The Children’s Hospital of Philadelphia

Hakon Hakonarson, MD, PhD,Professor of Pediatrics CHOP’s Endowed Chair in Genetic Research Director, Center for Applied GenomicsThe Children’s Hospital of Philadelphia University of Pennsylvania, School of Medicine

Duke Center for Applied Genomics and Precision Medicine 2019 Genomic and Precision Medicine Forum

Nov 07, 2019

Integrative Genomics Discoveries and Development at The Center for Applied Genomics at CHOP

Novel Genome-based Therapeutic Approaches

Genomics in the 21st Century

Disclosures

Dr. Hakonarson and CHOP own stock in Aevi Genomic Medicine Inc. developing anti-LIGHT therapy for IBD.

Dr. Hakonarson is an inventor of technology involving therapeutic development of ADHD, GLA and HCCAA

Novel Therapeutic Stem Cell/Gene Editing Approaches

§ iPS and stem cell therapy shows early promise

§ Gene therapy for LCA (RPE65) at CHOP via AAV

§ Targeted T cell therapy for cancer (UPENN/CHOP)

§ CRISPR-cas9 gene editing

§ Single cell sequencing

The Center for Applied Genomics (CAG) at CHOP

u Founded in June 2006

u Staff of 70

u Over 100 active disease projects with CHOP/Penn collaborators

u TARGET: Genotype 100,000 children

u ~450k GWAS samples >130k kids

u IC - participation in future studies >85%

u Databaseu Electronic Health Records u extensive information on each

childu >1.2 million visits per year to

CHOP

u High-level of automation

Population Genomics ResearchRecruitment of CHOP/PENN HealthCare Network Patients

ADHD, Autism, Diabetes, IBD, Autoimmunity, Asthma/Atopy, Cancer, RDs- all high priority

Children's Hospital of Philadelphia

Center for Injury Research and Prevention

Center for Cellular and Molecular Therapeutics

Center for Applied Genomics

PolicyLab

Center for Pediatric Clinical Effectiveness

Center of Mitochondrial and Epigenomic Medicine

Center for Developmental Biology and Pediatric Disorders

Center for Childhood Cancer Research

Center for Biomedical Informatics

Center for AutismResearch

CAG Repository (Major Disease Areas)

§ Major pediatric and adult diseases are represented

§ EHR have unlimited potential regarding9 Longitudinal f/u9 Medication use9 Development9 AEs/SAEs/DDIs

§ We have established over one hundred collaborations world-wide for discovery and replication purposes

Category # of samples

Total CAG/CHOP (internal) 132,752• Healthy Controls (0-21) 18,423• Psychiatric 19,351• Autism/Developmental Delay 13,972• Autoimmune/Inflammatory 31,643• Cancer 9,585• Metabolic 13,760• Malformations 8,954• Rare/Mendelian Diseases 22,436Samples through collaboration 334,534Total number of samples @ CAG 467,286

CAG Biobank – biomarker driven research§ 450,000 unique patients in CAG

biobank§ Over 100,000 children from CHOP

(>85% re-contact permission)§ Blood samples from >95%§ High quality DNA from with

over 2/3 of samples GWAS genotyped

§ PBMCs - opportunity for EBV, iPS and tissue/organ differentiation

§ RNAseq, single cell sequencing

§ Cell based assays§ Epigenetics

§ Plasma – for target driven biomarker measures

§ Thousands of different phenotypes in CAG§ Clinical disease traits in the

multi-hundred’s§ Other: blood, chemistry,

radiology, cardiology, sleep, PFTs, meds etc.

§ Consortium networking collaborations

§ Multiple partnerships with biotech and pharma

u 2 Million Samples in tubes u 10 Million Plated Samples

CAG CLIA certificate

CAG Discovery and Development PipelineObjective: To identify biomarkers indicative of the genetic underpinnings of a disease for novel innovative therapies

u Three databases:1. Family-based Samples2. Genomic Database 3. Health Records & Disease

Phenotypes

u We integate these databases together in search for new target biomarkers and diagnostic products

u High level of IT and Technology Structureu All cloud basedu Highly integrative u Cost effective discovery platform

Family-based

Analysis

Genomic Database

HealthRecords

and Disease

Phenotypes

New drug disovery and development of biomarkers and diagnositc

products

Precision Medicine in the Context of “Big Data“

Creating and turning data assets into insights.

• CAG database has >450k patients GWAS genotyped and imputed to >30M variants• 15,000 whole genomes are sequenced and growing• 20,000 whole exomes are sequenced and growing• Thousands of phenotypes to mine for new targetsCells, RNA, Plasma available from these patients for biomarker development

Representative CAG Milestones

Research is enabled by our unique, scalable biobank with sample collections that are highly enriched for rare disease causing variants

Reference, https://scholar.google.com/citations?user=nLerrWsAAAAJ&hl=en

Personalized/Precision Medicine Paradigm

u There are 6 billion bases (3 million pairs) in the human genome

u SNPs occur every 100-300 base pairs

u The greatest number of DNA variations that are associated with diseases or traits are

u Missense mutationsu Nonsense mutationsu Deletions (structural

variants)

u Today we can run an exome for <$350 and a genome for <$1000 and identify all know PGx and disease causing variants

Impact of pediatric age on genetic risk

u Early Onset Disease:

u Distinct, genetically driven

u Severe burden of illness

u Aggressive progression

u Less responsive to standard of care

Genetic Influence in Pediatric Diseases

Pediatric studies generally produce much larger genomic signals than studies in adults.

Neuroblastoma

u Embryonal canceru Misappropriation of normal

sympathetic neurodevelopment pathways

u Common pediatric cancer u Median age diagnosis 17 months

u 15% of childhood cancer mortalityu Approximately 2/3 of patients cured

Familial Neuroblastoma LocusChromosome 2p23-24

TELOMERE--NAG - DDX1 - MYCN - FAM49A - VSNL1 -KCNS3 - RDH14 - NT5C1B - OSR1 - TTC32 - WDR35 -MATN3 - LAPTM4A - SDC1 - PUM2 - RHOB - HS1BP3 - GDF7- C2orf43 - APOB - ATAD2B - UBXD4 - LOC388931 - C2orf44 -FKBP1B - SF3B14 - TP53I3 - PFN4 - FLJ30851 - ITSN2 -NCOA1 - LOC391356 - CENPO - ADCY3 - RBJ - POMC -DNMT3A - DTNB - ASXL2 - KIF3C - RAB10 - HADHA -HADHB - GPR113 - SELI - C2orf39 - OTOF - LOC339778 -CIB4 - KCNK3 - C2orf18 - CENPA - DPYSL5 - MAPRE3 -FLJ20254 - AGBL5 - EMILIN1 - KHK - CGREF1 - ABHD1 -PREB - C2orf53 - TCF23 - SLC5A6 - C2orf28 - CAD -SLC30A3 - DNAJC5G - TRIM54 - UCN - MPV17 - GTF3C2 -EIF2B4 - SNX17 - ZNF513 - PPM1G - NRBP1 - KRTCAP3 -IFT172 - FNDC4 - GCKR - C2orf16 - ZNF512 - CCDC121 -XAB1 - SUPT7L - SLC4A1AP - MRPL33 - RBKS - BRE -FOSL2 - PLB1 - PPP1CB - SPDYA - FLJ20628 - WDR43 -LOC165186 - FLJ 34931 - CLIP4 - ALK - YPEL5 - LBH -LYCAT - CAPN13 - GALNT14--CENTROMERE

Identification of Heritable Mutations in the ALKTyrosine Kinase Domain

Obligate carrier mother

Unaffected father 1

Affected child

Affected child

Affected child

Unaffected father 2

Unaffected grandfather

Affected grandmother

*

*

*

*

*

Mosse, Nature 2008

ALK is the Major Familial Neuroblastoma Gene

Familial NB

u A germline mutations in the anaplastic lymphoma kinase (ALK) gene explain most hereditary neuroblastomas

u Resequencing in high-risk neuroblastoma samples showed somatically acquired mutations in the tyrosine kinase domain in 12.4% of samples.

u Nine of the ten mutations map to critical regions of the kinase domain (oncogenic drivers).

u Several companies have ALK inhibitors in development (preclin to Phase I)

u CHOP is pursuing clinical development and with first patient to be enrolled in April

Neuroblastoma: Crizotinib

Personalization and Repositioning Case Study

CHOP Identified Mutations

• ALK

• mGluR

Repositioned Compound

• Lung cancer

• Dementia

Target New Patient

Population

• Lymphoma

• Neuroblastoma

• ADHD

• Schizophrenia• Autism

GenomicallyEnriched

Clinical Trials

• Phase I n=18

• Phase I n=23

• Phase I n=18• Phase IIa n=60

• Future trial• Future trial

u The tumors have responded in a robust way in those who are ALK mutation positive and receive Xalkori

u Both germ-line and somatic mutation cases are responsive to Xalkori

Discovery of Mutations (copy number variations/CNVs) in ADHD

Elia, Glessner et al. Nature Genetics, 2012GRM: Glutamate receptors metabotropicCNVs: Copy number variants

u CNVs affecting glutamatergicneurotransmission genes observed to be over-represented in multiple ADHD cohorts (~10 fold)

u 3,500 ADHD cases and 12,000 controls

u Replication accomplished across multiple ADHD cohorts

üAn mGluR agonist

üWell tolerated in previous preclinical and clinical studies

üShown to ameliorate cognitive impairment and slow down locomotor activity in animal models

üStructure-similar compounds have good safety profiles

The Drug NFC1 Activates the mGluR Pathway

NFC-1 (fasoracetam):

Genotyping or resequencing methods identify the ADHD children at risk

Prior Studies conducted on NFC-1

Preclinical(N=21)

Clinical(N=7)

Clinical Trial Design ADHD (GREAT STUDY)

Week 1 Week 2 Week 3 Week 4 Week 5 Week 9Day 7 (±2) Day 14 (±2) Day 21(±2) Day 28 (±2) Day 35 (±2) Day 75 (±2)

Adverse event monitoring X X X X X phoneLaboratory Safety Tests (blood and urine)A X X X X XPhysical Examination X X X X XVital Signs: BP, HR, RR X X X X XBody Weight (all points) & Height (week 1only)

X X X X X

12-lead ECG X X X X XUrine b-hCG test (menstruating females only) X X X X XContraception verification (selected females) X X X X XVanderbilt Parent Rating Scale X X X X XBREIF (Parent; Self) X X X X XQuotientâADHD test X X X X XPERMP-Math test X X X X XActigraphy (continuous monitoring) X X X X XCGI-I & CGI-S X X X X XDispense study drugB X X X XNFC-1 or placebo administration at homeC Placebo bid 50 mg bid 100 mg bid 200 mg bid 400 mg bidRetrieve pill bottle/pill count X X X X XA: Blood draws for hematology (RBC, WBC with differential, platelet count) and clinical chemistry (electrolytes, albumin, ALT, AST,

alkaline phosphatase, bilirubin, BUN, creatinine, glucose, B: Study drug for Week 1 administered at end of PK study; study drug for next week dispensed at each clinic visitC: Dose escalations to be determined by CGI-S and CGI-I scores at end of each week of treatment; maximum doses indicated

Thirty mGluR pos ADHD children completed a 24 hr PK study and 5 wks on drugPlacebo was given for 1 week – parents were blinded to placebo therapy

New IND filed to the FDA and approval obtained to treat 30 mGluR biomarker positive children for 5 weeks age 12-17 year old

Week 1 Week 2 Week 3 Week 4 Week 5

Mean 29.1 26.4 24.0 23.3 22.5

Week 1 Week 2 Week 3 Week 4 Week 5

Mean 3.79 3.13 2.79 2.79 2.21P < 0.001

P < 0.001

Average Vanderbilt score at each week – all patients

Average CGI - I score at the end of each week – all patients

ADHD Study: Repeated Measures Analysis increases confidence in demonstration of Treatment Effect

NFC-1 ADHD Study Results

u Seven kids had co-morbid autism demonstrating marked improvement in social behavior and language skills

u Two kids had 22q (one deletion/one duplication syndrome): Among the most robust responders

u Anxiety and mood symptoms improved across the board

u PERMP improved in Tier-1/Tier-2 mutation group

u Actigraphy measures of hyperactivity improved across all tiers (most robust in rapid repetitive movements, P<0.01)

(Elia et al Nature Communication, 2018)

mGluR enrichment in idiopathic ASD

u Enrichment of CNVs affecting glutamatergic neurotransmission genes also observed to be over-represented in multiple Autism

u Enrichment also observed in schizophrenia, anxiety, anorexia and mood disorders

Pharmacogenomic Model in ADHD/ASDs

Genetic Screening

Intervention at Biochemical Pathways

Neurological Disorder

New Drug Candidates

Fast-Track Clinical Trials

Market Test-and-Treat Product

Stratify Disease using

Biomarkers

ADHD

ASD22QSCZ

SNPs/CNVs Gene network mGluR agonistmGluR pathway

Precision Medicine

Re-sequencing panel designed for diagnostic

u We identified and replicated significantly associated, previously unreported loci on chromosomes 20q13 and 21q22 located close to the TNFRSF6B (DcR3) and PSMG1 genes, respectively

u Gene discovery studies in childhood-onset disease have unveiled genetic factors that are less likely to surface in adult studies

u We have identified multiple other novel IBD loci and replicated the vast majority of the pediatric-onset disease loci in the adult onset form of IBD

First Pediatric IBD GWAS

Expression of DcR3 is altered in IBD

• The level of TNFRSF6B/DcR3 mRNA is elevated in intestinal biopsies from IBD

• Serum levels of DcR3 by ELISA are correlated with the haplotype

DcR3 plays diverse roles in the immune system

Zhan C, Patskovsky Y, Yan Q, Li Z, Ramagopal U, Cheng H, et al. Decoy strategies: the structure of TL1A:DcR3 complex. Structure. 2011 Feb 9;19(2):162-71.

Meta-analysis tags the DcR3 pathway

DcR3 TL-1A

Fas ligand

Franke A, McGovern DP, Barrett JC, Wang K, Radford-Smith GL, Ahmad T, et al. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci. Nat Genet. 2010 Nov 21.

Sequencing of TNFRSF6B in pediatric IBD cases and controls

Case/Ctrl status HomozygousWT

Missense carriers

IBD 521 11Controls 555 3

Odds ratio = 3.9, p < 0.025 by Χ2

Case/Ctrl status HomozygousWT

Missense carriers

Crohn’s 521 11Exome seqproject

4200 24

Odds ratio = 3.7, p < 1.37×10–4

Secretion of DcR3 from IBD cases (293T cells)

Comparison of non-secretors

Normal secretor Non-secretorCrohn’s cases 388 5Controls 554 1

Odds ratio = 7.1, p < 0.037 by Χ2

Cardinale et al, Genes and Immunity, 2013

Decoy Receptor 3 (DCR3) is a Natural “Off-Switch” for Immune and Inflammatory Response

Normal PatientDCR3 Turns Off Immune Response

No InflammationInflammation

Normal PatientLIGHT Activates Immune Response

No Inflammation

Normal Patient

LIGHTDCR3

Certain IBD Patients Have Mutations that Result in LOF of DcR3

Chronic Inflammation

DCR3- IBD PatientNo “Off” Switch

DCR3- IBD PatientReplacement Tx with anti-LIGHT mAb

No Inflammation

Our Approach: Replace Deficiency with anti-Light Ab Therapeutic Antibody that “Mimics” DCR3

LIGHTAnti-LIGHTAntibody

u First-in-Class Biologic from Japanu Initial development in

Severe Pediatric Onset IBD

u Phase 2 ready human monoclonal antibody

u Program Updateu Successfully transferred

and approved INDu Requalified clinical trial

materialu Cleared by FDA to start

trialu Study began at CHOP

and 3 other sitesu Initial data expected Q4

Anti-LIGHT mAb Program

Decoy Receptor 3DcR3 LIGHT

ImmuneCell

HVEM LTbR

Therapeutic antibody which mimics DcR3 regulation by binding LIGHT

Heritability and Shared Genetic Architecture in Pediatric Autoimmune Diseases (pAIDs)

u A: Details of the 10 pAID cohorts and common population-based controls

u B: Top pAIDassociation signals identified by performing a GWAS merging cases from the 10 pAIDs (Left) or by performing an inverse chi-square meta-analysis (Right)

u C: Novel and established pAIDassociation loci (n=47)

u IL2RA, IL12B, CD40LG, SMAD3

Ten pAID case cohorts and top pAID association loci

Li et al, Nat Med, 2015

46 Loci Are Highly Associated (p < 1x10-6) with 27 of These Reaching Genome-Wide Significance (p < 5x10-8)

Pharmacogenomic Model in IBD/pAID

Genetic Screening

Intervention at Biochemical Pathways

Inflammatory Disorder

New Drug Candidates

Fast-Track Clinical Trials

Market Test-and-Treat Product

Stratify Disease using

Biomarkers

IBDpAIDs

SNPs Gene network aLIGHT-AbDcR3 pathway

Precision Medicine

CAG RARE DISEASE PROGRAM

Disease Area Breakdown 2019 2012%

ChangeMusculosketal 4,920 2,459 +50%Nervous System 4,855 2,652 +45%Cardiovascular 3,289 2,225 +32%Endocrine/Immune 2,846 1,337 +53%Blood Diseases 1,649 816 +51%Circulatory 1,418 795 +44%Genitourinary 1,027 645 +37%Eye/Face/Neck 909 571 +37%Chromosomal Abnormality 662 347 +48%Other 316 125 +60%Mitochondrial 165 76 +54%Skin 121 76 +37%Digestive 112 64 +43%Perinatal 92 15 +84%Respiratory 55 42 +24%

Total 22,436 12,245 +45%

Mendelian Disease Project at CAG• Number of WE sequenced samples:

6,740 (>1500 families)• Hundreds of additional families in

pipeline

Generalized Lymphatic Anomalies (GLAs)

u Orphan disease(s) with pleiomorphic presentation: some malignant/fatal

u Defined defects in mTOR/PI3KA pathwayu Somatic mutations been identified in a

subset of patientsu Sirolimus been used anecdotally as

therapy (subset of patients)

u Molecular diagnosis missing in most cases

Generalized Lymphatic Anomaly (GLA)Gorham-Stout Disease (GSD)Central Collecting Lymphatic Anomalies (CCLA)Klippel-Trenaunay Syndrome (KTS/CLVM)Kaposiform Lymphangiomatosis (KLA)Kaposiform Hemangioendothelioma (KHE)

Family 1 enrolled

III

IV

II

I1 2

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8 9

6

u Lead proband with history of complex lymphovascular disease since birth, manifesting primarily as a right-sided chylous effusion

u At birth - near complete opacifications of his lungs

u 4 months- pulmonary lymphangiomatosis

u 11 years - chylous effusion was drained and the fluid re-accumulated within 12 hours

u Family history – venous stasis was a consistent feature and variability in abdominal distension

u A stillbirth baby was affected by hydrops

Li, D. et al. Hum Mol Genet 438, 946 (2018)

Proband 1

A A'

A'' A'''

Central Conducting Lymphatic Anomaly (CCLA)

Li, D. et al. Hum Mol Genet 438, 946 (2018)

WES revealed splice-altering mutation in EPHB4

Tested positive EPHB4: *Tested negative EPHB4: **

** **

*

** ******

**

III

IV

II

I1 2

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8 9

1**

2**

3*

4**

5**

6

c.2334+1G>C

Li, D. et al. Hum Mol Genet 438, 946 (2018)

EphB4 signaling in ECs controls various cellular responses

Füller et al. Journal of Cell Science (2003) vol. 116 (Pt 12) pp. 2461-70

Knockdown study - lymphatic vessel misbranching phenotype

A B

C DControl ephb4a MO

ephb4a MOControl

54 hpf 54 hpf

4 dpf4 dpf

Li, D. et al. Hum Mol Genet 438, 946 (2018)

Lymphatic misbranching can be rescued by mTORC1 inhibitors

Rap

amyc

in

b-actin

p-mTOR (S2448)

p-p70S6K (T389)

Con

trol

EPH

B4

Morpholino:

Treatment:

BEZ2

35C

ontro

l

EPH

B4

Con

trol

Con

trol

EPH

B4

p-ERKs (T202/Y204)

u Perturbation of EPHB4 in developing zebrafishactivates mTORC1 signaling

u Activation of mTORC1 can be inhibited by treatment

Li, D. et al. Hum Mol Genet 438, 946 (2018)

Summary I

u Splice-altering mutation in EPHB4 leads to nonframeshift 4 amino acids insertion in the highly conserved catalytic loop of protein kinase domain

u The insertion decreases the phosphorylation state of EPHB4 protein

u Modeling the splicing-altering mutations in zebrafish results in vessel misbranching and deformities in the lymphatic vessel development

u Drugs that inhibit mTOR or PIK3A kinase signaling effectively rescued the misbranchingphenotype in a comparable manner

a) b) c)

a) T2 weighted MRI demonstrating large pericardial effusion (arrow). b) Dilated and tortuous thoracic duct (TD; arrow) retrograde perfusion of the periportal lymphatic networks (arrowhead) and abnormal mediastinal and pulmonary perfusion (boxed region). C) Contrast lymphangiogram of the boxed region in (b) demonstrates dilated and tortuous terminal TD with multiple dilated ducts perfusing both lung fields (arrows).

Patient 2: Central Conducting Lymphatic anomaly

Recurrent ARAF mutation Patient P1 Patient P2

T C C A C G T C C A C T C

T C C A C G Y C C A C T C

T C C A C G T C C A C T C T C C A C G T C C A C T C

T C C A C G Y C C A C T C T C C A C G T C C A C T C

ARAF c.640T>C (p.S214P)

ARAF c.640T>C (p.S214P) in patient P2

Daughter of patient P2

Mother of patient P2

Human PLQRIRST S TPNVHMVRhesus PLQRIRST S TPNVHMVMouse PLQRIRST S TPNVHMVDog PLQRIRST S TPNVHMVElephant PLQRIRST S TPNVHMVX_tropicalis SLQRHRST S TPNVHIDZebrafish --QRLRST S TPNVTMLB-RAF LSQRDRSS S APNVH-IC-RAF FGQRQRST S TPNVHMV

ARAF c.640T>C (p.S214P)

*CR1 CR2 CR3

wtmt

Molzan, M. et al. Mol Cell Biol 30, 4698–4711 (2010)

Wellbrock, C., Karasarides, M. & Marais, R. Nat Rev Mol Cell Biol 5, 875 (2004)

ARAF signaling cascade

14-3-3 (Pan)

b-actin

E VFLAG (ARAF)

E V

ARAF

-WT

ARAF

-S21

4P

Whole Cell lysate

a-FLAG-IP

p-ERKss.e.

l.e.

ARAF

-W

T

ARAF

-S21

4P

HEK293T

b-actin

EV

p-mTOR (S2448)

HEK293T

p-AKT (S473)

p-p70S6K (T389)

s.e.

l.e.

FLAG (ARAF)

ARAF

-WT

ARAF

-S21

4P

Transfection:

p-ERKs (T202/Y204)

p-p38 (T180/Y182)

The mutated serine is part of the motif that interacts with 14-3-3. The S214P mutation disrupts the interaction between ARAF and 14-3-3, resulting in dramatic ERK activation in 293T cells.

ARAF mutations induce ERK activation

FLAG (ARAF)

ERK

ARAF-WT ARAF-S214P

[Trametinib]

p-ERKs (T202/Y204)

30 nM

100 nM300 nM

DMSO

1000 nM

3000 nM30 nM

100 nM300 nM

DMSO

1000 nM

3000 nM

When expressed in primary human dermal lymphatic endothelial cells (HDLECs), ARAF S214P induces potent ERK phosphorylation, which is inhibited by the MEK inhibitor trametinib.

ARAF mutations induce ERK activation

ARAF-WT

ARAF-S214P

Trametinib 30 nM 100 nM 300 nM0 nM

ARAF S214P induces spontaneous sprouting in spheroids formed from HDLECs

ARAF mutations induce sprouting formation

ARAF-S214P + DMSO

ARAF-WT + DMSO

ARAF-S214P + 100 nM Trametinib

ARAF S214P induces elongation of HDLECs in monolayers and internalization of VE-cadherin. Changes are reversed by Trametinib.

VE-cadherin staining

ARAF mutations result in internalization of VE-cadherins reversed by MEK inhibitors

ARAF-S214P + DMSO

ARAF-WT + DMSO

DMSO

ARAF S214P + 100 nMTrametinib

Morphology changes induced by ARAF S214P are reflected in actin arrangement as well. Multiple, roughly parallel actin filaments are

observed in the interior cell bodies of ARAF-WT expressing cells, while ARAF-S214P expressing cells lack clear filaments except along the cell

perimeter (cortical actin). Changes are reversed by Trametinib.

Phalloidin staining

ARAF mutations result in actin filament derailment reversed by MEK inhibitors

Overexpression of human ARAFmutant in zebrafish results in dilated lymphatics

Mrc

1a:A

raf

S21

4P-v

2a-

mch

erry

Disrupted normal vasculature can be reversed by MEK inhibitor

TDPCV

mrc1a:ARAFwt

mrc1a:ARAFS214P

mrc1a:ARAFS214P

No treatment

Cobimetinib

TDPCV

Baseline Post Therapy03/17/2017 04/04/2017 05/04/2017 10/23/2017 03/08/2018

Parameter Unit Pre % Ref Pre % Ref Pre % Ref Pre % Ref Pre % RefWeight kg 38 40 42 40 39Height cm 142 142 143 145 148FVC L 0.58 23 0.88 35 0.80 31 0.95 35 1.18 40FEV1 L 0.52 23 0.77 34 0.72 31 0.85 35 1.09 42FEV1/FVC % 89.8 105 87 102 89.3 105 89.0 105 92.0 108FEF25-75 L/s 0.8 29 1.12 41 1.10 39 1.46 50 1.79 58TLC L 0.93 29 1.23 38 1.28 39 1.51 45 1.98 56RV L 0.27 31 0.35 40 0.48 55 0.56 62 0.80 86RV/TLC % 28.94 107 28 104 37.67 140 37 137 40 154DLCO [Hb] mL/min/mmHg - - - - - - 9.99 54 9.9 52

DLCO/VA mL/min/mmHg/L - - - - - - 9.08 135 7.67 116

MIP cmH2O 51.6 71 - - 62.2 82 70.0 95 85.0 115MEP cmH2O 69.4 66 - - 77.6 74 89.0 82 83.0 75O2 Sat % 92 - 97 100 97

Improvements in ARAF S214P patient following trametinib treatments

Wei

ght (

kg)

22.5

25

35

45

55

Age (years)8.9 10 12 14

Wei

ght (

lbs)

55

77

99

121

Clinical Response of ARAF Proband to a MEK Inhibitor

GLA patient with end-stage disease unresponsive to Rapamycin

MRI after 4 months therapyMRI lymphangiogram before therapy

•We discovered a new mutation guiding us to a new therapy in a 12 year old child who was dying from the disease

• Zebrafish studies expressing the human gene mutation developed lymphatic abnormalities, rescued with a drug blocking the abnormal gene function

• Endothelial cell studies showing uninhibited growth that was fully rescued with inhibitor therapy

•Patient had rapid improvement, fluid resolved, lung function reversed in 4 months; Off supplemental oxygen, walking to school

•Total remodeling of his lymphatic system

Li et al, Nature Medicine, 2019

Patient Gene Mutation Origin Main Phenotypic Features

1 ARAF c.640T>C:p.S214P Somatic Central Conducting Lymphatic Anomaly

2 ARAF c.640T>C:p.S214P Somatic Lymphangiomatosis

3 KRAS c.35G>A:p.(G12D) Somatic Lymphatic malformation with cardiofaciocutaneous syndrome and chylothorax

4 BRAF .1403T>C:p.(F468S) Germline Lymphatic malformation with cardiofaciocutaneous syndrome and chylothorax

5 RASA1 c.475_476del:p.(L159Gfs*20) Germline de novo Lymphatic disorder with chylous pericardial effusion and non-immune

hydrops

6 SOS1 c.2536G>A:p.(E846K) Germline de novo Noonan syndrome with lymphatic maformation and left pleural effusion

7 PTPN11 c.1504T>G:p.(S502A) Germline de novo Noonan syndrome with lymphatic disorder and chronic severe chylothorax

8 PTPN11 c.1510A>G:p.(M504V) Germline Noonan syndrome with lymphatic disorder and acute onset of chylousascites

9 PTPN11 c.1530G>C:p.(Q510H) Germline de novo LEOPARD syndrome with lymphangiectasia and respiratory distress

10 PTPN11 c.1507G>C:p.(G503R) Germline Plastic bronchitis and chylothorax with heart defects

11 ITGA9 c.1236+4A>G and c.289T>G:p.C97G Recessive Lymphatic anomaly, chylothorax, lymphedema

12 RAF1 c.433A>C:p.Thr145Pro (n) De novo Noonan; Chylothorax, Lymphatic disorder, Valvular pulmonary stenosis

13 RASA1 c.2246G>C p.Arg749Pro (n) inherited Baby Boy is a 2 month old male with congential lymphatic disorder

14 RIT1 c.270G>T:p.M90I unknown Noonan syndrome. In infancy her course was complicated by pulmonary lymphangiectasis and plastic bronchitis.

15 PIEZO1 c.7289C>T:p.P2430L Recessive Lymphedema and lymphatic conduction disorder

16 EPHB4 c.2288G>A:p.R763Q M inherited Central Conducting Lymphatic Anomaly

17 EPHB4 c.2654A>G:p.K885R M inherited prenatal onset nonimmune hydrops, ascites, and subcutaneous edema

Summary II

u A recurrent mutation in ARAF was identified in the conserved phosphorylation site in two unrelated patients

u ARAF mutant leads to enhanced MAPK activityu Transgenic expression of human ARAF mutation in

zebrafish leads to dilation of thoracic ductu This lymphatic defect is treatable with a MEK inhibitoru Submitted for a Frontier program this year and was

awarded

Background:

Patients suffering from HCCAA have amyloid fibrils precipitate in the cerebral arteries, resulting in cerebral hemorrhage, dementia and death in early adulthood (Wahlbom et al 2007)

The molecular pathophysiological process involves the transformation of a soluble functional monomeric Cystatin C/Amyloid complex into potentially toxic aggregates (Ostner et al 2013).

L68Q cystatin C, the mutated form causing HCCAA, has an increased propensity to form dimers compared with that of wt cystatin C.

Objective: To identify therapeutics agents capable of stopping the production of oligomers and fibrils of L68Q Cystatin C/Amyloid, thereby preventing vascular events and dementia.

Wahlbom et al 2007

Hereditary Cystatin-C Amyloid AngiopathyCystatin C amyloid deposition in samples from the cerebrum, cerebellum, midbrain and thalamus in HCCAA.

Mono

dimer

HMW Oligo

Trimer

DTT

50

20

25

Cystatin C L68Q

GSH(mM)

3

250

1

NAC(mM)

3 101

150

100

75

10

37

a-Cyst-Cun

treate

dDTT

Glu 1m

M

Glu 3m

M

Glu 10

mM

NAC 1 mM

NAC 3 mM

NAC 10 m

M0

50

100HMW

untre

ated

DTT

Glu 1m

M

Glu 3m

M

Glu 10

mM

NAC 1 mM

NAC 3 mM

NAC 10 m

M0

50

100

Mono

*

+

*

**

*

* *

+ +

+

% Oligomer in supernatant

% Monomer in supernatant

N-acetylcysteine blocks oligomerization of Cyst-C L68Q

Incubation for 24 h at 37C with indicated concentrationsDrug can be given orally

•We found a drug, GSH, that completely blocked the amyloid aggregation

•The drug is well tolerated and a precursor version of it (NAC) can be given as a tablet

•The oligomers essentially disappeared so there is no new precipitation of amyloid in the brain and brain vessels.

•A skin biomarker confirmed response to therapy in patients

Proband

~70% reduction in proband’s amyloid precipitation in skin biopsy in 15 months of NAC therapy

~50% reduction in the father’s skin biopsy following 6 months of therapy

Father

Cystatin C/amyloid protein aggregate in the skin in HCCAA following NAC therapy

u Clinical trial in Iceland with 50 patients with HCCAA to be treated for 12 months with NAC (FPI July 01, 2019)

u Dementia Rating Scale (DRS) administered every 3 months

u Biomarker measures – skin and bloodu Results will inform on therapy of

vascular dementia and Alzheimer disease (next trial)

Genome-Based Diagnosis And Development at CAG

CAG Biobank• Common

Diseases• Rare

Diseases

Phenotyping• EPIC• CHOP

Clinicians• Recontact

Genotyping• GWAS• Sequencing

• WES/WGS• RNAseq• ChIPseq

Variant Identification• ANNOVAR• PennCNV• SNVer

Targeted therapies• ADHD• Autism• IBD, pAIDs• GLAs• HCCAA

POC clinical trials

Test&Treat

Highly feasible and cost-effective to perform POC studies (Rx/Dx) within academic infrastructure with focus on genetic/genomic biomarker-stratified patient populations

Appropriately consented biobanking efforts are a pre-requisite for these opportunities

AcknowledgementsCAG staff:Patrick Sleiman,Dong LiAvni Santani Charlly KaoLifeng TianRenata PellegrinoSarah ShepardJoseph GlessnerElizabeth BhojJohn ConnollyHuiqi QuXiang WangAlvaro GutierrezMichael MarchBerta CastilloKenny NguyenMargaret HarrMike GonzalezStruan GrantFrank MentchHaijun QiuXiao ChangJonathan BradfieldYiran GuoChristopher CardianleMarina BakayRahul PandeyFernanda Marfa Zhi WeiGeorge OtienoAmber benderSandra DeliardMaria GarrisCuiping HouAdam Wenocur

Genetics:Donna McDonald-McGinnElaine ZackaiMarni FalkMatt DeardorfBeverly EmanuelDonna McDonald-McGinnMarcella Devoto

Larus HermannsonAsbjorn KristbjornssonJanine LarrJennifer Lebold

Collaborators:Yoav DoriRaquel GurBob BaldassanoJohn MarisJo EliaJean BelascoChristoph SeidlerTara WengerJonathan PerkinsAthena ZuppaBetsy GoldmuntzBob SchultzMichael GrunsteinJonathan SpergelJulian AllenNancy SpinnerDennis DlugosEric RappaportSusan LevyBob BerkowitzDimitri MonosBabette ZemelCharlie StanleyCharles BaileyGarrett BrodeurEdward AttyehYael Moss Bill GaynorScott Cook-Sather… and many others

Collaborators:Russell BuonoTom FarraroMichael SperlingMaja BucanHongzhe LiMingyao LiJason ChristieAllan PackDan RaderMuredach ReilyGarret FitzgeraldKate NathansonGholson LyonSteven AlbeldaHans BisgaardKathleen BarnesBill CooksonAdnan CustovicEric TopolJohn TrojanovskiVirginia LeeJohn AsplinDavid Golfarb….and many others

Management:Madeline BellBryan Wolf

Clinical CAG staff:Mcckenzie BehrAlexandria ThomasNora RobinsonShanell HarrisonCarlita GibbonsKisha HardenIris Ortiz-RiveraSalley FredaTyeisha RexDaniel SenicolaDebra Abrams