Background

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological tumor arising from the malignant transformation of hematopoietic progenitor cells primed towards T-cell development. T-ALL accounts for up to 15% of pediatric and 25% of adult ALL (acute lymphoblastic leukemia) and occurs more frequently in males than females (Goldberg et al., 2003). Despite being characterized by a high level of heterogeneity and molecular complexity (Liu et al., 2017), more than 65% of T-ALL patients carry gain-of-function mutations in the NOTCH1 gene (Weng et al., 2004). The resulting constitutive activation of NOTCH1 signaling is therefore the most prominent oncogenic pathway in T-cell transformation.

NOTCH1 proteins are developmentally conserved type I transmembrane receptors that play a prominent instructive role in T-cell lineage commitment and cell growth and proliferation during thymocyte development (Radtke et al., 1999; Defto and Bevan, 2000). NOTCH1 signaling is initiated upon binding of transmembrane ligands expressed on the surface of neighboring cells. This interaction prompts cleavage of the extracellular domain of the receptor by the ADAM10 metalloprotease followed by γ-secretase cleavage in the transmembrane domain of the receptor. Subsequent release from the membrane results in translocation of the intracellular domain (ICN1) to the nucleus and transcriptional activation of target genes (Kopan and Ilagan, 2009; Andersson et al., 2011). Finally, termination of NOTCH1 signaling is regulated by phosphorylation of the C-terminal PEST domain, which targets ICN1 for ubiquitination and FBXW7-mediated proteasomal degradation (O'Neil et al., 2007; Thompson et al., 2007). Constitutive activation of NOTCH1 in human T-ALL results from disruption of the mechanisms stringently regulating this multi-step process. The majority of T-ALL-associated mutations result either in disruption of the negative regulatory region, resulting in NOTCH1-signaling activation in the absence of ligand binding, or truncation of the C-terminal PEST domain which allows ICN1 to evade proteasomal degradation, thus impairing termination of signaling activity (Weng et al., 2004). In the past decade, the introduction of intensive combination chemotherapy approaches has increased the cure rate of pediatric T-ALL to nearly 90%. Nonetheless, the prognosis for patients with primary refractory or relapse T-ALL remains very poor (Goldberg et al., 2003; Litzow and Ferrando, 2015), underscoring the need to further decipher the molecular pathology underlying T-ALL transformation, identification of more specific therapeutic targets, and development of more effective and less toxic multi-drug therapies.

To date, in vitro human T-ALL-derived cell-line cultures remain the most commonly employed experimental approach to the study of T-ALL. Notably, this model does not correct for genetic drift associated with decade-long periods of culture and fails to account for the crosstalk between leukemia cells and the tumor microenvironment (Passaro et al., 2015; Pitt et al., 2015). Animal models therefore provide an alternative approach to functional studies that more accurately recapitulate human T-ALL in vivo. The mouse hematopoietic stem cell (HSC) retroviral transduction and transplantation model has been extensively applied to mimic initiation and progression of NOTCH1-driven T-ALL (Wendorff et al., 2010; Medyouf et al., 2011; Gachet et al., 2013). It has been shown that retroviral expression of ICN1 or ΔE-NOTCH1 (the extracellular domain-truncated form of the receptor) in murine bone marrow progenitors drives thymus-independent T-cell development and rapidly induces T-ALL development (Pear et al., 1996; Perez-Garcia et al., 2013). However, it should be noted that these aggressively truncated variants of NOTCH1, which result in supraphysiological levels of signaling activity, are rarely found in human T-ALL (Chiang et al., 2008). In fact, ΔE-NOTCH1 truncated alleles are expressed in only a small subset of patients carrying chromosomal translocations (1%) (Ellisen et al., 1991; Palomero et al., 2006). Much more commonly, human T-ALLs harbor acquired gain-of-function point mutations, which occur predominantly within the extracellular HD and the C-terminal PEST domains (Weng et al., 2004). Of note, 20% of T-ALL patients exhibit co-occurrence of these two mutation-driven activating mechanisms, which generate levels of activity well below those of ΔE-NOTCH1 (Chiang et al., 2008). As the outcome of NOTCH signaling is highly dose dependent in many developmental settings (Artavanis-Tsakonas et al., 1999; Bray, 2016), we hypothesized that the level of activity has a similar impact on the process of leukemogenesis. The current protocol describes the method for mouse hematopoietic stem cell (HSC) retroviral transduction with an oncogenic mutant NOTCH1 receptor (NOTCH1-L1601P-ΔP) that closely resembles the gain-of-function mutations found in patient samples. The L1601P-ΔP mutant contains a common HD point mutation (L1601P) that enables ligand-independent activation (Malecki et al., 2006), together with a frameshift mutation that results in truncation of amino acids 2,473-2,555, which comprise the negative regulatory PEST domain (ΔP). We outline the method for isolation of bone marrow cells from mice, enrichment for hematopoietic stem and progenitor cells, followed by retroviral transduction and transplantation into isogenic recipients (Figure 1). In this model, forced expression of constitutively active mutant NOTCH1 typically results in a wave of extrathymic T-cell lymphopoiesis 3 weeks after transplantation, which manifests as the transient appearance of GFP⁺ preleukemic CD4⁺ CD8⁺ double-positive (DP) immature T cells in peripheral blood (Figure 4). Transformation resulting in lethal leukemia progression occurs 12-15 weeks following transplantation (Wendorff et al., 2019) (Figure 4). When combined with constitutive- or inducible-knockout transgenic models, the protocol can be easily modified to address the role of candidate genes in NOTCH1-driven T-ALL transformation, tumor initiation and tumor progression (Wendorff et al., 2019). Furthermore, this approach provides a rapid and efficient model system for pre-clinical assessment of potential novel anti-leukemic drugs (Herranz et al., 2015; Sanchez-Martin et al., 2017).