Positive Cell Lines Research Articles

HGG-48. THE BLOOD-BASED TELOMERIC DNA C-CIRCLE ASSAY CAN DETECT ALTERNATIVE LENGTHENING OF TELOMERES (ALT) IN PEDIATRIC HIGH-GRADE GLIOMAS

Abstract BACKGROUND High-grade gliomas (HGG) comprise ~10% of pediatric brain tumors; 40% of pediatric HGG use the alternative lengthening of telomeres (ALT) mechanism to maintain replicative immortality. ALT cancers share a unique biology providing potential therapeutic targets. Extrachromosomal telomere DNA repeats, termed C-circles, can be detected by a unique PCR assay, providing a sensitive and specific biomarker for ALT cancers. C-circles have been detected in ALT tumors and serum of ALT patients. We have adapted the C-circle assay (CCA) to provide a sensitive and specific assay with cfDNA in patient plasma. Here we determined if cfDNA in plasma can be used to detect patients with ALT-positive HGG. METHODS Frozen plasma samples were collected in Streck tubes at time of surgery prior to resection from patients with HGG. DNA was extracted and quantified. Isothermal rolling C-circle amplification and real-time telomere PCR was performed followed by analysis. The C-circle positive neuroblastoma cell line, CHLA-90, and C-circle negative neuroblastoma cell line, CHLA-20, served as the positive and negative controls respectively. CCA values in plasma of 2 and above indicate positivity for C-circles. RESULTS Plasma samples were analyzed by CCA from three pediatric patients whose brain tumors were previously biopsied and identified as ATRX mutation positive. ATRX mutation indicates ALT however, not all ALT cancers have ATRX mutations. All plasma samples were above the 2.0 cutoff at 3.37, 3.13, and 4.59 and thus classified as C-circle positive. CONCLUSIONS This preliminary cohort shows that the CCA has potential for identifying ALT pediatric brain tumors using cfDNA from plasma. As novel therapies effective against ALT HGG are developed, the plasma CCA may allow accelerated initiation of neoadjuvant therapy to shrink tumor prior to surgical resection and lessen or prevent the need for tumor biopsy. Expansion of the initial test cohort is underway. Supported by NCI UO1 CA63988.

Exploring Raloxifene-Based Metallodrugs: A Versatile Vector Combined with Platinum(II), Palladium(II) and Nickel(II) Dichlorides and Carborates against Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) poses challenges in therapy due to the absence of target expression such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Frequently, the treatment of TNBC involves the combination of several therapeutics. However, an enhanced therapeutic effect can be also achieved within a single molecule. The efficacy of raloxifene can be improved by designing a raloxifene-based hybrid drug bearing a 2,2'-bipyridine moiety (2). Integration of platinum(II), palladium(II), and nickel(II) complexes into this structure dramatically changed the cytotoxicity. The platinum(II) dichloride complex 3 did not demonstrate any activity, while palladium(II) and nickel(II) dichloride complexes 4 and 5 exhibited various cytotoxic behavior towards different types of hormone-receptor positive (HR+) cancer and TNBC cell lines. The replacement of the two chlorido ligands in 3-5 with a dicarbollide (carborate) ion [C2B9H11]2- resulted in reduced activity of compounds 6, 7, and 8. However, the palladacarborane complex 7 demonstrated higher selectivity towards TNBC. Furthermore, the mechanism of action was shifted from cytotoxic to explicitly cytostatic with detectable proliferation arrest and accelerated aging, characterized by senescence-associated phenotype of TNBC cells. This study provides valuable insights into the development of hybrid therapeutics against TNBC.

A Novel Recombinant Modified Vaccinia Ankara Virus expressing Interleukin-13 Receptor α2 Antigen for Potential Cancer Immunotherapy.

Genetically altered recombinant poxviruses hold great therapeutic promise in animal models of cancer. Poxviruses can induce effective cell-mediated immune responses against tumor-associated antigens. Preventive and therapeutic vaccination with a DNA vaccine expressing IL-13Rα2 can mediate partial regression of established tumors in vivo, indicating that host immune responses against IL-13Rα2 need further augmentation. The aim of the study is developing a recombinant modified vaccinia Ankara (MVA) expressing IL-13RΑ2 (rMVA-IL13RΑ2) virus and study in vitro infectivity and efficacy against IL-13Rα2 positive cell lines. We constructed a recombinant MVA expressing IL-13Rα2 and a green fluorescent protein (GFP) reporter gene. Purified virus titration by infection of target cells and immunostaining using anti-vaccinia and anti-IL-13Rα2 antibodies was used to confirm the identity and purity of the rMVA-IL13Rα2. Western Blot analysis confirmed the presence of IL-13Rα2 protein (~52 kDa). Flow cytometric analysis of IL-13Rα2 negative T98G glioma cells when infected with rMVA-IL13Rα2 virus demonstrated cell-surface expression of IL-13Rα2, indicating the infectivity of the recombinant virus. Incubation of T98G-IL13α2 cells with varying concentrations (0.1-100 ng/ml) of interleukin-13 fused to truncated Pseudomonas exotoxin (IL13-PE) resulted in depletion of GFP+ fluorescence in T98G-IL13Rα2 cells. IL13-PE (10-1000 ng/ml) at higher concentrations also inhibited the protein synthesis in T98G-IL13Rα2 cells compared to cells infected with the control pLW44-MVA virus. IL13-PE treatment of rMVA-IL13Rα2 infected chicken embryonic fibroblast and DF-1 cell line reduced virus titer compared to untreated cells. rMVA-IL13Rα2 virus can successfully infect mammalian cells to express IL-13Rα2 in a biologically active form on the surface of infected cells. To evaluate the efficacy of rMVA-IL13Rα2, immunization studies are planned in murine tumor models.

#2200 Kidney stem/progenitor cells can be isolated from the urine of neonates independently of their gestational age at birth

Abstract Background and Aims In the human kidney, nephron structures derive from a population of SIX2 positive kidney stem/progenitor cells. These cells are only present during kidney development, which is reported to terminate at approximately 36 weeks of gestation. We have previously described a non-invasive strategy to isolate the native SIX2 positive kidney stem/progenitor cells from the urine of neonates born before 36 weeks of gestation, named the neonatal kidney/stem progenitor cells (nKSPC) [1]. In preterm neonates, nephrogenesis is still ongoing at the time of birth and continues postnatally, enabling isolation of kidney stem/progenitor cells from the voided urine. In this study, we aimed to determine the efficiency of nKSPC isolation from the urine of neonates and which gestational age (GA) results in the highest yield of nKSPC. We hypothesized that the lower the gestational age, the higher the probability of isolating nKSPC. Method 37 fresh urine samples were obtained from 36 neonates (9 female, 27 male) at day 1 after birth at the Neonatology department of University Hospitals Leuven. Five urine samples were collected from extreme preterm (< 28 weeks GA), 6 samples from very preterm (28–32 weeks GA), 18 samples from moderate to late preterm (32-37 weeks GA) and 8 samples from term neonates (>37 weeks). When a sample yielded cell growth, cell colonies were subcultured to achieve clonal expansion. Cell lines were characterized for the SIX2 stem cell marker using RT-qPCR and immunofluorescence (IF) staining. SIX2 positive cell lines were further evaluated for their potential to differentiate into kidney epithelial cells (i.e. proximal tubular epithelial cells (PTEC) and podocytes) in 2D cultures using our previously established protocols [1]. Results From the 37 urine samples collected, 28 samples yielded cell growth (76%). After subcloning, 147 cell lines were characterized for the expression of SIX2, of which 42 were SIX2 positive. Four SIX2 positive cell lines were derived from extreme preterm, 12 from very preterm, 13 from moderate to late preterm and 13 from term neonates. SIX2 positive cell lines isolated from term neonates exhibited similar cells growth and differentiation potential compared to those isolated from extreme preterm neonates. Additionally, we observed a dose-response effect with regard to expression levels of SIX2 and the differentiation potential: cell lines with higher levels of SIX2 maintained their undifferentiated, uninduced state while lower levels of SIX2 enabled successful differentiation to PTEC and/or podocytes. Conclusion This study demonstrates that SIX2 positive nKSPC can be isolated from the urine of neonates, independently of their GA at birth. This could indicate that nephrogenesis persists longer than what has previously been reported (i.e. 36 weeks). Furthermore, the nKSPC exhibit a dose-response effect with regard to levels of SIX2 and induction of differentiation. This effect has previously been observed in an in vivo mice model [2]. To further investigate the timing of nephrogenesis cessation, we aim to perform a SIX2 staining on human fetal kidney tissue across different GA. Furthermore, we aim to further characterize in detail the isolated nKSPC using single cell RNA sequencing.

Laminin I mediates resistance to lapatinib in HER2-positive brain metastatic breast cancer cells in vitro

The role of extracellular matrix (ECM) prevalent in the brain metastatic breast cancer (BMBC) niche in mediating cancer cell growth, survival, and response to therapeutic agents is not well understood. Emerging evidence suggests a vital role of ECM of the primary breast tumor microenvironment (TME) in tumor progression and survival. Possibly, the BMBC cells are also similarly influenced by the ECM of the metastatic niche; therefore, understanding the effect of the metastatic ECM on BMBC cells is imperative. Herein, we assessed the impact of various ECM components (i.e., Tenascin C, Laminin I, Collagen I, Collagen IV, and Fibronectin) on brain metastatic human epidermal growth factor receptor 2 (HER2)-positive and triple negative breast cancer (TNBC) cell lines in vitro. The highly aggressive TNBC cell line was minimally affected by ECM components exhibiting no remarkable changes in viability and morphology. On the contrary, amongst various ECM components tested, the HER2-positive cell line was significantly affected by Laminin I with higher viability and demonstrated a distinct spread morphology. In addition, HER2-positive BMBC cells exhibited resistance to Lapatinib in presence of Laminin I. Mechanistically, Laminin I-induced resistance to Lapatinib was mediated in part by phosphorylation of Erk 1/2 and elevated levels of Vimentin. Laminin I also significantly enhanced the migratory potential and replicative viability of HER2-positive BMBC cells. In sum, our findings show that presence of Laminin I in the TME of BMBC cells imparts resistance to targeted therapeutic agent Lapatinib, while increasing the possibility of its dispersal and clonogenic survival.

Intelligent Cell Profiling and Precision Release: Multimolecular Marker-Activated Transmembrane DNA Computing Nanosystem.

Accurate classification of tumor cells is of importance for cancer diagnosis and further therapy. In this study, we develop multimolecular marker-activated transmembrane DNA computing systems (MTD). Employing the cell membrane as a native gate, the MTD system enables direct signal output following simple spatial events of "transmembrane" and "in-cell target encounter", bypassing the need of multistep signal conversion. The MTD system comprises two intelligent nanorobots capable of independently sensing three molecular markers (MUC1, EpCAM, and miR-21), resulting in comprehensive analysis. Our AND-AND logic-gated system (MTDAND-AND) demonstrates exceptional specificity, allowing targeted release of drug-DNA specifically in MCF-7 cells. Furthermore, the transformed OR-AND logic-gated system (MTDOR-AND) exhibits broader adaptability, facilitating the release of drug-DNA in three positive cancer cell lines (MCF-7, HeLa, and HepG2). Importantly, MTDAND-AND and MTDOR-AND, while possessing distinct personalized therapeutic potential, share the ability of outputting three imaging signals without any intermediate conversion steps. This feature ensures precise classification cross diverse cells (MCF-7, HeLa, HepG2, and MCF-10A), even in mixed populations. This study provides a straightforward yet effective solution to augment the versatility and precision of DNA computing systems, advancing their potential applications in biomedical diagnostic and therapeutic research.

Abstract PO4-06-10: Targeting necroptosis in breast cancer induced by Ophiobolin A

Abstract Abstract Necroptosis or programmed necrosis is a form of cell death distinct from apoptosis. Necroptotic cells display the character of rounding of the cell, gain in cell volume, rupture of plasma membrane and release of intra-cellular contents. Although few key necroptosis regulators including receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like pseudokinase (MLKL) has been identified, their role/mechanism in inducing cell death in triple-negative breast cancer (TNBC) which lack RIPK3 expression remains elusive. Ophiobolin A is a compound naturally made by fungus and potently kills cancer cells especially TNBC. Our current study investigated the cell death mechanism(s) induced by Ophiobolin A (natural product) in breast cancer cell lines. The mechanism of cell death elicited by OpA was analyzed by applying inhibitors for specific pathways including apoptosis, necroptosis, ferroptosis and paraptosis, by analyzing markers of cell death using flow cytometry and western blotting. The formation of necrosomes was analyzed with exogeneous expression of RIPK3 tagged with green fluorescence protein (GFP) in HeLa, MDA-MB-231, MDA-MB-468 cell lines which lacks endogenous RIPK3 expression and in MCF7 estrogen receptor (ER) positive cell line. OpA induced necrosome formation was identified by formation of puncta in TNBC and HeLa cells which lack RIP3 expression, however, necrosomes were not detectable in ER positive cells. Clinical relevance of RIPK3 and MLKL has been analyzed using bioinformatic tools. Effect of OpA In vivo has been analyzed using Immune compromised SCID mice. The data obtained in the present study indicates that OpA has an increased anticancer effect in TNBC compared to the estrogen receptor (ER) positive breast cancer cells. Cells having undergone EMT were shown to have more sensitivity towards OpA when compared to uninduced cell lines, which is sensitive to Necrostatin-1, an inhibitor of necroptosis. However, apoptosis, ferroptosis and paraptosis inhibitors failed to rescue cell death which clearly confirms that OpA induces necroptosis cell death pathway in EMT undergone cells. OpA treatment significantly increased puncta in TNBC compared non-TNBC cells, further confirming the necroptotic cell death in TNBC. Bioinformatic analysis revealed that RIPK3 is negatively corelated with breast cancer and has least over-all survival in patients who lack RIPK3 expression. OpA treatment showed a significant tumor regression in tumor bearing SCID mice. All together our findings identify that OpA induce necroptosis in EMT-enriched TNBC. The study further needs an in-depth analysis of molecular mechanism induced by OpA and involvement of EMT in necroptotic cell death. Citation Format: Tolulope Ojo, Santhalakshmi Ranganathan. Targeting necroptosis in breast cancer induced by Ophiobolin A [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 PO4-06-10.

Discovery and pharmacological characterization of 1,2,3,4-tetrahydroquinoline derivatives as RORγ inverse agonists against prostate cancer.

The retinoic acid receptor-related orphan receptor γ (RORγ) is regarded as an attractive therapeutic target for the treatment of prostate cancer. Herein, we report the identification, optimization, and evaluation of 1,2,3,4-tetrahydroquinoline derivatives as novel RORγ inverse agonists, starting from high throughput screening using a thermal stability shift assay (TSA). The representative compounds 13e (designated as XY039) and 14a (designated as XY077) effectively inhibited the RORγ transcriptional activity and exhibited excellent selectivity against other nuclear receptor subtypes. The structural basis for their inhibitory potency was elucidated through the crystallographic study of RORγ LBD complex with 13e. Both 13e and 14a demonstrated reasonable antiproliferative activity, potently inhibited colony formation and the expression of AR, AR regulated genes, and other oncogene in AR positive prostate cancer cell lines. Moreover, 13e and 14a effectively suppressed tumor growth in a 22Rv1 xenograft tumor model in mice. This work provides new and valuable lead compounds for further development of drugs against prostate cancer.

Abstract PO4-24-09: Grape seed extract inhibits EGFR signaling pathway as a main factor of tamoxifen resistant in hormone receptor positive breast cancer

Abstract Purpose Because more than 60% of breast cancers are hormone-receptor (HR) positive, endocrine therapy plays an important role. Tamoxifen is an antagonist of estrogen receptor-α and main treatment drug for HR-positive breast cancers. However, efficacy is not satisfactory because of the development of resistance. EGFR expression was associated with poor prognosis in HR-positive breast cancers. Loss of ER by EGFR activation induced tamoxifen resistance. And extracellular signal-regulated kinase (ERK) is another important factor for tamoxifen resistance. Activation of ERK signaling in breast cancer is not only associated with augmented tumor growth and metastasis but also with tamoxifen resistance. Therefore, the inhibition of ERK/EGFR signaling associated with tamoxifen resistance could improve the survival rate. Grape seed extract (GSE) is which contains a high content of flavonoid polyphenolic compounds. GSE is known to have various pharmacological effects, including antioxidant, anti-inflammatory, antitumor activity. In this study, we explored the activity of GSE in tamoxifen resistant breast cancer cells by inhibiting EGFR signaling pathway. Method We examined the suppressive effect of GSE on various signaling pathways. To confirm the pharmacological activity of GSE, we treated EGFR positive breast cancer cells with GSE. Levels of mRNA and protein expression were analyzed by real-time PCR and western blot. And cell proliferation and invasiveness were analyzed by the colony-forming assay and the Boyden chamber assay. Results 1. GSE down regulates EGFR and ERK in EGFR positive breast cancer cell line. The cells were treated with the indicated concentrations GSE for 48 hrs. The levels of p- and t-ERK and t-EGFR were measured through Western blotting. The levels of phosphorylated EGFR and ERK were decreased by GSE at the indicated doses for 48hrs. 2. EGF, a ligand of EGFR, upregulates the MMP-9 and GSE suppresses MMP-9. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are upregulated in most cancers and play crucial roles in modulating invasion and metastasis. EGFR with its ligand epidermal growth factor (EGF) regulates Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). After treating EGFR positive breast cancer cells with EGF and TGF, MMP-9 levels were increased. However, MMP-9 level was decreased with GSE as indicated doses for 48hrs. Conclusion Taken together, GSE inhibited the ERK/EGFR and EGF/MMP-9 signaling pathway in EGFR positive breast cancer cells. Therefore, we propose the possibility of GSE as an effective adjuvant for tamoxifen resistant breast cancer. Citation Format: Seung Pil Jung, Eun-Shin Lee, Ji Young You, Jung Hwa Chun, Hyunyou Kim. Grape seed extract inhibits EGFR signaling pathway as a main factor of tamoxifen resistant in hormone receptor positive 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 PO4-24-09.

Construction of a stable expression HEK293T engineered cell line adapted to high-density suspension culture

The HEK293 cell line has received FDA approval and is increasingly used to produce therapeutic glycoproteins. However, the cell line faces the challenge of long-term expression instability. This problem could be solved by specifically integrating exogenous genes into stable sites and developing a HEK293 cell platform to efficiently and stably express recombinant proteins with large-scale suspension culture. The study uses CRISPR/Cas9 and recombinase-mediated cassette exchange (RMCE) technologies to integrate a variety of exogenous genes into specific chromosomal sites, such as the hROSA26 site, AAVS1 site, and CCR5 site. We evaluated the stability and expression intensity of exogenous proteins at different insertion sites under suspension culture conditions. A monoclonal cell line expressing enhanced green fluorescent protein (EGFP) is obtained using CRISPR/Cas9-mediated specific integration. The positive monoclonal cell lines express EGFP after 60 consecutive passages, and no significant differences in protein expression titer are observed. These results show that, under suspension culture conditions, the hROSA26 locus could stably express exogenous proteins after 60 generations. HEK293 cell lines integrating HSA (68 kDa) and IFNβ-HSA (89 kDa) genes at the above three sites were obtained respectively using Bxb1-mediated RMCE technology, and batch experiments demonstrated their expression capacity. In summary, a stable and efficient protein expression platform of HEK293 cells is successfully constructed and could be adapted to serum-free suspension culture.

Abstract LB128: A novel tri-specific T cell engager targeting BCMA and GPRC5D for treatment of multiple myeloma

Abstract Introduction Multiple myeloma (MM) accounts for 10% of all hematologic cancers. Recent advances in MM therapy have greatly increased the overall response and survival rate. However, almost all patients eventually relapse. The prognosis still remains poor. BCMA and GPRC5D are overexpressed in myeloma cells. Although CAR-T and T cell engager (TCE) targeting BCMA or GPRC5D have been efficacious in MM patients, resistance does occur. Since the expression of BCMA and GPRC5D in MM are heterogeneous, to further improve the overall response and survival, we have recently generated a novel tri-specific T-cell engager, GBD218, targeting both BCMA and GPRC5D. GBD218 has demonstrated potent in vitro and in vivo activity against myeloma cells. Methods Anti-BCMA and GPRC5D nanobodies were screened from alpaca immune libraries, and anti-CD3 antibody was engineered from mouse hybridoma clone. The tri-specific antibodies were constructed in a “1+1+1” format through “knob into hole” technology fused with silenced IgG1 Fc. The format of the tri-specific antibodies was optimized by multiple rounds of in vitro activity and druggability evaluation. The in vivo tumor growth inhibition effects were evaluated in PBMC-humanized xenograft mouse models. Results GBD218 has been designed to potently bind hBCMA (KD=0.4nM) and hGPRC5D (cell binding EC50 ~ 2nM). To reduce CRS and other potential AEs associated with TCEs, a low affinity of anti-CD3 Fab was used. In cell-based functional assays, GBD218 showed efficient cytotoxicity against single and double positive MM cell lines with various expression levels of BCMA and GPRC5D. T cell activation and cytokine release induced by GBD218, in the presence or absence of MM cancer cells, is nicely balanced for great killing efficacy and the low risk of CRS. Importantly, the results showed that GBD218 exhibited superior in vitro killing activity compared to benchmarks, including teclistamab, talquetamab, the combination of teclistamab and talquetamab, suggesting a synergistic effect of GBD218 by targeting BCMA and GPRC5D. In xenograft models, GBD218 showed excellent anti-tumor activity, indicating great potential for GBD218 as a promising therapeutics for MM. Conclusion GBD218 is a novel tri-specific antibody that showed potent in vitro and in vivo anti-tumor activity. GBD218 efficiently kills both BCMA and/or GPRC5D expressing MM cells, which may hold promise to increase response rate and improve survival in MM patients in clinic. Citation Format: Yongcong Tan, Xuezhen Li, Fan Yu, Juehua Xu, Zhen Qian, Yiqi Cao, Xueyan Yang, Qinglin Du, Fei Peng, Shuhua Han, Qian Ding. A novel tri-specific T cell engager targeting BCMA and GPRC5D for treatment of multiple myeloma [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 LB128.

Abstract LB059: Multimeric linker-exatecan-based ADC targeting guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal cancer

Abstract Background: The approval of sacituzumabgovitecan (Trodelvy) and trastuzumab deruxtecan (Enhertu) demonstrated the potential of topoisomerase I inhibitors in cancer therapy. Both ADCs rely on payload conjugation to interchain cysteines of an unmodified wildtype antibody coming along with unfavorable properties like instability in circulation, premature payload release and unspecific uptake via Fc gamma receptors. Heidelberg Pharma uses a proprietary, site-specific conjugation to engineered cysteines andFcγR silencing mutations (LALA) to obtain a homogeneous and stable ADC void of Fc-mediated uptake. To achieve a DAR of 4, a multimeric linker comprisingexatecan and a solubility enhanceris used to obtain more than one payload per conjugation site. In the current study, in vitro and in vivo data of an ADC targeting GCC (guanylyl cyclase C) for the treatment of colorectal cancer with favorable potency and tolerability are presented. Material and methods: Cell lines: GCC+cell line: HEK293-GUCY2C mono2; GCC-cell line: HEK293 ADC: The cysteine-reactive multimeric linker-exatecan construct was synthesized at Heidelberg Pharma and conjugated site-specifically to engineered cysteine residues of a proprietary anti-GCC antibody yielding ADCs with a DAR of 4.0. Animal models: Female NOD SCID mice were subcutaneously inoculated with GCC-overexpressing HEK293-GUCY2C mono2 tumor cells. Animals were treated with anti GCC ADC at single or multiple doses. Tolerability was assessed in female NODSCID mice. Results: The anti-GCC ADC with multimeric linker-exatecan payload showed favorable in vitro cytotoxicity with nanomolar activity on the GCC+ cell line and no cytotoxic activity on target-negative cells. In mouse xenograft models, the anti-GCCADC caused dose-dependent tumor regression in HEK293-GUCY2C mono2 s.c. xenografts. Multiple dosing improved this anti-tumor efficacy even further without negative impact on toxicity. The MTD of the ADC in mice was determined to be ≥200 mg/kg. Conclusions: Targeted cytotoxic drug delivery to GCC positive cell lines was achieved by using an anti-GCC ADCwith multimeric linker-exatecan payload. The use of solubility enhancers within the structure of the multimeric linker facilitated site-specific coupling to cysteines resulting in a stable, potent, and well tolerated ADC. The use of anti-GCC ADCs with multimeric linker-exatecan payloads in the therapy of GCC positive colorectal cancer, represents a promising approach in cancer therapy. Citation Format: Sarah-Jane Neuberth, Christian Orlik, Aniko Palfi, Christoph Mueller, Hendrik Gruss, Torsten Hechler. Multimeric linker-exatecan-based ADC targeting guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal cancer [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 LB059.

Sialyl-Tn serves as a potential therapeutic target for ovarian cancer

BackgroundOvarian cancer remains the deadliest of the gynecologic cancers in the United States. There have been limited advances in treatment strategies that have seen marked increases in overall survival. Thus, it is essential to continue developing and validating new treatment strategies and markers to identify patients who would benefit from the new strategy. In this report, we sought to further validate applications for a novel humanized anti-Sialyl Tn antibody-drug conjugate (anti-STn-ADC) in ovarian cancer.MethodsWe aimed to further test a humanized anti-STn-ADC in sialyl-Tn (STn) positive and negative ovarian cancer cell line, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. Furthermore, we sought to determine whether serum STn levels would reflect STn positivity in the tumor samples enabling us to identify patients that an anti-STn-ADC strategy would best serve. We developed a custom ELISA with high specificity and sensitivity, that was used to assess whether circulating STn levels would correlate with stage, progression-free survival, overall survival, and its value in augmenting CA-125 as a diagnostic. Lastly, we assessed whether the serum levels reflected what was observed via immunohistochemical analysis in a subset of tumor samples.ResultsOur in vitro experiments further define the specificity of the anti-STn-ADC. The ovarian cancer PDO, and PDX models provide additional support for an anti-STn-ADC-based strategy for targeting ovarian cancer. The custom serum ELISA was informative in potential triaging of patients with elevated levels of STn. However, it was not sensitive enough to add value to existing CA-125 levels for a diagnostic. While the ELISA identified non-serous ovarian tumors with low CA-125 levels, the sample numbers were too small to provide any confidence the STn ELISA would meaningfully add to CA-125 for diagnosis.ConclusionsOur preclinical data support the concept that an anti-STn-ADC may be a viable option for treating patients with elevated STn levels. Moreover, our STn-based ELISA could complement IHC in identifying patients with whom an anti-STn-based strategy might be more effective.

Unveiling the signaling network of FLT3-ITD AML improves drug sensitivity prediction.

Currently, the identification of patient-specific therapies in cancer is mainly informed by personalized genomic analysis. In the setting of acute myeloid leukemia (AML), patient-drug treatment matching fails in a subset of patients harboring atypical internal tandem duplications (ITDs) in the tyrosine kinase domain of the FLT3 gene. To address this unmet medical need, here we develop a systems-based strategy that integrates multiparametric analysis of crucial signaling pathways, and patient-specific genomic and transcriptomic data with a prior knowledge signaling network using a Boolean-based formalism. By this approach, we derive personalized predictive models describing the signaling landscape of AML FLT3-ITD positive cell lines and patients. These models enable us to derive mechanistic insight into drug resistance mechanisms and suggest novel opportunities for combinatorial treatments. Interestingly, our analysis reveals that the JNK kinase pathway plays a crucial role in the tyrosine kinase inhibitor response of FLT3-ITD cells through cell cycle regulation. Finally, our work shows that patient-specific logic models have the potential to inform precision medicine approaches.

Unveiling the signaling network of FLT3-ITD AML improves drug sensitivity prediction

Currently, the identification of patient-specific therapies in cancer is mainly informed by personalized genomic analysis. In the setting of acute myeloid leukemia (AML), patient-drug treatment matching fails in a subset of patients harboring atypical internal tandem duplications (ITDs) in the tyrosine kinase domain of the FLT3 gene. To address this unmet medical need, here we develop a systems-based strategy that integrates multiparametric analysis of crucial signaling pathways, and patient-specific genomic and transcriptomic data with a prior knowledge signaling network using a Boolean-based formalism. By this approach, we derive personalized predictive models describing the signaling landscape of AML FLT3-ITD positive cell lines and patients. These models enable us to derive mechanistic insight into drug resistance mechanisms and suggest novel opportunities for combinatorial treatments. Interestingly, our analysis reveals that the JNK kinase pathway plays a crucial role in the tyrosine kinase inhibitor response of FLT3-ITD cells through cell cycle regulation. Finally, our work shows that patient-specific logic models have the potential to inform precision medicine approaches.

Asciminib Maintains Antibody-Dependent Cellular Cytotoxicity against Leukemic Blasts.

B cell acute lymphoblastic leukemia (B-ALL) is characterized by an accumulation of malignant precursor cells. Treatment consists of multiagent chemotherapy followed by allogeneic stem cell transplantation in high-risk patients. In addition, patients bearing the BCR-ABL1 fusion gene receive concomitant tyrosine kinase inhibitor (TKI) therapy. On the other hand, monoclonal antibody therapy is increasingly used in both clinical trials and real-world settings. The introduction of rituximab has improved the outcomes in CD20 positive cases. Other monoclonal antibodies, such as tafasitamab (anti-CD19), obinutuzumab (anti-CD20) and epratuzumab (anti-CD22) have been tested in trials (NCT05366218, NCT04920968, NCT00098839). The efficacy of monoclonal antibodies is based, at least in part, on their ability to induce antibody-dependent cellular cytotoxicity (ADCC). Combination treatments, e.g., chemotherapy and TKI, should therefore be screened for potential interference with ADCC. Here, we report on in vitro data using BCR-ABL1 positive and negative B-ALL cell lines treated with rituximab and TKI. NK cell activation, proliferation, degranulation, cytokine release and tumor cell lysis were analyzed. In contrast to ATP site inhibitors such as dasatinib and ponatinib, the novel first-in-class selective allosteric ABL myristoyl pocket (STAMP) inhibitor asciminib did not significantly impact ADCC in our settings. Our results suggest that asciminib should be considered in clinical trials.

Surface modification of hollow gold nanoparticles conducted by incorporating cancer cell membrane and AS1411 aptamer, aiming to achieve a dual-targeted therapy for colorectal cancer

Due to its inherent membrane structure, a nanostructure enveloped by an active cell membrane possesses distinctive characteristics such as prolonged presence in the bloodstream, precise identification capabilities, and evasion of immune responses. This research involved the production of biomimetic nanoparticles, specifically hollow gold nanoparticles (HGNPs) loaded with methotrexate (MTX), which were further coated with cancer cell membrane. These nanoparticles were then adorned with AS1411 aptamer to serve as a targeting agent (Apt-CCM-HG@MTX). The nanoplatform demonstrated precise targeting towards cancer cells due to its dual-targeting characteristic (AS1411 aptamer and C26 cancer cell membrane), exhibiting uniformity in distribution. It also displayed a desirable response to photothermal stimulation, controlled release of drugs, and exceptional properties for fluorescence imaging. The system was composed of spherical HGNPs measuring 51.33 ± 5.70 nm in diameter, which were effectively loaded with MTX using a physical absorption method. The encapsulation efficiency achieved was recorded at 79.54 %, while the loading efficiency reached 38.21 %. The targeted formulation demonstrated a noteworthy mortality of approximately 45 % in the nucleolin positive cell line, C26, as determined by in vitro cytotoxicity assays. As a result of the functionalization process applied to the homologous binding adhesion molecules found in cancer cell membranes and targeting ability of AS1411 aptamer, Apt-CCM-HG@MTX demonstrated a substantial enhancement in targeting tumors and facilitating cellular uptake during in vivo experiments. Furthermore, under NIR radiation the photothermal effect exhibited by Apt-CCM-HG@MTX in the tumor area was notably robust due to the distinctive attributes of HGNPs. The conclusions obtained from this study have the potential to assist in adopting a bioinspired strategy that will significantly improve the effective management of MTX and therapy for individuals with colorectal cancer.

Abstract 3142: NN3201, a novel c-Kit targeting ADC, exhibits robust preclinical anti-tumor efficacy in SCLC and GIST models

Abstract c-Kit (CD117) is a type III receptor tyrosine kinase (RTK), which is known to be activated upon binding to its cognate ligand stem cell factor (SCF) to induce critical signaling pathways involved in cell proliferation, survival, and differentiation. Indicative of poor prognosis, overexpression and activating mutations of c-Kit have been implicated in a variety of human cancers including small cell lung cancer (SCLC), gastrointestinal stromal tumors (GIST), and acute myelogenous leukemia (AML). While imatinib, a small molecule c-Kit inhibitor, has been a beneficial treatment for GIST, resistance eventually develops to the drug, moreover, shows limited efficacy in c-Kit overexpressing tumors such as SCLC. Herein, we developed NN3201, a novel c-Kit targeting antibody-drug conjugate (ADC), comprised of fully human 2G4 antibody with a picomolar binding affinity to c-Kit and cytotoxic monomethyl auristatin E (MMAE) as the payload. NN3201 displayed potent in vitro cytotoxicity in various wild-type and mutant c-Kit positive SCLC and GIST cell lines with IC50 in the nanomolar range. In addition, NN3201 was rapidly internalized and inhibited c-Kit-mediated signaling., NN3201 treatment resulted in the increased proportions of the sub G1 and G2/M in the c-Kit positive cancer cell lines, indicating that release of MMAE induced cell cycle arrest. Moreover, bystander effect was confirmed by co-culturing c-Kit high and negative cell lines. Most importantly, in mouse xenograft models, NN3201 showed a remarkable tumor growth inhibition of several wild type, mutant c-Kit positive, and imatinib resistant SCLC and GIST cell lines. Enhanced anti-tumor activity and delayed tumor growth were observed in an SOC-treated (etoposide and carboplatin) SCLC xenograft model, by NN3201 in comparison to second line SOC (topotecan or irinotecan). In the GLP study, repeated intravenous administration of NN3201 was well tolerated at all doses (0.5, 1, 2 mg/kg Q3Wx3) without unscheduled death. Dose-dependent, yet transient, cytotoxicity of NN3201 was observed in the bone marrow. We confirmed HNSTD (Highest Non-Severely Toxic Dose) of NN3201 to be 2 mg/kg through exploratory and GLP repeat dose toxicity studies as no serious irreversible toxicity was observed. Accumulating data suggest that NN3201 is a promising therapeutic alternative for the treatment of SCLC and GIST regardless of wild-type or activating mutations in c-Kit. Citation Format: Chansik Kim, Jin Gu Cho, TaeMin Wi, Jiwoo Moon, Han-Jik Ko, Jina Lee, Jin Woo Park, Yeonjy Lee, Sanghee Kim, Jin-Ock Kim, Jaeyoung Song, Sun-Hwa Lee, Sang Gyu Park. NN3201, a novel c-Kit targeting ADC, exhibits robust preclinical anti-tumor efficacy in SCLC and GIST 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 3142.

Abstract 402: Aneuploidy-associated SQLE gain promotes prostate cancer aggressiveness by altering lipid metabolism

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death in men in the US. Epidemiology studies on primary PCa cohorts in Physicians' Health Study and Health Professionals Follow-up Study (PHS and HPFS) have shown that high levels of whole-genome aneuploidy, featured by imbalanced chromosome numbers, correlate with lethal progression in PCa. However, details of the mechanisms of how aneuploidy drives PCa aggressiveness are still unclear. Here, we used the case of chromosome 8q (chr 8q, the long arm of chr 8) gain to study aneuploidy-associated prostatic malignancies. Chr 8q gains are the most frequent gain events that occur in approximately 23% of PCa cases. By using the PHS and HPFS cohorts, we modeled the increased expression of each gene located on chr 8q, for predicting the risks for lethal progression, and obtained each corresponding gene’s odds ratio (OR). By ranking the ORs, we revealed that a cholesterol biosynthesis gene, squalene monooxygenase (SQLE), is one of the top associators with lethal progression, amongst all chr 8q genes. SQLE plays a pivotal role in cholesterol synthesis. Previous lymphoma studies have shown that loss of SQLE contributes to cholesterol auxotrophy, and squalene build-up protects against oxidative cell death. In our experimental study, we have used normal and cancerous TMPRSS2-ERG-driven organoid models and found that over-expression of SQLE promotes formation of invasive structures and proliferation in cancer organoids. Interestingly, overexpression of SQLE decreased the protein levels of TMPRSS2-ERG, which appeared to be independent of androgen receptor levels. We also utilized the TMPRSS2-ERG positive VCaP cell line, which harbors gains of SQLE gene copies. We found that knocking down SQLE expression significantly upregulated ERG protein levels. Our recent study has shown that ERG (or other ETS) positive prostate cancers have a strong correlation with downregulation of fatty acid metabolism signature. We speculate that gain of SQLE can drive aggressiveness of prostate cancer by modulating lipid metabolism for growth and migration. Inhibition of SQLE could translate to better clinical outcomes regarding prostate cancer lethality. Citation Format: Thomas Walter Janas, Xiaofeng A. Su, Konrad H. Stopsack, Daniel R. Schmidt, Duanduan Ma, Zhe Li, Kathryn L. Penny, Tamara L. Lotan, Lorelei A. Mucci, Matthew G. Vander Heiden, Elise DeArment, Angelika Amon, Paul A. Scheet. Aneuploidy-associated SQLE gain promotes prostate cancer aggressiveness by altering lipid metabolism [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 402.

Abstract 1733: SET-NUP214 rearranges the DNA-methylation landscape to upregulate the HOX-gene cluster in acute myeloid leukemia

Abstract The SET-NUP214 (S/N) fusion gene, which results from either cryptic t(9:9) (q34;q34) or del(9) (q34.11q34.13), is found in a very narrow spectrum of patients diagnosed with Acute Myeloid Leukemia (AML) or T-cell acute lymphoblastic leukemia (T-ALL). AML patients with S/N fusion were reported to have relatively poor prognostic outcomes despite receiving myeloablative conditioning. While previously, the fusion has been described to overrepresent HOX cluster genes, the underlying mechanism of gene regulation that contributes to the pathology remains elusive due to the rare incidence of the disease. The current study used the MEGAL (ACC719, DSMZ) cell line, identified as the solo megakaryocytic AML line expressing the S/N-fusion, for determining genome-wide DNA methylation (DNAm) and gene expression using 935K Infinium MethylationEPIC v2.0 BeadChip array and RNA-sequencing, respectively. The differential DNAm and expression data were analyzed and compared to the hematopoietic stem progenitor cells (HSPC) or normal megakaryocytes (MK). The ChIP-sequencing data from the S/N positive T-ALL cell line LOUCY has been used to represent the coincidence of regulatory chromatin marks and differentially methylated CpG (mC) sites. We observed n=1934 genes upregulated and n=2396 downregulated in MEGAL, compared to both HSPC and MK. The direction of expression changes between MEGAL and HSPC and MEGAL and MK showed a high correlation (r=0.92, p< 0.01). Per the previous literature, we observed an overrepresentation of HOX genes (n=19) in MEGAL. Based on DNAm profiling, we noticed a propensity of hypermethylation in MEGAL across the promoters (93%±0.6), bodies (93%±0.8), and intergenic (85%±0.3) regions, compared to HSPC/MK. Next, we integrated the mC and expression at the promoters and identified that the majority (75%±3) of the genes belong to the hyper-down (n=2563 against HPSC; n=1907 against MK) or hyper-up (n=965 against HPSC; n=923 against MK) clusters. We further investigated the HOXB (HOXB7, HOXB8, HOXB9) and HOXC (HOXC10, HOXC12, HOXC13, HOXC13-AS, HOXC4, HOXC5, HOC6, HOXC8, HOXC9, HOXC-AS1, HOXC-AS2, and HOXC-AS3) cluster genes, where hypermethylated CpGs mainly were concentrated at the CpG islands and overlapped with active promoter (H3K4me3, H3K4me1) signatures or enhancer-like (CTCF bound) elements. We also observed frequent overlaps between mC and H3K36me3 across the observed genes. The differentially expressed genes in this AML subtype were enriched for the hippo-signaling pathway, calcium signaling, focal adhesion, and PI3-Akt pathways. Collectively, our data showed that S/N favors genome-wide hypermethylation, which, in conjunction with the cis-regulatory elements, induces overrepresentation of the HOX cluster genes. These novel epigenetic vulnerabilities are worth pursuing for investigating the cellular function and pathophysiology of the disease. Citation Format: Samrat Roy Choudhury, Akhilesh Kaushal. SET-NUP214 rearranges the DNA-methylation landscape to upregulate the HOX-gene cluster in acute myeloid leukemia [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 1733.