The impact of NOTCH1, FBW7 and PTEN mutations on prognosis and downstream signaling in pediatric T-cell acute lymphoblastic leukemia: a report from the Children's Oncology Group

Abstract

We explored the impact of mutations in the NOTCH1, FBW7 and PTEN genes on prognosis and downstream signaling in a well-defined cohort of 47 patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). In T-ALL lymphoblasts, we identified high-frequency mutations in NOTCH1 (n=16), FBW7 (n=5) and PTEN (n=26). NOTCH1 mutations resulted in 1.3- to 3.3-fold increased transactivation of an HES1 reporter construct over wild-type NOTCH1; mutant FBW7 resulted in further augmentation of reporter gene activity. NOTCH1 and FBW7 mutations were accompanied by increased median transcripts for NOTCH1 target genes (HES1, DELTEX1 and cMYC). However, none of these mutations were associated with treatment outcome. Elevated HES1, DELTEX1 and cMYC transcripts were associated with significant increases in transcript levels of several chemotherapy relevant genes, including MDR1, ABCC5, reduced folate carrier, asparagine synthetase, thiopurine methyltransferase, BCL2 and dihydrofolate reductase. PTEN transcripts positively correlated with HES1 and cMYC transcript levels. Our results suggest that (1) multiple factors should be considered with attempting to identify molecular-based prognostic factors for pediatric T-ALL, and (2) depending on the NOTCH1 signaling status, modifications in the types or dosing of standard chemotherapy drugs for T-ALL, or combinations of agents capable of targeting NOTCH1, AKT and/or mTOR with standard chemotherapy agents may be warranted.

Figure 1. Potencies of clinically relevant NOTCH1 and FBW7 mutations, as measured by reporter gene assays

Human U2OS cells were transiently co-transfected in 35 mm dishes with 0.9 μg of the indicated NOTCH1 expression plasmid alone (A), or with 0.9 μg of both a NOTCH1 expression plasmid and FBW7 expression plasmid (B). For (A), 1 μg of HES1-Luc reporter gene construct and 30 ng of Renilla luciferase (pRL-SV40) internal control were used, whereas for (B), 500 ng HES1-Luc/30 ng pRL-SV40 were used. For all transfections, constant plasmid was maintained at 0.9 μg of pcDNA3 plasmid per well. Results represent normalized luciferase activities of whole cell lysates, relative to a control in which HES1-luc was co-transfected with 0.9 μg pcDNA3 vector in lieu of NOTCH1/FBW7 (assigned a value of 1). Results are presented as mean values ± standard errors [n=6 for (A); n=6 for (B)]. For (A), p-values were calculated using paired t tests, comparing the luciferase activities of the different NOTCH1 mutations to wild-type NOTCH1 (*, p≤0.05; **, p≤0.005). For (B), p-values were calculated using paired t-tests, comparing the clinically relevant NOTCH1 and FBW7 mutants as shown in the figure. For (A), the sample numbers designate the patient samples listed in Table 2. For (B), NOTCH1 and FBW7 forms refer to the sample numbers in Table 2. For sample 1, (a) is the early termination at position 322 and (b) is R465L. Abbreviations are: Wt, wild type; Δ, ICN (activated NOTCH1); NA, no addition.

Figure 1. Potencies of clinically relevant NOTCH1 and FBW7 mutations, as measured by reporter gene assays

Human U2OS cells were transiently co-transfected in 35 mm dishes with 0.9 μg of the indicated NOTCH1 expression plasmid alone (A), or with 0.9 μg of both a NOTCH1 expression plasmid and FBW7 expression plasmid (B). For (A), 1 μg of HES1-Luc reporter gene construct and 30 ng of Renilla luciferase (pRL-SV40) internal control were used, whereas for (B), 500 ng HES1-Luc/30 ng pRL-SV40 were used. For all transfections, constant plasmid was maintained at 0.9 μg of pcDNA3 plasmid per well. Results represent normalized luciferase activities of whole cell lysates, relative to a control in which HES1-luc was co-transfected with 0.9 μg pcDNA3 vector in lieu of NOTCH1/FBW7 (assigned a value of 1). Results are presented as mean values ± standard errors [n=6 for (A); n=6 for (B)]. For (A), p-values were calculated using paired t tests, comparing the luciferase activities of the different NOTCH1 mutations to wild-type NOTCH1 (*, p≤0.05; **, p≤0.005). For (B), p-values were calculated using paired t-tests, comparing the clinically relevant NOTCH1 and FBW7 mutants as shown in the figure. For (A), the sample numbers designate the patient samples listed in Table 2. For (B), NOTCH1 and FBW7 forms refer to the sample numbers in Table 2. For sample 1, (a) is the early termination at position 322 and (b) is R465L. Abbreviations are: Wt, wild type; Δ, ICN (activated NOTCH1); NA, no addition.

Figure 2. Expression of HES1, DELTEX1 and cMYC transcripts in patients harboring NOTCH1 and/or FBW7 mutations

Transcript levels were measured using real-time RT-PCR and normalized to those for GAPDH. Primers and PCR conditions are summarized in the Supplement (Table S2). Results are shown for HES1, DELTEX1, and cMYC transcript levels in T-ALL specimens exhibiting NOTCH1 and/or FBW7 mutations and T-ALL specimens characterized by wild type NOTCH1 and FBW7. Data were were analyzed using the non-parametric Wilcoxon test. Horizontal bars represent median values. Abbreviations: DTX, DELTEX.

Figure 3. Expression of relevant chemotherapy genes in relation to HES1

Patients with HES1 transcript levels (Figure 2) below the median value were considered to have low HES1 expression, and those with HES1 transcript expression above HES1 median values were considered to have high HES1 expression. Relative transcript levels for 22 chemotherapy-related genes were measured by real-time RT-PCR, as described in the Materials and Methods section, and are summarized in Table S4 (Supplement). Horizontal bars represent median values. Twelve of 22 genes were significantly over-expressed in samples with high HES1 transcripts (p<0.05 by non-parametric Wilcoxon test) (Table S4, Supplement) and of these, the 7 gene targets in the figure also showed a statistically significant association with levels of DELTEX1 and cMYC transcripts (Tables S5 and S6, Supplement). Abbreviations: ABCC5, Multidrug resistance-associated protein 5 (MRP5); ASNS, asparagine synthetase; BCL2, B-cell leukemia/lymphoma 2; DHFR, dihydrofolate reductase; hRFC, human reduced folate carrier; MDR1, multidrug resistance 1; TPMT, thiopurine-S-methyltransferase.