Combined expression of pTalpha and Notch3 in T cell leukemia identifies the requirement of preTCR for leukemogenesis

Abstract

Notch receptors are conserved regulators of cell fate and have been implicated in the regulation of T cell differentiation and lymphomagenesis. However, neither the generality of Notch involvement in leukemia, nor the molecules with which Notch may interact have been clarified. Recently, we showed that transgenic mice expressing the constitutively active intracellular domain of Notch3 in thymocytes and T cells developed early and aggressive T cell neoplasias. Although primarily splenic, the tumors sustained features of immature thymocytes, including expression of pTalpha, a defining component of the pre T cell receptor, known to be a potent signaling complex provoking thymocyte survival, proliferation, and activation. Thus, enforced expression of Notch3, which is ordinarily down-regulated as thymocytes mature, may sustain pre T cell receptor expression, causing dysregulated hyperplasia. This hypothesis has been successfully tested in this article by the observation that deletion of pTalpha in Notch3 transgenic mice abrogates tumor development, indicating a crucial role for pTalpha in T cell leukemogenesis. Parallel observations were made in humans, in that all T cell acute lymphoblastic leukemias examined showed expression of Notch3 and of the Notch target gene HES-1, as well as of pTalpha a and b transcripts, whereas the expression of all these genes was dramatically reduced or absent in remission. Together, these results suggest that the combined expression of Notch3 and pTalpha sustains T cell leukemogenesis and may represent pathognomonic molecular features of human T-ALL.

Figure 1

(A) RT-PCR expression of Notch3, Notch1, and pTα a and b transcripts in early, intermediate, and mature (early-T, interm-T, and mature-T, respectively) human T cell acute lymphoblastic leukemias, classified according to ref. . Peripheral blood samples obtained from patients affected by T cell leukemias of different phenotypes express Notch3, Notch1, and both pTα transcripts, irrespective of the differentiation phenotype. Notch3, Notch1, and pTα expression data are representative of RT-PCR reactions monitored during the exponential phase of amplification (PCR at 25 cycles is shown) normalized to coamplified β-actin internal control to quantitate mRNA expression levels. (C-, no RNA; Thy, RNA from normal unfractionated thymocytes; PTL, RNA from enriched peripheral T lymphocytes). (B) Expression of Notch3, Notch1, and pTα mRNA (assessed by RT-PCR) in human non-T cell leukemias, classified according to ref. . Notch3 and pTα expression was measured at the end point of amplification (PCR at 35 cycles is shown), whereas Notch1 was monitored along the exponential amplification phase (PCR at 25 cycles is shown). Lack of Notch3 expression versus Notch1 did not seem to be caused by difference in sensitivity of Notch3 primers in comparison with Notch1, because similar amounts of Notch1 and Notch3 amplicons were detected starting from the addition of similar amounts of Notch1 and Notch3 templates in PCR reaction (not shown).

Figure 2

(A) Northern Blot analysis of Notch3 and pTα mRNAs in a representative case of T-ALL, in normal unfractionated thymocytes (Thy), in enriched peripheral T lymphocytes (PTL), and in common ALL (C-ALL). (B) RT-PCR of Notch3 mRNA with different primers pairs spanning different regions throughout the Notch3 transcript (primers 1, C terminus, Notch3 C-COOH; 2, transmembrane, Notch3 TM-COOH; 3, N terminus, Notch3 NH₂-TM). Notch3 and Notch1 expression was monitored along the exponential amplification phase (PCR at 25 cycles is shown) and normalized to internal β-actin coamplification. Ratios of hybridization intensity between Notch3 expression in T-ALL versus thymocytes accounted for 1.1, 1.0, and 0.95, by using primers 1, 2, and 3, respectively. (C) RT-PCR expression of HES 1 RNA in three representative cases of T-ALL (T-ALL) and in enriched peripheral T lymphocytes (PTL) (Thy, RNA from unfractionated thymocytes; c-, no RNA). HES-1 expression was monitored along the exponential amplification phase (PCR at 25 cycles is shown) and normalized to internal β-actin coamplification.

Figure 3

Expression of Notch3, Notch1, pTα, and HES-1 mRNAs (assessed by RT-PCR) in normal thymocytes (Thy), peripheral T lymphocytes from normal subjects (PTL), and bone marrow samples from T-ALL patients (T-ALL) in different stages of human T cell leukemias (ex, exordium; rem, remission; C-, no RNA). mRNA expression was monitored during the exponential amplification phase (PCR at 25 cycles is shown) and normalized to internal β-actin coamplification.

Figure 4

(A) Macroscopic aspect of spleen and mesenteric lymph nodes (Mes-LN) isolated from Notch3-IC (N3-IC/pTα^+/+), Notch3-IC/pTα^−/− (N3-IC/pTα^−/−) transgenic and wild-type (wt) 10-week-old mice. (B) Mortality curve of several lines of transgenic mice. The numbers of spontaneously dead mice were plotted against their age. Results are indicated as the percentage of surviving mice at each age. The follow-up of Notch3-IC (N3-IC/pTα^+/+) and of Notch3-IC/pTα^−/+ (N3-IC/pTα^−/+) mice was stopped at 30 and 40 weeks, respectively, being 95% of the mice dead at these ages. (n, 160 for N3-IC/pTα^+/+; n, 60 for N3-IC/pTα^−/−; n, 30 for N3-IC/pTα^−/+ transgenic; and n, 100 for wild-type (wt) mice.

Figure 5

Total cell yield and immunophenotype of lymphocyte from thymus, spleen, and mesenteric lymph nodes from 4-week-old mice. (A) Total cell yield and subset distribution of thymocytes from wild-type (wt), pTα mutant (pTα^−/−), and Notch3-IC/pTα^−/− (N3-IC/pTα^−/−) double-transgenic mice. Numbers in the quadrants indicate the percentage of different subsets. (B) Expression of TCRβ chain on thymocytes from the same mice of A. Numbers indicate the percentage of TCRβ high-expressing thymocytes. (C) Absolute number of CD4, CD8, and TCRβ chain-expressing splenic lymphocytes (×10⁵) and total cell yield (×10⁶) from spleen of pTα^−/− mice (pTα^−/−, open bars) and Notch3-IC/pTα^−/− double-transgenic mice (N3-IC/pTα^−/−, shaded bars). (D) Absolute number of CD4, CD8, and TCRβ chain-expressing lymphocytes (×10⁵) and total cell yield (×10⁶) from mesenteric lymph nodes of pTα^−/− mice (pTα^−/−, open bars) and Notch3-IC/pTα^−/− double-transgenic mice (N3-IC/pTα^−/−, shaded bars). Results are representative of three different experiments with animals of the same age.