Mecanismo de acción del nuevo fármaco antitumoral abtl0812papel de la pseudoquinasa trib3, el estrés reticular y los esfingolípidos en la muerte mediada por autofagia

Resumen

ABTL0812 is a polyunsaturated fatty acid derivative with antitumoral activity licensed by the biopharmaceutical company Ability Pharmaceuticals. ABTL0812 shows cytotoxicity in a wide panel of human tumor cell lines and induces tumor growth inhibition in human tumor cell-derived xenografts. This have encouraged its pre-clinical and clinical development. ABTL0812 recently started Phase 2 Clinical trials in patients with advanced endometrial cancer and squamous NSCLC, as a first line of treatment in combination with paclitaxel and carboplatin (ClinicalTrials.gov: NCT03366480). Our laboratory is involved in the characterization of ABTL0812’s antitumoral mechanism of action, using MiPaca2 (pancreatic adenocarcinoma) and A549 (lung adenocarcinoma) cell lines as models. Previous studies carried out in our laboratory uncovered that in cells ABTL0812 activates peroxisome proliferator-activated receptors PPAR/ receptors, inducing autophagy-mediated cancer cell death without activating apoptosis. Here, by using gene reporter luciferase assay, qRT-PCR mRNA quantification and immunoblot analysis, we show that ABTL0812 activates PPAR-mediated transcription of Tribbles 3 (TRIB3) pseudokinase. Over-expressed TRIB3 then binds and inhibits Akt, preventing its activation by PDK1 and mTORC2 upstream kinases, resulting in inhibition of the oncogenic Akt/mTORC1 axis. However, we found that mTORC1 inhibition is not enough to induce robust autophagy in MiaPaca2 and A549 cell lines, as it does ABTL0812. Thus, we investigated whether endoplasmic reticulum (ER) stress could also account for ABTL0812-induced TRIB3 overexpression, as it has been proposed for other antitumoral drugs such as tetrahydrocannabinol. We found ABTL0812 induces endoplasmic reticulum dilatation and over-expression of the endoplasmic reticulum stress markers BiP, ATF4 and CHOP (both mRNA and protein levels), indicating that ABTL0812 induces ER stress in these tumor cells. We also show preliminary pharmacological data suggesting that ER stress has a role in mediating ABTL0812-induced cytotoxicity. Remarkably, we have optimized a protocol for the quantification of TRIB3 and CHOP mRNAs (qRT-PCR) in blood and purified PBMCs from patients enrolled in Clinical Phase 2. We describe increased levels of CHOP and TRIB3 mRNAs in response to ABTL0812 treatment. Furthermore, we have proposed and optimized RT quantification of the ER stress genes TRIB3 and CHOP as reliable pharmacodynamic biomarkers for the ongoing Phase 2 Clinical trials. Given the role of sphingolipids in initiating the ER stress, we undertook a comprehensive mass-spec analysis of cellular sphingolipids to show that ABTL0812 induces long-chain dihydroceramides accumulation without affecting ceramide levels. In cells, dihydroceramides are converted into ceramides by the desaturase-1 (Des-1) enzyme. Enzymatic analysis demonstrated that ABTL0812 treatment results in inhibition of Des-1 activity in vitro and in cellular assays, provoking an accumulation of long-chain dihydroceramides, analogously to that induced by Des-1 specific inhibitor GT11. Interestingly, we show that treatment with the dideuterated short-chain dihydroceramide d2c8DhCer (which induces an increase of cellular dihydroceramides) results in endoplasmic reticulum stress, autophagy and cytotoxicity in MiaPaca2 and A549 cell lines. Preliminary characterization of ABTL0812-induced cell death shows that this compound alters mitochondrial function (induces a drastic depletion of cellular ATP) and it activates the canonic mitophagy pathway PINK1/parkin/ubiquitin. Furthermore, ABTL0812 induces cytosolic release lysosomal cathepsin B, suggesting a lysosomal membrane permeabilization (LMP) that would explain the necrotic cell death observed in tumor cells. Finally, this work proposes that ABTL0812 exerts its antitumoral activity acting simultaneously on two axes. On one hand, ABTL0812 activates PPARa/g receptors, which induce TRIB3 over-expression and the subsequent Akt and mTORC1 inhibition. On the other hand, ABTL0812 inhibits Des-1 enzyme, resulting in accumulation of long-chain dihydroceramides and the subsequent activation of ER stress. Both axes synergize to activate a robust autophagy which ultimately leads to lysosomal membrane permeabilization and necrosis. In agreement with this, we show that the inhibition of mTORC1 (Everolimus) synergizes with Des-1 inhibition (GT11) to promote autophagy and cytotoxicity.