Srinivas Vinod Saladi Ph.D. Chabner Colloquium, 2019 Squamous Cell Carcinoma Genomic Landscape: Largely Tumor Suppressors

Targeting SWI/SNF/Hippo pathway in Squamous Cancers Srinivas Vinod Saladi Ph.D. Chabner Colloquium, 2019 Squamous cell carcinoma genomic landscape: largely tumor suppressors

Squamous NSCLC Hammerman et al, Nature, 2012

Head & Neck SCC Stransky et al, Science, 2011 Lung adenocarcinoma genotypes: many potential therapeutic opportunities

AKT 1% ROS1 1.5% NRAS 1% BRAF 2% IDH1 <1% HER2 2% CTNNB1 2% ALK 3% PIK3CA 4% TP53 5% No Mutation ~40%

EGFR 15%

KRAS 23% Excision of p63 in SCC in vivo results in rapid tumor regression

Oral SCC

L/L

p63 ; K14-CreER, Tamoxifen

Controls

CreER -

Tamoxifen

K14

+/+ +/+ p63

Day 0 Day 11

Tumor Volume

CreER

K14 L/L L/L

Days p63 Day 0 Day 13 100mg/kg tamoxifen daily days 1-5 p63 regulates genes by association with distinct epigenetic cofactors/complexes

Cistrome analysis p63 ChIP-Seq (JHU029)

Factors TP53 TCFP2L1 NRF1 TFCP2 SMARCC1 FOS E2F1 BCL11A

FOSL2 (Ramsey, MR, He L, (Corrie LG, Ramsey SMARCA4 Forster N, et al, Cancer MR, et al, Genes & Dev, 2012) FOSL1 Research, 2011) BACH2 HDAC ACTL6A SMARCB1 RUVBL 1/2 p63 1/2 p63 TRIM28 H2AZ RUNX1 PUMA, NOXA SAMD9L p63 regulates genes by association with distinct epigenetic cofactors/complexes

Cistrome analysis p63 ChIP-Seq (JHU029)

Factors TP53 TCFP2L1 NRF1 TFCP2 SMARCC1 FOS E2F1 BCL11A Glycerol Gradient FOSL2 Smaller 440 kDa Larger SMARCA4

FOSL1

Empty

1 5 9 11 15 19

Input 7 13 17 21 3 BACH2 p63 SMARCB1 TRIM28 ACTL6A RUNX1 SWI/SNFBR-GATP1 –is dependentthe cent rchromatinal ATPas remodelinge in PBAF enzymes compl earexe highlys heterogeneous

PBRM1 BRD7 P-BAF ARID2 BRG1

ATPase subunits

PBAF-specific subunits

BAF-specific subunits

BRM SS18 Core subunits BRD9 ARID1A/ ARID1B BAF SWI/SNF complexes control nucleosome position

DPF 1/2/3 BCL11 BRD9 A/B SS18 BAF BCL7 47 A/B/C ARID1A/ BRG1/ β-actin ARID1B BAF 60 BRM A/B/C SWI/SNF binding at BAF 53 SWI/SNF BAF specific loci BAF A/B BAF 57 170 155 ATP ADP

BAF complexes Homo sapiens, Mus musculus

PHF 10 BCL11 BRD7 A/B BAF BCL7 PBRM1 47 A/B/C β-actin ARID2 BAF 60 BRG1 A/B/C BAF 53 BAF Disruption of DNA-histone contacts BAF A/B BAF 57 170 155 Eviction Sliding

PBAF complexes Homo sapiens, Mus musculus

Core subunits BAF-specific subunits

BAF & PBAF subunits PBAF-specific subunits SWI/SNF SWI/SNF complex mediated Transcriptional Regulation

Kinetochore Transcriptional regulation Chromosome Chromatin (in highly condensed form)

Anaphase Bridge Compact SWI/SNF Kinetochore è NO transcription factor accessibility Chromosome (ATP dependant)

TF Open è Transcription factor accessibility Unwinding of Chromatin

Proliferation Binding of Transcription Cell migration Factors Retinoblastoma pathway

Immune response Transcription of Genes

Embryonic stem cell and diffenciation programs

Polycomb silencing

Brownlee et al. DNA Repair 2015 Functions of SWI/SNF

SWI/SNF enzymes play a key role in transcriptional regulation, replication, DNA repair, and cell cycle control.

SWI/SNF enzymes are critical for cellular differentiation and embryonic development

SWI/SNF subunits are mutated or deleted in many forms of cancer and one of the subunits BAF47 may act as tumor suppressor.

Gastric carcinomas with lymph node metastasis over-express BRG1 and are depleted of BRM expression.

BRG1 expression is high in invasive forms of prostate cancer.

SWI/SNF complex altered in multiple cancers

BAF/PBAF subunits mSWI/SNF complex (BAF) ARID1A PBRM1 Colorectal cancer SMARCA4 Leukemia-T-ALL Synovial sarcoma ARID2 ARID1B Leukemia/lymphoma Malignant SMARCA2 Myeloma rhabdoid tumors SMARCC2 Epithiloid sarcoma SMARCC1 Ovarian clear cell carcinoma SMARCB1 BRD7 Endometriod carcinoma Medulloblastoma ACTL6B Bladder cancer Breast cancer BCL11A Hepatocellular carcinoma Lung cancer BCL11B Neuroblastoma DPF2 Squamous cell carcinoma ACTL6A Small cell lung cancer Head and neck cancer SMARCD2 Clear cell BCL7C Pancreatic cancer meningioma SMARCD1 BRD9 PHF10 PHD3 Polybromo-containing BAF (PBAF) DPF3 SMARCE1 In-frame Head and neck Clear cell renal SMARCD3 Splice cancer carcinoma SS18L1 BCL7A Missense SS18 Truncating Hepatocellular DPF1 carcinoma BCL7B Head and neck 200 400 600 800 cancer Mutations in cBioPortal 1000

Kadoch et al. Human Cancer 2015 Kadoch et al. Biochemistry 2016 SWI/SNF complex widely considered as tumor suppressor

Hodges & Crabtree, 2016 ACTL6A (Baf53a) subunit of SWI/SNF complex is

amplified in multiple cancers

Alteration Frequency Alteration ACTL6A (Baf53a) subunit of SWI/SNF complex is amplified in multiple cancers

Immunodepletion

ACTL6A SWI/SNFcomplex subunits ARID1B

ARID2

ACTL6B ACTL6A PBRM1

SMARCA2

BCL11b

SMARCE1

ACTL6A (Baf53a): Progenitor State SMARCC1 ACTL6B (Baf53b): Differentiated State β-Tubulin

(Saladi SV, et al, Epigenetics, 2011) ACTL6A is required for maintaining the progenitor state in epithelium

(Bao X et al., Cell Stem Cell ,2013) ACTL6A is co-amplified with p63 and confers poor prognosis in HNSCC

3q26 TP63 ACTL6A Genetic Alteration (n=530)

P63 ACTL6A

Epidermis

HNSCC ACTL6A is co-amplified with p63 and confers poor prognosis in HNSCC

3q26 TP63 ACTL6A Genetic Alteration (n=530)

TCGA HNSCC Kaplan-Meier Survival for

P63 ACTL6A ACTL6A Expression

Epidermis

HNSCC ACTL6A is required for tumor progression in vivo

x" x" Harvest -Dox Tumors x" x" Harvest FaDu +Dox Tumors Dox inducible Inject Tumors (Day 8) (Day 20) shACTL6A Cells (Day 0)

**** ***

202000

)

3 3 m 1500

m 15

(

m u

l 101000

r o

(X100mm 500

m 5

Tumor Volume Tumor T 00 - Dox + Dox shACTL6A #1 #1 #2 Dox - + + ACTL6A is required for tumor progression in vivo

- Dox + Dox

x" x" Harvest -Dox Tumors x" x"

Harvest Ki67 FaDu +Dox Tumors Dox inducible Inject Tumors (Day 8) (Day 20) shACTL6A Cells (Day 0)

6 **** Control

*** shp63

202000 4

)

3 K13 m 1500

m 15 (

m u

l 101000 o

V 0

o (X100mm

500

m 5

50 u Tumor Volume Tumor 80 50 T 80 **** 80 ** 00 4040 - Dox + Dox shACTL6A 6060 #1 #1 #2 3030 Dox - + + 60 4040 2020 40

2020 1010 % K13 positive K13 %

% Ki67 positive Ki67 % 20 00 00 Dox - Dox - + + 0 ACTL6A promotes transformation of mouse keratinocytes via activation of YAP

Ras ) 150 RasRas + Vector (N=21) 150 3 Ras+p63 RasRaRas++ A+pCT 6ΔNp63αL36A (N=7) RasRas+ +A CTACTL6AL6A (N=7) 100 Ras+ACTL6A+p6320 40 60 80

0 ****

100 50 ***

Tumor Volume (mm Volume Tumor 0 8 10 14 17 20 22 25 27 29 32 36 38 40 42 44 47 49 51 Days 50

0 8 10 14 17 20 22 25 27 29 32 36 38 40 42 44 47 49 51 ACTL6A promotes transformation of mouse keratinocytes via activation of YAP

Ras ) 150 RasRas + Vector (N=21) 150 3 Ras+p63 RasRaRas++ A+pCT 6ΔNp63αL36A (N=7) RasRas+ +A CTACTL6AL6A (N=7) 100 Ras+ACTL6A+p6320 40 60 80

0 ****

100 50 ***

Tumor Volume (mm Volume Tumor 0 8 10 14 17 20 22 25 27 29 32 36 38 40 42 44 47 49 51 Days 50

H&E DAPI Total-YAP Merged

ACTL6A 0 ACTL6A 8 10 14 17 20 22 25 27 29 32 36 38 40 42 44 47 49 51

YAP1

p63 ACTL6A promotes Tumorigenesis in HNSCC

P Cytoplasmic YAP1 retention

Cytoplasm Regenerative YAP1 Proliferation SWI/SNF WWC11 ACTL6A p63 GPRC5A (?)

Differentiation Nucleus

Cancer Cell, 2017 ACTL6A inhibits SMARCA4 (BRG1) and correlates to high H3K27me3 (EZH2) at its target genes SWI/SNF antagonistic to PRC2 complex

MCB, 2008

ARID1B is a specific vulnerability in ARID1A-mutant cancers. Helming KC, Wang X, Wilson BG, Vazquez F, Haswell JR, Manchester HE, Kim Y, Kryukov GV, Ghandi M, Aguirre AJ, Jagani Z, Wang Z, Garraway LA, Hahn WC, Roberts CW. Nat Med. 2014 Mar;20(3):251-4. doi: 10.1038/nm.3480. Epub 2014 Feb 23. SWI/SNF antagonizes PRC2 complex

cSCC

Kia SK,et al, Mol Cell Bio, 2008 Wilson B, et al, Cancer Cell, 2010 Saladi SV, et al, Pigment Cell & Mel Res, 2013 Poly Comb Repressor (PRC) complex

Herviou et al. Oncotarget - 2016 SWI/SNF complex subunits alterations renders cells sensitive to EZH2 inhibitors

ACTL6A ACTL6A expression correlates strongly with EZH2 expression

5,000 Pearson: 0.35 ACTL6A mutated Spearman: 0.38 EZH2 mutated Neither mutated 4,500

4,000

) M

E 3,500

e 3,000

(

2,500

s e

r 2,000

EZH2

A N

R 1,500

H Z

E 1,000

500

0 1,000 2,000 3,000 4,000 5,000 6,000 7,000

ACTL6A, mRNA expression (RNA Seq V2 RSEM) ACTL6A EZH2 in HNSCC patients

PDX (frozen) HNSCC (FFPE)

EZH2 EZH2

Scale bar represents 50μM

(PDX samples from Sara Pai) ACTL6A cooperates with EZH2 to repress differentiation programs Data 3 EZH2 0.20 0.20 IgG EZH2

0.15 0.15 IgG * * 0.10 0.10 * 0.05Input % of 0.05

0.00 0.00

1 9 4 C A F 0 L W 0 1 K W S

WWC1 WWC1 ControlS100A9 S100A9 Control 55 5 5 shACTL6A shACTL6A Control WWC1 ControlControl 4 4 4 4 shACTL6A shACTL6AshACTL6A

5 33 3 3 4 22 2 2

3 % of Input % of

% of Input % of * 11 1 1 * 2 * 00 * 0 0 1

A 7 3 G 60 2 H g A e 3 G L K I 6 M H g T 3 L 7 I C H T 2 A A 7 3 G C K 6 2 H g A 3 L K I H T 3 C H A ACTL6A cooperates with EZH2 and promotes H3K27me3 globally

ACTL6A loci Global

Control shACTL6A Control shACTL6A

(normalized input) to (normalized 0

-2 H3K27me3 log2 counts in 10Kb windows in H3K27me3 10Kb log2 windows counts SWI/SNF complex subunits alterations renders cells sensitive to EZH2 inhibitors EZH2 regulates differentiation program in SCC

FaDu UT

6 100nM * 500nM 125 FaDu 125 125 Cal33 4 SCC4 100100 SCC4 * 100 HaCaTHaCaT * 75 2 *

y 75

i l i 50 b 75 50

a 0

i Relative mRNA levels Relative

% % Viable Cells 2525 A 1 V 5 C C 50 R W W % 0 0 P 0 0 1 1 102 1003 10004 100005 G FaDu 25 Log nM Tazemetostat 60 UT 100nM 0 HaCaT to FaDu:p= 0.043 * 500nM 0 1 2 HaCaT to3 SCC4: p=0.0134 40 * Conc (Log10 Scale) * 20 * * *

0 Relative mRNA levels Relative L 0 9 V 1 A I K 0 0 1 S One way ANOVA, multiple comparisions High mutation load in HNSCC

Lawrence M, et al Nature, 2013 ACTL6A correlates to high mutation load in HNSCC

800 Rho = 0.64

700 p = 1.4e-04

600 More Mutation Load 500 400 Better response to Immune 300 check point inhibitors

Total mutations # Total 200 100 0 -5 -4.5 -4 -3.5 -3 ACTL6A expression ACTL6A correlates to high mutation load in HNSCC

EV BRG1

800 Rho = 0.64

700 p = 1.4e-04 Control

600 500 400 30 min post 300 Cisplatin

Total mutations # Total 200 100 0 -5 -4.5 -4 -3.5 -3 2 hr post ACTL6A expression Cisplatin

Comet Assay High Mutation Load Predicts Better Clinical Outcome to Immune Check Point Inhibitors

Riaz N, Cell, 2017 High Mutation Load Predicts Better Clinical Outcome to Immune Check Point Inhibitors

Only 15-20% respond to Immune Check Point Inhibitors in HNSCC

Ferris R, et al , NEJM, 2016 ACTL6A mediated immune modulation in HNSCC

Repressed Programs HNSCC Cells

BROWNE_INTERFERON_RESPONSIVE_GENES Predicts PD1/PD-L1 response in HNSCC HLA-DRA CXCL-10 STAT1/2 PDL-2 Cytotoxic T Cell Factors Control/shACTL6A; HIGH LOW Granzyme A,B

HNSCC Primary Tumors IL-2R, 9R, 21R PD1_LIGATION_VS_CTRL_IN_ACT_TCELL_LINE_UP ICOS co-stimulator HLA Components HLA-B, C, E HLA-DRA, B6, B1; -DPA1, B1 TNF Family Members Fas Ligand Correlation with ACTL6A; HIGH LOW Lymphotoxin Alpha, Beta ACTL6A suppresses the InterferonG gene subset thereby evading the tumor immune response

Ctrl shACTL6A Predictive Treatment Model for HNSCC patients: Epigenetic therapy coupled with Immunotherapy

BRG1/BRM

) EZH2 ACTL6A Immunologically Cold Tumors

EZH2i

BRG1/BRM

) Reactivation of Immune program EZH2 (Interferon gamma responsive genes, ACTL6A T Cell responsive genes)

Immune Checkpoint Blockade

Better Clinical response (Saladi SV, et al., Manuscript in prep) EZH2 and DNMT inhibitors reactivate Immune program

TLR3

20 30 4 STAT1 CXCL10 Ctrl Ctrl

15 A EZH2 100 EZH2 100 N 3

20 R EZH2 1uM EZH2 1uM m Decitabine 100nm

10 e 2 Decitabine 100nm

v i t Decitabine 1uM

a Decitabine 1uM

10 l e

Relative mRNA Relative 5

R 1

0 0 0 l l l tr 0 M m M tr 0 M m M tr 0 M m M C 0 u n u 0 u n u 0 u n u 1 1 0 1 C 1 1 0 1 C 1 1 0 1 2 0 2 0 2 0 2 1 e 2 1 e 2 1 e H H in H H in H H in Z Z e b Z Z e b Z Z e b E in a E in a E in a E b it E it E b it a c b c c it e ta e ita e c ci c e D e D e D D D D Predictive Treatment Model for HNSCC patients: Epigenetic therapy coupled with Immunotherapy

BRG1/BRM

) EZH2 HDAC1/2 ACTL6A Immunologically Cold Tumors

HDACi EZH2i

BRG1/BRM

) Reactivation of Immune program

HDAC1/2 EZH2 (Interferon gamma responsive genes, ACTL6A T Cell responsive genes)

Immune Checkpoint Blockade

Better Clinical response (Saladi SV, et al., Manuscript in prep) ACTL6A, subunit of SWI/SNF complex is a driver oncogene in HNSCC

Tumors Regenerative Proliferation ACTL6A ACTL6A Immune evasion ACTL6A Differentiation EZH2 SMARCA4/A2 H3K27Me3

Differentiated Epithelium ACTL6AX Differentiation EZH2X SMARCA4/A2 H3K27Me3 ACTL6A/YAP-dependent regenerative program in squamous carcinomas

GSEA analysis with BRD4 inhibition(JQ1 treatment) in all cancer cell line

CORDENONSI_YAP_ EPITHELIAL_MESENCHYMAL INTERFERON_GAMMA APOPTOSIS )

S CONSERVED_SIGNATURE _TRANSITION _RESPONSE E ( 0.0 e 0.0 r 0.4 o -0.2

c -0.2 0.0

t -0.4 0.2 n -0.4 -0.2 e -0.6 m -0.6 0.0 h -0.8

c -0.4

n E Acknowledgements

Saladi Lab MEEI MGH Cancer Center Nana Chen Mark Varvares Michael Lawrence Rozenn Riou Kevin Emerick Leif Ellisen Nicholas Abt Derrick Lin Pavan Kannan Genevieve Boland MGH CRM Muna Alhusban Jayaraj Rajagopal Bradley Bernstein Brian Lin Soldano Ferrone Robert Morris Dana Farber Martin Sattler David Frank Funding