Abstract Bladder cancer (BC) is the 9th most common cancer worldwide. Most bladder tumors are detected and treated at the non-muscle invasive (NMIBC) stage. However, tumors recur frequently, many progress to MIBC and metastasize contributing to significant mortality. Rising incidence and mortality in BC underscore the need to develop new regimen for its management. Transcriptional factor Signal transducer and activator of transcription 3 (STAT3) regulates cell proliferation through inflammation and immune responses. Its activation is highly regulated in normal cells but in cancers including BC, it is constitutively active. Expression of dominant-negative STAT3 was shown to inhibit tumor formation in nude mice. Earlier we and others reported that STAT3 inhibitors reduces BC cell survival and proliferation. In the present study, we determined pharmacodynamic pSTAT3 inhibitory effect of STAT3 inhibiting small molecules (GLG-302, SH5-07, TTI-101) in-vitro BC cells and in-vivo rat tumors. TTI-101 was further evaluated for its potential to intercept BC in a N-butyl-N-(4-hydroxyl)-nitrosamine (BBN)-rat BC model. Female F344 rats were randomized into a placebo, STAT3 (n=24) and Sulindac (n=18) intervention groups. Eight-weeks of age, rats were given BBN by oral gavage (150mg/dose; 2x/week for 8 weeks). Drug treatment was initiated at papilloma stage i.e., ∼10 weeks after last BBN. Rats in intervention groups received TTI-101 at 25 or 75mg/kg body weight by oral gavage (5x/week) for 20 weeks. Sulindac was administered at 10mg/kg BW as a comparator. All drugs were formulated using 60% labrasol:40% PEG400. Bladder tumors were assessed at ∼45 weeks of age. TTI-101 did not cause any overt-toxicities. BBN-induced bladder tumors in all rats, resulting in larger bladders in placebo group (0.31±0.06g; Mean±SEM). Importantly, bladder weights in TT1-101 25mg/kg and 75mg/kg groups were 0.21±0.06mg (32% inhibition; p>0.05) and 0.13±0.01g (58% inhibition; p<0.01) when compared to the placebo. Sulindac treatment had non-significant 46% inhibition (0.17±0.02g; p=0.07). In the placebo group, about half of all rats developed large bladder tumors (>200mg; 50% incidence). Interestingly, significantly fewer rats developed such tumors with TTI-101 25-mg/kg (17% incidence; p<0.05) and at 75mg/kg BW dose (8% incidence; p<0.005) in a dose dependent manner (67%-83% less respectively). Thus, STAT3 inhibition with TTI-101 resulted in a dose dependent inhibition of bladder tumors growth. Biomarker and gene expression analysis suggested modulation of critical tumor promoting pathways with decrease in pSTAT3 expression. Collectively, our study demonstrated that STAT3 inhibition using TTI-101 can intercept bladder tumor growth and warrants further investigation in clinical trials. (Project funded in whole with Federal funds from the NCI-NIH, DHHS, under Contract No. 75N91019D00020-75N91020F00005). Citation Format: Venkateshwar Madka, Gopal Pathuri, Anil Singh, Nicole Stratton, Anh Bao, David J. Tweardy, Shizuko Sei, Vignesh Gunasekharan, Chinthalapally V. Rao. STAT3 inhibitor TTI-101 effectively intercepts bladder cancer in a carcinogen-induced rat bladder tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 951.

Abstract Colorectal Cancer (CRC) remains the leading cause of cancer-related mortality worldwide. Inflammation is a key hallmark of many cancers, including CRC. Pro-inflammatory lipid mediators play a key role, while COX-2 inhibiting NSAIDs are promising, their chronic use is linked with unwanted side-effects. In this context, mechanistic studies suggest that targeting microsomal prostaglandin synthase-1 (mPGES-1) and 5-lipoxygenase (5-LOX) with natural products (NP) presents a valuable opportunity to intercept CRC and mitigate those side effects. Here we aimed to perform high-throughput screening (HTS) of NCI NPs library (∼500,000 semi-purified fractions) to identify potential inhibitors of 5-LOX and mPGES1; and further validate the purified compounds using secondary assays. To establish the enzyme activity inhibitory assays, first we developed a stable Human Embryonic Kidney (HEK) 293 cell lines with mPGES-1 and 5-LOX overexpression as well as 5-LOX overexpressing insect cells. Proteins expression was confirmed using western blotting. Lysates from human 5-LOX expressed in Sf9 insect cells and HEK-293 cells, were used to generate an assay format in 384-well microplates suitable for HTS. In this assay format, lysates are preincubated with inhibitors for 20 minutes, stimulated with arachidonic acid (AA) for 5 minutes. The production of free radicals, as a result of the conversion of AA to 5HPETE and LTA4 by 5-LOX activity, is detected upon the addition of 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA). The non-fluorescent H2DCFDA when oxidized by the free radicals, generates a highly fluorescent compound, which can be measured to quantify enzyme activity. The reaction was stopped after 15 minutes upon the addition of acetonitrile. Finally, enzyme activity was calculated by measuring “total relative fluorescence units (RFU) at 485-nm excitation and 530-nm emission spectra. The assay has been optimized using a final volume of 15ul and has a Z’ Factor score of 0.65 and a S/B of 3.5 in 384-well microplates. The 5-LOX activity was completely inhibited by 20uM NDGA, a known inhibitor of 5-LOX. Additionally, a pre-plated NCI library of semi-purified NP fractions (5mg/ml stock in DMSO) has been assessed to validate the assay and identify potential fractions that demonstrate 5-LOX inhibitory activity. The fractions with the most promising activity based on this evaluation will be presented at the meeting. We have also optimized the mPGES1 activity assay using HEK293 cell line overexpressing COX2 and mPGES-1. In this assay format, treatment with AA results in elevated levels of PGE2, as detected in a PGE2 HTRF assay. In summary, these optimized assays will be employed for large scale robotic HTS of NCI NP library to discover and develop safer inhibitors of proinflammatory targets mPGES1 and 5-LOX for intercepting inflammation associated cancers. (Funded by NCI-UG3CA290310-01). Citation Format: Krishnendu Goswami, Nataliya Smith, Ravi Manjhi, Gopal Pathuri, Brandon Somerville, Yurong Song, Venkateshwar Madka, Kajal Biswas, Altaf Mohammed, Robert H. Shoemaker, Matthew J. Hart, Chinthalapally V. Rao. Screening of natural products library against 5-lipoxygenase (5-LOX) and mPGES1 proinflammatory targets for CRC interception [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3653.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide and carries substantial morbidity. Metformin, a widely used antidiabetic agent, shows promise for HNSCC prevention, but resistance arises in a subset of tumors. In a recent issue of Cancer Prevention Research, Hoang and colleagues use CRISPR screening to identify key mediators of metformin resistance, including AMPK and protein kinase A (PKA), and demonstrate that the cyclooxygenase 2-prostaglandin E2 axis acts upstream of PKA. Because this pathway is readily inhibited by common nonsteroidal anti-inflammatory drugs (NSAID), the findings support clinical evaluation of combined metformin and NSAID therapy to improve HNSCC chemoprevention. See related article by Hoang et al., p. 79 .

BACKGROUND AND AIMS:Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of HCC, particularly in obesity, yet mechanisms linking hepatocyte dysfunction to tumorigenesis remain unclear. Mixed lineage kinase domain-like protein (MLKL), the effector of necroptosis, is elevated in MASLD, but its hepatocyte-intrinsic role in obesity-driven MASLD-HCC is unknown. APPROACH AND RESULTS:Using a long-term western diet (WD)-induced MASLD-HCC model in hepatocyte-specific MLKL knockout ( MlklHepKO ) mice, we defined MLKL's hepatocyte-intrinsic function. WD increased hepatocyte MLKL protein expression without detectable necroptosis activation, indicating a necroptosis-independent role. MLKL deficiency did not alter WD-induced inflammation, fibrosis, or liver injury but increased hepatic lipid accumulation while reducing lipotoxic lipid species and preserving mitochondrial function. WD-fed MlklHepKO mice developed fewer and smaller tumors with reduced incidence, multiplicity, proliferation, and stemness. Transcriptomic analysis revealed upregulation of mitochondrial oxidative phosphorylation pathways in MlklHepKO livers. WD suppressed the mitochondrial fusion protein and tumor suppressor mitofusin 2 (MFN2), whereas MLKL deficiency restored MFN2 expression post-translationally. In HCC cells, MLKL deletion reduced proliferation, improved mitochondrial respiration, and decreased glycolysis; these effects were reversed by MFN2 deletion. MLKL is localized to nuclear and mitochondrial compartments, consistent with organelle-intrinsic functions. The human MLKL inhibitor necrosulfonamide (NSA) suppressed HepG2 xenograft growth, and elevated MLKL expression in human HCC correlated with poorer overall survival. CONCLUSIONS:Hepatocyte MLKL promotes MASLD-associated HCC through a non-necroptotic mechanism involving MFN2 suppression, impaired mitochondrial function, and increased tumor proliferation and stemness. These findings identify MLKL as a potential therapeutic target in MASLD-associated HCC.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide, accounting for approximately 10% of all cases, and is the second leading cause of cancer-related deaths in the United States. In patients with Familial Adenomatous Polyposis (FAP), APC mutations drive c-MYC overexpression, promoting metabolic and immune dysregulation that contributes to CRC development, making c-MYC a potential target for FAP-associated chemoprevention. Although no c-MYC inhibitors are currently available, recent evidence indicates that a uropathogenic E. coli protease can degrade c-MYC protein. This study evaluated recombinant LON protease (rLONP) for chemopreventive efficacy in PIRC rats, a model of FAP. Rat pups were genotyped, baseline polyp burden was established by colonoscopy, and animals were randomized to receive saline or rLONP twice daily (2.5 mg/kg, p.o.) for 2 or 4 weeks. Serum analyses showed no significant changes in liver or kidney function markers in rLONP-treated rats compared with controls. rLONP-treated male rats exhibited a ∼40% reduction in colonic polyps at both time points, and small intestinal polyp counts were also lower in treated males and females. Combined analysis demonstrated a ≥60% reduction in total intestinal polyp multiplicity after 2-4 weeks of treatment (p<0.001). Protein expression analyses of colonic polyps revealed decreased c-MYC protein levels following rLONP treatment. Immunohistochemistry further showed reduced Ki-67, Cyclin D1, and c-MYC expression in treated polyps compared with controls. Overall, rLONP was well tolerated and significantly reduced intestinal polyp burden in PIRC rats in this short-term study, supporting further long-term efficacy evaluation.

Abstract Lung cancer is one of the leading cancers worldwide. Around 226,000 people will be diagnosed with lung cancer in the US during 2025. Almost one-fourth of all lung cancers are KRAS-mutated tumors, which are very challenging to treat and have poor prognosis. Interception of lung cancer is of utmost importance to reduce the burden and mortality. However, there are currently no interception agents approved by FDA. TCGA database revealed that the apoptosis mediating TRAIL expression is lost during the progression of lung tumor stages. ONC201 (Dordaviprone) is an orally active TRAIL inducing small molecule compound with proven preclinical efficacy and is being clinically evaluated against multiple cancers. Previously we reported the cancer prevention potential of ONC201 in an NNK-induced lung cancer model in A/J mice. Here, we evaluated the efficacy of ONC201 in an aggressive KRASG12V lung tumor mouse model. Male and female KRASG12V mice were generated by inhouse breeding. Six-week aged mice were randomized (n=20/sex) into placebo and interception groups. Beginning at 8 weeks of age (early adenoma stage) mice were gavaged one of the doses of ONC201 (0, 25, 50, 100mg/kg in PBS vehicle) twice weekly for 28 weeks. All mice were euthanized at 36 weeks of age, and lungs were evaluated to determine tumor incidence and multiplicity. Both the male and female KRASG12V mice in placebo group developed lung tumors (∼100% incidence) with average tumor multiplicity of 12.2±1.5 (male) and 11.7±0.8 (female) per mouse. ONC201 treatment resulted in significant reduction in total lung tumors multiplicity in both male (34%-63% less; p<0.05-p<0.0001) and female (33%-55% less; p<0.001-p<0.0001) mice in a dose dependent-manner as compared to their control group. H&E stained lung tumor sections were histologically classified as adenomas (AD), and adenocarcinomas (ADCA). Placebo mice showed multiplicity of 9.3±1.3 ADCA, and 12±1.2 AD+ADCA in male; 8.3±0.6 ADCA, and 11.61±0.87 AD+ADCA in female mice. Histopathology results indicated dose-dependent reduction in ADCA multiplicity in ONC201 treated mice in male (53%-86%; p<0.001 - p<0.0001) and female mice (54%-79%; p<0.0001) when compared to their respective control mice. There was no clinical sign of toxicities observed in all three doses tested in both male and female mice. Immunoblotting and IHC results revealed that TRAIL inducing ONC201 triggering the downstream signaling molecules (TRAIL, DR5, FADD, Apaf1), elevated apoptosis markers (Caspases), concurrently reduces the proliferation markers (PCNA, Cyclin D1, EGFR, Ki67). In conclusion, ONC201 demonstrated strong efficacy in preventing lung cancer in two preclinical mouse models and warrants further clinical development for the prevention of lung tumor in high risk populations. (Project funded 100% with Federal funds from NCI, NIH, DHHS, under Contract 75N91019D00020_75N91022F00003) Citation Format: Karthikkumar Venkatachalam, Gopal Pathuri, Nicole Stratton, Anil Singh, Nandini Kumar, Shizuko Sei, Vignesh Gunasekharan, Chinthalapally V. Rao, Venkateshwar Madka. TRAIL inducing drug, ONC201 prevents adenocarcinoma in transgenic KRASG12V mouse lung cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 948.

Abstract Background Natural products (NPs) represent a rich source of bioactive compounds with cancer prevention/interception potential. NPs offer unique chemical diversity and a history of safe human use, making them attractive candidates for long-term use. High-throughput screening and mechanistic studies are key to discovering promising leads and translating them into effective and safe cancer prevention and interception strategies. To support the Discovery and Development of Natural Products for Cancer Interception and Prevention (DDNP-CIP) Initiative, we aimed to validate potentially clinically relevant molecular targets (SKP2, TEAD2, 5-LOX and RUNX1) and establish and optimize assays to enable high-throughput NPs library screening for cancer prevention. Methods Target validation was performed using tissue microarrays. SKP2, a component of the SKP2-SCF E3 ligase complex, and TEAD2, a DNA-binding transcription factor, were evaluated for their expression levels in TMAs of prostate and liver cancer, respectively. In addition, cell-based platforms were developed or adapted to identify NPs exhibiting immune-modulating activity or targeting either 5-LOX, a lipid-peroxidizing enzyme, or RUNX1, a transcription factor critical for hematopoietic differentiation and frequently mutated in hematologic malignancies. These assays utilized stably expressing reporter cell lines. Results SKP2 was significantly over expressed in 68.6% of prostate hyperplasia, 97.6% of prostate intraepithelial neoplasia, and 81.9% of adenocarcinoma compared with normal tissue. TEAD2 was highly expressed in liver hyperplasia and significantly upregulated in hepatitis (p = 0.0041), hepatocellular carcinoma (p = 0.0012), and intrahepatic cholangiocarcinoma (p < 0.0001). To identify NPs with immune-modulating activity, an assay using the THP-1 ISRE FRET reporter cell line was adapted and optimized. Reference compounds and initial challenge plates were tested, with several samples eliciting positive responses. A cell line expressing 5-LOX was generated and validated. An inhibition assay using a positive compound nordihydroguaiaretic acid demonstrated dose-dependent inhibition (29% at 0.1uM to 92% at 1uM) upon arachidonic acid treatment without toxicity. To screen NPs for splicing modulating activity, nano-luciferase reporter cell lines with RUNX1 mutations identified in patients with familial platelet disorder with associated myeloid malignancy were generated. Several reference compounds showed positive responses in selected mutations. Summary Validated targets and assay platforms establish a foundation for high-throughput NPs screening. These efforts advance the DDNP-CIP’s mission to identify and develop NPs for cancer prevention and interception. Funded partly by the National Cancer Institute under Contract No. HHSN261201500003I Citation Format: Yurong Song, Brandon Somerville, Kajal Biswas, Karim Baktiar, Liankun Song, Sara Sanders, Tanja Grkovic, Ligia A. Pinto, Ana Catarina Menezes, Paul P. Liu, Matthew J. Hart, Chinthalapally V. Rao, Shugeng Cao, Xin Chen, Xu Wu, Xiaolin Zi, Mark J. Henderson, Barry R. O'Keefe, Altaf Mohammed, Robert H. Shoemaker. Target validation and high-throughput screening assay development for natural product discovery for cancer prevention and interception [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 942.

Purpose. To assess the chemopreventive effect of oleanolic acid (ONA) and its synthetic analog 18 alpha-olean-12-ene-3 beta-23,28-triol (OT) on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in F344 rats and understand anti-inflammatory properties and apoptosis effects in HT29 colon cancer cells and Raw 264.7 macrophage cell lines.Methods. Five week-old male F344 rats were fed a control diet or experimental diets containing two doses of ONA (750 and 1,500 ppm) and OT (250 and 500 ppm). After 1 week, all animals were s.c. injected with AOM (15 mg/kg body weight, once weekly for 2 weeks). At 14 weeks of age, all rats were killed and colons were evaluated for ACF. Cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) expressions and apoptosis were assessed in cell lines exposed to OT using western blots and 4',6-diamidino-2-phenylindole staining.Results. Administration of ONA and OT inhibited mean colonic ACF and multi-crypt AC/foci in a dose dependent manner (p < 0.001-0.0001). OT blocked the COX-2 expression induced by phorbol 12-myristate 13-acetate in a dose-dependent manner and induced apoptosis in HT-29 cancer cells, and suppressed iNOS activation in RAW264.7 macrophages.Conclusions. ONA and OT possess chemopreventive activity against colon carcinogenesis in rat and OT inhibits the COX-2 and iNOS and induces apoptosis in cell lines.

Bladder cancer (BCa) is the second most common cancer of the genitourinary tract globally. It has limited treatment options, high recurrence rate, and acquires resistance to platinum-based therapy. Therefore, identifying novel therapeutic targets is urgently needed. Analysis of the TCGA data revealed that the enzyme galactokinase-1 (GALK1) is overexpressed (p < 0.0001) in bladder tumors compared to normal tissue. Our data also confirmed GALK1 protein upregulation in multiple human BCa cell lines and rodent bladder tumors. However, the precise role of GALK1 in BCa progression and effects of its specific inhibitor remain unexamined. In this study, we demonstrate that GALK1 gene silencing using shRNA resulted in a significant reduction in BCa cell proliferation, migration, and invasion. Pharmacological inhibition of GALK1 using small molecule Cpd36 resulted in anticancer efficacy against BCa. Cpd36 inhibited proliferation, migration, and invasion of BCa cells. Further, Cpd36 induced G1 phase cell cycle arrest, apoptosis, mitochondrial membrane depolarization, and ROS production in the BCa cells. Mechanistically, Cpd36-induced reduction in cell proliferation was associated with a decrease in expression of GALK1, PCNA proteins. Inhibition of metastatic potential was accompanied by decreased migration, invasion, and MMP-9 expression. Cell cycle arrest was associated with decrease in Cyclin D1 and increased expression of p21 and p27. Induction of apoptosis was linked with increased expression of cleaved caspase-3 and cleaved PARP, while downregulating p-AKT. Additionally, Cpd36 in combination with cisplatin or gemcitabine showed a strong synergistic effect on BCa cells. Taken together, our findings suggest that GALK1 plays a significant role in BCa cell survival and validates its inhibitors as promising therapeutic options for managing this disease.

Abstract Bladder cancer (BC) is the second-most diagnosed genitourinary cancer. Most tumors are detected at the non-muscle invasive (NMIBC) stage and treated; however high recurrence rate, treatment resistance, tumor progression and metastasis contribute to significant mortality annually. Many studies have indicated significantly higher incidence rates and BC progression to be more predominant in men. Experimental data have also showed strong association of BC tumorigenesis with androgen receptor (AR) signaling , making AR a promising target for cancer interception. In this study, AR antagonist, apalutamide (APA) was evaluated for BC preventive efficacy in a N-butyl-N-(4-hydroxyl)-nitrosamine (BBN)-rat BC model. Male and female Fischer rats were randomized into placebo and intervention groups (30 rats/group/sex: 24 BBN+6 Saline). At 8 weeks of age, rats in carcinogen groups received BBN by oral gavage (150mg/dose; 2x/week for 8 weeks) to induce BC. APA was given to rats in intervention groups at 7.5, 15, or 30mg/kg body weight by oral gavage (5x/week) until termination. Early intervention with APA started at carcinogenesis stage i.e., a week after last BBN, while delayed intervention began at papilloma stage i.e., ∼10 weeks after last BBN. Bladder tumors were assessed at 40 and 50 weeks of age in male and female rats respectively. APA did not cause any overt-toxicities. BBN treatment induced bladder tumors in all rats, resulting in significantly larger bladders in placebo group compared to normal bladders in saline group. Importantly, APA significantly reduced bladder weights suggesting tumor growth inhibition in intervention groups compared to placebo. With early intervention, bladder weights were reduced by 60%-65% in male rats (p<0.05) and by 54%-63% in female rats (p<0.05) when compared to the placebo group. Incidence of large bladder tumors was also significantly decreased with APA treatment by 30%-47% in males (p<0.05-p<0.01) and by 32%-50% in females (p<0.05) in a dose dependent manner. Delayed intervention also resulted in 21%-53% (p<0.01) reduction of bladder weights and 28%-66% less incidence of large tumors in male rats only. Histopathological analysis of the tumor sections demonstrated suppression of tumor progression in APA treated rats as indicated by the significant decrease in multiplicity of papillomas, NMIBC, and MIBC when compared to placebo. Biomarker and gene expression analysis suggested modulation of critical tumor promoting pathways with APA treatment. In summary, this preclinical study demonstrated that an AR antagonist, apalutamide, can intercept bladder tumor growth and progression and warrants further investigation in clinical trials. (Project funded in whole with Federal funds from the NCI-NIH, DHHS, under Contract No. 75N91019D00020 - 75N91022F00002). Citation Format: Venkateshwar Madka, Gopal Pathuri, Anil Singh, Surya P. Singh, Anh Bao, Nicole Stratton, Shizuko Sei, John Clifford, Chinthalapally V. Rao. Efficacy of androgen receptor inhibitor, Apalutamide, in intercepting bladder cancer in a BBN-induced rat bladder tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 950.

Cancer initiation and progression are associated with numerous somatic mutations, genomic rearrangements, and structure variants. The transformation of a normal cell into a cancer cell involves spatio-temporal changes in the regulation of different gene networks. The accessibility of these genes within the cell nucleus is manipulated via nucleosome remodeling ATPases, comprising one of the important mechanisms. Here, we reviewed studies of an ATP-dependent chromatin remodulator, chromodomain helicase DNA-binding 4 (CHD4), in cancer. Multiple domains of CHD4 are known to take part in nucleosome mobilization and histone binding. By binding with other proteins, CHD4 plays a vital role in transcriptional reprogramming and functions as a key component of Nucleosome Remodeling and Deacetylase, or NuRD, complexes. Here, we revisit data that demonstrate the role of CHD4 in cancer progression, tumor cell proliferation, DNA damage responses, and immune modulation. Conclusively, CHD4-mediated chromatin accessibility is essential for transcriptional reprogramming, which in turn is associated with tumor cell proliferation and cancer development.

In the original publication [...].

Urinary bladder cancer (BCa) is the second most prevalent cancer of the genitourinary tract. Although BCa affects both genders, it is more predominant among men. Most of the tumors are non-invasive at diagnosis, however, high rates of recurrence, heterogeneity, and treatment resistance lead to significant mortality. Therefore, it is necessary to identify and understand the role of novel metabolic targets and explore their therapeutic potential against BCa. Rapidly proliferating cancer cells have high nutritional demand which necessitates metabolic reprogramming and reliance on the alternative nutrients in the tumor microenvironment. Based on the human BCa transcriptome data analysis (TCGA data), we identified that the Galactokinase-1 (GALK1) gene is significantly overexpressed (p<0.0001) in human bladder tumors compared to normal tissue, and its expression also correlates with disease stage and poor prognosis. GALK1 is a member of the Leloir pathway that facilitates the initial step of galactose breakdown. Interestingly, we observed abundant expression of GALK1 in various human and rodent BCa cell lines as well as rodent bladder tumor tissues. Hence, we hypothesized that GALK1 may contribute to BCa survival by contributing to the cancer cell proliferation and could serve as a novel target for the management of this malignancy. To understand this, we abrogated GALK1 gene expression in MB49 cells using siRNA and found a significant decrease in cell proliferation with increased apoptosis. Further treatment of various human bladder cancer cells, in vitro with a small-molecule GALK1 inhibitor (Cpd36), resulted in reduced proliferation (IC50 = 29.09 - 43.67μM), migration and invasion potential. A similar growth inhibitory effect was also observed in BCa spheroids and tumoroids. Cell cycle analysis indicated that Cpd36 induced significant G1 arrest (p<0.0001) in BCa cells and pronounced G2/M arrest (p<0.0001). GALK1 inhibition increased mitochondrial ROS production while lowering the mitochondrial membrane potential and triggered apoptosis in BCa cells. GALK1 inhibition induced cell growth arrest was associated with decrease in protein levels of GALK1, PCNA, and Cyclins. Additionally, the induction of apoptosis was associated with increase in cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Further, we investigated the effect of GALK1 abrogation on chemotherapeutic drugs sensitivity. Invitro drug combination data suggested that targeting GALK1 sensitized the BCa cells to cisplatin or gemcitabine as evident from significant reduction in the IC50 of these chemotherapeutic drugs. Based on these findings, GALK1 plays a tumor promoting role in BCa and its inhibition may be a promising therapeutic target for its prevention and treatment. (Funding supported in part by NCI-P30CA225520 and ACS-134128-IRG-19-142-01 grants). Surya P. Singh, Chinthalapally V. Rao, Venkateshwar Madka. Galactokinase 1 is a novel metabolic target against bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5602.

TRIP13 is a member of the large AAA+ ATPase protein superfamily that plays a crucial role in the precise segregation of chromosomes during mitosis. The abnormal function of TRIP13 has diverse functions, including mitotic processes, DNA repair pathways, and spindle assembly checkpoints, which may contribute to chromosomal instability (CIN). Emerging evidence suggests that the overexpression of TRIP13, observed in many cancers, plays a significant role in drug resistance, autophagy, and immune invasion. Recently, significant advances have been made in identifying TRIP13-associated signaling pathways that have been implicated in cancer progression. Several small molecules that specifically inhibit TRIP13 function and reduce cancer cell growth have been developed. Combination treatments, including TRIP13 inhibitors and other anticancer drugs, have shown promising results. While these findings are promising, TRIP13 inhibitors are awaiting clinical trials. This review discusses recent progress in understanding the oncogenic function of TRIP13 and its possible therapeutic targets, which could be exploited as an attractive option for cancer management.

Bladder cancer (BC) is the 2nd most commonly diagnosed genitourinary cancer. While the majority of cancers are diagnosed and treated at the non-muscle invasive stage; high recurrence rate, treatment resistance, and tumor progression cause significant mortality. Epidemiologic and preclinical data have long demonstrated clear differences in incidence and progression of BC between genders, suggesting strong association with hormonal pathways with development and progression in these tumors. Based on these studies, androgen receptor (AR) signaling pathway emerged as a critical player and a promising target for BC interception. Thus, in the current study, BC preventive efficacy of the nonsteroidal AR antagonist, apalutamide, was evaluated in a N-butyl-N-(4-hydroxyl)-nitrosamine (BBN)-rat BC model. At 7 weeks of age, fisher rats were randomized by weight into 4 groups (30 rats/group/gender: 24 BBN + 6 Vehicle). Starting at 8 weeks of age, rats were administered BBN by oral gavage (150mg/dose; twice weekly for 8 weeks) to induce BC. A week after BBN treatment, rats in each group received apalutamide by oral gavage (5 days/wk) at increasing doses, 0, 7.5, 15, or 30mg/kg BW, until termination. Male and female bladder tumors were assessed at 40 & 50 weeks of age, respectively. Body and organ weights, blood profile, liver and kidney enzyme function analysis suggested non-significant differences, indicating safety of the tested doses. BBN treatment induced bladder tumors in both genders, resulting in significantly larger bladders (∼0.75±0.25g) compared to vehicle group (0.12±0.08g) with the incidence of large tumors (≥200mg) in 87% and 75% of male and female rats, respectively. Importantly, apalutamide treatment led to a significant reduction of bladder weights suggesting tumor growth inhibition in treated rats compared to control. Bladder weights were reduced by 60%-65% in males (p<0.05) and by 54%-63% in females (p<0.05) when compared to the untreated group. Moreover, the incidence of large bladder tumors was also significantly decreased with apalutamide treatment by 30%-47% in males (p<0.05-p<0.01) and by 32%-50% in females (p<0.05) in a dose dependent manner. Histopathological analysis of the tumor demonstrated a significant decrease in papillomas (40%-60% less vs control; p<0.01-p<0.0001), NMIBC (48%-76% less vs control; p<0.001-p<0.0001) and MIBC (>77% less vs control; p<0.001-p<0.0001) at all three doses tested. Biomarker analysis showed reduction in the proliferation markers Ki67 and cyclin D1 in apalutamide treated rats tumors. In conclusion, our study demonstrated that an AR antagonist, apalutamide, can intercept bladder tumor growth and progression in a preclinical rat model and warrants further investigation in clinical trials. (Project funded 100% with Federal funds from NCI, NIH, DHHS, under Contract 75N91019D00020_75N91022F00002) Venkateshwar Madka, Gopal Pathuri, Surya Pratap Singh, Anil Singh, Anh Bao, Nicole Stratton, Shizuko Sei, John L. Clifford, Chinthalapally V. Rao. Intercepting bladder cancer progression using androgen receptor inhibitor, apalutamide, in a carcinogen BBN-induced rat bladder tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6324.

Lung cancer is the 2nd most commonly diagnosed cancer in both genders in the US. Most lung tumors are diagnosed at late stages and distant metastasis drastically reduces 5-year survival to <10%. Hence, intercepting lung tumor progression would aid in reducing tumor burden and cancer mortality. Preclinical and TCGA data demonstrated that the TNF Related Apoptosis Inducing Ligand (TRAIL/TNFSF10) is incrementally downregulated with lung tumor progression. Hence we evaluated ONC201 (Dordaviprone), a orally active TRAIL-inducing small molecule for its cancer preventive efficacy in the tobacco carcinogen Nicotine-derived Nitrosamine Ketone (NNK) driven A/J mouse lung cancer model. Prior to the efficacy studies, five different oral doses of ONC201 (0-200mg/kg BW) were tested in A/J mice (n=6/gender/group) for optimal dose range selection. For efficacy evaluation, A/J mice (6 weeks age; n=20/sex/group) were randomized into vehicle control and intervention groups; and lung tumors were induced by a single i.p. injection of NNK (2.07mg/mouse). Six-weeks after NNK injection, mice were given 0, 25, 50, or 100mg/kg BW ONC201 (in PBS) by oral gavage twice weekly for 28 weeks. Mice were then euthanized and lungs were evaluated for tumor incidence and multiplicity. All of the NNK-injected control mice developed lung tumors (100% incidence); but significant difference (p<0.001) was observed in tumor multiplicity between genders (17±2.18 in females (Mean±SEM) Vs 7.7±0.89 in males). ONC201 treatment led to significant decrease in tumor multiplicity at all 3 tested doses in both genders (25-43% less tumors; p<0.05-p<0.001). Lung tumors were classified as adenomas (AD), and adenocarcinomas (AdCa) based on histological criteria. Control mice developed 2.25±0.19 AD, and 5.45±0.69 AdCa in male; 3.89±0.57 AD, and 11±1.24 AdCa in female mice. While ONC201 treatment reduced the adenomas moderately, AdCa was significantly inhibited at all 3 doses in both genders in a dose dependent manner. In the female mice, AdCa multiplicity was reduced by 27% (p<0.05), 53% (p<0.001), and 70% (p<0.0001) in 25 mg/kg, 50 mg/kg, and 100 mg/kg BW ONC201 respectively. Similarly, AdCa was inhibited by 55% (p<0.001), 67% (p<0.0001), and 84% (p<0.0001) in male mice at the 3 dose levels as compared to controls. No toxicities were observed in all doses tested in both genders. Biomarker analysis of the lung tumors using IHC and Western blotting analysis suggested an induction of TRAIL signaling pathway markers, increased apoptosis, and a decrease in proliferation markers (PCNA, Cyclin D1, EGFR, Ki67). In conclusion, our data indicate a strong preventive efficacy of ONC201 in a preclinical model of lung cancer and warrant further development towards clinical applications for lung cancer prevention in high-risk cohorts. (Project funded 100% with Federal funds from NCI, NIH, DHHS, under Contract 75N91019D00020_75N91022F00003) Karthikkumar Venkatachalam, Venkateshwar Madka, Gopal Pathuri, Anil Singh, Anh Bao, Nicole Stratton, Shizuko Sei, Vignesh Gunasekharan, Chinthalapally V. Rao. Preclinical efficacy of TRAIL inducing ONC201 against carcinogen (NNK)-induced lung adenocarcinoma in A/J mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6336.

In the original publication [...].

Hereditary Familial Adenomatous Polyposis (FAP) patients develop large number of colonic polyps that can progress to aggressive carcinomas. In the FAP patients, Apc or β-catenin mutations lead to c-Myc overexpression, which in turn drives the metabolic and immune alterations leading to colorectal cancer, making it a potential target for CRC prevention. Unfortunately, finding c-Myc inhibitors has been difficult and only recently studies show that uropathogenic E. Coli rLON protease (rLONP) can degrade the c-Myc in tumor tissues. In this study, PIRC rats (10- or 12- weeks age) were treated with rLONP (0.375mg/rat/day dose) in PBS by oral gavage twice daily for either 2- or 4- weeks and assessed for toxicity and intestinal tumor burden. Organ morphology and weight were not significantly different from control PIRC rats. Also, hematological profile was very similar in control and rLONP treated male rats, however in female PIRC rats, rLONP treatment for 4 weeks showed significant differences in WBC, neutrophils, and lymphocytes count compared to control. Serum analysis indicated a non-significant (p>0.05) increase in AST (297.6±74.3 u/L) and ALT (365.3±86.8 u/L) levels with 4-weeks rLONP treatment when compared to control group (133.9±27.5 u/L & 184.5±30.3 u/L respectively). ALKP levels were slightly lower (187.6±14.4 u/L) in rLONP treated rats compared to control (221.3±16.7 u/L). BUN (17.6±0.7 mg/dL) and CREA (0.15±0.03 mg/dL) of the rLONP treated rats were non-significantly different from control group (16.6±1.1 mg/dL & 0.15±0.02 mg/dL respectively). Histopathology of all major organs of rLONP treatment appeared normal, with no significant differences as compared to untreated groups. Colonic and small intestinal polyps were compared between control and rLONP treated rats. Average colonic polyps were less in rLONP treated male rats at both 2- and 4- week endpoints (40% reduction) compared to their respective control rats while this effect was not distinguishable in the females due to low polyp number and slow rate of polyp and tumor development. Small intestinal polyps also appeared to be less in number in rLONP treated male and female rats as compared to control. Total intestinal polyps showed ≥50% reduction of multiplicity, male (p<0.012; p<0.009) and female (p=0.06; p<0.022) with 2- and 4- week rLONP treatment, respectively, as compared to control group. Protein expression analysis of control and treated colonic polyp tissues suggested reduction of c-MYC expression with rLONP treatment. Also, IHC analysis showed reduction in Ki-67, Cyclin D1, and c-Myc expression in the rLONP treated rat colonic polyps when compared with control polyps. Based on these observations, it is concluded that rLONP treatment did not cause any overt-toxicities and reduced intestinal polyps in PIRC rats in a short-term study. (Project funded in whole with Federal funds from the NCI, NIH, DHHS, under Contract 75N91019D00020_75N91022F00001). Venkateshwar Madka, Karthikkumar Venkatachalam, Gopal Pathuri, Nicole Stratton, Anil Singh, Shizuko Sei, Mark S. Miller, Vignesh Gunasekharan, Chinthalapally V. Rao. Colonic polyp preventive effect of rLON protease in FAP-rat intestinal polyposis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2373.

Colorectal cancer (CRC) is one of the major reasons for cancer-related deaths around the world. Constitutive activation of WNT pathway, due to APC gene mutation, is the characteristic feature of most human colon tumors. Familial adenomatous polyposis (FAP) patients inherit APC mutations and pose an absolute risk of developing CRC in their lifetime. The genetically modified APC mouse models have paved the way to study various aspects of the hereditary human CRC, including biochemical, molecular, and histological aspects. Preclinical and clinical data suggest that certain dietary supplements, NSAIDs, natural products, and chemically synthesized compounds, can help in intercepting CRC incidence and progression by modulating various hallmarks of cancer. In this review, we have provided a summary of promising natural and synthetic agents that demonstrated chemopreventive efficacy against CRC in the FAP-mimicking APCMin/+ mouse model.

The high incidence and mortality rates of colorectal cancer (CRC) in Alabama African Americans (AAs) and Oklahoma American Indians (AIs) are recognized as cancer disparities, yet the underlying causes have been poorly demonstrated. Our previous study of transcriptomic profiles of CRCs of Alabama AAs, Oklahoma AIs, and white people from both states revealed molecular disparities and differentially expressed genes (DEGs) in the racial groups. By evaluating CRC whole-exome sequencing and mutational profiles, we report sets of mutated genes whose frequencies differed significantly (p<0.05) in a race-specific manner. Secondary screening with a CRC database (cBioportal database for bowel cancer) showed 42, 38, and 35 “survival-critical genes (SCGs)” (i.e., genes whose mutations/alterations are associated with significant differences in the patients’ survival rates) among the differentially mutated genes, suggesting that the mutations have a functional impact on cancer. Notable SCGs with race-pronounced variants were different from DEGs and their involved pathways included nucleotide catabolism (GDA, NT5M, XDH) and cell cycle checkpoints (CLIP1, PSMB2, PSMD3, TP53) for AAs, and extracellular matrix organization (COL4A4, LAMA1, LAMA2, NRXN1) for AIs (Benjamini-Hochberg adjusted p<0.05). Most of the SCGs with race-pronounced variants (35 SCGs out of 42 in whites, 29 of 38 AAs, and 30 of 35 AI) are under-investigated with less than ten CRC-related publications in PubMed. We considered multitudes of factors to select gene(s) for further validation, such as (i) statistical significance, (ii) being a SCG, (iii) type of variant/mutation (variants occurring in open reading frame may be prioritized with an assumption of direct effects on the protein function, compared with likely indirect effects of intron variants), (iv) amount and quality of existing work, and (v) available resources, then proceeded to validate the SCGs with race-pronounced variants. Among the candidate genes, ANGEL2 with Alabama African American-pronounced variant is involved in 3'-UTR-mediated mRNA stabilization and negative regulation of mitotic cell cycle. ANGEL2 expression increases in rat colon tumor (IHC) and in human CRCs in TCGA (vs normal; <0.0001), implicating a role in regulating CRC development. Cancer-influencing genes with minority race-pronounced variants have evaded discovery by being diluted in all-inclusive tumor data analysis. The inclusion of these SCGs with race-pronounced variants in the clinical CRC next-generation sequencing panels and the development of targeting drugs will serve as refinements for precision medicine to overcome racial disparities in health outcomes of CRC. Hiroshi Y. Yamada, Madhusmita Rout, Chao Xu, Gary Sanghera, Surya Singh, Venkateshwar Madka, Farrukh Afaq, Katherine T. Morris, Dharambir K. Sanghera, Upender Manne, Chinthalapally V. Rao. Mutational disparities in colorectal cancers of White Americans, Alabama African Americans, and Oklahoma American Indians revealed ANGEL2 as an African American CRC proneness candidate gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4958.