Translational Development of Preclinical Models and Therapies in MDS
MDS is a heterogeneous group of myeloid disorders characterized by bonemarrow failure, cytopenia and dysplasia. BoneMarrow transplantation(BMT) is the only curative treatment, however due to advanced age of disease and patient’s comorbidity, few are eligible for BMT and physicians following the watch and wait system for further disease development. On these grounds, animal-modelling studies are proposed to improve understanding of the disease and extend treatment options. In order to generate MDS animal model, we used the MDS92 cell line as the only cell line established for MDS studies. During the course of study we realized that MDS92 has two daughter cells that proliferate significantly different from each other in in vitro and in vivo studies. MDS-L (DA) extensively engrafted in immunodeficient mice and cells infiltrated quickly from femur to tibias and spine i.e. more representative of an AML/MDS phenotype, while MDS-L (SA) only showed transient engraftment (annual report 2015) more typical of MDS. These novel observations prompted us to evaluate these two daughter cells and elucidate differences between them, which may aid clinical prognostic or therapeutic difference. In 2016, the two daughter cells were compared regarding immune phenotype, cytogenetic features, engraftment ability in mice and disease phenotype. Results confirmed that both MDS-L (DA) and MDS-L (SA) are derived from a same parental cell by using DNA finger printing. Both sub-lines have shown complex karyotypes, which is indicative of high risk MDS, yet they harbored different aberrations. MDS-L (DA) carried more monosomies than MDS-L (SA) and also was seen with additional mutations in several tumor suppressor genes. According to these databases and several other observations, we concluded that MDS-L (DA) is more like an AML phenotype than MDS-L (SA). Therefore MDS-L (SA) is more indicative of human MDS. Thus establishment of an MDS preclinical model continued with MDS-L (SA). To improve transient engraftment of MDS-L (SA), we employed a novel 3D structure that modulates an extra-medullary bone marrow (BM) niche sub-cutaneously. Hydrogel Scaffold as unique structure in addition to use of stromal cells remarkably supports engraftment of MDS-L (SA) over 30 weeks. Although this structure aided generating an MDS-L(SA) animal model for the very first time, due to long term of MDS-L(SA) engraftment, complexity in manufacturing scaffolds and also stromal cells expenses, we required an improvement in the system. Thus, we tailored a humanized bone marrow niche using stromal cells, extracted from healthy donors (Performed in Bergen University Hospital) that co-cultured with MDS-L (SA) cells and supported with degradable form of extracellular matrix. Humanized BM niche, implanted in immunodeficient mice and engraftment monitored using molecular imaging. Outcomes showed a very steady proliferation of MDS-L (SA) cells over 12 weeks, without progressive features and consistency in time and number of mice with engraftments (n=5/5). This reveals an appropriate MDS pre-clinical mouse model that has a great potential to be used in drug development. In addition, the modified humanized BM niche suggests having a high impact in engraftment of primary MDS patient cells from indolent to aggressive range of disease. In order to take such an important step, we already started several studies recently, which will continue in the following year (2017). Considering this complexity and uniqueness of the project and models being developed I believe extension of the funding into 2017 will have the most positive impact on the work and result in publications of much higher impact.
Myelodysplastic syndrome (MDS) is a heterogeneous group of disease with disability of myeloid progenitor cells in production of normal blood cell lineages. Myelodysplastic syndrome causes bone marrow failure that eventually results in cytopenia and death. MDS become more challenging when it progress to other sever leukemic diseases, like Acute Myeloid Leukemia . The only curative therapy is the hematopoietic stem cell transplantation (HSCT). However, most of the patients will not qualify for the procedure due to their age. Several preclinical studies were performed based on different “MDS patient derived cell lines”, but only one cell line (MDS92) is associated with MDS; based on phenotype and genetic lesions in MDS. MDS92, potentially can differentiate into several myeloid lineages such as; neutrophils, macrophages, eosinophils, and megakaryocytes . Of all lineages, two blastic sublines (CD34+ cells) were sorted for MDS studies . In 2015 our studies emphasized on differences between these two daughter cells based on genetic content and MDS preclinical animal models. In addition, we focused on a novel therapeutic agent named BGB324. This drug is a selective Axl inhibitor, express in all the cell types. Axl tyrosine kinas receptors induce cell survival, migration and differentiation. High expression of Axl observed in 50% of AML . This gave us an idea to study BGB324 on MDS cell line and MDS patient cells. In some studies, Axl shown to be under the regulation of the tumour suppressor p53 in other cancers [5, 6], and the progression of MDS has been correlated to the mutational status of TP53 [7, 8]. In our studies we emphasised on the hypothesis weather expression of Axl in MDS patients is correlate with activation of P53. We investigated the effect of drug in vitro, however in the future we plan to use this as a new therapeutic option in preclinical xenograft MDS model based on cell line and patient cells. 1. Drexler, H.G., W.G. Dirks, and R.A. Macleod, Many are called MDS cell lines: one is chosen. Leukemia research, 2009. 33(8): p. 1011-6. 2. Tohyama, K., et al., A Novel Factor-Dependent Human Myelodysplastic Cell-Line, Mds92, Contains Hematopoietic-Cells of Several Lineages. British Journal of Haematology, 1995. 91(4): p. 795-799. 3. Tohyama, K., et al., Establishment and characterization of a novel myeloid cell line from the bone marrow of a patient with the myelodysplastic syndrome. British journal of haematology, 1994. 87(2): p. 235-42. 4. Janning, M., I. Ben-Batalla, and S. Loges, Axl inhibition: a potential road to a novel acute myeloid leukemia therapy? Expert Rev Hematol, 2015. 8(2): p. 135-8. 5. Vaughan, C.A., et al., Gain-of-Function Activity of Mutant p53 in Lung Cancer through Up-Regulation of Receptor Protein Tyrosine Kinase Axl. Genes Cancer, 2012. 3(7-8): p. 491-502. 6. Boysen, J., et al., The tumor suppressor axis p53/miR-34a regulates Axl expression in B-cell chronic lymphocytic leukemia: implications for therapy in p53-defective CLL patients. Leukemia, 2014. 28(2): p. 451-5. 7. Kulasekararaj, A.G., et al., TP53 mutations in myelodysplastic syndrome are strongly correlated with aberrations of chromosome 5, and correlate with adverse prognosis. Br J Haematol, 2013. 160(5): p. 660-72. 8. Wong, T.N., et al., Role of TP53 mutations in the origin and evolution of therapy-related acute myeloid leukaemia. Nature, 2015. 518(7540): p. 552
MDS as a pre-leukemic disease involving defunct colonies of Myeloid Progenitor Cells, resulting in bone marrow failure and insufficient production of normal blood cell. Starting in 2014, we have endeavored to evolve a greater understanding of MDS through xenograft modeling in immunodeficient mice Studies performed on 2014 are in two different phases: A. In our previous studies, MDS cell line after co-culturing with normal stromal cells shows increase in engraftment of MDS resulting in a novel MDS animal model. To build up better strategies in MDS modeling, we explore the potential development of MDS patient derived xenograft models. We hypothesized that in order to engraft MDS patients cells, the interaction of these cells with (in co-culture) with it’s own stromal cells vs normal stromal cells may be critical. Therefore in in vitro and iv-vivo studies, MDS patients cells proliferation was examined with in co-culture assay with their own stromal cells and compared with co-culture of stromal cells from healthy donors. In vitro studies showed better growth of MDS patients cells when they are in co-culture with normal stromal cells. Cytokine analysis of the supernatant from both co-cultures support the idea that healthy stromal cells induce MDS proliferation with elevation in hematopoietic growth factor (HGF)cytokines such as IL-6, GM-CSF, G-CSF, whereas the other condition with patient stromal cells high expression of pro-inflammatory cytokines such as IL-8, IL-10 and VEGF reduction, suppress proliferation of the MDS cells. These result match clinical findings. Consequently, we used healthy stromal cells in co-engrafted with an MDS patient in vivo part of the study,. Analysis shows consistency of 6/6 Immunodeficient NSGS mice with engraftment of MDS cells over 100 days by optical imaging using conjugated mAb with nearinfrared fluorophores (CD33, CD45, CD13). B. Employing an MDS-L cell line from collaborators in the USA (Dr. Daniel Starczynowski, Cincinnati) we, transducted with a vector that contains Luc and GFP gene to permit optical imaging of a cell line model and screen for chemotherapeutics agents. Subsequently, MDS-L transduced cells, stromal cells and matrigel matrix co-cultured were used in in vivo study. Molecular images of all immunodeficient NSGS implanted mice appear to have similar levels of engraftment. This brings the idea of reproducibility of our MDS xenograft model based on stromal cells, and cytokine expression that help MDS-L cell engraftment in immunodeficient mice. we will subsequently use this model to monitor the effect of drugs in the next in vivo studies with MDS-L cells. In vitro studies performed thus far have demonstrated that: 1. this cell line is sensitive to Lenalidomide and 5-Azacytidine and, 2. appear to have HGF (IL-3) independent effect on MDS-L proliferation, assessed by cell counting, WST-1 and Bioluminescence imaging. Also Lenalidomide in all concentrations were more likely to stop the cell cycle and cell proliferation in the beginning phase and then induce cell apoptosis, whereas 5-Aza provoke MDS-L cells to enter into cell death program in all conditions. The half maximal inhibitory concentration (IC50) of both medicines will found in further in vitro studies and this will be applied in upcoming in vivo studies.
Novel pre-clinical models of myelodysplastic syndrome (MDS)
August 2014, Experimental Hematology, Volume 42, Issue 8, Supplement, Page S59