Abstract Proliferating cell nuclear antigen (PCNA) plays essential roles in DNA replication, repair, transcription, and cell-cycle regulation, making it an attractive yet historically difficult target in cancer therapy. Building on our development of AOH1996, a first-in-class small molecule currently in Phase I clinical evaluation for solid and liquid tumors, we sought to identify additional PCNA-interacting chemotypes that could expand therapeutic opportunities and inform next-generation inhibitor design. Using an artificial intelligence-guided computer-aided drug discovery workflow, we screened more than ten million drug-like compounds and prioritized candidates through differential scanning fluorimetry. This approach led to the identification of COH005, a previously unreported small-molecule scaffold that binds PCNA. X-ray crystallography demonstrated that COH005 engages the major PCNA-interacting-protein (PIP)-box binding pocket, a critical interface for PCNA-mediated protein-protein interactions. Cellular thermal shift assays (CETSA) validated direct COH005-PCNA engagement in cells similar to clinical IND, AOH1996. To assess selectivity, we performed protein-structure frustration analysis comparing COH005 interactions with PCNA against an unrelated anti-target, revealing energetically favorable binding unique to PCNA. Together, these studies establish COH005 as a novel PCNA-interacting scaffold with strong potential for therapeutic development. This work provides a structural and mechanistic foundation for designing next-generation PCNA-targeted agents with improved specificity and pharmacological properties. Citation Format: Jennifer Jossart, Ning Ma, Pouya Haratipour, Caroline Li, Long Gu, Terrence O'Brien, Nagarajan Vaidehi, Linda H. Malkas, Robert Hickey, Jeff J. Perry. Identification and structural characterization of a novel PCNA-interacting small molecule scaffold [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 5124.

AOH1996 disrupts centrosome organization and spindle integrity. A, Representative immunofluorescence microscopy images of HFK, HeLa, and CaSki cells treated with DMSO or 1 μmol/L AOH1996 for 24 hours, stained for β-tubulin (red), γ-tubulin (green), and DNA (DAPI, blue). In DMSO-treated cells, mitotic spindles and centrosomes appear organized and bipolar. In contrast, AOH1996-treated cells exhibit multipolar, shortened, and disorganized spindles with supernumerary centrosomes. B, Quantification of spindle length shows a significant reduction in AOH1996-treated cells across all lines, confirming spindle collapse or failure. C, Quantification of centrosome number per cell reveals a significant increase following AOH1996 treatment, most pronounced in cervical cancer (CaCx) cells. D, Chemical structures of AOH1996 and its structurally related analog AOH1996-8Nq, which contains a nitrogen substitution in the aromatic ring and lacks cytotoxic or mitosis-disrupting activity. E, Protein thermal shift assay of recombinantly expressed 6xHis PCNA with AOH1996-8Nq. A PCNA-stabilizing shift of 0.5°C at each concentration indicates binding interaction. F, Brightfield and immunofluorescence microscopy images of HeLa cells treated with 200 nmol/L AOH1996 or AOH1996-8Nq. AOH1996 causes cell rounding and centrosome/spindle disruption, whereas AOH1996-8Nq does not. G, Immunofluorescence microscopy of β-tubulin (red), γ-tubulin (green), and DAPI (blue) in HeLa cells. AOH1996 significantly decreases colocalization between γ-tubulin and β-tubulin, as shown by quantification (right). AOH1996-8Nq has no effect. Bottom, pixel-based colocalization maps show overlapping γ/β-tubulin signal (white). H, Western blot analysis of centrosome components (pericentrin and γ-tubulin), spindle protein (β-tubulin), and mitotic marker (p-H3) from HeLa cells treated with DMSO, AOH1996, or AOH1996-8Nq. AOH1996 decreases centrosomal protein levels and increases p-H3 abundance. AOH1996-8Nq does not alter these levels. Nucleolin serves as a loading control. Bar graph represents corresponding quantification. Bar graphs represent the mean ± SEM from three independent experiments. Statistical comparisons were performed using two-way ANOVA. ****, P < 0.0001; ***, P < 0.001; **, P < 0.0001.

With over 27,100 papers currently written about Proliferating Cell Nuclear Antigen (PCNA) and more than 11,700 papers describing a role for the protein and its relationship to cancer, this chapter will just touch upon some of the important role's PCNA has within cells and what types of proteins might interact with PCNA. This chapter is not meant to provide a thorough review of the multitude of protein complexes utilizing PCNA as a cofactor but rather highlight only some of those direct or indirect interactions between PCNA and its binding partners which together are involved in maintaining the genome, regulating cytoplasmic activities, and participating in immune surveillance. Additionally, this chapter will provide a high-level overview of how the caPCNA isoform is being used as a platform for a new class of anticancer therapeutic agent that selectively kills cancer cells, while leaving non-cancer cells unharmed.

AOH1996 binds to tubulin and disrupts PCNA–γ-tubulin interactions. A, Tubulin polymerization assay performed in vitro using purified tubulin. AOH1996 partially inhibits polymerization relative to DMSO and paclitaxel (positive control) but more effectively than the inert analog AOH1996-8Nq and the microtubule-depolymerizing agent colcemid (negative control). B, SPR analysis of AOH1996 and AOH1996-8Nq binding to purified tubulin. Sensorgrams (top) and fitted binding curves (bottom) demonstrate that AOH1996 binds tubulin with higher affinity than AOH1996-8Nq. C, Apparent dissociation constants (Kd) derived from SPR data. AOH1996 binds tubulin with a mean Kd of ∼30 μmol/L, whereas AOH1996-8Nq shows modest, yet significantly weaker, binding with a mean Kd of ∼70 μmol/L. Graphs represent the mean and range of nine independent experiments. D, Co-immunoprecipitation (co-IP) of endogenous PCNA from HeLa lysates treated with or without AOH1996 or AOH1996-8Nq, followed by immunoblotting for α-, β-, or γ-tubulin. AOH1996 treatment reduces the interaction between PCNA and γ-tubulin by ∼50%, whereas AOH1996-8Nq has no effect. Normal rabbit IgG was used as a negative IP control. Right, quantification of γ-tubulin co-IP signal normalized to DMSO. Bar graphs represent the mean and range from at least two biological replicates. E, Dose–response curve from MTT assay shows reduced impact of AOH1996-8Nq (IC50 value not reached) on HeLa viability compared with AOH1996-treated (IC50 = 125 nmol/L) cells. Statistical comparisons were made using unpaired two-tailed t tests. **, P < 0.01.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy driven predominantly by oncogenic KRAS mutations. Proliferating cell nuclear antigen (PCNA) is a ring-shaped clamp protein that encircles DNA and regulates replication, repair, and resolution of transcription-replication conflicts; processes hyperactivated in PDAC. AOH1996 is a first-in-class, selective PCNA inhibitor currently in Phase I clinical trials. PCNA has predicted synthetic lethal interactions with KRAS, suggesting combination potential with emerging KRAS inhibitors. This study evaluated AOH1996 alone and in combination with KRAS-targeted agents in PDAC models. Methods: In this study, we investigated the use of AOH1996 in preclinical models of KRAS-mutant PDAC. We determined cell viability and growth inhibition by MTT, colony formation and spheroid assays. Apoptosis and the cell cycle were analyzed by flow cytometry. RNA-seq, RT-qPCR and western blot were performed for mechanistic evaluations. Drug combination synergy modeling was performed using SynergyFinder. In vivo efficacy was assessed in PDAC xenograft models treated with AOH1996, KRAS inhibitors (MRTX1133, sotorasib, and RMC-6236), or combinations. Residual tumors were analyzed for pERK and pAKT signaling changes. Results: AOH1996 showed potent, dose-dependent cytotoxicity in multiple PDAC cell lines and 3D spheroids (IC50: 0.5-1.5 μM). RNA-seq revealed broad transcriptional alterations, with enrichment of MAPK, PI3K-Akt and Hippo signaling pathways. Across KRAS G12C and G12D models, AOH1996 exhibited strong synergy with KRAS inhibitors, including MRTX1133, sotorasib, adagrasib, and RMC-6236. Combination therapy caused marked G1 and G2/M phase arrest, increased Annexin V-positive apoptosis, and dual suppression of pERK and pAKT. In patient-derived tumoroids, AOH1996 plus RMC-6236 significantly reduced viability compared to single agents. In vivo, AOH1996 combined with MRTX1133 or with sotorasib produced robust tumor regressions with no significant weight loss, supporting tolerability. Conclusions: AOH1996 is a promising therapeutic candidate for PDAC, demonstrating potent single-agent activity and strong synergy with clinically relevant KRAS inhibitors across in vitro, ex vivo, and in vivo models. The combination induces profound apoptotic and cell-cycle effects and disrupts key KRAS effector pathways. These results support further translational development of AOH1996-based combination regimens for patients with KRAS-mutant PDAC. Citation Format: Sahar F. Bannoura, Husain Y. Khan, Md Hafiz Uddin, Amro Aboukameel, Yin Wan, Bin Bao, Adeeb Aboukameel, Rafic Beydoun, Pouya Haratipour, Long Gu, Muhammad Wasif Saif, Robert J. Hickey, Linda H. Malkas, Yang Shi, Mohammed Najeeb Al Hallak, Ramzi M. Mohammad, Boris C. Pasche, Asfar S. Azmi. Novel PCNA inhibitor AOH1996 synergizes with KRAS-targeted therapies in pancreatic ductal adenocarcinoma [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 4580.

Abstract Acute myeloid leukemia or AML encompasses diverse molecular subtypes driven by distinct transcriptional and genetic programs that influence therapeutic response. AOH1996 which is a PCNA-dependent replication stress pathway inhibitor has demonstrated preclinical activity in AML. However biomarkers that may predict sensitivity across heterogeneous AML subgroups remain undefined. We first applied integrative multiomic analysis to begin defining the molecular contexts in which AOH1996 may be most effective. Transcriptomic, genomic, and proteomic datasets from PRISM, DepMap, and other curated subtype annotations were integrated with AOH1996 response metrics also known as AUC. Initial analyses focused on quantitative assessment of MYC RNA expression relative to AOH1996 sensitivity using a quadrant based visualization framework, and ongoing multi-omic analyses incorporating mutational backgrounds. Pathway activity scores, and protein level features are being used to explore broader biomarker patterns across AML models. Guided by these computational findings we are developing complementary in vitro studies in a panel of molecularly annotated AML models to functionally probe AOH1996 response, with planned readouts broadly focused on cell growth, cell cycle behavior, and stress associated phenotypes consistent with replication stress and innate immune pathway engagement. Preliminary computational results reveal subtype dependent variation in the relationship between MYC expression and AOH1996 response which aligns with MYC’s established role in driving transcriptional load and replication stress and nominating MYC expression as a biologically plausible candidate biomarker. Together, this integrated multiomics and emerging experimental framework supports the feasibility of biomarker-guided evaluation of AOH1996 across AML subtypes and provides a foundation for future translational studies aimed at molecularly informed therapeutic stratification. Citation Format: Hamsini Kala, Dana Abou Abbas, Robert J. Hickey, Linda H. Malkas, Caroline M. Li, Robert G. Lingeman, Guido Marcucci, Le Xuan Truong Nguyen, Brian Ball, Amanda Blackmon, Melissa Ronan, Jennifer Roth, Matthew G. Rees. Integrative multiomics and functional studies to identify biomarkers of AOH1996 sensitivity across AML subtypes [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 3749.

AOH1996 synergizes with cisplatin in vivo and reduces cisplatin-associated toxicity. A, HeLa xenograft tumor volumes after monotherapy with mock treatment (black), cisplatin (blue), or AOH1996 (red). B, Corresponding mouse body weights. C, Kaplan–Meier survival analysis of mice treated with vehicle control, 8 mg/kg cisplatin (blue), or 100 mg/kg AOH1996 (red). Both AOH1996 and cisplatin significantly reduce tumor growth and extend survival, though cisplatin is more effective. D, Volume analysis of HeLa xenografts treated with varying doses of AOH1996 reveals that doses below 50 mg/kg exhibit limited monotherapy efficacy. E, Determination of subtherapeutic cisplatin dose. HeLa xenograft volumes of mock (black), subtherapeutic 4 mg/kg (beige), and therapeutic 8 mg/kg (gray) are shown. F, HeLa xenograft volume measurements in mice treated with vehicle, therapeutic-dose cisplatin (8 mg/kg), subtherapeutic-dose cisplatin (4 mg/kg), or the combination of 25 mg/kg AOH1996 and 4 mg/kg cisplatin. G, CaSki xenograft volume measurements in mice treated with vehicle, therapeutic-dose cisplatin (8 mg/kg), subtherapeutic-dose cisplatin (4 mg/kg), or the combination of 25 mg/kg AOH1996 and 4 mg/kg cisplatin. H, Survival analysis of HeLa xenograft–bearing mice. Cutoff set at 290 mm3. I, Survival analysis of CaSki xenograft–bearing mice. J, Final tumor weights at necropsy from HeLa xenografts. K, Final tumor weights at necrophsy from CaSki xenografts. L, Relative body weights of HeLa xenograft–bearing mice throughout treatment. M, Relative body weights of CaSki xenograft–bearing mice throughout treatment. N, Urinary NGAL, a marker of kidney injury, levels in mice treated as indicated. O, Uric acid levels in mice treated as shown. P, Kidney weight at necrophsy in mice treated as indicated. Data represent the mean ± SEM from at least eight mice per group. Statistical comparisons were made using two-way ANOVA for tumor volumes and NGAL levels and log-rank (Mantel–Cox) tests for survival. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Cisplatin pretreatment impairs AOH1996 efficacy by blocking mitotic entry in cervical cancer cells.

AOH1996 induces mitotic arrest and mitotic death in cervical cancer cells. A, Representative images of nuclear phenotypes observed by live-cell imaging of HFK, HeLa, and HeLa POLη cells stably expressing GFP-LaminB1 (green) and mCherry-H2B (red). Cells were classified as normal, mitotic, irregular, or dead based on nuclear envelope integrity and chromatin morphology. Scale bars = 5 μm. B, Time-lapse microscopy of cells treated with 1 μmol/L AOH1996 or DMSO control. HFK, HeLa, and HeLa POLη cells undergo timely mitotic progression in control conditions. AOH1996 treatment causes extended mitotic arrest and death, particularly in transformed cells. Scale bars = 10 μm. C, Relative cell growth over a 72-hour imaging window. AOH1996 halts proliferation in HFKs and leads to significant cell loss in HeLa and HeLa POLη cells. D, Quantification of time spent in mitosis. AOH1996 significantly prolongs mitosis in all lines, with the greatest delay observed in HeLa POLη cells. E, Time-course of mitotic entry following AOH1996 treatment. All cell types enter mitosis at similar rates. F, Analysis of mitotic outcomes. HFKs primarily undergo single-cell mitotic exit (∼71%), whereas mitotic death is the predominant fate in HeLa and HeLa POLη cells (∼70% to 74%). G, Temporal distribution of single-cell mitotic exits. Most events occur between 20 and 30 hours after treatment in HFKs. H, Temporal profile of mitotic death. HeLa and HeLa POLη cells undergo mitotic death between 30 and 50 hours after treatment. Data represent the mean ± SEM from three independent experiments. Statistical analysis was performed using one-way or two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Abstract Background: AOH1996 is a small molecule that selectively targets the cancer-associated isoform of proliferating cell nuclear antigen (caPCNA), disrupting DNA replication and repair, and promoting DNA strand breaks through inhibition of effective resolution of transcription-replication conflicts. Building on its promising preclinical efficacy, we are also developing and testing next-generation caPCNA inhibitors with improved potency and druglikeness. We have developed two new inhibitors that exhibit up to 10-fold higher cellular potency relative to the parent compound, and an inhibitor with similar potency as the parent compound but 2.4-fold more soluble. Given PCNA’s diverse role in processes that cancer cells depend upon for survival, e.g., coordinating DNA synthesis, damage tolerance, cell cycle control, chromatin assembly, and stress response signaling, it is hard to predict what other anticancer drugs would combine productively with AOH1996 and next-generation inhibitors. To that end, we screened a panel of 179 anticancer drugs with AOH1996 and three next-generation inhibitors to identify potential combinations that exhibit synergy. Methods: A systematic combination drug screen was performed using AOH1996 and next-generation inhibitors and a drug panel of 179 different anticancer drugs. Synergy evaluation: We performed initial screens using the human neuroblastoma cell line SK-N-AS. We assessed the synergy of the combinations using the Chou-Talalay method for drug combination analysis. For favorable drug combinations, we tested additional cell lines including cancer cell types where the drug identified would be used clinically in a treatment regimen. Mechanistic studies: We analyzed cell cycle effects and apoptosis by flow cytometry. We also assessed DNA damage and checkpoint activation by using immunoblotting and immunofluorescence to measure γH2AX, cleaved PARP, and phospho-Chk1. Results: Both AOH1996 and its next-generation analogs demonstrated synergy with some DNA-damaging and repair inhibiting agents such as cisplatin. Tyrosine kinase inhibitors (TKIs) in particular EGFR TKIs synergized well with AOH1996 and next-generation inhibitors. Inhibitors of the mitochondrial apoptosis pathway (e.g., venetoclax) also synergized as well. Conclusions: AOH1996 and its next-generation analogs exhibit potent synergistic activity with targeted therapies and DNA repair inhibitors, supporting PCNA inhibition as a promising strategy to enhance therapeutic efficacy and overcome resistance. These findings provide a preclinical rationale for clinical development of AOH1996 combination regimens, as well as continued optimization of caPCNA-targeting analogs with superior potency and tolerability. Citation Format: Robert G. Lingeman, Long Gu, Caroline Li, Pouya Haratipour, Robert J. Hickey, Linda H. Malkas, Maryam Zangi. Antitumor synergy of AOH1996 and next-generation caPCNA Inhibitors in combination with targeted and chemotherapeutic agents [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 6506.

AOH1996 synergizes with cisplatin to enhance cytotoxicity in cervical cancer cells. A, Dose–response matrix of AOH1996 and cisplatin combinations in HFKs, HeLa, and CaSki cells measured by MTT viability assay. Heatmaps show normalized cell viability after 96 hours of treatment. Green indicates reduced viability, and red indicates higher survival. B, Synergy contour maps generated from matrix data in A, analyzed using SynergyFinder 3.0. A synergy score >10 indicates synergy, and <−10 indicates antagonism. C, AOH1996 and cisplatin were strongly synergistic in HeLa, CaSki, and HFK 16E6E7 cells antagonistic in HFKs. Table denotes absolute synergy scores. D, Flow cytometry histogram of HeLa cells treated with 1 μmol/L AOH1996 followed by 1 μmol/L cisplatin at indicated time points. Increased sub-G1 peak at 72 and 96 hours indicates enhanced cell death. E, Quantification of cell cycle profiles confirms that AOH1996 pretreatment followed by cisplatin induces a G2/M arrest at 48 hours and a progressive increase in the sub-G1 population at 72 and 96 hours, consistent with apoptotic cell death. Bar graphs show the mean ± SEM from three independent experiments. Statistical analysis was performed using two-way ANOVA. ****, P < 0.0001.

Xenograft Sizes, Study Arms and Treatment Regimen.

Abstract Proliferating cell nuclear antigen (PCNA) is a key regulator of at least 11 essential cellular processes, including DNA replication and repair, transcription, cell cycle progression, apoptosis induction, transcription-replication conflict resolution, energy metabolism, chromosome maintenance, mitosis, and immune surveillance. In tumor cells, PCNA exists as a cancer-associated acidic isoform (caPCNA), while non-malignant cells express only a basic isoform (nmPCNA). The absence of caPCNA in normal cells makes it an attractive target for selective cancer therapy. We previously developed AOH1996, a first-in-class small molecule that selectively inhibits caPCNA, inducing apoptosis in cancer cells without detectable toxicity in non-malignant cells. AOH1996 binds to caPCNA, stabilizes its trimeric structure, decreases chromatin association, and suppresses tumor cell proliferation. AOH1996 is orally bioavailable and is currently being evaluated in two first-in-human Phase I clinical trials: one for treatment refractory solid tumors (NCT05227326) and the other for acute myeloid leukemia (NCT06763341). No dose-limiting toxicities have been observed to date, with ongoing dose escalation currently reaching 1110 mg PO BID. In this study, we aimed to enhance the aqueous solubility and pharmacokinetic properties of AOH1996 through strategic single-point heteroatom substitutions. By replacing selected aromatic carbons with nitrogen atoms, we generated a series of quinoline, isoquinoline, and pyridine analogues optimized for liquid oral formulations that will be ultimately compatible with pediatric administration for AML. Structure-activity relationship (SAR) analyses across these three heterocyclic scaffolds identified several analogues with improved solubility, potency, selectivity, and metabolic stability when compared to the clinical lead. For example, introducing two nitrogen atoms into the aromatic regions of one analogue reduced the CLogP to 3.35 (vs. 4.46 for AOH1996) while maintaining IC50 values comparable to the parent compound. This reduction in lipophilicity corresponds to an estimated ∼13-fold increase in aqueous solubility, making this analogue more amenable to liquid formulation for oral administration. Over 30 novel AOH analogues were synthesized and evaluated for antiproliferative activity across eight tumor cell lines (including Molm-13, THP-1, HTB-0225, SK-N-AS, HCT116, U251, and MDA-MB-468), as well as in AML mouse models. Synthetic routes, biological data, and preliminary in vivo results will be presented. Together, these findings lay the groundwork for the next generation of AOH-based therapeutics with improved physicochemical and pharmacological properties suitable for pediatric oncology applications. Citation Format: Pouya Haratipour, Melissa Valerio, Michaela R. Jacobs, Maryam Zangi, Long Gu, Caroline Li, Robert Lingeman, Jennifer Jossart, Jefferson J. Perry, Le Xuan Truong Nguyen, Linda H. Malkas, Robert J. Hickey. Development of hydrophilic and metabolically stable heterocyclic AOH1996 analogues for pediatric AML oral liquid formulations [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 5140.

AOH1996 selectively induces apoptosis in cervical cancer (CaCx) cells. A, Dose–response curves of AOH1996 in primary HFKs, HeLa (HPV18+), CaSki (HPV16+), C33A (HPV−), and HeLa cells overexpressing POLη following 72 hours of treatment, assessed via MTT assay. HFKs show resistance, whereas all CaCx cell lines are sensitive to nanomolar concentrations. B, Calculated GI50 values confirm significantly greater sensitivity of CaCx lines to AOH1996 compared with HFKs. C, Representative Annexin V/PI flow cytometry plots after 48 hours of AOH1996 treatment (1 μmol/L) show live (Annexin V−/PI−), apoptotic (Annexin V+/PI−), and dead (Annexin V+/PI+ or Annexin V−/PI+) populations. D, Quantification of flow cytometry data indicates robust induction of apoptosis and cell death in all CaCx lines, with negligible effects in HFKs. E, CaSki organoid cultures treated with 3 μmol/L AOH1996 show increased cell death and reduced proliferation. Hematoxylin and eosin (H&E) staining highlights stratified epithelial structure and a dying cell zone upon AOH1996 or cisplatin treatment. F, Quantification of H&E-stained rafts by a pathologist. G, TUNEL (green) and γH2AX (red) staining indicate increased apoptosis and DNA damage in response to AOH1996. Graphs show intensity quantification and quantification of relative thickness. Data represent the mean ± SEM from at least three replicates. Statistical analysis by one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Abstract Cervical cancers remain a significant health burden. Limitations on cervical cancer chemotherapeutic intervention caused by toxic side effects are a persistent barrier to care. In this study, we show that the human papillomavirus oncogenes that cause most cervical cancers also increase the levels of a cancer-associated isoform of proliferating cell nuclear antigen (PCNA) known as caPCNA. The abundance of caPCNA is specifically elevated in cervical cancer. Similar to observations in other cancers, we found that a small-molecule inhibitor of caPCNA (AOH1996) selectively killed cell line, organoid, and xenograft models of cervical cancer. Our subsequent molecular analysis identified a novel ability of AOH1996 to induce cell death by disrupting the interaction between PCNA and γ-tubulin, resulting in mitotic arrest. We show AOH1996 selectively induces mitotic death in transformed cells, because these cells attempt to progress through mitosis, rather than decondensing their chromosomes and reforming their nuclear membranes like untransformed control cells. Furthermore, we show that these differences allow AOH1996 to specifically sensitize cervical cancer cells to cisplatin, a frontline chemotherapeutic used to treat cervical cancer. We found that subtherapeutic doses of AOH1996 and cisplatin could reduce cervical cancer xenograft growth and improve survival, similarly to a therapeutic dose of cisplatin without the cisplatin-induced toxicity that restricts care. To our knowledge, this study provides the first evidence that AOH1996 can function as a cisplatin-sensitizing agent in cervical cancer models. Significance: We identify a novel mechanism by which the small-molecule inhibitor AOH1996 targets cancer-associated PCNA to induce mitotic death in cervical cancer cells. By disrupting PCNA:γ-tubulin interactions, AOH1996 selectively sensitizes tumors to a lower dose of cisplatin, enabling effective therapy with reduced toxicity and suggesting a potential strategy to reduce treatment-associated toxicity.

AOH1996 induces mitotic arrest in cervical cancer (CaCx) cells. A, Cell-cycle analysis of HFKs and CaCx cell lines (HeLa, HeLa POLη, CaSki, and C33a) treated with 1 μmol/L AOH1996 for 24 or 48 hours. DNA content was measured by PI staining and flow cytometry. AOH1996 induces a G2/M arrest at 24 hours in all cell types. At 48 hours, CaCx cells accumulate in sub-G1 and >G2 populations, indicative of apoptosis and rereplication. B, Immunoblot of p-H3, a marker of mitotic chromatin condensation, from lysates of cells treated with 1 μmol/L AOH1996 for 24 hours. AOH1996 significantly increases p-H3 levels in CaCx cell lines but only modestly in HFKs, indicating selective induction of mitotic arrest in transformed cells. GAPDH is used as a loading control. Bar graph shows fold change relative to DMSO-treated controls. C, Mitotic spread assay of HFK, HeLa, and HeLa POLη cells treated with DMSO, 1 μmol/L AOH1996, or colcemid for 24 hours. Representative brightfield images show chromatin condensation characteristic of mitosis. Insets highlight mitotic figures. Quantification (right) shows the percentage of cells in mitosis. AOH1996 induces mitotic arrest in CaCx cells but not HFKs, whereas colcemid arrests both. D, Confocal immunofluorescence microscopy of HeLa and CaSki cells treated with DMSO or 1 μmol/L AOH1996 for 24 hours. Cells were stained with DAPI (blue) and anti–p-H3 (red) to identify mitotic cells (white arrows). Quantification (right) shows that >60% of AOH1996-treated cells are p-H3–positive, confirming widespread mitotic arrest. Bar graphs represent the mean ± SEM from at least three independent experiments. Statistical analysis by one-way ANOVA. **, P < 0.01; ***, P < 0.001; **, P < 0.0001.

PCNA and its cancer-associated isoform caPCNA are overexpressed in cervical cancer (CaCx). A, PCNA mRNA expression across 35 cancer types and matched normal tissues was analyzed using the GENT2 database (34). CaCx exhibited the highest overall PCNA expression and the largest differential between tumor and normal tissues. Average PCNA mRNA expression in cancer (pink dashed line) and normal tissues (blue dashed line) are indicated. B, Representative IHC images from tissue microarrays showing PCNA protein expression in normal cervical tissue (left) and cervical cancer tissue (right). Scoring of PCNA staining intensity indicates significantly elevated PCNA protein levels in CaCx compared with normal tissue (bar graph inset, ****P < 0.0001). Red arrow in normal tissue indicates expected PCNA-positive basal layer. C, IHC staining for the normal (PC10) and cancer-associated isoform of PCNA (caPCNA) in normal and CaCx tissue with adjacent nonmalignant cervical epithelium. caPCNA is strongly expressed in tumor regions but undetectable in adjacent normal basal epithelial cells or healthy cervical epithelium. D, 2D gel electrophoresis of PCNA from lysates of primary HFKs and HFKs transduced with HPV16 E6E7. Signal on the left is caPCNA, and signal on the right is normal PCNA. These findings support a model in which PCNA and caPCNA are selectively upregulated in CaCx and represent candidate therapeutic targets.

Cisplatin induces S-phase arrest and delayed apoptosis in HeLa cells.

AOH1996 induces G2/M arrest followed by apoptosis in cervical cancer cells.

Proliferating cell nuclear antigen (PCNA) protein is an emerging therapeutic target for several diseases including cancers, and certain viral infections. It was previously regarded as being undruggable, due to its functions as a molecular hub that regulates a multitude of essential cellular pathways. However, we recently developed a PCNA inhibitor, AOH1996, which acts as a molecular glue between PCNA and RNA Pol II promoting selective killing of cancer cells through the induction of transcription-replication conflicts. We have now moved this investigational new drug into Phase 1 clinical trials. Yet, the discovery of new PCNA inhibitors is still of notable importance, because of the potential for further improving the potency and overall in vivo stability relative to AOH1996, and to potentially provide more treatment options for differing patient populations and for overcoming drug resistance mechanisms that may arise from AOH1996 treatment. Moreover, targeting other PCNA mediated disease states could require the identification of novel compounds that have non-identical mechanisms of action or differing pharmokinetic and pharmodynamic profiles. We coupled artificial intelligence-based computational screens with thermal shift assays in a search for novel hit compounds, and characterized a key hit through protein crystallography, a cellular thermal shift assay and protein frustration calculations. Notably, we discovered that the Hsp90α inhibitor, SNX-2112, binds to PCNA within the major PIP-box binding pocket, revealing a new chemical scaffold for the potential development of novel PCNA-targeting inhibitors.