Regulation of CD9 Expression by Runx1 in Acute Myeloid Leukemia.
Authors: Racke Frederick , Baird Maureen E , Kowalski Jeanne , McDevitt Michael A 
Published: October 2019

Abstract
CD9 is a tetraspanin molecule that is expressed on precursor B cells, megakaryocytes, and certain acute myeloid leukemias. CD9 has been show to regulate important integrin-mediated functions such as adhesion and motility and has been found to be an independent adverse risk factor in the prognosis of AML. CD9 is also typically expressed in precursor B cell ALL. However, in pediatric precursor B cell ALL harboring the t(12;21) translocation, CD9 is often absent or its expression is very low. This translocation results in the presence of a fusion protein between TEL and Runx1, which is believed to exert a dominant negative effect on normal Runx1 function. This raises the possibility that CD9 may be a Runx1 transcriptional target. In order to study the role of Runx1 in the regulation of CD9 expression, we examined the role of Runx1 in the regulation of CD9 expression in K562 erythroleukemia cells. CD9 expression is induced by PKC signaling which can be blocked by the ERK signaling inhibitor U0126. The proximal CD9 promoter has previously been shown to have PKC-inducible activity. This core promoter has a canonical Runx1 binding site in it. Using chromatin immunoprecipitation assay, we show that Runx1 is recruited to this region of the CD9 promoter in K562 cells by PKC activation. In order to further evaluate the role of Runx1 in CD9 expression, we expressed Runx1-KRAB-ER in K562 cells. This artificial dominant-negative form of Runx1 utilizes the Runx1 DNA binding domain, a KRAB repressor domain, and a modified human estrogen receptor. In K562 cells expressing Runx1-KRAB-ER, CD9 induction by PKC activation was significantly reduced in the presence of 4-hydroxy-tamoxifen, which releases ER-fused proteins sequestered with heat shock proteins. This data demonstrates that inhibition of normal Runx1 activity interferes with induction of CD9 expression.
Similar to precursor B cell ALL with the t(12;21), AML with the t(8;21) has Runx1 fused to ETO and the Runx1/ETO fusion protein has also been reported to repress normal Runx1 function. In order to evaluate CD9 expression in different cytogenetically defined subgroups of AML, we interrogated two large, publicly available AML microarray gene expression data bases (referenced below). We evaluated the expression of CD19, known to be upregulated in t(8;21) AMLs, and IL-3, a known target of Runx1 transcription, as well as CD9. As compared with expression in t(15;17), inv(16;16), and those with normal karyotypes, expression of IL-3 and CD9 were significantly reduced in t(8;21) AML as compared with the other subgroups. In contrast, CD19 expression was greater in t(8;21) AML than the other subgroups. These findings show that CD9 expression is specifically reduced in t(8;21) AMLs as compared to the other cytogenetically-defined subgroups. These findings suggest that the Runx1/ETO protein may be actively repressing CD9 expression in t(8;21) AML. In order to test this hypothesis, we examined the expression of CD9 in Kasumi-1 cells which harbor this translocation. We could not detect CD9 expression at the mRNA or protein levels, nor could PKC activation induce its expression. However, if Kasumi-1 cells were treated with valproic acid, which has been previously shown to disrupt the Runx1/ETO-HDAC1 repressor complex, then CD9 induction by PKC activation is restored. Taken together, these data provide strong evidence that Runx1 regulates CD9 expression and that the absence of CD9 expression in acute leukemias harboring Runx1 fusion proteins likely relates to their repressive effects on normal Runx1 regulation of CD9 expression.