Drug resistance is a significant obstacle in the treatment of patients with multiple myeloma. The tumor immune microenvironment (TIME), consisting of various immune and stromal cells, is a major contributing factor to tumor relapse and drug resistance(27). Previous proteomic studies on multiple myeloma have focused mainly on molecular changes in myeloma cells, with limited investigation into the TIME(28–31). Here, we employed an integrated proteomics and metabolomics approach to examine plasma samples from pomalidomide-resistant/sensitive patients. Our results demonstrate a significantly lower level of complement components in the resistant group compared to the sensitive group. We found that the expression of complement activating proteins and complement terminal pathway proteins was significantly down-regulated in the drug-resistant group, suggesting that complement deficiency correlates with resistance to pomalidomide.
Patients with multiple myeloma have complex immunological defects(32). As an innate immune surveillance system, complement is a functional bridge between innate and adaptive immune responses, and plays a crucial role in almost every step of the immune reaction(33). Previous studies have shown abnormalities in complement levels and function in multiple myeloma(29, 34), leading to an imbalance in the immune system. Compared to the normal group, the catabolism of C1q in the plasma of myeloma patients was significantly accelerated(35). Yang et al. revealed that C1q levels are negatively correlated with MM tumor burden(36). A retrospective study showed that low levels of C4 were significantly correlated with poor prognosis in patients with MM(37). Our research suggests that the defect of complement in multiple myeloma should not be explained on the basis of a single complement component abnormality, but rather results from a group of complement abnormalities.
Previous studies have investigated the role of complement in drug resistance in MM. Daratumumab, a monoclonal antibody against CD38, has shown promising anti-tumor activity in the treatment of MM. It has been reported that resistance to daratumumab was associated with the upregulation of complement inhibitory proteins CD55 and CD59 on the MM cells(38). Complement deficiency may be a common feature in the development of resistance to immunomodulatory drugs, as highlighted by our findings.
Our study emphasizes the significance of the complement pathway in the development of drug resistance in MM, although the reasons for the complement deficiency remain unclear. Here we propose potential causes of complement deficiency in myeloma. The matrix metalloproteinases (MMPs) play an important role in tumor progression, including migration, invasion, metastasis and angiogenesis(39–41). Membrane-type 1 matrix metalloproteinase (MT1-MMP) interacts with the C1q and C3b components of the complement system and inactivates the complement propagation cascade, protecting tumor cells from the complement-mediated cytolysis(42, 43). In addition, it has been demonstrated that MT1-MMP and MT2-MMP are highly expressed in MM cells rather than in normal B cells and plasma cells, and contribute to the degradation of the extracellular matrix and the invasion of MM(44, 45). Further experimental work is required to define functional relationships of MMPs with complement contributing to pomalidomide resistance in multiple myeloma.
Furthermore, our targeted metabolomics analysis showed that glycine was significantly down-regulated in pomalidomide-resistant MM patient sera. Our results demonstrate that the addition of glycine increases the sensitivity of MM cell lines to pomalidomide. While glycine has been shown to promote MM proliferation through GSH synthesis(46, 47), our study found that glycine synergizes with pomalidomide to promote apoptosis and suppress proliferation in MM cells. The consumption and release profiles of the NCI-60 cancer cell lines revealed that glycine has a heterogeneous pattern of consumption and release, which may explain its paradoxical effects on cancer cells(48). In addition, dietary supplementation of glycine has been reported to inhibit the growth of certain types of tumors, such as liver tumors and melanoma tumors (49, 50). Additional experimental investigation is necessary to elucidate the precise mechanisms responsible for the impacts of glycine in MM.
Pomalidomide is a drug known to have a therapeutic effect through various mechanisms, including immune modulation by enhancing the function of T and NK cells(51). However, the contribution of immune cells to pomalidomide resistance remains poorly understood. In this study, we aimed to estimate the proportions of immune cells in peripheral blood and identify potential immune signatures associated with pomalidomide resistance using the ssGSEA algorithm. Our immune infiltration analysis revealed that resistant patients had a lower proportion of B cells and a higher proportion of Tfh cells in peripheral blood compared to the sensitive group, indicating the presence of an underlying immune signature that could predict the likelihood of benefiting from pomalidomide treatment.
B cell lineage is significantly altered in MM, characterized by a disrupted equilibrium towards the excessive proliferation of malignant plasma cells(52). Previous studies have shown that the level of B cells is inversely correlated with disease stage and is reduced in MM patients(53, 54). Our findings are consistent with these previous studies, indicating that the decreased level of B cells in peripheral blood of resistant patients may be indicative of a more heavily compromised B cell lineage. Further research is needed to elucidate the mechanisms behind the involvement of B cells in pomalidomide resistance.
T follicular helper (Tfh) cells play a crucial role in the formation of germinal centers (GCs) by providing necessary signals to B cells for their differentiation into plasmablasts and plasma cells that secrete high-affinity and isotype-switched antibodies(55). Circulating follicular helper T cells (cTfh) in peripheral blood have also been shown to induce plasmablast or plasma cell differentiation and antibody production(56). Resting PD-1 + CXCR3 − cTfh cells are a population of circulating memory Tfh cells that exhibit similar stimulating activity to GCTfh cells after activation(57), and transcriptionally and clonally resemble GC Tfh cells, suggesting that GCTfh can exit the GC and enter the pool of circulating memory T cells(58, 59). We hypothesize that cTfh share clonotypes with GCTfh cells that are associated with malignant plasma cells, potentially inducing the differentiation of B cells into malignant plasma cells. This proposed mechanism could provide insights into the relapse of multiple myeloma.
Moreover, our correlation analysis revealed significant correlations between B cells and Tfh cells with complement components. It has been known for 40 years that B cells express complement receptor CR2 (CD21), which interacts with C3d and iC3b on the surface of the antigen and induces an increase of B cell receptor (BCR) signaling(60, 61). Additionally, there is considerable evidence indicating that complement regulates T cell proliferation and differentiation (62–64). Our findings support the involvement of the complement pathway in the immune response underlying the resistance to pomalidomide.
Reactive oxygen species (ROS) production is a critical step in immune cell activation, and amino acids play a central role in regulating redox balance within immune cells(25, 65, 66). Glutathione (GSH) is a major cellular antioxidant molecule in cells(67). Upon activation, T cells increase GSH synthesis (68), while B cells and macrophages require GSH to regulate their redox status after ROS production(69, 70). Our WGCNA profiling results are consistent with previous studies, showing that glutathione derivative biosynthetic metabolism is significantly correlated with lymphocyte levels in MM patients. As glycine is a component of glutathione, its availability is a key determinant of glutathione levels. Recent evidence suggests that glycine has immunomodulatory effects(71). We also found that glycine levels were significantly correlated with complement components and immune cell proportions. Our results suggest that glycine may play a potential role in pomalidomide resistance through immunomodulatory effects, which require further investigation. Our analysis also highlighted the potential involvement of T follicular helper (Tfh) cells and B cells in the resistance to pomalidomide. These cells were found to be significantly correlated with complement levels, indicating their potential role in immune response and resistance to pomalidomide.
Limitations of the study
We acknowledge that this study has some limitations. First, the sample sizes in this study was relatively small, and the results need a larger sample size to verify. Second, the analysis of immune cells proportion is based on the estimation of the ssGSEA algorithm, which needs to be further confirmed by flow cytometry. Finally, this study lacks laboratory validation of the molecular mechanism regulating the resistance of pomalidomide, which can be further explored.