1. Introduction
Multiple myeloma (MM) is a relatively common and incurable hematological malignancy arising from post–germinal mature B cells and it is characterized by the presence of proliferating plasma cells in the bone marrow that secrete specific monoclonal immunoglobulin (also called M-protein) [ 1 ]. Given the exacerbated production of immunoglobulin, MM patients often suffer from a concomitant decrease in normal immunoglobulins [ 2 ] that causes immune dysfunction, increases susceptibility to opportunistic infections, and impacts on disease severity and prognosis [ 3 , 4 ].
Although the high production of monoclonal immunoglobulins occurring in MM invariably results in the presence of a high amount of unfolded or misfolded proteins that might be toxic for MM cells in the bone marrow, recent studies demonstrated that plasma cells are able to exploit molecular pathways to protect themselves from damage caused by toxic proteins [ 5 ]. These molecular pathways include the activation of unfolded protein response (UPR) [ 6 , 7 , 8 ], heat protein chaperones [ 9 ], aggresome formation [ 10 ], and the induction of cellular autophagy [ 5 ]. Autophagy is a lysosome-dependent catabolic degradation process by which cells remove toxic aggregated cytosolic proteins and malfunctioning organelles. It has been well-documented that autophagy not only is an autonomous mechanism that modulates cell homeostasis at basal level, but it is also involved in B-cell development and proliferation [ 11 ], cell survival [ 12 , 13 ], apoptosis [ 14 , 15 ], tumorigenesis, anti-tumoral immune responses [ 16 ], and resistance to chemotherapeutic agents. In this regard, a growing number of studies have suggested that autophagy shapes anti-tumoral immune responses by acting at multiple levels [ 17 ]. This includes the promotion of signals to activate phagocytosis of tumor cells [ 18 , 19 ], as well as signals to induce myeloid cell recruitment [ 20 ], MHC-class-I and -II presentation [ 21 ], B- and T-cell activation, development, maintenance [ 11 , 22 ], and self-tolerance [ 23 ]. Strikingly, experimental studies have demonstrated that the inhibition of autophagy enhances the sensitivity of MM cells to a number of anticancer agents and induces MM cell death [ 24 , 25 ]. Based on these findings, it has been suggested that autophagy could be targeted to treat MM [ 5 , 24 , 26 ] and recent studies and clinical trials have indeed confirmed the therapeutic implications of new and less toxic autophagy inhibitors in MM, used in combination with current anti-MM drugs [ 27 , 28 , 29 ].
In support of a possible role of autophagy in MM, it was reported that aberrant expression of autophagy-related genes is associated with cancer development [ 30 ] and that multiple activators of autophagy or specific autophagy-related genes are commonly found in cancer-associated regions [ 31 ]. Interestingly, it was also described that expression of autophagy-related genes influences the response to conventional treatments in MM [ 32 , 33 , 34 ] and, thereby, disease progression [ 35 , 36 ], arguing that they may be useful to predict disease risk [ 37 ]. However, despite these findings suggesting a key role of autophagy in the etiology of MM and the existence of a genetic component controlling this catalytic process in MM, so far only ULK4 , ATG5, and CDKN2A polymorphisms have been suggested to have an impact on the risk of MM [ 38 , 39 ]. Therefore, it is vital to perform a systematic analysis of autophagy-related markers to identify new susceptibility variants for MM and validate those associations already reported.
In this context, the aim of this study was to comprehensively evaluate the impact of common genetic variation of 234 autophagy-related genes in determining the risk of developing MM. We also assessed the influence of the most promising markers on modulating immune responses in whole blood, peripheral blood mononuclear cells (PBMCs), and monocyte-derived macrophages (MDM) from a large population of healthy donors from the Human Functional Genomic Project (HFGP). Additionally, we measured the autophagy flux in an independent population.
2. Results
2.1. Association of Autophagy-Related Polymorphisms with the Risk of Developing MM
This study included 8719 individuals (3916 MM cases and 4803 controls) from the German GWAS consisting of 1512 MM patients and 2107 controls and the InterLymph MM GWAS that included 2404 MM cases and 2696 healthy controls. Selected polymorphisms showed no deviation from HWE ( p < 0.001), either in the German GWAS or in the InterLymph MM GWAS. The association analysis of the German cohort showed that 440 independent SNPs (r 2 < 0.1) were significantly associated with MM risk at p ≤ 0.05. The association of these SNPs with MM risk was then validated through meta-analysis with data from the InterLymph MM GWAS. The meta-analysis of these large independent studies confirmed the association of 12 genetic variants within the ATG5 , CD46 , CDKN2A , CTSD , HSPB8 , IKBKE , PARK2 , RPTOR , ULK4 , and USP10 loci with MM risk ( Table 1 ), with loci in ULK4 and ATG5 reaching the highest level of statistical significance.
Although the lack of significant heterogeneity between both study populations suggested that the association found for these 12 SNPs might represent true associations, we decided to replicate these findings in a third independent population ascertained through the IMMEnSE consortium that included 2696 MM cases and 1701 controls. Results are reported in Table 2 . After correction for multiple testing ( p Bonferroni = 1.14 × 10 −4 ), we could confirm the previously reported association for ULK4 , ATG5 , and CDKN2A polymorphisms. Importantly, we also found that the association of the IKBKE rs17433804 SNP with the risk of MM was observed after multiple testing correction, which suggests that this gene could represent a new susceptibility locus for MM. In addition, although the associations were borderline significant after correction for multiple comparisons, we found of interest the associations of CD46 and PARK2 variants with the risk of developing MM. Although these associations need to be further validated, it was important to confirm that, with the exception of the CDKN2A , all the association signals included several SNPs (LD blocks), which reinforced the role of these loci in determining disease risk ( Supplementary Figure S1A–F ).
2.2. Functional Relevance of Autophagy-Related SNPs
Given the relatively strong association of the genetic variants within the CD46 , IKBKE , PARK2 , ULK4 , ATG5 , and CDKN2A loci on MM risk, we explored whether these variants could modulate host immune responses, serum steroid hormones, circulating immunological proteins, and blood-derived cell populations using data from the HFGP population. Although the genetic association of the USP10 rs7202154 SNP with MM risk was modest, we decided to include it in the functional analysis due to the known role of USP proteins on the modulation of MM cell apoptosis.
Interestingly, we found a significant association between the ULK4 rs6599175 SNP and circulating concentrations of vitamin D3 ( p = 4.0 × 10 −4 , Figure 1 A), which suggests a possible involvement of this SNP in modulating MM risk through a vitamin D-dependent mechanism. We also observed that the presence of the IKBKE rs17433804C allele was associated with increased numbers of transitional CD24 + CD38 + B cells ( p = 4.8 × 10 −4 , Figure 1 B), whereas carriers of the IKBKE rs17433804C/C genotype showed decreased circulating serum concentrations of MCP-2 ( p = 3.6 × 10 −4 ; Figure 1 C), a chemotactic molecule involved in the activation of multiple immune cells and linked to MM cell migration.
We also found that homozygous carriers of the CD46 rs1142469A allele, which was associated with an increased risk of MM, had decreased numbers of CD19 + B cells, CD19 + CD3 − B cells, CD5 + IgD − cells, IgM − cells, IgD − IgM − cells, and CD4 − CD8 − PBMCs compared with those subjects carrying the G allele ( p = 0.00025–0.00086; Figure 2 A–F).
Moreover, we found that homozygous carriers of the CD46 rs1142469A risk allele had increased circulating concentrations of IL20 compared with those subjects carrying the G allele ( p = 0.00082; Figure 2 G), which suggests a role of this angiogenic cytokine in modulating MM risk.
In addition, we observed that carriers of the CDKN2A rs2811710C allele had decreased levels of CD4 + EMCD45RO + CD27 − cells ( p = 0.00093; Figure 3 A), which constitutes a subset of memory T cells that does not require co-stimulation for T-cell receptors to display a high antigen recall response. Finally, we found that carriers of the USP10 rs7202154G/G genotype had decreased synthesis of p62 and LC3-II compared to non-carriers ( p = 0.005 and p = 0.047; Figure 3 B,C). Given that p62 serves as a useful marker for the induction of autophagy, clearance of protein aggregates, and the inhibition of autophagy and LC3-II serves to track the binding of p62 and subsequent recruitment of autophagosomes, this result might suggest that this genetic variant could be involved in determining autophagy activation. Given that the association of the USP10 SNP with autophagy flux markers (LC3-II/Actin ratio) did not remain statistically significant after correction for multiple testing, additional studies are needed to confirm whether the weak association of this SNP with MM risk might be mediated by the regulation of the autophagy flux. No functional effect on host immune responses or autophagy flux was detected for the remaining selected SNPs, which suggests that the effect of these variants on MM risk is not mediated by these biological processes.
3. Discussion
To the best of our knowledge this is the first population-based case-control study comprehensively assessing the role of autophagy-related SNPs in modulating the risk of developing MM. After a comprehensive meta-analysis of three large independent studies, we found three novel significant associations between MM risk and SNPs within CD46 , IKBKE , and PARK2 , in addition to confirming the known associations with MM-susceptibility genes ULK4 , ATG5 , and CDKN2A loci.
As the most interesting finding, we found an association of the CD46 rs1142469 SNP with the risk of developing MM. The CD46 locus is located on chromosome 1q32 and has been involved in pathogen recognition and in the differentiation of CD4 + cells into T-regulatory 1 cells that suppress immune responses by secreting IL10. The CD46 rs1142469 SNP is an intronic variant that acts as eQTL for the CD46 in multiple tissues [ 40 , 41 ] and lymphoblastoid cell lines [ 41 ] and it is in LD with three neighboring SNPs that confirm the association signal of the CD46 locus with MM risk. Our functional results also showed that homozygous carriers of the CD46 rs1142469A risk allele had decreased numbers of CD19 + B cells, CD19 + CD3 − B cells, CD5 + IgD − , IgM − cells, IgD − , IgM − cells, and CD4 − , CD8 − PBMCs, but also increased circulating concentrations of IL20. This latter observation is consistent with previous studies, which reported that IL20 is highly expressed in MM patients and correlates with levels of angiogenic cytokines and bone marrow microvascular density [ 42 ]. Although none of these functional results survived after correction for multiple testing and therefore need to be replicated, they are consistent with previous studies suggesting a relevant role of the CD46 locus in MM pathogenesis. Considering that the CD46 rs1142469 SNP also alters binding sites for Hoxa7 [ 43 ], a transcription factor that is frequently dysregulated in MM patients and plays a role in modulating hematopoiesis and cell differentiation [ 44 ], it seems conceivable to suggest that the CD46 rs1142469 SNP could influence the risk of developing MM by modulating not only CD46 expression, but also the number of specific subsets of B and T cells and IL20 − and Hoxa7-mediated signaling pathways.
We also found, for the first time, that carriers of the IKBKE rs17433804C allele had an increased risk of developing MM. Two additional neighboring SNPs in the same LD block confirmed the association of IKBKE SNPs with disease risk. The IKBKE locus is located at chromosome 1q32.1 and it acts as a modulator of multiple immune processes [ 45 ] including the necrosis factor (NF)-κB non-canonical pathway [ 46 ], a key process in MM pathogenesis. IKBKE expression is almost restricted to lymphoid tissue and Westra et al. reported that the IKBKE rs17433804 SNP is an eQTL marker for IKBKE in peripheral blood ( p = 9.65 × 10 −9 ) [ 43 ]. In addition, it has been described that the IKBKE locus is frequently mutated in MM patients [ 47 ], which reinforces the idea of a role of this gene in determining MM risk. Interestingly, we also found that carriers of the IKBKE rs17433804C allele had increased numbers of transitional CD24 + CD38+ B cells, which is a type of B cell linked to MM tumorigenesis. It has been reported that CD24+ cells are upregulated in MM and they have an increased expression of CXCR4 [ 48 ], which is a well-known chemokine receptor that plays a pivotal role in proliferation, invasion, dissemination, and drug resistance in MM [ 49 ]. However, CD24 expression has been also found to be associated to a more apoptotic and less tumorigenic phenotype by impaired capability to migrate and to create colonies as compared with CD24 − MM cells [ 48 ]. These controversial results might indicate that CD24 might have a dual effect on MM by contributing to disease onset but also by preventing disease progression. In line with this hypothesis, Gross Even-Zohar et al. reported that patients having high expression of CD24 + MM cells had longer overall survival and progression free survival [ 50 ]. In addition, we found that carriers of the IKBKE rs17433804C allele had decreased circulating serum concentrations of MCP-2, which has been linked to chemotaxis of myeloid and lymphoid cells, but also to other important processes such as autophagy, leukocyte behavior, cell adhesion and polarization, cell secretion, and cell survival of MM cells [ 51 ]. A recent study demonstrated that MCP-1, another chemokine from the MCP family, was increased in MM patients and correlated with clinical characteristics and enhanced angiogenesis in MM [ 52 ]. In line with these results, another study demonstrated that the inhibition of MCP-2, as well as its receptor CCR2, by neutralizing antibodies reduced migration of MM cells [ 53 ]. Altogether, these findings suggest that the IKBKE locus might influence MM risk by modulating absolute numbers of specific subsets of B-cells and MCP-2-mediated immune responses and MM cell migration. Nonetheless, it is also important to consider that the presence of this variant alters regulatory motifs for multiple transcription factors including AP-1, p300, TCF4, Myc, and PRMD1 [ 43 ]. These transcription factors which are highly expressed in MM [ 54 , 55 ] have been involved in B cell development and plasma cell differentiation, MM cell proliferation, cell survival, and drug resistance in the bone marrow microenvironment [ 56 , 57 , 58 ], and they even exhibit antitumoral activity in MM pathogenesis [ 54 , 59 ]. Even though the above reported results shed some light on the role of the IKBKE locus in MM, additional functional studies are still warranted to establish the exact mechanism by which the IKBKE locus is linked to MM onset and disease progression.
Another novel finding was the association between the PARK2 rs1884158 SNP (and four neighboring SNPs) and the risk of MM. In addition to its role in autophagy, PARK2 is involved in modulating stress response, mitochondrial biogenesis, stability of G1/S cyclins [ 60 ], cell growth (acting as tumor suppressor), and mitophagy, where it purges damaged organelles from the vital mitochondrial network [ 61 ]. Although the PARK2 gene was associated with the risk of solid tumors [ 62 ], little is known about its role in MM. Our functional experiments did not show any positive correlation between the PARK2 rs1884158 SNP and host immune responses and, to the best of our knowledge, no functional roles for this SNP have been reported in Haploreg or eQTL browsers. Therefore, additional studies are still warranted to replicate the association of the PARK2 rs1884158 SNP with MM risk and, subsequently, to characterize its functional role in MM pathogenesis.
As well as the novel association of CD46 , IKBKE , and PARK2 SNPs with MM risk, we could validate the association of SNPs within well-known MM susceptibility genes such as ULK4 , ATG5 , and CDKN2A [ 39 , 63 ]. The strongest association was found for the ULK4 rs6599175 polymorphism within the ULK4 gene. Each copy of the ULK4 rs6599175C allele increased the risk of developing MM by 31%. This result agreed with a previous study by Broderick et al. (2011) [ 63 ]. The ULK4 gene is located at chromosome 3p22.1 and encodes for a member of the unc-51-like serine/threonine kinase (STK) family that plays a role in modulating hypertension [ 64 ] and cardiologic disorders [ 65 ]. According to GTEx portal data and other eQTL studies [ 40 ], the ULK4 rs6599175C allele strongly correlates with higher levels of ULK4 mRNA expression levels in multiple tissues including whole blood ( p = 1.4 × 10 −59 ). However, outside of mRNA expression levels, little is known about the functional role of this genetic variant to modulate the risk of MM. In this regard, we found that carriers of the ULK4 rs6599175C risk allele correlated with increased serum concentrations of vitamin D3 in healthy donors, which might suggest that the effect of the ULK4 rs6599175C allele to increase the risk of MM might be mediated by a vitamin D-dependent mechanism likely affecting cell differentiation and proliferation, angiogenesis, immune responses, and apoptosis either in normal or malignant tissues [ 66 , 67 , 68 ]. In addition, this SNP has been linked to monoclonal gammopathy of undetermined significance (MGUS), a non-cancerous precursor condition that precedes MM [ 69 ], suggesting that the ULK4 rs6599175 SNP might exert its biological effect during the earliest stages of plasma cell dyscrasia progression from early to late-stage disease. Additional studies are required to decipher the interplay between the ULK4 locus and circulating vitamin D3 levels in the context of MM risk and progression.
On the other hand, the ATG5 is located on chromosome 6q21 and it has been related to autophagy vesicle formation and apoptosis [ 70 ]. A previous study reported the association between the ATG5 rs9372120 SNP (a variant in strong LD with the rs2299864) and MM risk [ 39 ], but also MGUS [ 69 ], which suggests that the ATG5 locus might also exert its biological effect on the risk of developing MM by acting at early and non-neoplastic stages. Although we could not find any correlation between the ATG5 rs2299864 SNP and host immune responses or autophagy flux, it was reported that this variant is located among histone marks for primary T helper naive and memory cells and primary T CD8 + naive cells from peripheral blood and that it disrupts regulatory motifs for several transcription factors including DMRT3, which has an altered methylation profile during early stages of myelomagenesis [ 71 ].
Finally, the CDKN2A gene is located on chromosome 9p21.3 and encodes for a cyclin-dependent kinase inhibitor 2A that has been involved in p53 stabilization, cell cycle arrest, and cell proliferation. Unlike the ULK4 rs6599175 or ATG5 rs2299864 SNPs, the CDKN2A rs2811710 SNP has not been linked to MGUS susceptibility, which suggests that the CDKN2A rs2811710 SNP might be involved in promoting the accumulation of tumorigenic MM cells rather than acting in earlier stages. In fact, several germline mutations within the CDKN2A have been consistently associated with MM predisposition [ 72 ], but also improved patient overall survival [ 73 ]. Interestingly, we found that carriers of the CDKN2A rs2811710C allele, which is associated with an increased risk of MM, had decreased numbers of CD4 + EMCD45RO + CD27 − T memory cells, which are cells that display a high antigen recall response and were reported to be poorly represented in MM patients compared to controls [ 74 ]. In addition, the CDKN2A rs2811710 SNP was associated with the expression of CDKN2B and STK16 mRNAs in monocytes that are involved in the modulation of cell cycle arrest through the TFG signaling pathway [ 75 ]. The CDKN2A rs2811710 SNP maps among histone marks for primary T helper memory cells and primary T regulatory cells from peripheral blood and alters binding sites for key transcription factors such as HDAC2, HNF1, OTX, and p300 that play important roles in epigenetic repression and transcriptional regulation, cell cycle progression, and developmental events, and are even involved in influencing overall survival [ 76 ].
It is worth mentioning that this study has both strengths and limitations. The major strengths of our study were the comprehensive analysis of autophagy-related SNPs and the inclusion of three large independent European populations of European ancestry including a total of 12,676 participants. Furthermore, we comprehensively analyzed the impact of autophagy-related SNPs in modulating blood cell counts, steroid hormones, serum and plasma metabolites, and immune responses in a large study of healthy subjects ascertained through the HFGP. Another important strength of this study was the experimental analysis assessing the effect of autophagy SNPs in modulating the autophagy flux in PBMCs left untreated or treated with metformin or bafilomycin. This approach allowed us to investigate, for the first time, the impact of susceptibility loci for MM on the autophagy flux. A limitation of this study was its multicentric nature that placed inevitable limitations such as the impossibility of uniformly collected mutation and/or cytogenetic profiles for a significant proportion of patients analyzed. In addition, this study included a subset of German participants, which overlapped with the GWAS used for discovering the ULK4 MM susceptibility locus. Finally, given that all study participants included in this study were of European ancestry, we could not determine the impact of autophagy variants in other ethnic or ancestral populations. Additional studies using other ethnic and/or ancestry populations are now warranted to confirm the results described herein.
4. Materials and Methods
A workflow diagram of the study is included in Figure 4 .
4.1. Study Populations
The discovery population consisted of 3169 subjects from GWAS conducted by the German-Speaking Multiple Myeloma Multicenter Study Group (GMMG), followed by meta-analysis with an independent GWAS on 5100 subjects by the International Lymphoma Epidemiology Consortium (InterLymph; https://epi.grants.cancer.gov/interlymph/ ; accessed on 12 February 2020). International Myeloma Working Group (IMWG) criteria were used by physicians to establish the diagnosis of MM [ 77 , 78 ]. Ethical approval for these respective studies was obtained from each participating institution and all participanting subjects provided written informed consent. The German GWAS coordinated by the University Clinic, Heidelberg (ISRCTN06413384: GMMG-HD3 http://www.isrctn.com/search?q=ISRCTN06413384 , accessed on 12 February 2020; ISRCTN64455289: GMMG-HD4 http://www.isrctn.com/search?q=ISRCTN64455289 , accessed on 12 February 2020; and ISRCTN05745813: GMMG-HD5 http://www.isrctn.com/search?q=ISRCTN05745813 , accessed on 12 February 2020) included 1512 MM patients that were genotyped using Illumina Human OmniExpress-12 v1.0 arrays and 2107 healthy individuals enrolled into the Heinz Nixdorf Recall (HNR) study that were genotyped using either the Illumina HumanOmni1-Quad_v1 or OmniExpress-12 v1.0 arrays [ 39 ]. The InterLymph MM GWAS included 2404 MM and 2696 controls that were genotyped using Affymetrix, Human660W-quad Beadchip, and Illumina arrays 610 Quad, Omni5, OmniExpress Beadchip, and OncoArray [ 69 ]. Both German and InterLymph GWAS were independently subjected to rigorous standard quality controls prior to imputation using the IMPUTE2 v2.3 software or the Michigan imputation server (based on the Haplotype Reference Consortium), respectively [ 79 ]. After imputation, each site filtered the data to include only high-quality imputed variants (information score > 0.8), and further quality-control checks were implemented including checks for missingness, duplicates, abnormal heterozygosity, cryptic relatedness, population outliers (evaluated by principal components analyses using Eigenstrat software), and genomic inflation (λ = 1.00) [ 39 , 69 ].
4.2. SNP Selection
A total of 234 autophagy-related genes were selected on the basis of their presence in the autophagy database ( http://autophagy.lu/index.html , accessed on 12 February 2020; Supplementary Table S1 ). Estimates of association with MM risk for all genotyped or imputed SNPs within or near these genes (5 Kb up-stream and 3 Kb downstream) were extracted from the German GWAS [ 39 ]. After testing Hardy-Weinberg Equilibrium (HWE) in the control group by a standard observed-expected chi-square (χ 2 ) test ( p < 10 −5 ), 3018 SNPs were selected according to their level of association with MM risk ( p ≤ 0.05), but also considering linkage disequilibrium values (r 2 < 0.8) and minor allele frequency (MAF) of > 0.01. Among them, 440 SNPs were independent (r 2 < 0.1) according to LDLink information ( https://ldlink.nci.nih.gov/?tab=snpclip , accessed on 12 February 2020) and, therefore, the multiple testing significance threshold was set to 1.14 × 10 −4 (0.05/440 independent SNPs; Supplementary Table S2 ).
4.3. Replication Cohort, Genotyping, and Meta-Analysis
Following the same approach, these SNPs were then tested for association with MM risk in the InterLymph MM GWAS and a meta-analysis of both study populations was conducted using METAL [ 80 ]. The I 2 statistic was used to assess statistical heterogeneity between the studies and the pooled odds ratio (OR) was computed using the fixed-effect model for SNPs showing non-significant heterogeneity and the random-effect model for SNPs reporting significant heterogeneity. After the meta-analysis of both populations, 12 SNPs were advanced for replication in the International Multiple Myeloma (IMMEnSE) consortium, consisting of 2696 MM patients and 1701 controls. As for the GWAS populations, the Ethical Committee of the Faculty of Medicine of the University of Heidelberg approved the IMMEnSE study protocol and written informed consent from each study participant was obtained [ 81 ].
Genotyping of selected SNPs in the IMMEnSE study was carried out at DKFZ (German Cancer Research Center, Heidelberg, Germany) using KASPar ® (LGC Genomics, Hoddesdon, UK) or Taqman ® SNP Genotyping assays (Thermo Fisher Scientific, Foster City, CA, USA) according to previously reported protocols [ 82 ]. For internal quality control, ~5% of samples were randomly selected and included as duplicates. Concordance between the original and the duplicate samples for the 12 SNPs tested was ≥99.0%. All SNPs showed genotype frequencies in the control population similar to those found in the 1000 Genomes database and were in HWE.
4.4. Functional Effect of the Autophagy-Related Variants on Immune Responses
To provide insight into the functional role of the most interesting autophagy SNPs in modulating immune responses, we tested if any of these genetic markers correlated with cytokine expression quantitative trait loci (cQTL) data from in vitro stimulation experiments, but also absolute numbers of 91 blood-derived cell populations and 103 serum or plasmatic inflammatory proteins quantified in the approximately 500 volunteers from the 500 Functional Genomics cohort from the Human Functional Genomics Project (HFGP; http://www.humanfunctionalgenomics.org/site/ , accessed on 12 February 2020). The HFGP study was approved by the Arnhem-Nijmegen Ethical Committee (no. 42561.091.12) and biological specimens were collected after informed consent was obtained.
cQTL data included cytokine levels (IFNγ, IL1β, IL6, TNFα, IL17, and IL22) after the stimulation of peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages (MDM), or whole blood from 408 healthy subjects with LPS (1 or 100 ng/mL; Sigma Aldrich, St. Louis, MO, USA), PHA (10 µg/mL, Sigma, St. Louis, MO, USA), Pam3Cys (10 µg/mL, EMC microcollections, Tübingen, Germany), or CpG (100 ng/mL, InvivoGen, San Diego, CA, USA). Concentrations of human IFNγ, IL1β, IL6, TNFα, IL17, and IL22 were determined using specific commercial ELISA kits (PeliKine Compact, Amsterdam, or R&D Systems), in accordance with the manufacturers’ instructions. When values were below or above the detection limit of the ELISA, the corresponding limit was used. After log transformation, linear regression analyses adjusted for age and sex were used to determine the correlation of the selected SNPs with cQTL data. Details on PBMC isolation, macrophage differentiation, and stimulation assays have been reported elsewhere [ 83 ].
To evaluate the impact of autophagy SNPs on cell-level variation, 91 blood-derived cell populations were measured by 10-color flow cytometry (Navios flow cytometer, Beckman Coulter, Miami, FL, USA) after blood sampling (2–3 h), and cell count analysis was performed using Kaluza software (Beckman Coulter, v.1.3). In order to reduce inter-experimental noise and increase statistical power, cell count analysis was performed by calculating parental and grandparental percentages, which were defined as the percentage of a certain cell type within the subpopulation of the cells from which it was isolated [ 48 ]. Detailed laboratory protocols for cell isolation, reagents, gating, and flow cytometry analysis have been reported elsewhere [ 84 ]. The accession number for the raw flow cytometry data and analyzed data files are available upon request to the authors (accessed on 13 December 2019; Supplementary Table S3 ).
A proteomic analysis was also performed in serum and plasma samples from the HFGP study. Circulating proteins were measured using the commercial Olink ® Inflammation panel (Olink, Sweden) that resulted in the measurement of 103 different biomarkers ( Supplementary Table S4 ). Protein levels were expressed on a log2-scale as normalized protein expression values and normalized using bridging samples to correct for batch variation.
All analyses were performed using R software ( http://www.r-project.org/ , accessed on 12 February 2020), using custom scripts in the R programming language based on existing functions such as lm (stats). In order to account for multiple comparisons, we used Bonferroni corrected significant thresholds of 0.00069 (0.05/12 SNPs/6 cytokines), 4.57 × 10 −5 (0.05/12 SNPs/91 cell-derived populations), and 4.05 × 10 −5 (0.05/12 SNPs/103 proteins) for the cQTL, cell variability, and proteomic analyses, respectively.
4.5. Correlation between Autophagy-Related SNPs and Steroid Hormone Levels
In addition to the immunological data, we also assessed the association of autophagy SNPs with data on steroid hormone levels (androstenedione, cortisol, 11-deoxy-cortisol, 17-hydroxy progesterone, progesterone, testosterone, and 25 hydroxy vitamin D3) from 279 healthy controls of the HFGP study without hormone replacement or oral contraceptive therapies. Serum steroid hormone levels were determined by ELISA following the manufacturer’s instructions. Correlation between levels of seven serum steroid hormones and autophagy-related SNPs was evaluated by linear regression analysis adjusted for age and sex. The significance threshold was set to p = 7.1 × 10 −4 , considering the number of independent SNPs ( n = 12) and the number of hormones tested ( n = 7).
4.6. Impact of Autophagy-Related Variants on the Autophagy Flux
In order to accurately determine the role of autophagy SNPs in modulating autophagy, we investigated their impact on the autophagy flux in a population of 68 European healthy donors. For that purpose, we isolated peripheral blood mononuclear cells (PBMCs) from whole blood by density gradient centrifugation using Histopaque ® , and we treated them for 2 h with 10 µM of bafilomycin A1 or 10 mM of metformin to inhibit or induce autophagy, respectively. A total of 5 × 10 5 PBMCs were plated in each well for stimulatory and inhibitory experiments and treated with metformin or bafilomycin A1 alone or in combination. Untreated cells were used as experimental controls. After treatment, cells were harvested and protein extraction was performed with 50 µL of lysis buffer (1% NP-40, 500 mM Tris HCL, 2.5 M NaCl, 20 mM EDTA, phosphatase and protease inhibitors—from Roche—at pH 7.2). Then 20 µg of the total protein were resolved in a 12% SDS gel and transferred to a nitrocellulose membrane for 10 min in a Trans-Blot Turbo transfer system. Membranes were then blocked for 1 h using Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) containing 5% BSA and incubated overnight at 4 °C with the polyclonal primary antibodies at 1:1000 in 1% BSA (Rabbit anti-LC3A/B Antibody, Cell-Signaling and Mouse anti-Actin antibody, Merck Millipore, Darmstadt, Germany). After washing with tris-buffered saline-tween (TBS-T), nitrocellulose membranes were incubated with the corresponding secondary antibodies (IgG anti-Rabbit for LC3A/B and IgG anti-Mouse for Actin). Protein levels were detected after incubation with SuperSignal West Femto Maximum Sensitivity Substrate (Thermofisher, Waltham, MA, USA) or Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA). Digital images of the Western blots were obtained in a ChemiDoc XRS System (Bio-Rad) with Quantity One software V4.6.5 (Bio-Rad). Autophagy flux was determined as the difference in the LC3-II/Actin ratio between cells treated or not with bafilomycin A1 and/or metformin. Linear regression analyses adjusted for age and sex were used to determine the correlation between autophagy-related SNPs and autophagy flux values. A significance threshold of p = 0.0028 was set according to the number of SNPs tested ( n = 12) and the treatments administrated in vitro ( n = 2).
4.7. In Silico Functional Analysis
Haploreg ( http://www.broadinstitute.org/mammals/haploreg/haploreg.php , accessed on 12 February 2020) and ENCODE annotation data ( https://genome.ucsc.edu/ENCODE ) were also used to predict the functional role of the most interesting autophagy SNPs. We also analyzed whether selected SNPs could be expression quantitative loci (eQTL) for different cell types and tissues using data from public eQTL browsers such as GTex portal ( https://gtexportal.org/home/ , accessed on 12 February 2020), Blood eQTL browser ( https://genenetwork.nl/bloodeqtlbrowser/ , accessed on 12 February 2020), and Haploreg [ 43 ].
5. Conclusions
This study identifies novel associations for genetic polymorphisms within the CD46 , IKBKE , and PARK2 loci and MM risk and validates previously reported associations for SNPs within the ULK4 , ATG5 , and CDKN2A genes. Although the biological effect of the CD46 , IKBKE , ULK4 , and CDKN2A SNPs seemed to be mediated by alterations of absolute numbers of certain B- and T-cell subsets and MCP-2-, IL20-, and vitamin D3-dependent mechanisms affecting cell differentiation and proliferation, angiogenesis, immune responses, and apoptosis, we could not identify the biological mechanisms underscoring the ATG5 association.
Acknowledgments
We kindly thank all individuals who agreed to participate in this study, as well as all cooperating physicians and students.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24108500/s1 .
Click here for additional data file.
Author Contributions
J.S. and A.F. designed and coordinated the study. E.C., J.M.S.-M., A.M. and J.S. were involved in the generation of genetic data from the CRuCIAL cohort and drafted the manuscript. J.S. obtained funding. J.M.S.-M., F.C., D.C. and J.S. performed data quality control and genetic association analyses. R.T.H., Y.L. and M.G.N. provided the functional raw data from the HFGP cohort and J.S. and R.T.H. performed the statistical analysis of functional data. A.J. (Artur Jurczyszyn), A.C.-G., A.S., M.A.T.H., N.W., G.B., R.G.-S., W.T., U.V., A.J. (Andrés Jerez), D.Z., M.W., J.N.H., S.L., J.J.S., A.B., A.K., J.M.-L., S.G., M.E.S., E.S., E.I.-J., G.G.G., M.R.-R., M.K., N.A., F.G.V., P.S.R., M.I.d.S.F., K.K., M.R., W.C., M.P., M.J., P.B., M.D., A.D.-S., E.O., A.D.N., J.M.Z., R.M.R., M.M., J.J.R.S., V.A., K.J., K.H., S.I.B., V.R., G.M., S.K.K., A.N., S.J.C., C.D., M.J.M., J.V., N.J.C., E.Z., A.J.V., E.E.B., C.M.V. and A.F. provided biological samples and clinical and demographic data of CLL patients and/or healthy controls from the InterLymph and/or CRuCIAL consortia. A.C.-G., S.I.B., V.R., E.E.B., C.M.V., K.H. and A.F. provided GWAS data from the GMMG or InterLymph cohorts. B.S.-M. and P.L. performed the autophagy flux experiments. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Boards of all institutions participating in patient recruitment.
Informed Consent Statement
Informed consent was obtained from all subjects involved in this study.
Data Availability Statement
The genotype data used in the present study are available by the corresponding authors upon reasonable request. Functional data used in this project have been meticulously catalogued and archived in the BBMRI-NL data infrastructure ( https://hfgp.bbmri.nl/ , accessed on 12 February 2020) using the MOLGENIS open-source platform for scientific data. This allows flexible data querying and download, including sufficiently rich metadata and interfaces for machine processing (R statistics, REST API) and using FAIR principles to optimize Findability, Accessibility, Interoperability, and Reusability.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Palumbo A. Anderson K. Multiple myeloma N. Engl. J. Med. 2011 364 1046 1060 10.1056/NEJMra1011442 21410373
2. Kyle R.A. Gertz M.A. Witzig T.E. Lust J.A. Lacy M.Q. Dispenzieri A. Fonseca R. Rajkumar S.V. Offord J.R. Larson D.R. Review of 1027 patients with newly diagnosed multiple myeloma Mayo Clin. Proc. 2003 78 21 33 10.4065/78.1.21 12528874
3. Kastritis E. Zagouri F. Symeonidis A. Roussou M. Sioni A. Pouli A. Delimpasi S. Katodritou E. Michalis E. Michael M. Preserved levels of uninvolved immunoglobulins are independently associated with favorable outcome in patients with symptomatic multiple myeloma Leukemia 2014 28 2075 2079 10.1038/leu.2014.110 24637336
4. Bradwell A. Harding S. Fourrier N. Mathiot C. Attal M. Moreau P. Harousseau J.L. Avet-Loiseau H. Prognostic utility of intact immunoglobulin Ig’kappa/Ig’lambda ratios in multiple myeloma patients Leukemia 2013 27 202 207 10.1038/leu.2012.159 22699454 PMC3542628
5. Hoang B. Benavides A. Shi Y. Frost P. Lichtenstein A. Effect of autophagy on multiple myeloma cell viability Mol. Cancer Ther. 2009 8 1974 1984 10.1158/1535-7163.MCT-08-1177 19509276
6. Obeng E.A. Carlson L.M. Gutman D.M. Harrington W.J. Jr. Lee K.P. Boise L.H. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells Blood 2006 107 4907 4916 10.1182/blood-2005-08-3531 16507771 PMC1895817
7. Lee A.H. Iwakoshi N.N. Anderson K.C. Glimcher L.H. Proteasome inhibitors disrupt the unfolded protein response in myeloma cells Proc. Natl. Acad. Sci. USA 2003 100 9946 9951 10.1073/pnas.1334037100 12902539 PMC187896
8. Landowski T.H. Megli C.J. Nullmeyer K.D. Lynch R.M. Dorr R.T. Mitochondrial-mediated disregulation of Ca 2+ is a critical determinant of Velcade (PS-341/bortezomib) cytotoxicity in myeloma cell lines Cancer Res. 2005 65 3828 3836 10.1158/0008-5472.CAN-04-3684 15867381
9. Mitsiades C.S. Mitsiades N.S. McMullan C.J. Poulaki V. Kung A.L. Davies F.E. Morgan G. Akiyama M. Shringarpure R. Munshi N.C. Antimyeloma activity of heat shock protein-90 inhibition Blood 2006 107 1092 1100 10.1182/blood-2005-03-1158 16234364 PMC1895907
10. Catley L. Weisberg E. Kiziltepe T. Tai Y.T. Hideshima T. Neri P. Tassone P. Atadja P. Chauhan D. Munshi N.C. Aggresome induction by proteasome inhibitor bortezomib and alpha-tubulin hyperacetylation by tubulin deacetylase (TDAC) inhibitor LBH589 are synergistic in myeloma cells Blood 2006 108 3441 3449 10.1182/blood-2006-04-016055 16728695 PMC1895432
11. Arsov I. Adebayo A. Kucerova-Levisohn M. Haye J. MacNeil M. Papavasiliou F.N. Yue Z. Ortiz B.D. A role for autophagic protein beclin 1 early in lymphocyte development J. Immunol. 2011 186 2201 2209 10.4049/jimmunol.1002223 21239722 PMC3070473
12. Shimizu S. Kanaseki T. Mizushima N. Mizuta T. Arakawa-Kobayashi S. Thompson C.B. Tsujimoto Y. Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes Nat. Cell Biol. 2004 6 1221 1228 10.1038/ncb1192 15558033
13. Kroemer G. Levine B. Autophagic cell death: The story of a misnomer Nat. Rev. Mol. Cell Biol. 2008 9 1004 1010 10.1038/nrm2529 18971948 PMC2727358
14. Yu L. Alva A. Su H. Dutt P. Freundt E. Welsh S. Baehrecke E.H. Lenardo M.J. Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8 Science 2004 304 1500 1502 10.1126/science.1096645 15131264
15. Schwarten M. Mohrluder J. Ma P. Stoldt M. Thielmann Y. Stangler T. Hersch N. Hoffmann B. Merkel R. Willbold D. Nix directly binds to GABARAP: A possible crosstalk between apoptosis and autophagy Autophagy 2009 5 690 698 10.4161/auto.5.5.8494 19363302
16. Ma Y. Galluzzi L. Zitvogel L. Kroemer G. Autophagy and cellular immune responses Immunity 2013 39 211 227 10.1016/j.immuni.2013.07.017 23973220
17. Kuballa P. Nolte W.M. Castoreno A.B. Xavier R.J. Autophagy and the immune system Annu. Rev. Immunol. 2012 30 611 646 10.1146/annurev-immunol-020711-074948 22449030
18. Deretic V. Autophagy as an innate immunity paradigm: Expanding the scope and repertoire of pattern recognition receptors Curr. Opin. Immunol. 2012 24 21 31 10.1016/j.coi.2011.10.006 22118953 PMC3288884
19. Deretic V. Autophagy: An emerging immunological paradigm J. Immunol. 2012 189 15 20 10.4049/jimmunol.1102108 22723639 PMC3382968
20. Ma Y. Adjemian S. Mattarollo S.R. Yamazaki T. Aymeric L. Yang H. Portela Catani J.P. Hannani D. Duret H. Steegh K. Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells Immunity 2013 38 729 741 10.1016/j.immuni.2013.03.003 23562161
21. Dengjel J. Schoor O. Fischer R. Reich M. Kraus M. Muller M. Kreymborg K. Altenberend F. Brandenburg J. Kalbacher H. Autophagy promotes MHC class II presentation of peptides from intracellular source proteins Proc. Natl. Acad. Sci. USA 2005 102 7922 7927 10.1073/pnas.0501190102 15894616 PMC1142372
22. Jia W. He Y.W. Temporal regulation of intracellular organelle homeostasis in T lymphocytes by autophagy J. Immunol. 2011 186 5313 5322 10.4049/jimmunol.1002404 21421856
23. Nedjic J. Aichinger M. Emmerich J. Mizushima N. Klein L. Autophagy in thymic epithelium shapes the T-cell repertoire and is essential for tolerance Nature 2008 455 396 400 10.1038/nature07208 18701890
24. Yun Z. Zhichao J. Hao Y. Ou J. Ran Y. Wen D. Qun S. Targeting autophagy in multiple myeloma Leuk. Res. 2017 59 97 104 10.1016/j.leukres.2017.06.002 28599191
25. Dykstra K.M. Allen C. Born E.J. Tong H. Holstein S.A. Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells Oncotarget 2015 6 41535 41549 10.18632/oncotarget.6365 26595805 PMC4747172
26. Desantis V. Saltarella I. Lamanuzzi A. Mariggio M.A. Racanelli V. Vacca A. Frassanito M.A. Autophagy: A New Mechanism of Prosurvival and Drug Resistance in Multiple Myeloma Transl. Oncol. 2018 11 1350 1357 10.1016/j.tranon.2018.08.014 30196237 PMC6132177
27. Escalante A.M. McGrath R.T. Karolak M.R. Dorr R.T. Lynch R.M. Landowski T.H. Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo Cancer Chemother. Pharmacol. 2013 71 1567 1576 10.1007/s00280-013-2156-3 23572175 PMC3669633
28. Vogl D.T. Stadtmauer E.A. Tan K.S. Heitjan D.F. Davis L.E. Pontiggia L. Rangwala R. Piao S. Chang Y.C. Scott E.C. Combined autophagy and proteasome inhibition: A phase 1 trial of hydroxychloroquine and bortezomib in patients with relapsed/refractory myeloma Autophagy 2014 10 1380 1390 10.4161/auto.29264 24991834 PMC4203515
29. Tucci M. Stucci S. Savonarola A. Resta L. Cives M. Rossi R. Silvestris F. An imbalance between Beclin-1 and p62 expression promotes the proliferation of myeloma cells through autophagy regulation Exp. Hematol. 2014 42 897 908.e1 10.1016/j.exphem.2014.06.005 24971696
30. Klionsky D.J. Petroni G. Amaravadi R.K. Baehrecke E.H. Ballabio A. Boya P. Bravo-San Pedro J.M. Cadwell K. Cecconi F. Choi A.M.K. Autophagy in major human diseases EMBO J. 2021 40 e108863 10.15252/embj.2021108863 34459017 PMC8488577
31. Liang C. Jung J.U. Autophagy genes as tumor suppressors Curr. Opin. Cell Biol. 2010 22 226 233 10.1016/j.ceb.2009.11.003 19945837 PMC2854193
32. Huang X. Cao W. Yao S. Chen J. Liu Y. Qu J. Li Y. Han X. He J. Huang H. NEDD4L binds the proteasome and promotes autophagy and bortezomib sensitivity in multiple myeloma Cell Death Dis. 2022 13 197 10.1038/s41419-022-04629-8 35236820 PMC8891287
33. Jaganathan S. Malek E. Vallabhapurapu S. Vallabhapurapu S. Driscoll J.J. Bortezomib induces AMPK-dependent autophagosome formation uncoupled from apoptosis in drug resistant cells Oncotarget 2014 5 12358 12370 10.18632/oncotarget.2590 25481044 PMC4323002
34. Di Lernia G. Leone P. Solimando A.G. Buonavoglia A. Saltarella I. Ria R. Ditonno P. Silvestris N. Crudele L. Vacca A. Bortezomib Treatment Modulates Autophagy in Multiple Myeloma J. Clin. Med. 2020 9 552 10.3390/jcm9020552 32085480 PMC7073518
35. Cheng Y. Qi F. Li L. Qin Z. Li X. Wang X. Autophagy-related genes are potential diagnostic and prognostic biomarkers in prostate cancer Transl. Androl. Urol. 2020 9 2616 2628 10.21037/tau-20-498 33457234 PMC7807329
36. Wang L. Fang D. Liu Y. Autophagy-related genes are potential diagnostic biomarkers for dermatomyositis Ann. Transl. Med. 2022 10 228 10.21037/atm-22-70 35280393 PMC8908178
37. Zhu F.X. Wang X.T. Zeng H.Q. Yin Z.H. Ye Z.Z. A predicted risk score based on the expression of 16 autophagy-related genes for multiple myeloma survival Oncol. Lett. 2019 18 5310 5324 10.3892/ol.2019.10881 31612041 PMC6781562
38. Went M. Sud A. Forsti A. Halvarsson B.M. Weinhold N. Kimber S. van Duin M. Thorleifsson G. Holroyd A. Johnson D.C. Identification of multiple risk loci and regulatory mechanisms influencing susceptibility to multiple myeloma Nat. Commun. 2018 9 3707 Correction in Nat. Commun. 2019 , 10 , 213 10.1038/s41467-018-08107-8 30213928 PMC6137048
39. Mitchell J.S. Li N. Weinhold N. Forsti A. Ali M. van Duin M. Thorleifsson G. Johnson D.C. Chen B. Halvarsson B.M. Genome-wide association study identifies multiple susceptibility loci for multiple myeloma Nat. Commun. 2016 7 12050 10.1038/ncomms12050 27363682 PMC4932178
40. Consortium G.T. Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: Multitissue gene regulation in humans Science 2015 348 648 660 10.1126/science.1262110 25954001 PMC4547484
41. Lappalainen T. Sammeth M. Friedlander M.R. t Hoen P.A. Monlong J. Rivas M.A. Gonzalez-Porta M. Kurbatova N. Griebel T. Ferreira P.G. Transcriptome and genome sequencing uncovers functional variation in humans Nature 2013 501 506 511 10.1038/nature12531 24037378 PMC3918453
42. Alexandrakis M.G. Pappa C.A. Kokonozaki M. Boula A. Vyzoukaki R. Staphylaki D. Papadopoulou A. Androulakis N. Tsirakis G. Sfiridaki A. Circulating serum levels of IL-20 in multiple myeloma patients: Its significance in angiogenesis and disease activity Med. Oncol. 2015 32 42 10.1007/s12032-015-0488-z 25631632
43. Westra H.J. Peters M.J. Esko T. Yaghootkar H. Schurmann C. Kettunen J. Christiansen M.W. Fairfax B.P. Schramm K. Powell J.E. Systematic identification of trans eQTLs as putative drivers of known disease associations Nat. Genet. 2013 45 1238 1243 10.1038/ng.2756 24013639 PMC3991562
44. Hudlebusch H.R. Lodahl M. Johnsen H.E. Rasmussen T. Expression of HOXA genes in patients with multiple myeloma Leuk. Lymphoma 2004 45 1215 1217 10.1080/10428190310001625836 15360004
45. Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway Nat. Immunol. 2003 4 491 496 10.1038/ni921 12692549
46. Chau T.L. Gioia R. Gatot J.S. Patrascu F. Carpentier I. Chapelle J.P. O’Neill L. Beyaert R. Piette J. Chariot A. Are the IKKs and IKK-related kinases TBK1 and IKK-epsilon similarly activated? Trends Biochem. Sci. 2008 33 171 180 10.1016/j.tibs.2008.01.002 18353649
47. Leich E. Weissbach S. Klein H.U. Grieb T. Pischimarov J. Stuhmer T. Chatterjee M. Steinbrunn T. Langer C. Eilers M. Multiple myeloma is affected by multiple and heterogeneous somatic mutations in adhesion- and receptor tyrosine kinase signaling molecules Blood Cancer J. 2013 3 e102 10.1038/bcj.2012.47 23396385 PMC3584721
48. Gilad N. Zukerman H. Pick M. Gatt M.E. The role of CD24 in multiple myeloma tumorigenicity and effects of the microenvironment on its expression Oncotarget 2019 10 5480 5491 10.18632/oncotarget.27190 31534632 PMC6739209
49. Ullah T.R. The role of CXCR4 in multiple myeloma: Cells’ journey from bone marrow to beyond J. Bone Oncol. 2019 17 100253 10.1016/j.jbo.2019.100253 31372333 PMC6658931
50. Gross Even-Zohar N. Pick M. Hofstetter L. Shaulov A. Nachmias B. Lebel E. Gatt M.E. CD24 Is a Prognostic Marker for Multiple Myeloma Progression and Survival J. Clin. Med. 2022 11 2913 10.3390/jcm11102913 35629039 PMC9144978
51. Gschwandtner M. Derler R. Midwood K.S. More Than Just Attractive: How CCL2 Influences Myeloid Cell Behavior Beyond Chemotaxis Front. Immunol. 2019 10 2759 10.3389/fimmu.2019.02759 31921102 PMC6923224
52. Valkovic T. Babarovic E. Lucin K. Stifter S. Aralica M. Seili-Bekafigo I. Duletic-Nacinovic A. Jonjic N. Plasma Levels of Monocyte Chemotactic Protein-1 Are Associated with Clinical Features and Angiogenesis in Patients with Multiple Myeloma Biomed. Res. Int. 2016 2016 7870590 10.1155/2016/7870590 26925413 PMC4748063
53. Broek I.V. Asosingh K. Vanderkerken K. Straetmans N. Van Camp B. Van Riet I. Chemokine receptor CCR2 is expressed by human multiple myeloma cells and mediates migration to bone marrow stromal cell-produced monocyte chemotactic proteins MCP-1, -2 and -3 Br. J. Cancer 2003 88 855 862 10.1038/sj.bjc.6600833 12644822 PMC2377079
54. Sukhdeo K. Mani M. Zhang Y. Dutta J. Yasui H. Rooney M.D. Carrasco D.E. Zheng M. He H. Tai Y.T. Targeting the beta-catenin/TCF transcriptional complex in the treatment of multiple myeloma Proc. Natl. Acad. Sci. USA 2007 104 7516 7521 10.1073/pnas.0610299104 17452641 PMC1863489
55. Gyory I. Fejer G. Ghosh N. Seto E. Wright K.L. Identification of a functionally impaired positive regulatory domain I binding factor 1 transcription repressor in myeloma cell lines J. Immunol. 2003 170 3125 3133 10.4049/jimmunol.170.6.3125 12626569
56. Fan F. Podar K. The Role of AP-1 Transcription Factors in Plasma Cell Biology and Multiple Myeloma Pathophysiology Cancers 2021 13 2326 10.3390/cancers13102326 34066181 PMC8151277
57. Jovanovic K.K. Roche-Lestienne C. Ghobrial I.M. Facon T. Quesnel B. Manier S. Targeting MYC in multiple myeloma Leukemia 2018 32 1295 1306 10.1038/s41375-018-0036-x 29467490
58. Jin Z. Zhou S. Ye H. Jiang S. Yu K. Ma Y. The mechanism of SP1/p300 complex promotes proliferation of multiple myeloma cells through regulating IQGAP1 transcription Biomed. Pharmacother. 2019 119 109434 10.1016/j.biopha.2019.109434 31536933
59. Viziteu E. Grandmougin C. Goldschmidt H. Seckinger A. Hose D. Klein B. Moreaux J. Chetomin, targeting HIF-1alpha/p300 complex, exhibits antitumour activity in multiple myeloma Br. J. Cancer 2016 114 519 523 10.1038/bjc.2016.20 26867162 PMC4782210
60. Gong Y. Zack T.I. Morris L.G. Lin K. Hukkelhoven E. Raheja R. Tan I.L. Turcan S. Veeriah S. Meng S. Pan-cancer genetic analysis identifies PARK2 as a master regulator of G1/S cyclins Nat. Genet. 2014 46 588 594 10.1038/ng.2981 24793136 PMC4251771
61. Tanaka A. Parkin-mediated selective mitochondrial autophagy, mitophagy: Parkin purges damaged organelles from the vital mitochondrial network FEBS Lett. 2010 584 1386 1392 10.1016/j.febslet.2010.02.060 20188730 PMC2843751
62. Kay D.M. Stevens C.F. Hamza T.H. Montimurro J.S. Zabetian C.P. Factor S.A. Samii A. Griffith A. Roberts J.W. Molho E.S. A comprehensive analysis of deletions, multiplications, and copy number variations in PARK2 Neurology 2010 75 1189 1194 10.1212/WNL.0b013e3181f4d832 20876472 PMC3013490
63. Broderick P. Chubb D. Johnson D.C. Weinhold N. Forsti A. Lloyd A. Olver B. Ma Y. Dobbins S.E. Walker B.A. Common variation at 3p22.1 and 7p15.3 influences multiple myeloma risk Nat. Genet. 2011 44 58 61 10.1038/ng.993 22120009 PMC5108406
64. Levy D. Ehret G.B. Rice K. Verwoert G.C. Launer L.J. Dehghan A. Glazer N.L. Morrison A.C. Johnson A.D. Aspelund T. Genome-wide association study of blood pressure and hypertension Nat. Genet. 2009 41 677 687 10.1038/ng.384 19430479 PMC2998712
65. Folkersen L. van’t Hooft F. Chernogubova E. Agardh H.E. Hansson G.K. Hedin U. Liska J. Syvanen A.C. Paulsson-Berne G. Franco-Cereceda A. Association of genetic risk variants with expression of proximal genes identifies novel susceptibility genes for cardiovascular disease Circ. Cardiovasc. Genet. 2010 3 365 373 10.1161/CIRCGENETICS.110.948935 20562444
66. Cross H.S. Lipkin M. Kallay E. Nutrients regulate the colonic vitamin D system in mice: Relevance for human colon malignancy J. Nutr. 2006 136 561 564 10.1093/jn/136.3.561 16484524
67. Pendas-Franco N. Aguilera O. Pereira F. Gonzalez-Sancho J.M. Munoz A. Vitamin D and Wnt/beta-catenin pathway in colon cancer: Role and regulation of DICKKOPF genes Anticancer Res. 2008 28 2613 2623 19035286
68. Rohan J.N. Weigel N.L. 1Alpha,25-dihydroxyvitamin D3 reduces c-Myc expression, inhibiting proliferation and causing G1 accumulation in C4-2 prostate cancer cells Endocrinology 2009 150 2046 2054 10.1210/en.2008-1395 19164469 PMC2671895
69. Clay-Gilmour A.I. Hildebrandt M.A.T. Brown E.E. Hofmann J.N. Spinelli J.J. Giles G.G. Cozen W. Bhatti P. Wu X. Waller R.G. Coinherited genetics of multiple myeloma and its precursor, monoclonal gammopathy of undetermined significance Blood Adv. 2020 4 2789 2797 10.1182/bloodadvances.2020001435 32569378 PMC7322948
70. Yousefi S. Perozzo R. Schmid I. Ziemiecki A. Schaffner T. Scapozza L. Brunner T. Simon H.U. Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis Nat. Cell Biol. 2006 8 1124 1132 10.1038/ncb1482 16998475
71. Heuck C.J. Mehta J. Bhagat T. Gundabolu K. Yu Y. Khan S. Chrysofakis G. Schinke C. Tariman J. Vickrey E. Myeloma is characterized by stage-specific alterations in DNA methylation that occur early during myelomagenesis J. Immunol. 2013 190 2966 2975 10.4049/jimmunol.1202493 23408834 PMC4581585
72. Dilworth D. Liu L. Stewart A.K. Berenson J.R. Lassam N. Hogg D. Germline CDKN2A mutation implicated in predisposition to multiple myeloma Blood 2000 95 1869 1871 10.1182/blood.V95.5.1869.005k09_1869_1871 10688850
73. Shah V. Boyd K.D. Houlston R.S. Kaiser M.F. Constitutional mutation in CDKN2A is associated with long term survivorship in multiple myeloma: A case report BMC Cancer 2017 17 718 10.1186/s12885-017-3715-5 29110637 PMC5674776
74. Kay N.E. Leong T. Bone N. Kyle R.A. Greipp P.R. Van Ness B. Oken M.M. T-helper phenotypes in the blood of myeloma patients on ECOG phase III trials E9486/E3A93 Br. J. Haematol. 1998 100 459 463 10.1046/j.1365-2141.1998.00609.x 9504626
75. Zeller T. Wild P. Szymczak S. Rotival M. Schillert A. Castagne R. Maouche S. Germain M. Lackner K. Rossmann H. Genetics and beyond--the transcriptome of human monocytes and disease susceptibility PLoS ONE 2010 5 e10693 10.1371/journal.pone.0010693 20502693 PMC2872668
76. Rios-Tamayo R. Lupianez C.B. Campa D. Hielscher T. Weinhold N. Martinez-Lopez J. Jerez A. Landi S. Jamroziak K. Dumontet C. A common variant within the HNF1B gene is associated with overall survival of multiple myeloma patients: Results from the IMMEnSE consortium and meta-analysis Oncotarget 2016 7 59029 59048 10.18632/oncotarget.10665 27437873 PMC5312293
77. International Myeloma Working Group Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: A report of the International Myeloma Working Group Br. J. Haematol. 2003 121 749 757 10.1046/j.1365-2141.2003.04355.x 12780789
78. Rajkumar S.V. Dimopoulos M.A. Palumbo A. Blade J. Merlini G. Mateos M.V. Kumar S. Hillengass J. Kastritis E. Richardson P. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma Lancet Oncol. 2014 15 e538 e548 10.1016/S1470-2045(14)70442-5 25439696
79. Howie B.N. Donnelly P. Marchini J. A flexible and accurate genotype imputation method for the next generation of genome-wide association studies PLoS Genet. 2009 5 e1000529 10.1371/journal.pgen.1000529 19543373 PMC2689936
80. Willer C.J. Li Y. Abecasis G.R. METAL: Fast and efficient meta-analysis of genomewide association scans Bioinformatics 2010 26 2190 2191 10.1093/bioinformatics/btq340 20616382 PMC2922887
81. Martino A. Sainz J. Buda G. Jamroziak K. Reis R.M. Garcia-Sanz R. Jurado M. Rios R. Szemraj-Rogucka Z. Marques H. Genetics and molecular epidemiology of multiple myeloma: The rationale for the IMMEnSE consortium (review) Int. J. Oncol. 2012 40 625 638 10.3892/ijo.2011.1284 22159523
82. Manuel Sanchez-Maldonado J. Martinez-Bueno M. Canhao H. Ter Horst R. Munoz-Pena S. Moniz-Diez A. Rodriguez-Ramos A. Escudero A. Sorensen S.B. Hetland M.L. NFKB2 polymorphisms associate with the risk of developing rheumatoid arthritis and response to TNF inhibitors: Results from the REPAIR consortium Sci. Rep. 2020 10 4316 10.1038/s41598-020-61331-5 32152480 PMC7062729
83. Li Y. Oosting M. Smeekens S.P. Jaeger M. Aguirre-Gamboa R. Le K.T.T. Deelen P. Ricano-Ponce I. Schoffelen T. Jansen A.F.M. A Functional Genomics Approach to Understand Variation in Cytokine Production in Humans Cell 2016 167 1099 1110.e14 10.1016/j.cell.2016.10.017 27814507
84. Aguirre-Gamboa R. Joosten I. Urbano P.C.M. van der Molen R.G. van Rijssen E. van Cranenbroek B. Oosting M. Smeekens S. Jaeger M. Zorro M. Differential Effects of Environmental and Genetic Factors on T and B Cell Immune Traits Cell Rep. 2016 17 2474 2487 10.1016/j.celrep.2016.10.053 27818087 PMC5130901