Salmon‐Divon, Mali

Abstract Introduction Systemic light chain (AL) amyloidosis is a clonal plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chains in vital organs, often resulting in irreversible organ damage. While plasma cell-directed therapies have improved clinical outcomes, the molecular mechanisms driving disease pathogenesis remain poorly understood. Our previous studies identified overactivation of the PI3K/AKT signaling pathway in AL amyloidosis (Zvida et al., Blood, 2024; Fishov et al., Cancer Med., 2023), a central regulator of cellular and lipid metabolism. Dysregulated lipid metabolism has been implicated in several hematologic malignancies, yet its role in AL amyloidosis remains largely unexplored. Objectives To characterize the lipidomic landscape in AL amyloidosis compared to multiple myeloma (MM) and healthy controls (HC) in the context of inflammatory signaling. The ultimate goal of this work is to uncover novel metabolic-immunologic targets for therapeutic intervention. Methods Lipidomic profiling was performed by liquid chromatography-mass spectrometry (LC-MS) on plasma samples from AL patients (n=30), MM (n=30), and HCs (n=15). Fatty acid synthase (FASN) expression was validated by qRT-PCR in CD138+ bone marrow (BM) samples. A lipogenesis index was calculated using the ratio of palmitoleic acid to linoleic acid as a surrogate marker of FASN activity. Additionally, RNA-seq was conducted on CD138⁺ BM cells from AL and HC samples (n=8). Cytokine profiling was performed on serum from AL, MM, and HC subjects (n=10 each). Functional assays and lipidomic analysis were conducted in ALMC1 cells using the FASN inhibitor TVB-2640 and the MEK inhibitor cobimetinib. Results Lipidomic analysis of pre-treatment plasma samples revealed a significant increase in the lipogenesis index in AL patients, indicating elevated FASN activity. This was consistent with increased FASN mRNA expression in bone marrow CD138⁺ cells, suggesting both transcriptional upregulation and enzymatic activation. In ALMC1 cells, dose- and time-dependent FASN inhibition with TVB-2640 (0-2 μM, 24-72h) significantly reduced cell viability (IC50=1.3 μM), suggesting that FASN activity supports AL plasma cell survival. In addition, AL patients demonstrated a significant decrease in polyunsaturated fatty acids (PUFAs) levels, including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and linoleic acid (LA), compared to MM and HC. As PUFAs are known suppressors of FASN expression and activity, their depletion may contribute to sustained FASN upregulation and a metabolic shift toward de novo lipogenesis. Pathway enrichment analysis of patient lipidomic data revealed significant enrichment of omega-3/6 fatty acid metabolism, ATP generation, and inflammation-related pathways. Notably, imbalances in omega-3 and omega-6 fatty acids ratio have been associated with heightened inflammatory responses. In parallel, RNA-seq analysis identified several upregulated pathways in AL amyloidosis patients compared to controls, including immune regulation, MAPK signaling, mitochondrial dysfunction, oxidative phosphorylation, and IL-13 signaling. Both IL-13 and MAPK are known to transcriptionally induce FASN and regulate immune responses, providing a possible mechanistic link between inflammation and lipid remodeling. Cytokine profiling revealed elevated levels of IL-1β, IL-13, IL-8, IFN-γ, MIP-1α (CCL3), G-CSF, MCP-1 (CCL2), and IP-10 (CXCL10) in AL serum samples, consistent with a systemic inflammatory phenotype. Pharmacologic inhibition of MAPK signaling (by cobimetinib) in ALMC1 cells led to restoration of PUFA levels and a concurrent reduction in cytokine secretion, suggesting that MAPK activity sustains both the inflammatory and lipogenic phenotype in AL. ConclusionsOur findings identify metabolic-inflammatory axis in AL amyloidosis, in which IL-13 and MAPK signaling may promote fatty acid reprogramming through FASN activation and PUFA suppression. This axis sustains a pro-inflammatory environment that may contribute to disease pathogenesis. Thus, FASN and MAPK may represent promising metabolic-immunologic targets for therapeutic intervention in AL amyloidosis.

Background: Systemic light chain (AL) amyloidosis is a clonal plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chain products in vital organs, leading to significant organ dysfunction. The molecular mechanisms underlying its pathogenesis remain incompletely elucidated. Identifying the molecular mechanisms and novel therapeutic targets is critical for improving treatment strategies and patient outcomes. Our previous research work identified that the PI3K/AKT signaling pathway is overexpressed in AL amyloidosis (Zvida et al., Blood, 2022, Fishov et al., Cancer Med., 2023). This pathway plays a critical role in regulating cellular metabolism. Specifically activated AKT can phosphorylate and regulate various target proteins involved in metabolic processes. One of the significant targets is fatty acid synthase (FASN), an enzyme responsible for the synthesis of fatty acids. AKT can enhance FASN expression and activity, thereby promoting lipid biosynthesis. Additionally, elevated FASN levels can drive inflammation through increased fatty acid synthesis, which activates inflammatory signaling pathways and enhances cytokine production (Acosta-Martinez et al.,IJMC, 2022). Elevated levels of FASN have been associated with hematological malignancies and cell survival (Vanauberg, et al., Oncogenesis, 2023). However, its role in AL amyloidosis is yet to be elucidated. Aim: To determine the role of FASN in the pathology and inflammatory response in AL amyloidosis, and to evaluate the potential of targeting FASN and the PI3K/AKT pathway for therapeutic intervention. Methods: We profiled FASN expression in CD138+ bone marrow (BM) samples from patients with AL amyloidosis (n=20), Multiple Myeloma (MM) (n=20), Monoclonal Gammopathy of Undetermined (MGUS) (n=8) and Healthy Controls (HC) (n=6), using qRT-PCR. To further explore the relationship between the PI3K/AKT pathway and FASN, we treated ALMC-1 cells with Capivasertib (an AKT inhibitor) and assessed FASN protein levels, cell proliferation by WST-1 assay, cell cycle distribution by flow cytometry, light chain secretion by ELISA, and expression of AKT and its downstream targets via western blot analysis. Furthermore, TVB-2640 (FASN inhibitor) was used to evaluate the cell response to FASN inhibition. Results: Our analysis revealed a significant upregulation of FASN in AL amyloidosis BM samples compared to MM, MGUS, and HCs. Capivasertib treatment resulted in a dose-dependent reduction in FASN protein levels. Additionally, Capivasertib treatment inhibited cell proliferation, induced G1 phase cell cycle arrest, reduced light chain secretion, and decreased AKT protein expression and its downstream targets p4EBP pS6 and pERK. Elevated levels of proinflammatory cytokines, including TNF and IP-10 (CXCL-10), were observed in AL amyloidosis serum samples, suggesting the involvement of immune mechanisms and the PI3K/AKT pathway, potentially induced by FASN. Finally, treatment with TVB-2640 reduced cell viability and decreased the protein expression of LIPIN-1, which is one of the key target genes regulated by FASN. Conclusions: Our findings highlight the significant upregulation of FASN in AL amyloidosis and its regulation by the PI3K/AKT pathway. The observed effects of Capivasertib on FASN expression and cell proliferation, along with the upregulation of inflammatory cytokines, underscore the potential for targeting the PI3K/AKT signaling pathway and FASN in developing more specific therapeutic strategies for AL amyloidosis.

Abstract Background Noncoding RNAs such as microRNAs (miRNAs) have attracted attention as biological pathway regulators, which differ from chromosomal translocations and gene point mutations. Their involvement in the molecular mechanisms underlying light chain (AL) amyloidosis pathogenesis is yet to be elucidated. Aims To decipher specific miRNA expression profile in AL‐amyloidosis and to examine how miRNAs are involved in AL pathogenesis. Methods The expression profile of miRNAs and mRNA from bone marrow (BM)‐derived CD138+ cells were determined using the NanoString nCounter assay and RNA‐Seq, respectively. The effect of aberrantly expressed miRNAs on potential molecular targets was analyzed by qRT‐PCR, Western blot, Mito‐potential assay, and Annexin‐PI staining. Results Genes which were significantly differentially expressed between AL‐amyloidosis and MM, were found to be involved in cell growth and apoptotic mechanisms. Specifically, BCL2L1, MCL1, and BCL2 were upregulated in AL‐amyloidosis compared with MM and controls. The levels of miR‐181a‐5p and miR‐9‐5p, which regulate the above‐mentioned genes, were lower in BM samples from AL‐amyloidosis compared with controls, providing a mechanism for BCL2 family gene upregulation. When miR‐9‐5p and miR‐181a‐5p were overexpressed in ALMC1 cells, BCL2L1, MCL1, and BCL2 were downregulated and induced apoptosis. Treatment of ALMC‐1 cells with venetoclax, (BCL‐2 inhibitor), resulted in the upregulation of those miRNAs, the downregulation of BCL2, MCL1, and BCL2L1 mRNA and protein levels, and subsequent apoptosis. Conclusion Our findings suggest that miR‐9‐5p and miR‐181a‐5p act as tumor‐suppressors whose downregulation induces anti‐apoptotic mechanisms underlying the pathogenesis of AL‐amyloidosis. The study highlights the post‐transcriptional regulation in AL‐amyloidosis and provides pathogenetic evidence for the potential use of BCL‐2 inhibitors in this disease.

Abstract Background: Systemic light chain (AL) amyloidosis is a clonal plasma cell disorder characterized by deposition of misfolded immunoglobulin light chain products in vital organs, causing their dysfunction. All therapies used to treat AL patients are adapted from multiple myeloma (MM) and customized to the typically frail AL population. Hence, it is important to identify novel therapeutic targets for these patients. MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression and have a role in cancer development and progression. MiRNAs can be purified from serum, plasma and bone marrow (BM) and may be used as biomarkers to distinguish between patients and healthy individuals. Moreover, miRNA-mRNA interactions may determine the molecular mechanism involved in AL pathogenesis and may suggest novel therapeutic approaches. To date, knowledge about circulating or BM miRNAs involved in AL amyloidosis is lacking. Aims: To decipher specific miRNA expression profiles in AL amyloidosis compared to MM patients and healthy controls (HC) and to examine how miRNAs are involved in AL pathogenesis. Methods: miRNA expression profile was determined using the nCounter assay (NanoString technologies). RNA-Seq data was downloaded from GEO database (GSE175384), and was used to detect potential miRNA-mRNA targets, and enriched biological pathways by the bioinformatics tool Ingunity Pathway Analysis (IPA). MiRNA and gene expression profiles were validated by qRT-PCR in 60 AL, 60 MM and 10 HC samples. The effect of aberrantly expressed miRNAs on potential molecular targets was analyzed in ALMC1 cells by transfecting the cells with miRNA mimic, following qRT-PCR, Western blot analysis and Annexin-PI staining. Results: BM and plasma miRNAs were differentially expressed in AL amyloidosis compared to MM or HC. MiRs-9a-5p, 181a-5p, 199a-3p, 130a-3p, 145-5p and 301a-3p were differentially expressed between AL and MM samples and may be used as biomarkers for distinguish AL amyloidosis from MM. Moreover, we found that the differentially expressed miRNAs and mRNA in AL patients regulates key signaling pathways related to cell cycle and anti-apoptosis mechanisms including cytokine signaling, oxidative phosphorylation (OXPHOS), NFkB signaling, activation of MAPK and PI3K/AKT pathways, which are all linked to cancer cell growth, proliferation and therapeutic resistance, and therefore may be used as a therapeutic targets. Specifically, our analysis showed that genes related to mitochondrial activity were upregulated in AL patients (Figure 1), particularly the anti-apoptotic BCL2 family genes (BCL2, MCL1, and BCL2L1). MCL1 and BCL2L1 are predicted targets of miR-181a-5p that was downregulated in BM and plasma samples of AL patients compared to MM patients, indicating a possible interaction between these molecules. MiR-9-5p, which was also found to be downregulated in AL BM and plasma samples compared to MM patients, is predicted to have an indirect effect on the BCL2 family members through CREB molecule (Figure 1). The biological significance of miR-9-5p and miR-181a-5p was evaluated, by transfecting ALMC1 cell line with miRNA mimics. Overexpression of these miRNAs led to downregulation of the BCL2 family anti-apoptotic genes and induced apoptosis by Annexin V staining. These findings might explain the biological mechanism by which AL patients respond to the BCL2 inhibitor, venetoclax, as recently reported (Sidiqi et al, BCJ, 2020). Conclusions: We provide insight into the molecular mechanisms mediated by miRNAs and the aberrant expression of oncogenic/tumor suppressor genes. The differential expression of miRNAs in AL amyloidosis may be used to understand disease pathogenesis and predict risk of progression to AL amyloidosis among patients with known plasma cell disorders. Additionally, signaling pathways involved in AL amyloidosis, mediated by miRNAs, may assist in tailoring more specific treatments. Figure 1 Figure 1. Disclosures Dispenzieri: Oncopeptides: Consultancy; Sorrento Therapeutics: Consultancy; Pfizer: Research Funding; Alnylam: Research Funding; Takeda: Research Funding; Janssen: Consultancy, Research Funding.