tbuckley/cabot-search huggingface dataset:
Abstract 93: Validation of an impedance-based <i>in vitro</i> potency assay for repeatability and precision
Authors: Danielle Califano, Jianjie Jiang, Dirk Windgassen, Caitlyn De La Flor, Stacie Chvatal, Denise Sullivan, Daniel Millard
Journal: Cancer Research (2024)

Abstract Cell-based immunotherapies, such as CAR T cells, are emerging as a promising approach to therapeutic intervention against cancer. Developing cell and gene therapy products requires reliable and reproducible methods of quantifying critical quality attributes, such as potency, to ensure product strength and consistency. A variety of assays can be used to assess potency; however, it is crucial that these tests are qualified for assay performance to ensure intra- and inter-assay precision. Although inherently variable, biological assays are often the best methods to investigate a product’s mechanism of action and predict clinical outcomes. Previous work has shown that impedance-based potency assays provide a non-invasive, real-time measure of effector cell-mediated cytotoxicity, thus reducing cell manipulation that impacts data reliability. Here, we describe an in vitro potency assay that demonstrates low variability and high precision across replicates, analysts, and days. Effector cell-mediated cytotoxicity was performed using a CD19-positive liquid tumor cell line (Raji) tethered to the well surface using an anti-CD40 antibody. CD19-specific CAR T effector cells were added 24 hours after the target cells and co-cultured with the target cells across a range of E: T ratios, and cytolysis was monitored for three days. Real-time measurement of cell-mediated cytotoxicity also allowed for the calculation of kill time 50 (KT50), defined as the time required for 50% cytolysis of the target cells. Repeatability was calculated across three replicates per E:T ratio. The co-culture experiment was performed in duplicate by a second analyst and on two subsequent days for a comparison of f three plates as well as two operators to evaluate inter-assay precision. In addition to this, CAR T-mediated cell killing in a multiplate impedance system was also investigated for plate-to-plate variability. The percent coefficient of variation (% CV), a statistical measure that describes the precision and repeatability of an assay, was calculated to assess variability between assay replicates, operators, and plate replicates. In all conditions tested, the percent CV for all E:T ratios was below 20%, illustrating the repeatability and precision of the assay. These data support the use of an impedance-based potency assay for evaluating and characterizing immunotherapy products. Citation Format: Danielle Califano, Jianjie Jiang, Dirk Windgassen, Caitlyn De La Flor, Stacie Chvatal, Denise Sullivan, Daniel Millard. Validation of an impedance-based in vitro potency assay for repeatability and precision [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 93.
Abstract 2680: Knockout on enhancer of zeste homolog 2 in triple-negative breast cancer model alters immune signaling and decreases tumor progression and lung metastasis
Authors: Lenore Monterroza, Maria Parrilla, Sarah Samaranayake, Dormarie Rivera, Brian Dobosh, Periasamy Selvaraj, Rabindra M. Tirouvanziam
Journal: Cancer Research (2024)

Abstract Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic subtype of breast cancer. Lack of known targets, patient-to-patient variations of target antigens and resistance to immune checkpoint inhibitors make TNBC one of the most challenging cancers to treat. TNBC is enriched in tumor-infiltrating neutrophils (TINs). TINs are key contributors to pro-tumorigenic processes linked to “immunologically cold” tumors, in part by preventing recruitment of T-lymphocytes (TILs). Prior studies identified the Enhancer of zeste homolog 2 (EZH2) as a prominent methyltransferase expressed in primary and metastatic TNBC cells. EZH2 is an upstream regulator of the stimulator of interferon genes (STING), a critical innate sensor mediating anti-tumor responses that is inhibited in cold tumors. To date, EZH2/STING signaling in TINs is relatively unknown. Here, we hypothesized that EZH2 inhibition and subsequent STING activation could exert anti-tumor activity by modulating TINs. We derived EZH2-knockout (KO) and EZH2-overexpressed (OX) cell lines from the well-established 4T1 TNBC mouse cell line. We demonstrate a 10-fold decrease in lung metastasis and significant reduction of 4T1 EZH2 KO tumor growth compared to the parent 4T1 cell line. Concomitantly, the TIN:TIL ratio was significantly reduced in 4T1 EZH2 KO vs. control tumors. Overexpression of EZH2 in 4T1 TNBC cells did not affect the primary tumor growth or lung metastasis. A therapeutic study using the repeated injection of the STING agonist MSA-2 after tumor engraftment in vivo also demonstrated a decrease in lung metastasis and tumor growth in MSA-2 vs. vehicle injection with both 4T1 parent and EZH2 KO cell lines. Together, our findings suggest a key role for STING pathway modulation and TIN/TIL poise in primary TNBC progression and lung metastasis. Citation Format: Lenore Monterroza, Maria Parrilla, Sarah Samaranayake, Dormarie Rivera, Brian Dobosh, Periasamy Selvaraj, Rabindra M. Tirouvanziam. Knockout on enhancer of zeste homolog 2 in triple-negative breast cancer model alters immune signaling and decreases tumor progression and lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2680.
Abstract 331: Ion TorrentTM NGS sequencing of the TERT promoter hotspots with Ion AmpliSeqTM HD technology
Authors: Anupma Sharma, Guobin Luo, Na Li, Giorgio Pea, Fiona Hyland
Journal: Cancer Research (2024)

Abstract Introduction: Recurrent mutations C228T and C250T in the TERT gene promoter are prevalent in cancer and serve as important biomarkers for aggressive disease. Ion Torrent™ NGS sequencing, combined with Ion AmpliSeqTM HD technology, enables comprehensive genomic profiling with ultra-high sensitivity for low-frequency variants in cell-free DNA (cf-DNA) and highly heterogeneous solid tumor samples. However, the high GC content (&amp;gt;80%) and the presence of G repeats that are known to adopt a compact stacked three-G-quadruplex conformation in the TERT gene promoter region pose sequencing challenges such as low read depth and strand-bias, i.e. prematurely truncated reads on one strand. Methods: We designed nine AmpliSeqTM HD panels with amplicon sizes of 70 to 104 bp, ideal for cf-DNA and FFPE samples, to target the recurrent TERT promoter hotspots. The designs were tested alone and combined with a larger Ion AmpliSeqTM HD DNA research panel resulting in highly multiplexed libraries. The libraries were used for Ion 540TM chip template preparation on the Ion ChefTM instrument and subsequently sequenced using the Ion GeneStudioTM S5 System. Sequencing data was used to evaluate coverage of the target hotspots by each design for each condition, and additional panels were designed to test single amplicons covering both TERT promoter hotspots. Results: Through performance evaluation, we selected two overlapping amplicons, each effectively amplifying one of the two hotspots. Individually tested, these amplicons yielded approximately 3000 families each from a 10ng gDNA input and when combined into the larger Ion AmpliSeqTM HD DNA research panel, they produced several thousand reads across various libraries, providing molecular coverage of around 200 functional molecules. While capture efficiency was lower compared to TERT amplicon-only libraries, these improved amplicons allow downstream analysis of variants. A single-pool solution (single amplicon/s covering both hotspots) will be explored further using the second batch of amplicons/primers. Conclusions: The optimized amplicons in two pools developed in this study enable the detection of challenging yet clinically significant TERT promoter hotspot mutations using the ultra-sensitive Ion AmpliSeqTM HD technology. For Research Use Only. Not for use in diagnostic procedures. © 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Citation Format: Anupma Sharma, Guobin Luo, Na Li, Giorgio Pea, Fiona Hyland. Ion TorrentTM NGS sequencing of the TERT promoter hotspots with Ion AmpliSeqTM HD technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 331.
Abstract 3326: PMR-116, a second generation RNA Polymerase I inhibitor
Authors: Luc Furic, Nadine Hein, Rita Ferreira, Konstantin Panov, Alisee Huglo, Richard Rebello, Eric Kusnadi, Denis Drygin, Mustapha Haddach, Ross Hannan
Journal: Cancer Research (2024)

Abstract Introduction: Inhibition of RNA Polymerase I, the enzyme responsible for the synthesis of ribosomal RNAs, the nucleic scaffold of ribosomes, has been proven as a novel therapeutic target as most cancer cells are "addicted" to ribosome biogenesis. Methods: In collaboration with Pimera Inc, we developed and tested the efficacy of a second generation RNA polymerase I inhibitor, PMR-116, in various pre-clinical cancer models. On-target activity of PMR-116 was confirmed in peripheral blood cells isolated from patients with solid tumors enrolled in a phase I trial of PMR-116. Results: PMR-116 potently inhibits Pol I transcription with ~200 fold higher selectivity towards Pol I compared to Pol II transcription. PMR-116 inhibits rRNA synthesis by stalling the Pol I complex at the rDNA promoter preventing promoter escape and consequently elongation. PMR-116 anti-cancer activity was evaluated against a panel of over 100 cancer cell lines representative of 20 major types of solid and haematological malignancies. This new Pol I inhibitor exhibited broad anti-proliferative and cytotoxic activity with an IC50 ranging from 32-4500nM and a median IC50 of 300nM. In contrast, cells derived from normal tissues were significantly less sensitive (IC50 ranging from 6-33μM). In vivo, PMR-116 significantly improved survival and reduce tumor burden in several preclinical models such as Vk*MYC transgenic model of indolent multiple myeloma, CT26 xenograft model of colorectal cancer, MMTV-PyMT transgenic model of metastatic breast cancer, mixed-lineage leukemia (MLL)-eleven nineteen leukemia (ENL)+Nras acute myeloid leukemia (+/-p53 mutation) and patient-derived xenografts of prostate cancer and AML. In a phase I trial of PMR-116, on-target activity of PMR-116 was observed in cells derived from the peripheral blood. Conclusion: PMR-116 significantly reduced cancer growth in a wide range of tumor models, including GEMM, syngeneic and PDX models. PMR-116 is undergoing clinical testing in phase I in solid tumors with the objective to determine maximum tolerated dose and identify a phase II dose. Citation Format: Luc Furic, Nadine Hein, Rita Ferreira, Konstantin Panov, Alisee Huglo, Richard Rebello, Eric Kusnadi, Denis Drygin, Mustapha Haddach, Ross Hannan. PMR-116, a second generation RNA Polymerase I inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3326.
Abstract 33: Revitalizing CAR-T cells for TNBC: Targeting phosphorylation of TCF7 to overcome T cell exhaustion
Authors: Zhou Jiang, Yu-Yi Chu, Heng-Huan Lee, Mien-Chie Hung, Liuqing Yang, Chunru Lin
Journal: Cancer Research (2024)

Abstract Breast cancer stands as the second leading cause of cancer-related deaths among women in the United States, with triple-negative breast cancer (TNBC) representing the most aggressive subtype. Although immune checkpoint inhibitors (ICI) demonstrated durable response in some TNBC patients, the added benefit of current FDA-approved ICI in combination with chemotherapy is estimated to be only around 10%. Fortunately, TNBC displays the highest number of tumor-infiltrating lymphocytes (TILs), compared to other BC subtypes, indicating an optimal context for the survival of T cells. Therefore, CAR-T therapy emerges as a promising therapeutic option for TNBC patients. Expanding the application of CAR-T therapies to solid tumors, including breast cancer, remains challenging. One of the key hurdles lies in the immunosuppressive tumor microenvironment, where the T cell receptor experiences long-term exposure to tumor antigens, leading to T cell exhaustion. This state impedes the T cells' capacity to efficiently target and eliminate cancer cells. Identifying key regulatory mechanisms downstream of the T cell receptor holds the potential to revolutionize existing immunotherapeutic strategies. In our study, we discovered that the presence of the master transcriptional factor of T cell stemness, TCF1, in CD8+ T cells, is essential for controlling TNBC tumors. TCF1 undergoes phosphorylation by LCK, a crucial component of initiating T cell receptor signaling, upon T cell activation. We found this phosphorylation site is indispensable for TCF1's transcriptional activities, inhibiting the downstream program of T-cell persistence and causing T-cell exhaustion. Reversing this process, either through LCK inhibition or introducing a non-phosphorylated form of TCF1, results in a more sustained CAR-T cell-mediated anti-tumor response. Our findings reveal the significance of TCF1 post-translational modification in the development and longevity of stem-like CD8+ T cells, bridging the gap between TCR signaling and transcriptional and epigenetic programs of stem-like CD8+ T cells. This insight contributes to a potential cure for TNBC by maintaining T cell persistence. Citation Format: Zhou Jiang, Yu-Yi Chu, Heng-Huan Lee, Mien-Chie Hung, Liuqing Yang, Chunru Lin. Revitalizing CAR-T cells for TNBC: Targeting phosphorylation of TCF7 to overcome T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 33.
Abstract 175: Tumor-associated monocytes and neutrophils: Targets for immunotherapy
Authors: Keehoon Jung
Journal: Cancer Research (2024)

Abstract Cancer is the leading cause of deaths worldwide. However, current anti-VEGF therapies for solid cancers provide limited survival benefit as tumors rapidly develop resistance to these agents.We have uncovered an immunosuppressive role for non-classical Ly6Clow monocytes that mediates resistance to anti-VEGFR2 treatment. We found that the chemokine CX3CL1 was upregulated in both human and murine tumors following the VEGF signaling blockade, resulting in recruitment of CX3CR1+ Ly6Clow monocytes into the tumor. We also found that treatment with VEGF-A reduced expression of CX3CL1 in endothelial cells. Intravital microscopy revealed that CX3CR1 is critical for Ly6Clow monocyte transmigration across the endothelium in tumors. Moreover, Ly6Clow monocytes recruit Ly6G+ neutrophils via CXCL5 and produce IL-10, which inhibits adaptive immunity. Preventing Ly6Clowmonocyte or Ly6G+ neutrophil infiltration into tumors enhanced inhibition of tumor growth with anti-VEGFR2 therapy. Furthermore, we developed a gene therapy using a nanoparticle formulated with a siRNA against CX3CL1, which reduced Ly6Clow monocyte recruitment and improved outcome of anti-VEGFR2 therapy.Taken together, we identified immunosuppressive non-classical Ly6Clow monocytes as key players in tumor resistance to anti-angiogenic therapy in cancers. We also revealed molecular mechanisms underlying anti-angiogenic treatment resistance, suggesting potential immunomodulatory strategies to enhance the long-term clinical outcome of anti-VEGF therapies, proven by state-of-the-art in vivo imaging modalities. Citation Format: Keehoon Jung. Tumor-associated monocytes and neutrophils: Targets for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 175.
Abstract 330: FusionPlex™-HT and VariantPlex™-HT: Automation ready solutions for anchored multiplex PCR
Authors: David Knupp, Christine Troutman, Allison Hadjis, Dhruti Legare, Paula Kalavakur, Michael Washburn
Journal: Cancer Research (2024)

Abstract Introduction: Anchored Multiplex PCR (AMP™) assays, VariantPlex and FusionPlex are amplicon-based, next generation sequencing (NGS) workflows that identify genomic variations present in DNA or RNA input, respectively. AMP reagents are available in a lyophilized format which are user-friendly and convenient to store. However, lyophilized reagents are not suitable for liquid handlers or high-throughput environments. Thus, we developed AMP reagents in liquid format to satisfy the need for a platform-agnostic method of high-throughput (-HT) AMP library preparation. Methods: The performance of our liquid reagent-based VariantPlex-HT and FusionPlex-HT workflows were evaluated relative to legacy lyophilized reagents using several input of varying quantity, quality and type. In addition, we examined liquid reagent performance across multiple AMP panels, which target solid tumor and blood cancers variants. Prepared libraries were then sequenced and analyzed using Archer™ Analysis v7.0 software. Results: When using 50ng SeraSeq® Myeloid DNA input (LGC Clinical Diagnostics, Inc.) and the VariantPlex Core Myeloid panel (37 gene targets), both the lyophilized VariantPlex and VariantPlex-HT detected 100% (22/22) known variants ranging from 3.8% to 20.4% allele frequency (AF). When using low-quality FFPE and the VariantPlex Complete Solid Tumor panel (430 gene targets), both workflows captured 28/28 (AF: 1.4%-20.2%) and 11/11 (AF:1.2%-18.9%) single nucleotide variants (SNVs) and Insertion and Deletions (InDels) when 50ng of SeraSeq compromised FFPE or 200ng Horizon Severe FFPE was used as input, respectively. The FusionPlex-HT workflow consistently showed increased library complexity using the FusionPlex Lung V2 panel with 20ng or 50ng of Seraseq RNA Fusion Mix v4 input. This, coupled with greater on-target percentage resulted in an average increase in fusion supporting reads of 1.95x ± 0.53 and 2.2x ± 0.41 for 20ng and 50ng input, respectively. Next, we prepared a dilution series of SeraSeq RNA Fusion Mix v4 FFPE material at 100%, 25%, 10% and 5% dilutions. We utilized our FusionPlex Pan Solid Tumor panel (137 gene targets) which covers all expected fusions in this input material. At 100% and 25% dilutions, all expected fusions were detected by lyophilized FusionPlex and FusionPlex-HT workflows. However, at 10% and 5% dilutions FusionPlex-HT detected 100% and 91% of expected fusions, respectively, while legacy lyophilized reagents detected 80% and 60% of expected fusions. Citation Format: David Knupp, Christine Troutman, Allison Hadjis, Dhruti Legare, Paula Kalavakur, Michael Washburn. FusionPlex™-HT and VariantPlex™-HT: Automation ready solutions for anchored multiplex PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 330.
Abstract 3325: HBW-012-D and HBW-012-E are novel, potent, selective, safe, and bioavailable KRAS G12D inhibitors with superior anti-tumor efficacy in mice
Authors: Ning Lee, Yingfu Li, Guanfeng Liu, Jiang Li, Junfeng Ren
Journal: Cancer Research (2024)

Abstract Purpose: This study is to characterize newly identified KRAS G12D small molecule inhibitors HBW-012-D and HBW-012-E, both showing improved oral bioavailability than MRTX1133, the known clinical compound with poor bioavailability. Method: Through our medicinal chemistry efforts, we have aimed to design novel small-molecule KRAS G12D inhibitors that are orally bioavailable. The compounds were screened via pERK and cell viability (CTG) assays in multiple cell types. The optimized compounds were further evaluated based on their pharmacokinetic (PK) properties and toxicity. The anti-tumor efficacy of the leads was confirmed in the GP2D (colorectal carcinoma with KRAS G12D mutation) mouse xenograft tumor model. Result: As shown in the table, HBW-012-D and HBW-012-E have significantly better potency and PK characteristics than MRTX1133. In the mouse GP2D model, comparing head-to-head with the same vehicle and protocol (50 mg/kg, twice daily, orally), MRTX1133 hardly inhibited tumor growth, whereas HBW-012-D inhibited tumor growth well (58 % tumor growth inhibition), and HBW-012-E not only completely inhibited tumor growth but also further regressed tumor size by around 40%. Preliminary toxicity tests showed that HBW-012-D and HBW-012-E are safe: weak inhibition of CYP450 enzymes, negative mini-Ames test, excellent kinase selectivity, and no obvious abnormalities in the 14-day rat or mouse toxicity test. Conclusion: New KRAS G12D inhibitors, HBW-012-D and HBW-012-E, were identified. Their preclinical profile, including potency, efficacy, and PK data, are all superior to MRTX1133, and their safety meets the criteria of clinical development. They show potential as a preferred oral treatment option for patients with tumors mediated by the KRAS G12D mutation, such as pancreatic cancer that still lacks effective treatments. in vitro data IC 50 (nM) MRTX1133 HBW-012-D HBW-012-E AGS (G12D, stomach, pERK) 1 0.8 0.46 AsPC-1 (G12D, pancreas, 3D CTG) 10.88 3.09 GP2D (G12D, colon, 3D CTG) 1.38 0.67 0.69 MKN-1 (WT, stomach, 3D CTG) &amp;gt;3000 Rat PK @ 100 mg/kg oral Cmax (ng/mL) 9 82 1444 AUC0~t (mg.h/mL) 20 258 5724 Citation Format: Ning Lee, Yingfu Li, Guanfeng Liu, Jiang Li, Junfeng Ren. HBW-012-D and HBW-012-E are novel, potent, selective, safe, and bioavailable KRAS G12D inhibitors with superior anti-tumor efficacy in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3325.