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Key Concepts in Targeting Cancer Stem Cells to Manage Disease Moderator Howard S. Hochster, MD Professor of Medicine Associate Director for Clinical Sciences Clinical Research Program Leader, Gastrointestinal Cancers Program Yale Cancer Center New Haven, Connecticut Panelists Max S. Wicha, MD Madeline and Sidney Forbes Professor of Oncology Founding Director Emeritus University of Michigan Comprehensive Cancer Center Ann Arbor, Michigan Manish A. Shah, MD Associate Professor of Gastrointestinal Oncology Director, Gastrointestinal Oncology Program Co-Director, Center for Advanced Digestive Care Weill Cornell Medical College of Cornell University/ New York-Presbyterian Hospital New York, New York Program Goals • Define cancer stem cells (CSCs) • Understand the role of CSCs in tumor development, metastasis, and disease progression • Describe how CSCs resist chemotherapy and other conventional treatments • Review advances in CSC-directed therapies This program will discuss investigational agents that the FDA has not approved for use in the United States. What Are CSCs? "CSCs are cells within a tumor that possess the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor." - AACR Consensus Workshop, 2006a • CSCs (sometimes called tumor-initiating cells) are a small subpopulation of cells in a tumorb • CSCs were first identified in AML and have since been found in most solid tumors and hematologic cancersb,c • According to the CSC hypothesis, CSCs drive tumorigenesis and metastasisa a. Clarke MF, et al. Cancer Res. 2006;66:9339-9344; b. Bonnet D, Dick JE. Nat Med. 1997;3:730737; c. Al-Haji M, et al. Proc Natl Acad Sci U S A. 2003;100:3983-3988. Types of Human Stem Cells Embryonic stem cells • Pluripotent stem cells that can give rise to any type of cell in the body • Found in embryos • Capable of self-renewal Adult stem cells • Multipotent stem cells that can differentiate into all the specialized cells needed to maintain their dedicated tissue • Found in all organs and in the blood • Capable of self-renewal Cancer stem cells • Multipotent stem cells that can differentiate into all the cells within a tumor • Found in the tumor microenvironment or "niche" • May arise from a mutated ASC or progenitor cell or from a reprogrammed differentiated cell De Los Angeles A, et al. Nature. 2015;525:469-478. Detecting CSCs Serial Transplantation Assays • This is the gold standard for definitively identifying CSCsa • Suspected CSCs are injected into immunodeficient mice to see whether they generate tumorsa − Only a few CSCs are needed to initiate a tumor − Drawback is differences in mouse vs human immune system Cell Surface Markers • Fluorescence-activated cell sorting and conjugating antibodies to magnetic beads can identify CSC markers • Colony-forming assays show whether a cell can self-renew • Sphere-forming assays measure how many tumor spheres a CSC produces in vitro (tumorigenic efficiency)b • Cell surface markers are good surrogates but are not definitive a. Bonnet D, Dick JE. Nat Med. 1997;3:730-737; b. Ajani JA, et al. Semin Oncol. 2015;42:S3-S17. CSC Markers in Different Tumor Types • CD133+ is 1 of the most common CSC markersa,b − CD133+ CSCs were isolated first in brain tumors and later in cancers of the colon, liver, breast, lung, ovary, prostate, pancreas, eye, skin, and bone/soft tissue − CD133+ may predict chemoresistance and worse survival • Breast CSC markers include CD44+, CD24-, and ALDH − Markers vary depending on histologic subtypec − CD44+/CD24-/low phenotype predicts poor prognosis • Some markers (eg, CD44 and CD24) are observed in CSCs across many tumor typesa a. Ajani JA, et al. Semin Oncol. 2015;42:S3-S17; b. Grosse-Gehling P, et al. J Pathol. 2013;229:355-378; c. De Beca FF, et al. J Clin Pathol. 2013;66:187-191. CSC Model vs Stochastic Model CSC (Hierarchical) Model Asymmetric CSC division = oncogenic event CSC = cancer stem cell P = progenitor cell M = mature cell P CSC P M M M CSC P CSC M Stochastic (Clonal Evolution) Model = oncogenic event All tumor cells can self-renew or differentiate. CSC M CSC P M CSC Symmetric division CSC P P CSC M CSC M P CSC Tumor Heterogeneity • Both the stochastic and CSC models start with 1 cell that produces a tumor with genetically distinct cells • The stochastic model attributes genetic heterogeneity in tumors to clonal evolution, with no cell hierarchy • The CSC model also accounts for tumor heterogeneity − Mutations or epigenetic changes cause CSC subclones to produce different progenitor cells from their parents − Mature cells with acquired mutations dedifferentiate into stemlike cells and proliferate − Migration of a CSC into another tissue microenvironment can cause further diversification Shackleton M, et al. Cell. 2009;138:822-829. Seed-and-Soil Hypothesis • In 1889, Paget noticed metastasis was more frequent in certain organs and proposed it was not randoma-c • Studies suggest metastasis results from signaling between the cancer cell (seed) and the organ microenvironment at metastatic sites (soil)b • The microenvironment regulates angiogenesis, an important component of metastasisd "When a plant goes to seed, its seeds are carried in all directions; but they can only live and grow if they fall on congenial soil." – Stephen Paget, 1889a,b a. Paget S. Cancer Metastasis Rev. 1989;8:98-101; b. Langley RR, Fidler IJ. Int J Cancer. 2011;128:2527-2535; c. Chambers AF, et al. Nat Rev Cancer. 2002;2:563-572; d. Folkman J. N Engl J Med. 1971;285;1182-1186. Epithelial-to-Mesenchymal Transition (EMT) Epithelial Cells Mesenchymal Cells Transition Basement membrane • EMT is important during embryogenesis and wound repaira • Loss of epithelial markers like E-cadherin (CDH1) causes CSCs to undergo EMT and lose their adhesive propertiesb − The cells detach from the extracellular matrix and from each othera,b − They progressively acquire mesenchymal-like traitsa,b − The now free-floating mesenchymal cells enter the blood stream or lymph nodes and metastasize to distant sitesa a. Radisky DC, LaBarge MA. Cell Stem Cell. 2008;2:511-512; b. Thiery JP. Nat Rev Cancer. 2002;2:442-454. Plasticity of CSCs and EMT The plasticity of CSCs allows the cells to transition between epithelial and mesenchymal states, which is what gives them the ability to invade tissue, disseminate, and grow at metastatic sites. Qualities of CSCs in Epithelial-like vs Mesenchymal-like State Epithelial-like State • Express epithelial markers • Have polarity • Proliferate extensively • Found more in tumor interior Liu S, et al. Stem Cell Reports. 2013;2:78-91. Mesenchymal-like State • Express mesenchymal markers • Remain relatively quiescent • Found at tumor invasive front • Invade the bloodstream • Establish micrometastases Drivers of Epithelial-Mesenchymal and Mesenchymal-Epithelial Transitions • Reversible EMT/MET transitions are regulated by the tumor microenvironment • Adverse tumor conditions that drive EMT includea,b − Hypoxia (sometimes caused by antiangiogenic agents) − Nutrient deprivation − Mutations that cause E-cadherin loss • microRNA regulates EMT/MET via multiple pathwaysb • Myeloid precursor cells at the pre-metastatic site (niche) send homing signals to attract CSCsc a. Liu S, et al. Stem Cell Reports. 2013;2:78-91; b. Thiery JP. Nat Rev Cancer. 2002;2:442-454; c. Kaplan RN, et al. Nature. 2005;438:820-827. Therapeutic Targets for CSCs • Therapies should target stemness pathways (internal pathways that induce or maintain stemness properties in CSCs)a − Wnt receptors and ligands − Notch receptors and ligands − Hedgehog (ligands, Patched-Smoothened complex) − JAK/STAT pathway, including STAT3 − TGF-β family of cytokines − Hippo − Crosstalk between Notch and other oncogenic pathways • Inflammatory mediators (extracellular pathways), like IL-6 and IL-8, promote CSC self-renewal and are also good treatment targetsb,c a. Ajani JA, et al. Semin Oncol. 2015;42:S3-S17; b. Iliopoulous D, et al. Cell. 2009;139:693-706; c. Ginestier C, et al. J Clin Invest. 2010;120:485-497. Response of CSCs to Chemotherapy 20 CSC Population, % • In preclinical models, the CSC population increased after chemotherapya • Clinical studies have observed enrichment of CSCs in patients treated with chemotherapyb • One proposed mechanism is the cytokines that dying cells release (eg, IL8) stimulate development of CSCsa In Vitro Assessment of CSCs After Chemotherapya 16 12 8 4 0 a. Ginestier C, et al. J Clin Invest. 2010;120:485-487; b. Li X, et al. J Natl Cancer Inst. 2008;100:672-679. Pilot Study of Reparixin Plus Paclitaxel • Phase 1b study in HER2-negative MBC (N = 33) − Reparixin loading dose (400, 800, or 1200 mg) 3 times/d for 3 days was followed by paclitaxel (80 mg/m2/wk) + reparixin for 21 days − Treatment continued until disease progression or toxicity • Safety was assessed after 1 cycle − 15 serious AEs occurred, none related to reparixin − 30% of patients experienced a grade 3/4 AE • Efficacy assessment included 18 patients − 2 CR and 6 PR − Duration of response: 2-20 mo Schott AF, et al. SABCS 2014. Abstract P6-03-01. Co-Targeting CSCs and Bulk Tumor Cells • Targeting CSCs − CSCs that survive treatment will repopulate the tumor − CSCs can cause recurrence even years later − Surviving CSCs have the potential to metastasize • Targeting bulk non-tumorigenic cells − In advanced cancer, the bulk tumor population is large enough to constitute a huge burden or death − Plasticity may allow mature tumor cells to dedifferentiate and acquire stem-like properties Effective treatment strategies must target the CSCs and the cells that make up the bulk of the tumor. Resistance of CSCs to Conventional Therapy CSCs have several pro-survival mechanisms that help them resist chemotherapy, radiotherapy, and targeted drugs. Intrinsic Mechanisms Indirect Mechanisms • Enhanced DNA repair • Induction of EMT or acquisition of CSC markers via • High expression of drug signaling pathways in the efflux pumps microenvironment • Upregulation of anti• Hypoxia-triggered adaptive apoptotic factors and changes via HIF activation detoxification enzymes • Quiescence during treatment (eg, formation of new blood vessels that are poor drug to protect self-renewal ability transporters) a. Maugeri-Saccà M, et al. Clin Cancer Res. 2011;17:4942-4947; b. Skvortsova I, et al. SeminCancer Biol. 2015 Sep 22. [Epub ahead of print]; c. Hashida S, et al. Cancer Sci. 2015 Jul 21. [Epub ahead of print] Tumor Regression: Inadequate End Point Tumor shrinkage by RECIST may not be useful for evaluating response to CSC-directed therapies. Little tumor shrinkage Tumor cannot make new cells Mature cells die, tumor shrinks, patient is cured. CSC CSC CSC Notable tumor shrinkage Reya T, et al. Nature. 2001;414:105-111. Patient has a relapse. Efficacy End Points for CSC-Directed Agents Possible End Points for Trials of CSC-Directed Agents End Point Time to progression (TTP) CSC agents delay TTP, so patients will have to take them until they have significant progression to determine their effectiveness Pathologic CR to neoadjuvant therapy This is a well-defined predictor of recurrence; a biopsy before and after neoadjuvant treatment would show the direct effects of therapy on CSCs Immune-related response criteria Measurable new lesions are incorporated into total tumor burden because tumors may grow before response is evident Reduction in CSCs A decrease in CSCs may predict better prognosis Overall survival CSC-directed agents must be shown to prolong survival without compromising patient safety CSCs as a Prognostic Marker • Studies have linked CSC markers with an increased risk of metastasis or recurrence, worse survival outcomes, and treatment resistancea • CSC markers have been found to have prognostic significance in many solid tumors, including breast, colon, brain, and head and neck cancers; and in hematologic malignancies such as AMLa • Breast cancer studies have shown higher proportions of cells expressing CSC marker ALDH1+ after neoadjuvant chemotherapy predicts early metastasis and worse survival outcomesb-d a. Ajani JA, et al. Semin Oncol. 2015;42:S3-S17; b. Charafe-Jauffret E, et al. Clin Cancer Res. 2010;16:45-55; c. Sakakibara M, et al. Cancer. 2012;118:3899-3910; d. Khoury T, et al. Mod Pathol. 2012;25;388-397. Circulating Tumor Cells and CSCs • Circulating tumor cells (CTCs) can predict the risk of metastasis and relapse in patients treated for cancera • Analyzing the portion of CTCs that are CSCs may offer clues to the efficacy of CSC-directed therapies • The only FDA-approved method for isolating CTCs uses the epithelial adhesion molecule EpCAMa − ~50% of CTCs have undergone EMT and have low expression of EpCAM or other epithelial markersb − This method may fail to detect CSCs that survive therapy • New technologies are being studied that use microfluidics to isolate single cells by biomarkerc a. Balic M, et al. Expert Rev Mol Diagn. 2012;12:303-312; b. Aktas B, et al. Breast Cancer Res. 2009;11:R46; c. Karabacak NM, et al. Nat Protoc. 2014;9:694-710. CSC-Directed Agents in Clinical Trials Several CSC-directed agents with various targets were shown to be safe in phase 1 clinical trials. Ipafricept (OMP-54F28) Demcizumab (anti-DLL4) DLL/JAG Tarextumab (OMP-59R5) BBI608 WNT Vantictumab (anti-FZD) NOTCH LPR/FZD β-CAT β-CAT, STAT3, Nanog TARGET DNA IIIIIII IL-8 IIIIIII CXCR CXCR1 1 IIIIIiI IIiiiiiii iiiiiiiii FAK FAK iiiiiiiI L-8II Reparixin Defactinib Self-renewal, drug resistance, metastasis Cancer stem cell a. Liu S, Wicha MS. J Clin Oncol. 2010;28:4006-4012; b. Prud'homme GJ. Curr Pharm Des. 2012;18:2838-2849. Forced Differentiation of CSCs • CSC-directed agents that inhibit signaling along the self-renewal pathway have been found to activate quiescent CSCs, causing them to differentiatea • Inducing CSCs to differentiate may represent a viable CSC-directed treatment strategy − Once differentiated, the CSCs would be susceptible to chemotherapy − You could potentially exhaust the population of CSCs • Effective cancer treatment will likely require a combination regimen with a CSC-directed agent and an agent that targets the bulk non-tumorigenic cells a. Guessous F, et al. Cell Cycle. 2010;9:1031-1036. Immunotherapy and CSCs • A small percentage of patients with cancer treated with immunotherapy have durable remission • Preclinical models suggest the immune system in these patients eradicates the CSCs • Anti-CSC vaccines are in development − In a proof-of-concept study, a dendritic cell-based vaccine gave immunocompromised mice antitumor immunitya − Preliminary evidence from a phase 1/2 study (N = 90) of a novel lung cancer CSC vaccine found it safe and effectiveb • Combining vaccines with checkpoint inhibitors may improve cure rates in some cancers a. Teitz-Tennenbaum S, et al. Oncoimmunology. 2012;1:1401-1403; b. Lin M, et al. Immunol Res. 2015;62:16-22. Summary • Growing evidence shows CSCs contribute to the development, growth, and metastasis of cancer • CSCs may originate from a mutated ASC or a mature cell that acquires stem-like properties • Many novel agents that target CSC signaling pathways or cytokines are being studied in clinical trials, and early data suggest these drugs are relatively safe • More research is needed to identify appropriate end points for CSC-directed therapies and to identify safe combinations that target CSCs and bulk tumor cells Abbreviations AE = adverse event ALDH = aldehyde dehydrogenase AML = acute myelogenous leukemia ASC = adult stem cell CDH1 = cadherin-1 CR = complete response CSC = cancer stem cells CTC = circulating tumor cell EMT = epithelial-to-mesenchymal transition EpCAM = epithelial cell adhesion molecule ERK= extracellular signal-regulated kinase IL = interleukin GI = gastrointestinal HER2 = human epidermal growth factor receptor 2 JAK = Janus kinase M = mature cell MAPK = mitogen-activated protein kinase Abbreviations (cont) MBC = metastatic breast cancer MSI = microsatellite instability P = progenitor cell PD-1 = programmed cell death 1 PD-L1 = programmed death ligand 1 PR = partial response RECIST = Response Evaluation Criteria In Solid Tumors STAT = signal transducer and activator of transcription TGF-β = transforming growth factor-β TTP = time to progression Abbreviations (cont) MBC = metastatic breast cancer MSI = microsatellite instability P = progenitor cell PD-1 = programmed cell death 1 PD-L1 = programmed death ligand 1 PR = partial response RECIST = Response Evaluation Criteria In Solid Tumors STAT = signal transducer and activator of transcription TGF-β = transforming growth factor-β TTP = time to progression References 1. 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