Induction of a Leukemic Bone Marrow Niche Mediated By TGFB1 of Leukemic Blasts in Pediatric Acute Mekaryoblastic Leukemia: Results of an in Vitro and In Vivo Analysis

Background: An increasing knowledge about the bone marrow niche demonstrates the high complexity of leukemogenesis. Mesenchymal stromal cells (MSC) are important members of the bone marrow niche and source of fibrosis. Further, the microenvironment seems to be regulated by megakaryocytes and platelets via cytokines, such as transforming growth factor beta 1 (TGFB1). Despite extensive research, the pathogenesis of the bone marrow niche in childhood leukemia and the therapeutic potential is still unclear.

We focus on acute childhood megakaryoblastic leukemia (AMKL) as a disease model and include patients with (ML-DS) and without Down syndrome. Based on similar clinical progressions - myelofibrosis occurs as a side-effect of both leukemia subtypes; these two diseases suit to characterize the leukemic bone marrow niche.

Methods: We performed a comprehensive characterisation of MSC from ML-DS (n=9), AMKL patients (n=5) and healthy donors (HD; n=6) via e.g. differentiation assays (adipogenic, osteogenic), gene expression profiles and western blot analysis. In addition, we established an in vivo model with humanized ossicles, representing a human bone marrow microenvironment (as described by Chen et al. 2012; Reinisch et al. 2015): We injected MSC mixed with pooled human umbilical vein endothelial cells (HUVEC) and Matrigel subcutaneously into NOD scid gamma (NSG) mice. After 8 weeks, the engrafted ossicles were injected with megakaryoblastic cells (CMK cell line); injected ossicles (n=16); uninjected ossicle (n=27), MSC from ML-DS (n=19 ossicles), AML M1 (n=15 ossicles) and HD (n=9 ossicles). After 4 more weeks, histopathology evaluation of fibrosis in the ossicles was performed in accordance with the European Consensus on Grading Bone Marrow criteria from an independent pathologist.

Results: The detailed characterisation of MSC with ML-DS and AMKL demonstrated a high similarity to MSC of HD: morphology, osteogenic differentiation potential, colony forming unit-fibroblast assay, proliferation and gene expression profiles.

However, two differences emerged in our analysis: MSC showed a decreased adipogenic differentiation potential in ML-DS and AMKL compared to HD (ML-DS vs. HD=0.26-fold, p<0.05; AMKL vs. HD=0.50-fold). Gene expression profiling identified an upregulation of IGF2BP3, an oncofetal RNA binding protein, in MSC of ML-DS compared to HD confirmed by qRT-PCR (2.6-fold, p<0.05). IGF2BP3 is known to be highly expressed in many cancers and seems to be associated with proliferation. The increased level of IGF2BP3 (protein: IF2B3) was confirmed at protein-level detected by western blot analysis (ML-DS vs HD: 37.3-fold, p<0.05 and AMKL-MSC vs HD: 13.1-fold, p<0.05).

TGFB1 - known to be secreted by leukemic megakaryoblasts - induced a fibrotic state in MSC regardless of their origin indicated by decreased adipogenic differentiation potential (treated vs. untreated: ML-DS 0.22-fold; AMKL 0.08-fold; HD 0.06-fold, p<0.05) and increased expression of collagen genes (qRT-PCR; COL1A1: ML-DS=1.63-fold, AMKL=1.80-fold (p<0.01), HD=1.66-fold (p<0.05); COL3A1: ML-DS=1.31-fold, AMKL=1.52-fold (p<0.05), HD=1.24-fold).

The humanized bone marrow niche in our mouse model demonstrated a development of myelofibrosis after injection of the megakaryoblastic cell line (CMK): Grade 1 or 2 in 81% of the ossicles. The induction was independent of the MSC entity (HD/ML-DS). Of note, a monocytic subpopulation, which engrafted unexpectedly in ossicle from HD-MSC (n=3 ossicle), did not induce fibrotic fibers.

Conclusion: Our data impressively illustrate the mutual influence between MSC and leukemic blasts that leads to a fibrotic microenvironment. This correlation has been observed in vitro but also in a unique mouse model. The interaction of MSC and leukemic blasts seems to be the key factor for the development of the leukemic niche in AMKL mediated inter alia by the TGFB pathway. However, we could identify several disease specific characteristics of MSC.

Our model offers a unique opportunity to fundamentally examine of the leukemic niche in order to subsequently evaluate the potential therapeutic use in further studies.