Cancer Cell Line-derived Xenografts Research Articles

Targeting undruggable phosphatase overcomes trastuzumab resistance by inhibiting multi-oncogenic kinases

AimsResistance to targeted therapy is one of the critical obstacles in cancer management. Resistance to trastuzumab frequently develops in the treatment for HER2+ cancers. The role of protein tyrosine phosphatases (PTPs) in trastuzumab resistance is not well understood. In this study, we aim to identify pivotal PTPs affecting trastuzumab resistance and devise a novel counteracting strategy. MethodsFour public datasets were used to screen PTP candidates in relation to trastuzumab responsiveness in HER2+ breast cancer. Tyrosine kinase (TK) arrays were used to identify kinases that linked to protein tyrosine phosphate receptor type O (PTPRO)-enhanced trastuzumab sensitivity. The efficacy of small activating RNA (saRNA) in trastuzumab-conjugated silica nanoparticles was tested for PTPRO upregulation and resistance mitigation in cell models, a transgenic mouse model, and human cancer cell line-derived xenograft models. ResultsPTPRO was identified as the key PTP which influences trastuzumab responsiveness and patient survival. PTPRO de-phosphorated several TKs, including the previously overlooked substrate ERBB3, thereby inhibiting multiple oncogenic pathways associated with drug resistance. Notably, PTPRO, previously deemed “undruggable,” was effectively upregulated by saRNA-loaded nanoparticles. The upregulated PTPRO simultaneously inhibited ERBB3, ERBB2, and downstream SRC signaling pathways, thereby counteracting trastuzumab resistance. ConclusionsAntibody-conjugated saRNA represents an innovative approach for targeting “undruggable” PTPs.

Abstract PR001: KIF18A inhibition, via ATX020, leads to mitotic arrest and robust anti-tumor activity through a synthetic lethal interaction with chromosome instability

Abstract KIF18A is a plus-end directed kinesin known to play a role in mitosis by facilitating chromosome alignment and spindle microtubule dynamics. Knockdown or knockout of KIF18A has been shown to inhibit proliferation in cells with whole genome doubling or aneuploidy, cellular states characterized by chromosomal rearrangements. These alterations render cells particularly vulnerable to disrupted mitosis upon KIF18A loss. As such, KIF18A is a compelling synthetic lethality target in a subset of tumors with chromosomal instability (CIN), which have ongoing chromosomal segregation defects. We have identified potent and selective small molecule inhibitors of KIF18A. A proprietary tool compound, ATX020, inhibits KIF18A ATPase activity with an IC50 of 0.014 µM; KIF18A is selectively inhibited over other kinesins including CENPE (IC50 > 10 µM) and EG5 (IC50 5.87 µM). ATX020 exhibits robust cellular activity, with anti-proliferative activity observed in chromosomally instable ovarian cancer cell lines such as OVCAR-3 (IC50 0.053 µM) and OVCAR-8 (IC50 0.54 µM). Upon treatment with ATX020, Annexin V is robustly induced in sensitive cell lines, demonstrating that KIF18A-dependent cell lines undergo apoptosis upon KIF18A inhibition. In KIF18Ai sensitive cells, a dose-dependent upregulation of phospho-Histone H3 and gamma-H2AX is observed upon ATX020 treatment in OVCAR-3 and OVCAR-8 cells, consistent with the induction of mitotic arrest and DNA damage. In these sensitive cell lines, ATX020 induces a dose-dependent cell cycle arrest in G2/M, consistent with the role of KIF18A in facilitating mitosis. OVCAR-3 cells treated with ATX020 exhibit fragmented nuclei and malformed mitotic spindles. In contrast, CIN negative Ovarian cancer cells that are insensitive to KIF18A loss, such as A2780 and OVK18, proliferate normally upon ATX020 treatment, cell division is unaffected, and no induction of Annexin V, phospho-Histone H3, and gamma-H2AX is observed. This selective dependency is also observed in vivo, where administration of ATX020 leads to dose-dependent tumor growth inhibition in an OVCAR-3 cancer cell line-derived xenograft model, with significant tumor regression observed at 100 mpk/day. In contrast, OVK18 tumors are unaffected by ATX020 administration, as expected based on the lack of cell cycle and proliferation effects in that cell line. Together, these data demonstrate a critical role for KIF18A in facilitating mitosis in a subset of chromosomally instable cells. These effects are selective, with KIF18A-independent cell lines proceeding through the cell cycle normally in the presence of inhibitors. Small molecule inhibition of KIF18A is therefore expected to have robust efficacy in solid tumors with high levels of chromosomal instability such as Ovarian cancer. Citation Format: Maureen S. Lynes, Laura Ghisolfi, Sanjoy Khan, Taylor Hotz, Brian A. Sparling, Mary-Margaret Zablocki, Deepali Gotur, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Stephen J. Blakemore, Serena J. Silver, Robert A. Copeland. KIF18A inhibition, via ATX020, leads to mitotic arrest and robust anti-tumor activity through a synthetic lethal interaction with chromosome instability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR001.

Sensitivity of magnetic resonance elastography to extracellular matrix and cell motility in human prostate cancer cell line-derived xenograft models

Prostate cancer (PCa) is a significant health problem in the male population of the Western world. Magnetic resonance elastography (MRE), an emerging medical imaging technique sensitive to mechanical properties of biological tissues, detects PCa based on abnormally high stiffness and viscosity values. Yet, the origin of these changes in tissue properties and how they correlate with histopathological markers and tumor aggressiveness are largely unknown, hindering the use of tumor biomechanical properties for establishing a noninvasive PCa staging system.To infer the contributions of extracellular matrix (ECM) components and cell motility, we investigated fresh tissue specimens from two PCa xenograft mouse models, PC3 and LNCaP, using magnetic resonance elastography (MRE), diffusion-weighted imaging (DWI), quantitative histology, and nuclear shape analysis. Increased tumor stiffness and impaired water diffusion were observed to be associated with collagen and elastin accumulation and decreased cell motility. Overall, LNCaP, while more representative of clinical PCa than PC3, accumulated fewer ECM components, induced less restriction of water diffusion, and exhibited increased cell motility, resulting in overall softer and less viscous properties. Taken together, our results suggest that prostate tumor stiffness increases with ECM accumulation and cell adhesion - characteristics that influence critical biological processes of cancer development.MRE paired with DWI provides a powerful set of imaging markers that can potentially predict prostate tumor development from benign masses to aggressive malignancies in patients. Statement of significanceXenograft models of human prostate tumor cell lines, allowing correlation of microstructure-sensitive biophysical imaging parameters with quantitative histological methods, can be investigated to identify hallmarks of cancer.

Abstract 6579: DS-3939a, a novel TA-MUC1-targeting antibody-drug conjugate (ADC) with a DNA topoisomerase I inhibitor DXd, exhibits potent antitumor activity in preclinical models

Abstract Background: Tumor-associated mucin-1 (TA-MUC1) is a tumor-specific transmembrane glycoprotein with aberrant glycosylation. It is highly expressed in various human epithelial cancers, making it an attractive target for cancer therapy. DS-3939a is a TA-MUC1 targeting antibody-drug conjugate (ADC) structurally composed of a humanized anti-TA-MUC1 antibody, an enzymatically cleavable tetrapeptide-based linker, and a DNA topoisomerase I inhibitor (DXd) with a high drug-to-antibody ratio (DAR). In this study, the pharmacological activity of DS-3939a was evaluated using preclinical in vitro and in vivo models. Methods: TA-MUC1 expression was assessed by immunohistochemistry and flow cytometry. Binding specificity of DS-3939a was confirmed by enzyme-linked immunosorbent assay. Induction of DNA damage and apoptosis to cancer cells by DS-3939a treatment were detected by Simple Western System. In vitro cell growth inhibitory and in vivo antitumor activities of DS-3939a were evaluated using various cancer cell lines and patient-derived xenograft (PDX) models. Results: Many specimens across multiple cancer types showed positive staining for TA-MUC1, with a particularly high positive rate observed in bladder, lung, and breast cancers. DS-3939a specifically bound to aberrantly glycosylated MUC1 peptides by recognizing both the glycan and backbone peptide moiety of MUC1. In vitro, DS-3939a inhibited the growth of TA-MUC1-positive cancer cells but not of TA-MUC1-negative cancer cells. Neither a parental anti-TA-MUC1 antibody nor isotype control ADC inhibited the growth of these cells. In addition, treatment with DS-3939a and DXd alone induced DNA damage and apoptosis in cancer cells. These results demonstrate that DS-3939a exhibits TA-MUC1-dependent cell growth inhibition through DXd-mediated cytotoxic effect. Moreover, DS-3939a exhibited significant antitumor effects in the several TA-MUC1-positive cancer cell line-derived xenograft models and various PDX models. DS-3939a also induced strong tumor regression even after treatment of other cytotoxic ADCs in several xenograft models. Conclusion: Based on these preclinical results, DS-3939a could provide valuable therapy with potential benefits for patients with TA-MUC1-expressing tumors. A first-in-human phase 1/2 study in patients with advanced solid tumors is currently in progress (NCT05875168). Citation Format: Mayuko Yukiura, Kohei Takano, Kazuki Takahashi, Akiko Zembutsu, Michiko Kitamura, Yoshinobu Shiose, Kokichi Honda, Kazunori Oyama, Wataru Obuchi, Makiko Yamada, Riki Goto, Ken Sakurai, Kazuyoshi Kumagai, Takashi Kagari, Yuki Abe, Toshinori Agatsuma. DS-3939a, a novel TA-MUC1-targeting antibody-drug conjugate (ADC) with a DNA topoisomerase I inhibitor DXd, exhibits potent antitumor activity in preclinical models [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 6579.

Abstract 3337: Inhibition of KIF18A leads to mitotic arrest and robust anti-tumor activity in chromosomally instable tumors

Abstract KIF18A is a plus-end directed kinesin known to play a role in mitosis by facilitating chromosome alignment and spindle microtubule dynamics. Knockdown or knockout of KIF18A has been shown to inhibit proliferation in cells with whole genome doubling or aneuploidy, cellular states characterized by chromosomal rearrangements. These alterations render cells particularly vulnerable to disrupted mitosis upon KIF18A loss. As such, KIF18A is a compelling target in a subset of tumors with chromosomal instability (CIN), which have ongoing chromosomal segregation defects. We have identified potent and selective small molecule inhibitors of KIF18A. A proprietary tool compound, ATX-21020, inhibits KIF18A ATPase activity with an IC50 of 14.5 nM; KIF18A is selectively inhibited over other kinesins including CENPE (IC50 > 10 µM) and EG5 (IC50 5.87 µM). ATX-21020 exhibits robust cellular activity, with anti-proliferative activity observed in chromosomally instable ovarian cancer cell lines such as OVCAR-3 (IC50 53.3 nM) and OVCAR-8 (IC50 0.54 µM). Upon treatment with ATX-21020, Annexin V is robustly induced in sensitive cell lines, demonstrating that KIF18A-dependent cell lines undergo apoptosis upon KIF18A inhibition. In KIF18Ai sensitive cells, a dose-dependent upregulation of phospho-Histone H3 and gamma-H2AX is observed upon ATX-21020 treatment in OVCAR-3 and OVCAR-8 cells, consistent with the induction of mitotic arrest and DNA damage. In contrast, CIN negative Ovarian cancer cells that are insensitive to KIF18A loss, such as A2780 and OVK18, proliferate normally upon ATX-21020 treatment, and Annexin V, phospho-Histone H3, and gamma-H2AX are not induced. In sensitive cell lines, ATX-21020 induces a dose-dependent cell cycle arrest in G2/M, consistent with the role of KIF18A in facilitating mitosis. OVCAR-3 cells treated with ATX-21020 exhibit fragmented nuclei and malformed mitotic spindles, in contrast to A2780 cells where cell division is unaffected, suggesting that KIF18A is dispensable in CIN negative cells. This selective dependency is also observed in vivo, where administration of ATX-21020 leads to dose-dependent tumor growth inhibition in an OVCAR-3 cancer cell line-derived xenograft model, with significant tumor regression observed at 100 mpk/day. In contrast, OVK18 tumors are unaffected by ATX-21020 administration, as expected based on the lack of cell cycle and proliferation effects in that cell line. Together, these data demonstrate a critical role for KIF18A in facilitating mitosis in a subset of chromosomally instable cells. These effects are selective, with KIF18A-independent cell lines proceeding through the cell cycle normally in the presence of inhibitors. Small molecule inhibition of KIF18A is therefore expected to have robust efficacy in solid tumors with high levels of chromosomal instability such as Ovarian cancer. Citation Format: Maureen S. Lynes, Sanjoy Khan, Taylor Hotz, Brian A. Sparling, Mary-Margaret Zablocki, Deepali Gotur, P Ann Boriack-Sjodin, Kenneth W. Duncan, Stephen J. Blakemore, Serena J. Silver, Robert A. Copeland. Inhibition of KIF18A leads to mitotic arrest and robust anti-tumor activity in chromosomally instable tumors [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 3337.

Fucosyltransferase 3 and 8 promote the metastatic capacity of cancer stem-like cells via CD15s and E-cadherin in esophageal cancer.

Esophageal cancer stem cells (ECSCs) have been identified as the subset of cells within esophageal squamous cell carcinoma that possess tumorigenic, invasive, and metastatic properties. One important aspect of cancer metastasis is the binding of sialyl-Lewis X (CD15s) with E- or P-selectin, which facilitates the adhesion and migration of cancer cells to distant sites. This study was conducted to investigate the impact of fucosylation processes on the metastatic behavior of ECSCs. The esophageal cancer cell line (KYSE-30) was cultured and divided into control and 2F-peracetyl fucose (2F-PerAcFuc) treated groups. Spheres were harvested from these cultures. Cell invasion assay and qPCR were conducted to examine migration and marker expression in both groups. Cancer cell line-derived xenografts were established in nude mice to validate findings in vivo. Our results initially indicated that the addition of 2F-PerAcFuc, an inhibitor of fucosylation, resulted in the down-regulation of the Fut3/CD15s pathway in both cancer stem-like cells and the xenograft model. Measurements of subcutaneous xenograft tumor volume revealed a significant decrease in tumor size among nude mice after treatment with 2F-PerAcFuc. Additionally, a reduction in Fut8/E-cadherin levels was observed in the xenograft model of nude mice. Furthermore, the administration of 2F-PerAcFuc lowered the levels of fucosylated glycoconjugates in nude mice. Our data suggest that inhibition of fucosyltransferase 3 and 8 can reduce the metastatic capacity of cancer stem-like cells by down-regulating CD15s and E-cadherin in a mouse model of esophageal cancer.

Abstract C034: First in Human phase 1/2a trial of a macrocyclic ATR inhibitor (ATRN-119) in patients with advanced solid tumors

Abstract Background: ATRN-119 is an oral and highly selective nanomolar inhibitor of Ataxia Telangiectasia and Rad3- related (ATR) kinase, an enzyme that is activated by replication stress and DNA damage and prevents the collapse of DNA replication forks into double strand breaks. ATRN-119 is being studied in advanced solid tumors with dysregulated components of the DNA damage response (DDR) based on the hypothesis that these tumors will be more susceptible to ATR inhibition. Single agent ATRN-119 demonstrates increased cytotoxicity in vitro against a broad spectrum of human colon, breast, pancreatic, sarcoma, ovarian and prostate cancer cell lines that harbor genomic alterations of DDR genes. In vivo, ATRN-119 has demonstrated tumor growth inhibition in human castration-resistant prostate cancer and human colon cancer cell line-derived xenografts (CDX) mice models, and in BRCA mutant high grade serous ovarian cancer patient derived xenografts (PDXs). Trial design: This is a first-in-human Phase 1/2a open-label study (NCT04905914) to evaluate safety, tolerability, pharmacokinetics (PK), and preliminary e!cacy of oral daily ATRN-119 in subjects with advanced solid tumors, conducted in 2 parts. Part 1 consists of a 3+3 dose escalation design at up to 6 dose levels to evaluate the tolerability, PK, dose limiting toxicity (DLT) and maximum tolerated dose (MTD) of repeated daily doses of ATRN-119. Enrollment in Part 1 is ongoing. Subjects must be ≥12 years old with advanced solid tumor, have documented DDR mutations, or advanced Merkel cell carcinoma, must have failed at least one prior standard treatment, have ECOG performance status of 0 or 1, with a life expectancy > 3 months. Primary objective is safety/tolerability and secondary objective is efficacy. After determination of the MTD or maximum feasible dose (MFD) in Part 1, Part 2 will enroll up to 30 additional subjects. The trial is currently recruiting across 4 locations in the United States. Clinical trial identification: ClinicalTrials.gov Identifier: NCT04905914 Citation Format: Fiona Simpkins, Reva Schneider, Amit Mahipal, Patricia LoRusso, Crystal Miller, Eric Brown, Mike Carleton, Joachim Gullbo, Nadeem Q Mirza. First in Human phase 1/2a trial of a macrocyclic ATR inhibitor (ATRN-119) in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C034.

Abstract 5501: Vertical inhibition of the MAPK pathway with D3S-002, a potent and selective ERK1/2 inhibitor, to improve anti-tumor activity of and overcome resistance to KRAS G12C inhibitors

Abstract While covalent KRAS G12C inhibitors have demonstrated encouraging clinical efficacy in patients with KRAS G12C mutation, the overall response rate and progression-free survival appear to be suboptimal. Achieving deeper and more durable responses in these patients remain as major challenges. Preclinical and clinical translation research highlights adaptive feedback reactivation of the RAS-RAF-MEK-ERK signaling cascade as key molecular mechanisms driving primary and acquired resistance to KRAS G12C inhibitors. Concurrent inhibition of KRAS G12C and downstream MAPK pathway is thus an attractive combination strategy to improve clinical outcomes for KRAS G12C-mutant population. D3S-002 is a potent and selective kinase inhibitor targeting ERK1/2, the final node of the RAS-MAPK pathway and the central mediator of the feedback activation loop. To investigate the combination effect of D3S-002 with KRAS G12C inhibitor D3S-001, this study utilized multiple preclinical in vivo models including cancer cell line-derived xenograft (CDX) tumors that are sensitive to, or primarily resistant to KRAS G12C inhibitors, and patient-derived xenograft (PDX) tumors that have progressed on KRAS G12C inhibitor treatment in the clinic. In NCI-H358 NSCLC and SW837 CRC models that are sensitive to KRAS G12C inhibitor monotherapy, adding D3S-002 to D3S-001 or to D3S-001 and cetuximab doublet regimen led to faster, deeper, and more durable anti-tumor responses at dose levels that are well tolerated in mice with no body weight loss observed. In NCI-H2122 NSCLC model that is primarily resistant to KRAS G12C inhibitor alone, the addition of D3S-002 resulted in near complete tumor growth inhibition. Furthermore, CR9537 PDX model was established from tumor biopsy of a CRC patient who had progressed on combination treatment of KRAS G12C inhibitor MRTX849 and a SHP2 inhibitor in a clinical trial. Next generation sequencing (NGS) analysis identified secondary KRAS Q61H mutation as an acquired resistance mechanism. In this PDX model, KRAS G12C inhibitor, D3S-001 or MRTX849, monotherapy only achieved approximately 50% tumor growth inhibition, highlighting the acquired resistant phenotype. Adding D3S-002 to D3S-001 or MRTX849 resulted in over 80% tumor growth inhibition and significantly prolonged survival, suggesting the combination treatment can be beneficial for KRAS G12C inhibitor pre-treated patient population. Collectively, vertical inhibition by concurrently targeting KRAS G12C and ERK1/2 with respective inhibitor D3S-001 and D3S-002 demonstrated enhanced anti-tumor activity in NSCLC and CRC models with different sensitivity to KRAS G12C inhibitor monotherapy. These results provide a strong rationale for further investigation of the combination strategy of D3S-001 and D3S-002 in KRAS G12C-mutant solid tumors in the clinic. Citation Format: Jing Zhang, Jingtao Lu, Zhiqiang Zheng, Jiang Lu, Allison Wang, Jia Wang, Janet Chen, Haopeng Rui, Cheng Chen, Zhi Jian Chen. Vertical inhibition of the MAPK pathway with D3S-002, a potent and selective ERK1/2 inhibitor, to improve anti-tumor activity of and overcome resistance to KRAS G12C inhibitors. [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 5501.

Abstract 6276: SKL27969, an oral selective PRMT5 inhibitor, sensitizes the effect of DNA-damaging agents in preclinical models of multiple solid tumors

Abstract Background: Protein arginine methyltransferase 5 (PRMT5) is overexpressed in various human malignancies and induces epigenetic and post-translational changes, which have a significant impact on cell growth, proliferation, apoptosis, and DNA damage repair. SKL27969 is a potent and selective brain-penetrating PRMT5 inhibitor demonstrating antitumor activity in the preclinical models of glioblastoma (GBM) and non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC) brain metastasis models. In this study, we evaluated the antitumor activity of SKL27969 alone or in combination with DNA-damaging agents in various cancer models. Methods: Cell cycle analysis, apoptosis, proliferation, and invasion assays were conducted to determine in vitro profiles of SKL27969. In vivo efficacy of SKL27969 was evaluated in human cancer cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) models. Immunohistochemistry and RNA-sequencing analysis in tumor samples from animal models were conducted to confirm the in vivo mechanism of action (MoA). In vitro synergistic effects of SKL27969 with combination partner drugs were analyzed using Combenefit software. Results: SKL27969 exhibits strong anti-proliferative effect by inducing cell cycle arrest and apoptosis and also inhibits invasion of cancer cells. An evaluation of SKL27969 in a panel of over 100 cancer cell lines revealed a broad spectrum of anti-proliferative activity in various cancer types, and an especially more sensitive response in cell lines harboring genetic alterations in the mitotic DNA damage checkpoint, DNA repair, or RNA processing pathways. Administration of SKL27969 demonstrated significant tumor growth inhibition, with a decrease in tumor symmetric dimethylarginine (SDMA) in NSCLC and TNBC subcutaneous xenograft models. Transcriptome and immunohistochemical analysis of SKL27969-treated tumors revealed that the cell cycle checkpoint pathways and DNA damage repair genes were downregulated, supporting the rationale for combination treatment with DNA-damaging agents such as chemotherapeutic agents. Robust antitumor effects were observed after treatment with SKL27969 in combination with paclitaxel or gemcitabine in NSCLC PDX or pancreatic cancer CDX models. Further ex vivo studies to confirm the in vivo MoA of this synergism, and in vivo combination studies of SKL27969 with standard chemotherapeutic agents in other solid cancer CDX models, are ongoing. Conclusion: This is the first preclinical study that suggests the therapeutic potential of SKL27969 in combination with currently approved DNA-damaging agents, and supports further clinical investigation of SKL27969’s role in improving the therapeutic benefit of standard therapies. Citation Format: Mijin Moon, Yongje Shin, Soyoung Ki, Jungtae Na, Ho Yeon Lee, Ilkyoo Koh, Beomjin Hong, Jiyeon Yun, Janice Laramy, Vijaykumar Vashi, Sook-Kyung Park. SKL27969, an oral selective PRMT5 inhibitor, sensitizes the effect of DNA-damaging agents in preclinical models of multiple solid tumors. [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 6276.

Targeting tumor vasculature to improve antitumor activity of T cells armed ex vivo with T cell engaging bispecific antibody

BackgroundSuccess of T cell immunotherapy hinges on the tumor microenvironment (TME), and abnormal tumor vasculature is a hallmark of most solid tumors and associated with immune evasion. The efficacy of...

Construction of a Humanized PBMC-PDX Model to Study the Efficacy of a Bacterial Marker in Lung Cancer Immunotherapy.

Commensal microbiome is a key factor of lung cancer immunotherapy efficacy. Elucidating the role of specific strains as bacterial markers in immunotherapy has drawn great attention from the academia. At present, most preclinical studies about the relationship between bacterial markers and immunotherapy rely on the syngeneic mouse models. However, mice differ greatly from humans in immune system and tumor characteristics. In this study, humanized mouse models based on peripheral blood mononuclear cells (PBMCs) immune reconstitution and lung cancer cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) were constructed. The PBMC-PDX model was shown to be superior to the PBMC-CDX model in preserving tumor heterogeneity and construction time-saving. Through optimizing the experimental process, the time it took for humanized models to evaluate the effect of cancer treatment was reduced to 42 days. Next, by utilizing PBMC-PDX mice treated with antibiotics (ATB), the role of Bifidobacterium longum in lung cancer immunotherapy was studied. It was found that although both Bifidobacterium longum and immunotherapy drug pembrolizumab alone showed suppressing tumor growth, the efficacy of pembrolizumab was attenuated when administrated to mice colonized with Bifidobacterium longum. Further exploration revealed that Bifidobacterium longum caused significant changes in the proportion of human CD45+ cells in the PBMC-PDX model. The PBMC-PDX model has the potential to be applied as an efficient platform to support evaluation of bacterial markers in immunotherapy research and facilitate development of precision medicine targeting human commensal bacteria.

A proof of concept for targeting the PrPC - Amyloid β peptide interaction in basal prostate cancer and mesenchymal colon cancer

The cellular prion protein PrPC partners with caveolin-1 (CAV1) in neurodegenerative diseases but whether this interplay occurs in cancer has never been investigated. By leveraging patient and cell line datasets, we uncover a molecular link between PrPC and CAV1 across cancer. Using cell-based assays, we show that PrPC regulates the expression of and interacts with CAV1. PrPC additionally controls the expression of the amyloid precursor protein APP and of the Aβ generating enzyme BACE1, and regulates the levels of Aβ, whose accumulation is a central event in Alzheimer’s disease. We further identify DKK1 and DKK3, involved in both Alzheimer’s disease and cancer progression, as targets of the PrPC-dependent axis. Finally, we establish that antibody-mediated blocking of the Aβ-PrPC interaction delays the growth of prostate cancer cell line-derived xenografts and prevents the development of metastases. Our data additionally support an enrichment of the Aβ-PrPC-dependent pathway in the basal subtype of prostate cancer, associated with anti-hormonal therapy resistance, and in mesenchymal colon cancer, associated with poor prognosis. Thus, based on a parallel with neurodegenerative diseases, our results bring to light an Aβ-PrPC axis and support the potential of targeting this pathway in patients with selected subtypes of prostate and colon cancer.

Abstract 330: Targeting B7-H3 in prostate cancer: Preclinical proof of concept with MGC018, an investigational anti-B7-H3 antibody-drug conjugate

Abstract Introduction: Prostate cancer is the second most common cancer among men worldwide. In 2021, it is estimated that 248,530 men in the United States will be diagnosed with prostate cancer, and 34,130 will die from the disease. Although current treatments have success initially, development of resistance commonly leads to recurrence of an incurable castrate-resistant form of the disease. Thus, significant need for novel therapies to improve the outcome of castrate-resistant prostate cancer remains. B7-H3 (CD276), a member of the B7 family of immunomodulatory molecules, is overexpressed in primary and metastatic prostate cancer, and correlates with disease severity and poor clinical outcome. MGC018, a duocarmycin-based B7-H3 antibody-drug conjugate, is currently being evaluated in clinical studies. Here, MGC018 was explored preclinically to assess the potential for targeting B7-H3 in prostate cancer. Methods: Immunohistochemistry studies were performed to define the expression of B7-H3 in prostate cancer tissue microarrays (TMA). In vivo efficacy studies were conducted with human prostate cancer cell line-derived xenograft (CDX) models to explore the antitumor activity of MGC018 as a single agent and in combination with Poly (ADP-ribose) polymerase (PARP) and androgen receptor (AR) inhibitors. Based on the results in the CDX studies, in vivo efficacy studies were extended to a panel of metastatic prostate cancer patient-derived xenograft (PDX) models, which exhibit heterogenous expression of B7-H3 and more closely mimic the biological characteristics of patient tumors. Results: Staining of prostate tumor TMAs revealed high expression of B7-H3 in primary and metastatic prostate cancer. Of the prostate samples evaluated, 95% (38/40) of the tumor samples were positive for B7-H3 (H-score ≥ 20): 65% had H-scores greater than 200, while 20% and 10% had H-scores between 101-200 and 1-100, respectively. MGC018 demonstrated in vitro cytotoxicity toward B7-H3-positive human prostate cancer cell lines. The in vitro cytotoxicity translated to potent antitumor activity in vivo toward prostate cancer CDX models, and the antitumor activity of MGC018 was enhanced when combined with inhibitors of PARP or AR. In PDX models of metastatic prostate cancer, MGC018 was active as a single agent toward heterogeneous B7-H3-expressing tumors, and combining MGC018 with inhibitors of PARP or AR led to a greater response in some models. Conclusion: B7-H3 is frequently overexpressed in prostate cancer. MGC018 demonstrated potent antitumor activity in vivo toward CDX and PDX models of prostate cancer, and enhanced antitumor activity when combined with inhibitors of PARP or AR. These results support prostate cancer as an indication that may be responsive to ADC-based treatments directed toward B7-H3. MGC018 is being investigated in metastatic prostate cancer in a Phase 1/2 clinical study. Citation Format: Juniper A. Scribner, Francine Z. Chen, Anushka De Costa, Ying Li, Michael Chiechi, Thomas Son, Jeff Hooley, Jonathan Li, Scott Koenig, Chet Bohac, Ezio Bonvini, Paul A. Moore, Deryk Loo. Targeting B7-H3 in prostate cancer: Preclinical proof of concept with MGC018, an investigational anti-B7-H3 antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 330.

Abstract 2126: Therapeutic potential of CPI-0209

Abstract Second-generation EZH2 inhibitor CPI-0209 has therapeutic potential for androgen receptor-dependent prostate cancer Rentian Wu, Feng Zhao, Patricia J. Keller, Jennifer A. Mertz, John P. McGrath, Barbara Bryant, Andrew R. Conery, Kaiming Sun, Jing Wang, and Patrick Trojer Post-translational modification of histones plays critical roles in epigenetic regulation of gene expression. Enhancer of Zeste Homolog 2 (EZH2), a subunit of Polycomb Repressive Complex 2 (PRC2), catalyzes trimethylation of histone H3 on lysine 27 (H3K27me3). Chromatin methylation by EHZ2 promotes chromatin architectural changes and silencing of gene expression. EZH2-mediated gene silencing was shown to contribute to tumor initiation and progression. Amplification and overexpression of EZH2 along with down regulation of its target genes correlate with treatment resistance and poor prognosis in various tumor types including metastatic castration resistant prostate cancer (mCRPC). While most mCRPC patients are initially responsive to agents targeting the androgen receptor (AR) signaling pathway, tumors eventually develop resistance and progress. Other than EZH2 overexpression, mechanisms of resistance involve AR alterations, including AR amplification, alteration of AR splice variants which constitutively activate ARS, and AR mutations that affect native ligand-binding specificity. CPI-0209 is an orally bioavailable and selective EZH2 and EZH1 inhibitor with highly enhanced potency and extended residence time compared to first-generation EZH2 inhibitors. CPI-0209 is currently being clinically developed as monotherapy and in combination with other cancer therapeutic agents [NCT04104776]. Here we demonstrated that AR-dependent prostate cancer cell lines including LNCaP, 22Rv1 and VCaP were sensitive to CPI-0209 treatment, while AR-independent cell lines like PC3 and DU145 were insensitive to CPI-0209. Sensitivity to CPI-0209 were also observed in AR-dependent cell line-derived xenografts (CDX) and patient-derived xenografts (PDX) models. In AR-dependent cells, CPI-0209 synergized with enzalutamide and overcame the anti-androgens resistance induced by AR alterations. In prostate cancer CDX and PDX models, the combination treatment of CPI-0209 and enzalutamide showed greater tumor growth inhibition compared to enzalutamide monotherapy. Transcriptomic analysis revealed that CPI-0209 treatment modulated both AR-related and AR-independent pathways, which reveals a potential mechanism to explain the synergy of CPI-0209 and AR inhibitors. Thus, our results indicate that CPI-0209 may have the potential to be effective for AR-dependent mCRPC that are resistant to current AR inhibitors, supporting potential clinical development in mCRPC. Keywords: EZH2, prostate cancer, epigenetics, resistant Citation Format: Rentian Wu, Feng Zhao, Patricia J. Keller, Jennifer A. Mertz, John P. McGrath, Barbara Bryant, Andrew R. Conery, Kaiming Sun, Jing Wang, Patrick Trojer. Therapeutic potential of CPI-0209 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2126.

Identification of ferroptosis as a novel mechanism for antitumor activity of natural product derivative a2 in gastric cancer

Ferroptosis is a type of cell death accompanied by iron-dependent lipid peroxidation, thus stimulating ferroptosis may be a potential strategy for treating gastric cancer, therapeutic agents against which are urgently required. Jiyuan oridonin A (JDA) is a natural compound isolated from Jiyuan Rabdosia rubescens with anti-tumor activity, unclear anti-tumor mechanisms and limited water solubility hamper its clinical application. Here, we showed a2, a new JDA derivative, inhibited the growth of gastric cancer cells. Subsequently, we discovered for the first time that a2 induced ferroptosis. Importantly, compound a2 decreased GPX4 expression and overexpressing GPX4 antagonized the anti-proliferative activity of a2. Furthermore, we demonstrated that a2 caused ferrous iron accumulation through the autophagy pathway, prevention of which rescued a2 induced ferrous iron elevation and cell growth inhibition. Moreover, a2 exhibited more potent anti-cancer activity than 5-fluorouracil in gastric cancer cell line-derived xenograft mice models. Patient-derived tumor xenograft models from different patients displayed varied sensitivity to a2, and GPX4 downregulation indicated the sensitivity of tumors to a2. Finally, a2 exhibited well pharmacokinetic characteristics. Overall, our data suggest that inducing ferroptosis is the major mechanism mediating anti-tumor activity of a2, and a2 will hopefully serve as a promising compound for gastric cancer treatment.

The antitumor capacity of mesothelin-CAR-T cells in targeting solid tumors in mice

Since the approval of chimeric antigen receptor (CAR) T cell therapy targeting CD19 by the FDA, CAR-T cell therapy has received increasing attention as a new method for targeting tumors. Although CAR-T cell therapy has a good effect against hematological malignancies, it has been less effective against solid tumors. In the present study, we selected mesothelin (MSLN/MESO) as a target for CAR-T cells because it is highly expressed by solid tumors but only expressed at low levels by normal tissues. We engineered a third generation MSLN-CAR comprising a single-chain variable fragment (scFv) targeting MSLN (MSLN-scFv), a CD8 transmembrane domain, the costimulatory domains from CD28 and 4-1BB, and the activating domain CD3ζ. In vitro, MSLN-CAR-T cells killed various solid tumor cell lines, demonstrating that it could specifically kill MSLN-positive cells and release cytokines. In vivo, we investigated the effects of MSLN-CAR-T cell therapy against ovarian, breast, and colorectal cancer cell-line-derived xenografts (CDX) and MSLN-positive colorectal and gastric cancer patient-derived xenografts (PDX). MSLN-CAR decreased the growth of MSLN-positive tumors concomitant with significantly increased T cells and cytokine levels compared to the control group. These results indicated that modified MSLN-CAR-T cells could be a promising therapeutic approach for solid tumors.

Rhenium N-heterocyclic carbene complexes block growth of aggressive cancers by inhibiting FGFR- and SRC-mediated signalling

BackgroundPlatinum-based anticancer drugs have been at the frontline of cancer therapy for the last 40 years, and are used in more than half of all treatments for different cancer types. However, they are not universally effective, and patients often suffer severe side effects because of their lack of cellular selectivity. There is therefore a compelling need to investigate the anticancer activity of alternative metal complexes. Here we describe the potential anticancer activity of rhenium-based complexes with preclinical efficacy in different types of solid malignancies.MethodsKinase profile assay of rhenium complexes. Toxicology studies using zebrafish. Analysis of the growth of pancreatic cancer cell line-derived xenografts generated in zebrafish and in mice upon exposure to rhenium compounds.ResultsWe describe rhenium complexes which block cancer proliferation in vitro by inhibiting the signalling cascade induced by FGFR and Src. Initially, we tested the toxicity of rhenium complexes in vivo using a zebrafish model and identified one compound that displays anticancer activity with low toxicity even in the high micromolar range. Notably, the rhenium complex has anticancer activity in very aggressive cancers such as pancreatic ductal adenocarcinoma and neuroblastoma. We demonstrate the potential efficacy of this complex via a significant reduction in cancer growth in mouse xenografts.ConclusionsOur findings provide a basis for the development of rhenium-based chemotherapy agents with enhanced selectivity and limited side effects compared to standard platinum-based drugs.

Π electron-stabilized polymeric micelles potentiate docetaxel therapy in advanced-stage gastrointestinal cancer

Gastrointestinal (GI) cancers are among the most lethal malignancies. The treatment of advanced-stage GI cancer involves standard chemotherapeutic drugs, such as docetaxel, as well as targeted therapeutics and immunomodulatory agents, all of which are only moderately effective. We here show that Π electron-stabilized polymeric micelles based on PEG-b-p(HPMAm-Bz) can be loaded highly efficiently with docetaxel (loading capacity up to 23 wt%) and potentiate chemotherapy responses in multiple advanced-stage GI cancer mouse models. Complete cures and full tumor regression were achieved upon intravenously administering micellar docetaxel in subcutaneous gastric cancer cell line-derived xenografts (CDX), as well as in CDX models with intraperitoneal and lung metastases. Nanoformulated docetaxel also outperformed conventional docetaxel in a patient-derived xenograft (PDX) model, doubling the extent of tumor growth inhibition. Furthermore, micellar docetaxel modulated the tumor immune microenvironment in CDX and PDX tumors, increasing the ratio between M1-and M2-like macrophages, and toxicologically, it was found to be very well-tolerated. These findings demonstrate that Π electron-stabilized polymeric micelles loaded with docetaxel hold significant potential for the treatment of advanced-stage GI cancers.

Targeting epidermal growth factor-overexpressing triple-negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor.

ObjectivesTraditional cancer therapy and regular immunotherapy are ineffective for treating triple‐negative breast cancer (TNBC) patients. Recently, chimeric antigen receptor‐engineered natural killer cells (CAR NK) have been applied to target several hormone receptors on different cancer cells to improve the efficacy of immunotherapy. Furthermore, epidermal growth factor receptor (EGFR) is a potential therapeutic target for TNBC. Here, we demonstrated that EGFR‐specific CAR NK cells (EGFR‐CAR NK cells) could be potentially used to treat patients with TNBC exhibiting enhanced EGFR expression.Materials and methodsWe investigated the cytotoxic effects of EGFR‐CAR NK cells against TNBC cells in vitro and in vivo. The two types of EGFR‐CAR NK cells were generated by transducing lentiviral vectors containing DNA sequences encoding the single‐chain variable fragment (scFv) regions of the two anti‐EGFR antibodies. The cytotoxic and anti‐tumor effects of the two cell types were examined by performing cytokine release and cytotoxicity assays in vitro, and tumor growth assays in breast cancer cell line‐derived xenograft (CLDX) and patient‐derived xenograft (PDX) mouse models.ResultsBoth EGFR‐CAR NK cell types were activated by TNBC cells exhibiting upregulated EGFR expression and specifically triggered the lysis of the TNBC cells in vitro. Furthermore, the two EGFR‐CAR NK cell types inhibited CLDX and PDX tumors in mice.ConclusionsThis study suggested that treatment with EGFR‐CAR NK cells could be a promising strategy for TNBC patients.

Faithful preclinical mouse models for better translation to bedside in the field of immuno-oncology.

The success of immunotherapy using immune checkpoint inhibitors has changed the practice of cancer treatment tremendously. However, there are still many clinical challenges, such as drug resistance, predictive biomarker development, exploration of combination therapies, and prediction of immune-related adverse events in preclinical settings. To overcome these problems, it is essential to establish faithful preclinical mouse models that recapitulate the clinical features, molecular genetics, biological heterogeneity, and immune microenvironment of human cancers. Here we review the advantages and disadvantages of current preclinical mouse models, including syngeneic murine tumor cell lines, autochthonous tumor models, cancer cell line-derived xenografts, patient-derived-xenografts, and various kinds of immunologically humanized mice. We discuss how these models should be characterized and applied in preclinical settings, and how we should prepare preclinical studies for successful translation from bench to bedside.