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