ERG Overexpression Induces Cell Adhesion Mediated Drug Resistance in Acute Leukemia

Abstract 1389

Overexpression of the ETS transcription factor ERG in subtypes of acute lymphoblastic and myeloid leukemias has been correlated with a poor prognosis. The underlying ERG mediated chemotherapy resistance of the leukemic cells may explain the link of poor outcome to ERG expression. We recently observed that ERG overexpression in K562 cells induced adhesion and morphological changes with elongated with bi-directional protrusions. This morphological transformation was mediated by WNT11, a direct target of ERG in acute leukemia. Herein, we determined the ERG transcriptional program responsible for cell adhesion and drug resistance potential. A genome wide transcriptional profiling was performed to identify candidate genes responsible for the observed cell shape changes. mRNA from doxycycline induced K562 cells harbouring tet-on inducible ERG expression constructs (pTRE-Tight-BI-DsRed-ERG) were analyzed with Affymetrix GeneChip (U133 2.0 plus). Non induced clones were used as a control. Genes significantly upregulated after ERG induction included WNT11 and genes associated with biological adhesion included CD44, ITGA10, FLT4, SELP, CD24, TYROBP and SHANK3. The upregulation (≥ 2-fold) of these potentially novel targets of ERG were also validated by RT-PCR. Interestingly, cell cultures of ERG induced cells incubated with a combination of both WNT11 and CD44 antibodies to block cell adhesion showed inhibition of the ERG induced morphological transformation. Furthermore, FACS analysis of ERG induced cells stained for Annexin V showed an increase in apoptosis (24%) by the addition of CD44 and WNT11 antibodies (dilutions 1: 250) whereas the no antibody control was measured at <3%. Non induced cultures were unaffected by the addition of both antibodies compared to the no antibody control. Based on these experiments, we further investigated the contribution of ERG induced cell adhesion to stroma given that leukemia cells interact with the bone marrow microenvironment. Co-cultures of HS5 stroma cells and ERG inducible K562 cells unexpectedly augmented the cell count of ERG induced cells by 2–3-fold on a stromal monolayer versus a non stromal monolayer control. Interestingly, ERG induced cells distinctively had a high induction of apoptosis (> 60%) with the addition of CD44 and WNT11 (dilutions 1: 250) blocking antibodies to co-culture assays. Cell culture of ERG induced K562 cells with a stromal monolayer bed resulted in resistance to cytarabine (Ara-C) and to midostaurin (PKC412). Apoptosis evaluation of DsReD ERG/Annexin V double positive cells treated with either Ara-C (10–30μg/ml) or PKC412 (10–30μM) were measured by FACS at only 14% and 20%, respectively. In contrast, non induced K562 cultures (absent of ERG) had a significantly higher apoptosis induction range: 46% to 55% after exposure to Ara-C and 56% to 65% after exposure to PKC412. We conclude that direct contact and cell adhesion with stromal cells promote an ERG-dependent survival mechanism and mediate resistance to Ara-C or PKC412. In addition, the observed cell death effects by WNT11 and CD44 antibodies in cultures with ERG induced cells strongly indicate their involvement in ERG-dependent cellular adhesion. We propose that, in acute leukemia patients with aberrant ERG overexpression, ERG may mediate genetic and morphological transformation (i.e. cell adhesion) of leukemic cells resulting in an escape mechanism and the development of resistance to chemotherapy.