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positive vesicles were calculated per cell with a 3D object analyzer using Huygens Professional software (Online Supplementary Figure S6D). Knockdown of ETV6-RUNX1 resulted in a 45% reduction in the number of LC3B posi- tive vesicles and 50% reduction in the volume of LC3B- positive vesicles per cell (P≤0.001 and P≤0.05, respectively) (Figure 3C-E). In addition, knockdown of Vps34 reduced the number of LC3B-positive vesicles and the volume of LC3B-positive vesicles with 79% (P≤0.001) and 84% (P≤0.05), respectively (Figure 3C-E). These results suggest that autophagy levels are high in ETV6-RUNX1 positive BCP-ALL cells. In addition, both ETV6-RUNX1 and Vps34 are important for maintaining high levels of autophagy in ETV6-RUNX1-positive leukemic cells.
Vps34 is essential for the survival of ETV6-RUNX1-positive leukemic cells
To determine the functional role of Vps34 in ETV6- RUNX1-positive leukemic cells, lentiviral knockdown studies were performed in two ETV6-RUNX1-positive cell lines (REH and REHS1). For these studies, four independ- ent short hairpin RNAs were used. Short hairpins shVps34#1 and shVps34#2 were directed against the main Vps34 transcript variants, whereas shVps34#3 and shVps34#4 were directed against the two full length tran- script variants only (Figure 4A and Online Supplementary Figure S7C). Vps34 knockdown of at least 80% was achieved with all four individual shRNAs (Figure 4B and C and Online Supplementary Figure S7D). Knockdown of only the full-length Vps34 transcript variants with shVps34#3 and shVps34#4 significantly reduced proliferation of the ETV6-RUNX1-positive cell lines REH and REHS1 (P≤0.01) (Figure 4A and Online Supplementary Figure S7E and F). Knockdown of all main Vps34 transcript variants with shVps34#1 and shVps34#2 resulted in a complete growth arrest of these ETV6-RUNX1-positive cell lines (P≤0.001) (Figure 4D and Online Supplementary Figure S7A). In con- trast, knockdown of either all main Vps34 transcript vari- ants or the full-length Vps34 transcripts in ETV6-RUNX1- negative NALM6 cells decreased proliferation to a signifi- cantly lesser extent (P≤0.001) (Figure 4D and Online Supplementary Figure S7A, E and F).
To investigate whether the observed growth arrest in ETV6-RUNX1-positive cells was due to a block in cell cycle progression or enhanced apoptosis, flow cytometric analysis was performed using DyeCycle and Annexin V. Cell cycle analysis revealed that knockdown of Vps34 modestly reduces the percentage of cycling ETV6-RUNX1 positive cells (Figure 4E and Online Supplementary Figure S7B and G). In contrast, a remarkable reduction in survival was observed upon Vps34 knockdown. While shRNA mediated knockdown of the full length Vps34 transcript variants in ETV6-RUNX1 positive cells (shRNA#3 and shRNA#4) already resulted in 40-50% apoptotic cells (Online Supplementary Figure S7H), targeting of all main Vps34 transcript variants resulted in even higher levels of apoptosis (80-90%, P<0.01) (Figure 4F).
In conclusion, knockdown of Vps34 completely arrests cell growth of ETV6-RUNX1-positive cells by modestly reducing cell cycle progression and strongly inducing apoptosis. Although Vps34 knockdown also affects cell cycle progression and survival in ETV6-RUNX1-negative cells (data not shown), these cells were still able to prolifer- ate.
ETV6-RUNX1-positive ALL cells are relatively sensitive to hydroxychloroquine
To date, no agents are clinically available that specifical- ly inhibit Vps34 activity. However, the efficacy of autophagy inhibitors is currently being examined in clini- cal cancer treatment trials. To investigate the effect of autophagy inhibition on ETV6-RUNX1-positive leukemia, we exposed leukemic cells to hydroxychloroquine (HCQ). This agent has favorable pharmacological properties and has been safely used for decades in the treatment of malar- ia and rheumatoid arthritis.32 HCQ accumulates within and de-acidifies the lysosome, resulting in increased LC3B-II levels, which here is indicative of impaired autophagy (Figure 5A). MTT assays were performed to determine the effect of HCQ on cell viability of ETV6- RUNX1-positive and ETV6-RUNX1-negative ALL cell lines (Figure 5B and C and Online Supplementary Figure S8A and B). While treatment with 20 mg/mL HCQ resulted in 82% and 95% reduced cell viability of ETV6-RUNX1-pos- itive BCP-ALL cell lines (REH and REHS1), the viability of ETV6-RUNX1-negative cell lines was reduced to a lesser extent (NALM6: 43%, TOM1: 50%, Loucy: 40%, Jurkat: 0%) (Figure 5B). The IC50 of HCQ was significantly lower in ETV6-RUNX1-positive ALL cell lines compared to ETV6-RUNX1-negative cell lines (P≤0.001) (Online Supplementary Figure S8A and B). In addition, the effect of HCQ on the survival of primary BCP-ALL samples was determined. Primary BCP-ALL samples were cultured for five days upon which flow cytometric analysis was per- formed (see Online Supplementary Figure S8C for flow cyto- metric gating strategy). Survival of primary ETV6-RUNX1-positive BCP-ALL samples was significant- ly reduced after treatment with 10 mg/mL HCQ (26%, P≤0.05) (Figure 5D and E). In contrast, this treatment did not affect cell viability of primary ETV6-RUNX1-negative BCP-ALL samples (Online Supplementary Figure S8D). The survival of primary ETV6-RUNX1-positive ALL samples was even further reduced after treatment with 20 mg/mL HCQ (79%, P≤0.01) (Figure 5E). Co-culture of these ALL samples in the presence of primary bone marrow-derived mesenchymal stromal cells (MSCs) significantly rescued the HCQ-mediated induction of apoptosis in primary ETV6-RUNX1-positive ALL samples (P≤0.05) (Figure 5E). These results demonstrate that although ETV6-RUNX1- positive BCP-ALL cells are relatively sensitive to HCQ treatment, this sensitivity is abrogated by primary MSCs.
Autophagy inhibition sensitizes ETV6-RUNX1-positive acute lymphoblastic leukemia cells to L-Asparaginase
As our results indicate that inhibition of autophagy reduces survival of ETV6-RUNX1-positive ALL samples, we investigated whether HCQ-mediated inhibition of autophagy could sensitize primary BCP-ALL samples to commonly used chemotherapeutics. To investigate the potential of HCQ treatment in sensitization to chemother- apeutics, the percentage of apoptotic cells was determined by flow cytometry after five days of culture either in absence or presence of IC50 values of the chemotherapeu- tic drug. Our results indicate that HCQ treatment (in clin- ically relevant concentrations33,34) selectively sensitizes ETV6 RUNX1-positive leukemic cells to L-Asparaginase treatment (Figure 6A and Online Supplementary Figure S9A). Treatment of primary ETV6 RUNX1-positive ALL sam- ples with 5 mg/mL or 10 mg/mL HCQ resulted in a 48%
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haematologica | 2019; 104(4)