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The long-standing perception of Protein Kinase C (PKC) as a family of oncoproteins has increasingly been challenged by evidence that some PKC isoforms may act as tumor suppressors. To explore the hypothesis that activation, rather than inhibition, of these isoforms is critical for anticancer activity, we isolated and characterized a family of 16 novel phorboids closely-related to tigilanol tiglate (EBC-46), a PKC-activating epoxytigliane showing promising clinical safety and efficacy for intratumoral treatment of cancers. While alkyl branching features of the C12-ester influenced potency, the 6,7-epoxide structural motif and position was critical to PKC activation in vitro. A subset of the 6,7-epoxytiglianes were efficacious against established tumors in mice; which generally correlated with in vitro activation of PKC. Importantly, epoxytiglianes without evidence of PKC activation showed limited antitumor efficacy. Taken together, these findings provide a strong rationale to reassess the role of PKC isoforms in cancer, and suggest in some situations their activation can be a promising strategy for anticancer drug discovery.

SummaryThe five-year survival rate for patients with head and neck squamous cell carcinoma (HNSCC) has remained at ~50% for the past 30 years despite advances in treatment. Tigilanol tiglate (TT, also known as EBC-46) is a novel diterpene ester that induces cell death in HNSCC in vitro and in mouse models, and has recently completed Phase I human clinical trials. The aim of this study was to optimise efficacy of TT treatment by altering different administration parameters. The tongue SCC cell line (SCC-15) was identified as the line with the lowest efficacy to treatment. Subcutaneous xenografts of SCC-15 cells were grown in BALB/c Foxn1nu and NOD/SCID mice and treated with intratumoral injection of 30 μg TT or a vehicle only control (40% propylene glycol (PG)). Greater efficacy of TT treatment was found in the BALB/c Foxn1nu mice compared to NOD/SCID mice. Immunohistochemical analysis indicated a potential role of the host’s innate immune system in this difference, specifically neutrophil infiltration. Neither fractionated doses of TT nor the use of a different excipiant led to significantly increased efficacy. This study confirmed that TT in 40% PG given intratumorally as a single bolus dose was the most efficacious treatment for a tongue SCC mouse model.

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/ER upregulation) 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.

Anthocyanin-rich black rice powder was incorporated into bread, and the stability of two specific anthocyanins, cyanidin-3-glucoside and cyanidin-3-rutinoside, during baking was investigated. Various baking conditions included different baking temperatures (200, 220, and 240 °C) and baking durations (0, 2, 4, 6, 8, 10, and 12 min). Non-isothermal kinetic models were successfully established for the two anthocyanins in both bread crumb and crust. The derived degradation rate ( k ref ) of cyanidin-3-glucoside and cyanidin-3-rutinoside in bread crumb at the reference temperature ( T ref ) of 65 °C were 2.49 × 10 −5 and 1.93 × 10 −5  s −1 , respectively. The k ref values of cyanidin-3-glucoside and cyanidin-3-rutinoside in bread crust ( T ref  = 125 °C) were 5.37 × 10 −4 and 5.72 × 10 −4  s −1 , respectively. The color development of bread crust and crumb was measured and expressed as L * C * H ° values. While the color of bread crust was significantly subjected to variations in oven operating parameters, the color of bread crumb was relatively less affected by baking conditions. The antioxidant capacity and total phenolic content of bread samples were measured using DPPH and Folin-Ciocalteu assays, respectively. Results showed that in bread crumb, both the antioxidant capacity and the total phenolic content decreased; however, an increase in both was observed in bread crust.

BackgroundTigilanol Tiglate (TT) is a novel small molecule under development for local treatment of solid tumours via intratumoral (I.T.) injection. TT is a protein kinase C (PKC)/C1 domain activator that disrupts tumour vasculature and causes direct oncolysis of tumour cells. Together, these activities lead to haemorrhagic necrosis of injected tumours with enduring ablation of >70% of target tumours in both pre-clinical xenograft/syngeneic mouse models and cutaneous tumours presenting in the veterinary clinic.1–3 TT has completed a Phase I/IIa dose-escalation trial in humans (ACTRN12614000685617), with strong evidence of local anti-cancer efficacy and signs of abscopal effects in some patients.4 However, the underlying mechanism of action (MOA) of TT, together with its immunotherapeutic potential in oncology, is not fully understood.MethodsA combination of microscopy, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, LDH release assays and mixed lymphocyte reactions were used to probe the MOA of TT in vitro. TT-mediated damage associated molecular pattern (DAMP) release/externalization was assessed using luciferase (ATP), ELISA (HMGB1), flow cytometry and immunohistochemical (HMGB1, calreticulin) approaches. In vivo experimentation with TT utilized CT-26 and B16-F10 tumor bearing mice. Analysis of DAMP release and immune cell infiltration into TT treated human head and neck tumours (ACTRN12619001407189) was performed by immunohistochemistry.ResultsOur data reveal that therapeutic concentrations of TT induce the death of cancer and endothelial cell lines via a pathway involving caspase activation and cleavage of the pore forming protein gasdermin E. TT promotes this mechanism of cell death by interacting with ER membranes, causing an ER stress response that results in loss of mitochondrial membrane potential, ATP depletion, organelle swelling and oncosis/pyroptosis. Treatment of cells with TT also led to the release of damage associated molecular patterns (DAMPs), indicative of an immunogenic cell death (ICD) pathway that also resulted in the generation of tumour-specific T cells in CT-26 tumor bearing mice. Whilst the induction of ICD is largely PKC-independent in vitro, PKC/C1 domain signaling appears necessary for efficacious tumour ablation in vivo. Consistent with our pre-clinical data, immunohistochemical analysis of TT-treated head and neck tumors found that drug stimulated DAMP release/externalisation and the recruitment of immune cells, principally CD8+ T cells, into remnant tumour mass.ConclusionsThese data indicate that TT is an oncolytic small molecule with the potential to ablate target tumours and enhance immunotherapy combinations through promoting immune cell infiltration. TT is currently undergoing Phase II trials in head and neck cancer (NCT05234437) and soft tissue sarcoma (NCT05755113).ReferencesBoyle GM, D’Souza MMA, Pierce CJ, Adams RA, Cantor AS, Johns JP, Maslovskaya L, Gordon VA, Reddell PW, Parsons PG. Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models. PLOS ONE 2014;9:e108887.Cullen JK, Boyle GM, Yap PY, Elmlinger S, Simmons JL, Broit N, Johns J, Ferguson B, Maslovskaya LA, Savchenko AI, Mirzayans PM, Porzelle A, Bernhardt PV, Gordon VA, Reddell PW, Pagani A, Appendino G, Parsons PG, Williams CM. Activation of PKC supports the anticancer activity of tigilanol tiglate and related epoxytiglianes. Sci. Rep. 2021;11:207.De Ridder TR, Campbell JE, Burke-Schwarz C, Clegg D, Elliot EL, Geller S, Kozak W, Pittenger ST, Pruitt JB, Riehl J, White J, Wiest ML, Johannes CM, Morton J, Jones PD, Schmidt PF, Gordon VA, Reddell PW. Randomized controlled clinical study evaluating the efficacy and safety of intratumoral treatment of canine mast cell tumors with tigilanol tiglate (EBC-46). J. Vet. Intern. Med. 2021;35:415–429.Panizza BJ, de Souza P, Cooper A, Roohullah A, Karapetis CS, Lickliter JD. Phase I dose-escalation study to determine the safety, tolerability, preliminary efficacy and pharmacokinetics of an intratumoral injection of tigilanol tiglate (EBC-46). EBioMedicine. 2019;50:433–441.Ethics ApprovalThe study obtained ethics approval from the following commitees and boards: Metro South Human Research Ethics Committee, 199 Ipswich Road, Woolloongabba, QLD, 4102, Australia. Ethics approval number: HREC/2019/QMS/54004. Bellberry Limited, 123 Glen Osmond Road, Eastwood, SA 5063, Australia. Ethics approval number: 2019–10-846 (REGIS 2019/ETH13063). Tata Memorial Hospital - Institutional Review Board, IRB Office, Dr. E. Borges Marg, Parel, Mumbai - 400 012, India. Ethics approval number: IEC/1219/3370/001. Tata Medical Center - Institutional Review Board, 14 Major Arterial Road (EW), New Town, Rajarhat, Kolkata - 700 160, India. Ethics approval number: 2019/PHARMA/57/IRB39.

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

BackgroundTigilanol Tiglate (TT) is a novel small molecule under development for local treatment of solid tumours via intratumoral (I.T.) injection. TT is a protein kinase C (PKC)/C1 domain activator that disrupts tumour vasculature, leading to haemorrhagic necrosis of the lesion.1 Strikingly, in both preclinical syngeneic mouse models and cutaneous/subcutaneous tumours presenting in the veterinary clinic, I.T. injection of TT results in complete and enduring ablation of target tumours in >70% of patients.1,2,3 TT has completed a Phase I/IIa dose-escalation trial in humans (ACTRN12614000685617), with strong evidence of local anti-cancer efficacy and signs of abscopal effects in some patients.4 However, the underlying mechanism of action (MOA) of TT, together with its immunotherapeutic potential in oncology, is not fully understood.MethodsA combination of microscopy, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, LDH release assays and mixed lymphocyte reactions were used to probe the MOA of TT in vitro. TT-mediated damage associated molecular pattern (DAMP) release/externalization was assessed using luciferase (ATP), ELISA (HMGB1), flow cytometry and immunohistochemical (calreticulin) approaches. In vivo experimentation with TT utilized CT-26 and B16-F10-OVA tumor bearing mice, with or without anti-PD1/anti-CTLA4 treatment.ResultsOur data demonstrates that therapeutic concentrations of TT induce death of cancer and endothelial cell lines, both in vitro and in vivo, via oncosis. Whilst largely PKC-independent, PKC/C1 domain signaling appears necessary for timely oncolysis in vitro and efficacious tumor ablation in vivo. Our results also show that TT binds to ER membranes, causing ER stress with subsequent activation of the integrated stress response. This is followed by mitochondrial membrane potential loss, ATP depletion, organelle swelling, oncosis and terminal necrosis. We also found that TT treatment promoted the release/externalization of DAMPs (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 rechallenge experiments using CT-26 tumour bearing mice, which also demonstrated that TT promoted the development of tumour-specific T cells. In addition to stimulating immune cell infiltration into tumours, TT significantly improved treatment response in the B16-F10-OVA mouse melanoma model when combined with immune checkpoint blockade.ConclusionsThese data indicate that TT is an oncolytic small molecule with the potential to enhance responses to immunotherapy. TT is currently undergoing Phase I/II trials in head and neck cancers (ACTRN12619001407189), soft tissue sarcomas, Stage III melanoma in-transit (NCT05234437) and non-resectable Stage IIIB to IV M1c melanoma (TT/pembrolizumab combination: NCT04834973).5ReferencesBoyle GM, D’Souza MMA, Pierce CJ, Adams RA, Cantor AS, Johns JP, Maslovskaya L, Gordon VA, Reddell PW, Parsons PG. Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models. PLOS ONE 2014;9:e108887.Cullen JK, Boyle GM, Yap PY, Elmlinger S, Simmons JL, Broit N, Johns J, Ferguson B, Maslovskaya LA, Savchenko AI, Mirzayans PM, Porzelle A, Bernhardt PV, Gordon VA, Reddell PW, Pagani A, Appendino G, Parsons PG, Williams CM. Activation of PKC supports the anticancer activity of tigilanol tiglate and related epoxytiglianes. Sci Rep 2021;11:207.De Ridder TR, Campbell JE, Burke-Schwarz C, Clegg D, Elliot EL, Geller S, Kozak W, Pittenger ST, Pruitt JB, Riehl J, White J, Wiest ML, Johannes CM, Morton J, Jones PD, Schmidt PF, Gordon VA, Reddell PW. Randomized controlled clinical study evaluating the efficacy and safety of intratumoral treatment of canine mast cell tumors with tigilanol tiglate (EBC-46). J Vet Intern Med 2021;35:415–429.Panizza BJ, de Souza P, Cooper A, Roohullah A, Karapetis CS, Lickliter JD. Phase I dose-escalation study to determine the safety, tolerability, preliminary efficacy and pharmacokinetics of an intratumoral injection of tigilanol tiglate (EBC-46). EbioMedicine 2019;50:433–441.QBiotics Group Ltd. website. https://qbiotics.com/Ethics ApprovalAll animal procedures were approved in accordance with NHMRC guidelines (Australian Code for the Care and Use of Animals for Scientific Purposes 8th Edition, 2013; National Health and Medical Research Council of Australia) by the QIMR Berghofer Animal Ethics Committee: A0106-042M, A0404-606M and A01047M.

Background: Bone tissue decalcification is essential for histopathological evaluation, but conventional methods using inorganic acids degrade nucleic acids, limiting molecular testing. EDTA is known to better preserve DNA, but its suitability for next-generation sequencing (NGS) in clinical settings remains to be validated. Methods: This retrospective study evaluated 752 formalin-fixed paraffin-embedded (FFPE) tissue samples undergoing NGS between January 2022 and October 2024. Of these, 31 were decalcified using EDTA (Osteosoft, Merck, Germany). DNA was extracted using the Qiagen AllPrep® kit and quantified using Qubit and NanoDrop. Libraries were prepared with a custom 30-gene Ampliseq panel and sequenced on the Ion Torrent platform. Sequencing was deemed suboptimal if <95% of target regions reached ≥250X depth. Results were compared to 721 non-decalcified FFPE samples. Results: Suboptimal sequencing occurred in 9.7% of EDTA-decalcified and 9.0% of non-decalcified cases (p = 0.9). DNA concentration (Qubit) and NanoDrop 260/280 ratios were not significantly different (p = 0.4 and p = 0.8, respectively), though EDTA cases had lower DNA concentrations (NanoDrop, p = 0.006) and 260/230 ratios (p = 0.002). Mutation detection in decalcified samples was consistent with known mutation profiles for respective tumor types. Conclusions: EDTA-decalcified FFPE bone tissues produce NGS results comparable to non-decalcified specimens, with similar sequencing success rates and acceptable DNA quality. These findings support the use of EDTA as a suitable decalcification method for molecular diagnostics, enabling broader inclusion of bone specimens in clinical testing.

The utility of neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) utility in predicting immune-related adverse events (irAEs) and survival have not been well studied in the context of treatment with immune checkpoint inhibitors (ICIs). We performed a case-control study of cancer patients who received at least one dose of ICI in a tertiary hospital. We examined NLR and PLR in irAE cases and controls. Logistic and Cox regression models were used to identify independent risk factors for irAEs, progression-free survival (PFS), and overall survival (OS). The study included 91 patients with irAEs and 56 controls. Multiple logistic regression showed that NLR < 3 at baseline was associated with higher occurrence of irAEs. Multivariate Cox regression showed that development of irAEs and reduction in NLR from baseline to week 6 were associated with longer PFS. Higher NLR values at baseline and/or week 6 were independently associated with shorter OS. A reduction in NLR from baseline to week 6 was associated with longer OS. In this study of cancer patients treated with ICIs, NLR has a bidirectional relationship with adverse outcomes. Lower NLR was associated with increased occurrence of irAEs while higher NLR values were associated with worse clinical outcomes.

Background Currently, there are no standard guidelines for recommending re-administration of a chemotherapeutic drug to a patient after a serious adverse drug reaction (ADR) incident. The decision on whether to rechallenge the patient is based on the experience of the clinician and is highly subjective. Thus the aim of this study is to develop a decision support tool to assist clinicians in this decision making process. Methods The inclusion criteria for patients in this study are: (1) had chemotherapy at National Cancer Centre Singapore between 2004 to 2009, (2) suffered from serious ADRs, and (3) were rechallenged. A total of 46 patients fulfilled the inclusion criteria. A genetic algorithm attribute selection method was used to identify clinical predictors for patients' rechallenge status. A Naïve Bayes model was then developed using 35 patients and externally validated using 11 patients. Results Eight patient attributes (age, chemotherapeutic drug, albumin level, red blood cell level, platelet level, abnormal white blood cell level, abnormal alkaline phosphatase level and abnormal alanine aminotransferase level) were identified as clinical predictors for rechallenge status of patients. The Naïve Bayes model had an AUC of 0.767 and was found to be useful for assisting clinical decision making after clinicians had identified a group of patients for rechallenge. A platform independent version and an online version of the model is available to facilitate independent validation of the model. Conclusion Due to the limited size of the validation set, a more extensive validation of the model is necessary before it can be adopted for routine clinical use. Once validated, the model can be used to assist clinicians in deciding whether to rechallenge patients by determining if their initial assessment of rechallenge status of patients is accurate.