K562 Cells Research Articles

Abstract 670: Preclinical candidate LAE120, a novel selective USP1 inhibitor shows effective anticancer and combination activity with PARP inhibitor

Abstract The Fanconi anemia (FA) and translesion synthesis (TLS) are DNA damage tolerance and repair pathways that have been found to be regulated by reversible ubiquitination in which ubiquitin-specific protease 1 (USP1) plays a key role. Numerous studies have indicated that USP1 inhibitors have therapeutic effects on tumors with homologous recombination deficiency (HRD), including mutations in BRCA1/2. Due to the emergence of resistance to PARP inhibitors in clinical applications, new strategies are needed to address this issue. Here we present LAE120, a novel, allosteric and highly potent USP1 inhibitor, displayed monotherapy potency and combination activity with PARP inhibitor in HRD cancers. LAE120 has a unique tail group distinct from all the published USP1 inhibitors and is predicted to induce a different conformational change at the USP1 allosteric site. In the biochemical assay, LAE120 strongly inhibited USP1 enzymatic activity with IC50 of 5.6 nM. LAE120 potently inhibited proliferation of BRCA-mutated MDA-MB-436 breast cancer cell (IC50:19 nM) and exhibited anti-proliferative activity in NCI-H1792 (IC50: 69 nM) and K562 (IC50: 38 nM) cell lines. In the xenograft model of MDA-MB-436 cells with BRCA mutations, LAE120 demonstrated robust single-agent tumor inhibitory activity (TGI: 88%, 10 mg/kg BID) and a favorable dose-dependent response (TGI: 106%, 25 mg/kg BID). We also observed strong tumor-inhibition efficacy of LAE120 as a single agent in the K562 xenograft model with Bcr-Abl expression (TGI: 86%, 10 mg/kg BID). Current research indicates that USP1 inhibitors exhibit synergistic activity in combination with PARP inhibitors and re-sensitizes PARP refractory tumors. Similar results have been confirmed in the xenograft model of BRCA-mutated MDA-MB-436 cells. The combination of LAE120 and Olaparib (PARP inhibitor) yielded a more robust and durable anti-tumor efficacy at low dose (TGI: 110%, LAE120, 5 mg/kg BID + Olaparib, 50 mg/kg QD). LAE120 also displayed favorable ADME properties and PK profiles. Preclinical-tox studies indicated the molecule was well tolerated without significant toxicities and hematological observations. Both in vitro and in vivo data strongly support further investigation of LAE120, whether as a single agent or in combination therapy. Citation Format: Jintao Wang, Yan Chen, Junyan Chen, Ling Jiang, Xiaofen Lin, Chaojun Cai, Minhua Zhang, Ming Li, Justin Gu. Preclinical candidate LAE120, a novel selective USP1 inhibitor shows effective anticancer and combination activity with PARP 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 670.

Abstract 4620: Exploring the role of GHRH antagonist MIA-602 in overcoming doxorubicin resistance in acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is characterized by the uncontrolled proliferation of immature hematopoietic progenitors, often associated with a poor prognosis. Conventional therapies involve chemotherapy and bone marrow stem cell transplantation. The growth hormone releasing hormone receptor (GHRH-R) antagonist MIA-602 has exhibited inhibitory effects on the growth of various human cancer cell lines, including AML cells. This study aimed to investigate the impact of MIA-602 alone and in combination with doxorubicin on three doxorubicin-resistant AML cell lines, K-562, U-937, and KG-1A. Initial examinations confirmed the presence of GHRH receptors in both wild-type and doxorubicin-resistant cell lines through western blot analysis. Doxorubicin was administered at concentrations of 0.005, 0.01, and 0.05 μg/ml, while MIA-602 was used at 5 μmol/L. Subsequent experiments involved culturing both wild-type and doxorubicin-resistant clones with MIA-602 at concentrations ranging from 0.05 μmol/L to 5 μmol/L for 24 and 48 hours. The results revealed a significant, dose- and time-dependent reduction in cell proliferation across all six cell lines. Both wild-type and doxorubicin-resistant clones exhibited a comparable decrease in cell proliferation when exposed to MIA-602 at concentrations greater than 0.05 μmol/L (p < 0.05) after 24 and 48 hours. In conclusion, our study demonstrates that these three AML cell lines and their doxorubicin-resistant clones remain susceptible to GHRH antagonist MIA-602. These findings may pave the way for the development of novel therapies or drug combinations using GHRH antagonists such as MIA-602 for treating AML. This is of particular interest for those who are resistant to conventional chemotherapy, further broadening the spectrum of treatment options available. Additional investigations are warranted to progress these findings to in vivo xenograft models. Citation Format: Simonetta I. Gaumond, Joel Costoya, Andrew V. Schally, Joaquin J. Jimenez. Exploring the role of GHRH antagonist MIA-602 in overcoming doxorubicin resistance in acute myeloid leukemia [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 4620.

Abstract 5236: Adoptive natural killer therapy is effective for synovial sarcoma cell lines

Abstract Despite the development of multimodal therapy consisting of surgery, radiotherapy, and chemotherapy, the prognosis of patients with metastatic or relapsed synovial sarcoma (SS) remains poor. A novel treatment option including immunotherapy is urgently required. Natural killer (NK) cells are innate immune cells that can recognize and kill tumor cells. The cytolytic activity of NK cells is determined by the balance of signals from both inhibitory and activating receptors expressed on cell surface. Activating receptors include NKp30, NKp44, NKp46, NKG2D, and DNAM-1. Furthermore, tumor cells with low HLA class I expression are potentially even more sensitive to NK-mediated cytotoxic killing due to a lack of inhibitory signals. We previously reported that HLA class I expression was down regulated in one third of patients with synovial sarcoma, suggesting that NK cell therapy may be effective for patients with SS. However, the efficacy of NK cells treatment in SS was not well established. In this study, we evaluated surface ligands for NK cell activation on SS cell lines and demonstrated the effectiveness of activated allogenic NK cells for SS cell lines in vitro.The surface expression of ligands for NK cell-activating receptors including NKp30, NKp44, NKp46, NKG2D, and DNAM-1 in three SS cell lines (SYO-I, HS-SY-II, and Yamato-SS) was evaluated using flow cytometry. Peripheral blood from two healthy donors were ficoll-separated to collect peripheral blood mononuclear cells (PBMCs). Then, PBMCs were stimulated with irradiated K562 cells expressing the NK-stimulatory molecules 4-1BB ligand and membrane-bound Interleukin 15 in the presence of human Interleukin-2 in RPMI1640 with 10% FBS. The cells were expanded for 14 days, and the expanded NK cells are CD3-CD56+ NK cells. These expanded cells are reproducible in their function, and we have utilized these cells for in vitro cytotoxicity assays. 2x10E4 of SS cell lines (SYO-I, HS-SY-II, and Yamato-SS) were seeded in a 96 well plates and cultured overnight. Then, allogenic NK cells or activated T cells as a control at various tumor effector ratio. The cells were co-cultured with activated T cells or NK cells in the presence of low dose interleukin2. Forty-eight hours later, cell viabilities were measured using WST-8 assay.Ligands for NKp44, NKp30, NK2GD, were expressed in all three SS cell lines. It suggested that SS cells upregulate activating receptor ligands for NK cells. In addition, cytotoxicity of allogenic NK cells for SS cell lines was significantly higher than that of activated T cells. To determine whether cytotoxic activity was effect of allogenic NK cells, CD107a mobilization assay using flowcytometry was performed. CD107a mobilization assay demonstrated degranulation of NK cells, leading us that NK cells had an antitumoral effect on SS cell lines in vitro. Based on these initial findings, we were able to demonstrate that allogenic NK cells may be a promising immune therapy for SS. Citation Format: Naoki Oike, Tomohiro Miyazaki, Yudai Murayama, Takashi Ariizumi, Akira Ogose, Minori Baba, Hiroyuki Kawashima, Chihaya Imai. Adoptive natural killer therapy is effective for synovial sarcoma cell lines [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 5236.

Abstract 1829: Brusatol overcomes tyrosine kinase inhibitors resistance in myeloid leukemia cells by inhibiting translation of fast turnover protein cyclin D1 and Mcl-1

Abstract Myeloid leukemia is characterized with fast proliferation and apoptosis evasion, which are controlled by mutant or activated tyrosine kinase signalings. Tyrosine kinase inhibitors (TKI) targeting BCR-ABL in chronic myeloid leukemia (CML) and FLT3-ITD in acute myeloid leukemia (AML) significantly improved the therapy, but not cure, due to the general resistance. Overactivated mTOR protein translation pathway is believed to be one of the mechanisms causing resistance and that targeting protein translation should be one effective approach to overcome resistance. Brusatol is a quassinoid extracted from the fruit of Brucea javanica and an inhibitor of protein translation. We tested its antiproliferation and apoptosis induction in a variety of AML and CML cell lines as well as in TKI resistant cell lines. Brusatol effectively inhibits the growth of seven leukemia cell lines with GI50 values less than 20 nM. Brusatol induces apoptosis in AML cell lines and induces G0/G1 phase arrest in CML cell lines. Brusatol decreases the levels of fast turnover protein cyclin D1 and Mcl-1. In FLT3-ITD inhibitor sorafenib resistant MOLM-13/Sor cell line brusatol exhibits similar growth inhibition and apoptosis induction with the parental MOLM-13 cells, associated with inhibition of Mcl-1 and FLT3-ITD proteins. In BCR-ABL inhibitor imatinib resistant K562/STI cell line brusatol is effective as in parental K562 cells, associated with inhibition of cyclin D1 and BCR-ABL proteins. Brusatol at 2 mg/kg effectively inhibit tumor growth of K562 xenografts without causing toxicity to normal organs. These data indicate that brusatol inhibits oncogenic protein translation and has potential to be used to treat TKI resistant leukemia. Citation Format: Huiming Hua, Ying Yang, Guangfuxin Lin, Jingyi Zhang, Samuel Waxman, Yongkui Jing. Brusatol overcomes tyrosine kinase inhibitors resistance in myeloid leukemia cells by inhibiting translation of fast turnover protein cyclin D1 and Mcl-1 [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 1829.

Abstract 4586: ITF3756 increases NKG2D expression and cytotoxic activity of activated human CD8 T cells

Abstract Effector cytolytic CD8 T cells play a major role in the recognition and killing of cancer cells. These cells are short living due to the loss of their self-renewal capacity and rapid exhaustion. Enduring antitumor immune response relies on the capacity of regenerating effector cells upon antigen re-encounter by memory cells. Focusing on molecules that could modulate the differentiation of T cells towards a memory subset with a prolonged anti-tumor activity is strategic for an efficient anti-tumor immunotherapy. Thus, we developed a selective HDAC6 inhibitor currently in Phase I clinical trial, ITF3756, which in vitro drives the differentiation of human CD8 T cells towards a central memory phenotype with reduced expression of exhaustion markers and higher expression of IFNγ and Granzyme b upon tumor cells coculture. This suggests that ITF3756-treated CD8 T cells could have a greater tumor killing activity. Moreover, if this augmented cytotoxic activity is directed not only towards tumor cells expressing MHC I but also towards MHC I negative cells, the anti-tumor efficacy could be improved. We thus selected the MHC I negative K562 leukemic cell line as target in an in vitro model of CD8 T cell killing assay. CD8 T cells were stimulated with anti CD3CD28 beads with or without ITF3756, and then co-cultivated with K562 cells for 4h. ITF3756 treatment improved significantly CD8 mediated K562 killing over this short period of time. However, since the effectiveness of memory cells is based on their capacity of maintaining a long-lasting immune response, we monitored the cytolytic activity over a period of 2 weeks. After 14 days of co-culture, ITF3756 treated CD8 T cells showed a higher cytotoxic capacity compared to control cells. These results indicate that ITF3756-treated CD8 T cells can kill a target tumor cell in a more efficient way independently of MHC I restricted tumor recognition, both after short and long term in vitro co-culture. Since it has been described that CD8 T cells can also kill MHC I-negative tumor cells through the NKG2D-NKG2DL axis, we assessed if ITF3756 could modulate the expression of NKG2D on CD8 T cells. Indeed, ITF3756-treated CD8 T cells had a significantly higher expression of NKG2D compared to untreated cells. In conclusion, we show that ITF3756 promotes the differentiation of cells that possess better effector activity when co-cultured with tumor cells for both short and long periods of time. ITF3756 increased the expression of NKG2D and this could be a driver of the increased cytolytic activity. One mechanism of tumor immune escape is the downregulation of MHC I. Our results suggest that ITF3756 could be used to improve the efficacy of T cell therapy by enhancing the cytolytic activity even in the absence of MHC I. Citation Format: Chiara Ripamonti, Valeria Spadotto, Christian Steinkuhler, Gianluca Fossati. ITF3756 increases NKG2D expression and cytotoxic activity of activated human CD8 T cells [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 4586.

Abstract 1742: A universal spike-in normalization strategy for CUT&RUN, CUT&Tag and ATAC-seq

Abstract The development of simple and efficient genome-wide approaches has recently democratized the study of epigenetics. For instance, CUT&RUN and CUT&Tag were developed as fast and affordable alternatives to ChIP-seq, whereas ATAC-seq readily identifies regions of open chromatin. These methods are ideal to look for genome-wide alterations in chromatin structures but are only semi-quantitative and can miss global changes when performed without normalization spike-ins. In fact, many standard computational pipelines fail to detect a reportedly substantial global H3K27me3 decrease in the presence of GSK126, a therapeutic target for lymphoma and potent inhibitor of the EZH2 methyltransferase. To overcome this challenge, we developed a spike-in approach using Drosophila melanogaster nuclei for normalization of ATAC, CUT&RUN and CUT&Tag experiments using human cells. The addition of the spiked-in nuclei allowed for the detection of the expected global changes in open regions of chromatin (ATAC-seq), H3K27me3 (CUT&Tag), R-loops (CUT&Tag), EED and p53 binding (CUT&RUN) upon treatment of K562 cells with GSK126. By contrast, these same changes were all missed when omitting the spike-in nuclei. This streamlined spike-in normalization strategy can be easily applied to any number of cells and on a large variety of conditions. Citation Format: Sarah Traynor, Bradley Townsley, Julianna Weber, Mary Anne Jelinek, Brian Egan, Benjamin Delatte. A universal spike-in normalization strategy for CUT&RUN, CUT&Tag and ATAC-seq [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 1742.

Abstract 2778: Influence of the differentiation strategy on the phenotypic and genotypic features of iPSC derived NK cells

Abstract The iPSC-derived NK (iNK) cells offer new perspectives to produce large-scale of homogeneous immunotherapeutic cellular products and open the way towards manufacturing off-the-shelf cancer immunotherapies. Numerous strategies and protocols of differentiation of iPSCs toward NK lineage have been published in the last decade. Nevertheless, the embryological development of the NK cells and the impact of the differentiation strategy on the activity of the NK cells are still not clearly understood. Furthermore, very few studies have compared different NK differentiation strategies from iPSC. this study, we compared two previously reported protocols for NK cell generation from the same iPSC line, one feeder-based and the other feeder-free. In the first protocol, 3D hematopoietic differentiation was followed by lymphoid differentiation, using an OP9 DLL4 expressing feeder cell line. The second protocol employed a clinical-grade approach, utilizing the “spin embryoid body” method to generate primitive hematopoietic progenitors. Subsequently, these progenitors were differentiated towards the NK lineage in a feeder-free environment. Hematopoietic progenitors produced by these protocols have distinct phenotypes, suggesting variations in their maturity levels. Furthermore, the protocols have different kinetics of differentiation revealed by CD56 and CD7 phenotypes. Although, both protocols were able to efficiently generate functional and mature NK cells, characterized by the expression of activating and maturity ligands but with a distinct transcriptomic profile. iNK cells generated using feeders exhibit a more pronounced inflammatory profile with the expression of hallmark NFKB and STAT3 signaling pathways. In contrast, feeder-free iNK cells have a more proliferative profile as evidenced by increased expression of cell cycle factors such as E2F or Myc. Feeder-based iNK cells exhibit increased expression of transcription factors involved in NK cell differentiation, such as GATA2, STAT1, and IRF1. These transcriptional variances are reinforced by superior functionality observed in feeder-based NK cells compared to feeder-free NK cells, evidenced by increased degranulation (p-value < 0.0001) and cytotoxicity (p-value = 0.0004) at a 1:2 (effector-to-target) ratio against the K562 cell line. Our findings indicate that, despite the advantages of the feeder-free strategy, the feeder-based protocol remains more efficient and produces iPSC-derived NK cells with an enhanced cytotoxic profile in vitro. Our comprehensive transcriptional analysis offers significant insights into the biological and functional properties of iNK cells. This information has the potential to oncover novel mechanisms for NK differentiation strategies from iPSCs. Citation Format: Matthias Huyghe, Christophe Desterke, Jusuf Imeri, Nathan Belliard, Ali G. Turhan, Annelise Bennaceur Griscelli, Frank Griscelli. Influence of the differentiation strategy on the phenotypic and genotypic features of iPSC derived NK cells [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 2778.

Abstract 7416: GeneSetR: A web server for gene set analysis based on genome-wide Perturb-Seq data

Abstract Identification of genotype-phenotype relationships is of utmost importance and a core effort in biology. Recent developments in efficient and precise gene targeting approaches coupled to omics methods have significantly improved deciphering of molecular interactions and relationships. However, many single gene perturbations can affect the expression of hundreds of other genes and analysis of the resulting omics-derived gene lists currently remains a significant challenge. Here we present Perturb-Seq based Gene Set Analyzer (GeneSetR), a user-friendly web-server that can analyze user-defined gene lists based on the data from a recently published genome-wide Perturb-Seq study, which targeted 9,866 genes with 11,258 sgRNAs in the K562 cell line. Through this tool, users can cluster gene lists following dimensionality reduction by various algorithms, undertake network analysis from RNA sequencing data, identify key nodes among the submitted genes, perform gene signature analyses, and generate heatmaps based on perturbation or gene expression data. GeneSetR enables researchers to readily identify gene clusters associated with specific phenotypes or biological processes, providing insights into the potential functional roles of these clusters and the role of single genes in them. With robust analysis capabilities, GeneSetR is a powerful resource to facilitate the exploration of genotype-phenotype relationships. Citation Format: Omer Faruk Kuzu, Fahri Saatcioglu. GeneSetR: A web server for gene set analysis based on genome-wide Perturb-Seq data [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 7416.

Abstract 1800: Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis in human cells

Abstract The genes that are required for the growth of mammalian cells can depend on an interplay of cell-intrinsic factors and environmental context. However, there is often little investigation into how nutrient availability impacts gene essentiality. Moreover, efforts such as DepMap have catalogued genes that contribute to cancer cell fitness using in vitro CRISPR screens in hundreds of cancer cell lines cultured in traditional media that poorly recapitulate the nutrient availability of human blood. Previously, we developed Human Plasma-Like Medium (HPLM), a physiologic medium designed to better model the nutrient availability of human blood. We recently tested the hypothesis that nutrient availability influences gene essentiality. By performing paired CRISPR-based screens in human blood cancer cells, we identified sets of genes that are differentially required to support cell growth in HPLM versus traditional media such as RPMI. Among the strongest scoring HPLM-essential genes was NAD kinase (NADK), which encodes an enzyme critical for the generation of cytosolic NADPH. NADK has been previously suggested as an anti-cancer therapeutic target, given the central importance of NADPH for enabling proliferative metabolism through macromolecule synthesis and ROS management. Interestingly, NADK has been annotated as an essential gene in only 1% of the cell lines included in DepMap, suggesting that the essentiality of NADK may have been largely masked by the nutrient conditions used to generate the DepMap. To investigate the conditional CRISPR phenotype for NADK, we engineered NADK-knockout cells and found that they showed a 50% stronger growth defect versus control cells in HPLM relative to RPMI. Next, we used localization and activity studies to confirm that cytosolic NAD+ kinase activity is required for the conditionally essential role of NADK. We then employed systematic approaches in media engineering and metabolomics to identify the medium component(s) that contribute to the NADK gene-nutrient interaction and to gain insights into the conditionally essential role of NADK. Through these studies, we traced the cause of NADK essentiality to an inhibition of folate-dependent nucleotide synthesis linked to differential folic acid availability. Finally, we examined the pre-translational potential of NADK as an anti-cancer therapeutic target by demonstrating that the reported NADK inhibitor thionicotinamide largely phenocopies NADK-knockout in a folic-acid dependent manner in the K562 cell line. Collectively, our work improves understanding of how NADK contributes to human cell growth and reveals how the relative importance of cytosolic NAD+ kinase activity can depend on nutrient availability. Citation Format: Kyle M. Flickinger, Kimberly S. Huggler, Jason R. Cantor. Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis in human cells [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 1800.

Abstract 7565: Tumor classification and deconvolution in liquid biopsy using enriched methylation sequencing

Abstract Background: Cell-free DNA (cfDNA) detected in proximal body fluids has demonstrated potential for cancer detection using minimally invasive methodology. Our past work showed that tumor cfDNA is present in the cerebrospinal fluid (CSF) and other body fluids of patients with inconclusive standard of care testing. However, past work measuring copy number aberrations or somatic mutations was limited in cancer classification. To facilitate reliable classification, even at low tumor fractions and with fragmented DNA, we developed XR-methylSeq, a methylation sequencing platform to enrich for cell type-specific markers. Methods: We benchmarked XR-methylSeq with the K562 cell line and correlated the methylation values with gold standard measurement − whole genome bisulfite sequencing (WGBS). Methylation classifiers were applied for at least 22 cytology-positive body fluids, incorporating methylation array data from public references. T-distributed stochastic neighbor embedding (t-SNE) analysis was used for visualization in R. Deconvolution of cell type fractions for at least 29 (seven cytology-negative) body fluids and plasma samples was conducted using wgbstools. Cell type-specific markers were identified from a human DNA methylation atlas. Results: Benchmarks: XR-methylSeq has a 5-fold enrichment of the cell type-specific markers compared with WGBS. XR-methylSeq at 20 ng input highly correlates with WGBS at 2 μg (Pearson’s r = 0.97). Body Fluids: Thirteen cytology-positive CSF samples had copy number aberrations, 77% of them had concordant tumor classification, while the remaining 23% clustered with low tumor fraction samples. All nine lung primaries, including a low tumor fraction case that did not originally classify, showed a consistent cell-of-origin through deconvolution, as indicated by increased contributions from lung alveolar epithelial cells. Among six other body fluids, three exhibited the highest fractions aligning with the clinically identified cancer cell-of-origin. Additionally, the plasma cfDNA of a patient with acute liver injury had a higher fraction of hepatocyte signatures (22%) than the healthy control (8%). Conclusions: This research highlights the potential of XR-methylSeq as an enriched methylation profiling method useful for liquid biopsy applications. Citation Format: Jingru Yu, Lauren S. Ahmann, Yvette Y. Yao, Angus Toland, Alicia Snowden, Chandler Ho, Benjamin Pinsky, Hannes Vogel, Ruben Y. Luo, Linlin Wang, Brooke Howitt, Brittany Holmes, Alarice C. Lowe, Wei Gu. Tumor classification and deconvolution in liquid biopsy using enriched methylation sequencing [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 7565.

Abstract 1335: Generating functional CAR-NK cells for cancer immunotherapy

Abstract Chimeric Antigen Receptor-Natural Killer (CAR-NK) cells are rapidly emerging as a promising cellular therapeutic overcoming the potential life-threatening adverse events that may result from CAR-T therapy, including cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD). Due to their intrinsic characteristics, NK cells can kill tumor cells without the requirement for recognition of antigen in the context of MHC. This removes the constraint of HLA matching and supports antigen independent cytotoxicity through NK cell intrinsic mechanisms. As such CAR-NK cells kill via both CAR-dependent and -independent mechanisms. For these reasons, they offer the advantage of being an “off-the-shelf” product; satisfying the requirement for large-scale production of cancer immunotherapies, with the potential to reach more patients at a reduced cost. The frequency of NK cells in the blood is low (~ 2-5%), therefore improving their in vitro expansion is key to generating sufficient material to offer a viable CAR-NK cell therapy. Here we outline an in-vitro method to generate, expand, and test primary human CAR-NK cells using engineered feeder cells, and go on to demonstrate the efficacy of the CAR-NK against target cancer cells. Primary human NK cells were cultured and expanded in the presence of irradiated K562 cells (iK562) modified to express 4-1BBL and membrane bound IL-21. Following initial expansion, NK cells were transduced with a clinically tested CD19-targeting CAR lentiviral vector (LV) or GFP expressing LV; as an empty vector transduced control. Following transduction, CD19 CAR-NK and GFP-NK were enriched by FACS sorting to reach >80% purity and further expanded in the presence of 4-1BBL/mbIL21 iK562 feeder cells. Following sufficient expansion, cytotoxicity of fresh or cryopreserved CD19 CAR-NK cells was tested in vitro in a Tumor Killing Assay (TKA) against CD19+ or CD19- target cells. As expected, CD19 CAR-NK cells showed increased on-target cytotoxicity, increased degranulation and IFNꝩ secretion compared to controls. This effect was not observed with CD19 negative cancer cells, strongly suggesting CD19 CAR engagement of the target antigen to mediate killing. Taken together these data show the generation, expansion, and enrichment of CD19 CAR-NK cells with enhanced cytotoxic activity towards CD19+ target cells. This approach provides a platform for in vitro characterization of novel CAR-constructs, allied with in-vitro safety assessment and in vivo pharmacology testing. Together, this offers the potential to further develop new and more widely available cellular therapeutics to treat cancer. Citation Format: Karina Di Gregoli, Henry Leonard, Dan Rocca, Sabrina de Munnik, Gemma Moiset, Robert Nunan, Lauren Schewitz-Bowers, Louise Brackenbury. Generating functional CAR-NK cells for cancer 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 1335.

Abstract 227: Novel and easy-to-use approach to triple-negative breast cancer tumoroid culture

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer diagnoses in the United States. Patients with metastatic TNBC have a low survival rate, driven in part by limited treatment options for the disease. Tumoroids, also known as cancer organoids, have been shown to better maintain tumor-specific mutational and gene expression profiles over long-term culture, and evidence is growing that the functional response of tumoroids to therapeutics better recapitulates in vivo response. These improved preclinical models of TNBC may facilitate the development of new therapies, but tumoroids are yet to be widely adopted due to challenging culture methods. In this study, we show that two TNBC tumoroid models publicly available through the National Cancer Institute Patient-Derived Models Repository can be expanded in suspension culture using Gibco™ OncoPro™ Tumoroid Culture Medium supplemented with FGF10 and beta-estradiol. We compared the bulk tumoroid morphologies, growth rates, maintenance of mutational and gene expression profiles, and molecular phenotypes of cells grown in the NCI-recommended medium in embedded culture, and the OncoPro medium in embedded culture and suspension culture. We saw comparable morphologies and growth rates in all culture conditions. A multivariate analysis of the allelic frequencies of single nucleotide variants and ploidy values of copy number variants for cells expanded in all experimental conditions revealed >95% and >88% correlation, respectively, with the initial material. Interestingly, despite the change in the medium formulation, gene expression was also highly correlated between the initial material and the OncoPro medium conditions for >20,000 RefSeq genes (Pearson r>0.94) and for a cancer-gene specific panel of 1,423 genes (r>0.87). Further analysis showed unchanged expression levels in 97% of total genes analyzed and the basal molecular subtype was maintained for both lines in all conditions. The OncoPro media system was also used to derive a new TNBC tumoroid line, which demonstrates consistent growth rate and maintains the patient-specific mutational profile of the original breast tumor. Differential gene expression analysis revealed that changes in the gene expression profile were largely due to the loss of non-malignant cell populations from the original tumor. Finally, we utilized this new tumoroid line to examine donor-specific killing efficiencies of primary natural killer cells in a co-culture experiment. The discrepancy in the dynamics of killing a tumoroid line versus the standard K562 cell line indicates that tumoroids may offer an improved approach for screening new cell therapies. In summary, the easy-to-adopt OncoPro medium and culture method does not change the characteristics of existing models and may be used to derive new tumoroid lines, both of which can be leveraged for preclinical studies to develop critical new therapies for TNBC. Citation Format: Brittany Balhouse, Chris Yankaskas, Colin Paul, Shyanne Salen, Sylvia Beam, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. Novel and easy-to-use approach to triple-negative breast cancer tumoroid culture [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 227.

Synthesis and biological activity of thiophene bioisosteres of natural styryl lactone goniofufurone and related compounds

Ten new thiophene derivatives related to goniofufurone have been obtained by multistep synthesis starting from d-glucose. The critical step of the synthesis was the Grignard reaction of 2-thienyl magnesium bromide with a protected dialdose, yielding the C-5 epimeric thiophene derivatives 9 and 10. The mixture was oxidized to the 5-keto derivative 11, which after deprotection was converted to the corresponding keto-lactone 14. Stereoselective reduction of 14 afforded the thiophene mimic of goniofufurone 3. Esterification of 3 with cinnamic or 4-fluorocinnamic acid gave hybrids 5–7. Synthesized analogues were evaluated for their in vitro cytotoxicity against several tumour cell lines. The vast majority of them showed better activity than lead 1. In the culture of K562 cells, compound 3 was more active than the commercial antitumour drug doxorubicin. Structural features of analogues important for their antiproliferative activities were identified by SAR analysis. Pro-apoptotic potential examination of compound 3 on the K562 cell line was performed using flow cytometry, double fluorescence staining and apoptotic morphology screening. Results show that this derivative induces cell membrane disruptions attributable to apoptosis and induces the apoptotic morphology, but decreasing simultaneously the population of cells in the subG1 phase of the cell cycle. The results further suggest that analogue 3 achieves strong cytotoxicity without causing DNA fragmentation. This is clearly indicated by the relatively low incidence of micronuclei, as well as the SAR analysis of all biological effects.

The role of TIM3+ NK and TIM3- NK cells in the immune pathogenesis of severe aplastic anemia.

Natural killer (NK) cells play important immunoregulatory roles in the immune pathogenesis of severe aplastic anemia (SAA). Our previous research showed that SAA caused a decrease in T cell immunoglobulin mucin-3 (TIM3) expression on NK cells. Here we investigated the expression of surface receptors, and the cytotoxicity of peripheral TIM3+ NK and TIM3- NK cells in patients with SAA. The expressions of surface receptors and cytoplasmic protein of TIM3+ NK and TIM3- NK cells from peripheral blood were detected by FCM. The functions of mDCs, and apoptosis rate of K562 cells after co-culture with TIM3+ NK and TIM3- NK cells were maesured by FCM. Westren-blot was used to detect the changes of TIM3+ NK and TIM3- NK signaling pathway proteins (AKT, P-AKT) and compare the functional activity of the two groups. Activating receptors NKG2D and Granzyme B were higher, while inhibiting receptors NKG2A, CD158a and CD158b were lower on TIM3- NK cells compared with TIM3+ NK cells in patients with SAA. In SAA, the expression of CD80 and CD86 on mDCs (Myeloid dendritic cells) was significantly decreased after incubation with TIM3- NK cells. The apoptosis rate (AR) of K562 cells was significantly increased after being incubated with TIM3- NK cells in SAA. The level of signal pathway protein AKT of TIM3- NK cells in SAA was similar to that of TIM3+ NK cells, and the levels of P-AKT and P-AKT/AKT ratio of TIM3- NK cells were significantly higher than those of TIM3+ NK cells. Therefore, TIM3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. Low expression of TIM3 contributes to the enhancement of NK cell activity which in turn inhibits the immune activation state of SAA and improves the disease state. Our research may aid the development of new therapeutic strategies based on TIM3-NK cells infusion for the treatment of SAA.

Use of CRISPR/Cas9 with Homology-Directed Repair to Gene-Edit Topoisomerase IIβ in Human Leukemia K562 Cells: Generation of a Resistance Phenotype.

DNA topoisomerase IIβ (TOP2β/180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2β/180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2β/180 (and the paralog TOP2α/170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2β/180, a premature stop codon was generated at the TOP2β/180 gene exon 19/intron 19 boundary (AGAA//GTAA→ATAG//GTAA) in parental K562 cells (which contain four TOP2β/180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2β/180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2β/180 gene-edited clones, with one or all four TOP2β/180 alleles mutated, revealed partial or complete loss of TOP2β mRNA/protein, respectively. The loss of TOP2β/180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2β/180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents. SIGNIFICANCE STATEMENT: Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2β/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2β/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.

The Effects of the Steroids 5-Androstenediol and Dehydroepiandrosterone and Their Synthetic Derivatives on the Viability of K562, HeLa, and Wi-38 Cells and the Luminol-Stimulated Chemiluminescence of Peripheral Blood Mononuclear Cells from Healthy Volunteers.

In order to evaluate the role of substituents at 3-C and 17-C in the cytotoxic and cytoprotective actions of DHEA and 5-AED molecules, their derivatives were synthesized by esterification using the corresponding acid anhydrides or acid chlorides. As a result, seven compounds were obtained: four DHEA derivatives (DHEA 3-propionate, DHEA 3-butanoate, DHEA 3-acetate, DHEA 3-methylsulfonate) and three 5-AED derivatives (5-AED 3-butanoate, 5-AED 3,17-dipropionate, 5-AED 3,17-dibutanoate). All of these compounds showed micromolar cytotoxic activity toward HeLa and K562 human cancer cells. The maximum cytostatic effect during long-term incubation for five days with HeLa and K562 cells was demonstrated by the propionic esters of the steroids: DHEA 3-propionate and 5-AED 3,17-dipropionate. These compounds stimulated the growth of normal Wi-38 cells by 30-50%, which indicates their cytoprotective properties toward noncancerous cells. The synthesized steroid derivatives exhibited antioxidant activity by reducing the production of reactive oxygen species (ROS) by peripheral blood mononuclear cells from healthy volunteers, as demonstrated in a luminol-stimulated chemiluminescence assay. The highest antioxidant effects were shown for the propionate ester of the steroid DHEA. DHEA 3-propionate inhibited luminol-stimulated chemiluminescence by 73% compared to the control, DHEA, which inhibited it only by 15%. These data show the promise of propionic substituents at 3-C and 17-C in steroid molecules for the creation of immunostimulatory and cytoprotective substances with antioxidant properties.

Multicenter integrated analysis of noncoding CRISPRi screens

The ENCODE Consortium’s efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE–gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.

Nilotinib Exerts a Therapeutic Approach via JAK/STAT Pathway and Cytokine Network in Chronic Myeloid Leukemia Cells

Aim: Chronic myeloid leukemia (CML) displays a constitutive tyrosine kinase (TK) activity which in turn leads to the activation of various signaling pathways and the outcome of leukemic phenotype. Activated STAT5A and STAT5B from JAK/STAT pathway induce cell growth, proliferation, differentiation, and survival of leukemic cells which are promoted by a cytokine network. Since the second-generation tyrosine kinase inhibitor nilotinib has the advantage of inhibiting this oncogenic TK activity; we aimed to investigate the underlying mechanism of its therapeutic approach and how it induced apoptosis via analyzing the forthcoming molecular targets of the pathway. 
 Methods: By Nilotinib treatments, cell viability and proliferation assays, apoptotic analysis, expressional regulations of STAT5A&5B mRNA transcripts, protein expression levels, and also cytokines’ expressional assessments were determined in CML model K562 cells, in vitro.
 Results: Nilotinib treatment in a time and dose-dependent manner assessed a therapeutic approach by decreasing leukemic cell proliferation and survival; inducing leukemic cell apoptosis, down-regulating STAT5A&5B mRNA, and protein expression levels, and regulating cytokine expressional network. 
 Conclusion: Nilotinib-mediated therapeutics could be dependent on targeting JAK/STAT pathway members STAT5A and STAT5B, besides; regulating the cytokine network might be another underlying mechanism for sensitization and response of K562 cells to nilotinib in leukemia pathogenesis.

Accumulation of antitumor polyketides by fermentation of Rubus delavayi Franch. with Clonostachys rogersoniana

The aim of this work is to explore the effects of herbal medicine on secondary metabolites of microorganisms during fermentation. Clonostachys rogersoniana was found to metabolize only small amounts of polyketide glycosides rogerson B and C on fresh potatoes, but after replacing the medium to the medicinal plant Rubus delavayi Franch., the type and content of the metabolized polyketones showed significant changes. The sugars and glycosides in R. delavayi are probably responsible for the changes in secondary metabolites. Six polyketide glycosides including a new metabolite, rogerson F, and two potential antitumor compounds, TMC-151C and TMC-151D, were isolated from the extract of R. delavayi fermented by C. rogersoniana. In addition, 13C labeling experiments were used to trace the biosynthesis process of these compounds. TMC-151C and TMC-151D showed significant cytotoxic activity against PANC-1, K562 and HCT116 cancer cells but had no obvious cytotoxic activity against BEAS-2B human normal lung epithelial cells. The yields of TMC-151C and TMC-151D reached 14.37 ± 1.52 g/kg and 1.98 ± 0.43 g/kg, respectively, after fermentation at 28 °C for 30 days. This is the first study to confirm that herbal medicine can induce microbes to metabolize active compounds. And the technology of fermenting medicinal materials can bring more economic value to medicinal plants.

Antiproliferative and Apoptosis-Inducing Effects of Ethanolic Extract of Morus Alba Leaves on Human Chronic Myeloid Leukaemia K-562 Cell Lines

The rate of occurrence and prevalence of chronic myeloid leukaemia (CML) are increasing globally, despite it being a rare type of cancer. Morus alba also known as mulberry, has been used in Chinese traditional medicine for a long time to treat various conditions and diseases like hypertension and arthritis. Modern scientific studies have also recognized its medicinal properties, including antioxidant, antidiuretic, antidiabetic, antimicrobial and anticancer. However, its antiproliferative effects on specifically human CML K-562 cell lines remain poorly elucidated. This study aims to determine the antiproliferative effects of M. alba on K-562 cells. In vitro cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Acridine orange and propidium iodide (AO/PI) dual staining were then employed to assess the method of cell death. The ethanolic extract of M. alba leaves exhibited significant antiproliferative activity, with the lowest maximal half inhibitory concentration (IC50) value observed at 72 hours being 23 µg/mL. Notably, AO/PI staining revealed significant morphological changes, such as membrane blebbing, nuclear fragmentation, and indicator of early apoptosis, and late apoptosis. These results demonstrated that ethanolic extract of M. alba leaves induces apoptotic cell death in K-562 cells. Overall, these findings suggested that ethanolic extract of M. alba leaves exerts potent antiproliferative effects on human CML K-562 cells a in time- and dose-dependent manner.