Myelodysplastic Syndrome Mice Research Articles

Mice with Myelodysplastic Syndrome Respond to Treatment with Azanucleosides: A Preclinical Model for MDS Therapies

AbstractAbstract 3829Therapies for the treatment of myelodysplastic syndromes (MDS) are limited. The only known curative therapy for MDS is allogeneic hematopoietic stem cell transplantation. MDS is frequently associated with epigenetic gene silencing via methylation of cytosine residues in gene regulatory regions. Clinical trials with DNA methyl-transferase inhibitors 5'azacytidine and 5-aza-2'-deoxycytidine (Decitabine, DAC) have shown hematologic responses and a survival benefit. Both azanucleosides have recently been approved for use in the clinic. However, responses are typically not durable, thus further characterization of response to treatment is required, as is the identification of new drugs to better treat MDS.Using bone marrow cells from NUP98-HOXD13 (NHD13) transgenic mice, which have previously been shown to faithfully recapitulate key features of MDS, we co-transplanted MDS and wild type (WT) bone marrow cells into WT irradiated recipient mice. The chimaeric bone marrow transplant (BMT) produces a recipient that mimics the human condition, with patient bone marrow comprised of hematopoietic cells derived from both the MDS clone as well as normal hematopoietic precursors. WT and MDS cells in the mice can be distinguished by differential CD45 alleles (CD45.1 and CD45.2, respectively), which enables analysis and purification of the MDS and normal cells; this feat is not easily achieved with human MDS patient samples, which lack cell surface antigens specific for the MDS clone. We treated mice with 0.018mg DAC (or saline) daily for 5 days, given every 4–5 weeks, to approximate MDS patient dosing schedules. Three trial groups were divided into saline (total n=19) and DAC (total n=24) treatment cohorts. Successful treatment of the chimeric MDS mice resulted in increasing levels of WT cells in the peripheral blood, with a concomitant reduction or eradication of the MDS cells. For instance, in trial #1, DAC treated chimaeric WT/MDS mice showed less severe anemia (2.0 g/dL higher Hgb), normalized neutrophil counts (57– 85% of mice vs 20–60%) and a significant survival benefit (median 27 vs 20 weeks, p=0.004) compared to saline treated mice. The response to treatment varied within individual mice and between trials, analogous to the variability in response seen in human MDS patients.We purified WT and MDS cells from treated mice and evaluated CpG island (CGI) DNA methylation in Chd13, a gene aberrantly methylated in MDS patients. Chd13 was hypermethylated in the murine MDS cells compared to WT cells (42–59% vs 10–16%, respectively). Of note, there was considerable variability between mice in the response to DAC treatment; half of the DAC treated mice showed a normalization of Chd13 methylation (14–20%) and half had a modest decrease in Chd13 methylation (38–40%). We next assayed global CGI methylation using a deep sequencing technique (DREAM, Digital Restriction Enzyme Analysis of Methylation). 8.2% of all CGI sites assayed were hypermethylated in MDS cells compared to WT; this increase in methylation was reduced to only 2.0% following DAC treatment of the MDS cells. This genome wide approach documented extensive hypermethylation in the NHD13 mice, similar to findings in a subset of MDS patients.Chimaeric WT/MDS mice demonstrated variable hematologic outcomes and cytosine demethylation in response to DAC treatment, as seen in human MDS patients. Successful treatment resulted in depletion of MDS cells allowing reconstitution of the bone marrow with WT cells as would happen in the successful treatment of patients. Durable responses were seen in several recipients, including one long-term survivor with no evidence of MDS cells in the bone marrow at 24 months of age (16 months post treatment). The chimaeric WT/MDS mice therefore represent a useful pre-clinical model of human MDS, which can be used to better characterize current treatments and test novel therapies. Disclosures:Issa:Johnson & Johnson: Consultancy; Astex: Consultancy. Aplan:NIH Office of Technology Transfer: Patents & Royalties.

Increased Mutation Frequency Induced by Oxidative Stress in the NUP98-HOXD13 MDS Mouse Model

Abstract 1705The myelodysplastic syndromes (MDS) comprise a group of hematologic disorders characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML) in a subset of cases. The causative agents for de novo or therapy-related MDS (t-MDS) include exposure to known genotoxins, such as anti-cancer chemotherapy, or ionizing radiation. It is known that MDS is associated with chromosomal aberrations including deletions, amplifications, inversions, and translocations. However, the molecular mechanisms that lead to progression of MDS to AML have not been completely elucidated. Recently, several studies have reported that oxidative stress contributes to disease progression of MDS. Reactive oxygen species (ROS) play a role in regulating several biologic phenomena including activation of signaling pathways in response to cytokines, and the gene expression pattern induced by this signaling. ROS is also known to induce oxidative DNA damage, which can lead to activation of proto-oncogenes or inactivation of tumor suppressor genes. A mouse model for MDS, generated by expression of a NUP98-HOXD13 (NHD13) fusion gene, was exploited to investigate ROS levels. Lineage negative (Linneg) bone marrow mononuclear cell (BMNC) from NHD13 mice with MDS had a 6.0 fold increased level of ROS compared with wild-type (WT) Linneg BMNC. In order to address potential mechanism(s) leading to this increased ROS, a Real-Time Quantitative PCR array, which profiles the expression of 84 genes related to oxidative stress, was utilized and the data demonstrated down regulation of Myeloperoxidase (Mpo; −9.9 fold), Eosinophil peroxidase (Epx; −8.8 fold) and Lactoperoxidase (Lpo; −5.2 fold) in the Linneg BMNC from NHD13 mice. The increased ROS level in NHD13 Linneg BMNC was associated with a 2 fold increase in G2/M phase cells along with increased expression of p16INK4A (43 fold) and p21 (10 fold), possibly to allow for DNA repair at cell cycle checkpoints. In order to investigate genomic DNA damage by ROS, immunocytochemistry for γH2AX foci was performed and demonstrated a 2 fold increase in γH2AX foci in the NHD13 BMNC compared to WT BMNC. To determine if mis-repair of the ROS-induced DNA damage led to mutations of genomic DNA in vivo, mutation frequencies (MF) were determined using the Big Blue® cII mutation detection assay. Big Blue mice are transgenic mice that have a λLIZ shuttle vector, which is derived from the coliphage lambda. Selection of mutation is based on the ability of the λ phage to multiply through either the lytic or lysogenic cycle in E. coli host cells. Big Blue mice were crossed to NHD13 mice, and NHD13/Big Blue® mice were compared with littermate controls that were positive for the BigBlue transgene but negative for the NHD13 transgene. BMNC from the NHD13/BigBlue mice showed a 1.8-fold increased MF compared to control BMNC (p value = 0.0371). Mutations were comfirmed by sequencing the cII target gene, and revealed that the NHD13/BigBlue mice had a 3-fold higher frequency of frameshift mutations compared to the control animals. These results suggest that the oxidative stress induced by the NHD13 fusion may contribute to disease progression of MDS to AML through DNA damage and mutation. Disclosures:No relevant conflicts of interest to declare.

DNA and All-Trans Retinoic Acid as Immunotherapy or Add-on Adjuvants to 5-Azacytidine In Myelodysplastic Syndromes

AbstractAbstract 2938 Background:Myelodysplastic syndromes (MDS) are clonal stem cell hematological disorders characterized by ineffective hematopoiesis leading to cytopenia, which evolve to acute myeloid leukemia (AML). The ability of the DNA vaccination to induce effective immune responses has been demonstrated in different preclinical models of diseases. Having previously shown that DNA vaccination with a PMLRARaFrC plasmid in combination with all-trans retinoic acid (ATRA) induced long term remissions with appropriate immune responses in a mouse model of acute promyelocytic leukemia (APL), our aim was to use this strategy in a mouse model of MDS we have created using transgenic mice bearing a mutant NRAS and overexpression of human BCL-2. Methods:We used our MDS-like triple transgenic mice bearing NRASD12/MMTVLTRtTA/TetoBCL-2 (Omidvar et al Cancer Res 67:11657-67, 2007). We cloned part of the kanamycin resistance gene and adjacent pVax1 plasmid sequences flipped so the antisense sequences were inserted into the pVax1 vector (named Flipper). These sequences code for 5 polypeptides. We treated the MDS mice with either DNA alone, ATRA alone (10mg daily release for 21 days) or a combination of ATRA+DNA or as add-on therapy with 5-azacytidine (5-aza). 5-aza (5mg/kg) was administered 3 times a week and continued until death. Treatment with ATRA and/or DNA was initiated after 12 courses of 5-aza injections. Results and Conclusions:Sequencing of the Flipper insert predicted 5 peptides, which were synthesized and shown not to affect growth in vitro of myeloid leukemic NB4, K562 and p39 cell lines. In vivo injections of healthy mice with these peptides had no effect on mortality. The inserted sequences did not appear to have detrimental effects. The studies on our MDS mouse model show that DNA vaccination, alone or in combination with ATRA induces a long-term survival of the treated mice compared to untreated controls (p<0,0001). DNA vaccination results in an increase of interferon-gamma secretion as measured by ELISPOT, increased CD4+/CD44hi/CD62-Llo memory T cells in the peripheral blood and maintains stable disease as determined by the persistence of low peripheral blood platelet counts and the of the Mac-1hi/GR-1lo populations, which mark the primitive cells and the ERK phosphorylation signatures measured by the NanoPro (Cell Biosciences). Memory T-cell levels increased 3-fold (at 6% compared to 2% in healthy controls) and remained high. The DNA vaccine stabilized the disease and its protective effect may persist for up to 10 months with increased expression of MYD88, which is downstream of toll-like receptors, in long-term survivors, suggesting the activation of this DNA pathway. ATRA alone also had efficacy in this model. As there was no evidence of differentiation as the Mac-1hi/Gr-1lo blast cell population remained unchanged after treatment compared to the levels before treatment, it is possible that in this setting where the leukemic stem cells are pro-apoptotic, that ATRA is acting as an immuno-adjuvant and the antigen shedding that occurs allows cross presentation and induces immune responses potentiated by ATRA. However, the disease in this group progresses and the mice die earlier than the other treatment groups (mean survival of 4.5 months compared with the untreated group of 1 month, p=0.0016). Vaccination with an adjuvant DNA alone or in combination with ATRA, results in a significant extension of lifespan in MDS mice. As add-on therapy to 5-aza the response rate increases from 20% with 5-aza alone to 50% with either ATRA or DNA and 100% with the 5-aza, ATRA and DNA combination. These studies suggest that adjuvant therapy with ATRA+DNA may have a role in addition to conventional therapy in prolonging remissions and may be promising for clinical trials. Disclosures:Fenaux:Novartis, Janssen, Cilag, Roche, Amgem, GSK, Merck and Cephalon: Honoraria, Research Funding. Chomienne:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Padua:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Increased Reactive Oxygen Species Induced by NUP98-HOXD13 Fusion Gene in MDS Mouse Model

The myelodysplastic syndromes (MDSs) comprise a group of pre-malignant hematologic disorders characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML). The causative agents for de novo or therapy-related MDS (t-MDS) include exposure to known genotoxins, such as benzene and other solvents, anti-cancer chemotherapy, and ionizing radiation. It is known that MDS is closely related to chromosomal aberrations including deletions, amplifications, inversions, and translocations, however, the molecular mechanism of disease progression from MDS to AML not been completely elucidated. Recently, several studies have reported that oxidative stress contributes to the disease progression mechanism in MDS. Reactive oxygen species (ROS) play a role in regulating several biologic phenomena involving activation of signaling pathways in response to cytokines, and the gene expression induced by this signaling. ROS is also known to induce oxidative DNA damage which generates DNA base modifications and DNA helix alterations. Therefore, increased intracellular levels of ROS can induce mutations which lead to transformation of MDS to AML. A mouse model for MDS, generated by expressing a NUP98-HOXD13 (NHD13) fusion gene was exploited to investigate ROS levels in this study. Lineage negative (Linneg) bone marrow mononuclear cell (BMNC) from NHD13 mice with MDS had a 7.8±5.6 fold increased level of ROS compared with wild-type (WT) Linneg BMNC, while ROS levels of unfractionated BMNC were similar in NHD13 and WT mice. The increase ROS level in NHD13 Linneg BMNC was associated with an increase in G2/M phase (2 fold). Interleukin 3 (IL-3) dependent NHD13 ‘knock-in’ cell lines also showed an increased level of ROS (3 to 5 fold), in comparison with other IL-3 dependent cell lines, BaF3 and 32D. To determine if the increase in ROS could be generated by an NHD13 fusion, BaF3 and 32D cell were transfected with an NHD13 expression vector. The BaF3 and 32D transfectant showed 2 fold (2.1±0.3 and 1.9±0.6 fold) increase ROS level compared to controls transfected with an empty vector. These findings suggest that expression of an NHD13 fusion gene can induce ROS in hematopoietic progenitor cells, which may contribute to the development of MDS and subsequent disease progression to AML through DNA damages.

A NUP98-HOXD13 Fusion Gene Induces a Transplantable Myelodysplastic Syndrome in Mice.

The myelodysplastic syndromes (MDSs) are a group of hematologic stem cell disorders characterized by ineffective hematopoiesis and dysplasia. A large number of chromosomal aberrations including deletions, amplifications, inversions, and translocations, some of which involve the NUP98 gene, have been associated with MDS. Recently an MDS mouse model expressing a NUP98-HOXD13 (NHD13) fusion gene was developed, which faithfully recapitulates all of the key features of MDS. Although it is well-established that acute myeloid leukemia (AML) is transplantable, there is no evidence that MDS is a transplantable condition. Therefore, in order to develop evidence for MDS as a hematopoietic stem cell (HSC) disease, we attempted to transfer MDS to normal recipients through bone marrow transplantation (BMT). All the recipients transplanted with bone marrow (BM) cells from NHD13 mice with MDS showed anemia, leukopenia, lymphopenia, and neutropenia when compared to recipients of wild-type (WT) littermates. The homing efficiency of the NHD13 primitive progenitor cells (Lineage negative [Lin−], Sca-1+) was about 2 fold higher than WT, and there was no significant difference in BM cellularity between the recipients of NHD13 and WT BM, indicating that the NHD13 recipients had ineffective hematopoiesis. These phenomena were reproduced in secondary recipients using primary recipients of NHD13 BM as donor mice. In secondary transplantation assays, 3 out of 5 recipients developed acute myeloid leukemia (AML) at 16 weeks post-transplantation. Morphological features of MDS, including nuclear-cytoplasmic asynchrony, binucleate cells, hypersegmented neutrophils, and giant platelets were detected in BM and peripheral blood of NHD13 donor, primary and secondary recipients by cytospin preparations. In competitive repopulation assays, mice transplanted with equal numbers of WT and NHD13 BM cells showed a decreased percentage of NHD13 cells in the peripheral blood, but an increased percentage of NHD13 cells in the BM, again providing evidence of ineffective hematopoiesis of the NHD13 cells. The transplantation of lineage depleted cells from BM has shown that the transplantable cells for MDS reside in the Lin− population of NHD13 BM. These findings demonstrate that MDS can be transferred to healthy recipients by BMT, supporting the concept that MDS originates in a transplantable multilineage hematopoietic stem cell.