Fatty acid synthase (FASN) is the only human lipogenic enzyme available for de novo fatty acid synthesis and is often highly expressed in cancer cells. We found that FASN mRNA levels were significantly higher in acute myeloid leukemia (AML) patients than in healthy granulocytes or CD34+ hematopoietic progenitors. Accordingly, FASN levels decreased during all-trans retinoic acid (ATRA)-mediated granulocytic differentiation of acute promyelocytic leukemia (APL) cells, partially via autophagic degradation. Furthermore, our data suggest that inhibition of FASN expression levels using RNAi or (-)-epigallocatechin-3-gallate (EGCG) accelerated the differentiation of APL cell lines and significantly re-sensitized ATRA refractory non-APL AML cells. FASN reduction promoted translocation of transcription factor EB (TFEB) to the nucleus, paralleled by activation of CLEAR network genes and lysosomal biogenesis. Together, our data demonstrate that inhibition of FASN expression in combination with ATRA treatment facilitates granulocytic differentiation of APL cells and may extend differentiation therapy to non-APL AML cells.

Switzerland 2 TRANSAUTOPHAGY: European network for multidisciplinary research and translation of autophagy knowledge, COST Action CA15138 Abstract Acute myeloid leukemia (AML) is a blood cancer characterized by a block in differentiation and increased survival of the resulting AML blast cells. While standard chemotherapy for AML cures only 30% of patients, differentiation-inducing therapy using all-trans retinoic acid (ATRA) in combination with arsenic trioxide achieves 90% cure rates in acute promyelocytic leukemia (APL). A better understanding of the molecular mechanisms underlying ATRA therapy in APL may provide new perspectives in the treatment of additional AML subtypes.Fatty acid synthase (FASN) is the only human lipogenic enzyme available for de novo fatty acid synthesis. While FASN levels are very low in healthy adult tissues, it is often highly expressed in cancer cells, thus representing a potential therapeutic target. We found that FASN mRNA levels were significantly higher in AML patients than in healthy granulocytes or CD34 + hematopoietic progenitors in two AML cohorts (n=68, n=203) (p<0.05). Accordingly, FASN levels decreased during ATRA-mediated granulocytic differentiation of APL cells, partially via autophagic degradation. Furthermore, our data suggests that inhibition of FASN expression levels using RNAi or (-)-epigallocatechin-3-gallate (EGCG), accelerates the differentiation of APL cell lines and significantly resensitized ATRA refractory non-APL AML cells. Decreasing FASN expression reduced mTOR activity and promoted translocation of transcription factor EB (TFEB) to the nucleus. Lysosomal biogenesis was activated, consistent with TFEB transcriptional activation of CLEAR network genes.Together, our data demonstrates that inhibiting FASN expression in combination with ATRA treatment promotes granulocytic differentiation of APL cells and may extend differentiation therapy to non-APL AML cells. BioRXiv

Acute myeloid leukemia (AML) is characterized by a block in myeloid differentiation and an aberrant cell proliferation of leukemia blasts. While other AML therapies cure only 30% of the patients, acute promyelocytic leukemia (APL) is successfully treated by differentiation therapy using all-trans retinoic acid (ATRA) in combination with chemotherapy or arsenic trioxide. A better understanding of the molecular mechanisms underlying ATRA therapy in APL may offer new perspectives in the treatment of additional AML subtypes. Fatty acid synthase (FASN) is the only human lipogenic enzyme able of de novo fatty acid synthesis. Its expression is very low in healthy adult tissues, whereas increased expression is commonly found in a variety of cancerous tissues. Therefore, FASN represents an attractive potential drug target. To investigate if a similar expression pattern is found in normal versus malignant myeloid cells, we determined FASN expression in fresh leukemic blast cells from untreated AML patients obtained at the Inselspital Bern (Switzerland) (n=68) and expression data available online from the Bloodspot server (n=203). In both AML cohorts, we found that FASN expression is significantly higher in AML patients than in healthy granulocytes (p<0.05). Accordingly, FASN protein levels are reduced in NB4 and HT93 APL cells upon ATRA-induced granulocyte differentiation. Additionally, silencing FASN in NB4 cells led to an acceleration of differentiation upon ATRA treatment. Surprisingly, solely inhibiting the catalytic function of FASN using C75 or Orlistat did not affect granulocyte differentiation nor FASN expression. To validate that FASN levels and not activity attenuate granulocyte differentiation, we took advantage of the green tea polyphenol epigallocatechin-3-gallate (EGCG) that reduces FASN protein expression. Indeed, similar to knocking down FASN, EGCG treatment of NB4 cells resulted in increased differentiation paralleled by decreased FASN protein levels. We and others demonstrated the importance of autophagy for successful ATRA-induced APL differentiation. Autophagy is an intracellular degradation system that ensures a dynamic recycling of cytoplasmic contents. This process is regulated by the so-called autophagy related genes or ATGs. ATG1, or ULK1, is a key autophagy gene of the initiation complex that is inhibited by mTOR-mediated phosphorylation leading to reduced autophagy activity. FASN expression correlates with mTOR activity in several solid cancers, therefore we hypothesized that FASN activates mTOR resulting in decreased autophagy in AML cells. Indeed, lowering FASN protein expression by either shRNA or EGCG resulted in increased autophagy together with a decreased mTOR-mediated phosphorylation of ULK1. Next, we aimed at understanding how FASN expression is regulated during ATRA treatment. We found a negative correlation between KLF5 and FASN expression in both AML patient cohorts (r=-0.3) and 4 potential KLF binding sites on the FASN promoter, suggesting that KLF5 inhibits FASN transcription. However, since the protein half-life of FASN is around 24 hours, transcriptional inhibition is not sufficient to fully explain the reduced protein levels upon ATRA treatment. We therefore tested if FASN protein itself is degraded via autophagy. Co-treatment of NB4 cells with ATRA and the lysosome inhibitor Bafilomycin A1 led to a dose dependent accumulation of FASN protein suggesting that FASN is an autophagy substrate during ATRA treatment. Furthermore, using mCherry-LC3B expressing NB4 cells, we were able to show FASN and LC3B co-localization by fluorescence microscopy. Finally, we asked whether inhibiting FASN expression levels would increase ATRA sensitivity in non-APL AML cells. To this end, we treated KG-1, OCI-AML2 and MOLM-13 AML cells with EGCG in combination with ATRA. Importantly, combining ATRA with EGCG resulted in increased differentiation of these non-APL cells paralleled by reduced FASN levels, decreased mTOR activity and ULK1 phosphorylation. Together, our data suggest that EGCG treatment is beneficial for differentiation therapy of non-APL AML cells by activating autophagy via degradation of FASN and thereby inactivating mTOR. Furthermore, high FASN expression in AML is likely due to low expression of its positive transcriptional regulator KLF5 and low autophagy activity. Disclosures No relevant conflicts of interest to declare.