Targeting CXCR6 Disrupts β-Catenin Signaling and Enhances Sorafenib Response in Hepatocellular Carcinoma

Cancers · 2025-11-28

Background/Objectives: Hepatocellular carcinoma (HCC) therapies are limited by poor response, rapid resistance, and recurrence of aggressive disease. Sorafenib, a multi-tyrosine kinase inhibitor, can trigger β-catenin stabilization and activation, contributing to resistance. Overexpression of the chemokine receptor CXCR6 and its ligand CXCL16 and hyperactivation are implicated in HCC progression and β-catenin stabilization. We hypothesized that SBI-457, a small-molecule CXCR6 antagonist we developed, could disrupt CXCR6/β-catenin crosstalk and enhance sorafenib sensitivity. Methods: We tested SBI-457 alone and in combination with sorafenib in SK-Hep-1 xenograft models and a panel of human HCC cell lines. Tumor burden, β-catenin activation, and CXCR6 expression were assessed by tumor volume measurements, immunohistochemistry, Western blotting, and immunofluorescence. Soluble CXCL16 levels were quantified by ELISA, and cell death responses were evaluated using MTT assays. Results: In vivo, SBI-457 combined with sorafenib reduced normalized tumor volume by 55% compared to vehicle controls, modestly exceeding monotherapy effects, and attenuated sorafenib-induced β-catenin upregulation. In vitro, SBI-457 blocked nuclear accumulation of β-catenin and reversed sorafenib-induced increases in β-catenin levels. Enhanced cell death was observed in specific “responder” HCC cell lines (Hep-3B, SNU-398, JHH-5), which correlated with high intracellular β-catenin, secretion of soluble CXCL16, and expression of a high molecular weight form of CXCR6. In contrast, “non-responder” cell lines with conventional CXCR6 expression and low CXCL16 secretion showed no enhanced cell death response. Conclusions: CXCR6 antagonism with SBI-457 can modulate β-catenin activation and may help overcome sorafenib resistance in selected HCC models. These findings support further development of CXCR6 antagonists as single agents or combination therapies to improve treatment outcomes in HCC.

Abstract 4355: Investigating the role of CXCR6 signaling in sorafenib resistance in HCC

Cancer Research · 2025-04-21

Abstract Introduction: Hepatocellular carcinoma (HCC), one of the most common liver cancers and a leading cause of cancer-related deaths globally, is often diagnosed at late stages limited to systemic treatment. Sorafenib, an FDA-approved first in line treatment, efficacy is hindered due to innate or developed resistance in patients. Hyperactivation of CXCR6/CXCL16 signaling has been detected and shown to contribute to a proinflammatory and pro-survival tumor microenvironment driving drug resistance in HCC. Wnt/β-catenin signaling pathway is dysregulated in 50% of HCC and has shown to be involved in sorafenib resistance due to accumulation of its core component, β-catenin. Our hypothesis posits that CXCR6 antagonism will restore potency of sorafenib in HCC cell lines via inhibition of β-catenin-mediated signaling. Experimental Procedure: Basal sorafenib sensitivity assessed via MTT assay, and β-catenin and CXCR6 protein expression via western blot were compared across five HCC cell lines. An orally bioavailable small molecule antagonist, SBI-457, was used to probe CXCR6 signaling. We evaluated sorafenib-, SBI-457- and combination-induced modulation of β-catenin expression via western blot and IF imaging in SKHep1. CXCL16- and SBI-457-induced modulation of pCREB protein expression post forskolin activation in both SKHep1 and CXCR6-overexpressed CHOK1 cells were compared. Secretion of soluble CXCL16 was measured via ELISA in SKHep1 cells. Results: Sorafenib dose-dependently increased β-catenin protein expression via immunofluorescence imaging and whole cell lysates of SKHep1 cells. SBI-457 in combination showed trends towards attenuating sorafenib-induced increases, yet lacked significance. We demonstrate that high CXCR6 protein expression levels match sorafenib growth inhibitory (GI50) values across all five HCC cells tested. 200ng/ml CXCL16 significantly attenuated forskolin-induced increase in pCREB expression and SBI-457 dose-dependently restored in a CXCR6 overexpressed CHOK1 cell line. However, both CXCL16 and SBI-457 were unable to clearly modulate forskolin-induced pCREB in CXCR6 endogenously expressed HCC cell lines. SKHep1 cells significantly cleave increasing levels of soluble CXCL16 as measured up to 24h, of which was blocked by ADAM 10 and 17 inhibition. Conclusion: Sorafenib treatment increased total β-catenin levels in SKHep1 cells. Though we show clear CXCR6-mediated signaling in an overexpressed cell line, it is inconsistent in HCC cells, despite CXCR6 expression. SKHep1 cells secrete significantly increasing CXCL16 and this was attenuated by combined inhibition of ADAM10and17. This data suggests SKHep1 cleave soluble CXCL16 from its transmembrane form and potentially alter traditional forward signaling. Future studies aim to evaluate tmCXCL16 expression levels across HCC cells and evaluate the role this plays in CXCR6-mediated signaling via our small molecule inhibitor to further understand the therapeutic potential of CXCR6 inhibition in HCC. Citation Format: Morgan Reeves, Satyamaheshwar Peddibhotla, Siobhan Malany. Investigating the role of CXCR6 signaling in sorafenib resistance in HCC [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 4355.

Discovery of small molecule guanylyl cyclase B receptor positive allosteric modulators

PNAS Nexus · 2024-05-31 · 2 citations

Myocardial fibrosis is a pathological hallmark of cardiovascular disease (CVD), and excessive fibrosis can lead to new-onset heart failure and increased mortality. Currently, pharmacological therapies for myocardial fibrosis are limited, highlighting the need for novel therapeutic approaches. The particulate guanylyl cyclase B (GC-B) receptor possesses beneficial antifibrotic actions through the binding of its natural ligand C-type natriuretic peptide (CNP) and the generation of the intracellular second messenger, cyclic guanosine 3',5'-monophosphate (cGMP). These actions include the suppression of fibroblast proliferation and reduction in collagen synthesis. With its abundant expression on fibroblasts, the GC-B receptor has emerged as a key molecular target for innovative CVD therapeutics. However, small molecules that can bind and potentiate the GC-B/cGMP pathway have yet to be discovered. From a cell-based high-throughput screening initiative of the NIH Molecular Libraries Small Molecule Repository and hit-to-lead evolution based on a series of structure-activity relationships, we report the successful discovery of MCUF-42, a GC-B-targeted small molecule that acts as a positive allosteric modulator (PAM). Studies herein support MCUF-42's ability to enhance the binding affinity between GC-B and CNP. Moreover, MCUF-42 potentiated cGMP levels induced by CNP in human cardiac fibroblasts (HCFs) and notably also enhanced the inhibitory effect of CNP on HCF proliferation. Together, our findings highlight that MCUF-42 is a small molecule that can modulate the GC-B/cGMP signaling pathway, potentially enhancing the antifibrotic actions of CNP. Thus, these data underscore the continued development of GC-B small molecule PAMs as a novel therapeutic strategy for targeting cardiac fibrosis and CVD.

Triazolothiadiazine derivative positively modulates CXCR4 signaling and improves diabetic wound healing

Biochemical Pharmacology · 2023-08-25 · 7 citations

An Optimized Dihydrodibenzothiazepine Lead Compound (SBI-0797750) as a Potent and Selective Inhibitor of Plasmodium falciparum and P. vivax Glucose 6-Phosphate Dehydrogenase 6-Phosphogluconolactonase

Antimicrobial Agents and Chemotherapy · 2022-03-10 · 18 citations

In Plasmodium , the first two and rate-limiting enzymes of the pentose phosphate pathway, glucose 6-phosphate dehydrogenase (G6PD) and the 6-phosphogluconolactonase, are bifunctionally fused to a unique enzyme named GluPho, differing structurally and mechanistically from the respective human orthologs. Consistent with the enzyme’s essentiality for malaria parasite proliferation and propagation, human G6PD deficiency has immense impact on protection against severe malaria, making Pf GluPho an attractive antimalarial drug target.

Discovery of Anthranilic Acid Derivatives as Difluoromethylornithine Adjunct Agents That Inhibit Far Upstream Element Binding Protein 1 (FUBP1) Function

Journal of Medicinal Chemistry · 2022-11-16 · 7 citations

Polyamine biosynthesis is regulated by ornithine decarboxylase (ODC), which is transcriptionally activated by c-Myc. A large library was screened to find molecules that potentiate the ODC inhibitor, difluoromethylornithine (DFMO). Anthranilic acid derivatives were identified as DFMO adjunct agents. Further studies identified the far upstream binding protein 1 (FUBP1) as the target of lead compound 9. FUBP1 is a single-stranded DNA/RNA binding protein and a master controller of specific genes including c-Myc and p21. We showed that 9 does not inhibit 3H-spermidine uptake yet works synergistically with DFMO to limit cell growth in the presence of exogenous spermidine. Compound 9 was also shown to inhibit the KH4 FUBP1–FUSE interaction in a gel shift assay, bind to FUBP1 in a ChIP assay, reduce both c-Myc mRNA and protein expression, increase p21 mRNA and protein expression, and deplete intracellular polyamines. This promising hit opens the door to new FUBP1 inhibitors with increased potency.

Discovery of Small Molecule Activators of Chemokine Receptor CXCR4 That Improve Diabetic Wound Healing

International Journal of Molecular Sciences · 2022-02-16 · 15 citations

Diabetes produces a chronic inflammatory state that contributes to the development of vascular disease and impaired wound healing. Despite the known individual and societal impacts of diabetic ulcers, there are limited therapies effective at improving healing. Stromal cell-derived factor 1α (SDF-1α) is a CXC chemokine that functions via activation of the CXC chemokine receptor type 4 (CXCR4) receptor to recruit hematopoietic cells to locations of tissue injury and promote tissue repair. The expression of SDF-1α is reduced in diabetic wounds, suggesting a potential contribution to wound healing impairment and presenting the CXCR4 receptor as a target for therapeutic investigations. We developed a high-throughput β-arrestin recruitment assay and conducted structure–activity relationship (SAR) studies to screen compounds for utility as CXCR4 agonists. We identified CXCR4 agonist UCUF-728 from our studies and further validated its activity in vitro in diabetic fibroblasts. UCUF-728 reduced overexpression of miRNA-15b and miRNA-29a, negative regulators of angiogenesis and type I collagen production, respectively, in diabetic fibroblasts. In vivo, UCUF-728 reduced the wound closure time by 36% and increased the evidence of angiogenesis in diabetic mice. Together, this work demonstrates the clinical potential of small molecule CXCR4 agonists as novel therapies for pathologic wound healing in diabetes.

Abstract 11943: MCUF-651: A Novel Natriuretic Peptide Receptor A (NPRA) Small Molecule Positive Allosteric Modulator With Blood Pressure Lowering and Renal Enhancing Properties in Experimental Hypertension

Circulation · 2022-11-08

Introduction: Hypertension (HTN) represents one of the greatest burdens in human health and is associated with increased morbidity and mortality. Despite the availability of several antihypertensive drugs, most patients have suboptimal blood pressure (BP) control, thus underscoring the need for new therapeutic drugs. The natriuretic peptide receptor A (NPRA) possesses potent vasodilating, natriuretic and diuretic actions via activation by ANP or BNP and generation of its second messenger, cGMP. Recently, we reported the discovery of a small molecule positive allosteric modulator (PAM) for NPRA, MCUF-651, which enhances the ANP/BNP/NPRA/cGMP pathway (PNAS,2021). To date, the in vivo actions of MCUF-651 remains unknown. Here we tested the hypothesis that MCUF-651 would have BP lowering and renal enhancing properties in experimental HTN. Methods: A single IV bolus of MCUF-651 (10mg/kg; n=4) or vehicle (V; n=5) was injected into anesthetized spontaneous hypertensive rats (SHRs). Plasma and urinary cGMP (PcGMP and UcGMP), mean arterial pressure (MAP) and urinary volume (UV) and sodium (UNa) excretion were assessed at baseline (BL) and over 60-mins post-bolus. Results: In SHRs, PcGMP and UcGMP were significantly elevated with MCUF-651 compared to the vehicle group (ΔPcGMP MCUF-651: 16±5, ΔPcGMP V: -2±3, pmol/mL; P =0.003; ΔUcGMP MCUF-651: 78±29, ΔUcGMP V: 30±8, pmol/min, P =0.01), thus supporting NPRA target engagement. Notably, the elevations in cGMP with MCUF-651 were associated with a significant reduction in MAP at 15-mins post bolus compared to vehicle (ΔMAP MCUF-651: -29±14, ΔMAP V: -6±2 mmHg; P =0.04) and this MAP reduction was sustained over 60 mins. Further, UV and UNa excretion were significantly increased, from BL, with MCUF-651 (BL UV: 6 ± 2, 60 mins post bolus UV: 56± 18 μL/min, P <0.001; BL UNa: 1±0.2, 60 mins post bolus UNa: 8±2 μmol/min, P <0.001). Conclusions: We report for the first time that MCUF-651, a novel NPRA small molecule PAM, engages NPRA, increases cGMP and reduces MAP while enhancing sodium and water excretion in experimental HTN. These findings support the development of MCUF-651 as a potential new therapeutic modality to optimize BP lowering goals and reduce adverse outcomes in human HTN.

Optimization of a urea-containing series of nicotinamide phosphoribosyltransferase (NAMPT) activators

Bioorganic & Medicinal Chemistry Letters · 2021-03-31 · 17 citations

Discovery of small molecule guanylyl cyclase A receptor positive allosteric modulators

Proceedings of the National Academy of Sciences · 2021 · 19 citations

• Chemistry
• Computational biology
• Biophysics

The particulate guanylyl cyclase A receptor (GC-A), via activation by its endogenous ligands atrial natriuretic peptide (ANP) and b-type natriuretic peptide (BNP), possesses beneficial biological properties such as blood pressure regulation, natriuresis, suppression of adverse remodeling, inhibition of the renin-angiotensin-aldosterone system, and favorable metabolic actions through the generation of its second messenger cyclic guanosine monophosphate (cGMP). Thus, the GC-A represents an important molecular therapeutic target for cardiovascular disease and its associated risk factors. However, a small molecule that is orally bioavailable and directly targets the GC-A to potentiate cGMP has yet to be discovered. Here, we performed a cell-based high-throughput screening campaign of the NIH Molecular Libraries Small Molecule Repository, and we successfully identified small molecule GC-A positive allosteric modulator (PAM) scaffolds. Further medicinal chemistry structure-activity relationship efforts of the lead scaffold resulted in the development of a GC-A PAM, MCUF-651, which enhanced ANP-mediated cGMP generation in human cardiac, renal, and fat cells and inhibited cardiomyocyte hypertrophy in vitro. Further, binding analysis confirmed MCUF-651 binds to GC-A and selectively enhances the binding of ANP to GC-A. Moreover, MCUF-651 is orally bioavailable in mice and enhances the ability of endogenous ANP and BNP, found in the plasma of normal subjects and patients with hypertension or heart failure, to generate GC-A-mediated cGMP ex vivo. In this work, we report the discovery and development of an oral, small molecule GC-A PAM that holds great potential as a therapeutic for cardiovascular, renal, and metabolic diseases.