Quantitative single cell determination of ERK phosphorylation and regulation
in relapsed and refractory primary acute myeloid leukemia
Running Title: p-ERK expression in refractory/relapsed AML samples
Maria R. Ricciardi1, Teresa McQueen1, David Chism1, Michele Milella1,
Eric Kaldjian2, Judith Sebolt-Leopold2, Marina Konopleva1, Michael Andreeff1.
1
Departments of Blood and Marrow Transplantation, Section of Molecular Hematology and
Therapy, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 USA;
2
Cancer Molecular Sciences Department, Pfizer Global Research & Development,
Ann Arbor, Michigan 48105, USA
Keywords: ERK, MEK/ERK inhibitors, acute myelogenous leukemia, hematopoietic progenitor
cells, G-CSF
Supported in part by grants from the National Cancer Institute (PO1 CA55164 and CA16672) (to
M.A.).
Correspondence should be addressed to:
Michael Andreeff, M.D., Ph.D.
Section of Molecular Hematology and Therapy
Department of Blood and Marrow Transplantation
The University of Texas, M. D. Anderson Cancer Center
1400 Holcombe Blvd, Unit 448, Houston, Texas 77030
Phone: 713-792-7260, Fax: 713-794-4747, E-mail: mandreef@mdanderson.org
ON-LINE SUPPLEMENTARY INFORMATIONS
SUPPLEMENTARY RESULTS
Time dependent analysis of p-ERK levels in whole blood samples from AML patients.
Signal transduction pathways are typically regulated by kinases and phosphatases, which
modulate the phosphorylation state of various phospho-proteins. We examined the effects of
prolonged storage on levels of p-ERK in whole blood samples from AML patients. The
samples were stored for up to 72 hr at RT. Aliquots from these samples were fixed every 24 hr
to measure p-ERK levels by FCM. The time-course experiments revealed that initial levels of
p-ERK declined in 3 of the 5 samples examined. As shown in Fig. 1a, p-ERK levels decreased
after 24 hr in 1 out of 5 samples and after 48 hr in 3 samples. In one sample, there was a
strong increase in the mean fluorescence intensity of p-ERK after 24 hr, which was followed
by a dramatic decline after 48 hr. Two of the samples with low constitutive p-ERK expression
remained unchanged for up of 72 hr. Furthermore, after stimulation with PMA there was an
approximately 10-fold increase in p-ERK level as evidenced by the increased mean
fluorescence intensity in all of the patient samples examined (Fig. 1b). These levels also
diminished over time in much the same fashion observed in the unstimulated samples.
Ficoll separation enhances fluorophore labeling
We performed FCM analysis on different cell separation systems to establish the most
sensitive method to detect p-ERK levels. We measured p-ERK levels in whole blood alone or
after processing with rapid hypotonic lysis or Ficoll separation (Fig. 2). The hypotonic lysis
was performed incubating 10 ml of whole blood for 5 minutes with 40 ml of not-fixing lysis
2
solution containing 1.5 M NH4Cl, 0.1 M KHCO3, 1 mM EDTA disodium salt. The major
problem using whole blood for MAP kinase activation analysis lies in the requirement to
preserve the phosphorylation status of ERK while eliminating red blood cells. Fixation in 2%
formaldehyde stabilizes p-ERK levels but makes red cells resistant to lysis. Hypotonic lysis of
red cells following PMA stimulation before formaldehyde fixation gave acceptable results.
Nevertheless sample preparation with Ficoll separation was superior to the others preparation
systems in detecting p-ERK expression for its ability to show the greatest difference in PE
fluorescence intensity compared to controlss. PE-labeled secondary antibodies resulted in
improved detection of p-ERK levels compared to FITC-labeled antibodies. This was likely
due to phycoerythrin’s higher quantum efficiency as compared to FITC (data not shown).
Optimization of intracellular staining methods in combination with surface staining
One of the most important advantages of using FCM rather than Western Blot analysis to
examine cellular proteins is the opportunity to readily combine intracellular with surface
staining, especially in heterogeneous populations such as PB or BM. To test the possibility of
combining indirect conjugated intracellular phospho-specific mAb with direct conjugated
surface antigen-specific mAbs, unstimulated and PMA-stimulated MNC cells from normal PB
were labeled with either anti-CD3-APC or anti-CD14-FITC. These molecules are both bright,
photostable and suitable for flow cytometry. In addition, APC can be used without
compensation due to the large Stoke’s shift in its emission spectrum. As shown in Fig. 3,
unstimulated CD3+ lymphocytes did not express p-ERK, whereas monocytes showed
constitutive ERK activation. We were unable to perform simultaneous staining of p-ERK-PE
3
with anti-CD34-APC and anti-CD38-FITC in AML samples because of the cross-reactivity of
CD38 Ab with the PE-labeled secondary Ab (data not shown).
DISCUSSION OF SUPPLEMENTARY RESULTS
In order to address some methodological issues and ascertain whether a FCM assay may
be employed for clinical investigations, preliminary experiments were performed on patients
not included in the pharmacological study. First, because samples might be collected
randomly, we evaluated the stability of constitutive p-ERK levels in whole blood samples
from AML patients before fixation and permeabilization. We observed that prolonged storage
considerably decreased signal intensity of p-ERK and therefore concluded that the time
interval between sample harvesting and processing is crucial in order for analysis of the
phosphorylation status of proteins. In a previous report, Chow et al.(13) reported that specific
and non-specific antibody labeling intensity for PMA-stimulated and control samples were
similar when examined on fresh samples or after up to 48 h storage at room temperature.
However, they studied normal resting T-lymphocytes that did not express constitutively
activated ERK. In this study, we evaluated AML samples that exhibited variable expression of
p-ERK. Because our goal was to detect differences in p-ERK levels, it was important to
identify any changes related to sample handling.
The ability to analyze signaling pathways can be compromised by the cell separation
technique used. We found it to be important to monitor the response to PMA stimulation and
p-ERK inhibition in undiluted whole blood, because of the possible selective loss of cellular
subpopulations or of cellular targets during cell separation. Although it was reported that
hypotonic lysis of red cells following PMA stimulation yielded satisfactory results, we prefer
4
Ficoll separation because it yielded the highest fluorescence ratios. We also tested the relative
efficacy of two different fluorophores as a variable in the labeling of phosphospecific mAb
(data not shown). The higher quantum efficiency of PE compared to that of FITC allows us to
better detect low levels of p-ERK and to better quantify small changes induced by CI-1040.
The conditions described here optimized the quantitation of p-ERK in primary patient samples
by FCM.
5
Supplementary Table 1: Patients characteristics.
No. Gender Blast% PB
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
M
M
M
M
F
F
M
F
F
M
M
F
M
F
M
F
M
F
M
F
M
F
M
M
M
F
M
M
F
F
M
M
M
M
F
M
M
F
F
M
M
M
25
22
75
29
60
65
87.3
9.3
20.5
96
68
88
87
13
20
67
81
76
78
46
85
66
91
97
45
90
12.6
8
80
16
15
98
99
50
65.2
3
5
97
96
85
70
4.9
Blast% BM
Status
Treatment at time of harvest
Kariotype
46
85.5
80
80
ND
ND
88
61
77
82
90
94
94
50
51
80
91
80
ND
87
75
ND
ND
53
ND
79
59
32
92
67
53
91.4
84
64
83
62
93
92
ND
61.4
ND
67
At diagnosis
At diagnosis
At diagnosis
At diagnosis
At diagnosis
At diagnosis
At diagnosis
At diagnosis
At diagnosis
At diagnosis
At diagnosis
At diagnosis
At diagnosis
At diagnosis
At diagnosis
At diagnosis
Relapse
Relapse
Relapse
Relapse
Relapse
Relapse
Relapse
Relapse
Relapse
Relapse
Relapse
Relapse
Relapse
Relapse
Relapse
Refactory
Refactory
Refactory
Refactory
Refactory
Refactory
Refactory
Rel-Ref
Rel-Ref
Rel-Ref
Rel-Ref
Ida/Ara-C/IL-11
Untreated
Hydrea
Untreated
Untreated
Untreated
Untreated
Untreated
Untreated
Untreated
Untreated
Untreated
Untreated
Untreated
Untreated
Untreated
Clofarabine
Mylotarg
Topotecan/Ara-C/Fludarabine
Idarubicin/Ara-C
No treatment
Tripaline
Investigational Drug (CEP 701)
Hydrea
No treatment
FLT-3 Inhibitor
Idarubicin/Ara-C
SAHA
No treatment
No treatment
No treatment
Hydrea
No treatment
Idarubicin/Ara-C
No treatment
Fludarabine/Ara-C
No treatment
Hydrea
Hydrea/ Topotecan/Ara-C
Clofarabine
Hydrea
No treatment
46 xy, inv 16, (p13.2q22) [19]
none done
46,XY[18]
46,XY[20]
none done
46,XX[20]
46,XY[20]
46,XX,t(9;11)(p22;q23)[20]
46,XX[20]
46,XY[20]
46,XY[20]
46 XX, [20]
45 x,-y, t(9,11)
46 xx t(1,3)
45 x,-y
inv 16
46,XY[20] on 5/28/02
none done
complex
complex
47,XY,del(9)(q22),+21[20]
46,XY,t(11;19)(q13;q13.3)[20] on10/15/02
none done
46,XY,-2,-17,+2mar[2] on07/23/02
none done
complex
complex
46 xy
complex
none done
46,XY,del(5)(q31q35),del(7)(q21),del(12)(p11.2)[15]
complex
46,XY,-2,-17,+2mar[2]
45,XY,-7[20]
complex
complex
del (9)(q22)
46 xx t(1,3)
46,XX[19]
complex
46,XY,del(16)(q22)[20]
46,XY[18] on 10/28/02
6
LEGENDS TO SUPPLEMENTAL FIGURES
Figure 1. Changes of p-ERK mean fluorescence intensity (MFI) in whole blood samples
from AML patients stored at RT for 3 days. (a) Unstimulated or (b) PMA-stimulated
aliquots from AML samples were fixed with formaldehyde and permeabilized with methanol
for flow cytometric analysis each 24 hr for 3 days. Samples collected were then
simultaneously stained for p-ERK as described in Materials and Methods. Constitutive and
PMA-induced p-ERK levels gradually decrease in a time-dependent fashion.
Figure 2. Comparison of different separation systems to optimize p-ERK expression
evaluation in primary cells. Flow cytometric analysis of p-ERK levels was performed in
undiluted whole blood, hypotonic lysed whole blood and after Ficoll separation. Sample
preparation with Ficoll separation was superior to whole blood after rapid hypotonic lysis for
the detection of p-ERK expression.
Figure 3. Dual parameter of p-ERK/surface marker flow cytometry. MNC from normal
peripheral blood were stained with anti-CD3 and anti-CD14 antibodies for 30 min on ice,
washed with PBS, fixed and stained for p-ERK as described in Materials and Methods. Left
panels show p-ERK levels in CD3+ T-lymphocytes, right panels in monocytes in unstimulated
(top panels) and PMA-stimulated (bottom panels) cells, respectively.
7