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What’s new in the biology of CML?

What’s new in the biology of CML?. John Goldman Imperial College London Newcastle, 1 st March 2013. Hammersmith Hospital London. Some outstanding biological issues in CML. Molecular features of BCR-ABL1 BCR-ABL1 as a clinical target Signal transduction pathways

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What’s new in the biology of CML?

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  1. What’s new in the biology of CML? John Goldman Imperial College London Newcastle, 1st March 2013

  2. Hammersmith Hospital London

  3. Some outstanding biological issues in CML • Molecular features of BCR-ABL1 • BCR-ABL1 as a clinical target • Signal transduction pathways • Signal transduction molecules as clinical targets • Stem cells as clinical targets

  4. Some outstanding biological issues in CML • Molecular features of BCR-ABL1 • BCR-ABL1 as a clinical target • Signal transduction pathways • Signal transduction molecules as clinical targets • Stem cells as clinical targets

  5. Smo AHI1 JAK2 P P P mTOR GSK3 BCL6 BCLX eIF4E P Cat GLI-1 BCR-ABL1 activates a myriad of signaling pathways Tyr177 STAT1 LYN STAT5 Goldman and Melo, NEJM 2003; 349: 1451, modified by van Etten, 2010

  6. Some outstanding biological issues in CML • Molecular features of BCR-ABL1 • BCR-ABL1 as a clinical target • Signal transduction pathways • Signal transduction molecules as clinical targets • Stem cells as clinical targets

  7. Smo AHI1 JAK2 P P P mTOR GSK3 BCL6 BCLX eIF4E P Cat GLI-1 BCR-ABL1 activates a myriad of signaling pathways Tyr177 STAT1 LYN STAT5 Goldman and Melo, NEJM 2003; 349: 1451, modified by van Etten, 2010

  8. Approaches to increasing the efficacy of TKI Exploitable differences Epigenetics HLA class II Cell cycle regulation Chemokines PML/PTEN PP2A FoxO BCL6 Cytokines CXCR4 Wnt Hedgehog Autophagy Alox 5/15 Immune based Genomic instability ROS Actual approaches HDAC-inh. Arsenic FTY720 TGFinh. RI-BP1 Jak2-inh. Plerixafor GSK3inh. Smo inhibitors 0H-chloroquine Zileuton Immunotherapy PARP-inh.

  9. Approaches to increasing the efficacy of TKI Exploitable differences Epigenetics HLA class II Cell cycle regulation Chemokines PML/PTEN PP2A FoxO BCL6 Cytokines CXCR4 Wnt Hedgehog Autophagy Alox 5/15 Immune based Genomic instability ROS Actual approaches HDAC-inh.& Demeth. agents Arsenic FTY720 TGFinh. RI-BP1 Jak2-inh. Plerixafor GSK3inh. Smo inhibitors 0H-chloroquine Zileuton Immunotherapy PARP-inh.

  10. Capacity of catenin to enhance LSC self renewal

  11. Capacity of catenin to enhance LSC self renewal

  12. Possible role of PI3 kinase and AKT in signal transduction

  13. Possible role of PI3 kinase and AKT in signal transduction

  14. Role of protein phosphatase 2A in CML

  15. Cancerous inhibitor of PP2A (CIP2A) predicts progression to BT IM BCR-ABL1 • PP2A is a tumor suppressor that is inactivated by BCR-ABL1 signaling, • CIP2A also renders protein phosphatase 2A (PP2A) inactive • Patients with high levels of CIP2A progress to BT, whereas patients with lower levels do not, and • CIP2A may act through altered phosphorylation of MYC on S62 JAK2 CIP2A SET PP2A c-Myc Ser62 Lucas et al, Blood 2011; 117: 6660-6668

  16. CIP2A at diagnosis as a predictive marker for response to TKI 25 20 15 10 5 0 CIP2A (MFI) Diagnosis Diagnosis Diagnosis CCyR No CCyR BC Lucas et al, Blood 2011; 117: 6660-6668

  17. Probability of disease progression stratified by CIP2A level at diagnosis Low High Patients with a high diagnostic CIP2A protein level have 100% probability of progressing to blast crisis (p=<0.0001). Lucas CM et al. Blood 2011

  18. Sonic hedgehog (Shh or HH) in CML

  19. Shh Patched Smo Gli1 Expression of Shh and its receptors in different phases of CML Activation of the Sonic hedgehog pathway in chronic myeloid leukemia The levels of Shh, Smo and Gli are increased in chronic phase and considerably more increased in advanced phases of CML Bing Long et al, J Exper Clin Cancer Research 2011; 30:8

  20. Sonic hedgehog (Shh or HH) in CML

  21. Proposed cross-talk between Shh and WNT pathways in CML SHH sFRP-1 + PTCH1 P Wnt + Frizzled ? SMO Sufu GSK3 GLI1 -catenin GLI1+GLI3 -catenin + TCF Target Genes (e.g. cyclinD1, CD44) Target Genes (e.g. PTCH1, GLI1, Myc)

  22. Sabutoclax, a pan BCL2 inhibitor renders bone marrow resident leukemia stem cell sensitive to TKI Goff et al, Cell Stem Cell, 2013; 12: 1-13

  23. Peptide inhibition of BCL6 with retroinverso BCL6 peptide inhibitor (RI-BPI) compromises colony formation in CML and selectively eradicates CD34+ CD38- leukemia initiating cells Hurtz et al. J Exper Med 2011; 208: 2016 BCL6-mediated repression of p53 is critical for CML LSC survival

  24. BCL6-mediated repression of p53 is critical for CML LSC survival Inhibitory effect of a retro-inverso BCL6 peptide Inhibitor (RI-BPI) Hurtz et al, J Exper Med 2011;11: 2163-2174

  25. ADAR1 promotes myeloid progenitor re-programming in CML Isoform diversity can be generated by RNA editing by adenosine deaminase activation of RNA (ADAR) enzymes that regulate stem cell maintenance. Jiang et al used serially transplantable blast crisis progenitors in a mouse model and showed BCR-ABL amplification and enhanced expression of an IFN-responsive ADAR1 p150 isoform The authors suggest that techniques directed against ADAR1 might be useful in eradication of CML LSC Jiang et al, PNAS 2013; 110: 1041

  26. Nilotinib and MEK inhibitors induce synthetic lethality through paradoxical activation of RAF in drug-resistant CML Imatinib, nilotinib and dasatinib all possess weak off-target activity against BRAF and CRAF in a RAS dependent manner. Because RAS is activated by BCR-ABL RAS activity may persist in the presence of TKIs. The authors have shown that nilotinib synergises with MEK inhibitors to kill drug resistant CML cells and block leukemia growth in mice Packer et al, Cancer Cell 2011; 20: 715-727

  27. Nilotinib and MEK inhibitors induce synthetic lethality through paradoxical activation of RAF in drug-resistant CML PD = PD184352

  28. Nilotinib and MEK inhibitors induce synthetic lethality through paradoxical activation of RAF in drug-resistant CML Packer et al, Cancer Cell 2011; 20: 715-727

  29. Can we target the quiescent stem cell without targeting as signal transduction molecule?

  30. 12-prostglandin J3, an omega 3 fatty acid-derived metabolite, selectively ablates leukemia stem cell in mice 12-prostglandin J3 was identified by screening publically acquired databases of cyclopentenone prostaglandins. The agent alleviates the development of leukemia in a murine model system – both in mice infected with a Friend erythro- leukemia virus and mice expressing a BCR-ABL oncogene in their haematopoietic compartment Hegde et al, Blood 2011;118: 6909

  31. 12-prostaglandin J3, an omega 3 fatty acid-derived metabolite, selectively ablates leukemia stem cell in mice Hegde et al, Blood 2011;118: 6909

  32. Possible role of constitutional factors in dictating responses to TKIs

  33. A common polymorphism in the BIM gene contributes to intrinsic resistance to IM • Used massively parallel DNA sequencing of paired end ditags • Identified a novel deletion polymorphism in the BIM gene that is associated with deletion of exon 4 and the BH3 domain • 12.3% of East Asian persons have this BIM deletion which is not seen in Africans or Caucasians • The BIM deletion inhibits imatinib-induced apoptosis in CML cells and so results in resistance to imatinib as well as to other TKIs Ng, Tiong-Ong et al, Nat Med 2012;18: 521-528

  34. Resistance to TKI results from deletion of exon 4 that contains BIMi2+/+ encoding BH3 transcripts that activate apoptosis x Ng, Tiong-Ong et al, Nat Med 2012;18: 521-528

  35. Ng, Tiong-Ong et al, Nat Med 2012;18: 521-528

  36. Association of a BIM deletion polymorphism with clinical resistance to imatinib in CML-CP Ng, Tiong-Ong et al, Nat Med 2012;18: 521-528

  37. Have we neglected study of possible epigenetic factors in dictating disease progression ?

  38. Targeting epigenetic mechanisms in CML • Histone tail modifications • DNA methylation • Chromatin remodeling • Non-coding RNAs Chromatin remodeller Transcription Transcription Histones Histone tails Non coding RNAs Chromosome AACR, Nature 2008; 454: 711

  39. Targeting epigenetic mechanisms in CML • DNA methylation • Histone tail modifications • Chromatin remodeling • Non-coding RNAs Chromatin remodeller Transcription Transcription Histones Histone tails Non coding RNAs Chromosome AACR, Nature 2008; 454: 711

  40. Questions that can now be addressed with epigenome wide studies to identify aberrant DNA methylation • Are there specific differences in the pattern of DNA methylation in CML and normal cells? • Does the pattern of methylation differ in different phases of CML? • Does the pattern of DNA methylation differ within chronic phase? • Does the pattern of DNA methylation differ in different sub-populations of Ph+ cells, eg CD34+ cells vs. GMP?

  41. Are there specific differences in the pattern of DNA methylation in CML and normal cells? • Bazeos et al. used Illumina 450 array based technology to study the methylation status CpG dinucleotides in DNA from CD34+ cells from 46 patients with CML-CP and normal controls • Results were compared with methylation patterns of 29 of the patients after they had achieved CCyR • DNA methylation analysis was clearly able to distinguish CML-CP, CML-CCyR and normal CD 34+ cells Bazeos et al, poster presented at EHA-Amsterdam – June 2012

  42. DNA methylation patterns in CML Unsupervised hierarchical clustering (Bazeos et al, 2013) Height DIAGNOSIS NORMALS CCyR

  43. Do we rely too much on measurement of BCR–ABL transcripts?

  44. Molecular monitoring of residual in CML – RT-PCR compared with genomic PCR 28.22 Residual disease: % BCR-ABL/ABL mRNA % BCR-ABL DNA 2.08 2.228 1.82 0.09 0.04 0.06 0.054 0.03 0.016 UND 0.009 UND 0.006 0.005 3 0.003 0.002 6 12 0.001 15 18 21 27 36 42 Months Low Level Positivity of g-PCR When RT-PCR Is Negative Mattarucchi E, et al. J Mol Diagn. 2009;11:482-487.

  45. DNA-based PCR may be more sensitive than RQ-PCR Ross et al, Leukaemia 2010

  46. BCR-ABL breakpoint cloning strategy Alikian et al, 2013 paper submitted

  47. DNA-based MRD (NGS) Alikian et al, 2013 submitted

  48. What about blastic transformation?

  49. Pathways to blastic transformation Adapted from: Perrotti D, et al. J Clin Invest 2010; 120;2254

  50. Conclusions (1) • One approach to improving the efficacy of TKI should be to add a signal transduction inhibitor, but the ideal agent as not yet been identified • It should be possible to identify agents that act directly on the LSC, regardless of whether the LSC is quiescent or active and regardless of whether it is or is not oncogene addicted

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