bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2222: The molecular landscape of acral lentiginous melanoma in Mexican patients
Authors: Vázquez-Cruz Martha E. , Basurto-Lozada Patricia , Molina-Aguilar Christian , Castaneda-Garcia Carolina , Ferreira Ingrid , Martínez-Said Héctor , Álvarez-Cano Alethia , Tavares-de la Paz Luis Alberto , Hinojosa-Ugarte Diego , García-Ortega Dorian Y. , Martínez-Gómez Julia M. , Levesque Mitchell P. , Adams David J. , Robles-Espinoza Carla D.
Published: July 2021

Acral lentiginous melanoma (ALM) is the most common melanoma subtype in several countries in Africa, Asia, and Latin America, including Mexico. Comparatively fewer studies have been published of ALM than other subtypes of the disease. However, it is becoming clear that complex DNA structural aberrations, as well as several genes, such as NOTCH2, KIT, PTEN and TYRP1, in addition to the established melanoma drivers, play a role in its development. To date, no comprehensive genomic characterization has been performed on tumors from Latin American patients. The causes of ALM are unknown, and little is known about its transcriptomic profile and immune landscape. Because of these factors, therapeutic options are limited, while approaches such as immunotherapy are too costly for most patients in Latin American countries, and are of unknown efficacy in rarer forms of melanoma such as ALM. A better understanding of the molecular and immune profile of ALM would allow us to identify potential therapeutic targets and treatment strategies in this population.

We have sequenced the exomes and transcriptomes of 113 ALM tumors (primaries, metastases and recurrences) from 86 patients being treated at the National Cancer Institute of Mexico, a national reference hospital. We have performed analyses to define the genomic landscape of the disease, its mutational signatures, the biological pathways involved in its development, and its immune profile. From these analyses, we have identified recurrently amplified and deleted genomic regions, potential causal agents, and the involvement of specific interleukins and cell-adhesion proteins in ulcerated and metastatic tumors.

This study is one of the first to profile the molecular landscape of ALM in a Latin American population. We hope that it will contribute to increasing the diversity of cancer genomics datasets, help highlight differences in disease biology, and provide insights that may inform therapeutic strategies for all patients with this lethal malignancy.

Citation Format: Martha E. Vázquez-Cruz, Patricia Basurto-Lozada, Christian Molina-Aguilar, Carolina Castaneda-Garcia, Ingrid Ferreira, Héctor Martínez-Said, Alethia Álvarez-Cano, Luis Alberto Tavares-de la Paz, Diego Hinojosa-Ugarte, Dorian Y. García-Ortega, Julia M. Martínez-Gómez, Mitchell P. Levesque, David J. Adams, Carla D. Robles-Espinoza. The molecular landscape of acral lentiginous melanoma in Mexican patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2222.
Abstract 2234: Detection of structural variation and analysis on HuKemia models by Using Bionano optical mapping
Authors: Chen Xiaobo , Tian Huan , Qian Wubin , Li Henry Q. , Guo Sheng
Published: July 2021

Background: Genomic Structural variation (SV) refer to abnormalities in chromosome structure, is a normal part of variation in the human genome. Now Genomics SVs are recognized as the largest source of interindividual genetic variation and are closely associated with oncogenesis. The ability to identify constitutive and low-allelic fraction SVs is crucial. Standard SV detection method include chromosome banding, fluorescence in situ hybridization (FISH), and array comparative genome hybridization (CGH), which are manually intensive and have trouble finding low-frequency mutations. In recent year, genome physical mapping technologies have received increasing attention and optical mapping-based methods can well-accomplish sufficient to detect large size SVs or SVs within repetitive regions. Lately, Bionano optical mapping technology rapidly expanded its applications in the detection of structural variations. As well known, Acute Leukemia show distinct patterns of genetic aberrations including chromosome translocations, mutations, and aneuploidies in genes responsible for cell cycle regulation and lymphoid cell development.

Method: We use our liver cancer model LI6671 to establish the Bionano Saphyr Gen2 genome imaging platform. DNA >100kbp is extracted, “CTTAAG” sequences were labelled across the entire genome by using Direct-Label-Enzyme (DLE) and linearized through chips for visualizations. Saphyr was loaded, linearized, and imaged labeled DNA in repeated cycles. Assembly Algorithms covert collected DNA images into constructing consensus genome maps. And this assay platform will be used to comprehensively identify SVs for studying our 21 HuKemia models.

Results: By using this fluorescence labels distribution patterns, we find different structure variants among genome. We used filtered data for genome map assembly and compared with human genome hg38 for SV detection. In LI6671, we found out 3952 SVs in LI667 including 1166 deletion, 2497 insertion, 101 duplication, 78 inversion breakpoints, 66 interchr. translocation breakpoints and intrachr. translocation. Oncogenesis related SVs, such as a ~13Kb heterozygous deletion at KCNQ1 region, EPC1::SP1 gene fusions have be founded.

Conclusion: Saphyr is a comprehensive platform, can discover a broad range of Genomic SVs and further improves our understanding of Acute Lymphocytic Leukemia (ALL) and Acute Myelocytic Leukemia (AML).

Citation Format: Xiaobo Chen, Huan Tian, Wubin Qian, Henry Q. Li, Sheng Guo. Detection of structural variation and analysis on HuKemia models by Using Bionano optical mapping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2234.
Abstract 2259: Single-cell multi-omics analysis of SNV, CNV, and protein expression
Authors: Ooi Aik , Dhingra Dalia , Sciambi Adam , Thompson Kate , Marin Jacqueline , Parikh Saurabh , Manivannan Mani , Ruff David
Published: July 2021

Recent advancements in precision medicine, while highly promising, presents a major technical challenge to researchers due to disease heterogeneity. The emergence of single-cell technologies has greatly refined the resolution in which sample diversity can be investigated, enhancing the efficiency of selecting appropriate molecular targets. Additionally, applying multi-omics analysis on single cells would further improve the understanding of cell-to-cell heterogeneity by providing unique insights on cellular and genetic composition. Using a two-step droplet microfluidic technology, the Mission Bio Tapestri Platform enables multiplex-PCR based high-throughput targeted DNA sequencing in single cells to obtain single-nucleotide variation (SNV) and copy number variation (CNV) information. By leveraging this technology, a new workflow is developed to detect protein expression in addition to DNA genotype in the same single cells. In this approach, cells are labeled with a pool of oligonucleotide-conjugated antibodies prior to loading the cells into the Tapestri Instrument. Sequencing libraries are then prepared from both antibody oligonucleotides and the amplified DNA sequences, followed by identification of single-cell DNA genotypes and protein signatures from the sequencing readout. The number of protein targets can be in the range from 6 to over 40, which is beyond the limit for a single flow cytometry run. This method has been successfully performed on cell lines, fresh and frozen PBMCs, as well as clinical samples. In an acute myeloid leukemia (AML) sample, combined single-cell SNV, CNV, and protein expression data illustrated the heterogeneity within the sample. The data clearly identified CD3+ T cells and CD19+ B cells without pathogenic SNVs and CNVs. CD34hiCD11blo and CD34loCD11bhi subpopulations were also identified within the cells carrying the same pathogenic SNVs and CNVs. We believe that this novel multi-omics technology will facilitate new discoveries in the complex relationship between genotype and phenotype, enable a better understanding of disease biology, and subsequently improve the design of diagnostics and therapies.

Citation Format: Aik Ooi, Dalia Dhingra, Adam Sciambi, Kate Thompson, Jacqueline Marin, Saurabh Parikh, Mani Manivannan, David Ruff. Single-cell multi-omics analysis of SNV, CNV, and protein expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2259.
Abstract 2261: A multimodal single-cell workflow to interrogate cellular responses to cancer therapy
Authors: Dhingra Dalia , Ooi Aik , Li Alex , Sciambi Adam , Wang Shu , Gulati Saurabh , Ruff David
Published: July 2021

Understanding the genomic landscape of cancer in single cells can be valuable for the characterization of molecular events that drive evolution of tumorigenesis and fostering progress in identifying druggable regimens for patient treatment scenarios. We report a new approach to measure multiple modalities simultaneously from up to 10,000 individual cells using microfluidics paired with next generation sequencing. Our procedure determines targeted protein levels, mRNA transcript levels and somatic gDNA sequence variations including copy number variants. This approach can resolve over 20 proteins, 100s of targeted transcripts and DNA amplicons. We employ oligo-conjugated antibody panels to probe cell surface markers and targeted RNA and DNA amplification to resolve gene expression levels and genomic variants. Cell suspensions are first stained with the antibody panel then loaded onto the Mission Bio Tapestri for generation of a droplet. This droplet biochemistry allows for concurrent cell lysis and release of gDNA/mRNA. A second droplet formation event brings together a barcoded bead and multiplex PCR amplification reagents. After amplification, the combined libraries are sequenced yielding a multimodal readout. We applied this technique to several dynamic immunology and oncology in vitro models. As fusion gene detection has long been a focus of cancer research, we demonstrate this multi-modal workflow on cell lines containing variants of BCR-ABL transcripts with high sensitivities and specificities. We further explored the relationship between genotype-to-phenotype in imatinib impact on retinoblastoma and BCR-ABL driven leukemic cells. Our pilot studies demonstrate the utility of multimodal resolution in further illuminating cellular states and biological responses in cancer cells.

Citation Format: Dalia Dhingra, Aik Ooi, Alex Li, Adam Sciambi, Shu Wang, Saurabh Gulati, David Ruff. A multimodal single-cell workflow to interrogate cellular responses to cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2261.
Abstract 2262: Comprehensive characterization of cancer drivers and therapeutic targets
Authors: Li Yize ,   Oncogenic Drivers and Pathways GroupClinical Proteomic Tumor Analysis Consortium
Published: July 2021

Cancer is a family of diseases caused primarily by genetic “driver” mutations. Building upon a large-scale Pan-cancer study of ours, we expand our investigation to the proteogenomic level (including global proteome and phosphoproteome data) drawing >1,000 cases over 10 cancer types (BR, ccRCC, CO, EC, GBM, HNSCC, LSCC, LUAD, OV, PDAC) from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). We concentrate on 6 aspects of drivers: (1) We identify genomic & epigenomic drivers (mutations, SVs, CNVs, fusions, etc.), estimate their frequencies, and investigate their exclusivities & co-occurrences. (2) We investigate the proteogenomic impact of drivers, especially recurrent ones, e.g. cis/trans effects, on miRNA, RNA, and protein expression. (3) We investigate their impact on Post Transcriptional Modifications (PTMs), comparing levels of protein and PTM, e.g. for one- and two-hit events. For example, we found upregulation of PTPN11 phosphorylation sites in EGFR-altered LUAD, GBM, and HNSCC samples, suggesting a pan-cancer effect, and observed high NRF2 phosphorylation and activation of antioxidant pathways in KEAP1 or NRF2-altered samples in lung cancer (LSCC and LUAD); (4) We compare tumors and normal adjacent tissues (NATs) to identify key protein level changes and regulations for oncogenic pathways and further examine cell type composition, proteomic variation, and association with driver mutations. For instance, we found VHL mutations often signify enrichment of endothelial cells. (5) We capture potential Significant Protein Drivers (SPRDs), independent of DNA level changes, to uncover hidden drivers in cause-ambiguous tumors. (6) We examine actionable driver alterations and expression changes across proteogenomic levels and associations with immune infiltration and immunogenic peptides to understand targeting, therapeutic implications and, influence on tumor micro-environment. We comment upon the clinical utility of comprehensive proteogenomics, especially in cases where a strictly genomic assessment is inconclusive. Proteomics can further prioritize gene targets with additional layers of evidence for differential expression among features such as cell-type composition, anatomical location, disease stage, and immune infiltration. Our approach has the potential for identifying cancer-type-specific and non-specific novel druggable targets, which would provide new biological and clinical insights into cancer.

Citation Format: Yize Li, Oncogenic Drivers and Pathways GroupClinical Proteomic Tumor Analysis Consortium. Comprehensive characterization of cancer drivers and therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2262.
Abstract 2270: Membrane organization of KRas4B with regulatory domain of Raf-1
Authors: Jang Hyunbum , Zhang Mingzhen , Nussinov Ruth
Published: July 2021

A major pathway in RAS-driven cell proliferation is the MAPK (Raf/MEK/ERK) pathway. In cancer, membrane-anchored, proximally located, oncogenic Ras isoforms promote Raf dimerization and fully activate MAPK signaling. Among Ras isoforms, KRas4B is frequently mutated in cancer. In the MAPK pathway, active KRas4B dimer or nanocluster can lead to dimerization of Raf kinase domain enhancing the affinity of the KRas4B-Raf interaction. Recently, we modeled the binary KRas4B-Raf-1 complex and its quaternary assembly with the regulatory domain of Raf-1 containing the Ras binding domain (RBD) and cysteine-rich domain (CRD), and suggested that Raf-1 CRD bolsters the high-affinity interaction of Raf-1 with KRas4B by reducing the KRas4B-RBD fluctuations. For the quaternary assembly, recent in silico studies suggested that KRas4B dimer is aligned through the α3 and α4 helical interface, not the α4 and α5 helical interface due to hindrance to the membrane contact of Raf-1 CRD. The Raf-1 regulatory domain contains the N-terminal region (residues 1-55), RBD, and CRD. However, previous modeling efforts were conducted for the KRas4B-Raf-1 complex in the absence of the N-terminal region. Here, computational studies were performed for the KRas4B-Raf-1 complex with full sequence of Raf-l regulatory domain interacting with monomeric and dimeric KRas4B-GTP at the membrane. Increasing experimental evidences suggested that the N-terminal region of Raf plays a significant role in the Ras interaction and the membrane anchorage of Raf. The membrane interaction of isolated N-terminal segment was investigated as the first step toward the complex model construction. Since oncogenic KRas4B mutants form dimers/nanoclusters at the membrane microdomains that provide the signaling platform for Raf, KRas4B in different mutation states interacting with the membrane microdomains with different lipid composition was also studied. The presence of the N-terminal segment of Raf-1 regulatory domain at the membrane can enhance the stability of KRas4B-Raf-1 and thereby relieving Raf's autoinhibition toward a kinase domain-accessible state, providing a complete mechanistic picture of the KRas4B-Raf-1 interaction. Our recent work uncovers the mechanism of Raf-1 activation by KRas4B at atomic resolution and highlight how this may help in elucidating vital mechanistic questions in Ras biology. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E.

Citation Format: Hyunbum Jang, Mingzhen Zhang, Ruth Nussinov. Membrane organization of KRas4B with regulatory domain of Raf-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2270.
Abstract 2271: Potential small-molecule inhibitors of the Tiam1 oncogene product
Authors: Fritz Claire , Stamper Michaela , Valentino Stephen , Ulatowski Lynn , Gupta Meghana , Thakur Varsha , Chung Stacey , DeHann Nick , Laggner Christian , Manor Danny
Published: July 2021

T-cell lymphoma invasion and metastasis-inducing factor (Tiam)1 is an established proto-oncogene that drives cancer cell migration and metastasis. The correlation between Tiam1 integrity and patient survival, epithelial-mesenchymal transition, and angiogenesis, render it as an important prognostic factor in multiple human malignancies. Tiam1 is a guanine nucleotide exchange factor (GEF) that facilitates the activation of the small GTPase Rac1, thereby controlling cytoskeletal organization, cell polarity, motility, and invasion. In light of the clinical relevance of Tiam1 signaling, we investigated the utility of small molecule inhibitors that target Tiam1 as a therapeutic intervention strategy. We used an in silico computational approach to screen 10 million compounds for candidate drugs that bind to a select patch on Tiam1's surface. We then identified 13 compounds that inhibit the migration of Tiam1-expressing A549 cells without affecting overall viability. Our studies open the door for a new targeted therapy approach in Tiam1-relevant cancers.

Citation Format: Claire Fritz, Michaela Stamper, Stephen Valentino, Lynn Ulatowski, Meghana Gupta, Varsha Thakur, Stacey Chung, Nick DeHann, Christian Laggner, Danny Manor. Potential small-molecule inhibitors of the Tiam1 oncogene product [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2271.
Abstract 2273: MAPK signaling pathway disruption explains the anticancer mechanism for polyisoprenylated cysteinyl amide inhibitors (PCAIs)
Authors: Tawfeeq Nada , Jin Yonghao , Lamango Nazarius
Published: July 2021

The Mitogen-activated protein kinase (MAPK) pathway is one of the key signaling pathways that regulates normal cell proliferation, differentiation, and survival. Abnormalities of the pathway play vital roles in cancer initiation and progression. Ras small GTPases that are key upstream mediators of the MAPK pathway are mutated in 20%-30% of human tumors. KRAS is the most frequently mutated isoform of the RAS proteins, accounting for up to 86% of RAS mutant cancer cases. Mutant KRAS is prevalent in lung, colon, and pancreatic cancers, which considered to be the most aggressive and account for the most cancer deaths in the US. To help mitigate this problem and fill the therapeutic need that has lingered for decades, polyisoprenylated cysteinyl amide inhibitors (PCAIs) were designed and synthesized to target the hyperactivity of aberrant monomeric G-Proteins. Recently, the PCAIs underwent a synthetic optimization to mitigate the excessive hydrophobicity of the earlier analogs. While the newer agents are more polar and have maintained the effectiveness of the earlier PCAIs against cancer cell lines driven by mutant KRAS proteins, the specific anticancer mechanisms are still unclear. Thus, we explored the effects of the newer PCAIs on the MAPK pathway using lung cancer A549 cells which harbor the KRAS mutation. When the cells were treated with NSL-YHJ-2-27 for 48 h, the KRAS protein levels dropped by 40% at 5μM compared to the controls. However, no significant difference was observed in the levels of total or phosphorylated B- and C-Raf proteins. Moreover, it was observed that the PCAIs immensely stimulated the MAPK pathway in a concentration-dependent manner following treatment with 1, 2, and 5μM of NSL-YHJ-2-27. The phosphorylation of MEK1/2, ERK1/2, and p90RSK significantly increased by 84%, 59%, and 160%, respectively, relative to controls following treatment for 48h at 5 µM of NSL-YHJ-2-27. Interestingly, a non-farnesylated analog, NSL-YHJ-2-62, did not elicit similar effects on the MAPK pathway. Although ERK stimulation has mostly been associated with cell survival and proliferation, various studies show that ERK activation can also mediate apoptosis depending on the intensity and duration of the stimulation. These data reveal that the Ras-MAPK signaling axis is involved in the anticancer effects of the PCAIs. The PCAIs may thus be considered as possible agents for the unmet therapeutic needs of patients with aberrantly hyperactive G-proteins.

Citation Format: Nada Tawfeeq, Yonghao Jin, Nazarius Lamango. MAPK signaling pathway disruption explains the anticancer mechanism for polyisoprenylated cysteinyl amide inhibitors (PCAIs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2273.
Abstract 2278: Analysis of primary and secondary EGFR-TKI resistance mutations evolution in 835 EGFR-mutant Chinese lung cancers
Authors: Dong Xiaorong , Zeng Dongqiang , Liu Xinyi , Huang Mengli
Published: July 2021

Background: EGFR gene is the most frequent mutated gene in lung cancer. EGFR-Ex19del and EGFR-L858R mutations account for 90% mutations in EGFR mutations and these two kinds of genomic alterations are both sensitive to EGFR-TKIs. However, resistance to EGFR-TKIs would always happen after a period of treatment. EGFR-T790M and EGFR-ex20ins alterations had been identified as alterations result in resistance. The evolution of EGFR-T790M and EGFR-ex20ins in lung cancers with EGFR-mutant baseline features have not been systemically investigated.

Method: We analyzed 835 EGFR-mutant Chinese lung cancers. Each patient had received treatment containing EGFR-TKIs and at least two longitudinal follow-up evaluations based on NGS testing. The median follow-up time was 278 days. We systemically analyzed the evolution of EGFR primary and secondary mutations as previously defined.

Result: EGFR-Ex19del, EGFR-L858R, EGFR-G719X, EGFR-L861Q and EGFR-Ex20ins were basically mutually exclusive. Out of 835 cases, 401 were with EGFR-Ex19del, 348 were with EGFR-L858R, 27 were with EGFR-G719X, 22 were with EGFR-L861Q and 9 were with EGFR-Ex20ins at baseline time. EGFR-T790M were co-mutated in 16.6% EGFR-19del population and in 11.3% EGFR-L858R population. In EGFR-G719X, EGFR-L861Q and EGFR-Ex20ins population, the proportion of patients with EGFR-T790M co-mutations were all about 10%. EGFR-S768I was the most frequent co-occurring EGFR mutation in EGFR-G719X subgroup while was not occur in EGFR-L861Q subgroup. In follow-up time, 26.8% EGFR-Ex19del patients loss the T790M mutation and 51.4% EGFR-L858R patients loss the T790M mutation. Interestingly, the EGFR-T790M and the driver EGFR mutation in EGFR-G719X, EGFR-L861Q and EGFR-Ex20ins subgroups keep all the time in EGFR-TKI treatment.

Conclusion: We investigated the resistance EGFR alterations evolution longitudinally in a large Chinese lung cancer cohort. We found that EGFR-T790M was a common co-mutation in lung cancers with different baseline EGFR driver mutations. Although the co-mutant frequency of T790M was similar (about 10%) in each subgroup, it was most likely to disappear in EGFR-L858R baseline subgroup after EGFR-TKI treatment.

Citation Format: Xiaorong Dong, Dongqiang Zeng, Xinyi Liu, Mengli Huang. Analysis of primary and secondary EGFR-TKI resistance mutations evolution in 835 EGFR-mutant Chinese lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2278.
Abstract 2279: Improving cell type and state classification in CITE-seq using probabilistic models and hierarchical cell ID matrix
Authors: Fakharzadeh Lila , Tessier Julien , McGrath Shannon , Wang Emma , Biancotto Angelique , Protopopov Alexei , Pollard Jack , Lee Joon Sang
Published: July 2021

Accurate classification of cells in a population is important for most immune phenotyping applications. Surface protein marker expression measured with multiplexed platforms like Flow Cytometry and sequential gating strategies are typically employed to classify cells. However, new single-cell RNA sequencing (scRNA-seq) methods that measure RNA expression alone or Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) methods that concurrently measure both surface protein and RNA expression in single cells are providing new data and opportunities to develop methods both to classify cells and to assess their states of activation.

Traditional manual gating methods using biaxial plots, where one designates a threshold separating a bimodal distribution through visual approximation, are subjective and can vary widely between individuals. Instead of manual, sequential gating for cell classification, we have implemented a probabilistic Gaussian Mixture Model (GMM) and K-means clustering to separate cell populations based on a library of marker thresholds for a variety of cell types present in human peripheral blood mononuclear cells (PBMCs) using a CITE-seq ADT expression matrix that has been preprocessed with centered-log-ratio (CLR) normalization.

Furthermore, we coupled this GMM method with a hierarchical approach for cell typing. We have developed a Cell ID Matrix that outlines over 40 different immune cell types using consensus markers applicable to immune cell types across various studies. In this typing method, major cell types are identified following positive and negative marker expression as described in the Cell ID Matrix. Hierarchical gating of cells can then be used to identify subpopulations in a principled manner. The information in the Cell ID matrix is input as a binary matrix for each hierarchy level and is matched to the binarized expression values for classifying the cells. This approach can also be extended to CITE-seq datasets from disassociated tumor cells (DTCs).

We conclude that automated cell gating with CITE-seq data may enhance the classification of numerous cell types in addition to significantly decreasing the amount of human intervention and time allocated for gating cells. Identifying cell states is a work in progress and will supplement this technique in the future.

Citation Format: Lila Fakharzadeh, Julien Tessier, Shannon McGrath, Emma Wang, Angelique Biancotto, Alexei Protopopov, Jack Pollard, Joon Sang Lee. Improving cell type and state classification in CITE-seq using probabilistic models and hierarchical cell ID matrix [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2279.
Abstract 2285: The capability of NTRK fusion detection by next-generation sequencing/FISH/IHC in 25976 solid tumor patients
Authors: Peng Yuan , Chen Hui , Zhao Xiaochen , Han Zhijun , Bai Yuezong
Published: July 2021

Purpose: Despite the low prevalence of oncogenic neurotrophin receptor kinase (NTRK) fusion among most solid tumors, the encouraging results of tropomyosin receptor kinase (Trk) inhibitors such as larotrectinib and entrectinib have prompted the screening of eligible patients. Here, we aimed to report the prevalence of NTRK fusion in the Chinese solid tumor patients and to compare NTRK fusion evaluation with targeted next-generation sequencing (NGS), fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC).

Methods: A total of 25976 tumor samples from Chinese solid tumor patients were collected between January 2017 and June 2020. All samples were assessed with targeted NGS in a CLIA-certified laboratory. NTRK genes fusions were confirmed by FISH (ZytoLight SPEC NTRK1/2/3 Dual Color Break Apart Probe) and/or pan-TRK IHC (clone EPR17341).

Results: In total, 47/25976 (0.2%) patients harbored one or more NTRK fusions. NTRK fusions were found in 15 cancer types including thyroid (1/92, 1.1%), sarcoma (3/547, 0.5%), neuroendocrine (1/183, 0.5%), GIST (1/265, 0.4%), endometrium (1/274, 0.4%), esophagus (1/293, 0.3%), colorectum (14/4548, 0.3%), head neck (1/375, 0.3%), lung (16/8950, 0.2%), ovary (1/625, 0.2%), breast (1/706, 0.1%), biliary tract (2/1530, 0.1%), liver (2/2282, 0.1%), stomach (1/1505, 0.1%), and others (1/3801, 0.03%). Among these 47 patients, 11 were further confirmed by both FISH and IHC and 3 were confirmed only by IHC. 36.4% (4/11) of the patients had completely concordant NTRK status (NGS+/FISH+/IHC+). 45.5% (5/11) of the patients had consistency between NGS and FISH (NGS+/FISH+). 71.4% (10/14) of the patients had consistency between NGS and IHC (NGS+/IHC+). Moreover, there were two novel fusions includingTEX10-NTRK2 (NGS+/FISH+/IHC+) in head and neck cancer and PTPN22-NTRK1 (NGS+/IHC+) in ovarian cancer, which is the first case of ovarian cancer with NTRK fusion.

Conclusions: NGS was effectively help to screen patients with novel or reported NTRK fusion, who may benefit from NTRK inhibitor therapy. And NGS showed good consistency with IHC. Further prospective study is needed to compare the impact of the NTRK status identified by NGS and IHC on NTRK inhibitors efficacy.

Citation Format: Yuan Peng, Hui Chen, Xiaochen Zhao, Zhijun Han, Yuezong Bai. The capability of NTRK fusion detection by next-generation sequencing/FISH/IHC in 25976 solid tumor patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2285.
Abstract 2291: A novel TGF-βRI inhibitor ASKC852, possessing antitumor and PD-L1 synergistic effect activities
Authors: Song Tingting , Sun Min , Chen Hongyu , Fu Xiangyu , Hu Lihong , Han Shuhua , Tong Haijun , Xia Yuanfeng , Zuo Jian , Qin Shaohua , Chen Ying , Ding Charles , Chan Chichung , Chen Shuhui
Published: July 2021

Background: Transforming growth factor-β (TGF-β) plays a significant role in multiple physiological signaling pathways, thus occupies a pivotal position in balancing immunity and antigen tolerance. Activation of TGFβ type I receptor (TGF-βRI) recruits and phosphorylates SMAD transcription factors and promotes EMT gene expression, leading to tumor progression and invasion. Inhibition of this pathway therefore has potential to treat cancer. The preclinical evaluations of a novel TGF-βRI inhibitor ASKC852 is described.

Results: ASKC852 has been shown to be a potent and highly selective inhibitor of TGF-βRI. ASKC852 inhibited SMAD phosphorylation in HEK293 cells (IC50 < 0.1 μM), a down-stream PD marker of TGF-β signaling pathway. This compound showed metabolic stability, oral bioavailability in mice and rats, with anti-tumor activity when given alone (TGI: 81.2%) in CT26 colon cancer model. In EMT6 breast carcinoma model, ASKC852 demonstrated significant synergistic effect when combined with a mouse PD-L1 antibody. Greater than 50% p-SMAD inhibition was achieved at low doses in in vivo PD studies. In addition, ASKC852 effectively inhibited tumor metastasis in 4T1-luc2 model. In a 28-days pre-clinical toxicology study, ASKC852 showed no effect on rat cardiovascular system @ 30 mg/kg.

Conclusion: Our data demonstrates ASK852 is a potent and highly selective TGF-βRI inhibitor. It effectively blocked the TGF-β-SMAD signal pathway, with anti-tumor effect in syngeneic mouse models. Pharmacokinetic profile and safety properties support ASKC852 as a potential anticancer agent for the clinical treatment of solid tumors.

Citation Format: Tingting Song, Min Sun, Hongyu Chen, Xiangyu Fu, Lihong Hu, Shuhua Han, Haijun Tong, Yuanfeng Xia, Jian Zuo, Shaohua Qin, Ying Chen, Charles Ding, Chichung Chan, Shuhui Chen. A novel TGF-βRI inhibitor ASKC852, possessing antitumor and PD-L1 synergistic effect activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2291.
Abstract 2293: Role of phosphatidylinositol 4-kinase IIIα in chemokine signaling
Authors: Govindarajan Barani , Chinni Sreenivasa , Sbrissa Diego
Published: July 2021

The CXCR4-CXCL12 chemokine signaling axis plays a key role in migration and bone metastasis in prostate cancer (PC). Androgens regulate CXCR4 expression and its receptor activation in lipid-raft micro domains of PC cells, resulting in higher protease expression and invasion. In order to identify some novel CXCR4 co-regulators associated within the lipid-raft, a SILAC (Stable isotope labeling using amino acid in cell culture)-based proteomic analysis was performed with PC3 stable cell-lines over-expressing or knocking-down CXCR4. Phosphatidylinositol 4-kinase III alpha (PI4KIIIα or PI4KA) and SAC1 lipid phosphatase were identified as candidate proteins enriched in CXCR4 expressing cells. PI4KA is an evolutionarily conserved mammalian kinase that converts PI to PI4P; and SAC1 dephosphorylates PI4P to PI. PI4K is required for the maintenance and functioning of the plasma-membrane and vesicular trafficking in the Golgi apparatus. PI4KA is also needed to maintain a steady pool of PI(4,5)P2 in the plasma-membrane, to be utilized in various signaling pathways. We show that CXCR4 interacts with PI4KA through its adaptor proteins EFR3B and TTC7B, recruiting it to the plasma-membrane for PI4P generation. Similarly PI4KA was closely linked to CXCR4 induced PC cell invasion, and knockdown of PI4KA in CXCR4 over-expressing PC3 cell lines reduced cell invasion. Also localized productions of PI4P was evident in invasive projections of CXCR4 over-expressing cell lines through immunofluorescence microscopy. PC tumor microarray data show increased PI4K expression in metastatic PC tissue vs localized or normal adjacent tissue. These data suggest a novel interaction between PI4KA and CXCR4, promoting tumor cell invasion and metastasis. Pharmacological targeting of PI4KA, its adaptor proteins or its signaling-related proteins might prove therapeutically beneficial and enhance survival in PC patients.

Citation Format: Barani Govindarajan, Sreenivasa Chinni, Diego Sbrissa. Role of phosphatidylinositol 4-kinase IIIα in chemokine signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2293.
Abstract 2299: An unbiased in vivo screen for activating EGFR mutations
Authors: Chakroborty Deepankar , Kurppa Kari J. , Elenius Klaus
Published: July 2021

Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites across different samples. Most of these somatic mutations are expected to be inconsequential passenger mutations that reflect the general instability of the tumors. The discovery of most of the currently known driver mutations has been facilitated by their accumulation in mutation hot-spots within their respective genes. However, a vast majority of mutations in cancer tissues are rare and their functional significance remains unknown. Several lines of in vitro and clinical evidence also indicate that there is a significant number of, as yet unidentified, activating driver mutations which could serve as predictive markers in oncology. We are developing our in vitro Screen for Activating Mutations (“iSCREAM”)[1] further and advancing it to better recapitulate natural intratumor heterogeneity by taking the system in vivo.

To establish the in vivo model for screening activating mutations we utilize the SALE-Y cells which are immortalized human small airway epithelial cells harboring an activating YAP1 variant[2]. In immunocompromised mice, SALE-Y cells fail to form tumors up to 120 days, but they become tumorigenic after transduction of mutations activating the EGFR-MAPK signaling pathway[2]. We will use the SALE-Y cell background for transduction of an expression library encoding thousands of different randomly mutated variants of the kinase to be tested. EGFR will be used to set up the model because: i) EGFR mutants are known to of transforming the SALE-Y cells[2] and ii) we have previously used EGFR as a model to set up the in vitro screen[1]. The SALE-Y expressing the random collection of EGFR variants will be inoculated subcutaneously into immunocompromised mice, and the activating mutations will be given an opportunity to outcompete passenger mutations during subsequent expansion and suvival of the xenograft. Genomic DNA will be harvested from the tumor tissue and subjected to targeted next-generation sequencing allowing unbiased identification of enriched activating mutations promoting tumor growth.

References:1. Chakroborty D, Kurppa KJ, Paatero I, Ojala VK, Koivu M, Tamirat MZ, et al. An unbiased in vitro screen for activating epidermal growth factor receptor mutations. J Biol Chem. 2019.2. Berger AH, Brooks AN, Wu X, Shrestha Y, Chouinard C, Piccioni F, et al. High-throughput Phenotyping of Lung Cancer Somatic Mutations. Cancer Cell. 2015.

Citation Format: Deepankar Chakroborty, Kari J. Kurppa, Klaus Elenius. An unbiased in vivo screen for activating EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2299.
Abstract 2306: The ubiquitin-binding non-receptor tyrosine kinase TNK1 is regulated by a MARK-mediated interaction with 14-3-3
Authors: Egbert Christina Marie , Chan Tsz Yin , Larsen Logan , Balasooriya Eranga Roshan , Kohler Kristina , Tsang Jeremy , Frey Madison , Maxson Julia , Huang David , Christensen Kenneth A. , Moody James , Tyner Jeffrey W. , Andersen Joshua L.
Published: July 2021

Tyrosine non-receptor kinase 1 (TNK1) is a poorly characterized member of the ACK family of non-receptor tyrosine kinases. We identified TNK1 as a mediator of cell survival in subsets of primary hematological malignancies. Previous studies have indicated a role for TNK1 in driving oncogenic potential; however, the function, regulation, and substrates of TNK1 still remain unknown. We identified an interaction between TNK1 and 14-3-3 that is promoted by a MARK-mediated phosphorylation at S502 on TNK1. 14-3-3 binding sequesters TNK1 to the cytosol. Upon the release of 14-3-3 binding, TNK1 moves to perinuclear puncta and is in a highly active state. This highly active form of TNK1 is able to drive growth factor independent proliferation of lymphoid cells in culture and in mice more potently than TNK1 WT. A unique feature of TNK1 is its C-terminal ubiquitin association domain (UBA) domain. In vitro ubiquitin pulldowns indicate a high affinity between the TNK1 UBA and polyubiquitin of varying linkages, with dissociation constants in the low nanomolar range. Mutation of residues within ubiquitin-binding interfaces of the UBA and ubiquitin leads to a reduction of ubiquitin association and a marked shift of TNK1 from perinuclear puncta to a diffuse cytosolic localization. Importantly, we show that loss of ubiquitin binding reduces TNK1 activity and oncogenic signaling. Taken together, our data uncovers a 14-3-3 and UBA-mediated mechanism of TNK1 regulation.

Citation Format: Christina Marie Egbert, Tsz Yin Chan, Logan Larsen, Eranga Roshan Balasooriya, Kristina Kohler, Jeremy Tsang, Madison Frey, Julia Maxson, David Huang, Kenneth A. Christensen, James Moody, Jeffrey W. Tyner, Joshua L. Andersen. The ubiquitin-binding non-receptor tyrosine kinase TNK1 is regulated by a MARK-mediated interaction with 14-3-3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2306.
Abstract 2312: Identification of tumor associated genes using transcriptome analysis
Authors: Liang Guoqiang , Sun Jinqiu , Liu Yiwei , Liu Dongxue , Liang Shengxian , Guo Rui , Zhong Li
Published: July 2021

Background: Lung cancer is the leading cause of cancer death worldwide. Identification of tumor associated genes is an effective approach for early cancer detection, prognosis prediction and target therapy development. With the rapid growth in high-throughput sequencing techniques, big data regarding gene expression of lung cancer has been generated and deposited in public data banks. In this study, we used bioinformatic and statistic techniques to analyze big data from public data banks in identifying novel genes in association with prognosis of non-small cell lung cancer.

Methods: Transcriptome data and clinical information of 1020 non-small cell lung cancer (NSCLC) cases were downloaded from The Cancer Genome Atlas (TCGA). After removing the incomplete data, we obtained 1008 NSCLC cases. All the gene expression data was normalized using differential analysis software DESeq2 in R language. The normalized data was further analyzed using weighted gene co-expression network to screen out the differentially expressed tumor associated genes. After applying Protein-Protein Interaction Networks (PPI) analysis in combination with clinical information, we have identified some hub genes. Using univariate/multivariate Cox regression analysis we identified several genes associated with NSCLC prognosis.

Results: After DESeq2 analysis, we obtained 2873 differentially expressed genes when the threshold padj < 0.01 and Fold change > 4. Among them, 1955 were upregulated and downregulated genes. When the weighted gene co-expression network analysis was applied, we achieved 50 blocks after data clustering (7 cases were outliers and removed). After linked with the clinical information, we identified that the yellow block contained the most NSCLC related genes. This block contains a total of 2803 genes, and 417 of them were differentially expressed tumor genes. Further analysis using univariate/multivariate Cox regression, we identified SPP1, PKMYT1, PRB11, LINC0116 and PTPRM 5 genes that were associated with poor prognosis of NSCLC. After combined analysis, we found that the 5-year survival rate is 31.3% for early stages of NSCLC and 4.1% with late stages of NSCLC.

Conclusion: transcriptome analysis is a good approach in identifying disease associated genes, which can lead to a quick and reliable approach in developing clinical applications.

Citation Format: Guoqiang Liang, Jinqiu Sun, Yiwei Liu, Dongxue Liu, Shengxian Liang, Rui Guo, Li Zhong. Identification of tumor associated genes using transcriptome analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2312.
Abstract 2318: EP300 selectively controls the enhancer landscape of high risk neuroblastoma
Authors: Durbin Adam D. , Wang Tingjian , Wimalasena Virangika K. , Zimmerman Mark W. , Li Deyao , Dharia Neekesh V. , Mariani Luca , Park Paul M. , Sigua Logan H. , Morita Ken , Conway Amy S. , Robichaud Amanda L. , Perez-Atayde Antonio , Bikowitz Melissa , Quinn Taylor , Wiest Olaf , Schonbrunn Ernst , Bulyk Martha , Abraham Brian J. , Stegmaier Kimberly , Look A. Thomas , Qi Jun
Published: July 2021

Gene expression is regulated by promoters and enhancers marked by histone H3-lysine-27 acetylation (H3K27ac) and established by the paralogous histone acetyltransferases (HATs), EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma depend on EP300, while CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2β, a transcription factor member of the lineage-defining core-regulatory transcriptional circuitry (CRC). To disrupt EP300, we developed a proteolysis-targeted-chimaera (PROTAC) compound “JQAD1” that selectively targets EP300 for degradation. This results in loss of H3K27ac at CRC enhancers and neuroblastoma apoptosis, with limited toxicity to untransformed cells where CBP compensates. EP300 physically interacts with the MYCN oncoprotein, promoting its chromatin localization in neuroblastoma. EP300 degradation therefore capitalizes on EP300 dependency in neuroblastoma by controlling MYCN localization and preventing MYCN from sustaining the malignant cell state.

Citation Format: Adam D. Durbin, Tingjian Wang, Virangika K. Wimalasena, Mark W. Zimmerman, Deyao Li, Neekesh V. Dharia, Luca Mariani, Paul M. Park, Logan H. Sigua, Ken Morita, Amy S. Conway, Amanda L. Robichaud, Antonio Perez-Atayde, Melissa Bikowitz, Taylor Quinn, Olaf Wiest, Ernst Schonbrunn, Martha Bulyk, Brian J. Abraham, Kimberly Stegmaier, A. Thomas Look, Jun Qi. EP300 selectively controls the enhancer landscape of high risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2318.