Targeting Aberrant Expression of CD81 Impacts Cell Adhesion and Migration, Drug Resistance and Prognosis of Acute Myeloid Leukemia

Introduction: Treatment outcome of acute myeloid leukemia (AML) remains dismal due to toxicity of current therapies and poor response rates to standard chemotherapy. Therefore, better methods to monitor treatment response and novel targets to develop new therapeutic strategies against, are urgently needed. Recently, we have reported CD81, also known as TAPA-1, as a novel prognostic marker in AML. CD81 belongs to the transmembrane tetraspanin family, and is implicated in a variety of cellular functions. Notably, CD81 regulates cell migration and invasion and in solid cancers has been shown to contribute to tumor growth and metastasis. In this study, we aimed to investigate how these cell functions may be altered by CD81 and how this could impact treatment outcome in AML. To validate the prognostic impact of CD81, we have increased the number of AML patients of our prior cohort (n=169) and the median follow-up time was updated to 2.8 years. Furthermore, we modified CD81 expression in established AML cell lines for in vitro and in vivo functional studies. Finally, using our defined AML model, we tested in vivo a blocking mouse antibody against hCD81 as a therapeutic option.

Materials and Methods: CD81 expression was determined on a cohort of 169 de novo AML patients by flow cytometry analysis as we have described previously. We have used three AML cell lines with varying degree of CD81 expression (HNT-34, Oci-AML3, U937). To modify CD81 expression, we used either overexpression by full-length cDNA transfection or shRNA mediated knock-down. Drug resistance to standard chemotherapy agents was tested in vitro . Cell adhesion was measured using fibronectin coated plates and using a HS5 stromal co-culture assay. Differential in cell migration ability was examined by HS5 co-culture Transwell^® plates. Finally, NSG mice were intravenously injected with 1x10^{^6} AML cells with varying CD81 expression levels to evaluate its impact on blast homing, engraftment efficiency, tumor invasion and overall survival.

Results: We have validated the prognostic impact of CD81 expression on AML blasts from adult patients; showing a significant association with disease-free, event-free and overall survival even after adjusting for age at diagnosis and European Leukemia Net (ELN) classification. Accordantly, CD81 positive cells were 30 to 50% more resistant to daunorubicin and cytarabine. Overexpression of CD81 increased AML cell adhesion and migration and concordantly, shRNA knock-down had the inverse effect. Furthermore, in vivo higher CD81 expression increased blast homing and engraftment efficiency. Tumor invasion measured in peripheral blood, bone marrow and spleen was higher in mice engrafted with CD81 overexpressing AML cells and subsequently survival was significantly shorter in those mice. More importantly, treatment with the blocking antibody vice versa decreased tumor invasion and improved survival of both moderate and high CD81 expressing AML engrafted mice compared to isotype control treated mice.

Conclusion: In this study, we provide functional evidence for therapeutic targeting of CD81 in AML. In our AML cohort, we observed increased relapse rates and poorer overall survival in AML patients with higher proportion of CD81 positive cells. Our controlled cell line models provided functional evidence on how altered CD81 expression maybe an underlying mechanism explaining variance in clinical outcome in AML.