Abstract
Acute myeloid leukemia (AML) is the most common acute leukemia in adults, characterized by high rate of relapse even in recent days. AML is organized in a hierarchy of heterogeneous cell populations and relapse is generally attributable to chemoresistant leukemia stem cells (LSCs). Anthracyclins and cytarabine, which cannot eliminate LSCs, have remained the mainstay of AML therapy for almost 40 years and novel drugs targeting LSCs more efficiently are urgently needed. Toward that end, a potential strategy is to identify cell surface markers that can be differentially regulated in LSCs within the bulk of leukemic cells. Here in this study, focusing on heterogeneity of AML cells, we exploited single-cell gene expression analysis to find a novel cell surface antigen differentially expressed in LSCs, which might be missed by analyses as a whole.
Firstly, we proposed "single-cell leukemia initiating cell (LIC) score", defined as sum of mRNA expression levels of 15 genes specifically expressed in functionally-determined LICs according to public transcriptome datasets (GSE30375), and graded primary AML samples on a single-cell basis. Single CD34+ CD38- cells (n = 66) and CD34+ CD38+ cells (n = 30) from bone marrow (BM) of two AML patients were analyzed. Single-cell gene expression analysis using Fluidigm technology revealed that within 50 candidate cell surface molecules highly expressed in LICs according to public datasets, expression of 19 genes were positively related to the LIC score. Among them, we focused on ALCAM (Activated Leucocyte Cell Adhesion, CD166), a cell adhesion molecule implicated in tumorigenesis, because survival analyses using public datasets showed that high expression of ALCAM was associated with shorter overall survival in AML.
When we examined cell-surface expression of ALCAM in undifferentiated AML cells, ALCAM was highly expressed on CD34+ AML cells compared with CD34+ normal hematopoietic cells. We separated BM cells from AML patients into CD34+ ALCAM high fraction and CD34+ ALCAM low fraction. Colony-forming activities were enriched in CD34+ ALCAM high fraction in vitro (n = 5). And then, we did xenotransplantation model by intravenous injection of human AML BM cells to NSG mice. We show that LICs tended to be enriched within CD34+ ALCAM high fraction in some primary AML samples by using xenotransplantation model.
To elucidate whether ALCAM exerts any biological functions and could be a novel therapeutic target in AML, we established human THP-1 AML cells in which expression of ALCAM was silenced by short hairpin RNA (shRNA). Knockdown of ALCAM reduced proliferation and enhanced chemosensitivity of THP-1 cells to cytarabine by curbing genotoxic stress-induced apoptosis in vitro. THP-1 cells expressing shRNA against ALCAM also showed decreased leukomogenic capacity in a xenotransplantation model. When we suppressed activities of ALCAM using a soluble isoform of ALCAM (soluble ALCAM), which binds to ALCAM and inhibits intercellular ALCAM-ALCAM hemophilic interactions, THP-1 cells expressing soluble ALCAM also showed decreased proliferation, enhanced chemosensitivity and decreased leukomogeneic capacity. Furthermore, we transplanted human primary AML cells transduced with a tetracycline-inducible lentiviral construct encoding soluble ALCAM into NSG mice. After engraftment of human cells, expression of soluble ALCAM was induced. BM engraftment of human primary AML cells expressing soluble ALCAM was reduced, which indicated that decreased BM engraftment was not due to impaired homing, but to leukemogenic ability.
These findings implicate that LICs with high clonogenic capacity were enriched within CD34+ ALCAM high fraction and ALCAM could be a novel therapeutic target of AML cells. Furthermore, our results also suggests single-cell gene expression analysis and a scoring method such as "LIC score" could be applicable to other hematological malignancies and other cancers to identify and exploit a property of heterogenous cancer cells.
Disclosures
No relevant conflicts of interest to declare.
P20. TACI-signalling in multiple myeloma – From the identification as potential therapeutic target by gene expression analysis and functional testing to clinical trials
