Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities

Abstract

Aneuploidy, the incorrect number of whole chromosomes, is a common feature of tumors that contributes to their initiation and evolution. Preventing aneuploidy requires properly functioning kinetochores, which are large protein complexes assembled on centromeric DNA that link mitotic chromosomes to dynamic spindle microtubules and facilitate chromosome segregation. The kinetochore leverages at least two mechanisms to prevent aneuploidy: error correction and the spindle assembly checkpoint (SAC). BubR1, a factor involved in both processes, was identified as a cancer dependency and therapeutic target in multiple tumor types; however, it remains unclear what specific oncogenic pressures drive this enhanced dependency on BubR1 and whether it arises from BubR1's regulation of the SAC or error-correction pathways. Here, we use a genetically controlled transformation model and glioblastoma tumor isolates to show that constitutive signaling by RAS or MAPK is necessary for cancer-specific BubR1 vulnerability. The MAPK pathway enzymatically hyperstimulates a network of kinetochore kinases that compromises chromosome segregation, rendering cells more dependent on two BubR1 activities: counteracting excessive kinetochore-microtubule turnover for error correction and maintaining the SAC. This work expands our understanding of how chromosome segregation adapts to different cellular states and reveals an oncogenic trigger of a cancer-specific defect.

Keywords: BubR1; MAPK; aneuploidy; kinetochore; mitosis.

Fig. 1.

Laboratory-transformed cells recapitulate RAS-dependent behaviors observed in tumor-isolated cells. (A) Schematic of signaling pathways downstream of RAS and their connections to mitosis. Proteins outlined in blue or brown localize to the kinetochore or mitotic spindle, respectively. (B) Schematic of viral-mediated genomic modifications to transform retinal pigment epithelial cells with different oncogenes. (C) Ability of genetically modified cells to proliferate as colonies suspended in a matrix of soft agar. Three biological replicates are shown with the SD indicated. (D) RNA-seq data from three biological replicates analyzed through PCA reveals parental cell lines ARPE19 and ARPE19^T53D4 are essentially indistinguishable from each other, and are clearly partitioned from the three oncogenically stressed cell lines. (E) Transcripts overexpressed in ARPE19^T53D4-AKT, ARPE19^T53D4-MEK, and ARPE19^T53D4-RAS cells relative to parental ARPE19 cells were tested for their enrichment in published gene signatures of AKT, MEK, and RAS activation. (F) Distances between kinetochores on duplicated chromosomes (interkinetochore distances) were measured in unperturbed cells and those treated for 1 h with MEK or ERK inhibitors. Rest length refers to mean interkinetochore distances of prophase kinetochore pairs prior to microtubule attachment. Scale bars are 5 μm (Top) and 1 μm (Bottom). Below the images, the points on the graph represent the average interkinetochore distance per cell, and bars represent mean and 95% CIs. For each condition, at least 20 kinetochore pairs were measured from at least 28 cells from three independent experiments. (G) Chromosome alignment assay with and without short-term inhibition of MEK using drug U0126 (Left). Three biological replicates were measured, and the SD is indicated (Right). All P values in figure were calculated with Tukey’s multiple comparisons test.

Fig. 2.

RAS-transformed cells require BubR1-mediated recruitment of PP2A-B56α. (A) Diagram of BubR1 mutants/fusions used to manipulate PP2A-B56α recruitment to kinetochores. KEN, KEN boxes; TPR, Tetratricopeptide Repeat motif; BUB-BD, BUB Binding Domains (Bub3 and Bub1); KARD, kinetochore attachment regulatory domain. (B) Chromosome alignment assay in ARPE19^T53D4-RAS cells depleted of endogenous BubR1 and rescued with transient expression of empty vector (E.V.), BubR1^WT, BubR1^3A that cannot bind PP2A-B56α, or Mis12-KARD^3D that constitutively recruits PP2A-B56α to kinetochores independent of BubR1. ARPE19 data are replotted from Fig. 1 (green bar) as a reminder that BubR1 is dispensable for chromosome alignment in these cells. Three biological replicates are shown, and the SD is indicated. Numbers above each bar in the graph indicate P value when compared to E.V. transfection alone. (C) Interkinetochore distances measured in ARPE19^T53D4-RAS cells when PP2A-B56α is constitutively recruited through expression of Mis12-KARD^3D. Points represent the average distance per cell (at least 20 kinetochores were measured per cell) collected from three biological replicates (n = 34 cells for control cells; n = 35 cells for Mis12-KARD^3D-expressing cells), and bars represent mean and 95% CIs. (D) Cell proliferation of ARPE19 and ARPE19^T53D4-RAS cells depleted of endogenous BubR1 and rescued with stable expression of E.V., BubR1^WT, or BubR1^3A. Three biological replicates are shown with the mean and SD reported. All P values in figure were calculated with Tukey’s multiple comparisons test.

Fig. 3.

MAPK signaling in ARPE19^T53D4-RAS cells hyperactivates a network of kinetochore kinases. (A) Published connections between BubR1/PP2A-B56α and RAS/MAPK that could be implicated in the observed chromosome alignment and interkinetochore distance phenotypes. (B–F) Immunostaining of prometaphase ARPE19 and ARPE19^T53D4-RAS cells ± MEK inhibitor U0126 with corresponding antibodies (Left), whose kinetochore levels were quantified and normalized to those measured in ARPE19^T53D4-RAS cells (Right). Points represent the average kinetochore intensity per cell, and bars represent mean and 95% CIs. For all panels, at least 30 kinetochores were measured per cell from at least 26 cells, totaled from three biological replicates. All P values were calculated with Tukey’s multiple comparisons test. For panel B, levels of pHec1/Hec1 in prometaphase and metaphase were independently normalized.

Fig. 4.

Inhibition of Mps1 relieves RAS/MAPK-dependent mitotic phenotypes. (A) lnterkinetochore distances in ARPE19^T53D4-RAS cells treated with increasing concentrations of the Mps1 inhibitor, Reversine. MG132 was included to prevent mitotic exit. The average interkinetochore distance measured in ARPE19 cells shown in Fig. 1 is indicated on the graph (green dotted line). For each concentration of Reversine, at least 20 kinetochore pairs were measured from at least 24 cells. (B) Interkinetochore distance values for ARPE19 and ARPE19^T53D4-RAS cells treated with 750 nM Reversine (data from ARPE19^T53D4-RAS cells replotted from A; data from ARPE19 cells with no drug are replotted from Fig. 1F). For each condition, at least 20 kinetochore pairs were measured from at least 33 cells. (C) Chromosome alignment phenotypes following small interfering RNA depletion of BubR1 in the presence or absence of 750 nM Reversine (n = 3 independent experiments).

Fig. 5.

ARPE19^T53D4-RAS cells exhibit chromosome alignment delays and chromosome segregation errors in a MAPK-dependent manner. (A) Mitotic duration (nuclear envelope breakdown [NEB] to anaphase onset) was quantified in asynchronous ARPE19 and ARPE19^T53D4-RAS cells expressing histone H2B-EGFP. The delay observed in ARPE19^T53D4-RAS cells (Top) was reduced when cells were treated with MEK inhibitor U0126 (Bottom). Points represent the percent of cells in mitosis binned by image acquisition intervals (3 min) from three biological replicates. Data are fitted with a sigmoidal curve, and dotted lines represent 95% CI for the fitted line. (B) Mitotic duration in A was separated into time to alignment (Left, NEB to chromosome alignment) and SAC silencing (Right, chromosome alignment to anaphase onset). Timing for each cell is represented as a point on the graph, and P values were calculated using Dunn’s multiple comparisons test. Cell n values are as follows for chromosome alignment timing: ARPE19, 175; ARPE19 + U0126, 166; ARPE19^T53D4-RAS, 515; ARPE19^T53D4-RAS + U0126, 251; and for SAC silencing timing: ARPE19, 170; ARPE19 + U0126, 165; ARPE19^T53D4-RAS, 515; and ARPE19^T53D4-RAS + U0126, 520. (C) Number of cell divisions with chromosome segregation errors in ARPE19 and ARPE19^T53D4-RAS cells when untreated or in the presence of MEK inhibitor U1026.

Fig. 6.

Oncogenic RAS/MAPK signaling is associated with BubR1 sensitivity in patient-derived glioblastoma stem-like cells. (A) Genes overexpressed in aneuploid compared with diploid tumor cells (17) were interrogated for enrichment of previously published signatures of RAS, MEK (–39), and AKT activation. (B) Sets of genes expressed at elevated levels in BubR1-resistant (827 and 1502) and BubR1-sensitive (131, G155, and G179) glioblastoma patient isolates compared with nontransformed neural stem cells (CB660) were generated using published RNA-seq data (58) and represent each column. Fold enrichment and P values for the enrichment of previously published gene signatures of RAS, MEK, and AKT signaling within glioblastoma isolates are displayed in each row. The number of genes significantly elevated in patient isolates is listed in parentheses at the bottom of the plot. (C and D) Average metaphase interkinetochore distance ± MEK inhibition in (C) neural stem cells (CB660) and RAS-transformed neural stem cells (CB660^T53D4/RAS) and in (D) glioblastoma patient isolates with or without the enhanced BubR1 requirement. (C and D) Points represent the average distance per cell collected from three biological replicates, and bars represent mean and 95% CIs. P values were calculated with Tukey’s multiple comparisons test. For each condition in C and D, at least 20 kinetochore pairs were measured from at least 22 cells. (E) Copy number gains through aneuploidy and/or activating mutations of RAS/MAPK components alter the balance of phosphatase and kinase activity at kinetochores. This weakens kinetochore–microtubule attachments and renders the phosphatase recruitment activities of BubR1 a possible target for therapeutics.