Nek2A prevents centrosome clustering and induces cell death in cancer cells via KIF2C interaction.

Unlike normal cells, cancer cells frequently exhibit supernumerary centrosomes, leading to formation of multipolar spindles that can trigger cell death. Nevertheless, cancer cells with supernumerary centrosomes escape the deadly consequences of unequal segregation of genomic material by coalescing their centrosomes into two poles. This unique trait of cancer cells presents a promising target for cancer therapy, focusing on selectively attacking cells with supernumerary centrosomes. Nek2A is a kinase involved in mitotic regulation, including the centrosome cycle, where it phosphorylates linker proteins to separate centrosomes. In this study, we investigated if Nek2A also prevents clustering of supernumerary centrosomes, akin to its separation function. Reduction of Nek2A activity, achieved through knockout, silencing, or inhibition, promotes centrosome clustering, whereas its overexpression results in inhibition of clustering. Significantly, prevention of centrosome clustering induces cell death, but only in cancer cells with supernumerary centrosomes, both in vitro and in vivo. Notably, none of the known centrosomal (e.g., CNAP1, Rootletin, Gas2L1) or non-centrosomal (e.g., TRF1, HEC1) Nek2A targets were implicated in this machinery. Additionally, Nek2A operated via a pathway distinct from other proteins involved in centrosome clustering mechanisms, like HSET and NuMA. Through TurboID proximity labeling analysis, we identified novel proteins associated with the centrosome or microtubules, expanding the known interaction partners of Nek2A. KIF2C, in particular, emerged as a novel interactor, confirmed through coimmunoprecipitation and localization analysis. The silencing of KIF2C diminished the impact of Nek2A on centrosome clustering and rescued cell viability. Additionally, elevated Nek2A levels were indicative of better patient outcomes, specifically in those predicted to have excess centrosomes. Therefore, while Nek2A is a proposed target, its use must be specifically adapted to the broader cellular context, especially considering centrosome amplification. Discovering partners such as KIF2C offers fresh insights into cancer biology and new possibilities for targeted treatment.

Prolonged overexpression of PLK4 leads to formation of centriole rosette clusters that are connected via canonical centrosome linker proteins.

Centrosome amplification is a hallmark of cancer and PLK4 is one of the responsible factors for cancer associated centrosome amplification. Increased PLK4 levels was also shown to contribute to generation of cells with centriole amplification in mammalian tissues as olfactory neuron progenitor cells. PLK4 overexpression generates centriole rosette (CR) structures which harbor more than two centrioles each. Long term PLK4 overexpression results with centrosome amplification, but the maturation of amplified centrioles in CRs and linking of PLK4 induced amplified centrosomes has not yet been investigated in detail. Here, we show evidence for generation of large clustered centrosomes which have more than 2 centriole rosettes and define these structures as centriole rosette clusters (CRCs) in cells that have high PLK4 levels for 2 consecutive cell cycles. In addition, we show that PLK4 induced CRs follow normal centrosomal maturation processes and generate CRC structures that are inter-connected with canonical centrosomal linker proteins as C-Nap1, Rootletin and Cep68 in the second cell cycle after PLK4 induction. Increased PLK4 levels in cells with C-Nap1 and Rootletin knock-out resulted with distanced CRs and CRCs in interphase, while Nek2 knock-out inhibited separation of CRCs in prometaphase, providing functional evidence for the binding of CRC structures with centrosomal linker proteins. Taken together, these results suggest a cell cycle dependent model for PLK4 induced centrosome amplification which occurs in 2 consecutive cell cycles: (i) CR state in the first cell cycle, and (ii) CRC state in the second cell cycle.

Keep Calm and Carry on with Extra Centrosomes.

Aberrations in the centrosome number and structure can readily be detected at all stages of tumor progression and are considered hallmarks of cancer. Centrosome anomalies are closely linked to chromosome instability and, therefore, are proposed to be one of the driving events of tumor formation and progression. This concept, first posited by Boveri over 100 years ago, has been an area of interest to cancer researchers. We have now begun to understand the processes by which these numerical and structural anomalies may lead to cancer, and vice-versa: how key events that occur during carcinogenesis could lead to amplification of centrosomes. Despite the proliferative advantages that having extra centrosomes may confer, their presence can also lead to loss of essential genetic material as a result of segregational errors and cancer cells must deal with these deadly consequences. Here, we review recent advances in the current literature describing the mechanisms by which cancer cells amplify their centrosomes and the methods they employ to tolerate the presence of these anomalies, focusing particularly on centrosomal clustering.

SC1 limits tube formation, branching, migration, expansion and induce apoptosis of endothelial cells.

Endothelial cells (ECs) are essential in the growth and progression of the tumor cells by supplying nutrition and angiogenesis factors. Targeting ECs emerged as a major strategy to prevent the growth of tumors. Studies suggest that ERK1/2 signaling is important for endothelial cells, which could be specifically targeted by small molecule SC1. We aimed to study the effects of SC1 treatments on endothelial cell proliferation, angiogenesis, and death. To this end, we performed viability, apoptosis, cell cycle, gene expression, wound closure, tube formation, and western blot analysis in endothelial cells post SC1 treatments. Intriguingly, we found that SC1 has an antiangiogenic effect on endothelial cells, which limits the endothelial cell expansion, tube formation, branching, and migration. The proliferation is especially limited in dose dependent manner by SC1. In addition, we found that SC1 elevates the apoptosis of endothelial cells and associated pathways including BAK1, Stat1, Sox4, and Caspase1. We believe that these findings could contribute to the development of improved therapies based on the SC1 as an attractive candidate for anticancer clinical studies targeted to tumor angiogenesis.

Development of Small Molecule MEIS Inhibitors that modulate HSC activity.

Meis1, which belongs to TALE-type class of homeobox gene family, appeared as one of the key regulators of hematopoietic stem cell (HSC) self-renewal and a potential therapeutical target. However, small molecule inhibitors of MEIS1 remained unknown. This led us to develop inhibitors of MEIS1 that could modulate HSC activity. To this end, we have established a library of relevant homeobox family inhibitors and developed a high-throughput in silico screening strategy against homeodomain of MEIS proteins using the AutoDock Vina and PaDEL-ADV platform. We have screened over a million druggable small molecules in silico and selected putative MEIS inhibitors (MEISi) with no predicted cytotoxicity or cardiotoxicity. This was followed by in vitro validation of putative MEIS inhibitors using MEIS dependent luciferase reporter assays and analysis in the ex vivo HSC assays. We have shown that small molecules named MEISi-1 and MEISi-2 significantly inhibit MEIS-luciferase reporters in vitro and induce murine (LSKCD34l°w cells) and human (CD34+, CD133+, and ALDHhi cells) HSC self-renewal ex vivo. In addition, inhibition of MEIS proteins results in downregulation of Meis1 and MEIS1 target gene expression including Hif-1α, Hif-2α and HSC quiescence modulators. MEIS inhibitors are effective in vivo as evident by induced HSC content in the murine bone marrow and downregulation of expression of MEIS target genes. These studies warrant identification of first-in-class MEIS inhibitors as potential pharmaceuticals to be utilized in modulation of HSC activity and bone marrow transplantation studies.

CASIN and AMD3100 enhance endothelial cell proliferation, tube formation and sprouting.

Endothelial dysfunction is prominent in atherosclerosis, hypertension, diabetes, peripheral and cardiovascular diseases, and stroke. Novel therapeutic approaches to these conditions often involve development of tissue-engineered veins with ex vivo expanded endothelial cells. However, high cell number requirements limit these approaches to become applicable to clinical applications and highlight the requirement of technologies that accelerate expansion of vascular-forming cells. We have previously shown that novel small molecules could induce hematopoietic stem cell expansion ex vivo. We hypothesized that various small molecules targeting hematopoietic stem cell quiescence and mobilization could be used to induce endothelial cell expansion and angiogenesis due to common origin and shared characteristics of endothelial and hematopoietic cells. Here, we have screened thirty-five small molecules and found that CASIN and AMD3100 increase endothelial cell expansion up to two-fold and induce tube formation and ex vivo sprouting. In addition, we have studied how CASIN and AMD3100 affect cell migration, apoptosis and cell cycle of endothelial cells. CASIN and AMD3100 upregulate key endothelial marker genes and downregulate a number of cyclin dependent kinase inhibitors. These findings suggest that CASIN and AMD3100 could be further tested in the development of artificial vascular systems and vascular gene editing technologies. Furthermore, these findings may have potential to contribute to the development of alternative treatment methods for diseases that cause endothelial damage.

Development of gene editing strategies for human ß-globin (HBB) gene mutations.

Recent developments in gene editing technology have enabled scientists to modify DNA sequence by using engineered endonucleases. These gene editing tools are promising candidates for clinical applications, especially for treatment of inherited disorders like sickle cell disease (SCD). SCD is caused by a point mutation in human ß-globin gene (HBB). Clinical strategies have demonstrated substantial success, however there is not any permanent cure for SCD available. CRISPR/Cas9 platform uses a single endonuclease and a single guide RNA (gRNA) to induce sequence-specific DNA double strand break (DSB). When this accompanies a repair template, it allows repairing the mutated gene. In this study, it was aimed to target HBB gene via CRISPR/Cas9 genome editing tool to introduce nucleotide alterations for efficient genome editing and correction of point mutations causing SCD in human cell line, by Homology Directed Repair (HDR). We have achieved to induce target specific nucleotide changes on HBB gene in the locus of mutation causing SCD. The effect of on-target activity of bone fide standard gRNA and newly developed longer gRNA were examined. It is observed that longer gRNA has higher affinity to target DNA while having the same performance for targeting and Cas9 induced DSBs. HDR mechanism was triggered by co-delivery of donor DNA repair templates in circular plasmid form. In conclusion, we have suggested methodological pipeline for efficient targeting with higher affinity to target DNA and generating desired modifications on HBB gene.