DPAGT1 Inhibition as a Mechanism-Based Strategy for Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a rare, aggressive, and generally lethal human malignancies, with a five-year survival rate of 13.7% [1] poorer when diagnosed advanced metastatic which is a majority of cases. Standard regimens such as FOLFIRINOX with gemcitabine & nab-paclitaxel provide only modest benefit, largely due to early metastasis, profound intrinsic immune checkpoint & chemoresistance [2,3] and enrichment of cancer stem cells (CSCs) [4-5] that further reinforce these hallmark oncogenic behaviors. Increasing evidence implicates aberrant N-linked glycosylation, driven by overexpression of dolichylphosphate N-acetylglucosamine-phosphotransferase 1 (DPAGT1), as a “central molecular hub” connecting synergistic, hallmark cancer characteristics that include the epithelial-to-mesenchymal transition (EMT) driven invasion, immune evasion, CSC effects, and multidrug resistance in PDAC [8-13].

DPAGT1 is an essential enzyme of post-translational protein synthesis. DPAGT1 is the first, rate limiting step in the attachment of complex oligosaccharides to asparagine residues to facilitate proper protein folding, function, and location in a process known as N-linked glycosylation. Upregulated DPAGT1 expression has been identified as important step in the evolution of several cancers, including PDAC [9-12]. Importantly, these effects are reversible with the application of DPAGT1 inhibitors, however, the severe toxicity of these compounds limits their clinical utility [8-13]. In PDAC, DPAGT1 inhibition confers profound, multi-pathway effects and the discovery of safer alternatives represents an important research objective. These effects include the ability to suppress EMT [10-12], disrupt downstream KRAS proliferative pathways [8] and potentiate chemotherapies at substantially lower dosages [10-12]. The FDA has validated the importance of this multimechanistic approach by awarding DPAGT1 inhibitor aminouridyl phenoxypiperidinbenzyl butanamide (APPB - renamed ANV221) Orphan Drug Designation for the treatment of pancreatic cancer [14] due to its high efficacy and strong safety signals in vivo [12].

DPAGT1 catalyzes the first committed step of N-glycan biosynthesis, transferring GlcNAc-1-phosphate from UDP-GlcNAc to dolichyl-phosphate [15]. Overexpression of DPAGT1 has been documented in multiple solid tumours and in pancreatic cancer models [16], where high DPAGT1 correlates with increased migratory and invasive behaviour [8,9]. Mechanistically, overexpression of DPAGT1 increases complex N glycans on E cadherin, reduces its membrane localization, and promotes cellular discohesion and a more mesenchymal morphology. This releases sequestered β-catenin which accumulates in the nucleus, activating the canonical Wnt pathway followed by transcription of EMT drivers (i.e. Snail) that further degrades E-cadherin in an oncogenic, positive feedback loop [17-19].

In PDAC, EMT is characterized by loss of E-cadherin, gain of N-cadherin and vimentin, and activation of MMP-2/-9. Snail, ZEB, and Twist also act as core transcriptional regulators of this program, and Snail expression in particular has also been linked to drug resistance, recurrence, and poor prognosis [21]. We suggest that DPAGT1 overactivity in PDAC contributes to Snail upregulation via Wnt/β-catenin and destabilization of epithelial adhesion, thereby coupling N-glycosylation directly to EMT, metastasis, CSC sustainment, and drug resistance. CSC biology amplifies this pathology. CD133-positive CSCs preferentially express ABC transporters, especially ABCG2, which effluxes cytotoxic drugs (gemcitabine & cisplatin) [13], along with survival pathways (e.g. PI3K/Akt) in PDAC [22]. Chemotherapy agents such as cisplatin and 5-fluorouracil can paradoxically enrich CSCs in some cancers, increasing stemness markers (OCT4, Nanog, Notch1) and ABCG2 expression in another oncogenic positive feedback loop [23,24]. Thus, any effective and durable PDAC therapy must confront EMT and CSCs together, not as separate issues. Kurosu et al found that inhibition of DPAGT1 in PDAC resulted in normalization of mesenchymal phenotypes and restoration of E-cadherins leading to the sequestration of β-catenin and the general disruption of this positive feedback loop [10,12,20]. This is an important signal that the EMT/CSS programs in PDAC can be simultaneously disrupted therapeutically.

The major MDR-related ABC transporters, ABCB1, ABCC1, and ABCG2, are heavily N-glycosylated and require glycan structures for proper folding, membrane trafficking, and stability [13]. Experimental disruption of N-glycosylation by tunicamycin or other inhibitors retains ABC transporters in intracellular compartments and accelerates their proteasomal degradation, leading to increased intracellular drug accumulation and partial reversal of the multidrug resistance (MDR) phenotype [13]. In PDAC and hepatocellular carcinoma, ABCG2 is preferentially expressed in CD133-positive CSCs, and its activity is modulated by PI3K/Akt signaling and N-glycosylation [13,25].

Hou and colleagues demonstrated that tunicamycin reduced ABCG2 protein levels, altered its subcellular localization, decreased the side-population fraction, and synergized with cisplatin and 5-fluorouracil to suppress tumor growth in xenograft models via the DPAGT1–Akt–ABCG2 pathway [5]. DPAGT1 knockdown reproduced the chemo-sensitizing effect of tunicamycin, confirming that DPAGT1 is a tractable molecular target [13]. Study of DPAGT1 inhibitors effects on ABC transporters in PDAC is a provocative line of inquiry for this intensely chemo-resistant disease.

KRAS is the most frequently upregulated oncogenic driver in PDAC, >85% of diagnosis [26]. In PDAC, KRAS mutations activate several cooperatively proliferative downstream programs PI3K– Akt–mTOR, PI3K-Akt-mTOR, releasing the eukaryotic translation initiation factor 4E (eIF4E) resulting in sustained proliferative signaling [27]. KRAS was long considered undruggable and a viable strategy is to limit the downstream effects of KRAS mutations. DPAGT1 inhibition is a compelling strategy against KRAS based on its known effects against these downstream pathways. Further, DPAGT1 inhibitors are known to increase ER stress and induce apoptosis in high DPAGT1 expressing cells. KRAS cells are already under chronic metabolic stress, thus DPAGT1 inhibition potentially amplifies apoptotic effects in KRAS cancers [28,29].

The acquisition of immune evasion behaviors is an important evolutionary hallmark for many cancers. Immune checkpoint blockade (ICB) is a proven approach to activate the immune responses against these cancers. This involves the disruption of the PD-1/PDL1 interaction that prevents T-cell activation through therapeutic inhibition of PD-1 or its ligand PD-L1. PDAC is stubbornly refractory to immune checkpoint blockade therapeutics and N-glycosylation of the PD-L1 is a compelling hypothesis. N-glycosylation can be an important oncological strategy for securing oncogenic proteins that are normally degraded. For example, in HER2-positive breast cancer it was found that heavily glycosylated ADAM10 proteases allowed them to survive degradation, migrate to the cell surface, and cleave the HER2 receptor from the HER2 protein, in a process known as HER2 shedding, by depriving anti-HER2 drugs with a receptor to attach, HER2-positive breast cancers become resistant followed by aggressive recurrence [30].

This oncogenic strategy is also found with PD-1 binding. Researchers (Li et al) found that heavily glycosylated programmed death ligand (PD-L1) on tumor surfaces improved their binding with the corresponding PD-1 receptors resulting in immune evasion [31]. Importantly, both of these effects were reversible with the application of DPAGT1 inhibitors. This evidence suggests DPAGT1 inhibition as a potential strategy in ICB refractory PDAC to resensitize PDAC tumours to a robust immune response.

In various studies, tunicamycin provided the initial proof-ofconcept that DPAGT1 inhibition can attenuate EMT, CSC markers, and drug resistance. However, its systemic toxicity (mouse LD50 approximately 2.0 mg/kg single dose) and lack of selectivity for tumor versus normal cells have precluded clinical development of tunicamycin and similarly toxic substitutes. Further, tunicamycin displays low IC50 values across both cancer and normal cell lines, causes significant hepatotoxicity and nephrotoxicity, and exhibits hemolytic activity, reflecting broad disruption of membranes and additional off-target effects beyond DPAGT1 inhibition [9-12,20]. Structure-based design against DPAGT1 has yielded capuramycinderived and muramycin-derived analogues with markedly improved selectivity [10,11]. Mitachi et al. reported inhibitors such as CPPB and APPB (ANV221) that inhibit DPAGT1 with sub-micromolar IC50 values, yet show minimal cytotoxicity toward healthy cells, even at 70X concentrations [10,11].

APPB’s water solubility, low hemolytic activity, and negligible hemolysis compare favorably against other DPAGT1 inhibitors while mouse LD50 values are vastly improved at least ten-fold, indicating a widened therapeutic window and realistic potential for systemic administration [12,20]. Structural modelling suggests these next-generation inhibitors occupy DPAGT1’s dolicholphosphate tunnel and UDP-GlcNAc binding site with higher specificity than tunicamycin, thereby further reducing off-target membrane disruption.

Importantly for PDAC, DPAGT1 inhibitors show robust synergy with standard chemotherapies. Our research has confirmed that APPB synergizes with paclitaxel in patient-derived metastatic PDAC cells, reducing the IC50 of paclitaxel by more than two orders of magnitude in checkerboard assays [12,20]. DPAGT1 inhibition has been further shown to potentiate cisplatin and 5-fluorouracil activity in hepatocellular carcinoma and reversing chemotherapyinduced upregulation of stemness genes [13]. The mechanistic basis of this synergy is clear: Safer DPAGT1 inhibition is a viable strategy to blunt CSC survival, prevent EMT-driven dissemination, and counteract chemotherapy-induced CSC enrichment, thereby transforming cytotoxic drugs from transient debulking agents into more durable treatments.

Anviron, in partnership with academic institutions, has exclusively licensed a family of low toxicity DPAGT1 inhibitors (i.e. APPB & CPPB) for development in orphan oncology indications [12]. The United States FDA granted Orphan Drug Designation to APPB (ANV221) for the treatment of pancreatic cancer [14], recognizing both the rarity and severity of PDAC and the plausibility of DPAGT1 inhibition as a novel therapeutic strategy.

Collectively, available data support DPAGT1 as a high-value, underexploited target in PDAC. Selective small-molecule DPAGT1 inhibitors offer the potential to suppress EMT and metastasis via Snail/Wnt/E-cadherin modulation, deplete CSCs, reverse multidrug resistance through the DPAGT1–ABCG2 axis, enabling synergies with existing chemotherapies, particularly paclitaxel, cisplatin, and gemcitabine, and actuating immune vulnerability via PD-L1 binding disruption, and potentially disrupting downstream effects of the historically undruggable KRAS; achieving this with a promising safety profile. With FDA Orphan Drug validation, APPB/ ANV221 is in the pole position to make a transformative leap in the fight against one of medicine’s most difficult to treat diseases.

None.

I’m the Founder and CEO of Anviron, we own the licenses for the drugs we mention in the article. Michio Kurosu is on our Board of Advisors.

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