Cell Viability In Response Research Articles

Icariin alleviates cellular injury induced by cardiac ischemia-reperfusion injury by inhibiting IRE1/JNK-induced ferroptosis

BackgroundIschemia-induced cellular damage and stress responses significantly impact cellular viability and function. Icariin (ICA), known for its protective effects, has been studied to understand its role in mitigating oxygen-glucose deprivation/reperfusion (OGD/R)-induced endoplasmic reticulum (ER) stress and ferroptosis in H9C2 cardiomyoblast cells. MethodsWe employed an in vitro OGD/R model using H9C2 cells. ICA's effects were analyzed across multiple concentrations. Key indicators of ER stress, autophagy, and ferroptosis—including markers like Bip, PERK, IRE1, ATF6, P62, FTH1, LC3II/LC3I, and NCOA4—were assessed using Western blotting, electron microscopy, and biochemical assays. Additionally, the role of the IRE1/JNK pathway in mitochondrial dynamics and its influence on mitochondrial dynamics protein was explored through specific inhibition and activation experiments. ResultsICA significantly reduced the activation of UPR pathways, decreased autophagic vacuole formation, and maintained cell viability in response to OGD/R and Erastin-induced ferroptosis. These protective effects were associated with modulated autophagic processes, reduced lipid peroxidation, and decreased ferrous ion accumulation. Inhibition of the IRE1/JNK pathway and subsequent Drp1 activity demonstrated reduced mitochondrial recruitment and mitophagy, correlating with decreased ferroptosis markers and improved cell survival. ConclusionOur findings highlight ICA's potential in modulating IRE1/JNK pathway, autophagy, providing a therapeutic avenue for mitigating ferroptosis in myocardial ischemia-reperfusion injury (MIRI).

Rosmarinic Acid Potentiates Cytotoxicity of Cisplatin against Colorectal Cancer Cells by Enhancing Apoptotic and Ferroptosis.

Rosmarinic acid (RA) has demonstrated anticancer effects on several types of malignancies. However, whether RA promotes the anticancer effect of cisplatin on colorectal cancer cells remains sketchy. This study aimed to explore whether RA potentiates the cytotoxicity of cisplatin against colon cancer cells and the underlying mechanism. Cell viability, cell cycle progression, and apoptosis was evaluated using sulforhodamine B (SRB) assay, flow cytometric analysis, and propidium iodide/Annexin V staining, respectively. Western blotting was utilized to analyze signaling pathways. Our findings showed that RA significantly enhanced the inhibitory effect on cell viability and the induction of apoptosis on the colon cancer cell lines DLD-1 and LoVo. Signaling cascade analysis revealed that the combination of RA and cisplatin jointly induced Bax and caspase activation while downregulating Bcl-2, glutathione peroxidase 4 (GPX4), and SLC7A11 in DLD-1 cells. Moreover, caspase inhibitor and ferroptosis inhibitor significantly reversed the inhibition of cell viability in response to RA combined with cisplatin. Collectively, these findings demonstrate that RA enhances the cytotoxicity of cisplatin against colon cancer cells, attributing to the promotion of apoptosis and ferroptosis.

Annexin-A1 tripeptide enhances functional recovery and mitigates brain damage in traumatic brain injury by inhibiting neuroinflammation and preventing ANXA1 nuclear translocation in mice.

This study explores the role and mechanism of Annexin-A1 Tripeptide (ANXA1sp) in mitigating neuronal damage and promoting functional recovery in a mouse model of traumatic brain injury (TBI). Our goal is to identify ANXA1sp as a potential therapeutic drug candidate for TBI treatment.Adult male C57BL/6J mice were subjected to controlled cortical impact (CCI) to simulate TBI, supplemented by an in vitro model of glutamate-induced TBI in HT22 cells. We assessed neurological deficits using the Modified Neurological Severity Score (mNSS), tested sensorimotor functions with beam balance and rotarod tests, and evaluated cognitive performance via the Morris water maze. Neuronal damage was quantified using Nissl and TUNEL staining, while microglial activation and inflammatory responses were measured through immunostaining, quantitative PCR (qPCR), Western blotting, and ELISA. Additionally, we evaluated cell viability in response to glutamate toxicity using the Cell Counting Kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) release.Intraperitoneal administration of ANXA1sp significantly enhanced neurological outcomes, markedly reducing sensorimotor and cognitive impairments caused by TBI. This treatment resulted in a significant reduction in lesion volume and decreased neuronal cell death in the ipsilateral cortex. Moreover, ANXA1sp effectively diminished microglial activation around the brain lesion and decreased the levels of pro-inflammatory markers such as IL-6, IL-1β, TNF-α, and TGF-β in the cortex, indicating a significant reduction in neuroinflammation post-TBI. ANXA1sp also offered protection against neuronal cell death induced by glutamate toxicity, primarily by inhibiting the nuclear translocation of ANXA1, highlighting its potential as a neuroprotective strategy in TBI management.Administration of ANXA1sp significantly reduced neuroinflammation and neuronal cell death, primarily by blocking the nuclear translocation of ANXA1. This treatment substantially reduced brain damage and improved neurological functional recovery after TBI. Consequently, ANXA1sp stands out as a promising neuroprotective agent for TBI therapy.

The Incorporation of Chitosan in the Antibacterial Capability and Biocompatibility of a Protein-Repellent Orthodontic Cement.

This study aimed to develop an orthodontic cement containing chitosan and 2-methacryloyloxyethyl phosphorylcholine (MPC) and to investigate its antibacterial properties and biocompatibility. Chitosan and MPC were incorporated into commercial cement. The enamel bonding strength and biocompatibility of the new cement were evaluated. The antibacterial properties were assessed by examining biofilm metabolic activity and colony-forming units (CFU). An evaluation of the protein repellency of the cement was also conducted. The new cement containing chitosan and MPC had clinically acceptable bonding strength. In comparison to the control, the novel cement demonstrated enhanced protein-repellent properties (p < 0.05), inhibited biofilm metabolic activity (p < 0.05), and reduced CFU counts (p < 0.05) without diminishing cell viability in response to cement extracts (p > 0.05). The synergistic application of chitosan and MPC endows the cement with potent antibacterial abilities, protein repellency, and favorable biocompatibility.

HEBP2 affects sensitivity to cisplatin and BCNU but not to paclitaxel in MDA-MB-231 breast cancer cells.

Breast cancer has the highest incidence of all cancer types in women. Triple-negative breast cancer (TNBC) accounts for 15% of all breast cancer cases and is the most aggressive type, with a poor prognosis and limited treatment. Treatment failure in patients is largely due to resistance to chemotherapy. In this study, we aimed to identify the novel factors contributing to chemoresistance in TNBC using cisplatin and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). We found that transactivation of the heme-binding protein 2 (HEBP2) gene was common in surviving colonies of cells after exposure to two types of chemotherapeutic agents, namely cisplatin and BCNU, from genome-scale transcriptional activation library screening in the TNBC cell line MDA-MB-231. Analysis of a public database (Proteogenomic Landscape of Breast Cancer, CPTAC) indicated that HEBP2 mRNA expression was elevated in TNBC tissues compared to that in non-TNBC tissues. HEBP2 facilitates necrotic cell death under oxidative stress; however, it is not yet known whether HEBP2 affects cancer cell survival following chemotherapy. Therefore, we investigated the effects of HEBP2 expression on the sensitivity to cisplatin and BCNU in MDA-MB-231 cells. Overexpression of HEBP2 significantly enhanced the viability of MDA-MB-231 cells in response to cisplatin and BCNU, but not methyl methanesulfonate (MMS) and paclitaxel. In contrast, CRISPR/Cas9-mediated HEBP2-knockout greatly reduced cell viability in response to cisplatin and BCNU, but not to MMS and paclitaxel, in MDA-MB-231 cells. Moreover, the exogenous introduction of HEBP2 restored the resistance of HEBP2-deficient cells to cisplatin and BCNU to wild-type levels. These findings suggest that HEBP2 may play a significant role in resistance to cisplatin and BCNU, which induce intrastrand and interstrand DNA crosslinks, but not to monoalkylating or microtubule-stabilizing agents in TNBC cells. The possibility exists that HEBP2 serves as a biomarker for predicting response or a therapeutic target for overcoming resistance to platinum-based and alkylating anticancer agents in TNBC.

Mechanisms of mammalian orthoreovirus-induced cell death and NF-kB activation in ER+ breast cancer.

e13171 Background: Estrogen receptor positive (ER+) breast cancer is one of the most commonly diagnosed types of breast cancer in the United States. Although the five-year survival rate of ER+ breast cancer is 90% or higher, ER+ breast cancer can recur and metastasize 10-20+ years after initial diagnosis. Novel therapeutic strategies are needed to prevent and treat breast cancer recurrence and metastasis. A possible solution is mammalian orthoreovirus (MRV), a replication-competent natural oncolytic virus. The strain T3D (Reolysin) is in current clinical trials to treat metastatic breast cancer; however, the mechanisms of MRV-induced death are not well elucidated. Furthermore, other MRV strains have not been well studied for their oncolytic potential in ER+ breast cancer. Our studies aim to explore the different cell death pathways elicited by MRV. Prior studies have reported that MRV induces cell death in L929 cells (murine fibroblasts, permissive to MRV infection) by activating apoptosis, necroptosis, and inhibiting the NFκB pathway to enhance viral replication. Methods: Our goal is to compare the effects of MRV in L929 cells and ER+ breast cancer cell line MCF7 using chemical inhibitors of necroptosis, apoptosis, and the NFκB pathway. Results: Our results indicate that necroptosis inhibitors do not impact cell viability in response to MRV infection, but apoptosis inhibitors increase cell viability, suggesting that MRV-induces apoptotic cell death in ER+ breast cancer cells. Previous work has shown that the NFκB pathway may be activated or inhibited by MRV. Our cell viability studies in MCF7 cells with the IKK inhibitor BAY-11-7082 indicate that several MRV strains likely activate the NFκB pathway to potentially enhance viral replication. Conclusions: Future studies are aimed at further defining mechanisms of MRV-induced cell death in ER+ breast cancer. We anticipate future studies will lead to an improved MRV virotherapeutic that may be combined with clinically relevant treatments, such as CDK4/6 inhibitors and anti-estrogens, to prevent and treat therapy-resistant ER+ breast cancer.

Establishing mesothelioma patient-derived organoid models from malignant pleural effusions

ObjectivesPleural mesothelioma is a cancer arising in the cells that line the lungs and chest wall with poor survival and poor response to first-line therapy. Organoid models of cancer can faithfully recapitulate the genetic and histopathological characteristics of individualized tumors and have potential to be used for precision medicine, however methods of establishing patient-derived mesothelioma organoids have not been well established in the published literature. Materials and methodsLong-term mesothelioma patient-derived organoids were established from ten malignant pleural effusion fluids. Mesothelioma patient-derived organoids were compared to the corresponding biopsy tissue specimens using immunohistochemistry labelling for select diagnostic markers and the TruSight Oncology-500 sequencing assay. Cell viability in response to the chemotherapeutic drug cisplatin was assessed. ResultsWe established five mesothelioma patient-derived organoid cultures from ten malignant pleural effusion fluids collected from nine individuals with pleural mesothelioma. Mesothelioma patient-derived organoids typically reflected the histopathological and genomic features of patients’ matched biopsy specimens and displayed cytotoxic sensitivity to cisplatin in vitro. ConclusionThis is the first study of its kind to establish long-term mesothelioma organoid cultures from malignant pleural effusions and report on their utility to test individuals’ chemotherapeutic sensitivities ex vivo.

Abstract 1085: Exploiting bioenergetic vulnerabilities in chemotherapy resistant osteosarcoma

Abstract BACKGROUND: Osteosarcoma (OS) is the most common bone cancer in children and young adults. Standard treatment involves chemotherapy and surgical resection, yet nearly 4 in 10 patients experience disease relapse, and almost all of these patients are resistant to additional chemotherapy. Therefore, there is an urgent need to understand the biology of chemo-resistant OS and identify new therapeutic strategies to treat this therapy-resistant disease state. METHODS: Using a CRISPR-Cas9 based lineage tracing system, RNA sequencing, and Seahorse XF assays in chemo-naïve, -resistant, and -released human 143b and patient-derived 173x OS cell lines, we modeled the evolution of chemoresistance and release from the selective pressure of chemotherapy in osteosarcoma. The phenotype of each cell state was assessed through in vitro and ex vivo cell proliferation assays, a comparison of cell viability upon re-challenge to standard-of-care chemotherapy, and comparison of cell viability in response to novel therapies targeting alterations in cell respiration. RESULTS: Chemo-resistant OS cells demonstrate a decrease in expression of proliferation-associated gene pathways and shift their resources toward expression and maintenance of multi-drug resistance ABC transporters. Chemo-released cells maintain elevated expression of ABC transporters while re-activating proliferation. Across the spectrum of chemotherapy exposure, OS cells demonstrate changes in respiratory capacity, with unique bioenergetic phenotypes observed in chemo-naïve, -resistant, and -released cells. CONCLUSIONS: Chemo-resistant and chemo-released OS cells demonstrate unique bioenergetic patterns that correlate with gene expression signatures and growth phenotypes. Evolution imposed by the selective pressure of chemotherapy may render OS cells vulnerable to therapies that disrupt cellular energetics and exploit multi-drug resistant ABC transporters. Citation Format: Valerie U. Nguyen, Laurie Graves, Veronica Colmenares, Abol Macabangon, Casey Syal, Kelsey Fisher-Wellman, William C. Eward, So Young Kim, Jason A. Somarelli. Exploiting bioenergetic vulnerabilities in chemotherapy resistant osteosarcoma [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 1085.

Anti-cancer agent piperlongumine is an inhibitor of transient receptor potential melastatin 7 channel in oral squamous cell carcinoma

ObjectivesTo elucidate the association between the anticancer activities of piperlongumine (PL) and its potential target, transient receptor potential melastatin 7 channel (TRPM7), in oral squamous cell carcinoma (OSCC). MethodsThe expression levels and electrical characteristics of TRPM7 as well as cell viability in response to various PL treatments were investigated in the OSCC cell line Cal27. ResultsPL treatment resulted in a concentration- and time-dependent reduction in TRPM7 mRNA and protein expression in Cal27 cells. Furthermore, PL treatment inhibited TRPM7-like rectifying currents in Cal27 cells; however, this inhibition was less effective than that of the TRPM7 antagonist waixenicin A. Rapid perfusion and washout experiments revealed an immediate inhibitory effect of PL on TRPM7-like currents. The antagonistic effect of PL occurred within 1 min and was not completely reversed following washout. Notably, the extracellular Ca2+ concentration still influenced PL-induced changes in the TRPM7-like current, indicating that PL can directly but gently antagonize the TRPM7 channel. Functional changes in TRPM7 correlated with the observed antiproliferative and cytotoxic effects of PL in Cal27 cells. ConclusionsThese findings suggest that PL exhibits potent inhibitory effects on TRPM7 and exerts its anti-cancer effects by downregulating TRPM7 expression and antagonizing channel currents.

Abstract C093: Unlocking the potential of Metformin: Revealing its impact on colorectal cancer cells at a molecular level

Abstract Metformin is a drug commonly used to treat type-2 diabetes, but recent studies suggest that it may also have anti-cancer effects by influencing cell cycle arrest and apoptosis. While previous studies have focused on metformin's effect on mTor, the downstream genetic pathways underlying its anti-proliferation mechanism remain unclear. Identifying these pathways may help predict patient response and allow for tailored treatment plans. This study aims to investigate the early genetic pathways involved in colon cancer using an in-vitro cell model and assess the impact of metformin on cell cycle and cancer proliferation in colon cancer cells, taking into account the p53/p21 status. Four different metformin concentrations were used on the HCT116 colon cancer cell line and its genetic modified forms. To determine cell viability and to measure cell cycle and apoptosis, MTT assay and flow cytometer were used. RNA-seq was performed on HCT116 after 24 hours of metformin treatment and pathway analysis was conducted using the Pathway Studio software. Results showed a significant decline in cell viability in response to metformin treatment, with an increase in the apoptosis phase. Transcriptomic analysis revealed that over 3,000 genes were significantly down-regulated, while 1,200 genes were up-regulated. Metformin promotes cell death and fragmentation at the SubG0 level, particularly in the case of p21 null, but p53 likely plays a primary role in driving apoptosis in colorectal cancer cells. In conclusion, metformin has potential in inhibiting proliferation and inducing apoptosis in colorectal cancer cells, affecting molecular pathways involved in the disease. However, further investigation through next-generation sequencing and molecular targeting is necessary to fully understand the disease at different levels. Citation Format: Jamila Hijazi, Pierre Khoueiry, Assaad Eid, Nadine Darwiche, Georges Nemer. Unlocking the potential of Metformin: Revealing its impact on colorectal cancer cells at a molecular level [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C093.

Class IIa HDACs inhibit cell death pathways and protect muscle integrity in response to lipotoxicity

Lipotoxicity, the accumulation of lipids in non-adipose tissues, alters the metabolic transcriptome and mitochondrial metabolism in skeletal muscle. The mechanisms involved remain poorly understood. Here we show that lipotoxicity increased histone deacetylase 4 (HDAC4) and histone deacetylase 5 (HDAC5), which reduced the expression of metabolic genes and oxidative metabolism in skeletal muscle, resulting in increased non-oxidative glucose metabolism. This metabolic reprogramming was also associated with impaired apoptosis and ferroptosis responses, and preserved muscle cell viability in response to lipotoxicity. Mechanistically, increased HDAC4 and 5 decreased acetylation of p53 at K120, a modification required for transcriptional activation of apoptosis. Redox drivers of ferroptosis derived from oxidative metabolism were also reduced. The relevance of this pathway was demonstrated by overexpression of loss-of-function HDAC4 and HDAC5 mutants in skeletal muscle of obese db/db mice, which enhanced oxidative metabolic capacity, increased apoptosis and ferroptosis and reduced muscle mass. This study identifies HDAC4 and HDAC5 as repressors of skeletal muscle oxidative metabolism, which is linked to inhibition of cell death pathways and preservation of muscle integrity in response to lipotoxicity.

Exploring the Mechanisms of Venetoclax Resistance Via Drug Screening and Genome-Wide CRISPR Screening

Introduction: Combining Venetoclax, a selective BCL-2 inhibitor, with hypomethylating agents has revolutionized frontline acute myeloid leukemia (AML) treatment. Despite its success, the occurrence of resistance remains a significant concern, limiting the range of available treatment alternatives. Little is known about the mechanism by which the cells escape the treatment. Recent studies have observed MCL1 upregulation in AML cells to promote resistance, and the rationale to use an MCL1 inhibitor became attractive. Although MCL1 inhibitors work exceptionally well in vitro their clinical trials were unsuccessful due to their toxicity. Here, we aim to identify other genes responsible for acquiring resistance and tackle the problem through screening for clinically approved drugs to rapidly benefit the patients with our study. Methods: First, we generated a model of the venetoclax resistance from 3 AML cell lines: MOLM-13, HL-60 and MV4-11. Resistance was achieved through chronic administration of the compound, inhibiting cell viability to 80-90% in multiple rounds by gradually increasing concentrations until the cells developed resistance. We have then characterized these Venetoclax-resistant (VeR) cells using quantitative RT-PCR and RNA sequencing to reveal the mechanisms behind their resistance. VeR cell lines were subjected to drug screening with a library of 859 FDA and EMA-approved compounds with various clinical indications. Cell viability in response to the drug library was assessed after 72h by Cell-Titer Glo and evaluated against the wild-type counterparts. Top-performing compounds were validated in dose-response curves, and synergy scores were identified from combinational treatments. Simultaneously, we performed genome-wide CRISPR/Cas9 knockout screening using Brunello CRISPR knockout library on both MOLM-13 wild-type (WT) and VeR cell lines to identify genes that maintain the resistance to venetoclax and genes that may potentially synergize with targeting BCL-2. Results: Results from the CRISPR screen pointed out multiple genes whose loss of function may contribute to venetoclax resistance in MOLM-13 cells, among which BAX, NOXA, ELAVL1 and TP53 ranked as top hits. On the contrary, we also identified several genes whose loss of function sensitized the cells to venetoclax (e.g., MCL1, OPA1, CDK2, MCAT), suggesting them to be promising therapeutic targets. Among these hits, CDK2 correlates with our drug screening data indicating Flavopiridol as an effective compound in treating VeR cell lines. Moreover, we confirmed the elevated MCL1 gene expression for the MOLM-13 VeR cell line through RNAseq (and qPCR) and detected an altered expression of genes mainly in metabolic pathways and PI3K-Akt signaling pathway via differential gene expression analysis. Our drug screening results revealed overall high efficacy of DNA-damaging agents, proteasome and HDAC inhibitors for both WT and VeR cell lines. We also included an MCL1 inhibitor as a positive control in our drug library and confirmed a synergistic effect when combined with venetoclax. Conclusion: Integrating a range of screening approaches may lead to discovering previously unknown therapeutic targets for venetoclax-resistant AML. Our screens confirmed previously described involvement of the apoptotic pathway genes in venetoclax resistance as well as identified some novel promising targets. Our ongoing efforts involve thorough validations of the identified hits from CRISPR and drug screening to enhance the reliability and translatability of the results. This project was partly supported by grants MUNI/A/1330/2021, MUNI/A/1419/2021 OPRDE (No.CZ.02.2.69/0.0/0.0/19_073/0016943) and project NICR (EU program EXCELES, No. LX22NPO5102).

DDDR-21. A TEMOZOLOMIDE-INDUCED LONG NONCODING RNA, LINC02454, REGULATES CHEMOSENSITIVITY IN GLIOBLASTOMA VIA MODULATION OF CXCR4

Abstract INTRODUCTION Chemoresistance is one of the primary factors influencing the poor prognosis in glioblastoma (GBM) patients. Long noncoding RNAs (lncRNAs) are a novel class of RNAs representing up to 80% of the genome, and noted to induce profound alterations in transcriptional regulation and phenotype, including chemotherapy response. We therefore set out to identify novel lncRNAs induced by chemotherapy that regulate GBM chemosensitivity. We further evaluated the mechanism by which a candidate chemoresistance-associated lncRNA exerts function. METHODS We evaluated gene expression following temozolomide treatment in multiple glioma lines (via RNA-seq). Candidate lncRNAs demonstrating significant expression upon TMZ treatment were assessed for association with patient survival. We further evaluated the effect of knockdown of candidate lncRNAs upon resulting TMZ sensitivity and downstream gene expression. RESULTS Linc02454 was among the most highly upregulated genes following TMZ treatment. Linc02454 resides on chromosome 12q14 within a locus of genes amplified in 10% of GBM patients. Elevated linc02454 expression was associated with decreased survival in TCGA data, while linc02454 knockdown reduced in vitro GBM cell viability in response to TMZ. RNA-seq identified CXC-chemokine receptor 4 (CXCR4) among the most heavily downregulated genes following lncRNA knockdown. Finally, TMZ induces similar increases in linc02454 and CXCR4, while CXCR4 KD chemosensitivity phenotypes closely parallel linc02454 KD phenotypes. CONCLUSION TMZ-induced increases in long noncoding RNAs, particularly in linc02454, regulate expression of well-known and previously targeted chemokines, including CXCR4. Subsequent studies, evaluating the manner by which chemotherapy increases linc02454, and the manner by which linc02454 affects CXCR4 and treatment sensitivity may improve GBM temozolomide sensitivity.

The Combination of Lanraplenib, a Selective SYK Inhibitor, and Gilteritinib, a FLT3 Inhibitor, Targets Aberrant Proliferation and Differentiation Blockade in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is an aggressive disease characterized by uncontrolled clonal proliferation of poorly differentiated myeloid progenitor cells in the bone marrow and blood. Deregulated spleen tyrosine kinase (SYK) impairs myeloid differentiation and has been implicated in the pathogenesis of AML driven by FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutation (FLT3-ITD). While SYK is rarely mutated, it is highly activated in FLT3-ITD-mutated AML and directly phosphorylates FLT3-ITD, resulting in aberrant expression of multiple oncogenic pathways. Lanraplenib (LANRA) is a selective, next-generation, SYK inhibitor that has shown favorable PK, PD, and safety in healthy volunteers and patients with autoimmune diseases. LANRA is currently being evaluated in combination with gilteritinib, a FLT3 inhibitor, in patients with relapsed or refractory (R/R) FLT3-mutated AML (NCT05028751). By jointly inhibiting the leukemogenic network created by SYK and mutant FLT3, LANRA and gilteritinib potentially provide a new treatment regimen in a patient population with few treatment options. Method: AML cell viability in response to LANRA, gilteritinib or their combination was assessed by CellTiter Glo (CTG) at 5 days. Apoptosis was evaluated by measuring Annexin V and propidium iodide staining by flow cytometry. Myeloid differentiation was assessed by flow cytometry using CD11b and CD14 cell surface markers and/or immunohistochemistry. In addition, RNA-seq was performed to evaluate differential changes in gene expression in response to LANRA. Gene set enrichment analysis (GSEA) was performed to evaluate perturbation in leukemogenic signaling pathways. In vivo studies were conducted using the MV4;11 cell line-derived xenograft model. LANRA was administered twice daily at a dose of 75 mg/kg via subcutaneous injections, either alone or in combination with gilteritinib at a dose of 2 mg/kg dosed once daily by oral gavage, for 28 days. Results: Treatment of FLT3-ITD mutated AML cells with LANRA results in dose- and time-dependent increase in myeloid differentiation and reductions in leukemic cell viability. RNA-seq analysis of MV4;11 cells revealed that LANRA abrogates multiple downstream FLT3-ITD leukemogenic signaling pathways including JAK/STAT3/5, MAPK, mTOR and OXPHOS. Combined pharmacologic inhibition of SYK with LANRA and FLT3-ITD with gilteritinib results in robust antileukemic effects in preclinical models of FLT3 ITD-driven AML. Additionally, the combination of LANRA and gilteritinib demonstrated increased differentiation and apoptosis induction in vitro. LANRA in combination with gilteritinib showed significant tumor growth inhibition compared to either single agent in a subcutaneous FLT3-ITD AML cell line-derived xenograft mouse model. In line with these observations, a retrospective analysis of bone marrow-engrafted AML cells from a disseminated FLT3-ITD AML patient-derived xenograft mouse model showed evidence of increased myeloid differentiation and inhibition of cell proliferation. Conclusion: In preclinical models, LANRA demonstrates compelling anti-leukemic activity in combination with gilteritinib. By jointly targeting hyperproliferation and differentiation blockade, LANRA and gilteritinib may provide a new therapeutic approach that can be exploited in AML. Additionally, LANRA disrupts downstream signaling of key pathways often associated with resistance to gilteritinib, providing additional mechanistic support for continued testing of LANRA in combination with gilteritinib in FLT3-ITD-mutated AML patients. These studies helped motivate the ongoing clinical combination with gilteritinib in patients with (R/R) FLT3-mutated AML (NCT05028751).

Targeting VEGF/Neuropilin-2 as a Novel Approach to Induce Radiosensitivity in Triple Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive phenotype with patients having more limited survival compared with other breast cancer subtypes. The high rate of locoregional/distant recurrence after primary management implies that additional treatment is necessary to optimize the sensitivity of TNBC tumors to therapy including radiation. Our lab studies the role of VEGF/Neuropilin-2 (NRP2) in mediating cancer stem cell properties such as self-renewal and therapy resistance. We sought to identify a new therapeutic approach that can induce radiosensitization in TNBC. Cell viability in response to radiotherapy in patient-derived organoids (PDO) and xenografts (PDX) was determined using CellTiter-Glo. The VEGF/NRP2 pathway was disrupted in human BT549 and murine 4T1 models using RNA interference and a function blocking antibody (aNRP2-10) provided by aTyr Pharma. Nitrite levels, a surrogate marker for NOS2 activity, were measured using Measure-IT Nitrite Assay Kit. H2DCFDA was used to measure intracellular reactive oxygen species (ROS). Clonogenic survival assays were used to calculate radiosensitivity enhancement ratios (rER) in TNBC cell lines. For our in vivo models, 4T1 xenografts were given antibody treatment every 48 hours starting a day prior to 10Gy irradiation. We identified a novel radioresistant population expressing high levels of NRP2 and NOS2 and observed that NOS2 expression was dependent on VEGF/NRP2 via Gli1 transcription. Interestingly, reducing NRP2 expression increased radiation-induced ROS because of reduced NOS2/NO levels. The downstream consequence of higher NOS2/NO was increased Nrf2 activation via KEAP1 S-nitrosylation; thus, inducing expression of antioxidant response elements. TNBC cells were radiosensitized by knocking down NRP2 (rER 1.19 - 1.34). We saw similar results when using aNRP2-10 vs IgG (rER 1.30 - 1.37). Ectopic expression of NOS2 and constitutively active Nrf2 rescued the radioresistance phenotype in the NRP2 knock-down cells (rER 0.73 - 0.82). Consistent with the cell line data, the clinically relevant models (PDOs and PDXs) had a synergistic reduction in cell viability in response to aNRP2-10 prior to radiotherapy. We also observed a significant reduction in tumor volume and increase in necrosis in vivo in response to VEGF/NRP2 inhibition during radiotherapy. Inhibiting VEGF/NRP2 induces radiosensitization of TNBC by decreasing NOS2/NO levels and, consequently, Nrf2-mediated antioxidant gene expression. The role of Gli1 as a transcription factor for NOS2 is a novel finding that provides an unexpected link between VEGF/NRP2 and redox homeostasis in response to radiation. Current Nrf2 inhibitors are non-specific and have high toxicity; thus, making VEGF/NRP2 inhibition more favorable. Our data provide a novel therapeutic strategy for targeting TNBC breast cancer.

MiR-1-3p Inhibits Osteosarcoma Cell Proliferation and Cell Cycle Progression While Promoting Cell Apoptosis by Targeting CDK14 to Inactivate Wnt/Beta-Catenin Signaling.

Osteosarcoma (OS) is a common bone malignancy and is diagnosed frequently in children and young adults. According to previous RNA sequencing, miR-1-3p is downregulated in OS clinical samples. Nevertheless, the functions of miR-1-3p in OS cell process and the related mechanism have not been revealed yet. In the current study,miR-1-3p expression in OS tissues and cells were evaluated using quantitative polymerase chain reaction. CCK-8 assays were conducted to measure OS cell viability in response to miR-1-3p overexpression. Colony forming assays and EdU staining were conducted for measurement of cell proliferation, and flow cytometry analysis was performed to determine cell apoptosis and cell cycle progression. Protein levels of apoptotic markers, beta-catenin, and Wnt downstream targets were quantified using western blotting. The binding relation between miR-1-3p and cyclin dependent kinase 14 (CDK14) was validated utilizing luciferase reporter assays. Experimental results revealed thatmiR-1-3p expression was decreased in OS tissues and cells. Additionally,miR-1-3p inhibited cell proliferation and cell cycle progression while enhancing OS cell apoptosis. Moreover, miR-1-3p directly targeted CDK14 and inversely regulated CDK14 expression in OS cells. Furthermore, miR-1-3p inactivated the Wnt/beta-catenin signaling. CDK14 overexpression partially rescued the inhibitory impact of miR-1-3p on OS cell growth. Overall,miR-1-3p inhibits OS cell proliferation and cell cycle progression while promoting cell apoptosis by targeting CDK14 and inactivating the Wnt/beta-catenin signaling.

CD317 maintains proteostasis and cell survival in response to proteasome inhibitors by targeting calnexin for RACK1-mediated autophagic degradation

Unbalanced protein homeostasis (proteostasis) networks are frequently linked to tumorigenesis, making cancer cells more susceptible to treatments that target proteostasis regulators. Proteasome inhibition is the first licensed proteostasis-targeting therapeutic strategy, and has been proven effective in hematological malignancy patients. However, drug resistance almost inevitably develops, pressing for a better understanding of the mechanisms that preserve proteostasis in tumor cells. Here we report that CD317, a tumor-targeting antigen with a unique topology, was upregulated in hematological malignancies and preserved proteostasis and cell viability in response to proteasome inhibitors (PIs). Knocking down CD317 lowered Ca2+ levels in the endoplasmic reticulum (ER), promoting PIs-induced proteostasis failure and cell death. Mechanistically, CD317 interacted with calnexin (CNX), an ER chaperone protein that limits calcium refilling via the Ca2+ pump SERCA, thereby subjecting CNX to RACK1-mediated autophagic degradation. As a result, CD317 decreased the level of CNX protein, coordinating Ca2+ uptake and thus favoring protein folding and quality control in the ER lumen. Our findings reveal a previously unrecognized role of CD317 in proteostasis control and imply that CD317 could be a promising target for resolving PIs resistance in the clinic.

Abstract 1370: Overexpression of the small GTPase Rab27B regulates autophagy flux and contributes to tumor growth in colorectal cancer

Abstract Rab27B is a member of the Rab small GTPases that belong to the RAS-like GTPase superfamily. Rab27B has been shown to regulate endosomal trafficking, exosome secretion, and delivery of secretory granules. However, the role of Rab27B in colorectal cancer (CRC) tumor biology is unknown. Using CRC tissue microarrays, a significant upregulation of Rab27B was detected in staged adenocarcinoma compared to normal colon tissue. Interestingly, microsatellite unstable (MSI) tumors showed a higher increase in Rab27B (2.4-fold) than microsatellite stable (MSS) tumors (1.6-fold), consistent with TCGA data. To study the role of Rab27B in CRC, we targeted Rab27B in MSI HCT116 cell line by CRISPR-Cas9 deletion and siRNA knockdown. In both cases, loss of Rab27B resulted in a dysregulation of cellular autophagy and reduction in extracellular vesicle secretion. Rab27B-depleted cells showed an abnormal accumulation of autophagy vesicles and increase in autophagy markers LC3-II and p62, indicating a defect in autophagy flux. To characterize this autophagy defect, we used eGFP-mCherry-LC3 reporter, lysotracker and Cyto-ID staining to identify that autophagy flux is blocked at the autophagosome/lysosome fusion step when Rab27B is lost. To test whether the GTPase activity of Rab27B is needed for the autophagy role, we added back constitutively active and dominant negative GTPase mutants of Rab27B in Rab27B KO cells and observed autophagy phenotype rescue with only wildtype and constitutively active GTPase forms. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that this defect in autophagy flux resulting from Rab27B deletion will impact cellular stress response and reduce tumor growth. While Rab27B knockout (KO) cells show similar in vitro 2D-growth characteristics as wildtype cells under normal conditions, loss of Rab27B resulted in a 60% reduction in cell viability in response to starvation conditions. Similarly, a 94% reduction in colony formation was observed when grown in soft agar, along with deficiencies in spheroid formation. In vivo, Rab27B KO cells showed a major reduction (p&amp;lt;0.0001) in tumor growth xenografts compared to wildtype HCT116 cells. Taken together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Futures studies will focus on further characterizing the relationship among Rab27B overexpression, microsatellite instability, and autophagy in vitro and in vivo using Rab27B knockout mouse model, as well as investigating whether Rab27B can be targeted as a potential new therapeutic strategy. Citation Format: Sahida Afroz, Ranjan Preet, Vikalp Vishwakarma, Dan A. Dixon. Overexpression of the small GTPase Rab27B regulates autophagy flux and contributes to tumor growth in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1370.

Abstract 2796: Tumor pharmacokinetics, pharmacodynamics and efficacy analysis of WEE1 inhibitor, zn-c3 in patient-derived xenograft models of glioblastoma

Abstract WEE1 kinase regulates G2/M checkpoint transition via phosphorylation of CDK1 and prevents entry into mitosis. WEE1 inhibition increases genomic instability due to G2/M checkpoint inactivation and can result in mitotic catastrophe as monotherapy or when combined with DNA damaging agents. In this study, we evaluated the efficacy as well as pharmacokinetic and pharmacodynamic properties of ZN-c3, a best-in-class WEE1 inhibitor, in orthotopic patient-derived xenograft (PDX) models of GBM. Mice with intracranial tumors were randomized to 80 mg/kg ZN-c3 PO QD x 2 weeks vs. placebo. ZN-c3 levels in plasma and contrast-enhancing tumor tissue were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Unbound fractions were determined by equilibrium dialysis. Immunohistochemistry was performed to assess levels of pCDK1, phospho-Histone H3 (pHH3), and cleaved caspase 3 (CC3). The median unbound concentrations of ZN-c3 were 315 nmol/L and 15 nmol/kg in plasma and tumor tissue, respectively - higher than the biochemical IC50 of ZN-c3 for inhibition of WEE1 (3.8nM). An increase in pHH3(+) cells (0.40% vs. 6.57%, p=0.03) indicated drug-induced mitotic entry upon ZN-c3 treatment. ZN-c3 was potently cytotoxic across multiple patient-derived xenograft (PDX)-derived cell lines (IC50- 150-10000nM). Time and dose-escalation experiments exhibited target engagement at concentrations lower than IC50 values within one hour of treatment. Basal levels of WEE1 and pCDK1 were correlated with cell viability in response to ZN-c3 across multiple PDX cell lines. Overall, ZN-c3 is well tolerated, achieves pharmacologically-relevant unbound concentrations in GBM PDX models, and is associated with significant checkpoint modulation. Preclinical evaluation of ZN-c3 in combination with radiation therapy and DNA damage response pathway inhibitors is currently underway, which will support future combinatorial strategy for glioblastoma treatment. Citation Format: Shwetal Mehta, An-Chi Tien, Sonam Patel, Tigran Margaryan, Anilkumar Thaghalli Shivanna, Leonel Elena, Jennifer Molloy, William Knight, Ruiheng Hon, Mackenna Elliott, Mariya Stavnichuk, Zorana Opachich, Yu-Wei Chang, Chelsea Montgomery, Sarah Himes, Barbara Hopkins, Artak Tovmasyan, Nader Sanai. Tumor pharmacokinetics, pharmacodynamics and efficacy analysis of WEE1 inhibitor, zn-c3 in patient-derived xenograft models of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2796.

Cytotoxic n-Hexane Fraction of the Egyptian Pteris vittata Functions as Anti-breast Cancer Through Coordinated Actions on Apoptotic and Autophagic Pathways.

We investigated the possible anticancer mechanisms of Pteris vittata [PV] n-hexane extract on MCF-7 [breast cancer cell line]. Cultured cell lines were treated with various concentrations of this extract ± Baf-A1 [autophagic inhibitor]. Cells' viability, apoptotic markers [caspase-7, Bax, and Bcl-2], autophagic markers [light chain 3 [LC-3] and P62/SQSTM1]], and the tumor suppressor P53 and its mRNA were checked by their corresponding methods. Treated cell lines showed significant concentration and time-dependent reductions in cell viability in response to PV-n-hexane extract and also exhibited a concomitant induction of apoptosis [increased chromatin condensation, nuclear fragmentation, and pro-apoptotic Bax, and cleaved caspase-7 levels while decreased Bcl-2 levels] and autophagy [increased autophagosomes vacuoles, and LC3B II levels while decreased P62/SQSTM1 levels]. Moreover, PV-n-hexane extract-treated cells showed significant increases in the P53 and its mRNA levels. The addition of Baf-A1 reversed the PV-n-hexane extract autophagic effects and increased apoptotic cell percentage with a much increase in the cleaved caspase-7 and P53 protein and its mRNA levels. We concluded that the PV-n-hexane extract exhibits cytotoxic effects on the MCF-7 cell line with significant reductions in cell viability and concomitant autophagy and apoptosis induction. Inhibition of autophagy in the PV-treated MCF-7 cells enhances apoptosis via a p35-dependent pathway.