CellTiter-Glo Assay Research Articles

A proposed in vitro cytotoxicity test battery to detect and predict hepatotoxicity via multiple mechanisms and pathways: a minireview.

The 21st-century toxicity testing program recommends the use of cytotoxicity data from human cells in culture for rapid in vitro screening focusing on biological pathways of potential toxicants to predict in vivo toxicity. Liver is the major organ for both endogenous and exogenous chemical metabolism of xenobiotics. Therefore, this review was undertaken to evaluate side by side five different currently used commercial cytotoxicity assay kits for purpose of rapid predictive screening of potential hepatotoxicants. The test compounds for this review were selected from the NIH LiverTox and FDA Liver Toxicity Knowledge Base (LTKB) databases. Human liver HepG2, HepaRG, and rat liver Clone 9 cell cultures were used as the in vitro liver models. Five commercial assay kits representing different biomarkers or pathways were selected for this review. These kits are Vita-Orange Cell Viability Assay Kit (Sigma-Aldrich), CellTiter-Glo Cell Viability Assay Kit (Promega), CytoTox-ONE Homogeneous Membrane Integrity Assay Kit (Promega), DNA Quantitation Fluorescence Assay Kit (Sigma-Aldrich), and Neutral Red Based In Vitro Toxicology Assay Kit (Sigma-Aldrich). This review found that these kits can all be used for rapid predictive cytotoxicity screening of potential hepatotoxicants in human liver HepG2 and rat liver Clone 9 cells in culture as in vitro liver models without compromising quality and accuracy of endpoint measurements as well as the length of toxicity screening time. Unraveling the structure-activity relationship of potential hepatotoxins would help to classify their hepatotoxic effects. Therefore, in addition to the current regulatory hepatotoxicity testing strategies, development and regulatory approval of hepatotoxins need to be discussed in order to identify potential gaps in the safety assessment. The overall results of our study support the hypothesis that a battery of rapid, simple, and reliable assays is an excellent tool for predicting in vivo effects of suspected liver toxins. The human liver HepaRG cells do not appear to be an ideal in vitro liver model for this purpose.

Forskolin-mediated cAMP activation upregulates TNF-α expression despite NF-κB downregulation in LPS-treated Schwann cells.

Although Schwann cells have been found to play a key role in inflammation and repair following nerve injury, the exact pathway is still unknown. To explore the mechanism by which Schwann cells exert their effects in the neuron microenvironment, we investigated two main inflammatory pathways: the NF-κB and cAMP pathways, and their downstream signaling molecules. In this study, lipopolysaccharide (LPS), a bacterial endotoxin, was used to activate the NF-κB pathway, and forskolin, a plant extract, was used to activate the cAMP pathway. The rat RT4-D6P2T Schwann cell line was treated with 0.1, 1, or 10 μg/mL of LPS, with or without 2 μM of forskolin, for 1, 3, 12, and 24 hours to determine the effects of elevated cAMP levels on LPS-treated cell viability. To investigate the effects of elevated cAMP levels on the expression of downstream signaling effector proteins, specifically NF-κB, TNF-α, AKAP95, and cyclin D3, as well as TNF-α secretion, RT4-D6P2T cells were incubated in the various treatment combinations for a 3-hour time period. Overall, results from the CellTiter-Glo viability assay revealed that forskolin increased viability in cells treated with smaller doses of LPS for 1 and 24 hours. For all time points, 10 μg/mL of LPS noticeably reduced viability regardless of forskolin treatment. Results from the Western blot analysis revealed that, at 10 μg/mL of LPS, forskolin upregulated the expression of TNF-α despite a downregulation of NF-κB, which was also accompanied by a decrease in TNF-α secretion. These results provide evidence that cAMP might regulate TNF-α expression through alternate pathways. Furthermore, although cAMP activation altered AKAP95 and cyclin D3 expression at different doses of LPS, there does not appear to be an association between the expression of AKAP95 or cyclin D3 and the expression of TNF-α. Exploring the possible interactions between cAMP, NF-κB, and other key inflammatory signaling pathways might reveal a potential therapeutic target for the treatment of nerve injury and inflammation.

Abstract LB219: DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays

Abstract Gastrointestinal cancers, including gastric cancer (GC) and colorectal cancer (CRC), continue to be devastating diseases despite advances in screening, early detection, and removal of pre-neoplastic and early cancerous lesions. Even with advancements in targeted chemotherapy and immunotherapies, advanced and metastatic diseases continue to be responsible for the high mortality associated with GC and CRC. Newer, safer, and more effective therapies to prevent, inhibit, or reverse advanced disease progression are urgently needed. Doublecortin-like kinase 1 (DCLK1) is a microtubule associated protein kinase that has been reported to drive tumor initiation, progression, metastasis, and drug resistance in many solid tumor cancers including GC and CRC, where it is upregulated in patient tumors and associated with poor overall survival. An anti-DCLK1 monoclonal antibody has demonstrated potential therapeutic activity against CRC. In this study, we established patient-derived organoids (PDOs) to evaluate the effects of inhibiting DCLK1 kinase activity (DCLK1-IN-1) or targeting DCLK1 with a highly specific CAR-T cells on the treated PDOs. Two GC PDOs were treated with various concentrations of DCLK1 kinase inhibitor DCLK1-IN-1 (0-32 μM) for 72 hrs, viability of the PDOs were determined, histochemistry and immunohistochemistry staining were used to image the live cells and DCLK1 expression. For CRC PDOs, five CRC PDOs were cultured in the presence or absence of PBMC, treated with Mock CAR-T cells or DCLK1 CAR-T cells (three doses) for 4 days. Tumor size, tumor total area, and viability of these PDOs were determined. Here, we demonstrate that treating GC PDOs with DCLK1 kinase inhibitor resulted in a significant dose-dependent inhibition in PDO numbers in both PDOs (two-way ANOVA P<0.0001), a decrease of cell viability, revealing IC50 values of 7.566 and 9.958 μM respectively, and a near total loss of GC PDO viability at 10 μM using CellTiterGlo assay. In CRC PDOs, two of five CRC PDOs showed a significant decrease in total tumor size, tumor areas, and an increase in cell death measured by the DRAQ7 method. Compared to Mock CAR-T cells, treatment with DCLK1 CAR-T cells resulted in a dose dependent decrease of tumor size and total tumor area, with more than a 50% reduction at the 50000 cells dose (p<0.05). Treatment with DCLK1 CAR-T cells also resulted in a dose dependent increase in cell death, with approximately 20% cell death without PBMC, and 50% cell death with PBMC at the 50000 cells dose (p<0.05). These results suggest that targeting DCLK1 represents a potential novel therapeutic strategy for GI cancers, especially in combination with FDA approved drugs: 5-fluorouracil (5-FU) or Gefitinib. Citation Format: Dongfeng Qu, Jinsen Shi, Zhiyun Cao, Eddie BAnnerman-Menson, Nathaniel Weygant, Courtney Houchen. DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays [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 LB219.

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.

Matched Three-Dimensional Organoids and Two-Dimensional Cell Lines of Metastatic Melanoma to the Brain Mirrors Response to Targeted Molecular Therapy

INTRODUCTION: Metastatic melanoma to the brain (MMB) represents a significant treatment challenge. Limitations in preclinical models, namely the lack of heterogeneity in cell lines and low throughput of xenograft models, have thwarted novel therapeutic development. Surgically eXplanted Organoids (SXOs) are ex vivo, three-dimensional models that recapitulate genotypic and phenotypic features of parent tumors with minimal extracranial processing. We aim to develop the first known matched patient-derived SXO and cell line of BRAF-mutant MMB to investigate responses to targeted therapy. METHODS: MMB SXOs were created by a novel protocol incorporating techniques for creating glioma and cutaneous melanoma organoids. A BRAFV600K-mutant MMB sample was collected and processed within 30 minutes of resection. SXOs were created using a 1.0 mm biopsy punch. Organoids were cultured in low-adherence plates in an optimized culture medium under continuous orbital rotation without an artificial extracellular scaffold. Organoid media was changed every 48 hours. Concurrently, matched patient-derived cell lines were created and passaged weekly. Drug screens were conducted with the CellTiter-Glo Luminescence Viability Assay (cell line) and ReadyProbe Cell Viability Imaging Kit (SXOs). RESULTS: After an initial shedding period, organoid growth was observed within 3-4 weeks. MMB SXOs retained histological phenotypes of the parent tissue, including pleomorphic epithelioid cells with abundant cytoplasm, large nuclei, focal melanin accumulation, strong Sox10 positivity, and HMB45 positivity. After sufficient growth, organoids could be manually parcellated to increase the number of replicates. Matched SXOs and cell lines demonstrated sensitivity to BRAF-V600 and MEK inhibitors. CONCLUSIONS: Here, we outline the creation of the first known scaffold-free organoid model of MMB. Further study using SXOs will improve the translational relevance of preclinical studies and enable the study of the melanoma tumor microenvironment.

Abstract 3134: SDP03923-000-9106, a novel and optimized LIV-1 ADC with superior anti-tumor efficacy in breast cancer xenograft models

Abstract Background: LIV-1, also known as SLC39A6 or ZIP6, is a transmembrane protein belonging to the zinc transporter family. LIV-1 is a promising target for antibody-drug conjugate (ADC) therapy due to its broad expression in tumors and limited normal tissue expression. Ladiratuzumab vedotin, a LIV-1 targeting ADC with payload of MMAE, is now in phase I clinical trial stage and is the only LIV-1 ADC under clinical development worldwide. The preliminary clinical results demonstrated promising efficacy in TNBC (ORR=32%) but no response in HR+/HER2- breast cancer (ORR=0%), and typical MMAE-related adverse events were observed. Herein, we presented a novel LIV-1 directed ADC, SDP03923-000-9106, consisting of a LIV-1 mAb developed in house conjugated to a proprietary topoisomerase I inhibitor via a cleavable linker with an optimized average drug-to-antibody ratio (DAR) of 6. Method: The in vitro binding affinity of SDP03923-000-9106 against LIV-1 protein was measured by Surface Plasmon Resonance (SPR) assays. The expression levels of LIV-1 in different cancer cell lines were detected by flow cytometry and IHC staining. In vitro activity against cancer cell lines was analyzed by CellTiter Glo assays following 10 days of treatment. The in vivo anti-tumor activities were investigated in both CDX and PDX models of breast cancer. Results: SDP03923-000-9106 is a novel anti-LIV-1 ADC comprised of a humanized LIV-1 monoclonal antibody conjugated to the potent topoisomerase I inhibitor (a novel exatecan derivative designed by Hengrui with a better liposolubility and cellular permeability) by a cleavable linker. SDP03923-000-9106 had an optimal drug-to-antibody ratio (DAR) of 6 and showed strong binding affinity to LIV-1 and favorable internalization. SDP03923-000-9106 exhibited robust anti-tumor activity in cell lines across breast cancer, esophageal squamous cancer, gastric cancer, prostate cancer and non-small cell lung cancer. In the LIV-1 highly expressed breast cancer cell line of MCF-7, SDP03923-000-9106 demonstrated a strong inhibitory effect with IC50 of 5.36 nM. Moreover, in a LIV-1 high-expressing patient-derived xenograft model of breast cancer, SDP03923-000-9106 showed pronounced tumor regression at both doses of 3 and 10 mg/kg and showed stronger anti-tumor activity (TGI: 109%) at the same dose of 3 mg/kg versus BMK-MMAE (TGI: 75%; BMK-MMAE is synthesized by Hengrui using the published structure of Ladiratuzumab vedotin). Conclusion: In summary, SDP03923-000-9106 is a novel LIV-1 targeted ADC with a highly permeable payload demonstrating great anti-tumor activity across multiple types of tumors in preclinical studies. The promising preclinical data support advancing SDP03923-000-9106 into clinical testing, and Investigational New Drug (IND) application to NMPA has been submitted. Citation Format: Yanling Gong, Xing Sun, Changyong Yang, Simeng Chen, Wei Zhang, Cheng Liao. SDP03923-000-9106, a novel and optimized LIV-1 ADC with superior anti-tumor efficacy in breast cancer xenograft models [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 3134.

Abstract 2035: A Benzenesulfornamide with potential to improve chemoresponse of ovarian cancer through activation of AMPK and immunogenic cell death

Abstract Introduction: Ovarian cancer is a leading cause of death among women globally often characterized by poor prognosis and aggressive tumor growth. The therapeutic outcomes of ovarian cancer patients are limited by the development of acquired chemoresistance and the lack of targeted therapies. This study aimed to understand the mechanism in which a benzenesulfornamide named Y3 enhances the efficacy of chemotherapeutic agents. Y3 was previously identified by our team as an activator of AMPK with the activity to induce immunogenic cell death of ovarian cancer cells. Material and Methods: Patient-derived ovarian cancer cell lines were pretreated with Y3 for 3 h before treatment of carboplatin or paclitaxel. Celltiter Glo assay was performed to determine the impact of Y3 pretreatment on the IC50 values of carboplatin or paclitaxel after 48 h. Apoptosis was evaluated using Annexin VI/PI staining and Caspase 3 activity assays. The role of AMPK pathway was assessed using AMPK siRNAs and western blot. The potential of Y3 to enhance immunogenic cell death induced by carboplatin or paclitaxel was evaluated in a syngeneic mouse model of ovarian cancer using a vaccination assay. The control group mice were vaccinated by subcutaneously injecting mouse ovarian cancer cells treated by carboplatin in vitro for 24 h into the left flank. The test group mice were vaccinated by injecting Y3-carboplatin co-treated cells. One week after vaccination, untreated live cancer cells were injected into the right flank of both groups. Tumor growth was monitored. Results: Ovarian cancer cell lines pretreated with Y3 showed decreased IC50 values for carboplatin and paclitaxel treatments. The apoptotic cell populations stained by Annexin VI were increased in the Y3 pretreated in comparisons with the single treatment groups. Caspase-3 activity validated the results of Annexin VI/PI staining assay. AMPK knockdown by siRNAs effectively reduced the levels of phospho-AMPK that was induced by Y3, which also dampened the Y3-promoted chemotherapeutic effects on ovarian cancer cells. In the in vivo vaccination assay, tumor growth in the right flank was suppressed in the mice vaccinated with Y3-carboplatin co-treated cells to an extend that is more significant than the control group. Conclusion: Y3 pretreatment increased the sensitivity of ovarian cancer cell lines to carboplatin and paclitaxel. The effects of Y3 on ovarian cancer cells depend on the activation of AMPK. Y3 enhances the immunogenicity of carboplatin-treated ovarian cancer cells. Citation Format: Juhyun Kim, Nia Montero, Shefali Sharma, Miranda Mansolf, Kevin Yang, Tobias Hartwich, Yang Yang-Hartwich. A Benzenesulfornamide with potential to improve chemoresponse of ovarian cancer through activation of AMPK and immunogenic cell death [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 2035.

Abstract 636: Coculture model for investigating cellular interactions between colorectal tumor organoids and cancer-associated fibroblasts

Abstract The tumor microenvironment (TME) is well recognized for its role in tumor progression, chemoresistance, and recurrence. The TME consists of various components, such as extracellular matrix (ECM), chemical and mechanical cues, as well as stromal cells and their interactions. As the most abundant cells in tumor stroma, cancer-associated fibroblasts (CAFs) have drawn a lot of attention. It has been reported that CAFs not only directly interact with cancer cells via surface proteins and cytokines, but also influence their behaviors by remodeling the ECM. However, due to their heterogenous characteristics and diverse functions, the influence of tumor-CAF interactions on drug response is not fully understood. Here, we present a coculture model for investigating CAF effects on patient-derived colorectal tumor organoids (CTOs) growth, differentiation, and drug response. It consists of normal colon fibroblasts, CCD18-Co, or patient-derived CAFs seeded with CTOs in a mixed hydrogel containing 90% basement membrane extract (BME) and 10% collagen type I. Media conditions were optimized (1:1 mix of CTO culture media plus reduced media excluding growth factors and inhibitors) to achieve similar organoid sizes in the monoculture and coculture conditions at the onset of drug testing. To support higher throughput investigations, our workflow incorporates a liquid handler for automated CTO seeding, media and drug exchange, and an automated microscope with a robotic arm for multi-plate imaging. Briefly, single cell CTOs and fibroblasts were seeded at a 1:1 ratio (3000 total cells/well) in 96-well plates. After 4 days in culture, anti-cancer drugs were administrated in reduced media to the samples. Brightfield images were acquired on days 0, 3, and 5 after drug administration. The collected images were analyzed with a neural network (NN) to dynamically monitor the growth and viability of individual organoids. The CellTiter-Glo assay was performed on day 5 to quantify the total CTO cell viability. The normalized drug response (NDR) was calculated from the NN processed images to determine the drug response. An immunostaining panel, which includes protein markers such as CK20, Ki67, α-SMA, Vimentin, and FAP, was used to evaluate the fibroblast phenotypes and CTO differentiation. Additionally, the cultures were subjected to fluorescent lifetime imaging microscopy (FLIM), a label-free imaging technique, to measure the metabolic adaptations of each cell population. Preliminary studies revealed a CAF-dependent increase in CTO growth and glycolytic signatures in the co-culture setting; however, minimal effects on drug response to common chemotherapy agents such as 5-fluorouracil and irinotecan were observed. In the future, we will use this integrated pipeline as a screening method to identify new treatment strategies aimed at targeting the stromal microenvironment in colorectal cancer. Citation Format: Yuyuan Zhou, Yu-Kai Huang, Emma J. Fong, Elizabeth Elton, Erin McManus, Brandon Choi, Seungil Kim, Shannon M. Mumenthaler. Coculture model for investigating cellular interactions between colorectal tumor organoids and cancer-associated fibroblasts [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 636.

Abstract 4569: APG-2449, a novel focal adhesion kinase (FAK) inhibitor, inhibits metastasis and enhances the antitumor efficacy of PEGylated liposome doxorubicin (PLD) in epithelial ovarian cancer (EOC)

Abstract Ovarian cancer is the leading cause of cancer-related death in women and most cases are diagnosed at later stages, with distant metastasis. Standard-of-care therapy is surgery followed by platinum and paclitaxel; however, most patients who initially respond to therapy eventually relapse after developing metastatic disease and chemoresistance. FAK overexpression or activation occurs in a substantial proportion of EOCs and is predictive of poor clinical outcomes. Recent studies have reported that FAK plays an important role in cell migration and chemoresistance, rendering FAK inhibition a promising treatment approach to reduce metastasis of tumor cells and sensitize them to chemotherapy. The aim of this study was to evaluate the antitumor efficacy of investigational APG-2449, a novel FAK inhibitor, combined with PLD, a commonly used chemotherapy, in relapsed or refractory ovarian cancer. Four EOC cell lines (OVCAR-3, SK-OV-3, A2780, and Kuramochi) were used for in vitro experiments. Cell proliferation was measured by CellTiter-Glo assays. Cell migration ability was assessed by wound healing assays. OVCAR-3 xenograft and luciferase-tagged ID8 (ID8-Luc) peritoneal syngeneic models were established to evaluate the synergistic antitumor efficacy of combined APG-2449 and PLD in vivo. APG-2449 combined with doxorubicin showed synergistic antiproliferative effects in both platinum-resistant (OVCAR-3 and SK-OV-3) and platinum-sensitive (A2780 and Kuramochi) cell lines. In wound healing assays, FAK inhibition via APG-2449 alone attenuated migration of ovarian cancer cells (SK-OV-3, OVCAR-3, and ID8-Luc) in a dose-dependent manner. In the OVCAR-3 xenograft model, combined APG-2449 and PLD demonstrated enhanced antitumor effects compared with either APG-2449 or PLD monotherapy (synergistic index, 1.33). In the ID8-Luc peritoneal syngeneic model, the combination regimen outperformed PLD or APG-2449 monotherapy in tumor growth inhibition. This inhibitory effect persisted more than 1 month after stopping combination treatment, whereas the monotherapy groups experienced rapid disease relapse. The combination group also exhibited the longest ascites-free and survival times compared with PLD or APG-2449 monotherapy groups. Overall, our data suggest that inhibition of FAK by APG-2449 enhances the antitumor effect of PLD in EOC cell lines and the OVCAR-3 xenograft mouse model. The combination regimen can also prolong ascites-free and survival times, as shown in the ID8-Luc peritoneal syngeneic model. These promising results support future clinical development of this combination treatment for ovarian cancer. Citation Format: Zhou Yu, Eric Liang, Daojie Liu, Bingxing Wu, Xinyi Yao, Yanqiu Ji, Dajun Yang, Yifan Zhai. APG-2449, a novel focal adhesion kinase (FAK) inhibitor, inhibits metastasis and enhances the antitumor efficacy of PEGylated liposome doxorubicin (PLD) in epithelial ovarian cancer (EOC) [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 4569.

Abstract 4535: JAB-26766: A small-molecule, orally bioavailable PARP7 inhibitor with high potency and selectivity

Abstract Background: PARP7 (also referred to as TIPARP or ARTD14) is a mono-ADP-ribosyl transferase that inhibits type I interferon (IFN) signaling. Targeting PARP7 to restore antitumor immunity represents a promising treatment strategy. We have developed JAB-26766, an orally bioavailable, highly potent, and selective PARP7 inhibitor. Methods: A time-resolved fluorescence energy transfer (TR-FRET) assay was applied to determine the binding and inhibition of PARP7 by JAB-26766. IFN-β secretion assay, STAT1 phosphorylation assay, and ISG (interferon stimulated gene) mRNA assay were performed to evaluate the inhibitory activities of JAB-26766 on the downstream signaling of PARP7. CellTiter-Glo assay was performed to evaluate cell viability upon treatment of JAB-26766. In vivo PK-PD study was conducted to evaluate the relationship between JAB-26766 concentration and the level of tumor STAT1 phosphorylation. The antitumor activity of JAB-26766 either as a single agent or in different drug combinations was evaluated in multiple mouse models. Results: High potency of JAB-26766 on PARP7 inhibition as well as >1800-fold selectivity over PARP2 was observed through biochemical assays. At the cellular level, JAB-26766 induced IFN-β secretion, up-regulated ISG mRNA levels, increased phosphorylation of STAT1, and inhibited cell viability of multiple tumor cell lines (with most IC50 values below 50 nM). JAB-26766 combined with a STING agonist resulted in synergistic activation of STING pathway, as evidenced by increased CXCL10 secretion, and inhibited tumor cell proliferation. In vivo PK-PD study showed that JAB-26766 exposure in plasma and tumor has good correlation with induction of tumor STAT1 phosphorylation. Furthermore, JAB-26766 showed potent antitumor activity and good tolerability either as monotherapy or combined with STING agonist or anti-PD-1 antibody in mouse models of non-small cell lung cancer, breast cancer and colorectal cancer. Conclusions: JAB-26766 is an orally bioavailable, highly potent, and selective PARP7 inhibitor that shows early promising anti-tumor activities. Citation Format: Di Kang, Yanping Wang, Xin Sun, Man Yan, Haijun Li, Mingming Chen, Yiwei Lin, Wei Long. JAB-26766: A small-molecule, orally bioavailable PARP7 inhibitor with high potency and selectivity [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 4535.

Abstract 606: The PLK1 inhibitor, onvansertib, synergizes with paclitaxel in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with rapid disease progression and poor patient survival. Therapeutic options for SCLC remain a challenge due to limited understanding of both the biology of the disease and the major determinants of treatment response. Key transcription factors associated with unique biological phenotypes of SCLC include ASCL1, NEUROD1, YAP1, and POU2F3. Previously, we demonstrated that onvansertib, a highly specific inhibitor of the PLK1 Ser/Thr kinase, was effective in SCLC preclinical models, leading to its ongoing clinical development for relapsed/refractory SCLC patients (NCT05450965). Here we further explored potential strategies to enhance onvansertib activity by assessing its in vitro and in vivo activity in combination treatments. Methods: Human SCLC cell lines were treated for 72 hr with onvansertib in combination with paclitaxel, lurbinectedin or the ATR inhibitors BAY1895344 and AZD6738. Effect on cell proliferation was analyzed by WST1 and CellTiterGlo assays. In vivo activity of onvansertib in combination with paclitaxel was tested in 3 SCLC patient-derived xenograft (PDX) models either cisplatin-sensitive (TKO5- ASCL1 subtype) or cisplatin-resistant (TKO2-ASCL1 subtype, TKO8-NEUROD1 subtype). Tumor-bearing nude mice were treated with vehicle, onvansertib (50 mg/kg, oral, 4 times/week), paclitaxel (15 mg/kg, IP, once a week) or the combination for 5 weeks. Mice were followed for tumor growth and survival (defined as time to reach tumor volume greater than 2000 mm3). Results: The combination of onvansertib and paclitaxel inhibited cell proliferation synergistically in cell lines from 3 SCLC subtypes (ASCL1, NEUROD1, and YAP1), while the combinations with lurbinectedin or ATR inhibitors were not synergistic. We further examined the combination of onvansertib and paclitaxel in SCLC PDXs. The combination exhibited potent anti-tumor activity, resulting in significantly greater tumor growth inhibition and increase in survival compared to the monotherapies in all 3 PDXs. The combination was well tolerated with no observed body weight loss. While onvansertib and paclitaxel had negligible single agent activity in cisplatin-resistant TK02 PDX and TKO8 PDX models, the combination induced durable complete responses in 25% and 100% of treated mice respectively. Conclusion: The PLK1 inhibitor onvansertib in combination with paclitaxel showed synergy in multiple SCLC cell lines in vitro and potent anti-tumor activity in cisplatin-sensitive and -resistant SCLC PDX models in vivo. Tumor tissue analysis is underway to detail the mechanisms of this interaction. Citation Format: John C. Schmitz, Guojing Zhang, Abbe Pannucci, Tod Smeal, Chu-Chiao Wu, Maya Ridinger, Taofeek K. Owonikoko. The PLK1 inhibitor, onvansertib, synergizes with paclitaxel in small cell lung cancer [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 606.

Abstract 3173: Impact of hypoxia on the integrated stress response activated by imipridones ONC201 and ONC206 in pediatric diffuse midline glioma cells

Abstract Diffuse midline gliomas (DMGs) are highly aggressive, high-grade gliomas which typically arise in children and young adults. With currently approved treatments the median survival from time of diagnosis for pediatric DMG is 8-10 months with only 10% of children living to 2 years post diagnosis. Despite being only 15% of the cases it makes up 40% of the pediatric brain cancer deaths making it the leading cause of death for all pediatric glioma cases. Two recent clinical studies, NCT03416530 and NCT03134131, have shown the clinical efficacy of Dordaviprone (ONC201/TIC10) for the treatment of DMG; increasing median survival to 22 months in patients following radiation treatment prior to recurrence. Imipridone ONC206, a chemical derivative of ONC201, is under clinical development for treatment of pediatric and adult patients with primary brain tumors (NCT04732065 and NCT04541082). To further improve treatment, we must continue to study how the tumor microenvironment impacts the efficacy of imipridones. One crucial aspect of all brain cancers is hypoxia, so the IC50s of the SU-DIGP-25, SU-DIPG-XIII and SU-DIPGIV pediatric DMG cell lines treated with ONC201 or ONC206 were measured using the CellTiter-Glo assay under conditions of hypoxia and normoxia. Imipridones mediate apoptosis through the upregulation of the TRAIL death receptor DR5 and the activation of the integrated stress response (ISR), so western blots were used to show changes in the ISR protein expression in ONC201 treated DMG cells at multiple different degrees of hypoxia. To understand how hypoxia inducible factors (HIFs) play a role in resistance and susceptibility to ONC201 or ONC206, HIF-1a, HIF-2a, and HIF-3a were knocked down showing altered ISR protein expression after treating with the imipridones under hypoxic and normoxic conditions. Citation Format: Tyler J. Roady, Nolan Stubbs, Josephine Chen, Yutong Xia, Ashley Sanchez Sevilla Uruchurtu, Xiaobing Tian, Lanlan Zhou, Wafik S. El-Deiry. Impact of hypoxia on the integrated stress response activated by imipridones ONC201 and ONC206 in pediatric diffuse midline glioma 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 3173.

Abstract 1816: In silico design of novel multitarget small molecule inhibitor LCI139 for the treatment of PTEN-mutant endometrial carcinoma

Abstract Background: Previously we described multitarget CDK4/6-CDK9 inhibitor, LCI133 for the treatment of endometrial carcinoma (EC). We have optimized LCI133’s chemical properties to increase PI3K p110α potency to better target epithelial-derived cancers, resulting in nanomolar potent LCI139. We characterized LCI139’s in vitro potency, target specificity, activity in in vitro EC models, safety against normal cell lines. Methods: LCI139’s chemical structure was determined using nuclear magnetic resonance and high-resolution mass spectrometry. Specific target inhibition was assessed by cell-free kinase hotspot/lipid kinase assay and selectivity profile. LCI139 (0.001-10μM) was screened against EC cell lines AN3CA, RL95-2, Ishikawa, KLE, HEC-1-A and -B for 48 hours; IC50 values determined by CellTiter-Glo Assay. EC cells were treated with LCI139 or single agent control for 24 hours and stained with Annexin V/7-AAD to assess apoptosis by cytometer. Representative PTEN wildtype (HEC-1) and mutant (AN3CA) EC cell lines were chosen for mechanistic studies and treated 6 to 24 hours with multitarget or single agent inhibitor. RNA was isolated to determine gene expression by qRT-PCR, cells were stained with Vybrant DyeCycle Violet to determine cell cycle arrest by cytometer, or cellular lysates were prepared for Western blot (WB). Results: LCI139’s has a molecular weight of 502.57Da and is a highly potent PI3Kα-CDK4/6-CDK9 inhibitor by cell-free kinase assays: 0.070µM (PI3Kα), 0.461µM (PI3Kγ), 0.214µM (PI3Kδ), 0.0015µM (CDK4), 0.0036µM (CDK6), 0.000039µM (CDK9). KINOMEscan revealed a favorable S(35) selectivity score of 0.221 screened against 403 non-mutant kinases. PTEN mutant AN3CA (IC50 243.6nM), RL95-2 (IC50 22.8nM), Ishikawa (IC50 9.8nM) had nanomolar LCI139 IC50 values, while PTEN-wild type cell lines were insensitive up to 10μM; confirmed by dose-dependent apoptosis and colony-forming assays. WB revealed LCI139 treatment ablated MCL-1 expression, decreased phosphorylation of AKT, Rb, Rpb1 (Ser2), induced significant amounts of cPARP. MCL-1 ablation correlated to decreased Mcl1 expression (p<0.0010) assessed 6 hours post treatment. LCI139 generated heterogenous cell line specific effects on cell cycle progression and arrest. LCI139 displayed a favorable toxicity profile against normal human kidney cells (HEK-293) compared to single agent controls. Conclusions: Triple PI3K-CDK4/6-CDK9 inhibitor, LCI139, is nanomolar potent against PTEN mutant EC cell lines and displays decreased toxicity compared with CDK9 inhibitor reference compounds, AZD4573 and flavopiridol. Our results merit further mechanistic dissection of PTEN interaction with PI3K pathway signaling, cell cycle machinery, CDK9-mediated transcriptional control. These data support extensive in vivo potency, toxicity, PK/PD studies to assess LCI139’s clinical utility in treating EC. Citation Format: Vaidehi Mujumdar, Ritchie Delara, Hailey Dryden, Dhananjaya Pal, Krishnaiah Maddeboina, Page Mangum Arditti, Bharath Yada, Robert Wendel Naumann, Yovanni Casablanca, Erin Crane, Jubilee Brown, Donald Durden, Cody McHale. In silico design of novel multitarget small molecule inhibitor LCI139 for the treatment of PTEN-mutant endometrial carcinoma [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 1816.

Abstract 7226: Synergy between ONC201 and temozolomide on ATF4 integrated stress response in glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of 15 months. The standard of care involves surgical resection followed by chemoradiotherapy. Even with the full course of treatment, GBM is uncurable, and patients eventually relapse. Although temozolomide (TMZ) is the first-choice chemotherapeutic for GBM, tumor drug resistance limits the effects of the treatment. There is a growing need for new therapies with enhanced efficacy on GBM tumors. Dordaviprone (ONC201) is a TNF-related apoptosis inducing ligand (TRAIL)-inducing compound that is increasingly being studied for GBM treatment. This imipridone small molecule induces apoptosis by activating the integrated stress response, which involves transcription factors ATF4 and CHOP. Subsequently, these transcription factors upregulate proapoptotic protein TRAIL and its receptor, Death Receptor 5 (DR5). This study aims to investigate the combinatory effects of chemotherapeutics TMZ and ONC201 on GBM. Preliminary studies examined the cell viability of GBM cell line U251MG after dual treatment of 0 to 10uM of ONC201 and 0 to 200uM of TMZ using the CellTiter-Glo assay. Synergy was observed between the two drugs at higher concentrations of TMZ. This synergistic interaction was further characterized using western blots. When 0, 2, and 4uM of ONC201 were administered alone or in combination with 0, 60, and 120uM of TMZ, greater ATF4 expression was observed with the dual treatments than with the monotherapies. Subsequent western blots were conducted using three GBM cell lines (U251MG, T98G, and SNB19-GFP) at three time points (8, 24, and 48 hours). All cell lines were treated with 0 and 5uM of ONC201 alone or in combination with 0 and 200uM of TMZ. For all three cell lines, ATF4 expression increased in the combined presence of TMZ and ONC201. However, the effects of TMZ on ONC201-induced ATF4 expression were most pronounced in U251MG and least conclusive in T98G. Additionally, the time points in which this increase in ATF4 expression occurred varied among cell lines. CHOP expression was also probed and was seen to increase with the combination treatment in U251MG. These observations suggest a potential synergistic relationship between chemotherapeutics ONC201 and TMZ in the ATF4 integrated stress response pathway in GBM. By exploring the effects of this dual treatment on upstream and downstream factors in the pathway, we can identify, characterize, and potentially target the root cause of this synergy. Citation Format: Josephine Chen, Tyler J. Roady, Lanlan Zhou, Wafik S. El-Deiry. Synergy between ONC201 and temozolomide on ATF4 integrated stress response in glioblastoma [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 7226.

Abstract 3318: GFH375 (VS-7375): An oral, selective KRAS G12D (ON/OFF) inhibitor with potent anti-tumor efficacy

Abstract Background: KRAS is the best-known oncogene but had long been considered ‘undruggable’ until the approval of the first KRAS G12C inhibitor sotorasib in 2021. However, most current clinical-stage KRAS inhibitors target the KRAS G12C mutation. Inhibitors of other KRAS mutants such as KRAS G12D, the most frequent KRAS mutation in human cancer, are needed for patients. We have developed GFH375 (VS-7375), an oral, selective KRAS G12D inhibitor targeting both “ON” (GTP-bound) and “OFF” (GDP-bound) states of KRAS proteins. The in vitro potency, selectivity, and in vivo efficacy of monotherapy and combination therapy with avutometinib, a unique RAF/MEK clamp, were evaluated in preclinical studies. Methods: Biochemical and cellular assays were used to investigate inhibition to KRAS cycling and signaling. CellTiter-Glo assay was performed to determine the effects on proliferation of tumor cell lines. Several KRAS G12D CDX tumor models were employed to study the in vivo pharmacodynamic and anti-tumor effects. Results: GFH375 inhibited both nucleotide exchange on GDP-bound KRAS G12D and interaction between GMPPNP-bound KRAS G12D with RAF1 with single-digit nanomolar IC50 values. GFH375 suppressed phosho-ERK1/2 (p-ERK) level with sub-nanomolar IC50 values and potently inhibited cell proliferation across a panel of KRAS G12D tumor cell lines. GFH375 showed high selectivity for KRAS G12D relative to non-G12D KRAS variants, KRAS wild type cells, and NRAS, HRAS, or BRAF mutated cells. Following a single oral dose, GFH375 produced deep and durable inhibition of p-ERK in KRAS G12D CDX tumors. GFH375 demonstrated dose-dependent anti-tumor activity with tumor regressions at 10 or 30 mg/kg given orally twice daily in multiple KRAS G12D PDAC and CRC CDX tumor models. GFH375 also showed strong anti-tumor efficacy in an intracranial KRAS G12D tumor model starting as low as 10 mg/kg orally twice daily. Strong synergy between GFH375 and avutometinib was observed in vitro and was validated in vivo as anti-tumor activity of GFH375 was further enhanced by avutometinib. Conclusions: GFH375 is a highly potent and selective orally active inhibitor of KRAS G12D (ON/OFF) and demonstrated promising anti-tumor activity in multiple KRAS G12D tumor models. GFH375 was also effective in an intracranial tumor model. GFH375 showed strong synergy with avutometinib in vitro and in vivo. GFH375 is currently in IND-enabling development in preparation for clinical studies of monotherapy and potentially in combination with other agents for patients with KRAS G12D mutant tumors. Citation Format: Feng Yan, Tao Jiang, Tao Liang, Lijian Cai, Leitao Zhang, Xiaoming Xu, Yanhui Zhao, Xiaoling Lan, Xiaohui Zhang, Meng Liu, Qiang Liu, Jinting Gao, Fubo Xie, Xueyan Gao, Li Wang, Jingyang Zhang, Hongcan Ren, Dong Liu, Siyuan Le, Lili Tang, Silvia Coma, Yaofeng Cheng, Nathan Sanburn, Jonathan A. Pachter, Fusheng Zhou, Jiong Lan, Qiang Lu. GFH375 (VS-7375): An oral, selective KRAS G12D (ON/OFF) inhibitor with potent anti-tumor efficacy [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 3318.

Abstract 5940: JAB-30355: A highly potent, orally bioavailable p53-Y220C reactivator

Abstract Background: As a key tumor suppressor, p53 precisely regulates cellular events such as cell cycle arrest, apoptosis, senescence, and DNA repair under physiological conditions. In 50-60% of human cancers, TP53 gene is mutated. The TP53 Y220C is a hotspot loss-of-function mutation occurring in around 1% of solid tumors. Application of p53-Y220C reactivator for restoration of p53 function represents a promising treatment strategy for patients with this mutation. Methods: The Induced Allosteric Drug Discovery Platform (IADDP), which integrates medicinal chemistry, biochemical and biophysical studies, and computational techniques, was applied for the development of JAB-30355. The proportion of p53-Y220C with wild-type protein-like structure after JAB-30355 treatment was determined by immunoprecipitation. Thermal shift assay was applied to assess the thermal stability of JAB-30355-reactivated p53-Y220C protein. DNA binding assay was developed to evaluate the in vitro DNA binding activity. p53-luciferase reporter system was developed to evaluate the cellular transcriptional activity. qPCR and Western blotting were performed to evaluate the transcriptional and translational levels of p53 downstream targets, respectively. CellTiter-Glo assay was performed to evaluate the inhibitory activity of JAB-30355 on the viability of tumor cell lines harboring wild-type or Y220C mutated TP53. In vivo PK-PD study was conducted to evaluate the relationship between JAB-30355 concentration and expression of p53 target genes. Multiple CDX and PDX mouse models harboring TP53 Y220C were used to evaluate the antitumor activity of JAB-30355. Results: Immunoprecipitation assay and thermal shift assay results demonstrated that JAB-30355 efficiently restored the wild-type p53-like structure and enhanced protein stability of p53-Y220C in a dose-dependent manner. In addition, JAB-30355 significantly enhanced the DNA binding activity of p53-Y220C and subsequently increased the expression of p53 target genes, such as CDKN1A, MDM2 and PUMA. JAB-30355 inhibited cell viability of multiple TP53 Y220C-mutated cancer cell lines with IC50s ranging from 0.2 to 0.7 μM, and exhibited good selectivity against TP53 wild-type cells. Overall, the in vitro data demonstrated that JAB-30355 is a potent and selective p53-Y220C reactivator. In vivo PK-PD study showed good correlation between JAB-30355 exposure and activation of p53 target genes. Furthermore, JAB-30355 exhibited dose-dependent anti-tumor activity, inducing tumor stasis or regression in multiple CDX and PDX models of ovarian cancer, pancreatic cancer, gastric cancer, and small cell lung cancer, with overall good tolerability. Conclusions: JAB-30355 is a highly potent, selective, and orally bioavailable p53-Y220C reactivator that will be tested in clinical space soon. Citation Format: Qian Zheng, Peng Wang, Chen Liang, Yao Li, Xin Sun, Amin Li, Wei Zhang, Wei Long, Yanping Wang. JAB-30355: A highly potent, orally bioavailable p53-Y220C reactivator [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 5940.

Abstract 518: The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation

Abstract Richter transformation (RT), characterized by the transformation of chronic lymphocytic leukemia (CLL) to an aggressive large-cell lymphoma, remains a challenge for the therapy. Genetic aberrations, such as Myc activation, TP53 abnormality, NOTCH1 gene mutation or loss of CDKN2A are associated with uncontrolled RT cell proliferation and escaping of apoptosis. Among those changes, gain of Myc activity, found in around 70% of RT, plays a nexus role in the pathogenesis of RT. As a transcription factor, Myc activation has a profound effect on cell survival, proliferation, adhesion and metabolism. Because of its essential role in lymphoma transformation and aggressiveness, Myc is a vital target for therapy toward RT. Fadraciclib (CYC065) is a second-generation CDK inhibitor with selective potency toward CDK2 and CDK9. We have shown previously that by inhibiting CDK9-mediated transcription, fadraciclib reduced expression of the short-lived anti-apoptotic protein Mcl-1 to initiate apoptosis in primary CLL cells. Like Mcl-1, Myc is a quintessential example of an oncogene with rapid turnover in both its mRNA and protein, therefore a promising target of CDK9 inhibition. Thus, we proposed that fadraciclib would target both the survival and proliferation pathways of RT pathogenesis through collaborative inhibition of Mcl-1, Myc and CDK2. First, our data showed that fadraciclib effectively induced apoptosis in the RT cell line HPRT1, as well as the lymphoma cell lines with Myc amplification, including Raji, Ramos and SU-DHL-2. The IC50 for apoptosis was 0.3 µM at 24h in the HPRT1 cells, which is less than the average IC50 for the primary CLL cells (0.8 µM). The IC50 for the lymphoma lines ranged from 0.8 µM to 1.3 µM. Immunoblots showed reduction of RNA Pol2 phosphorylation by fadraciclib, consistent with the inhibition of CDK9. As a result of loss of RNA Pol2 activity and pausing of transcription, there was a clear reduction of both Mcl-1 and Myc mRNA, measured by real-time RT-PCR, that was associated with the reduction of their protein levels. CDK2 was also inhibited by fadraciclib, shown by the reduction of the phosphorylation of both RB and NPM. Second, Fadraciclib inhibited cell proliferation of the HPRT1 cells at an IC50 of 0.25 µM measured by the CellTiter-Glo assay. The IC50 for growth inhibition in the lymphoma lines ranged from 0.5 to 0.7 µM. CFSE tracing demonstrated halting of cell division upon fadraciclib incubation. Cell cycle analysis by Click-iT EdU showed reduction of S-phase population and arresting of cells in both G1 and G2. We are currently working on dissecting the contributions of inhibiting Myc, Mcl-1 and CDK2 individually and together, in apoptosis induction and proliferation blockade. Taken together, these data suggest that fadraciclib is active in RT cells and Myc-dependent lymphoma cell lines and support expansion of its clinical testing in patients with RT. Citation Format: Rong Chen, Yuling Chen, Ping Xiong, William Plunkett. The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation [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 518.

Abstract 7153: PHI-101 synergizes with chemotherapy and venetoclax in preclinical models of acute myeloid leukemia

Abstract FLT3 mutations are one of the most frequent genetic alterations in AML, resulting in poor prognosis. Although the development of FLT3 tyrosine kinase inhibitors (TKIs) provided new treatment options, patients with FLT3-mutant AML still suffer from high relapse rates when treated with FLT3 TKI alone. Accumulating evidence supports a combination treatment strategy to lower the recurrence rate. We have previously shown that PHI-101 demonstrated potent activity in various FLT3 mutant AML cell lines when used alone. Here we explore the use of PHI-101 in combination with other therapeutics in preclinical models of AML. We investigated the preclinical efficacy of the combination of PHI-101 with FDA-approved AML therapeutic agents in FLT3 mutant (MV4-11, Molm13, Molm14) and wild-type (THP-1) AML cell lines. Cell growth inhibition was assessed by CCK-8 and/or CellTiter-Glo assays. The Chou-Talalay method was used to calculate the combination index (CI) to test for synergy. Western blotting analysis was performed to explore the potential mechanisms underlying the combination effects. AML xenograft mouse models were used to assess the in vivo efficacy of PHI-101 combination therapy. Statistical comparisons included unpaired t-tests or one-way ANOVA. Combination treatment of PHI-101 with venetoclax or azacytidine showed strong synergistic effects on growth inhibition against both FLT3-ITD mutant (CI<0.3) and wild-type AML cells (CI <0.6). It is noteworthy that the FLT3-ITD homozygous cell line (MV4-11) showed especially strong synergism (CI <0.1). In the xenograft mouse model experiments, 14-day treatment with either PHI-101 alone or in combination with venetoclax or azacytidine showed significant suppression of tumor growth compared to venetoclax or azacytidine alone (in average tumor growth inhibition (TGI) 88.3% vs 8.6%)(p<0.05). In particular, PHI-101 3 mg/kg/day (mpk) with venetoclax 80mpk resulted in a stronger anti-tumor effect (TGI 94.2%) compared to PHI-101 (TGI 87.4%) or venetoclax alone (-9.6% of TGI) (p<0.05) even 21 days after stopping the treatment. In addition, PHI-101 treatment showed a survival advantage in AML animal models, and the combination with venetoclax led to significantly longer survival compared to the control groups. Our preclinical data showed that the combination of PHI-101 with venetoclax or azacytidine resulted in a highly effective anti-leukemic response against AML cells by inducing DNA damage, BCL-2 inhibition, and MCL-1 degradation. Additionally, treatment of mice with low-dose PHI-101 inhibited tumor regrowth and further potentiated venetoclax or azacytidine response in vivo. Our results suggest that the combination of PHI-101 and venetoclax or azacitidine may have the advantage of improving clinical responses in FLT3-ITD AML patients and offers a potential therapeutic option to treat patients with newly diagnosed or relapsed/refractory FLT3-mutant AML. Citation Format: Hee-sun Hwang, Gi-Jun Sung, Kyung-Ah Kim, Ky-Youb Nam, Kyu-Tae Kim, June H. Han, Jeong-Hyeok Yoon, Bao Nguyen, Li Li, Tessa Seale, Donald Small. PHI-101 synergizes with chemotherapy and venetoclax in preclinical models of 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 7153.

Abstract 4752: Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling

Abstract Introduction Relapsed acute lymphoblastic leukemia (ALL) portends a poor prognosis. Glucocorticoids (GCs) are a central component of a multi-agent regimen to treat ALL. GCs mediate ligand-dependent global transcriptional changes activating feedback loop that increases GC receptor (GR) expression and pro-apoptotic signaling. GC resistance is a common mechanism in relapsed/refractory ALL. Altered function of Enhancer of Zeste Homolog 2 (EZH2), a histone methyl transferase (HMT), including increased expression and activating mutations have been reported in ALL. Previously, EZH2 inhibitors have been shown to increase GC efficacy in non-ALL B-cell lymphoid malignancies including non-Hodgkin lymphoma. Furthermore, EZH2 inhibitors reversed GC resistance and increased efficacy of GCs in an ALL subtype with an activating point mutation in an HMT called nuclear set domain 2. Overcoming GC resistance by novel therapeutic strategies may help improve outcomes in relapsed/refractory ALL. Hypothesis EZH2 inhibitors may enhance GC efficacy by augmenting downstream effects of GCs. Methods We performed in-vitro studies of EZH2 inhibitors, EPZ-6438 and CPI-1205, at graded doses in GC resistance human-derived B-cell (SUPB15) and T-cell (SUPT1, KOPTK1, PEER) ALL cell lines followed by graded doses of dexamethasone (dex). Cell viability was performed using CellTiter-Glo assay. Induction of apoptosis was measured with Annexin V flow cytometry. Pro-apoptotic BIM mRNA and protein levels were measured using real-time PCR and immunoblot. Global H3K27me3 protein levels were assessed using immunoblot. Results GC resistant B-cell ALL and T-cell ALL treated with EZH2 inhibitors for 3-7 days followed by the addition of dex for 48-72 hours led to decreased cell viability and increased apoptosis of the ALL cells compared to cells treated with vehicle (DMSO) and dex alone. EZH2 inhibitors on their own did not cause apoptosis. SynergyFinder revealed Bliss synergy scores between 23- 31 in B-cell ALL and T-cell ALL cell lines with greater than 10 considered a synergistic drug interaction. Immunoblotting of cells treated with EZH2 inhibitors alone showed an increase in pro-apoptotic protein, BIM. BIM level was further enhanced when dex was added in combination with EZH2 inhibitors compared to vehicle and dex alone suggesting that EZH2 inhibitors were augmenting BIM expression. Further assessment of the mechanism of how EZH2 inhibitors lead to improved GC efficacy will be performed by integrating transcriptomic and epigenomic profiling in the presence and absence of EZH2 inhibitors. Furthermore, in-vivo testing of EZH2 inhibitors in patient derived xenografts will be performed. Conclusion EZH2 inhibitors can overcome GC resistance in ALL posing as a potential novel therapeutic agent to treat relapsed/refractory ALL. Citation Format: Mansi Dalal, Joanna Yi, Jianping Li, Amin Shobh, Daphne DupereRicher, Richard Bennett, Jonathan Licht. Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling [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 4752.

Abstract 490: Significant anti-proliferative effects of targeted magnetoelectric nanoparticles as potential theragnostic tools

Abstract Although current electroporation therapies have been demonstrated to be effective not only in ablating tumor tissue, but also in eliciting an anti-tumor immune response, they are limited in their application due to the strong risk factors associated with the high voltages required. Current methods of interacting with the body’s electrical currents are limited to traditional electrodes, which cause damage to surrounding tissue upon insertion, and require high voltages to create responses at the cellular level. Recent efforts at non-invasive interaction with the body’s electric fields have led to the fabrication of magnetoelectric nanoparticles (MENPs), a novel technology which has the ability to convert harmless magnetic fields into strong local electric fields, all while maintaining a diameter of ~70nm. These particles have been demonstrated to trigger neuronal firing under magnetic stimulation both in vitro and in vivo, and have been able to release chemotherapeutic agents on command in vitro and in vivo. This demonstrates the ability for MENPs to wirelessly generate electric fields within the body on demand with high temporal and spatial resolution. This suggests the potential for non-invasive electroporation therapy which could be triggered by external magnetic fields. Given the particles are themselves magnetic, this could show potential as a theragnostic tool as their magnetic properties suggest they may act simultaneously as an MRI contrast and an ablative therapy. To explore this possibility, we fabricated MENPs consisting of a magnetostrictive CoFe2O4 spinel core coupled with a piezoelectric BaTiO3 perovskite shell. These particles were then PEGylated to reduce agglomeration which is typical of magnetic nanoparticles. 50ul of a 1mg/ml PEG-MENP dispersion was deposited into a cell culture of SKOV-3 ovarian cancer cells. These cells were then stimulated by 120 10ms pulses of a 2kOe magnetic field over the course of 30 minutes. An cell-titre glo ATP assay was performed to indicate viability 24 hours after treatment. Treatment wells were compared to controls of no intervention, magnetic stimulation with no particles, and particles with no stimulation. The combination of particles and magnetic stimulation was the only group which showed significant decreases in viability, with a 32% reduction in viability. Particles on their own showed no effect on cell viability, neither did magnetic-only stimulation. This preliminary investigation into the use of MENPs as a non-invasive cancer treatment has shown some potential. The ability to wirelessly activate the particles provides an inherent targeting mechanism not found in traditional chemotherapeutics, while the nanometer diameter provides a non-invasive method to generate strong electric fields within the body. Citation Format: Max Shotbolt, Elric Zhang, Emily Zhu, Wael El-Rifai, John Bryant, Ping Liang, Sakhrat Khizroev. Significant anti-proliferative effects of targeted magnetoelectric nanoparticles as potential theragnostic tools [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 490.