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P.M. Le et al.
apoptotic pathways and downregulation of RNA regula- tors previously implicated in survival and differentiation of leukemic cells.46 Of particular interest, marked under- expression was observed for IGFBP5, an insulin-like growth factor binding protein that primarily inhibits osteoblast differentiation of MSCs.47 Moreover, TP53 was increased in AML-derived MSCs, resulting in senescence. We also found that the leukemia genotype, in particular the presence of FLT3-ITD mutations and lack of p53, induce both shared and leukemia genome-specific alter- ations in MSCs.48 These reports suggest that AML cells alter stromal development, and potentially their function- ality.
Previously, Hanoun et al. had reported that AML primed MSCs to commit to osteoblastic lineage.49 The endosteal surface of mice transplanted with MLL-AFL9 leukemic cells was packed with Osx-expressing osteo- progenitor cells yet lacking Osteocalcin-positive (Osc+) mature osteoblasts. It is important to note that despite this osteogenic potential, these mice showed deficient bone mineralization and a lack of terminally differentiat- ed osteoblasts compared with healthy controls. These observations were confirmed independently both in vitro and in vivo by our group.50 AML-MSCs displayed signifi- cantly higher alkaline phosphatase (ALP) expression and activity than did healthy donor-derived MSCs. In addi- tion, when cultured in osteogenic differentiation medi- um, AML-MSCs differentiated to mature osteoblasts (alizarin red-positive) within two weeks compared with the three weeks needed for normal MSCs. Remarkably, gene expression analysis of normal MSCs co-cultured with different leukemic cell lines for five days revealed 2- to 10-fold upregulation of osteogenic markers, such as Runt-related transcriptional factor (Runx2), Osx, Opn, and tissue non-specific ALP (Tnap), suggesting that this osteo- progenitor-priming pattern in MSCs resulted from AML exposure.50 To validate AML-induced osteoblast differen- tiation in vivo, we created a human BM implant mouse model and assessed osteogenic potential of BM MSCs after four weeks of leukemia engraftment. Human MSCs obtained from these transplanted mice showed a 5- to 7- fold increase in Osx and Runx2 expression compared with control mice.50 These experimental data were consistent with OSX and RUNX2 upregulation in BM biopsies of AML patients.
We also found that AML-MSCs became less multipo- tent since they differentiated poorly into adipocytes and chondrocytes, two mesodermal lineages that usually arise from MSCs; the Bhatia group confirmed this adipocyte suppression in the setting of AML by immunostaining within human BM and global transcrip- tome analysis of AML-MSCs.51 Note that in the same study, gene sets poised towards osteoblast, but not adipocyte lineage were enriched in AML-MSCs, under- scoring the need to understand the role of distinct mes- enchymal fractions in AML progression.
We asked whether this osteolineage-specific priming provided any advantage for leukemic growth. Indeed, AML cells up-regulated connective tissue growth factor (CTGF) in MSCs and activated BMP signaling via Smad1/5 phosphorylation, both of which have been asso- ciated with persistence of tumors and poor prognosis in patients with acute leukemia.52-54 Besides, AML-induced TNAP overexpression in MSCs was implicated in osteoblast-mediated protection of leukemia blasts against
apoptosis.55 By unraveling a feedback loop between stro- ma functionality and AML expansion, our study has high- lighted the dynamics of the endosteal niche in AML pathogenesis (Figure 2).
The reduced bone mineralization seen by Hanoun et al.49 could have resulted from altered osteolytic activity.56 A short-lived increase in osteoclasts and upregulation of CCL3, a pro-inflammatory cytokine with pro-osteoclastic action previously established in multiple myeloma,57 was found in a murine model of blast-crisis chronic myeloid leukemia (CML) phenotype.56 These acute leukemia-like mice showed a significant reduction in Osc+ osteoblasts and thinning of bone structures that could not be reversed completely by inhibition of osteoclasts. Bone deposition and resorption are tightly coupled processes that main- tain bone homeostasis; however, this evidence suggests that the leukemic condition distorts this balance. Of inter- est, excessive CCL3 production does not typically lead to osteolytic lesions or bone loss,58 as seen in Ptpn11-mutat- ed leukemic mice,39 although overexpression of this pro- tein is common in the BM of AML patients.56 It is possi- ble that monocyte differentiation into osteoclasts is defective in AML, yet the effects are masked by strong CCL3-driven recruitment of monocytes into the osteogenic niche.59 The extent to which osteoblasts and osteoclasts work in tandem to reconstruct an inhospitable microenvironment under AML burden needs further investigation.
Osteoprogenitors or mature osteoblasts: true 'partners-in-crime' in AML progression?
The osteogenic niche comprises a variety of cell types which differ in their maturation status, ranging from very immature multipotent MSCs to mature osteoblasts and osteocytes (Figure 1). However, the differentiation status of osteogenic cells supporting normal hematopoiesis or leukemogenesis is an emerging question that remains to be resolved. Several studies have shown that, compared with less mature osteoblasts, terminally differentiated osteoblasts regulate HSC lineage commitment, such as B lymphopoiesis and erythropoiesis, while having less effect on HSC proliferation.60-62 Whether this functional stratification applies to the context of malignancy is poor- ly understood. Accumulating evidence has demonstrated that defective osteolineage cells are potent initiators of leukemia in the BM. These findings led to the question: which osteolineage cells, osteoprogenitors or mature osteoblasts, play a major role in promoting leukemogen- esis?
As previously discussed, Raaijmakers et al.35 were the first to show that hematopoiesis could go awry as a result of a genetic alteration in osteoprogenitors. The Scadden group emphasized the differential leukemogenic capacity of immature and mature osteoblasts by comparing AML phenotype in Dicer1fl/fl mice with Osx- versus Osc-driven Cre recombinase. Mice with a Dicer1 defect in mature osteoblasts did not exhibit any hematologic problem besides bone-related deformities. Similarly, Dong et al.39 confirmed the distinct role of stage-specific osteoblasts in leukemic development by generating mice with Ptpn11 mutations at various stages of MSCs: mesenchymal pro- genitor/stem cells, differentiated MSCs, Osx+ osteoprog- enitors, and Osc+ mature osteoblasts. Of interest, the leukemogenic effect of this abnormality was observed in mice bearing the mutated form of either primitive MSCs
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haematologica | 2018; 103(12)