conceived, coordinated and supervised the project, designed experiments, analysed data and wrote the paper. Competing financial interests: The authors declare no competing financial interests.. but abolishes leukemia development. LAIR1 induces activation of SHP-1, which acts as a phosphatase-independent signaling adaptor to recruit CAMK1 for activation BIO-5192 of downstream CREB in AML cells. The LAIR1/SHP-1/CAMK1/CREB pathway sustains the survival and self-renewal of AML stem cells. Intervention in the signaling initiated by ITIM-containing receptors such as LAIR1 may result in successful treatment of AML. Introduction Leukemias are malignant blood diseases characterized by uncontrolled overproduction of hematopoietic progenitors or terminally differentiated leukocytes. Acute myeloid leukemia (AML) is the most common adult acute leukemia. Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and is also diagnosed in adults. Current chemotherapies are not particularly successful in treating AML and some ALL. For example, despite continuous treatment, the majority of the AML patients relapse within 5 years 1. It has been suggested that leukemia stem cells, a small population of stem-like cancer cells that have the capacity for indefinite self-renewal 2, 3, are responsible for initiation and relapse. To effectively inhibit the activity of leukemia stem cells and treat acute leukemia, new molecular targets and therapeutic approaches need to be identified. It is hypothesized that leukemia stem cells reside in a bone marrow BIO-5192 microenvironment or niche and play an important role in regulation of initiation, differentiation, migration, and chemoresistance of leukemia 4-6. In addition, systematic inflammatory and oxidative factors are critical extrinsic factors for leukemia development 7. Specific surface receptors on leukemia cells presumably interact with the extrinsic environment and regulate the fates of leukemia cells through unique signaling pathways. These include tyrosine kinase receptors 8, cytokine receptors 9, chemokine receptors 10, adhesion molecules and integrins (such as CD44, CD49d, integrin beta 3, CD47, CD96, CD33) 11-16, Notch 17, Wnt receptors 18, 19, Smoothened 20, receptors for TGF-beta family 21, and other surface molecules. Some of these receptors mediate signaling that differs in leukemia cells from that in normal hematopoietic cells, which should enable the development of novel anti-leukemia strategies 4, 16, 22-24. In our attempt to identify stem cell and leukemia related surface receptors, we isolated human leukocyte immunoglobulin BIO-5192 (Ig)-like receptor B2 (LILRB2) and mouse paired Ig-like receptor (PirB) as receptors for angiopoietin-like proteins (Angptls) 25. These receptors contain immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their intracellular domains and are classified as inhibitory receptors because ITIM motifs can recruit phosphatases like SHP-1, SHP-2, and SHIP to negatively regulate cell activation 26-28. We showed that PirB is expressed on AML cells and required for AML development in mouse leukemia models 25. Nevertheless, it is unknown whether ITIM-receptors have direct effects on leukemia cells. Here we demonstrated that some ITIM-receptors are expressed on leukemia cells and directly support leukemia development. We further discovered a signaling pathway initiated from the LAIR1, a representative ITIM-receptor. This identified ITIM-receptor signaling pathway may represent an ideal target for AML treatment. Our demonstration that some ITIM-receptors are not inhibitory but supportive of leukemia development will alter the current understanding of the mechanisms of cancer pathogenesis, cell signaling, and therapeutic approaches. Results The expression of Rabbit polyclonal to Neurogenin1 some ITIM-receptors inversely correlates with AML development To identify potential surface receptor genes that support leukemia development, we performed an analysis of the relationship between gene expression and the overall survival of AML patients. To our surprise, while the expression of 2 out of 58 ITIM-receptors positively correlated with the overall survival of acute myeloid leukemia (AML) patients, 20 of these receptors had negative correlation between expression and survival (Supplementary Fig. 1a, Supplementary Table 1). To determine the functions of these ITIM-receptors, we inhibited expression of these receptors individually in human leukemia cell lines using lentivirus-encoded small hairpin RNAs (shRNAs) and found that BIO-5192 cell growth was blocked when expression of certain receptors was silenced (Fig. 1A, Supplementary Fig. 1b). These results suggest that some ITIM-receptors directly support human leukemia cell growth. Open in a separate window Fig 1 Lair1, a representative ITIM-receptor, is essential for the growth of human leukemia cell linesA, Effects of shRNA-mediated silencing of expression of indicated ITIM receptors on growth of MV4-11 and NB4 AML cells. The cells were counted on day 6 post-infection, and normalized to cells treated with scrambled shRNAs (mean s.e.m., Student’s t-test; n=3 samples, sh-LAIR1 *** p 0.0001; sh-KIR3DL1 ** p = 0.0051; sh-PECAM1 ** p = 0.0061; sh-SIGLEC11 ** p = 0.0089; sh-SIGLEC6 * p = 0.0326; sh-LILRB1 * p = 0.0412; sh-LILRB2 ** p = 00059; sh-LILRB3 *** p 0.0001; sh-LILRB4 *** p 0.0001; sh-KIR2DL2 * p = 0.0182; sh-KIRG1 ** p = 0.0096). B, Flow cytometry analysis showing that LAIR1 is highly expressed on the cell surfaces of RCH-ACV,.