Aurora Kinase Inhibitors Research Articles

Combined inhibition of aurora kinases and Bcl-xL induces apoptosis through select BH3-only proteins

Aurora Kinases (AURK) are mitotic kinases important for regulating cell cycle progression. Small molecule inhibitors of AURK have shown promising antitumor effects in multiple cancers; however, the utility of these inhibitors as inducers of cancer cell death has thus far been limited. Here, we examined the role of the Bcl-2 family proteins in AURK inhibition-induced apoptosis in colon cancer cells. We found that Alisertib and Danusertib, two small molecule inhibitors of AURK, are inefficient inducers of apoptosis in HCT116 and DLD-1 colon cancer cells, the survival of which requires at least one of the two anti-apoptotic Bcl-2 family proteins, Bcl-xL and Mcl-1. We further identified Bcl-xL as a major suppressor of Alisertib or Danusertib-induced apoptosis in HCT116 cells. We demonstrate that combination of a BH3-mimetic inhibitor (ABT-737), a selective inhibitor of Bcl-xL, Bcl-2, and Bcl-w, with Alisertib or Danusertib potently induces apoptosis through the Bcl-2 family effector protein Bax. In addition, we identified Bid, Puma, and Noxa, three of the BH3-only proteins of the Bcl-2 family, as mediators of Alisertib/ABT-737-induced apoptosis. We show while Noxa promotes apoptosis by constitutively sequestering Mcl-1, Puma becomes associated with Mcl-1 upon Alisertib treatment. On the other hand, we found Alisertib treatment causes activation of caspase-2, which promotes apoptosis by cleaving Bid into tBid, a suppressor of both Bcl-xL and Mcl-1. Together, these results define the Bcl-2 protein network critically involved in AURK inhibitor-induced apoptosis, and suggest that BH3-mimetics targeting Bcl-xL may help overcome resistance to AURK inhibitors in cancer cells.

The inhibition of Aurora A kinase regulates phospholipid remodeling by upregulating LPCAT1 in glioblastoma.

Metabolic reprogramming is a common feature of glioblastoma (GBM) progression and metastasis. Altered lipid metabolism is one of the most prominent metabolic alterations in cancer. Understanding the links between phospholipid remodeling and GBM tumorigenesis may help develop new anticancer strategies and improve treatments to overcome drug resistance. We used metabolomic and transcriptomic analyses to systematically investigate metabolic and molecular changes in low-grade glioma (LGG) and GBM. We then re-established the reprogrammed metabolic flux and membrane lipid composition in GBM based on metabolomic and transcriptomic analyses. By inhibiting Aurora A kinase via RNA interference (RNAi) and inhibitor treatment, we investigated the effect of Aurora A kinase on phospholipid reprogramming LPCAT1 enzyme expression and GBM cell proliferation in vitro and in vivo. We found that GBM displayed aberrant glycerophospholipid and glycerolipid metabolism compared with LGG. Metabolic profiling indicated that fatty acid synthesis and uptake for phospholipid synthesis were significantly increased in GBM compared to LGG. The unsaturated phosphatidylcholine (PC) and phosphatidylethanolamine (PE) levels were significantly decreased in GBM compared to LGG. The expression level of LPCAT1, which is required for the synthesis of saturated PC and PE, was upregulated in GBM, and the expression of LPCAT4, which is required for the synthesis of unsaturated PC and PE, was downregulated in GBM. Notably, the inhibition of Aurora A kinase by shRNA knockdown and treatment with Aurora A kinase inhibitors such as Alisertib, AMG900, or AT9283 upregulated LPCAT1 mRNA and protein expression in vitro. In vivo, the inhibition of Aurora A kinase with Alisertib increased LPCAT1 protein expression. Phospholipid remodeling and a reduction in unsaturated membrane lipid components were found in GBM. Aurora A kinase inhibition increased LPCAT1 expression and suppressed GBM cell proliferation. The combination of Aurora kinase inhibition with LPCAT1 inhibition may exert promising synergistic effects on GBM.

AURORA KINASE A and related downstream molecules: A potential network for cancer therapy.

Aurora-A kinase (AURKA) belongs to the serine/threonine kinase family specific to cell division. In normal cells, activation of the AURKA protein is essential for regulating chromosomal segregation and centrosome maturation. The physiological concentration of AURKA accumulation has utmost importance during cell division. AURKA starts accumulating during the S phase of the cell cycle, gets functionally activated during the G2/M phase, attaches to the microtubule, and gets degraded during mitotic exit. Overexpression of AURKA could lead to deregulated cell cycle division, which is intrinsic to numerous cancers. Moreover, dysregulated AURKA affects various downstream molecules that aid in cancer pathogenesis. AURKA phosphorylates its substrates, including oncoproteins, transcriptional factors, tumor suppressor proteins, or other kinases central to various oncogenic signaling pathways critical to cancer. Considering the central role of AURKA in cell proliferation and tumorigenesis, targeting AURKA can be a novel alternative to cancer management. Several AURKA inhibitors have shown promising responses against different cancers either as a single agent or combined with various therapies. This chapter briefly discusses the role of AURKA and its downstream molecules in cancer vis-à-vis the role of AURKA inhibitor in chemoprevention.

TPX2 prompts mitotic survival via the induction of BCL2L1 through YAP1 protein stabilization in human embryonic stem cells

Genetic alterations have been reported for decades in most human embryonic stem cells (hESCs). Survival advantage, a typical trait acquired during long-term in vitro culture, results from the induction of BCL2L1 upon frequent copy number variation (CNV) at locus 20q11.21 and is one of the strongest candidates associated with genetic alterations that occur via escape from mitotic stress. However, the underlying mechanisms for BCL2L1 induction remain unknown. Furthermore, abnormal mitosis and the survival advantage that frequently occur in late passage are associated with the expression of BCL2L1, which is in locus 20q11.21. In this study, we demonstrated that the expression of TPX2, a gene located in 20q11.21, led to BCL2L1 induction and consequent survival traits under mitotic stress in isogenic pairs of hESCs and human induced pluripotent stem cells (iPSCs) with normal and 20q11.21 CNVs. High Aurora A kinase activity by TPX2 stabilized the YAP1 protein to induce YAP1-dependent BCL2L1 expression. A chemical inhibitor of Aurora A kinase and knockdown of YAP/TAZ significantly abrogated the high tolerance to mitotic stress through BCL2L1 suppression. These results suggest that the collective expression of TPX2 and BCL2L1 from CNV at loci 20q11.21 and a consequent increase in YAP1 signaling promote genome instability during long-term in vitro hESC culture.

HIV-1 Transcription Inhibitor 1E7-03 Decreases Nucleophosmin Phosphorylation

Transcription activation of latent human immunodeficiency virus-1 (HIV-1) occurs due to HIV-1 rebound, the interruption of combination antiretroviral therapy, or development of drug resistance. Thus, novel HIV-1 inhibitors, targeting HIV-1 transcription are needed. We previously developed an HIV-1 transcription inhibitor, 1E7-03, that binds to the noncatalytic RVxF-accommodating site of protein phosphatase 1 and inhibits HIV-1 replication in cultured cells and HIV-1–infected humanized mice by impeding protein phosphatase 1 interaction with HIV-1 Tat protein. However, host proteins and regulatory pathways targeted by 1E7-03 that contribute to its overall HIV-1 inhibitory activity remain to be identified. To address this issue, we performed label-free quantitative proteome and phosphoproteome analyses of noninfected and HIV-1–infected CEM T cells that were untreated or treated with 1E7-03. 1E7-03 significantly reprogramed the phosphorylation profile of proteins including PPARα/RXRα, TGF-β, and PKR pathways. Phosphorylation of nucleophosmin (NPM1) at Ser-125 residue in PPARα/RXRα pathway was significantly reduced (>20-fold, p = 1.37 × 10−9), followed by the reduced phosphorylation of transforming growth factor-beta 2 at Ser-46 (TGF-β2, >12-fold, p = 1.37 × 10−3). Downregulation of NPM1’s Ser-125 phosphorylation was further confirmed using Western blot. Phosphorylation mimicking NPM1 S125D mutant activated Tat-induced HIV-1 transcription and exhibited enhanced NPM1–Tat interaction compared to NPM1 S125A mutant. Inhibition of Aurora A or Aurora B kinases that phosphorylate NPM1 on Ser-125 residue inhibited HIV-1, further supporting the role of NPM1 in HIV-1 infection. Taken together, 1E7-03 reprogrammed PPARα/RXRα and TGF-β pathways that contribute to the inhibition of HIV-1 transcription. Our findings suggest that NPM1 phosphorylation is a plausible target for HIV-1 transcription inhibition.

Inhibitors of One or More Cellular Aurora Kinases Impair the Replication of Herpes Simplex Virus 1 and Other DNA and RNA Viruses with Diverse Genomes and Life Cycles.

We utilized a high-throughput cell-based assay to screen several chemical libraries for inhibitors of herpes simplex virus 1 (HSV-1) gene expression. From this screen, four aurora kinase inhibitors were identified that potently reduced gene expression during HSV-1 lytic infection. HSV-1 is known to interact with cellular kinases to regulate gene expression by modulating the phosphorylation and/or activities of viral and cellular proteins. To date, the role of aurora kinases in HSV-1 lytic infection has not been reported. We demonstrated that three aurora kinase inhibitors strongly reduced the transcript levels of immediate-early (IE) genes ICP0, ICP4, and ICP27 and impaired HSV-1 protein expression from all classes of HSV-1, including ICP0, ICP4, ICP8, and gC. These restrictions caused by the aurora kinase inhibitors led to potent reductions in HSV-1 viral replication. The compounds TAK 901, JNJ 7706621, and PF 03814735 decreased HSV-1 titers by 4,500-, 13,200-, and 8,400-fold, respectively, when present in a low micromolar range. The antiviral activity of these compounds correlated with an apparent decrease in histone H3 phosphorylation at serine 10 (H3S10ph) during viral infection, suggesting that the phosphorylation status of H3 influences HSV-1 gene expression. Furthermore, we demonstrated that the aurora kinase inhibitors also impaired the replication of other RNA and DNA viruses. These inhibitors significantly reduced yields of vaccinia virus (a poxvirus, double-stranded DNA, cytoplasmic replication) and mouse hepatitis virus (a coronavirus, positive-sense single-strand RNA [ssRNA]), whereas vesicular stomatitis virus (rhabdovirus, negative-sense ssRNA) yields were unaffected. These results indicated that the activities of aurora kinases play pivotal roles in the life cycles of diverse viruses. IMPORTANCE We have demonstrated that aurora kinases play a role during HSV-1 lytic infection. Three aurora kinase inhibitors significantly impaired HSV-1 immediate-early gene expression. This led to a potent reduction in HSV-1 protein expression and viral replication. Together, our results illustrate a novel role for aurora kinases in the HSV-1 lytic cycle and demonstrate that aurora kinase inhibitors can restrict HSV-1 replication. Furthermore, these aurora kinase inhibitors also reduced the replication of murine coronavirus and vaccinia virus, suggesting that multiple viral families utilize the aurora kinases for their own replication.

The Synergistic Anti-colon Cancer Effect of Aurora A Inhibitors and AKT Inhibitors Through PI3K/AKT Pathway.

Both AKT and Aurora inhibitors are a potential therapeutic agent for the treatment of malignant tumors. However, the role of combined inhibition of AKT and Aurora in colon cancer and its underlying mechanism have yet to be fully investigated. To investigate the role of combined AKT and Aurora inhibitors in colon cancer and its underlying mechanisms. CCK8 assay, colony formation assay, and flow cytometry were performed to analyze the proliferation and apoptosis of colon cancer cell line SW480 treated with combined AKT inhibitor MK2206 and Aurora inhibitor Alisertib, respectively. And tumor formation and growth were measured in tumor allograft model mice administered with the combined inhibitors. Western blot analysis was used to examine the expression levels of apoptosis-related proteins and signal transduction pathway components. The PI3K agonist 740Y-P and Overexpression of AKT are used to verify whether the PI3K/AKT pathway plays an anti-tumor effect when combined with inhibitory administration. Aurora A inhibitor Alisertib and AKT inhibitor MK2206 displayed consistent and synergistic antiproliferation and proapoptotic effects. Combined inhibition of Aurora A and AKT down-regulated the expression of Bcl-2/Bax and up-regulated the expression of cleaved-caspase-3 and cleaved-PARP. While single-drug treatment can significantly inhibit the expression of P-PI3K and P-AKT as well as increase the expression of P53 and H2A.X, the combined drugs had a more significant inhibitory effect than the single drug. Moreover, administration of PI3K agonist 740Y-P and AKT1 overexpression in experiments proved that the combined drugs exert an anticancer effect by inhibiting the PI3K/AKT pathway. Meanwhile, we showed that the combined administration had an anti-colon cancer effect on tumor allograft mice, and the underlying mechanism involved inhibition of the PI3K/AKT pathway. Combined administration of Aurora A inhibitor Alisertib and AKT inhibitor MK2206 can inhibit the proliferation of colon cancer cells and induce apoptosis, while inhibiting tumor growth in vivo. The underlying mechanism may involve the PI3K/AKT pathway and DNA damage pathway.

BI-847325, a selective dual MEK and Aurora kinases inhibitor, reduces aggressive behavior of anaplastic thyroid carcinoma on an in vitro three-dimensional culture.

Anaplastic thyroid carcinoma (ATC) is the most aggressive subtype of thyroid cancer. In this study, we used a three-dimensional in vitro system to evaluate the effect of a dual MEK/Aurora kinase inhibitor, BI-847325 anticancer drug, on several cellular and molecular processes involved in cancer progression. Human ATC cell lines, C643 and SW1736, were grown in alginate hydrogel and treated with IC50 values of BI-847325. The effect of BI-847325 on inhibition of kinases function of MEK1/2 and Aurora kinase B (AURKB) was evaluated via Western blot analysis of phospho-ERK1/2 and phospho-Histone H3 levels. Sodium/iodide symporter (NIS) and thyroglobulin (Tg), as two thyroid-specific differentiation markers, were measured by qRT-PCR as well as flow cytometry and immunoradiometric assay. Apoptosis was assessed by Annexin V/PI flow cytometry and BIM, NFκB1, and NFκB2 expressions. Cell cycle distribution and proliferation were determined via P16, AURKA, and AURKB expressions as well as PI and CFSE flow cytometry assays. Multidrug resistance was evaluated by examining the expression of MDR1 and MRP1. Angiogenesis and invasion were investigated by VEGF expression and F-actin labeling with Alexa Fluor 549 Phalloidin. Western blot results showed that BI-847325 inhibits MEK1/2 and AURKB functions by decreasing phospho-ERK1/2 and phospho-Histone H3 levels. BI-847325 induced thyroid differentiation markers and apoptosis in ATC cell lines. Inversely, BI-847325 intervention decreased multidrug resistance, cell cycle progression, proliferation, angiogenesis, and invasion at the molecular and/or cellular levels. The results of the present study suggest that BI-857,325 might be an effective multi-targeted anticancer drug for ATC treatment.

The Aurora Kinase Inhibitor CYC116 Promotes the Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in applications such as regenerative medicine, cardiac disease modeling, and in vitro drug evaluation. However, hPSC-CMs are immature, which limits their applications. During development, the maturation of CMs is accompanied by a decline in their proliferative capacity. This phenomenon suggests that regulating the cell cycle may facilitate the maturation of hPSC-CMs. Aurora kinases are essential kinases that regulate the cell cycle, the role of which is not well studied in hPSC-CM maturation. Here, we demonstrate that CYC116, an inhibitor of Aurora kinases, significantly promotes the maturation of CMs derived from both human embryonic stem cells (H1 and H9) and iPSCs (induced PSCs) (UC013), resulting in increased expression of genes related to cardiomyocyte function, better organization of the sarcomere, increased sarcomere length, increased number of mitochondria, and enhanced physiological function of the cells. In addition, a number of other Aurora kinase inhibitors have also been found to promote the maturation of hPSC-CMs. Our data suggest that blocking aurora kinase activity and regulating cell cycle progression may promote the maturation of hPSC-CMs.

Abstract B022: CHD1 promotes sensitivity to aurora kinase inhibitors by modulating the interaction of AURKA with TPX2

Abstract Clinical studies have shown that subsets of cancer patients achieve a significant benefit from Aurora kinase inhibitors, suggesting an urgent need to identify biomarkers for predicting drug response. Chromodomain-helicase-DNA-binding protein 1 (CHD1) is involved in chromatin remodeling, DNA repair, and transcriptional plasticity. Prior studies have demonstrated that CHD1 has distinct expression patterns in cancers with different molecular features, but its impact on drug responsiveness remains understudied. Here, we show that CHD1 promotes the susceptibility of prostate cancer cells to inhibitors targeting Aurora kinases, while depletion of CHD1 impairs their efficacy in vitro and in vivo. Pan-cancer drug sensitivity analyses revealed that high expression of CHD1 was associated with increased sensitivity to Aurora kinase A (AURKA) inhibitors. Mechanistically, KPNA2 served as a direct target of CHD1 and suppressed the interaction of AURKA with the co-activator TPX2, thereby rendering cancer cells more vulnerable to AURKA inhibitors. Consistent with previous research reporting that loss of PTEN elevates CHD1 levels, studies in a genetically engineered mouse model, patient-derived organoids, and patient samples showed that PTEN defects are associated with a better response to AURKA inhibition in advanced prostate cancer. These observations demonstrate that CHD1 plays an important role in modulating Aurora kinases and drug sensitivities, providing new insights into biomarker-driven therapies targeting Aurora kinases for future clinical studies. Citation Format: Haoyan Li, Yin Wang, Kevin Lin, Varadha Balaji Venkadakrishnan, Martin Bakht, Kaitlyn Tremble, Yue Lu, Himisha Beltran, Di Zhao. CHD1 promotes sensitivity to aurora kinase inhibitors by modulating the interaction of AURKA with TPX2. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B022.

The Aurora Kinase Inhibitor TAK901 Inhibits Glioblastoma Growth by Blocking SREBP1-Mediated Lipid Metabolism

Glioblastoma (GBM) is the most common and lethal malignant primary brain tumor. The standard treatment for GBM including surgical resection followed by radiation therapy and adjuvant chemotherapy with temozolomide remains unsatisfactory. In this study, we investigated the effects of the Aurora kinase inhibitor, TAK901, in GBM both in vitro and in vivo, and explored its key downstream targets. The effects of TAK901 were investigated using cell viability, cell apoptosis, live/dead, cell cycle, Transwell, 3D cell invasion, neuro-sphere, and self-renewal assays. Mechanistic studies were conducted using RNA-seq, lipid measurements, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Western blotting. The in vivo efficacy of TAK901 was validated using orthotopic xenograft GBM mouse models. In both GBM cells and GSCs, TAK901 remarkably reduced cell viability, self-renewal, migration and invasion and induced apoptosis and cell cycle arrest. Treatment with TAK901 considerably inhibited GBM growth in vivo. RNA-seq and RT-qPCR analyses showed that TAK901 downregulated the expression and activation of SREBP1. Moreover, SREBP1 overexpression alleviated the TAK901-mediated suppression of cell viability and apoptosis in GBM cells. Our results provide evidence that TAK901 inhibits GBM growth by suppressing SREBP1-mediated lipid metabolism.

Computational Biology Dynamics of Mps1 Kinase Molecular Interactions with Isoflavones Reveals a Chemical Scaffold with Potential to Develop New Therapeutics for the Treatment of Cancer.

The protein kinase Mps1 (monopolar spindle 1) is an important regulator of the Spindle Assembly Checkpoint (SAC), the evolutionary conserved checkpoint system of higher organisms that monitors the proper bipolar attachment of all chromosomes to the mitotic spindle during cell division. Defects in the catalytic activity and the transcription regulation of Mps1 are associated with genome instability, aneuploidy, and cancer. Moreover, multiple Mps1 missense and frameshift mutations have been reported in a wide range of types of cancer of different tissue origin. Due to these features, Mps1 arises as one promising drug target for cancer therapy. In this contribution, we developed a computational biology approach to study the dynamics of human Mps1 kinase interaction with isoflavones, a class of natural flavonoids, and compared their predicted mode of binding with that observed in the crystal structure of Mps1 in complex with reversine, a small-sized inhibitor of Mps1 and Aurora B kinases. We concluded that isoflavones define a chemical scaffold that can be used to develop new Mps1 inhibitors for the treatment of cancer associated with Mps1 amplification and aberrant chromosome segregation. In a broader context, the present report illustrates how modern chemoinformatics approaches can accelerate drug development in oncology.

EP0042, a Dual FLT3 and Aurora Kinase Inhibitor: Preliminary Results of an Ongoing Phase I/IIa First in Human Study in Patients with Relapsed/Refractory Acute Myeloid Leukemia

EP0042, a Dual FLT3 and Aurora Kinase Inhibitor: Preliminary Results of an Ongoing Phase I/IIa First in Human Study in Patients with Relapsed/Refractory Acute Myeloid Leukemia

The Progress of Growth and Apoptosis Are Associated with Both of AURKA and Aurkb in Marimo Cells with Calr Gene Mutation

Objective: Mutations of calreticulin (CALR) are a breakthrough discovery in exon 9 as most common driver mutations in janus kinase 2 (JAK2) or thrombopoietin receptor (MPL)-negative mutant essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. Several new inhibitors have displayed a relatively good therapeutic prospect after the JAK inhibitors, but still not completely cure the patients on their own. Thus, improvement of the therapeutic effect of these new inhibitors is particularly important through exploration of the related targets, which is probably compensated the function of original target in patients. Methods: The protein level of aurora was detected in CALR mutant patients, and was quantified by calculating the gray value of WB band. The biological function was examined in CALR mutant cell line (MARIMO) after aurora inhibition by EdU staining, colony assay, western blot, Annexin V, and flow cytometry assay, respectively. Transcriptomic analyzed the enriched genes and related signaling pathway in MARIMO cells with knockdown of Aurora A (AURKA) or Aurora B (AURKB). Results: Here, we found that Aurora B, similarly with Aurora A, was activated in CALR mutant patients. Selective inhibition of AURKA with MLN 8237 induced cell-growth arrest, maturation and apoptosis, and this cell phenotype was further enhanced when the AURKB was also inhibited as the increase of inhibitors' concentration. Specific downregulation of AURKB alone showed obvious inhibition on cell growth, and produced a similar effect on CALR mutant cells treated with MLN 8237 or interfered AURKA. Transcriptomic analyses revealed a replication reaction on cells caused by knockdown of AURKA or AURKB, which also induced a similar inhibition for mitogen-activated protein kinase (MAPK) signaling, but activation for metabolic and apoptosis pathway. Conclusion: These results suggested that both of AURKA and AURKB were associated with the progress of growth and apoptosis in MARIMO cells with CALR gene mutation, and provided a reference for recently proposed complementary therapies with Aurora kinase inhibitors in CALR mutated MPNs.

EPCO-14. ISOSCELES: AN INTEGRATIVE FRAMEWORK FOR THE PREDICTION OF TREATMENT RESISTANT GLIOBLASTOMA CELLS

Abstract Glioblastoma (GBM) remains the most common and lethal adult primary brain cancer. Two of the most significant issues preventing the development of effective GBM treatments are inter- and intra-tumor heterogeneity. To address these issues, we developed a novel platform termed ISOSCELES (Inferred cell Sensitivity Operating on the integration of Single-Cell Expression and L1000 Expression Signatures). ISOSCELES integrates single-cell gene expression data in individual GBM tumors with perturbation-response data derived from the NIH Library of Integrated Network-Based Cellular Signatures (LINCS) L1000 dataset to predict sensitive and resistant tumor cell populations. Importantly, we analyzed the predictive power of ISOSCELES in an in vivo xenograft model and demonstrated that ISOSCELES reveals the GBM cell identities primed for lineage expansion during treatment with the aurora kinase inhibitor alisertib. These studies suggest that ISOSCELES can be used to identify sensitive and resistant cell populations to targeted therapies in GBM, which can inform treatment decisions in ongoing and future clinical trials.

Loss of transcription factor EB dysregulates the G1/S transition and DNA replication in mammary epithelial cells

Triple-negative breast cancer (TNBC) poses significant challenges for treatment given the lack of targeted therapies and increased probability of relapse. It is pertinent to identify vulnerabilities in TNBC and develop newer treatments. Our prior research demonstrated that transcription factor EB (TFEB) is necessary for TNBC survival by regulating DNA repair, apoptosis signaling, and the cell cycle. However, specific mechanisms by which TFEB targets DNA repair and cell cycle pathways are unclear, and whether these effects dictate TNBC survival is yet to be determined. Here, we show that TFEB knockdown decreased the expression of genes and proteins involved in DNA replication and cell cycle progression in MDA-MB-231 TNBC cells. DNA replication was decreased in cells lacking TFEB, as measured by EdU incorporation. TFEB silencing in MDA-MB-231 and noncancerous MCF10A cells impaired progression through the S-phase following G1/S synchronization; however, this proliferation defect could not be rescued by co-knockdown of suppressor RB1. Instead, TFEB knockdown reduced origin licensing in G1 and early S-phase MDA-MB-231cells. TFEB silencing was associated with replication stress in MCF10A but not in TNBC cells. Lastly, we identified that TFEB knockdown renders TNBC cells more sensitive to inhibitors of Aurora Kinase A, a protein facilitating mitosis. Thus, inhibition of TFEB impairs cell cycle progress by decreasing origin licensing, leading to delayed entry into the S-phase, while rendering TNBC cells sensitive to Aurora kinase A inhibitors and decreasing cell viability. In contrast, TFEB silencing in noncancerous cells is associated with replication stress and leads to G1/S arrest.

Efficient Synthesis of Densely Functionalized Pyrido[2,3-d]Pyrimidines via Three-component One-pot Domino Knoevenagel aza-Diels Alder Reaction and Induces Apoptosis in Human Cancer Cell Lines via Inhibiting Aurora A and B Kinases

The identification of novel aurora kinase inhibitors is one of the most attractive directions in the field of anticancer research and development. In our ongoing efforts to pursue the class of inhibitors, a series of pyrido[2,3-d]pyrimidines were synthesized in DIPEAc/EtOH media. The advantages of the present methodology include a one-pot multicomponent environmentally friendly approach, cost effectiveness, broad substrate scope, operational simplicity, short reaction times, easy workup procedure and high yields. Synthesized compounds were screened against human lung carcinoma A549 cells, human hepatocellular liver carcinoma HepG2 cells and human cervical carcinoma epithelial HeLa cells. Compound 6 b i.e. diethyl 5-(4-methoxyphenyl)-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-6,7-dicarboxylate was found to be equipotent than the standard drug VX-680 against selected cancer cell lines. The selected compounds (6b, 6d and 6h) exhibit potent inhibition against Aurora A and B with IC50 values (9.4–25 mg/L). Molecular docking model showed that the compounds can bind well to the target by interacting with amino acid residues. It will provide some valuable information for the commercial Aurora Kinase inhibitors.

Aurora A phosphorylates Ndel1 to reduce the levels of Mad1 and NuMA at spindle poles.

Dynein inactivates the spindle assembly checkpoint (SAC) by transporting checkpoint proteins away from kinetochores toward spindle poles in a process known as "stripping." We find that inhibition of Aurora A kinase, which is localized to spindle poles, enables the accumulation of the spindle checkpoint activator Mad1 at poles where it is normally absent. Aurora kinases phosphorylate the dynein activator NudE neurodevelopment protein 1 like 1 (Ndel1) on Ser285 and Mad1 accumulates at poles when Ndel1 is replaced by a nonphosphorylatable mutant in human cells. The pole focusing protein NuMA, transported to poles by dynein, also accumulates at poles in cells harboring a mutant Ndel1. Phosphorylation of Ndel1 on Ser285 is required for robust spindle checkpoint activity and regulates the poles of asters inXenopusextracts. Our data suggest that dynein/SAC complexes that are generated at kinetochores and then transported directionally toward poles on microtubules are inhibited by Aurora A before they reach spindle poles. These data suggest that Aurora A generates a spatial signal at spindle poles that controls dynein transport and spindle function.

2-Phenoxy-3, 4′-bipyridine derivatives inhibit AURKB-dependent mitotic processes by disrupting its localization

Activity-based drug screens have successfully led to the development of various inhibitors of the catalytic activity of aurora kinases (AURKs), major regulatory kinases of cell division. Disrupting the localization of AURKB, rather than its catalytic activity, represents a largely unexplored alternative approach to disabling AURKB-dependent processes. Localization disruptors could be just as specific as direct inhibitors of AURKB activity, may bypass their off-target and select on-target toxicities, and are likely less susceptible to drug resistance resulting from mutations of the AURKB catalytic site. In this study, we demonstrate that the pan-AURK inhibitor AMG900 works at a low concentration not by inhibiting the phosphorylation of H3 at Ser10, an AURKB substrate, but by disrupting the mitotic localization of AURKB. Structural deletion studies pinpoint this undescribed activity to the 2-phenoxy-3,4′-bipyridine moiety of AMG900. Guided by a mechanism-informed phenotypic screening (MIPS) assay, the drug fragment is optimized into a novel class of inhibitors that, at low nanomolar concentrations, can disable AURKB through disruption of its mitotic localization and have desirable oral PK properties. Hierarchical clustering of cell fitness profiles reveals that these compounds cluster with each other, rather than with known AURK inhibitors such as AMG900 and VX-680. Validation studies in mice demonstrate that compound 15a elicits mitotic arrest and apoptosis in NCI–H23 human lung adenocarcinoma xenografts, resulting in a pronounced suppression of tumor growth. The discovery and optimization of compounds that disrupt AURKB localization are successfully facilitated by MIPS. Our findings suggest that 2-phenoxy-3, 4′-bipyridine derivatives have the potential to be further developed as effective therapeutics for the treatment of malignancy by delocalizing AURKB.

Unweaving the mitotic spindle: A focus on Aurora kinase inhibitors in lung cancer.

Lung cancer is one of the most aggressive malignancies, classified into two major histological subtypes: non-small cell lung cancer (NSCLC), that accounts for about 85% of new diagnosis, and small cell lung cancer (SCLC), the other 15%. In the case of NSCLC, comprehensive genome sequencing has allowed the identification of an increasing number of actionable targets, which have become the cornerstone of treatment in the advanced setting. On the other hand, the concept of oncogene-addiction is lacking in SCLC, and the only innovation of the last 30 years has been the introduction of immune checkpoint inhibitors in extensive stage disease. Dysregulation of cell cycle is a fundamental step in carcinogenesis, and Aurora kinases (AURKs) are a family of serine/threonine kinases that play a crucial role in the correct advance through the steps of the cycle. Hyperexpression of Aurora kinases is a common protumorigenic pathway in many cancer types, including NSCLC and SCLC; in addition, different mechanisms of resistance to anticancer drugs rely on AURK expression. Hence, small molecule inhibitors of AURKs have been developed in recent years and tested in several malignancies, with different results. The aim of this review is to analyze the current evidences of AURK inhibition in lung cancer, starting from preclinical rationale to finish with clinical trials available up to now.