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Acute Myeloid Leukemia |
Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands Key words: acute myeloid leukemia, mutations, CBL
Correspondence: Peter J.M. Valk, Department of Hematology Ee1391a, Erasmus University Medical Center Rotterdam, Dr. Molewaterplein 50 3015 GE Rotterdam, The Netherlands. Phone: international +31.10.704 39 75. Fax: international +31.10.7044745. E-mail:p.valk{at}erasmusmc.nl
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
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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).
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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.
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  F. H. Grand, C. E. Hidalgo-Curtis, T. Ernst, K. Zoi, C. Zoi, C. McGuire, S. Kreil, A. Jones, J. Score, G. Metzgeroth, et al. Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms Blood, June 11, 2009; 113(24): 6182 - 6192. [Abstract] [Full Text] [PDF]
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 C. Reindl, H. Quentmeier, K. Petropoulos, P. A. Greif, T. Benthaus, B. Argiropoulos, G. Mellert, S. Vempati, J. Duyster, C. Buske, et al. CBL Exon 8/9 Mutants Activate the FLT3 Pathway and Cluster in Core Binding Factor/11q Deletion Acute Myeloid Leukemia/Myelodysplastic Syndrome Subtypes Clin. Cancer Res., April 1, 2009; 15(7): 2238 - 2247. [Abstract] [Full Text] [PDF]
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