Characterization of a MOB1 Homolog in the Apicomplexan Parasite Toxoplasma gondii

Monopolar spindle One Binder1 (MOB1) proteins are conserved components of the tumor-suppressing Hippo pathway, regulating cellular processes such as cytokinesis. Apicomplexan parasites present a life cycle that relies on the parasites’ ability to differentiate between stages and regulate their proliferation; thus, Hippo signaling pathways could play an important role in the regulation of the apicomplexan life cycle. Here, we report the identification of one MOB1 protein in the apicomplexan Toxoplasma gondii. To characterize the function of MOB1, we generated gain-of-function transgenic lines with a ligand-controlled destabilization domain, and loss-of-function clonal lines obtained through CRISPR/Cas9 technology. Contrary to what has been characterized in other eukaryotes, MOB1 is not essential for cytokinesis in T. gondii. However, this picture is complex since we found MOB1 localized between the newly individualized daughter nuclei at the end of mitosis. Moreover, we detected a significant delay in the replication of overexpressing tachyzoites, contrasting with increased replication rates in knockout tachyzoites. Finally, using the proximity-biotinylation method, BioID, we identified novel members of the MOB1 interactome, a probable consequence of the observed lack of conservation of some key amino acid residues. Altogether, the results point to a complex evolutionary history of MOB1 roles in apicomplexans, sharing properties with other eukaryotes but also with divergent features, possibly associated with their complex life cycle.

Mps1 contributes to tamoxifen resistance in Breast Cancer through phosphorylation of ERα

Overexpression of mitotic kinase monopolar spindle 1 (Mps1) has been identified in many tumor types and targeting Mps1 for tumor therapy has been shown great promise in multiple preclinical cancer models. However, the role of Mps1 in tamoxifen resistance in breast cancer has never been reported. Here in this study, we report that Mps1 determined the sensitivity of breast cancer cells to tamoxifen treatment. Mps1 overexpression rendered breast cancer cells more resistant to tamoxifen, while Mps1 inhibitor or siMps1 oligos could overcome tamoxifen resistance. Mechanistically, Mps1 interacted with ERα and stimulated its transcriptional activity in kinase activity-dependent manner. Mps1 was responsible for ERα phosphorylation at S559 and T224 sites. Importantly, Mps1 failed to enhance the transcriptional activity of ERα in the presence of ERα S559A or T224A mutant. Collectively, our findings suggest that Mps1 contributes to tamoxifen resistance in breast cancer and is a potential therapeutic to overcome tamoxifen resistance in breast cancer.

Multiple motors cooperate to establish and maintain acentrosomal spindle bipolarity in C. elegans oocyte meiosis

ABSTRACTWhile centrosomes organize spindle poles during mitosis, oocyte meiosis can occur in their absence. Spindles in human oocytes frequently fail to maintain bipolarity and consequently undergo chromosome segregation errors, making it important to understand mechanisms that promote acentrosomal spindle stability. To this end, we have optimized the auxin-inducible degron system in C. elegans to remove factors from pre-formed oocyte spindles within minutes and assess effects on spindle structure. This approach revealed that dynein is required to maintain the integrity of acentrosomal poles; removal of dynein from bipolar spindles caused pole splaying, and when coupled with a monopolar spindle induced by depletion of kinesin-12 motor KLP-18, dynein depletion led to a complete dissolution of the monopole. Surprisingly, we went on to discover that following monopole disruption, individual chromosomes were able to reorganize local microtubules and re-establish a miniature bipolar spindle that mediated chromosome segregation. This revealed the existence of redundant microtubule sorting forces that are undetectable when KLP-18 and dynein are active. We found that the kinesin-5 family motor BMK-1 provides this force, uncovering the first evidence that kinesin-5 contributes to C. elegans meiotic spindle organization. Altogether, our studies have revealed how multiple motors are working synchronously to establish and maintain bipolarity in the absence of centrosomes.

Studies of Interaction Mechanism between Pyrido [3,4-d] Pyrimidine Inhibitors and Mps1

Monopolar spindle 1 (Mps1), a dual-specific kinase, is related to the proper execution of chromosome biorientation and mitotic checkpoint signaling. The overexpression of Mps1 promotes the occurrence of cancer or the survival of aneuploid cancer cells, in other words, the reduction of Mps1 will severely reduce the viability of human cancer cells. Therefore, Mps1 is a potential target for cancer treatment. Recently, a series of novel pyrido [3,4-d] pyrimidine derivatives targeting Mps1 with high biological activity were synthesized. The crystal structure of Mps1 in complex with pyrido [3,4-d] pyrimidine derivatives was also reported, but there were no specific mechanism studies for this series of small molecule inhibitors. In this study, complexes binding modes were probed by molecular docking and further validated by molecular dynamics simulations and the molecular mechanics/generalized Born surface area (MM/GBSA) method. The results indicated that the van der Waals interactions and the nonpolar solvation energies were responsible to the basis for favorable binding free energies, all inhibitors interacted with residues I531, V539, M602, C604, N606, I607, L654, I663, and P673 of Mps1. By analyzing the hydrogen bonds, we found the residues G605 and K529 in Mps1 formed stable hydrogen bonds with compounds, it was more conducive to activities of Mps1 inhibitors. According to the above analysis, we further designed five new compounds. We found that compounds IV and V were better potential Mps1 inhibitors through docking and ADMET prediction. The obtained new insights not only were helpful in understanding the binding mode of inhibitors in Mps1, but also provided important references for further rational design of Mps1 inhibitors.

EMBR-23. KIF11 DEPENDENCY ON P53 MUTATIONAL STATUS IN MEDULLOBLASTOMA

Introduction KIF11, a mitotic kinesin, is a component responsible for assembly and maintenance of mitotic spindle during mitosis. Tumor cells can upregulate KIF11. Inhibition of KIF11 results monopolar spindle formation, resulting in monoastral mitosis in cells. This activates the spindle assembly checkpoint, cells are arrested and prevented from entering cell cycle, resulting in cell death via apoptosis or necrosis, cell division with aneuploidy or mitotic slippage without division into tetraploid G1 phase. Methods We hypothesized that the effect of KIF11 inhibition on medulloblastoma (MB) is dependent of its p53 mutational status. Results Our findings on Hoechst staining demonstrated a small molecule inhibitor of KIF11 which induced apoptosis in p53-wildtype MB cells at 48h (p<0.0001), was able to trigger mitotic catastrophe (p = 0.0010) in p53-mutant MB cells at 24h and subsequent necrosis (p=0.0039) at 48h. KIF11 inhibitor exerted anti-proliferative effects on five MB cell lines at nanomolar concentration range, independent of its p53 mutational status. Cells treated with KIF11 inhibitor were arrested in G2/M phase. Apoptosis was observed on Annexin V flow cytometry 24h after treatment, followed by necrosis after 48h in p53-wildtype cells. In contrast, treated p53-mutant cells underwent necrosis at 24h. Differences in cell death mechanisms upon KIF11 inhibition was confirmed on immunoblotting by upregulated p53 expression and presence of cleaved-PARP and DNA-damage marker in p53-wildtype cells, indicative of apoptosis. While inhibition of KIF11 and increased p53 expression were observed only after 48h, cleaved-PARP expression was detected as early as 24h in p53-wildtype, suggesting KIF11-independent, cleaved-PARP-mediated cell death at 24h. In contrast, treated p53-mutant cells showed decreased p53 expression and absence of cleaved-PARP and DNA-damage marker after 24h. Conclusions Our results suggest that when mitotic arrest is induced, p53-mutant MB cells undergo mitotic catastrophe and necrosis while p53-wildtype MB cells predominantly undergo apoptosis.

The essential roles of Mps1 in spermatogenesis and fertility in mice

Monopolar spindle 1 (MPS1), which plays a critical role in somatic mitosis, has also been revealed to be essential for meiosis I in oocytes. Spermatogenesis is an important process involving successive mitosis and meiosis, but the function of MPS1 in spermatogenesis remains unclear. Here, we generated Mps1 conditional knockout mice and found that Ddx4-cre-driven loss of Mps1 in male mice resulted in depletion of undifferentiated spermatogonial cells and subsequently of differentiated spermatogonia and spermatocytes. In addition, Stra8-cre-driven ablation of Mps1 in male mice led to germ cell loss and fertility reduction. Spermatocytes lacking Mps1 have blocked at the zygotene-to-pachytene transition in the prophase of meiosis I, which may be due to decreased H2B ubiquitination level mediated by MDM2. And the expression of many meiotic genes was decreased, while that of apoptotic genes was increased. Moreover, we also detected increased apoptosis in spermatocytes with Mps1 knockout, which may have been the reason why germ cells were lost. Taken together, our findings indicate that MPS1 is required for mitosis of gonocytes and spermatogonia, differentiation of undifferentiated spermatogonia, and progression of meiosis I in spermatocytes.

Development of the First Covalent Monopolar Spindle Kinase 1 (MPS1/TTK) Inhibitor

Monopolar spindle kinase 1 (MPS1/TTK) is a key element of the mitotic checkpoint securing proper chromosome segregation. It is being evaluated as a target in the treatment of aggressive tumors such as triple-negative breast cancer with several reversible inhibitors currently undergoing clinical trials. While long drug–target residence times have been suggested to be beneficial in the context of therapeutic MPS1 inhibition, no irreversible inhibitors are known. Here we present the design and characterization of the first irreversible covalent MPS1 inhibitor <b>RMS-07</b> targeting a cysteine (Cys604) in the kinase's hinge region present only in few other protein kinases. The compound showed excellent MPS1 inhibitory potency and high selectivity against all protein kinases harboring an equivalent cysteine as well as in a larger differential scanning fluorimetry-based screening panel. Covalent binding was confirmed by mass spectrometry and X-ray crystal structure. We expect this tool compound to open new avenues for the design of MPS1-specific covalent chemical probes or drugs.

Development of the First Covalent Monopolar Spindle Kinase 1 (MPS1/TTK) Inhibitor

Monopolar spindle kinase 1 (MPS1/TTK) is a key element of the mitotic checkpoint securing proper chromosome segregation. It is being evaluated as a target in the treatment of aggressive tumors such as triple-negative breast cancer with several reversible inhibitors currently undergoing clinical trials. While long drug–target residence times have been suggested to be beneficial in the context of therapeutic MPS1 inhibition, no irreversible inhibitors are known. Here we present the design and characterization of the first irreversible covalent MPS1 inhibitor <b>RMS-07</b> targeting a cysteine (Cys604) in the kinase's hinge region present only in few other protein kinases. The compound showed excellent MPS1 inhibitory potency and high selectivity against all protein kinases harboring an equivalent cysteine as well as in a larger differential scanning fluorimetry-based screening panel. Covalent binding was confirmed by mass spectrometry and X-ray crystal structure. We expect this tool compound to open new avenues for the design of MPS1-specific covalent chemical probes or drugs.

Paternal chromosome elimination and X non-disjunction on asymmetric spindles in Sciara male meiosis

Meiosis in male Sciara is unique with a single centrosome. A monopolar spindle forms in meiosis I, but a bipolar spindle forms in meiosis II. The imprinted paternal chromosomes are eliminated in meiosis I; there is non-disjunction of the X in meiosis II. Despite differences in spindle construction and chromosome behavior, both meiotic divisions are asymmetric, producing a cell and a small bud. Observations of live spermatocytes made with the LC-PolScope, differential interference contrast optics and fluorescence revealed maternal and paternal chromosome sets on the monopolar spindle in meiosis I and formation of an asymmetric monastral bipolar spindle in meiosis II where all chromosomes except the X congress to the metaphase plate. The X remains near the centrosome after meiosis I and stays with it as the spindle forms in meiosis II. Electron microscopy revealed amorphous material between the X and the centrosome. Immunofluorescence with an antibody against the checkpoint protein Mad2 stains the centromeres of the maternal X dyad in late meiosis I and in meiosis II where it fails to congress to the metaphase plate. Mad2 is also present throughout the paternal chromosomes destined for elimination in meiosis I, suggesting a possible role in chromosome imprinting. If Mad2 on the X dyad mediates a spindle checkpoint in meiosis II, it may delay metaphase to facilitate formation of the second half spindle through a non-centrosomal mechanism.