All-Trans-Retinoic Acid Combined With Valproic Acid Can Promote Differentiation in Myeloid Leukemia Cells by an Autophagy Dependent Mechanism.

Acute myeloid leukemia (AML) is an aggressive blood cancer with an overall survival of 30%. One form of AML, acute promyelocytic leukemia (APL) has become more than 90% curable with differentiation therapy, consisting of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO). Application of differentiation therapy to other AML subtypes would be a major treatment advance. Recent studies have indicated that autophagy plays a key role in the differentiation of ATRA-responsive APL cells. In this study, we have investigated whether differentiation could be enhanced in ATRA resistant cells by promoting autophagy induction with valproic acid (VPA). ATRA sensitive (NB4) and resistant leukemia cells (NB4R and THP-1) were co-treated with ATRA and valproic acid, followed by assessment of autophagy and differentiation. The combination of VPA and ATRA induced autophagic flux and promoted differentiation in ATRA-sensitive and -resistant cell lines. shRNA knockdown of ATG7 and TFEB autophagy regulators impaired both autophagy and differentiation, demonstrating the importance of autophagy in the combination treatment. These data suggest that ATRA combined with valproic acid can promote differentiation in myeloid leukemia cells by mechanism involving autophagy.

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

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).

Lentiviral-Mediated shRNA Approaches: Applications in Cellular Differentiation and Autophagy.

Acute myeloid leukemia (AML) is characterized by the accumulation of immature white blood cell precursors in the bone marrow and peripheral circulation. In essence, leukemic cells fail to differentiate and are stalled at a particular step of hematopoietic maturation and are unable to complete their development into functional blood cells with a finite life cycle. They are thus said to possess a "differentiation block." Pharmacological override of this block is one attractive avenue of therapy, termed "differentiation therapy." The most successful example of this therapeutic strategy is the use of the physiologic retinoid all-trans-retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL). In this chapter, we will outline the methods used to characterize the mechanisms mobilized by retinoid signaling and will use the activation of a key regulator of autophagy, ATG7, as an example of the functional characterization of a retinoid regulated gene during differentiation. We will discuss how lentiviral delivery of shRNA constructs into cultured APL cells, such as NB4, can be used to functionally deplete key proteins. We will also describe how the effect of protein knockdown on ATRA-induced differentiation and autophagy can be assessed using quantitative PCR, Western blotting, and flow cytometry.