bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3138: Discovery of AT65474, a highly selective anti-CLDN6  ADC with a proprietary payload
Authors: Zhong Chen , Wu You Xue , Zhang Xi Yun , Cheng Yin Wen , Wang Qian , Du Ping , Liu Shao Jun , Zou Bin
Published: March 2024

CLDN6 is a membrane-bound oncofetal protein and a promising target for cancers such as ovarian and testicular cancer. CLDN6 shares high sequence homology with other CLDN family members which are expressed in normal tissues, making a high degree of selectivity required for CLDN6-targeting therapies. In particular, CLDN6 is closest to CLDN9 in sequence with a difference of only three residues in the extracellular loops, making it challenging to generate a highly selective antibody.

We report the development of a humanized anti-CLDN6 antibody with high affinity and high selectivity for targeted cells which displays > 800-fold higher binding affinity for CLDN6 over CLDN9. The antibody was refined for use as an ADC and incorporates AxcynCYSTM technology for site-specific conjugation which can reproducibly achieve high DAR4 ratios of greater than 97% by HIC analysis. AT65474 is an ADC candidate that incorporates a proprietary payload derived from an FDA approved drug that was optimized in-house for increased potency and improved PK properties to limit systemic exposure. In vitro, the payload displays broad sub-nanomolar activity across a wide range of cancer cell lines including those resistant to paclitaxel, DM1, MMAE and DXd. The payload is conjugated via an enzyme-cleavable linker optimized for solubility, stability, and high drug to antibody ratios.

We provide evidence characterizing AT65474 as a highly potent ADC with high DAR4 homogeneity and strong in vivo activity at low doses. Compared with a benchmark ADC in clinical development, AT65474 shows stronger binding, higher internalization, superior in vitro cytotoxicity, and stronger bystander killing effect. When evaluated in PA-1 and OV90 xenograft models of ovarian cancer, AT65474 displays significantly higher efficacy over the benchmark ADC. Furthermore, we show that AT65474 displays an ADCC effect that contributes to tumor suppression in the OV90 model. AT65474 was shown to be well tolerated in all xenograft mouse models and a pre-tox study in non-human primates. The combined data support further development of AT65474.​

Citation Format: Chen Zhong, You Xue Wu, Xi Yun Zhang, Yin Wen Cheng, Qian Wang, Ping Du, Shao Jun Liu, Bin Zou. Discovery of AT65474, a highly selective anti-CLDN6  ADC with a proprietary payload [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 3138.
Abstract 314: Fixative eXchange (FX)-seq reveals single nucleus transcriptional landscapes of archived clinical cancer FFPE specimens
Authors: Park Han-Eol , Lee Yu Tak , Lee Jaewon , Lee Junsuk , Ji Hyelin , Song Young-Lan , Han Yoon Dae , Kim Hyunki , Sohn Chang Ho
Published: March 2024

The wide availability of FFPE tissues stored in hospitals and biobanks could potentially be an invaluable resource to gain insights for improved medical treatment towards precision medicine by combining genomic and transcriptomic data with pathological annotations and medical records. Nevertheless, the majority of current single-cell sequencing methods depend on fresh or fresh-frozen samples to generate high-quality transcriptome data due to the limited reverse transcription (RT) yields in FFPE samples. Consequently, single-cell level transcriptomic studies of FFPE samples have been a longstanding challenge. Here, we present Fixative-eXchange (FX)-seq, a highly scalable snRNA-seq method for heavily paraformaldehyde (PFA)-fixed and/or FFPE samples. We successfully analyzed a total of nearly 320k nuclei from various samples, including PFA-fixed tissue, FFPE blocks, FFPE and hematoxylin and eosin (H&E)-stained sections from mouse brain and human clinical cancer specimens. In a human gastrointestinal stromal tumor (GIST) FFPE sample, we observed rare transcriptomic transitions toward increased epigenetic modifications affected by acquired resistance in response to prolonged imatinib treatment. Furthermore, in an archived human colorectal cancer (CRC) FFPE sample, spatially-contextualized FX-seq with pathologist annotation distinguishes tumor cells from epithelial progenitors along with identification of the tumor microenvironment (TME) such as cancer-associated fibroblasts (CAFs). We then proceeded to investigate transcriptional programs associated with drug resistance and lack of response in immunotherapy observed in CRC patients. With a hypothesis-driven curation, we compared transcription profiles at the single-nucleus level observed in MSS and MSI-H CRC patients from archived FFPE blocks and found distinctive transcriptional profiles from each group. Overall, FX-seq holds promise for clinical research, precision medicine, biomarker discovery, and molecular diagnosis using archived clinical FFPE specimens.

Citation Format: Han-Eol Park, Yu Tak Lee, Jaewon Lee, Junsuk Lee, Hyelin Ji, Young-Lan Song, Yoon Dae Han, Hyunki Kim, Chang Ho Sohn. Fixative eXchange (FX)-seq reveals single nucleus transcriptional landscapes of archived clinical cancer FFPE specimens [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 314.
Abstract 3140: BL-M05D1, a novel claudin 18.2-targeting ADC, demonstrates specific potent anti-tumor efficacy in preclinical evaluation
Authors: Wan Weili , Dinkel Kelcey , Sanford Austin , Zhao Shuwen , Zhuo Shi , Zhang Yong , Chen Lan , Li Ruigang , Khalili Jahan Salar , Sa Xiao , Yan Yongqi , Shen Xuejiao , Zhu Yi
Published: March 2024

Claudin 18.2, is a member of the tight junction protein family, and an isoform of claudin18. It is therapeutically targeted in cancer due to its limited expression in normal tissue and prevalent expression in a variety of human carcinomas. To develop a promising therapeutic anti-tumor agent, we generated BL-M05D1, an anti-Claudin 18.2-Ed-04 ADC. It is comprised of a novel, specific monoclonal antibody against Claudin 18.2 (5103F3), having a wt Fc that can mediate ADCC and CDC in vitro. BL-M05D1 is composed of (5103F3), a cathepsin B cleavable linker, and a novel topoisomerase I inhibitor agent (Ed-04). The novel Ed-04 is a derivative of the alkaloid camptothecin and mediates cell cycle arrest at the S phase and subsequent apoptosis. BL-M05D1 achieves a high drug-to-antibody-ratio (DAR=8) with a highly stable linker and is readily internalized and trafficked to lysosomal cellular compartments. The antitumor efficacy of BL-M05D1 was evaluated in xenograft tumor models. BL-M05D1 exhibited strong tumor inhibition capacity toward the human gastric cancer cell MKN-45-Claudin18.2 (#C11D) xenograft model, and human pancreatic cancer cell Patu8988s-Claudin18.2 (#C7G) xenograft model. In summary, these studies suggest BL-M05D1, a novel Claudin 18.2-targeting ADC, is potentially efficacious in the treatment of Claudin 18.2-expressing carcinomas. The clinical phase I is being undertaken to evaluate safety, dosing and observations of preliminary signs of efficacy.

Citation Format: Weili Wan, Kelcey Dinkel, Austin Sanford, Shuwen Zhao, Shi Zhuo, Yong Zhang, Lan Chen, Ruigang Li, Jahan Salar Khalili, Xiao Sa, Yongqi Yan, Xuejiao Shen, Yi Zhu. BL-M05D1, a novel claudin 18.2-targeting ADC, demonstrates specific potent anti-tumor efficacy in preclinical evaluation [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 3140.
Abstract 3146: HRA00130-C004, a novel anti-DLL3 ADC with bystander effect, high DAR and favorable safety profiles
Authors: Yao Qingqing , Xue Changding , Xu Zhibin , You Lingfeng , Xue Zhendong , Mao Yuchang , Lin Sophie , Feng Jun , Zhang Zhe , Ye Xin , Hu Min , He Feng
Published: March 2024

Small Cell Lung Cancer (SCLC) is a highly aggressive neuroendocrine tumor, accounting for ~15% of all new lung cancer cases1. It is estimated that there are 250,000 new SCLC cases and at least 200,000 deaths globally each year2. SCLC exhibits high recurrence after first-line treatment, poor response to subsequent therapies, and rare survival3. Lacking of recommended treatments for prolonging survival remains a problem over decades4. DLL3 is an inhibitory ligand of the Notch receptor. It binds to its Notch receptor through a cis-interaction, thus, mediates inhibition of Notch pathway to facilitate neuroendocrine tumorigenesis5. DLL3 is highly upregulated and aberrantly expressed on the cell surface in SCLC and other high-grade neuroendocrine tumors with limited expression in normal tissues6.Here we presented a DLL3-directed ADC, HRA00130-C004, which features a differentiated topoisomerase I inhibitor payload DXh conjugated via a cleavable linker to a humanized IgG1 antibody. HRA00130-C004 strongly inhibited the proliferation of cell lines with different DLL3 expression. In the bystander killing assays, HRA00130-C004 was able to kill both DMS53 cells (DLL3 over-expressed) and U-2OS (DLL3-negative) cells when they are co-cultured. The in vivo treatment of HRA00130-C004 resulted in a dramatic and sustained inhibition of tumor growth in H1184 (DLL3 high) and DMS53 (DLL3 low) xenograft models. No obvious weight loss was observed during the experiment. Furthermore, HRA00130-C004 demonstrated a favorable PK profile and satisfactory molecular integrity in rats with 3mg/kg dosing and cynomolgus monkeys with 10 mg/kg dosing. The exposure of total antibody and intact ADC was consistent. Moreover, HRA00130-C004 was well tolerated in rats and cynomolgus monkeys with no related adverse findings, which indicated its favorable safety profiles. In summary, taking advantage of Hengrui’s DXh platform, HRA00130-C004 has demonstrated great potency and good safety profiles. These data support the future clinical development of HRA00130-C004.

1.CA Cancer J. Clin., 2018, 68, 7; 2.J. Natl. Compr. Cancer Netw. 2018, 16, 1171; 3.Mol. Ther. Oncolytics, 2021, 20, 470; 4.Journal of Cancer, 2022, 13, 2945; 5.Cellular Oncology, 2019, 42, 261; 6.Journal of Hematology and Oncology 2019, 12, 61;

Citation Format: Qingqing Yao, Changding Xue, Zhibin Xu, Lingfeng You, Zhendong Xue, Yuchang Mao, Sophie Lin, Jun Feng, Zhe Zhang, Xin Ye, Min Hu, Feng He. HRA00130-C004, a novel anti-DLL3 ADC with bystander effect, high DAR and favorable safety profiles [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 3146.
Abstract 3168: Amphiphilic dendrimer-mediated siRNA delivery for precision regulation of cAMP pathway in cancer cells
Authors: Yuan Wanjun , Gao Zhengyin , Peng Ling , Lee Leo Tsz On
Published: March 2024

Exchange proteins directly activated by cAMP (EPACs) are involved in multiple cellular processes. The two EPAC isoforms (EPAC1 and EPAC2) have fundamental differences in structure, expression pattern, and signaling specificity, indicating that they may function independently. Therefore, the highly specific regulation of EPAC1 and EPAC2 could be beneficial in targeting cAMP pathway-related therapies. Small RNA molecules could theoretically target genetic components based on Watson-Crick base pairing, offering a unique and promising approach to achieve precision medicine. In this study, we successfully design small interfere (si)RNAs that specifically target EPAC1 and 2, without cross-reactivity with other isoforms. The use of the amphiphilic dendrimer (AD) efficiently delivers siRNAs, resulting in significant down-regulations of EPAC1 and 2 mRNA and protein expressions in multiple cancer cells, including ovarian, breast, colon, and pancreatic cancers. Functional analysis demonstrate that EPAC1 and EPAC2 regulate cancer progression depending on different mechanisms. siEPAC2-AD is more potent compared to siEPAC1-AD in suppressing cell proliferation by altering energy metabolism. Hence, this siEPACs-AD system not only enhances the drug specificity and reduces potential side effects, but also provides a new strategy for precision pharmaceutical control of the cAMP/PKA/EPAC signaling pathway for cancer treatment.

Keywords: EPAC1; EPAC2; siRNA; amphiphilic dendrimer; cAMP signaling; cancer

Citation Format: Wanjun Yuan, Zhengyin Gao, Ling Peng, Leo Tsz On Lee. Amphiphilic dendrimer-mediated siRNA delivery for precision regulation of cAMP pathway in cancer cells [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 3168.
Abstract 3176: Otopetrin 3 promotes cell proliferation and regulates glycolysis via interaction with MYC in colorectal cancer
Authors: Jang Hyeung-Jin , Jee Wona , Park So-Mi , Park Ye-rin , Kim Seok Woo , Jung Ji Hoon
Published: March 2024

Otopetrin 3 (OTOP3) stands out as a pivotal regulator of intracellular pH through its control over proton transportation into cells, affecting fundamental cellular processes. Previous studies have shown that OTOP3 is associated with the prognosis of colorectal cancer (CRC) patients. However, the mechanism underlying OTOP3 regulation remains unclear in cancer. Our investigation reveals OTOP3 as a potent oncogene in CRC, with higher OTOP3 levels correlating with diminished overall survival rates in CRC patients. Comparative analysis between cancerous and normal colon cells highlights a pronounced overexpression of OTOP3 in cancer cells. Suppression of OTOP3 expression significantly curtailed CRC cell proliferation, colony formation, migration, and invasion, concurrently inducing apoptosis and cell cycle arrest, ultimately leading to cancer cell death. Moreover, through CHX, FBS inactivation, and MG132 treatment experiments, it was confirmed that OTOP3 regulates c-Myc expression and stability through ubiquitination and has a critical impact on CRC cell proliferation and growth. Additionally, OTOP3's modulation of HK2, PKM2, and LDHA, underscores its role in impacting cancer cell behavior via glycolytic pathways. In summary, the overexpression of OTOP3 in CRC cells drives cell proliferation and growth through the regulation of c-Myc, impacting crucial cellular processes. Additionally, it promotes these processes by influencing glycolysis. These findings provide valuable insights into potential therapeutic targets for CRC treatment, possibly involving OTOP3 and its associated pathways.

Citation Format: Hyeung-Jin Jang, Wona Jee, So-Mi Park, Ye-rin Park, Seok Woo Kim, Ji Hoon Jung. Otopetrin 3 promotes cell proliferation and regulates glycolysis via interaction with MYC in colorectal cancer [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 3176.
Abstract 3185: Glatiramer acetate enhances tumor retention and mitigates systemic toxicity of toll-like receptor 9 agonists as intratumoral immunotherapy
Authors: Gong Huan , Griffin Daniel J. , Groer Chad E. , Wu Xiaoqing , Abdelaziz Moustafa M. , Wu Sa , Xu Liang , Forrest Laird M. , Berkland Cory J.
Published: March 2024

Pathogen-associated molecular patterns (PAMP) are conserved molecular structures produced by microorganisms that are recognized as foreign by receptors of the innate immune system. Unmethylated cytosine-guanine oligodeoxynucleotides (CpG ODNs) are a storied drug class from the PAMP family that ligates Toll-like receptor 9 (TLR9). CpG ODNs have shown promising antitumor effects in preclinical studies by inducing potent pro-inflammatory immune responses. However, there are still practical challenges that have hindered clinical success, including inefficient in vivo delivery and severe immune-related toxicities caused by systemic exposure to CpG. We previously identified that glatiramer acetate (GA), an FDA-approved, lysine-rich polypeptide, could be applied as a cationic drug delivery vector for CpG delivery proximal to the injection site while mitigating systemic release of pro-inflammatory cytokines. To dive into the mechanism and evaluate pharmacological efficacy in more pathological models that simulate colorectal carcinoma, we validated the biodistribution of GA-CpG complexes (or R4B) and explored their safety and antitumor immunity in multiple mouse colorectal carcinoma models. In the CT26 single tumor model, intratumoral R4B treatment displayed comparable antitumoral efficacy as CpG treatment. Genome-wide transcriptome analysis showed that R4B treatment elicited much more prominent immune system response pathways in both lesions and spleens. The biodistribution study proved that R4B localized and gradually released CpG around the injection site while ‘naked’ CpG diffused away from the injection site quickly. In the CT26 dual tumor-bearing model, intratumoral administration of R4B generated systemic immune efficacy that led to an abscopal effect on distant untreated cancers, comparable to treatment with CpG alone. Notably, R4B treatment accomplished these effects with greatly mitigated systemic pro-inflammatory cytokines compared to CpG alone. These data help further elucidate the noncanonical role of glatiramer acetate to serve as a nucleic acid delivery scaffold that potentiates the safety of CpG adjuvant for cancer immunotherapy.

Citation Format: Huan Gong, Daniel J. Griffin, Chad E. Groer, Xiaoqing Wu, Moustafa M. Abdelaziz, Sa Wu, Liang Xu, Laird M. Forrest, Cory J. Berkland. Glatiramer acetate enhances tumor retention and mitigates systemic toxicity of toll-like receptor 9 agonists as intratumoral immunotherapy [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 3185.
Abstract 3198: An analysis of breast cancer membrane lipid-modified nanoliposomes for enhanced targeting of triple-negative breast cancer
Authors: Ayertey Christiana , Amid Parmida , Almozain Mohammed H. , Campbell Robert B.
Published: March 2024

Background: Every breast cancer diagnosis is unique in its own way. Triple-negative breast cancer (TNBC), has a highly aggressive phenotype characterized by a rapid growth rate profile, increased risk of metastasis and recurrence. TNBC is devoid of epidermal growth factor receptor 2, estrogen, and progesterone receptors. All three are commonly present in other forms of breast cancer disease. The aim of this study was to develop a cell membrane lipid-extracted nanoliposomes (CLENs) for selective targeting compared to conventional nanoliposomal varieties. An additional objective was to investigate the role of microenvironment on targeting using cell model of TNBC, and representations of non-target tissue environments for comparison.

Method: Lipid extracts (LE) obtained from 4T1, a murine mammary carcinoma cell line from a BALB/cfC3H mouse was used to formulate the CLENs (cell membrane lipid-extracted nanoliposomes) with different ratios of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a phospholipid used in most conventional drug delivery liposomes. Different lipid compositions and ratios were employed to evaluate cellular uptake in target, non-target and off-target cells. The target cell line, 4T1, off-target and non-target cell populations included CRL-2089, normal breast fibroblasts, SKBR3 a (human breast cancer), and A549 a (lung cancer) cell lines. Other studies involved determination of molecular charge and zeta potential of various nanoliposomal preparations and corresponding fluorescence studies in vitro.

Results: The physicochemical properties of CLENs such as particle size and surface charge characteristics were vital when assessing benefit of the lipid-based preparations. The size of the CLENs typically ranged between 130 - 165 nm and zeta potential values were negatively-charged. The inclusion of 70 mol% of LE in preparation of 4T1-CLENs demonstrated increased cellular uptake and binding by the 4T1 target cells compared to the control DOPC (100%). The highest degree of selectivity was observed in early cell binding events, and with studies involving more extended incubation time points under different experimental conditions. Off-target cells demonstrated reduced uptake by comparison. Overall, 4T1-CLENs was most efficient when applied against intended target 4T1 cells, when compared to relevant control cell populations. In conclusion, studies performed to date suggest the notion that lipid extracts derived from TNBC improves targeting.

Citation Format: Christiana Ayertey, Parmida Amid, Mohammed H. Almozain, Robert B. Campbell. An analysis of breast cancer membrane lipid-modified nanoliposomes for enhanced targeting of triple-negative breast cancer [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 3198.
Abstract 3254: Development of novel luminescent assays for sensitive and specific quantitation of double-stranded RNA in mRNA vaccines
Authors: Moravec Rich , Wang Jun , Hartnett Jim , Cong Mei , Grailer Jamison
Published: March 2024

mRNA-based therapeutics have shown clinical efficacy, evidenced by the success of the mRNA vaccines for SARS-CoV-2. Personalized mRNA vaccines containing patient-derived tumor mutations are now entering the clinic and have shown promise. Delivery of mRNA-encoded therapeutic proteins (e.g., antibodies) is also under development. mRNA drug substance is typically generated by in vitro transcription (IVT). One byproduct and contaminant of IVT is the formation of double-stranded RNA (dsRNA). dsRNA is highly immunogenic, can induce inflammation and cell death, and inhibit protein translation, including the protein encoded by the therapeutic mRNA. As such, dsRNA must be measured for all IVT products to ensure safety and efficacy. However, existing dot bot and ELISA technology to detect dsRNA is neither sensitive nor quantitative. Herein, we describe the development of novel assays for sensitive and specific quantitation of dsRNA in IVT products and following encapsulation of IVT products in lipid nanoparticles (LNPs). First, NanoBiT® split luciferase technology was applied to detect dsRNA in IVT products. dsRNA binding domains were genetically attached to either SmBiT or LgBiT. In the presence of dsRNA, the SmBiT and LgBiT constructs bind via the dsRNA binding domains, resulting in dimerization and the complementation of functional NanoBiT® luciferase. NanoBiT® then generates light which is proportional to the amount of dsRNA in the sample. The assay detects dsRNA greater than or equal to 30 base pairs and is independent of the size and sequence of the dsRNA contaminant. The assay does not cross-react with single-stranded RNA or DNA and is highly sensitive, with a limit of detection less than 100 pg/ml. As a complementary tool to the biochemical assay, we developed a cell-based bioassay to detect dsRNA encapsulated in LNPs. dsRNA/LNPs are taken up by endocytosis where Toll-like receptor 3 (TLR3) detects dsRNA. Activation of TLR3 initiates a signaling pathway that is detected by a TLR3 pathway-specific promoter driving luciferase expression. This pathway likely represents the most physiologically-relevant readout of dsRNA bioactivity. Both assays are developed in “add-mix-read” format with no wash steps or media transfer, and exhibit robust, sensitive, and repeatable performance for specific detection of dsRNA in mixed solutions.

Citation Format: Rich Moravec, Jun Wang, Jim Hartnett, Mei Cong, Jamison Grailer. Development of novel luminescent assays for sensitive and specific quantitation of double-stranded RNA in mRNA vaccines [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 3254.
Abstract 3266: Plk1 promotes Pdcd4 degradation to enhance enzalutamide resistance in prostate cancer
Authors: Wang Qing , Zhang Qiongsi , Zhang Yanquan , Liu Xiaoqi , Yang Hsin-Sheng
Published: March 2024

The androgen receptor (AR) signaling pathway plays critical role in the development of prostate cancer (PCa), including castration-resistant prostate cancer (CRPC). As a result, androgen signaling inhibitors (ASIs) like abiraterone and enzalutamide (ENZ) have emerged as the first-line treatment for CRPC. Nevertheless, the limited success of ASIs underscores the urgency of developing new approaches for managing CRPC patients who have become unresponsive to ASIs. Programmed cell death 4 (Pdcd4) is a tumor suppressor, which is frequently down-regulated during PCa progression. It has been shown that reduced Pdcd4 expression promotes both AR-dependent and AR-independent growth in PCa. In this study, we found that down-regulation of Pdcd4 led to acquisition of ENZ resistance in PCa. We also observed that down-regulation of Pdcd4 resulted in activation of mTORC2 activity. Additionally, knockdown of Rictor, a component of mTORC2 complex, enhanced sensitivity to ENZ treatment in PCa. These findings suggested that activation of mTORC2 due to Pdcd4 loss contributes to ASI resistance in PCa. To understand how Pdcd4 is down-regulated in the PCa, we identified that polo-like kinase (Plk1) phosphorylated Pdcd4 at Ser239. Phosphorylation of Pdcd4 by Plk1 led to ubiquitin-mediated proteasomal degradation of Pdcd4. Over-expression of the phosphomimic Pdcd4(S239D) in 22Rv1 cells promoted tumor cell growth in cultured cells and mice. Taken together, our data suggest that targeting Plk1/Pdcd4/mTORC2 signaling axis holds promise as a strategy to overcome ENZ resistance in PCa.

Citation Format: Qing Wang, Qiongsi Zhang, Yanquan Zhang, Xiaoqi Liu, Hsin-Sheng Yang. Plk1 promotes Pdcd4 degradation to enhance enzalutamide resistance in prostate cancer [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 3266.
Abstract 3286: Synergistic impact of long non-coding RNA TUG1 depletion in overcoming temozolomide chemoresistance in glioblastoma
Authors: Kito Hidekazu , Kibe Yuji , Suzuki Miho , Shinjo Keiko , Kondo Yutaka
Published: March 2024

Glioblastoma (GBM) stands out as the most prevalent and aggressive primary brain tumor in adults, marked by a high mortality rate. Standard therapy involves temozolomide (TMZ) chemotherapy, an alkylating agent. However, most cases develop resistance to TMZ, necessitating the development of new treatment options to overcome TMZ-resistant GBM. Long non-coding RNAs (lncRNAs) constitute a class of RNA molecules that, despite not encoding proteins, play pivotal roles in regulating diverse cellular processes. Overexpression of specific lncRNAs in cancer is linked to crucial aspects of cancer progression, such as cell proliferation, apoptosis, and metastasis. Taurine upregulated gene 1 (TUG1) is a highly expressed lncRNA in GBM, playing a crucial role in preventing excessive DNA replication stress and associated DNA damage (Nat Commun 2023).This study aimed to investigate the combined effects of TUG1 depletion and TMZ in TMZ-resistant GBM. First, we established a TMZ-resistant U251MG cell line, U251MG-R, characterized by a low DNA methylation level of the O6-methylguanine-DNA methyltransferase (MGMT) promoter. We observed that TMZ increased DNA replication stress in the U251MG-R. Depleting TUG1 by using antisense oligonucleotide (TUG1-ASO) sensitized U251MG-R cells to TMZ. Treatment with TMZ or TUG1-ASO alone inhibited cell proliferation, with the most significant inhibition observed when both were combined. Next, we employed a xenograft mouse model to evaluate the efficacy of intravenous TUG1-ASO treatment coupled with a tumor-specific drug delivery system (antiTUG1-DDS). The combination of antiTUG1-DDS with TMZ displayed the most effective suppression of tumor growth compared to either TMZ or antiTUG1-DDS alone. Targeted therapy aimed at TUG1 may be a promising approach for overcoming TMZ resistance in GBM.

Citation Format: Hidekazu Kito, Yuji Kibe, Miho Suzuki, Keiko Shinjo, Yutaka Kondo. Synergistic impact of long non-coding RNA TUG1 depletion in overcoming temozolomide chemoresistance in glioblastoma [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 3286.
Abstract 331: Ion TorrentTM NGS sequencing of the TERT promoter hotspots with Ion AmpliSeqTM HD technology
Authors: Sharma Anupma , Luo Guobin , Li Na , Pea Giorgio , Hyland Fiona
Published: March 2024

Introduction: Recurrent mutations C228T and C250T in the TERT gene promoter are prevalent in cancer and serve as important biomarkers for aggressive disease. Ion Torrent™ NGS sequencing, combined with Ion AmpliSeqTM HD technology, enables comprehensive genomic profiling with ultra-high sensitivity for low-frequency variants in cell-free DNA (cf-DNA) and highly heterogeneous solid tumor samples. However, the high GC content (>80%) and the presence of G repeats that are known to adopt a compact stacked three-G-quadruplex conformation in the TERT gene promoter region pose sequencing challenges such as low read depth and strand-bias, i.e. prematurely truncated reads on one strand.

Methods: We designed nine AmpliSeqTM HD panels with amplicon sizes of 70 to 104 bp, ideal for cf-DNA and FFPE samples, to target the recurrent TERT promoter hotspots. The designs were tested alone and combined with a larger Ion AmpliSeqTM HD DNA research panel resulting in highly multiplexed libraries. The libraries were used for Ion 540TM chip template preparation on the Ion ChefTM instrument and subsequently sequenced using the Ion GeneStudioTM S5 System. Sequencing data was used to evaluate coverage of the target hotspots by each design for each condition, and additional panels were designed to test single amplicons covering both TERT promoter hotspots.

Results: Through performance evaluation, we selected two overlapping amplicons, each effectively amplifying one of the two hotspots. Individually tested, these amplicons yielded approximately 3000 families each from a 10ng gDNA input and when combined into the larger Ion AmpliSeqTM HD DNA research panel, they produced several thousand reads across various libraries, providing molecular coverage of around 200 functional molecules. While capture efficiency was lower compared to TERT amplicon-only libraries, these improved amplicons allow downstream analysis of variants. A single-pool solution (single amplicon/s covering both hotspots) will be explored further using the second batch of amplicons/primers.

Conclusions: The optimized amplicons in two pools developed in this study enable the detection of challenging yet clinically significant TERT promoter hotspot mutations using the ultra-sensitive Ion AmpliSeqTM HD technology.

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Citation Format: Anupma Sharma, Guobin Luo, Na Li, Giorgio Pea, Fiona Hyland. Ion TorrentTM NGS sequencing of the TERT promoter hotspots with Ion AmpliSeqTM HD technology [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 331.
Abstract 3324: Discovery of a novel SOS1 inhibitor, AIG01012, targeting pan-KRAS mutant cancers
Authors: Kim Jihye , Park Sejeong , Kim Sunkyu , Lee Sanghoon , Yoo Kiwoong , Lee Ho-Jeong , Kang Jaewoo , Lee Kwang-Ok
Published: March 2024

Targeting the SOS1-KRAS interaction represents a pivotal strategy in mitigating aberrant KRAS signaling, which is implicated in numerous malignancies. At AIGEN Sciences, Inc., we have harnessed an advanced human-in-the-loop (HITL) AI-based drug discovery platform (AIGEN ChemTailor). This innovative platform allows online learning through expert feedback, optimizing our drug development endeavors. We designed a potent SOS1 inhibitor, AIG01012 using AIGEN ChemTailor in combination with fragment-based drug discovery (FBDD) and molecular modeling techniques. We conceived numerous compounds and shortlisted the leading candidates for extensive testing after meticulous computer simulations. AIG01012 manifested substantial inhibitory effects against several KRAS mutant interactions with SOS1, specifically SOS1-KRAS WT, SOS1-KRAS G12C, SOS1-KRAS G12D, SOS1-KRAS G12V, and SOS1-KRAS G13D. However, its inhibitory capacity did not extend to the SOS2-KRAS G12C interaction. AIG01012 demonstrated remarkable inhibitory effects in 3D cell proliferation assays across various cell lines, including H358, MIA-PaCa2, SW837, AsPC-1, A427, SW620, Capan-2, A549, DLD-1, MKN-1, and H1666. Moreover, AIG01012 showed synergistic antiproliferative effects when combined with approved drugs such as sotorasib (for lung cancer with KRAS mutations), trametinib (a MEK inhibitor), and Osimertinib (targets specific EGFR mutations) in either H358 or PC-9 cell lines. Mouse studies further confirmed its favorable pharmacological properties, and its efficacy was evaluated in subsequent mouse tumor model. In summary, utilizing our human-in-the-loop (HITL) AI platform, AIGEN ChemTailor, we have efficiently derived AIG01012 as a potent SOS1 inhibitor that targets a wide range of KRAS interactions. With its notable in vitro and in vivo outcomes, AIG01012 is set for further exploration and prospective clinical trials as a significant contender in KRAS-targeted cancer treatments.

Citation Format: Jihye Kim, Sejeong Park, Sunkyu Kim, Sanghoon Lee, Kiwoong Yoo, Ho-Jeong Lee, Jaewoo Kang, Kwang-Ok Lee. Discovery of a novel SOS1 inhibitor, AIG01012, targeting pan-KRAS mutant cancers [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 3324.
Abstract 3326: PMR-116, a second generation RNA Polymerase I inhibitor
Authors: Furic Luc , Hein Nadine , Ferreira Rita , Panov Konstantin , Huglo Alisee , Rebello Richard , Kusnadi Eric , Drygin Denis , Haddach Mustapha , Hannan Ross
Published: March 2024

Introduction: Inhibition of RNA Polymerase I, the enzyme responsible for the synthesis of ribosomal RNAs, the nucleic scaffold of ribosomes, has been proven as a novel therapeutic target as most cancer cells are "addicted" to ribosome biogenesis.

Methods: In collaboration with Pimera Inc, we developed and tested the efficacy of a second generation RNA polymerase I inhibitor, PMR-116, in various pre-clinical cancer models. On-target activity of PMR-116 was confirmed in peripheral blood cells isolated from patients with solid tumors enrolled in a phase I trial of PMR-116.

Results: PMR-116 potently inhibits Pol I transcription with ~200 fold higher selectivity towards Pol I compared to Pol II transcription. PMR-116 inhibits rRNA synthesis by stalling the Pol I complex at the rDNA promoter preventing promoter escape and consequently elongation. PMR-116 anti-cancer activity was evaluated against a panel of over 100 cancer cell lines representative of 20 major types of solid and haematological malignancies. This new Pol I inhibitor exhibited broad anti-proliferative and cytotoxic activity with an IC50 ranging from 32-4500nM and a median IC50 of 300nM. In contrast, cells derived from normal tissues were significantly less sensitive (IC50 ranging from 6-33μM).

In vivo, PMR-116 significantly improved survival and reduce tumor burden in several preclinical models such as Vk*MYC transgenic model of indolent multiple myeloma, CT26 xenograft model of colorectal cancer, MMTV-PyMT transgenic model of metastatic breast cancer, mixed-lineage leukemia (MLL)-eleven nineteen leukemia (ENL)+Nras acute myeloid leukemia (+/-p53 mutation) and patient-derived xenografts of prostate cancer and AML.

In a phase I trial of PMR-116, on-target activity of PMR-116 was observed in cells derived from the peripheral blood.

Conclusion: PMR-116 significantly reduced cancer growth in a wide range of tumor models, including GEMM, syngeneic and PDX models. PMR-116 is undergoing clinical testing in phase I in solid tumors with the objective to determine maximum tolerated dose and identify a phase II dose.

Citation Format: Luc Furic, Nadine Hein, Rita Ferreira, Konstantin Panov, Alisee Huglo, Richard Rebello, Eric Kusnadi, Denis Drygin, Mustapha Haddach, Ross Hannan. PMR-116, a second generation RNA Polymerase I inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3326.
Abstract 3338: Development of a CK2α inhibitor for treatment of wnt-driven colorectal cancer
Authors: Cawkill Darren
Published: March 2024

A novel, potent and highly selective orally bioavailable small molecule inhibitor of Casein Kinase 2α (CK2α) has been developed and validated in pre-clinical models of wnt-driven colorectal carcinoma; and is poised to enter clinical development.CK2α is considered as a positive regulator of wnt signaling and tumorigenesis. The wnt pathway is known to be sensitive to and amplified by CK2α activity at multiple nodes and can be inhibited by loss of CK2α function. The human genetics of colorectal cancer (CRC) have been well characterized and approximately 80% tumors are identified as being wnt pathway mutation driven. CRC tumors can be further stratified into consensus molecular subtypes (CMS), with iCMS2 representing 37% of CRC patients, being characterized by ongoing wnt pathway upregulation. Development of CK2α inhibitors as a targeted therapy for CRC is therefore mechanistically well aligned to the wnt signaling drive and may serve as a promising therapeutic strategy for this cancer. A novel small molecule binding site on CK2α has been exploited to design and develop an inhibitor with Ki of 95 pM and exquisite selectivity over the kinome. This inhibitor has been comprehensively characterized using in vitro, ex vivo and in vivo pharmacology studies. Cellular mechanism of action studies in CRC cell lines have quantified the potency of target engagement, inhibition of known CK2α phospho-sites, inhibition of tumorigenic wnt pathway activity, and induction of apoptosis. This CK2α inhibitor was also profiled in a high content screen of CRC patient-derived organoids established from various driver mutation backgrounds. Use of these ex vivo models has validated the potent cytotoxic activity of CK2α inhibition in tumor samples with wnt-driven characteristics and highlights the potential therapeutic efficacy of CK2α inhibition in this setting. In vivo CRC xenograft studies have confirmed anti-tumor efficacy and developed the understanding of pharmacokinetic pharmacodynamic relationship between free plasma concentration of drug and inhibition of CK2α-specific biomarker in the tumor. The preclinical data presented establishes the strong rationale for clinical development of a novel CK2α inhibitor for the treatment of locally advanced or metastatic solid tumors, with an emphasis on wnt-driven CRC.

Citation Format: Darren Cawkill. Development of a CK2α inhibitor for treatment of wnt-driven colorectal cancer [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 3338.
Abstract 336: Genome integrity assessment by optical genome mapping for cell manufacturing/bioprocessing applications
Authors: Hastie Alex , Pang Andy W. , Chaubey Alka
Published: March 2024

Cells and cell lines are used for multiple applications such as bioprocessing, therapy, and research. To ensure quality of cells and downstream applications, appropriate quality control (QC) methods are critical. Historically, karyotyping has been employed, but it is limited by its very low resolution and tedious laboratory workflow. Other methods such as PCR and targeted sequencing can characterize only small genomic variants at specific loci. Whole genome sequencing can detect small variants genome-wide but has limited sensitivity in detecting structural variants (SVs). Optical genome mapping (OGM) is a novel genome analysis technique that can fill many of the gaps in current capabilities for assessing genome integrity. To find clonal variants, 400 Gbp of data is collected from the parental/control and test samples. For both samples, a de novo assembly is constructed, and homozygous or heterozygous SVs are assessed. Subsequently, the dual variant annotation pipeline identifies unique SVs in the test sample compared to the parental sample. To discover subclonal SVs down to 5% variant allele fraction (VAF), the workflow leverages the generation of 1.5 Tbp of data, requiring a simple adjustment to longer data collection time. Comparison of test sample to parental sample enables easy determination of acquired variants. Finally, generation of ~5 Tbp of data, requiring 2-3 flowcells to be run, enables the detection of SVs at ultra-low VAF down to ~1%. In this study, several dilutions and simulations were performed to examine OGM’s limit of detection. Targeting a coverage of 5 Tbp and analysis using the somatic SV-analysis workflow revealed that OGM has the sensitivity to detect deletions >50kbp, insertions >20kbp, duplications >100 kbp, and translocations at ~1% VAF. We have applied the clonal and somatic workflows to verify genomes’ integrity after cell immortalization, induced pluripotency, transgene-integration, and gene-editing. The data using the robust and sensitive workflows demonstrate that the OGM platform is a cost-effective solution for cell manufacturing/bioprocessing QC applications.

Citation Format: Alex Hastie, Andy W. Pang, Alka Chaubey. Genome integrity assessment by optical genome mapping for cell manufacturing/bioprocessing applications [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 336.
Abstract 3366: FoRx-06-428 is a novel PARG inhibitor with potent anti-tumor efficacy in preclinical cancer models
Authors: Querolle Olivier , Lücking Ulrich , Iacovino Luca , Freudenmann Alena , Rossetti Giacomo , Gysin Marina , Bologna Serena , Dionellis Vasilis , Malattia Marta , Potenza Alessandro , Bocquet Nicolas , Konstantinova Irena , Goutopoulos Andreas , Sotiriou Sotirios , Halazonetis Thanos , Bashir Tarig , Zenke Frank
Published: March 2024

The formation of poly(ADP-ribose) (PAR) chains from NAD+ precursors by PARP enzymes is a characteristic posttranslational modification during DNA damage repair. PAR chains function as docking platforms for DNA repair proteins that are required to resolve DNA lesions. Equally important is the subsequent removal of PAR chains from protein substrates to conclude the repair process and restore genomic integrity. Poly(ADP-ribose) glycohydrolase (PARG) is the key enzyme in this process and has recently garnered significant attention as a novel cancer target. FoRx-06-428 is a novel, orally bioavailable and highly potent, low molecular weight PARG inhibitor. It binds to the catalytic domain of human PARG with high affinity, as evidenced by surface plasmon resonance (SPR), and displays potent inhibition of enzymatic activity. FoRx-06-428 shows selective reduction of viability in a panel of cancer cell lines highlighting therapeutic potential in multiple cancer indications. Pharmacological inhibition of PARG leads to accumulation of PAR chains and is indicative of target engagement, accompanied by modulation of DNA damage response markers, such as phosphorylation of replication protein A (pRPA) and phosphorylation of H2AX (γH2AX). Compelling anti-tumor efficacy as single agent was observed in several xenograft models (breast and gastric cancer) with excellent tolerability. Dose-dependent accumulation of PAR chains was demonstrated in vivo and constitutes a tractable target engagement biomarker for clinical exploration. Sensitivity to PARG inhibition is partly driven by distinct genomic instabilities, such as defects in homologous recombination (HR) repair, however the activity of PARG inhibitors is also influenced by aberrations in other cancer-relevant pathways. This report represents the first disclosure of the pharmacological characterization of FoRx-06-428 and highlights the value of PARG inhibition as a novel targeted approach to treat DNA repair-deficient cancers.

Citation Format: Olivier Querolle, Ulrich Lücking, Luca Iacovino, Alena Freudenmann, Giacomo Rossetti, Marina Gysin, Serena Bologna, Vasilis Dionellis, Marta Malattia, Alessandro Potenza, Nicolas Bocquet, Irena Konstantinova, Andreas Goutopoulos, Sotirios Sotiriou, Thanos Halazonetis, Tarig Bashir, Frank Zenke. FoRx-06-428 is a novel PARG inhibitor with potent anti-tumor efficacy in preclinical cancer 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 3366.