{"paper_id":"1cd483fd-4890-47b2-95e6-9dcae08054b9","body_text":"ADT-030, a novel PDE10 inhibitor, demonstrates potent antitumor activity in pancreatic ductal 1 \nadenocarcinoma 2 \nDhana Sekhar Reddy Bandi 1 , Ganji Purnachandra Nagaraju 1 , Sujith Sarvesh 1 , Jeremy B. Foote 2 , 3 \nAdam B. Keeton 3,1 1 , Xi Chen 3, 11 , V eronica Ramirez-Alcantara 4 , Thomas Holmes 3 , Mehmet Ak ce 1 , 4 \nAnju Singh 5 , Craig M. Powell 5 , Santosh Behera 6 , Asfar S. Azmi 7 ,  Elmar Nurmemmedov 8 , Ivan 5 \nBabic 8 , Gregory S. Gorman 9 , Lori Coward 9 , Donald J. Buchsbaum 10 , Y ulia Y . Maxuitenk o 3 , Gary A. 6 \nPiazza 3,1 1 , and Bassel F . El-Rayes 1*  7 \n1 Division of Hematology and Oncology , University of Alabama at Birmingham, Birmingham, AL 8 \n35233, USA.  9 \n2 Department of Microbiology , University of Alabama at Birmingham, Birmingham, AL 35294, 10 \nUSA. 11 \n3 Drug Discovery and Development Department, Harrison College of Pharmacy , Auburn 12 \nUniversity , Auburn, AL 36849, USA. 13 \n4 Health Biobank and Histology Core F acility , Mitchell Cancer Institute, University of South 14 \nAlabama, Mobile, AL 36604, USA. 15 \n5 Department of Neurobiology , University of Alabama at Birmingham, Birmingham, AL 35294, 16 \nUSA. 17 \n6 Department of Biotechnology, National Institute of Pharmaceutical Education and Research, 18 \n(NIPER), Ahmedabad, Gujarat- 382355, India. 19 \n7 Department of Oncology , Karmanos Cancer Institute, W ayne State University School of  20 \nMedicine, Detroit, MI 48201, USA.  21 \n8 CellarisBio LLC, San Diego, CA 92121, USA 22 \n9 Department of Pharmaceutical, Social and Administrative Sciences, McWhorter School of 23 \nPharmacy, Samford University, Birmingham, AL 35229, USA. 24 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n10 Department of Obstetrics and Gynecology , University of Alabama at Birmingham, Birmingham, 25 \nAL 35233, USA. 26 \n11 ADT Pharmaceuticals, LLC, Orange Beach, AL 31691, USA. 27 \n*Corresponding Author:  Bassel F . El-Rayes, MD , Albert F . LoBuglio Endowed Chair for 28 \nT ranslational Cancer Research, Division Director , Hematology and Oncology , Deputy Director , 29 \nO'Neal Comprehensive Cancer Center , Heersink School of Medicine, University of Alabama at 30 \nBirmingham. belrayes@uabmc.edu 31 \n 32 \nAbstract 33 \nPhosphodiesterase 10 (PDE10) was previously reported to be overexpressed in various cancers 34 \nand essential for cancer cell prolif eration and survival. Here, we studied a novel PDE10 inhibitor , 35 \nADT-030, and found it to potently and selectively inhibit KRAS mutant PDAC cell prolif eration 36 \nand clonogenicity by inducing G2/M arrest and apoptosis. ADT-030 also inhibited motility of 37 \nPDAC cells  in vitro . These eff ects were mediated by increased cAMP /cGMP levels and activation 38 \nof PKA/PK G. The growth inhibitory activity of ADT-030 was associated with reduced β- catenin  39 \nand RAS signaling. Notably , ADT-030 also inhibited the growth of KRAS G12D  and KRAS G12 C  mutant 40 \nPDAC cells resistant to allele-specific KRAS inhibitors. Oral administration of ADT-030 41 \nsignificantly suppressed tumor growth, reduced lung and liver metastasis, and increased 42 \nsurvival without systemic toxicity in syngeneic and patient-derived xenograft (PDX) PD AC 43 \nmodels. ADT-030 also increased chemotherapy response in orthotopic PDAC models. Immune 44 \nphenotyping and single-cell RNA sequencing revealed remodeling of the tumor 45 \nmicroenvironment by ADT-030 with a more favorable i mmune suppressive profile to activate 46 \nanti-tumor immunity . These results show that ADT-030 is a promising drug development 47 \ncandidate  for the treatment of KRAS-mutant PDAC capable of simultaneously targeting k ey 48 \noncogenic  signaling pathways, r esulting in tumor-intrinsic and immunomodulatory eff ects.  49 \nKey words: KRAS-mutant PDAC, targeted therapy , immune checkpoint inhibition, T and NK cell 50 \nactivation, PKA/PK G signaling, myeloid polarization, PDE10, β-catenin. 51 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n 52 \n 53 \nIntroduction 54 \nPancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer-55 \nrelated mortality in the U.S., with a five-year survival of under 13% 1 . Sur vival of patients with 56 \nmetastatic PDAC r emains poor even with the current chemotherapeutic regimens such as 57 \nFOLFIRINO X or gemcitabine and nab-paclitax el 2 . The aggressive nature of PDAC is mainly 58 \nattributed to activation of multiple compensatory signaling path ways driv ing proliferation an d  59 \nsurvival, along with a hypoxic microenviroment driven by dense desmoplastic stroma and 60 \ndecreased vascular perfusion 3- 7 . Although allele-specific KRAS inhibitors have demonstrated  61 \npromising activity in early-phase clinical trials in patients with PDAC, the development of 62 \nresistance remains a major challenge and highlights the need to identify new therapeutic  63 \ntargets and agents with broader activity 8-1 0 . A better understanding of the complexity of 64 \noncogenic signaling, the importance of stroma, and the role of immune evasion in PDAC 65 \nprogression is critical for the development of more effective target-directed drugs for the 66 \ntreatment of PDAC 11 .  67 \nMutations in the KRAS gene have been reported in about 90% of PDAC patients, with the 68 \nmajority at the 12 th  codon (KRAS G12 D , KRAS G12V , and KRAS G12 C ) 12 . These mutations result in the 69 \nconstitutive activation of downstream pathways such as RAS/RAF /MEK and PI3K/ AKT /mT OR 70 \nsignaling to promote the proliferation, survival, and metabolic reprogramming of PDAC 13 . 71 \nAlthough the mutation frequency of CTNNB1 is relatively low in PDAC , β-catenin signaling is 72 \naberrantly activated from WNT overexpression, which, along with KRAS mutations, contributes 73 \nto the aggressive behavior of PDAC 6 . KRAS has been reported to form a complex with β- cateni n  74 \nto modulate the phosphorylation of the transcription factor TCF4, leading to crosstalk between 75 \nthese two oncogenic signaling pathways 14 , 15 . In addition, both β-catenin and RAS signaling have 76 \nbeen reported to be activated with gemcitabine treatment, suggesting that these pathways play 77 \na major role in therapy resistance in PDAC 16 . Given the interactions between RAS and β- cateni n  78 \nin PDAC, a strategy that targets both pathways with single inhibitor could off er more robust 79 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\ntherapeutic approach. Emerging evidence also suggests that simultaneous inhibition of multiple 80 \noncogenic  pathways  not only increases the potential for efficacy of target-directed anticancer 81 \ndrugs but also reduces the potential for resistance by overcoming compensatory signaling 82 \nmechanisms 17,1 8 . KRAS mu tated tum ors can u til ize β-caten in si gnaling to maintain a stem-lik e, 83 \nimmune-depleted, niche within the tumor immune microenvironment (TiME) resulting in 84 \nrelapse following chemotherapy . T arg eting both pathways with one inhibitor could also sensitize 85 \ncancer cells to undergo apoptosis while modulating the TiME to favor immune activation 86 \nleading to inhibition of tumor growth 19 .  87 \nPhosphodiesterase (PDE) isoenzymes hydrolyze and inactivate the second messengers, 88 \ncyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) 20 . 89 \nAlthough understudied, PDEs have been reported to play a role in the initiation and progression 90 \nof PDAC and other cancers  21 , 22 . Notably , the cAMP /PKA and cGMP /PK G signaling ax es have 91 \nbeen reported to suppress MAPK signaling downstream from KRAS 23 , 2 4 . In addition, PDE 92 \nisoenzymes have been shown to regulate β-catenin signaling, which can also influence RAS 93 \nsignaling 25- 27 . Several PDE isozyme families, most notably , PDE4, PDE5, and PDE10 have been 94 \ninvestigated as anti-cancer targets inhibition of which can impact cancer cell prolif eration, 95 \nsurvival, and immune responses 22, 28 . Notably , isozyme-specific inhibitors of the dual 96 \ncAMP /cGMP degrading PDE10 isozyme and gene silencing approaches have been reported to 97 \nselectively inhibit the prolif eration and induce apoptosis of cells from colon, lung, and ovarian 98 \ncancers through activating cGMP /PKG signaling and disrupting RAS signaling and WNT /β-99 \ncatenin-mediated transcription  25, 27 ,2 9 ,30 . These findings established the basis for our hypothesis 100 \nthat ADT-030, a novel PDE10 inhibitor  with properties distinct from known PDE10 inhibitors 101 \ndeveloped for CNS disorders, can block both RAS and β-catenin signaling and result in tumor 102 \ninhibition and modulation of the TiME in PDAC.  103 \n 104 \n 105 \n 106 \n 107 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n 108 \n 109 \n 110 \nMaterials and methods 111 \nADT-030 synthesis   112 \nThe synthesis of ADT-030 [(S,Z)-2-(5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-113 \ninden-3-yl)-N-(1-methylpyrrolidin-3-yl)acetamide] is based on a procedure originally described 114 \nin US patent 20200223815 using 3-(4-methoxyphenyl)-2-methylacrylic acid as the starting 115 \nmaterial. 116 \nHuman scRNA-seq datamining 117 \nThe Single Cell RNA seq Pancr eatic Cancer A tlas R Data Serialization (RDS) file 118 \n( https://zenodo.org /records/14199536 ) was downloaded 31 . This dataset has normalized and 119 \nscaled scRNA-seq (10x genomics sequencing) data from 12 studies containing 229 patients 120 \nacross the groups. The ductal cells were identified from the main dataset and the expression 121 \nlevels of PDE10 was queried across the tissue samples (donor , adjacent normal, primary tumor , 122 \nand meta static  lesi on) as described in the previously published paper 31 . 123 \nCellular target engagement assay 124 \nHEK293 cells expressing PDE10 fused to the MICRO-T AG reporter were subjected to a 125 \ntemperature-series denaturation assay to determine the aggregation midpoint under cellular 126 \nconditions as previously described  32 . Cells were heated for 10 min across a defined 127 \ntemperature range, followed by non-denaturing lysis and fluorescence complementation 128 \nquantification. Fitting the resulting thermal curve yielded a T agg₅₀ of 44°C for PDE10. This 129 \ndefined T agg₅₀ provided the fix ed challenge temperature for subsequent experiments to 130 \ndetermine if ADT-030 binds PDE10 in intact cells. 131 \nCell culture 132 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nPanc-1 (A TCC# CRL-1469; RRID:CVCL_0Q68), AsPC-1 (A TCC# CRL-1682; RRID:CVCL_0152), Panc 133 \n02.03 (A T CC# CRL-2553; RRID:CVCL_1633), Panc 10.05 (A T CC# CRL-2547; RRID:CVCL_1639), MIA 134 \nPaCa-2 (A T CC# CRL-1420; RRID:CVCL_0428), BxPC-3 (A TCC# CRL-1687; RRID:CVCL_0186), and 135 \nKLE (A TCC# CRL-1622; RRID:CV CL_1329) cell lines were obtained from American T ype Culture 136 \nCollection (A TCC, Manassas, V A, USA) and maintained as recommended. Mouse PDAC cell line 137 \n2838c3 (Kerafast# EUP013-FP; RRID:CVCL_YM18) was purchased from Kerafast (Boston, MA, 138 \nUSA). MKN1 (Accegen#  ABC-T C0685; RRID:CVCL_1415) cell line was purchased from Accegen 139 \nInc (F airfield, NJ, USA). Dr . Gregory Lesinski, Emory University , USA, gifted the KPC cell line. Dr . 140 \nDenis C Guttridge, Medical University of South Carolina, USA, gifted the KPCML1 cell line. All 141 \ncells were grown in appropriate medium as recommended by the A TCC and Kerafast with either 142 \nDulbecco’ s Modified Eagle Medium (DMEM; A TCC# 30-2002) or Roswell Park Memorial Institute 143 \n(RPMI)-1600 Medium (RPMI; A T CC# 30-2001) supplemented with 10% f etal bovine serum (FBS; 144 \nA TCC# 30-2020) and 1% penicillin/streptomycin (A TCC# 30-2300) under 5% CO 2 . Additionally , 145 \nMIA PaCa-2 cells received 2.5% horse serum (Thermo# 26050088). 146 \nPhosphodiesterase assay  147 \nThe enzymatic activity of recombinant PDE10 was measured using the Immobilized Metal 148 \nAffinity Particle (IMAPTM) fluorescence polarization (FP) progressive binding system (Molecular 149 \nDevices; San Jose, CA; USA) as previously described to determine the inhibitory eff ect of ADT-150 \n030  33 . FP was measured using a Synergy H4 Hybrid plate reader (BioT ek; Santa Clara, CA; USA). 151 \nRecombinant PDE10 was purchased from BPS Biosciences (San Diego, CA; USA). 152 \nProliferation assa y 153 \nHuman and murine PDAC cells with KRAS mutations (KRAS G12D  and KRAS G12 C ) and wild-type cells 154 \n(BxPC-3) were plated at a density of 5×10 3  cells/well in 96-well plates. After 20 hrs, cells were 155 \ntreated with ADT-030 or PF-2545920 (a known PDE10 inhibitor)  34  in a dose-dependent manner . 156 \nAfter 72 hrs, the medium was removed, 10-μL methylthiazole tetrazolium (MTT ; 5 mg /mL in 157 \nPBS; Sigma-Aldrich# 475989) was added, and cells were incubated for another 2 hrs at 37 °C. 158 \nThe resulting formazan crystals were solubilized in 100 μL DMSO (Sigma-Aldrich# D2438), and 159 \nabsorbance was measured at 570 nm with a ref erence wavelength of 630 nm. 160 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nClonogenic as say 161 \nMouse derived PDAC cell line 2838c3 (KRAS G12D ) and human derived cell line MIA PaCa-2 162 \n(KRAS G12C ) were plated in 6-well culture plates (2 × 10 3  cells/well). The cells were then treated 163 \nwith either DMSO , ADT-030 at 0.5, 1, 2, and 5 µM, or PF-2545920 at 5, 10, 25, 50, and 100 µM 164 \nevery 3 days. After 10 days, the cells were stained with a 0.005% Coomassie Brilliant Blue R-250 165 \nsolution, and plates were imaged using an Epson Perf ection V850 Pro Photo Scanner (USA). The 166 \nresulting colonies were counted using ImageJ (RRID:SCR_003070).  167 \nMotility assa y 168 \nT o measure eff ects on cell motility , 2838c3 and MIA PaCa-2 cells were grown in 6-well plates 169 \nuntil they reached confluence. The cells were treated with DMSO or ADT-030 (0.5, 1, 2, and 5 170 \nµM). A scratch was created using a sterile 10-µL pipette tip, and cell migration was monitored 171 \ndaily using light microscopy . Quantification of cell movement was performed using ImageJ 172 \nsoftware.  173 \nApoptosis as say 174 \nThe binding of annexin V to cells was measured using the PE-Annexin V Apoptosis Detection Kit 175 \nI (BD Biosciences# 559763), according to the manufacturer ’ s protocol. Briefly , 2838c3 and MIA 176 \nPaCa-2 cells were treated with either DMSO or ADT-030 (at 2 and 5 µM) for 72 hrs. After 177 \ntreatment, cells were collected, washed twice with cold 1x PBS (A T CC# 30-2200), and suspended 178 \nin 1x Binding Buff er . The cells were then stained with 300 µL PE Annexin V F ITC and 5-µL of 179 \npropidium iodide (PI) and incubated for 15 min in the dark. Flow cytometry analysis was 180 \nperformed using a BD LSR Fortessa Flow Cytometer and data were analyzed using FlowJo 181 \n(RRID:SCR_008520). 182 \nCell cycle assay 183 \n2838c3 and MIA PaCa-2 cells were treated with either DMSO or ADT-030 (2 and 5µM) for 24 hrs 184 \nand the cells were trypsinized and centrifuged at 1,000x g for 3 min at 4 °C. Cells were then 185 \nwashed with PBS and fix ed using 70% ethanol at 4 °C overnight. The following day , the cells 186 \nwere incubated with 1 mL of RNAse solution for 30 min in the dark and stained with PI for 30 187 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nmin. The cells were then analyzed for cell cycle arrest using a BD LSR Fortessa Flow Cytometer . 188 \nThe experiment was repeated thrice independently , and the results were analyzed using FlowJo.  189 \nImmunoblotting  199 \nWhole-cell protein extracts were prepared using RIP A L ysis Buff er (Pierce Chemical, Rockford, IL, 200 \nUSA) containing protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase 201 \ninhibitor cocktail (Sigma-Aldrich, St. Louis, MO , USA). L ysed samples were centrifuged at 12,000 202 \nrpm for 40 min, and clarified supernatants were stored at −80 °C. Protein concentrations were 203 \ndetermined using the Pierce Bicinchoninic Acid (BCA) protein assay kit. Equal amounts of 204 \nprotein samples were electrophoresed on 4-20% sodium dodecyl sulfate (SDS)- polyacrylamide 205 \ngels (BIO-RAD , #4568096) and transferred onto PVDF membranes (Invitrogen, #IB34001). The 206 \nmembr anes were then incubated with antibodies diluted in 2% Bovine Serum Albumin (BSA, 207 \nFisher Scientific, #BP1600) for 2 hrs at room temperature. Primary antibodies were pERK, ERK, 208 \npAKT , AKT , pmT OR, mT OR, pP70s6 kinase, p70s6 kinase, pCREB, CREB, Bcl-2, VEGF A, PDE3B, 209 \nPDE4C, PDE4D , LC3A/B, cleaved P ARP , cleaved caspase 3, non phospho β-catenin, β-catenin, 210 \npV ASP , V ASP , and anti-β-actin. Incubation with HRP-link ed secondary antibodies (CST , 211 \n#7074/7076;) at a dilution of 1:3000 in a 2% BSA solution was carried out for 1 hr at room 212 \ntemperature. The signal was then detected on a LI-COR Odyssey DLx Imager using the ECL 213 \nchemiluminescence detection system (Thermo Fisher Scientific, #34577). 214 \nMeasurement of intracellular cAMP and cGMP levels  215 \n2838c3 and MIA PaCa-2 cells were treated with ADT-030 at varying doses, harvested, and the 216 \nintracellular cAMP and cGMP levels were measured. Enzyme immunoassay kits were used to 217 \ndetect cAMP (Cat# 581001, Cayman) and cGMP (Cat# 581021, Ca yman) by following 218 \nmanufacturer ’ s instructions. The results were expressed as picomoles/µg of total protein.  219 \nImmunohistochemistry (IHC) and immunofluor escence (IF) 220 \nPar affin-embedded tumor tissue slides from 2838c3 and KPC orthotopic studies were used for 221 \ndetecting the expression patterns of extracellular matrix (ECM) remodeling, apoptosis and 222 \nautophagy mark ers through IHC and IF . H&E-stained sections from lungs, liver , and primary 223 \ntumor were evaluated by a board-certified veterinary anatomic pathologist (JBF) to quantify the 224 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nnumber of metastatic lesions in a blinded fashion. For IHC, tumor slides were deparaffinized 225 \nwith xylene for 20 min and rehydrated using 100% and 90% ethanol for 20 min each. The slides 226 \nwere then washed twice with deionized water for 5 min. Antigen retrieval was performed in 10 227 \nmM citrate bu ffer by microwaving for  10 min followed by two w ashes with deionized water for 5 228 \nmin each. The slides were then quenched in BLO XALL blocking solution (Cat# PK-8200, V ector 229 \nLabs) for 15 min to block endogenous peroxidase activity and the slides were block ed with 2.5% 230 \nnormal horse serum for 30 min. Primary antibodies were diluted in 2.5% normal horse serum 231 \nand added to the slides and incubated overnight at 4°C in a humidified chamber . The slides 232 \nwere then washed twice with 1% serum in PBS-T for 10 min each. For IF , the secondary 233 \nantibodies were diluted in 1% serum in PBS-T and incubated for 2 hrs at room temperature. The 234 \nslides were washed twice with 1% serum in PBS-T , and nuclear labelling was performed with 235 \nDAPI containing anti-fade mounting medium. A coverslip was placed and sealed with nail polish. 236 \nFor IHC, the slides were incubated with prediluted biotinylated horse anti-mouse/rabbit IgG 237 \nsecondary antibody for 30 min and washed in PBS-T for 15 min. Then slides were incubated with 238 \nVECT AST AIN elite ABC reagent for 30 mins and washed in PBS-T for 15 mins. DAB staining was 239 \nperformed until the intensities were reached and then the counterstaining was performed with 240 \nhematoxylin (cat# 51275, Sigma). The slides were then washed with deionized water and 241 \ndehydration was performed in 90% and 100% ethanol for 1 min each followed by 1 min in 242 \nxylene incubation. The slides were then mounted using the mounting medium (Cat# 1442, 243 \nePredia). Stained slides were imaged using the Echo Revolution automated microscope (ECHO , 244 \nUSA) at 20× magnification, and quantified using ImageJ (RRID:SCR_003070) with the same 245 \nthreshold for each stain. The results were expressed as percent staining per visual field. 246 \nActive RAS detection assay  247 \nRAS activation (RAS-GTP) levels were measured using the active RAS activation assay kit (Cell 248 \nSignaling Technology, Cat# 8821). L ysates were prepared from cell lines or tumor tissues from 249 \nvarious in vivo  experiments by performing the steps provided by the manufacturer ’ s protocol. 250 \nT umors were lysed using the provided lysis buff er supplemented with protease and 251 \nphosphatase inhibitors. A total of 1 mg/mL lysate in 1x lysis buff er was employed for the 252 \nexperiment. Equal amounts of protein were then incubated with the GST-Raf1-RBD protein, and 253 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nthe reaction mixture was loaded onto a RAS affinity resin to capture activated RAS. Following 254 \nextensive washing to remove unbound proteins, bound protein was eluted in sample buffer and 255 \nsubjected to immunoblotting using a mouse RAS mAb (1:200 dilution) with gentle agitation 256 \novernight at 4 °C. The membrane w as then probed with anti-mouse IgG, HRP-link ed antibody 257 \n(Cell Signaling T echnology , Cat# 7076, RRID:AB_330924; 1:2000), and HRP-conj ugated ant i-258 \nbiotin antibody (Cell Signaling T echnology Cat# 7075, RRID:AB_10696897; 1:1000) to detect 259 \nbiotinylated protein mark ers. Activated RAS levels were measured using chemiluminescent 260 \nreagents and quantified using the ImageJ system. 261 \nPharmacokinetics, tissue distribution and histopathological examination of AD T-030 in mice  262 \nPathogen-free 8-week-old f emale C57BL/6J mice (Envigo#044; RRID:IMSR_ENV :HSD-044) were 263 \nhoused in the Biologic Research Labo rato ry at t he Univers ity of South Alabama (U of SA), 264 \nCollege of Medicine. Following acclimatization, mice were treated with ADT-030 at a dose of 265 \n100 mg /kg once daily for 14 days by oral gavage. Bl ood was collected at 0.5, 1, 2, 4, 8, and 24 266 \nhrs (n=4 per time point) following the last treatment into K 2 EDT A-microtainer tubes (BD 267 \nBiosciences; Franklin Lak es, NJ; USA) to obtain plasma. Major organs (lungs, kidneys, spleen, 268 \nheart, liver , brain, colon, and ovaries) were collected at 8 hrs (n=4). ADT-030 levels in plasma 269 \nand organs were determined by LC-MS/MS. The study followed established guidelines and 270 \nadhered to the approved protocol of the U of SA Institutional Animal Care and Use Committee 271 \n(IACUC). 272 \nIn another study , following acclimatization at the University of Alabama at Birmingham (U AB) 273 \nanimal facility , 5-6-week-old male C57BL/6J mice (The Jackson Labo rato ry #000664; 274 \nRRID:IMSR_JAX:000664) were randomly assigned to two groups (n=5) and received ADT-030 275 \n(150 mg /kg) by oral gavage for 2 weeks. At the end of the treatment, blood was drawn for 276 \nserum biochemical analysis, and mice were necropsied, organs were collected and fixed for 277 \nhistopathological analysis, and bone marrow smears were prepared for cytology . A board-278 \ncertified veterinary anatomic pathologist (JBF) performed blinded assessment of organ viscera 279 \n(heart, lung, kidney , liver , duodenum, pancreas, colon, spleen, thymus, testes, and brain) 280 \nfollowing standard procedures. The study followed established guidelines and adhered to the 281 \napproved protocol of the UAB IACUC.  282 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nOpen field locomotor activity 283 \nMice had ad libitum  access to food and water throughout the experiments. Behavioral 284 \nexperiments were performed during the light cycle (between 8 a.m. and 6 p.m.). Before 285 \nevaluation, mice were habituated for at least 30 min in the testing room. Mice were placed in 286 \nan open-field arena (44×44×30 cm) in a dimly lit room (7 lx) and allowed to freely explore for 10 287 \nmin as previously described  35 . Locomotor activity was identified as the cumulative distance 288 \ntraveled du ring the entire 10 min. Statistical analyses were performed using GraphPad Prism 289 \n(V ersion 10.4.1) using Student ’ s t-test for 2 groups and one-way ANOV A for comparing more 290 \nthan 2 treatment groups. Experimenters were blinded to treatment for all comparisons.  291 \nOrthotopic gr afting of PD AC cells in mice  292 \nIn vivo studies followed established guidelines and adhered to the approved protocol of the 293 \nUAB IACUC. 4-5-weeks-old male C57BL/6J mice (The Jackson Labo rato ry #000664; 294 \nRRID:IMSR_JAX:000664) were subjected to isoflurane anesthesia, followed by an intra-295 \nabdominal incision to access the spleen and pancreas. A matrigel suspension (40 μL), containing 296 \nKPC- f -luc (1 × 10 5 ), 2838c3-f- luc (1 × 10 5 ), or KPCML1- f  -luc (1 × 10 5 ) cells was injected into the 297 \npancreas. The skin and abdominal wall were then closed by suturing. Successful engraftment of 298 \nthe tumor cells was confirmed by PerkinElmer IVIS Lumina III In Vivo  Imaging System 299 \n(RRID:SCR_025239) one week later , and mice with tumors were randomized into four groups 300 \n(n=5 per group) for KPC and 2838c3, and two groups for KPCML1 for monotherapy studies, and 301 \nfive groups for KPCML1 for chemotherapy combination study . Mice in PK C and 2838c3 studies 302 \nwere given oral dosages as follows: the first group received a vehicle, and the other three 303 \ngroups received ADT-030 at varying oral doses (50, 100, and 150 mg /kg). In the KPCM1 study , 304 \nmice received vehicle or ADT-030 at 150 mg /kg. In the combination study with KPCML1 cells 305 \nimplanted, mice received vehicle, ADT-030 (150 mg/kg), or PF-2545920 (10 mg /kg, IP) daily for 306 \n4 weeks, gemcitabine (50 mg /kg, IP) plus nab-paclitax el (30 mg /kg, IP), weekly twice (GPT x), and 307 \nADT-030 plus GPT x. T umor tracking and response to therapy were monitored using D-lucif erin 308 \ninjection and were conducted bi-weekly throughout the studies. The total luminescence from 309 \ntumor-bearing regions was quantified using the Living Image in vivo  imaging software. Body 310 \nweights of the animals were measured twice a week. A t termination, all animals were subjected 311 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nto imaging of the whole body , followed by euthanasia, at which time tumors were collected, 312 \nweighed, and used for subsequent experiments.  313 \nSingle-cell tumor processing 314 \nFFPE blocks of the tumor tissues from KPC orthotopic experiments treated with vehicle or ADT-315 \n030 (150 mg /kg) were used for sc-RNA sequencing, as per the standard protocol used at 316 \nAdmera Health (South Plainfield, NJ, USA). After sequencing, the data were analyzed by 317 \ndemultiplexing and aligned to the mouse ref erence genome (GRCm39) for gene expression 318 \nquantification, and processed with Cell Ranger 9.0.1. The count matrices were then analyzed in 319 \nSeurat (v .5.3.0) R package v .4.5.1. Cells with more than 200 genes and less than 5% 320 \nmitochondrial content were k ept for downstream analysis. After SC T ransform analysis, PCA and 321 \nUMAP were used for dimensionality adjustment and clustering. Diff erentially expressed genes 322 \n(DEGs) were identified with FindallMark ers, and clusters were labeled using known mark ers. 323 \nT umor gr owth inhibition studies using PD AC PD X tumors  324 \nMouse experiments were conducted using PDAC PD X models with KRAS G12D  and KRAS G12 C  325 \nmutations as described previously 36 . 4-5-week-old male NSG mice (The Jackson Laboratory# 326 \n005557; RRID:IMSR_JAX:005557) were utilized for the experiments. In brief , F1 generation 327 \ntumors were cut into 2-mm × 2-mm fragments and subcutaneously implanted through a small 328 \nincision made in the right flanks of NSG mice while they were anesthetized. T umor size and 329 \nbody weight were monitored biweekly . T umor volume was calculated using the formula: length 330 \n× width 2  × 0.5. Once tumors reached approximately 80–100 mm 3 , mice (n=5 per group) were 331 \nrandomly assigned into two groups. The first group received vehicle, and the second group 332 \nreceived ADT-030 (150 mg /kg). A survival study was conducted for 70 days after 28 days of 333 \ntreatment. The UAB IACUC approved the experimental protocol for these mouse studies.  334 \nFlow cytometry  335 \nT umors derived from 2838c3 -f- luc and KPC-f- luc PD AC cells implanted into the pancreas of 336 \nC57BL/6J mice were digested using a solution containing 0.1 mg /mL DNase 1 and 1 mg /mL 337 \ncollagenase IV (W orthington Biochemical, Lak ewood, NJ) in Hank's Balanced Salt Solution (HBSS) 338 \nat 37 °C with shaking for 45 min. Following digestion, the samples were rinsed, and enzymes 339 \nwere quenched with RPMI-1640-supplemented with 10% FBS, and a 70 μm strainer was used to 340 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nfilter them to produce single-cell suspensions. The separated cells were labeled for 60 min at 4 341 \n°C using primary antibodies conjugated to fluorophores and a live/dead dye ( Supplementary 342 \nT able 1 ). Cells were then rinsed and suspended in a F ACS buff er (PBS + 2% FBS). After labeling 343 \nthe cell surface, the cells were fix ed at room temperature in 4% paraformaldehyde or FoxP3 344 \ntranscription buff er set (eBioscience# 00-5523-00) for 45 min, then washed with 1x Perm/W ash 345 \n(BD , 554723) followed by resuspension in F ACS buff er for data acquisition. Flow cytometry was 346 \nperformed. Data acquisition was conducted using a Symphony A5 flow cytometer , and analysis 347 \nwas performed using FlowJo. 348 \nStatistical analysis 349 \nStatistical analyses and data visualization were done using GraphPad Prism (RRID:SCR_005375). 350 \nThe data are represented as means accompanied by either standard deviation (SD) or standard 351 \nerror of the mean (SEM). A repeated measures analysis of variance (ANOV A) or ANOV A with 352 \nBonf erroni correction was conducted to evaluate and apply multiple corrections for assessing 353 \nstat ist ica l signifi c ance between groups. A stati sti cal s ignifi c ance threshold was set at p < 0.05.  354 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nResults 355 \nPDE10 over expr ession in PD AC  356 \nT o investigate the role of PDE10 in PDAC development, we used 10x Genomic sequencing and 357 \nmeasured PDE10 expression from 229 patients across 12 diff erent study groups. Employing sc-358 \nRNA seq data, we found that PDE10 mRNA is significantly enriched in primary tumors and 359 \nmetastatic  lesio ns c ompared to tissues from normal donors and adjacent uninvolved tissues  360 \n(Figure 1A). We then tested the PDAC cell lines used in our experiments and found that all 361 \nexpressed PDE10 protein ( Figure 1B ).  362 \nADT-030 inhibition and binding of PDE10 363 \nADT-030 is an indene chemically related to the nonsteroidal anti-inflammatory drug, sulindac, 364 \ndesigned to block cyclooxygenase (COX) inhibitory activity while targeting PDE10 ( Figure 1C ).  365 \nThe potency of ADT-030 to inhibit the enzymatic activity of recombinant PDE10 was 366 \ndetermined by measuring cGMP and cAMP hydrolysis using a fluorescence polarization assay. 367 \nADT-030 inhibited cAMP and cGMP hydrolysis with IC 50  values of 0.95 and 1.15 µM, respectively 368 \n( Figure 1D ). Molecular modeling studies using the PDE10 (2OUN) structure were performed by 369 \ninduced-fit molecular dynamics and simulation interaction analysis to identify a potential 370 \nbinding site on PDE10 for ADT-030. An optimal GLIDE docking score of -10.3 was calculated with 371 \nADT-030 bound in the PDE10 catalytic domain with the tri-methoxy benzylic moiety oriented 372 \ntoward the deep hydrophobic region of the pocket, while the more polar substituents were 373 \noriented towards the pocket entrance ( Supplementary Figure 1A ). The amide carbonyl and 374 \nneighboring heteroatoms are predicted to form hydrogen bonds with His525 through bridging 375 \nwater molecules ( Supplementary Figure 1B ). Hydrophobic and aromatic contacts with residues 376 \nTyr524, Leu635, Phe639, Ile692, Tyr693, Phe696, Met714, Phe729, Val733, and Ala734 help to 377 \nanchor the aromatic system within the binding site. These results suggest a favorable binding 378 \nfor ADT-030 in the PDE10 catalytic domain, which is supported by the docking score and a 379 \nnetwork of direct and water-mediated interactions, and consistent with a competitive 380 \nmechanism of enzyme inhibition as previously reported for an analog, ADT-061  30 . 381 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nWe next evaluated the antiproliferative activity of ADT-030 against a series of PDAC cell lines 382 \nharboring various KRAS mutations, as well as wild-type RAS, by performing cell viability 383 \nmeasurements using the MTT assay and determining potency (IC 50 ) values. As shown in Figure 384 \n1E, ADT-030 inhibited the proliferation of all KRAS G12D  and KRAS G12C  mutant PDAC cell lines 385 \ntested with IC 50  values in the low micromolar range (1.8-4.5 µM). Notably, the KRAS wild-type 386 \nPDAC cell line, BxPC-3, was found to be essentially insensitive to ADT-030, suggesting that ADT-387 \n030 selectively inhibits the proliferation of KRAS mutant PDAC cells ( Figure 1E ).  388 \nExperiments were also conducted to confirm that ADT-030 binds PDE10 in intact cells. In brief , 389 \nHEK-293 cells expressing PDE10 Micro-T ag were treated with ADT-030. PDE10 thermal stability 390 \nwas measured by Micro-T ag enzyme compleme ntat ion as  described in the Materials and 391 \nMethods section ( Supplementary Figure 1C-F ). The results revealed an EC 50  value of 0.9 µM for 392 \nADT-030 to bind PDE10, which paralleled the potency ranges of ADT-030 to inhibit the 393 \nenzymatic activity of PDE10 and the prolif eration of PDAC cells.  394 \nADT-030 inhibits the clonog enicity and mi gration of KRAS mutant PDAC cells  395 \nA mouse PD AC cell line,  2838c3 (KRAS G12D  mutant), and a human PDAC cell line, MIA 396 \nPaCa-2 (KRAS G12C  mutant), were selected to further study the anti-cancer activity of ADT-030. 397 \nThe long-term inhibitory eff ect of ADT-030 on cancer cell survival was evaluated in both PDAC 398 \ncell lines by colony formation assays. ADT-030 treatment significantly reduced the number and 399 \nsize of colonies in both PDAC cell lines across a concentration range comparable to the potency 400 \n(IC 50 ) values to inhibit prolif eration ( Figure 1F-G ). In addition, 2838c3 and MIA PaCa-2 cells 401 \nshowed significant impairment in motility after treatment with ADT-030 at non-cytotoxic 402 \nconcentrations ( Figure 1H - I). T ogether , these results indicate that ADT-030 inhibits the 403 \nprolif eration, colony formation, and motility of PDAC cell lines harboring KRAS G12D  and KRAS G12 C  404 \nmutations within the same concentration range as that is required to inhibit recombinant PDE10 405 \nand bind PDE10 in cells.  406 \nADT-030 induces apoptosis and G2/M cell cycle arr est in PD AC cells 407 \nT o determine the eff ect of ADT-030 on apoptosis and cell cycle progression, the 2838c3 and MIA 408 \nPaCa-2 PDAC cell lines were treated with ADT-030 for 24 and 72 hrs, respectively . Flow 409 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\ncytometry analysis of apoptosis as measured by Annexin V /PI staining showed that ADT-030 410 \nincreased both early and late apoptotic cells at concentrations of 2 and 5 µM. In vehicle-treated 411 \n2838c3 cells, apoptotic cells comprised 1.6% of the population, whereas treatment with ADT-412 \n030 increased the percentage to 3.5% (2 µM) and ~8% (5 µM) ( Supplementary Figures 2A and  413 \n2C). For MIA PaCa-2 cells, vehicle-treated cells had 2.3% apoptotic cells within the population, 414 \nwhereas ADT-030 treatment increased the number to 14.5% (2 µM) and 32% (5 µM) 415 \n( Supplementary Figur es 2B and  2 D ). Analysis of cell cycle distribution revealed that ADT-030 416 \ntreatment increased the percentage of cells arrested in the G2/M phase in both PDAC cell lines 417 \n( Supplementary Figures 2E-H ).  418 \nADT-030 inhibits PDE10 and activates PKA/PK G signaling  419 \nSince PDE10 inhibition by ADT-030 is expected to increase intracellular levels of cAMP and 420 \ncGMP , we measured both levels in 2838c3 and MIA PaCa-2 PD AC cell lines following treatment 421 \nwith ADT-030 by ELISA. Consistent with a PDE10 inhibitor , ADT-030 significantly increased the 422 \nlevels of cAMP and cGMP in a concentration-dependent manner in both PDAC cell lines. 423 \nNotably , the eff ect was apparent at concentrations that paralleled the concentration range 424 \neff ective for inhibiting recombinant PDE10 and prolif eration of both PDAC cell lines, as well as 425 \nfor inducing cell cycle arrest and apoptosis ( Figure 2A-D ). T o determine if increased cyclic 426 \nnucleotide levels by ADT-030 activated downstream protein kinases PKA and PK G,  in 2838c3 427 \nand MIA PaCa-2 cells, we measur ed the phosphorylation of V ASP (vasodilator-stimulated 428 \nphosphoprotein), a known subst rate for PKA and PK G  37 . Consistent with a PDE10 inhibitor , ADT-429 \n030 increased V ASP phosphorylation in both PD AC cell lines ( Figure 2E-F) , demonstrating that 430 \nADT-030 induced elevation of intracellular cAMP and cGMP levels results in the activation of 431 \nPKA and PK G. Finally , it should be noted that ADT-030 treatment did not aff ect the expression o f 432 \nPDE10 ( Figur e 2E ) or other cGMP and cAMP-degrading PDE isozymes, PDE3 or PDE4, 433 \nrespectively ( Supplementary Figur e 3A ). W e also determined if ADT-030 can activate canonical 434 \ndownstream signaling from PKA/PK G activation commonly reported in normal cells. T reatment 435 \nof 2836c3 and MIA PaCa-2 cells with ADT-030 did not have any eff ect on level of activated 436 \nphospho-CREB, VEGF-A, and Bcl-2 ( Supplementary Figur e 3B-C ). This suggests that the 437 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nactivation of PKA and/or PK G by AD T-030 in PD AC cells may involve downstream targets unique 438 \nto cancer cells. 439 \nADT-030 attenuates RAS and β-catenin signaling to promot e apoptosis 440 \nPrevious reports suggest that known PDE10 inhibitors and activation of PKG can phosphorylate 441 \nthe oncogenic pool of β-catenin in cell lines from various cancers 2 5, 27, 29, 30 . W e therefore 442 \nperformed W estern blot analysis to measure levels of the unphosphorylated (stable) form of β-443 \ncatenin, representing the oncogenic pool of β- caten in requ ired for TCF/LEF tr anscriptional 444 \nactivity in PDAC cells treated with ADT-030. AD T-030 treatment significantly reduced levels of 445 \nthe unphosphorylated form of β-catenin in 2838c3 and MIA PaCa-2 cell lines ( Figure 2G ). 446 \nPrevious research also suggested that PDE10 inhibitors and activation of PKG could suppress 447 \nMAPK and AKT signaling in lung and ovarian cancer cells 25 , 27 . Hence, we determined if ADT-030 448 \nhas a similar eff ect in PDAC cells by measuring phosphorylated levels of ERK (pERK) and mT OR 449 \n(pmT OR) within the MAPK and AKT signaling nodes, respectively . ADT-030 decreased pERK 450 \nlevels at its activating phosphorylation sites, Thr 20 2  and T yr 20 4 , as well as pmT OR at its activation 451 \nsite (Ser 24 48 ) ( Figure 2G ). These results suggest that the PDE10 inhibitory activity of ADT-030 can 452 \nsimultaneously suppress RAS and β-catenin signaling in PDAC cells. 453 \nT o further study the eff ects of ADT-030 on RAS signaling, RAS-GTP pulldown assays were 454 \nperformed to measure activated RAS levels following the treatment of PDAC cells with ADT-030 455 \nfor 24 hrs at concentrations of 2 and 5 µM. ADT-030 treatment did not reduce activated RAS 456 \nlevels in KRAS wild-type BxPC-3 and P anc 02 cells, as well as in KRAS amplified KLE (endometrial 457 \nadenocarcinoma) and MKN1 (gastric adenocarcinoma) cancer cell lines ( Figure 2H - I and  458 \nSupplementary Figure 4A-D ). Conversely , ADT-030 reduced activated RAS levels in 2838c3 cells 459 \nexpressing KRAS G12D  and MIA PaCa-2 cells expressing KRAS G12C mutations at concentrations 460 \neff ective for inhibiting prolif eration and PDE10 ( Figure 2J and  Supplementary Figure 4E-F ).  461 \nThese results suggest that ADT-030 c an inhibit activated RAS levels in KRAS-mutant PDAC cells 462 \nbut not in KRAS wild-type or KRAS amplified cells. This may explain the selective growth 463 \ninhibitory activity of ADT-030 observed between RAS-mutated and RAS-wild-type PDAC cells. 464 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nW e also analyzed the eff ect of ADT-030 treatment on autophagy in 2838c3 and MIA PaCa-2 465 \nPDAC cell lines, given previous studies reporting that RAS signaling and autophagy are 466 \ninterconnected and that RAS can modulate autophagy to promote tumorigenicity 38 . Autophagy 467 \nwas assessed by W estern blotting using LC3A/B as a mark er . ADT-030 treatment increased the 468 \nexpression of LC3A/B, indicating its capacity to disrupt autophagic flux ( Figure 2G ). The cells 469 \nwere also treated with ADT-030 alone or in combination with hydroxychloroquine (HCQ), a 470 \nknown autophagy inhibitor . ADT-030 in combination with HCQ did not increase the levels of 471 \nLC3A/B, suggesting that ADT-030, lik e HCQ , inhibits autophagic flux ( Supplementary Figure 5A ), 472 \nand are consistent with a previously reported analog of ADT-030  39 . Furthermore, ADT-030 473 \ntreatment reduced the expression of p70s6 kinase in 2838c3 and MIA P aCa-2 cells, which is 474 \nassociated with reduced mig rato ry ca pacity (Figure 2G )  40 . 475 \nHematologic, clinical chemistry , histopathologic and behavioral assessment of ADT-030 476 \ntreated mice  477 \nOur next objective was to evaluate the tolerance of mice to ADT-030 treatment. T en mice were 478 \nrandomly assigned to vehicle (n=5) or ADT-030 (150 mg /kg, n=5) treatment by oral gavage for  479 \ntwo weeks. Complete blood counts (WBC, RBC, HGB, HCT , MCV , MCH, MCHC, RDW , PL T , MPV , 480 \nneutrophils, lymphocytes, monocytes, eosinophils, and basophils) and serum biochemistry 481 \n(albumin, AL T , ALP , amylase, total bilirubin, BUN, phosphorus, creatinine, glucose, electrolytes 482 \n(calcium, sodium and potassium), total protein, and globulin were measured following two 483 \nweeks of treatment (Supplementary Figure 6A-B) . W e observed no significant differences 484 \nbetween the vehicle and ADT-030-tr eated mice, except for a slight reduction of total bilirubin 485 \nlevels in the treatment group. In addition, gross examination of multiple organs, including lungs, 486 \nliver , kidney , pancreas, heart, duodenum, colon, spleen, thymus, brain, and testis, showed no 487 \nhistopathological abnormalities in ADT-030-treated mice compared with vehicle-treated mice 488 \n(Figure 3A-B) .  489 \nSedation is a well-known side effect of conventional PDE10 inhibitors that were designed to 490 \ncross the blood-brain barrier and developed for the treatment of CNS disorders (schizophrenia 491 \nand Huntington’ s disease). W e therefore performed an open-field locomotor test to determine 492 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nif ADT-030 causes sedation. These behavioral experiments revealed no significant diff erences 493 \nbetween vehicle and ADT-030-treated mice, suggesting that ADT-030 does not cause sedation, a 494 \ncommon side eff ect from previously developed PDE10 inhibitors (Supplementary Figure 7A) .  495 \nPharmacokinetics and tissue distribution of ADT-030  496 \nA PK study was conducted in f emale C57BL/6J mice after oral gavage administration of 100 497 \nmg /kg ADT-030 once daily for 14 days. ADT-030 generated plasma levels that exceeded those 498 \nrequired to inhibit PDE10 and PD AC cell growth in vitro  ( Supplementary Figur e 7B ). Plasma 499 \nlevels of ADT-030 reached a Cmax of 7 µM  by 1 hr post-treatment and remained unchanged for 500 \nan additional hour before decreasing by 4 hrs post-treatment to the level (5 µM). High levels of 501 \nADT-030 were also detected in various organs (lungs, kidneys, spleen, heart, liver , ovaries, and 502 \ncolon) 8 hrs after administration, but low levels were measured in brain ( Supplementary Figure 503 \n7C). The low concentration of ADT-030 measured in the brain following oral administration 504 \nlik ely account for the absence of sedation, a known side eff ect of conventional PDE10 inhibitors 505 \ndeveloped for the treatment of CNS disorders that were designed to cross the blood-brain 506 \nbarrier to achieve high concentrations in the brain  41 . 507 \nADT-030 suppresses tumor growth in orthotopic PD AC model and r ep rograms the TiME 508 \nA mouse model of PDAC involving orthotopically implanted 2338c3- f-luc PDAC cells in the 509 \npancreas was initially used to assess the in vivo antitumor activity of ADT-030. Mice that 510 \nestablished palpable tumors one week following implantation were randomized into four 511 \ngroups and treated by oral g avage administration with vehicle or ADT-030 at dosages of 50, 100, 512 \nand 150 mg /kg once daily 5x/week for 23 days. T umor progression was monitored using 513 \nbioluminescence imaging ( Figure 3C ). All dosages of ADT-030 were eff ective, with the highest 514 \ndose of ADT-030 tested showing tumor regression in all mice in the group. Quantitation of 515 \nbioluminescence confirmed a significant reduction in tumor mass in ADT-030-treated mice 516 \ncompared to vehicle treatment ( Figure 3D ). Both tumor images and tumor weight 517 \nmeasurements confirmed tumor shrinkage in ADT-030-treated groups in a dose-dependent 518 \nmanner compared to vehicle treatment ( Figure 3E-F and  Supplementary Figure 8A-B ). AD T-030 519 \ntreatment did not cause apparent systemic toxicity , as evidenced by no effect on body weight 520 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\ngain during treatment, suggesting the potential for greater efficacy at higher dosages or a more 521 \nfrequent dosing schedule ( Figure 3G ).  522 \nT o study the immunomodulatory eff ects of ADT-030 relevant to PDAC, we performed 523 \nmultiparametric flow cytometry on orthotopic 2838c3 tumors from vehicle and ADT-030-524 \ntreated mice. The analysis revealed that ADT-030 induced profound shifts in immune cell 525 \ncomposition in the TiME, favoring a more immunostimulatory phenotype. ADT-030 treatment 526 \nenhanced the immune cell infiltration within the TiME, resulting in a significant increase in  527 \noverall populations of CD45 +  leuk ocytes compared with vehicle treatment ( Supplementary 528 \nFigure 9A ). Further characterization of the T-cell compartment revealed an increase in CD3 +  T  529 \ncells ( Supplementary Figure 9B ), observed with both CD4 +  ( Supplementary Figur e 9C ) and CD8 +  530 \nT cells ( Supplementary Figur e 9D ). T reatment with ADT-030 induced higher levels of several 531 \nimmune checkpoint mark ers, including CTLA-4 ( Supplementary Figure 9E ), PD-1 532 \n( Supplementary Figur e 9F ), LAG-3 ( Supplementary Figur e 9G ), and TIGIT ( Supplementary 533 \nFigure 9H ) in the total T cell populations. In another experiment involving the KPC -f- luc model, 534 \nthe immune checkpoint mark ers were diff erentially regulated with a decrease in CD8 +  T  c e l l s ,  535 \nand an increase in CD4 +  T cells. These results indicate that there was a concurrent adaptive  536 \nimmune regulatory response, lik ely representative of an acute but low , probably exhausted, T 537 \ncell phenotype within the TiME. Apart from modulating the T cell compartment, ADT-030 also 538 \nelevated NK cell infiltration within the TiME. Flow cytometry results revealed a pronounced 539 \nincrease in the NK1.1 +  cell population in the tumors of the ADT-030-treated mice compared to 540 \nvehicle-treated mice, indicating enhanced activation of the innate immune system 541 \n( Supplementary Figur e 9I ). In addition to augmenting NK cell numbers, increased expression of 542 \nimmune checkpoint receptors was measured on NK cells by ADT-030 treatment. These 543 \nobservations were paralleled within the CD3 +  T cell population, where ADT-030 enhanced 544 \ninfiltration of CD4 +  and CD8 +  T cells while upregulating CTLA-4, PD-1, LAG-3, and TIGIT 545 \n( Supplementary Figure 9E-H ). These data demonstrate that ADT-030 has the capacity to broadly 546 \nremodel the immune landscape, attracting both adaptive and innate eff ector cells into the 547 \ntumor while simultaneously engaging checkpoint regulatory pathways.  548 \nADT-030 alters RAS and β-catenin signaling in tumor s 549 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nT o determine the activity of ADT-030 treatment to inhibit oncogenic signaling in vivo , 2838c3 550 \ntumors were har vested from mice treated with vehicle or ADT-030 and analyzed for k ey 551 \nsignaling and apoptosis mark ers. W estern blot analysis revealed a mark ed decrease in pERK 552 \n( Figure 4A-B ) and pAKT ( Figure 4A and  4 C ) levels with no effect on total ERK or AKT levels in 553 \ntumors from ADT-030-treated mice, indicating the concurrent inhibition of the MAPK and 554 \nPI3K/ AKT pathw ays, respectively , reflective of upstream RAS inhibition. In addition, ADT-030 555 \ntreatment reduced levels of the non-phosphorylated form of β-catenin, indicative of the stable 556 \npool of β-catenin driving transcription of proteins involved in oncogenesis, for example, from 557 \naberrant activation of WNT signaling ( Figure 4A and  4 D ). Along with these alterations in k ey 558 \nsignaling node proteins, ADT-030 treatment also increased the expression of LC3A/B, consistent 559 \nwith in vitro  experiments, indicating that ADT-030 inhibits autophagic flux ( Figure 4A and  4 E ). 560 \nIncreased levels of cleaved P ARP ( Figure 4A and  4 F ) and cleaved caspase 3 ( Figure 4A and  4 G ) 561 \nwere also observed in the ADT-030-treated group, again consistent with in vitro  experiments 562 \nshowing apoptosis induction by ADT-030 treatment. Also consistent with in vitro  experiments, 563 \nADT-030 treatment reduced levels of activated (GTP-bound) RAS as measured by RAS-RBD 564 \npulldown assays ( Figure 4H and  Supplementary Figur e 9J ). IHC showed significantly reduced 565 \nexpression of the prolif eration mark er , Ki-67, in tumors from AD T-030-treated mice, 566 \ncorroborating the antiprolif erative activity of ADT-030 as observed in vitro  ( Figure 4I and  4 M ). IF 567 \nmicroscopy evaluation was used to analyze the treatment impact on autophagy and 568 \nmesenchymal-to-epithelial transition (MET). Mice treated with ADT-030 at 150 mg /kg showed 569 \nan increased level of LC3A/B in tumors, indicative of a disruption of autophagic flux ( Figure 4J 570 \nand  4 N ). ADT-030 also reduced vimentin expression ( Figure 4K and  4 O ) and increased 571 \nexpression of E-cadherin ( Figure 4L and  4 P ), which are associated with MET , and indicative of 572 \ntransforming cancer cells to a more normal epithelial phenotype with lower invasive capability .  573 \nADT-030 enhances antitumor immune r esponses and inhibits metastasis in an orthotopic 574 \nPD AC model 575 \nT o confirm the anti-tumor eff ects of ADT-030  observed in the orthotopic 2838c3 tumors, we 576 \nevaluated ADT-030 in the KPC orthotopic mouse model of PDAC to study specific immune cell 577 \nsubsets and functional T cell responses. T o accomplish this, we implanted KPC -f- luc cells in 578 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nC57BL/6J mice followed by treatment with ADT-030 (50, 100, and 150 mg/kg) or vehicle by oral 579 \ngavage. T u mor growth was monitored by bioluminescence imaging on Day 0 and on Day 23 580 \nprior to euthanasia ( Figure 5A ).  The KPC model recapitulated the major findings from the 581 \n2838c3 model. Normalized bioluminescence intensities showed statistically significant 582 \ndiff erences between the vehicle and treatment groups on Day 23 ( Figure 5B ). Both tumor size 583 \n( Figure 5C)  and tumor weights ( Figure 5D ) at the end of the experiment showed substantial 584 \nshrinkage in a dose-dependent manner in the treated groups compared to the vehicle-treated 585 \ngroup. W e then performed IHC analysis for prolif eration using the Ki-67 antibody in tumor 586 \nsections from the KPC orthotopic model. T umors treated with ADT-030 showed a mark ed 587 \ndecrease in the number of Ki-67 positive cells compared to the vehicle group, confirming the 588 \nanti-prolif erative activity of ADT-030 in vivo  ( Supplementary Figures 10A and  10E ). T o further 589 \nsubstantiate the above findings, we performed immunofluorescence microscopy with the tumor 590 \ntissues using autophagy (LC3A/B) and the MET mark ers, E-cadherin and vimentin. These 591 \nanalyses showed a significant increase in LC3A/B, suggestive of disrupted autophagy flux 592 \n( Supplementary Figures 10B and  10F ) and elevated levels of E-cadherin ( Supplementary 593 \nFigures 10C and  1 0 G ), along with a decrease in vimentin expression, reflective of MET 594 \n( Supplementary Figures 10D and  10H ).  595 \nADT-030 promotes anti-tumor immunity in a mouse model of PD AC  596 \nW e then performed multiparametric flow cytometry on the excised KPC- f -luc tumors to assess 597 \nthe immune changes underlying ADT-030 treatment. A significant elevation in overall immune 598 \ninfiltration was observed, following ADT-030 treatment, as determined by increased frequency 599 \nof CD45 +  cells ( Supplementary Figur e 11A ). Among the infiltrating pool of immune cells, there 600 \nwas an increase in αβ +  T  c e l l s  ( Supplementary Figure 11B ), γδ +  T  c e l l s  ( Supplementary Figur e 601 \n11C ), TNK cells ( Supplementary Figure 11D ), and conventional NK cells ( Supplementary Figur e 602 \n11E ), suggesting the activation of a broad-nature innate and adaptive immune response. The 603 \nreduced expression of immune checkpoint molecules PD-1 ( Supplementary Figure 11F ) and 604 \nCTLA-4 ( Supplementary Figure 11G ) on NK1.1 +  cells, indicates NK cell exhaustion. In contrast, a 605 \nmark ed increase in the frequencies of CD4 +  T cells ( Supplementary Figure 11H ), as well as 606 \nhigher expression levels of PD-1, TIGIT , CTLA4 + , PD-1 +  CTLA4 +  and F ASr subsets ( Supplementary 607 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nFigure 11I ), were observed in the CD4 +  T cell compartment of ADT-030-treated tumors. The 608 \nnumber of effec tor CD4 +  T cells increased in the ADT-030-treated mice, signifying the 609 \ninvolvement of helper T cell activation and functionality diff erences ( Supplementary Figure 610 \n11J ). Similar significant increases were present in overall CD8 +  T cell numbers after ADT-030 611 \ntreatment ( Supplementary Figur e 11K ). In comparison, we measured decreased expression of 612 \nimmune checkpoint mark ers, including PD-1, CTLA-4, PD-1 + CTLA-4 + , and  LAG-3, as well as lower 613 \nexpression of PD-1 + CTLA-4 + LAG-3 +  triple-positive subsets ( Supplementary Figur e 11L )  in the 614 \nCD8+ T cell compartment. These data indicate a reversal of T cell exhaustion and re-engagement 615 \nof cytotoxic potential. In addition, increased eff ector CD8 +  T cells were observed with ADT-030 616 \ntreatment, reflecting improved anti-tumor immunity ( Supplementary Figure 11M ).  617 \nBased on these results, we further explored the myeloid cell compartment in the TiME after 618 \nADT-030 treatment. W e observed an enhanced influx of myeloid cells, as evidenced by 619 \nincreased total macrophages (F4/80 + ) ( Supplementary Figure 11N ). Another characteristic 620 \nindicating myeloid infiltration was the increased expression of PD-L1 on macrophages after ADT-621 \n030 treatment, thereby enhancing antigen presentation and potential interaction with eff ector 622 \nT cells ( Supplementary Figur e 11O ). Phenotypic characterization of macrophages showed an 623 \nincreased M1-type characterized by MHC-II + CD86 + being more frequently expressed, while M2-624 \nlik e macrophages (CD206 + ) were less frequent from ADT-030 treatment compared to vehicle 625 \n( Supplementary Figures 11P-Q ). Additionally , an increased M1/M2 ratio was observed after 626 \nADT-030 treatment, signifying an enhanced immune-stimulatory TiME ( Supplementary Figure 627 \n11R ). Moreover , there was also an increase in the overall frequency of dendritic cells (DCs) post 628 \nADT-030 treatment ( Supplementary Figure 11S ). Both conventional subsets of dendritic cells, 629 \ncDC1, and cDC2, were increased in frequency , suggesting enhanced antigen processing and 630 \npresentation ( Supplementary Figur es 11T-U ). This further rise in functional antigen-presenting 631 \ncells, together with T and NK cell infiltration and activation, shows the wide immunomodulatory  632 \ncapacity of ADT-030 in remodeling of the pancreatic TiME toward an anti-tumor immune state.  633 \nSingle cell RNA-seq (scRNA-seq) of orthotopic KPC tumors tr eated with ADT-030  634 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nW e also performed scRNA-seq on the resected tumors from this orthotopic KPC mouse 635 \nexperiment to further investigate the eff ects of ADT-030 on signaling pathways and immune 636 \nmicroenvironment. A uniform manifold approximation and projection (UMAP) analysis was 637 \nperformed ( Figure 6A ), including 10 diff erent cellular populations identified as pericytes, 638 \ngMDSCs, CAFs, endothelial cells, m yocytes, T NK and B cells, dendritic cells, macrophages, 639 \nacinar to ductal metaplasia (ADM), and PD AC ( Figur e 6A-B ). These populations were identified 640 \nbased on the expression of canonical mark er genes for mature terminal lineages 641 \n( Supplementary Figur es 12A-B ). W e then identified 7 PDAC sub-clusters ( Figure 6C-D ) in which 642 \npathway analysis revealed that ADT-030-treated mice had significant downregulation in EMT , 643 \napical junction, KRAS signaling, and m yogenesis signaling ( Figure 6E ). W e then focused on MAPK 644 \nsignaling, as this pathway plays a major role in driving PDAC. In ADT-030-treated mice, there 645 \nwas a significant reduction in the expression of Raf1, a downstream mediator of activated RAS, 646 \nsuggesting the functional downregulation of the RAS-MAPK pathway in response to ADT-030 647 \ntreatment ( Figure 6F-H ). Although an increase in upstream RAS signaling was observed, the 648 \nMAPK signaling flux analysis revealed a significant reduction in the expression of Raf1 and 649 \nMapk3 ( Figure 6I-J ), suggesting that downstream RAS signaling was completely inhibited. 650 \nFurthermore, deep analysis revealed a reduction in the expression of several MAPK pathway 651 \ngenes, including Map2k2, Mapk3, Dusp6, and Elk4 ( Figur e 6K ). W e then analyzed the EMT 652 \npathway and found the concurrent reduction in the expression of mesenchymal mark ers such as 653 \nvimentin and fibronectin 1 (FN1) ( Figure 6L-M ). Next, we analyzed the impact of ADT-030 654 \ntreatment on WNT signaling and found that WNT pathway mark ers were also suppressed 655 \n( Figure 6N ), including APC, AXIN2, Lrp5 and Lrp6 ( Figure 6O ). Mechanistic investigation 656 \nconfirmed that ADT-030 treatment reduced expression of several MAPK and WNT pathway 657 \ngenes, supporting the similar observations at the protein level ( Figure 6P ).  658 \nNext, we focused on identifying the role of ADT-030 on the immune microenvironment. T o this 659 \nend, we sub-clustered the UMAP into four groups, including CD8 T cells, TNK cells, T regs and NK 660 \ncells ( Figure 6Q ). A concurrent increase in the TNK cells was identified after ADT-030-treated 661 \nmice, suggesting that ADT-030-treatment may enhance anti-tumor immune responses ( Figure 662 \n6R). Although the total number of CD8 T cells was reduced, higher numbers of activated CD8 T 663 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\ncells were present after ADT-030-treated mice compared to vehicle-treated mice ( Figure 6S ). 664 \nW e also analyzed several mark ers of CD8 T cell activation, exhaustion, and stem-lik e properties. 665 \nA significant increase in activation mark ers, including CD69, Prf1, IFNγ, and granzyme A (Gzma) 666 \nwas observed in ADT-030-treated mice. These findings show that ADT-030 increases CD8 T cell 667 \nactivation and enhances cytotoxic potential towards an eff ector state despite an overall 668 \nreduction in total CD8 T cell numbers ( Figure 6T ). E valuation of the transcriptomic signature of 669 \nCD8 T cells clearly showed that ADT-030 treatment increased the expression of several early-670 \nactivation genes, eff ector diff erentiation factors, cytotoxicity mediators, and chemokine ligands 671 \nin ADT-030-treated mice, suggesting increased CD8 T cell functionality ( Figure 6U ). 672 \nADT-030 treatment induced a similar but broader remodeling of the TNK compartment, 673 \nextending the CD8 T-cell-specific effects to encompass both T and NK cells within the TiME 674 \n( Figure 7A ). In line with enhanced activation and reduced dysfunction of CD8 T cells, the TNK 675 \nglobal state trajectory demonstrated that ADT-030 shifted TNK cells towards higher pan-676 \nactivation scores with relatively lower pan-dysfunction scores compared to vehicle treatment, 677 \nindicating a coordinated reinforcement of an activated, less dysfunctional state across cytotoxic 678 \nlymphocytes. This was accompanied by increased expression and prevalence of k ey eff ector and 679 \nactivation genes such as Gzmb, Nkg7, and Prf1 ( Figur e 7B-C ). Additionally , ADT-030 treatment 680 \ninduced a mark ed shift in NK cell functional state toward an activated phenotype compared to 681 \nvehicle treatment (Supplementary Figure 12C) . NK cell trajectory analysis revealed that ADT-682 \n030-treated mice showed higher activation and lower dysfunction signatures, indicating 683 \ncoordinated enhancement of activation programs. Concordantly , dot-plot analysis revealed 684 \nincreased expression and prevalence of activation and maturation mark ers, including Zeb2, Bcl2, 685 \nKlrg1, Itgam, Cd160, Havcr2, Prf1, IFNγ, and Gzmb (Supplementary Figures 12D-E) . T ogether , 686 \nthese results demonstrate that ADT-030 enhances cytotoxic lymphocyte activation within the 687 \nTiME, driving CD8 T and TNK compartments towards a sustained, less dysfunctional eff ector 688 \nstate to enhance anti-tumor immunity .      689 \nE ffects of ADT-030 on metastasis  690 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nT o  invest igate th e po tent ial o f ADT-030 to bloc k metastasis, we  used an established metastatic 691 \nPDAC cell line, KPCML1, derived from the KPC mouse model 42 . KPCML1 cells have a high 692 \npropensity for liver and lung metastasis, representative of patients with meta static pancreatic 693 \ncancer  42 . Using similar tumor inoculation methods and treatment (vehicle vs. AD T-030 at 150 694 \nmg /kg daily) as described in the Materials and Methods section, we examined the impact of 695 \nADT-030 treatment on mice orthotopically implanted with KPCML1 P DAC cells.  On day  23 afte r  696 \ntumor impla ntati on, ADT- 030 treatment decreased the size and weight of the primary 697 \northotopic tumor compared to vehicle treatment ( Figure 5E-F ). Lucif erase levels were measured 698 \nusing ex vivo  imaging, which revealed that while mice in the vehicle group implanted 699 \northotopically with KPCML1 cells developed liver and lung metastasis, there was a complete 700 \nabsence of liver ( Supplementary Figures 13A-B ) and lung ( Supplemen tary Figures 13C-D ) 701 \nmetastasis in mice treated with ADT-030. W e also analyzed lung and liver sections histologically 702 \nby H&E staining, which confirmed metastasis in the vehicle group and supported the obser ved 703 \nanti-meta stat ic act ivit y of AD T-030 ( Figure 5G - J).  704 \nADT-030 enhances the antitumor efficacy of chemotherapy in orthotopic PD AC models  705 \nKPCML1 cells were implanted orthotopically and treated with vehicle, ADT-030 (150 mg /kg), 706 \nstandard-of-care chemotherapy (a combination of gemcitabine, 50 mg /kg and nab-pacli taxe l, 10 707 \nmg /kg, GPT x), or a combination of ADT-030 with GPT x. ADT-030 produced a better therapeutic 708 \neff ect than chemotherapy as indicated by tumor size and tumor weight measurements ( Figure 709 \n7D-F ). Interestingly , the combination of ADT-030 with chemotherapy showed better efficacy 710 \ncompared to chemotherapy or ADT-030 alone, demonstrating that ADT-030 has the potential to 711 \nenhance standard-of-care chemotherapy efficacy for the treatment of PDAC.  712 \nADT-030 has increased potency and improv ed therapeutic window compared to other PDE10 713 \ninhibitors  714 \nPF-2545920 is a known PDE10 inhibitor developed for CNS disorders such as schizophrenia and 715 \nHuntington’ s disease. The potency IC 50  values for PF-2545920 to inhibit the prolif eration of MIA 716 \nPaCa-2 and 2838c cells were measured to be 25.02 and 24.1 µM, respectively ( Supplementary 717 \nFigure 14A ) compared with AD T-030 having IC 50  values of 3.01 and 1.79 µM, respectively 718 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n( Figure 1E ). Similarly , colony formation assays revealed that PF-2545920 had IC 50  concentrations 719 \nexceeding 25 µM ( Supplementary Figure 14B-C ) whereas AD T-030 showed significant inhibition 720 \nof colony formation at 5 µM ( Figure 1G-H ).  721 \nW e then compared PF-2545920 with ADT-030 in the KPCML1 orthotopic mouse model of PDAC. 722 \nMice were treated with vehicle, ADT-030 (150 mg/kg), or PF-2545920 (10 mg /kg) in which each 723 \nwere once daily by oral gavage. PF- 2545920 failed to show antitumor activity , while ADT-030 724 \nsignificantly inhibited tumor growth as evidenced by tumor images and measurement of tumor 725 \nweight ( Figure 7D-F ). Additionally , we evaluated the eff ects of PF-2545920 and ADT-030 726 \ntreatments on mice using open-field locomotor tests. There were no significant diff erences in 727 \nbehavior or mobility between ADT-030 and vehicle-treated mice, whereas PF-2545920-treated 728 \nmice displayed significantly reduced mobility throughout the test, reflective of sedation, a 729 \nknown side eff ect of conventional PDE10 inhibitors ( Supplementary Figur e 15A ). These data led 730 \nus to conclude that ADT-030, but not PF-2545920 displays antitumor activity without causing 731 \nsedation, which lik ely reflects differences in brain and systemic levels between ADT-030 and 732 \nknown PDE10 inhibitors. 733 \n ( Supplementary Figur e 15A ). This data led us to conclude that ADT-030 displays superior 734 \nantitumor activity compared to a known PDE10 inhibitor without causing sedation. 735 \nADT-030 suppr esses tumor gr owth and induces prolong ed r esponses in KRAS G12D  and 736 \nKRAS G12C  PD AC PD X models 737 \nT o further evaluate the efficacy of ADT-030 in suppressing PDAC tumor growth in vivo , we used 738 \ntwo clinically annotated subcutaneously implanted PDX models of PDAC harboring KRAS G12D  and 739 \nKRAS G12C  mutations. ADT-030 was administered orally at a dose of 150 mg/kg for 23 days, once 740 \ndaily , 5x/week. T umor dimensions and body weight were measured twice/week. The results 741 \ndisplayed a strong anti-tumor response in both KRAS G12D  ( Figure 8A ) and KRAS G12C PDX  models 742 \n( Figure 8D ) with no apparent systemic toxici ty  in terms of reduction in body weight ( Figure 8B 743 \nand  8 E ). The treatment was stopped after four weeks, and mice were monitored for tumor 744 \nrecurrence and survival. Mice treated with ADT-030 did not develop tumor regrowth over 70 745 \ndays of follow-up ( Figure 8C and  8 F ), while vehicle-treated mice progressively died during the  746 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\npost-treatment period. These data demonst rate bo th r obust and durable antitumor activity of 747 \nADT-030 in clinically relevant PDX models of PDAC. 748 \nADT-030 suppresses the growth of KRAS G12D  and KRAS G12C  - resistant PD AC cells  749 \nFinally , we tested the anti-proliferative activity of ADT-030 in KRAS G12D  and KRAS G12C resi stan t  750 \ncell lines grown in vitro . AsPC-1 cells resistant to MRTX1133 (AsPC-1-MRTX-R) and parental cells 751 \nwere treated with ADT-030 or MRTX1133, a KRAS G12D  inhibitor  43 . Cell viability measurements  752 \nusing the MTT assay revealed that ADT-030 showed comparable antiprolif erative activity in both 753 \nparental (IC 50  = 1.75 µM) and AsPC-1-MRTX-R cells (IC 50  = 1.47 µM) ( Figure 8G-I ) , whereas 754 \nMRTX1133 inhibited the prolif eration of parental AsPC-1 cells (IC 50  = 43.74 nM), but not of the 755 \nAsPC-1-MRTX-R cells (IC 50  > 25 µM), confirming that these cells are resistant to MRTX1133. W e 756 \nalso determined if this eff ect is sustained with longer treatment durations by performing colony 757 \nformation assays. The results showed activity of ADT-030 similar as proliferation assays, where  758 \nADT-030 inhibited colony formation in both parental and AsPC-1-MRTX-R cells, while MRTX1133 759 \ninhibited colony formation only in parental cells ( Supplementary Figur es 16A-B ). W e also 760 \ntreated MIA PaCa-2 G12C  parental and MIA PaCa-2 resistant to MRTX849 and AMG-510 (MIA-761 \nAMG-R) cells with ADT-030 and MRTX849, a KRAS G12C  inhibitor  44 . ADT-030 inhibited the 762 \nprolif eration of both MIA PaCa-2 parental and MIA-AMG-R cells with comparable potency . In 763 \ncontrast, MRTX849 inhibited the prolif eration of MIA PaCa-2 parental cells, but not MIA-AMG-R  764 \n( Figure 8J-L ) . Similar results were found in colony formation assays, whereas ADT-030 reduced 765 \nthe number and size of colonies in both MIA PaCa-2 parental and MIA-AMG-R cells, while 766 \nMRTX849 reduced the colony formation only in MIA PaCa-2 parental cells ( Supplementary 767 \nFigures 16C-D ). T og ether , these results show that ADT-030 exhibits a broad spectrum of RAS 768 \ninhibitory activity and has the potential to escape acquired resistance that limits the efficacy of 769 \nmutant-specific KRAS G1 2D  and KRAS G12 C  inhibitors. 770 \nDiscussion 771 \nAberrant activation of MAPK/ AKT signaling from KRAS mutations, along with the activation of 772 \nWNT /β-catenin mediated transcription, plays a major role in driving cancer cell prolif eration, 773 \nsurvival, and metastasis in PDAC, and other cancers, including colorectal, liver , lung, and breast 774 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\ncancer  45 -4 8 . KRAS is mutated in over 90% of PDAC, while mutations or increased expression of β-775 \ncatenin or other pathway components (e.g., W nt ligands, FZD receptors, LRP co-receptor , APC, 776 \nAxin) are also observed in a high percentage of patients with PDAC and other cancers 49, 50 . 777 \nCompensatory or cooperative interactions between these signaling pathways lik ely also  778 \ncontribute to aggressiveness of the disease as well as therapy resistance 51 . Phosphodiesterase 779 \nisozymes have been previously studied in the context of cancer , but no particular isozyme has 780 \nbeen targeted by an inhibitor , and  no PDE inhibitor has received FDA approval for the treatment 781 \nof cancer 22 . Recently , several publications have reported that isozyme-specific PDE10 inhibitors  782 \nor genetic silencing of PDE10 can block RAS and β-catenin by activating PKG  25, 27, 2 9,3 0 .  Similarly , 783 \ncAMP-activated PKA can inhibit signaling downstream of RAS by disrupting interaction with Raf1  784 \n52 .  Our data revealed increased expression of PDE10 in PDAC cells as compared to adjacent 785 \nnormal pancreatic tissue, which provided an initial rationale to targeti this pathway for the 786 \ntreatment of PDAC. AD T-030 is a non-CO X inhibitory sulindac derivative and a second-787 \ngeneration analog of ADT-061 (aka MCI-030), previously reported to selectively inhibit PDE10 788 \nand the prolif eration of colorectal cancer and ovarian cancer cell lines  25 , 30 . In these studies, 789 \nPDE10 knock down resulted in reduced sensitivity of the cancer cells to ADT-061, as well as 790 \nknown PDE10 inhibitors, which confirmed the selectivity of this class of agents to PDE10 and 791 \nsuggested that PDE10 is a understudied vulnerability of cancer cells. Molecular docking 792 \nsimulations and cellular thermal stability assays presented in this study provide structural 793 \ninsight into the interaction between ADT-030 and PDE10 and confirmation of target 794 \nengagement, respectively . These findings support an mechanism of action f or ADT-030 involving 795 \nPDE10 inhibition, elevation of cyclic nucleotides, and protein kinase activation and support  796 \nfuture research are needed to further study the oncogenic role of PDE10. 797 \nHere, we show that ADT-030 inhibits the enzymatic activity of recombinant PDE10 and acti vates  798 \ncyclic nucleotide signaling in PD AC cells at concentrations that selectively inhibit the 799 \nprolif eration of KRAS mutant PDAC cells. Of clinical relevance, we found that ADT-030 also 800 \ninhibits the prolif eration of PDAC cells that develop resistance to KRAS inhibitors and can 801 \nenhance the efficacy of standard-of-care chemotherapy , suggesting that ADT-030 has the 802 \npotential to be a front-line treatment for patients with PDAC. ADT-030 is distinct from other 803 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nKRAS inhibitors, FDA-approved or in development, by its capacity to escape resistance, which 804 \nwe attribute to cell cycle arrest and the induction apoptosis, resulting from the dual blockage of 805 \nβ-catenin and RAS signaling. The observed inhibition of MAPK and AKT /PI3K pathways by ADT-806 \n030 is particularly significant for PDAC treatment, as  both pathways are known for their 807 \nextensive crosstalk and compensatory activation to drive cancer cell prolif eration and survival  808 \n53, 54 . Compensation from β-catenin may also contribute to resistance to monospecific inhibitors 809 \nof KRAS or β-catenin where by d ual blockage of RAS or β- caten in  pathways through PDE10 810 \ninhibition may prevent the development of resistance to KRAS inhibitors, FDA-approved or in 811 \ndevelopment 55 . T o corroborate these findings using gene expression profiling, we evaluated 812 \ntumors excised from mice treated with ADT-030 or vehicle using single-cell transcriptomics. The 813 \nresults confirmed suppressive effects of ADT-030 on k ey oncogenic signaling pathways, 814 \nincluding RAS-MAPK, EMT , and WNT , as evidenced by reduced expression of Raf1, Mapk3, 815 \nMap2K2, vimentin, FN1, APC, and Axin2. W e also conducted assays on RAS activation in RAS 816 \nwild-type and KRAS mutant PDAC cell lines and found that ADT-030 selectively inhibited RAS 817 \nactivation in KRAS mutant PDAC cell lines. This interesting observation needs further study to 818 \nunderstand the diff erential effects of PDE10 inhibition and impact of cyclic nucleotide signaling 819 \nin KRAS mutant versus RAS wild-type PDAC cells.  820 \nADT-030 is orally bioavailable with attractive drug-lik e properties and appears to be well 821 \ntolerated at dosages that exhibit robust and durable antitumor activity . W e found that ADT-030 822 \ninhibits both primary tumor growth and metastasis without discernible toxicity in several mouse 823 \nmodels of PDAC, including PDX and orthotopic models. ADT-030 also potentiated the efficacy of 824 \nstandard-of-care chemotherapy regimens for PD AC. These findings support the rationale for 825 \ndeveloping ADT-030 as a front-line treatment for PDAC as a monotherapy or in combination 826 \nwith standard-of care chemotherapy .  827 \nPDE10 inhibitors have been previously developed for the treatment of CNS disorders such as 828 \nschizophrenia and Huntington’ s disease. W e the refore c ompared ADT-030 to the known PDE10 829 \ninhibitor , PF-2545920, and found that ADT-030 displayed appreciably greater potency than PF-830 \n2545920 to inhibit PDAC cell proliferation in vitro . This observation suggests that although 831 \nPDE10 is a cancer target, PF-2545920 has low potency to inhibit cancer cell prolif eration, which 832 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nmay be attributed to compensation by other co-expressed PDE isozymes (e.g., PDE5). When 833 \ncompared to ADT-030 in vivo, PF-2545920 failed to demonstrate anti-tumor activity . In addition, 834 \nADT-030 displayed improved tolerance without the side eff ects (sedation) observed with PF-835 \n2545920  56 .   836 \nPDAC is also known to be associated with an immunosuppressive TiME, a major factor 837 \nresponsible for resistance to immunotherapy 57 , 58 . The immune infiltration in PDAC is 838 \ncharacterized by the abundance of immune suppressive cells and the lack of anti-tumor 839 \nimmune cells 59 . Activation of the immune system by ADT- 030 treatment is another intriguing 840 \nand clinically relevant finding as we report. ADT-030 treatment increased CD4 + and  CD8 +  T cells, 841 \nas well as NK cell infiltration, resulting in a shift towards M1-lik e macrophage polarization. The 842 \ninhibition of expression of CTLA-4, PD-1, and LAG-3 on CD8 +  T cells by ADT-030 treatment 843 \nsuggests that ADT-030 alleviates T cell exhaustion and reestablishes cytotoxic T cell function 844 \nwithin the TiME 60 . Aside from maintaining antitumor T cells, ADT-030 also enhanced myeloid 845 \ncell infiltration by increasing the number of total macrophages (F4/80 + ) within the TiME. In 846 \nparticular , these macrophages displayed enhanced antigen-presenting potential as evidenced by 847 \nincreased expression of PD-L1, crucial for eff ector T cell interaction. In addition, phenotypic 848 \nanalysis revealed a shift in macrophage polarization toward a pro-inflammatory , M1-lik e 849 \nphenotype (MHCII +  CD86 + ), which was accompanied by a decrease in M2-lik e macrophages 850 \n(CD206 + ) 61 . These results were further supported by the identification of a high M1/M2 ratio. In 851 \naddition to macrophages, ADT-030 treatment led to an increase in conventional dendritic cells 852 \n(cDC1 and cDC2), contributing to activated T and NK cells 62 . Our single-cell RNA sequencing 853 \ndata demonstrated that the cytotoxic lymphocyte compartment is reprogrammed, with CD8 T 854 \ncells and TNK cells exhibiting enhanced activation, eff ector function, and reinvigoration. 855 \nActivation of cytotoxic genes (IFNγ, Gzma, and Prf1) and activation mark ers (CD69 and Cxcr3), 856 \nalongside modulation of exhaustion path ways (Lag3 and CTLA4), indicates that ADT-030 not 857 \nonly suppresses tumor cell prolif eration but also potentiates anti-tumor immunity . These 858 \nconvergent tumor-cell and immune-cell eff ects provide a strong mechanistic rationale for 859 \nevaluating ADT-030 in combination with immune checkpoint blockade or other 860 \nimmunomodulatory strategies, with the goal of converting immunologically cold PDAC into a 861 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nmore treatment-responsive state. This will be a future direction for preclinical studies 862 \ncombining ADT-030 with immune checkpoint blockade and possible clinical trials, given the 863 \nlimitations of immunotherapy for the treatment of PDAC.  864 \nWhile mutant-selective KRAS G12 C  and KRAS G12D  inhibitors have demonstrated promising efficacy 865 \nfor KRAS mutant cancers 63 , 64 , acquired resistance remains a major clinical limitation. V arious 866 \nmechanisms of resistance have been reported, including secondary RAS mutations, activation of 867 \nco-expressed RAS wild-type isozymes, and compensatory receptor tyrosine kinase mutations, all 868 \nof which frequently emerge in recurrent tumors and contribute to treatment failure, disease 869 \nrelapse, and death of the patient 65 . In the current study , we investigated whether ADT-030 may 870 \nbe less susceptible to the same mechanisms of resistance that limit the efficacy of KRAS G12C  and 871 \nKRAS G12D  inhibitors using PDAC cell lines developed to be resistant to such drugs. Strikingly , ADT-872 \n030 demonstrated potent anti-proliferative activity in both MRTX1133 and MRTX849 resistant 873 \nPDAC cell lines, highlighting its broad-spectrum pan-RAS inhibitory activity and its ability to 874 \nbypass diverse mechanisms of acquired resistance to allele-specific KRAS inhibitors.  875 \nThe therapeutic potential of ADT-030 is supported by the antitumor results observed in 876 \nclinically and genetically relevant PDAC PD X models harboring KRAS G12D  and KRAS G12C  mutations 877 \nas well as several orthotopic mouse models. In these experiments, efficacy and tolerability of 878 \nADT-030 were assessed following oral administration at a dose of 150 mg/kg for 4 weeks. This 879 \ndosage caused no discernible toxicity and is a human equivalent dosage of 12 mg/kg, or 840 mg 880 \nonce daily for a 70 kg human, a mode rate  dose for many drugs. T reated mice showed tumor 881 \nregression and no tumor regrowth for over 70 days after stopping treatment, highlighting the 882 \ndurability in maintaining long-term tumor control. These findings provide compelling evidence 883 \nof robust and clinically relevant antitumor activity of ADT-030 by inhibiting PDE10 that support 884 \nIND-enabling studies. This activity and capacity of ADT-030 to simultaneously block RAS and β-885 \ncatenin signaling also supports further mechanistic studies of the oncogenic role of PDE10  886 \n( Supplementary Figure 17 ). Future studies focusing on identifying the activity of ADT-030 in 887 \ngenetically engineered mouse models as a monotherapy and in combination with immune 888 \ncheckpoint inhibitors will further help in the translation of this agent to the clinic. In conclusion, 889 \nthe ability of ADT-030 to inhibit PDE10 and block multiple aspects of malignant progression, 890 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nincluding cancer cell prolif eration, survival, and metastasis, as well as creating a more favorable  891 \nTiME, while having the potential to escape resistance that limits the efficacy of other RAS 892 \ninhibitors, mak es ADT-030 a highly desirable drug development candidate for clinical trials in 893 \npatients with metastatic PDAC and other RAS-driven malignancies.  894 \nRef erences:  895 \n1 Siegel, R. L., Giaquinto, A. N. & Jemal, A. Cancer statistics, 2024. CA: a cancer journal for 896 \nclinicians  74, 12-49 (2024).  897 \n2 Balsano, R., T ommasi, C. & Garajova, I. State of the art for metastatic pancreatic cancer 898 \ntreatment: Where are we now? Anticancer research  39, 3405-3412 (2019).  899 \n3 Eser , S., Schniek e, A., Schneider , G. & Saur , D. Oncogenic KRAS signalling in pancreatic cancer . 900 \nBritish journal of cancer  111 , 817-822 (2014).  901 \n4 Leonhardt, L. & Hebrok, M. KRAS degradation averts PDAC chemoresistance. Nature Cancer  5 , 902 \n375-377 (2024).  903 \n5 Zhang, Y .  e t  a l .  β-Catenin mediated T AM phenotype promotes pancreatic cancer metastasis via 904 \nthe OSM/ST A T3/LO XL2 axis. Neoplasia 60 , 101096 (2025).  905 \n6 Sano, M.  e t  a l .  Activation of WNT /β-catenin signaling enhances pancreatic cancer development 906 \nand the malignant potential via up-regulation of Cyr61. Neoplasia 18, 785-794 (2016).  907 \n7 Ram Mak ena, M.  e t  a l .  W nt/β-catenin signaling: the culprit in pancreatic carcinogenesis and 908 \ntherapeutic resistance. International journal of molecular sciences  20, 4242 (2019).  909 \n8 Manegold, P .  e t  a l .  Diff erentiation therapy targeting the β-catenin/CBP interaction in pancreatic 910 \ncancer . Cancers  10, 95 (2018).  911 \n9 Dominguez, A. A.  e t  a l .  Unveiling the Promise: Navigating Clinical T rials 1978–2024 for PD AC. 912 \nCancers  16, 3564 (2024).  913 \n10 Jiménez, D. J., Javed, A., Rubio-T omás, T ., Seye-Loum, N. & Barceló, C. Clinical and preclinical 914 \ntargeting of oncogenic pathways in PDAC: targeted therapeutic approaches for the deadliest 915 \ncancer . International journal of molecular sciences  25, 2860 (2024).  916 \n11 Huber , M.  e t  a l .  The immune microenvironment in pancreatic cancer . International journal of 917 \nmolecular sciences  21, 7307 (2020).  918 \n12 Y ousef , A.  e t  a l .  Impact of KRAS mutations and co-mutations on clinical outcomes in pancreatic 919 \nductal adenocarcinoma. NP J Precision Oncology  8 , 27 (2024).  920 \n13 Zhang, Z., Zhang, H., Liao, X. & T sai, H.-i. KRAS mutation: The booster of pancreatic ductal 921 \nadenocarcinoma transformation and progression. Frontiers in Cell and Developmental Biology  922 \n11, 1147676 (2023).  923 \n14 Li, J., Mizukami, Y ., Zhang, X., Jo, W .-S. & Chung, D. C. Oncogenic K-ras stimulates W nt signaling in 924 \ncolon cancer through inhibition of GSK-3β. Gastroenterology  128 , 1907-1918 (2005).  925 \n15 Shetu, S. A. & Bandyopadhyay , D. Small-molecule RAS inhibitors as anticancer agents: discovery , 926 \ndevelopment, and mechanistic studies. International Journal of Molecular Sciences  23, 3706 927 \n(2022).  928 \n16 Ryu, W .-J., Han, G., Lee, S.-H. & Choi, K.-Y . Suppression of W nt/β-catenin and RAS/ERK pathways 929 \nprovides a therapeutic strategy for gemcitabine-resistant pancreatic cancer . Biochemical and 930 \nBiophysical Research Communications  549 , 40-46 (2021).  931 \n17 W ang, W .  e t  a l .  Dual-targeted therapy circumvents non-genetic drug resistance to targeted 932 \ntherapy . Frontiers in Oncology  12, 859455 (2022).  933 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n18 Raghavendra, N. M., Pingili, D., Kadasi, S., Mettu, A. & Prasad, S. Dual or multi-targeting 934 \ninhibitors: The next generation anticancer agents. European journal of medicinal chemistry  143 , 935 \n1277-1300 (2018).  936 \n19 Lemieux, E., Cagnol, S., Beaudry , K., Carrier , J. & Rivard, N. Oncogenic KRAS signalling promotes 937 \nthe Wnt/β-catenin pathway through LRP6 in colorectal cancer . Oncogene 34, 4914-4927 (2015).  938 \n20 Samidurai, A.  et al.  Role of phosphodiesterase 1 in the pathophysiology of diseases and potential 939 \ntherapeutic opportunities. Pharmacology & therapeutics  226 , 107858 (2021).  940 \n21 Gross, N. E.  e t  a l .  Phosphodiesterase-5 inhibition collaborates with vaccine-based 941 \nimmunotherapy to reprogram myeloid cells in pancreatic ductal adenocarcinoma. JCI insight  9 , 942 \ne179292 (2024).  943 \n22 Kelly , M. P .  e t  a l .  Cyclic nucleotide phosphodiesterases as drug targets. Pharmacological reviews  944 \n77 (2025).  945 \n23 F aure, M. & Bourne, H. Differential eff ects on cAMP on the MAP kinase cascade: evidence for a 946 \ncAMP-insensitive step that can bypass Raf-1. Molecular biology of the cell  6 , 1025-1035 (1995).  947 \n24 Suhasini, M., Li, H., Lohmann, S. M., Boss, G. R. & Pilz, R. B. Cyclic-GMP-dependent protein 948 \nkinase inhibits the Ras/Mitogen-activated protein kinase pathway . Molecular and cellular biology  949 \n18, 6983-6994 (1998).  950 \n25 Borneman, R. M.  e t  a l .  Phosphodiesterase 10A (PDE10A) as a novel target to suppress β-catenin 951 \nand RAS signaling in epithelial ovarian cancer . Journal of Ovarian Research  15, 120 (2022).  952 \n26 Jeong, W .-J., Ro, E. J. & Choi, K.-Y . Interaction between Wnt/β-catenin and RAS-ERK pathways and 953 \nan anti-cancer strategy via degradations of β-catenin and RAS by targeting the W nt/β-catenin 954 \npathway . NP J Precision oncology  2 , 5 (2018).  955 \n27 Zhu, B. e t  a l .  Phosphodiesterase 10A is overexpressed in lung tumor cells and inhibitors 956 \nselectively suppress growth by blocking β-catenin and MAPK signaling. Oncotarget  8 , 69264 957 \n(2017).  958 \n28 Peng, T .  e t  a l .  Inhibitors of phosphodiesterase as cancer therapeutics. European journal of 959 \nmedici\nnal chemistry 150 , 742-756 (2018).  960 \n29 Li, N.  e t  a l .  Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell 961 \ngrowth and β-catenin-dependent TCF transcriptional activity . Oncogene 34, 1499-1509 (2015).  962 \n30 Lee, K. J.  et al. Suppression of colon tumorigenesis in mutant apc mice by a novel PDE10 inhibitor 963 \nthat reduces oncogenic β-catenin. Cancer Prevention Research  14, 995-1008 (2021).  964 \n31 Loveless, I. M.  e t  a l .  Human Pancreatic Cancer Single-Cell Atlas Reveals Association of CX CL10+ 965 \nFibroblasts and Basal Subtype T umor Cells. Clinical cancer research  31, 756-772 (2025).  966 \n32 Babic, I. e t  a l .  MICRO-T AG enzyme complementation enables quantification of cellular drug-967 \ntarget engagement in temperature series. SLAS Discovery , 100291 (2025).  968 \n33 Tinsley , H. N.  e t  a l .  Sulindac sulfide selectively inhibits growth and induces apoptosis of human 969 \nbreast tumor cells by phosphodiesterase 5 inhibition, elevation of cyclic GMP , and activation of 970 \nprotein kinase G. Molecular cancer therapeutics  8 , 3331-3340 (2009).  971 \n34 Kim, D.-Y ., Park, J.-S., Leem, Y .-H., Park, J.-E. & Kim, H.-S. The potent PDE10A inhibitor MP-10 (PF-972 \n2545920) suppresses microglial activation in LPS-induced neuroinflammation and MPTP-induced 973 \nParkinson’ s disease mouse models. Journal of Neuroimmune Pharmacology  16, 470-482 (2021).  974 \n35 Seibenhener , M. L. & W ooten, M. C. Use of the open field maze to measure locomotor and 975 \nanxiety-lik e behavior in mice. Journal of visualized experiments: JoVE, 52434 (2015).  976 \n36 Bandi, D. S. R.  e t  a l .  ADT-1004: A promising pan-RAS inhibitor for targeting KRAS mutations in 977 \npancreatic ductal adenocarcinoma. Cancer Research  84, 5915-5915 (2024).  978 \n37 Reinhard, M. et al.  The 46/50 kDa phosphoprotein V ASP purified from human platelets is a novel 979 \nprotein associated with actin filaments and focal contacts. The EMBO journal  11, 2063-2070 980 \n(1992).  981 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n38 Üffing, A., Attridge, E. & T ooze, S. A. T argeting an alternative route: autophagy in RAS-driven 982 \ncancers. Cell Research 35, 389-390 (2025).  983 \n39 Gurpinar , E.  e t  a l .  A novel sulindac derivative inhibits lung adenocarcinoma cell growth through 984 \nsuppression of Akt/mT OR signaling and induction of autophagy . Molecular cancer therapeutics  985 \n12, 663-674 (2013).  986 \n40 Berven, L. A. & Crouch, M. F . Cellular function of p70S6K: a role in regulating cell motility . 987 \nImmunology and cell biology  78, 447-451 (2000).  988 \n41 T u, Z.  e t  a l .  Radiosynthesis and in vivo evaluation of [11C] MP-10 as a PET probe for imaging 989 \nPDE10A in rodent and non-human primate brain. Bioorganic & medicinal chemistry  19, 1666-990 \n1673 (2011).  991 \n42 Spadafora, V . e t  a l .  Optimization of a mouse model of pancreatic cancer to simulate the human 992 \nphenotypes of metastasis and cachexia. BMC cancer  24, 414 (2024).  993 \n43 Hallin, J.  e t  a l .  Anti-tumor efficacy of a potent and selective non-covalent KRASG12D inhibitor . 994 \nNature medicine  28, 2171-2182 (2022).  995 \n44 Hallin, J.  e t  a l .  The KRASG12C inhibitor MRTX849 provides insight toward therapeutic 996 \nsusceptibility of KRAS-mutant cancers in mouse models and patients. Cancer discovery  10 , 54-71 997 \n(2020).  998 \n45 Bahar , M. E., Kim, H. J. & Kim, D. R. T argeting the RAS/RAF /MAPK pathway for cancer therapy: 999 \nfrom mechanism to clinical studies. Signal transduction and targeted therapy  8 , 455 (2023).  1000 \n46 Choucair , K.  e t  a l .  T argeting KRAS mutations: orchestrating cancer evolution and therapeutic 1001 \nchallenges. Signal T ransduction and T argeted Therapy  10, 385 (2025).  1002 \n47 Y u, F .  e t  a l .  W nt/β-catenin signaling in cancers and targeted therapies. Signal T ransduction and 1003 \nT argeted Therapy  6 , 307 (2021).  1004 \n48 Xu, H., Ren, S., W ang, Y ., Zhang, T . & Lu, J. Abnormal activation of the Ras/MAPK signaling 1005 \npathway in oncogenesis and progression. Cancer Adv  8 , e25002 (2025).  1006 \n49 \nKim, E.  e t  a l .  Promotion of growth factor signaling as a critical function of β-catenin during HCC 1007 \nprogression. Nature communications  10, 1909 (2019).  1008 \n50 Zeng, G.  e t  a l .  Aberrant Wnt/β-catenin signaling in pancreatic adenocarcinoma. Neoplasia 8 , 1009 \n279-289 (2006).  1010 \n51 Lee, S.-K., Hwang, J.-H. & Choi, K.-Y . Interaction of the W nt/β-catenin and RAS-ERK pathways 1011 \ninvolving co-stabilization of both β-catenin and RAS plays important roles in the colorectal 1012 \ntumorigenesis. Advances in biological regulation  68, 46-54 (2018).  1013 \n52 Dumaz, N. & Marais, R. Protein kinase A blocks Raf-1 activity by stimulating 14-3-3 binding and 1014 \nblocking Raf-1 interaction with Ras. Journal of Biological Chemistry  278 , 29819-29823 (2003).  1015 \n53 Bye, B. A. e t  a l .  Combined PI3K and MAPK inhibition synergizes to suppress PDAC. bioRxiv , 1016 \n2023.2008. 2015.553438 (2023).  1017 \n54 Ouissam, A. J., Hind, C., Sami Aziz, B. & Said, A. Inhibition of the PI3K/ AKT /mT OR pathway in 1018 \np a n c r e a t i c  c a n c e r :  i s  i t  a  w o r t h w h i l e  e n d e a v o r ?  Therapeutic advances in medical onc ology  16 , 1019 \n17588359241284911 (2024).  1020 \n55 Awasthi, N.  e t  a l .  Dual inhibition of the PI3K and MAPK pathways enhances nab-1021 \npaclitax el/gemcitabine chemotherapy response in preclinical models of pancreatic cancer . 1022 \nCancer Letters  459 , 41-49 (2019).  1023 \n56 Uthayathas, S.  e t  a l .  Phosphodiesterase 10A inhibitor MP-10 eff ects in primates: comparison 1024 \nwith risperidone and mechanistic implications. Neuropharmacology  77, 257-267 (2014).  1025 \n57 Ju, Y . e t  a l .  Barriers and opportunities in pancreatic cancer immunotherapy . NP J Precision 1026 \nOncology 8 , 199 (2024).  1027 \n58 Y e, N. e t  a l .  A multi-omic approach reveals utility of CD45 expression in prognosis and novel 1028 \ntarget discovery . Frontiers in Genetics 13, 928328 (2022).  1029 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n59 Pollini, T .  et al.  The tumour immune microenvironment and microbiome of pancreatic intraductal 1030 \npapillary mucinous neoplasms. The Lancet Gastroenterology & Hepatology  7 , 1141-1150 (2022).  1031 \n60 Kabacaoglu, D., Ciecielski, K. J., Ruess, D. A. & Algül, H. Immune checkpoint inhibition for 1032 \npancreatic ductal adenocarcinoma: current limitations and future options. Frontiers in 1033 \nimmunology  9 , 1878 (2018).  1034 \n61 W ang, L.  e t  a l .  PD-L1-expressing tumor-associated macrophages are immunostimulatory and 1035 \nassociate with good clinical outcome in human breast cancer . Cell Reports Medicine  5  (2024).  1036 \n62 Böttcher , J. P .  e t  a l .  NK cells stimulate recruitment of cDC1 into the tumor microenvironment 1037 \npromoting cancer immune control. Cell 172 , 1022-1037. e1014 (2018).  1038 \n63 T anaka, N.  e t  a l .  Clinical acquired resistance to KRASG12C inhibition through a novel KRAS 1039 \nswitch-II pock et mutation and polyclonal alterations converging on RAS–MAPK reactivation. 1040 \nCancer discovery  11, 1913-1922 (2021).  1041 \n64 Li, Y ., Zhao, J. & Li, Y . New exploration of KRASG12D inhibitors and the mechanisms of resistance. 1042 \nExperimental Hematology & Oncology  14 , 39 (2025).  1043 \n65 Awad, M. M.  et al. Acquired resistance to KRASG12C inhibition in cancer . New England Journal of 1044 \nMedicine  384 , 2382-2393 (2021).  1045 \n 1046 \n 1047 \nConflict of interest: A.B.K, X.C., and G.A.P are affiliated with ADT Pharmaceuticals, LLC. 1048 \nData and materials a vailability: All data associated with this study are present in the paper or 1049 \nthe Supplementary Materials. 1050 \nFunding decl aration:  This work was supported by the University of Alabama at Birmingham (UAB), 1051 \nBirmingham, AL, USA. B.E was also supported by the UAB O’Neal Comprehensive Cancer Center, 1052 \nNational Institutes of Health (NIH) core support grant 5P30CAO13148-47 and 1R01CA294647. This work 1053 \nwas also supported by the NIH grants R01CA254197 (Piazza) and R01CA238514 (Zhou and Piazza) 1054 \nContributions 1055 \nConcept and design: BE and GP; acquisition, analysis, or interpretation of data: DSRB, VRA, GSG, 1056 \nLC; drafting of the manuscript: DSRB; critical revision of the manuscript for important 1057 \nintellectual content: all authors; statistical analysis: DSRB; administrative, technical, or material 1058 \nsupport: BE; validation: DSRB; supervision: BE. sc-RNA data analysis: SS; Modeling: TH; 1059 \nHistopath: JBF; Thermal assay: EN and IB; In vitro testing: ABK; All authors have read and 1060 \napproved the article. 1061 \n 1062 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n 1063 \n 1064 \n 1065 \n 1066 \n 1067 \n 1068 \n 1069 \n 1070 \n 1071 \n 1072 \n 1073 \n 1074 \nFigure leg ends 1075 \nFigure 1:  ADT-030 inhibits PDE10 at concentrations that inhibit proliferation, colony 1076 \nformation, and motility , while inducing apoptosis and cell cycle arr est of KRAS mutant PD AC 1077 \ncells .  1078 \nA. Violin plot of PDE10 expression in human donor , adjacent normal tissue, primary tumor and  1079 \nmetastatic l esion as det ermined by sc-RNAseq analysis of human PD AC.  B .  Baseline expression 1080 \nof PDE10 across indicated PDAC cell lines. β-actin was used as a loading control.  C .  Chemical 1081 \nstructure of ADT-030. D. ADT-030 inhibits the enzymatic activity of recombinant PDE10 using 1082 \ncAMP and cGMP as substrates. Data are expressed as mean ± SD , n = 2 samples/concentration. 1083 \nE. PDAC cell lines were treated with various concentrations of ADT-030 f or 3 days followed by 1084 \ndetermining viable cell number using MTT assays. Relative percentage cell viability was plotted 1085 \nwith respect to vehicle (DMSO) treated cells. The table lists the IC 50  values for the PD AC cell 1086 \nlines treated with ADT-030.  F - G .  The indicated PDAC cell lines were treated with various 1087 \nconcentrations of ADT-030 for 2–4 weeks, and long-term cell survival was measured using 1088 \nclonogenic assays. Representative images are shown in F and quantification plotted in G. H.  1089 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nIndicated PDAC cell lines were treated with vehicle or the indicated concentrations of ADT-030, 1090 \nand cell migration was analyzed using cell motility assays. Representative images under 1091 \nindicated treatment conditions for indicated PDAC cell lines are shown.  I. Bar diagrams are 1092 \npresented to show relative migration (%) from the experiment presented in H. Data represents 1093 \nthe meanLi44±Li44SEM of three biological replicates. nsLi44=Li44not significant, *pLi44<Li440.05, **pLi44<Li440.01, 1094 \n***pLi44<Li440.001, ****pLi44<Li440.0001. (one-way ANOV A).  1095 \nFigure 2.  ADT-030 blocks PDE10 and activates PKA/PK G to reduce β-c atenin levels and inhibit 1096 \nRAS signaling.  1097 \nA-D.  ADT-030 increases cyclic nucleotide levels in a concentration-dependent manner in 2838c3 1098 \n( A-B ) and MIA PaCa-2 cells ( C-D). E-F .  T reatment with ADT-030 for 4 hrs did not aff ect the 1099 \nexpression of PDE10 but induced V ASP phosphorylation at serine 157 (PKA site) and serine 239 1100 \n(PKG site) in a concentration-dependent manner in 2838c3 and MIA PaCa-2 cells. β-actin was 1101 \nused as a loading control. G.  ADT-030 decreased phosphorylation of ERK, mT OR, and levels of 1102 \nactive (oncogenic) β-catenin in 2838c3 and MIA PaCa-2 cells in a concentration-dependent 1103 \nmanner . β-actin was used as a loading control. H-J.  Indicated cell lines were treated with 1104 \nincreasing concentrations of ADT-030 for 24 hrs and RAS pulldown was performed. Data 1105 \nrepresent the meanLi44±Li44SEM of three biological replicates. nsLi44=Li44not significant, *p < 0.05, 1106 \n**pLi44<Li440.01, ***pLi44<Li440.001, and ****pLi44<Li440.0001 (one -way ANOV A). 1107 \nFigure 3.  Effect of ADT-030 treatment on tumor growth and modulation of TME in 2838c3 cell-1108 \nimplanted C57BL/6J mice.  1109 \nA-B. Histopathological examination of vital organs (heart, lung, kidney, liver, duodenum, 1110 \npancreas, colon, spleen, thymus, testes, and brain) from mice treated with ADT-030 at 150 1111 \nmg/kg as assessed by H&E staining. C. 2838c3 -f- luc cells were injected orthotopically into the 1112 \npancreas of C57BL/6J mice. Representative bioluminescence images at the indicated time 1113 \npoints are shown. D. Relative normalized whole-body bioluminescence intensities in mice under 1114 \nthe indicated conditions (nLi44=Li445). Statistical significance was determined using one-way ANOVA. 1115 \nE. Images of the pancreatic tumors in the vehicle- and the 150 mg/kg ADT-030-treated groups 1116 \nat termination. F.  Tumor weights at the end of the experiment for the indicated doses. G.  1117 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nAverage body weights of mice treated with vehicle and ADT-030 (50, 100, and 150 mg/kg). 1118 \nStatistical significance was determined using one-way ANOVA. nsLi44=Li44not significant, 1119 \n**pLi44<Li440.01, and ****pLi44<Li440.0001. 1120 \nFigure 4. ADT-030 reduces β-catenin levels, inhibits RAS/AKT signaling, and induces 1121 \nautophagic cell death. 1122 \nA. Western blots showing levels of pERK, total ERK, pAKT, total AKT, non-phospho-β-catenin, 1123 \ntotal β-catenin, LC3A/B, cleaved PARP, and cleaved caspase 3 in 2838c3 -f- luc tumors after 1124 \nvehicle or ADT-030 treatment. B-G. Bar graphs representing the quantifications of western 1125 \nblots  from  panel A :  pERK ( B), p-AKT (C ), non-phospho-β-catenin ( D), LC3A/B ( E ), cleaved PARP 1126 \n( F), and cleaved caspase 3 ( G) in tumor tissues after ADT-030 vs. vehicle treatments. Welch t-1127 \ntest was used for statistical analysis. H. The inhibitory effect of ADT-030 on activated (GTP-1128 \nbound) RAS in  tumors after vehicle or ADT-030 treatments was assessed by RAS-RBD pull-down 1129 \nassay. I. Representative Ki-67 IHC results in tumors after vehicle or ADT-030 treatment. J-L.  1130 \nRepresentative IF images of LC3A/B (J) , vimentin (K) , and E-Cadherin (L) in tumors after vehicle 1131 \nor ADT-030 treatment. M. Bar graph representing the quantification of IHC staining for KI-67.  N-1132 \nP. Dot-plot graphs representing the IF quantifications of LC3A/B  (N), vimentin (O), and E-1133 \nCadherin (P)  in tumors after vehicle or ADT-030 treatment. Welch t-test was used for statistical 1134 \nanalysis. ns, non-significant, ∗ pLi44<Li440.05, ∗∗ pLi44<Li440.01, and ∗∗∗∗ pLi44<Li440.0001.  1135 \nFigure 5.  ADT-030 induces tumor growth arrest and regression in KPC and KPCML1 cells-1136 \nimplanted C57BL/6J mice and reduces liver and lung metastasis.  1137 \nA. KP C -f- luc cells were injected orthotopically into the pancreas of C57BL/6J mice. 1138 \nRepresentative bioluminescence images at the indicated time points are shown. B. Relative 1139 \nnormalized whole-body bioluminescence intensities in mice under the indicated conditions 1140 \n(nLi44=Li445). Statistical significance was determined using one-way ANOVA. C-D.  Tumor images 1141 \nafter treatment with vehicle or ADT-030 at 50, 100, and 150 mg/kg ( C ) and bar graph 1142 \nrepresenting tumor weights ( D) from KPCML1 orthotopic model  at termination. Statistical 1143 \nsignificance was determined using one-way ANOVA. E-F. Tumor images after treatment with 1144 \nvehicle or ADT-030 at 150 mg/kg (E)  and bar graph representing tumor weights  (F) from 1145 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nKPCML1 orthotopic model at termination. Statistical significance was determined using one-1146 \nway ANOVA.  G .  Hematoxylin and eosin (H&E) staining of KPC-derived lungs after 4 weeks is 1147 \nshown. Representative images of H&E stained sections of metastasis in lung are displayed (20x). 1148 \nH. H&E staining of KPCML1-derived liver metastasis after 4 weeks is shown. Representative 1149 \nimages of H&E staining sections from metastasis in lung are displayed (20x). I-J. Graph 1150 \nrepresenting the quantification of lung (I)  and liver (J) metastatic nodules in lung sections after 1151 \nvehicle or ADT-030 treatment from KPC and KPCML1 orthotopic experiments. Welch t-test was 1152 \nused for statistical analysis.  ns, non-significant, ∗ pLi44<Li440.05, ∗∗ pLi44<Li440.01, ∗∗∗pLi44<Li440.001, and 1153 \n∗∗∗∗ pLi44<Li440.0001  1154 \nFigure 6.  ADT-030 remodels tumor cell states and reinvigorates CD8 T cell in PDAC  1155 \nA-B. UMAP visualization of all single cells isolated from orthotopic PDAC tumors treated with 1156 \neither vehicle or ADT-030, colored by major cell types as indicated. C.  UMAP reclustering 1157 \nshowing 7 transcriptionally distinct PDAC cell types. D. Split UMAPs of PDAC cells from vehicle 1158 \nand ADT-030 tumors demonstrating treatment-associated shifts. E. Bar graph showing top 1159 \nsignificantly downregulated pathways in ADT-030 treated PDAC cells compared to vehicle as 1160 \ndemonstrated by GSEA. F-H. Violin plots showing expression or module scores for RAF1 1161 \nsuppression (F), upstream RAS (G) , downstream MAPK (H)  related signatures in PDAC cells, 1162 \nincluding upstream WNT activation score (F), a downstream Wnt target gene score (G) , and 1163 \nMAPK/ERK pathway score (H), comparing vehicle and ADT-030. I-J.  UMAP features plots 1164 \ndemonstrating single-cell MAPK signaling flux for Raf1 (I), and Map2k2 (ERK) (J) with 1165 \ncorresponding dot plots summarizing average pathway activity (color scale) and fraction of  1166 \nPDAC cells expressing each gene set (dot size) in vehicle and ADT-030 tumors.  K-L. Dot plots 1167 \nsummarizing GSEA-derived pathway scores in PDAC cells highlighting RAS/MAPK pathway (K) , 1168 \nand reduced EMT (L). M-N.  Violin plots showing stemness-associated EMT-related module 1169 \nscores for fibronectin in PDAC cells (M), and WNT/β-catenin-dependent gene signatures (N). O.  1170 \nDot plot of canonical WNT/β-catenin pathway genes across PDAC clusters. P. Dot plot of 1171 \nproposed mechanism-related genes illustrating transcriptional repression. Q.  UMAPs of T and 1172 \nNK cell compartments in vehicle and ADT-030 tumors. R. Stacked bar plot quantifying the 1173 \nproportion of CD8 T cell, NK cell, T regs and TNK cell states among total T cells in vehicle vs. 1174 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nADT-030. S. Scatter plot showing CD8 T cell state trajectory scores, with each point representing 1175 \na single CD8 T cell positioned according to activation and exhaustion signature scores in vehicle 1176 \nand ADT-030 treatments. T. Dot plot summarizing the expression of representative activation, 1177 \nexhaustion, and stem-like genes in CD8 T cells. U.  Heat map of CD8 T cell exhaustion-associated 1178 \ngenes across individual CD8 T cells by trajectory state comparing vehicle and ADT-030 treated 1179 \ntumors depicting broad downregulation of exhaustion markers.  1180 \nFigure 7. ADT-030 enhances global TNK activation and sensitizes chemotherapy to suppress 1181 \nPDAC tumor growth in vivo 1182 \nA. TNK global state trajectory plot displaying pan-activation vs. pan-dysfunction signatures in 1183 \nvehicle and ADT-030 treated tumors. B. Dot plot showing expression of activation, dysfunction, 1184 \nand effector genes in TNK cells from vehicle and ADT-030 treated tumors with dot size  1185 \nrepresenting the percentage of expressing cells.  C .  Heat map of differentially expressed genes 1186 \nin TNK cells representing a global transcriptional shift toward an activated, cytotoxic program in 1187 \nADT-030 treated tumors compared to vehicle. D. Representative images of tumors harvested 1188 \nfrom the mice treated with vehicle, PF-2545920, ADT-030, gemcitabine paclitaxel (GPTx), and 1189 \nthe combination of ADT-030+GPTx.  E .  Quantification of tumor weights across all treatment 1190 \ngroups showing tumor growth inhibition with ADT-030 and further significant reduction in ADT-1191 \n030+GPTx combination groups compared to monotherapies and vehicle.  F .  Comparison of 1192 \ntumor weights between known PDE10 inhibitor (PF-2545920) and ADT-030 as monotherapy 1193 \ndemonstrating superior efficacy of ADT-030. ANOVA and Welch t-test was used for statistical 1194 \nanalysis. ns, non-significant, ∗ pLi44<Li440.05, ∗∗ pLi44<Li440.01, ∗∗∗pLi44<Li440.001, and ∗∗∗∗ pLi44<Li440.0001. 1195 \nFigure 8. ADT-030 induces tumor regression and extends survival in KRAS G12D  and KRAS G12C  1196 \nPDX models with potential to escape resistance to KRAS G12D  and KRAS G12C inhibitors. A. Tumor 1197 \ngrowth in NOD.Cg-Prkdc scid  Il2rg tm1 Wjl /SzJ mice implanted with KRAS G12D  PDX and treated with 1198 \nvehicle or 150 mg/kg ADT-030. B. Average body weights of mice treated with vehicle or ADT-1199 \n030. C. Survival curves of mice after the treatment with the vehicle or ADT-030. D. Tumor 1200 \ngrowth in NOD.Cg-Prkdc scid  Il2rg tm1 Wjl /SzJ mice implanted with KRAS G12 C  PDX and treated with 1201 \nvehicle or ADT-030. E. Average body weights of mice treated with vehicle or ADT-030. F. 1202 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nSurvival curves of mice after the treatment with the vehicle or ADT-030. G-H. The indicated 1203 \nPDAC cell lines were treated with various concentrations of ADT-030 (G)  or  MRTX1133 ( H) and 1204 \nsubjected to MTT assays. Relative percentage cell viability was plotted relative to vehicle 1205 \ntreated cells.  I. Table showing the IC 50  values for the cell lines used in panels G-H.  J-K. The 1206 \nindicated PDAC cell lines were treated with various concentrations of ADT-030 ( J)  or MRTX849 1207 \n( K) and subjected to MTT assays. Relative percentage cell viability was plotted relative to 1208 \nvehicle treated cells.  L. Table listing the IC 50  values for each PDAC cell line shown in panels J-K.   1209 \nSupplementary figure leg ends: 1210 \nSupplementary Figure 1:  A. Docking of ADT-030 to PDE10 (2OUN) resulted in an optimal 1211 \ndocking score of -10.325. Representative surface rendering of 2OUN with electrostatic potential 1212 \nmapped onto the surface shows ADT-030 bound in the PDE10 cataly t ic pock et. ADT-030 is 1213 \nrendered as a ball-and-stick representation. Pink and grey spheres illust rate magnesium and 1214 \nzinc ions in the pock et, respectively .  B .  Interaction diagram showing molecular interactions of 1215 \nADT-030 with PDE10.  B inding affinity of ADT-030 to PDE10 as determined by treating HEK293 1216 \ncells (45 min) expressing PDE10-Micro-T ag with ADT-030.  C .  W estern blot analysis showing 1217 \nPDE10 expression in HEK293 cells transf ected with PDE10-Micro-T ag and detected using anti- 1218 \nMicro-T ag antibody .  D .  Quantification of Micro-T ag enzyme compleme ntati on in ce ll s  1219 \ntransf ected with PDE10-Micro-T ag c onstruct vs untransf ected HEK293 cells.  E .  PDE10 thermal 1220 \ncurve yielding a T agg₅₀ of 44°C, providing the fix ed challenge temperature to determine ADT-1221 \n030 binding to PDE10 in transf ected HEK293 cells . F . AD T-030 binding to PDE10 in transf ected 1222 \nHEK293 cells. The curve is graphed as the average of two replicates ± SEM. 1223 \nSupplementary Figure 2:  A-B.  Representative F ACS analysis showing ADT-030 induced apoptosis 1224 \nin 2838c3 (A), and MIA PaCa-2 (B)  cells after treatment with ADT-030 (2 and 5 µM) or vehicle 1225 \nfor 24 hrs. After double-staining with annexin V and PI, cells were subjected to flow cytometry 1226 \nanalysis. C-D.  The indicated PDAC cell lines were treated with ADT-030 at varying concentrations 1227 \nfor 24 hrs, and apoptosis was measured following annexin V /propidium iodide labeling. E-F. 1228 \nRepresentative DNA histogram showing cell cycle arrest in 2838c3 (E) , and MIA PaCa-2 (F)  cells 1229 \nafter treatment with the indicated concentrations of ADT-030 or vehicle controls for 72 hrs. G-1230 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nH. Flow cytometry analysis of cell cycle distribution in 2838c3 (G) and MIA P aCa-2 (H) cells 1231 \ntreated with vehicle or ADT-030 at the indicated concentrations. Data represents the 1232 \nmeanLi44±Li44SEMr of three biological replicates. nsLi44=Li44not significant, *pLi44<Li440.05, **pLi44<Li440.01, 1233 \n***pLi44<Li440.001, ****pLi44<Li440.0001. (one-way ANOV A).  1234 \nSupplementary Figur e 3: A-C. W estern blot analysis showing the expression of indicated PDE 1235 \nisozymes, pCREB CREB, VEGF A, and Bcl-2 in 2838c3 and MIA PaCa-2 cells treated with DMSO , 1236 \nand varied time points/concentrations of ADT-030.  1237 \nSupplementary Figure 4: A-F .  Quan tification of RAS-GTP activation in BxPC-3, Panc 02, KLE, 1238 \nMKN1, 2838c3, and MIAPaCa-2 cells treated with either DMSO or increasing concentrations of 1239 \nADT-030.  1240 \nSupplementary Figure 5. A. W estern blot analysis showing the expression of LC3 A/B in 2838c3 1241 \nand MIA PaCa-2 cells treated with DMSO , HCQ , ADT-030, and the combination of HCQ+ADT-1242 \n030. β-actin was used as a loading control.  1243 \nSupplementary Figure 6: A-B.  Serum biochemical analysis of mice treated with ADT-030. Male 1244 \nC57BL/6J mice were treated with vehicle or ADT-030 (150 mg /kg) orally , 5 days/week for 2 1245 \nweeks.  A.  Serum was collected at the end of the treatment (n=5). Complete blood counts (WBC, 1246 \nRBC, HGB, HCT , MCV , MCH, MCHC, RDW , PL T , MPV , neutrophils, lymphocytes, monocytes, 1247 \neosinophils, and basophils) revealed no diff erence between vehicle and ADT-030 treatment. B. 1248 \nBiochemical analysis indicated unchanged all measured parameters (total protein, albumin, ALP , 1249 \nAL T , amylase, urea nitrogen, calcium, creatinine, phosphorus, glucose, sodium, potassium, and 1250 \nglobulin) except for an increase in total bilirubin because of ADT-030 tr eatment compared to 1251 \nvehicle treatment. ns: not significant and **pLi44<Li440.01.  1252 \nSupplementary Figure 7: A.  Open field and locomotor assay revealed no significant diff erent in 1253 \nthe overall mobility between vehicle and ADT-030 treated mice (n=5). B. ADT-030 plasma 1254 \nconcentrations after daily repeated oral administration of 100 mg /kg. C. Drug concentrations in 1255 \nlung, kidneys, spleen, heart, liver , brain, ovaries, and colon after oral administration of 100 1256 \nmg /kg dose. ns: not significant. 1257 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nSupplementary Figur e 8. A-B. T umor images from 2838c3 cell-implanted C57BL/6J mice after 1258 \ntreatment with vehicle or ADT-030 at 50 mg/kg (A)  and 100 mg/kg (B) .  1259 \nSupplementary Figure 9. A. Percentage of CD45 +  cells in 2838c3 tumors after treatment with 1260 \nADT-030 at 150 mg/kg vs. vehicle.  B-H. Increased percentage of total CD3 +  T  c e l l s  ( B),  CD4 +  T  1261 \ncells ( C ), CD8 +  T  c e l l s  ( D), CD3 + CTLA4 +  cells ( E ), CD3 + PD-1 +  cells ( F), CD3 + LAG3 +  cells ( G), 1262 \nCD3 + TIGIT + cells ( H) after treatment with ADT-030 vs. vehicle. I. Increased percentage of  NK 1263 \n(CD3 -  NK1.1 + ) cells in vehicle vs. ADT-030 treatment. All quantitative data represent the 1264 \nmeanLi44±Li44SEM. Welch t-test was used for statistical analysis. J. Quantification of RAS-GTP levels 1265 \nin tumor tissues of 2838c-implanted mice treated with either vehicle or 150 mg/kg dose of ADT-1266 \n030. Welch t-test was used for statistical analysis. ns, non-significant, ∗ pLi44<Li440.05, ∗∗ pLi44<Li440.01, 1267 \n∗∗∗pLi44<Li440.001, and ∗∗∗∗ pLi44<Li440.0001. 1268 \nSupplementary Figur e 10. A. Representative Ki-67 IHC results in tumors after vehicle or ADT-1269 \n030 treatment from the KPC- f -luc orthotopic model.  B-D.  Representative IF images of LC3A/B 1270 \n(B), E-Cadherin (C) , and vimentin  (D)  in tumor tissues after ADT-030 vs. vehicle treatments.  E .  1271 \nBar graph representing the quantification of IHC staining for KI-67.  F-H. Dot-plot graphs 1272 \nrepresenting the immunofluorescence quantifications of LC3A/B  (F), E-cadherin (G) , and 1273 \nvimentin  (H) in tumor tissues after ADT-030 vs. vehicle treatment. W elch t-test was used for 1274 \nstat ist ica l analysis. ∗∗ pLi44<Li440.01, ∗∗∗pLi44<Li440.001, and ∗∗∗∗ pLi44<Li440.0001.  1275 \nSupplementary Figure 11. ADT-030 modulates tumor immunity in the PD AC TIME in KPC cell-1276 \nimplanted C57BL/6J mice . A. Percentage of CD45 +  immune cells in KPC tumors after vehicle or 1277 \nADT-030 treatment.  B .  Percentage/mg tumor of total αβ T cells,  C .  γδ (T CRγδ + ) T cells,  D .  TNK 1278 \n(CD3 +  NK1.1 + ) cells, E. NK  (CD3 -  NK1.1 + ) cells, F.  NK1.1 + PD-1 +  cells G.  NK1.1 + CTLA4 +  cells,  H. CD4 + 1279 \nT cells,  I. CD4 + PD-1 +  cells, CD4 + TIGIT +  cells, CD4 + CTLA4 +  cells,  CD4 + PD-1 + CTLA4 +  cells, and 1280 \nCD4 + F ASr +  cells, J. CD4 + T cell subpopulations in tumors from vehicle or ADT-030 treatment.  K .  1281 \nCD8 + T cells,  L. CD8 + PD-1 +  cells, CD8 + CTLA4 +  cells, CD8 + PD-1 + CTLA4 +  cells, CD8 + LAG3 +  cells, and 1282 \nCD8 + PD-1 + CTLA4 + LAG3 +  cells, M. CD8 + T cell subpopulations in tumors from vehicle or ADT-030 1283 \ntreatment. N. macrophages, O . F4/80+ PD-L1+ macrophages, P.  M1 macrophages, Q.  M 2  1284 \nmacrophages,  R .  M1/M2 ratio in KPC tumors after vehicle or ADT-030 treatment. S. Percentage  1285 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nof total dendritic cells/mg tumor , percentage of T.  cDC1, and U. cDC2/mg tumor after ADT-030 1286 \nvs. vehicle treatment. All quantitative data represents the meanLi44±Li44SEM. W elch t-test was used 1287 \nfor stat ist ica l analysis. ns, non-significant, ∗ pLi44<Li440.05, ∗∗ pLi44<Li440.01, ∗∗∗pLi44<Li440.001, and 1288 \n∗∗∗∗ pLi44<Li440.0001. 1289 \nSupplementary Figure 12. A. Dot plot showing the mark ers used to identify various clusters 1290 \nsuch as PD AC, ADM, macrophages, dendritic cells, T NK & B cells, myocytes, endothelial cells, 1291 \nCAFs, pericytes, and gMDCs.  B .  Dot plot showing the mark ers used to identify NK cells, T regs, 1292 \nTNK cells, and CD8 T cells.  C .  NK global state trajectory plot displaying pan-activation vs. pan-1293 \ndysfunction signatures in vehicle and ADT-030 treated tumors. D.  Dot plot showing expression 1294 \nof activation, dysfunction, and maturation genes in NK cells from vehicle and ADT-030 treated 1295 \ntumors with dot size representing the percentage of expressing cells. E. Heat map of 1296 \ndiff erentially expressed genes in NK cells representing a global transcriptional shift toward an 1297 \nactivated, cytotoxic program in ADT-030-treated tumors compared to vehicle.  1298 \nSupplementary Figur e 13: A-B. Ex vivo imaging of livers of KPCML-1-implanted mice treated 1299 \nwith vehicle or ADT-030 ( A) and bar graph representing the bioluminescence quantification (B). 1300 \nC-D.  Ex vivo imaging of lungs in vehicle or ADT-030-treated mice showing reduced metastasis (C)  1301 \nand bar graph representing number of distant metastases ( D) . W elch t-test was used for 1302 \nstat ist ica l analysis. pLi44values are listed on bar graphs.  1303 \nSupplementary Figur e 14.  A. Indicated PDAC cell lines were treated with various concentrations 1304 \nof PF-25465920 for three days followed by determining viable cell number using MTT assays. 1305 \nRelative percentage cell viability was plotted with respect to vehicle (DMSO) treated cells. B-C.  1306 \nThe indicated PDAC cell lines were treated with various concentrations of PF-2545920 for 2–4 1307 \nweeks, and long-term cell survival was measured using clonogenic assays. Representative 1308 \nimages are shown in ( B) and quantification plotted in ( C). 1309 \nSupplementary Figur e 15. A.  Open field and locomotor assay revealed that mice treated with 1310 \nADT-030 did not show diff erences in their mobility compared to vehicle while PF-2545920 1311 \nproduced significant reduction in mobility revealing CNS toxicity . ns, non-significant, ∗ pLi44<Li440.05, 1312 \nand ∗∗∗pLi44<Li440.001.  1313 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nSupplementary Figure 16.  The indicated PDAC cell lines were treated with various 1314 \nconcentrations of ADT-030 (A & C) , MRTX1133  (B), or MRTX849  (D) for 2–4 weeks, and long-1315 \nterm cell survival was measured using clonogenic assays.  1316 \nSupplementary Figure 17. Schematic illustration of the proposed mechanism of action of ADT-1317 \n030. Inhibition of PDE10 by ADT-030 leads to accumulation of cAMP /cGMP and concomitant 1318 \nactivation of PKA/PK G, resulting in both direct antitumor activity and stimulation of antitumor 1319 \nimmunity . Direct eff ects on growth inhibition, induction of apoptosis, and inhibition of 1320 \nmetastasis are mediated by suppression of β-catenin/TCF-LEF transcriptional activity and 1321 \ninhibition of both ERK1/2 and PI3K signaling downstream of RAS. Anti-tumor immune eff ects of 1322 \nADT-030 are characterized by CD8 T cell-mediated cytotoxicity and immunologic cell death. 1323 \n 1324 \n 1325 \n 1326 \n 1327 \n 1328 \n 1329 \n 1330 \n 1331 \nMark e r  Clone Supplie r -Cat #  Dilution \nPD-1 F ITC 29F .1A12 Bi oLegend, 135214 1-200 \nCD206 FIT C C068C2 BioLegend, 141703 1-200 \nTIM-3 PE ID4B BioLegend, 121607 1-1000 \nF4/80 PE BM8 BioLegend, 123110 1-800 \nLAG-3 PE Cy7 9D3,1C8 BioLegend, 517008 1-200  \nL y6C PECy7 HK1.4 BioLegend, 128017 1-3000 \nCD11b PE CF594 M1/70 BioLegend, 101255 1-3000 \nCD45 PerCP 30-F11 BioLegend, 103130 1-800 \nFoxp3 APC F JK-16s Invitrogen, 17-5773-82 1-200 Nuclear \nCD11c APC N418 BioLegend, 117309 1-400 \nCTLA4 APC R700 MP6-XT22 BD , 565778 1-1000 Cytoplas m  \nCD172α AF700 P84 BioLegend, 144022 1-1000 \nCD62L APC Cy7 ME L -14 BioLegend, 104428 1-800 \nCD86 APC Cy7 GL1 BioLegend, 105045 1-200 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\nCD3 BV 421 145-2C11 BioLegend, 100341 1-800 \nX CR1 BV 421 ZE T  BioLegend, 148216 1-1000 \nLive/Dead Aqua  Invitrogen, L34966 1-500 \nTCR γδ BV 605 GL3 BioLegend, 118219 1-800 \nPD-L1 BV 605 MIH5 BioLegend, 153606 1-400 \nCD4 BV 650 GK1.5 BioLegend, 100469 1-800 \nCD103 BV 711 2E7 BioLegend, 121435 1-800 \nNK1.1 BV 711 PK136 BioLegend, 108475 1-400 \nCD8 BV 785 53-6.7 BioLegend, 100750 1-800 \nCCR7 BV 785 4B12 BioLegend, 120217 1-250 \nCD69 BUV 395 H1.2F3 BD , 569367 1-400 \nB220 BUV 395 RA3.6B2 BD , 563793 1-400 \nCD44 BUV 737 IM7 BD , 612799 1-800 \nCD16/CD32 93 BioLegend, 101320 1-500 \n 1332 \n 1333 \nSupplementary T able 1 . Details on antibodies used for multi-parameter flow cytometry .  1334 \n 1335 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 14, 2026. ; https://doi.org/10.64898/2026.02.11.705411doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}