PMC

Kaplan-Meier analysis conducted with SurvExpress. Analysis of RUNX1 status alone, or in conjunction with genes identified in our screen (NBEAL2, ITPKB*, CCDC12*). *p = < 0.05

Vector-chromosome junctions were mapped to the mouse genome using the UCSC BLAT genome browser. The first identified integration occurred within the inositol triphosphate 3-Kinase B (Itpkb) gene. The second integration occurred within a CpG island between gene regions for Coiled-coil domain containing 12 (Ccdc12) and Neurobeachin-like2 (Nbeal2).

Identified genes were analyzed using cBioPortal on 166 fully annotated patients from the 2013 TCGA human AML data set. The percentage of total altered cases in the data set is listed, and only cases with genetic alterations are shown. Cases are sorted by the French, American, and British (FAB) subtype.

Copy number alterations were observed for overall survival and also disease free survival post initial diagnosis/treatment A. CNA ITPKB demonstrated reduced survival in patients with gains or amplifications. B. CNA CCDC12 demonstrated reduced survival in patients with shallow deletions. C. CNA NBEAL2 demonstrated reduced survival in patients with shallow deletions. D–F. Disease free survival was also explored by CNA demonstrating all gene alterations (ITPKB, CCDC12, NBEAL2) lead to higher incidence of disease recurrence.

A. K562 cells were transduced with pCL-SD171N- PGW3-KO and sorted for purity reaching 97% EGFP positive cells. B. K562 cells were induced towards megakaryoblasts with TPA treatment. Differentiation was measured by the expression of megakaryocytic differentiation-specific membrane antigen (CD41a) for control K562, TPA treated K562, and D171N expressing TPA treated K562 cells. C. Erythroid differentiation of K562 cells was induced with sodium butyrate. Hemoglobin expression was measured using benzidine staining. Control K562 cells (<1%), Sodium Butyrate treated K562 cells (12%), and D171N Sodium Butyrate treated K562 cells (<1%).

Replication incompetent gammaretroviral vector pCL-SD171N-PGW3-KO. Genomic DNA containing vector provirus is sheared, forming genomic DNA fragments containing the R6Kγ origin of replication, kanamycin resistance gene (KO), and viral LTR B). Shuttle vector rescue of genomic plasmids grown in E. coli, picked, and sequenced for vector-chromosome junctions using a 3′ LTR specific primer. Sequences are trimmed removing the LTR and aligned to the human genome to identify integration sites.

A. Schematic of RUNX1 (WT), and D171N mutant. B. Schematic of gammaretroviral vector pCL-SD171N-PGW-KO. Flag epitope tagged [F] AML1-D171N is expressed from an internal spleen focus forming viral promoter [S]. Enhanced green fluorescent protein [G] is expressed from a second internal phosphoglycerate kinase-1 promoter [P]. The vector contains a modified woodchuck hepatitis response element [W] for increased stability. The R6Kγ bacterial origin of replication and kanamycin resistance gene [KO] allow for efficient shuttle vector rescue in bacteria. C. Transduced HEK293T cells. D. Protein expression of mutant D171N is confirmed by Western blot. HEK293T cells do not express AML1b, and serve as a negative control. Positive E. coli FLAG protein lysates were used as a positive FLAG epitope control. Actin was used as an equal loading control.

Left panels are overall survival and right panels are disease free survival after initial treatment A. ITPKB. Overall survival time for patients that do not harbor alterations in ITPKB is significantly longer than patients with alterations in ITPKB (log rank test P-Value: 5.5 × 10⁻⁴) (Cases with alteration in query gene: total cases: 12, deceased: 5, median months survival: 5.2, cases without alteration in query gene: total cases: 154, deceased: 98, median months survival: 20.5). Patients with ITPKB alterations had significantly lower disease free survival (log rank test P-Value: 0.01) (Cases with alteration in query gene: total cases: 12, deceased 5 median months survival 5.2, Cases without alteration in query gene: total cases: 152, deceased: 72, median months survival: 17). B. NBEAL2. The overall survival time for patients that do not harbor alteration in NBEAL2 is significantly longer than patients with alterations in NBEAL2 (log rank test P-Value: 0.004) (Cases with alteration in query gene: total cases: 8, deceased: 11, median months survival 11.8, cases without alteration in query gene: total cases: 154, deceased: 98, median months survival: 18.5). Effect of NBEAL2 alteration on disease free survival after initial treatment. Disease free survival was not significantly different for patients with or without NBEAL2 alterations (log rank test P-Value: 0.53) (Cases with alteration in query gene: total cases: 2, deceased: 1, median months survival: 8.2, cases without alteration in query gene: total cases: 162, deceased: 76, median months survival: 17). C. CCDC12. The overall survival time for patients that do not harbor alteration in CCDC12 is significantly longer than patients with alterations in CCDC12 (log rank test P-Value: 0.03) although there were few patients harboring this mutation in the 2013 TCGA cohort (Cases with alteration in query gene: total cases: 2, deceased: 2 median months survival: 0.8, cases without alteration in query gene: total cases: 164, deceased: 107, median months survival: 18.5). The disease free survival was not significantly different for patients with or without CCDC12 alterations (log rank test P-Value: 0.18) (Cases with alteration in query gene: total cases: 2, deceased: 1, median months survival: 8.2, cases without alteration in query gene: total cases: 162, deceased: 76, median months survival: 17).