Large-scale RNA Interference Research Articles

The quest for the best target genes for RNAi-mediated pest control.

RNA interference (RNAi) has emerged as an eco-friendly alternative to classic pesticides for pest control. This review highlights the importance of identifying the best target genes for RNAi-mediated pest control. We argue that the knowledge-based approach to predicting effective targets is limited by our current gaps of knowledge, making unbiased screening a superior method for discovering the best target processes and genes. We emphasize the recent evidence that suggests targeting conserved basic cellular processes, such as protein degradation and translation, is more effective than targeting the classic pesticide target processes. We support these claims by comparing the efficacy of previously reported RNAi target genes and classic insecticide targets with data from our genome-wide RNAi screen in the red flour beetle, Tribolium castaneum. Finally, we provide practical advice for identifying excellent target genes in other pests, where large-scale RNAi screenings are typically challenging.

SF3B14 is involved in the centrosome regulation through splicing of TUBGCP6 pre-mRNA

Splicing precursor messenger RNA (pre-mRNA) is a critical step to produce physiologically functional protein. Splicing failure not only gives rise to dysfunctional proteins but also generates abnormal protein function, which causes several diseases. Several pre-mRNA splicing factors are reported to regulate mitosis directly at mitotic structures and/or indirectly through controlling the pre-mRNA splicing for mitotic proteins. In this study, we described the mitotic functions of SF3B14, a component of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), which we identified as a candidate involved in mitosis based on the large-scale RNA interference (RNAi) screen of the nucleolar proteome database. We observed that SF3B14 depletion caused prolonged mitosis and several mitotic defects, such as monopolar spindle and chromosome misalignment during metaphase. Although SF3B14 was found in the nucleolar proteome database, our immunofluorescent stainings demonstrated that SF3B14 was predominantly localized in the nucleoplasm and excluded from the nucleolus during interphase. In addition, SF3B14 did not colocalize with specific mitotic structures during mitosis, which is not in line with its direct mitotic function. Notably, we found that the SF3B14 depletion reduced protein levels of TUBGCP6, required for centrosome regulation, and increased the unspliced/spliced ratio of its mRNA. Taken together, we propose that the pre-mRNA of TUBGCP6 is one of the targets for SF3B14 splicing through which SF3B14 controls mitotic chromosome behavior.

Chromosome-level assembly of the brown planthopper genome with a characterized Y chromosome.

Hundreds of insect genome sequences have been reported; however, most sequencing projects have not determined the sex chromosomes, and no Y chromosomes from a heterometabolous insect have been identified and characterized to date. The brown planthopper (Nilaparvata lugens Stål) is the most economically damaging pest to rice and is also an ideal research subject for paddy ecology and functional genomics. We previously assembled a draft female genome mainly using second-generation sequencing technologies, with a contig N50 of only 24kb, due to the large size and excessive repetitive regions in the N.lugens genome. Here, we utilize third-generation sequencing technologies and Hi-C data to generate a high-quality male N.lugens assembly with a contig N50 of 1.01Mb, a scaffold N50 of 69.96Mb and more than 95.6% of the assembled bases located on 16 chromosomes. Fourteen autosomes and two sex chromosomes (X+Y) were identified, filling in the gap related to the Y chromosome in heterometabolous insects. A total of 18,021 protein-coding genes and 6423 long-noncoding RNAs were predicted with full-length cDNA sequencing data. All 315 of the Y chromosome genes (Y-genes) were derived from autosomal and X-chromosome duplications. Large-scale RNA interference (RNAi) experiments were conducted against the N.lugens Y-genes, demonstrating that 7 Y-genes were essential for normal BPH development or male organ development, suggesting the importance of Y-genes. The first identified Y chromosome in heterometabolous insects will help gain more insight into sex determination, fertility and chromosome evolution.

Overexpression of FKH-2/FOXG1 is neuroprotective in a C. elegans model of Machado-Joseph disease

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is the most common form of dominantly inherited ataxia worldwide. This disease is caused by an expanded CAG repeat in the coding region of ATXN3. Due to our incomplete understanding of mechanisms and molecular pathways related to this disease, there are no therapies that successfully treat core MJD patients. Therefore, the identification of new candidate targets related to this disease is needed.In this study, we performed a large-scale RNA interference (RNAi) screen of 387 transcription factor genes leading to the identification of several modifiers (suppressors and enhancers) of impaired motility phenotypes in a mutant ATXN3 transgenic C. elegans model. We showed that inactivation of one particular gene, fkh-2/FOXG1, enhanced the motility defect, neurodegeneration and reduced longevity in our MJD models. Opposite to genetic inactivation, the overexpression of fkh-2 rescued the impaired motility, shortened-lifespan, and neurodegeneration phenotypes of mutant ATXN3 transgenics. We found that overexpression of FKH-2/FOXG1 in ATXN3 mutant worms is neuroprotective.Using our transgenic ATXN3 C. elegans models and the screening of an RNAi library, we gained insights into the pathways contributing to neurodegeneration, and found that FKH-2/FOXG1 has neuroprotective activity. These findings may aid the development of novel therapeutic interventions for MJD.

Large-scale RNAi screening uncovers therapeutic targets in the parasite Schistosoma mansoni.

Schistosome parasites kill 250,000 people every year. Treatment of schistosomiasis relies on the drug praziquantel. Unfortunately, a scarcity of molecular tools has hindered the discovery of new drug targets. Here, we describe a large-scale RNA interference (RNAi) screen in adult Schistosoma mansoni that examined the function of 2216 genes. We identified 261 genes with phenotypes affecting neuromuscular function, tissue integrity, stem cell maintenance, and parasite survival. Leveraging these data, we prioritized compounds with activity against the parasites and uncovered a pair of protein kinases (TAO and STK25) that cooperate to maintain muscle-specific messenger RNA transcription. Loss of either of these kinases results in paralysis and worm death in a mammalian host. These studies may help expedite therapeutic development and invigorate studies of these neglected parasites.

Schistosome biology illuminated

Parasite Genetics Schistosomiasis is caused by a parasitic flatworm about which little is known. Therefore, options to combat human disease caused by schistosome infection are limited. To aid in our quest to develop treatments, two studies undertook molecular investigations of the parasite Schistosoma mansoni . By generating a single-cell atlas, Wendt et al. identified the developmental trajectory of the flatworm, including the blood-feeding gut required for its survival in the host. From these data, they found a gene required for gut development that, when knocked out through RNA interference, confers reduced pathology in infected mice. Wang et al. performed a large-scale RNA interference survey of S. mansoni and identified an essential pair of protein kinases that can be targeted by approved pharmacological intervention (see the Perspective by Anderson and Duraisingh). These molecular investigations add to our understanding of the schistosome parasite and provide biological information that may help to combat this neglected tropical disease. Science , this issue p. [1644][1], p. [1649][2]; see also p. [1562][3] [1]: /lookup/doi/10.1126/science.abb7709 [2]: /lookup/doi/10.1126/science.abb7699 [3]: /lookup/doi/10.1126/science.abe0710

Identification of β-catenin target genes in colorectal cancer by interrogating gene fitness screening data.

β-catenin regulates its target genes which are associated with proliferation, differentiation, migration and angiogenesis, and the dysregulation of Wnt/β-catenin signaling facilitates hallmarks of colorectal cancer (CRC). Identification of β-catenin targets and their potential roles in tumorigenesis has gained increased interest. However, the number of identified targets remains limited. The present study implemented a novel strategy, interrogating gene fitness profiles derived from large-scale RNA interference and CRISPR-CRISPR associated protein 9 screening data to identify β-catenin target genes in CRC cell lines. Using these data sets, pair wise gene fitness similarities were determined which highlighted a total of 13 genes whose functions were highly correlated with β-catenin. It was further demonstrated that the expression of these genes were altered in CRC, illustrating their potential roles in the progression of CRC. The present study further demonstrated that these targets could be used to predict disease-free survival in CRC. In conclusion, the findings provided novel approaches for the identification of β-catenin targets, which may become prognostic biomarkers or drug targets for the management of CRC.

Full-Length Hairpin RNA Accumulates at High Levels in Yeast but Not in Bacteria and Plants.

Hairpin-structured (hp) RNA has been widely used to induce RNA interference (RNAi) in plants and animals, and an in vivo expression system for hpRNA is important for large-scale RNAi applications. Bacterial expression systems have so far been developed for in vivo expression of hpRNA or double-stranded (ds) RNA, but the structure of the resulting RNAi molecules has remained unclear. Here we report that long hpRNAs expressed in the bacteria Escherichia coli and Sinorhizobium meliloti were largely processed into shorter dsRNA fragments with no or few full-length molecules being present. A loss-of-function mutation in the dsRNA-processing enzyme RNase III, in the widely used E. coli HT115 strain, did not prevent the processing of hpRNA. Consistent with previous observations in plants, the loop sequence of long hpRNA expressed in Agrobacterium-infiltrated Nicotiana benthamiana leaves was excised, leaving no detectable levels of full-length hpRNA molecule. In contrast to bacteria and plants, long hpRNAs expressed in the budding yeast Saccharomyces cerevisiae accumulated as intact, full-length molecules. RNA extracted from hpRNA-expressing yeast cells was shown to be capable of inducing RNAi against a β-glucuronidase (GUS) reporter gene in tobacco leaves when applied topically on leaf surfaces. Our results indicate that yeast can potentially be used to express full-length hpRNA molecules for RNAi and perhaps other structured RNAs that are important in biological applications.

TRRAP is essential for regulating the accumulation of mutant and wild-type p53 in lymphoma

AbstractTumors accumulate high levels of mutant p53 (mutp53), which contributes to mutp53 gain-of-function properties. The mechanisms that underlie such excessive accumulation are not fully understood. To discover regulators of mutp53 protein accumulation, we performed a large-scale RNA interference screen in a Burkitt lymphoma cell line model. We identified transformation/transcription domain-associated protein (TRRAP), a constituent of several histone acetyltransferase complexes, as a critical positive regulator of both mutp53 and wild-type p53 levels. TRRAP silencing attenuated p53 accumulation in lymphoma and colon cancer models, whereas TRRAP overexpression increased mutp53 levels, suggesting a role for TRRAP across cancer entities and p53 mutations. Through clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 screening, we identified a 109-amino-acid region in the N-terminal HEAT repeat region of TRRAP that was crucial for mutp53 stabilization and cell proliferation. Mass spectrometric analysis of the mutp53 interactome indicated that TRRAP silencing caused degradation of mutp53 via the MDM2-proteasome axis. This suggests that TRRAP is vital for maintaining mutp53 levels by shielding it against the natural p53 degradation machinery. To identify drugs that alleviated p53 accumulation similarly to TRRAP silencing, we performed a small-molecule drug screen and found that inhibition of histone deacetylases (HDACs), specifically HDAC1/2/3, decreased p53 levels to a comparable extent. In summary, here we identify TRRAP as a key regulator of p53 levels and link acetylation-modifying complexes to p53 protein stability. Our findings may provide clues for therapeutic targeting of mutp53 in lymphoma and other cancers.

A large-scale RNA interference screen identifies genes that regulate autophagy at different stages

Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed a large-scale RNA interference screen in K562 human chronic myeloid leukemia cells using monodansylcadaverine staining, an autophagy-detecting approach equivalent to immunoblotting of the autophagy marker LC3B or fluorescence microscopy of GFP-LC3B. By coupling monodansylcadaverine staining with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays revealed that 57 autophagy-regulating genes suppressed autophagy initiation, whereas 21 candidates promoted autophagy maturation. Our RNA interference screen identified genes that regulate autophagy at different stages, which helps decode autophagy regulation in cancer and offers novel avenues to develop autophagy-related therapies for cancer.

High-throughput screening by RNA interference and its application in breast cancer research

RNA interference (RNAi) is a gene silencing process induced by double-stranded RNA molecules’ binding to message RNA, at both transcriptional level and post-transcriptional level. Recently, high-throughput screening (HTS) with large-scale RNAi has been applied to the field of cancer research, including investigation of cancer development and progression. Especially, HTS with RNAi library was used to identify deregulated signaling pathway, to discover novel drug targets, and to optimize combination therapy and dosing schedule. Furthermore, synthetic lethality strategy is also combined with RNAi HTS to identify new drug targets. The development of breast cancer involves multiple factors, and is associated with multiple genetic alterations, and is a multi-step carcinogenesis process. The RNAi HTS might help to understand this complex process and underlying mechanism that collectively attribute to cancer cell invasion and metastasis. This article reviews the advancement of RNAi HTS assays, and focuses on its applications in both basic and translational research in breast cancer. A promising breakthrough is expected with RNAi HTS technology in the future. Key words: Breast cancers; RNA interference; High-throughput screening; Therapy

A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors.

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4+ T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention.

Large-Scale RNA Interference Screening to Identify Transcriptional Regulators of a Tumor Suppressor Gene.

RNA interference (RNAi) is a powerful research tool that can be used to silence the expression of a specific gene. In the past several years, RNAi has provided the opportunity to identify factors and pathways involved in complex biological processes by performing unbiased loss-of-function screens on a genome-wide scale. Here we describe a genome-wide RNAi screening strategy to identify factors that regulates epigenetic silencing of a specific tumor suppressor gene, using RASSF1A as an example. The approach we describe is a general RNAi screening strategy that can be applied to identify other factors that drive and/or maintain epigenetic modifications on specific genes, including cancer-related genes.

MARCH1 regulates insulin sensitivity by controlling cell surface insulin receptor levels.

Insulin resistance is a key driver of type 2 diabetes (T2D) and is characterized by defective insulin receptor (INSR) signalling. Although surface INSR downregulation is a well-established contributor to insulin resistance, the underlying molecular mechanisms remain obscure. Here we show that the E3 ubiquitin ligase MARCH1 impairs cellular insulin action by degrading cell surface INSR. Using a large-scale RNA interference screen, we identify MARCH1 as a negative regulator of INSR signalling. March1 loss-of-function enhances, and March1 overexpression impairs, hepatic insulin sensitivity in mice. MARCH1 ubiquitinates INSR to decrease cell surface INSR levels, but unlike other INSR ubiquitin ligases, MARCH1 acts in the basal state rather than after insulin stimulation. Thus, MARCH1 may help set the basal gain of insulin signalling. MARCH1 expression is increased in white adipose tissue of obese humans, suggesting that MARCH1 contributes to the pathophysiology of T2D and could be a new therapeutic target.

The histone H3K9 demethylase KDM3A promotes anoikis by transcriptionally activating pro-apoptotic genes BNIP3 and BNIP3L.

Epithelial cells that lose attachment to the extracellular matrix undergo a specialized form of apoptosis called anoikis. Here, using large-scale RNA interference (RNAi) screening, we find that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase, plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. We find that KDM3A promotes anoikis through transcriptional activation of BNIP3 and BNIP3L, which encode pro-apoptotic proteins. Using mouse models of breast cancer metastasis we show that knockdown of Kdm3a enhances metastatic potential. Finally, we find defective KDM3A expression in human breast cancer cell lines and tumors. Collectively, our results reveal a novel transcriptional regulatory program that mediates anoikis.

Protein synthesis and degradation are essential to regulate germline stem cell homeostasis in Drosophila testes.

The homeostasis of self-renewal and differentiation in stem cells is controlled by intrinsic signals and their niche. We conducted a large-scale RNA interference (RNAi) screen in Drosophila testes and identified 221 genes required for germline stem cell (GSC) maintenance or differentiation. Knockdown of these genes in transit-amplifying spermatogonia and cyst cells further revealed various phenotypes. Complex analysis uncovered that many of the identified genes are involved in key steps of protein synthesis and degradation. A group of genes that are required for mRNA splicing and protein translation contributes to both GSC self-renewal and early germ cell differentiation. Loss of genes in the protein degradation pathway in cyst cells leads to testis tumors consisting of overproliferated germ cells. Importantly, in the Cullin 4-RING E3 ubiquitin ligase (CRL4) complex, we identified multiple proteins that are crucial to GSC self-renewal: pic/DDB1, a CRL4 linker protein, is not only required for GSC self-renewal in flies but also for maintenance of spermatogonial stem cells (SSCs) in mice.

Design and Methods of Large-Scale RNA Interference Screens in Drosophila.

Drosophila is an ideal model system for addressing important questions in biology. The use of RNA interference (RNAi) to knockdown gene expression in fly tissues is both very effective and relatively simple. In the past few decades, genome-wide UAS-RNAi transgenic libraries and thousands of Gal4 strains have been generated and have facilitated large-scale in vivo RNAi screening. Here, we discuss methods for the design and performance of a large-scale in vivo RNAi screen in Drosophila. Furthermore, methods for the validation of results and analysis of data will be introduced.

RNAi profiling of primary human AML cells identifies ROCK1 as a therapeutic target and nominates fasudil as an antileukemic drug

AbstractAcute myeloid leukemia (AML) is characterized by a marked genetic heterogeneity, which complicates the development of novel therapeutics. The delineation of pathways essential within an individual patient's mutational background might overcome this limitation and facilitate personalized treatment. We report the results of a large-scale lentiviral loss-of-function RNA interference (RNAi) screen in primary leukemic cells. Stringent validation identified 6 genes (BNIPL1, ROCK1, RPS13, STK3, SNX27, WDHD1) whose knockdown impaired growth and viability of the cells. Dependence on these genes was not caused by mutation or overexpression, and although some of the candidates seemed to be rather patient specific, others were essential in cells isolated from other AML patients. In addition to the phenotype observed after ROCK1 knockdown, treatment with the approved ROCK inhibitor fasudil resulted in increased apoptosis and decreased viability of primary AML cells. In contrast to observations in some other malignancies, ROCK1 inhibition did not foster growth of immature malignant progenitors but was toxic to this cell fraction in feeder coculture and xenotransplant experiments, indicating a distinct effect of ROCK1 inhibition on leukemic progenitors. We conclude that large-scale RNAi screens in primary patient–derived cells are feasible and can complement other methods for personalized cancer therapies, such as expression and mutation profiling.

Isocitrate dehydrogenase 1 and 2 mutations induce BCL-2 dependence in acute myeloid leukemia.

Mutant isocitrate dehydrogenase (IDH) 1 and 2 proteins alter the epigenetic landscape in acute myeloid leukemia (AML) cells through production of the oncometabolite (R)-2-hydroxyglutarate (2-HG). Here we performed a large-scale RNA interference (RNAi) screen to identify genes that are synthetic lethal to the IDH1(R132H) mutation in AML and identified the anti-apoptotic gene BCL-2. IDH1- and IDH2-mutant primary human AML cells were more sensitive than IDH1/2 wild-type cells to ABT-199, a highly specific BCL-2 inhibitor that is currently in clinical trials for hematologic malignancies, both ex vivo and in xenotransplant models. This sensitization effect was induced by (R)-2-HG-mediated inhibition of the activity of cytochrome c oxidase (COX) in the mitochondrial electron transport chain (ETC); suppression of COX activity lowered the mitochondrial threshold to trigger apoptosis upon BCL-2 inhibition. Our findings indicate that IDH1/2 mutation status may identify patients that are likely to respond to pharmacologic BCL-2 inhibition and form the rational basis for combining agents that disrupt ETC activity with ABT-199 in future clinical studies.

Isocitrate Dehydrogenase Mutations Induce BCL-2 Dependence in Acute Myeloid Leukemia through Inhibition of Cytochrome C Oxidase Function

IDH1 and IDH2 are two of the most frequently mutated genes in acute myeloid leukemia at an overall frequency of about 15-20%. The genes encode enzymes in the citric acid cycle that normally catalyze the oxidative decarboxylation of isocitrate, producing α-ketoglutarate (α-KG). The mutant enzymes gain a neomorphic activity that catalyzes the conversion of α-KG to (R)-2-hydroxyglutarate (2-HG). The intracellular concentration of (R)-2-HG is over 100-fold higher in IDH-mutated cells than in wildtype cells. (R)-2-HG has been shown to be a competitive inhibitor of multiple α-KG dependent dioxygenases including TET2, and the Jumonji-C domain containing histone demethylases. These enzymes are thought to be the main targets through which (R)-2-HG exerts its effects on leukemogenesis.We previously reported that inhibition of the anti-apoptotic BCL-2 protein is synthetic lethal against mutant IDH which we discovered through a large-scale pooled lentiviral RNA interference screen. We confirmed that expression of mutant IDH1 or IDH2 strikingly sensitized AML cells to shRNA-mediated BCL-2 knockdown and pharmacologic BCL-2 inhibition with ABT-199, a highly specific BH3 mimetic. Importantly, we found that primary human AML blasts harboring IDH mutations were significantly more sensitive to ABT-199 than blasts with wildtype IDH ex vivo and in xenograft transplant models. Furthermore, we showed that ABT-199 was able to target the leukemic stem cell compartment in IDH-mutated samples.Here, we present our work to uncover the synthetic lethal mechanism. An important clue to the mechanism surfaced with the finding that treatment with a cell-permeable precursor of (R)-2-HG sensitized AML cells to BCL-2 inhibition, indicating that the intracellular accumulation of (R)-2-HG found in IDH-mutated cells is sufficient to mediate the phenotype. In addition, we found that (R)-2-HG was able to sensitize isolated mitochondria to ABT-199 with collapse of the mitochondrial transmembrane potential as a surrogate marker for commitment to apoptosis. This finding indicates that 1) the target mediating the synthetic lethal phenotype is localized to the mitochondria, and 2) changes in the epigenome and expression of nuclear-encoded genes are not required for the synthetic lethal phenotype.To identify the potential mitochondrial molecular target, we focused our analysis on the effect of (R)-2-HG on the enzymatic activity of individual complexes in the electron transport chain (ETC), given that ETC dysfunction can potentially alter the threshold for apoptosis. We found that (R)-2-HG at concentrations found in IDH mutated cells inhibited the in vitro enzymatic activity of complex IV (cytochrome C oxidase (COX)) in a dose-dependent manner, but had no effect on the remaining ETC complexes. This finding has in vivo significance as COX activity in intact IDH-mutated primary AML cells was found to be significantly decreased compared with non-mutated AML cells.Next, we investigated the possibility that COX inhibition is sufficient to induce BCL-2 dependence. We found that suppression of COX activity with chemical inhibitors or genetically through shRNA-mediated knockdown of a COX subunit (COX-IV) was sufficient to sensitize AML cells to ABT-199. Furthermore, treatment with tigecycline, a FDA-approved antibiotic that has previously been shown to disrupt ETC function through inhibition of mitochondrial translation resulting in decreased expression of mitochondrial-encoded proteins including the catalytic subunits of the COX complex, reproduced the sensitization effect in non-IDH mutated AML cells. Based on the above findings, we propose a mechanistic model in which (R)-2-HG accumulation in IDH mutant cells directly inhibits COX, thereby lowering the mitochondrial threshold for triggering apoptosis upon BCL-2 inhibition.In summary, we discovered that in addition to the previously described inhibition of α-KG dependent dioxygenases through (R)-2-HG production, IDH mutations also affect mitochondrial bioenergetics. This finding opens up the intriguing possibility that IDH mutations contribute to leukemogenesis not only through epigenetic changes but also through metabolic dysregulation. Lastly, our findings form the rational basis for combining agents that disrupt ETC function such as tigecycline with ABT-199 to target resistant cancer cells and maximize the clinical utility of this promising drug. DisclosuresMedeiros:Agios: Consulting - Ad board Other.