Abstract

Background

   Proliferating cell nuclear antigen (PCNA), a well-documented anticancer
   target, is critical for DNA synthesis, replication, and repair.
   AOH1996, a small-molecule PCNA inhibitor, is currently undergoing
   clinical trials for the treatment of advanced solid tumors. However,
   the therapeutic effect of AOH1996 on head and neck squamous cell
   carcinoma (HNSCC) remains unclear.

Methods

   The effects of AOH1996 on HNSCC biological behaviors and cancer
   stemness were tested in HNSCC cells and nude mice. The combination
   treatment of AOH1996 and anti-PD1 was performed in a 4-nitroquinoline
   N-oxide (4NQO)-induced HNSCC mouse model. RNA sequencing, Western
   Blotting, immunofluorescence staining, comet assays, and qRT‒PCR were
   conducted for mechanistic studies.

Results

   Our results showed that AOH1996 effectively inhibited HNSCC
   proliferation and invasion both in vitro and in vivo. AOH1996
   suppressed HNSCC stemness, development, and metastasis. Moreover,
   AOH1996 altered the tumor immune microenvironment into an inflamed
   state with increased CD8^+ T-cell infiltration, rendering it a
   favorable partner for combination therapy with immune checkpoint
   inhibitors. Mechanistically, AOH1996 induced cellular DNA damage,
   suppressed cancer stemness through the upregulation of p-TBK1, and
   promoted the secretion of CD8^+ T-cell-recruiting chemokines by
   stimulating IRF3-mediated transcription.

Conclusions

   Taken together, our results demonstrated that AOH1996 suppressed tumor
   growth, eliminated cancer stem cells (CSCs), and synergistically
   enhanced the efficacy of anti-PD1 immunotherapy in HNSCC.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s13287-025-04607-9.

   Keywords: Head and neck squamous cell carcinoma (HNSCC), AOH1996, PCNA,
   Cancer stem cell (CSC), Immunotherapy

Background

   Head and neck squamous cell carcinoma (HNSCC) is the most common
   malignant tumor of the head and neck [[32]1]. HNSCC is derived mainly
   from the mucosal epithelium of the oral cavity, pharynx, and larynx
   [[33]2]. HNSCC is highly aggressive and frequently metastasizes to
   cervical lymph nodes, resulting in a poor prognosis [[34]2]. The
   conventional treatments include surgery, chemotherapy, radiotherapy,
   and multimodal approaches [[35]2–[36]4]. However, the 5-year survival
   rate for HNSCC, which ranges from 35 to 70% [[37]5, [38]6], has not
   improved in recent years, attributed to HNSCC metastasis, recurrence,
   and drug resistance [[39]7, [40]8].

   For metastatic and recurrent HNSCC, the Food and Drug Administration
   has approved immune checkpoint inhibitor (ICI) therapy, including
   pembrolizumab and nivolumab, which target the programmed cell death
   protein 1 (PD1) receptor [[41]2, [42]9, [43]10]. However, only
   approximately 20% of patients with HNSCC achieve a durable response to
   ICI treatment, indicating that most patients with HNSCC are resistant
   to PD1 blockade-based immunotherapy [[44]11–[45]13]. Thus, more
   effective therapeutic targets and strategies for HNSCC are needed.

   Proliferating cell nuclear antigen (PCNA) reflects the status of cell
   proliferation and is located mostly in the nuclei of normal
   proliferating cells and tumor cells [[46]14]. In the form of a
   homotrimeric ring structure, PCNA encircles DNA and is essential for
   DNA synthesis, replication, and repair [[47]15–[48]17]. PCNA is highly
   expressed in HNSCC tumors compared with adjacent normal epithelium and
   serves as a cell proliferation marker in HNSCC [[49]18, [50]19]. Thus,
   PCNA is a potential target for cancer treatment [[51]20]. Recently,
   AOH1996, a small-molecule PCNA inhibitor that targets transcription
   replication conflict, has been reported [[52]21]. AOH1996 is lethal to
   malignant tumors but not normal cells and inhibits cancer-associated
   PCNA [[53]22]. AOH1996 suppresses tumor growth as a monotherapy or in
   combination with chemotherapy without discernible side effects
   [[54]21]. AOH1996 is orally active, metabolically stable, and currently
   in clinical trials for the treatment of advanced solid tumors. However,
   the therapeutic effect of AOH1996 on HNSCC remains unclear.

   Cancer stem cells (CSCs) are a subpopulation of cancer cells capable of
   self-renewal, infinite proliferation, and multiple differentiation
   potential [[55]23–[56]25]. CSCs are closely associated with HNSCC
   initiation, development, metastasis, recurrence, and drug resistance
   [[57]26, [58]27], and serve as promising targets for HNSCC treatment
   [[59]28–[60]30].

   In this study, we aimed to evaluate the therapeutic potential of
   AOH1996, with a particular focus on its effects on cancer stemness and
   the tumor immune microenvironment, and to elucidate the underlying
   mechanisms involved. These findings may provide a better understanding
   of the therapeutic potential of AOH1996 in HNSCC and promote the
   development of more effective treatment strategies targeting PCNA and
   its associated pathways in cancer.

Methods

Cell culture and treatment

   The human HNSCC cell lines CAL27 [[61]31] and SCC15 [[62]32] (American
   Type Culture Collection, ATCC, Manassas, VA, USA), both HPV-negative,
   were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
   with 10% fetal bovine serum (FBS; Vazyme, Nanjing, China; Cat# F101-01)
   at 37 °C in a humidified atmosphere containing 5% CO[2]. The culture
   medium contained 1% penicillin‒streptomycin solution (Aoqing
   Biotechnology Co., Ltd., Beijing, China; Cat# AQ512). AOH1996 (Selleck,
   Shanghai, China; Cat# E159601) was dissolved in dimethyl sulfoxide
   (DMSO; Aoqing Biotechnology Co., Ltd.; Cat# AQ62650). After reaching
   70%‒80% confluence, HNSCC cells were treated with 1 µM DMSO or AOH1996.

Cell counting Kit-8 (CCK-8) assay

   The proliferation of HNSCC cells was evaluated using a CCK-8 assay
   (Dojindo, Shanghai, China). The cells treated with AOH1996 were added
   to 96-well plates (2 × 10^3 cells/well). Ten microliters of CCK-8
   reagent was added to each well at 0, 24, 48, and 72 h and incubated at
   37 °C for 1 h before detection. The absorbance at 450 nm was measured
   using a microplate spectrophotometer (Bio-Tek Instruments Inc.,
   Winooski, VT, USA). The IC50 and EC50 values of the HNSCC cells were
   calculated.

Transwell assay

   Transwell chambers with 8 μm pore-size membranes coated with Matrigel
   (Corning Inc., NY, USA; Cat# 3422) were placed in 24-well plates. HNSCC
   cells were resuspended in 200 µL of serum-free DMEM (1 × 10^5 cells)
   and added to the upper chamber. DMEM containing 20% FBS (600 µL) was
   added to the lower chamber. After 24 h of incubation at 37 °C, the
   cells on the upper side of the membranes were removed using a cotton
   swab. Invaded cells were fixed with 4% paraformaldehyde and stained
   with 0.1% crystal violet aqueous solution (Solarbio, Beijing, China;
   Cat# G1064). The membranes were then washed with phosphate-buffered
   saline (PBS). Images were acquired using an optical microscope
   (Olympus, Tokyo, Japan). The number of invaded cells was randomly
   counted from three fields and analyzed using ImageJ (NIH, Bethesda, MD,
   USA).

Terminal deoxynucleotidyl transferase nick-end-labeling (TUNEL) assay

   A TUNEL Apoptosis Assay Kit (Solarbio; Cat# T2130) was used to detect
   apoptotic cells following the manufacturer’s instructions. Briefly, the
   cells were incubated with TUNEL working solution for 1 h at 37 °C after
   AOH1996 treatment for 24 h. The cells were fixed with 4%
   paraformaldehyde for 20 min and then stained with
   4′,6-diamidino-2-phenylindole (DAPI; Solarbio; Cat# C0065). Images were
   acquired using a fluorescence microscope.

CSC isolation and flow cytometry assay

   After AOH1996 treatment, an ALDEFLUOR Kit (STEMCELL Technologies,
   Vancouver, Canada; Cat# 01700) was used to isolate CSCs from HNSCC
   cells expressing high levels of aldehyde dehydrogenase (ALDH^high).
   DEAB is a specific inhibitor of ALDH and is used as an ALDH^low control
   for background fluorescence. A fluorescence-activated cell sorting
   (FACS) assay was used to separate ALDH^high and ALDH^low subpopulations
   in HNSCC cells. To isolate CSCs from human HNSCC patient-derived
   xenograft (PDX) model #1, a human Tumor Dissociation Kit (Miltenyi
   Biotec, Bergisch Gladbach, Germany; Cat# 130-095-929) was used to
   digest the HNSCC PDX tumors into single cells. The single-cell
   suspension was incubated with an EpCAM antibody (Miltenyi Biotec; Cat#
   130-111-116) to isolate EpCAM^+ tumor cells. An ALDEFLUOR Kit was then
   used to separate ALDH^high and ALDH^low subpopulations in EpCAM^+ tumor
   cells. The flow cytometry results were analyzed using FlowJo software
   (Becton, Dickinson & Company, New Jersey, USA).

Tumorsphere formation assay

   Ultralow attachment 6-well plates (Corning Inc.; Cat# 3471) were used
   to analyze tumorsphere formation. FACS-isolated HNSCC cells treated
   with AOH1996 were plated. Serum-free DMEM/F12 (Thermo Fisher
   Scientific, Shanghai, China; Cat# 8122659) supplemented with 1% N2
   (Thermo Fisher Scientific; Cat# 17502048) and 1% B27 supplement (Thermo
   Fisher Scientific; Cat# 17504044) was used to culture the cells. A
   total of 20 ng/mL recombinant human epidermal growth factor (EGF; R&D
   Systems, Minneapolis, MN, USA; Cat# 236-EG-01 M) and 10 ng/mL human
   basic fibroblast growth factor (FGF; R&D Systems; Cat# 233-FB/CF)
   [[63]33] were added to the medium. Microscopy images of tumorspheres
   with diameters greater than 70 μm were acquired, and the tumorsphere
   volumes were evaluated after 14 days.

Western blot

   HNSCC cells were lysed using radioimmunoprecipitation assay (RIPA)
   buffer (Solarbio; Cat# R0010) containing phenylmethylsulfonyl fluoride
   (PMSF; Beyotime, Shanghai, China; Cat# ST506). Protein samples were
   separated on 10% SDS polyacrylamide gels and transferred to
   polyvinylidene difluoride (PVDF) membranes. The membranes were blocked
   with 5% nonfat milk for 1 h and then incubated with primary antibodies
   at 4 °C overnight. The primary antibodies used included the following:
   anti-BMI1 (1:1000; Cell Signaling Technology, Shanghai, China; Cat#
   6964), anti-SOX2 (1:1000; Cell Signaling Technology; Cat# 14962),
   anti-ALDH1 (1:1000; Cell Signaling Technology; Cat# 54135), anti-MYC
   (1:1000; Cell Signaling Technology; Cat# 18583), anti-phosphorylated
   H2A.X (anti-p-H2A.X; 1:1000; Cell Signaling Technology; Cat# 9718),
   anti-p-STING (1:1000; Cell Signaling Technology; Cat# 19781),
   anti-STING (1:1000; Cell Signaling Technology; Cat# 13647), anti-p-TBK1
   (1:1000; Cell Signaling Technology; Cat# 5483), anti-TBK1 (1:1000; Cell
   Signaling Technology; Cat# 3504), anti-p-IRF3 (1:1000; Cell Signaling
   Technology; Cat# 29047), and anti-IRF3 (1:1000; Cell Signaling
   Technology; Cat# 4302). GAPDH (1:1000; Cell Signaling Technology; Cat#
   5174) was used as the internal control. The membranes were then
   incubated with secondary horseradish peroxidase (HRP)-labeled goat
   anti-rabbit or goat anti-mouse antibodies (Beyotime; Cat# A0208 or
   #A0216) at room temperature for 1 h. Immunoreactive protein bands were
   visualized using NcmECL Ultra (NCM Biotech, Suzhou, China; Cat#
   [64]P10100) reagent.

RNA-seq and pathway enrichment analysis

   Total RNA was isolated from SCC15 cells using TRIzol Reagent (Thermo
   Fisher Scientific; Cat# 15596026). The quality of total RNA was
   evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies,
   Santa Clara, CA, USA). An Illumina TruSeq RNA sample preparation kit
   (Illumina, San Diego, CA, USA) was used for RNA-sequencing library
   construction [[65]34]. An Illumina HiSeq 2500 sequencer was used to
   sequence single-ended RNA. Kyoto Encyclopedia of Genes and Genomes
   (KEGG) pathway analysis was performed using the DAVID Bioinformatics
   platform ([66]https://davidbioinformatics.nih.gov/tools.jsp). The
   RNA-seq data were deposited in the Gene Expression Omnibus (GEO)
   database of the National Center for Biotechnology Information under
   accession number [67]GSE289770.

Comet assay

   A Comet SCGE Assay Kit (Enzo, NY, USA; Cat# ADI-900-166) was used for
   single-cell gel electrophoresis [[68]35]. After HNSCC cells were
   treated with AOH1996 for 24 h, the cells (1 × 10^5 cells/ml) were mixed
   with molten LM agarose at a 1:10 (v/v) ratio. Prewarmed slides were
   loaded with 75 µl aliquots and placed at 4 °C in the dark for 10 min.
   The slides were immersed in prechilled lysis solution for 1 h, followed
   by prechilled alkaline solution for 40 min and TBE buffer for 10 min.
   Slides in TBE buffer were subjected to horizontal electrophoresis at
   25 V for 20 min. Slides were immersed in 70% ethanol for 5 min, dried
   at room temperature, and stained with CYGREEN dye solution for 30 min.
   Images were acquired using a fluorescence microscopy. At least 50 cells
   per sample were analyzed using the CASP analysis tool Version 1.2.2
   (CASPlab, Wroclaw, Poland).

Cytosolic DsDNA staining

   After AOH1996 treatment for 24 h, HNSCC cells were stained with
   PicoGreen (Thermo Fisher Scientific; Cat# [69]P11496) for 1 h and
   MitoTracker (Thermo Fisher Scientific; Cat# M7512) for 30 min. The
   cells were fixed with 4% paraformaldehyde for 15 min, and the nuclei
   were stained with DAPI. Images were acquired using a confocal laser
   scanning microscope (Olympus FV3000, Tokyo, Japan).

Quantitative reverse transcription polymerase chain reaction (qRT‒PCR)

   Total RNA was extracted from HNSCC cells using TRIzol Reagent (Thermo
   Fisher Scientific; Cat# 15596026), and then 500 ng of RNA was reverse
   transcribed to cDNA using a reverse transcription kit (Takara, Beijing,
   China; Cat# RR036A). A SYBR Green Kit (Roche, Basel, Switzerland; Cat#
   04913914001) was used to quantify the cDNA by quantitative real-time
   PCR, which was conducted at 95 °C for 10 min, followed by 40 cycles of
   95 °C for 15 s and 60 °C for 1 min using an ABI Prism 7500 real-time
   PCR System (Applied Biosystems, Foster City, CA, USA). The primers used
   for qRT‒PCR are listed in Table [70]S1.

In vivo HNSCC tumor growth, ELDA, patient-derived xenografts (PDXs), and the
orthotopic model

   Female BALB/c nude mice (6–8 weeks old) were purchased from SPF
   Biotechnology Co., Ltd (Beijing, China). All the animal studies
   strictly conformed to the regulations of the Institutional Animal Care
   and Use Committee of Peking University Health Science Center. In this
   study, the mice were euthanized by cervical dislocation after
   isoflurane anesthesia. The work has been reported in line with the
   ARRIVE guidelines 2.0.

   For the subcutaneous tumor models, twelve mice were randomly divided
   into two groups (n = 6/group). SCC15 cells were suspended in a 1:1
   mixture of PBS and Matrigel matrix (Corning Inc., Bedford, MA, USA;
   Cat# 354234), and then subcutaneously injected (5 × 10^6 cells/mouse)
   into the flanks of the mice. 5% AOH1996 was dissolved in 95% corn oil.
   One week after injection, vehicle or AOH1996 (50 mg/kg) was used to
   treat the mice orally twice a week for three weeks. Tumor size was
   measured weekly. After four weeks, the tumors were harvested,
   photographed, and weighed (g).

   For the extreme limiting dilution assay (ELDA), ALDH^high cells were
   obtained from SCC15 cells using FACS. The FACS-sorted CSCs were
   suspended in a 1:1 mixture of PBS and Matrigel matrix, and then
   subcutaneously injected into the flanks of the mice. One week after
   injection, vehicle or AOH1996 (50 mg/kg) was used to treat the mice
   orally twice a week for three weeks. The results were analyzed using
   ELDA software: [71]https://bioinf.wehi.edu.au/software/elda/index.html.

   For the HNSCC PDX and orthotopic models, CSCs were obtained from the
   HNSCC PDX #1 model using FACS with EpCAM^+ ALDH^high markers, as
   previously described [[72]36]. FACS-sorted HNSCC CSCs were injected
   orthotopically into the tongues of mice (1 × 10^6 cells/mouse). One
   week after injection, thirty mice were randomly divided into two groups
   (n = 15/group) and treated with vehicle or AOH1996 (50 mg/kg) orally
   twice a week for three weeks.

   After the mice were sacrificed, the tongues and cervical lymph nodes
   were collected. The tumor volume (mm^3) was calculated as V = 1/2 × D ×
   d^2, where D is the longer diameter and d is the shorter diameter.

HNSCC 4-nitroquinoline 1-oxide (4NQO) mouse model, treatment and histology

   For 4NQO-induced HNSCC, 6–8-week-old Bmi1^CreER, Rosa^tdTomato mice
   were given 50 µg/ml 4NQO (Santa Cruz, CA, USA; Cat# 256815) in the
   drinking water for 16 weeks, followed by normal drinking water for 6
   weeks. For lineage tracing, tamoxifen (9 mg/40 g weight, Sigma–Aldrich,
   St. Louis, MO, USA; Cat# T5648), which labels BMI1^+ CSCs, was
   intraperitoneally injected into the mice 2 days before sacrifice.

   For HNSCC treatment, after 22 weeks, the mice were randomly divided
   into four groups and treated as follows: (1) control vehicle; (2)
   AOH1996 (50 mg/kg, twice a week); (3) anti-PD1 (BioXcell, Lebanon, NH,
   USA; Cat# BE0146, 200 µg/mouse, twice a week); and (4) AOH1996 combined
   with anti-PD1, which lasted four weeks. To assess the tolerability of
   anti-PD1, AOH1996, and combination treatments in mice, the heart,
   liver, lung, kidney, and spleen were collected for histopathological
   analysis, and H&E-stained images were captured. The blood of the mice
   was collected for routine blood tests and biochemical indicator
   analysis. HNSCC tumor growth and lymph node metastasis were evaluated
   as previously described [[73]36]. Tongues and lymph nodes were
   collected after sacrifice, and tongue lesion areas were measured.

   For histological analysis and immunostaining, longitudinally cut
   tongues (dorsal/ventral) and lymph nodes were sectioned into 4-µm-thick
   tissues, fixed with 4% paraformaldehyde, and paraffin-embedded
   overnight. Tongue sections were stained with hematoxylin and eosin
   (H&E). The number of squamous cell carcinomas (SCCs) was counted, and
   SCC areas were measured as previously described [[74]36]. The HNSCC
   invasion score was based on the following criteria. Grade 1: normal or
   epithelial dysplasia; Grade 2: distinct invasion, unclear basement
   membrane, and drop or diffuse infiltration into the superficial muscle
   layer; Grade 3: basement layer loss and extensive invasion into the
   deep muscle layer.

Immunostaining

   For immunohistochemistry (IHC), after antigen repair, the lymph node
   sections were incubated with the primary antibody anti-pan-cytokeratin
   (anti-PCK; 1:200; Santa Cruz Biotechnology, Shanghai, China; Cat#
   sc-8018) at 4 °C overnight. Anti-PCK can be used to specifically test
   epithelial tumor cells in lymph nodes. The tongue sections were
   incubated with the primary antibody anti-CD8 (1:200; Cell Signaling
   Technology; Cat# 98941) at 4 °C overnight. The sections were then
   incubated with goat anti-mouse IgG polymer (ZSGB-BIO; Cat# PV-6002) or
   goat anti-rabbit IgG polymer (ZSGB-BIO; Cat# PV-6001) secondary
   antibodies for 1 h at room temperature. The percentages of lymph node
   metastasis, metastatic areas, and CD8^+ T cells were quantified.

   For cell immunofluorescence staining, HNSCC cells were fixed with 4%
   paraformaldehyde for 15 min after AOH1996 treatment at room
   temperature. The cells were incubated with the primary antibody
   anti-p-H2A.X (1:100; Cell Signaling Technology; Cat# 9718) at 4 °C
   overnight, followed by incubation with a secondary antibody conjugated
   with the fluorescent marker Cy3 (Jackson ImmunoResearch Laboratories,
   West Grove, PA, USA). The nuclei were stained with DAPI.

   All immunostaining images were acquired using a fluorescence
   microscope.

Statistical analysis

   Statistical analyses were performed using GraphPad Prism 10.4.1 for
   Windows (GraphPad Software Inc., La Jolla, CA, USA). In vitro
   experiments were performed at least three times, and in vivo
   experiments were performed at least twice. The data are expressed as
   the mean ± standard deviation (SD) or standard error of the mean (SEM)
   [[75]28]. Differences between two groups were analyzed using Student’s
   t test. Differences among multiple groups were analyzed by one-way
   analysis of variance (ANOVA) [[76]33]. A two-tailed value of P < 0.05
   was considered statistically significant.

Results

AOH1996 inhibits cell proliferation and invasion while promoting apoptosis in
HNSCC cells

   To investigate the therapeutic effect of AOH1996 on HNSCC, two HNSCC
   cell lines, CAL27 and SCC15, were treated with AOH1996. AOH1996 reduced
   the relative cell viability of CAL27 and SCC15 cells (Fig. [77]1A).
   Moreover, AOH1996 increased the relative cell inhibition of CAL27 and
   SCC15 cells (Fig. [78]1B). Based on the IC50 and EC50 values of
   AOH1996, the concentration of 1 µM AOH1996 was selected for further
   study (Fig. [79]1C-D). A CCK-8 assay revealed that AOH1996 inhibited
   the proliferation of CAL27 and SCC15 cells (Fig. [80]1E). A Matrigel
   invasion assay revealed that AOH1996 inhibited the invasion of CAL27
   and SCC15 cells (Fig. [81]1F). Moreover, AOH1996 increased the
   percentages of apoptotic CAL27 and SCC15 cells (Fig. [82]1G). To assess
   whether AOH1996 could inhibit tumor growth in vivo, AOH1996 was given
   to nude mice with SCC15 cell-derived tumors. Compared with the control
   treatment, AOH1996 treatment reduced the weight and volume of HNSCC
   tumors (Fig. [83]1H-J).

Fig. 1.

   [84]Fig. 1
   [85]Open in a new tab

   AOH1996 inhibits HNSCC cell proliferation and invasion, promotes
   apoptosis, and suppresses tumor growth

   (A-B) Relative cell viability and relative cell inhibition of CAL27 and
   SCC15 cells treated with AOH1996 determined by CCK-8 analysis. Values
   and error bars represent the means ± SDs. (C-D) The IC50 and EC50
   values of CAL27 and SCC15 cells treated with AOH1996 determined by
   CCK-8 analysis. Values and error bars represent the means ± SDs with an
   unpaired Student’s t test. ns, not significant. (E) Effects of AOH1996
   on the proliferation of CAL27 and SCC15 cells after 0–72 h, as
   determined in a CCK-8 assay. Optical density (OD) 450 values and error
   bars represent the means ± SDs with an unpaired Student’s t test.
   **P < 0.01. (F) Numbers of invaded CAL27 and SCC15 cells treated with
   AOH1996 determined by the Transwell assay. Values and error bars
   represent the means ± SDs with an unpaired Student’s t test. Scale bar
   = 100 μm. **P < 0.01. (G) Apoptosis rates of CAL27 and SCC15 cells
   treated with AOH1996. Values and error bars represent the means ± SDs
   with an unpaired Student’s t test. Scale bar = 20 μm. **P < 0.01. (H)
   Images of subcutaneous tumors formed by SCC15 cells treated with
   AOH1996 in nude mice. Scale bar = 1 cm. (I) Weights of subcutaneous
   tumors formed by SCC15 cells treated with AOH1996 in nude mice. Values
   and error bars represent the means ± SDs with an unpaired Student’s t
   test (n = 6). **P < 0.01. (J) Volume growth curves of subcutaneous
   tumors formed by SCC15 cells treated with AOH1996 in nude mice for four
   weeks. Values and error bars represent the means ± SDs with an unpaired
   Student’s t test (n = 6). **P < 0.01

AOH1996 inhibits the cancer stemness signature of HNSCC

   We further investigated whether AOH1996 could inhibit cancer stemness
   in HNSCC. Aldehyde dehydrogenase (ALDH) is a specific marker on the
   surface of CSCs [[86]23, [87]37]. Using an ALDEFLUOR Kit, we sorted
   ALDH^low nonstem tumor cells and ALDH^high CSC-like cells with the
   specific ALDH inhibitor diethylaminobenzaldehyde (DEAB) as a control.
   Fluorescence-activated cell sorting (FACS) analysis revealed that the
   percentages of ALDH^high CSC-like cells among CAL27 and SCC15 cells
   after AOH1996 treatment were significantly lower than those in the
   control group (Fig. [88]2A). AOH1996 treatment inhibited the
   tumorsphere formation of CAL27 and SCC15 ALDH^high CSC-like cells,
   reducing the number and volume of tumorspheres (Fig. [89]2B-C). BMI1,
   SOX2, ALDH1, and MYC are classical markers of CSCs [[90]30, [91]38,
   [92]39]. Western blot analysis revealed that BMI1, SOX2, ALDH1, and MYC
   protein levels were significantly reduced in CAL27 and SCC15 cells
   treated with AOH1996 (Fig. [93]2D). Moreover, an in vivo extreme
   limiting dilution assay (ELDA) indicated that AOH1996 treatment reduced
   the tumor incidence/injection ratio and inhibited the tumorigenic
   potential of ALDH^high SCC15 cells (Fig. [94]2E-F). These results
   demonstrated that AOH1996 inhibited the cancer stemness of HNSCC.

Fig. 2.

   [95]Fig. 2
   [96]Open in a new tab

   AOH1996 inhibits the cancer stemness of HNSCC

   (A) Representative fluorescence-activated cell sorting (FACS) images of
   ALDH^high CAL27 and SCC15 cells treated with AOH1996.
   N-Diethylaminoazobenzene (DEAB) was used as a control for background
   fluorescence. The percentage of ALDH^high cells is indicated in the
   gate. (B-C) Representative images and quantification of tumorsphere
   formation in ALDH^high and ALDH^low CAL27 and SCC15 cells treated with
   AOH1996. Values and error bars represent the means ± SDs with an
   unpaired Student’s t test. Scale bar = 100 μm. **P < 0.01. (D) Western
   blot showing the protein levels of BMI1, SOX2, ALDH1, and MYC in CAL27
   and SCC15 cells treated with AOH1996. GAPDH was used as the internal
   control. The full-length blots are presented in Supplementary Material
   1. (E-F) Extreme limiting dilution analysis (ELDA) of ALDH^high
   CSC-like SCC15 cells treated with AOH1996 in vivo (n = 6). Scale bar
   = 1 cm. The table shows the frequency of tumor formation at each cell
   dose injected. The data were analyzed using ELAD software

AOH1996 inhibits the progression and metastasis of CSCs cultured from
patient-derived xenograft (PDX) models

   PDX models and human cancers share similar genetic landscapes, and PDXs
   have been widely used as preclinical models in cancer studies [[97]40].
   Here, we used a human HNSCC PDX model to evaluate the therapeutic
   efficacy of AOH1996. CSCs were isolated from the HNSCC PDX #1 model
   using FACS with EpCAM^+ ALDH^high markers, as previously described
   [[98]36]. The FACS-sorted HNSCC CSCs from the PDX model were injected
   orthotopically into the tongues of the mice (Fig. [99]3A). The results
   showed that AOH1996 treatment suppressed the growth of HNSCC tumors
   (Fig. [100]3B-C). Consistently, AOH1996 treatment significantly reduced
   the tumor volume and area (Fig. [101]3D-E). Metastasis is a complex
   process in which cancer cells migrate and invade from the primary tumor
   to distant sites [[102]41]. CSCs are associated with this process
   because of their ability to self-renew and differentiate into various
   cell types within the tumor [[103]37, [104]42]. Immunohistochemistry
   (IHC) staining with an anti-pan-cytokeratin (anti-PCK) antibody
   indicated that, compared with vehicle treatment, AOH1996 treatment
   inhibited the cervical lymph node metastasis of CSCs (Fig. [105]3F-H).
   Thus, AOH1996 treatment effectively inhibited the tumorigenic potential
   and prevented the progression and metastasis of HNSCC CSCs derived from
   the PDX model.

Fig. 3.

   [106]Fig. 3
   [107]Open in a new tab

   AOH1996 inhibits the tumorigenic potential and lymph node metastasis of
   HNSCC CSCs

   (A) Schematic diagram showing the timeline of BALB/c-nude mice injected
   with HNSCC CSCs cultured from patient-derived xenografts (PDXs) #1,
   AOH1996 treatment, and sacrifice (Sac). (B) Representative images of
   orthotopic tongue tumors from nude mice treated with vehicle or
   AOH1996. The white dashed circles mark the tumor areas. Scale bar
   = 2 mm. (C) Representative hematoxylin and eosin (H&E) staining images
   of orthotopic tongue tumors from nude mice treated with vehicle or
   AOH1996. Scale bar = 500 μm. Magnified images are shown in the lower
   panels. Scale bar = 100 μm. (D) Quantification of orthotopic tongue
   tumor volume in nude mice treated with vehicle or AOH1996 as indicated.
   Values and error bars represent the means ± SDs with an unpaired
   Student’s t test (n = 15). **P < 0.01. (E) Quantification of the
   squamous cell carcinoma (SCC) area in nude mice treated with vehicle or
   AOH1996 as indicated. Values and error bars represent the means ± SDs
   with an unpaired Student’s t test (n = 15). **P < 0.01. (F)
   Representative images of metastatic cells in the cervical lymph nodes
   from nude mice treated with vehicle or AOH1996 and immunostained with
   anti-pan-cytokeratin (anti-PCK). Scale bar = 200 μm. (G) Percentages of
   metastatic lymph nodes from nude mice treated with vehicle or AOH1996.
   Metastatic lymph nodes (LN) numbers are indicated in the histogram. The
   result represents the average of two independent experiments according
   to the chi-square test. **P < 0.01. (H) Quantification of the
   metastatic area in cervical lymph nodes from nude mice treated with
   vehicle or AOH1996. Values and error bars represent the means ± SEMs
   from two independent experiments; unpaired Student’s t tests were
   performed. **P < 0.01

AOH1996 eliminates CSCs and enhances the response to anti-PD1 immunotherapy
in HNSCC

   In our previous study, we successfully established a Bmi1^CreER,
   Rosa^tdTomato mouse model of HNSCC using 4-nitroquinoline 1-oxide
   (4NQO) induction. This model leverages the specific labeling of Bmi1^+
   tumor cells (CSCs) with tdTomato fluorescence, enabling precise
   tracking and analysis of CSC dynamics in vivo [[108]28]. The
   4NQO-induced carcinogenesis model closely mimics the histopathological
   features of human HNSCC, providing a robust approach for investigating
   whether AOH1996 treatment could eliminate CSCs and synergize with
   anti-PD1 immunotherapy (Fig. [109]4A).

Fig. 4.

   [110]Fig. 4
   [111]Open in a new tab

   AOH1996 enhances the therapeutic effect of anti-PD1 immunotherapy in
   HNSCC

   (A) Schematic diagram showing the timeline of Bmi1^CreER, Rosa^tdTomato
   mouse lineage tracing of primary HNSCC, treatment, tamoxifen (Tam)
   injection, and sacrifice (Sac). (B) Representative images of HNSCC
   orthotopic tongue lesions in the different groups. The white dashed
   circles mark the lesion areas. Scale bar = 2 mm. (C) Quantification of
   the HNSCC lesion area. Values and error bars represent the means ± SDs
   (n = 12) according to one-way analysis of variance (ANOVA). *P < 0.05
   and **P < 0.01. (D) Representative H&E staining images of HNSCC
   orthotopic tongue tumors. Scale bar = 500 μm. Magnified images are
   shown in the lower panels. Scale bar = 100 μm. (E-F) Quantification of
   the SCC number and area. Values and error bars represent the
   means ± SDs of one-way ANOVA (n = 12). *P < 0.05 and **P < 0.01. (G)
   Quantification of the HNSCC invasion grade by the Cochran–Armitage
   test. *P < 0.05 and **P < 0.01. (H) Representative anti-PCK
   immunostaining images of metastatic cells in cervical lymph nodes.
   Scale bar = 200 μm. (I) Percentage of metastatic lymph nodes. The
   number of metastatic lymph nodes is indicated in the histogram by the
   chi-square test. *P < 0.05 and **P < 0.01. (J) Quantification of the
   metastatic area in the cervical lymph nodes. Values and error bars
   represent the means ± SEMs, as determined by one-way ANOVA. *P < 0.05
   and **P < 0.01. (K) Representative immunofluorescence images and
   quantification of Tomato^+ BMI1^+ CSCs in HNSCC. The white dashed lines
   mark the tumor-stromal boundary. Scale bar = 20 μm. Values and error
   bars represent the means ± SDs of one-way ANOVA. *P < 0.05 and
   **P < 0.01. (L) Representative immunostaining images and quantification
   of CD8^+ T cells infiltration in HNSCC. Scale bar = 20 μm. Values and
   error bars represent the means ± SDs of one-way ANOVA. *P < 0.05 and
   **P < 0.01

   Compared with either anti-PD1 monotherapy or AOH1996 monotherapy,
   combination treatment with AOH1996 and anti-PD1 significantly reduced
   the tongue lesion area (Fig. [112]4B-C). Compared with either treatment
   alone, histological analysis demonstrated that the combination
   treatment significantly reduced the number, area, and invasion of HNSCC
   cells (Fig. [113]4D-G). The combination therapy effectively suppressed
   cervical lymph node metastasis in HNSCC, as evidenced by anti-PCK
   immunostaining (Fig. [114]4H-J).

   In vivo lineage tracing revealed that AOH1996 reduced Bmi1^+ CSCs in
   HNSCC, whereas the combination treatment of AOH1996 and anti-PD1 led to
   increased elimination of Bmi1^+ CSCs (Fig. [115]4K). Moreover, we
   investigated the impact of the combination treatment on the tumor
   immune microenvironment. Immunostaining revealed that the combination
   treatment significantly increased the infiltration of CD8^+ T cells
   into the tumor microenvironment (Fig. [116]4L). These results
   demonstrated that the combination treatment of AOH1996 and anti-PD1 not
   only inhibited the CSCs and metastasis of HNSCC but also enhanced tumor
   cell-intrinsic immune responses, highlighting its potential as a
   synergistic therapeutic strategy for HNSCC.

   To evaluate the drug safety of the anti-PD1, AOH1996, and the
   combination treatment of AOH1996 and anti-PD1, we conducted
   histopathological and hematological analyses in C57BL/6J mice.
   Histopathological examination revealed no significant morphological
   alterations in major organs, including the heart, liver, lung, kidney,
   and spleen (Fig. [117]5A). Routine blood tests, including assessments
   of white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), and
   platelet (PLT) levels, revealed no notable abnormalities after
   anti-PD1, AOH1996, or combination treatment (Fig. [118]5B).
   Additionally, blood biochemical analyses, in which alanine
   aminotransferase (ALT), aspartate aminotransferase (AST), creatinine
   (CREA), and urea levels were measured, revealed no significant changes,
   confirming the absence of systemic toxicity (Fig. [119]5C).
   Collectively, these results demonstrated that anti-PD1, AOH1996, and
   combination treatment were well tolerated in vivo, supporting the
   safety and potential clinical applicability of this therapeutic
   strategy.

Fig. 5.

   [120]Fig. 5
   [121]Open in a new tab

   Systematic effect of AOH1996 combined with anti-PD1 treatment in HNSCC
   in vivo

   (A) Representative hematoxylin and eosin (H&E) staining images of
   heart, liver, lung, kidney, and spleen from C57BL/6J mice treated with
   vehicle, AOH1996, anti-PD1, or AOH1996 combined with anti-PD1. Scale
   bar = 200 μm. (B) The diagram shows the results of routine blood
   analysis in different treatment groups, which included tests of white
   blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), and platelet
   (PLT) levels in the mice. Values and error bars represent the
   means ± SDs of one-way ANOVA. ns, not significant. (C) Diagram showing
   the blood biochemical indicators in different treatment groups, which
   include the test of alanine aminotransferase (ALT), aspartate
   aminotransferase (AST), creatinine (CREA), and urea levels in the mice.
   Values and error bars represent the means ± SDs of one-way ANOVA. ns,
   not significant

AOH1996 induces cellular DNA damage, activates the cGAS–STING signaling
pathway, inhibits cancer stemness, and stimulates antitumor immune responses
in HNSCC

   To investigate the mechanisms underlying the therapeutic potential of
   AOH1996 in HNSCC, RNA sequencing (RNA-seq) was performed on HNSCC
   cells. Comprehensive transcriptome alterations were analyzed in SCC15
   cells after treatment with AOH1996. KEGG pathway analysis suggested
   that AOH1996 treatment modulated the regulation of transcription by RNA
   polymerase II, DNA-templated transcription, and DNA damage response
   signaling pathways (Fig. [122]6A). Given that PCNA is pivotal in DNA
   synthesis, replication, and repair, previous studies have shown that
   AOH1996 can induce DNA double-strand breaks [[123]21]. We focused the
   subsequent experiments on the DNA damage signaling pathway.

Fig. 6.

   [124]Fig. 6
   [125]Open in a new tab

   AOH1996 induces cellular DNA damage, activates the cGAS-STING signaling
   pathway, and stimulates antitumor immune responses

   (A) Histogram showing the top 10 enriched KEGG pathways in SCC15 cells
   treated with AOH1996 according to RNA sequencing. Fold change > 1.5.
   Y-axis: categories of the KEGG pathway; X-axis: the statistical
   significance of the enrichment. The number of genes in each KEGG
   pathway category is listed on the right. (B) Western blot showing the
   protein levels of p-H2A.X in CAL27 and SCC15 cells treated with
   AOH1996. The full-length blots are presented in Supplementary Material
   1. (C) Immunofluorescence staining and quantification of p-H2A.X (red)
   in CAL27 and SCC15 cells treated with AOH1996. The nuclei were stained
   with DAPI (blue). Values and error bars represent the means ± SDs with
   an unpaired Student’s t test. Scale bar = 20 μm. **P < 0.01. (D)
   Representative images and quantification of AOH1996-treated CAL27 and
   SCC15 cells by the DNA comet assay. Values and error bars represent the
   means ± SDs with an unpaired Student’s t test. Scale bar = 20 μm.
   **P < 0.01. (E) Representative images and quantification showing the
   accumulation of cytosolic DNA in CAL27 and SCC15 cells treated with
   AOH1996. The nuclei, double-stranded DNAs (dsDNAs), and mitochondrial
   DNAs (mtDNAs) were stained with DAPI (blue), PicoGreen (green), and
   MitoTracker (red), respectively. The white arrows indicate cytosolic
   dsDNA. Values and error bars represent the means ± SDs with an unpaired
   Student’s t test. Scale bar = 20 μm. **P < 0.01. (F) Western blot
   showing the protein levels of p-STING, STING, p-TBK1, TBK1, p-IRF3, and
   IRF3 in CAL27 and SCC15 cells treated with AOH1996. The full-length
   blots are presented in Supplementary Material 1. (G-H) qRT‒PCR analysis
   showing the relative expression levels of IFNβ and CXCL9 mRNAs in CAL27
   and SCC15 cells treated with AOH1996. Values and error bars represent
   the means ± SDs with an unpaired Student’s t test. **P < 0.01

   Phosphorylated H2A.X (p-H2A.X) has been used as a DNA damage marker in
   previous studies [[126]43, [127]44]. Western blot analysis revealed
   that AOH1996 increased p-H2A.X protein levels in HNSCC cells
   (Fig. [128]6B). Consistently, immunofluorescence staining of the
   p-H2A.X marker indicated that AOH1996 treatment caused the accumulation
   of DNA damage in CAL27 and SCC15 cells (Fig. [129]6C). Nuclear DNA
   damage in single eukaryotic cells can be evaluated in a comet assay
   [[130]45]. Damaged DNA fragments separated from intact cellular DNA are
   shown as comet tails under a fluorescence microscope. The results
   revealed that AOH1996 treatment increased the comet tail moments of
   CAL27 and SCC15 cells, suggesting that AOH1996 induced DNA damage in
   HNSCC cells (Fig. [131]6D). To investigate whether increased nuclear
   DNA damage could cause the accumulation of cytosolic DNA, CAL27 and
   SCC15 cells after AOH1996 treatment were stained with PicoGreen and
   MitoTracker. PicoGreen is a specific double-stranded DNA (dsDNA) marker
   that can also stain mitochondrial DNA (mtDNA) [[132]46]. MitoTracker
   was used to stain the mtDNA to avoid visual interference when the
   cytosolic dsDNA was quantified. Confocal microscopy images revealed the
   increased cytosolic DNA accumulation in HNSCC cells treated with
   AOH1996 (Fig. [133]6E).

   The cyclic GMP–AMP synthase-stimulator of interferon genes (cGAS–STING)
   signaling pathway can be activated by accumulated cytosolic dsDNA,
   inducing the phosphorylation of STING, TANK binding kinase 1 (TBK1) and
   interferon regulatory factor 3 (IRF3). Phosphorylated IRF3 regulates
   the expression of IFN [[134]47–[135]49]. Western blot analysis revealed
   that AOH1996 increased the phosphorylated STING (p-STING), p-TBK1, and
   p-IRF3 protein levels in CAL27 and SCC15 cells (Fig. [136]6F). The
   total protein levels of STING, TBK1, and IRF-3 remained unchanged.
   Thus, AOH1996 activated the cGAS–STING signaling pathway in HNSCC. A
   previous study showed that increased expression of p-TBK1 inhibited the
   proliferation and maintenance of CSCs in cancer [[137]50]. Our results
   indicated that AOH1996 inhibited cancer stemness by increasing the
   protein level of p-TBK1.

   qRT–PCR analysis revealed that AOH1996 increased the relative
   expression of IFNβ mRNA in CAL27 and SCC15 cells (Fig. [138]6G). The
   expression of C-X-C motif chemokine ligand (CXCL) is regulated by IFN
   [[139]51]. qRT–PCR analysis revealed that AOH1996 increased the
   relative expression of the CD8^+ T-cell-recruiting chemokine CXCL9 mRNA
   in CAL27 and SCC15 cells (Fig. [140]6H). Thus, AOH1996 stimulated
   antitumor immune responses in HNSCC.

Discussion

   In recent years, targeted therapies that focus on specific molecules
   have been widely applied in the treatment of various cancers, which
   potentiate precise therapeutic strategies [[141]52]. The identification
   and validation of biomarkers has driven the advancement of targeted
   therapy, improving the outcomes and prognosis of patients with HNSCC
   [[142]53]. Previous studies have shown that PCNA is essential for cell
   proliferation and maintenance of genomic stability [[143]14, [144]54].
   PCNA is closely associated with cancer initiation and progression and
   serves as a target in anticancer therapy [[145]54]. PCNA reflects the
   rapid proliferation of HNSCC and is a biomarker for carcinogenesis
   [[146]18, [147]19]. A novel small-molecule PCNA inhibitor, AOH1996, has
   shown great therapeutic potential in many cancers, including non-small
   cell lung cancer, colon cancer, and breast cancer [[148]21]. Compared
   with other PCNA inhibitors, T2AA, PCNA-I1, and AOH1160 [[149]22],
   AOH1996 selectively kills cancer cells but not normal cells [[150]21].
   Here, we investigated the therapeutic effect of AOH1996 on HNSCC.

   In this study, our results demonstrated that AOH1996 inhibited the
   biological behaviors of HNSCC both in vitro and in vivo. AOH1996
   suppressed HNSCC stemness, development, and metastasis. Moreover,
   AOH1996 eliminated CSCs, increased CD8^+ T-cell infiltration, and
   enhanced the efficacy of anti-PD1 immunotherapy. Mechanistically,
   AOH1996 induced cellular DNA damage, activated the cGAS–STING signaling
   pathway, suppressed cancer stemness by upregulating p-TBK1 expression,
   and promoted CD8^+ T-cell-recruiting chemokines by stimulating
   IRF3-mediated transcription (Fig. [151]7A).

Fig. 7.

   [152]Fig. 7
   [153]Open in a new tab

   Mechanism of AOH1996 treatment in HNSCC

   (A) Schematic diagram (created with Biorender.com) illustrating the
   mechanism by which AOH1996 combined with anti-PD1 treatment eliminates
   CSCs and enhances the efficacy of anti-PD1 immunotherapy in HNSCC

   The therapeutic effect of AOH1996 on regulating HNSCC cancer stemness
   remains unclear. A previous study demonstrated that PCNA is essential
   for maintaining cancer stemness through Y211 phosphorylation [[154]14].
   Loss of Y211 phosphorylation in PCNA impairs cancer stemness, showing
   reduced ALDH activity and inhibited tumorsphere formation. In contrast,
   cells with intact Y211 phosphorylation maintain cancer stemness,
   showing increased ALDH activity and enhanced tumorsphere formation
   [[155]14]. Thus, we speculated that PCNA inhibition could impair cancer
   stemness in HNSCC. We further explored whether AOH1996 could inhibit
   CSCs in HNSCC. Here, we used HNSCC PDX CSC-injected nude mice to
   explore the therapeutic effect of AOH1996 in vivo. PDX models can
   reflect the original characteristics of tumor progression in patients
   with cancer and are genetically similar to human cancers, providing a
   promising model for clinical translation in cancer treatment [[156]55,
   [157]56]. Our results showed that AOH1996 eliminated and targeted CSCs
   in HNSCC. Moreover, AOH1996 suppressed HNSCC development and
   metastasis, suggesting the possibility of the clinical application of
   AOH1996 in patients with HNSCC.

   Cancer stemness and immune evasion are important characteristics of
   HNSCC initiation, development, and metastasis [[158]57, [159]58]. The
   interaction between CSCs and the tumor microenvironment enables CSCs to
   evade immune surveillance in HNSCC [[160]26, [161]33]. The tumor
   microenvironment in HNSCC is immunosuppressive with low levels of
   lymphocyte infiltration [[162]59]. Here, we investigated the effect of
   AOH1996 on the tumor microenvironment of HNSCC. Immune checkpoint
   inhibitor therapy has been approved for treating metastatic and
   recurrent HNSCC. However, most patients with HNSCC are resistant to PD1
   blockade-based immunotherapy [[163]11–[164]13]. CSCs can create
   immunosuppressive niches in the tumor microenvironment and are
   associated with resistance to PD1 blockade-based immunotherapy
   [[165]42, [166]51]. Previous studies have found that CSCs induce
   immunosuppression through high expression of PD-L1, TGF-β, and the
   recruitment of regulatory T cells (Tregs) [[167]60]. Thus, the AOH1996
   treatment, which inhibits CSCs in HNSCC, may improve the efficacy of
   immunotherapy.

   The 4NQO-induced HNSCC Bmi1^CreER, Rosa^tdTomato mouse model enables
   CSC lineage tracing in vivo [[168]28]. We used this model to
   investigate the effect of AOH1996 on CSCs in HNSCC. Our results
   suggested that AOH1996 eliminated CSCs and enhanced the therapeutic
   effect of anti-PD1 immunotherapy, suppressing the development and
   metastasis of HNSCC. Both the AOH1996 treatment alone and the
   combination treatment with anti-PD1 increased the recruitment of CD8^+
   T cells in the tumor microenvironment of HNSCC. The increased
   infiltration of CD8^+ T lymphocytes can directly identify, target, and
   destroy cancer cells in the HNSCC tumor microenvironment, which is
   related to a favorable prognosis [[169]59, [170]61]. Thus, AOH1996
   targets CSCs in HNSCC and improves the efficacy of anti-PD1
   immunotherapy. Taken together, AOH1996 prevented the immune evasion of
   CSCs, activated the tumor immune response, and inhibited HNSCC
   progression and metastasis. Moreover, combination treatment with
   AOH1996 and anti-PD1 exhibited therapeutic effects on HNSCC without
   causing discernible toxicity or side effects in vivo.

   Mechanistically, AOH1996 can cause DNA double-strand breaks and genome
   instability [[171]21]. Previous studies have shown that the DNA damage
   response activates innate immunity through the cGAS–STING pathway in
   cancers [[172]62–[173]65]. In this study, we explored the underlying
   mechanism of AOH1996 in HNSCC by bioinformatic analysis. The results
   showed that AOH1996 induced HNSCC cellular DNA damage. Moreover,
   AOH1996 increased the expression levels of p-STING, p-TBK1, and p-IRF3
   in HNSCC, activating the cGAS–STING signaling pathway. A previous study
   revealed that in glioblastoma cancer, TLR4 overexpression increases the
   phosphorylation of TBK1, and increasing p-TBK1 inhibits the expression
   of the CSC markers SOX2, OCT4, and NANOG [[174]50]. Thus, p-TBK1
   upregulation inhibits CSC proliferation and maintenance [[175]50]. Our
   results indicated that AOH1996 eliminated CSCs by upregulating p-TBK1
   expression in HNSCC. Moreover, IFN regulates the expression of CXCL
   chemokines [[176]51]. Our results showed that AOH1996 increased the
   expression of IFNβ and induced the recruitment of the CD8^+
   T-cell-recruiting chemokine CXCL9 by stimulating IRF3-mediated
   transcription. Thus, AOH1996 stimulated antitumor immune responses of
   HNSCC cells by inducing DNA damage and activating the cGAS–STING
   signaling pathway.

   However, there are limitations in this study. The translational
   potential of AOH1966 is constrained by its unknown effects in human
   HNSCC samples or patient-derived immune microenvironments. Future
   studies should further validate the therapeutic efficacy of AOH1996 in
   human HNSCC samples. The molecular mechanism by which AOH1996 modulates
   PCNA in HNSCC CSCs requires further investigation. Moreover, how
   AOH1996 modulates immune checkpoints such as PD-L1 and Tregs also needs
   to be explored.

Conclusions

   Our study demonstrated that the PCNA inhibitor AOH1996 suppressed HNSCC
   biological behaviors, impaired cancer stemness, prevented development
   and metastasis, enhanced the effect of anti-PD1 immunotherapy, promoted
   cellular DNA damage, and stimulated antitumor immune responses. This
   study provides a novel therapeutic strategy for HNSCC.

Supplementary Information

   Below is the link to the electronic supplementary material.
   [177]Supplementary Material 1^ (34.4KB, pdf)
   [178]Supplementary Material 2^ (314.8KB, pdf)

Acknowledgements