ITIM (immunoreceptor tyrosine-based inhibition motifs) motifs in the cytoplasmic tail of inhibitory receptors recruit inhibitory phosphatases. Upon ligation, these phosphatases inhibit signal transduction from tyrosine kinases including BCR-ABL1. ITIM-receptors are critical in the control of immune responses of B and T cells. Surprisingly, we found that a number of inhibitory ITIM-receptors are expressed at very high levels on the surface of patient-derived Ph+ acute lymphoblastic leukemia (ALL) cells compared to normal pre-B cells. In this study, three central ITIM-receptors were identified and analyzed for their function in Ph+ALL: PECAM1 (platelet/endothelial cell adhesion molecule 1/CD31), CD300A and LAIR1 (leukocyte-associated immunoglobulin-like receptor 1).
Microarray data showed mRNA levels of PECAM1, CD300A and LAIR1 were about 5-fold higher (p<0.005) in Ph+ ALL (n=15) than normal human pre-B cells (n=8). We confirmed this difference at the protein level by flow cytometry studying patient-derived Ph+ALL (n=11) and pre-B cells from normal bone marrow samples (n=2).
These findings seem counterintuitive because signaling from these receptors would attenuate the signaling strength downstream of BCR-ABL1. Importantly, we found that high expression levels of ITIM-receptors are predictive of poor outcome in two clinical trials. In the COG trial (P9906; n=207) for children with high-risk ALL, we found that mRNA levels of PECAM1, CD300A and LAIR1 at diagnosis positively correlated with early minimal residual disease (MRD) findings on day 29 (p<0.0005), and negatively correlated with overall survival (OS) rate (p<0.02) or relapse free survival (RFS) rate (p<0.05). In ECOG trial E2993 for adults with ALL (n=215), we found that PECAM1 mRNA level in ALL patients negatively correlated with OS rate (p=0.0285). These results collectively indicate that inhibitory ITIM-receptors contribute to the course of human ALL disease.
To study the role of PECAM1, CD300A and LAIR1 in Ph+ ALL in genetic experiments, we transformed pre-B cells from Pecam1−/−, Cd300a−/− and wildtype mice with BCR-ABL1. Compared to wildtype ALL cells, Pecam1−/− or Cd300a−/− ALL cells showed increased ROS levels. Consistent with higher levels of ROS, the Pecam1−/− and Cd300a−/− ALL cells accumulate p53, p21 and p27 protein, are prone to G0/G1cell cycle arrest and cellular senescence. Colony forming assays revealed that Pecam1- and cd300-deficient leukemia cells also formed 10-fold fewer colonies in methyl cellulose compared to wildtype ALL cells (p<0.0001). For Lair1, we performed genetic loss-of-function experiments by inducible activation of Cre in Lair1fl/fl leukemia cells. Inducible deletion of Lair1 resulted in drastic upregulation of ROS, accumulation of Arf, p53 and p21, cellular senescence and subsequent leukemia cell death. Transplanting Lair1fl/fl ALL cells into NOD-SCID mice, we found that Lair1 deletion resulted in rapid leukemia regression and prolonged survival of recipient mice. Leukemia cell death caused by Lair1 deletion could be rescued by overexpression of the inhibitory phosphatase Ptpn6 (SHP1) indicating that the protective effect of the Lair1 is indeed mediated by ITIM-based recruitment of inhibitory phosphatases. To test whether these findings are also relevant to other subtypes of ALL, we used a model for NRASG12D-driven ALL. BCR-ABL1 (∼25%) and NRAS lesions (∼30%) account for more than half of cases of ALL. Consistent with our findings with BCR-ABL1, NRASG12D Pecam1−/−, Cd300a−/−leukemia cells were prone to G0/G1 cell cycle arrest and cellular senescence (p<0.01).
These results indicated that inhibitory ITIM-receptors are critical regulators of oncogenic signaling strength in BCR-ABL1 and NRAS-driven ALL. Deficiency of ITIM-receptor signaling can be rescued by overexpression of the PTPN6 phosphatase. These findings are of particular relevance, because they identify ITIM-receptors and inhibitory phosphatases as members of a fundamentally novel class of therapeutic targets. The concept of pharmacological perturbance of oncogenic signaling equilibrium in leukemia cells by inhibition (e.g. TKI-treatment) or exaggeration of signaling strength (e.g. blockade of ITIM-receptors) may lead to the discovery of multiple additional therapeutic targets and broaden our repertoire of currently available pathways for therapeutic intervention.
No relevant conflicts of interest to declare.
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