Download 1Department of Hematology, 2Department of Dermatology, Leiden

LOSS OF PIGH EXPRESSION FREQUENTLY RESULTS IN A GPI-NEGATIVE SUBCLONE
LACKING CD52 MEMBRANE EXPRESSION, CONFERRING ALEMTUZUMAB RESISTANCE
TO B CELL ACUTE LYMPHOBLASTIC LEUKEMIA
 In a previous study (Nijmeijer et al, 2010), outgrowth of CD52
negative escape variants after alemtuzumab treatment was
shown in a mouse model engrafted with human B-ALL. These
variants expressed normal CD52 mRNA levels, but lacked
CD52 membrane expression due to loss of GPI anchor
expression
 The aim of the current study was to further unravel the
mechanism underlying the loss of CD52/GPI anchor
expression in adult B-ALL
GPIneg and GPIpos B-ALL cultures
Cell cultures generated from two B-ALL patient samples were
phenotypically highly comparable to the original patient sample
Patient
VBK
Patient
MHX
CD52
PIGH
Leiden BVALL
Leiden HPALL
PBGD
•
•
•
Frequency of CD52/GPI anchor negative cells was analyzed in blood and
bone marrow samples from treatment naïve B-ALL, chronic lymphocytic
leukemia (CLL), mantle cell lymphoma (MCL), and hairy cell leukemia
(HCL) patients and healthy donors by flow cytometry with a fluorescently
labeled GPI-specific aerolysin FLAER and CD52 and CD19
counterstaining (detection limit 0.01%)
[email protected]
CD52/GPI negative cell populations were present in 10 out of 18 B-ALL
samples, but not in other B cell malignancies such as CLL (n=5), MCL
(n=5), HCL (n=6), or in healthy donors (n=5)
KYE9115
GPI positive and GPI negative B-ALL populations were purified by
fluorescence activated cell sorting (FACS) and from these populations
mRNA was isolated, cDNA synthesized, and expression of the 26 genes
that comprise the GPI anchor biosynthesis pathway was analyzed by
conventional PCR using specific primers and visualized by gel
electrophoresis
Two different B-ALL samples were cultured, and subsequently divided
using FACS into pure GPI negative and GPI positive subcultures to
perform retroviral transduction studies with constructs encoding PIGH,
PIGA or an empty vector
Flaer (GPI anchor)
GPI negative and positive subcultures were used to compare the
promoter CpG methylation by bisulfite sequencing. GPI negative
subculture was used for a demethylation assay using 5-Azacitidine in a
14 day culturing regimen
wtPIGH restores GPI expression
Restored GPI anchor expression
in the GPI negative B-ALL
subcultures upon retroviral
transduction with the PIGH
construct, but not PIGA or empty
vector
NGFR
(marker)
T2A
PIGH, PIGA, or
empty
Construct used for transductions encodes
for a markergene coupled to PIGH or PIGA
via the autocleaving T2A peptide
Flow cytometric analysis of the transduced
GPI negative cell subcultures
HP-ALL
GPIneg
BV-ALL
GPIneg
Example of a flow cytometric analysis of
CD52/GPI-anchor negative cells within the
CD19+ B cell compartment in a B-ALL sample
Sample
nr
Patient ID
Sample
type**
# B-cells GPI negative
screened (%)
1
HRG7504
PB
385,018
4.98
2
MEP6179
PB
274,959
2.14
3
KYE9115
PB
36,783
0.91
4
PGZ0747
BM
769,559
0.65
5
JHO8262
BM
25,465
0.28
6
EBG6102
BM
938,627
0.18
7
HBZ8516
PB
31,171
0.11
8
MHJ8932
PB
331,127
0.04
9
AOR6995
BM
628,534
0.03
10
HBP6230
PB
795,546
0.03
11
LOW9484
BM
40,682
0.00
12
LMJ5903
PB
226,151
0.00
13
ASC5968
PB
103,698
0.00
14
CPH6760
BM
356,142
0.00
15
JWK9219
BM
45,701
0.00
16
MGR9751
PB
21,305
0.00
17
MRQ3306
PB
514,778
0.00
18
PMX8367
BM
674.114
0.00
** BM, Bone marrow; PB, Peripheral blood
Detailed analysis of the GPI negative and GPI positive ALL subcultures
showed:
• Loss of PIGH mRNA was not caused by genetic aberrations
• Higher level of promoter methylation in GPI negative subcultures
• Demethylation using azacytidine resulted in GPI anchor reexpression
Promoter methylation assay by bisulfite sequencing
CpG 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
TSS
HP GPI+ L L L L L L L L L L L L L L L L
HP GPI- L L L M M L M L L L L L L L L L
+ PIGA
MOCK
25
BV GPI+ L L L L L L L L L L L L L L L L
BV GPI- L M L M M M M L L L M M L L L L
20
Healthy
B-cells U U U U U U U U U U U U U U U U
Demethylation assay
U Unmethylated
15
10
BV 0.5nM azacytidine
5
0
Flaer
L Partially methylated
HP 0.5nM azacytidine
HP 0nM azacytidine
Highly or fully
M
methylated
No PIGH expression in
cells
Recurrent loss of PIGH mRNA expression, but of none of the other genes
involved in GPI anchor biosynthesis, in purified GPI negative B-ALL cells
DPM1
DPM2
DPM3
GPAA1
PIGA
PIGB
PIGC
PIGF
PIGH
PIGK
PIGL
PIGM
PIGN
PIGO
PIGP
PIGQ
PIGS
PIGT
PIGU
PIGV
PIGW
PIGX
PIGY
PIGZ
PGAP1
PGAP2
PGAP3
SL15
GPI: + -- + -EBG 6102
KYE 9115
HRG 7504
MEP 6179
PGZ 0747
JHO 8262
AOR 6995
Example of a B-ALL sample screened for
mRNA expression for the 26 GPI genes (n=3)
Epigenetic downregulation of PIGH
+ PIGH
GPI positive and negative subcultures
were generated from the Leiden-ALL
cell cultures by FACS. mRNA
expression analysis showed that the
GPI negative subcultures also lack
expression of PIGH.
Flaer
B-ALL cells at diagnosis
neg
GPI
DOI: 10.3252/pso.eu.21EHA.2016
 In contrast, alemtuzumab which targets the glycophosphatidylinositol (GPI) anchored CD52 protein has shown modest
clinical efficacy, which could be caused by the outgrowth of
CD52 negative B-ALL escape variants
neg
GPI
CD52
 The development of novel antibody based therapies targeting
CD19 or CD20 antigens has improved treatment outcomes in
B cell acute lymphoblastic leukemia (B-ALL)
Inge
of Dermatology, Leiden University Medical Center, The Netherlands.
Material and methods
•
Constantijn J.M.
1
Jedema
PIGH mRNA expression analysis in B-ALL
samples, with GAPDH as a loading control
Conclusions
In the majority of B-ALL patients CD52/GPI negative cell
populations were present at diagnosis
These cells lost CD52/GPI expression due to loss of PIGH
mRNA expression, a key component in GPI anchor synthesis
The CD52/GPI negative phenotype of B-ALL cell subcultures
was stable, but could be corrected by enforced PIGH
expression by retroviral transduction
Loss of PIGH mRNA was not due to DNA mutations, but
possibly due to epigenetic down regulation by methylation of
the promoter region
Combining epigenetic modulatory drugs with alemtuzumab
might be a promising therapeutic strategy to prevent outgrowth
of CD52/GPI negative escape variants in B-ALL
Inge JEDEMA
Background
J.H.Frederik
1
Halkes ,
Poster presented
at EHA 21 on:
10th June 2016
of Hematology,
2Department
Maarten H.
1
Falkenburg ,
Acute lymphoblastic leukemia - Biology 2
1Department
Wim H.
2
Vermeer ,
BV 0nM azacytidine
0
5
10
15
162-P
H.M. Esther van
2
Zoutman ,
% GPI positive cells
Kevin
1
Egmond ,
NGFR (marker gene)
Floris C
1
Rijs ,
Time (days)
There are no conflicts of interest to disclose
21eha
1
Loeff ,