Motor Proteins Research Articles

Directional change during active diffusion of viral ribonucleoprotein particles through cytoplasm

A mesh of cytoskeletal fibers, consisting of microtubules, intermediate filaments, and fibrous actin, prevents the Brownian diffusion of particles with a diameter larger than 0.10 μm, such as vesicular stomatitis virus ribonucleoprotein (RNP) particles, in mammalian cells. Nevertheless, RNP particles do move in random directions but at a lower rate than Brownian diffusion, which is thermally driven. This nonthermal biological transport process is called “active diffusion” because it is driven by ATP. The ATP powers motor proteins such as myosin II. The motor proteins bend and cross-link actin fibers, causing the mesh to jiggle. Until recently, little was known about how RNP particles get through the mesh. It has been customary to analyze the tracks of particles like RNPs by computing the slope of the ensemble-averaged mean-squared displacement of the particles as a signature of mechanism. Although widely used, this approach “loses information” about the timing of the switches between physical mechanisms. It has been recently shown that machine learning composed of variational Bayesian analysis, Gaussian mixture models, and hidden Markov models can use “all the information” in a single track to reveal that that the positions of RNP particles are spatially clustered. Machine learning assigns a number, called a state, to each cluster. RNP particles remain in one state for 0.2–1.0 s before switching (hopping) to a different state. This earlier work is here extended to analyze the movements of a particle within a state and to determine particle directionality within and between states.

Label-free 3D tracking of biological micro-objects using RGB pinholes

Defocusing digital particle image velocimetry is a method developed within the field of fluidics to measure the velocities of particles within a volume through image analysis. For three-dimensional (3D) tracking, approaches utilizing defocusing techniques have been proposed to enable depthwise positional measurements. Recently, there have been suggestions to simplify this approach by employing a triple pinhole mask and to further enhance it with color filters that allow distinguish particles, even under dense conditions. This paper presents an attempt to apply the defocusing method using a triple pinhole mask with red, green, and blue (RGB) color-coding filters, demonstrating the successful label-free 3D tracking of biological micro-objects at multiple positions within a volume. The application of this method in fluidics and its potential for biological analysis are highlighted through successful examples of 3D tracking involving micro-particles influenced by motor proteins, dividing cells, and swimming microorganisms.

Cooperation of Various Cytoskeletal Components Orchestrates Intercellular Spread of Mitochondria between B-Lymphoma Cells through Tunnelling Nanotubes.

Membrane nanotubes (NTs) are dynamic communication channels connecting spatially separated cells even over long distances and promoting the transport of different cellular cargos. NTs are also involved in the intercellular spread of different pathogens and the deterioration of some neurological disorders. Transport processes via NTs may be controlled by cytoskeletal elements. NTs are frequently observed membrane projections in numerous mammalian cell lines, including various immune cells, but their functional significance in the 'antibody factory' B cells is poorly elucidated. Here, we report that as active channels, NTs of B-lymphoma cells can mediate bidirectional mitochondrial transport, promoted by the cooperation of two different cytoskeletal motor proteins, kinesin along microtubules and myosin VI along actin, and bidirectional transport processes are also supported by the heterogeneous arrangement of the main cytoskeletal filament systems of the NTs. We revealed that despite NTs and axons being different cell extensions, the mitochondrial transport they mediate may exhibit significant similarities. Furthermore, we found that microtubules may improve the stability and lifespan of B-lymphoma-cell NTs, while F-actin strengthens NTs by providing a structural framework for them. Our results may contribute to a better understanding of the regulation of the major cells of humoral immune response to infections.

Matrix mechanics regulates muscle regeneration by modulating kinesin-1 activity

Sarcopenia, a prevalent muscle disease characterized by muscle mass and strength reduction, is associated with impaired skeletal muscle regeneration. However, the influence of the biomechanical properties of sarcopenic skeletal muscle on the efficiency of the myogenic program remains unclear. Herein, we established a mouse model of sarcopenia and observed a reduction in stiffness within the sarcopenic skeletal muscle in vivo. To investigate whether the biomechanical properties of skeletal muscle directly impact the myogenic program, we established an in vitro system to explore the intrinsic mechanism involving matrix stiffness control of myogenic differentiation. Our findings identify the microtubule motor protein, kinesin-1, as a mechano-transduction hub that senses and responds to matrix stiffness, crucial for myogenic differentiation and muscle regeneration. Specifically, kinesin-1 activity is positively regulated by stiff matrices, facilitating its role in transporting mitochondria and enhancing translocation of the glucose transporter GLUT4 to the cell surface for glucose uptake. Conversely, the softer matrices significantly suppress kinesin-1 activity, leading to the accumulation of mitochondria around nuclei and hindering glucose uptake by inhibiting GLUT4 membrane translocation, consequently impairing myogenic differentiation. The insights gained from the in-vitro system highlight the mechano-transduction significance of kinesin-1 motor proteins in myogenic differentiation. Furthermore, our study confirms that enhancing kinesin-1 activity in the sarcopenic mouse model restores satellite cell expansion, myogenic differentiation, and muscle regeneration. Taken together, our findings provide a potential target for improving muscle regeneration in sarcopenia.

Kinesin KIFC3 is essential for microtubule stability and cytokinesis in oocyte meiosis

KIFC3 is a member of Kinesin-14 family motor proteins, which play a variety of roles such as centrosome cohesion, cytokinesis, vesicles transportation and cell proliferation in mitosis. Here, we investigated the functional roles of KIFC3 in meiosis. Our findings demonstrated that KIFC3 exhibited expression and localization at centromeres during metaphase I, followed by translocation to the midbody at telophase I throughout mouse oocyte meiosis. Disruption of KIFC3 activity resulted in defective polar body extrusion. We observed aberrant meiotic spindles and misaligned chromosomes, accompanied by the loss of kinetochore-microtubule attachment, which might be due to the failed recruitment of BubR1/Bub3. Coimmunoprecipitation data revealed that KIFC3 plays a crucial role in maintaining the acetylated tubulin level mediated by Sirt2, thereby influencing microtubule stability. Additionally, our findings demonstrated an interaction between KIFC3 and PRC1 in regulating midbody formation during telophase I, which is involved in cytokinesis regulation. Collectively, these results underscore the essential contribution of KIFC3 to spindle assembly and cytokinesis during mouse oocyte meiosis.

Ruxolitinib: A new hope for ventilator-induced diaphragm dysfunction.

Mechanical ventilation (MV) results in diminished diaphragm size and strength, termed ventilator-induced diaphragm dysfunction (VIDD). VID increases dependence, prolongs weaning, and increases discharge mortality rates. The Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway is implicated in VIDD, upregulated following MV. JAK/STAT inhibition alleviates chronic muscle wasting conditions. This study aimed to explore the therapeutic potential of Ruxolitinib, an FDA approved JAK1/2 inhibitor (JI) for the treatment of VIDD. Rats were subjected to 5 days controlled MV (CMV) with and without daily Ruxolitinib gavage. Muscle fiber size and function were assessed. RNAseq, mitochondrial morphology, respirometry, and mass spectrometry were determined. CMV significantly reduced diaphragm size and specific force by 45% (p < 0.01), associated with a two-fold P-STAT3 upregulation (p < 0.001). CMV disrupted mitochondrial content and reduced the oxygen consumption rate (p < 0.01). Expression of the motor protein myosin was unaffected, however CMV alters myosin function via post-translational modifications (PTMs). Daily administration of JI increased animal survival (40% vs. 87%; p < 0.05), restricted P-STAT3 (p < 0.001), and preserved diaphragm size and specific force. JI was associated with preserved mitochondrial content and respiratory function (p < 0.01), and the reversal or augmentation of myosin deamidation PTMs of the rod and head region. JI preserved diaphragm function, leading to increased survival in an experimental model of VIDD. Functional enhancement was associated with maintenance of mitochondrial content and respiration and the reversal of ventilator-induced PTMs of myosin. These results demonstrate the potential of repurposing Ruxolitinib for treatment of VIDD.

Motor properties of Myosin 5c are modulated by tropomyosin isoforms and inhibited by pentabromopseudilin.

Myosin 5c (Myo5c) is a motor protein that is produced in epithelial and glandular tissues, where it plays an important role in secretory processes. Myo5c is composed of two heavy chains, each containing a generic motor domain, an elongated neck domain consisting of a single α-helix with six IQ motifs, each of which binds to a calmodulin (CaM) or a myosin light chain from the EF-hand protein family, a coiled-coil dimer-forming region and a carboxyl-terminal globular tail domain. Although Myo5c is a low duty cycle motor, when two or more Myo5c-heavy meromyosin (HMM) molecules are linked together, they move processively along actin filaments. We describe the purification and functional characterization of human Myo5c-HMM co-produced either with CaM alone or with CaM and the essential and regulatory light chains Myl6 and Myl12b. We describe the extent to which cofilaments of actin and Tpm1.6, Tpm1.8 or Tpm3.1 alter the maximum actin-activated ATPase and motile activity of the recombinant Myo5c constructs. The small allosteric effector pentabromopseudilin (PBP), which is predicted to bind in a groove close to the actin and nucleotide binding site with a calculated ΔG of -18.44kcal/mol, inhibits the motor function of Myo5c with a half-maximal concentration of 280nM. Using immunohistochemical staining, we determined the distribution and exact localization of Myo5c in endothelial and endocrine cells from rat and human tissue. Particular high levels of Myo5c were observed in insulin-producing β-cells located within the pancreatic islets of Langerhans.

Dynein Light Intermediate Chains Exhibit Different Arginine Methylation Patterns.

The motor protein dynein is integral to retrograde transport along microtubules and interacts with numerous cargoes through the recruitment of cargo-specific adaptor proteins. This interaction is mediated by dynein light intermediate chain subunits LIC1 (DYNC1LI1) and LIC2 (DYNC1LI2), which govern the adaptor binding and are present in distinct dynein complexes with overlapping and unique functions. Using bioinformatics, we analyzed the C-terminal domains (CTDs) of LIC1 and LIC2, revealing similar structural features but diverse post-translational modifications (PTMs). The methylation status of LIC2 and the proteins involved in this modification were examined through immunoprecipitation and immunoblotting analyses. The specific methylation sites on LIC2 were identified through a site-directed mutagenesis analysis, contributing to a deeper understanding of the regulatory mechanisms of the dynein complex. We found that LIC2 is specifically methylated at the arginine 397 residue, a reaction that is catalyzed by protein arginine methyltransferase 1 (PRMT1). The distinct PTMs of the LIC subunits offer a versatile mechanism for dynein to transport diverse cargoes efficiently. Understanding how these PTMs influence the functions of LIC2, and how they differ from LIC1, is crucial for elucidating the role of dynein-related transport pathways in a range of diseases. The discovery of the arginine 397 methylation site on LIC2 enhances our insight into the regulatory PTMs of dynein functions.

A model for cooperativity of kinesin-4 motors by communicating through the microtubule track

Kinesin-4 motor proteins at nanomolar concentrations can accumulate at microtubule plus ends, regulating the microtubule length. Recent experimental studies showed that at picomolar motor concentrations, under which nearly no motor crowding can occur, the run length of the kinesin-4 motor increases whereas the velocity decreases with the motor concentration, indicating the presence of a long-range communication between the motors through the microtubule track. However, the mechanism of the long-range communication is illusive. Here, based on the proposal that large conformational changes of the tubulin induced by the strong interaction with a kinesin head can propagate along the microtubule such that influence the tubulin-binding energy of other motors distant away, the dynamics of the kinesin-4 motor at the picomolar motor concentrations is studied theoretically, explaining quantitatively the experimental data. Moreover, the lifetime of the motor when multiple motors are jammed near the plus ends at high motor concentrations is studied theoretically.

A novel in-frame deletion in KIF5C gene causes infantile onset epilepsy and psychomotor retardation.

Motor proteins, encoded by Kinesin superfamily (KIF) genes, are critical for brain development and plasticity. Increasing studies reported KIF's roles in neurodevelopmental disorders. Here, a 6 years and 3 months-old Chinese boy with markedly symptomatic epilepsy, intellectual disability, brain atrophy, and psychomotor retardation was investigated. His parents and younger sister were phenotypically normal and had no disease-related family history. Whole exome sequencing identified a novel heterozygous in-frame deletion (c.265_267delTCA) in exon 3 of the KIF5C in the proband, resulting in the removal of evolutionarily highly conserved p.Ser90, located in its ATP-binding domain. Sanger sequencing excluded the proband's parents and family members from harboring this variant. The activity of ATP hydrolysis in vitro was significantly reduced as predicted. Immunofluorescence studies showed wild-type KIF5C was widely distributed throughout the cytoplasm, while mutant KIF5C was colocalized with microtubules. The live-cell imaging of the cargo-trafficking assay revealed that mutant KIF5C lost the peroxisome-transporting ability. Drosophila models also confirmed p.Ser90del's essential role in nervous system development. This study emphasized the importance of the KIF5C gene in intracellular cargo-transport as well as germline variants that lead to neurodevelopmental disorders and might enable clinicians for timely and accurate diagnosis and disease management in the future.

Abstract 2007: Neurolysin: A novel architect of the prostate cancer microenvironment and therapeutic resistance

Abstract Introduction: Prostate cancer is the second leading cause of cancer-related death in American men, primarily driven by its ability to evade therapeutic interventions. This study uncovers a novel paradigm where neurolysin, a peptidase previously ascribed to neuronal function, orchestrates the tumor microenvironment (TME) by interfacing with cellular motor proteins. This interaction precipitates a series of events that not only fortify the cancer cells' survival but also underpin the resistance to antiandrogen therapies. Experimental Procedures: We deployed advanced prostate cancer models to delve into the extra-neuronal influence of neurolysin, probing its role in the orchestration of the TME. An exhaustive suite of transcriptomic and proteomic analyses, complemented by comprehensive patient cohort studies, laid the foundation for unraveling the complex interplay between neurolysin, motor proteins, and the TME dynamics. Results: Our data elucidates that neurolysin's depletion correlates with an altered TME, conducive to cancer proliferation and resilience against targeted therapies. This shift is mediated through neurolysin's interaction with cellular motor proteins, a liaison that appears to be a cornerstone in TME modulation. A significant subset of prostate cancer cases exhibits a neurolysin deficiency, paralleling more aggressive disease and therapeutic defiance, highlighting its role as a critical biomarker and a novel target for intervention. Conclusions: Neurolysin steps into the limelight as a pivotal modulator of the TME, operating through mechanisms distinct from its known neuronal functions. Its interplay with motor proteins opens a novel avenue in prostate cancer biology, providing a unique perspective on the regulation of the TME and subsequent therapeutic resistance. Targeting this newly discovered regulatory axis presents a promising therapeutic strategy that could potentially redefine the clinical approach to managing prostate cancer and may have implications for other malignancies driven by similar TME alterations. Citation Format: Choushi Wang, Ping Mu. Neurolysin: A novel architect of the prostate cancer microenvironment and therapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2007.

Abstract 4421: Intracellular cytomatrix actin and the Warburg effect

Abstract Introduction: The cytoplasm of living cells is dynamic and the motion arising from fluctuating forces in cells is an active process, rather than being thermally induced. Motor proteins could be the primary causative force of intracellular movement. However, which motor protein is involved in the intracellular dynamics, what process requires fluctuational intracellular activity, and where the energy source comes from is unknown. To address the impact of intracellular motion in cellular processes, we chemically separated the viscous fluid (cytosol) and elastic solid (cytomatrix) phases of cytoplasm. The multiomic analysis was used to dissect the compositional differences between the liquid and solid phases. LC-MS/MS-based proteome analysis was used to identify the composition of the HCT-15 colon cancer cell cytomatrix. The mRNA translation was measured by Ribosome footprinting. Results: Since the kinesis of the solid phase can trigger liquid phase flux, the composition of the cytomatrix was the primary focus of the study of cytoplasmic motion. The systemic analysis of the cytomatrix constituency allowed the identification of a diverse functional group of proteomes that included cytoskeletal and structural elements of organoids, metabolic enzymes, and normal- and proto-oncogenic signaling pathways. The two-phase system elucidated how diverse chemical reactions can occur simultaneously within the cell cytoplasm. Namely, the immobilization of catalytic complexes on the solid cytomatrix physically segregates biochemical reactants, thereby overcoming spatial impediments and allowing diverse metabolic reactions to coincide. Actomyosin and other actin-binding proteins of cytomatrix were the most abundant and diverse proteome, indicating that the primary machinery driving cytomatrix mobility consists of actomyosin, actin-binding, and actin-regulating proteins of cytomatrix. The cytomatrix dynamics trigger the cytosolic motion that carries substrates to immobilized catalytic complexes of the cytomatrix, thereby increasing product output, which appears to be a more intense process in cancer cells due to genetic and epigenetic aberrations. Under normal physiological conditions, mitochondrial respiration can deliver the energy requirements of cytomatrix micromechanics, but in cancer cells, additional energy of ATP is supplied by the aerobic glycolysis known as the Warburg effect to provide the ATP surplus for actomyosin mechanics. Conclusion: The intracellular motion is supported through non-muscle actomyosin action of the solid phase (cytomatrix), permitting increased glycolytic pathway activity in tumor cells. This novel concept of cancer links the Warburg effect with the bioenergetics of cytomatrix micromechanics and the dynamics of metabolic processes in tumors. Thus, two of the mysteries in cancer cell biology, how chemical reactions occur in the cytoplasm and energy is generated by tumor cells, have now been linked. Citation Format: Tattym E. Shaiken. Intracellular cytomatrix actin and the Warburg effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4421.

Abstract 527: Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification

Abstract Therapy resistance is one of the most common underlying causes of poor prognosis in cancer patients, ultimately driving fatal outcome. We have previous demonstrated that following chemotherapeutic stress, a subset of cancer cells undergo a mitotic skip and enter an endocycle, causing whole-genome duplication without division. This endocycling cell state is resistant to cytotoxic therapies and thus is an actuator of therapy resistance. Cells in the endocycling state eventually undergo depolyploidization and repopulate the tumor, observed clinically as a recurrence. We also demonstrated that the minus-end directed motor protein KIFC1, a mediator of centrosome clustering, has higher expression in cells following treatment. Centrosome amplification (CA) involves aberrations in centrosome number, where cells have 3 centrosomes. Cells with extra centrosomes create a multi-polar spindle during mitosis, which presents high risk for cell death and arrest, but cancer cells with CA avoid this by clustering extra centrosomes to create a pseudo-bipolar spindle. Based upon this data, we hypothesize that studying centrosome dynamics and abnormalities presents a window into better understanding endocycle biology and this novel mechanism of therapy resistance. Cancer cells were treated with LD50 dose of cisplatin and released from treatment for varying lengths of time. CA in endocycling cells increased from 59.2% to 100% during the treatment recovery period while maintaining identical levels of clustering. Endocycling cancer cells have a nearly 5-fold increase in declustered centrosomes compared to proliferative cells. Centrosomes in endocycling cells are displaced from the perinuclear region and toward the cell periphery. We hypothesize that centrosome number may indicate the number of times a cell has endocycled and provide insight into the timing and mechanism of mitotic bypass. Centrosomes play a major role in microtubule nucleation, affecting cell motility and polarity, and act as signaling platforms, enhancing signaling activation and specificity. Because of this, we hypothesize that CA may be a critical mechanism for endocycling cancer cells to survive and maintain this resistant cell state. Citation Format: Madison Purkerson, Sarah Amend, Kenneth J. Pienta. Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 527.

Abstract 4945: Computational modeling-based discovery of novel anticancer drug candidates targeting centromere-associated protein E

Abstract Purpose: Centromere-associated protein E (CENP-E), a mitotic motor protein, is crucial for cell division. Inhibition of CENP-E can lead to chromosome misalignment and aneuploidy. Our recent studies revealed that the CENP-E inhibitor (CENP-Ei), Compound A, induces aneuploid-mediated cGAS-STING pathway activation in cancer cell lines, which is expected to trigger innate immune response and induce immunological conversion of the tumor microenvironment from cold to hot. However, known CENP-Eis exhibited limited efficacy, and none have gone beyond phase I trials since 2012. The aim of this study is to develop a workflow integrating structure and artificial intelligence-based modeling approaches to accelerate the discovery of novel CENP-Eis with improved efficacy and overcome related challenges. Method: The inhibitor-bound CENP-E structure was modeled using chimeric homology modeling followed by induced fit docking with known CENP-Eis. Structure-based virtual screening (VS) was performed on large compound libraries in Enamine REAL, Synthetically Accessible Virtual Inventory, and MolPort databases. Consensus scoring methods were applied by averaging ranks of individual molecules obtained from VS against different CENP-E conformations. An active learning-enhanced VS pipeline was developed using ATOM Modeling PipeLine (AMPL) to find the best-ranked ligands more efficiently. CENP-E ligand candidates were further filtered based on their safety properties predicted by machine learning models trained with curated databases of adverse drug reactions using AMPL. The success of this study was measured by its ability to reveal novel chemotypes that could modulate CENP-E in kinesin ATPase assays. Result: Conformations of CENP-E’s ligand binding site were predicted for VS using the homology model. Structure-based VS was performed to select high-ranked compounds based on consensus scoring. The active learning-enhanced VS pipeline achieved 75% accuracy in ranking the top 20% of hit compounds. The graph convolutional network model with a ROCAUC, 0.76, was created using AMPL to filter ligand candidates based on their predicted safety properties. Seventy-three potential CENP-E ligands computationally prioritized from the Molport database were experimentally tested using kinesin ATPase assays from which 4 potential inhibitors and 8 potential activators were identified indicating overlapping binding sites of inhibitors and activators. Conclusion: Our integrated workflow is effective in discovering novel CENP-E modulators. The discovery of CENP-E activators provides an opportunity to identify novel therapeutics in hepatocellular carcinoma malignancies known to experience decreased CENP-E activity leading to aneuploidy. The predictions generated by computational models combined with experimental validations could further accelerate anticancer drug discovery. Citation Format: Pinyi Lu, Ryo Kamata, Michael R. Weil, Naomi Ohashi, Akihiro Ohashi, Eric A. Stahlberg. Computational modeling-based discovery of novel anticancer drug candidates targeting centromere-associated protein E [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4945.

Genome-wide identification of the key kinesin genes during fiber and boll development in upland cotton (Gossypium hirsutum L.).

Kinesin is a kind of motor protein, which interacts with microtubule filaments and regulates cellulose synthesis. Cotton fiber is a natural model for studying the cellular development and cellulose synthesis. Therefore, a systematic research of kinesin gene family in cotton (Gossypium spp.) will be beneficial for both understanding the function of kinesin protein and assisting the fiber improvement. Here, we aimed to identify the key kinesin genes present in cotton by combining genome-wide expression profile data, association mapping, and public quantitative trait loci (QTLs) in upland cotton (G. hirsutum L.). Results showed that 159 kinesin genes, including 15 genes of the kinesin-13 gene subfamily, were identified in upland cotton; of which 157 kinesin genes can be traced back to the diploid ancestors, G. raimondii and G. arboreum. Using a combined analysis of public QTLs and genome-wide expression profile information, there were 29 QTLs co-localized together with 28 kinesin genes in upland cotton, including 10 kinesin-13 subfamily genes. Genome-wide expression profile data indicated that, among the 28 co-localized genes, seven kinesin genes were predominantly expressed in fibers or ovules. By association mapping analysis, 30 kinesin genes were significantly associated with three fiber traits, among which a kinesin-13 gene, Ghir_A11G028430, was found to be associated with both cotton boll length and lint weight, and one kinesin-7 gene, Ghir_D04G017880 (Gh_Kinesin7), was significantly associated with fiber strength. In addition, two missense mutations were identified in the motor domain of the Gh_Kinesin7 protein. Overall, the kinesin gene family seemingly plays an important role in cotton fiber and boll development. The exploited kinesin genes will be beneficial for the genetic improvement of fiber quality and yield.

Abstract 3347: KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe

Abstract Cancer cells often have more than two centrosomes (supernumerary centrosomes) and this is one of the hallmarks of cancer. We previously demonstrated that CDK2 antagonism inhibits clustering of supernumerary centrosomes at mitosis leading to multipolar cell division and apoptotic death in non-small cell lung cancer. This antineoplastic process was designated anaphase catastrophe. Anaphase catastrophe can preferentially affect cancer cells sparing normal cells with two centrosomes. On the other hand, as CDK2 functions in normal cell cycle progression, its inhibition can also affect normal and malignant cells. Identification of a novel drug target that would preferentially cause anaphase catastrophe is needed. The motor protein KIFC1 affects centrosome clustering. Small cell lung cancer (SCLC), known for its poor prognosis with limited therapeutic options, displays disruptions in the cell cycle checkpoint mechanism due to inactivation of p53 and RB. We hypothesized SCLC was susceptible to mitotic abnormalities, including anaphase catastrophe. In this study, we explored the possibility of KIFC1 as a novel therapeutic target that triggers anaphase catastrophe in SCLC. In-silico GEO database analysis revealed elevated levels of KIFC1 and PLK4 in SCLCs compared with other cancers and normal tissues. PLK4 plays a key role in centrosome amplification. Our in-silico analysis results imply that centrosome amplification and their clustering frequently occur in SCLC. We performed in-vitro functional analysis of KIFC1 inhibition in SCLC cell lines (NCI-H146, SHP77, NCI-H209, and NCI-H524 cells), using siRNAs and CRISPR-Cas9 system for genetic inhibition and AZ82, a specific KIFC1 inhibitor, for pharmacologic inhibition of KIFC1. Cell proliferation was measured by the CellTiter-Glo Luminescent Assay and apoptosis was scored by annexin V and PI staining followed by flow cytometry analysis. Cell growth was reduced and apoptosis was statistically significantly increased after genetic inhibition of KIFC1 by siRNAs and by use of the CRISPR-Cas9 system in SCLC cell lines. These antineoplastic effects also occurred after AZ82 treatments, in a dose-dependent manner in these SCLC cell lines. To examine the mitotic status of SCLC cells after KIFC1 inhibition, SCLC cells were stained with α-tubulin antibody and DAPI before analysis with a fluorescence microscope. Besides normal bipolar mitotic cells, multipolar mitotic cells were observed, and their population was increased after KIFC1 knockdown with siRNAs. This indicated that clustering of supernumerary centrosomes was inhibited by targeting KIFC1 in SCLC cells. Taken together, KIFC1 is a potential therapeutic target to induce anaphase catastrophe in cancer cells. Targeting KIFC1 is a novel therapeutic strategy to consider for SCLC that currently has limited treatment options. Citation Format: Natsuki Nakagawa, Masakatsu Tokunaga, Mirei Ka, Yuriko Sugiura, Takahiro Iida, Takahiro Ando, Kousuke Watanabe, Xi Liu, Ethan Dmitrovsky, Hidenori Kage, Masanori Kawakami. KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3347.

Abstract 664: GSC000190, a highly potent inhibitor of KIF18A, for tumors with chromosome instability

Abstract Chromosome instability (CIN) and Genetic instability is a common feature of malignant tumor cells, which may contribute to tumor progression and lead to drug resistance and tumor metastasis. KIF18A, a member of the kinesin-8 family, is an ATP dependent plus end microtubule motor protein. Studies have found that genetic depletion of KIF18A greatly impacts the chromosome alignment, elongation of mitotic spindles, activation spindle assembly checkpoint of CIN+ tumor cell and leads to cell death, while have little impact towards the survival of normal cells, making KIF18A an attractive therapeutic target for targeting the vulnerability of CIN related tumors. Here we report the pharmacology characterization of a “best in class” potential KIF18A inhibitor for CIN+ tumors. In vitro, GSC000190 inhibited KIF18A ATPase activity with a single nM potency and has great potency inhibiting growth of a wide range of TP53 mutant/CIN+ tumor cells. In vivo, GSC000190, at approximately one tenth of systematic exposure comparing to that of AMG650, the leading clinical KIF18A inhibitor, causes tumor shrinkage in multiple CDX models including ovarian, colon and lung cancers. In an HGSOC PDX model which is resistant to platinum and olaparib, GSC000190 induced strong tumor regression both as single agent and through combination without significant body weight reduction. PD study confirms a more elevated mitotic phosphorylated histone 3 (PH3) staining in GSC000190 treated tumor tissues, correlating with a better in vivo efficacy than that of AMG650. GSC000190 has a favorable ADME profile in rodents, dogs and NHPs, as well as a much better safety profile. GSC000190 is now under IND-enabling study and P1 study is planned in the second quarter of 2024. Legal entity responsible for the study: The authors. Disclosure: All authors have declared no conflicts of interest. Citation Format: Peng Tu, Chen Zhang, Biao Lu, Yuanfeng Xia, Fanglong Yang. GSC000190, a highly potent inhibitor of KIF18A, for tumors with chromosome instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 664.

Abstract 5723: Selectivity profiling of small molecule kinesin inhibitors using microplate-based ATPase activity assay

Abstract Kinesin superfamily proteins (KIFs) are a large family of molecular motor proteins, that share a highly conserved motor domain. KIFs play an important role in cell division and transport of vesicles and organelles within cells. Mitotic kinesins hydrolyze ATP through its ATPase activity to move along the spindle microtubules and carry out several functions during mitosis to facilitate precise chromosome segregation. Altered expressions of several kinesins are shown in various cancers, fueling the aggressive nature of cancers leading to genomic instability. KIF11/Eg5 overexpression is shown in various cancers including hepatic carcinoma, lung, prostate, colorectal, gastric, and pancreatic cancers. Small molecule inhibitors of KIF11/Eg5 and CENP-E are shown to result in mitotic arrest leading to apoptosis in tumor cell lines. Despite the poor clinical outcome of several Eg5 and CENP-E inhibitors entered in clinical trials, targeting kinesins remains an attractive anti-cancer therapeutic approach. Recently, an inhibitor of KIF18A has entered clinical trial for treatment of solid tumors with high chromosomal instability. Here we show development of a microplate based biochemical screening assays for a panel of kinesin motor domain proteins. We summarize kinetic characterization of kinesin proteins and study the selectivity of various previously reported KIF18A, Eg5, CENP-E inhibitors across a kinesin selectivity panel. Citation Format: Brenna P. Lee, Sung Won Oh, Kurumi Y. Horiuchi, Safnas F. AbdulSalam. Selectivity profiling of small molecule kinesin inhibitors using microplate-based ATPase activity assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5723.

Gliding motility proteins GldJ and SprB contribute to Flavobacterium columnare virulence.

Flavobacterium columnare causes columnaris disease in fish. Columnaris disease is incompletely understood, and adequate control measures are lacking. The type IX secretion system (T9SS) is required for F. columnare gliding motility and virulence. The T9SS and gliding motility machineries share some, but not all, components. GldN (required for gliding and for secretion) and PorV (involved in secretion but not required for gliding) are both needed for virulence, implicating T9SS-mediated secretion in virulence. The role of motility in virulence is uncertain. We constructed and analyzed sprB, sprF, and gldJ mutants that were defective for motility but that maintained T9SS function to understand the role of motility in virulence. Wild-type cells moved rapidly and formed spreading colonies. In contrast, sprB and sprF deletion mutants were partially defective in gliding and formed nonspreading colonies. Both mutants exhibited reduced virulence in rainbow trout fry. A gldJ deletion mutant was nonmotile, secretion deficient, and avirulent in rainbow trout fry. To separate the roles of GldJ in secretion and in motility, we generated gldJ truncation mutants that produce nearly full-length GldJ. Mutant gldJ563, which produces GldJ truncated at amino acid 563, was defective for gliding but was competent for secretion as measured by extracellular proteolytic activity. This mutant displayed reduced virulence in rainbow trout fry, suggesting that motility contributes to virulence. Fish that survived exposure to the sprB deletion mutant or the gldJ563 mutant exhibited partial resistance to later challenge with wild-type cells. The results aid our understanding of columnaris disease and may suggest control strategies.IMPORTANCEFlavobacterium columnare causes columnaris disease in many species of freshwater fish in the wild and in aquaculture systems. Fish mortalities resulting from columnaris disease are a major problem for aquaculture. F. columnare virulence is incompletely understood, and control measures are inadequate. Gliding motility and protein secretion have been suggested to contribute to columnaris disease, but evidence directly linking motility to disease was lacking. We isolated and analyzed mutants that were competent for secretion but defective for motility. Some of these mutants exhibited decreased virulence. Fish that had been exposed to these mutants were partially protected from later exposure to the wild type. The results contribute to our understanding of columnaris disease and may aid development of control strategies.

Identification and validation of neutrophils-related subtypes and prognosis model in triple negative breast cancer

BackgroundNeutrophils are considered to be crucial players in the initiation and progression of cancer. However, the complex relationship between neutrophils and cancer prognosis remains elusive, mainly due to the significant plasticity and diversity exhibited by these immune cells.MethodsAs part of our thorough investigation, we examined 38 Neutrophils-Related Genes (NRGs) and the associated copy number variations (CNV), somatic mutations, and gene expression patterns in relation to triple negative breast cancer (TNBC). The interactions between these genes, their biological roles, and their possible prognostic significance were then examined. With the NRGs as our basis, we applied Lasso and Cox regression analyses to create a predictive model for overall survival (OS). Furthermore, TNBC tissue and a public database were used to assess changes in MYO1D expression (MYO1D is characterized as a member of the myosin-I family, a group of motor proteins based on actin), its connection to neutrophil infiltration, and the clinical importance of MYO1D in TNBC.ResultsFour neutrophil-related genes were included in the development of a prognostic model based on neutrophils. The model was further shown to be an independent predicted factor for overall survival by multivariate Cox regression analysis. According to this study, neutrophil subtype B as well as gene subtype B, were associated with activated cancer immunity and poor prognosis of TNBC patients. Furthermore, considering that poor OS was linked to increased MYO1D expression, MYO1D was increased in TNBC tissues and associated with neutrophil infiltration. In vitro experiments also confirmed that MYO1D facilitates breast cancer invasion and metastasis.ConclusionBased on the degree of gene expression linked to neutrophils, a unique prognostic model was created. MYO1D could be a potential prognostic biomarker in TNBC patients and also a prospective target for therapy.