A549 Xenograft Research Articles

Functional genomics pipeline identifies CRL4 inhibition for the treatment of ovarian cancer.

The goal of precision oncology is to find effective therapeutics for every patient. Through the inclusion of emerging therapeutics in a high-throughput drug screening platform, our functional genomics pipeline inverts the common paradigm to identify patient populations that are likely to benefit from novel therapeutic strategies. Utilizing drug screening data across a panel of 46 cancer cell lines from 11 tumor lineages, we identified an ovarian cancer-specific sensitivity to the first-in-class CRL4 inhibitors KH-4-43 and 33-11. CRL4 (i.e., Cullin-4 RING E3 ubiquitin ligase) is known to be dysregulated in a variety of cancer contexts, making it an attractive therapeutic target. Unlike proteasome inhibitors that are associated with broad toxicity, CRL4 inhibition offers the potential for tumor-specific effects. We observed that CRL4 inhibition negatively regulates core gene signatures that are upregulated in ovarian tumors and significantly slowed tumor growth as compared to the standard of care, cisplatin, in OVCAR8 xenografts. Building on this, we performed combination drug screening in conjunction with proteomic and transcriptomic profiling to identify ways to improve the antitumor effects of CRL4 inhibition in ovarian cancer models. CRL4 inhibition consistently resulted in activation of the mitogen-activated protein kinase (MAPK) signaling cascade at both the transcriptomic and protein levels, suggesting that survival signaling is induced in response to CRL4 inhibition. These observations were concordant with the results of the combination drug screens in seven ovarian cancer cell lines that showed CRL4 inhibition cooperates with MEK inhibition. Preclinical studies in OVCAR8 and A2780 xenografts confirmed the therapeutic potential of the combination of KH-4-43 and trametinib, which extended overall survival and slowed tumor progression relative to either single agent or the standard of care. Together, these data demonstrate the prospective utility of functional modeling pipelines for therapeutic development and underscore the clinical potential of CRL4 inhibition in the ovarian cancer context. A precision medicine pipeline identifies ovarian cancer sensitivity to CRL4 inhibitors. CRL4 inhibition induces activation of MAPK signalling as identified by RNA sequencing, proteomics, and phosphoproteomics. Inhibitor combinations that target both CRL4 and this CRL4 inhibitor-induced survival signalling enhance ovarian cancer sensitivity to treatment.

Identification of narciclasine as a novel NRF2 inhibitor

Cancer genome sequencing studies have identified somatic mutations in the KEAP1/NRF2 pathway. In an effort to identify novel NRF2 small molecule inhibitor(s), we have screened a natural compound library comprising 1,330 chemicals in A549-ARE-GFP-luciferase cells and identified that narciclasine significantly inhibits NRF2-dependent luciferase activity. Narciclasine suppressed the expression of NRF2 and NRF2 target genes, caused significant oxidative stress, and sensitized cisplatin-mediated apoptosis in A549 cells. In addition, we have observed that WD Repeat Domain 43 (WDR43) serves as a direct target of narciclasine for the inhibition of NRF2 as narciclasine binds to recombinant WDR43 in vitro and silencing WDR43 attenuated the inhibition of NRF2 by narciclasine in A549 cells. Finally, we observed that administration of narciclasine significantly decreased the growth of A549 xenografts. Together, our results demonstrate that the inhibition of NRF2 by narciclasine is mediated by WDR43 and future studies are necessary to elucidate the exact mechanism how WDR43 mediates the inhibition of NRF2 by narciclasine.

Preclinical studies on the antitumor and non-toxic effect of combining pirfenidone with vinorelbine and carboplatin in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) shows limited therapeutic response to vinorelbine and carboplatin. Combining these drugs with an antifibrotic drug may enhance their antitumor effect. Pirfenidone is an antifibrotic drug whose antitumor activity has been described in different types of cancer. This work aimed to perform preclinical studies on the combination of pirfenidone with vinorelbine, or with vinorelbine plus carboplatin, in NSCLC. Our data revealed that pirfenidone sensitized three NSCLC cell lines to vinorelbine by reducing cell growth, viability and proliferation, inducing alterations in the cell cycle profile, and increasing cell death (%). Importantly, pirfenidone increased the sensitivity of the three NSCLC cell lines to the combined treatment of vinorelbine plus carboplatin. This combined drug treatment (triplet) did not induce cytotoxicity against non-tumorigenic cells. Notably, the triplet drug combination significantly reduced the growth and proliferation of A-549 xenografts in nude mice, as also reduced vimentin and collagen expression. Most interestingly, the triplet treatment exhibited a safer toxicological profile than the doublet (vinorelbine plus carboplatin) currently applied in the clinical practice. Altogether, these preclinical data support the possibility of repurposing pirfenidone in combination with vinorelbine or with vinorelbine plus carboplatin for NSCLC perioperative treatment, improving therapeutic efficacy while reducing toxicity.

Experimental study of early evaluation of radiosensitivity in mouse models of lung cancers using 89Zr-anti-γH2AX-TAT PET imaging

BackgroundEarly evaluation of radiation sensitivity in lung cancer patients can facilitate the transition to personalized treatment strategies. To this end, we assessed the capability of 89Zr-anti-γH2AX-TAT microPET imaging in determining the radiosensitivity of lung cancer xenograft models. We prepared and conducted quality control on 89Zr-anti-γH2AX-TAT. The radiosensitivity of human non-small cell lung cancer cells (H460) and adenocarcinoma cells (A549) was analyzed through clonogenic survival experiments. Additionally, the role of γH2AX as a biomarker for radiosensitivity was validated by quantifying γH2AX foci via fluorescence staining. Subsequently, the H460 and A549 xenograft mouse models were subjected to irradiation, followed by 89Zr-anti-γH2AX-TAT microPET imaging. Concurrently, we performed immunofluorescence staining for γH2AX in tumor tissues to establish a correlation between the uptake of 89Zr-anti-γH2AX-TAT and γH2AX expression.ResultsThe surviving fraction 2 Gy (SF2) values of H460 and A549 indicating that A549 adenocarcinoma has higher radiosensitivity. The cell immunofluorescence experiment showed that the repair of γH2AX foci in H460 cells after irradiation was significantly higher than that in A549 cells, which also confirmed that A549 has higher radiosensitivity. The microPET imaging results showed the uptake of 89Zr-anti-γH2AX-TAT in the tumor of the A549 models after radiotherapy was higher than H460 models. The immunofluorescence staining of tumor tissue confirmed that the expression level of γH2AX was higher and the correlation with microPET imaging uptake was good.Conclusion89Zr-anti-γH2AX-TAT allows PET imaging of radiosensitivity in lung cancer xenograft models, and is expected to become an early evaluation method for lung cancer radiosensitivity.

DDDR-04. PRECLINICAL INVESTIGATION OF A NOVEL BRAIN PENETRANT COMBINATION THERAPY TARGETING RAF AND MEK FOR MELANOMA BRAIN METASTASIS

Abstract INTRODUCTION Up to 75% of patients with advanced melanoma develop brain metastases. BRAF mutations are found in 50-55% of melanoma brain metastases. Current BRAF/MEK inhibitors, such as dabrafenib and vemurafenib, have shown responses on the order of 50-60% in patients with BRAF-mutant melanoma; however, responses are not durable. Better therapies are needed with improved penetration of the blood brain barrier. This study aims to evaluate the efficacy of two novel brain-penetrant inhibitors, KIN-7136 (MEK inhibitor) and KIN-8391 (RAF inhibitor), in preclinical mouse models of melanoma brain metastases driven by aberrant BRAF-MEK signaling. METHODS CellTiter-Glo® 2.0 Cell Viability Assay (Promega) was used for in vitro cell viability assays. Intracranial A375 melanoma models were generated in athymic nude mice to assess the pharmacodynamics, safety, and efficacy of combination therapy of KIN-7136 and KIN-8391. RESULTS In mice, the brain-to-plasma unbound partition coefficients (Kpuu) of KIN-7136 and KIN-8391 were 0.67 and 0.7 respectively, higher than the comparators binimetinib and belvarafenib, demonstrating improved brain exposure. The IC50s of KIN-7136/ KIN-8391 in melanoma cell lines were 27.0/36.5 nM in A375 (BRAF V600E), 78.6/35.4 nM in HMV2 (BRAF G469V), 183.0/699.0 nM in SK-MEL-2 (NRAS Q61R), and 53.9/110.0 nM in SK-MEL30 (BRAF D287H and E275K), respectively. KIN-7136 and KIN-8391 synergistically inhibited HMV2, SK-MEL-2, and SK-MEL-30 cells. Pharmacodynamic studies showed that both KIN-7136 and KIN-8391 suppressed phospho-ERK, a downstream component of BRAF-MEK signaling, in A375 xenografts in mice. Daily oral treatment with a combination of KIN-7136 and KIN-8391 was well-tolerated and extended overall survival to 64 days (median survival), compared to the vehicle control and monotherapies with KIN-7136 or KIN-8391 (28, 58, and 46.5, respectively) in the A375 intracranial tumor model. CONCLUSION These preclinical data confirm the synergistic effect of KIN-7136 and KIN-8391 in BRAF-mutant melanoma brain metastases models, supporting further research to advance its clinical translation.

Combination of ataxia telangiectasia and Rad3-related inhibition with ablative radiotherapy remodels the tumor microenvironment and enhances immunotherapy response in lung cancer

We investigated the combined effects of ataxia telangiectasia and Rad3-related (ATR) inhibition, ablative radiotherapy, and immune checkpoint inhibitor (ICI) therapy against lung cancer. ATR inhibitor was administered combined with ablative radiotherapy to assess its radiosensitizing effect on lung cancer cells. Treatment response and survival were evaluated in vivo using A549 xenograft flank tumor and synchronous LLC lung and flank tumor mouse models. Mice received ablative radiotherapy (12 Gy/d for 2 d), ATR inhibitor, and ICI. The tumor microenvironment was assessed in irradiated flank and non-irradiated lung tumors. Programmed death-ligand 1 expression was upregulated after irradiation. ATR inhibition attenuated this upregulation. ATR inhibitor pretreatment decreased cell survival after irradiation by inhibiting DNA double-strand break repair, inducing mitotic cell death, and altering cell cycle progression. ATR inhibition enhanced radiation-induced damage-associated molecular patterns determined by high mobility group box 1 quantification and activated the cyclic GMP-AMP synthase-stimulator of interferon genes pathway. Combined ATR inhibition and ablative radiotherapy inhibited tumor growth and improved survival in mice. Adding ICI therapy further enhanced local antitumor effects, reducing the metastatic lung tumor burden and remodeling the tumor microenvironment through immunogenic cell death induction and enhanced immune cell infiltration. Triple therapy increased immune cell infiltration in distant non-irradiated lung tumors and stimulated the generation of protective T-cell immunity in splenocytes. Safety analysis showed minimal toxicity. ATR inhibition enhanced the efficacy of ablative radiotherapy and immunotherapy in lung cancer. These findings underscore the importance of combination therapies for enhancing systemic antitumor immune responses and outcomes.

A PD-L1-Targeted Probe Cy5.5-A11 for In Vivo Imaging of Multiple Tumors.

PD-L1 is an immune checkpoint molecule mediating cancer immune escape, and its expression level in the tumor has been used as a biomarker to predict response to immune checkpoint inhibitor (ICI) therapy. Our previous study reveals that an 11 amino acid-long ANXA1-derived peptide (named A11) binds and degrades the PD-L1 protein in multiple cancers and is a potential peptide for cancer diagnosis and treatment. Near-infrared fluorescence (NIF) optical imaging of tumors offers a noninvasive method for detecting cancer and monitoring therapeutic responses. In this study, an NIF dye Cy5.5 was conjugated with A11 peptide to develop a novel PD-L1-targeted probe for molecular imaging of tumors and monitor the dynamic changes in PD-L1 expression in tumors. In vitro imaging studies showed that intense fluorescence was observed in triple-negative breast cancer MDA-MB-231, nonsmall cell lung cancer H460, and melanoma A375 cells incubated with Cy5.5-A11, and the cellular uptake of Cy5.5-A11 was efficiently inhibited by coincubation with unlabeled A11 or knockdown of cellular PD-L1 by shRNA. In vivo imaging studies showed accumulation of Cy5.5-A11 in the MDA-MB-231, H460, and A375 xenografts with good contrast from 0.5 to 24 h after intravenous injection, indicating that Cy5.5-A11 possesses the strong ability for in vivo tumor imaging. Moreover, the fluorescent signal of A11-Cy5.5 in the xenografts was successfully blocked by coinjection of unlabeled A11 peptide or knockdown of cellular PD-L1 by shRNA, indicating the specificity of Cy5.5-A11 targeting PD-L1 in tumor imaging. Our data demonstrate that Cy5.5-A11 is a novel tool for tumor imaging of PD-L1, which has the potential for detecting cancer and predicting ICI therapeutic responses.

Real-Time Fluorescence Monitoring System for Optimal Light Dosage in Cancer Photoimmunotherapy.

Background/Objectives: Near-infrared photoimmunotherapy (NIR-PIT) was recently approved for the treatment of unresectable locally advanced or recurrent head and neck cancers in Japan; however, only one clinical dose has been validated in clinical trials, potentially resulting in excessive or insufficient dosing. Moreover, IRDye700X (IR700) fluorescence intensity plateaus during treatment, indicating a particular threshold for the antitumor effects. Therefore, we investigated the NIR laser dose across varying tumor sizes and irradiation methods until the antitumor effects of the fluorescence decay rate plateaued. Methods: Mice were subcutaneously transplanted with A431 xenografts and categorized into control, clinical dose (cylindrical irradiation at 100 J/cm², frontal irradiation at 50 J/cm²), and evaluation groups. The rate of tumor IR700 fluorescence intensity decay to reach predefined rates (-0.05%/s or -0.2%/s) until the cessation of light irradiation was calculated using a real-time fluorescence imaging system. Results: The evaluation group exhibited antitumor effects comparable to those of the clinical dose group at a low irradiation dose. Similar results were observed across tumor sizes and irradiation methods. Conclusions: In conclusion, the optimal antitumor effect of NIR-PIT is achieved when the fluorescence decay rate reaches a plateau, indicating the potential to determine the appropriate dose for PIT using a real-time fluorescence monitoring system.

Investigating the In Vivo Biodistribution of Extracellular Vesicles Isolated from Various Human Cell Sources Using Positron Emission Tomography.

Positron emission tomography (PET) is a powerful tool for investigating the in vivo behavior of drug delivery systems. We aimed to assess the biodistribution of extracellular vesicles (EVs), nanosized vesicles secreted by cells isolated from various human cell sources using PET. EVs were isolated from mesenchymal stromal cells (MSCs) (MSC EVs), human macrophages (Mϕ EVs), and a melanoma cell line (A375 EVs) by centrifugation and were conjugated with deferoxamine for radiolabeling with Zr-89. PET using conjugated and radiolabeled EVs evaluated their in vivo biodistribution and tissue tropisms. Our study also investigated differences in mouse models, utilizing immunocompetent and immunocompromised mice and an A375 xenograft tumor model. Lastly, we investigated the impact of different labeling techniques on the observed EV biodistribution, including covalent surface modification and membrane incorporation. PET showed that all tested EVs exhibited extended in vivo circulation and generally low uptake in the liver, spleen, and lungs. However, Mϕ EVs showed high liver uptake, potentially attributable to the intrinsic tissue tropism of these EVs from the surface protein composition. MSC EV biodistribution differed between immunocompetent and immunodeficient mice, with increased spleen uptake observed in the latter. PET using A375 xenografts demonstrated efficient tumor uptake of EVs, but no preferential tissue-specific tropism of A375 EVs was found. Biodistribution differences between labeling techniques showed that surface-conjugated EVs had preferential blood circulation and low liver, spleen, and lung uptake compared to membrane integration. This study demonstrates the potential of EVs as effective drug carriers for various diseases, highlights the importance of selecting appropriate cell sources for EV-based drug delivery, and suggests that EV tropism can be harnessed to optimize therapeutic efficacy. Our findings indicate that the cellular source of EVs, labeling technique, and animal model can influence the observed biodistribution.

DNA self-assembly-mediated high drug-antibody ratio ADC platform for targeted tumor therapy and imaging

Antibody-drug conjugates (ADCs) achieve therapeutic effects through toxin delivery inside cells, targeting high-abundance antigens. However, tumor heterogeneity and the low abundance of tumor-specific antigens underscore the urgent requirement for developing a flexible, multifunctional, and high-capacity drug-delivery platform. The current study developed a self-assembled ADC platform called the high drug–antibody-ratio ADC (HD-ADC). Cytotoxic payloads were efficiently conjugated into a 12-arm deoxyribonucleic acid (DNA) branched junction via DNA-mediated precise self-assembly to achieve a high drug–antibody ratio (DAR). Anti-EGFR HD-ADC showed strong efficacy in causing cytotoxicity and suppressing tumor growth in an A431 xenograft mouse model. Furthermore, the Cy5-conjugated HD-ADC platform was a simple and effective method for improving fluorescent signal detection, enabling the detection of targets—such as neoantigens—with ultralow-expression levels. The HD-ADC platform supports the assembly of various functional components, providing the foundation for usage across multiple antibody-mediated targeted therapies and diagnostics.

5 A Phase 1b, Open-Label, Safety, Tolerability, and Efficacy Study of HC-7366 in Combination with Belzutifan (WELIREGTM) in Patients with Advanced or Metastatic Renal Cell Carcinoma, NCT06234605

Abstract Background HC-7366 is a novel, orally administered, highly selective and potent activator of general control nonderepressible 2 (GCN2) kinase, a core regulator of metabolic stress through activation of the integrated stress response (ISR). Activation of GCN2 promotes cell survival, whereas prolonged activation induces apoptosis. GCN2 activation also suppresses general protein synthesis and induces cell cycle arrest, thereby preventing cell growth during nutrient scarcity. Additionally, HC-7366 decreases HIF expression and inhibits glycolysis, oxidative phosphorylation, and TCA cycle function. HC-7366 also inhibits HIF expression in immunosuppressive myeloid cells, including macrophages. These effects of HC-7366 on metabolism, HIF signaling, and immune suppression suggest therapeutic benefit in ccRCC with clear rationale for combinations with HIF2a antagonists and immune checkpoint inhibitors. HC-7366 (0.5-1 mg/kg), combined with belzutifan (1 mg/kg), exhibited combination benefit in HIF-2 dependent A-498 and 786-O RCC xenografts, yielding 90% tumor growth inhibition and a three-fold increase in complete responses, respectively. Additionally, HC-7366 drives significant monotherapy antitumor activity in PDX models that demonstrated belzutifan resistance. Mechanism of action studies have identified several pathway engagement and potential efficacy biomarkers (Stokes, et al., 2024). Trial design/schema This multicenter, open-label phase 1b study will identify the Maximum Tolerated Dose (MTD) and/or the Recommended Phase 2 Dose (RP2D) of HC-7366 in combination with fixed-dose belzutifan, 120 mg po qd, in patients with advanced or metastatic RCC with renal cell histology irrespective of VHL gene mutation. Monotherapy HC-7366 will be evaluated in parallel. The monotherapy arm, HC-7366, 60 mg p.o. qd, includes patients who have relapsed after 1 to 4 prior lines of standard of care that may include belzutifan or another HIF-2α inhibitor. The combination arm includes patients who have received 1 to 3 prior lines of standard of care and are belzutifan or HIF-2α naïve. HC-7366 dose escalation arm evaluates combination fixed dose belzutifan, 120 mg po qd plus HC-7366 at 20, 40, or 60 mg po qd. Enrollment will start with the HC-7366 monotherapy arm. Subsequently, patients will begin to enroll in the combination therapy HC-7366 dose escalation arm, while the monotherapy arm will continue to enroll. Expansion will evaluate the combination of fixed dose belzutifan with two doses of HC-7366 selected from escalation. Assessments include safety, PK, and anti-tumor activity. The study will enroll up to 80 patients at US study sites. Significance and vision Combination HC-7366 plus belzutifan, supported by preclinical evidence, is being studied to assess antitumor activity in the RCC relapsed setting. Determining safety and evaluation of dose are foundational to this study. Reference Stokes M, Tameire F, Wojnarowicz P, et al. HC-7366, a potent GCN2 activator, complements belzutifan, a HIF-2⍺ antagonist, by providing combination benefit in belzutifan-sensitive models and monotherapy activity in belzutifan-resistant models. Meeting of the American Association for Cancer Research; 2024 Apr 5-10; San Diego (CA); AACR; 2024. Abstract 4615. This study is in collaboration with Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA

Preclinical ImmunoPET Imaging Using a Zr-89-Labeled Anti-CD146 Monoclonal Antibody for Diagnosis of Melanoma.

The aim of this study was to evaluate the preclinical efficacy of [89Zr]Zr-DFO-Ab253 as a novel positron emission tomography (PET) tracer for CD146-positive malignant melanoma imaging. Considering the high expression of CD146 in malignant melanoma, this study investigated the effect of different CD146 expression levels on the tumor uptake of [89Zr]Zr-DFO-Ab253. CD146 selectivity was investigated by using the CD146-positive human melanoma cell A375 and the CD146-negative human alveolar epithelial cell A549. The cell uptake of [89Zr]Zr-DFO-Ab253 tracers was investigated, and receptor-binding affinities were measured by radioactive enzyme-linked immunosorbent assay. Biodistribution studies and micro-PET imaging of the radiotracers were performed on mice bearing A375 and A549 xenografts under baseline and blocking conditions. An immunohistochemical test was performed using A375 and A549 tissue sections for CD146 expression level analysis. [89Zr]Zr-DFO-Ab253 was obtained with a high radiochemical yield (87.86 ± 4.66%) and a satisfactory radiochemical purity (>98.0%). The specificity and affinity of [89Zr]Zr-DFO-Ab253 were confirmed in melanoma A375 cells and in vivo PET imaging of A375 tumor models. [89Zr]Zr-DFO-IgG and A549 lung tumors were prepared as control radiotracers and negative models to verify the specificity of [89Zr]Zr-DFO-Ab253 on CD146. [89Zr]Zr-DFO-Ab253 has a Kd of 4.01 ± 0.50 nM. PET imaging and biodistribution showed a higher uptake of [89Zr]Zr-DFO-Ab253 in A375 melanomas than that in A549 tumors (42.1 ± 4.04% vs 7.87 ± 1.30% ID/g at 120 h, P < 0.05). A low tumor uptake of [89Zr]Zr-DFO-IgG was observed with uptakes of 1.91 ± 0.41 and 2.80 ± 0.14 ID%/g when blocked at 120 h. The radiation-absorbed dose was calculated to be 0.13 mSv/MBq. This study demonstrates the synthesis and preclinical evaluation of [89Zr]Zr-DFO-Ab253 and indicates that the novel tracer has promising applications in malignant melanoma-specific PET imaging because of its high uptake and long-time retention in malignant melanoma. It also provides feasibility for the development of integrated molecular probes for diagnosis and treatment based on the CD146 target.

A Food Odorant, α-Ionone, Inhibits Skin Cancer Tumorigenesis by Activation of OR10A6.

This study aims to investigate the anticancer properties of α-ionone in squamous cell carcinoma (SCC). The expression of OR10A6 together with olfactory receptor signaling components is demonstrated in A431 human SCC cells via RT-PCR and qRT-PCR analysis. OR10A6 activation in A431 cells using the ligand α-ionone inhibits proliferation and migration but induces apoptosis which is confirmed by proliferation assay, colony formation, and western blotting. The mechanism involves the core proteins of the Hippo pathway, where the phosphorylation of large tumor suppressor kinase (LATS), yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ) is confirmed by western blotting. However, the anticancer effects of α-ionone are abrogated in A431 cells with OR10A6 gene knockdown. In A431 xenograft mouse model, the injection of α-ionone suppresses tumor growth, induces apoptosis, and increases phosphorylation of the LATS-YAP-TAZ signaling axis in the Hippo pathway. None of these effects are observed in xenografted tumors with OR10A6 gene knockdown. These findings collectively demonstrate that activation of ectopic OR OR10A6 by α-ionone in SCC cells stimulates the Hippo pathway and suppresses tumorigenesis both in vitro and in vivo, suggesting a novel therapeutic candidate for the treatment of SCC.

68Ga]Ga-labeled FAPI Conjugated with Gly-Pro Sequence for PET Imaging of Malignant Tumors.

To improve tumor uptake and prolong tumor retention, a novel fibroblast activation protein (FAP) ligand based on a quinoline-based FAP inhibitor (FAPI) conjugated with the Gly-Pro sequence and 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA) was radiolabeled with [68Ga]GaCl3 ([68Ga]Ga-DOTA-GPFAPI-04). Due to the tumor heterogeneity, this study aimed to further validate the preclinical value of [68Ga]Ga-DOTA-GPFAPI-04 PET imaging in tumor mice models with different FAP expression levels. [68Ga]Ga-DOTA-GPFAPI-04 was synthesized and its partition coefficient was measured. The stability of [68Ga]Ga-DOTA-GPFAPI-04 was tested in phosphate-buffered saline (PBS, pH 7.4) and fetal bovine serum (FBS). Small animal PET and semi-quantitative studies were conducted in Panc-1 and A549 xenograft tumor mice models compared with [68Ga]Ga-DOTA-FAPI-04. Immunofluorescent and immunohistochemical staining and western blot assay were performed to confirm FAP expression in xenograft tumors. [68Ga]Ga-DOTA-GPFAPI-04 exhibited a radiochemical purity of > 99% and high stability in PBS and FBS. [68Ga]Ga-DOTA-GPFAPI-04 had higher hydrophilic property than [68Ga]Ga-DOTA-FAPI-04 (-4.09 ± 0.05 vs -3.45 ± 0.05). Small animal PET and semi-quantitative analysis revealed Panc-1 xenograft tumor displayed higher tumor uptake of [68Ga]Ga-DOTA-GPFAPI-04 and tumor-to-background ratios compared to A549 xenograft tumor, consistent with the results of immunofluorescence, immunohistochemistry, and western blot. Moreover, [68Ga]Ga-DOTA-GPFAPI-04 demonstrated higher tumor accumulation and longer tumor retention than [68Ga]Ga-DOTA-FAPI-04 in both Panc-1 and A549 xenograft tumors. Furthermore, the FAP-binding specificity of [68Ga]Ga-DOTA-GPFAPI-04 was confirmed in vivo by co-injection of unlabeled GPFAPI-04. [68Ga]Ga-DOTA-GPFAPI-04 showed more favorable in vivo tumor imaging and longer tumor retention compared to [68Ga]Ga-DOTA-FAPI-04, which has high potential to be a promising PET probe for detecting FAP-positive tumors.

Pharmacologic ascorbate induces transient hypoxia sensitizing pancreatic ductal adenocarcinoma to a hypoxia activated prodrug

Hypoxic tumor microenvironments pose a significant challenge in cancer treatment. Hypoxia-activated prodrugs like evofosfamide aim to specifically target and eliminate these resistant cells. However, their effectiveness is often limited by reoxygenation after cell death. We hypothesized that ascorbate's pro-oxidant properties could be harnessed to induce transient hypoxia, enhancing the efficacy of evofosfamide by overcoming reoxygenation.To test this hypothesis, we investigated the sensitivity of MIA Paca-2 and A549 cancer cells to ascorbate in vitro and in vivo. Ascorbate induced a cytotoxic effect at 5 mM that could be alleviated by endogenous administration of catalase, suggesting a role for hydrogen peroxide in its cytotoxic mechanism. In vitro, Seahorse experiments indicated that the generation of hydrogen peroxide consumes oxygen, which is offset at later time points by a reduction in oxygen consumption due to hydrogen peroxide's cytotoxic effect.In vivo, photoacoustic imaging showed pharmacologic ascorbate treatment at sublethal levels triggered a complex, multi-phasic response in tumor oxygenation across both cell lines. Initially, ascorbate generated transient hypoxia within minutes through hydrogen peroxide production, via reactions that consume oxygen. This initial hypoxic phase peaked at around 150 s and then gradually subsided. However, at longer time scales (approximately 300 s) a vasodilation effect triggered by ascorbate resulted in increased blood flow and subsequent reoxygenation. Combining sublethal levels of i. p. Ascorbate with evofosfamide significantly prolonged tumor doubling time in MIA Paca-2 and A549 xenografts compared to either treatment alone. This improvement, however, was only observed in a subpopulation of tumors, highlighting the complexity of the oxygenation response.

A Novel Fluorescent Gemcitabine Prodrug That Follows a Nucleoside Transporter-Independent Internalization and Bears Enhanced Therapeutic Efficacy With Respect to Gemcitabine.

The multiplexity of cancer has rendered it the second leading cause of mortality worldwide and theragnostic prodrugs have gained popularity in recent years as a means of treatment. Theragnostic prodrugs enable the simultaneous diagnosis and therapy of tumors via high-precision real-time drug release monitoring. Herein, we report the development of the small theragnostic prodrug GF, based on the nucleoside anticancer agent gemcitabine and the fluorescent dye 5(6)-carboxyfluorescein. We have successfully demonstrated its efficient internalization in tumor cells, showing localization throughout both the early and late endocytic pathways. Its mechanism of cell internalization was evaluated, confirming its independence from nucleoside transporters. Its cellular localization via confocal microscopy revealed a clathrin-mediated endocytosis mechanism, distinguishing it from analogous compounds studied previously. Furthermore, GF exhibited stability across various pH values and in human blood plasma. Subsequently, its in vitro cytotoxicity was assessed in three human cancer cell lines (A549, U87 and T98). Additionally, its pharmacokinetic profile in mice was investigated and the consequent drug release was monitored. Finally, its in vivo visualization was accomplished in zebrafish xenotransplantation models and its in vivo efficacy was evaluated in A549 xenografts. The results unveiled an intriguing efficacy profile, positioning GF as a compelling candidate warranting further investigation.

The deubiquitinating protein OTUD6B promotes lung adenocarcinoma progression by stabilizing RIPK1

BackgroundThere is growing evidence indicating that deubiquitinating enzymes may contribute to tumor progression and can serve as promising therapeutic targets.MethodsThe overexpression of deubiquitinase OTUD6B in lung adenocarcinoma (LUAD) and its adjacent tissues was analyzed by immunohistochemistry and TCGA/GO database. Survival analysis further supported OTUD6B as a potential target for LUAD treatment. We assessed the effect of OTUD6B on LUAD cell growth using cell viability assays and conducted TUNEL staining, migration, and invasion experiments to investigate the impact of OTUD6B on the apoptosis and metastasis of LUAD cells. Additionally, we established a transplanted tumor model in nude mice to validate our findings in vivo. Finally, using IP mass spectrometry and co-IP experiments, we screened and confirmed the influence of RIPK1 as a substrate of OTUD6B in LUAD.ResultsOTUD6B is highly overexpressed in human LUAD and predicts poor prognosis in LUAD patients. OTUD6B knockdown inhibited the proliferation of LUAD cells and enhanced apoptosis and inhibited metastasis in LUAD cells suppressed. A549 xenografts revealed that OTUD6B deletion can slow down tumour growth. Additionally, OTUD6B can bind to RIPK1, reduce its ubiquitination level and increase its protein stability.ConclusionsOur results suggest that OTUD6B is a promising clinical target for LUAD treatment and that targeting OTUD6B may constitute an effective anti-LUAD strategy.

Antitumor activity of nab-sirolimus versus mTOR inhibitors temsirolimus, sirolimus, and everolimus in A549 NSCLC xenografts.

e15096 Background: The mTORC1 pathway, often activated by mutations in genes like TSC1, TSC2, PIK3CA, PTEN, STK11, and KEAP1, plays a pivotal role in cancer progression; in some settings, alterations in STK11 and KEAP1 may be associated with treatment resistance and poor prognosis. Despite the broad importance of the mTORC1 pathway in cancer cell growth and survival, mTOR inhibitors (mTORis) temsirolimus (TEM), sirolimus (SIRO), and everolimus (EVE) have limited clinical application in the cancer setting. nab-Sirolimus, an injectable form of albumin-bound SIRO that leverages unique transport properties of albumin known to increase tumor drug accumulation, is approved for treatment of malignant PEComa. Here, we report the antitumor activity of nab-sirolimus in comparison to other mTORis in A549 NSCLC ( KRAS G12S, STK11 Q37*, and KEAP1 G333C) xenografts, and the correlation of antitumor activity, tumor PK profile, and mTOR pathway suppression. Methods: Athymic mice bearing subcutaneous A549 NSCLC xenografts were treated with either saline or equal weekly doses of nab-sirolimus (administered intravenously [IV]; 5 or 15 mg/kg/week), TEM (IV; 5 mg/kg/week), or SIRO or EVE (both oral; 15 mg/kg/week). Tumors were harvested at different time points after mTORi treatment and analyzed for tumor drug levels (LC-MS/MS) and mTOR pathway biomarkers (pS6 and p4EBP1) via western blot (WB). Results: In A549 xenografts, nab-sirolimus (IV) treatment resulted in significantly greater suppression of tumor growth compared with TEM (IV), SIRO (oral), and EVE (oral) (Table). Average intratumoral drug concentrations 24 hours after IV mTORi treatment were significantly higher with nab-sirolimus (420-539 ng/g) compared with TEM (TEM [parent] 34.9 ng/g; SIRO [active metabolite measured from TEM] 13.2 ng/g) and compared with 7-day steady-state concentrations for oral SIRO (17 ng/g) and EVE (10 ng/g). WB confirmed that nab-sirolimus consistently inhibited mTOR targets pS6 and p4EBP1, whereas TEM, SIRO, and EVE were less effective. Conclusions: nab-Sirolimus resulted in significantly higher intratumoral drug concentration, stronger inhibition of mTOR targets, and greater antitumor activity compared to other IV and oral mTORis in a KRAS/STK11/KEAP1 mutated NSCLC xenograft model. These results support further clinical evaluation of nab-sirolimus as a single agent or in combination with other therapeutic agents in oncology. [Table: see text]

Discovery of Novel p53-MDM2 Inhibitor (RG7388)-Conjugated PlatinumIV Complexes as Potent Antitumor Agents.

While a number of p53-MDM2 inhibitors have progressed into clinical trials for the treatment of cancer, their progression has been hampered by a variety of problems, including acquired drug resistance, dose-dependent toxicity, and limited clinical efficiency. To make more progress, we integrated the advantages of MDM2 inhibitors and platinum drugs to construct novel PtIV-RG7388 (a selective MDM2 inhibitor) complexes. Most complexes, especially 5a and 5b, displayed greatly improved antiproliferative activity against both wild-type and mutated p53 cancer cells. Remarkably, 5a exhibited potent in vivo tumor growth inhibition in the A549 xenograft model (66.5%) without apparent toxicity. It arrested the cell cycle at both the S phase and the G2/M phase and efficiently induced apoptosis via the synergistic effects of RG7388 and cisplatin. Altogether, PtIV-RG7388 complex 5a exhibited excellent in vitro and in vivo antitumor activities, highlighting the therapeutic potential of PtIV-RG7388 complexes as antitumor agents.

Development of erythrina-based PARP-1/FTase dual-target inhibitors against lung cancer epithelial-mesenchymal transition (EMT) in vivo and in vitro

A novel series of erythrina derivatives as PARP-1/FTase inhibitors were synthesized, and evaluated for their biological activities. Compound T9 had excellent inhibitory effects on cell viability (A549: IC50 = 1.74 μM; A549/5-Fu: IC50 = 1.03 μM) and in vitro enzyme activities (PARP-1: IC50 = 0.40 μM; FTase: IC50 = 0.067 μM). Molecular docking and point mutation assays demonstrated the interaction of compound T9 with key amino acid residues. The compound T9 exhibited potent anti-proliferation and anti-migration capabilities against A549 and A549/5-Fu cells. PCR array and western blot results showed that compound T9 could effectively inhibit EMT-related proteins in A549 and A549/5-Fu cells, thereby inhibiting the development of lung cancer. Importantly, compound T9 could significantly inhibit tumor growth in the A549 xenograft tumor model (TGI = 65.3 %). In conclusion, this study was the first presentation of the concept of dual-target inhibitors of the PARP-1/FTase enzymes. It also provides the basis for further research and development of novel PARP-1/FTase inhibitors.