A powerful molecular synergy between mutant Nucleophosmin and Flt3-ITD drives acute myeloid leukemia in mice

Acute myeloid leukemia (AML) is the commonest myeloid malignancy, yet there has been little therapeutic progress for this disease in decades, and only 25–30% of patients survive long term.1 This reflects its pathogenetic complexity and the fact that the molecular basis of its largest cytogenetic subgroup, AML with a normal karyotype (AML-NK), was obscure until recently. Recent advances in DNA sequencing have revealed that AML-NK is molecularly heterogeneous with >30 genes recurrently targeted by somatic mutations in this disease.2 What is also evident is that each individual case of AML-NK appears to harbor only a small number of coding driver mutations, often as few as three and usually no more than five.2, 3 Furthermore, it is manifest that the precise combination of driver mutations in the genome of each AML impacts on its salient features, including responsiveness to treatments and prognosis.3

Acute myeloid leukemia (AML) is the commonest myeloid malignancy, yet there has been little therapeutic progress for this disease in decades, and only 25-30% of patients survive long term. 1 This reflects its pathogenetic complexity and the fact that the molecular basis of its largest cytogenetic subgroup, AML with a normal karyotype (AML-NK), was obscure until recently. Recent advances in DNA sequencing have revealed that AML-NK is molecularly heterogeneous with 430 genes recurrently targeted by somatic mutations in this disease. 2 What is also evident is that each individual case of AML-NK appears to harbor only a small number of coding driver mutations, often as few as three and usually no more than five. 2,3 Furthermore, it is manifest that the precise combination of driver mutations in the genome of each AML impacts on its salient features, including responsiveness to treatments and prognosis. 3 These observations provide a sound starting point for systematic mechanistic studies to understand the pathogenesis and improve the treatment of AML-NK. Carefully designed mouse models are the gold standard in the study of normal and malignant hemopoiesis, and are already instructing our understanding of AML-NK. 4,5 Here, we report that the two most commonly cooccurring somatic mutations in AML, namely Nucleophosmin (NPM1) exon 12 mutations (NPM1c) and internal tandem duplications of FLT3 (FLT3-ITD), cooperate explosively to induce AML in knock-in mice. In revealing this striking molecular synergy, our work offers a basis for the frequent co-occurrence of these two mutations and provides a valuable model for in-depth studies of the pathogenesis and treatment of this large subgroup of AML.
NPM1 is a nucleolar phosphoprotein involved in many cellular processes. For many of its roles, it relies on its ability to shuttle between the nucleolus, nucleus and cytoplasm using subcellular localization signals. 6 This ability is impaired in 30% of AMLs as a result of NPM1c mutations, which disrupt the nucleolar localization signal of NPM1 and generate a nuclear export signal in its place. 7 Mutant NPM1 is known to bind to and alter the subcellular distribution of several proteins, including HEXIM1, p19Arf and nuclear factor-kB; 8 however, the relevance of these interactions to AML is unclear. FLT3-ITD mutations occur in 20-25% of AML 9 and result in ligand-independent receptor dimerization and constitutive FLT3 signaling, 10 and are associated with an increased risk of relapse. Moreover, patients with low or absent levels of wild-type (WT) FLT3, consistent with loss-of-heterozygosity (LOH) for this locus, have a particularly poor outcome. 9 Recently, we described a conditional knock-in mouse model of NPM1c mutations and demonstrated that one-third of mice developed delayed-onset AML, suggesting a requirement for cooperating mutations. We went on to show that insertional mutagenesis with transposons led rapidly to AML in 80% of Npm1c mice, in association with specific recurrent mutations including activating insertions in Csf2 and Flt3. 4 Flt3-ITD homozygous mutant mice exhibit enhanced proliferation and survival properties in hemopoietic progenitors and develop a lateonset disease akin to chronic myelomonocytic leukemia. 11 To study the combined effects of NPM1c with FLT3-ITD we crossed conditional Npm1 flox À cA/ þ with constitutive Flt3 ITD/ þ to generate Npm1 flox À cA/ þ ; Flt3 ITD/ þ double heterozygous mice, then crossed into Mx1-Cre transgenic mice to induce recombination of Npm1 flox À cA in hematopoietic stem cells. 4 The Mx1-Cre allele requires induction by interferon, usually achieved by intraperitoneal injection of polyinosinic-polycytidylic acid (pIpC). However, we observed universal and rapid emergence of AML (myeloid leukemia with maturation) in uninjected Npm1 flox À cA ;FLT3 ITD/ þ ;Mx1-Cre þ mice (hereafter referred to as 'Npm1c/Flt3-ITD mice'). Mx1-Cre is known to 'leak' in 2-4% of hemopoietic stem/progenitor cells, 12 and this was sufficient to rapidly generate AML from double mutant cells signifying a striking cooperativity between Npm1c and Flt3-ITD. The presence of the cytoplasmic NPM1 was confirmed on protein blots ( Figure 1a).
Interestingly, Npm1c/Flt3-ITD siblings/littermates often progressed to AML at different rates or developed more/less aggressive disease. To explain this observation we hypothesized that, as seen in human AML, LOH for Flt3-ITD may be responsible. We found evidence for significant spontaneous loss of the WT Flt3 allele in blood samples from Npm1c/Flt3-ITD mice and a tendency for higher blood leukocyte counts (Figure 1d) when LOH was present. LOH was also seen in bone marrow and spleen but not tail DNA, in keeping with somatic loss of the WT allele in leukemic cells (Figure 1d). At the time mice became sick with AML, LOH was detected in 12 of 15 spleen samples tested.
Flow cytometric analysis of blood samples demonstrated, in Npm1c/Flt3-ITD mice, a population of blasts/immature cells with low side scatter (SSC) and CD45 dim ( Figure 2a) and a large number of single Mac1 þ precursors (Figure 2b). In addition, we also observed an increased number of mature myeloid (Gr1 þ / Mac1 þ ) cells in Npm1c/Flt3-ITD mice, indicating that any maturation block was incomplete (Figure 2b). The relative numbers of circulating B (B220 þ ) and T (CD3 þ ) lymphocytes were reduced (data not shown). To assay their self-renewal potential, bone marrow cells from Npm1c (n ¼ 4), Flt3-ITD (n ¼ 4), WT (n ¼ 4) and Npm1c/Flt3-ITD (n ¼ 4) were studied in serial replating assays. Npm1c/Flt3-ITD cells gave rise to significantly more colonies at first and subsequent platings than any other genotype (Figure 2c), demonstrating a significantly increased selfrenewal potential.
AML is a molecularly and clinically heterogeneous disease and recent studies have revealed that this heterogeneity is derived, to a large extent, from the specific combinations of somatic driver mutations present in individual cases. Here, we show that the combination of Npm1c and Flt3-ITD, the two most commonly co-occurring AML mutations, is rapidly and universally leukemogenic in knock-in mice. These findings are particularly striking in light of the fact that, in isolation, both Npm1c 4 and Flt3-ITD 11 mutations have relatively subtle effects on mouse hemopoiesis and lead to leukemia or a myeloproliferative disorder only after prolonged latencies and in a minority of mice.
What is most remarkable about our findings is the very short latency of AML in Npm1c/Flt3-ITD mice, which suggests either: (i) that the two mutations are sufficient to promote AML in this strain of mice (C57BL6/N) or (ii) that additional mutations are acquired very rapidly in the pool of cells susceptible to leukemic transformation. The later possibility is supported by the fact that at least one type of somatic mutation, namely LOH for Flt3-ITD, was frequently observed in our mouse AMLs over this short time span. We recently reported that Npm1c can generate AML in collaboration with, amongst others, activating insertions of the GrOnc transposon in intron 9 of mouse Flt3. These insertions led to aberrant expression of a Flt3 messenger RNA predicted to code for an amino-terminal truncated version of Flt3 4 which, like Flt3-ITD, was thought to be constitutively active. Most of these murine AMLs harbored additional transposon insertions thought to be important in leukemogenesis. Thus, at this stage it appears more likely that additional mutations may be required for leukemogenesis in our Npm1c/Flt3-ITD mice, but this cannot be stated unequivocally.
In interesting contrast to our present work, a recent report demonstrated that the combination of Flt3-ITD with NUP98-HOXD13 in mice led to AML after a much longer latency (median 95 days), 14 despite the fact that, unlike Npm1c, NUP98-HOXD13 alone leads to a highly penetrant myelodysplastic syndrome with a high risk of leukemic transformation. This relative delay is particularly intriguing as NUP98-HOXD13 can promote leukemic transformation in association with simple overexpression of WT FLT3. 15 By contrast, in two large transposon-mediated insertional mutagenesis screens, one published 4 and one ongoing, we never observed transposon insertions leading to simple Flt3 overexpression amongst 4100 mouse Npm1c þ ve AMLs.
Notwithstanding the above, our observations emphasize the remarkable complementarity between Npm1c and Flt3-ITD. In the context of a stochastic model for AML pathogenesis, 2 this potent molecular synergy goes some way toward explaining why NPM1c and FLT3-ITD co-occur so frequently and make the model described here a valuable tool for the study of the pathogenesis and treatment of one of the largest molecularly defined subgroups of AML.

CONFLICT OF INTEREST
The authors declare no conflict of interest. Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are incurable B-cell malignancies usually responsive to initial immunochemotherapy, but virtually all patients experience relapse. Salvage therapy choices for relapsed/refractory disease are often limited by resultant cytopenias and acquired drug resistance. The current priority in these B-cell malignancies, therefore, is to develop agents with novel mechanisms of action that are selective for tumor cells, overcome shared patterns of acquired drug resistance and exhibit limited toxicities. Styrylbenzylsulfones are a new family of non-ATP-competitive anticancer agents that induce apoptosis in a variety of tumor cell lines, including those resistant to many chemotherapy agents. 1,2 As a class, styrylbenzylsulfones inhibit cell cycle progression and induce mitotic arrest of tumor cells with less toxicity to normal human cells. 3,4 ON 01910.Na (rigosertib) is a styryl sulfonyl compound that demonstrated inhibition of phosphatidylinositol-3-kinase (PI3K), preferentially targeting the PI3Ka and PI3Kb isoforms, and triggered apoptosis via the release of cytochrome c from mitochondria in MCL cell lines. 3 Rigosertib's mechanism of action was initially considered to include inhibition of polo-like 1 kinase, 4 but evidence for direct inhibition was not confirmed in subsequent studies 5 and its antimitotic activity may rely on the phosphorylation of mitosis coordinator RanGAP1 SUMO1. 6 First-in-man studies of rigosertib in solid tumors demonstrated excellent tolerability with limited hematologic toxicity. 7 Rigosertib has also demonstrated preclinical and early clinical activities in myelodysplastic syndromes (MDS), 8,9 and it is currently being tested in a randomized phase III trial in patients with relapsed/refractory MDS (NCT01241500).
We have previously reported that rigosertib induces rapid apoptosis in CLL cells with the relative sparing of normal B-cells and T-cells. 10 We demonstrated that the in vitro activity of rigosertib involved a dual mechanism of inhibition of PI3K pathway signaling coupled with the induction of an oxidative stress response. Importantly, activity of rigosertib was equally observed against Accepted article preview online 14 March 2013; advance online publication, 12 April 2013