PS1439 ENHANCED SUPPORT OF MYELOFIBROSIS STEM CELLS IN NEXT GENERATION HUMANIZED MICE
Authors: V. Lysenko, N. Wildner, K. Zimmermann, M.‐C. Weller, P. Schürch, C. Fritz, L. Calabresi, R.A. Flavell, A.M. Vannucchi, P.J. Wild, S. Dirnhofer, M.G. Manz, A. Theocharides
Journal: HemaSphere (2019)

Background: Myelofibrosis (MF) is a hematopoietic stem cell (HSC) neoplasm characterized by an expansion of myeloid cells followed by deposition of fibers in the bone marrow (BM) and leukemic transformation depending on the clonal evolution of MF stem cells (MF SCs). Mutations in the receptor tyrosine kinase JAK2 , the endoplasmic reticulum chaperone CALR or the thrombopoietin receptor ( MPL ) drive the disease phenotype through cytokine‐independent activation of the JAK‐STAT signaling pathway. Patient‐derived xenograft (PDX) models have emerged as powerful tools for investigating aggressive malignancies, such as acute leukemia's. However, the development of less‐aggressive malignancies, like MF, is often limited in PDX models. We hypothesized that the constitutive expression of human cytokines and growth factors in immunocompromised mice (i.e. MISTRG mice) may provide a supportive microenvironment for MF SC development. Aims: To develop a pre‐clinical MF PDX model that faithfully recapitulates the disease phenotype and the genetic heterogeneity observed in patients. Methods: Purified peripheral blood (PB) stem and progenitor (CD34+) cells from 14 MF patients were transplanted intra‐hepatically into sub‐lethally irradiated newborn MISTRG mice and NSG mice (controls). For secondary transplantations, human CD45+ cells purified from primary animals were transplanted intra‐hepatically. Mice were sacrificed 5–26 weeks (median: 9.8 weeks) post‐transplantation and characterized by flow cytometry, immunohistochemistry, and mutational profiling. Results: MISTRG mice supported significantly higher human MF engraftment in the BM (29.20% vs 4.175%, p &lt; 0.0001), the PB (48.70% vs. 0.73%, p &lt; 0.0001) and the spleen (8.19% vs. 0.33%, p &lt; 0.0001) compared to NSG mice. All 14 investigated patient samples engrafted in MISTRG mice, while 9/14 patient samples engrafted in NSG mice. MISTRG mice exhibited superior engraftment independent of risk categories (DIPSS, MIPSS70, and MYSEC), disease stage (chronic, accelerated) and diagnoses (primary, secondary MF). Both NSG and MISTRG mice supported robust monocytic and granulocytic engraftment in the BM. Furthermore, immunohistochemistry revealed human megakaryocytes in both strains. An increase in reticulin fibers was detected in MISTRG mice transplanted with 1 patient sample that was analyzed after 18 weeks. To determine whether the engrafted human cells were derived from the MF clone, NSG and MISTRG mice that showed &gt;10% human engraftment were analyzed for driver mutations ( JAK2 or CALR ). JAK2 or CALR mutations were preserved in all NSG (7/7) and MISTRG (39/39) mice transplanted with the respective sample. Next, we performed targeted sequencing of 54 myeloid genes frequently mutated in myeloid neoplasms on 7 NSG mice transplanted with 4 patient samples and 22 MISTRG mice transplanted with 8 patient samples to determine whether the mutational profile of the primary sample was preserved in the corresponding engrafted xenografts. This data showed maintenance of the primary patient sample clonal composition in both NSG and MISTRG mice. Finally, purified human MF cells isolated from primary mice showed myeloid reconstitution in secondary recipients. Summary/Conclusion: These results show that MISTRG mice support robust engraftment of MF SCs across all disease stages and categories and faithfully reproduce the genetic complexity observed in patients. This novel MISTRG MF PDX model is currently being used to assess the effect of the JAK inhibitor (JAKi) Ruxolitinib on engrafted human MF cells.