Immune checkpoint blockade (ICB) has transformed cancer therapy, yet many tumors remain refractory to treatment due to diverse immune evasion mechanisms. To uncover novel drivers of ICB resistance, we performed an in vivo CRISPR-Cas9 screen in mice with increasing levels of immune pressure. This screen identified phosphatidylserine (PS) externalization as a potent mediator of immune escape. Tumors with elevated surface PS exposure prevented reinvigoration of CD8+ T cells upon PD-1 blockade, which was associated with increased recruitment of immunosuppressive myeloid cells. Targeting of PS Synthase 1 (PTDSS1) or either of the scramblases, XKR8 or ANO6, sensitized tumors to ICB in vivo. These findings demonstrate that elevated PS exposure constitutes a mechanism of resistance to ICB, and that targeting regulators of PS externalization represents a promising strategy to enhance the efficacy of immunotherapy in refractory tumors.
Immune checkpoint inhibitors (ICIs) have improved cancer outcomes; however, many patients fail to respond, highlighting the need for novel targets. HVEM (Herpes Virus Entry Mediator) is an immune regulator with both inhibitory and stimulatory functions, making it a promising therapeutic candidate. We have developed Anti-4CB1, a fully human monoclonal antibody (mAb) that selectively blocks HVEM interactions with BTLA and CD160. Its activity was evaluated in-vitro using human tumor-infiltrating lymphocytes (TILs), peripheral blood mononuclear cells (PBMCs), and M1 macrophages, as well as in ex-vivo patient-derived tumor samples and in-vivo transgenic and humanized mouse models. HVEM expression was also assessed in serum and tumor tissues. Anti-4CB1 enhanced T-cell activation and cytotoxicity, evidenced by increased tumor cell killing, upregulation of activation markers (41BB, CD107a), and elevated IFNγ and TNFα secretion. It also promoted macrophage-mediated phagocytosis. In ex-vivo analyses of 49 patient-derived tumor samples, Anti-4CB1 increased cytotoxicity in 28.5% of cases, including samples unresponsive to anti-PD1. In-vivo, Anti-4CB1 demonstrated significant anti-tumor activity as monotherapy and showed enhanced efficacy in combination with anti-PD1. Additionally, higher tumor HVEM expression correlated with improved response to checkpoint blockade, while elevated soluble HVEM levels were associated with reduced responsiveness to Anti-HVEM. Anti-4CB1 enhances both adaptive and innate anti-tumor immunity and shows activity in anti-PD1 resistant settings. These findings support its potential as a novel therapeutic agent and suggest HVEM as a predictive biomarker for immunotherapy response.
Pancreatic adenocarcinoma (PDAC) is a leading cause of cancer-related morbidity, with oncogenic KRAS/MAPK/ERK signalling arising from activating KRAS mutations being the key feature of this malignancy. Dysregulation of RNA polymerase II (Pol II)-dependent transcription is a universal hallmark of cancers including PDAC, with aberrant gene expression programs driving tumorigenesis and influencing therapy response. Pol II progression through multiple transcription cycle checkpoints depends on transcriptional cyclin-dependent-kinases (tCDKs), and targeted inhibition of distinct tCDKs has been shown to be a promising anti-cancer strategy. In this study we profile biological and molecular PDAC cell responses to selective blockade of tCDK kinase activity at all Pol II transcription cycle checkpoints, alone and in combination with inhibitors of mutant-KRAS or MEK. We identify post-transcription-initiation checkpoints controlled by CDK9, CDK11, and CDK12/13 as critical for nascent gene expression and cell proliferation in PDAC. However, while dual targeting of tCDK and KRAS/MAPK/ERK networks amplifies transcriptomic phenotypes this resulted in limited cooperative therapeutic impacts.
While HER2-directed antibody-drug conjugates (ADCs), such as ENHERTU® (DS-8201), represent a major therapeutic advance, treatment options for tumors with low HER2 expression remain limited, and acquired resistance continues to pose a significant clinical challenge. Given the frequent co-expression of EGFR and HER2 across multiple tumor types, we hypothesized that dual targeting could improve therapeutic efficacy and overcome resistance mechanisms. This approach may achieve broader tumor coverage, synergistic enhancement of binding and internalization, and concurrent blockade of two key oncogenic pathways. To test this, we developed GenSci139, an EGFR×HER2 bispecific ADC (BsADC). It is conjugated via a novel, proprietary hydrophilic peptide-cleavable linker to a potent topoisomerase I inhibitor payload. In preclinical studies, GenSci139 exhibited remarkable plasma stability and favorable pharmacokinetics, enabling efficient tumor-specific payload delivery. Compared to monospecific ADCs, it demonstrated superior binding affinity and enhanced internalization across a panel of cancer cell lines with diverse EGFR/HER2 expression profiles. In vitro, GenSci139 mediated potent cytotoxic activity and induced a robust bystander-killing effect. Furthermore, its parental bispecific antibody effectively inhibited both EGF-induced signaling and cell proliferation. In vivo, GenSci139 demonstrated superior antitumor efficacy over DS-8201 in multiple cell-line-derived xenograft and patient-derived xenograft models. Mechanistic studies revealed that GenSci139 induced DNA damage, apoptosis, and hallmarks of immunogenic cell death, suggesting potential for synergistic combination with immunotherapies. Collectively, our preclinical data position GenSci139 as a promising novel therapeutic candidate with the potential to address the unmet medical need in a broad spectrum of solid tumors, including those of the lung, breast and stomach.
Abstract Antiangiogenic tyrosine kinase inhibitors (TKI) targeting vascular endothelial growth factor receptor (VEGFR) remain the backbone of therapy in advanced renal cell carcinoma (RCC). However, durability of responses is limited and resistance typically arises. Thus, there is an urgent need for therapeutic agents that enhance responses to TKIs, including in patients who progress on prior TKI therapies. In this study, we show that the farnesyl transferase inhibitor (FTI) KO-2806 inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling in endothelial cells to enhance the antiangiogenic properties of TKIs. This translates to tumor regressions and robust inhibition of tumor neovascularization in preclinical models of RCC exposed to the combination of KO-2806 and anti-VEGFR TKIs. KO-2806 also sensitizes tumors previously progressing on anti-VEGFR TKIs, suggesting potential benefits of KO-2806 as a combination partner across the treatment continuum in RCC.
Abstract Venetoclax (VEN), a selective BCL-2 inhibitor, effectively induces apoptosis in a wide range of malignancies. VEN-based regimens, which combine VEN with either hypomethylating agents or low-dose cytarabine, have markedly improved treatment outcomes in elderly patients with acute myeloid leukemia (AML). However, approximately one third of patients exhibit intrinsic resistance to these regimens, and the majority of initial responders eventually develop acquired resistance. Therefore, intrinsic and acquired resistance to VEN-based regimens remains a major barrier to achieving durable clinical responses in patients with AML. In this study, we aimed to identify effective treatment strategies to overcome VEN resistance. Among the drugs tested in this study, we found that bortezomib (BTZ), a proteasome inhibitor, showed potent synergy with VEN in inducing apoptosis in a wide range of AML cell lines, irrespective of RAS or TP53 mutation status. Mechanistically, BTZ upregulates proapoptotic proteins such as NOXA, BIM, and PUMA, which neutralize MCL1 and promote apoptosis. Notably, NOXA upregulation plays a critical role in the efficacy of the combination of VEN and BTZ. Moreover, BTZ resensitized AML cell lines with acquired resistance to VEN, further supporting its role in overcoming therapeutic resistance. Importantly, the combination of BTZ and VEN significantly prolongs the survival of mice inoculated with a VEN-resistant AML cell line harboring BAX mutations, which are commonly observed in relapsed AML following VEN-based regimens and confer resistance to VEN by inhibiting the BAX-dependent apoptotic pathway. Collectively, this study provides a rationale for the VEN–BTZ combination as a potential strategy to overcome VEN resistance in certain AML subsets.
Abstract ABL209 (NEOK002) is a bispecific antibody–drug conjugate (ADC) designed to enhance efficacy and therapeutic window through dual targeting of the epidermal growth factor receptor (EGFR) and mucin 1 (MUC1). EGFR is a key oncogenic driver in multiple tumor types; however, clinical targeting of EGFR is limited by dose-dependent skin toxicity. MUC1 is a tumor-associated antigen characterized by aberrant glycosylation and overexpression, but its expression can be heterogeneous, and the MUC1 extracellular domain is shed from the tumor, limiting monospecific targeting of MUC1. ABL209 is a heterodimeric 1 + 1 bispecific ADC with a drug-to-antibody ratio of 4, conjugated with tavatecan. ABL209 demonstrated enhanced cell binding and internalization compared with monospecific EGFR or MUC1 ADCs. ABL209 did not seem to inhibit the proliferation of human epidermal keratinocytes in vitro, unlike cetuximab-based ADCs. In vivo, ABL209 resulted in complete regression of tumors with single doses as low as 1.5 mg/kg in a CFPAC-1 pancreatic cell line–derived xenograft model. ABL209 demonstrated tumor growth inhibition across all 36 tested patient-derived xenograft models, inducing tumor regressions in 78% of models and showing efficacy in 6 of 10 KRAS-mutant tumors. Cotreatment with sotorasib prolonged tumor regression in a KRAS-mutated NCI-H1373 model for 58 days following treatment. ABL209 showed a favorable pharmacokinetic profile with a half-life of 5.2 days at 10 mg/kg in monkeys. ABL209 was well tolerated in monkeys up to 40 mg/kg. Our data suggest that a bispecific ADC leverages the benefits of cotargeting two antigens, resulting in enhanced antitumor activity while reducing liabilities through attenuated target-related toxicities.
Anti-GD2 monoclonal antibodies (aGD2 mAbs) are the standard immunotherapy for patients with high-risk neuroblastoma. This treatment has improved 5-year overall survival; however, long-term efficacy still requires improvement. The "don't eat me" signal CD47 is upregulated on neuroblastoma tumor cells and inhibits aGD2 mAb effector mechanisms. Although GD2 is restrictively expressed on neuroblastoma tumor cells, CD47 expression is ubiquitous, resulting in an antigen sink and on-target off-tumor-related cytotoxicities. Recently, we developed two aGD2-SIRPα fusion mAbs for the murine and human settings. In vitro, these aGD2-SIRPα fusion mAbs restrict CD47 blockade toward GD2+ neuroblastoma tumor cells. In this study, we explored the tumor-targeting ability of aGD2-SIRPα fusion mAbs in a syngeneic 9464D-Luc-GFP and SK-N-AS xenograft neuroblastoma tumor model. Conventional aCD47 and aSIRPα mAbs encountered a dominant antigen sink in the 9464D-Luc-GFP model. Surprisingly, although aGD2 mAbs preferentially targeted tumors, murine aGD2-mSIRPα fusion mAbs accumulated in similar organs as aCD47 mAbs. Binding analysis of murine aGD2-mSIRPα and human aGD2-hSIRPα fusion mAbs to red blood cells (RBC) revealed strong binding of murine aGD2-mSIRPα to RBCs, whereas their human counterparts showed negligible binding. These data indicate species-specific CD47-SIRPα binding patterns. Utilizing a SK-N-AS xenograft model, we show effective tumor targeting of the human aGD2-hSIRPα fusion mAbs with hSIRPα in a C-terminal configuration. These data provide the first proof of principle for neuroblastoma tumor-targeted blockade of CD47 by aGD2-hSIRPα fusion mAbs in vivo and support the further development of aGD2-hSIRPα mAbs as attractive therapeutics to improve aGD2-based neuroblastoma immunotherapy.
Anti-CD19 chimeric antigen receptor T (CAR T) cells have promising therapeutic potential for diffuse large B-cell lymphoma (DLBCL), yet many treated patients relapse due to progressive disease driven by CD19-negative clones. Tandem CAR (TCAR) designs co-targeting CD19 and CD20 may overcome this problem. However, murine and primate models do not fully recapitulate human disease and clinical responses to CAR T therapy, limiting preclinical optimization. Moreover, clinical trials are lengthy and costly, further delaying the evaluation of new strategies. By contrast, canine DLBCL closely resembles the human disease, and CAR T trials in canines are feasible, with clinical sequelae that mirror those observed in human CAR T therapy. We previously reported CD20 loss in canine DLBCL patients treated with CD20-specific CAR T cells, consistent with mechanisms of antigen escape in humans. We therefore hypothesized that canine DLBCL could be leveraged to accelerate the translation of more effective TCAR-based strategies, benefiting both canine and human patients. To test this, we first assessed the expression of CD19 and CD20 in canine lymphoma patient samples. We then developed canine TCARs directed against CD19 and CD20 and evaluated their efficacy and specificity against canine DLBCL cells. We show that canine B-cell lymphoma co-expresses CD19 and CD20 with heterogeneous expression patterns similar to those observed in humans, and that TCAR-engineered canine T cells effectively and specifically eliminate cells expressing CD19 and/or CD20. Our TCAR platform holds promise to improve outcomes in canine DLBCL and to further optimize next-generation CAR-based strategies before entering human trials.
Proteogenomic analyses have identified an association between LIG1 (DNA Ligase I) loss and chemotherapy resistance in a subset of triple negative breast cancer (TNBC) enriched for TP53 mutations. Here, we demonstrate that co-occurrence of TP53 mutations and LIG1 loss is associated with upregulated DDR activity, including homologous recombination, likely contributing to reduced platinum sensitivity. Unbiased genetic and monotherapy drug screens identified PARP inhibitors (PARPi) as a potential treatment for LIG1-depleted tumors; however, the increase in sensitivity was modest and lower than that observed in TNBC models with homologous recombination deficiency. Subsequently, a screen of PARP inhibition in combination with each of 120 clinically relevant DDR inhibitors revealed that PARPi sensitivity in LIG1-loss cells was significantly enhanced by the addition of an ATR inhibitor (ATRi). Olaparib and ceralasertib demonstrated synergistic cytotoxicity in LIG1-loss cell line models; the combination significantly reduced tumor volume in a LIG1-low PDX model compared to either monotherapy, and showed greater ex vivo cytotoxicity in a LIG1-low PDXO model versus a LIG1-high control. Hence, this study highlights LIG1 status as a stratification factor for ongoing and future clinical trials of DDR-targeted combinations in TNBCs.
Mutations in β-catenin, together with recurrent genetic alterations affecting the WNT signaling pathway, define one of the most prevalent oncogenic axes, collectively occurring in approximately 10% of human cancers. Thus, β-catenin is a prime target for precision oncology. Over the three decades following the pathway's discovery, substantial progress has been made in elucidating how aberrant WNT/β-catenin signaling can drive cancer initiation, progression and maintenance. Attempts to therapeutically address this pathway have until recently been unsuccessful. Approaches to block the pathway upstream of relevant mutations were ineffective while progress in directly targeting β-catenin, either by blocking its interaction with key complex partners, most notably T cell factor (TCF) proteins, or by inducing β-catenin degradation has long remained an unreachable goal for conventional drug discovery approaches. In this review, we describe opportunities and challenges in the development of therapies that directly aim to target β-catenin. We highlight recent progress based on novel approaches, suggesting that cracking this previously considered "undruggable" central oncogenic driver is becoming a reality. Effective targeting of β-catenin has the potential to address extensive unmet patient needs in hard-to-treat cancer types such as colorectal cancer, hepatocellular carcinoma as well as other cancers driven by WNT/β-catenin pathway alterations.
Sarcomas are rare, heterogeneous tumors with poor prognoses, underscoring the urgent need for novel therapies. Liposomal eribulin (E7389-LF) was developed to improve the efficacy and safety profile of eribulin; however, its preclinical evaluation in sarcomas remains limited. This study aimed to evaluate the clinical applicability of E7389-LF. E7389-LF was comprehensively evaluated and compared with eribulin using 12 patient-derived xenograft (PDX) models representing six sarcoma subtypes. Antitumor activity was assessed across five treatment groups, including independently scaled, clinically relevant regimens of eribulin (0.47 mg/kg) and E7389-LF (0.77 mg/kg). Pharmacokinetic analyses were performed using liquid chromatography-tandem mass spectrometry and fluorescence immunostaining. In addition, correlations between antitumor activity and clinical efficacy were descriptively investigated. Treatment with E7389-LF (0.77 mg/kg) induced tumor regression in 7 of 12 models (58%), compared with 4 of 12 models (33%) treated with eribulin (0.47 mg/kg). Notable responses were observed in rhabdomyosarcoma, Ewing sarcoma, and leiomyosarcoma models. No apparent weight loss was observed in either treatment group. Pharmacokinetic analyses indicated increased systemic and intratumoral drug exposure with E7389-LF. In three PDX models derived from patients treated with eribulin, tumor responses reflected the corresponding clinical outcomes, providing preliminary evidence for the translational relevance of these models. This study demonstrates that E7389-LF exerts antitumor activity broadly comparable to eribulin and exerts antitumor activity in several models across sarcoma PDX models, with favorable tolerability and improved intratumoral drug retention. These findings support the clinical development of E7389-LF for patients with sarcoma who have limited treatment options.
Somatostatin receptor 2 (SSTR2) is overexpressed in neuroendocrine tumors (NETs) and meningiomas. The objective of this study was to develop an SSTR2-targeted therapy to treat both tumors. We developed a humanized anti-SSTR2 monoclonal antibody demonstrating strong cancer cell binding, internalization in cancer cells, and tumor specificity, as evidenced by flow cytometry, confocal microscopy, and live-animal imaging. Antibody-drug conjugates were generated by conjugating the SSTR2 mAb with potent payloads, including monomethyl auristatin F or mertansine. In vitro assessments revealed high cytotoxicity across NET subtypes and meningioma cell lines. In vivo efficacy was confirmed in two mouse models, i.e. subcutaneous NET xenografts and intracranial meningioma xenografts, where treatment inhibited proliferation, induced apoptosis and cell death, exhibited minimal toxicity, and extended survival. The mechanism of action was further elucidated through bulk RNA sequencing after treatment. These findings highlight the therapeutic potential of our humanized SSTR2 mAb for targeted payload delivery in NETs and meningiomas.
Caseinolytic protease proteolytic subunit (ClpP) is part of the mitochondrial ClpXP protease responsible for degrading damaged proteins in the matrix, thus maintaining metabolic homeostasis. Small molecule activators of ClpP (ClpP agonists) have recently shown great promise against metabolically active cancers. TR107, a novel ClpP activator, demonstrates potent and selective cytotoxicity against glioma cells at nanomolar concentrations. Compared to the FDA-approved ClpP agonist ONC201 (Dordaviprone), TR107 shows greater efficacy across patient-derived and isogenic glioma models. TR107 effects are ClpP-dependent and result in widespread mitochondrial dysfunction evident by extensive protein degradation, impaired OxPhos, reduced mtDNA copy number, and ATP depletion. These disruptions are more severe in IDH-mutant than IDH-wildtype glioma cells, leading to enhanced cell cycle arrest, apoptosis, and inhibition of mTOR/AKT/4EBP1 signaling pathway. In this study, we show that ClpP agonism demonstrates preferential anti-tumor activity against IDH-mutant glioma in in vitro, ex vivo, and in vivo models. These findings support TR107 as a promising new targeted therapeutic for IDH-mutant gliomas.
H3K27-altered diffuse midline glioma (DMG) is an aggressive and treatment-resistant form of pediatric high-grade glioma (pHGG). The disease is defined by point mutations in histone H3 that convert lysine 27 to methionine (termed H3K27M), resulting in genome-wide epigenetic changes that drive tumorigenesis. While radiation therapy is the standard of care, subsequent recurrence, often within the high dose radiation field, is universal. We found that the apical DNA damage response kinase Ataxia Telangiectasia-Mutated (ATM) was uniquely upregulated in H3K27Mexpressing patient tumor samples compared to pHGG expressing only wild type histone H3. Using a panel of H3K27 isogenic cell lines, we further found that H3K27M was associated with reduced H3K27me3 within the ATM promoter, increased ATM mRNA levels, and elevated DNA damage response (DDR) signaling, even in the absence of radiation-induced DNA damage. In assessing the effect of these changes on the ability of AZD1390, a clinical-grade, CNS-penetrant ATM inhibitor, to sensitize cells to radiation, we found that while exogenous expression of H3K27M conferred preferential radiosensitization by AZD1390, CRISPR/Cas9 deletion of H3K27M did not attenuate the efficacy of AZD1390 in patient derived DMG cell lines. Finally, AZD1390 sensitized orthotopic H3K27M mutant tumors to radiotherapy in both immune deficient and syngeneic immune competent hosts with minimal adverse effects. Taken together, these data provide a direct mechanistic link between the H3K27M mutation and ATM expression and support the recently initiated clinical investigation of AZD1390 with radiotherapy in newly diagnosed pHGG.
Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive form of pancreatic cancer, with a 5-year survival rate below 10%, largely due to late diagnosis, high metastatic capacity, and resistance to standard therapies. Drug repurposing guided by polypharmacology offers a time- and cost-effective strategy to identify new treatments with reduced clinical risk. Fedratinib, a JAK2 inhibitor approved for myelofibrosis, was previously identified through our bioinformatics analysis as a compound capable of reversing KRAS-driven transcriptional programs in PDAC cells. This study investigates the therapeutic potential and underlying mechanisms of fedratinib in PDAC. Two KRAS-mutant PDAC cell lines were used for in vitro analyses. Cell viability, protein expression, and cell-cycle distribution or apoptosis were assessed using MTT assays, Western blot analysis, and flow cytometry, respectively. Cellular thermal shift assays (CETSA) were performed to evaluate drug-target engagement. In vivo efficacy was examined using a PANC-1 xenograft mouse model treated with fedratinib alone or in combination with mitomycin C. Fedratinib significantly reduced PDAC cell viability, which was associated with caspase-independent cell death, and induced a non-canonical G2/M phenotype characterized by G2/M accumulation, reduced expression of mitotic markers, and accelerated mitotic exit after release from prometaphase arrest. Kinome profiling revealed off-target inhibition of multiple mitotic kinases, which was confirmed by CETSA. Combination screening identified strong synergy between fedratinib and mitomycin C, which was validated in vitro and in vivo. Overall, these findings highlight fedratinib as a promising repurposed therapeutic agent for PDAC and support its combination with mitomycin C as a potential new treatment strategy.
Proteolysis-targeting chimeras (PROTAC) leverage the ubiquitin-proteasome system to selectively degrade oncogenic proteins, including those previously seen as undruggable. Recent preclinical studies indicate that PROTACs may represent a novel therapeutic strategy in lymphoma and myeloma. Indeed, preclinically, PROTACs have shown high efficacy and remarkable selectivity, a favorable safety profile, and lower toxicity compared with conventional therapies. Their catalytic, reusable mechanism enables drug dosing and offers the perspective of long-term low-dose treatment. PROTACs have demonstrated their ability to overcome drug resistance by targeting and degrading overexpressed or mutant proteins that are responsible for refractory disease. This review aims to offer a comprehensive evaluation of the currently existing PROTACs that have been tested in lymphoma and myeloma to highlight the need for drug optimization and further translational research that could translate PROTACs to clinical trials.
The immune checkpoint protein B7H4 is overexpressed in tumors compared to normal tissues, making it an attractive target for cancer immunotherapy. Here, we compare two B7H4-targeting CD3 bispecific antibodies (bsAbs) with different CD3 affinities across in vitro and in vivo characterization studies. In vitro, the CD3xB7H4 bsAb variant with higher CD3 affinity showed greater potency for tumor cell killing and cytokine secretion than the lower affinity variant. In nonclinical toxicology assessments in cynomolgus monkeys, the CD3xB7H4 bsAb variant with lower CD3 affinity induced less cytokine secretion and was tolerated at a higher maximum plasma concentration. Both CD3xB7H4 bsAbs demonstrated antitumor activity in vivo in an ovarian cancer patient-derived xenograft (PDX) mouse model and ex vivo in dissociated primary human ovarian tumor samples. These results demonstrate that increased CD3 affinity enhances cytotoxic potency but results in cytokine secretion at lower antibody concentrations in vitro and in vivo, highlighting an important design trade-off in CD3 bsAb development. The therapeutic potential of CD3xB7H4 bsAbs, particularly in relation to CD3 affinity, warrants further investigation in the clinic.
Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic pattern recognition receptor that senses short double-stranded RNA with uncapped 5'-triphosphate (3p-RNA). Upon activation, RIG-I induces type I interferons and proinflammatory cytokines, thereby promoting adaptive immunity. Thus, RIG-I activation is a promising approach for creating a proinflammatory tumor microenvironment. In this study, we investigated its therapeutic potential in hepatocellular carcinoma (HCC). We explored and confirmed RIG-I expression and signaling in human HCC samples and cell lines. The therapeutic potential of RIG-I activation by 3p-RNA for the treatment of HCC was investigated in vitro and in syngeneic murine orthotopic tumor models. In vivo, 3p-RNA treatment significantly reduced the tumor burden, delayed disease progression, and achieved partial complete remission of RIL-175 tumors with durable immune memory. However, no therapeutic effects were observed in the Hep-55.1C model. Tumor clearance depended on CD4⁺ and CD8⁺ T cells, but not NK cells. Additionally, 3p-RNA induced PD-L1 expression on HCC cells, enhancing their sensitivity to anti-PD-1 immune checkpoint therapy in vivo. RIG-I activation via 3p-RNA therapy shows promise as an immunotherapeutic strategy for hepatocellular carcinoma (HCC). Future investigations need to focus on tumor-intrinsic factors to understand heterogeneity between tumors and to overcome resistance mechanisms.
