bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 376: Targeting the convergence on HIF-1α of CDK4/6 and MAPK pathway: Implications for enhanced anticancer strategies
Authors: Zhao Shuai , Zhou Lanlan , Zhang Shengliang , El-Deiry Wafik S. 
Published: March 2024

Abstract
Hypoxia-inducible factor 1-alpha (HIF-1α) plays a pivotal role in orchestrating cellular responses to hypoxia, influencing cancer cell survival and progression. Our previous work identified a non-canonical mechanism wherein Smurf2 mediates HIF-1α degradation under CDK4/6 inhibition. Through proteomic analysis, we discovered that serine 451 phosphorylation occurs on HIF-1α but not in palbociclib-treated samples. Point mutations at this site, substituting serine with alanine, resulted in decreased HIF-1α levels and enhanced interaction with Smurf2. Intriguingly, under palbociclib treatment, we observed phosphorylation at the serine 643 site. This site has been previously associated with MAPK-dependent regulation of HIF-1α localization and activity (Ilias Mylonis, et al., 2006), particularly relevant given the reported MAPK reliance in acquired CDK4/6 inhibitor-resistant scenarios (Renée de Leeuw, et al., 2018). To explore therapeutic implications, we investigated the impact of combined CDK4/6 (palbociclib) and MEK1/2 inhibition (trametinib, selumetinib, PD98059, U0126). Dual inhibition robustly reduced HIF-1α expression in colorectal cancer cells (HCT116, SW480) and suppressed HIF-1α activity in luciferase reporter assays. This effect extended to synergistic inhibition of cell viability under both normoxia and hypoxia in HCT116 and SW480 cells. Such effect is also applicable to other cancer types and cell lines (e.g. U251). In summary, our findings unveil a phosphorylation site on HIF-1α associated with CDK4/6 activity, influencing its protein stabilization. This discovery supports the rationale for combining CDK4/6 and MEK1/2 inhibition as a promising strategy in the treatment of solid tumors.
Citation Format: Shuai Zhao, Lanlan Zhou, Shengliang Zhang, Wafik S. El-Deiry. Targeting the convergence on HIF-1α of CDK4/6 and MAPK pathway: Implications for enhanced anticancer strategies [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 376.
Abstract 3769: Spatial biology in situ hybridization and multiplexed immunohistochemistry detection compatibility using a modified protocol
Authors: Clarke Scott T. , Voeun Mae , Montoya Leticia , Vonnegut Chris , Mandavilli Bhaskar 
Published: March 2024

Abstract
In the field of spatial-omics imaging, combining mRNA in situ hybridization (ISH) detection with multiplexed protein immunohistochemistry detection (IHC) is desirable. We present a simplified workflow in FFPE tissue sections where ISH is detected using branched ssDNA signal amplification visible by brightfield imaging combined with multiplexed IHC using directly labeled antibodies detected by fluorescence. In multiple studies mRNA expression of PCNA (Proliferating Cell Nuclear Antigen) shows moderate to high nuclear positivity observed in most cancer cells, with gliomas and lymphomas being the few negative exceptions. In contrast, expression of PCNA in adult non-cancerous tissue has low to no expression, with tonsil and bone marrow being the exceptions. We first show the relationship between proliferation as measured by EdU detection, PCNA antibody and PCNA mRNA expression in the embryonic mouse model where PCNA expression is prevalent in developing tissue. Then we demonstrate a simplified working protocol for combining ISH and IHC in FFPE human cancer tissue sections for the co-detection of PCNA mRNA together with IHC using a panel of six protein markers relevant to the tumor microenvironment. For IHC, we directly labeled validated unconjugated antibodies with fluorophores using amine-reactive chemistry and select the degree of labeling (DOL) and titer for optimal signal/background most useful for multiplexing on FFPE tissue. We found important protocol steps to consider for compatibility between IHC and ISH detection method. Loss of IHC protein epitope due to ISH protocol use of proteases and loss of ISH signal due to presence of RNase commonly present in IHC antibody relevant reagents can prevent combining IHC with ISH. Stabilizing IHC through refixation prior to ISH workflow is also required. Modifying the protocols to avoid these incompatible treatments and using directly labeled antibodies provides useful strategies for multiplexing IHC with ISH.
Citation Format: Scott T. Clarke, Mae Voeun, Leticia Montoya, Chris Vonnegut, Bhaskar Mandavilli. Spatial biology in situ hybridization and multiplexed immunohistochemistry detection compatibility using a modified protocol [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 3769.
Abstract 3823: Clinical outcomes of uveal melanoma patients in the emergency department
Authors: Beneke Alice A. , Taneja Kamil , Ladehoff Lauren , Root Kevin , Toloza Eric 
Published: March 2024

Abstract
Purpose: Uveal melanoma is a rare ocular malignancy that has not been well characterized at a large scale. Specifically, uveal melanoma diagnosis and outcomes in the emergency department (ED) has not been characterized.
Methods: The Nationwide Emergency Department Sample was queried from the last quarter of 2015 to 2019 to identify patients with a primary diagnosis of uveal melanoma. Multivariable logistic regression, with patient- and hospital-level characteristics as covariates, was used to identify factors associated with increased risk of inpatient admission.
Results: From the last quarter of 2015 to 2019, there were 1,274 ED encounters with a primary diagnosis of uveal melanoma. Most patients were less than 11 years old (43.4%), male (51.1%), or a Medicaid beneficiary (37.8%), and most belonged to a family in the lowest income quartile (32.0%). The median age of these patients was 35 years (IQR: 2-64). Most patients presented to hospitals in the South (53.9%), with a Trauma Level I designation (51.4%), and most to a metropolitan teaching hospital (89.1%). The most common ED outcomes were same-day discharge (61.6%) and admission to the inpatient setting (31.2%). Patients presenting to Trauma Level I hospitals were most likely to be admitted (odds ratio [OR]=2.21; p=0.031). The factors that were most significantly associated with decreased odds of inpatient admission were: patients who were not charged for services (often due to charity) (OR=0.13; p=0.040), Medicaid beneficiaries (OR=0.27; p=0.031), and private insurance beneficiaries (OR=0.34; p=0.047). Among the admitted patients, patients can be discharged to home with self-care (76.2%) or with home-health care (14.9%) or can be transferred to an intermediate-care facility (8.1%). The mean hospital length of stay was 5.7±0.6 days. In-hospital mortality rate for admitted patients was very low (0.8%).
Conclusion: Uveal melanoma patient outcomes are closely related to insurance status, indicating socioeconomic factors as a modulator of patient outcomes.
Citation Format: Alice A. Beneke, Kamil Taneja, Lauren Ladehoff, Kevin Root, Eric Toloza. Clinical outcomes of uveal melanoma patients in the emergency department [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 3823.
Abstract 3862: Identification, profiling and clonotypic tracking of tumor-reactive T cells derived from bronchoalveolar lavage in lung cancer patients
Authors: Ryu Heeju , Sun Wei , Wurscher Michelle A. , Nair Viswam S. , Newell Evan W. 
Published: March 2024

Abstract
Immunotherapeutic strategies for disinhibiting T cell responses have revolutionized lung cancer treatment. However, the identification of tumor-reactive T cells and the acquisition of adequate tissue for molecular and immune profiling remains a challenge that hinders our ability to adequately characterize the mechanisms of immunotherapy response versus resistance. To address this gap, we are investigating the use of bronchoalveolar lavage (BAL) as an alternative to blood or tumor biopsy samples to identify tumor reactive T cells.
We collected cells from a prospective bronchoscopic biorepository of patients undergoing lung cancer evaluation that were subjected to bilateral BAL, phlebotomy, and tissue sampling. We then employed flow cytometry and single-cell multi-omic analyses to identify, characterize and clonotypically track tumor-reactive T cells obtained from BAL, blood and tumor samples from lung cancer patients. Analysis of BAL samples revealed a significant enrichment of terminally exhausted CD8+CD39+CD103+ T cells in BAL from tumor-bearing lungs compared to non-tumor bearing lung BAL and blood PBMCs in 14 patients. We then used single-cell sequencing-based multi-omics to deeply profile bilateral BAL cells, blood PBMCs and tumor tissues from 5 of these patients.
By tracking T cell receptor (TCR) sequences across patient-matched samples, we found BAL derived T cell clonotypes that expressed markers of terminal exhaustion, such as CD39 and CXCL13 in the tumor, which strongly implicates them as being tumor reactive and relatively abundant in BAL. Further validation through functional assays is being used to confirm the tumor reactivity of these clones.
These data demonstrate the feasibility of capturing tumor-specific T cells during bronchoscopy, providing promising opportunities for patient outcome characterization and exploration of novel therapies. This relatively non-invasive and practical test during routine bronchoscopy offers a cost-effective and safe alternative to identifying tumor-reactive T cells from surgical specimens or biopsies.
Citation Format: Heeju Ryu, Wei Sun, Michelle A. Wurscher, Viswam S. Nair, Evan W. Newell. Identification, profiling and clonotypic tracking of tumor-reactive T cells derived from bronchoalveolar lavage in lung cancer patients [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 3862.
Abstract 3875: Glioblastoma mutational profiles drive cancer cell signaling and immune evasion
Authors: Pourmaleki Maryam , Greenstein Brian D. , Jones Caitlin J. , Nandakumar Subhiksha , Navarrete Daniel A. , Mehta Smrutiben , Campos Carl , Hollmann Travis J. , Socci Nicholas D. , Bale Tejus , Shah Sohrab P. , Mellinghoff Ingo K. 
Published: March 2024

Abstract
Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with a median survival below two years. Characterization of GBM through gene expression profiling and DNA sequencing has identified disease subgroups and commonly altered genes and pathways, including alterations in the genes encoding neurofibromin-1 (NF1) and the epidermal growth factor receptor (EGFR). NF1 and EGFR are involved in receptor tyrosine kinase (RTK), RAS/MEK/ERK, and PI3K signaling, and together, are genetically altered in over 60% of adult GBMs. Interestingly, NF1 and EGFR alterations in GBM are almost always mutually exclusive. Here, we designed a multimodal approach integrating spatial proteomics, transcriptomics, and genomics to measure cell composition, immune cell function, cancer cell signaling, cellular spatial organization, gene expression, transcriptional subtypes, and genome alterations in 80 NF1 and EGFR altered human GBMs with extensively curated treatment and survival data. We quantified the spatial expression of 42 proteins in over 31 million single cells measuring RTK/RAS/PI3K and Rb signaling and features of the TME, including 23 cell types covering tumor cells, immune cells, and brain resident cells. In addition to tumor genotype labels, we classified each GBM tumor by its transcriptional subtype (i.e., mesenchymal, classical, or proneural). We identified genotype-phenotype associations in GBM including hyperactive RAS signaling cellular niches, a macrophage-rich TME, and an epithelial-mesenchymal signature in NF1 altered tumors and hyperactive RTK signaling and an immune desert TME in EGFR amplified tumors. Furthermore, we observed spatial heterogeneity in cancer cell signaling patterns suggestive of the presence of different tumor cell clones within different regions of each GBM tumor. Together, we identify several genotype-associated features of GBMs, reinforcing the value of integrating spatial multi-omic data for generation of genotype-driven therapeutic hypotheses, with implications for GBM genotype-based therapeutic stratification.
Citation Format: Maryam Pourmaleki, Brian D. Greenstein, Caitlin J. Jones, Subhiksha Nandakumar, Daniel A. Navarrete, Smrutiben Mehta, Carl Campos, Travis J. Hollmann, Nicholas D. Socci, Tejus Bale, Sohrab P. Shah, Ingo K. Mellinghoff. Glioblastoma mutational profiles drive cancer cell signaling and immune evasion [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 3875.
Abstract 4009: Discovery of inhibitory CAR target DSG1 for damping NECTIN4 on-target off-tumor toxicity in iPSC-derived CAR-T cell therapy
Authors: Hall Matthew S. , Dower Christopher M. , Miller Michael , Wheeler John , Carton Jill , Manor Ohad , Levitsky Hyam 
Published: March 2024

Abstract
Century Therapeutics is developing NECTIN4 targeted induced Pluripotent Stem Cell (iPSC) derived Chimeric Antigen Receptor (CAR) T cell therapy. NECTIN4 targeted antibody drug conjugate enfortumab vedotin, while generally well tolerated, causes severe skin adverse events in some patients, thought to be driven by on-target off-tumor toxicity against NECTIN4 displaying skin keratinocytes. We include a safety switch in our first clinical-stage iPSC-derived CAR-NK cell therapy, CNTY-101, which provides an antibody target for elimination of the engineered cells should that be necessary. An additional method for reducing the incidence and severity of this type of adverse event is through the incorporation of an inhibitory CAR, also known as a NOT-gate, directed against antigen expressed on normal skin keratinocytes, but the optimal surface protein to target with such an inhibitory CAR for a NECTIN4 targeted therapy is unknown. Here, we identify DSG1 (Desmoglein-1) as a top inhibitory CAR target for NECTIN4 targeted CAR-T cell therapy by conducting a multi-omics analysis of public single-cell RNAseq, bulk RNAseq, and protein microarray immunohistochemistry datasets. We find that DSG1 is constitutively and uniformly displayed on the surface of skin keratinocytes in the layers of epidermis where they display NECTIN4, and DSG1 gene expression in normal skin is uniformly high across individuals. Conversely, DSG1 mRNA and protein are rarely expressed in NECTIN4 expressing cancer indications including bladder, breast, non-small cell lung, ovarian, and pancreatic. Further, autoimmune antibodies specific to DSG1 have been described, suggesting that DSG1 is targetable by a CAR in situ. Finally, DSG1 is not expressed by our iPSC-derived CAR-T cells. While NECTIN4 expression is greatest in skin keratinocytes, epithelial cells in other tissues express NECTIN4 at lower levels. In this study, we compare the expression levels of NECTIN4 to targets associated with tissue and cell-type specific on-target off-tumor toxicity in primary CAR-T cell clinical trials. Our findings support the strategy of DSG1-directed inhibitory CAR incorporation into NECTIN4 specific CAR-T cell therapeutic candidates.
Citation Format: Matthew S. Hall, Christopher M. Dower, Michael Miller, John Wheeler, Jill Carton, Ohad Manor, Hyam Levitsky. Discovery of inhibitory CAR target DSG1 for damping NECTIN4 on-target off-tumor toxicity in iPSC-derived CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4009.
Abstract 409: Deciphering chromosomal instability in consensus molecular subtypes (CMS) in CRC: Insights from an integrative multi-omics approach
Authors: Bischof Julia , Woodsmith Jonathan , Church David 
Published: March 2024

Abstract
CRC is a heterogeneous disease with different molecular subtypes, which can have a profound impact on treatment response and patient outcomes. CMS classification based on gene expression profiles has been shown to differentiate patients into clinically relevant sub-groups. However, deep multi-omics characterization of the CMS classes based on high quality patient data is lacking. Through the integration of multi-omics data, we aim to enhance our understanding of CRC’s molecular heterogeneity and thereby facilitate the development of personalized therapeutic strategies.
We performed a multi-omics analysis of CRC patients to identify potential new features of CMS groups. 890 fresh-frozen, surgically resected CRC tumor and adjacent normal samples were used to analyze whole genome and RNA sequencing data. DNA and RNA was prepared using Qiagen AllPrep Universal and KAPA Hyper Prep kit (DNA), as well as TruSeq Stranded Total mRNA kits and sequenced on a NovaSeq6000 system. We identified CMS subtypes based on RNA-Seq data and performed all statistical analyses using R.
Our analysis revealed distinct molecular characteristics across the four CMS subtypes, including unique driver mutations, signaling cascades, and immune cell infiltration profiles. The poor prognosis associated with CMS4 patients was corroborated by our clinical data. Notably, we observed high global chromosomal instability (CIN) profiles for both CMS2 and CMS4 and identified recurrent global aneuploidy patterns within these subtypes that link alterations of known cancer genes to these large-scale structural events. We found higher levels of CIN in CMS2 compared to CMS4, which appears to be predominantly driven by numerical, rather than structural CIN. Specifically, in CMS2, SMAD2/4 is commonly impacted by deletion on chromosome arm 18q, while PLCG1 is more influenced by amplifications on 20q respectively, suggesting potential values as CRC CMS biomarkers.
In conclusion, our integrative multi-omics analysis offers a comprehensive understanding of CRC molecular subtypes, which can inform personalized treatment strategies. Our findings underscore the importance of considering not only specific driver mutations but also large-scale structural rearrangements in the context of CMS subtypes.
Citation Format: Julia Bischof, Jonathan Woodsmith, David Church. Deciphering chromosomal instability in consensus molecular subtypes (CMS) in CRC: Insights from an integrative multi-omics approach [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 409.
Abstract 4137: Feasibility of a novel image guided injection tool for non-surgical implantation of orthotopic tumors
Authors: Kierski Thomas M. , Rojas Juan D. , Beaumont Nathan , Charity Theresa , Marek Robert D. , Young Kenneth H. , Palmer Gregory M. , Gessner Ryan C. , Czernuszewicz Tomek J. 
Published: March 2024

Abstract
The purpose of this study was to evaluate a novel device for non-surgical implantation of tumor cells using ultrasonic (US) image guidance. There are a variety of mouse model cancer types (e.g. sub-cu, orthotopic, genetically engineered). Orthotopic tumors are increasingly prevalent but usually require surgery, which reduces throughput and injures the surrounding anatomy. Injections are an alternative to surgery, but usually require image guidance or extensive training. US guided injection systems exist, but are suboptimal because they require significant training, as the US beam is not necessarily coplanar with needle location or trajectory, reducing accuracy and throughput. We tested a new approach to image guided injection which ensures that the US beam is coplanar with the needle. To test the efficacy of the image guided injection device across a variety of common orthotopic tumors, we delivered luc-tagged cells to the prostate (2.5e5 cells in 40 µL, PC3, N=8), liver (2e6 cells in 100 µL, Hep G2, N=5), and brain (1.75e5 cells in 100 µL, U87-MG, N=3). The brain tumor model was introduced via intracardiac injection to simulate metastases. Between 3 and 8 mice were injected for each tumor model. For injection, animals were anesthetized and positioned on motion stage. A 1 mL syringe was mounted to the adjustable injection gantry with the needle co-planar with the US beam, and the target organ was visualized with US. Cells were delivered to the targets using the US live stream as a guide. Between 1 to 3 weeks after the injections, tumors were confirmed via BLI with an IVIS (Revvity, Waltham, MA, USA). For each of the three tumor cell lines, animal preparation and injections could be performed in 10 mins by recently-trained novice users. All three cell lines could be effectively delivered to the orthotopic host tissues, and this was confirmed by using BLI of the cells. This study represents the first time a co-planar US beam and needle delivery mechanism was used for the purpose of non-surgical implantation of orthotopic tumors. Future developments of this platform could include improvements to automation of needle placement and trajectory to ensure successful payload delivery by novice users.
Citation Format: Thomas M. Kierski, Juan D. Rojas, Nathan Beaumont, Theresa Charity, Robert D. Marek, Kenneth H. Young, Gregory M. Palmer, Ryan C. Gessner, Tomek J. Czernuszewicz. Feasibility of a novel image guided injection tool for non-surgical implantation of orthotopic tumors [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 4137.
Abstract 418: BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response
Authors: Krishnan Aravind M. , Jiang Haoyang , Zhang Tianpeng , Kaur Hardeep , Shi Tao , Kwon Youngho , Sung Patrick , Greenberg Roger A. 
Published: March 2024

Abstract
The Bloom Syndrome helicase (BLM) is critical for Alternative Lengthening of Telomeres (ALT), a homology directed repair mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT positive cells to assemble a replication stress associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2 commensurate with the appearance of telomere C-strand specific single-stranded DNA. Moreover, DNA2 nuclease deficiency increased 5’-lagging strand flap formation and ALT in a BLM dependent manner, and BLM promoted ALT in response to telomere C-strand, but not G-strand, single-stranded nicks. These findings define the seminal events in the ALT DNA damage response, linking aberrant lagging strand DNA replication with a BLM directed HDR mechanism that sustains telomere length in a subset of human cancers.
Citation Format: Aravind M. Krishnan, Haoyang Jiang, Tianpeng Zhang, Hardeep Kaur, Tao Shi, Youngho Kwon, Patrick Sung, Roger A. Greenberg. BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response [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 418.