A microprotein N1DARP encoded by LINC00261 promotes Notch1 intracellular domain (N1ICD) degradation via disrupting USP10-N1ICD interaction to inhibit chemoresistance in Notch1-hyperactivated pancreatic cancer

The extensively activated Notch signaling pathway in pancreatic cancer cells is important in carcinogenesis, chemoresistance, and recurrence. Targeting this pathway is a promising therapeutic strategy for pancreatic cancer; however, few successful approaches have been reported, and currently used molecular inhibitors of this pathway exhibit limited clinical benefits. In this study, we identified a previously uncharacterized microprotein, Notch1 degradation-associated regulatory polypeptide (N1DARP), encoded by LINC00261. N1DARP knockout accelerated tumor progression and enhanced stem cell properties in pancreatic cancer organoids and LSL-Kras, LSL-Trp53, and Pdx1-Cre (KPC) mice. Mechanistically, N1DARP suppressed canonical and non-canonical Notch1 pathways by competitively disrupting the interaction between N1ICD and ubiquitin-specific peptidase 10 (USP10), thereby promoting K11- and K48-linked polyubiquitination of N1ICD. To evaluate the therapeutic potential of N1DARP, we designed a cell-penetrating stapled peptide, SAH-mAH2-5, with a helical structure similar to that of N1DARP that confers remarkable physicochemical stability. SAH-mAH2-5 interacted with and promoted the proteasome-mediated degradation of N1ICD. SAH-mAH2-5 injection provided substantial therapeutic benefits with limited off-target and systemic adverse effects in Notch1-activated pancreatic cancer models. Taken together, these findings confirm that N1DARP acts as a tumor suppressor and chemosensitizer by regulating USP10-Notch1 oncogenic signaling, and suggest a promising therapeutic strategy targeting the N1DARP–N1ICD interaction in Notch1-activated pancreatic cancer.

gRNAs targeted exon 4 of LINC00261 and full-length N1DARP.The highest-scoring designs were synthesized using in silico prediction and screening.The sequences were as follows: gLINC00261-1:

Establishment of stably expressed cell lines and organoids
Lentiviral vectors expressing wild-type LINC00261, LINC00261 with start codon mutant (ATG to ATT) of N1DARP (LINC00261-N1DARPmut), wild-type N1DARP, and start codon mutant (ATG to ATT) of N1DARP (N1DARPmut) were transfected into HEK293T cells together with packaging vectors psPAX2 (Addgene, Watertown, MA, USA) and pMD2G (Addgene) for lentivirus generation through Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's instructions.Wild-type Capan-1 or Capan-1 with LINC00261 knockout were transfected with the corresponding lentiviruses mentioned above, along with polybrene (5 mg/ml).After incubation for 24 h, the cells were selected using 10 mg/ml blasticidin for 5 days.Similarly, to establish stably transfected tumor organoids, 3D cultured patient-derived organoids (PDAC-1 and PDAC-2) were digested into single cells or fragments and plated in a 2D culture environment.After adherence, the cells were transfected with the aforementioned lentiviruses, along with polybrene (5 mg/ml), and selected using 10 mg/ml blasticidin for 5 days.The remaining organoids were digested again and maintained in Matrigel until further passaging.This process is illustrated in Fig 2a.

Quantitative real-time PCR (qRT-PCR)
Total RNA from pancreatic tissues and cell lines was extracted using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA).RNA from nuclear and cytoplasmic fractions was separated using a PARIS Kit.

Cell proliferation assay
Equal numbers of stably transfected pancreatic cancer cells were plated in 96-well plates (2000 cells/well).Cell viability was measured using a CCK8 (Dojindo, Kumamoto, Japan).A colony formation assay (1000 cells/well in 6-well plates) was performed to determine the proliferative capacity of pancreatic cancer cells.In addition, a Cell-Light EdU DNA cell proliferation kit (RiboBio, Guangzhou, China) was used to detect cell proliferation potential, following the manufacturer's instructions.

Tumor sphere formation assay
Stably transfected pancreatic cancer cells (Capan1 or Panc1) were digested and gently resuspended in a single-cell suspension of StemXVivo Serum-Free Tumorsphere Media (R&D Systems, Minneapolis, MN, USA) containing heparin and hydrocortisone.Ten thousand cells were resuspended in 1 ml complete StemXVivo medium and transferred to each well of an ultra-low adhesion 12-well plate.The cell culture plate was then moved into a 37℃ cell culture incubator and maintained for 7 days.

Immunofluorescence (IF) and colocalization assay
For pancreatic cancer cell lines, Capan1 and Panc1 seeded on coverslips were briefly washed with PBS and fixed with 4% buffered paraformaldehyde for 15 min, permeabilized with 0.5% Triton X100 for 15 min, blocked with 3% BSA for 30 min at 25℃, and then stained with N1DARP, USP10, and N1ICD antibodies followed by corresponding secondary antibodies.Nuclei were counterstained with DAPI.To prepare pancreatic cancer organoids, PDAC-1 cells cultured in Matrigel were briefly washed with PBS, dispersed, and fixed in 4% buffered paraformaldehyde.The cells were then embedded in agarose, fixed, and sliced.The sections were blocked with 3% BSA and stained with N1DARP, USP10, and N1ICD antibodies, followed by incubation with the corresponding secondary antibodies.Nuclei were counterstained with DAPI.The images were captured using a confocal fluorescence microscope (Olympus Microsystems, Tokyo, Japan).Quantitative image analysis was performed using ZEN 2.0 software.

Immunohistochemistry (IHC) analysis and tissue microarray
Pancreatic tumor tissue sections were analyzed by two pathologists who were blinded to the patient's identity.The sections were deparaffinized with xylene, rehydrated in an ethanol series, submerged in EDTA antigenic retrieval buffer (pH 8), and microwaved for antigenic retrieval.Subsequently, the sections were treated with 3% hydrogen peroxide in methanol to quench endogenous peroxidase activity, followed by incubation with 1% goat serum albumin to block non-specific binding.Tissue sections were incubated with the corresponding antibodies overnight at 4°C.After washing, the tissue sections were treated with goat anti-mouse/rabbit IgG horseradish peroxidase polymer for 20 min.3, 3'-Diaminobenzidine was used as the chromogen.Tissue microarrays of seventy-five paraffinembedded pancreatic cancer tissue sections were prepared according to manufacturer's instructions.
Briefly, tissue cylinders (diameter, 0.6 mm) were obtained from selected regions of the donor block and punched precisely into a recipient paraffin block using a tissue arraying instrument (Beecher Instruments, Sun Prairie, WI, USA).Consecutive 5 μm sections of the microarray blocks were made with a microtome.The H-scores were determined by combining the intensity of staining with the proportion of positively stained tumor cells.The intensity was graded as follows: 0 = negative; 1 = weak; 2 = moderate; and 3 = strong.The proportion of positive tumor cells was graded: 0, 0%-5%; 1, 5%-25%; 2, 26%-50%; 3, 51%-75%; 4, 75%-100%.The final score was calculated by multiplying the two primary scores.Final scores of 0-4 were defined as negative, and final scores of 8-12 as positive.

Flow cytometry
Cells were seeded in 6-well plates overnight to reach 70%-80% confluence.After the indicated treatments, the cells were digested using trypsin, washed twice with phosphate-buffered saline, and stained using the Annexin Ⅴ-FITC/PI or Annexin Ⅴ-633/PI Apoptosis Detection Kit (Dojindo) according to the manufacturer's instructions.Stained cells were analyzed using a FACSCelesta multicolor flow cytometer (Becton Dickinson, Franklin Lakes, NJ USA) and the data were processed using FlowJo software.

In situ proximity ligation assay (PLA) assay
The Duolink In Situ Red Kit (Sigma-Aldrich, St. Louis, MO, USA) was used to visualize direct protein-protein interactions in Panc1 cells.Briefly, cells were seeded onto 6-well plates overnight at 70%-80% confluence.The cells were washed twice with cold PBS and fixed with 4% paraformaldehyde for 15 min at room temperature.The fixed cells were then permeabilized with PBS containing 1% Triton X-100 for 30 min, subsequently blocked in Blocking Solution (Sigma-Aldrich) at 37°C for 1 h and incubated with primary antibodies (anti-N1ICD [CST, #4147]; anti-USP10 [CST, #8501] and anti-N1DARP [generated and validated by Abcam]) overnight at 4°C.The next day, cells were washed twice with washing buffer, and incubated with PLA probes in a ratio of 1:4 in antibody diluent for 1 h at 37°C.Subsequently, the cells were incubated with the ligation solution at 37°C for 30 min and then with amplification solution at 37°C for 100 min.Duolink in situ mounting medium mixed with DAPI was added to the cells and incubated at room temperature for 15 min.Images were captured using a confocal microscope (FV1000; Olympus).

Preparation of fusion proteins and in vitro pulldown assay
Glutathione S-transferase (GST)-fused N1ICD was cloned into a pGEX-2T vector, which was then expressed in Escherichia coli and affinity-purified using glutathione-Sepharose (Pharmacia and Upjohn, London, UK).Ten micrograms of purified GST and GST-N1ICD fusion protein was mixed with Panc1 cell lysates transfected with 10 μg of N1DARP-Flag or USP10-Flag and incubated in binding buffer (0.8% BSA in PBS in the presence of the protease inhibitor mixture) overnight at 4°C under gentle rotation.Subsequently, the binding reaction was added to 30 μL of glutathione-Sepharose beads and mixed at 4°C for 2 hours.The beads were washed five times with binding buffer, resuspended in 50 μL of 2 × SDS-PAGE loading buffer, and resolved on a 10% gel.Protein bands were detected with anti-N1DARP or anti-USP10 antibodies by western blotting.
Biotin pull-down analysis was performed as previously described 3 .Biotin-labelled SAH-mAH2-5 and SAH-CTRL were generated using EZ-Link™ NHS-Biotin (Thermo Fisher Scientific).Biotinylated polypeptides were incubated with the PDAC-R organoid cell lysates at 37 °C for 30 min.Interacting protein complexes were isolated using streptavidin-conjugated Dynabeads (Thermo Fisher Scientific).
The precipitated components were subjected to SDS-PAGE, followed by Coomassie blue staining.
Differentially expressed proteins were cut for mass spectrometry (LTQ Orbitrap XL).

Immunoprecipitation and Mass Spectrometry (MS)
Pancreatic cancer cells transfected with the indicated plasmids or lentiviruses were collected and lysed on ice for 10 min.Centrifugation was performed to obtain the supernatant, followed by incubation with the appropriate antibody and Protein A/G Plus-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.The immunocomplex was washed 4-6 times and boiled in 2×SDS sample buffer for 5 min.The coprecipitates were resolved by SDS-PAGE and blotted with specific antibodies.
Bound proteins were dissolved in SDS sample buffer and analyzed by immunoblotting.For mass spectrometry (MS), Capan1 cells were treated with Flag-N1DARP for 48h and lysed.Whole cell lysates were extracted and immunoprecipitated with an anti-FLAG antibody using a Pierce CO-IP Kit (Thermo Fisher Scientific).The eluents were separated using SDS-PAGE, followed by Coomassie blue staining, as shown in Fig. 4C.The bands were extracted from the gel and subjected to LC-MS/MS sequencing and data analysis by QL Bio Biotechnology Co., Ltd.(Beijing, China).MS/MS data were searched against the human FASTA sequence from UniProt using an in-house Proteome Discoverer (Version PD1.4,Thermo Fisher Scientific).Peptides assigned only to a given protein group were considered unique.

Tumor xenograft assay
All animal experimental procedures were performed in compliance with the institutional ethical requirements and approved by the Shanghai Jiao Tong University School of Medicine Committee for the Use and Care of Animals.Nude male BALB/c mice (4-6 weeks old) were purchased from the Chinese Academy of Sciences (Shanghai, China) and maintained in a specific pathogen-free facility.
For the subcutaneously injected tumor model, pancreatic cell lines (Capan1 and Panc1) (1×10 7 cells/site) and patient-derived organoids (PDAC1 and PDAC2) (5×10 6 cells/site) transfected with the empty vector and N1DARP or LINC00261 were injected subcutaneously into the right flank of each mouse.Tumor volumes were measured every 7 days for 35 days from the first injection using the formula: tumor volume (mm 3 ) = 1/2 (a×b 2 ), in which "a" represents the longest longitudinal diameter, and "b" is the longest transverse diameter.
In vivo imaging was performed to monitor the development of Capan1 cells and organoids in BALB/c nude mice treated with peptides using an IVIS Spectrum optical imaging system every week after inoculation.
To evaluate the toxicity of SAH-mAH2-5, healthy BALB/c nude mice (5 weeks) were intravenously administered SAH-CTRL or SAH-mAH2-5 (2 mg/kg) once a week for 4 weeks.Mice were then sacrificed, and some organs, including the pancreas, lungs, spleen, kidneys, liver, and colon, were extracted and HE stained to screen for morphological abnormalities.In addition, serum was collected for the biochemical analysis of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) using a Chemistry Analyzer TBA-40FR (Toshiba Medical System, Tokyo, Japan).