The Interaction between Gut Microbiota and Host Amino Acids Metabolism in Multiple Myeloma

Simple Summary Multiple myeloma (MM) is the second most common hematological malignancy and remains incurable. Recent evidence substantiates the interaction of gut microbiota and MM, together with abnormal amino acid metabolism and MM. Moreover, the association between gut microbiota and host amino acid metabolism on MM has been highlighted. This article presents a review of the literature on the relationship between gut microbiota, metabolism, and MM, together with strategies to modulate the microbiota. Abstract Although novel therapies have dramatically improved outcomes for multiple myeloma (MM) patients, relapse is inevitable and overall outcomes are heterogeneous. The gut microbiota is becoming increasingly recognized for its influence on host metabolism. To date, evidence has suggested that the gut microbiota contributes to MM, not only via the progressive activities of specific bacteria but also through the influence of the microbiota on host metabolism. Importantly, the abnormal amino acid metabolism, as well as the altered microbiome in MM, is becoming increasingly apparent, as is the influence on MM progression and the therapeutic response. Moreover, the gut-microbiota–host-amino-acid metabolism interaction in the progression of MM has been highlighted. Modulation of the gut microbiota (such as fecal microbiota transplantation, FMT) can be modified, representing a new angle in MM treatment that can improve outcomes. In this review, the relationship between gut microbiota, metabolism, and MM, together with strategies to modulate the microbiota, will be discussed, and some unanswered questions for ongoing and future research will be presented.


Introduction
Multiple myeloma (MM) is a hematologic malignancy with abnormal proliferation of clonal plasma cells in the bone marrow, which accounts for the second most common hematological malignancy [1] and is characterized by the manifestations of destructive bone lesions, kidney injury, anemia, and hypercalcemia [2]. Tremendous progress has been made in both the pathogenesis and treatment of MM, thereby largely improving clinical outcomes with a median survival of 5-7 years [3]. Nevertheless, the inevitable occurrence of treatment resistance and relapse seriously threatens patients' lives, and heterogeneous overall outcomes remain challenging for researchers [1], which requires us to explore new angles for a better understanding of MM. Recent evidence substantiates the association However, the concentration of ammonia could be regulated by the host to reduce its toxicity, probably by assimilation into microbe for microbial amino acid biosynthetic processes, or through conversion to citrulline and glutamine in host intestinal epithelial cells, together with slowing the release into the bloodstream [31,32]. Furthermore, SCFAs can be produced by the function of amino acid fermentation. The ketogenic and glucogenic metabolic pathways are processed to produce SCFAs (butyrate and propionate) (Figure 1), and then utilized for the metabolic needs of the colon and the body [33,34]. Several studies have shown the remarkable interactions between SCFAs and anti-inflammatory and antiapoptotic effects on cancer [35]. The recent studies mainly aimed at the end-products of amino acids, while the role of secondary metabolites on MM during the metabolic process of amino acids is still unclear. A deeper understanding of this topic in the future may show this to be a potential mechanism and modulation in MM. mucosal damage and further cause colorectal adenocarcinoma, showing potential ca cinogenic effects [30]. However, the concentration of ammonia could be regulated by th host to reduce its toxicity, probably by assimilation into microbe for microbial amino aci biosynthetic processes, or through conversion to citrulline and glutamine in host intes tinal epithelial cells, together with slowing the release into the bloodstream [31,32]. Fu thermore, SCFAs can be produced by the function of amino acid fermentation. The ke togenic and glucogenic metabolic pathways are processed to produce SCFAs (butyrat and propionate) (Figure 1), and then utilized for the metabolic needs of the colon and th body [33,34]. Several studies have shown the remarkable interactions between SCFAs an anti-inflammatory and anti-apoptotic effects on cancer [35]. The recent studies mainl aimed at the end-products of amino acids, while the role of secondary metabolites o MM during the metabolic process of amino acids is still unclear. A deeper understandin of this topic in the future may show this to be a potential mechanism and modulation i MM. Figure 1. Key metabolic pathways of amino acids in the gut. Through the function of fermentatio the ketogenic and glucogenic metabolic pathways are processed to produce ammonia and SCFA (butyrate and propionate). In detail, as for the ketogenic process, the amino acids are converted t lysine, and some bacteria then cause the production of ammonia (Lysine pathway). In additio lysine is metabolized to butyryl-CoA for the production of butyrate (CoA-transferase, succinat and butyrate kinase pathway). Concerning the glucogenic pathway, the microbiota is mainly fo the final synthesis of butyrate, and the production of propionate by the succinate pathway.
The fermentation of amino acids could produce a variety of potentially bioactiv products by several Bacteroides spp. and some Firmicutes [30,[36][37][38]. Some nitrogenou products, especially N-nitroso compounds (NOCs), have emphasized a significant feca increase in high-protein dietary individuals [38]. Moreover, the increase in the dietar Figure 1. Key metabolic pathways of amino acids in the gut. Through the function of fermentation, the ketogenic and glucogenic metabolic pathways are processed to produce ammonia and SCFAs (butyrate and propionate). In detail, as for the ketogenic process, the amino acids are converted to lysine, and some bacteria then cause the production of ammonia (Lysine pathway). In addition, lysine is metabolized to butyryl-CoA for the production of butyrate (CoA-transferase, succinate, and butyrate kinase pathway). Concerning the glucogenic pathway, the microbiota is mainly for the final synthesis of butyrate, and the production of propionate by the succinate pathway.
The fermentation of amino acids could produce a variety of potentially bioactive products by several Bacteroides spp. and some Firmicutes [30,[36][37][38]. Some nitrogenous products, especially N-nitroso compounds (NOCs), have emphasized a significant fecal increase in high-protein dietary individuals [38]. Moreover, the increase in the dietary intake of NOCs has been reported to be positively correlated with colorectal cancer [39], and exert carcinogenic effects potentially via DNA alkylation [40]. In addition, polyamines synthesis also occurs in gut bacteria [41], together with up-regulated production by host cells promoted by certain gut bacteria, such as enterotoxigenic Bacteroides fragilis [42]. Polyamines are involved in varieties of physiological functions [35]. However, high levels of polyamines have been reported to be associated with cancer, probably by oxidative stress that results from polyamine catabolism [42]. In addition, some bacteria exhibit toxicity through polyamines [43].

Abnormal Amino Acid Metabolism and Multiple Myeloma
Given this growing development of the metabolic function of gut microbiota, it is becoming increasingly clear that gut microbiota participates in host metabolic processes and exerts influences on the carcinogenesis and development of cancer. Studies to further dissect the gut microbiota-metabolic interactions underlying the myelomagenesis, progression, and anti-myeloma responses were discussed. Among them, myeloma-related metabolic reprogramming has been defined.

The Effects of Abnormal Amino Acid Metabolism on MM
Different from other solid tumor cells, MM cells produce abundant ineffective monoclonal immunoglobulin. In line with this, different metabolic characteristics may exist in MM. Studies have provided strong evidence for this speculation. Aided by highperformance liquid chromatography (HPLC) and mass spectrometry detection, metabonomics analysis of MM patients and healthy individuals was performed and demonstrated that the differential metabolites were mainly enriched in amino acid metabolism [44]. Additional cohorts have also been studied by untargeted/targeted metabolomics, showing the abnormal amino acid metabolisms in MM, rather than the traditional views of abnormal glucose metabolism in a majority of cancers, which may be related to the biological function of MM [25,[44][45][46][47][48]. Therefore, differential amino acid metabolic profiles in MM patients have been brought to this topic and are summarized based on the insights gained from the results.
Accounting for the onset of MM, the stage of MGUS has been studied to explore myelomagenesis. A substantially different metabolomic profile of MGUS and MM patients and healthy individuals was determined, which is characterized by a significant difference in glutamate between MM and MGUS, with the potential involvement of amino acid metabolism disorders in the myelomagenesis [48]. The results were further supplemented by multiple studies demonstrating the role of amino acids in MM patients. Our inspirational findings involved substantiating the higher concentrations of glycine, serine, proline, and glutamate in MM patients [25]. Differential metabolites of leucine, tryptophan, and valine have also been drawn in MM patients; these metabolites are considered evidence for the pathogenesis and development of MM [44]. In addition, a disorder of glutamate metabolism in the bone marrow microenvironment of MM patients has been highlighted, demonstrating increased glutamate and decreased glutamine [49]. The group also studied and defined serum aspartic acid as a candidate biomarker for diagnosis, while serum threonine was for risk prediction [49].

The Effects of Abnormal Amino Acid Metabolism on MM Progression
Importantly, the impacts of the amino acids on MM progression have also been studied in the setting of association with MM tumor burden. Our findings involved substantiating the accelerative function, demonstrating that the MM group with high glycine concentration in the bone marrow had a more serious ISS stage and higher plasma cell percentages. Our further investigations into mechanisms confirmed that glycine contributed to MM cell proliferation through glutathione synthesis. Effectively blocking the utilization of glycine may inhibit proliferation, suggesting that targeted amino acid metabolism (such as glycine metabolism) may have therapeutic potential in MM [25]. Another study assessed serine synthesis on MM progression, demonstrating that the increase of serine synthesis was related to the proliferation of MM cells, and higher expression of the related gene PHGDH was associated with a poor prognosis of MM [50]. Furthermore, abnormal amino acid metabolism promoted MM progression through the immunosuppressive microenvironment [51].
In addition to these studies on MM progression, the impact of abnormal amino acids has been studied in myeloma-related end-organ dysfunction. As reported, higher glycine in the bone marrow was significantly correlated with bone destruction in MM patients. Conversely, osteolysis had been alleviated in MM mice by glycine-free diets [25]. Our group also proposed the notion of a positive correlation between higher glutamine and pneumonia in MM. This has demonstrated the aggravative function of increased inflammatory factors by glutamine [52]. Given the importance of abnormal amino acids in MM progression, it is possible that targeting them could serve as a potential strategy to improve clinical outcomes. However, efforts to identify ideal targets remain to be defined.

The Effects of Abnormal Amino Acid Metabolism on MM Therapy
Based on the available literature, it is clear that amino acid metabolism is associated with drug response and toxicity of MM. To be specific, glycine [25], as well as proline [53], has demonstrated an increase in the resistance of Bortezomib (BTZ). Furthermore, inhibition of glycine utilization and PHGDH inhibitors respectively may enhance the effects of BTZ on MM cells. Moreover, inhibition of the L-glutamine transporter ASCT2 has been reported to enhance the sensitivity of MM cells to proteasome inhibitors [54]. In line with this, amino acid depletion triggered by L-asparaginase has been reported to sensitize MM cells to carfilzomib by inducing mitochondria ROS-mediated cell death [55]. Furthermore, targeting MM glutamine metabolism has been confirmed to enhance the binding of the BIM gene to BCL-2, thereby increasing the sensitivity of MM cells to Venetoclax, providing an effective treatment strategy for relapsed refractory MM [56].
Overall, these data provide evidence that the metabolic pattern in MM is mainly characterized by abnormal amino acid metabolism, especially abnormal glycine and serine metabolism. The impact of the abnormal amino acids on MM progression and the therapeutic response has also been defined. Thus, it is increasingly clear that targeted amino acid metabolism may represent a novel and important adjunct to current anti-myeloma therapeutic modalities. However, major challenges remain. Furthermore, efforts to identify the origin of abnormal amino acid metabolism of MM to better understand MM are underway.

Gut Microbiota and Multiple Myeloma
Increasing evidence suggests an important role of microbes and microbial functions in cancer, ranging from carcinogenesis and progression to response to cancer therapy. With recent studies demonstrating the influence of the gut microbiome on hematological cancers, the correlation between gut microbiota and MM, together with their impact on responses to MM therapy, will be discussed herein (Table 1).

Gut Microbiota Composition and MM
Concerning increasingly developed methods for profiling (16S rRNA sequencing and metagenomic shotgun sequencing), the diversity, along with compositional differences in the gut microbiota, have been studied in MM individuals; this is characterized by a lower diversity of microbiota [26,57] and potential enrichment of opportunistic pathogenic bacteria. Specifically, MM patients had significant enrichment of Raoultella ornithinolytica, Enterobacter cloacae, Citrobacter freundii, Klebsiella pneumoniae, etc., in the fecal sample [26]. Another cohort of MM patients also demonstrated significant differences at the stages of healthy individuals and MGUS and MM patients. MM patients had a higher abundance of Odoribacter and Lactobacillus and a lower abundance of Blautia and Faecalibacterium than healthy patients; MM patients also had a higher abundance of Kluyvera and Bacteroides and a lower abundance of Blautia and Parabacteroides compared with MGUS patients [58]. Furthermore, these dominant forces in the development of MM will be reviewed.

Gut Microbiota and MM Progression
Once the diversity and compositional differences in the gut microbiota have been identified, the next focus would be how they affect MM. Unlike the composition of the gut microbiota between MGUS and MM patients, it has been suggested that the imbalance of gut microbiota may be related to the development of MGUS into MM [59]. In addition, the influence of gut microbiota on hematopoiesis has been explored, demonstrating that Rag-1 deficient mice had a different composition of gut microbiota compared with wildtype mice, and transferable fecal bacteria from wild-type mice may significantly increase hematopoiesis [60]. This suggests that gut microbiota may play an important role in myelomagenesis. Meanwhile, a more direct correlation was studied by Calcinotto et al. [61], which showed that the level of IL-17 in the bone marrow of VK*MYC mice increased at the MGUS stage, and was then closely related to the progression of smoldering MM to MM, suggesting that the higher levels of IL-17 in bone marrow increase the susceptibility of smoldering MM to MM. Moreover, the IL-17 may be related to the stimulation of Th17 cells by gut microbiota. However, the effects of Th17 and its product IL-17 on MM cells are not yet clear. It has been reported that IL-17 promotes the progression of clonal plasma cells, but another study alleviates the progression of MM [62,63].
Furthermore, additional studies have aimed at understanding the gut microbiota and MM progression. In our work, a higher abundance of Enterobacteriaceae and Streptococcaceae was positively correlated with the ISS stage of MM patients, and enrichment of Klebsiella pneumoniae was confirmed to promote MM progression, underlying the effects of the microbiota on MM progression [26]. Meanwhile, intestinal Prevotella heparinolytica was reported to accelerate disease progression by promoting intestinal Th17 cell differentiation and migration to bone marrow in MM [61].

Gut Microbiota and MM Therapy
Clinically, the most significant advancements in MM therapy have been the introduction of proteasome inhibitors (bortezomib and carfilzomib), immunomodulatory agents, monoclonal antibodies directed against MM cell surface antigens, and autologous HSCT [2], which significantly prolonged patients' survival time. However, resistance to therapy and the chances of recurrence are still inevitable. The emerging knowledge of the ability of the gut microbiota to modulate the response to anti-cancer therapy offers new possibilities to improve anti-myeloma efficacy.

Gut Microbiota and Chemotherapy
Huang et al. [64] have discovered that the usage of corticosteroids may cause a change in the composition of gut microbiota, demonstrating a higher abundance of Bifidobacterium and Lactobacillus and a lower abundance of Mucispirillum in mice exposed to corticosteroids. The exposure to dexamethasone also led to substantial shifts in gut microbiota. Additionally, gut microbiota could shape responses to effective treatment with conventional chemotherapy. The effects of cyclophosphamide were abrogated in the antibiotic-treated mice, and the presence of particular bacterial species could increase the sensitivity to cyclophosphamide [65]. In addition to the efficacy of anti-cancer therapy, adverse effects regulated by gut microbiota have also been studied. SCFAs produced by intestinal microbes were reported to alleviate gastrointestinal toxicity caused by proteasome inhibitors [66].

Gut Microbiota and Hematopoietic Stem Cell Transplantation
High-dose chemotherapy combined with auto-HSCT has been considered an important treatment for MM patients [67]. Treated with concurrent therapies that significantly alter the composition of the gut microbiota, including immunosuppressants, broad-spectrum antibiotics, etc, these patients commonly had intestinal dysbiosis [68,69]. Indeed, analyses of longitudinal fecal samples demonstrated a loss of bacterial diversity after HSCT [70][71][72][73][74], demonstrating that the diversity of the gut microbiota of MM patients with auto-HSCT lowered before transplantation, and then lowered further during transplantation [71]. Moreover, MM patients with a low diversity of gut microbiota during transplantation had an impaired transplantation response [72], while those with a high diversity of gut microbiota had better transplantation results and lower transplant-related mortality [71]. In addition, the influence of the gut microbiota on the toxicity to HSCT therapies was investigated. During the pretreatment stage of melphalan, a higher abundance of Bacteroides was discovered to be associated with the reduced incidence of severe diarrhea, while a higher abundance of Blautia and Ruminococcus was closely related to severe diarrhea and post-transplant nausea and vomiting [73]. Meanwhile, the significant susceptibility to bloodstream infections (BSI) remained another toxicity of HSCT with lowered diversity and intestinal dysbiosis [74]. Studies have reported that with low diversity of gut microbiota and dominance of specific bacteria (mainly enterococcus, streptococcus, and various proteobacteria), bacteremia remarkedly increased during HSCT [75].

Gut Microbiota and CAR-T Therapy
Recently, chimeric antigen receptor (CAR) T-cell therapy has changed the therapeutic landscape in MM. Studies have revealed the potential role of gut microbiota in the efficacy and toxicity of CAR-T therapy for B-cell malignancies [76,77]. Hu et al. have found that a higher abundance of Sutterella was associated with a complete response and prolonged survival in MM patients with BCMA-CART treatment. The study has also defined the higher abundance of Bifidobacterium, Leuconostoc, Stenotrophomonas, and Staphylococcus in patients with severe cytokine release syndrome (CRS), which is reproducible in an independent cohort of 38 MM patients [78], suggesting that gut microbiota play a role in regulating efficacy and CRS of BCMA-CART therapy. However, there is still a great deal to learn more about the gut microbiota and CAR-T therapy, from the inherent mechanisms to optimal strategies to enhance therapeutic responses.

Gut Microbiota and Therapeutic Response
Based on the evidence of the association between gut microbiota and clinical outcomes of MM [71,73], efforts have focused on identifying predictors of the therapeutic response, which reveals that the higher abundance of butyrate-producing bacteria, such as Eubacterium hallii and Faecalibacterium prausnitzii, associated with negative minimal residual disease (MRD) [79], and together with fecal butyrate, are related to persistent negative MRD [80]. Thus, butyrate-producing bacteria may play a significant role in the evaluation of MRD status and prediction of efficacy in MM patients.
Summarily, with the change in the diversity and composition of the gut microbiota, insights have been gained into the influence of the gut microbiota on MM, not only from the aspects of myelomagenesis and progression but also with regard to the response and toxicity of therapy. Thus, valuable information on how gut microbiota affects MM has drawn the attention of researchers.

Gut Microbiota-Host Amino Acid Metabolism Interaction on Multiple Myeloma
As reviewed above, the gut microbiota is becoming increasingly recognized for its influence on host metabolism. Meanwhile, gut microbiota contributes to MM, not only via the progressive activities of specific bacteria but also through the influence of the microbiota on host metabolism. In particular, the application of high throughput multi-omics analysis to the study of MM (Figure 2) brought a deeper understanding of the association of gut microbiota and host metabolism. Therefore, we further review evidence for gut microbiotahost amino acid metabolism interaction in the progression of MM (Figure 3).

Gut Microbiota-Host Amino Acid Metabolism Interaction on Multiple Myeloma
As reviewed above, the gut microbiota is becoming increasingly recognized for its influence on host metabolism. Meanwhile, gut microbiota contributes to MM, not only via the progressive activities of specific bacteria but also through the influence of the microbiota on host metabolism. In particular, the application of high throughput multi-omics analysis to the study of MM (Figure 2) brought a deeper understanding of the association of gut microbiota and host metabolism. Therefore, we further review evidence for gut microbiota-host amino acid metabolism interaction in the progression of MM (Figure 3).

Gut Microbiota-Host Amino Acid Metabolism Interaction on Multiple Myeloma
As reviewed above, the gut microbiota is becoming increasingly recognized for its influence on host metabolism. Meanwhile, gut microbiota contributes to MM, not only via the progressive activities of specific bacteria but also through the influence of the microbiota on host metabolism. In particular, the application of high throughput multi-omics analysis to the study of MM ( Figure 2) brought a deeper understanding of the association of gut microbiota and host metabolism. Therefore, we further review evidence for gut microbiota-host amino acid metabolism interaction in the progression of MM (Figure 3).  Interaction of gut microbiota and host amino acid metabolism in multiple myeloma. The amino acids are converted to ammonia. Conversely, ammonia is re-utilized by nitrogen bacteria to produce glutamine, wherein the bone marrow, glutamine, which is addicted to MM cells, is utilized by MM cells for proliferation. When it accumulates in the lung, glutamine contributes to the Figure 3. Interaction of gut microbiota and host amino acid metabolism in multiple myeloma. The amino acids are converted to ammonia. Conversely, ammonia is re-utilized by nitrogen bacteria to produce glutamine, wherein the bone marrow, glutamine, which is addicted to MM cells, is utilized by MM cells for proliferation. When it accumulates in the lung, glutamine contributes to the proliferation of lung normal fibroblast cells and elevated secretion of TNF-α for inflammatory infiltration. In addition, the SCFAs produced in the gut are adsorbed and distributed in the bone marrow, alleviating the proliferation of MM cells.

Gut Microbiota-Host Glutamine Metabolism Interaction on MM Progression
The crosstalk between microbiota and host metabolism is critical. Microbes encode related genes to metabolize dietary nutrients in the gut [24] and produce varieties of metabolites. Among them, L-glutamine (Gln) is one of the microbial metabolites, which is also synthesized from glutamate (Glu) and ammonium (NH 4 + ) by glutaminase [81] to meet the requirements of both energy generation and as a source of carbon and nitrogen for biomass accumulation in physiological conditions [82]. The maintenance of high levels of glutamine has also been shown to provide a ready source of carbon and nitrogen to drive tumor growth [82][83][84]. As for MM, glutamine has been proven to be important in the growth of cells. Furthermore, researchers have revealed that the gut dominant bacteria were significantly correlated with the serum differential metabolites in MM. Specifically, Klebsiella pneumoniae promoted MM progression via de novo synthesis of glutamine [26]. Then, addicted to Gln, MM cells metabolized Gln, leading to an accumulation of NH 4 + in the bone marrow and subsequent release into circulation [26,81,85]. Vice versa, abnormal accumulation of NH 4 + promoted the proliferation of nitrogen-source circulating bacteria, such as Klebsiella pneumoniae, thereby promoting the synthesis of glutamine [26]. The data presently available suggest that the crosstalk between gut microbiota and host glutamine metabolism in the MM progression, giving hints that manipulation of gut microbiotaglutamine interaction could be a novel treatment strategy. Another study has focused on pneumonia in MM patients, which accounts for a significant cause of morbidity and mortality [86], demonstrating that the dominant bacteria of Klebsiella pneumoniae synthesized glutamine to promote the expression of pulmonary inflammatory factors, and thereby contribute to pneumonia in MM [52].

Gut Microbiota-Host Short-Chain Fatty Acids Metabolism Interaction on MM Progression
A better understanding of the roles of microbes in SCFAs metabolism provides an opportunity to explore gut microbiota-SCFA metabolism interaction in MM. As the most common bacterial end-product involved in amino acid metabolism [87], SCFAs exert beneficial biological activities on MM. Researchers have reported a lower abundance of SCFAs-producing bacteria in MM compared with healthy controls [26,88,89]. The alleviation of progression in MM mice with oral gavage of Clostridium butyricum has also been observed [26]. Nonetheless, the inherent mechanisms of SCFAs on MM progression remain poorly understood, SCFAs were reported to skew the balance of inflammatory cytokines locally and systemically or modulate immunity regarding cancer development and progression or response to therapy [90,91]. In fact, the functions of the anti-tumor role have been extended to recognize mechanisms that alleviate MM progression, including SCFAs that either inhibit inflammatory factors (such as IL-1β, IL-6, and TNF-a) or induce the expression of anti-inflammatory factors (such as IL-10), all of which were considered to be at a high level in the bone marrow microenvironment [74].
These studies have highlighted a role in the interaction between gut microbiota and host amino acid metabolism (mainly glutamine and SCFAs) in the MM progression. Furthermore, additional studies on the interaction of gut microbiota and host amino acid metabolism in MM remain to be defined.

Clinical Translational Insights of the Microbiota-Host Amino Acid Metabolism Interactions
Given the above presentation, the clinical translations of the interactions have attracted the attention of scientists. Based on the MM-enriched microbiota, host metabolites, such as creatinine, aminomalonic acid, and L-proline, were reported to be positively associated with Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella variicola; their abundance was positively reflected with ISS stages in MM patients [26]. There are still limited studies that examine the interaction between gut microbiota and host amino acid metabolism in MM. As the field moves forward, there may be renewed interest in these clinical translations.
Clinical translations made by modulating the gut microbiota may represent a novel adjunct of modalities of MM patients. The prophylactic antibiotics were presumed to alle-viate MM progression or improve therapeutic efficacy by intervening in the gut microbiota to eliminate pathogenic bacteria. However, after the first demonstration that administration of antibiotics abrogated anti-tumor activity in a murine model of melanoma [92], the TEAMM results showed similar results, which demonstrated the higher mortality of MM patients with 12-week treatment of prophylactic levofloxacin [93]. Subsequent studies drew the same conclusion, that an imbalance of gut microbiota caused by prophylactic antibiotic treatment may promote MM progression [94,95]. In addition, more relevant reports showed that disrupting the gut microbiota by antibiotic use could impair the anti-tumor efficacy [65,[96][97][98].
Fecal bacteria transplantation (FMT) is considered the most direct and effective means to manipulate gut microbiota. To our knowledge, various factors, including conditioning regimens, infections, antibiotics use, and immune response, lead to drastic and rapid perturbations of the gut microbiota during HSCT. FMT has been proven to be an optimal modulation for patients with HSCT to restore the diversity of gut microbiota [99][100][101][102], as well as an effective strategy for HSCT-associated Clostridium difficile infection [103,104] and steroid-resistant acute GVHD [105,106]. With the potential transmission of antibioticresistant pathogens, engineered microbial transplantation is another approach in the preclinical stage. In addition, intervening with dietary and pre-/probiotic supplementation is also of important consideration to improve the outcomes of cancer [107]. From this aspect, a study has reported that MM patients with healthier prediagnosis dietary habits may have longer survival than those with less healthy diets [108], suggesting the potentiality of the dietary intervention for MM.

Limitations of Microbiome Analysis and Studies
The profiling of microbiome and metabolome has been increasingly yielding new information on the altered microbiota and metabolites by the utility of omics. However, with the complexities and the unknowns of microbiota, the concern of optimal sequence methods and the responding analytical methods (such as 16S rRNA sequencing or metagenomic shotgun sequencing, the choice of which analytical databases) still exist, especially for the interpretation of analytic results that will be translated into clinical applications. In addition, the microbiota imbalance, which influences the phenotype of the observed mice, may not be entirely attributable to bacteria, but may involve the other microbiota (fungi, viruses, etc.) [109,110], hinting at the need for cautious interpretation in clinical translations. Then, inconsistency existed in different studies. Studies on the altered microbiome and metabolome of MM patients have been identified. However, without enough consistency across different studies and considerations of differences in host interaction with environment and diet, which may vary substantially, the limitation of cross-study comparison inevitably exists. Furthermore, recognizing the specific bacteria involved, as they vary by disease and patient population, can create a better fundamental understanding of the heterogeneity of MM as well as an ability to predict the effects of gut microbiota-host amino acid metabolism interaction on MM; as of now, however, this topic is poorly understood. Finally, regarding the interaction between gut microbiota and host amino acid metabolism, there is not yet a great deal of literature to draw more general conclusions, and it is unclear why this interaction contributes to MM (especially myelomagenesis). Overall, although many challenges remain, building a better understanding of the roles of microbes and metabolites may enable a powerful new tool for improving the outcomes of MM.

Challenges of the Microbiome in Future Clinical Translations
Most current studies focusing on the modulation of gut microbiota have been in mice, and therefore, the translation of these achievements to clinical applications remains challenging. First, the differences between mice and humans should not be ignored. The similarities between mice are amplified for several reasons, such as relative homogeneity (identical genetic backgrounds, diet, and similar environment), and the substantial cage effects [111]. Conversely, the many inherent inter-subject variabilities, especially for disease activity and medication use, lead to biological outcomes that have obscure meanings. Second, to identify the correlation of different bacterial species with a clinical response by FMT, these humanized mice mainly reproduce the bacterial diversity of the donor microbiota [112,113]. However, these mice are not identical to humans in the aspect of physiological and immune responses [114]. Indeed, studies have demonstrated the differences [115,116]. Finally, FMT has been shown to successfully benefit patients in some diseases. However, the process of FMT is difficult and expensive; it also has the potential to transfer to other diseases. These factors necessitate careful considerations of donor selection, conditioning regimen, and banking for potential future autologous transplant.

Future Directions
Preclinical studies on the role of gut microbiota and host metabolism in MM have brought us to focus on this potentially dominant mediator in MM and therapy. However, there is still a great deal to learn concerning the inherent mechanisms, predictive significance, and efficacy to modulate the interaction of gut microbiota and host metabolism, probably ranging from definitive cause-effect relationships, screening of biomarkers in the aspect of diagnosis and dynamic surveillance, to the selection of modulation. In the process, the standardized approaches of collection and analyses of specimens, together with the integration of available data, will bring more valuable information and strategies to target MM therapy. Especially for the modulation, clinical trials targeting this approach for MM patients are in development. Importantly, multicenter studies are critical to minimize geographic differences. Although many challenges remain, building a better understanding of the roles of microbes and metabolites may enable a powerful new tool for improving the outcomes of MM.

Conclusions
There is compelling evidence that the gut microbiota affects host amino acid metabolism in MM, and that manipulation of microbiota may augment response to anti-myeloma therapy in preclinical models. Based on the influence of gut microbiota on host amino acid metabolism, the abnormal amino acid metabolism, as well as the altered microbiome in MM have been first defined, and these items influence MM progression and the therapeutic response. Then, the gut-microbiota-host-amino-acid metabolism interaction (mainly glutamine and SCFAs) in the MM progression, together with modulation of the gut microbiota (such as FMT) as a novel adjunct regimen of MM patients, has been highlighted. Overall, only through a comprehensive understanding of these interactions can we learn to optimally modulate the gut microbiota to enhance the clinical outcomes of MM.

Conflicts of Interest:
The authors declare no conflict of interest.