bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 646: CRISPR/Cas9 screening to identify mechanisms of azacitidine failure in myelodysplastic syndrome
Authors: Schieber Michael , Crispino John D.
Published: September 2020

Myelodysplastic syndrome (MDS) is a heterogenous myeloid lineage malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of secondary transformation to acute myeloid leukemia (sAML). The mainstay of treatment in patients with higher-risk MDS are the hypomethylating agents (HMAs) azacitidine and decitabine, which extend overall survival by approximately 12 to 18 months. Unfortunately, disease relapse and HMA resistance are inevitable and carry a very poor prognosis. Genomic sequencing of large MDS cohorts has led to the identification of recurrent genetic abnormalities in MDS, some of which predict sensitivity to HMA therapy. For example, abnormalities in TET2, but not ASXL1, are associated with a favorable response to the HMAs. However, it is unknown why some patients with otherwise similar clinical features have variable responses to HMA therapy. We hypothesize that there are common cellular mechanisms promoting resistance to HMA therapy and that these can be therapeutically targeted to enhance treatment responses.

To uncover cellular processes promoting HMA treatment failure we performed duplicate genome-wide CRISPR/Cas9 screen in the human MDS-L cell line using resistance to azacitidine as a positive selection readout. MDS-L was established from bone marrow mononuclear cells in a 52-year-old male patient with high-risk MDS. We transduced the Brunello sgRNA CRISPR library into Cas9 expressing MDS-L cells and subjected the cells to azacitidine treatment for 7 days. Cells surviving azacitidine treatment were then expanded and harvested for genomic DNA. High throughput sequencing of the barcoded DNA produced raw reads that were analyzed using the PinAPL-Py web-based software. sgRNAs appearing in duplicate with over 10-fold enrichment compared to the control library were considered significant.

We identified 125 candidate genes involved in HMA resistance from the CRISPR-Cas9 screen. Pathway analysis revealed a significant enrichment of genes involved in lipid metabolism, DNA methylation, and response to small molecule catabolism. Future experiments to validate these candidates as well as test whether they contribute in vivo to HMA resistance are underway. We conclude genome-wide CRISPR-Cas9 screens for drug-resistance in the MDS-L model are feasible and may uncover future therapeutic targets to augment HMA therapy or utilize upon HMA failure.

Citation Format: Michael Schieber, John D. Crispino. CRISPR/Cas9 screening to identify mechanisms of azacitidine failure in myelodysplastic syndrome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 646.
Abstract 6462: Radiomics features of 18F-fluorodeoxyglucose positron-emission tomography as a novel prognostic signature in colorectal cancer
Authors: Kang Jeonghyun , Clemenceau Jean Rene , Park Sunho , Xu Hongming , Hong Changjin , Hwang Tae Hyun
Published: September 2020

Background: The aim of this study was to investigate the prognostic value of radiomics signatures derived from 18F-fluorodeoxyglucose (18F-FDG) positron-emission tomography (PET) in patients with colorectal cancer (CRC).

Methods: This was a retrospective, single-center study. From April 2008 to Jan 2014, we identified CRC patients who underwent 18F-FDG-PET before starting any neoadjuvant treatments and surgery. Radiomics features were extracted from the primary lesions identified on 18F-FDG-PET. Patients were divided into a training and a validation set by random sampling. A least absolute shrinkage and selection operator (LASSO) Cox regression model was applied for prognostic signature building with progression-free survival (PFS) using the training data set. Using the calculated radiomics score, a nomogram was developed, and the clinical utility of this nomogram was assessed in the validation set.

Results: Three-hundred-and-eight-one patients with surgically resected CRC patients (training set 228 vs. validation set 153) were included. In the training set, a radiomics signature called a rad_score was generated using two PET-derived features such as Gray Level Run Length Matrix_Long-Run Emphasis (GLRLM_LRE) and Grey-Level Zone Length Matrix_Short-Zone Low Gray-level Emphasis (GLZLM_SZLGE). Patients with a high-rad_score in the training and the validation set had shorter PFS (P < 0.001 in the training set and P = 0.008 in the validation set). In the multivariable analysis with forward selection, presence of lymphovascular invasion (LVI) (hazard ratio [HR] 3.73; 95% confidence interval [CI], 1.63-8.47; P = 0.001), pathological node-positivity (HR 2.52; 95% CI, 1.01-6.26; P = 0.046), and a high rad_score (HR 7.82; 95% CI, 2.36-25.85; P < 0.001) remained independent predictors of a worse prognosis in the training set. The rad_score was also identified as an independent prognostic factor in the validation and the overall set. A radiomics nomogram was developed using the rad_score, pathological node stage, and lymphovascular invasion, and showed good performance in the calibration curve in the validation set.

Conclusions: Textural features, such as GLRLM_LRE and GLZLM_SZLGE, derived from 18F-FDG-PET images may enable more detailed stratification of prognosis in patients with CRC.

Citation Format: Jeonghyun Kang, Jean Rene Clemenceau, Sunho Park, Hongming Xu, Changjin Hong, Tae Hyun Hwang. Radiomics features of 18F-fluorodeoxyglucose positron-emission tomography as a novel prognostic signature in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6462.
Abstract 6474: FAT1 and CASP8 co-mutations are associated with poor prognosis in HPV negative head and neck squamous cell carcinoma
Authors: Yilmaz Emrullah , Lee David , Cowan Andrew , Gan Gregory , Ozbun Michelle , Guo Yan
Published: September 2020

INTRODUCTION: FAT1 alterations with somatic mutations or deletions are observed in head and neck squamous cell carcinomas (HNSCC). These alterations lead to loss of function of FAT1. FAT1 loss of function was known to be associated with aberrant Wnt activation, cell migration and invasion. In this study, we present analysis and outcomes of co-mutations seen in FAT1-mutation positive HNSCC.

METHODS: TCGA data were analyzed for mutation, gene expression data and overall survival. We examined mutation data in 415 HPV negative samples and 72 HPV positive samples. The Fisher's Exact test was used to determine if differences in mutation frequency existed between the different cohorts. Mantel-Cox method was used for overall survival (OS) comparison between the cohorts.

RESULTS: FAT1 somatic mutation was the third most commonly mutated gene in HPV-negative HNSCC while it was not common in HPV-positive HNSCC. FAT1 mutation was seen in 25.8% of HPV negative HNSCC (107/415), while 4.2% (3/72) HPV positive HNSCC had FAT1 mutation. We then analyzed the frequency of co-mutations in FAT1-mutated HPV-negative HNSCC. CDKN2A, NOTCH1, CASP8, FBXW7, ZFHX4, HRAS, DCC, ATR mutations were more common in FAT1-mutated cancers. CASP8 mutation was seen in 24.30% (26 out of 107 samples) of FAT1-mutations positive HPV negative HNSCC, while it was less common in FAT1-mutation negative HNSCC (9.08% 28/308) (P=0.002). The patients with FAT1 and CASP8 co-mutation were associated with shorter overall survival when compared with patients with FAT1-mutation (19.8 months vs 52.3 months P=0.02). FAT1 only or CASP8 only mutation without co-mutation did not affect survival. To determine the mutation impact of the FAT1 and CASP8 mutations in HNSCC, differentially expressed genes between mutated and non-mutated genes were analyzed in HNSCC. CASP8 mutation was the top gene among the genes with somatic mutation in HNSCC and associated with gene expression changes in more than 6000 genes. FAT1 was ranked 11th resulting in differential gene expression in more than 1000 genes.

DISCUSSION: FAT1 somatic mutations are commonly seen in HPV negative HNSCC. We have demonstrated for the first time that CASP8 and FAT1 co-mutations are associated with poor prognosis with decreased overall survival in HPV negative HNSCC. Both CASP8 and FAT1 mutations are among the top genes with mutation impact on differential gene expression in HNSCC suggesting significance of these mutations in HNSCC.

Citation Format: Emrullah Yilmaz, David Lee, Andrew Cowan, Gregory Gan, Michelle Ozbun, Yan Guo. FAT1 and CASP8 co-mutations are associated with poor prognosis in HPV negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6474.
Abstract 6484: Cross-linked multimeric pro-peptides of type III collagen (PC3X) in hepatocellular carcinoma - A biomarker that provides additional prognostic value in AFP positive patients
Authors: Jensen Christina , Nielsen Signe H. , Eslam Mohammed , Genovese Federica , Nielsen Mette J. , Vongsuvanh Roslyn , Uchila Raj , van der Poorten David , George Jacob , Karsdal Morten A. , Leeming Diana J. , Willumsen Nicholas
Published: September 2020

Background: Non-invasive biomarkers for diagnosing and prognosing hepatocellular carcinoma (HCC) are urgently needed. Cirrhosis, a consequence of deposition and cross-linking of collagens, underlies HCC in 80-90% of cases, and associates with HCC initiation and progression. We evaluated circulating cross-linked pro-peptides of type III collagen (PC3X) as a diagnostic and prognostic biomarker for HCC.

Methods: PC3X was measured by ELISA in plasma from patients with HCC (n=79), cirrhosis (n=86), non-cirrhotic hepatitis-B infection (n=74) and from healthy controls (n=44). PC3X was compared to the liver fibrosis marker PRO-C3 and the HCC tumor-cell derived marker alpha-fetoprotein (AFP). Diagnostic and prognostic potential was evaluated by AUROC and by calculating hazard ratios (HR) for progression free survival (PFS) and overall survival (OS).

Results: PC3X, PRO-C3 and AFP were significantly elevated in patients with HCC compared to other liver diseases and healthy controls (p=0.0002, p<0.0001). In patients with normal AFP (<20 IU/ml), PC3X and PRO-C3 separated HCC from cirrhosis with an AUROC of 0.69 and 0.67, respectively. High PC3X and AFP predicted for poor PFS (HRPC3X=1.80, p=0.032; HRAFP=1.70, p=0.031) and OS (HRPC3X=2.12, p=0.024; HRAFP=2.55; p=0.003), whereas PRO-C3 did not (PFS: HR=1.19, p=0.059 and OS: HR=1.12, p=0.324). PC3X was independent of AFP (PFS: HR=1.74, p=0.045 and OS: HR=2.21, p=0.018) and combining the two improved prognostic value (PFS: HR=2.66, p=0.004 and OS: HR=5.86, p<0.0001).

Conclusions: PC3X is associated with HCC independent of AFP and provides diagnostic and prognostic value for HCC patients. If validated, this suggests that PC3X has biomarker potential for HCC.

Citation Format: Christina Jensen, Signe H. Nielsen, Mohammed Eslam, Federica Genovese, Mette J. Nielsen, Roslyn Vongsuvanh, Raj Uchila, David van der Poorten, Jacob George, Morten A. Karsdal, Diana J. Leeming, Nicholas Willumsen. Cross-linked multimeric pro-peptides of type III collagen (PC3X) in hepatocellular carcinoma - A biomarker that provides additional prognostic value in AFP positive patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6484.
Abstract 6512: Tissue engineered metastases to improve cancer immunotherapy
Authors: Sultanpuram Nikhila Reddy
Published: September 2020

Background: Radiotherapy can induce the abscopal effect when combined with immune checkpoint inhibition. The main limitations to this treatment are the lack of control over the tumor microenvironment and the tumor is not amendable for radiosurgery. To overcome this, we hypothesize that using ex-vivo engineered cancer metastases treated with radiotherapy can improve the impact of the abscopal effect, as the immune cascade reaction is radiotherapy dose dependent and tumors can be exposed to higher amounts of radiation ex-vivo without the risk of radiation toxicity using our tissue engineered metastases model to boost immune response.

Methods: Using B16F10 as a model, we first engineered B16F10 lung metastasis ex-vivo using decellularized rat lung biomatrix scaffolds. The engineered lung metastases were then lethally irradiated with 100 Gy of radiotherapy. The cells were then injected subcutaneously into tumor bearing C57BL6 mice in combination with anti PD-1 checkpoint inhibitor. Experimental groups included: no treatment, anti PD-1 only, cell lysate with and without anti PD-1, and irradiated engineered metastases with and without anti PD-1. All the mice were inoculated with 50,000 plastic grown B16F10 cells. On days 3, 6, and 9 the mice received immunotherapy and radiotherapy combinations based on their respective groups. The tumors were then analyzed by flow cytometry and IHC staining for different T cell populations.

Results: We successfully engineered B16F10 lung metastasis ex vivo. We then demonstrated that mice treated irradiated engineered lung metastasis had significantly better survival than any other treatment arms with median survival twice that of irradiated cells (P <0.01). Correlative studies showed that mice that received irradiated engineered metastasis had increased CD8+ cells and lower Treg cells when compared to other experimental groups. This indicates that engineered metastasis increased immune activation and response to tumor.

Conclusions: Our data demonstrates that engineered metastases can indeed improve cancer immunotherapy and induce a robust abscopal/vaccine effect.

Citation Format: Nikhila Reddy Sultanpuram. Tissue engineered metastases to improve cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6512.
Abstract 6524: A novel HER2/4-1BB bispecific antibody, YH32367 (ABL105) shows potent anti-tumor effect through tumor-directed T cell activation
Authors: Song Moo Young , Lee Eun-Jung , Chung Hyejin , Lee Yangsoon , Park Young Bong , Jee Min Hyeok , Kim KyeongBae , Ju Mikyeong , Lee Jun Kyung , Choi Byung Hyun , Park Ju Young , Kim DaeWon , Kim Junhwan , Ahn Sujin , Ahn Kyoung Kyu , Park Kyeongsu , Jung Uijung , Son Wonjun , Eom Jaehyun , Kim Eunjung , Yeom Donghoon , Ahn Jinhyung , Song Daehae , Sung Byungje , You Weon-kyoo , Kim Jong Gyun , Oh Se-Woong , Jung Jaeho
Published: September 2020

4-1BB (TNFSF9, CD137) is a member of the tumor necrosis factor receptor superfamily that functions as a potent costimulator to immune cells, especially T-cell. Although current 4-1BB-targeted strong agonistic antibody exhibit a considerable anti-tumor effect through T cell activation, it has a limitation of liver toxicity. To overcome the challenges of targeting the 4-1BB, we developed a bispecific antibody that can induce tumor-directed T cell activation.

A novel HER2/4-1BB bispecific antibody, YH32367 (ABL105) targets human epidermal growth factor receptor 2 (HER2) and 4-1BB simultaneously and binds to both targets with high affinity. YH32367 (ABL105) exhibited a strong activation of 4-1BB signal in tumor cells expressing HER2 such as NCI-N87, BT474 and HCC1954 cell lines. In co-culture assay using human peripheral blood mononuclear cell (hPBMC) and HER2-expressing tumor cells, YH32367 (ABL105) stimulated IFN-γ secretion from hPBMC and thereby induced tumor cell lysis. The anti-tumor efficacy of YH32367 (ABL105) was evaluated in HCC1954-bearing hPBMC engrafted humanized mice and MC38/hHER2-bearing h4-1BB knock-in mice. YH32367 (ABL105) had more potent efficacy on tumor growth inhibition in both mouse models compared to the equimolar dosing of trastuzumab. Peripheral T cell increase was not observed in these studies. In particular, YH32367 (ABL105) showed a tumor regression in MC38/hHER2-bearing h4-1BB knock-in mice.

In conclusion, YH32367 (ABL105) significantly inhibited tumor growth in a HER2 expression dependent manner and showed potent in vivo efficacy through tumor-directed T cell activation without non-specific systemic T-cell activation.

Citation Format: Moo Young Song, Eun-Jung Lee, Hyejin Chung, Yangsoon Lee, Young Bong Park, Min Hyeok Jee, KyeongBae Kim, Mikyeong Ju, Jun Kyung Lee, Byung Hyun Choi, Ju Young Park, DaeWon Kim, Junhwan Kim, Sujin Ahn, Kyoung Kyu Ahn, Kyeongsu Park, Uijung Jung, Wonjun Son, Jaehyun Eom, Eunjung Kim, Donghoon Yeom, Jinhyung Ahn, Daehae Song, Byungje Sung, Weon-kyoo You, Jong Gyun Kim, Se-Woong Oh, Jaeho Jung. A novel HER2/4-1BB bispecific antibody, YH32367 (ABL105) shows potent anti-tumor effect through tumor-directed T cell activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6524.
Abstract 6566: Defining the effects of inhibiting the beta-catenin-CBP signaling axis with the small molecule E7386 by integrative analysis of omics data
Authors: Federico Anthony , Owa Takashi , Nomoto Kenichi , Varelas Xaralabos , Kukuruzinksa Maria , Monti Stefano
Published: September 2020

Head and neck squamous cell carcinoma (HNSCC) is associated with high morbidity, poor survival rates, and limited treatment options. We previously reported that aberrant activation of nuclear β-catenin/cAMP-responsive element binding (CREB)-binding protein (CBP) signaling in primary HNSCC tumors was associated with progression to advanced disease and poor patient survival. Further, inhibition of β-catenin-CBP interaction with the first-generation inhibitor, ICG-001, greatly attenuated aggressive properties of HNSCC cells and tumor growth and metastases in murine and zebrafish models. Our recent findings show that a novel β-catenin/CBP modulator, E7386, displays an activity profile that closely overlaps with that of ICG-001, but exhibits ~50-100-fold lower EC50 values.

In this study, we used a novel method for gene regulatory network (GRN) reconstruction which analyzes publicly available high-throughput transcriptomics data from HNSCC to further elucidate the mechanisms of β-catenin-CBP inhibition by E7386. A Bayesian framework was used to reconstruct high-dimensional GRNs using constraint-based and shared structure learning from bulk RNA-seq data from TCGA. HNSCC patients were grouped into two subtypes, based on their “high” vs. “low” response to E7386 inhibition as predicted by the activation - or enrichment by Gene Set Variation Analysis (GSVA) - of the transcriptional signature we derived.

GRNs for the two subtypes were built, and their comparison highlighted new interactions in the “high” responders network between the β-catenin/CBP network and genes associated with histone modifications (ASH1L) and amino acid metabolism (MTR) concomitant with loss of interactions with regulators of cell cycle (ERCC6L) and protein secretion (SCAMP1) identified in the “low” responders network. Additionally, Kaplan-Meier survival plots showed that HNSCC patients with high activation of the E7386 signature had a significantly lower probability of survival. These results suggest that our GRN-based approach represents an important novel tool for assessing changes in regulatory network interactions in HNSCC and cancer.

Citation Format: Anthony Federico, Takashi Owa, Kenichi Nomoto, Xaralabos Varelas, Maria Kukuruzinksa, Stefano Monti. Defining the effects of inhibiting the beta-catenin-CBP signaling axis with the small molecule E7386 by integrative analysis of omics data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6566.
Abstract 657: Inhibition of mTORC1 by dihydroartemisinin
Authors: Liu Wang , Luo Jun , Odaka Yoshinobu , Huang Shile
Published: September 2020

mTOR functions as two complexes, mTORC1 and mTORC2, and is a central controller for cell growth and metabolism. Both mTORC1 and mTORC2 are activated in numerous cancers, and have become the focus of anticancer drug development. Rapalogs and mTOR kinase inhibitors (TORKIs) have been developed. However, the anticancer efficacy of rapalogs is clinically weak or mild to most cancers, and the adverse effects of rapalogs and TORKIs are greatly concerned. So, it is imperative to discover and develop new generation of mTOR inhibitors. Dihydroartemisinin (DHA) is an anti-malarial agent showing good safety in both children and adults. Recently, we have shown that DHA, at pharmacological concentrations, inhibits proliferation and induces apoptosis in cells derived from various cancer types but not in normal or immortalized cell lines, implying that DHA is potentially a selective anticancer agent and may be repurposed for cancer therapy. Of interest, DHA potently suppresses mTORC1-mediated phosphorylation of S6K1 and 4E-BP1, but does not obviously affect mTORC2-mediated phosphorylation of Akt, which is often activated by classic mTOR inhibitors (rapalogs) via a feedback mechanism. The results suggest that DHA is a novel mTORC1 inhibitor. This study was set to determine how DHA inhibits mTORC1. Here we found that DHA did not bind to any mTORC1 components (mTOR, mLST8 or raptor) directly, as determined by in vivo [3H]-DHA radiolabeling. Besides, our molecular docking revealed that DHA did not have the ability to bind FKBP12 or mTOR kinase domain, in contrast to the positive controls rapamycin (binding FKBP12) and INK128 (binding mTOR kinase domain). These data suggest that DHA inhibits mTORC1 via indirect mechanism(s). Further research revealed that DHA altered neither the phosphorylation of IGF-1R/PI3K/PTEN and ERK, nor the expression of REDD1, but induced phosphorylation of AMPKα (T172). Interestingly, DHA-induced mTORC1 inhibition was profoundly attenuated by inhibition with Compound C (AMPK inhibitor) or ectopic expression of dominant negative AMPKα2 (AMPKα2-DN), indicating that activation of AMPK links to inhibition of mTORC1. Collectively, the results support that DHA inhibits mTORC1 by activating AMPK. (Supported by the Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA, USA.)

Citation Format: Wang Liu, Jun Luo, Yoshinobu Odaka, Shile Huang. Inhibition of mTORC1 by dihydroartemisinin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 657.
Abstract 6570: Predicting drug effects on breast cancer cell shapes by converging image and transcriptomic profiles
Authors: Yin Zheng , Bakal Chris , Wong Stephen T.
Published: September 2020

Cell shapes have long served as hallmarks for cancer diagnostics and therapeutics evaluations. This work converges public available image and transcriptome profiles to build a drug-gene-shape networks, thus facilitate the usage of cell shape features as drug targets during the development of novel cancer therapeutics. We took advantage of a previous work (Sailem et. al., 2017) of image-omits modeling where 504 shape-correlated genes were identified for 20 cell shape features across 18 breast cancer cell lines. Transcriptome profiles from Broad Institute Clue.io database were used to identify small molecular compounds that can significantly impact those shape-correlated genes. As a result, 22 compound families of at least two members were identified as candidates that can significantly impact the shape-correlating gene sets for 13 shape features. Our work re-opens the doors for using cell shape as drug target and considering the impact on cell shapes during drug repurposing and design of combination therapies for breast cancer.

Compound families with the most significant correlations with gene sets regulating average cell areaCompound famileAverage scoremTOR inhibitors94.975Tubulin inhibitors94.615Adrenergic receptor antagonist94.545PKC inhibitor-93.580MEK inhibitor-94.137HDAC inhibitor-94.153

Citation Format: Zheng Yin, Chris Bakal, Stephen T. Wong. Predicting drug effects on breast cancer cell shapes by converging image and transcriptomic profiles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6570.
Abstract 6575: A novel AI-driven spatial genomics platform enables tumor enrichment and analysis of heterogeneity
Authors: Chaudhuri Bidhan , Konigsfeld Katie , Sanchez Marisa , Smith Jason , Laris Casey , Weston Claire , Butler John
Published: September 2020

Spatial genomics and digital pathology continue to advance at a rapid pace, generating large amounts of data. There is a critical unmet need for advanced tools to expedite spatial data generation, visualization, and analysis, to facilitate an improved understanding of health and disease, especially with the advancement of personalized cancer treatments. In this study we present a novel method for the extraction and analysis of genomic information from specific regions of interest (ROI) within a series of formalin-fixed paraffin embedded (FFPE) breast cancer adenocarcinoma tissue sections. An artificial intelligence-based tumor profiling algorithm first identified tumor, stroma, inflammatory, and necrotic regions within the tissue, and segmentation results were confirmed by a Board Certified Pathologist. Automated lysate extraction was then performed in the selected ROIs using ink-jet technology and novel ink chemistry. The process was performed directly on the tissue slide and the crude lysates generated were transferred to a standard collection tube for subsequent sequencing. DNA was extracted from the selected areas before being purified and analyzed using a multi-cancer panel. Library QC showed optimal coverage at an average of >95% uniformity. Sequencing results showed an average coverage of 95% at 250x across all amplicons of the panel. Variant calling was successfully performed. Sequencing data is presented to demonstrate the high coverage achieved using this workflow as well as the resulting tumor heterogeneity. This novel technology provides an efficient solution for the enrichment of tumor content with high sequencing coverage from FFPE tissue sections. Novel spatial data visualization and analysis techniques allowed a comprehensive assessment of tumor heterogeneity. Further studies on both DNA and RNA should be conducted to validate this approach for clinical molecular testing.

Citation Format: Bidhan Chaudhuri, Katie Konigsfeld, Marisa Sanchez, Jason Smith, Casey Laris, Claire Weston, John Butler. A novel AI-driven spatial genomics platform enables tumor enrichment and analysis of heterogeneity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6575.
Abstract 6585: Systems biology for investigating drug resistance mechanism of melanoma
Authors: Su Yapeng , Li Guideng , Ko Melissa , Cheng Hanjun , Zhu Ronghui , Xue Min , Robert Lidia , Levine Raphael , Ribas Antoni , Nolan Garry , Wei Wei , Plevritis Sylvia , Baltimore David , Heath James R.
Published: September 2020

Integrated proteomic and metabolic single-cell assays reveal multiple independent adaptive responses to drug tolerance in a BRAF-mutant melanoma cell line

Cancers commonly develop resistance against chemotherapeutics or targeted therapies through various types of genetic or non-genetic mechanisms. Non-genetic mechanisms have been shown to occur early on and can provide a latent reservoir of cells for the emergence of various different type of mechanisms, yet very limited understanding of process were resolve main from bulk analysis. Considering the heterogeneous nature of the tumor cells, a single-cell level characterization of the process worth detailed further investigation. Using MAPK inhibition of BRAF-mutant melanomas as a model system, we resolved that cells take different paths to go from drug-sensitive to drug-resistant state. Using a microfludic-based single-cell integrated proteomic and metabolic assay, we assayed for a panel of signaling, phenotypic, and metabolic regulators at four time points during the first five days of drug treatment. Dimensional reduction of the resultant data set, coupled with information theoretic analysis, uncovered a complex cell state landscape and identified two distinct paths connecting drug-naïve and drug-tolerant states. Cells are shown to exclusively traverse one of the two pathways depending on the level of the lineage restricted transcription factor MITF in the drug-naïve cells. The two trajectories are associated with distinct signaling and metabolic susceptibilities, and are independently druggable. Our results update the paradigm of adaptive resistance development in an isogenic cell population and offer insight into the design of more effective combination therapies.

Citation Format: Yapeng Su, Guideng Li, Melissa Ko, Hanjun Cheng, Ronghui Zhu, Min Xue, Lidia Robert, Raphael Levine, Antoni Ribas, Garry Nolan, Wei Wei, Sylvia Plevritis, David Baltimore, James R. Heath. Systems biology for investigating drug resistance mechanism of melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6585.
Abstract 6591: Antibody-based redirection of meditope-CAR T cells selectively kill antigen bearing tumor cells
Authors: Kuo Yi-Chiu , King Jeremy D. , Kuo Cheng-Fu , Kenyon Victor , Park Miso , Chang Wen-Chung , Stern Lawrence , Brown Christine E. , Williams John C.
Published: September 2020

Adoptive T cell transfer of engineered T cells with a chimeric antigen receptor (CAR) can harness the patients' immune system to recognize a specific antigen and redirect T cells to target disease. Conventional CAR T cells, however, are specific to a single antigen. While remarkably effective, antigen escape, due in part to the heterogeneous nature of a tumor, renders these CAR T cells ineffective. In order to address the disadvantage of conventional CAR T cells, we developed a switchable CAR T cell system. Specifically, we replaced the antigen-binding domain of a conventional CAR with a meditope and used meditope-enabled monoclonal antibodies (memAbs) to alter antigen specificity. As the T cell decoration is similar to how a bike rack is used to transport bicycles on cars, we have coined the technology a Fabrack. To fully examine our switchable CAR T cell system, Fabrack CAR Jurkat-NFAT-Luc cells were established to quantitate T cell activation through NFAT-regulated luciferase expression. Multiple memAbs with unique antigen specificity were combined with the Fabrack CAR Jurkat cells and target tumor cells and demonstrated the feasibility, specificity and robustness of the in vitro system. Next, native human T cells transduced with Fabrack showed dramatic tumor-killing effect when coupled with an antigen specific memAb. The engaged Fabrack T cells proliferated with remarkably increased CD107a and IFNγ, indicating functional activation of T cells. Live cell imaging demonstrated selective killing of target cells in the presence of corresponding memAbs. Furthermore, tumor burden in immunocompromised mice with OVCAR3 xenografts was largely reduced when mice were treated with Fabrack T cells and memAbs. Collectively, these studies suggest the feasibility of switchable CAR T cell and support further development for future clinical use in cancer immunotherapy.

Citation Format: Yi-Chiu Kuo, Jeremy D. King, Cheng-Fu Kuo, Victor Kenyon, Miso Park, Wen-Chung Chang, Lawrence Stern, Christine E. Brown, John C. Williams. Antibody-based redirection of meditope-CAR T cells selectively kill antigen bearing tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6591.
Abstract 6595: Targeting T cell lymphomas with CRISPR/Cas9-generated anti-CD70 allogeneic CAR-T cells
Authors: Karnik Sushant , Pham Minh Thu , Keerthipati Pooja , Padalia Zinkal , Carson Julie , Ho Tony , Morawa Ewelina , Will Matthias , Terrett Jonathan , Dequeant Mary-Lee
Published: September 2020

T cell lymphomas account for 10% to 15% of non-Hodgkin lymphomas and are diverse biologically and clinically. Unlike B-cell lymphomas, T cell lymphomas are rather resistant to conventional therapies, such as chemotherapy and antibody-based therapeutics. This resistance coupled with the diversity of the T cell diseases means that there is a significant unmet need across the T cell lymphoma subtypes. CD70 (CD27 ligand) is a candidate target antigen for T cell lymphomas and has been is the subject of clinical trials using an enhanced ADCC antibody (ARGX-110). Using flow cytometry and immunohistochemistry (IHC) methods, we analyzed the expression of CD70 in cell lines and clinical samples representing T cell lymphomas and found significant expression of CD70 in multiple types of T cell lymphoma, but at highly variable antigen density. CAR-T cells targeting CD70 may thus be a potent new therapy to tackle these diseases. However, the use of autologous CAR-T therapy against T cell lymphomas is complicated by the likelihood of creating lymphoma cells carrying the CAR construct. Thus, we sought to examine the potency of our allogeneic anti-CD70 CAR-T cells (CTX130) against T cell lymphoma cells. CTX130 has previously been shown to be active across a range of CD70 expression levels in other cell types representing other malignancies. Consistent with these prior observations, CTX130 exhibited high potency in vitro and in vivo against T cell lymphoma cells across a range of CD70 antigen density and representing different types of T cell lymphomas such as Sezary syndrome and cutaneous T cell lymphoma (CTCL). CTX130 may thus be a valid therapeutic to evaluate in T cell lymphoma patients.

Citation Format: Sushant Karnik, Minh Thu Pham, Pooja Keerthipati, Zinkal Padalia, Julie Carson, Tony Ho, Ewelina Morawa, Matthias Will, Jonathan Terrett, Mary-Lee Dequeant. Targeting T cell lymphomas with CRISPR/Cas9-generated anti-CD70 allogeneic CAR-T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6595.
Abstract 660: Combination of mTOR kinase inhibitors with Src inhibitors in PTEN-mt prostate cancer cells
Authors: Dai Yao , Siemann Dietmar
Published: September 2020

Although Src inhibitors are able to suppress malignant functions, their clinical outcomes are modest for the treatment of advanced prostate cancer, many of which harbor mutated PTEN. mTOR pathway is commonly hyperactivated in prostate cancer due to frequent PTEN mutation, and it is known that two mTOR complexes are both critical in prostate cancer progression. However, the functional role of mTOR kinase in Src inhibitor-mediated response remains unknown. In the current study, we combined mTOR kinase inhibitor AZD8055 and Src inhibitor dasatinib to evaluate functional behaviors in PTEN-deleted prostate cancer cells. Gene silencing was achieved by siRNA or shRNA transfection. Cell proliferation and clonogenic survival were performed by MTT and colony formation assay, respectively. “Wound-healing” scratch assay was conducted to monitor cell migration, and Matrigel-based transwell assay was applied to assess cell invasion. Protein expression was detected by Western blotting. We found that in PTEN-mt cells, C4-2B showed greater expression of p-Akt, p-S6, p-4EBP1 and p-eIF4E, all of which are mTOR complexes downstream components. Such molecular profile is consistent with functional observation that C4-2B cells are more resistant to dasatinib. Interestingly, single knockdown of either 4EBP1, S6RP or AKT showed little effects of inhibiting cell survival, while double knockdown of both 4EBP1 and AKT, cell survival was significantly inhibited. Consistently, two agents together showed significantly greater inhibition on proliferation and clonogenic survival than either single agent. Such combination effects were either additive or synergistic depending on the cell types, as reflected by combination index calculation. In addition, cell migration and invasion were inhibited in a greater extent by AZD8055 plus dasatinib, compared to either treatment alone. At the molecular level, co-targeting mTOR and Src kinases leads to strong inhibition of mTOR and Src pathways, such as FAK, Akt, S6RP and 4E-BP1. These data indicate that adding AZD8055 to dasatinib may achieve better treatment outcomes in PTEN-mt prostate cancer models. Such combination modality hold potential for future development as a novel strategy for PTEN-mutated prostate cancer patients who are less responsive to Src inhibitors.

Citation Format: Yao Dai, Dietmar Siemann. Combination of mTOR kinase inhibitors with Src inhibitors in PTEN-mt prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 660.
Abstract 6621: A combined VHH and small molecule approach to modulate the antitumor immunity function of ERAP1
Authors: Basse Nicolas
Published: September 2020

ERAP1 is a multifunctional enzyme trimming N-terminal amino acids of peptides. Its most characterised function is its role in generating peptide ligands in the endoplasmic reticulum for loading onto MHC class I (MHCI) molecules for presentation at the cell surface. Here it acts as an antigenic peptide editor influencing the peptide repertoire displayed at the cell surface to circulating CD8+ T cells and NK cells. ERAP1 modulates the immunopeptidome and could play a key role in tumor antigenicity.

ERAP1 is a highly dynamic enzyme and its conformation is crucial for intracellular immuno-signals and also various extracellular functions. The precise molecular mechanisms behind the secretion and conformational status of ERAP1 remain unclear. To study ERAP1, we combined a VHH and a small molecule approach. After Lama immunization, the selected ERAP1 binders were filtered for functional activity. To measure ERAP1 activity with relevant substrates, we developed a mass spectrometry assay and a fluorogenic substrate mimicking ERAP1 peptidic substrates. Nanobodies modulators of ERAP1 activity are currently used for structural studies.

In addition to the mechanistic studies, the team also developed ERAP1 small molecule modulators to induce or restore tumor immunogenicity. Herein, we report a chemical family of potent ERAP1 inhibitors that demonstrate structure activity relationship.

The nanobodies and small molecule modulators we developed provide useful tools to better understand the biology of ERAP1 in immune responses.

Citation Format: Nicolas Basse. A combined VHH and small molecule approach to modulate the antitumor immunity function of ERAP1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6621.
Abstract 6625: Cancer neoepitope identification in soluble HLA from patient plasma
Authors: Rumble Julie M. , Pisano Michael , Jones Richard , Ford Michael
Published: September 2020

The roles of the Major Histocompatibility Complexes (MHC) in anti-cancer immunity are well established. Cytotoxic immune cells can recognize MHC presentation of unique peptides from altered proteins on the surface of tumor cells (tumor neoantigens) or the downregulation of MHC itself and kill the affected cells. One of the hallmarks of cancer progression is the evasion of anti-tumor immunity by various mechanisms, including the upregulation of checkpoint proteins. Checkpoint inhibitors, which have recently seen dramatic success in the clinic in some cancers, act by inducing potent immune responses to tumor neoantigens. In addition, the promise of personalized medicine lies in the theoretical ability to teach the immune system through vaccination to recognize and kill tumor cells bearing MHC-presented neoantigens. Aside from MHC on cell surfaces, the presence of soluble MHC (sMHC) has been shown in a variety of bodily fluids, including blood. Soluble MHC may be an important component of the tumor arsenal for inhibiting anti-tumor immune responses. The precise function of sMHC may be disease-dependent, but it has been postulated to play an inhibitory role, presumably through signaling cognate TCRs in the absence of a cellular signal (signal 2). Presence of sMHC carrying cancer-associated neoantigens could predict success of vaccination therapy or checkpoint therapy. Furthermore, sMHC-associated peptides could help to identify neoantigens that have already induced T cell anergy and would hence not be good targets. Here, data are presented describing the plasma immunopeptidome of advanced non small-cell lung cancer subjects under a variety of treatment paradigms.

Citation Format: Julie M. Rumble, Michael Pisano, Richard Jones, Michael Ford. Cancer neoepitope identification in soluble HLA from patient plasma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6625.
Abstract 665: Discovery and preclinical characterization of PQR626: A potent, orally available, and brain-penetrant mTOR inhibitor for the treatment of tuberous sclerosis complex
Authors: Borsari Chiara , Keles Erhan , Rageot Denise , Melone Anna , Bohnacker Thomas , Batchelor Lucinda Kate , De Pascale Martina , Hebeisen Paul , Hillmann Petra , Fabbro Doriano , Wymann Matthias
Published: September 2020

Mechanistic target of rapamycin (mTOR), a key player in cell proliferation, growth and survival, is overactivated in tumors and neurological disorders.[1] Rapalogs have recently been explored to alleviate epileptic seizures in Tuberous Sclerosis Complex (TSC).[2] Herein, we combined pharmacophore features of PQR620[3], the first-in-class brain penetrant ATP-competitive mTOR kinase inhibitor showing efficacy in a TSC mouse model, and PQR617[4], a potent, highly selective mTOR inhibitor. An extensive chemical exploration of the morpholine ring led to the discovery of PQR626, a highly potent, selective, brain penetrant inhibitor of mTORC1/2 kinase. In male Sprague Dawley rats and female C57BL/6J mice, PQR626 displayed an excellent brain penetration compared to everolimus, which possessed a limited ability to cross the blood-brain-barrier (brain/plasma levels ~ 1.4:1 vs 1:92). An additional pharmacokinetic comparative study with everolimus and AZD2014 confirmed the superiority of PQR626 [brain/plasma levels: ~ 1:25 (AZD2014), ~ 1:61 (everolimus), ~ 1.8:1 (PQR626)]. PQR626 showed very good tolerability in mice (MTD 100-150 mg/kg). Efficacy studies, using mice with conditional inactivation of the Tsc1 gene primarily in glia (Tsc1GFAPCKO mice), were performed in a dose-range finding study. PQR626 (50 mg/kg, BID - twice a day) showed a significant effect on survival and significantly prevented/decreased mortality as compared to the vehicle. On the basis of its favorable pharmacological parameters, excellent brain penetration, safety profile and efficacy in Tsc1GFAPCKO mice, PQR626 qualifies as a novel mTOR inhibitor with potential application in the treatment of epilepsy and neurological disorders. [1] Wymann M. P. and Schneiter R. Nat. Rev. Mol. Cell Biol. 2008, 9, 162-176. [2] Krueger D. A. et. al. Neurology 2016, 87, 2408-2415. [3] Rageot D. et. al. J Med Chem. 2018, 61 (22), 10084-10105. [4] Borsari C. et. al. J Med Chem. 2019, 62 (18), 8609-8630.

Citation Format: Chiara Borsari, Erhan Keles, Denise Rageot, Anna Melone, Thomas Bohnacker, Lucinda Kate Batchelor, Martina De Pascale, Paul Hebeisen, Petra Hillmann, Doriano Fabbro, Matthias Wymann. Discovery and preclinical characterization of PQR626: A potent, orally available, and brain-penetrant mTOR inhibitor for the treatment of tuberous sclerosis complex [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 665.
Abstract 6674: Multi-dimensional time-series analysis to reveal pharmacodynamic changes induced by immunotherapy
Authors: Wang Jingjing , Wang Panpan , Zhang Wenli , Zhang Qiyao , Gu Qingyang , Ji Qunsheng
Published: September 2020

Background: Immune checkpoint inhibitors currently represent the most promising cancer therapeutics to produce durable responses. Many studies have highlighted checkpoint inhibitors' targets for future monotherapies or combination therapies. However, due to the complexity of tumor microenvironment, different types of cells exhibit different reaction time and order. To understand the MoA of immune checkpoint inhibitors in cell or animal models, time-series analysis will help select proper PD sampling time, as well as administration time & order in combination therapy. However, such analysis has limited application in syngeneic tumor models in the early stage of drug treatment due to the small tumor volumes.

Methods and Results: In this study, we combined NanoString technology and flow cytometry in an in-vivo efficacy study to explore the therapeutic response mechanism in mouse breast cancer EMT-6 model by the combinational treatment with anti-PD1 and an undisclosed compound A. Through time-course sampling of tumor tissues of EMT-6 xenograft tumors in the early stage of treatment, we found that anti-PD1 could enhance antigen-presentation and T cell priming as a single agent, and this effect could be enhanced by combination with compound A. In addition, we found the change of antigen-presentation induced by anti-PD1 occurs at RNA level, but not at protein level by flow cytometry analysis, which might imply to employ different time points to combine with RNA-targeted or protein-targeted drugs on T cell priming.

Summary: We have developed a time-series analysis combining in vivo efficacy study and NanoString technology with flow cytometry, to evaluate the dynamic changes of TME after immune checkpoint blocker treatment. This can be a powerful method to reveal time-dependent drug-induced pharmacodynamic changes, and help us to select the best time course for combination therapy.

Citation Format: Jingjing Wang, Panpan Wang, Wenli Zhang, Qiyao Zhang, Qingyang Gu, Qunsheng Ji. Multi-dimensional time-series analysis to reveal pharmacodynamic changes induced by immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6674.