Silencing c-Myc Translation as a Therapeutic Strategy ... 2016 Deng Final.pdf · PDF file...

Click here to load reader

  • date post

    22-Jul-2020
  • Category

    Documents

  • view

    4
  • download

    0

Embed Size (px)

Transcript of Silencing c-Myc Translation as a Therapeutic Strategy ... 2016 Deng Final.pdf · PDF file...

  • Silencing c-Myc Translation as a Therapeutic

    Strategy through Targeting PI3Kδ and CK1ε in

    Hematological Malignancies

    Changchun Deng1,2,3,*, Mark M. Lipstein2, Luigi Scotto2,

    Xavier O. Jirau Serrano2, Michael A. Mangone2, Shirong Li3,

    Jeremie Vendome4, Yun Hao5, Xiaoming Xu2, Xiaopin Liu2,

    Ipsita Pal2, Shi-Xian Deng2, Nicholas P. Tatonetti5, Suzanne

    Lentzsch3, Barry Honig4, Donald W. Landry2, and Owen A.

    O'Connor1,2

    Columbia University Medical Center

  • Disclosure

    • Research funding from TG Therapeutics, Inc.

  • • No c-Myc targeting drugs have been approved.

    • C-Myc protein has a short half life, 30 min.

    • C-Myc mRNA has complex secondary structures in the 5’

    untranslated region (UTR), which negatively regulate cap

    dependent translation of c-Myc.

    • Translation of c-Myc is potently inhibited by silvestrol, a

    selective inhibitor of the eukaryotic initiation factor 4A (eIF4A).

    Targeting of c-Myc Translation as a Novel

    Therapeutic Strategy

    Andresen et al., Nucleic Acids Res 2012

    Wolfe et al., Nature 2014

  • Targeting Translation of c-Myc through Inhibiting

    Phosphorylation of 4E-BP1

    Dibble CC and Cantley LC.

    Trends Cell Biol, 2015

    Suraweera, A., et al., Mol Cell, 2012

    Quy, P.N., et al., J Biol Chem, 2013

    Hutter, G., et al., Leukemia, 2012 Zhang, Y., et al., Nature, 2014

    c-Myc

    eIF4E 4E-BP1

    eIF4E

    eIF4A

    eIF4G

    P P

    4E-BP1

    (eIF4F)

    Translation

    mTOR

    PI3Kd

    Proteasome

    Amino acids

    AKT

    ?Other?

    Kinases

  • Combining PI3K and Proteasome Inhibitors May

    Synergistically Inhibit Translation of c-Myc and Kill

    Lymphoma Cells

    Drugs TGR-1202 (TG)

    Idelalisib (Ide)

    Carfilzomib (Cfz)

    TC IC

    Bortezomib (Bz)

    TB IB

    x

    x

    eIF4F c-MycmTORC1PI3K

    Proteasome

    pp-4E-BP1

    PI3Kδ Inhibitors

    P ro

    te a

    s o

    m e

    In h

    ib it o

    rs

    x

  • C a

    rf il

    z o

    m ib

    (n M

    ) B

    o rt

    e z o

    m ib

    (n M

    )

    Excess over Bliss (EOB) > 0: Synergy

    Idelalisib (μM)TGR-1202 (μM)

    TC Is Highly Synergistic and Superior to Other

    Combinations of PI3K and Proteasome Inhibitors

  • TC Is Highly Synergistic and Superior to Other

    Combinations

    • TC is highly synergistic in 12 cell line models of DLBCL, MCL,

    MM, T-ALL, and CTCL.

    • TC is highly synergistic in primary CLL, MCL, and MZL cells.

    • TC synergistically induces apoptosis.

  • TC Uniquely and Synergistically Inhibits Translation

    of c-Myc and Phosphorylation of 4E-BP1

    LY10

    4EBP1*T70

    4EBP1 c-Myc

    Actin

    LY7

    4EBP1*T70

    c-Myc

    B-Actin

    4EBP1

    SLL CLL

    • TC does not inhibit the mRNA level of c-Myc.

    • A reporter of MYC 5’UTR confirms TC inhibits translation of c-Myc.

  • Effects of TC on Global mRNA Translation

    Ribosome footprinting & RNAseq

    Gel Purify Ribosomal Footprints

    & Generate Library Sequence

    (Translation Rate)

    mRNA

    RNAseq

    (mRNA expression level )

    Polysomes

    Translation Efficiency (TE) =

    Translation Rate / mRNA level

  • TC Inhibits Global mRNA Translation

    F re

    q u

    e n

    c y

    Log2 (TETreated/TEControl)

    Genome Wide Effects of TC on Translation Efficiency (TE)

    -6 -4 -2 0 2 4 6 8

    TE decreased TE increased

  • TC Selectively Inhibits Translation of Genes Involved

    in Translation

    Decrease With Treatment Increase With Treatment

    Measure of Change In Translation Efficiency +/-(1-p)

    iPAGE analysis of the ontology of translationally altered genes

    Extracellular matrix

    Translation factor

    RNA splicing

    Mitochondrial membrane

    Nucleolus

    Constituents of ribosome

    Mitotic cell cycle

    Proteasome complex

  • R u

    n n

    in g

    E n ri

    c h m

    e n t

    S c o re

    ( R

    E S

    )

    GS52: Schuhmacher

    Gene List Index

    NES q-val

    -7.70 0.00

    Gene List Index

    GS70: Dang

    R E

    S

    NES q-val

    -6.56 0.00

    TC Inhibits the Transcription of c-Myc Target Genes

    • Cytotoxicity of TC is recued by forced overexpression of c-Myc.

    • Cytotoxicity of TC is recued by forced overexpression of eIF4E.

  • TGR-1202 and carfilzomib, but not combinations of other

    drugs in the same classes, synergistically inhibit c-Myc

    translation and c-Myc dependent gene transcription, by

    potently inhibiting phosphorylation of 4E-BP1.

    TGR-1202 and carfilzomib synergistically induce apoptosis

    In lymphoma cells through targeting c-Myc.

  • Idelalisib DuvelisibTGR-1202

    TGR-1202 Is Structurally Distinct from Idelalisib

    and Duvelisib

  • Kinase #1 #2 #1 #2 #1 #2

    CK1a1 111 111 110 107 112 111

    CK1a1L 105 103 102 101 104 99

    CK1delta 105 98 96 104 100 97

    CK1epsilon 40 40 93 93 93 91

    CK1g1 99 98 105 105 102 98

    CK1g2 104 104 102 100 99 99

    CK1g3 96 95 94 93 93 93

    CK2a 83 78 97 96 95 84

    CK2a2 86 86 94 92 102 100

    TGR-1202, but not Idelalisib or Duvelisib, Inhibits

    Casein Kinase 1 Epsilon (CK1ε)

    Kinase activity (% of control) using the Reaction Biology

    Kinome Profiling platform

    TGR-1202 Idelalisib Duvelisib

  • L85

    E83

    L84

    L135

    M82

    S88

    PF4800567 (X-Ray) TGR-1202 (In silico docking)

    TGR-1202 and the CK1ε Inhibitor PF4800567

    Share an Identical Structural Moiety

    N

    N N

    N

    NH2 O

    Cl

    O

    3

    1

    2 4

    56 7

    8

    9

    Central pyrazolo-

    pyrimidine moiety

    PF4800567

    N

    N N

    N

    NH2

    O

    F

    O

    O

    F

    F

    TGR-1202

    3

    1

    2 4

    56 7

    8

    9

  • TGR-1202 and Its Analogs Inhibit CK1ε

    1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2 0

    2 5

    5 0

    7 5

    1 0 0

    lo g [A g o n is t], M

    C K

    1 e

    A c

    ti v

    it y

    (% o

    f v

    e h

    ic le

    c o

    n tr

    o l)

    P F -4 8 0 0 5 6 7

    C U X -0 3 1 7 3

    C U X -0 3 1 6 6

    Id e la lis ib

    T G R -1 2 0 2

    N

    N N

    N

    NH2

    O

    F

    N

    N

    O F

    CUX-03173

    N

    N N

    N

    NH2

    O

    F

    N

    N

    O F

    CUX-03166

    Kinase activity (% of control) using recombinant CK1ε

    • Two analogs of TGR-1202, CUX-

    03173 and CUX-03166, demonstrate

    markedly different potency targeting

    CK1ε, despite they differ by only one

    methyl group.

    • Idelalisib does not inhibit CK1ε.

  • 0 0 3 3 5 5

    0 0 5 5 5 5

    - + - + - +shRNA

    Ide (M)

    Cfz (nM)

    B-Actin

    CK1e

    4EBP1

    4EBP1 *T70

    c-Myc

    4EBP1 *S65

    4EBP1 *T70

    4EBP1

    B-Actin

    Dual Targeting of PI3Kδ and CK1ε Underscores the

    Unique Activity of TGR-1202 in DLBCL

    • 38% (6/16) Combo

    Responders.

    • 30% (3/10) single

    responders.

    • CR only in combo

    responders.

    -50%

  • eIF4F

    c-Myc

    Translation

    mTOR

    PI3Kd

    Proteasome

    pp-4EBP1

    TGR-1202

    Carfilzomib,

    Bortezomib

    CK1e Id

    e la

    li s ib

    TGR-1202 as the First CK1ε Inhibitor Available for

    Patients May Have a Unique Therapeutic

    Role in c-Myc Driven Lymphoma

    NCT02867618: actively enrolling patients

    Phase I/II Study of TGR-1202 and Carfilzomib in the Treatment

    of Patients with Relapsed or Refractory Lymphoma

  • Thank You!!

    Center for Lymphoid Malignancies

    Owen A. O’Connor, M.D., Ph.D. Mark Lipstein, B.S. Xavier Jirau, B.S. Luigi Scotto, Ph.D. Michael Mangone, Ph.D. Xiaoping Liu, M.D., Ph.D. Ipsita Pal, Ph.D.

    Nicholas Hornstein M.D., Ph.D. and Peter Sims, Ph.D. Biochemistry & Mol. Biophysics; Systems Biology

    Shi-Xian Deng, Ph.D. Xiaoming Xu, Ph.D. Donald W. Landry, M.D., Ph.D. Experimental Therapeutics

    Jeremie Vendome, Ph.D. and Barry Honig, Ph.D. Biochemistry & Mol. Biophysics; Systems Biology Howard Hughes Medical Institute

    Yun Hao and Nicholas Tatonetti, Ph.D. Biomedical informatics

    Shirong Li, Ph.D. and Suzanne Lentzsch, M.D., Ph.D. Hematology & Oncology

    Funding Support Lymphoma Research Fund Irving Institute CTO Pilot Award Precision Medicine Pilot Award TG The