Kinase Cdk Research Articles

Abstract 3191: Chk1/2 inhibition enhances response to lurbinectedin treatment in small cell lung cancer

Abstract Small cell lung cancer (SCLC) remains a significant clinical concern, with only a 7% 5 year survival rate; therefore, there remains an unmet need for efficacious treatments. Lurbinectedin was FDA approved in 2019 to treat platinum-resistant SCLC. Preliminary experiments in SCLC cell lines indicated an increase in intracellular pChk1 when treated with lurbinectedin. Based on these data and our understanding of the role of checkpoint kinases in the DNA damage response (DDR), we hypothesize that the activity of Chk1 is indicative of upregulated DDR activity as a result of double stranded breaks and replicative stress induced by lurbinectedin. In subsequent experiments, we sought to identify the role of Chk1 and Chk2 in the tumor cell response to lurbinectedin by inhibiting their activity using prexasertib (LY2606368, Acrivon Therapeutics). Cell viability experiments indicate decreased tumor cell viability when three SCLC cell lines (H1048, H1105, H1417) are treated with prexasertib as a single agent and in combination with lurbinectedin. Western blot analysis of intracellular proteins from the same SCLC cell lines treated with both drugs demonstrate dose-dependent effects on cell death marker cPARP, DNA damage marker γH2AX, and DDR/cell cycle pathway kinases cdk1 and 2. Ongoing experiments seek to replicate results using alternative Chk1/2 inhibitors, and to explore the effect of lurbinectedin-Chk1/2 inhibitor combination treatment on antitumor immune effector function by in vitro co-culture. Citation Format: Ashley Sanchez Sevilla Uruchurtu, Tyler Roady, Agenxnie Anderson, Wafik S. El-Deiry. Chk1/2 inhibition enhances response to lurbinectedin treatment 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 3191.

Abstract 1634: Cyclin D1 induces Akt1 activity in RB deficient prostate cancer

Abstract Background: Prostate cancer (PCa), the second leading cause of death in American men, is a genetically heterogeneous disease. The aberrant activation of AKT is one of the most frequent alterations in human cancers, and an elevated level of phosphorylated AKT at Ser473 site (pSer473) is associated with metastatic cancers (1). In prostate cancer (PCa), PI3K/AKT activation induces tumorigenesis (2), androgen receptor (AR)-mediated signaling, enzalutamide resistance and stemness, synergizing with other pathways including the notch intracellular domain (NICD). The cyclin D1 gene (CCND1), which is overexpressed in PCa, conveys canonical function by encoding the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma gene (RB) product, and non-canonical functions through phosphorylating other substrates and transcriptional activities. Cyclin D1 expression augments growth of some castrate-resistant PCa (CRPC) and clinical trials are targeting the cyclin D1 related CDK kinase activity in pRB+ PCa. Several Rb-independent mechanisms of action have been described (3). In recent studies the effect of CDK inhibitors was similar in patients with Rb+ vs. Rb− PCa (4). Whether Rb expression is required for the antiproliferative effects of CKD4/6 inhibition in PCa remains to be established. Methods: The kinetics of Akt1 induction by mitogens was assessed using the phosphorylation of the dichromic fluorescent (DCF) dye substrate LS456. We used cyclin D1 shRNA to show endogenous cyclin D1 augmented AKT1 signaling in response to mitogens. Results: Cyclin D1 reintroduction into cyclin D1−/− cells augmented Akt1 phosphorylation, requiring the cyclin D1 K112 residue. CDK inhibitors reduced Akt activity in RB+ and RB− human prostate cancer cell lines. Prostate-specific epithelial cell deletion of cyclin D1 (probasin-CRE-cyclin D1fl/fl) reduced prostate epithelial cell Akt1 activity assessed by immunohistochemical (IHC) staining. pAktSer473 was reduced in cell nuclei and the cytoplasm of both epithelial cells and stromal fibroblasts without a significant change in the abundance of Akt1. The downstream targets of Akt signaling (pTSC2Ser939, and pFKHRSer319) and ARSer213P were reduced in both epithelial cells and stromal fibroblasts. Genetic deletion of both epithelial cell and stromal cyclin D1 further reduced Akt1 activity, prostate epithelial cell proliferation, and markers of cancer stem cells. Proteomic analysis of cyclin D1-expressing stroma identified heterotypic chemokine signals that augment PCa AKT activity. Conclusions: Cyclin D1 induces Akt1 activity in both RB+ and RB− PCa cells which may contribute to tumor progression and therapy resistance. Reference: (1) Chen YL, Law PY, Loh HH. Curr Med Chem Anticancer Agents 2005;5:575-892. (2) Stoyanova T, et al. Proc Natl Acad Sci U S A 2013;110:20111-63. (3) Chen K, et al. Cell Rep 2020;32:1081514. (4) de Kouchkovsky I, et al. Clin Cancer Res 2022;28:1531-9 Citation Format: Xuanmao Jiao, Anthony W. Ashton, Zhiping Li, Ritika Harish, Danni Li, Gordon Robertson, Duanwen Shen, Xiaoming Ju, Ke Chen, Wei Zhang, Samuel Archilefu, Kenneth A. Iczkowski, Hallgeir Rui, Beatrice Knudsen, Paul Timpson, Max Nobis, Richard G. Pestell. Cyclin D1 induces Akt1 activity in RB deficient prostate 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 1634.

Comprehensive multi-omics analysis reveals WEE1 as a synergistic lethal target with hyperthermia through CDK1 super-activation

Hyperthermic intraperitoneal chemotherapy’s role in ovarian cancer remains controversial, hindered by limited understanding of hyperthermia-induced tumor cellular changes. This limits developing potent combinatory strategies anchored in hyperthermic intraperitoneal therapy (HIPET). Here, we perform a comprehensive multi-omics study on ovarian cancer cells under hyperthermia, unveiling a distinct molecular panorama, primarily characterized by rapid protein phosphorylation changes. Based on the phospho-signature, we pinpoint CDK1 kinase is hyperactivated during hyperthermia, influencing the global signaling landscape. We observe dynamic, reversible CDK1 activity, causing replication arrest and early mitotic entry post-hyperthermia. Subsequent drug screening shows WEE1 inhibition synergistically destroys cancer cells with hyperthermia. An in-house developed miniaturized device confirms hyperthermia and WEE1 inhibitor combination significantly reduces tumors in vivo. These findings offer additional insights into HIPET, detailing molecular mechanisms of hyperthermia and identifying precise drug combinations for targeted treatment. This research propels the concept of precise hyperthermic intraperitoneal therapy, highlighting its potential against ovarian cancer.

Unpacking Immune Modulation as a Site of Therapeutics Innovation for Nematode Parasite Wuchereria bancrofti: A Temporal Quantitative Phosphoproteomics Profiling of Macrophage Migration Inhibitory Factor 2.

Nematode infections are common in both humans and livestock, with major adverse planetary health and economic impacts. Wuchereria bancrofti is a parasitic nematode that causes lymphatic filariasis, a neglected tropical disease that can lead to severe disability and deformity worldwide. For the long-term survival of the bancroftian parasites in the host, a complex immune invasion strategy is involved through immunomodulation. Therefore, immunomodulation can serve as a site of research and innovation for molecular targets. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine crucial to the host antimicrobial alarm system and stress response. Interestingly, the nematode parasite W. bancrofti also produces two homologs of MIF (Wba-MIF1 and 2). Using a mass spectrometry-based phosphoproteomics approach, we report new findings on the immunomodulatory effect and signaling mechanism of Wba-MIF2 in macrophage cells. Accordingly, we observed 1201 phosphorylated sites on 467 proteins. Out of the 1201 phosphorylated sites, 1075, 117, and 9 were found on serine (S), threonine (T), and tyrosine (Y) residues, respectively. Our bioinformatics analysis led to identification of major pathways, including spliceosomes, T cell receptor signaling pathway, Th17 differentiation pathway, interleukin-17 signaling pathway, and insulin signaling pathway upon Wba-MIF2 treatment. Wba-MIF2 treatment also enriched CDK4, CDK1, and DNAPK kinases. The comparison of the signaling pathway of Wba-MIF2 with that of human-MIF suggests both share similar signaling pathways. These findings collectively offer new insights into the role and mechanism of Wba-MIF2 as an immunomodulator and inform future diagnostics and drug discovery research for W. bancrofti.

Comprehensive Parameter Space Mapping of Cell Cycle Dynamics under Network Perturbations.

Studies of quantitative systems and synthetic biology have extensively utilized models to interpret data, make predictions, and guide experimental designs. However, models often simplify complex biological systems and lack experimentally validated parameters, making their reliability in perturbed systems unclear. Here, we developed a droplet-based synthetic cell system to continuously tune parameters at the single-cell level in multiple dimensions with full dynamic ranges, providing an experimental framework for global parameter space scans. We systematically perturbed a cell-cycle oscillator centered on cyclin-dependent kinase (Cdk1), enabling comprehensive mapping of period landscapes in response to network perturbations. The data allowed us to challenge existing models and refine a new model that matches the observed response. Our analysis demonstrated that Cdk1 positive feedback inhibition restricts the cell cycle frequency range, confirming model predictions; furthermore, it revealed new cellular responses to the inhibition of the Cdk1-counteracting phosphatase PP2A: monomodal or bimodal distributions across varying inhibition levels, underscoring the complex nature of cell cycle regulation that can be explained by our model. This comprehensive perturbation platform may be generalizable to exploring other complex dynamic systems.

Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics.

Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based Förster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC-FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.

Modulation of neuronal morphology by antipsychotic drug: Involvement of serotonin receptor 7

Antipsychotic drugs (APDs) are the primary pharmacological treatment for schizophrenia, a complex disorder characterized by altered neuronal connectivity. Atypical or second-generation antipsychotics, such as Risperidone (RSP) and Clozapine (CZP) predominantly block dopaminergic D2 and serotonin receptor 2A (5-HT2A) neurotransmission. Both compounds also exhibit affinity for the 5-HT7R, with RSP acting as an antagonist and CZP as an inverse agonist. Our study aimed to determine whether RSP and CZP can influence neuronal morphology through a 5-HT7R-mediated mechanism. Here, we demonstrated that CZP promotes neurite outgrowth of early postnatal cortical neurons, and the 5-HT7R mediates its effect. Conversely, RSP leads to a reduction of neurite length of early postnatal cortical neurons, in a 5-HT7R-independent way.Furthermore, we found that the effects of CZP, mediated by 5-HT7R activation, require the participation of ERK and Cdk5 kinase pathways. At the same time, the modulation of neurite length by RSP does not involve these pathways.In conclusion, our findings provide valuable insights into the morphological changes induced by these two APDs in neurons and elucidate some of the associated molecular pathways. Investigating the 5-HT7R-dependent signaling pathways underlying the neuronal morphogenic effects of APDs may contribute to the identification of novel targets for the treatment of schizophrenia.

The SIAMESE family of cell-cycle inhibitors in the response of plants to environmental stresses.

Environmental abiotic constraints are known to reduce plant growth. This effect is largely due to the inhibition of cell division in the leaf and root meristems caused by perturbations of the cell cycle machinery. Progression of the cell cycle is regulated by CDK kinases whose phosphorylation activities are dependent on cyclin proteins. Recent results have emphasized the role of inhibitors of the cyclin-CDK complexes in the impairment of the cell cycle and the resulting growth inhibition under environmental constraints. Those cyclin-CDK inhibitors (CKIs) include the KRP and SIAMESE families of proteins. This review presents the current knowledge on how CKIs respond to environmental changes and on the role played by one subclass of CKIs, the SIAMESE RELATED proteins (SMRs), in the tolerance of plants to abiotic stresses. The SMRs could play a central role in adjusting the balance between growth and stress defenses in plants exposed to environmental stresses.

The development of a custom RNA-sequencing panel for the identification of predictive and diagnostic biomarkers in glioma

PurposeVarious molecular profiles are needed to classify malignant brain tumors, including gliomas, based on the latest classification criteria of the World Health Organization, and their poor prognosis necessitates new therapeutic targets. The Todai OncoPanel 2 RNA Panel (TOP2-RNA) is a custom-target RNA-sequencing (RNA-seq) using the junction capture method to maximize the sensitivity of detecting 455 fusion gene transcripts and analyze the expression profiles of 1,390 genes. This study aimed to classify gliomas and identify their molecular targets using TOP2-RNA.MethodsA total of 124 frozen samples of malignant gliomas were subjected to TOP2-RNA for classification based on their molecular profiles and the identification of molecular targets.ResultsAmong 55 glioblastoma cases, gene fusions were detected in 11 cases (20%), including novel MET fusions. Seven tyrosine kinase genes were found to be overexpressed in 15 cases (27.3%). In contrast to isocitrate dehydrogenase (IDH) wild-type glioblastoma, IDH-mutant tumors, including astrocytomas and oligodendrogliomas, barely harbor fusion genes or gene overexpression. Of the 34 overexpressed tyrosine kinase genes, MDM2 and CDK4 in glioblastoma, 22 copy number amplifications (64.7%) were observed. When comparing astrocytomas and oligodendrogliomas in gene set enrichment analysis, the gene sets related to 1p36 and 19q were highly enriched in astrocytomas, suggesting that regional genomic DNA copy number alterations can be evaluated by gene expression analysis.ConclusionsTOP2-RNA is a highly sensitive assay for detecting fusion genes, exon skipping, and aberrant gene expression. Alterations in targetable driver genes were identified in more than 50% of glioblastoma. Molecular profiling by TOP2-RNA provides ample predictive, prognostic, and diagnostic biomarkers that may not be identified by conventional assays and, therefore, is expected to increase treatment options for individual patients with glioma.

Hijacking of nucleotide biosynthesis and deamidation-mediated glycolysis by an oncogenic herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS) and multiple types of B cell malignancies. Emerging evidence demonstrates that KSHV reprograms host-cell central carbon metabolic pathways, which contributes to viral persistence and tumorigenesis. However, the mechanisms underlying KSHV-mediated metabolic reprogramming remain poorly understood. Carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) is a key enzyme of the de novo pyrimidine synthesis, and was recently identified to deamidate the NF-κB subunit RelA to promote aerobic glycolysis and cell proliferation. Here we report that KSHV infection exploits CAD for nucleotide synthesis and glycolysis. Mechanistically, KSHV vCyclin binds to and hijacks cyclin-dependent kinase CDK6 to phosphorylate Ser-1900 on CAD, thereby activating CAD-mediated pyrimidine synthesis and RelA-deamidation-mediated glycolytic reprogramming. Correspondingly, genetic depletion or pharmacological inhibition of CDK6 and CAD potently impeded KSHV lytic replication and thwarted tumorigenesis of primary effusion lymphoma (PEL) cells in vitro and in vivo. Altogether, our work defines a viral metabolic reprogramming mechanism underpinning KSHV oncogenesis, which may spur the development of new strategies to treat KSHV-associated malignancies and other diseases.

Rational Design of Selenophene‐infused BODIPY Molecules as Fluorogenic Probes for Lung Tumor Imaging – A Computational Approach

AbstractThe BODIPY based molecules show promising properties towards bioimaging applications due to its structural flexibility and versatile optoelectronic characteristic features. Among many strategies in the development of such molecules, fusion of heterocyclic units into the BODIPY core structure is vital in tuning the π‐delocalization and spectral properties. Recently reported Thieno [3,2b] Thiophene based BODIPY (TTB) attracts a lot of attention while the caliber of their Selenophene derivatives are interesting and yet to be explored. Therefore, a systematic replacement of Sulphur by Selenium has been done on TTB and seven new selenophene fused TTB molecules are computationally designed and evaluated for their optoelectronic properties towards bioimaging of CDK2 kinase tumor cells. The optimized geometries indicate that all these molecules are showing excellent π‐delocalization and further stabilized by a range of intra‐molecular C−H−F and B−F−S/Se interactions. Molecular docking studies show that these TTB molecules are potential inhibitors and can be used for lung tumor imaging in biomedical applications. Molecular Dynamics analysis confirms their preferential binding inside the active site up to 100 ns. In summary, this study highlights the importance of Se replacement in designing new BODIPY‐based bioimaging candidates with excellent near‐IR luminescence and photo sensitizers for future biomedical applications.

Novel Bipyrazole Targeting Dual CDK-2 and VEGFR-2 Kinases: Synthesis, Anticancer Evaluation, and In Silico Studies

Novel Bipyrazole Targeting Dual CDK-2 and VEGFR-2 Kinases: Synthesis, Anticancer Evaluation, and In Silico Studies

An Antiherpesviral Host-Directed Strategy Based on CDK7 Covalently Binding Drugs: Target-Selective, Picomolar-Dose, Cross-Virus Reactivity.

The repertoire of currently available antiviral drugs spans therapeutic applications against a number of important human pathogens distributed worldwide. These include cases of the pandemic severe acute respiratory coronavirus type 2 (SARS-CoV-2 or COVID-19), human immunodeficiency virus type 1 (HIV-1 or AIDS), and the pregnancy- and posttransplant-relevant human cytomegalovirus (HCMV). In almost all cases, approved therapies are based on direct-acting antivirals (DAAs), but their benefit, particularly in long-term applications, is often limited by the induction of viral drug resistance or side effects. These issues might be addressed by the additional use of host-directed antivirals (HDAs). As a strong input from long-term experiences with cancer therapies, host protein kinases may serve as HDA targets of mechanistically new antiviral drugs. The study demonstrates such a novel antiviral strategy by targeting the major virus-supportive host kinase CDK7. Importantly, this strategy focuses on highly selective, 3D structure-derived CDK7 inhibitors carrying a warhead moiety that mediates covalent target binding. In summary, the main experimental findings of this study are as follows: (1) the in vitro verification of CDK7 inhibition and selectivity that confirms the warhead covalent-binding principle (by CDK-specific kinase assays), (2) the highly pronounced antiviral efficacies of the hit compounds (in cultured cell-based infection models) with half-maximal effective concentrations that reach down to picomolar levels, (3) a particularly strong potency of compounds against strains and reporter-expressing recombinants of HCMV (using infection assays in primary human fibroblasts), (4) additional activity against further herpesviruses such as animal CMVs and VZV, (5) unique mechanistic properties that include an immediate block of HCMV replication directed early (determined by Western blot detection of viral marker proteins), (6) a substantial drug synergism in combination with MBV (measured by a Loewe additivity fixed-dose assay), and (7) a strong sensitivity of clinically relevant HCMV mutants carrying MBV or ganciclovir resistance markers. Combined, the data highlight the huge developmental potential of this host-directed antiviral targeting concept utilizing covalently binding CDK7 inhibitors.

A new treatment for breast cancer using a combination of two drugs: AZD9496 and palbociclib

In this study, we examined a mathematical model of breast cancer (BC) treatment that combines an oral oestrogen receptor inhibitor, AZD9496 with Palbociclib, a selective inhibitor of cyclin- dependent kinases CDK4 and CDK6. Treatment is described by analytical functions that enable us to control the dosage and time interval of the treatment, thus personalising the treatment for each patient. Initially, we investigated the effect of each treatment separately, and finally, we investigated the combination of both treatments. By applying numerical simulations, we confirmed that the combination of AZD9496 with palbociclib was the optimal treatment for BC. The dosage of AZD9496 increased and decreased throughout the treatment period, while the intervals were constant between treatments. Palbociclib changed almost cyclically, whereas the time intervals remained constant. To investigate the mathematical model, we applied the singularly perturbed homotopy analysis method, which is a numerical algorithm. The significant advantage of this method is that the mathematical model does not have to contain a small parameter (as is standard in perturbation theory). However, it is possible to artificially introduce a small parameter into the system of equations, making it possible to study the model using asymptotic methods.

Design, synthesis and in vitro anti-proliferative evaluation of new pyridine-2,3-dihydrothiazole/thiazolidin-4-one hybrids as dual CDK2/GSK3β kinase inhibitors.

Herein, the molecular hybridization drug discovery approach was used in the design and synthesis of twelve novel pyridine-2,3-dihydrothiazole hybrids (2a,b-5a,b and 13a,b-14a,b) and fourteen pyridine-thiazolidin-4-one hybrids (6a,b-12a,b) as anti-proliferative analogues targeting CDK2 and GSK3β kinase inhibition. Almost all of the newly synthesized hybrids, including their precursors (1a,b), were evaluated for their anti-proliferative activity against three human cancer cell lines-MCF-7, HepG2 and HEp-2-as well as normal Vero cell lines. Both compounds 1a (pyridine-thiourea precursor) and 8a (pyridine-5-acetyl-thiazolidin-4-one hybrid) exhibited excellent anti-proliferative activity against HEp-2 (IC50 = 7.5 μg mL-1, 5.9 μg mL-1, respectively). Additionally, 13a (pyridine-5-(p-tolyldiazenyl-2,3-dihydrothiazole)) hybrid demonstrated excellent anti-proliferative activity against HepG2 (IC50 = 9.5 μg mL-1), with an acceptable safety profile against Vero (<45% inhibition at 100 μg mL-1) in the cases of 8a and 13a alone. The three promising anti-proliferative hybrids (1a, 8a, 13a) were selected for the assessment of their in vitro inhibitory kinase activity against CDK2/GSK3β using roscovitine (IC50 = 0.88 μg mL-1) and CHIR-99021 (IC50 = 0.07 μg mL-1) as references, respectively. Compound 13a was the most potent dual CDK2/GSK3β inhibitor (IC50 = 0.396 μg mL-1, 0.118 μg mL-1, respectively) followed by 8a (IC50 = 0.675 μg mL-1, 0.134 μg mL-1, respectively), and the weakest was 1a. To elucidate the mechanism of the most potent anti-proliferative 13a hybrid, further cell cycle analysis was performed revealing that it caused G1 cell cycle arrest and induced apoptosis. Moreover, it resulted in an increase in Bax and caspase-3 with a decrease in Bcl-2 levels in HepG2 cells compared with untreated cells. Finally, in silico drug likeness/ADME prediction for the three potent compounds as well as a molecular docking simulation study were conducted in order to explore the binding affinity and interactions in the binding site of each enzyme, which inspired their usage as anti-proliferative leads for further modification.

A recent tactic for searching CDK-7 kinase inhibitor by NCI database screening

The present study was based on an exploration of NCI database for searching specific CDK-7 kinase inhibitor by HTVS, SP, XP, molecular dock-ing, molecular dynamic simulation, and ADMET evaluation. The best CDK-7 kinase inhibitors (NCI613391, NCI169676, NCI281246, NCI339580) were identified via NCI database screening. The stability of binding interaction between receptor protein and protein-ligand complex of potent finding com-pounds (NCI613391) was further confirmed by dynamics simulations and MM- -GBSA. The RMSD value of receptor and receptor?ligand complexes was ana-lysed, and it revealed the stability of binding interactions and remained stable throughout the simulation. The RMSF values and gyration radius of the unbound receptor and backbone atoms of the complex were found to be equal, which indicates that the drug molecule inside the CDK7 receptor is also stable. The study of MM-GBSA data also revealed stronger binding interactions of ligands to CDK7 receptors. With the exception of NCI169676, all compounds were shown to be substrates for CYP450 2D6, CYP450 3A4, inhibitors of CYP450 2C9, and non-inhibitors of p-glycoprotein. All compounds were quali-fied and found suitable to be as drug-likeness according to the Lipinski rule, Ghose filter, MDDR like rule and CMC-like rule. The compound (NCI613391) exhibited human intestinal absorption (76.08%), displayed negative AMES and T.E.S.T (US-EPA) toxicity with OSIRIS property and found to be a promising CDK-7 kinase inhibitor and its efficacy may be further explored in clinical trials.

Cdk5-mediated oligodendrocyte myelin breakdown and neuroinflammation: Implications for the link between Type 2 Diabetes and Alzheimer's disease

Oligodendrocytes, crucial myelinating glia in the central nervous system, play a vital role in maintaining axonal integrity and facilitating efficient nerve impulse conduction. The degradation of myelin in oligodendrocytes has been implicated in Alzheimer's disease (AD) and cognitive dysfunction. Interestingly, individuals with Type 2 Diabetes (T2D) have a significantly higher likelihood of developing cognitive impairment, possibly due to insulin resistance and glucose toxicity within the central nervous system (CNS). However, the precise relationship between these two disorders remains elusive. Our study proposes a potential link between T2D and AD, involving Cdk5-mediated breakdown of oligodendrocyte myelin and neuroinflammation. In the context of T2D, glucose toxicity in oligodendrocytes leads to heightened Cdk5 kinase activity and cPLA2 hyperactivation, resulting in chronic inflammation and myelin deterioration. This myelin breakdown in oligodendrocytes is thought to contribute to the development of AD and cognitive dysfunction. Notably, the administration of a Cdk5 inhibitor (TFP5) effectively alleviates neuroinflammation and myelin degradation. Moreover, our findings demonstrate heightened activity of Cdk5, cPLA2, and phospho-cPLA2 levels in the brain of a mouse model with Type 2 Diabetes (T2D). Hence, our findings suggest that targeting Cdk5 could be a promising therapeutic strategy to counteract AD pathogenesis in T2D-related conditions.

Alternative cleavage and polyadenylation of the Ccnb1 mRNA defines accumulation of cyclin protein during the meiotic cell cycle.

Progression through the mitotic and meiotic cell cycle is driven by fluctuations in the levels of cyclins, the regulatory subunits controlling the localization and activity of CDK1 kinases. Cyclin levels are regulated through a precise balance of synthesis and degradation. Here we demonstrate that the synthesis of Cyclin B1 during the oocyte meiotic cell cycle is defined by the selective translation of mRNA variants generated through alternative cleavage and polyadenylation (APA). Using gene editing in mice, we introduced mutations into the proximal and distal polyadenylation elements of the 3' untranslated region (UTR) of the Ccnb1 mRNA. Through in vivo loss-of-function experiments, we demonstrate that the translation of mRNA with a short 3' UTR specifies Cyclin B1 protein levels that set the timing of meiotic re-entry. In contrast, translation directed by a long 3' UTR is necessary to direct Cyclin B1 protein accumulation during the MI/MII transition. These findings establish that the progression through the cell cycle is dependent on the selective translation of multiple mRNA variants generated by APA.

Abstract B057: Preclinical development of PKMYT1 and ATR inhibitor combinations

Abstract Background: Inactivation of the PKMYT1 kinase is synthetically lethal in tumor cells with genomic alterations leading to high replication stress, such as CCNE1 amplification frequently found in high-grade serous ovarian, uterine, and gastro-esophageal cancers. On this basis, we developed lunresertib (RP-6306), a first-in-class, potent and selective oral PKMYT1 inhibitor currently in Phase I clinical trials (NCT04855656). We have shown previously that PKMYT1 inhibition abrogates an inhibitory phosphorylation of the cyclin-dependent kinase CDK1 at threonine 14 (CDK1 pT14), leading to premature mitotic entry and catastrophic chromosome shattering specifically in cyclin E-high tumor cells, but not in cells with normal cyclin E levels. This leads to robust tumor growth inhibition by lunresertib in cyclin E-high pre-clinical models. We hypothesized that inhibition of the DNA damage response kinase ATR may further potentiate the tumor cytotoxicity of lunresertib, as ATR is known to regulate CDK1 pT14 levels in a parallel fashion to PKMYT1 through inhibition of the CDC25 family of phosphatases. Here we present the pre-clinical validation of this hypothesis. Methods: We used in vitro cell viability studies to determine the level of synergy between lunresertib and the ATR inhibitor camonsertib (RP-3500) in various engineered as well as tumor-derived models. We investigated the mechanism of action of the lunresertib/camonsertib combination using high content imaging and immunoblotting for DNA damage and cell cycle markers in cultured cells. Finally, we have evaluated the pre-clinical efficacy and tolerability of lunresertib/camonsertib combinations in vivo using mouse xenograft models. Results: In CCNE1-overexpressing cells, combining lunresertib with camonsertib led to a synergistic increase in cytotoxicity and catastrophic DNA damage at doses that did not affect wild type counterparts. Increased DNA damage resulted primarily from enhanced CDK1 dephosphorylation when lunresertib was combined with camonsertib, leading to robust and rapid induction of premature mitosis. Altogether these data suggest that camonsertib synergistically potentiates lunresertib’s mechanism of action. Consistent with that, we observed synergy between camonsertib and lunresertib in models carrying genomic biomarkers predictive of lunresertib sensitivity (CCNE1 amplification, FBXW7 loss-of-function), and not unrelated DNA repair defects (ATM or BRCA1 loss-of-function). In mouse xenograft models of tumors carrying predictive molecular alterations, the combination of lunresertib/camonsertib was well tolerated, enhanced CDK1 dephosphorylation and DNA damage, and was highly efficacious, including generating tumor regressions at doses sub-therapeutic as monotherapy. Conclusions: Lunresertib and camonsertib elicit synergistic activity in pre-clinical models of CCNE1-amplified and cyclin E-overexpressing tumors due to a complementary mechanism of action. These data provide a rationale for clinical development of PKMYT1 and ATR inhibitor combinations. Citation Format: David Gallo, Marc L. Hyer, Giovanni Martino, Adam Pertrone, Elia Aguado-Fraile, Rosie Kryczka, Jimmy Fourtounis, Rino Stocco, Stephen J. Morris, C. Gary Marshall, Anne Roulston, Michael Zinda, Michal Zimmermann. Preclinical development of PKMYT1 and ATR inhibitor combinations [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 B057.

Single Cell Transcriptome Comparisons of Diagnosis-Relapse Pairs Uncover Programs Associated with Chemoresistance in Pediatric AML

Background: Relapse rates for pediatric AML remain unacceptably high at nearly 50% after frontline therapy. Once relapse occurs, the disease is often refractory to further chemotherapy. Prior studies have shown that acquired mutations conferring chemoresistance are uncommon, and most relapses are thought to arise from the selection of clones already present at diagnosis. Therefore, characterizing chemoresistant disease at the gene expression level could lead to new insights and novel therapy targets. Profiling cases by bulk RNA sequencing has revealed important disease features such as novel gene fusions. However, identifying resistant subpopulations by bulk sequencing is challenging, particularly if they constitute small fractions of the leukemia cells at diagnosis. We hypothesize that by applying single-cell transcriptome profiling (scRNA-seq) to diagnosis-relapse pairs we can identify and characterize the subclones selected by treatment. Additionally, we will discover pathways and gene-expression programs that are associated with chemoresistance. Methods: We profiled thirteen diagnosis-relapse pairs from pediatric AML patients who were treated on or similarly to the Children's Oncology Group (COG) study AAML1031 and consented to banking of cells for future research. Six pairs were banked at Texas Children's Cancer Center and 7 were obtained from the COG biobank. The COG cases were flow-sorted to enrich for blasts before undergoing scRNA-seq with 10X-Chromium and Illumina NovaSeq systems. In addition, two local normal bone marrow cases were profiled, one with prior sorting and one with and without prior sorting. We included scRNA-seq profiles of 8 pediatric AML patients and 4 non-cancer bone marrow samples from Bailur et al. (JCI insight, 2020) as additional controls. Sequencing data were analyzed using CellRanger and Seurat. Four of the COG cases were profiled at the protein level by mass cytometry (CyTOF) and analyzed in Cytobank. Results: In total, for the 13 diagnosis-relapse pairs, 17,233 to 34,246 cells were profiled per patient, and 22,800 transcripts were detected. Unsupervised clustering of our samples and controls yielded 43 clusters (Figure 1) that could be confidently identified as either malignant or non-malignant (normal). We further classified all clusters into inferred hematopoietic developmental stages based on comparisons to publicly available expression reference data (Figure 2). We distinguished between patients with cell types at relapse that were also identified at diagnosis (Group 1), albeit often at a lower proportion, and patients whose cell types at relapse were not detected at diagnosis (Group 2). We also characterized 4 of the diagnosis-relapse pairs by CyTOF with an antibody panel that quantified surface lineage markers and intracellular signaling proteins. The protein expression data validated the RNA data in both the degree of differentiation of AML cells and the degree of similarity between diagnosis and relapse. We identified a Relapse-Up signature composed of 139 differentially expressed genes that were upregulated in AML clusters that expanded between diagnosis and relapse, compared to clusters that diminished between diagnosis and relapse. The Relapse-Up gene signature is enriched in genes that are co-expressed with FLT3, CDK6, and ILK kinase genes. With publicly available TARGET gene expression data, we confirmed that this gene signature is upregulated in relapsed AML samples when compared to diagnosis samples. We further used the profiles of the cell types that expanded between diagnosis and relapse to construct a risk-predictive function that predicted patient outcomes based on bulk RNA-Seq profiles of their diagnostic samples with significant accuracy. Conclusion: Single-cell RNA-seq of diagnosis-relapse pairs from pediatric patients with AML revealed distinct patterns of relapse that were also apparent at the protein level. The Relapse-Up signature was enriched in pediatric AML relapse samples in a large bulk RNA-seq data set, confirming its broader applicability. The kinase co-expression programs present in the signature are likely to be functionally associated with chemotherapy resistance and represent targets for future preclinical therapeutic studies.