DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation.

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

radioGWAS links radiome to genome to discover driver genes with somatic mutations for heterogeneous tumor image phenotype in pancreatic cancer.

Addressing the significant level of variability exhibited by pancreatic cancer necessitates the adoption of a systems biology approach that integrates molecular data, biological properties of the tumors, medical images, and clinical features of the patients. In this study, a comprehensive multi-omics methodology was employed to examine a distinctive collection of patient dataset containing rapid autopsy tumor and normal tissue samples as well as longitudinal imaging with a focus on pancreatic cancer. By performing a whole exome sequencing analysis on tumor and normal tissues to identify somatic gene variants and a radiomic feature analysis to tumor CT images, the genome-wide association approach established a connection between pancreatic cancer driver genes and relevant radiomic features, enabling a thorough and quantitative assessment of the heterogeneity of pancreatic tumors. The significant association between sets of genes and radiomic features revealed the involvement of genes in shaping tumor morphological heterogeneity. Some results of the association established a connection between the molecular level mechanism and their outcomes at the level of tumor structural heterogeneity. Because tumor structure and tumor structural heterogeneity are related to the patients' overall survival, patients who had pancreatic cancer driver gene mutations with an association to a certain radiomic feature have been observed to experience worse survival rates than cases without these somatic mutations. Furthermore, the association analysis has revealed potential gene mutations and radiomic feature candidates that warrant further investigation in future research endeavors.

Cancer-associated fibroblast-derived acetate promotes pancreatic cancer development by altering polyamine metabolism via the ACSS2-SP1-SAT1 axis.

The ability of tumour cells to thrive in harsh microenvironments depends on the utilization of nutrients available in the milieu. Here we show that pancreatic cancer-associated fibroblasts (CAFs) regulate tumour cell metabolism through the secretion of acetate, which can be blocked by silencing ATP citrate lyase (ACLY) in CAFs. We further show that acetyl-CoA synthetase short-chain family member 2 (ACSS2) channels the exogenous acetate to regulate the dynamic cancer epigenome and transcriptome, thereby facilitating cancer cell survival in an acidic microenvironment. Comparative H3K27ac ChIP-seq and RNA-seq analyses revealed alterations in polyamine homeostasis through regulation of SAT1 gene expression and enrichment of the SP1-responsive signature. We identified acetate/ACSS2-mediated acetylation of SP1 at the lysine 19 residue that increased SP1 protein stability and transcriptional activity. Genetic or pharmacologic inhibition of the ACSS2-SP1-SAT1 axis diminished the tumour burden in mouse models. These results reveal that the metabolic flexibility imparted by the stroma-derived acetate enabled cancer cell survival under acidosis via the ACSS2-SP1-SAT1 axis.

The MUC1-HIF-1α signaling axis regulates pancreatic cancer pathogenesis through polyamine metabolism remodeling.

Dysregulation of polyamine metabolism has been implicated in cancer initiation and progression; however, the mechanism of polyamine dysregulation in cancer is not fully understood. In this study, we investigated the role of MUC1, a mucin protein overexpressed in pancreatic cancer, in regulating polyamine metabolism. Utilizing pancreatic cancer patient data, we noted a positive correlation between MUC1 expression and the expression of key polyamine metabolism pathway genes. Functional studies revealed that knockdown of spermidine/spermine N1-acetyltransferase 1 (SAT1), a key enzyme involved in polyamine catabolism, attenuated the oncogenic functions of MUC1, including cell survival and proliferation. We further identified a regulatory axis whereby MUC1 stabilized hypoxia-inducible factor (HIF-1α), leading to increased SAT1 expression, which in turn induced carbon flux into the tricarboxylic acid cycle. MUC1-mediated stabilization of HIF-1α enhanced the promoter occupancy of the latter on SAT1 promoter and corresponding transcriptional activation of SAT1, which could be abrogated by pharmacological inhibition of HIF-1α or CRISPR/Cas9-mediated knockout of HIF1A. MUC1 knockdown caused a significant reduction in the levels of SAT1-generated metabolites, N1-acetylspermidine and N8-acetylspermidine. Given the known role of MUC1 in therapy resistance, we also investigated whether inhibiting SAT1 would enhance the efficacy of FOLFIRINOX chemotherapy. By utilizing organoid and orthotopic pancreatic cancer mouse models, we observed that targeting SAT1 with pentamidine improved the efficacy of FOLFIRINOX, suggesting that the combination may represent a promising therapeutic strategy against pancreatic cancer. This study provides insights into the interplay between MUC1 and polyamine metabolism, offering potential avenues for the development of treatments against pancreatic cancer.

Interrogating the Theranostic Capacity of a MUC16-Targeted Antibody for Ovarian Cancer.

Aberrantly expressed glycans on mucins such as mucin-16 (MUC16) are implicated in the biology that promotes ovarian cancer (OC) malignancy. Here, we investigated the theranostic potential of a humanized antibody, huAR9.6, targeting fully glycosylated and hypoglycosylated MUC16 isoforms. Methods: In vitro and in vivo targeting of the diagnostic radiotracer [89Zr]Zr-DFO-huAR9.6 was investigated via binding experiments, immuno-PET imaging, and biodistribution studies on OC mouse models. Ovarian xenografts were used to determine the safety and efficacy of the therapeutic version, [177Lu]Lu-CHX-A″-DTPA-huAR9.6. Results: In vivo uptake of [89Zr]Zr-DFO-huAR9.6 supported in vitro-determined expression levels: high uptake in OVCAR3 and OVCAR4 tumors, low uptake in OVCAR5 tumors, and no uptake in OVCAR8 tumors. Accordingly, [177Lu]Lu-CHX-A″-DTPA-huAR9.6 displayed strong antitumor effects in the OVCAR3 model and improved overall survival in the OVCAR3 and OVCAR5 models in comparison to the saline control. Hematologic toxicity was transient in both models. Conclusion: PET imaging of OC xenografts showed that [89Zr]Zr-DFO-huAR9.6 delineated MUC16 expression levels, which correlated with in vitro results. Additionally, we showed that [177Lu]Lu-CHX-A″-DTPA-huAR9.6 displayed strong antitumor effects in highly MUC16-expressing tumors. These findings demonstrate great potential for 89Zr- and 177Lu-labeled huAR9.6 as theranostic tools for the diagnosis and treatment of OC.

Structural Basis for Multivalent MUC16 Recognition and Robust Anti-Pancreatic Cancer Activity of Humanized Antibody AR9.6.

Mucin-16 (MUC16) is a target for antibody-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC) among other malignancies. The MUC16-specific monoclonal antibody AR9.6 has shown promise for PDAC immunotherapy and imaging. Here, we report the structural and biological characterization of the humanized AR9.6 antibody (huAR9.6). The structure of huAR9.6 was determined in complex with a MUC16 SEA (Sea urchin sperm, Enterokinase, Agrin) domain. Binding of huAR9.6 to recombinant, shed, and cell-surface MUC16 was characterized, and anti-PDAC activity was evaluated in vitro and in vivo. HuAR9.6 bound a discontinuous, SEA domain epitope with an overall affinity of 88 nmol/L. Binding affinity depended on the specific SEA domain(s) present, and glycosylation modestly enhanced affinity driven by favorable entropy and enthalpy and via distinct transition state thermodynamic pathways. Treatment with huAR9.6 reduced the in vitro growth, migration, invasion, and clonogenicity of MUC16-positive PDAC cells and patient-derived organoids (PDO). HuAR9.6 blocked MUC16-mediated ErbB and AKT activation in PDAC cells, PDOs, and patient-derived xenografts and induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. More importantly, huAR9.6 treatment caused substantial PDAC regression in subcutaneous and orthotopic tumor models. The mechanism of action of huAR9.6 may depend on dense avid binding to homologous SEA domains on MUC16. The results of this study validate the translational therapeutic potential of huAR9.6 against MUC16-positive PDACs.

Engrailed-1 Promotes Pancreatic Cancer Metastasis.

Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, it is reported that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating roles in the activation of MAPK pathways and the acquisition of mesenchymal cell properties. Gain- and loss-of-function experiments demonstrated that EN1 promoted PDA transformation and metastasis in vitro and in vivo. The findings nominate the targeting of EN1 and downstream pathways in aggressive PDA.