Tomoyuki U. Tanaka

Chromosomes must efficiently and properly interact with the mitotic spindle during prometaphase for correct segregation in anaphase. Chromosomes at the nuclear periphery or behind the spindle poles interact less efficiently with the mitotic spindle, increasing the risk of missegregation. The mechanisms that mitigate such risks in unperturbed cells are unknown. An actomyosin network (PANEM) forms around the nucleus during prophase. While the myosin-II-dependent PANEM contraction immediately after nuclear envelope breakdown (NEBD) facilitates chromosome interaction with the mitotic spindle, the mechanism by which it does so remains unclear. Here, using human cell lines, we show that immediately after NEBD, PANEM contraction directly pushes chromosomes at the nuclear periphery or behind spindle poles toward the center of cells. Detailed tracking of kinetochore movements following light-induced activation of a myosin II inhibitor reveals that this inward movement of chromosomes facilitates kinetochores’ initial interaction with spindle microtubules. It also promotes the onset of kinetochores’ congression toward the spindle mid-plane, but not congression itself once it starts. Thus, PANEM contraction ensures high-fidelity chromosome segregation by relocating chromosomes from unfavorable locations. Since some chromosomally unstable cancer cells fail to establish PANEM during early mitosis, the absence of PANEM may contribute to numerical chromosomal instability in these cells.

Chromosomes must efficiently and properly interact with the mitotic spindle during prometaphase for correct segregation. Chromosomes at the nuclear periphery or behind the spindle poles interact less efficiently with the mitotic spindle, increasing the risk of missegregation during anaphase. The mechanisms that mitigate such risks in unperturbed cells are unknown. An actomyosin network (PANEM) forms around the nucleus during prophase. While the myosin-II-dependent PANEM contraction facilitates chromosome interaction with the mitotic spindle following nuclear envelope breakdown (NEBD), the mechanism by which it does so remains unclear. Here, using human cell lines, we show that immediately after NEBD, PANEM contraction directly pushes chromosomes at the nuclear periphery toward the centre. Detailed tracking of kinetochore movements following light-induced activation of a myosin-II inhibitor reveals that this inward movement facilitates kinetochores’ initial interaction with spindle microtubules. PANEM contraction also promotes the onset of their congression towards the spindle mid-plane, but not congressional motion itself. Furthermore, PANEM contraction reduces the volume of polar regions, helping reposition chromosomes and initiate their congression. Thus, PANEM contraction ensures high-fidelity chromosome segregation by relocating chromosomes from unfavorable locations. Some chromosomally unstable cancer cells fail to establish PANEM during early mitosis, implying its absence in cancer progression.

Supplementary Movie 2 from Elraglusib Induces Cytotoxicity via Direct Microtubule Destabilization Independently of GSK3 Inhibition

Supplementary Movie 1 from Elraglusib Induces Cytotoxicity via Direct Microtubule Destabilization Independently of GSK3 Inhibition

Chromosomes must efficiently and properly interact with the mitotic spindle during prometaphase for correct segregation in anaphase. Chromosomes at the nuclear periphery or behind the spindle poles interact less efficiently with the mitotic spindle, increasing the risk of missegregation. The mechanisms that mitigate such risks in unperturbed cells are unknown. An actomyosin network (PANEM) forms around the nucleus during prophase. While the myosin-II-dependent PANEM contraction immediately after nuclear envelope breakdown (NEBD) facilitates chromosome interaction with the mitotic spindle, the mechanism by which it does so remains unclear. Here, using human cell lines, we show that immediately after NEBD, PANEM contraction directly pushes chromosomes at the nuclear periphery or behind spindle poles toward the center of cells. Detailed tracking of kinetochore movements following light-induced activation of a myosin-II inhibitor reveals that this inward movement of chromosomes facilitates kinetochores’ initial interaction with spindle microtubules. It also promotes the onset of kinetochores’ congression towards the spindle mid-plane, but not congression itself once it starts. Thus, PANEM contraction ensures high-fidelity chromosome segregation by relocating chromosomes from unfavorable locations. Since some chromosomally unstable cancer cells fail to establish PANEM during early mitosis, the absence of PANEM may contribute to numerical chromosomal instability in these cells. Impact statement: An actomyocin network (PANEM) forms around the nucleus in prophase, and its contraction repositions peripheral and polar chromosomes to facilitate their interaction with the mitotic spindle, ensuring their correct segregation.

Supplementary Movie 2 from Elraglusib Induces Cytotoxicity via Direct Microtubule Destabilization Independently of GSK3 Inhibition

Supplementary Movie 1 from Elraglusib Induces Cytotoxicity via Direct Microtubule Destabilization Independently of GSK3 Inhibition

Elraglusib (9-ING-41) is an ATP-competitive inhibitor of glycogen synthase kinase-3 (GSK3) with preclinical studies demonstrating broad activity against many tumor types. Promising early-phase clinical trial data led to FDA orphan drug status, and a randomized phase II study in combination with cytotoxic chemotherapy in pancreatic cancer has recently completed its recruitment. Similarly, single-agent responses in adult T-cell leukemia/lymphoma and melanoma and combination treatment data in several other tumor types have been encouraging. The elraglusib mechanism of action is unknown, but it is unlikely to act through GSK3 inhibition because cytotoxicity is observed below the IC50 for GSK3, and other small molecule GSK3 inhibitors do not produce cytotoxic effects, at least in lymphoma cells. We show here that elraglusib perturbs chromosomal alignment to cause a mitotic arrest in multiple tumor lines. This arrest is caused by direct microtubule (MT) depolymerization, which prevents the attachment of kinetochores to MTs. At clinically relevant doses, these mitotically arrested cells eventually undergo mitotic slippage, leading to gross chromosome missegregation, DNA damage, and apoptosis. These effects explain the cytotoxicity of elraglusib because temporarily pausing cell-cycle progression with the CDK4/6 inhibitor palbociclib abolishes any drug-induced genotoxicity and apoptosis. In summary, elraglusib acts as a direct MT destabilizer both in vitro and across multiple cancer types, resulting in mitotic arrest, DNA damage, and apoptosis. These effects likely account for its broad pan-cancer activity, which does not rely upon GSK3 inhibition as they are not replicated by other GSK3 inhibitors.Significance:Elraglusib was designed as a GSK3 inhibitor and is currently in clinical trials for several cancers. We show conclusively that the target of elraglusib that leads to cytotoxicity is MTs and not GSK3. This has significant implications for ongoing clinical trials of the compound and will help in understanding off-target side effects, inform future clinical trial design, and facilitate the development of biomarkers to predict response.

Elraglusib impairs proliferation and reduces cell viability in stimulated primary human T cells but does not affect unstimulated cells (A and B). Primary human T cells were isolated from PBMCs from healthy volunteers. Unstimulated controls (A) remained in CM, and stimulated cells (B) were treated with a CD3/CD28-co-stimulation kit. Cells were treated with DMSO control or elraglusib (Elr). Cell viability was assayed using Promega RealTime-Glo MT assay and the plate read daily for 120 hours. Data combine three independent experiments with T cells from three individual volunteers. Data normalized to the experimental control peak luminescence. Elraglusib increases the soluble tubulin fraction and decreases the insoluble tubulin fraction in stimulated primary human T cells (C and D). Proliferating stimulated primary human T cells were treated for 5 hours and assayed for soluble and insoluble tubulin as described above. Blots are representative of three independent experiments, and quantification combines three independent experiments. Fold change calculated vs. control, error bars represent SEM, and statistical significance was determined using one-way ANOVA, with P ≤ 0.05. ns, not significant; *, P < 0.05; ****, P < 0.0001.

Elraglusib causes a mitotic arrest. A and B, Example flow cytometry plots (A) and quantifications (B) showing the effect of 24-hour elraglusib (Elr) treatment on the percentage of 2N, 4N, and 4N-mitotic K299 WT cells. C and D, Example flow cytometry plots (C) and quantifications (D) following 24-hour treatments with two structurally distinct GSK3 inhibitors, CT99021 and LY2090314 in K299 WT cells. E and F, Example flow cytometry plots (E) and quantifications (F) showing the effect of 24-hour elraglusib (Elr) treatment on the percentage of 2N, 4N, and 4N-mitotic K299 GSK3A/B dual paralog shRNA knockdown cells (DKD). G and H, Total GSK3 immunoblot (G) with quantification confirming knockdown efficiency in K299 DKD compared with K299 WT cells (H). Cells were treated for 24 hours prior to cell-cycle analysis by flow cytometry using propidium iodide and anti–phospho-histone-H3 (Ser10) staining. Ten thousand events were analyzed per sample. Cell-cycle histograms are representative of three independent experiments, and quantifications combine three independent experiments. Immunoblot representative of three independent experiments; quantification was performed by normalizing to actin loading control and combines three independent experiments. Fold change calculated vs. control, error bars represent SEM, and statistical significance was determined using one-way ANOVA, with P ≤ 0.05. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.