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.

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.

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.

Investigation of the activity of a novel tropolone in osteosarcoma.

Osteosarcoma (OS) is a primary malignant bone tumor characterized by frequent metastasis, rapid disease progression, and a high rate of mortality. Treatment options for OS have remained largely unchanged for decades, consisting primarily of cytotoxic chemotherapy and surgery, thus necessitating the urgent need for novel therapies. Tropolones are naturally occurring seven-membered non-benzenoid aromatic compounds that possess antiproliferative effects in a wide array of cancer cell types. MO-OH-Nap is an α-substituted tropolone that has activity as an iron chelator. Here, we demonstrate that MO-OH-Nap activates all three arms of the unfolded protein response (UPR) pathway and induces apoptosis in a panel of human OS cell lines. Co-incubation with ferric chloride or ammonium ferrous sulfate completely prevents the induction of apoptotic and UPR markers in MO-OH-Nap-treated OS cells. MO-OH-Nap upregulates transferrin receptor 1 (TFR1) protein levels, as well as TFR1, divalent metal transporter 1 (DMT1), iron-regulatory proteins (IRP1, IRP2), ferroportin (FPN), and zinc transporter 14 (ZIP14) transcript levels, demonstrating the impact of MO-OH-Nap on iron-homeostasis pathways in OS cells. Furthermore, MO-OH-Nap treatment restricts the migration and invasion of OS cells in vitro. Lastly, metabolomic profiling of MO-OH-Nap-treated OS cells revealed distinct changes in purine and pyrimidine metabolism. Collectively, we demonstrate that MO-OH-Nap-induced cytotoxic effects in OS cells are dependent on the tropolone's ability to alter cellular iron availability and that this agent exploits key metabolic pathways. These studies support further evaluation of MO-OH-Nap as a novel treatment for OS.

Vitamin B6 Competition in the Tumor Microenvironment Hampers Antitumor Functions of NK Cells.

Nutritional factors play crucial roles in immune responses. The tumor-caused nutritional deficiencies are known to affect antitumor immunity. Here, we demonstrate that pancreatic ductal adenocarcinoma (PDAC) cells can suppress NK-cell cytotoxicity by restricting the accessibility of vitamin B6 (VB6). PDAC cells actively consume VB6 to support one-carbon metabolism, and thus tumor cell growth, causing VB6 deprivation in the tumor microenvironment. In comparison, NK cells require VB6 for intracellular glycogen breakdown, which serves as a critical energy source for NK-cell activation. VB6 supplementation in combination with one-carbon metabolism blockage effectively diminishes tumor burden in vivo. Our results expand the understanding of the critical role of micronutrients in regulating cancer progression and antitumor immunity, and open new avenues for developing novel therapeutic strategies against PDAC. SIGNIFICANCE: The nutrient competition among the different tumor microenvironment components drives tumor growth, immune tolerance, and therapeutic resistance. PDAC cells demand a high amount of VB6, thus competitively causing NK-cell dysfunction. Supplying VB6 with blocking VB6-dependent one-carbon metabolism amplifies the NK-cell antitumor immunity and inhibits tumor growth in PDAC models. This article is featured in Selected Articles from This Issue, p. 5.

Oral Delivery of Nucleic Acid Therapies for Local and Systemic Action.

Nucleic acid (NA) therapy has gained importance over the past decade due to its high degree of selectivity and minimal toxic effects over conventional drugs. Currently, intravenous (IV) or intramuscular (IM) formulations constitute majority of the marketed formulations containing nucleic acids. However, oral administration is traditionally preferred due to ease of administration as well as higher patient compliance. To leverage the benefits of oral delivery for NA therapy, the NA of interest must be delivered to the target site avoiding all degrading and inhibiting factors during its transition through the gastrointestinal tract. The oral route presents myriad of challenges to NA delivery, making formulation development challenging. Researchers in the last few decades have formulated various delivery systems to overcome such challenges and several reviews summarize and discuss these strategies in detail. However, there is a need to differentiate between the approaches based on target so that in future, delivery strategies can be developed according to the goal of the study and for efficient delivery to the desired site. The goal of this review is to summarize the mechanisms for target specific delivery, list and discuss the formulation strategies used for oral delivery of NA therapies and delineate the similarities and differences between local and systemic targeting oral delivery systems and current challenges.

ENT1 blockade by CNX-774 overcomes resistance to DHODH inhibition in pancreatic cancer.

Inhibitors of dihydroorotate dehydrogenase (DHODH), a key enzyme for de novo synthesis of pyrimidine nucleotides, have failed in clinical trials for various cancers despite robust efficacy in preclinical animal models. To probe for druggable mediators of DHODH inhibitor resistance, we performed a combination screen with a small molecule library against pancreatic cancer cell lines that are highly resistant to the DHODH inhibitor brequinar (BQ). The screen revealed that CNX-774, a preclinical Bruton tyrosine kinase (BTK) inhibitor, sensitizes resistant cell lines to BQ. Mechanistic studies showed that this effect is independent of BTK and instead results from inhibition of equilibrative nucleoside transporter 1 (ENT1) by CNX-774. We show that ENT1 mediates BQ resistance by taking up extracellular uridine, which is salvaged to generate pyrimidine nucleotides in a DHODH-independent manner. In BQ-resistant cell lines, BQ monotherapy slowed proliferation and caused modest pyrimidine nucleotide depletion, whereas combination treatment with BQ and CNX-774 led to profound cell viability loss and pyrimidine starvation. We also identify N-acetylneuraminic acid accumulation as a potential marker of the therapeutic efficacy of DHODH inhibitors. In an aggressive, immunocompetent pancreatic cancer mouse model, combined targeting of DHODH and ENT1 dramatically suppressed tumor growth and prolonged mouse survival. Overall, our study defines CNX-774 as a previously uncharacterized ENT1 inhibitor and provides strong proof of concept support for dual targeting of DHODH and ENT1 in pancreatic cancer.

Design and Evaluation of Short Bovine Lactoferrin-Derived Antimicrobial Peptides against Multidrug-Resistant Enterococcus faecium.

Enterococcus faecium has become an important drug-resistant nosocomial pathogen because of widespread antibiotic abuse. We developed short and chemically simple antimicrobial peptides (AMPs) with a selective amino acid composition, fixed charge, and hydrophobicity ratio based on the core antimicrobial motif of bovine lactoferrin (LfcinB6). Among these peptides, 5L and 6L (both 12 residues long) demonstrated a narrow spectrum and high antibacterial activity against drug-resistant E. faecium isolates with a minimal inhibitory concentration (MIC) that ranged from 4-16 µg/mL. At 32 µg/mL, peptides 5L and 6L inhibited E. faecium strain C68 biofilm formation by 90% and disrupted established biofilms by 75%. At 40 µg/mL, 5L reduced 1 × 107E. faecium persister cells by 3 logs within 120 min of exposure, whereas 6L eliminated all persister cells within 60 min. At 0.5× MIC, 5L and 6L significantly downregulated the expression of a crucial biofilm gene ace by 8 folds (p = 0.02) and 4 folds (p = 0.01), respectively. At 32 µg/mL, peptides 5L and 6L both depolarized the E. faecium membrane, increased fluidity, and eventually ruptured the membrane. Physiologically, 5L (at 8 µg/mL) altered the tricarboxylic acid cycle, glutathione, and purine metabolism. Interestingly, in an ex vivo model of porcine skin infection, compared to no treatment, 5L (at 10× MIC) effectively eliminated all 1 × 106 exponential (p = 0.0045) and persister E. faecium cells (p = 0.0002). In conclusion, the study outlines a roadmap for developing narrow-spectrum selective AMPs and presents peptide 5L as a potential therapeutic candidate to be explored against E. faecium.

Ecdysoneless Overexpression Drives Mammary Tumorigenesis through Upregulation of C-MYC and Glucose Metabolism.

Ecdysoneless (ECD) protein is essential for embryogenesis, cell-cycle progression, and cellular stress mitigation with an emerging role in mRNA biogenesis. We have previously shown that ECD protein as well as its mRNA are overexpressed in breast cancer and ECD overexpression predicts shorter survival in patients with breast cancer. However, the genetic evidence for an oncogenic role of ECD has not been established. Here, we generated transgenic mice with mammary epithelium-targeted overexpression of an inducible human ECD transgene (ECDTg). Significantly, ECDTg mice develop mammary hyperplasia, preneoplastic lesions, and heterogeneous tumors with occasional lung metastasis. ECDTg tumors exhibit epithelial to mesenchymal transition and cancer stem cell characteristics. Organoid cultures of ECDTg tumors showed ECD dependency for in vitro oncogenic phenotype and in vivo growth when implanted in mice. RNA sequencing (RNA-seq) analysis of ECDTg tumors showed a c-MYC signature, and alterations in ECD levels regulated c-MYC mRNA and protein levels as well as glucose metabolism. ECD knockdown-induced decrease in glucose uptake was rescued by overexpression of mouse ECD as well as c-MYC. Publicly available expression data analyses showed a significant correlation of ECD and c-MYC overexpression in breast cancer, and ECD and c-MYC coexpression exhibits worse survival in patients with breast cancer. Taken together, we establish a novel role of overexpressed ECD as an oncogenesis driver in the mouse mammary gland through upregulation of c-MYC-mediated glucose metabolism. IMPLICATIONS: We demonstrate ECD overexpression in the mammary gland of mice led to the development of a tumor progression model through upregulation of c-MYC signaling and glucose metabolism.

Hypoxia-Driven Oncometabolite L-2HG Maintains Stemness-Differentiation Balance and Facilitates Immune Evasion in Pancreatic Cancer.

In pancreatic cancer, the robust fibroinflammatory stroma contributes to immune suppression and renders tumors hypoxic, altering intratumoral metabolic pathways and leading to poor survival. One metabolic enzyme activated during hypoxia is lactate dehydrogenase A (LDHA). As a result of its promiscuous activity under hypoxia, LDHA produces L-2 hydroxyglutarate (L-2HG), an epigenetic modifier, that regulates the tumor transcriptome. However, the role of L-2HG in remodeling the pancreatic tumor microenvironment is not known. Here we used mass spectrometry to detect L-2HG in serum samples from patients with pancreatic cancer, comprising tumor cells as well as stromal cells. Both hypoxic pancreatic tumors as well as serum from patients with pancreatic cancer accumulated L-2HG as a result of promiscuous activity of LDHA. This abnormally accumulated L-2HG led to H3 hypermethylation and altered gene expression, which regulated a critical balance between stemness and differentiation in pancreatic tumors. Secreted L-2HG inhibited T-cell proliferation and migration, suppressing antitumor immunity. In a syngeneic orthotopic model of pancreatic cancer, inhibition of LDH with GSK2837808A decreased L-2HG, induced tumor regression, and sensitized tumors to anti-PD1 therapy. In conclusion, hypoxia-mediated promiscuous activity of LDH produces L-2HG in pancreatic tumor cells, regulating the stemness-differentiation balance and contributing to immune evasion. Targeting LDH can be developed as a potential therapy to sensitize pancreatic tumors to checkpoint inhibitor therapy. SIGNIFICANCE: This study shows that promiscuous LDH activity produces L-2HG in pancreatic tumor and stromal cells, modulating tumor stemness and immune cell function and infiltration in the tumor microenvironment.

USP11 mediates repair of DNA-protein cross-links by deubiquitinating SPRTN metalloprotease.

DNA-protein cross-links (DPCs) are toxic DNA lesions that interfere with DNA metabolic processes such as replication, transcription, and recombination. USP11 deubiquitinase participates in DNA repair, but the role of USP11 in DPC repair is not known. SPRTN is a replication-coupled DNA-dependent metalloprotease that cleaves proteins cross-linked to DNA to promote DPC repair. SPRTN function is tightly regulated by a monoubiquitin switch that controls SPRTN auto-proteolysis and chromatin accessibility during DPC repair. Previously, VCPIP1 and USP7 deubiquitinases have been shown to regulate SPRTN. Here, we identify USP11 as an SPRTN deubiquitinase. USP11 interacts with SPRTN and cleaves monoubiquitinated SPRTN in cells and in vitro. USP11 depletion impairs SPRTN deubiquitination and promotes SPRTN auto-proteolysis in response to formaldehyde-induced DPCs. Loss of USP11 causes an accumulation of unrepaired DPCs and cellular hypersensitivity to treatment with DPC-inducing agents. Our findings show that USP11 regulates SPRTN auto-proteolysis and SPRTN-mediated DPC repair to maintain genome stability.