bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6258: Inference of gene expression using fragmentation patterns from targeted high-depth sequencing of cell-free DNA
Authors: Leff Emily , Tseng Alexander , Cheung Victoria , Tabari Ehsan , Walter Kimberly , Lovejoy Alex , Lin Cheng-Ho J.
Published: March 2024

Cell-free DNA (cfDNA) circulating in blood plasma arises primarily from cellular chromatin fragmentation and release due to cell death. Studies have demonstrated that the assessment of fragmentomic features of cfDNA enables gene expression inference and tissue-of-origin classification with potential applications for noninvasive cancer detection. However, due to low depth of coverage of sites of interest, current whole genome sequencing (WGS) methods have not been able to infer expression of individual genes or limited gene sets. Therefore, we developed a targeted sequencing approach that enables individual gene expression inference from cfDNA fragmentation patterns based on transcriptional start-site gene activation probability (TSS-GAP).

To assess the sensitivity of this method for cfDNA-inferred gene expression profiling, we applied TSS-GAP to blends of micrococcal nuclease (MNase)-digested DNA from a cancer cell line (LS180) and sorted peripheral blood mononuclear cells (PBMC), with LS180 DNA at varying concentrations (0.1-10%). This enabled the determination of the limit of detection (LoD), relative to healthy donor background levels, on the basis of the genes’ activation probabilities (TSS-GAP scores).

We found 936 genes with high inferred expression in the LS180 cancer cell line and low inferred expression in non-cancer samples (i.e. CD4, CD14, and plasma cfDNA from 4 healthy donors) and used those for healthy donor background and LoD assessment. Using these gene-level background values as cutoffs, blends were assessed to determine the lowest blend level at which expression of the gene was able to be differentiated from background. Out of the 936 genes, 326 (34.8%) were detectable at a blend level of 0.1%, 59 (6.3%) at 0.3%, 31 (3.3%) at 1%, 19 (2.0%) at 3%, and 39 (4.2%) at 10%. In total, 416 genes could be detected by TSS-GAP at levels 1% of or lower above background, highlighting the potential of our method for detecting expression of certain genes even from a small fraction of cells contributing cfDNA to liquid biopsy samples. Further expansion of this work could consider the utility of such a method for circulating tumor DNA (ctDNA) detection, cfDNA tissue-of-origin classification, and deconvolution of cell types contributing to cfDNA found in blood.

Citation Format: Emily Leff, Alexander Tseng, Victoria Cheung, Ehsan Tabari, Kimberly Walter, Alex Lovejoy, Cheng-Ho J. Lin. Inference of gene expression using fragmentation patterns from targeted high-depth sequencing of cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6258.
Abstract 6276: In vitro neuroendocrine differentiation of BPH-1 cells under beta-adrenergic stimulation
Authors: Pigat Natascha , Garrido Laura N. , Lefevre Coline , Baures Manon , Goffin Vincent , Armand Anne-Sophie , Capiod Thierry
Published: March 2024

Recent studies have shown that adrenergic fibers from the sympathetic nervous system (SNS), acting through stromal β2-adrenergic receptors, play an important role in the initial phases of cancer development by promoting tumor cell survival. Recent epidemiological data suggest that β-blocker intake is associated with improved survival of prostate cancer patients. Hypertension and high blood pressure have been suggested to increase prostate cancer development suggesting that the use of β-blockers in these patients could also reduce prostate cancer risk. β-adrenoceptors signal through the cAMP/PKA pathway that is known to promote neuroendocrine differentiation (NED) in LNCaP prostate cancer cell line. While neuroendocrine (NE) cells expressing synaptophysin (SYN) represent a minor cell population in the epithelial compartment of normal prostate glands, the population of NE-like cells, exhibiting NE phenotypes and expressing NE markers, is increased in, and correlates with, cancer progression, androgen-independent state and poor prognosis. Expression of the routine tissue biomarker of prostate cancer AMACR in NE-like cells discriminates them from NE cells. Our aim was to investigate whether NED could also be associated with benign prostatic hyperplasia (BPH) as recent studies showed that an increase in neuroendocrine cell density occurred before development of BPH in hypertensive rats. Our results showed that a 10-day treatment with 100µM IBMX (blocking cAMP degradation) and 10µM forskolin (FSK, adenylate cyclase activator) to trigger sustained activation of the cAMP/PKA pathway led to an increase in SYN and AMACR expression in BPH-1 cells (qPCR and WB). Similar results were observed by combining the effect of the β-adrenoceptor agonist isoproterenol (1µM) and FSK. The same treatments reduced BPH-1 cell proliferation rates by 40% and 60%, respectively, as expected for NED. Effects on AMACR and SYN expression and cell proliferation were fully reversed by the β-blocker carvedilol (5µM). This data suggests that β-adrenoceptors could be linked to the development of NE-like cells in BPH, a non-lethal disease affecting the transition zone (TZ). As 20% of the prostate cancer occur in this TZ, this observation may need further investigation.

Citation Format: Natascha Pigat, Laura N. Garrido, Coline Lefevre, Manon Baures, Vincent Goffin, Anne-Sophie Armand, Thierry Capiod. In vitro neuroendocrine differentiation of BPH-1 cells under beta-adrenergic stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6276.
Abstract 6281: Context dependent role of glucocorticoids in breast cancer
Authors: Effah Wendy , Ponnusamy Suriyan , Asemota Sarah , Khosrosereski Yekta , Narayanan Ramesh
Published: March 2024

Glucocorticoids bind to the transcription factor glucocorticoid receptor (GR) and regulate genes in response to cellular metabolism, inflammation, and stress. Although GR has been extensively evaluated in inflammation and metabolic diseases, its role as a therapeutic target in cancer has not been thoroughly studied. Results from studies with another member of the steroid receptor family, androgen receptor (AR), demonstrated that the AR is an oncogene in estrogen receptor (ER)-negative breast cancer, but a tumor suppressor in ER-positive breast cancer. We expected a comparable mechanism with GR in different breast cancer subtypes. In this study, we evaluated the role of GR in various breast cancer subtypes and discerned the mechanism of action. Six breast cancer cell lines and seven patient-derived xenografts (PDXs) were characterized by Western blot for their expression of GR, ER and AR. Proliferation assays were performed in cell lines with a dose-response of a synthetic glucocorticoid, dexamethasone. Subsequently, we performed xenograft studies in cell line xenografts (CDXs) and PDXs. GR was ubiquitously expressed in various cell lines and PDX models. Dexamethasone reduced cell proliferation in ER-positive models, while it increased the proliferation in ER-negative cell lines, suggesting that the GR might have a mechanism of action that is similar to AR. GR antagonist, mifepristone, reversed the effects of dexamethasone. CDX studies in an ER-positive cell line, MCF7 suggested that dexamethasone (10 mpk) inhibited the tumor growth significantly that was better than the standard of care fulvestrant. This result was confirmed in an ER positive PDX model HCI013. Study by Obradović et al, indicates that glucocorticoids have an oncogenic role and furthermore promote breast cancer metastasis in TNBC model thereby validating the subtype and context dependent role of the glucocorticoid receptor. We are currently exploring the mechanism of GR’s context-dependent tumor suppressor and oncogenic role in distinct breast cancer subtypes.

Citation Format: Wendy Effah, Suriyan Ponnusamy, Sarah Asemota, Yekta Khosrosereski, Ramesh Narayanan. Context dependent role of glucocorticoids in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6281.
Abstract 6285: Defining androgen receptor function and the effects of PARP inhibition in BRCA1/2-deficient vs. proficient prostate cancer
Authors: Traphagen Nicole A. , Wheeler Esme , Li Rong , Qiu Xintao , Brown Myles
Published: March 2024

Mutation or loss of the tumor suppressor genes BRCA1 and BRCA2 promotes the development of aggressive prostate cancer with poor outcomes and a high risk of metastatic relapse. Approximately 15% of metastatic prostate tumors are BRCA1/2-deficient. These tumors are frequently resistant to standard-of-care treatments that block androgen receptor (AR) function, although the mechanisms underlying this effect remain unclear. PARP inhibitors are synthetically lethal with BRCA1/2-deficiency and are commonly used as a late-line treatment in metastatic prostate cancer; however, both preclinical and clinical data have provided conflicting results on whether PARP inhibitors can be effective in BRCA1/2-proficient prostate tumors. BRCA1, BRCA2, PARP-1, and PARP-2 all have proposed roles in regulating transcription and the chromatin landscape. We therefore hypothesized that loss of BRCA1, BRCA2, and/or inhibition of PARP modulates AR function through changes in global chromatin structure and transcriptional pathways. We used patient-derived xenograft models and next-generation sequencing techniques to profile AR function in BRCA1/2-deficient vs. proficient prostate tumors. We found that loss of BRCA1 and BRCA2 is associated with chromatin accessibility changes, and differentially accessible regions correlate with DNA binding motifs for AR and AR cofactors. ChIP-seq for AR in these tumors demonstrated that BRCA1/2-deficiency is associated with an altered AR cistrome and a global decrease in AR genomic binding. Corresponding RNA-seq data confirmed a downregulated androgen response in BRCA1/2-deficient tumors. In cell line models, inhibition of PARP1 and PARP1/2 inhibited AR-driven transcription. Combining PARP inhibition with either androgen deprivation or the AR antagonist enzalutamide dampened the growth-inhibitory effects of PARP inhibitor treatment. Together, these results indicate that BRCA1/2-deficiency and PARP inhibition modulate AR function in prostate cancer.

Citation Format: Nicole A. Traphagen, Esme Wheeler, Rong Li, Xintao Qiu, Myles Brown. Defining androgen receptor function and the effects of PARP inhibition in BRCA1/2-deficient vs. proficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6285.
Abstract 630: The role of AKT pathway in β-catenin mutated hepatocellular carcinoma
Authors: Wang Xue , Chen Xin
Published: March 2024

The Wnt/β-catenin and PI3K/AKT pathways are pivotal contributors to the development of the malignant phenotype in cancer. The activated Wnt/β-catenin pathway synergizes with various signaling cascades to propel HCC formation, operating through its downstream effectors. It has been well-established that the mammalian target of rapamycin complex 2 (mTORC2) serves as an upstream regulator of AKT, orchestrating the phosphorylation and activation of AKT. Once activated, AKT phosphorylates and inhibits canonical downstream targets like forkhead box O protein (FOXO) and tuberous sclerosis complex 2 (TSC2). Studies indicated that AKT-mediated phosphorylation of TSC2 activates mTORC1, subsequently suppressing autophagy. However, there are limited data exist on the interplay between Wnt/β-catenin and AKT pathways in HCC. In this study, we sought to explore the role of the AKT pathway in β-catenin activated HCC tumors. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of β-catenin (N90-β-catenin) and c-MET in the livers of mice to establish the HCC model (c-Met/β-catenin model). Notably, the knockout of Rictor, a component of the mTORC2 complex, significantly impeded tumor growth, underscoring the pivotal role of AKT in c-Met/β-catenin tumor development. AKT1 and AKT2 are the two main isoforms expressed in the liver. We determined that both AKT1 and AKT2 isoforms are critical for c-Met/β-catenin tumor growth. Intriguingly, simultaneous knockout of Rictor and activation of mTORC1 in the c-Met/β-catenin model resulted in delayed tumor growth, indicating that mTORC1 is not the sole downstream determinant of AKT signaling during c-Met/β-Catenin tumor development. Furthermore, overexpression of the activated FOXO1 (FOXO1AAA) significantly delayed the tumor growth, which highlighted the importance of FOXO1. Given the limited understanding of FOXO1 in HCC, we extended our investigation on FOXO1 to human HCC cell lines. Overexpression of FOXO1AAA notably delayed tumor growth in vitro. RNAseq analysis of FOXO1AAA-overexpressed human HCC cell lines revealed a significant upregulation of "metabolic pathways", suggesting metabolic reprogramming post FOXO1 activation. This study, for the first time, unveils the role of the AKT pathway in β-catenin-mutated HCC growth and highlights the potential therapeutic value of targeting the AKT pathway in patients with β-catenin-mutated HCC.

Citation Format: Xue Wang, Xin Chen. The role of AKT pathway in β-catenin mutated hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 630.
Abstract 6316: Exploring the resistant mechanism of CAR T cell therapy targeting CLDN18.2 by single-cell sequencing
Authors: Ma Mingyang , Qi Changsong , Liu Chang , Jiang Lei , Tao Min , Liu Dan , Zhang Panpan , Peng Zhi , Zhang Xiaotian , Gong Jifang , Chen Yang , Zheng Chunhong , Deng Mi , Shen Lin
Published: March 2024

Chimeric antigen receptor T cell (CAR T) therapy has achieved remarkable success in treating hematological malignancies but still faces overwhelming challenges for solid tumors. Our previous clinical trial (NCT03874897) has demonstrated promising results in patients with advanced CLDN18.2-positive gastric after receiving CT041 CAR T cell product. Though the disease control rate (DCR) reached 73% in 37 patients, it was unclear who would benefit from this therapy. Here in this study, we dynamically collected peripheral blood and ascites samples from 5 patients in this clinical trial before and at 3 and 7 days after the infusion of CT041, respectively. Utilizing single-cell sequencing analysis, we found that patients with a high proportion of naive-like T cells were more likely to benefit from CT041. Furthermore, we discovered that the high expression of CLDN18 in the ascites epithelial cells was correlated with a favorable prognosis, while ascites epithelial cells showing high MYC expression and the strong interaction between tumor cells and T cells were identified as adverse prognostic factors for CT041 treatment. These findings may provide theoretical evidence for subsequent screening of CAR T cell therapy benefitable population and improving the efficacy of CAR T cell therapy.

Citation Format: Mingyang Ma, Changsong Qi, Chang Liu, Lei Jiang, Min Tao, Dan Liu, Panpan Zhang, Zhi Peng, Xiaotian Zhang, Jifang Gong, Yang Chen, Chunhong Zheng, Mi Deng, Lin Shen. Exploring the resistant mechanism of CAR T cell therapy targeting CLDN18.2 by single-cell sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6316.
Abstract 6347: To develop a tumor selective INF-γ-Yervoy fusion antibody to increase the therapeutic effect and safety of current Yervoy therapy in cancer
Authors: Chuang Chih-Hung
Published: March 2024

Introduction: Immune checkpoint inhibitor therapy is a highly promising strategy for clinical treatment of cancer. Among these inhibitors, yervoy stands out for its ability to induce cytotoxic T cell proliferation and activation by binding to CTLA-4. However, FDA has reported that yervoy would induce the over-activation of immune cells and cause the serious side effects. In additional, the therapeutic efficacy and application of Yervoy were still limited by the MHC expression of patient's tumor.

Strategy: Therefore, we have developed a protease specific pro INF-γ-Yervoy to increase the therapeutic effect and safety of current immune checkpoint blockade therapy in cancer. We used the MMP-2/9 protease substrate as a linker between INF-γ and Yervoy. Using the concept of spatial shielding, IFNγ and Yervoy can block their functions from each other. Once the INF-γ-Yervoy arrives to the tumoral region, the INF-γ-Yervoy will be removed from Hinge Yervoy by the over-expressed MMPs protease locally so that the Yervoy will recruit its binding ability in order to increase the drug selectivity and improve the side effects. The INF-γ also can increase the MHC I expression in tumor to overcome the MHC-limitation of Yervoy.

Result: On activation by MMP, IFNγ-yervoy not only restored yervoy’s binding ability but also released biologically active IFNγ. This finding confirms the reliability of IFNγ-yervoy’s functionality. In vivo studies demonstrated that IFNγ-yervoy enhances the therapeutic effect of yervoy against colorectal cancer by increasing CD8+ and CD4+ lymphocyte infiltration into the tumor area and inducing MHC-I expression in tumor cells. Mice treated with IFNγ-yervoy showed higher survival rates and body weight, as well as lower CD4+ and CD8+ lymphocyte activation rates in the blood and reduced organ damage.

Conclusion: These results provide strong evidence that tumor-selective IFNγ-yervoy can improve the effectiveness of yervoy while reducing its side effects. It is likely that future immunotherapies would rely on such antibodies to activate local cancer cells or immune cells, thereby increasing the therapeutic effectiveness of cancer treatments and ensuring their safety. Keyword: Immune checkpoint inhibitor therapy, IFNγ, yervoy, rectal cancer, CTLA4

Citation Format: Chih-Hung Chuang. To develop a tumor selective INF-γ-Yervoy fusion antibody to increase the therapeutic effect and safety of current Yervoy therapy in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6347.
Abstract 6355: Urokinase plasminogen activator receptor-associated protein (uPARAP) is overexpressed in human glioblastomas and is a therapeutic target for antibody-drug conjugates (ADC)
Authors: Gregersen Ida , Løkke Cecillie R. , Lange Jette B. , Barkholt Pernille , Côme Christophé , Broberg Tina , Petersen Marianne M. , Lynch Carmel M. , Mumberg Dominik , Behrendt Niels , Engelholm Lars H.
Published: March 2024

Background: Glioblastoma Multiforme (GBM), primarily arising in the brain's frontal and temporal lobes, is a devastating malignancy originating from astrocytes. Despite treatment advancements, the prognosis remains bleak, with a median survival of 15-18 months and a 10% five-year survival rate.Our study explored a novel targeted approach to combat this challenge. uPARAP is an endocytic receptor expressed at low levels on selected mesenchymal cell types but highly overexpressed on the majority of Soft-Tissue- and Bone Sarcomas. It internalizes fragments of collagen and transfers them to the lysosome for degradation.

Methods & Results: First, we analyzed formalin-fixed paraffin-embedded (FFPE) tumors from GBM patients through immunohistochemistry (IHC) staining. Interestingly, a majority of the samples showed strong but varying uPARAP expression. In parallel, we detected and quantified uPARAP receptor expression in GBM cell lines by flow cytometry, underscoring its potential as a therapeutic target.Next, we generated an antibody-drug conjugate (ADC) linked to monomethyl auristatin E (MMAE), a potent antimitotic agent. Confocal microscopy showed rapid internalization of the ADC in several GBM cell lines in vitro. Cellular cytotoxicity was demonstrated in Incucyte and Alamar Blue in vitro assays. Finally, anti-tumor activity of the uPARAP-ADC was studied in a GBM patient-derived xenograft (PDX) model in vivo, where it resulted in significant tumor regression with a tumor growth inhibition (TGI) of 92 and 97% at 3 and 6 mg/kg, respectively.

Conclusions: Our findings show that uPARAP is overexpressed on GBM tumors and that a uPARAP-targeted ADC has anti-tumor activity in a PDX model of GBM. This research provides initial evidence that uPARAP-ADCs could become a novel treatment option for this highly aggressive disease.

Citation Format: Ida Gregersen, Cecillie R. Løkke, Jette B. Lange, Pernille Barkholt, Christophé Côme, Tina Broberg, Marianne M. Petersen, Carmel M. Lynch, Dominik Mumberg, Niels Behrendt, Lars H. Engelholm. Urokinase plasminogen activator receptor-associated protein (uPARAP) is overexpressed in human glioblastomas and is a therapeutic target for antibody-drug conjugates (ADC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6355.
Abstract 6366: The triple-specific antibody KA-3009 against EGFR, B7H3, and 4-1BB specifically enhances T cell activity within the tumor
Authors: Wu Guojin , Liu Tongtong , Liang Jiabei , Peng Hao , Hao Feng , Ning Jinying
Published: March 2024

Cancer immunotherapy with 4-1BB agonists faces limited further clinical development due to dose-limiting toxicity. Moreover, EGFR and B7H3 are highly expressed in various solid tumors, as well as in different normal tissues. In this study, we developed a trispecific antibody, KA-3009, targeting EGFR, B7-H3, and human 4-1BB, with the aim of restricting 4-1BB stimulation within tumors. KA-3009 was developed based on our common light chain bispecific antibody discovery platform and nanobody antibody discovery platform using knob-into-hole technology. It is in IgG1 format with mutations in the Fc domain to eliminate ADCC, ADCP, and CDC activity. Featuring two identical nanobodies at the C-terminal against 4-1BB, KA-3009 binds to EGFR and B7H3 respectively with two Fabs at the N-terminal, sharing an identical light chain. KA-3009 exhibits low avidity binding to tumor cells expressing only EGFR or B7H3, while binding to tumor cells co-overexpressing EGFR and B7H3 with increased avidity by more than ten times. Additionally, KA-3009 binds to 4-1BB expressed on 293T cells and activated human T cells. In the presence of a low concentration of CD3 antibody to stimulate T cell activation, co-culture of tumor cells co-expressing EGFR and B7H3 with primary T cells isolated from human PBMC enhances T cell activation and proliferation. In PBMC humanized immunodeficient mouse tumor models, KA-3009 effectively suppresses the growth of NCI-H1568 tumors expressing both EGFR and B7H3 but does not affect the growth of MS751 and NCI-H661 tumors, which only express EGFR or B7H3, respectively. Moreover, KA-3009 has a half-life of more than five days in the blood of Balb/c mice. Finally, we assessed the developability of this antibody with different treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature, and KA-3009 shows neither aggregation nor degradation. In conclusion, the triple-specific antibody KA-3009, developed with our common light chain antibody discovery platform and nanobody antibody discovery platform, is a promising pre-clinical candidate drug for the treatment of different solid cancers that co-overexpress EGFR and B7H3.

Citation Format: Guojin Wu, Tongtong Liu, Jiabei Liang, Hao Peng, Feng Hao, Jinying Ning. The triple-specific antibody KA-3009 against EGFR, B7H3, and 4-1BB specifically enhances T cell activity within the tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6366.
Abstract 6429: Critical role of elevated TMEM176B gene expression in humanized patient-derived xenografts mouse models of triple negative breast cancer
Authors: Her Yujeong , Yun Jihui , Heo Woohang , Kim Jong-Il , Lee Han-Byoel , Han Wonshik , Moon Hyeong-Gon
Published: March 2024

Objective: In the cancer research, a highly promising in vivo platform for personalized treatment involves the adoption of an advanced approach—specifically, the grafting of patient-derived xenografts (PDX) onto humanized mouse models. These models capture distinctive aspects of interaction with the human immune system and the tumor microenvironment (TME). In this study, we have engineered humanized triple-negative breast cancer (TNBC) PDX models to observe alterations in differentially expressed genes, thereby enhancing our understanding of the intricacies of personalized treatment in this context.

Method: We utilized two TNBC PDX models established for the humanized models, where CD34+ hematopoietic stem cells were transplanted into immunodeficient mice. RNA sequencing was performed to identify altered gene expression in humanized PDX models compared to NSG PDXs. Additionally, we generated stable cell lines overexpressing TMEM176B in MDAMB231 and Hs578T cells, and we knocked down TMEM176B in 4T1 cells using recombinant techniques

Results: In the humanized model, the growth of PDX tumors did not show a significantly faster or markedly different rate compared to previous studies. However, upon conducting RNA sequencing in the PDX model, the expression of the TMEM176B gene was consistently downregulated. Consequently, we transfected and observed that the increased expression of TMEM176B in MDAMB231 and Hs578T cell lines resulted in enhanced cell proliferation in vitro. Transplanting MDAMB231 cells overexpressing TMEM176B into athymic nude mice resulted in accelerated tumor growth. The study further investigates the expression of TMEM176B in an immune-competent environment by knocking down the gene in 4T1 cells or transplanting the control group into a balb/c mouse model. Tumors with TMEM176B knockdown exhibited inhibited growth, increased CD3+ T cell infiltration, and significant inhibition when treated with anti-PD-L1 compared to the control group.

Conclusion: This research demonstrates the utility of PDX models, particularly humanized PDX models, in studying the complexities of cancer biology and the tumor microenvironment. The findings regarding TMEM176B and its role in tumorigenesis and immune responses in triple-negative breast cancer provide valuable insights for further research and potential therapeutic interventions.

Citation Format: Yujeong Her, Jihui Yun, Woohang Heo, Jong-Il Kim, Han-Byoel Lee, Wonshik Han, Hyeong-Gon Moon. Critical role of elevated TMEM176B gene expression in humanized patient-derived xenografts mouse models of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6429.
Abstract 6477: Modulators of hospital length of stay after intracranial neoplasm excision
Authors: Hsiung Jayla , Taneja Kamil , Ladehoff Lauren , Patel Karan , Toloza Eric
Published: March 2024

Purpose: Intracranial neoplasms are the leading cause of cancer death among men under 40 years old. A major part of the standard of care is excision of the tumor, but there is a lack of robust large-patient studies of outcomes for these patients.

Methods: The Nationwide Inpatient Sample was queried from 2018 to 2020 to identify patients with an intracranial neoplasm. Multivariable linear regression with patient- and hospital-level characteristics as covariates was used to identify factors associated with long hospital stays.

Results: From 2018 to 2020, there were 97,795 hospital encounters with a primary diagnosis of an intracranial neoplasm. Of these patients, 64.6% underwent a tumor excision procedure during their hospital visit. The average age of these patients undergoing excision was 57.80.2 years. Most patients were male (58.6%), white (79.1%), private insurance beneficiaries (46.3%), and of the highest income quartile (26.8%). These patients most commonly presented to hospitals in the West (34.9%), with a medium number of beds (36.4%), and presented to rural hospitals (41.7%). The most common locations of these neoplasms were the frontal (29.1%), temporal (21.3%), and parietal (12.1%) lobes. Neoplasms in more than one location in the brain occurred in 4.5% of patients. Multivariable linear regression identified the following factors as significantly associated with longer hospital stays: identifying as Black (beta=2.08; p<0.001), identifying as an Asian or Pacific Islander (beta=1.22; p=0.017), being a Medicaid beneficiary (beta=2.25; p<0.001), and being uninsured (beta=1.63; p<0.001). Having a neoplasm in the cerebral ventricle (beta=4.93; p<0.001), cerebellum (beta=2.81; p<0.001), or brain stem (beta=3.45; p<0.001), and having multiple neoplasms (beta=1.85; p=0.008) were also associated with longer hospital stays. Interestingly, having a parietal (beta=-0.54; p=0.011) or occipital (b=-0.99; p<0.001) lobe neoplasm was associated with shorter hospital stays.

Conclusion: Intracranial neoplasms are deadly condition and excision of tumor is a important life-saving treatment option. In this study, we demonstrated that location of neoplasm, race, and insurance status are significant modulators of the length of a hospital stay after surgery.

Citation Format: Jayla Hsiung, Kamil Taneja, Lauren Ladehoff, Karan Patel, Eric Toloza. Modulators of hospital length of stay after intracranial neoplasm excision [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6477.
Abstract 6489: Paradoxical activation of kinases occurs directly with ATP-competitive kinase inhibitors and is observable biochemically at physiologically relevant drug concentrations
Authors: Loch Christian M. , Eason Mia , Liang Shuguang , Goupil Alison , Acinapura Anthony , Purcell Jamie , Domiguez Maia
Published: March 2024

We sought to screen all FDA approved, cancer-indicated, kinase targeting drugs using the gold standard radiometric biochemical activity assay (HotSpot) against the largest collection of kinases in the world (>100 drugs against 735 total kinases including 395 wild type and 340 mutants). One of these drugs, mitapivat, is an activator of Pyruvate Kinase. Here, we show mitapivat-driven biochemical inhibition of several, but not all, mutants of c-KIT. We also observed the equal and opposite phenomenon whereby many ATP-competitive kinase inhibitors demonstrate biochemical activation of certain kinases, several of which seem particularly susceptible to the effect. Previous work by other investigators (at Genentech and the Rosen lab, MSKCC) demonstrated that PLX4720 (precursor to Vemurafenib) paradoxically activated wild type BRAF, but not BRAF mutants, by driving dimerization of the kinase in drug concentrations insufficient to inhibit both members of the dimerized pair. Our data suggests this phenomenon may be more widespread than previously appreciated and fully observable in vitro. Such paradoxical activation could be driving both toxicities and inefficacy against cancers expressing some of these activatable kinases. This information could suggest novel opportunities for combination therapy and improve outcomes by contraindicating certain medication from use based on tumor expression profile.

Citation Format: Christian M. Loch, Mia Eason, Shuguang Liang, Alison Goupil, Anthony Acinapura, Jamie Purcell, Maia Domiguez. Paradoxical activation of kinases occurs directly with ATP-competitive kinase inhibitors and is observable biochemically at physiologically relevant drug concentrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6489.
Abstract 6516: Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound
Authors: White Erin , Wei Hong-Jian , Fernández Ester Calvo , Minns Hanna , Pavisic Jovana , Gartrell Robyn , Wu Cheng-Chia , Califano Andrea , Obradovic Aleksandar
Published: March 2024

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, located in the brainstem, and traditionally thought to be immune-excluded with no significant infiltration by cytotoxic T-cells, and therefore not amenable to immunotherapies. Focused ultrasound technology (FUS) for drug delivery is being investigated for DIPG in both preclinical and clinical trial settings. In this study, we sought to study the effects of FUS and radiation therapy (RT) on immune cell infiltration in DIPG tumors to assess their ability to alter the immune microenvironment and potentially enhance combination immunotherapies. Specifically, we generated single-cell RNAseq profiles for 27,780 cells from syngeneic orthotopic DIPG tumors (KPAP cell line containing H3K27M mutation, ATRX and P53 loss, and PDGFRA overexpression) after 14 days of tumor growth across the following treatment arms: vehicle control, FUS, and RT (n=2 each group). FUS was applied by 4-point sonication around the tumor with a pressure of 0.6 Mpa at days 8 and 11 post tumor implantation (PTI). RT was administered in fractions of 3 Gy daily from days 8 to 11 PTI (12Gy total). We leveraged a set of algorithms for network-based inference of regulatory protein activity to infer a set of context-matched gene regulatory networks from which protein activity can be inferred by expression of downstream targets. By this approach, we identified distinct clusters of immune cells segregating by cell type as inferred by singleR and activity of known lineage markers. Strikingly, we found that both RT and FUS significantly depleted a TGFb-expressing population of immune-suppressive macrophages as compared to vehicle controls. Importantly, FUS only additionally increased T-cell infiltration into the tumor micro-environment over three-fold as compared to vehicle controls, which was not observed in response to RT. Further protein validation is underway. This has significant implications for therapeutic combinations of FUS-mediated drug delivery with immune checkpoint inhibitors in DIPG, such that in addition to drug delivery, FUS-mediated immune modulatory effects may provide more proinflammatory response to enhance immunotherapy.

Citation Format: Erin White, Hong-Jian Wei, Ester Calvo Fernández, Hanna Minns, Jovana Pavisic, Robyn Gartrell, Cheng-Chia Wu, Andrea Califano, Aleksandar Obradovic. Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6516.
Abstract 6543: A map of osteosarcoma cell architecture reveals convergence of pediatric cancer mutations on protein assemblies
Authors: Hu Mengzhou , Schaffer Leah V. , Huttlin Edward L. , Qian Gege , Latham Andrew , Pal Abantika , Vaites Laura Pontano , Le Trang , Qin Yue , Churas Christopher , Pratt Dexter , Bachelder Robin , Zage Peter , Echeverria Ignacia , Šali Andrej , Harper J. Wade , Gygi Steven P. , Lundberg Emma , Ideker Trey
Published: March 2024

Pediatric tumors are characterized by a low incidence of somatic mutations, which occur in different genomic patterns from adult cancers. For these reasons, interpreting the functional impact of mutations in pediatric cancer patients has remained difficult. One approach to interpreting rare coding mutations is to analyze the convergence of these mutations on larger structural and functional units, such as protein complexes, super assemblies, biomolecular condensates, and organelles. Towards this aim, we created a cancer cell map in the osteosarcoma cell line based on proteome-wide integration of protein interactions and immunofluorescent imaging in U-2 OS pediatric bone cancer cells. This multiscale integrated cell map of U-2 OS cell organizes 5,254 proteins into 270 protein assemblies. Using this multiscale cell map, we analyze 772 genomes from 18 cancer types reported in the DKFZ pan-pediatric cancer study to identify 25 protein assemblies in the map under mutational pressure. This analysis reveals mutated protein assemblies involved with chromatin maintenance and remodeling as well as cell integrity and mobility. For example, we identify a Collagen Biosynthesis and Remodeling Complex, as mutated in 9 cancer types and 15 patients in the dataset, indicating extracellular matrix remodeling is associated with pediatric cancer progression. We also identify a putative biological condensate, which we call the Nuclear Transcription Suppression Complex (NTSC). This protein assembly is found mutated in 20 patients across 10 cancer types, implicating intrinsically disordered proteins in development of pediatric cancer. Overall, this work illustrates how a global multiscale map of cell architecture enables a mechanistic understanding of clinical patient genomes.

Citation Format: Mengzhou Hu, Leah V. Schaffer, Edward L. Huttlin, Gege Qian, Andrew Latham, Abantika Pal, Laura Pontano Vaites, Trang Le, Yue Qin, Christopher Churas, Dexter Pratt, Robin Bachelder, Peter Zage, Ignacia Echeverria, Andrej Šali, J. Wade Harper, Steven P. Gygi, Emma Lundberg, Trey Ideker. A map of osteosarcoma cell architecture reveals convergence of pediatric cancer mutations on protein assemblies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6543.
Abstract 655: Fungal extract screening reveals malformin C to preferentially kill glioblastoma stem-like cells via concerted induction of proteotoxic stress and autophagic flux blockade
Authors: Phillips Emma L. , van Enk Sizèd , Kildgaard Sara , Schlue Silja , Göttmann Mona , Jennings Victoria , Bethke Frederic , Müller Gabriele , Herold-Mende Christel , Pastor-Flores Daniel , Schneider Martin , Helm Dominic , Larsen Thomas Ostenfeld , Goidts Violaine
Published: March 2024

Glioblastoma is a highly aggressive brain tumor for which there is no cure. The dire prognosis of this disease is largely attributable to a high level of heterogeneity, including the presence of a sub-population of tumor initiating glioblastoma stem-like cells (GSCs), which are refractory to chemo- and radiotherapy. Here, in an unbiased marine-derived fungal extract screen, together with bioguided dereplication based on high resolution mass spectrometry, we identified malformin C to preferentially induce cell death in patient-derived GSCs, and explore the potential of this cyclic peptide as a therapeutic agent for glioblastoma. Malformin C significantly reduced tumor growth in an in vivo xenograft model of glioblastoma. Using transcriptomics and chemoproteomics, we found that malformin C binds to many proteins leading to aggregation, and rapidly induces unfolded protein response, including autophagy, in GSCs. Malformin C also appears to accumulate in lysosomes, disrupting autophagic flux and driving cells to death. Supporting this, malformin C synergizes with chloroquine, an inhibitor of autophagy. Strikingly, we observed that autophagic flux is differentially regulated in GSCs compared with normal astrocytes. The sensitivity of GSCs to malformin C highlights the relevance of proteostasis and autophagy as a therapeutic vulnerability in glioblastoma.

Citation Format: Emma L. Phillips, Sizèd van Enk, Sara Kildgaard, Silja Schlue, Mona Göttmann, Victoria Jennings, Frederic Bethke, Gabriele Müller, Christel Herold-Mende, Daniel Pastor-Flores, Martin Schneider, Dominic Helm, Thomas Ostenfeld Larsen, Violaine Goidts. Fungal extract screening reveals malformin C to preferentially kill glioblastoma stem-like cells via concerted induction of proteotoxic stress and autophagic flux blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 655.
Abstract 6584: Aberrant TRIM7 expression potentiates RACO-1 mediated proliferation and dysregulated interferon responsiveness in the setting of anti-PD-1 acquired resistance in cancer
Authors: Nguyen Thuy-Ai Tran , Morra Marc , Li Kevin , Bhatt Ulhas , Mwangi Joseph , Rivera-Molina Yisel , Oien Nathan , Medina Julio , Schreiber Taylor H. , Fromm George
Published: March 2024

While checkpoint inhibitor treatment has extended the survival of many cancer patients, most initial responders will later develop disease progression through a variety of poorly understood acquired resistance mechanisms (CPI-AR). One such recently described mechanism involves the transcriptional hyperactivation of genes associated with interferon (IFN) signaling, JAK/STAT, and antigen processing/presentation pathways. Aberrant IFN regulation in the setting of AR suggests that tumor cell intrinsic mechanisms have conferred a growth advantage despite the presence of immune-mediated pressure unleashed by PD-1/L1 antibody blockade. Analysis of transcripts progressively upregulated during acquisition of AR in vivo and transcripts upregulated by IFN exposure of isolated AR cell lines in vitro identified the E3 ubiquitin ligase TRIM7 as a candidate mediator of the AR phenotype. A specific pocket on the PRY/SPRY domain of TRIM7 has been shown to bind viral peptides, resulting in ubiquitination and degradation as a mechanism to mitigate viral propagation. Here we describe the evaluation of small molecule inhibitors (SMI) designed to the TRIM7 PRY/SPRY viral-pocket domain, where binding specificity was confirmed using mass spec analysis. These SMI competitively inhibited TRIM7 mediated stabilization of RACO-1, resulting in decreased cancer cell proliferation which was associated with altered c-Jun/AP-1 transcription. TRIM7 SMI also disrupted STING and MAVS ubiquitination and restored IFN responsiveness and sensitivity to PD-1 inhibitor treatment. Finally, given the position of TRIM7 downstream of growth factor receptor and kras signaling, combination of TRIM7 inhibitors with kras, braf, and/or MEK inhibitors was shown to additively suppress proliferation of tumor cells with kras mutations. These results provide evidence that upregulation of TRIM7 mediates both a pro-proliferative and IFN resistant phenotype in the setting of CPI-AR, and that inhibition of TRIM7 with small molecule inhibitors may prevent or reverse AR to PD-1/L1 blockade.

Citation Format: Thuy-Ai Tran Nguyen, Marc Morra, Kevin Li, Ulhas Bhatt, Joseph Mwangi, Yisel Rivera-Molina, Nathan Oien, Julio Medina, Taylor H. Schreiber, George Fromm. Aberrant TRIM7 expression potentiates RACO-1 mediated proliferation and dysregulated interferon responsiveness in the setting of anti-PD-1 acquired resistance in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6584.
Abstract 6599: 3D genomic analysis reveals novel enhancer hijacking mechanisms caused by complex structural alterations that drive oncogene overexpression
Authors: Mortenson Katelyn L. , Dawes Courtney , Wilson Emily R. , Patchen Nathan E. , Johnson Hailey , Gertz Jason , Bailey Swneke D. , Liu Yan , Varley Katherine E. , Zhang Xiaoyang
Published: March 2024

Enhancer hijacking, caused by structural alterations on chromosomes as well as extrachromosomal DNA (ecDNA), is a common cancer driver event. The complexity and ubiquity of structural alterations in cancer genomes make it difficult to identify enhancer hijacking with genome sequencing alone. Here we describe a 3D genomics-based analysis called HAPI (Highly Active Promoter Interactions) to characterize enhancer hijacking caused by various types of structural alterations. Applying HAPI analysis to HiChIP data from 34 cancer cell lines, we identified novel enhancers hijacked through chromosomal rearrangements to activate both known and potentially novel oncogenes such as MYC, CCND1, ETV1, CRKL, and ID4, which we validated using CRISPRi assays and RNA-seq analysis. Furthermore, we found that ecDNAs often contain multiple oncogenes from different chromosomes, which cause nested enhancer hijacking among them. For instance, we found that MYC ecDNAs relocate additional oncogenes from other chromosomes such as CDX2, ERBB2, or CD44 near the MYC locus, co-opting MYC’s enhancers for their overexpression, which we validated using dual-color DNA FISH and CRISPRi assays. This multiple oncogenes-involved enhancer hijacking mechanism may suggest novel therapeutic strategies such as targeting either the co-opting oncogenes or the hijacked enhancers for ecDNAs. Our study provides a robust strategy to detect enhancer hijacking events using our publicly available HAPI analysis tool and reveals novel mechanisms underlying oncogene activation caused by chromosomal and extrachromosomal structural alterations.

Citation Format: Katelyn L. Mortenson, Courtney Dawes, Emily R. Wilson, Nathan E. Patchen, Hailey Johnson, Jason Gertz, Swneke D. Bailey, Yan Liu, Katherine E. Varley, Xiaoyang Zhang. 3D genomic analysis reveals novel enhancer hijacking mechanisms caused by complex structural alterations that drive oncogene overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6599.