Oncogenic activation of the Notch1 gene by deletion of its promoter in Ikaros-deficient T-ALL

Abstract

The Notch pathway is frequently activated in T-cell acute lymphoblastic leukemias (T-ALLs). Of the Notch receptors, Notch1 is a recurrent target of gain-of-function mutations and Notch3 is expressed in all T-ALLs, but it is currently unclear how these receptors contribute to T-cell transformation in vivo. We investigated the role of Notch1 and Notch3 in T-ALL progression by a genetic approach, in mice bearing a knockdown mutation in the Ikaros gene that spontaneously develop Notch-dependent T-ALL. While deletion of Notch3 has little effect, T cell-specific deletion of floxed Notch1 promoter/exon 1 sequences significantly accelerates leukemogenesis. Notch1-deleted tumors lack surface Notch1 but express γ-secretase-cleaved intracellular Notch1 proteins. In addition, these tumors accumulate high levels of truncated Notch1 transcripts that are caused by aberrant transcription from cryptic initiation sites in the 3' part of the gene. Deletion of the floxed sequences directly reprograms the Notch1 locus to begin transcription from these 3' promoters and is accompanied by an epigenetic reorganization of the Notch1 locus that is consistent with transcriptional activation. Further, spontaneous deletion of 5' Notch1 sequences occurs in approximately 75% of Ikaros-deficient T-ALLs. These results reveal a novel mechanism for the oncogenic activation of the Notch1 gene after deletion of its main promoter.

Figure 1

RBP-J deletion delays T-ALL development. (A) Survival curves of Ik^L/LRBP-J^f/fCD4-Cre⁺ (IRC⁺) mice and Ik^L/LRBP-J^f/fCD4-Cre⁻ (IRC⁻) mice. The statistical significance was calculated by log-rank test. Note that the 2 IRC⁺ mice killed at 52 weeks did not show signs of disease. (B) Thymocyte CD4/CD8 profiles as determined by flow cytometry (top) and photos of the thoracic cavity (bottom) from 18-week-old IRC⁺ and IRC⁻ mice and a 7-week-old WT mouse. (C) Western blot of RBP-J and ICN1 (Val1744 antibody) expression in a panel of thymic tumors from IRC⁺ mice. Control samples are an IRC⁻ tumor and sorted CD4⁺CD8⁺ (DP) cells from 4-week-old IRC⁺ and WT mice. α-Tubulin was used as a loading control. The variable sizes of the ICN1 proteins are likely due to C-terminal truncations. Note that the deletion of the RBP-J floxed sequences effectively leads to loss of RBP-J proteins in nontransformed IRC⁺ DP cells. (D) PCR analysis of the deletion of the floxed sequences in the samples shown in C. The left panel shows amplification of control samples consisting of mixes of DNA from IRC⁺ and IRC⁻ thymocytes, at the indicated ratio. (E) CD4/CD8 profiles of samples shown in panel C, except for T22 where the FACS profile was not available.

Figure 2

Notch3 is dispensible for leukemogenesis in Ik^L/L mice. (A) Survival curves of Ik^L/LNotch3^+/+ and Ik^L/LNotch3^−/− mice. (B) Representative CD4/CD8 profiles of thymic lymphomas from Ik^L/LNotch3^+/+ and Ik^L/LNotch3^−/− mice.

Figure 3

Deletion of Notch1 accelerates T-ALL development. (A) Survival curves of Ik^L/LNotch1^f/fCD4-Cre⁺ (IN1C+), Ik^L/LNotch1^f/fCD4-Cre⁻ (IN1C⁻), and Ik^L/LNotch1^+/+CD4-Cre⁺ (IC⁺) mice. The P value corresponds to the statistical difference between the survival of IN1C⁺ and IN1C⁻ mice by log-rank test. (B) PCR analysis of the deletion of the floxed sequences in a panel of IN1C⁺ tumors. (C) Thymocyte CD4/CD8 profiles (top) and photos of the thoracic cavity (bottom) from 7-week-old WT, IN1C⁻, and IN1C⁺ mice. (D) Surface Notch1 and CD25 expression of IN1C⁺ and IN1C⁻ tumor cells and WT thymocytes. The immunoglobulin G isotype control is shown in the left panels. Similar results were observed in all Ik^L/L/IN1C⁻ and IN1C⁺ mice analyzed (n > 10). (E) Transcriptome profiling of Notch target genes in 3 IN1C⁺ and 3 IN1C⁻ tumors using Affymetrix 430 2.0 arrays. The data were normalized with those from leukemic T cells of Tel-Jak2 tg mice and from WT DN3, DN4, and DP thymocytes using the Robust Microarray Analysis algorithm. Red and green colors indicate high and low expression, respectively. (F) Proliferation of IN1C⁺ and IN1C⁻ cell lines in the absence or presence of γ-secretase inhibitor over 6 days. Representative of 3 independent experiments. (G) Western blot of ΙCN1 expression in IN1C⁺ tumors using the Val1744 antibody. β-actin was used as a loading control. T99 is a IRC⁺ tumor that expresses ICN1 proteins of the normal 120 kDa size. The asterisk in the right panel points to likely degradation products. (H) PEST region sequences of IN1C⁺ tumors. The bold nucleotides correspond to insertions in the WT sequence. ICN1 proteins from samples T34, T6, and T49 are shown in supplemental Figure 1C; T51, T70 and T110 are shown in Figure 3G; T1 and T2 correspond to IN1C⁺ cell lines described in supplemental Figure 2. Numbering according to the Notch1 reference sequence NM_008714.

Figure 4

Oncogenic effect of deleting floxed Notch1 sequences from a single allele. (A) Survival curves of Ik^L/LNotch1^+/Δf (IN1^+/Δf) and Ik^L/LNotch1^+/+ (IN1^+/+) mice. The statistical significance was calculated by log-rank test. (B) Western blot of ICN1 expression in IN1^+/Δf tumors. The control sample is the IRC⁺ T99 tumor shown in Figure 1C, which expresses ICN1 proteins of normal size. α-Tubulin was used as a loading control. (C) Strategy to identify the allele harboring the PEST domain mutation. A single nucleotide polymorphism in exon 26 (rs27201809; Mouse Genome Informatics database) distinguishes the WT and deleted alleles, which are derived from the C57Bl/6 and 129/Sv strains, respectively. RT-PCR amplification and sequencing of exon 26 and the PEST region in exon 34 identifies the allele carrying the mutation. (D) Association of PEST region mutations with the N1^Δf allele in the 3 tumors shown in B. T3 had a single nt insertion. T7a had a duplication of 19 nt (in bold and italic). T123 had 2 separate mutations, both in the N1^Δf allele: a deletion of 6 nt, which were replaced by a single T, and a single nt insertion. Numbering according to the Notch1 reference sequence NM_008714.

Figure 5

Truncated Notch1 transcripts and proteins in IN1C+ tumors. (A) Northern blot of total RNA (10 μg) from primary tumors and cell lines of the indicated genotypes using a probe for exon 34 (top). Methylene blue staining of 18S and 28S ribosomal RNA was used as a loading control (bottom). (B) RT-quantitative PCR of Notch1 transcripts, using primers to amplify exons 23-24 (encoding the extracellular EGF repeats 33/34) and exons 30-31 (encoding the first intracellular ankyrin repeat). Results were normalized to hypoxanthine-guanine phosphoribosyltransferase levels and to those of WT thymocytes, for which the exon 23-24 mRNA level was arbitrarily fixed at 1. Data represent the mean of 2 experiments. (C) Scheme summarizing the results of the 5′-RACE experiments performed on the T1 and T34 IN1C⁺ cell lines. The organization of the Notch1 transcript in the region of interest is shown, with the position of the S1, S2, and S3 cleavage sites, and the putative methionines that could be used for translation initiation. cDNAs identified by 5′-RACE are shown (see supplemental Figure 5 for sequence details). (D) Northern blot of Notch1 transcripts in poly(A)⁺ RNA (1.5 μg each) from the T1 cell line, hybridized with probes from the indicated exons/introns. E25(5′) corresponds to nt 4279-4741; E25(3′)/26 corresponds to nt 4758-5246; E30/31 corresponds to nt 5715-6179 (reference sequence NM_008714). E27b corresponds to a 628 nt region from intron 27 that includes the 113 nt sequence of exon 27b (see supplemental Figure 5). Autoradiograms for E25(3′)/26 and E30/31 were exposed for 18 hours; those for E25(5′) and E27b were exposed for 44 hours. Asterisks indicate transcripts initiating from 5′ promoters; black arrowheads indicate transcripts initiating from exon 25; white arrowheads indicate transcripts initiating downstream of exon 26 (likely in exon 27); white asterisks indicate transcripts containing exon 27b. (E) RT-PCR of exon 27b–containing transcripts in the indicated samples. cDNA was amplified using a forward primer located within exon 27b and a reverse primer from exon 31. The arrowhead indicates the correctly spliced transcripts; the asterisk indicates likely splicing intermediates that had not excised intron 27. (F) Western blot of Notch1 expression in total cell extracts from the T1 IN1C⁺ and T7 IN1C⁻ cell lines, cultured in the presence or absence of GSI for 3 days. The membrane was first analyzed with the Val1744 antibody, and then with the mN1A Ab. α-tubulin was used as a loading control. Long (10 minutes) and short (30 seconds) exposures are shown for the mN1A Ab. The lines between the top 2 panels indicate the positions of the molecular weight markers used to align the blots. Asterisks indicate the γ-secretase–cleaved proteins from the T7 cell line; arrowheads indicate the γ-secretase–cleaved proteins from the T1 cell line. Note that the ICN1 proteins in the T7 line do not completely disappear after GSI treatment, probably due to the increased stability of the truncated proteins in this cell line. All data are representative of > 2 independent experiments.

Figure 6

Transcriptional reprogramming of the Notch1 locus after deletion of proximal promoter sequences. (A) Chromatin immunoprecipitation-sequence analysis of histone H3 acetylation in the Notch1 locus of T7 IN1C⁻ and the T2 IN1C⁺ cells and WT and Notch1^f/fCD4-Cre⁺ (N1C⁺) thymocytes. Top 4 histograms have a vertical scale of 150. The enlarged histograms in the inset have a vertical scale of 50. The regions identified as I1 and I2 correspond to islands of enriched tag density in the IN1C⁺ thymocytes, which were predicted by the “statistical model for identification of chip-enriched regions” algorithm in the N1C⁺, but not in the WT or input samples (see also Table 1). The gap in the T2 IN1C⁺ sample corresponds to the location of the floxed deletion. (B) Real-time PCR measurement of H3 acetylation in the WT and N1C⁺ samples shown in panel A at several positions along the Notch1 locus. Amplicons in intron 2 and islands I1 and I2 were located, respectively, at 29.3, 36.5, and 41.7 kb downstream of the transcription start site. (C) Northern blot of Notch1 transcripts in WT and N1C⁺ thymocytes and in the T1 IN1C⁺ cell line. An amount of 2 μg of poly(A)⁺ RNA was loaded for each sample, and the blots were hybridized with the E30/31 probe (see Figure 6D). The left panel shows a 42-hour exposure; the right panel shows a 7-day exposure of the WT and N1C⁺ lanes. Arrowheads indicate transcripts likely to have initiated from 3′ promoters in the N1C⁺ sample. A photo of the methylene blue staining of the membrane is shown as a loading control in the bottom panel. (D) RT-PCR of exon 27b–containing transcripts in nontransformed thymocytes from 3- to 4-week-old mice with the indicated genotypes. See Figure 5E for details. Samples were defined as nontransformed according to their CD4/CD8 profile and normal CD25 expression. (E) RT-PCR of exon 27b–containing transcripts in the T7.D5 Cre-ERT2⁺ clone cultured in the presence of 4OHT or vehicle for the indicated times (bottom). Cells were also analyzed for the deletion of floxed Notch1 sequences by PCR (top). T2 corresponds to a IN1C⁺ leukemic cell line. Similar results were obtained in 4 independent experiments. In panels D and E, the arrowhead indicates the specific product from a correctly spliced transcript; the asterisk indicates products that may correspond to splicing intermediates of transcripts initiated at upstream locations.

Figure 7

Presence of 3′ Notch1 transcripts and 5′ genomic deletions in primary Ik^L/L tumors. The indicated RNA or DNA samples were analyzed for the presence of exon 27b–containing transcripts by RT-PCR (top) and genomic deletion of sequences between 2 recombination signal sequences (RSS) present in the 5′ region of the Notch1 locus by PCR (bottom). RAG-mediated deletion of the sequences between the RSS sites moves the sequences of the PCR primers P1 and P2 closer together, allowing amplification of a 500-bp fragment. The Ik^L/L sample was extracted from premalignant thymocytes of a 7-week-old mouse; T7 and T29 are cell lines derived from IN1C⁻ and Ik^L/L tumors, respectively; T49 and T90 are the primary tumors from Figure 5A. β-Actin and exon 34 of Notch1 served as control RT-PCR and PCR reactions, respectively. *Products that may correspond to splicing intermediates of transcripts initiated upstream.