Non-Canonical Autophagy during APL Therapy

Autophagy is an intracellular degradation system that ensures a dynamic recycling of cytoplasmic contents. Autophagy is required for self-renewal and cell survival under stress caused by a variety of stimuli including starvation and chemotherapy. There is accumulating evidence for additional functions of autophagy during myeloid development and hematopoietic stem cell maintenance. In this study, we used primary acute myeloid leukemia (AML) samples and human APL/AML cell lines to investigate the autophagy pathway active in all-trans retinoic acid (ATRA) mediated neutrophil differentiation. By characterizing the autophagic pathway during neutrophil differentiation of APL cells in more detail, we identified a non-canonical autophagy pathway, which not necessarily requires a hierarchal involvement of all autophagy-related (ATG) proteins. In addition to previous findings, from us and others, showing that ATRA-induced autophagy in APL cells is Beclin-1 independent, we discovered that ATRA-induced autophagy during APL differentiation is dependent on only one ATG16L isoform. The ATG16L proteins ATG16L1 and L2 are part of the ubiquitin-like conjugation systems ATG12-ATG5-ATG16L1 and ATG8/LC3 that are essential for phagophore elongation and autophagosome maturation. ATG16L2 is an isoform of ATG16L1, which is dispensable for starvation-induced autophagy despite forming an ATG12-ATG5-ATG16L2 complex in COS-7 cells. By investigating ATG16 gene expression in acute myeloid leukemia (AML) blast cells, we found that ATG16L1 as well as L2 are significantly downregulated in primary AML patient samples. In addition, neutrophil differentiation of APL/AML cell lines and CD34⁺ myeloid progenitor cells resulted in a significant induction of ATG16L1 and ATG16L2 expression. Induction of ATG16L2 was clearly more prominent than that of ATG16L1. Importantly, knocking down ATG16L2 but not ATG16L1 significantly attenuated neutrophil differentiation of AML cells as evidenced by decreased expression of the differentiation markers CD11b, GCSFR and CEBPE. Moreover, inhibition of ATG16L2 but not ATG16L1 resulted in decreased autophagy induction upon ATRA-treatment. Conversely, silencing ATG16L1 but not ATG16L2 was able to inhibit canonical starvation but not ATRA-induced differentiation associated autophagy in APL cells. Our data reveal distinct functions of ATG16L1 and ATG16L2 in starvation and ATRA-induced autophagy.

To investigate the transcriptional regulation of ATG16L2 during neutrophil differentiation, we screened the ATG16L2 promoter region for putative transcription factor binding sites. We identified PU.1 as a transcriptional regulator of ATG16L2 using chromatin immunoprecipitation, PU.1 knockdown APL cells and a PU.1 inducible AML cell line model. These findings are in line with our earlier findings that PU.1 activates transcription of the ATG genes WIPI1, ATG3, MAP1S and ATG4C during APL differentiation.

Our data provide strong evidence for a particular, non-canonical subtype of autophagy operative during neutrophil differentiation of APL cells. ATG16L2, in contrast to ATG16L1 is essential for successful ATRA-induced neutrophil differentiation and autophagy. This is in sharp contrast to its lack of function during starvation-induced autophagy. Deciphering the particular autophagy pathway active during APL differentiation is a prerequisite to develop novel differentiation therapies that are based on autophagy modulation. Since our findings have been validated in non-APL cells, activation of autophagy might support neutrophil differentiation of AML cells in a more general way.