SPARC contributes to Leukemia Growth and Aggressive Disease in Acute Myeloid Leukemia (AML)

Abstract 773

Altered expression of SPARC, [secreted protein, acidic, cysteine-rich (osteonectin)] a gene encoding a protein that modulates cell-matrix interactions, has been reported in cancer and leukemia, but its precise contribution to malignant transformation is unknown. We previously reported that SPARC was among the most upregulated genes in the gene-expression profile (GEP) associated with IDH2-R172 mutations (mut; Marcucci et al. JCO 2010;28:2348) and among the most downregulated genes associated with NPM1 mut (Becker et al. JCO 2010;28:596) in cytogenetically normal (CN) AML. Both mutations have strong but opposite impact (IDH2-R172 mut, adverse; NPM1 mut, favorable) on clinical outcome. Thus, we hypothesized that SPARC may act as an oncogene and contribute to AML aggressiveness. To determine functional and mechanistic activities of SPARC in AML, we conducted gain- and loss-of-function experiments in cell lines and primary blasts collected from patients under the OSU Leukemia Tissue Bank protocol. In vitro, ectopic expression and knockdown of SPARC in THP-1 and Kasumi-1 AML cells led to increase (2.5-fold P=0.03) and decrease (45%, P=0.03) in colony forming ability, respectively. Clonogenic assays in SPARC- vs empty vector (EV)-infected AML blasts also showed 2-fold increase in colony-formation (P=.01) supporting a role of SPARC overexpression in promoting leukemia growth. The leukemogenic activity of the SPARC protein was likely mediated by its binding to αvβ3 integrin and in turn by the activation of the kinase ILK. This induced GSK3β phosphorylation (p) and in turn activation of β-catenin, previously reported to contribute to AML. Indeed, while ILK knockdown abolished SPARC-induced p-GSK3β, ectopic SPARC expression led to a 4-fold increased p-GSK3β, measured by p-GSK3β (Ser-9) immunoblot assay, and to stabilization of β-catenin, measured by p-β-catenin(Ser-552) and p-β-catenin(Ser-675) immunoblot assays in AML blasts. As a result, we observed increase in translocation of β-catenin to the nucleus in SPARC overexpressing cells by confocal microscopy. Nuclear β-catenin led to increased activity of the TCF/LEF transcription factors as shown by a TCF/LEF luciferase reporter assay (5.8-fold, P<.001) and the expression of MYC, a TCF/LEF target gene. In contrast, SPARC knockdown decreased MYC expression (50%; P=0.05) in AML blasts. In vivo, mice engrafted with THP-1 cells overexpressing SPARC exhibit more aggressive disease, significant splenomegaly (P=.008), and hepatomegaly with increased number and size of hepatic granulocytic sarcoma-like lesions when compared with mice engrafted with THP-1-EV cells.

The results in the preclinical models prompted us to evaluate whether higher SPARC expression associated with aggressive human disease. Thus, SPARC expression levels were measured by NanoString nCounter assay in a cohort of CN-AML patients (pts; n=362; age, 18–83 years) comprehensively characterized for mutations in the NPM1, FLT3, CEBPA, WT1, TET2, MLL, IDH1, IDH2, RUNX1, ASXL1 and DNMT3A genes, and treated with cytarabine-anthracycline based regimens. Higher SPARC expressers were more likely classified in the European Leukemia Net (ELN) Intermediate-I (CEBPA wt, NPM1 mut with FLT3-ITD, or NPM1 wt) than the ELN Favorable Genetic Group (CEBPA mut and/or NPM1 mut without FLT3-ITD; P<.001). Higher expression of SPARC associated with lower complete remission (CR) rates (P<.001; OR .75), shorter disease-free (DFS; P<.001; HR 1.17) and overall (OS; P<.001; HR 1.16) survival. In multivariable analyses, higher SPARC expression remained associated with worse CR rates (P<.001), once adjusted for RUNX1, white blood count (WBC) and age group (<60 vs >60 years); shorter DFS (P=.07), once adjusted for FLT3-ITD, RUNX1, WBC and age group; and shorter OS (P<.001), once adjusted for FLT3-ITD, WT1, ASXL1 and age group. Gene and microRNA (miR)-expression profiling associated SPARC with CD34, BAALC and MN1 among the most upregulated and the HOX genes among the most downregulated genes, and with the hematopoietic stem cell-specific miR-126 and miR-130a among the most upregulated miRs.

In conclusion, high SPARC expression contributes to aggressive AML growth via β-catenin activation-mediated mechanisms. SPARC overexpression independently predicted worse outcome in CN-AML pts, and may represent a potentially novel prognostic marker and therapeutic target in AML.