Glioblastoma Multiforme!

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1 Glioblastoma Multiforme! Highly malignant, invasive, difficult-to-treat primary brain tumor" " Frequency: 9,000 cases/year (peak age, years)" " Recurrence: rapid growth; size may double every 10 days" " Median survival: ~ 1 year"

2 Survival of adult patients with glioblastoma multiforme" Kaplan-Meier Survival Curves! Survival %! 100! 90! 80! 70! 60! 50! 40! 30! 20! 10! 0! 0! 3! 6! 9! 12! 15! 18! 21! 24! 28! 32! 36! 40! 44! Months from Surgery!

3 Pediatric Brain Tumors" Frequency : 3000 cases/year"

4 Pediatric brainstem glioma" Brainstem location represents 8-15% of all brain tumors in the pediatric population" Usually inoperable tumors because of the particular location in the brain "

5 Survival for Children with Diffuse Pontine Gliomas (CCG 9941)" J Clin Oncol 20: , 2002!

6 Tumor cells multiply which results in growth"

7 Normal growth is controlled"

8 Why do tumor cells grow?" Tumor cells receive the instructions to grow but are insensitive to instructions to stop"

9 Propagation of neural stem cells" Blue: nucleus" Green: nestin" Nestin: marker of stem cells"

10 Differentiation of neural stem cells " in neurons and glia" Blue: nucleus" Green: GFAP" astrocytes" Red: β-iiitub" neurons"

11 Brain development requires a controlled switch from proliferation to differentiation!?"?" Stem Cells! Differentiated " neurons!

12 Neurogenesis and Brain tumors! " Disruption of pathways essential for neurogenesis have been implicated in childhood and adult brain cancers, for which immature progenitor cells have been proposed as cells-of-origin"

13 Id proteins: inhibitors of differentiation" Undifferentiated state! Differentiated state! High growth potential! High amounts of Id proteins! Low growth potential! Low amounts of Id proteins! Iavarone and Lasorella, 2003

14 ! No Id proteins! Id proteins are antagonists " of transcription factors! bhlh heterodimer! CANNTG! E-box! Activation of" transcription" and differentiation!! Id proteins in functional excess! Id2! Id2! Id2! Id2! CANNTG! E-box! Inhibition of" transcription and" block of differentiation!

15 The Rb-Id2-bHLH pathway in pediatric tumors! Myc IGF EWS-Ets Rb! Id2! bhlh p57 Kip2! G 1! M! S! G 2!

16 Normal cells Cancer cells Cancer cells invade normal tissues Rb! Id2 Rb! Id2! Wild type Rb Id2 inactive Mutant Rb Id2 hyperactive

17 Id2 loss impairs tumor growth and " angiogenesis in tumors from Rb+/- mice" Rb+/-! Id2-/-;Rb+/-! Microfil-orange " Latex perfusion " Pecam-1"

18 Id proteins are coexpressed with HIF1α in human glioblastoma" Id1" Id2" HIF1α#

19 Id2 overexpression in neuroblastoma" is associated with reduced survival! Survival (percentage)! 100" 80" 60" 40" 20" Overall study population! Id2" negative, n=18" Id2"positive, n=29" P!=0.0046! 0" 0" 20" 40" 60" 80" 100" 120" Time (months after diagnosis)!

20 Id proteins involved in all processes associated with development of neural tumors! VEGF Signaling Integrins, MMP2 Id Metalloproteinases Angiogenesis Tissue Invasion Anaplasia Lineage Specific bhlh Id Proliferation Rb, bhlh, Ets, Pax

21 THE FUTURE : Anti Id2 therapeutics" Growth arrest! Id2! Id2! Id2! Id2! Id2! Id2! Id2! Id2! Id2! Differentiation! Increased cell death! Inhibition of angiogenesis!

22 Developmental lineages derived from the neural crest and the genesis of neuroblastoma"

23 Underlying challenge: how to control stem cells!?"?"

24 Control brain tumor/neural stem cell behavior! Glia! Stem cell! N-Myc! Huwe1! N-Myc! Neuron!

25 Loss of Huwe1 expands " the neural stem cell population" Pax6/DAPI! Huwe1 F/Y! Huwe1 F/Y Nes! Pax6/DAPI! IZ" IZ" SVZ" SVZ"

26 Loss of Huwe1 impairs" neural stem cell differentiation" Nestin/DAPI" Huwe1 F/Y! Nestin/DAPI" Huwe1 F/Y Nes! 50 µm! βiii-tubulin/dapi" βiii-tubulin/dapi"

27 Focal deletions and decreased expression of Huwe1 in GBM! TCGA! Oncomine! Normal brain! GBM! n=23! n=77!! P-value: 9.3E-10!

28 Expression of Huwe1 is lost in primary neuroblastomas displaying accumulation of N-Myc protein" Huwe1! N-Myc! #3524! #3629!

29 Neural stem cell Growth factors N-Myc N-Myc Growth factors Huwe1 Lineage commitment Differentiation Cell cycle arrest Tumor stem cell N-Myc Huwe1 N-Myc Tumor growth CD2 p27 Growth arrest Maturation

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31 Malignant gliomas invade the normal brain

32 The mesenchymal signature of high-grade glioma" Unsupervised clustering of 76 high grade tumors by expression of 108 genes that are positively or negatively associated with survival reveals 3 tumors classes - Proneural (PN), Mesenchymal (Mes) and Proliferative (Prolif)." Malignant(gliomas(belonging(to(the(mesenchymal(sub2class(express(genes(linked(to( the(most(aggressive(proper9es(of(glioblastoma((migra9on,(invasion(and( angiogenesis)(and(mark(the(worst(clinical(outcome.(

33 The mesenchymal network of six major hubs " of transcription factors in high-grade gliomas" Mesenchymal genes( Activator" Repressor"

34 STAT3 and C/EBPβ inhibit neuronal differentiation and induce mesenchymal transformation in neural stem cells" Tau( SMA( Tau/SMA/Dapi" FN1" Ctgf" Vector" Stat3C/CEBPβ E" 5" 10" E" 5" 10" Vector" Olig2! βiiitubulin" βactin" Stat3C/ CEBPβ Vector" Stat3C/ CEBPβ Stat3C/ CEBPβ" Untreated" 20 days"

35 Knockdown of Stat3 and C/EBPβ cooperates to inhibit tumor cell invasion and angiogenesis" shctr" shstat3" shc/ebpβ# shstat3+shc/ebpβ# shctr" Vimen9n( shstat3+shc/ebpβ# Vimen9n( T! T! B! B! 10000(µm( CD31( CD31(

36 Loss of Stat3 and C/EBPβ in human glioma cells inhibits tumorigenesis in the mouse brain" Human glioma-shctr" Human glioma-shstat3+shc/ebpβ# 100" Cumulative Survival" 80" 60" 40" 20" 0" 40" 60" 80" 100" Days post-injection" **! 120" shstat3+shc/ebpβ# shctr"

37 The combined expression of Stat3 and C/EBPβ correlates with the poorest outcome of glioma patients" Negative Stat3 + C/EBPβ " Positive Stat3 + C/EBPβ "

38 Glioblastoma From systems biology to prognosis to personalized therapy" Mesenchymal Signature Of High Grade Glioma ARACNe Regulatory Network CEBPB STAT3 bhlh-b2 FOSL2 RUNX1 Stat3 and C/EBPβ are Master Regulators Stat3 and C/EBPβ are Transforming Oncogenes of Neural Stem Cells Stat3 and C/EBPβ are Predictors of Negative Clinical Outcome

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