bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1505: Unraveling diepafitaxis: Investigating ECM stiffness and microarchitecture in tumor spheroid migration
Authors: Chen Hsiang-Pei , Tsai Ya-Chu , Tu Ting-Yuan
Published: March 2024

Background: Metastasis, accounting for up to 90% of cancer-related deaths, remains one of the least understood aspects of cancer progression. Recent research has shifted focus from solely biological hallmarks to the physical characteristics of cancer, such as the stiffness and microarchitecture of the extracellular matrix (ECM), which play pivotal roles in metastatic advancement. We previously introduced the concept of 'diepafitaxis' to describe a unique mechanism driving the interface-mediated tropism of tumor spheroids at matrix-surface boundaries. This phenomenon suggests that cancer cells preferentially migrate along a 2D 'interfacial' track formed by heterogeneous 3D ECM structures, facilitating invasion and dispersion. However, the exact mechanisms by which interfacial components, stiffness, and structural attributes influence cell movement remain debated. To investigate this, we have developed a microfluidic chip with a precisely controlled stiffness of the hydrogel matrix-matrix interface (MMI), aiming to shed light on specific diepafitatic behaviors.

Method: We developed a polydimethylsiloxane (PDMS)-based microfluidic chip using a traditional photolithographic process. This chip features three channels, with the center channel designed to precisely control various MMI stiffness combinations: stiff vs soft (t-o), stiff vs stiff (t-t) and soft vs soft (o-o). MDA-MB-231 breast cancer spheroids (BCS), representing an in vitro tumor model, were embedded in the top gel. We observed the dynamic behavior of these cancer spheroids over a 24-h period using differential interference contrast (DIC) microscopy.

Result: The study revealed that BCS exhibited more pronounced dispersion in a soft homogeneous ECM compared to a stiff ECM in 3D environments. Interestingly, when BCS were near an MMI, a stiffer MMI (t-t) significantly enhanced BCS invasiveness and single-cell migration, more so than t-o, and notably more than o-o. We previously described this phenomenon as '2.5D migration,' where cells migrate in a ventral 2D manner within a sandwiched 3D gel structure. This diepafitatic behavior was further analyzed for its response to gravitational effects and the influence of chemotaxis, induced by introducing VEGF on one side of the microfluidic channel.

Conclusion: Conclusively, our study demonstrates that the stiffness and structure of the ECM, particularly at MMI, significantly influence the invasive behavior and migration patterns of BCS, underscoring the critical role of mechanical microenvironments in cancer metastasis.

Citation Format: Hsiang-Pei Chen, Ya-Chu Tsai, Ting-Yuan Tu. Unraveling diepafitaxis: Investigating ECM stiffness and microarchitecture in tumor spheroid migration [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 1505.
Abstract 1555: Nuclear accumulation of cell surface programmed cell death ligand 1 promotes metastatic outgrowth in triple-negative breast cancer cells
Authors: Gohara Yuma , Tomonobu Nahoko , Kinoshita Rie , Yamamoto Kenichi , Murata Hitoshi , Sakaguchi Masakiyo
Published: March 2024

PD-L1 presented on the cancer cell surface is well known for its role in immune checkpoint regulation by interacting with PD-1 on immune cells. On the other hand, it has been reported that PD-L1 is localized in the cancer cells’ nuclei, where the nuclear PD-L1 (nPD-L1) is conducive to cancer outgrowth and motility. However, there has yet to be reported about the mechanistic insight into the event. In this study, we, therefore, aimed to clarify still unidentified points in the nPD-L1-mediated event. Our efforts revealed that the upregulation is induced by a functional suppression of nuclear protein X (tentative name), which we identified as a novel binding protein with nPD-L1 at its cytoplasmic tail. Their nuclear interaction also abolished an aggressive cancer proclivity, epithelial-mesenchymal transition (EMT). The mechanism is helpful to the complex nature of PD-L1 in plural cancer cell behaviors.

Citation Format: Yuma Gohara, Nahoko Tomonobu, Rie Kinoshita, Kenichi Yamamoto, Hitoshi Murata, Masakiyo Sakaguchi. Nuclear accumulation of cell surface programmed cell death ligand 1 promotes metastatic outgrowth in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1555.
Abstract 1561: Identification of tumor-associated macrophages promoting early-stage metastasis during peritoneal dissemination in gastric cancer
Authors: Kawase Wataru , Hiroshima Yukihiko , Hashimoto Itaru , Onuma Shizune , Furuta Mitsuhiro , Machida Nozomu , Oshima Takashi , Miyagi Yohei
Published: March 2024

Peritoneal dissemination (PD) is the most frequent metastasis pattern in gastric cancer (GC) and is a significant predictor of poor prognosis. However, the mechanisms underlying PD formation remain unclear, and a PD-specific signature has not been identified. PD development is thought to be influenced by various changes occurring in GC tumor cells and immune cells present in the peritoneal cavity. We previously performed RNA-seq analysis of cell components in cytology-positive (CY1) gastric cancer ascites. The results suggested that tumor-associated macrophages (TAMs) infiltrate the abdominal cavity together with GC tumor cells and might be involved in PD development. In this study, we aimed to identify the specific TAMs responsible for promoting metastasis during the early stage of PD in GC. We demonstrated the abundant presence of TAMs in CY1 GC ascites using flow cytometry analysis and identified several genes that were significantly expressed in CY1 TAMs using single-cell RNA-seq analysis. Furthermore, we evaluated the potential of these genes to serve as therapeutic targets for the treatment of PD in GC.

Citation Format: Wataru Kawase, Yukihiko Hiroshima, Itaru Hashimoto, Shizune Onuma, Mitsuhiro Furuta, Nozomu Machida, Takashi Oshima, Yohei Miyagi. Identification of tumor-associated macrophages promoting early-stage metastasis during peritoneal dissemination in gastric 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 1561.
Abstract 1572: POU5F1B is a human-restricted ROCK inhibitor-sensitive oncoprotein that restructures membrane nanodomains to increase cell adhesion
Authors: Simó-Riudalbas Laia , Offner Sandra , Abrami Laurence , Unterauer Eduard M. , Duc Julien , Planet Evarist , Jungmann Ralf , van der Goot Gisou , Trono Didier
Published: March 2024

Evolution confers new species with distinctive biological features, which can translate in purely mechanistic speciation or in novel phenotypic traits. POU5F1B arose by retrotransposition of POU5F1 (OCT4) in the last common ancestor of modern Hominoidae. Its human product is endowed with oncogenic properties, notably promoting gastrointestinal cancer growth and metastasis. Here, we reveal that the oncogenic action of POU5F1B requires ubiquitination of lysine residues found only in the human protein. This post-translational modification is essential for POU5F1B to localize to the cytoplasm and become acylated by the DHHC17 palmitoyltransferase. This leads to POU5F1B association with detergent-resistant membrane subdomains, where it triggers the accumulation of integrins and signaling molecules, and to a stimulation of cell focal adhesion. Finally, we determined that POU5F1B stability is critically dependent on ROCK, a crucial regulator of cytoskeletal dynamics and cell motility.

Citation Format: Laia Simó-Riudalbas, Sandra Offner, Laurence Abrami, Eduard M. Unterauer, Julien Duc, Evarist Planet, Ralf Jungmann, Gisou van der Goot, Didier Trono. POU5F1B is a human-restricted ROCK inhibitor-sensitive oncoprotein that restructures membrane nanodomains to increase cell adhesion [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 1572.
Abstract 1615: Balanced molecular feature selection unveils universally tumor-lineage informative methylation sites in colorectal cancer
Authors: Li Xuan Cindy , Liu Yuelin , Schäffer Alejandro A. , Mount Stephen M. , Sahinalp Süleyman C.
Published: March 2024

In the era of precision medicine, performing comparative analysis over diverse patient populations is a fundamental step toward tailoring healthcare interventions. However, the critical aspect of equitably selecting molecular features across multiple patients is often overlooked. To address this challenge, we introduce FALAFL (FAir muLti-sAmple Feature seLection), a novel algorithmic approach based on integer linear programming. FALAFL is designed to bridge the gap between molecular feature selection and algorithmic fairness, ensuring a balanced selection of molecular features across different patient samples.

We have applied FALAFL to the problem of selecting lineage-informative CpG sites within a cohort of 9 colorectal cancer patients subjected to low-coverage single-cell methylation sequencing. Our results demonstrate that FALAFL excels at rapidly determining the optimal set of CpG sites, which are well covered by cells across the vast majority of the patients, while ensuring that each patient contributes a similar number of sites to the final selection. An analysis of the FALAFL-selected CpG sites reveals that their lineage informativeness exhibits a strong association across diverse patient profiles, suggesting the non-stochasticity and universality of methylation changes in tumor evolution. Furthermore, these universally lineage-informative sites exhibit subclonal level alterations in the colorectal cancer primary tumors and are strongly associated with lineage-specific gene expression patterns.

By integrating equity considerations into the molecular feature selection process, FALAFL is poised to propel equitable healthcare data science practices and advance our understanding of cancer and other complex diseases.

Citation Format: Xuan Cindy Li, Yuelin Liu, Alejandro A. Schäffer, Stephen M. Mount, Süleyman C. Sahinalp. Balanced molecular feature selection unveils universally tumor-lineage informative methylation sites in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1615.
Abstract 1648: RB represses cohesin-dependent loop formation and activates E2F-independent transcription
Authors: Lee Hanjun , Gkotinakou Ioanna-Maria , Krishnan Badri , Dyson Nicholas J. , Lawrence Michael S. , Sanidas Ioannis
Published: March 2024

The retinoblastoma protein (RB) suppresses the activity of the E2F transcription factor family, controlling cellular proliferation. Recent studies, however, indicate that RB's role in chromatin organization, which is not yet fully understood, might be distinct from this E2F-dependent regulation. Employing chromosome conformation capture at a single nucleosome resolution, we identified RB as a cell cycle-regulated repressor of cohesin-dependent loop formation at topologically associating domain (TAD) boundaries. RB depletion increased the number and size of cohesin-dependent loops and strengthened topologically associating domains (TADs). This phenomenon was specific to the G1 phase and was not observed in the S phase, indicating that this novel RB function is regulated by the cell cycle. Mechanistically, RB showed extensive colocalization with cohesin in the human genome, and it impacted cohesin’s distribution on the chromatin. Active RB reduced cohesin from RB-bound TAD boundaries and decreased cohesin activity therein, as assessed by the increased K105/106 acetylation of the cohesin subunit SMC3. This led to reduced insulation in chromosome conformation capture assays. Importantly, by weakening the insulation activity of the adjacent insulators, RB non-canonically enhanced the expression of non-E2F target genes salient for cell adhesion and extracellular matrix organization. When RB was lost, cells showed a more rapid cellular detachment rate and an elevated migration rate, indicating that this novel RB function controls specific transcriptional programs rather than arbitrary genes to regulate the process of cell adhesion and migration. Overall, we conclude that RB has a central role in the interplay between cell cycle and chromatin organization, by repressing the cohesin-dependent loop formation at TAD boundaries. This RB function safeguards E2F-independent transcriptional programs driven by active enhancers and helps maintain cellular adhesion to the extracellular matrix.

Citation Format: Hanjun Lee, Ioanna-Maria Gkotinakou, Badri Krishnan, Nicholas J. Dyson, Michael S. Lawrence, Ioannis Sanidas. RB represses cohesin-dependent loop formation and activates E2F-independent transcription [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 1648.
Abstract 1672: PKC-ι and 14-3-3 are crucial regulators of c-Raf and the Erk pathway in human medulloblastoma
Authors: Lajmi Luke , Ratnayake Wishrawana S. , Palathinkal Joseph , Breedy Sloan , Todman Aaron , Acevedo-Duncan Mildred
Published: March 2024

Medulloblastoma is the second most common childhood cancer, behind leukemia, and the most common pediatric brain tumor, comprising 20% of all childhood brain cancers. Current treatment options are limited to radiation therapy, surgical resection, and chemotherapy, which are extremely invasive. This underscores the need for more targeted and effective therapeutic treatments to improve patient outcomes. The following study will focus on exploring the PKC-ι specific inhibitor 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and its effects on medulloblastoma cell lines Daoy and ONS-76. Our in-vitro treatments showed that PKC-ι functions an oncogenic protein that is necessary for Daoy and ONS-76 cell proliferation and that ICA-1S was found to be cytostatic to both Daoy and ONS-76 cells. The inhibition of PKC-ι in both cell lines resulted in a decreased phosphorylation of PKC-ι as well as downregulation of 14-3-3 and crucial components of the c-Raf/Erk pathway including total and phosphorylated levels of c-Raf, Mek1/2, and Erk1/2. This comes as a result of ICA-1S blocking the association of PKC-ι with 14-3-3 while PKC-ι and c-Raf were not found to directly associate with each other, indicating indirect modulation of c-Raf by PKC-ι. Additionally, PKC-ι inhibition decreased its association with Stat3. Expression levels of Stat1, Stat3, c-Fos, and c-Myc transcription factors were significantly downregulated, which is likely the result of reduced transcriptional activation due to upstream inhibition of PKC-ι and Erk signaling. Treatment with ICA-1S was also shown to substantially decrease cellular invasion of Daoy and ONS-76 cells and induce apoptosis. Taken together, these data suggest that PKC-ι likely plays a critical role in the regulation of the c-Raf/Erk pathway, which controls the proliferation and invasion of human medulloblastoma.

Citation Format: Luke Lajmi, Wishrawana S. Ratnayake, Joseph Palathinkal, Sloan Breedy, Aaron Todman, Mildred Acevedo-Duncan. PKC-ι and 14-3-3 are crucial regulators of c-Raf and the Erk pathway in human medulloblastoma [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 1672.
Abstract 1689: CRISPR functional genetic screening for immune enhancers of inhibitor receptors in human primary CD8 T cells
Authors: Wang Ping , Giles Josephine R. , Huang Hua , Shi Junwei , Wherry E.John
Published: March 2024

The restrained efficacy of T cell therapies in cancer treatment is associated with T cell exhaustion (TEX), an impaired effector function state that is marked by distinct epigenetic landscape. These unique epigenetic features promoted the progression of TEX, hindered reinvigoration of TEX state and controlled the abnormal expression of inhibitor receptors (IRs), which could serve as target sites in cancer treatment. Thus, deciphering the regulatory elements in primary human T cells will deeply reveal the mechanism controlling TEX and provide novel therapeutic strategies to promote clinical response. However, how enhancers can be effectively applied in anti-cancer treatment is still poorly understood because high throughput identifying the functional enhancers of inhibitor receptors at scale is technically limited in human T cells. Here, we first developed a pooled screening platform that employs a compact CRISPRi system and enabled efficient one-step delivery of pooled screening libraries to primary human T cells. Leveraging this platform, we screened large genomic regions (~300kb) at saturation around key TEX genes, two IRs—PDCD1, HAVCR2 and one transcription factor (TF)—TBX21 to interrogate functional enhancers in human primary CD8 T cells. We identified two enhancers for PDCD1, three enhancers for HAVCR2 and three enhancers for TBX21, all of which are located over 12kb away from each gene’s transcription start sites. Then, to parse the essential sequences that fulfill enhancer function, we employed Cas9-based in-situ saturation mutagenesis screening to the identified enhancers of PDCD1 and HAVCR2. We found disrupt essential sequences impaired the entire enhancer function, quantitatively phenocopied the effect of targeting the entire enhancer with CRISPRi. Transcription factor motifs analyses of these sequences revealed potential regulation of ZFX for controlling PDCD1 expression and STAT for HAVCR2 expression. Finally, we explored the functional consequences of applying enhancers to anti-cancer treatment. Indeed, targeting the identified enhancers of PDCD1 and HAVCR2 in CAR T cells lead to enhanced cancer cell killing efficiency in vitro. Together, our study provides a platform and paradigm to interrogate functional enhancers in human T cells, illuminated the great potential of manipulating enhancers in T cell therapies.

Citation Format: Ping Wang, Josephine R. Giles, Hua Huang, Junwei Shi, E.John Wherry. CRISPR functional genetic screening for immune enhancers of inhibitor receptors in human primary CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1689.
Abstract 1695: Genomic clustering tendency of transcription factors reflects phase-separated transcriptional condensates at cancer super-enhancers
Authors: Wang Zhenjia , Wang Shengyuan , Zang Chongzhi
Published: March 2024

Many transcription factors (TFs) have been shown to bind to super-enhancers, forming transcriptional condensates to activate transcription in various cellular systems including cancer. However, the genomic and epigenomic determinants of phase-separated transcriptional condensate formation remain poorly understood. Questions regarding which TFs tend to associate with transcriptional condensates and what factors influence their association are largely unanswered. Here we systematically analyzed 528 DNA sequence motifs across the human genome and 6,650 TF binding profiles across different cell types to identify the molecular features contributing to the formation of transcriptional condensates. We found that the genomic distributions of sequence motifs for different TFs exhibit distinct clustering tendencies. Notably, TF motifs with a high genomic clustering tendency are significantly associated with super-enhancers. TF binding profiles showing a high genomic clustering tendency are further enriched at cell-type-specific super-enhancers. TFs with a high binding clustering tendency also possess high liquid-liquid phase separation abilities. Compared to non-clustered TF binding, densely clustered TF binding sites are more enriched at cell-type-specific super-enhancers with higher chromatin accessibility, elevated chromatin interaction, and stronger association with cancer outcomes. Our results indicate that the clustered genomic binding patterns and the phase separation properties of TFs collectively contribute to the formation of transcriptional condensates at cancer super-enhancers.

Citation Format: Zhenjia Wang, Shengyuan Wang, Chongzhi Zang. Genomic clustering tendency of transcription factors reflects phase-separated transcriptional condensates at cancer super-enhancers [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 1695.
Abstract 171: Chemotherapeutic agent, cisplatin, influences the proportion of TREM2 (+) M2 macrophages in the TME of LUAD
Authors: Cha Yoon Jin , Park Min Kyung , Chang Yoon Soo
Published: March 2024

Background: Triggering receptor expressed on myeloid cells (TREM2) serves as an immune signaling hub that senses and interacts with a wide range of ligands arising from damaged tissues in pathologic conditions. Despite TREM2’s diverse role in casting immune suppressive environment, the effects of chemotherapy on tumor microenvironment (TME), TREM2 and their interactions are not well established. In this study, we aimed to investigate the characteristics of TREM2 (+) Mφ within the lung TME (Tu), focusing on the impact of chemotherapy on the immune dynamics of lung adenocarcinoma (LUAD).

Method: Changes in the number and distribution of Mφ subclusters in normal appearing lung tissue (NL) and Tu were examined using open-source single cell RNA sequencing (scRNA-seq) datasets and immunofluorescent staining of lung cancer-induced Kras-G12D mouse tissue. Clinical measures affecting the expression of TREM2 were explored using the TCGA-LUAD and -LUSC datasets, and the effects of anticancer drugs and the apoptotic cells on Mφ were observed by differentiating THP1 cells into M0, M1, and M2.

Results: scRNA-seq analysis on LUAD datasets showed that Mφ were the second most abundant cell population that constitutes the lung TME, and the proportion of Mφ in the Tu decreases compared to NL. Among the Mφ subclusters in Tu, a subcluster which specifically increased in Tu shows enrichment of apoptotic cell clearance gene sets, including TREM2. In lung cancer induced Kras-G12D mouse, compared to NL the Tu has an increase in M2 proportion, showing the characteristics of interstitial Mφ in addition with overexpression of TREM2 and enrichment of monocyte chemotaxis gene set enrichment. Among TCGA-LUAD, the high TMB group has low TREM2 expression, and the proportion of TREM2 (+) M2 in the Tu decreases in cisplatin-treated Kras-G12D mouse Tu, suggesting the expression of TREM2 in M2 is suppressed by either cisplatin treatment itself or active immunity in Tu. Additionally, we are going to observe the effect on TREM2 expression by adding cisplatin treated A549-GFP cells or cisplatin alone, to the THP-1 cells that were differentiated into M0, M1, or M2 state.

Conclusion: In addition to changes in the lung immune environment due to the tumor, anticancer treatment changes in the characteristics of M2 in TME, in terms of decrease in TREM2 (+) Mφ fraction. Considering that TREM2 plays a central inhibitory role in the tumor immune environment, the effect of anticancer chemotherapeutic agents should be considered in the development TREM2 targeting agents.

Citation Format: Yoon Jin Cha, Min Kyung Park, Yoon Soo Chang. Chemotherapeutic agent, cisplatin, influences the proportion of TREM2 (+) M2 macrophages in the TME of LUAD [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 171.
Abstract 1720: 53BP1 regulates heterochromatin through liquid-liquid phase separation (LLPS)
Authors: Geng Xinran , Zhang Youwei
Published: March 2024

As compacted DNA, heterochromatin represses abnormal gene expression by inhibiting DNA transcription and maintains genome integrity by protecting aberrant chromosome segregation. Therefore, abnormalities in heterochromatin function are commonly found in cancer. However, how exactly heterochromatin preserves the genome integrity remains poorly understood due largely to the complex structure of heterochromatin. Recently, increasing evidence suggests that heterochromatin factors, including HP1α, KAP1, and SUV39H1/2, could drive the soluble heterochromatin into phase-separated droplets/condensates to promote gene silencing, which is referred to as liquid-liquid phase separation (LLPS). TP53-binding protein 1 (53BP1) has traditionally been a critical factor in DNA double-strand break (DSB) repair. We recently reported an unexpected finding that under normal growth conditions, 53BP1 regulates heterochromatin function through LLPS, which is independent of its DSB repair function. Our studies reveal a new paradigm in studying 53BP1, heterochromatin, and genome stability. However, an important question remains unanswered: how 53BP1 is recruited to heterochromatin to undergo LLPS and the associated broad biological impact. To address this question, we performed a special crosslinking coupled mass spectrometry to uncover potential factors in the 53BP1 condensates assembled on heterochromatin. We identified many factors associated with DNA methylation and histone modification, which are the two main steps in restoring heterochromatin epigenetic modification. Their interactions with 53BP1 at heterochromatin were confirmed by different approaches. We further focused on one of the key factors, PCNA, and carried out a detailed analysis of the interaction between PCNA and 53BP1 and the role of such interaction in 53BP1 LLPS, DSB repair, and heterochromatin function. These findings reveal the previously uncharacterized function of 53BP1 at heterochromatin during normal DNA replication.

Citation Format: Xinran Geng, Youwei Zhang. 53BP1 regulates heterochromatin through liquid-liquid phase separation (LLPS) [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 1720.
Abstract 1737: METTL3-mediated m6A modification promotes tumor progression in upper tract urothelial carcinoma
Authors: LIN LI-JIE , PENG PEI-HUA , HSU KAI-WEN
Published: March 2024

Upper tract urothelial carcinoma (UTUC), comprising 5% of urothelial neoplasms, is a rare malignancy arising from the transitional epithelium of the renal pelvis and ureter. Its rarity and diverse histology pose diagnostic and therapeutic challenges, often lead to delayed diagnoses. The absence of targeted therapies, coupled with a limited understanding of molecular pathogenesis, underscores the ongoing research in urological oncology for effective UTUC management. N6-methyladenosine (m6A) is the predominant RNA methylation in mammalian cells, with its inscription primarily orchestrated by METTL3, a crucial methyltransferase. METTL3 has been implicated in various cancers, affecting critical processes like proliferation, migration, and tumor progression. However, the specific mechanisms and biological function of how METTL3/m6A regulates the progression of UTUC remain unclear. In our study, we found that m6A modification and METTL3 expression were elevated in aggressive UTUC, both are negatively correlated with unfavorable clinical outcomes and poor patient survival. By overexpressing METTL3 in less aggressive UTUC cell lines BFTC909 and KTCC28, the abilities of cell proliferation, migration, invasion, and colony formation increased. Notably, overexpression of METTL3 also increased tumor growth and metastasis in the in vivo orthotopic mouse model. In contrast, knockdown of METTL3 decreases tumor progression in a highly aggressive UTUC cell line KTCC28M. Furthermore, our comparative analysis of RNA-seq and m6A MeRIP-seq datasets unveiled the regulation of 1660 genes by METTL3. This diverse set includes genes associated with tumor growth and metastasis (such as MYC, JAK2, and STAT3), as well as RNA processing-related genes which their critical roles in modulating mRNA stability and splicing (such as GEMIN5 and PABPC1L). Together, we noticed METTL3/m6A is a crucial protagonist in the oncogenic landscape of UTUC, which suggested that it can be used as a target for novel therapeutic strategy for UTUC treatment.

Citation Format: LI-JIE LIN, PEI-HUA PENG, KAI-WEN HSU. METTL3-mediated m6A modification promotes tumor progression in upper tract urothelial carcinoma [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 1737.
Abstract 1767: Molecular subclassification of gastrointestinal stromal tumors by genomic backgrounds
Authors: Yamamoto Hideki , Nakada Eiji , Kawano Seiji , Kato Fumino , Fukano Chika , Futagawa Mashu , Urakawa Yusaku , Ohsumi Risa , Ueno Sayaka , Yanai Hiroyuki , Hirasawa Akira
Published: March 2024

Gastrointestinal stromal tumors (GISTs) are the most common gastrointestinal mesenchymal tumors. While about 10% of GISTs are known to be developed from the KIT or PDGFRA gain-of-function mutations, the rest, which is the majority of GISTs, is so-called as wild-type GISTs. There are attempts to make subclassification of wild-type GISTs in syndromic or non-syndromic group as promising for understanding of heterogeneity or treatment strategy. Genomic states of succinate dehydrogenase B (SDHB) and neurofibromatosis type 1 (NF1) have been raised as candidates for subclassification of wild-type GISTs. In this presentation, we will discuss our recent three cases of wild-type GISTs, carrying germline pathogenic variants in SDHB or NF1. Two cases with SDHB germline pathogenic variants showed stomach GISTs with good clinical course, the maintained performance status and long survival nevertheless of the recurrence. The other case with an NF1 germline pathogenic variant showed multiple GISTs in small intestine which was controlled surgically and the late onset of other clinical symptoms of NF1. These cases might clinically suggest potential prognostic and predictive markers in genomic alterations of SDHB or NF1 for the cases with wild-type GISTs.

Citation Format: Hideki Yamamoto, Eiji Nakada, Seiji Kawano, Fumino Kato, Chika Fukano, Mashu Futagawa, Yusaku Urakawa, Risa Ohsumi, Sayaka Ueno, Hiroyuki Yanai, Akira Hirasawa. Molecular subclassification of gastrointestinal stromal tumors by genomic backgrounds [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 1767.
Abstract 1834: Discovery of a novel and highly selective PARP7 inhibitor for cancer immunotherapy
Authors: Liu Xiaohui , Wang Ying , Xia Dongliang , Zhang Wei , Tian Shijie , He Yan , Feng Xuerong , Yang Xia , Zhang Qi , Jiang Shiyi , Wu Meifeng , Deng Jianfeng , Wang Yeye , Su Yuxi , Yao Min , Yuan Weijiao , You Xuedan
Published: March 2024

Poly-ADP-ribose polymerase 7 (PARP7) is a member of the wider PARP enzyme family that modulate protein function by using nicotinamide adenine dinucleotide (NAD+) as a substrate to transfer an ADP-ribose monomer onto specific amino acid acceptor residues of target proteins. PARP7 acts as a key repressor of type I interferon (IFN) signaling. Its inhibition combats tumor regression by enhancing antitumor immunity that depends on STING pathway. Therefore, PARP7 is emerging as a promising drug target for new immunotherapy. Using structure-based drug design and optimization, we discovered a novel PARP7 inhibitor, HSN002066, with high inhibitory potency (Enzyme assay IC50 = 0.96 nM) and high selectivity for PARP7 over PARP1/2/12/5A/5B/10 (IC50 =163.4/4.7/345.2/1790/110.2/>10000 nM, respectively). In addition, HSN002066 has excellent ADMET profiles with CYPs inhibition, IC50 >10 μM, human LMS T1/2 > 60 min, hERG inhibition, IC50 > 30 μM,mouse oral bioavailability (F) 98.1%. Notably, HSN002066 and RBN-2397, the only PARP7 inhibitor currently in Ph 1/2 clinical development, displayed differentiated antiproliferative activities in cell line NCI-H1373 (IC50 are 387.8 nM and 15.2 nM, respectively). However, HSN002066 revealed remarkable antitumor effects as monotherapy (TGI 77% at 100 mg/kg) or combination with PD-1 antibody in CT-26 allograft model.

The structure of HSN002066 was not presented and will not be disclosed at the time of presentation at AACR meeting.

Citation Format: Xiaohui Liu, Ying Wang, Dongliang Xia, Wei Zhang, Shijie Tian, Yan He, Xuerong Feng, Xia Yang, Qi Zhang, Shiyi Jiang, Meifeng Wu, Jianfeng Deng, Yeye Wang, Yuxi Su, Min Yao, Weijiao Yuan, Xuedan You. Discovery of a novel and highly selective PARP7 inhibitor for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1834.
Abstract 184: M1-like macrophage infiltration, CD8+ T cell activation, and increased cancer cell apoptosis in immunologically cold Lewis lung carcinoma treated with tumor-trophic Salmonella Typhimurium expressing anticancer proteins in immunocompetent C57BL/6 mice
Authors: He Qiuhong , Brncick Lindsay R. , Gong Boxi , Flowers Julia L.
Published: March 2024

Immunotherapy has revolutionized cancer treatment with Immune checkpoint inhibitors (ICIs) and genetically engineered T-Cells expressing chimeric antigen receptors (CARs). CAR-T treatment of hematologic malignancies has achieved outstanding patient response rate. CAR T therapy, however, is thwarted in solid tumor treatment due to two major factors: the immune-suppressive tumor microenvironment that Inhibits T-cell infiltration and T-cell function. In addition, cancer resistance to ICIs treatment also limits the solid tumor response rate. This research aims to activate proinflammatory anticancer immune responses in immunologically cold solid tumors using the genetically engineered Salmonella typhimurium VNP20009 strains that express anticancer proteins. Previously, we have shown that the Salmonella Typhimurium VNP20009 strain expressing methioninase (METase) and/or Tumor Necrosis Factor (TNFα) caused rapid tumor destruction of cold Lewis Lung Carcinoma (LLC) grown on immunocompetent C57BL/6 mice. In this investigation, several mechanisms of this tumor tropic anti-cancer bacteria were studied including the innate and adaptive immune system responses and cancer cell apoptosis. In a pilot study, we selected eight tumor tissues treated with several different bacteria strains and performed histological analysis to observe the effectiveness of each. We found that in the tumor tissues treated with VNP20009_rMETase_TNFα, there was infiltration and activation of CD8+ T cells coupled with reduced amounts of T regulatory cells, as well as an increased ratio of M1-like to M2-like macrophages. We also observed increased tumor apoptosis associated with an elevated detection of an apoptosis biomarker and a suppressed amount of the Ki67 tumor growth signal. These findings indicate that VNP20009_rMETase_TNFα anticancer bacteria can disrupt the tumor microenvironment in the cold LLC tumors and active innate and adaptive anti-cancer immune response in immunocompetent mice. Further, we are investigating additional cold solid mouse tumor models to validate our findings and how this can apply to bacteria-based immunotherapy.

Citation Format: Qiuhong He, Lindsay R. Brncick, Boxi Gong, Julia L. Flowers. M1-like macrophage infiltration, CD8+ T cell activation, and increased cancer cell apoptosis in immunologically cold Lewis lung carcinoma treated with tumor-trophic Salmonella Typhimurium expressing anticancer proteins in immunocompetent C57BL/6 mice [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 184.
Abstract 1840: MYBBP1A AMPylated by SELO to influence its function
Authors: Li Wu
Published: March 2024

Introduction: MYBBP1A, named Myb-binding protein 1A, is a protein that mainly locates in nucleolar and shuttles between the nucleus and cytoplasm, nuclear import may be mediated by KPNA2, however export depend partially on XPO1/CRM1. It activates or represses transcription via interactions with sequence specific DNA-binding proteins. Repression may be mediated at least in part by histone deacetylase activity (HDAC activity). Acts as a corepressor and in concert with CRY1, represses the transcription of the core circadian clock component PER2. Preferentially binds to di-methylated histone H3 'Lys-9' (H3K9me2) on the PER2 promoter. MYBBP1A overexpressed in tumor than normal and have a significantly negative correlation with patient overall survival. We aim to explore the role of MYBBP1A in PDAC progression and underlying mechanism. In our research, we find and prove the novel AMPylating enzyme SELO-AMPylate MYBBP1A inhibits PDAC progression and modified site in S1353 and further influence downstream relative RNA, such as 18s rRNA, 28S rRNA.

Methods and Materials:

1. The function of MYBBP1A in tumorigenesis at the organismal and cellular levels.

MYBBP1A is widely expressed in different tissues. Firstly, we explored the correlation between MYBBP1A expression level and PDAC through TCGA database and Kaplan-Meier plotter. We then established MYBBP1A gene knockout mice mating with PDX/P53K172H/KRASG12C to induced a primary PDAC model to analyze the progression of pancreatic cancer. The effect of MYBBP1A on tumor cell growth and proliferation was explored through cell proliferation analysis and a nude mouse tumor xenograft model.

2. The MYBBP1A AMPylated by SELO influence MYBBP1A function in cell proliferation was investigated at the molecular and cellular levels.

AMPylation-specific antibodies were used to confirmed MYBBP1A AMPylated by SELO in both cell lysates and purified proteins through experiments such as CO-IP and WB. Stably expressed MYBBP1A WT/S1353A were constructed to verify the function in cell proliferation or migration by SELO. The signaling molecules were further explored through CO-IP and WB to identify the changed molecular by SELO-AMPylate-MYBBP1A, revealing the entire signaling pathways of AMPylation-mediated regulation of PDAC proliferation.

Conclusion: We have demonstrated the function of MYBBP1A in the formation of pancreatic cancer, and further revealed SELO-AMPylate-MYBBP1A importance in the process of reversing MYBBP1A’s role, which has played a certain significance for the treatment of pancreatic cancer and the exploration of related therapeutic targets.

Citation Format: Wu Li. MYBBP1A AMPylated by SELO to influence its function [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 1840.
Abstract 1981: Profiling of protein interactome with cysteine-reactive libraries led to the discovery of EML4ALK dimerizers
Authors: Yang Diane , Harry Stefan , Codd Elizabeth , Kaluziak Stefan T. , Carlin Alexander D. , Zhang Edwin , Oppong-Holmes Zavontae , Yang Wenxin , Smith Abigail , Mitchell Dawn , Hara Mariko , Zhang Siwen , Onozato Maristela , Bar-Peled Liron , Iafrate Anthony J.
Published: March 2024

Cysteine covalent binders enable direct interactions with previously deemed undruggable protein targets, revolutionizing cancer therapeutics development. The primary focus has been on tailoring single-warhead compounds to target specific cysteine residues within the functional domains of target proteins. However, the biochemical properties including dimerization, induced conformational changes, and interactome disruption have been underexplored, presenting an ongoing limitation in current drug discovery approaches. In this study, we generated a library of 32 cysteine-reactive warhead compounds using various combinations of scaffolds and warheads to represent diverse reactivities. A combinatory assay approach, including cysteine druggability mapping (CDM) to discover reactive residues and SDS-PAGE fractionation assays to identify disrupted protein interaction events, was employed. Characterization of the library revealed distinct reactive cysteines and dimerization profiles among the 32 unique compounds. Compound 1C9, identified as a lead compound, demonstrated significant dimerization of EML4, a common fusion gene partner in lung cancer. Using the EML4ALK fusion gene as a proof of concept, we explored potential mechanisms underlying the compound-induced dimerization of EML4. Subsequent structure-activity relationship (SAR) studies and screening of 70 additional compounds were conducted to optimize ALK dimerization and minimize off-target effects. The enhanced compound disrupted the ALK cellular signaling pathways AKT and ERK upon dimerization of the EML4ALK fusion gene. Additionally, we discovered that covalent dimerization induced EML4ALK degradation without requiring a ubiquitin ligase like a PROTAC. In conclusion, our work has uncovered a novel class of cysteine covalent binders, which we believe are valuable tools to modulate protein-protein interactions in previously undruggable proteins in cancer. This discovery holds significant therapeutic potential for future drug development.

Citation Format: Diane Yang, Stefan Harry, Elizabeth Codd, Stefan T. Kaluziak, Alexander D. Carlin, Edwin Zhang, Zavontae Oppong-Holmes, Wenxin Yang, Abigail Smith, Dawn Mitchell, Mariko Hara, Siwen Zhang, Maristela Onozato, Liron Bar-Peled, Anthony J. Iafrate. Profiling of protein interactome with cysteine-reactive libraries led to the discovery of EML4ALK dimerizers [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 1981.
Abstract 201: Morphological changes to the vulvar squamous carcinoma cell line, A431, resulting from clobetasol-induced upregulation of vimentin
Authors: Lewis Jani E. , Haering Kia , Yokoyama Kazushige
Published: March 2024

Vimentin is recognized as an important marker in the epithelial-mesenchymal transition (EMT) of several cancer cell types. The overexpression of vimentin appears to coincide with increased tumor growth, migration, and poor prognosis. The vulvar cancer cell line, A431, is of squamous cell origin and displays hallmarks of a squamous cell including expression of cytokeratins 8 and 18 as well as E- and P-cadherin. We have found that treatment of A431 cells with the glucocorticoid, clobetasol, results in upregulation of vimentin which is accompanied by change to a more mesenchymal-like morphology. Despite this change in morphology, the cells continue to express cytokeratins 8 and 18. This suggests that the vimentin has a greater impact on cell morphology than does cytokeratins 8 and 18. However, we previously reported that the analogous glucocorticoid, dexamethasone, also downregulates E- and P-cadherin in these cells which may account for the observed change in morphology. Here we examine more closely the impact of vimentin on cellular morphology in the clobetasol treated A431 cells by both immunofluorescence microscopy and Raman imaging.

Citation Format: Jani E. Lewis, Kia Haering, Kazushige Yokoyama. Morphological changes to the vulvar squamous carcinoma cell line, A431, resulting from clobetasol-induced upregulation of vimentin [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 201.
Abstract 2010: TRIM27 Interacts with KLF12 to regulate L1CAM expression, influencing cisplatin resistance and cancer metastasis in esophageal squamous cell carcinoma
Authors: Zhang Hao , Li Chunxiang , He Jie
Published: March 2024

Esophageal squamous cell carcinoma (ESCC) accounts for 85% of cases of esophageal cancer worldwide and is the predominant form of esophageal cancer in Asia. A significant number of patients with ESCC are diagnosed at an advanced stage, resulting in poor prognosis. Cisplatin resistance and cancer metastasis are the main clinical challenges in the treatment of advanced ESCC. KLF12 has been extensively characterized as a transcriptional repressor, and previous studies have unveiled its roles in angiogenesis, neural tube defect, and NK cell proliferation. However, the precise contribution of KLF12 to cancer treatment remains undefined. Here, we show that KLF12 is downregulated in various cancer types, and KLF12 downregulation promotes cisplatin resistance, cancer metastasis and recruitment of regulatory T cells (Tregs) in ESCC cell lines. Mechanistically, KLF12 binds to the promoters of L1CAM and represses its expression. Depletion of L1CAM abrogates cisplatin resistance, cancer metastasis, and Treg recruitment caused by KLF12 loss. Moreover, the E3 ubiquitin ligase TRIM27 binds to the N-terminal region of KLF12 and ubiquitinates KLF12 at K326 via K33-linked polyubiquitination. Notably, TRIM27 depletion enhances the transcriptional activity of KLF12 and consequently inhibits L1CAM expression. Overall, our study has firstly elucidated a novel regulatory mechanism involving TRIM27, KLF12 and L1CAM, which plays a substantial role in cisplatin resistance and cancer metastasis in ESCC, and targeting KLF12/L1CAM axis may be a promising approach for ESCC treatment.

Citation Format: Hao Zhang, Chunxiang Li, Jie He. TRIM27 Interacts with KLF12 to regulate L1CAM expression, influencing cisplatin resistance and cancer metastasis in esophageal squamous cell carcinoma [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 2010.