A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation.

BACKGROUND: CAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit of CAR T cell therapy for patients with solid tumors. Novel manufacturing and engineering approaches show great promise to enhance CAR T cell function against solid tumors. However, similar to single agent chemotherapy approaches, CAR T cell monotherapy may be unable to achieve high cure rates for patients with difficult to treat solid tumors. Thus, combinatorial drug plus CAR T cell approaches are likely required to achieve widespread clinical success. METHODS: We developed a novel, confocal microscopy based, high-content screen to evaluate 1114 FDA approved drugs for the potential to increase expression of the solid tumor antigen B7-H3 on the surface of osteosarcoma cells. Western blot, RT-qPCR, siRNA knockdown and flow cytometry assays were used to validate screening results and identify mechanisms of drug-induced B7-H3 upregulation. Cytokine and cytotoxicity assays were used to determine if drug pre-treatment enhanced B7-H3-CAR T cell effector function. RESULTS: Fifty-five drugs were identified to increase B7-H3 expression on the surface of LM7 osteosarcoma cells using a novel high-content, high-throughput screen. One drug, ingenol-3-angelate (I3A), increased B7-H3 expression by up to 100%, and was evaluated in downstream experiments. Validation assays confirmed I3A increased B7-H3 expression in a biphasic dose response and cell dependent fashion. Mechanistic studies demonstrated that I3A increased B7-H3 (CD276) mRNA, total protein, and cell surface expression via protein kinase C alpha activation. Functionally, I3A induced B7-H3 expression enhanced B7-H3-CAR T cell function in cytokine production and cytotoxicity assays. CONCLUSIONS: This study demonstrates a novel high-content and high-throughput screen can identify drugs to enhance CAR T cell activity. This and other high-content technologies will pave the way to develop clinical trials implementing rational drug plus CAR T cell combinatorial therapies. Importantly, the technique could also be repurposed for an array of basic and translational research applications where drugs are needed to modulate cell surface protein expression.

Identification and validation of a glycolysis-related taxonomy for improving outcomes in glioma.

BACKGROUND: Reprogramming of glucose metabolism is a prominent abnormal energy metabolism in glioma. However, the efficacy of treatments targeting glycolysis varies among patients. The present study aimed to classify distinct glycolysis subtypes (GS) of glioma, which may help to improve the therapy response. METHODS: The expression profiles of glioma were downloaded from public datasets to perform an enhanced clustering analysis to determine the GS. A total of 101 combinations based on 10 machine learning algorithms were performed to screen out the most valuable glycolysis-related glioma signature (GGS). Through RSF and plsRcox algorithms, adrenomedullin (ADM) was eventually obtained as the most significant glycolysis-related gene for prognostic prediction in glioma. Furthermore, drug sensitivity analysis, molecular docking, and in vitro experiments were utilized to verify the efficacy of ADM and ingenol mebutate (IM). RESULTS: Glioma patients were classified into five distinct GS (GS1-GS5), characterized by varying glycolytic metabolism levels, molecular expression, immune cell infiltration, immunogenic modulators, and clinical features. Anti-CTLA4 and anti-PD-L1 antibodies significantly improved the prognosis for GS2 and GS5, respectively. ADM has been identified as a potential biomarker for targeted glycolytic therapy in glioma patients. In vitro experiments demonstrated that IM inhibited glioma cell progression by inhibiting ADM. CONCLUSION: This study elucidates that evaluating GS is essential for comprehending the heterogeneity of glioma, which is pivotal for predicting immune cell infiltration (ICI) characterization, prognosis, and personalized immunotherapy regimens. We also explored the glycolysis-related genes ADM and IM to develop a theoretical framework for anti-tumor strategies targeting glycolysis.

Comparative efficacy of treatments for molluscum contagiosum: A systematic review and network meta-analysis.

BACKGROUND AND OBJECTIVES: Various interventions have been applied to treat molluscum contagiosum, but benefits and efficacy remain unclear. To assess the comparative efficacy and safety of interventions for molluscum contagiosum, a network meta-analysis was performed. PATIENTS AND METHODS: Embase, PubMed, and the Cochrane Library were searched for articles published between January 1, 1990, and November 31, 2020. Eligible studies were randomized clinical trials (RCTs) of interventions in immunocompetent children and adults with genital/non-genital molluscum contagiosum lesions. RESULTS: Twelve interventions from 25 RCTs including 2,123 participants were assessed. Compared with the placebo, ingenol mebutate had the most significant effect on complete clearance (odds ratio [OR] 117.42, 95% confidence interval [CI] 6.37-2164.88), followed by cryotherapy (OR 16.81, 95% CI 4.13-68.54), podophyllotoxin (OR 10.24, 95% CI 3.36-31.21), and potassium hydroxide (KOH) (OR 10.02, 95% CI 4.64-21.64). Data on adverse effects were too scarce for quantitative synthesis. CONCLUSIONS: Ingenol mebutate, cryotherapy, podophyllotoxin, and KOH were more effective than the other interventions in achieving complete clearance, but safety concerns regarding ingenol mebutate have recently been reported. Due to the possibility of spontaneous resolution, observation is also justified for asymptomatic infection. Factors including adverse effects, cost, patient preference, and medical accessibility should be considered.

Ingenol mebutate inhibits the growth of pancreatic cancer cells in vitro via STING with an efficacy comparable to that of clinically used anticancer agents.

Pancreatic cancer is associated with a poor prognosis; thus, there is an urgent need to develop new and effective treatments. Ingenol mebutate (IM), which is isolated from the latex of Euphorbia peplus, was recently shown to be effective against pancreatic cancer cell lines; however, its mechanism of action has not been fully elucidated. In this study, we focused on the less drug-sensitive pancreatic cancer cell line Panc-1 and compared IM to commercially available anticancer drugs using cell survival assays. In addition, we aimed to identify novel biomolecules that may be involved in the mechanism of action of IM using RNA sequencing, western blotting, and inhibition assays. The IC50 values after 72 h of exposure to IM and SN-38, drugs to which the Panc-1 cells are most sensitive among the tested anticancer agents, were 43.1 ± 16.8 nM and 165 ± 37 nM, respectively. IM showed a cytostatic effect equal to or greater than that of the clinically used pancreatic cancer therapeutic drugs. RNA sequencing and protein expression analysis revealed that expression of stimulator of interferon genes (STING) increased at low IM concentration, whereas cell viability decreased. Co-exposure of IM and STING inhibitor, H-151, to Panc-1 or MIA PaCa-2 cell lines canceled the growth-inhibitory effects of IM alone. In conclusion, IM may have an efficacy comparable to that of existing pancreatic cancer therapeutic agents on the less drug-sensitive Panc-1 cell line and the immune-related molecule STING plays a role in the mechanism of action of IM.

Ingenol Mebutate Is Associated With Increased Reporting Odds for Squamous Cell Carcinoma in Actinic Keratosis Patients, a Pharmacovigilance Study of the FDA Adverse Event Reporting System (FAERS).

BACKGROUND: Recently the production and marketing of ingenol mebutate in the European Union (EU) and Canada was halted due to a possible increased risk of squamous cell carcinoma (SCC) in patients with actinic keratosis (AK). OBJECTIVE: To investigate the relationship between SCC and topical AK medications including ingenol mebutate in the FDA Adverse Event Reporting System (FAERS). METHODS: Case/non-case analyses were performed in FAERS using data from 2012 to 2020 to examine the reporting odds ratio (ROR) signal for SCC for ingenol mebutate and all classes of topical AK medications under multiple conditions: i. comparison to all other drugs in FAERs, ii. comparison to other topical AK medications, iii. comparison to all other topical AK medications where only a single agent was implicated, iv. comparison of ingenol mebutate vs. imiquimod. RESULTS: A statistically significant ROR for SCC was found for ingenol mebutate under all conditions (i. 31.57 (25.45, 39.16), ii. 50.35 (32.21, 78.82), iii 61.09 (35.36, 105.56), iv. 2.53 (1.27, 5.05). A significant but substantially smaller signal was observed for imiquimod (i. 12.38 (6.42, 32.84), ii. 5.18 (2.61, 10.26), iii 5.42 (2.49, 11.78), but not for fluorouracil or diclofenac. When compared to imiquimod directly, ingenol mebutate had a statistically significant ROR for SCC (2.53 (1.27, 5.05). CONCLUSION: Our findings support an association between SCC and ingenol mebutate. This association is maintained under controls to limit bias and falsely elevated signal including controlling for disease state and cases with multiple drug exposures and when compared to imiquimod as in Phase IV studies of ingenol mebutate.

Topical Treatment of Actinic Keratosis and Metalloproteinase Expression: A Clinico-Pathological Retrospective Study.

Actinic keratosis is an intraepithelial proliferation of atypical keratinocytes that could progress into invasive squamous cell carcinoma. Most evidence suggests an important role of the dermal matrix metalloproteinases in the progression of atypical skin epithelial lesions. We evaluated the clinical efficacy of three different therapeutic modalities (a medical device containing 0.8% piroxicam cream and 50+ sunscreen, photodynamic therapy, and ingenol mebutate gel) to treat suspicious actinic keratoses, which were biopsied for histopathological examination and then analyzed for the expression of matrix metalloproteinases by immunohistochemistry. Clinical, dermoscopic, and reflectance confocal microscopy evaluations revealed a gradual decrease in all standard scores validated for actinic keratosis assessment at the end of the treatments. From a histopathological point of view, we documented the substantial restoration of normal skin architecture, while the immunohistochemical evaluation of matrix metalloproteinases showed a reduction in expression in the treated skin lesions compared to the baseline. As actinic keratoses are considered the precursors of squamous cell carcinoma, their treatment is crucial to prevent the development of a more aggressive disease. Our study monitored the evolution of actinic keratoses subjected to three different topical therapies, with the value of correlating clinical and histopathological findings. Moreover, as the matrix metalloproteinases are largely recognized factors involved in the pathogenesis and evolution of actinic keratosis to squamous cell carcinoma, the demonstration by immunohistochemistry of a reduction in their expression after the treatments adds new valuable concern to the field.

Influence of serum vitamin D level in the response of actinic keratosis to ingenol mebutate.

Vitamin D (VD) serum levels, and keratinocytic basal expression of vitamin D receptor (VDR) before treatment of actinic keratoses (AK) have been previously reported as possible biomarkers of the response of AK to treatments. We intended to evaluate the association between these and other serum and immunohistochemical parameters with the response of AK to treatment with topical ingenol mebutate (IM). Twenty-five patients with AK on the head were treated with topical IM 0.015% gel once daily for 3 days. Biopsies were taken at baseline and 6 weeks after treatment. Immunohistochemical staining was performed for VDR, P53, Ki67, Aurora B, Survivin and ß-catenin. Basal serum 25(OH)D levels were determined. IM was more effective for KIN I and II AKs than in KIN III, and histological responders showed significantly higher serum VD levels (30.278 [SD 8.839] ng/mL) than nonresponders (21.14 [SD 7.079] ng/mL, p = 0.023). In addition, mean basal expression of VDR (45.63 [SD 16.105] %) increased significantly (57.92 [SD 14.738] %, p = 0.003) after treatment with IM. A significant decrease after treatment in the expression of several markers of aggressiveness and progression to squamous cell carcinoma, namely P53, Ki-67, aurora B kinase and survivin, was also observed. Our results support a relationship between VD status and the response of AK to treatment with topical IM, suggesting that its previous correction to proper serum levels in VD-deficient patients could improve the response of AK to the treatment.

Repurposing of ingenol mebutate for treating human colorectal cancer by targeting S100 calcium-binding protein A4 (S100A4).

Colorectal cancer (CRC) is the world's second most common cause of cancer-related death. Novel treatments are still urgently needed. S100 calcium-binding protein A4 (S100A4) was demonstrated to be an anticancer therapeutic target. Herein, we found that higher S100A4 expression was associated with a poorer prognosis in publicly available cohorts and a Taiwanese CRC patient cohort. To identify repurposed S100A4 inhibitors, we mined the Connectivity Map (CMap) database for clinical drugs mimicking the S100A4-knockdown gene signature. Ingenol mebutate, derived from the sap of the plant Euphorbia peplus, is approved as a topical treatment for actinic keratosis. The CMap analysis predicted ingenol mebutate as a potent S100A4 inhibitor. Indeed, both messenger RNA and protein levels of S100A4 were attenuated by ingenol mebutate in human CRC cells. In addition, CRC cells with higher S100A4 expressions and/or the wild-type p53 gene were more sensitive to ingenol mebutate, and their migration and invasion were inhibited by ingenol mebutate. Therefore, our results suggest the repurposing of ingenol mebutate for treating CRC by targeting S100A4.

Approaches to Field Therapy for Actinic Keratoses: Relating Clinical Trial Results to Real-world Practice-A Commentary.

There have been multiple direct and indirect comparison studies evaluating different field therapies used in the treatment of actinic keratosis (AK). A recent clinical trial directly compared 5% fluorouracil (5-FU), imiquimod, ingenol mebutate, and methyl aminolevulinate photodynamic therapy (MAL-PDT), reporting that 5-FU was superior to the other treatments in achieving sustained clearance of 75 percent or greater of AK lesions compared to baseline. In this commentary, the author reviews and discusses the methods and results of this comparison study and propose these results are limited by a number of factors, such as the selected primary % clearance endpoint, grade range of included AKs, and treatments included in the comparison, when considered in the context of other clinical and real-world comparison studies evaluating AK field therapies. The author postulates that patient acceptance of and adherence to field therapy regimens for the treatment of AK may be better evaluated in a real-world setting. Additionally, the author suggests that selection of field therapy in the treatment of AK should be driven by consideration of relevant patient-, disease-, and treatment-related factors, and what is considered best may differ from patient to patient, depending on each patient's individual needs and expectations.

Discovery of a novel megakaryopoiesis enhancer, ingenol, promoting thrombopoiesis through PI3K-Akt signaling independent of thrombopoietin.

Thrombocytopenia, a most common complication of radiotherapy and chemotherapy, is an important cause of morbidity and mortality in cancer patients. However, there are still no approved agents for the treatment of radiation- and chemotherapy-induced thrombocytopenia (RIT and CIT, respectively). In this study, a drug screening model for predicting compounds with activity in promoting megakaryocyte (MK) differentiation and platelet production was established based on machine learning (ML), and a natural product ingenol was predicted as a potential active compound. Then, in vitro experiments showed that ingenol significantly promoted MK differentiation in K562 and HEL cells. Furthermore, a RIT mice model and c-MPL knock-out (c-MPL-/-) mice constructed by CRISPR/Cas9 technology were used to assess the therapeutic action of ingenol on thrombocytopenia. The results showed that ingenol accelerated megakaryopoiesis and thrombopoiesis both in RIT mice and c-MPL-/- mice. Next, RNA-sequencing (RNA-seq) was carried out to analyze the gene expression profile induced by ingenol during MK differentiation. Finally, through experimental verifications, we demonstrated that the activation of PI3K/Akt signaling pathway was involved in ingenol-induced MK differentiation. Blocking PI3K/Akt signaling pathway abolished the promotion of ingenol on MK differentiation. Nevertheless, inhibition of TPO/c-MPL signaling pathway could not suppress ingenol-induced MK differentiation. In conclusion, our study builds a drug screening model to discover active compounds against thrombocytopenia, reveals the critical roles of ingenol in promoting MK differentiation and platelet production, and provides a promising avenue for the treatment of RIT.

Cascade targeting codelivery of ingenol-3-angelate and doxorubicin for enhancing cancer chemoimmunotherapy through synergistic effects in prostate cancer.

Immunotherapy has led to an expansion of the treatment of malignancies, but its effect in prostate cancer (PCa) patients is modest. Chemoimmunotherapy is a promising approach that has attracted substantial attention. Although the widely used clinical chemotherapeutic drug doxorubicin (DOX) elicits immunogenic cell death (ICD), its weak ICD effect and the abnormal vasculature of tumors severely limit its efficacy in chemoimmunotherapy. Ingenol-3-angelate (I3A), an emerging antitumor drug with dual chemotherapeutic and immune response-eliciting effects, is expected to exert synergistic effects when administered in combination with DOX. I3A induces the ICD of PCa cells by triggering mitophagy and apoptosis and promotes the normalization of tumor vessels, resulting in sufficient infiltration of immune cells into tumors. A synergistic effect of I3A and DOX was observed in vitro at a molar ratio of 1:4. To codeliver this ratio of I3A and DOX to tumor and ensure their uptake, we designed a dual-targeting delivery system, polylactide-poly(ethylene) glycol-2-(3-((S)-5-amino-1-carboxypentyl)-ureido) pentanedioate/triphenylphosphonium (PLA-PEG-ACUPA/TPP), which targets prostate-specific membrane antigen (PSMA) and mitochondria. Delivery of these nanomedicines led to inhibited tumor growth and a strong antitumor immune response. This study sheds light on the mitophagic and antiangiogenic mechanisms underlying I3A treatment of PCa and provides a strategy for combining vascular normalization and chemoimmunotherapy for PCa treatment.

Histone deacetylase inhibition reduces deleterious cytokine release induced by ingenol stimulation.

Latency reversing agents (LRAs), such as protein kinase C (PKC) agonists, constitute a promising strategy for exposing and eliminating the HIV-1 latent reservoir. PKC agonists activate NF-κB and induce deleterious pro-inflammatory cytokine production. Adjuvant pharmacological agents, such as ruxolitinib, a JAK inhibitor, have previously been combined with LRAs to reduce deleterious pro-inflammatory cytokine secretion without inhibiting HIV-1 reactivation in vitro. Histone deacetylase inhibitors (HDACi) are known to dampen pro-inflammatory cytokine secretion in the context of other diseases and synergize with LRAs to reactivate latent HIV-1. This study investigates whether a panel of epigenetic modifiers, including HDACi, could dampen PKC-induced pro-inflammatory cytokine secretion during latency reversal. We screened an epigenetic modifier library for compounds that reduced intracellular IL-6 production induced by the PKC agonist Ingenol-3,20-dibenzoate. We further tested the most promising epigenetic inhibitor class, HDACi, for their ability to reduce pro-inflammatory cytokines and reactivate latent HIV-1 ex vivo. We identified nine epigenetic modulators that reduced PKC-induced intracellular IL-6. In cells from aviremic individuals living with HIV-1, the HDAC1-3 inhibitor, suberohydroxamic acid (SBHA), reduced secretion of pro-inflammatory cytokines TNF-α, IL-5, IL-2r, and IL-17 but did not significantly reactivate latent HIV-1 when combined with Ingenol-3,20-dibenzoate. Combining SBHA and Ingenol-3,20-dibenzoate reduces deleterious cytokine production during latency reversal but does not induce significant viral reactivation in aviremic donor PBMCs. The ability of SBHA to reduce PKC-induced pro-inflammatory cytokines when combined with Ingenol-3,20-dibenzoate suggests SBHA can be used to reduced PKC induced pro-inflammatory cytokines but not to achieve latency reversal in the context of HIV-1.

Ingol and Ingenol-Type Diterpenes from Euphorbia trigona Miller with Keratinocyte Inhibitory Activity.

Ingenol mebutate, isolated from Euphorbia peplus, is an ingenane-type diterpenoid, primarily used for the topical treatment of actinic keratosis, a premalignant skin condition. The aim of our work was to investigate other Euphorbia species to find structurally similar diterpenes that can be used as alternatives to ingenol mebutate. Pharmacological investigation of Euphorbia candelabrum, Euphorbia cotinifolia, Euphorbia ramipressa, and Euphorbia trigona revealed the potent keratinocyte (HPV-Ker cell line) inhibitory activity of these spurge species. From the methanolic extract of the aerial parts of Euphorbia trigona Miller, the most active species, five ingol (1-5) and four ingenane-type diterpenoids (6-9) were isolated by various chromatographic separation techniques, including open column chromatography, vacuum liquid chromatography, thin-layer chromatography, and high-performance liquid chromatography. The structures of the compounds were determined by NMR spectroscopic analysis and by comparison of the assignations with the literature data. The cytotoxic activity of the compounds against keratinocytes was tested in vitro by using ingenol mebutate as a positive control. Among the isolated compounds, two ingenane derivatives (6 and 7) exhibited remarkably stronger cytotoxic activity (IC50 values 0.39 µM and 0.32 µM, respectively) on keratinocytes than ingenol mebutate (IC50 value 0.84 µM). These compounds could serve as starting materials for further investigations to find alternatives to Picato® (with active substance ingenol mebutate), which was withdrawn from marketing authorization in the European Union.

Safety of topical interventions for the treatment of actinic keratosis.

Introduction: Actinic keratosis (AK) are proliferations of atypical keratinocytes that may eventually progress to cutaneous squamous cell carcinoma. Therefore, AK requires consequent and early treatment. Areas covered: A variety of effective approaches is currently available for the clearance of AK. These interventions may be applied either in a lesion-directed or field-directed mode as AK can occur as single or multiple lesions. Field-directed approaches typically comprise topical drug-mediated interventions which aim at eliminating all visible lesions and also at clearing subclinical changes of the actinically damaged field. However, most treatment options are associated with local adverse events such as erythema, scaling, pain, and rarely with systemic symptoms. This expert review provides a comprehensive and up-to-date overview of the safety considerations of the commonly prescribed topical treatment agents cyclooxygenase inhibitors, 5-fluorouracil, imiquimod, ingenol mebutate, and photodynamic therapy. All these therapies have been proven efficient, yet they differ considerably regarding their safety profile. Expert opinion: In the future, safety concerns will relate to long-term and irreversible adverse drug events instead of application site reactions. In particular, the rate of treatment-associated non-melanoma skin cancers will increasingly come into focus and warrant investigation in postmarketing surveillance trials with a long-term follow-up.