Education
Clinical Care
Research
Molecular markers in NKTL
A/Prof Chng Wee Joo
Department of Haematology-Oncology
National...
Extranodal nasal-type Natural
Killer/T-cell lymphoma (NKTL)
• Distinct clinicopathologic entity most commonly
affecting As...
Clinical Spectrum of Extra-nodal Natural Killer / T-cell Lymphoma
Kwong YL. Leukemia 2005; 19:2186
EBV infection
LMP-1 or other
factors
MYC
activation
P53 mutations
Induce Survivin
Proliferation Anti-apoptotic
Regulate
Pr...
microRNAs: small molecules with a big impact
• MicroRNAs (miRNA) are a class of small,
non-coding RNAs (~20 nts long) that...
• Samples:
– 30 cases of NKTL FFPE
– 6 NK cell lines (KHYG-1, NK-92, HANK-1, SNT-8, SNK-6 and
NK-YS)
– 3 paired samples of...
miRNA are predominantly downregulated
in NKTL
• miRNA deregulation in NKTL
– In both NK cell lines and FFPE NKTL samples
c...
quantitative-PCR validation of selected miRNAs
consistent with MEP data
Upregulated
miRNA
Downregulated miRNA
miR-155
Norm...
Selection of high probability predicted target
genes of deregulated miRNA
Intersect with our previous GEP data to
narrow d...
Validation of miRNA targets: Re-expression
of miRNA using lentiviral vector
• Target genes of miR-101, miR-26a, and miR-26...
mRNA expression changes upon overexpression of miRNAs by Lentiviral transduction
mir-26b
precursor
control control control...
Immunohistochemistry reveals overexpression
of target proteins of suppressed miRNAs in NKTL
• IHC for selected target prot...
Mechanism of miRNA deregulation in
NKTL
Role of MYC
•MYC is known to cause extensive repression of miRNA
expression (Chang...
EZH2 overexpression in majority of NKTL.
NKTLcelllines
NormalNKcells
NKTL
Normaltissue
681012
P=0.003 P=0.002
A
B
Inhibition of EZH2 with DZNep induced cell growth inhibition and
apoptosis in NK malignant cells.
EBV infection
LMP-1 or other
factors
MYC
activation
NF-KB
activation
P53 mutations
P53
Deregulation
Induce
Survivin
Prolif...
Future Studies
• MYC-EZH2 in about 50%, JAK3 mutation in
about 35%
– Do they signify 2 molecular groups of NKTL i.e are th...
Acknowledgement
YAN Junli Viknes Koh Tze Loong
Ng Siok Bian
Acknowledgement and thanks
National University Health
System, Singapore
Jim Liang-Seah Tay
Baohong Lin
Chonglei Bi
Joy Tan...
 MOLECULAR MARKERS IN NKTL
 MOLECULAR MARKERS IN NKTL
 MOLECULAR MARKERS IN NKTL
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MOLECULAR MARKERS IN NKTL

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  • Based on our findings, we propose a model of NKTL pathogenesis involving the activation of Myc and NF-KB pathways, possibly driven by the EBV LMP-1 protein ( Figure 6 ). In addition, the tumor acquires p53 mutations that lead to deregulated p53 function. The cumulative consequence of these oncogenic pathways is the up-regulation of survivin
  • MiRNAs are ~20-nucleotide, highly conserved RNAs with a primary role in post-transcriptional silencing through base pairing to partially or fully complementary sites in the 3’UTR of protein-coding transcripts. They regulate most cellular processes including development, cell differentiation, proliferation, stress response and apoptosis. 10 New miRNAs are continuously being identified. To date, more than 1500 human mature miRNAs are registered in miRBase, a centralized database of published miRNA sequences and annotation. 11
  • Figure 1. EZH2 is over-expressed in NKTL. (A) EZH2 mRNA levels are elevated in NKTL and cell lines. Expression score for EZH2 mRNAs in NKTL GEP dataset. (B) EZH2 protein expressions in NKTL FFPE samples were compared with that in respective normal FFPE tissue controls.
  • Figure 6. DZNep inhibits cell growth and induces apoptosis in NKTL tumor cells. (A) Western blot analysis of NK tumor cells exposed to increasing concentrations of DZnep showed a dose dependent decrease in EZH2 protein, decrease in Cyclin D1 protein and PARP cleavage in response to DZNep treatment. (B) Reduction of CCND1 mRNAs in DZnep treated cells. (C) Apoptosis assay showing cell death response of KHYG and NKYS cells to DZNep.D) Quantification of cell viability in KHYG and NKYS cells treated with DZnep. (E) The rescued effects by EZH2 or EZH2∆ overexpression on DZNep- induced cell growth inhibition.
  • Based on our findings, we propose a model of NKTL pathogenesis involving the activation of Myc and NF-KB pathways, possibly driven by the EBV LMP-1 protein ( Figure 6 ). In addition, the tumor acquires p53 mutations that lead to deregulated p53 function. The cumulative consequence of these oncogenic pathways is the up-regulation of survivin
  • MOLECULAR MARKERS IN NKTL

    1. 1. Education Clinical Care Research Molecular markers in NKTL A/Prof Chng Wee Joo Department of Haematology-Oncology National Cancer Institute of Singapore National University Health System Senior Principle Investigator Cancer Science Institute, Singapore National University of Singapore
    2. 2. Extranodal nasal-type Natural Killer/T-cell lymphoma (NKTL) • Distinct clinicopathologic entity most commonly affecting Asians and Central and South Americans • characterized by a clonal proliferation of NK or T cells with a cytotoxic phenotype. • There is a strong association with Epstein-Barr Virus (EBV), which manifests a type II latency – expression of LMP-1 and EBNA-1, – absence of EBNA-2. • EBV detected in the neoplastic cells in a clonal episomal form, supporting the role of the virus in tumor pathogenesis
    3. 3. Clinical Spectrum of Extra-nodal Natural Killer / T-cell Lymphoma Kwong YL. Leukemia 2005; 19:2186
    4. 4. EBV infection LMP-1 or other factors MYC activation P53 mutations Induce Survivin Proliferation Anti-apoptotic Regulate Proposed model of NKTL pathogenesis NF-KB activation p53 Deregulation Ng SB, et al. J Pathol. 2011 Mar;223(4):496-51
    5. 5. microRNAs: small molecules with a big impact • MicroRNAs (miRNA) are a class of small, non-coding RNAs (~20 nts long) that repress gene expression (in most cases) – Degrading or repressing mRNAs – Important class of gene regulators that controls most biological processes. – Latest in human: 1527 precursors, 1921 mature miRNAs (miRbase 19) • Each miRNA can have hundreds of different conserved or nonconserved targets
    6. 6. • Samples: – 30 cases of NKTL FFPE – 6 NK cell lines (KHYG-1, NK-92, HANK-1, SNT-8, SNK-6 and NK-YS) – 3 paired samples of normal NK cells (unstimulated and stimulated) isolated from buffy coat packs of whole blood samples from blood bank – 2 cases each of normal skin, intestinal, nasal and lymph node FFPE tissue were also included as control tissue
    7. 7. miRNA are predominantly downregulated in NKTL • miRNA deregulation in NKTL – In both NK cell lines and FFPE NKTL samples compared to normal NK cells, among the miRNAs showing at least 2-fold and statistically significant difference (p<0.05) in expression: • 2 upregulated (miR-155 and miR-378) • 39 were down-regulated: miR-342-5p, miR-26b, miR- 363, miR-150 and miR28-5p – Validation of MEP results • Real-time RT–PCR quantification of miRNAs • Correlation with microRNA transcriptome of NK cell using sequencing method
    8. 8. quantitative-PCR validation of selected miRNAs consistent with MEP data Upregulated miRNA Downregulated miRNA miR-155 Normal NKTL NK Cell Lines 0.1 1 10 100 miR-378 Normal NKTL NK Cell lines 0.1 1 10 100 1000 miR-26b Normal NKTL NK Cell Lines 0.001 0.01 0.1 1 10 miR-363 Normal NKTL NK Cell Lines 0.001 0.01 0.1 1 10 miR-342-5p Normal NKTL Nk Cell lines 0.001 0.01 0.1 1 10
    9. 9. Selection of high probability predicted target genes of deregulated miRNA Intersect with our previous GEP data to narrow down target genes whose expression is inversely correlated with expression of deregulated miRNAs pictar mirBase targetScan miRanda tarBase mirtarget2 Predicted targets Gene expression profile list (J Pathol. 2011 Mar;223:496- 51) Final list 226 target genes of 41 deregulated miRNA
    10. 10. Validation of miRNA targets: Re-expression of miRNA using lentiviral vector • Target genes of miR-101, miR-26a, and miR-26b selected (STMN1, BCL2, IGF1, EZH2) • Lentiviral vectors used to re-express these miRNAs in NKYS cell line • Results – reduced growth of NKYS – modulated the expression of their predicted target genes – suggesting the potential functional role of the deregulated miRNAs in the oncogenesis of NKTL
    11. 11. mRNA expression changes upon overexpression of miRNAs by Lentiviral transduction mir-26b precursor control control controlmir-26b precursor Mir-101 precursor mir-101 precursor control BCL2 IGF1
    12. 12. Immunohistochemistry reveals overexpression of target proteins of suppressed miRNAs in NKTL • IHC for selected target proteins (MUM1/IRF4, BLIMP1, STMN1) of deregulated miRNAs performed on 38 cases of NKTL for validation BLIMP1 MUM1 STMN1 17/34 (50%) 20/38 (53%) 20/35 (57%)
    13. 13. Mechanism of miRNA deregulation in NKTL Role of MYC •MYC is known to cause extensive repression of miRNA expression (Chang TC, et al. Nat Genet. 2008;40:43-50) •Indeed, in our cohort, tumor samples with increase expression of BLIMP1, MUM1 and STMN1 proteins, regulated by their underexpressed miRNAs, showed higher MYC nuclear expression, consistent with MYC activation
    14. 14. EZH2 overexpression in majority of NKTL. NKTLcelllines NormalNKcells NKTL Normaltissue 681012 P=0.003 P=0.002 A B
    15. 15. Inhibition of EZH2 with DZNep induced cell growth inhibition and apoptosis in NK malignant cells.
    16. 16. EBV infection LMP-1 or other factors MYC activation NF-KB activation P53 mutations P53 Deregulation Induce Survivin Proliferation Anti-apoptotic Regulate Proposed model of NKTL pathogenesis P53 Deregulation  miRNA  EZH2 JAK3 mutations STAT activation
    17. 17. Future Studies • MYC-EZH2 in about 50%, JAK3 mutation in about 35% – Do they signify 2 molecular groups of NKTL i.e are these abnormalities mutually exclusive or overlapping ? – What are the clinical implications if such subtypes exist? – Opportunity to answer these questions as collaborative projects within the Asian lymphoma study group • STAT3 and p53 mutation • EZH2, MYC and NFKB protein by IHC
    18. 18. Acknowledgement YAN Junli Viknes Koh Tze Loong Ng Siok Bian
    19. 19. Acknowledgement and thanks National University Health System, Singapore Jim Liang-Seah Tay Baohong Lin Chonglei Bi Joy Tan Gaofeng Huang Queen Mary Hospital, Hong Kong Yok-Lam Kwong Tokyo Medical and Dental University, Japan Norio Shimizu Osaka University Graduate School of Medicine, Japan Katsuyuki Aozasa Funding from NMRC, NRF, MOE

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