Junia Melo - COLT 2014

Views:
 
Category: Others/ Misc
     
 

Presentation Description

No description available.

Comments

Presentation Transcript

PowerPoint Presentation:

Junia V. Melo University of Adelaide , Australia Key biological questions in CML

PowerPoint Presentation:

BCR-ABL p210 Y412 Y177 Oligom. Domain P-S/T (SH2-bind.) rho -GEF SH3 SH2 SH1 bind. Actin NLS DNA bind. t(9;22)(q34;q11) 9q+ Ph CML

PowerPoint Presentation:

ATP-binding competitors Y P Y P P P BCR-ABL Substrate BCR-ABL Y Substrate Y N N N H N H N N N O Imatinib mesylate

PowerPoint Presentation:

TKI therapy – The pitfalls 1) Secondary resistance 2) Inability to eradicate the stem cell

PowerPoint Presentation:

CML CD34+ cells expand in liquid culture without GFs TKI treatment Why? Incomplete inhibition of Bcr-Abl? Cells not dependent on Bcr-Abl for survival, i.e. not oncogene addicted? Survival & G1 arrest = induced quiescence TKIs induce apoptosis in mature CD34+ cells, quiescence in progenitors, and little effect on CML stem cells Holyoake , Eaves, Deininger, Bhatia, Perrotti

PowerPoint Presentation:

10% of CML cells survive 10 fold enrichment for stem cells following dasatinib treatment Dasatinib and Growth Factor Withdrawal No drug 150nM dasatinib Time-point (day) 0 4 8 12 0.1 1 10 100 Cell number (x10 9 ) Hamilton et al, Blood 2012

PowerPoint Presentation:

No drug dasatinib p-CrkL Actin Complete Bcr-Abl Inhibition Achieved No drug 150nM dasatinib Time-point (day) 0 4 8 12 0.1 1 10 100 Cell number (x10 9 ) CML stem cells may be independent from Bcr-Abl kinase activity → need to find alternative targets ! Hamilton et al, Blood 2012

PowerPoint Presentation:

Shaoguang Li Worcester, MA 1) Molecular differences between normal HSCs and LSCs 2) Specific target genes for eradicating LSCs Since BCR-ABL kinase inhibitors do not eradicate CML stem cells Mouse models of CML

PowerPoint Presentation:

DNA microarray using sorted GFP+Lin-Sca-1+c-Kit+ (BCR-ABL+/-) expressing LSK BM cells Retrovirus: GFP BCR-ABL-GFP GFP BCR-ABL-GFP Imatinib: - - + + pre-stimulation (IL-3, IL-6, SCF) and ex vivo transduction pMSCV- BCR-ABL Vector: MPSV GFP BCR-ABL PBSQ  LTR Donor BM cells Short list (10-20 genes) Functional test Shaoguang Li Worcester , MA

PowerPoint Presentation:

Blk (B-lymphoid kinase) Tumour suppressor gene Downregulated in CML LSCs Downregulation not reversed by imatinib (= kinase independent)

Blk and CML development:

Blk and CML development BCR-ABL Blk GFP Zhang et al. Nature Genetics , 2012 Worse survival without Blk Better survival with Blk

Blk suppresses CML development:

Blk suppresses CML development BCR-ABL Blk GFP Zhang et al. Nature Genetics , 2012 Worse survival without Blk Better survival with Blk

PowerPoint Presentation:

B C D E F Blk does not suppress normal HSCs A

PowerPoint Presentation:

B C D E F Blk does not suppress normal HSCs A Comparison between normal HSCs: Expressing Blk ( WT ) vs . not expressing Blk ( Blk -/- ) Expressing Blk ( vector ) vs . Over expressing Blk ( Blk ) Expect / hope / bet / hypothesise  No difference

PowerPoint Presentation:

B C D E F Blk does not suppress normal HSCs A Comparison between normal HSCs: Expressing Blk ( WT ) vs . not expressing Blk ( Blk -/- ) Expressing Blk ( vector ) vs . Over expressing Blk ( Blk ) Expect / hope / bet / hypothesise  No difference

PowerPoint Presentation:

B C D E F Blk does not suppress normal HSCs A

PowerPoint Presentation:

Blk does not suppress normal HSCs

PowerPoint Presentation:

How is Blk regulated? BCR-ABL Blk ?

PowerPoint Presentation:

Libermann et al., JBC 1999 Pax5 Blk promoter How is Blk regulated?

Pax5 suppresses CML development…:

Pax5 suppresses CML development… Blk-/- WT BCR-ABL BCR-ABL- Pax5 BCR-ABL BCR-ABL-Pax5 CML mice Retrovirus

Pax5 suppresses CML development…:

Pax5 suppresses CML development… Blk-/- WT BCR-ABL BCR-ABL-Pax5 BCR-ABL BCR-ABL-Pax5 CML mice Retrovirus … and Blk deletion partially rescues Pax5 function

PowerPoint Presentation:

How is Pax5 regulated? Pax5 BCR-ABL Blk ?

PowerPoint Presentation:

How is Pax5 regulated? Myc EBF1 -306 -1638 Pax5 promoter (+) (-)

PowerPoint Presentation:

Myc & EBF1 mediate down-regulation of Pax5 by BCR-ABL BCR-ABL Blk Pax5 ? EBF1 Myc Myc EBF1 -306 -1638 Pax5 promoter

PowerPoint Presentation:

How does Blk suppress LSC proliferation? BCR-ABL Blk Pax5 EBF1 Myc ???

PowerPoint Presentation:

Blk upregulates p27 (Cdkn1b) Blk Pax5 EBF1 Myc p27 Cyclin-dependent kinase inhibitor

PowerPoint Presentation:

Blk upregulates p27 (Cdkn1b) BCR-ABL Blk Pax5 ? EBF1 Myc p27 Cyclin-dependent kinase inhibitor

PowerPoint Presentation:

BCR-ABL Positive regulatory pathway (Alox5, Hif1a……) Negative regulatory pathway (Msr1, Scd1, Blk ……) Survival, self-renewal and differentiation of LSCs Critical molecular pathways in LSCs

PowerPoint Presentation:

Quiescence Dormancy Self-renewal Make new stem cells Stem Cell Fates Apoptosis Programmed cell death Differentiation Become specialised & functional

PowerPoint Presentation:

Quiescence Dormancy Self-renewal Make new stem cells Leukaemic Stem Cells Apoptosis Programmed cell death Differentiation Become specialised & functional

PowerPoint Presentation:

Apoptosis Programmed cell death Differentiation Become specialised & functional Quiescence Dormancy Self-renewal Make new stem cells Leukaemic Stem Cells

PowerPoint Presentation:

Apoptosis Programmed cell death Quiescence Dormancy Differentiation Become specialised & functional Self-renewal Make new stem cells Leukaemic Stem Cells Strategies Inhibit self-renewal Reverse quiescence Induce differentiation Induce apoptosis

PowerPoint Presentation:

Targeting CML stem cells Ahmed, W. & van Etten, R., Hematology 2013: 189-200, 2013

PowerPoint Presentation:

HSC LSC BCR-ABL BCR-ABL dependent transcriptional changes e.g. 1200 genes either up or down BCR-ABL dependent BUT kinase independent transcriptional changes LSC Most aberrant gene expression normalises with TKI exposure e.g. 1000 normalise = BCR-ABL kinase dependent TKI 200 genes remain aberrantly expressed when BCR-ABL kinase activity is completely suppressed WHAT ARE THEY? (Copland & Holyoake) Human Cells

PowerPoint Presentation:

Other key biological questions Do we NEED to target CML stem cells to ‘ cure ’ CML? What are the biological markers that dictate the probability of response, relapse, cure? Can we target the genomic instability of CML cells to prevent blast crisis? Why does BCR-ABL induce predominantly CML, but can also give rise to ALL? And why………? And how………?

What to Target / How to Target:

What to Target / How to Target Knowledge Systems biology Gene expression miRNA expression Proteomics p-Proteomics Epigenetic signatures Whole genome sequencing 3. Exploit differences vs. toxicity HDAC inhib. Arsenic FTY720 TGF b inhib. RI-BP1 Jak inhib. Plerixafor GSK3 b inhib. Smo inhib. HCQ Zileuton Immunotherapy PARP inhib. 2. Identify differences Epigenetics HLA class II Cell cycle regulation Chemokines Blk PP2A TGF b , Foxo BCL6 Cytokines CXCR4 Wnt, Hedgehog Autophagy Alox 5/15 IL1RAP, CD26 Immune based Genomic instability ROS, Rad52

PowerPoint Presentation:

Special thanks Mhairi Copland (Glasgow, UK) Tessa Holyoake (Glasgow, UK) Rick van Etten (Univ. Irvine, CA, USA) Shaoguang Li (Univ. Massachusetts, USA) Sue Branford (Molecular Pathology, CCB)

authorStream Live Help