Introduction
Multiple myeloma (MM) is a hematologic malignancy defined by the clonal expansion of plasma cells within the bone marrow (BM) [ 1 ]. As the disease progresses, malignant plasma cells develop pronounced metabolic plasticity and resistance to apoptosis, both of which are tightly linked to mitochondrial function [ 2 ].
The complex and heterogeneous genomic landscape of multiple myeloma (MM), characterized by prognostically significant cytogenetic abnormalities, gene mutations, and dysregulated transcriptional networks, drives the emergence of diverse and dynamic metabolic alterations throughout the disease course [ 3 – 5 ]. Metabolically, MM cells display a wide range of alterations, including elevated glucose uptake, augmented aerobic glycolysis, and increased lactate production; in parallel, they exhibit a marked dependency on tricarboxylic acid cycle activity and oxidative phosphorylation (OXPHOS) to support the increased demands of protein synthesis, folding, and secretion typical of malignant plasma cells [ 6 , 7 ].
Over the past decade, accumulating evidence has highlighted the importance of mitochondrial morphology regulation, governed by the dynamic equilibrium between fusion and fission events, as a key determinant of cellular homeostasis and survival [ 2 ]. Disruption of this finely tuned system, collectively termed mitochondrial dynamics, promotes mitochondrial dysfunction, thereby facilitating oncogenic transformation and progression of a wide variety of tumors [ 8 – 12 ].
In MM, mitochondrial dynamics not only serves as a central regulatory node in metabolic reprogramming, but also represents a potential therapeutic vulnerability amenable to pharmacologic intervention [ 13 , 14 ]. Alterations in mitochondrial dynamics have been increasingly documented in hematological malignancies, including MM, and associated with enhanced tunneling nanotube-mediated trafficking of plasma cell-derived post-fission mitochondria within the BM microenvironment [ 15 ], promoting cell survival and drug resistance. In the context of mitochondrial dynamics, MARCH5 (Membrane-Associated RING-CH-type Finger 5), a mitochondrial outer membrane E3 ubiquitin ligase, has emerged as a critical regulator of mitochondrial morphology and quality control [ 16 ]. MARCH5 has been shown to govern the turnover of key mitochondrial fusion and fission proteins, including Drp1, Mfn1, and Fis1, via ubiquitination, thereby affecting mitochondrial structure, bioenergetics, and stress responses. In addition, MARCH5 has been implicated in the regulation of apoptosis, autophagy, and endoplasmic reticulum (ER) stress responses, pathways frequently hijacked by cancer cells to promote survival and therapeutic resistance [ 17 ].
In hematologic malignancies such as leukemia, recent findings suggest a critical role for MARCH5 in maintaining the survival and metabolic flexibility of cancer cells. Acute myeloid leukemia (AML) [ 18 ] and chronic lymphocytic leukemia (CLL) [ 19 ], in particular, are characterized by a heavy reliance on mitochondrial OXPHOS and a tightly regulated mitochondrial network, which is finely tuned by MARCH5. Despite this emerging evidence in leukemias, the role of MARCH5 in the context of mitochondrial dynamics and metabolic flexibility remains largely unexplored in plasma cell disorders. Given the reliance of MM cells on mitochondrial functions for survival, proliferation, and drug resistance [ 3 ], understanding the impact of MARCH5 to mitochondrial dynamics could reveal novel mechanisms of disease progression and therapeutic vulnerability.
This study aims to elucidate the role of MARCH5 in modulating mitochondrial dynamics in MM, with a focus on its regulatory function in maintaining mitochondrial integrity, orchestrating apoptotic signaling pathways, and influencing cellular responsiveness to therapeutic interventions.
Methods
MM cell lines
The MM cell lines used in this study were: AMO, AMO-BZB, NCI-H929, NCI-H929-BZB, NCI-H929-CFZ, KMS26, OPM2, RPMI-8226, JJN3. AMO and AMO-BZB cell lines were provided by Dr. C. Driessen (University of Tubingen, Germany). NCI-H929 cell line was purchased from DSMZ (Braunschweig, Germany). Proteasome inhibitor resistant NCI-H929 cells were selected by incubation with increasing concentrations of Bortezomib (BZB) and Carfilzomib (CFZ). All the other cell lines were a gift of Dr. Eugenio Morelli from Dana Farber Cancer Institute (Harvard, Boston, MA). Human MM cell lines were cultured in RPMI-1640 medium (Corning) supplemented with 10% of heat-inactivated fetal bovine serum (FBS) (Gibco®, Life Technologies, Carlsbad, CA), 100 U/mL of Penicillin and 100 µg/mL of Streptomycin (P/S) (Gibco®, Life Technologies, Carlsbad, CA) and incubated at 37 °C in a 5% CO 2 atmosphere. HS-5 human stromal cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in Dulbecco’s modified eagle medium (Corning, New York, NY, USA) supplemented with 10% FBS (Gibco®, Life Technologies, Carlsbad, CA), 100 U/mL of Penicillin and 100 µg/mL of Streptomycin (P/S) (Gibco®, Life Technologies, Carlsbad, CA) and incubated at 37 °C in a 5% CO 2 atmosphere.
Drugs and oligonucleotides
Leflunomide (HY-B0083), Melphalan (#S8266) and Uridine (#2029) powders, Bortezomib (#S1013) and Carfilzomib (#2853) DMSO solutions were purchased from Selleck Chemicals LLC (Munich, Germany). Venetoclax (HY-15531) was purchased from MedChemExpress (MCE). Oroboros reagents, including Oligomycin (#O4876), FCCP (#C2920), and Antimycin A (#A8674), were purchased from Sigma Aldrich (USA). Silencer® Select siRNA for MARCH5 (siMARCH5, cat #4392420, ID: s29332, ID: s29333, ID: s29334), or control siRNA (siNC, cat #AM4611) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Transfection and transduction of MM cells
To obtain MM cells stably overexpressing MARCH5 cDNA, HEK-293Ta cells were co-transfected with 10 µg of the lentiviral Lenti-hCMV-ORF-TAG-IRES-puro for Homo sapiens MARCH5 (or the empty vector, both from Transomics), along with 4 µg of p-CMV-VSV G, and 10 µg of Delta8.9 plasmids. After 24 h, supernatants containing lentiviral particles were collected and sterile 0.45 µm filtered. AMO and H929 cells were spinoculated for 1 h with media containing lentiviral particles in the presence of 8 µg/mL polybrene (Sigma Aldrich, St. Louis, MO). After 48 h, stable clones were selected using puromycin at a concentration of 0.5 μg/ml. Human MFN2 cDNA cloned into pLVX-TetOne-Puro vector was kindly provided by Prof. C.M. Taniguchi (University of Texas MD Anderson Cancer Center, Houston, Texas, USA).
Transmission electron microscopy (TEM)
Cell pellets were fixed in 3% glutaraldehyde (Merck Sigma-Aldrich) solution in 0.1 M phosphate buffer (pH 7.4; ClinicalSciences) for 2 h at 4 °C. After washing in 1 × PBS at 4 °C, samples were fixed in 1% osmium tetroxide in phosphate buffer (0.1 M, pH 7.4) to preserve the lipid structures, and then dehydrated through acetone-graded series and progressively embedded in acetone/resin with a final embedment in epoxy resin (Epon). Afterward, ultrathin sections were cut using an RMC PowerTome series ultramicrotome with a Diatome diamond knife, mounted on copper grids (G300 Cu) and observed with a Jeol JEM-1400 Plus transmission electron microscope operating at 80 kV. Mitochondrial size measurements were obtained using Image J software.
Cell viability and proliferation assay
Cell viability was assessed using the CellTiter-Glo (CTG) assay kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Briefly, MM cells were seeded in 24-well plates following electroporation, and the effects of siRNAs on cell viability were evaluated after 48 h. MM cell lines were treated with Venetoclax, Leflunomide, Bortezomib, Carfizomib or Melphalan, and collected at various time points. Luminescence was measured using the GloMax-Multi Detection System (Promega, Madison, WI, USA).
Cell proliferation of siMARCH5-transfected or doxycycline-inducible MFN2-overexpressing cells was evaluated using the trypan blue exclusion assay. siMARCH5-transfected MM cells were plated in 24-well plates, and viable cells were counted after 0, 48 and 72 h using an automated cell counter. Similarly, doxycycline-inducible MFN2-overexpressing MM cells were plated in 6-well plates, and viable cells were determined on 0, 6 and 9 days.
Drug combination studies and their synergy quantification followed the Chou-Talalay method [ 20 ], a mathematical method commonly used to quantify drug interactions. Combination indexes (CI) were calculated by CalcuSyn software (BIOSOFT, Cambridge, UK). CI < 1 synergic; CI = 1 additive effect; CI > 1 antagonism.
For co-culture experiments, AMO cells were seeded onto HS5 stromal cells at a 1:2 ratio in 24-well plates and maintained in indirect contact using Transwell® permeable supports (Corning, USA). After 48 h of co-culture, cell viability was assessed using the CTG, and luminescence was quantified using the GloMax® plate reader (Promega, USA).
For the colony-forming assay, doxycycline-inducible MFN2-expressing MM cells were plated in triplicate in 24-well plates, in 1 mL of a semi-solid medium composed of 1.1% methylcellulose (MethoCult, STEMCELL Technologies) in RPMI-1640 supplemented with 10% fetal bovine serum (FBS). After 10 days of incubation at 37 °C in a humidified atmosphere, colonies were fixed with methanol, stained with 0.04% crystal violet, and subsequently counted under a light microscope (Leica DM IL LED).
Apoptosis assay
Apoptotic cell death was assessed using a flow cytometry-based assay (BD Biosciences) by Annexin V conjugated to phycoerythrin (PE) and 7-Aminoactinomycin D (7-AAD), as described [ 21 ]. Briefly, MM cells were stained in 5 mL polystyrene tubes according to the manufacturer’s instructions provided with the PE/Annexin V Apoptosis Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA). After treatments, MM cells were collected, washed with a binding buffer, and exposed to Annexin V-PE and 7-AAD probes for 15 min at room temperature in the dark. Data acquisition was performed using a BD FACS Fortessa X-20 flow cytometer (BD Biosciences, San Jose, CA, USA), and data analysis was carried out with FlowJo software (version 10).
Immunofluorescence
AMO-BZB cells were fixed and permeabilized using BD Cytofix™ Fixation Buffer (#554722) on poly-L-lysine-coated coverslips for 20 min at room temperature in the dark. Following fixation, cells were blocked for 1 h with 3% BSA in PBT (0.05% Triton X-100 in PBS). Coverslips were then incubated overnight at 4 °C with primary antibodies against Mfn2 (1:100; MA5-27647, Thermo Fisher Scientific, Waltham, MA, USA) and MARCH5 (1:100, #PA5-25584, Cell Signaling Technology, Denvers, MA, USA). The following day, cells were washed in PBS and incubated for 1 h with fluorescent secondary antibodies: Alexa Fluor 488-conjugated goat anti-mouse (Invitrogen, 1:1000) and Alexa Fluor-conjugated goat anti-rabbit (Invitrogen, 1:1000). After washing, coverslips were mounted onto glass slides using a DAPI-containing mounting medium (Santa Cruz Biotechnology, Dallas, TX, USA) for nuclear staining. Images were acquired using a Leica DM4 B fluorescence microscope.
Antibody-dependent cellular cytotoxicity assays
MM cells were stained with CellTrace Far Red (# C34564 ) and co-cultured overnight at 37 °C with Peripheral Blood Mononuclear Cells (PBMCs) at an effector-to-target (E:T) ratio of 30:1. The co-culture was carried out in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL Penicillin, and 100 µg/mL Streptomycin (P/S), in the presence of Daratumumab, Isatuximab, or a vehicle control (DMSO). Residual CD8⁺ T lymphocytes (CD3⁺) were analyzed by flow cytometry using a BD FACS Fortessa. Surface antigen detection was performed using an anti-human CD3 antibody (#555340; BD Biosciences).
Western blotting (WB) and immunoprecipitation (IP) assays
Total protein extracts were prepared using NP-40 Cell Lysis Buffer supplemented with Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA), as reported [ 13 ].
Protein samples (30 µg per lane) were resolved on 10% SDS-PAGE gels and subsequently transferred onto nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were then blocked and incubated with the following primary antibodies: MARCH5 (#PA5-25584) (Thermo Fisher Scientific, Waltham, MA, USA); MFN2 (# 9482S), FLAG(#F3165), MFF(#84580S), DRP1 (#14647S), UBIQUITIN (#3933S), PARP (#9532), CASPASE 3 (#9665P), CASPASE 8 (#9746P), BAD (#9239), BIK (#4592), BAK(#12105), BID(#2002), BIM (#2933), NOXA (#14766), NDUFS1 (#70264), NDUFAB1 (#71814), UQCRFS1 (#95231), COX1 (#55159), COX IV (#4850) were from Cell Signaling Technology (Denvers, MA, USA); PUMA (SC-374223) was from Santa Cruz Biotechnology (Dallas, TX, USA). γ-Tubulin (T6557; Sigma-Aldrich, St. Louis, MO, USA), GAPDH (#5174; Cell Signaling Technology, Denvers, MA, USA) and α-TUBULIN (#2125S; Cell Signaling Technology, Denvers, MA, USA) were used as loading controls. Protein bands were visualized using the Clarity Western ECL Substrate (Bio-Rad Laboratories, USA), and chemiluminescence signals were quantified using the UVITEC Imaging System (Cleaver Scientific, UK). For immunoprecipitation experiments, 1.5 mg of total protein lysate was incubated with anti-FLAG M2 Affinity Gel (A2220; Sigma-Aldrich, St. Louis, USA). Prior to incubation, beads were washed three times with an NP40 buffer supplemented with Halt™ Protease Inhibitor Cocktail. The antibody-bead-cell lysate mixture was incubated overnight at 4 °C under gentle agitation. Immune complexes were washed three times with NP40 buffer supplemented with Halt™ Protease Inhibitor Cocktail, eluted in denaturing buffer, and subsequently analyzed by Western blotting as described above.
Reverse transcription and quantitative real-time amplification (qRT-PCR)
RNA extraction, reverse transcription (RT), and quantitative real-time PCR (qRT-PCR) were performed as previously described [ 14 ]. Total RNA was isolated from multiple myeloma (MM) cells using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol. RNA concentration and purity were assessed using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific). To assess the expression of NDUFA2, NDUFA8, NDUFB8, NDUFS2, SDHA, UQCR11 and COX5A, SYBR Green chemistry (Applied Biosystems) was employed, following standard protocols. Reactions were run in triplicate on a QuantStudio 12 K Flex Real-Time PCR System (Thermo Fisher Scientific), and included no-template controls. Relative gene expression was calculated using the comparative Ct (ΔΔCt) method, with normalization to human β-actin.
The primer sequences (purchased from Eurofins) were as follows:
NDUFA2 fwd (5’-CTACGTGGAGCTGAAGAAGGCGA-3’)
NDUFA2 rev (5’-AGAACGTTCTCCAGGGCTCTGGTTA-3’)
NDUFA8 fwd (5’-CAAACTGGTCAACAAGTGTGCTTTGG-3’)
NDUFA8 rev (5’-CACACTCGTCAAACTTTGCCTGC-3’)
NDUFB8 fwd (5’-CCACACCTGTTTCTTGGCATGTCA-3’)
NDUFB8 rev (5’-TCACCGCCTCGTTCCAGGTAC-3’)
NDUFS2 fwd (5’-TACCAAGTTCCTCCAGGAGCCA-3’)
NDUFS2 rev (5’-GGCAAAACCAGGAGCCTTGATCT-3’)
SDHA fwd (5’-GAGATGTGGTGTCTCGGTCCAT-3’)
SDHA rev (5’-GCTGTCTCTGAAATGCCAGGCA-3’)
UQCR11 fwd (5’-TGGTGTGGGCCACCGATTGG-3’)
UQCR11 rev (5’-CAGAGCAGTCTGTGAAGGGTTTGTG-3’)
COX5A fwd (5’-CAGATGAGGAGTTTGATGCTCGC-3’)
COX5A rev (5’-GCAGCATCAATGATTTTGGGCTCTG-3’)
β-Actin fwd (5ʹ-CAAGGCCAACCGCGAGAAGATGAC-3ʹ)
β-Actin rev (5ʹ-GCCAGAGGCGTACAGGGATAGCACA-3ʹ)
To assess the expression of MARCH5, a TaqMan mRNA assay (ID: Hs01546967; Applied Biosystems) was employed, following standard protocols.
RNA-seq, library construction, sequencing and analysis
Total RNA from AMO cells transfected with siMARCH5 was isolated with RNeasy Micro Kit according to the manufacturer’s instructions (QIAGEN, Hilden, Germany). Concentration and quality of RNA were determined using the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and 4200 TapeStation System (Agilent Technologies, Santa Clara, CA, USA). Libraries were generated from 1 µg of RNA derived from AMO cultures with the TruSeq mRNA V2 sample preparation kit with Ribo-Zero Gold (Illumina, San Diego, CA, USA). The quality and yield of the prepared libraries were assessed using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and 4200 TapeStation System (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed on a NextSeq™ 550 (Illumina, San Diego, CA, USA) with 2 × 76 cycles, following the manufacturer’s instructions. FastQC (v0.11.9, Babraham Institute) was used to conduct quality control checks of the sequencing raw data while trimmomatic (v0.32) to perform both low-quality read removal and adapter-trimming [ 22 ] STAR (v2.7.0) [ 23 ] was used to map the remaining reads to human reference genome hg19, and transcript abundances were measured with RSEM (v1.3.3). Gene expression levels were normalized by transcripts per kilobase million (TPM) and fold change of differentially expressed genes (DEGs) was set ≥ 1.5 or ≤ 0.6666. For functional gene annotation, the obtained sequences were annotated by searching Gene Ontology (GO) databases. These deregulated genes were subjected to GO significant enrichment analyses to identify the biological functions in which these genes are involved. In figures, we only reported the significant top 10 ranked terms.
Immunohistochemistry
Tumors excised from treated mice were fixed in 4% buffered formaldehyde and, after 24 h, washed, dehydrated, and embedded in paraffin. Hematoxylin–eosin (H&E) staining was performed on 4 μm sections mounted on polylysine-coated slides. Tissue sections were examined using an OLYMPUS BX51 optical microscope (Olympus Corporation, Tokyo, Japan). For immunohistochemistry (IHC), 2 μm tumor sections were deparaffinized and subjected to antigen retrieval using Epitope Retrieval Solution 2 (EDTA buffer, pH 8.8) at 98 °C for 20 min. After washing, endogenous peroxidase activity was blocked for 10 min using Bond Polymer. All IHC procedures were conducted with the Benchmark XT Automated Immunohistochemistry System (Ventana Medical Systems, Oro Valley, AZ, USA). Sections were then washed and incubated with a primary antibody against Ki67 (Dako, clone MIB-1; dilution 1:150). This was followed by a 10-min incubation with the polymer and subsequent development using DAB-Chromogen for 10 min. All experiments were independently repeated at least three times.
Assessment of mitochondrial ROS and membrane potential
Mitochondrial reactive oxygen species (ROS) production was assessed using the MitoSOX™ Red mitochondrial superoxide indicator (Thermo Fisher Scientific), while mitochondrial membrane potential was evaluated using the TMRM probe (Thermo Fisher Scientific), following the manufacturer's protocols. After treatment with Venetoclax, OE MARCH5 cells (or the EV) were stained with 5 μM MitoSOX Red or 100 nM TMRM and incubated at 37 °C for 30 min in the dark. Stained cells were analyzed using a FACS Fortessa X-20 flow cytometer (BD Biosciences). All experiments were conducted in triplicate, and for each sample, a minimum of 1 × 10 4 events were acquired. Data analysis was performed using FlowJo software, version 10.
Oroboros O2k-high resolution respirometer
The oxygen consumption was analyzed in 2 mL chambers at 37 °C using the Oroboros Oxygraph-2 k high resolution respirometer (Oroboros Instrument, Innsbruck, Austria), following the procedures of the SUIT protocol D009. After instrumental calibration, MM cells were collected and loaded in the O2k-closed chambers at a density of 1 × 10 6 per mL, for a total of 2 mL of complete medium per chamber, by maintaining cell suspension continuously mixed. First, the basal respiration was recorded as routine respiration (before adding reagents). Then, variations of respirations were measured after sequential addition of oligomycin, FCCP, and antimycin A, evaluating the LEAK state, the maximal respiration and the residual oxygen consumption, respectively. Reagents were added into the closed chamber by Hamilton syringes (Oroboros Instruments, Innsbruck, Austria). Data acquisition and analysis were performed using DatLab software (Oroboros Instrument, Innsbruck, Austria). Measurements were normalized for the cell count and protein quantification. O 2 flow is expressed per cell [amol s −1 x −1 ] equivalent to [pmol·s −1 ·(10 6 cells) −1 ].
In vivo studies
Male NOD.SCID mice (6–8 weeks old; Envigo, Indianapolis) were housed and monitored in the Animal Research Facility at the University Magna Graecia of Catanzaro. All experimental procedures were conducted in compliance with Italian Ministry of Health authorization no. 887/2021-PR and approved by both the National Directorate of Veterinary Services and the Institutional Ethical Committee of the University Magna Graecia of Catanzaro (Italy). In the first experimental animal model, mice were subcutaneously injected with 5.0 × 10 6 AMO-BZB cells carrying an inducible MFN2 gene. When tumors became palpable (100–200 mm 3 , approximately three weeks post-injection; n = 5 mice per group), Doxycycline (Sigma, 200 μg/mL) was added to the drinking water every other day for mice in the treatment group. In the second model, 5.0 × 10 6 AMO cells were subcutaneously implanted in NOD.SCID mice. When palpable tumors developed (100–200 mm 3 ), mice were divided into two groups (n = 5 per group) and treated intraperitoneally with either leflunomide (20 mg/kg) or DMSO as a vehicle, twice a week for two weeks. Tumor volume was measured using an electronic caliper and calculated with the formula: V = (a 2 × b)/2, where a is the width and b is the length of the tumor. During measurements, the investigator was blinded to group allocation. In accordance with institutional guidelines, mice were euthanized when tumors reached a volume of 2000 mm 3 , or earlier in cases of paralysis or a substantial decline in quality of life, to prevent unnecessary suffering.
Statistical analysis
All in vitro experiments were independently performed at least three times. Data are presented as mean ± standard deviation (SD). Statistical comparisons between two groups were conducted using the unpaired Student’s t -test, while differences among multiple groups were assessed using appropriate statistical tests implemented in GraphPad Prism software (version 8.0; GraphPad Software, La Jolla, CA, USA). A p -value < 0.05 was considered statistically significant. Graphs and data visualizations were generated using GraphPad Prism.
Results
MARCH5 reduces mitochondrial fusion via MFN2 ubiquitylation
Previous studies have reported overexpression of MARCH5 in cancers [ 8 ], including MM [ 24 ], although the functional role of this E3 ubiquitin ligase remains only partially understood. In our study, we further assessed MARCH5 protein expression in MM cell lines compared to healthy PBMCs, and confirmed elevated expression levels in the cancer cell lines (Supplementary Fig. 1A). Notably, MARCH5 expression was further upregulated in proteasome inhibitor (PI)-resistant isogenic cell lines (AMO-BZB, H929-BZB, and H929-CFZ) compared to their drug-sensitive counterparts (AMO, H929) (Fig. 1 A; (Supplementary Fig. 1B). Given the reported role of MARCH5 as a regulator of mitochondrial dynamics across various tumor types [ 16 ], we investigated whether modulation of MARCH5 levels may influence mitochondrial morphology. First, we screened different siRNAs for their ability to knock-down (KD) MARCH5 expression (Supplementary Fig. 1C). TEM analysis of AMO-BZB cells transfected with two independent siRNAs targeting MARCH5 demonstrated a pronounced elongation of mitochondrial structures, indicative of increased mitochondrial fusion (Fig. 1 B). Consistent with a shift toward mitochondrial fusion, WB analysis showed that MARCH5 KD decreased the expression of key mitochondrial fission mediators, including DRP1 and MFF, alongside an upregulation of the fusion protein MFN2 (Fig. 1 C; Supplementary Fig. 1D). Conversely, ectopic overexpression (OE) of MARCH5 via a lentiviral vector resulted in increased DRP1 and MFF expression and a concomitant reduction in MFN2 levels (Fig. 1 D; Supplementary Fig. 1E).
To explore the potential interaction between MARCH5 and MFN2, we performed co-immunoprecipitation experiments, demonstrating that exogenously expressed MARCH5 physically associates with MFN2 (Fig. 2 A). Supporting this interaction, immunofluorescence analyses showed co-localization of endogenous MARCH5 and MFN2 in discrete subcellular domains of AMO-BZB cells (Fig. 2 B).
Given these findings, we hypothesized that MARCH5 may negatively regulate MFN2 protein stability. Cycloheximide (CHX) chase assays revealed that MARCH5 overexpression significantly reduced MFN2 protein half-life (Fig. 2 C). Furthermore, in vivo ubiquitination assays in AMO cells indicated that MARCH5 OE increased MFN2 ubiquitination (Fig. 2 D), whereas MARCH5 silencing produced the opposite effect (Supplementary Fig. 2A). Collectively, these data support a model in which MARCH5 negatively regulates mitochondrial fusion by promoting the ubiquitination and proteasomal degradation of MFN2, thereby contributing to altered mitochondrial dynamics.
MARCH5 activates ETC and OXPHOS metabolism in MM cells
To investigate the molecular alterations associated with MARCH5 activity, we performed RNA sequencing on MARCH5 KD cells. Gene Ontology (GO) pathway enrichment analyses identified OXPHOS and related mitochondrial pathways as the most significantly downregulated upon MARCH5 KD (Fig. 3 A). Consistent with these findings, quantitative RT-PCR analysis revealed a marked reduction in the expression of several electron transport chain (ETC) components, including NDUFA2, NDUFS2, NDUFA8, SDHA, UQCR11, and COX5A, both in AMO and AMO-BZB cells (Fig. 3 B, C). Western blot analysis further confirmed decreased protein levels of NDUFS1, COX1, UQCRFS1, NDUFAB1 and COX4 in MARCH5-depleted cells (Fig. 3 D). To functionally validate mitochondrial impairment, we conducted high-resolution respirometry (Oroboros) in MM cells following MARCH5 KD. Consistent with ETC inhibition, these analyses revealed a significant reduction in basal oxygen consumption rate (OCR), spare respiratory capacity, maximal respiration, and ATP production (Fig. 3 E). These mitochondrial dysfunctions were accompanied by a decrease in viable cell number upon MARCH5 silencing by siRNAs (Fig. 3 F).
To explore the mechanisms of cell death associated with MARCH5 inhibition, we performed flow cytometric assays, that demonstrated an increase in Annexin V/7-AAD-positive cells, indicative of apoptosis (Fig. 3 G). A concomitant reduction in TMRM fluorescence supported the loss of mitochondrial membrane potential occurring during mitochondrial apoptosis (Supplementary Fig. 3). Western blot analysis of MARCH5 KD cells further revealed increased levels of cleaved PARP and Caspase 8, a reduction of pro-Caspase 3, upregulation of BAK, and elevated expression of pro-apoptotic BH3-only family proteins including BAD, BIK, BID, PUMA, and NOXA, supporting its activation (Fig. 3 G). Collectively, these findings indicate that MARCH5 silencing induces mitochondrial dysfunction, impairs OXPHOS, and triggers intrinsic apoptosis in MM cells.
Genetic or pharmacologic induction of MFN2 recapitulates the effects of MARCH5 knockdown
We next investigated whether the phenotypic and molecular effects observed following MARCH5 KD could be recapitulated by enforced expression of MFN2. To this end, MFN2 was overexpressed in MM cells using an inducible lentiviral system, and two independent clones (Clones 15 and 19), exhibiting robust MFN2 upregulation, were selected for further analysis.
Notably, MFN2 induction led to a marked reduction in ETC protein levels (Fig. 4 A), which was accompanied by decreased mitochondrial respiration and ATP production, as determined by high-resolution respirometry (Oroboros) (Fig. 4 B). Similar to the effects of MARCH5 KD, inducible MFN2 expression exerted anti-MM activity in vitro, as evidenced by reduced cell proliferation (Fig. 4 C), impaired clonogenic capacity (Supplementary Fig. 4), and increased apoptotic cell death (Fig. 4 D). This apoptotic response was further supported by the upregulation of the pro-apoptotic BH3-only proteins PUMA and NOXA (Fig. 4 E).
To assess the in vivo relevance of these findings, we utilized an MFN2-inducible xenograft model in which doxycycline administration leads to MFN2 overexpression. In this model, MFN2 induction resulted in a significant reduction in tumor growth over time (Fig. 4 F), along with increased survival of mice bearing MFN2-overexpressing xenografts (Fig. 4 G). Analysis of tumor xenografts confirmed MFN2 upregulation in tumors from doxycycline-treated mice (Fig. 4 H), accompanied by decreased Ki67 expression as determined by immunohistochemistry (F i g. 4 I), and increased levels of BAD, BID, NOXA and PUMA as shown by Western blot analysis (Fig. 4 L), consistent with apoptosis induction in vivo.
Previous studies have repurposed the anti-arthritic drug leflunomide as an anticancer agent capable of inducing MFN2 expression [ 25 ]. In line with this, leflunomide treatment could upregulate MFN2 protein levels in MM cells (Fig. 5 A), and consistently reduced the viability of different MM cell lines (Fig. 5 B; Supplementary Fig. 5A), inducing apoptosis (Supplementary Fig. 5B, C). Notably, leflunomide remained effective in co-culture systems with HS5 stromal cells, suggesting its ability to overcome stromal-mediated protection (Fig. 5 C). Moreover, leflunomide anti-MM activity was not attenuated by uridine supplementation (Supplementary Fig. 5D), indicating that it exerts anti-MM effects through a mechanism independent of DHODH, a previously reported leflunomide molecular target [ 26 ].
Similar to the effects observed with genetic MFN2 induction, leflunomide treatment downregulated the expression of key ETC components, including NDUFS1, NDUFAB1, and COX4 (Fig. 5 D), reduced mitochondrial respiration (Fig. 5 E), and promoted apoptosis, as evidenced by the upregulation of the pro-apoptotic proteins NOXA and PUMA (Fig. 5 F).
In vivo, intraperitoneal administration of leflunomide led to a significant reduction in MM xenograft growth (Fig. 5 G), and prolonged the survival of treated animals (Fig. 5 H), without any gross architectural change in animal vital organs (Supplementary Fig. 5E). Analysis of tumor tissues confirmed increased MFN2 protein expression (F i g. 5 I), a reduction in proliferative index (Ki67) (Fig. 5 L), and upregulation of BH3-only proteins, including BAK, BIK, BIM, BID, NOXA, and PUMA, in leflunomide-treated xenografts (Fig. 5 M).
Collectively, these findings demonstrate that modulation of mitochondrial dynamics toward fusion, via MFN2 upregulation, phenocopies MARCH5 KD, eliciting anti-MM effects both in vitro and in vivo.
Imbalance of mitochondrial dynamics toward fusion sensitizes MM cells to venetoclax
We next investigated whether promoting mitochondrial fusion could sensitize MM cells to anti-MM therapies. Building on previous studies indicating that low ETC activity is associated with increased venetoclax sensitivity in MM [ 27 ], we evaluated whether targeting the MARCH5-MFN2 axis-through its impact on ETC function-could modulate venetoclax responsiveness. Consistent with higher MARCH5 expression, bortezomib-resistant cell lines upregulated multiple ETC components (Figure Supplementary 6A), and demonstrated reduced sensitivity to venetoclax (Fig. 6 A; Supplementary Fig. 6B). KD of MARCH5 enhanced venetoclax-induced cytotoxicity (Fig. 6 B), effects that were also observed after leflunomide treatment (Fig. 6 C). Conversely, enforced MARCH5 expression antagonized the anti-MM effects of venetoclax, as evidenced by diminished inhibition of cell viability (Fig. 6 D), impaired mitochondrial respiration, ATP production (Fig. 6 E) and mitochondrial dysfunction (Fig. 6 F), along with attenuated apoptosis (Fig. 6 G), confirmed by reduced PARP1, Caspase 3 cleavage (Fig. 6 H), and TMRM staining (F i g. 6 I). Importantly, MARCH5 OE did not alter the efficacy of other anti-MM agents (Supplementary Fig. 6C, D), including proteasome inhibitors (bortezomib, carfilzomib), alkylating agents (melphalan), or monoclonal antibodies (daratumumab, isatuximab), suggesting that the MARCH5 is primarily involved in the mechanism of action of venetoclax.
Discussion
Multiple myeloma (MM) is a malignancy of plasma cells that presents with considerable clinical heterogeneity, spanning a continuum from asymptomatic, biologically indolent precursor states, such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), to fully symptomatic disease and aggressive extramedullary forms like plasma cell leukemia [ 28 – 30 ].
Emerging evidence underscores the central role of mitochondrial dysfunction in MM pathogenesis. Diverse and multifactorial mitochondrial defects contribute to key aspects of disease biology by enhancing metabolic plasticity, promoting therapeutic resistance, and enabling immune evasion. Consequently, the mitochondrial network has emerged as a promising therapeutic target, with increasing interest in strategies aimed at modulating mitochondrial pathways to overcome drug resistance and improve clinical outcomes [ 4 , 29 ], particularly in advanced disease settings [ 31 , 32 ], where hyperactive kinase signaling may prompt dysregulated mitochondrial dynamics. The maintenance of a healthy mitochondrial network, crucial for meeting cellular energy demands, relies on mitochondrial dynamics, a process involving alternating fission and fusion events that impinge on mitochondrial size, shape, growth, redistribution and cristae remodeling [ 8 ]. This “mitochondrial quality control” mechanism is often deregulated in various pathological conditions, including cancer, where cells disrupt the delicate balance of mitochondrial dynamics to gain proliferative and survival advantages [ 2 ].
An imbalance in mitochondrial dynamics toward fission, resulting in a characteristic fragmented mitochondrial pattern, has been reported in several solid tumors [ 10 , 33 – 35 ] and hematological malignancies [ 13 , 14 ]. This imbalance contributes to metabolic reprogramming, evasion of cell death, and increased migration and invasiveness of tumor cells. Based on these findings, selective inhibition of mitochondrial fission has been shown to exert anti-MM activity in preclinical models, highlighting the therapeutic relevance of the GTPases that regulate this process.
MARCH5 is an E3 ubiquitin ligase localized to the outer mitochondrial membrane, where it plays pivotal roles in regulating mitochondrial fission dynamics [ 36 , 37 ], maintaining mitochondrial quality control [ 38 ], and facilitating mitophagy [ 39 , 40 ]. MARCH5 plays a critical role in maintaining mitochondrial function by ubiquitinating key mitochondrial proteins involved in cell cycle regulation [ 41 ], mitochondrial morphology [ 42 ], outer mitochondrial membrane integrity [ 43 ], and cellular senescence [ 44 ]. It also exerts context-dependent effects by modulating mitochondrial dynamics through fission and fusion effectors [ 45 , 46 ], regulating stress-responsive proteins such as IRE1 and NOXA to protect against cellular stress [ 46 , 47 ], and dampening innate immune responses by inhibiting mitochondrial antiviral signaling pathways [ 48 ].
Several solid and hematological cancers upregulate MARCH5 to promote malignant transformation and metastasis via derangement of mitochondrial fitness [ 16 ]. Previous analysis of public MM gene expression profile datasets revealed a significant correlation between MARCH5 overexpression and poor patient outcomes [ 24 ]. In our study, we confirmed that MARCH5 expression is significantly elevated in MM cells, with the highest levels observed in drug-resistant subpopulations. This suggests that MARCH5 may play a critical role in supporting survival mechanisms under therapeutic stress. To investigate the metabolic consequences of MARCH5 loss, first we conducted ultrastructural analysis of mitochondria in MARCH5-depleted cells, which revealed a markedly hyperfused mitochondrial network. This morphological phenotype is indicative of enhanced mitochondrial fusion activity, implying that MARCH5 may act to restrain excessive fusion under normal conditions. Supporting this hypothesis, ectopic overexpression of MARCH5 led to a reduction in MFN2 levels, a key driver of mitochondrial fusion. This reduction was linked to enhanced ubiquitylation of MFN2, suggesting that MARCH5 promotes its degradation. Conversely, MARCH5 KD resulted in accumulation of MFN2 protein, due to reduced ubiquitylation and prolonged protein half-life, reinforcing MARCH5's role as a negative regulator of mitochondrial fusion machinery.
To decipher the broader biological pathways modulated by MARCH5, we performed transcriptomic profiling using RNA sequencing. This analysis revealed a consistent downregulation of genes involved in the ETC upon MARCH5 depletion, a finding that was corroborated at the protein level. These data suggest that changes in mitochondrial dynamics triggered by MARCH5 KD impair mitochondrial bioenergetic function.
Functional metabolic assays confirmed these findings: MARCH5 KD led to reduced basal and maximal oxygen consumption rates, as measured by high-resolution respirometry in live cells, pointing to a defect in OXPHOS. The resulting metabolic stress ultimately triggered mitochondrial apoptosis, as confirmed by the increase in several BCL-2 homology 3 (BH3) domain-containing pro-apoptotic proteins, highlighting the critical link between MARCH5, mitochondrial dynamics, and MM cell survival. These results are in line with previous findings in AML, where it has been reported that MARCH5 inhibition is sufficient to trigger the canonical mitochondrial apoptosis pathway [ 49 ]. To directly evaluate whether mitochondrial fusion alone could recapitulate these effects, we used an inducible MFN2 overexpression system. Upon MFN2 induction, cells exhibited reduced ETC gene expression, along with metabolic and apoptotic phenotypes that mimicked the consequences of MARCH5 KD, both in vitro and in vivo. These results validate MFN2 as a key downstream effector of MARCH5 in the regulation of mitochondrial function and MM cell fate.
Building on these insights, we explored a therapeutic strategy to pharmacologically mimic MARCH5 depletion. Leflunomide, an FDA-approved anti-arthritic drug previously repurposed as an anti-cancer agent [ 25 ], was identified as a compound capable of inducing MFN2 expression. The tolerable safety profile of leflunomide observed in a recent phase I study, along with its potential to stabilize disease, underscores its promise as an anti-MM agent [ 50 ]. Treatment of MM cells with leflunomide resulted in upregulation of MFN2 protein, inhibition of ETC and OXPHOS, and induction of mitochondrial apoptosis, regardless of the underlying genetic background of the MM cells. Notably, these effects were observed both in vitro and in vivo in a MM xenograft model, where leflunomide exhibited significant anti-MM activity without causing observable toxicity in treated animals.
We next investigated whether promoting mitochondrial fusion could sensitize MM cells to anti-MM therapies, focusing specifically on venetoclax. Venetoclax acts by competitively binding to the BCL-2 homology 3 (BH3) domain of BCL-2, thereby preventing BCL-2 from sequestering and inactivating pro-apoptotic proteins, ultimately inducing apoptotic cell death [ 47 ]. In MM, venetoclax has paved the way for biomarker-driven interventions, owing to its significant clinical activity both as a monotherapy or in combination with other agents, particularly in patients with relapsed/refractory MM harboring the t(11;14) translocation [ 51 – 57 ]. Numerous intrinsic and extrinsic factors influence the therapeutic response to venetoclax. Notably, resistance to venetoclax has been primarily linked to reduced mitochondrial priming and altered expression of BCL-2 family proteins in MM cells, leading to broad resistance to standard-of-care anti-myeloma therapies. Moreover, other predictive features-such as B cell–like epigenetic signatures-have been associated with increased sensitivity to venetoclax, even in the absence of t(11;14) [ 27 , 58 , 59 ].
Notably, previous studies also reported that reduced expression and activity of ETC complexes correlate with heightened sensitivity to venetoclax in MM [ 27 ]. Based on this, and given that induction of mitochondrial fusion is associated with reduced ETC and OXPHOS activity, we hypothesized that MFN2 overexpression could enhance sensitivity to venetoclax. As expected, both genetic overexpression and pharmacological induction of MFN2 via leflunomide increased venetoclax’s anti-MM activity; conversely, MARCH5 overexpression abrogated venetoclax-induced mitochondrial dysfunction and apoptosis.
Conclusion
The MARCH5–MFN2 axis plays a key role in regulating mitochondrial fusion in MM. Shifting the mitochondrial dynamics machinery toward fusion results in metabolic changes that increase mitochondrial apoptosis and enhance the anti-MM activity of venetoclax. Overall, these findings support the development of mitochondrial fusion inducers, such as leflunomide, as novel mitochondria-targeted therapeutics for the treatment of MM, particularly in advanced, drug-resistant disease settings.
Supplementary Information
Supplementary Material 1: Figure S1. A Western Blot analysis of MARCH5 protein expression in PBMCs from three different healthy donors, and in MM cell lines; GAPDH served as a loading control; densitometric analysis of protein levels represents the mean ± SD from PBMCs after normalization. B Densitometric analysis of MARCH5 protein levels in MM cell lines; mean ± SD after normalization is reported in the densitometric analysis. The value was set at 100 for AMO and H929 cells. C Western blot analysis of MARCH5 expression in AMO-BZB cells, 48 h after electroporation with 100 nM of siMARCH5#1, siMARCH5#2, siMARCH5#3 or negative control siRNA (NC); GAPDH was used as loading control. D Densitometric analysis of DRP1, MFF and MFN2 protein levels in AMO-BZB cells transfected with siRNA MARCH5#1, siRNA MARCH5#2, siRNA MARCH5#3 or siRNA control (siNC). E Densitometric analysis of DRP1, MFF and MFN2 protein levels in MM cell lines transduced with MARCH5-Flag cDNA (OE MARCH5) or the empty vector (EV). * p < 0.05.
Supplementary Material 2: Figure S2. Western blot analysis of ubiquitin-conjugated MFN2, total MFN2, and MARCH5 in AMO-BZB cells electroporated with 100 nM siMARCH5#1 or negative control (NC) siRNA for 24 h, treated with the proteasome inhibitor MG132 (20 μmol/L) during the last 4 h, and immunoprecipitated with an anti-MFN2 antibody. GAPDH served as a loading control for input samples.
Supplementary Material 3: Figure S3. Flow cytometry analysis of mitochondrial TMRM levels in AMO-BZB cells stained with the TMRM probe after electroporation with 100 nM siMARCH5#1 or negative control (NC) siRNA. The histogram is representative of three independent experiments. Mean fluorescence intensity (MFI) values are reported. ** p < 0.001.
Supplementary Material 4: Figure S4. Colony formation assay performed on doxycycline-inducible AMO-BZB (CL.19) cells after seeding in methylcellulose for 10 days. Histogram bars represent the mean ± SD. * p < 0.05.
Supplementary Material 5: Figure S5. A Half-maximal inhibitory concentration (IC 50 ) of leflunomide determined for AMO, AMO-BZB, H929, H929-BZB, H929-CFZ, JJN3, KMS26, OPM2, and 8226 cell lines. IC 50 values were calculated using GraphPad Prism software from three independent experiments. B Flow cytometry analysis of Annexin V/7-AAD-stained AMO cells after treatment with increasing concentrations of Leflunomide or DMSO as vehicle control. C Western blot analysis of pro-Caspase 3 and PARP in AMO cells, 72 h after leflunomide treatment; GAPDH was used as a loading control. D Cell viability assessed by CellTiter-Glo (CTG) assay in AMO cells, 48 h after combined treatment with uridine and leflunomide. Viable cells are reported as a percentage relative to vehicle control. E H&E staining (20× magnification) of liver, kidney, lung, heart, and spleen tissues from mice after treatment. Representative images are shown.
Supplementary Material 6: Figure S6. A Quantitative RT-PCR analysis of NDUFA2, NDUFS2, NDUFA8, NDUFB8, SDHA, UQCR11, and COX5A mRNA expression levels in AMO and AMO-BZB cells. Results represent the mean ± SD of three independent experiments after normalization to β-actin using the ΔΔCt method. ** p < 0.01. B Half-maximal inhibitory concentration (IC 50 ) values of Venetoclax, determined for AMO, AMO-BZB, H929, and H929-BZB cell lines. IC 50 values were calculated using GraphPad Prism software from three independent experiments. C Cell viability assessed by CellTiter-Glo (CTG) assay in AMO cells following lentiviral expression of MARCH5 cDNA (OE MARCH5) or empty vector (EV), alone or in combination with Bortezomib, Carfilzomib, or Melphalan. Viable cells are expressed as a percentage relative to vehicle control. D Cytotoxicity analysis in AMO cells overexpressing MARCH5 cDNA (OE MARCH5) or the empty vector (EV) control, in co-culture with PBMCs in the presence of Daratumumab or Isatuximab at a 30:1 effector-to-target ratio for 12 h. ** p < 0.01.
Abbreviations
Bortezomib
Cell titer glo
Dynamin-related protein 1
Electron transport chain
Leflunomide
E3 ubiquitin ligase membrane-associated ring-CH-type finger 5
Mitofusin 1
Mitofusin 2
Mitochondrial fission factor
Multiple Myeloma
Mitochondrial outer membrane
Oxygen consumption rate
Oxidative phosphorylation
Proteasome inhibitor
Transmission electron microscopy
Tetramethylrhodamine methyl ester
Venetoclax
Kioto Encyclopedia of Genes and Genomes
Gene Ontology
Author contributions
NA was responsible for the overall design of the study, experimental validation, and writing of the manuscript. IV, MEGC, RT, PM, LG and MM performed in vitro and in vivo experiments. AG and NC performed transcriptomic experiments. IP performed TEM experiments. FT performed IHC experiments. MG and GV provided critical comments. All the authors read and approved the final manuscript.
Funding
This research was supported by a grant from the Italian Association for Cancer Research (IG24449) to NA, and partially by: the PRIN PNRR (code: P2022THN5N; CUP: F53D23012310001) to NA, and the PNRR CN00000041 “Sviluppo di terapia genica e farmaci con tecnologia a RNA” to MEGC, GV and NA.
Data availability
Transcriptomic data have been deposited to NCBI BioProject database and are available through the BioProject with Accession Number: PRJNA1281010 ( https://www.ncbi.nlm.nih.gov/bioproject/ ). All the other data supporting the findings of this study are available within the article.
Declarations
Ethics approval and consent to participate
Peripheral blood mononuclear cells (PBMCs) were isolated following informed consent and Institutional Review Board (University of Catanzaro, Catanzaro, Italy) approval (institutional approval: n. 266/2021). Animal experimentation was carried out at University Magna Graecia of Catanzaro under the Italian MOH authorization n. 887/2021-PR.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Footnotes
The original version of this article was revised: “Figure has been corrected”.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Maria Eugenia Gallo Cantafio and Roberta Torcasio have equally contributed to this work.
Change history
11/11/2025
A Correction to this paper has been published: 10.1186/s12967-025-07052-7
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Associated Data
Supplementary Materials
Supplementary Material 1: Figure S1. A Western Blot analysis of MARCH5 protein expression in PBMCs from three different healthy donors, and in MM cell lines; GAPDH served as a loading control; densitometric analysis of protein levels represents the mean ± SD from PBMCs after normalization. B Densitometric analysis of MARCH5 protein levels in MM cell lines; mean ± SD after normalization is reported in the densitometric analysis. The value was set at 100 for AMO and H929 cells. C Western blot analysis of MARCH5 expression in AMO-BZB cells, 48 h after electroporation with 100 nM of siMARCH5#1, siMARCH5#2, siMARCH5#3 or negative control siRNA (NC); GAPDH was used as loading control. D Densitometric analysis of DRP1, MFF and MFN2 protein levels in AMO-BZB cells transfected with siRNA MARCH5#1, siRNA MARCH5#2, siRNA MARCH5#3 or siRNA control (siNC). E Densitometric analysis of DRP1, MFF and MFN2 protein levels in MM cell lines transduced with MARCH5-Flag cDNA (OE MARCH5) or the empty vector (EV). * p < 0.05.
Supplementary Material 2: Figure S2. Western blot analysis of ubiquitin-conjugated MFN2, total MFN2, and MARCH5 in AMO-BZB cells electroporated with 100 nM siMARCH5#1 or negative control (NC) siRNA for 24 h, treated with the proteasome inhibitor MG132 (20 μmol/L) during the last 4 h, and immunoprecipitated with an anti-MFN2 antibody. GAPDH served as a loading control for input samples.
Supplementary Material 3: Figure S3. Flow cytometry analysis of mitochondrial TMRM levels in AMO-BZB cells stained with the TMRM probe after electroporation with 100 nM siMARCH5#1 or negative control (NC) siRNA. The histogram is representative of three independent experiments. Mean fluorescence intensity (MFI) values are reported. ** p < 0.001.
Supplementary Material 4: Figure S4. Colony formation assay performed on doxycycline-inducible AMO-BZB (CL.19) cells after seeding in methylcellulose for 10 days. Histogram bars represent the mean ± SD. * p < 0.05.
Supplementary Material 5: Figure S5. A Half-maximal inhibitory concentration (IC 50 ) of leflunomide determined for AMO, AMO-BZB, H929, H929-BZB, H929-CFZ, JJN3, KMS26, OPM2, and 8226 cell lines. IC 50 values were calculated using GraphPad Prism software from three independent experiments. B Flow cytometry analysis of Annexin V/7-AAD-stained AMO cells after treatment with increasing concentrations of Leflunomide or DMSO as vehicle control. C Western blot analysis of pro-Caspase 3 and PARP in AMO cells, 72 h after leflunomide treatment; GAPDH was used as a loading control. D Cell viability assessed by CellTiter-Glo (CTG) assay in AMO cells, 48 h after combined treatment with uridine and leflunomide. Viable cells are reported as a percentage relative to vehicle control. E H&E staining (20× magnification) of liver, kidney, lung, heart, and spleen tissues from mice after treatment. Representative images are shown.
Supplementary Material 6: Figure S6. A Quantitative RT-PCR analysis of NDUFA2, NDUFS2, NDUFA8, NDUFB8, SDHA, UQCR11, and COX5A mRNA expression levels in AMO and AMO-BZB cells. Results represent the mean ± SD of three independent experiments after normalization to β-actin using the ΔΔCt method. ** p < 0.01. B Half-maximal inhibitory concentration (IC 50 ) values of Venetoclax, determined for AMO, AMO-BZB, H929, and H929-BZB cell lines. IC 50 values were calculated using GraphPad Prism software from three independent experiments. C Cell viability assessed by CellTiter-Glo (CTG) assay in AMO cells following lentiviral expression of MARCH5 cDNA (OE MARCH5) or empty vector (EV), alone or in combination with Bortezomib, Carfilzomib, or Melphalan. Viable cells are expressed as a percentage relative to vehicle control. D Cytotoxicity analysis in AMO cells overexpressing MARCH5 cDNA (OE MARCH5) or the empty vector (EV) control, in co-culture with PBMCs in the presence of Daratumumab or Isatuximab at a 30:1 effector-to-target ratio for 12 h. ** p < 0.01.
Data Availability Statement
Transcriptomic data have been deposited to NCBI BioProject database and are available through the BioProject with Accession Number: PRJNA1281010 ( https://www.ncbi.nlm.nih.gov/bioproject/ ). All the other data supporting the findings of this study are available within the article.