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P. Giannoni et al.
osteoclasts and infliximab significantly blocked the enhancement in formation of fully mature osteoclasts treated with CLL-cm. This finding suggests that TNFα, released by leukemic cells, stimulates osteoclastogenesis. As previously reported, malignant CLL cells release TNFα,38 and the levels of plasma TNFα, higher in CLL patients than in healthy controls, correlate with disease stage, CD38 expression and chromosomal abnormalities (17p and 11q deletion).39 Interestingly, TNFα levels, signif- icantly higher in sera from patients with advanced disease than in patients with stable disease (Binet C vs. Binet A; 36 cases studied), were directly proportional with the degree of appendicular bone erosion.2 Among CLL patients enrolled in our osteoclastogenesis studies, we further determined a direct correlation between TNFα serum levels and the unmutated IGVH status of the assessed patients (serum cut-off level: 9.0 pg/mL; χ2 test, P=0.0046). We also found that the same unmutated IGVH status of the CLL-cell donors correlated with an increased number of trinucleated TRAP+ cells when pre-activated monocytes were cultured with their CLL-derived condi- tioned media (χ2 test, P=0.0147) (Online Supplementary Figure S11).
TNFα plays a key role in many inflammatory diseases characterized by bone damage and enhanced osteoclast differentiation (e.g., rheumatoid arthritis and psoriatic arthritis).40-42 It has been recently demonstrated that TNFα-promoted osteoclastogenesis is exerted solely in the contemporary presence of RANKL,43 as we also showed. Other cytokines released by leukemic cells, such as IL-6 and IL-11, may however contribute to monocyte differentiation and participate in the redundancy of osteoclastogenesis control. In the absence of RANKL pre- stimulation, neutralizing anti-IL-11 or anti-IL-6R and anti- GP130 monoclonal antibodies reduced the number of small tri-nucleated TRAP+ cells derived from monocytes cultured with MCSF and CLL-cm only. Hence cytokines present in CLL-cm (i.e., IL-6, IL-11) might prompt mono- cytes toward an early step of osteoclast differentiation, thus stimulating the expansion of precursor cells: low lev- els of soluble RANKL may further limit their ability to complete osteoclast differentiation. Observations by McCoy et al.44 appear to be in accordance with this sug- gestion: although IL-11 derived from breast tumor pro- moted the development of osteoclast progenitors, the enhancement of fully differentiated osteoclasts appeared to be dependent on subsequent RANKL addition. When provided alone, MCP1 also increased differentiation of monocytes in multinuclear TRAP+ cells without bone resorption activity. Nonetheless the same cells, after RANKL exposure, differentiated into authentic bone- resorbing osteoclasts.45 The complex process of osteoclast differentiation involves the regulated expression of vari- ous molecules and their linked transduced signal. The interaction between RANKL, residing on osteoblasts, and RANK, expressed on activated monocytes, results in the recruitment of TNF receptor-associated factor-6 (TRAF-6) which in turn activates NF-κB, JNK, ERK, p38, Akt and NFATc1. In particular NFATc1 is considered the master transcription factor that regulates the expression of mole- cules such as MMP9, OSCAR, DC-stamp, TRAP and cathepsin K, which altogether define the functional osteoclast phenotype.46 We have shown here higher cathepsin K, MMP9, and NFATc1 mRNA expression in RANKL-activated monocytes cultured with CLL-cm than
in controls: this finding may parallel the observed incre- ment in the number of fully differentiated osteoclasts found after the addition of CLL-cm. The reduced levels of cathepsin K mRNA observed in monocytes stimulated with CLL-cm only may further suggest a blockade of their differentiation stage.
A high incidence of bone alterations, in various B-cell malignancies including chronic lymphocytic leukemia, has been previously reported.47 In bone biopsies from patients with such malignancies, a consistent number of TRAP+ monocytes are located near bone trabeculae and are smaller than the osteoclasts found in multiple myelo- ma. These small cells induce areas of micro-resorption and are found in the proximity of malignant lymphoid cells. Further evidence of the presence of minute, single bays facing small TRAP+ osteoclasts in eroded bone sur- faces of patients with B-cell malignancies was subse- quently provided.48 These findings suggest that, in B-cell malignancies, higher numbers of active small osteoclasts are needed to cause rates of resorption similar to those observed in patients with multiple myeloma. Interestingly, evaluation of the frequency distribution profile of osteoclast dimensions in bone sections from patients with B-cell malignancies has demonstrated the existence of a bimodal distribution of TRAP+ osteoclasts, characterized by two different average diameters, peak- ing at 10-15 mm and at 20-25 mm. In contrast, in multiple myeloma the distribution appeared homogenous and uni- modal, with a single peak around 20-25 mm.49 Data obtained from our in vitro model of osteoclast generation, under different conditions, resemble these in vivo findings: consistent numbers of small tri-nucleated TRAP+ cells and only limited numbers of large mature osteoclasts could be derived when MCSF-activated monocytes were cultured with CLL-cm alone. In contrast, pre-stimulation with RANKL allowed the differentiation of monocytes into authentic, fully mature osteoclasts. Bone structure alter- ations might therefore be relatively common in CLL patients, but usually surmised, because extensive bone lesions do not occur frequently. Reasonably, in CLL patients, the multi-cytokine panel produced by leukemic cells stimulates the generation of osteoclast progenitors which, however, usually fail to mature completely. Hence the impaired bone homeostasis in CLL differs from that previously determined in multiple myeloma. In the for- mer malignant plasma cells release RANKL, while in the latter CLL cells are incapable of shedding it.17 Exceptionally, some CLL cases, hypothetically character- ized by more advanced disease stage and possibly by the presence of higher levels of RANKL and TNFα, might dis- play osteolysis. The presence of neoplastic B cells, often close to osteoclasts, also appears of interest:50 small areas of bone micro-resorption could represent small niches of recovery and protection for neoplastic B cells. We con- firmed that in bone biopsies from CLL patients, leukemic cells are detectable in strict vicinity of osteoclasts, thus suggesting their mutual interactions. The proof that fully differentiated osteoclasts, or their precursors, protect leukemic B cells from apoptosis, upregulate Ki-67 and stimulate their proliferation sustains the idea of an active interplay between these two populations of cells. Moreover, the disruption of CLL-cell-mediated osteoclast support by the BTK inhibitor, ibrutinib, is a novel finding. Finally, the observations in the present study highlight new relationships between neoplastic CLL cells and nor-
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haematologica | 2021; 106(10)