Figure 4.

AML samples with the NPMc mutation are associated with increased abundance of several nuclear importins, and NPMc interacts directly with several family members. (A) Volcano plot showing protein abundance in NPM1-mutated vs wt AML samples. P values are calculated using the t test and corrected for multiple-hypothesis testing with Benjamini-Hochberg method. Dashed red line shows P = .05. (B-C) Normalized abundance of the nuclear importins organized by NPM1 mutation status in TMT data (B) and bulk RNA sequencing data (C). *P < .05 by t test between groups. (D) TurboID vectors were created with no fused complementary DNA (cDNA) (“TurboID only”) or fused at either the N or C terminus of wt NPM1 or mutant NPMc (T-NPM1 and T-NPMc indicate N-terminal fusions, whereas NPM1-T and NPMc-T indicate C-terminal fusions). Each vector was stably transduced into primary mouse hematopoietic stem/progenitor cells and, after 4 days, cultured in the presence of biotin for 4 hours. Biotin-labeled proteins were then enriched with streptavidin beads and stringently washed, and tryptic peptides were released from the beads and identified by mass spectrometry. Z-scores are calculated based on spectral counts across 10 TurboID-only biological replicates and 3 biological replicates for each of the other indicated vectors. The 30 interacting proteins with the greatest fold change for the NPM1-TurboID constructs are shown; all display significant differences (t test, multiple-hypothesis correction by Benjamini-Hochberg method with P < .05) from samples expressing TurboID only and NPMc-TurboID. Similarly, 30 proteins with the greatest fold change were selected for the NPMc-TurboID vector, and all displayed significant differences from both wt NPM1 and TurboID only. KPNA3 and KPNA4, 2 members of the nuclear importin family, are highlighted in green. (E) Spectral counts detected in the TurboID experiments for each of the displayed nuclear importins are normalized for display between 0 and 1. *P < .05 by t test between groups. N- and C-terminal TurboID constructs are analyzed together for the NPM1 and NPMc vectors. (F) Western blotting of protein abundance for the indicated proteins in cell lysates created from the stably transduced mouse bone marrow cells prior to streptavidin pulldown. Normalized total protein for each lane is shown as a loading control as determined on the Jess western blotting system. In this short-term expression system, the abundance of the nuclear importins is not increased, suggesting that the detection of interactions with NPMc is not due to an increase in total importin protein abundance. One representative example from 3 biologic replicates is shown. mut, mutant; NS, not significant.