4119 Background: Lenvatinib is a first-line treatment for advanced hepatocellular carcinoma (HCC), but its efficacy is frequently limited by intrinsic and acquired resistance. The underlying molecular mechanisms remain incompletely understood, and strategies to overcome resistance are urgently needed. Methods: We integrated genome-wide CRISPR/Cas9 screening, transcriptomic profiling of lenvatinib-resistant HCC cells, and proteomic analysis of patient tumors to identify key mediators of resistance. Results: Heat shock protein family A (Hsp70) member 6 (HSPA6) emerged as a central driver of both intrinsic and acquired resistance, was consistently upregulated in resistant models, and was associated with poor response and reduced survival in lenvatinib-treated patients. Functional studies demonstrated that HSPA6 knockdown sensitized HCC cells and xenograft tumors to lenvatinib, whereas HSPA6 overexpression conferred resistance both in vitro and in vivo . Mechanistically, HSPA6 recruited the deubiquitinase ubiquitin-specific protease 9X (USP9X) to stabilize thioredoxin reductase 1 (TXNRD1), thereby suppressing lenvatinib-induced ferroptosis. Moreover, lenvatinib enhanced HSPA6 liquid–liquid phase separation (LLPS) through its intrinsically disordered region 1 (IDR1), facilitating the formation of HSPA6–USP9X–TXNRD1 condensates that reinforced TXNRD1 stability. Through structure-based virtual screening, we identified canagliflozin, an FDA-approved sodium–glucose cotransporter 2 (SGLT2) inhibitor, as a direct HSPA6 binder that disrupted this complex, restored ferroptosis sensitivity, and synergized with lenvatinib in preclinical models. Conclusions: Our study defines a novel HSPA6-driven resistance axis that integrates chaperone function, phase separation, and redox homeostasis to suppress ferroptosis in HCC. Targeting this axis with canagliflozin represents a promising therapeutic strategy to overcome lenvatinib resistance.

Microsatellite stable (MSS) rectal cancer exhibits intrinsic resistance to immunotherapy. Although radiotherapy is frequently combined with immune checkpoint inhibitors (ICI) to augment immunotherapy responses, numerous immunologically cold tumors remain unresponsive. In this study, we observed a significant increase in electron transport chain activity, acetyl-CoA levels, and global lysine acetylation levels in patients achieving a pathologic complete response following immunotherapy administered after radiotherapy. Transcriptomic screening and in vivo experiments revealed that SIRT1, a key regulator of protein acetylation, restricted the immunostimulatory effects of radiotherapy. Mechanistically, SIRT1 deacetylated DDX5, promoting the unwinding of irradiation-induced R-loops and inhibiting the accumulation of cytoplasmic RNA:DNA hybrids to suppress cGAS/STING pathway activation and T-cell infiltration. Moreover, radiotherapy induced a tryptophan-SIRT1-SLC36A4 positive feedback loop that enhanced SIRT1 activity and promoted competitive tryptophan uptake from the microenvironment, thereby inhibiting tertiary lymphoid structure (TLS) formation and radioimmunotherapy efficacy. Finally, combining both an SIRT1 inhibitor and aspirin with radiotherapy converted ICI-unresponsive rectal cancer into immunogenic tumors that were sensitive to ICI. Together, this study identifies SIRT1 as a potential biomarker and therapeutic target to overcome radioimmunotherapy resistance in MSS rectal cancer. SIGNIFICANCE:Radiotherapy activates a tryptophan-SIRT1 metabolic feedback loop in microsatellite stable rectal cancer that suppresses T cell infiltration and tertiary lymphoid structures formation, which can be overcome with SIRT1 inhibition and aspirin.

e16211 Background: Increasing evidence showed atezolizumab plus bevacizumab (A+B) with transarterial therapies (ABT therapy) was a highly effective regimen for unresectable hepatocellular carcinoma (uHCC). While, it is still unclear whether dynamic changes of alpha-fetoprotein (AFP) can early predict tumor response, residual tumor activity, and prognosis of HCC patients in the era of combined therapy. This study aimed to explore the AFP trajectory patterns and its impact on clinical outcomes following ABT therapy. Methods: This retrospective study enrolled uHCC patients with baseline AFP levels ＞25 ng/mL who achieved normalization following ABT treatment. Analyses included time to first AFP-complete response (AFP-CR, AFP normalization measured from treatment initiation) and AFP-progression-free survival (AFP-PFS, defined as the time from the first normalization of AFP to the first documented recurrence of AFP). Results: Among the 276 patients who received ABT as first-line therapy, 91 patients had elevated baseline AFP level that normalized after treatment. The median time to first AFP-CR was 2.4 months. At the time of first AFP-CR, tumor response assessed by mRECIST criteria showed CR in 13 patients (14.3%), partial response (PR) in 58 patients (63.7%), and stable disease (SD) in 20 patients (22.0%), whereas assessment by RECIST 1.1 criteria recorded PR in 37 patients (40.7%) and SD in 54 patients (59.3%). Ultimately, disease progression occurred in 27 (29.7%) patients. Among the 44 patients who achieved a mRECIST-based radiological CR (rCR), it occurred later than AFP-CR in 29 patients (65.9%), with a median lag time of 3.0 months (IQR: 2.0-5.1). Of the remaining 15 patients with mRECIST-rCR, it was concurrent with AFP-CR in 10 patients (22.7%) and occurred earlier in 5 patients (11.4%). The median AFP-PFS was 24.5 months, closely aligning with median mRECIST-PFS of 27.4 months. A total of 73 patients maintained sustained normal AFP level until the last follow-up. Conclusions: AFP trajectories provide valuable information complementary to radiological assessment in the combined modality era. Further prospective study is warranted to solidify the utility of AFP trajectory patterns in optimizing management for uHCC.

Background/Aims Lenvatinib resistance remains a critical barrier in advanced hepatocellular carcinoma (HCC) therapy. However, the underlying mechanisms and strategies for reversing resistance remain incompletely understood. Methods Integrated transcriptomics of lenvatinib-resistant patient tumors and an acquired-resistance murine model identified a novel macrophage subpopulation. Functional validation employed CRISPR-SAM screening, conditioned medium (CM) assays, subcutaneous/orthotopic xenografts, patient-derived organoids (PDOs), and patient-derived xenografts (PDXs). Mechanistic studies included ChIP-qPCR, co-immunoprecipitation, and pharmacologic targeting. Clinical relevance was assessed in a retrospective cohort. Results Resistant HCC exhibited significant enrichment of a COLEC12high TAM subset, which correlated with poor survival and treatment response. These TAMs secreted neuregulin-1 (NRG1), activating HER2/HER3-AKT signaling in tumor cells to drive cancer stemness and lenvatinib resistance. Mechanistically, in TAMs COLEC12 sequestered STAT1 in the cytoplasm, preventing its phosphorylation, and thereby derepressing STAT3-mediated NRG1 transcription. Depletion of NRG1 reversed the stemness phenotypes and resensitized tumors to lenvatinib both in vitro and in vivo. Clinically, high NRG1 expression predicted an inferior lenvatinib response and shorter survival. Crucially, the bispecific anti-HER2/HER3 antibody zenocutuzumab restored lenvatinib efficacy in PDOs, PDXs, and murine models. Conclusions Our work establishes the COLEC12high TAM/NRG1 axis as a master regulator of therapeutic resistance and identifies NRG1 as a predictive biomarker, providing a clinically actionable strategy to overcome lenvatinib resistance in HCC.

SIRT1 inhibitor and aspirin enhances the radiotherapy-induced immune priming in MSS rectal cancer

HSPA6 drives lenvatinib resistance in vitro and in vivo

BACKGROUND:Transarterial chemoembolization (TACE) or systemic treatments are recommended for unresectable hepatocellular carcinoma (uHCC) but have low conversion-to-resection rates. This single-arm phase II trial (PLATIC) aims to investigate the conversion efficacy of PD-1 inhibitor sintilimab, lenvatinib plus TACE and hepatic arterial infusion chemotherapy (TACE-HAIC) in uHCC. METHODS:57 uHCC patients received 200 mg of sintilimab and lenvatinib (8/12 mg for body weight <60 kg or ≥60 kg) and TACE-HAIC every 3-4 weeks. The primary endpoint was conversion-to-resection rate. A historical-based cohort treated with TACE-HAIC was included as control. FINDINGS:After a median of three treatment cycles, 44 (77.2%; 95% confidence interval [CI], 0.64-0.87]) patients underwent conversion resection. The objective response rates were 80.7% (95% CI, 0.68-0.90) according to modified Response Evaluation Criteria in Solid Tumors (mRECIST) and 43.8% (95% CI, 0.31-0.58) according to RECIST 1.1. Grade ≥ 3 treatment-related adverse events (TRAEs) occurred in 37 (64.9%) patients; no treatment-related deaths were reported. Among the 44 patients receiving resection, the median progression-free survival (PFS) was 17.3 months, and the median overall survival (OS) was unavailable. After inverse probability of treatment weighting analysis, the PLATIC cohort exhibited longer PFS (hazard ratio [HR] = 0.55, 95% CI, 0.33-0.94; p = 0.020) and OS (HR, 0.50, 95% CI, 0.28-0.92; p = 0.029) than the historical cohort. CONCLUSIONS:Sintilimab, lenvatinib, and TACE-HAIC demonstrated favorable conversion-to-resection rates and acceptable TRAEs in uHCC. FUNDING:Supported by grants from the National Natural Science Foundation of China (no. 82172815 and 82272887) and Cancer Innovative Research Program of Sun Yat-sen University Cancer Center (no. PT22040201).

Vessels encapsulating tumor clusters (VETC), a distinct vascular pattern in hepatocellular carcinoma (HCC), facilitates noninvasive metastasis in whole clusters. The interaction between VETC and the tumor microenvironment requires exploration. Here, we found that, compared with human non-VETC-HCCs, VETC-tumors exhibited more PD1+CD8+ T cells and Tregs, especially TNFRSF4+ Tregs and Ki67+ Tregs, which showed increased immunosuppressive and proliferative activity. Such immunosuppressive status was also detected in tumor emboli of VETC-HCCs, and Treg density in emboli was positively associated with metastatic cell proliferation. VETC-HCCs revealed abundance correlation, closer spatial proximity, and stronger immunosuppressive ligand-receptor interactions between TNFRSF4+ Tregs/Ki67+ Tregs and PD1+CD8+ T cells. Depleting Tregs in mice reduced PD1+CD8+ T cells in primary lesions, tumor emboli, and metastatic foci of VETC-allografts, and attenuated allograft metastasis. TGF-β1 levels were upregulated in endothelial cells of VETC-HCCs and associated with TNFRSF4+ Tregs/Ki67+ Tregs enrichment. Disrupting VETC formation decreased endothelial TGF-β1 expression and reduced TNFRSF4+ Tregs, Ki67+ Tregs, PD1+CD8+ T cells, and Treg/CD8+ T cell ratios. Collectively, VETC may enhance Treg activity via TGF-β1, while Tregs promote and sustain CD8+ T cell exhaustion through immune inhibitory ligand-receptor interaction, thereby shaping the immunosuppressive microenvironment and enabling tumor clusters to retain such niche to disseminate. These findings disclose mechanisms of tumor immune microenvironment formation and provide rationales for precision medicine.

Integrated multi-omics identifies HSPA6 as a key mediator of lenvatinib resistance in HCC

Proteins detected by IP in CT26 cell lysates

Significantly differentially expressed genes identified in pCR group compared to non-pCR group after radioimmunotherapy

SIRT1-mediated deacetylation of DDX5 resolves R-loops induced by irradiation to prevent RNA:DNA hybrids accumulation

e16169 Background: Advanced intrahepatic cholangiocarcinoma (ICC) remains a highly aggressive malignancy with limited treatment options and poor prognosis. Systemic chemotherapy and immune therapies have shown modest efficacy, highlighting the need for novel combination strategies. This single-arm phase 2 study evaluated the efficacy and safety of tislelizumab (a PD-1 inhibitor) combined with apatinib (a VEGFR-2 inhibitor) and hepatic arterial infusion chemotherapy (HAIC) in patients with advanced ICC. Methods: Eligible patients with histologically confirmed advanced ICC were enrolled between Jan 2022 and Jun 2025. Treatment consisted of HAIC (oxaliplatin 130 mg/m²,5-Fu 400mg/ m², 5-Fu 1200mg/ m² via hepatic artery on day 1/2 each 3-week cycle), tislelizumab (200 mg intravenously every 3 weeks) and apatinib (250 mg orally daily). The primary endpoint was objective response rate (ORR) per RECIST v1.1, assessed by independent radiological review. Secondary endpoints included disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and safety. Results: A total of 17 patients were enrolled, with a median follow-up of 22 months. The confirmed ORR was 41.2% (95% CI: 18.4%-67.1%). The DCR was 88.3% (95% CI: 65.6%-98.5%). Median PFS was 8.8 months (95% CI: 4.33 - NA months), and the median OS was 17.6 months (95% CI: 15.0 - NA months). Notably, 3 of 17 patients (17.6%) subsequently underwent liver tumor resection with curative intent, achieving R0 resection. Eight patients (47.1%) had at least one any-grade treatment-related adverse event (TRAE); grade 3-4 TRAEs occurred in 3 patients (17.6%), with elevated transaminases (47.1%) being most common. No treatment-related deaths occurred. Conclusions: The combination of HAIC, tislelizumab and apatinib demonstrated promising efficacy with a manageable safety profile in advanced ICC, supporting further investigation in larger controlled trials.

PURPOSE:Image guided stereotactic body radiation therapy (SBRT) serves as a key local treatment for liver metastases. As a next-generation technology, magnetic resonance guided SBRT (MRgRT) offers theoretical advantages over computed tomography guided SBRT for the management of liver metastases. However, clinical evidence regarding the efficacy of MRgRT for colorectal cancer liver metastases (CRLM) remains lacking. METHODS AND MATERIALS:This retrospective study included patients with CRLM who underwent MRgRT at a single institution between September 2021 and December 2023. Local control (L), distant hepatic control, progression-free survival, and overall survival (OS) were estimated using the Kaplan-Meier method and analyzed using statistical software (R and SPSS). Adverse events were graded according to the Common Terminology Criteria for Adverse Events version 5.0. RESULTS:Overall, 51 targets (planning target volumes) in 49 patients with CRLM were treated with MRgRT. The median lesion diameter was 1.9 cm (range, 0.5-6.4 cm), whereas the median prescribed dose to the gross tumor volume was 57 Gy (range, 48-60 Gy). The median follow-up duration was 20.1 months. Among the 51 targets, 3 experienced local failure. None of the patients developed grade >2 radiation-related toxicity. The LC rates at 12 and 24 months were 93.8% and 86.6%, respectively. The distant hepatic control rates at 12 and 24 months were 52.5% and 49.2%, respectively. The progression-free survival rates at 12 and 24 months were 31.5% and 25.5%, whereas the OS rates at 12 and 24 months were 91.4% and 84.0%, respectively. CONCLUSIONS:MRgRT achieved favorable LC and OS rates in the treatment of CRLM with minimal toxicity, demonstrating its potential for broader clinical application. Further studies with larger sample sizes and long-term follow-up are warranted to validate survival outcomes and assess late-onset toxicities.

HSPA6 drives lenvatinib resistance in vitro and in vivo

Abstract The development of therapy resistance compromises the long-term efficacy of lenvatinib in advanced hepatocellular carcinoma (HCC), highlighting the need to characterize the molecular drivers of resistance. Through integrated multiomics analysis combining genome-wide CRISPR screening, transcriptomics, and proteomics, we identified Heat shock protein family A member 6 (HSPA6) as a critical driver of lenvatinib resistance. HSPA6 was consistently upregulated in resistant cell lines and patient tumors, and high expression correlated with poor treatment response and survival. HSPA6 recruited the deubiquitinase USP9X to stabilize the antioxidant enzyme thioredoxin reductase 1 (TXNRD1), thereby suppressing lenvatinib-induced ferroptosis. Furthermore, HSPA6 underwent drug-enhanced liquid–liquid phase separation through its intrinsic disordered region 1 domain, facilitating the assembly of biomolecular condensates that reinforce TXNRD1 stability and ferroptosis resistance. Leveraging these insights, canagliflozin, an FDA-approved sodium-glucose cotransporter 2 inhibitor, was repurposed as a direct HSPA6-targeting compound that disrupted this resistance axis, restored ferroptosis sensitivity, and synergized with lenvatinib in patient-derived models. This work unveils a dynamic, condensate-driven mechanism of drug resistance and offers a readily translatable strategy to overcome lenvatinib resistance in HCC. Significance: HSPA6 drives lenvatinib resistance and can be targeted by repurposing canagliflozin to restore ferroptosis sensitivity and overcome therapy resistance in hepatocellular carcinoma.

Protein modifications detected by IP in HT29 cell lysates