Modeling Myc Inhibition In Vitro

Alex Yu

Mentor: Nicole M. Sodir

Gerard I. Evan Lab

MYC

PROLIFERATION

CELL GROWTH

CELL CYCLE PROGRESSION

METABOLISM

APOPTOSIS

Aberrantly high and/or deregulated expression of Myc is implicated in the majority of cancers

However, in most tumors, aberrant Myc expression appears not to be due to

mutation in the c-Myc gene itself

Therapy?

CANCER

MYC

Upstream Oncogenic Signals

Heterologous Repressor Targeting Strategy of c-Myc Gene

c-Myc

c-Myc

c-Myc

c-MycTRE/TRE

TREc-Myc susceptible to tetracycline-regulatable repression

Targeted allele in TET-Myc mice

~10.1 kb

Not I

E1 E2 TRE E3 L2 Neo

Hind III

R1

x

Targeting of the c-Myc Locus with a Tetracycline Responsive Element (TRE)

c-Myc allele in wild-type

mouse

E1 E2 E3

Not I

~6.1 kb

R1 R1Hind III

TET-Myc repressor targeting vector

E1 E2 TRE E3 L2 Neo HSV-TK

Not I

pBluescript

Hind III

x

Not I

Heterologous Repressor Targeting Strategy of c-Myc Gene

c-Myc

c-MycTRE/TRE

TRE

tTSKid Hybrid Repressor

-actin tTSKid

TS

KRAB

-actin-tTSKid

Modified TET transactivator

Transcriptional repressor

c-Myc susceptible to tetracycline-regulatable repression

Heterologous Repressor Targeting Strategy of c-Myc Gene

Presence of Tetracycline

tTSkid is inactivated, normal expression of c-Myc

TET TS

KRABTET

E1 E2 TRE E3 L2 Neo

R1

x

Hind III

Not I

Absence of Tetracycline

c-Myc expression is blocked by the super-repressor

tTSKid Hybrid Repressor

TET Operator Sequence

E1 E2 TRE E3 L2 Neo

R1

x

Hind III

Not I

TS

KRAB

Questions

• How would we model this in vitro?

• Does this system function properly?

Mouse Adult Lung Fibroblasts (MALFs) Isolation MethodEuthanize adult mouse on tetracycline

Isolate lungs

Mince lungs

Keep isolated MALFs on tetracycline until confluency

Extract cells onto 2 tissue culture plates

(+) Tetracycline (-) Tetracycline

Count & seed 100,000/well

Growth Curve in c-MycTRE/TRE; -actin-tTSKid MALFs

Num

ber o

f cel

ls X

105

Days

+ Tetracycline

- Tetracyclin

e

+ Tetracyclin

e

Conclusions and Future Directions

• Myc inhibition represses cell proliferation

• Ability of reversibly inhibiting Myc at will

• Basic approach towards cancer therapy

• Method is to be tested in vivo

Acknowledgments

John Schlauraff

Jean MacCormack

Ben Koo

All UCSF SEP Staff

Nicole M. Sodir

Gerard I. Evan Lab