Definition and Clinical Significance of the Monoclonal Gammopathy of Undetermined Significance-Like Phenotype in Patients With Monoclonal Gammopathies.

PURPOSE: The existence of patients with multiple myeloma (MM) and light-chain (AL) amyloidosis who present with a monoclonal gammopathy of undetermined significance (MGUS)-like phenotype has been hypothesized, but methods to identify this subgroup are not standardized and its clinical significance is not properly validated. PATIENTS AND METHODS: An algorithm to identify patients having MGUS-like phenotype was developed on the basis of the percentages of total bone marrow (BM) plasma cells (PC) and of clonal PC within the BM PC compartment, determined at diagnosis using flow cytometry in 548 patients with MGUS and 2,011 patients with active MM. The clinical significance of the algorithm was tested and validated in 488 patients with smoldering MM, 3,870 patients with active MM and 211 patients with AL amyloidosis. RESULTS: Patients with smoldering MM with MGUS-like phenotype showed significantly lower rates of disease progression (4.5% and 0% at 2 years in two independent series). There were no statistically significant differences in time to progression between treatment versus observation in these patients. In active newly diagnosed MM, MGUS-like phenotype retained independent prognostic value in multivariate analyses of progression-free survival (PFS; hazard ratio [HR], 0.49; P = .001) and overall survival (OS; HR, 0.56; P = .039), together with International Staging System, lactate dehydrogenase, cytogenetic risk, transplant eligibility, and complete remission status. Transplant-eligible patients with active MM with MGUS-like phenotype showed PFS and OS rates at 5 years of 79% and 96%, respectively. In this subgroup, there were no differences in PFS and OS according to complete remission and measurable residual disease status. Application of the algorithm in two independent series of patients with AL predicted for different survival. CONCLUSION: We developed an open-access algorithm for the identification of MGUS-like patients with distinct clinical outcomes. This phenotypic classification could become part of the diagnostic workup of MM and AL amyloidosis.

Tumor cells in light-chain amyloidosis and myeloma show distinct transcriptional rewiring of normal plasma cell development.

Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation-related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies.

Minimal residual disease negativity by next-generation flow cytometry is associated with improved organ response in AL amyloidosis.

Light chain (AL) amyloidosis is caused by a small B-cell clone producing light chains that form amyloid deposits and cause organ dysfunction. Chemotherapy aims at suppressing the production of the toxic light chain (LC) and restore organ function. However, even complete hematologic response (CR), defined as negative serum and urine immunofixation and normalized free LC ratio, does not always translate into organ response. Next-generation flow (NGF) cytometry is used to detect minimal residual disease (MRD) in multiple myeloma. We evaluated MRD by NGF in 92 AL amyloidosis patients in CR. Fifty-four percent had persistent MRD (median 0.03% abnormal plasma cells). There were no differences in baseline clinical variables in patients with or without detectable MRD. Undetectable MRD was associated with higher rates of renal (90% vs 62%, p = 0.006) and cardiac response (95% vs 75%, p = 0.023). Hematologic progression was more frequent in MRD positive (0 vs 25% at 1 year, p = 0.001). Altogether, NGF can detect MRD in approximately half the AL amyloidosis patients in CR, and persistent MRD can explain persistent organ dysfunction. Thus, this study supports testing MRD in CR patients, especially if not accompanied by organ response. In case MRD persists, further treatment could be considered, carefully balancing residual organ damage, patient frailty, and possible toxicity.

Maturation of NAA20 Aminoterminal End Is Essential to Assemble NatB N-Terminal Acetyltransferase Complex.

Protein lifespan is regulated by co-translational modification by several enzymes, including methionine aminopeptidases and N-alpha-aminoterminal acetyltransferases. The NatB enzymatic complex is an N-terminal acetyltransferase constituted by two subunits, NAA20 and NAA25, whose interaction is necessary to avoid NAA20 catalytic subunit degradation. We found that deletion of the first five amino acids of hNAA20 or fusion of a peptide to its amino terminal end abolishes its interaction with hNAA25. Substitution of the second residue of hNAA20 with amino acids with small, uncharged side-chains allows NatB enzymatic complex formation. However, replacement by residues with large or charged side-chains interferes with its hNAA25 interaction, limiting functional NatB complex formation. Comparison of NAA20 eukaryotic sequences showed that the residue following the initial methionine, an amino acid with a small uncharged side-chain, has been evolutionarily conserved. We have confirmed the relevance of second amino acid characteristics of NAA20 in NatB enzymatic complex formation in Drosophila melanogaster. Moreover, we have evidenced the significance of NAA20 second residue in Saccharomyces cerevisiae using different NAA20 versions to reconstitute NatB formation in a yNAA20-KO yeast strain. The requirement in humans and in fruit flies of an amino acid with a small uncharged side-chain following the initial methionine of NAA20 suggests that methionine aminopeptidase action may be necessary for the NAA20 and NAA25 interaction. We showed that inhibition of MetAP2 expression blocked hNatB enzymatic complex formation by retaining the initial methionine of NAA20. Therefore, NatB-mediated protein N-terminal acetylation is dependent on methionine aminopeptidase, providing a regulatory mechanism for protein N-terminal maturation.

N-terminal acetylation mutants affect alpha-synuclein stability, protein levels and neuronal toxicity.

Alpha-synuclein (aSyn) protein levels are sufficient to drive Parkinson's disease (PD) and other synucleinopathies. Despite the biomedical/therapeutic potential of aSyn protein regulation, little is known about mechanisms that limit/control aSyn levels. Here, we investigate the role of a post-translational modification, N-terminal acetylation, in aSyn neurotoxicity. N-terminal acetylation occurs in all aSyn molecules and has been proposed to determine its lipid binding and aggregation capacities; however, its effect in aSyn stability/neurotoxicity has not been evaluated. We generated N-terminal mutants that alter or block physiological aSyn N-terminal acetylation in wild-type or pathological mutant E46K aSyn versions and confirmed N-terminal acetylation status by mass spectrometry. By optical pulse-labeling in living primary neurons we documented a reduced half-life and accumulation of aSyn N-terminal mutants. To analyze the effect of N-terminal acetylation mutants in neuronal toxicity we took advantage of a neuronal model where aSyn toxicity was scored by longitudinal survival analysis. Salient features of aSyn neurotoxicity were previously investigated with this approach. aSyn-dependent neuronal death was recapitulated either by higher aSyn protein levels in the case of WT aSyn, or by the combined effect of protein levels and enhanced neurotoxicity conveyed by the E46K mutation. aSyn N-terminal mutations decreased E46K aSyn-dependent neuronal death both by reducing protein levels and, importantly, by reducing the intrinsic E46K aSyn toxicity, being the D2P mutant the least toxic. Together, our results illustrate that the N-terminus determines, most likely through its acetylation, aSyn protein levels and toxicity, identifying this modification as a potential therapeutic target.

Transcriptional profiling of circulating tumor cells in multiple myeloma: a new model to understand disease dissemination.

The reason why a few myeloma cells egress from the bone marrow (BM) into peripheral blood (PB) remains unknown. Here, we investigated molecular hallmarks of circulating tumor cells (CTCs) to identify the events leading to myeloma trafficking into the bloodstream. After using next-generation flow to isolate matched CTCs and BM tumor cells from 32 patients, we found high correlation in gene expression at single-cell and bulk levels (r ≥ 0.94, P = 10-16), with only 55 genes differentially expressed between CTCs and BM tumor cells. CTCs overexpressed genes involved in inflammation, hypoxia, or epithelial-mesenchymal transition, whereas genes related with proliferation were downregulated in CTCs. The cancer stem cell marker CD44 was overexpressed in CTCs, and its knockdown significantly reduced migration of MM cells towards SDF1-α and their adhesion to fibronectin. Approximately half (29/55) of genes differentially expressed in CTCs were prognostic in patients with newly-diagnosed myeloma (n = 553; CoMMpass). In a multivariate analysis including the R-ISS, overexpression of CENPF and LGALS1 was significantly associated with inferior survival. Altogether, these results help understanding the presence of CTCs in PB and suggest that hypoxic BM niches together with a pro-inflammatory microenvironment induce an arrest in proliferation, forcing tumor cells to circulate in PB and seek other BM niches to continue growing.

The Mechanism of Action of the Anti-CD38 Monoclonal Antibody Isatuximab in Multiple Myeloma.

PURPOSE: Knowledge about the mechanism of action (MoA) of monoclonal antibodies (mAb) is required to understand which patients with multiple myeloma (MM) benefit the most from a given mAb, alone or in combination therapy. Although there is considerable research about daratumumab, knowledge about other anti-CD38 mAbs remains scarce. EXPERIMENTAL DESIGN: We performed a comprehensive analysis of the MoA of isatuximab. RESULTS: Isatuximab induces internalization of CD38 but not its significant release from MM cell surface. In addition, we uncovered an association between levels of CD38 expression and different MoA: (i) Isatuximab was unable to induce direct apoptosis on MM cells with CD38 levels closer to those in patients with MM, (ii) isatuximab sensitized CD38hi MM cells to bortezomib plus dexamethasone in the presence of stroma, (iii) antibody-dependent cellular cytotoxicity (ADCC) was triggered by CD38lo and CD38hi tumor plasma cells (PC), (iv) antibody-dependent cellular phagocytosis (ADCP) was triggered only by CD38hi MM cells, whereas (v) complement-dependent cytotoxicity could be triggered in less than half of the patient samples (those with elevated levels of CD38). Furthermore, we showed that isatuximab depletes CD38hi B-lymphocyte precursors and natural killer (NK) lymphocytes ex vivo-the latter through activation followed by exhaustion and eventually phagocytosis. CONCLUSIONS: This study provides a framework to understand response determinants in patients treated with isatuximab based on the number of MoA triggered by CD38 levels of expression, and for the design of effective combinations aimed at capitalizing disrupted tumor-stroma cell protection, augmenting NK lymphocyte-mediated ADCC, or facilitating ADCP in CD38lo MM patients.See related commentary by Malavasi and Faini, p. 2946.

Flow cytometry for fast screening and automated risk assessment in systemic light-chain amyloidosis.

Early diagnosis and risk stratification are key to improve outcomes in light-chain (AL) amyloidosis. Here we used multidimensional-flow-cytometry (MFC) to characterize bone marrow (BM) plasma cells (PCs) from a series of 166 patients including newly-diagnosed AL amyloidosis (N = 94), MGUS (N = 20) and multiple myeloma (MM, N = 52) vs. healthy adults (N = 30). MFC detected clonality in virtually all AL amyloidosis (99%) patients. Furthermore, we developed an automated risk-stratification system based on BMPCs features, with independent prognostic impact on progression-free and overall survival of AL amyloidosis patients (hazard ratio: ≥ 2.9;P ≤ .03). Simultaneous assessment of the clonal PCs immunophenotypic protein expression profile and the BM cellular composition, mapped AL amyloidosis in the crossroad between MGUS and MM; however, lack of homogenously-positive CD56 expression, reduction of B-cell precursors and a predominantly-clonal PC compartment in the absence of an MM-like tumor PC expansion, emerged as hallmarks of AL amyloidosis (ROC-AUC = 0.74;P < .001), and might potentially be used as biomarkers for the identification of MGUS and MM patients, who are candidates for monitoring pre-symptomatic organ damage related to AL amyloidosis. Altogether, this study addressed the need for consensus on how to use flow cytometry in AL amyloidosis, and proposes a standardized MFC-based automated risk classification ready for implementation in clinical practice.

NatB-mediated protein N-α-terminal acetylation is a potential therapeutic target in hepatocellular carcinoma.

The identification of new targets for systemic therapy of hepatocellular carcinoma (HCC) is an urgent medical need. Recently, we showed that hNatB catalyzes the N-α-terminal acetylation of 15% of the human proteome and that this action is necessary for proper actin cytoskeleton structure and function. In tumors, cytoskeletal changes influence motility, invasion, survival, cell growth and tumor progression, making the cytoskeleton a very attractive antitumor target. Here, we show that hNatB subunits are upregulated in in over 59% HCC tumors compared to non-tumor tissue and that this upregulation is associated with microscopic vascular invasion. We found that hNatB silencing blocks proliferation and tumor formation in HCC cell lines in association with hampered DNA synthesis and impaired progression through the S and the G2/M phases. Growth inhibition is mediated by the degradation of two hNatB substrates, tropomyosin and CDK2, which occurs when these proteins lack N-α-terminal acetylation. In addition, hNatB inhibition disrupts the actin cytoskeleton, focal adhesions and tight/adherens junctions, abrogating two proliferative signaling pathways, Hippo/YAP and ERK1/2. Therefore, inhibition of NatB activity represents an interesting new approach to treating HCC by blocking cell proliferation and disrupting actin cytoskeleton function.

Phenotypic, transcriptomic, and genomic features of clonal plasma cells in light-chain amyloidosis.

Immunoglobulin light-chain amyloidosis (AL) and multiple myeloma (MM) are 2 distinct monoclonal gammopathies that involve the same cellular compartment: clonal plasma cells (PCs). Despite the fact that knowledge about MM PC biology has significantly increased in the last decade, the same does not apply for AL. Here, we used an integrative phenotypic, molecular, and genomic approach to study clonal PCs from 24 newly diagnosed patients with AL. Through principal-component-analysis, we demonstrated highly overlapping phenotypic profiles between AL and both monoclonal gammopathy of undetermined significance and MM PCs. However, in contrast to MM, highly purified fluorescence-activated cell-sorted clonal PCs from AL (n = 9) showed almost normal transcriptome, with only 38 deregulated genes vs normal PCs; these included a few tumor-suppressor (CDH1, RCAN) and proapoptotic (GLIPR1, FAS) genes. Notwithstanding, clonal PCs in AL (n = 11) were genomically unstable, with a median of 9 copy number alterations (CNAs) per case, many of such CNAs being similar to those found in MM. Whole-exome sequencing (WES) performed in 5 AL patients revealed a median of 15 nonrecurrent mutations per case. Altogether, our results show that in the absence of a unifying mutation by WES, clonal PCs in AL display phenotypic and CNA profiles similar to MM, but their transcriptome is remarkably similar to that of normal PCs.

Image analysis for the quantitative comparison of stress fibers and focal adhesions.

Actin stress fibers (SFs) detect and transmit forces to the extracellular matrix through focal adhesions (FAs), and molecules in this pathway determine cellular behavior. Here, we designed two different computational tools to quantify actin SFs and the distribution of actin cytoskeletal proteins within a normalized cellular morphology. Moreover, a systematic cell response comparison between the control cells and those with impaired actin cytoskeleton polymerization was performed to demonstrate the reliability of the tools. Indeed, a variety of proteins that were present within the string beginning at the focal adhesions (vinculin) up to the actin SFs contraction (non-muscle myosin II (NMMII)) were analyzed. Finally, the software used allows for the quantification of the SFs based on the relative positions of FAs. Therefore, it provides a better insight into the cell mechanics and broadens the knowledge of the nature of SFs.

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB.

Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

Characterization of the human Nalpha-terminal acetyltransferase B enzymatic complex.

BACKGROUND: Human Nalpha-acetyltransferase complex B (hNatB) is integrated by hNaa20p (hNAT5/hNAT3) and hNaa25p (hMDM20) proteins. Previous data have shown that this enzymatic complex is implicated in cell cycle progression and carcinogenesis. In yeast this enzyme acetylates peptides composed by methionine and aspartic acid or glutamic acid in their first two positions respectively and it has been shown the same specificity in human cells. METHODS: We have silenced hNAA20 expression in hepatic cell lines using recombinant adenoviruses that express specific siRNAs against this gene and analyzed cell cycle progression and apoptosis induction after this treatment. Immunopurified hNatB enzymatic complexes from human cell lines were used for analyzing hNatB in vitro enzymatic activity using as substrate peptides predicted to be acetylated by NatB. RESULTS: hNAA20 silencing in hepatic cell lines reduces cell proliferation in a p53 dependent and independent manner. At the same time this treatment sensitizes the cells to a proapototic stimulus. We have observed that the hNatB complex isolated from human cell lines can acetylate in vitro peptides that present an aspartic or glutamic acid in their second position as has been described in yeast. CONCLUSION: hNatB enzymatic complex is implicated in cell cycle progression but it exerts its effects through different mechanisms depending on the cellular characteristics. This is achievable because it can acetylate a great number of peptides composed by an aspartic or glutamic acid at their second residue and therefore it can regulate the activity of a great number of proteins.