Abstract

Therapy-related acute myeloid leukemia and myelodysplastic syndrome (t-AML and MDS) are severe late complications of treatment with genotoxic chemotherapeutic agents. Children with neurofibromatosis type 1 (NF1) are predisposed to malignant myeloid disorders that are associated with inactivation of the NF1 tumor suppressor gene in the leukemic clone. Recent clinical data suggest that NF1 might be also associated with an increased risk of t-AML after treatment with alkyating agents. To test this hypothesis, we administered cyclophosphamide or etoposide to cohorts of wild-type and heterozygousNf1 knockout mice. Cyclophosphamide exposure cooperated strongly with heterozygous inactivation of Nf1 in myeloid leukemogenesis, while etoposide did not. Somatic loss of the normalNf1 allele correlated with clinical disease and was more common in 129/Sv mice than in 129/Sv × C57BL/6 animals. Leukemic cells showing loss of heterozygosity at Nf1 retained a structural allele on each chromosome 11 homolog. These studies establish a novel in vivo model of alkylator-induced myeloid malignancy that will facilitate mechanistic and translational studies.

IN 1977, ROWLEY ET AL1described a group of patients who developed a myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) as a second malignant neoplasm after treatment for another cancer. Two forms of therapy-related (t) myeloid malignancies are now recognized; these subtypes are associated with distinct clinical and biologic features and differ with respect to the prior anticancer treatment. Most patients with t-AML and t-MDS previously received chemotherapeutic agents that alkylate DNA. This subtype typically involves a latency of 3 to 7 years between genotoxin exposure and disease onset, a myelodysplastic prodrome, and frequent loss of chromosomes 5 and/or 7 (−5 and/or −7) or deletions involving the long arms of these chromosomes [del(5q)/del(7q)].1-4 The second subtype of t-AML develops after therapy with drugs that inhibit topoisomerase II. These cases are characterized by a shorter interval between cytotoxic therapy and clinical signs, overt leukemia at presentation, and balanced translocations that usually involve the MLL gene located on chromosome 11, band q23.5-8 The prognosis is poor for patients with t-AML or t-MDS. Importantly, as aggressive multi-agent regimens are used increasingly to treat many primary cancers, the incidence of therapy-related myeloid malignancies is expected to increase over the next few years.

Individuals with neurofibromatosis type 1 (NF1) are predisposed to specific benign and malignant neoplasms, which arise primarily in cells derived from the embryonic neural crest.9 In addition, children (but not adults) with NF1 show a 200 to 500-fold increase in the incidence of de novo malignant myeloid disorders, particularly juvenile myelomonocytic leukemia (JMML).10-12 TheNF1 gene encodes neurofibromin, a guanosine triphosphatase (GTPase) activating protein that accelerates the slow intrinsic rate of GTP hydrolysis on p21ras (Ras) proteins.13 Genetic and biochemical data strongly support the hypothesis that NF1 functions as a tumor suppressor gene in human and murine hematopoietic cells by negatively regulating Ras output.13-20 For example, approximately 10% of heterozygous Nf1 knockout mice (Nf1+/−) spontaneously develop myeloid leukemia beginning around age 15 months, with tumor cells exhibiting loss of the wild-type Nf1allele.16 

A few cases of t-AML associated with monosomy 7 were recently reported in children with NF1, all of whom received alkylating agents to treat primary cancers including anaplastic astrocytoma, glioblastoma, Wilms’ tumor, or acute lymphoblastic leukemia.21 However, because these patients were not ascertained in a systematic way, it is uncertain if the risk of therapy-related myeloid malignancies is increased over children without NF1 who received similar therapies. Moreover, loss of heterozygosity (LOH) at NF1 was not detected in the leukemic cells of children with NF1 who developed t-AML.21 If germline inactivation of NF1 cooperates with genotoxic agents that are used to treat human cancers in leukemogenesis, we reasoned that exposing Nf1+/− mice to these drugs might produce an in vivo model of therapy-induced myeloid disease. To test this hypothesis, we administered two chemotherapeutic agents that have been implicated in human t-AML and t-MDS toNf1 mice. Here we show that the alkylating agent cyclophosphamide, but not the toposiomerase II inhibitor etoposide, efficiently induces a myeloproliferative disorder (MPD) inNf1+/− mice, and we present correlative cytogenetic and molecular data.

MATERIALS AND METHODS

Animal care.

Mice were housed in the University of California, San Francisco (UCSF) Animal Care facility and were examined regularly by one of the investigators. Cyclophosphamide and etoposide were prepared by the UCSF pharmacy and were administered by one of the investigators. Mice were weighed at the beginning of the study and weekly thereafter to adjust the drug doses. Complete blood counts (CBCs) were performed on blood samples collected from tail veins in an automated cell counter. The accuracy of abnormal blood counts was verified by direct examination of stained smears. The study procedures were reviewed and approved by the UCSF Committee for Animal Research.

Treatment and monitoring.

We mated Nf+/− and wild-type (Nf+/+) mice and genotyped the offspring by Southern blot analysis of tail DNA. The initial group of mice were from the inbred 129/Sv strain in which theNf1 mutation was created.16 To perform LOH analysis at loci other than Nf1 in alkylator-treated mice, F1 offspring of a cross between the 129/Sv and C57BL/6 strains were used in the latter part of the study. Nf1+/− and Nf1+/+ littermates were assigned to observation (control group) or to receive treatment with either etoposide or cyclophosphamide beginning at 6 to 10 weeks of age. These agents were selected because they are used widely in human cancer therapy. Treated mice received a single 6-week course of 100 mg/kg/wk of either agent, a schedule which approaches the maximally tolerated doses. Cyclophosphamide (CY) was administered by intraperitoneal injection whereas etoposide was administered through an orogastric tube.

CBCs with white blood cell (WBC) differentials were performed every 3 months in mice that appeared well, and whenever a mouse showed signs of systemic illness. The CBC was repeated immediately whenever the WBC count was >20,000/μL. All mice that appeared moribund and animals with WBC counts >20,000/μL on two consecutive determinations were killed, the spleens were weighed, and hematopoietic tissues were collected for morphologic and genetic analysis.

Nf1 genotyping and LOH analysis.

Genomic DNA was prepared from tail clippings or from hematopoietic tissues (spleen or bone marrow) by standard procedures.22,Nf1 genotypes and loss of heterozygosity were determined by digesting DNA samples with NcoI + HindIII followed by gel electrophoresis, blotting to nylon membranes, and hybridization with an NcoI-PstI fragment from intron 31 ofNf1 as described previously.16 LOH was scored by comparing the relative intensities of restriction fragments derived from paired normal and leukemic tissues.

LOH analysis with microsatellite markers.

These procedures have been described in detail.14 Briefly, DNA samples were amplified in a DNA Thermocycle Machine (Perkin Elmer Cetus, Norwalk, CT). Polymerase chain reaction (PCR) was performed in reaction mixtures that include 0.66 μmol/L of respective 3′ and 5′ primers, 100 ng of target genomic DNA, 1 U of Taq polymerase (AmpliTaq; PE Applied Biosystems, Foster City, CA), and 0.4 μmol/L final concentrations of deoxynucleotides in a final reaction volume of 25 μL. The forward primer was kinase-labeled with γ-33P adenosine triphosphate (ATP). Labeled PCR products were separated on (6 mol/L urea, 8% polyacylamide) sequencing-type gels and run at 60 to 80 W constant power for 2 to 4 hours. The gels were dried, placed in Saran wrap (Dow Brands L.P., Indianapolis, IN), and exposed to x-ray film at −70°C. The polymorphic markers tested included D18Mit55, D18Mit13, and D13Mit13, which are syntenic to human 5q31 and D6Mit48, D5Mit40, and D12Mit64, which are syntenic to genes within human 7q22-31.

Cytogenetic analysis and fluorescence in situ hybridization (FISH).

A trypsin-Giemsa banding technique was used to analyze cells from bone marrow and spleen. Metaphase cells from short-term (24 to 72 hours) unstimulated cultures were examined. Ten metaphase cells were examined each from the bone marrow and spleen cultures for each mouse. Chromosomes were identified using the standardized mouse karyotype as described by Cowell.23 FISH was performed as described previously.24 Briefly, a biotin-labeled Nf1 probe was prepared by nick-translation using Bio-16-dUTP (Enzo Diagnostics). Hybridization was detected with fluorescein-conjugated avidin (Vector Laboratories), and chromosomes were identified by staining with 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI). Images were obtained using a Zeiss Axiophot microscope coupled to a cooled charge coupled device (CCD) camera. Separate images of DAPI-stained chromosomes and the hybridization signal were merged using image analysis software (NU200, Photometrics Inc, Phoenix, AZ and NIH Image 1.57, National Institutes of Health, Bethesda, MD). TheNf1 probe used for FISH was a 10.6 kb genomic lambda clone containing exon 31 and flanking intron sequences.

RESULTS

Leukemia in Nf1+/+ and Nf1+/− mice.

Myeloid disorders developed in 4 of 101 Nf1+/+ mice, 2 of which received CY (Table 1). In contrast, myeloid malignancies were diagnosed in 14% of the untreated Nf1+/− mice (8 of 58), in 25% of the etoposide-treated animals (8 of 32), and in 38% (14 of 37) of the mice assigned to the CY group (Table 1). Kaplan-Meier plots comparing disease incidence over time inNf1+/+ and Nf1+/− mice that received no treatment, etoposide, or CY are shown in Fig 1.Nf1+/− mice that received either drug had a significantly higher rate of disease than wild-type animals treated in parallel (Fig1). Treated and untreated Nf1+/− mice were also compared to ascertain the relative contributions of Nf1 genotype and chemotherapy exposure to leukemia susceptibility. This analysis showed that the incidence of disease was significantly higher, and the latency period shorter, in the Nf1+/− mice that received CY (0.004v untreated Nf1+/− mice by pairwise logrank statistics), but not in the etoposide group (P = .2 vthe untreated group). The in vivo leukemogenic effect of CY was restricted to Nf1+/− mice as Nf1+/+ animals in the control and CY-treated groups had similar rates of leukemia (Table 1). The incidence of leukemia was higher in Nf1+/− mice from the inbred 129/Sv background than in 129/Sv × C57BL/6 animals (Table 1), although these differences did not achieve statistical significance.

Table 1.

Incidence of Leukemia in Nf1 Mice

Genotype and Treatment Genotype No. of Mice No. (%) with Leukemia
129/Sv  
 None  Nf1+/+  31 2 (6%)  
 Nf1+/−  46  8 (17%) 
 Etoposide  Nf1+/+  26  0 (0%) 
 Nf1+/−  32  8 (25%)  
 CY Nf1+/+  5  0 (0%) 
 Nf1+/−  12  7 (58%) 
129/Sv × C57BL/6  
 None  Nf1+/+  14 0 (0%)  
 Nf1+/−  12  0 (0%)  
 CY Nf1+/+  25  2 (8%)  
 Nf1+/−  25 7 (28%) 
Genotype and Treatment Genotype No. of Mice No. (%) with Leukemia
129/Sv  
 None  Nf1+/+  31 2 (6%)  
 Nf1+/−  46  8 (17%) 
 Etoposide  Nf1+/+  26  0 (0%) 
 Nf1+/−  32  8 (25%)  
 CY Nf1+/+  5  0 (0%) 
 Nf1+/−  12  7 (58%) 
129/Sv × C57BL/6  
 None  Nf1+/+  14 0 (0%)  
 Nf1+/−  12  0 (0%)  
 CY Nf1+/+  25  2 (8%)  
 Nf1+/−  25 7 (28%) 
Fig. 1.

Kaplan-Meier plots showing the proportion ofNf1+/+ and Nf1+/− mice surviving without leukemia. Nf1+/+ mice are shown as an unbroken line andNf1+/− mice as a dotted line. (A) Data from untreated mice; (B) data from the etoposide group; (C) data from the CY group.

Fig. 1.

Kaplan-Meier plots showing the proportion ofNf1+/+ and Nf1+/− mice surviving without leukemia. Nf1+/+ mice are shown as an unbroken line andNf1+/− mice as a dotted line. (A) Data from untreated mice; (B) data from the etoposide group; (C) data from the CY group.

A myeloproliferative phenotype was observed in most diseased mice that was similar in control and chemotherapy-treated animals. This MPD was characterized by elevated peripheral blood leukocyte counts with a high percentage of mature neutrophils and monocytes (Fig2). The mean WBC count was 31,000/μL (range, 20,000 to 98,000), and the mean myeloid cell count was 28,000/μL (range, 14,000 to 88,000). Blood smears showed a variable degree of myeloid differentiation with some containing greater than 80% mature neutrophils and others showing 30% to 40% monocytes and monocytoid cells. Some smears showed rare blasts. Platelet counts and hemoglobin values were normal in mice with MPD and immature erythroid lineage cells were not seen in the peripheral blood. There was no consistent relationship between treatment group, WBC count, and the degree of myeloid maturation visible on blood smears. The bone marrows of mice with MPD showed an overwhelming predominance of myeloid cells with a shift toward immature elements, and sections of the spleen showed expansion of red pulp with infiltration of myeloid cells at various stages of differentiation admixed with areas of erythropoiesis (Fig 2). This MPD is similar to the JMML-like disorder that arises after adoptive transfer of Nf1−/− fetal liver cells into irradiated recipient mice.17 A disease phenotype more consistent with acute leukemia was seen in one CY-treatedNf1+/− mouse and in one mouse that received etoposide. Both animals had WBC counts >150,000/μL with large numbers of blasts and few mature neutrophils in the peripheral blood. The CY-treated mouse also had anemia (hemoglobin level, 5.7 g/dL) and thrombocytopenia.

Fig. 2.

Tissue sections from CY-treated Nf1+/− mice with MPD. (A and B) Blood and bone marrow smears showing immature and well differentiated myeloid cells. (C) A cytocentrifuge preparation of spleen cells stained with the myeloid lineage marker nonspecific esterase demonstrates many positive cells (brown stain). (D) A spleen section shows a dense infiltrate of myeloid cells within the red pulp.

Fig. 2.

Tissue sections from CY-treated Nf1+/− mice with MPD. (A and B) Blood and bone marrow smears showing immature and well differentiated myeloid cells. (C) A cytocentrifuge preparation of spleen cells stained with the myeloid lineage marker nonspecific esterase demonstrates many positive cells (brown stain). (D) A spleen section shows a dense infiltrate of myeloid cells within the red pulp.

Laboratory investigation of murine leukemias.

LOH at Nf1 correlated with clinical evidence of leukemia inNf1+/− mice (Table 2) and this invariably involved loss of the wild-type Nf1 allele (Fig3). Within the CY-treated group, leukemic cells from 129/Sv × C57BL/6 mice showed a much lower incidence of LOH than cells from 129/Sv animals (Table 2). Both animals with evidence of acute leukemia had LOH in hematopoietic tissues. In mice with MPD, LOH was not consistently associated with higher leukocyte counts or with increased numbers of immature myeloid cells. Unexpectedly, we detected LOH at sacrifice in the hematopoietic tissues of 18% of mice that did not fulfill the criteria used to diagnose leukemia. Most of these animals appeared well and WBC counts <10,000/μL and absence of prominent myeloid infiltrates in splenic sections. These results implicate inactivation of Nf1 as an early event that confers an in vivo proliferative advantage upon a clone of cells, but also suggest that additional mutations are required to produce the characteristic MPD. LOH in the absence of leukemia was much more common in 129/Sv mice than in 129/Sv × C57BL/6 animals (Table 2). Among mice without leukemia, LOH was relatively common in control animals but infrequent in the etoposide-treated cohort (Table 2).

Table 2.

Loss of Heterozygosity in Nf1+/− Mice

Genotype and Treatment No. of Mice No. With LOH/ No. With Leukemia No. With LOH/ No. Without Leukemia
129/Sv 
 None  46  5/8 (62%)  10/38 (26%)  
 Etoposide 32  7/8 (87%)  1/24 (4%)  
 CY  12  5/7 (71%) 4/5 (80%)  
129/Sv × C57BL/6  
 None  12  0/0 1/12 (8%)  
 CY  25  2/7 (28%)  1/18 (5%)  
All mice   19/30 (63%)  17/97 (17%) 
Genotype and Treatment No. of Mice No. With LOH/ No. With Leukemia No. With LOH/ No. Without Leukemia
129/Sv 
 None  46  5/8 (62%)  10/38 (26%)  
 Etoposide 32  7/8 (87%)  1/24 (4%)  
 CY  12  5/7 (71%) 4/5 (80%)  
129/Sv × C57BL/6  
 None  12  0/0 1/12 (8%)  
 CY  25  2/7 (28%)  1/18 (5%)  
All mice   19/30 (63%)  17/97 (17%) 
Fig. 3.

Southern blot analysis of tissues from Nf1+/− mice with MPD. The 3.2-kb restriction fragment corresponds to the targeted Nf1 allele, and the 2.4-kb band is derived from the wild-type allele. DNA extracted from the bone marrows or spleens of five mice with leukemia (L) show absence or a marked reduction in the wild-type Nf1 allele compared to paired tail (T) DNA specimens.

Fig. 3.

Southern blot analysis of tissues from Nf1+/− mice with MPD. The 3.2-kb restriction fragment corresponds to the targeted Nf1 allele, and the 2.4-kb band is derived from the wild-type allele. DNA extracted from the bone marrows or spleens of five mice with leukemia (L) show absence or a marked reduction in the wild-type Nf1 allele compared to paired tail (T) DNA specimens.

Cytogenetic analysis of bone marrow and spleen cells from 6 mice with MPD (5 CY-treated mice and 1 from the etoposide group) revealed a normal karyotype (Fig 4A). To ascertain if LOH on Southern blots was associated with submicroscopic deletions ofNf1 or with duplication of the mutant allele, we used a genomicNf1 probe from the disrupted segment of the gene to perform FISH analysis of hematopoietic cells from 3 of these 6 mice. FISH showed 2 structural copies of the Nf1 gene in each case (Fig4B). We also used six polymorphic microsatellite markers to examine bone marrow DNA from mice with t-ML for LOH at loci syntenic to regions of human chromosomes 5 and 7 that are frequently deleted in humans with t-MDS and t-AML, but found none (data not shown). Similarly, Southern blot analysis of specimens from etoposide-treated mice did not show rearrangements of Mll when hybridized with a probe from the human MLL breakpoint cluster region that detects virtually all of the breakpoints in human leukemias (data not shown).

Fig. 4.

(A) Cytogenetic analysis of spleen cells from a CY-treated mouse with LOH at Nf1 shows a normal diploid karyotype. (B) FISH of the same specimen shown in (A) with a genomicNf1 probe shows 1 copy of the gene on each chromosome 11 homolog.

Fig. 4.

(A) Cytogenetic analysis of spleen cells from a CY-treated mouse with LOH at Nf1 shows a normal diploid karyotype. (B) FISH of the same specimen shown in (A) with a genomicNf1 probe shows 1 copy of the gene on each chromosome 11 homolog.

DISCUSSION

This study establishes an in vivo model of therapy-induced myeloid malgnancies in Nf1+/− mice that will facilitate basic and translational research studies of this important clinical disorder. In human t-MDS/t-AML, frequent deletions involving chromosomes 5 and 7 have implicated loss of gene function in genotoxin-induced leukemogenesis. How alkylating agents actually cause leukemia is unknown; however, CY increases the frequency of somatic inactivation of target genes in a variety of assays.25 In contrast, leukemias that arise after exposure to topoisomerase II inhibitors are associated with recurring chromosomal translocations involving theMLL gene that result in the production of dominantly acting chimeric proteins. If the leukemias that develop after treatment with alkylating agents predominately involves the inactivation of specific target genes, it is possible that some human patients who develop t-ML after alkylator-based chemotherapeutic regimens are highly susceptible because of germline mutations of undiscovered tumor-suppressor genes that, like NF1, restrain the growth of immature myeloid cells.

We used clinical criteria to diagnose leukemia because the bone marrows of some children with NF1 who develop malignant myeloid disorders do not show LOH at NF1.14,19,21 In this study, mice with clinical evidence of MPD or AML had a threefold higher rate of LOH at Nf1 than mice without these findings. The presence of somatic LOH in hematopoietic tissues supports the clonal nature of these myeloid disorders. MPDs with and without LOH had similar features to the myeloid disorder that emerges after adoptive transfer ofNf1−/− fetal liver cells into irradiated recipient mice.17 Together with the extraordinary increase in the incidence of leukemia in Nf1+/− versus Nf1+/+ animals, these observations provide evidence that inactivation ofNf1 is a central event in leukemogenesis even in the absence of LOH. If this is true, it is likely the wild-type Nf1 allele is inactivated in bone marrows without LOH by subtle somatic mutations. An alternative consideration is that some Nf1+/− mice develop myeloid malignancies through a genetic pathway that does not involve biallelic inactivation of Nf1, as has recently been shown for a subset of tumors from heterozygous p53 knockout mice.26 Experiments using techniques that can identify point mutations will be required to distinguish between these possibilities. Nf1 is a very large gene, and protein truncation has proven to be the most efficient method for detecting subtle mutations in normal and leukemic cells from NF1 patients.20,27,28 

Adoptive transfer of Nf1−/− fetal liver cells into irradiated mice consistently induces a MPD with features of JMML.17 Inasmuch as these data suggested that inactivation of Nf1 in early hematopoietic cells might be both necessary and sufficient to induce clinical disease, we were surprised to detect LOH at Nf1 in hematopoietic tissues from 18% of Nf1+/− mice with normal WBC counts. This idea that genetic alterations in addition to inactivation of Nf1 are required for clinical disease is consistent with the relatively long latency between CY exposure and the onset of t-ML in Nf1+/− mice (Fig 1). Cooperating somatic mutations such as bone marrow monosomy 7 and epigenetic events have also been identified in human NF1-associated myeloid disorders.21,29 It will be of interest to determine if LOH can be detected in circulating blood cells some months before the onset of leukocytosis and splenomegaly in Nf1+/− mice.

We did not inject bone marrow cells from Nf1+/− mice that acquired myeloid disorders associated with LOH into secondary hosts. In our hands, transferring marrow from recipients previously engrafted with Nf1−/− fetal liver cells consistently induces MPD in irradiated, but not in unirradiated, mice (data not shown). In an interesting experiment, Largaespada et al17 crossed a mutant Nf1 allele into the BXH2 line of mice in which a leukemogenic retrovirus is transmitted vertically from mother to pups. They observed preferential viral integration into the wild-typeNf1 allele, shortened latency, and a change in disease phenotype from MPD to AML.17 Their finding of other somatically acquired leukemia-specific viral integrations within these clones implicated alterations in addition to inactivation ofNf1 in progression from MPD to AML. Adoptive transfer into secondary recipients provides a way of further characterizing therapy-induced myeloid disorders arising in Nf1 mice and may be especially informative in rare cases that show features of acute leukemia.

LOH at Nf1 and clinical leukemia were more common in homozygous 129/Sv mice than in 129/Sv × C57BL/6 animals. This was true in both control and CY-treated mice. Thus, 129/Sv hematopoietic cells are unexpectedly prone to spontaneously undergo LOH at Nf1 followed by clonal expansion. Rates of cancer in the F1 progeny of crosses between two inbred mouse strains often correlate poorly with parental rates and may be higher, lower, or unchanged.30 The net effect of our having assigned disproportionate numbers of 129/Sv × C57BL/6 mice to the CY group is to understate the magnitude of the leukemogenic effect of this agent. CY-treated mice showed a higher incidence of clinical leukemia than the control group irrespective of genotype (58% v 17% in strain 129/Sv and 28% v 0% in strain 129/Sv × C57BL/6; Table 1).

LOH was less frequent in CY-treated 129/Sv × C57BL/6 mice with MPD than in any of the 129/Sv cohorts. This low incidence suggests that the mechanism of Nf1 inactivation in 129/Sv × C57BL/6 hematopoietic cells involves subtle alkylator-induced mutations rather than loss of the wild-type allele. Consistent with this, Shoemaker et al31 recently identified somatic Apc point mutations caused by transitions or transversions in intestinal tumors from multiple intestinal neoplasia (Min) mice that had been exposed to the alkylating agent N-ethyl-N-nitrosourea (ENU). Interestingly, other tumors from this ENU-exposed cohort showed LOH at Apc. Taken together with our data from CY-treated mice, these data suggest that mechanisms of alkylator-induced tumor suppressor gene inactivation in colonic and hematopoietic cells include somatic rearrangements that result in LOH as well as subtle intragenic events.

Intestinal tumors that spontaneously arise in Min mice show LOH atApc with apparent loss of an entire chromosome 18 homolog.32 However, in a line of mice that carried mutations of the Apc and Dpc4 tumor suppressor genes incis, intestinal tumorigenesis was associated with apparent loss of one entire chromosome homolog with duplication of the mutant chromosome.33 Consistent with this, FISH analysis of murine leukemias with LOH showed an Nf1 allele on each chromosome 11 homolog. Although deletion of the chromosome containing the normal tumor suppressor gene allele followed by duplication of the mutant homolog has been proposed as a likely underlying mechanism,33 other models are also plausible. Mitotic nondisjunction resulting in two copies of the mutant homolog might occur first, with subsequent loss of the normal chromosome. Alternatively, the DNA segment that contains the normal allele might be replaced by a homologous segment from the mutant chromosome by a double mitotic recombination event, as has been reported in a human NF1-associated leukemia.34 

Haran-Ghera et al35 previously observed a weak leukemogenic effect of multiple doses of CY when this agent was administered with radiation and dexamethasone to SJL/J mice, a strain that is susceptible to radiation-induced AML. However, CY did not induce leukemia in the absence of radiation, and only cooperated with radiation when it was combined with dexamethasone.35 In contrast, we have developed a murine model of t-ML based on clinical observations in NF1 patients21 in which CY alone efficiently induces myeloid leukemia in Nf1+/− mice.

Our data provide direct experimental evidence that exposure to a commonly used cancer chemotherapeutic agent can cooperate with a genetic predisposition in the development of myeloid malignancies. Although human patients with t-MDS/t-AML frequently show peripheral blood cytopenias when they seek medical attention, their bone marrows are hypercellular and the disease typically evolves into a frankly proliferative phase with time. Similarly, Nf1+/− mice only exhibit overproliferation of myeloid cells months after exposure to CY. As in humans, LOH in murine hematopoietic cells is associated with a copy of the mutant Nf1 allele on each chromosomal homolog. The relevance of this model to human leukemia is further suggested by the presence of genetic alterations that deregulate Ras signaling in many human myeloid leukemias,36,37 and by the finding of activating RAS mutations in the bone marrows of some patients with t-AML.38,39 Although our data implicating mutations of genes in addition to Nf1 in murine leukemogenesis are also consistent with observations in human patients, we did not detect LOH with polymorphic markers from regions of the murine genome that are syntenic to human 5q31 and 7q22. There are a number of potential explanations for these findings, including: (1) the probes used might be some distance from the critical murine loci, (2) the relevant murine genes may be inactivated by somatic mutations which do not result in LOH, (3) loss of DNA sequences synteic to human 5q31 and 7q22 could be late events in progression of MPD to AML that had not occurred by the time of sacrifice, and/or (4) a different spectrum of cooperating genes might be mutated in human and murine leukemias. The nature of the alterations that are involved in alkylator-related leukemias awaits identification of additional target genes in both species.

This novel model provides a rigorous in vivo system to address a number of important (and in some cases controversial) issues in therapy-related myeloid disorders including the relative leukemogenic potential of different alkylating agents, the role of dose intensity, and the additive effects (if any) of alkylating agents and external beam radiotherapy. Furthermore, molecular analysis at Nf1 may elucidate the mechanistic basis of genetic damage induced by specific alkylating agents in immature hematopoietic cells. Nf1+/− knockout mice will also be useful for testing the utility of surrogate markers of gene mutation such as inactivation of Hprt to ascertain if exposure to specific mutagens portends an elevated risk of leukemia and to investigate chemopreventive strategies. Finally, these results have implications for the care of individuals with NF1 who develop neoplasms, because they suggest that alkylator-based regimens should be avoided whenever possible.

ACKNOWLEDGMENT

We thank Peter Houghton for suggesting the chemotherapy drug schedules used in this study, Rafael Espinosa III for assistance with FISH analysis, Anthony Fernald for assistance with cytogenetic studies, Nancy Zeleznik-Le and Janet Rowley for the MLL probe, Alexander Muller and Arnold Levine for information about polymorphic loci in 129/Sv × C57BL/6 hybrid mice, Sandra-Luna Fineman and Nancy Swigonski for assistance with statistical analyses, and Ben Yen and Ivy Hseih for preparing sections of murine tissues.

N.M. and B.R.T. contributed equally to this work.

Supported by American Cancer Society Research Grant No. DB-80030 and by National Institutes of Health (NIH) Grants No. R01 CA72614 and P01 CA40046. N.M. was supported by NIH Training Grant No. DK07636. T.J. is an Investigator of the Howard Hughes Medical Institute.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

REFERENCES

REFERENCES
1
Rowley
JD
Golomb
HM
Vardiman
J
Nonrandom chromosomal abnormalities in acute nonlymphocytic leukemia in patients treated for Hodgkin disease and non-Hodgkin lymphoma.
Blood
50
1977
759
2
Le Beau
M
Albain
KS
Larson
RA
Vardiman
J
Davis
E
Blough
R
Golomb
H
Rowley
J
Clinical and cytogenetic correlations in 63 patients with therapy-related myelodysplastic syndromes and acute nonlymphocytic leukemia: Further evidence for characteristic abnormalties of chromosomes 5 and 7.
J Clin Oncol
3
1986
325
3
Pedersen-Bjergaard
J
Phillip
P
Cytogenetic characteristics of therapy-related acute nonlymphocytic leukemia, preleukemia, and acute myeloproliferative syndrome: Correlation with clinical data in 61 consecutive cases.
Br J Hematol
66
1987
199
4
Pedersen-Bjergaard
J
Rowley
JD
The balanced and unbalanced chromosomal aberrations of acute myeloid leukemia may develop in different ways and may contribute differently to malignant transformation.
Blood
83
1994
2780
5
Ratain
MJ
Kaminer
LS
Bitran
JD
Larson
RA
Le Beau
MM
Skosey
C
Rowley
JD
Acute nonlymphoblastic leukemia following etoposide and cisplatin combination chemotherapy for advanced small cell carcinoma of the lung.
Blood
70
1987
1212
6
Levine
E
Bloomfeld
C
Leukemias and myelodysplastic syndromes secondary to drug, radiation, and environmental exposure.
Semin Oncol
19
1992
47
7
Pui
C-H
Behm
FG
Raimondi
SC
Dodge
RK
George
SL
Rivera
GK
Mirro
JJ
Kalwinsky
DK
Dahl
GV
Murphy
SB
Crist
WM
Williams
DL
Secondary acute myeloid leukemia in children treated for acute lymphoid leukemia.
N Engl J Med
321
1989
136
8
Smith
MA
Rubinstein
L
Ungerleider
RS
Therapy-related acute myeloid leukemia following treatment with epipodophyllotoxins: Estimating the risks.
Med Pediatr Oncol
23
1994
86
9
Riccardi
VM
Eichner
JE
Neurofibromatosis.
1986
Johns Hopkins University Press
Baltimore, MD
10
Bader
JL
Miller
RW
Neurofibromatosis and childhood leukemia.
J Pediatr
92
1978
925
11
Stiller
CA
Chessells
JM
Fitchett
M
Neurofibromatosis and childhood leukemia/lymphoma: A population-based UKCCSG study.
Br J Cancer
70
1994
969
12
Arico
M
Biondi
A
Pui
C-H
Juvenile myelomonocytic leukemia.
Blood
90
1997
479
13
Boguski
M
McCormick
F
Proteins regulating Ras and its relatives.
Nature
366
1993
643
14
Shannon
KM
O’Connell
P
Martin
GA
Paderanga
D
Olson
K
Dinndorf
P
McCormick
F
Loss of the normal NF1 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders.
N Engl J Med
330
1994
597
15
Kalra
R
Paderanga
D
Olson
K
Shannon
KM
Genetic analysis is consistent with the hypothesis that NF1 limits myeloid cell growth through p21ras.
Blood
84
1994
3435
16
Jacks
T
Shih
S
Schmitt
EM
Bronson
RT
Bernards
A
Weinberg
RA
Tumorigenic and developmental consequences of a targeted Nf1 mutation in the mouse.
Nature Genet
7
1994
353
17
Largaespada
DA
Brannan
CI
Jenkins
NA
Copeland
NG
Nf1 deficiency causes Ras-mediated granulocyte-macrophage colony stimulating factor hypersensitivity and chronic myeloid leukemia.
Nat Genet
12
1996
137
18
Bollag
G
Clapp
DW
Shih
S
Adler
F
Zhang
Y
Thompson
P
Lange
BJ
Freedman
MH
McCormick
F
Jacks
T
Shannon
K
Loss of NF1 results in activation of the Ras signaling pathway and leads to aberrant growth in murine and human hematopoietic cells.
Nat Genet
12
1996
144
19
Miles
DK
Freedman
MH
Stephens
K
Pallavicini
M
Sievers
E
Weaver
M
Grunberger
T
Thompson
P
Shannon
KM
Patterns of hematopoietic lineage involvement in children with neurofibromatosis, type 1, and malignant myeloid disorders.
Blood
88
1996
4314
20
Side
L
Taylor
B
Cayouette
M
Connor
E
Thompson
P
Luce
M
Shannon
K
Homozygous inactivation of the NF1 gene in bone marrow cells from children with neurofibromatosis type 1 and malignant myeloid disorders.
N Engl J Med
336
1997
1713
21
Maris
JM
Wiersma
SR
Mahgoub
N
Thompson
P
Geyer
RJ
Lange
BJ
Shannon
KM
Monosomy 7 myelodysplastic syndrome and other second malignant neoplasms in children with neurofibromatosis type 1.
Cancer
79
1997
1438
22
Shannon
KM
Turhan
AG
Chang
SSY
Bowcock
AM
Rogers
PCJ
Carroll
WL
Cowan
MJ
Glader
BE
Eaves
CJ
Eaves
AC
Kan
YW
Familial bone marrow monosomy 7: Evidence that the predisposing locus is not on the long arm of chromosome 7.
J Clin Invest
84
1989
984
23
Cowell
JK
A photographic representation of the variability in the G-banded structures of the chromosome in the mouse karyotype.
Chromosoma
89
1984
294
24
Rowley
JD
Diaz
MO
Espinosa
R
Patel
YD
van Melle
E
Ziemin
S
Le Beau
MM
Mapping chromosome band 11q23 in acute leukemia with biotinylated probes: Identification of 11q23 breakpoints within a yeast artifical chromosome.
Proc Natl Acad Sci USA
87
1990
9358
25
Anderson
D
Bishop
JB
Garner
RC
Ostrosky-Wegman
P
Selby
PB
Cyclophosphamide: Review of its mutagenicity for an assessment of potential germ cell risks.
Mutation Res
330
1995
115
26
Venkatachalam
S
Shi
YP
Jones
SN
Vogel
H
Bradley
A
Pinkel
D
Donehower
LA
Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.
EMBO J
17
1998
4657
27
Heim
R
Silverman
L
Farber
R
Kam-Morgan
L
Luce
M
Screening for truncated NF1 proteins.
Nat Genet
8
1994
218
28
Heim
R
Kam-Morgan
L
Binnie
C
Corns
D
Cayouette
M
Farber
R
Aylsworth
A
Silverman
L
Luce
M
Distribution of 13 truncating mutations in the neurofibromatosis 1 gene.
Human Mol Genet
4
1995
975
29
Shannon
KM
Watterson
J
Johnson
P
O’Connell
P
Lange
B
Shah
N
Kan
YW
Priest
JR
Monosomy 7 myeloproliferative disease in children with neurofibromatosis, type 1: Epidemiology and molecular analysis.
Blood
79
1992
1311
30
Smith
GS
Walford
RL
Mickey
MR
Lifespan and incidence of cancer in selected long-lived inbred mice and their F1 hybrids.
J Natl Cancer Inst
50
1973
1195
31
Shoemaker
AR
Luongo
C
Moser
AR
Marton
LJ
Dove
WF
Somatic mutational mechanisms involved in intestinal tumor formation in Min mice.
Cancer Res
57
1997
1999
32
Luongo
C
Moser
AR
Gledhill
S
Dove
WF
Loss of Apc+ in intestinal adenomas from Min mice.
Cancer Res
54
1994
5847
33
Takaku
T
Oshima
M
Miyoshi
M
Matsui
M
Seldin
MF
Taketo
M
Intestinal tumorigenesis in compound mutant mice of both Dpc4 and Apc genes.
Cell
92
1998
645
34
Stephens
K
Weaver
M
Leppig
K
Side
LE
Shannon
KM
Maruyama
K
Somatic confined uniparental disomy of the NF1 gene region in myeloid leuekmic cells of an NF1 patient mimics a loss of hetrozygosity due to deletion (abstract).
Am J Human Genet
59
1996
A5
(suppl)
35
Haran-Ghera
N
Peled
A
Krautghamer
R
Resnitzkt
P
Initiation and promotion of radiation-induced myeloid leukemia.
Leukemia
6
1992
689
36
Sawyers
CL
Denny
CT
Chronic myelomonocytic leukemia: tel-a-kinase what ets all about.
Cell
77
1994
171
37
Shannon
KM
The Ras signaling pathway and the molecular basis of myeloid leukemogenesis.
Curr Opin Hematol
3
1995
305
38
Pedersen-Bjergaard
J
Janssen
JWG
Lyons
J
Point mutations of the ras protooncogenes and chromosome aberrations in acute nonlymphocytic leukemia and preleukemia related to therapy with alkylating agents.
Cancer Res
48
1988
1812
39
Side
LE
Teel
K
Wang
P
Mahgoub
N
Larson
R
Le Beau
MM
Shannon
KM
Activating RAS mutations in therapy-related myeloid disorders associated with deletions of chromosomes 5 and 7 (abstract).
Blood
88
1996
566a
(abstr, suppl 1)

Author notes

Address reprint requests to Kevin M. Shannon, MD, Department of Pediatrics, University of California, Room HSE-302, Box 0519; San Francisco, CA 94143-0519; e-mail: kevins@itsa.ucsf.edu.