Relating Activating K-Ras Mutations to Small Molecule Sensitivity in Non-Small-Cell Lung Cancer

Flavian D. BrownCarleton CollegeClass of 2009

Lung Cancer

Leading cause of death from cancer in the world

Over 90% of NSCLC contain mutations in EGFR, BRAF and K-Ras

Discovery of Gefitnib

Ras Signaling

Schubbert et al. (2007) Hyperactive Ras in developmental disorders and cancer. Nature Review of Cancer, Vol. 7 295-307.

Oncogenic Mutation

Oncogenic Mutation

←

←

←

Hypothesis

NSCLC tumors are genetically sensitized due to changes in cellular state secondary to activating K-Ras mutations.

- Different drug targets - Oncogene Addiction

Small Molecule Screens

+ DMSO Control

100nl

Pin Transfer

250500

1000

48Hrs 72Hrs2-10Hrs

cell adherence

24 Hrs

A549: Hits Highlighted

Color coding on the images:

Red = unbiased commercial compound Forma setGreen = bioactives (including kinase inhibiting drugs)Magenta = HDAC biased DOSBlue = commercial kinase biased (CBkinase)Yellow = analyticon purified natural productsBlack = DMSO control plateGray = +con dose plate

Hits From Primary Screen

Assay Development

H1792

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

0 200 400 600 800 1000 1200

Cells/Well

Fluo

resc

ence

(535

/595

nm)

2hr

4hr

6hr

8hr

Small Molecule Sensitivity

0

2

4

6

8

10

12

14

16

A549 (K-Ras) H460 (K-Ras) H1792 (K-Ras) H1975 (EGFR) H1650 (EGFR) H1395 (BRAF)

Cell Lines

IC50

(µm

ol)

Small Molecule Sensitivity

0

2

4

6

8

10

12

14

16

18

A549 (K-Ras) H460 (K-Ras) H1792 (K-Ras) H1975 (EGFR) H1650 (EGFR) H1395 (BRAF)

Cell Lines

IC50

(µm

ol)

Small Molecule Sensitivity

0

2

4

6

8

10

12

14

16

A549 (K-Ras) H460 (K-Ras) H1792 (K-Ras) H1975 (EGFR) H1650 (EGFR) H1395 (BRAF)

Cell Lines

IC50

(um

ol)

Structural Activity Relationship

OHNH

OO

NH

N

I

OHNH

O

O

HN

N

Br

N

O

O

NH

OH

HN

Aromatic group at the opposite end of structures

Carbon spacer can be rigid or flexible

Hydroxamic acids attached to a 4 or 5 carbon chain

Future Investigations

Analyze signaling downstream of the activating mutation -Immunofluorescence

-Western Blotting

Target Identification

- Pull down assay

Correlate phenotypic data with genetic data

- SNP copy number

Impact

Genotype specific inhibitors for K-Ras mutants

Paradigm for investigating genotype-phenotype relationships in other malignancies

- WGAS for somatic alterations

Molecularly targeted cancer therapeutics.

AcknowledgementsPrinciple Investigator - Stuart L.Schreiber, Ph.D

Mentor - Gopal S. Ramachandran, Ph.D

Summer Research Program in Genomics- Shawna Young- Lucia Vielma- Maura L. Silverstein- Bruce Birren, Ph.D

Collaborators- Jordi Barretina, Ph.D- Damian W. Young, Ph.D

Broad Institute Screening- Nicola Tolliday, Ph.D- Josh Bittker, Ph.D- Melanie de Silva- Kate Hartland

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