BMF deletions and functional consequences of reduced BMF expression in E/R-positive ALL. (A) Log2-ratio copy numbers of genomic deletions encompassing BMF (indicated by an arrow) are shown for 2 patients with a deletion at 15q15.1. (B) Segmental deletions at 15q15.1 in 3 cases (3, 10, and 15). (Top) A partial ideogram of the 15q region and the position of the FISH probe used for confirmation of the deletions. The scale indicates genomic positions of the deletions in megabases according to the University of California Santa Cruz Genome Browser (NCBI Build, Version 36.1). Each black horizontal bar represents a deletion in a single patient as inferred by SNP array. Vertical lines indicate the overlapping region encompassing the only commonly deleted gene (BMF) (bottom). (C) Verification of the BMF deletion by FISH in patient 10 in 57% of nuclei (green represents BMF; and red, chromosome 15 centromer). (D) Genomic breakpoint of the BMF deletion from case 10 was cloned (details provided in supplemental data; accession code #GU984796) and sequence information used for nested PCR. The resulting PCR products from initial diagnosis (middle lane) and relapse (right lane) are shown. Left lane indicates size marker. (E) Quantification of BMF expression in E/R-positive (E/R+, n = 15) and E/R-negative (E/R−, n = 15) BCP ALL cases without relapse. Data represent relative expression values of BMF compared with GUS. Boxes include values between the 25th and 75th percentile, and whiskers indicate the minimal and maximal values. The black line indicates the median BMF expression of all samples. For statistical evaluation, Welch unpaired t test was used. (F) Western blot analysis of REH cells on BMF repression by RNAi. Protein lysates were prepared from green fluorescent protein-sorted cells expressing the shRNA vector targeting human BMF (KD) or a nontargeting control shRNA (C). BMF isoforms (BMF1, BMF2, and BMF3, top-down) are marked with arrows. GAPDH levels were used as a loading control. (G) Glucocorticoid-induced apoptosis on BMF repression in REH cells. BMF silenced cells were treated with prednisolone for the indicated times, and apoptosis was assessed by annexin V/propidium iodide staining in green fluorescent protein-positive cells carrying the lentiviral shRNA construct. Apoptosis was calculated by subtraction of annexin single-positive cells in untreated from prednisolone-treated cells. Percentages of 4 independent experiments from 3 separate infections are shown. Bars represent mean ± SD. *P < .05 (paired Student t test). **P < .01 (paired Student t test).
