bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 384: Lipid nanoparticle aided delivery of a STING agonist to treat pancreatic cancer
Authors: Shaji Sherin George , Patel Pratikkumar , Cheng Kun
Published: June 2022

The tumor microenvironment (TME) of pancreatic cancer is highly immunosuppressive and characterized by a huge population of cancer-associated fibroblasts, myeloid-derived suppressor cells, regulatory T cells, and a scarce number of effector immune cells. Stimulator of interferon genes (STING) is an endoplasmic reticulum receptor that plays a critical role in immunity. Agonists of STING have been shown to inflame the TME, reduce tumor burden and confer anti-tumor activity in mouse models. 2’, 3’ cyclic guanosine monophosphate adenosine monophosphate (cGAMP) is a high-affinity endogenous ligand of STING. However, the cytosolic delivery of exogenous cGAMP to antigen-presenting cells and tumor cells remains a challenge due to poor cellular permeability, poor stability, and rapid clearance. We address these limitations by encapsulating cGAMP in a lipid nanoparticle (cGAMP-LNP) engineered for efficient cytosolic delivery. The size and morphology of the cGAMP-LNP were characterized using dynamic light scattering and TEM, respectively. The in-vitro activity of the cGAMP-LNP was determined in the THP-1 Blue࣪ ISG reporter cell line, whereas the endosomolytic activity of the LNP was evaluated by red blood cell hemolysis assay. The cGAMP-LNP activates the IRF3 pathway in-vitro and shows concentration- and pH-dependent endosomolytic activity. Moreover, the cGAMP-LNP was evaluated for cytotoxicity in-vitro and were found non-toxic to human pancreatic duct epithelial cell line at bioactive concentrations. Collectively, the cGAMP-LNP represents a promising strategy to convert immunosuppressive pancreatic cancer to immune-inflamed pancreatic cancer.

Citation Format: Sherin George Shaji, Pratikkumar Patel, Kun Cheng. Lipid nanoparticle aided delivery of a STING agonist to treat pancreatic 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 384.
Abstract 3862: Microbial immunomodulation for enhancing immunotherapeutic efficacy in a patient-specific colorectal cancer chip
Authors: Shin Yong Cheol , Wu Alexander , Min Soyoun , Kim Daesung , Shin Woojung , Eckhardt S. Gail , Fleming R. Y. Declan , Kim Hyun Jung
Published: June 2022

Immune checkpoint inhibitors (ICI) targeting PD-1, PD-L1, or CTLA-4 have shown remarkable therapeutic outcomes in various cancers. However, the therapeutic efficacy of ICI-based immunotherapy in microsatellite stable (MSS) colorectal cancer (CRC) has been extremely limited. Recent studies strikingly revealed that the human gut microbiome controls the efficacy of immune checkpoint blockades. Yet, the effect of the gut microbiome on reinforcing the ICI efficacy in MSS tumors in CRC is unknown. Here, we suggest a microengineered CRC Chip that recapitulates the tumor microenvironment of individual CRC patients by introducing biopsy-derived colonic organoid epithelium. We have built a three-dimensional (3D) lumen-capillary interface under peristalsis-like motions and flow in an oxygen-controlled microenvironment. We leverage this pathomimetic CRC Chip to recapitulate the patient-specific tumor-microbiome-immune axis to demonstrate microbial immunomodulation on the tumor cells and cytotoxic tumor immunity. We co-cultured various human intestinal bacteria in a CRC Chip to assess the immunomodulatory contribution to the tumor epithelium. We found that living Bifidobacterium sp. significantly reduces the PD-L1 on the tumor surface, followed by elevated anti-tumor activity after CD8 T cell-mediated cytotoxic interactions. We envision that our CRC Chip model can potentially reduce the knowledge gap between the bench and bedside. Furthermore, our innovative CRC Chip model may potentially offer a platform to validate the “Druggable microbiome” and the utility of the patient’s avatar model for Precision Medicine.

Citation Format: Yong Cheol Shin, Alexander Wu, Soyoun Min, Daesung Kim, Woojung Shin, S. Gail Eckhardt, R. Y. Declan Fleming, Hyun Jung Kim. Microbial immunomodulation for enhancing immunotherapeutic efficacy in a patient-specific colorectal cancer chip [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 3862.
Abstract 3876: The importance of spatial analysis and multispectral imaging when evaluating tumor-immune interactions in glioblastoma
Authors: Haggerty Agnes E. , Mammadova Najiba , Barish Michael E. , Brown Christine E.
Published: June 2022

Glioblastoma (GBM) is an aggressive and almost universally fatal brain cancer. Glioblastoma tumor masses are highly vascularized and infiltrated with cells mediating both innate and adaptive immunity. The presence and organization of various immune cells within the tumor microenvironment varies between patients and within individual tumors. This variability in the immune landscape influences responses to different treatment modalities, including immunotherapies. Understanding reciprocal immune-tumor cell interactions will enhance design and implementation of novel therapies. Currently, review of GBM patient tissues by neuropathologists is done visually using single or low-plex biomarker panels whose complexity is constrained by limitations of traditional immunohistochemistry, including the lack of well characterized primary antibody clones, limited antibody selection due to species-dependent workflows, and inaccuracies such as false signal intensities introduced by autofluorescence (AF) and spectral overlap from traditional immunofluorescence imaging methods. Multiplex immunofluorescence (mIF) and multispectral imaging (MSI) allows for fast, accurate imaging on a single tissue section of up to eight fluorophores (using Phenoptics technology), cell type-specific AF spectra, and DAPI/Hoechst. It has been shown in other cancer types (i.e. lung cancer, breast cancer, melanoma) that spatial biology techniques including multiplex immunofluorescence is necessary to determine cellular densities and interactions in tissue context to better predict outcomes and stratify patient populations. Here, we have used pre-validated mIF kits that use Opal fluorophores together with the Vectra Polaris Automated Quantitative MSI system on human formalin-fixed, paraffin embedded GBM tissue samples to further characterize immune-tumor microenvironments, with consideration for spatial distributions of interacting cells. Pre-optimized immunofluorescent kits targeting immune cell subsets, checkpoint inhibitors, and other biomarkers have been shown to provide clinically relevant information in other cancer types (such as lung cancer and melanoma). Our study indicates that widespread use of these reagents, coupled with spatial analysis and complex phenotyping, would better characterize the complex immune landscape of tumors and enhance design and implementation of therapeutics for GBM.

Citation Format: Agnes E. Haggerty, Najiba Mammadova, Michael E. Barish, Christine E. Brown. The importance of spatial analysis and multispectral imaging when evaluating tumor-immune interactions in glioblastoma [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 3876.
Abstract 388: AI/ML-driven discovery of a novel proteoglycan for precision targeting of ADCs for disruption of stromal barriers and direct anti-tumor activity
Authors: Koch Elizabeth , London Max , Berkley Amy , Nixon Allison , Phippen Sean , White Kerry , Hanson Amanda , Cooper Samuel , Harvey Christopher , Briskin Michael
Published: June 2022

Background: While checkpoint inhibitors (CPIs) such as anti-CTLA-4 and anti-PD-1/L1 have demonstrated efficacy in a number of solid tumor indications, those with high stromal presence have been difficult to treat with minimal response observed. We aimed to use a proprietary machine learning/artificial intelligence platform to identify novel stromal targets to relieve this immunosuppressive barrier and increase CPI responsiveness in difficult to treat indications.

Methods: Based on bioinformatic analysis using our single cell RNA atlas, we assessed cancer-associated fibroblasts (CAFs)/fibroblastic cells in cancer tissue for identification of novel targets, including proteoglycans. Antibodies were generated by immunization of humanized mice, and lead antibodies were tested for activity in inhibiting cell adhesion and were further characterized for staining of both CAFs as well as tumor cells. ADCs were developed and tested in vitro for selective tumor cell killing.

Results: Bioinformatic analysis identified a unique subset of cancer-associated fibroblasts, termed ecmCAFs, which demonstrated selective expression of Collagen Triple Helix Repeat Containing 1 (CTHRC1). This highly-selective expression pattern suggests it may be ideal as a target for alternative modalities, including ADC targeting or specific T cell activation. In addition, we identified that in certain tumor types, such a triple negative breast cancer and pancreatic ductal adenocarcinoma (PDAC), CTHRC1 is also highly expressed by cancer cells within the tumor and shows a more favorable expression profile for ADC targeting when compared to other stromal proteins such as FAP and LRRC15. We have confirmed surface expression and binding of CTHRC1 by our lead antibodies and have observed robust internalization on both human and mouse cancer cell lines. In vitro killing of tumor cells by ADCs and in vivo PD and efficacy will be presented on both ADCs and naked antibodies.

Conclusions: We have identified CTHRC1 as a novel proteoglycan expressed by both ecmCAFs and tumor cells that appears to be an ideal target for both direct inhibition of stromal barrier function as well as targeting of cytotoxic payloads as an ADC. CTHRC1 expression is more selective than the classical markers FAP and LRRC15, both of which have been previously developed as ADCs.

Citation Format: Elizabeth Koch, Max London, Amy Berkley, Allison Nixon, Sean Phippen, Kerry White, Amanda Hanson, Samuel Cooper, Christopher Harvey, Michael Briskin. AI/ML-driven discovery of a novel proteoglycan for precision targeting of ADCs for disruption of stromal barriers and direct anti-tumor activity [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 388.
Abstract 3891: Efficacy of dinaciclib in hepatoblastoma through inhibition of cyclin dependent kinases
Authors: Srivastava Rohit K. , Patel Roma , Espinoza Andres F. , Yu Yang , Whitlock Richard S. , Larson Samuel , Badachhape Andrew , Yang Jianhua , Woodfield Sarah E. , Vasudevan Sanjeev A.
Published: June 2022

Introduction: Hepatoblastoma (HB) is the most frequent liver malignancy of childhood with an annual incidence of 1.5 per million and a survival rate less than 50%. Due to significant side effects and ineffectiveness of standard chemotherapy for relapse refractory disease, the treatment options are now shifting towards targeted therapies. Cyclin-dependent kinases (CDKs) are potential cancer therapeutic targets because of their critical role in promoting cell growth and regulating transcriptional processes. This study aims to assess the therapeutic efficacy of dinaciclib, cyclin-dependent kinase inhibitor, with HB in preclinical in vitroand in vivo models.

Method: Dinaciclib was tested in patient derived cell lines (PDCL) from different patient derived xenograft (PDX) models using CellTiter-Glo Luminescent Viability assays. Dinaciclib was also assayed in vitro for cytotoxicity (MTT assay) and proliferation (CCK-8 assay). Differential expression of CDKs was checked by microarray analysis between normal liver and HB patients. Results were confirmed by immunoblotting assays. Immunoblotting assays were also used to see the effect of dinaciclib on CDK 1/2/5/9 and to assess the induction of apoptosis (PARP cleavage) in vitro. Dinaciclib was also tested in our orthotopic PDX models of high-risk HB. MRI and alpha-fetoprotein (AFP) ELISA were done to evaluate tumor growth. Tumor weights and volumes, as well as, immunohistochemistry were also used to assess effects of dinaciclib in vivo.

Result: Dinaciclib suppressed cell proliferation and viability in a dose-dependent manner in all HB cell lines tested with IC50 in the low micromolar range (HepG2- 3.75; HepT1- 0.007; Huh6- 1.42; HB17- 0.72). Microarray analysis showed significant upregulation of CDK1 (p=0.0003), CDK2 (p=0.00023) and CDK9 (p=0.015) in HB patient samples, however, immunoblotting with dinaciclib-treated cells showed decreased expression of CDKs 1/2/5/9 as well as induction of apoptosis. Dinaciclib also showed in vivo inhibition of tumor growth compared to placebo in both PDX models (HB47: relative tumor volume p = 0.03, T/C ratio= 0.36, tumor weight p = 0.02; HB52: relative tumor volume p = 0.06, T/C ratio= 0.16, tumor weight p = 0.07).

Conclusion: Dinaciclib treatment showed promising effects with preclinical HB cell lines and PDX models.

Citation Format: Rohit K. Srivastava, Roma Patel, Andres F. Espinoza, Yang Yu, Richard S. Whitlock, Samuel Larson, Andrew Badachhape, Jianhua Yang, Sarah E. Woodfield, Sanjeev A. Vasudevan. Efficacy of dinaciclib in hepatoblastoma through inhibition of cyclin dependent kinases [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 3891.
Abstract 3892: High-throughput combination screen for identifying novel therapies against high-risk neuroblastoma
Authors: Radke Katarzyna , Hansson Karin , Saarela Jani , Ianevski Aleksandr , Ianevski Philipp , Baudet Aurélie , Magnusson Mattias , Bexell Daniel
Published: June 2022

Neuroblastoma is a childhood solid tumor commonly located in the adrenal gland. High-risk neuroblastoma patients undergo multidrug and multi-interventional treatment but despite these efforts many patients develop resistance to chemotherapy and eventually relapse. Neuroblastoma patients face an urgent need of novel therapeutic strategies that could effectively combat the disease.

In a recent study we identified kinesin spindle protein (KSP) as a highly effective target in high-risk neuroblastoma. Pharmacological inhibition of KSP resulted in complete regression of a subset of neuroblastoma subcutaneous PDX tumors and tumor growth delay in orthotopic PDXs (Hansson, Radke et al, Sci. Transl. Med 2020). The purpose of the present study is to identify novel synergistic drug combinations effective against neuroblastoma and investigate their translational potential. We designed, optimized, and performed a high-throughput combination screen using 3D tumor organoids that represent three MYCN-amplified neuroblastoma tumors. KSP inhibition was tested in combination with the FIMM drug set of 527 approved and investigational drugs.

Screening was performed stepwise:

• Step 1: High-throughput drug screening 527 drug combinations and machine learning prediction (DECREASE tool) of drug combination synergy based on minimal required input.

• Step 2: Screening of 6x6 dose-response matrices of top 26 predicted hits in high-risk neuroblastoma models.

• Step 3: Screening of 6x6 dose-response matrices of top 26 hits in CD34+ cord blood controls to identify compounds that have low toxicity towards proliferating fraction of hematopoietic cells.

Overall, we identified seven drug candidates (P1-P7). They classify as: kinase inhibitors, differentiating modifiers, and one hormone therapy. Screened combinations were chosen based on selective efficacy (difference between anti-neuroblastoma efficacy and efficacy towards CD34+ cord blood controls). Identified drug combinations are common among three high-risk neuroblastoma models and have most synergistic area score (MSA) over five. We further plan to validate seven hits in neuroblastoma models in vitro, investigate MOA of the most promising combination and test it in PDX models in vivo.

Citation Format: Katarzyna Radke, Karin Hansson, Jani Saarela, Aleksandr Ianevski, Philipp Ianevski, Aurélie Baudet, Mattias Magnusson, Daniel Bexell. High-throughput combination screen for identifying novel therapies against high-risk neuroblastoma [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 3892.
Abstract 3921: Precise spatial multiplexing for immune profiling in clinical FFPE tumor samples with ChipCytometry࣪
Authors: Gamber Kevin , Christians Arne , Brooks Jennifer , Kwarta Karen , Winkeler Crystal
Published: June 2022

Highly multiplexed spatial biomarker analysis has the potential to inform the discovery of novel biomarkers and treatments with the potential to improve clinical outcomes. Clinical diagnoses and treatment decisions have traditionally been made with spatially resolved techniques like immunohistochemistry, that offer a wealth of information about cellular morphology and the spatial context of cells but are limited to a single plex. Newer technologies, such as single-cell RNA sequencing, identify cell subpopulations within tissue but do not capture their spatial distribution or intercellular interactions in situ. Here we present the analysis of clinical FFPE tumor samples using ChipCytometry, a novel precise spatial multiplexing technology that offers high-plex biomarker analysis, without compromising spatial context. ChipCytometry combines iterative immuno-fluorescent staining with high-dynamic range imaging to facilitate quantitative phenotyping with single-cell resolution. Standard FCS files are generated from multichannel OME-TIFF images, enabling identification of cellular phenotypes via flow cytometry-like hierarchical gating. In this study, we identify and quantify immune and epithelial cell phenotypes and subtypes in melanoma and non-small cell lung cancer FFPE samples. The results show precise expression levels for each marker in the assay in each individual cell in the sample, while maintaining spatial information about each cell. Spatial analysis of the samples reveals quantifiable heterogeneity of immune cell infiltration within the tumor samples, demonstrating the utility of the ChipCytometry platform for the in-depth immune profiling in clinical FFPE samples.

-Bruker Confidential-

Citation Format: Kevin Gamber, Arne Christians, Jennifer Brooks, Karen Kwarta, Crystal Winkeler. Precise spatial multiplexing for immune profiling in clinical FFPE tumor samples with ChipCytometry࣪ [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 3921.
Abstract 3943: Discovery of a novel menin-MLL inhibitor for potential treatment of MLLr leukemias and NPM1c AML
Authors: Hu Taishan , Deng Zhilin , Li Honghai , Ma Xiaochu , Shen Quanrong , Zhang Lei , Sun Peihua , Hua Ye , Huang Byran
Published: June 2022

Rearrangement of the mixed lineage leukemia (MLL) gene occurs in up to 10% of acute leukemias and is particularly prevalent in infant acute leukemias (~80% of infant ALL cases). MLL-rearranged (MLLr) leukemia is one of the high-risk types of leukemia with aggressive nature, resistance to therapy, and high frequency of early relapse, and currently there are no targeted therapies for its treatment. So, there is huge unmet medical needs for MLLr leukemias. The protein protein interaction (PPI) between menin and MLLr is critical to the pathogenesis of MLLr-driven leukemias by deregulation of the HOXA and MEIS1 genes. It has been demonstrated by both preclinical and clinical studies that blockage of this PPI with menin inhibitors has potential therapeutic effects on MLLr associated acute leukemias. NPM1 mutation (NMP1c) is a frequently mutated gene found in AML, and NMP1 mutated AML depends on menin-MLL interaction for leukemogenesis. Given the fact that wild type MLL and MLLr have similar leukemogenesis-driven target genes like HOXA and MEIS1, as well as the same binding pocket on menin, it was therefore conceived that menin inhibitor could be useful to the treatment of NPM-mutated AML, which was again supported by both preclinical and clinical data. Herein, we would like to report the discovery of our proprietary menin inhibitor. Taking advantage of a couple of key interactions with menin protein, our compounds has relatively low molecular weight for a challenging PPI target, however, demonstrated comparable to or even better potency than those menin inhibitors in clinical trials. Besides in vitro pharmacology data, the DMPK, PK/PD and in vivo efficacy in animal model will be discussed too.

Citation Format: Taishan Hu, Zhilin Deng, Honghai Li, Xiaochu Ma, Quanrong Shen, Lei Zhang, Peihua Sun, Ye Hua, Byran Huang. Discovery of a novel menin-MLL inhibitor for potential treatment of MLLr leukemias and NPM1c AML [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 3943.
Abstract 3946: Development of selective small molecule AR-V7 inhibitors for prostate cancer treatment
Authors: Au CheukMan Cherie , Kim Seaho , Vatsa Prerna , Jamalruddin Mohd Azrin Bin , Miller Michael , Meinke Peter T. , Giannakakou Paraskevi
Published: June 2022

Castration resistance prostate cancer (CRPC) is a lethal disease in which the expression of ligand-independent androgen receptor (AR) splice variants (AR-Vs) is associated with worse clinical outcomes. AR-V7 is the most prevalent variant in CRPC, it lacks the ligand binding domain and is constitutively active in the nucleus. We and others have showed that, AR-V7 expression confers resistance to the AR signaling inhibitors (abiraterone/enzalutamide) and to taxanes in vivo and in patients with metastatic CRPC. Since abiraterone/enzalutamide and taxanes represent the two most effective therapeutic modalities for men with CRPC, the development of selective AR-V7 inhibitors is a high priority, clinically unmet need. AR-V7 shares with full-length AR (AR-fl), high sequence homology, largely overlapping cistromes and gene transactivation profiles. To develop selective AR-V7 inhibitors, we sought to identify unique biological features of AR-V7, that differentiate it from AR-fl and therapeutically exploit them. Mechanistic studies showed that AR-V7 utilizes a unique nuclear import pathway, not shared by AR-fl. Using fluorescently tagged-AR-fl or AR-V7 proteins in conjunction with live cell imaging, FRAP assays and pathway inhibitors, we showed that AR-V7 exhibits fast nuclear import kinetics partially mediated by the dimerization D-box domain, independently of microtubules and importin α/β. Taken together, these data suggest that AR-V7 nuclear import mechanism is distinct providing a window of therapeutic opportunity to selectively target it. To identify AR-V7 selective inhibitors, we designed and performed a high throughput enzyme complementation screening (HTS) assay using nuclear AR-V7 as a surrogate for AR-V7 activity. We screened a chemical library of ~170K compounds and identified hit compounds inhibiting AR-V7 by proteasomal degradation. Among the degraders, we observed 2 main modes of action: I. compounds selectively degrading AR-V7 and II. compounds degrading both AR-fl and AR-V7. Ongoing efforts include medicinal chemistry for lead compound optimization and target validation experiments.In conclusion, we identified first-in-class selective AR-V7 inhibitors, with the potential to be clinically combined with existing, but mechanistically unrelated AR signaling inhibitors. Further mechanistic studies will elucidate their potential for future clinical development.

Citation Format: CheukMan Cherie Au, Seaho Kim, Prerna Vatsa, Mohd Azrin Bin Jamalruddin, Michael Miller, Peter T. Meinke, Paraskevi Giannakakou. Development of selective small molecule AR-V7 inhibitors for prostate cancer treatment [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 3946.
Abstract 3957: Receptor Activator of NF-κB (RANK) confers resistance to chemotherapy in AML and associates with dismal disease course
Authors: Clar Kim L. , Weber Lisa M. , Schmied Bastian J. , Heitmann Jonas S. , Marconato Maddalena , Tandler Claudia , Schneider Pascal , Salih Helmut R.
Published: June 2022

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Untreated, AML leads to death within months after first symptoms. Combinatorial chemotherapy, mostly using a pyrimidine analog together with an anthracycline, made this previously incurable disease medicable, and response to remission-inducing chemotherapy strongly correlates with patient outcome. Despite advances in treatment over the recent years, AML still has a poor prognosis with an average 5-year survival rate of 30%. A better understanding of the molecular mechanisms predicting and affecting treatment efficacy is key to improve disease control and outcome. Here we studied the expression, prognostic relevance and functional role of the tumor necrosis factor receptor (TNFR) family member Receptor Activator of Nuclear Factor (NF)-κB (RANK) in AML. We conducted an experimental ex vivo study using leukemic cells of 54 AML patients. Substantial surface expression of RANK was detected on the primary AML cells in 35% of the analyzed patients. We further found that RANK signaling induced the release of the cytokines IL-6, IL-8, TNF and IL-10 acting as growth and survival factors for the leukemic cells and mediated resistance of AML cells to treatment with doxorubicin and cytarabine, the most commonly used cytostatic compounds in AML treatment. In line, prevalence of RANK expression on leukemic cells correlated with dismal disease course of patients as revealed by reduced overall survival. Together, our results show that RANK plays a yet unrecognized role in AML pathophysiology and resistance to treatment. Moreover, the association of RANK expression with adverse disease course identifies RANK as potential “functional” prognostic marker and putative target for therapeutic intervention to improve treatment response of AML patients.

Citation Format: Kim L. Clar, Lisa M. Weber, Bastian J. Schmied, Jonas S. Heitmann, Maddalena Marconato, Claudia Tandler, Pascal Schneider, Helmut R. Salih. Receptor Activator of NF-κB (RANK) confers resistance to chemotherapy in AML and associates with dismal disease course [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 3957.
Abstract 3992: Modular ddPCR assay design for highly sensitive single nucleotide variant detection
Authors: Heine Sarah , Beams Ashley , Riccitelli Nathan , Tangri Shabnam
Published: June 2022

Introduction: Single-nucleotide variants (SNVs) are key drivers of both oncogenesis and emergent resistance to targeted therapies. Although high-throughput Identification of SNVs can be achieved with Next Generation Sequencing (NGS) technologies, the cost is significant, and proportional to the desired sensitivity of variant detection. Conversely, while only small numbers of SNVs can be queried in an individual quantitative PCR (qPCR) or droplet digital PCR (ddPCR) reaction, a fully optimized qPCR or ddPCR assay can detect SNVs at very low frequencies for a fraction of the cost of sequencing. Herein, we present a generalizable approach to PCR assay design, using a combination of mutation-sensitive induced mismatch primers, wild-type suppressors, and TaqMan probes, that permits detection of SNVs below 0.1%.

Method: Known oncogenic hotspot regions were selected for development of mutation-detecting PCR assays. For each hotspot, a mutation-specific-mismatch primer, suppressor, and probe (MSMPSP) cassette was designed consisting of 1) a mutation-bearing forward primer with engineered mismatches, 2) a wild-type, extension-locked blocker oligo, 3) a fluorescent TaqMan probe, and 4) a downstream reverse primer. Designed mutation assays were evaluated using plasmid control sequences, remnant clinical specimens, and healthy donor peripheral blood samples spiked with known quantities of mutation-bearing plasmid or cell lines and processed on the BioRad AutoDG/QX200 droplet digital PCR system.

Results: Variant detection below 0.1% was achieved for all tested mutations, including those residing in homopolymeric and GC-rich regions, from as low as 100 ng DNA input, in a single day. At higher variant frequencies accessible to multiple platforms, results with MSMPSP-based ddPCR assays exhibited excellent concordance with NGS and qPCR results and yielded linear signal decline down to 0.01% variant allele frequency. Overall assay sensitivity was proportional to DNA input, and improved detection limits were achieved by increased DNA input and combined well analysis.

Conclusion: Utilizing the MSMPSP approach, coupled with ddPCR, variant detection for targeted mutations below 0.1% is achievable in diverse genomic contexts. Furthermore, simple design principles and flexible probe-binding sites permit rapid development of new assays, allowing critical mutations to be tracked as they are discovered. While multiplexing capabilities are reduced compared with NGS, short time-to-results, coupled with low cost-per-sample for MSMPSP ddPCR, permits faster treatment decisions at reduced cost to the healthcare system.

Citation Format: Sarah Heine, Ashley Beams, Nathan Riccitelli, Shabnam Tangri. Modular ddPCR assay design for highly sensitive single nucleotide variant detection [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 3992.
Abstract 4031: PRMT1 regulates EGFR and Wnt signaling pathways and is a promising target for combinatorial treatment of breast cancer
Authors: Suresh Samyuktha , Huard Solène , Némati Fariba , Dakroub Rayan , Nicolas André , Meseure Didier , Decaudin Didier , Roman-Roman Sergio , Dubois Thierry
Published: June 2022

Identifying new therapeutic strategies for triple-negative breast cancer (TNBC) patients is a priority as these patients are highly prone to relapse after chemotherapy. We found that protein arginine methyltransferase 1 (PRMT1) is highly expressed in all breast cancer subtypes. Its depletion decreases cell survival by inducing DNA damage and apoptosis in various breast cancer cell lines. Transcriptomic analysis revealed that PRMT1 regulates the epidermal growth factor receptor (EGFR) and the Wnt signaling pathways, reported to be activated in TNBC. The enzymatic activity of PRMT1 is also required to stimulate the canonical Wnt pathway. Type I PRMT inhibitors decrease breast cancer cell proliferation and show anti-tumor activity in a TNBC xenograft model. These inhibitors display synergistic interactions with some chemotherapies used to treat TNBC patients, as well as erlotinib, an EGFR inhibitor. Therefore, targeting PRMT1 in combination with these chemotherapies may improve the existing treatments for TNBC patients.

Citation Format: Samyuktha Suresh, Solène Huard, Fariba Némati, Rayan Dakroub, André Nicolas, Didier Meseure, Didier Decaudin, Sergio Roman-Roman, Thierry Dubois. PRMT1 regulates EGFR and Wnt signaling pathways and is a promising target for combinatorial treatment of 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 4031.
Abstract 4054: Investigation of antitumor activity induced by antifungal agents in human metastatic melanoma cells
Authors: Jira Lukas R. , Foster Hattie M. , Hall Hannah K. , Koh David W.
Published: June 2022

Melanoma remains as the most aggressive and lethal form of skin cancer for which new therapies are still needed. Current treatment options include conventional chemotherapy, immunotherapy, and targeted therapy. Disadvantages of these therapies, as well as other factors such as refractiveness, have contributed to poor outcomes in melanoma patients. Antifungal drugs potentially represent a novel class of anticancer agents. Our objective here was to analyze the ability of these agents to eradicate primary human metastatic melanoma cells. Antitumor effects were investigated using cell growth and proliferation assays, flow cytometry (FACS), and cell death assays. Wild-type human melanoma cell lines or those possessing the BRAF V600E mutation or the NRAS Q61R mutation were analyzed. Clotrimazole treatment led to profound decreases in cell growth in wild-type and NRAS mutant melanoma cells. Significant, but lesser effects were observed in BRAF mutants. However, treatment with econazole caused substantial reductions in cell growth in wild-type and BRAF mutant cells. These results were corroborated by cell death analyses and FACS, where clotrimazole treatment caused greater than 80% cell death in wild-type and NRAS mutant cells, while econazole caused greater than 75% cell death in BRAF mutants. Taken together, these preliminary studies indicated that clotrimazole and econazole were effective in inducing antitumor effects in human metastatic melanoma cell lines, but with differing degrees of efficacy. This data suggests that melanoma tumors of each known genotype can potentially be treated with antifungal agents. In conclusion, this study demonstrated the ability of antifungal agents to successfully treat human metastatic melanoma cells. Since each agent displayed differential effects for each type of mutation, it is possible that these antifungal agents affect different molecular targets in each melanoma cell line. These antifungal agents thus appear to be potential alternative treatments, as well as affordable and widely accessible therapies, for melanoma patients in the future.

Citation Format: Lukas R. Jira, Hattie M. Foster, Hannah K. Hall, David W. Koh. Investigation of antitumor activity induced by antifungal agents in human metastatic melanoma cells [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 4054.
Abstract 4069: The potent quadruplex-binding compound SOP1812 shows anti-tumor activity in patient-derived in vivo models of pancreatic cancer
Authors: Neidle Stephen , Ahmed Ahmed , Angell Richard , Oxenford Sally
Published: June 2022

The compound SOP1812, a tetra-substituted naphthalene diimide derivative has been previously disclosed to have single-digit nM anti-proliferative activity in a panel of human pancreatic ductal adenocarcinoma (PDAC) cell lines (Ahmed et al., ACS Med Chem Lett, 2020, 11, 1634-1644). It also has significant anti-tumor activity in the MIA-PACA2 xenograft model for PDAC as well as in the more demanding KPC model. We now report that this compound also shows significant anti-tumor activity in three patient-derived xenograft (PDX) models for PDAC. One is from a primary PDAC tumor at stage I and the two others from patients with stage IV PDAC (PDX tumor studies undertaken under contract by Champions Oncology). Transcriptome studies on cellular responses to SOP1812 have previously demonstrated that expression of a number of oncogenes and related signaling pathways are down-regulated by this agent. The promoter regions of these genes have an over-representation of quadruplex-containing sequences and several have also been shown by us to be also down-regulated in treated tumor xenografts. Our working hypothesis that the mechanism of action of SOP1812 in vivo involves targeting of quadruplex-containing genes, is also supported by the PDX data since these tumors have elevated levels of several quadruplex-containing genes previously identified by us in cells and in vivo. SOP1812 is bio-available at therapeutic doses and is well tolerated at these levels in these animal models. It is currently being evaluated as a candidate for clinical development by Qualigen Therapeutics Inc.

Citation Format: Stephen Neidle, Ahmed Ahmed, Richard Angell, Sally Oxenford. The potent quadruplex-binding compound SOP1812 shows anti-tumor activity in patient-derived in vivo models of pancreatic 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 4069.
Abstract 4070: Caveolin-1 functions as a potential predictive biomarker for JNTX-101, an albumin encapsulated gemcitabine prodrug
Authors: Cui Tiantian , Corrales-Guerrero Sergio , Maneval Dan , Monnig Curtis , Kearney Patrick , Ellis Sam , Williams Terence M.
Published: June 2022

Purpose: Human serum albumin (HSA) is an attractive candidate as a carrier in developing novel albumin-bound nanoparticle therapeutic drugs. Gemcitabine has been used in the treatment of solid tumors, including non-small cell lung, pancreatic, bladder, and breast cancers. Our previous work in pancreatic and lung cancer models demonstrates that caveolin-1 (Cav-1) enhances uptake of albumin and albumin-conjugated drugs. The aim of this study is to investigate whether the presence of Cav-1 expression alters therapeutic efficacy of JNTX-101, a novel albumin encapsulated gemcitabine prodrug.

Experimental design: We first determined the treatment efficacy of JNTX-101 in a panel of 4 pancreatic/lung cancer cell lines (MIAPaCa-2, PANC-1, H23, and HCC15) by alamarBlue cell viability assay. We further established 4 pairs of Cav-1 isogenic cells stably expressing shRNA targeting Cav-1 (shCav-1) or scrambled control shRNA (shCtrl). We investigated whether the absence of Cav-1 expression alters sensitivity of JNTX-101 using alamarBlue cytotoxicity assay and western blotting in both high and low serum conditions. In addition, we tested whether JNTX-101 treatment can alter expression and cellular distribution of Cav-1 as well as other caveolae resident proteins, such as Cav-2 and Cavin-1.

Results: Differences in Cav-1 expression resulted in different uptake of albumin prodrug conjugate and Cav-1 depletion attenuated sensitivity of the cells to JNTX-101. Decreased Cav-1 expression markedly reduced JNTX-101-induced apoptosis in a panel of cells in both high and low serum conditions, but particularly in low serum conditions. In addition, our data showed both serum starvation and JNTX-101 independently increased Cav-1 expression in a time-dependent manner (24hrs, 48hrs, 72hrs) similar to gemcitabine, with peak level at 48 hrs post treatment, while other caveolae resident proteins were minimally affected.

Conclusions: Our preliminary results suggest that JNTX-101 effectively inhibits cell viability in pancreatic/lung cancer cells and Cav-1 expression dictates sensitivity to JNTX-101, at least partially through reduced apoptotic cell death. These data indicate that Cav-1 may be a potential predictive biomarker of JNTX-101 sensitivity, especially in nutrient deprived conditions. Further study will be performed to explore the impact of Cav-1 expression on JNTX-101 sensitivity in mouse models of pancreatic/lung cancer.

Citation Format: Tiantian Cui, Sergio Corrales-Guerrero, Dan Maneval, Curtis Monnig, Patrick Kearney, Sam Ellis, Terence M. Williams. Caveolin-1 functions as a potential predictive biomarker for JNTX-101, an albumin encapsulated gemcitabine prodrug [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 4070.
Abstract 4081: Leveraging single-cell RNA sequencing data to design multi-targeting SeekRRNA therapeutics
Authors: Massey Susan , Hsiao Richard , Yadav Shweta , Azorsa David , Mousses Spyro , Kiefer Jeffrey A.
Published: June 2022

SeekR™ chimeric molecules consist of dual flanking RNA aptamers designed to bind specific proteins (BINDERS), joined by a double stranded RNA bridge that encodes multiple siRNAs designed to silence select disease target genes (SILENCERS). Aptamers are folded nucleic acid chains that act much like chemical antibodies in that they can bind proteins in a sequence-specific manner. By identifying RNA aptamers capable of seeking and binding to specific cell surface protein receptors, SeekR™ RNA therapeutics can be engineered to self-deliver to a particular receptor defined tissue type. For example, a SeekR™ can be directed to prostate tumors that selectively express PSMA. The combination of two RNA aptamer binders with two siRNA silencers thus enables the selective delivery of the siRNAs to tumor cells expressing the binder target, wherein it directs sequence-specific silencing of cancer target genes. To identify and prioritize the right combination of SeekR targets, we have taken advantage of single-cell sequencing (scRNA-seq) data sets. By analyzing scRNA-seq data, we can identify specific cell populations within tumors to co-target with an aptamer and siRNA. We leveraged several publicly available colon and non-small cell lung cancer scRNA data sets for this analysis. A custom analysis pipeline was applied to these data to identify specific cell types within tumor samples. We then profiled specific cancer targets and determined percentages of target co-expression within a single tumor compared to normal cells. This provides information for cell-type-specific targeting to reduce potential toxicity within non-tumor tissue. In addition to targeting aptamers, we performed ligand-receptor analyses to prioritize specific siRNA targets in particular cell types within the tumor environment. This analysis revealed novel combinations of binders and silencers that will more precisely deliver the right multi-targeted gene silencing to the correct cell types. In summary, scRNA-seq data provide a rich resource of data to direct SeekR™ development and optimization.

Citation Format: Susan Massey, Richard Hsiao, Shweta Yadav, David Azorsa, Spyro Mousses, Jeffrey A. Kiefer. Leveraging single-cell RNA sequencing data to design multi-targeting SeekRRNA therapeutics [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 4081.
Abstract 4106: Curating protein complexes from multiple resources and validate their co-regulations at RNA/protein level in pan-cancer
Authors: Song Yizhe , Zhou Daniel Cui , Li Yize , Wang Liang-Bo , Wendl Michael C. , Getz Gad , Ding Li
Published: June 2022

Protein complexes are sets of proteins that not only physically interact with each other, but also play important roles in progression of diseases like cancer. However, little is known about the co-regulation patterns of complexes and how many of them could be potential therapeutic targets. We collected 16,680 complexes from CORUM, Reactome, Complex Portal, and Havugimana PC. Harmonization of subunits of complexes by UniProt ID removed 4,112 redundant complexes. We further found that 3,018/12,559 complexes have at least one cancer driver gene involved, of which 2,408 complexes were found to have druggable genes, according to CiVic and OncoKB. Co-regulation patterns of 12,559 complexes were annotated utilizing gene expression and protein expression data from 1,101 cases across 10 cancer types (BR, COAD, OV, UCEC, HNSCC, PDAC, LSCC, LUAD, GBM, and ccRCC) from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Pairwise correlations across subunits of each complex at protein and RNA levels were calculated separately. Correlation >0.8 would suggest strong interaction of the subunits of the complex. Protein-only co-regulation complex was declared for protein pairwise correlation >0.8 and RNA pairwise correlation <0.5. RNA-only co-regulation complex was defined vice versa. We found more protein-only co-regulation complexes than RNA-only co-regulation complexes. This validated that RNA expression doesn’t always correlate with protein expression and few complexes were co-regulated at both RNA and protein levels. Our study suggests the value of utilizing proteome data to understand the formation of complex and inner relationships between subunits of complexes.

Citation Format: Yizhe Song, Daniel Cui Zhou, Yize Li, Liang-Bo Wang, Michael C. Wendl, Gad Getz, Li Ding. Curating protein complexes from multiple resources and validate their co-regulations at RNA/protein level in pan-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 4106.
Abstract 4112: Patient derived organoids predict clinical response: A patient in the lab
Authors: Verissimo Carla S. , Smabers Lidwien , Wensink Emerens , Koedoot Esmee , Huismans Maarten , Barón Celia Higuera , Korporaal Ricardo , Teal Emma , Pitsa Katerina-Chara , van Oosten Edwin , Nair Roshni , Valkenburg Liselot , Cirkel Geert , Brousali Anneta , Salij Jorieke , Overmeer René , Braat Manon , Elias Sjoerd , Vries Robert , Kranenburg Onno , Koopman Miriam , Boj Sylvia F. , Roodhart Jeanine
Published: June 2022

Colorectal cancer (CRC) is the third leading cause of cancer and cancer-related death. A large fraction of the CRC patients diagnosed with de novo metastatic disease do not benefit from the standard of care but still experience substantial side effects. Therefore, there is the urgency for a new model to predict clinical response.

Adult epithelial stem cell (ASC) -derived organoids are proving to be a major breakthrough in pre-clinical models. ASC-derived organoids can be developed from healthy as well as diseased tissue, including cancer lesions and therefore are often referred to as patient-derived organoids (PDOs or HUB Organoids࣪). These model is established directly from patient tissue, represent the tissue of origin and faithfully recapitulate patient disease in vitro and can be propagated for drug testing in a matter of weeks. PDOs bridges the gap between the lab and the clinic and effectively bring a “patient in the lab.”

In this study we aimed to validate the predictive value of PDOs in the stratification of metastatic CRC (mCRC) patients for treatment with chemotherapeutic agents. PDOs from mCRC tissues were established following our optimized procedures and drug screening was performed. Clinical response was compared with PDO drug response and the best predicting drug response parameters (growth rate (GR), GRmax, GR50 and area under the curve) were identified. The patient response was evaluated by the percentage of change in size of the target lesions on response scans (% size change), best Response Evaluation Criteria in Solid Tumors (RECIST) response and progression-free survival (PFS). Importantly, we validated the predictive value of organoids towards fluorouracil (5-FU). We are validating these results in a large trial.

Current efforts to further expand our PDO and drug sensitivity biobank will enable the implementation of the personalized HUB Organoid Technology to accurate and fast predict of the treatment response to improve clinical outcome of mCRC patients.

Citation Format: Carla S. Verissimo, Lidwien Smabers, Emerens Wensink, Esmee Koedoot, Maarten Huismans, Celia Higuera Barón, Ricardo Korporaal, Emma Teal, Katerina-Chara Pitsa, Edwin van Oosten, Roshni Nair, Liselot Valkenburg, Geert Cirkel, Anneta Brousali, Jorieke Salij, René Overmeer, Manon Braat, Sjoerd Elias, Robert Vries, Onno Kranenburg, Miriam Koopman, Sylvia F. Boj, Jeanine Roodhart. Patient derived organoids predict clinical response: A patient in the lab [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 4112.