Background Screening for colorectal cancer (CRC) reduces its mortality but has limited sensitivity and specificity. Aims We aimed to explore potential biomarker panels for CRC and adenoma detection and to gain insight into the interaction between gut microbiota and human metabolism in the presence of these lesions.Methods This multicenter case-control cohort was performed between February 2016 and November 2019. Consecutive patients ≥18 years with a scheduled colonoscopy were asked to participate and divided into three age, gender, body-mass index and smoking status-matched subgroups: CRC (n = 12), adenomas (n = 21) and controls (n = 20). Participants collected fecal samples prior to bowel preparation on which proteome (LC-MS/MS), microbiota (16S rRNA profiling) and amino acid (HPLC) composition were assessed. Best predictive markers were combined to create diagnostic biomarker panels. Pearson correlation-based analysis on selected markers was performed to create networks of all platforms.Results Combining omics platforms provided new panels which outperformed hemoglobin in this cohort, currently used for screening (AUC 0.98, 0.95 and 0.87 for CRC vs controls, adenoma vs controls and CRC vs adenoma, respectively). Integration of data sets revealed markers associated with increased blood excretion, stress- and inflammatory responses and pointed toward downregulation of epithelial integrity.Conclusions Integrating fecal microbiota, proteome and amino acids platforms provides for new biomarker panels that may improve noninvasive screening for adenomas and CRC, and may subsequently lead to lower incidence and mortality of colon cancer.

Sulfate is the fourth most abundant anion in human plasma but is not measured in clinical practice and little is known about the consequences of sulfate deficiency. Nevertheless, sulfation plays an essential role in the modulation of numerous compounds, including proteoglycans and steroids. We report the first patient with a homozygous loss-of-function variant in the SLC13A1 gene, encoding a renal and intestinal sulfate transporter, which is essential for maintaining plasma sulfate levels. The homozygous (Arg12Ter) variant in SLC13A1 was found by exome sequencing performed in a patient with unexplained skeletal dysplasia. The main clinical features were enlargement of joints and spondylo-epi-metaphyseal radiological abnormalities in early childhood, which improved with age. In addition, autistic features were noted. We found profound hyposulfatemia due to complete loss of renal sulfate reabsorption. Cholesterol sulfate was reduced. Intravenous N-acetylcysteine administration temporarily restored plasma sulfate levels. We conclude that loss of the SLC13A1 gene leads to profound hypersulfaturia and hyposulfatemia, which is mainly associated with abnormal skeletal development, possibly predisposing to degenerative bone and joint disease. The diagnosis might be easily missed and more frequent.

The risk of recurrent dysplastic colonic lesions is increased following polypectomy. Yield of endoscopic surveillance after adenoma removal is low, while interval colorectal cancers occur. To longitudinally assess the dynamics of fecal microbiota and amino acids in the presence of adenomatous lesions and after their endoscopic removal. In this longitudinal case–control study, patients collected fecal samples prior to bowel preparation before scheduled colonoscopy and 3 months after this intervention. Based on colonoscopy outcomes, patients with advanced adenomas and nonadvanced adenomas (0.5–1.0 cm) who underwent polypectomy during endoscopy (n = 19) were strictly matched on age, body-mass index, and smoking habits to controls without endoscopic abnormalities (n = 19). Microbial taxa were measured by 16S RNA sequencing, and amino acids (AA) were measured by high-performance liquid chromatography (HPLC). Adenoma patients were discriminated from controls based on AA and microbial composition. Levels of proline (p = .001), ornithine (p = .02) and serine (p = .02) were increased in adenoma patients compared to controls but decreased to resemble those of controls after adenoma removal. These AAs were combined as a potential adenoma-specific panel (AUC 0.79(0.64–0.94)). For bacterial taxa, differences between patients with adenomas and controls were found (Bifidobacterium spp.↓, Anaerostipes spp.↓, Butyricimonas spp.↑, Faecalitalea spp.↑ and Catenibacterium spp.↑), but no alterations in relative abundance were observed after polypectomy. Furthermore, Faecalitalea spp. and Butyricimonas spp. were significantly correlated with adenoma-specific amino acids. We selected an amino acid panel specifically increased in the presence of adenomas and a microbial signature present in adenoma patients, irrespective of polypectomy. Upon validation, these panels may improve the effectiveness of the surveillance program by detection of high-risk individuals and determination of surveillance endoscopy timing, leading to less unnecessary endoscopies and less interval cancer.

BACKGROUND:Fecal metabolomic profiles differ between pediatric inflammatory bowel disease (IBD) patients and controls and may provide new insights in the pathophysiology of IBD. The role of amino acids, however, is not fully elucidated. We aimed to assess fecal amino acid profiles in pediatric IBD.METHODS:In this case-control study, treatment-naïve, newly diagnosed pediatric IBD patients and a non-IBD control group, matched based on sex and age, were included in 2 tertiary centres. Fecal amino acid profiles were assessed using a targeted high-performance liquid chromatography technique. A random forest classifier method was used to develop a prediction model differentiating IBD from controls and predicting IBD phenotype. The association between IBD localization and amino acid concentrations was tested with ordinal regression models.RESULTS:We included 78 newly diagnosed IBD patients (40 Crohn's disease [CD], 38 ulcerative colitis [UC]) and 105 controls. Patients with IBD could be differentiated from controls with an accuracy of 82% (sensitivity 63%, specificity 97%). Twenty-nine out of the 42 measured unique amino acids were included in the prediction model. Increased levels of tryptophan, taurine, alanine, ornithine, valine, histidine, and leucine were the most differentiating features. Children with CD and UC could be differentiated from the controls with an accuracy of 80% and 90%, respectively. Inflammatory bowel disease phenotype could not be predicted. Tryptophan, valine, and histidine levels were positively associated with more extended disease in UC patients (P < .05).CONCLUSIONS:Fecal amino acids may enhance understanding of the role of host-microbial interactions in the pathophysiology of IBD and may evolve into biomarkers for pediatric IBD diagnostic and personalized medicine.

Background Pyridoxine-dependent epilepsy (PDE) is due to biallelic variants in ALDH7A1 (PDE-ALDH7A1). ALDH7A1 encodes α-aminoadipic semialdehyde dehydrogenase in lysine catabolism. We investigated the gamma aminobutyric acid (GABA) metabolism and energy production pathways in human PDE-ALDH7A1 and its knock-out aldh7a1 zebrafish model. Methods We measured GABA pathway, and tricarboxylic acid cycle metabolites and electron transport chain activities in patients with PDE-ALDH7A1 and in knock-out aldh7a1 zebrafish. Results We report results of three patients with PDE-ALDH7A1: low paired complex I+II and complex II+III and individual complex IV activities in muscle biopsy in patient 1 (likely more severe phenotype); significantly elevated CSF glutamate in the GABA pathway and elevated CSF citrate, succinate, isocitrate and α-ketoglutarate in the TCA cycle in patient 3 (likely more severe phenotype); and normal CSF GABA pathway and TCA cycle metabolites on long-term pyridoxine therapy in patient 2 (likely milder phenotype). All GABA pathway metabolites (γ-hydroxybutyrate, glutamine, glutamate, total GABA, succinic semialdehyde) and TCA cycle metabolites (citrate, malate, fumarate, isocitrate, lactate) were significantly low in the homozygous knock-out aldh7a1 zebrafish compared to the wildtype zebrafish. Homozygous knock-out aldh7a1 zebrafish had decreased electron transport chain enzyme activities compared to wildtype zebrafish. Discussion We report impaired electron transport chain function, accumulation of glutamate in the central nervous system and TCA cycle dysfunction in human PDE-ALDH7A1 and abnormal GABA pathway, TCA cycle and electron transport chain in knock-out aldh7a1 zebrafish. Central nervous system glutamate toxicity and impaired energy production may play important roles in the disease neuropathogenesis and severity in human PDE-ALDH7A1.

((S)-(+)/(R)-(-)) vigabatrin (Sabril(R); gamma-vinyl GABA), an antiepileptic irreversibly inactivating GABA-transaminase, was administered to male C57Bl6 J mice via continuous infusion (0, 40, 80 mg/kg/d) for 12 days. Our study design pooled retina, eye (minus retina), whole brain and plasma from n = 24 animals for each dose to provide n = 8 triplicates per treatment group. Hypothesizing that (S)-(+) VGB (active isomer) would preferentially accumulate in retina, we determined VGB isomers, comprehensive amino acids, and pharmacokinetic parameters. In brain, eye and plasma, the ((S)-(+)/(R)-(-)) ratio varied from 0.73 to 1.29 and 13.3 in retina, accompanied by a partition coefficient (tissue/plasma, ((S)-(+);(R)-(-))) of 5.8;0.34, 0.63;0.49, and 0.51;0.34 in retina, eye and brain, respectively. Racemic VGB (nmol/g; plasma, nmol/mL, range of means for dose) content was: retina, 25-36; eye (minus retina), 4.8-8.0; brain, 3.1-6.8 and plasma, 8.7-14.9. GABA tissue content (nmol/g) was 1246-3335, 18-64 and 2615-3200 as a function of VGB dose for retina, eye (minus retina) and brain, respectively. The retinal glial cell toxin 2-aminoadipic acid also increased with VGB dose (76-96 nmol/g). Partitioning of active (S)-(+) VGB to retina suggests the involvement of a stereospecific transporter, the identification of which could reveal new therapeutic paradigms that might mitigate VGB's wellknown retinal toxicity and expand its clinical utility.

Purpose: Vigabatrin (VGB) is an effective antiepileptic that increases concentrations of inhibitory γ-aminobutyric acid (GABA) by inhibiting GABA transaminase. Reports of VGB-associated visual field loss limit its clinical usefulness, and retinal toxicity studies in laboratory animals have yielded conflicting results. Methods: We examined the functional and morphologic effects of VGB in C57BL/6J mice that received either VGB or saline IP from 10 to 18 weeks of age. Retinal structure and function were assessed in vivo by optical coherence tomography (OCT), ERG, and optomotor response. After euthanasia, retinas were processed for immunohistochemistry, and retinal GABA, and VGB quantified by mass spectrometry. Results: No significant differences in visual acuity or total retinal thickness were identified between groups by optomotor response or optical coherence tomography, respectively. After 4 weeks of VGB treatment, ERG b-wave amplitude was enhanced, and amplitudes of oscillatory potentials were reduced. Dramatic rod and cone bipolar and horizontal cell remodeling, with extension of dendrites into the outer nuclear layer, was observed in retinas of VGB-treated mice. VGB treatment resulted in a mean 3.3-fold increase in retinal GABA concentration relative to controls and retinal VGB concentrations that were 20-fold greater than brain. Conclusions: No evidence of significant retinal thinning or ERG a- or b-wave deficits were apparent, although we describe significant alterations in ERG b-wave and oscillatory potentials and in retinal cell morphology in VGB-treated C57BL/6J mice. The dramatic concentration of VGB in retina relative to the target tissue (brain), with a corresponding increase in retinal GABA, offers insight into the pathophysiology of VGB-associated visual field loss.

Metabolomic characterization of post-mortem tissues (frontal and parietal cortices, pons, cerebellum, hippocampus, cerebral cortex, liver and kidney) derived from a 37 y.o. male patient with succinic semialdehyde dehydrogenase deficiency (SSADHD) was performed in conjunction with four parallel series of control tissues. Amino acids, acylcarnitines, guanidino- species (guanidinoacetic acid, creatine, creatinine) and GABA-related intermediates were quantified using UPLC and mass spectrometric methods that included isotopically labeled internal standards. Amino acid analyses revealed significant elevation of aspartic acid and depletion of glutamine in patient tissues. Evidence for disruption of short-chain fatty acid metabolism, manifest as altered C4OH, C5, C5:1, C5DC (dicarboxylic) and C12OH carnitines, was observed. Creatine and guanidinoacetic acids were decreased and elevated, respectively. GABA-associated metabolites (total GABA, γ-hydroxybutyric acid, succinic semialdehyde, 4-guanidinobutyrate, 4,5-dihydroxyhexanoic acid and homocarnosine) were significantly increased in patient tissues, including liver and kidney. The data support disruption of fat, creatine and amino acid metabolism as a component of the pathophysiology of SSADHD, and underscore the observation that metabolites measured in patient physiological fluids provide an unreliable reflection of brain metabolism.

ABSTRACTIn this prospective intention‐to‐diagnose pilot study, we aimed to assess accuracy of serum and fecal amino‐acids to discriminate de novo pediatric inflammatory bowel disease (IBD) and non‐IBD children. Patients with suspected IBD were allocated the IBD (n = 11) or non‐IBD group (n = 8) following laboratory testing or endoscopy according to the revised Porto‐criteria. Fecal calprotectin levels were obtained, an additional blood and fecal sample were collected. Fecal and serum amino‐acid profiles were analyzed using high performance‐liquid chromatography. Nine fecal amino‐acids (alanine [area under the curve 0.94], citrulline [0.94], glutamine [0.89], leucine [0.98], lysine [0.89], phenylalanine [0.99], serine [0.91], tyrosine [0.96], and valine [0.95]) differed significantly between IBD and non‐IBD. In serum, no significant differences were observed. This study underlines the potential of fecal amino‐acids as novel, adjuvant noninvasive, and low‐cost biomarkers in the diagnostic work‐up of pediatric IBD detection.

Stability of the cerebrospinal fluid (CSF) composition under different pre-analytical conditions is relevant for the diagnostic potential of biomarkers. Our aim was to examine the pre-analytical stability of promising CSF biomarkers that are currently evaluated for their discriminative use in various neurological diseases. Pooled CSF was aliquoted and experimentally exposed to delayed storage: 0, 1, 2, 4, 24, 72, or 168 h at 4 degrees C or room temperature (RT), or 1-4 months at - 20 degrees C; or up to 7 freeze/thaw (f/t) cycles, before final storage at - 80 degrees C. Eleven CSF biomarkers were screened using immunoassays, liquid chromatography, or enzymatic methods. Levels of neurogranin (truncP75), chitinase-3-like protein (YKL-40), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), acetylcholinesterase (AChE) enzymatic activity, theobromine, secreted protein acidic and rich in cysteine-like 1 (SPARCL-1) and homovanillic acid (HVA) levels were not affected by the applied storage conditions. 3-Methoxy-4-hydroxyphenylglycol (MHPG) levels linearly and strongly decreased after 4 h at RT ( - 10%) or 24 h at 4 degrees C ( - 27%), and with 6% after every f/t cycle. 5-Methyltetrahydrofolate (5MTHF) ( - 29% after 1 week at RT) and 5-hydroxyindoleacetic acid levels (5-HIAA) ( - 16% after 1 week at RT) were reduced and 3,4-dihydroxyphenylacetic acid (DOPAC) levels ( + 22% after 1 week at RT) increased, but only after > 24 h at RT. Ten out of eleven potential CSF novel biomarkers showed very limited change under common storage and f/t conditions, suggesting that these CSF biomarkers can be trustfully tested under the pre-analytical conditions present across different cohorts.

The anticonvulsant vigabatrin (VGB; SabrilR) irreversibly inhibits GABA transaminase to increase neural GABA, yet its mechanism of retinal toxicity remains unclear. VGB is suggested to alter several amino acids, including homocarnosine, β-alanine, ornithine, glycine, taurine, and 2-aminoadipic acid (AADA), the latter a homologue of glutamic acid. Here, we evaluate the effect of VGB on amino acid concentrations in mice, employing a continuous VGB infusion (subcutaneously implanted osmotic minipumps), dose-escalation paradigm (35–140 mg/kg/d, 12 days), and amino acid quantitation in eye, visual and prefrontal cortex, total brain, liver and plasma. We hypothesized that continuous VGB dosing would reveal numerous hitherto undescribed amino acid disturbances. Consistent amino acid elevations across tissues included GABA, β-alanine, carnosine, ornithine and AADA, as well as neuroactive aspartic and glutamic acids, serine and glycine. Maximal increase of AADA in eye occurred at 35 mg/kg/d (41 ± 2 nmol/g (n = 21, vehicle) to 60 ± 8.5 (n = 8)), and at 70 mg/kg/d for brain (97 ± 6 (n = 21) to 145 ± 6 (n = 6)), visual cortex (128 ± 6 to 215 ± 19) and prefrontal cortex (124 ± 11 to 200 ± 13; mean ± SEM; p < 0.05), the first demonstration of tissue AADA accumulation with VGB in mammal. VGB effects on basic amino acids, including guanidino-species, suggested the capacity of VGB to alter urea cycle function and nitrogen disposal. The known toxicity of AADA in retinal glial cells highlights new avenues for assessing VGB retinal toxicity and other off-target effects.

D-2-hydroxyglutaric aciduria Type I (D-2-HGA Type I), a neurometabolic disorder with a broad clinical spectrum, is caused by recessive variants in the D2HGDH gene encoding D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). We and others detected 42 potentially pathogenic variants in D2HGDH of which 31 were missense. We developed functional studies to investigate the effect of missense variants on D-2-HGDH catalytic activity. Site-directed mutagenesis was used to introduce 31 missense variants in the pCMV5-D2HGDH expression vector. The wild type and missense variants were overexpressed in HEK293 cells. D-2-HGDH enzyme activity was evaluated based on the conversion of [2H4]D-2-HG to [2H4]2-ketoglutarate, which was subsequently converted into [2H4]L-glutamate and the latter quantified by LC-MS/MS. Eighteen variants resulted in almost complete ablation of D-2-HGDH activity and thus, should be considered pathogenic. The remaining 13 variants manifested residual activities ranging between 17% and 94% of control enzymatic activity. Our functional assay evaluating the effect of novel D2HGDH variants will be beneficial for the classification of missense variants and determination of pathogenicity.

D-glycerate 2 kinase (DGK) is an enzyme that mediates the conversion of D-glycerate, an intermediate metabolite of serine and fructose metabolism, to 2-phosphoglycerate. Deficiency of DGK leads to accumulation of D-glycerate in various tissues and its massive excretion in urine. D-glyceric aciduria (DGA) is an autosomal recessive metabolic disorder caused by mutations in the GLYCTK gene. The clinical spectrum of DGA is highly variable, ranging from severe progressive infantile encephalopathy to a practically asymptomatic condition. We describe a male patient from a consanguineous Arab family with infantile onset of DGA, characterized by profound psychomotor retardation, progressive microcephaly, intractable seizures, cortical blindness and deafness. Consecutive brain MR imaging showed an evolving brain atrophy, thinning of the corpus callosum and diffuse abnormal white matter signals. Whole exome sequencing identified the homozygous missense variant in the GLYCTK gene [c.455 T > C, NM_145262.3], which affected a highly conserved leucine residue located at a domain of yet unknown function of the enzyme [p.Leu152Pro, NP_660305]. In silico analysis of the variant supported its pathogenicity. A review of the 15 previously reported patients, together with the current one, confirms a clear association between DGA and severe neurological impairment. Yet, future studies of additional patients with DGA are required to better understand the clinical phenotype and pathogenesis.

Murine succinic semialdehyde dehydrogenase deficiency (SSADHD) manifests with high concentrations of gamma-aminobutyric acid (GABA) and gamma-hydroxybutyrate (GHB) and low glutamine in the brain. To understand the pathogenic contribution of central glutamine deficiency, we exposed aldh5a1(-/-) (SSADHD) mice and their genetic controls (aldh5a1(+/+)) to either a 4% (w/w) glutamine-containing diet or a glutamine-free diet from conception until postnatal day 30. Endpoints included brain, liver and blood amino acids, brain GHB, ataxia scores, and open field testing. Glutamine supplementation did not improve aldh5a1(-/-) brain glutamine deficiency nor brain GABA and GHB. It decreased brain glutamate but did not change the ratio of excitatory (glutamate) to inhibitory (GABA) neurotransmitters. In contrast, glutamine supplementation significantly increased brain arginine (30% for aldh5a1(+/+) and 18% for aldh5a1(-/-) mice), and leucine (12% and 18%). Glutamine deficiency was confirmed in the liver. The test diet increased hepatic glutamate in both genotypes, decreased glutamine in aldh5a1(+/+) but not in aldh5a1(-/-), but had no effect on GABA. Dried bloodspot analyses showed significantly elevated GABA in mutants (approximately 800% above controls) and decreased glutamate (approximately 25%), but no glutamine difference with controls. Glutamine supplementation did not impact blood GABA but significantly increased glutamine and glutamate in both genotypes indicating systemic exposure to dietary glutamine. Ataxia and pronounced hyperactivity were observed in aldh5a1(-/-) mice but remained unchanged by the diet intervention. The study suggests that glutamine supplementation improves peripheral but not central glutamine deficiency in experimental SSADHD. Future studies are needed to fully understand the pathogenic role of brain glutamine deficiency in SSADHD.

AbstractVigabatrin (VGB; (S)‐(+)/(R)‐(‐) 4‐aminohex‐5‐enoic acid), an antiepileptic irreversibly inactivating GABA transaminase (GABA‐T), manifests use‐limiting ocular toxicity. Hypothesizing that the active S enantiomer of VGB would preferentially accumulate in eye and visual cortex (VC) as one potential mechanism for ocular toxicity, we infused racemic VGB into mice via subcutaneous minipump at 35, 70, and 140 mg/kg/d (n = 6‐8 animals/dose) for 12 days. VGB enantiomers, total GABA and β‐alanine (BALA), 4‐guanidinobutyrate (4‐GBA), and creatine were quantified by mass spectrometry in eye, brain, liver, prefrontal cortex (PFC), and VC. Plasma VGB concentrations increased linearly by dose (3 ± 0.76 (35 mg/kg/d); 15.1 ± 1.4 (70 mg/kg/d); 34.6 ± 3.2 μmol/L (140 mg/kg/d); mean ± SEM) with an S/R ratio of 0.74 ± 0.02 (n = 14). Steady state S/R ratios (35, 70 mg/kg/d doses) were highest in eye (5.5 ± 0.2; P < 0.0001), followed by VC (3.9 ± 0.4), PFC (3.6 ± 0.3), liver (2.9 ± 0.1), and brain (1.5 ± 0.1; n = 13‐14 each). Total VGB content of eye exceeded that of brain, PFC and VC at all doses. High‐dose VGB diminished endogenous metabolite production, especially in PFC and VC. GABA significantly increased in all tissues (all doses) except brain; BALA increases were confined to liver and VC; and 4‐GBA was prominently increased in brain, PFC and VC (and eye at high dose). Linear correlations between enantiomers and GABA were observed in all tissues, but only in PFC/VC for BALA, 4‐GBA, and creatine. Preferential accumulation of the VGB S isomer in eye and VC may provide new insight into VGB ocular toxicity.

#Michael Heuser and Anuhar Chaturvedi share senior authorship Background: Isocitrate dehydrogenase-1 (IDH1) is mutated in about 6% of AML patients. Mutant IDH produces R-2-hydroxyglutarate (R-2HG), which induces histone and DNA hypermethylation through inhibition of epigenetic regulators, thereby linking metabolism to tumorigenesis. We recently reported that at comparable intracellular R-2HG levels, mice receiving transplants of IDH1 mutant cells died significantly earlier than R-2HG treated mice in the context of HOXA9 overexpression. This suggests oncogenic functions of mutant IDH1 beyond R-2HG production. We employed a splice variant of mutated IDH1 that does not produce R-2HG (IDH1mutantΔ7) to decipher R-2HG independent signaling pathways that may contribute towards leukemogenesis. Methods: Bone marrow cells from mice were immortalized with HoxA9, and IDH1wildtype (IDH1wt), IDH1mutant (IDH1mut), IDH1wildtypeΔ7 (IDH1wtΔ7) and IDH1mutΔ7, were constitutively expressed and the leukemogenic potential was evaluated in vivo. Intracellular R-2HG was measured by enantiomer-specific quantification. Deletion of exon 7 from IDH1mut leads to a frameshift that creates a premature stop codon in the 9th exon, finally producing a 119 amino acids truncated protein, IDH1mutΔ7. This splice variant does not produce increased levels of R-2HG. The signaling pathways were explored by immunoblotting and immunofluorescence. Results: Mice receiving cells with IDH1mutΔ7 had the same short latency to leukemia as mice receiving cells with full-length mutant IDH1, while IDH1wt and IDH1wtΔ7 cells died with significantly longer latency. The WBC count increased over time in IDH1mutΔ7 mice similar to IDH1mut mice, whereas WBC counts in IDH1wtΔ7 mice remained normal. IDH1mutΔ7 mice died from monocytic leukemia that was phenotypically and morphologically indistinguishable from IDH1mut mice. HoxA9 IDH1mutΔ7 cells were readily transplantable into secondary recipients. During in vivo cell cycle analysis, we observed that the proportion of cells in S/G2/M phases was significantly higher in bone marrow cells transduced with IDH1mut or IDH1mutΔ7 when compared to cells transduced with IDH1wt or CTL. These data suggest that mutant IDH1 enhances myeloproliferation even in the absence of R-2HG. To identify R-2HG independent signaling pathways mediated by the mutant IDH1 protein, we first analyzed the gene expression of important regulators of cell cycle, differentiation, cell signaling and transcription by quantitative RT-PCR. Several genes (Ccnd1, Slc2a, Hdac3, Tgif2,and c-myc) were upregulated in IDH1mut and IDH1mutΔ7 cells compared to IDH1wt cells. Interestingly, we found a specific up-regulation of Ctnnb1 and Nfkb genes in IDH1mutΔ7 cells over both IDH1mut and IDH1wt cells. We next validated our mRNA expression results by immunoblotting and found that NFKB and ERK signaling were upregulated in both IDH1mut and IDH1mutΔ7 compared to IDH1wt and IDH1wtΔ7 cells. Interestingly, the protein level of β-catenin, STAT3 and STAT5 were many fold higher in IDH1mutΔ7 compared to IDH1mut and IDH1wt cells. β-catenin is known to be transactivated via c-Src, which is phosphorylated by EGFR to promote β-catenin nuclear localization and signaling. We traced this pathway for its relevance in our cells and found that IDH1mutΔ7 cells indeed showed higher levels of both EGFR and c-Src phosphorylation compared to IDH1mut cells. We performed immunofluorescence and cellular fractionation for β-catenin and found it to be partially localized in the nucleus in IDH1mutΔ7 but not in IDH1mut cells. We also observed an up-regulated STAT3 phosphorylation in IDH1mutΔ7 cells over IDH1mut. Conclusions: In summary, mutant IDH1 activates ERK and NFKB signaling, which is attributed to both R-2HG dependent and independent mechanisms of leukemogenesis. Interestingly, IDH1mutΔ7 employs R-2HG independent EGFR/β-catenin and JAK/STAT signaling for oncogenesis. This R-2HG-independent leukemogenesis reveals a novel signaling dynamic of IDH1mut which should be evaluated for its therapeutic potential. Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Heuser:Astellas: Research Funding; Karyopharm: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; StemLine Therapeutics: Consultancy; Bayer Pharma AG: Consultancy, Research Funding; Sunesis: Research Funding; BergenBio: Research Funding; Tetralogic: Research Funding; Daiichi Sankyo: Research Funding.