Exon 8 splice site mutations in the gene encoding the E3-ligase CBL are associated with core binding factor acute myeloid leukemias

Acute myeloid leukemia (AML) is a heterogeneous disease with diverse genetic abnormalities and variable response to treatment. In the last decade the diverse genetic abnormalities have refined risk-stratification of AML.1 Recently, mutations in the Casitas B-cell lymphoma gene encoding the E3-ligase CBL2 were identified in de novo AML.3,4 In one study, a single case with an inactivating point mutation in exon9 of the CBL gene was identified in a cohort of 150 de novo AML cases.3 In a second study, exon8 missense mutations were demonstrated in 3 out of 12 randomly selected AML cases.4 In an additional AML case, a DNA insertion/deletion mutation in intron7 of the CBL gene resulted in the expression of a CBL splice variant, i.e., a CBL mRNA lacking exon8.4

All published CBL mutations are located within the conserved linker region (LR) and ring finger (RF) of the CBL protein.2 In fact, the mutant CBL splice variant without exon8 results in an inframe deletion, which encodes a CBL protein lacking part of the LR, including two essential tyrosine residues, and almost the entire RF, which is critical for E3 activity.2 This suggests that mutant CBL may act as a dominant negative protein by inhibiting proper downregulation of critical activated tyrosine kinases, such as KIT and FLT3 in AML.5

It is still not clear how frequently mutations in the CBL gene occur in newly diagnosed AML. In a diverse population of primary AML (n=319, Table 1) we assessed the frequency of CBL mutations, i.e., point mutations in exon84 and exon93, and mutations affecting proper splicing of CBL exon8.4

Patients had a diagnosis of primary AML, confirmed by cytological examination of blood and bone marrow, and were treated according to the HOVON protocols (http://www.hovon.nl). After obtaining patients’ informed consent, bone marrow aspirates or peripheral blood samples were taken at diagnosis (n=319). CBL exon8 mRNA splice variants, as well as point mutations in exon8, were determined by cDNA amplification using the primer set CBLe6F 5'-AAACCTCTCTTCCAAGCACTG-3' and CBLe9R 5'-TCCCTCTAGGATCAAACGGA-3' or CBL-exon7-FOR 5’-GTGAACCAACTCCCCAAGAC-3’ and CBL-exon9-REV 5’-GGACAGCCCTGACCTTCTG-3’. Mutations in genomic DNA were determined by amplification using CBL-intron7-FOR 5’-GGACCCAGACTA-GATGCTTTC-3’ and CBL-exon8-REV 5’-GTGCACAT-GAGGTGTCCACAG-3’ (mutations 5’ of exon8) or CBL-exon7-FOR and CBL-exon9-REV (mutations 3’ of exon8). (0.25 mM dNTP, 15 pmol primers, 2 mM MgCl2, Taq polymerase and 1xbuffer (Invitrogen Life Technologies, Breda, The Netherlands); 1 cycle at 94°C for 5 mins., 35 cycles at 94°C for 1 min, 60°C for 1 min., 72°C for 1 min., and 1 cycle at 72°C for 7 mins.). Samples showing aberrant patterns were sequenced by using forward and reverse primers on the ABI PRISM3100 genetic analyzer (Applied Biosytems, Foster City, CA, USA).

Table 1.Clinical and molecular characteristics of the 319 patients* with de novo acute myeloid leukemia.

All AML cases were screened by RT-PCR for CBL exon8 splice variants, whereas a randomly selected subset of 183 out of the 319 AML cases was examined by sequence analysis for the presence of exon84 or exon93 point mutations. We did not find any point mutation in exon8 or exon9 of CBL in the subset of 183 AML cases. However, out of the 319 AML cases, we did identify two AML cases expressing a CBL mRNA splice variant (#2274 and #6717, Figure 1A), which lacked exon8. This aberrant CBL mRNA is similar to the splice variant previously identified in the leukemic cell line MOLM13 (Figure 1A).4 CBL transcripts lacking exon8 were not present in 5 normal bone marrow samples and 3 fluorescence activated cell sorted CD34-positive progenitor cell samples (data not shown).

Interestingly, the two primary AML cases showing aberrant splicing carried an inversion of chromosome 16 (inv(16)) suggesting that CBL mutations might be associated with core-binding factor (CBF) leukemias, i.e., AML and inv(16) or t(8;21). In a selected screen of 39 inv(16) AML and 40 t(8;21) AML we did not detect point mutations in CBL exon8 or exon9. However, the CBL exon8 splice variant was present in 2 additional cases with a t(8;21) and one additional case with an inv(16) (Figure 1A). The CBL exon8 splice variant was absent in 4 independent remission samples of AML patient #2549 (data not shown).

Figure 1.CBL mRNA splice variant mutations in core-binding factor acute myeloid leukemia. (A) RT-PCR with primers CBL-exon7-FOR and CBL-exon9-REV of primary acute myeloid leukemia cases showing aberrant splice variants (#2254 (inv(16)), #2549 (t(8;21)), #2274 (inv(16)), #6717 (inv(16)), #7056 (t(8;21)), wild-type CBL (#2173 and #2214) and cell lines MOLM13 (FLT3 internal tandem duplication (ITD)), Kasumi-1 (t(8;21)) and ME-1 (inv(16)). The presence of these CBL exon8 splice mutants in the primary AML were confirmed using various primer sets (data not shown). AML #7056 consistently expresses two additional CBL mRNA splice variants. Of note, both these CBL splice variants are inframe ((exon7) – ACC CAG ATG GGC TCC (exon8) and (exon7) ACC CAG GAT GTA AAG (exon8)) (B) Schematic representation of exon 7-to-9 of the CBL gene to indicate the insertion (I)/deletion (D) mutations in the CBL gene of the 5 CBF acute myeloid leukemia cases (#2254, #2549, #2274, #6717, #7056).

By nucleotide sequencing of the flanking sequences of CBL exon8 in all AML cases expressing the CBL mRNA splice variant, we identified various insertion/deletion mutations in the CBL gene (Figure 1B). All insertion/deletion mutations affect the splice acceptor or donor sites of exon8 of CBL. In fact, the G to C point mutation in case #7056 is located within the splice acceptor site of CBL exon8, resulting in two additional aberrant inframe CBL transcripts (Figure 1A).

These results indicate that there is a preferential association between CBL exon8 splice variant mutations and CBF leukemias. This raises the possibility of a functional association between impaired CBL function and the CBF-related fusion proteins CBFB-MYH11 and AML1-ETO. Activating point mutations in receptor tyrosine kinase KIT are strongly associated with CBF leukemias.6–8 In fact, we recently screened 500 cases of AML for exon8/exon17 KIT mutations and mutations were present in 25 AML cases (5%). Of the KIT mutant cases, 88% carried a proven CBF mutation, such as t(8;21), inv(16) or t(3;21) (data not shown). CBL proteins mediate ubiquitination and degradation of KIT upon stimulation with stem cell factor.9 Thus, the expressed mutant CBL protein, which is still able to bind KIT by its N-terminal tyrosine kinase binding domain, but impaired in downstream ubiquitination,2 may act as a dominant negative protein. Expression of this dominant negative protein could result in impaired routing of KIT and sustained activation, similar to KIT activating mutations in CBF leukemias.

No other AML-specific mutations, such as those affecting FLT3, NRAS, KRAS, CEBPA and NPM1, were present in the CBL mutant AML cases. Interestingly, however, CBL mutant t(8;21) AML case #2549 also carried a KIT D816 mutation. In this AML, impaired function of the CBL protein would potentially result in prolonged constitutive activation of KIT.

Our results demonstrate that CBL mutations are rare in AML. However, the strong association of these mutations with CBF leukemias suggests that there may be a co-operative activity of mutant CBL with the CBF-related fusion proteins CBFB-MYH11 and AML1-ETO in CBF leukemogenesis, most likely by impaired ubiquitination of KIT.

Footnotes

• Funding: the research described was supported by grants from the Erasmus University Medical Center (Revolving Fund) and the Dutch Cancer Society "Koningin Wilhelmina Fonds".