Journal: bioRxiv

Article Title: Non-Genetic Mechanisms of Fractional Resistance to Abemaciclib in Dedifferentiated Liposarcoma

doi: 10.64898/2026.05.22.727236

Figure Lengend Snippet: (A) Experimental workflow of the operating room-to-lab pipeline used for single-cell, multiplexed immunofluorescence profiling. Lipo246 cells were cultured and dedifferentiated primary tumor cells were resected and dissociated to be treated with increasing concentrations of abemaciclib for 24 h. Cells were fixed and subjected to iterative indirect immunofluorescence imaging (4i) consisting of iteration of antibody labeling (block → 1°/2° Ab → elution) and imaging. Images from all 4i rounds are processed through a computational pipeline to perform single-cell segmentation, signal quantification, and feature extraction. (B) Lipo246 and primary tumor cells were stained for 17 cell cycle regulators pRB, RB, Ki-67, CDK2, CDK4, cyclin D1 (cycD1), cyclin E1 (cycE1), Cdt1, E2F1, cyclin A2 (cycA2), cyclin B1 (cycB1), p21, Mdm2, pS6, S6, p53, p16, and integrated DNA. Representative immunofluorescence images from sequential rounds of 4i demonstrate a subset of these regulators for repeated labeling of distinct protein targets within the same cells. Nuclear DNA staining is used for segmentation, image registration, and integration across imaging rounds. (C) Single-cell matrix of 4i-measured cell cycle features. Heatmap of normalized protein expression across all quantified regulators, with individual cells ordered by increasing ratio of phosphorylated to total RB (pRB/RB), computed from 4i measurements. The corresponding single-cell pRB/RB distribution is shown at right and exhibits a bimodal structure used to define low pRB/RB (blue; nonproliferating) and high pRB/RB (pink; proliferating) cell populations. This organization reveals coordinated variation in cell cycle regulators across the pRB/RB-defined axis of cell cycle state.

Article Snippet: To evaluate sensitivity to combined CDK4/6 and CDK2 inhibition, Lipo246 cells were cotreated with abemaciclib (Selleckchem, catalog no. S5716) and the CDK2 inhibitor tagtociclib (MedChem Express, catalog no. HY-137894A).

Techniques: Single Cell, Immunofluorescence, Cell Culture, Imaging, Antibody Labeling, Blocking Assay, Extraction, Staining, Labeling, Expressing

Journal: bioRxiv

Article Title: Non-Genetic Mechanisms of Fractional Resistance to Abemaciclib in Dedifferentiated Liposarcoma

doi: 10.64898/2026.05.22.727236

Figure Lengend Snippet: (A) Distribution of the ratio of phosphorylated to total RB protein (pRB/RB) across individual Lipo246 cells under control (0 nM), 50 nM, and 250 nM abemaciclib treatment. pRB/RB exhibited a bimodal distribution, with a low pRB/RB population corresponding to hypophosphorylated RB and cell cycle arrest, and a high pRB/RB population corresponding to RB hyperphosphorylation and active proliferation. A conservative threshold (vertical red line) was defined to distinguish low pRB/RB and high pRB/RB cells and was used to classify proliferating cells for all subsequent single-cell analyses. (B) 95% CI of proliferating cells for the differences in mean expression (normalized z-scores) between either untreated cells and 50 nM abemaciclib (blue); or untreated and 250 nM abemaciclib (orange). CI overlapping with the dashed line at 0 indicate a lack of statistical significance. (C) Lipo246 cells were quantified, separated into high pRB/RB (pink) or low pRB/RB (Blue) levels. The top panel shows a dose-dependent reduction in cell cycle activity by abemaciclib as demonstrated by a reduction in pRB/RB positivity. While the middle and bottom panel shows an enrichment of CDK2 and cyclin B1 (cycB1) (respectively) in fractionally resistant cells. This enrichment is shown to be statistically significant by ANOVA analysis as seen in the corresponding violin plots. (D) Representative images of the effect of abemaciclib on Lipo246. Cells were treated for 24 hours with 0 (control), 50 nM or 250 nM abemaciclib. Cells were labeled for phosphorylated RB (pRB) which is a marker for actively cycling cells (top panel), cyclin dependent kinase 2 (CDK2)(middle panel) or DAPI for DNA and cycB1 (bottom panel). DAPI staining in the bottom panels shows roughly equal amounts of cells in each drug condition. We can visually observe the quantified depletion of pRB over increasing doses while a fraction of high pRB/RB cells persisting at high drug concentration with of upregulated CDK2 and cycB1.

Article Snippet: To evaluate sensitivity to combined CDK4/6 and CDK2 inhibition, Lipo246 cells were cotreated with abemaciclib (Selleckchem, catalog no. S5716) and the CDK2 inhibitor tagtociclib (MedChem Express, catalog no. HY-137894A).

Techniques: Control, Single Cell, Expressing, Activity Assay, Labeling, Marker, Staining, Concentration Assay

Journal: bioRxiv

Article Title: Non-Genetic Mechanisms of Fractional Resistance to Abemaciclib in Dedifferentiated Liposarcoma

doi: 10.64898/2026.05.22.727236

Figure Lengend Snippet: (A) Distribution of the ratio of phosphorylated to total RB protein (pRB/RB) across individual primary tumor cells under control (0 nM), 50 nM, and 250 nM abemaciclib treatment. A conservative pRB/RB threshold (vertical red line) was applied to identify cells retaining RB phosphorylation and active proliferation under treatment. (B) Differences in mean expression (normalized z-scores) for cell cycle regulators within the high pRB/RB proliferative fraction, comparing untreated cells with 50 nM (blue) or 250 nM (orange) abemaciclib. Points indicate mean differences and error bars denote 95% confidence intervals; intervals overlapping zero indicate nonsignificant differences. (C) Violin plots showing the distributions of selected cell cycle regulators in low pRB/RB (nonproliferating, blue) and high pRB/RB (proliferating, pink) primary tumor cells across treatment conditions. CDK2 and cyclin B1 (cycB1) exhibit selective enrichment within the proliferative fraction under high dose abemaciclib, consistent with fractional resistance. This enrichment is shown to be statistically significant by ANOVA analysis as seen in the corresponding violin plots. D) Representative images of the effect of dedifferentiated liposarcoma primary tumor cells were treated for 24 hours with 0 (control), 50 nM or 250 nM abemaciclib. Cells were labeled for phosphorylated RB (pRB) which is a marker for actively cycling cells (top panel), cyclin dependent kinase 2 (CDK2)(middle panel) or DAPI for DNA and cycB1 (bottom panel). DAPI staining in the bottom panels shows roughly equal amounts of cells in each drug condition. We can visually observe the colocalization of the upregulation of CDK2 and cycB1 in cells expressing pRB at high abemaciclib dose.

Article Snippet: To evaluate sensitivity to combined CDK4/6 and CDK2 inhibition, Lipo246 cells were cotreated with abemaciclib (Selleckchem, catalog no. S5716) and the CDK2 inhibitor tagtociclib (MedChem Express, catalog no. HY-137894A).

Techniques: Control, Phospho-proteomics, Expressing, Labeling, Marker, Staining

Journal: bioRxiv

Article Title: Non-Genetic Mechanisms of Fractional Resistance to Abemaciclib in Dedifferentiated Liposarcoma

doi: 10.64898/2026.05.22.727236

Figure Lengend Snippet: Lipo246 cells were cotreated with the CDK4/6 inhibitor abemaciclib (control, 10 nM, and 50 nM) and a range of CDK2 inhibitor concentrations (0, 0.25, 0.5, 1, 2.5, 5, 10 µM tagtociclib). (A) Proportion of pRB/RB positive cells following treatment with CDK2 inhibitor, tagtociclib. (B) Fold changes in the proportion of pRB/RB positive cells under treatment with tagtociclib. For each abemaciclib condition, fold changes were computed by normalizing the proportion of pRB/RB positive cells to the average proportion of the first three doses of tagtociclib (0, 0.25, and 0.5 µM). Error bars represent the SD across three technical replicates. Dashed lines indicate (A) the proportion or (B) fold change of pRB/RB positive cells without treatment with the CDK2 inhibitor.

Article Snippet: To evaluate sensitivity to combined CDK4/6 and CDK2 inhibition, Lipo246 cells were cotreated with abemaciclib (Selleckchem, catalog no. S5716) and the CDK2 inhibitor tagtociclib (MedChem Express, catalog no. HY-137894A).

Techniques: Control

Journal: bioRxiv

Article Title: Cryo-EM structures of the CDK11-cyclin L-SAP30BP complex reveal mechanisms of CDK11 regulation

doi: 10.64898/2026.03.24.713564

Figure Lengend Snippet: ( a ) Bound AMP-PNP in the active site of CDK11 (green). Residues involved in interactions with the nucleotide are shown as sticks, hydrogen bonds are indicated as black dashed lines, and magnesium ions are shown as green spheres. ( b ) The pseudo-substrate density occupying the substrate binding site of CDK11 is shown in light green (CDK11 green, T-loop blue, SAP30BP yellow, cyclin L purple). ( c ) Cryo-EM map shown as grey mesh and semi-transparent surface, with fitted model coloured as in b. ( d ) Molecular model of the CDK11 pseudo-substrate segment (residues 751-762, light green) occluding the substrate binding site. ( e ) CDK2-cyclin A (cyan and yellow) in complex with a substrate peptide (sand) in the same view as panel C (PDB ID 3QHR) . ( f ) Kinase assay using an SF3B1 1-463 substrate and trimeric kinase complexes containing CDK11B p110 wild type (WT) and the 1-743, S752E, and S752A variants. SF3B1 phospho-T313 (P-T313) was detected using Western blot. A loading control stained with Ponceau S is provided. ( g ) Quantification of Western blot band intensities, presented as the mean ± standard deviation and individual data points of N = 4 kinase assay technical replicates. The relative activity of the wild-type complex at 60 min was set to 1. The significance level for activity comparisons between complexes, determined by 2-way ANOVA and Tukey’s multiple comparisons test, is provided (*: p < 0.05; **: p < 0.01). ( h ) Schematic illustration of the effect of pseudo-substrate phosphorylation on the efficiency of binding of a simple model substrate to CDK11. Hypothetical models for effects on substrate specificity in the context of more complex substrates are shown in Supplementary Fig. 4e, f. The substrate binding site is schematically represented as a cleft, and the active site is indicated by a stylised ATP molecule.

Article Snippet: CDK2 was expressed with an N-terminal His 10 -tag (Addgene plasmid #79726; http://n2t.net/addgene:79726 ; RRID:Addgene_79726, provided by John Chodera, Nicholas Levinson, and Markus Seeliger) , and cyclin A2 was expressed with an N-terminal His 6 -tag.

Techniques: Binding Assay, Cryo-EM Sample Prep, Kinase Assay, Western Blot, Control, Staining, Standard Deviation, Activity Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Cryo-EM structures of the CDK11-cyclin L-SAP30BP complex reveal mechanisms of CDK11 regulation

doi: 10.64898/2026.03.24.713564

Figure Lengend Snippet: ( a ) Rendering of CDK11-bound OTS964 (blue) in the cryo-EM map (shown as semi-transparent grey mesh and surface). ( b , c ) Active site views of the CDK11-cyclin L-SAP30BP-OTS964 complex in the two alternative conformations observed in the cryo-EM reconstruction. Hydrogen bonds (donor-acceptor distance 3.5 Å or less) are indicated by black dashed lines. The shortest distance between inhibitor and gatekeeper residue (M516 in CDK11) is indicated and shown by a white dashed line. The change in position of Y449 between panels b and c is indicated with a dashed arrow in panel b. ( d ) Comparison of the structure of CDK11-cyclin L-SAP30BP-OTS964 (CDK11 in green, cyclin L in purple with residues 173-181 in pink, OTS964 in blue) to the structure of isolated CDK11B-OTS964 (PDB ID 7UKZ ; light green and light blue, respectively). The site of steric incompatibility of the isolated CDK11B conformation with the presence of cyclin L is indicated by a black triangle. Shifts in adjacent loops because of cyclin binding are indicated by arrows. ( e ) Active site view of the CAK-OTS964 complex (CDK7 is shown in grey, water molecules as red spheres). Only the hydrogen bonds to the hinge region are preserved because several hydrogen bonding partners are absent or beyond hydrogen bonding distance (distance to D97 indicated by a yellow dashed line). The distance to the gatekeeper residue (F91 in CDK7) is enlarged (yellow dashed line). ( f ) Active site view of the CDK2-cyclin A2-OTS964 complex (CDK2 is shown in cyan). OTS964 forms three hydrogen bonds to CDK2, as it is within hydrogen bonding distance of CDK2 D86. Y15 is tucked under the dimethylamino-propyl group of OTS964.

Article Snippet: CDK2 was expressed with an N-terminal His 10 -tag (Addgene plasmid #79726; http://n2t.net/addgene:79726 ; RRID:Addgene_79726, provided by John Chodera, Nicholas Levinson, and Markus Seeliger) , and cyclin A2 was expressed with an N-terminal His 6 -tag.

Techniques: Cryo-EM Sample Prep, Residue, Comparison, Isolation, Binding Assay