of leukemia cells are specifically reacted against the anti-LR11 antibody. Thus, LR11 is expressed both in the cytoplasm and on the cell surface of acute leukemia cells.

Conclusion:
LR11 is specifically and highly expressed on cell surface of acute leukemia cells in addition to normal leukocytes. Together with our finding that sLR11 is a novel marker for acute leukemia, the identification of novel surface antigen sheds light on leukocyte biology and leukemia cell development.

Disclosures:
No relevant conflicts of interest to declare.