Hasan M K, Yu J, Chen L, Cui Bing, Widhopf Ii G F, Rassenti L, Shen Z, Briggs S P, Kipps T J
Moores Cancer Center, University of California San Diego, La Jolla, CA, USA.
Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA.
Leukemia. 2017 Dec;31(12):2615-2622. doi: 10.1038/leu.2017.133. Epub 2017 May 3.
ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL). We found that ROR1 associates with hematopoietic-lineage-cell-specific protein 1 (HS1) in freshly isolated CLL cells or in CLL cells cultured with exogenous Wnt5a. Wnt5a also induced HS1 tyrosine phosphorylation, recruitment of ARHGEF1, activation of RhoA and enhanced chemokine-directed migration; such effects could be inhibited by cirmtuzumab, a humanized anti-ROR1 mAb. We generated truncated forms of ROR1 and found its extracellular cysteine-rich domain or kringle domain was necessary for Wnt5a-induced HS1 phosphorylation. Moreover, the cytoplamic, and more specifically the proline-rich domain (PRD), of ROR1 was required for it to associate with HS1 and allow for F-actin polymerization in response to Wnt5a. Accordingly, we introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1 PRD at positions 784, 808, 826, 841 or 850 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1 mutants, ROR1 had impaired capacity to recruit HS1 and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1 to phosphorylate HS1 or activate ARHGEF1, and was unable to enhance CLL-cell motility. Collectively, these studies indicate HS1 plays an important role in ROR1-dependent Wnt5a-enhanced chemokine-directed leukemia-cell migration.
ROR1（受体酪氨酸激酶样孤儿受体1）是一种保守的癌胚表面抗原，在慢性淋巴细胞白血病（CLL）中表达。我们发现，在新鲜分离的CLL细胞或用外源性Wnt5a培养的CLL细胞中，ROR1与造血谱系细胞特异性蛋白1（HS1）相关联。Wnt5a还诱导HS1酪氨酸磷酸化、ARHGEF1的募集、RhoA的激活并增强趋化因子导向的迁移；这些效应可被人源化抗ROR1单克隆抗体cirmtuzumab抑制。我们构建了ROR1的截短形式，发现其富含半胱氨酸的胞外结构域或kringle结构域对于Wnt5a诱导的HS1磷酸化是必需的。此外，ROR1的胞质结构域，更具体地说是富含脯氨酸的结构域（PRD），是其与HS1结合并允许响应Wnt5a发生F-肌动蛋白聚合所必需的。因此，我们在ROR1 PRD中潜在的SH3结合基序的第784、808、826、841或850位将脯氨酸（P）替换为丙氨酸（A）进行单氨基酸替换。与野生型ROR1或其他ROR1突变体相比，ROR1响应Wnt5a将HS1和ARHGEF1募集到ROR1的能力受损。此外，Wnt5a不能诱导表达ROR1的细胞使HS1磷酸化或激活ARHGEF1，并且无法增强CLL细胞的运动性。总的来说，这些研究表明HS1在ROR1依赖的Wnt5a增强趋化因子导向的白血病细胞迁移中起重要作用。
