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Loss of heterozygosity and promoter hypermethylation of APC is frequently observed in human endometrial cancer, which is the most common gynecological cancer in the USA, but its carcinogenic driver status in the endometrial epithelium has not been confirmed. We have identified a novel population of progenitor endometrial epithelial cells (EECs) in mice that express lysozyme M (LysM) and give rise to approximately 15% of all EECs in adult mice. LysM is a glycoside hydrolase that is encoded by Lyz2 and functions to protect cells from bacteria as part of the innate immune system. Its expression has been shown in a subset of hematopoietic stem cells and in specialized lung and small intestinal epithelial cells. Conditional deletion of Apc in LysM + EECs results in significantly more epithelial cells compared to wild-type mice. At 5 months of age, the ApccKO mice have enlarged uterine horns with pathology that is consistent with endometrial hyperplasia with cystic endometrial glands, non-villous luminal papillae and nuclear atypia. Nuclear accumulation of β-catenin and ERα, both of which are known to induce endometrial hyperplasia, was observed in the EECs of the ApccKO mice. These results confirm that loss of APC in EECs can result in a phenotype similar to endometrial hyperplasia.

Desmoplastic small round cell tumor (DSRCT) is a rare pediatric sarcoma with poor overall survival. This tumor is absolutely dependent on the continued expression and activity of its defining molecular lesion, the EWS–WT1 transcription factor. Unfortunately, the therapeutic targeting of transcription factors is challenging, and there is a critical need to identify compounds that inhibit EWS–WT1. Here we show that the compound lurbinectedin inhibits EWS–WT1 by redistributing the protein within the nucleus to the nucleolus. This nucleolar redistribution interferes with the activity of EWS–WT1 to reverse the expression of over 70% of the transcriptome. In addition, the compound blocks the expression of the EWS–WT1 fusion protein to inhibit cell proliferation at the lowest GI50 ever reported for this compound in any cell type. The effects occur at concentrations that are easily achievable in the clinic and translate to the in vivo setting to cause tumor regressions in multiple mice in a xenograft and PDX model of DSRCT. Importantly, this mechanism of nucleolar redistribution is also seen with wild-type EWSR1 and the related fusion protein EWS–FLI1. This provides evidence for a “class effect” for the more than 18 tumors driven by EWSR1 fusion proteins. More importantly, the data establish lurbinectedin as a promising clinical candidate for DSRCT.

Supplementary Data from Lurbinectedin Inhibits the EWS–WT1 Transcription Factor in Desmoplastic Small Round Cell Tumor

Supplementary Figure from Lurbinectedin Inhibits the EWS–WT1 Transcription Factor in Desmoplastic Small Round Cell Tumor

Laboratory values

Desmoplastic small round cell tumor (DSRCT) is a rare pediatric sarcoma with poor overall survival. This tumor is absolutely dependent on the continued expression and activity of its defining molecular lesion, the EWS–WT1 transcription factor. Unfortunately, the therapeutic targeting of transcription factors is challenging, and there is a critical need to identify compounds that inhibit EWS–WT1. Here we show that the compound lurbinectedin inhibits EWS–WT1 by redistributing the protein within the nucleus to the nucleolus. This nucleolar redistribution interferes with the activity of EWS–WT1 to reverse the expression of over 70% of the transcriptome. In addition, the compound blocks the expression of the EWS–WT1 fusion protein to inhibit cell proliferation at the lowest GI50 ever reported for this compound in any cell type. The effects occur at concentrations that are easily achievable in the clinic and translate to the in vivo setting to cause tumor regressions in multiple mice in a xenograft and PDX model of DSRCT. Importantly, this mechanism of nucleolar redistribution is also seen with wild-type EWSR1 and the related fusion protein EWS–FLI1. This provides evidence for a “class effect” for the more than 18 tumors driven by EWSR1 fusion proteins. More importantly, the data establish lurbinectedin as a promising clinical candidate for DSRCT.

There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor–targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1–targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic “class” effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC50 and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the BTG2 gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1–targeted combination therapy for Ewing sarcoma.

Supplementary Table S2 and Supplementary Figures S1 to S10

Supplementary Figure S6 A) In vitro validation of NR0B1 immunofluorescent assay. Data shows images and quantitation of TC32 control cells or TC32 cells following siRNA silencing of NR0B1. B) ki67 and cleaved caspase 3 staining of Ewing sarcoma xenograft tissue. C) Histology from tumor xenograft showing zone of differentiation of Ewing sarcoma cells into benign fat cells. Alu-ish staining (blue) marks human cells.

There is a great need to develop novel approaches to target oncogenic transcription factors with small molecules. Ewing sarcoma is emblematic of this need, as it depends on the continued activity of the EWS-FLI1 transcription factor to maintain the malignant phenotype. We have previously shown that the small molecule trabectedin interferes with EWS-FLI1. Here, we report important mechanistic advances and a second-generation inhibitor to provide insight into the therapeutic targeting of EWS-FLI1. We discovered that trabectedin functionally inactivated EWS-FLI1 by redistributing the protein within the nucleus to the nucleolus. This effect was rooted in the wild-type functions of the EWSR1, compromising the N-terminal half of the chimeric oncoprotein, which is known to be similarly redistributed within the nucleus in the presence of UV light damage. A second-generation trabectedin analogue lurbinectedin (PM01183) caused the same nuclear redistribution of EWS-FLI1, leading to a loss of activity at the promoter, mRNA, and protein levels of expression. Tumor xenograft studies confirmed this effect, and it was increased in combination with irinotecan, leading to tumor regression and replacement of Ewing sarcoma cells with benign fat cells. The net result of combined lurbinectedin and irinotecan treatment was a complete reversal of EWS-FLI1 activity and elimination of established tumors in 30% to 70% of mice after only 11 days of therapy. Our results illustrate the preclinical safety and efficacy of a disease-specific therapy targeting the central oncogenic driver in Ewing sarcoma. Cancer Res; 76(22); 6657–68. ©2016 AACR.

Supplementary Table S3 Table showing list of EWS-FLI1 targets with corresponding references.

Supplementary Data S1A-S9

Purpose:The successful clinical translation of compounds that target specific oncogenic transcription factors will require an understanding of the mechanism of target suppression to optimize the dose and schedule of administration. We have previously shown trabectedin reverses the gene signature of the EWS-FLI1 transcription factor. In this report, we establish the mechanism of suppression and use it to justify the reevaluation of this drug in the clinic in patients with Ewing sarcoma.Experimental Design: We demonstrate a novel epigenetic mechanism of trabectedin using biochemical fractionation and chromatin immunoprecipitation sequencing. We link the effect to drug schedule and EWS-FLI1 downstream target expression using confocal microscopy, qPCR, Western blot analysis, and cell viability assays. Finally, we quantitate target suppression within the three-dimensional architecture of the tumor in vivo using 18F-FLT imaging.Results:Trabectedin evicts the SWI/SNF chromatin-remodeling complex from chromatin and redistributes EWS-FLI1 in the nucleus leading to a marked increase in H3K27me3 and H3K9me3 at EWS-FLI1 target genes. These effects only occur at high concentrations of trabectedin leading to suppression of EWS-FLI1 target genes and a loss of cell viability. In vivo, low-dose irinotecan is required to improve the magnitude, penetrance, and duration of target suppression in the three-dimensional architecture of the tumor leading to differentiation of the Ewing sarcoma xenograft into benign mesenchymal tissue.Conclusions:These data provide the justification to evaluate trabectedin in the clinic on a short infusion schedule in combination with low-dose irinotecan with 18F-FLT PET imaging in patients with Ewing sarcoma.

Legends for supplementary figures and tables.

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. However, the metabolic pathways critical for optimal T cell responses remain poorly understood. Here, we identify ketone bodies (KBs) – including β-hydroxybutyrate (βOHB) and acetoacetate (AcAc) – as essential fuels supporting CD8+ T cell metabolism and effector function. Ketolysis is an intrinsic feature of highly functional CD8+ T effector (Teff) cells and βOHB directly increases CD8+ Teff cell IFN-γ production and cytolytic activity. Using metabolic tracers, we establish that CD8+ Teff cells preferentially use KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boost the respiratory capacity of CD8+ T cells and TCA cycle-dependent metabolic pathways that fuel T cell growth. Mechanistically, we find that βOHB is a major substrate for acetyl-CoA production in CD8+ T cells and regulates effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses.One Sentence summaryKetone bodies promote CD8+ T cell metabolism and effector function through regulation of epigenetic programming

Background: Ewing sarcoma (ES) is dependent on the oncogenic transcription factor EWS-FLI1 for oncogenesis and progression. EWS-FLI1 binds to chromatin to both induce and repress gene expression. These gene expression changes drive proliferation, disrupt the cell cycle, and block cellular differentiation. One approach to target EWS-FLI1 is to reverse the gene signature. However, we have observed an underappreciated but striking heterogeneity of the EWS-FLI1 transcriptome among cell lines. While many common downstream targets exist, at least an equal number of cell-line specific large magnitude gene expression changes are also present. In this study, we evaluate the phenotypic importance of these cell-line specific changes and evaluate potential mechanisms that EWS-FLI1 may use to drive divergent transcription. Methods: The EWS-FLI1 transcriptome was characterized by RNA sequencing, following siRNA silencing of EWS-FLI1 at two time points in 6 cell lines. Effects on specific genes was confirmed by qPCR and western blot analysis. Cellular migration was evaluated using scratch assays on the Incucyte Zoom. EWS-FLI1binding was evaluated using Cleavage Under Targets and Tagmentation assay (CUT&Tag) and the transcriptome of Ewing subpopulations was determined by single cell RNA sequencing. Results: Next generation RNA sequencing of highly optimized siRNA knockdowns of EWS-FLI1 in 6 ES cell lines showed striking differences in gene expression. Conditions were optimized to capture the same magnitude change in EWS-FLI1 expression at the shortest time point ever reported. Fewer common targets were identified across all cell lines than previously reported, perhaps due to the short time frame and focus on direct targets. Independently generated well-established lists of targets were enriched in the data. However, there were 184 genes that changed by a Log2FC &gt 3 (P &lt 0.0001) in only one cell line including important genes like WNT7B and CTLA4. Marked heterogeneity was observed in a previously described migratory transcriptional signature including the key effectors Zyxin and Integrin Alpha-5; an observation confirmed by unbiased pathway analysis and cellular migration assays. Importantly, increased cellular migration was only seen in one cell line with siRNA silencing and not with any EWS-FLI1 targeted small molecules. To characterize the source of heterogeneity, we evaluated EWS-FLI1 binding at Zyxin and Alpha-5 integrin and single cell RNA sequencing of the migratory sub-signature. CUT&Tag demonstrates that EWS-FLI1 is found bound at these genes even in non-migratory cell lines. Conclusion: Careful consideration of Ewing sarcoma model-specific gene expression changes is important to future therapeutic approaches as these unique changes influence cellular phenotype. Citation Format: Seneca S. Kinn-Gurzo, Susan M. Kitchen-Goosen, Maggie Chasse, Marie Adams, Rebecca Kaufman, Patrick J. Grohar. Heterogeneity of the EWS-FLI1 transcriptome drives differences in the cellular phenotype of Ewing sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1466.

There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor–targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1–targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic “class” effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC50 and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the BTG2 gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1–targeted combination therapy for Ewing sarcoma.

How environmental nutrient availability impacts T cell metabolism and function remains poorly understood. Here, we report that the presence of physiologic carbon sources (PCSs) in cell culture medium broadly impacts glucose utilization by CD8+ T cells, independent of transcriptional changes in metabolic reprogramming. The presence of PCSs reduced glucose contribution to the TCA cycle and increased effector function of CD8+ T cells, with lactate directly fueling the TCA cycle. In fact, CD8+ T cells responding to Listeria infection preferentially consumed lactate over glucose as a TCA cycle substrate in vitro, with lactate enhancing T cell bioenergetic and biosynthetic capacity. Inhibiting lactate-dependent metabolism in CD8+ T cells by silencing lactate dehydrogenase A (Ldha) impaired both T cell metabolic homeostasis and proliferative expansion in vivo. Together, our data indicate that carbon source availability shapes T cell glucose metabolism and identifies lactate as a bioenergetic and biosynthetic fuel for CD8+ effector T cells.

How environmental nutrient availability impacts T cell metabolism and function remains poorly understood. Here, we report that the presence of physiologic carbon sources (PCSs) in cell culture medium broadly impacts glucose utilization by CD8

Purpose:The successful clinical translation of compounds that target specific oncogenic transcription factors will require an understanding of the mechanism of target suppression to optimize the dose and schedule of administration. We have previously shown trabectedin reverses the gene signature of the EWS-FLI1 transcription factor. In this report, we establish the mechanism of suppression and use it to justify the reevaluation of this drug in the clinic in patients with Ewing sarcoma.Experimental Design: We demonstrate a novel epigenetic mechanism of trabectedin using biochemical fractionation and chromatin immunoprecipitation sequencing. We link the effect to drug schedule and EWS-FLI1 downstream target expression using confocal microscopy, qPCR, Western blot analysis, and cell viability assays. Finally, we quantitate target suppression within the three-dimensional architecture of the tumor in vivo using 18F-FLT imaging.Results:Trabectedin evicts the SWI/SNF chromatin-remodeling complex from chromatin and redistributes EWS-FLI1 in the nucleus leading to a marked increase in H3K27me3 and H3K9me3 at EWS-FLI1 target genes. These effects only occur at high concentrations of trabectedin leading to suppression of EWS-FLI1 target genes and a loss of cell viability. In vivo, low-dose irinotecan is required to improve the magnitude, penetrance, and duration of target suppression in the three-dimensional architecture of the tumor leading to differentiation of the Ewing sarcoma xenograft into benign mesenchymal tissue.Conclusions:These data provide the justification to evaluate trabectedin in the clinic on a short infusion schedule in combination with low-dose irinotecan with 18F-FLT PET imaging in patients with Ewing sarcoma.

Rhabdoid tumor (RT) is a pediatric cancer characterized by the inactivation of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex. Although this deletion is the known oncogenic driver, there are limited effective therapeutic options for these patients. Here we use unbiased screening of cell line panels to identify a heightened sensitivity of rhabdoid tumor to mithramycin and the second‐generation analogue EC8042. The sensitivity of MMA and EC8042 was superior to traditional DNA damaging agents and linked to the causative mutation of the tumor, SMARCB1 deletion. Mithramycin blocks SMARCB1‐deficient SWI/SNF activity and displaces the complex from chromatin to cause an increase in H3K27me3. This triggers chromatin remodeling and enrichment of H3K27ac at chromHMM‐defined promoters to restore cellular differentiation. These effects occurred at concentrations not associated with DNA damage and were not due to global chromatin remodeling or widespread gene expression changes. Importantly, a single 3‐day infusion of EC8042 caused dramatic regressions of RT xenografts, recapitulated the increase in H3K27me3, and cellular differentiation described in vitro to completely cure three out of eight mice., EC8042 is identified as an inhibitor of oncogenic SWI/SNF and a promising therapeutic candidate for rhabdoid tumor. The mechanism of target inhibition is elucidated and used to optimize compound administration, characterize an associated biomarker, and cure mice bearing rhabdoid tumor xenografts.

Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive soft-tissue malignancy with a poor overall survival and no effective therapeutic options. The tumor is believed to be dependent on the continued activity of the oncogenic EWS-WT1 transcription factor. However, the dependence of the tumor on EWS-WT1 has not been well established. In addition, there are no studies exploring the downstream transcriptional program across multiple cell lines. In this study, we have developed a novel approach to selectively silence EWS-WT1 without impacting either wild-type EWSR1 or WT1. We show a clear dependence of the tumor on EWS-WT1 in two different cell lines, BER and JN-DSCRT-1. In addition, we identify and validate important downstream target pathways commonly dysregulated in other translocation-positive sarcomas, including PRC2, mTOR, and TGFB. Surprisingly, there is striking overlap between the EWS-WT1 and EWS-FLI1 gene signatures, despite the fact that the DNA-binding domain of the fusion proteins, WT1 and FLI1, is structurally unique and classified as different types of transcription factors. This study provides important insight into the biology of this disease relative to other translocation-positive sarcomas, and the basis for the therapeutic targeting of EWS-WT1 for this disease that has limited therapeutic options.

Summary Cancer cells display metabolic plasticity to survive stresses in the tumor microenvironment. Cellular adaptation to energetic stress is coordinated in part by signaling through the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Here, we demonstrate that miRNA-mediated silencing of LKB1 confers sensitivity of lymphoma cells to mitochondrial inhibition by biguanides. Using both classic (phenformin) and newly developed (IM156) biguanides, we demonstrate that elevated miR-17∼92 expression in Myc+ lymphoma cells promotes increased apoptosis to biguanide treatment in vitro and in vivo. This effect is driven by the miR-17-dependent silencing of LKB1, which reduces AMPK activation in response to complex I inhibition. Mechanistically, biguanide treatment induces metabolic stress in Myc+ lymphoma cells by inhibiting TCA cycle metabolism and mitochondrial respiration, exposing metabolic vulnerability. Finally, we demonstrate a direct correlation between miR-17∼92 expression and biguanide sensitivity in human cancer cells. Our results identify miR-17∼92 expression as a potential biomarker for biguanide sensitivity in malignancies., Graphical Abstract, Highlights IM156 is a newly developed biguanide with more potency than phenformin IM156 inhibits mitochondrial respiration to cause bioenergetic stress in lymphoma miR-17-dependent silencing of LKB1 reduces adaptation to bioenergetic stress miR-17∼92 expression correlates with biguanide sensitivity in human cancer cells, Izreig et al. show that miR-17∼92 expression in cancer cells sensitizes them to biguanide treatment by disrupting bioenergetic stability. These data implicate miR-17∼92 expression as a potential biomarker for biguanide sensitivity in hematological malignancies and possibly solid tumors.

Purpose: The goal of this study was to identify second-generation mithramycin analogues that better target the EWS-FLI1 transcription factor for Ewing sarcoma. We previously established mithramycin as an EWS-FLI1 inhibitor, but the compound's toxicity prevented its use at effective concentrations in patients. Experimental Design: We screened a panel of mithralogs to establish their ability to inhibit EWS-FLI1 in Ewing sarcoma. We compared the IC50 with the MTD established in mice to determine the relationship between efficacy and toxicity. We confirmed the suppression of EWS-FLI1 at the promoter, mRNA, gene signature, and protein levels. We established an improved therapeutic window by using time-lapse microscopy to model the effects on cellular proliferation in Ewing sarcoma cells relative to HepG2 control cells. Finally, we established an improved therapeutic window using a xenograft model of Ewing sarcoma. Results: EC-8105 was found to be the most potent analogue and was able to suppress EWS-FLI1 activity at concentrations nontoxic to other cell types. EC-8042 was substantially less toxic than mithramycin in multiple species but maintained suppression of EWS-FLI1 at similar concentrations. Both compounds markedly suppressed Ewing sarcoma xenograft growth and inhibited EWS-FLI1 in vivo. Conclusions: These results provide a basis for the continued development of EC-8042 and EC-8105 as EWS-FLI1 inhibitors for the clinic. Clin Cancer Res; 22(16); 4105–18. ©2016 AACR.

The text of this abstract has not been printed at the request of the authors due to intellectual property matters. Citation Format: Maggie H. Chasse, Benjamin K. Johnson, Elissa A. Boguslawski, Katie M. Sorensen, Ian Beddows, Zachary Madaj, Gabrielle E. Foxa, Kristin J. Rybski, Susan M. Kitchen-Goosen, Bart O. Williams, Timothy J. Triche Jr., Patrick J. Grohar. Mithramycin evicts SWI/SNF from chromatin to induce epigenetic reprogramming in rhabdoid tumor [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A75.

Megakaryocytes are highly specialized precursor cells that produce platelets via cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and actin organization. In this work, we demonstrated that DIAPH1 (mDia1), a mammalian homolog of Drosophila diaphanous that works as an effector of the small GTPase Rho, negatively regulates PPF by controlling the dynamics of the actin and microtubule cytoskeletons. Moreover, we showed that inhibition of both DIAPH1 and the Rho-associated protein kinase (Rock)/myosin pathway increased PPF via coordination of both cytoskeletons. We provide evidence that 2 major effectors of the Rho GTPase pathway (DIAPH1 and Rock/myosin II) are involved not only in Rho-mediated stress fibers assembly, but also in the regulation of microtubule stability and dynamics during PPF.

BACKGROUND: Rhabdoid tumor (RT) is an aggressive pediatric malignancy defined by the genetic deletion of SMARCB1 (BAF47/SNF5/INI1), a subunit of the SWI/SNF chromatin remodeling complex. SMARCB1 loss redistributes the SWI/SNF complex to favor a proliferative non-differentiated cellular state. Nevertheless, while EZH2, the catalytic subunit of PRC2, is an established, synthetically lethal therapeutic target, other vulnerabilities that result from this imbalance between the SWI/SNF and PRC2 complexes are relatively unexplored. METHODS: In a screen of multiple pediatric cancer cell lines, we identified a heightened sensitivity of RT cells to mithramycin (MMA), a small molecule transcription inhibitor. We hypothesized the hypersensitivity of rhabdoid tumor to mithramycin was due to the disruption of SWI/SNF and PRC2 dynamics. Here, we describe an epigenetic mechanism of action for mithramycin in rhabdoid tumor. We characterize KDM6A, a H3K27me3 histone demethylase, as a novel therapeutic vulnerability in rhabdoid tumor with live cell imaging, western blot, small molecule inhibition, and chromatin immunoprecipitation (ChIP) sequencing. We identify SWI/SNF eviction from chromatin as a novel mechanism of action for mithramycin with biochemical fractionation, ChIP-seq, and ATAC-seq. Finally, we demonstrate these findings in vivo using RT xenograft models. RESULTS: Mithramycin induced apoptosis and a striking increase in H3K27me3, the catalytic mark of PRC2, in rhabdoid tumor cell lines in a dose and time-dependent manner. This amplification of H3K27me3 preceded suppression of cellular proliferation and induction of apoptosis suggesting H3K27me3 amplification may drive the apoptotic phenotype. In addition, knockdown of KDM6A, a H3K27me3 histone demethylase, potentiates the effects of MMA providing further evidence that KDM6A loss and H3K27me3 amplification lead to apoptosis. We have identified SP1 as the transcription factor driving KDM6A and mithramycin interferes with SP1-dependent activation of KDM6A by evicting SWI/SNF from chromatin. We are currently working on the mechanism of action for mithramycin in rhabdoid tumor cells, including H3K27me3 ChIP-seq and ATAC-seq to elucidate global transcription and chromatin changes. We have recapitulated these results in vivo with xenograft mouse models. CONCLUSIONS: Mithramycin inhibits KDM6A, a histone demethylase, and SWI/SNF leading to apoptosis through the amplification of H3K27me3. Eviction of SWI/SNF from chromatin is a novel mechanism of action for mithramycin. Overall, this study indicates KDM6A and SWI/SNF loss are novel therapeutic vulnerabilities in rhabdoid tumor and mithramycin may be a promising clinical candidate for the treatment of rhabdoid tumor. Citation Format: Maggie H. Chasse, Elissa Boguslawski, Katie Sorensen, Ian Beddows, Kristin Rybski, Zachary Madaj, Susan M. Kitchen-Goosen, Patrick J. Grohar. Mithramycin inhibits KDM6A (UTX) and SWI/SNF to induce apoptosis in malignant rhabdoid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2883.

There is a great need to develop novel approaches to target oncogenic transcription factors with small molecules. Ewing sarcoma is emblematic of this need, as it depends on the continued activity of the EWS-FLI1 transcription factor to maintain the malignant phenotype. We have previously shown that the small molecule trabectedin interferes with EWS-FLI1. Here, we report important mechanistic advances and a second-generation inhibitor to provide insight into the therapeutic targeting of EWS-FLI1. We discovered that trabectedin functionally inactivated EWS-FLI1 by redistributing the protein within the nucleus to the nucleolus. This effect was rooted in the wild-type functions of the EWSR1, compromising the N-terminal half of the chimeric oncoprotein, which is known to be similarly redistributed within the nucleus in the presence of UV light damage. A second-generation trabectedin analogue lurbinectedin (PM01183) caused the same nuclear redistribution of EWS-FLI1, leading to a loss of activity at the promoter, mRNA, and protein levels of expression. Tumor xenograft studies confirmed this effect, and it was increased in combination with irinotecan, leading to tumor regression and replacement of Ewing sarcoma cells with benign fat cells. The net result of combined lurbinectedin and irinotecan treatment was a complete reversal of EWS-FLI1 activity and elimination of established tumors in 30% to 70% of mice after only 11 days of therapy. Our results illustrate the preclinical safety and efficacy of a disease-specific therapy targeting the central oncogenic driver in Ewing sarcoma. Cancer Res; 76(22); 6657–68. ©2016 AACR.

Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined. Here, we show that the migration of immature DCs depends on two main actin pools: a RhoA-mDia1-dependent actin pool located at their rear, which facilitates forward locomotion; and a Cdc42-Arp2/3-dependent actin pool present at their front, which limits migration but promotes antigen capture. Following TLR4-MyD88-induced maturation, Arp2/3-dependent actin enrichment at the cell front is markedly reduced. Consequently, mature DCs switch to a faster and more persistent mDia1-dependent locomotion mode that facilitates chemotactic migration to LVs and lymph nodes. Thus, the differential use of actin-nucleating machineries optimizes the migration of immature and mature DCs according to their specific function.

BACKGROUND: Rhabdoid tumor (RT) is an aggressive pediatric malignancy defined by the genetic deletion of BAF47 (SMARCB1/SNF5/INI1), a subunit of the BAF chromatin remodeling complex. Loss of BAF47 leads to BAF complex redistribution and overexpression of EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2) to favor a proliferative non-differentiated cellular state. Nevertheless, while EZH2 itself is an established, synthetically lethal therapeutic target, other vulnerabilities that result from this imbalance between the BAF and PRC2 complexes are relatively unexplored. EXPERIMENTAL DESIGN: In a screen of multiple pediatric cancer cell lines, we identified a heightened sensitivity of RT cells to mithramycin (MMA), a small molecule transcription inhibitor. We hypothesized the hypersensitivity of rhabdoid tumor to mithramycin was due to the disruption of BAF and PRC2 dynamics. Here, we characterized the activity of mithramycin in three RT cell lines. To understand the effect of MMA, we performed in vitro qRT-PCR, immunoblot assays, and viability assays to characterize the expression and activity of the PRC2 complex, including a H3K27me3 histone demethylase. We further investigate the kinetics of cell viability and induction of apoptosis with live cell imaging. Finally, we demonstrate these findings in vivo using rhabdoid tumor xenograft models. RESULTS: Mithramycin inhibited mRNA and protein expression of all four PRC2 subunits in a dose-dependent manner in two different rhabdoid tumor cell lines. Even though EZH2 is suppressed with MMA treatment, we found a striking increase in H3K27me3 that correlated with EZH2 trafficking into the nucleus. This amplification of H3K27me3 preceded suppression of cellular proliferation and induction of apoptosis suggesting H3K27me3 amplification may drive the apoptotic phenotype. In addition, knockdown of KDM6A potentiates the effects of MMA providing further evidence that KDM6A loss and H3K27me3 amplification lead to apoptosis. We are currently working on the mechanism of action for mithramycin in RT cells, including H3K27me3 ChIP-seq and RNA-seq to elucidate global transcription and chromatin changes. We have recapitulated these results in vivo with xenograft mouse models. CONCLUSIONS: Mithramycin represses KDM6A, a histone demethylase, leading to apoptosis through the amplification of H3K27me3. Here, H3K27me3 amplification, in addition to depletion, is detrimental to rhabdoid tumor proliferation and progression. Overall, this study indicates KDM6A loss and H3K27me3 amplification are novel therapeutic vulnerabilities in rhabdoid tumor. Citation Format: Maggie H. Chasse, Elissa Boguslawski, Courtney E. Wernette, Susan Kitchen-Goosen, Patrick J. Grohar. Mithramycin amplifies the imbalance between the BAF and PRC2 complexes to drive apoptosis in rhabdoid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4634.

BACKGROUND: Ewing sarcoma (ES) is the second most common pediatric bone cancer. ES cells are dependent on the activity of the EWS/FLI1 transcription factor for cell survival. EWS/FLI1 drives proliferation, dysregulates the cell cycle and establishes a block in differentiation. Despite the known dependence of Ewing sarcoma on EWS/FLI1, the exact identity of all the downstream targets of EWS/FLI1 is not known. While gene signatures have been reported for EWS/FLI1, these fail to capture the marked heterogeneity in the EWS/FLI1 transcriptome or correlate these expression changes with cellular phenotype. METHODS: Next generation RNA sequencing with deep coverage was used to generate a comprehensive analysis of the EWS/FLI1 transcriptome by silencing EWS/FLI1 in 5 different ES cell lines at two different time points. These results were organized into gene expression networks and signaling nodes were identified by weighted correlation network analysis (WGCNA). The networks were validated by a 500-gene signature of EWS/FLI1 targets using a next generation sequencing capture assay and the Illumina platform. The cellular endpoint of EWS/FLI1 silencing was evaluated by caspase 3,7 cleavage to measure apoptosis, BGAL staining to measure senescence and scratch assays to capture migration. RESULTS: Next generation RNA sequencing with siRNA silencing of EWS/FLI1 revealed marked heterogeneity in the EWS/FLI1 transcriptome among cell lines. Targets of EWS/FLI1 that were common across cell lines exhibited small magnitude gene expression changes (log fold 2). When the log fold change in expression was restricted to greater than 2, only 13 common repressed targets and 4 common induced targets were identified in all 5 cell lines. In addition, silencing of EWS/FLI1 induced dramatically different phenotypes across cell lines, where p53 wild-type cell lines underwent senescence and the p53 mutant cell lines did not. Most cell lines did not continue to proliferate with EWS/FLI1 silencing with the exception being the widely used A673 cell line. Cellular migration did not change with EWS/FLI1 silencing in many of the cell lines in contrast to A673 cells. CONCLUSIONS: EWS/FLI1 drives different gene expression signatures and different phenotypes in unique cell lines. This diversity needs to be considered as EWS/FLI1 directed therapies are developed. Citation Format: Susan M. Kitchen-Goosen, Megan J. Bowman, Marie Adams, Penny Berger, Patrick J. Grohar. The EWS/FLI1 transcriptome is characterized by marked heterogeneity across Ewing sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3356.

Although the cancer cell cytoskeleton is a clinically validated target, few new strategies have emerged for selectively targeting cell division by modulating the cytoskeletal structure, particularly ways that could avoid the cardiotoxic and neurotoxic effects of current agents such as taxanes. We address this gap by describing a novel class of small-molecule agonists of the mammalian Diaphanous (mDia)-related formins, which act downstream of Rho GTPases to assemble actin filaments, and their organization with microfilaments to establish and maintain cell polarity during migration and asymmetric division. GTP-bound Rho activates mDia family members by disrupting the interaction between the DID and DAD autoregulatory domains, which releases the FH2 domain to modulate actin and microtubule dynamics. In screening for DID–DAD disruptors that activate mDia, we identified two molecules called intramimics (IMM-01 and -02) that were sufficient to trigger actin assembly and microtubule stabilization, serum response factor-mediated gene expression, cell-cycle arrest, and apoptosis. In vivo analysis of IMM-01 and -02 established their ability to slow tumor growth in a mouse xenograft model of colon cancer. Taken together, our work establishes the use of intramimics and mDia-related formins as a new general strategy for therapeutic targeting of the cytoskeletal remodeling machinery of cancer cells. Cancer Res; 73(22); 6793–803. ©2013 AACR.

Corrigendum: Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells

While the murine equivalent to “5q– syndrome” should map to the distal arms of murine chromosomes 11 and 18, molecular attempts have been unsuccessful in generating a reliable mouse model of myelodysplasia leading to exacerbated myeloid leukemia. Genetic studies suggest interactions between individual proteins in this locus suppress tumor initiation. More specifically, cellular and biochemical studies demonstrated collaboration between two proteins in the commonly deleted regions of 5q– syndrome: mDia1 (mammalian Diaphanous-related Rho GTPase effector formin) and APC (adenomatous polypolis coli). Therefore, we generated mice compounding these two putative disease alleles. Because both mDia1 knockout [Drf1(–/–)] mice and Apcmin (min = multiple intestinal neoplasia; haploinsufficient) mice develop age-dependent myelodysplastic syndrome (MDS), we hypothesized that a mouse strain lacking both proteins will exhibit a more severe MDS phenotype than either mutant allele alone. To define the disease caused by mutation in Drf1 and Apcmin, we monitored erythropoiesis, myelopoiesis, and overall survival. At all ages, mice with both mutant alleles exhibit granulocytosis. Anemia occurs in aged mice (>270 days), exhibiting drastic differences in blood cell morphology that corresponded to differences in long-term survival in comparison to either Drf1–/– or Apcmin animals. Furthermore, immunophenotyping of all subpopulations indicate an accumulation of CD71+/TER-119+ cells in mice with both mutant alleles, however no significant differences in CD11b expression appeared among CD45+ bone marrow subpopulations. Our studies indicate that combined loss of mDia1 and APC expression and function direct disease progression. We are currently defining the hematopoietic precursor cell responsible for anemia and bone marrow failure. This enhanced mouse model also serves as molecular laboratory to study disease progression and to test potential therapeutics under development in our laboratory. Citation Format: Julie Davis Turner, Susan Kitchen-Goosen, Andrew M. Howard, Heather Schumacher, Art S. Alberts. Modeling MDS in mice through precise molecular lesions in APC and mDia1. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B42.

Myelodysplastic syndromes (MDS) are characterized by complex cytogenetic abnormalities, including loss of all or part of the long arm of chromosome 5 (5q). Known 5q tumor suppressors include APC1 and EGR1. APC is associated with familial colon cancer where it functions to suppress canonical Wnt signaling. Egr1 is a transcription factor that governs expression of tumor suppressor genes ranging from the p53 family to PTEN1. Adjacent to EGR1 is the DIAPH1 gene for the Rho GTPase effector formin DIAPH1. Knockout of Diaph1, Egr, or Apc in mice creates a range of MDS phenotypes. Likewise, mice with the multiple intestinal neoplasia (min/Apcmin) allele develop anemia and MDS most similar to the Diaph1 knockout phenotype. DIAPH1 builds linear actin filaments and stabilizes microtubule dynamics to establish and maintain cell polarity during adhesion, migration, and asymmetric cell division when activated by Rho GTPases, such as RhoB. Combinatorial deficiencies in RhoB (also a known tumor susceptibility gene) with Diaph1 exacerbates the Diaph1 MDS phenotype but not the rate of pathogenesis. RhoB-activated DIAPHs also signal and activate new gene expression by the myocardin-related (MRTF) and Mcm10/Agamous deficiens/ [serum response factor (SRF)] (MADS)-box transcription factors. MRTF-MADS registers changes in actin dynamics downstream of DIAPH1 to activate EGR1 expression through multiple serum response elements (SREs; recognized by SRF) within the EGR1 promoter. DIAPH1 also collaborates with APC in cytoskeletal remodeling as both proteins physically associate with and stabilize microtubules. APC can also nucleate new actin filaments that become processively elongated by DIAPH1. We examined interactions between APC and DIAPH1 signaling to SRF and in suppressing MDS pathogenesis. First, we found that both DIAPH1 and APC activate SRF- regulated gene expression. Consistent with biochemical data showing that DIAPH1 elongates APC-nucleated filaments, activation of SRF by APC relies upon DIAPH1. We bred Apcmin mice to our Diaph1 knockout mice and found that the MDS phenotype is significantly accelerated in Apcmin/Diaph1 knockout mice when compared to either the Apcmin or Diaph1 mutant phenotype. Pathological defects (dysplasia and anemia in peripheral blood and myeloid organs (spleen/bone marrow) requires at least 200 days to develop in either single mutant; combined mutations create aberrant peripheral blood cell pathology by 100 days of age. Consistent with early disease onset, mortality is accelerated in Apcmin /Diaph1-deficient mice. These novel studies show that genetic defects in Diaph1 and Apc synergize to effectively mimic 5q- MDS. Further, the data supports the idea that RhoB - DIAPH1/APC and MRTF-MADS comprise a novel tumor suppression pathway that is essential for hematopoiesis. Apcmin/Diaph1-deficient mice may prove to be a useful pre-clinical model for the development of more effective theranostic tools to attack MDS. Citation Format: Julie D. Turner, Susan M. Kitchen-Goosen, Andrew M. Howard, Heather L. Schumacher, Arthur S. Alberts. APC and DIAPH1 collaborate to suppress myelodysplastic syndrome (MDS) pathogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4194. doi:10.1158/1538-7445.AM2015-4194

Rho small GTP-binding proteins locally manage Diaphanous-related formins as they construct the cytoskeletal infrastructure that supports essential changes in cell morphology. Rho binding unlatches formin intramolecular autoinhibition. The autoinhibition is mediated by the Dia-inhibitory and -autoregulatory domains (DID & DAD) which flank the formin homology-2 (FH2) domain. The free FH2 domain guides the assembly of the linear actin and microtubule structures required for adhesion, migration, division, and polarity establishment. Microtubule stabilization is already known to be an effective anti-cancer strategy. Combined with the knowledge that formins can act as haploinsufficient tumor suppressor genes, a screen was conducted for agents that could impair autoinhibition in order to activate formins for therapeutic benefit. We have focused on two of the primary “hits” for development. VARI01 and VARI02 are nearly identical thioamide inhibitors of DID-DAD binding (IC50 values ≤ 140 nM). Exposure of cultured tumor cells to either compound triggered the expected changes in cell structure and signaling that were consistent with formin activation. These included microtubule stabilization, F-actin assembly, the activation of the transcription factor SRF, cell-cycle arrest, and eventual apoptosis. SRF senses changes in actin dynamics to activate the expression of genes that reinforce cell structure and monitor genome integrity. VARI01 and VARI02 both exhibit anti-tumor activity in xenograft studies. Ongoing studies are aimed at identifying VARI01/VARI02-sensitive tumor types and to define other potential therapeutic applications. Significance: Microtubule stabilization by taxol has proven to be an effective anti-cancer strategy, but it has significant deleterious side effects. Small-molecule formin activation serve as a novel class of anti-cancer agents that may provide tools for unlocking the biological roles of formins in signalling and disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4764. doi:1538-7445.AM2012-4764

The Adenomatous polyposis coli (APC1) tumor suppressor is a multi-function protein that is involved in both the Wnt signaling cascade and cytoskeletal remodeling. Mutations or loss of APC1 can result in defects in cell migration, proliferation, and differentiation. APC frequently collaborates with the canonical Diaphanous-related formin, mDia1 (DIAPH1), which also functions to regulate cytoskeletal dynamics. APC1 and DIAPH1 are genetically linked on Chromosome 5q in a region frequently disrupted in myeloproliferative neoplasms (MPNs). Mice lacking mDia1 (Drf1) and the Apc min mice develop myelodysplastic phenotypes. The physical and functional linkage of APC and mDia1 as 5q tumor suppressors and in cytoskeletal remodeling led us to explore the role of APC in tumor suppression in myeloid-derived cell lineages. We created a mouse which lacks APC in myeloid lineage cells by breeding Apc-flox mice with mice expressing Cre recombinase under the control of lysozyme M (LyzsM) gene promoter. Lysozyme M is expressed in myeloid lineage cells including monocytes, macrophages and granulocytes. Surprisingly, the female mice develop cystic endometrial hyperplasia and are unable to sustain pregnancy. Lysozyme M expression appears to be limited to tissue resident macrophages within the uterus and was not detected in uterine epithelial cells. However, there is a small subset of uterine epithelial cells with increased β-catenin expression, suggesting that these cells lack APC. This subset of cells has elevated expression of cyclin D1, a regulator of cell cycle progression and a downstream effector of Wnt signaling, resulting in increased proliferation. Interestingly, the uterine epithelial cells with increased β-catenin expression also have increased estrogen receptor ≤ (ERα) expression. Estrogen stimulates uterine growth and unopposed estrogen signaling is a hallmark of Type I endometrial cancer. It is unclear if the endometrial hyperplasia seen in these mice is due to lysozyme M expression early on in uterine epithelial cell development or perhaps in a progenitor cell population. A second alternative is that loss of APC in tissue resident myeloid cells residing in the uterine stroma affects epithelial morphology. Regardless, these data suggest that APC plays a role in uterine tissue homeostasis, perhaps even regulating ERα expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3280. doi:1538-7445.AM2012-3280

The Adenomatous polyposis coli (APC1) tumor suppressor is a multi-function protein with a central role in Wnt-regulated cell growth and differentiation. Additionally, APC functions to modulate microtubule and microfilament dynamics to establish polarity in epithelia during directed cell migration and asymmetric cell division. APC frequently collaborates with the canonical Diaphanous-related formin, mDia1 (DIAPH1), which also binds microfilaments and microtubules to modulate cytoskeletal dynamics. APC1 and DIAPH1 are genetically linked on chromosome 5q in a region frequently disrupted in myeloproliferative neoplasms (MPNs). mDia1 knockout mice develop an age-dependent outgrowth of myeloid-lineage cells. The physical and functional linkage of APC and mDia1 as 5q tumor suppressors and in cytoskeletal remodeling led us to explore the role of APC in tumor suppression in myeloid-derived cell lineages affected by mDia1 knockout. To test this hypothesis in a genetic model, we created a mouse in which APC was knocked out in myeloid lineage cells by breeding the Apc-flox mice with mice expressing Cre recombinase under the control of lysozyme M (LyzsM) gene promoter. The resulting phenotype was unexpected and 100% penetrant: Female mice developed cystic endometrial hyperplasia and were unable to sustain pregnancy. Embryo implantation occurred, but embryos were resorbed mid-gestation irrespective of progeny genotype. Two hypothesized mechanisms to explain the initiation of endometrial hyperplasia are being pursued. The first hypothesis is that loss of APC impairs the homeostasis between growth and differentiation of myeloid lineage stem/progenitor cells that transdifferentiate into uterine epithelia. An alternate hypothesis is that loss of APC expression affects the inflammatory response of resident uterine macrophages that lead to defects in epithelial morphology. In either case, these genetic studies imply that APC gene defects in myeloid lineage cells may be an initiating event in endometrial cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4330. doi:10.1158/1538-7445.AM2011-4330

Summary Cancer cells display metabolic plasticity to survive stresses in the tumor microenvironment. Cellular adaptation to energetic stress is coordinated in part by signaling through the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Here, we demonstrate that miRNA-mediated silencing of LKB1 confers sensitivity of lymphoma cells to mitochondrial inhibition by biguanides. Using both classic (phenformin) and newly developed (IM156) biguanides, we demonstrate that elevated miR-17∼92 expression in Myc+ lymphoma cells promotes increased apoptosis to biguanide treatment in vitro and in vivo. This effect is driven by the miR-17-dependent silencing of LKB1, which reduces AMPK activation in response to complex I inhibition. Mechanistically, biguanide treatment induces metabolic stress in Myc+ lymphoma cells by inhibiting TCA cycle metabolism and mitochondrial respiration, exposing metabolic vulnerability. Finally, we demonstrate a direct correlation between miR-17∼92 expression and biguanide sensitivity in human cancer cells. Our results identify miR-17∼92 expression as a potential biomarker for biguanide sensitivity in malignancies.