The anti-cancer drug ABTL0812 induces ER stress-mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer cells.

ABSTRACT

ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased DDIT3 and TRIB3 mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer.Abbreviations 4-PBA 4-phenylbutyrate; AKT AKT serine/threonine kinase; ATG autophagy related; ATF4 activating transcription factor 4; Cer ceramide; DDIT3 DNA damage inducible transcript 3; DEGS1 delta 4-desaturase, sphingolipid 1; dhCer dihydroceramide; EIF2A eukaryotic translation initiation factor 2 alpha; EIF2AK3 eukaryotic translation initiation factor 2 alpha kinase 3; ER endoplasmic reticulum; HSPA5 heat shock protein family A (Hsp70) member 5; MAP1LC3B microtubule associated protein 1 light chain 3 beta; MEF mouse embryonic fibroblast; MTORC1 mechanistic target of rapamycin kinase complex 1; NSCLC non-small cell lung cancer; THC Δ9-tetrahydrocannabinol; TRIB3 tribbles pseudokinase 3; XBP1 X-box binding protein 1; UPR unfolded protein response.