Leukemia-associated NOTCH1 alleles are weak tumor initiators but accelerate K-ras–initiated leukemia

Abstract

Gain-of-function NOTCH1 mutations are found in 50%-70% of human T cell acute lymphoblastic leukemia/ lymphoma (T-ALL) cases. Gain-of-function NOTCH1 alleles that initiate strong downstream signals induce leukemia in mice, but it is unknown whether the gain-of-function NOTCH1 mutations most commonly found in individuals with T-ALL generate downstream signals of sufficient strength to induce leukemia. We addressed this question by expressing human gain-of-function NOTCH1 alleles of varying strength in mouse hematopoietic precursors. Uncommon gain-of-function NOTCH1 alleles that initiated strong downstream signals drove ectopic T cell development and induced leukemia efficiently. In contrast, although gain-of-function alleles that initiated only weak downstream signals also induced ectopic T cell development, these more common alleles failed to efficiently initiate leukemia development. However, weak gain-of-function NOTCH1 alleles accelerated the onset of leukemia initiated by constitutively active K-ras and gave rise to tumors that were sensitive to Notch signaling pathway inhibition. These data show that induction of leukemia requires doses of Notch1 greater than those needed for T cell development and that most NOTCH1 mutations found in T-ALL cells do not generate signals of sufficient strength to initiate leukemia development. Furthermore, low, nonleukemogenic levels of Notch1 can complement other leukemogenic events, such as activation of K-ras. Even when Notch1 participates secondarily, the resulting tumors show "addiction" to Notch, providing a further rationale for evaluating Notch signaling pathway inhibitors in leukemia.

Cooperation between somatic Ikaros and Notch1 mutations at the inception of T-ALL

“…Similar HD mutations and mutations that lead to truncation of the Notch1 intracellular domain have been identified in other animal models bearing Ikaros mutations suggesting that Notch1 is a frequent target of mutations in these T cell derived tumors [23,24]. This is consistent with the conclusions from retroviral introduction of Notch1 HD or truncating mutations into bone marrow reconstituted mice [11].…”

“…Potentially activating mutations in Notch1 itself can be detected even earlier, and these are strongly enriched concurrent with Ikzf1 LOH and flow cytometric abnormalities in thymocyte growth or differentiation. These results extend the view emerging from human T-ALL and associated experimental studies [10,11] that multiple mutations acting in concert upon the Notch pathway are needed to exaggerate Notch1 transcriptional activity above a threshold that can promote neoplastic T cell growth in vivo.…”

“…Loss of DNA-binding Ikaros appears to be a critical collaborator in this inception phase, relieving the ICN-RBPJk-MAML transcriptional complex from repressive effects of Ikaros [23,42] and correlating closely with a dramatic increase in Notch target gene expression in pre-leukemic DP cells. This multi-step increase in Notch signalling may be required to achieve a threshold for inducing Myc, repressing p19Arf, and initiating neoplastic DP cell growth in vivo, as suggested by Chiang et al [11]. Mutant Notch1 receptors can still bind and respond to Notch ligands and this may be important in the engraftment of the mutant cells into the bone marrow and secondary lymphoid tissues [44].…”

“…The earliest stages of abnormal thymic differentiation were accompanied by somatic mutations in the HD and PEST domains of the Notch1 gene that are known to exaggerate ICN production, induce expression of Myc [11] and Cyclin-D1 (26) and inhibit the expression of tumour suppressor genes (e.g. p19ARF -p53) or growth regulators such as p27kip1 and Nur77 [43].…”

“…The extent to which these individual NOTCH1 mutations in T-ALL confer a growth advantage or malignant transformation is unclear. T-ALL have been described with multiple NOTCH1 mutations in cis altering the HD and C-terminal domains, and these have been shown to exaggerate NOTCH1 activity in tissue culture in an additive manner and to function cooperatively for leukemogenesis in vivo [7,[9][10][11][12]. Some T-ALL NOTCH1 mutations are sufficient for promoting leukemogenesis while others are only sufficient for leukemogenesis in conjunction with other mutations, raising the question of how many cooperating events precede the first outgrowth of a leukemic T cell clone [13][14][15][16].…”