Haploinsufficiency of the c-myc transcriptional repressor FIR, as a dominant negative-alternative splicing model, promoted p53-dependent T-cell acute lymphoblastic leukemia progression by activating Notch1

FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIR.exon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIR.exon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIR.exon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR+/-) C57BL6 mice were generated. FIR complete knockout (FIR-/-) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR(+/-)mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIR.exon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR(+/-)TP53(-/-) generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR(+/-)TP53(-/-) compared with that in FIR+/+ TP53(-/-) mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIR.exon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIR.exon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion, the alternative splicing of FIR, which generates FIR.exon2, may contribute to both colorectal carcinogenesis and leukemogenesis.