Pten/Ship

Because of the influence that Ship (src homology 2- containing 5′ phosphoinositol phosphatase) and Pten (phosphatase and tensin homolog) have on the regulation of second messenger PIP3 and the fact that Ship-deficient mice develop a lethal myeloproliferative condition, Pten+/−Ship−/− mice were developed. Although hypothesized that these mice would develop leukemia, instead they displayed features consistent with aberrant hematopoiesis. Pten+/−Ship−/− mice survived only about 5 weeks and during that time, anemia, leukocytosis, and thrombocytopenia were observed. There was no evidence of leukemia; however, extramedullary hematopoiesis in the liver was found. Although increased numbers of neutrophils were present in the peripheral blood, decreases in myeloid progenitors were revealed following methocellulose culture of the BM. The BM from Pten+/−Ship−/− mice failed to reconstitute the peripheral blood of irradiated wild-type mice in a transplantation assay.

Evi1 Overexpression

Transgenic Evi1 mice were developed by Louz and colleagues by using the Sca-1 promoter for expression in hematopoietic stem cells. The expression of Evi1 in the hematopoietic tissues of these mice resulted in a gender-specific response in one of the transgenic lines whereby females remained healthy during an 18-month period in contrast to their male littermates who manifested reduced colony-forming unit erythroid formation. These male mice became severely ill because of impaired erythropoiesis and died within 7 to 11 weeks. Although these mice did not develop MDS or leukemia, the potential for Evi1 to collaborate in tumor formation was demonstrated by the ability of retrovirally infected transgenic Evi1 mice to develop myeloid leukemia more quickly than infected wild-type mice.

Expression of Evi1 in a BM transduction-transplantation model resulted in a progressive pancytopenia, however, which was inevitably fatal by 12 months. On closer examination, necropsy revealed splenomegaly in Evi1 -positive mice with an increase in erythroid precursors, iron deposition, and activated caspase-3 compared with spleens from control animals. Similar to clinical MDS, the BM displayed hyperplasia of erythroid cells and megakaryocytes. In contrast to MDS in humans, the Evi1 -induced MDS did not transform to leukemia in this study. Before the onset of overt disease, Evi1 -positive mice at 4 months had low levels of EpoR and c-Mpl in BM cells, which correlates with the defect in erythropoiesis and platelet generation observed in moribund mice months later. Follow-up studies observed that binding of Evi1 to Gata1 resulted in impaired expression of the EpoR , whereas Evi1 association with PU.1 altered myelopoiesis.

Npm1 Deletion

Npm1 was of interest for study because of its frequent mutation in AML patients (35%), location on chromosome 5q, and ability to regulate tumor suppressor genes One allele of Npm1 was inactivated in mouse embryonic stem cells by homologous recombination that produced a deletion of exons 2 through 7. After generation of chimeric mice and germline transmission, the investigators noted that Npm1 −/− ( Npm1 knockout) mice were not viable. The Npm1 −/− mice died between embryonic day E11.5 and E16.5 because of severe anemia and aberrant organogenesis. Npm1 +/− mice were viable and demonstrated some features that were consistent with MDS. Most mice aged 6 to 18 months presented at least one blood abnormality commonly associated with MDS. Npm1 +/− mice often demonstrated an elevated mean corpuscular volume and had a wider range of platelet counts, both increased and decreased, than that of wild-type mice; however, the hemoglobin levels did not differ between groups. Approximately two thirds of the Npm1 +/− mice had morphologic evidence of erythroid or megakaryocytic dysplasia, including binucleated erythroblasts and hypolobated megakaryocytes. In a follow-up study, prolonged (up to 24 months) monitoring of Npm1 +/− mice revealed an increased incidence of malignancy compared with wild-type mice, including lung and liver tumors, myeloproliferative neoplasms, AML, B-cell lymphoma, and T-cell lymphoma.

Dido1 Deficiency

Dido1 is a gene that was initially identified in a screen for genes whose expression was up-regulated during the initial stages of apoptosis. Homozygous deletion of Dido1 in mice led to a hematopoietic disease manifested by a variable degree of mild anemia and a variable degree of granulocytosis, in combination with decreased hematopoietic colony formation in vitro. Dysplastic cells in the spleen and BM were reported, but not characterized in detail, and progression to acute leukemia was not noted. The disease was thought to be consistent with a diagnosis of myelodysplastic syndrome/myeloproliferative disease (MDS-MPD) and it was transplantable to recipient mice, indicating that it was cell-autonomous.

NUP98-HOXD13

Over 20 chromosomal translocations involving the NUP98 gene associated with hematopoietic malignancy have been identified. Pineault and colleagues described a NUP98-HOXD13 mouse model in which primary murine BM cells were transduced with a retrovirus carrying a NUP98-HOXD13 fusion gene, which was initially identified in a patient with MDS. These cells were subsequently transplanted into irradiated recipient mice. Significant decreases in white blood cells were observed in mice expressing the NUP98-HOXD13 fusion, which was further characterized by a reduction in B220+ and CD4+/CD8+ lymphocytes. In contrast, significant increases in myeloid BM cells ( Gr1+/Mac1+ ) were identified. Results from colony-forming unit spleen assays with NUP98-HOXD13 –transduced BM cells indicated a decrease in erythroid precursors (identified by the Ter119 cell surface marker), an increase in myeloid Gr1+ cells, and an overall increase in the number of colony-forming unit spleen colonies at day 12. Although some of these mice developed a myeloproliferative disease as early as 4 weeks following transplantation, they did not transition to AML except when mice were engineered to simultaneously express Meis1.

In comparison, NUP98-HOXD13 ( NHD13 ) transgenic mice that used Vav 1 regulatory elements to direct expression of the NHD13 transgene in hematopoietic tissues developed anemia, neutropenia, and lymphopenia at 4 to 7 months. These mice also had a variable degree of thrombocytopenia and macrocytosis; however, these differences were not statistically significant. Despite the peripheral blood cytopenias, these mice had hypercellular or normocellular BMs, indicating ineffective hematopoiesis. Morphologically, the NHD13 mice showed dysplasia in the form of binucleate erythroblasts, megaloblastosis, pseudo Pelger-Huët cells, and micromegakaroyctes. Similar to the progression pattern observed in patients with MDS, approximately half of the NHD13 mice with MDS developed an acute leukemia, typically at 10 to 14 months of age. A variety of leukemic phenotypes were identified, most commonly AML. Some animals developed pre–T-lymphoblastic leukemia-lymphoma (pre–T-LBL), however, which is rarely seen in secondary leukemia that evolves from patients with MDS. Although the development of pre–T-LBL was somewhat surprising, it was not completely unexpected given that NUP98 fusion genes have been associated with both T-cell and myeloid malignancies. Of note, although the initial studies of the NHD13 fusion used FVB/N mice, the entire study was subsequently repeated with C57Bl/6 mice. The results were remarkably similar in terms of peripheral blood cytopenias, dysplastic morphology, and progression to acute leukemia, demonstrating that the effect of the transgene was not mouse strain-dependent. There are both similarities and differences between the transgenic and retroviral transduction-transplantation models. Both lead to anemia, lymphopenia, and neutropenia initially; however, the retrovirally transduced model quickly transforms to a myeloproliferative disease, whereas the transgenic mice transform more slowly, usually to an AML. The differences between the transgenic and retroviral transduction models could be caused by differences in mouse strain, ex vivo manipulation of cells, effects from BM reconstitution, or differential susceptibility of the BM stem and progenitor cells by the NUP98-HOXD13 retrovirus.

SALL4 Overexpression

SALL4B is a zinc finger transcription factor expressed early during embryonic development, and is involved in the transcriptional regulation of Oct4 and Nanog . SALL4B was constitutively expressed in mouse tissues under the control of the cytomegalovirus promoter. These mice developed some classic features of MDS within a few months of life, including peripheral blood leukopenia and dysplasia, and a mild anemia. BM from these mice was hypercellular and showed an increased number of Mac1+/Gr1+ cells, and increased apoptosis when compared with wild-type counterparts. Half of the transgenic mice transformed to AML by an average of 14.5 months of age and the disease was transplantable. In addition to the hematopoietic abnormalities, most of the transgenic mice were also diagnosed with polycystic kidneys and a minority was identified as having cerebral spinal fluid accumulation in the brain that affected motor function.

BCL-2 and NRAS

To study the process of MDS transformation to AML, overexpression of an inducible BCL-2 transgene along with simultaneous expression of an NRAS mutant ( NrasD12 ) were selected for examination in a mouse model. In collaboration with the increased proliferative effects attributed to mutant NRAS , it was predicted that a complementary mutation, induced by overexpression of antiapoptotic BCL-2 , would induce MDS or AML. The authors found that mice that expressed BCL-2 and mutant NRAS developed a mild leukocytosis and thrombocytopenia, without anemia. The mice also displayed hepatosplenomegaly, dysplastic neutrophils, and increased BM blasts (mean of 15%). Serial transplantations of the lin ^neg but not the lin ^pos cells from mice with MDS into immunodeficient mice were successful.

RUNX1 Mutations

Watanabe-Okochi and colleagues infected mouse BM with retroviral constructs that encoded one of two different RUNX1 (also know as AML1 ) gene mutations that were initially identified in patients with MDS or AML. The D171N mutation is a missense mutation involving the runt homology domain, and the AML1-S291fsX300 mutation is a frameshift mutation that results in truncation of the C-terminal region of the AML1 protein. Most mice that expressed the D171N RUNX1 mutation developed MDS with an increased red blood cell mean corpuscular volume, dysplasia, anemia, leukopenia, and progression to AML ( Table 1 ). Of note, a fraction of the RUNX1 -D171N mice had integrations of the retroviral vector used to express the RUNX1 mutant at the Evi1 locus, and transplantation of BM cells coinfected with RUNX1 -D171N and Evi1 reduced the latency of the disease to 3 to 5 months. This study provides support for the cooperation of RUNX1 mutants and Evi1 for inducing MDS with progression to AML.

Arid4a Deficiency

ARID4A and ARID4B (also known as “RBBP1” and “RBBP1L1”) are proteins that contain an AT-rich interaction domain (ARID). ARID4A, but not ARID4B, interacts with RB and recruits histone deacetylases to E2F-dependent promoters. Arid4a deficient ( Arid4a−/− ) mice were identified by a gene trap mutagenesis screen. Young (2–5 month old) Arid4a−/− mice developed lymphopenia, neutropenia, thrombocytopenia, and a mild anemia, with a normocellular BM. The peripheral blood cytopenias described previously became progressively more severe, with a marked increase in monocytes, as the mice aged. These changes were accompanied by BM fibrosis with increased reticulin fibers and hepatosplenomegaly secondary to a compensatory extramedullary hematopoiesis. These features are reminiscent of a chronic myelo-monocytic leukemia (CMML) that has evolved from a MDS/MPD. In 12% of the mice, the disease transformed to a frank AML, with high peripheral blood white blood cell, at ages between 12 and 22 months. BM from the Arid4a−/− mice had decreased levels of several Hoxb cluster genes ( Hoxb3 , 5 , 6 , and 8 ) that are normally expressed in BM, but levels of Hoxa7 , Hoxa9 , and Meis1 , which are up-regulated in some MDS cases in mice and patients (see previously), were normal. Although mice that had deleted both Arid4a and Arid4b were not viable ( Arid4a−/− Arid4b−/− ), Arid4a −/− Arid4b+/− developed AML with a higher frequency (83%) at an early age (7–15 months).

DNA Polymerase Gamma (Polg) Mutations

Although controversial because of the inexorable accumulation of mitochondrial DNA mutations with age, some studies have suggested that MDS patients have an increased frequency of mitochondrial DNA mutations. DNA polymerase gamma (Polg), a protein that is encoded by the nuclear genome, is responsible for replication of mitochondrial DNA. Polg has two enzymatic activities: a C-terminal polymerase and an N-terminal “proofreading” exonuclease. Mice with targeted instability of the mitochondrial genome were produced by selective elimination of the N-terminal proofreading activity through gene-targeted creation of a missense mutation ( Polg D257A); the mutant allele is designated Polg ^A . Homozygous mutant mice (designated Polg ^A/A ) show an increase in the frequency of mitochondrial DNA mutations and exhibit features of premature aging, including hair loss, thymic involution, testicular atrophy, and loss of intestinal crypts. In addition, the mice showed a progressive, fatal severe megaloblastic anemia and impaired lymphopoiesis, but there was no evidence for transformation to acute leukemia. Of note given the loss of intestinal crypts in the Polg ^A/A mice, the megaloblastic anemia was demonstrated to be caused by mitochondrial defects in the hematopoietic compartment, as opposed to malabsorption, by transplantation of Polg ^A/A cells in wild-type recipient mice.

Clinical Implications of Modeling MDS in Mice

Mouse models are developed to study, in detail, questions that cannot easily (or are impossible to) be addressed in the clinic. A useful animal model for MDS would accurately represent genetic aberrations in patients, provide similar microenvironmental conditions for reproducing hematopoietic phenotypes (eg, impaired differentiation and increased apoptosis), recapitulate patient symptoms and responses to existing treatment regimens, and progress to AML with similar kinetics and features as seen in the clinic. Although a model that satisfies all of these criteria may not exist, cancer biologists have several different models to choose from, and can select an available animal model, being cognizant of its caveats. Once an MDS model is developed and validated, it can be used to advance the understanding of the molecular biology of the disease and provide a platform for cultivation of new therapeutic approaches. This section focuses on mice with NUP98-HOX fusions, because there have been a number of follow-up studies performed with these mice beyond the initial description, providing examples for the types of additional studies that are possible with these animal models.

Although rare, many of the NUP98 translocations, including NUP98-TOP1 , NUP98-DDX10 , and NUP98-HOX fusions, have been seen in patients with MDS, especially younger patients. Furthermore, the NUP98-HOXA9 and NUP98-HOXD13 translocations both lead to overexpression of HOXA9 in mice; marked (10-fold–1000-fold greater than normal BM or CD34+ cells) overexpression of HOXA9 is a common finding in patients with AML and MDS. NHD13 mice develop a variety of leukemias including myeloid, erythroid, megakaryocytic, and undifferentiated; however, although unexpected, about 15% to 25% of NHD13 mice developed pre–T- or pre–B-LBLs. This is in contrast to reports of human MDS transformation to lymphoid leukemias, which are rare. Despite this difference between MDS patients and mouse models of the disease, 60% of NHD13 mice developed acute leukemias and 20% died because of severe MDS, statistics that are similar with patient progression.

Additional studies based on the NHD13 translocation have sought to describe the implications of this mutation and to understand its mechanistic role in MDS. The NHD13 protein can inhibit maturation of the K562 cell line, suggesting that NHD13 expression plays a role in hematopoietic precursors by impairing their differentiation. To study the impaired differentiation attributed to the NHD13 protein, colony-forming cell assays were performed with BM from the NHD13 transgenic mice. Although the number of colonies formed by plating whole BM from wild-type and NHD13 mice were similar, 10-fold fewer total colonies were observed from the NHD13 lin ^neg BM population (which contains the stem and progenitor cells) than from the wild-type lin ^neg BM cells. Lin ^neg cells from NHD13 mouse BM also showed dramatic increases in apoptosis following treatment with hematopoietic cytokines that promote survival and differentiation of wild-type murine hematopoietic stem and progenitor cells. Moreover, most of the surviving NHD13 cells were undifferentiated lin ^neg cells. Taken together, these findings indicate that the NHD13 fusion impairs differentiation of hematopoietic stem and progenitor cells and predisposes these cells to apoptotic death; these findings are reminiscent of the maturation arrest and increased apoptosis seen in patients with MDS.

Subsequent studies in the lymphoid system of NHD13 mice revealed impaired B-lymphocyte differentiation at the pro-B to pre-B transition and impaired differentiation of T lymphocytes during the transition from double negative (DN) DN2 to DN3 thymocytes. Hoxa7 , Hoxa9 , and Hoxa10 were overexpressed in the thymus of NHD13 mice compared with that of wild-type mice, an association that has previously been observed with impaired thymocyte differentiation.

Additional studies have identified a long-term repopulating MDS-initiating cell by demonstration of successful engraftment of lin ^neg NHD13 -expressing BM cells into primary and secondary lethally irradiated wild-type recipients. Importantly, mice that received the NHD13 BM developed signs and symptoms of MDS, including peripheral blood cytopenias caused by ineffective hematopoiesis, morphologic dysplasia, and transformation to acute leukemia. These studies also demonstrated that, in a competitive repopulation assay, the NHD13 BM cells were able to out-compete normal wild-type BM cells, even when a 10-fold excess of wild-type BM was transplanted. Finally, limiting dilution experiments identified the frequency of the MDS-initiating cell to be similar to the frequency of hematopoietic stem cells from wild-type mice (approximately 1 per 20,000).

In addition to elucidating the role of NHD13 in MDS, its contribution to the development of leukemia is also of interest. In the context of the recently developed two-class mutation hypothesis for AML, the NUP98-HOX fusions can be classified as Type II, or differentiation-impairing, mutations. Infection of primary BM cells by replication-competent retroviral particles predisposes mice to develop leukemia through a process known as “retroviral insertional mutagenesis.” This happens because of integration of the retrovirus, with the retroviral LTR serving as a powerful promoter-enhancer, near to cellular proto-oncogenes. Activation of two or more collaborative proto-oncogenes leads to leukemia or lymphoma in the infected mice. When NHD13 mice were infected with the MOL4070LTR retrovirus, the time from MDS to leukemic transformation was shortened by 6 months, providing further support that a second event is needed in collaboration with the NHD13 fusion for progression to leukemia. Potential collaborators with NHD13 identified by retroviral insertional mutagenesis include Meis1 , Mn1 , Gata2 , miR29a , and miR29b1 .

To identify spontaneous mutations that might collaborate with NHD13 as the MDS converts to an AML, genes frequently mutated in human AML were resequenced in leukemic tissue from NHD13 mice that had developed AML. In NHD13 mice with AML, 32% of mice assessed were found to have a Nras or Kras mutation of codon 12. Interestingly, no codon 13 or 61 mutations were found, although a codon 61 Nras mutation was identified in a NHD13 mouse with a pre–T-LBL. One mouse was found to have a mutation in Cbl , specifically an exon 8 deletion. These results, along with the lack of mutations observed in Npm1 and Runx1 (both classified as Type II, or differentiation-blocking mutations), are again consistent with NHD13 being a Type II mutation. Given that KIT mutations are most commonly found in patients with core-binding factor translocations, it may not have been surprising that Kit mutations were not identified in the NHD13 AMLs, because the NHD13 fusion does not seem to affect the core-binding factor pathways. It was curious that no Flt3 mutations were identified, however, because Flt3 mutations are commonly associated with lesions (eg, fusions of the MLL gene) that up-regulate HOXA cluster genes.

In addition to verifying that mutations (eg, Runx1 point mutations or NUP-HOX gene fusions) found in MDS patients are important in the disease process, mouse models provide a platform that can be used for preclinical testing. In addition to testing small molecules, singly or in combination, these animal models can be used to study the mechanisms by which allogeneic hematopoietic stem cell transplant is curative for patients with MDS. Hematopoietic stem cell transplant is the only known definitive treatment for MDS patients, and is thought to be effective by a combination of myeloablative radiochemotherapy and adoptive immunotherapy leading to a graft-versus-tumor effect. Inbred strains of mice with complete or partial major histocompatibility complex mismatches afford the ability to test the relative contributions of these two effects. In addition, these animal models may also be useful for examining possible autoimmune contributions to MDS initiation as proposed when immunosuppressive therapy resulted in positive responses in certain patient subsets.