1. IMI CONFIDENTIAL KLF4 regulates adult lung tumor-initiating cells and represses K-Ras-mediated lung cancer Cell Death and Differentiation (26 June 2015 ； IF= 8.184) 报告人：董浩 2015-8-20

2. www.xianimi.com Background IMI CONFIDENTIAL 2 KLF4 A zinc finger transcription factor Enriched in the epithelium of various tissues In normal lung tissue (fibroblasts and airway epithelial cells) Development and cell differentiation Has role in cell fate and selfrenewal of embryonic stem cells Been identified as a tumor suppressor in many types of cancer In the lung and lung cancer are still not known

3. www.xianimi.com Background IMI CONFIDENTIAL 3 K-RAS GTP-binding protein Promotes proliferation and cell survival Mutations result in constitutive activation Mutations are linked to lung tumorigenesis, limited to non- small-cell lung cancer Mutations are found in more than one-third of human lung adenocarcinomas

4. www.xianimi.com Background IMI CONFIDENTIAL 4 Lung tumor-initiating cells Lung Bronchiolar epithelium Alveolar epithelium Clara cells (CC10) AT1 and AT2 cells (SP-C) functions as stem cells Clara and AT2 cells are the origin of lung tumorigenesis

5. www.xianimi.com Materials and Methods IMI CONFIDENTIAL 5 Clinical Date: TCGA (The Cancer Genome Atlas) Animal studies: Cre-inducible Klf4 fl/fl mice Cre-inducible K-ras LSL-G12D/+ mice Cell: Human lung cancer cell lines A549 and H460 HEK293T cells, Lung epithelial cell line BEAS-2B Proliferation assay (cell viability analyzer) Adenovirus transfection Western blotting RT-PCR real-time PCR H&E IHC Immunofluorescent Luciferase reporter assays

6. www.xianimi.com Materials and Methods IMI CONFIDENTIAL 6 诱导性基因敲除： Cre/LoxP 、 Flp/Frt 同源重组系统 Cre: 位点特异性重组酶，介导 LoxP 序列间同源重组 LoxP ：两个 13bp 反向重复序列 中间 8bp 序列

7. www.xianimi.com IMI CONFIDENTIAL 7

8. www.xianimi.com IMI CONFIDENTIAL 8

9. www.xianimi.com Results IMI CONFIDENTIAL 9 1.Klf4 is a putative tumor suppressor for lung cancer 2.Klf4 ablation altered the differentiation pattern in mouse lung 3.Klf4 deletion facilitates lung tumor formation and progression 4.Multiple mechanisms of KLF4 downregulation in lung cancer

10. www.xianimi.com 1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL 10 1.KLF4 mutations ： 9 out of 488 patients Small-cell lung carcinoma Non-small-cell lung carcinoma (adenocarcinoma) <2%

11. www.xianimi.com 1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL 11 These mutations have no significant effect on KLF4 function in repressing cyclinB1 promoter activity.

12. www.xianimi.com 1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL 12 2. KLF4 mRNA in human lung tumor tissues Non-small-cell lung carcinoma (adenocarcinoma) Small-cell lung carcinoma KLF 1, 2, 6, 8, 9, 10, 11, 12, 13, 14, 15 and 17 were also significantly lower in tumor

13. www.xianimi.com 1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL 13 3. The role of KLF4 in lung cancer cells Small-cell lung carcinoma KLF1, 2, 6, 8, 9, 10, 11, 12, 13, 14, 15 and 17 were also significantly lower in tumor

14. www.xianimi.com 1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL 14 3. The role of KLF4 in lung cancer cells No significant effect on proliferation of these cells, probably because of the endogenous KLF4 expression

15. www.xianimi.com 1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL 15 4. Effects of KLF4 on target genes

16. www.xianimi.com 1. Klf4 is a putative tumor suppressor for lung cancer IMI CONFIDENTIAL 16 4. Effects of KLF4 on target genes KLF4 represses lung cancer cell growth by regulating cell cycle and cell proliferation.

17. www.xianimi.com 2. Klf4 ablation altered the differentiation pattern in mouse lung IMI CONFIDENTIAL 17 1. Cre-induced Klf4 deletion mouse

18. www.xianimi.com 2. Klf4 ablation altered the differentiation pattern in mouse lung IMI CONFIDENTIAL 18 2. Effects of Klf4 deletion on AT2 cells Klf4 deletion mouse lung WT mouse lung

19. www.xianimi.com 2. Klf4 ablation altered the differentiation pattern in mouse lung IMI CONFIDENTIAL 19 2. Effects of Klf4 deletion on other cells Klf4 deletion could be a driving force of lung tumorigenesis through stimulating AT2 cells in mouse lung. AT1 Clara cell Ciliated cell

20. www.xianimi.com 3. Klf4 deletion facilitates lung tumor formation and progression IMI CONFIDENTIAL 20 1. K-ras activation and Klf4 deletion in mouse Klf4 fl/fl mouse K-ras LSL-G12D/+ mouse Klf4 fl/fl /K-ras LSL-G12D/+ mouse

21. www.xianimi.com 3. Klf4 deletion facilitates lung tumor formation and progression IMI CONFIDENTIAL 21 2. Klf4 deletion contributed to tumor formation in mouse lung Klf4 fl/fl WT 8 weeks after Cre-induction

22. www.xianimi.com 3. Klf4 deletion facilitates lung tumor formation and progression IMI CONFIDENTIAL 22 3. Effect of Klf4 deletion and K-ras activation on lung tumor

23. www.xianimi.com 3. Klf4 deletion facilitates lung tumor formation and progression IMI CONFIDENTIAL 23 3. Effect of Klf4 deletion and K-ras activation on lung tumor

24. www.xianimi.com 3. Klf4 deletion facilitates lung tumor formation and progression IMI CONFIDENTIAL 24 3. Effect of Klf4 deletion and K-ras activation on lung tumor Klf4 deletion synergized with K-ras activation to facilitate lung tumor

25. www.xianimi.com 4. Multiple mechanisms of KLF4 downregulation in lung cancer IMI CONFIDENTIAL 25 1. Correlation between KLF4 expression and methylation r=−0.03 Mutation Epigenetics TCGA

26. www.xianimi.com 4. Multiple mechanisms of KLF4 downregulation in lung cancer IMI CONFIDENTIAL 26 2. Histone deacetylases (HDACs) expressed in lung cancer Class I HDACs Histone acetylation TCGA

27. www.xianimi.com 4. Multiple mechanisms of KLF4 downregulation in lung cancer IMI CONFIDENTIAL 27 3. HDACs inhibit KLF4 expression

28. www.xianimi.com 4. Multiple mechanisms of KLF4 downregulation in lung cancer IMI CONFIDENTIAL 28 3. HDACs inhibit KLF4 expression

29. www.xianimi.com 4. Multiple mechanisms of KLF4 downregulation in lung cancer IMI CONFIDENTIAL 29 4. HDACs inhibitors repressed lung cancer cell proliferation

30. www.xianimi.com 4. Multiple mechanisms of KLF4 downregulation in lung cancer IMI CONFIDENTIAL 30 5. HDAC3 has an inhibitory role on KLF4 promoter activity

31. www.xianimi.com S. KLF4 regulate lung homeostasis and tumorigenesis through multiple mechanisms IMI CONFIDENTIAL 31 1. Bmi1 is overexpressed in lung tumor stem cell marker AT2 cell

32. www.xianimi.com S. KLF4 regulate lung homeostasis and tumorigenesis through multiple mechanisms IMI CONFIDENTIAL 32 2. Myeloperoxidase (MPO) is overexpressed in lung tumor neutrophils marker inflammation Klf4 depletion triggered lung inflammation, which may contribute to lung tumorigenesis in the mice

33. www.xianimi.com Highlights IMI CONFIDENTIAL 33 Clinical Date: TCGA (The Cancer Genome Atlas) Proliferation assay (cell viability analyzer) Multiple mechanisms of KLF4 downregulation

34. www.xianimi.com IMI CONFIDENTIAL 34