USCAP 2017 · 3/24/2017 7 PD0325901 Reduces Myeloproliferationand Enhances Erythropoiesis in Mx1‐Cre, Nf1flox/flox Mice Chang, JCI 2013 WT Veh WT 901 Nf1-/-Veh

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Juvenile Myelomonocytic Leukemia: From Bedside to Bench and Back

Mignon Loh, M.D.UCSF Benioff Professor of Children’s Health

Deborah and Arthur Ablin Chair of Pediatric Molecular Oncology

Benioff Children’s Hospital

University of California, San Francisco

Chair, Acute Lymphoblastic Leukemia Committee

Children’s Oncology Group

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Disclosure of Relevant Financial Relationships

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MIGNON LOH: NO RELEVANT FINANCIAL RELATIONSHIPS

There are two clinical challenges in JMML

1. JMML is difficult to diagnose

Category 1 Category 2

ALL of the following At least 2 of the following

AMC >1000/uL Circulating myeloid precursors

Blasts in PB/BM < 20%

WBC >10,000/uL

Absence of the t(9;22) or BCR/ABL fusion gene

Increased Hgb F for age

GM-CSF hypersensitivityudingon7

Here’s the second challenge…

1. JMML is difficult to diagnose…

2. JMML is hard to treat

Category 1 Category 2

ALL of the following At least 2 of the following

AMC >1000/uL Circulating myeloid precursors

Blasts in PB/BM < 20%

WBC >10,000/uL

Absence of the t(9;22) or BCR/ABL fusion gene

Increased Hgb F for age

GM-CSF hypersensitivityudingon7

What strategies can weuse to solve these problems?

• Genetics approach

– Identify novel genetic mutations

– Incorporate mutations into diagnostic criteria

– Correlate genotype with phenotype

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What strategies can weuse to solve these problems?

• Genetics approach

– Identify novel genetic mutations

– Incorporate mutations into diagnostic criteria

– Correlate genotype with phenotype

• Functional approach

– Determine how mutations contribute to pathogenesis

– Determine if perturbed signaling networks exist in JMML that can be harnessed therapeutically

– Use specific agents to reverse these abnormalities and test in preclinical models

– Take new agents into clinical trial

The patients inform our science and we hope the science will lead to improved therapies for

patients…

Juvenile myelomonocytic leukemia

• WHO classification: Mixed MDS/MPN of young children

• Shares clinical and biological features with CMML and CML

• Boys > girls• Median age: 1.7 years

• Splenomegaly: 93%

• Lymphadenopathy: 59%


0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 1 2 3 4 5 6 7 8 9 10 11 12

Log‐Rank p=0.0001 years

Plt 33x109/L, age 2 yrs: 0.08 Plt 33x109/L, age 2 yrs: 0.05Plt 33x109/L, : 0.00



• Boys > girls• Median age: 1.7 years



• Survival following conventional chemotherapy is dismal

Niemeyer, Blood 1997




• Boys > girls• Median age: 1.7 years• Splenomegaly: 93%• Lymphadenopathy: 59%• Only hematopoietic stem cell

transplantation is curative• Relapse remains the most

common cause of treatment failuretreatment failure

Stieglitz, Peds. Blood and Cancer 2015

COG AAML0122

0

0.25

0.5

0.75

1

0 2 4 6 8 10 12

Event‐free survival

Years from study entry




• Boys > girls

• Median age: 1.7 years



• Hallmark laboratory feature is hypersensitivity of myeloid progenitors to CM-CSF

• Hematopoetic stem cell transplant is curative but relapse remains the most common cause of treatment failure

Emanuel, Blood 1991

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Cooke, J. Pediatrics 1953

Patients with NF1 are at Increased Risk of Developing Leukemia

Leukemia Relative risk Confidence Interval

“CMML” (JMML) 221 71‐514

ALL 5.4 2.8‐9.4

NHL 10.9 3.3‐23.4

Neurofibromatosis Type 1 (NF1)

• dominant genetic disorder– Spontaneous germline

mutations can occur in up to 50% of cases

• pigmentary lesions • benign and malignant tumors

usually affect cells derivedfrom the embryonic neural crest

• greatly increased risk of developing JMML

rasGAP(mammalian, p120)

NF1(mammalian, p280)

IRA1/2(S.cerevisiae, p320)

GAP1(Drosophila, p130)

gap 1(S.pombe, p80)

NF1 Encodes a GTPase Activating Protein (GAP)

Ras

Courtesy of F. McCormick, UCSF

“The beating heart of signal transduction”

A. Wittinghofer

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Ward, Blood 2012

Ras Proteins Act as Molecular Switches NF1 associated JMML demonstrates NF1 is aclassic tumor suppressor gene

Shannon, NEJM, 1994

Mo Fa GL JMML

Bollag, Nat. Genetics 1994

Blood, April 1994

Blood, November 1994

ShcGrb2 SOS Ras‐GDP

Neurofibromin

Effector Pathways

SHP‐2

Gab2

Ras‐GTP*

BCR‐ABL

JAK

STAT

JMML is a disease of hyperactive Ras signaling

Gene %

NF1 15%

RAS 25%

Facial anomalies

Short stature

Pulmonic stenosis

Noonan syndrome (Jackie Noonan, 1963)

Courtesy of Zenker/Kratz

Hypertrophiccardiomyopathy

“Myeloid disorders of Noonan Syndrome and a resident’s rule recalled” Shannon and Side, J. Pediatrics, 1997

Nature Genetics 34, 148 ‐ 150 (2003)

Missense somatic mutations in PTPN11occur in de novo JMML

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Neurofibromin

Effector Pathways

SHP‐2

Gab2

Ras‐GTP*

BCR‐ABL

JAK

STAT


Gene %

NF1 15%

RAS 25%

PTPN11 (SHP2)

35%

Loh, ML et al. Blood 2009;114:1859

Harnessing SNP arrays demonstrated region on 11q with copy neutral LOH

Analysis of deleted region suggested CBL as a likely candidate gene


Wildtype

Diagnosis

* 2 heterozygous mutations

Location Nucleotide change Amino acid change No of Patients

Intron 7 1096-1G>C Splice site 1

Exon 8 1111T>C 371 Tyr>His 10

1111T>G 371 Tyr>Asp 2

1112A>G 371 Tyr>Cys 1

1139T>C 380 Leu>Pro 1

1141T>C 381 Cys>Arg 1

1150T>C 384 Cys>Arg 4

1186T>C 396 Cys>Arg 1

1190 del99bp* deletion 1

1210 T>C 404 Cys>Arg 1

1222 T>C 408 Trp>Arg 1

Intron 8 1227+4C>T* Splice site 1

1228-2A>G Splice site 1

Exon 9 1244G>T 415 Gly>Val 1

27 CBL mutations detected in 68 JMML patientswithout RAS/PTPN11/NF1


Is this a new germline syndrome?

Wildtype

Cord Blood

Diagnosis

Loh, Blood, 2009, 114: 1859

Lung Ca, 50s

6 mos.6 mos.

*7 mos.

1111T>C

1111T>C

1111T>C

1111T>C

1111T>CWT

WT

WT

WT

WT

1111T>C

WTWT

CBL Mutations in JMML are Frequently Inherited in an Autosomal Dominant Fashion

Niemeyer, Nature Genetics, 2010, 42: 794‐800

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Neurofibromin

Effector Pathways

SHP‐2

Gab2

Ras‐GTP*

BCR‐ABL

JAK

STAT


Gene %

NF1 15%

RAS 25%

PTPN11 (SHP2)

35%

CBL 10%

CBL

Rasopathies Support Hypothesis that PediatricCancer is Development Gone Awry

Chang, T Blood 2014©2014 by American Society of Hematology

Can Ras Signaling Networks be Therapeutically Harnessed?

Shc

Grb2 SOS Ras‐GDP

Neurofibromin

SHP‐2

Gab2

Ras‐GTP

JAK2

STAT5

Survival, Proliferation

Ral‐GDS

RAFPI3K

Ral‐A

MEKAkt

ERK mTor

S6

CBL

Can we harness perturbed signaling networks for novel therapeutics?

Shc

Grb2 SOS Ras‐GDP

Neurofibromin

SHP‐2

Gab2

Ras‐GTP*

JAK2

STAT5

Survival, Proliferation

Ral‐GDS

RAFPI3K

Ral‐A

MEKAkt

ERK mTor

S6

Ruxolitinib

PD0325901TrametinibBinimetinibSelumetinibPimasertibCobimetinib

PictilisibCopanlisib

Dactolisib

SirolimusEverolimus

Pre-clinical MPN mouse models

• Mx1-Cre ,Nf1flox/flox (Parada, Shannon, Le)

• Mx1-Cre, KrasG12D (Jacks, Shannon, Braun)

• Mx1-Cre, NrasG12D (Jacks, Shannon, Haigis)

• Ptpn11D61Y (Neel, Araki)

• Mx1-Cre, Ptpn11E76K (Qu, Xu)

• cCblC379A/- (Langdon, Thien, Rathinam)

• MMTV-Cre, Cblflox/flox;Cbl-b-/- (Band, Naramura)

No JMML cell lines, xenografts difficult to establish

PD0325901 Results in MEK Inhibition In Vivo

PD03259015mg/kg/day

+Mx1‐Cre, Nf1 flox/flox

pERK pSTAT5

Myeloid progenitors

Chang, T, JCI 2013

Nf1flox/flox Nf1wt

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PD0325901 Reduces Myeloproliferation and Enhances Erythropoiesis in Mx1‐Cre, Nf1flox/floxMice

Chang, JCI 2013

WT Veh

WT 901

Nf1-/- Veh

Nf1-/- 901

0 20 40 600

10

20

30

40

50

Days From Enrollment

WB

C (

K/

L)

0 15 30 45 60 750

5

10

15


HB

(g

/dL

)

0 30 600

5

10

15


% R

etic

ulo

cyte

s

0

1

2

3

4

5

Nf1-/-WT

901 901Vehicle Vehicle

Sp

lee

n w

eig

ht

(g) ***

** ***

**

** **

**

PD0325901 Reduces Splenic Hematopoiesis In Vivo

WT Nf1-/- Veh Nf1-/- 901

d e d0

100

200

300

CFU-E

Col

ony

num

ber

Vehicle 901 Vehicle 901

WT Nf1-/-

e d e d0

100

200

300

400

Col

ony

num

ber

BFU-E

Vehicle 901 Vehicle 901

WT

Nf1-/-

Chang, JCI 2013

WT Veh

WT 901

Nf1-/- Veh

Nf1-/- 901

Primary human JMML cells vs. MEK inhibition

J. Weng, Loh lab

0

20

40

60

80

100

120

0 0.01 0.1 1 10

% M

axim

al c

olo

ny

gro

wth

GM-CSF dose

No drug (n=6)

Normals (n=13)

JMML (n=23)

0

20

40

60

80

100

120

0 0.01 0.1 1 10

% M

axim

al c

olo

ny

gro

wth

GM-CSF dose

No drug (n=6)

100nM PD-901(n=5)Normals (n=13)

JMML (n=23)

• Selective allosteric inhibitor of MEK1/2• OG daily dose • Long circulating t1/2

• Sustained suppression of pERK1/2 for more than 24h

• FDA approved for melanoma• MEK116540:Phase 1 in pediatric solid

tumors currently enrolling

Trametinib

Borthakur, G, Cancer 2016

Part A: Phase 2 Trametinib In JMML

Population: Relapsed/Refractory JMML• Age 2 years‐21 years

Endpoints: • Objective Response• Safety/Toxicity

ADVL1521

Dose Level Trametinib

‐1 One dose < RP2D

0 100% RP2D

Genotyping

Colony Assays

Pharmacodynamics

Will MEK inhibition Monotherapy Work?

PTPN11 PTPN11

What Additional Genetic or Epigenetic Modifiers Occur in JMML?

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The grand fishing expedition?

Stieglitz, Taylor‐Wiener, Nature Genetics 2015

Genomic Landscape of JMML


Mutations Identified in Exome and TCA


No Prognostic Significance for Initiating Ras pathway alteration


More Alterations Confer Worse Outcome


PTPN11 PTPN11 + SH2B3

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Summary

• JMML is a genetically more complex disease than previously documented, with implications for mono and combinatorial therapies (?will MEK inhibition work?)

• Documentation of multiple genetic events can clarify diagnosis and identify patients at highest risk of relapse

• Discovery of additional genetic events informs future mechanistic studies of how Ras signaling is affected by protein X, Y, Z….

Acknowledgements

Elliot Stieglitz

Ernesto Diaz‐Flores

Laura Gelston

Thai Tran

Tiffany Chang

Kyle Beckman

Kailyn Kim

Emilio Esquivel

Sophie Archambeault

Michelle Kang

Debbie Sakai

Kevin Shannon

Ben Braun

Michael WangScott Olsen

Adam AbateJohn Haliburton

Amaro Taylor‐WienerTodd Golub

Kim Stegmaier

Future Directions

“Deep Sequencing” Misses Subclonal Mutations

Ras Pathway Mutations SETBP1 Mutation

Gene Nucleotide Amino Acid

Allelic Fraction Nucleotide

Amino Acid

Allelic Fraction

J259 NRAS c.35G>T p.G12V 0.38 c.2602G>A p.D868N 0.47

J264 PTPN11 c.226G>A p.E76K 0.21 c.2609G>A p.G870D <0.02*

J276 NF1 c.1722C>G p.S574R 0.55 c.2602G>A p.D868N <0.02*

J286 PTPN11 c.182A>T p.D61V 0.57 c.2609G>A p.G870D <0.02*

J295 PTPN11 c.226G>A p.E76K 0.46 c.2602G>A p.D868N <0.02*

J313 NRAS c.38G>A p.G13D 0.28 c.2602G>A p.D868N 0.38

J322 PTPN11 c.182A>T p.D61V 0.52 c.2602G>A p.D868N <0.02*

J342 PTPN11 c.226G>A p.E76K 0.48 c.2602G>A p.D868N <0.02*

J397 - - - - c.2602G>A p.D868N <0.02*

J403 NRAS c.38G>A p.G13D 0.21 c.2602G>A p.D868N <0.02*

J404 CBL c.1112A>C p.Y371S 0.82 c.2609G>A p.G870D <0.02*

NRAS c.35G>A p.G12D 0.07

J405 NRAS c.38G>A p.G13D 0.36 c.2602G>A p.D868N <0.02*

Does SETBP1 Occur in a Progenitor or Initiating Cell?

Modified from Sakaguchi et al. (2013)

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SETBP1 Affects Putative Stem Cells

Laura GelstonJon Akutagawa

Human HSCs (Lin‐CD34+CD38+CD45RA‐CD90+)

USCAP 2017 · 3/24/2017 7 PD0325901 Reduces Myeloproliferationand Enhances Erythropoiesis in Mx1‐Cre, Nf1flox/flox Mice Chang, JCI 2013 WT Veh WT 901 Nf1-/-Veh

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