Putative Autophagy Protein VMP1 Amplifies Protective Autophagy Under Stress Conditions in AML Cells

Autophagy is a multi-step catabolic process orchestrated by a large set of specific proteins. We previously reported that autophagy is essential for normal and AML cells. However, how (basal) autophagy is regulated in these cells remains elusive. Vacuole membrane protein (VMP1) is a putative autophagy protein residing in the endoplasmic reticulum membrane. Upon autophagy induction, VMP1 can recruit and activate the PI3K complex, thereby facilitating and initiating the biogenesis of autophagy vesicles.

Transcriptome analyses of autophagy-associated genes was performed on a previously established gene expression dataset of a cohort of patient AML CD34⁺ cells (n=62) in comparison with normal bone marrow CD34⁺ cells (n=22). VMP1 mRNA expression was upregulated in 66% of AML cases, with an average increase of 1.4 fold (p<0.001), which was confirmed by qPCR. Within the panel of tested AMLs, VMP1 was significantly higher expressed in AML cells belonging to the monocytic (M4-M5) compared to myeloid (M1-M2) lineage. Moreover, in publicly available expression data sets (R2: Genomics Analysis and Visualization Platform), VMP1 expression was significantly lower in AMLs harbouring C/EBPα mutations.

Functional studies demonstrated that downregulation of VMP1 by shRNAs in cord blood (CB)-derived CD34⁺ HSPCs resulted in a decline of autophagic-flux, coinciding with reduced progenitor frequencies (2.9 fold reduction, p=<0.05). Similar, in vitro expansion was reduced under both myeloid (~2.5 fold reduction, p=<0.05) and erythroid (~7.6 fold reduction, p=<0.01) culture conditions. The reduced cell expansion was due to delayed differentiation and increased apoptosis. Notably, the strongest effects were observed in the erythroid lineage. Co-culturing of shVMP1-transduced CD34⁺ cells on MS5 bone marrow stromal cells did not rescue the observed negative phenotype. Next, shVMP1-GFP or shSCR-GFP-transduced CD34⁺ HSPCs were transplanted in immunodeficient NSG mice after sublethal irradiation. Chimerism levels were defined by the percentage of human CD45 in peripheral blood. While GFP levels for shSCR remained stable at around ~60%, the contribution of the shVMP1-transduced cells to the engrafted HSPCs was severely reduced (<2%, p<0.0001). Similar to normal HSPCs, knockdown of VMP1 in leukemic cell lines (n=6) or primary patient CD34⁺ AML cells (n=3) resulted also in a strong reduction in expansion compared to control. Moreover, knockdown of VMP1 resulted in accumulation of reactive oxygen species (ROS) and mitochondrial mass as determined by FACS, which might be due to impaired mitophagy.

In order to model high VMP1 expressing AMLs, we constructed a lentiviral VMP1 overexpression vector. In contrast to VMP1 knockdown, overexpression of VMP1 did not affect cell expansion, differentiation or autophagy flux under normal culture conditions in CB CD34⁺ cells. However, under hypoxia or during glucose starvation normal CD34⁺ or AML cells overexpressing VMP1 showed an increased autophagic-flux. Of interest, the VMP1 expression in CB 34⁺ cells increased (~1.5 fold, p=<0.05) under hypoxia compared to normoxia. Finally to enhance autophagic-flux, leukemic TF-1 and NB4 cells were serum starved (1% FCS) or cultured overnight under hypoxic conditions (1% oxygen). Both serum starvation and hypoxia triggered cell death in TF-1 and NB4 cells, which could be in part rescued by ectopic overexpression of VMP1 (~2 fold increased survival, p=<0.05).

In conclusion, our data demonstrate that in normal hematopoietic- and leukemic cells VMP1 is associated with mitochondrial quality control and increased VMP1 expression amplifies protective autophagy under adverse growth conditions such as hypoxia or starvation.