Preclinical Models Of Ewing Sarcoma Research Articles

Enhancing Standard of Care Chemotherapy Efficacy Using DNA-Dependent Protein Kinase (DNA-PK) Inhibition in Preclinical Models of Ewing Sarcoma.

Disruption of DNA damage repair via impaired homologous recombination is characteristic of Ewing sarcoma (EWS) cells. We hypothesize that this disruption results in increased reliance on nonhomologous end joining to repair DNA damage. In this study, we investigated if pharmacologic inhibition of the enzyme responsible for nonhomologous end joining, the DNA-PK holoenzyme, alters the response of EWS cells to genotoxic standard of care chemotherapy. We used analyses of cell viability and proliferation to investigate the effects of clinical DNA-PK inhibitors (DNA-PKi) in combination with six therapeutic or experimental agents for EWS. We performed calculations of synergy using the Loewe additivity model. Immunoblotting evaluated treatment effects on DNA-PK, DNA damage, and apoptosis. Flow cytometric analyses evaluated effects on cell cycle and fate. We used orthotopic xenograft models to interrogate tolerability, drug mechanism, and efficacy in vivo. DNA-PKi demonstrated on-target activity, reducing phosphorylated DNA-PK levels in EWS cells. DNA-PKi sensitized EWS cell lines to agents that function as topoisomerase 2 (TOP2) poisons and enhanced the DNA damage induced by TOP2 poisons. Nanomolar concentrations of single-agent TOP2 poisons induced G2M arrest and little apoptotic response while adding DNA-PKi-mediated apoptosis. In vivo, the combination of AZD7648 and etoposide had limited tolerability but resulted in enhanced DNA damage, apoptosis, and EWS tumor shrinkage. The combination of DNA-PKi with standard of care TOP2 poisons in EWS models is synergistic, enhances DNA damage and cell death, and may form the basis of a promising future therapeutic strategy for EWS.

Abstract 1294: Dual targeting of PDK1 and Aurora A using first-in class OXID-pyridonyl compounds in preclinical models of Ewing sarcoma

Abstract Background. PDK1, is a master activator of multiple prosurvival and oncogenic protein kinases such as AKT, PKC, RSK and SGK families in oncology[1]. On the other hand, overexpression and aberrant activation of Aurora-A kinase (AurA) have been functionally linked to oncogenic transformation mainly through development of centrosome amplification and chromosomal instability[2]. In this study, the molecular mechanisms at the simultaneous PDK1/AurA inhibition using OXID-pyridonyl[3] compounds were explored in panel of tumor cell lines carrying different oncogenic mutations (PI3KCA, K-RAS, PTEN, RB-1, EGFR and TP53). Materials and Methods. Established human cancer cell lines from breast, glioblastoma, renal prostate, colon, lung and Ewing sarcoma were studied. Anti-proliferative effects of OXID-pyridonyl compounds (SA16, IB35, VI8, VI18) were assessed by soft agar clonogenic formation and MTT assays after 72h-exposure with increasing concentrations (from 0.014 to 30 µM). Protein levels were analyzed by Western Blot using commercial antibodies. OSU-03012 (PDK1 inhibitor) and MK-5108 (AurA inhibitor) were used as reference compounds to investigate the functional crosstalk between the two pathways in sensitive cancer cell lines. Results. We assessed anti-proliferative activity of single-agents SA16, IB35, VI8 and VI18 in a panel of 15 cancer cell lines and observed that three Ewing sarcoma and one colon carcinoma cell lines were sensitive (6647,TC71, SKNMC and HCT116), with IC50s values between 2.6 and 25 µM after 72h-exposure, whereas all the studied cell lines were resistant to IB35 and VI18. These results were confirmed by soft agar clonogenic formation studies. AurA, PDK-1, p-PDK1 ser241, AKT, p-AKT ser473 protein levels were characterized but no differences in their basal levels were observed between OXID-pyridonyl compounds -sensitive or -resistant cell lines. SA16 effect at the protein level was evaluated after 5 to 120 min in sensitive cell lines. In SKNMC, TC71 and HCT116 cells, we didn't observe a modulation of AurA or p-PDK1 ser241 while a decrease of p-AKT was observed in SKNMC and HCT116 cells. In addition, a decrease of the p-p44/42 MAPK was observed after 30 min-exposure in SKNMC cells. On the other hand, p-RB1, p-Cdc2, P21 and C-MYC were not regulated at the protein level with SA16 treatment up to 120 min-exposure. In resistant cell lines, U87MG and PC3, both p-PDK1 ser241 and AurA levels were not modulated up to 2h of treatment with SA16. Conclusion. Here we report the characterization of two potent first-in-class dual PDK1-AurA inhibitors, SA16 and VI8. Our findings suggest that innovative PDK1/AurA dual-target molecules which could represent an attractive lead scaffold for the design and synthesis of a new multitarget strategy for Ewing sarcoma. [1] Nagashima K et al. J Biol Chem. 2011. [2] Yan M et al. Med Res Rev. 2016. [3] Sestito, S et al. Proceedings 2019. Citation Format: Maria Eugenia Riveiro, Simona Rapposelli, Samuel Huguet, Ramiro Vazquez, Mohamed Bekradda, Guido Puricelli, Simona Sestito, Olivier Madar, Keyvan Rezai. Dual targeting of PDK1 and Aurora A using first-in class OXID-pyridonyl compounds in preclinical models of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1294.

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) with OT-82 induces DNA damage, cell death, and suppression of tumor growth in preclinical models of Ewing sarcoma

NAMPT mediates the rate-limiting step of the NAD salvage pathway, which maintains cellular bioenergetics and provides a necessary substrate for functions essential to rapidly proliferating cancer cells. In this study, we evaluated the efficacy and mechanisms of action of OT-82, a novel, high-potency NAMPT inhibitor with a favorable toxicity profile, in preclinical models of Ewing sarcoma (EWS), an aggressive pediatric malignancy with previously reported selective sensitivity to NAMPT inhibition. We show that OT-82 decreased NAD concentration and impaired proliferation of EWS cells in a dose-dependent manner, with IC50 values in the single-digit nanomolar range. Notably, genetic depletion of NAMPT phenocopied pharmacological inhibition. On-target activity of OT-82 was confirmed with the addition of NMN, the product of NAMPT, which rescued NAD concentration and EWS cellular viability. Mechanistically, OT-82 treatment resulted in impaired DNA damage repair through loss of PARP activity, G2 cell-cycle arrest, and apoptosis in EWS cells. Additional consequences of OT-82 treatment included reduction of glycolytic and mitochondrial activity. In vivo, OT-82 impaired tumor growth and prolonged survival in mice bearing EWS xenografts. Importantly, antitumor effect correlated with pharmacodynamic markers of target engagement. Furthermore, combining low-dose OT-82 with low doses of agents augmenting DNA damage demonstrated enhanced antitumor activity in vitro and in vivo. Thus, OT-82 treatment represents a potential novel targeted approach for the clinical treatment of EWS.

Abstract 5714: Targeting serine metabolism impairs tumor growth in preclinical models of Ewing sarcoma

Abstract Despite a growing body of knowledge about the genomic landscape of Ewing sarcoma (ES), translation of basic discoveries into targeted therapies and significant clinical gains has remained elusive. Recent insights have revealed that the oncogenic transcription factor EWS-FLI1 can impact ES cellular metabolism, raising the possibility of targeting metabolic dependencies as a novel therapeutic strategy. Here, we show that 3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme in de novo serine synthesis, is highly expressed in human ES cells and that EWS-FLI1 regulates PHGDH expression and de novo serine synthesis. PHGDH knockdown resulted in decreased ES cell proliferation, especially under serine limitation, and significantly inhibited xenograft tumorigenesis in preclinical orthotopic models of ES. Additionally, the PHGDH inhibitor NCT-503 caused a dose-dependent decrease in cellular proliferation. Moreover, we report a novel drug combination in which nicotinamide phosphoribosyltransferase (NAMPT) inhibition, which blocks production of the PHGDH substrate NAD+, synergized with NCT-503 to abolish ES cellular proliferation and tumor growth. Furthermore, we show that serine deprivation inhibited ES cell proliferation and tumorigenesis, indicating that ES cells depend on exogenous serine in addition to de novo serine synthesis. Our findings suggest that serine metabolism is critical for ES tumorigenesis, and that targeting serine metabolism should be further investigated as a potential therapeutic strategy for ES. Citation Format: Sameer Issaq, Arnulfo Mendoza, Tracy Rosales, Christine Heske, Craig Thomas, Ralph DeBerardinis, Lee Helman. Targeting serine metabolism impairs tumor growth in preclinical models of Ewing sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5714.

EWS-FLI1-regulated Serine Synthesis and Exogenous Serine are Necessary for Ewing Sarcoma Cellular Proliferation and Tumor Growth.

Despite a growing body of knowledge about the genomic landscape of Ewing sarcoma, translation of basic discoveries into targeted therapies and significant clinical gains has remained elusive. Recent insights have revealed that the oncogenic transcription factor EWS-FLI1 can impact Ewing sarcoma cellular metabolism, regulating expression of 3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme in de novo serine synthesis. Here, we have examined the importance of serine metabolism in Ewing sarcoma tumorigenesis and evaluated the therapeutic potential of targeting serine metabolism in preclinical models of Ewing sarcoma. We show that PHGDH knockdown resulted in decreased Ewing sarcoma cell proliferation, especially under serine limitation, and significantly inhibited xenograft tumorigenesis in preclinical orthotopic models of Ewing sarcoma. In addition, the PHGDH inhibitor NCT-503 caused a dose-dependent decrease in cellular proliferation. Moreover, we report a novel drug combination in which nicotinamide phosphoribosyltransferase (NAMPT) inhibition, which blocks production of the PHGDH substrate NAD+, synergized with NCT-503 to abolish Ewing sarcoma cell proliferation and tumor growth. Furthermore, we show that serine deprivation inhibited Ewing sarcoma cell proliferation and tumorigenesis, indicating that Ewing sarcoma cells depend on exogenous serine in addition to de novo serine synthesis. Our findings suggest that serine metabolism is critical for Ewing sarcoma tumorigenesis, and that targeting metabolic dependencies should be further investigated as a potential therapeutic strategy for Ewing sarcoma. In addition, the combination strategy presented herein may have broader clinical applications in other PHGDH-overexpressing cancers as well.

Integrated human organ-on-a-chip model for predictive studies of anti-tumor drug efficacy and cardiac safety.

Traditional drug screening models are often unable to faithfully recapitulate human physiology in health and disease, motivating the development of microfluidic organs-on-a-chip (OOC) platforms that can mimic many aspects of human physiology and in the process alleviate many of the discrepancies between preclinical studies and clinical trials outcomes. Linsitinib, a novel anti-cancer drug, showed promising results in pre-clinical models of Ewing Sarcoma (ES), where it suppressed tumor growth. However, a Phase II clinical trial in several European centers with patients showed relapsed and/or refractory ES. We report an integrated, open setting, imaging and sampling accessible, polysulfone-based platform, featuring minimal hydrophobic compound binding. Two bioengineered human tissues - bone ES tumor and heart muscle - were cultured either in isolation or in the integrated platform and subjected to a clinically used linsitinib dosage. The measured anti-tumor efficacy and cardiotoxicity were compared with the results observed in the clinical trial. Only the engineered tumor tissues, and not monolayers, recapitulated the bone microenvironment pathways targeted by linsitinib, and the clinically-relevant differences in drug responses between non-metastatic and metastatic ES tumors. The responses of non-metastatic ES tumor tissues and heart muscle to linsitinib were much closer to those observed in the clinical trial for tissues cultured in an integrated setting than for tissues cultured in isolation. Drug treatment of isolated tissues resulted in significant decreases in tumor viability and cardiac function. Meanwhile, drug treatment in an integrated setting showed poor tumor response and less cardiotoxicity, which matched the results of the clinical trial. Overall, the integration of engineered human tumor and cardiac tissues in the integrated platform improved the predictive accuracy for both the direct and off-target effects of linsitinib. The proposed approach could be readily extended to other drugs and tissue systems.

Targeting Glycolysis through Inhibition of Lactate Dehydrogenase Impairs Tumor Growth in Preclinical Models of Ewing Sarcoma.

Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both in vitro and in vivo by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis. SIGNIFICANCE: LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.

Combinatorial BRD4 and AURKA inhibition is synergistic against preclinical models of Ewing sarcoma

AbstractBackgroundEwing sarcoma (ES), a bone cancer affecting children, adolescents, and young adults, is driven by the EWS‐FLI1 fusion protein in the majority of cases, but the mechanisms of tumorigenesis are still being elucidated. Consequentially, therapeutic advances have been limited in the last 25 years. Two therapeutic targets have been recently examined. Aurora kinase A (AURKA) promotes cell cycling and posttranslational stabilization of the oncoprotein MYC, while bromodomain‐containing protein 4 (BRD4) promotes gene expression epigenetically. Studies of AURKA and BRD4 inhibitors showed some impairment of tumorigenesis in ES preclinical models, but this efficacy was limited in single‐agent use. AURKA and BRD4 activate common oncogenic pathways through different mechanisms, so we hypothesized dual inhibition would be synergistic against tumorigenesis.Aims(a) Define the synergistic antineoplastic effects of BRD4 inhibitor I‐BET151 and AURKA inhibitor alisertib against ES cell lines in vitro. (b) Confirm the mechanism of activity of each agent in these cell line models. (c) Define the efficacy of I‐BET 151 and alisertib alone and in combinations with each other, and with the chemotherapeutic drug vincristine against ES tumor xenografts in vivo.Methods and resultsI‐BET151 and alisertib synergistically inhibit viability in ES cell lines SK‐ES, TC71, and ES2 in vitro. Alisertib alone upregulates transcriptional expression of its targets, but combined use of I‐BET151 mitigates that upregulation, likely contributing to the drug synergy and downregulating RNA and protein expression of key oncogenic pathways as shown by RT‐qPCR and western blot. Alisertib and I‐BET151 significantly prolong survival in three ES xenograft models in vivo, and this efficacy is augmented by the addition of vincristine.ConclusionDual targeting of oncogenic drivers in ES epigenetically and posttranslationally, specifically by use of BRD4 and AURKA inhibitors, may have significant clinical efficacy in ES alone and/or in combination with current chemotherapy regimens.

Abemaciclib Is Active in Preclinical Models of Ewing Sarcoma via Multipronged Regulation of Cell Cycle, DNA Methylation, and Interferon Pathway Signaling.

Ewing sarcoma (ES) is a rare and highly malignant cancer that occurs in the bone and surrounding tissue of children and adolescents. The EWS/ETS fusion transcription factor that drives ES pathobiology was previously demonstrated to modulate cyclin D1 expression. In this study, we evaluated abemaciclib, a small-molecule CDK4 and CDK6 (CDK4 and 6) inhibitor currently under clinical investigation in pediatric solid tumors, in preclinical models of ES. Using Western blot, high-content imaging, flow cytometry, ELISA, RNA sequencing, and CpG methylation assays, we characterized the in vitro response of ES cell lines to abemaciclib. We then evaluated abemaciclib in vivo in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models of ES as either a monotherapy or in combination with chemotherapy. Abemaciclib induced quiescence in ES cell lines via a G1 cell-cycle block, characterized by decreased proliferation and reduction of Ki-67 and FOXM1 expression and retinoblastoma protein (RB) phosphorylation. In addition, abemaciclib reduced DNMT1 expression and promoted an inflammatory immune response as measured by cytokine secretion, antigen presentation, and interferon pathway upregulation. Single-agent abemaciclib reduced ES tumor volume in preclinical mouse models and, when given in combination with doxorubicin or temozolomide plus irinotecan, durable disease control was observed. Collectively, our data demonstrate that the antitumor effects of abemaciclib in preclinical ES models are multifaceted and include cell-cycle inhibition, DNA demethylation, and immunogenic changes.

Abstract 1162: The evaluation of INCB059872, an FAD-directed covalent inhibitor of LSD1, in preclinical models of Ewing sarcoma

Abstract Ewing sarcoma is a rare bone cancer affecting predominantly children. The chromosomal translocation of chromosomes 11 and 22 results in the EWS/FLI gene fusion oncoprotein that is associated with ~85% of Ewing sarcoma cases. The EWS/FLI fusion protein is involved in deregulating gene expression and consequently causing cellular transformation. It was previously reported that lysine specific demethylase 1 (LSD1) regulates EWS/FLI transcriptional activity via its functional interaction through the NuRD co-repressor complex. We therefore evaluated whether inhibition of LSD1 could have anti-tumor effects in Ewing sarcomas that express the EWS/FLI fusion oncoprotein. INCB059872 is a potent, selective, and orally available FAD-directed covalent inhibitor of LSD1. To investigate the potential utility of INCB059872 in Ewing sarcoma, the A673 cell line having the characteristic chromosomal translocation was chosen as the experimental model system. INCB059872 inhibition of LSD1 did not significantly alter A673 proliferation in vitro. However, INCB059872 inhibited oncogenic transformation as determined by colony formation clonogenicity assays. NKX.2.2 was previously identified as a critical downstream target molecule of the EWS-FLI fusion oncoprotein that is required for transformation. A significant downregulation of NKX2.2 was observed in A673 cells treated with INCB059872, suggesting that INCB059872 mediates its effects through modulation of the EWS/FLI -NKX2.2 axis. Oral administration of INCB059872 significantly suppressed the growth of both A673 and SK-ES Ewing sarcoma xenografts in vivo. In addition, in vivo efficacy was evaluated in patient derived xenograft (PDX) models that were developed from relapsed tumor tissues of Ewing sarcoma patients. Notably, a subset of PDX models having EWS/FLI translocations (3/6) exhibited significant tumor growth inhibition at well-tolerated doses of INCB059872. Molecular signatures obtained from RNA-Seq data with these PDX models exhibited intrinsic differences between responders and non-responders, suggesting additional molecular or genetic variations may contribute to their sensitivity to INCB059872. Studies identifying potential candidate molecular mechanisms are underway. Together, these data suggest that INCB059872 may be therapeutically efficacious in a subset of Ewing sarcoma patients. Citation Format: Valerie Dostalik Roman, Min Ye, Huiqing Liu, Melody Diamond, Antony Chadderton, Yvonne Lo, Xuesong M. Liu, Jin Lu, Chunhong He, Liangxing Wu, Timothy Burn, Richard Wynn, Wenqing Yao, Gregory Hollis, Gregory Hollis, Peggy Scherle, Bruce Ruggeri, Sang Hyun Lee. The evaluation of INCB059872, an FAD-directed covalent inhibitor of LSD1, in preclinical models of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1162. doi:10.1158/1538-7445.AM2017-1162

Abstract 5054: Nanoformulation of talazoparib to increase efficacy when combined with temozolomide for the treatment of Ewing sarcoma xenografts

Abstract Ewing Family of tumors (EFT) comprises the fourth most common highly malignant childhood cancer. Although sustained event-free survival (EFS) can be achieved with intensive chemo-radiation therapy for patients with local-regional disease, this therapy is relatively ineffective in the treatment of metastatic disease with EFS of 12% at 5 years. Ewing sarcoma is characterized by a reciprocal translocation between chromosomes 11 and 22 that encodes a chimeric oncoprotein resulting from the fusion of EWSR1 to the FLI1 transcription factor in ~85% of tumors. Therapy for patients with EFT comprises surgery, intensive use of cytotoxic agents and radiation therapy. Dose intensification and dose compression has resulted in some improvement in outcome, but patents with advanced or metastatic disease at diagnosis still represent a challenge. Further, patients alive at 5 years from diagnosis still have a high probability of subsequent relapse. Further, long-term consequences of treatment included cardiac dysfunction, and secondary malignancies. Thus, more effective and less toxic therapies are required to treat patients with advanced disease. Our studies, as part of the Pediatric Preclinical Testing Program (PPTP), identified the combination of the PARP inhibitor, talazoparib, with the DNA damaging agent temozolomide, as being highly synergistic in xenograft models of Ewing sarcoma, but not against other tumor types. In this study 5 of 10 Ewing tumor xenografts models showed dramatic regressions to the combination, while administered as single agents neither talazoparib or temozolomide were active. We have studied the talazoparib-temozolomide synergy in vitro, and results indicate that in models where there is no synergy as xenografts, the cell lines have either intrinsic resistance to talazoparib, temozolomide or both drugs. In mice, and in the clinical trial (NCT02116777), the talazoparib-temozolomide combination is toxic requiring a reduction in temozolomide dose to ~15% of its single maximum-tolerated dose. We are exploring the use of nanoparticle-formulated talazoparib (npTLZ) developed by Nanomaterials Synthesis Laboratory at Northeastern University without tumor targeting or with antibody-mediated targeting to increase the tumor-drug delivery, reduce normal tissue toxicity (mainly thrombocytopenia), and potentially allow escalation of temozolomide dose. In the PPTP study, the MTD for temozolomide combined with free talazoparib (0.25 mg/kg PO BID daily x 5) was 12 mg/kg. Our recent data showed no toxicity of temozolomide at 66 mg/kg (PO daily x 5) in mice treated with npTLZ (0.5 mg/kg IV daily x 5), suggesting that npTLZ does not potentiate TMZ toxicity to normal mouse tissues. We anticipate that nanoparticle delivery of talazoparib combined with temozolomide will allow reduced toxicity while increasing the response rate for this combination in preclinical models of Ewing sarcoma. Citation Format: Nabeela Baig, Rostislav Likhotvorik, Paige Baldwin, Srinivas Sridhar, Raushan Kurmasheva. Nanoformulation of talazoparib to increase efficacy when combined with temozolomide for the treatment of Ewing sarcoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5054. doi:10.1158/1538-7445.AM2017-5054

Abstract 1654: The small molecule YK-4-279 shows anti-lymphoma activity in pre-clinical models

Abstract We have reported that up to 20% of diffuse large B cell lymphomas (DLBCL) present recurrent 11q23 gains leading to deregulated expression of the two transcription factors ETS1 and FLI1 and silencing of FLI1 was shown to lead to cell death in 6/6 DLBCL cell lines (Blood 2013). YK-4-279 is a small molecule that inhibits the binding of EWS1-FLI1 to RHA, leading to growth arrest and apoptosis of pre-clinical models of Ewing Sarcoma (Nat Med 2009). RHA is transcriptional co-activator, part of the EWS1-FLI1 transcription complex, but also involved in the activity of NFKB and STAT transcription complexes. Indeed, the compound also presented anti-cancer activity in a few additional cancer cell lines perhaps targeting not EWS1-FLI1 or FLI1 but other ETS factors, which are very similar to FLI1 in terms of DNA binding site (PLoS One 2011). Here, we assessed the anti-proliferative activity of YK-4-279 in a panel of human cell lines derived from B- and T-cell lymphomas. Methods. 48 cell lines [27 derived from DLBCL, 10 from mantle cell lymphoma (MCL), 3 from splenic marginal zone lymphoma (SMZL), 8 from anaplastic large cell lymphoma (ALCL)] were treated with increasing doses of YK-4-279) and IC50s were calculated with the MTT assays after 72 hrs exposure. Baseline gene expression profiling (GEP) was obtained on the cell lines with the Illumina HumanHT-12 Expression BeadChips and integrated with the anti-proliferative effect. Results. YK-4-279 showed potent dose-dependent anti-proliferative activity in most of the cell lines tested, with the majority of IC50 values being below 1000 nM. The median IC50 value was 393 nM (95% C.I., 272 - 3622 nM). There were no apparent differences among the different lymphoma subtypes: DLBCL (median IC50 = 386 nM; 95% C.I., 223-497), MCL (596 nM; 95% C.I., 230-1305), SMZL (217 nM; 95% C.I., 183-524) and ALCL (403 nM; 95% C.I., 170-3387). Results were confirmed by performing further MTT experiments using the R-YK-4-279 and S-YK-4-279, showing activity only when cells were exposed to the latter compound. The comparison of baseline GEP between 14 less sensitive (IC50 > 500 nM) and 9 highly sensitive (IC < 200 nM) B-cell lines highlighted that the expression signature of the latter group was enriched of E2F1 and PAX5 targets (including FLI1), genes involved in germinal center, cell cycle, sequence-specific DNA binding, and RNA processing. Conversely, the low sensitivity was associated with NFKB-related genes. Genes such as BCL6, BACH2, LMO2, CXCR4 and FAK were among the 50 most up-regulated transcripts in the sensitive cells. Conclusion. YK-4-279 shows strong anti-proliferative activity in lymphomas. Its activity appears positively correlated with the presence of germinal center B cell-related expression signatures and negatively with NFKB activity. The compound appears worth of further investigations in the lymphoma setting. Citation Format: Elain YL Chung, Valdemar Priebe, Eugenio Gaudio, Ivo Kwee, Chiara Tarantelli, Andrea Rinaldi, Laura Carrassa, Monica Testoni, Luciano Cascione, Massimo Broggini, Jeffrey A. Toretsky, Emanuele Zucca, Francesco Bertoni. The small molecule YK-4-279 shows anti-lymphoma activity in pre-clinical models. [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 1654. doi:10.1158/1538-7445.AM2015-1654