Abstract

Conclusions

A

Acknowledgements

Figure 1. Imetelstat Treatment of SUM149PT Cells Inhibits Telomerase Activity in a Dose-dependent Manner.

Telomerase Products

IC

[Imetelstat], uM

Figure 3. Three-week Imetelstat Pretreatment Inhibits Clonogenic Survival in BRCA1 Deficient Cells.

Culture cells in presence of drug for 2-3 passages (2-3

weeks)

Experimental Set-up

Plate cells at a density to passage 1 x per week

Treat with Imetelstat or mismatch after cell attachment (Day 2) and

refresh drug every 3 days

Plate at low density and allow to grow undisturbed

for 11-18 days

24hr treatment

Investigating the Role of BRCA1 in Sensitivity to Imetelstat, a Telomerase Template Antagonist Elizabeth A. Phipps1, Sergei M. Gryaznov2 and Brittney-Shea Herbert1

1Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN USA 2Geron Corporation, 230 Constitution Drive, Menlo Park, CA 94025, USA

BRCA1 Mutant Cell Lines Used

Domain Ring finger BRCT

Interacting Proteins

BARD1 UbcH5c BAP1 E2F1

Rad50 Mre11 Nbs1

BRCC complex

BRIP/FANCJ HADC1/2

P53 pRB

Function E3 ubiquitin ligase activity Phospho-peptide binding,

transcriptional activation; BRCA1 localization to damage sites

Adapted from Tomlinson et al. (1998) Cancer Research

HCC1937 5382insC

MDA.MB.436 5396+1G>A

SUM149PT 2288delT

Table 1. Summary of Morphologic Changes Induced by Imetelstat Treatment

This work was supported by DOD BCRP predoctoral fellowship BC093058 (award #W81XWH-10-1-0165). We also thank the Indiana University Simon Cancer Center (IUSCC) for providing a 2012 IUSCC Travel Award to attend the AACR annual meeting. We thank Jillian Koziel for technical

assistance and all Herbert Lab members for helpful discussions. SUM149PT cells were a kind gift from Dr. George Sledge (IUSCC). We thank Dr. Stephen Elledge for the HCC1937+BRCA1 cells and

Dr. Sergei Gryaznov (Geron Corp.) for providing the Imetelstat.

0

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100

% in

hibi

tion

vs. u

ntre

ated

[Imetelstat], uM

Telomerase Products

IC

Δ - MM + - + - + - + - + - + - + - +

Figure 2. Imetelstat Treatment Inhibits Telomerase Activity in BRCA1 Deficient Cells.

DNA input 1.5ug

21.2 kb

8.6 kb 7.4 kb 6.1 kb 5.1/5.0/4.9 kb 4.2kb 3.6/3.5 kb 2.7 kb 2.0/1.9/1.8 kb 1.6/1.5 kb 1.4/1.3 kb 1.1 kb

Figure 6. Long-term Imetelstat Treatment Induces Complete Cell Death in HCC1937 pBp Cells.

UWB1.289

[GRN163L], uM [CCDP], uM CI

0.85 0.01 6.19E+09

0.85 0.1 0.501

0.85 1 0.243

0.85 10 0.155

UWB1.289+BRCA1

[GRN163L], uM [CCDP], uM CI

0.85 0.01 4.87E+14

0.85 0.1 0.782

0.85 1 0.108

0.85 10 0.601

HCC1937

HCC1937+BRCA1

UT 3.4uM Imetelstat

UWB1.289

UWB1.289+BRCA1

All cell lines tested exhibited a dose-dependent response to treatment with Imetelstat, but not a mismatch oligonucleotide. We observed differential sensitivity among cell lines tested to Imetelstat, at clinically relevant concentrations, in terms of morphology and both TRAP and clonogenic survival assays. UWB1.289 cells exhibit a shorter average telomere length at baseline compared to UWB1.289+BRCA1 cells, and both cell lines show telomere shortening over a three-week period due to Imetelstat treatment. Long-term treatment with Imetelstat preferentially induces cell death in BRCA1 mutant HCC1937 pBp cells. Population doubling study using UWB1.289 and UWB1.289+BRCA1 cells are ongoing. Cis-platin pretreatment synergizes with Imetelstat in UWB cells. Concurrent Doxorubicin treatment synergizes with Imetelstat in HCC cells.

50

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100

% in

hibi

tion

vs. u

ntre

ated

24hr treatment

Figure 4. Baseline Telomere Lengths Among BRCA1 Cell Lines. Figure 5. Imetelstat-Treated Ovarian Cancer Cells Exhibit

Progressive Telomere Shortening. UWB1.289 2594delC

*P<0.05 ***P<0.001

0 1 2 3 1 2 3 1 2 3 0 1 2 3 1 2 3 1 2 3 Week

UWB1.289 UWB1.289+BRCA1

Untreated MM GRN163L Untreated MM GRN163L

Mar

ker

Con

trol

MC

F7

MD

A.M

B.4

68

LB #245

BRCA1 is a tumor suppressor gene with a variety of functions related to safeguarding genomic integrity. Regulation of telomere length is crucial in maintaining genomic stability, with critically short telomeres leading to telomere uncapping, end-to-end fusions, activation of the DNA damage response, and cell cycle arrest. Recent evidence suggests knockdown of BRCA1 in cell lines increases both telomerase activity and average telomere length, but likely results in more unstable telomeres compared to BRCA1 wild-type cell lines. The objective of this study was to determine whether GRN163L, a telomerase template antagonist currently in clinical trials, has enhanced activity in BRCA1 mutant breast or ovarian cancer cell lines compared to paired BRCA1 wild-type breast/ovarian cancer cell lines. In addition, we sought to determine whether BRCA1 cell lines have shorter average telomere lengths than other cancer cell lines, making them more amenable to inhibition of telomerase activity. We utilized a panel of breast and ovarian cancer cell lines harboring both N-terminal and C-terminal BRCA1 mutations, in addition to matched cells expressing exogenous wild-type BRCA1, and assessed the effects of GRN163L or a mismatch oligonucleotide on telomerase activity and telomere length using the TRAP (Telomere Repeat Amplification Protocol) and TeloTAGGG assays, respectively. All cell lines tested exhibited a dose-dependent response to treatment with GRN163L, but not a mismatch control oligonucleotide. In addition, we observed differential sensitivity between BRCA1 mutant and BRCA1 wild-type cell lines tested to clinically relevant concentrations of GRN163L in both TRAP and clonogenic survival assays. Of interest, the majority of the cell lines tested showed gross morphological changes as early as 12 hours following inhibitor treatment. We found UWB1.289 cells have a shorter average telomere length at baseline compared to their BRCA1 wild-type counterpart cells, and saw progressive telomere shortening over a three-week period of treatment with GRN163L, but not with mismatch oligonucleotide. Furthermore, long-term (12 week) treatment with GRN163L preferentially induced complete cell death in HCC1937 (BRCA1 mutant) breast cancer cell population compared to HCC1937 cells reconstituted with wild-type BRCA1. Finally, we demonstrate that pretreatment with GRN163L acts synergistically with cis-platin in UWB1.289 and UWB1.289+BRCA1 ovarian cancer cells. In summary, this work provides insight into the role of BRCA1 mutations in cancer cells and their impact on sensitivity to the cellular effects of telomerase antagonism.

8.6 kb 7.4 kb 6.1 kb 5.1/5.0/4.9 kb 4.2kb 3.6/3.5 kb 2.7 kb 2.0/1.9/1.8 kb 1.6/1.5 kb 1.4/1.3 kb 1.1 kb

21.2 kb

Figure 7. Cis-platin Pretreatment (UWB cells) or Concurrent Doxorubicin Treatment (HCC cells)

Synergizes with Imetelstat.

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Cum

ulat

ive

PDL

Days

UWB1.289

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120

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Cum

ulat

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PDL

Days

UWB1.289+BRCA1

0

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50

60

0 20 40 60 80

Cum

ulat

ive

PDL

Days

HCC1937 pBp

UT MM 3.4uM GRN163L 1.7uM GRN163L 0.85uM GRN163L

0

10

20

30

40

50

60

0 20 40 60 80

Cum

ulat

ive

PDL

Days

HCC1937+BRCA1

HCC1937 pBp [GRN163L], uM [Doxorubicin], uM CI

0.325 0.081 0.397 0.625 0.156 0.236 1.25 0.313 0.369 2.5 0.625 0.469 5 1.25 0.611

10 2.5 1.161 20 5 2.571

HCC1937+BRCA1 [GRN163L], uM [Doxorubicin], uM CI

0.325 0.081 0.066 0.625 0.156 0.111 1.25 0.313 0.189 2.5 0.625 0.328 5 1.25 0.574 10 2.5 1.223 20 5 2.471

A.

B.