Novel c-CBL and CBL-b ubiquitin ligase mutations in human acute myeloid leukemia

Abstract

The CBL ubiquitin ligase targets a variety of activated tyrosine kinases (TKs) for degradation. Many TKs are mutationally or autocrine activated and/or often overexpressed at the mRNA and protein levels in acute leukemias. We hypothesized that CBL is mutated in patients with acute myeloid leukemia (AML). Four of 12 patients and the MOLM-13 cell line harbored c-CBL mutations, either RNA splicing mutations, missense mutations, or a nucleotide insertion. Additionally, 1 of the 12 patients harbored a missense mutation in the related CBL-b gene. Each c-CBL mutation involves the structurally important alpha-helix within the linker region, while the mutation in CBL-b was located in the Ub-E2 protein-binding RING finger. Short-interfering RNA knockdown of mutant c-CBL present in MOLM-13 cells was growth inhibitory. In summary, novel mutations in c-CBL and CBL-b have been identified in human AML and may represent potential targets for novel therapeutics.

Figure 1

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.

Figure 2

siRNA-mediated reduction in MOLM-13-expressed mutant c-CBL transcript and protein is associated with reduced cell proliferation. (A) MOLM-13 cells were transfected (Nucleofector; Amaxa, Gaithersburg, MD) with siRNAs targeting the MOLM-13 c-CBL mutant transcript fusing exon 7 and exon 9 (siMolD8CBL) or a scrambled sequence (scrMolD8CBL) or transfected with “control siRNA” that has been validated to not affect mRNA levels in mammalian cells (Ambion, Austin, TX) and incubated at 37°C for 24 hours. Relative real-time RT-PCR was carried out using total RNA and mutant-specific primers and primers common to mutant and wild-type c-CBL transcripts (sequences available upon written request). 18S rRNA was used to normalize for starting template amounts. Expression of c-CBL WT or mutant transcript in each transfected sample was measured. Data are depicted as percent change in the mutant transcript level relative to the level of mutant in the control siRNA-transfected cells. No difference in c-CBL WT mRNA levels was observed (not shown). siRNA sequences are available upon request. (B) Immunoblot analysis of c-CBL protein in MOLM-13 cells transfected with no siRNA or either Scr or MolD8CBL siRNAs. Detection of actin was used to control for well loading. (C) MTS cell proliferation assay was carried out at 48 hours after transfection of MOLM-13 cells without (no siRNA) or with 1 nmol MolD8CBL or Scr siRNA. Viability is indicated as the average absorbance at 492 nm of triplicate wells corrected for background and relative to the 0-hour time point.