The CXCL12gamma chemokine immobilized by heparan sulfate on stromal niche cells controls adhesion and mediates drug resistance in multiple myeloma

The uncontrolled growth of cancer cells is driven by mutations in essential growth control genes, but their growth and survival are also strongly dependent on signals from the tumor microenvironment. In multiple myeloma (MM), a clonal expansion of malignant plasma cells in the bone marrow (BM), the interaction with specific BM niches plays an important role in tumor cell proliferation and survival. This interaction involves signaling via cell surface receptors, including adhesion molecules, as well as by soluble factors secreted by various cells in the BM niche [1‐3]. Despite improved survival due to the introduction of proteasome inhibitors, immunomodulatory drugs, and, more recently, monoclonal antibodies targeting MM cells [4‐6], MM is generally still incurable, which is largely due to the development of therapy resistance. MM cell interaction with the BM niche is believed to play a key role in this resistance; hence, targeting this interaction presents a promising therapeutic strategy [1, 7, 8].

The homing of hematopoietic stem cells (HSCs) as well as plasma cell precursors to the BM is controlled by the chemokine CXCL12 [9, 10]. This chemokine also regulates the adhesion, transendothelial migration, and homing of MM cells to the BM by binding its receptor CXCR4 on the MM cells [11‐13]. In the BM microenvironment, CXCL12 is mainly produced by specialized reticular BMSCs, also referred to as ‘CXCL12-abundant reticular (CAR)’ cells. Several splice variants of CXCL12 have been identified [14], which all contain the CXCR4-binding motif but are differentially expressed in various murine and human tissues [15]. To date, the functional differences and biological significance of these distinct isoforms have remained largely unexplored. Virtually all in vitro functional studies, including those on MM cell migration and adhesion, have exclusively employed the CXCL12α isoform. Moreover, reported in vivo studies do not allow conclusions concerning the specific functions of the distinct CXCL12 isoforms, since the mice employed carried either a full deletion of CXCL12 or a deletion of CXCR4, the cognate receptor for all isoforms [16‐19]. Interestingly, the recently characterized gamma isoform of CXCL12 (CXCL12γ) has been shown to promote leukocyte accumulation and angiogenesis with a much higher efficacy than the ‘canonical’ CXCL12α isoform [15]. This enhanced biological activity of CXCL12γ is mediated by its extended C-terminal domain, which binds heparan sulfate proteoglycans (HSPGs) with an unprecedentedly high affinity [15, 20, 21]. Notably, in mouse BM, CXCL12γ was reported to be the dominant CXCL12 isoform. Furthermore, mice with a partial deletion in the HSPG-binding motives of CXCL12 showed increased numbers of circulating HSCs, suggesting a role for CXCL12-HSPG interaction in the retention of HSCs in the BM [22].

HSPGs are membrane-bound or extracellular matrix proteins, consisting of a core protein decorated by covalently linked HS side chains composed of repeating disaccharide units. These HS chains undergo complex enzymatic modifications, which determine their binding capacity and specificity [23, 24] for a wide variety of morphogens, growth factor, and chemokines, thereby controlling the spatial distribution and activity of these ligands [25‐27]. Given these properties, HSPGs appear well equipped to act as organizers of growth and survival niches. Indeed, studies in Drosophila have shown a crucial role for HSPGs in the germ cell as well as hematopoietic stem cell niches, controlling the activity of bone morphogenetic proteins (BMPs) [28, 29]. In addition, HSPGs are known to bind a variety of proteins like Wnts, fibroblast growth factor (FGF), Midkine, and CXCL12, involved in the control of intestinal, neural, and hematopoietic niches [25, 26, 30].

The extraordinary high affinity of CXCL12γ for HS, and its strong expression in mouse BM, prompted us to hypothesize that CXCL12γ could have a specific role in the organization of BM niches, including the plasma/MM cell niche. To explore this notion, we investigated the expression of this CXCL12 isoform in human BM and studied its functional role in the interaction of MM cells with BMSCs cells.

The CXCL12/CXCR4 axis plays a key role in the homing of normal plasma cell precursors and MM cells to the BM [9, 10], but the expression and specific role of CXCL12γ, a recently characterized CXCL12 isoform, which binds HSPGs with an extremely high affinity, have not been addressed. Here, we show that CXCL12γ is expressed in situ by reticular stromal cells in the human bone marrow niche as well as by BMSC lines and primary BMSC isolates. Unlike CXCL12α, CXCL12γ is immobilized on the cell surface of BMSCs by HSPGs, upon secretion. Functionally, this membrane-bound CXCL12γ promotes adhesion of MM cells to the stromal niche cells, thereby protecting MM cells from drug-induced cell death.

Our study of the in situ expression of CXCL12γ shows that it is expressed by CAR-like reticular stromal cells in the BM. In normal BM, distinct CXCL12γ expression was present on stromal cells with long cytoplasmic processes, scattered among hematopoietic cells, as well as around adipocytes and capillaries, and in the endosteal zone (Fig. 1a), areas with putative niche functions [33‐38]. In BM sections of MM patients, CXCL12γ was also observed on stromal cells in areas infiltrated by MM cells. Notably, employing an antibody against an epitope shared by all CXCL12 isoforms, Abe-Suzuki et al. [37] recently reported a similar expression pattern, which also resembles the distribution of CAR cells in mouse bone marrow [9]. Study of isolated primary BMSCs and BMSC lines corroborates these findings, demonstrating that CXCL12γ is specifically expressed by isolated primary BMSCs and BMSC lines (Fig. 1b).

CXCL12γ possesses an extraordinarily high affinity for HSPGs due to its unique C-terminal domain [15, 20]. Interestingly, we observed that both primary BMCSs and HS5 cells constitutively express CXCL12γ on their cell surface, suggesting that this chemokine is retained by HSPGs upon secretion (Fig. 1c). Indeed, we observed that KO of the HS-chain co-polymerase EXT1 in HS5 BMSCs results in a complete loss of membrane-bound CXCL12γ. Importantly, immobilization by cell surface HS was a specific feature of the CXCL12γ isoform, since overexpression of CXCL12α in HS5 did not result in detectable membrane retention, notwithstanding substantial intracellular expression (Fig. 2).

We observed that specific deletion of CXCL12γ strongly reduces the capacity of HS5 BMSCs to mediate adhesion of MM cells to their cell surface. This result extends the previous observation that a total (i.e., non-isoform specific) knockdown of CXCL12 reduces the capacity of BMSCs to mediate adhesion of MM cells [8], pinpointing this effect to the CXCL12γ isoform. Similar to CXCL12γ deletion, EXT1 deletion also attenuated MM cell adhesion to the BMSCs. Importantly, whereas the defective adhesion to HS5-CXCL12γKO cells could be overcome by exogenous expression of CXCL12γ, this could not correct the adhesion defect in HS5-EXT1KO cells, indicating that CXCL12γ immobilization by HSPGs is critically required (Fig. 5). In line with this notion, in experiments employing recombinant CXCL12 to induce MM cell adhesion to VCAM-1 plastic, we observed that only immobilized (i.e. coated) CXCL12 effectively induced adhesion (Fig. 3b).

Interaction of MM cells with BMSCs plays a central role in MM cell homing/retention and can also confer drug resistance [1, 7]. We observed that co-culture with HS5 BMSCs of the HMCLs XG1 and MM1.S and of primary MM cells did hardly or not affect tumor cell viability per se, but significantly reduced their sensitivity to the proteasome inhibitors bortezomib and carfilzomib. Interestingly, this resistance was largely annulled by specific deletion of CXCL12γ in BMSCs, identifying CXCL12γ as a major factor in the BMSC-mediated drug resistance. HS5 BMSCs cells with a deletion of EXT-1 showed a similarly reduced capacity to protect MM cells, showing the essential role of membrane retention of CXCL12γ by HSPGs (Fig. 6).

Drug resistance mediated by BMSCs can be caused either by soluble factors or by interactions via cell adhesion molecules [1, 42, 43]. We observed that the protective effect of BMSCs to MM cells was largely abolished by physical separation of the MM and BMSCs, implying that this protection requires direct cell–cell contact (Fig. 7a, b). This suggests that BMSCs might convey MM drug resistance via direct integrin-mediated signals, rather than by soluble growth and survival factors, although such factors are abundantly expressed by BMSCs [30, 41, 44]. However, recombinant CXCL12γ (or CXCL12α)-induced adhesion to VCAM-1-coated plastic did not protect MM cells against bortezomib-induced cell death (Additional file 1: Figure S6), indicating that integrin-mediated cell adhesion per se is not sufficient to instigate bortezomib resistance. Conceivably, CXCL12γ-controlled adhesion serves to retain MM cells in close physical contact with the BMSCs, providing MM cells with growth and survival signals through integrin receptors as well as with access to short-range growth and survival factors, such as Wnts and vascular endothelial growth factor [45, 46], which may act in concert to mediate drug resistance.

Our data suggest targeting CXCL12γ and/or its interaction with HSPGs, as a potential therapeutic strategy. Notably, MM cells express high levels of the HSPG syndecan-1, which is crucial for MM cell survival [47, 48] and promotes Wnt-mediated cell proliferation [30] as well as hepatocyte growth factor (HGF), FGF, epidermal growth factor (EGF), and a proliferation-inducing ligand (APRIL)-mediated signaling [49‐51]. Hence, targeting HSPGs or the HS biosynthesis machinery may disconnect the interaction of MM cells with the BM microenvironment at various levels [52]. Our studies corroborate previous studies, showing that disruption of the interaction between MM cells and BMSCs by the CXCR4 inhibitor AMD3100 enhances MM sensitivity to multiple therapeutic agents such as bortezomib, dexamethasone, and melphalan [1, 7, 41]. Furthermore, targeting pan-CXCL12 by olaptesed pegol (ola-PEG), which neutralizes CXCL12 irrespective of the isoform, prevented MM progression in a murine model [8], while a recent phase IIa clinical trial showed that patients with relapsed/refractory MM respond favorably to a combination of bortezomib or dexamethasone with ola-PEG [53]. Apart from CXCR4, MM cells also express CXCR7, an alternative receptor of CXCL12, which may also be involved in CAM-DR in MM [7] as well as in MM progression [54]. Targeting the ligand CXCL12(γ) will simultaneously inhibit signaling through both chemokine receptors. It will also be of interest to explore whether CXCL12γ plays similar roles in the interaction of other hematological malignancies with the BM microenvironment, including acute myeloid leukemia (AML) and non-Hodgkin lymphomas.

Taken together, our data suggest a scenario in which CXCL12γ functions as a ‘niche chemokine’ that, in conjunction with HSPGs, plays a key role in controlling adhesion, BM retention, and CAM-DR of MM cells (Fig. 7c). These findings identify this unique membrane-bound chemokine, and associated HSPGs, as potential therapeutic targets in MM.