Tigilanol tiglate is an oncolytic small molecule that induces immunogenic cell death and enhances the response of both target and non-injected tumors to immune checkpoint blockade
Authors: Cullen Jason K , Yap Pei-Yi , Ferguson Blake , Bruce Zara C , Koyama Motoko , Handoko Herlina , Hendrawan Kevin , Simmons Jacinta L , Brooks Kelly M , Johns Jenny , Wilson Emily S , de Souza Marjorie M A , Broit Natasa , Stewart Praphaporn , Shelley Daniel , McMahon Tracey , Ogbourne Steven M , Nguyen Tam Hong , Lim Yi Chieh , Pagani Alberto , Appendino Giovanni , Gordon Victoria A , Reddell Paul W , Boyle Glen M , Parsons Peter G 
Published: April 2024

BackgroundTigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that intratumoral (I.T.) injection of TT causes vascular disruption with concomitant tumor ablation in several preclinical models of cancer, in addition to various (sub)cutaneous tumors presenting in the veterinary clinic. TT has completed Phase I dose escalation trials, with some patients showing signs of abscopal effects. However, the exact molecular details underpinning its mechanism of action (MoA), together with its immunotherapeutic potential in oncology remain unclear.MethodsA combination of microscopy, luciferase assays, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, phagocytosis assays and mixed lymphocyte reactions were used to probe the MoA of TT in vitro. In vivo studies with TT used MM649 xenograft, CT-26 and immune checkpoint inhibitor refractory B16-F10-OVA tumor bearing mice, the latter with or without anti-programmed cell death 1 (PD-1)/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) mAb treatment. The effect of TT at injected and non-injected tumors was also assessed.ResultsHere, we show that TT induces the death of endothelial and cancer cells at therapeutically relevant concentrations via a caspase/gasdermin E-dependent pyroptopic pathway. At therapeutic doses, our data demonstrate that TT acts as a lipotoxin, binding to and promoting mitochondrial/endoplasmic reticulum (ER) dysfunction (leading to unfolded protein responsemt/ERupregulation) with subsequent ATP depletion, organelle swelling, caspase activation, gasdermin E cleavage and induction of terminal necrosis. Consistent with binding to ER membranes, we found that TT treatment promoted activation of the integrated stress response together with the release/externalization of damage-associated molecular patterns (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through vaccination and rechallenge experiments using CT-26 colon carcinoma tumor bearing mice. Furthermore, TT also reduced tumor volume, induced immune cell infiltration, as well as improved survival in B16-F10-OVA tumor bearing mice when combined with immune checkpoint blockade.ConclusionsThese data demonstrate that TT is an oncolytic small molecule with multiple targets and confirms that cell death induced by this compound has the potential to augment antitumor responses to immunotherapy.