bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1881: Screening of Rac1 and Cdc42 inhibitors as anti-metastatic compounds
Authors: González Jessica Colón , Fuentes María C. Santa María , Vlaar Cornelis , Dharmawardhane Suranganie
Published: March 2024

Metastasis is responsible for >90% of cancer-related deaths and is a key cause of failure of cancer therapy. The development of effective targeted therapy to prevent and treat metastasis has been challenging. Homologous Rho GTPases Rac and Cdc42 are viable metastasis targets because they have a crucial role in the signaling pathways that regulate cell survival, proliferation, and migration, so dysregulation of these proteins leads to cancer progression and metastasis of diverse cancer types. Therefore, targeting these regulatory proteins results in anticancer and antimetastatic activity, as observed with our dual Rac/Cdc42 inhibitor, MBQ-167, in triple-negative breast cancer. MBQ-167 has an IC50 of ~100nM for Rac and Cdc42 activation inhibition and a GI50 of 130nM. MBQ-167 inhibits the binding of guanine nucleotides to Rac and Cdc42 with similar efficacy. The purpose of this study was to identify MBQ-167 derivatives with similar or greater efficacy and specificity, with a range of inhibitory mechanisms, that may be specific to the different guanine nucleotide exchange factors expressed in distinct cancer cell types. Currently, over 30 different compounds with potential Rac1 and Cdc42 inhibitory activity are being evaluated. So far, some of the evaluated compounds have shown comparable GI50 values to MBQ-167 in metastatic breast cancer cell lines. From this screening, CPV-337 was selected as a compound with a GI50 of ~55nM and ~70% Rac activation inhibition at 50nM, therefore being 2X more effective than our leading compound MBQ-167. However, evaluation of CPV-337 in the viability of the MCF-10 non-tumorigenic epithelial cell line demonstrated a GI50 of ~63nM, which is similar to that of breast cancer cells. Therefore, CPV-337 may have cytotoxic effects on breast cancer. We are continuing to screen the library of MBQ-167 derivatives in breast, pancreatic, lung, and prostate cancer cell lines.

Citation Format: Jessica Colón González, María C. Santa María Fuentes, Cornelis Vlaar, Suranganie Dharmawardhane. Screening of Rac1 and Cdc42 inhibitors as anti-metastatic compounds [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 1881.
Abstract 1946: Elucidating the mechanisms of acquired resistance to AMG510 in cancer models harboring KRAS G12C mutations
Authors: Ni Ting , Huo Tongxin , Zhang Yawen , Wang Jingjing , Liu Gaoxiang , Shi Wenting , Gu Qingyang
Published: March 2024

GTPase KRas (KRAS), a critical oncogene, is known for its frequent mutations across a wide array of human cancers, most notably in pancreatic, lung, and colorectal cancers. Recent advancements in the field have led to the successful development of numerous inhibitors targeting the mutant KRAS. Clinical application of KRAS G12C inhibitors, such as AMG510 and MTRX849, represents major breakthroughs in addressing previously 'undruggable' cancer targets. Nevertheless, a subset of patients initially responsive to these inhibitors eventually exhibit treatment resistance. Uncovering the underlying mechanism would help to develop novel therapies that overcome the acquired drug resistance.

To elucidate the underlying mechanisms, we generated AMG510-resistant tumor models, namely AMG510-R-MIA PaCa-2 and AMG510-R CT26 KRAS G12C, by continuous administration of AMG510 to tumor-bearing mice. Upon comparison of sensitive parental tumors and resistant tumors, we found that highly expressed KRAS and constant activation of RAS-MAPK signaling pathway are critical for resistant tumor progression following AMG510 treatment. In addition, resistant tumor cells are responsible for the elevated secretion of CXCL2/3/5, which dramatically reconditions the tumor immune microenvironment and recruits immunosuppressive cells.

In summary, the exploration of the resistance mechanism in AMG510-induced resistant models provides insight into the development of new-generation KRAS-G12C inhibitors and novel combinatorial therapies.

Citation Format: Ting Ni, Tongxin Huo, Yawen Zhang, Jingjing Wang, Gaoxiang Liu, Wenting Shi, Qingyang Gu. Elucidating the mechanisms of acquired resistance to AMG510 in cancer models harboring KRAS G12C mutations [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 1946.
Abstract 1970: Discovery and characterization of potent and selective HER2 exon20 insertion mutant inhibitors
Authors: Nam Ho Yeon , Jang Sun Young , Jeon Jiyoung , Yoo HyungSeok , Byun Jooyun , Lee Gunwoo , Lee Youngjoo , Kim Yu-Yon , Jeon Soye , Ahn Young Gil
Published: March 2024

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for over 85% of all cases. Among NSCLC, approximately 2-4% of patients have mutations in the HER2, a member of the ErbB family of receptor tyrosine kinases and key proliferation driver, with exon 20 insertions making up about 90% of these mutations. HER2 exon mutations can have various alterations, and among them, the most common HER2 mutation is in-frame insertions in exon 20, especially the A775_G776insYVMA insertion/duplication. These mutations can affect the structure and function of the HER2 protein and may contribute to tumor development and growth. HER2 mutations have often shown resistance to EGFR tyrosine kinase inhibitors (TKIs), further highlighting the need for HER2-specific treatments. Also, inhibitors targeting HER2 mutations are selective, meaning they specifically target HER2 alterations without causing toxicities or affecting EGFRWT. Therefore, there is a need for the development of selective HER2 exon20 insertion mutant inhibitors. Herein, Hanmi has identified selective HER2 exon20 insertion mutant small molecule inhibitors, designed based on affinity and binding mode starting from a known crystal structure using GLIDE (Schrödinger), and the screening for the viability of cell lines including HER2A775_G776insYVMA, HER2G776delinsVC , HER2WT and EGFRWT was investigated. Hanmi's HER2 exon20 insertion mutant inhibitors selectively target mutant HER2 receptor tyrosine domains through shared binding, sparing EGFRWT and minimizing EGFR-related toxicities. With highly selectivity against EGFRWT and desirable pharmacokinetic properties, the Hanmi compounds inhibit HER2 mutant cells and HER2 mutant enzymes. Additionally, the compounds have an oral administration-compatible DMPK profile and demonstrated anti-tumor activity in HER2 mutant and HER2WT tumor xenograft mice. In conclusion, based on our study, Hanmi compounds, a novel HER2 exon20 insertion mutant inhibitors could be suggested as an appropriate therapeutic agent for inhibitory activity against HER2 exon20 insertion mutant NSCLC.

Citation Format: Ho Yeon Nam, Sun Young Jang, Jiyoung Jeon, HyungSeok Yoo, Jooyun Byun, Gunwoo Lee, Youngjoo Lee, Yu-Yon Kim, Soye Jeon, Young Gil Ahn. Discovery and characterization of potent and selective HER2 exon20 insertion mutant inhibitors [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 1970.
Abstract 1975: Discovery of I-1000233, a potent, selective and brain permeable SHP2 allosteric inhibitor
Authors: Puca Francesca , Fabbrini Danilo , Ciammaichella Alina , Missineo Antonino , Bisbocci Monica , Nibbio Martina , Marco Annalise Di , Apicella Claudia , Colaceci Fabrizio , Sasso Romina , Natale Daniela , Scalabrì Francesco , Alli Cristina , Pucci Vincenzo , Montalbetti Christian , Petrocchi Alessia , Carugo Alessandro , Toniatti Carlo
Published: March 2024

Background: SHP2 (Src homology region 2-containing protein tyrosine phosphatase 2) is a target of interest for cancer therapy due to its key role in the regulation of RAS/MAPK signal transduction downstream of Receptor Tyrosine Kinases (RTKs). We report here the identification of I-1000233, a novel, highly potent, orally available and brain permeable SHP2 allosteric inhibitor, with potential for the treatment of tumors with dysregulated RTK/RAS/ERK signaling pathways.

Material and Methods: High-throughput biological, biochemical and pharmacodynamic (PD) assays were used to inform Structure Activity Relationship studies which led to the identification of a chemical series of potent SHP2 inhibitors. Iterative optimization of physicochemical and pharmacological properties resulted in the identification of I-1000233, an orally available SHP2 inhibitor that showed excellent drug-like properties in preclinical studies.

Results: I-1000233 is a potent inhibitor of the SHP2 enzyme in vitro (IC50 = 2 nM) with an exceptionally long residence time on the target. When tested in cell lines, it exhibits significant anti-proliferation activity against brain tumor cells and multiple RAS or EGFR mutant models. It strongly inhibits pERK, a downstream marker of MAPK pathway activity, in a dose dependent manner. Further, in vivo preclinical data confirmed that I-1000233 penetrates the BBB and shows anti-tumor activity, supporting its potential in the treatment of CNS and brain-metastasized tumors.

Conclusions: I-1000233 is a potent, selective and orally available SHP2 inhibitor with potential for once-a-day dosing and its brain permeability in humans makes it a promising therapeutic agent against SHP2i responsive brain tumors and metastases.

Citation Format: Francesca Puca, Danilo Fabbrini, Alina Ciammaichella, Antonino Missineo, Monica Bisbocci, Martina Nibbio, Annalise Di Marco, Claudia Apicella, Fabrizio Colaceci, Romina Sasso, Daniela Natale, Francesco Scalabrì, Cristina Alli, Vincenzo Pucci, Christian Montalbetti, Alessia Petrocchi, Alessandro Carugo, Carlo Toniatti. Discovery of I-1000233, a potent, selective and brain permeable SHP2 allosteric 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 1975.
Abstract 2044: A live cell PRMT5 NanoBRET Target Engagement Assay querying competitive and uncompetitive modes of inhibition
Authors: Michaud Ani , Rothweiler Elisabeth , Corona Cesear , Beck Michael , Wilkinson Jennifer , Vasta James , Huber Kilian , Robers Matthew
Published: March 2024

PRMT5 is an essential arginine methyltransferase that regulates a wide spectrum of cellular processes through the methylation of histone and non-histone substrates. In PRMT5 catalysis, SAM serves as the cofactor and methyl group donor, generating a methylated guanidinium moiety on the target substrate. In normal cells, the SAM pools are maintained through the methionine salvage pathway. In 15% of cancers, a key enzyme in this pathway (MTAP) is deleted, leading to an accumulation of the intermediate MTA, which inhibits PRMT5 activity. This genetic loss of function has therefore been pursued as a collateral vulnerability in MTAP deleted cancers. To exploit this synthetic lethality, a number of novel inhibitors have been developed that bind cooperatively at the PRMT5/MTA complex, offering a compelling pathway to precision medicine. Here we describe a novel, live cell NanoBRET Target Engagement assay that enables mechanistic studies on a variety of PRMT5 inhibitors. Using a cell-permeable NanoBRET probe directed to the substrate pocket of PRMT5, both substrate- and cofactor-competitive engagement can be quantified in cells. Moreover, this method can be used to quantify MTA-uncompetitive target engagement in cells, providing a platform to measure the potency of PRMT5-MTA-drug ternary complex formation. This method serves as a first-in-class method to quantify uncompetitive target engagement in live cells, which can be applied to other model systems.

Citation Format: Ani Michaud, Elisabeth Rothweiler, Cesear Corona, Michael Beck, Jennifer Wilkinson, James Vasta, Kilian Huber, Matthew Robers. A live cell PRMT5 NanoBRET Target Engagement Assay querying competitive and uncompetitive modes of inhibition [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 2044.
Abstract 205: Adaptive membrane trafficking activated by epithelial-to-mesenchymal transition drives lung adenocarcinoma progression
Authors: Obaleye Oluwafunminiyi E. , Fulp Kevin L. , Xiao Guan-Yu
Published: March 2024

Cancer cells can become metastatic and develop resistance to targeted therapies through epithelial-to-mesenchymal transition (EMT), which is regulated by epigenetic factors. Our ultimate objective is to understand the molecular mechanisms behind EMT-driven cancer progression and identify potential targets for preventing cancer metastasis. Recently, we made groundbreaking discoveries indicating that EMT can modify membrane trafficking machinery to coordinate cancer cell invasion and immunosuppression in the tumor microenvironment (TME) of lung adenocarcinoma (LUAD). To dissect the “adaptive” membrane trafficking program, we initiated a CRISPRi in vivo screen to assess more than 2000 trafficking-related genes in syngeneic mouse LUAD models for their requirements for tumorigenesis both with and without immune selective pressure. We identified a panel of membrane trafficking regulators that specifically “dropped out” in immunocompetent mice, suggesting their essential roles in the immunosuppressive TME. Of particular interest is REEP2. REEPs are an evolutionarily conserved protein family that is critical to the endoplasmic reticulum (ER) function and potentially regulates secretory trafficking. We discovered that REEP2 is specifically linked to poor prognosis in LUAD patients and is highly correlated with a z-normalized, 16-gene EMT score (P = 1.32e-27) that was previously reported. Our previous studies demonstrated that the EMT activator, ZEB1, accelerates membrane trafficking dynamics by silencing a microRNA network that targets multiple membrane trafficking components. Analysis of REEP2 3’-untranslated regions (3’-UTRs) identified predicted binding sites for several microRNAs, which are silenced by ZEB1. Thus, we hypothesize that ZEB1 coordinates a microRNA network to activate REEP2-driven ER trafficking. We show that REEP2 depletion significantly reduces the invasion of cancer cells but does not affect their migration and proliferation. These findings suggest that REEP2 plays a significant role in extracellular matrix (ECM) modification to enhance the invasion of metastatic cells, conferring a vulnerability in EMT-driven LUAD metastasis.

Citation Format: Oluwafunminiyi E. Obaleye, Kevin L. Fulp, Guan-Yu Xiao. Adaptive membrane trafficking activated by epithelial-to-mesenchymal transition drives lung adenocarcinoma progression [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 205.
Abstract 2069: High content imaging without sacrificing coverage, resolution or speed: Functional validation through multiple assays
Authors: Boisvert Matthew , Kieler Josh , DiSepio Dan , Weldon Don
Published: March 2024

Traditionally, high content screening (HCS) required compromise: sacrificing resolution, sample coverage or speed. This has limited high content screening’s utility as a high throughput tool, as speed is essential to cancer research at scale. Using a new approach, Araceli Endeavor® offers a solution to this compromise. This system images 96, 384 or 1536 -well high content microplates in under 10 minutes with full well coverage and sub-micron resolution, enabling high content screening at high throughput speeds without compromising resolution or coverage. Paired with the Clairvoyance™ analysis software, plates are analyzed at similar speeds, going from images to object-level data in 5-25 minutes. As with any new technology, validation is needed. Here we validate Endeavor and Clairvoyance at the level of the bioassay, using a range of common high content assays across different modalities. Assays are chosen to quantifiably demonstrate sensitivity, breadth, and broad applicability. Performance was benchmarked by using well-characterized compounds, matching published EC50/IC50 values when possible. We examined submicron-level nuclear spot counting, measuring histone yH2Ax and micronuclei formation after genotoxic insult. Intensity-based assays include cellular positivity, here live/dead, and subcellular translocation with transcription factor NFΚB after induction with cytokines Il1b and TNFα. Looking at cytosolic vesicle aggregation, we measured autophagic flux with two distinct modalities, comparative imaging and analysis of live cell dye and immunohistochemistry-based detection yielded consistent EC50 values between methods. Overall, these assays yield consistent, robust results corresponding to literature values, showing the importance of full well coverage and resolution. We demonstrate how assays that formerly took hours can be effectively imaged and analyzed without sacrificing quality in 10-30 minutes.

Citation Format: Matthew Boisvert, Josh Kieler, Dan DiSepio, Don Weldon. High content imaging without sacrificing coverage, resolution or speed: Functional validation through multiple assays [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 2069.
Abstract 2077: Exploring the multifaceted efficacy of de novo lipogenesis inhibitor in cancer therapy
Authors: Lowder Dallin , Shin Jin Na , Ruiz-Echartea Maria E. , Deng Jenny , Rusin Aleksandra , Skapura Darlene , Ittmann Michael , Kaochar Salma
Published: March 2024

Cancer cells frequently exhibit altered lipid metabolism to sustain their rapid growth and proliferation.Sterol Regulatory Element-Binding Proteins (SREBPs) are key transcription factors that orchestratelipid biosynthesis and uptake in cells. Dysregulation of SREBPs is a common feature of many cancertypes, making them attractive targets for therapeutic intervention. In this study, we present acomprehensive exploration of the potential and rationale for targeting lipid metabolism in cancerthrough the use of a small molecule inhibitor that targets activation of SREBP. We illustrate that oursmall molecule inhibitor (SMI) exhibits potent anticancer activity and effectively targets SREBPsignaling in a variety of prostate cancer models as well as in non-prostate cancer models that arelipogenesis driven. Our novel SMI induces apoptosis and reduces PCa cell proliferation, suppressescell invasion and migration. Furthermore, we illustrate that in addition to suppressing the transcriptionfactor activity of SREBPs, these novel SMIs are highly effective in suppressing both the full length andvariant androgen receptor and their signaling in prostate cancer cells. Notably, in cell and organoidmodels, these novel inhibitors exhibit synergistic anticancer activity in combination with the ARsignaling inhibitor, Enzalutamide. The anticancer activity of these inhibitors extend beyond ARdependent prostate cancer and are also effective in organoid models of neuroendocrine PCa, aparticularly drug-resistant and aggressive PC subtype. Lastly, we illustrate excellent pharmacokineticsand potent anti-tumor effects of these novel SREBP activation inhibitors using in vivo castration-resistant prostate cancer CDX tumors. Given the urgent need for novel targets in PCa, our first-in-class inhibitor represents a promising therapeutic strategy with the potential to effectively targetdysregulated lipogenesis in cancer patients.

Citation Format: Dallin Lowder, Jin Na Shin, Maria E. Ruiz-Echartea, Jenny Deng, Aleksandra Rusin, Darlene Skapura, Michael Ittmann, Salma Kaochar. Exploring the multifaceted efficacy of de novo lipogenesis inhibitor in cancer therapy [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 2077.
Abstract 2080: SOS1 inhibition is an effective therapeutic strategy in SOS1-mutant cancer
Authors: Sale Matthew J. , Mukherjee Neal , Koch Martín , Ruzicka Regina , Jacob Robin , Albrecht Christoph , Zanin Teresa , Madensky Thomas , Schenk Robyn L. , Baum Anke , Kostyrko Kaja , Gerlach Daniel , Hofmann Marco H. , McCormick Frank
Published: March 2024

SOS1 is a principal guanine nucleotide exchange factor for canonical RAS GTPases and activates RAS signaling. Mutations in SOS1 are found in several prominent cancer types, including 2-4% of NSCLC, CRC, and melanoma. The recent development of novel SOS1 inhibitors (SOS1i), including BI-3406, that block interaction between SOS1 and RAS, presents a potential therapy for SOS1-driven cancers. Here we show that the majority of SOS1 mutations observed in patients enhance RAS guanine nucleotide exchange in cells and activate RAS-MAPK signaling. We treated 11 SOS1 mutant cancer cell lines without RAS co-mutation with a SOS1i and found that 10 were sensitive to SOS1 inhibition in 2D and/or 3D cell culture. In contrast, cell lines with SOS1 and RAS co-mutations were resistant to SOS1 inhibition. In vitro treatment of SOS1 mutant cells with a SHP2 inhibitor, which is presumed to inhibit the activity of both SOS1 and SOS2, revealed a subset of four cell lines that were more sensitive to SHP2 inhibition than SOS1 inhibition. This implied that, in certain contexts, SOS2 can compensate for the loss of SOS1 activity. Accordingly, these cells exhibited a SOS2-dependent rebound in RAS-MAPK signaling after treatment with SOS1i. SOS2 knock-down further sensitized these cells to SOS1 inhibition, resulting in responses equivalent to SHP2 inhibition. Thus, upon SOS1 inhibition relief of ERK1/2-dependent negative regulation likely contributes to receptor tyrosine kinase-driven SOS2 activation that may mediate SOS1 inhibitor resistance in some contexts. In vivo, SOS1i efficiently inhibited tumor growth in four SOS1-mutant NSCLC, CRC, and AML xenograft models. The tumor-suppressive effects of SOS1i in these models were comparable to or stronger than the effects of SHP2i or MEKi. Thus, our results suggest that SOS1 inhibition could be an effective therapeutic approach in RAS wild-type cancer patients with SOS1 mutations.

Citation Format: Matthew J. Sale, Neal Mukherjee, Martín Koch, Regina Ruzicka, Robin Jacob, Christoph Albrecht, Teresa Zanin, Thomas Madensky, Robyn L. Schenk, Anke Baum, Kaja Kostyrko, Daniel Gerlach, Marco H. Hofmann, Frank McCormick. SOS1 inhibition is an effective therapeutic strategy in SOS1-mutant 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 2080.
Abstract 2085: Targeting the translation initiation complex component eIF4G1 in melanoma
Authors: Feng Yongmei , Radaeva Mariia , Kim Hyungsoo , Deshpande Anagha , Deshpande Ani , Jovanovic Predrag , Murad Rabi , Topisirovic Ivan , Olson Steven , Cherkasov Artem , Ronai Ze'ev
Published: March 2024

The translation initiation factor 4F (eIF4F) complex assembly is a rate-limiting step in mRNA translation. eIF4F subunits including eIF4A, eIF4E, and eIF4G, are often upregulated in cancer and neurodegeneration diseases. Elevated eIF4F level/activity has been correlated with poor prognosis and drug resistance. Leveraging our findings with the small molecule SBI-756, which interacts with eIF4G1 and impairs eIF4F complex assembly, we set to map domains that are required for SBI-756 activity. A CRISPR screen using sgRNAs that target different sequences on eIF4G1 led to the identification of the MA3 domain, as a putative binding site for SBI-756. Deletion/mutation of the eIF4G1 MA3 domain attenuated melanoma cells and spheroids growth. Polysome profiling assays confirmed attenuated translation activity, which resembled those seen with SBI-756. In silico virtual screen identified 64 small molecules (out of >10 million) that interact with the MA3 domain. Of these, we have selected four that effectively attenuated melanoma growth in culture. Analogs developed for these four compounds were more potent in impairing the assembly of the eIF4F complex, inhibition of protein translation and the 2D and 3D growth of melanoma cells. RNA-sequencing analysis highlighted altered expression of genes implicated in apoptosis, UPR, cell cycle, and ROS pathways, leading us to test possible combination with pathways that may complement the above. Among those, autophagy inhibitors synergized with our lead compound, M19-6, resulting in efficient melanoma cell death, using notably lower concentrations of these inhibitors. Our findings identify the eIF4G1 MA3 domain as an important player in eIF4F assembly and a potential target for cancer therapy.

Citation Format: Yongmei Feng, Mariia Radaeva, Hyungsoo Kim, Anagha Deshpande, Ani Deshpande, Predrag Jovanovic, Rabi Murad, Ivan Topisirovic, Steven Olson, Artem Cherkasov, Ze'ev Ronai. Targeting the translation initiation complex component eIF4G1 in melanoma [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 2085.
Abstract 2093: Co-treatment with KRAS G12D inhibitor MRTX1133 plus TTFields against human pancreatic and Colorectal cancer cell lines results in synergistic up-regulation of cleaved PARP in KRAS G12D & unexpectedly in KRAS G12V as well
Authors: Tajiknia Vida , Srinivasan Praveen , PInho-Schwermann Maximilian , MacDonald William , Purcell Connor , El-Deiry Wafik
Published: March 2024

Background: Both colorectal and pancreatic cancers are major global health issues and among deadliest cancers with great disease burden. The most commonly mutated oncogenic alteration in human cancers is KRAS, which is prevalent in pancreatic malignancies. The KRAS G12D mutation subtype is present in more than 40% of pancreatic ductal adenocarcinoma (PDAC). To this date there is no available targeted therapy options for patients with KRAS subtype mutations. MRTX1133 has been identified as a non-covalent, potent, and selective inhibitor of KRASG12D. This small molecule inhibitor has been shown to suppress KRASG12D signaling in cells and in vivo. Tumor Treating Fields (TTFields) therapy is a novel approach to treating cancer, the electric fields alter the behavior of cancer cells and prevent them from growing and dividing. We hypothesize that the co-application of MRTX1133 with TTFields will enhance the effects of MRTX1133 in PDAC and colorectal cancer (CRC).

Martials and Methods: PDAC cell line CFPAC1 with KRAS G12 V mutation and LS513 CRC cell line with KRAS G12D Mutation were treated with same dose of MRTX1133 (500 nM) and were co-treated with 150kHz TTFields. After 48 hours western blot was used to probe for cleaved PARP, cleaved C3, phosphor-ERK and DUSP6.

Results: In both KRAS G12D & KRAS G12V cell lines, synergistic upregulation of cPARP was observed following 48-hour co-treatment. Synergistic inhibition of pERK happened in KRAS G12D cell line but no change in pERK level following TTFields or MRTX1133 treatment was seen. Upregulation of cC3 in KRAS G12D happened in only co-treatment conditions. DUSP6 levels increased following MRTX1133 treatment in KRAS G12V cell line.

Conclusions: The surprising synergistic upregulation of cPARP in both KRAS G12V and G12D cells following co-treatment of KRAS G12D inhibitor MRTX1133 and TTFields can bring hope for other KRAS mutation subtypes as well as G12D. the increase in cPARP in KRAS G12V with no effect on pERK is particularly interesting and demonstrates the need of more studies to investigate the mechanisms of this synergy.

Citation Format: Vida Tajiknia, Praveen Srinivasan, Maximilian PInho-Schwermann, William MacDonald, Connor Purcell, Wafik El-Deiry. Co-treatment with KRAS G12D inhibitor MRTX1133 plus TTFields against human pancreatic and Colorectal cancer cell lines results in synergistic up-regulation of cleaved PARP in KRAS G12D & unexpectedly in KRAS G12V as well [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 2093.
Abstract 211: Applying cellular thermal shift assays to uncover novel regulators of epithelial to mesenchymal transition in triple negative breast cancer
Authors: Lidster Jessica A. , Loo Ser Yue , Wu Zhengwei , Dai Lingyun , Ng Seow Qi , Prabhu Nayana , Kappei Dennis , Tam Wai Leong
Published: March 2024

Metastatic Breast Cancer is accountable for over half a million deaths worldwide each year, the majority of which are from the triple negative breast cancer (TNBC) subtype, known to be highly aggressive, metastatic and resistant to targeted therapy. Metastasis is driven by the ability of cancer cells to undergo epithelial to mesenchymal transition (EMT), a cell state transition involving a gain of stemness, cell motility and chemoresistance. EMT has been well characterized using a range of techniques based upon quantitative proteomics and transcription profiling, yet targeting EMT to prevent tumor metastasis and chemoresistance is an obstacle that remains to be fully overcome. Using a cellular thermal shift assay coupled with mass spectrometry (CETSA-MS), we have measured changes in protein stability upon induction of EMT and have uncovered key enzymes in the citric acid (TCA) cycle that are stabilized during EMT of human mammary epithelial cells. Such enzymes are responsible for the conversion of TCA cycle intermediates required for a recently discovered post-translational modification. In TNBC cells, we find that loss of these enzymes causes not only global changes to crucial post-translational modification of mitochondrial proteins, but very specific alterations in several cytoskeletal proteins, leading to altered EMT status as well as reduced tumor growth and initiation both in vitro and in vivo. This demonstrates the importance of TCA cycle-controlled post-translational modification of cytoskeletal proteins in tumorigenesis and establishes basis for further investigation into their role in EMT and metastasis of breast cancer. Through pharmacological inhibition and immunofluorescence imaging, we discovered regulators of post-translational modifications that may be viable drug targets to overcome chemoresistance and relapse in TNBC patients with limited and inefficacious options.

Citation Format: Jessica A. Lidster, Ser Yue Loo, Zhengwei Wu, Lingyun Dai, Seow Qi Ng, Nayana Prabhu, Dennis Kappei, Wai Leong Tam. Applying cellular thermal shift assays to uncover novel regulators of epithelial to mesenchymal transition in 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 211.
Abstract 2129: Efficacy and underlying mechanisms of surufatinib combined with PD-1 monoclonal antibody and chemotherapy in pancreatic cancer
Authors: Zhang Nan , Jia Ru , Dai Guanghai
Published: March 2024

Background: A phase 1b/2 study (NCT05218889) of surufatinib combined with camrelizumab, nab-paclitaxel, and S-1 (NASCA) as first-line therapy for metastatic pancreatic adenocarcinoma has shown promising results, potentially overcoming the limitations of current pancreatic immunotherapies. Animal studies are underway to further explore the synergistic effects and mechanisms of this combined therapeutic approach.

Methods: Genetically engineered KPC mouse cells were used to establish a subcutaneous transplantation tumor model in female mice, which were then divided into control group, AG group (nab-paclitaxel + gemcitabine), and NASCA group. Tumor metrics were recorded until euthanasia after 21 days. Blood and tumor samples were analyzed using Flow Cytometry.

Results: Compared with the control group, the NASCA and AG groups demonstrated tumor volume reductions of 14.7% (p=0.172) and -0.4% (p=0.972) on day 7, respectively. At day 14, the reductions were 48.8% and 29.9% (p<0.001*, p=0.002*). At day 21, the reductions were 37.4% and 21.5% (p=0.001*, p=0.144). The tumor growth inhibitions (TGIs) for NASCA and AG group were 56.7% (p<0.001*) and 21.2% (p=0.36), respectively. Compare to AG group, the tumor volume reduction in NASCA group were 15.1% (p=0.050*), 26.9% (p=0.001*) and 20.3% (p=0.116), respectively. And the TGIs for NASCA group compare AG group was 45.0% (p=0.020*). Distinguishingly, the NASCA group, when compared to the AG group, had a heightened proportion of M1 macrophages (28.5% vs. 15.5%, p=0.019*), mCD68+ Macrophages (99.7% vs. 99.1%, p=0,014*) and mPD-1+/mCD8+ T cells (24.8% vs. 9.8%, p=0.03*). Furthermore, T-regulatory cells (T-regs) in peripheral blood demonstrated uniformity across the groups, were no statistically. RNA-seq analysis revealed that the NASCA significantly suppressed multiple anti-angiogenic pathways while enhancing the cytokine pathway (p=0.005*), indicating potential immune system activation.

Conclusion: The NASCA group outperformed the AG group in controlling tumor growth and early tumor shrinkage, potentially due to macrophage polarization regulation. Further research is vital to understand the benefits of integrating targeted therapy, immunotherapy, and chemotherapy for pancreatic cancer.

Citation Format: Nan Zhang, Ru Jia, Guanghai Dai. Efficacy and underlying mechanisms of surufatinib combined with PD-1 monoclonal antibody and chemotherapy in pancreatic 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 2129.
Abstract 2130: Glioblastoma-derived extracellular vesicle cargo as a reporter of drug delivery and effect
Authors: DeLuca Valerie , Tang Nanyun , Hansen Nathaniel , Raju Anjali , Shaffer Charles , Hao Yue , Burns Terry C. , Sarkaria Jann N. , Parney Ian , Pirrotte Patrick , Van Keuren-Jensen Kendall , Berens Michael E.
Published: March 2024

The selectivity of the blood-brain-barrier (BBB) can prevent therapeutics from reaching the brain, and this may contribute to the high failure rate of new drugs for tumors like glioblastoma (GBM) and brain metastasis. The capacity to detect BBB penetrance in real-time in a noninvasive manner would arguably aid the rate of therapeutic development. Extracellular vesicles, which are membrane bound particles secreted by all cell types in the body, have potential as a neuro pharmacodynamic reporter given that their protein and RNA cargo reflect the originating cell population. We hypothesized that the exposure of GBM cells to therapy results in altered EV cargo, and that the detection of this shift can be leveraged as a liquid biopsy from patients during early clinical trials to determine BBB penetrance and tumor engagement. To identify cargo shifts indicative of treatment, we exposed four short-term patient derived glioma cell lines to arsenic trioxide (an idiopathic toxin), MLN4924 (a neddylation inhibitor), and the alkylating agent temozolomide, then harvested EVs for unbiased mass spectrometry and RNA sequencing. Interestingly, we identified conserved upregulated and downregulated protein and RNA species across cell lines and across drug treatments, despite the diverse mechanisms of action. Candidate proteins were further validated by mesoscale immunoassays and exoview evaluation of tetraspanin-captured EVs. RNA species were validated by qRT-PCR. Overall, we demonstrate that GBM-derived EV cargo changes in response to therapy, supporting the development of EVs as a reporter of drug delivery in GBM. Ongoing and future work is focused on identifying enrichment handles in order to selectively evaluate GBM-derived EV cargo from patient biofluids. Thus far, B7H3, PTPRZ1, EGFRvII and IL13Ra2 have been screened as putative handles. Success in this pursuit could substantially enhance findings and accelerate drug testing in early-stage clinical trials in brain tumor patients by reporting drug accessibility to the tumor and engagement with drug targets.

Citation Format: Valerie DeLuca, Nanyun Tang, Nathaniel Hansen, Anjali Raju, Charles Shaffer, Yue Hao, Terry C. Burns, Jann N. Sarkaria, Ian Parney, Patrick Pirrotte, Kendall Van Keuren-Jensen, Michael E. Berens. Glioblastoma-derived extracellular vesicle cargo as a reporter of drug delivery and effect [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 2130.
Abstract 2264: Identifying and analyzing independent clonal populations in single Barrett’s esophagus biopsies
Authors: Kazachkova Mariya , He Phoebe , Alexandrov Ludmil B.
Published: March 2024

Barrett’s esophagus (BE) is a non-obligate precursor of esophageal adenocarcinoma (EAC), often measuring multiple centimeters in length and characterized by columnar epithelium replacing normal squamous epithelium in the distal esophagus. Early studies in BE suggested that all cells within a patient’s Barrett’s segment are progenies of a single founder cell. However, in recent years this previously accepted model of BE as a monoclonal disease has been retired in favor of a multiclonal model with new evidence suggesting that a patient’s Barrett’s segment can be composed of multiple independent clones with independent founder cells. Nonetheless, single BE samples are still routinely assumed to be monoclonal, leading to independent sets of genomic aberrations erroneously being analyzed as a single set in the case of multiclonal samples. Here, we utilize a previously generated whole-genome sequenced BE dataset and repurpose an established pipeline for detecting subclonal populations to identify BE samples that are comprised of multiple independent clones. Additionally, we present a novel approach for validating multiclonality from sequencing data by phasing pair-end sequencing reads using germline variants. We demonstrate that 20/80 patients have at least one multiclonal sample and that the independent clones contained in these single biopsies can have drastically different genomic profiles. Further, we speculate that these multiclonal samples capture a snapshot of clonal competition and evolution within BE. These findings highlight the importance of accounting for multiclonality when studying BE and provide the first systematic analysis of multiclonal BE samples.

Citation Format: Mariya Kazachkova, Phoebe He, Ludmil B. Alexandrov. Identifying and analyzing independent clonal populations in single Barrett’s esophagus biopsies [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 2264.
Abstract 2278: Unraveling immune dynamics of CD4+ in non-small cell lung cancer through single-cell analysis and gene expression profiling
Authors: Woo Hyunmin , Yang JiHye , Yang Siyoung , Eyun Seong-il
Published: March 2024

Non-small cell lung cancer (NSCLC) is a type of epithelial lung cancer other than small cell lung cancer and represents the majority of cases in lung cancer. The immune response against NSCLC relies significantly on the vital role by CD4+ T cells, contributing to the regulation of immune system and the suppression of tumor growth. Various CD4+ T cell subtypes, such as T conventional cells (Tconv cells) and T regulatory cells (Treg cells), perform distinct functions within the immune system. Tconv cells boost immune responses against specific pathogens, while Treg cells play a role in suppressing immune reactions. Investigating the correlation between Tconv cells and Treg cells in NSCLC could serve a valuable biomarker for evaluating the immune status of lung cancer patients. We examine the connection between particular CD4+ T cells in the lung and specific gene expressions associated with NSCLC. Through single-cell analysis, we classified the proportions of Treg cells and Tconv cells within CD4+ T cells in both the lungs and spleen of mice. Our discovery revealed that the proportion of Tconv cells in the lungs exceeded that in the spleen. From the differential gene expression (DEG) analysis using RNA-Seq data for Treg cells and Tconv cells in NSCLC, compared to the control group of Peripheral Blood Mononuclear Cells (PMBC), we identified 1823 genes uniquely expressed in NSCLC Treg cells and 711 genes in Tconv cells. Within these genes, we verified the expression of IL2RA and CTLA4, known to be expressed in NSCLC. Additionally, we identified high expression of genes TM4SF1 and KRT6A. By comparing the genes specific to Tconv and Treg cells in the lungs with those significantly expressed in NSCLC, we were able to identify 114 co-expressed genes in Treg cells and 71 in Tconv cells. We conducted GO and KEGG pathway analyses on these genes and examined the interaction networks among the associated genes. Furthermore, we found receptor interactions between Tconv cell subtypes (Th1, Th2, Th17, and Tfh) and Treg cells through cell-cell interaction analysis. We explored the mouse phenotypes associated with these genes. These discoveries unveiled connections within lung-specific gene networks of Treg and Tconv cells associated with NSCLC, highlighting candidate genes for targeted therapy in cancer.

Citation Format: Hyunmin Woo, JiHye Yang, Siyoung Yang, Seong-il Eyun. Unraveling immune dynamics of CD4+ in non-small cell lung cancer through single-cell analysis and gene expression profiling [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 2278.
Abstract 2302: Innovative blood-based cell-free RNA whole transcriptome sequencing for comprehensive RNA profiling in patient plasma samples and its clinical applications
Authors: Liu Fang , Huang Yong , Wang Min , Sun Xiangying , Xiang Binggang , Jia Shidong , Du Pan , Luo Shujun
Published: March 2024

Background: Plasma cell-free RNA (cfRNA) holds promise for non-invasive cancer biomarker discovery, particularly in diseases like prostate cancer where specific biomarkers (AR splicing variants and TMPRSS2:ERG fusion) are detectable at the RNA level. Despite its potential, the technical challenges posed by blood cell-originated cfRNA abundance necessitates the development of robust and reliable assays for non-invasive cancer diagnostics. This study establishes and demonstrates the clinical utility of an innovative protocol for isolating and identifying tumor-originated cfRNA in plasma samples.

Methods: A novel methodology was developed for the simultaneous extraction of cfDNA and cfRNA from a single blood sample tube, enabling whole transcriptome sequencing (WTS) analysis with minimal input as low as 1ng. A proprietary method was developed to selectively deplete blood cell-originated transcripts in the library. An enrichment-based WTS assay was conducted, and a customized RNASeq analysis pipeline was employed to enable expression profiling and cancer biomarker testing from the cfRNA samples.

Results: The developed extraction method yielded substantial amounts of cfDNA and cfRNA from a 4 mL blood sample. Efficient depletion of blood cell-originated transcripts, achieving over 99% removal, enhanced the detection of a broader range of gene expressions in plasma. Implementation of the cfRNA WTS assay across healthy donor and cancer samples demonstrated consistency with documented biomarker status. The assay revealed significant differential clustering between cancer samples and healthy donors, with characteristic cancer genes contributing to the distinction. Reproducibility and analytical sensitivity of the developed cfRNA WTS assay were successfully validated.

Conclusions: This study introduces Predicine's proprietary cfRNA WTS assay, enabling concurrent expression profiling and RNA biomarker detection in plasma samples. Overcoming technical challenges associated with blood cell-originated cfRNA, this assay represents a significant advancement in liquid biopsy-based cancer diagnostics.

Citation Format: Fang Liu, Yong Huang, Min Wang, Xiangying Sun, Binggang Xiang, Shidong Jia, Pan Du, Shujun Luo. Innovative blood-based cell-free RNA whole transcriptome sequencing for comprehensive RNA profiling in patient plasma samples and its clinical 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 2302.
Abstract 2304: Detection of early-disseminated cancer cells with deep learning-enabled holographic imaging
Authors: Heller Nicholas , Mallery Kevin , Bristow Nathaniel , Travadi Yash , Lee Eng Hock , Hong Jiarong
Published: March 2024

Recent research increasingly validates the early dissemination of tumor cells in peripheral blood during the nascent stages of cancer, often preceding clinical identification of the primary tumor. This emerging evidence emphasizes the potential of Circulating Tumor Cells (CTCs) as early indicators for cancer. Despite their significance, CTCs are exceptionally rare compared to normal blood cells, which presents a considerable challenge in detection, particularly during early stages of cancer. Traditional detection methods, such as those based on nucleated cell enrichment, antibody labeling and fluorescence imaging, encounter notable limitations when applied in the context of early detection. These limitations arise from the inherent heterogeneity of CTCs, and the multistep process involved in labeling and manual enumeration, compromising cell viability and resulting in cell loss. This loss becomes particularly problematic in the context of early cancer detection scenarios where the tumor burden is extremely low, and any cell loss can significantly impact assay sensitivity. To overcome these challenges, we propose an innovative CTC detection approach employing a deep learning framework combined with holographic imaging. This technique utilizes coherent light to generate holograms from cell samples, capturing detailed 3D morphological and optical properties of individual cells. We developed a custom convolutional neural network designed to facilitate high-throughput cell sorting in real-time. Trained on healthy blood samples and diverse cancer cell lines, the dataset consists of more than 50 million cellular holograms enhanced by proprietary Gaussian annotation and image augmentation strategies. Moreover, by combining the morphological signatures from holography with antigen signatures (e.g. HER2/ER/PR) in real-time, our model demonstrates the ability to detect less than 1 false positive per 1 million nucleated cells, highlighting its potential as a robust tool for targeted early-stage breast cancer detection. Our technology represents highly sensitive alternative to traditional screening methods, with the promise of enhancing patient outcomes.

Citation Format: Nicholas Heller, Kevin Mallery, Nathaniel Bristow, Yash Travadi, Eng Hock Lee, Jiarong Hong. Detection of early-disseminated cancer cells with deep learning-enabled holographic imaging [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 2304.