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Pancreatic Cancer Research

Jonathan Brody, Ph.D.

Co-director of the Jefferson Pancreatic,

Biliary, andRelated Cancer Center

Director of Surgical Research

Kimmel Cancer Center Member

Wallace Davis Professor of Surgery

Thomas Jefferson University

Pancreatic Cancer Action Network

Educational Session

04/14/2016

May 25, 1961

“I believe we should go to the moon…

It is a most important decision that we

make as a nation.”

And 8 years later…

July 20, 1969

1. Genetic mutations: We know a lot about the

genes involved or mutated in pancreatic.

2. Treat every aspect of the tumor (the complex

TME): We know that this is a complex tumor

compared to other tumor systems.

3. Update on early detection, monitoring disease

4. Optimize agents with existing and proven

activity (even gemcitabine)

5. Understand and identify new targets beyond

genetic lesions

Key facts, concerns, and hope in the field of

pancreatic cancer research

Knudsen ES, O'Reilly EM, Brody JR, Witkiewicz AK. Gastroenterology. 2015 Sep 15.

1. Personalized

approach

The progression model has not changed…. August, 2008

PanIN-1 PanIN-2 PanIN-3 Normal ductal epithelium

KRAS mutations Telomere shortening

CDKN2A loss TP53 loss SMAD4 loss BRCA2 loss

Winter, Yeo, Brody: Devita, Rosenberg: Cancer Principles & Practice of Oncology

Gleevec

Philadelphia chromosome (P. Nowell & D. Hungerford)

Novartis

(High Throughput Screen)

Game changer:

CML, GIST

Average development time: 15 years

Average cost: approximately $1 billion

*Studies in humans

6 years 7 years

2 years

+ +

Preclinical

Research

Preclinical

Development

Pivotal

Trials*

Clinical

Research*

FDA Review

and Approval*

Developing a targeted therapy against these mutated pathways takes time and money….

Single-dose

treatment with

MMC (5 mg/kg)

Complemented

Empty vector

Clin Cancer Res 2005;11:7508-7515

Best example….

Case study: BRCA2

• Mrs. Smith: Prior tongue cancer;

uterine cancer and breast cancer

• Surgery—pancreatic ductal

adenocarcinoma (PDA) by Dr. Yeo

early January 2007

Platinum-based adjuvant therapy

Patient alive and well today (~8 years)

BRCA2 (mutation, inactivation)

1. The cause of her tumor:

via a defect in genome

maintenance

2. An Achilles Heal of the tumor:

giving Mrs. Smith a great response

Team: Charles Yeo, MD,

Sarah Charles, Ph.D.

An example (rare)

Gemcitabine

Radiation

FOLFORINOX

0 20 40 600

20

40

60

80

100

Days

Clin

ical

ben

efit

World with no biomarkers….

World with an effective Biomarker….

0 20 40 60 800

20

40

60

80

100

+ Biomarker A

negative biomarker A

Days

Clin

ical b

en

efit

Surgery or

Biopsy Laboratory work:

analysis to predict patient therapy

Predictive biomarkers

Optimized

adjuvant therapy

Early detection

Sequence:

BRCA2 mutation Immunohistochemistry Expression Heatmap

Long survival Short survival

What are Biomarkers?

Future of medicine: Personalized Medicine

WE HAVE BANKED >400 specimens to date!

Roa et al, Under Review Cancer Research Subha Madhavan

ERCC1 and

RRM1

might be

Useful…

Chemotherapy Predictive Markers

• Pilot Trial in Pancreatic Cancer • Patient “Tailored” Therapy (*2000 version)

Tumor Biopsy

Low RRM1

Gem - Based

High RRM1

No Gem

Low ERCC1

Platinum - Based

High ERCC1

No Platinum

Low ERCC1

GemOX

High ERCC1

No Platinum

Predictive Markers

Gemcitabine

RRM1

Platinum

ERCC1

5-FU

TS Low TS

Gem 5FU

High TS

Gem Tax

Low TS

FOLFOX

High TS

Ox - Tax

Low TS

5 - FU Tax

High TS

Tax - Iri

Michael Pishvaian, MD, Ph.D., LCC, Georgetown University

Clinical trial over 4 institutions: TJU the lead institution

Is the pancreatic cancer field ready for “personalized” therapy?

Multi-Omic analysis

• NGS (DNA)

• Expression (RNA)

• Immunohistochemistry

• Phosphoprotein

Biopsy while on Front line therapy (prior to progression)

(Overview of the grant) Patient chronology: Metastatic pancreatic cancer patients

1st Disease

progression

R

A

N

D

O

M

I

Z

E

Physician’s discretion (SOC)

Molecular Tailored Therapy

2nd line

therapy

Aim 1: Clinical trial

Aim 2: V 2.0

Biopsy Biopsy upon

Progression

ORGs

ORGs

O

U

T

C

O

M

E

S

Molecular Tailored Therapy

Version 2.0 for Phase III trial

Aim 3: Resistance Pathways

ORGs

ORGs

Resistant CRCs

Additional Omic analysis

• Focused genomic sequencing

• RNA seq

• Phosphoprotein

• Post-transcriptional

New targets and Resistance pathways

Front line

therapy

Systems Biology

2nd Disease

progression

Multi-Omic Analysis

*2016

5 core Biopsies

• Validate sequencing • SNPs

• Drug sensitivity • MOP

• Protein • RNA • NGS

Develop CRCs 2D

• Orthotopic

mouse

• Xenograft

• Zebratars

David Tuveson, Cold Spring

Harbor

Correlative science

Sue Chand, a graduate student,

actively plating cancer cells for

organoid growth

On top of the 1 million dollar grant we raised >500K for the organoid lab.

SURGICAL

BIOPSY

GENERATE

ORGANOIDS

WHOLE

EXOME

SEQUENCE

FOCUSED DRUG

SCREENING

PERTHERA TESTING

PERTHERA & Organoids: Trial Design (stage I & 2)

Jordan Winter

Talar Tatarian

SURGICAL

RESECTION

GENERATE

ORGANOIDS

DRUG SCREENING

DEVELOP A

LIVE BIOBANK

Organoids and

Pharmacologic Tailored

Therapy (PTT) PTT

Jordan Winter

Talar Tatarian

KYT-Perthera Workflow

Patient 576: IDH1 Mutation

• 48 year-old female diagnosed with metastatic pancreatic cancer

• Started FOLFIRINOX – progression after nine months

• Started on a clinical trial of gemcitabine plus nab-paclitaxel plus

pembrolizumab

• Biopsy of a liver metastasis was performed while on study

FM identified an established IDH1 R132H mutation in found in GBMs

(frequency of 12% in GBM - Cosmic)

From a separate sample from the tumor we confirmed via

targeted next-generation sequencing pan cancer panel

Mutant IDH1 specific Ab H & E

IHC confirmation with an Ab against mutant IDH1

Part II: Treat every aspect of the tumor (the

complex TME): We know that this is a

complex tumor compared to other tumor

systems.

Fibroblasts

Collagen and elastic fibers

T cells

The many facets of targeting pancreatic cancer...

Weinberg BA, Yabar CS, Brody JR, Pishvaian MJ.

Oncology (Williston Park). 2015 Nov;29(11):809-20, 886.

2. Know your entire tumor

IDO

IDO

T cells

T cells Regs

T cells

Cancer cells

T cells

T cells

T cells

Genotyping and expression analysis of IDO2 in human pancreatic

cancer: a novel, active target.

Witkiewicz AK1, Costantino CL, Metz R, Muller AJ, Prendergast GC,

Yeo CJ, Brody JR.

IDO2 cooperates with Kras

IDO2 -/- genotype

Wild type genotype

IDO2-null genotype

aids therapy

Part III: early detection and monitoring

T1 T2 T3

-5 -10 -15 -20

yrs

interval

clinical relevance

histologic progression

Death

t3 t2 t1 t0

Surgical intervention

Detectable By CT

Disseminated pancreatic

cancer

Nature. 2010; 467; 1114 Canc Res. 2000; 60: 2002. Clin Can Res. 2008; 14: 412. Clin Im. 2010; 34:277.

X

T1: tumor initiation to parental clone T2: parental clone to metastastic clone T3: dissemination to death Nature 2010; 467: 1114.

KRAS TP53

Genetic events

Pancreatic tumorigenesis Infiltrating/metastasis

Treatment/ tumor microenvironment

Lethality

microRNAs/Chromatin changes

Darwinian selection

12 years 2 years

Nature. 2010; 467; 1114 Canc Res. 2000; 60: 2002. Clin Can Res. 2008; 14: 412. Clin Im. 2010; 34:277.

Disseminated pancreatic

cancer

Part IV: Optimize agents with existing

and proven activity (even gemcitabine)

Pencil sketch of the DNA double helix

by Francis Crick in 1953

>RNA>Protein

Central Dogma

13104 publications

80

publications

RNA processing

2001

Microscopic Chronic Pancreatitis* (Non-Smoker) (Smoker) Any Fam. Hx of Cancer (Non-Smoker) (Smoker) Fam. Hx of Colon Cancer* (Non-Smoker)§ (Smoker) Fam. Hx of Prostate Cancer (Non-Smoker) (Smoker)

N/A

N/A

Fam. Hx of Breast Cancer (Non-Smoker) (Smoker) Fam. Hx of Pancreas Cancer (Non-Smoker) (Smoker)

Group 1* (Non-Smoker) (Smoker) Group 2§ (Non-Smoker)§ (Smoker)

Fam. Hx of Lung Cancer (Non-Smoker)

(Smoker) N/A

0.06 0.25 1.00 4.00 16.00

Odds Ratio (95% CI)

Risks Associated with WEE1 INDEL vs. WEE1 WT (N=99)

Smokers (N=37), Non-Smokers(N=51).

Risk of Cancer in 1st degree relatives, Odds Ratio (95% Confidence Interval).

* P < 0.05; § P = 0.06

Group 1: Colon, Rectal, Gastric, Endometrial, Ovarian

Group 2: Colon, Rectal, Gastric, Breast, Hepatobiliary, Pancreas, Endometrial, Ovary, Melanoma, Brain, Urinary

OR P

Microscopic Chronic Pancreatitis 2.403846 0.042

Microscopic Chronic Pancreatitis (Non-

Smoker) 2.285714 0.178

Microscopic Chronic Pancreatitis (Smoker) 2.428571 0.21

Familial History of Colon Cancer 6.323529 0.012

Familial History of Colon Cancer (Non-

Smoker) 5.294118 0.057

Familial History of Colon Cancer (Smoker) 0.144

Group 1 4.298387 0.016

Group 1 (Non-Smoker) 3.733333 0.095

Group 1 (Smoker) 0.132

Group 2 2.384868 0.057

Group 2 (Non-Smoker) 3.340659 0.055

Avi Nevler, MD

Struan Grant, Ph.D. (CHOP)

James Eshleman, MD/PhD (Johns

Hopkins)

Blo

od

ve

ss

el

Tumor cells

pO2

0.1%

Hypoxia

pO2 Drug Resistance

Poor Prognosis Nature, 379 (1996),

Cancer Res, 60 (2000)

1. Lower microvessel density: drug penetration

2. Hypoxic niches: Arrested or quiescent cells become

refractory to agents targeting rapidly-dividing cells

The Hypoxia-Inducible Proto-Oncogene PIM1

Promotes Chemoresistance

PIM1

a-Tubulin

0 6 12 24

MiaPaCa2 cells

PIM1 is a serine-threonine kinase that regulates DNA repair and therapeutic response

PIM1

Hypoxia

Inhibition of

apoptosis

e.g., BAD

Cell Cycle

Arrest

DNA Repair

Chemoresistance

The role of PIM1 expression in hypoxia-induced

chemoresistance to Oxaliplatin

0

20

40

60

80

100

120

0.01 0.1 1 10 100

Oxaliplatin (mM)

Rela

tive

Cell

Su

rviv

al (%

)

Normoxia

0

20

40

60

80

100

120

0.01 0.1 1 10 100

Oxaliplatin (mM)

Hypoxia

Hours in Hypoxia, 1%O2:

Blanco et al Oncogene 2015

Sensitivity to Oxaliplatin in Normoxia and Hypoxia

Blanco et al Oncogene 2015

HuR PIM1

Nuclear/cytoplasmic

Blanco et al Oncogene 2015

In patient samples cyto HuR correlates with PIM1 levels

Nat Chem Biol. 2007 Aug;3(8):508-15.

Blanco et al Oncogene 2015

Phase I/II Trial ofvABT-888 in

Pancreatic Cancer • Phase I – ABT-888 dose escalation

• Mixture of untreated and previously treated patients

• Phase II - two strata • Untreated vs. previously treated

Georgetown Lombardi

TJU

1 2 3 4 5 6 7 29 15 43 56

Cycle 4 Cycle 1 Cycle 3 Cycle 2

Day

Response

Assessment

5-FU CI

2400mg/m2

ABT-888

Orally BID

5-FU CI

2400mg/m2

ABT-888

Orally BID

5-FU CI

2400mg/m2

ABT-888

Orally BID

Oxaliplatin

85mg/m2

Leucovorin

400mg/m2

Oxaliplatin

85mg/m2

Leucovorin

400mg/m2

Oxaliplatin

85mg/m2

Leucovorin

400mg/m2

Oxaliplatin

85mg/m2

Leucovorin

400mg/m2

5-FU CI

2400mg/m2

ABT-888

Orally BID

Based on some of

the preliminary data

obtained from the

Pascal collaboration

Pishvaian MJ, Brody JR…. PARP inhibitor + mFOLFOX for the treatment of

pancreatic cancer, GI ASCO and ASCO presentations

Cancer Res. 2015 Sep 29;6(29):27312-31.

PARP inhibitors translocate HuR

Untreated Veliparib Olaparib Rucaparib Niraparib Talazoparib

HuR HuR HuR HuR HuR HuR

DAPI

HuR

DAPI

HuR

DAPI

HuR

DAPI

HuR

DAPI

HuR

DAPI

HuR

Cyto

pla

sm

ic

Nucle

ar

Lamin A/C

α-Tubulin

HuR

Tota

l

Xenograft studies with Mia.sh290 and Olaparib treatment

Vehicle only shHuR + Olap + Olap,

shHuR

PARP1 PARP1

Trapped PARP1

PARP1

PARP1

DNA damage repair

PARP1 released

HuR cytoplamic expression

(expression and function)

Targeting HuR

Chand et al., unpublished

HuR regulates Poly ADPribose Glycohydrolase (PARG) to

modulate PARPi resistance

Veliparib Olaparib Untreated

PARP1

Histone H3

PAR

GAPDH

CH

RO

MA

TIN

-BO

UN

D F

RA

CT

ION

Abdelmohsen and Gorospe, WIRES Res 2010

Chemotherapeutic

Efficacy:

PIM1, PARG, WEE1

TME:

PIM1, IDH1

A novel, alternative non-genetic target

for pancreatic cancer.

Part V: Understand and identify new targets beyond genetic lesions

Would targeting RNA regulation be more effective than targeting DNA?

A Global Synthetic Lethal approach: Multiple targets at once

Chemotherapeutic

Efficacy:

PIM1, PARG, WEE1

TME:

PIM1, IDH1

• Discovered in bacteria as a key part of a precise immune

reaction

• Short DNA sequences are recognized and put back together

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system

Wave of the future: Latest in DNA/RNA tech

CRISPR plasmid was transfected into MiaPaCa2 cells in 6-well plate

48 h later GFP

positive cells were

single sorted by FACS in 96-well

plate

2-3 weeks later single cell

colonies were expanded into 24-well plate

Cells were then expanded into T-75 flask

Cells were then expanded into

6-well plate

Expanded colonies were assessed for mutations with sequencing, qPCR and Western

CRISPR

Technology

Shruti Lal, Ph.D.

N/A

No Tumor

N/A

No Tumor

HuR -/- cells

Xenograft lethality

Lal et al, unpublished Novartis, HuR inhibitor (Nature 2007)

Univ. of Kansas, HuR inhibitor

Genisphere, DNA dendrimer (Cancer Research, In press, Ovarian)

Take home points

• High throughput DNA sequencing of pancreatic cancer

genomes has provided us with some validating pearls,

BRCAness: Best example for personalized therapy

• Alternative approach: Post-transcriptional gene

regulation can provide us with information about the

biology of the tumor now (not yesterday but when the

tumor is treated): Personalized therapy=Present focus

Therapy: “Pancreatic cancer Transcriptionally

addicted”

• Understanding these acute, stress response mechanisms

by tumor cells will allow us to NOW:

• 1: Optimize current therapies

• 2: Attack and find new targets

How to make a difference against this foe?

• Multi-disciplinary conferences (surgeons, PhDs, medical

oncologists, Radiation Oncology, nutrition, geneticists, basic

scientists, medicinal chemists, bioinformatics): patient

centric

• Programmatic initiatives with authentic questions: think out

of the box type strategies

• Development/philanthropic strategies to support pilot

awards and bridge funding

• LiveBiobank: How many important questions have been

answered from a frozen BioBank (except for genetics)

Summary of key concepts

1. Genetic mutations: We know a lot about the

genes involved or mutated in pancreatic.

2. Treat every aspect of the tumor (the complex

TME): We know that this is a complex tumor

compared to other tumor systems.

3. Update on early detection, monitoring disease

4. Optimize agents with existing and proven

activity (even gemcitabine)

5. Understand and identify new targets beyond

genetic lesions

4/14/2016 61

Lab Members

Mahsa Zarei

Saswati Chand

Joseph Cozzitorto

Rick Burkhart

Carmella Romeo

Masaya Jimbo

Kevin O’Hayer

Shruti Lal

Fernando Blanco

Edwin Cheung

Amanda Grigoli

Avinoam Nevler

Nava Azimzadeh

Cinthya Yabar

Collaborators

Michael Pishvaian (GU)

Chip Albanese (GU)

Peter Kozuch (MS) Dan Dixon (Kansas)

George Prendergast(LIMR)-R01

Janet Sawicki (LIMR)

David Tuveson (Cold Spring)

Jonathan Sachs (Minn)- R01

Struan Grant (CHOP)

Jim Eshleman (JHU)

Tim Yen (FC)

Igor Astsaturov (FC)

TJU

Jordan Winter

Charles J. Yeo

Mark Tykocinski

Isidore Rigoutsos

Scott Waldman

Matt Weber

Ashwin Sama

Peter McCue

Paolo Fortina

John Pascal

Wei Jiang

Theresa Yeo

Support from: NCI; NIH; PanCAN-AACR; American Cancer Society; WW Smith; Fund A

Cure, Sarah Parvin Foundation; Hirshberg; Fund A CURE; Mary Halinski Pancreatic

Cancer Research Fund ; Linda Katz; Van Vance