Using Mouse Models of Acute Lymphoblastic Leukemia...
Transcript of Using Mouse Models of Acute Lymphoblastic Leukemia...
Using Mouse Models of Acute Lymphoblastic Leukemia to Test Novel Therapies
David T. Teachey, MD
Tumor Models Boston 2017
July 20, 2017
Disclosures for
David T. Teachey
Employee N/A
Consultant N/A
Major Stockholder N/A
Speakers Bureau N/A
Honoraria/Grant Funding Novartis (Institution)
Scientific Advisory Board Amgen
Hunger S P et al. JCO 2012;30:1663-1669
©2012 by American Society of Clinical Oncology
ALL OS: COG studies
Dismal Outcome for 2nd+ Relapse of ALL
Resimuller et al. JPHO 2013
10 year EFS
Leukemia is the #2 cause of pediatric cancer mortality: NOVEL THERAPIES ARE NEEDED
How to tackle the problem?
• Intensify therapy for high risk disease
• Reduce therapy for low risk disease
• Introduce targeted therapies
• Addressing clonal evolution in chemoresistance
• Incorporate immunotherapies
• Modify therapy based on
host polymorphisms that
can affect drug metabolism
NSG Xenograft Model of ALL
Spectral Unmixing of Click Beetle Green (CBG) and Click Beetle Red (CBR) Luciferase
CBG Leukemia only
EMpeak 540 nm
CBR T-cells only EMpeak 640 nm
Barrett
CHOP ALL Xenografts
• > 400 patients – 75% 1o engraftment – 66% >85% replacement bm and
spleen (usable samples) – 100%: 2o engraftment – Start with 1 million
lymphoblasts – Gain 10-960 million/mouse
• Cytotoxics – DEX, VCR, PRED, ASNase, ARA-C,
DOX, VP-16, MTX, MITO
• Biologic agents – Sirolimus, Temsirolimus – Bortezomib, Ruxolitinib – R04929097, MLN8237 – ENZ3042, CEP-701 – Dasatinib, AT9283, Trametinib – Triptolide, iCRT and many more
• Immunotherapies – moABs – CAR T – BITEs – DARTs
Targeted Therapies
Somatic Alterations Are Associated With Prognosis and Have Therapeutic Implications
Pui CH, et al. Blood. 2012;120(6):1165-1174. Schultz KR, et al. J Clin Oncol. 2009;27(31):5175-5181.
Ph-like ALL
Roberts, et. al. NEJM 2014
12.6% 3.9%/7.4% 50% 12.6% 4.3% 9%
Targeting JAK pathway in vivo
0.0E+00
1.0E+08
2.0E+08
3.0E+08
4.0E+08
5.0E+08
6.0E+08
7.0E+08
BC
R-J
AK
2
0
1000
2000
3000
4000
5000
6000
Vehicle
Ruxolitinib
Maude, et. al. Blood 2012
Ruxolitinib for Ph-like ALL
USI JAK2 mut CRLF2r Other kinase lesion Rux activity
PALKTY QGinsR683 P2RY8 Yes
PAMDRM GPinsR683 IGH@ No
PAKHZT R687Q IGH@ Yes (Partial)
PAMDKS R683G IGH@ Yes (Partial)
PALLSD R683G IGH@ Yes
PAKRSL R683G IGH@ Yes
PAKMZM I682F IGH@ No
PALNTB P933R IGH@ Yes
PALJCF JAK1 L624_R629>W P2RY8 Yes (Partial)
PAKSWW JAK1 V658F IGH@ Yes
PAKMVD JAK1 S646F Yes (Partial)
PALTWS IGH@ Yes
PAKKXB IGH@ FLT3 N609ins23aa No
PAKYEP BCR-JAK2 Yes
PALJDL IL7R, SH2B3 Yes
PAKKCA EBF1-PDGFRB No
PAKVKK NUP214-ABL1 Yes (Partial)
PAKTAL STRN3-JAK2 Yes
PANSFD ETV6-ABL1 no
PAPDFU SSBP2-CSF1R no
Engrafted >35 samples
EXPERIMENTAL DESIGN SCHEMA AALL08B1
Risk Assessment & LDA testing
NCI HR B-precursor ALL and SR B-
precursor ALL (CNS3, Testicular disease, Steroid pre-treated)
AALL1131 (Study Entry)
Patients receive 3-drug Induction on AALL0932 –
Post Induction risk assessment determined as HR-ALL or VHR-ALL
4-Drug Induction
(< 10 years-DEX for 14 days) (≥10 years-PRED for 28 days)
VHR-ALL1
Randomization
Control Arm Exp Arm 1
Consolidation 3 (Day 1-28) MBFM
Consolidation (Day 29-57)
MBFM
Consolidation (Day 29-57) CPM/ETOP
Evaluation (MRD Flow) 4
Interim Maintenance I (MBFM-IMHDM)
Delayed Intensification (Day 1-28)
Delayed Intensification
(Day 29-57) MBFM
Delayed Intensification
(Day 29-57) CPM/ETOP
Interim Maintenance II (MBFM- CMTX)
Maintenance5#(MBFM)
HR-ALL1
Randomization
Consolidation 3
MBFM (+ ITT)
Consolidation 3
MBFM (+ IT MTX)
Interim Maintenance
MBFM-IMHDM
(+ IT MTX)
Interim Maintenance
MBFM-IMHDM
(+ ITT)
Delayed
Intensification
MBFM (+ IT MTX)
Delayed Intensification
MBFM (+ ITT)
Maintenance5
MBFM
(+ ITT)
Maintenance5
MBFM
(+ IT MTX)
Maintenance
+ dasatinib
Delayed Intensification
MBFM
+ dasatinib
Interim Maintenance I
MBFM-IMHDM
+ dasatinib
BM Evaluation (MRD Flow)
Consolidation
MBFM
+ dasatinib
Dasatinib Arm
Interim Maintenance II
MBFM-CMTX
+ dasatinib
Ph-Like
with drug
targetable
kinase
mutation
Ph-Like
dasatinib
sensitive
kinase
mutation
Ph-Like
ruxolitinib
sensitive
kinase
mutation
AALL1521
ETP ALL
• AALL1231 Clinical Trial – 1200 patient randomized trial aBFM
vs bortezomib plus aBFM – Proteomics, RNAseq, Xenografts on
~40% of patients – >150 samples injected into mice – >80 have engrafted; 15% ETP
• DNM2, JAK3 • PTPN11 • GATA3, JAK1, SH2B3 • FLT3 • EZH2, NOTCH1, RUNX1, SH2B3 • Biallelic TRGD, EED, PRC2_SETD2 • CDKN2SA, RB1, GATA3, JAK1, PTEN
Coustan-Smith et al, Lancet Oncology 2009 Woord, et. al., ASH 2015
Targeting JAK/STAT in ETP ETP 12 Absolute Blast Count x 10 in Spleen
Control RuxDay 0 Day 7 Day 14 Day 21 Day 28
500
100
ETP 12 Absolute Blast Count Peripheral Blood
500
1,500
6
300
2,500Ruxolitinib
Control
p <0.01 p <0.01
ETP 13 Absolute Blast Count x 10 in Spleen
Control RuxDay 0 Day 7 Day 14 Day 21 Day 28
250
50
ETP 13 Absolute Blast Count Peripheral Blood
400
1,000
6
150
1,600 Ruxolitinib
Control
p <0.01
p <0.01
ETP 14 Absolute Blast Count x 10 in Spleen
Control RuxDay 0 Day 7 Day 14 Day 28
300
100
ETP 14 Absolute Blast Count Peripheral Blood
500
2,500
6
2001,500
Ruxolitinib
Control
p <0.01p <0.01
Maude, et. al. Blood 2015
Clonal Evolution
• Ma, et. al. Nat Commun. 2015
Clonal Evolution
Sometimes, evolution stinks
Frequently mutated pathways at relapse Ras signaling (65%) JAK-STAT signaling (25%) Epigenetic modification (65%) Cell cycle regulation (60%) B-cell development (85%) Nucleoside metabolism (45%)
0.0 0.5 1.0 1.5 2.0 2.50
20000
40000
60000
80000
Matched Relapsed Sample
Trametinib
Control
P=0.005
Weeks
Ab
solu
te b
last
co
un
t p
er µ
L
0.0 0.5 1.0 1.5 2.0 2.50
10000
20000
30000
Matched Diagnosis Sample
Trametinib
Control
P=NS
Weeks
Ab
solu
te b
last
co
un
t p
er µ
L
MEK inhibition in relapsed ALL
Jones, et. al., Blood 2015
NT5C2 activating mutations • Mutations NT5C2
– ~19% relapsed T-ALL (Tzoneva, et al Nat Med 2013)
– ~20% early relapse B-ALL (Meyer, et al Nat Gen 2013)
– Resistance to nucleoside analogues: 6MP and 6TG
0
200000
400000
600000
800000
1000000
1200000
SPN Diagnosis vs Relapse Blood
R-Veh
R-6MP
R-Cyt
D-Veh
D-6MP
D-Cyt
Immunotherapies
CAR T cell Engineering
Lentiviral vector
T cell
CD19
Native TCR
Tumor cell
CTL019 cell
Dead tumor cell
Anti-CD19 CAR construct
• T cells collected from patient
• Lentiviral vector introduces gene encoding CAR
• CAR links extracellular antibody to intracellular T cell signaling domains
• T cells expanded ex vivo
• Reinfused come in contact with antigen engage CAR cytotoxic response and in vivo proliferation
• Persistent CTL019 cells may allow long-term disease control
93CAR T cells effective in r/r ALL
• 60 r/r Pediatric ALL patients – 93% CR rate (56/60)
– 12 mo RFS: 60%; 12 mo OS: 79%
– 24 mo RFS: 53%; 24 mo OS: 61%
– 98% of pts have CTL019 detectable in CSF
>200 patients with CLL, ALL, NHL, MM have received CTL019
Grupp/Maude
1.0E+06
1.0E+07
1.0E+08
1.0E+09
1.0E+10
1.0E+11
1 2 3 5 6 7 13 16 21Days
Time Course of Nalm-6 Expansion and Distribution Using Bioluminescence
Barrett
Preclinical Models to Test Efficacy
19-z Saline
Day 7 Day 10
72 hours after single T cell infusion
19-z Saline
5.0E+05
2.1E+07
4.1E+07
6.1E+07
8.1E+07
1.0E+08
1.2E+08
1.4E+08
1.6E+08
1.8E+08
2 hours 24 hours 48 hours 72 hours
Photo
ns/s
econd/c
m2
mock
meso-41BB-zeta
19-41BB-zeta
19-28-41BB-zeta
20-28-stop
19-41BB-zeta/20-28-stop
Saline
p/s/c
m2/sr
Mock
Meso-41BB-z
19-41BB-z
19-28-41BB-z
20-28-*stop
19-41BB-z + 20-28-*stop
Saline
Compare T cell Migration & Expansion
Compare Different Co-stimulatory Domains
Milone, et. al. Molecular Therapy 2009
Compare cell culturing methods
CD3/CD28 beads vs OKT3 and IL2 CD3/CD28 bead stimulated cells Proliferated earlier More effective Longer telomeres Central memory phenotype Effective against re-challenge
Barrett, et. al. Cytotherapy 2014
Epitope Matters
Haso, et. al. Blood 2013
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
1.00E+12
Day 5 Day 8 Day 14 Day 18 Day 21 Day 27 Day 34
saline
19-41bb-zeta
*
* * * *
Saline
19-41BB-z
Day 5 8
A
B
C
mRNA Transfected CAR+ T cells Effective
Overcoming CD19 escape
Ruella, et. al. JCI 2016
Toxicity
• Cytokine Release Syndrome (CRS) – Correlates with T cell proliferation and efficacy
– Severity related to disease burden
– Reversed with novel approach – cytokine blockade
• Tumor Lysis Syndrome – Not a prominent feature, but may be with high WBC
• Neurotoxicity – Seen in several CD19 immunotherapy trials: NCI,
CHOP/UPENN, MSKCC, Blinatumomab – In our experience - generally untreated, fully resolves
• Chronic B cell aplasia requiring IgG replacement
Cytokine profile: 30 days
10
50100
5001000
500010000
GCSFpk35
0−3 4−5
p<0.001 125
102050
100200500
GMCSFpk35
0−3 4−5
p<0.0015
10
50100
5001000
500010000
IFGpk35
0−3 4−5
p<1e−05 20
50
100
200
IFNApk35
0−3 4−5
p<0.001 51020
50100200
50010002000
IL10pk35
0−3 4−5
p=0.001
50100200500
100020005000
100002000050000
IL1RApk35
0−3 4−5
p=0.0012
5
10
20
50
100
200
IL4pk35
0−3 4−5
p<0.001 1
100
10000
IL6pk35
0−3 4−5
p<1e−07 10
50100
5001000
500010000
IL8pk35
0−3 4−5
p<1e−07 5e+011e+02
5e+021e+03
5e+031e+04
5e+041e+05
IP10pk35
0−3 4−5
p<1e−06
5e+021e+032e+03
5e+031e+042e+04
5e+041e+052e+05
MCP1pk35
0−3 4−5
p<1e−05 10
50100
5001000
500010000
MIGpk35
0−3 4−5
p<1e−05 50100200
50010002000
5000
MIP1Bpk35
0−3 4−5
p<0.00120
50
100
200
500
1000
MIP1apk35
0−3 4−5
p<1e−04 12
51020
50100
TNFApk35
0−3 4−5
p<1e−04
0.51.02.0
5.010.020.0
50.0100.0
VEGFpk35
0−3 4−5
p<1e−04
2e+05
3e+05
4e+05
5e+05
sgp130pk35
0−3 4−5
p<0.001100
200
500
sIL_1RIpk35
0−3 4−5
p<1e−045e+031e+042e+04
5e+041e+052e+05
5e+05
sIL_1RIIpk35
0−3 4−5
p<1e−055e+021e+032e+03
5e+031e+042e+04
5e+041e+052e+05
IL2RApk35
0−3 4−5
p<1e−07
2e+04
5e+04
1e+05
2e+05
sIL_6Rpk35
0−3 4−5
p<1e−05 50
100
200
500
1000
sRAGEpk35
0−3 4−5
p<0.001 1000
2000
5000
10000
20000
sTNFRIpk35
0−3 4−5
p<1e−051e+04
2e+04
5e+04
1e+05
2e+05
5e+05
sTNFRIIpk35
0−3 4−5
p<1e−08
1e−01
1e+00
1e+01
1e+02
1e+03
BFGFpk35
0−3 4−5
p=0.642 5e−011e+00
5e+001e+01
5e+011e+02
5e+021e+03
EGFpk35
0−3 4−5
p=0.942
50
100
200
Eotaxinpk35
0−3 4−5
p=0.041200
500
1000
2000
5000
10000
HGFpk35
0−3 4−5
p=0.0150
100
200
500
IL12pk35
0−3 4−5
p=0.117
2
5
10
20
50
100
IL13pk35
0−3 4−5
p=0.0235
10
50100
5001000
500010000
50000
IL15pk35
0−3 4−5
p=0.0062
5
10
20
50
100
IL17pk35
0−3 4−5
p=0.1 5e−011e+00
5e+001e+01
5e+011e+02
5e+021e+03
5e+03
IL1Bpk35
0−3 4−5
p=0.282 1
510
50100
5001000
IL2pk35
0−3 4−5
p=0.011
0.5
1.0
2.0
5.0
10.0
20.0
50.0
100.0
200.0
IL5pk35
0−3 4−5
p=0.017 0.5
1.0
2.0
5.0
10.0
20.0
50.0
100.0
200.0
IL7pk35
0−3 4−5
p=0.011 500
1000
2000
5000
10000
20000
50000
RANTESpk35
0−3 4−5
p=0.397 1
2
5
10
20
50
100
200
500
sCD30pk35
0−3 4−5
p=0.421 40000
60000
80000
100000
120000
140000
sEGFRpk35
0−3 4−5
p=0.731
200
300
400
500
600
700800900
sIL_4Rpk35
0−3 4−5
p=0.127
50
100
200
500
1000
2000
5000
10000
20000
sVEGFR1pk35
0−3 4−5
p=0.397
10000
20000
50000
sVEGFR2pk35
0−3 4−5
p=0.6992000
5000
10000
20000
sVEGFR3pk35
0−3 4−5
p=0.003
10
50100
5001000
500010000
GCSFpk35
0−3 4−5
p<0.001 125
102050
100200500
GMCSFpk35
0−3 4−5
p<0.0015
10
50100
5001000
500010000
IFGpk35
0−3 4−5
p<1e−05 20
50
100
200
IFNApk35
0−3 4−5
p<0.001 51020
50100200
50010002000
IL10pk35
0−3 4−5
p=0.001
50100200500
100020005000
100002000050000
IL1RApk35
0−3 4−5
p=0.0012
5
10
20
50
100
200
IL4pk35
0−3 4−5
p<0.001 1
100
10000
IL6pk35
0−3 4−5
p<1e−07 10
50100
5001000
500010000
IL8pk35
0−3 4−5
p<1e−07 5e+011e+02
5e+021e+03
5e+031e+04
5e+041e+05
IP10pk35
0−3 4−5
p<1e−06
5e+021e+032e+03
5e+031e+042e+04
5e+041e+052e+05
MCP1pk35
0−3 4−5
p<1e−05 10
50100
5001000
500010000
MIGpk35
0−3 4−5
p<1e−05 50100200
50010002000
5000
MIP1Bpk35
0−3 4−5
p<0.00120
50
100
200
500
1000
MIP1apk35
0−3 4−5
p<1e−04 12
51020
50100
TNFApk35
0−3 4−5
p<1e−04
0.51.02.0
5.010.020.0
50.0100.0
VEGFpk35
0−3 4−5
p<1e−04
2e+05
3e+05
4e+05
5e+05
sgp130pk35
0−3 4−5
p<0.001100
200
500
sIL_1RIpk35
0−3 4−5
p<1e−045e+031e+042e+04
5e+041e+052e+05
5e+05
sIL_1RIIpk35
0−3 4−5
p<1e−055e+021e+032e+03
5e+031e+042e+04
5e+041e+052e+05
IL2RApk35
0−3 4−5
p<1e−07
2e+04
5e+04
1e+05
2e+05
sIL_6Rpk35
0−3 4−5
p<1e−05 50
100
200
500
1000
sRAGEpk35
0−3 4−5
p<0.001 1000
2000
5000
10000
20000
sTNFRIpk35
0−3 4−5
p<1e−051e+04
2e+04
5e+04
1e+05
2e+05
5e+05
sTNFRIIpk35
0−3 4−5
p<1e−08
Activated T cells
Activated Macrophages
Chemotactic for Macrophages Tissue Damage and Inflammation
Negative Regulators
CRS = ?MAS/HLH
Cytokine level by CRS grade
pg/m
L
IFG
pk3
5
IL1
0p
k3
5
IL6
pk3
5
IL8
pk3
5
IP1
0p
k3
5
MC
P1
pk3
5
MIG
pk3
5
MIP
1B
pk3
5
IL2
RA
pk3
5
GM
CS
Fp
k3
5
TN
FA
pk3
5
IL1
2p
k3
5
IL1
3p
k3
5
IL1
7p
k3
5
IL1
Bp
k3
5
IL2
pk3
5
IL4
pk3
5
IL5
pk3
5
IL7
pk3
50
50000
100000
150000
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●
CRS grade 0−3
CRS grade 4−5
* Holm Significance
* * * * * * * * * * * *
Splenomegaly Gr 0-3: 0 of 25 pts Gr 4-5: 5 of 10 pts Hepatomegaly Gr 0-3: 0 of 25 pts Gr 4-5: 7 of 10 pts Fibrinogen <150mg/dl Gr 0-3: 2 of 15 pts Gr 4-5: 9 of 10 pts
Teachey, et.al., Cancer Discovery 2016
CRS Prediction
sgp130+IFNg+IL1RA IFNg + IL13 + MIP1a
Combined cohort Pediatric cohort
CRS: Different CARs
Turtle, et.al, JCI 2017
Davilla, et.al., Science Trans Med 2014
Tocilizumab and CRS after CTL019
Different drugs to block cytokines
• IL-1a: Anakinra
• IL2R (CD25): Basiliximab
• IL6: Siltuximab
• IL6R: Tocilizumab
• TNFa: Etanercept, Infliximab
• MCP1, MIP1b, sgp130, IFNg: Clinical trials
• Jak/Stat: Ruxolitinib, Tofacitinib, others
• BTK: Ibrutinib
• Corticosteroids
MCL NSG model of CRS
Ruella, et. al. Leukemia 2017
Immunotherapy for B-ALL
B-ALL
• Naked Monoclonals • Rituximab
• Epratuzumab
• Conjugated Monoclonals • Inotuzumab
• Moxetumomab
• BiTEs • Blinatumomab
• CARs
T-ALL
• UM…..
CD5 CAR T cells
Mamonkin, et al, Blood 2015 CD5 quickly down-regulated in normal T cells
Daratumumab
0
25000
50000
75000
T-A
LL
cells x
10e6 in
blo
od p= 0.0112
0
50
100
150
T-A
LL
cells x
10e6 in
sp
leen p = 0.0003
Peripheral blood blasts Splenic blasts
Karen Bride
Thanks
• Stephan Grupp • David Barrett • Stephen Hunger • Jim Whitlock • Mignon Loh • Elizabeth Raetz • William Carroll • Mini Devidas • Terzah Horton • Michelle Hermiston • Brent Wood • Patrick Zweidler-Mckay • Karen Rabin • Richard Lock • Charles Mullighan • Cheryl Willman • Richard Harvey • Peter Adamson • Malcolm Smith • Stuart Winter • Kim Dunsmore • John K. Choi • Jos Melenhorst • Simon Lacey • Pam Shaw • Sara Tasian
Teachey Lab • Shannon Maude • Tiffaney Vincent • Junior Hall • Theresa Ryan • Karen Bride • Tori Fuller • Abanoub Gab