bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2003: Evaluation of liposome encapsulated sunitinib efficacy and immune modulation on renal cell carcinoma
Authors: Yueh Po-Fu , Chang Yuan , Lan Keng-Li , Hsu Fei-Ting
Published: April 2023

Renal cell carcinoma (RCC) is one of the deadliest malignant tumors worldwide. The RCC’s standard treatments are including surgery, radiation, and chemotherapy. SUTENT® (sunitinib) was approved by US Food and Drug Administration (FDA) in 2016 to treat advanced RCC. According to the results from the clinical trial, sunitinib is associated with a higher objective response rate and survival rate than IFN-α, which indicates that it is an effective multiple tyrosine kinase inhibitor (TKi) against advanced RCC. Meanwhile, recent studies demonstrated sunitinib could cause tumor immune surveillance by upregulated autophagy to inhibit tumor PD-L1 expression. However, sunitinib targets not only tumor tissue but also multiple organs in the human body, which causes many serious side effects and even leads to death. In order to reduce the toxicity of sunitinib, our group design a novel nanocarrier by PEGylated liposome to encapsulate sunitinib (lipo-sunitinib). The PEGylated liposome could passively target tumor tissue by extravasation of nanoparticles through increased permeability of the tumor vasculature (enhanced permeability and retention, EPR effect). Moreover, the PEGylation on the liposome surface could prolong liposome half-life in the body through increased particle size. Through the EPR effect and PEGylation of lipo-sunitinib to target tumor cells precisely, we successfully increased RCC orthotopic tumor inhibition in mouse models compared with vehicle and sunitinib groups. Furthermore, the RCC tumor immunofluorescence staining revealed lipo-sunitinib had higher activated effector T cells (CD8+IL-2+IFN-γ+) and type 1 dendritic cells (CD11c+CD24+MHCII+) infiltration and lower vessel formation than sunitinib group. The flow cytometry also demonstrated that lipo-sunitinib had increased anti-tumor immune cells and inhibited immune suppressors in the systemic system. More importantly, lipo-sunitinib showed lower toxicity in mouse livers, hearts, and kidneys compared to the sunitinib group. Our results suggested lipo-sunitinib could inhibit tumor growth and prolong survival rate by modulating a positive anti-tumor microenvironment and anti-angiogenesis effects. In sum, with the polish of the delivery system, our novel nanodrug lipo-sunitinib could be a potential new treatment strategy for RCC patients.

Citation Format: Po-Fu Yueh, Yuan Chang, Keng-Li Lan, Fei-Ting Hsu. Evaluation of liposome encapsulated sunitinib efficacy and immune modulation on renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2003.
Abstract 2074: Genome Integrity assessment and verification by optical genome mapping for cell manufacturing/bioprocessing applications
Authors: Hastie Alex R.
Published: April 2023

Cells and cell lines are used for multiple applications such as bioprocessing, therapy, and research. To ensure quality of cells and downstream applications, appropriate quality control (QC) methods are critical. Historically, karyotyping has been employed, but it is limited by its very low resolution and tedious laboratory workflow. Other methods such as PCR and targeted sequencing can characterize only small genomic variants at specific loci. Whole genome sequencing can detect small variants genome-wide but has limited sensitivity in detecting structural variants (SVs). Optical genome mapping (OGM) is a novel genome analysis technique that can fill many of the gaps in current capabilities for assessing genome integrity. To find clonal variants, 400 Gbp of data is collected from the parental/control and test samples. For both samples, a de novo assembly is constructed, and homozygous or heterozygous SVs are assessed. Subsequently, Bionano Access software’s dual variant annotation pipeline identifies unique SVs in the test sample compared to the parental sample. To discover subclonal SVs down to 5% variant allele fraction (VAF), the workflow leverages the generation of 1.5 Tbp of data, requiring a simple adjustment to longer data collection time. Comparison of test sample to parental sample easily enables the determination of acquired variants. Finally, generation of ~5 Tbp of data, requiring 2-3 flow cells to be run, enables the detection of SVs at ultra-low VAF down to ~1%. In this study, several dilutions and simulations were performed to examine OGM’s limit of detection. Targeting a coverage of 5 Tbp and analysis using the somatic SV-analysis workflow revealed that OGM has the sensitivity to detect deletions >50kbp, insertions >20kbp, duplications >100 kbp, and translocations at ~1% VAF.

We have applied the clonal and somatic workflows to verify genomes’ integrity after cell immortalization, induced pluripotency, transgene-integration, and gene-editing. The data using the robust and sensitive workflows demonstrate that the OGM platform is a cost-effective solution for cell manufacturing/bioprocessing QC applications.

Citation Format: Alex R. Hastie. Genome Integrity assessment and verification by optical genome mapping for cell manufacturing/bioprocessing applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2074.
Abstract 2134: Microsatellite instability status and high risk features in decision making for adjuvant chemotherapy in stage 2 colon cancer
Authors: Bae Sung Uk , Jeong Woon Kyung , Baek Seong Kyu
Published: April 2023

Background: The impact of adjuvant chemotherapy for patients with stage II colon cancer has been addressed in several clinical trials. NCCN guideline recommends that patients with stage II disease that is MSS/pMMR and at high risk for systemic recurrence can be considered adjuvant chemotherapy. However, research on whether these high-risk features or status of microsatellite instability (MSI) are predictive markers for adjuvant chemotherapy (ACT) is insufficient.

Materials and Methods: We assessed the efficacy of ACT and possible interaction with MSI status and high-risk features for recurrence in patients with resected stage II colon diagnosed at 16 tertiary referral hospitals between January 2010 and December 2017.

Results: According to MSI status, 394 patients (21.8%) had MSI-high tumors and 1416 (78.2%) had MSI-low or microsatellite stable (MSS) tumors. There were 881 patients (48.7%) with one or more high-risk feature for recurrence. Among 161 patients with MSI-high and high-risk features, the 5-year disease-free survival (DFS) and overall survival (OS) of patients who

received no ACT, 5-FU chemotherapy, and oxaliplatin-based chemotherapy were 65.5% and 72.3%, 89.4% and 97.9%, and 88.2% 92.0%, respectively (DFS and OS: 5-FU vs. Oxaliplatin, p=0.972 and p=0.233, Oxaliplatin vs. no chemotherapy, p=0.016 and p=0.032, 5-FU vs. no chemotherapy, p=0.025 and p=0.003, respectively). Among 720 patients with MSI-low or MSS and high-risk features, the 5-year DFS and OS of patients who received no ACT, 5-FU, and oxaliplatin-based were 77.1% and 87.2%, 90.4% and 95.6%, and 86.9% 93.5%, respectively (DFS and OS: 5-FU vs. Oxaliplatin, p=0.580 and p=0.492, Oxaliplatin vs. no chemotherapy, p=0.037 and p=0.077, 5-FU vs. no chemotherapy, p=0.009 and p=0.012, respectively). In patients without risk factors, there was no benefit from ACT regardless of MSI status.

Conclusions: 5-FU chemotherapy can be recommended in patients with one or more high-risk features for recurrence regardless of MSI status in stage II colon cancer.

Keyword: Colonic Neoplasms, Adjuvant Chemotherapy, Microsatellite Instability, Risk Factors

Citation Format: Sung Uk Bae, Woon Kyung Jeong, Seong Kyu Baek. Microsatellite instability status and high risk features in decision making for adjuvant chemotherapy in stage 2 colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2134.
Abstract 2354: Regulation of TIMPs through MMP dependent cleavage in breast cancer
Authors: Coates-Park Sasha , Gurung Sadeechya , Rich Josh , Stetler-Stevenson William , Peeney David
Published: April 2023

Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) are a broadly expressed family of matrisome proteins that are the primary regulators of matrix metalloproteinases (MMPs). In addition to their originally described function, TIMPs display a broad range of MMP independent biological functions associated with proliferation, apoptosis, migration, and differentiation. MMPs are zinc containing endopeptidases that play an essential role in maintaining the extracellular matrix (ECM) through proteolytic turnover; a balancing act maintained (in part) by the local MMP:TIMP ratio. Disturbances in the balance between MMPs and TIMPs is associated with various disease states, including cancer, heart disease, arthritis, and cognitive dysfunction. While TIMPs are classically characterized as regulators of MMP proteolytic activity, we describe a novel mode of MMP-dependent regulation of TIMP function. We show that active MMP9 (aMMP9) in molar excess can cleave TIMP proteins within their C-terminal domains. As a result of this cleavage, the MMP-dependent and MMP-independent functions of the TIMP proteins are predicted to be altered. MMP9 has been found to contribute to the pathogenesis of many cancer types including breast cancer. Additionally, MMP9 is described as a breast cancer biomarker, with high levels of MMP9 being associated with a worse prognosis. Previous research from our lab has shown recombinant TIMP2 may represent a novel biological therapeutic in triple negative breast cancer models. Utilizing murine models of breast cancer, we describe the prevalence of TIMP cleavage in 4T1 breast tumors as well as the functional outcomes of TIMP cleavage within this model system. Studying the outcomes of TIMP regulation by MMPs may allow researchers to target this model system more effectively in translational studies of breast cancer.

Citation Format: Sasha Coates-Park, Sadeechya Gurung, Josh Rich, William Stetler-Stevenson, David Peeney. Regulation of TIMPs through MMP dependent cleavage in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2354.
Abstract 2416: Uncovering the protective neurological mechanisms of hypofractionated FLASH radiotherapy
Authors: Limoli Charles L. , Allen Barrett D. , Kramár Eniko A. , Alaghband Yasaman , Vozenin Marie-Catherine
Published: April 2023

Implementation of ultra-high dose-rate FLASH radiotherapy (FLASH-RT) is rapidly gaining traction as a unique cancer treatment modality able to dramatically minimize normal tissue toxicity while maintaining anti-tumor efficacy compared to standard of care radiotherapy at conventional dose rate (CONV-RT). The resultant improvements in the therapeutic index have sparked intense investigations in pursuit of the underlying mechanisms. As a preamble to clinical translation, we exposed non-tumor bearing male and female mice to hypofractionated (3x10 Gy) whole brain FLASH- and CONV-RT to evaluate differential neurological responses using a comprehensive panel of functional and molecular outcomes over a 6-month follow up. In each instance, extensive and rigorous behavioral testing showed FLASH-RT to preserve cognitive indices of learning and memory that corresponded to a similar protection of synaptic plasticity as measured by long-term potentiation (LTP). These beneficial functional outcomes were not found after CONV-RT and were linked to a preservation of synaptic integrity at the molecular (synaptophysin) level and to reductions in neuroinflammation (CD68+ microglia) throughout specific brain regions known to be engaged by our selected cognitive tasks (hippocampus, medial prefrontal cortex). Ultra-structural changes in pre/post-synaptic bouton (Bassoon/Homer-1 puncta) within these same regions of the brain were not found to differ in response to dose rate. With this clinically relevant dosing regimen, we provide a mechanistic blueprint from synapse to cognition detailing how FLASH-RT reduces normal tissue complications in the irradiated brain.

Citation Format: Charles L. Limoli, Barrett D. Allen, Eniko A. Kramár, Yasaman Alaghband, Marie-Catherine Vozenin. Uncovering the protective neurological mechanisms of hypofractionated FLASH radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2416.
Abstract 2569: Investigation of the effect of chronic IL-1 exposure on breast cancer cell line response to external signaling factors
Authors: Dhar Roopal , Olaleye Ola M. , Abdelaziz Rana Tarig , Reddy Jyotsna Tera , Delk Nikki
Published: April 2023

As part of our immune response, the body produces anti-tumorigenic inflammatory proteins to kill cancer cells. However, cancer cells can usurp chronic inflammation for their own survival. Interleukin-1 (IL-1) is an inflammatory protein found in breast cancer (BCa) tumors that is elevated in the BCa patient tumors and serum and promotes metastasis, indicating IL-1 is clinically relevant. But the molecular mechanisms underlying the effect of chronic IL-1 on BCa progression remain unknown. We previously discovered that acute exposure to IL-1 represses essential hormone receptors in cancers cells, but concomitantly upregulates other compensatory pro-survival proteins that contribute to tumor progression. To determine if chronic IL-1 inflammation has the same effect, we generated cell subline models using the human breast cancer cell line, T47D, by exposing T47D cells chronically to IL-1 for several months. Notably, we found that the T47D chronic IL-1 sublines stably evolve insensitivity to exogenous IL-1. This result suggests that chronic IL-1 causes stable molecular changes that may affect cancer cell response to exogenous factors.Therapeutic strategies include blocking exogenous hormone or growth factor signals in the tumor microenvironment to attenuate cancer cell growth, proliferation, and survival. Therefore, we grew T47D parental and chronic IL-1 sublines in the absence of serum to determine the effect of chronic IL-1 exposure on BCa cell response to hormone and growth factor inhibition. Preliminarily, we do not observe differences in parental versus subline response to serum starvation, suggesting that differential response to exogenous factors is specific to IL-1 signaling. To better understand how chronic IL-1 -induced molecular changes effect BCa cell response to the tumor microenvironment, we will perform sequencing and bioinformatics analysis to identify IL-1 -dependent and/or independent signaling pathways stably altered in the chronic IL-1 sublines.

Citation Format: Roopal Dhar, Ola M. Olaleye, Rana Tarig Abdelaziz, Jyotsna Tera Reddy, Nikki Delk. Investigation of the effect of chronic IL-1 exposure on breast cancer cell line response to external signaling factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2569.
Abstract 257: CDX model based genome-scale CRISPR KO screens for modulators of KRAS (G12C) inhibitor sensitivity
Authors: Zhao Haixin , Zhai Fangzheng , Li Bin , Fan Qiangqiang , Peng Zhengang
Published: April 2023

KRAS is the most frequently mutated oncogene in human cancers and had long been considered an undruggable target for cancer therapy, until the FDA approval of Sotorasib (AMG 510) for the treatment of patients with non-small cell lung cancer (NSCLC) harboring the KRAS (G12C) mutation. To gain more insights into AMG 510 therapeutic mechanisms, herein we carried out a genome-scale CRISPR Cas9 knock-out screen in a KRAS G12C-harboring NCI-H2122 xenograft model. We first generated a stable NCI-H2122 cell line overexpressing Cas9 and confirmed its Cas9 editing efficiency by a BFP-GFP dual reporter system. Referring to our previous study for in vivo subcutaneous xenograft growth and response to AMG 510 treatment of NCI-H2122 cell line in immunodeficient mice, we decided to conduct the in vivo screens using 10M cell inoculation each mouse. We infected NCI-H2122 Cas9 cells with the genome-wide Brunello guide RNA library. After puromycin selection and two passages of cell recovery, the resultant cells were inoculated into Nu/nu mice subcutaneously, with part of cells collected as baseline. Mice bearing tumors were grouped into three groups at average tumor size of around 200 mm3 for vehicle, 10 or 30 mpk AMG510 treatment. Tumors were collected while the average tumor size of vehicle treatment group reached around 800 mm3. Genomic DNA was extracted from both baseline cell samples and tumor samples followed by Next-generation sequencing. We have performed QC analysis for all the samples and will discuss the hits from the screen and their implication in AMG 510-induced cancer sensitivity and resistance.

Citation Format: Haixin Zhao, Fangzheng Zhai, Bin Li, Qiangqiang Fan, Zhengang Peng. CDX model based genome-scale CRISPR KO screens for modulators of KRAS (G12C) inhibitor sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 257.
Abstract 2940: Use of NK humanized mouse models for the in vivo evaluation of anti-tumor NK-cell therapies
Authors: Rettman Pauline , Meyer Laure-Marie , Rangara Ravi , Ponchet-Lac Anna , Moindrot Nicolas , Biancotto Angélique , Lefranc Céline , Delahaye Fabien , Rabia Emilia , Cesaroni Matteo , Sidhu Sukhvinder , Nicolazzi Céline
Published: April 2023

Novel immunotherapeutic strategies targeting human Natural Killer (huNK) cells using monoclonal or multi-specific antibodies and immune modulator molecules are under development and there is a pressing need to have suitable mouse models for the evaluation of these therapeutics. Existing humanized mouse models have limitations and do not show an optimal development of innate immune cells such as NK, dendritic or other myeloid cells. We previously described the development of robust mouse models that allow the engraftment and maintenance of fully functional huNK cells using several strains of immunodeficient transgenic mice for human cytokines (IL-15). The optimal parameters (source of immune cells, pre-conditioning regimen, route of administration) were identified, and the models were validated by flow cytometry and in vivo efficacy studies using an anti-CD20 Ab.

Here, we report further development and characterization of these models and highlight their value to evaluate a variety of NK based therapeutics. We show that huNK cells can infiltrate a subcutaneous B-cell lymphoma model after treatment with an anti-CD20 Ab. We show that SAR444245 (non-alpha IL2) can activate huNK cells in vivo in all developed huIL-15 transgenic models and confirmed the minimal effective dose by flow-cytometry PD studies. We show the efficacy of both reference and internal ADCC-enhanced antibodies in disseminated tumor models in our humanized huIL-15 transgenic mice. In addition, we performed a deep characterization of huNK cells in huIL-15 transgenic models, using a 28-color flow cytometry panel centered on NK cell biology and profiled them transcriptionally by single cell RNA Seq in a disseminated B-cell lymphoma model.

Overall, we have developed robust models sustaining fully functional huNK cells that can be proficiently recruited in vivo by various NK-based therapeutics and can be used to evaluate the efficacy of combination therapies.

Citation Format: Pauline Rettman, Laure-Marie Meyer, Ravi Rangara, Anna Ponchet-Lac, Nicolas Moindrot, Angélique Biancotto, Céline Lefranc, Fabien Delahaye, Emilia Rabia, Matteo Cesaroni, Sukhvinder Sidhu, Céline Nicolazzi. Use of NK humanized mouse models for the in vivo evaluation of anti-tumor NK-cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2940.
Abstract 2963: CDX-585, a novel bispecific antibody targeting PD-1 and ILT4
Authors: Thomas Lawrence J. , Vitale Laura A. , Murphy Michael , Xia Collin , Peng Zeyu , Ejas Asma , Patel Montu , Boyer James , Baronas April R. , O'Neill Thomas , Widger Jenifer , Mills-Chen Laura , Crocker Andrea , Ma Mark , Chen Mingjiu , Davis Hugh M. , Hammond Russ A. , Taglienti Cherie , Yellin Michael , Goldstein Joel , Alvarado Diego , Marsh Henry C. , Keler Tibor
Published: April 2023

Both PD-1 and ILT4 (LILRB2, CD85d) have well established immunosuppressive effects that enable tumors to evade anti-tumor immunity. Tumor expression of PD-L1 and ILT4 has been associated with poor clinical outcomes in several cancer types. Recently, ILT4 activation has been postulated as a resistance mechanism for PD-1/PD-L1 blockade prompting the investigation of dual blockade of these pathways. Siu, L.L. et al. (2022 Clin Cancer Res 28: 57-70) reported encouraging results of the initial clinical study combining the ILT4 antagonist mAb MK-4830 with pembrolizumab. The combination was generally well tolerated and led to several durable responses including in PD-1/PD-L1 refractory patients. We have combined novel PD-1 (mAb E1A9) and ILT4 (mAb 7B1) antagonist antibodies into a tetravalent IgG-scFv format bispecific antibody (bsAb, CDX-585). CDX-585 was engineered to avoid effector functionality by eliminating interaction with Fcγ receptors, and to increase its half-life by enhancing binding to FcRn. As expected, CDX-585 inhibits interaction of PD-1 and ILT4 with their ligands, results in potent inhibition of PD-1 signaling and enhances myeloid cell inflammatory responses to stimulation through toll like receptors (LPS) or CD40 (CD40 agonist mAb). In mixed lymphocyte reactions CDX-585 promoted greater T cell activation than the combination of the parental mAbs. Similarly, CDX-585 demonstrated superior anti-tumor activity over the combination of the parental mAbs in a humanized mouse model of melanoma. CDX-585 has completed GMP manufacturing and IND-enabling activities. Doses of CDX-585 up to 60 mg/kg were well tolerated in cynomolgus macaques and displayed a favorable pharmacokinetic profile. Together these data demonstrate that CDX-585 effectively combines PD-1 and ILT4 blockade into one molecule with favorable biophysical and functional characteristics supporting the initiation of a dose-escalation clinical trial in patients with advanced solid tumors.

Citation Format: Lawrence J. Thomas, Laura A. Vitale, Michael Murphy, Collin Xia, Zeyu Peng, Asma Ejas, Montu Patel, James Boyer, April R. Baronas, Thomas O'Neill, Jenifer Widger, Laura Mills-Chen, Andrea Crocker, Mark Ma, Mingjiu Chen, Hugh M. Davis, Russ A. Hammond, Cherie Taglienti, Michael Yellin, Joel Goldstein, Diego Alvarado, Henry C. Marsh, Tibor Keler. CDX-585, a novel bispecific antibody targeting PD-1 and ILT4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2963.
Abstract 3277: In vivo CRISPR screening identifies PTDSS1 as a potential combinatorial target to improve immune checkpoint therapy response
Authors: Liu Jielin , Anandhan Swetha , Basu Sreyashi , Chen Yulong , Goswami Sangeeta , Nagarajan Ashwat , Herbrich Shelley , Xiong Liangwen , Guan Baoxiang , Sharma Padmanee
Published: April 2023

Immune checkpoint therapy (ICT) has recently revolutionized the treatment paradigm for various types of cancer. However, despite the unprecedented safety and efficacy profile compared to traditional treatment options, many patients do not respond to ICT, highlighting the urgent need for novel strategies to improve ICT efficacy. Here, we used a pooled in vivo CRISPR knockout screening in a mouse bladder cancer model treated with immune checkpoint inhibitor to discover targets that may improve immunotherapy response. Our data identified Ptdss1, which encodes phosphatidylserine synthase 1, as a potential regulator of anti-PD-1 response. Depletion of Ptdss1 not only increased tumor cell immunogenicity and response to interferon gamma, but also facilitated development of an anti-tumor microenvironment. Ptdss1 depletion in tumor cells increased effector CD8+ T cells cytolytic function, decreased regulatory T cells activation and inhibitory function, increased frequency of iNOS+ myeloid cells and induced macrophage M2-to-M1 polarization. Ptdss1 knockdown induced changes in the tumor microenvironment were further amplified when combined with anti-PD-1 treatment, which translates into better tumor control and prolonged animal survival in mouse bladder cancer model. Together, our results provide insights on a new therapeutic strategy for overcoming resistance to immune checkpoint therapy through combination of PTDSS1 inhibition and PD-1 blockade.

Citation Format: Jielin Liu, Swetha Anandhan, Sreyashi Basu, Yulong Chen, Sangeeta Goswami, Ashwat Nagarajan, Shelley Herbrich, Liangwen Xiong, Baoxiang Guan, Padmanee Sharma. In vivo CRISPR screening identifies PTDSS1 as a potential combinatorial target to improve immune checkpoint therapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3277.
Abstract 3484: Oncohistone H2B-E76K alters gene expression & cellular phenotype in a bronchial epithelial cell line
Authors: Pereira Kimberly Espinoza , Bennett Richard L. , Shan Jixiu , Sarthy Jay , Licht Jonathan D.
Published: April 2023

The nucleosome is the basic unit of chromatin, comprising 147 bp of DNA wrapped around an octamer of core histone proteins H2A, H2B, H3 and H4. Compact chromatin acts as a barrier to gene expression and is a key property governing DNA replication and gene expression. Analyzing over 46,000 in cancer patient samples in cBioPortal, we characterized mutations located in canonical histone genes and found several recurrent missense mutations. Foremost among the identified mutations was a glutamate to lysine missense mutation at amino acid 76 of histone H2B (H2B-E76K) that disrupts histone H2B-H4 interaction. Previously, we demonstrated that exogenous expression of H2B-E76K disrupted nucleosome stability, altered chromatin accessibility, and gene expression. We developed a new model utilizing CRISPR “knock-in” technology to integrate the E76K mutation into the endogenous H2BC4 gene of the lung epithelial cell line BEAS-2B to create a homozygous and heterozygous H2B-E76K cell line. Although the mutant histone only accounts for about 2-5% of total histone H2B, expression of H2B-E76K catalyzed distinct changes in cell migration, proliferation, colony formation and gene expression. CUT&RUN technology revealed mutant histones broadly incorporate throughout the genome. Gene set enrichment analysis (GSEA) revealed that H2B-E76K homozygous cells displayed expression changes similar to polycomb repressive complex 1 (PRC1) disruption or oncogenic KRAS expression. Using available datasets, we discovered that expression of H2B-E76K caused increased expression of PTN, PKIB, and CDH2, genes important in migration, epithelial-mesenchymal transition (EMT), and adhesion pathways. Furthermore, analysis of chromatin accessibility changes indicated that increased accessibility was significant in and around genes governing cell migration. Notably, H2B-E76K significantly increased heterogeneity of gene expression as measured using scRNA-seq. Together these results indicate that mutations disrupting histone structure may create millions of dysfunctional nucleosomes and new sites of “open” chromatin, resulting in loss of nucleosome-mediated gene repression and/or altered interactions with regulators of chromatin, fundamentally changing cell growth and migration properties to favor tumorigenesis.

Citation Format: Kimberly Espinoza Pereira, Richard L. Bennett, Jixiu Shan, Jay Sarthy, Jonathan D. Licht. Oncohistone H2B-E76K alters gene expression & cellular phenotype in a bronchial epithelial cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3484.
Abstract 3543: Mesenchymal stromal cells and tumor-associated macrophages modulate adrenergic to mesenchymal state switching in neuroblastoma
Authors: Lee Meng-Hua , Louault Kevin , Giannikou Krinio , Ding Xiangming , Yanovsky Alice , Park Jin-Seok , DeClerck Yves A. , Asgharzadeh Shahab
Published: April 2023

Neuroblastoma (NB), a common solid tumor of children, can adopt a lineage-committed adrenergic (ADRN) or an immature mesenchymal (MES) tumor cell type, which differs in phenotype, epigenetic landscape, transcription factors, and core regulatory circuitries. These cell types can spontaneously interconvert, but the mechanism remains largely unknown. Here, we hypothesize that the tumor-associated macrophages (TAM) and cancer-associated fibroblast (CAF) within the tumor microenvironment contribute to pro-tumorigenic factors production and drive switching from ADRN to MES state in neuroblastoma. We first demonstrate the ADRN and MES lineage identity of NB cell lines by Western blot and compared the control and NB cell lines co-cultured with mesenchymal stromal cells (MSC), the precursor cell of CAF, and monocyte in transwell. Initial experiments demonstrated the morphology of NB cell line CHLA255 and CHLA136 showing increased spreading area and spindle shape in TAM-CAF co-cultures compared to control cells. As compared to control cells, monocytes and MSC-CAF co-culture induced the MES lineage markers SOX9 and Notch1 of NB cell lines that had low basal SOX9 expression, with a reduction in protein levels of ADRN lineage markers PHOX2B, and GATA3. Next, single-cell RNA-sequencing was utilized to analyze the expression of TAM and CAF, and MES- and ADRN-signature genes of NB cells in NB-TAM-CAF co-cultures. Single-cell analyses of TAM-CAF co-cultured CHLA255 and CHLA136 demonstrated enrichment of MES signature over time providing clear evidence of mesenchymal differentiation of NB cells within a TME-rich environment. Pathway enrichment analyses reveal enrichment of pathways associated with extracellular matrix (ECM) deposition and epithelial to mesenchymal of TAM-CAF co-cultured NB cell lines. Our results suggested that the presence of CAF and TAM in the tumor microenvironment drives neuroblastoma ADRN: MES switch akin to that observed in epithelial: mesenchymal switch in adult tumors. ADRN-MES state switching has been shown relevant for tumor relapse and therapy resistance. Our studies point to a therapeutic vulnerability by targeting cells and pathways activated in the NB-TAM-CAF axis.

Citation Format: Meng-Hua Lee, Kevin Louault, Krinio Giannikou, Xiangming Ding, Alice Yanovsky, Jin-Seok Park, Yves A. DeClerck, Shahab Asgharzadeh. Mesenchymal stromal cells and tumor-associated macrophages modulate adrenergic to mesenchymal state switching in neuroblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3543.
Abstract 3902: Glutathione as a potential marker of tamoxifen resistance in breast cancer
Authors: Hamadneh Yazan I. , AlWahsh Mohammad , Alrawabdeh Jawad , Hergenröder Roland , Dahabiyeh Lina A. , Hamadneh Lama
Published: April 2023

Glutathione (GSH) is an antioxidant with an important protective intracellular role against reactive oxygen species. Increased glutathione level is associated with higher rates of metastases and a more aggressive behavior in breast cancer. This study aims to study the molecular and metabolic changes in glutathione synthesis in tamoxifen resistant (TAM-R) MCF-7 breast cancer cell lines.Methods: Three tamoxifen resistant MCF-7 cell lines were produced by using two methods. The first was achieved by treating the cells with tamoxifen and gradually increasing the amount until reaching a predetermined concentration of tamoxifen. The second was achieved by giving multiple fixed concentrations of tamoxifen, the third model was produced from the second by treating the cells with continuous 1µM of tamoxifen. mRNA and metabolites were extracted from the cell lines. cDNA was synthesized from the extracted mRNA, and gene expression of glutathione synthetase (GSS) was measured using real-time PCR. Nuclear Magnetic Resonance technique with PABO probe was used for metabolic profiling of extracted metabolites from the resistant and sensitive MCF-7cells and Chenomix software for quantification. Additionally, Kaplan Meier plotter (https://kmplot.com/) was used to predict the significance of GSS gene expression to overall survival of breast cancer patients. Results: A significant increase in glutathione accompanied with significant decrease in cysteine levels were found in TAM-R cells compared to control TAM sensitive cells. However, there was a 2 times reduction in the expression of GSS in TAM-R cells. Correlation of GSS gene expression among ER +ve, PR +ve patients who received tamoxifen in their therapy showed that its down regulation is significantly linked to poor overall survival (p-value 0.022 and HR of 0.7). Conclusion: The significant increase of glutathione in TAM-R cells may contribute to their increased resistance to oxidative stress and exhibition of a more aggressive behavior. Moreover, the decrease in GSS expression is a strong indicator of decreased glutathione synthesis in TAM-R cells as a result of cysteine depletion in the cells that was correlated to poor overall survival among BC patients. However, high glutathione levels in TAM-R cells could be due to increased glutathione regeneration in TAM-R cell lines compared to control.

Citation Format: Yazan I. Hamadneh, Mohammad AlWahsh, Jawad Alrawabdeh, Roland Hergenröder, Lina A. Dahabiyeh, Lama Hamadneh. Glutathione as a potential marker of tamoxifen resistance in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3902.
Abstract 3954: Evaluation of long mononucleotide repeat markers for detection of microsatellite instability
Authors: Chen Suping , Lin John H. , Pallavajjala Aparna , Lotan Tamara L. , Bacher Jeffery W. , Eshleman James R.
Published: April 2023

Microsatellite instability (MSI) and mismatch repair immunohistochemistry are biomarkers of defective MMR (dMMR) that predict response to immune checkpoint inhibitor therapy in solid tumors. The microsatellites in the Promega MSI Analysis System Version 1.2 may not be ideal to detect MSI in certain cancer types. Long mononucleotide repeat (LMR) markers may improve detection of microsatellite instability in early colorectal lesions and non-colorectal cancers.In this study, we compared the performance of the current gold standard, MSI Analysis System Version 1.2 (Promega), which consists of 5 mononucleotide repeat markers (21-27 bases), with a new LMR kit (Promega), which consists of 4 markers from the MSI V1.2 panel and 4 LMR loci (52-60 bases). We studied five cohorts including 24 MMR proficient (pMMR) and 24 dMMR colorectal cancer (CRC) samples, 24 pMMR and 42 dMMR endometrial cancer (EC) samples, 12 dMMR prostate cancer (PC) samples, 22 MSI-high (MSI-H) samples of other cancer types, and 12 MSI-low (MSI-L) samples, where MMR status was confirmed by immunohistochemical (IHC) staining and/or MSI Analysis System Version 1.2.The specificity and sensitivity of the LMR MSI panel for dMMR detection were both 100% in CRC. The specificity of the MSI V1.2 and LMR MSI panels in EC was both 100%, and the sensitivity was 88% versus 98%, respectively. The 22 MSI-high (MSI-H) samples of other cancer types, these include cholangiocarcinoma, appendix, duodenal, pancreatic and gastric cancer, which were previously classified as MSI-H by using the MSI V1.2 panel were also classified as MSI-H by using the LMR MSI panel. Among 12 samples that were previously classified as MSI-L by the MSI V1.2 panel, 9 of the samples were classified as MSI-L, and 3 of the samples were diagnosed as MSI-H using the MSI LMR panel. The LMR panel has performed well on 80 samples over the past 6 months.The LMR MSI panel is highly concordant with the MSI V1.2 panel for dMMR detection in colorectal cancer and showed increased sensitivity in endometrial cancer. The LMR MSI panel showed improved dMMR detection in non-colorectal cancers.

Citation Format: Suping Chen, John H. Lin, Aparna Pallavajjala, Tamara L. Lotan, Jeffery W. Bacher, James R. Eshleman. Evaluation of long mononucleotide repeat markers for detection of microsatellite instability. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3954.
Abstract 3969: Screening of kinase inhibitors as bioenergetic metabolism modulator using the XF Real-Time ATP Rate Assay
Authors: Kam Yoonseok , Winer Lisa , Romero Natalia
Published: April 2023

Cancer cells are metabolically reprogrammed through tumor initiation and progression, and there are increasing developments in cancer therapies exploiting the potential metabolic vulnerability. A better understanding of cancer cell metabolic regulation can also contribute to modulating the harsh tumor microenvironment, which strongly affects the efficacy of emerging anti-cancer immunotherapies. As one of the critical contributors to cancer therapy design, there is increasing demand for time-efficient and reliable analytic tools when searching for drugs and genes modulating cancer cell metabolism. The XF Real-Time ATP Rate Assay measures ATP production from glycolysis and mitochondrial respiration simultaneously and provides a quantitative overview of the bioenergetic phenotype of the cells. By using ATP production rate as a universal unit of bioenergetic metabolism, this assay quickly detects metabolic modulations induced by chemical stimuli or genetic modifications. It allows the identification of potential targets for therapy design. In this study, we used the Seahorse XF Pro Analyzer combined with the XF ATP Rate assay to screen 80 kinase inhibitors for the suppressive effects on energy metabolism and/or inducing a metabolic phenotype shift in THP-1 cells, a monocytic leukemia cell line, using PBMC as the control. We identified four mitochondrial and two glycolytic suppressors using the screening and dose-response views available in Seahorse Analytics, a cloud-based data analysis tool for Seahorse XF assays. These compounds showed moderate or no effects on the net ATP production rates but induced a significant shift in the metabolic phenotype of THP-1 cells. Among the selected kinase inhibitors, AG-879 (ErbB2 inhibitor), SU-1498 (VEGFR inhibitor), and U-0126 (MEK1/2 inhibitor) showed the most potent inhibitory effect on mitochondrial energy production in THP-1 cells. Dose-response studies indicated that SU-1498 and U-0126 have higher potency in THP-1 than PBMC, and thus they can be considered good cancer-cell targets for mitochondrial suppressors. In contrast, AG-879 showed a similar or higher inhibitory effect on PBMC even though it is the most potent mitochondrial suppressor among the selected compounds. These results demonstrate that the XF Real-Time ATP Rate Assay can be used as a primary assay for screening and validating drug candidate(s) targeting the bioenergetic metabolism of cancer cells.

Citation Format: Yoonseok Kam, Lisa Winer, Natalia Romero. Screening of kinase inhibitors as bioenergetic metabolism modulator using the XF Real-Time ATP Rate Assay. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3969.
Abstract 3970: NCG-hIL15 humanized mice: an ideal model for human immune reconstitution of NK cells
Authors: Liu Xing , Wu Meirong , Wang Huiyi , Yu Weiwei , Xu Jianming , Sun Hongyan , Ju Cunxiang , Wang Hongyu , Chen Santi Suryani , Li Zhiying , Moore Mark Wade , Zhao Jing , Gao Xiang
Published: April 2023

Introduction: IL-15 is a four α-helix bundle cytokine which is produced by dendritic cells, monocytes, and epithelial cells. IL-15 is necessary for the development, survival, and activation of natural killer (NK) cells. NK cells are unable to persist in immunodeficient NCG mice reconsituted with human NK cells, but after knocking in human IL-15 (hIL-15), NCG-hIL15 mice can support the reconstitution and survival of human NK cells.

Results: Compared with NCG mice, the level of hIL-15 was significantly increased in NCG-hIL15 mice. After transplanting human PBMC (huPBMC) or NK (huNK) cells into NCG-hIL15 mice, the peripheral blood level of NK cells in huNK-NCG-hIL15 mice was much higher than in huPBMC-NCG-hIL15 mice. The expression of perforin in peripheral blood of huNK-NCG-hIL15 mice was significantly higher than that in huPBMC-NCG-hIL15 mice. Considering the obvious ADCC of rituximab acting on Raji cells in vitro, we evaluated the efficacy of rituximab in huNK-NCG-hIL15 mice subcutaneously engrafted with Raji cells. Rituximab significantly inhibited the tumor growth in huNK-NCG-hIL15 mice.

Conclusion: NCG-hIL15 is a great mouse model to evaluate the anti-tumor efficacy of drugs targeting human NK cells without the interference of T cells.

Citation Format: Xing Liu, Meirong Wu, Huiyi Wang, Weiwei Yu, Jianming Xu, Hongyan Sun, Cunxiang Ju, Hongyu Wang, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao. NCG-hIL15 humanized mice: an ideal model for human immune reconstitution of NK cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3970.
Abstract 4004: Targeting drug-resistant acute myeloid leukemia (AML) cells using novel casein kinase II (CK2) inhibitor
Authors: Duke Koby , Mercer Katherine , Rajaiah Rajesh , Daniyal Muhammad , Qiu Yi , Purandare Ashok , Uzun Yasin , Zhang Lijun , Klink Morgann , Gowda Chandrika
Published: April 2023

Introduction: Protein Kinase CK2 level and activity are high in Acute Myeloid Leukemia (AML) stem cells. Genetic inhibition of CK2 promotes apoptosis and shows synergistic cytotoxic activity with cytotoxic therapy. Here we report the anti-leukemia efficacy of a novel and potent small molecule inhibitor of CK2, BMS-135, in AML mouse models.

Methods: AML cell lines (n=8) and primary AML cells (n=3) representing various AML genetic subtypes (MLL rearranged, FLT3-ITD, TP53 mutation) were tested in vitro. Cells were treated with serial dilution of BMS-135 for 24 to 48hrs, and cell viability was measured using calorimetric cell viability (WST) assay. Similarly, treated cells were analyzed for cell cycle distribution, apoptosis, and colony formation. We measured the mRNA and protein levels of known CK2 targets. RNA sequencing and gene expression analysis was done to evaluate gene expression changes following treatment with BMS-135. Cell line-derived xenograft and patient-derived xenograft of AML were treated with BMS-135 at a dose of 7.5mg/kg oral gavage twice daily for 21 days or vehicle.

Results: Combined in vitro and in vivo experiments establish the efficacy of BMS-135. Cytotoxicity at inhibitory concentrations of 30-800nM, G0-G1 cell cycle arrest, increased apoptosis, and poor colony formation following treatment were noted consistently in all cells. Single-drug treatment with BMS-135 achieved 50-80% leukemia inhibition and significantly prolonged survival in treated mice after 3 weeks of therapy. Following in vivo treatment with BMS-135, we confirmed the inhibition of known CK2 targets (AKT, PI3K, Bcl-xL) in the bone marrow and spleen AML cells. No significant myelosuppression or organ toxicity was noted in tumor-bearing mice following 3 weeks of treatment when compared to vehicle-treated mice. BMS-135 shows synergistic cytotoxic activity with daunorubicin and cytarabine.

Conclusions: Selective inhibitor of CK2 is well tolerated and shows superior in vivo efficacy and target inhibition in a series of AML cells and xenografts. BMS-135 works synergistically with cytotoxic agents like daunorubicin and cytarabine. These results support further pre-clinical characterization and clinical development of BMS-135 for treating AML in combination with cytotoxic therapy.

Citation Format: Koby Duke, Katherine Mercer, Rajesh Rajaiah, Muhammad Daniyal, Yi Qiu, Ashok Purandare, Yasin Uzun, Lijun Zhang, Morgann Klink, Chandrika Gowda. Targeting drug-resistant acute myeloid leukemia (AML) cells using novel casein kinase II (CK2) inhibitor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4004.