Page 119 - 2019_04-Haematologica-web

Autophagy drives ETV6-RUNX1-positive leukemia
and 71% reduction in cell survival during L-Asparaginase exposure, respectively (P≤0.01 and P≤0.001) (Figure 6B), while ETV6-RUNX1-negative BCP-ALL cells were not sensitized to L-Asparaginase (Figure 6B and Online Supplementary Figure S9D). Co-culture of primary BCP- ALL samples with primary MSCs significantly reversed the HCQ-mediated sensitization to L-Asparaginase (P≤0.01) (Figure 6C). To investigate whether these primary samples could still be sensitized, similar experiments were performed with a higher dose of HCQ (20 mg/mL). Inhibition of autophagy with 20 mg/mL HCQ was indeed sufficient to significantly sensitize primary ETV6-RUNX1- positive BCP-ALL samples to L-Asparaginase both in absence or presence of primary MSCs (80% reduced sur- vival, P≤0.05 and P≤0.01, respectively) (Figure 6D and Online Supplementary Figure S9B and C). In contrast to L-Asparaginase, HCQ did not significantly induce apopto- sis of primary BCP-ALL samples upon treatment with prednisolone or 6-mercaptopurine (Online Supplementary Figure S9E and F).
These data show that HCQ-mediated inhibition of autophagy results in sensitization of ETV6-RUNX1-posi- tive BCP-ALL cells, but not ETV6-RUNX1-negative BCP- ALL cells, to L-Asparaginase.
Discussion
In this study, we show that the ETV6-RUNX1 fusion gene induces a transcriptional network regulating pre- leukemic features in hematopoietic progenitors. We show that this network facilitates the induction of autophagy by up-regulating Vps34 expression in ETV6-RUNX1-positive BCP-ALL (Figure 7). In addition, our data show for the first time that inhibition of autophagy is a promising strategy for sensitization of ETV6-RUNX1-positive BCP-ALL cells to the important anti-leukemic agent L-Asparaginase.
The importance of the ETV6-RUNX1 fusion protein for modulation of proliferation, survival and cell cycle distri- bution has already been shown in cell lines14,17,31 and mouse models.10 Similarly, expression of the ETV6- RUNX1 fusion gene induces survival properties in human cord blood-derived progenitors transplanted in NOD/SCID mice or co-cultured in the presence of murine MS-5 stromal cells.11,20 However, to date, the downstream effectors of this pro-survival and pro-proliferative pheno- type have not been elucidated. In this study, we uncov- ered the transcriptional network regulating these pheno- types in a human progenitor population by analyzing the gene expression profile after ectopic expression of ETV6- RUNX1. This approach allowed us to examine early effects of ETV6-RUNX1 expression in human hematopoi- etic progenitors. These data, therefore, provide a compre- hensive and functional list of ETV6-RUNX1 target genes (Online Supplementary Table S2). In addition to a pro-sur- vival and pro-proliferative phenotype, genes involved in cytoskeleton rearrangements and cellular homeostasis were found to be regulated by the ETV6-RUNX1 fusion protein (Online Supplementary Figure S2). Importantly, our results reveal that autophagy is induced in ETV6-RUNX1- positive cells because of transcriptional activation of Vps34, a member of the core (macro)autophagy-regulating complex (Figures 1-3).22
In the present study, we show that the ETV6-RUNX1 fusion gene can directly up-regulate the level of autophagy
in leukemic cells in absence of cellular stress. Our results demonstrate that these enhanced levels of autophagy are important to maintain proliferation and survival of ETV6- RUNX1-positive leukemic cells (Figures 4-6). Knockdown of Vps34 and inhibition of autophagy with HCQ reduced the proliferation and survival of ETV6 RUNX1-positive BCP-ALL cells, confirming the importance of induced autophagy in these cells (Figures 4 and 5). These results are in line with a recently published study showing sensitiza- tion of REH cells to L-Asparaginase during chloroquine treatment in a xenograft model.35
Importantly, we found that primary ETV6-RUNX1-neg- ative BCP-ALL samples were not affected by autophagy inhibition. Autophagy might play an important role in protecting leukemic cells during chemotherapeutic treat- ment with nutrient-modulating drugs like L-Asparaginase that actively inhibits protein biosynthesis by asparagine depletion, which leads to nutritional deprivation and effective killing of leukemic cells.36 Here, we show that autophagy selectively protects ETV6-RUNX1-positive leukemic cells against L-Asparaginase treatment, whereas this effect is absent in ETV6-RUNX1-negative leukemic cells (Figure 6). This is in contrast with the study of Takashi et al.,35 highlighting the importance of the use of primary human leukemic cells in studies investigating tar- geted therapy. HCQ-mediated inhibition of autophagy did not sensitize cells to prednisolone or 6-mercaptop- urine, two other chemotherapeutics often used in treat- ment of BCP-ALL. These results highlight the importance of the cellular and molecular context in which autophagy inhibition is embedded and show that caution is warrant- ed before the general introduction of autophagy inhibitors in the treatment of leukemia.
The leukemic microenvironment or niche has been shown to protect leukemic cells from elimination by immune responses and chemotherapeutic agents and facilitates the development of drug resistance to classic and targeted chemotherapy.37 Here, we show that MSCs can abrogate the effects of autophagy inhibition in ETV6- RUNX1-positive BCP-ALL cells. This highlights the crucial role of the leukemic niche in induction of resistance to chemotherapy, including autophagy inhibition. However, MSC-induced resistance of ETV6-RUNX1-positive cells could still be overcome when adequate concentrations of the autophagy inhibitor HCQ were used (Figure 6).
The efficacy of autophagy inhibitors during cancer treatment is currently being examined in clinical trials (reviewed, for example, by White25). Initial results indicate that HCQ treatment is safe and tolerated at high concen- trations and might be effective in a subset of patients (reviewed, for example, by Vogl et al.38). In addition, autophagy independent “off-target” effects of chloro- quines, resulting in enhanced response to chemotherapy have been reported.39 This strengthens the rationale to use HCQ in clinical practice. However, more specific and potent autophagy inhibitors are currently being developed and preclinical studies with these novel inhibitors (e.g. Lys05) show promising results.40 In addition, the recent determination of the crystal structure of Vps3441 will enable the development of Vps34 inhibitors for clinical use in the near future.42,43 Our observation that the ETV6- RUNX1 fusion protein induces Vps34 expression and sub- sequently autophagy, strongly indicates that Vps34/autophagy inhibitors should be considered in future protocols of ETV6-RUNX1-positive BCP-ALL.
haematologica | 2019; 104(4)
747