Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation

Abstract

NUP98 fusion oncoproteins (FO) are drivers in pediatric leukemias and many transform hematopoietic cells. Most NUP98 FOs harbor an intrinsically disordered region from NUP98 that is prone to liquid-liquid phase separation (LLPS) in vitro. A predominant class of NUP98 FOs, including NUP98-HOXA9 (NHA9), retains a DNA-binding homeodomain, whereas others harbor other types of DNA- or chromatin-binding domains. NUP98 FOs have long been known to form puncta, but long-standing questions are how nuclear puncta form and how they drive leukemogenesis. Here we studied NHA9 condensates and show that homotypic interactions and different types of heterotypic interactions are required to form nuclear puncta, which are associated with aberrant transcriptional activity and transformation of hematopoietic stem and progenitor cells. We also show that three additional leukemia-associated NUP98 FOs (NUP98-PRRX1, NUP98-KDM5A, and NUP98-LNP1) form nuclear puncta and transform hematopoietic cells. These findings indicate that LLPS is critical for leukemogenesis by NUP98 FOs.

Significance: We show that homotypic and heterotypic mechanisms of LLPS control NUP98-HOXA9 puncta formation, modulating transcriptional activity and transforming hematopoietic cells. Importantly, these mechanisms are generalizable to other NUP98 FOs that share similar domain structures. These findings address long-standing questions on how nuclear puncta form and their link to leukemogenesis. This article is highlighted in the In This Issue feature, p. 873.

Figure 1.

LLPS by the N-terminal FG motif–rich IDR governs the puncta-forming behavior of G-NHA9 in cells and in vitro. A, Scheme depicting the hypothesized role of both homotypic and heterotypic interactions in the formation of aberrant transcriptional condensates by NHA9 via LLPS. B, Representative confocal microscopy images of live HEK293T cells expressing EGFP empty vector (top, green) or G-NHA9 (bottom, green). DNA is stained with Hoechst dye (blue). C, The number of puncta per 10³ μm³ nuclear volume (puncta #,/10³ μm³) versus the total nuclear concentration of the G-NHA9 construct [G-NHA9]. The dotted lines represent linear fitting for visualization purposes, and the gray area indicates 95% confidence interval. Number of cells (n) = 935, including the cells with zero punctum. D, Confocal micrographs of fluorescence recovery (inside yellow box) of a single G-NHA9 punctum in HEK293T cells at different times after photobleaching (FRAP, left). FRAP recovery curve for a photobleached punctum (green, right) and an unbleached punctum (black, right). Individual puncta were manually tracked at different times, and recovery was plotted as the mean ± SD (n = 20). E, The average puncta volume (V_p, μm³) versus the total nuclear concentration of the G-NHA9 construct [G-NHA9]. The dotted lines represent linear fitting for visualization purposes, and the gray area indicates 95% confidence interval. Number of cells (n) = 378, excluding the cells with zero punctum. F, Confocal fluorescence micrographs of Alexa 488–labeled NHA9 condensates prepared in vitro with increasing protein concentration. The micrographs are presented as maximum intensity projections of 13 confocal planes offset by 0.5 μm per plane. Saturation concentration (C_sat) is less than 10 nmol/L. G, Confocal micrographs of fluorescence recovery of 20 μmol/L NHA9 (mixed with 200 nmol/L Alexa 488–labeled NHA9) within condensates at multiple time points acquired within 2 minutes of initiation of phase separation (left). FRAP recovery curves for NHA9 condensates within 2 (green), 4 (brown), and 8 (cyan) minutes of formation (right). Data are plotted as mean ± SD (n = 15). T_1/2 represents the half-life of the fluorescence recovery, and M_f represents the mobile fraction.

Figure 2.

DNA binding by the HOXA9 homeodomain of NHA9 influences puncta morphology and behavior. A, Representative confocal microscopy image of live HEK293T cells expressing G-NHA9–ΔDNA (green). DNA is stained with Hoechst dye (blue). An overlay of the G-NHA9–expressing cell from Fig. 1B is included for comparison (right). B and C, Plots of puncta # (/10³ μm³; B) and V_p (μm³; C) versus [G-NHA9 construct] for G-NHA9 (green) and G-NHA9–ΔDNA (red) from data represented in A. Data are plotted on a semi-log (y-axis: log₁₀) scale. D, Still images of multiple time points taken from a time-lapse confocal fluorescence microscopy video (Supplementary Video S2) of a fusion event in an HEK293T cell expressing G-NHA9–ΔDNA. E, Confocal micrographs of FRAP of a G-NHA9–ΔDNA punctum in HEK293T cells at different time points after photobleaching (left). Fluorescence recovery curves are shown for bleached (red, right) and unbleached puncta (black, right). The recovery curve for G-NHA9 is also provided for comparison (green). Individual puncta were manually tracked at different times, and the G-NHA9–ΔDNA fluorescence intensity versus recovery time was plotted as the mean ± SD (n = 20). The pairwise P value for the recovery curves between G-NHA9 and G-NHA9–ΔDNA is 2.2 × 10⁻¹⁶ using the t test. F–H, Plots of the concentration of the NHA9 construct in the nuclear light phase ([LP], μmol/L; F) and within puncta (termed the dense phase; [DP], μmol/L; G), and the K_p ( (H) versus [G-NHA9 construct] for G-NHA9 (green) and G-NHA9–ΔDNA (red). Data are plotted on a semi-log (y-axis: log₁₀) scale. I, 1D-density distribution of PCC per cell for G-NHA9 and G-NHA9–ΔDNA to analyze the linear relationship of the signal between mEGFP and Hoechst. Refer to Supplementary Table S2 for mean values ± standard error. The pairwise P values between G-NHA9 and G-NHA9–ΔDNA are shown in each plot (B, C, F–I; see Methods; n = 935 and 780 in B, F, and I including the cells with zero punctum, and n = 378 and 254 in C, G, and H excluding the cells with zero punctum, respectively, for G-NHA9 and G-NHA9–ΔDNA).

Figure 3.

Mutation of multiple F and FG residues in the FG-rich IDR of NHA9 alters puncta formation in cells. A, Schematic of NHA9 and mutant constructs used in this study. FG motif valence is shown on the left. B, Representative image of live HEK293T cells expressing G-NHA9–8FA (top, green) and G-NHA9–21FGAA (bottom, green). DNA is stained with Hoechst dye (blue). C–F, Plots of puncta # (/10³ μm³; C), V_p (μm³; D), K_p ( (E), and ΔG_Tr (kcal/mol; F) versus [G-NHA9 construct] for G-NHA9 (green), G-NHA9–8FA (purple), and G-NHA9–21FGAA (blue) from data represented in B. Data are plotted on a semi-log (y-axis: log₁₀) scale in C–E. Refer to Supplementary Table S2 for mean values ± standard error. The pairwise P value between G-NHA9 versus G-NHA9–8FA and G-NHA9 versus G-NHA9–21FGAA is shown in each plot (C–F; n = 935, 683, and 865 in C including the cells with zero punctum and n = 378, 273, and 159 in D–F excluding the cells with zero punctum, respectively, for G-NHA9, G-NHA9–8FA, and G-NHA9–21FGAA).

Figure 4.

Mutation of F and FG residues in the FG-rich IDR of NHA9 alters LLPS behavior in vitro. A–C, Confocal fluorescence micrographs of Alexa 488–labeled NHA9–8FA (A), NHA9–21FGAA (B), and NHA9_Midi (C) condensates in vitro with increasing protein concentration. Micrographs are presented as maximum intensity projections of 13 Z-stack images acquired over 6 μm with 0.5 μm resolution. D, Confocal micrograph of Alexa 488–labeled NHA9_Midi–21FGAA at a concentration of 20 μmol/L. Saturation concentration (C_sat) is less than 10 nmol/L (A), between 160 nmol/L and 315 nmol/L (B), and between 40 nmol/L and 80 nmol/L (C).

Figure 5.

The NHA9 constructs form puncta in lin⁻ HSPCs. A–G, Representative images of live lin⁻ HSPCs expressing EGFP empty vector as a control (A), G-NHA9 (B), G-NHA9–ΔDNA (C), G-NHA9–8FA (D), G-NHA9–21FGAA (E), G-NHA9_Midi (F), and G-NHA9_Midi–21FGAA (G).

Figure 6.

Expression of high FG motif valence NHA9 constructs in lin⁻ HSPCs leads to hematopoietic cell transformation and aberrant expression of Hox family and other genes. A, Average number of colonies per 2,000 cells for lin⁻ HSPCs expressing negative control empty vector and mEGFP-tagged NHA9 and mutant constructs. The values of colony numbers shown are mean ± SD from triplicate technical replicates of a representative experiment. B–D, RNA-seq was performed for lin⁻ HSPCs expressing empty vector, G-NHA9, or mutants after 1 week of growth in methylcellulose containing myeloid and erythroid growth factors (n = 5 for each condition). B, Heat map for differentially expressed genes of interest. C, PCA of the 500 most variable genes. D, Gene set enrichment analysis for cells expressing G-NHA9, G-NHA9–8FA, or G-NHA9_Midi—each versus empty vector. Pathways of interest are shown, with a complete list of significantly upregulated or downregulated gene sets in Supplementary Table S3. The most significantly dysregulated genes from each pathway are marked in B.

Figure 7.

Additional leukemia-associated NUP98 FOs form nuclear puncta and transform lin⁻ HSPCs. A, Schematic of NHA9 and three additional NUP98 FOs (NUP98–PRRX1, NUP98–KDM5A, and NUP98–LNP1). Numbers indicate the amino acid residue. Numbers above the schematic reflect NUP98 residues, whereas numbers beneath reflect the fusion partner's residues. B, Representative images of live lin⁻ HSPCs expressing G-NUP98–PRRX1, G-NUP98–KDM5A, and G-NUP98–LNP1. EGFP empty vector and G-NHA9 are included for comparison. C, Average number of colonies per 2,000 cells for lin⁻ HSPCs expressing negative control empty vector and NUP98 FOs. Data shown are mean ± SD from triplicate technical replicates of a representative experiment. D, Representative images of a fixed, nontransduced human CD34⁺ (hCD34⁺) cell (top) and a NUP98–KDM5A PDX cell (bottom) stained with an antibody against NUP98. NUP98 is magenta, and DNA is blue. The heat map is a normalized representation of NUP98 fluorescence intensity across the PDX cell. E, Conceptual scheme illustrating how LLPS by NHA9 mediates the formation of aberrant transcriptional condensates in hematopoietic cells. NHA9 (top, left cell image) undergoes LLPS to form many small, chromatin-associated puncta that drive aberrant expression of Hox and other genes and transform hematopoietic cells. LLPS is driven by both homotypic and heterotypic interactions. Mutation of FG motifs (NHA9–21FGAA, bottom left) weakens both homotypic and heterotypic protein–protein interactions, yielding less numerous, larger, and less dense puncta that do not activate Hox gene expression or transform HSPCs. Mutation of residues in the HOXA9 homeodomain (HD; NHA9–ΔDNA, bottom right) weakens heterotypic interactions with DNA, yielding less numerous, larger, and more dense puncta that also do not induce the leukemogenic phenotype in HSPCs.