bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6193: TWIST2 promotes tumor progression and represses myogenic differentiation in rhabdomyosarcoma
Authors: Shah Akansha , Guo Lei , Jaichander Priscilla , Chen Kenian , Hernandez Karla Cano , Xu Lin , Bassel-Duby Rhonda , Olson Eric , Liu Ning
Published: June 2022

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma of myoblast origin. RMS tumors are unable to terminally differentiate into syncytial muscle despite elevated expression of the muscle master regulators, MyoD and Myf5. Fusion-positive RMS (FP-RMS) and fusion-negative RMS (FN-RMS) have emerged as the two major RMS subtypes. The pathogenesis of FP-RMS is commonly defined by the balanced translocation of chromosomes 2 and 3 resulting in the misexpression of a chimeric PAX3/7-FOXO1 oncoprotein that derails normal differentiation pathways. However, the molecular basis of FN-RMS is poorly understood as single nucleotide variations in genes like KRAS, NRAS, and TP53 have limited recurrence (less than 10% of all patient tumors). In contrast, DNA copy number variations are highly recurrent and the genes they encode represent vulnerabilities in FN-RMS. Strikingly, we discovered that 72% of FN-RMS patient tumors harbor copy number amplifications at loci of TWIST1, TWIST2, or both, resulting in the upregulation of TWIST gene expression. We demonstrated that depletion of TWIST2 using a doxycycline inducible shRNA system caused cell cycle arrest and increased myogenic differentiation in xenograft models of FN-RMS. To interrogate whether TWIST2 can directly regulate the expression of these genes, we performed RNA-seq and ChIP-seq for TWIST2, H3K27Ac, and MYOD in TWIST2 knockdown and control FN-RMS cells. Utilizing this genome-wide data of chromatin binding, we comprehensively demonstrated that TWIST2 competes with MYOD preferentially at a subset of enhancers required for myogenic differentiation, causing repression of these loci. Interestingly, while MYOD binding is gained at myogenic loci in the absence of TWIST2, binding of MYOD is not lost at growth-promoting, oncogenic enhancers despite a decrease in their associated gene expression. This suggests that TWIST2 interacts with a different slew of transcription factors or chromatin regulators to activate gene expression. To interrogate this possibility, we are characterizing the TWIST2 protein interactome in FN-RMS cells using BioID as well as motifs enriched at TWIST2-regulated peaks. By studying TWIST2-associated pathways that affect RMS growth and differentiation, we see an opportunity to unlock novel therapeutic strategies for RMS.

Citation Format: Akansha Shah, Lei Guo, Priscilla Jaichander, Kenian Chen, Karla Cano Hernandez, Lin Xu, Rhonda Bassel-Duby, Eric Olson, Ning Liu. TWIST2 promotes tumor progression and represses myogenic differentiation in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6193.
Abstract 6200: MANGO: pipeline for identifying genomic translocations with key biological significance in inflammatory breast cancer
Authors: Morales-Rivera Carlos S. , Driscoll Emilia R. , Peterson-Peguero Esther A.
Published: June 2022

Genomic instability in cancer is essential for aberrant gene expression and translocation events that potentiate oncogenesis. Thus gene fusions, nonsense mutations and gene deregulation are key contributors to oncogenic phenotypes. Certain genomic translocations have been linked to cancer subtypes, such BCR—ABL1 for chronic myeloid leukemia (CML) serving as potential onco-biomarkers. Inflammatory breast cancer (IBC) is the rarest and most lethal subtype of breast cancer with a 5-year survival rate below 50%. Ethnic disparities for breast cancer exist; negative estrogen (ER) and progesterone (PR) hormone receptor status is more frequent in Hispanic and African American women. In addition, inflammatory breast cancer lacks defined molecular markers limiting its early onset detection which leads to poorer overall prognosis. Thus, we hypothesized that profiling IBC translocations, including its subtypes, show valuable insights to better understand this complex disease. We developed MANGO, a pipeline that leverages STAR, STAR-Fusion, FusionInspector, and Trinity to identify, visualize and assemble gene fusions from IBC sequencing data. Using the SUM149PT cell line as a model for IBC, we identified and experimentally validated a model fusion gene ZDHHC5—EPB41L5. Fusion unaware sequence assemblies cross-validated the fusion and its multiple splice sites, one of which encodes a fusion peptide. Differential gene expression between SUM149PT and SUM190PT, an IBC cell line without evidence for the identified translocation, showed loss of expression of ZDHHC5 but no change in EPB41L5, suggesting the latter retained its expression in its new genomic localization. Furthermore, in-silico functional predictions for experimental results reveals an in-frame fusion peptide with transmembrane signals in EPB41L5. Genomic translocation mapping reveals triple-negative IBC diverges from traditional breast cancer translocation patterns. In short, MANGO serves as a powerful tool to identify biologically significant translocations involved in IBC and other cancer classifications.

Citation Format: Carlos S. Morales-Rivera, Emilia R. Driscoll, Esther A. Peterson-Peguero. MANGO: pipeline for identifying genomic translocations with key biological significance in inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6200.
Abstract 624: Classifying cGAS-STING activity links chromosomal instability with immunotherapy response in metastatic bladder cancer
Authors: Sokac Mateo
Published: June 2022

The cGAS-STING pathway serves a critical role in anti-cancer therapy. Particularly, response to immunotherapy is likely driven by both active cGAS-STING signaling that attracts immune cells, and by the presence of cancer neoantigens that presents as targets for cytotoxic T-cells. Chromosomal instability (CIN) is a hallmark of cancer, but also leads to an accumulation of cytosolic DNA that in turn results in increased cGAS-STING signaling. To avoid triggering the cGAS-STING pathway, it is commonly disrupted by the cancer cells, either through mutations in the pathway or through transcriptional silencing. Given its effect on the immune system, determining the cGAS-STING activation status prior to treatment initiation is likely of clinical relevance, but to date no robust method has been developed. Here, we used combined expression data from 2307 tumors from five cancer types from The Cancer Genome Atlas (TCGA) to define a novel cGAS-STING activity score based on eight genes with a known role in the pathway. Using unsupervised clustering, four distinct categories of cGAS-STING activation were identified. In multivariate models, the cGAS-STING active tumors show improved prognosis. Importantly, in an independent bladder cancer immunotherapy treated cohort, patients without cGAS-STING activation showed very limited response to treatment, while patients with strong cGAS-STING activation showed improved response and improved prognosis, particularly among patients with high CIN and high numbers of neoantigens. In a multivariate model, a significant interaction was observed between CIN, neoantigens, and cGAS-STING activation. Together, this supports a role of cGAS-STING activity as a predictive biomarker in combination with measures of CIN and neoantigens for the application of immunotherapy.

Citation Format: Mateo Sokac. Classifying cGAS-STING activity links chromosomal instability with immunotherapy response in metastatic bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 624.
Abstract 6241: Macc1 regulates breast cancer progression and metastasis via remodeling tumor immune microenvironment
Authors: Son Hye-Youn , Heo Woo Hang , Li Song Bin , Quan Mingji , Her Yu Jeong , Lee Kyu-Jin , Kim Ju Hee , Lee Han-Byoel , Han Wonshik , Moon Hyeong-Gon
Published: June 2022

Although the established cancer cell lines exhibit less degrees of intratumoral heterogeneity when compared to human solid tumors, studies have suggested that, even within the same cancer cell line, substantial degree of heterogeneity may exist. In this study, we have performed single cell cloning experiment using 4T1 mouse mammary carcinoma cell line and compared the phenotypes of different clones. When compared to the parental 4T1 cell line, one clone (C12) showed markedly different phenotype by exhibiting less aggressive features in vitro and significantly slower tumor growth and metastasis in vivo. The tumor tissue of the C12 showed significant alteration in immune microenvironment as higher number of recruited immune cells including T and B cells. RNA sequencing of the C12 clones and parental 4T1 cells identified Macc1 as a significantly up-regulated gene in parental 4T1 cells. Metastasis-associated colon cancer 1 (MACC1) has been associated with poor prognosis and the promotion of metastasis within several types of cancer, especially colon cancer. The gene encoding the hepatocyte growth factor (HGF) receptor, MET, is a transcriptional target of MACC1. Next, we established Macc1-silenced 4T1 cells using the parental 4T1 cells and observed that Macc1-silencing resulted in a similar phenotype seen in the C12 clone; decreased cell proliferation and migration in vitro and slow tumor growth and metastasis in vivo. To mimic the metastasis in vitro, intravasation was determined as which tumor cells were co-cultured with HUVEC, human umbilical vein endothelial cells. We discovered tumor cell penetration into HUVEC cells was blocked in Macc1-silencing 4T1 group. According to CD45, CD3, and CD19 immunohistochemistry, we also defined immune cells, especially T and B cells, were highly infiltrated in Macc1-silencing tumor like as C12 clone tumor. These results were also reconstructed in EMT6, another TNBC mouse cell line model. In this study, we show that MACC1 can regulate tumor progression and metastasis by remodeling of immune microenvironment.

Citation Format: Hye-Youn Son, Woo Hang Heo, Song Bin Li, Mingji Quan, Yu Jeong Her, Kyu-Jin Lee, Ju Hee Kim, Han-Byoel Lee, Wonshik Han, Hyeong-Gon Moon. Macc1 regulates breast cancer progression and metastasis via remodeling tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6241.
Abstract 6243: RAR gamma interactome and cistrome guide the androgen receptor enhancer usage in prosate cancer
Authors: Wani Sajad A. , Gray Jaimie S. , Campbell Moray J.
Published: June 2022

Epigenetic mechanisms in prostate cancer (PCa) alter androgen receptor (AR) enhancer access resulting in re-wired AR signaling and cancer progression and/or therapy resistance. Previously, we established miR-96 targets RARγ and its coactivator TACC1, which in turn impacts AR signaling associated with PCa progression. In the current study we sought to define the RARγ complex, its interactions with the AR, and in turn how this controls gene expression. Increased RARγ levels through miR-96 treatments increased both ENZA antiproliferative sensitivity associated with an increase in G1 and G2/M. IMPACT-seq identified miR-96 recognition elements (MREs) in HPr1AR (~1950 at 24h) and LNCaP cells (~ 350) with ~ 150 shared. In parallel, m6A-Seq revealed that the epitranscriptome mark m6A differed significantly between cells. For example, m6A was more significantly enriched on TACC1 mRNA in LNCaP compared to HPr1AR and m6A-marked MRE resulted in genes being more signficantly down-regulated by miR96, including TACC1. RIME revealed that RARγ significantly interacts miR-96 differentially expressed transcripts (DETs) and proteins (DEPs) including m6A regulators (METTL3) and QSER1 complex that protects against enhancer methylation. Reflecting this, restoring RARγ levels in 22Rv1 cells, increased AR genomic binding 12-fold to greater than 9,500 sites and further by TACC1. These gained AR sites were enriched for GATA and Homeobox motifs, significantly overlapped with H3K27ac and super-enhancers, and also ChromHMM-defined Poised Enhancers (logPV = 163) and Transcribed regions (logPV = 179). RNA-Seq experiments revealed that RARγ/TACC1 significantly altered the frequency and fold change of DHT-regulated genes. Finally, miR-96 antagomir/ENZA treatments drove a unique transcriptome that triggered cell cycle arrest and significantly associated with worse survival in the SU2C cohort. To summarize these data suggest that miR-96 is directed to control the RARγ/TACC1 complex in part by m6A marks. Restoring the RARγ/TACC1 complex increased association with potential bookmarking factor like QSER1 and redirects AR to active enhancers that promote ENA-regulated genes and increase propensity for cells to undergo cell arrest.

Citation Format: Sajad A. Wani, Jaimie S. Gray, Moray J. Campbell. RAR gamma interactome and cistrome guide the androgen receptor enhancer usage in prosate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6243.
Abstract 6244: A T cell engaging bispecific antibody utilizing a bivalent tumor-selective folate receptor alpha binding arm for the treatment of ovarian cancer
Authors: Avanzino Brian C. , Prabhakar Kirthana , Dalvi Pranjali , Hartstein Sharon , Kehm Hannes , Balasubramani Aarti , Boudreau Andrew A. , Buelow Ben , Chang Karen , Davison Laura M. , Iyer Suhasini , Kalwit Vidyut , Malik-Chaudhry Harbani , Pierson Will , Pineda Geovanni , Rangaswamy Udaya S. , Saiganesh Sowmya , Schellenberger Ute , Ugamraj Harshad S. , Buelow Roland , Chapman Jocelyn , Trinklein Nathan D. , Harris Katherine E.
Published: June 2022

The use of T cell engagers (TCEs) to treat solid tumors in humans is challenging, and several have been limited by narrow therapeutic windows due to substantial toxicity. Dose-limiting toxicities of TCEs include cytokine release syndrome (CRS) and on-target, off-tumor toxicities since many solid tumor target antigens are also expressed in healthy tissues, albeit at lower levels. While there have been improvements in the clinical management of immune-mediated toxicities such as CRS, on-target, off-tumor effects remain one of the primary challenges of using TCEs for treatment of solid tumors. Here, we describe TNB-928B, a novel fully human TCE that is bivalent for folate receptor alpha (FRα) that selectively targets FRα overexpressing tumor cells yet avoids targeting cells that express low levels of FRα found in normal human cells. Selectivity is conferred by an avidity effect of the FRα targeting arm in combination with a unique low-affinity CD3 arm and is anticipated to reduce the risk of adverse events in normal cells expressing low levels of FRα. TNB-928B induces preferential effector T cell activation, proliferation, and cytotoxic activity with minimal cytokine release, compared to a positive control containing OKT3, thereby improving the therapeutic index. Moreover, TNB-928B exhibits substantial ex vivo tumor cell lysis using endogenous T cells and is effective in vivo promoting T cell infiltration and tumor regression in mouse models of ovarian cancer. Together, these results demonstrate TNB-928B represents a novel next generation immunotherapy modality with enhanced safety and specificity for the treatment of ovarian cancer.

Citation Format: Brian C. Avanzino, Kirthana Prabhakar, Pranjali Dalvi, Sharon Hartstein, Hannes Kehm, Aarti Balasubramani, Andrew A. Boudreau, Ben Buelow, Karen Chang, Laura M. Davison, Suhasini Iyer, Vidyut Kalwit, Harbani Malik-Chaudhry, Will Pierson, Geovanni Pineda, Udaya S. Rangaswamy, Sowmya Saiganesh, Ute Schellenberger, Harshad S. Ugamraj, Roland Buelow, Jocelyn Chapman, Nathan D. Trinklein, Katherine E. Harris. A T cell engaging bispecific antibody utilizing a bivalent tumor-selective folate receptor alpha binding arm for the treatment of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6244.
Abstract 6248: Modulation of CD22 protein expression in childhood leukemia by pervasive splicing aberrations: Implications for CD22-directed immunotherapies
Authors: Zheng Sisi , Gillespie Elisabeth , Naqvi Ammar S. , Hayer Katharina E. , Ang Zhiwei , Torres-Diz Manuel , Quesnel-Vallières Mathieu , Hottman David A. , Bagashev Asen , Chukinas John A. , Schmidt Carolin , Asnani Mukta Mukta , Shraim Rawan , Taylor Deanne M. , Rheingold Susan R. , M. O’Brien Maureen , Singh Nathan , Lynch Kristen W. , Ruella Marco , Barash Yoseph , Tasian Sarah K. , Thomas-Tikhonenko Andrei
Published: June 2022

Downregulation of surface epitopes causes post-immunotherapy relapses in B-lymphoblastic leukemia (B-ALL). Here we demonstrate that mRNA encoding CD22 undergoes aberrant splicing in B-ALL. We describe the plasma-membrane-bound CD22 Δex5-6 splice isoform resistant to CAR-T cells targeting the third immunoglobulin-like domain of CD22. We also describe splice variants skipping the AUG-containing exon 2 and failing to produce any identifiable protein; therefore, this event is rate-limiting for epitope presentation. Indeed, forcing exon 2 skipping with Morpholino oligonucleotides reduced CD22 protein expression and conferred resistance to the CD22-directed antibody-drug conjugate inotuzumab in vitro. Furthermore, among inotuzumab-treated pediatric B-ALL patients, we identified one non-responder in whose blasts Δex2 isoforms comprised the majority of CD22 transcripts. In a second patient, a sharp reduction in CD22 protein levels during relapse was driven entirely by increased CD22 exon 2 skipping. Thus, dysregulated CD22 splicing is a major mechanism of epitope downregulation and ensuing resistance to immunotherapy.

Citation Format: Sisi Zheng, Elisabeth Gillespie, Ammar S. Naqvi, Katharina E. Hayer, Zhiwei Ang, Manuel Torres-Diz, Mathieu Quesnel-Vallières, David A. Hottman, Asen Bagashev, John A. Chukinas, Carolin Schmidt, Mukta Mukta Asnani, Rawan Shraim, Deanne M. Taylor, Susan R. Rheingold, Maureen M. O’Brien, Nathan Singh, Kristen W. Lynch, Marco Ruella, Yoseph Barash, Sarah K. Tasian, Andrei Thomas-Tikhonenko. Modulation of CD22 protein expression in childhood leukemia by pervasive splicing aberrations: Implications for CD22-directed immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6248.
Abstract 6262: Emergence of tumor mismatch repair deficiency and increased mutational burden in blood and tissue of metastatic colorectal cancer patients treated with temozolomide
Authors: Crisafulli Giovanni , Sartore-Bianchi Andrea , Lazzari Luca , Pietrantonio Filippo , Amatu Alessio , Macagno Marco , Barault Ludovic , Cassingena Andrea , Bartolini Alice , Luraghi Paolo , Mauri Gianluca , Battuello Paolo , Personemi Nicola , Pessei Valeria , Vitiello Pietro Paolo , Tosi Federica , Idotta Laura , Valtorta Emanuele , Bonoldi Emanuela , Germano Giovanni , Nicolantonio Federica Di , Marsoni Silvia , Siena Salvatore , Bardelli Alberto
Published: June 2022

The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient (MMRp) and unresponsive to immunotherapy, while MMR deficient (MMRd) tumors often respond to immune checkpoint blockade (ICB). We previously reported that treatment of CRC preclinical models with temozolomide (TMZ) leads to MMR deficiency, increased tumor mutational burden (TMB) and, sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-Methylguanine-DNA-methyltransferase (MGMT) deficient, MMR proficient and KRAS mutant mCRC patients receive priming therapy with TMZ. Analysis of solid tissue biopsies and circulating tumor DNA (ctDNA) obtained after TMZ treatment revealed the emergence of TMZ mutational signature, alterations in MMR genes and increased TMB in 14 out of 16 patients. Genetic mutations induced by TMZ were dose-dependent and multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes such as the MSH6 T1219I variant which was detected in ctDNA and tissue of 13/14 (93%) of the cases. A subset of the patients whose tumors after TMZ priming displayed the MSH6 mutation, the TMZ mutational signature and increased TMB, achieved disease stabilization upon pembrolizumab treatment. Overall, we provide proof-of-concept that treatment of MGMT deficient/MMR proficient KRAS mutant mCRCs with TMZ can be tracked by mutational signature analysis and lead to inactivation of the MMR pathway, emergence of the TMZ mutational signature, TMB increase, and, in some cases, to disease stabilization during ICB.

Citation Format: Giovanni Crisafulli, Andrea Sartore-Bianchi, Luca Lazzari, Filippo Pietrantonio, Alessio Amatu, Marco Macagno, Ludovic Barault, Andrea Cassingena, Alice Bartolini, Paolo Luraghi, Gianluca Mauri, Paolo Battuello, Nicola Personemi, Valeria Pessei, Pietro Paolo Vitiello, Federica Tosi, Laura Idotta, Emanuele Valtorta, Emanuela Bonoldi, Giovanni Germano, Federica Di Nicolantonio, Silvia Marsoni, Salvatore Siena, Alberto Bardelli. Emergence of tumor mismatch repair deficiency and increased mutational burden in blood and tissue of metastatic colorectal cancer patients treated with temozolomide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6262.
Abstract 6270: Integrating spatial transcriptomics and single-cell RNA-seq reveal the tumorigenesis and heterogeneity of esophageal squamous cell carcinoma
Authors: Yin Xiaoyang , Li Baosheng
Published: June 2022

Background: Esophageal cancer is the sixth leading cause of cancer death globally and esophageal squamous cell carcinoma (ESCC) is the major histological type in China. The heterogeneity of ESCC resulted in poor therapeutic effect and tumor recurrence. To investigate the heterogeneity of ESCC at the single-cell level and the oncogenic evolution, we performed single-cell RNA-seq and spatial transcrtiptomics analysis in ESCC samples from different patients and position.

Methods: We performed single-cell RNA-seq of tumor, adjacent normal tissue and the junction of cancer and normal tissues from 4 ESCC patients. We also performed spatial transcriptomics of paired cancer, normal and junction of ESCC patients. Unsupervised clustering was performed to find different cell clusters of ESCC and marker genes of different clusters were determined. Inferred copy number variation was performed to identify malignant cells and pseudotime analysis to investigate the development of different cell clusters. Combined analysis of single-cell and spatial data was performed to reveal the spatial characteristics of different cell types.

Results: We successfully determined different cell types including epithelial cells, immune cells, fibroblasts, myeloid cells and so on. And we also reclustered epithelial, myeloid and T/NK cells to investigate different subtypes and their marker genes. We also distinguished malignant cells from heterogeneous cellular populations using inferred copy number variation analysis. Pseudotime analysis revealed the evolution of different cell types of ESCC. We identified key signatures associated with oncogenic evolution of ESCC and the spatial characteristics of key cell clusters.

Conclusion: These findings reveal the heterogenesis of ESCC and key signatures in ESCC tumorigenesis.

Keywords: ESCC, single cell sequencing, spatial transcriptomics, heterogeneity, tumorigenesis

Citation Format: Xiaoyang Yin, Baosheng Li. Integrating spatial transcriptomics and single-cell RNA-seq reveal the tumorigenesis and heterogeneity of esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6270.
Abstract 6280: Preclinical development of a bispecific antibody-trap selectively targeting CD47 and HER2 for the treatment of breast as well as gastric cancer
Authors: Tian Wenzhi , Li Song , Chen Dianze , Liu Dandan , Guo Huiqin , Yang Chunmei , Zhang Li , Zhang Wei , Tu Xiaoping , Peng Liang , Zhao Gui , Zhang Ruliang , Zhang Fan
Published: June 2022

Trastuzumab executes anti-tumor efficacy mainly via antibody-dependent cell cytotoxicity (ADCC). However due to the fact that majority of the populations including patients with cancers carry a CD16A-158F allele which has low binding affinity to the Fc fragment of trastuzumab, response to trastuzumab treatment is hindered. Furthermore, overexpression of CD47 on cancer cells prevent macrophages from activation via Fc-FcγRIIa interaction due to the so called “Don’t eat me” signal. We therefore designed a bispecific mAb-trap designated as IMM2902. Extensive in vitro as well as in vivo characterization demonstrated that IMM2902 binds to both CD47 and HER2 with high affinity (KD = 5.8x10-10M for HER2; KD = 8.9x10-9M for CD47) and has stronger ADCC activity against NCI-N87 (EC50 = 0.054nM for IMM2902, v.s. 0.868nM for trastuzumab). In vivo efficacy studies in several xenograft tumor models revealed a robust anti-tumor activity as reflected by the complete elimination of established tumors in BT-474 breast cancer and NCI-N87 gastric xenograft models even at the dose as low as 3.5mg/kg. Interestingly, even in Herceptin-resistant breast tumor model, when administered at a dose of 10mg/kg, the established HCC-1954 tumors were also eliminated. Immunohistochemistry staining of the tumor tissues demonstrated an accelerated HER2 degradation which is highly correlated with tumor volume. IMM2902 has no impact on hemagglutination, nor does it have significant hemotoxicity following single as well as multiple administrations in non-human primate animals at different dosage. Our study suggests that IMM2902 has the potential to be an alternative promising treatment option for patients with her2-positive cancers refractory to trastuzumab treatment.

Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Dandan Liu, Huiqin Guo, Chunmei Yang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Gui Zhao, Ruliang Zhang, Fan Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD47 and HER2 for the treatment of breast as well as gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6280.
Abstract 6281: Preclinical development of a bispecific antibody-trap selectively targeting CD47 and CD24 for cancer immunotherapy
Authors: Tian Wenzhi , Li Song , Chen Dianze , Liu Dandan , Guo Huiqin , Yang Chunmei , Zhang Li , Zhang Wei , Tu Xiaoping , Peng Liang , Zhao Gui , Zhang Ruliang , Zhang Fan
Published: June 2022

Limited treatment beneficial rate and market saturation of the first generation of immune checkpoint inhibitors (Such as PD-(L)1 inhibitors) have turned the attention of pharmaceutical industry to the next generation of innate immune checkpoints such as CD47/SIRPα and CD24/Siglec-10. Various cancers of lung, breast, colon, prostate, ovarian, stomach, pancreas, myeloid and lymphoid tissues are known to highly express CD47 and/or CD24, which confer cancer cells means of immune-evasion via induction of “Don’t eat me” signals. Precisely and simultaneously neutralizing CD47 and CD24 signaling on tumors, when coupled with an IgG1 antibody’s potent phagocytosis and cytotoxicity activity, may serve as potent immunotherapy for multiple cancer types. We have developed a humanized CD24 neutralizing antibody which has been revealed to neutralize CD24-Siglec-10 interaction and completely eliminate established colon cancer in syngeneic mouse models via proposed mechanisms of antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and subsequent tumor-specific immunity as a result of macrophage activation. Using this mAb as backbone, we developed an CD24 mAb-CD47 trap by conjugating the SIRPα decoy receptor to the N-terminal of the antibody heavy chain. Initial in vitro studies indicated that this mAb-trap has simultaneous two-target binding as well as blocking activities. More promisingly, eight administrations of the mAb-trap at 3mg/kg dose in mice bearing triple-negative breast cancer resulted in a complete tumor growth inhibition. Furthermore, in a Herceptin-resistant xenograft tumor model, the mAb-trap is also efficacious and resulted in over 60% of tumor growth inhibition when administrated at 7.2mg/kg. Taken together, our data show that precise neutralization of CD47 and CD24 signaling on tumors using IgG1 antibody may serve as potent monotherapy for cancers.

Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Dandan Liu, Huiqin Guo, Chunmei Yang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Gui Zhao, Ruliang Zhang, Fan Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD47 and CD24 for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6281.
Abstract 6310: Designing genomic test reports using a user-centered approach for clinicians in Pakistan
Authors: Agha Momal , Khan Faisal
Published: June 2022

In the developed world, precision medicine and genomics are transforming traditional healthcare approaches. In spite of rapid technological progress being made in genomics, a 'genomic divide' is emerging between developed and developing countries. This is partly because of the limited genomic literacy of healthcare professionals and a scarcity of available genomics workforce that reportedly deters them from proposing genomic testing. We aim to help reduce this 'genomic divide' by proposing a genomic test report design for clinicians in Pakistan, that incorporates user-centered design principles. We tested two commonly used genomic reportswiththat varied in language, content and layout. Report A was a one-page genomic report from the Laboratory for Molecular Medicine, of a patient with suspected hypertrophic cardiomyopathy. Report B was a report from FoundationOneCDx, of a breast carcinoma specimen with confirmed findings mentioned. We employed a qualitative descriptive study design, including a survey for trainees, non-specialists, and specialists. The parameters recorded were: subject comprehension, overall visual impression, level of difficulty of the language, and ease of understanding, along with, actionability and degree of trust. A total of 49 medical professionals across 11 institutes in Pakistan participated in the survey, of which 11 were specialists and 38 were non-specialists. We extracted 19 recommendations from the survey and broadly grouped them into three categories: communication style, report content, and layout. About 25% of the respondents commented that they would prefer a short and concise report, 20.8% wanted the language to be ‘doctor-friendly’ and 8.3% preferred data to be presented in tables. All recommendations were incorporated into our proposed genomic test report template. The template displays the results and what they mean for the patient (in layman's terms for ease of access to non-specialists), what possible action is to be taken by the doctor, and details of further information and support regarding the results. We propose the addition of a box on technical methodology at the end of the report template that may help in assisting with refreshing the genomic knowledge of non-specialists. Our findings have shown that in order to design impactful genomic reports for clinicians in Pakistan, we will have to start with a user-centered design approach. This can be instrumental in helping us improve the adoption of genomic testing in clinics around Pakistan.

Citation Format: Momal Agha, Faisal Khan. Designing genomic test reports using a user-centered approach for clinicians in Pakistan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6310.
Abstract 6314: TME subtype impacts response to combined thermal ablation and immunotherapy
Authors: Wang James , Fite Brett Z. , Kare Aris , Wu Bo , Raie Marina N. , Tumbale Spencer , Davis Ryan R. , Tepper Clifford G. , Ferrara Katherine
Published: June 2022

The tumor microenvironment (TME) subtype is a critical factor when combining immunotherapy with tumor debulking strategies such as ultrasound ablation. To understand the effects of the TME subtype, we studied two multi-site murine cancer models, an immune-suppressed KPC (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) pancreatic adenocarcinoma (MT4) model and a neu deletion HER2+ (NDL) mammary adenocarcinoma model with a larger naïve lymphocyte population. After profiling the two models with histology and single-cell sequencing, we designed immunotherapy combinations with Magnetic Resonance-guided Focused Ultrasound (MRgFUS) thermal ablation that were specific to each TME subtype. To understand the impact of the molecular and cellular differences upon each model, we characterized the treatment effect with transcriptomic analysis and digital cytometry. We found that combining MRgFUS ablation with immunotherapy reduced tumor growth and extended survival by at least 2-fold in the MT4 pancreatic cancer model. Gene set enrichment analysis (GSEA) of bulk RNA sequencing data indicated that the combination of ablation with immunotherapy preferentially enriched functional annotations for leukocyte migration in the MT4 model and wound healing and inflammatory cytokines in the NDL model (e.g. for IL6: 1.6 fold increase in MT4 vs 10.3 fold in NDL tumors). We then particularly focused on whether dendritic cell (DC) activation and CD4 T cell populations could be enhanced when ablating pancreatic tumors. By CIBERSORTx digital cytometry, aCD40 + aPD-1 combinations increased the presence of activated DCs in MT4 tumors by 2.4-fold. Activated DCs were also increased by CP4 (aCD40 + aPD-1 + CTLA-4) alone 1.7 fold or in combination with ablation in both treated (2.4 fold) and distant (2.2 fold) lesions. Ablation combined with aPD-1 alone did not significantly impact DCs in the MT4 model. In addition, CD4 lymphocytes increased 5 and 8 fold in treated and distant tumors by ablation combined with CP4 in the MT4 model. Taken together, inflammatory cytokines were preferentially elevated by ablation in a dense epithelial breast cancer as compared with a pancreatic tumor model. Further, the results suggest that aCD40 immunotherapy retains efficacy when combined with thermal ablation in pancreatic tumors. This work can inform clinical translation of ablative and immunotherapy combination protocols.

Citation Format: James Wang, Brett Z. Fite, Aris Kare, Bo Wu, Marina N. Raie, Spencer Tumbale, Ryan R. Davis, Clifford G. Tepper, Katherine Ferrara. TME subtype impacts response to combined thermal ablation and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6314.
Abstract 6320: Connecting TET2 deficiency with inflammation in CMML and anti-tumor responses
Authors: Singh Anup Kumar , Onodera Atsushi , Sepulveda Hugo , Angel Carlos , Rao Anjana
Published: June 2022

Chronic Myelomonocytic Leukemia (CMML) is a clonal hematologic disorder of older individuals characterized by monocytosis and increased risk of progression to acute myeloid leukemia (AML). Although CMML is characterized by relatively low genetic complexity consistent with clonal expansion, loss-of-function mutations in ten-eleven translocation 2 (TET2) protein are frequent and reported in almost 60% of CMML cases. Consistent with this finding, deletion of the Tet2 gene in mice leads to CMML-like disease. TET2 restrains inflammatory signals through various means, for instance by directly suppressing the expression of the IL6 gene in mouse myeloid cells by recruiting histone deacetylases. Conversely, the levels of pre-inflammatory cytokines including IL6 correlate with pre-leukemic proliferation of Tet2-deficient mouse monocytes and other myeloid cells, suggesting that Tet2 deficiency increases IL-6 expression and increased IL-6 expression in turn drives the proliferation of Tet2-deficient myeloid-lineage cells in a positive feedback loop. This scenario may operate in CMML patients, who show increased levels of inflammatory cytokines and increased expression of interferon-induced genes compared to age-matched healthy individuals. To gain insight into the connection between inflammation caused by TET deficiency and monocyte hyperproliferation, we set up cultures of bone marrow derived macrophages (BMDMs) from inducible TET deficient mice. Using this model, we found that TET-deficient BMDMs show increased proliferation compared to wild type controls; moreover, LPS stimulation leads to significantly increased expression of IL6 and IL1b in TET-deficient BMDMs. Our previous RNA sequencing of expanded TET-deficient myeloid cells in vivo showed that TET deficiency correlated with increased expression of transposable elements (TEs) and induced a DNA damage response, leading us to hypothesize that TET deficiency and increased TE expression may activate DNA and RNA sensing pathways and induce sterile inflammation. I am currently using several molecular approaches to delineate the potential connection of TET deficiency with endogenous inflammation in CMML. There is growing evidence that TET deficiency or non-specific inhibition of TET enzymes in CD8 T cells can potentiate anti-tumor responses. I am now asking whether the TET deficiency-inflammation axis I observe in BMDMs might also enhance anti-tumor responses by increasing proinflammatory responses and hence T cell recruitment and anti-tumor activity.

Citation Format: Anup Kumar Singh, Atsushi Onodera, Hugo Sepulveda, Carlos Angel, Anjana Rao. Connecting TET2 deficiency with inflammation in CMML and anti-tumor responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6320.
Abstract 6322: Dynamic, stage-course protein interaction network using high power CpG sites in head and neck squamous cell carcinoma
Authors: Riaz Arsalan , Khan Faisal
Published: June 2022

Head and neck cancer is the most incident and prevalent cancer in men in South Asian countries, especially Pakistan. Prediction of pathological stages of cancer can play a critical role in early diagnosis and personalized medicine. This project ventures into the prediction of different stages of head and neck squamous cell carcinoma (HNSCC) using prioritized DNA methylation patterns. DNA methylation profiles for each HNSCC stage (stage-I-IV) were used to extensively analyze 485,577 methylation CpG sites and prioritize them on the basis of the highest predictive power using a wrapper-based feature selection method, along with different classification models. We identified 68 high-power methylation sites which predicted the pathological stage of HNSCC samples with 90.62% accuracy using a Random Forest classifier. We set out to construct a protein-protein interaction network for the proteins encoded by the 67 genes associated with these sites to study its network topology and also undertook enrichment analysis of nodes in their immediate neighborhood for GO and KEGG Pathway annotations revealing their role in cancer-related pathways, cell differentiation, signal transduction, metabolic and biosynthetic processes. With information on the predictive power of each of the 67 genes in each HNSCC stage, we unveil a dynamic stage-course network for HNSCC that evolves over the course of the four cancer stages. We intend to further study these genes in light of functional datasets from CRISPR, RNAi, drug screens for their putative role in HNSCC initiation and/or progression.

Citation Format: Arsalan Riaz, Faisal Khan. Dynamic, stage-course protein interaction network using high power CpG sites in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6322.
Abstract 6323: Differential analysis of HPV16-human and EBV1-human protein interaction networks to identify new protein candidates in head and neck squamous cell carcinomas
Authors: Riaz Arsalan , Ilyas Yusra , Khan Faisal
Published: June 2022

Oro-pharyngeal squamous cell carcinoma (OPSCC) cases are rising worldwide specifically among young individuals with risk factors including smoking and alcohol. HPV infection specifically the subtype HPV16 is a significant risk factor in the initiation and progression of OPSCC, more prevalent in the western part of the world. This study carries out a computational analysis using protein interactions between HPV16 proteins and protein products of key OPSCC and Oral Squamous Cell Carcinoma (OSCC) cancer genes, which aims to investigate the influence of HPV16 on OPSCC and OSCC in terms of protein-protein interactions (PPIs).We created a protein interaction network of HPV16 and human proteins followed by retrieval of mutated genes in OPSCC and OSCC from online databases including COSMIC, cBioPortal and the GDC Portal, along with literature mining. We used MCODE and ClueGo tools for cluster and enrichment analysis, respectively. Drug candidates against the common interacted proteins were found using drug databases like PanDrugs and CLUE Repurposing. After annotating the HPV16-Human PPI network with proteins encoded by genes found to be mutated in both cancers, 31 proteins in OPSCC and 17 proteins in OSCC were in direct interaction with the viral proteins especially, E5, E6, and E7. The immediate network (of first neighbors only) appeared to be sparse, but after looking at the broader HPV16-Human network we found 5 highly connected modules, 4 of which were dominated by proteins encoded by genes mutated in OPSCC as compared to OSCC, indicating the influence of HPV16 on OPSCC. Based on module integrity and topological characteristics we identified 4 significant candidates for HPV16-driven OPSCC i.e. MYC, CREBBP, PML, and RB1 which are significantly enriched in cancer pathways. In the South Asian countries, Epstein Bar Virus type 1 (EBV-1) is a more prevalent risk factor for virus-driven Head and Neck Squamous Cell Carcinoma (HNSCC) as compared to HPV-16, however, knowledge regarding the EBV induced OPSCC and OSCC is scarce. We created a protein interaction network for EBV1 and Human proteins to analyze the topological characteristics especially of proteins encoded by genes mutated in HNSCC using the same methodology and we identified 5 candidate proteins i.e. EP300, CUL3, RB1, DDB1, and CREBBP for further in vitro validation.

Citation Format: Arsalan Riaz, Yusra Ilyas, Faisal Khan. Differential analysis of HPV16-human and EBV1-human protein interaction networks to identify new protein candidates in head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6323.
Abstract 6325: Identification of genes responsible for osteosarcomas metastatic evolution by active super enhancers profiling
Authors: Lavaud Mélanie , Tesfaye Robel , Charrier Céline , Thépault Rose Anne , Golias Maxime , Page Sarah Le , Mullard Mathilde , Brounais Bénédicte , Verrecchia Franck , Baud'huin Marc , Lamoureux François , Georges Steven , Ory Benjamin
Published: June 2022

Super enhancers are strong spatiotemporal transcriptional regulators. They can be mapped on genomes thanks to their astounding enrichment for well-known enhancers markers, such as acetylation of the lysine 27 of type 3 histones (H3K27Ac). Their target genes, confined in their so-called topologically associated domains, present exceptionally high expression levels, depending on associated super enhancers activation status, and are responsible for defining cells identity whether physiological or pathological. Osteosarcomas are the most common primary malignant bone tumors. Patients suffering from osteosarcomas, on average 15 years old, mostly develop pulmonary metastasis, responsible for drastic decrease in their survival. We have conducted chromatin immunoprecipitation sequencing against H3K27Ac and RNA sequencing experiments on osteosarcoma cells presenting high propensity to metastatic dissemination or devoid of this capacity, in order to unveil genes implicated in metastasis development through super enhancers genomic mapping. We have highlighted exclusive active super enhancers profiles between metastatic and non-metastatic osteosarcoma cells. Among target genes of spotted super enhancers, three of them caught our attention. Implication of these genes in metastatic development have already been demonstrated in various cancers evolution, except in osteosarcomas. Osteosarcoma cells having high capacity for metastatic development present identified genes over expressions compared to cells incompetent in this pathological process. Artificial down expressions of identified genes decrease osteosarcoma cells metastatic capacity in vitro. In vivo experiments, that correspond to paratibial implantation of modified osteosarcoma cells and analysis of primary tumor metastatic development, are currently under way. Our strategy may allow identification of new therapeutic targets to fight against osteosarcoma metastatic evolution and open new perspectives to improve patients prognostic.

This work is funded by Ligue contre le cancer, ANR, SFCE and Région Pays de la Loire.

Citation Format: Mélanie Lavaud, Robel Tesfaye, Céline Charrier, Rose Anne Thépault, Maxime Golias, Sarah Le Page, Mathilde Mullard, Bénédicte Brounais, Franck Verrecchia, Marc Baud'huin, François Lamoureux, Steven Georges, Benjamin Ory. Identification of genes responsible for osteosarcomas metastatic evolution by active super enhancers profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6325.