ATRA-Induced Activation of the Autophagy Regulator Tfeb Regulates Myeloid Differentiation in Acute Promyelocytic Leukemia

Introduction

Acute promyelocytic leukemia (APL) is characterized by gene fusions involving RARα, which disrupt retinoid signaling and lead to the clonal expansion of myeloid precursors halted at the promyelocyte stage of maturation. Treatment with all-trans-retinoic acid (ATRA) can overcome this differentiation block, restore granulocytic differentiation in APL cells and improve APL patient outcome. ATRA treatment of APL cells induces autophagy, a catabolic process whereby redundant proteins and organelles are degraded using lysosomal machinery. ATRA-induced autophagy is important for successful granulocytic differentiation of APL cells through the degradation of fusion oncoproteins and executing the structural protein remodeling necessary for cellular differentiation.

The molecular mechanisms linking retinoid signaling and autophagy pathways are poorly understood and are the focus of our study. By integrating available chromatin immunoprecipitation, coupled with next-generation sequencing (ChIPseq) datasets and transcriptomic approaches, we examined the transcriptional effects of ATRA treatment on autophagy-related genes during APL cell differentiation. We identified transcription factor EB (TFEB), a master transcriptional regulator of autophagy, as a retinoid target and have evaluated the consequences of TFEB knockdown on both ATRA-induced autophagy and APL cell differentiation.

Methods & Results

A list of 557 autophagy-related genes was generated from 3 public autophagy databases. Cluster analysis of expression of these 557 genes in the APL TCGA RNAseq dataset, suggest autophagy is associated with patient survival (Cluster 1 = 26.4 months / Cluster 2 = 60 months). We therefore examined whether the 557 autophagy related genes may be direct ATRA targets. Accessing a public ChipSeq database (GSE18886), increased RARα binding was detected within the TFEB gene in NB4 cells (APL cell line, t15:17) treated with ATRA for 24 hours. Increased RNAPolII binding and transcriptionally active histone markers were also detected, consistent with a direct transcriptionally activating retinoid effect.

To test the effects of retinoid therapy on APL gene expression, we treated NB4 cells with ATRA or ethanol control for 72 hours. Isolated RNAs were sequenced using the Illumina HiSeq 2000 platform. Reads were aligned and annotated to the hg19 genome using Tophat and subsequent differential expression analysis performed using Cufflinks/Cuffdiff and Cluster/Treeview. We observed changes in autophagy gene expression upon ATRA treatment and recorded a significant >2 fold increase in expression in >50 autophagy-related genes including TFEB, p62 and DRAM1. We then carried out lentivirally-mediated shRNA knockdown of TFEB in NB4 cells and treated both knockdown and scrambled control cells with ATRA for 72 hours. Transcriptomic analysis demonstrated that TFEB knockdown blocked ATRA-induced TFEB induction, affected the regulation of key autophagy-related genes and impaired CD11b expression. These results were validated by qRT-PCR (n=6)(P<0.001).

We confirmed TFEB upregulation by qRT-PCR in primary human APL samples treated with ATRA ex vivo (n=1) and in primary peripheral blood mononuclear cells isolated from tumour patients treated with ATRA as part of clinical trial NCT00195156 (n=3). We also found that TFEB was only minimally induced by ATRA in the differentiation-resistant NB4R cell line (qRT-PCR n=6), suggesting an important role for TFEB in ATRA-induced APL differentiation. We evaluated the ability of two clinically used differentiating agents - valproic acid (VPA) and arsenic trioxide (ATO), alone and in combination with ATRA, to induce TFEB expression in NB4 cells. While VPA alone induced TFEB (5 fold), combined therapy with ATRA and VPA resulted in a >20 fold induction of TFEB expression, which correlated with enhanced differentiation (n=6)(P<0.001). ATO alone did not induce TFEB nor did it enhance the induction seen with ATRA. Notably, combined therapy with ATRA and ATO did not significantly enhance differentiation over levels seen with ATRA alone.

Conclusion

Retinoid signaling promotes APL differentiation in part through transcriptionally activating autophagy. Novel or existing therapies, which enhance autophagy signaling, may promote retinoid-induced differentiation and circumvent resistance to retinoid therapy in acute myeloid leukemia.