silencing c-myc translation as a therapeutic strategy ... 2016 deng final.pdf · silencing c-myc...
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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, 2012Zhang, 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
Pro
tea
so
me
Inh
ibito
rs
x
Ca
rfil
zo
mib
(nM
)B
ort
ezo
mib
(nM
)
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
SLLCLL
• 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
Fre
qu
en
cy
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
Ru
nn
ing
Enri
chm
ent
Score
(R
ES
)
GS52: Schuhmacher
Gene List Index
NES q-val
-7.70 0.00
Gene List Index
GS70: Dang
RE
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
NN
N
NH2
O
Cl
O
3
1
2 4
56 7
8
9
Central pyrazolo-
pyrimidine moiety
PF4800567
N
NN
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
CK
1e
Ac
tiv
ity
(% o
f v
eh
icle
co
ntr
ol)
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
NN
N
NH2
O
F
N
N
OF
CUX-03173
N
NN
N
NH2
O
F
N
N
OF
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
CK1eId
ela
lisib
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 BiologyHoward 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 SupportLymphoma Research FundIrving Institute CTO Pilot AwardPrecision Medicine Pilot AwardTG Therapeutics