CSE Strasbourg

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1. Karl Munger, Ph.D. karl.munger@tufts.edu Human Papillomaviruses - From Warts to Cancer
2. Munger Lab Tufts University School of Medicine Department of Developmental, Cellular and Chemical Biology 150 Harrison Ave Boston, MA 02111 NIH/NCI Chris Crum (BWH) Kristian Helin (U. Copenhagen) Jackie Lees (MIT) Martin McMahon (UCSF) Paul Lambert (UWI) Margaret McLaughlin-Drubin (BWH) Jordan Meyers Miranda Grace Mallory Harden Tyshia Wellman Surendra Sharma Katie Mattaini Elana Sanford
3. Papillomaviruses Infect squamous epithelial cells Cause hyperplastic lesions (warts) Circular 8 kB dsDNA genomes One coding strand HPV16 7904bp E6 E7 E1 E2 E4 E5 L1 L2 LCR P97 P670 7904/1 1000 2000 3000 4000 5000 6000 7000 polyAE polyAL
4. Human Papillomaviruses beta HPVs gamma HPVs cutaneous warts? cutaneous warts SCCs in “special” patients ~5% of all human cancers >99% of cervical carcinomas ~90% anal carcinomas ~40% vulvar and vaginal carcinomas ~20% of oral cancers (60% OPSCC) alpha HPVs “low-risk” – genital warts “high-risk” – premalignant lesions ~200 HPVs
5. Productive Infection HPV infections HPV16 7904bp E6 E7 E1 E2 E4 E5 L1 L2 LCR P97 P670 7904/1 1000 2000 3000 4000 5000 6000 7000 polyAE polyAL Life cycle depends of host cell machinery Linked to epithelial differentiation Productive life cycle in terminally differentiated cells Withdrawn from cell cycle
6. Productive Infection HPV16 7904bp E6 E7 E1 E2 E4 E5 L1 L2 LCR P97 P670 7904/1 1000 2000 3000 4000 5000 6000 7000 polyAE polyAL Life cycle depends of host cell machinery Linked to epithelial differentiation Productive life cycle in terminally differentiated cells Withdrawn from cell cycle Papillomaviruses uncouple epithelial differentiation from cell cycle withdrawal HPV infections
7. Most high-risk HPV infections asymptomatic and cleared (~180 days) 80% have contracted mucosal HPV infection by age 50 Productive Infection 0-1 Year 0-5 Years HPV Infection CIN2/3 CIN1 Regression/Clearance HPV infections
8. Invasive Cervical Cancer 1–40 Years LCR E6 E7 E1 L1 Non-productive Infection Cancer: a rare outcome of a frequent infection and not part of viral life cycle Productive Infection 0-1 Year 0-5 Years HPV Infection CIN2/3 CIN1 Regression/Clearance HPV-associated cancers
9. Expression in cells recapitulates CINs Causes cervical cancer in transgenic mice Required for proliferation of cervical cancer cell lines LCR E6 E7 E1 L1 HPV E6/E7
10. Small, non-enzymatic No cellular homologues No specific DNA binding Associate with and modify cellular protein complexes HPV E6/E7 - X29 - CXXC CXXC 1 151 E6 - X29 - CXXC CXXC LXCXE 1 98 E7 - X29 - CXXC CXXC
11. Genomic Instability E6 E7 Cellular Immortalization TERT E6 Extended proliferation E6 UBE3A TP53 Aberrant proliferation E7 RB1/E2F CUL2 Figure 6. Therapeutic Targeting of the Hallmarks of Cancer Drugs that interfere with each of the acquired capabilities necessary for tumor growth and progression have been developed and are in clinical trials or cases approved for clinical use in treating certain forms of human cancer. Additionally, the investigational drugs are being developed to target eac enabling characteristics and emerging hallmarks depicted in Figure 3, which also hold promise as cancer therapeutics. The drugs listed are but illu examples; there is a deep pipeline of candidate drugs with different molecular targets and modes of action in development for most of these hallmark E5 ? E5 E7 E7 E7 E7 E7 E7 E6 E6 E6 E6 E6 E6 E7 E6 E6 Hanahan & Weinberg Cell 144: 646-74, 2011 Mesri, Feitelson & Munger Cell Host &Microbes 15: 266-82, 2014 High-risk alpha HPV carcinogenesis
12. Is RB1 degradation the initiating carcinogenic hit? Genomic Instability E6 E7 Cellular Immortalization TERT E6 Extended proliferation E6 UBE3A TP53 Aberrant proliferation E7 RB1/E2F CUL2
13. The RB1 pathway E2F G1 S G2 M RB1 CDK4/6 CCND p16
14. p16 The RB1 pathway E2F G1 S G2 M RB1 CDK4/6 CCND CCND E2F RB1P E2F G1 S G2 M RB1 CDK4/6 G1/S transition E2F G2 M RB1 CCND CDK4/6 p16 G1 S Senescence Growth factors
15. The RB1 pathway E2F G1 S G2 M RB1 CDK4/6 CCND CCND E2F RB1P E2F G1 S G2 M RB1 CDK4/6 G1/S transition E2F G2 M RB1 CCND CDK4/6 p16 G1 S Senescence Cell Stress p16
16. E7 triggers p16INK4A expression E7 Cell Stress p16 p16INK4A is a cervical cancer biomarker
17. RB1 HR E7 expression triggers senescence E2F G1 S G2 M CDK4/6 CCND CCND E2F RB1P E2F G1 S G2 M RB1 CDK4/6 G1/S transition E2F G2 M RB1 CCND CDK4/6 p16 G1 S Senescence Cell Stress p16 E7
18. 0 20 40 60 80 100 120 % SA-ß-gal positive SAOS2 cells + RB1 - RB1 bdg RB1 degr HPV16 E7 HPV16 E7Δ21-24 - - HPV16 E7C24G - - HPV16 E7Δ6-10 - HPV1 E7 + - + + + Gonzalez et al., J Virol 75:7583-91, 2001 RB1 degradation is necessary for evading senescence RB1 E2F G1 S G2 M CDK4/6 CCND E7 Cell Stress p16
19. RB1 HFF (p 6) 0 10 20 30 40 7.8±4.2 5.8±3.4 35±7.7 Ctrl HPV16 E7 HPV16 E7Δ21-24 % SA ß-Gal positive cells C E7 E7Δ21-24 RB1 degradation defective HPV16 E7 mutant causes OIS E2F G1 S G2 M CDK4/6 CCND E7 Cell Stress p16 RB1 degradation is necessary for evading senescence
20. RB1 E7 evades RB1 senescence E2F G1 S G2 M CDK4/6 CCND CCND E2F RB1P E2F G1 S G2 M RB1 CDK4/6 G1/S transition G2 M RB1 CCND CDK4/6 p16 G1 S Senescence E2F Aberrant S-Phase E7 Cell Stress p16
21. Is running red lights a good idea? G2 M RB1 CCND CDK4/6 p16 G1 S Senescence E2F Aberrant S-Phase
22. RB1 Stress-induced p16INK4A expression McLaughlin-Drubin et al PNAS 108(5):2130-5, 2011. E2F G1 S G2 M CDK4/6 CCND E7 Cell Stress p16
23. RB1 E7 induces p16INK4A through KDM6B KDM6B HFKs 16E7 HFKs McLaughlin-Drubin et al PNAS 108(5):2130-5, 2011. E2F G1 S G2 M CDK4/6 CCND E7 p16
24. KDM6B reverses gene silencing McLaughlin-Drubin et al PNAS 108(5):2130-5, 2011. McLaughlin-Drubin et al PNAS 108(5):2130-5, 2011. SUZ12 EED EZH2 RBBP7 K27 me3 K27 me3 silenced chromatin de-repressed chromatin KDM6B
25. Decreased H3K27me3 mark by E7 McLaughlin-Drubin et al PNAS 108(5):2130-5, 2011. McLaughlin-Drubin et al PNAS 108(5):2130-5, 2011. SUZ12 EED EZH2 RBBP7 K27 me3 K27 me3 silenced chromatin de-repressed chromatin KDM6B E7 HFKs rect con are reve To co INK4A- perform results ( INK4A- There w scriptio when K we obs transcri cause o the INK with the creased KDM6A a Cervic pression formed KDM6A cinoma colonie was dep Discuss We disc duction Fig. 3. HPV16 E7-mediated induction of KDM6B is critical for the expression of the cervical cancer biomarker p16INK4A. (A) Coimmunofluorescence stain- ing of p16INK4A and H3K27me3 in an HPV16-positive cervical intraepithelial neoplasia (CIN) specimen. Upper and Lower are from different areas of the same specimen. Similar staining patterns were detected in three additional CIN specimens. Hoechst staining is shown to visualize nuclei, and a phase picture shows cellular morphology. (B) Monolayer cultures of HPV16- immortalized HFKs were transfected with KDM6B-specific siRNA duplexes or INK4A ARF normal tissue 16E7 HFKs rect con are reve To co INK4A- perform results ( INK4A- There w scriptio when K we obs transcri cause o the INK with the creased KDM6A CIN3
26. E7 triggers p16INK4A through KDM6B McLaughlin-Drubin et al PNAS 108(5):2130-5, 2011. McLaughlin-Drubin et al PNAS 108(5):2130-5, 2011. p16 SUZ12 EED EZH2 RBBP7 K27 me3 K27 me3 silenced chromatin de-repressed chromatin KDM6B E7 Senescence
27. HPV E7 expressing cells are “epigenetically reprogrammed” McLaughlin-Drubin et al PNAS 108(5):2130-5, 2011. McLaughlin-Drubin et al PNAS 108(5):2130-5, 2011. p16 Senescence PHF20 Differentiation HOX Cell identity SUZ12 EED EZH2 RBBP7 K27 me3 K27 me3 silenced chromatin de-repressed chromatin KDM6B E7
28. 0 10 20 30 -10 40 Ctrl% 16%E7% 16%E6% 16%E6/E7% % Decrease in Viability **% **% ,%16%E7% +%16%E7% **% % Decrease in Viability 0 10 20 30 40 50 -10 -20 % Decrease in Viability 40 60 70 -10 Ctrl% 18%E7% 18%E6% 18% E6/E7% ** 20 0 50 10 30 ** HPV E7 expression generates dependence on: KDM6B&is&required&for&cervical&cancer&cell&survival % Decrease in Viability 0 10 20 30 40 50 60 70 -10 -20 **" **" **" 80 SiHa" CaSki" Me+180" HeLa" E7 KDM6B KDM6B Collateral damage - Synthetic Lethality
29. HPV E7 expression generates dependence on: KDM6B Collateral damage - Synthetic Lethality U2OS Ctrl% 16%E7% 16%E6% 16%E6/E7% **% **% ,%16%E7% +%16%E7% **% % Decrease in Viability 0 10 20 30 40 50 -10 -20 Ctrl% 18%E7% 18%E6% 18% E6/E7% ** **
30. p16INK4A % Decrease in Viability 0 10 20 30 40 50 -10 -20 -30 Ctrl% 16%E7% 16%E6% 16%E6/E7% ** ** ** ** * ** 0 10 20 30 40 50 -10 % Decrease in Viability +%16%E7% +%16%E7% *% *% 30 40 50 ** * Viability 30 40 20 Viability * Collateral damage - Synthetic Lethality HPV E7 expression generates dependence on: KDM6B
31. p16INK4A % Decrease in Viability 0 10 20 30 40 50 -10 -20 -30 Ctrl% 16%E7% 16%E6% 16%E6/E7% ** ** ** ** * ** 0 10 20 30 40 50 -10 % Decrease in Viability +%16%E7% +%16%E7% *% *% 30 40 50 20 ** * ease in Viability 10 30 40 20 ease in Viability * Collateral damage - Synthetic Lethality HPV E7 expression generates dependence on: KDM6B 0 10 20 30 40 50 60 70 -10 % Decrease in Viability SiHa% CaSki% HeLa% **% **% **% **% **% **%
32. KDM6B lethality is rescued by p16INK4A * 0 10 20 -10 % Decreas * 0 10 30 40 -10 20 % Decrease in Viability * * p < 0.05 **p< 0.01 p16INK4A HPV E7 expression generates dependence on: KDM6B Collateral damage - Synthetic Lethality U2OS
33. KDM6B lethality is dependent on CDK4/6 % Decrease in Viability 20 30 10 0 * se in Viability * 20 30 40 * 40 % Decrease in Viability 20 30 10 0 40 -10 * e in Viability 20 30 40 U2OS * p < 0.05 Collateral damage - Synthetic Lethality p16INK4A HPV E7 expression generates dependence on: KDM6B
34. p16INK4A resistant CDK4/6 causes lethality % Decrease in Viabili 20 30 10 0 % Decrease in Viability * 20 30 10 0 40 * % Decrease in Viabili 20 30 10 0 -10 % Decrease in Viability 20 30 10 0 40 -10 U2OS * p < 0.05 Collateral damage - Synthetic Lethality p16INK4A HPV E7 expression generates dependence on: KDM6B
35. Summary HR HPV E7 expression triggers OIS
36. HR HPV E7 expression triggers OIS HR HPV E7 degrade RB1 to evade OIS Summary
37. HR HPV E7 expression triggers OIS HR HPV E7 degrade RB1 to evade OIS RB1 E7/CUL2 E7 TP53 E6/UBE3A p16 RB1 CUL2 TP53 E6/UBE3A Aberrant proliferation Extended proliferation Telomere Erosion E6/TERT Immortalization Extended proliferation Telomere Erosion E6/TERT Immortalization Summary
38. HR HPV E7 expression triggers OIS HR HPV E7 degrade RB1 to evade OIS HR HPV E7 cells are epigenetically reprogrammed Summary
39. HR HPV E7 cells are epigenetically reprogrammed “Addiction” to KDM6B and p16INK4A HR HPV E7 expression triggers OIS HR HPV E7 degrade RB1 to evade OIS Summary
40. “Addiction” to KDM6B and p16INK4A HR HPV E7 expression triggers OIS HR HPV E7 degrade RB1 to evade OIS HR HPV E7 cells are epigenetically reprogrammed p16INK4A CDK4/6 inhibition is necessary in RB1 inactive cells Summary
41. Oncogenic Tumor suppressive Normal Cells RB1 E2F G1 S G2 M CDK4/6 CCND p16 RB1 KDM6B E2F G1 S G2 M CDK4/6 CCND p16 Oncogenic Tumor suppressive RB1 defective Cells Summary
42. “Addiction” to KDM6B and p16INK4A HR HPV E7 expression triggers OIS HR HPV E7 degrade RB1 to evade OIS KDM6 addiction may be exploited therapeutically p16INK4A CDK4/6 inhibition is necessary in RB1 inactive cells HR HPV E7 cells are epigenetically reprogrammed Summary
43. 0 10 20 30 40 50 60 70 -10 -20 -30 SiHa% CaSki% HeLa% J4% J4% J4% % Decrease in Viability JARID1A (KDM5A) JARID1B (KDM5B) JARID1C (KDM5C) JARID1D (KDM5A) JMJD2B (KDM4B) JMJD3 (KDM6B) JMJD1A (KDM3A) JMJD2C (KDM4C) JMJD2A (KDM4A) JMJD2D (KDM4D) JARID2 UTY UTX (KDM6A) JMJD2E (KDM4E) JMJD1B (KDM3B) JHD2C HAIR JMJD4 JMJD6 EGLN1 JMJD7 JMJD5 JMJD8 CB060 HBAP1 PHF2 PHF8 JHD1D FBXL11 (KDM2A) FBXL10 (KDM2B) <1 °C 1–5 °C >5 °C HIF1N b M) 10 100 a b GSK-J4 GSK-J5 Vehicle c GSK-J4 N N N N H N O O GSK-J5 N N N N H N O O IL IL FAM TNF I TNFS IFN I IL I INH IL C IFN T I I GD TNF FAS IFN TNFS Summary
44. HR HPV E7 cells have acquired vulnerabilities “Addiction” to KDM6B and p16INK4A HR HPV E7 expression triggers OIS HR HPV E7 degrade RB1 to abrogate OIS KDM6 addiction may be exploited therapeutically p16INK4A addiction is related to CDK4/6 inhibition Summary
45. PRC2 KDM6A/B PRC2 KDM6A/B K27 me3 K27 me3 K4 me3 K4 me3 K27 me3 K27 me3 K4 me3 K4 me3 The complexity of PRC – additional synthetic lethalities?
46. PRC1 DUBs MLL KDM5A KDM5A MLL K27 me3 K27 me3 K119 Ub K119 Ub K27 me3 K27 me3 K4 me3 K4 me3 K27 me3 K27 me3 K4 me3 K4 me3 Repressed Active Cancer Discov. 1:391-407 Cancer Discov. 1:391-407 The complexity of PRC – additional synthetic lethalities with E7? PRC2 KDM6A/B PRC2 KDM6A/B
47. RB1 E2F G1 S G2 M CDK4/6 CCND p16 Oncogenic Tumor suppressive p16 defective Cells RB1 KDM6B E2F G1 S G2 M CDK4/6 CCND p16 Oncogenic Tumor suppressive RB1 defective Cells ? Are p16INK4A defective cells RB1 addicted?
48. 0 10 30 % Decrease in Viability Non-HPV cancers with p16INK4A expression SAOS2 osteosarcoma cells: RB1/TP53 defective express high p16INK4A undergo senescence when RB1 is re-expressed
49. + pRB 0 10 30 -10 -30 -50 % Decrease in Viability SAOS2 osteosarcoma cells: RB1/TP53 defective express high p16INK4A undergo senescence when RB1 is re-expressed Non-HPV cancers with p16INK4A expression
50. Ovarian cancers (High grade serous adenocarcinomas) Breast cancers (Basal-like, triple neg) Lung cancers Herschkowitz et al Breast Cancer Research 10:R75 (2008) Jarrard et al Cancer Lett 185:191 (2002) Kommoss et al BJC 96:306 (2007) Andujar et al Lung Cancer 67:23 (2010) Prostate cancers p16INK4A expressing tumors often are RB1 mutated Non-HPV cancers with p16INK4A expression RB1 KDM6B E2F G1 S G2 M CDK4/6 CCND p16 Oncogenic Tumor suppressive