Mutant Ikzf1, KrasG12D, and Notch1 cooperate in T lineage leukemogenesis and modulate responses to targeted agents

Abstract

Mice that accurately model the genetic diversity found in human cancer are valuable tools for interrogating disease mechanisms and investigating novel therapeutic strategies. We performed insertional mutagenesis with the MOL4070LTR retrovirus in Mx1-Cre, Kras(G12D) mice and generated a large cohort of T lineage acute lymphoblastic leukemias (T-ALLs). Molecular analysis infers that retroviral integration within Ikzf1 is an early event in leukemogenesis that precedes Kras(G12D) expression and later acquisition of somatic Notch1 mutations. Importantly, biochemical analysis uncovered unexpected heterogeneity, which suggests that Ras signaling networks are remodeled during multistep tumorigenesis. We tested tumor-derived cell lines to identify biomarkers of therapeutic response to targeted inhibitors. Whereas all T-ALLs tested were sensitive to a dual-specificity phosphoinosityl 3-kinase/mammalian target of rapamycin inhibitor, biochemical evidence of Notch1 activation correlated with sensitivity to gamma-secretase inhibition. In addition, Kras(G12D) T-ALLs were more responsive to a MAP/ERK kinase inhibitor in vitro and in vivo. Together, these studies identify a genetic pathway involving Ikzf1, Kras(G12D), and Notch1 in T lineage leukemogenesis, reveal unexpected diversity in Ras-regulated signaling networks, and define biomarkers of drug responses that may inform treatment strategies.

Fig. 1.

Incidence, latency, and clonality of MOL4070LTR-induced T-ALL. (A) Incidence of T-ALL vs. myeloid leukemia. (B) Time to myeloid leukemia. (C) Time to T-ALL. (D) Overview of T-ALLs generated by transplanting bone marrow from virus-injected mice (VIM) with MPD into primary and secondary recipients. (E) Southern blotting with a MOL4070LTR probes does not reveal a dominant clone in VIM but shows a unique pattern of insertions in each T-ALL. Paired VIM and recipient T-ALLs are separated by space between the blots. (F) Southern blotting demonstrates the same integration pattern in secondary recipients (R1 and R2).

Fig. 2.

Notch1 is deregulated in MOL4070LTR-induced T-ALL. Western blot analysis of NICD expression in (A) primary T-ALLs and (B) T-ALL cell lines. (C) Expression of Deltex1 and Hes1 were assessed by quantitative RT-PCR in cell lines without a Notch1 mutation or detectable NICD by Western blotting (Notch1 WT/-NICD; n = 2); with a Notch1 mutation and detectable NICD (Notch1 MUT/+NICD; n = 2); and with a Notch1 mutation but not detectable NICD (Notch1 MUT/-NICD; n = 3). Asterisks (*) indicate undetectable levels.

Fig. 3.

Ras signaling in T-ALL. (A) Ras-GTP levels are elevated in Kras^G12D cell lines deprived of serum for 16 h. (B and C) PTEN p-Akt, p-S6, and p-ERK levels in cell lines under normal growth conditions (B) and in primary T-ALLs (C).

Fig. 4.

Effects of small molecule inhibitors on T-ALL cell lines and primary tumors. Cell lines were grown for 5–7 days in the presence of each compound, and the number of viable cells was determined. (A) Effects of Compound E, a GSI. The solid line displays the growth of NICD negative cell lines (n = 6), and dashed line shows NICD positive lines (n = 10; P = 0.04–<0.001). (B) Effects of PI-103, a dual-specificity PI3K/mTOR inhibitor. Kras^WT cell lines are summated in the solid line (n = 2), and Kras^G12D cell lines are shown as a dashed line (n = 8; P = 0.03–0.8). (C) Effects of PD0325901, a MEK inhibitor. The Kras^WT cell lines are shown in the solid line (n = 5), and the Kras^G12D cells are presented in the dashed line (n = 11; P = 0.0009–<0.0001). Error bars represent SEM. (D) Combined inhibitor treatment. A cell line was grown in increasing concentrations of both (Left) GSI and PD0325901 or (Right) GSI and PI-103. The blue line (GSI alone) shifts to the left (green and purple lines) when combined with PD0325901 or PI-103, thereby reducing the IC₅₀ of GSI. (E) PD0325901 prolongs survival in mice transplanted with Kras^G12D T-ALLs. Mice were transplanted with three independent primary Kras^WT (Left) or six independent Kras^G12D (Right) T-ALLs and were treated with vehicle (dashed line) or PD0325901 (solid line). There was no significant difference in the survival (in days) of mice engrafted with Kras^WT leukemias (P = 0.13). In contrast, PD0325901 treatment increased survival in recipients of Kras^G12D T-ALLs (P < 0.0005). Kras^WT vehicle n = 7, Kras^WT PD0325901 n = 13, Kras^G12D vehicle n = 15, and Kras^G12D PD0325901 n = 30.

Fig. 5.

Models. (A) RIM-induced T-ALL in Kras^G12D mice. Pups are injected with MOL4070LTR at 3–5 days of age, followed by induction of Kras^G12D expression at 21 days. Virus-injected mice (VIM) die of Kras^G12D-induced MPD at ≈90 days. Bone marrow is then transplanted into sublethally irradiated recipients. Myeloid malignancies develop with short latency. By contrast, an individual VIM can give rise to multiple independent T-ALLs with different retroviral insertion sites and Notch1 mutations. (B) Relationship of MEK and γ-secretase inhibitor responses to mutations found in T-ALL. (Left) The pharmacologic target of PD0325901 (MEK) is downstream of oncogenic K-Ras-GTP, which allows for negative feedback and remodeling (dotted lines). As a result, ERK phosphorylation may not accurately reflect pathway activation and/or dependence. (Right) Notch1 PEST domain mutations result in the production of mutant Notch1 proteins that remain dependent on γ-secretase for proteolytic cleavage and biologic activity. NICD levels therefore provide a direct biochemical readout of the deleterious effects of a Notch1 mutation, and the ability of GSI treatment to reduce these levels is a robust marker of drug response.