Fig 2: CRYBMIM is an improved peptidomimetic chimera that specifically binds the KIX domain of CBP/P300 in vitro and in cells.(A) Heatmap of the top 10 and bottom 10 gene dependencies for survival and proliferation of 652 cancer cell lines in the DepMap Cancer Dependency Map Project, ranked by the greatest dependency for 37 leukemia lines, 20 of which are AML cell lines, as indicated by the red color gradient; *p=1.1e-15, t-test of leukemia versus other tumor types. (B) Retro-inverso amino acid sequences of MYBMIM, CREBMIM and CRYBMIM, with amino acids derived from MYB, CREB, and TAT marked in blue, green, and black, respectively. (C) Molecular model of the CRYBMIM:KIX complex. Residues making contact with KIX are labeled, with portions derived from MYB and CREB marked in blue and green, respectively. (D) Binding of FITC-conjugated CREBMIM (blue), CRYBMIM (red), and MYBMIM (black), as measured using microscale thermophoresis; Kd = 2.9 ± 0.7, 5.7 ± 0.2, and 17.3 ± 1.6, respectively. Error bars represent standard deviations of three biological replicates; p<1e-15, ANOVA, for CRYBMIM versus MYBMIM. (E) Western blot showing binding of nuclear CBP/P300 isolated from AML cells to biotinylated CRYBMIM or CREBMIM, specifically competed by the excess free peptides as indicated. (F) Quantification of CBP/P300 binding to CRYBMIM and CREBMIM by fluorescence densitometry, with black, gray, and red denoting PBS control, TAT control, and peptide competition, respectively.

Fig 3: CRYBMIM remodels MYB and CBP/P300 chromatin complexes in AML cells.(A–B) Volcano plots of relative MYB chromatin occupancy in MV411 cells changes after 1 hr (A) and 4 hr (B) of 20 µM CRYBMIM treatment compared to PBS control, as analyzed by ChIP-seq. Sequence motifs found in CRYBMIM-induced MYB-depleted (left) and MYB-enriched loci (right) are shown. p-Values denote statistical significance of three technical replicates. (C–D) Volcano plots of CBP/P300 chromatin occupancy changes after 1 hr (C) and 4 hr (D) of 20 µM CRYBMIM treatment as compared to control, as analyzed by ChIP-seq. Sequence motifs found in CRYBMIM-induced CBP/P300-depleted (left) and CBP/P300-enriched loci (right) are shown. p-Values denote statistical significance of three technical replicates.Figure 6—source data 1.Differentially occupied chromatin loci measured by MYB and CBP/P300 ChIP-seq in CRYBMIM vs PBS treated MV411 cells (1 hr and 4 hr).

Fig 4: MYB assembles aberrant nuclear transcription factor complexes in AML cells.(A) Volcano plot of nuclear MYB-associated proteins compared to IgG control, as analyzed by affinity purification-mass spectrometry of MV411 cells. Red symbols denote specifically MYB-associated proteins, as defined by association with CBP (MYB/IgG log2 >1). p-Values denote statistical significance of three biological replicates. (B) Enrichment of MYB-associated proteins (red) as a function of their corresponding CRISPR DepMap dependency scores in MV411 cells. Red symbols denote functionally required proteins, as defined by the genetic dependency of CBP (score <-0.18). (C) Network of BioGRID protein interactions for MYB-associated nuclear AML proteins as a function of their respective hematopoietic expression aberrancy scores, based on their relative gene expression in AML cells as compared to normal bone human bone marrow progenitor cells (white to red color gradient indicates increasingly aberrant gene expression). (D) Comparison of the genetic dependency scores in leukemia cell lines as compared to all other non-hematopoietic cancer cell lines for MYB-associated nuclear AML proteins, with red symbols denoting proteins that are required in leukemia as compared to non-hematopoietic cancers.Figure 7—source data 1.Lists of proteins identified by IP-MS of IgG control and MYB or CBP/P300 complex purifications from MV411 cell nuclei.

Fig 5: Potent and broad-spectrum activity of CRYBMIM against diverse AML cell lines but relatively sparing of normal hematopoietic progenitor cells.(A) Representative live cell confocal microscopy images of MV411 cells treated with 100 nM FITC-conjugated peptides as indicated for 1 hr, counterstained with Mitotracker (red) and Hoechst 33342 (blue). Scale bar indicates 20 µm, with z-stack of 1.5 µm. (B) Western blots showing immunoprecipitated nuclear CBP/P300 co-purified with MYB and CREB from MV411 cells treated with 10 µM peptides as indicated for 1 hr. (C–D) Cell viability of MV411 cells as a function of increasing concentrations of 48 hr peptide treatment, comparing (C) CRYBMIM to MYBMIM, CREBMIM and MLLMIM (IC50 = 6.88 ± 3.39, 13.1 ± 3.3 29.15 ± 3.79, and 24.22 ± 2.00, p<1e-15, ANOVA); (D) TG3 and CG3 (IC50 = 16.65 ± 1.00 and 48.91 ± 2.55, p<1e-15, ANOVA). Error bars represent standard deviation of three biological replicates. (E) Colony forming ability of CD34+ cells isolated from human umbilical cord blood (CD34+ CB, gray) and MV411 AML (red) cells following CRYBMIM or MYBMIM treatment. Data represent three biological replicates; *p=7.4e-5, t-test of normal CD34+ CB versus MV411 AML cells. (F) Preservation of clonogenic capacity of CD34+ CB cells in differentiating into erythroid blast forming units (BFU-E, light gray) and granulocyte macrophage colony forming units (CFU-GM, gray) as a function of CRYBMIM treatment. (G) Cell viability of AML cell lines treated with control PBS (black), 20 µM CREBMIM (blue), MYBMIM (gray), or CRYBMIM (red) as indicated for 6 days with media replacement every 48 hr in three biological replicates (p=8.6e-3, t-test for CRYBMIM versus control).