MECHANISM OF MYELOID-DERIVED SUPPRESSOR CELL ACCUMULATION IN CANCER AND SUSCEPTIBILITY TO REVERSAL BY SUNITINIB by JENNIFER SUSAN KO, M.D. Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Adviser: Dr. James H. Finke Department of Pathology CASE WESTERN RESERVE UNIVERSITY January, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis of Jennifer Susan Ko candidate for the Doctor of Philosophy degree*. (signed) Alan Levine Ph.D. David Kaplan M.D., Ph.D. Clark Distelhorst M.D. James Finke Ph.D. Charles Tannenbaum Ph.D. (date) October 12th, 2009 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 TABLE OF CONTENTS Title Page 1 Signature Sheet 2 Table of Contents 3 List of Tables 6 List of Figures 7 Acknowledgements 9 List of Abbreviations 10 Abstract 14 Chapter 1: Introduction 16 Overview: Myeloid-derived suppressor cells in cancer: a novel therapeutic target. 16 Immunotherapy in cancer 16 Myeloid-derived suppressor cells limit immunotherapy 22 Myeloid-derived suppressor cells limit anti-angiogenic therapy 28 Multiple factors are implicated in MDSC formation 30 Vascular Endothelial Growth Factor 30 Stem Cell Factor 32 Granulocyte- and Granulocyte/Monocyte Colony Stimulating Factors 33 S100A9 and Inflammation 34 Intracellular signaling implicated in MDSC programming 36 3 Chapter 2: Sunitinib Mediates Reversal of Myeloid-Derived Suppressor Cell Accumulation in Renal Cell Carcinoma Patients 44 Statement of Clinical Relevance 44 Abstract 45 Introduction 46 Materials and Methods 48 Results 53 Discussion 71 Chapter 3: Direct and Differential Suppression of Myeloid-derived Suppressor Cell Subsets by Sunitinib is Compartmentally Constrained 75 Abstract 75 Introduction 76 Materials and Methods 77 Results 81 Discussion 97 Chapter 4: Tumor-Derived Products Dynamically Activate CD15+ Myeloid-Derived Suppressor Cells and are Partially Modulated by Sunitinib 102 Abstract 102 Introduction 103 Materials and Methods 106 Results 111 Discussion 129 Chapter 5: Dissertation Discussion and Future Directions 134 Rationale 134 4 Summary of Critical Findings 135 Future Directions 144 Sunitinib’s Impact on MDSC Expansion 144 The Role of GM-CSF and Gangliosides in MDSC Activation and the Impact of Sunitinib on MDSC Activation 146 Sunitinib’s Impact on MDSC Viability 150 Further Defining the RTK Targets of Sunitinib in MDSC Inhibition 156 Regarding Intratumoral Resistance to Sunitinib 159 Bibliography 163 5 LIST OF TABLES TABLE PAGE 1-1 MDSC Phenotype and Subsets 25 1-2 MDSC Suppressive Mechanisms 27 2-1 Sunitinib Study Patient Characteristics 54 2-2 Sunitinib-induced Alterations in White Blood Cell Parameters 59 3-1 Characteristics of RCC Patients for Tumor Analysis 95 6 LIST OF FIGURES FIGURE PAGE 2-1 Elevated MDSC in mRCC patients decline in response to sunitinib 57 2-2 Sunitinib mediated normalization of MDSC is associated with sunitinib-mediated enhancement in T cell production of IFNγ in mRCC patients 61 2-3 In vitro depletion of MDSCs restores patient T cell production of IFN-γ 63 2-4 Effect of sunitinib on MDSC suppressive function in vitro 65 2-5 Effect of sunitinib on MDSC viability and differentiation in vitro 68 2-6 Patient MDSCs correlate with Tregs in response to sunitinib treatment 70 3-1 Sunitinib reverses immune suppression in tumor-bearing hosts 82 3-2 Anti-proliferative and pro-apoptotic effects of sunitinib on MDSC subsets in the spleen 85 3-3 MDSC in 4T1 tumor bed are relatively protected from sunitinib- Mediated downregulation 88 3-4 GM-CSF is unique in its ability to protect MDSC in the presence of sunitinib, possibly via Stat3 repression 91 3-5 Relatively high levels of GM-CSF are unique to tumor microenvironment 93 7 3-6 Tumor microenvironment limits local anti-MDSC effect of sunitinib in RCC patients 96 4-1 N-MDSC comprise the predominant MDSC subset within RCC tissue 113 4-2 Tumor conditioned media (TCM) from RCC lines induce N-MDSC from whole blood 116 4-3 N-MDSC induced from whole blood by TCM express markers of activated neutrophils 119 4-4 Tumor conditioned media prolongs survival of cultured N-MDSC 121 4-5 GM-CSF reproduces effect of TCM on N-MDSC activation and viability 123 4-6 Sunitinib treatment of tumor cells causes a partial reduction in the ability of TCM to induce N-MDSC 125 4-7 RCC derived gangliosides induce N-MDSC and sunitinib reduced expression of gangliosides 128 5-1 Proposed model of MDSC accumulation and sunitinib-mediated MDSC reversal 143 8 ACKNOWLEDGEMENTS The PhD process has perhaps been one of the most difficult in my life up until this point, but its success was highly attributable to several individuals beyond myself. For their patience, understanding, and unconditional love, I thank my husband, Tim, my daughter, Eva, and my parents and brothers. Life could have been easier, but thank you for understanding why I took the road less traveled by medical doctors. For their encouragement, inspiration, and teaching, I thank Dr. Finke, Dr. Borden, and Dr. Cohen. Thank you for seeing the potential in me, and my project, especially at times when I leant toward self doubt. I especially thank Dr. Finke for the intellectual freedom he was brave enough to bestow me with from day one. For their support, friendship, tolerance, and technical guidance I thank the members of the Finke lab – Pat, Joanna, Kaushik, Soumika, Cyndi, and Li, and other past and rotating members. Your patience and good humor helped make all the learning fun. I especially thank Pat and Joanna, two people who know no technical or organizational obstacle, even when it comes to patient studies with clinical samples. Thank you to Drs. Levine, Kaplan, Tannenbaum, Distelhorst, and Hamlin for your intellectual discussions and support of this project. I especially thank Dr. Levine, for always being available to me as a teacher and as a chairman. Finally, I thank God, who created all this beauty and wonder which sustains me with the excitement of a child, even as I continue to age. 9 LIST OF ABBREVIATIONS 7AAD – 7 aminoactinomycin D ACT – adoptive cellular therapy AECCM – activated endothelial cell conditioned medium Ag – antigen AMN – age-matched normals APC – antigen presenting cell ARG1 – arginase 1 ATRA – all-trans retinoic acid ATT – adoptive T cell therapy Bcl-2 – B-cell lymphoma 2 Bcl-xl – B-cell lymphoma – extra large BCR/abl–breakpoint cluster region/Abelson murine leukemia viral oncogene homolog1 BM – bone marrow CD – cellular differentiation CD40L – CD40 ligand COX2 – cyclooxygenase 2 CSF-1R – colony stimulating factor-1 receptor CT – computed tomography DC – dendritic cell EGOG – Eastern Cooperative Oncology Group FACS – fluorescence-activated cell sorting G-CSF – granulocyte colony stimulating factor 10 GFP – green fluorescent protein GM-CSF – granulocyte/monocytes colony stimulating factor HLA – human leukocyte antigen HPC – hematopoietic progenitor cell HPLC – high-performance liquid chromotography IFNα – interferon-alpha IFNγ – interferon-gamma Ig - immunoglobulin IL – interleukin IRB – Institutional Review Board JAK – Janus kinase KO – knock out M-CSFR – macrophage colony stimulating factor receptor MDSC – myeloid-derived suppressor cells MHC – major histocompatibility complex m-MDSC – monocytic myeloid-derived suppressor cells mMM – metastatic malignant melanoma MMP – matrix metalloproteinase mRCC – metastatic renal cell carcinoma MRP – myeloid related protein MSKCC – Memorial Sloan-Kettering Cancer Center NAC – N-acetylcysteine NADPH – nicotinamide adenine dinucleotide phosphate 11 NK – natural killer n-MDSC – neutrophilic myeloid-derived suppressor cells NO – nitric oxide NOS2 – nitric oxide synthase 2 PBMC – peripheral blood mononuclear cells PDGFR – platelet-derived growth factor receptor PGE2 – prostaglandin E2 RAG- recombination activating gene RCC – renal cell carcinoma RECIST – response evaluation criteria in solid tumors ROS – reactive oxygen species RTK – receptor tyrosine kinase RTKI – receptor tyrosine kinase inhibitor SCF – stem cell factor SOCS – suppressor of cytokine signaling STAT – signal transducer and activator of transcription TAA – tumor-associated antigen TCR – T cell receptor TCM – tumor-conditioned media TGFβ – transforming growth factor- beta TKI – tyrosine kinase inhibitor TLR – toll-like receptor TNFα- tumor necrosis factor-alpha 12 Treg – regulatory T cells VEGF – vascular endothelial growth factor VEGFR – vascular endothelial growth factor receptor VHL – Von Hippel-Lindau 13 Mechanism of Myeloid-Derived Suppressor Cell Accumulation in Cancer and Susceptibility to Reversal by Sunitinib ABSTRACT by Jennifer Susan Ko, M.D. Tumor-driven accumulation of myeloid-derived suppressor cells (MDSC) facilitates tumor immune evasion via T-cell inhibition, therefore limiting therapeutic approaches. MDSC accumulate in tumor-bearing hosts via several factors, and suppress type-1 T-cell function via multiple mechanisms. Elevated MDSC in the blood of renal cell carcinoma patients, were first shown to significantly decline following treatment with sunitinib (inhibits VEGFr, ckit, flt3, PDGFr) treatment. This decline was correlated with a recovery in patients’ T-cell function, an effect which could be reproduced with in vitro MDSC depletion. Sunitinib induced MDSC apoptosis in vitro. Sunitinib-mediated declines in MDSC occurred even in non-responder patients and were not correlated with tumor shrinkage. Studies in several murine tumor models confirmed sunitinib’s ability to universally