Lefamulin Overcomes Acquired Drug Resistance via Regulating Mitochondrial Homeostasis by Targeting ILF3 in Hepatocellular Carcinoma

Abstract Acquired resistance represents a critical clinical challenge to molecular targeted therapies such as tyrosine kinase inhibitors (TKIs) treatment in hepatocellular carcinoma (HCC). Therefore, it is urgent to explore new mechanisms and therapeutics that can overcome or delay resistance. Here, a US Food and Drug Administration (FDA)‐approved pleuromutilin antibiotic is identified that overcomes sorafenib resistance in HCC cell lines, cell line‐derived xenograft (CDX) and hydrodynamic injection mouse models. It is demonstrated that lefamulin targets interleukin enhancer‐binding factor 3 (ILF3) to increase the sorafenib susceptibility of HCC via impairing mitochondrial function. Mechanistically, lefamulin directly binds to the Alanine‐99 site of ILF3 protein and interferes with acetyltransferase general control non‐depressible 5 (GCN5) and CREB binding protein (CBP) mediated acetylation of Lysine‐100 site, which disrupts the ILF3‐mediated transcription of mitochondrial ribosomal protein L12 (MRPL12) and subsequent mitochondrial biogenesis. Clinical data further confirm that high ILF3 or MRPL12 expression is associated with poor survival and targeted therapy efficacy in HCC. Conclusively, this findings suggest that ILF3 is a potential therapeutic target for overcoming resistance to TKIs, and lefamulin may be a novel combination therapy strategy for HCC treatment with sorafenib and regorafenib.


Animals
All animal care and experimental procedures were approved by the University Committee on Use and Care of Animals of the China Pharmaceutical University (Nanjing, China) (Resolution Number 2022-03-024).4-week-old male Balb/c nude mice and C57BL/6J mice used for this study (16-20 g, specific pathogen-free class) were purchased from Charles River Laboratories Co., Ltd (Beijing, China).
For the subcutaneous tumor model, 4 × 10 6 HepG2 cells suspended in 100 μL serum-free DMEM and Matrigel (1:1) were subcutaneously injected into the right flank of Balb/c nude mice.
When the xenografts were palpable, measuring the tumor size every 2 or 3 days using a digital caliper, calculating the tumor volumes with the formula: tumor volume = length × width 2 × 0.5.
When tumor volume reached approximately 100 mm 3 , the mice were randomized into treatment groups, including vehicle control, sorafenib (30 mg/kg/day, orally), lefamulin(25 or 50 mg/kg/day, intraperitoneally) or the combination, 6 mice per groups.Blood samples were collected before sacrificing the mice, then all the tumors were removed, weighed, and frozen in liquid nitrogen for further studies.
To generate hydrodynamic injection liver cancer mouse model, C57BL/6J mice were used and the procedure was performed as described previously.In brief, 2 μg plasmids encoding human C-Myc, 40 μg plasmids encoding human N-RasV12, along with 4 μg sleeping beauty transposase (SBT) , were diluted in 2 mL saline, filtered through 0.22 μm filter and injected into mouse livers via the tail vein in 5 to 7 seconds.Five weeks later, drug administration with sorafenib was initiated to mimic clinical sorafenib resistance process of advanced HCC patients.
3 weeks later, the mice were randomized into different treatment groups, including vehicle control, sorafenib (30 mg/kg/day, orally) or regorafenib (10 mg/kg/day, orally), lefamulin(50 mg/kg/day, intraperitoneally) or the combination, 7 mice per groups.After completing the therapy, tumor-bearing mice were sacrificed, and livers were harvested for further analysis.

Cell lines and cell culture
In this study, human HCC cell lines (HepG2, HCCLM3, Huh7, SNU449, MHCC-97H, SK-Hep-1, Bel7402 and Hep3B) and normal cell lines (L02, WRL68 and HEK293T) were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences.These cells were cultured in the 10% fetal bovine serum (FBS; Gibco) basal medium supplemented with 1% penicillin-streptomycin (NCM) at 37 ℃ in the presence of 5% CO2 in an incubator and routinely detected for mycoplasma.

Establishment and culture of sorafenib-resistant cells in vitro and in vivo
The isogenic resistant cell lines resistant to sorafenib (HepG2 SR and HCCLM3 SR) in vitro were established by exposing cells to escalating doses of sorafenib for approximately 6 months and maintained with low dose of sorafenib, and authenticated.
The sorafenib-resistant in vivo tumor cells were isolated from xenografts of sorafenib-resistant HCC mouse.A subcutaneous mouse model was constructed firstly, as follows described.In total, 4 × 10 6 HepG2 cells were implanted into the flank of Balb/c nude mice.After 4 weeks, mice with similar tumor burdens were subjected to receiving 30 mg/kg/day sorafenib (MCE) or vehicle.
After 8 weeks of treatment, mice were sacrificed, and tumor samples were cut into small pieces to isolate tumor cells for further investigation.The isolation process includes the following steps: subcutaneous HepG2-derived xenografts were excised and mechanically dissociated by gentle pipetting, digested with collagenase (Thermo Fisher Scientific), filtered through 70 µm cell strainer (BD Bioscience), and centrifuged at 1500 g for 5 min, cell sediments were exposed to red cell lysis buffer (Beyotime) for 10 min to remove the red blood cells, tumor cells were washed with D-Hanks (Solarbio) 3 times and resuspended in Dulbecco's modified eagle's medium (DMEM; Hyclone) supplemented with 10% FBS.

Cell growth and viability assay
Cell growth curves and cell viability were determined using the Cell Counting Kit-8 (CCK-8; MCE) assay according to the manufacturer's instructions.
For cell growth curves analysis, cells were seeded at a density of 3000 cells per well in 96-well plates and treated with different concentrations of lefamulin (MCE), and/or sorafenib, regorafenib and lenvatinib (MCE) for 24, 48, 72 and 96 hours.For cell viability assay, cells were seeded at a density of 8000 cells per well in 96-well plates and treated with different concentrations of lefamulin, and/or sorafenib and regorafenib for 48 hours.The absorbance was measured at 450 nm after incubation with 10 μL of CCK-8 reagent for 3 hours using Spectra-Max Plus 384 (Molecular Devices).

Combination effect analysis
Combinational treatments will result in synergistic, additive or antagonistic effects.The combination index (CI) values were calculated using CompuSyn software according to the Chou-Talalay method: CI < 1, synergistic; CI = 1, additive; CI > 1, antagonistic.

Colony formation assay
Cells were seeded at a density of 1 × 10 4 cells per well in 6-well plates and treated with different concentrations of lefamulin, and/or sorafenib, regorafenib and lenvatinib for 24 hours.The cells were allowed to grow in complete media for another 14 days.The colonies were washed with PBS and fixed in 4% paraformaldehyde (Servicebio) for 15 minutes at room temperature and then stained with crystal violet (Beyotime).Finally, the colony numbers (>50 cells) were counted.

EDU incorporation assay
Cells were seeded at a density of 8000 cells per well in 96-well plates and treated with different concentrations of lefamulin, and/or sorafenib, regorafenib and lenvatinib for 24 hours.The cell proliferation was determined by 5-ethynyl-2'-deoxyuridine (EDU) incorporation assay using BeyoClick™ EdU-488 imaging detection kit (Beyotime) according to the manufacturer's instructions.

Cell apoptosis assays
Cell apoptosis was determined by flow cytometry using Annexin V-Fluorescein isothiocyanate (FITC)/PI Apoptosis Detection Kit (Beyotime) according to manufacturer's instructions.In brief, 3 × 10 5 cells were seeded in 6-well plates and treated with different concentrations of lefamulin, and/or sorafenib for 48 hours.Then the cells were double stained with FITC-conjugated Annexin V and PI for 15 minutes at room temperature in the dark and analyzed by flow cytometer (BD).

RNA extraction, reverse transcription and RT-qPCR assay
Total RNA from cells or tumors was extracted using an RNA extraction kit (ES Science) according to the manufacturer's instructions.The concentrations were measured by measuring absorbance at 260 and 280 nm with NanoDrop (Thermo Fisher Scientific), and 1 µg of total RNA was subjected to synthesize cDNA using HiScript ® II Q Select RT SuperMix (Vazyme).RT-qPCR was conducted in biological triplicates using SYBR Green reagent (Vazyme) on a LightCycler 480 II system (Roche).Expression levels were normalized to the expression of GAPDH.PCR primer sequences are listed in Table S3.A melting curve of each amplicon was determined to verify its specificity.

Co-immunoprecipitation (Co-IP) and western blot analysis
Cells and tumor samples were lysed by RIPA lysis buffer (YEASEN) with 1% PMSF (Beyotime) to extract the total proteins by centrifugation at 12,000 g for 10 minutes at 4 ℃, and the protein concentrations were determined by BCA kit (Beyotime).
For Co-IP, 1 mg proteins were incubated with the appropriate antibodies overnight at 4 ℃ and then the complexes were incubated with Protein A/G Plus Agarose (Santa Cruz Biotechnology) for 4 hours at 4 ℃, immune-complexes were washed five times with PBS, suspended in 2 × SDS loading buffer and boiled for 10 minutes.The immune-complexes were subjected to subsequent western blot analysis.
For western blot analysis, equal amounts of immune-complexes or lysates were subjected to 8 to 12% SDS-PAGE gel separation and transferred to PVDF membranes (Bio-Rad).Membranes were blocked with 5% skim milk (Beyotime) in PBST buffer (PBS containing 0.2% Tween 20) for 2 hours at room temperature and immunoblotted with diluted primary antibodies overnight at 4 °C.The specific antibodies used in this study are listed in Table S4.After incubation with peroxidase-conjugated secondary antibodies (YEASEN) for 2 hours at room temperature, the immunoblots were subjected to electrochemiluminescence using an ECL kit (Vazyme) according to the manufacturer's instructions with a ChemiDoc XRS + imaging system (Bio-Rad), and quantified using Image Lab software.Actin was served as an internal control.

siRNA, plasmids, transfection, and site-directed mutagenesis
The plasmids expressing human ILF3 and MRPL12, as well as the different length of MRPL12 promoter plasmids, were constructed by standard subcloning separately.The small interfere RNA (siRNA) specifically targeting human ILF3, MRPL12, GCN5 or CBP were purchased from Ribobio.The primers used for plasmid construction and the sequences of siRNA are listed in Table S3.Cells were transfected with siRNA or plasmids using Lipofectamine 3000 (Invitrogen) in Opti-MEM (Invitrogen) according to the manufacturer's instructions.After approximately 10 to 12 hours of incubation, the medium was changed to complete medium, gene knockdown or overexpression were detected by western blot or RT-qPCR analysis after transfection for 48 hours.
Cells transfected with pcDNA or siNC duplexes were used as control.

Measurement of ROS and superoxide in the mitochondria
Cells were seeded in 6-well plates (3 × 10 5 cells per well) and treated with drugs for 48 hours.
Cells were harvested and washed with PBS, then incubated with 10 mM DCFH-DA (Beyotime) or MitoSOX Red (YEASEN) for 20 min at 37 °C without lighting.Celluar ROS or mitochondrial ROS was determined by flow cytometry.Experimental data were analyzed using FlowJo software.

Transmission electron microscopy
HepG2 cells were harvested after treatment and fixed with 2.5% glutaraldehyde fixative buffer (Beyotime).Then, cells were subjected to postfixation, dehydration and embedding to obtain the ultrathin sections.Sections were stained with 1% uranyl acetate and/or lead citrate.The morphology of mitochondria was acquired on a transmission electron microscope.

Mitochondrial mass determination
The mitochondrial mass was analyzed by MitoTracker Green staining (Beyotime).Cells were seeded in 6-well plates overnight and subjected to different treatments.MitoTracker Green was then incubated with cells for 15 minutes at 37 ℃ according to the manufacturer's instructions, and subjected to flow cytometric analysis.

Mitochondrial DNA quantification
Total DNA was extracted from cells using Genomic DNA Mini Preparation Kit with Spin Column (Vazyme).Relative mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) were determined by RT-qPCR with D-Loop2 gene to represent mtDNA and G6PC gene to represent nDNA.The mitochondrial DNA copy numbers were indicated by the ratio of mtDNA to nDNA.

Immunofluorescence staining
For mitochondrial morphological observation, L02 and WRL68 cells were plated on glass coverslips in a dish and treated with different agents, then the cells were washed with PBS buffer and stained with MitoTracker @ Deep Red FM (Thermo Fisher Scientific) to examine the mitochondrial morphology for 40 min at 37 ℃，fixed with 4% paraformaldehyde for 15 minutes.
For determination of intracellular distribution of ILF3, HepG2 and HCCLM3 cells were plated on glass coverslips in a dish and treated with different agents, fixed with 4% paraformaldehyde for 15 minutes.The fixed cells were then permeabilized with 0.5% Triton X-100 for 30 min, followed by a blocking step in 1% bovine serum albumin for 2 hours at room temperature.And incubated with primary antibodies at 4 ℃ overnight, followed by incubated with fluorochrome-labelled secondary antibody for 2 hours at room temperature (YEASEN).Next, the cells were then washed with PBS 3 times, and the nuclei were counterstained with DAPI (Beyotime) for 5 min at room temperature.Images were captured using an ImageXpress Micro Confocal Platform (Molecular Devices).

Oxygen consumption rate detection
The oxygen consumption rate of cells was analyzed by a Seahorse XF96 Extracellular Flux Analyzer (Seahorse Biosciences) to monitor mitochondrial respiration in real time using Seahorse XF Cell Mito Stress Test Kit (Seahorse Biosciences) as described.In brief, 1 × 10 4 cells in 3 replicates were seeded into each well, and treated by different drugs or transfected with siRNA or plasmids.OCR was measured before and after the injection of 1.5 µM oligomycin, 1.0 µM FCCP, and 0.5 µM rotenone/antimycin A.

Cellular thermal shift assay (CETSA)
Briefly, HepG2 cells or HEK293T cells transfected with Flag-ILF3 was seeded in 10 cm culture dishes and treated with lefamulin or DMSO for 6 hours.Then cells were harvested and washed with PBS, then resuspended to a density of 5 × 10 6 cells/mL in PBS containing protease inhibitor and dispensed into 8 PCR tubes (100 μL/tube) and heated at 46 to 60 ℃ by a thermal cycler (eppendorf) for 3 minutes.Subsequently, the cells were lysed by freeze-thaw in liquid nitrogen, cell lysates were obtained by centrifugation at 15,000 g for 15 minutes at 4 °C and analyzed by western blot.

Drug affinity responsive target stability (DARTS)
Briefly, HepG2 cells or HEK293T cells transfected with Flag-ILF3 was seeded in 10 cm culture dishes and collected.The total protein was extracted using NP40 lysis buffer (Sigma-Aldrich), cell lysates were centrifuged at 18,000 g for 10 minutes at 4 °C.The supernatants from independent biological replicates were aliquoted in equivalent volumes containing 100 µg of protein and incubated for 1 hour at 37 °C with or without lefamulin.Pronase was added simultaneously into all samples and incubated at 37 °C for 30 minutes, then protease inhibitor cocktail was added to stop reactions and SDS-PAGE was carried out to analyze the protein expression.

Bio-layer interferometry (BLI) analysis
The dose-dependent binding affinities between lefamulin and ILF3 was assessed by BLI with Octet RED96 (ForteBio).Lefamulin was prepared at different concentrations in kinetic buffer [PBS, 0.05% bovine serum albumin, 0.01% Tween 20].WT ILF3 or mutant ILF3 was the ligand and coupled to the equilibrated Ni-NTA biosensor tips (ForteBio, Menlo Park, CA), sensors that incubated in buffer without proteins background were binding controls.Assays were performed according to a standard protocol with a total volume of 200 μL per well in 96-well black plates at 30 ℃ and datas were analyzed by Octet data analysis software using a double reference subtraction method.

RNA-Sequencing (RNA-seq) analysis
The total RNA was extracted using TRIzol (Vazyme) reagent and reverse transcribed into cDNA from HepG2 cells treated with DMSO or lefamulin for 48 hours, and HepG2-derived xenografts treated with vehicle, sorafenib, lefamulin, or the combination.Then RNA-seq of cells and tumors were accomplished with the assistance of Shanghai Majorbio Biotechnology Co., Ltd.(Shanghai, China) and Beijing Novogene Co., Ltd.(Beijing, China), respectively.A fold change > 1.2 and P-adj value < 0.05 were defined as differential expression to screen downregulated genes using DESeq2 software to perform DEG analysis and enrichment analysis for further study.Then we conducted KEGG pathways and GO enrichment analysis.The transcriptome sequencing data have been deposited in NCBI Gene Expression Omnibus (GEO) under the following accession number: GSE252987 and GSE252988 .

Mass spectrum analysis
We identified the different band between DMSO and lefamulin treated cell lysates under the assistance of CETSA and SDS-PAGE.Protein reduction alkylation, enzymatic hydrolysis, followed by peptide desalt, and mass spectrometry detection were conducted in turn.The mass spectrum analysis was accomplished with the assistance of Beijing Novogene Co., Ltd.(Beijing, China).

Chromatin immunoprecipitation (ChIP) assay
Chromatin immunoprecipitation (ChIP) assays were performed by EpiQuik TM Chromatin Immunoprecipitation Kit (Epigentek) according to the manufacturer's instructions.Briefly, 2 × 10 6 HepG2 cells were harvested after 48 hours treatment of DMSO, sorafenib, lefamulin, or the combination and crosslinked with 10 mL of PBS containing 1% formaldehyde.Sheared chromatin fragments (200 to 600bp) was immunoprecipitated with 3 μg of the anti-IgG, anti-RNA Polymerase II, or anti-ILF3 antibody for 1.5 hours at room temperature.
Immunoprecipitated DNA and input DNA were analyzed by RT-qPCR to examine the occupancy of ILF3 in the promoters of MRPL12.

EMSA
The purified His-ILF3 protein and 25bp WT-or MUT-MRPL12-5'FAM probe (Tsingke Biotechnology) were used for EMSA assays.Approximately 10 μM of probe and 50 μM protein were used per reaction, reactions were carried out in a 10 μL volume at 20 °C for 30 min.
The reaction products were loaded on a 8% EMSA gel and run at 4 °C and 80 V for 2 hours.DNA bands were visualized using UV light at 260 nm.The sequences of the probes used to show ILF3-MRPL12 binding were WT-MRPL12 promoter oligonucleotide (5'-GAGGCTCTGCCTGTTCTGATCTGAA-3') and MUT-MRPL12 promoter oligonucleotide (5'-GAGGCTCTGCAAAAACTGATCTGAA-3') which located at -445 to -421 of MRPL12 promoter.

Luciferase reporter assay
The different promoter regions of MRPL12 were ligated into the pGL4 luciferase reporter vector, then several pGL4-MRPL12-Luc reporter plasmids were constructed to further explore the binding site of ILF3 on the promoters of MRPL12.HepG2 cells were grown in 24-well plates and co-transfected with luciferase reporter plasmids and a renilla reporter plasmid and then conducted to different treatments for 48 hours.Cells were lysed and subjected to luciferase reporter assay to examine firefly and renilla luciferase signals (considered as a negative control) using Dual Luciferase® Reporter Assay System (Vazyme) by SpectraMax Paradigm (Molecular Devices ).

Plasma biochemistry
Blood samples supplemented with anticoagulant from animals were centrifugated at 4000 rpm for 10 minutes at 4 °C to obtain plasma.Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE) and urea nitrogen (BUN) levels were measured in animals using standard enzymic procedures to evaluate liver or kidney injury according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute).

Histology and IHC analysis
Tumors and livers were fixed with 4% paraformaldehyde overnight, embedded in paraffin, and then cut into 5 μm sections used for H&E staining and IHC staining with specific antibodies against ILF3, MRPL12 and Ki67.Histological sections were scanned with a light microscope (Olympus) and the percentage of stained positive areas were quantified using Image J. At least six randomly chosen fields from each section were analyzed.

Human HCC samples
The human HCC tissue microarrays (LVC1609), consisting of 64 pairs of HCC tissues with comprehensive clinicopathological and follow-up data, were purchased from Shanghai WEIAOBIO Biotech.Tissue microarrays were stained with anti-ILF3 antibodies and scanned with a light microscope.Kaplan-Meier survival curves were generated and analyzed using the Log-rank test.

Public database analysis
The expression of MRPL12 or ILF3 in HCC and non-tumor tissues was investigated in GEPIA database (http://gepia.cancer-pku.cn/).
The Kaplan-Meier analysis was performed using HCC patients data retrieved from using the TCGA datasets in the GEPIA database and Kaplan-Meier plotter database Firstly, we constructed the pET-28a plasmids expressing His-tagged wild-type or mutant ILF3 by transforming into Escherichia coli BL21 Star (DE3) cells.Then, ILF3 protein expression was induced by 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) (Sangon Biotech) and incubated for 20 hours at 16 °C with shaking at 200 rpm.Next, bacterial cells were collected and washed with double distilled water, lysed in non-denatured lysis buffer by sonication, and collected the supernatant by centrifugation at 15,000 g for 30 minutes at 4 °C, the supernatant was filtered through 70 µm strainer and loaded onto a His-tag Purification Resin (Beyotime) overnight at 4 ℃ shaking slowly.Finally, the His-tagged ILF3 fusion protein was eluted with 50 mM imidazole (Sangon Biotech) analyzed by SDS-PAGE.

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Figure S1.Identification of pleuromutilin class of antibiotics as sensitizers for sorafenib

Figure S2 .
Figure S2.Combination of lefamulin and sorafenib significantly inhibits HCC growth in

Figure
Figure S3.Lefamulin-mediated mitochondrial dysfunction augmented the susceptibility of

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Figure S6.ILF3 is a transcriptional activator of MRPL12.