All‐trans retinoic acid (ATRA)‐induced TFEB expression is required for myeloid differentiation in acute promyelocytic leukemia (APL)

Abstract

Objective In acute promyelocytic leukemia (APL), normal retinoid signaling is disrupted by an abnormal PML‐RARα fusion oncoprotein, leading to a block in cell differentiation. Therapeutic concentrations of all‐trans‐retinoic acid (ATRA) can restore retinoid‐induced transcription and promote degradation of the PML‐RARα protein. Autophagy is a catabolic pathway that utilizes lysosomal machinery to degrade intracellular material and facilitate cellular re‐modeling. Recent studies have identified autophagy as an integral component of ATRA‐induced myeloid differentiation. Methods As the molecular communication between retinoid signaling and the autophagy pathway is not defined, we performed RNA sequencing of NB4 APL cells treated with ATRA and examined autophagy‐related transcripts. Results ATRA altered the expression of >80 known autophagy‐related transcripts, including the key transcriptional regulator of autophagy and lysosomal biogenesis, TFEB (11.5‐fold increase). Induction of TFEB and its transcriptional target, sequestosome 1 (SQSTM1, p62), is reduced in ATRA‐resistant NB4R cells compared to NB4 cells. TFEB knockdown in NB4 cells alters the expression of transcriptional targets of TFEB and reduces CD11b transcript levels in response to ATRA. Conclusions We show for the first time that TFEB plays an important role in ATRA‐induced autophagy during myeloid differentiation and that autophagy induction potentiates leukemic cell differentiation (Note: this study includes data obtained from NCT00195156, https://clinicaltrials.gov/show/NCT00195156).

Reducing FASN expression sensitizes acute myeloid leukemia cells to differentiation therapy

“…Interestingly, the majority of the TFEB downstream targets from the different categories (lysosomal hydrolases and accessory proteins, lysosomal membrane, lysosomal acidification, non-lysosomal proteins involved in lysosomal biogenesis and autophagy) are negatively associated with FASN transcript levels in primary AML patient blasts from TCGA analyzed using the UCSC Xena platform (Goldman et al, 2019) and the Blood spot gene expression profiles data base (Bagger et al, 2016) ( Figure 5A, Supplementary Figures 4B-C Supplementary Table 1-2). Furthermore, analyzing RNA-seq data of NB4 cells treated with ATRA confirmed a reduction of FASN expression paralleled by increased TFEB and TFEB target gene transcript levels (Orfali et al, 2019) ( Figure 5B). To test if the FASN-mTOR pathway is involved in regulating TFEB activity, we analyzed the cellular localization of TFEB upon ATRA treatment in NB4 control and FASN depleted cells.…”

“…We and others have demonstrated that autophagy gene expression is repressed in AML samples compared to granulocytes from healthy donors and that autophagy activity is essential for successful ATRA-induced APL differentiation (Isakson et al, 2010;Wang et al, 2011;Jin et al, 2018;Humbert et al, 2017;Brigger et al, 2014b;Watson et al, 2015;Orfali et al, 2015Orfali et al, , 2019. The decrease in FASN expression upon ATRA-induced differentiation cannot be explained solely by transcriptional regulation due to the long half-life of this protein (1-3 days) (Volpe & Vagelos, 1976;Weiss et al, 1986).…”

“…Figure 4A). A recent study demonstrated the key role of TFEB during ATRA induced differentiation (Orfali et al, 2019). We therefore investigated the relationship between FASN and CLEAR network gene expression.…”

“…Deciphering the mechanisms active during ATRA-mediated differentiation at the molecular level will support the translation of differentiation therapy to non-APL AML patients. We and others have demonstrated the importance of autophagy in ATRA induced granulocytic differentiation of APL cells (Isakson et al, 2010;Wang et al, 2011;Jin et al, 2018;Humbert et al, 2017;Brigger et al, 2014b;Orfali et al, 2019). Autophagy is an intracellular degradation mechanism that ensures dynamic recycling of various cytoplasmic contents (Feng et al, 2014).…”

Inhibition of UBE2L6 attenuates ISGylation and impedes ATRA‐induced differentiation of leukemic cells

“…Our RNA sequencing data showed a 17.54‐fold increase in ISG15 gene expression in NB4 cells with ATRA treatment (Orfali et al , ) (Table ). We validated this finding by qPCR measurement of ISG15 expression in ATRA‐treated NB4 cells at 72 h, which showed a 23‐fold increase in expression in differentiating cells (**** P ≤ 0.0001).…”