Human Xenografts Research Articles

Possible pathogenic mechanisms for doxorubicin-induced splenic atrophy in a human breast cancer xenograft mouse model.

Doxorubicin-based chemotherapy is a widely used first-line treatment for breast cancer, yet it is associated with various side effects, including splenic atrophy. However, the pathogenic mechanisms underlying doxorubicin-induced atrophy of the spleen remain unclear. This study investigates that doxorubicin treatment leads to splenic atrophy through several interconnected pathways involving histological changes, an inflammatory response, and apoptosis. Immunohistochemical and western blot analyses revealed reduced size of white and red pulp, decreased cellularity, amyloidosis, and fibrotic remodeling in the spleen following doxorubicin treatment. Additionally, increased secretion of pro-inflammatory cytokines was detected using an antibody array and enzyme-linked immunosorbent assay (ELISA), which triggers inflammation through the regulation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-κB) signaling pathways. Further analysis revealed that the loss of regulators and effectors of the oxidative defense system, including sirtuin (Sirt)3, Sirt5, superoxide dismutase (SOD)1, and SOD2, was implicated in the upstream regulation of caspase-dependent cellular apoptosis. These findings provide insights on the pathogenic mechanisms underlying doxorubicin-induced splenic atrophy and suggest that further investigation may be warranted to explore strategies for managing potential side effects in breast cancer patients treated with doxorubicin.

Zurletrectinib is a next-generation TRK inhibitor with strong intracranial activity against NTRK fusion-positive tumours with on-target resistance to first-generation agents

BackgroundWhile NTRK fusion-positive cancers can be exquisitely sensitive to first-generation TRK inhibitors, resistance inevitably occurs, mediated in many cases by acquired NTRK mutations. Next-generation inhibitors (e.g., selitrectinib, repotrectinib) maintain activity against these TRK mutant tumors; however, there are no next-generation TRK inhibitors approved by the FDA and select trials have stopped treating patients. Thus, the identification of novel, potent and specific next-generation TRK inhibitors is a high priority.MethodsIn silico modeling and in vitro kinase assays were performed on TRK wild type (WT) and TRK mutant kinases. Cell viability and clonogenic assays as well as western blots were performed on human primary and murine engineered NTRK fusion-positive TRK WT and mutant cell models. Finally, zurletrectinib was tested in vivo in human xenografts and murine orthotopic glioma models harboring TRK-resistant mutations.ResultsIn vitro kinase and in cell-based assays showed that zurletrectinib, while displaying similar potency against TRKA, TRKB, and TRKC WT kinases, was more active than other FDA approved or clinically tested 1st- (larotrectinib) and next-generation (selitrectinib and repotrectinib) TRK inhibitors against most TRK inhibitor resistance mutations (13 out of 18). Similarly, zurletrectinib inhibited tumor growth in vivo in sub-cute xenograft models derived from NTRK fusion-positive cells at a dose 30 times lower when compared to selitrectinib. Computational modeling suggests this stronger activity to be the consequence of augmented binding affinity of zurletrectinib for TRK kinases. When compared to selitrectinib and repotrectinib, zurletrectinib showed increased brain penetration in rats 0.5 and 2 h following a single oral administration. Consistently, zurletrectinib significantly improved the survival of mice harboring orthotopic NTRK fusion-positive, TRK-mutant gliomas (median survival = 41.5, 66.5, and 104 days for selitrectinib, repotrectinib, and zurletrectinib respectively; P < 0.05).ConclusionOur data identifies zurletrectinib as a novel, highly potent next-generation TRK inhibitor with stronger in vivo brain penetration and intracranial activity than other next-generation agents.

HGG-51. INTRANASAL DELIVERY OF PDGFRA-IMMUNOLIPOSOMAL SN-38 FOR THE TREATMENT OF PEDIATRIC HIGH-GRADE GLIOMAS

Abstract INTRODUCTION pHGGs are the most common cause of cancer-related death in children, of which the most malignant and devastating tumors include diffuse midline glioma (DMG). Its anatomical location in the braistem, infiltrative nature, and blood-brain barrier (BBB) limit surgical resection and the distribution of systemically administered drugs. Intranasal delivery (IND) is a noninvasive method that bypasses the BBB, by leveraging the unique anatomic connections of the olfactory and trigeminal nerve pathways with the nasal cavity. We previously demonstrated the efficacy of IND of liposomal (LS) formulation of the active metabolite of the DNA topoisomerase I inhibitor, irinotecan, LS-SN-38, in orthotopic human brainstem xenograft models. The specificity of liposomes can be enhanced by using immunoliposome (iLS) formulation, which are coated with antibodies specific to the tumor cells leading to targeted drug delivery and reduced toxicity to normal cells. Platelet-derived growth factor receptor alpha (PDGFRA) plays a role in DMG oncogenesis and is expressed in as much as 70% of DMG by immunohistochemistry. In this study, we evaluated the specificity of anti-PDGFRA monoclonal antibody conjugated immunoliposome (PDGFRA-iLS) in pHGG cell lines. METHODS PDGFRA signals were assessed in pHGG cells using western blotting. Cells with high and low expression of PDGFRA were treated with rhodamine (Rho) labeled PDGFRA-iLS and imaged with fluorescent microscope. RESULTS DIPG007, SF7761 and KNS42 expressed high levels of PDGFRA, while SF8628 and DIPG36 cells showed low expression. Fluorescence microscopy revealed high PDGFRA-rhodamine (Rho)-iLS expression in DIPG007 cells (PDGFRA high), relative to SF8628 and DIPG36 cells (PDGFRA low). CONCLUSION PDGFRA-Rho-iLS shows increase cellular uptake in DIPG cells. Work is underway to determine the biodistribution, host toxicity, and anti-tumor activity of IND of PDGFRA-iLS-SN- 38 alone and in combination with radiation therapy in pHGG xenograft models.

Combining the constitutive TRAIL-secreting induced neural stem cell therapy with the novel anti-cancer drug TR-107 in glioblastoma

Tumor-homing neural stem cell therapy (tNSC) is emerging as a promising treatment for aggressive cancers of the brain. Despite their success, developing tNSC therapies that maintain durable tumor suppression remains a challenge. Herein, we report a synergistic combination regimen where the novel small molecule TR-107 augments NSC-TRAIL therapy (hiNeuroS-TRAIL) in models of incurable brain cancer glioblastoma (GBM) in vitro. We report that the combination of hiNeuroS-TRAIL and TR-107 synergistically upregulated caspase markers and restored sensitivity to the intrinsic apoptotic pathway by significantly downregulating inhibitory pathways associated with chemoresistance and radioresistance in the TRAIL-resistant LN229 cell line. This combination also showed robust tumor suppression and enhanced survival of mice bearing human xenografts of both solid and invasive GBM. These findings elucidate a novel combination regimen and suggest combination of these clinically relevant agents may represent a new therapeutic option with increased efficacy for GBM patients.

A Compound That Inhibits Glycolysis in Prostate Cancer Controls Growth of Advanced Prostate Cancer.

Metastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Nonspecific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small-molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. We tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell-cycle, metabolic, and enzymatic assays were used to demonstrate their mechanism of action. A human patient-derived xenograft model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. We demonstrate a new class of small-molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. This work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer.

Self-assembling dendrimer nanosystems for specific fluorine magnetic resonance imaging and effective theranostic treatment of tumors

Fluorine magnetic resonance imaging (19F-MRI) is particularly promising for biomedical applications owing to the absence of fluorine in most biological systems. However, its use has been limited by the lack of safe and water-soluble imaging agents with high fluorine contents and suitable relaxation properties. We report innovative 19F-MRI agents based on supramolecular dendrimers self-assembled by an amphiphilic dendrimer composed of a hydrophobic alkyl chain and a hydrophilic dendron. Specifically, this amphiphilic dendrimer bears multiple negatively charged terminals with high fluorine content, which effectively prevented intra- and intermolecular aggregation of fluorinated entities via electrostatic repulsion. This permitted high fluorine nuclei mobility alongside good water solubility with favorable relaxation properties for use in 19F-MRI. Importantly, the self-assembling 19F-MRI agent was able to encapsulate the near-infrared fluorescence (NIRF) agent DiR and the anticancer drug paclitaxel for multimodal 19F-MRI and NIRF imaging of and theranostics for pancreatic cancer, a deadly disease for which there remains no adequate early detection method or efficacious treatment. The 19F-MRI and multimodal 19F-MRI and NIRF imaging studies on human pancreatic cancer xenografts in mice confirmed the capability of both imaging modalities to specifically image the tumors and demonstrated the efficacy of the theranostic agent in cancer treatment, largely outperforming the clinical anticancer drug paclitaxel. Consequently, these dendrimer nanosystems constitute promising 19F-MRI agents for effective cancer management. This study offers a broad avenue to the construction of 19F-MRI agents and theranostics, exploiting self-assembling supramolecular dendrimer chemistry.

Aqueous VEGF-A Levels as a Liquid Biopsy Biomarker of Retinoblastoma Vitreous Seed Response to Therapy.

Regression of retinoblastoma vitreous seeds (VS) during intravitreal chemotherapy can be delayed, resulting in supernumerary injections. Similarly, VS relapse may not be clinically evident at first. A predictive biomarker of tumor regression and relapse could help guide real-time clinical decision making. Retinoblastoma is an oxygen-sensitive tumor; paradoxically, VS survive in the hypoxic vitreous. We hypothesized that VS elaborate pro-angiogenic cytokines. The purpose was to determine if pro-angiogenic cytokine signatures from aqueous humor could serve as a biomarker of VS response to treatment. Multiplex ELISA was performed on aqueous from rabbit eyes with human retinoblastoma VS xenografts to identify expressed proangiogenic cytokines and changes in aqueous cytokine levels during intravitreal treatment were determined. Confirmatory RNAscope in situ hybridization for VEGF-A was performed on human retinoblastoma tumor sections and VS xenografts from rabbits. For human eyes undergoing intravitreal chemotherapy, serial aqueous VEGF-A levels measured via VEGF-A-specific ELISA were compared to clinical response. VEGF-A was highly expressed in human retinoblastoma VS in the xenograft model, and was the only proangiogenic cytokine that correlated with VS disease burden. In rabbits, aqueous VEGF-A levels decreased in response to therapy, consistent with quantitative VS reduction. In patients, aqueous VEGF-A levels associated with clinical changes in disease burden (regression, stability, or relapse), with changes in VEGF-A levels correlating with clinical response. Aqueous VEGF-A levels correlate with extent of retinoblastoma VS, suggesting that aqueous VEGF-A may serve as a predictive molecular biomarker of treatment response.

Comparative responses to demethylating therapy in animal models of osteosarcoma.

Background The demethylating agent decitabine (DAC) effectively inhibits tumor growth and metastasis by targeting ESR1 methylation to restore estrogen receptor alpha (ERα) signaling and promoting cellular differentiation in models of human osteosarcoma (OSA). Whether this pathway can be targeted in canine OSA patients is unknown. Methods Canine OSA tumor samples were tested for ERα expression and ESR1 promoter methylation. Human (MG63.3) and canine (MC-KOS) OSA cell lines and murine xenografts were treated with DAC in vitro and in vivo , respectively. Samples were assessed using mRNA sequencing and tissue immunohistochemistry. Results ESR1 is methylated in a subset of canine OSA patient samples and the MC-KOS cell line. DAC treatment led to enhanced differentiation as demonstrated by increased ALPL expression, and suppressed tumor growth in vitro and in vivo . Metastatic progression was inhibited, particularly in the MG63.3 model, which expresses higher levels of DNA methyltransferases DNMT1 and 3B. DAC treatment induced significant alterations in immune response and cell cycle pathways. Conclusion DAC treatment activates ERα signaling, promotes bone differentiation, and inhibits tumor growth and metastasis in human and canine OSA. Additional DAC-altered pathways and species- or individual-specific differences in DNMT expression may also play a role in DAC treatment of OSA.

Establishment and characterization of cMYB-expressing human salivary adenoid cystic carcinoma cell lines (UM-HACC-14, UM-HACC-6) and matching patient-derived xenograft model (UM-PDX-HACC-14)

Limited availability of authentic human adenoid cystic carcinoma (ACC) cell lines has hindered progress in understanding mechanisms underpinning the biology of this disease and the development of safe and effective therapies. Surgical human ACC specimens (UM-HACC-6, UM-HACC-14) were dissociated into single cell suspensions and cultured in fibronectin-coated flasks. Alternatively, tumor fragments were transplanted subcutaneously into female immunodeficient (SCID) mice to establish patient-derived xenograft tumors (PDX; UM-PDX-HACC-14). Both ACC cell lines showed continuous growth in monolayers for over 100 passages. Total RNA-Seq, RT-PCR, and FISH analysis revealed that both are MYB-NFIB fusion negative. Western blots revealed passage-dependent expression of E-Cadherin, PCNA, p63, phospho-c-MYB, and NFIB. Both, UM-HACC-14 and UM-HACC-6 cells exhibited tumorigenic potential when injected orthotopically into mouse submandibular glands. UM-HACC-14, patient-matching UM-PDX-HACC-14, and the UM-HACC-6 cell line are new, authenticated preclinical models of ACC that are well suited for mechanistic and developmental therapeutics studies.

Hypoxia-induced activation of HIF-1alpha/IL-1beta axis in microglia promotes glioma progression via NF-κB-mediated upregulation of heparanase expression

BackgroundGlioma is a common tumor that occurs in the brain and spinal cord. Hypoxia is a crucial feature of the tumor microenvironment. Tumor-associated macrophages/microglia play a crucial role in the advancement of glioma. This study aims to illuminate the detailed mechanisms by which hypoxia regulates microglia and, consequently, influences the progression of glioma.MethodsThe glioma cell viability and proliferation were analyzed by cell counting kit-8 assay and 5-ethynyl-2’-deoxyuridine assay. Wound healing assay and transwell assay were implemented to detect glioma cell migration and invasion, respectively. Enzyme-linked immunosorbent assay was conducted to detect protein levels in cell culture medium. The protein levels in glioma cells and tumor tissues were evaluated using western blot analysis. The histological morphology of tumor tissue was determined by hematoxylin-eosin staining. The protein expression in tumor tissues was determined using immunohistochemistry. Human glioma xenograft in nude mice was employed to test the influence of hypoxic microglia-derived interleukin-1beta (IL-1β) and heparanase (HPSE) on glioma growth in vivo.ResultsHypoxic HMC3 cells promoted proliferation, migration, and invasion abilities of U251 and U87 cells by secreting IL-1β, which was upregulated by hypoxia-induced activation of hypoxia inducible factor-1alpha (HIF-1α). Besides, IL-1β from HMC3 cells promoted glioma progression and caused activation of nuclear factor-κB (NF-κB) and upregulation of HPSE in vivo. We also confirmed that IL-1β facilitated HPSE expression in U251 and U87 cells by activating NF-κB. Hypoxic HMC3 cells-secreted IL-1β facilitated the proliferation, migration, and invasion of U251 and U87 cells via NF-κB-mediated upregulation of HPSE expression. Finally, we revealed that silencing HPSE curbed the proliferation and metastasis of glioma in mice.ConclusionHypoxia-induced activation of HIF-1α/IL-1β axis in microglia promoted glioma progression via NF-κB-mediated upregulation of HPSE expression.

A Platinum-Protein hybrid nanostructure enables both targeted cancer theragnosis and platinum clearance

Considering the fundamental advantages of photothermal therapy (PTT) and photoacoustic imaging (PAI) of cancer such as precise spatiotemporal control-based, non-invasive therapy with negligible drug resistance and side effects and sensitive, specific diagnosis with high spatial resolution at substantial depth, an advanced, or clinically feasible material that enables both PTT and PAI surely needs to be developed but remains a major challenge yet. Here we developed a hybrid nanostructure (PtNC@ABP-HBVC) between plasmonic platinum nanocluster (PtNC) comprised of many tiny Pt nanoparticles (PtNPs, ∼ 3 nm that is small enough to pass through glomerular filtration in kidney) and a specially designed protein scaffold (ABP-HBVC) capable of binding to human serum albumin (HSA). PtNC is beneficial for efficient light-to-heat conversion and acoustic wave generation due to plasmonic coupling effect among the small PtNPs, and the HSA binding to ABP-HBVC significantly lowers immunogenicity and enables PtNC to be effectively delivered to tumor. Using human breast cancer-bearing xenograft mice, we demostrated that PtNC@ABP-HBVC has the superior efficacy in targeted cancer theragnosis (PTT + PAI) and is rapidly cleared from the body through urinary excretion, which shows its great potential as a promising, clinically attractive agent for cancer theragnosis.

Discovery of a Novel and Potent LCK Inhibitor for Leukemia Treatment via Deep Learning and Molecular Docking.

The lymphocyte-specific protein tyrosine kinase (LCK) plays a crucial role in both T-cell development and activation. Dysregulation of LCK signaling has been demonstrated to drive the oncogenesis of T-cell acute lymphoblastic leukemia (T-ALL), thus providing a therapeutic target for leukemia treatment. In this study, we introduced a sophisticated virtual screening strategy combined with biological evaluations to discover potent LCK inhibitors. Our initial approach involved utilizing the PLANET algorithm to assess and contrast various scoring methodologies suitable for LCK inhibitor screening. After effectively evaluating PLANET, we progressed to devise a virtual screening workflow that synergistically combines the strengths of PLANET with the capabilities of Schrödinger's suite. This integrative strategy led to the efficient identification of four potential LCK inhibitors. Among them, compound 1232030-35-1 stood out as the most promising candidate with an IC50 of 0.43 nM. Further in vitro bioassays revealed that 1232030-35-1 exhibited robust antiproliferative effects on T-ALL cells, which was attributed to its ability to suppress the phosphorylations of key molecules in the LCK signaling pathway. More importantly, 1232030-35-1 treatment demonstrated profound in vivo antileukemia efficacy in a human T-ALL xenograft model. In addition, complementary molecular dynamics simulations provided deeper insight into the binding kinetics between 1232030-35-1 and LCK, highlighting the formation of a hydrogen bond with Met319. Collectively, our study established a robust and effective screening strategy that integrates AI-driven and conventional methodologies for the identification of LCK inhibitors, positioning 1232030-35-1 as a highly promising and novel drug-like candidate for potential applications in treating T-ALL.

Oxygen Sensor-Guided Fine Needle Biopsy Studies of Human Cancer Xenografts in Mice.

An oxygen sensor-mounted fine-needle biopsy tool was used for in vivo measurement of oxygen levels in tumor xenografts. The system provides a means of measuring the oxygen content in harvested tumor tissue from specific locations. Oxygen in human tumor xenografts in a murine model was observed for over 1 min. Tissues were mapped in relation to oxygen tension (pO2) readings and sampled for conventional cytological examination. Careful modeling of the pO2 readings over 60 seconds yielded a diffusion coefficient for oxygen at the sensor tip, providing additional diagnostic information about the tissue before sampling. Oxygen level measurement may provide a useful adjunct to the use of biomarkers in tumor diagnosis.

Reduced Tumor Volume and Increased Necrosis of Human Breast Tumor Xenograft in Mice Pretreated by a Cocktail of Three Specific Anti-HER2 scFvs.

We aimed to assess the effects of a cocktail comprising three specific anti- HER2 scFvs on breast tumor formation in a xenograft mouse model and to evaluate quantitative changes in the tumor using stereological analysis. Three specific anti-HER2 phage antibodies were produced from a scFv-library using phage display technology. The cell binding capacities of the antibodies were assessed via FACS analysis. Soluble forms of the antibodies were prepared by infecting HB2151-E. coli cells and purified using a centrifugal ultrafiltration method. The purification process was evaluated by SDSPAGE analysis. Two forms of scFv cocktails were prepared, soluble scFv and phage-scFv cocktail, which contained an equal amount/phage of each of the three antibodies. Inbred female BALB/c mice were pretreated with 5 and 20 mg/kg of the soluble scFv cocktail and 1011 phage-scFv cocktail/ kg. The mice were then injected with 2×106 SKBR-3 human breast cancer cells. Total tumor, inflammatory and non-inflammatory volumes were estimated using the Cavalieri principle after preparing photomicrograph slides. The anti-HER2 scFvs showed significantly higher binding to SKBR-3 cells compared to the isotype control. SDS-PAGE analysis confirmed the high purification of the scFvs. Stereological analysis revealed that the group pretreated with 20 mg/kg of the soluble scFv cocktail exhibited the highest reductions in total tumor volume, non-inflammatory volume, and inflammatory volume, with reductions of 73%, 78%, and 72%, respectively, compared to PBS-pretreated mice (P-value < 0.0001). The volumetric ratio of necrotic tissue to total tumor volume increased by 2.2-fold and 2- fold in the 20 mg/kg of soluble scFv cocktail and phage-scFv cocktail groups, respectively, compared to the PBS-treated mice (P-value < 0.05). Pre-treatment with a 20 mg/kg anti-HER2 scFv cocktail resulted in a significant reduction in tumor volume and increased necrotic area in a human breast cancer xenograft model, indicating the remarkable anti-tumor effect of the cocktail in vivo.

Effect of a novel anti-PD-1-proIL-2 bifunctional fusion protein on potent anti-tumor activity via PD-1 checkpoint inhibition and conditional IL-2R agonism.

e14697 Background: HD IL-2 was approved by FDA for metastatic melanoma (mM) and renal cell carcinoma (mRCC) but the treatment is associated with severe toxicities. PD-1 blockade has improved the overall survival, but only in 20-30% of cancer patients and many of them experience frequent relapses. Interestingly, HD IL-2 therapy displayed durable anti-tumor activity in mM and mRCC patients who progressed on anti-PD-1. Furthermore, combination therapy of IL-2 and pembrolizumab in mRCC has shown durable 70% response rate, while the ORR of monotherapy is 20% and 33% for IL-2 and pembrolizumab, respectively. Thus, combining IL-2 receptor activation and PD-1 blockade may represent a promising strategy in the post PD-1 era to overcome the resistance and improve the clinical efficacy. PTX-912 is a novel bifunctional PD-1-proIL-2v fusion protein designed to simultaneously harness PD-1 blockade and PD-1-cis directed IL-2R agonism conditionally in TME without the peripheral toxicities associated with IL-2. Methods: The biological functions were demonstrated in human PBMC assays. Anti-tumor efficacy and biomarker studies were evaluated in mouse / human PBMC xenograft models. Tox studies were performed in NHP. Results: PTX-912 shows dose-dependent inhibition of PD-1 pathway measured by GM-CSF and IFNgamma production in an MLR assay. Protease activated PTX-912, but not the intact drug, stimulates the phosphorylation of STAT5 of NK and T cells, the proliferation of CD8 T cells as well as the cytokine production in human whole blood. The potency of pSTAT5 by protease activated product is enhanced by &gt; 10 folds on PD-1+ T cells than T cells without PD-1. When tested in mouse tumor models resistant to PD-1 blockade, PTX-912 demonstrated high potency of tumor inhibition at as low as 0.1 mg/kg dose. In contrast, non-targeted proIL-2v at molar equivalent or higher dose showed minimal antitumor effect in either monotherapy or combination with anti-PD-1. Analysis of PTX-912 treated mice showed increased tumor infiltrating CD4+ and CD8+ T cells, including PD-1+Tcf-1+ and PD-1+ IL-18Ra+TIM3- CD8+ T cells, referred to “stem-like” progenitor effector cells and “better” effector cells, respectively. Activation of the IL-2v in PTX-912 was observed in mouse tumor tissues but not in the plasma. In monkey GLP-compliant 4-week repeat-dose toxicity study, PTX-912 demonstrated excellent safety profiles without overt irAEs that are characteristic of IL-2. Conclusions: Our preclinical data demonstrated that PTX-912 effectively stimulated the expansion and reinvigoration of antigen specific T cells in the tumor tissue resulting in strong anti-tumor efficacy while it did not cause significant peripheral toxicity. Phase 1 clinical trial of PD-1-proIL-2v is planned to start in early 2024 to evaluate the safety, pharmacokinetics and anti-tumor activity in patients with advanced solid tumors.

Effect of oncolytic herpes simplex virus on fusion-positive rhabdomyosarcoma to radiotherapy.

10063 Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite multimodal interventions including surgery, chemotherapy, and radiotherapy, nearly a third of patients with localized disease will experience tumor recurrence. Patients with tumors harboring an oncogenic FOXO1 fusion are at greater risk of relapse and are most in need of novel therapies. Oncolytic viruses are being explored as treatments for pediatric tumors, and data suggest that oncolytic viruses may enhance responses to chemoradiotherapy. M002 is a genetically engineered oncolytic herpes simplex virus (oHSV) that exhibits aneurovirulence in normal cells. M002 selectively replicates in malignant cells, eliminating these cells through cellular burst during the lytic cycle. In the present study, we explored the efficacy of the M002 virus alone and in combination with low-dose ionizing radiotherapy in a human patient-derived xenograft (PDX) model of fusion-positive RMS (FP-RMS). Methods: A human FP-RMS PDX was established and maintained by injection into the quadriceps femoris muscle of athymic nude mice under Institutional Animal Care and Use Committee guidelines (IACUC-09363). Tumors were dissociated and cells were treated with increasing multiplicity of infection (MOI) of either the oHSV, R3659 or its genetically engineered variant, M002. A colorimetric assay evaluated the cytotoxicity of oHSV treatments on the RMS PDX cells after 24, 48, and 72 hours of infection. In separate experiments, cells treated with vehicle (control) or infected with M002 were exposed to irradiation (2 Gy), and viability was determined at 24, 48, 72, and 96 hours. Results: Treatment with M002 or its parent construct, R3659, significantly reduced RMS PDX cell viability in a dose-dependent manner at all time points. M002 showed slightly higher cytotoxicity than R3659 as measured by lethal dose 50% (LD50, 37±1.25 vs 44±0.95 PFU/cell, M002 vs R3659, at 24 hours, p=0.02). When sub-lethal doses of M002 were combined with low-dose irradiation, RMS PDX cell viability was significantly decreased compared to cells treated with either M002 or irradiation alone (26% decrease at 10 PFU/cell). Conclusions: Our preliminary data highlight that M002 exhibits cytotoxicity in a PDX model of FP-RMS. Treatment with M002 further increased the cytotoxicity of ionizing radiation compared to individual treatments, suggesting that oHSV sensitized the cells to radiotherapy. Future studies in vivo may provide more pre-clinical evidence for the radio-sensitizing and therapeutic potential of M002 in FP-RMS.

Synergistic effects of RPT1G, a first-in-class small molecule NAMPT inhibitor, with venetoclax and olaparib in hematological malignancies.

e18512 Background: MultipleBCL-2 and PARP inhibitors are currently in clinical trials, or have been approved for use, in hematological malignancies and solid tumors. Venetoclax, an inhibitor of the apoptosis regulator B-cell lymphoma-2 (BCL-2), has activity in patients with chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). PARP inhibitors, such as Olaparib, exploit synthetic lethality in homologous recombination-deficient tumors, notably in breast and ovarian cancers. However, patients treated with BCL-2 or PARP inhibitors often face relapse and refractory disease, requiring alternative strategies to counter adaptive responses. Nicotinamide Phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway, is upregulated in malignancies and plays a key role in cancer metabolism (e.g., Warburg effect). NAMPT makes nicotinamide mononucleotide which is then converted to NAD+, while PARP enzymes consume NAD+, placing NAMPT directly upstream of PARP. NAMPT overexpression is associated with Venetoclax resistance in leukemia patients, highlighting the link between NAD+, mitochondrial metabolism, and BCL-2 inhibition. Consequently, NAMPT inhibition may synergize with BCL-2 or PARP inhibitors, potentially enhancing 1st-line of treatment outcomes and re-sensitizing refractory cancer cells to BCL-2 or PARP inhibition. Methods: In this study, we investigated the combination effects of Venetoclax and Olaparib with RPT1G, a novel hyperbolic NAMPT inhibitor that simultaneously inhibits and stabilizes NAMPT in a catalytically active state. RPT1G disrupts NAMPT activity without ever turning it off completely avoiding the severe on-target toxicities seen with complete NAMPT inhibitors. Several cell lines, including OCI-AML3 and MV4;11 (AML), RS4;11 (ALL), and RL (diffuse large B-cell lymphoma) were treated with RPT1G and either Venetoclax or Olaparib to determine the degree of drug interactions. In a follow up in vivo study, we treated mice in a human xenograft model using semi-resistant MV4;11 cells with RPT1G alone or in combination with Venetoclax to measure the ability of RPT1G to enhance the effects of Venetoclax. Results: RPT1G achieves significant single-agent efficacy in leukemia and lymphoma mouse xenograft models. When RTP1G is used in combination with either Venetoclax or Olaparib significant synergy is seen across a broad range of concentrations in vitro. Consistent with in vitro synergy results, in a xenograft model of human AML, mice treated with a combination of RPT1G and Venetoclax show enhanced efficacy as compared to Venetoclax alone. Conclusions: Combination of RPT1G with either Venetoclax or Olaparib synergistically enhances killing of leukemic cells and presents as an effective strategy to improve treatment outcomes for patients with diverse malignancies.

A phase 1 study to evaluate the safety and preliminary efficacy of minnelide given alone or in combination with paclitaxel in advanced gastric cancer.

4033 Background: Minnelide is a water-soluble disodium salt variant of triptolide, an HSP70 inhibitor. Studies using pancreatic cancer cell lines and human xenograft transplants demonstrated that Minnelide prevents tumor progression, increases survival, and causes tumor regression. This study aims to evaluate the safety and antitumor activity of Minnelide and its combination with paclitaxel in patients with advanced gastric cancer (AGC). Methods: This open-label, single-center, dose-escalation phase I study recruited adult patients with previously treated advanced gastric cancer. Patients received Minnelide per oral (PO) daily starting with 1.0 mg for 3weeks and 1week rest schedule, escalating to 1.25mg, and 1.5mg. In combination Regimen, patients received Minnelide starting at a dose of 0.25 mg and escalating by 0.25mg to 1.25mg plus paclitaxel 60-80 mg/m2 intravenously on Day 1, 8, and 15 of each cycle, administered in 4-week cycles. The primary objective was to assess the safety and determine the recommended phase II dose (RP2D). Secondary objectives included preliminary efficacy using RECIST v1.1, and survival analysis. Results: Among Regimen A (n = 11), two patients experienced dose-limiting toxicity (DLTs) (Minnelide 1.5 mg: Grade 3 abdominal pain). No DLTs occurred in combination treatment, Regimen B &amp; C (n=25). The MTD was Minnelide 1.25 mg once daily for 21days Q4 weeks as monotherapy. The most common Grade ≥3 AEs were neutropenia (19.4%) and abdominal pain (11.1%). In Regimen C, 71.5% achieved either a partial response or a stable disease with the median PFS of 4.5 months, and the median OS of 10.7 months. Notably, patients without previous exposure to paclitaxel (n = 4) in the last 2 cohorts of Regimen C, had a confirmed ORR of 50.0%, DCR of 75.0%, the median PFS was 5.3 months (95% CI NE-NE) and the median OS has not reached as some of the patients are still receiving treatment. Conclusions: The combination of Minnelide plus paclitaxel as salvage treatment in AGC patients showed meaningful clinical activity with a manageable safety profile. Based on this Phase I experience a Phase II will be initiated in AGC patients. Clinical trial information: NCT05566834 .

A Technique to Generate a Human PBMC-Engrafted Humanized Xenograft Mouse Model

A Technique to Generate a Human PBMC-Engrafted Humanized Xenograft Mouse Model

Clinically Relevant Humanized Mouse Models of Metastatic Prostate Cancer Facilitate Therapeutic Evaluation.

There is tremendous need for improved prostate cancer models. Anatomically and developmentally, the mouse prostate differs from the human prostate and does not form tumors spontaneously. Genetically engineered mouse models lack the heterogeneity of human cancer and rarely establish metastatic growth. Human xenografts are an alternative but must rely on an immunocompromised host. Therefore, we generated prostate cancer murine xenograft models with an intact human immune system (huNOG and huNOG-EXL mice) to test whether humanizing tumor-immune interactions would improve modeling of metastatic prostate cancer and the impact of androgen receptor-targeted and immunotherapies. These mice maintain multiple human immune cell lineages, including functional human T-cells and myeloid cells. Implications: To the best of our knowledge, results illustrate the first model of human prostate cancer that has an intact human immune system, metastasizes to clinically relevant locations, responds appropriately to standard-of-care hormonal therapies, and can model both an immunosuppressive and checkpoint-inhibition responsive immune microenvironment.