One reason for the difficulty in developing effective treatments for myelodysplastic syndrome is that there are no myelodysplastic syndrome cell lines which can be used to model or study the disease. Although numerous investigators have attempted to develop xenograft models for myelodysplastic syndrome, these attempts have met with little success. Given that the NUP98-HOXD13 (NHD13) mice develop a highly penetrant myelodysplastic syndrome that closely resembles the human disease, we have begun studies to determine if these mice are a useful pre-clinical model for myelodysplastic syndrome. Our initial studies have used the DNA-methyltransferase inhibitor 5-azacytidine. Our pilot study included 3 groups of mice [NHD13 mice injected with 5-azacytidine (n=6), NHD13 mice injected with saline (n=4), and WT mice injected with 5-azacytidine (n=4)]. After 16 weeks of therapy, the results appeared promising, as the treated NHD13 mice showed a significant increase in hemoglobin compared to the saline treated NHD13 mice (2.21 +/- 1.47 g/dL vs 0.13 +/- 0.66 g/dL, p=0.02). Unfortunately, three of the NHD13 treated mice were found dead in the following two months, and we were not able to determine the cause of death for these mice. The three remaining mice had gradual decreases in hemoglobin over the next 12 weeks, and were close to baseline hemoglobin levels. Nonetheless the observation that these mice had stable hemoglobin levels after 28 weeks was encouraging. To determine whether we were achieving levels of 5-azacytidine adequate to cause cytosine demethylation, we examined global and gene-specific methylation status, in collaboration with Dr. J.P. Issa (M.D. Anderson);these experiments demonstrated hypermethylation in the NHD13 mice compared to controls, and partial reversal of the hypermethylation with 5-azacytidine treatment. Because the experiments discussed above used transgenic mice, effective treatment with 5-azacytidine could not replace the MDS bone marrow with completely normal (ie, wildtype or WT) bone marrow, since all of the bone marrow was transgenic. Therefore, in order to distinguish improvement in peripheral blood cytopenia due to differentiation of the MDS clone from elimination of the MDS clone, we have repeated the experiments using chimeric mice, that have both WT and NHD13 bone marrow. These repeat experiments have been performed using Decitabine (DAC), a related DNA methyltransferase inhibitor. Mice treated with DAC showed hematologic improvement and a survival advantage compared with saline-treated control mice;this experiment has now been repeated three times. We have sorted BM cells from treated mice into WT and NHD13 populations, and sent DNA from these samples to our collaborator (Dr. J.P. Issa), who has shown clear differences in global cytosine methylation between the NHD13 and WT samples, and partial reversal of this hypermethylation with DAC treatment, using a deep sequencing technique. The major portion of this data is unpublished, a small amount has been published in 2010. A manuscript describing these findings is currently being prepared. Despite the recent FDA approval of three drugs for MDS, the only curative option for patients with MDS remains allogeneic hematopoietic stem cell transplantation (HSCT). Allogeneic HSCT has two essential components, high-dose cytotoxic chemo-radiotherapy, and an immune-mediated graft versus host (GVH), or graft versus leukemia (GVL) effect. However, the relative contributions of high dose cytotoxic therapy and GVL are not well established in MDS patients. We propose to use transplantation of the NHD13 mice as a means to investigate this question. Our initial experiments, using either 650 or 1000 CGy of radiation, indicated that 650 CGy was ineffective, but that 1000 CGy could induce a remission of 26-38 weeks, defined by normalization of peripheral blood counts and less than 2% circulating host cells. However, despitethis period of prolonged remission, and prolonged survival compared to non-transplanted mice, all of the mice have ultimately relapsed, indicating that this myeloablative therapy was not curative. To address the question of a GVL effect, we crossed C57bl6 mice with C3h.SW mice. C3H.SW mice are identical to C57Bl6 mice at the major histocompatibility loci, but have numerous mismatches at minor histocompatibility loci108. For this reason, transplantation of C3H.SW donor cells into C57Bl6 host mice has been used to study GVH and GVL. We transplanted BM from C57Bl6/C3HSW mice into C57Bl6 NHD13 recipients. The mice developed little GVH or GVL under these conditions. Subsequent experiments using higher doses of BM and 5 x 10E06 peripheral T cells showed severe GVH, that was lethal to 3 of 5 mice. A third trial, using a higher dose of bone marrow cells (10E07) is promising, as the recipients have developed mild GVH, and have survived up to 36 weeks post-transplant. These studies have not yet been published. In addition to the experiments outlined above, we have transferred NHD13 mice to colleagues at several academic institutions, and have licensed NHD13 mice to at least two separate biotech companies for pre-clinical studies. These colleagues have plans to treat NHD13 mice with a variety of agents, including histone deacetylase inhibitors, apoptosis inhibitors, and angiogenesis inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010983-05
Application #
8552913
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2012
Total Cost
$312,971
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Monlish, Darlene A; Bhatt, Sima T; Duncavage, Eric J et al. (2018) Loss of Toll-like receptor 2 results in accelerated leukemogenesis in the NUP98-HOXD13 mouse model of MDS. Blood 131:1032-1035
Bhagat, Tushar D; Chen, Si; Bartenstein, Matthias et al. (2017) Epigenetically Aberrant Stroma in MDS Propagates Disease via Wnt/?-Catenin Activation. Cancer Res 77:4846-4857
Ma, Hayley S; Greenblatt, Sarah M; Shirley, Courtney M et al. (2016) All-trans retinoic acid synergizes with FLT3 inhibition to eliminate FLT3/ITD+ leukemia stem cells in vitro and in vivo. Blood 127:2867-78
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Gough, Sheryl M; Lee, Fan; Yang, Fan et al. (2014) NUP98-PHF23 is a chromatin-modifying oncoprotein that causes a wide array of leukemias sensitive to inhibition of PHD histone reader function. Cancer Discov 4:564-77
Gough, Sheryl M; Chung, Yang Jo; Aplan, Peter D (2012) Depletion of cytotoxic T-cells does not protect NUP98-HOXD13 mice from myelodysplastic syndrome but reveals a modest tumor immunosurveillance effect. PLoS One 7:e36876
Lin, Ying-Wei; Beharry, Zanna M; Hill, Elizabeth G et al. (2010) A small molecule inhibitor of Pim protein kinases blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma. Blood 115:824-33
Beachy, Sarah H; Aplan, Peter D (2010) Mouse models of myelodysplastic syndromes. Hematol Oncol Clin North Am 24:361-75
Estécio, Marcos R H; Gallegos, Juan; Vallot, Céline et al. (2010) Genome architecture marked by retrotransposons modulates predisposition to DNA methylation in cancer. Genome Res 20:1369-82

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