Aurora Kinase Inhibitors Research Articles

Novel, highly potent PROTACs targeting AURORA-A kinase

The family of AURORA kinases is essential for cell cycle progression and dysregulation of AURORA-A in cancer led to a large number of clinical and pre-clinical inhibitors. However, ATP competitive AURORA-A inhibitors usually do not target non-catalytic functions that have also been identified as mechanisms promoting tumorigenesis. To target non-catalytic as well as catalytic functions, we developed a series of PROTACs (PROteolysis TArgeting Chimeras) based on the selective AURORA-A kinase inhibitor MK-5108 (VX-689) and the CEREBLON E3-ligase ligand thalidomide. The most potent PROTAC, JB301, had good physicochemical properties and cell penetration resulting in degradation of AURORA-A in leukemic cells at single digit nM concentration.

Co-treatment with miR-21-5p inhibitor and Aurora kinase inhibitor reversine suppresses breast cancer progression by targeting sprouty RTK signaling antagonist 2

ABSTRACT Numerous studies have reported the regulatory effects of miR-21-5p and reversine in human breast cancer (HBC). However, the mechanism of reversine and miR-21-5p has not been fully investigated in HBC. The aim of the current study was to assess the mechanism of action of reversine, with or without miR-21-5p, in HBC progression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot results confirmed the upregulation of miR-21-5p and downregulation of sprouty RTK signaling antagonist 2 (SPRY2) in HBC. Bioinformatics analysis and luciferase assay identified the correlation between miR-21-5p and SPRY2. Cell function experiment results indicated a decrease in migration, proliferation, and invasion of HBC cells treated with miR-21-5p inhibitor and reversine; however, an increase in apoptosis was observed in these cells. Apoptotic ability was more enhanced and migration, proliferation, and invasion were more impaired in HBC cells treated with both miR-21-5p inhibitor and reversine than in those treated individually with either inhibitors. SPRY2, downstream of miR-21-5p, participated in HBC progression with reversine. Overall, our study proved that combining the miR-21-5p inhibitor with reversine produced a synergistic effect by regulating SPRY2, thereby limiting HBC progression. This knowledge might offer insights into the clinical therapy of HBC.

E-cadherin is a robust prognostic biomarker in colorectal cancer and low expression is associated with sensitivity to inhibitors of topoisomerase, aurora, and HSP90 in preclinical models.

Cell–cell and cell–matrix adhesion proteins that have been implicated in colorectal epithelial integrity and epithelial‐to‐mesenchymal transition could be robust prognostic and potential predictive biomarkers for standard and novel therapies. We analyzed in situ protein expression of E‐cadherin (ECAD), integrin β4 (ITGB4), zonula occludens 1 (ZO‐1), and cytokeratins in a single‐hospital series of Norwegian patients with colorectal cancer (CRC) stages I–IV (n = 922) using multiplex fluorescence‐based immunohistochemistry (mfIHC) on tissue microarrays. Pharmacoproteomic associations were explored in 35 CRC cell lines annotated with drug sensitivity data on > 400 approved and investigational drugs. ECAD, ITGB4, and ZO‐1 were positively associated with survival, while cytokeratins were negatively associated with survival. Only ECAD showed independent prognostic value in multivariable Cox models. Clinical and molecular associations for ECAD were technically validated on a different mfIHC platform, and the prognostic value was validated in another Norwegian series (n = 798). In preclinical models, low and high ECAD expression differentially associated with sensitivity to topoisomerase, aurora, and HSP90 inhibitors, and EGFR inhibitors. E‐cadherin protein expression is a robust prognostic biomarker with potential clinical utility in CRC.

Improving Generation of Cardiac Organoids from Human Pluripotent Stem Cells Using the Aurora Kinase Inhibitor ZM447439.

Drug-induced cardiotoxicity reduces the success rates of drug development. Thus, the limitations of current evaluation methods must be addressed. Human cardiac organoids (hCOs) derived from induced pluripotent stem cells (hiPSCs) are useful as an advanced drug-testing model; they demonstrate similar electrophysiological functionality and drug reactivity as the heart. How-ever, similar to other organoid models, they have immature characteristics compared to adult hearts, and exhibit batch-to-batch variation. As the cell cycle is important for the mesodermal differentiation of stem cells, we examined the effect of ZM447439, an aurora kinase inhibitor that regulates the cell cycle, on cardiogenic differentiation. We determined the optimal concentration and timing of ZM447439 for the differentiation of hCOs from hiPSCs and developed a novel protocol for efficiently and reproducibly generating beating hCOs with improved electrophysiological functionality, contractility, and yield. We validated their maturity through electro-physiological- and image-based functional assays and gene profiling with next-generation sequencing, and then applied these cells to multi-electrode array platforms to monitor the cardio-toxicity of drugs related to cardiac arrhythmia; the results confirmed the drug reactivity of hCOs. These findings may enable determination of the regulatory mechanism of cell cycles underlying the generation of iPSC-derived hCOs, providing a valuable drug testing platform.

Abstract P078: Aurora A kinase inhibition with VIC-1911 overcomes intrinsic and acquired resistance to KRASG12C inhibition in KRAS(G12C)-mutated lung cancer

Abstract Oncogenic KRAS mutation is common in non-small cell lung cancer (NSCLC) and portends poor outcome. Direct KRASG12C inhibitors have clinical activity; however, intrinsic and acquired resistance to these drugs limits their utility. Aurora A Kinase (AURKA), a mitotic cell cycle regulator, has been considered as a key druggable KRAS effector and mediates adaptive resistance to direct KRASG12C inhibitors. We found that AURKA expression correlated with poor outcome of lung cancer patients in caBIG, GEO and TCGA, and its expression was greater in KRAS-mutated NSCLC cells resistant to KRASG12C inhibitor sotorasib (AMG510) compared to normal human tracheobronchial epithelial (NHTBE), lung fibroblast, and KRAS wild-type cells. We have previously demonstrated synergistic antitumor effects for combination AURKA and WEE1 inhibition. Here, we explored a novel combination of AURKA and KRASG12C inhibition in KRAS(G12C)-mutated NSCLC cells intrinsically resistant to the KRASG12C inhibitor, along with a combination of AURKA and WEE1 inhibition in mutant KRAS(G12C) lung cancer cells with acquired resistance to the inhibitor. To overcome resistance to the KRASG12C inhibitor sotorasib, we tested combination treatment with VIC-1911, a newly synthesized selective AURKA inhibitor, with sotorasib in sotorasib-resistant human lung cancer cells. Cooperative screening, Loewe plotting, and clonogenic survival assays were performed to determine synergistic antitumor effects. In addition, we established KRAS(G12C)-mutated human lung cancer cell lines with acquired resistance to sotorasib through the means of escalated incremental dosing or sorting non-quiescent cells after drug exposure. Synergy was further confirmed with cell cycle distribution, phenotypic mitotic catastrophe on confocal microscopy, and induction of apoptosis. Interestingly, cells intrinsically resistant to sotorasib showed profound synergistic suppressive effect on cancer cell survival with addition of VIC-1911 compared to sotorasib-sensitive cells (Loewe synergy scores: NCI-H1792=16.03; HCC44=14.37; NCI-H358=1.48). Further, the combination of AURKA and WEE1 inhibition synergistically induced greater cell death in NCI-H358 lung carcinoma cells harboring acquired resistance to sotorasib, with dramatic induction of Bim. Moreover, the combination of AURKA and WEE1 inhibition resulted in a synergistic tumor control in KRAS/TP53-mutated lung cancer xenograft models. Our findings suggest AURKA activation leads to both intrinsic and acquired resistance to sotorasib in KRAS(G12C)-mutated NSCLC; therefore, addition of AURKA inhibition to sotorasib may be a promising therapeutic approach in NSCLC with intrinsic resistance to direct KRASG12C inhibitors, while the combination of AURKA and WEE1 inhibition merits exploration in acquired resistance to these agents. Citation Format: Jong Woo Lee, Sundong Kim, Cindy Yang, Barbara Burtness. Aurora A kinase inhibition with VIC-1911 overcomes intrinsic and acquired resistance to KRASG12C inhibition in KRAS(G12C)-mutated lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P078.

Abstract P164: Identification of the p53 negative feedback loop as a target for enhancing selinexor activity in neuroblastoma

Abstract Neuroblastoma (NB) is the most common extra-cranial pediatric solid tumor and accounts for 15% of cancer-related deaths in children, highlighting an unmet need for novel therapies. Selinexor is a small molecule inhibitor of XPO1. XPO1 shuffles cargo proteins with a nuclear export sequence from the nucleus to the cytosol, many of which are essential for cancer growth and cell maintenance. XPO1 also has a prognostic significance in patients with NB since those with high XPO1 levels have an inferior event-free (P = 1.2e-5, Bonferroni P = 5.4e-3) and overall survival (P = 4.5e-7, Bonferroni P = 2.1e-4) compared to patients with low XPO1 expression. To characterize the global transcriptomic consequences of XPO1 inhibition, we performed RNA-sequencing in the NB cell line IMR-5 where we performed a knockdown of XPO1 or treated cells with selinexor. Conducting a gene set enrichment analysis, we found genes encoding MYCN targets were significantly downregulated by selinexor (normalized enrichment score [NES] = -2.7; nominal P = <0.001) or XPO1 knockdown (NES = 2.2; nominal P = <0.001). Genes associated with the p53 pathway were significantly enriched (NES = 2.7; nominal P = <0.001 [selinexor treatment]; NES = 2.2; nominal P = <0.001 [XPO1knockdown]). Given the large number of XPO1-targets, we applied an advanced tandem mass tag (TMT)-liquid chromatography (LC)/LC-MS/MS based pipeline for deep whole-cell proteomic and phospho-proteomic analyses to interrogate unknown mechanisms of selinexor’s action. We found that selinexor induced its cytotoxic effects in NB through the nuclear accumulation of p53. Drug sensitivity assays with selinexor against seven human and one murine NB cell lines yielded good (median EC50=28.7nM) and intermediate responses (median EC50=133.5nM) with all intermediate responders harboring a TP53 mutation while good responders had TP53 wild type status. Further, phosphoproteomic analysis showed that nuclear accumulation of p53 led to an increase in p53 phosphorylation at site S315 marking p53 for MDM2-mediated ubiquitination. Since this phosphorylation step is undertaken predominantly by aurora kinase A (AURKA), we used a clinically available AURKA inhibitor, alisertib, in combination with selinexor and found synergistic killing activity in vitro and enhanced growth inhibition in two orthotopic xenograft mouse models. These findings suggest a potential therapeutic benefit using selinexor and alisertib to synergistically increase p53-mediated cytotoxicity of high-risk NB. Given the known toxicity profiles and potential therapeutic benefits, these findings support a clinical trial of selinexor and alisertib in children with high-risk NB. Citation Format: Rosa Nguyen, Hong Wang, Ming Sun, Dong Geun Lee, Junmin Peng, Carol J. Thiele. Identification of the p53 negative feedback loop as a target for enhancing selinexor activity in neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P164.

Discovery of potential Aurora-A kinase inhibitors by 3D QSAR pharmacophore modeling, virtual screening, docking, and MD simulation studies

The Aurora-kinase family comprises of cell cycle-regulated serine/threonine kinases playing a vital role during mitosis. Aurora-A kinase is involved in multiple mitotic events in cell cycle and is a major regulator of centrosome function during mitosis. Aurora-A is overexpressed in breast, lung, colon, ovarian, glial, and pancreatic cancer. Hence, Aurora-A kinase is a promising target in cancer therapy. In our current study, a four-point 3D QSAR pharmacophore model has been generated using substituted pyrimidine class of Aurora-A kinase inhibitors. It had a fixed cost value 88.7429. The model mapped well to the external test set comprising of clinically active molecules, with a correlation coefficient r = 0.99. From the mapping, it was found that the hydrophobic features (HY) of a molecule play an important role for Aurora-A kinase inhibitory activity, whereas the ring aromatic feature provides geometric constraint for spatial alignment of different functional group. The hypothesis, with one hydrogen bond acceptor, two ring aromatic features, and one hydrophobic feature, was selected to screen miniMaybridge database. The screened ligands were filtered on the basis of activity, shape, and drug likeliness. This led to the identification of five top hits. These identified potential leads were further subjected to docking with the ATP-binding site of Aurora-A kinase. The molecular dynamic simulation studies of top lead molecules having diverse scaffolds endorsed that the identified molecules had distinctive ability to inhibit Aurora-A kinase. Thus, this study may facilitate the medicinal chemists to design promising ligands with various scaffolds to inhibit Aurora-A kinase. Communicated by Ramaswamy H. Sarma

Aurora kinase A regulates liver regeneration through macrophages polarization and Wnt/β-catenin signalling.

Liver regeneration is a complex process regulated by a variety of cells, cytokines and biological pathways. Aurora kinase A (AURKA) is a serine/threonine kinase that plays a role in centrosome maturation and spindle formation during the cell division cycle. The purpose of this study was to further explore the mechanism of AURKA on liver regeneration and to identify new possible targets for liver regeneration. The effect and mechanism of AURKA on liver regeneration were studied using a 70% hepatectomy model. Human liver organoids were used as an in vitro model to investigate the effect of AURKA on hepatocyte proliferation. AURKA inhibition significantly reduced the level of β-catenin protein by reducing the phosphorylation level of glycogen synthase kinase-3β (GSK-3β), leading to the inhibition of liver regeneration. Further studies showed that AURKA co-localized and interacted with GSK-3β in the cytoplasm of hepatocytes. When phosphorylation of GSK-3β was enhanced, the total GSK-3β level remained unchanged, while AURKA was not affected, and β-catenin protein levels were increased. In addition, AURKA inhibition affected the formation and proliferation of human liver organoids. Furthermore, AURKA inhibition led to the polarization of M1 macrophages and the release of interleukin-6 and Tumour necrosis factor α, which also led to reduced liver regeneration and increased liver injury. These results provide more details on the mechanism of liver regeneration and suggest that AURKA is an important regulator of this mechanism.

EPCO-18. A MESENCHYMAL CELL POPULATION MEDIATES RESISTANCE TO AURORA KINASE INHIBITORS IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) remains the most common adult brain cancer, with a dismal average patient survival of less than two years. No new treatments have been approved for GBM since the introduction of the alkylating agent temozolomide in 2005. Even then, temozolomide treatment only increases the average survival of GBM patients by a few months. Thus, novel therapeutic options are direly needed. The aurora kinases A and B are targetable and overexpressed in GBM, and their expression is highly correlated with patient survival outcomes. Our lab has found that small molecule aurora kinase inhibition reduces GBM tumor growth in vitro and in vivo, however, eventually tumors still grow. Computational analysis integrating compound transcriptional response signatures from the LINCS L1000 dataset with the single-cell RNA-sequencing data of patient GBM tumors resected at the University of Miami predicts that aurora inhibition targets a subset of cells present within any GBM tumor. Results of in vivo single-cell perturbation experiments with the aurora kinase inhibitor alisertib coincide with our predictions and reveal a cellular transcriptional phenotype resistant to aurora kinase inhibition, characterized by a mesenchymal expression program. We find that small molecules that are predicted to target different cell populations from alisertib, including this resistant mesenchymal population, synergize with alisertib to kill GBM cells. As a whole, we have identified the cellular population resistant to aurora kinase inhibition and have developed an analytical framework that identifies synergistic small molecule combinations by identifying compounds that target transcriptionally distinct cellular populations within GBM tumors.

EXTH-09. TUMOR PHARMACOKINETICS, PHARMACODYNAMICS AND RADIATION SENSITIZATION IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA TREATED WITH THE AURORA KINASE A INHIBITOR LY3295668

Abstract Cell-cycle deregulation is at the crux of all malignancies, including glioblastoma (GBM). Aurora Kinase A (AURKA) plays a central role in G2/M transition and faithful chromosome segregation. In this study, we evaluated the pharmacokinetics, pharmacodynamics, and radiation sensitization properties of LY3295668, a highly specific AURKA inhibitor, in orthotopic patient-derived xenograft (PDX) models of GBM. Mice with intracranial tumors were randomized to 50 mg/kg LY3295668 PO BID x 4 days vs. placebo. LY3295668 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 pAURKA (T288), phospho-Histone H3 (pHH3), and cleaved caspase 3 (CC3). For survival studies, mice with intracranial tumors were randomized to four cohorts – vehicle, radiotherapy, LY3295668 monotherapy, and LY3295668 plus radiotherapy. The median unbound concentration of LY3295668 was 270.88 nmol/L and 22.33 nmol/kg in plasma and tumor tissue, respectively – significantly higher than the biochemical IC50 of LY3295668 for AURKA inhibition (0.8 nM). A decrease in pHH3(+) cells (0.8% vs. 6.4%, p=0.036) indicated drug-induced mitotic arrest and was accompanied by an increase in CC3(+) cells (6.4% vs. 8.0%, p=0.67). Combination of LY3295668 with radiotherapy prolonged survival compared to either therapy alone in orthotopic GBM PDX models. LY3295668 is well tolerated, achieves pharmacologically-relevant unbound concentrations in GBM PDX models, and is associated with significant target modulation. Preclinical combination of LY3295668 with radiation therapy leads to synergistic effects and supports future clinical study of this multimodal strategy in glioblastoma patients.

Treatment of RB-deficient retinoblastoma with Aurora-A kinase inhibitor.

Retinoblastoma, also known as ocular cancer, usually affects children under the age of five. The standard of care for managing early-stage retinoblastoma is a combination of vincristine, carboplatin, and etoposide. However, this combination-based modality has limited applications owing to its side and late effects. Moreover, in advanced tumor stages, nearly 50% of patients would suffer a partial or full loss of vision. Therefore, therapies that preserve vision and reduce side effects are urgently required. Here, we focused mainly on the common loss-of-function (LOF) mutation of retinoblastoma gene 1 (RB1) in advanced retinoblastoma and investigated the synthetic lethality between RB1-LOF and Aurora kinase inhibition. We showed that Aurora kinase A inhibition could lead to cell mitotic abnormality and apoptosis, and demonstrated in vivo efficacy in a mouse model xenografted with RB1-deficient retinoblastoma. Our findings provide a promising druggable molecular target and potential clinical strategy for tackling retinoblastoma disease.

Alisertib Synergistically Strengthens the Anti-Leukemia Activity of Venetoclax in TCF3-Hlf B-ALL

Background: Despite maximally-intensive chemotherapy and stem cell transplantation, survival of patients with the very rare t(17;19)/TCF3-HLF B-acute lymphoblastic leukemia (B-ALL) subtype remains effectively 0%. Prior studies have demonstrated association of the oncogenic TCF3-HLF fusion protein with multi-drug resistance via increased expression of ABC and P-glycoprotein drug efflux transporters, as well as via upregulation of pro-survival Ras and BCL-2 pathways. Preclinical studies and small clinical case series of targeted inhibitor addition to chemotherapy or antibody-based and cellular immunotherapies have aimed to improve outcomes for children with TCF3-HLF ALL. Unfortunately, targeting of these activated pathways with the BCL-2 inhibitor venetoclax or other small molecule inhibitors (SMIs) has been minimally or only transiently effective, suggesting more complex mechanism(s) of chemoresistance. In recent years, many patients with relapsed TCF3-HLF ALL have enrolled on clinical trials of CD19- or CD22-targeted immunotherapies. However, TCF3-HLF ALL frequently harbours deactivating mutations in PAX5, a major B-cell regulator and indispensable CD19 transcription factor, placing immunotherapy-treated patients at higher risk of CD19 antigen-loss relapse. New therapies remain needed to prevent relapse and attempt cure.Methods: We designed an unbiased kinome-wide CRISPR/Cas9 library to identify essential drivers in TCF3-HLF leukemogenesis. We screened the human TCF3-HLF ALL cell line HAL-01 and our TCF3-HLF ALL patient-derived xenograft (PDX) model ALL1807 (Hurtz JCI 2020, Schultz Genome Biol 2021), then validated identified targets using 49 SMIs targeting receptor tyrosine kinases (RTK), MEK signaling, and cell cycle pathways. We selected promising candidate inhibitor pairings with non-overlapping mechanisms of action and assessed for in vitro drug synergy via SynergyFinder analyses. Finally, we assessed the in vivo activity of targeted inhibitors in ALL1807 and two newly established TCF3-HLF ALL PDX models (CPCT-0002, CPCT-0003) created from primary pediatric specimens obtained via the LEAP Consortium (Pikman Cancer Disc 2021).Results: RNA-sequencing of HAL-01 and ALL1807 cells followed by functional protein association (STRING) analysis confirmed a network of significantly upregulated (>3-fold) plasma membrane and cytoplasm components of RTK pathways as well as BCL-2. The intersection of the results of the SMI drug library screen with the top 1% targets identified in CRIPSR/Cas9 screen determined p120-RasGAP and Aurora kinase A (AURKA) as therapeutic targets in TCF3-HLF ALL. In vitro treatment of HAL-01 or ALL1807 cells with the RasGAP inhibitor, pluripotin, or the AURKA inhibitor, alisertib, across a range of concentrations demonstrated robust anti-ALL activity. AURKA and RasGAP co-immunoprecipitated and this protein complex was disrupted with alisertib or pluripotin treatment. The AURKB inhibitor barisertib had minimal activity against TCF3-HLF ALL cells, confirming preferential dependency of these cells upon AURKA. Treatment of TCF3-HLF ALL cells with the BCL-2i venetoclax did not disrupt the AURKA/RasGAP complex, suggesting its different mechanism of action and potential for combinatorial drug therapy. Next, we found that alisertib and venetoclax synergistically killed TCF3-HLF ALL cells. Finally, we observed superior inhibition of in vivo leukemia with dual AURKA and BCL-2 inhibitor treatment of three TCF3-HLF ALL PDX models compared to single-agent alisertib or venetoclax (Figure 1).Conclusions: We identified AURKA as a critical new driver in TCF3-HLF ALL via orthogonal genetic and functional assays and confirmed prior observations of BCL-2 dependency in our models. We validated these key targets via in vitro and in vivo pharmacologic inhibition studies with drug synergy detected with combined alisertib and venetoclax in human TCF3-HLF ALL cell lines and PDX models. We posit that dual AURKA and BCL-2 inhibition is a clinically-pragmatic and potentially effective therapeutic strategy for patients with this rare, but highly fatal, leukemia subtype that merits formal clinical investigation. [Display omitted] DisclosuresCarroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy. Stegmaier: Auron Therapeutics, Kronos Bio, AstraZeneca, Novartis Institute of Biomedical Research: Consultancy, Research Funding. Tasian: Incyte Corporation: Research Funding; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Aleta Biotherapeutics: Consultancy.

Changes in ABC Transporter Expression during Hematopoiesis Cause Lineage-Biased Cytopenias in Patients Treated with Aurora Kinase Inhibitors

Chemotherapy-induced cytopenias are a prevalent and significant issue that worsens clinical outcomes and hinders the effective treatment of cancer. While they are classically associated with traditional cytotoxic chemotherapies, they also occur with newer targeted small molecule drugs and the factors that determine the hematotoxicity profiles of chemotherapies are not fully understood. Here, we explore why Aurora kinase inhibitor drugs cause preferential neutropenia when compared to the cytopenic profiles of targeted small molecule cancer drugs that are FDA approved. By studying drug responses of healthy human hematopoietic cells in vitro and analyzing existing published clinical datasets, we provide evidence that the enhanced vulnerability of neutrophil-lineage cells to Aurora kinase inhibitors is acquired at an early stage of differentiation and is caused by developmental changes in the expression pattern of ATP-binding cassette (ABC) transporters. These data show that hematopoietic cell-intrinsic expression of ABC transporters may be an important factor that determines how some chemotherapies affect the bone marrow. DisclosuresDavid: AstraZeneca: Current Employment. Randle: AstraZeneca: Current Employment. Polanska: AstraZeneca: Current Employment. Urosevic: AstraZeneca: Current Employment. Travers: AstraZeneca: Ended employment in the past 24 months. Ingber: Emulate: Membership on an entity's Board of Directors or advisory committees; BOA Biomedical: Membership on an entity's Board of Directors or advisory committees; Freeflow Medical Devices Inc: Membership on an entity's Board of Directors or advisory committees.

Aurora Kinase Inhibition Overcomes Primary Venetoclax Failure and Leads to Synthetic Lethality in BCL2-Positive Lymphomas Via Upregulation of P53/P21/BAX Axis

Aurora Kinase Inhibition Overcomes Primary Venetoclax Failure and Leads to Synthetic Lethality in BCL2-Positive Lymphomas Via Upregulation of P53/P21/BAX Axis

Biological and Clinical Significance of PIM1 Genetic Alterations in Diffuse Large B-Cell Lymphoma

Background: PIM1 is a somatic hypermutation gene in diffuse large B-cell lymphoma (DLBCL) and its inhibitors exhibit a great value in application in multiple types of lymphoma. Nevertheless, investigation on its genetic alterations, biological characteristics, clinical significance and response to drugs is still lacking.Methods: We integrated the genome sequencing (discovery cohort, n=162; validation cohort, n=1001) and transcriptome sequencing (discovery cohort, n=140; validation cohort, n=928) to capture more detailed insights into the potentially biological functions of PIM1 genetic alterations, and analyzed their relationship with biological characteristics and clinical value which provide a possibility for risk stratification and therapeutic exploitation for patients with DLBCL.Results: PIM1 mutations were identified in 28.4% of DLBCL patients and significantly correlated with higher IPI scores (P=0.013), disease relapse (P=0.031) and CNS and/or testis involvement (P=0.001), as well as inferior PFS (P=0.022) and OS (P=0.0022). Multivariate analysis revealed that PIM1 mutation status was an independent poor prognostic factor (HR=2.86; 95% CI, 1.40-5.84; P=0.004). The most frequent PIM1 mutation type was missense mutations (84.1%), followed by frameshift deletions and nonsense mutations. During the distribution of base substitutions, C > T base substitution was predominant mutation type (54.4%), followed by C >G transversion (29.3%). During different exons, exon 4 of PIM1 was most often mutated. PIM1 mutations significantly co-occurred with the mutations of SETD1B (P<0.001) and CD79B (P=0.001), and was mutually exclusive to the SPEN mutation (P=0.024). We also explored the relationship between PIM1 mutations and genes distributed on previously reported signaling pathways in DLBCL and uncovered that mutations in MYD88 (P<0.001) and PRDM1 (P<0.001) involved in the NF-κB pathway were significantly enriched in the patients with PIM1 mutations. Unsurprisingly, patients with PIM1 mutations exhibited higher mutation frequencies in CD79B (P=0.001) involved in BCR pathway. PIM1 mutations were involved in immunoglobulin-related immune response, complement activation, B cell mediated immunity, B cell activation, antigen binding, and cytokine activity, which contributed to the signaling pathways of tumor microenvironment (e.g. cytokine-cytokine receptor interaction, chemokine signaling pathway, IL-17 signaling pathway, TNF signaling pathway), JAK-STAT and NF-κB. By MCODE analysis, five significant modules were obtained from the protein-protein interaction (PPI) network. Finally, a PIM1 mutation-related gene signature consisting of some independent prognostic factors was developed. According to the risk score, patients with high-risk score exhibited significantly shorter OS (P=0.0016) and PFS (P<0.001). The areas under the curve (AUCs) for the predictions of 1-, 3-, and 5-year OS were respectively 0.69, 0.70, and 0.72, suggesting that the risk score based on the PIM1 mutation-related gene signature had satisfactory sensitivity and specificity. We further found that compared to patients with low-risk score, patients with high-risk score had higher sensitivity to some drugs of targeting the immune microenvironment, including TGFβ receptor inhibitors SB525334 (P<0.0001) and SB505124 (P<0.0001) and immunomodulator Lenalidomide (P=0.041), as well as NF-κB inhibitors Parthenolide (P<0.0001) and TPCA-1 (P<0.0001) and JAK inhibitors Ruxolitinib (P=0.014) and TG101348 (P=0.0053), accompanying with significantly lower IC50 values. In addition, another common chemotherapeutic drug Gemcitabine was also predicted to be more sensitive for patients with high-risk score (P=0.047). Other targeted drugs such as Aurora kinase inhibitors VX-680 (P<0.0001) and ZM-447439 (P=0.014), Bcl-2 inhibitors Obatoclax Mesylate (P=0.00036) and Navitoclax (P<0.0001), and CDK inhibitors Roscovitine (P=0.0012), AT-7519 (P=0.0033), PHA-793887 (P<0.0001) and THZ2-49 (P=0.0053) also exhibited higher drug sensitivity for patients with high-risk score.Conclusions: PIM1 mutations play a vital role in patient risk stratification and provide novel insights into therapeutic decision making for DLBCL patients with high-risk score. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Aurora kinase B inhibits aurora kinase A to control maternal mRNA translation in mouse oocytes.

Mammalian oocytes are transcriptionally quiescent, and meiosis and early embryonic divisions rely on translation of stored maternal mRNAs. Activation of these mRNAs is mediated by polyadenylation. Cytoplasmic polyadenylation binding element 1 (CPEB1) regulates mRNA polyadenylation. One message is aurora kinase C (Aurkc), encoding a protein that regulates chromosome segregation. We previously demonstrated that AURKC levels are upregulated in oocytes lacking aurora kinase B (AURKB), and this upregulation caused increased aneuploidy rates, a role we investigate here. Using genetic and pharmacologic approaches, we found that AURKB negatively regulates CPEB1-dependent translation of many messages. To determine why translation is increased, we evaluated aurora kinase A (AURKA), a kinase that activates CPEB1 in other organisms. We find that AURKA activity is increased in Aurkb knockout mouse oocytes and demonstrate that this increase drives the excess translation. Importantly, removal of one copy of Aurka from the Aurkb knockout strain background reduces aneuploidy rates. This study demonstrates that AURKA is required for CPEB1-dependent translation, and it describes a new AURKB requirement to maintain translation levels through AURKA, a function crucial to generating euploid eggs.

Disease Modeling on Tumor Organoids Implicates AURKA as a Therapeutic Target in Liver Metastatic Colorectal Cancer.

Background & AimsPatient-derived tumor organoids recapitulate the characteristics of colorectal cancer (CRC) and provide an ideal platform for preclinical evaluation of personalized treatment options. We aimed to model the acquisition of chemotolerance during first-line combination chemotherapy in metastatic CRC organoids.MethodsWe performed next-generation sequencing to study the evolution of KRAS wild-type CRC organoids during adaptation to irinotecan-based chemotherapy combined with epidermal growth factor receptor (EGFR) inhibition. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 protein (Cas9)-editing showed the specific effect of KRASG12D acquisition in drug-tolerant organoids. Compound treatment strategies involving Aurora kinase A (AURKA) inhibition were assessed for their capability to induce apoptosis in a drug-persister background. Immunohistochemical detection of AURKA was performed on a patient-matched cohort of primary tumors and derived liver metastases.ResultsAdaptation to combination chemotherapy was accompanied by transcriptomic rather than gene mutational alterations in CRC organoids. Drug-tolerant cells evaded apoptosis and up-regulated MYC (c-myelocytomatosis oncogene product)/E2F1 (E2 family transcription factor 1) and/or interferon-α–related gene expression. Introduction of KRASG12D further increased the resilience of drug-persister CRC organoids against combination therapy. AURKA inhibition restored an apoptotic response in drug-tolerant KRAS–wild-type organoids. In dual epidermal growth factor receptor (EGFR)– pathway blockade-primed CRC organoids expressing KRASG12D, AURKA inhibition augmented apoptosis in cases that had acquired increased c-MYC protein levels during chemotolerance development. In patient-matched CRC cohorts, AURKA expression was increased in primary tumors and derived liver metastases.ConclusionsOur study emphasizes the potential of patient-derived CRC organoids in modeling chemotherapy tolerance ex vivo. The applied therapeutic strategy of dual EGFR pathway blockade in combination with AURKA inhibition may prove effective for second-line treatment of chemotolerant CRC liver metastases with acquired KRAS mutation and increased AURKA/c-MYC expression.

Viability fingerprint of glioblastoma cell lines: roles of mitotic, proliferative, and epigenetic targets

Despite the use of multimodal treatment combinations, the prognosis of glioblastoma (GB) is still poor. To prevent rapid tumor recurrence, targeted strategies for the treatment of GB are widely sought. Here, we compared the efficacy of focused modulation of a set of signaling pathways in two GB cell lines, U-251 MG and T98-G, using a panel of thirteen compounds targeting cell cycle progression, proliferation, epigenetic modifications, and DNA repair mechanism. In parallel, we tested combinations of these compounds with temozolomide and lomustine, the standard chemotherapy agents used in GB treatment. Two major trends were found: within individual compounds, the lowest IC50 values were exhibited by the Aurora kinase inhibitors, whereas in the case of mixtures, the addition of DNA methyltransferase 1 inhibitor azacytidine to lomustine proved the most beneficial. The efficacy of cell cycle-targeting compounds was further augmented by combination with radiation therapy using two different treatment regimes. The potency of azacytidine and lomustine mixtures was validated using a unique assay pipeline that utilizes automated imaging and machine learning-based data analysis algorithm for assessment of cell number and DNA damage extent. Based on our results, the combination of azacytidine and lomustine should be tested in GB clinical trials.

Synthesis, biological evaluation and molecular docking of new sulfonamide-based indolinone derivatives as multitargeted kinase inhibitors against leukemia

Series of novel sulfonamide-based 3-indolinones 3a-m and 4a-f were designed, synthesized and then their cytotoxic activity was evaluated against a panel of sixty cancer cell lines. This screening indicated that 4-(2-(5-fluoro-2-oxoindolin-3-ylidene)acetyl)phenyl benzenesulfonate (4f) possessed promising cytotoxicity against CCRF-CEM and SR leukemia cell lines with IC50 values 6.84 and 2.97 µM, respectively. Further investigation of the leukemic cytotoxicity of compound 4f was carried out by performing PDGFRα, VEGFR2, Aurora A/B and FLT3 enzyme assays and CCRF-CEM and SR cell cycle analysis. These investigations showed that compound 4f exhibited pronounced dual inhibition of both kinases PDGFRα and Aurora A with potency of 24.15 and 11.83 nM, respectively. The in vitro results were supported by molecular docking studies in order to explore its binding affinity and its key amino acids interactions. This work represents compound 4f as a promising anticancer agent against leukemia.

Reconstitution of muscle cell microtubule organization in vitro.

Skeletal muscle differentiation occurs as muscle precursor cells (myoblasts) elongate and fuse to form multinucleated syncytial myotubes in which the highly-organized actomyosin sarcomeres of muscle fibers assemble. Although less well characterized, the microtubule cytoskeleton also undergoes dramatic rearrangement during myogenesis. The centrosome-nucleated microtubule array found in myoblasts is lost as the nuclear membrane acquires microtubule nucleating activity and microtubules emerge from multiple sites in the cell, eventually rearranging into a grid-like pattern in myotubes. In order to characterize perinuclear microtubule organization using a biochemically tractable system, we isolated nuclei from mouse C2C12 skeletal muscle cells during the course of differentiation and incubated them in cytoplasmic extracts prepared from eggs of the frog Xenopus laevis. Whereas centrosomes associated with myoblast nuclei gave rise to radial microtubule arrays in extracts, myotube nuclei produced a sun-like pattern with microtubules transiently nucleating from the entire nuclear envelope. Perinuclear microtubule growth was suppressed by inhibition of Aurora A kinase or by degradation of RNA, treatments that also inhibited microtubule growth from sperm centrosomes. Myotube nuclei displayed microtubule motor-based movements leading to their separation, as occurs in myotubes. This in vitro assay therefore recapitulates key features of microtubule organization and nuclear movement observed during muscle cell differentiation.