Aberrant CBL transcripts in AML. (A) RT-PCR screening of AML cell lines and the RS(4;11) acute lymphoblastic leukemia (ALL) cell line was carried out as described in “Patients, materials, and methods.” Gel electrophoresis of resulting amplification products using a primer set covering human c-CBL exons 2 through 9 revealed major and minor transcript variants (gray arrows) in the MOLM-13 cell line. MW indicates 1 kb + DNA size ladder; NTC, no template control reaction. (B) RT-PCR screening for c-CBL transcript was performed using AML patient samples (lanes 1-12). A representative agarose gel is shown. (Lane 6) UPN03149 expresses a smaller transcript variant (arrow) and the wild-type mRNA for c-CBL. MW indicates 1 kb + DNA size marker; NTC, no template control reaction. (C) Partial sequence alignment of gDNA PCR amplification products in MOLM-13 compared with the normal c-CBL gene sequence. MOLM-13 is missing 14 bp at the exon 8-intron 8 boundary, including the normal “G” donor site. (D) Partial sequence alignment of gDNA PCR amplification products in UPN03149 relative to wild-type c-CBL DNA. UPN03419 is missing 4 bp that is replaced with a 12-bp sequence. (E) The predicted amino acid sequences derived from the region of interest of mutant CBL transcripts are shown in comparison with the corresponding CBL WT sequence. FLT3 TK allelic status, that is, absence (WT/WT) or presence of internal tandem duplication (FLT3-ITD) or activating domain mutations (FLT3-TKD), was determined for each case of AML and is shown on the right. Asterisk indicates location of missense mutation or nucleotide insertion that gives rise to an amino acid change.