Cell-derived Xenograft Models Research Articles

Discovery of Novel Non-nucleoside DOT1LR231Q Inhibitors with Improved Pharmacokinetic Properties and Anti-lung Cancer Efficacy.

Given the considerable potential of DOT1LR231Q inhibitors in lung cancer therapy and the problematic pharmacokinetics of nucleoside inhibitors, our group launched a development program of non-nucleoside DOT1LR231Q inhibitors to improve the pharmacokinetic properties. Herein, two series of non-nucleoside compounds bearing piperidine or 3-(aminomethyl)pyrrolidin-3-ol as "ribose mimics" were designed and evaluated through antiproliferation assay and western blot analysis. The optimal TB22 inhibited the proliferation of H460R231Q cells with an IC50 value of 2.85 μM, about 13-fold more potent than SGC0946. Notably, TB22 demonstrated significant in vivo efficacy (TGI = 60.57%) in H460R231Q cell-derived xenograft models and improved pharmacokinetic properties (t1/2 = 6.06 ± 2.94 h and CL = 55.18 ± 8.56 mL/kg/min). Moreover, a mechanism study validated that TB22 suppressed malignant phenotypes of lung cancer cells harboring R231Q mutation via the MAPK/ERK signaling pathway. This work provides a promising molecule for lung cancer therapy in favor of clinical patients.

ZCCHC3 and Efp coordinately contribute to the pathophysiology of triple-negative breast cancer by modulating NCAPH

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited targeted therapies and high rates of recurrence. We previously showed that Efp promotes TNBC cell proliferation by regulating cell cycle-related gene expression. Recent studies showed that ZCCHC3 interacts with Efp, promoting Efp signaling in innate immune responses. We here characterize whether ZCCHC3 plays a pathophysiological role in TNBC tumorigenesis. We showed that ZCCHC3 silencing significantly repressed the proliferation of TNBC conventional cultured cells and three-dimensional patient-derived spheroid culture, which we established from a clinical TNBC tissue. RNA-sequencing in TNBC cells defined that “cell division” was a major pathway commonly downregulated by ZCCHC3 and Efp silencing, and NCAPH was a cell division-related gene highly downregulated by ZCCHC3 silencing. In a TNBC cell-derived xenograft model, ZCCHC3-specific siRNA injection successfully reduced in vivo TNBC tumor growth and downregulated NCAPH expression. Overall, our findings demonstrate that ZCCHC3 and Efp coordinately promote TNBC progression by regulating NCAPH expression and that ZCCHC3/Efp/NCAPH pathway can be applied to clinical TNBC management.

The expansion of MDSCs induced by exosomal PD-L1 promotes the progression of gastric cancer

BackgroundMyeloid-derived suppressor cells (MDSCs) are the major factor in gastric cancer (GC) immune evasion. Nevertheless, the molecular process underlying the expansion of MDSCs induced by tumor-derived exosomes (TDEs) remains elusive.MethodsThe levels of exosomal and soluble PD-L1 in ninety GC patients were examined via enzyme-linked immunosorbent assay (ELISA) to determine their prognostic value. To investigate the correlation between exosomal PD-L1 and MDSCs, the percentage of MDSCs in the peripheral blood of 57 GC patients was assessed via flow cytometry. Through ultracentrifugation, the exosomes were separated from the GC cell supernatant and detected via Western blotting, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). The function of exosomal PD-L1 in MDSCs was evaluated via immunofluorescence, Western blotting and flow cytometry in a GC cell-derived xenograft (CDX) model.ResultsThe overall survival (OS) of GC patients in the high exosomal PD-L1 group was significantly lower than that of patients in the low exosomal PD-L1 group (P = 0.0042); however, there was no significant correlation between soluble PD-L1 and OS in GC patients (P = 0.0501). Furthermore, we found that the expression of exosomal PD-L1 was positively correlated with the proportions of polymorphonuclear MDSCs (PMN-MDSCs, r = 0.4944, P < 0.001) and monocytic MDSCs (M-MDSCs, r = 0.3663, P = 0.005) in GC patients, indicating that exosomal PD-L1 might induce immune suppression by promoting the aggregation of MDSCs. In addition, we found that exosomal PD-L1 might stimulate MDSC proliferation by triggering the IL-6/STAT3 signaling pathway in vitro. The CDX model confirmed that exosomal PD-L1 could stimulate tumor development and MDSC amplification.ConclusionsExosomal PD-L1 has the potential to become a prognostic and diagnostic biomarker for GC patients. Mechanistically, MDSCs can be activated by exosomal PD-L1 through IL-6/STAT3 signaling and provide a new strategy against GC through the use of exosomal PD-L1 as a treatment target.

Identification of an m6A Natural Inhibitor, Lobeline, That Reverses Lenvatinib Resistance in Hepatocellular Tumors.

Hepatocellular carcinoma (HCC) is an aggressive cancer that has an effect on human health. As a first-line drug for HCC, despite its excellent efficacy, lenvatinib (Len) is prone to developing drug resistance in HCC patients. The N6-methyladenosine (m6A) modification is not only related to the development of HCC but also shows great potential in overcoming HCC resistance. Using Dot Blot, our group first screened a small molecule m6A regulator, lobeline (Lob), from a library of 390 compounds (mostly natural products). In vitro experiments demonstrated that Lob could significantly enhance the sensitivity to Len of Len-resistant HCC (HCC/Len) and inhibit migration of resistant cells. In Len-resistant cell-derived and patient-derived xenograft models, Lob could reverse the resistant phenotype, with reductions in tumor volume of 68% and 60%, respectively. Furthermore, MeRIP-m6A sequencing results indicated that the underlying molecular mechanism of Lob reversal of HCC drug resistance was related to UBE3B. Taken together, this study highlighted that Lob, a plant derived natural product, could reverse the resistance of HCC to Len by regulating the m6A levels. It is hoped that this will provide a pharmacological research basis for the clinical treatment of HCC patients.

Therapeutic targeting ERRγ suppresses metastasis via extracellular matrix remodeling in small cell lung cancer

Small-cell lung cancer (SCLC) is the most aggressive and lethal type of lung cancer, characterized by limited treatment options, early and frequent metastasis. However, the determinants of metastasis in SCLC are poorly defined. Here, we show that estrogen-related receptor gamma (ERRγ) is overexpressed in metastatic SCLC tumors, and is positively associated with SCLC progression. ERRγ functions as an essential activator of extracellular matrix (ECM) remodeling and cell adhesion, two critical steps in metastasis, by directly regulating the expression of major genes involved in these processes. Genetic and pharmacological inhibition of ERRγ markedly reduces collagen production, cell-matrix adhesion, microfilament production, and eventually blocks SCLC cell invasion and tumor metastasis. Notably, ERRγ antagonists significantly suppressed tumor growth and metastasis and restored SCLC vulnerability to chemotherapy in multiple cell-derived and patient-derived xenograft models. Taken together, these findings establish ERRγ as an attractive target for metastatic SCLC and provide a potential pharmacological strategy for treating this lethal disease.

Off-the-shelf CAR-NK cells targeting immunogenic cell death marker ERp57 execute robust antitumor activity and have a synergistic effect with ICD inducer oxaliplatin

BackgroundChimeric antigen receptor natural killer (CAR-NK) therapy holds great promise for treating hematologic tumors, but its efficacy in solid tumors is limited owing to the lack of suitable targets and...

Harnessing Decellularized Extracellular Matrix for Enhanced Fidelity in Colorectal Cancer Organoid and Cell-Derived Xenograft Models.

This study evaluates the efficacy of a decellularized intestine tissue-derived extracellular matrix (Intestine ECM) as a scaffold for culturing colorectal cancer (CRC) organoids and establishing cell-derived xenograft (CDX) models, comparing its performance to traditional Matrigel. Intestine ECM demonstrates comparable support for organoid formation and cellular function, highlighting its potential as a more physiologically relevant and reproducible platform. Our findings suggest that Intestine ECM enhances the mimetic environment for colon epithelium, supporting comparable growth and improved differentiation compared to Matrigel. Moreover, when used as a delivery carrier, Intestine ECM significantly increases the growth rate of CDX models using patient-derived primary colorectal cancer cells. This enhancement demonstrates Intestine ECM's role not only as a scaffold but also as a vital component of the tumor microenvironment, facilitating more robust tumorigenesis. These findings advocate for the broader application of Intestine ECM in cancer model systems, potentially leading to more accurate preclinical evaluations and the development of targeted cancer therapies.

Development of GPC3-CAR-NK cells and optimization as a therapy for HCC.

Hepatocellular carcinoma (HCC) is a highly malignant tumor characterized by insidious onset and rapid progression, with limited treatment choices. One treatment modality, chimeric antigen receptor (CAR)-modified natural killer (NK) cell immunotherapy, has shown promise for various cancers. In this study, we developed two GPC3-specific CAR-NK-92 cell lines (GPC3-CAR-NK) and explored their antitumor efficacy for the treatment of HCC. Significant levels of cytokine production and in vitro cytotoxicity were produced following co-culture of GPC3+ HCC cells with the developed GPC3-CAR-NK cells. GC33-G2D-NK cells with NK cell-specific signaling domains showed better activation and killing abilities than GC33-CD28-NK cells containing T cell-specific signaling domains. Moreover, GC33-G2D-NK cells efficiently eliminated tumors in cell-derived xenograft and patient-derived xenograft mouse models. In an abdominal metastasis model, intraperitoneally delivered GC33-G2D-NK cells showed better antitumor ability than intravenously injected cells. Finally, the combination of microwave ablation with GC33-G2D-NK cell administration showed greater CAR-NK infiltration and tumor regression in ablated tumors than monotherapy alone. These findings indicate that administration of GPC3-CAR-NK cells may be a potential strategy for the treatment of HCC, and regional delivery or their combination with microwave ablation may optimize their efficacy against HCC and may have translational value.

IFIT1 + neutrophil is a causative factor of immunosuppressive features of poorly cohesive carcinoma (PCC)

The importance of the immune microenvironment in poorly cohesive carcinoma (PCC) has been highlighted due to its limited response rate to conventional therapy and emerging treatment resistance. A combination of clinical cohorts, bioinformatics analyses, and functional/molecular experiments revealed that high infiltration of Interferon Induced Protein with Tetratricopeptide Repeats 1 (IFIT1) + tumor-associated neutrophils (TANs) is a distinguishing feature of PCC patients. Upregulation of IFIT1 + TANs promote migration and invasion of gastric cancer (GC) cell lines (MKN45 and MKN74) and stimulates the growth of cell-derived xenograft models. Besides, by promoting macrophage secreted phosphoprotein 1 (SPP1) expression and facilitating cancer-associated fibroblast and endothelial cell recruitment and activation through TANs, IFIT1 promotes a mesenchymal phenotype, which is associated with a poor prognosis. Importantly, compared to non-PCC (NPCC), PCC tumors is more immunosuppressive. Mechanistically, IFIT1 can be stimulated by IFN-γ and contributes to the expression of Programmed Cell Death 1 Ligand (PDL1) in TANs. We demonstrated in mouse models that IFIT1 + PDL1 + TANs can induce acquired resistance to anti-PD-1 immunotherapy, which may be responsible for the difficulty of PCC patients to benefit from immunotherapy. This work highlights the role of IFIT1 + TANs in mediating the remodeling of the tumor immune microenvironment and immunotherapeutic resistance and introduces IFIT1 + TANs as a promising target for precision therapy of PCC.

Establishment of a novel small bowel adenocarcinoma cell line using patient‑derived xenografts, which produces CEA and CA19‑9.

Small bowel adenocarcinoma (SBA) is a rare tumor with a poor prognosis. Due to its rarity, the research infrastructure for SBA, including cell lines, is inadequate. The present study established a novel SBA cell line, SiCry-15X, using patient-derived xenografts of SBA. The following criteria were defined for establishment: Long-term culturability, tumorigenicity and similarity with the original tumor. The biological characteristics of the cell line, its sensitivity to anticancer drugs and its ability to produce tumor markers carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) were evaluated. SiCry-15X cells adhered and grew as a monolayer, with a population doubling time of 37 h. Polymerase chain reaction results confirmed the human origin of the cell line, and short tandem repeat analysis revealed that the cells were genetically identical to the original tumor. The 50% inhibitory concentrations of 5-fluorouracil, paclitaxel, irinotecan, oxaliplatin and cisplatin for SiCry-15X were 104.05, 0.24, 63.3, 146.55 and 49.29 µM, respectively. CEA and CA19-9 concentrations in the culture media were markedly elevated. In addition, CEA and CA19-9 levels in the serum of cell-derived xenograft model mice were elevated. Moreover, CEA and CA19-9 were produced by SiCry-15X cells and distributed throughout the blood. Furthermore, increases in serum CEA and CA19-9 of cell-derived xenograft model mice were consistent with the clinical course of the disease. The newly established SBA cell line, SiCry-15X, could be an effective tool for conducting further studies on SBA.

Preclinical studies of an innovative 5T4-ADC ACR246 with the potential to better treat 5T4-positive solid tumors.

e15005 Background: 5T4 oncofetal antigen shows limited expression in normal adult tissues but highly expressed by many different cancers. It drives spread of cancer cells through epithelial mesenchymal transition (EMT), potentiation of CXCL12/CXCR4 chemotaxis and favoring non-canonical Wnt pathway, leading to poor prognosis of cancer patients. Besides, 5T4 has a highly efficient endocytosis ability which enables it to be an ideal target for the development of ADC drugs. A handful of 5T4-targeted ADCs have been evaluated in early phase clinical trials for several years showing only preliminary anti-tumor activity. However, uncertainty of their safety profiles has also raised concerns due to high toxic payloads, MMAF and duocarmycin, being used. Thus, additional approaches to this target with the potential of improved efficacy and reduced toxicity are needed urgently. ACR246, an innovative 5T4-targeted ADC consisting of specific humanized monoclonal antibody site-specifically conjugated with a cell penetrating and moderate toxic payload (DNA topoisomerase I inhibitor, D2102) that has a robust bystander effect through a stable and cleavable linker, with a drug-to-antibody ratio (DAR) of 8, was designed to improve the safety and efficacy in treating tumor patients. Methods: The pharmacologic profiles of ACR246 were assessed in vitro and in vivo including efficacy, pharmacokinetics-pharmacodynamics (PK-PD), safety, and tissue distribution, in 5T4-positive cell lines, cell-derived xenograft (CDX) models and cynomolgus monkeys. Results: ACR246 bound specifically to 5T4 and was internalized into tumor cells followed by D2102 release, which induced DNA damage, cell cycle arrest and apoptosis in 5T4 positive tumor cells in vitro. In vivo low systemic exposure and tumor-specific accumulation of D2102 rendered ACR246 superior efficacy and safety property. Accordingly, ACR246 exhibited robust in vivo anti-tumor activity across various types of 5T4 positive CDX models in a dose-dependent manner, with DNA damage induced by the accumulated D2102 in tumor tissue. GLP toxicity studies indicated that ACR246 was well tolerated in cynomolgus monkeys. The estimated therapeutic index was 24. Conclusions: The overall preclinical profile marks ACR246 has the potential to become the best in class (BIC) 5T4-ADC, showing more favorable benefit-risk ratio in clinic.

Natural compound Alternol exerts a broad anti-cancer spectrum and a superior therapeutic safety index in vivo.

Alternol is a natural compound isolated from the fermentation of a mutated fungus. We have demonstrated its potent anti-cancer effect via the accumulation of radical oxygen species (ROS) in prostate cancer cells in vitro and in vivo. In this study, we tested its anti-cancer spectrum in multiple platforms. We first tested its anti-cancer spectrum using the National Cancer Institute-60 (NCI-60) screening, a protein quantitation-based assay. CellTiter-Glo screening was utilized for ovarian cancer cell lines. Cell cycle distribution was analyzed using flow cytometry. Xenograft models in nude mice were used to assess anti-cancer effect. Healthy mice were tested for the acuate systemic toxicity. Our results showed that Alternol exerted a potent anti-cancer effect on 50 (83%) cancer cell lines with a GI50 less than 5µM and induced a lethal response in 12 (24%) of those 50 responding cell lines at 10µM concentration. Consistently, Alternol displayed a similar anti-cancer effect on 14 ovarian cancer cell lines in an ATP quantitation-based assay. Most interestingly, Alternol showed an excellent safety profile with a maximum tolerance dose (MTD) at 665mg/kg bodyweight in mice. Its therapeutic index was calculated as 13.3 based on the effective tumor-suppressing doses from HeLa and PC-3 cell-derived xenograft models. Taken together, Alternol has a broad anti-cancer spectrum with a safe therapeutic index in vivo.

Abstract PO3-18-08: Rational therapeutic combination of Bromodomain and Extra-Terminal domain (BET) inhibitor and Fibroblast Growth Factor Receptor (FGFR) inhibitor for treatment of invasive lobular carcinoma

Abstract Background: Invasive lobular carcinoma (ILC) is the second most pervasive subtype after invasive ductal carcinoma (IDC), accounting for approximately 10-15% of all breast cancers. It is characterized by loss of E-cadherin expression and non-adherent tumor cells that invade the stroma in a “single-file” pattern. Women with ILC are typically diagnosed at an older age and later stage with ER-positive disease. ILC is more likely to exhibit late recurrence and metastasize to the gastrointestinal tract and urogenital tract compared with IDC. It is routinely treated with anti-endocrine therapy and chemotherapy, however, while not entirely chemo-refractory, displays a poor response to chemotherapy compared with IDC. As such, options for recurrent disease are limited and there is an urgent need to develop tailored therapy for ILC, especially for those patients that recur. The family of bromodomain and extra-terminal domain (BET) proteins, comprising BRD2/3/4/T, are epigenetic readers that bind to acetylated lysine residues on histones and recruit transcription factors to drive the expression of oncogenes. Previously. we discovered that BRD3 is a marker of poor prognosis in ILC and there is emerging evidence that BET inhibitors (iBET) are effective in diverse types of breast cancer. Here, we investigated the therapeutic potential of iBET in ILC, alone and in combination with FGFR inhibitors. Methods: IC50s for a panel of iBET using two typical ILC cell lines MDA-MB-134VI (MM134) and SUM44PE (SUM44) were determined. RNA-sequencing and Genexplain analysis was applied to reveal transcriptional networks, master regulators and potential resistance mechanisms. BRD3 and FGFR3 were knocked down using siRNA to evaluate their function in ILC cell lines. Furthermore, we utilized ILC cell-derived xenograft (CDX) models in SCID-beige mice established by mammary intraductal (MIND) implantation to evaluate the therapeutic potency of iBET alone or in combination with fibroblast growth factor receptor (FGFR) inhibitor in vivo. Results: We demonstrated that iBET significantly inhibited ILC cell growth in both 2D and 3D culture, with the greatest potency demonstrated by JQ1 and Mivebresib (ABBV-075). RNA-sequencing revealed dysregulated pathways in cell cycle division, DNA damage, apoptosis and MAPK signaling following iBET treatment. Reverse engineering of transcriptional profiles using Genexplain revealed that FGFR3 is a significantly upregulated master regulator (MTR) among 142 MTRs across both cell lines and both iBETs. Upregulation of FGFR3 after iBET treatment was verified at the protein level by Western blotting. We also show that BRD3 and FGFR3 knockdown significantly inhibited cell growth, which supports the key role both play in ILC progression. Further, we analyzed the iBET therapeutic effect when combined with the FGFR inhibitor, erdafitinib, as a strategy to overcome potential resistance due to FGFR upregulation post iBET treatment. This revealed that the combination of iBET and erdafitinib could inhibit ILC cell growth more effectively compared to using either agent alone. Furthermore, our in vivo study showed that JQ1 could inhibit tumor growth in a SUM44-MIND model and alleviate metastasis to peritoneum, bone and ovary compared with the vehicle group. Moreover, we also assessed the combination of mivebresib and erdafitinib in vivo. This revealed that iBET and an FGFR inhibitor work synergistically to decrease tumour burden and metastatic potential in both MM134-MIND and SUM44-MIND models. Conclusion: Our results provide evidence that iBET, either alone or in combination with erdafitinib, is remarkably effective at inhibiting ILC growth, both in vitro and in vivo and represents a rational therapeutic strategy for recurrent ILC patients in the future. Citation Format: Binbin Gao, Elspeth Ward, Anna Blümel, Emer Conroy, Rachel Moore, Grainne Cremin, Rachel Bleach, Kathryn Haley, Tríona Ní Chonghaíle, Andreas Lindner, Jochen Prehn, Yi Zhang, Idalia Cruz, Leena Hilakivi-Clarke, Georgios Sflomos, Cathrin Brisken, William Gallagher, Darran O'Connor. Rational therapeutic combination of Bromodomain and Extra-Terminal domain (BET) inhibitor and Fibroblast Growth Factor Receptor (FGFR) inhibitor for treatment of invasive lobular carcinoma [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-18-08.

Abstract PO5-27-05: Computational design and validation of a novel peptide-drug conjugate for treatment of triple negative breast cancer

Abstract Sortilin (SORT1) is a member of the vacuolar protein sorting 10 protein (Vps10p) family that functions as a receptor regulating peptide and protein trafficking between the plasma membrane, lysosomes, and trans-golgi network. As a cell surface receptor, SORT1 is able to mediate efficient endocytosis of extracellular ligands to the lysosomal compartment. Numerous reports have identified enriched SORT1 expression in a variety of tumor types, including triple-negative breast cancer (TNBC), a subtype of breast cancer associated with aggressive clinical behavior and poor disease outcomes. We sought to exploit SORT1-dependent internalization of peptides as a platform for rapid and specific chemotherapy delivery into TNBC cells. Using ProteinStudio (our proprietary computation-enabled design capabilities), we generated high-affinity SORT1 targeting peptides that exhibit efficient receptor dependent internalization and lysosomal localization. Alternative computational approaches such as AlphaFold2 failed to recapitulate the peptide design. Peptide drug conjugates (PDCs) were generated via a linkage strategy that combines our designed peptides to the antimitotic agent monomethyl auristatin E (MMAE). Our PDC molecules exhibit potent tumor regression in a MDA-MB-231 TNBC cell derived xenograft model, thereby highlighting the potential of SORT1-engaging PDCs as an efficacious targeted chemotherapeutic delivery strategy. Citation Format: Francine Liu, Andrew Zhai, Ozge Yoluk, Aron Broom, Tracy Stone, Glenn Butterfoss, Serban Popa, Tianyu Li, Lucas Siow, Christopher Ing, David White. Computational design and validation of a novel peptide-drug conjugate for treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-05.

AHNAK2 Promotes the Progression of Pancreatic Ductal Adenocarcinoma by Maintaining the Stability of c-MET.

Pancreatic ductal adenocarcinoma (PDAC) is extremely malignant and rapidly progresses. The overall response rate of PDAC to current treatment methods is still unsatisfactory. Thus, identifying novel targets and clarifying the underlying mechanisms associated with PDAC progression may potentially offer additional treatment strategies. AHNAK2 is aberrantly expressed in a variety of tumors and exerts pro-tumorigenic effects. However, the biological role of AHNAK2 in PDAC remains poorly understood. The expression of AHNAK2 in PDAC and paired non-tumor tissues was detected by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). Lentivirus knockdown was performed to investigate the impact of AHNAK2 on the biological function of pancreatic cancer cells. The subcutaneous cell-derived xenograft (CDX) model and the KPC spontaneous mouse model with AHNAK2 silencing were used to observe the effects of AHNAK2 on tumor growth and prognosis. The expression of c-MET at protein level in response to HGF treatment was assessed using western blot. Our results demonstrated that AHNAK2 was highly expressed in PDAC clinical samples and associated with poor prognosis. Knockdown of AHNAK2 significantly inhibited the proliferation, migration, and invasion of pancreatic cancer cells. AHNAK2 knockdown or knockout resulted in tumor growth suppression and prolonged survival in mice with PDAC. In addition, AHNAK2 and c-MET expression levels showed a significant positive correlation at the post-transcriptional level. Mechanistically, AHNAK2 promoted tumor progression by preventing c-MET degradation and persistently activating the HGF/c-MET signaling pathway. Overall, our study revealed that AHNAK2 plays an important role in PDAC progression by modulating the c-MET signaling pathway, and targeting AHNAK2 may be an effective therapeutic strategy for PDAC.

Metabolic difference between patient-derived xenograft model of pancreatic ductal adenocarcinoma and corresponding primary tumor

BackgroundPatients-derived xenograft (PDX) model have been widely used for tumor biological and pathological studies. However, the metabolic similarity of PDX tumor to the primary cancer (PC) is still unknown.MethodsIn present study, we established PDX model by engrafting primary tumor of pancreatic ductal adenocarcinoma (PDAC), and then compared the tumor metabolomics of PC, the first generation of PDX tumor (PDXG1), and the third generation of PDX tumor (PDXG3) by using 1H NMR spectroscopy. Then, we assessed the differences in response to chemotherapy between PDXG1 and PDXG3 and corresponding metabolomic differences in drug-resistant tumor tissues. To evaluate the metabolomic similarity of PDX to PC, we also compared the metabolomic difference of cell-derived xenograft (CDX) vs. PC and PDX vs. PC.ResultsAfter engraftment, PDXG1 tumor had a low level of lactate, pyruvate, citrate and multiple amino acids (AAs) compared with PC. Metabolite sets enrichment and metabolic pathway analyses implied that glycolysis metabolisms were suppressed in PDXG1 tumor, and tricarboxylic acid cycle (TCA)-associated anaplerosis pathways, such as amino acids metabolisms, were enhanced. Then, after multiple passages of PDX, the altered glycolysis and TCA-associated anaplerosis pathways were partially recovered. Although no significant difference was observed in the response of PDXG1 and PDXG3 to chemotherapy, the difference in glycolysis and amino acids metabolism between PDXG1 and PDXG3 could still be maintained. In addition, the metabolomic difference between PC and CDX models were much larger than that of PDX model and PC, indicating that PDX model still retain more metabolic characteristics of primary tumor which is more suitable for tumor-associated metabolism research.ConclusionsCompared with primary tumor, PDX models have obvious difference in metabolomic level. These findings can help us design in vivo tumor metabolomics research legitimately and analyze the underlying mechanism of tumor metabolic biology thoughtfully.

An oncolytic virus delivering tumor-irrelevant bystander T cell epitopes induces anti-tumor immunity and potentiates cancer immunotherapy

Tumor-specific T cells are crucial in anti-tumor immunity and act as targets for cancer immunotherapies. However, these cells are numerically scarce and functionally exhausted in the tumor microenvironment (TME), leading to inefficacious immunotherapies in most patients with cancer. By contrast, emerging evidence suggested that tumor-irrelevant bystander T (TBYS) cells are abundant and preserve functional memory properties in the TME. To leverage TBYS cells in the TME to eliminate tumor cells, we engineered oncolytic virus (OV) encoding TBYS epitopes (OV-BYTE) to redirect the antigen specificity of tumor cells to pre-existing TBYS cells, leading to effective tumor inhibition in multiple preclinical models. Mechanistically, OV-BYTE induced epitope spreading of tumor antigens to elicit more diverse tumor-specific T cell responses. Remarkably, the OV-BYTE strategy targeting human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory efficiently inhibited tumor progression in a human tumor cell-derived xenograft model, providing important insights into the improvement of cancer immunotherapies in a large population with a history of SARS-CoV-2 infection or coronavirus disease 2019 (COVID-19) vaccination.

Combined therapy of dabrafenib and an anti-HER2 antibody–drug conjugate for advanced BRAF-mutant melanoma

BackgroundMelanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma.MethodsWe evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy.ResultsOur results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway.ConclusionThese findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.

Birinapant Reshapes the Tumor Immunopeptidome and Enhances Antigen Presentation.

Birinapant, an antagonist of the inhibitor of apoptosis proteins, upregulates MHCs in tumor cells and displays a better tumoricidal effect when used in combination with immune checkpoint inhibitors, indicating that Birinapant may affect the antigen presentation pathway; however, the mechanism remains elusive. Based on high-resolution mass spectrometry and in vitro and in vivo models, we adopted integrated genomics, proteomics, and immunopeptidomics strategies to study the mechanism underlying the regulation of tumor immunity by Birinapant from the perspective of antigen presentation. Firstly, in HT29 and MCF7 cells, Birinapant increased the number and abundance of immunopeptides and source proteins. Secondly, a greater number of cancer/testis antigen peptides with increased abundance and more neoantigens were identified following Birinapant treatment. Moreover, we demonstrate the existence and immunogenicity of a neoantigen derived from insertion/deletion mutation. Thirdly, in HT29 cell-derived xenograft models, Birinapant administration also reshaped the immunopeptidome, and the tumor exhibited better immunogenicity. These data suggest that Birinapant can reshape the tumor immunopeptidome with respect to quality and quantity, which improves the presentation of CTA peptides and neoantigens, thus enhancing the immunogenicity of tumor cells. Such changes may be vital to the effectiveness of combination therapy, which can be further transferred to the clinic or aid in the development of new immunotherapeutic strategies to improve the anti-tumor immune response.

Abstract 4592: Preclinical characterization of ATG-042-198, a novel MTAP null-selective PRMT5 inhibitor

Abstract Background: PRMT5 inhibitors are considered as a promising approach for cancer therapy due to their crucial role in the initiation and progression of many types of malignancies. Nevertheless, the crucial involvement of PRMT5 in controlling hematopoiesis, even in normal cells, resulted in significant on-target-off-tumor hematological toxicity when using first-generation, non-selective PRMT5 inhibitors in clinical settings, which hampered the clinical development of these drugs. The MTAP gene is deleted in around 10-15% of all human malignancies. As a result, the loss of MTAP leads to the buildup of MTA, which can bind to PRMT5 and partially inhibit PRMT5 activity. Inhibitors targeting the PRMT5-MTA complex become a promising strategy for treating MTAPnull cancer in a synthetically lethal manner. Herein, we report the preclinical feature of ATG-042-198, a novel MTAPnull-selective PRMT5 inhibitor. Methods: The in vitro activity and MTAP selectivity of ATG-042-198 on cell proliferation and cellular symmetric dimethylarginine (SDMA) expression were profiled using HCT116 MTAP wild type (wt) cells and HCT116 MTAP knock out (ko) cells, as well as in five MTAPnull cell lines. The in vivo efficacy was tested in cell derived xenograft (CDX) mouse models with HCT116 MTAP wt cells, HCT116 MTAP ko cells and LU99 cells (MTAPnull). The in vitro and in vivo pharmacokinetic properties were assessed with corresponding assay methods. Results: ATG-042-198 demonstrated potent and selectively inhibitory effect on HCT116 MTAP ko cells’ proliferation (IC50=44nM) and SDMA expression (IC50=0.146nM), but very weak effect on HCT116 MTAP wt cells’ proliferation (IC50=4550nM) and SDMA expression (IC50&amp;gt;500nM). Consistently, ATG-042-198 showed excellent anti-proliferation activities on multiple endogenous MTAPnull cell lines (A549, IC50=2nM; LU99, IC50=6nM; U87MG, IC50=11nM; NALM-6, IC50=37nM; MIA PaCa-2, IC50=43nM). ATG-042-198 demonstrated high permeability (Caco2 assay, ER=2), good metabolic stability (Human liver microsomes assay, T1/2&amp;gt;145min), and low risk of drug interaction (CYP inhibition assay, 3A4/2C19/2D6, all IC50&amp;gt;10μM). In vivo PK study showed that ATG-042-198 was well absorbed, with a dose-dependent increase in plasma distribution and high oral bioavailability (F=108%). Plasma free drug concentrations were continuously higher than the IC50 of cell proliferation inhibition within 24 hours after 30mpk administration. In three CDX models (HCT116 MTAP wt, HCT116 MTAP ko and LU99), ATG-042-198 at a dose of 30 mg/kg QD potently and selectively inhibited tumor growth (TGI=-1%, 89% and 106%) without inducing body weight loss. Conclusions: In summary, ATG-042-198 is an oral MTAP null-selective PRMT5 inhibitor with potent efficacy against MTAPnull tumor. It also demonstrated good tolerability and preclinical PK profiles. Citation Format: Ya Kong, Ming Zhang, Lulu Jiang, Ruiting Xu, Bin Jiang, Guoqiang Dai, Yulong He, Huiling Liu, Jay Mei, Bing Hou, Bo Shan. Preclinical characterization of ATG-042-198, a novel MTAP null-selective PRMT5 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4592.