Targeted Therapy for ETV6-RUNX1-Driven B-Cell Precursor Acute Lymphoblastic Leukemia

Background: Translocation t(12;21), resulting in the ETV6-RUNX1 fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite the favorable prognostic parameters of this B-ALL subgroup, relapse and resistance to chemotherapeutics occur and treatment-induced side effects are considerable. The molecular mechanisms underlying ETV6-RUNX1-driven leukemia are largely unknown. Increased knowledge of these mechanisms is essential to develop novel therapeutic strategies to selectively target ETV6-RUNX1-positive leukemia.

Objectives: This study aims to identify and target the molecular drivers behind ETV6-RUNX1-positive BCP-ALL.

Results: Gene expression profiling of leukemic blasts of 654 ALL patients revealed that the class III PI3-kinase Vps34, an important regulator of autophagy, was exclusively up-regulated in ETV6-RUNX1-positive compared to ETV6-RUNX1-negative BCP-ALL patients (2.7-fold; p ≤ 10^-30). In addition, ectopic expression of ETV6-RUNX1 in cord blood-derived hematopoietic progenitor cells (CB-HPCs) significantly induced expression of Vps34 1.3-fold already 40 hours after transduction (p ≤ 0.05). This suggests that the Vps34-autophagy pathway is activated by ETV6-RUNX1, which may mechanistically explain the leukemogenic and pro-survival properties ascribed to ETV6-RUNX1. In correspondence, Ingenuity Pathway Analysis (IPA) predicted a pro-survival and pro-proliferative phenotype in ETV6-RUNX1 transduced CB-HPCs and highlighted a network of up-regulated transcription factors, including HEY1, EGR1, GATA1 and GATA2 (2 – 25-fold up-regulation; p ≤ 0.05). Luciferase reporter assays revealed that not only the ETV6-RUNX1 fusion protein, but also the ETV6-RUNX1-induced target genes HEY1, EGR1 and GATA1 positively regulate Vps34 promoter activity (5 – 13-fold up-regulation; p ≤ 0.01).Lentiviral knockdown experiments were performed to elucidate the importance of Vps34 expression in ETV6-RUNX1-positive BCP-ALL cells. Knockdown of all Vps34 transcript variants, with two independent constructs, led to complete growth arrest of the ETV6-RUNX1-positive cell lines REH and AT2, while this only led to a decrease in proliferation of the ETV6-RUNX1-negative cell line NALM6. This growth arrest was caused by a significant induction of apoptosis (more than 4-fold 7 days after transduction; p ≤ 0.001) and a significantly reduced percentage of cycling cells (1.3-fold 7 days after transduction; p ≤ 0.05). Analysis of p62 protein expression by western blot and reverse phase protein arrays revealed that the levels of autophagy were significantly higher in ETV6-RUNX1-positive compared to ETV6-RUNX1-negative BCP-ALL patients (p ≤ 0.001). In addition, knockdown of ETV6-RUNX1 and Vps34 significantly reduced autophagy, quantified with confocal microscopy, in ETV6-RUNX1-positive cells with 50% and 84%, respectively (p ≤ 0.01). Furthermore, pharmacological inhibition of autophagy with hydroxychloroquine (HCQ) significantly reduced cell viability of BCP-ALL cell lines and primary patient-derived BCP-ALL cells (p ≤ 0.001). Treatment of the ETV6-RUNX1-positive BCP-ALL cell lines REH and AT2 with 20 µg/mL HCQ resulted in a 82% and 95% reduced cell viability, while the viability of ETV6-RUNX1-negative BCP-ALL cell lines and T-ALL cell lines were reduced to a lesser extent (NALM6: 43%; TOM-1: 50%; Loucy: 40%; Jurkat: 0%). Importantly, HCQ selectively sensitized ETV6-RUNX1-positive leukemic cells to L-asparaginase treatment in clinically relevant concentrations. Treatment of primary ETV6-RUNX1-positive patient cells with 10 µg/mL HCQ resulted in a 70% reduction in cell survival during L-asparaginase exposure (p ≤ 0.01). This sensitization was not observed in ETV6-RUNX1-negative BCP-ALL cells.

Conclusion: The ETV6-RUNX1 fusion protein activates autophagy via Vps34, which is essential for survival and proliferation of ETV6-RUNX1-positive cells. Inhibition of autophagy in primary ETV6-RUNX1-positive leukemic cells inhibited cell survival and sensitized these cells to L-asparaginase treatment. These results indicate that autophagy inhibition may provide a novel means to sensitize L-asparaginase-resistant ETV6-RUNX1-positive BCP-ALL patients.