Tumor-derived Xenograft Research Articles

Cyclohexanediamine Triazole (CHDT) Functionalization Enables Labeling of Target Molecules with Al18F/68Ga/111In.

The Al18F-labeling approach offers a one-step access to radiofluorinated biomolecules by mimicking the labeling process for radiometals. Although these labeling conditions are considered to be mild compared to classic radiofluorinations, improvements of the chelating units have led to the discovery of (±)-H3RESCA, which allows Al18F-labeling already at ambient temperature. While the suitability of (±)-H3RESCA for functionalization and radiofluorination of proteins is well established, its use for small molecules or peptides is less explored. Herein, we advanced this acyclic pentadentate ligand by introducing an alkyne moiety for the late-stage functionalization of biomolecules via click chemistry. We show that in addition to Al18F-labeling, the cyclohexanediamine triazole (CHDT) moiety allows stable complexation of 68Ga and 111In. Three novel CHDT-functionalized PSMA inhibitors were synthesized and their Al18F-, 68Ga-, and 111In-labeled analogs were subjected to a detailed in vitro radiopharmacological characterization. Stability studies in vitro in human serum revealed among others a high kinetic inertness of all radiometal complexes. Furthermore, the Al18F-labeled PSMA ligands were characterized for their biodistribution in a LNCaP derived tumor xenograft mouse model by PET imaging. One radioligand, Al[18F]F-CHDT-PSMA-1, bearing a small azidoacetyl linker at the glutamate-urea-lysine motif, provided an in vivo performance comparable to that of [18F]PSMA-1007 but with even higher tumor-to-blood and tumor-to-muscle ratios at 120 min p.i. Overall, our results highlight the suitability of the novel CHDT moiety for functionalization and radiolabeling of small molecules or peptides with Al18F, 68Ga, and 111In and the triazole ring seems to entail favorable pharmacokinetic properties for molecular imaging purposes.

Disruption of NADPH homeostasis by total flavonoids from Adinandra nitida Merr. ex Li leaves triggers ROS-dependent p53 activation leading to apoptosis in non-small cell lung cancer cells

Ethnopharmacological relevanceAdinandra nitida Merr. ex Li leaves serve as a herbal tea and hold a significant role in traditional Chinese medicine, being applied to assist in tumor treatment. Flavonoids present the primary bioactive constituents in Adinandra nitida Merr. ex Li leaves. Aim of the studyTo explore the potential of total flavonoids from Adinandra nitida Merr. ex Li Leaves (TFAN) in inhibiting non-small cell lung cancer (NSCLC) and further elucidate the underlying mechanisms. Materials and methodsHuman NSCLC cell lines and normal lung cell line were employed to assess the impact of TFAN (0–160 μg/mL for 24, 28 and 72 h) on cell proliferation in vitro. Immunofluorescence (IF) staining gauged p53 expression changes in NSCLC cells under TFAN present condition (150 μg/mL for 24 h). In vivo study utilized NSCLC cell derived xenograft tumors in nude mice, administering TFAN orally (200 and 400 mg/kg) for 14 days. Immunohistochemistry assessed Cleaved Caspase 3 expression change in A549 xenograft tumors treated with TFAN (400 mg/kg for 14 days). RNA-seq and KEGG analysis identified gene expression changes and enriched processes in A549 xenograft tumors treated with TFAN. CM-H2DCFDA and metabolomics assessed ROS level and GSH/GSSG pool changes in A549 cells under TFAN present condition. Cell viability assay and IF staining assessed A549 cell proliferation and p53 expression changes under H2O2-induced oxidative stress (0–40 μM for 24 h) and TFAN present conditions. GSEA and N-Acetyl-L-cysteine (NAC) rescue (0–1 μM for 24 h) analyzed the impact of TFAN on GSH de novo synthesis. NADPH/NADP+ pool measurement and NADPH rescue (0–10 μM for 24 h) analyzed the impact of TFAN on GSH salvage synthesis. GC-FID and HPLC-MS were utilized to detect ethanol and ethyl acetate residues, and to characterize the chemical constituents in TFAN, respectively. The total flavonoid content of TFAN was determined using a 330 nm wavelength. ResultsTFAN significantly inhibited A549 cells (wild-type p53) but not NCI–H1299 cells (p53-deficient), NCI–H596 cells (p53-mutant) or BEAS-2B in vitro. IF staining validated p53 genotype for the cell lines and revealed an increase in p53 expression in A549 cells after TFAN treatment. In vivo, TFAN selectively inhibited A549 xenograft tumor growth without discernible toxicity, inducing apoptosis evidenced by Cleaved Caspase 3 upregulation. RNA-seq and KEGG analysis suggested ROS biosynthesis was involved in TFAN-induced p53 activation in A549 cells. Elevated ROS level in TFAN-treated A549 cells were observed. Moreover, TFAN sensitized A549 cells to H2O2-induced oxidative stress, with higher p53 expression. Additionally, A549 cells compensated with GSH de novo synthesis under TFAN present condition, confirmed by GSEA and NAC rescue experiment. TFAN disrupted NADPH homeostasis to impair GSH salvage biosynthesis, supported by NADPH/NADP+ change and NADPH rescue experiment. The chemical constituents of TFAN, with acceptable limits for ethanol and ethyl acetate residues and a total flavonoid content of 68.87%, included Catechin, Epicatechin, Quercitroside, Camellianin A, and Apigenin. ConclusionThe disruption of NADPH homeostasis by TFAN triggers ROS-dependent p53 activation that leads to apoptotic cell death, ultimately suppressing NSCLC growth. These findings offer potential therapeutic implications of Adinandra nitida Merr. ex Li leaves in combating NSCLC.

Deep learning generation of preclinical positron emission tomography (PET) images from low-count PET with task-based performance assessment.

Preclinical low-count positron emission tomography (LC-PET) imaging offers numerous advantages such as facilitating imaging logistics, enabling longitudinal studies of long- and short-lived isotopes as well as increasing scanner throughput. However, LC-PET is characterized by reduced photon-count levels resulting in low signal-to-noise ratio (SNR), segmentation difficulties, and quantification uncertainties. We developed and evaluated a novel deep-learning (DL) architecture-Attention based Residual-Dilated Net (ARD-Net)-to generate standard-count PET (SC-PET) images from LC-PET images. The performance of the ARD-Net framework was evaluated for numerous low count realizations using fidelity-based qualitative metrics, task-based segmentation, and quantitative metrics. Patient Derived tumor Xenograft (PDX) with tumors implanted in the mammary fat-pad were subjected to preclinical [18F]-Fluorodeoxyglucose (FDG)-PET/CT imaging. SC-PET images were derived from a 10min static FDG-PET acquisition, 50min post administration of FDG, and were resampled to generate four distinct LC-PET realizations corresponding to 10%, 5%, 1.6%, and 0.8% of SC-PET count-level. ARD-Net was trained and optimized using 48 preclinical FDG-PET datasets, while 16 datasets were utilized to assess performance. Further, the performance of ARD-Net was benchmarked against two leading DL-based methods (Residual UNet, RU-Net; and Dilated Network, D-Net) and non-DL methods (Non-Local Means, NLM; and Block Matching 3D Filtering, BM3D). The performance of the framework was evaluated using traditional fidelity-based image quality metrics such as Structural Similarity Index Metric (SSIM) and Normalized Root Mean Square Error (NRMSE), as well as human observer-based tumor segmentation performance (Dice Score and volume bias) and quantitative analysis of Standardized Uptake Value (SUV) measurements. Additionally, radiomics-derived features were utilized as a measure of quality assurance (QA) in comparison to true SC-PET. Finally, a performance ensemble score (EPS) was developed by integrating fidelity-based and task-based metrics. Concordance Correlation Coefficient (CCC) was utilized to determine concordance between measures. The non-parametric Friedman Test with Bonferroni correction was used to compare the performance of ARD-Net against benchmarked methods with significance at adjusted p-value ≤0.01. ARD-Net-generated SC-PET images exhibited significantly better (p ≤ 0.01 post Bonferroni correction) overall image fidelity scores in terms of SSIM and NRMSE at majority of photon-count levels compared to benchmarked DL and non-DL methods. In terms of task-based quantitative accuracy evaluated by SUVMeanand SUVPeak, ARD-Net exhibited less than 5% median absolute bias for SUVMean compared to true SC-PET and lower degree of variability compared to benchmarked DL and non-DL based methods in generating SC-PET. Additionally, ARD-Net-generated SC-PET images displayed higher degree of concordance to SC-PET images in terms of radiomics features compared to non-DL and other DL approaches. Finally, the ensemble score suggested that ARD-Net exhibited significantly superior performance compared to benchmarked algorithms (p ≤ 0.01 post Bonferroni correction). ARD-Net provides a robust framework to generate SC-PET from LC-PET images. ARD-Net generated SC-PET images exhibited superior performance compared other DL and non-DL approaches in terms of image-fidelity based metrics, task-based segmentation metrics, and minimal bias in terms of task-based quantification performance for preclinical PET imaging.

Discovery of immunotherapy targets for pediatric solid and brain tumors by exon-level expression

Immunotherapy with chimeric antigen receptor T cells for pediatric solid and brain tumors is constrained by available targetable antigens. Cancer-specific exons present a promising reservoir of targets; however, these have not been explored and validated systematically in a pan-cancer fashion. To identify cancer specific exon targets, here we analyze 1532 RNA-seq datasets from 16 types of pediatric solid and brain tumors for comparison with normal tissues using a newly developed workflow. We find 2933 exons in 157 genes encoding proteins of the surfaceome or matrisome with high cancer specificity either at the gene (n = 148) or the alternatively spliced isoform (n = 9) level. Expression of selected alternatively spliced targets, including the EDB domain of fibronectin 1, and gene targets, such as COL11A1, are validated in pediatric patient derived xenograft tumors. We generate T cells expressing chimeric antigen receptors specific for the EDB domain or COL11A1 and demonstrate that these have antitumor activity. The full target list, explorable via an interactive web portal (https://cseminer.stjude.org/), provides a rich resource for developing immunotherapy of pediatric solid and brain tumors using gene or AS targets with high expression specificity in cancer.

Abstract LB157: Discovery of HP537, a potent and selective p300/CBP inhibitor for the treatment of hematologic malignancies

Abstract E1A-binding protein P300 (p300) and its paralog CREB binding protein (CBP) are key acetyl transferases (HAT) and transcriptional co-factors. p300/CBP bromodomain (BRD) inhibition reduces acetylation of H3K27 at enhancers, leading to suppression of enhancer-driven gene expression, such as MYC and IRF4 that are critical for the initiation, progression, and chemoresistance of hematological malignancies. Thus, p300/CBP inhibition is a highly potential therapeutic strategy for the treatment of hematological malignancies, including multiple myeloma (MM), non-Hodgkin lymphoma (NHL) and leukemia. We have discovered HP537 that competitively inhibits an acetylated peptide substrate to bind BRD of p300/CBP with IC50 < 5 nM. HP537 does not inhibit the enzymatic activity of HAT domain of p300/CBP and HAT1, nor has binding affinity for other BRD containing proteins including BRD4 and BRD9. HP537 inhibits proliferation of a wide range of cell lines derived from MM, leukemia and lymphoma with IC50 values ranged from 0.01 μM to 0.4 μM. Protein expression levels of acetylated H3K27, c-Myc and IRF4 are significantly decreased by HP537 in MM, NHL and acute myelogenous leukemia (AML) cell lines in a dose- and time-dependent manner. HP537 robustly and dose-dependently inhibits tumor growth in OPM-2 (MM), MOLM-16 (AML), and KARPAS-422 (NHL) cell line derived tumor xenograft (CDX) mouse models. Down-regulation of c-Myc and IRF4 are also observed in the tumor samples. In conclusion, HP537 is a novel, potent and selective inhibitor of p300/CBP BRD. The pre-clinical data supports the clinical development of HP537 for the treatment of hematological malignancies. A FIH study of HP537 in cancer patients is planned to start in 2024. HP537 structure will not be disclosed. Citation Format: Jing Li, Zhilin Tu, Jian Peng, Lei Fan, Hua Yu, Fei Wang, Xinghai Li. Discovery of HP537, a potent and selective p300/CBP inhibitor for the treatment of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB157.

Abstract LB178: HP518, an oral AR-targeting PROTAC for the treatment of AR positive triple negative breast cancer with a novel mechanism of action

Abstract Triple negative breast cancer (TNBC) accounts for 15-20% of all breast carcinomas and has poor prognosis. Chemotherapy is the mainstay treatment for TNBC patients, yet with limited clinical benefit. Androgen receptor (AR) is expressed in approximately 50% of TNBC tumors and thus the anti-androgen therapy could be a promising solution for the treatment of AR+ TNBC. Nevertheless, AR inhibitors including enzalutamide and bicalutamide or the androgen biosynthesis inhibitor abiraterone have failed to demonstrate satisfying anti-tumor activity in clinical trials. We have discovered HP518, an AR-targeting PROTAC (proteolysis targeting chimera) that induces potent degradation of both the wild-type AR and AR mutants. HP518 is currently under the clinical development for the treatment of mCRPC. Here we report the preclinical data on HP518 for the treatment of AR+ TNBC. HP518 robustly degrades AR in multiple TNBC cell lines with a half-maximal degradation concentration (DC50) < 1 nM. HP518 strongly inhibits AR+ TNBC cell growth, whereas the AR inhibitor enzalutamide does not show significant anti-proliferative effect on these cells in the presence or absence of androgen. Consistent with the in vitro results, HP518 significantly and dose-dependently inhibits tumor growth in MDA-MB-453 cell line derived tumor xenograft (CDX) and patient-derived xenograft (PDX) mouse models. PI3KCA activating mutations are frequently expressed in AR+ TNBC. HP518 in combination with alpelisib, a PI3Kα inhibitor shows a synergistic anti-tumor activity. Mechanistically, HP518, but not AR inhibitor enzalutamide downregulates the mRNA expression levels of a set of DNA replication-related genes in AR+ TNBC cells, indicating a novel and ligand-independent role for AR in regulating growth of AR+ TNBC cells. Therefore, HP518, an AR-targeting PROTAC degrader holds great potential to fulfill the unmet clinical needs for the AR+ TNBC therapy. Selected pre-clinical data will be presented. HP518 structure will not be disclosed. Citation Format: Jing Li, Hongwei Yuan, Kefan Chen, Yongxu Huo, Guoqing Liu, Wu Du, Xinghai Li. HP518, an oral AR-targeting PROTAC for the treatment of AR positive triple negative breast cancer with a novel mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB178.

Abstract LB177: Discovery of HC-4955, a novel AR-V7-targeting PROTAC for the treatment of mCRPC

Abstract Prostate cancer is the second most commonly diagnosed cancer and the second leading cause of cancer death among men worldwide. Androgens play an important role in the development and progression of prostate cancers and targeting androgen receptor (AR) signaling axis over decades has remained a mainstay of prostate cancer therapy. Next generation hormonal agents (NHAs) enzalutamide and abiraterone have brought clinical benefits for metastatic castrate-resistant prostate cancer (mCRPC) patients, AR axis-targeted therapy resistance is inevitable due to AR gene amplification or mutations. AR-V7 is the most clinically relevant AR splice variant associated with endocrine resistance and poor prognosis. AR-V7 constitutively activates AR signaling and lacks the ligand binding domain (LBD) where hormones and AR antagonists interact, resulting in resistance to AR signaling inhibitors (ARSi). Thus, AR-V7 is a promising therapeutic target to address ARSi resistance in mCRPC. We have discovered a highly potent AR-V7 proteolysis targeting chimera (PROTAC), HC-4955 that degrades both the wild-type AR (WT-AR) and AR-V7 mutant. HC-4955 degrades AR-V7 with DC50 < 5 nM. In silico molecular docking analysis predicts the binding of HC-4955 to the N-terminal domain of AR and then the essential amino acids contributed to HC-4955 binding are validated by mutational analysis studies. HC-4955 also potently degrades mutants of AR-LBD mutations including L702H and T878A and these mutations are also associated with ARSi resistance. HC-4955 strongly inhibits proliferation of ARSi-resistant prostate cancer cell lines with IC50 < 10 nM. HC-4955 shows a robust anti-tumor activity in the AR-V7-bearing 22Rv1 cell line derived tumor xenograft (CDX) and patient-derived xenograft (PDX) mouse models, with majority of the tumors completely regressed and > 90% AR-V7 expression decreased at 1-5 mg/kg (QD, PO) in these models. The pre-clinical results support the clinical development of HC-4955 for the treatment of mCRPC. Selected pre-clinical data along with the chemical structure of HC-4955 will be presented. Citation Format: Jing Li, Zhilin Tu, Wu Du, Xi Xiang, Zeyu Wen, Yang You, Lijuan Zhao, Yin Chen, Chengcheng Yang, Hongwei Yuan, Xinghai Li. Discovery of HC-4955, a novel AR-V7-targeting PROTAC for the treatment of mCRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB177.

Abstract LB159: Discovery of HP560, a novel BET inhibitor for the treatment of myelofibrosis

Abstract Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by defective clonal hematopoiesis, bone marrow fibrosis, splenomegaly, severe anemia, and the propensity for transformation to acute myeloid leukemia (AML). Inhibitors targeting JAKs (Janus kinases) have revolutionized the treatment of MF, but are limited by modest efficacy or acquired resistance after prolonged treatment. Here, we report a novel inhibitor, HP560 that targets BET (bromodomain and extra-terminal) proteins and shows promising therapeutic activities against MF and AML. Through engaging the bromodomain pocket of BET proteins in a competitive manner with acetylated histones, HP560 causes the disruption of BET proteins from chromatin. HP560 has high binding affinities for all BET proteins and strong anti-proliferative activity as well as synergistic anti-proliferative effect in combination with ruxolitinib, a JAK1/2 inhibitor. In an MF cell line derived tumor xenograft (CDX) mouse model, HP560 prolongs survival and improves splenomegaly and anemia, and these effects are enhanced when combined with ruxolitinib. HP560 also significantly and dose-dependently inhibits tumor growth in an AML CDX mouse model. Together, HP560 is a potent pan-BET inhibitor and can be developed as a monotherapy or in combination with a JAK inhibitor for the treatment of MF. HP560 structure will not be disclosed. Citation Format: Jing Li, Lei Fan, Hongwei Yuan, Yongxu Huo, Hua Yu, Fei Wang, Xinghai Li. Discovery of HP560, a novel BET inhibitor for the treatment of myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB159.

The therapeutically actionable long non-coding RNA ‘T-RECS’ is essential to cancer cells’ survival in NRAS/MAPK-driven melanoma

Finding effective therapeutic targets to treat NRAS-mutated melanoma remains a challenge. Long non-coding RNAs (lncRNAs) recently emerged as essential regulators of tumorigenesis. Using a discovery approach combining experimental models and unbiased computational analysis complemented by validation in patient biospecimens, we identified a nuclear-enriched lncRNA (AC004540.4) that is upregulated in NRAS/MAPK-dependent melanoma, and that we named T-RECS. Considering potential innovative treatment strategies, we designed antisense oligonucleotides (ASOs) to target T-RECS. T-RECS ASOs reduced the growth of melanoma cells and induced apoptotic cell death, while having minimal impact on normal primary melanocytes. Mechanistically, treatment with T-RECS ASOs downregulated the activity of pro-survival kinases and reduced the protein stability of hnRNPA2/B1, a pro-oncogenic regulator of MAPK signaling. Using patient- and cell line- derived tumor xenograft mouse models, we demonstrated that systemic treatment with T-RECS ASOs significantly suppressed the growth of melanoma tumors, with no noticeable toxicity. ASO-mediated T-RECS inhibition represents a promising RNA-targeting approach to improve the outcome of MAPK pathway-activated melanoma.

Preparation of protein nanoparticles using diselenide functionalized gelatin for redox responsive release of doxorubicin

The present study demonstrates the fabrication of a nanocarrier system using diselenide conjugated gelatin (G-Se-Se-G) for the redox responsive delivery of a wide spectrum anticancer drug, doxorubicin (Dox). The method involves nanoprecipitation of the aqueous solution of G-Se-Se-G employing ethanol and F68 as the desolvating and stabilizing agents respectively. The drug, Dox is loaded within the nanocarrier by entrapment method during the nanoparticle synthesis. The physico-chemical characterizations confirm the formation of nanoparticles of regular spherical shape with hydrodynamic size, surface charge and loading efficiency of 181 ± 12.6 nm, -13.10 ± 0.83 mV and ∼9 % respectively. Notably, Dox loaded G-Se-Se-G carrier shows significantly higher drug release in response to acidic (pH = ∼5.5) and reducing (10 mM glutathione) sink conditions. Dox release kinetic data fitted to a universal Korsmeyer–Peppas model suggests that irrespective of the nature of stimuli, the release of Dox from G-Se-Se-G carrier follows non-Fickian mechanism which is attributed to the diffusion and erosion. The cytotoxicity studies employing human lung adenocarcinoma (A549) and human normal lung fibroblast (WI38) cells confirm the potency as well as selectively of Dox loaded G-Se-Se-G carrier towards cancer cells. The intracellular drug release studies through fluorescence imaging indeed corroborate the redox (reducing environment) mediated preferential release of Dox from G-Se-Se-G carrier in A549 cells as compared to WI38 cells. Finally, in vivo studies using A549 derived xenograft tumor model establishes that Dox loaded G-Se-Se-G nanocarrier is better tolerable than free Dox with almost similar efficacy.

Acid and Hypoxia Tandem-Activatable Deep Near-Infrared Nanoprobe for Two-Step Signal Amplification and Early Detection of Cancer.

The early detection of cancers can significantly change outcomes even with existing treatments. However, ~50% of cancers still cannot be detected until they reach an advanced stage, highlighting the great challenges in the early detection. Here, an ultrasensitive deep near-infrared (dNIR) nanoprobe that is successively responsive to tumor acidity and hypoxia is reported. It is demonstrated that the new nanoprobe specifically detects tumor hypoxia microenvironment based on deep NIR imaging in ten different types of tumor models using cancer cell lines and patient-tissue derived xenograft tumors. By combining the acidity and hypoxia specific two-step signal amplification with a deep NIR detection, the reported nanoprobe enables the ultrasensitive visualization of hundreds of tumor cells or small tumors with a size of 260µm in whole-body imaging or 115µm metastatic lesions in lung imaging. As a result, it reveals that tumor hypoxia can occur as early as the lesions contain only several hundred cancer cells.

Abstract 3402: In vivo efficacy of RAF/MEK clamp avutometinib (VS-6766) in combination with FAK inhibition in low grade serous ovarian cancer

Abstract Objective: Low grade serous carcinoma (LGSC) of the ovary is characterized by a high recurrence rate, resistance to most chemotherapeutic agents, and limited effective therapeutic options. Recently, LGSC has been found to often contain mutations in KRAS (~30%), NRAS (~10%), and BRAF (~8%). Avutometinib (VS-6766) is a RAF/MEK clamp that induces dominant negative complexes of RAF with MEK, and inhibits proliferation of KRAS, NRAS, BRAF, and CRAF mutant cell lines. VS-4718 is a focal adhesion kinase (FAK) inhibitor that has shown synergistic anti-tumor activity with avutometinib in other cancer models, as it blocks the potential of FAK to function as an adaptive resistance mechanism to RAF/MEK inhibition. This study aimed to determine the efficacy of the RAF/MEK clamp avutometinib with FAK inhibition in vivo in LGSC patient derived tumor xenograft (PDX) models. Methods: Whole-exome sequencing was used to evaluate mutations in two primary ovarian LGSC PDX models (OVA(K)250 and PERIT(M)17). OVA(K)250 and PERIT(M)17 cells were injected into SCID mice and animals were treated with either vehicle control (CTRL), RAF/MEK clamp avutometinib (MEKi), FAK inhibitor VS-4718, or the combination (MEKi/FAKi). Tumor volumes were measured twice per week. Results: Both the OVA(K)250 and PERIT(M)17 PDX models express wildtype KRAS and express RAF1 mutations at position 12641679 A>C, V207G. In PDX OVA(K)250 mice treated with MEKi/FAKi, day 9 tumor volume was significantly lower compared to mice treated with CTRL (p=0.0002). At day 9, tumor volume was also significantly lower in mice treated with MEKi alone compared to CTRL (p=0.0007). Mice treated with FAKi alone had tumor volumes that were significantly smaller than CTRL by day 12 (p=0.009). By day 23, CTRL mice had reached tumor volume characteristics requiring sacrifice. Combination MEKi/FAKi treatment resulted in improved overall survival (OS); median OS for combination or MEKi alone was not reached by 60 days. Treatment with FAKi led to median OS of 35 days; CTRL mice had median OS of 23 days (p=0.001). In vivo experiments with PDX PERIT(M)17 and PDX OVA(K)250 are ongoing. Conclusion: Combination of avutometinib with FAK inhibition showed improved in vivo preclinical anti-tumor efficacy relative to either agent alone in an LGSC PDX model with wildtype KRAS and with a RAF1 mutation. These data support an ongoing registration-directed study with avutometinib ± the FAK inhibitor defactinib for patients with recurrent LGSC (NCT04625270). Citation Format: Blair McNamara, Cem Demirkiran, Stefania Bellone, Levent Mutlu, Margherita Zipponi, Miguel S. Verzosa, Justin Harold, Tobias M. Hartwich, Gary Altwerger, Elena Ratner, Gloria Huang, Mitchell Clark, Vaagn Andikyan, Masoud Azodi, Peter E. Schwartz, Peter Dottino, Alessandro D. Santin. In vivo efficacy of RAF/MEK clamp avutometinib (VS-6766) in combination with FAK inhibition in low grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3402.

Abstract B026: SIWA318H, a novel advanced glycation end product (AGE) specific antibody, targets cancer and senescent cells in the tumor microenvironment and exhibits potent antitumor activity in a humanized mouse model for pancreatic cancer. [R

Abstract The aging-related and cancer cell derived oxidatively stressed tumor microenvironment drives cell senescence. Senescent cells (SCs), in turn, promote immune suppression, tumor progression, and metastasis. Hence, targeting SCs presents an attractive approach for cancer therapy. SIWA318H is a monoclonal antibody that selectively targets an advanced glycation end product biomarker on the surface of dysfunctional cells exhibiting both (a) aerobic glycolysis and (b) oxidative stress. The SIWA318H biomarker is associated with cancer cells, SCs, oxidatively damaged cells, and infected cells. SIWA318H has been shown to bind to an ADCC inducing human IgG FcγR3 receptor as a mechanism of action. SIWA318H exhibits strong binding to pancreatic cancer patient derived xenograft tumors as demonstrated by immunohistochemistry. In an in vivo study using a humanized mouse xenograft model for pancreatic cancer, we showed that treatment with SIWA318H (both high and low dose groups) statistically significantly reduced tumor growth compared to the isotype antibody control group (P < 0.0001). In addition, there was a significantly higher number of mice had complete remission in the treated groups compared to the isotype control group: 37.5% in high dose group (P = 0.0325) and 43.8% in low dose group (P = 0.0143). Moreover, mice treated with 318H had a median overall survival of > 45 days compared to 26 days for the isotype control mice. There was no difference in animal body weight between the SIWA318H-treated mice and the isotype control mice. Immunohistochemical analysis of tumor tissues taken from the in vivo study showed that SIWA318H treatment significantly reduced (a) tumor fibrosis as measured by the area of αSMA+ staining and (b) the number of senescent cells in the tumor stroma as measured by the number of p16INK4a positive cells in αSMA+ areas. In summary, our results demonstrate that SIWA318H is a novel senolytic antibody that exhibits potent preclinical antitumor activity against pancreatic cancer. SWA318H is currently under development for a first in human clinical trial. Citation Format: Gabriela Rossi, Lewis Gruber, Misty Gruber, Haiyong Han. SIWA318H, a novel advanced glycation end product (AGE) specific antibody, targets cancer and senescent cells in the tumor microenvironment and exhibits potent antitumor activity in a humanized mouse model for pancreatic cancer. [R] [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B026.

Long noncoding RNA LINC01132 enhances immunosuppression and therapy resistance via NRF1/DPP4 axis in hepatocellular carcinoma

BackgroundLong noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression and play fundamental roles in various types of cancer. Current developments in transcriptome analyses unveiled the existence of lncRNAs; however, their functional characterization remains a challenge.MethodsA bioinformatics screen was performed by integration of multiple omics data in hepatocellular carcinoma (HCC) prioritizing a novel oncogenic lncRNA, LINC01132. Expression of LINC01132 in HCC and control tissues was validated by qRT-PCR. Cell viability and migration activity was examined by MTT and transwell assays. Finally, our results were confirmed in vivo mouse model and ex vivo patient derived tumor xenograft experiments to determine the mechanism of action and explore LINC01132-targeted immunotherapy.ResultsSystematic investigation of lncRNAs genome-wide expression patterns revealed LINC01132 as an oncogene in HCC. LINC01132 is significantly overexpressed in tumor and associated with poor overall survival of HCC patients, which is mainly driven by copy number amplification. Functionally, LINC01132 overexpression promoted cell growth, proliferation, invasion and metastasis in vitro and in vivo. Mechanistically, LINC01132 acts as an oncogenic driver by physically interacting with NRF and enhancing the expression of DPP4. Notably, LINC01132 silencing triggers CD8+ T cells infiltration, and LINC01132 knockdown combined with anti-PDL1 treatment improves antitumor immunity, which may prove a new combination therapy in HCC.ConclusionsLINC01132 functions as an oncogenic driver that induces HCC development via the NRF1/DPP4 axis. Silencing LINC01132 may enhance the efficacy of anti-PDL1 immunotherapy in HCC patients.

999P JIN-A02, a fourth-generation, highly effective tyrosine kinase inhibitor with intracranial activity, targeting EGFR C797S mutations in NSCLC

Even though epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have improved treatment outcomes for EGFR-driven NSCLC, resistance inevitably emerges and disease often progresses due to brain metastasis. The C797S is the most significant resistance mutation to occur after 3rd generation TKIs treatment. JIN-A02 is an orally available EGFR-TKI targeting C797S mutation with high brain penetration. Cellular activities of JIN-A02 were evaluated on phosphorylation-EGFR expression with AlphaLISA assay and on EGFR mutants with patients-derived cell (PDC) and patients-derived organoid (PDO). Different type of allelic relationship was also assessed in all models by whole exome sequencing. Patients-derived xenografts (PDX) was used to assess antitumor activities of JIN-A02. For in vivo intracranial activity, NCI-H1975 cell line derived tumor xenograft was used. In AlphaLISA assay, JIN-A02 showed high potency in EGFRex19del/T790M/C797S (IC50=4.7 nM, Ba/F3), EGFRL858R/T790M/C797S (IC50=12.8 nM, NCI-H1975 LTC). JIN-A02 strongly inhibited cellular activity of in trans model (IC50=89.7 nM, PDO) and in cis model (IC50=92.1 nM, PDC) of EGFRex19del/T790M/C797S. It appears to be superior to osimertinib (IC50 >4,000 nM for both cells). In addition, JIN-A02 showed comparable potency to osimertinib in EGFRex19del/T790M (IC50=84.4 nM for PC-9GR), and EGFRL858R/T790M (IC50=73.3 nM for NCI-H1975). In the PDX mouse model (YHIM-1094, EGFRex19del/T790M/C797S), JIN-A02 delayed tumor growth at a dose of 30 mg/kg. In the brain metastases model, antitumor activity of JIN-A02 was observed with intracranial implanted NCI-H1975 tumors. JIN-A02 is highly potent EGFR-TKI for various EGFR targeted mutations including in cis and trans EGFRex19del/T790M/C797S and shows intracranial activity. Based on these robust activities for EGFR mutants, JIN-A02 is expected to provide a potential therapeutic opportunity for NSCLC.

Endothelial cell–leukemia interactions remodel drug responses, uncovering T-ALL vulnerabilities

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and often incurable disease. To uncover therapeutic vulnerabilities, we first developed T-ALL patient–derived tumor xenografts (PDXs) and exposed PDX cells to a library of 433 clinical-stage compounds in vitro. We identified 39 broadly active drugs with antileukemia activity. Because endothelial cells (ECs) can alter drug responses in T-ALL, we developed an EC/T-ALL coculture system. We found that ECs provide protumorigenic signals and mitigate drug responses in T-ALL PDXs. Whereas ECs broadly rescued several compounds in most models, for some drugs the rescue was restricted to individual PDXs, suggesting unique crosstalk interactions and/or intrinsic tumor features. Mechanistically, cocultured T-ALL cells and ECs underwent bidirectional transcriptomic changes at the single-cell level, highlighting distinct “education signatures.” These changes were linked to bidirectional regulation of multiple pathways in T-ALL cells as well as in ECs. Remarkably, in vitro EC-educated T-ALL cells transcriptionally mirrored ex vivo splenic T-ALL at single-cell resolution. Last, 5 effective drugs from the 2 drug screenings were tested in vivo and shown to effectively delay tumor growth and dissemination thus prolonging overall survival. In sum, we developed a T-ALL/EC platform that elucidated leukemia-microenvironment interactions and identified effective compounds and therapeutic vulnerabilities.

Genotype-to-Phenotype Associations in the Aggressive Variant Prostate Cancer Molecular Profile (AVPC-m) Components.

Simple SummaryWe treat prostate cancer like it is one disease, but it is clear that it behaves like different diseases in different patients, with some having excellent responses to hormonal therapies and prolonged survivals, and others having virulent, aggressive courses and short survivals. These differences reflect different tumor biologies and therapeutic sensitivities, but the absence of molecular markers that identify each of these subsets makes it difficult to develop treatments specific to each. We found that alterations in two or more of the tumor suppressors TP53, RB1 and PTEN characterize the more virulent prostate cancers, and that patients with this molecular profile (called the aggressive variant prostate cancer molecular profile) appear to benefit more from combination chemotherapies than those without. The alterations in these markers can be determined either by staining tumor tissues or by examining their DNA. In this study, we used 28 mouse models of the human disease to assess the performance of various assays in determining these alterations. We found that although both staining and DNA sequencing are complementary, staining (which is a broadly available technique) is likely sufficient to make these determinations. Our results will inform the use of this molecular signature in clinical research and clinical practice.The aggressive variant prostate cancer molecular profile (AVPC-m), composed of combined defects in TP53, RB1 and PTEN, characterizes a subset of prostate cancers linked to androgen indifference and platinum sensitivity. To contribute to the optimization of the AVPC-m assessment for inclusion in prospective clinical trials, we investigated the status of the AVPC-m components in 28 patient tumor-derived xenografts (PDXs) developed at MDACC. We subjected single formalin-fixed, paraffin-embedded (FFPE) blocks from each PDX to immunohistochemistry (IHC), targeted next-generation genomic sequencing (NGS) and Clariom-S Affymetrix human microarray expression profiling. Standard validated IHC assays and a 10% labeling index cutoff resulted in high reproducibility across three separate laboratories and three independent readers for all tumor suppressors, as well as strong correlations with loss-of-function transcriptional scores (LOF-TS). Adding intensity assessment to labeling indices strengthened the association between IHC results and LOF-TS for TP53 and RB1, but not for PTEN. For TP53, genomic alterations determined by NGS had slightly higher agreement scores with LOF-TS than aberrant IHC, while for RB1 and PTEN, NGS and IHC determinations resulted in similar agreement scores with LOF-TS. Nonetheless, our results indicate that the AVPC-m components can be assessed reproducibly by IHC using various widely available standardized assays.

Abstract A015: Inferring phylogenetic trees based on copy number aberrations from methylation array

Abstract Background: The development of methylation array platforms has allowed large scale studies on cancer epigenomes. Apart from intended analyses on methylation patterns, it is also possible to infer copy number profiles by leveraging the signal from methylation array data. To disentangle complex mixtures of phylogenetically related clones, it is best to jointly analyze different samples (different regions, timepoints, etc.) from the same tumor. However, current methods for studying copy number aberrations (CNAs) from methylation arrays consider tumor samples independently. In this project, we introduce Multi-Sample EPIC Phylogenetic Reconstruction (MultiSEPhyR), aiming to investigate the copy number profiles and clonal compositions from multi-sample methylation array data. Material and methods: As part of the GCGR (Glioma Cellular Genetic Resource; gcgr.org.uk) project, we analyze 53 samples from 26 cases with multiple methylation array data of primary glioblastomas, tumor-derived cell lines and xenograft re-derived cell lines. Copy Number Aberrations (CNAs) called from the methylation array data are validated using WGS data from a subset of the cohort. By jointly clustering the segmented LogR ratios from related samples, we can define tumor clones based on clustered CNA events. We use a two-step approach, where we firstly identify clonal events to obtain sample purity and tumor ploidy and use these values to subsequently infer the copy number state and the clonality of the remaining events. Results and Discussions: On a simulated dataset, MultiSEPhyR could accurately estimate sample purity and identify both clonal and sub-clonal CNA events. We then apply our method on GCGR dataset and show similar extent of intra-tumor heterogeneity in primary glioblastomas and derived cell lines. We also investigate clonal dynamics in each case. In the cell lines, while the heterogeneity is well captured, some sub-clonal CNA events may exhibit significantly different clonality compared to the primary glioblastomas. For example, we observe a clonal expansion of the clones harboring a deletion in chromosome 6 in xenograft re-derived cell lines. Conclusion We present a systematic way to call CNAs and resolve clonal compositions from methylation array data, leading to a cost-efficient way to study tumor evolution. Citation Format: Chuling Ding, Javier Herrero. Inferring phylogenetic trees based on copy number aberrations from methylation array [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A015.

BOXR1030, an anti-GPC3 CAR with exogenous GOT2 expression, shows enhanced T cell metabolism and improved anti-cell line derived tumor xenograft activity.

PurposeThe solid tumor microenvironment (TME) drives T cell dysfunction and inhibits the effectiveness of immunotherapies such as chimeric antigen receptor-based T cell (CAR T) cells. Early data has shown that modulation of T cell metabolism can improve intratumoral T cell function in preclinical models.Experimental designWe evaluated GPC3 expression in human normal and tumor tissue specimens. We developed and evaluated BOXR1030, a novel CAR T therapeutic co-expressing glypican-3 (GPC3)-targeted CAR and exogenous glutamic-oxaloacetic transaminase 2 (GOT2) in terms of CAR T cell function both in vitro and in vivo.ResultsCell surface expression of tumor antigen GPC3 was observed by immunohistochemical staining in tumor biopsies from hepatocellular carcinoma, liposarcoma, squamous lung cancer, and Merkel cell carcinoma patients. Compared to control GPC3 CAR alone, BOXR1030 (GPC3-targeted CAR T cell that co-expressed GOT2) demonstrated superior in vivo efficacy in aggressive solid tumor xenograft models, and showed favorable attributes in vitro including an enhanced cytokine production profile, a less-differentiated T cell phenotype with lower expression of stress and exhaustion markers, an enhanced metabolic profile and increased proliferation in TME-like conditions.ConclusionsTogether, these results demonstrated that co-expression of GOT2 can substantially improve the overall antitumor activity of CAR T cells by inducing broad changes in cellular function and phenotype. These data show that BOXR1030 is an attractive approach to targeting select solid tumors. To this end, BOXR1030 will be explored in the clinic to assess safety, dose-finding, and preliminary efficacy (NCT05120271).

Docetaxel and 5-FU enhanced the inhibitory effects of apatinib and ramucirumab on growth and migration of gastric cancer

BackgroundGastric cancer (GC) is one of the most common malignant tumors in the world. The clinical benefit of anti-angiogenic strategy as a single drug is limited. Some studies showed that the combination of anti-angiogenic therapy and chemotherapy exhibited synergistic effect and reduced the side effects of chemotherapy drugs. We investigated the combined effects of these two types of drugs in gastric cancer cells in vitro and in vivo. Methodscell viability, migration, invasion, and apoptosis were evaluated by CCK-8, wound-healing, transwell, and Annexin V-FITC/PI assay, respectively. In vivo anti-cancer efficacy was tested for the cell proliferation and metastasis in cell line derived tumor xenograft (CDX) model and patient derived tumor xenografted (PDX) model based on Tg (fli-1: EGFP) zebrafish embryos; ResultsIn the cell experiments, the combination of the two types of drugs could inhibit the proliferation and metastasis of gastric cancer cells and promote apoptosis through VEGFR-2/AKT/ERK1/2 signal. In the zebrafish CDX (zCDX) model and zebrafish PDX (zPDX) model, the combination of the two treatment also showed a synergistic effect in inhibiting gastric cancer cell metastasis and cell proliferation. ConclusionsApatinib/ramucirumab targeted therapy combined with docetaxel or 5-fluorouracil (5-FU) may serve as an effective treatment strategy for patients with advanced gastric cancer.