1. Ribosomal Dysfunction Results in an Immunologic Susceptibility to Lung Cancer Stephanie Chang MD, Ryuji Higashikubo PhD, Saeed Arefanian MD, Andrew Gelman PhD, Daniel Kreisel MD, PhD, Alexander Krupnick MD April 27 th,2015 Department of Surgery Division of Cardiothoracic Surgery

2. Department of Surgery Division of Cardiothoracic Surgery No financial disclosures Disclosures

3. Department of Surgery Division of Cardiothoracic Surgery Susceptibility to Lung Cancer Lung Cancer Resistant Lung Cancer Susceptible C3H Previous work from our lab demonstrates natural killer (NK) cells play a role in this process Cancer Research 2012 Smoker with Bullous Emphysema Non-Smoker with Lung Cancer

4. Department of Surgery Division of Cardiothoracic Surgery Sly1 380 AA 55 kD SH3SAM SH3SAM X chromosome linked protein with unknown function Expression of Sly 1 Correlates with Immunologic Resistance to Lung Cancer Protein protein binding domains

5. Department of Surgery Division of Cardiothoracic Surgery The aim of this study was to determine the role of Sly 1 in the susceptibility to lung cancer development Objective

6. Department of Surgery Division of Cardiothoracic Surgery Expression of Sly 1 Correlates with Immunologic Resistance to Lung Cancer Lung Cancer Resistant Lung Cancer Susceptible Sly 1 Level by RT-PCR (normalized to  - Actin/standardized patient) % TNF  + NK Cells Humans

7. Department of Surgery Division of Cardiothoracic Surgery Expression of Sly 1 Correlates with Immunologic Resistance to Lung Cancer Lung Cancer Resistant Lung Cancer Susceptible Sly 1 Level by RT-PCR (normalized to  - Actin/standardized patient) % TNF  + NK Cells Human Sly 1 Function

8. Department of Surgery Division of Cardiothoracic Surgery Sly 1 Contributes to NK Cytotoxicity Sly1- (knockout of the gene) Wild-type littermates

9. Department of Surgery Division of Cardiothoracic Surgery Activating Receptors Expression Sly 1 Contributes to NK Cytotoxicity Cytokine Expression Sly1- (knockout of the gene) Wild-type littermates

10. Department of Surgery Division of Cardiothoracic Surgery Days After Injection Lewis Lung Carcinoma Tumor Growth Tumor Volume (mm 3 ) Control Group Days After Injection Tumor Volume (mm 3 ) NK Depletion Group Sly 1 Expression Influences Lung Cancer Growth Via NK Cells

11. Department of Surgery Division of Cardiothoracic Surgery Lung Cancer Lysis in vitro Sly 1 Contributes to NK Cytotoxicity of Lung Cancer

12. Department of Surgery Division of Cardiothoracic Surgery Immunoprecipitation of WT and Sly 1 KO NK cell lysates with anti-Sly 1 Ab to evaluate Sly 1 binding partners No detection of signaling proteins from activating receptors or cytokine pathways Sly 1 is a Ribosome Associated Protein

13. Department of Surgery Division of Cardiothoracic Surgery Sly 1 Affects mRNA levels

14. Department of Surgery Division of Cardiothoracic Surgery How Could a Ribosomal Protein Impair Gene Expression? 1)mRNA binding - No RNA-binding capacity ribosome mRNA 40s 60s 2) Ribosomal instability - Affects the transcription factor E2F as a protective mechanism

15. Department of Surgery Division of Cardiothoracic Surgery mRNA RB P P P P P P P P P kinase E2F promoter ribosome Normal Cell How Could a Ribosomal Protein Impair Gene Expression?

16. Department of Surgery Division of Cardiothoracic Surgery E2FRB P P P P P P P P kinase ribosome Ribosomal Stress Associated with Malignant Transformation or Cell Stress X p53 p27 p21 Stop cellular proliferation and activation How Could a Ribosomal Protein Impair Gene Expression?

17. Department of Surgery Division of Cardiothoracic Surgery E2FRB P P P P P P P P kinase ribosome X p53 p27 p21 phospho-RB RB Free E2F Wild-type littermate Sly1 - Actin How Could a Ribosomal Protein Impair Gene Expression? Ribosomal Stress Associated with Malignant Transformation or Cell Stress

18. Department of Surgery Division of Cardiothoracic Surgery High-dose IL-2 Can Stabilize the Ribosome Lewis Lung Carcinoma Lysis in vitro Activating Receptors Expression At Rest+ IL-2At Rest+ IL-2

19. Department of Surgery Division of Cardiothoracic Surgery Sly1 is a ribosomal protein that affects NK ribosome stability at rest o Ribosomal stress is a sign of malignant transformation which can destroy the cell or decrease its activation/viability Prior to our research, most X linked ribosomopathies were considered severely disabling but we now demonstrate that subtle defects can manifest such as NK dysfunction and immunologic susceptibility to lung cancer IL2 can alleviate ribosomal stress and may be useful in lung cancer immunotherapy Conclusions

20. Department of Surgery Division of Cardiothoracic Surgery Thoracic Surgery Foundation for Research and Education NIH Ruth L Kirschstein NRSA Thoracic Immunology Lab Eberhard-Karls University Tuebingen Sandra Beer-Hammer, PhD Daniel Schall, PhD Acknowledgements

21. Department of Surgery Division of Cardiothoracic Surgery Questions?