idh-1 neomorphic mutation confers sensitivity to vitamin B12 via increased dependency on one-carbon metabolism in Caenorhabditis elegans

idh-1 neomorphic mutation confers sensitivity to vitamin B12 via increased dependency on one-carbon metabolism in Caenorhabditis elegans | 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 idh-1 neomorphic mutation confers sensitivity to vitamin B12 via increased dependency on one-carbon metabolism in Caenorhabditis elegans View ORCID Profile Olga Ponomarova , Alyxandra N. Starbard , Alexandra Belfi , Amanda V. Anderson , Meera V. Sundaram , View ORCID Profile Albertha J.M. Walhout doi: https://doi.org/10.1101/2024.03.13.584865 Olga Ponomarova 1 Department of Systems Biology, University of Massachusetts Chan Medical School , Worcester, MA, USA 2 Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine , Albuquerque, NM, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Olga Ponomarova For correspondence: oponomarova{at}salud.unm.edu marian.walhout{at}umassmed.edu Alyxandra N. Starbard 1 Department of Systems Biology, University of Massachusetts Chan Medical School , Worcester, MA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alexandra Belfi 3 Department of Genetics, University of Pennsylvania Perelman School of Medicine , PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amanda V. Anderson 2 Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine , Albuquerque, NM, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Meera V. Sundaram 3 Department of Genetics, University of Pennsylvania Perelman School of Medicine , PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Albertha J.M. Walhout 1 Department of Systems Biology, University of Massachusetts Chan Medical School , Worcester, MA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Albertha J.M. Walhout For correspondence: oponomarova{at}salud.unm.edu marian.walhout{at}umassmed.edu Abstract Full Text Info/History Metrics Preview PDF Abstract The isocitrate dehydrogenase neomorphic mutation ( idh-1neo ) generates increased levels of cellular D-2-hydroxyglutarate (D-2HG), a proposed oncometabolite. However, the physiological effects of increased D-2HG and whether additional metabolic changes occur in the presence of an idh-1neo mutation are not well understood. We created a C. elegans model to study the effects of the idh-1neo mutation in a whole animal. Comparing the phenotypes exhibited by the idh-1neo to Δdhgd-1 (D-2HG dehydrogenase) mutant animals, which also accumulate D-2HG, we identified a specific vitamin B12 diet-dependent vulnerability in idh-1neo mutant animals that leads to increased embryonic lethality. Through a genetic screen we found that impairment of the glycine cleavage system, which generates one-carbon donor units, exacerbates this phenotype. Additionally, supplementation with an alternate source of one-carbon donors suppresses the lethal phenotype. Our results indicate that the idh-1neo mutation imposes a heightened dependency on the one-carbon pool and provides a further understanding how this oncogenic mutation rewires cellular metabolism. Introduction Increased levels of D-2-hydroxyglutarate (D-2HG), a metabolite derived from the structurally similar hub metabolite alpha-ketoglutarate (αKG), are associated with multiple disorders indicating that tight regulation of this metabolite is important ( 1 )( 2 )). For instance, D-2-hydroxyglutaric aciduria, a rare inborn error of metabolism (IEM), is associated with elevated D-2HG levels due to loss-of-function mutations in the D-2HG dehydrogenase enzyme ( 3 ). This IEM often results in neurological dysfunctions and delayed development. Previously, we found that loss of the C. elegans D-2HG dehydrogenase dhgd-1 causes a high rate of embryonic lethality due to reduced ketone body production ( 4 ). Additionally, we found that dhgd-1 activity is necessary for the regulation of the propionate shunt, an alternate vitamin B12-independent breakdown pathway for this short chain fatty acid (Watson et al., 2016). In this shunt, the enzymes DHGD-1 and HPHD-1 are coupled via D-2HG metabolism: HPHD-1 transfers a hydride from 3HP to α-ketoglutarate (αKG), producing D-2HG, while DHGD-1 oxidizes D-2HG back to αKG ( 4 , 5 ). The propionate shunt is transcriptionally repressed in the presence of vitamin B12 ( 6 ). Vitamin B12 rescues the embryonic lethality of Δdhgd-1 mutants by generating energy via the canonical propionate degradation pathway, alleviating the need for ketone bodies to distribute an energy source across tissues ( 4 ). D-2HG is also known as an oncometabolite and is linked to various cancers. D-2HG accumulates due to neomorphic mutants of either one of two isocitrate dehydrogenase (IDH) enzymes (IDH1 and IDH2). These mutations primarily affect catalytic arginine residues ( 7 – 9 ) and are associated with the development of cancers such as glioma, cholangiocarcinoma, and AML ( 10 – 12 ). Neomorphic mutations in IDH1 and IDH2 enzymes lead to abnormal D-2HG production from αKG ( 13 ), thereby disrupting cell function. Effects of D-2HG are multifaceted and can drive cancer development by several different mechanisms ( 1 , 14 ). D-2HG acts as a potent competitive inhibitor of αKG-dependent enzymes, including histone demethylases and hypoxia-inducible factor (HIF) prolyl hydroxylase, often leading to dysregulated oncogene expression ( 15 ). Abnormal D-2HG production also disturbs the balance between NADPH and NADP+, crucial for cellular redox equilibrium ( 16 ). This disruption can cause oxidative stress, leading to DNA damage. High levels of D-2HG also have been shown to inhibit succinate dehydrogenase ( 17 ) and αKG-dependent transaminases ( 16 ), disrupt chromosomal topology ( 18 ), and activate the mTOR pathway ( 19 ). D-2HG also affects the immune system, particularly T cells, potentially creating a tumor-friendly environment by suppressing immune response. Malignant cells with IDH mutations release D-2HG, which can suppress T-cell function by inhibiting lactate dehydrogenase and disrupting other metabolic pathways ( 20 – 22 ). While many effects of D-2HG are well-documented, the complete implications of dysregulated D-2HG metabolism remain unclear. Its versatile effects range from supporting oncometabolism to causing developmental and psychomotor defects in D-2-hydroxyglutaric aciduria patients. Understanding the diverse toxic effects of D-2HG is crucial for unraveling disease progression mechanisms and developing new treatments. To gain a better understanding of how D-2HG impacts cellular metabolic function, we generated C. elegans idh-1neo mutant animals to use as a comparative model for studying the effects of increased D-2HG levels. We find that while some shifts in metabolism are shared with what we found previously in our studies of Δ dhgd-1 mutant animals, differences exist. These differences led us to uncover a unique diet-dependent, vitamin B12-induced vulnerability in idh-1neo mutant animals. Whereas vitamin B12 rescues embryonic lethality in Δ dhgd-1 mutant animals, it exacerbates lethality of idh-1neo mutant animals. We find that this difference is due to decreased one carbon metabolism in idh-1neo mutant animals. Overall, our results provide a further understanding of how the idh-1neo oncogenic mutation may rewire cellular metabolism. Results C. elegans with neomorphic idh-1 mutation accumulate D-2HG We previously found that when the function of C. elegans D-2HG dehydrogenase dhgd-1 is disrupted, there is a marked increase in D-2HG levels in the animal ( Figure 1A , Model 1 ) ( 4 ). Seeking to further understand the metabolic implications of D-2HG accumulation, we aimed to increase D-2HG levels through a distinct mechanism; by introducing an exogenous D-2HG-producing enzyme ( Figure 1A , Model 2 ). Neomorphic mutations in IDH, whether cytosolic (IDH1) or mitochondrial (IDH2), alter their function. Rather than converting isocitrate to αKG, these mutated enzymes convert αKG to D-2HG ( Figure 1B ). We separately introduced four idh alleles with missense mutations that mirror human variants into C. elegans ( Figure 1C, D ), while keeping the endogenous wild type idh genes intact. Using these mutant strains, we assessed 2HG (D- and L-2HG) accumulation in the animals by gas chromatography - mass spectrometry (GC-MS). While neither idh-2neo allele resulted in 2HG accumulation, animals harboring an idh-1neo allele did show an increased accumulation of 2HG, with the highest levels found in animals expressing the idh-1 (R156C) allele ( Figure 1E ). Approximately 50% of idh-1 (R156C) animals also showed developmental abnormalities, including dilations in the excretory system, larval lethality, and a smaller proportion of embryonic lethality, suggesting systemic disruptions to animal physiology ( Figure S1 , Table S1 ). Therefore, we chose this strain for detailed exploration, and hereafter refer to it as idh-1neo . Download figure Open in new tab Figure 1. Introduction of exogenous idh-1 with neomorphic mutation leads to D-2HG accumulation in C. elegans A. Models of D-2HG accumulation. Mutation in D-2-hydroxyglutarate dehydrogenase dhgd-1 prevents D-2HG recycling (Model 1). Neomorphic mutation in isocitrate dehydrogenase 1 ( idh-1neo ) creates a new source of D-2HG (Model 2). B. Reactions catalyzed by wild type and neomorphic IDH. C. Protein sequences of IDH1 and IDH2 from human and C. elegans . Alignment was performed using Clustal Omega software. Blue color highlights conserved residues. Arginine residues homologous to those that typically mutate in human cancers are shown in pink. D. Amino acid substitutions in C. elegans IDH-1 and IDH-2 that correspond to common human cancer-associated mutations. E. GC-MS quantification of 2HG (D- and L-2HG) in C. elegans expressing neomorphic idh-1 and idh-2 . Boxplot midline represents median of independent biological replicates (dots). F. idh-1neo animals predominantly accumulate the D isoform of 2HG. D- and L-2HG enantiomers were measured by GC-MS after chiral derivatization. 2HG exists as either the D-2HG or L-2HG enantiomer, and neomorphic IDH mutations cause production of D-2HG ( 23 ). Using a specific derivatization technique to distinguish 2HG enantiomers ( 24 ), we confirmed that idh-1neo animals accumulate D-2HG ( Figure 1F ). These combined results show that we have generated a new mode of D-2HG accumulation in C. elegans , distinct from that in Δ dhgd-1 animals ( 4 ). Using these two models we went on to further understand the metabolic implications of D-2HG accumulation. Vitamin B12 supplementation increases D-2HG levels and causes embryonic lethality in idh-1neo mutants We next investigated whether metabolites other than D-2HG change in abundance in idh-1neo animals. GC-MS metabolomics revealed that, much like Δ dhgd-1 animals, idh-1neo animals exhibited elevated levels of 3-hydroxypropionate (3HP) and β-alanine, along with reduced levels of αKG and glutamate ( Figure 2A ) ( 4 ). Download figure Open in new tab Figure 2. Vitamin B12 increases D-2HG levels and induces embryonic lethality in idh-1neo C. elegans A. GC-MS profiling of metabolic changes in idh-1neo C. elegans compared to wild type (WT) animals. P-values are Benjamini–Hochberg adjusted. B. Schematic of the propionate degradation shunt pathway. C. GC-MS profiling of metabolic changes in idh-1neo C. elegans supplemented with vitamin B12 compared to non-supplemented idh-1neo animals. P-values are Benjamini– Hochberg adjusted. D. 3HP abundance in idh-1neo mutants with and without supplemented vitamin B12. E. Brood size and hatching rate of idh-1neo mutants. Bars represent mean and standard deviation of three biological replicates. F. Schematic of predicted IDH-1neo, HPHD-1, and DHGD-1 contributions to D-2HG accumulation. IDH-1neo is an introduced and HPHD-1 is an endogenous source of D-2HG. DHGD-1 recycles D-2HG by converting it to αKG. In idh-1neo animals, vitamin B12 causes an increase in D-2HG accumulation by repressing the expression of dhgd-1 , which encodes the enzyme that converts D-2HG into αKG in the propionate shunt. Vitamin B12 also represses the expression of hphd-1 , which is the enzyme that generates D-2HG in the propionate shunt. G. 2HG abundance in idh-1neo mutants with and without supplemented vitamin B12. H. 2HG abundance in idh-1neo mutants upon RNAi of dhgd-1 with and without supplemented vitamin B12. I. Embryonic lethality of idh-1neo mutants upon RNAi of dhgd-1 with and without supplemented vitamin B12. All panels show data for idh-1neo animals on a diet of E. coli OP50 or RNAi competent E. coli OP50 (xu363). Boxplot midline in panels C, F and G represents median of independent biological replicates (dots). In Δ dhgd-1 mutants, 3HP accumulates because D-2HG inhibits HPHD-1, an enzyme that produces D-2HG while oxidizing 3HP in the propionate shunt pathway ( 4 , 25 ) ( Figure 2B ). To determine if 3HP accumulation in idh-1neo also originates from this shunt pathway, we supplemented the animals with vitamin B12. Vitamin B12 transcriptionally inhibits the propionate shunt pathway while promoting the activity of the canonical, B12-dependent propionate degradation pathway ( 5 , 6 ). We reasoned that if suppressed HPHD-1 activity is the cause of 3HP accumulation, then inhibiting the entire shunt pathway should prevent it. Indeed, vitamin B12 supplementation led to reduced 3HP levels in idh-1neo animals, similar to Δ dhgd-1 mutant animals ( Figures 2C and D ). Interestingly, in contrast to Δ dhgd-1 mutant animals, vitamin B12 supplementation significantly increased the rate of embryonic but not larval lethality in the F1 generation of idh-1neo animals ( Figure 2E , Figure S1 , Table S1 ) ( 4 ). We hypothesized that since vitamin B12 suppresses the expression of the shunt pathway, which includes dhgd-1 , its supplementation may hinder DHGD-1 dependent D-2HG recycling, thereby further elevating D-2HG levels in idh-1neo animals ( Figure 2F ). Indeed, adding vitamin B12 to the diet of the idh-1neo significantly increased their D-2HG levels ( Figure 2G , Figure S2 ). To test this hypothesis further, we asked if suppressing dhgd-1 expression would elevate D-2HG in idh-1neo animals. As predicted, dhgd-1 RNAi was sufficient to drive further increase in D-2HG levels in idh-1neo animals ( Figure 2H ). Importantly, dhgd-1 RNAi also led to 100% penetrant embryonic lethality among the F1 generation of idh-1neo animals ( Figure 2I ). The opposite response to vitamin B12 supplementation highlighted key differences between the two models of D-2HG accumulation. The embryonic lethality observed in Δ dhgd-1 animals arises from a lack of energy source (ketone bodies) and can be rescued by vitamin B12, which activates an alternative energy production pathway ( 4 ). In contrast, embryonic lethality of idh-1neo animals is induced by vitamin B12 and cannot be mitigated by ketone body supplementation ( Figure S3 ). We therefore conclude that idh-1neo mutation causes embryonic lethality through a different molecular mechanism. Knockdown of the glycine cleavage system exacerbates lethality of idh-1neo animals supplemented with vitamin B12 To identify the molecular mechanism underlying the lethality of idh-1neo animals in the presence of vitamin B12, we conducted a reverse genetic screen. We used an RNAi library targeting 2,104 predicted metabolic genes ( 26 ) to identify those that are essential for idh-1neo animals but not required for wild-type C. elegans survival in the presence of B12 ( Figure 3A ). The screen identified five metabolic genes whose depletion is specifically lethal to idh-1neo animals ( Figure 3B ). Among these, two genes, T04A8.7 and W07E11.1, encode a glycogen branching enzyme and glutamate synthase, respectively. The other three identified genes - gldc-1 , gcst-1, and gcsh-1 – all belong to the glycine cleavage system (GCS)( 27 ). Given the strong enrichment for this pathway in our screen results, we next considered possible connections between GCS, vitamin B12, and idh-1neo . Download figure Open in new tab Figure 3. Metabolic RNAi screen for synthetic lethality with idh-1neo A. Experimental design to screen metabolic RNAi library for synthetic (embryonic) lethal interactions with idh-1neo . B. ‘Hits’ identified in metabolic RNAi library screen. *P-value < 0.05, compared to vector control. idh-1neo mutation confers sensitivity to perturbations of one-carbon metabolism The GCS breaks down glycine, thereby generating ammonia, carbon dioxide, and reducing NAD+ to NADH, while also methylating tetrahydrofolate, a one-carbon (1C) unit donor used for different biosynthetic reactions ( Figure 4A ). 1C metabolism, similar to the canonical propionate breakdown pathway, also depends on vitamin B12: in the methionine/S-adenosylmethionine (Met/SAM) cycle, METR-1 (methionine synthase) methylates homocysteine to regenerate methionine using vitamin B12 as a cofactor. The Met/SAM cycle utilizes 1C units provided by the enzyme methylenetetrahydrofolate reductase MTHF-1 ( 28 ) ( Figure 4A ). Both the GCS and the Met/SAM cycle influence the 1C pool of methylene tetrahydrofolate: GCS contributes to its synthesis, while the Met/SAM cycle utilizes it. Therefore, we hypothesized that idh-1neo animals are sensitive to depletion of the 1C pool ( Figure 4B ). To test this hypothesis, we supplemented B12-treated idh-1neo animals with formate, an alternative 1C donor ( 29 ). This supplementation restored the survival of idh-1neo embryos to wild-type levels on a regular diet of E. coli OP50 as well as the diet of RNAi-competent E. coli HT115 ( Figure 4C ) . Further, we posited that if vitamin B12 induces lethality in idh-1neo animals by depleting the 1C pool via its utilization in the Met/SAM cycle, then suppressing Met/SAM cycle genes in idh-1neo should prevent this depletion and restore availability of 1C units for other reactions ( Figure 4A ). Indeed, RNAi depletion of mthf-1 and sams-1 (S-adenosylmethionine synthetase) rescued the embryonic lethality of idh-1neo animals supplemented with vitamin B12 ( Figure 4D ). These findings demonstrate that lack of 1C units underlies the embryonic lethality observed in idh-1neo animals. Download figure Open in new tab Figure 4. idh-1neo animals are sensitive to perturbing one-carbon metabolism A. Glycine cleavage system contributes to a pool of one carbon units, and methionine/SAM cycle draws from this pool. B. Hypothesized interaction of idh-1neo with vitamin B12 and GCS via 1C pool. GCS or supplemented formate replenish 1C pool. Vitamin B12 depletes it by promoting Met/SAM cycle activity. C. Embryonic lethality of idh-1neo on vitamin B12 is rescued by supplementing formate on diets of E. coli OP50 and HT115. *P-value < 0.05, compared to no supplement condition. D. Suppressing Met/SAM cycle activity rescues embryonic lethality of idh-1neo supplemented with vitamin B12. *P-value < 0.05, compared to vector control. E. Model for the idh-1neo interaction with 1C metabolism. Discussion By comparing two models of D-2HG accumulation in C. elegans we have gained deeper insight into the metabolic perturbations caused by D-2HG in a whole animal. Similarities between Δdhgd-1 and idh-1neo include the perturbed function of the propionate shunt enzyme HPHD-1, evident from an increase in levels of its substrate 3HP. Other similarities include elevated β-alanine and reduced αKG and glutamate. The differences in the metabolic phenotypes of the two models include changes in lysine, 2-aminoadipate and glutarate levels, and can be linked to the compartmentalization of D-2HG production and the different subcellular origins of D-2HG: DHGD-1 recycles D-2HG produced by HPHD-1 in mitochondria, while IDH-1neo generates D-2HG in the cytosol. DHGD-1 dysfunction is thus more likely to affect mitochondrial enzymes while IDH-1neo may have a stronger impact on cytosolic metabolism. Consistent with this theory, 3HP levels, indicative of HPHD-1 inhibition, are several-fold higher in Δ dhgd-1 mutants than in idh-1neo animals. In further support of subcellular stratification, mitochondrial lysine degradation pathway intermediates (lysine and 2-aminoadipate) change levels in Δdhgd-1 mutants, but not in idh-1neo animals ( Figure 2A ). These lysine catabolism intermediates, however, become perturbed in idh-1neo when vitamin B12, a transcriptional suppressor of dhgd-1 , is supplemented ( Figure 2B ). 1C units in the form of methylated tetrahydrofolate are essential metabolic intermediates used for nucleotide biosynthesis and various methylation reactions ( 30 ). A lack of these building blocks results in embryonic lethality ( 29 ). Formate, a one-carbon donor exchanged between mitochondria and cytosol, has been demonstrated to rescue these detrimental effects ( 31 ). Our results show that idh-1neo C. elegans rely on GCS to supply one-carbon units. We propose that the metabolic rewiring caused by the idh-1neo mutation reduces the availability of methylated tetrahydrofolate. This limitation, in turn, causes sensitivity of idh-1neo to vitamin B12 and GCS knockdown, both of which can drain the 1C pool ( Figure 4E ). Wild-type C. elegans can generate 1C via cytosolic serine hydroxymethyltransferase MEL-32, whose loss causes embryonic lethality ( 27 , 28 , 32 ). Why would the MEL-32 route for 1C unit generation not be available in idh-1neo animals? One possibility is inhibition of this pathway through accumulated D-2HG. The phosphoglycerate dehydrogenase C31C9.2 functions upstream of MEL-32 ( 27 ), and its human ortholog PHGDH was found to produce D-2HG ( 33 ). A recent study demonstrated that D-2HG accumulation in Δdhgd-1 animals suppresses the activity of the D-2HG-producing enzyme HPHD-1 ( 25 ). A similar mechanism of end-product inhibition could cause the excess D-2HG produced by idh-1neo to suppress C31C9.2 activity, limiting the downstream generation of 1C by MEL-32. Overall, our results uncover metabolic perturbations induced by the idh-1neo mutation and highlight the differences in the pathogenicity mechanism of idh-1neo and Δdhgd-1 models. While both mutants accumulate D-2HG and incur embryonic lethality, the Δ dhgd-1 phenotype is caused by a lack of ketone bodies, while idh-1neo suffers from a 1C deficiency. Comparing the two models offers a unique tool for mechanistic insight. These findings may help navigate metabolic reprogramming that occurs in IDH-driven oncogenic transformations. For instance, future studies may explore 1C metabolism as a potential target in the therapy of cancers with the idh-1neo mutation. Methods Bacterial strains E. coli HT115, E. coli OP50 (xu363) ( 34 ) and E. coli OP50 from Caenorhabditis Genetics Center (CGC) were cultured overnight in Luria-Bertani Broth (Miller) at 37°C, plated and incubated overnight on assay plates before adding C. elegans larvae. For RNAi experiments E. coli HT115 was used, and assay plates were supplemented with µg/mL 50 ampicillin and 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). C. elegans cultures C. elegans strains were maintained at 20°C on nematode growth medium (NGM) seeded with E. coli HT115 or OP50. Supplements were added to NGM agar as specified. Vitamin B12 (adenosylcobalamin) was used at 64 nM throughout. N2 strain was obtained from CGC and mutant strains were constructed as described below. C. elegans strains used in this study View this table: View inline View popup Download powerpoint Constructing C. elegans strains Transgenic C. elegans strains with neomorphic mutations in idh-1 and idh-2 were created by inserting a mutated gene in an intergenic region on chromosome II at position 8420158..8420158 using Mos1-mediated single copy insertion (MosSCI) technique ( 36 ). We used expression of an added allele to ensure that endogenous idh-1 remains functional since wild type IDH1 activity was demonstrated to be necessary for efficient D-2HG production in cells with monoallelic neomorph mutations of IDH1 ( 37 ). Wild type idh genes, together with their promotor regions, were amplified from C. elegans genomic DNA using a high-fidelity polymerase. Neomorphic missense mutations were introduced using QuikChange Lightning site-directed mutagenesis kit (Agilent). C. elegans strain EG6699 with mos1 site on Chromosome II was used for a direct insertion. 50 animals in the L4/young adult stages were injected with a mix of vectors carrying transgene, Mos1 transposase and selection markers. Injection mix contained 2.5 µg/mL of pCFJ90 (Pmyo-2::mCherry), 5 µg/mL pCFJ104 (Pmyo-3::mCherry), 10 µg/mL pGH8 (Prab-3::mCherry), 50 µg/mL of pCFJ601 (Peft-3::Mos1 transposase), 10 µg/mL of pMA122 (Phsp16.41::peel-1), and pCFJ150 with mutated idh sequence. Progeny of individual P0 animals were allowed to starve at 25°C and heat shocked at 34°C for two hours in a water bath. After 4 hours of recovery at 20°C wild type moving animals without mCherry expression were picked onto individual plates. Resulting lines with full transmittance were verified for transgene integration by PCR. C. elegans synchronization Synchronized L1 populations were obtained by treating gravid adult animals with 1% sodium hypochlorite solution buffered with sodium hydroxide. Released embryos were washed with M9 buffer four times and incubated on a rocker for 18-20 hours. GC-MS metabolomics Targeted quantification of metabolites by GC-MS was done as described previously ( 4 ). Gravid adult animals were washed three times with filter-sterilized saline (0.9% NaCl). 50 µL of washed animal pellet were transferred into a FastPrep tube (MP Biomedicals), flash frozen in ethanol/dry ice bath and stored at −80°C. Samples were homogenized in 1 ml of 80% cold methanol with 0.5 ml of acid-washed glass beads (Sigma) using FastPrep24 bead beater (MP-Bio). Supernatant was cleared by centrifugation for 10 min at 10,000g. For each sample, 250 ul of cleared extract were transferred into a glass insert and dried under vacuum. Dry residues were derivatized with 20 μL of 20 mg/mL methoxyamine hydrochloride (Sigma-Aldrich) in pyridine for one hour at 37°C. This step was followed by adding 50 μL of N -methyl- N -(trimethylsilyl) trifluoroacetamide (Sigma-Aldrich) and a subsequent three-hour incubation at 37°C. After additional 5-hour room temperature incubation the samples were analyzed on an Agilent single quadrupole mass spectrometer 5977B coupled with gas chromatograph 7890B. HP-5MS Ultra Inert capillary column (30 m × 0.25 mm × 0.25 μm) was used with a constant 1 mL/min flow rate of helium gas. Temperature settings were as follows: inlet at 230°C, transfer line at 280°C, MS source at 230°C, and quadrupole at 150°C. A 1 μl sample was injected in split mode with a 5 mL/min split flow. The initial oven temperature was 80°C, rising to 310°C at a 5°C/min rate. MS parameters included 3 scans/s across a 30–500 m/z range and an electron impact ionization energy of 70 eV. Each metabolite’s identification relied on its retention time, a quantifier ion, and two qualifier ions, all manually selected using a reference compound. Peak integration and peak area quantification were executed using Agilent’s MassHunter software (v10.1). Blank subtraction and normalization relative to total quantified metabolites were performed using R software. Relative quantification of D- and L-2HG A previously published method ( 24 ) was adapted to differentiate the D-and L-enantiomers of 2HG. Initially, 300 μl of C. elegans metabolite extract were dried in glass inserts. 50 μl of R-(-)-butanol and 5 μl of 12N hydrochloric acid were then introduced into each insert and heated to 90°C for 3 hours. The samples were cooled to room temperature and extracted with 400 μl hexane. 250 μl of the organic phase were dried, the residue was re-suspended in 30 μl of pyridine and 30 μl of acetic anhydride and incubated for 1 hour at 80°C. The samples were dried once again, resuspended in 60 μl of hexane and immediately analyzed by GC-MS. The analytical method settings were identical to the targeted metabolomics method described above, with few modifications. The oven ramp was set from 80 to 190°C at a rate of 5°C/min and then to 280°C at 15°C/min. D- and L-2HG peaks were quantified using the 173 m/z ion. Brood size assay Animals in the L4 larval stage were singled on 35 mm petri dishes. Every 24 hours animals were moved to fresh plates until egg laying ceased. The remaining plates with embryos were incubated at 20°C for 24 hours. Subsequently, L1 larvae and unhatched embryos were counted. Brood counts from animals that died or left the plate were excluded. For each biological replicate data from at least seven animals were collected. The experiment was conducted three times. Hatching assay Approximately 30 synchronized L1 animals were placed on seeded 35 mm NGM agar plates. Animals were incubated at 20°C and allowed to lay eggs. Before eggs start hatching, adults were washed away and approximately 300 embryos were transferred onto new plates. After 24 hours of incubation hatched larvae and unhatched embryos were counted to determine the rate of embryonic lethality. Imaging Differential interference contrast (DIC) images were captured with a Zeiss Axioskop fitted with a Leica DFC360 FX camera. Confocal z-stacks were captured with a Leica TCS SP8 confocal microscope. Images were processed using ImageJ. RNAi screen RNAi clones of 2,104 C. elegans metabolic genes ( 26 ) were cultured in deep 96-well plates in LB (Miller) containing 50 µg/ml ampicillin and grown to stationary phase at 37°C. Cultures were concentrated 20-fold, and 15 µL were plated onto a shallow 96-well plate containing NGM agar supplemented with 64 nM vitamin B12 (adenosylcobalamin), 50 µg/ml ampicillin, and 1 mM IPTG. Plates were dried and stored overnight at 20°C. The next day 15 synchronized L1 animals were added to each well. Plates were screened for strong hatching defects on the fourth and fifth day of incubation at 20°C. The screen was performed three times. All hits were re-tested by performing a hatching assay on 35 mm NGM agar plates. Supplemental Figures and Tables Download figure Open in new tab Figure S1. Excretory system of idh-1neo animals form dilations. A. idh-1neo L1 larva with excretory dilation. B, B’. Two different confocal z-slices of the same B12-supplemented VL1409 idh-1neo L1 larva are shown. AJM-1::GFP and RDY-2::GFP (both green) mark apical junctions and the apical membrane of the excretory canal, duct, and pore tubes. lin-48pro: :mRFP (red) marks the duct tube cytoplasm. Dilations were present within the canal tube lumen and in another unidentified cell that may be the associated excretory gland. Arrowhead points to the secretory junction that links the canal, duct, and gland ( 38 ). Images are representative of n=17 animals imaged after B12 supplementation (3/4 L1 larvae and 12/13 elongated embryos had spherical dilations within and adjacent to the excretory canal tube). Download figure Open in new tab Figure S2. idh-1neo animals supplemented with vitamin B12 predominantly accumulate the D isoform of 2HG. D- and L-2HG enantiomers were measured by GC-MS after chiral derivatization. Download figure Open in new tab Figure S3. Embryonic lethality of idh-1neo animals supplemented with vitamin B12 cannot be rescued by ketone body 3-hydroxybutyrate. View this table: View inline View popup Download powerpoint Table S1. Quantification of lethal arrest stage in idh-1neo animals with or without B12 supplementation. Exc, excretory failure, as judged by the rod-like appearance of arrested larvae. Acknowledgements We thank Dr. Ralph DeBerardinis for advice on formate supplementation experiments. This work was funded by grants from the National Institutes of Health DK068429 to AJMW and R35GM136315 to MVS. References 1. ↵ X. Du , H. Hu , The Roles of 2-Hydroxyglutarate . Front Cell Dev Biol 9 , 651317 ( 2021 ). OpenUrl 2. ↵ D. Ye , K. L. Guan , Y. Xiong , Metabolism, Activity, and Targeting of D-and L-2-Hydroxyglutarates . Trends Cancer 4 , 151 – 165 ( 2018 ). OpenUrl 3. ↵ E. A. Struys et al. , Mutations in the D-2-hydroxyglutarate dehydrogenase gene cause D-2-hydroxyglutaric aciduria . Am J Hum Genet 76 , 358 – 360 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 4. ↵ O. Ponomarova et al. , A D-2-hydroxyglutarate dehydrogenase mutant reveals a critical role for ketone body metabolism in Caenorhabditis elegans development . PLoS Biol 21 , e3002057 ( 2023 ). OpenUrl 5. ↵ E. Watson et al. , Metabolic network rewiring of propionate flux compensates vitamin B12 deficiency in C. elegans . Elife 5 , pii : e17670 ( 2016 ). OpenUrl CrossRef PubMed 6. ↵ J. T. Bulcha et al. , A persistence detector for metabolic network rewiring in an animal . Cell Rep 26 , 460 – 468 ( 2019 ). OpenUrl CrossRef 7. ↵ A. Green , P. Beer , Somatic mutations of IDH1 and IDH2 in the leukemic transformation of myeloproliferative neoplasms . N Engl J Med 362 , 369 – 370 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 8. A. K. Andersson et al. , IDH1 and IDH2 mutations in pediatric acute leukemia . Leukemia 25 , 1570 – 1577 ( 2011 ). OpenUrl CrossRef PubMed 9. ↵ F. E. Bleeker et al. , IDH1 mutations at residue p.R132 (IDH1(R132)) occur frequently in high-grade gliomas but not in other solid tumors . Hum Mutat 30 , 7 – 11 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 10. ↵ S. Han et al. , IDH mutation in glioma: molecular mechanisms and potential therapeutic targets . Br J Cancer 122 , 1580 – 1589 ( 2020 ). OpenUrl CrossRef PubMed 11. G. C. Issa , C. D. DiNardo , Acute myeloid leukemia with IDH1 and IDH2 mutations: 2021 treatment algorithm . Blood Cancer J 11 , 107 ( 2021 ). OpenUrl 12. ↵ A. K. Murugan , A. S. Alzahrani , Isocitrate Dehydrogenase IDH1 and IDH2 Mutations in Human Cancer: Prognostic Implications for Gliomas . Br J Biomed Sci 79 , 10208 ( 2022 ). OpenUrl 13. ↵ L. Dang et al. , Cancer-associated IDH1 mutations produce 2-hydroxyglutarate . Nature 462 , 739 – 744 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 14. ↵ P. Jezek , 2-Hydroxyglutarate in Cancer Cells . Antioxid Redox Signal 33 , 903 – 926 ( 2020 ). OpenUrl CrossRef 15. ↵ A. M. Intlekofer et al. , Hypoxia Induces Production of L-2-Hydroxyglutarate . Cell Metab 22 , 304 – 311 ( 2015 ). OpenUrl CrossRef PubMed 16. ↵ S. K. McBrayer et al. , Transaminase Inhibition by 2-Hydroxyglutarate Impairs Glutamate Biosynthesis and Redox Homeostasis in Glioma . Cell 175 , 101 – 116 e125 ( 2018 ). OpenUrl CrossRef PubMed 17. ↵ F. Li et al. , NADP(+)-IDH Mutations Promote Hypersuccinylation that Impairs Mitochondria Respiration and Induces Apoptosis Resistance . Mol Cell 60 , 661 – 675 ( 2015 ). OpenUrl CrossRef 18. ↵ W. A. Flavahan et al. , Insulator dysfunction and oncogene activation in IDH mutant gliomas . Nature 529 , 110 – 114 ( 2016 ). OpenUrl CrossRef PubMed 19. ↵ M. Carbonneau et al. , The oncometabolite 2-hydroxyglutarate activates the mTOR signalling pathway . Nat Commun 7 , 12700 ( 2016 ). 20. ↵ G. Notarangelo et al. , Oncometabolite d-2HG alters T cell metabolism to impair CD8(+) T cell function . Science 377 , 1519 – 1529 ( 2022 ). OpenUrl 21. L. Zhang et al. , D-2-Hydroxyglutarate Is an Intercellular Mediator in IDH-Mutant Gliomas Inhibiting Complement and T Cells . Clin Cancer Res 24 , 5381 – 5391 ( 2018 ). OpenUrl Abstract / FREE Full Text 22. ↵ M. Bottcher et al. , D-2-hydroxyglutarate interferes with HIF-1alpha stability skewing T-cell metabolism towards oxidative phosphorylation and impairing Th17 polarization . Oncoimmunology 7 , e1445454 ( 2018 ). OpenUrl CrossRef 23. ↵ S. J. Parker , C. M. Metallo , Metabolic consequences of oncogenic IDH mutations . Pharmacol Ther 152 , 54 – 62 ( 2015 ). OpenUrl 24. ↵ H. Li , J. M. Tennessen , Quantification of D-and L-2-Hydroxyglutarate in Drosophila melanogaster Tissue Samples Using Gas Chromatography-Mass Spectrometry . Methods Mol Biol 1978 , 155 – 165 ( 2019 ). 25. ↵ J. Zhou et al. , A feedback loop engaging propionate catabolism intermediates controls mitochondrial morphology . Nat Cell Biol 24 , 526 – 537 ( 2022 ). OpenUrl CrossRef 26. ↵ S. Bhattacharya et al. , A metabolic regulatory network for the Caenorhabditis elegans intestine . iScience 25 , 104688 ( 2022 ). 27. ↵ M. D. Walker et al. , WormPaths: Caenorhabditis elegans metabolic pathway annotation and visualization . Genetics 219 ( 2021 ). 28. ↵ G. E. Giese et al. , Caenorhabditis elegans methionine/S-adenosylmethionine cycle activity is sensed and adjusted by a nuclear hormone receptor . Elife 9 ( 2020 ). 29. ↵ Y. J. Pai et al. , Glycine decarboxylase deficiency causes neural tube defects and features of non-ketotic hyperglycinemia in mice . Nat Commun 6 , 6388 ( 2015 ). OpenUrl CrossRef PubMed 30. ↵ I. Amelio , F. Cutruzzola , A. Antonov , M. Agostini , G. Melino , Serine and glycine metabolism in cancer . Trends Biochem Sci 39 , 191 – 198 ( 2014 ). OpenUrl CrossRef PubMed 31. ↵ J. Momb et al. , Deletion of Mthfd1l causes embryonic lethality and neural tube and craniofacial defects in mice . Proc Natl Acad Sci U S A 110 , 549 – 554 ( 2013 ). OpenUrl Abstract / FREE Full Text 32. ↵ G. P. Vatcher et al. , Serine hydroxymethyltransferase is maternally essential in Caenorhabditis elegans . J Biol Chem 273 , 6066 – 6073 ( 1998 ). OpenUrl Abstract / FREE Full Text 33. ↵ J. Fan et al. , Human phosphoglycerate dehydrogenase produces the oncometabolite D-2-hydroxyglutarate . ACS Chem Biol 10 , 510 – 516 ( 2015 ). OpenUrl CrossRef PubMed 34. ↵ R. Xiao et al. , RNAi interrogation of dietary modulation of development, metabolism, behavior, and aging in C. elegans . Cell Rep 11 , 1123 – 1133 ( 2015 ). OpenUrl CrossRef PubMed 35. H. K. Gill et al. , Integrity of Narrow Epithelial Tubes in the C. elegans Excretory System Requires a Transient Luminal Matrix . PLoS Genet 12 , e1006205 ( 2016 ). OpenUrl CrossRef 36. ↵ C. Frokjaer-Jensen et al. , Single-copy insertion of transgenes in Caenorhabditis elegans . Nat Genet 40 , 1375 – 1383 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 37. ↵ P. S. Ward et al. , The potential for isocitrate dehydrogenase mutations to produce 2-hydroxyglutarate depends on allele specificity and subcellular compartmentalization . J Biol Chem 288 , 3804 – 3815 ( 2013 ). OpenUrl Abstract / FREE Full Text 38. ↵ M. V. Sundaram , M. Buechner , The Caenorhabditis elegans Excretory System: A Model for Tubulogenesis, Cell Fate Specification, and Plasticity . Genetics 203 , 35 – 63 ( 2016 ). OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted March 14, 2024. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following idh-1 neomorphic mutation confers sensitivity to vitamin B12 via increased dependency on one-carbon metabolism in Caenorhabditis elegans Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share idh-1 neomorphic mutation confers sensitivity to vitamin B12 via increased dependency on one-carbon metabolism in Caenorhabditis elegans Olga Ponomarova , Alyxandra N. Starbard , Alexandra Belfi , Amanda V. Anderson , Meera V. Sundaram , Albertha J.M. Walhout bioRxiv 2024.03.13.584865; doi: https://doi.org/10.1101/2024.03.13.584865 Share This Article: Copy Citation Tools idh-1 neomorphic mutation confers sensitivity to vitamin B12 via increased dependency on one-carbon metabolism in Caenorhabditis elegans Olga Ponomarova , Alyxandra N. Starbard , Alexandra Belfi , Amanda V. Anderson , Meera V. Sundaram , Albertha J.M. Walhout bioRxiv 2024.03.13.584865; doi: https://doi.org/10.1101/2024.03.13.584865 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biochemistry Subject Areas All Articles Animal Behavior and Cognition (7651) Biochemistry (17746) Bioengineering (13928) Bioinformatics (42066) Biophysics (21499) Cancer Biology (18650) Cell Biology (25579) Clinical Trials (138) Developmental Biology (13409) Ecology (19947) Epidemiology (2067) Evolutionary Biology (24374) Genetics (15633) Genomics (22557) Immunology (17775) Microbiology (40505) Molecular Biology (17217) Neuroscience (88796) Paleontology (667) Pathology (2845) Pharmacology and Toxicology (4836) Physiology (7664) Plant Biology (15179) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9839) Zoology (2272)