The role played by lipids in the process of granulocytic differentiation activated by all-trans retinoic acid (ATRA) in Acute-Promyelocytic-Leukemia (APL) blasts is unknown. The process of granulocytic differentiation activated by ATRA in APL blasts is recapitulated in the NB4 cell-line, which is characterized by expression of the pathogenic PML-RARα fusion protein. In the present study, we used the NB4 model to define the effects exerted by ATRA on lipid homeostasis. Using a high-throughput lipidomic approach, we demonstrate that exposure of the APL-derived NB4 cell-line to ATRA causes an early reduction in the amounts of cardiolipins, a major lipid component of the mitochondrial membranes. The decrease in the levels of cardiolipins results in a concomitant inhibition of mitochondrial activity. These ATRA-dependent effects are causally involved in the granulocytic maturation process. In fact, the ATRA-induced decrease of cardiolipins and the concomitant dysfunction of mitochondria precede the differentiation of retinoid-sensitive NB4 cells and the two phenomena are not observed in the retinoid-resistant NB4.306 counterparts. In addition, ethanolamine induced rescue of the mitochondrial dysfunction activated by cardiolipin deficiency inhibits ATRA-dependent granulocytic differentiation and induction of the associated autophagic process. The RNA-seq studies performed in parental NB4 cells and a NB4-derived cell population, characterized by silencing of the autophagy mediator, ATG5, provide insights into the mechanisms underlying the differentiating action of ATRA. The results indicate that ATRA causes a significant down-regulation of CRLS1 (Cardiolipin-synthase-1) and LPCAT1 (Lysophosphatidylcholine-Acyltransferase-1) mRNAs which code for two enzymes catalyzing the last steps of cardiolipin synthesis. ATRA-dependent down-regulation of CRLS1 and LPCAT1 mRNAs is functionally relevant, as it is accompanied by a significant decrease in the amounts of the corresponding proteins. Furthermore, the decrease in CRLS1 and LPCAT1 levels requires activation of the autophagic process, as down-regulation of the two proteins is blocked in ATG5-silenced NB4-shATG5 cells.

A hallmark of acute myeloid leukemia (AML) is a differentiation block of hematopoietic precursors at different developmental stages. In acute promyelocytic leukemia (APL) this block can be overcome by treating the patients with retinoic acid (RA) and low dose chemotherapy allowing for neutrophil differentiation. APL is characterized by the translocation t(15;17) leading to the expression of the oncogenic PML-RARA. In contrast to the wild-type RARA protein PML-RARA acts as a transcriptional repressor causing inhibition of genes important for neutrophil differentiation. Recent data strongly suggest a role for autophagy, a self-digesting recycling mechanism, in RA-induced neutrophil differentiation by for example supporting the degradation of aggregated PML-RARA. Interestingly, the Autophagy-Linked FYVE domain-containing protein (ALFY/WDFY3) is involved in the autophagy-dependent degradation of aggregated proteins. This prompted us to investigate the role of ALFY during RA-induced neutrophil differentiation of APL cells. Firstly, we found 3-fold lower ALFY mRNA levels in a large cohort of primary AML samples as compared to normal granulocytes. Secondly, ALFY mRNA levels significantly increased following RA-induced neutrophil differentiation of NB4 and HT93 APL cells. In line with these findings, knocking down ALFY NB4 cells significantly attenuated neutrophil differentiation. Direct binding of ALFY to PML-RARA was found and is needed for the degradation of PML-RARA. Moreover, we found that RA-induced autophagy in APL cells depends on an alternative non-canonical mechanism, independent of Beclin1, but dependent on additional key autophagy genes such as ULK1, VPS34, WIPI-1, ATG5, ATG7 and MAP1S as shown by knockdown experiments. Similar to ALFY 5/6 of these key autophagy genes are significantly downregulated in primary AML patient samples as compared to healthy granulocytes. Since myeloid development is orchestrated by a panel of lineage specific transcription factors including the myeloid master regulators PU.1 and CEPBA, and since these transcription factors are often inactivated in AML, we asked if they might regulate ATG genes. Indeed, we discovered that several ATG genes are transcriptional targets of PU.1 and CEBPA. Thus, low ATG gene expression in particular AML subtypes can be attributed to aberrant expression and function of PU.1 or CEBPA in this disease. Together, our results suggest a crucial role for autophagy in APL differentiation and we identified first transcriptional regulators of ATG genes during myeloid differentiation further supporting a critical role of autophagy in this process. Lastly, our data indicate that in APL a combination of differentiation- and autophagy-inducing agents might be favorable. Citation Format: Mario P. Tschan, Anna M. Schlaefli, Daniel Brigger, Aladin Haimovici, Elena A. Federzoni, Deborah Shan, Martin F. Fey. Autophagy pathways in acute promyelocytic leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1663. doi:10.1158/1538-7445.AM2013-1663

A series of pre-clinical studies showed promising anti-cancer effects of retinoic acids (RA) in breast cancer models. However, clinical studies were mostly disappointing possibly also due to the trial design and emerging resistances. One way to confer resistance to a given treatment is activation of protective autophagy. Autophagy is a cellular recycling mechanism that is characterized by the formation of double-membraned autophagosomes. Autophagosomes engulf bulk cytoplasm or selected contents thereof and transfer them to lysosomes for degradation. Activation of autophagy upon anti-cancer treatment may allow prolonged survival of the tumor cells. We therefore asked if autophagy is activated by all-trans retinoic acid (ATRA), a pan-retinoic acid receptor (RAR) agonist, in mammary tumor cells and if autophagy might protect the cells from the pro-apoptotic effects of ATRA. We found that ATRA induces autophagic flux in ATRA-sensitive SKBR3 but not in -resistant MD-MBA- 453 human breast cancer cells as seen by increased LC3B-II protein abundance and turnover of long-lived proteins. Moreover, using RA receptor alpha (RARA), RARB and RARG agonists as well as RARA knockdown breast cancer cells we demonstrated that activation of autophagy by RA depends on RARA expression. RA-treatment also caused induction of the transcription factor FOXO3A, a known transcriptional target of RARA. Since the expression of several autophagy genes namely the ATG8 family members, Beclin-1 and PI3KC3 are controlled by FOXO3A, we tested if FOXO3A might link ATRA-mediated signaling to autophagy activation. Indeed, depleting FOXO3A in SKBR3 cells resulted in significantly decreased autophagic activity and autophagy gene expression upon RA treatment. Importantly, blocking autophagy by knocking down the key autophagy genes ATG5 or ATG7 in breast cancer cells resulted in significantly increased apoptosis under ATRA treatment. Of note, inhibition of autophagy also attenuated cyto-differentiation similarly to the phenotype seen in RARA knockdown breast cancer cells. In summary, our data demonstrate for the first time that retinoic acids induce autophagy in breast cancer cells via the FOXO3A-RARA axis. Furthermore, inhibition of autophagy results in enhanced ATRA-mediated apoptosis representing a potential new treatment strategy for a selected group of breast cancer patients. Citation Format: Anna M. Schlaefli, Enrico Garattini, Mario P. Tschan. Retinoic acids activate autophagy in breast cancer cells through the FOXO3A-RARA pathway and blocking autophagy enhances retinoic acid-mediated cell death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 996. doi:10.1158/1538-7445.AM2015-996