Hepatocyte MLKL Drives Obesity-Driven Hepatocellular Carcinoma Progression via Mitochondrial Dysfunction Independent of Necroptosis in MASLD

Hepatocyte MLKL Drives Obesity-Driven Hepatocellular Carcinoma Progression via Mitochondrial Dysfunction Independent of Necroptosis in MASLD | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Hepatocyte MLKL Drives Obesity-Driven Hepatocellular Carcinoma Progression via Mitochondrial Dysfunction Independent of Necroptosis in MASLD Phoebe Ohene-Marfo , Sabira Mohammed , Chao Jiang , Shylesh Bhaskaran , Constantin Georgescu , Megan John , Kevin Pham , Albert Tran , Chinthalapally V Rao , Tae Gyu Oh , Michael Kinter , View ORCID Profile Willard M Freeman , Courtney Houchen , Jonathan D Wren , Sathyaseelan S. Deepa doi: https://doi.org/10.1101/2025.11.26.690789 Phoebe Ohene-Marfo 1 Department of Biochemistry & Physiology, The University of Oklahoma Health Campus Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sabira Mohammed 1 Department of Biochemistry & Physiology, The University of Oklahoma Health Campus 2 Stephenson Cancer Center, The University of Oklahoma Health Campus Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chao Jiang 1 Department of Biochemistry & Physiology, The University of Oklahoma Health Campus 2 Stephenson Cancer Center, The University of Oklahoma Health Campus Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shylesh Bhaskaran 1 Department of Biochemistry & Physiology, The University of Oklahoma Health Campus 2 Stephenson Cancer Center, The University of Oklahoma Health Campus Find this author on Google Scholar Find this author on PubMed Search for this author on this site Constantin Georgescu 3 Genes and Human Disease Research Program, Oklahoma Medical Research Foundation , Oklahoma City, Oklahoma, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Megan John 4 University of Oklahoma , Norman Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kevin Pham 3 Genes and Human Disease Research Program, Oklahoma Medical Research Foundation , Oklahoma City, Oklahoma, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Albert Tran 2 Stephenson Cancer Center, The University of Oklahoma Health Campus Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chinthalapally V Rao 5 Department of Medicine, The University of Oklahoma Health Campus 9 The Oklahoma City Veterans Affairs Medical Center , Oklahoma City, Oklahoma, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tae Gyu Oh 6 Oncology Science Department, and The University of Oklahoma Health Campus Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michael Kinter 8 Aging & Metabolism Research Program, Oklahoma Medical Research Foundation , Oklahoma City, Oklahoma, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Willard M Freeman 3 Genes and Human Disease Research Program, Oklahoma Medical Research Foundation , Oklahoma City, Oklahoma, USA 9 The Oklahoma City Veterans Affairs Medical Center , Oklahoma City, Oklahoma, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Willard M Freeman Courtney Houchen 5 Department of Medicine, The University of Oklahoma Health Campus 9 The Oklahoma City Veterans Affairs Medical Center , Oklahoma City, Oklahoma, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jonathan D Wren 3 Genes and Human Disease Research Program, Oklahoma Medical Research Foundation , Oklahoma City, Oklahoma, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sathyaseelan S. Deepa 1 Department of Biochemistry & Physiology, The University of Oklahoma Health Campus 2 Stephenson Cancer Center, The University of Oklahoma Health Campus 7 Oklahoma Center for Geroscience & Healthy Brain Aging, The University of Oklahoma Health Campus Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: Deepa-Sathyaseelan{at}ou.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Background and Aims Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of hepatocellular carcinoma (HCC), particularly in obesity. Mixed Lineage Kinase Domain Like (MLKL), the effector of proinflammatory cell death pathway necroptosis, is consistently elevated in human and experimental MASLD, yet the hepatocyte-specific role of MLKL in the neoplastic progression of MASLD to HCC in obesity is unknown. Approach and Results We used a long-term WD-induced, MASLD-driven HCC model in which mice develop liver cancer at ages comparable to human HCC onset. Hepatocyte-specific MLKL knockout ( Mlkl HepKO ) mice were generated to define MLKL’s liver-intrinsic function. WD robustly induced hepatic MLKL, whereas necroptosis markers were undetectable. MLKL loss did not alter WD-induced inflammation, fibrosis, or liver injury but markedly increased lipid droplet accumulation. Strikingly, Mlkl HepKO mice developed significantly fewer and smaller tumors with reduced stemness markers (DCLK1, OCT4). Transcriptomic analysis revealed upregulation of mitochondrial oxidative phosphorylation pathways in WD-fed Mlkl HepKO livers. WD suppressed the mitochondrial fusion protein and tumor suppressor Mfn2, while MLKL deficiency restored its expression. In HepG2 cells, MLKL knockdown or inhibition reduced proliferation and clonogenicity and enhanced oxidative metabolism. Human HCC datasets and tissue microarrays showed elevated MLKL in tumors and its association with poor survival. Conclusions Hepatocyte MLKL drives MASLD-driven HCC progression by an intrinsic, non-necroptotic mechanism. The mechanism involves MLKL-mediated suppression of the Mfn2 tumor suppressor protein, impaired mitochondrial respiration, and increase in both tumor cell proliferation and tumor stemness. All together these data position MLKL as a novel, druggable target in MASLD-associated HCC. Download figure Open in new tab GRAPHICAL ABSTRACT: Graphical illustration of the proposed mechanism of the role of MLKL in WD-induced MASLD driven HCC. INTRODUCTION Metabolic dysfunction-associated steatotic liver disease (MASLD), driven primarily by obesity and metabolic disorders, has emerged as the most prevalent liver disease, affecting nearly 30% of adults and 10% of children in the United States. [ 1 , 2 ] MASLD comprises a spectrum of liver diseases from hepatic steatosis, steatohepatitis (MASH), to cirrhosis. Approximately 30% of MASLD cases progress to MASH, and among those, a similar fraction develops advanced fibrosis or cirrhosis, with 7-13% of cirrhotic cases ultimately progressing to HCC, the 5th leading cause of cancer-related death worldwide. [ 3 ] Although most HCC in MASLD arises from cirrhosis, a surprisingly large fraction of MASLD-HCC progression (15-46%) occurs in the absence of cirrhosis, making prediction and surveillance more challenging. [ 4 ] Despite these observations, the molecular mechanisms driving MASLD-HCC progression remain poorly understood, particularly in the context of obesity, which increases HCC risk nearly twofold and HCC-related mortality fourfold compared to individuals with normal BMI. [ 5 ] This knowledge gap continues to limit the development of effective therapies for obesity-driven HCC. Chronic inflammation is a central driver of MASH pathogenesis and progression to HCC. [ 6 ] Necroptosis, a regulated form of inflammatory cell death, has been implicated in both human and experimental MASH. [ 7 ] Necroptosis occurs through the sequential activation of Receptor-Interacting serine/threonine-Protein Kinase 1 (RIPK1), RIPK3, and Mixed Lineage Kinase domain-Like protein (MLKL) through phosphorylation, in response to stimuli such as TNFα. Phosphorylated MLKL (Ser345) undergoes oligomerization and attaches to the plasma membrane to promote cell membrane rupture and release of proinflammatory damage-associated molecular patterns (DAMPs) that activate innate immune cells to promote inflammation. [ 8 ] Genetic or pharmacological inhibition of necroptosis pathway proteins reduce hepatic inflammation and injury in MASLD and HCC mouse models, [ 9 – 12 ] supporting a role of necroptosis in these liver diseases. While hepatocytes, comprising nearly 80% of liver mass, are traditionally viewed as the main cell type undergoing necroptosis, emerging evidence challenges this notion by showing that RIPK3 is epigenetically silenced in hepatocytes and liver cancer cells, preventing MLKL activation and canonical necroptosis. [ 13 ] Nevertheless, MLKL expression is consistently elevated in the livers of humans and mice with MASH, [ 7 , 14 , 15 ] although its functional relevance is unclear. Although MLKL is classically viewed as the executioner of necroptosis, recent studies show that it also mediates non-necroptotic functions, including extracellular vesicle biogenesis and autophagy, independent of RIPK3. [ 16 , 17 ] Prior work linking necroptosis to MASLD or HCC has relied on whole-body MLKL knockout mice, making it difficult to define the hepatocyte-specific role of MLKL, particularly given that RIPK3 is epigenetically silenced in hepatocytes. [ 18 , 19 ] Thus, the functional relevance of MLKL in hepatocytes and liver cancer cells, where canonical necroptosis is often impaired, remains unclear. Moreover, no studies have examined how hepatocyte MLKL contributes to liver tumor development under obesity-induced metabolic stress, such as Western diet feeding. Addressing these gaps is essential to define MLKL’s context-specific role in MASLD progression and tumorigenesis. Here, we examined the hepatocyte-specific role of MLKL in obesity-driven MASH-HCC using a Western diet, a well-established model of MASLD-associated HCC. [ 20 ] Extended WD feeding (∼15 months) induces obesity, MASH, fibrosis, and spontaneous HCC, closely reflecting human disease. Notably, WD-induced HCC in mice arises around 17 months of age, roughly equivalent to 50 years in humans, when HCC incidence begins, making this model highly relevant for interrogating hepatocyte-intrinsic MLKL function. Our data show that hepatocyte MLKL accelerates WD-induced HCC by impairing mitochondrial function/dynamics and enhancing cell-cycle progression, independent of canonical necroptosis. METHODS Animals, Diets, and Hepatocyte-Specific Mlkl Deletion All procedures were approved by the University of Oklahoma Health Campus IACUC. Male Mlkl fl/fl mice (originally generated by Murphy et al. 2013) [ 21 ] on a C57BL/6J background were injected with AAV8-TBG-Cre (hepatocyte-specific) or AAV8-TBG-Null (Vector Biolabs, PA,USA) via tail vein at 1.5 months of age to generate hepatocyte-specific Mlkl knockout ( Mlkl HepKO ) or control mice. Beginning at 2.5 months of age, mice were fed ad libitum either a control diet (CD, Teklad 7013 NIH-31, Research Diets) or a Western diet (WD, Research Diets, D22090208i custom made to match SF-11-078) for 4, 8, or 15 months. Five mice were housed per ventilated cage under a 12-h light/dark cycle at 20 ± 2°C. Diet composition is provided in Supplementary Table S1. Hepatocyte Isolation Hepatocytes and non-parenchymal cells were isolated by in situ perfusion and collagenase digestion. [ 22 ] Cell fractions were confirmed by lineage marker expression. Seahorse Extracellular Flux Analysis Mitochondrial respiration was measured using the Agilent Seahorse XF96 Mito Stress Test. HepG2 or AML12 cells were transfected with siControl or siMLKL and plated in Seahorse XF96 plates. OCR was recorded following sequential injection of oligomycin, FCCP, and antimycin A/rotenone. Basal respiration, ATP-linked respiration, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration were calculated using Wave software (Agilent). Fatty acid oxidation was assessed in AML12 cells treated with BSA control or palmitate. RNA Sequencing RNA-seq libraries were prepared by the University of Oklahoma Health Campus Institutional Core Facility and sequenced on an Illumina NextSeq 2000. Differential expression was assessed using limma-voom. Pathway enrichment was performed using fgsea, ReactomePA, and viewPathway. Statistics Data are represented as mean ±SEM. Two-tailed unpaired t-test or One-way ANOVA was used to analyze data with GraphPad Prism. P< 0.05 is considered as statistically significant. For RNA sequencing data analysis, moderate t-test p-values were adjusted for multiple testing using the false discovery rate (FDR) method. FDR (q-value) <0.05 and absolute log2 fold change above 1 were used as criteria to filter significantly differentiated genes. Complete procedural details and reagent lists are provided in Supplementary Methods. RESULTS Western diet induces MLKL expression in hepatocytes without activating necroptosis Western blot analysis of liver tissues from Mlkl HepKO mice confirmed efficient MLKL deletion specifically in the liver ( Figure S1A ), with no changes in other tissues ( Figure S1B ). Hepatocyte-specific loss of MLKL was validated in isolated hepatocytes, whereas non-parenchymal cells retained MLKL expression ( Figures 1A , S1C ). Control and Mlkl HepKO mice were fed either a control diet (CD) or a Western diet (WD) beginning at 2.5 months of age and sacrificed after 4, 8, or 15 months of diet feeding ( Figure 1B ) . WD feeding significantly increased body weight ( Figure S1D , 1.5-fold at 4 or 8 months and 1.2-fold at 15 months) and liver weight ( Figure 1C 1.6-, 2.6-, and 1.8-fold increase at 4, 8 or 15 months) in both control and Mlkl HepKO mice, with comparable levels of upregulation between genotypes. Notably, WD feeding markedly upregulated MLKL expression in the liver of control mice at all time points (2-fold at 4 or 8 months and 4-fold at 15 months; Figure 1D ). Consistently, MLKL expression was also increased in isolated hepatocytes from WD-fed mice, confirming that hepatocytes are a primary source of this induction ( Figure S1E ). Despite elevated MLKL expression, markers of necroptosis, phosphorylated MLKL (P-MLKL, S345) and MLKL oligomers, were not elevated in the liver tissues ( Figure 1D ). Together, these findings indicate that WD induces robust upregulation of MLKL in hepatocytes without activating canonical necroptosis. Download figure Open in new tab FIGURE 1. (A) Immunoblots showing the expression of MLKL and β-actin Control or Mlkl HepKO hepatocytes and NPCs ( Top ); Graphical representation of quantified blots normalized to β-actin ( bottom ), White bar: Control mice; Dark green bar: Mlkl HepKO mice. (B) Schematic of experimental design. (C) The ratio of liver weight normalized to the percentage of body weight after 4, 8 and 15 months of diet feeding. (D) Top : Immunoblots showing expression of MLKL, P-MLKL, and MLKL oligomer in liver tissues of control and Mlkl HepKO mice fed either CD or WD for 4, 8, or 15 months. Positive controls: Sod1 -/- for MLKL oligomer blot. *represents non-specific bands. Bottom: Graphical representation of quantified immunoblots for MLKL normalized to β-tubulin (loading control). (E) Left: Immunoblots showing expression of GPX4 and Ponceau stain (loading control) in the livers of control and Mlkl HepKO mice 15 months post-diet feeding. Right: Graphical representation of quantified GPX4 blot normalized to Ponceau. (F) Transcript levels of inflammatory cytokines TNFa, IL1 β, and IL6, and chemokines CCL2, CXCL9, CXCL10 in the livers of control and Mlkl HepKO mice 15 months post diet feeding. In C-F, white bars represent CD-fed mice; beige, coral, or red bars: mice fed WD for 4, 8, or 15 months, respectively. n=4-9 mice per group. Data represented as mean + SEM. Data points marked with asterisks (*) indicate statistically significant differences comparing the mean of each group with that of every other group. (A) Two-tailed unpaired t-test, (C-F) One-way ANOVA P< 0.05; *p<0.05, **p< 0.01, ***p<0.001. To investigate whether loss of MLKL shifts cell death toward alternative pathways, we examined markers of apoptosis, pyroptosis, and ferroptosis in livers of 15-month CD and WD-fed mice. Cleaved PARP/PARP ratio, a marker of apoptosis, was significantly reduced in WD-fed Mlkl HepKO mice compared to WD-fed control mice, whereas levels of the pro-apoptotic protein BAX were similar between the groups ( Figure S1F ). Pyroptotic signaling, assessed by cleaved-gasderminD (GSDMD)/GSDMD ratio, was not different between WD-fed control and Mlkl HepKO mice; however, CD-fed Mlkl HepKO mice displayed a significant increase compared to CD-fed controls ( Figure S1F ). The ferroptosis marker GPX4, which was significantly elevated by WD feeding in control mice (2-fold), was further increased in the absence of MLKL (3.4-fold) ( Figure 1E ). In 15-month WD-fed control mice, transcript levels of proinflammatory cytokine ILβ, but not TNFα or IL6, were upregulated, whereas chemokines (CCL2, CXCL9, CXCL10) were significantly elevated. Strikingly, MLKL knockdown did not alter these inflammatory responses, except for TNFα and CCL2 that were elevated ( Figure 1F ). Comparable results were observed at 8 months of WD feeding ( Figure S1G ). Consistent results were obtained when the protein levels of these cytokines and chemokines were measured ( Figure S1H ). Collectively, these findings suggest that hepatocyte MLKL deficiency does not engage compensatory apoptotic or pyroptotic programs but instead induces a cellular defense mechanism against ferroptotic stress while leaving WD-induced hepatic inflammation largely unaffected. Hepatocyte MLKL deficiency enhances lipid droplet storage without altering WD-induced liver injury or fibrosis H&E staining of liver sections showed that WD induced steatosis in both genotypes, however, WD-fed Mlkl HepKO mice exhibited significantly larger lipid droplets at 4 months and 8 months, but this difference was reduced at 15 months post diet feeding ( Figures 2A , S2A ). Quantification of hepatic triglycerides showed similar WD-induced increases in both control and Mlkl HepKO mice at 4- and 8-months post diet feeding. However, at 15 months post diet feeding, while WD-fed control mice did not show an increase in liver triglycerides, Mlkl HepKO mice exhibited significantly higher triglyceride accumulation ( Figure 2B ). Consistent with increased lipid storage, lipid droplet-associated protein Perilipin 2 (Plin2) was markedly elevated in WD-fed Mlkl HepKO mice livers (1.6-fold at 8 months and 2-fold at 15 months), compared with WD-fed controls ( Figure S2B ). Additionally, levels of CD36 and FATP2 (fatty acid uptake), CPT1a (fatty acid oxidation), and ACC, SCD1, and PPARγ (lipid synthesis and storage) were significantly upregulated in WD-fed Mlkl HepKO mice livers compared to WD-fed controls at 15 months post diet feeding ( Figure 2C ). To assess the hepatocyte-intrinsic effects of MLKL on lipid metabolism, MLKL was knocked down in mouse AML12 hepatocytes (80% KD; Figure S2C ) and steatosis was induced. MLKL knockdown increased lipid accumulation assessed by Oil Red O staining ( Figure S2D ) and upregulated fatty acid transport (CD36, FATP5), lipid droplet formation/storage (Plin2, Cidec), and fatty acid utilization/oxidation genes (Pnpla2, LIPE, Cpt1a) ( Figure S2E ). Download figure Open in new tab FIGURE 2. Data from liver tissues of control and Mlkl HepKO mice fed CD or WD: (A) Top: Representative images of H&E-stained liver sections (10X objective; n=3 per group), Bottom: Mean lipid droplet surface area (μm 2 ) (n=3 per group). (B) Quantification of triglyceride levels. (C) Transcript levels of genes involved in: Fatty acid transport ( CD36 , FATP2 ); Fatty acid oxidation ( Cpt1a ), and Fatty acid synthesis and storage ( Srebp1, ACC1, SCD1, PPARγ) , 15 months post diet feeding. (D ) Representative PSR staining of liver sections [(n=3 per group), 10X objective]. (E) Estimation of total hydroxy proline content (μg/g) (n=5-8 per group). (F) Serum ALT levels (IU/L). White bars represent CD-fed mice; beige, coral, or red bars represent Mlkl HepKO mice fed WD for 4, 8, or 15 months, respectively. Data represented as mean +SEM. Data points marked with asterisks (*) indicate statistically significant differences comparing the mean of each group with that of every other group. One-way ANOVA P< 0.05; *p<0.05, **p< 0.01, ***p<0.001. Assessment of fibrosis by picrosirius red (PSR) staining and hydroxy proline assay of liver tissues at 4-, 8-, and 15-months post diet feeding showed similar levels of fibrosis in WD-fed control and Mlkl HepKO mice ( Figure 2D , 2E ). Transcript levels of fibrosis-related genes followed a similar pattern ( Figure S2F ). Additionally, analysis of plasma ALT levels, a marker of liver injury, showed comparable elevations in WD-fed control and Mlkl HepKO mice ( Figure 2F ). Overall, these data indicate that while loss of hepatocyte MLKL does not influence WD-driven fibrosis or liver injury, it exacerbates steatosis through coordinated dysregulation of lipid uptake, storage, and oxidation pathways. Hepatocyte MLKL deficiency reduces WD-induced HCC Gross inspection revealed multiple large tumors in WD-fed control mice, whereas Mlkl HepKO mice developed fewer and smaller tumors ( Figure 3A ). Quantification confirmed a higher tumor incidence in control mice (7 out of 8 mice) compared to Mlkl HepKO mice (3 out of 8 mice) ( Figure 3B ). Similar results were observed for tumor multiplicity, i.e. number of tumors per mouse ( Figure 3C ). While the total number of tumor foci was comparable between the two groups, the numbers of both small (1-4 mm) and large (>5 mm) tumors were significantly reduced in Mlkl HepKO mice ( Figure S3A ). These data suggest that hepatocyte MLKL deficiency does not prevent the initiation of tumors but significantly impairs their growth and progression. Download figure Open in new tab FIGURE 3. Data from control or Mlkl HepKO mice fed WD for 15 months: (A) Representative images of liver tumors (white arrows). Graphical representation of tumor incidence (B) tumor multiplicity (C). (D) Plasma AFP levels (n= 6-8 mice per group). (E) Left: Representative IHC images for Ki67 staining; Right: Quantified Ki67 positive cells per 10x field. Dark brown spots in tumor regions represent positive stains for Ki-67 (20x objective; Scale Bar: 200 μm); dotted lines separate tumor from non-tumor region. (F) Left: Immunoblots showing levels of cancer stem cell markers DCKL1 (long or short isoform) and Ponceau (loading control) ( top ) and OCT4 (middle) and β-actin (loading control) ( bottom ); Right : Graphical representation of quantified blots normalized to Ponceau or β-actin. White or red bars represent CD or WD-fed mice, respectively, for 15 months in control or Mlkl HepKO mice. Data represented as mean +SEM. Data points marked with asterisks (*) indicate statistically significant differences comparing the mean of each group with that of every other group. (E) Two-tailed unpaired t-test, (B-D, F) One-way ANOVA P< 0.05. *p<0.05, **p< 0.01, ***p<0.001. Consistent with gross and histological findings, levels of alpha-fetoprotein (AFP), a clinically relevant biomarker of HCC, were significantly elevated in WD-fed control mice (7.3-fold), whereas Mlkl HepKO mice showed markedly reduced AFP ( Figure 3D ). Immunostaining revealed robust staining of Glypican-3 (GPC3, a diagnostic marker of HCC) in tumors from WD-fed control mice, while its expression was substantially reduced in tumors from Mlkl HepKO tumors mice ( Figure S3B ). Ki67 staining revealed that Mlkl HepKO tumors had markedly fewer Ki67-positive nuclei than controls, with quantification confirming a significant reduction in proliferating cells ( Figure 3E ). Additionally, expression of cancer stem cell markers DCLK1 and OCT4 were significantly reduced in Mlkl HepKO livers compared to livers from control mice ( Figure 3F ). Together, these findings demonstrate that hepatocyte MLKL promotes WD-induced hepatocarcinogenesis by enhancing tumor growth, proliferation, and stemness, whereas its deficiency protects against HCC progression. MLKL promotes proliferation and clonogenicity in liver cancer cells and is elevated in human HCC Effect of MLKL on tumor cell proliferation and stemness was further validated using the human liver cancer cell line HepG2. MLKL protein levels were elevated in HepG2 cells compared to the normal human liver cell line THLE2 ( Figure 4A ). Across multiple human liver cancer cell lines and a mouse liver cancer cell line (Hepa1-6), MLKL was expressed at varying levels, whereas RIPK3 was undetectable ( Figure S4A ). MLKL knockdown in HepG2 cells achieved nearly 90% reduction in protein expression ( Figure 4B ), and RNA-seq analysis of HepG2 cells revealed that MLKL knockdown reduced pathways associated with cell cycle ( Figure 4C ), whereas pathways associated with tissue organization and immune responses were upregulated ( Figure S4B ). Flow cytometric analysis confirmed that MLKL knockdown increased the proportion of cells in S phase while reducing cells in G2/M phase ( Figure S4C ). Functionally, MLKL knockdown in HepG2 cells significantly suppressed cell proliferation, compared to control cells. Similar results were obtained with pharmacological inhibition using the human MLKL inhibitor necrosulfonamide (NSA) ( Figure 4D ). Colony formation assay demonstrated that MLKL knockdown markedly reduced both the number and size of colonies, effects that were recapitulated with NSA treatment ( Figures S4D, S4E ). Similar results were obtained by spheroid formation assay ( Figure 4E , S4F ). Download figure Open in new tab FIGURE 4. (A) Immunoblot showing the levels of MLKL and β-actin (loading control) in normal human liver cell line (THLE2) and HCC cell line (HepG2). (B-G) Data from Control siRNA (siControl) or MLKL siRNA (siMLKL) treated HepG2 cells: (B) Left : Immunoblots of MLKL and β-actin in siControl or siMLKL HepG2 cells; Right : Graphical representation of quantified blot normalized to β-actin, siControl (blue) or siMLKL (red). (C) Schematic representation of significantly downregulated pathways obtained from RNA seq analysis in siMLKL compared to siControl HepG2 cells. (D) Left : Representative images from Incucyte live-cell imaging show changes in cell confluence from 0-96 hours. Top : siControl or siMLKL cells, Bottom : DMSO (vehicle) or necrosulfonamide (NSA, 5μM) treated HepG2 cells; Right : Proliferation curves representing cell confluency (in percentage) from time 0-96h. siControl (black line) or siMLKL or NSA (red line). (E) Left : Representative images of spheroids on day 10 in siControl or siMLKL HepG2 cells; Right : Real-time spheroid growth monitoring curve representing spheroid area (in μm 2 ) over 10 days. Control (black line) or Mlkl knockdown (red line). (F) Representative IHC images of human liver tissue microarray showing MLKL expression in normal liver or HCC tumors. Black arrows represent positive staining for MLKL. (G) Kaplan-Meier curve showing the survival probability of liver cancer patients with either low (black line) or high (red line). Data represented as mean + SEM. Two-tailed unpaired; P< 0.05. *p<0.05, **p< 0.01, ***p<0.001. To extend these findings to human HCC, analysis of The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) datasets revealed that MLKL expression is significantly upregulated in primary HCC tumors compared to adjacent normal tissue ( Figure S4G ). Immunohistochemical staining of patient samples confirmed elevated MLKL protein within tumor regions relative to surrounding non-tumor tissue ( Figure 4F ). Kaplan-Meier survival analysis demonstrated that patients with high MLKL expression had significantly worse overall survival than those with low MLKL expression ( Figure 4G ). Thus, MLKL promotes hepatocyte proliferation, stemness, and clonogenicity in vitro , is upregulated in human HCC, and correlates with poor patient survival, underscoring its role as a driver of tumor progression. Loss of MLKL restores the mitochondrial fusion and tumor-suppressor protein Mfn2 and enhances mitochondrial respiratory capacity Transcriptomic profiling of 15-month WD-fed livers revealed distinct genotype-specific clustering, with Mlkl HepKO mice showing broad transcriptomic remodeling compared to controls ( Figure 5A ). Pathway enrichment analysis revealed significant upregulation of oxidative phosphorylation, respiratory electron transport, mitochondrial translation, and complex I biogenesis in Mlkl HepKO livers compared to WD-fed controls ( Figure 5B ). Conversely, pathways related to immune and inflammatory responses, including cytokine storm signaling, T cell receptor signaling, and neutrophil extracellular trap formation, were reduced ( Figure 5B ). Upstream regulator analysis and causal network predictions are provided in Supplementary Results ( Figures S5A-D ). To test whether enhanced mitochondrial pathway activation in WD-fed Mlkl HepKO livers reflected increased mitochondrial biogenesis, mtDNA copy number ( Figure 5C ) and electron transport chain protein abundance ( Figure S5E ) were assessed and found to be unchanged between control and Mlkl HepKO mice under both CD and WD conditions. Download figure Open in new tab FIGURE 5. (A-D) Data from livers of control or Mlkl HepKO mice fed either CD or WD for 15 months: (A) Heat map showing differential gene expression; Red and blue colors represent genes that are significantly upregulated or downregulated. (B) Graphical representation of canonical pathways which are significantly upregulated (red) or suppressed (blue). (C) Mitochondrial DNA copy number. CD (white) and WD (red). (D) Left : Immunoblots showing P-Drp1 (Ser616), Drp1, Fis1, Mfn2, Mfn1, OPA1 and Ponceau (loading control). Right: Quantification of P-Drp1 (Ser616)/ Drp1, and Fis1, Mfn2, Mfn1, and OPA1 normalized to ponceau. (E) Left : Immunoblots showing Mfn2, Mfn1, and Ponceau (loading control) in siControl and siMLKL AML12 cells treated with BSA (control) or BSA-PA:OA (250:125μM) for 48 hours. Right: Quantification of the Mfn2 and Mfn1 expression. (F) Mitochondrial respiration analysis in siControl (blue) or siMLKL (red) HepG2 cells. Left: Representative oxygen consumption rate (OCR) curve, with sequential addition of oligomycin, FCCP, and rotenone/antimycin A. Right : Bar graph summarizing the quantified respiratory parameters; siControl (blue) and siMLKL (red). Data are represented as mean ± SEM. Statistical significance was determined by One-way ANOVA (D) or two-tailed unpaired t-test (F); P-value <0.05; *p<0.05, **p< 0.01, ***p<0.001. Analysis of the expression of mitochondrial fission/fusion proteins that regulate mitochondrial dynamics showed that in control livers WD significantly increased phospho-DRP1 (p-Drp1, Ser616)/Drp1 ratio, a marker of fission, and reduced mitofusin 2 (Mfn2), a marker of fusion. Notably, in WD-fed Mlkl HepKO livers, P-Drp1/Drp1 was significantly downregulated while Mfn2 was significantly upregulated compared with WD-fed controls ( Figure 5D ). While fission protein, fission mitochondrial 1 (Fis1) or fusion proteins Mitofusin 1(Mfn1) or Optic Atrophy 1(OPA1) were not impacted by WD in CD-fed mice livers, absence of hepatocyte MLKL significantly reduced Fis1 and Mfn1 levels ( Figure 5D ). Consistent with this, electron microscopy images showed significantly altered mitochondrial structure on CD- and WD-fed Mlkl HepKO mice livers ( Figure S5F ). In AML12 hepatocytes, fatty acid treatment reduced Mfn2 protein, but not Mfn1, and MLKL knockdown preserved Mfn2 expression( Figure 5E ) . Additionally fatty acid treatment reduced P-Drp1/Drp1 ratio and OPA1 protein in fatty acid treated MLKL knockdown cells compared to control cells ( Figure S5G ). Analysis of Mfn2 transcript levels showed that WD feeding did not alter Mfn2 expression in control mice livers. In contrast, Mlkl HepKO mice exhibited a significant reduction in hepatic Mfn2 transcripts following WD feeding. Consistent with the in vivo findings, MLKL knockdown AML12 hepatocytes treated with fatty acids also displayed reduced Mfn2 expression. ( Figure S5H ). To assess the impact of MLKL knockdown on mitochondrial respiration, we performed a Seahorse XF Cell Mito Stress Test in HepG2 cells transfected with control or MLKL siRNA. MLKL knockdown markedly increased OCR following FCCP treatment, indicating improved maximal respiratory capacity, and significantly elevated basal, ATP-linked, spare respiratory, and non-mitochondrial respiration ( Figure 5F ). ECAR was reduced in MLKL-deficient cells ( Figure S6A ), consistent with a metabolic shift from glycolysis toward oxidative metabolism. Similar increases in OCR were observed with the MLKL inhibitor NSA ( Figure S6B ). Together, these findings demonstrate that loss of MLKL upregulates the tumor-suppressor protein Mfn2, potentially promoting mitochondrial fusion, enhancing oxidative metabolism, and reducing glycolysis, revealing a previously unrecognized role for MLKL in regulating mitochondrial function under metabolic stress. DISCUSSION Our findings identify a hepatocyte-intrinsic, non-necroptotic role for MLKL in MASLD-driven HCC. Although WD robustly increased hepatic MLKL, canonical necroptosis was not detected (absence of P-MLKL/oligomers). Hepatocyte MLKL loss did not alter WD-induced cytokines, fibrosis, or liver injury, but increased lipid droplet accumulation and Plin2 protein, and notably reduced WD-induced HCC incidence, tumor size, stemness markers, and proliferation. MLKL knockdown and pharmacologic inhibition reduced clonogenicity and proliferation in HepG2 cells, and MLKL was elevated in human HCC and associated with poorer survival. These data position MLKL as a hepatocyte-intrinsic tumor-progression factor in metabolic liver cancer, independent of canonical necroptosis and MASH severity. Our results align with reports that hepatocytes and liver cancer cells express little RIPK3 due to hypermethylation, thereby blunting necroptosis [ 23 ] and that MLKL exerts non-canonical functions in membrane trafficking and metabolism, independent of RIPK3. [ 16 , 18 ] Prior studies across various liver-injury models support a pro-disease role for hepatocyte MLKL: in fibrogenesis models (CCl 4 or bile-duct ligation), knocking out MLKL in hepatocytes reduces hepatocyte death, limits inflammatory infiltration, and attenuate fibrosis. [ 24 ] In DEN-initiated carcinogenesis, hepatocyte MLKL loss suppresses HCC by relieving MLKL-mediated inhibition of autophagy and enabling tumor-restraining AMPK activation. [ 25 ] In ischemia–reperfusion injury, hepatocyte MLKL deficiency lowers ALT/AST and dampens intrahepatic inflammation, effects linked to enhanced mitophagy and reduced macrophage activation. [ 19 ] Our work extends these insights to a MASLD-driven HCC and is novel in showing that hepatocyte MLKL supports tumor growth and stemness under chronic dietary stress without necessarily amplifying inflammation or fibrogenesis. The dissociation between exacerbated steatosis and reduced tumor burden observed with MLKL loss, suggests that MLKL promotes cancer progression via cell-intrinsic signaling rather than by worsening steatohepatitis per se. Consistent with our hepatocyte data, macrophage-specific MLKL deletion increases lipid accumulation in plaques and foam cells in Apoe knockout mice [ 26 ] , supporting a model in which MLKL influences lipid trafficking. It is possible that in our system, greater lipid-droplet storage in Mlkl HepKO livers could buffer lipotoxic cues, uncoupling fat accumulation from mitogenic signaling that fuels HCC outgrowth. However, this hypothesis needs to be tested. Mechanistically, WD-fed Mlkl HepKO livers showed enrichment of mitochondria-associated pathways, while inflammation that is traditionally associated with MASLD-HCC progression was not significantly altered, suggesting a metabolic mechanism is dominant here. MLKL loss increased basal and maximal respiration in HepG2 cells, consistent with prior reports in hepatocytes. [ 18 , 27 ] This functional finding aligns with the observation that WD lowered Mfn2 expression in controls but not Mlkl HepKO livers. Mfn2 expression is frequently reduced in HCC, and lower Mfn2 levels correlate with poorer patient outcomes. [ 28 – 32 ] Mfn2-mediated fusion restrains proliferation and metastasis by promoting OXPHOS and limiting glycolysis. [ 33 ] We propose that MLKL promotes HCC progression by reducing Mfn2 protein in hepatocytes, thereby suppressing mitochondrial fusion and oxidative capacity and favoring a glycolytic state permissive for tumor growth ( Figure 6 ). Although Mfn2 transcript levels were not altered by WD, WD selectively reduced Mfn2 protein, consistent with prior reports that Mfn2 is primarily regulated post-translationally. [ 34 ] Further studies are warranted to delineate the molecular mechanisms by which MLKL regulates MFN2 expression and to test whether restoring Mfn2 could possibly rescue mitochondrial defects, thus inhibiting MLKL-mediated tumorigenesis in obesity. In WD-driven MASLD, where necroptosis is inactive, loss of hepatocyte MLKL does not reroute death to apoptosis or pyroptosis. By contrast, GPX4 was elevated with WD and was further increased in Mlkl HepKO livers, the key enzyme that detoxifies lipid peroxides [ 35 ] , indicating greater lipid-peroxidation pressure and a compensatory anti-ferroptotic response. A possible explanation for GPX4 upregulation is that MLKL deficiency elevates mitochondrial respiration, increasing reactive oxygen species and mitochondrial lipid peroxidation, thereby further upregulating GPX4. Thus, MLKL loss did not simply substitute one cell death program for another, rather protected from ferroptosis-mediated cell death. GPX4 is frequently upregulated in HCC and often supports tumor metastasis and drug resistance by preventing lipid peroxidation-driven ferroptosis. In contrast, studies have also shown that overexpression of GPX4 in HCC cell lines reduced ROS, slowed xenograft growth, and correlated with better survival in patient datasets, suggesting context-dependent anti-tumor effects. [ 36 – 38 ] Finally, analysis of human cohorts and tissues underscores clinical relevance and a druggable axis. In TCGA LIHC and our patient specimens, MLKL is elevated in tumors versus adjacent liver and high expression correlates with poor survival. Functionally, the human-specific MLKL inhibitor NSA phenocopied genetic knockdown in HepG2 cells, suppressing proliferation and clonogenic growth and recapitulating the respiration phenotype. These findings suggest MLKL as both a biomarker and a therapeutic target in HCC. In summary, our data identify MLKL as a hepatocyte-intrinsic driver of tumor promotion in WD-driven MASLD, acting independently of canonical necroptosis, coupling mitochondrial dynamics/respiration to HCC progression. Consistent with this mechanism, both genetic loss-of-function and pharmacologic inhibition attenuate HCC cell proliferation, positioning MLKL as a druggable target. Limitations include reliance on terminal timepoints, absence of acute necroptosis flux measurements, and whole-liver readouts that cannot exclude contributions from non-parenchymal cells. Mechanistic resolution of how MLKL regulates mitochondrial dynamics, and how specific MLKL domains or post-translational modifications contribute, remain areas for future study. Additional work will require primary hepatocytes, cell-type-specific deletions, and PTM-resolved MLKL variants to define the scaffolding functions that underlie the non-canonical metabolic activities of MLKL, and to determine whether restoring Mfn2 rescues MLKL-dependent tumor phenotypes. ACKNOWLEDGMENTS The authors would like to acknowledge Prof. James Murphy, Walter and Eliza Hall Institute of Medical Research for generously providing the Mlkl HepKO mice model used in the study. The H&E staining and PSR staining service provided by the Stephenson Cancer Center tissue pathology core, supported partly by the National Institute of General Medical Sciences Grant P30GM154635 and National Cancer Institute Grant P30CA225520 of the National Institutes of Health. The authors thank imaging core facility at Oklahoma Medical Research Foundation for the electron microscopy imaging. The Incucyte live cell imaging service provided by the cancer functional genomics core was supported partly by the National Institute of General Medical Sciences Grant P30GM154635 and National Cancer Institute Grant P30CA225520 of the National Institutes of Health. Funder Information Declared National Institutes of Health, https://ror.org/01cwqze88 , R01AG059718 , R03 CA262044 Harold Hamm Diabetes Center-Stephenson Cancer Center , Harold Hamm Diabetes Center-Stephenson Cancer Center Team Science Grant Oklahoma Center for Adult Stem Cell Research , Oklahoma Center for Adult Stem Cell Research grant United States Department of Veterans Affairs, https://ror.org/05rsv9s98 , IK6BX006033 National Institutes of Health, https://ror.org/01cwqze88 , P30GM149376 Footnotes FINANCIAL SUPPORT AND SPONSORSHIP: This work was supported by NIH grants R01AG059718 and R03 CA262044, the Harold Hamm Diabetes Center-Stephenson Cancer Center Team Science Grant, and the Oklahoma Center for Adult Stem Cell Research (OCASCR) grant to Sathyaseelan S Deepa; VA Research Career Scientist award (IK6BX006033) to Willard M Freeman; Jonathan D Wren and Constantin Georgescu were supported by NIH grant P30GM149376. CONFLICTS OF INTEREST: The authors have no conflicts of interest to report. LIST OF ABBREVIATIONS: AAV8, Adeno-associated virus serotype 8; ACC, Acetyl-CoA carboxylase; AFP, alpha-fetoprotein; ALT, Alanine transaminase; AML12, Alpha mouse liver 12; AMPK, AMP-activated protein kinase; BAX, Bcl-2-associated X protein; Cidec, Cell Death Inducing DFFA Like Effector C; Cpt1a, Carnitine palmitoyltransferase 1a; DAMPs, Damage-associated molecular patterns; DCLK1, Doublecortin Like Kinase 1; ETC, Electron transport chain; FATP, Fatty Acid Transport Protein; Fis1, Mitochondrial fission 1 protein; GPC3, Glypican-3; GPX4, Glutathione peroxidase 4; HCC, Hepatocellular carcinoma; LIPE, Lipase E; MASH, Metabolic dysfunction-associated steatohepatitis; MASLD, Metabolic dysfunction-associated steatotic liver disease; Mfn2, Mitofusin 2; MLKL, Mixed lineage kinase domain-like protein; NSA, Necrosulfonamide; OCR, Oxygen Consumption Rate; PARP, Poly(ADP-ribose) polymerase; PLIN1, Perilipin 1; Pnpla, Patatin-like phospholipase domain containing 2; PPAR, Peroxisome proliferator-activated receptor; PSR, Picro-Sirius Red Stain; PTM, Post-Translational Modifications; RIPK, Receptor interacting serine/threonine kinase; TBG, Thyroxin Binding Globulin; TCGA-LIHC, The Cancer Genome Atlas Liver Hepatocellular Carcinoma; WD, Western diet. 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Share Hepatocyte MLKL Drives Obesity-Driven Hepatocellular Carcinoma Progression via Mitochondrial Dysfunction Independent of Necroptosis in MASLD Phoebe Ohene-Marfo , Sabira Mohammed , Chao Jiang , Shylesh Bhaskaran , Constantin Georgescu , Megan John , Kevin Pham , Albert Tran , Chinthalapally V Rao , Tae Gyu Oh , Michael Kinter , Willard M Freeman , Courtney Houchen , Jonathan D Wren , Sathyaseelan S. Deepa bioRxiv 2025.11.26.690789; doi: https://doi.org/10.1101/2025.11.26.690789 Share This Article: Copy Citation Tools Hepatocyte MLKL Drives Obesity-Driven Hepatocellular Carcinoma Progression via Mitochondrial Dysfunction Independent of Necroptosis in MASLD Phoebe Ohene-Marfo , Sabira Mohammed , Chao Jiang , Shylesh Bhaskaran , Constantin Georgescu , Megan John , Kevin Pham , Albert Tran , Chinthalapally V Rao , Tae Gyu Oh , Michael Kinter , Willard M Freeman , Courtney Houchen , Jonathan D Wren , Sathyaseelan S. 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