CellTiter-Glo Luminescent Cell Viability Assay Research Articles

Novel 1,3,4-trisubstituted Pyrazolopyrimidine Derivatives Show Potent Antiproliferative Activity in Mantle Cell Lymphoma

Background: Pyrazolopyrimidine scaffold is an important pharmacophore in drug discovery. This pharmacophore has been reported to produce numerous biological activities, of which anticancer is an important one. The development of novel pyrazolopyrimidine derivatives is of great importance for antitumor drug research. Objective: Compound 6, a pyrazolopyrimidine derivative reported by our group, showed weak antiproliferative activity with IC50 values of over 30 μM against mantle cell lymphoma (MCL) cell lines. In this study, we will further perform the structural optimization of compound 6 to screen highly active pyrazolopyrimidine derivatives. Methods: A novel series of 1,3,4-trisubstituted pyrazolopyrimidine derivatives were synthesized and their structures were elucidated by 1H-NMR, 13C-NMR, and HRMS. The antiproliferative activities of target compounds against MCL cell lines (Mino, Jeko-1, and Z138) were evaluated by the CellTiter-Glo luminescent cell viability assay. The effect of representative compounds to induce apoptosis was evaluated by Annexin V/Propidium Iodide (PI)-binding assay. Mitochondrial membrane potential and reactive oxygen species (ROS) levels in 15c-treated Z138 cells were tested by JC-1 and DCFH-DA probes, respectively. Results: Most compounds demonstrated improved antiproliferative activity against MCL cell lines compared to the lead compound 6, especially 15c, 15f, 15g, 15j, and 15o, with IC50 values at low micromolar levels. In addition, compound 15c could induce apoptosis in a dose-dependent manner in Z138 cells through reduction of mitochondrial membrane potential and enhancing reactive oxygen species production. Conclusion: The results showed that 1,3,4-trisubstituted pyrazolopyrimidine derivatives could be valuable lead compounds for the further development of anti-lymphoma agents.

Novel Piperazine Derivatives of Vindoline as Anticancer Agents.

A series of novel vindoline-piperazine conjugates were synthesized by coupling 6 N-substituted piperazine pharmacophores at positions 10 and 17 of Vinca alkaloid monomer vindoline through different types of linkers. The in vitro antiproliferative activity of the 17 new conjugates was investigated on 60 human tumor cell lines (NCI60). Nine compounds presented significant antiproliferative effects. The most potent derivatives showed low micromolar growth inhibition (GI50) values against most of the cell lines. Among them, conjugates containing [4-(trifluoromethyl)benzyl]piperazine (23) and 1-bis(4-fluorophenyl)methyl piperazine (25) in position 17 of vindoline were outstanding. The first one was the most effective on the breast cancer MDA-MB-468 cell line (GI50 = 1.00 μM), while the second one was the most effective on the non-small cell lung cancer cell line HOP-92 (GI50 = 1.35 μM). The CellTiter-Glo Luminescent Cell Viability Assay was performed with conjugates 20, 23, and 25 on non-tumor Chinese hamster ovary (CHO) cells to determine the selectivity of the conjugates for cancer cells. These compounds exhibited promising selectivity with estimated half-maximal inhibitory concentration (IC50) values of 2.54 μM, 10.8 μM, and 6.64 μM, respectively. The obtained results may have an impact on the design of novel vindoline-based anticancer compounds.

Abstract LB444: Identification of a novel and selective Transglutaminase 2 (TGM2) inhibitors modulate tumor microenvironment in Glioblastoma

Abstract Background: TGM (Transglutaminase) family enzymes are Ca2+ dependent and engaged in specific posttranslational modifications by cross-linking extracellular matrix (ECM) proteins. ECM protein complex is more stable and resistant to enzymatic degradation. These enzymes modify proteins by cross-linking epsilon-(gamma-glutamyl), lysine, or (gamma-glutamyl) polyamine bonds. TGM2 (Transglutaminase 2) is a ubiquitous enzyme, and its expression is identified in the cytoplasm, plasma membrane, and the nucleus of various cells. The membrane-bound form of TGM2 binds GTP and functions as a G protein. TGM2 regulates cell survival, proliferation, migration, and modulation through ECM organization. We explored the role of TGM2 in Glioblastoma cells and its role in modulating the tumor microenvironment, cell proliferation, and survival. In addition to TGM2's role in GBM tumors, its expression has been linked to resistance to certain therapies in glioblastoma was investigated. Targeted modulation of macrophages with TGM2 inhibitors impacts the immune response against GBM, potentially enhancing the efficacy. Understanding the intricate interplay between TGM2, and GBM-associated macrophages is a key component of the tumor microenvironment (TME) and crucial for the development of targeted TGM2 inhibitors. Materials and Methods: Human Transglutaminase 2 (hTGM2) activity of inhibitors was assayed using the fluorescent transamination assay incorporating dansylcadaverine into glutamine-donor substrate N, N-dimethyl casein, and DCC. Cellular efficacies of selected TGM2 inhibitors evaluated in U138 and U87 glioblastoma cells in CellTiter-Glo Luminescent cell viability assay. Results: We have identified a series of novel small molecule TGM2 inhibitors based on our initial lead fragment hits with an IC50s 6-29 uM from our in-house MolecuLern fragment library. Fragments2Lead (F2L) optimizations strategies, synthesis, and SAR studies provided a lead SLX-9029 and SLX-9031 inhibitors with IC50s of 1.2, 1.5 uM in inhibiting TGM2 activity in our established cell-free fluorescent transamination assay. The initial TMG2 inhibitor leads further screening for TGM2-expressed U138 and U87 glioblastoma cells demonstrating promising cellular efficacies as singles agents. Additional GBM cell profiling, combination studies along with safety secondary pharmacology, cellular toxicities, and PK results will be discussed. Conclusion: In summary, we identified promising lead TGM2 inhibitors that show activity in both cell-free and cell-based TGM2-expressed GMB cells. The TGM family members, selectivity, additional GBM cellular profiling, ADME, cellular toxicity, and in vivo PK studies are underway, and these results will be presented. Citation Format: Hariprasad Vankayalapati, Chenyu Lin, Zhaoang li, Kyle Medley, David J. Bearss. Identification of a novel and selective Transglutaminase 2 (TGM2) inhibitors modulate tumor microenvironment in Glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB444.

The Reversible BTK Inhibitor Nemtabrutinib Demonstrates Favorable Antitumor Efficacy and Enhances the Function of CAR T Cells in Mantle Cell Lymphoma

Background Covalent Bruton's tyrosine kinase (BTK) inhibitors have revolutionized the therapy of mantle cell lymphoma (MCL). In January 2023, the reversible BTK inhibitor pirtobrutinib was approved by the FDA for the treatment of relapsed or refractory MCL. Nemtabrutinib is another reversible BTK inhibitor which also targets other kinases. The objective of this study was to evaluate the efficacy of nemtabrutinib alone and in combination with anti-CD19 CAR T cells against MCL. Methods Cell viability was measured following a 72-hour treatment with nemtabrutinib in a panel of MCL cell lines using CellTiter-Glo luminescent cell viability assay (Promega). Annexin V/PI staining was utilized to determine whether nemtabrutinib induces cell death through apoptosis. Patient-derived organoids (PDOs) were generated by culturing patient primary cells in 50% Matrigel (Corning) and cultured in cytokine-containing medium. The PDOs were treated for 72 hours, and viability assays were performed. Western blotting was employed to investigate the drug impact on kinase signaling pathways. Nemtabrutinib treated Mino cells were subjected to bulk RNA sequencing to interrogate the transcriptome profiling. The anti-MCL efficacy of nemtabrutinib was tested in an ibrutinib-resistant patient derived xenograft (PDX) mouse model in vivo. Additionally, the potential synergistic effects of nemtabrutinib and anti-CD19 CAR T cells were examined using the luciferase-expressing MCL cells. Results Nemtabrutinib demonstrated comparable growth inhibitory activity to ibrutinib in MCL cell lines with IC 50 values at micromolar concentrations (IC 50 = 0.7-10.1 μM). Remarkably, in the 3D PDO screening system that mimics the tumor microenvironment for MCL, nemtabrutinib displayed superior anti-MCL efficacy compared to ibrutinib (p < 0.01). The apoptotic potential of nemtabrutinib was further investigated using Annexin V/PI staining, revealing a dose-dependent induction of apoptosis, and demonstrated a similar sensitivity profile across cell lines observed with ibrutinib. Western blotting revealed that nemtabrutinib effectively inhibited phosphorylation at both Tyr223 and Tyr551 sites of BTK, and also dramatically suppressed the activation of Src family kinases, Syk and ERK. Bulk RNA-seq was performed to examine the transcriptome profile following nemtabrutinib treatment. 449 genes were identified to be significantly upregulated and 460 genes were downregulated. Gene set enrichment analysis uncovered a significant decrease in TNFα signaling via NF-κB, inflammatory response, and IFNγ response signalings (FDR < 0.05). In an ibrutinib-resistant PDX mouse model, treatment with nemtabrutinib resulted in a significant reduction in tumor burden and effectively attenuated the tumor involvement in spleen, liver and bone marrow. Furthermore, to enhance efficacy, we investigated the combination of nemtabrutinib with anti-CD19 CAR T cells in luciferase-expressing MCL cells. The result demonstrated that nemtabrutinib enhanced the effector function and anti-MCL activity of anti-CD19 CAR T cells. Conclusion Nemtabrutinib demonstrated favorable anti-MCL efficacy in both in vitro and in vivo studies, the promising synergistic effects observed in combination with CAR T cells warrants further investigation in both preclinical and clinical settings.

Synergistic Effect of Lenvatinib and Chemotherapy in Hepatocellular Carcinoma Using Preclinical Models.

The current study aimed to evaluate the synergistic efficacy of lenvatinib and FOLFOX (infusional fluorouracil (FU), folinic acid, and oxaliplatin) in hepatocellular carcinoma (HCC) using patient-derived xenograft (PDX) and PDX-derived organotypic spheroid (XDOTS) models in vivo and in vitro. PDX and matched XDOTS models originating from three patients with HCC were established. All models were divided into four groups and treated with drugs alone or in combination. Tumor growth in the PDX models was measured and recorded, and angiogenesis and phosphorylation of the vascular endothelial growth factor receptor (VEGFR2), rearranged during transfection (RET), and extracellular signal-regulated kinase (ERK) were detected using immunohistochemistry and Western blot assays. The proliferative ability of XDOTS was evaluated through active staining and immunofluorescence staining, and the effect of the combined medication was evaluated using the Celltiter-Glo luminescent cell viability assay. Three PDX models with genetic characteristics similar to those of the original tumors were successfully established. Combining lenvatinib with FOLFOX led to a higher tumor growth inhibition rate than individual therapies (P < 0.01). Immunohistochemical analysis demonstrated that the combined treatment significantly inhibited the proliferation and angiogenesis of PDX tissues (P < 0.05), and Western blot analysis showed that the combined treatment significantly inhibited the phosphorylation of VEGFR2, RET, and ERK compared with single-agent treatment. Additionally, all three matched XDOTS models were successfully cultured with satisfactory activity and proliferation, and the combined therapies led to better suppression of XDOTS growth compared with individual therapy (P < 0.05). Lenvatinib combined with FOLFOX had a synergistic antitumor effect in HCC PDX and XDOTS models by inhibiting the phosphorylation of VEGFR, RET, and ERK.

Validation of a Novel Rac1 Inhibitor As Potent Secdrug Against Activating Ras Mutant and Drug-Resistant Multiple Myeloma

Multiple myeloma is the second-most common hematopoietic malignancy in the United States. Although several drugs have so far been approved by the FDA, myeloma still remains an incurable disease with dose-limiting toxicities and resistance to primary drugs like proteasome inhibitors (PIs) and Immunomodulatory drugs (IMiDs). Furthermore, activating Ras gene (NRAS, KRAS, and HRAS) mutations are known to occur in >50% of myeloma patients, which are also associated with greater tumor burden, cancer aggressiveness, high recurrence rates, and poor survival. We have recently established a novel drug development/re-purposing pipeline called secDrug that incorporates a pharmacogenomics data-driven computational algorithm to introduce several new drug candidates (secDrugs), including EHT1864 (a Rac1 (Ras superfamily GTPase) inhibitor), CCT018159 (Hsp90 inhibitor), and CP466722 (a reversible ATM inhibitor) as synergistic partners of primary drugs against drug-resistant myeloma. The overall objective of this study is to validate EHT1864 using in vitro and ex vivo models of relapsed and refractory myeloma (RRMM) patients. First, we used single-cell RNA sequencing (scRNAseq) as a novel screening tool to demonstrate that EHT1864 is potentially effective against myeloma subclones based on the enrichment of target genes (Figure 1). For in vitro validation, we used a human myeloma cell line (HMCL) panel representing drug-sensitive (FLAM76), innate/refractory resistance (LP1), and acquired/relapsed resistance (parental and clonally derived PI-resistant and IMiD-resistant HMCL pairs U266 P/VR, RPMI P/VR, MM1S P/VR/LenR). FLAM76 KRas (FLAM76-K12) and FLAM76 Nras (FLAM76-N12) cell lines were generated using Adeno-associated viral (AAV) vector-mediated delivery of CRISPR-Cas9 for genome editing in humans. Ras mutations were confirmed using Sanger DNA sequencing method. Cell viability measured using CellTiter-Glo luminescent cell viability assay showed the potency of EHT1864 as a single-agent (IC50 range 15-40µM) as well as in combination with PIs (represented by Ixazomib) and IMiDs (represented by Lenalidomide). Combination index (CI) values calculated using Chou-Talalay's CI theorem were consistently less than 0.9, while all the four synergy scores (HSA, Bliss, Loewe, and ZIP) were higher than 5, demonstrating high synergy. Cell death due to apoptosis was confirmed using flow cytometry (Annexin-V-Propidium Iodide staining), Caspase 3/7 activity assay, and immunoblotting. Interestingly, EHT1864 was most potent against drug-resistant and Ras-mutant HMCLs. Further, we showed that EHT1864 treatment also affects ROS generation and mitochondrial membrane potential, indicating that EHT1864 induces apoptosis via mitochondria-mediated pathway. Finally, we performed pre-vs-post-treatment genome-wide transcriptome analysis (tumor mRNAseq) and single-cell multi-ome analysis (single-cell RNA sequencing/scRNAseq + single-cell Assay of Transposase Accessible Chromatin sequencing/scATACseq) analysis to derive molecular signatures and pathways representing the basis of (on-target and off-target) efficacy of EHT1864 and drug synergy at the bulk tumor and subclonal levels. Finally, we will perform single-cell proteomics (high-dimensional immunophenotyping using mass cytometry/CyTOF/Cytometry time of flight) in CD138+ bone marrow cells derived from newly-diagnosed and relapsed myeloma patients (ex vivo) to demonstrate efficacy in RRMM. Thus, our work lays a framework to establish EHT1864 as a clinical trial-ready therapeutic option for the management of drug-resistant myeloma. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The SPATA20 Gene Modifies Rectal Cancer Radiosensitivity

INTRODUCTION: Rectal cancer response to neoadjuvant chemoradiotherapy correlates with outcomes, and is highly variable between patients, ranging from a complete response to minimal or no response. We have previously identified an association between SPATA20 gene expression and response in patients. The goal of this study was to establish and test a rectal cancer cell model to study SPATA20. METHODS: HRT18 rectal cancer cell lines were transfected with a lentiviral construct containing small interfering RNAs (siRNA) targeting SPATA20 mRNA to create knockdown cell lines. Western blot was subsequently used for knockdown validation. Wildtype HRT18 and knockdown HRT18 cell lines were plated in 96 well plates and subsequently irradiated with 10 Gy. Cell viability was assessed at 24 hours interval using CellTiter-Glo Luminescent Cell Viability Assay for both irradiated and non-irradiated controls (Figure 1). RESULTS: Decreased expression of SPATA20 was confirmed in knockdown HRT18 cell lines compared with wildtype HRT18 using Western blot. SPATA20 knockdown by itself did not significantly affect cell viability (p = 0.18). Knockdown SPATA20 cells were significantly more sensitive to radiation when compared with wildtype cells, resulting in a 17% decrease in cell viability (p < 0.0001). CONCLUSION: We established a SPATA20 knockdown rectal cancer model to study the effects of radiation. Manipulation of SPATA20 correlates with radiation response, signifying it as a possible modulator of radiosensitivity in rectal cancer.

Abstract 1859: VIP152, a selective CDK9 inhibitor, demonstrates sensitivity in gynecologic cell lines that are cisplatin sensitive or resistant and delivers in vivo antitumor efficacy

Abstract Background: The Myc transcription factor has a short half-life of 20-30 minutes (Ramsay et al. 1984) and oncogenic activation and transcriptional addiction leads to sustained levels of Myc, (Gabay et al. 2014). Mechanisms of activation include MYC gene amplification, mutation or rearrangement and have been reported to be prevalent in gynecologic malignancies, such as serous ovarian (39.2%), uterine (28.1%) and endometrial (19.5%) (https://www.cancer.gov/tcga). Since CDK9 phosphorylation of RNA polymerase II is required for transcription of Myc mRNA, we evaluate VIP152, a highly selective CDK9 inhibitor (Lücking et al 2021), to deliver antitumor responses in preclinical models of gynecologic malignancies. Methods: Gynecologic cell lines were treated with 9 dose levels of VIP152 with DMSO and cisplatin as controls. IC50 were calculated using CellTiter-Glo luminescent cell viability assay after 72 hours treatment. A preliminary analysis of 19 cell lines was undertaken to determine whether response to VIP152 is associated with baseline mutation, gene expression, or copy number variation data. Monotherapy efficacy of VIP152 was evaluated in a cisplatin sensitive A2780 in vivo xenograft mouse model. Results: Screening of gynecologic cell lines demonstrates a 3-log range of sensitivity with VIP152 IC50s ranging from 38-593nM. Simultaneous cisplatin screening identified sensitive and resistant cell lines in the same panel. Low VIP152 IC50s were observed in cell lines sensitive or resistant to cisplatin. Cell lines were assigned as sensitive and resistant based on the VIP152 IC50 values. Preliminary analysis suggests a gene signature could predict response to VIP152 in gynecologic malignancies. VIP152 was evaluated in an ovarian cancer A2780 in vivo xenograft model. Monotherapy treatment with 4 doses of VIP152 across 5 dose levels from 5- to 15-mg/kg weekly regimens provided increasing tumor growth inhibition compared with vehicle control. Conclusions: VIP152 demonstrates sensitivity in gynecologic cell lines independent of cisplatin sensitivity. An interim gene signature that is associated with VIP152 sensitivity was defined and we plan to optimize this signature with a larger cell line panel. Dose-dependent tumor growth inhibition in an in vivo xenograft model is demonstrated. VIP152 is currently being evaluated in the clinic (ClinicalTrials.gov Identifiers: NCT02635672 and NCT04978779).

Abstract 1143: ATG-022, an antibody-drug conjugate targeting Claudin 18.2, demonstrated potent in vivo efficacy in gastric cancer patient-derived xenografts

Abstract Background: Antibody-drug conjugates (ADCs) have become promising antitumor agents in recent years. ADCs are comprised of a monoclonal antibody, targeting antigens expressed at higher levels on tumor cells than on normal cells, cross-linked to small molecule payloads with cytotoxic activity. Human Claudin 18.2 (CLDN18.2) is overexpressed in a large proportion of gastric and pancreatic cancers, with restricted normal tissue expression. The monoclonal antibody targeting CLDN18.2 (IMAB362) has demonstrated promising clinical benefit in combination with chemotherapies for gastric cancer patients. However, the same antibody shows suboptimal efficacy in patients with low CLDN18.2 expression. Here we report the potent in vivo efficacy of ATG-022, a CLDN18.2 ADC, in multiple gastric cancer patient-derived xenograft (PDX) models, including those with low CLDN18.2 expression. Methods: The binding affinity of ATG-022 with CLDN18.2 was detected by SPR and FACs analysis. The in vitro 50% inhibition concentration (IC50) of ATG-022 was determined in CLDN18.2 positive cell lines using CellTiter-Glo luminescent cell viability assay. In vivo efficacy of ATG-022 was evaluated in a series of gastric cancer PDX models with different expression level of CLDN18.2 The expression level of CLDN18.2 was determined using IHC staining. ATG-022 was dosed twice at 1 mg/kg, 3 mg/kg or 10 mg/kg every two weeks. Results: ATG-022 binds to CLDN18.2 protein with sub-nM affinity. It induced potent in vitro cytotoxicity in CHOK1 cells overexpressing CLDN18.2, with an IC50 of 5-7nM. Bystander killing by ATG-022 was observed. ATG-022 demonstrated potent in vivo antitumor efficacy in a gastric cancer PDX model with high CLDN18.2 expression. Intravenous dosing of 1 mg/kg, 3 mg/kg, and 10 mg/kg ATG-022 induced 35.41%, 71.56% tumor growth inhibition (%TGI) and tumor regression respectively. In a PDX model with low CLDN18.2 expression, 3 mg/kg ATG-022 or 10 mg/kg clinical benchmark CLDN18.2-ADC did not inhibit tumor growth, while 10 mg/kg ATG-022 induced tumor regression. Conclusions: ATG-022 demonstrated potent in vitro and in vivo antitumor effects, with in vivo efficacy observed in CLDN18.2-low expression PDX models, suggesting a promising therapeutic strategy for gastric cancer patients with a broad range of CLDN18.2 expression levels. Citation Format: Peng Chen, Yun Liu, Min Deng, Linjie Tian, Kevin Lynch, Bo Shan, Jay Mei, Bing Hou. ATG-022, an antibody-drug conjugate targeting Claudin 18.2, demonstrated potent in vivo efficacy in gastric cancer patient-derived xenografts [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 1143.

Abstract 5430: Inhibition of de novo and salvage pathways for dNTP synthesis enhances sensitivity to ionizing radiation in pancreatic neuroendocrine tumor cells

Abstract Introduction: The treatment of gastroenteropancreatic neuroendocrine tumors (GEP-NET) with ionizing radiation (IR) is a promising treatment modality for a disease that carries a poor prognosis. However, these tumors confer high levels of radioresistance based on their ability to repair DNA damaged by IR. Deoxynucleoside triphosphates (dNTPs) are needed for DNA repair and are produced by two pathways: de novo and salvage, which depend on ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK), respectively. Ataxia telangiectasia and Rad3-related protein (ATR) activates dCK and can be targeted with inhibitors. We hypothesized that blocking both pathways for dNTP synthesis with an ATR inhibitor (ATRi) and the RNR inhibitor (RNRi), triapine, will sensitize GEP-NETs to IR in vitro. Methods: (i) The CellTiter-Glo (Promega) luminescent cell viability assay established drug sensitivities of two pancreatic NET cell lines (BON and QGP-1) for triapine and three ATRi (AZD6738, VX-970, BAY1895344). Assays were performed with at least three replicates. Data were fit using a four-parameter log-logistic model, and IC50 values were calculated with R statistical software. (ii) Loewe synergy models were used to calculate the two-drug combination effects in both cell lines with synergy scores greater than 0 indicating synergistic effects and scores less than 0 representative of antagonistic effects. (iii) Clonogenic assays were performed and analyzed via colorimetry with sulforhodamine B to assess the effect of each drug alone and in combination with IR. (iv) Immunoblots were used to assess ATR activation following IR, RNRi, and ATRi treatments. Results: (i) Cell viability assays showed lower IC50 values in the BON cell line compared to the QGP-1 cell line (BON cell IC50 : triapine 2.6µM ± 0.6µM, AZD6738 1.0µM ± 0.2µM, VX-970 204nM ± 20nM, BAY1895344 139nM ± 15nM; IC50 in QGP-1 cells: triapine 6.1µM ± 2.7µM, AZD6738 7.9µM ± 2.9µM, VX-970 1.7µM ± 0.5µM, BAY1895344 1.1µM ± 2.8µM). (ii) Loewe synergy models estimated the combination of BAY1895344 and triapine to be synergistic with scores greater than 0 in both cell lines (BON 9.89, p-value 4.07e-11; QGP-1 15.91, p-value 1.96e-7). (iii) Clonogenic assays showed an increase in efficacy of BAY1895344 when combined with IR in both BON (IC50 s: 150nM ± 8nM without radiation versus 18nM ± 35nM with 2Gy) and QGP-1 cells (IC50 values: 277nM ± 48nM without radiation versus 93nM ± 4nM with 2Gy). (iv) Western blot analysis showed strong activation of the ATR pathway with IR and triapine alone with subsequent inhibition by an ATRi. Conclusion: Our findings show that treatment of GEP-NET cell lines with inhibitors that block the de novo and salvage pathways for dNTP production markedly sensitize these cells to subsequent IR. The combination of IR with both an ATRi and an RNRi has the potential to be an effective treatment modality for GEP-NETs. Citation Format: Jennifer Castle, J. Robert McCorkle, Jeremy Johnson, Aman Chauhan, Percy Ivy, Susanne Arnold, William Carson, B. Mark Evers, Piotr Rychahou, Jill Kolesar. Inhibition of de novo and salvage pathways for dNTP synthesis enhances sensitivity to ionizing radiation in pancreatic neuroendocrine tumor 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 5430.

SIPL1, Regulated by MAZ, Promotes Tumor Progression and Predicts Poor Survival in Human Triple-Negative Breast Cancer.

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer owing to a lack of effective targeted therapy and acquired chemoresistance. Here, we explored the function and mechanism of shank-interacting protein-like 1 (SIPL1) in TNBC progression.MethodsSIPL1 expression was examined in human TNBC tissues and cell lines by quantitative reverse transcription PCR, western blot, and immunohistochemistry. SIPL1 overexpression and silenced cell lines were established in BT-549 and MDA-MB-231 cells. The biological functions of SIPL1 in TNBC were studied in vitro using the CCK-8 assay, CellTiter-Glo Luminescent Cell Viability assay, caspase-3/8/9 assay, wound healing assay, and transwell assay and in vivo using a nude mouse model. The potential mechanisms underlying the effects of SIPL1 on TNBC progression were explored using bioinformatics analysis, luciferase reporter assays, and chromatin immunoprecipitation followed by qPCR.Results SIPL1 expression was higher in human TNBC tissues and cell lines than in adjacent normal tissues and a breast epithelial cell line (MCF10A). High expression of SIPL1 was positively correlated with poor overall and disease-free survival in patients with TNBC. SIPL1 overexpression elevated and SIPL1 silencing repressed the malignant phenotypes of TNBC cells in vitro. SIPL1 overexpression promoted xenograft tumor growth in vivo. Myc-associated zinc-finger protein (MAZ) transcriptionally activated SIPL1. Finally, we found that SIPL1 promoted TNBC malignant phenotypes via activation of the AKT/NF-κB signaling pathways.ConclusionsThese results indicate that the MAZ/SIPL1/AKT/NF-κB axis plays a crucial role in promoting the malignant phenotypes of TNBC cells.

Abstract P209: Collaborative crosstalk between two apoptosis pathways drives synergy of dual inhibition of BCL-2/MDM2 in preclinical models of neuroblastoma

Abstract Introduction: The prognosis of patients with neuroblastoma is poor, and novel therapeutic strategies are needed to improve clinical outcomes. Neuroblastoma often exhibits a low TP53 mutation rate and overexpresses MDM2 and BCL-2 genes. Dual targeting of the MDM2-P53 and BCL-2 apoptotic pathways is an attractive treatment strategy (Vernooij et al, Mol Cancer Ther 2021; Dalton et al, Mol Cancer Ther 2021) and is under clinical development using idasanutlin and venetoclax. However, the mechanisms of action (MOAs) of this combination in neuroblastoma are not fully understood. Alrizomadlin (APG-115) is a potent, orally bioactive MDM2 inhibitor. Lisaftoclax (APG-2575) is a novel BCL-2 selective inhibitor. Both agents have shown clinical activity in solid and hematologic malignancies (Tolcher et al, J Clin Oncol 2021:2506; Ailawadhi et al, J Clin Oncol 2021:7502). We investigated the effects of combined alrizomadlin and lisaftoclax treatment in preclinical models of neuroblastoma and examined novel MOAs associated with this combination. Materials and Methods: Antiproliferative activity was measured by CellTiter-Glo luminescent cell viability assay. Apoptosis was evaluated by annexin V/propidium iodide staining and flow cytometry. Meso-Scale Discovery ELISA and western blot analyses characterized the mechanisms of this combination’s synergistic effects. In animal studies, clinically relevant doses of lisaftoclax were employed. A pulsed high-dose regimen of alrizomadlin was used to achieve best efficacy and manage on-target toxicity. Results: In TP53WT neuroblastoma cell lines IMR-32 and SH-SY5Y, the combination of lisaftoclax and alrizomadlin synergistically inhibited proliferation and induced apoptosis, as evidenced by elevations in caspase-3/7, cleaved PARP, and annexin V+ subpopulations. Tumor growth was synergistically inhibited in 3 distinct subcutaneous mouse xenograft tumor models derived from neuroblastoma cell lines or patient tumors. In the cell lines, alrizomadlin treatment upregulated proapoptotic BCL-2 family member expression (NOXA, BAX, and PUMA) and downregulated antiapoptotic BCL-2 family member MCL-1, likely sensitizing cancer cells to lisaftoclax-induced apoptosis. Treatment of these cells with lisaftoclax and alrizomadlin reduced all 3 major BIM-related complexes, thereby increasing free BIM and triggering cellular apoptosis. Lisaftoclax and alrizomadlin downregulated NMYC and its transcription target MDM2, preventing the development of alrizomadlin resistance mediated by a feedback upregulation of MDM2, and inhibition of the MDM2-P53 apoptotic pathway by alrizomadlin primed cancer cells to BCL-2 inhibition. Conclusions: Alrizomadlin synergizes with lisaftoclax to achieve synthetic lethality through a regulated interaction at multiple molecular nodules, and inhibition of BCL-2 apoptotic pathway by lisaftoclax may prevent cancer cells from developing resistance to MDM2-P53 inhibition. These data warrant clinical development of combined alrizomadlin and lisaftoclax treatment in patients with neuroblastoma. Citation Format: Douglas D. Fang, Qiuqiong Tang, Qixin Wang, Yanhui Kong, Na Li, Feng Zhou, Ran Tao, Dajun Yang, Yifan Zhai. Collaborative crosstalk between two apoptosis pathways drives synergy of dual inhibition of BCL-2/MDM2 in preclinical models of neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P209.

Abstract P208: Synergistic antitumor activity of lisaftoclax (APG-2575) and alrizomadlin (APG-115) through dual targeting of BCL-2/MDM2-P53 apoptotic pathways in preclinical models of acute myeloid leukemia

Abstract Background: In many hematologic malignancies, BCL-2 is overexpressed and is a viable therapeutic target. In relapsed or refractory acute myeloid leukemia (R/R AML), BCL-2 inhibitor venetoclax as a single agent has limited activity, thereby necessitating combination treatments, some of which are actively under clinical development. In addition, the mutation rate of TP53 is rare in AML, and the frequency of MDM2 overexpression is high, representing an opportunity for pharmacologic intervention of the MDM2-P53 apoptosis pathway. Synergistic effects of MDM2 inhibitor idasanutlin and venetoclax have been reported in preclinical models of TP53 wild-type AML (Pan et al, Cancer Cell 2017). Lisaftoclax (APG-2575) and alrizomadlin (APG-115) are novel BCL-2 and MDM2 inhibitors, respectively. They show unique pharmacological properties as well as clinical activity and tolerable safety profiles in patients with hematologic or solid malignancies (Tolcher et al, J Clin Oncol 2021:2506; Ailawadhi et al, J Clin Oncol 2021:7502). Here, we examined the effects of combined lisaftoclax and alrizomadlin in preclinical models of AML. Materials and Methods: AML cell lines and mouse xenograft models were employed. Antiproliferative activity was measured by CellTiter-Glo luminescent cell viability assay. Apoptosis was evaluated by annexin V/propidium iodide staining and flow cytometry. Meso-Scale Discovery ELISA assay and western blot analyses were used to characterize the mechanisms of the synergistic effects of this combination. In animal studies, clinically relevant doses of lisaftoclax were employed. A pulsed high-dose regimen of alrizomadlin was used to achieve best efficacy and manage on-target toxicity. Results: In TP53 wild-type AML cell lines MOLM-13, MV-4-11, and OCI-AML-3, both single-agent lisaftoclax and alrizomadlin exerted apoptogenic activity. The combination had synergistic apoptogenic and antiproliferative activity. In subcutaneous xenograft models derived from MV-4-11 and OCI-AML-3 cells, the combination substantially inhibited tumor growth relative to the impact observed with either single agent. In a systemic xenograft model derived from MOLM-13 cells, the combination significantly (P &amp;lt; .05) extended the life span of tumor-bearing mice by 135% to 237% compared to that of tumor-bearing mice treated with lisaftoclax or alrizomadlin alone, which resulted in extensions of 4% to 26% and 41% to 95%, respectively. Synergistic anti-leukemic activity was confirmed in a patient-derived systemic AML model. Mechanistically, the combination synergistically downregulated anti-apoptotic proteins MCL-1 and BCL-xL and primed AML cells to apoptosis induced by lisaftoclax. Conclusions: We show a significant synergy of lisaftoclax and alrizomadlin and novel mechanisms associated with the combination of these agents in preclinical models of TP53 wild-type AML. These data support investigation into the clinical outcomes of the combination treatment in patients with TP53 wild-type AML. Citation Format: Douglas D. Fang, Qiuqiong Tang, Qixin Wang, Na Li, Feng Zhou, Guoqin Zhai, Yan Yin, Dajun Yang, Yifan Zhai. Synergistic antitumor activity of lisaftoclax (APG-2575) and alrizomadlin (APG-115) through dual targeting of BCL-2/MDM2-P53 apoptotic pathways in preclinical models of acute myeloid leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P208.

EXTH-57. EFFICACY OF SOLUBLE PANOBINOSTAT (MTX110) IN PRECLINICAL MODELS OF ADULT GLIOBLASTOMA

Abstract INTRODUCTION One of the biggest challenges in treating glioblastoma is achieving effective local drug concentrations, and trials using systemic administration of therapeutics have failed in GBM despite compelling pre-clinical evidence. MTX110 (Midatech Pharma PLC) is a water-soluble form of panobinostat currently in clinical development for DIPG and medulloblastoma using direct tumor delivery. We have previously reported a significant positive benefit of MTX110 in a subcutaneous GBM mouse model in an IDH1 mutated cell line. Here we present follow on data on the potential for MTX110 synergy with radiation in pre-clinical in vivo models. We also present data on efficacy of MTX110 in a panel of GBM cell lines. METHODS In vitro: Cell lines were incubated with MTX110 for 72h over a range of concentrations (1nM-10µM). Each concentration was tested in triplicate with the appropriate blank controls included in each plate. After 72 hours, cell viability was measured via luminescence signal (Promega CellTiter-Glo Luminescent Cell Viability Assay kit (Promega-G7573) read via 2104 EnVision Multilabel Reader, PerkinElmer. In vivo: Tumor-bearing mice were stratified to either the vehicle control or treatment group based on median tumor volume and were treated with MTX110 at a dose of 15mg/kg IP, 5 days consecutively for 2 consecutive weeks, following radiation treatment at a dose of 4Gy delivered as a single fraction on day 1. RESULTS In vitro, MTX110 demonstrated cytotoxic activity in 4 GBM cell lines (U87MG, U251MG, U118MG and T98G) with an average IC50 value in the region of 40nM (17-43nM). In vivo MTX110 in combination with radiation treatment showed an enhanced delay in tumor growth of approximately 50% compared to MTX110 or radiation alone. The results are supportive of the planned exploratory trial in GBM with MTX110.

Abemaciclib in Combination with Copanlisib to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

IntroductionAlthough novel therapeutic strategies including BTK and Bcl-2 inhibitors have dramatically improved the prognosis of MCL patients, resistance to these treatments is inevitable. We recently reported that the tumor suppressor gene CDKN2A were commonly deleted in ibrutinib-resistant tumors, leading to upregulation of CDK4/6 signaling. Among the other hallmarks are the mTOR/PI3K, Myc and OXPHOS pathways. Therefore, we attempt to exploit combinatory targeting of CDK4/6 and PI3K pathways to overcome therapy resistance using in vitro and PDX models.MethodsIbrutinib or venetoclax sensitive and resistant MCL cell lines were used in this preclinical study. 1x10 4 cells per well are seeded in 96-well plates and treated with abemaciclib monotherapy or in combination with copanlisib (PI3K inhibitor) in triplicate for 72h and then mixed with CellTiter-Glo Luminescent Cell viability Assay Reagent. For cell cycle assay, cells were seeded in 6 well plates and treated with vehicle or abemaciclib for 24h. Cells were fixed in 70% pre-cold ethanol and stained with propidium iodide. The cell cycle stages were quantified through the Novocyte Flow Cytometer. The molecular events at the protein level after treatment were determined by immunoblotting. For in vivo experiment, the combination of abemaciclib (25mg/kg, oral, daily) and copanlisib (5mg/kg, IP, three times a week) was assessed in Mino-venetoclax-resistant xenograft model. IC50 values were calculated using GraphPad Prism 8 for each cell line. Student's t-test was performed to compare the difference between vehicle and treated groups. Two-way analysis of variance (ANOVA) was conducted to analyze the tumor growth in vivo experiments. P values less than 0.05 were considered statistically significant.ResultsOur previous studies have identified a subset of MCL cells that were resistant to venetoclax (JeKo-1) or ibrutinib treatment (Maver-1 and Z-138). To overcome the resistance, we first treated MCL cell lines with abemaciclib and the result showed that abemaciclib as a single agent showed potent anti-MCL activity in a subset of MCL cell lines (IC 50 = 70-952 nM) including venetoclax sensitive- (Mino, Rec-1, Maver-1, and Z138) and primary resistant- MCL cells (JeKo-1). However, the cell lines Mino-ven-R and Rec-ven-R with acquired venetoclax resistance are highly resistant to abemaciclib treatment (IC 50 = 6.0 and 4.4 µM). PI3K/ATK pathway has been reported to be highly upregulated in Mino-ven-R and Rec-ven-R cells compared to their parental cells. To further increase the efficacy of the targeted therapy, we treated the resistant MCL cells with a combination of abemaciclib and copanlisib and the result showed synergistically enhanced cytotoxicity in ibrutinib or venetoclax-resistant MCL cell lines. Consistent with the role of CDK4/6 in cell cycle progression, inhibition of CDK4/6 with abemaciclib resulted in the cell cycle arrest at G1 phase in MCL cell lines.To validate whether abemaciclib in combination with copanlisib can overcome venetoclax resistance in vivo, we assessed the antitumor effect of abemaciclib in combination with copanlisib using a venetoclax-resistant xenograft models derived from Mino-ven-R cell line in immunodeficient NSG mice. As a result, abemaciclib (25 mg/kg, oral, daily), but not venetoclax (5 mg/kg, oral, daily) or copanlisib (5 mg/kg, IP, three times a week), significantly reduced tumor volume compared to the vehicle control (n = 5, p < 0.0001). Remarkably, the combination of abemaciclib and copanlisib also exhibited significantly in vivo synergistic efficacy compared with single-agent treatment (p<0.0001). Of note, the combination did not cause major decreases in body weight. Taken together, these results suggest that the combinatory therapy is effective in overcoming venetoclax resistance in MCL.ConclusionsCombinatory treatment with abemaciclib and copanlisib may achieve clinical actionable efficacy through overcoming the venetoclax-resistance in MCL that may become an effective treatment regimen for refractory/relapsed MCL patients in the future. DisclosuresWang: Dava Oncology: Honoraria; Imedex: Honoraria; CStone: Consultancy; Hebei Cancer Prevention Federation: Honoraria; OMI: Honoraria; Chinese Medical Association: Honoraria; Newbridge Pharmaceuticals: Honoraria; Moffit Cancer Center: Honoraria; Bayer Healthcare: Consultancy; Kite Pharma: Consultancy, Honoraria, Research Funding; Clinical Care Options: Honoraria; Physicians Education Resources (PER): Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; Mumbai Hematology Group: Honoraria; InnoCare: Consultancy, Research Funding; Epizyme: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genentech: Consultancy; DTRM Biopharma (Cayman) Limited: Consultancy; Acerta Pharma: Consultancy, Honoraria, Research Funding; Scripps: Honoraria; BGICS: Honoraria; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Anticancer Association: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Juno: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; Oncternal: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; VelosBio: Consultancy, Research Funding; BioInvent: Research Funding; Celgene: Research Funding; Lilly: Research Funding; Molecular Templates: Research Funding.

The Synergistic Anti-Tumor Activity of EZH2 Inhibitor SHR2554 and HDAC Inhibitor Chidamide through ORC1 Reduction of DNA Replication Process in Diffuse Large B Cell Lymphoma.

Simple SummaryThe EZH2-targeted drugs have demonstrated notable therapeutic effects in EZH2 mutant B-cell lymphoma patients. In this study, we demonstrated that the combination of EZH2 inhibitor SHR2554 and HDAC inhibitor HBI8000 exert synergistic anti-proliferative activity in both EZH2 wide-type and mutation B-cell lymphoma. More importantly, gene expression profile analysis revealed simultaneous treatment with these agents led to dramatic inhibition of DNA replication initiator protein ORC1, which might contribute to great efficacy of combination strategy. The combination of EZH2 inhibitor and HDAC inhibitor could provide a potential therapeutic treatment for both EZH2 wide-type and mutation B-cell lymphoma patients.Background: Upregulation of H3K27me3 induced by EZH2 overexpression or somatic heterozygous mutations were implicated in lymphomagenesis. It has been demonstrated that several EZH2-target agents have notable therapeutic effects in EZH2-mutant B-cell lymphoma patients. Here we present a novel highly selective EZH2 inhibitor SHR2554 and possible combination strategy in diffuse large B-cell lymphoma (DLBCL). Methods: Cell proliferation, cell cycle and apoptosis were analyzed by CellTiter-Glo Luminescent Cell Viability Assay and flow cytometry. Western Blot was used to detect the expression of related proteins. The gene expression profiling post combination treatment was analyzed by RNA-Seq. Finally, CDX and PDX models were used to evaluate the synergistic anti-tumor effects of the combination treatment in vivo. Results: The novel EZH2 inhibitor SHR2554 inhibited proliferation and induced G1 phase arrest in EZH2-mutant DLBCL cell lines. The combination of EZH2 inhibitor SHR2554 with histone deacetylase (HDAC) inhibitor chidamide (hereafter referred to as HBI8000) exerted synergistic anti-proliferative activity in vitro and in vivo. Gene expression profile analysis revealed dramatic inhibition of the DNA replication process in combined treatment. Conclusions: SHR2554, a potent, highly selective small molecule inhibitor of EZH2, inhibited EZH2-mutant DLBCL more significantly in vitro and in vivo. The combination of HDAC inhibitor HBI8000 with EZH2 inhibitor SHR2554 exhibited dramatic anti-tumor activity in both mutant and wild-type DLBCL, which may become a potential therapeutic modality for the treatment of DLBCL patients.

Abstract 1380: Synergistic effect of the combination of XPO1 and mTORC1/2 inhibition for the treatment of triple-hit DLBCL

Abstract Background Double/triple-hit lymphoma is an aggressive form of DLBCL with particularly poor outcome. XPO1 (exportin 1) is a well characterized nuclear export protein which is overexpressed in multiple tumor types. XPO1 exports many tumor-suppressor proteins and thus acts as a protooncogene by removing tumor suppressor protein and growth regulatory factors from the nucleus, where they are active, to the cytoplasm. First-in-class, single agent oral XPO1 inhibitor, Selinexor (ATG-010), was recently approved for the treatment of patients with DLBCL (de novo or transformed from follicular NHL) after at least two prior therapies. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K-AKT pathway. Signaling by the PI3K/AKT/mTOR pathway is frequently deregulated in NHL. Inhibition of mTOR has shown preclinical and clinical efficacy in treating DLBCL. This study tested the antitumor effects induced by the combination of the XPO1 inhibitor, Selinexor and the dual mTORC1/2 kinase inhibitor, ATG-008 (Onatasertib) on triple-hit DLBCL cells, DoHH2. Methods Firstly, the in vitro 50% inhibition concentration (IC50) of two compounds was determined in DoHH2 cell line using CellTiter-Glo luminescent cell viability assay. The synergy effect of compound ATG-008 in combination with ATG-010 were evaluated by combination index (CI). The synergy effect is calculated by the Chou-Talalay Method [1]. The CI score ranging from 0.1-0.9 suggests very strong to slight synergism. The in vivo combination of the drugs were tested in DoHH2 CDX mouse model. The tumor bearing mouse were treated with vehicle control, Selinexor (5mg/kg, MWF), ATG-008 (10mg/kg, QD) or the combination for 21 days. The tumor size was measured twice a week and tumor growth inhibition (TGI) was evaluated compared with vehicle control group. Results Potent in vitro and in vivo anti-tumor efficacy and synergy has been observed for the combination of the two drugs. The in vitro IC50 for DOHH-2 cell line were 0.85µM and 0.08µM for ATG-008 and Selinexor, respectively. Synergy has been observed for most combo concentrations tested with the strongest synergism observed for ATG-008 (1.7µM) + Selinexor (0.16µM). The combination index was 0.48. In the DoHH2 CDX in vivo study, the mono therapy of ATG-008 showed 41% TGI at day 19 after grouping (p value&amp;lt;0.001), and 19% TGI for Selinexor (p value= 0.147) mono therapy treatment group. The combination of ATG-008 and Selinexor showed 75% TGI (p value&amp;lt;0.01). Conclusions Strong synergism has been observed for the combination of XPO1 and mTORC1/2 inhibition by Selinexor and ATG-008, respectively, suggesting promising therapeutic strategies for double/triple-hit lymphoma patients that warrants further investigation. [1]Chou TC. Cancer Res. 2010 Jan 15;70(2):440-6. Citation Format: Bing Hou, Aihua Wang, Bo Shan, Jay Mei. Synergistic effect of the combination of XPO1 and mTORC1/2 inhibition for the treatment of triple-hit DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1380.

FoxM1: A New Therapeutic Target for Mantle Cell Lymphoma

IntroductionMantle cell lymphoma (MCL) is an aggressive B-cell lymphoma subtype with elevated B-cell receptor activity. Ibrutinib (IBN), the Bruton's tyrosine kinase (BTK) inhibitor, has been shown to have an overall response rate of 68% in relapsed or refractory MCL patients. However, with the emergence of IBN resistance, novel therapies to thwart resistance are urgently needed. FOXM1 (Forkhead box M1) is a proliferation-associated transcription factor that stimulates cell proliferation and exhibits a proliferation-specific expression pattern. FOXM1 has recently been classified as a human proto-oncogene and we previously found this gene to be associated with IBN resistance in our gene expression analysis; therefore, the prognostic significance of FOXM1 and its potential as a new MCL therapeutic target was investigated.MethodsBone marrow aspirates and peripheral blood clinical specimens were obtained from MCL patients. FOXM1 gene expression was assessed in 50 primary MCL patient samples using Quantitative real-time PCR (Q-PCR). To conduct dose-dependent cell viability assays, MCL cell lines (ATCC) were cultured in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (Millipore). The drug screening was performed in a 96-well format in which MCL cells were seeded at 10,000 cells per well and were treated with the FOXM1 inhibitor thiostrepton or ibrutinib at the following concentrations: 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 uM. Cell viability was tested using the CellTiter-Glo luminescent cell viability assay (Promega) after a 72-hour incubation period. To determine the effects of inhibiting FOXM1 on apoptosis, MCL cells (200,000 cells) were incubated with IBN or thiostrepton at varying doses (1, 3, 5 uM) for 24 hours, and apoptosis was detected using the Annexin V-binding assay. Furthermore, caspase-3/7 activity was assessed in MCL cells with the Caspase-Glo-3/7 Assay (Promega) after 24 hours of treatment with thiostrepton or IBN.ResultsTo evaluate the possibility that FOXM1 is important for MCL, we conducted an analysis of FOXM1 expression in MCL patient samples at the time of diagnosis using Q-PCR. FOXM1 expression was detected in 50 MCL patient samples, and FOXM1 expression levels were significantly higher (82.3%, fold change=2) than the levels observed in peripheral blood mononuclear cells (PBMCs). The association between FOXM1 expression in MCL and clinical pathology was also analyzed by examining the relationship between FOXM1 and Ki-67 expression (a cell-proliferation marker) in 40 diagnosed MCL patient samples, and a significant correlation was noted between the two markers (p<0.05).The effects of inhibiting FOXM1 on MCL cell lines (Jeko-1, Jeko-1-BTK-KO-#1, Jeko-1-BTK-KO-#2, Mino, Maver-1, Z-138, JVM-2, and JVM-13) in vitro were examined by transfection of siRNA targeting FOXM1 or treatment with the FOXM1 inhibitor thiostrepton. Thiostrepton reduced the cell viability of the MCL cell lines in a dose-dependent manner compared with the DMSO control. Likewise, siFOXM1-transfected MCL cell lines showed reduced cell viability compared with the control siRNA-transfected cells (P < 0.01). Annexin V/PI dual staining showed that thiostrepton induced apoptosis in the MCL cell lines in a dose-dependent manner. Furthermore, activated-caspase3-7 expression was increased in a dose-dependent manner in MCL cell lines after treatment with thiostrepton.ConclusionWe have shown that FOXM1 inhibition may be a potential candidate treatment for MCL based on the results of our clinicopathological assessment and in vitro studies. Clinical studies are warranted to further validate FOXM1 as a potential therapeutic target in high-risk MCL. DisclosuresWang:Celgene: Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Oncternal: Research Funding; BeiGene: Research Funding; Juno Therapeutic: Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Consultancy, Honoraria, Research Funding; Oncoceutics: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Asana: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Karus: Research Funding.

CDK9 Inhibition with LDC526 Is an Effective Treatment for T-Cell Prolymphocytic Leukemia (T-PLL) in Vitro

▪IntroductionT-cell prolymphocytic leukemia (T-PLL) is a rare leukemia with an aggressive disease course, characterized by chemotherapy resistance and a median overall survival of less than two years. Apart from defects in DNA damage response genes like ATM and genes of JAK/STAT signaling, overexpression of MYC, often caused by gain of chromosomal material, is frequently observed in T-PLL. Intravenous application of the anti-CD52 antibody Alemtuzumab has been demonstrated to improve progression-free survival and is currently considered the standard of care. In younger patients remissions can be consolidated by allogeneic stem cell transplantation, which results in long-term remission in a small subgroup of patients. The prognosis in the majority of patients relapsing from their disease is dismal and thus novel treatment options are urgently needed. Cyclin-dependent kinase 9 (CDK9), a serine/ threonine kinase which is ubiquitously expressed, is the catalytic unit of the positive transcription elongation factor (P-TEFb). In the presence of cyclin T, p-TEFb stimulates transcription elongation by phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II (RNA Pol II). CDK9 inhibition by LDC526, a specific CDK9 inhibitor, leads to reduced phosphorylation of CTD-Ser2, Ser5 and Ser7 of RNA Pol II and subsequently to the downregulation of MYC and MCL-1.Methods</spanCD3+CD4+ tumor cells from cryopreserved peripheral blood samples of 11 T-PLL patients were enriched to a purity of >95% by fluorescence-activated cell sorting (FACS) or magnetic activated cell separation (MACS). RNA and DNA were isolated using the RNeasy Mini Kit® or the QIAamp DNA blood midi kit® (Qiagen, Hilden, Germany), respectively. To determine IC50 values, we performed cell viability assays for T-PLL cells of 11 patients treated with LDC526 and a panel of reference compounds compared to DMSO controls (CellTiter-Glo Luminescent Cell Viability Assay; Promega, Fitchburg, WI, USA). Gene expression profiling was performed in primary T-PLL cells treated with the CDK9 inhibitor LDC526 in a concentration of 10µM compared to DMSO (n=3) on Clariom S Pico Assays (Affymetrix, Santa Clara, CA, USA) and analyzed by gene set enrichment analysis (GSEA) and GeneTrail analysis. Gene expression measurement was performed with quantitative real-time PCR (qPCR) for MYC (Taqman, Thermo Fisher Scientific, Waltham, MA, USA). Protein expression was analyzed for pCTD Ser2, Ser5 and Ser7, MYC, p-STAT3 and MCL-1 by Western Blot (n=4) after LDC526-treatment in three different concentrations (10, 30 and 100 µM).ResultsLDC526 inhibited T-PLL cell survival in vitro in a dose-dependent manner. The median IC50 was 1.7 µM (range: 0.9 -3.5) which compared favourably to a panel of reference compounds including fludarabine, clofarabine, methotrexate, cytarabine and ruxolitinib (median IC50 values 6.4, 6.2, 29.9, 8.3, and 29.2, respectively). Similar experiments with BAY1143572, a structurally closely related CDK9 inhibitor, which is currently investigated in phase I studies of acute myeloid leukemia, revealed an even lower median IC50 of 0.9 µM (range: 0.5 - 1.7). Of note, the purine analogues fludarabine and clofarabine were only active in some of the patients tested, whereas both CDK9 inhibitors showed activity in all samples analyzed.Gene expression profiles of LDC526 treated T-PLL cells compared to DMSO controls revealed an enrichment of genes in gene sets associated with interferon and TNFA signatures, MYC targets as well as JAK/STAT genes, which were subsequently confirmed by qPCR experiments. At the protein level LDC526 dose-dependently reduced phosphorylation of CTD-Ser2, Ser7 and to a lesser extent of Ser5 compared to DMSO controls resulting in efficient down-regulation of MYC, MCL-1 and p-STAT3.ConclusionsOur experiments identify CDK9 as a novel therapeutic target in T-PLL which can be efficiently inhibited by LDC526 and BAY1143572 at clinically meaningful concentrations. CDK9 inhibition resulted in both reduced T-PLL cell survival and down-regulation of MYC and JAK/STAT signaling, which have a key role in the molecular pathogenesis of T-PLL. DisclosuresDürig:Lead Discovery Center: Research Funding.

RRM2 protects against ferroptosis and is a tumor biomarker for liver cancer

BackgroundFerroptosis is the process of cell death triggered by lipid peroxides, and inhibition of glutathione (GSH) synthesis leads to ferroptosis. Liver cancer progression is closely linked to ferroptosis suppression. However, the mechanism by which inhibition of GSH synthesis suppresses potential ferroptosis of liver cancer cells and whether ferroptosis-related liver cancer biomarkers have a promising diagnostic value remain unknown.MethodsRibonucleotide reductase regulatory subunit M2 (RRM2) levels were measured using an enzyme linked immunosorbent assay (ELISA), quantitative RT-PCR (qPCR), immunoblotting (IB) and immunochemistry (IHC). Cell viability and cell death were measured by a CellTiter-Glo luminescent cell viability assay and staining with SYTOX Green followed by flow cytometry, respectively. Metabolites were measured using the indicated kits. The Interaction between glutathione synthetase (GSS) and RRM2 was measured using immunofluorescence (IF), co-immunoprecipitation (co-IP) and the proximal ligation assay (PLA). The diagnostic value was analyzed using the area under the receiver operating characteristic curve (AUC-ROC). Bioinformatics analysis was performed using the indicated database.ResultsRRM2 showed specifically elevated levels in liver cancer and inhibited ferroptosis by stimulating GSH synthesis via GSS. Mechanistically, phosphorylation of RRM2 at the Threonine 33 residue (T33) was maintained at normal levels to block the RRM2–GSS interaction and therefore protected RRM2 and GSS from further proteasome degradation. However, under ferroptotic stress, RRM2 was dephosphorylated at T33, thus the RRM2–GSS interaction was promoted. This resulted in the translocation of RRM2 and GSS to the proteasome for simultaneous degradation. Clinically, serum RRM2 was significantly associated with serum alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT), albumin (ALB) and total bilirubin. The AUC-ROC for the combination of RRM2 with AFP was 0.947, with a sensitivity of 88.7% and a specificity of 97.0%, which indicates better diagnostic performance compared to either RRM2 or AFP alone.ConclusionRRM2 exerts an anti-ferroptotic role in liver cancer cells by sustaining GSH synthesis. Serum RRM2 will be useful as a biomarker to evaluate the degree to which ferroptosis is suppressed and improve diagnostic efficiency for liver cancer.