Citations

Abstract Chimeric antigen receptor T (CART) cell therapy has shown clinical successes in B cell hematological malignancies. Two B cell maturation antigen (BCMA)-directed CART cell therapies treating multiple myeloma (MM) were approved by FDA but most patients relapse within 1-2 years. Ex vivo-expanded antigen specific T cells as cancer treatment rely on TCR-mediated killing of tumors. We hypothesized that arming ex vivo enriched tumor-specific T cells with CARs targeting a different antigen improves T cell functions. First, we expanded mucin 1 (MUC1)-activated T cells from patients with MM through ex vivo stimulation by MUC1 peptides-loaded autologous PBMCs for 19 days. Then, we engineered MUC1-activated T cells with 4-1BB-co-stimulated BCMA CAR to generate MUC1 TCR+BCMA CAR+ T cells. Baseline T cells without MUC1 stimulation were engineered with BCMA CAR as control. Baseline T cells, MUC1 T cells, baseline BCMA CART cells, or MUC1 BCMA CART cells were cocultured with luc+ RPMI-8226, a MUC1+BCMA+ MM cell line, for 3 days. MUC1 BCMA CART cells showed stronger tumor killing than BCMA CART or MUC1 T cells in all 3 patients (p<0.0001). MUC1 BCMA CART showed improved proliferation than BCMA CART or MUC1 T cells (p=0.0765, p<0.0001, respectively) after a 5-day stimulation by RPMI-8226. To exclude possibility of metabolic alterations by 19-day PBMC incubation leading to better T cell functions in MUC1 BCMA CART, baseline and MUC1-stimulated T cells from 3 healthy donors were processed for the same assays. Day-19 BCMA CART cells showed inferior tumor killing compared to baseline BCMA CART in all 3 donors (p<0.0001). Lower T cell proliferation was also observed in day-19 BCMA CART (p=0.0838). Our data indicate that improved T cell functions in MUC1 BCMA CART were unlikely due to metabolic changes and required stimulation by tumor antigens prior to CAR engineering likely through MUC1 TCR. Additionally, MUC1 T cells, BCMA CART, or MUC1 BCMA CART cells from tested patients were stimulated by RPMI-8226 for bulk RNA sequencing. Both BCMA CART and MUC1 T cells had distinct transcriptomic features from MUC1 BCMA CART cells. Differential gene expression analysis showed upregulation of T cell function-associated genes (RYR2, ITGA1, GNLY, GZMA, etc.) in MUC1 BCMA CART vs BCMA CART. Enrichment of T cell function-associated pathways was also observed in MUC1 BCMA CART. Analysis between MUC1 T cell and MUC1 BCMA CART showed upregulation of T cell function-associated genes (CSF2, CRTAM, IFNG, GZMB, etc.) and enrichment of immune response pathways in MUC1 BCMA CART. Our findings suggest that equipping ex vivo-enriched tumor-specific T cells with CARs targeting a different antigen improves T cell functions in vitro. Importantly, this provides a rationale for a therapeutic regimen where patients receive ex vivo-enriched tumor-induced T cells followed by CAR engineering. Persistence of the engineered T cells is being investigated Citation Format: Kun Yun, R. Leo Sakemura, Ismail Can, Omar Gutierrez Ruiz, Ekene J. Ogbodo, Truc N. Huynh, Claudia Manriquez Roman, Olivia Sirpilla, Carli M. Stewart, Jennifer M. Feigin, Grace E. DeFranco, Sophia Y. Goldberg, Dominic A. Skeele, Mariam Stein, Dara Missan, Latha Pathangey, Long Mai, Hong Xia, Brooke Kimball, Lionel A. Kankeu Fonkoua, Mehrdad Hefazi, Michael W. Ruff, Elizabeth L. Siegler, Leif Bergsagel, Michael Gustafson, Sandra Gendler, Saad S. Kenderian. BCMA CAR engineering arms T cells with superior functions post ex vivo stimulation by MUC1 peptide-loaded autologous PBMCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6103.

Abstract Histopathology has been a cornerstone of biomedical tissue assessment for decades, involving a labor-intensive and multi-step process that includes biopsy, gross examination, sampling, and subsequent tissue processing into snap-frozen or formalin-fixed paraffin-embedded (FFPE) blocks. These blocks are then sectioned, stained, and examined microscopically to diagnose and grade tissues. Despite its effectiveness, the traditional workflow is time-consuming, resource-intensive, and reliant on chemical dyes and expert interpretation. Recently, innovative chemical imaging techniques, such as Stimulated Raman Scattering (SRS) microscopy, have begun to redefine this field by enabling direct measurement of the molecular composition of tissues, thereby bypassing conventional tissue processing and staining protocols. This study demonstrates the integration of SRS microscopy into pathology workflows to rapidly acquire high-resolution chemical data from minimally processed fresh-frozen tissues. Unlike traditional methods that rely on thin-sectioned slides, this approach collects data from intact thick tissues using "optical sectioning, " enabling the generation of three-dimensional datasets across multiple planes. These chemical images are processed through a generative adversarial network (GAN) pipeline to produce virtual hematoxylin and eosin (H&E) images that are visually and diagnostically comparable to traditionally stained specimens. Furthermore, the generative model is extended to simulate the morphology and staining patterns of FFPE-processed tissues, offering a versatile tool for creating archival-quality images. This method significantly enhances diagnostic capabilities by preserving lipids and small molecules that are often lost during conventional processing. Within minutes, it generates high-quality stained images that demonstrated high inter-pathologist agreement in tissue grading, proving diagnostically reliable for clinical use. We evaluate our approach through a comprehensive survey of pathologists, assessing its diagnostic utility and reliability. Our results demonstrate that the generated images are comparable in quality to real histological images, showing no discernible inferiority in diagnostic utility. In prostate pathology, the integration of SRS microscopy and AI significantly streamlines traditional workflows, reducing complexity and processing time while preserving diagnostic precision. Citation Format: Kianoush Falahkheirkhah, Sudipta Mukherjee, John Cheville, Rohit Bhargava. Enhancing pathology workflows with chemical imaging and AI-driven generative models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2469.

Temozolomide (TMZ) is the main chemotherapy for GBM, but resistance develops in most patients, highlighting the need for sensitizing strategies. PARP inhibitors (PARPi), which disrupt DNA repair, are being investigated as TMZ-sensitizing agents. Here we evaluated the efficacy and explored molecular mechanisms of pamiparib-mediated TMZ sensitization. In vitro, low concentrations of pamiparib (0.1-1.0 μM) synergized with TMZ and enhanced DNA damage, particularly during the S- and G2/M phases. In vivo, pamiparib monotherapy (0.75 or 3 mg/kg, twice daily) was ineffective, however, TMZ afforded survival benefit in orthotopic patient-derived xenograft (PDX) models (GBM12, GBM22, and GBM84) of MGMT-methylated primary GBM. Pamiparib combined with TMZ (25 or 50 mg/kg daily) prolonged survival in GBM12 [median survival 272 vs. 114 days with TMZ (P=0.02), 30 days with placebo (P<0.001)] and GBM22 (median survival 88 vs. 60 days with TMZ (P=0.05), 26 days with placebo, (P<0.001)] but not in GBM84 [median survival 355 vs. 404 days with TMZ (P=0.44), 37 days with placebo (P<0.001)]. These findings align with prior studies indicating that PARPi can potentiate TMZ efficacy in a subset of primary GBM models sensitive to TMZ. TMZ induces cytotoxic DNA adducts, N7MeG, N3MeA, and O6MeG. While N7MeG and N3MeA are repaired by base-excision repair (BER), MGMT removes O6MeG. Suppressed MGMT expression, due to promoter methylation, drives TMZ sensitivity in ∼40% of primary GBM cases. In MGMT-methylated tumors, O6MeG-thymine mispairs trigger futile mismatch repair, causing replication stress. Although BER is PARP-dependent, BER inhibition did not replicate the TMZ-sensitizing effects of pamiparib. Beyond DNA repair, PARP regulates replication stress by recruiting translocases like SMARCAL1 and controlling endonucleases such as MRE11 and EXO1, protecting stressed forks and nascent DNA. We targeted these components using siRNA and small-molecule inhibitors to elucidate their roles in TMZ sensitization. Unexpectedly, silencing SMARCAL1 or MRE11, or inhibiting MRE11 with mirin, did not affect pamiparib-mediated sensitization in U251 cells. While EXO1 silencing attenuated DNA damage signaling, it had minimal impact on TMZ sensitivity or pamiparib-mediated sensitization. These results suggest that fork protection mechanisms may be epistatic to PARP but redundant for TMZ sensitivity. Alternatively, replication stress may be mitigated by lesion bypass via translesion synthesis (TLS), where replicative polymerases are replaced with low-fidelity polymerases. Consistent with this, silencing RAD18, involved in TLS, enhanced sensitivity to both TMZ and pamiparib, with the combination being more effective than either drug alone. In conclusion, pamiparib enhances TMZ efficacy in a subset of primary GBM. While further in vivo validation is needed, TLS deficiency may be a key determinant of pamiparib-mediated sensitization. Citation Format: Shiv K. Gupta, Ann C. Mladek, Sonia Jain, Katrina K. Bakken, Zeng HU, Brett L. Carlson, Danielle M. Burgenske, Jann N. Sarkaria. Pamiparib-induced replication stress augments the efficacy of temozolomide in GBM PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2910.

Background: Prostate cancer is the most common cancer in men in the United States with an estimated 288,300 new cases diagnosed in 2023. The treatment strategies include androgen deprivation therapies (ADTs) for metastatic prostate cancer (mPC) but 10-20% of patients eventually progress to castration-resistant prostate cancer (CRPC). However, nearly all patients develop primary and acquired resistance overtime, and developing therapies and identifying novel molecular targets are essential in enhancing the clinical outcomes of patients with prostate cancer. In this study, we report a small molecule that belongs to a class of tricyclic quinolones, QL47 that was previously screened among various other compounds in prostate PDX organoid models and showed a potent anti-proliferative activity. Methods: QL47 was tested on prostate cancer cell lines (22Rv1, 22RV1 abiraterone-resistant and DU145) for its anti-proliferative potential. Further, we used the biotinylated QL47 (QL47B) to pulldown its potential targets and we identified the targets through immunoprecipitation-mass spectrometry (IP-MS) technique followed by Western blot analysis. Cell cycle and cell death analyses were performed after staining the cells with propidium iodide and Annexin V followed by flow cytometry analysis. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was investigated using the Seahorse assay. Results: The initial screening of QL47 in prostate cancer cell lines showed that it inhibits the proliferation at an IC50 of 50-200 nM. Through IP-MS and Western blot analysis, we identified a member of the pyruvate dehydrogenase complex dihydrolipoamide S-acetyltransferase (DLAT) as a direct target of QL47. Mechanistically, we found that QL47 does not increase cell death or affect cell cycle, but inhibits metabolic function, with a decrease in both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Conclusion: We have identified a novel mechanism of action of QL47 on the pyruvate dehydrogenase complex, and we plan to further validate the safety and efficacy for the treatment of prostate cancer. Citation Format: Grace Nirmala Jesuthankaraj, August John, Huanyao Gao, Arun Kanakkanthara, Richard Weinshilboum, Liewei Wang. Targeting metabolic dysfunction in prostate cancer: the anti-proliferative effects of ql47 through inhibition of dlat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4297.

Total body irradiation for bone marrow transplantation (BMT) conditioning is commonly used in immunologic research. Historically, cesium-137 (137Cs) irradiators were predominantly used as a radiation source for these studies. However, 137Cs has a moderate half-life (30 years), is highly water soluble, and poses significant environmental and radiological safety concerns. As a result, the Department of Energy is promoting the replacement of 137Cs irradiation systems with orthovoltage x-ray cabinet systems, which pose no such concerns. Because the physical characteristics of x-ray and 137Cs photon beams have slightly different energy deposition characteristics, the relatively biological effectiveness (RBE) for x-rays is slightly higher than 137Cs in cell survival studies. This study aims to compare the RBE of orthovoltage and 137Cs irradiators with regards to BMT efficiency. Total body irradiation was performed on C57BL6 mice using 137Cs or 320 kVp x-rays across six dose levels to establish a RBE for lethality. Cohorts of five mice were irradiated using split-dose radiation delivered four hours apart. Daily observations and body weights were captured over a 30-day period. Mice with poor body condition scores or weight loss ≥20% were euthanized. With x-rays, 50% lethality was observed ∼750 cGy while 137Cs required ∼850 cGy, which results in an RBE of 750 cGy/850 cGy = 0.88. Bone marrow ablation and subsequent engraftment efficacy was compared in a parallel study. Cohorts of five C57BL6 CD45.1 recipient mice received total body irradiation with an established single dose 1000 cGy 137Cs regimen or a range of x-ray doses (910-1030 cGy single-dose) and then were infused with CD45.2 donor bone marrow. Ten weeks after BMT, bone marrow, spleen, thymus, and peripheral blood were analyzed via multi-color flow cytometry and the percentage of CD45.1 and CD45.2-positive cells were analyzed to determine marrow engraftment efficiency across various immune compartments. A clear dose-response relationship with bone marrow ablation and engraftment was observed in the stem cell and progenitor compartments. LSK (Lineage- Sca-1+ c-Kit+) cells and other hematopoietic progenitors in the bone marrow were efficiently engrafted (>99%) except for the lowest dose of 910 cGy (around 98%). Similarly, in the spleen, B cells, myeloid, megakaryocyte/erythrocyte, and granulocyte/macrophage lineages were also efficiently engrafted similar to that of 137Cs. Developing thymocytes were efficiently replaced by all radiation regimens, while replacement of peripheral T cell populations varied but were similar to traditional 137Cs at the highest doses. Taken together, these data demonstrate comparable BMT efficiencies with orthovoltage irradiation after appropriate dose-optimization. Citation Format: Danielle M. Burgenske, Brett L. Carlson, Matthew J. Rajcula, Michael J. Shapiro, Virginia M. Shapiro, Jann N. Sarkaria. An in vivo comparison of 137Cs and orthovoltage irradiation for murine bone marrow transplantation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4684.

Abstract Introduction: Multiple myeloma (MM) is a B-cell-derived malignancy associated with immune dysregulation. Anti-CD38 monoclonal antibodies (mAbs) therapies have shown improved outcomes in patients with MM. Silbermann et al. showed that anti-CD38 mAb-containing therapies lead to profound hypogammaglobulinemia and reduction in Ig gene repertoire. The effect of modern therapies such as CAR-T and bispecific antibodies (BsAbs) on B-cell repertoire has not been well described. In this study, we sought to investigate the effect of these therapies on B-cell repertoire in patients with MM. Methods: This is a single-institution retrospective observational study evaluating MM patients treated with anti-BCMA CAR-T and BsAbs. Baseline characteristics, laboratory data, treatment history, were extracted from medical records. Total unique sequences (TUS) and total sequences of IgH, IgK, and IgL were identified and quantified using clonoSEQ. Patients were classified in two groups: CAR-T and BsAbs. The primary objectives were (1) characterize changes in TUS and total sequences within IgH, IgK, and IgL before and after treatment, and (2) examine how the sequences evolve over time following CAR-T. Results: 76 MM patients received CAR-T or BsAbs. Among those, 22 CAR-T and 5 BsAbs patients had pre- and post-treatment sequences available. Median age at initiation of treatment was 66 years in the CAR-T group (42-81 y) and 65 years in the BsAbs group (48-71 y). Based on mSMART v3 criteria, 72% of CAR-T and 60% of BsAbs recipients were classified as high-risk. Median number of prior lines of therapy was 5 (2-8) in the CAR-T group and 6 (5-13) in the BsAbs group.CAR-T recipients were classified into early (<40 days) and late (>40 days), based on when the post-CAR-T treatment clonoSEQ assay was performed. Changes in TUS and total sequences of IgH, IgK, and IgL were calculated, and the averages were assessed independently. The mean early and late change in total unique IgH sequences were (-75, 586 and 131, 038, p=0.052), unique IgK (-57, 797 and 52, 586, p=0.020), and unique IgL (-22, 354 and 25, 056, p=0.011). A subgroup of 8 patients with sequential MRD reports post-CAR-T therapy were identified, six patients showed improvement in B-cell sequence dynamics and two patients showed a decrease over time. For patients who received BsAbs, results indicated a similar pattern, with an early decrease in B-cell repertoires, characterized by reductions in sequence diversity and quantity. Conclusions: Our analysis of B-cell repertoires following CAR-T revealed changes before and after treatment. Patients receiving CAR-T therapy showed a decline in the number of TUS and total sequences of IgH, IgK, and IgL, likely due to the lymphodepletion regimen, followed by a gradual restoration reflecting immune recovery. Longer follow-up and a larger cohort will be necessary to assess the implications of these B-cell repertoire changes on patient outcomes. Citation Format: Feba Miriam Jacob, Rodolfo Garza-Morales, Joseph G Hentz, Alex Wonnaparhown, Julia E. Wiedmeier-Nutor, Mariano Arribas, Gabriela Alatorre, Sharene Best, Jayden Poulignot, Udit Yadav, Saurabh Chhabra, P. Leif Bergsagel, Rafael Fonseca, Talal Hilal. Dynamics of B-cell repertoires following CAR-T and bispecific antibody therapy in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4537.

Abstract Background: The transcriptional cofactor Yes-associated protein (YAP) is activated in CCA pathophysiology. While canonical regulation by the Hippo pathway resulting in phosphorylation at serine 127 (S127) is inhibitory, SRC-family kinases (SFKs) phosphorylate YAP at tyrosine 357 (Y357), causing activation. Previously, cholangiocytes, hepatic progenitor cells (HPCs), and even mature hepatocytes were suggested as potential cells of origin of CCA, which highlights the plasticity in the liver. However, the role of YAP activation in selecting the cell of origin and lineage commitment during hepatobiliary oncogenesis is unknown. Aim: To determine if YAP Y357 phosphorylation is required for CCA tumorigenesis, we utilized an in vivo model. Unexpectedly, a shift in tumor phenotype to hepatocellular carcinoma (HCC) was identified. Subsequently, we further explored cell signaling and cell of origin contributions to this phenotypic switch. Method: C57BL/6 mice underwent biliary transfection with Sleeping Beauty transposase and transposons encoding myr-AKT and either YAP S127A (YAP-S) or YAP S127A/Y357F (YAP-SY), followed by IL-33 treatment. Tumor nodule count, histologic classification, and liver-to-body ratio were assessed at necropsy after 10 weeks. RNA sequencing and ingenuity pathway analysis (IPA) were performed on HCC and CCA nodules. To determine the cell of origin, tumors were generated in lineage tracing mice (Rosa26 mT/mG mice treated with AAV8-TBG-Cre). Finally, two tumor-derived cell lines with YAP-SY and constitutive AKT expression were isolated. Results: Both YAP-S and YAP-SY models developed similar tumor numbers and bulk. YAP-S mice generated frequent CCA (25.3%) in addition to combined HCC-CCA (cHCC-CCA) tumors (10.7%) and HCC (64%), whereas YAP-SY mice developed predominantly HCC (86.7%), with rare CCA (8.8%) and mixed tumors (4.5%). Transcriptome analysis identified 3,367 differentially expressed genes (DEGs) between HCC from YAP-SY mice and CCA from YAP-S mice. Notably, YAP-regulated genes had decreased expression in the HCC from YAP-SY mice. IPA revealed key upstream regulators, including cytokines and growth factors (e.g., TGFB1, TNF, INFG, CSF2, IL6, IL1B, IL4) and transcription factors (e.g., TP53, JUN, SP1, NFKBIA, STAT3). Lineage tracing demonstrated HCC originated from hepatocytes and CCA from cholangiocytes in both models. Finally, gene expression analysis of novel syngeneic cancer cell lines from YAP-SY mice showed mixed features of both HCC and CCA. Conclusion: In this murine model, we found that sub-maximal YAP activation is associated with increased formation of HCC from hepatocytes, while a higher level of YAP activation due to YAP Y357 phosphorylation is associated with efficient formation of CCA. We identified cytokines and growth factors that may orchestrate this phenotypic switch. Finally, we developed novel syngeneic cancer cell lines with cHCC-CCA characteristics. Citation Format: Muhammed D. Aksu,Jayla T. Millender,Ryan D. Watkins,Jennifer A. Yonkus,EeeLN H. Buckarma,Nathan W. Werneburg,Daniel R. O'Brien,Chantal E. McCabe,Rondell P. Graham,Gregory J. Gores,Rory L. Smoot,Caitlin B. Conboy. YAP tyrosine phosphorylation regulates lineage commitment in liver tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1457.

Rab32 is a small GTPase and molecular switch implicated in vesicular trafficking. Rab32 is also an A-Kinase Anchoring Protein (AKAP), which anchors cAMP-dependent Protein Kinase (PKA) to specific subcellular locations and specifies PKA phosphorylation of nearby substrates. Surprisingly, we found that a form of Rab32 deficient in PKA binding (Rab32 L188P) relocalized away from the Golgi apparatus and induced a marked disruption in Golgi organization, assembly, and dynamics. Although Rab32 L188P did not cause a global defect in PKA activity, our data indicate that Rab32 facilitates the phosphorylation of a specific PKA substrate. We uncovered a direct interaction between Rab32 and the adaptor protein optineurin (OPTN), which regulates Golgi dynamics. Further, our data indicate that optineurin is phosphorylated by PKA at Ser342 in a Rab32-dependent manner. Critically, blocking phosphorylation at OPTN Ser342 leads to Golgi fragmentation, and a phospho-mimetic version of OPTN rescues Golgi defects induced by Rab32 L188P. Finally, Rab32 AKAP function and OPTN phosphorylation are required for Golgi repositioning during cell migration, contributing to tumor cell invasion. Together, these data reveal a role for Rab32 in regulating Golgi dynamics through PKA-mediated phosphorylation of OPTN.

Abstract Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a fast-rising global public health problem. About 10% of patients with MASLD will develop hepatocellular carcinoma (HCC), but molecular markers for predicting future risk of HCC development in patients with MASLD are lacking. We investigated whether protein biomarkers in pre-malignant plasma samples can predict susceptibility to HCC in the setting of MASLD. Methods: A nested case-control study was performed among patients with MASLD who later developed HCC (cases) and MASLD patients who never progressed to HCC (controls), matched on age at MASLD diagnosis (± 1-year), sex, the year of blood sample collection, and length of follow-up (± 1-year). MASLD at baseline was determined by the presence of hepatic steatosis (assessed by imaging or biopsy) plus at least one cardiometabolic condition (obesity, diabetes mellitus, hypertension, or dyslipidemia). Archival baseline plasma samples stored at -80oC were used to perform the SomaScan 7k v4.0 assay. We also performed genotyping of the well-established HCC susceptibility variant, PNPLA3-rs738409 (I148M), among the cases and controls. The study sample was split into discovery (70%) and validation (30%) sets, and we selected promising protein biomarkers in the discovery set using differential expression analysis with volcano plots and Bonferroni-corrected p-values. A final set of significant proteins was used for prediction modeling in the validation sample, calculating area under the receiver operating characteristic curve (AUC-ROC), sensitivity, specificity, and accuracy. Results: The study sample included 54 MASLD to HCC cases and 54 MASLD controls, split into discovery and validation sets. We identified five significantly differentially expressed proteins in the discovery set, four overexpressed (TSP2, GPR56, MIC-1, TIMP-1) and one under-expressed (IGFALS) in the cases versus controls. Fitting the five proteins in the validation sample yielded an AUC=0.88, sensitivity=0.87, specificity=0.75, and accuracy=0.81. Adding PNPLA3-rs738409 to the model improved the model’s specificity and accuracy (AUC=0.88, sensitivity=0.87; specificity=0.81, accuracy=0.84). Conclusions: Our findings show that a five-protein biomarker panel can robustly predict future HCC development in patients with MASLD. Adding a well-established HCC genetic risk variant, PNPLA3-rs738409, slightly improved the model’s performance. These initial findings provide evidence of HCC prediction with baseline plasma biomarkers and genetic risk variants, toward improving risk stratification and enhancing early detection of HCC in the setting of metabolic perturbations. Citation Format: Ampem Darko Jnr Siaw, Sebastian M. Armasu, Jacob A. Frank, Irene K. Yan, Kirk J. Wangensteen, Tushar Patel, Samuel O. Antwi. Protein biomarkers of HCC development in patients with metabolic liver disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2350.