Multiparameter Flow Cytometric Analysis Reveals Aberrant Activation of Signaling Molecules in Define

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Multiparameter Flow Cytometric Analysis Reveals
Aberrant Activation of Signaling Molecules in
Defined Populations of Kras Mutant Bone Marrow
Van Meter MEM1, Diaz-Flores E1, Archard JA1,
Kotecha N2, Sabnis AJ1, Irish JM2, Nolan GP2,
Shannon KM1, Braun BS1 1University of
California, San Francisco 2Stanford University
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Overview of Ras Signaling

• RAS Mutations
• encode Ras proteins that accumulate in the
  GTP-bound conformation
• found in 30 of human cancers
• Lung, colorectal, pancreatic
• Myeloid Leukemias AML, CMML, JMML

Ras-GDP
GAPs
GEFs
Ras-GTP
Effector Pathways
Proliferation
Survival
Differentiation
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Ras-GDP
SOS
Grb2
Shc
GAPs
Gab2
JAK2
SHP-2
STAT5
Ras-GTP
PI3K
RAF
Ral-GDS
Akt
MEK
Ral-A
mTor
ERK
S6
Survival, Proliferation
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Ras-GDP
SOS
Grb2
Shc
GAPs
Gab2
JAK2
SHP-2
STAT5
Ras-GTP
PI3K
RAF
Ral-GDS
Akt
MEK
CI-1040
Ral-A
mTor
ERK
S6
Survival, Proliferation
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Kras Conditional Mutant Allele
KrasWT allele KrasG12D-LSL allele
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Mx1-Cre
STOP
1
1
(inducible)
Fatal Myeloid Disease Resembling Human JMML,CMML
loxP
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Advantages of FACS Analysis
Krutzik et al., Clinical Immunol 2004
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Gating Strategy

• c-kit/lin-/lo population
• 1-2 of bone marrow cells
• contains most clonogenic cells

c-kit
Lineage Markers
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Unique Signaling Profile of Progenitors
pERK
Whole Marrow
c-kit/lin- Progenitors
GM-CSF
-
wild type

-
KrasG12D

0
1
2
3
4
0
1
2
3
4
10
10
10
10
10
10
10
10
10
10
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Differential Responses of Progenitors to GM-CSF
and Stem Cell Factor (SCF)
pERK
GM-CSF
SCF
-
wild type
pERK

-
KrasG12D

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CI-1040 Inhibits ERK Activation in vivo
CI-1040 (MEK inhibitor)
pERK
Vehicle
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Summary

• FACS analysis gives results that are similar to
  Western blotting in cultured cells
• In diverse primary cell populations, FACS can
  detect events in rare, pathogenic populations
• in Kras mutant mice, ERK signaling is enhanced
  with GM-CSF, suppressed with SCF
• Kras mutant mice also have aberrant activation of
  pS6, pSTAT5
• responses to targeted therapy can be monitored

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Acknowledgements

• Ernesto Diaz-Flores
• Joehleen Archard
• Ben Braun
• Kevin Shannon
• Everyone in the Shannon lab!
• Genentech Foundation

Research Support