Fig 2: Localization of iDAb LMO2 mutations and the corresponding affected amino acids on LMO2 structure.The crystal structure of LMO2-iDAb PDB 4KFZ was used to simulate the mutation effect for the dematuration of the anti–LMO2 iDAb. (A) Amino acid sequence alignment of the original iDAb LMO2 [wild type (WT)] and the various iDAb mutants (iDAb LMO2dm(1–6)). The framework is in black, and the CDRs regions are colored in green (CDR1), orange (CDR2), and blue (CDR3). Amino acids mutated in iDAb LMO2dm(1–6) are underlined, and the black dots indicate the amino acids within the CDRs making contact with LMO2 (23). (B) Localization of iDAb LMO2dm3 mutations (S28G, H31G, S55A, E102A, and T107A) are shown in yellow on the parental iDAb LMO2 structure, with the affected LMO2 residues shown in red on LMO2 structure (displayed in gray). (C) Localization of iDAb LMO2dm6 mutations (S28G, H31G, S55A, E102A, S103A, and T107A) are shown in yellow on the parental iDAb LMO2 structure, with the affected LMO2 residues shown in red on LMO2 structure (displayed in gray). LMO2-iDAb LMO2 structure used is PDB 4KFZ. Each panel has a table listing the interacting amino acids of LMO2 and iDAb.

Fig 3: Structure-activity relationship of Abd LMO2-binding compounds.Hits identified from the HTS library screen were classified and a family of related compounds identified as the basis of SAR analysis. The chemical structures of the hit matter from the BRET screen were examined and a family of compounds was identified. (A) Chemical structures of the 8 re-synthesized hits (Abd-L5 to Abd-L12) with their respective molecular weight (MW). (B) Dose response inhibition of LMO2-iDAb LMO2dm3 interaction by compounds Abd-L5 to Abd-L12 (concentration range: 1, 10, and 20 µM). (C) SAR study of Abd-L9 compound as template. The compound was divided into four substituents (named A to D) that were substituted by various other chemical groups to give new compounds. The colored boxes (red, pink, blue, and green) represent the different moieties substituted in the SAR study in positions A, B, C, and D, respectively. Structures are shown of representative compounds from Abd-L15 to Abd-L25. The new compounds were tested by BRET assays with LMO2-iDAb LMO2dm3 interaction. The different SAR-derived compounds are shown with their MW and the percentage of BRET inhibition of the interaction LMO2-iDAb LMO2dm3 at 20 µM including Abd-L24 and Abd-L25 modified on their positions B and C, respectively, which do not affect the BRET interaction LMO2-iDAb LMO2dm3. (D) Dose response effect of the Abd-L15 to Abd-L25 compounds on LMO2-iDAb LMO2dm3 interaction (Abd concentration used: 5, 10, and 20 µM). Experiments in (B) and (D) were performed twice. Error bars presented in (B) and (D) correspond to mean values ± SD of biological repeats.

Fig 4: RNAseq analyses of WT and Lmo2-/- Flk-1+ and HE1 cells. (A) Screenshots from the UCSC browser illustrating the distribution of reads over the Eomes; Ets2, and Erg loci. Uniform y-axis scales were used for each genomic region and positive and negative values correspond to the direction of the transcripts. The screenshots are flanked by gene expression levels of Eomes and Erg, measured by qPCR. Data represent the mean of three independent samples measured in duplicate ± SEM relative to Hprt. (B) Pairwise comparison of the significantly differentially expressed genes. Shown are the total number of up or down regulated genes for each comparison and their overlap. Quantitation and analyses of RNAseq data are based on biological triplicate samples for Flk-1+ cells and duplicates for HE1 cells. (C) Heat map showing hierarchical clustering of differentially expressed genes at the Flk-1+ stage. (D) Gene ontology enrichment analyses for biological process performed on the two clusters identified in C. (E) Heat map showing hierarchical clustering of all differentially expressed genes. Scale bar represents colour index for the log2 FPKM values. Self-organizing tree analysis identified six clusters, which are numbered 1 to 6. (F) Gene ontology enrichment analyses for biological process was performed on clusters 2, 5, 6 identified in E. Terms were ordered according to their Modified Fisher Extract P-value and only terms with P < 0.05 were considered significant.

Fig 5: Dynamic Alterations in Cofactor Binding upon RUNX1/ETO Knockdown(A) Western blot detecting RUNX1/ETO, RUNX1, C/EBPa, LMO2, PU.1, p300, HDAC2, LYL1, LDB1, and HEB protein in Kasumi-1 cells treated for 48 hr with mismatch control siRNA (siMM) and with RUNX1/ETO siRNA (siRE). GAPDH served as the loading control.(B) UCSC genome browser screenshot of the NFE2 locus showing changes in the RNA expression and binding pattern of p300, RUNX1/ETO (R/E), RUNX1, and DHS upon RUNX/ETO knockdown in Kasumi-1 cells.(C) Increase of p300 binding and decrease of HDAC2 binding upon RUNX1/ETO knockdown.(D) Global changes of p300 binding peaks shared between RUNX1/ETO and RUNX, peaks exclusively bound by RUNX1, and PU.1 peaks not associated with RUNX1/ETO or RUNX1 binding. qPCR data represent the mean ± SD of three to five independent experiments. For other control analyses, see Figure S3B.