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.