Cyclin-dependent Kinase Research Articles

CKS2 induces autophagy-mediated glutathione metabolic reprogramming to facilitate ferroptosis resistance in colon cancer.

Ferroptosis, a form of cell death characterized by lipid peroxidation, plays a crucial role in tumor suppression, offering novel avenues for cancer therapy. Previous studies have indicated that high levels of cyclin-dependent kinase subunit 2 (CKS2) promote the progression of various cancers. However, the potential interplay between CKS2 and ferroptosis in colon cancer (CC) remains unclear. Bioinformatics and RNA-seq analyses were employed to study genes associated with the ferroptosis signaling pathway. CKS2 expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot (WB). The in vitro and in vivo effects of CKS2 on CC cells were assessed through the CCK-8 assay, colony formation assay, propidium iodide (PI) staining, BODIPY staining, DCFH-DA staining, and animal experiments. Additionally, the impact of CKS2 on autophagy and glutathione (GSH) metabolism was investigated using a transmission electron microscope (TEM), immunofluorescence (IF) assays, WB experiments, and relevant assay kits. CKS2 expression was elevated in CC, indicating a poor clinical outcome. Knockdown of CKS2 significantly enhanced Erastin-induced ferroptosis in CC cells, leading to reduced GSH metabolism. Conversely, CKS2 overexpression produced opposite effects. Mechanistically, CKS2-induced autophagy reinforced GSH metabolism, thereby increasing resistance to ferroptosis in CC cells. Furthermore, inhibiting CKS2 promoted tumor ferroptosis by downregulating GPX4 expression. Additionally, CKS2 knockdown effectively increased sorafenib-induced ferroptosis both in vitro and in vivo. CKS2 suppresses ferroptosis in CC by modulating GSH metabolism in both in vitro and in vivo settings. These findings offer new insights into targeting CKS2 for CC treatment and shed light on the mechanism of ferroptosis in CC.

The deleterious effects of CDK4/6 inhibition on renal recovery post-acute kidney injury.

Acute kidney injury (AKI) is a common clinical problem, and patients who survive AKI have a high risk of chronic kidney disease (CKD). The acute protective effects of Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors in AKI have been examined, there is still relatively little known regarding the impact of acute CDK4/6 inhibition on the chronic sequence of AKI. Therefore, we utilized the CDK4/6 inhibitor Palbociclib to examine the long-term effects of CDK4/6 inhibition in a rodent model of ischemic AKI. Palbociclib (Palb) was administered during the acute stage or post the acute stage of AKI and mice were sacrificed 21 days post injury. We found that Palb could cause renal senescence and renal fibrosis. Furthermore, dasatinib (D) plus quercetin (Q) were used to eliminate senescent cells in ischemic AKI murine model and Palb was administered post the treatment of D + Q. We found that Palb could reverse the senolytic and antifibrotic effects induced by D + Q, which indicates that the profibrotic effect of Palb could be ascribed to its pro-senescent effects. Our results demonstrate that CDK4/6 inhibitors treatment might be deleterious on the chronic sequence of AKI.

Cyclin-dependent kinase 4/6 inhibitors and cardiotoxic events in breast cancer: A pharmacovigilance study based on the FAERS database.

Recent trials have highlighted the cardiotoxicity of ribociclib, a CDK4/6 inhibitor, particularly its association with QT prolongation. However, studies on the link between CDK4/6 inhibitors and cardiotoxic events show inconsistent results, and the factors influencing these events and related drug interactions remain underexplored. To address these uncertainties, our study utilizes the FDA adverse event reporting system database (Q1 2015 to Q1 2024) to examine the cardiotoxic events of CDK4/6 inhibitors in breast cancer patients. We employed a comprehensive analytical framework, applying disproportionality methods including Bayesian confidence propagation neural network, proportional reporting ratio, and reporting odds ratio. Our findings highlight significant variability in cardiotoxic events among different CDK4/6 inhibitors, with ribociclib (IC: 0.26, 95%CI: 0.18-0.34) exhibiting pronounced cardiotoxicity. Notably, ribociclib was associated with serious cardiotoxic events such as torsade de pointes/QT prolongation (IC: 2.11, 95% CI: 1.90-2.29) and conduction defects (IC: 2.07, 95% CI: 1.87-2.23). For the first time, palbociclib has been identified with positive signals for cardiotoxic events at the preferred terms level, including pulmonary oedema, increased blood pressure, myocardial infarction, and cardiac flutter. Moreover, multivariable logistic regression and Bayesian network analyses reveal that age, geographic location, and the number of concomitant medications significantly influence cardiotoxic events. Our study also highlights significant drug interactions that increase the probability of specific cardiotoxic outcomes, notably with drugs like sertraline, lansoprazole, capecitabine, and torasemide. These findings highlight the need for personalized treatment plans to mitigate cardiotoxic events and improve patient safety.

Encapsulation of soybean lunasin and amaranth unsaponifiable matter in liposomes induces cell cycle arrest in an allograft melanoma mouse model.

Melanoma is the most aggressive type of skin cancer and can metastasize during primary tumor formation. This research aimed to determine the relationship between the prevention of melanoma development in a mouse model treated with liposomes loaded with soybean lunasin and amaranth unsaponifiable matter (UM + LunLip) and cell cycle arrest. Tumors excised from C57BL/6 mice treated topically or subcutaneously with UM + LunLip were subjected to immunohistochemistry. Markers related to cell cycle inhibition (p16, p21, p27, and p53) and markers involved in cell cycle progression (cyclin-dependent kinase, CDK6, and cyclin D1) were assessed. The results showed that UM + LunLip had antitumor activity in C57BL/6 mice treated either topically or subcutaneously by p16, p21, p27, and p53 overexpression (up to 572-, 134-, 30-, and 57-fold change, FC, respectively) in the tumors of mice treated with 30mg UM + LunLip/kg body weight compared with the tumor-bearing untreated control. However, CDK6 and cyclin D1 expression was not inhibited (up to 1.37 FC and 2.09 FC, respectively), which is a typical behavior of cyclin D in melanoma. Therefore, melanoma tumor development was prevented by the overexpression of cell cycle inhibitors p16, p21, p27, and p53 due to UM + LunLip treatments. Since the topical application was effective, less invasive, and more practical for the user, this application will be recommended for future steps in in vivo studies.

HNF4α-CDKL3 axis restricts MASLD progression by targeting FoxO1 via non-canonical phosphorylation

Background and Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a worldwide disease with a broad spectrum of symptoms. Though being mild at early stages, further development of MASLD causes steatohepatitis, cirrhosis, liver cancers and accompanied diabetes. Discovery of the critical regulators in MASLD progression hold great values in both basic and translational medicine. Approach and Results: Herein, we identified Cyclin-dependent kinase like 3 (CDKL3) as a primary guardian against MASLD progression. Mice with liver-specific Cdkl3 ablation developed severe MASLD-related hepatic inflammation, fibrosis and diabetes. Mechanism-wise, CDKL3 directly phosphorylates FoxO1 on an unconventional site for the ubiquitination-dependent degradation of FoxO1, thereby remarkably alleviating glycogen and lipid accumulation and essentially preventing the onset of higher MASLD stages. Moreover, hepatic CDKL3 is a direct target gene of HNF4α. HNF4α is inhibited during MASLD, which led to diminished CDKL3 expression. The CDKL3-mediated crosstalk of HNF4α and FoxO1 hence forms a feedback loop in MASLD progression. Conclusions: We unearthed an alternative but critical regulatory path of FoxO1 by HNF4α-CDKL3 axis. CDKL3 serves as a guardian against MASLD and also may function as a prognosis indicator of FoxO1 inhibitor in MASLD treatment.

Abstract B072: Molecular targets of caffeine in breast cancer treatment: A network pharmacology approach

Abstract In Ghana, breast cancer leads in female cases (31.8%), demanding advanced treatments. Rising caffeine-filled energy drinks raise concerns. Yet, caffeine's role in breast cancer lacks understanding, urging more research. Our study uses network pharmacology and molecular docking to unravel the intricate molecular targets of caffeine in breast cancer, offering new therapies for this widespread disease We identified breast cancer-related targets from GEO datasets and TCGA databases, while caffeine targets were obtained from the SwissTarget Prediction database and literature. Common targets of both caffeine and breast cancer were considered using Venny software. PPI networks were generated using STRING and Cytoscape, identifying hub genes. Target proteins were annotated using the GO database, and pathway enrichment analysis was performed using KEGG. To investigate the binding affinity target proteins were retrieved from the Protein DataBank and docking studies were conducted using AutoDock Vina to explore the binding interactions between caffeine and potential target proteins identified. We identified 108 targets, and 6 common targets, including Cyclin-Dependent Kinase 1, Cyclin B1, Epidermal Growth Factor Receptor, Cyclin B2, Monoamine Oxidase A, and Carbonic Anhydrase 12 in relation to breast cancer. Gene Ontology (GO) and KEGG pathway enrichment analysis revealed their interactions in key pathways such as the P53 signaling pathway and Foxo signaling pathway, suggesting their critical roles in anti-breast cancer processes. Notably, caffeine exhibited the best overall binding affinity when interacting with CDK1, CCNB1, EGFR, and CCNB2 These findings provide valuable insights into the potential molecular targets and pathways associated with caffeine's anti-breast cancer effects, offering new strategies for therapeutic strategies. Citation Format: Kingsley A Osei, Justina S Owusu. Molecular targets of caffeine in breast cancer treatment: A network pharmacology approach [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B072.

Using bioinformatics and artificial intelligence to map the cyclin-dependent kinase 4/6 inhibitor biomarker landscape in breast cancer.

A cyclin-dependent kinase 4/6 (CDK4/6) inhibitor combined with endocrine therapy is the standard-of-care for patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer. However, not all patients respond to the treatment, resistance often occurs and efficacy outcomes from early breast cancer trials have been mixed. To identify biomarkers associated with CDK4/6 inhibitor response or resistance, we combined bioinformatic-database analyses, artificial intelligence-assisted literature review, and manual literature review (Embase and OVID Medline; search window: January 2012-October 2022) to compile data to comprehensively describe the CDK4/6 inhibitor biomarker landscape. Based on these results, and validation by external experts, we identified 15 biomarkers of clinical importance (AR , AURKA, ERBB2, ESR1, CCNE1, CDKN1A/B, CDK2, CDK6, CDK7, CDK9, FGFR1/2, MYC, PIK3CA/AKT, RB1 and STAT3) that could guide future breast cancer research.

Advancements in breast cancer management: a comprehensive review of ribociclib combined with endocrine therapy

Background: In this review, the complicated landscape of breast cancer management is explored with a focus on the promising synergies between ribociclib and endocrine therapy. Ribociclib mainly acts as a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, which disrupts cell cycle progression necessary for tumor growth. This, in combination with endocrine therapy, aims to produce hormone receptor-positive breast cancers, which is a very relevant subtype with challenging therapeutics. Methods: A comprehensive review was conducted using multiple databases, PubMed, Embase, Scopus, Cochrane Library, and Web of Science, covering the period from January 1990 to May 2024. Results: Pharmacokinetic studies underscore the efficacy and tolerability of ribociclib, thus providing vital information for dose adjustments, particularly among patients with renal and hepatic impairments. Ribociclib’s value in extending progression-free survival and improving overall survival has been shown by clinical trials such as the MONALEESA series. Quality of life considerations and patient-reported outcomes from these trials indicate that ribociclib has a broader effect on the well-being of the patients. However, despite the success experienced by this drug in clinical practice, it still has some side effects, including hematologic toxicity, hepatotoxicity, and thromboembolism associated with it. Ribociclib resistance mechanisms are multifaceted mixtures comprising genetic variations or mutations, compensatory signaling pathways, and epigenomic changes. While overcoming resistance remains challenging, ongoing research seeks to reconcile. Conclusion: Ribociclib combined with endocrine therapy represents a significant advancement in breast cancer treatment, albeit with challenges that necessitate ongoing research and holistic patient care approaches.

Physiological and pathological roles of the transcriptional kinases CDK12 and CDK13 in the central nervous system.

The cyclin-dependent kinases 12 (CDK12) and 13 (CDK13) govern several steps of gene expression, including transcription, RNA processing and translation. The main target of CDK12/13 is the serine 2 residue of the carboxy-terminal domain of RNA polymerase II (RNAPII), thus influencing the directionality, elongation rate and processivity of the enzyme. The CDK12/13-dependent regulation of RNAPII activity influences the expression of selected target genes with important functional roles in the proliferation and viability of all eukaryotic cells. Neuronal cells are particularly affected by the loss of CDK12/13, as result of the high dependency of neuronal genes on RNAPII processivity for their expression. Deregulation of CDK12/13 activity strongly affects brain physiology by influencingthe stemness potential and differentiation properties of neuronal precursor cells. Moreover, mounting evidence also suggest the involvement of CDK12/13 in brain tumours. Herein, we discuss the functional role(s) of CDK12 and CDK13 in gene expression regulation and highlight similarities and differences between these highly homologous kinases, with particular attention to their impact on brain physiology and pathology. Lastly, we provide an overview of CDK12/13 inhibitors and of their efficacy in brain tumours and other neoplastic diseases.

CCRG-04. RIBOCICLIB RESPONSIVE MENINGIOMA IN THE TREATMENT OF BREAST CANCER

Abstract BACKGROUND Meningiomas are the most common primary intracranial tumors, typically managed with surgical resection and/or radiation therapy. Systemic therapy options are limited and reserved for cases refractory to conventional interventions. Cyclin dependent kinase (CDK) proteins are key cell cycle regulators. CDK inhibitors are clinically available, approved for treatment of breast cancer and in clinical trials for meningioma. METHODS We present a case where a patient with incidentally-discovered intracranial meningioma undergoing CDK inhibitor therapy with ribociclib for breast cancer experienced a radiographic response. RESULTS A 73-year-old female was diagnosed with estrogen receptor (ER) positive, progesterone receptor (PR) positive, HER2 negative metastatic invasive ductal carcinoma of the breast. She was treated with estrogen receptor antagonist fulvestrant and CDK 4/6 inhibitor ribociclib. MRI of the brain performed for disease staging demonstrated an extraaxial, dural-based, homogenously enhancing mass along the left posterior petrous ridge consistent with a meningioma. Two-month interval scan demonstrated a marginal decrease in size of the mass (15.6x18.3 mm to 15.4x17.5 mm). Systemic disease related to breast cancer concurrently improved. Two years later, with ongoing fulvestrant and ribociclib therapy, a repeat MRI brain demonstrated further decrease in size of the meningioma (13.4x16.8 mm). CONCLUSIONS Hormonally-directed therapies previously demonstrated limited benefit for meningiomas despite PR/ER positivity reported for these tumors. However, CDK 4/6 inhibitors abemaciclib and ribociclib have demonstrated activity in treatment of recurrent grade 2/3 meningiomas. The radiographic response noted in our case suggests there may be a role for systemic therapy even in treatment-naive cases that may not be amenable to surgical resection or radiation therapy.

BIOM-12. ALLIANCE A071401: GENOMICALLY GUIDED PHASE II TRIAL OF ABEMACICLIB IN PATIENTS WITH GRADE 2/3 MENINGIOMAS HARBORING SOMATIC NF2 OR CDK PATHWAY ALTERATIONS

Abstract BACKGROUND Patients with meningiomas that have progressed after surgery and/or radiation have limited treatments. Loss of NF2 and CDKN2A/B are common in higher-grade meningiomas and promote meningioma progression. We evaluated the efficacy of abemaciclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, as part of Alliance A071401, a genomically-driven phase II trial in recurrent or progressive meningiomas. METHODS Eligible patients (pts) with grade 2/3 tumors and NF2 mutations or CDK pathway alterations were treated with abemaciclib 200 mg orally twice daily. Two co-primary endpoints were used: progression-free survival at 6 months (PFS6) and response rate (RR); the trial would be declared positive if either endpoint was met. Twenty-four evaluable pts provided ≥85% power to detect a PFS6 ≥41.5% (vs. null 15%; alpha=0.02), and ≥89% power to detect RR ≥20% (vs. null 2.5%; alpha=0.021). RESULTS Of 83 pts screened while the abemaciclib arm was open between September 15, 2021 and October 3, 2022, 36 eligible pts received abemaciclib. The observed PFS6 rate in the first 24 evaluable pts was 54% (95% CI 33-75%), thus the study met PFS6 endpoint. Of all 35 evaluable pts, median PFS was 7.6 months (95% CI 3.6-17.1 months), estimated 6-month PFS was 55% (95% CI 40-75%) and estimated 6-month OS was 88% (95% CI 77-100%). No objective responses were observed. Of the 36 pts who started treatment, two had a grade 4 adverse event at least possibly related to treatment which included ALT elevation(1), AST elevation(1) and vomiting(1). Patients with NF2 alterations (n=24) had favorable PFS6 outcomes (58%, 95% CI 37-78%) compared to those with CDK (n=4, 25%, 95% CI 1-81%) and CDK+NF2 alterations (n=7, 29% 95% CI 4-71%), (Fisher’s Exact p=0.036). CONCLUSIONS Abemaciclib was well-tolerated and resulted in improved PFS6, meeting the trial’s co-primary endpoint. Abemaciclib warrants further investigation for the treatment of patients with progressive meningiomas.

Molecular modeling and cytotoxic activity studies of oxirane-2-carboxylate derivatives

In this study, five 3-aryloxirane-2-carboxylate derivatives were prepared, and the antiproliferative activities of molecules were screened in lung and colon cancer cell lines. The results showed that molecules had antiproliferative activity on cancerous cells with IC50 values under 100 µM. Furthermore, all of the molecules were found to have a much higher cytotoxic effect than cisplatin in colon cancer cells. The interactions of the relatively active compounds to the crucial enzyme in cancer cell proliferation, cyclin-dependent kinase 1 (CDK1), were investigated using molecular docking. The stability of the resulting CDK1-compound complexes procured from the docking was also assessed through molecular dynamics (MD) simulations. Then, the binding affinity of compounds 2-3a and 2-3c to the target enzyme was computed by MMPBSA. The molecular docking study demonstrated that the two most active compounds could bind to the enzyme. The binding potential of 2-3a is anticipated to be higher as it had one more conventional hydrogen bond and a slightly lower binding energy than compound 2-3c. The MD simulation study exhibited that the two compounds formed a stable complex with the enzyme. On the other hand, the MMPBSA energy computation implicated a slightly higher binding affinity for compound 2-3c toward the enzyme. Furthermore, electrical and frontier molecular orbital analysis of all of the tested compounds was conducted by density functional theory (DFT) studies. Compound 2-3a is anticipated to be the most chemically stable as it gave the highest energy gap value in the DFT analysis.

Preclinical metabolism and disposition of [14C]GFH009, a novel selective CDK9 inhibitor

1. GFH009 is a potent and highly selective cyclin-dependent kinase 9 (CDK9) inhibitor currently under phase II clinical trials. In this study, we investigated the metabolism and disposition of GFH009 in Sprague-Dawley (SD) rats, as well as in vitro metabolism of CD-1 mouse, SD rat, beagle dog, cynomolgus monkey and human. 2. A radiolabelled study indicated that [14C]GFH009 was quickly and widely distributed throughout the body, but presented low levels in brain, testis and epididymis after a single intravenous dose of 6 mg (100 µCi)/kg to SD rats. 3. GFH009 undergoes systemic metabolic changes, primarily throughO-demethylation, oxidation to carboxylic acid and N-dealkylation, cleavage off the methoxyisopropyl moiety being a minor pathway. These metabolic pathways were found to be mainly consistent both in vitro and in vivo. 4. In SD rats, GFH009 was rapidly and completely eliminated, with feces serving as the major excretion pathway and urine serving as the minor one. Besides, the major clearance pathway for GFH009 was excretion and the minor one was metabolism. 5. GFH009 exhibits favourable drug metabolism and pharmacokinetics (DMPK) properties, which provides valuable insights into the disposition of GFH009 and can be used to guide future clinical studies.

BIOM-59. INDIVIDUALIZED TUMOR-SPECIFIC AMPLIFIED DNA JUNCTIONS FOR PLASMA-BASED MONITORING IN PATIENTS WITH HIGH-GRADE GLIOMAS

Abstract Accurate response assessment in patients with high-grade gliomas (HGGs) is complicated by post-treatment radiographic changes that are equivocal for treatment effect versus progression. Plasma-based liquid biopsy assays for circulating tumor DNA (ctDNA) have had limited sensitivity to date, due in part to the presence of the blood-brain barrier that limits the diffusion of tumor-derived analytes out of the central nervous system. To address this challenge, we developed a ctDNA plasma-based assay to detect individualized tumor-derived amplified junctions in patients with HGGs. These junctions occur due to large-scale chromosomal rearrangements, such as those associated with epidermal growth factor receptor (EGFR) or cyclin-dependent kinase 4 (CDK4) amplification. To identify these amplified junctions, whole genome sequencing was performed on resected tissue. Primers were designed to flank junction breakpoints and were then utilized on each patient’s plasma samples to detect the amplified junction via qPCR. Tumor-specific amplified junctions were robustly detected in plasma from 14/14 (100%) patients with IDH-mutant or wild-type grade 4 astrocytomas whose tumor genomes demonstrated amplification-related junctions, located at chromosomes 7, 11, or 12. In comparison, ctDNA could not be detected in three control cases that did not have an amplification or did not have high copy number junctions. Importantly, serial plasma samples obtained prior to and at disease recurrence in 4 patients demonstrated an increasing abundance of amplified junctions that accurately correlated with disease progression in 4/4 patients (100%). In two of these patients, increasing abundance of amplified junctions preceded radiographic changes compatible with disease progression by approximately 2 or 4 months, respectively. In conclusion, tumor-specific amplified junctions could reliably be detected in plasma from patients with IDH-mutant or wild-type grade 4 astrocytomas and correlated with changes in disease burden. Further work is currently ongoing to evaluate these promising results in a larger cohort of patients with serial plasma samples.

DDDR-19. PHARMACOSCOPY FOR BRAIN METASTASES FROM NON-SMALL CELL LUNG CANCER (NSCLC)

Abstract Brain metastases remain a common and important complication in the course of disease in patients with non-small cell lung cancer (NSCLC). While targeted therapies and immunotherapies have led to prolonged disease control in subsets of patients with brain metastases from NSCLC, there is still an urgent need for novel treatments after failure of these standards and after exhaustion of local therapies including surgery and radiotherapy. Here we performed an explorative feasibility study to profile ex vivo drug sensitivities across a large panel of classical anti-cancer as well as neuroactive drugs, using an automated imaging platform called “pharmacoscopy”. The anti-cancer drug panel included chemotherapies and targeted therapies such as receptor tyrosine kinase, cyclin dependent kinase, and BCL2 inhibitors. The neuroactive drug panel included antidepressants and antipsychotics. A total of 19 samples from 17 patients were dissociated after surgery and incubated with drugs for 48 hours ex vivo to measure drug sensitivities. Patient cancer cells were identified by immunofluorescence using EpCAM, TTF-1, or CK7 as markers whereas immune cells were identified by CD45. On-target drug activity (pharmacoscopy score) was measured by quantifying the specific reduction of cancer cells upon drug treatment. Among the top ranking drugs ex vivo, several patient samples displayed on-target responses to candidate targeted therapies such as the CDK4/6 inhibitor abemaciclib, the BCL2 inhibitor venetoclax, and the MEK inhibitor cobimetinib as well as unexpected sensitivies to certain neuroactive drugs. Integration of pharmacoscopy results with molecular data from next generation sequencing in the respective brain metastases tissues is ongoing. Overall, this study demonstrates the feasibility of pharmacoscopy-based functional drug testing in NSCLC brain metastases, and highlights novel opportunities of personalized treatment for future exploratory clinical studies in patients with brain metastases from NSCLC.

DNAR-06. EPITRANSCRIPTOMIC TRNA MODIFICATIONS TARGETING ADENOSINE (A) AT POSITION 37 IN GBM

Abstract GBM stands as the most aggressive brain tumor in adults. The median overall survival is less than 2 years despite conventional treatments. Recent advancements in DNA analysis have delineated the molecular characteristics of gliomas, leading to revisions in the WHO classification based on DNA profiles. However, the role of RNA modifications, epitranscriptomics, remains poorly understood in glioma. In this study, we underscore the significance of tRNA modifications targeting adenosine at position 37 in GBM. Transfer RNA isopentenyl transferase 1 (TRIT1) is identified as an isopentenyl transferase catalyzing the conversion of A into i6A at position 37 of tRNA, a nucleotide adjacent to the anticodon region. Additionally, i6A is further modified to 2-methylthio i6A (ms2i6A) by cyclin-dependent kinase 5 regulatory subunit associated-protein 1 (CDK5RAP1) within mitochondrial tRNA. Analysis of TCGA data reveals elevated TRIT1 mRNA expression levels in GBM compared to non-tumor samples. Furthermore, a low expression group of TRIT1 in gliomas demonstrates improved survival outcomes according to data from the CGGA, implicating TRIT1 in glioma progression. Functional assays involving TRIT1 knockdown in human GBM cell lines result in decreased cell viability, indicating its functional significance. Moreover, our data suggest that this phenotype may be mediated through translational control of selenoprotein W (SEPW1) via tRNA^(Ser)Sec modification. GO analysis of TRIT1 and SEPW1 in TCGA data further supports this proposed mechanism. At position 37 of tRNA, we also found that CDK5RAP1 in mitochondrial tRNAs is indispensable for sustaining glioblastoma initiating cell (GIC)-related traits. CDK5RAP1 maintains the self-renewal capacity, undifferentiated state, and tumorigenic potential of GICs, independent of translational control of mitochondrial proteins. Notably, CDK5RAP1 abrogates the antitumor effect of i6A by converting i6A to ms2i6A and shields GICs from excessive autophagy triggered by i6A. This work underscores the critical role of tRNA modifications in glioma pathogenesis. We will report these findings alongside RNA modification profile data and clinical data from glioma patients.

DDDR-44. REPURPOSING SSRI/SNRI TARGETING NEUROTRANSMITTER SIGNALING TO OVERCOME RESISTANCE TO CDK9 INHIBITORS IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma (DMG) is a subset of aggressive brain tumors with few effective therapies. Cyclin-dependent kinase 9 (CDK9i) inhibitors such as zotiraciclib (ZTR) are in clinical trials for glioma, but CDK9i treatment alone may be ineffective due to the development of resistance. To identify CDK9i resistance mechanisms, we analyzed the transcriptomes of DMG xenograft tumors after three weeks of treatment with ZTR, which revealed that transcripts encoding synaptic signaling regulators such as glutamate receptors were overexpressed in surviving cells, suggesting that altered regulation of synaptic signaling may mediate CDK9i resistance. We also performed drug synergy screens using a library of FDA-approved and bioactive compounds and found that drugs that modulate neurotransmitter signaling, such as SSRIs and SNRIs (selective serotonin and serotonin/norepinephrine reuptake inhibitors), increased DMG sensitivity to an IC25 dose of ZTR. Follow-up studies confirm that combinations of structurally distinct CDK9i (ZTR, AZD4573) and SSRI/SNRI (sertraline, duloxetine) synergistically reduce DMG growth in neurospheres by BLISS analysis and show that co-treatment with CDK9i and SSRI/SNRI strongly induces the apoptotic marker cleaved caspase 3 and reduces the CDK9 target phos-Pol2Ser2. Additional phospho-proteomic analyses identified differentially phosphorylated proteins following CDK9i+SSRI treatment, including chromatin regulators, Pol2 elongation factors, and splicing regulators. Further RNAseq studies reveal that CDK9i+SSRI treatment resulted in more robust transcriptional changes than either drug alone, including the downregulation of transcripts associated with ZTR resistance such as GRIA3/4, and SYT12. These observations suggest that CDK9i +SSRI/SNRI drug combinations may overcome some of the transcriptional changes underlying CDK9i resistance. Finally, preclinical studies show that co-treatment with low doses of AZD4573 and sertraline reduces the growth of orthotopic DMG xenografts and prolongs overall survival. Taken together, these data suggest that SSRI/SNRI and CDK9i might be used in combination to synergistically reduce DMG growth and overcome CDK9i resistance.

CSIG-32. MODULATION OF HALLMARKS OF GLIOBLASTOMA MULTIFORME BY BIOACTIVE PHYTOCHEMICALS: A PRECLINICAL MECHANISTIC EVALUATION

Abstract Glioblastoma multiforme (GBM) is an aggressive malignancy with extensive neovascularization, an immunosuppressive tumor microenvironment, and high recurrence rates. Bioactive plant secondary metabolites (phytochemicals), many from fruits and vegetables, have been extensively studied for GBM prevention and treatment. The objective of this study is to determine the effects of phytochemicals on GBM hallmarks and associated molecular mechanisms of action through evaluation of in vitro and in vivo studies. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed to collect articles using PubMed, ScienceDirect and Scopus. While 1,344 articles were screened, 115 in vitro and 36 in vivo studies met our inclusion criteria. The Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) review of bias tool assessed in vivo study bias. In vitro, 49 alkaloids, 10 phenolics, 2 sulfur- compounds, and 5 terpenoids modulated GBM hallmarks of cell growth, invasion, cell cycle progression, neovascularization, and apoptosis. The PI3K/Akt/mTOR pathway was suppressed by 13 phytochemicals. Apoptosis was induced by 15 phytochemicals activating caspase-3, whereas 11 compounds inhibited Bcl-2. Cell cycle modulation occurred through decreased cyclin-dependent kinases and increased p53, p21, and p27. In vivo, 17 alkaloids, 5 phenolics, 2 sulfur-compounds, and 2 terpenoids had effects on GBM hallmarks. Invasion and neovascularization were inhibited through MMP, HIF-1α, and VEGF modulation. Six phytochemicals induced apoptosis in vivo through caspases and Bcl-2. Utilizing the SYRCLE review of bias tool, the average score quality of animal studies is 10% with many indicators of bias unclear. Various phytochemicals show promise to prevent and treat GBM due to their effects on numerous GBM hallmarks. Study limitations include the inclusion of literature reporting effects of single agents and lack of clinical studies. Future research is warranted to fill gaps in our analysis before recommending phytochemicals for prevention and treatment of GBM, a perilous malignancy.

PRMT5/WDR77 Enhances the Proliferation of Squamous Cell Carcinoma via the ΔNp63α-p21 Axis

Protein arginine methyltransferase 5 (PRMT5) is a critical oncogenic factor in various cancers, and its inhibition has shown promise in suppressing tumor growth. However, the role of PRMT5 in squamous cell carcinoma (SCC) remains largely unexplored. In this study, we analyzed SCC patient data from The Cancer Genome Atlas (TCGA) and the Cancer Dependency Map (DepMap) to investigate the relationship between PRMT5 and SCC proliferation. We employed competition-based cell proliferation assays, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, flow cytometry, and in vivo mouse modeling to examine the regulatory roles of PRMT5 and its binding partner WDR77 (WD repeat domain 77). We identified downstream targets, including the p63 isoform ΔNp63α and the cyclin-dependent kinase inhibitor p21, through single-cell RNA-seq, RT-qPCR, and Western blot analyses. Our findings demonstrate that upregulation of PRMT5 and WDR77 correlates with the poor survival of head and neck squamous cell carcinoma (HNSCC) patients. PRMT5/WDR77 regulates the HNSCC-specific transcriptome and facilitates SCC proliferation by promoting cell cycle progression. The PRMT5 and WDR77 stabilize the ΔNp63α Protein, which in turn, inhibits p21. Moreover, depletion of PRMT5 and WDR77 repress SCC in vivo. This study reveals for the first time that PRMT5 and WDR77 synergize to promote SCC proliferation via the ΔNp63α-p21 axis, highlighting a novel therapeutic target for SCC.

CSIG-09. BEYOND SINGLE AGENTS: EXPLORING THE POTENTIAL OF ABEMACICLIB IN COMBINATION WITH PANOBINOSTAT FOR IMPROVED GLIOMA TREATMENT

Abstract BACKGROUND Glioblastoma is the most common primary central nervous system tumour with a median survival of less than 15 months. Addressing the underlying redundant, and converging signaling pathways necessitates the utilization of small molecule inhibitors capable of simultaneous targeting and inhibition. In glioma, the aberrant CDK4/6-Rb pathway dysregulation resulting in disruption of cell cycle checkpoint progression can lead to uncontrolled cell division, a hallmark of cancer. The neuro-pharmacokinetics of abemaciclib, a Cyclin-dependent kinase inhibitor, is being currently investigated in phase 1 clinical trials by National Cancer Institue, [NCT05413304]. The importance of panobinostat as a Histone deacetylase inhibitor and potentiating the effects of various small molecular inhibitors in published literature is paramount. This study is based on investigating ways to overcome resistance by exploring the synergistic cytotoxic effects of abemaciclib in combination with panobinostat in glioma. METHODS We investigated the antiproliferative effects of abemaciclib and panobinostat on glioma cells using an MTS cell proliferation assay. Inhibitor combination analysis was done using, SYNERGYFINDER via zip-model. Flow cytometry with Annexin V staining further assessed cell death and apoptosis induced. RESULTS The antiproliferative effects of abemaciclib, alone and in combination with panobinostat, in pediatric lines (KNS 42, SJG2), and adult human glioblastoma line, SF188 exhibited lower IC50 as compared to adult high grade glioma lines (LN 18, LNZ 308). Synergy analysis revealed a strong cytotoxic effect in KNS 42 and SF 188 cells treated with the combination. Flow cytometric analysis indicated that the combination therapy induced significant cell death only in a subset of cell lines- KNS 42 and SF 188, suggesting underlying genetic heterogeneity influences treatment response, warranting further mechanistic studies. CONCLUSION Combination therapies using abemaciclib and panobinostat, targeting CDK4/6 and histone deacetylation pathways, show promise against aggressive and treatment-resistant gliomas. Further investigation into these combination approaches is crucial to understand resistant mechanisms, optimize efficacy, and establish their clinical efficacy for improving glioma patient outcomes.