bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 54: Regulation of luminal epithelial cell identity in the pre-tumorigenic mammary gland
Authors: Breindel Jerrica , Gross Kayla M. , Kuperwasser Charlotte
Published: June 2023

The transcription factor MYC is frequently altered in a large portion of human breast cancers; the gene is amplified in ~15% of breast cancers and overexpressed in another 30-50%. Mouse mammary tumor models driven by overexpression of MYC, such as MMTV-Myc, also exhibit a rapid onset and high frequency of mammary tumors indicating that MYC overexpression plays an important role in driving breast carcinogenesis. However, the role of MYC in breast cancer initiation and subtype determination is not well understood. In the normal mammary gland, MYC is a critical regulator of and is essential for alveolar luminal epithelial cell differentiation during pregnancy. Aberrant MYC expression can promote improper alveolar development and milk production and is a potent inducer of cellular proliferation. Based on these known functions of MYC during development, we hypothesized that its overexpression may induce early changes in epithelial cell differentiation leading to pre-tumorigenic shifts in cell lineages that contribute to the formation of luminal breast tumors. To investigate this, we analyzed the mammary glands of MMTV-Myc (MYC) mice prior to any signs of tumor formation. As early as 5 weeks of age, MYC overexpression led to alterations at both the histological and cellular levels. Interestingly, pre-tumorigenic MYC glands phenotypically resembled mid-pregnant glands of WT mice. Specifically, pre-tumorigenic MYC glands demonstrated budding along the mammary ducts and expansion of a Sca1−/CD49b− luminal epithelial cell subset not typically seen in nulliparous mice. RNA-seq was performed on luminal epithelial cells isolated from WT or pre-cancerous MYC mammary glands to define the luminal differentiation states upon MYC overexpression. While there was an overlap in twenty-nine genes that control milk production and cellular metabolism between pre-cancerous MYC luminal cells and those from mid-pregnant WT mice, luminal cells from MYC mice also exhibited a unique fingerprint of forty-one genes that overlap with cell states found in breast cancer. These uniquely expressed luminal cell genes may be important for MYC-regulated tumorigenesis. Ongoing and future experiments will investigate the role of specific MYC-target genes in mammary gland development versus tumor formation to clarify the role of MYC in these divergent processes.

Citation Format: Jerrica Breindel, Kayla M. Gross, Charlotte Kuperwasser. Regulation of luminal epithelial cell identity in the pre-tumorigenic mammary gland [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 54.
Abstract 5404: Predicting protein receptor status from H&E-stained images in breast cancer
Authors: Lee Geongyu , Kim Chungyeul , Kwak Tae-Yeong , Kim Sun Woo , Chang Hyeyoon
Published: June 2023

Correct prediction of cancer molecular subtype plays a very important role in determining the treatment of cancer patients. In determining the molecular subtype of breast cancer, protein receptors such as estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor type 2 (HER2) are used as key factors. These protein receptors also play an important role in determining the treatment method or predicting the prognosis of breast cancer. To confirm this, a test should be performed by immunohistochemical (IHC) staining. In this study, we investigated the morphological relationship between the molecular subtypes and hematoxylin and eosin (H&E) stained whole slide images (WSIs) without IHC stained images, and performed an experiment to evaluate that the protein receptors can be predicted by these morphological features. The TCGA-BRCA data were utilized in this study. There were 728 cases out of the total 1097 cases where the IHC status for ER, PR, and HER2 was either positive or negative. Each individual case is scanned using different scanners and magnifications. In some cases with multiple slides, we use only the first scanned image. The entire WSI was randomly split into 3:1:1, and used for training, tuning, and test, respectively. Individual WSIs was tiled into 1024 × 1024 patches for this study. The multi-task deep learning model predicts the protein receptor status of individual patches. In this study, a confidence measure was added to remove the uncertainty of the deep learning model. When learning WSIs, patches of less than 70% based on these confidence scores did not affect WSIs. Specifically, when learning morphological features, we used strong augmentation such as grayscale, gaussian blur, color jitter, and posterization to ensure that predictions were not biased by color alone. Based on individual patches, ER accuracy of 76%, PR accuracy of 65%, and HER2 accuracy of 79% are shown. As a result of prediction by majority voting on WSI images through patch predicted results, ER accuracy of 74.6% PR accuracy of 66%, and HER2 accuracy of 76.6% Through this study, it was confirmed that the sufficiently trained deep learning model predicted ER, PR, and HER2, which are important factors of molecular subtype, relatively well. Through this, it was found that there was some correlation between morphological characteristics and molecular subtypes in H&E stained WSIs. If more data are collected through future experiments, a molecular subtype prediction model can be developed.

Citation Format: Geongyu Lee, Chungyeul Kim, Tae-Yeong Kwak, Sun Woo Kim, Hyeyoon Chang. Predicting protein receptor status from H&E-stained images 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 5404.
Abstract 5456: Application of a multiplex urinalysis test for predicting treatment response in patients with BCG unresponsive bladder cancer: a pilot study
Authors: Furuya Hideki , Murakami Kaoru , Pagano Ian , Chen Runpu , Sun Yijun , Kim Nari , Kadel Edward E. , Wong Cheryl V. , Davarpanah Nicole , Rosser Charles J.
Published: June 2023

Introduction and Objective: Recently, we reported that OncuriaTM, a multiplex urinalysis test, could be predictive of BCG treatment response. Furthermore, high-grade BCG unresponsive bladder cancer have limited treatment options. Such patients are offered either radical cystectomy or systemic therapy. One of the systemic therapies include immune-oncology agents, e.g., PD-1 or PD-L1 inhibitors. In this study, we tested the performance of OncuriaTM in a BCG unresponsive cohort to determine if it could predict response to a PDL1 inhibitor.

Methods: OncuriaTM data was evaluated in voided urine samples obtained from a prospectively collected cohort of 18 subjects who have BCG-unresponsive NMIBC with treatment of atezolizumab monotherapy or atezolizumab BCG combination (provided by Genentech, NCT02792192). The urine samples were collected prior to treatment in both arms. The OncuriaTM test, which measures 10 cancer-associated biomarkers was performed in an independent clinical laboratory. Predictive models were previously developed using supervised learning and cross-validation analyses. Model performance was validated using ROC curves.

Results: Pre-treatment urinary concentrations of MMP9, VEGFA, CA9, SDC1, PAI1, APOE, A1AT, ANG and MMP10 were increased in patients who developed disease recurrence. A combinatorial predictive model of treatment outcome achieved sensitivity and specificity of >90%.

Conclusion: Previous pilot study found that monitoring the urinary levels of a cancer-associated biomarker panel enabled the discrimination of patients who did not respond to intravesical BCG therapy. In this study, we noted the performance of OncuriaTM for the prediction of systemic PDL1 inhibitor treatment response. A limitation of this study includes its small sample size. With further study, the multiplex OncuriaTM test may be applicable for the clinical evaluation of bladder cancer patients who have not previously responded to intravesical BCG treatment and is considering systemic immune-oncology options.

Citation Format: Hideki Furuya, Kaoru Murakami, Ian Pagano, Runpu Chen, Yijun Sun, Nari Kim, Edward E. Kadel, Cheryl V. Wong, Nicole Davarpanah, Charles J. Rosser. Application of a multiplex urinalysis test for predicting treatment response in patients with BCG unresponsive bladder cancer: a pilot study. [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 5456.
Abstract 546: Targeting BRAF mutations in head and neck cancer
Authors: Law Chun Ho , Chan Franky Leung , Lui Vivian Wai Yan
Published: June 2023

The mitogen-activating protein kinase (MAPK) pathway [RAS/RAF/MEK/MAPK] is a well-known mitogenic pathway essential for regulation of cell proliferation, survival, differentiation, invasion, and apoptosis, etc. In normal cells, upon the binding of external stimuli, activated Ras relays the signals through a RAF-MEK-ERK (MAPKKK-MAPKK-MAPK) phosphorylation cascade, leading to MAPK pathway activation. On the other hand, in cancer cells, aberrations or mutations of the pathway components usually could lead to constitutive activation of the pathway, resulting in dysregulation of these cellular processes. Notably, mutations of MAPK pathway components are found in 18% of head and neck cancer (HNC). Among these, mutations of the serine/threonine-protein kinase B-Raf (BRAF gene) account for about 1.8% of HNC (based on TCGA-HNSCC cohort, USA). Serving as one of the key kinase members of the MAPK pathway, BRAF is considered as a precision drug target in various cancer types, including melanoma, thyroid cancer, and non-small cell lung cancer (NSCLC). While the significance of BRAF mutations remains underexplored in HNC, exceptionally favorable responses to BRAF and MEK inhibition had been reported in HNC patients carrying BRAF-mutations. Based on this, we hypothesize that BRAF mutations may confer drug sensitivity and serve as a predictive biomarker for personalized therapy in HNC. Here, we demonstrated that ectopic overexpression of mutated BRAF resulted in MAPK pathway activation in HNC cell models. We also showed that ectopic BRAFV600E could promote the in vitro growth of head and neck cancer cells in both 2D and 3D culture conditions as well as enhance anoikis resistance, which is a prerequisite for metastasis. Further preliminary results showed that the mutated BRAFV600E mutant could promote the invasiveness of HNC cells. In summary, our findings support that targeting the BRAF mutants could be a potential therapeutic approach or precision treatment for HNC. (This study was supported by the Research Impact Fund (R4015-19F in the period of 6/29/2020-9/7/2021) from the Research Grants Council, University Grants Committee, Hong Kong SAR.)

Citation Format: Chun Ho Law, Franky Leung Chan, Vivian Wai Yan Lui. Targeting BRAF mutations in head and neck 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 546.
Abstract 5523: Impact of COVID-19 on longitudinal breast cancer research studies involving TNBC patients
Authors: Bates Cherie , Kong Esther , Zaikova Elena , Aparicio Samuel , Gelmon Karen
Published: June 2023

Background Triple-negative breast cancer (TNBC) accounts for ~15% of breast cancer diagnoses but is linked to worse outcomes and comprises a disproportionate number of breast cancer deaths. The TNBC pilot study is a prospective longitudinal study to provide a critical resource for understanding TNBC disease. However, the pandemic impacted the collection of samples.

Objective To highlight the impacts of COVID-19 on this longitudinal cancer translational research study including the patient's perspective and to develop recommendations to avoid future disruptions.

Methods 389 participants were enrolled in the prospective longitudinal cohort, which collected serial blood samples for up to 5 years. Due to the pandemic, research was curtailed for 6 months due to concerns about patient safety, halting the collection of blood samples. Missed samples and data gaps were documented. To complement this, we initiated a survey capturing the patient perspective on their experience of the study disruption due to COVID.

Results 217 enrolled participants missed a blood draw or had a collection outside the study window. 158 patients missed 1 time-point collection, and 59 patients missed ≥ 2 collections. Of the 217 participants who missed a collection, 6 disease recurrence diagnoses and 3 deaths occurred during research curtailment. The collection of survey responses from participants is ongoing and will be presented at the AACR Annual Meeting.

Conclusion Missed samples resulted in irreplaceable data gaps critical to monitoring patient outcomes, and reduced cohort sampling during the pandemic. Our current knowledge of the risks suggests that with proper informed consent, collections could have continued. To mitigate disruption in future clinical studies, clear plans should be part of study design to provide continuity. The participants’ experience to be reported will also help researchers understand their issues and help develop policies.

Missed collections and clinical events during COVID-19 research curtailment Number of patients Total with missed or late blood draws 217 Missed 1 collection 158 Missed ≥ 2 collections 59 Diagnosed with disease recurrence 6 Deaths 3

Citation Format: Cherie Bates, Esther Kong, Elena Zaikova, Samuel Aparicio, Karen Gelmon. Impact of COVID-19 on longitudinal breast cancer research studies involving TNBC patients. [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 5523.
Abstract 56: Unlocking primary cell culture for more relevant in vitro models
Authors: Theek Benjamin , Franke Anne , Agorku David , Lamfried Katharina , Jelveh Nojan , Linnebacher Michael , Carrera Lilian Martinez , Hardt Olaf
Published: June 2023

Tumor cell lines are commonly used to investigate biological and translational questions. However, many cell lines have been cultured in serum-containing medium for decades, continuously accumulating genetic and epigenetic aberrations. Without a doubt, these cell lines have very little in common with the tumor they originate from, resulting in low biological and clinical relevance. Despite these drawbacks, cell lines are still the most frequent research tool when it comes to the use of ex vivo models. Yet, in light of higher consistency, reproducibility, and biological relevance, more and more researchers are paying attention to which cells and culture medium to use. Although primary cell cultures certainly reflect cellular and genetic heterogeneity of the original tumor, finding a suitable medium that supports their growth and maintains genetic heterogeneity remains challenging. To this end, we developed serum-free cell culture media to cultivate primary epithelial tumor cells of pancreatic, renal, and ovarian origin. Notably, generating in vitro models with high biological relevance was successful when using our media during the derivation and expansion of tumor cell lines from primary and xenotransplanted tumors. Here, we present another serum-free cell culture media supporting the in vitro growth of primary colon adenocarcinoma. Using this optimized medium, we had a 42% success rate in establishing a primary cell culture for a given tumor sample, higher than for any other media tested in our hands. Next to 2D cell culture, our formulation supports 3D tumoroid formation when cells are embedded in a basement membrane matrix. Furthermore, targeted sequencing of 486 tumor-associated genes revealed that more than 99% of the detected genetic variants in an early passage (p11) were also present after extended time in culture (p53). In summary, our new serum-free medium enables the efficient derivation and expansion of primary tumor and xenograft-derived colon adenocarcinoma cell cultures in 2D and 3D, yet retaining their genetic identity.

Citation Format: Benjamin Theek, Anne Franke, David Agorku, Katharina Lamfried, Nojan Jelveh, Michael Linnebacher, Lilian Martinez Carrera, Olaf Hardt. Unlocking primary cell culture for more relevant in vitro models [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 56.
Abstract 5602: Markers of hypoxic cells: Testing a pre-clinical detection assay
Authors: Zayas Beatriz , Rios Karoline , Ortiz Luis , Cox Osvaldo
Published: June 2023

The proposed project aims to test the applicability of a novel set of patented organic compounds as markers of hypoxic circulating tumor cells (H-CTC) with potential pre-clinical relevance. Circulating tumor cells (CTC) are individual cells or clusters that can move from tumors to circulation invading other tissues. Often cells in the tumor need to adapt to low oxygen levels, generating a population of cells known as hypoxic cells that can survive low oxygen levels. These hypoxic cells can produce more cell junction proteins, augmenting connections between cells causing them to break away in clusters rather than individually. Clustered CTCs are much more efficient at metastasis formation. Release of CTC can also be stimulated by cancer treatments such as radiation and chemotherapy. To date, our research has generated in-vitro studies with a set of 3-nitrobenzazolo[3,2- a]quinolinium chloride salts (NBQS) demonstrating its capacity as markers of hypoxic human cancer cells in vitro. Comparison with the control Pimonidazole demonstrated the advantages of novel NBQ-TOM compounds as hypoxic marker. We here, aim to test the applicability of NBQS to identify hypoxic cells among circulating tumor cells (H-CTC). To date limited commercially CTC detection methods are available. Our application can be described as pre-clinical detection technology of hypoxic cancer cells serving also in cancer management. To determine the capacity of novel compound, NBQ-345 TOM as hypoxic fluorescent marker, colon tumor cells (COLO 205) are culture under hypoxic and aerobic environment and treated with NBQ-345TOM (25uM) for 24 hours. After treatment fluorescence is measured with an OPTIMA BMG Fluorimeter. Fluorescence emission results demonstrated significant formation of the NBQ-234 TOM fluorescent metabolite on hypoxic tumor cells in contrast to cells treated under aerobic environment. Also, fluorescence microscopy analysis of colon cancer cells treated for 24 hours with NBQ-345 TOM at hypoxic and aerobic conditions confirmed the stronger fluorescence generation at hypoxic conditions in contrast to cells at aerobic conditions.

Citation Format: Beatriz Zayas, Karoline Rios, Luis Ortiz, Osvaldo Cox. Markers of hypoxic cells: Testing a pre-clinical detection 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 5602.
Abstract 5671: MB201, a CD8+ T cell-selective anti-PD-L1 x IL-2 variant fusion protein, exhibits a potent anti-tumor efficacy without peripheral toxicity
Authors: Park Bom , Bang Hyojoo , Jung Youngjun , Cho Sunjung , Kim Mi Seong , Kang Seok Chan , Jung Sungyoub , Park Youngjin
Published: June 2023

Interleukin-2 (IL-2), a cytokine with pleiotropic immune effects, is the first approved cancer immunotherapy. However, the clinical application of IL-2 is limited by systemic activation of unexpected immune cells, leading to severe toxicities including vascular leakage syndrome (VLS). These adverse effects were mainly caused by the activation of Treg and endothelial cells bearing IL-2 receptor (IL-2R) αβγ. Recently, studies have mainly focused on the optimization of IL-2Rs binding affinity to selectively invigorate CD8+ T cells in tumors, but the toxicity risk has yet to be solved. To overcome these limitations, we developed MB201, a novel anti-PD-L1 x IL-2v fusion protein, which selectively activates CD8+ T cells by attenuating binding affinity to IL-2Rβγ with no binding to IL-2Rα.

MB201 showed a strong binding affinity to PD-L1 (KD value is 0.5 nM) and a weak binding affinity to IL-2Rβγ and IL-2Rαβγ (KD value is 23.8 nM and 30.6 nM, respectively), indicating the preferential targeting PD-L1+ tumor cells. MB201 exhibited a preferential activation of CD8+ T cells than Treg cells, which showed remarkably reduced activation compared to rhIL-2 (more than 100-fold based on EC50). Consistently with the result of T cell activation, MB201 only increased the expansion of CD8+ T cells rather than Treg cells in CD3-stimulated PBMC. Indeed, MB201 induced negligible expansion of immune cells and lower pro-inflammatory cytokine release compared to the rhIL-2 in CD3-unstimulated PBMC, indicating the reduced peripheral toxicity risk. When administered to MC38 syngeneic mice, MB201 showed superior anti-tumor efficacy without both body weight reduction and lung weight gain compared to the avelumab monotherapy or combination therapy with rhIL-2. These preclinical observations support that MB201 has characteristics of a superb safety profile and excellent anti-tumor efficacy, and it may offer an advantageous therapeutic strategy for the treatment of cancer.

Citation Format: Bom Park, Hyojoo Bang, Youngjun Jung, Sunjung Cho, Mi Seong Kim, Seok Chan Kang, Sungyoub Jung, Youngjin Park. MB201, a CD8+ T cell-selective anti-PD-L1 x IL-2 variant fusion protein, exhibits a potent anti-tumor efficacy without peripheral toxicity. [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 5671.
Abstract 5823: Uncovering the mechanisms of matrix metalloproteinase-3 (MMP-3) in the cisplatin resistance of ovarian cancer
Authors: Rivera-Serrano Mariela , Vivas-Mejia Pablo E.
Published: June 2023

Seventy percent of women with ovarian cancer will develop chemotherapy-resistance to cisplatin. Regardless of the advancements in technology, mortality rates for ovarian cancer have declined only slightly in the last forty years, reiterating the need to generate novel treatment options targeting cisplatin resistance. In RNA-seq and microarrays experiments we observed that Matrix-metalloproteinase-3 (MMP3) was highly abundant in cisplatin-resistant when compared to cisplatin-sensitive cells. MMP3 is a member of the matrix metalloproteinases (MMPs) family of proteolytic enzymes, involved in the degradation of proteins in the extracellular matrix (ECM). Deregulation of MMPs alters the extracellular matrix and contributes to proliferation, invasion, and metastasis of cancerous cells. A typical MMP structure consists of a pro-domain, a catalytic, and a hemopexin domain. The catalytic domain is responsible for MMPs proteolytic activity, while the hemopexin domain contributes to substrate and ligand specificity. Inhibitors targeting the catalytic domain of MMPs were unsuccessful in clinical trials due to a lack of selectivity and specificity. When we used a chemical inhibitor, we did not observe reduced cell-proliferation or invasiveness. However, MMP3-targeted small interference RNA (siRNA) was able to reduce cell proliferation and invasive capabilities in cisplatin resistant ovarian cancer cells. Additionally, MMP3-targeted siRNA significantly reduced the sensitivity of ovarian cancer cells to cisplatin treatment. Cisplatin also increased MMP3 mRNA levels in ovarian cancer mice models and human cell lines. This evidence suggests that the MMP3 levels correlate with the sensitivity of ovarian cancer cells to cisplatin treatment. We hypothesize that the hemopexin domain will bind to proteins promoting cisplatin-resistance. We validated our expression profile studies at mRNA and protein levels via western blot, ELISA and qRT-PCR. Additionally, survival analysis using the Kaplan-Meier plotter database showed that ovarian cancer patients with low MMP3 levels live longer than patients with higher MMP3 levels. Furthermore, when we measured MMP3 activity, we observed decreased activity in cisplatin-resistant versus cisplatin-sensitive cells. Taken together, these data suggest that other domains of MMP3 (i.e., hemopexin domain) may have a function in the cisplatin resistance of ovarian cancer cells.

Citation Format: Mariela Rivera-Serrano, Pablo E. Vivas-Mejia. Uncovering the mechanisms of matrix metalloproteinase-3 (MMP-3) in the cisplatin resistance of ovarian 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 5823.
Abstract 5825: TM4SF19 regulates oxidative stress-dependent YAP transcription in HNSCC
Authors: Shin Eunbie , Kim Joon
Published: June 2023

Head-and-neck squamous cell carcinoma (HNSCC), which arises from epithelial cells in mucosal surfaces of the upper aerodigestive tract, is the sixth leading cancer worldwide with 40-50% mortality rates. YAP is a transcriptional regulator that plays important roles in cancer cell proliferation and survival. HNSCC shows the highest levels of YAP gene expression among various types of human cancers. However, the molecular mechanisms explaining upregulation of YAP expression in HNSCC are poorly understood. Excessive consumption of tobacco and alcohol are classical risk factors of HNSCC. Tobacco and alcohol generates metabolites that increase reactive oxygen species (ROS) to abnormally high levels. It is not known whether YAP expression and function are interlinked with the oxidative stress response in HNSCC. Here, we aimed to elucidate the mechanism of YAP upregulation involved in the progression of HNSCC under conditions of oxidative stress. To identify candidates for YAP expression regulators in HNSCC, we analyzed whole-genome siRNA library screening data and TCGA genomics data. We performed loss-of-function and gain-of-function experiments to elucidate the molecular mechanism of TM4SF19, which is the top candidate for mediating transcriptional regulation of YAP in HNSCC. TM4SF19 is one of the transmembrane 4 L six family proteins that have topological similarities to tetraspanins, but little is known about its biological function. We found TM4SF19 controls the expression of YAP gene by regulating GABPβ1, a subunit of the GABP transcription factor complex, which is known to bind and activate YAP gene promoter. We observed that the depletion of TM4SF19 reduced GAPBPβ1, thereby decreasing the transcriptional activity of GABP complex. Importantly, we observed that TM4SF19 was dimerized in response to oxidative stress and consequently mediates the upregulation of YAP transcription by ROS elevation. We further show that TM4SF19 knockdown significantly reduces key characteristics of malignant cells, such as active proliferation and migration. Our results suggest that TM4SF19 is not only a novel transcriptional regulator of YAP but also a strong therapeutic target to suppress the oncogenic activity of YAP, and also provide new clue to understand HNSCC tumorigenesis involving YAP and oxidative stress.

Citation Format: Eunbie Shin, Joon Kim. TM4SF19 regulates oxidative stress-dependent YAP transcription in HNSCC. [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 5825.
Abstract 6296: Preclinical study of a novel anti-CLEC12A antibody-drug conjugate with a glucuronide-protected pyrrolobenzodiazepine payload
Authors: Jung Jinwon , Kim Juhee , Lee Bora , Kwon Jung A , Lee Suyoun , Yoo Byeongmin , Ko Minji , Ryu Ilhwan , Yeom Donghoon , Lee Kyoungjae , Eom Jaehyun , Lee Hanbyul , Ahn Jinhyung , Sung Eunsil , You Weonkyoo , Lee Sang Hoon , Kim Myeong Joo , Kwon Keon Woo , Bae Hyun Joo , Park Yun-Hee , Song Ho Young , Chung Chul-Woong
Published: June 2023

C-type lectin domain family 12 member A (CLEC12A, CLL-1) is a single-pass transmembrane protein of 265 amino acids that is found on monocytes and AML blasts. We have created and characterized antibody-drug conjugates (ADCs) based on a humanized CLEC12A specific antibody to determine whether CLEC12A may be exploited as a therapeutic target for AML. The payload is a proprietary PBD prodrug with a beta-glucuronide trigger, and the linker-payload was conjugated through farnesyltransferase-mediated functionalization. The ADC demonstrated sub-nM cytotoxicity in vitro against several CLEC12A-positive cell lines including HL-60 and PL21. Leu234Ala/Leu235Ala (LALA) mutations were employed to reduce Fc gamma receptor binding and to avoid on-target toxicity. The LALA mutation-bearing ADC displayed a nearly 50-fold decrease in cytotoxicity towards CD34+ hematopoietic stem cells. Its potency was reduced but still sufficient with IC50 values of 61 pM and 15 pM against HL60 and PL21, respectively. Regardless of LALA mutation, the ADCs demonstrated potent antitumor activities with a complete regression at a dose of 0.5 mpk against a subcutaneous HL-60 SCID mouse xenograft model. When the LALA-mutated ADC was tested against a disseminated NSGA mouse model of HL-60-luc, all treated animals survived without clinical symptoms for three weeks after treatment, whereas vehicle-treated animals exhibited morbidity 19 days after treatment or 35 days after tumor implantation. The bone marrow of ADC-treated animals appeared to have nearly fully recovered, whereas that of vehicle-treated animals showed necrosis or tumor growth. In cynomolgus monkeys, the LALA-mutated ADC had a half-life of 82 hours at a dose of 0.2 mpk, and target-mediated drug disposition appeared weak or negligible. Our preclinical studies have shown that CLEC12A targeting ADC can be used as a therapeutics for treating AML.

Citation Format: Jinwon Jung, Juhee Kim, Bora Lee, Jung A Kwon, Suyoun Lee, Byeongmin Yoo, Minji Ko, Ilhwan Ryu, Donghoon Yeom, Kyoungjae Lee, Jaehyun Eom, Hanbyul Lee, Jinhyung Ahn, Eunsil Sung, Weonkyoo You, Sang Hoon Lee, Myeong Joo Kim, Keon Woo Kwon, Hyun Joo Bae, Yun-Hee Park, Ho Young Song, Chul-Woong Chung. Preclinical study of a novel anti-CLEC12A antibody-drug conjugate with a glucuronide-protected pyrrolobenzodiazepine payload [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 6296.
Abstract 6298: A novel topoisomerase I inhibitor based anti-ICAM-1 antibody drug conjugate for the treatment of hematologic malignancies and solid tumors
Authors: Wong Oi Kwan , Jackson David , Shi Lei , Fei Qi , Chen Xiaocheng , Post Leonard
Published: June 2023

Intercellular Cell Adhesion Molecule 1 (ICAM-1, CD54) is a type I transmembrane protein and a member of the immunoglobulin superfamily. ICAM-1 is involved in many key processes such as cell-cell interactions, signal transduction and leukocytes trans-endothelial migration. ICAM-1 is constitutively present at low levels on healthy tissues but highly expressed in some lymphoma, myeloma and some types of solid tumors including melanoma, non-small cell lung cancers and liver cancers. Previous attempt to target ICAM-1 with a monoclonal antibody bersanlimab (BI505, BioInvent) showed that it was well-tolerated but with limited clinical efficacy. To improve the effectiveness of targeting ICAM-1, we developed a novel ICAM-1 antibody drug conjugate (ADC). The ICAM-1 ADC is comprised of a humanized anti-ICAM-1 hIgG1 antibody conjugated to a novel topoisomerase I inhibitor through a cleavable linker at a drug-to-antibody ratio (DAR) of about 8. The antibody moiety of the ADC targets a unique epitope on ICAM-1 which is far from its ligand binding domain. Potent in vitro cytotoxicity of ICAM-1 ADC, with nanomolar to sub-nanomolar EC50, was observed on a panel of hematologic and solid tumor cell lines. In preclinical studies, one or two doses of ICAM-1 ADC at 5-6 mg/kg were generally sufficient to induce tumor regression in multiple cell line-derived xenograft models, including those of lung and liver cancers. In all models tested so far, the novel topoisomerase I inhibitor conjugated ICAM-1 ADC is at least as efficacious as the same ICAM-1 antibody conjugated to the benchmark topoisomerase I inhibitor linker payload deruxtecan (ICAM-1-DXd). Furthermore, in a repeat dose exploratory non-human primate safety study, the benchmark conjugate ICAM-1-DXd (DAR 8) was well-tolerated at 41 mg/kg, the highest dose tested. In conclusion, ICAM-1 is an attractive target for topoisomerase I inhibitor based ADC and warrants further investigation.

Citation Format: Oi Kwan Wong, David Jackson, Lei Shi, Qi Fei, Xiaocheng Chen, Leonard Post. A novel topoisomerase I inhibitor based anti-ICAM-1 antibody drug conjugate for the treatment of hematologic malignancies and solid tumors [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 6298.
Abstract 63: Machine learning identifies experimental brain metastasis subtypes based on their influence on neural circuits
Authors: Masmudi-Martín Mariam , Sanchez-Aguilera A , Navas-Olive A , Baena P , Hernández-Oliver C , Martínez S , Lafarga M , Cerebral Renacer Red Nacional de Metástasis , Lin MZ , de la Prida L Menendez , Valiente M
Published: June 2023

A high percentage of patients with brain metastases frequently develop neurocognitive symptoms, however understanding how brain metastasis co-opt the function of neuronal circuits beyond a mass effect remains unknown. We report a comprehensive multidimensional modelling of brain functional analysis in the context of brain metastasis. By testing different pre-clinical models of brain metastasis from various primary sources and oncogenic profiles we dissociated the heterogeneous impact on brain function that we detected from the homogeneous inter-model tumor size or glial response. In contrast we report a potential underlying molecular program responsible for impairing neuronal crosstalk in a model-specific manner. Additionally, measurement of various brain activity readouts matched with machine learning strategies confirmed model-specific alterations that could help to predict the presence and subtype of metastasis.

Citation Format: Mariam Masmudi-Martín, A Sanchez-Aguilera, A Navas-Olive, P Baena, C Hernández-Oliver, S Martínez, M Lafarga, Renacer Red Nacional de Metástasis Cerebral, MZ Lin, L Menendez de la Prida, M Valiente. Machine learning identifies experimental brain metastasis subtypes based on their influence on neural circuits [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 63.
Abstract 6302: Identification of OASEP1 as a biomarker and therapeutic target for oral cancer
Authors: Takano Atsushi , Yoshitake Yoshihiro , Shinohara Masahiro , Daigo Yataro
Published: June 2023

This study aims to identify new biomarkers and therapeutic targets for oral cancer. Our strategies are as follows: i) Identification of overexpressed genes in oral cancers by gene expression array analysis, ii) Validation of clinicopathologic significance of their protein expression by tissue microarray, iii) Examination of their growth effect on cancer cell by siRNAs. We identified a secreted protein, OASEP1 (oral cancer-associated serum protein 1) as a candidate. Immunohistochemical staining showed that OASEP1 protein expression was observed in 120 (75.4%) of 159 oral cancers that had undergone curative surgery. High level of OASEP1 expression was associated with poor prognosis for oral cancer patients (P = 0.0054, by log-rank test). Multivariate analysis revealed that strong OASEP1 expression was an independent prognostic factor. Suppression of OASEP1 expression by siRNA significantly inhibited the growth of oral cancer cell lines through apoptosis as detected by apoptosis assays and time lapse imaging. Transient OASEP1 overexpression significantly enhanced the growth of oral cancer cells. In addition, knock down of OASEP1 receptor by siRNAs also significantly inhibited the growth of oral cancer cells. Addition of OASEP1 protein significantly increased the growth of oral cancer cells in a dose dependent manner, suggesting that the interaction between OASEP1 and its receptor regulated the growth of oral cancer cells. Microarray analysis coupled with siRNAs for OASEP1 demonstrated various OASEP1-related oncogenic pathways in oral cancer cells. Our data suggest that OASEP1 is a possible prognostic biomarker and therapeutic target for oral cancer.

Citation Format: Atsushi Takano, Yoshihiro Yoshitake, Masahiro Shinohara, Yataro Daigo. Identification of OASEP1 as a biomarker and therapeutic target for oral 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 6302.
Abstract 6316: In vitro and in vivo studies of human granzyme B fusion constructs targeting mesothelin
Authors: Mohamedali Khalid A. , Cheung Lawrence H. , DePalatis Louis R. , Thuning-Roberson Claire , Rosenblum Michael G.
Published: June 2023

Mesothelin is highly expressed on tumor cells and promotes tumor growth and drug resistance. Pancreatic, ovarian, mesothelioma, and lung cancers highly express mesothelin and are cancers of unmet needs with ≤16% relative 5-year survival using standard of care. When delivered to tumor cells by immune effector cells, granzyme B (GrB) is a highly cytotoxic serine protease which induces apoptosis through both caspase-dependent and -independent mechanisms. We developed a fusion protein (GrB-Fc-SD1) composed of human GrB and containing SD1, a novel human single-domain antibody that uniquely targets Region III of the glycoprotein, mesothelin. The SD1 and GrB domains are connected by a human IgG heavy chain fragment which enables dimerization. Thus, the increase in total construct molecular weight leads to an increased in vivo circulation time and anti-tumor efficacy. GrB-Fc-SD1 was transiently expressed in HEK-293E cells under serum-free conditions and purified to homogeneity to a final yield of approximately 5 mg/L. Anti-GrB western blot analysis confirmed the expression of GrB-Fc-SD1 exclusively as a dimer. Comparison of GrB-Fc-SD1 with its expected molecular weight suggests significant glycosylation when produced in HEK-293E cells. The enzymatic activity of granzyme B in GrB-Fc-SD1 was comparable to that of commercially available human granzyme B. In vitro cytotoxicity against a limited panel of lung, pancreatic and ovarian cancer cell lines expressing surface mesothelin indicate cytotoxicity in the nanomolar range, while cytotoxicity against mesothelin-negative cells was at micromolar levels. GrB-Fc-SD1 bound specifically to mesothelin extra-cellular domain compared to a non-specific control, as determined by ELISA. Biacore analysis confirmed binding with a dissociation constant between 10−7 to 10−8 M. These data warrant continued investigation of the protein as a therapeutic candidate. Accordingly, ex vivo serum stability studies are underway. In addition, screening of an expanded panel of tumor cell lines for in vitro cytotoxicity is currently in progress and will be reported. In vivo efficacy studies against nude mice bearing Capan-2 xenograft models are currently underway and will also be reported. Research supported by H2Biologics. Research conducted, in part, by the Clayton Foundation for Research.

Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Louis R. DePalatis, Claire Thuning-Roberson, Michael G. Rosenblum. In vitro and in vivo studies of human granzyme B fusion constructs targeting mesothelin [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 6316.
Abstract 6325: A novel EGFR x MUC1 bispecific antibody-drug conjugate, BSA01, targets MUC1 transmembrane cleavage products and improves tumor selectivity
Authors: Zhang Yifu , Shang Chenzhang , Wang Anqi , Zhang Jia , Liu Yuji , Li Hao , Li Xiaopeng , An Gao , Hui Li , An Frank , Yang Yi
Published: June 2023

Epidermal growth factor receptor (EGFR) and mucin 1 (MUC1) are tumor-associated antigens that are highly co-expressed in esophageal squamous cell carcinomas, non-small cell lung cancers (NSCLC), and triple negative breast cancers, among others. MUC1, a glycoprotein essential for the formation of the epithelial mucous barrier, is hypoglycosylated and dimerizes with EGFR, a potent oncoprotein, in transformed cells. Dual-targeting of both tumor-associated antigens (TAAs) represents a promising therapeutic strategy to treat common malignancies. We generated a fully human anti-EGFR/MUC1 bispecific antibody-drug conjugate (BSA01) from RenLite® humanized, common light chain mice. BSA01 is conjugated with Monomethyl auristatin E (MMAE). BSA01 targets the extracellular domain of MUC1-C (C-terminal domain of MUC1), which is the membrane-bound MUC1 cleavage product that remains after MUC1 is cleaved, providing more access to the tumor antigen in situ. In addition, we generated affinity and internalization optimized binders targeting EGFR that increases the tumor selectivity, to avoid potential skin toxicity due to ubiquitous basal EGFR expression. Internalization assays demonstrate that the anti-EGFR/MUC1 bispecific antibody used to generate BSA01 was endocytosed in tumor cells co-expressing EGFR/MUC1 more efficiently than mono- or bivalent antibodies targeting EGFR or MUC1 alone. When conjugated with payload, BSA01 also exhibited strong anti-tumor efficacy in vitro against gastric, NSCLC and pancreatic cancer cell lines. Moreover, BSA01 showed similar activity in vivo, effectively inhibiting tumor growth in multiple cell-derived xenograft and patient-derived xenograft models. These results suggest that BSA01 has great clinical potential for treating tumors co-expressing EGFR and MUC1.

Citation Format: Yifu Zhang, Chenzhang Shang, Anqi Wang, Jia Zhang, Yuji Liu, Hao Li, Xiaopeng Li, Gao An, Li Hui, Frank An, Yi Yang. A novel EGFR x MUC1 bispecific antibody-drug conjugate, BSA01, targets MUC1 transmembrane cleavage products and improves tumor selectivity [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 6325.
Abstract 6336: Heterodimeric Fc-fused IL12 shows potent antitumor activity and low toxicity
Authors: Huang Yang , Liu Wenting , Zhang Dayan , Zhou Pengfei , Zhou Weiming , Zeng Xiaoli , Zhao Qun , Li Liangwei , Shen Guodong , Cheng Liansheng
Published: June 2023

Background: Interleukin-12 (IL12) is a heterodimeric pro-inflammatory cytokine (70 kDa) composed of two different polypeptide chains: an α-chain (p35 subunit) and a β-chain (p40 subunit). IL12 could induce the proliferation and cytotoxicity of T and NK cell and production of interferon-γ (IFN-γ), promote the differentiation of T helper 1 (TH1) cells and regulate innate resistance and adaptive immunity. However, the clinical efficacy of IL12 has been hindered for its high dose-related toxicities and short serum half-life. Here we have developed a heterodimeric Fc-fused IL12, HK054-V, with reduced potency to improve tolerability and prolong half-life.

Methods: We assessed the binding, bioactivity, anti-tumor efficacy and safety of HK054-V. The binding ability of IL12-Fc lead variants to activated PBMCs was analyzed by flow cytometry. The bioactivity of HK054-V was determined by assessing the activated T-cell proliferation and IFN-γ production in human PBMCs, and STAT-4 phosphorylation in a reporter gene assay. A murine surrogate, mHK054-V, was engineered to mimic HK054-V to evaluate the antitumor efficacy in MC38 and CT26 syngeneic tumor mouse models. Cytokine release assay was conducted by incubating HK054-V with peripheral blood mononuclear cells (PBMCs) from healthy donors. The safety profiles and pharmacokinetic (PK) behavior of HK054-V were characterized in non-tumor-bearing Balb/c mice.

Results: HK054-V is a heterodimeric Fc-fused IL12, which was designed with reduced potency. HK054-V was observed to stimulate the proliferation of activated T cells and NK cells and induce production of IFN-γ in a dose-dependent manner. HK054-V could activate pSTAT4 signal in luciferase reporter gene assay and showed up to 100-fold reduction of potency compared to wild-type IL12-Fc. Human IL12 is inactive in mice, so in vivo anti-tumor activity was assessed by a murine surrogate, mHK054-V. mHK054-V demonstrated anti-tumor efficacy in syngeneic mouse models and was better tolerated than WT mIL12-Fc. Furthermore, mHK054-V has improved PK and therapeutic index in mice.

Conclusion: These data demonstrate that HK054-V, a heterodimeric Fc-fused IL12 with reduced potency, retain strong anti-tumor efficacy with a favorable tolerability profile, which may provide a practical alternative to the systemic administration of IL12 for antitumor therapy.

Citation Format: Yang Huang, Wenting Liu, Dayan Zhang, Pengfei Zhou, Weiming Zhou, Xiaoli Zeng, Qun Zhao, Liangwei Li, Guodong Shen, Liansheng Cheng. Heterodimeric Fc-fused IL12 shows potent antitumor activity and low toxicity. [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 6336.