Key Concepts in Targeting Cancer Stem Cells

to Manage Disease

Key Concepts in Targeting Cancer Stem Cells

to Manage Disease

ModeratorHoward S. Hochster, MD Professor of MedicineAssociate Director for Clinical SciencesClinical Research Program Leader, Gastrointestinal Cancers ProgramYale Cancer CenterNew Haven, Connecticut

Max S. Wicha, MDMadeline and Sidney Forbes Professor of OncologyFounding Director EmeritusUniversity of Michigan Comprehensive Cancer CenterAnn Arbor, Michigan

Manish A. Shah, MD Associate Professor of Gastrointestinal OncologyDirector, Gastrointestinal Oncology ProgramCo-Director, Center for Advanced Digestive CareWeill Cornell Medical College of Cornell University/New York-Presbyterian HospitalNew York, New York

Panelists

Program GoalsProgram 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?What Are CSCs?

• 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;3:730-737; c. Al-Haji M, et al. Proc Natl Acad Sci U S A. 2003;100:3983-3988.

"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

Types of Human Stem CellsTypes of Human Stem Cells

• Pluripotent stem cells that can give rise to any type of cell in the body

• Found in embryos• Capable of self-renewal

Embryonic 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

Adult 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

Cancer stem cells

De Los Angeles A, et al. Nature. 2015;525:469-478.

• 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

Detecting CSCsDetecting CSCs

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.

Serial Transplantation Assays

CSC Markers in Different Tumor TypesCSC Markers in Different Tumor Types

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.

• 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

CSC Model vs Stochastic ModelCSC Model vs Stochastic Model

CSC

CSC

CSC CSC

CSC

CSC

CSC

CSC

CSC

CSC

CSC

P

P

P P

P P

P

M M

M M M M M M

Asymmetricdivision

Symmetricdivision

Stochastic (Clonal Evolution) Model

= oncogenic event

CSC (Hierarchical) Model

= oncogenic eventCSC = cancer stem cellP = progenitor cellM = mature cell

All tumor cells can self-renew or differentiate.

Tumor HeterogeneityTumor Heterogeneity

Shackleton M, et al. Cell. 2009;138:822-829.

• 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 stem-

like cells and proliferate− Migration of a CSC into another tissue microenvironment can

cause further diversification

Seed-and-Soil HypothesisSeed-and-Soil Hypothesis

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.

• 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

Epithelial-to-Mesenchymal Transition (EMT)Epithelial-to-Mesenchymal Transition (EMT)

a. Radisky DC, LaBarge MA. Cell Stem Cell. 2008;2:511-512; b. Thiery JP. Nat Rev Cancer. 2002;2:442-454.

• 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

Epithelial Cells

Transition

Mesenchymal Cells

Basement membrane

Plasticity of CSCs and EMTPlasticity of CSCs and EMT

Liu S, et al. Stem Cell Reports. 2013;2:78-91.

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.

Epithelial-like State Mesenchymal-like State• Express epithelial markers• Have polarity• Proliferate extensively• Found more in tumor interior

• Express mesenchymal markers• Remain relatively quiescent• Found at tumor invasive front• Invade the bloodstream• Establish micrometastases

Qualities of CSCs in Epithelial-like vs Mesenchymal-like State

Drivers of Epithelial-Mesenchymal and Mesenchymal-Epithelial TransitionsDrivers of Epithelial-Mesenchymal and Mesenchymal-Epithelial Transitions

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.

• 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

Therapeutic Targets for CSCsTherapeutic Targets for CSCs

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.

• 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

Response of CSCs to ChemotherapyResponse of CSCs to Chemotherapy

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.

• 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

CSC

Popu

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n, %

Control

Repar

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Repar

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Doceta

xel

20

16

12

8

4

0

In Vitro Assessment of CSCs After Chemotherapya

Pilot Study of Reparixin Plus PaclitaxelPilot Study of Reparixin Plus Paclitaxel

Schott AF, et al. SABCS 2014. Abstract P6-03-01.

• 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

Co-Targeting CSCs and Bulk Tumor CellsCo-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 TherapyResistance of CSCs to Conventional Therapy

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]

CSCs have several pro-survival mechanisms that help them resist chemotherapy, radiotherapy, and targeted drugs.

Intrinsic Mechanisms Indirect Mechanisms• Enhanced DNA repair• High expression of drug

efflux pumps• Upregulation of anti-

apoptotic factors and detoxification enzymes• Quiescence during treatment

to protect self-renewal ability

• Induction of EMT or acquisition of CSC markers via signaling pathways in the microenvironment• Hypoxia-triggered adaptive

changes via HIF activation (eg, formation of new blood vessels that are poor drug transporters)

Tumor Regression: Inadequate End PointTumor Regression: Inadequate End Point

Reya T, et al. Nature. 2001;414:105-111.

CSC-directed

therapy

Conventional therapy

Tumor cannot make new cells Mature cells die,

tumor shrinks, patient is cured.

CSC regenerates the tumor Patient has a relapse.

CSC

CSC

CSCNotable tumor

shrinkage

Little tumor shrinkage

Tumor shrinkage by RECIST may not be useful for evaluating response to CSC-directed therapies.

Efficacy End Points for CSC-Directed AgentsEfficacy End Points for CSC-Directed Agents

End PointTime 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

Possible End Points for Trials of CSC-Directed Agents

CSCs as a Prognostic MarkerCSCs as a Prognostic Marker

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.

• 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

Circulating Tumor Cells and CSCsCirculating Tumor Cells and CSCs

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.

• 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

CSC-Directed Agents in Clinical TrialsCSC-Directed Agents in Clinical Trials

a. Liu S, Wicha MS. J Clin Oncol. 2010;28:4006-4012; b. Prud'homme GJ. Curr Pharm Des. 2012;18:2838-2849.

Tarextumab(OMP-59R5)

β-CAT

IIIIIIII

IIIIIIII

IIIiIIIii

iiiiiiiiii

iiiiiiiiii

iIL-

8II

CXCR1

Self-renewal, drug resistance, metastasisTARGET DNA

Cancer stem cell

NOTCH

DLL/JAG WNT

LPR/FZD

Demcizumab(anti-DLL4)

Vantictumab(anti-FZD)

Ipafricept(OMP-54F28)

Reparixin

FAK Defactinibβ-CAT, STAT3, NanogBBI608

IL-8

CXCR1

FAK

Several CSC-directed agents with various targets were shown to be safe in phase 1 clinical trials.

Forced Differentiation of CSCsForced Differentiation of CSCs

a. Guessous F, et al. Cell Cycle. 2010;9:1031-1036.

• 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

Immunotherapy and CSCsImmunotherapy and CSCs

a. Teitz-Tennenbaum S, et al. Oncoimmunology. 2012;1:1401-1403; b. Lin M, et al. Immunol Res. 2015;62:16-22.

• 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

SummarySummary

• 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

AbbreviationsAbbreviationsAE = adverse eventALDH = aldehyde dehydrogenase AML = acute myelogenous leukemia ASC = adult stem cellCDH1 = cadherin-1CR = complete responseCSC = cancer stem cellsCTC = circulating tumor cellEMT = epithelial-to-mesenchymal transitionEpCAM = epithelial cell adhesion moleculeERK= extracellular signal-regulated kinase IL = interleukinGI = gastrointestinal HER2 = human epidermal growth factor receptor 2JAK = Janus kinaseM = mature cellMAPK = mitogen-activated protein kinase

Abbreviations (cont)Abbreviations (cont)MBC = metastatic breast cancerMSI = microsatellite instabilityP = progenitor cellPD-1 = programmed cell death 1PD-L1 = programmed death ligand 1PR = partial responseRECIST = Response Evaluation Criteria In Solid TumorsSTAT = signal transducer and activator of transcriptionTGF-β = transforming growth factor-βTTP = time to progression

Abbreviations (cont)Abbreviations (cont)MBC = metastatic breast cancerMSI = microsatellite instabilityP = progenitor cellPD-1 = programmed cell death 1PD-L1 = programmed death ligand 1PR = partial responseRECIST = Response Evaluation Criteria In Solid TumorsSTAT = signal transducer and activator of transcriptionTGF-β = transforming growth factor-βTTP = time to progression

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