Lung Cancer Stem Cells and Targeting of Embryonic Signaling Pathways

Claudio Dansky Ullmann, MD Cancer Therapy Evaluation Program, National Cancer Institute

I HAVE NO DISCLOSURES OR CONFLICTS

Lung Cancer: There Are Some Changes But Not Enough

Plateau of effectiveness of doublet chemotherapy Advanced NSCLC : 10 months OS Addition of bevacizumab: 12 months OS Maintenance: OS? SCLC: MST 8-10 months / 5-10% 2 y SV EGFR mutant NSCLC EGFR TKI >70% will respond 12 mo PFS 2 y OS EML-4/ALK fusion NSCLC Crizotinib 64% ORR 90% DCR Median PFS not reached

Despite profound responses, patients eventually develop disease progression (Are reemergent/resistant cells enriched in CSCs?)

Why Target Cancer Stem Cells? The CSC Hypothesis

Tumors have a subpopulation of cells with Stem Cell features among a predominant differentiated population Standard treatment targets mainly the bulk cells and re-emerging cells are consistent with CSC with increased signaling of embryonic pathways, resistance and survival mechanisms Better characterization of CSC compartment and interactions is needed

Current Anticancer Therapies vs. CSC-Based Therapies

R Gangemi et al. Curr Med Chem, 2009

TheCancerStemCellHypothesis:Implications
ResponseRate:Maybelargelydeterminedbybehaviorofthe(large) chemosensitive cellpopulation Survival:Maybedeterminedprimarilybybehaviorofthe(small) chemoresistant cellpopulation Implications:
Newanticanceragentsthatkillmoreofthesamechemosensitivepopulationmaynot leadtofurtherimprovementinsurvival Analysisofthepropertiesofthesmallchemoresistantpopulationmaybeinformative

response
rd nda s Sta toxic o cyt

recurrence

noresponse
Ste the mcel rap l ies

resorption/necrosis

Rudinetal.,JNCCN, 2008

Normal stem cells and cancer cells

Dontu, Gabriela, Al-Hajj, Muhammad, Abdallah, Wissam M., Clarke, Michael F. & Wicha, Max S. Stem cells in normal breast development and breast cancer. Cell Proliferation 36 (s1), 59-72.

Features of Normal Stem Cells and Cancer Initiating Cells

R Gangemi et al. Curr Med Chem, 2009

RP Hill and R Perris. JNCI, 2007

CSC:CanWeReallyDefineThem?
PutativeCSCidentifiedinseveralhematologicandsolidtumors:
Leukemia,MultipleMyeloma,Breast,Colorectal,Ovarian,Head&Neck,Pancreatic, Brain,Melanoma,Liver,Lung

Butmanyquestionsstillunresolved:
MurineCSCvs.HumanCSC:Notallcounterpartsareclearfordifferenttumortypes CSCorigin:NormalSC?RestrictedProgenitors?Dedifferentiatedcells? OneCSCtypepertumortype? CSCmarkersstilltobewelldefined lackofvalidation

NSCLC: CD133, SP-C, CCA, Sca-1, ALDH 1&3?, Sulf-2? SCLC: CD87 (uPar), CD133, ALDH 1&3?

CA OBrien et al. Clin Can Res, 2010

Epithelial Organization of the Murine Distal Lung

DM Raiser et al. CSHS on QB, 2008

Model of BASC Function and Distal Lung Homeostasis

Quiescent normal lung epithelia

Ablated Clara Cells

BASC stimulated to self-renew and generate Clara Cells to repair epithelium

Naphthalene lung injury model

DM Raiser et al. CSHS on QB, 2008

Proposed Sites of Self-Renewal and Tumor Initiation in Lung Epithelia

JP Sullivan et al. Ca Met Rev, 2010

Human CSC assayed in Immunodeficient Mice In Vivo Transplantation Models to Detect and Characterize True CSCs

CA OBrien et al. Clin Can Res, 2010

Importance of Tumor Genotype as a Determinant of Lung Cancer Propagating Cells

All Develop Lung Adenocarcinomas

SJ Curtis and CF Kim. Cell Stem Cell, July 2, 2010

Tumor Propagating Capacity of Sca-1+ Lung Cancer Cells

Conclusion: The identity of the L-CSC population promoting adenocarcinogenesis is likely dependent on the oncogenotype driving the malignancy Implications: Histologically distinct tumors may contain more than one TPC or CSC populations The same marker or set of markers may not identify TPC in all patient samples of a specific tumor type Oncogene phenotype specificity CSC heterogeneity CSC may or may not respond equally to one CSC-targeted TX different L-CSCs may need to be targeted differently
SJ Curtis and CF Kim. Cell Stem Cell, July 2, 2010 JP Sullivan and JD Minna. Cell Stem Cell, July 2, 2010

Lung Cancer Stem Cells Complex Challenge

Overall, need further development of:

Lung CSC identification and functional characterization Better in vivo models - challenging Biomarker validation Assay development Understanding how critical pathways in lung cancer (i.e. EGFR, Ras, P 53, PI3 K AKT-mTO R, c-Met, IGFR, etc) affect or are affected in this context Cross-talk with non-CSC cells and with TME Combined targeting of CSC pathways

Targeting Embryonic Developmental Pathways

CSC driven by pathways that promote self-renewal of normal stem cells

WNT, BMI-1, SHh, Notch, etc

In the absence of well-accepted CSC markers:

The targeting of a small chemoresistant population of clonogenic cells driven by aberrant expression/function of embryonic pathways may be able to eliminate the CSC population

Signaling Pathways: Normal Stem Cell Development/Transformation

Embryonic Signaling Cross-Talk

N Takebe and S Percy Ivy. Clin Can Res, 2010

CTEP Clinical Drug Development Agents Targeting Embryonic pathways / Cancer Initiating Cells
Key to successful translation to clinical use:
To test these therapies in the appropriate patient population

Challenge: Marker and clinical assay development, qualification/validation

For identification of CIC (not widely available/ Lab investigators experience) PD markers for CIC targeting therapeutics

Current CTEP clinical programs:

Targeting of Hedgehog and Notch pathways
GDC-0449 and RO 4929097

Actively pursuing agents targeting Wnt pathway

Clincial trials of Hh inhibitor in SCLC (ongoing), Notch inhibitor in NSCLC (in development), and pathway combinations (in development) Collaboration CTEP / DTP / Pharma / Academia to support pre-clinical translational development

TheHedgehogsignalingpathway

Cyclopamine

Pasca di Magliano & Hebrok, Nature Rev CA, 2003

Hedgehogsignalinginlungdevelopment
Mouse embryonic lung, dpc 11.5
Shh (ligand) Ptc1(receptor)

SP-C Shh transgenic mouse

Bellusci et al., Devel, 1997

Lungdevelopmentrequires SonicHedgehog
Shh knock-out mouse
Ptc1(receptor)

dpc 12.5

dpc 18.5

Pepicelli et al., Curr Biol, 1998

Hedgehogsignalinginlunginjuryrepairandincancer
Napthalene lung injury model

Small cell lung cancer

Expression of both in 5 of 10 tumors

H249 xenografts
Watkins et al., Nature, 2003

The Hedgehog Pathway Is Mentioned As Operative In Nearly Every Type of Cancer

1. HH pathway is mentioned as an important signaling pathway

prostate cancer ovarian cancer breast cancer Ewing's SCCHN

pancreatic cancer esophagus biliary tract multiple myeloma

hepatoma colon cancer stomach NSCLC

glioblastoma rhabdo myosarcoma ALL

hepatoblastoma

Burkitts lymphoma SCLC

melanoma cholangiocarcinoma

Hedgehog Signaling Is Important (? Essential) for Maintenance of Tumor Stem Cells Hematologic and Solid Malignancies
Tumor type CML Comment Author Zhao et al. Nature 458: 776-779, 2009 Peacock et al. PNAS 104: 4048-4053, 2007 Clement et al. Current Biology 17: 165-172, 2007 Bar et al. Stem Cells 25: 2524-2533, 2007 Liu et al. Cancer Res. 66: 6603-6071, 2006 Lee et al. J. Clin Oncol. 26: 2806-2811, 2008 Watkins et al. Cell Cycle. 2:196-8, 2003 Required for maintenance of stem cells and imatinib resistant cells HH pathway blockade inhibits clonal expansion HH-Gli signaling required for sustained growth HH blockade eliminates tumorigenicity HH signaling pathway is activated Sonic HH upregulated HH pathway activated

Myeloma Glioblastoma multiforme Glioblastoma multiforme Breast cancer Pancreatic cancer SCLC

OpportunitiesforHhpathwayinhibition
Ligand Independent Ligand Dependent

Genetic Mutations

Paracrine Signaling

Signaling to tumor cell

BCC Medulloblastoma

Pancreatic, CRC ovarian, prostate, bladder SCLC?

CML , MM, CLL, chondrosarcoma

Hedgehogdysregulationvariesbycancertype
State Normal Basal cell carcinoma*1,2 Medulloblastoma*3 Pancreatic cancer4,5,6 Prostate cancer7,8 Small cell lung cancer9 Hepatocellular cancer10 Breast cancer11 Hedgehog OFF OFF OFF Turned ON Turned ON Turned ON Turned ON Turned ON
* Mutation in Patched

Patched ON Mutant - OFF Mutant - OFF OFF OFF OFF OFF OFF

Smoothened OFF ON ON ON ON ON ON ON

Frequency --95% 30-40% 100% 100% 50% ? 100%

1 2

Hahn et al., Cell, 1996 Bale & Yu, Human Mol Gen, 2001 3 Berman et al., Science, 2002 4 Berman et al., Nature, 2003 5 Kayed et al., Int J Cancer, 2004

6 7

Thayer et al., Nature, 2003 Karhadkar et al., Nature, 2004 8 Fan et al., Endocrinology, 2004 9 Watkins et al.,, Nature, 2003 10 Sicklick ASCO 2005; Mohini, AACR 2005 11 Kubo et al., Cancer Res, 2004

Hedgehog Signaling in Lung Cancer

Hedgehog signaling in normal lung
Role in the maintenance and expansion of the progenitor (stem) cells. Essential for the growth and differentiation of trachea and lung. Normal airway epithelial compartment uses the Hh pathway to repopulate itself following acute injury

Hedgehog signaling progenitor phenotype in SCLC

Ligand dependent Hh pathway activated in lung development during normal differentiation of pulmonary neuroendocrine precursor cells and in SCLC Increased Hh ligand and overexpression of Gli in SCLC Hh signaling is a frequent in vivo but rare in vitro event in SCLC

Hedgehog signaling in NSCLC

NSCLC cells express Shh protein GLI1 overexpression less frequent Reduced expression of HIP (natural antagonist of Hh signaling)

SCLC may be more dependent on Hh signaling than NSCLC

AA Merchant and W Matsui, Clin Can Res, 2010

PreclinicaleffectsofHhinhibitorIPI926inminimalresidualdisease Delayintumorrecurrencepostcytoreduction
SCLCpostetoposide/cisplatin NSCLCpostgefitinib

UpregulationofHhligandandGli1withcytoreduction
IncreaseintumorstromaHhsignaling InhibitedbyIPI926TX

InhibitsserialSCLCxenografttransplantion
?effectonatumorstemcellpopulation

 Small molecule, oral inhibitor of SMOOTHENED

IC50 in Gli-Luciferase assay = ~ 3 nM Molecular weight 421.30 g/mol Preclinical pharmacokinetics favor once daily dosing
H HN H

GDC-0449

O H H HO H

Cyclopamine

GDC-0449

Developed by Genentech under a collaboration agreement with Curis.

AtargetedHedgehoginhibitorhasactivityagainst PTCH1 mutanttumors

Basal cell carcinoma Medulloblastoma

Von Hoff et al., N Engl J Med, 2009 Rudin et al., N Engl J Med, 2009 Von Hoff D et al. N Engl J Med 2009;361:1164-1172

Most Common Adverse Events in Patients with BCC (n=33)

Von Hoff D et al. N Engl J Med 2009;361:1164-1172

GDC- 0 449 Phase 1: Summary

GDC-0449 has an unusual pharmacokinetic profile High sustained micromolar plasma concentrations Long terminal half-life Dose-limiting toxicities were not seen with GDC-0449 Reversible grade 3 fatigue and asymptomatic hyponatremia reported beyond the DLT window Objective responses seen in patients with basal cell carcinoma Phase II studies using GDC-0449 at 150 mg once daily are ongoing

E1508:arandomizedphaseIIstudyofC/E+/ HHinhib GDC0449orIGF1RMoAbA12

Chemotherapy Maintenance

Hedgehog inhib

Hedgehog inhib

IGF-1R moAb

IGF-1R moAb

Belani&Rudin

Notch Signaling and Proteolytic Processing

Notch receptor is a cell surface protein. Interaction with the Notch ligand, i.e. Jagged, initiates proteolytic cleavage at the extracellular site by {}secretase followed by cleavage at the intracellular site by {}-secretase, with release of Notch-IC Notch-IC is then translocated into the nucleus where it interacts with CSL and recruits coactivators (to form a transcription-activating complex Notch-IC can be polyubiquitylated and targeted for degradation

Shih, I.-M. et al. Cancer Res 2007

Notch Pathway Role Targeting Notch Axis Inhibits Tumor Growth

Fundamental role in cell fate decisions (stem cell maintenance, cell proliferation, differentiation, and apoptosis) Notch ligand (DLL-4) cooperates to alter tumor angiogenesis. Notch and VEGF signaling cooperate to drive tumor angiogenesis by modulating endothelial cell fate Notch signaling axis is disrupted in primary human cancer Notch signaling promotes tumor proliferation and survival Inhibition of Notch signaling inhibits tumor proliferation and survival
leads to a less transformed phenotype with loss of soft agar growth Inhibits tumor angiogenesis Inhibits cancer stem cell proliferation and survival

Altered Notch expression and function associated with driving a tumor phenotype

T-ALL Pancreatic Lung carcinoma Breast Cervical Colon Melanoma Ovarian Medulloblastoma Testicular germ cell tumors Head and Neck Glioblastoma HCC

Notch and NSCLC

NSCLC cell lines commonly express: Notch Notch ligand (Jagged 1) Notch transcriptional target genes (HES1, HEY1) Notch signaling in: ALDH + NSCLC stem cells CD133 + NSCLC stem cells (Kurie et al)

Notch and NSCLC

Chromosome 19 translocation leads to dysregulation and overexpression of Notch-3 in Squamous NSCLC Tumors overexpressing Notch-3 are highly sensitive to Notch inhibition Dominant negative Notch-3 inhibits MAPK and growth of lung cancer Lung adenocarcinomas: Notch-3 overxpression Oncogenic role of Notch-1, potentiated by hypoxia. Very sensitive to GSI in low oxygen conditions. Rationale for GSI / anti-angiogenic combinations Notch inhibition increases sensitivity to EGFR inhibition (rationale for combination) Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells

Notch in EGFR Mutant NSCLC

EGFR mutant NSCLC cell lines have Jag 1 expression ( J. Kurie et al.) Resected NSCLC (I. Wistuba et al): Notch 3 expression with EGFR mutation (p = 0.0001) Nuclear pEGFR staining correlated with Jag 1 staining

Notch targeting and Lung Cancer Stem Cells ( Michigan Group, WCLC 2009)
Notch inhibition in NSCLC and SCLC cell lines
Notch pathway components expressed in both NSCLC and SCLC GSI induces growth (-) and apoptosis in NSCLC and SCLC Tumors from Notch GSI-treated NSCLC xenografts without tumorigenic capacity when reimplanted in NOD /SCID mice Key result suggesting the CSC is being targeted

Conclusion: Notch pathway may be driving the CSC compartment and its inhibition can eliminate tumorigenic potential

A Pannuti et al, Clin Can Res, 2010

Attractiveness of RO 4929097 -secretase inhibitor

A potent and selective inhibitor of - secretase Active in human xenograft models of NSCLC
Sustained activity after 7 or 14 days dosing supports intermittent dosing regimen in the clinic

Excellent brain penetration Good oral bioavailability Long half life, mean ~ 20 -25 h Low toxicity observed in phase 1 studies (mainly secretory diarrhea and fatigue) Responses seen in melanoma, STS, NSCLC

NSCLC GSI studies at MDACC

Phase I with expansion cohorts: Roche GSI + Erlotinib
Preference to E pre-TX (dose escalation) Expansion with stratification by mutation and 2 RO GSI sequences (start at C1 or C3). No prior EGFR TX

Phase II pharmacodynamic study of GSI RO 4929097 in nonprogressors advanced NSCLC after 1st line induction chemotherapy
Assessment of further tumor shrinkage Time to progression

All patients on both studies will have fresh tumor biopsies to assess:
Notch pathway markers Stem cell markers

Wnt/-Catenin Signaling

J Kim et al. JTCS 2007

J Mazieres et al. Cancer Letters 222 (2005) 1-10

F Takahashi- Yanaga and M Kahn, Clin Can Res, 2010

Lung Cancer Stem Cells and Embryonic Pathways Lung CSC For sure they are there But not all the suspects are the real ones Need to get better at identifying and characterizing them Field is rapidly evolving Therapeutic targeting of Embryonic Pathways (and potentially CSC) already in the clinic

South Africa 2010

Im not talking about this. Todavia no me recupero

Diego ya no esta
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Hay Que Ganar el Apertura!

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THANK YOU!

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