Notch is oncogenic dominant in T-cell acute lymphoblastic leukemia

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic neoplasm characterized by malignant expansion of immature T cells. Activated NOTCH (Notch(IC)) and c-MYC expression are increased in a large percentage of human T-ALL tumors. Furthermore, c-MYC has been shown to be a NOTCH target gene. Although activating mutations of Notch have been found in human T-ALL tumors, there is little evidence that the c-MYC locus is altered in this neoplasm. It was previously demonstrated that Notch and c-Myc-regulated genes have a broadly overlapping profile, including genes involved in cell cycle progression and metabolism. Given that Notch and c-Myc appear to function similarly in T-ALL, we sought to determine whether these two oncogenes could substitute for each other in T-ALL tumors. Here we report that NOTCH(IC) is able to maintain T-ALL tumors formed in the presence of exogenous NOTCH(IC) and c-MYC when exogenous c-MYC expression is extinguished. In contrast, c-MYC is incapable of maintaining these tumors in the absence of NOTCH(IC). We propose that failure of c-MYC to maintain these tumors is the result of p53-mediated apoptosis. These results demonstrate that T-ALL maintenance is dependent on NOTCH(IC), but not c-MYC, demonstrating that NOTCH is oncogenic dominant in T-ALL tumors.

Figure 1

Coexpression of ectopic c-MYC does not cooperate in Top-NOTCH tumor formation or prevent tumor regression. (A) Schematic of the tumor formation protocol. (B) Diagram of retroviral constructs. (C) Death curve of mice reconstituted with Top-NOTCH bone marrow infected with MIT-c-MYC (left panel) and MIT-GFP (right panel). (D) Top-NOTCH/MIT-c-MYC mouse before and after treatment with Dox. *Reduction of lymph nodes.

Figure 2

T-ALL tumors are not maintained by c-MYC in the absence of NOTCH^IC. (A) Spleens from Top-NOTCH/MIT-c-MYC (left panel) and Top-NOTCH/MIT-NOTCH^IC (right panel) tumors treated for the indicated times with Dox. (B) FACS analysis using anti-CD4-PE and anti-CD8-PerCP-Cy5.5 antibodies to detect double-positive tumor cells in the spleen before and after Dox treatment of mice reconstituted with Top-NOTCH/MIT-c-MYC bone marrow (upper and lower left panels, respectively), wild-type (WT) thymus, and wild-type spleen (upper and lower right panels, respectively).

Figure 3

Spleen size of moribund mice reconstituted with Top-NOTCH/MIT-c-MYC bone marrow is significantly reduced after Dox treatment. (A) Area (length × width) of moribund mice reconstituted with Top-NOTCH/MIT-c-MYC bone marrow, untreated with Dox (No Dox) or treated for 96 hours with Dox. *P = .000009 (Student t test). n = 10. (B) Quantification of CD4⁺CD8⁻ and CD4⁺CD8⁺ T cells in spleens of mice with Top-NOTCH/MIT-c-MYC tumors untreated or treated with Dox for 96 hours. *P = .00022 (Student t test). n = 7.

Figure 4

Expression of p53 is induced in T-ALL tumors when NOTCH^IC expression is extinguished. Each primary tumor was transplanted into multiple syngeneic mice, and tumors were allowed to form. When mice were moribund, Dox was added and tissue collected at various time points for analysis. RT-PCR (A), quantitative RT-PCR (B), and Western blot (C) analyses were performed with the indicated specific primers or antibodies on single-cell suspensions from spleens of Top-NOTCH^IC-/MIT-c-MYC tumors.

Figure 5

NOTCH^IC expression can maintain Tet-o-MYC/MIT-NOTCH^IC T-ALL tumors. (A) Schematic for generation of Tet-o-MYC/MIT-NOTCH^IC tumors. (B) Diagram of retroviral constructs. (C) Death curves for mice reconstituted with Tet-o-MYC bone marrow infected with MIT-NOTCH^IC (left panel) and MIT-GFP (right panel). (D) Spleens from nonregressing Tet-o-MYC/MIT-NOTCH^IC. (E) FACS analysis using α-CD4-PE and α-CD8-PerCP-Cy5.5 antibodies on single-cell suspensions from spleens of moribund nonregressing Tet-o-MYC/MIT-NOTCH^IC treated with Dox for the indicated times.

Figure 6

Spleen size of moribund mice reconstituted with Tet-o-MYC/MIT-NOTCH^IC bone marrow does not significantly reduce after Dox treatment. (A) Area (length × width) of moribund mice reconstituted with Tet-o-MYC/MIT-NOTCH^IC bone marrow, untreated with Dox (No Dox) or treated for 2 weeks with Dox. N.S. indicates not significant. P = .8257 (Student t test). n = 6. (B) Quantification of CD4⁺CD8⁻ and CD4⁺CD8⁺ T cells in spleens of mice with Tet-o-MYC/MIT-NOTCH^IC tumors untreated or treated with Dox for 2 weeks. There was no significant change (N.S.) in the size of spleens of mice with Tet-o-MYC/MIT-NOTCH^IC treated with Dox (P = .8574; Student t test). n = 6.

Figure 7

NOTCH^IC expression is maintained in nonregressing Tet-o-MYC/MIT-NOTCH^IC tumors treated with Dox. (A) RT-PCR analysis of spleens from nonregressing Tet-o-MYC/MIT-NOTCH^IC tumors treated with Dox for the indicated times using the indicated specific primers. (B) Quantitative RT-PCR analysis on single-cell suspensions from spleens of nonregressing Tet-o-MYC/MIT-NOTCH^IC tumors treated with Dox. (C) Western blot analysis with antibodies against the indicated proteins in nonregressing Tet-o-MYC/MIT-NOTCH^IC tumors treated with Dox.