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Temozolomide resistance in glioblastoma multiforme (GBM) and its application

Oncology Nursing 2016. Temozolomide resistance in glioblastoma multiforme (GBM) and its application for drug development. Sang Y. Lee, Ph.D. Assistant Professor Pennsylvania State University College of Medicine. Conflict of interest. ■ None. Contents. ■ Brain tumor & GBM

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Temozolomide resistance in glioblastoma multiforme (GBM) and its application

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  1. Oncology Nursing 2016 Temozolomide resistance in glioblastoma multiforme (GBM) and its application for drug development Sang Y. Lee, Ph.D. Assistant Professor Pennsylvania State University College of Medicine

  2. Conflict of interest ■ None

  3. Contents ■ Brain tumor & GBM ■ Temozolomide (TMZ) & its resistance ■ TMZ resistant GBM cell lines (intrinsic, acquired) ■ Drug development for TMZ resistant GBM

  4. Top 10 leading causes of death in the US (2014) 1. Heart disease: 614,348 (23.5% of total deaths)2. Cancer: 591,699 (22.5% of total deaths)3. Chronic lower respiratory diseases: 147,101 (5.7% of total deaths)4. Accidents (unintentional injuries): 136,053 (5.0% of total deaths)5. Stroke (cerebrovascular diseases): 133,103 (5.0% of total deaths)6. Alzheimer's disease: 93,541 (3.3% of total deaths)7. Diabetes: 76,488 (2.9% of total deaths)8. Influenza and pneumonia: 55,227 (2.2% of total deaths)9. Kidney disease (Nephritis, nephrotic syndrome, and nephrosis): 48,146 (1.8% of total deaths)10. Intentional self-harm (suicide): 42,773 (1.6% of total deaths) Centers for Disease Control and Prevention (www.cdc.gov)

  5. 4 1 The top 10 cancers in the US (2016) 2 3 Brain 23,770 (1.4%) 16,050 (2.7%) American Cancer Society: Cancer Facts and Figures 2016. Atlanta, Ga: American Cancer Society, 2016.

  6. Brain tumor (BT) : statistics (incidence, death) - BTs are the most common cancer among those age 0-19 (leukemia is the second) - BTs are thesecond leading cause of cancer-related deaths in children (males and females) under age 20 (leukemia is the first) American Brain Tumor Association (www.abta.org)

  7. Brain tumor (BT) : general information • BT is an abnormal growth of tissue in the brain or central spine that can disrupt proper brain function • BTs may be primary (starting in the brain) or secondary (spreading to the brain from another area of body). Secondary BT (Metastatic BT) is about 5-10 times more common than primary BT • Primary BT may be benign or malignant. Benign BT grows slowly, has distinct boundaries, and rarely spreads. Malignant (cancerous) BT grows quickly, has irregular boundaries, and spreads to nearby brain areas • BTs are named after the cell type which they grow. There are more than 120 histologically distinct types of primary brain and central nervous system tumors

  8. Brain tumor : general information cont’d • The median age at diagnosis for all primary brain tumors is 59 years old • Survival after diagnosis with a primary BT varies significantly by age, histology, molecular markers and tumor behavior. Central Brain Tumor Registry of the United States (CBTRUS) (1995 – 2010) (www.cancer.org)

  9. Brain tumor : statistics (cancer type) • Meningiomas represent 34-36% of all primary brain tumors, making them the most common primary brain tumor.  There will be an estimated 24,880 new cases in 2016 • Gliomas, a broad term which includes all tumors arising from the gluey or supportive tissue of the brain, represent 27-30% of all brain tumors and 80% of all malignant tumors - Glioblastomas represent 15.1% of all primary brain tumors, and 55.1% of all gliomas • Glioblastoma has the highest number of cases of all malignant tumors • Astrocytomas, including glioblastoma, represent approximately 75% of all gliomas American Brain Tumor Association (www.abta.org)

  10. Brain tumor treatments Brain tumor treatment depends on a number of factors, including the type, location, size, and grade of the tumor, as well as the age and health of the patient.  • (Image guided) Surgery • Radiation therapy (e.g., conventional radiation therapy, stereotactic radiosurgery) • Chemotherapy • Targeted therapy (e.g., anti-angiogenesis therapy) • Alternating electric field therapy (tumor treating fields) for recurrent GBM www.cancer.net

  11. FDA approved treatments for GBM www.cancer.org

  12. Stupp R et al. N Engl J Med 2005

  13. Overall survival according to treatment group(total 573 patients from 85 centers) Stupp R et al. N Engl J Med 2005

  14. Effect of MGMT promoter methylation status on survival in glioblastoma (n=206) EORTC/NCIC Trial 100 80 60 Probability of OS (%) UnmethylatedMGMTpromoter MethylatedMGMTpromoter 40 20 P < .001 0 6 24 30 0 36 42 12 18 Mos Pts at Risk, nUnmethylatedMethylated 11492 10084 5964 1646 724 47 11 Hegi ME et al. N Engl J Med 2005

  15. Standard care for glioblastoma For people with glioblastoma, the latest standard of care is radiation therapy with daily low-dose temozolomide (Temodar), followed by monthly doses of temozolomide after radiation therapy for six months to one year However, the development of TMZ resistance during adjuvant therapy occurs in >30% of patients In addition, it is a challenge in overcoming dose-limiting myelo-suppressive toxicity of TMZ while maintaining its efficacy

  16. Temozolomide (Temodar) • Temozolomide (C6H6N6O2, MW 194) is an alkylating agent prodrug • MOA: alkylate/methylate DNA, which most often occurs at the N-7 or O-6 positions of guanine residues. The methylation damages the DNA and triggers the death of tumor cells • Metabolism: spontaneously hydrolyzed at physiologic pH to the active species, 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) and to temozolomide acid metabolite • Usage: Anaplastic astrocytoma (AA), Anaplastic oligodendroglioma (AO), GBM, Melanoma, Metastatic melanoma • Side effects: bone marrow suppression, nausea, vomiting, genotoxic

  17. Mechanism of TMZ and TMZ resistance MGMT/APNG/BER N3-methyladenine N7-methylguanine O6-methylguanine MMR TMZ Cell cycle arrest Nucleic acid 70% mG T MGMT/APNG/BER MMR Non-cytotoxic (TMZ resistant) Cell death (TMZ sensitive) Alkylpurine-DNA-N-glycosylase (APNG), Base excision repair (BER), DNA mismatch repair (MMR), O6-methylguanine-DNA methyltransferase (MGMT)

  18. Example of intrinsic (pre-existing) TMZ resistant GBM cell lines Lee SY Genes & Diseases 2016 Bold indicates TMZ sensitive/resistant cell lines which reported in the at least two papers

  19. CCF-STTG1 human GBM cell lines are TMZ resistant A. Cytotoxicity to TMZ B. MGMT methylation CCF-STTG1 SW1088 U87-MG LN-18 T98G U251 H2O U C U C U C U C U C U C C. MGMT expression CCF-STTG1 SW1088 U87-MG LN-18 T98G U251 Lee SY et al. Int J Cancer 2011

  20. Effect of MGMT and HFE on TMZ resistance in human glioma cell lines

  21. Apo-Tf Iron Tf TfR HFE HFE (Hemochromatosis protein) WT H63D C282Y (Hanson EH et al. 2001. Am J Epidemiol; n=6,203) (Adams PC et al. 2005. N Engl J Med; n=44,082) (Allen KJ et al. 2008. N Engl J Med; n=29,676)  Iron homeostasis gene  Binds to transferrin receptor and reduces its affinity for iron-loaded transferrin  Two major mutation sites : : H63D (histidine  aspartic acid, 1 domain) : C282Y (cysteine  tyrosine, 3 domain)  H63D and C282Y mutant cells have iron overload • HFE polymorphisms (H63D and C282Y) are the common genetic variants in Caucasians : Genotype frequency of H63D : 23.6% ~ 26.4% : Genotype frequency of C282Y : 9.6% ~ 11.8%

  22. Example of acquired TMZ resistant GBM cell lines 6 fold 10-16 fold ~4 fold 40 fold 5.6 fold 4.7 fold Lee SY Genes & Diseases 2016 Bold indicates TMZ sensitive cell lines which reported in the at least two papers

  23. Molecular events of intrinsic and acquiredTMZ resistant GBM cell lines NAMPT: nicotinamide phosphoribosyl transferase Lee SY Genes & Diseases 2016

  24. Treatments to intrinsic (pre-existing)TMZ resistant GBM cell lines Lee SY Genes & Diseases 2016 Bold indicates TMZ resistant cell lines which reported in the at least two papers

  25. Treatments of acquired TMZ resistant GBM cell lines Lee SY Genes & Diseases 2016 Bold indicates TMZ sensitive cell lines which reported in the at least two papers

  26. Intrinsic or acquired TMZ resistant xenograft animal models Lee SY Genes & Diseases 2016

  27. Effects of TMZ on survival on GBM orthotopic xenografts methylated MGMTlow level MGMT unmethylated MGMT higher level of MGMT TMZ: 120 mg/kg/d for 5 days Arrow: treatment initiated • GBM12 – response well to TMZ • GBM14 – response well to TMZ • GBM43 – resistant to TMZ (?) • GBM44 – resistant to TMZ MS: 70 d MS: 105 d MS: 18 d MS: 41 d unmethylated MGMT higher level of MGMT unmethylated MGMT higher level of MGMT MS: 48 d MS: 62 d MS: 24 d MS: 67 d TMZ is response well to methylated MGMT GBM. Response of TMZ to unmethylated MGMT GBM is diverse Kitange GJ et al. Neuro Oncol 2009

  28. Glioblastoma orthotopic xenografts P < 0.001 P < 0.001 Clarke MJ et al. Mol Cancer Ther 2009

  29. TMZ resistance, MGMT, and HFE mutations in human patient-derived xenograft (PDX) lines GS: Gliosarcoma Collaboration with Dr. Gasper Kitange & Dr. Jann Sarkaria

  30. Acquired TMZ resistant GBM12 orthotopic xenografts adjuvant TMZ : 66 mg/kg/d, day 1-5, administered in one, two, or three 28-day cycles TMZ: 33 mg/kg/d, 5 of 7 d × 2 wk TMZ : 66 mg/kg/d, day 1-5 Clarke MJ et al. Mol Cancer Ther 2009

  31. Acquired TMZ resistant GBM22 orthotopic xenografts adjuvant TMZ : 66 mg/kg/d, day 1-5, administered in one, two, or three 28-day cycles TMZ; 33 mg/kg/d, 5 of 7 d × 2 wk TMZ : 66 mg/kg/d, day 1-5 Clarke MJ et al. Mol Cancer Ther 2009

  32. Testing TMZ in animal models of glioma: 60 papers up to 2013 Rat Gliosarcoma Human Glioblastoma Rat Glioma Human Glioblastoma Rat Glioblastoma Human Glioma Red line: TMZ resistant Blue line: TMZ sensitive Rat Glioblastoma Human Glioblastoma Human Glioblastoma Human Glioblastoma Human Glioblastoma Hirst TC et al. Br J Cancer 2013

  33. Meta-analysis of TMZ in animal models of glioma: 60 papers up to 2013 TMZ prolonged survival by a factor of 1.88 (95% CI 1.74-2.03) and reduced tumor volume by 50.4% (41.8-58.9) compared with untreated controls Hirst TC et al. Br J Cancer 2013

  34. Cytotoxicity of selected compounds in gliomas TMZ sensitive TMZ resistant Cells were treated with 10 µg/ml of compounds for 6 days, followed by SRB assay. Cell viability was compared with DMSO vehicle control group.

  35. Dose response curve of compounds in CCF-STTG1

  36. Cytotoxicity of CC-I (#19) in GBM and normal cells A. B.    

  37. Cytotoxicity of similar structure compounds with CC-I in CCF-STTG1 cells

  38. Anti-tumor effect of CC-I in s.c. nude mouse brain tumor model SW1088 injected CCF-STTG1 injected Control (n=3)‏ Control (n=3)‏ CC-I (n=7)‏ CC-I (n=8)‏ i.p. injection (25 mg/kg) of CC-I once a week for 7 weeks CC-I treated mice do not loose body weight Lee SY et al. PLOS ONE 2014

  39. Anti-tumor effect of CC-I in intracranial brain tumor model C. A. B. Lee SY et al. PLOS ONE 2014 P<0.0001

  40. Anti-tumor effect of CC-I in T4121 glioma stem cell orthotopic BT nude mouse model P=0.03 (1x/w, 20 mg/kg, ip)

  41. Summary ■ We need a better understanding for the TMZ resistant (recurrent) GBM ■ There are distinct molecular events between intrinsic TMZ resistant GBM and acquired (recurrent) TMZ resistant GBM. Therefore, we should treat them different way. ■ TMZ resistance is not mediated by a single molecular event but by multiple ones. Therefore, identification of GBM patients based on the patient’s gene/protein profiling data could be beneficial for selecting drugs for their treatment ■ CC-I showed strong anti-tumor effect on GBM in in vivo tumor models

  42. Acknowledgements • Dr. James Connor • Ms. Becky Webb • Dr. Mohammed Alsaidi • Drug Discovery, Development and Delivery Core • MRI Imaging Core Facility, Solution Phase NMR Core Facility • Dr. Gasper Kitange & Dr. Jann Sarkaria (Mayo Clinic) • Penn State Hershey Cancer Institute Experimental Therapeutics Research initiative • NIH/NCI R21CA167406 • Elsa U. Pardee Foundation • Tara Leah Witmer Endowment

  43. Questions

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