Identifying Targetable Liabilities in Ewing Sarcoma The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Vallurupalli, Mounica. 2014. Identifying Targetable Liabilities in Ewing Sarcoma. Doctoral dissertation, Harvard Medical School. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12407620 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Abstract: Background: Despite multi-modality therapy, the majority of patients with metastatic or recurrent Ewing sarcoma (ES), the second most common pediatric bone malignancy, will die of their disease. ES tumors express aberrantly activated ETS transcription factors through translocations that fuse the EWS gene to ETS family genes FLI1 or ERG. The aberrant activation of ETS transcription factors promotes malignant transformation and proliferation. While, FLI1 or ERG cannot be readily targeted, there is an opportunity to deploy functional genomics screens, to develop novel therapeutic approaches by identifying targetable liabilities in EWS/FLI1 dependent tumors. Materials and Methods: We performed a near whole-genome pooled shRNA screen in a panel of five EWS/FLI1 dependent Ewing sarcoma cell lines and one EWS/ERG cell line to identify essential genes. Essential genes were defined as those genes whose loss resulted in reduced viability selectively in ES cells compared to non-Ewing cancer cell lines. Essential hits were subsequently validated with genomic knockdown and chemical inhibition in vitro, followed by validation of the on-target effect of chemical inhibition. Next, we determined the in vivo effects of small-molecule inhibition on survival and tumor growth in NOD scid gamma (NSG) mice with established subcutaneous ES xenografts. Results: Top hits in our screen that could be readily targeted by small-molecule inhibitors, and thus have potential for rapid clinical validation, were selected for further investigation. These hits included IKBKE, CCND1 and CDK4. IKBKΕ, a non-canonical IKK with an oncogenic role in breast cancer, was one of the top kinase hits in the screen. IKBKΕ shares significant homology to TBK1, another non-canonical IKK that is essential in k-RAS dependent lung cancer. We validated IKBKE through small-molecule inhibition of IKBKE/TBK1 and shRNA based knockdown. Ewing sarcoma cell lines are sensitive to low micromolar concentrations of ! 2 two IKBKE/TBK1 inhibitors (CYT387 and MRT67307). Additionally, in a panel of ES cell lines, knockdown of IKBKE resulted in decreased growth and impaired colony formation. These observations, paired with impairment of NF-κB nuclear localization following CYT387 treatment suggests that non-canonical IKK mediated signaling may be essential in Ewing sarcoma. We further validated these results through inhibition of IKBKE/TBK1 in in vivo xenograft models treated with 100 mg/kg/day of CYT387. Treatment over the course of twenty- nine days resulted in a significant increase in survival (p-value = 0.0231) and a significant decrease (p-value = 0.036) in tumor size after fifteen days of treatment. CDK4 and CCND1 are highly expressed in Ewing sarcoma as compared to other tumor types. shRNA mediated knockdown of CDK4 and CCND1 resulted in impaired viability and anchorage independent growth. Furthermore, treatment of Ewing sarcoma cell lines with a highly selective CDK4/6 inhibitor, LEE011, resulted in decreased viability (IC50 range of 0.26-18.06 µM), potent G1 arrest in six of eight EWS/FLI1 containing Ewing sarcoma lines tested and apoptosis in a panel of four highly sensitive lines. Administration of 75 mg/kg/day and 250 mg/kg/day of LEE011 in NSG mice with Ewing xenografts resulted in significant impairment of tumor growth, (p-value <0.001 for both treatment arms), as compared to vehicle control. Conclusions: These studies suggest a role for the targeting of IKBKE and CDK 4/6 in Ewing sarcoma, findings with immediate clinical relevance for patients with this malignancy, because small-molecule inhibitors of these proteins have already entered clinical trial for other disease indications. ! 3 Table of Contents: 1. Abstract 2. Table of Contents 3. Glossary of abbreviations 4. Introduction 5. Materials and Methods 6. Results 7. Discussion 8. Summary 9. Acknowledgements 10. References 11. Tables and Figures ! 4 Glossary of abbreviations: Akt: also known as protein kinase B ATP: Adenosine triphosphate CCND1: Cyclin D1 CDK4: Cyclin-dependent kinase 4 CD99: Cluster of differentiation 99, also known as MIC2 CYLD: cylindromatosis gene cIAP: cellular Inhibitor of Apoptosis EFT: Ewing sarcoma family of tumors ERG: ETS-related gene ES: Ewing sarcoma ETS: E26 Transformation-specific transcription factor or Erythroblast transformation-specific transcription factor EWS: Ewing sarcoma gene FLI1: Friend leukemia integration 1 transcription factor, also known as transcription factor ERGB IC50: Fifty percent inhibitory concentration ID2: Inhibitor of DNA binding 2 IκBα: Inhibitor of kappa B (κB) IKBKA: Inhibitor of kappa B (κB) kinase alpha, also known as IKKα IKBKB: Inhibitor of kappa B (κB) kinase beta, also known as IKKβ IKBKE: Inhibitor of kappa B (κB)kinase epsilon, also known as IKKε or IKK-i IKK: Inhibitor of kappa B (κB) kinase MEF: Mouse embryonic fibroblasts ! 5 NF-κB: Nuclear factor-kappa B (κB) NKX2.2: NK2 Homeobox 2 NR0B1: Nuclear Receptor Subfamily 0, Group B, Member 1, also known as DAX-1 NSG: Nod-SCID-gamma mice PARP-1: Poly [ADP-ribose] polymerase 1, also known as NAD+ ADP-ribosyltransferase 1 PI: Propidium iodide RPM: Revolutions per minute shRNA: small hairpin RNA TANK: TRAF family member NF-κB activator TBK1: TANK-binding kinase 1 TRAF2: TNF receptor associated factor 2 TRAF6: TNF receptor associated factor 6 ! 6 Introduction Ewing sarcoma (ES) is the second most common pediatric bone malignancy in the United States. Despite advances in treatment of primary ES, the disease continues to have very poor survival for patients with metastatic or relapsed disease, even with multi-modality therapy.1,2 ES represents a spectrum of tumors belonging to Ewing sarcoma family of tumors (EFT). These tumors are often located in diaphyses of long bones and appear as undifferentiated “small round blue cells” on histological examination. EFT also include chest wall tumors (Askin tumors) and atypical ES.3 EFT share similarities not only in histology, but also in molecular biology, as they are characterized by balanced chromosomal translocations that define the biology of the disease.4 Characteristic translocations involve fusion of EWS with ETS transcription factor-encoding genes. The most common translocation, present in 85% of Ewing sarcoma, involves chromosome t(11;22) which results in the fusion of the 5’ end of the transcriptional regulator encoding gene EWS with the 3’ region of the transcription factor encoding gene FLI1.5 The resultant, aberrantly expressed 72-kDa-fusion protein is a nuclear protein that retains DNA-binding activity, recognizing the GGAA Ets- family DNA binding site.6 Another 5-10% of ES and related tumors can also arise from a second chromosome translocation, t(21;22), that fuses EWS with the ETS family transcription factor encoding gene ERG, resulting in the fusion protein (EWS/ERG).7 EWS/FLI1 promotes transformation and tumorigenesis in Ewing sarcoma EWS/FLI1 expression is oncogenic with cDNA transcripts of EWS/FLI1 capable of transforming NIH-3T3 cells, while absence of the EWS or FLI1 domain from the cDNA transcript abrogates this phenotype.8 NIH-3T3 cells that express EWS/FLI1 result in accelerated tumor formation in immunocompromised mice. Additionally, dominant negative and RNA interference studies have demonstrated a dependence of Ewing sarcoma cells on the EWS/FLI1 ! 7 fusion protein.9,10 The fusion of EWS/FLI1 results in an aberrant transcription profile in which the N-terminal EWS domain promotes greater transcriptional activation than the native FLI1 protein alone and the intact DNA-binding domain of FLI1 within EWS/FLI1 results in localization of the protein to GGAA containing promoter or enhancer sites on target genes.11 Targets of EWS/FLI1 as mediators of Ewing sarcoma oncogenesis Several targets of EWS/FLI1 have been identified and among these regulated genes, some have been shown to promote the oncogenic phenotype of Ewing sarcoma. For example, EWS/FLI1 expression results in overall gene downregulation that has been attributed to the direct activation of transcriptional repressors, such as NKX2.2 and NR0B1, by EWS/FLI1.9,12,13 Additional direct targets of EWS/FLI1 that play a role in ES oncogenesis include the cell surface marker CD99 and inhibitor of DNA binding 2 (ID2). CD99, also known as MIC2, is a 32 kDa integral membrane glycoprotein that is used as a histologic marker for the diagnosis of Ewing sarcoma. It has been shown to be essential for Ewing transformation, with decreased expression of CD99 in patient derived Ewing cells resulting in abrogation of the transformation phenotype. The knockdown of CD99 also inhibits neural differentiation and results in G2 arrest with an increase in apoptosis.14 ID2 has been shown to be upregulated in several tumor types including colon and pancreatic adenocarcinoma as well as neuroblastoma. The upregulation of ID2 in these tumor types is mediated by c-Myc, N-myc or β-catenin,