Title: BDH2 is a poor novel independent maker in CN-AML
1BDH2 is a poor novel independent maker in
CN-AML
- Wen-Chi, Yang, MD, PhD
- Hematology/Medical Oncology
- Yuans General Hospital, Taiwan
2Cytogenetic normal AML
- approximately 40 to 50 of AML patients are
considered to be cytogenetically normal - molecular analysis of markers that have been
incorporated into both the WHO and ELN
classifications (such as NPM1, FLT3, and CEBPA)
is now routine - other mutated genes (such as WT1, IDH1/IDH2,
TET2, RUNX1, and MLL) or aberrantly expressed
genes (such as BAALC, ERG, EVI1, and miR-181a)
are likely to be useful for defining molecular
risk in CN-AML Class I, Class II, Class III, etc
Byrd, et al. Blood 20021004325-4336. Gaidzik,
et al. J Clin Oncol 2011, 291364-1372. Mithat
Gönen, et al. NEJM2012, 3661079-1089.
3Iron utility and mammalian cells
- The acquisition of iron by cells is critical for
survival, growth, and differentiation. -
Pantopoulos, et al.
Biochemistry 2012 51(29)5705-24 - Mammalian cells and tissues contain low molecular
weight siderophores that bind iron with high
affinity.
Fernandes-Pol et al. Cell 1978 14(3)
489-99 - Lipocalin24p3 can bind small molecular weight
ligands involved in multiple biological process
including apoptosis, innate immunity and renal
development - Devireddy et al. Cell 2005
1231293-305 Li et al. Cytokine 2011 56435-41
Flo et al. Nature 2004 432917-21.
4BDH2
- Siderophores are best known small iron binding
molecular that facilitate microbial iron
transport. - A member of short chain dehydrogenase family
reductases, BDH2, catalyzes a rate-limiting step
biogenesis of the mammalian siderophore. - Mitochondrial metabolism
- Depletion of BDH2 results in abnormal
accumulation of cellular iron and mitochondrial
iron deficiency.
Devireddy et al, Cell 2010 1411006-17
Liu et al , J mol Med 2012 901209-21.
5BDH 2
Kunde Guo et al 2006
6Metabolic pathway
7BDH2 is required for 24p3-mediated iron transport
Devireddy et al. Cell 2010 1411006-17
8Siderophore protects cells from oxidative stress
Devireddy et al. Cell 2010 1411006-17
CDDHCF-DA nonfluorescent probe CDCF-DA highly
fluorescent
9Iron regulation of siderophore controls
mitochondrial iron
Zhuomin Liu et al J Mol Med 2012901209-21
10Iron, BDH2 and BDH2 IRE
J Mol Med 2012901209-21
11- If BDH2 expression related to prognosis of
CN-AML? - If the presentation of genetic alternations, like
NPM1 mutation, FLT3-ITD, FLT3-TKD, CEBPA, IDH1/2,
and DNMT3A would be difference in different BDH2
expression group?
12Enrolled patients
- Enroll 113 newly diagnosed CN-AML patients
2000.2 2012.2 - 86 patients received conventional induction C/T
with I3A7 (followed by 70 I3A7 if not reach CR
at 7th14th days) - Twenty two patients with secondary CR, one with
first CR and five with refractory disease
received allogenetic PBSCT - 43 patient with normal bone marrow (most of them
are lymphoma without bone marrow involvement)
good risk (11 patients with AML-ETO () 3
patients with Inv (16)) and 25 poor risk patients
with multiple chromosome abnormality
13Methods-2
- Q-RT-PCR to analysis the mRNA expression in
patients bone marrow sample - Statistics two sample t test to analysis BDH2
expression difference ROC curve to estimate the
cut off point of BDH2 to predict death Two
sample t-tests and X2 square tests were used to
analyze the differences in age, sex, peripheral
white blood cells (WBC), hemoglobin (Hb),
platelet and blasts count, CD34 and blasts
percentage in bone marrow, percentage of
FLT3-ITD, FLT3-TKD, NPM1 mutations and CEBPA
mutation in the LCN2 low and high expression
groups Kaplan-Meier analysis was performed to
estimate the differences in overall survival
between patients with different BDH2 levels and
NPM1, FLT3, and CEBPA mutations and the
disease-free survival and relapse-free survival
between the higher and lower LCN2 expression
groups. Univariate and multivariate analysis.
14BDH2 mRNA expression
Yang et al. J Biomedical Sci 2013 2058
15Yang et al. J Biomedical Sci 2013 2058
16Difference of genetic alterations
Yang et al. J Biomedical Sci 2013 2058
17Yang et al. J Biomedical Sci 2013 2058
18Yang et al. J Biomedical Sci 2013 2058
19Yang et al. J Biomedical Sci 2013 2058
20Survival between BDH2 expression
Overall Survival
Yang et al. J Biomedical Sci 2013 2058
21Survival between 4 groups
Overall Survival
Yang et al. J Biomedical Sci 2013 2058
22BDH2 expression vs PBSCT
23How does it happen?
- Increase of BDH2 expression may cause leukemia
cells resistant to stress, like ROS and
chemotherapy, induced death, that is related to
poor response rate.
24Methods
- Leukemia cell lines THP1 (human AML, M4), and
HL60 - RNA interference-mediated BDH2-KD by shRNA.
- Annexin V Apoptosis assay
- Induce apoptosis by H2O2 and DFO
- Flowcytometry for caspase 3 activity Western
blot for apoptosis related protein analysis. - Mitochondrial membrane potential test
25BDH2 mRNA
shRNA-BDH2 (retrovirus)
THP1 HL60
Western Blot
Selected by puromycin
26BDH2 mRNA expression
Yang et al. J Biomedical Sci 2013 2058
27H2O2 50uM 2hours 200x
Yang et al. J Biomedical Sci 2013 2058
28Apoptosis assay with 50uM H2O2, 2hours in HL60
Yang et al. J Biomedical Sci 2013 2058
29(No Transcript)
30Caspase 3 cleavage
HL60
shRNA-BDH2-2 HL60
shRNA-BDH2-3 HL60
shRNAc HL60
31Apoptosis related protein expression
Yang et al. J Biomedical Sci 2013 2058
32The mitochondrial membrane potential change
Yang et al. J Biomedical Sci 2013 2058
33(No Transcript)
34Others?
- If higher BDH2 expression related to poor
differentiation and higher proliferation rate ?
35Methods
- Growth rate analysis in BDH2-KD THP1, control
empty vector and native THP1 cells. - 10-7M Vit D3 culture 72 hours for differentiation
- Surface markers CD11b, CD14, CD15, CD16, CD64
analysis by flow-cytometry - Cell cycle arrest by starving with serum free
medium - Cell cycle analysis by flowcytometry
36Growth rate
Unpublished data
37Differentiation
Unpublished data
38Differentiation
39Cell cycle retardantdelay into S phase
Unpublished data
40Day 3
41Day 4
42ROS
BDH2
survivin
43Conclusion
- Higher BDH2 expression is a poor prognostic
predictor for CN-AML, with lower CR rate and
shorter OS. - BDH2 works as a anti-apoptotic factor, mediated
by survivin. - BDH2 knock down THP1 cells showed higher
differentiation rate and lower proliferation rate
with cell cycle retardant. - Higher BDH2 gt chemo-resistant gt consider
allogenic hematological stem cell transplantation
44Thank you for your attention !!
45The mechanism of BDH2 increasing in CN-AML
patientspreliminary data
- DNA mutation or polymorphism?
- gt no DNA mutation or SNP in 4 high BDH2
expression and 4 low BDH2 expression patients. - Promoter problem?
- gt -782bp before CDS with one T insertion (one
high BDH2 expression patient) - -311bp before CDS G to A
- Epigenetic problem?
46Overall survival
Leukemia free survival