tbuckley/cabot-search huggingface dataset:
Abstract 6248: Engineered probiotics for local delivery of checkpoint blockade nanobodies
Authors: Candice Gurbatri, Ioana Lia, Rosa Vincent, Courtney Coker, Samuel Castro, Piper Treuting, Taylor Hinchliffe, Nicholas Arpaia, Tal Danino
Journal: Cancer Research (2020)

Abstract Checkpoint inhibitors have revolutionized cancer therapy but only work in a subset of patients and can lead to a multitude of toxicities, suggesting the need for more targeted delivery systems. Because of their preferential colonization of tumors, microbes are a natural platform for the local delivery of cancer therapeutics. Here, we engineer a probiotic bacteria system for the controlled production and intratumoral release of nanobodies targeting programmed cell death-ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated protein-4 (CTLA-4) using a stabilized lysing release mechanism. We used computational modeling coupled with experimental validation of lysis circuit dynamics to determine the optimal genetic circuit parameters for maximal therapeutic efficacy. A single injection of this engineered system demonstrated an enhanced therapeutic response compared to analogous clinically relevant antibodies, resulting in tumor regression in syngeneic mouse models. Supporting the potentiation of a systemic immune response, we observed a relative increase in activated T cells, an abscopal effect, and corresponding increases in systemic T cell memory populations in mice treated with probiotically delivered checkpoint inhibitors. Last, we leveraged the modularity of our platform to achieve enhanced therapeutic efficacy in a poorly immunogenic syngeneic mouse model through effective combinations with a probiotically produced cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). Together, these results demonstrate that our engineered probiotic system bridges synthetic biology and immunology to improve upon checkpoint blockade delivery. Citation Format: Candice Gurbatri, Ioana Lia, Rosa Vincent, Courtney Coker, Samuel Castro, Piper Treuting, Taylor Hinchliffe, Nicholas Arpaia, Tal Danino. Engineered probiotics for local delivery of checkpoint blockade nanobodies [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 6248.
Abstract 5490: Scales and dynamics of intratumor heterogeneity
Authors: Meghan C. Ferrall-Fairbanks, Gregory J. Kimmel, Philipp M. Altrock
Journal: Cancer Research (2020)

Abstract Current dogma stipulates that more heterogenous tumor cell populations results from compounding genetic and epigenetic changes and instability, ultimately driving unfavorable outcomes for these patients. Nonetheless, some cancers, including many pediatric cancers and some leukemias, have limited genomic diversity. As a result, we have a limited number of biomarkers available to stratify patients into high versus low-risk groups by their mutational cellular diversity or heterogeneity. For these cases, we have developed a method to quantify intratumor heterogeneity, for example at the transcriptomic level, using a generalized diversity index (GDI) from ecology. This index allows control of a scaling parameter called the order of diversity, q, to tailor the scale or resolution of the cellular diversity to a given context. However, the question remains, what role does the order of diversity play in the context of relative tumor age or disease stage? We show that the order of diversity parameter allows to either emphasize clonal richness (at low values of q), while high values shift the analysis toward the abundance of potential drivers of the tumor evolution. To gain deeper quantitative insight, we use evolutionary dynamics approaches that model the dynamics of heterogeneous cell populations over time, to understand how a patient's tumor diversity could emerge, persist, and change over time. Applying the replicator-mutator dynamics framework, we explore the effects and magnitude of frequency-dependent (positive or negative) selection on GDI. These analyses suggest that GDI often peaks at intermediate time points and, when run over a sufficiently long timescale, they reduce as the tumor population presumable becomes clonally dominant or otherwise stable before a major transition (e.g. metastasis). We explore GDI changes over time further in vitro in sequential single-cell RNA sequencing samples of fused breast cancer cells, where at earlier passages after fusion, the GDI peaks, and at later passages after fusion the GDI returns to levels similar to that of the parental breast cancer cell lines. Overall, GDI, as a means to quantify intratumor heterogeneity at select time points, can be a powerful tool to stratify high-risk patients. Our analyses show how computational analyses and mathematical modeling tools can be coupled with clinical and experimental data to understand eco-evolutionary dynamics of a patient's tumor. Citation Format: Meghan C. Ferrall-Fairbanks, Gregory J. Kimmel, Philipp M. Altrock. Scales and dynamics of intratumor 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 5490.
Abstract 506: A bespoke screening platform to study mono(ADP-ribosylation)
Authors: Tim J. Wigle, Danielle J. Blackwell, Laurie B. Schenkel, Yue Ren, William D. Church, Hetvi J. Desai, Kerren K. Swinger, Andrew G. Santospago, Christina R. Majer, Alvin Z. Lu, Mario Niepel, Nicholas R. Perl, Melissa M. Vasbinder, Heike Keilhack, Kevin W. Kuntz
Journal: Cancer Research (2020)

Abstract Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are post-translational modifications deposited on multiple amino acids by PARP enzymes using nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate. While there are approved drugs and clinical trials on-going for inhibitors of the polyPARP enzymes that deposit poly(ADP-ribose) (specifically PARP1 and PARP2 inhibitors), monoPARP enzymes that deposit mono(ADP-ribose) are only recently gaining recognition for their role in cellular stress signaling, inflammation and cancer. However, there is a lack of chemical probes to study their function in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop screening assays to enable determination of potency and selectivity of inhibitors during the hit finding and lead optimization phases. The development of enzyme assays is complicated by the fact that the substrates for the majority of the monoPARPs are unknown, and even for those with identified substrates, it is uncertain how they engage their substrates. Here we describe the development of robust high-throughput biochemical and cellular monoPARP assays that overcome the lack of knowledge around the substrates and construction of a family-wide screening panel. We highlight derivatized microplates that activate the enzymes to self-MARylate in dissociation enhanced lanthanide fluorescence assays (DELFIA), antibodies that recognize MARylation in in-cell western (ICW) and immunofluorescence (IF) assays, and NAD+-competitive molecular probes that are used to develop in vitro time-resolved fluorescence resonance energy transfer (TR-FRET) and cellular NanoLuc bioluminescence resonance energy transfer (NanoBRET) probe displacement assays. Additionally, we employ several methods to characterize inhibitor binding kinetics. These assays have been used in high-throughput screening campaigns of up to 500,000 compounds, as well as in the development of potent and selective inhibitors of multiple monoPARP enzymes including RBN012759, a tool compound for PARP14 that inhibits in vitro with an IC50 of 3 nM and in cells using with an IC50 of 9 nM, and is 300-fold selective over all other PARP enzymes. Citation Format: Tim J. Wigle, Danielle J. Blackwell, Laurie B. Schenkel, Yue Ren, William D. Church, Hetvi J. Desai, Kerren K. Swinger, Andrew G. Santospago, Christina R. Majer, Alvin Z. Lu, Mario Niepel, Nicholas R. Perl, Melissa M. Vasbinder, Heike Keilhack, Kevin W. Kuntz. A bespoke screening platform to study mono(ADP-ribosylation) [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 506.
Hypoxia-inducible factor signaling in pulmonary hypertension
Authors: Soni Savai Pullamsetti, Argen Mamazhakypov, Norbert Weissmann, Werner Seeger, Rajkumar Savai
Journal: Journal of Clinical Investigation (2020)

Pulmonary hypertension (PH) is characterized by pulmonary artery remodeling that can subsequently culminate in right heart failure and premature death. Emerging evidence suggests that hypoxia-inducible factor (HIF) signaling plays a fundamental and pivotal role in the pathogenesis of PH. This Review summarizes the regulation of HIF isoforms and their impact in various PH subtypes, as well as the elaborate conditional and cell-specific knockout mouse studies that brought the role of this pathway to light. We also discuss the current preclinical status of pan- and isoform-selective HIF inhibitors, and propose new research areas that may facilitate HIF isoform-specific inhibition as a novel therapeutic strategy for PH and right heart failure.
Abstract 4932: mTORC1 regulates FAK phosphorylation
Authors: Lin Li, Yan Luo, Lei Liu, Shile Huang
Journal: Cancer Research (2020)

Abstract The mammalian target of rapamycin (mTOR) functions as two complexes, mTORC1 and mTORC2, regulating cell growth, proliferation, survival, and motility. Previous studies have shown that mTORC2 controls cytoskeleton and cell motility. Recently, we have demonstrated that both mTORC1 and mTORC2 participate in the regulation of cell motility. Further studies have shown that rapamycin suppresses IGF-I induced cell motility by inhibiting S6K1-mediated phosphorylation of focal adhesion kinase (FAK), protein phosphatase 2A (PP2A)-mediated phosphorylation of Erk1/2, as well as expression and activities of the small GTPases (RhoA, Cdc42 and Rac1). As FAK plays a pivotal role in the regulation of cell motility, this study was set to determine how mTORC1 regulates the phosphorylation of FAK. By co-immunoprecipitation (co-IP) and immunoblotting (IB), we failed to detect a physical interaction between FAK and S6K1, suggesting that S6K1 may regulate the phosphorylation of FAK indirectly. Besides, knockdown of protein phosphatase 2A (PP2A) enhanced the phosphorylation of FAK and conferred resistance to rapamycin inhibition of IGF-I-stimulated phosphorylation of FAK. However, the co-IP/IB results revealed no direct interaction between FAK and PP2A either. Of interest, knockdown of protein phosphatase 5 (PP5) suppressed IGF-I stimulated cell motility, while knockdown of ATG13 increased IGF-I stimulated cell motility, suggesting that PP5 positively regulates cell motility, whereas ATG13 negatively regulates cell motility. It is well known that PP5 is an anti-apoptotic phosphatase, negatively regulating the ASK1-JNK cascade, and mTORC1 regulates autophagy through the ULK1/2-ATG13-FIP200 complex. Further research will determine how mTORC1 regulates cell motility in part by regulating the phosphorylation of FAK via S6K1, PP2A, PP5 and ATG13. (Supported by the Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA, USA.) Citation Format: Lin Li, Yan Luo, Lei Liu, Shile Huang. mTORC1 regulates FAK phosphorylation [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 4932.
Abstract 5176: A novel, completely human fusion construct containing active Granzyme B efficiently kills Folate Receptor alpha positive tumor cells
Authors: Ana Alvarez de Cienfuegos, Laurence H. Cheung, Khalid A. Mohamedali, Walter N. Hittelman, Michael G. Rosenblum
Journal: Cancer Research (2020)

Abstract Folate receptor alpha (FRα) is an attractive therapeutic target because it is highly overexpressed on several tumors such as ovarian, breast and lung cancers and demonstrates low or restricted distribution on normal tissues. In addition, FRα has been shown to play a tumor promoting role with its expression associated with poor prognosis of cancer patients. In our laboratory, we have generated two new molecules targeting FRα and containing active granzyme B (GrB) as the cytotoxic payload. We used as binding domains two different scFvs targeting the FRα from antibodies used in clinical trials. These human scFvs (designated mov003 and mov018) were fused to human GrB through an engineered IgG heavy-chain fragment (designated as Fc) which contains a dimerization domain. The constructs were cloned into the pSECTag vector containing an upstream 5 kDa purification tag with an enterokinase (EK) cleavage site for facile removal. The molecules were expressed in transiently-transfected HEK-293E suspension cells over 3 days at 37°C in 5% CO2. The molecules were isolated from culture media and purified by ion-exchange chromatography followed by EK cleavage to remove the free tag. The yields were 12-18 mg/L for both molecules. The assessment of the specific binding of each molecule to the antigen target is ongoing. The cytotoxicity of the constructs against a panel of cell lines expressing various levels of FRα showed a specific cytotoxic effect with an IC50 in the low nanomolar range (5-100 nM), particularly exhibiting very low IC50 values against breast (MDA-MB-231) and ovarian cell lines (IGROV-1). Receptor negative cells demonstrated IC50 levels in the micromolar range. Based on IC50 values, these data demonstrate suggest the fusion constructs targeting FRα are efficiently internalized and effectively deliver the active serine protease to the cytosol, activating the cytotoxic cascade. In vitro stability assays over 96 hours showed that both constructs are highly stable in mouse serum. Immunofluorescence and confocal studies to examine the time course of internalization and the pro-apoptotic events triggered by these agents are under examination and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez de Cienfuegos, Laurence H. Cheung, Khalid A. Mohamedali, Walter N. Hittelman, Michael G. Rosenblum. A novel, completely human fusion construct containing active Granzyme B efficiently kills Folate Receptor alpha positive 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 5176.
Abstract 480: Molecular mechanism of action of photoimmunotherapy with antibody-IR700 dye conjugates
Authors: Roger Heim, Lew Makings, Torsten Wiemann, Anthony Manibusan, Miguel Garcia-Guzman
Journal: Cancer Research (2020)

Abstract INTRODUCTION: Cancer cell-targeted photoimmunotherapy (PIT) is a platform technology under development for the treatment of various cancers. PIT is a drug and device combination that utilizes an antibody conjugated to the IR700 (IRDye 700DX®) light-excitable dye, which upon antigen binding and illumination with non-thermal red light, leads to necrotic cell death. The objective of these studies was to characterize the molecular pharmacology and the underlying mechanism of action of PIT. METHODS: In vitro PIT studies using human and mouse cancer cells were performed to evaluate conjugate (antibody + dye) binding, dose response and light dosimetry response. Mouse xenograft experiments assessed necrotic markers in tumor tissue and surrounding normal tissue post-PIT treatment. Oxidative damage to lipids was detected with a reporter dye. Mass spectrometry analysis was performed to assess protein oxidation, crosslinking and axial ligand dissociation from the dye. RESULTS: Binding of the conjugate to the target cancer cells was required for PIT treatment to induce cell death as measured by antibody competition studies, however, internalization of the conjugate was not required for cytotoxic effects. Administration of non-thermal red light to the target cells bound with the conjugate led to morphologic changes and membrane disruption within minutes, and necrotic cell death was observed in a conjugate- and light-dose dependent manner. Requirement of conjugate binding to the cancer antigen provided high level of tumor specificity as demonstrated by lack of damage to normal surrounding tissue after PIT treatment. Light excitation of the conjugate generated 1O2 with high quantum yield. In vitro PIT treatment led to cell membrane lipid peroxidation, mediated by 1O2 as shown by abrogation with sodium azide, a selective 1O2 quencher. In addition, protein oxidation, cross-linking and aggregation of the conjugate was observed. In low oxygen concentrations and in the presence of ascorbate, photo-induced dissociation of axial ligands from the dye was detected. CONCLUSIONS: These studies suggest the molecular mechanism of action of PIT includes generation of 1O2 that leads to lipid peroxidation and cell membrane disruption. Additional mechanisms may include protein oxidation and non-oxygen dependent mechanisms such as axial ligand loss of the IR700 dye. These results describe a biophysical process that damages and disrupts the cell membrane integrity resulting in necrotic cell death and immunogenic cell death. Citation Format: Roger Heim, Lew Makings, Torsten Wiemann, Anthony Manibusan, Miguel Garcia-Guzman. Molecular mechanism of action of photoimmunotherapy with antibody-IR700 dye conjugates [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 480.
Abstract 4479: Immune exhaustion of tumor microenvironment mediates crizotinib resistance in ALK rearranged advanced non small cell lung cancer
Authors: Yongchang Zhang
Journal: Cancer Research (2020)

Abstract Introduction: Previous study showed the correlation between immune tumor microenvironment and the efficacy of checkpoint inhibitor in non-small cell lung cancer (NSCLC). This study evaluated whether biomarkers for immune tumor microenvironment including programmed death ligand 1 (PD-L1), cluster of differentiation 8 (CD8), Granzyme B (GZMB) and T cell receptor (TCR) could predict the treatment response to the first-line crizotinib of patients with ALK-rearranged non-small cell lung cancer (NSCLC). Methods: We retrospectively evaluated patients with advanced NSCLC who received crizotinib at Hunan Cancer Hospital between January 2016 and December 2018. The patients' baseline expression of PD-L1, CD8 and GZMB was evaluated in one slide using immunofluorescence. TCR was also evaluated with paired blood samples simultaneously. Results: All the 108 eligible patients were stratified according to their expression profile into PD-L1+/CD8+/GZMB+ (n=21), PD-L1-/CD8+/GZMB+ (n=42), PD-L1+/CD8-/GZMB- (n=23), and PD-L1-/CD8-/GZMB- (n=22) group. Patients with strong PD-L1 expression have a significantly inferior objective response rate (36.5% versus 72.2%, p = 0.002), and shorter progression-free survival (PFS) (5.6 versus 16.5 months, p &amp;lt; 0.001) as compared to patients with weak or negative PD-L1 expression. Among the four groups, we found patients with PD-L1-/CD8+/GZMB+ had the best response (85.7% versus 26%, p=?), while PD-L1+/CD8-/GZMB- showed primary resistance to crizotinib had the worse outcomes (4.6 versus 18 months, p &amp;lt; 0.001). Furthermore, for TCR analysis, the counts of TCR and Shannon index was strongly correlated with longer PFS for crizotinib treatment (20.6 versus 8.2months, p &amp;lt; 0.001; 19.3 versus 6.1 months, p &amp;lt; 0.001, respectively). However, the clonality acts as poor predictive marker with worse PFS (6.8 versus 18.2 months, p &amp;lt; 0.001). Finally, three patients with de novo resistance to crizotinib experienced a favorable response to anti-PD-1 therapy. Conclusions: This study revealed immune exhaustion for tumor microenvironment as a poor predictive marker in first-line crizotinib-treated ALK-rearranged non-small cell lung cancer. The findings indicate the reshaping of an inflamed immune phenotype characterized by PD-L1, CD8, GZMB, and TCR expression and suggest potential therapeutic sensitivity to PD-1 blockade. Citation Format: Yongchang Zhang. Immune exhaustion of tumor microenvironment mediates crizotinib resistance in ALK rearranged advanced non small cell lung 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 4479.
Abstract 896: Enhanced sensitivity detection of replication competent lentivirus by qPCR
Authors: David Burke, Kristine Drafahl, Clark Fjeld, Chad Galderisi, Cindy Spittle
Journal: Cancer Research (2020)

Abstract Introduction: Chimeric antigen receptor T (CAR T) cells are an important emerging therapy for hematologic malignancies and are currently being investigated for other cancer types as well. CAR-T cells require a retrovirus or lentivirus to introduce the chimeric antigen receptor gene into the activated T cells. Safety measures are put in place by design to eliminate any chance to create replication competent virus. Multiple plasmids carrying specific components of the virus are used to reduce the chances of creating a replication competent lentivirus. Despite these measures, safety concerns have been posed by the FDA regarding use of a lentivirus for this treatment due to the potential for replication competent lentiviruses (RCL) to arise. FDA requires monitoring of both CAR T products as well as patients undergoing therapy. It has proven to be difficult to achieve acceptable detection sensitivity of the VSV-G sequence in a background of gDNA due to the high level of homology to many areas in gDNA. Methods: We have developed a new rapid and sensitive qPCR assay targeting the vesicular stomatitis virus G glycoprotein (VSV-G), an envelope protein on lentiviral vectors. Incorporation of the envelope gene would be required to generate a replication competent virus. The intended use of the validated assay is to detect and monitor for RCL presence in human blood samples collected during clinical monitoring. A validated reference assay targeting RPPH1 will be used to quantify the amount of amplifiable genomic DNA in each extracted DNA sample. The size of the amplicons are 66 and 87 base pairs for VSV-G and RPPH1 respectively. The VSV-G assay test includes one positive and one negative control per assay run. The VSV-G positive control is composed of plasmid containing the VSV-G target sequence spiked into a background of VSV-G negative human gDNA. The negative control is composed of negative gDNA without VSV-G plasmid. Results: Improved sensitivity without non-specific amplification was achieved by optimized primer and probe selection. Additionally sensitivity and specificity were enhanced by employing a step-down PCR protocol whereby the annealing and extension temperature is lowered in stepwise fashion over the first five cycles of PCR. Genomic DNA inputs up to 200ng per reaction showed high efficiency of amplification from 10 to 1e6 copies target sequence per reaction. Conclusion: This assay will provide a rapid and sensitive method for safety monitoring of patients undergoing CAR-T therapy with therapeutic cells generated using lentiviral vectors. Citation Format: David Burke, Kristine Drafahl, Clark Fjeld, Chad Galderisi, Cindy Spittle. Enhanced sensitivity detection of replication competent lentivirus by qPCR [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 896.