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G.I. Gavriilidis et al.
(si)RNA and analyzed Ki-67 positivity in three independ- ent biological replicates. In line with our data, SCF down- modulation by two of the four siRNA used (FD:1.2 for Si 9 and FD:2 for Si 8; P=0.05) was accompanied by decreases in the proliferative fraction of MEC1 cells (FD:1.3; P=0.05 for Si 9, FD:1.2; P=0.07 for Si 8) (Online Supplementary Figure S3F and G).
To further corroborate our findings, we used immuno- histochemistry to analyze the expression of SCF in biopsy samples from bone marrow (n=6) and lymph nodes (n=7) where the proliferative fraction of CLL cells reside; in two cases, concurrent, paired lymph node/bone marrow biopsy samples were available. In all cases, the vast majority of the CLL cells invading the lymph nodes and/or the bone mar- row showed SCF positivity, presenting a granular cytoplas- mic staining pattern; in some cases, membranous localiza- tion of the immunostain was also observed. There was no difference in staining pattern intensity between lymph nodes and bone marrow biopsies, including the two cases with matched biopsies from both sites. Endothelial cells and macrophages were also SCF immunopositive (Figure 2G, Online Supplementary Figure S4). As controls, we stud- ied reactive lymph nodes, in which SCF positivity was observed in mantle zones of the follicles, and in some inter- follicular lymphoid cells, whereas germinal centers were negative or showed moderate positivity. Moreover, in a non-CLL reactive bone marrow biopsy, sparse SCF immunopositivity was only traced in a small number of macrophages and endothelial cells (Figure 2H).
Mitochondrial reactive oxygen species, mesenchymal stroma and hypoxia regulate stem cell factor within the chronic lymphocytic leukemia microenvironment
Since SCF induction can be driven by oxidative stress, we investigated whether the observed induction of SCF expression after microenvironmental triggering in CLL cells might be related to changes in redox homeostasis. To this purpose, we quantified the mitochondrial mass as well as the membrane potential of activated CLL cells because aberrant ROS in CLL cells derive from the mitochondria.30
Of note, in tandem with augmented SCF expression, sig- nificant increases were found in both mitochondrial mass (n=6, FD: 1.7, P=0.01) and membrane potential (n=7, FD: 1.8; P=0.01) in TLR9-stimulated versus unstimulated CLL cells (Figure 3A, Online Supplementary Figure S5A). Moreover, in parallel to augmented proliferation/SCF lev- els, a significant increase in mitochondrial mass was also found in TLR9/CD40-stimulated versus unstimulated CLL cells (n=7, FD: 1.5; P=0.01). Interestingly, a similar but more pronounced effect was detected for the membrane potential of the activated CLL cells (n=7, FD: 2.8; P=0.01) (Figure 3B, Online Supplementary Figure S5B).
To further explore the possibility of a causal relationship between mitochondrial ROS and SCF regulation in CLL, we suppressed the oxidative stress of CLL cells in vitro and measured SCF levels. To this end, we used short-term (24 h) co-cultures of CLL cells with fully confluent HS-5 mes- enchymal cells which are a known source of precursors for the anti-oxidant glutathione.31,32 As expected, HS-5 cells conferred significant anti-apoptotic protection to co-cul- tured CLL cells (n=8, FD: 1.6; P=0.0156) (Online Supplementary Figure S5C) and also decreased their mito- chondrial mass, in keeping with the literature (n=6, FD: 1.5; P=0.03) (Figure 3C, Online Supplementary Figure S5D).31,32 Of note, short-term co-culture with HS-5 cells
triggered a significant reduction in the number of SCF+ CLL cells (n=15, FD: 2.9; P<0.001) (Figure 3D, Online Supplementary Figure S5E) which significantly correlated with the suppression of mitochondrial mass (n=6, r=0.73; P=0.0087) (Figure 3E). Moreover, we incubated co-cultures of CLL cells from ten cases with HS-5 cells with an oxida- tive agent (H2O2) in order to induce ROS production and determined the number of SCF+ cells by flow cytometry. As expected, this stimulation significantly increased the number SCF+ cells (FD=2; P=0.002); an even more pro- nounced effect was observed when HS-5/CLL cell co-cul- tures were stimulated in vitro by CpG/CD40L for 72 h which, as described above, induces mitochondrial ROS (n=6, FD: 3.2; P=0.03) (Figure 3F).
Next, we explored the association of HIF-1a with SCF protein expression. HIF-1a is a major hypoxia regulator that is abnormally expressed in CLL cells even under nor- moxia, critically relying on both mitochondrial ROS to avoid proteasomal degradation by the PHD/pVHL proteins but also the mesenchymal stroma.33-35 Western blotting analysis of healthy tonsillar B cells (n=5) and CLL cells (n=18) unveiled significant overexpression of HIF-1a pro- tein levels in the latter (FD: 5; P<0.001), in line with the lit- erature (Online Supplementary Figure S5F and G).36 Interestingly, in CLL a significant correlation emerged between HIF-1a and SCF protein levels (n=23, r=0.68; P<0.001) (Figure 3G). To further strengthen the correlation between SCF and HIF-1a, we also studied long-term co- cultures of SCFhigh U-CLL cells with HS-5 cells for 72 h, a setting known to simulate the bone marrow CLL milieu more closely, augmenting critical survival pathways such as hypoxia and glycolysis, and identified a significant increase in HIF-1 levels in CLL cells (n=5, FD:1.76; P=0.0192) (Figure 3H, Online Supplementary Figure S5H).35,37 The co-cultured CLL cells also manifested higher SCF pos- itivity than untreated controls (n=5, FD: 2.67; P=0.0026), in agreement with our immunohistochemical data (Figure 3I, Online Supplementary Figure S5I).
Ibrutinib downregulates stem cell factor expression in chronic lymphocytic leukemia
Recent literature suggests that the pharmacological mechanism of action of ibrutinib involves curtailment of inflammatory cytokines/chemokines as well as perturba- tions of mitochondrial dynamics.38,39 With this in mind, we assessed mitochondrial dynamics, hypoxia and SCF expression in ten CLL patients under ibrutinib therapy by profiling samples taken at two time points, namely before treatment initiation and at 1 month under treatment, and found that ibrutinib led to: (i) significant suppression of mitochondrial mass (n=6, FD: 1.6; P=0.0313) (Figure 4A, Online Supplementary Figure S6A), (ii) significant downregu- lation of HIF-1a expression (n=7, FD: 1.6; P=0.0156) (Figure 4B, Online Supplementary Figure S6B); and (iii) a sig- nificant decrease in SCF levels (n=10, FD: 2.4; P=0.002) (Figure 4C, Online Supplementary Figure S6C). Moreover, we analyzed longitudinal samples from four CLL patients who lost their response to ibrutinib therapy and observed that SCF expression showed a statistically significant increase at ibrutinib failure (FD:1.5, P=0.01) (Figure 4D, Online Supplementary Figure S6D).
The stem cell factor/c-kit signaling axis is active in chronic lymphocytic leukemia
To further explore the role of SCF in the CLL microen-
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haematologica | 2021; 106(3)