1) Potential Novel Therapeutic Agents 2) Aberrant Expression of HMGA1, HOX & WNT Genes

1. Novel Cell-Based Models of Pediatric Spinal Ependymoma: 1) Potential Novel Therapeutic Agents2) Aberrant Expression of HMGA1, HOX & WNT Genes Li Luo PhD, Kenneth Cohen MD, Charles Eberhart MD PhD & Linda Resar MD Johns Hopkins University School of Medicine Ewa Krawczyk PhD, Richard Schlegel MD, PhD Georgetown University Angela Waanders, MD Children’s Hospital of Philadelphia, University of Pennsylvania Anbarasu Lourdusamy PhD, Richard Grundy MD University of Nottingham, UK

2. Background • Spinal ependymomas (SPE) are exceedingly rare tumors of the central nervous system with limited treatment options which occur in both children and adults • Successful outcomes depend primarily upon the extent of surgical resection • Unfortunately significant impairment is common after surgery, particularly in children with developing nervous systems.  • There are no established tumor models, nor are there many studies to define underlying molecular lesions

3. Conditionally Reprogrammed Cells (CRCs) • Primary carcinoma or normal epithelial cells can be cultured indefinitely ex vivo on fibroblast feeder cells or in conditioned medium in the presence of a rho kinase (ROCK) inhibitor • These cells are denoted conditionally reprogrammed cells (CRCs) Plate cells onto irradiated feeder cells + ROCK inhibitor Mince & digest tissue from tumor resection • In prior studies, CRCs form colonies in vitro, replicate tumor pathology as xenograftsin vivo, and predict drug responses to the primary tumor • Liu X, et al. Am J Pathol. 2012 Feb; 180(2):599-607. • Yuan H, et al. N Engl J Med 2012 Sept 27; 367(13): 1220-7.

4. ` HMGA1 Chromatin Remodeling Proteins • “High mobility group” proteins (low molecular weight) • Bind to AT-rich regions in chromosomal DNA • Displace Histone H1 proteins (relieving transcription repression) • Highly expressed in rapidly dividing, undifferentiated tissues as a delayed-early gene • HMGA1 encodes the HMGA1a & HMGA1b isoforms (Ch 6p21); • HMGA2 encodes HMGA2 (Ch 12q14); • - Germline mutations in HMGA2 associated with cancer (<20 y/o) • - Polymorphisms in HMGA1/2 associated with height • HMGA1 is enriched in refractory hematologic malignancy, poorly differentiated \ • solid tumors, ESCs, ASCs, CSCs _ 1 107 T S HMGA1a A-T A-T A-T _ 96 1 S T HMGA1b A-T A-T A-T

5. Background • High Mobility Group A1 (HMGA1) • - Chromatin remodeling protein (low molecular weight, thus high mobility) • - Binds to AT-rich regions in chromosomal DNA • - Displaces Histone H1 proteins (relieving transcriptional repression) • Highly expressed in rapidly dividing, undifferentiated tissues • - early embryogenesis, aggressive tumors, cancer stem cells & adult stem cells 1 107 S HMGA1a A-T A-T A-T T 96 1 A-T S A-T HMGA1b T A-T 5

6. HMGA1 Networks HMGA1 Networks • Pathways Activated • by HMGA1 • Transformation • Proliferation(E2F1) • Anti-apoptotic (p53) • Tumor Inflammation (COX2/STAT3) • Invasion/metastasis • (MMPs, EMT) • Angiogenesis (COX-2/STAT-3) • Chromosomal • Instability (XPA) • Immune Evasion • Tumor Metabolism (Glut3, IGFB() • Development • Pluripotency &stem cell • survival • (Lin28. cMyc, Oct4) • Senescence • Factors Inducing HMGA1 • Growth Factors • Oncogenic Transcription Factors • (cMYC, AP1, Others) • Inflammation • Hypoxia • Viral Infection • Epigenetic Modifications L. Resar Cancer Res, (Review) Jan 15, 2010 Sumter, et al. Curr Mol Med, 2016 in press • Pathways Activated • by HMGA1 • Transformation • Proliferation • Anti-apoptosis • Inflammation • Invasion/metastasis • Angiogenesis • Chromosomal Instability • Immune evasion • Tumor Metabolism • Development • Pluripotency • Stem cell survival • Epithelial-mesenchymal transition • Senescence HMGA1 Networks • Pathways Activated • by HMGA1 • Transformation • Proliferation • Anti-apoptosis • Inflammation • Invasion/metastasis • Angiogenesis • Chromosomal Instability • Immune evasion • Tumor Metabolism • Development • Pluripotency • Stem cell survival • Epithelial-mesenchymal transition • Senescence HMGA1 Networks • Pathways Activated • by HMGA1 • Transformation • Proliferation • Anti-apoptosis • Inflammation • Invasion/metastasis • Angiogenesis • Chromosomal Instability • Immune evasion • Tumor Metabolism • Development • Pluripotency • Stem cell survival • Epithelial-mesenchymal transition • Senescence HMGA1 Networks • Pathways Activated • by HMGA1 • Transformation • Proliferation • Anti-apoptosis • Inflammation • Invasion/metastasis • Angiogenesis • Chromosomal Instability • Immune evasion • Tumor Metabolism • Development • Pluripotency • Stem cell survival • Epithelial-mesenchymal transition • Senescence HMGA1 Networks • Factors Inducing HMGA1 • Growth Factors • Oncogenic Transcription Factors • (cMYC, AP1, Others) • Inflammation • Hypoxia • Viral Infection • Epigenetic Modifications • Pathways Activated • by HMGA1 • Transformation • Proliferation • Anti-apoptosis • Inflammation • Invasion/metastasis • Angiogenesis • Chromosomal Instability • Immune evasion • Tumor Metabolism • Development • Pluripotency • Stem cell survival • Epithelial-mesenchymal transition • Senescence • Factors Inducing HMGA1 • Growth Factors • Oncogenic Transcription Factors • (cMYC, AP1, Others) • Inflammation • Hypoxia • Viral Infection • Epigenetic Modifications • Factors Inducing HMGA1 • Growth Factors • Oncogenic Transcription Factors • (cMYC, AP1, Others) • Inflammation • Hypoxia • Viral Infection • Epigenetic Modifications • Factors Inducing HMGA1 • Growth Factors • Oncogenic Transcription Factors • (cMYC, AP1, Others) • Inflammation • Hypoxia • Viral Infection • Epigenetic Modifications

7. THE HALLMARKS OF CANCER

8. HMGA1 is Enriched in High-Grade, Refractory Cancer & Stem Cells • Burkitt’s lymphoma cell lines • Acute Leukemia • Thyroid carcinomas • Lung cancer • Breast cancer • High grade prostate cancer • Uterine & cervical cancer • Skin cancer • Medulloblastoma • Stem cells (CSCs, hESCs, iPSCs, ASCs)

9. Hypothesis • Conditionally Reprogrammed Cells (CRCs) will be a useful strategy to derive cell lines from primary SPE tumors and normal developing spinal cord; • CRC-derived SPE cells will predict tumor responses in vivo in a high-throughput drug screen for effective anti-cancer agents • Primary tumors, CRCs, and other tumor-derived cell lines will provide useful models to elucidate underlying transcriptional networks and molecular mechanisms driving tumor initiation and progression

10. Primary SPE cell line was established using a modified CRC method Primary tumor sample from a 12 year-old girl with a spinal myxopapillary ependymoma Figure. 1: Primary SPE cells cultured under standard CRC conditions A B • Primary myxopapillary spinal ependymoma cells were cultured under standard CRC conditions. Day 1 after isolation and culture are shown (40X). • Cells after 26 days in culture. The primary tumor cells grew between weeks 1-4, but ultimately stopped replicating.

11. Primary Spinal Ependymoma (SPE) cell line was established using a modified CRC method Figure. 2: Primary SPE cells cultured under hypoxic conditions without feeders Primary myxopapillary SPE cells were cultured under hypoxic conditions without feeders in F medium with ROCK inhibitor (10 µM). After 4 weeks in culture, they began to expand. Bar: 400 µm

12. Authentication of SPE cells: STR analysis of SPE cells vs. tumor tissue SPT: primary tumor tissue SPE P2: CRC SPE cells Figure 3. STR analysis shows the primary SPE CRC cells have the same profile as the primary tumor tissue, with no matches in the ATCC or DMSZ databases.

13. Expanded SPE cells show high HMGA1/2, WNT and HOX gene expression levels • Figure 4. Compared to normal spinal cord, targeted gene expression profiling of SPE CRCs reveals: • Up-regulation in HMGA1, HMGA2, HOXB13,& HOXA10, • Similar ITIH2 • Down-regulation in CDKN2A, HOXB7, HOXD1, HOXD13.

14. Drug screening reveals potential hits cytotoxic to SPE Figure 5. A heat map of blind drug screening with Alamar Blue Assay. Red to blue shows low to high survival of SPE CRCs in response to FDA-approved drugs in collaboration with Johns Hopkins Drug Library (JHDL).

15. Blind drug screen with >3000 FDA- approved drugs reveals therapeutic candidates with CBTTC SPE cells 1. Received 3 pediatric SPE cell lines from CHOP on Dec 14/2016: • 7316-2170 7316-2170-CELN [552000] CELN NA0007508299 • 7316-2182 7316-2182-CELN [552063] CELN NA0007508296 • 7316-21387316-2138-CELN [557528] CELN NA0007508308 2. Completed the preliminary drug screening April 2017 for 2138 CBTTC line: 7316-2138 CELN_ALQ CELC_# CV 1.8 557528 31 drug hits with <10% survival at 10 um drug concentration

16. Top drug hits with <1% survival rate after 48 hour drug treatment

17. In vivo tumorigenicity test of SPE CRCs by Intracranial injection into mouse brain Figure 6. Intracranial injection of human SPE CRCs into immuno-deficient (NSG) mice. 6 male NSG mice (5 weeks old) survived injection of 250,000 cells (50,000/uL). Injections were intraventricular (n=3) or into the striatum (n=3) as shown.

18. HMGA1 expression is positively correlated with WNT pathway genes in primary SPE tumors Ependymoma sites: spinal (n=10; green) supratentorial (n=55; light purple) posterior fossa (n=80; darker purple) HMGA1 (scaled expression) HMGA1 (scaled expression)

19. HMGA1 is positively correlated with HOX genes in primary SPE tumors HMGA1 (scaled expression) HMGA1 (scaled expression) HMGA1 (scaled expression) HMGA1 (scaled expression)

20. HDAC4 is positively correlated with miR-10a and miR-10b in primary SPE tumors

21. Conclusions • We derived SPE cells using a modified reprogramming approach • We used a CHOP SPE cell line to screen for cytotoxic drugs • We identified 10 potential therapeutic agents • Genes encoding HMGA1 together with WNT and HOX developmental genes are up-regulaed in in primary pediatric SPE • Specific gene and microRNA signatures are characteristic of pediatric SPE compared to adult SPE or intracranial tumors 

22. Future Plans & Directions • Future studies are underway to validate hits in our our drug screen using different SPE lines • We also plan to test additional SPE lines in the drug screen • We hope to establish additional cell lines • We will perform transcriptomic (RNA-seq), exomic sequencing and epigenetic profiles on established lines and primary tumors to elucidate the functional significance of HMGA1, WNT, HOX and other genes in the pathogenesis of pediatric SPE.

23. Acknowledgements • Drs. Li Luo, Ken Cohen & Charles Eberhart at JHUSOM • Drs. EwaKrawczyk& Richard Schlegel at Georgetown University • Dr. Angela Waanders & her team at CHOP/U PENN • Drs. Richard Grundy, AnbarasuLourdusamy& their team at University of Nottingham in US

24. Acknowledgements:

25. The RESAR Gene Family