Inhibition of Key Autophagy Genes in Primary AML Patients and Their Functional Role in Neutrophil Differentiation of APL Cells

Abstract

Abstract 2456There is accumulating evidence that autophagic mechanisms are operative in the development of different hematopoietic lineages. Autophagy may support the remodelling processes that are taking place during differentiation and maturation of different cell types. Generally, autophagy is a bulk degradation mechanism mainly involved in cell survival responses to a variety of stress stimuli. Furthermore, autophagy-inducing drugs support degradation of aggregated PML-RARA protein in acute promyelocytic leukemia (APL). We used customized 384-well micro fluidic cards to determine the expression of key autophagy genes (ATGs) in a large cohort of primary acute myeloid leukemia (AML) samples (n=122) and normal neutrophils (n=14). In general, we found significantly lower ATG mRNA levels in AML as compared to neutrophils for 21/38 autophagy genes investigated. Among the most significantly downregulated genes in AML were ATG1, ATG4D, ATG14, ATG16L2, WDFY3, BNIP3L, FIP200, GATE-16 and GABARAPL1. Given the low expression of these ATGs in AML, a disease mainly characterized by a block in differentiation, we hypothesized that these genes are implicated in myeloid differentiation. We therefore analyzed expression of selected ATGs in two APL cell line models for neutrophil differentiation. We found a significant 2- to 40-fold increase of ATG1, ATG7, WDFY3, GATE16 and GABARAPL1 in NB4 and HT93 APL cell lines upon all-trans retinoic (ATRA)-induced neutrophil differentiation. The expression of these genes did not change in ATRA-resistant NB4-R2 cells upon neutrophil differentiation indicating that their increased expression is functional in neutrophil differentiation. Next, we knocked down ATG1, WDFY3, GATE16 and GAPARAPL1 in NB4 cells using lentivirally delivered shRNAs. Inhibition of ATG1, WDFY3 and GATE16 but not GABARAPL1 significantly attenuated ATRA-induced neutrophil differentiation as measured by reduced levels of the myeloid markers CD11b, CEBPE and G-CSF-R. Analysis of the autophagic markers p62/SQSTM1 and LC3B revealed that inhibiting ATG1, WDFY3 and GATE16 not only diminished neutrophil differentiation of APL cells but also myeloid differentiation-associated autophagy (MDAA). In summary, low ATG expression is generally associated with an immature AML phenotype. Furthermore, knocking down key autophagy genes attenuates ATRA-induced neutrophil differentiation and MDAA of APL cells. Our studies indicate that autophagy is a putative tumor suppressor mechanism in AML/APL and already available autophagy-inducing drugs such as lithium chloride may be used in combination with differentiation-inducing drugs. Disclosures:No relevant conflicts of interest to declare.