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.