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associated with this cytogenetic type of BCP-ALL,7 resist- ance to chemotherapeutic drugs and relapse occur in approximately 10% of these patients.7-9
The ETV6-RUNX1 fusion protein induces a silent pre- leukemic clone that requires additional genetic hits for the transition to leukemia.10-12 Although these pre-leukemic ETV6-RUNX1-positive hematopoietic stem cells (HSCs) still possess self-renewal properties and are capable of con- tributing to hematopoiesis, they fail to outcompete normal HSCs.11,12 In ETV6-RUNX1-positive leukemia, this early genetic lesion is followed by a number of ‘driver’ copy number alterations, including loss of ETV6 and alterations directed to genes regulating normal B-cell differentiation.13 These alterations are acquired independently without pref-
erential order, thereby generating a dynamic clonal archi- tecture.13 This genetic variation implies that targeted thera- py in ETV6-RUNX1-driven ALL should preferably be directed to targets that are present in all subclones, i.e. those being deregulated by the ETV6-RUNX1 fusion pro- tein itself. This concept is further supported by the obser- vation that ETV6-RUNX1-positive cell lines are highly dependent on the expression of the fusion protein for their survival.14,15 Previous reports revealed that enhanced levels of STAT3, heat-shock proteins, survivin, has-mir-125b-2, the erythropoietin receptor, cytoskeleton regulatory genes, and the PI3K/PKB/mTOR pathway, as well as aberrant reg- ulation of the TGFβ pathway, are important for ETV6- RUNX1-positive BCP-ALL.15-20 However, the molecular
Autophagy drives ETV6-RUNX1-positive leukemia
ABC
D
Figure 1. Vps34 is recurrently up-regulated in ETV6-RUNX1-positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and is driven by the ETV6-RUNX1 fusion protein. (A) 2log expression levels of the gene probe set mapped to Vps34 were analyzed in a cohort of 132 pediatric ALL patients published by Yeoh et al.26 Gene expression of ETV6-RUNX1-positive patients (green bar) was compared to gene expression of all other B-ALL patients (excluding T-ALL): ***False Discovery Rate (FDR)-adjusted P=3.45*10-15. (B) In addition, 2log expression levels of the gene probe set mapped to Vps34 were analyzed in a cohort of 653 pediatric ALL patients published by Van der Veer et al.30 Gene expression of ETV6-RUNX1-positive patients (green bar) was compared to gene expression of all other B-ALL patients (excluding T-ALL). Gray dashed line represents mean expression of all patients: ***FDR-adjusted P=7.24*10-39. (C) CB-CD34+ cells were transduced with ETV6- RUNX1-IRES-eGFP or with control EV-IRES-eGFP after which eGFP+ cells were sorted and Vps34 mRNA levels were determined by Q-PCR and normalized to hypoxan- thine-guanine phosphoribosyltransferase (HPRT). Gray bars represent the mean of 5 biological replicates. Gene expression of Vps34 was compared between ETV6- RUNX1+ CB-CD34+ cells and EV-control CB-CD34+ cells (n=5; P=0.03). (D) ETV6-RUNX1-positive cells were transfected with siRNAs directed to the ETV6-RUNX1 break- point or scrambled control siRNAs. Vps34 mRNA levels were determined in ETV6-RUNX1+ cells (REH (n=2) and ETV6-RUNX1 transduced CB-CD34+ cells (n=2) by Q- PCR, normalized to HPRT, and compared to the average expression of cells transfected with scrambled control siRNAs (n=3; P≤0.001). Vps34 protein expression was quantified in REH cells by western blot and compared to protein expression of REH cells transfected with scrambled control siRNAs (n=2; P≤0.05 and P≤0.01 for siRNA#1 and siRNA#5, respectively). Bars represent mean. Error bars represent Standard Error of Mean. *P≤0.05, **P≤0.01, ***P≤0.001. See also Online Supplementary Figures S1-S5.
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