bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2563: FAK inhibitor APG-2449 and CDK4/6 inhibitor palbociclib synergistically suppress mesothelioma tumor growth via autophagy induction
Authors: Tao Ran , Fang Douglas D. , Li Yuanbao , Zhai Guoqin , Zhou Feng , Wang Qixin , Deng Jing , Yang Dajun , Zhai Yifan
Published: June 2022

Malignant mesothelioma is a rare but highly lethal malignancy, with a 5-year survival rate of less than 10%. Current treatment, which combines surgery, chemotherapy, radiation, and immunotherapy, yields marginal benefit and ultimately results in drug resistance or recurrence. Therefore, developing more effective treatments is an urgent unmet need. Among the most common genomic abnormalities in mesothelioma are alterations of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) and neurofibromin 2 (NF2). Combined CDKN2A deletions and NF2 loss is a negative prognostic factor for both progression-free and overall survival. Mesothelioma cells lacking expression of CDKN2A/B or NF2 are reported to be sensitive to CDK4/6 or focal adhesion kinase (FAK) inhibition, respectively. This study explored the potential utility of combining CDK4/6 inhibitor palbociclib and APG-2449, an investigational, clinical-stage ALK/ROS1/FAK inhibitor, in preclinical models of mesothelioma. Compared to controls and either single agent, the combination of palbociclib and APG-2449 enhanced inhibition of growth of cultured mesothelioma cells. This binary combination also synergistically induced durable antitumor activity in multiple murine mesothelioma models, including both cell line- and patient-derived xenografts. Mechanistically, treatment of mesothelioma cells with this combination likely downregulated FAK phosphorylation and enhanced cellular senescence and autophagy, as evidenced by an increase in the percentage of β-galactosidase-positive cells and upregulation in protein expression levels of autophagy markers P62 and LC3B. The combination also downregulated DNA damage-relevant proteins high mobility group box protein 1 (HMGB1) and γ-H2A histone family member X (ϒ-H2AX). Taken together, the data implicate that an interplay between autophagy and DNA damage repair contributes to the synergistic antitumor effects. In summary, our results unveil a novel potential therapeutic strategy of targeting both FAK and CDK4/6 in order to synergistically inhibit tumor growth in mesothelioma. The synergistic effects are evidently mediated by induced autophagy and enhanced cellular senescence. This preclinical study lays a foundation for future clinical development of APG-2449 combined with CDK4/6 inhibitors for mesothelioma.

Citation Format: Ran Tao, Douglas D. Fang, Yuanbao Li, Guoqin Zhai, Feng Zhou, Qixin Wang, Jing Deng, Dajun Yang, Yifan Zhai. FAK inhibitor APG-2449 and CDK4/6 inhibitor palbociclib synergistically suppress mesothelioma tumor growth via autophagy induction [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 2563.
Abstract 2574: Q901; a highly selective covalent cdk7 inhibitor inducing substantial anti-tumor effect in a broad spectrum of solid tumor lineages
Authors: Yu Donghoon , Jeon Yeejin , Lee Seung-Joo , Kim Jaeseung , Nam Kiyean
Published: June 2022

As a key regulator of its dual role in transcription and cell cycle progression, CDK7 has been determined to be an attractive therapeutic target in a variety of tumor types driven by cell cycle dysregulation and aberrant control of the transcriptional processes. Q901 is an irreversible small molecule inhibitor with extreme selectivity and potent inhibition activity for CDK7, which forms a covalent bond with C312 of the CDK7 C-terminal domain. Q901 demonstrated selective cytotoxic activities in a broad spectrum of solid tumor lineages. TP53 mutation could be a potential predictive marker for the Q901 response as TP53 wild-type cancer cells demonstrates a significantly increased cytotoxic response compared to TP53 mutant cells in the bioinformatic analysis. In addition, gene expression profiling analysis showed a dose-response increase of POLR2A expression upon Q901 treatment and a correlation with tumor growth inhibition rate, indicating that POLR2A might be a potential PD marker for the Q901-induced response. Taken together, these data support the clinical development of Q901 for solid tumor indications. Q901 is progressing to IND-enabling studies to support the start of Phase 1 oncology clinical trials in early 2022.

Citation Format: Donghoon Yu, Yeejin Jeon, Seung-Joo Lee, Jaeseung Kim, Kiyean Nam. Q901; a highly selective covalent cdk7 inhibitor inducing substantial anti-tumor effect in a broad spectrum of solid tumor lineages [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 2574.
Abstract 2584: Development of an Fc gamma receptor fully-humanized mouse model to support the accurate preclinical study of therapeutic antibodies
Authors: Zhang Mingkun , Ju Cunxiang , Li Ying , Jiang Yunlong , Yu Weiwei , Li Shuai , Sun Hongyan , Li Zhiying , Zhao Jing , Gao Xiang
Published: June 2022

The Fc gamma receptor (FcγR), a member of the immunoglobulin superfamily, binds the IgG Fc segment of antibodies and is mainly expressed on the surface of immune cell. The FcγR binds to the Fc region of an antibody that is attached to target cells, transmitting an activation or inhibition signal to the FcγR expressing cells, resulting in various biological responses and functions. Many therapeutic antibodies are developed based on the engineering of the Fc region to fine-tune the FcγR mediated effects.

There are four subtypes of mouse FcγRs, including Fcgr1, Fcgr2b, Fcgr3, and Fcgr4. In contrast, human FcγRs consist of at least seven members, including FCGR1A, FCGR1B, FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B. Additionally, the expression pattern of even the orthologous FcγR genes varies between humans and mice. e.g., the distribution of mouse FCGR3 and FCGR2. Therefore, animals harboring the murine FcγRs are not ideal models for the accurate preclinical evaluation of human therapeutic antibodies. To address this problem, we established an FcγRs fully humanized mouse model by injecting an engineered human bacterial artificial chromosome construct carrying all seven human FcγR genes into mouse zygotes. The transgene carriers were mated with another strain in which all mouse FcγR genes were knocked out to establish the colony.

Unlike the endogenous mouse FcγR genes expression profiles, the transgenic human FcγR genes regulated under the control of the human control elements retain the human expression pattern. For example, the transgenic human FCGR3A was observed on mouse NK cells and macrophages, whereas the FCGR3B was detected on mouse neutrophils only, which were consistent with their expression patterns in humans. Antibody-dependent cellular cytotoxicity (ADCC) experiment indicated that a human therapeutic antibody could direct NK cells derived from the FcγRs fully-humanized mice to kill target tumor cells. Moreover, a significant anti-tumor effect could also be achieved by an ADCC based human antibody in tumor-bearing FcγRs humanized mice.

Taken together, the FcγR gene fully-humanized model established in this study demonstrates a human FcγR expression pattern and functionality, thus providing a valuable tool for accurate preclinical evaluation of human therapeutic antibodies.

Citation Format: Mingkun Zhang, Cunxiang Ju, Ying Li, Yunlong Jiang, Weiwei Yu, Shuai Li, Hongyan Sun, Zhiying Li, Jing Zhao, Xiang Gao. Development of an Fc gamma receptor fully-humanized mouse model to support the accurate preclinical study of therapeutic antibodies [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 2584.
Abstract 2593: Combined ATR+PARP inhibition exhibits potent synergy in colorectal and pancreatic cancer cytotoxicity
Authors: Ochsner Anna M. , Huntington Kelsey E. , Zhou Lanlan , Carneiro Benedito , El-Deiry Wafik
Published: June 2022

Precision medicine continues to expand treatment options for patients tailored to tumor phenotype. In homologous recombination (HR) deficient cancers like those with BRCA1/2-mutations, inhibition of poly (ADP-ribose) polymerase (PARP) has shown improved mortality compared to older gold standard therapies like DNA damaging agents. Unfortunately estimates upward of 40% of patients develop resistance to PARP inhibitor monotherapy during the course of their treatment. We explored potential synergistic drug combinations to re-sensitize tumors with acquired PARP inhibitor resistance in pancreatic and colorectal cancer. We hypothesized that dual inhibition of the DDR pathway through PARP and Ataxia telangiectasia and Rad3 related (ATR) inhibition would combine synergistically especially in cell lines with existing HR deficiencies through a synthetic lethality-mediated response. The ATR inhibitor Ceralasertib was selected for its potent antagonism of the DDR pathway, a key pathway associated with cell survival through DNA repair. Pancreatic and colorectal cell lines were carefully selected based on their varying mutational profiles to study differences in response to dual treatment. CellTiter-Glo® (CTG) was used after drug treatment to quantify IC-50. CTG synergy and combination indices were used to study drug combinations, and western blotting identified changes in protein expression among key mediators of cell survival and apoptosis. Colony forming assays and cytokine profiling using Luminex technology (and single cell cytokine profiling) were performed to study changes within the tumor microenvironment. We observed strong synergy and cytotoxicity in addition to increased apoptosis and cellular disassembly following treatment with dual ATR+PARP inhibition compared to monotherapy alone. Western Blot showed upregulation of cleaved PARP and cleaved Caspase 8 with combination therapy, and cytokine profiles provided insights into immune-suppressive versus stimulatory TME after drug treatment. There is potent synergy when ATR inhibitors are added to PARPi therapy in the context of pancreatic and colorectal cancers. Ongoing research efforts are further characterizing the mechanisms underlying these observed synergies in the same and additional tumor types such as prostate and ovarian cancer.

Citation Format: Anna M. Ochsner, Kelsey E. Huntington, Lanlan Zhou, Benedito Carneiro, Wafik El-Deiry. Combined ATR+PARP inhibition exhibits potent synergy in colorectal and pancreatic cancer cytotoxicity [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 2593.
Abstract 2607: Inhibition of HDAC3 induces BRCAness and potent synergy with PARP inhibition in neuroendocrine prostate and small cell lung cancers
Authors: Nagl Stephanie , Devore Kendra , Hirschfeld Gregg , Kotlarczyk Kari , Diaz Damaris , Wang Tong , Murray Maggie , Gonzales Paul , Gately Stephen Thomas
Published: June 2022

HDAC3 is essential for transcriptional repression and is required for the activity of NCOR1 & SMRT co-repressors complexes. Inhibition of HDAC3 results in the suppression of genes responsible for DNA damage repair and maintenance of genomic stability. HDAC3/NCOR1 also play critical roles in tumor metabolism and immune cell biology. GB-3103 is a novel and potent inhibitor of HDAC3 with an IC50 of 0.56nM that was tested for activity against human neuroendocrine prostate (NEPC) and small cell lung cancers (SCLC). RNA-seq analysis of GB-3103 treated human tumor cells revealed induction of BRCAness, inhibition of genes from DNA repair pathways and inhibition of PI3K-AKT1, MYC and p53 signaling. GB-3103 showed potent in vitro anticancer activity against the human H660 NEPC line, with an IC50 of 28nM, and 21nM against the murine PNEC30 line and potent IC50 of 16nM against NCI-H209 human lung neuroendocrine cancer. Because of the potent induction of BRCAness we used zero interaction potency (ZIP) model to test the activity of the combination of GB-3103 with a PARP inhibitor against human H660 and murine PNEC30 cell lines. These experiments revealed synergy for the combination of PARPi with GB-3103 in NEPC. GB-3103 was subsequently tested alone and in combination with olaparib in a xenograft model of H660. GB3103 (TGI=3%) showed minimal activity at the selected dose against H660 compared to single agent olaparib (TGI=67%). However, when combined with olaparib, GB-3103 revealed potent synergy (TGI=96%, p<0.01). In the NCI-H209 human lung neuroendocrine xenograft model, GB-3103 showed robust activity as a single agent (TGI=83%) compared to single agent olaparib (TGI=33%) and single agent talazoparib (TGI=69%). When combined with olaparib, GB-3103 induced synergistic antitumor effect (TGI=99%, p100%, p<0.001) when compared to talazoparib treatment alone. Taken together, these data confirm the important role of HDAC3 in neuroendocrine prostate and small cell lung cancers and suggest HDAC3 inhibition by GB-3103 could be an effective approach for patients with neuroendocrine cancers particularly when combined with inhibitors of PARP.

Citation Format: Stephanie Nagl, Kendra Devore, Gregg Hirschfeld, Kari Kotlarczyk, Damaris Diaz, Tong Wang, Maggie Murray, Paul Gonzales, Stephen Thomas Gately. Inhibition of HDAC3 induces BRCAness and potent synergy with PARP inhibition in neuroendocrine prostate and small cell lung cancers [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 2607.
Abstract 2608: Pre-clinical evaluation of small molecule CHK2 inhibitor, ART-122 combined with olaparib in BRCA wild type tumors
Authors: Baik Taegon , Park Chaeun , Kang Young Woo , Joh Danny , Lee Ha-Young , Park Sojung , Hwang Supyong , Kim In Ki , Kim Jong Oh
Published: June 2022

Background: PARP inhibitors are widely used for BRCA-mutant cancers, but often tumors become resistant to PARP inhibitors. The resistance develops through various mechanisms, including BRCA1/2 reversion mutation. Several publications have suggested that CHK2 inhibition could help overcome the resistance to PARP inhibitors. We discovered a novel small molecule CHK2 inhibitor, ART-122, and conducted a series of experiments to explore the rationale for combining a PARP inhibitor and ART-122 to treat the BRCA wild-type ovarian and breast tumors.

Results: In the LanthaScreen࣪ Kinase assay, ART-122 was shown to have the IC50 values of 0.57 nM and 10.85 nM, for CHK2 and CHK1, respectively. We observed the strong synergy between ART-122 and olaparib in the mouse xenograft model using the MDA-MB-231 cancer cells (BRCA1/2 wild type, triple-negative breast cancer cells). Animals were treated with 50 mg/kg olaparib on days 1-5, 30 mg/kg ART-122 on days 1-2 each week for 3 weeks, or both therapies. The result demonstrated approximately 41% reduction in tumor volume in mice treated with the combination therapy compared to the vehicle group. Animals treated with either of the single-agent therapies showed tumor growth similar to the vehicle group. We did not observe apparent toxicity or significant body weight differences among the animals. Additionally, the synergism between ART-122 and olaparib was confirmed in other cancer cell lines, including several BRCA wild-type and BRCA mutant cells lines such as CaOV3, SK-OV-3, UWB1.289, UWB1.289 with BRCA overexpression, HCC1937, MDA-MB-436, and MCF7.

Conclusion: The combination of ART-122 and Olaparib demonstrated a synergistic effect in BRCA wild-type ovarian and breast tumor cell lines. In the mouse xenograft model, the CHK2 inhibitor ART-122 sensitized BRCA1/2 wild-type tumor cells MDA-MB-231 to olaparib treatment. The data provide a strong rationale for the further development of ART-122 as a therapeutic agent.

Citation Format: Taegon Baik, Chaeun Park, Young Woo Kang, Danny Joh, Ha-Young Lee, Sojung Park, Supyong Hwang, In Ki Kim, Jong Oh Kim. Pre-clinical evaluation of small molecule CHK2 inhibitor, ART-122 combined with olaparib in BRCA wild type tumors [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 2608.
Abstract 2681: Novel and selective inhibitors of KRASG12D
Authors: Bhavar Prashant Kashinath , Surampudi Uday Kumar , Sarma Partha Pratim , Shidore Mahesh Mohan , Kshirsagar Anuj Ramesh
Published: June 2022

RAS proteins are family of small GTP-binding proteins that play a major role in regulating cellular responses such as proliferation, differentiation, migration, and cell growth. RAS proteins function as molecular switches, cycling between inactive (GDP-bound) and active (GTP-bound) states, which relay cellular signals in response to extracellular stimuli. Aberrant RAS signaling plays a role in up to 30% of all human cancers, with the highest incidence occurring in carcinomas of the pancreas (63–90%), colon (36–50%), and lung (19–30%). Targeting the inactive, GDP bound KRASG12C has been a promising approach for generating novel anti RAS therapies, which is validated by the approval of AMG 510 (Lumakras) by the FDA. It is also known that KRASG12D also retains GTPase activity. Consequently, KRASG12D is one of the most important chemotherapeutic drug targets. KRASG12D is commonly observed in pancreatic cancer. Here we describe preclinical profile of a potent, selective, in-vivo tool compound, VRTX144. VRTX144 was potent in HPAC cell line, representing pancreatic adenocarcinoma, and a panel of KRASG12D mutated cell lines. It was found to be several folds less active in non-KRASG12D mutant cell lines. In vitro combination strategies with other targeted therapies such as EGFR, SHP2 and CDK4/6 inhibitor is also being investigated. VRTX144 exhibited low propensity for metabolism in microsomes, and has high protein bound. VRTX144 was found to have no adverse effect in hERG upto 10 uM.

Citation Format: Prashant Kashinath Bhavar, Uday Kumar Surampudi, Partha Pratim Sarma, Mahesh Mohan Shidore, Anuj Ramesh Kshirsagar. Novel and selective inhibitors of KRASG12D [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 2681.
Abstract 2688: Bombesin receptors regulate transactivation of HER4
Authors: Moody Terry W. , Ramos-Alvarez Irene , Mantey Samuel A. , Jensen Robert T.
Published: June 2022

Bombesin (BB) is an autocrine growth factor for non-small cell lung cancer (NSCLC) cells. BB stimulates whereas the BB2 receptor (R) antagonist PD176252 inhibits NSCLC growth (Moody et al., Eur. J. Pharmacol; 2003; 474:21). The BB2R regulates the transactivation of the EGFR, HER2 and HER3 (Lee et al., BBAMCR 2020; 1867: 118625). HER4 increases the proliferation of NSCLC cells (Mota et al., Oncotarget 2017; 8: 89284). It is unknown if BB2R stimulation increases tyrosine phosphorylation of HER4 and this was investigated using NSCLC cells. BB or neuregulin (NRG1) addition to NSCLC cells increased the phosphorylation of Tyr1284-HER4. By RT-PCR and western blot, mRNA and protein for BB2R, HER4 and NRG1 was present in NCI-H522 and NCI-H661 NSCLC cells. The addition of BB to NCI-H522 or NCI-H661 cells increased phosphorylation of HER4, ERK and AKT which was blocked by PD176252. By immunoprecipitation, HER4 heterodimerized with HER2 and the EGFR after addition of BB to NSCLC cells. PP2 and N-acetylcysteine impaired the ability of BB to increase P-Tyr1284-HER4 in NSCLC cells, which indicates that SRC and reactive oxygen species are essential. Ibrutinib, a tyrosine kinase inhibitor (Rauf et al., Oncogene 2018; 37: 2237), impaired the ability of BB2Rs to regulate HER4 transactivation. Ibrutinib or PD176252 inhibited the growth of NSCLC cells whereas BB or NRG1 increased NSCLC colony formation. The results indicate that peptide receptors for BB regulate HER4 transactivation and the proliferation of NSCLC cells.

Citation Format: Terry W. Moody, Irene Ramos-Alvarez, Samuel A. Mantey, Robert T. Jensen. Bombesin receptors regulate transactivation of HER4 [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 2688.
Abstract 2716: Cancer study of germline carriers of Inborn Errors of Metabolism (IEMs) related genes
Authors: Kim Seokhyeon , Song Seulki , Koh Youngil , Yoon Sung-Soo
Published: June 2022

Inborn Errors of Metabolism (IEMs) are a hereditary disorder in which a gene fails to operate correctly as a result of a mutation in a metabolic-related gene passed down from the parents. Majority of IEMs are inherited in autosomal recessive manner. Recent research has been undertaken on mutations in numerous metabolic genes, including IEMs and cancer development. This study used Next Generation Sequencing (NGS) data to confirm the link between metabolic-related genes and cancer development in heterozygous carriers. The PCAWG (Pan-Cancer Analysis of Whole Genomes) cancer patient cohort and the 1000G (1000 Genomes) healthy cohort were used in this work, with the TCGA (The Cancer Genome Atlas) cancer patient cohorts being used for validation analysis. A regression analysis was used to determine the statistical significance of the cancer patient group versus the normal cohort. We analyzed RNA-seq data by separating heterozygous carrier and non-carrier groups of IEM related genes. The proportion of IEM gene heterozygote carriers was higher in cancer patients than in the normal population. We chose genes showing similar patterns of occurrence with statistical significance across several cancer patient cohorts. Accordingly, the most important gene, phenylalanine hydroxylase (PAH), has been established, and the carrier frequency was consistently high in lung squamous cell carcinoma (LUSC). RNA analysis results revealed that the PAH heterozygous carrier group was enriched with various immune response pathways suggesting inflammation related carcinogenesis in PAH related LUSC. Our finding suggest that heterozygote carrier status of IEM genes may lead to generation of aberrant metabolites and the breakdown of metabolic networks, which eventually leads to cancer.

Citation Format: Seokhyeon Kim, Seulki Song, Youngil Koh, Sung-Soo Yoon. Cancer study of germline carriers of Inborn Errors of Metabolism (IEMs) related genes [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 2716.
Abstract 2748: High-speed and high-precision FL-based cell count and viability assays using the Cellaca™ MX high-throughput cell counter
Authors: Bell Jordan , Huang Yongyang , Yung Sun , Qazi Henry , Hernandez Charles , Lin Bo , Qiu Jean , Chan Leo
Published: June 2022

With multiple FDA-approved cell therapy products available and others in pre-clinical and clinical trials, now, more than ever, it is critical to provide fast and accurate measurements of cell concentration and viability. For certain clinical products, cell concentration is synonymous with dosage, and proper cell viability is crucial to avoidance of potential harmful side effects. The complex nature of patient-derived samples makes legacy cell analysis methods such as the trypan blue exclusion assay difficult or impossible to perform. Nonspecific objects such as RBCs and cellular debris can significantly increase cell counting variation. Meanwhile, the need for cell-based products that are custom-tailored to each patient has resulted in the analysis of smaller and more numerous batches, leading to cell counting bottlenecks. With the challenges of messy samples and counting bottlenecks in mind, we investigate the use of the Cellaca™ MX, a cell counter that can increase throughput and employ fluorescence-based cell counting methods. The high-throughput cell counter can utilize 5 fluorescence imaging channels for cell count and viability analysis in less than 3 min for 24 samples. In addition, fluorescent cell-based assays such as apoptosis (Annexin V or Caspase 3/7), cell cycle, reactive oxygen species measurement can be performed to better characterize the target cell samples. In this work, we demonstrate the capabilities of Cellaca™ MX for high-throughput fluorescence-based cell counting. Comparison among multiple instruments using Jurkat cells and fluorescent beads reveals high consistency between replicate counts, between Cellaca consumable plates, and between instruments. We also compare cell counts made on the Cellaca™ MX with manual counts performed using a hemocytometer, as well as counts performed using other fluorescence-based automated cell counting methods. In addition, we illustrate the use of the ISO 20391-II cell counting standard to evaluate and compare the performance of cell counting methods. Finally, we include real-world primary T cell linearity results over a 4-log range of concentration. The results of the experiments confirm the suitability of the Cellaca™ MX for general fluorescence-based assays applicable to cell therapy-related workflows.

Citation Format: Jordan Bell, Yongyang Huang, Sun Yung, Henry Qazi, Charles Hernandez, Bo Lin, Jean Qiu, Leo Chan. High-speed and high-precision FL-based cell count and viability assays using the Cellaca™ MX high-throughput cell counter [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 2748.
Abstract 2763: Adaptation of therapeutic NK cell line to tumor microenvironment culture conditions increases potency and reduces cellular exhaustion
Authors: Lu Ann , Li Yunmin , Wong Albert , Lim James
Published: June 2022

NK-92 is a well-characterized human NK cell line that has demonstrated promising anti-cancer activities in clinical trials. However, the immunosuppressive solid tumor microenvironment (TME) can inhibit NK-92 function thereby limiting its cytolytic properties against tumor cells. To address this limitation, we serially passaged and maintained NK-92 cell lines under tumor microenvironment culture conditions (2.0 PSI + 1% O2) for a minimum of 5+ weeks. This adaptation period was used to precondition and expand NK cells with lower oxygen dependency, enriching for subset of cells that have metabolically adapted to pressurized and hypoxic culture conditions. NK cells maintained and expanded under TME culture conditions gradually reduced their cell-doubling times to reach parity with parental lines maintained under ambient oxygen (21% O2) culture conditions. This adaptation period coincided with improved tumor killing properties for TME-Adapted NK cells (TANK) against the non-small cell lung cancer cell line, A549. Enhanced cytolytic activity of TANK cells was two-fold greater than their non-TME adapted counterparts, with sustained potency observed in both ambient and TME screening conditions. Furthermore, serial exhaustion studies where TANK cells were repeatedly stimulated with tumor cells show sustained cytolytic activity, in contrast to non-TME adapted NK92 counterparts. TANK cells also exhibit higher levels of granzyme B and perforin as assessed by flow cytometry, while exhibiting distinct gene expression signatures as profiled with RNAseq. This initial study supports the hypothesis that immune cells can both react and adapt to different microenvironments. TME-adaptation strategies during cell expansion can enhance potency and efficacy of cell therapies designed to work in a solid tumor microenvironment.

Citation Format: Ann Lu, Yunmin Li, Albert Wong, James Lim. Adaptation of therapeutic NK cell line to tumor microenvironment culture conditions increases potency and reduces cellular exhaustion [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 2763.
Abstract 2767: Low baseline serum IFNg and TNFa are predictive of response to bexmarilimab
Authors: Koivunen Jussi , Jalkanen Juho , Björkman Mari , Mandelin Jami , Bono Petri
Published: June 2022

Introduction: Bexmarilimab is a novel macrophage re-programming agent targeting CLEVER-1 on tumor associated macrophages. CLEVER-1 is a highly immunosuppressive macrophage scavenger receptor. Inhibition of CLEVER-1 has been shown remove T cell exhaustion, induce clonal expansion of cytotoxic CD8 cells, and improve their infiltration into the tumor. Recently in a first-in-human Phase I/II study named MATINS (NCT03733990) bexmarilimab was reported to have a survival benefit in 30% of patients with advanced gastric cancer, cutaneous melanoma and cholangiocarcinoma as a single agent (ESMO 2021).

Methods & Aims: As a part of the mode of action of bexmarilimab, it is known to induce TNFa and IFNg responses, especially when IFNg levels are low. Using ROC analysis we aimed to test the performance of baseline cytokine serum IFNg and TNFa levels to identify patients that experienced a survival benefit from bexmarilimab compared to patients that did not benefit from bexmarilimab.

Results: 30 patients, 10 per each tumor type, were treated with bexmarilimab at 1mg/kg Q3W. Mean age 59 years (SD, 9 years), 73% male, with an average of 3 previous lines of therapy, median survival not yet reached. At baseline, patients that saw a clinical benefit (CBR) from bexmarilimab had average serum IFNg levels of 5.11 pg/ml (SD, +/- 4.99 pg/ml) compared to 13.07 pg/ml (SD, +/- 13.26) in patients that did not respond to the treatment. Similarly serum TNFa levels 0.96 pg/ml (SD, +/- 0.74 pg/ml) in patients that benefited from bexmarilimab compared to 2.37 pg/ml (SD, +/- 1.43 pg/ml) in patients that did not respond to the treatment. Log transformed values were used for ROC analyses. Used together IFNg and TNFa performed well to identify patients that are responsive to bexmarilimab (AUC 0.83, P < 0.0001).

Conclusion: Bexmarilimab works well in patients with low IFNg and TNFa levels, which is opposite to what is usually seen with checkpoint inhibitors and other T cell activating agents.

Citation Format: Jussi Koivunen, Juho Jalkanen, Mari Björkman, Jami Mandelin, Petri Bono. Low baseline serum IFNg and TNFa are predictive of response to bexmarilimab [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 2767.
Abstract 2769: Advance understanding of the tumor microenvironment with multiplex immunofluorescence
Authors: Patel Bhavika , Allen Stephanie , Boldt Brittney , Mammadova Najiba , Haggerty Agnes , Mehra Navi
Published: June 2022

Immunotherapy and precision medicine are rapidly developing approaches to cancer therapy. Biomarkers that detect the tumor and tumor microenvironment allow for the development of strategies that accelerate the advancement of treatments to enhance a patient’s immune system. Akoya’s MOTiF™ PD-1/PD-L1 Panel is a validated, multiplex immunoassay enabling detection of the 6 most clinically relevant immuno-oncology and spatial biomarkers: PD-1, PD-L1, FoxP3, CD8, CD68, and PanCK. The MOTiF™ PD-1/PD-L1 Panel was used to analyze the tumor microenvironment and specifically assess immune phenotypes of different types of cancers: non-small cell lung cancer (NSCLC), colon adenocarcinoma, head and neck squamous cell carcinoma (HNSCC), and breast cancer. We demonstrate the utility of Akoya’s MOTiF™ PD-1/PD-L1 panel kit in studying the cellular diversity of various cancers while retaining spatial context. Stained slides were analyzed using the InForm® and PhenoptrReports image analysis platforms to identify and better understand spatial relationships between a variety of simple and complex cell phenotypes. The MOTiF™ PD-1/PD-L1 panel kit provides reproducibility across different tissue types. These data provide insight into the innate and adaptive immune environment for targeted design of new immunotherapies and have implications for improving the treatment paradigm across many tumor types.

Citation Format: Bhavika Patel, Stephanie Allen, Brittney Boldt, Najiba Mammadova, Agnes Haggerty, Navi Mehra. Advance understanding of the tumor microenvironment with multiplex immunofluorescence [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 2769.
Abstract 2815: Exploring the role of telomerase in senescence and exhaustion in CAR T cell immunotherapy
Authors: Murty Tara , Ramello Maria C. , Sotillo Elena , Chen Lu , Artandi Steven A. , Mackall Crystal L.
Published: June 2022

Chimeric antigen receptor (CAR) T cell therapy has revolutionized oncology through engineered targeting of antigens on previously untreatable cancers. However, less than half of patients on CAR T cell therapy experience long-term disease control, with better outcomes observed in pediatric compared to adult populations. Senescent T cells have been shown to manifest defective killing abilities and the development of negative regulatory functions, with evidence suggesting that senescence may play a role in decreasing CAR T cell efficacy and persistence. Moreover, levels of telomerase have been shown to control the lifespan of human T cells, with increased levels delaying senescence. It has been shown that T cell exhaustion limits CAR T cell efficacy in the context of solid tumors, where CAR T cells have yet to demonstrate sustained responses. Using a model of CAR T cell exhaustion developed in our lab, and CAR T cells manufactured from younger and older donors, we have developed a comprehensive method to profile T cell functionality, phenotype, and proliferation. We are able to characterize features of senescence via cell surface markers, intracellular activity, telomere length, and telomerase activity as well as features of exhaustion. Using CRISPR/Cas9, we have inactivated TERT in primary human T cells and CAR T cells and interrogated the effects of hTERT knockout as well as of hTERT overexpression on key markers of T cell identity, exhaustion, senescence, and cytotoxic activity. Understanding telomerase in the context of CAR T cell therapy will provide mechanistic insights into senescence and exhaustion cellular programs and has the potential to inform increasingly effective CAR T cell cancer treatments.

Citation Format: Tara Murty, Maria C. Ramello, Elena Sotillo, Lu Chen, Steven A. Artandi, Crystal L. Mackall. Exploring the role of telomerase in senescence and exhaustion in CAR T cell immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2815.
Abstract 2818: Targeting solid tumors with GDT002, a first-in-class γδTCR-based T cell therapy
Authors: Drent Esther , Bisso Andrea , Baardman Sjoerd , Verweij Dagmar , Salcedo Estefania , Coomans Chris , Braem Steven , van de Weg Sander , Melief Sara , Norell Haakan , van Loenen Marleen , Gobessi Stefania , Throsby Mark
Published: June 2022

Broad application of cell therapies like CAR-T have been hampered by a lack of tumor-specific targets. Gadeta leverages the natural HLA-independent tumor recognition capabilities of γδTCRs combined with the proliferative capacity and robust tumor killing of αβT cells to develop tumor-specific cell therapies. γδT cells expressing Vγ9Vδ2 TCRs are the most common subtype found in peripheral blood. They sense the presence of phosphoantigens (pAgs) upregulated in malignant cells due to a dysregulated mevalonate pathway. The Vγ9Vδ2 TCR expressed by GDT002 was isolated from a PBMC-derived γδT cell of a healthy donor and was selected for its broad and strong tumor reactivity. GDT002 is currently being evaluated in a multicenter first-in-human phase 1/2 study for the treatment of Multiple Myeloma (NCT04688853). Here we describe studies carried out to identify potential solid tumor indications which could be effectively targeted by GDT002. Tumor reactivity was tested against cell lines from different cancer types in the presence of pamidronate, a clinically approved N-bisphosphonate that boosts pAgs levels. Significant GDT002 reactivity, in terms of cytotoxicity and cytokine release, was observed against most tumor targets. In a more complex ex vivo 3D co-culture system, GDT002 reactivity was also observed towards a broad panel of patient-derived tumor organoids. In contrast, there was no or very limited GDT002 reactivity against primary healthy cells. From our broad screening of solid tumor types, we identified ovarian cancer as a promising indication for GDT002. GDT002 displayed significant reactivity towards 4/5 ovarian cell lines and 9/10 patient-derived ovarian cancer organoids in the presence of pamidronate. To study GDT002 in vivo, we investigated the biodistribution of i.v. administered pamidronate. In tumor-bearing mice, intra-tumor pamidronate concentrations were detected during the 7-day observation period, allowing targeting by GDT002. Taken together, our results demonstrate that GDT002 has a potent anti-tumor activity across a broad spectrum of solid and liquid tumor types. We have identified ovarian cancer as a potential indication to explore the clinical effect of GDT002 against solid tumors.

Citation Format: Esther Drent, Andrea Bisso, Sjoerd Baardman, Dagmar Verweij, Estefania Salcedo, Chris Coomans, Steven Braem, Sander van de Weg, Sara Melief, Haakan Norell, Marleen van Loenen, Stefania Gobessi, Mark Throsby. Targeting solid tumors with GDT002, a first-in-class γδTCR-based T cell therapy [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 2818.
Abstract 2822: Enhanced effector activity of mediator CDK8 kinase module deficient CAR-T Cells
Authors: Freitas Katherine A. , Belk Julia A. , Sotillo Elena , Daniel Bence , Sandor Katalin , Klysz Dorota , Duong Vandon T. , Xu Peng , Malipatlolla Meena , Weber Evan W. , Majzner Robbie G. , Chang Howard Y. , Satpathy Ansuman T. , Mackall Crystal
Published: June 2022

Adoptive T cell immune therapies mediate impressive clinical benefit in a fraction of patients, but anti-tumor effects are often limited by inadequate T cell potency. To identify genes that limit T cell effector function, we conducted genome-wide CRISPR knock-out screens in human primary CAR-T cells. The top hits were components of the CDK8 kinase module of the Mediator complex, an evolutionarily conserved regulator of gene transcription. CDK8 kinase module deficient CAR-T cells manifest increased expansion, cytokine production, metabolic fitness, effector function, anti-tumor activity and reduced terminal effector differentiation. CDK8 kinase module deficient CAR-T cells showed widespread but selective increases in chromatin accessibility, MED1 chromatin occupancy, and H3K27 acetylation most notably involving transcription factors that play a critical role in T cell fate, including several STAT and AP1 family members. The most pronounced enhancement was observed for STAT5 which manifested as increased sensitivity to IL-2 in CDK8 kinase module deficient CAR-T cells. These results link Mediator induced transcriptional coactivation with T cell effector programming and identify the CDK8 kinase module as a target for enhancing the potency of anti-tumor T cell responses.

Citation Format: Katherine A. Freitas, Julia A. Belk, Elena Sotillo, Bence Daniel, Katalin Sandor, Dorota Klysz, Vandon T. Duong, Peng Xu, Meena Malipatlolla, Evan W. Weber, Robbie G. Majzner, Howard Y. Chang, Ansuman T. Satpathy, Crystal Mackall. Enhanced effector activity of mediator CDK8 kinase module deficient CAR-T 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 2822.
Abstract 2831: High-throughput T cell receptor cloning allows optimization of anti-miHA T cell therapies
Authors: Rowe Alexander M. , Didiano Dominic , Wei Wenzhong , Ito Sawa , Shlomchik Mark , Shlomchik Warren
Published: June 2022

Adoptive T cell therapy with CD8+ T cells engineered to express T cell receptors (TCRs) that recognize hematopoietically-restricted minor histocompatibility antigens (miHAs) is an attractive approach for improving the efficacy of allogeneic hematopoietic stem cell transplantation. We developed a mouse model to determine how to optimally implement anti-miHA T cell therapy, focusing on the key characteristics of anti-miHA TCRs that correlate with in vivo efficacy. We immunized B6 mice to the Kb-restricted miHA H60 and used a high throughput approach to amplify TCR α/β chains from single CD8+ MHC Multimer-H60+ (TetH60+) cells. Based on β chain sequencing of 472 single T cells, a broad repertoire was engaged by H60-vaccination, without a clear β chain preference. We randomly chose 178 unique TCRs, cloned them into retroviral backbones and expressed them in a TCR-CD8+ NFAT-reporter reporter cell lines and screened for H60-reactivity by NFAT-induction after stimulation with H60-pulsed antigen presenting cells (APCs). We chose 22 unique TCRs that had clear H60-specific NFAT-induction for deeper characterization. In similarly-transduced 4G4 reporter cells, these TCRs spanned a range of surface expression levels, affinities for H60 as measured by TetH60 binding, and EC50s in response to H60-pulsed APCs (between 1 and 12 μM peptide concentration). We focused initial functional studies on 2 high avidity TCRs (TCR 61 and TCR 167) with EC50s of 1.25 and 2.5 µM, respectively. These were cloned into an optimized retroviral vector and transduced into B6 CD8 cells concomitant with efficient CRISPR-knock out of the endogenous TCRα/β genes. Engineered T cells bound H60 loaded MHC multimers and killed H60+ targets in vitro. The mean TCR expression of transduced cells was 16-30% of that in unmanipulated T cells, though some had TCR expression similar to or greater than that in unmanipulated T cells. We used TCR 61 and 167 transduced T cell products to treat H60+ blast crisis leukemia cells (BC-CML). TCR 61 and 167 T cell products expanded and were equally effective at reducing BC-CML numbers in spleen and bone marrow (BM). Despite having a greater EC50 and equivalent surface expression in reporter cells, when recovered post-transplant, TCR167 cells had higher TetH60 binding, and produced more IFN-γ. Ongoing studies are testing these and additional TCRs alone and in competition to better elucidate TCR properties that predict for in vivo efficacy.

Citation Format: Alexander M. Rowe, Dominic Didiano, Wenzhong Wei, Sawa Ito, Mark Shlomchik, Warren Shlomchik. High-throughput T cell receptor cloning allows optimization of anti-miHA T cell therapies [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 2831.
Abstract 284: A 3D multicellular in vitro prostate cancer model featuring racially/ethnically diverse PDXs
Authors: Iyer Divya , Bonteanu Andrei , Shepherd Peter , Kittles Rick , Navone Nora , Harrington Daniel , Bircsak Kristin
Published: June 2022

Current in vitro prostate cancer (PCa) research tools do not incorporate the complexities of the tumor microenvironment including perfusion, multiple cell types, extracellular matrix (ECM) and 3D-orientation. Additionally, these models lack racial/ethnic (R/E) diversity, failing to address the cancer health disparity (CHD) observed in Black men with PCa who experience double the incidence and mortality rates as compared to White men. Population-based in vitro PCa models that support crosstalk between different cell types will improve our understanding of the underlying mechanisms contributing to this CHD and improve the prediction of drug response in specific populations. Here, we used the high-throughput, perfusion-based microfluidic platform called the MIMETAS OrganoPlate® to culture up to 96 individual, multicellular PCa-on-a-chip cultures in parallel. PCa PDX cells of various R/E were embedded together with E2Crimson-labeled stromal fibroblasts in a migration-permissive hyaluronic acid hydrogel in the gel compartment, alongside an endothelium-lined perfusion channel containing a targeted immune cell population. Using high content imaging, we confirmed the stability and viability of the cultures over 7 days. The cultures maintained a 3D structure with PCa cells and fibroblasts evenly dispersed throughout the height of the gel compartment and a blood vessel-like structure within the perfusion channel. Closer evaluation of the gel compartment revealed a close association between PCa cells and stromal fibroblasts in a core-shell structure with PCa clusters surrounded by fibroblasts. Further, all cells maintained expected expression of phenotypic markers (PCa: epCAM, PSMA; fibroblast: vimentin, endothelium: CD31). For the immune component, medium and cell tracker dye conditions were optimized to support immune cell viability. Over 72 hours, immune cells were monitored, revealing migration of PBMCs through the gel compartment. Finally, the value of the complex, population-based multicellular in vitro model of PCa will be evaluated in a drug screen comparing the system with simple, PCa PDX monocultures. This R/E diverse 3D prostate tumor model will enable the full incorporation of all cell types and ECM into a single model which will better recapitulate population specific PCa and drug response.

Citation Format: Divya Iyer, Andrei Bonteanu, Peter Shepherd, Rick Kittles, Nora Navone, Daniel Harrington, Kristin Bircsak. A 3D multicellular in vitro prostate cancer model featuring racially/ethnically diverse PDXs [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 284.