bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2764: Application of NanoBRET target engagement cellular assay for development of inhibitors against protein kinases, RAS, and epigenetic proteins in cancer therapy
Authors: Wu Jianghong , Wang Yuan , McGinley Shawn , Gallagher Peter , Liang Li , Lanni Alisha Di , Miles Ashley , Haque Mohammad , Wan Yong , Ma Haiching
Published: April 2023

NanoBRET target engagement (TE) is the first biophysical technique that broadly enables the quantitative determination of protein inhibitor occupancy in live cells, without disruption of cellular membrane integrity. This quantitative capability is achieved in live cells via BRET with an optimized set of cell-permeable tracers, allowing the measurement of compound binding to a selected cellular target protein. Our NanoBret assay is scaled to 384-well plate format and Z’ analysis demonstrated the assay to be High Throughput Screening compatible. Protein kinases, RAS, and epigenetic proteins are very important drug targets. NanoBRET TE cellular assay has enabled the development of live-cell quantitative compound binding assays for >360 individual wild type and mutant protein kinases including serine/threonine-specific kinases, receptor tyrosine kinases, and all 20 cyclin-dependent kinases (CDKs). RAS is a well-known oncogene that is frequently mutated in most lung, pancreatic and colorectal cancers and is associated with poor disease prognosis. The recent discovery of a unique switch II binding pocket and successful inhibition of the KRAS (G12C) mutant by covalent inhibitors have led to the resurgence of interest in the design of inhibitors targeting RAS directly. Our data demonstrate that NanoBRET TE cellular assay can measure the apparent affinity of RAS inhibitors by competitive displacement of a NanoBRET RAS switch I/II pocket tracer, reversibly bound to the LgBiT- and SmBiT- KRAS, KRAS (G12C), KRAS (G12D), KRAS (G12V), KRAS (G13D), KRAS (Q61H), KRAS (Q61L), KRAS (Q61R), KRAS-G12A, KRAS-G12S, HRAS, HRAS-G12C, and HRAS-G12V fusion constructs co-transfected in live HEK293 cells. Our data also show NanoBRET cellular assay can be used to measure compound binding affinity to epigenetic drug target proteins such as BRD4, BRD4-BD1, BRD4-BD2, HDAC6, HDAC1, HDAC2, and HDAC3. Taken together, our results suggest NanoBRET TE cellular assay can serve as a great tool to facilitate new drug discovery against human cancers.

Citation Format: Jianghong Wu, Yuan Wang, Shawn McGinley, Peter Gallagher, Li Liang, Alisha Di Lanni, Ashley Miles, Mohammad Haque, Yong Wan, Haiching Ma. Application of NanoBRET target engagement cellular assay for development of inhibitors against protein kinases, RAS, and epigenetic proteins in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2764.
Abstract 2790: Preclinical studies of ZG19018, a novel irreversible covalent inhibitor of KRAS G12C, for the treatment of advanced non-small cell lung cancer and other solid tumors
Authors: Zhu Bing , Cui Dawei , Liu Ruifeng , Feng Weidong , Sheng Zelin , Lv Binhua
Published: April 2023

Growing evidences of covalent irreversible inhibitors in clinical treatments of solid tumors with KRAS G12C mutation have recently emerged after FDA-approved AMG510 (Sotorasib) for NSCLC chemotherapies. ZG19018 is an irreversible KRAS G12C inhibitor with novel structure that shows promising preclinical results for in vitro molecular and biological studies and in vivo G12C-mutated cancer models and safety studies in animals. For instance, ZG19018 rapidly and selectively forms covalent bonds with G12C-GDP, which accounts for lock-in KRAS protein as the inactive GDP-bound state but inhibits the activation of numerous downstream signaling pathways like RAF/MEK on ERK phosphorylation. ZG19018 showed highly inhibition of a 3D growth panel for human cancer cell lines harboring the KRAS G12C mutation, e.g. H358 (NSCLCs), MIA PaCa-2 (pancreatic) and SW837 (colorectal), etc. For pharmacodynamics, a battery of in vitro and in vivo ADME/DMPK studies indicated a high plasma protein binding (>99.6%) of ZG19018 with over 40% bioavailability in rat and dog via oral administration for the adequate drug levels in various tissues including lung, crossing BBB to reach brain, and accumulation in tumors. For in vivo anti-tumor efficacy, ZG19018 exhibited significant dose-dependent inhibition of tumor growth of G12C-driven subcutaneous models in nude mouse like H358 NSCLC cells but induced further tumor regression in pancreatic cancer MIA PaCa-2 xenograft. ZG19018 also showed an enhanced anti-tumor activities as combined with chemo-reagent Donafinib, a broad tyrosine kinase inhibitor blocking RAF/MEK/ERK signaling pathways of tumor cells, in the same H358 xenograft model. The general toxicology studies in rat under 28 days repeated oral administration (q.d.) showed NOAEL and STD10 values at 75 and 150 mg/kg/day, respectively. The safety pharmacological studies showed no notable effects of ZG19018 on the cardiovascular, respiratory and CNS systems. The genotoxic potential studies indicated ZG19018 without mutagenesis. Collectively, ZG19018 is now in phase I/II clinical trials for the treatments of solid tumors with KRAS G12C mutation in China.

Citation Format: Bing Zhu, Dawei Cui, Ruifeng Liu, Weidong Feng, Zelin Sheng, Binhua Lv. Preclinical studies of ZG19018, a novel irreversible covalent inhibitor of KRAS G12C, for the treatment of advanced non-small cell lung cancer and other solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2790.
Abstract 2793: Preclinical pharmacokinetics in cynomolgus monkeys and first in human dose prediction of DB-1303, a HER2-targeting antibody-drug conjugate
Authors: Shi Rong , Lin Shengchao , Zhang Yu , Li Bing , Hua Haiqing , Qiu Yang
Published: April 2023

Background: DB-1303 is a novel antibody-drug conjugate comprised of trastuzumab biosimilar, enzymatically cleavable peptide-linker, and a proprietary topoisomerase I inhibitor P1003. It is designed to have high plasma stability, low free payload in circulation and wide therapeutic index. Herein, the preclinical pharmacokinetics (PK) characteristics of DB-1303 in cynomolgus monkeys is reported, and human PK exposure in a first in human study was predicted using population PK model with allometric scaling.

Material and Methods: The PK parameters of DB-1303 were obtained from single PK and repeated TK studies in female and male cynomolgus monkeys. Concentrations of DB-1303 ADC, total antibody and released payload P1003 were measured using ELISA or LC-MS/MS. In a PK study, single dose of DB-1303 at 1, 3 and 10 mg/kg were determined in cynomolgus monkeys via IV infusion. In TK studies, cynomolgus monkeys were intravenously administered with DB-1303 at 50, 80 mg/kg once every 3 weeks for 2 repeated doses, or 10, 40, 80 mg/kg once every 3 weeks (Q3W) for 3 repeated doses followed by a 4-week recovery. A population PK model was developed based on the collective PK/TK data in monkeys.

Results: The non-compartment analysis PK parameters of three analytes (DB-1303 ADC, total antibody and released payload P1003) are summarized following single and multiple doses of DB-1303 in cynomolgus monkeys. To predict the human PK for DB-1303, a target mediated drug disposition (TMDD) model was constructed, incorporating binding to serum HER2 and subsequent elimination of the complex into a standard 2-compartmental PK model. The 2-compartment linear parameters were scaled from cynomolgus monkey population PK parameters using allometric scaling exponents of 1 for volumes and 1 for clearance. The binding of DB-1303 to HER2 was incorporated into the model and assumed to be the same for HER2 ECD and transmembrane domain. The PK profiles for DB-1303 in human at the proposed doses in the first in human study are predicted. The safety margins from the monkey HNSTD for each proposed human dose are calculated.

Conclusions: DB-1303 exhibited favorable PK characteristics of DB-1303 in cynomolgus monkeys, and is predicted to have large safety margins at the selected doses in the first in human study. At the time of presentation, the first-in-human phase 1/2 study in patients with advanced solid tumors is in progress (NCT05150691).

Citation Format: Rong Shi, Shengchao Lin, Yu Zhang, Bing Li, Haiqing Hua, Yang Qiu. Preclinical pharmacokinetics in cynomolgus monkeys and first in human dose prediction of DB-1303, a HER2-targeting antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2793.
Abstract 28: In vivoscreening platform based on Ba</>F3 kinase engineered cell lines for discovery of next-generation kinase inhibitors
Authors: Liu Tongtong , He Feng , Duan Xuyang , Li shuliang , Liu Chang , Ning Jinying , Hao Feng
Published: April 2023

Protein kinases have become very popular targets for cancer and other diseases therapy, with more than 70 kinase inhibitor drugs approved by FDA since 2001. Due to innate or acquired drug resistance of tumors, most of these small-molecule inhibitors can only delay tumor progression. Next-generation kinase inhibitors with better specificity and lower drug resistance remain to be developed. Ba/F3 is a mouse pro-B cell line which is dependent on IL-3 for survival and proliferation. Upon transduction of a driver gene such as kinase genes or their mutants, Ba/F3 cells switch from IL3-dependent to driver gene-dependent, which makes Ba/F3 cells a powerful tool for the discovery of new kinase inhibitors. Our group has constructed more than 500 Ba/F3 engineered cell lines stably transfected with mutants of kinase genes. These Ba/F3 kinase cell lines are well validated by sequencing, western blot and inhibitor test, and cover many hot kinases, including EGFR (151 cell lines), FGFR (45 cell lines), ERBB2 (38 cell lines), KRAS (36 cell lines), BCR-ABL (36 cell lines), EML4-ALK (33 cell lines), KIF5B-RET (30 cell lines), FLT3 (26 cell lines) et.al. Most of these transformed Ba/F3 cell lines can be used in allograft models in immune-deficient mice. Based on these Ba/F3 kinase cell line allograft models, we established an in vivo screening platform to evaluate the efficacy and toxicity of drug candidatestargeting specific mutant types of kinases, as well as comparisons with previous generation drugs. Overall, our data indicate that Ba/F3 kinase cell line-derived allograft models are a powerful model for the discovery of next-generation kinase inhibitors.

Citation Format: Tongtong Liu, Feng He, Xuyang Duan, shuliang Li, Chang Liu, Jinying Ning, Feng Hao. In vivoscreening platform based on Ba</>F3 kinase engineered cell lines for discovery of next-generation kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 28.
Abstract 2805: Pharmacogenomic platform using patient-derived cells to delineate tumor heterogeneity and therapeutic response of refractory lung cancer
Authors: Park Charny , Hong Sehwa , Hwang Mihwa , Lee Youngjoo , Song Bo Ram , Kang Eun Hye , Sim Hanna , Ahn Beung-Chul , Hwang Kum Hui , Yu Namhee , Kim Sunshin , Han Ji-Youn
Published: April 2023

A pharmacogenomic platform using patient-derived cells (PDCs) was established to identify the underlying resistance mechanisms and tailored treatment for advanced or refractory lung cancer patients. Drug sensitivity screening was acquired from short-term PDC culture and multi-omics datasets were acquired from lung cancer PDCs (n = 199). Integrative analysis was performed to explore susceptible drug candidates from genetic variants, gene expression, and clinical profiles. To demonstrate PDC heterogeneity and its therapeutic response, cellular subpopulation was identified from single cell transcriptome from the patients (n = 8) harboring EGFR mutations with poor response to EGFR-TKIs. Mutation and gene expression profile of our PDC samples conserved with lung cancer tissue. Poor survival mutations were observed in KRAS, PDGFA, KEAP1, and MET. EGFR rare variants facilitated fast progression as well as poor susceptibility in EGFR-TKIs. PDC therapeutic subtypes classified patients into five groups: (1) epithelial-to-mesenchymal transition (EMT)-like (30.6 %), (2) EGFR-dominant (21.4 %), (3) stemness (19.4 %), (4) angiogenesis (17.3 %), and (5) inflammatory (11.2 %). The subtypes implicated in distinct pathway regulation activation,

variant enrichment, pathologic type, and survival as well as therapeutic candidates. Meanwhile, single cell transcriptome uncovered IDO1 and MET activation of EGFR-TKI resistance cancer cells. Stromal cell clusters exhibited the variety according to combinatorial immunotherapy response. While VEGFA+, and TOP2A+ clusters exhibited well response to angiogenesis and DNA damage target drug combinations of patients, CALB2+ cluster was clearly abundant to non-responder patients. We could detect angiogenesis (dasatinib) and DNA damage (irinotecan) drug candidates according to stromal clusters’ scoring from bulk PDC samples. Our PDC platform recapitulated the molecular characteristics of solid lung tumor tissues including tumor microenvironment. Uncovered molecular mechanism implied drug candidates.

Citation Format: Charny Park, Sehwa Hong, Mihwa Hwang, Youngjoo Lee, Bo Ram Song, Eun Hye Kang, Hanna Sim, Beung-Chul Ahn, Kum Hui Hwang, Namhee Yu, Sunshin Kim, Ji-Youn Han. Pharmacogenomic platform using patient-derived cells to delineate tumor heterogeneity and therapeutic response of refractory lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2805.
Abstract 2814: The mutational basis of cancer's vulnerability to ionizing radiation
Authors: Gopal Priyanka , Abazeed Mohamed E.
Published: April 2023

Introduction: Large-scale sequencing efforts have established that cancer-associated genetic alterations are highly diverse, posing a challenge to the identification of variants that regulate complex phenotypes like radiation sensitivity. The impact of the vast majority of rare or common genetic variants on the sensitivity of cancers to radiotherapy remains largely unknown.

Methods: We developed a scalable gene editing and irradiation platform to assess the role of categories of variants in cells. Variants were prioritized based on genotype-phenotype associations from a previously completed large-scale cancer cell line radiation profiling study. Altogether, 488 alleles (396 unique SNVs) from 92 genes were generated and profiled in an immortalized lung cell line, BEAS-2B. We validated our results in other cell lines (TRT-HU1 and NCI-H520), in vivo via the use of both cell line and patient-derived murine xenografts, and in clinical cohorts.

Results: We show that resistance to radiation is characterized by substantial inter- and intra-gene allelic variation. Some genes (e.g. KEAP1) demonstrated significant intragenic allelic variation in the magnitude of conferred resistance and other genes (e.g. CTNNB1) displayed both resistance and sensitivity in a protein domain-dependent manner. We combined results from our platform with gene expression and metabolite features and identified the upregulation of amino acid transporters that facilitate oxidative reductive capacity and cell cycle deregulation as key regulators of radiation sensitivity.

Conclusions: Our results reveal new insights into the genetic determinants of tumor sensitivity to radiotherapy and nominate a multitude of cancer mutations that are predicted to impact treatment efficacy.

Citation Format: Priyanka Gopal, Mohamed E. Abazeed. The mutational basis of cancer's vulnerability to ionizing radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2814.
Abstract 2831: Enhanced efficacy of HDAC inhibitor panobinostat and ONC206 combined with radiation therapy against human glioblastoma cell lines without H3K27M mutation
Authors: Tajiknia Vida , Deiry Wafik El , Zhou Lanlan
Published: April 2023

Background: Glioblastoma multiforme (GBM) is the most common form of malignant brain tumor in adults. The prognosis of GBM is still poor despite first-line therapy, and the median overall survival (OS) is 12-15 months, while the 5-year survival does not exceed 5%. Despite aggressive initial treatment, most patients develop recurrent diseases. Novel drug combinations for malignant CNS tumors are under investigation. Panobinostat is a histone deacetylase inhibitor (HDACi) with antineoplastic and antiangiogenic effects in glioma, and we previously noted synergy with combination of panobinostat and imipridone ONC206. ONC206 is under clinical investigation for treating adult and pediatric brain tumor patients. We decided to investigate the triple combination of ONC206, Panobinostat and radiation against glioma cells.

Materials & Methods: We investigated cell viability and drug synergies of ONC206 plus Panobinostat with or without radiation in 4 human GBM cell lines at 72 hours.

Results: We identified synergy between impridone ONC206 and Panobinostat which improved with radiation therapy IC50 values of panobinostat and ONC206 against cell lines U138, U87, T98G & U251MG ranges from 29-85 nM & 0.8-1.2 μM, respectively. Synergies between ONC206 and Panobinostat were observed across cell lines with combination indices of <0.5 indicating strong synergy. When treating with 4GY radiation after 48 h, combination index improved. documenting enhanced tumor cell killing of human glioma cells.

Conclusion: Our ongoing studies reveal synergy between ONC206, Panobinostat and radiation against GBM cell lines. The enhanced synergy with radiation provides an opportunity to develop a novel treatment combination for high grade glioma with a focus on recurrent GBM.

Citation Format: Vida Tajiknia, Wafik El Deiry, Lanlan Zhou. Enhanced efficacy of HDAC inhibitor panobinostat and ONC206 combined with radiation therapy against human glioblastoma cell lines without H3K27M mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2831.
Abstract 3011: Psychosocial stress, social support, and incident breast cancer by estrogen receptor status
Authors: Lawrence Wayne R. , McDonald Jasmine A. , Williams Faustine , Shiels Meredith S. , Freedman Neal D. , Lin Ziqiang , Magnani Jared W.
Published: April 2023

Introduction: Chronic stress affects immune function and hormonal signaling and has been hypothesized to be associated with breast cancer, although results from the few prior studies are mixed and have not examined potential differences by estrogen receptor (ER) status. We investigated the relationship between stressful life events and incident breast cancer by ER status among postmenopausal women. We further evaluated whether this association is modified by social support.

Methods: Our analysis included 76,951 postmenopausal women enrolled in the Women’s Health Initiative. Participants were followed for events for up to 26 years (median, 16.7). We generated Cox proportional hazards models adjusting for demographic, clinical, lifestyle/behavioral, and social factors to estimate hazard ratios (HR) and 95% confidence intervals (95%CI) for associations between baseline self-reported stressful life events and incident breast cancer by ER status.

Results: The mean age was 63 (SD 7.3), and majority of participants were White race (83.5%) and married or in a marriage-like relationship (63.0%). In analyses stratified by ER status, there was no relationship between stressful life events and ER-positive breast cancer. In contrast, compared to women in the lowest quartile, those in higher quartiles had an increased risk of ER-negative breast cancer, where those in quartile 4 had the highest risk (Quartile 4 vs Quartile 1; HR=1.30; 95%CI 1.01-1.68; Ptrend=0.050). Additionally, associations were stronger for the highest versus lowest quartile of stressful life events among widowed women (HR=2.39; 95%CI 1.29-4.44; Pinteraction<0.001). Moreover, while the interaction was not statistically significant, we observed an elevated risk for ER-negative breast cancer among women in the highest quartile of stressful life events reporting lower social support.

Conclusion: In this cohort of postmenopausal women, higher experiences of pre-diagnostic stressful life events were associated with increased risk of ER-negative breast cancer.

Citation Format: Wayne R. Lawrence, Jasmine A. McDonald, Faustine Williams, Meredith S. Shiels, Neal D. Freedman, Ziqiang Lin, Jared W. Magnani. Psychosocial stress, social support, and incident breast cancer by estrogen receptor status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3011.
Abstract 3063: A novel mechanism for therapeutic targeting of the glucocorticoid receptor in triple negative breast cancer
Authors: Khan Shagufta H. , Carbe Christian , Ling Jun , Kumar Raj
Published: April 2023

Triple negative breast cancer (TNBC) is frequently diagnosed in younger women and is prevalent in African American women. Most TNBCs are highly aggressive with early pattern of metastasis, and with limited treatment options and poor prognosis. Finding effective therapeutic targets for TNBC has proven difficult so far. Therefore, there is an urgent need to develop new and novel therapeutic management of these patients. Recent studies have shown that glucocorticoid receptor (GR) is a mediator of pro-survival genes in TNBC cells, and its expression is predictive of increased risk of metastasis in TNBC tumors and decreased rate of patient survival. Therefore, blocking the effects of GR should be beneficial for TNBC patients. However, GR antagonists such as RU486 (mifepristone) targeting AF2/coactivators have shown only partial success in TNBC cell lines as well as in animal models. Based on our previous data we hypothesize that since in TNBC cells, the GR is constitutively active; therefore, attempts to block/inhibit GR activity through classic antagonists such as mifepristone that bind to ligand binding pocket of the receptor will have a limited clinical efficacy and higher side effects. To circumvent this problem, in this study, we propose a novel and innovative therapeutic approach to target constitutively active AF1 domain of the GR, which exists in an intrinsically disordered (ID) conformation. In this study, we tested our hypothesis that inhibiting/blocking TATA-box binding protein (TBP)-mediated GR’s AF1 activity by our newly identified molecule, PepT will attenuate TNBC cell survival via altering associated endogenous gene expressions. Our binding kinetics of AF1 showed that PepT binds to AF1 with about 10-fold higher affinity when compared with TBP’s binding to AF1. AF1 pre-bound with PepT fails to bind to TBP suggesting that PepT competes and blocks AF1-TBP interaction. We also found that treatment of MDA-MB-231 cells with PepT results in significant attenuation of cell viability and proliferation. Together, these results support our hypothesis that PepT has potential to kill TNBC cells by blocking GR AF1:co-regulator interactions.

Citation Format: Shagufta H. Khan, Christian Carbe, Jun Ling, Raj Kumar. A novel mechanism for therapeutic targeting of the glucocorticoid receptor in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3063.
Abstract 3071: Pituitary adenylate cyclase activating polypeptide stimulates NSCLC growth by increasing HER4 tyrosine phosphorylation in a neuregulin-1 dependent manner
Authors: Moody Terry W. , Alvarez Irene Ramos , Jensen Robert T.
Published: April 2023

Pituitary adenylate cyclase activating polypeptide [PACAP] is a 27 amino acid peptide which stimulates the growth of numerous cancers including non-small cell lung cancer (NSCLC). PACAP binds with high affinity to the type B G protein-coupled receptor [GPCR] PAC1 (Moody et al., Peptides 2021; 137: 170480). PAC1 activation causes phosphatidyl inositol [PI] turnover stimulating matrix metalloprotease [MMP]. By ELISA, PACAP increased secretion of neuregulin [NRG1] from NSCLC cells. NRG1 binds with high affinity to HER4. Here the ability of PAC1 to regulate transactivation of HER4 was investigated. By RT-PCR and Western blot, PAC1, NRG1 and HER4 but not HER3 or NRG2 were detected in NCI-H522 and H661 cells. Adding PACAP-27 (0.1 μM) or NRG1 (0.01 μg/ml) to NCI-H522 or H661 cells increased P-Tyr1284-HER4 3-fold after 5 min. Using immunoprecipitation techniques, adding PACAP TO NSCLC cells increased formation of HER4 homodimers and HER4-EGFR as well as HER4-HER2 heterodimers. The increase in P-HER4 caused by PACAP-27 was impaired by PACAP(6-38) [PAC1 antagonist], HER4 siRNA, NRG1 antibody [Ab] or ibrutinib [TKI]. The PAC1 regulation of HER4 transactivation is impaired by GM6001 [MMP inhibitor]. The clonal growth of NSCLC cells was stimulated by PACAP-27 or NRG1 but inhibited by PACAP(6-38) or ibrutinib. The results indicate that PACAP-27 stimulates the growth of NSCLC due to release of NRG1 which activates receptor tyrosine kinases such as HER4.

Citation Format: Terry W. Moody, Irene Ramos Alvarez, Robert T. Jensen. Pituitary adenylate cyclase activating polypeptide stimulates NSCLC growth by increasing HER4 tyrosine phosphorylation in a neuregulin-1 dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3071.
Abstract 3103: Dual targeting MYC G-quadruplex and topoisomerase I by indenoisoquinolines for cancer therapy
Authors: Yang Danzhou , Cushman Mark , Wang Kai-Bo , Elsayed Mohamed S. , Wu Guanhui , Buric Adam J. , DeMoss Mercedes
Published: April 2023

MYC is one of the most important oncogenes and is overexpressed in a number of human cancers. The G-quadruplex formed in the MYC oncogene promoter (MycG4) is a transcriptional silencer and amenable to small molecule targeting. G-quadruplexes are noncanonical four-stranded DNA or RNA secondary structures that have emerged as novel therapeutic targets. Indenoisoquinolines are human topoisomerase I inhibitors with improved physicochemical and biological properties as compared to the clinically used camptothecin anticancer drugs. Three indenoisoquinolines, indotecan (LMP400), indimitecan (LMP776), and LMP744, have entered phase I clinical trials in adults with relapsed solid tumors and lymphomas. However, some indenoisoquinolines showed potent anticancer activity without being strong topoisomerase I inhibitors, suggesting additional mechanisms of action. Herein, we demonstrate that a large number of anticancer indenoisoquinolines strongly bind and stabilize MycG4 in vitro, using fluorescence, nuclear magnetic resonance (NMR), and circular dichroism (CD) spectroscopy, as well as gel electromobility shift assay (EMSA). In addition, MycG4-interactive indenoisoquinolines lower MYC mRNA and protein levels in vivo, as shown by western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) experiments. Structure-activity-relationships of MycG4 recognition by indenoisoquinolines were investigated to understand structural requirements for binding. Systemic analysis of indenoisoquinoline derivatives led to the discovery of a synergistic effect of MYC inhibition and topoisomerase I inhibition on anticancer activity. Therefore, these results indicate that targeting the MYC promoter G-quadruplex to downregulate MYC is a new mechanism of action for anticancer indenoisoquinolines. Moreover, our data reveals dual targeting of MycG4 and topoisomerase I as a novel strategy for cancer therapy.

Citation Format: Danzhou Yang, Mark Cushman, Kai-Bo Wang, Mohamed S. Elsayed, Guanhui Wu, Adam J. Buric, Mercedes DeMoss. Dual targeting MYC G-quadruplex and topoisomerase I by indenoisoquinolines for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3103.
Abstract 3110: Design, synthesis, and biological evaluation of transducin β-like protein 1 (TBL1) degraders
Authors: Yang Rui , Alinari Lapo , Li Pui-kai (Tom) , Cheng Xiaolin
Published: April 2023

Transducin β-like protein 1 (TBL1) is an essential scaffold protein that participates in multiple critical signaling pathways, such as the Wnt/β-catenin pathway, where it protects β-catenin from ubiquitination and proteasomal degradation. Only one compound, BC-2059 (tegavivint, Iterion Therapeutics), has been reported to promote apoptosis by disrupting the TBL1/β-catenin interactions and show promising therapeutic effects in Wnt-driven cancers, such as colorectal cancer, breast cancer, and leukemia. However, recent studies showed that TBL1 modulates Wnt-regulated genes in a β-catenin-independent manner in diffuse large B-cell lymphoma (DLBCL). Although the involvement of β-catenin is still under debate, TBL1’s engagement in the antitumor effects of BC-2059 is certain and critical. In this study, we aimed to develop proof-of-concept BC-2059-based TBL1 selective degraders against DLBCL using the Proteolysis Targeting Chimeras (PROTACs) strategy and use them as chemical probes to investigate TBL1-related pathways in DLBCL. Two series (N- and O-linked) of BC-2059-based PROTACs were synthesized, and their cytotoxicity and protein degradation profiles were tested in cellular assays. Several compounds showed low micromolar to nanomolar cytotoxic activity. The TBL1 degradation of these PROTACs is currently underway. In the meantime, we modeled the ternary complex formation by developing a workflow where two binary complexes and a library of PROTACs having varied linker lengths/chemical compositions were modeled by PRosettaC. The protein-protein interactions of the generated ternary complexes were analyzed by Rosetta and the interactions between the linkers and the protein were calculated by Autodock 4. The results suggested that short linkers (8-14 heavy atoms) are more suitable for N-linked series degraders in terms of PROTAC-induced ternary complex formation, while long linkers (>18 heavy atoms) are preferable for O-linked series.

Citation Format: Rui Yang, Lapo Alinari, Pui-kai (Tom) Li, Xiaolin Cheng. Design, synthesis, and biological evaluation of transducin β-like protein 1 (TBL1) degraders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3110.
Abstract 3115: Predicting in-depth mechanism of action of cancer drugs
Authors: Sinha Sanju , Sinha Neelam , Ruppin Eytan
Published: April 2023

Knowing a drug’s mechanism of action (MOA) is essential for its clinical success by selecting the best indications, likely responders, and combinations. Yet knowledge of many drugs’ MOA remains lacking. Here we present DeepTarget, a computational tool for deep characterization of cancer drugs’ MOA by integrating existing large-scale genetic and drug screens. Spanning ∼1500 drugs across ∼18K possible target genes, DeepTarget predicts: (1) a drug’s primary target(s), (2) whether it specifically targets the wild-type or mutated target forms, and (3) the secondary target(s) that mediate its response when the primary target is not expressed. We first tested and successfully validated DeepTarget in a total of eleven unseen gold-standard datasets, with an average AUC of 0.82, 0.77, and 0.92 for the above three prediction abilities, respectively. We then proceed to use it in a wide range of applications: First, we find that DeepTarget’s predicted specificity of a drug to its target is strongly associated with its success in clinical trials and is higher in its FDA-approved indications. Second, DeepTarget predicts candidate drugs for targeting currently undruggable cancer oncogenes and their mutant forms. Finally, DeepTarget predicts new targets for drugs with unknown MOA and new secondary targets of approved drugs. Taken together, DeepTarget is a new computational framework for accelerating drug prioritization and its target discovery by leveraging large-scale genetic and drug screens.

Citation Format: Sanju Sinha, Neelam Sinha, Eytan Ruppin. Predicting in-depth mechanism of action of cancer drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3115.
Abstract 3119: A novel tissue resident memory T cell (TRM) signature predicts prognosis and tumor microenvironment of patients with melanoma
Authors: Jiang Chongming , Li Jianrong , Helman Elena , Shen Xiling , Cheng Chao
Published: April 2023

Background: Melanoma is a prime example of how basic and translational research can improve cancer prognosis The tumor immune microenvironment (TIME) of melanoma is a dynamic, heterogeneous mixture of multiple immune cells and malignant and nonmalignant cells. Tissue resident memory T cells (TRM) may be prognostic for improved melanoma survival and proliferate during treatment with immune checkpoint inhibitors. The role of TRM in melanoma and rare melanoma subtypes is unknown.

Method: Our goal was to examine the relationship between TRM abundance and melanoma prognosis. We generated TRM signatures using multiple independent single-cell RNA sequencing data from human melanoma cells to infer TRM abundance. Lastly, we built a LASSO Cox regression model that predicts patient survival. The TCGA dataset was used as a test group. Model accuracy was validated using Kaplan-Meier survival analysis, receiver operating characteristic curves, principal component analysis, and t-distributed stochastic neighbor embedding.

Findings: We found a significantly longer overall survival in patients with high TRM infiltration. Furthermore, we found that TRM can interact with the tumor microenvironment and genomic features of melanoma, making TRM abundance positively correlate with the prognosis of melanoma patients. Melanoma abundance was also significantly associated with several common genomic aberrations, including the TP53 mutation. In addition, we developed a 20-gene risk score that significantly classifies patients into low- and high-risk categories. We found that in several independent datasets, patients with high risk scores had significantly worse overall survival than patients with low risk scores.

Conclusions: Altogether, our findings suggest that TRM abundance may positively correlate with melanoma survival. We developed a high-precision LASSO Cox regression model based on the new TRM abundance characteristics. Our study shows that this model effectively predicts the prognosis of melanoma patients and may provide new therapeutic targets and measure the effectiveness of melanoma-directed therapies.

Keywords: Tissue resident memory T cell, tumor immune microenvironment, melanoma, prognosis

Citation Format: Chongming Jiang, Jianrong Li, Elena Helman, Xiling Shen, Chao Cheng. A novel tissue resident memory T cell (TRM) signature predicts prognosis and tumor microenvironment of patients with melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3119.
Abstract 3131: Anaplastic transformation model in thyroid cancer revealed by single cell lineage and fate analysis
Authors: Lu Lina , Wang Jennifer Rui , Henderson Ying C. , Bai Shanshan , Yang Jie , Hu Min , Yan Yuanqing , Tran Tuan M , Li Jianzhuo , Shiau Cheng-Kai , Kieser Rachel , Zhao Xiao , Wang Jiping , Nurieva Roza , Williams Michelle D. , Cabanillas Maria E. , Dadu Ramona , Busaidy Naifa Lamki , Zafereo Mark , Navin Nicholas , Lai Stephen Y. , Gao Ruli
Published: April 2023

The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC), however the complex intra-tumor transformation process is poorly understood. We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single cell transcriptomes and genetic alterations data of patients with different subtypes of thyroid cancer. The resulting model started from stress-responsive DTC cells to inflammatory ATC cells, to mitotic defective ATC cells and extended all the way to mesenchymal ATC cells. In parallel with tumor cell evolution, macrophages shifted from anti-tumor to tumor-promoting states and T cells reprogrammed from cytotoxic to exhausted states. Further, our analysis identified two important milestones: 1) diploid stage, where ATC cells were commonly diploids with non-RAS mutations and inflammatory phenotypes. 2) aneuploid stage, where ATC cells gained aneuploidy with frequent RAS mutations and mesenchymal phenotypes leading to the extreme lethal stage of ATC progression.

Citation Format: Lina Lu, Jennifer Rui Wang, Ying C. Henderson, Shanshan Bai, Jie Yang, Min Hu, Yuanqing Yan, Tuan M Tran, Jianzhuo Li, Cheng-Kai Shiau, Rachel Kieser, Xiao Zhao, Jiping Wang, Roza Nurieva, Michelle D. Williams, Maria E. Cabanillas, Ramona Dadu, Naifa Lamki Busaidy, Mark Zafereo, Nicholas Navin, Stephen Y. Lai, Ruli Gao. Anaplastic transformation model in thyroid cancer revealed by single cell lineage and fate analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3131.
Abstract 3141: Epiregulon infers single-cell transcription factor activity to dissect mechanisms of lineage plasticity and drug response
Authors: Wlodarczyk Tomasz , Tan Jenille , Seidel Kerstin , Wu Diana , Chen Shang-Yang , Chlebowski Aleksander , Keyes Timothy , Guo Yu , Lun Aaron , Siebel Christopher , Xie Shiqi , Yao Xiaosai
Published: April 2023

Single-cell multiomic technologies enable holistic reconstruction of cell states and hold the potential to identify drivers of tumor evolution and drug resistance. However, the data sparsity of single-cell assays can preclude accurate estimation of transcription factor activity. In addition, gene expression alone cannot capture transcription factor activity, especially in the context of drug treatment which alters transcription factor function without suppressing gene expression. To circumvent these challenges, we have developed epiregulon, a R package that constructs gene regulatory networks and infers transcription factor (TF) activity in single cells by integrating single cell gene expression, chromatin accessibility and bulk TF occupancy data. Epiregulon applies tests of independence to identify likely transcription factor - regulatory element - target gene relationships occurring at joint probabilities exceeding the expected probabilities of independent events. With epiregulon, we are able to detect lineage factor activity at enhanced sensitivity and predict perturbations more accurately than gene expression could. We further applied this tool to understand transcription factor activity to enzalutamide treatment and identify potential drivers of enzalutamide resistance. Finally, we generated a ground truth dataset using reprogram-seq, a technology that captures multi-omic profiles of single cells upon expression of defined transcription factors. Epiregulon correctly predicts NKX2-1 targets and demonstrates that NKX2-1 expression reprograms the epigenome towards a neuroendocrine state in LNCaP cells. Epiregulon is a useful tool that constructs gene regulatory networks to model the underlying gene regulation hierarchies that drive gene expression and cell states.

Citation Format: Tomasz Wlodarczyk, Jenille Tan, Kerstin Seidel, Diana Wu, Shang-Yang Chen, Aleksander Chlebowski, Timothy Keyes, Yu Guo, Aaron Lun, Christopher Siebel, Shiqi Xie, Xiaosai Yao. Epiregulon infers single-cell transcription factor activity to dissect mechanisms of lineage plasticity and drug response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3141.
Abstract 3175: Comprehensive multi-omic characterization of hepatocellular carcinoma in Hispanic patients
Authors: Das Debodipta , Wang Xiaojing , Chiu Yu-Chiao , Bouamar Hakim , Chen Yidong , Zheng Siyuan , Cigarroa Francisco G. , Sun Lu-Zhe
Published: April 2023

Hispanics in the US experience a disproportionate burden of hepatocellular carcinoma (HCC) with an overall incidence and cancer-related mortality nearly two times higher than non-Hispanic whites. The genomic characteristics and underlying molecular mechanisms driving tumorigenesis associated with HCC in Hispanic patients remain unknown. Here, we used 31 paired tumor and adjacent non-tumor liver samples from Hispanic patients in South Texas to perform integrated genomic, transcriptomic, proteomic, and metabolomic analyses. Similar to HCC in other ethnic groups, CTNNB1 and TP53 were found to have the most frequent somatic mutations in the Hispanic HCC. On the other hand, AXIN2 and to a lesser degree MTOR appear more frequently mutated in the Hispanics compared to the various ethnic groups included in The Cancer Genome Atlas (TCGA) study. Somatic mutations in the Hispanic HCC showed signatures of tobacco chewing and aflatoxin exposure, which are different from the signatures found frequently in the liver cancer patients in the TCGA study. The WNT, TP53, & cell cycle were the most frequently altered oncogenic pathways in our cohort. Unsupervised clustering of patients, based on the enrichment scores from single sample Gene Set Enrichment Analysis (ssGSEA) using our tumor and nontumor paired RNA-Seq and proteomic data, revealed two clusters with striking differences in various signaling pathways and cellular functions. A similar clustering pattern with a significant difference in overall survival was seen in the patients from the TCGA study. Irrespective of ethnicity, immune infiltration in the tumor microenvironment and liver function-related pathways were significantly different between patients in these clusters. Integrated transcriptomic, proteomic, and metabolomic analyses identified significant negative enrichments in gluconeogenesis, TCA cycle, and glutamate metabolism in Hispanic HCC. These results suggest the existence of molecular mechanisms either unique in Hispanics or similar to non-Hispanic HCC, especially from the TCGA study. Our findings might provide insights to developing predictive biomarkers for the early diagnosis and novel therapeutics for the management of HCC.

Citation Format: Debodipta Das, Xiaojing Wang, Yu-Chiao Chiu, Hakim Bouamar, Yidong Chen, Siyuan Zheng, Francisco G. Cigarroa, Lu-Zhe Sun. Comprehensive multi-omic characterization of hepatocellular carcinoma in Hispanic patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3175.
Abstract 3176: Modeling the complex multi-cellular ecosystem of the tumor microenvironment in colorectal cancer using multi-omics data
Authors: Ildefonso Geena , Finley Stacey
Published: April 2023

The high inter-tumoral phenotypic heterogeneity of colorectal cancer exists within the tumor microenvironment (TME), which ultimately contributes to overall tumor survival. Tumor cells, as the center of the tumor microenvironment, employ sophisticated signaling networks to alter the cell fate decisions between cancerous and non-cancerous cells. A complex network of interactions is the source of this intercellular communication, resulting in tumor growth and progression, drug resistance, and ineffective treatment response. Recent experimental developments in colorectal cancer (CRC) have shown that understanding various underlying mechanisms of tumor growth and progression requires an in-depth understanding of the complex interactions between tumor cells and their surrounding microenvironment. Here, we analyze single-cell RNA sequencing data in CRC and construct gene regulatory networks to define interactions and identify cell states within the TME. The gene regulatory networks are incorporated as a multi-cellular ecosystem representing an experiment-based computational model of phenotypic crosstalk within the TME of CRC. The model simulates and predicts the multi-cellular responses to colorectal cancer to probe and identify mechanisms of cell state evolution and decisions resulting from chemo-agents, used to target TME components. This research will further our understanding of the multi-cellular crosstalk underlying the mechanisms within the TME and facilitate the development and optimization of cancer therapies that inhibit tumor growth in CRC.

Citation Format: Geena Ildefonso, Stacey Finley. Modeling the complex multi-cellular ecosystem of the tumor microenvironment in colorectal cancer using multi-omics data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3176.