Abstract: Despite initial responses to hormone treatment, metastatic prostate cancer invariably evolves to a lethal state. To characterize the intra-patient evolutionary relationships of metastases that evade treatment, we perform genome-wide copy number profiling and bespoke approaches targeting the androgen receptor (AR) on 167 metastatic regions from 11 organs harvested post-mortem from 10 men who died from prostate cancer. We identify diverse and patient-unique alterations clustering around the AR in metastases from every patient with evidence of independent acquisition of related genomic changes within an individual and, in some patients, the co-existence of AR -neutral clones. Using the genomic boundaries of pan-autosome copy number changes, we confirm a common clone of origin across metastases and diagnostic biopsies, and identified in individual patients, clusters of metastases occupied by dominant clones with diverged autosomal copy number alterations. These autosome-defined clusters are characterized by cluster-specific AR gene architectures, and in two index cases are topologically more congruent than by chance ( p -values 3.07 × 10 −8  and 6.4 × 10 −4 ). Integration with anatomical sites suggests patterns of spread and points of genomic divergence. Here, we show that copy number boundaries identify treatment-selected clones with putatively distinct lethal trajectories.

Abstract: Lung cancer is the leading cause of cancer-associated mortality worldwide 1 . Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS . We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET , ROS1 , ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource.

Abstract: Background: About 40% of IDH1 mutated (IDH1mut) acute myeloid leukemia (AML) patients respond to IDH1 inhibitors with a median duration of response of 8.2 months. A better understanding of the biology of IDH1mut leukemia may further improve the treatment of these patients. IDH1mut produces R-2-hydroxyglutarate (R-2HG), which activates PHD1 and PHD2 but have negligible effects on PHD3. In the present study we assessed whether PHD3 plays a role in the pathogenesis of IDH1 mutated leukemia and can be targeted in a patient-derived xenograft (PDX) model of IDH1 mutated AML. Methods: Bone marrow cells from Phdwt and Phd3ko mice were immortalized with HoxA9, and IDH1wildtype (IDH1wt) and IDH1mut respectively, were constitutively expressed. The effects on cell proliferation, apoptosis and colony formation were evaluated in vitro, whereas the leukemic potential was evaluated in vivo by transplantation in syngeneic mice. To show that PHD3 is a therapeutic target, either IDH1mut cells from AML patients were transduced with shRNA against PHD3 and transplanted in immunocompromised mice, or leukemic cells from an AML patient with mutated IDH1 were xenografted in immunocompromised mice and treated with the PHD inhibitor molidustat. Results: In in-vitro functional assays loss of Phd3 specifically impaired proliferation, apoptosis and clonogenic capacity of HoxA9-IDH1mut but not HoxA9-IDH1wt cells. Likewise, in mouse transplantation assays, loss of Phd3 eliminated HoxA9-IDH1mut induced leukemia. However, Phd3 was dispensable to the engraftment and proliferation of HoxA9-IDH1wt cells. Additionally, the IDH1-independent model of MN1-induced leukemia remained unaltered in the absence of Phd3, indicating the specificity of the role of Phd3 in mutant IDH1-induced transformation. To identify molecular pathways that might explain in vitro and in vivo phenotypes gene expression profiling was performed. Immune and stress-response pathways as well as metabolism-related genes were most prominently dysregulated in Phd3ko IDH1-mutant cells. Analysis of dysregulated transcription factors by gene set enrichment analysis revealed a depletion of key oncogenic transcription factors (Myc, Rb, Stk33, and Rps14) in Phd3ko IDH1mut cells compared to Phd3ko IDH1wt cells. To study if IDH1mut signals to Phd3 through R-2HG, we transduced Phd3kocells, with a splice variant of mutant IDH1, which does not produce R-2HG but causes leukemia in mice with similar kinetics as in mice with the full-length IDH1 mutant protein. Interestingly, loss of Phd3 also eliminated leukemia in these mice, which demonstrates that mutant IDH1 signals through Phd3 independently of R-2HG. To study the functional relevance of PHD3 inhibition in patients, cells from an IDH1 mutated AML patient were transduced with an shRNA against PHD3 and were transplanted in immunodeficient NSG mice. Inhibition of PHD3 depleted human AML cells in the IDH1-mutated PDX model. Moreover, the PHD inhibitor molidustat was 50-fold more active in IDH1mut (80 nM) compared to IDH1wt AML patient cells (4000 nM) in colony-forming unit assays. In a xenograft model of IDH1 mutated AML, molidustat significantly prolonged survival compared to control-treated mice (P 〈 .001). Conclusion: We demonstrate that the leukemogenic activity of the mutant IDH1 protein depends on PHD3 independently of R-2HG. We identified inhibition of PHD3 as a novel therapeutic strategy in IDH1 mutated AML. Since PHD3 can be targeted pharmacologically, combinatorial treatment of PHD3 and IDH1 inhibitors is warranted to improve eradication of leukemic stem cells in IDH1 mutated AML. #AC and MMAC share first authorship Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Heuser:Karyopharm: Research Funding; Daiichi Sankyo: Research Funding; Sunesis: Research Funding; Tetralogic: Research Funding; Bayer Pharma AG: Consultancy, Research Funding; StemLine Therapeutics: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; BergenBio: Research Funding; Astellas: Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Abstract: Background In a recent phase-III trial CPX-351 (Jazz Pharmaceuticals, Palo Alto, CA), a liposomal encapsulation of cytarabine and daunorubicin, has shown higher remission rates and longer overall survival (OS) in patients aged 60 to 75 years with AML with myelodysplasia-related changes (AML-MRC) or therapy-related AML (t-AML) in comparison to conventional 7+3 regimen. Based on this CPX-351 has been approved in the USA 2017 and in Europe 2018 for adult patients with newly-diagnosed AML-MRC or t-AML. Still, several issues such as age ( & lt;60 years), measurable residual disease (MRD), molecular subgroups and outcome after allo-HCT were not addressed in the phase-III trial. Aiming to investigate these open aspects and to provide more clinical experience with CPX-351, we performed a real-world analysis of patients with AML treated with CPX-351 as first-line therapy. Design/Methods: For this retrospective analysis, we collected data on baseline characteristics, treatment details including allo-HCT and outcome from patients with newly-diagnosed AML-MRC or t-AML, who were treated with CPX-351 according to the EMA label between 2018 and 2020 in 25 German centers participating in the Study Alliance Leukemia (SAL), German Cooperative Transplant Study Group and the AML Study Group (AMLSG). Results: A total of 188 patients (median age 65 years, range 26 to 80) with t-AML (29%) or AML-MRC (70%) including 46 patients (24%) & lt;60 years could be analyzed. Eigthy-six percent received one, 14% two induction cycles and 10% received consolidation with CPX-351. Following induction, CR/CRi rate was 47% including 64% of patients with available information achieving measurable residual disease (MRD) negativity ( & lt;10-3) as measured by flow cytometry at local laboratories. Additionally, 35 patients were categorized as MLFS at first remission control, which achieved CRi (n=16) or CR (n=10) in the further course without additional therapy. After median follow-up of 9.3 months, median overall survival (OS) was 21 months and 1-year OS rate was 64%. In multivariate analysis, complex karyotype predicted lower response (p=.0001), and pretreatment with hypomethylating agents (p=.02) and adverse European LeukemiaNet 2017 genetic risk (p & lt;.0001) were associated with lower OS. Allo-HCT was performed in 116 patients (62%) including 101 of these patients with CR prior transplant and resulted in 1-year OS of 73% (median survival not reached), especially in MRD negative patients (p=.048). With 69% of patients developing grade III/IV non-hematologic toxicity following induction and a day 30-mortality of 8% the safety profile was consistent with previous findings. Conclusion: The results from this real-world analysis confirm CPX-351 as an efficient treatment for these high-risk AML patients bridging to facilitating allo-HCT in many patients with encouraging outcome after transplantation. Disclosures Röllig: AbbVie: Honoraria, Research Funding; Amgen: Honoraria; Bristol-Meyer-Squibb: Honoraria, Research Funding; Janssen: Honoraria; Jazz: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Stelljes: Pfizer: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Medac: Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy, Speakers Bureau; Kite/Gilead: Consultancy, Speakers Bureau. Gaidzik: Janssen: Speakers Bureau; Pfizer: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Unglaub: Novartis: Consultancy, Other: travel costs/ conference fee; JazzPharma: Consultancy, Other: travel costs/ conference fee. Thol: Abbvie: Honoraria; Astellas: Honoraria; BMS/Celgene: Honoraria, Research Funding; Jazz: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Krause: Siemens: Research Funding; Takeda: Honoraria; Pfizer: Honoraria; art-tempi: Honoraria; Kosmas: Honoraria; Gilead: Other: travel support; Abbvie: Other: travel support. Haenel: Celgene: Consultancy, Honoraria; Amgen: Consultancy; Novartis: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Bayer Vital: Honoraria; Jazz: Consultancy, Honoraria; GSK: Consultancy. Vucinic: Novartis: Honoraria; Janssen: Honoraria, Other: Travel Sponsoring; Abbvie: Honoraria, Other: Travel Sponsoring; Gilead: Honoraria, Other: Travel Sponsoring; MSD: Honoraria. Fransecky: Novartis: Honoraria; Medac: Honoraria; Abbvie: Honoraria, Research Funding; Amgen: Honoraria; Takeda: Honoraria. Holtick: Celgene: Honoraria; Sanofi: Honoraria. Kobbe: Celgene: Research Funding. Holderried: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; MSD: Speakers Bureau; Daiichi Sankyo: Other: travel support; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Therakos: Other: Travel support; Janssen: Other: Travel support; Abbvie: Other: Travel support; Eurocept Pharmaceuticals: Other: Travel support; Medac: Other: Travel support. Heuser: Astellas: Research Funding; Bayer AG: Honoraria, Research Funding; BMS/Celgene: Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; BergenBio: Research Funding; Daichi Sankyo: Honoraria, Research Funding; Karyopharm: Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Research Funding; Tolremo: Honoraria; AbbVie: Honoraria; Janssen: Honoraria. Sauer: Pfizer: Consultancy, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Matterhorn Biosciences AG: Consultancy, Other: DSMB/SAB Member; Takeda: Consultancy, Other: DSMB/SAB Member. Goetze: Abbvie: Other: Advisory Board; BMS/Celgene: Other: Advisory Board, Research Funding. Döhner: Jazz Roche: Consultancy, Honoraria; Agios and Astex: Research Funding; Astellas: Research Funding; Abbvie: Consultancy, Honoraria; Janssen: Honoraria, Other: Advisory Board; Daiichi Sankyo: Honoraria, Other: Advisory Board; Novartis: Consultancy, Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding. Döhner: Jazz Pharmaceuticals: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Pfizer: Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Gilead: Honoraria; Janssen: Honoraria; Helsinn: Honoraria; GEMoaB: Honoraria; Amgen: Honoraria, Research Funding; Astellas: Honoraria, Research Funding; Astex Pharmaceuticals: Honoraria; AstraZeneca: Honoraria; Berlin-Chemie: Honoraria; Oxford Biomedica: Honoraria; Roche: Honoraria. Schliemann: Philogen S.p.A.: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Other: travel grants; Astellas: Consultancy; AstraZeneca: Consultancy; Boehringer-Ingelheim: Research Funding; BMS: Consultancy, Other: travel grants; Jazz Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy; Roche: Consultancy; Pfizer: Consultancy. Schetelig: Roche: Honoraria, Other: lecture fees; Novartis: Honoraria, Other: lecture fees; BMS: Honoraria, Other: lecture fees; Abbvie: Honoraria, Other: lecture fees; AstraZeneca: Honoraria, Other: lecture fees; Gilead: Honoraria, Other: lecture fees; Janssen: Honoraria, Other: lecture fees . Germing: Novartis: Honoraria, Research Funding; Janssen: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding; Celgene: Honoraria; Jazz Pharmaceuticals: Honoraria. Schroeder: JAZZ: Honoraria, Research Funding.

Abstract: IDH1 promotes leukemogenesis in vivo in cooperation with HoxA9. Pharmacologic inhibition of mutant IDH1 efficiently inhibits AML cells of IDH1-mutated patients but not of normal CD34+ bone marrow cells.

Abstract: Mutations in the metabolic enzymes IDH1 and IDH2 are frequently found in several tumors including glioma and acute myeloid leukemia (AML). Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. However, it is unknown whether R2HG alone is sufficient to recapitulate the biologic effects of mutant IDH1 in vivo. Recently, we have shown that IDH1mut cooperates with HoxA9 and induces a monocytic leukemia in mice. In order to evaluate the effects of R2HG independently of the mutated IDH1 protein and to determine whether the effects are specific to the R-enantiomer of 2HG, we treated mice transplanted with HoxA9 immortalised bone marrow cells with R2HG, S-2-hydroxyglutarate (S2HG), alpha-ketoglutarate (aKG) and phosphate buffered saline (PBS). The mice in the metabolite cohorts received an intraperitoneal dose of 1 mg per day. Mice treated with R2HG had higher engraftment levels at 16 and 20 weeks post transplantation than the mice treated with S2HG, αKG and PBS respectively (P 〈 .01). High WBC counts (70±16 /nl) and lower platelet counts than in control mice were observed in the cohort receiving R2HG after 16 to 20 weeks of treatment, while the S2HG, αKG and PBS cohorts had normal blood counts even at 20 weeks (P 〈 .05). Peripheral blood from R2HG treated mice revealed significantly more immature Mac1+Gr1- and less mature Mac1+Gr1+ cells at 12 and 16 weeks after treatment than S2HG, αKG and PBS treated mice (P 〈 .001). In addition, the R2HG treated mice died with a median latency of 137 days post transplantation from monocytic leukemia, while mice treated with S2HG, αKG and PBS died with a median latency of 223, 202 and 184 days respectively (P 〈 .001). Further, in order to assess whether R2HG alone was sufficient as a single hit to induce myeloproliferation, normal C57BL/6 mice (without HoxA9) were treated with R2HG, S2HG and PBS for eight months. No differences were observed for survival, blood counts, immunophenotype and frequencies of progenitor cells (lin-ckit+sca1+, CMP, GMP and MEP) between treatment groups and control. This data shows that the metabolite R2HG like the IDH1 mutant protein cooperates with HoxA9 to induce monocytic leukemia. We next compared mice receiving transplants of HoxA9+IDH1mut cells with mice receiving HoxA9 cells that were then treated with R2HG. Both cohorts developed monocytic leukemia, albeit with different kinetics. The Hoxa9+IDH1mut mice died with a median latency of 83 days while the R2HG cohort died with a median latency of 137 days post transplantation (P 〈 0.001). Also, while the former cohort developed severe leukocytosis, anemia and thrombocytopenia at 12 weeks, the R2HG treated mice had high WBC counts and lower platelet counts than control mice at 16 to 20 weeks after treatment. The faster disease kinetics in IDH1mut mice could be attributed to a significantly lower proportion of cells in G0/G1 and higher proportion of cells in S phase when compared to cells from mice treated with R2HG at 9 weeks after transplantation (P 〈 .001). This resulted from a marked downregulation of cyclin-dependent kinase inhibitors (Cdkn) 1A (p21), 1B (p27), 2A (p16), and 2B (p15) in HoxA9+IDH1mut cells as compared to HoxA9 cells treated with R2HG or PBS. In order to rule out an influence of differential R2HG levels on the disease progression between the two cohorts, levels of R2HG were quantified. IDH1mut expressing and R2HG treated bone marrow cells from mice had similarly high ratios of R2HG/S2HG. The quantified R2HG levels were also comparable to that of primary AML patient cells harbouring mutated IDH1. In unsupervised hierarchical clustering mutated cells clustered together with R2HG and separated from PBS treated mice. 69 of the top 100 enriched Gene Ontology gene sets of HoxA9+IDH1mut were also found in HoxA9+R2HG, suggesting largely redundant but also non-overlapping functions of the mutant IDH1 protein and the oncometabolite R2HG. In summary, we show that R2HG, similar to the mutant IDH1 protein, promotes leukemogenesis in cooperation with HoxA9, although with delayed kinetics. Our data proves that R2HG acts as an oncometabolite in vivo in a murine model of leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Abstract: High-grade serous ovarian cancer (HGSOC) is the most common subtype of ovarian cancer, accounting for more than 70% of all epithelial ovarian cancers. It is characterized by high degrees of genomic instability and heterogeneity, with the majority of patients eventually acquiring resistance to platinum chemotherapy. The diversity in platinum-resistance mechanisms and limited effective predictive biomarkers mean delivering the best treatment options for patient tumor remains challenging. The purpose of this study is to understand the extent of intratumoral heterogeneity (ITH) in advanced-stage HGSOC and how this changes over time at relapse, to describe the molecular mechanisms behind peritoneal dissemination, and to delineate the link between ITH at the molecular and phenotypic levels. Patients undergoing radical upfront debulking for advanced HGS ovarian cancer underwent tumor mapping of their tumor dissemination patterns (n=50). Biopsies were collected from disseminated tumors (range 4-15, median=9), snap frozen, and placed in short-term cultures. Tumor cultures were treated with cisplatin, apoptosis/viability assayed and IC50 for cisplatin determined. DNA was extracted from frozen tumors (5 tumors per patient plus germline) and Illumina Human OmniExpress genotyping performed. Allele-specific copy number (CN) was quantified using ASCAT. Genomic heterogeneity was quantified as the estimated number of CN aberration events distinct between each pair of deposits. Clonal diversity within a patient’s deposits was calculated using the difference between within-patient and between-patient heterogeneity. When relapsed, patients had paired biopsies collected for genomic and phenotypic analysis. Broad heterogeneity was observed in response to platinum treatment in vitro across cases at the phenotypic level (n=50), with higher variances in apoptosis induction observed in patients with platinum-resistant disease. Genomic analysis of copy number data revealed widespread variations in patterns of evolution for different patients’ tumors, including the relationship between primary deposits and relapsed disease. Variations in CCNE1 CN were observed across multiple tumors in the same patients, and overall higher CCNE1 CN associated with poorer patient outcome (p=0.041). Extensive heterogeneity is observed at the phenotypic and genomic levels in HGSOC patients, which correlates with the subsequent development of platinum-resistant disease. CCNE1 copy number variations across multiple intra-abdominal samples within patients indicate that single-site biopsies do not truly represent overall disseminated HGSOC biology and may have implications for overinterpretation of studies relating to outcome and platinum resistance. Citation Format: Paula Cunnea, Ed Curry, Katherine Nixon, Ratri Wulandari, Kerstin Thol, Chun Hei Kwok, Jennifer Ploski, Iain McNeish, Elizabeth Christie, David Bowtell, Christina Fotopoulou. Phenotypic and genomic characterization of intratumoral heterogeneity in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A10.

Abstract: #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. Disclosures 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.