Abstract

Simple Summary

   Calcitriol, the biologically active form of vitamin D, modulates a
   plethora of cellular processes following its receptor ligation, namely
   the vitamin D receptor (VDR). Epidemiological studies have linked low
   blood levels of vitamin D to adverse disease outcome in several B cell
   malignancies, including chronic lymphocytic leukemia (CLL), for as yet
   undetermined reasons. In this study, we sought to obtain deeper
   biological insight into the role of vitamin D in the pathophysiology of
   CLL. To this end, we investigated whether the calcitriol/VDR system is
   functional in CLL and analyzed key signaling pathways that are
   regulated by calcitriol supplementation, while also exploring the role
   of microenvironmental signals in the regulation of calcitriol/VDR
   system. Overall, we provide evidence that the calcitriol/VDR system is
   functional in CLL, regulating signaling pathways critical for cell
   survival/proliferation. Although microenvironmental triggers can
   modulate VDR expression and function, calcitriol appears to act
   independently, alluding to a potential clinical utility of vitamin D
   supplementation in CLL.

Abstract

   It has been proposed that vitamin D may play a role in prevention and
   treatment of cancer while epidemiological studies have linked vitamin D
   insufficiency to adverse disease outcomes in various B cell
   malignancies, including chronic lymphocytic leukemia (CLL). In this
   study, we sought to obtain deeper biological insight into the role of
   vitamin D and its receptor (VDR) in the pathophysiology of CLL. To this
   end, we performed expression analysis of the vitamin D pathway
   molecules; complemented by RNA-Sequencing analysis in primary CLL cells
   that were treated in vitro with calcitriol, the biologically active
   form of vitamin D. In addition, we examined calcitriol effects ex vivo
   in CLL cells cultured in the presence of microenvironmental signals,
   namely anti-IgM/CD40L, or co-cultured with the supportive HS-5 cells;
   and, CLL cells from patients under ibrutinib treatment. Our study
   reports that the calcitriol/VDR system is functional in CLL regulating
   signaling pathways critical for cell survival and proliferation,
   including the TLR and PI3K/AKT pathways. Moreover, calcitriol action is
   likely independent of the microenvironmental signals in CLL, since it
   was not significantly affected when combined with anti-IgM/CD40L or in
   the context of the co-culture system. This finding was also supported
   by our finding of preserved calcitriol signaling capacity in CLL
   patients under ibrutinib treatment. Overall, our results indicate a
   relevant biological role for vitamin D in CLL pathophysiology and
   allude to the potential clinical utility of vitamin D supplementation
   in patients with CLL.

   Keywords: calcitriol, microenvironment, vitamin D receptor, chronic
   lymphocytic leukemia, RNA-sequencing

1. Introduction

   Vitamin D is the precursor to the steroid hormone calcitriol, which is
   the biologically active form of vitamin D, and exerts its biological
   activities by regulating the transcriptional activation and/or
   repression of target genes. In order to achieve this, calcitriol binds
   to the vitamin D receptor (VDR), a nuclear transcription factor that is
   present in almost all tissues [[44]1]. After activation by calcitriol,
   VDR undergoes heterodimerization with the retinoic X receptor (RXR).
   The activated calcitriol-VDR-RXR complex enters the nucleus and binds
   to vitamin D response elements (VDREs) located in the promoter regions
   of target genes [[45]2]. A well-known target of VDR is CYP24A1, a
   24-hydroxylase enzyme responsible for the degradation of calcitriol via
   a negative feedback regulation loop [[46]3,[47]4]. Calcitriol can also
   act independently of the VDR, by rapidly activating signaling molecules
   such as PLC, PI3K and MAP kinases [[48]5,[49]6].

   The classical role of calcitriol concerns the physiological regulation
   of calcium and phosphate in the context of bone formation [[50]7].
   However, various studies have demonstrated that calcitriol also engages
   in multiple extra-skeletal actions, including, amongst others, the
   regulation of many cellular pathways that could play an important role
   in determining cancer risk and evolution, including the MAPK/ERK and
   PI3K/AKT pathways [[51]8].

   VDR is expressed by cells of the immune system, including B cells,
   where vitamin D was shown to inhibit the proliferation of activated B
   cells and induce their apoptosis [[52]3]. Moreover, calcitriol has been
   found to inhibit the differentiation of B cells to memory B cells and
   plasma cells, leading to the inhibition of immunoglobulin (IG)
   production [[53]9,[54]10]. Interestingly, evidence exists for an
   association between vitamin D insufficiency and adverse outcomes in
   several B cell malignancies, e.g., follicular lymphoma [[55]11],
   diffuse large B cell lymphoma [[56]12], and chronic lymphocytic
   leukemia [[57]13].

   Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells
   characterized by pronounced heterogeneity regarding both the underlying
   biology as well as the clinical presentation and eventual outcome
   [[58]14], that appear to be critically dependent on a complex interplay
   between microenvironmental triggering and cell-intrinsic aberrations
   [[59]15]. In CLL, observational studies have reported significantly
   shorter time-to-treatment (TTT) and overall survival (OS) for
   25(OH)D-insufficient patients [[60]13], even amongst Binet stage A
   cases [[61]16]. Interestingly, it has also been documented that vitamin
   D replenishment in CLL patients, including those undergoing
   chemotherapy, is safe and effective in restoring vitamin D levels
   [[62]17,[63]18]. Nevertheless, the above-mentioned studies concluded
   that thorough clinical testing will be required before reaching solid
   conclusions as to whether normalizing vitamin D levels may delay
   disease-progression of patients with early disease.

   There are limited experimental data concerning the role of vitamin D
   and its receptor, VDR, in malignant CLL B cells. Ex vivo
   supplementation of a vitamin D analog (EB1089) has been found to induce
   apoptosis in CLL cells via a p53-independent mechanism [[64]19].
   Moreover, it has been reported that vitamin D administration in vitamin
   D-deficient CLL patients suppresses chemokine CCL11 levels, that confer
   a survival advantage on CLL cells [[65]20]. Finally, vitamin D has also
   been shown to inhibit CLL cell-mediated induction of myeloid-derived
   suppressor cells [[66]21].

   With this background, here we sought to obtain deeper biological
   insight into the role of vitamin D in the pathophysiology of CLL. To
   this end, we investigated whether the calcitriol/VDR system is
   functional in CLL cells and analyzed the key molecules and signaling
   pathways that are regulated by calcitriol supplementation, while also
   exploring the role of microenvironmental signals in the regulation of
   calcitriol/VDR system. Our novel findings support that the
   calcitriol/VDR system is active in CLL and, moreover, that the effects
   of calcitriol appear unaffected by various microenvironmental triggers
   known to promote CLL cell survival and/or proliferation.

2. Results

2.1. The VDR Is Expressed and Acts as a Transcription Factor in CLL Cells

   Using qPCR analysis, we evaluated VDR gene expression in purified
   circulating malignant cells from 73 CLL patients as well as B cells
   from 29 healthy donors. VDR mRNA was found significantly underexpressed
   in CLL cells compared to healthy B cells (FD: 7.3; p < 0.0001)
   ([67]Figure 1A). In contrast, this expression pattern was not confirmed
   at the protein level by flow cytometry ([68]Figure 1B) disclosing
   marginally lower VDR levels in healthy donors versus CLL patients (FD:
   1.2; p < 0.05), hence overall suggesting the possibility of
   post-transcriptional modulation of VDR expression in CLL.
   Interestingly, CLL cases with unmutated IGHV genes (U-CLL) showed
   significantly higher VDR mRNA levels compared to those with mutated
   IGHV genes (M-CLL) (FD: 3.2, p < 0.01) ([69]Figure 1C). This
   association was also likely reflected in significantly shorter OS (p <
   0.05) and time-to-first treatment (TTFT) (p < 0.01) for patients with
   high VDR mRNA levels (VDR^high) versus those with low VDR mRNA levels
   (VDR^low) ([70]Figure 1D,E) ([71]Table S1).

Figure 1.

   [72]Figure 1
   [73]Open in a new tab

   Vitamin D receptor (VDR) is underexpressed in chronic lymphocytic
   leukemia (CLL), albeit functional as indicated by CYP24A1 induction.
   (A) VDR mRNA expression is significantly lower in CLL compared to
   healthy donors’ CD19^+ B cells. The y axis represents VDR relative
   expression to GAPDH as detected by RQ-PCR. (B) The opposite pattern is
   observed at the protein level, as validated by flow cytometry. The y
   axis represents VDR mean fluorescent intensity (MFI). (C) VDR mRNA
   expression is significantly increased in U-CLL cases in comparison with
   M-CLL. (D,E) Kaplan Meier OS and time-to-first treatment (TTFT) curves
   for CLL patients with high and low VDR mRNA expression. VDR^high cases
   show shorter overall survival (OS) (D) and shorter TTFT (E), compared
   to VDR^low cases. (F) RXRα is overexpressed in CLL cases in comparison
   with B cells from healthy donors. y axis represents RXRα relative
   expression to GAPDH as detected by RQ-PCR. (G) CLL cases show
   significantly lower percentage of CYP24A1^+ cells compared to healthy
   cases (A–C, F,G). Each individual data point represents a unique case
   and lines represent the median values. The Mann–Whitney test was
   performed to assess statistical significance. (H,I) Calcitriol
   treatment for 24 h led to increased percentage of CYP24A1^+ CLL cells
   (H) and no change in VDR expression (I) compared to unstimulated
   control cells as revealed by flow cytometric analysis. Bars represent
   the median values and the Wilcoxon test was performed to assess
   statistical significance. * p < 0.05, ** p < 0.01, **** p < 0.0001, FD:
   Fold Difference, ns: not significant.

   We then assessed the mRNA expression of RXRα, the VDR co-receptor, and
   found significantly increased levels in CLL compared to healthy B cells
   (FD: 7.2, p < 0.0001) ([74]Figure 1F), albeit with no differences
   between U-CLL and M-CLL cases ([75]Figure S1A). Finally, we evaluated
   by flow cytometry the protein expression of CYP24A1, a well-known
   target gene of the activated VDR, and found significantly lower
   percentages of CYP24A1^+ cells in CLL cases versus healthy donors (FD:
   1.4, p < 0.01) ([76]Figure 1G).

   Next, we sought to determine if the VDR receptor is functional after
   binding its ligand. To this purpose, we cultured CLL cells with
   calcitriol for 24 h and measured by flow cytometry the expression of
   CYP24A1. We found that the percentage of CYP24A1^+ CLL cells
   dramatically increased in the presence of calcitriol compared to
   non-treated cells (n = 16 cases: FD: 3.3, p < 0.0001; [77]Figure 1H),
   indicating that the VDR is functional in CLL. U-CLL cases (n = 9)
   displayed stronger induction of CYP24A1 (FD: 4, p < 0.01) after
   calcitriol treatment compared to M-CLL cases (n = 7) (FD: 1.9, p <
   0.05) ([78]Figure S1B,C). Interestingly, VDR expression remained
   unchanged after in vitro calcitriol supplementation, as revealed by
   flow cytometric analysis (n = 15 cases) ([79]Figure 1I); this was
   consistent in both U-CLL and M-CLL cases ([80]Figure S1D,E). Of note,
   CLL cell viability was not altered by calcitriol treatment ([81]Figure
   S2). Overall, these results indicate that, despite lower expression
   compared to normal B cells, the VDR is functional in CLL cells, as
   evidenced by calcitriol-mediated CYP24A1 induction.

2.2. Calcitriol Induces a Distinctive Transcriptional Program in CLL Cells

   RNA-Sequencing (RNA-Seq) was performed in six CLL cases after ex vivo
   treatment with calcitriol for 24 h. Differential expression analysis
   revealed 85 differentially expressed genes (DEGs) (log2FC ≥ |1| and p ≤
   0.05), of which 28 (32.9%) were overexpressed in calcitriol-treated
   cells versus unstimulated CLL cells, thus, contrasting the remaining 57
   (67.1%) which showed the opposite pattern ([82]Figure 2A, [83]Table
   S2). RNA-Seq analysis confirmed our aforementioned findings that the
   CYP24A1 gene is significantly upregulated (log2FC = 9.7, p = 6.12 ×
   10^−8) by calcitriol, being the top upregulated gene, whereas the VDR
   gene remained unaffected by this treatment ([84]Figure 2A).

Figure 2.

   [85]Figure 2
   [86]Open in a new tab

   Calcitriol-treated CLL cells display a distinct transcriptional
   profile. (A) Volcano plot indicating the differentially expressed genes
   in calcitriol-treated vs untreated CLL cells. The fold change of gene
   expression is displayed on the x-axis and the significance of
   difference in gene expression [(-log10(p value)] is shown on the
   y-axis. The significantly upregulated genes are highlighted in green,
   whereas, the significantly downregulated genes are highlighted in red.
   (B) GO enrichment analysis showing top 10 significantly enriched
   biological processes (BP) and molecular functions (MF) in
   calcitriol-treated CLL cells. (C) The figure presents enriched pathways
   from the pathway enrichment analysis (q value < 0.1) and the
   differentially expressed genes between calcitriol-treated and
   non-treated CLL cases that are implicated. Upregulated genes are
   illustrated in green, whereas downregulated genes are shown in red.
   Enrichment score for each cluster is considered the overall enrichment
   score for the group based on the p value of each term members.

   Supervised hierarchical clustering analysis was performed, based on the
   differentially expressed genes, revealing distinct gene expression
   patterns between calcitriol-treated versus untreated (control) CLL
   cells ([87]Figure S3). Gene ontology (GO) enrichment analysis unveiled
   pronounced enrichment (q value < 0.1) in biological processes related
   to the regulation of localization, cell migration and motility,
   locomotion, and secretion. Concerning the molecular functions, the most
   enriched terms related to binding, including protein binding, calcium
   ion binding, lipid binding, and Toll-like receptor (TLR) 4 binding
   ([88]Figure 2B).

   Moreover, pathway enrichment analysis revealed that, besides the VDR
   pathway, calcitriol-regulated genes are implicated in TLR cascades and
   the PI3K/AKT signaling pathway, both known to be deregulated in CLL
   biology [[89]22,[90]23]. Finally, degradation of the extracellular
   matrix and vesicle-mediated transport were also among the
   calcitriol-regulated pathways ([91]Figure 2C). Interestingly, genes
   known to be implicated in lymphoma biology, such as S100A8, S100A9 and
   KITLG [[92]24,[93]25], were included amongst those regulated by
   calcitriol.

   Altogether, transcriptome analysis supported VDR functionality in CLL
   and highlighted the possible implication of calcitriol in the
   regulation of immune signaling pathways relevant to CLL
   pathophysiology.

2.3. Calcitriol Affects Key Immune Signaling Cascades in CLL Cells

   We further investigated the impact of calcitriol on signaling pathways
   known to regulate CLL cell proliferation and survival. Considering that
   calcitriol regulates the TLR and PI3K/AKT signaling pathways, we sought
   to investigate the activation status of extracellular signal-regulated
   kinase 1/2 (ERK1/2) and NF-κB p65, both implicated in these pathways.
   To this end, using intracellular phospho-flow cytometry, we analyzed
   pERK1/2 and pNF-κΒ p65 levels in CLL cells supplemented with calcitriol
   for 24 h and observed a significant increase in pERK (n = 8, FD: 1.6, p
   < 0.01) ([94]Figure 3A,B), hence contrasting the phosphorylation of
   NF-κB that was significantly decreased (n = 6, FD: 3.4, p < 0.05)
   ([95]Figure 3C,D).

Figure 3.

   [96]Figure 3
   [97]Open in a new tab

   Calcitriol modulates key signaling molecules and inhibits CLL cell
   proliferation. (A) Calcitriol treatment for 24 h led to increased
   percentage of pERK^+ CLL cells. (B) Representative histograms from flow
   cytometry analysis for pERK^+ cells in control and calcitriol-treated
   CLL cells for one CLL case. (C) Decreased percentage of pNF-κB^+ CLL
   cells compared to unstimulated control cells after calcitriol
   supplementation; (D) representative case. (E) Flow cytometric analysis
   of proliferating cells (Ki-67^+) in U-CLL cases treated with CpG/CD40L
   for four days supplemented with calcitriol at 48 h of culture. (F)
   Representative dot plot showing the decrease in Ki-67^+ cells after
   calcitriol supplementation. Bars represent the median values and the
   Wilcoxon test was performed to assess statistical significance. * p <
   0.05, ** p < 0.01, FD: Fold Difference.

   Next, we investigated the impact of calcitriol on the functionality of
   the TLR pathway by performing concurrent stimulation of CD19^+ B cells
   from U-CLL cases via the TLR9 and CD40 receptors in order to induce CLL
   cell proliferation. On day 2, calcitriol was added to the culture
   medium and, on day 4, the percentage of proliferating (Ki-67^+) CLL
   cells was determined by flow cytometry. CpG/CD40L stimulation induced
   CLL cell proliferation as expected [[98]26]. CpG/CD40L-stimulated cells
   supplemented with calcitriol showed significantly reduced proliferation
   compared to cells treated with CpG/CD40L alone (n = 5, FD: 1.5, p <
   0.05) ([99]Figure 3E,F). Altogether, these results support the notion
   that calcitriol can regulate key signaling pathways implicated in CLL
   pathophysiology.

2.4. Mincroenvironmental Signals Affect VDR Signaling, yet Do not Impede the
Action of Calcitriol in CLL

   In order to investigate the impact of external triggering on VDR and
   CYP24A1 expression, we performed concurrent stimulation of CD19^+ B
   cells from U-CLL and M-CLL cases via the BcR and CD40 receptors for 24
   h. Flow cytometric analysis showed that the VDR was significantly
   induced by co-stimulation with anti-IgM/CD40 ligand in U-CLL (n = 6,
   FD: 1.6, p < 0.05) ([100]Figure 4A), while no changes were observed in
   M-CLL (n = 4; [101]Figure 4B). The combination of calcitriol with
   anti-IgM/CD40L induced changes similar to BcR/CD40 stimulation alone in
   both U-CLL and M-CLL ([102]Figure 4A,B). BcR/CD40 stimulation led to a
   significant increase in CYP24A1^+ cells in U-CLL (n = 4, FD: 5.9, p <
   0.01) but not in M-CLL (n = 3) ([103]Figure 4C), yet co-stimulation of
   CLL cells with calcitriol/anti-IgM/CD40 ligand did not confer any
   additional change compared to calcitriol alone both in U-CLL and M-CLL
   ([104]Figure 4D). Overall, BcR/CD40 stimulation appears to modulate the
   VDR signaling pathway exclusively in U-CLL upregulating the expression
   levels both the VDR and CYP24A1, though calcitriol seems to act
   independently of these microenvironmental triggers since its action was
   not impeded when combined with anti-IgM/CD40L stimulants.

Figure 4.

   [105]Figure 4
   [106]Open in a new tab

   Immune signals affect VDR/calcitriol system, yet calcitriol action
   remains unaffected. Flow cytometric analysis of VDR (MFI) in (A) CLL
   with unmutated IGHV genes (U-CLL) and (B) CLL with mutated IGHV genes
   (M-CLL) cases treated with calcitriol, anti-IgM/CD40L, and a
   combination of them for 24 h. (C) Flow cytometric analysis of CYP24A1
   expression in U-CLL and M-CLL cells treated with anti-IgM/CD40L for 24
   h. (D) U-CLL and M-CLL cases were incubated with calcitriol and
   combination of anti-IgM/CD40L/calcitriol for 24 h. The percentage of
   CYP24A1^+ CLL cells was determined by flow cytometry. (A–D) Bars
   represent the median values and the Wilcoxon test was performed to
   assess statistical significance. * p < 0.05, ** p < 0.01, FD: Fold
   Difference.

   Next, we co-cultured CLL cells with the HS5 bone marrow stromal cell
   line, that partially mimics the microenvironment providing stimuli that
   prevent apoptosis of CLL cells ex vivo [[107]27]. Considering the
   herein documented limited responsiveness of M-CLL cells to calcitriol,
   only U-CLL cases were selected for the co-culture system. Co-cultured
   CLL cells from nine U-CLL cases showed significantly higher viability
   in comparison to mono-cultured CLL cells for 48 and 72 h (FD: 1.8; FD:
   1.9, p < 0.01 respectively) ([108]Figure 5A) and significantly
   increased pNF-κB levels (FD: 2.2, p < 0.05) ([109]Figure 5B), as
   previously described [[110]28]. VDR expression was not altered in the
   co-culture system (n = 13), even when calcitriol was applied
   ([111]Figure 5C). On the contrary, the presence of stromal cells in the
   culture system resulted in a significant increase in CYP24A1^+ CLL
   cells at 24 h (n = 9, FD: 2, p < 0.05), which was further enhanced when
   calcitriol was added to the medium (FD: 1.2, p < 0.05) ([112]Figure
   5D), indicating that calcitriol action is maintained in the co-culture
   system. Supporting this argument, the addition of calcitriol to the
   co-culture system induced pERK levels (n = 8, FD: 1.3, p < 0.01)
   ([113]Figure 5E) and strongly reduced HS5-induced NF-κB p65
   phosphorylation (n = 6, FD: 1.6, p < 0.05) ([114]Figure 5B) as in
   mono-cultured CLL cells. Moreover, using our co-culture system, we
   performed a proliferation assay for two U-CLL cases in the presence of
   calcitriol. On day 2, calcitriol was added to the culture medium and,
   on day 4, the percentage of proliferating (Ki-67^+) CLL cells was
   determined by flow cytometry. CLL co-cultured cells treated with
   calcitriol showed reduced proliferation compared to untreated cells
   ([115]Figure S4).

Figure 5.

   [116]Figure 5
   [117]Open in a new tab

   Co-culture of CLL cells with HS5 mesenchymal cells affects VDR
   signaling pathway, but not calcitriol action. (A) Viability of mono- vs
   co-cultured CLL cells was assessed by flow cytometry at 48 and 72 h of
   cell culture. (B–E) CLL cells from U-CLL cases were co-cultured with
   the HS5 stromal cell line and both mono- and co-cultured CLL cells were
   treated with calcitriol for 24 h. (B) pNF-κB^+ cells, (C) VDR (MFI),
   (D) CYP24A1^+, and (E) pERK^+ cells were determined by flow cytometry.
   Bars represent the median values and the Wilcoxon test was performed to
   assess statistical significance. * p < 0.05, ** p <0.01, FD: Fold
   Difference.

2.5. Preserved Calcitriol/VDR Signaling Capacity in CLL Cases under Ibrutinib
Therapy

   Next, we investigated whether the BTK inhibitor ibrutinib, an agent
   interfering with microenvironmental interactions in CLL [[118]29],
   might modulate the function of the calcitriol/VDR system by assessing
   VDR and CYP24A1 expression in 12 CLL patients under ibrutinib
   treatment. Three consecutive time points were selected; before
   treatment initiation, at 1 month (+1 m), and at 3–6 months (+3–6 m)
   under treatment. Flow cytometric analysis revealed that ibrutinib
   treatment led to a significant decrease in VDR levels both at +1 m (FD:
   1.4, p < 0.01) and at +3–6 m (FD: 1.9, p < 0.01) in comparison to the
   respective pre-treatment samples ([119]Figure 6A). Consistent with
   this, the percentage of CYP24A1^+ cells was also reduced at +1 m (FD:
   1.5, p< 0.05), with further reduction noted at +3–6 m (FD: 4, p < 0.05)
   ([120]Figure 6B,D).

Figure 6.

   [121]Figure 6
   [122]Open in a new tab

   The calcitriol/VDR system appears unaffected by inhibition (ibrutinib)
   of microenvironmental signals. (A,B) CLL cells from patients before
   treatment initiation with ibrutinib and at +1m and +3–6 m under
   treatment were evaluated for VDR and CYP24A1 expression by flow
   cytometry. Bars represent the median values and the Wilcoxon test was
   performed to assess statistical significance. (C,D) Representative
   histograms from flow cytometry analysis for CYP24A1^+ cells (C) at
   pre-treatment versus at +1 m of ibrutinib treatment and (D) at
   pre-treatment versus at +3–6 m of ibrutinib treatment. (E) VDR (MFI),
   CYP24A1 (% +), pERK (% +) and pNF-κB (% +) of CLL cells treated with
   calcitriol. Individual data points represent the fold difference (FD)
   of calcitriol-treated cells to the untreated control cells and the
   horizontal lines show the median value. Asterisks above each time point
   represent statistically significant differences compared to the
   untreated controls. * p < 0.05, ** p < 0.01.

   Moreover, CLL cells from patients under ibrutinib treatment, at both
   +1m and +3–6 m, were ex vivo exposed to calcitriol for 24 h. VDR levels
   were not affected by the addition of calcitriol at either timepoint. On
   the contrary, calcitriol treatment led to significantly increased
   CYP24A1 levels at +1m (FD: 1.6, p < 0.05) and +3–6 m (FD: 1.6, p <
   0.01). Phosphorylated ERK was also increased after calcitriol
   administration at both timepoints (FD: 1.3; FD: 1.6, p < 0.05
   respectively), while the phosphorylation of NF-κB was reduced at both
   +1 (FD: 3, p < 0.05) and +3–6 m (FD: 2.5, p < 0.05) ([123]Figure 6E).
   Altogether, these findings indicate that inhibition of immune signaling
   by ibrutinib does not affect calcitriol action in CLL.

3. Discussion

   In the present study, we addressed the question whether the
   calcitriol/VDR system is functional in CLL cells, investigated the
   biological processes and signaling pathways that might be regulated by
   calcitriol supplementation, and also explored the role of
   microenvironmental signals in the regulation of the calcitriol/VDR
   system.

   VDR mRNA expression in CLL cells was lower compared to B cells from
   healthy donors. That said, U-CLL displayed stronger VDR expression
   compared to M-CLL and, most importantly, higher VDR expression was
   found to be significantly associated with reduced overall survival and
   time-to-first treatment. On the contrary, RXRα, the VDR co-receptor,
   was expressed at similar levels in U-CLL and M-CLL cases and
   overexpressed in comparison to healthy B cells, in line with the
   literature [[124]30].

   Stimulation of CLL B cells with calcitriol induced CYP24A1, a target
   gene of activated VDR, indicating that VDR has a functional classical
   action as a transcription factor in CLL cells. CYP24A1 induction was
   greater in U-CLL versus M-CLL cases, perhaps linked to the increased
   VDR levels in the former. However, VDR expression was not affected by
   calcitriol supplementation in either U-CLL nor M-CLL, in contrast with
   normal B cells, where published data show that calcitriol
   administration leads to increased VDR expression [[125]3].
   Additionally, calcitriol supplementation in CLL cells induced ERK
   phosphorylation whereas it downregulated NF-κB p65 phosphorylation,
   indicating that the classical action of calcitriol to modulate gene
   transcription might also be complemented by its ability to regulate
   cytoplasmic signaling cascades. Non genomic actions of calcitriol are
   hypothesized to involve binding to cytosolic VDR and membrane VDR, and
   speculated to activate the mitogen-activated ERK1/2 cascade [[126]8].
   Calcitriol-induced ERK phosphorylation has been previously reported in
   promyelocytic NB4 leukemia cells and T helper 17 cells
   [[127]31,[128]32], while in CLL an opposite effect has been observed,
   though a different vitamin D analog was used in that study (EB1089)
   [[129]19]. Vitamin D-regulated NF-κB inhibition has also been reported
   in macrophages, suppressing TLR-mediated inflammation [[130]33].

   Delving into the mechanisms of calcitriol/VDR modes of action, our
   transcriptome analysis revealed that several genes regulated by
   calcitriol administration are participating in biological processes
   related to cell migration and motility, both mandatory for cancer
   progression [[131]34]. Interestingly, these processes have been
   reported to be inhibited by vitamin D in breast, ovarian, and colon
   cancer [[132]35,[133]36,[134]37]. Transcriptome analysis also revealed
   TLR and PI3K/AKT signaling regulation by calcitriol; this is in line
   with our finding that calcitriol regulates MAPK and NF-κB cascades,
   since ERK is activated by TLR signaling [[135]38], while pNF-κΒ is
   implicated in both PI3K/AKT and TLR signaling cascades
   [[136]38,[137]39], and both regulate important cellular processes
   concerning cancer cell proliferation and inflammation
   [[138]40,[139]41]. Supporting this result, we here show that calcitriol
   can inhibit TLR/CD40-induced CLL cell proliferation. Furthermore,
   calcitriol-induced downregulation of S100A8 and S100A9 genes, that are
   known to promote inflammatory processes [[140]42], as well as
   upregulation of CD14, that recognizes pathogen-associated molecular
   patterns [[141]43], implies that calcitriol mediates anti-inflammatory
   responses in CLL. Relevant to mention, S100A9 is overexpressed in CLL,
   increases with disease progression, and correlates with NF-κB pathway
   activation [[142]24]. Our findings are also in accordance with
   published evidence for calcitriol-mediated inhibition of inflammation
   via impairment of TLR signaling [[143]44]. Moreover, calcitriol has
   been shown to inhibit PI3K/AKT/mTOR signaling in a diffuse large B cell
   lymphoma (DLBCL) cell line and the HL60 cell line [[144]45].
   Considering the above and the downregulation of the NF-κB p65
   phosphorylation, as a result of calcitriol supplementation, that we
   show here, it is not unreasonable to argue that calcitriol could act as
   a potential PI3K/AKT inhibitor in CLL.

   CLL cells are highly dependent on the tumor microenvironment for their
   survival and proliferation. Our work suggests that microenvironmental
   triggers affect also the calcitriol/VDR system, since the prosurvival
   stimulants anti-IgM/CD40L modulate both CYP24A1 and VDR levels. This
   effect was observed exclusively in U-CLL, perhaps related to our
   finding of higher VDR expression in U-CLL versus M-CLL. Moreover,
   co-culturing CLL cells with the HS5 stromal cell line also increased
   CYP24A1 levels, altogether indicating that various stimulants, besides
   calcitriol, may regulate CYP24A1 expression. In any case, our present
   findings suggest that calcitriol action is likely independent of the
   microenvironmental signals in CLL, since it was not impeded when
   combined with anti-IgM/CD40L or in the context of the co-culture
   system.

   The regulation of calcitriol/VDR system by external triggering in CLL
   is further supported by our finding that CLL patients under ibrutinib
   treatment show reduced VDR and CYP24A1 expression. Importantly,
   calcitriol/VDR signaling capacity was maintained in CLL cells under
   ibrutinib since calcitriol administration to ibrutinib-treated cells
   could upregulate CYP24A1 and pERK and decrease pNF-κB, further
   supporting our argument that microenvironmental signaling does not
   impede calcitriol action.

   From a clinical perspective, the findings reported herein also raise
   the intriguing hypothesis that calcitriol supplementation might have a
   place in CLL patients under ibrutinib and/or other treatment
   modalities. Support to this hypothesis is provided by (i) associations
   between pretreatment vitamin D deficiency and inferior progression-free
   and overall survival in Hodgkin lymphoma [[145]46]; and, (ii)
   preclinical evidence in non-Hodgkin lymphoma that vitamin D
   replenishment can lead to improved rituximab-mediated cellular
   cytotoxicity in vitro [[146]12]. In CLL, a clinical trial carried out
   by the Mayo Clinic showed that vitamin D substitution in CLL patients
   with insufficient vitamin D levels is safe and feasible, however could
   not provide evidence that vitamin D can delay disease progression
   [[147]17]. More recently, the combination of vitamin D with curcumin
   was reported to be safe in previously untreated patients with early
   stage CLL or small lymphocytic lymphoma (SLL), yet event-free and
   overall survival did not show any alteration [[148]47]. Obviously,
   therefore, further studies are required in order to reach definitive
   conclusions regarding the role, if any, of vitamin D in the management
   of patients with B cell malignancies, including CLL.

4. Materials and Methods

4.1. Study Group

   Peripheral blood samples from 94 patients diagnosed with CLL, according
   to the guidelines of the International Workshop Chronic Lymphocytic
   Leukemia/National Cancer Institute (iwCLL/NCI) [[149]48], were obtained
   with patients’ informed consent. Patients were either treatment-naïve
   or off treatment at least 6 months prior to sample collection,
   excepting the ibrutinib study group, which was analyzed at 1 month and
   3–6 months after treatment initiation. Clinicobiological data for the
   study group are listed in [150]Table S3. Moreover, peripheral blood
   samples were collected after informed consent from 29 age-matched
   healthy donors. The study was approved by the Ethics Committee of CERTH
   (decisions: ETH.COM-1| 2 July 2014; and ETH.COM-45 | 21 March 2019) and
   conducted in accordance with the Declaration of Helsinki.

4.2. Cell Isolation and Cell Cultures

   CD19^+ cells were negatively selected from whole blood from CLL
   patients and healthy donors using the RosetteSep B-cell enrichment kit
   (StemCell Technologies, Vancouver, Canada) according to the
   manufacturer’s instructions. The purity of the samples was assessed by
   flow cytometry and the percentage of CD19^+ cells always exceeded 95%.

   Both CLL cells and the HS5 stromal cell line (ATCC^® CRL-11882™) were
   cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA)
   supplemented with L-glutamine, 10% heat-inactivated fetal bovine serum
   (FBS), 50 μg/mL penicillin/streptomycin and 15 μg/mL gentamicin. All
   cultures were incubated at 37°C in a humidified 5% CO[2] atmosphere.
   The HS5 cell line was a kind gift from Prof. Asterios S Tsiftsoglou
   (Aristotle University of Thessaloniki, Thessaloniki, Greece).

   CLL cells were cultured at a density of 3 × 10^6 cells/mL in the
   presence of different stimulants for certain time points, as detailed
   below: (i) BcR stimulation: 10 μg/mL goat F(ab)2 anti-human IgM
   (ThermoFisher Scientific, Waltham, MA, USA); (ii) CD40 stimulation:
   soluble 0.1 μg/mL CD40L plus 1 μg/mL enhancer (Enzo Life Sciences);
   (iii) TLR9 stimulation: 2.5 μg/mL CpG ODN 2006 (Invivogen, San Diego,
   CA, USA); and (iv) VDR stimulation: 100 nM calcitriol (Selleckchem,
   Houston, TX, USA).

   Regarding the co-culture system, 5 × 10^4 HS5 cells/mL were seeded per
   well in a 24-well plate. Three days later, the confluence of the HS5
   cell layer was checked and CLL cells were added at a ratio of 60:1 (3 ×
   10^6 cells/mL). CLL cells were treated with DMSO (vehicle control) or
   calcitriol (100 nM). Cells were harvested at the indicated time points
   with mild trypsinization (ThermoFisher Scientific).

4.3. Quantification of VDR and RXRα mRNA Expression

   Total cellular RNA was isolated from purified CLL B cells and healthy
   donors’ B cells with the NucleoSpin RNA Kit (Macherey-Nagel, Düren,
   Nordrhein-Westfalen, Germany), including a DNAase digestion step.
   Quantification of VDR and RXRα mRNA levels were achieved by RQ-PCR.

   One microgram of total RNA was reverse transcribed to cDNA and a 2.5:50
   aliquot of the RT product was used as the template for RQ-PCR.
   Quantification of VDR and RXRα mRNA levels were achieved by RQ-PCR,
   using custom designed primers and EvaGreen qPCR mix (Solis Biodyne,
   Tartu, Estonia). The GAPDH gene was used as reference (housekeeping
   gene). Gene primer sequences used were VDR:
   5′-CCTTCTGTGACCCTAGAGCTGTCC-3′ and 5′-TCATCTTAGCAAAGCCAATGACCT-3′;
   RXRα: 5′-TTCGCTAAGCTCTTGCTCCG-3′ and 5′-AGGTGTCAATGGGTGTGTCC-3′; GAPDH:
   5′-ACTGTGGATGGCCCCTCCGG-3′ and 5′-ACGGCAGGTCAGGTCCACCA-3′. For RQ-PCR
   experiments all samples were run in triplicate. Data were analyzed
   using the 2^−ΔΔCt method [[151]49].

4.4. RNA Sequencing

   Total cellular RNA was extracted from calcitriol-treated and
   non-treated CLL cells from 6 patients. RNA was extracted using
   NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer’s
   instructions. One μg of total RNA was used for poly(A) mRNA selection
   using NEBNext Poly(A) mRNA Magnetic Isolation Module. Library
   preparation was performed with the NEBNext Ultra II Directional RNA
   Library Prep Kit. The libraries were paired-end sequenced on the
   NextSeq 500 Illumina platform using the NextSeq 550 System Mid-Output
   Kit (Illumina, San Diego, CA, USA). Data analysis was performed based
   on the ‘new tuxedo’ protocol [[152]50].

   Reads were trimmed using cutadapt (version 1.8.1) and then mapped to
   the hg19 UCSC reference genome using HISAT2 (version 2.0.5). The
   alignments were then passed to StringTie, which assembles and
   quantifies the transcripts in each sample. Additionally, read
   quantification at the gene level was performed using htseq-count
   (version 0.6.0) as well as featureCounts (subread suite, version
   1.6.4).

   Differential expression between calcitriol-treated and non-treated CLL
   cells was assessed using DESeq2 (Bioconductor,
   10.18129/B9.bioc.DESeq2). Genes with log2 fold change (log2FC) ≥ |1|
   and p ≤ 0.05 were considered as significantly differentially expressed.
   Hierarchical clustering was applied to assess distinct expression
   profiles based on the values of all significantly differentially
   expressed genes. “Heatmapper” software was used for that purpose
   [[153]51]. Gene ontology and pathway enrichment analyses were performed
   using the online tools “GenCLiP3” [[154]52] and “gProfiler” [[155]53].
   RNA sequencing datasets have been deposited in the GEO database
   (accession number: [156]GSE162427).

4.5. Flow Cytometry Studies

   Cell viability, proliferation, apoptosis, intracellular expression of
   VDR and CYP24A1 as well as the expression of phospho-ERK (pERK) and
   phospho-NF-κB p65 (pNF-κB) were determined by flow cytometry. In all
   experiments, the BD Horizon Fixable viability stain 660 (BD
   Biosciences, Franklin Lakes, NJ, USA) was used for gating viable cells.
   Co-cultured CLL cells were further stained for surface CD19 (PE-Cy5
   mouse anti-human CD19 antibody, BD Biosciences) in order to be gated
   against the HS5 cells. Intracellular staining of VDR and CYP24A1 was
   performed with the Fixation/Permeabilization Solution Kit (BD
   Biosciences) following the manufacturer’s instructions. Intracellular
   staining of pERK and pNF-κB was carried out following the phosflow
   protocol from BD Biosciences. The list of the antibodies used is given
   in [157]Table S4. Apoptosis was measured using the FITC Annexin V
   Apoptosis Detection Kit with PI (Immunostep, Salamanca, Spain), whereas
   cell proliferation was measured using FITC Mouse Anti-Ki-67 Set (BD
   Biosciences).

   Flow cytometric analysis was conducted on a BD FACSCalibur flow
   cytometer (BD Bioscience). For each sample, 10,000 events were acquired
   and nonspecific binding was excluded by using appropriate isotypic
   negative control antibodies. Gating analysis strategy included only
   viable cells and was performed using FlowJo software (BD Biosciences).

4.6. Statistical Analysis

   The statistical significance of differences between groups of variables
   and matched samples was assessed with the use of Mann-Whitney test and
   the Wilcoxon test, respectively. Descriptive statistics for
   quantitative variables included statistical measures like mean, median,
   and SD. Values less than 0.05 were considered statistically
   significant.

   Survival analysis was conducted to assess the impact of VDR on overall
   survival (OS) and TTFT. In particular, the “survminer” R package was
   used to determine the optimal cutpoint for VDR mRNA levels, leading to
   the generation of two groups of CLL patients: one with high VDR mRNA
   levels (VDR^high) and one with low VDR mRNA levels (VDR^low). To
   compare the OS and TTFT in both groups (high vs. low), Kaplan-Meier
   curves were plotted and p values were calculated. All statistical
   analyses were performed with the use of the GraphPad Prism 8 software
   (La Jolla, CA, USA), and R.

5. Conclusions

   Our study shows that the calcitriol/VDR system is functional in CLL
   cells, regulating signaling pathways critical for cell survival and
   proliferation. Although external triggers can modulate VDR expression
   and function in CLL, calcitriol acts independently and remains
   unaffected by microenvironmental signaling, prompting further
   investigations into the potential clinical utility of vitamin D
   supplementation in CLL patients under treatment.

Acknowledgments