Overt Toxicity Research Articles

In Vivo Biocompatibility of Synechococcus sp. PCC 7002-Integrated Scaffolds for Skin Regeneration.

Cyanobacteria, commonly known as blue-green algae, are prevalent in freshwater systems and have gained interest for their potential in medical applications, particularly in skin regeneration. Among these, Synechococcus sp. strain PCC 7002 stands out because of its rapid proliferation and capacity to be genetically modified to produce growth factors. This study investigates the safety of Synechococcus sp. PCC 7002 when used in scaffolds for skin regeneration, focusing on systemic inflammatory responses in a murine model. We evaluated the following three groups: scaffolds colonized with genetically engineered bacteria producing hyaluronic acid, scaffolds with wild-type bacteria, and control scaffolds without bacteria. After seven days, we assessed systemic inflammation by measuring changes in cytokine profiles and lymphatic organ sizes. The results showed no significant differences in spleen, thymus, and lymph node weights, indicating a lack of overt systemic toxicity. Blood cytokine analysis revealed elevated levels of IL-6 and IL-1β in scaffolds with bacteria, suggesting a systemic inflammatory response, while TNF-α levels remained unaffected. Proteome profiling identified distinct cytokine patterns associated with bacterial colonization, including elevated inflammatory proteins and products, indicative of acute inflammation. Conversely, control scaffolds exhibited protein profiles suggestive of a rejection response, characterized by increased levels of cytokines involved in T and B cell activation. Our findings suggest that Synechococcus sp. PCC 7002 does not appear to cause significant systemic toxicity, supporting its potential use in biomedical applications. Further research is necessary to explore the long-term effects and clinical implications of these responses.

Small-molecule GSDMD agonism in tumors stimulates antitumor immunity without toxicity

Gasdermin-mediated inflammatory cell death (pyroptosis) can activate protective immunity in immunologically cold tumors. Here, we performed a high-throughput screen for compounds that could activate gasdermin D (GSDMD), which is expressed widely in tumors. We identified 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB) as a direct and selective GSDMD agonist that activates GSDMD pore formation and pyroptosis without cleaving GSDMD. In mouse tumor models, pulsed and low-level pyroptosis induced by DMB suppresses tumor growth without harming GSDMD-expressing immune cells. Protection is immune-mediated and abrogated in mice lacking lymphocytes. Vaccination with DMB-treated cancer cells protects mice from secondary tumor challenge, indicating that immunogenic cell death is induced. DMB treatment synergizes with anti-PD-1. DMB treatment does not alter circulating proinflammatory cytokine or leukocyte numbers or cause weight loss. Thus, our studies reveal a strategy that relies on a low level of tumor cell pyroptosis to induce antitumor immunity and raise the possibility of exploiting pyroptosis without causing overt toxicity.

In vitro and in vivo evaluation of hypoglycemic potential and acute toxicity of microencapsulated turmeric instant powder

BackgroundCurcuma longa L. (turmeric), traditionally used to treat various ailments including diabetes, has demonstrated beneficial health effects. However, its potential in food formulations requires further investigation. PurposeThis study examined the hypoglycemic effects of an instant powder containing microencapsulated turmeric oleoresin both in vitro and in vivo, alongside an acute toxicity assessment in Holtzman rats. MethodsThis study encompassed both in vitro and in vivo evaluations. In vitro, the α-glucosidase (a digestive enzyme) inhibition assay was employed to determine the powder's effectiveness. In vivo assessments included: (1) evaluation of the hypoglycemic effect in mice; (2) conducted safety testing following international guidelines, administering a 2000 mg/kg dose to rats with subsequent monitoring of biochemical parameters; and (3) histological examination of liver, kidney, and spleen tissues for potential harmful effects. ResultsThe instant powder with turmeric oleoresin microencapsulation demonstrated significant α-glucosidase inhibition in vitro and hypoglycemic effects in vivo. The acute toxicity test at 2000 mg/kg showed no mortality but revealed significant alterations in erythrocyte parameters and hemoglobin percentage. Histological examinations of liver, kidney, and spleen tissues did not differ from those of the control group, though pancreatic tissue was not evaluated in this study. ConclusionAccording to this study, the microencapsulated turmeric oleoresin instant powder exhibited antidiabetic effects without signs of overt toxicity at doses up to 2000 mg/kg in rats.

Safety evaluation of curdlan as a food additive.

The EFSA Panel on Food Additives and Flavourings (FAF) provides a scientific opinion on the safety of curdlan as a new food additive used as firming and gelling agent, stabiliser, thickener. Curdlan is a high molecular weight polysaccharide consisting of β-1,3-linked glucose units, produced by fermentation from Rhizobium radiobacter biovar 1 strain NTK-u. The toxicological dataset consisted of sub-chronic, chronic and carcinogenicity, reproductive and developmental toxicity studies as well as genotoxicity. Invivo data showed that curdlan is not absorbed as such but is extensively metabolised by the gut microbiota into CO2 and other innocuous compounds. Curdlan was not genotoxic and was well-tolerated with no overt organ-specific toxicity. Effects observed at very high doses of curdlan, such as decreased growth and increased cecum weight, are common for indigestible bulking compounds and therefore considered physiological responses. In a combined three-generation reproductive and developmental toxicity study, decreased pup weight was observed during lactation at 7500 mg curdlan/kg body weight (bw) per day, the highest dose tested. The Panel considered the observed effects as treatment-related and adverse, although likely secondary to nutritional imbalance and identified a conservative no observed adverse effect level (NOAEL) of 2500 mg/kg bw per day. Despite the limitations noted in the dataset, the Panel was able to conclude applying the margin of exposure (MOE) approach. Given that curdlan and its break-down products are not absorbed and that the identified adverse effect is neither systemic nor local, no adjustment factor was deemed necessary. Thus, an MOE of at least 1 was considered sufficient. The highest exposure estimate was 1441 mg/kg bw per day in toddlers at the 95th percentile of the proposed maximum use level exposure assessment scenario. The Panel concluded that there is no safety concern for the use of curdlan as a food additive at the proposed uses and use levels.

Perfluorohexanesulfonic Acid (PFHxS) Impairs Lipid Homeostasis in Zebrafish Larvae through Activation of PPARα.

Perfluorohexanesulfonic acid (PFHxS), an emerging short-chain per- and polyfluoroalkyl substance, has been frequently detected in aquatic environments. Adverse outcome pathway studies have shown that perfluorinated compounds impair lipid homeostasis through peroxisome proliferator activated receptors (PPARs). However, many of these studies were performed at high concentrations and may thus be a result of overt toxicity. To better characterize the molecular and key events of PFHxS to biota, early life-stage zebrafish (Danio rerio) were exposed to concentrations detected in the environment (0.01, 0.1, 1, and 10 μg/L). Lipidomic and transcriptomic evaluations were integrated to predict potential molecular targets. PFHxS significantly impaired lipid homeostasis by the dysregulation of glycerophospholipids, fatty acyls, glycerolipids, sphingolipids, prenol lipids, and sterol lipids. Informatic analyses of the lipidome and transcriptome indicated alterations of the PPAR signaling pathway, with downstream changes to retinol, linoleic acid, and glycerophospholipid metabolism. To assess the role of PPARs, potential binding of PFHxS to PPARs was predicted and animals were coexposed to a PPAR antagonist (GW6471). Molecular simulation indicated PFHxS had a 27.1% better binding affinity than oleic acid, an endogenous agonist of PPARα. Antagonist coexposures rescued impaired glycerophosphocholine concentrations altered by PFHxS. These data indicate PPARα activation may be an important molecular initiating event for PFHxS.

Targeting vascular disrupting agent-treated tumor microenvironment with tissue-penetrating nanotherapy

Cancer treatment with vascular disrupting agents (VDAs) causes rapid and extensive necrosis in solid tumors. However, these agents fall short in eliminating all malignant cells, ultimately leading to tumor regrowth. Here, we investigated whether the molecular changes in the tumor microenvironment induced by VDA treatment sensitize the tumors for secondary nanotherapy enhanced by clinical-stage tumor penetrating peptide iRGD. Treatment of peritoneal carcinomatosis (PC) and breast cancer mice with VDA combretastatin A-4 phosphate (CA4P) resulted in upregulation of the iRGD receptors αv-integrins and NRP-1, particularly in the peripheral tumor tissue. In PC mice treated with CA4P, coadministration of iRGD resulted in an approximately threefold increase in tumor accumulation and a more homogenous distribution of intraperitoneally administered nanoparticles. Notably, treatment with a combination of CA4P, iRGD, and polymersomes loaded with a novel anthracycline Utorubicin (UTO-PS) resulted in a significant decrease in the overall tumor burden in PC-bearing mice, while avoiding overt toxicities. Our results indicate that VDA-treated tumors can be targeted therapeutically using iRGD-potentiated nanotherapy and warrant further studies on the sequential targeting of VDA-induced molecular signatures.

Small Molecule Inhibitors of Arylamine N-Acetyltransferase 1 Attenuate Cellular Respiration.

Arylamine N-acetyltransferase 1 (NAT1) expression has been shown to attenuate mitochondrial function, suggesting it is a promising drug target in diseases of mitochondrial dysfunction. Here, several second-generation naphthoquinones have been investigated as small molecule inhibitors of NAT1. The results show that the compounds inhibit both in vitro and in whole cells. A lead compound (Cmp350) was further investigated for its ability to alter mitochondrial metabolism in MDA-MB-231 cells. At concentrations that inhibited NAT1 by over 85%, no overt toxicity was observed. Moreover, the inhibitor decreased basal respiration and reserve respiratory capacity without affecting ATP production. Cells treated with Cmp350 were almost exclusively dependent on glucose as a fuel source. We postulate that Cmp350 is an excellent lead compound for the development of NAT1-targeted inhibitors as both experimental tools and therapeutics in the treatment of hypermetabolic diseases such as amyotrophic lateral sclerosis, cancer cachexia, and sepsis.

Therapeutic activity of a new KRASG12C inhibitor JMKX001899 in brain metastases: Preclinical models of KRASG12C-mutant non-small cell lung cancer.

8630 Background: Development of therapies targeting KRAS have transformed KRAS from undruggable to druggable. However, patients with KRASG12C-mutant non-small cell lung cancer (NSCLC) with brain metastasis (BM) have a poor prognosis. JMKX001899, a new potent oral small-molecule KRASG12C inhibitor, irreversibly and selectively binds KRASG12C, locking it in its inactive state. Its antitumor activity in the intracranial is unclear. Methods: In order to verify the activity of new KRASG12C inhibitor JMKX001899 in KRASG12C-mutant NSCLC with BM, murine models were employed for investigating the efficacy. 5-6 weeks old female Balb/c nude mice were implanted intracranially with H358-Luc and H23-Luc cells. Baseline bioluminescent imaging (BLI) was used to monitor the tumor burden and total flux was calculated by Living Image. Ten days after implantation, oral once-daily dosing of vehicle, sotorasib (AMG510) at 100 mg/kg, 30 mg/kg and JMKX001899 at 100 mg/kg, 30 mg/kg, 10 mg/kg was started and continued for 21 days. The tumor burden was monitored by BLI weekly. The mice were weighed per 3 days. Results: At the 30mg/kg and 100mg/kg dose level, the mean cerebrospinal fluid (CSF)-to-unbound plasma ratios (Kp,uu) was 0.18-0.50, which determined JMKX001899 CSF exposure was sufficient to mediate regression tumors in experimental BM models. In mice bearing intracranial xenografts of H358-Luc and H23-Luc, oral JMKX001899 100mg/kg once daily treatment for 21 days resulted in significant inhibition of brain tumor growth revealed by BLI, compared with vehicle and AMG510. H358-Luc xenografts responded particularly well to the JMKX001899 treatment, showed by the ratio of 14 days and 0 days total bioluminescence flux (vehicle vs. AMG510 100mg/kg vs. JMKX001899 100mg/kg, 0.85 vs. 0.89 vs. 0.01, p < 0.05). In addition, JMKX001899 at 100mg/kg once daily demonstrated an increase in over survival. JMKX001899 100 mg/kg once daily treatment was well tolerated with minimal sign of overt toxicity or animal weight loss. Conclusions: The preclinical models in vivo suggest the new KRASG12C inhibitor JMKX001899 penetrates the central nervous system and show great inhibition of KRASG12C-mutant NSCLC with BM, which demonstrates therapeutic activity of JMKX001899 in BM. These data support further development of JMKX001899 in patients with KRASG12C-mutant NSCLC with BM.

Abstract PO3-26-09: A novel class of RNR inhibitors that induces genomic instability and breast cancer cell death without inducing overt toxicities

Abstract Despite the development of screening techniques and therapies, nearly 30% of breast cancers progress to Stage IV metastatic disease. These advanced cancers are highly proliferative, engaging in ongoing DNA synthesis that requires ribonucleotide reductase (RNR), the rate-limiting enzyme for dNTP synthesis. RRM2, the small subunit of RNR, activates the enzyme, increases deoxynucleotide pools, and is overexpressed in many cancers, including breast cancer. Thus, RNR is an established therapeutic target in advanced breast cancer. Currently, gemcitabine is the only FDA-approved RNR inhibitor used to treat Stage IV disease. As a nucleoside analog, gemcitabine also causes severe dose-limiting toxicities (i.e. myelosuppression and hepatotoxicity) due to off-target DNA replication termination. We hypothesize that a novel, non-nucleoside, RNR inhibitor may overcome these limitations. To address this, we developed TMU27a, a derivative of naphthyl salicylic acyl hydrazone (NSAH), which was identified in an in silico screen for its high-affinity binding to the RNR catalytic site. To measure direct interactions between TMU27a and purified RNR protein, we used surface plasmon resonance, revealing on-target, tight binding (Kd = 150 nM). Unbiased docking and molecular dynamics simulations (using lowest energy calculations) predicted the binding sight of TMU27a to be the catalytic site of RNR, where it is stabilized through extensive interactions formed with conserved residues. Preliminary docking predictions also indicate multiple clash sites between TMU27a and the DNA polymerase lambda nucleotide pocket even in the lowest energy pose (unlike gemcitabine), suggesting that TMU27a inhibits RNR activity through a novel mechanism without significant off-target binding to other nucleotide binding proteins that contribute to toxicity. Demonstrating on-target inhibition, TMU27a reduces dATP levels and induces S and G2/M-phase stalling, leading to significant DNA damage and chromosomal instability. This excessive genomic damage causes growth repression, apoptosis, and senescence in several triple negative breast cancer (TNBC) cell lines. In xenograft mouse models of TNBC, TMU27a fully suppressed tumor growth without impacting mouse weight, suggesting strong drug efficacy in the absence of overt toxicity. Moreover, while short-term exposure to gemcitabine suppresses red and white blood cell populations, TMU27a does not. Together, these data indicate that TMU27a and its potential analogs are novel, non-toxic RNR inhibitors that overcome the limitations of gemcitabine for treating breast cancer. We are currently investigating how and where TMU27a binds human RNR using cryo-electron microscopy with the goal of generating a novel class of RNR inhibitors through structure-based drug design, improving binding affinity and selectivity. Citation Format: Taylor Baker, Kristen Weber-Bonk, Wei Huang, Derek Taylor, Ruth Keri. A novel class of RNR inhibitors that induces genomic instability and breast cancer cell death without inducing overt toxicities [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-09.

Genome-wide CRISPR screens identify PKMYT1 as a therapeutic target in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with an overall 5-year survival rate of <12% due to the lack of effective treatments. Novel treatment strategies are urgently needed. Here, PKMYT1 is identified through genome-wide CRISPR screens as a non-mutant, genetic vulnerability of PDAC. Higher PKMYT1 expression levels indicate poor prognosis in PDAC patients. PKMYT1 ablation inhibits tumor growth and proliferation in vitro and in vivo by regulating cell cycle progression and inducing apoptosis. Moreover, pharmacological inhibition of PKMYT1 shows efficacy in multiple PDAC cell models and effectively induces tumor regression without overt toxicity in PDAC cell line-derived xenograft and in more clinically relevant patient-derived xenograft models. Mechanistically, in addition to its canonical function of phosphorylating CDK1, PKMYT1 functions as an oncogene to promote PDAC tumorigenesis by regulating PLK1 expression and phosphorylation. Finally, TP53 function and PRKDC activation are shown to modulate the sensitivity to PKMYT1 inhibition. These results define PKMYT1 dependency in PDAC and identify potential therapeutic strategies for clinical translation.

Enhanced bacterial cancer therapy delivering therapeutic RNA interference of c-Myc

BackgroundBacterial cancer therapy was first trialled in patients at the end of the nineteenth century. More recently, tumour-targeting bacteria have been harnessed to deliver plasmid-expressed therapeutic interfering RNA to a range of solid tumours. A major limitation to clinical translation of this is the short-term nature of RNA interference in vivo due to plasmid instability. To overcome this, we sought to develop tumour-targeting attenuated bacteria that stably express shRNA by virtue of integration of an expression cassette within the bacterial chromosome and demonstrate therapeutic efficacy in vitro and in vivo.ResultsThe attenuated tumour targeting Salmonella typhimurium SL7207 strain was modified to carry chromosomally integrated shRNA expression cassettes at the xylA locus. The colorectal cancer cell lines SW480, HCT116 and breast cancer cell line MCF7 were used to demonstrate the ability of these modified strains to perform intracellular infection and deliver effective RNA and protein knockdown of the target gene c-Myc. In vivo therapeutic efficacy was demonstrated using the Lgr5creERT2Apcflx/flx and BlgCreBrca2flx/flp53flx/flx orthotopic immunocompetent mouse models of colorectal and breast cancer, respectively. In vitro co-cultures of breast and colorectal cancer cell lines with modified SL7207 demonstrated a significant 50–95% (P < 0.01) reduction in RNA and protein expression with SL7207/c-Myc targeted strains. In vivo, following establishment of tumour tissue, a single intra-peritoneal administration of 1 × 106 CFU of SL7207/c-Myc was sufficient to permit tumour colonisation and significantly extend survival with no overt toxicity in control animals.ConclusionsIn summary we have demonstrated that tumour tropic bacteria can be modified to safely deliver therapeutic levels of gene knockdown. This technology has the potential to specifically target primary and secondary solid tumours with personalised therapeutic payloads, providing new multi-cancer detection and treatment options with minimal off-target effects. Further understanding of the tropism mechanisms and impact on host immunity and microbiome is required to progress to clinical translation.

Abstract 4012: SOT302: Dual expression of exogenous glutamine oxaloacetate transaminase (GOT2) and TP53-induced glycolysis and apoptosis regulator (TIGAR) enhance CAR T cell activity in preclinical solid tumor models

Abstract Background: Despite success in hematological malignancies, CAR T therapy has had limited efficacy against solid tumors. The suppressive tumor microenvironment limits CAR T activity through various mechanisms including competition for available nutrients and chronic stimulation resulting in reduced effector functions or T cell exhaustion. CAR T cells with enhanced metabolic fitness or more durable memory phenotype could potentially improve the clinical outcome in solid tumors. GOT2 plays an important role in mitochondrial function and maintenance of redox homeostasis. We have previously demonstrated enhancement of CAR T cells overexpressing GOT2. TIGAR is an enzyme known to promote antioxidative activites and reduce reactive oxygen species and has a role in protecting against apoptosis. Here we tested SOT302 CAR T cells expressing both GOT2 and TIGAR in preclinical solid tumor models. Results: Expression of GOT2 and TIGAR transgenes were confirmed by qRT-PCR and western blot. Activity of GOT2 and TIGAR were confirmed by measuring aminotransferase activity (AST) and glutathione production, respectively. Compared to control CAR T cells, SOT302 is enriched for CD8+ Tscm cells and has a higher percentage functional, non-senescent (CD28+CD57-) cells. SOT302 has similar cytokine expression and cytotoxicity in standard co-culture assays compared to controls. SOT302 cells were chronically stimulated with plate-bound antigen every 3-4 days for 4 rounds. SOT302 had greater expansion of cells and were less exhausted and had better cytolytic capacity compared to controls. SOT302 cells were then evaluated in tumor xenograft mouse models. Consistent with our previous findings, CAR T cells expressing the single GOT2 transgene were more efficacious compared to CAR T cells alone. Expression of the single TIGAR transgene had no apparent benefit over CAR alone, however SOT302 cells expressing both GOT2 and TIGAR had superior anti-tumor activity compared to CAR alone and CAR+GOT2. No overt toxicity or significant body weight loss was observed. Ex vivo analyses to measure peripheral expansion, tumor infiltration and exhaustion were also performed. Preliminary findings indicate that SOT302 has better peripheral expansion compared to CAR alone and similar expansion compared to CAR+GOT2 controls. SOT302 cells had better tumor infiltration compared to both the CAR alone and CAR+GOT2 cells. Tumor infiltrating SOT302 cells also exhibited lower levels of exhaustion compared to CAR alone or CAR+GOT2 controls. Summary: SOT302 cells express exogenous GOT2 and TIGAR transgenes and have superior anti-tumor activity in preclinical solid tumor models. These data suggest that SOT302 may be a promising candidate for patients with solid tumor cancers. Citation Format: Emily Kuiper, Samyabrata Bhaduri, Daniel Garafola, Kshitij Sharma, Jennifer Coccia, Kathleen Whiteman, Amy Jensen-Smith. SOT302: Dual expression of exogenous glutamine oxaloacetate transaminase (GOT2) and TP53-induced glycolysis and apoptosis regulator (TIGAR) enhance CAR T cell activity in preclinical solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4012.

Abstract 41: Mitigating on-target off-tumor toxicity of mesothelin-targeting CAR T cells by affinity-tuning

Abstract Purpose: Mesothelin (MSLN) is a surface antigen associated with tumor invasion and is overexpressed in mesothelioma and lung, pancreatic, ovarian, and other cancers. Chimeric antigen receptor (CAR) T cells targeting MSLN have been tested in patients with MSLN-positive solid tumors. Despite reported patient fatalities in trials sponsored by PENN, Memorial Sloan Kettering/Atara Bio, and MD Anderson/TCR2, the design approach for MSLN CAR remains constructing high affinity CARs to maximize target cell killing. In this study, we designed MSLN CARs with two different antibodies: one targeting human MSLN specifically and another binding both human and mouse MLSN with comparable affinities. The objectives are to 1) elucidate the risk of fatal on-target off-tumor toxicity associated with high affinity MSLN CAR, and 2) demonstrate how optimizing the affinity of CARs can render CAR T cells to spare normal tissues while retaining cytotoxicity against tumors. Methods: To asses on-target off-tumor toxicity in animal models, we developed a CAR using a nanobody isolated from an immune alpaca library with nanomolar affinity for both human and mouse MSLN (labeled as hmMSLN CAR). For comparison, we constructed a CAR using a scFv specific only to human MSLN, designated hMSLN CAR. Each CAR is designed to co-express somatostatin receptor 2 to enable PET/CT imaging of CAR T cells with 18F-NOTA-Octreotide. Anti-tumor activities and toxicities were assessed in NSG mice using MSTO-211H cell line expressing human MSLN. Results: In NSG mice bearing subcutaneous MSTO-211H tumors, hMSLN CAR T cells eliminated tumors rapidly without inducing any overt toxicity. In contrast, hmMSLN CAR T cells not only failed to control tumor growth but also caused severe toxicity, resulting in significant body weight loss and necessitating euthanasia. PET/CT imaging revealed while hMSLN CAR T cells expanded exclusively within the tumor and subsequently contracted after tumor elimination, hmMSLN CAR T cells exhibited extensive infiltration and expansion in the lungs, liver, and bone marrow, indicating off-tumor, normal tissue-driven proliferation of CAR T cells. To test if affinity-tuned hmMSLN CAR T cells can selectively target tumors, we used a yeast display system to screen for affinity variants of the hmMSLN CAR. We identified one micromolar affinity CAR that binds approximately 1,000 times weaker to MSLN than the parental CAR. In stark contrast to the onset of severe toxicity soon after the administration of the parental hmMSLN CAR T cells, animals treated with micromolar affinity hmMSLN CAR T cells exhibited tumor regression without overt toxicity. Conclusions: Our findings underscore the risks of using high affinity CAR against MSLN, which may cause unpredictable on-target, off-tumor toxicities. Fine tuning the affinity of MSLN CAR offers a promising strategy for developing a potent and safe CAR for the treatment of MSLN-expressing solid tumors. Citation Format: Yanping Yang, Yogindra Vedvyas, Yago Alcaina, Ju Y. Son, Niloufar Salehi, Irene M. Min, Moonsoo M. Jin. Mitigating on-target off-tumor toxicity of mesothelin-targeting CAR T cells by affinity-tuning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 41.

Abstract 5701: AU2-94: A CDK4-specific inhibitor with the marked anti-tumor efficacy

Abstract Cyclin-dependent kinases (CDKs) are a vital family of proteins in the cell development. Several CDK4/6 inhibitor drugs significantly extended the lives of subtypes breast cancer patients. However, they have some shortcomings. Many patients do not respond to them well mainly due to off-target toxicity requiring dose reductions and treatment interruptions, and thus building resistance, which limit their effectiveness. Here, we describe AU2-94, a highly selective CDK4 inhibitor, for the treatment of CDK4-dependent tumours including breast cancer (ER+ and TNBC), colorectal cancer and glioblastoma (GBM). AU2-94 exhibits high selectivity for CDK4 (Ki = 2 nM) over CDK6 (Ki = 279 nM). Consistent with the CDK4-targeted mechanism, AU2-94 arrested the G1 cells and inhibited cancer cell proliferation. Interestingly, AU2-94 reduced cellular CDK2, Cyclin E2, Cyclin A2, Cyclin B1, and induced apoptosis via reduction of Bcl-2, FOXM1 etc. In contrast to cancer cells, AU2-94 has little impact on the cell cycle, senescence, or apoptosis of human bone marrow cells, whereas the known CDK4/6 inhibitors increased the G1 cells. AU2-94 showed little or no effect on lymphocyte and neutrophil counts whilst the clinical CDK4/6 inhibitors reduced them. This lower effect of AU2-94 on the bone marrow, neutrophils and lymphocytes might be due to its lesser effect on CDK6 compared to the clinical CDK4/6 inhibitors. AU2-94 was highly efficacious against multiple in vivo tumour models. In the ER+ breast cancer T47D xenografts, AU2-94 caused tumour regression. In the model of MDA-MB-453 AR+ luminal breast cancer (CDK4, KRASmut, PI3KCAmut), AU2-94 demonstrated remarkable efficacy, resulting in the 6 out of 7 (86%) mice tumour-free. In the colorectal cancer HCT116 (CDK4/6, KRASmut) xenografts, AU2-94 inhibited tumour growth markedly in a dose-dependent manner from 25 to 200 mg/kg without any overt toxicity. The pharmacokinetic and pharmacodynamic study revealed up to 500-fold higher concentration of AU2-94 in the tumours than in the plasma, suggesting its tumour targeting specificity. Several robust biomarkers/PD markers were identified. The Rb-CDK4-Cyclin D pathway is altered in the majority of GBM due to homozygous deletion of CDKN2A/B. AU2-94 has an excellent therapeutic potential as it crossed the blood-brain-barrier effectively. In the both U87 subcutaneous and orthotopic models, AU2-94 reduced the tumour burden and increased the survival of GBM bearing mice significantly compared to vehicle. Moreover, AU2-94 synergised the anti-cancer efficacy of PI3K/Akt/mTOR and HER2 inhibitors in combination. In summary, AU2-94 is a CDK4 inhibitor with high selectivity against a panel of 360 human kinases. This selectivity can be a reason for the better safety of AU2-94 compared to the clinical CDK4/6 inhibitors. The high oral bioavailability, robust antitumor efficacy and excellent safety profiles make AU2-94 a highly attractive candidate for clinical development. Citation Format: Laychiluh Bantie, Jimma Likisa, Muhammed Rahaman, Theodosia Teo, Ava Safaroghliazar, Sunita KC Basnet, Ramin Hassankhani, Benjamin Noll, Robert Milne, Shudong Wang. AU2-94: A CDK4-specific inhibitor with the marked anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5701.

Abstract 3924: Dual COX-2/sEH inhibition enhances immunotherapy and chemotherapy to induce bladder cancer regression

Abstract Unresolved inflammation plays a critical role in bladder cancer initiation and progression. Chemotherapy (e.g. gemcitabine and cisplatin), the standard of care for advanced bladder cancer, disrupts inflammation resolution and is only partially effective in preventing tumor recurrence after treatment. Immunotherapy has emerged as a potential treatment option for bladder cancer patients, however it is ineffective in 80% of patients and can induce a pro-inflammatory cytokine storm. Therefore, there is a critical unmet medical need to improve chemotherapy and immunotherapy in bladder cancer. To address this, we have developed dual COX-2/sEH inhibitors (e.g. PTUPB). This lead compound is representative of a newly patented class of “dual acting” molecules that target two important enzymes in the arachidonic acid cascade with nanomolar binding affinity: cyclooxygenase-2 (COX-2) and soluble epoxide hydrolase (sEH). Oral dosing of PTUPB is well-tolerated in animal models. Since chemotherapy and immunotherapy both induce tumor-promoting inflammation via an eicosanoid/cytokine storm, we hypothesized that dual COX-2/sEH inhibition would enhance immunotherapy in experimental bladder cancer via anti-inflammatory mechanisms. When syngeneic (MB49) bladder tumors reached ~200 mm3 in immunocompetent mice, treatment was initiated with PTUPB, anti-CTLA-4, anti-PD1, gemcitabine &amp; cisplatin, or various combinations thereof. Here we demonstrate that chemotherapy and/or immunotherapy stimulated sEH and COX-2 expression in tumor tissue which was counter-regulated by PTUPB. While monotherapy treatment with PTUPB, gemcitabine &amp; cisplatin, anti-PD1 or anti-CTLA-4 suppressed tumor growth compared to control, the tumors escaped single treatments by the end of the study. Remarkably, dual COX-2/sEH inhibition in combination with chemotherapy (gemcitabine and cisplatin) and/or immune checkpoint blockade (anti-CTLA-4 or anti-PD1) induced sustained tumor regression via synergistic anti-tumor activities. Chemotherapy and/or immunotherapy induced the expression of ER stress response genes (e.g. BiP and CHOP) and angiogenic factors (e.g. EGF and VEGF-C) in bladder cancer tissue, which were repressed by PTUPB. PTUPB also prevented chemotherapy-induced toxicity and prolonged survival in an orthotopic MB49 tumor model. Taken together, our results demonstrate dual COX-2/sEH inhibition as a novel therapeutic approach to enhance immunotherapy in bladder cancer without overt toxicity. Citation Format: Kimberly Lupita Vazquez, Eva Rothenberger, Weicang Wang, Sung Hee Hwang, Diane R. Bielenberg, Bruce D. Hammock, Dipak Panigrahy. Dual COX-2/sEH inhibition enhances immunotherapy and chemotherapy to induce bladder cancer regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3924.

Abstract 2461: Discovery of an orally available and liver-targeted ALPK1 agonist for cancer immunotherapy

Abstract Boosting anti-tumor immune responses can be achieved by activating the innate immune system. Despite the ongoing development of numerous STING and TLR agonists for this purpose, none of them have progressed to late-stage clinical evaluation. Alpha protein kinase-1 (ALPK1) is an intracellular innate immune receptor newly identified for its ability to detect ADP-heptose, a metabolite released during the biosynthesis and degradation of lipopolysaccharides (LPS). Since heptose is not naturally present in mammalian cells, the recognition of heptose metabolites by ALPK1 serves as a reliable mechanism to identify bacterial infections. When ADP-heptose binds to ALPK1, it initiates the phosphorylation of TIFA, thereby triggering a cascade of immune responses. Systemic administration with ADP-heptose induces robust ALPK1-dependent cytokine responses in mice. While high doses of ADP-heptose demonstrate anti-tumor effects, its instability and low in vivo potency hinder its clinical development. Through a SBDD medicinal chemistry effort, a novel, stable, and potent ADP-heptose analog, designated PTT-936, has been discovered and is progressing into clinical evaluation in cancer patients. PTT-936 exhibits remarkable nanomolar potencies for ALPK1 activation in various cellular assays. Notably, in human PBMCs, PTT-936 induces both concentration-dependent release of cytokines and activation of NK and T cells. Consistent with in vitro studies, oral administration of PTT-936 triggered significant cytokine responses in mice at microgram dose levels. However, overt toxicities were observed only at milligram dose levels, indicating a wide therapeutic window for this compound. In syngeneic tumor models, oral administration of PTT-936 induced dose-dependent tumor growth inhibition, which correlated with elevations in several cytokine biomarkers. Mechanistic studies show that PTT-936, working exclusively through the ALPK1 pathway, elicited strong innate and adaptive immune responses, as evidenced by robust cytokine induction and immune cell activation and recruitment. In syngeneic mouse models, the anti-tumor effects of PTT-936 were significantly hindered by either CD8+ T cell depletion or neutralization of select cytokines. Besides monotherapy studies, compelling evidence demonstrates strong synergistic anti-tumor effects when combining PTT-936 with immune checkpoint inhibitors. Intriguingly, PTT-936 exhibits favorable tolerability and a wider therapeutic index compared to certain clinical-stage STING and TLR agonists. Furthermore, PTT-936 does not enhance cytokine elevations induced by checkpoint inhibitors, which have been linked to adverse events (including CRS) observed in clinical combination studies. The dosing frequency of PTT-936 in the upcoming first-in-human clinical trial is carefully designed to prioritize both tolerability and patient compliance. Citation Format: Yupeng Wang, Nana Du, Xiaona Dong, Xuebing Sun, Yuan Hong, Xiaolong Yang, Zhijiang Ji, Qianyu Wang, Yun Lv, Zhichen Shi, Yajuan Gao, Xiaofu Hou, Yaxin Zhang, Juan Wang, Rui Mao, Yinxin Liu, Liang Li, Jingjin Ding, Hexiang Wang, Nan Hu, Counde O’Yang, Zhihong Li, Keith Bley, Feng Shao. Discovery of an orally available and liver-targeted ALPK1 agonist for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2461.

Abstract 3045: PPAR-α antagonist enhances immunotherapy and anti-angiogenic therapy to inhibit murine renal cancer

Abstract Peroxisome proliferator activated receptor alpha (PPARα) is a transcription factor and master regulator of fatty acid oxidation. PPARα ligands modulate the switch between co-activator and co-repressor transcription complexes at PPARα-controlled target genes. In addition, PPARα has been shown to transrepress NF-κB signaling and inhibit angiogenesis. Renal cell carcinoma (RCC) expresses high levels of PPAR-α and is a highly angiogenic cancer. Current frontline treatments for RCC include chemotherapy, anti-angiogenics, and immunotherapy but are limited by the immune suppressive state of the tumor microenvironment. TPST-1120 is a novel PPARα antagonist for the treatment of cancer patients that binds in the ligand binding domain, positioning the AF-2 activation helix in an inactive conformation. The inactive conformation has a lower affinity for co-activator motifs and reduces activation of PPARα regulated genes. In a CHO-based reporter cell line, TPST-1120 potently competed against the PPARα-specific agonist GW7647 (IC50 = 36 nM at 20 nM GW7647) and an endogenous PPARα agonist oleoylethanolamide (OEA; IC50 = 15 nM at 30 µM OEA). Furthermore, in an enzyme fragment complementation assay, we demonstrated that TPST-1120 inhibited agonist-induced binding of PPARα to MED1 by destabilizing the co-activator complex thereby stabilizing the inactive conformation of PPAR-α. In a murine model of renal cell adenocarcinoma (RENCA), TPST-1120 treatment for 12 days (30 mg/kg qd) reduced tumor growth by 52% as a monotherapy (p&amp;lt;0.0001). Consistent with these findings, TPST-1120 inhibited tumor angiogenesis and proliferation as quantified by Ki67 levels. Combination treatment with current frontline therapeutics resulted in synergistic tumor inhibition of 74% with anti-PD1 (200 ug Q3D for 12 days) and 81% with cabozantinib (15 mg/kg QD for 15 days) (p&amp;lt;.0001). Flow cytometry-based analysis of tumors demonstrated increased CD8+ T cell infiltration in the TME as well as decreases in M2 and elevations in M1 macrophages. Importantly, we demonstrate that TPST-1120 can reverse an immunosuppressive class of immune cells to promote anti-tumor immunity and anti-angiogenesis in kidney cancer in the absence of overt toxicity. These data support advancement of TPST-1120 into RCC. Citation Format: Michael Gillespie, Valerie Chen, Isabella Howard, Keira Smith, Dave Freund, Nathan Standifer, Dara Burdette, Thomas W. Dubensky, Sam Whiting, Dipak Panigrahy. PPAR-α antagonist enhances immunotherapy and anti-angiogenic therapy to inhibit murine renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3045.

Abstract 3575: The estrogenic endocrine disrupting compounds bisphenol-a (BPA) and alpha-zeranol (aZAL) induce early mammary hyperplasia in female ACI rats

Abstract Endocrine disrupting compounds (EDCs) found in food-grade plastics, drinking water, and foodstuffs, pose a threat to human health. Estrogenic EDCs are capable of binding and activating the estrogen receptor (ER). Since breast tissue relies on estrogen signaling for growth and renewal, excessive estrogen stimulation is associated with breast cancer development. Breast cancer is a leading cause of cancer-related death in women. Only 5-10% of all breast cancer cases can be attributed to genetic causes, and the proportion of breast cancers of the luminal subtype are increasing in the United States for reasons unknown. Estrogenic EDCs may be able to initiate breast cancers through their estrogenic activities. The present study assessed two EDCs: the plasticizer bisphenol-A (BPA) and the mycotoxin alpha-zeranol (aZAL). To assess the estrogenic effects of these EDCs in an in vivo model, we utilized the ACI rat strain due to its sensitivity to estrogen-induced mammary carcinogenesis. The hypothesis of this work was that BPA and aZAL will induce mammary gland proliferation and estrogen signaling at early timepoints post-exposure. Estrogen, BPA, or aZAL were implanted into the backs of female ACI rats in silastic tubes at either a 9 gram doses. At a timepoint of 5 days post implantation, we saw no overt toxicity of our treatment. Both BPA and aZAL resulted in increased glandular proliferation and hyperplasia in a comparable manner to estrogen. Immunohistochemistry and qPCR revealed that the estrogen signaling marker progesterone receptor (PGR) was significantly upregulated in the estrogen, BPA, and aZAL treatment groups compared to control. The percent positivity of PGR staining in the mammary glands were 21.5% +/- 4.2%, 25.7% +/- 1.7%, 27.9% +/- 3.8%, and 8.18% +/- 2.5% respectively. Additionally, PCNA, a marker of cell proliferation, was significantly upregulated in the estrogen and aZAL treatment groups. The percent positivity of PCNA staining in the mammary glands were 7.67% +/- 2.56%, 49.6% +/- 5.2%, and 41.0% +/- 11.5% respectively. Additionally, the weight of the pituitary gland, an estrogen-sensitive endocrine organ, significantly increased in both the estrogen and aZAL treatment groups. Overall, this study demonstrated that the estrogenic EDCs BPA and aZAL induced mammary gland proliferation and hyperplasia in a comparable manner to estrogen. This is concerning due to the routine detection of these two compounds in human biosamples. More information is needed to assess the risk of exposure to these compounds as it pertains to breast cancer and other hormonal cancers. Citation Format: Cassandra Winz, Ba Liu, Caroline Xie, Shlok Rohatagi, Eric Li, Philip Furmanski, Nanjoo Suh. The estrogenic endocrine disrupting compounds bisphenol-a (BPA) and alpha-zeranol (aZAL) induce early mammary hyperplasia in female ACI rats [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3575.

Abstract 6064: Discovery of potent and selective EP300 degraders with anti-cancer activity

Abstract E1A binding protein (EP300) and CREB binding protein (CBP) are paralog histone acetyltransferases involved in many cellular processes via their activity as transcriptional co-activators. Dysregulation of one or both proteins has been implicated in various cancers, and functional genomic screens have demonstrated a bidirectional synthetic lethal relationship between the two genes in tumor cells. Due to the high homology between EP300 and CBP, identifying chemical matter that selectively targets EP300 or CBP has proven challenging. Here, we describe a potent, highly selective heterobifunctional degrader of EP300 with biological activity in CBP-deficient and EP300-dependent tumor cells. Targeted degradation of EP300 protein resulted in a stronger suppression of cell growth and survival than targeting the bromodomain or HAT activity of EP300/CBP with small molecule inhibitors. Anti-proliferative effects have been demonstrated in multiple cancer types, including malignant lymphomas and castration-resistant prostate tumors, highlighting the essential role of EP300 in mediating oncogenic transcription required for tumor cell growth and survival. Degradation of EP300 in vivo attenuated androgen-driven transcription and inhibited tumor growth in VCAP (AR+) prostate tumor xenografts. Importantly, no evidence of overt toxicity or thrombocytopenia was observed at therapeutically efficacious doses. These findings indicate that selective targeting of EP300 with targeted protein degradation is a safe and effective treatment strategy for advanced tumors. Citation Format: Mark Zimmerman, Ammar Adam, Hafiz Ahmad, Benjamin Adams, Ketaki Adhikari, Wesley Austin, Breanna Bullock, Julie Di Bernardo, Thomas Dixon, Danette Daniels, Claudia Dominici, GiNell Elliot, Brian Ethell, Anais Gervais, Md Imran Hossain, David Huang, Dave Lahr, Mei Yun Lin, David Mayhew, Karolina Mizeracka, Solymar Negretti, Tyler Nguyen, Olga Prifti, Darshan Sappal, Shawn Schiller, Brenna Sherbanee, David Terry, Nihan Ucisik, Elizabeth Wittenborn, Qianhe Zhou, Laura La Bonte. Discovery of potent and selective EP300 degraders with anti-cancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6064.

Abstract 4701: A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile

Abstract Initial evaluation of the prenylated hydroxy-stilbene isolated from bee propolis, AUS_001, in the NCI 60 human cell line anticancer screen showed significant growth inhibitory effects with the most robust being observed in leukemia, central nervous system and breast cancer cell lines. The aim of the current study was to further assess the potency of AUS_001 in vitro and investigate its safety profile and mechanism of action. A commercially available cell-based profiling screen (OncoPanel®) identified significant AUS_001-induced growth inhibition in 273 out of 280 cancer cell lines with a concentration causing 50% cell growth inhibition in the range of 0.021-0.94 µM, while non-neoplastic cells required an average of 20x higher concentration to generate similar efficacy. These findings indicate that AUS_001 exerts a high degree of potency across 30 cancer types with a selectivity for cancer versus normal cells. Maximum tolerated dose studies in mice illustrated that orally administered AUS_001 is well tolerated at doses 4 times greater than those that conferred antitumor efficacy, with no myelosuppression or other overt toxicities observed. Predictive toxicology and safety screening indicated that AUS_001 exerts a strong safety profile as evidenced by absence of mutagenic potential in the Ames test using up to 100 µM drug doses and low hERG-blocking liability with a half-maximal inhibitory concentration of 65 µM. Notably, AUS_001 is a poor substrate of the drug efflux transporter, P-glycoprotein 1 (Pgp), as it does not inhibit drug-stimulated Pgp ATPase activity and retains efficacy in high Pgp-expressing models in vitro. Subsequently, the effects of AUS_001 were explored in the ToxTracker® assay, consisting of a panel of mammalian stem cell lines that contain fluorescent reporters for the exploration of DNA damage, oxidative stress, and protein damage using flow cytometry. Robust activation of the Rtkn genotoxicity reporter under low cytotoxicity conditions, in combination with accumulation of cells in G2/M phases of the cell cycle within 4 h and an increase in aneuploid cells after 24 h of drug treatment, suggested an aneugenic mode of action, typically caused by tubulin-targeting agents or mitotic kinase inhibitors. Biochemical tubulin polymerization assays then revealed that AUS_001 acts as a direct tubulin destabilizing agent and monitoring microtubule dynamics in GFP-tubulin reporter cells provided evidence that AUS_001 blocks microtubule assembly. Our studies have elaborated the mechanism of AUS_001 as an inhibitor of tubulin polymerization. The favorable safety profile of AUS_001, along with its ability to circumvent Pgp-mediated multidrug resistance, provides potential for efficacy against multiple cancers where microtubule destabilization is proven to be an effective target. Citation Format: Herman Lelie, Inger Brandsma, Giel Hendriks, Lee R. Cavedine, Brogan A. Epkins, Steven M. Garner, Andrew J. Cook, Muthukrishnan Renganathan, Yong Zhao, Alastair J. King, Peter Y.W. Chan, April Risinger, Marina Koutsioumpa. A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4701.