Supplementary Materials for

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1 Supplementary Materials for Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of the PI3K-Akt Pathway in Triple-Negative Breast Cancer Jessica J. Tao, Pau Castel, Nina Radosevic-Robin, Moshe Elkabets, Neil Auricchio, Nicola Aceto, Gregory Weitsman, Paul Barber, Borivoj Vojnovic, Haley Ellis, Natasha Morse, Nerissa Therese Viola-Villegas, Ana Bosch, Dejan Juric, Saswati Hazra, Sharat Singh, Phillip Kim, Anna Bergamaschi, Shyamala Maheswaran, Tony Ng, Frédérique Penault-Llorca, Jason S. Lewis, Lisa A. Carey, Charles M. Perou, José Baselga,* Maurizio Scaltriti* *Corresponding author. (M.S.); (J.B.) The PDF file includes: Published 25 March 214, Sci. Signal. 7, ra29 (214) DOI: /scisignal Fig. S1. EGFR and PTEN abundance in HCC7 and MDA-MB-468 xenografts. Fig. S2. Growth of HCC7 and MDA-MB-468 xenografts treated with MEHD7945A and PI3K-Akt pathway inhibitors. Fig. S3. CEER analysis of HCC7 xenografts. Fig. S4. Immunostaining for EGFR, PTEN, and Ki67 in patient-derived TNBC xenografts. Fig. S5. Imaging of EGFR and HER3 in patient-derived TNBC treated with GDC- 68. Fig. S6. Ki67 staining of tumors and CTCs from PDXs treated with MEHD7945A and PI3K-Akt pathway inhibitors. Fig. S7. HER3 knockdown in MDA-MB-468 cells treated with GDC-68 or GDC Fig. S8. Tumor growth of HCC7 and PDXs treated with cetuximab and GDC-68. Fig. S9. Correlation between EGFR mrna expression after cetuximab treatment and overall survival in TNBC metastatic patients. Fig. S1. Quantification of HER3 abundance in paired samples from patients treated with cetuximab. Fig. S11. Antibody specificity for FRET analysis. Table S1. CEER values (single replicas). Table S2. Patient clinicopathological information.

2 Supplementary Materials EGFR PTEN MDA468 HCC7 Figure S1: EGFR and PTEN abundance in HCC7 and MDA-MB-468 xenografts. Immunostaining for EGFR and PTEN in HCC7 and MDA-MB-468 (MDA468)-derived xenografts. Images are representative of three tumors. Scale bar, 5 µm.

3 A B Tumor Volume (mm 3 ) Control MEHD7945A GDC-941 GDC-68 Tumor Volume (mm 3 ) Control GDC-941 GDC-68 MEHD7945A 941+MEHD 68+MEHD Days after treatment * ** 68+MEHD 941+MEHD Time (days) * ** Figure S2: Growth of HCC7 and MDA-MB-468 xenografts treated with MEHD7945A and PI3K-Akt pathway inhibitors. Tumor growth of HCC7 (A) and MDA-MB-468 (B) xenografts treated as indicated. MEHD7945A was given 1 mg/kg twice weekly, GDC mg/Kg daily and GDC-58 4 mg/kg daily. N= 7-1 for each arm. HCC7: *p=.39 vs GDC-68; **p=.3 vs GDC MDA468: *p=.23 vs GDC-941; **p=.65 vs GDC-68.

4 pegfr pher * Control GDC-68 GDC-941 MEHD7945A 68+MEHD 941+MEHD Control GDC-68 GDC-941 MEHD7945A 68+MEHD 941+MEHD CUs CUs CUs CUs Control GDC-68 GDC-941 MEHD GDC-68+MEHD GDC-941+MEHD 2 15 ther3 Control GDC-68 GDC-941 MEHD GDC-68+MEHD GDC-941+MEHD 3 2 tegfr * ** Figure S3: CEER analysis of HCC7 xenografts. Quantification of active and total EGFR and HER3 in HCC7 xenografts treated as indicated. Data are shown as mean ± SEM from at least 3 independent tumors. P-value was calculated using two-sided student s t-test. *P=.25 vs GDC-941 (pegfr). **P=.54 vs GDC-941 (pher3).

5 EGFR PTEN KI67 PDX4 PDX3 PDX2 Figure S4: Immunostaining for EGFR, PTEN, and Ki67 in patient-derived TNBC xenografts. Detection of EGFR, PTEN, and Ki67 in three independent patient-derived TNBC xenografts. Scale bar, 5 µm.

6 Figure S5: Imaging of EGFR and HER3 in patient-derived TNBC treated with GDC-68. (A) Representative PET images obtained 12 h post-injection of 89Zr-MEHD7945A in mice treated with placebo (control) or 4mg/Kg GDC-68 (treated). Observed uptake in the liver (L) is rationalized as the main route of excretion. (B) Quantification of the regions-of-interest drawn on the tumors (n=6 for each group) confirming a two-fold increase in 89Zr-MEHD7945A uptake in treated (i.e. 7.2 ± 2.6 %ID/g at 24 h, 8.28 ± 1.93 %ID/g at 48, and 9.76 ± 1.51 %ID/g at 12 h p.i.) versus control (4.5 ±.98 %ID/g at 24 h, 4.99 ± 1.1 %ID/g at 48, and 5.74 ± 2.4 %ID/g at 12 h p.i.) mice. P=.6. For each cohort, 3 mice bearing bilateral tumors were analyzed.

7 A 1 CTRL GDC-68 GDC-941 MEHD GDC-68+MEHD GDC-941+MEHD Ki67 staining (%) P=.42 P=.47 B Total CTCs Ki67-pos CTCs CTCs / 5ul CTRL GDC-68 GDC-941 MEHD GDC-68+MEHD GDC-941+MEHD * ** Figure S6: Ki67 staining of tumors and CTCs from PDXs treated with MEHD7945A and PI3K-Akt pathway inhibitors. Ki67 staining and quantification of tumors (A) and CTCs (B) of mice bearing PDXs treated as indicated. MEHD7945A was given 1mg/Kg twice weekly, GDC-68 4mg/Kg daily and GDC mg/Kg daily. CTCs were quantified in three independent mice per condition. Data are shown as mean ± SEM. P value was calculated using two-sided student s t-test. *P=.2 vs CTRL. **P=.23 vs CTRL. Scale bar, 5 µm.

8 12 1 HER3 actin 12 1 Viability (%) * * sictr siher * Viability (%) * sictr siher3 * GDC68 (µm) GDC941 (µm) Figure S7: HER3 knockdown in MDA-MB-468 cells treated with GDC-68 or GDC-941. Cell viability of MDA-MB-468 cells treated as displayed with DMSO (CTR) or either GDC-68 (left) or GDC-941 (right) in the presence of HER3 knock-down. The experiments have been performed two times in triplicate. Western blot showing HER3 abundance after transfection with sirna for HER3 is shown between the graphs. Data are shown as mean ± SEM. P-value was calculated using two-sided student s t-test. *P<.1 siher3 vs control (for both conditions).

9 HCC7 HCC7 PDX 7 1 Mean Tumor Volume (mm 3 ) Vehicle Cetuximab GDC68 Combo Mean Tumor Volume (mm 3 ) Vehicle Vehicle Cetuximab Cetuximab GDC68 GDC68 Combo Combo Days of treatment Days of treatment Figure S8: Tumor growth of HCC7 and PDXs treated with cetuximab and GDC-68. HCC7- (left) and patient-derived xenografts (right) were treated as indicated. Cetuximab was given 1 mg/kg twice weekly and GDC-68 4 mg/kg daily. N=8-1 for each arm. Data are shown as mean ± SEM. P-value was calculated using two sided student s t-test. No statistical difference was observed between the combination arms and GDC-68 single agent.

10 Figure S9: Correlation between EGFR mrna expression after cetuximab treatment and overall survival in TNBC metastatic patients. (A) mrna expression of EGFR before (blue bar) and after treatments (red bar). Samples labeled as single indicate patients treated with cetuximab alone while Combo refers to patients treated with cetuximab in combination with carboplatin. (B) Kaplan Meier overall survival analysis of patients stratified in two groups based on the abundance of EGFR mrna after treatments (down vs no change (nc)/up) using the log-rank test.

11 3 Pre Post 2 1 HER3 Histoscore 12F514 12F154 11H H145 9H557 12R19 12F156A 9H211 9H H143 9H627 12F36 12H8 12H H1512 9H F155A 9H F157 12F24 Patients Figure S1: Quantification of HER3 abundance in paired samples from patients treated with cetuximab. Quantification of IHC for HER3 in baseline and residual tumors of the patients who did not achieve pcr upon treatment with cetuximab-based therapy (p=.88).

12 Figure S11: Antibody specificity for FRET analysis. (A) Cells expressing plasmid encoding EGFR (top) or HER3 (bottom) stained with anti-egfr-alexa546 and anti-her3-cy5, respectively. (B) Breast cancer tissue test-blocks co-stained with the same antibodies. Scale bar, 5 µm.

13 Table S1. CEER values (single replicas). (A) HCC7 xenografts from experiment shown in fig S3. (B) PDXs from experiment shown in Fig. 1D. (C) HCC7 xenografts from experiment shown in Fig. 2B. Cetux=cetuximab; L= lower than detectable range; TCK=total cytokeratins. A Samples ID pegfr tegfr pher3 ther3 CK CT 241RA CT CT GDC GDC GDC GDC GDC GDC MEHD7945A MEHD7945A MEHD7945A MEHD MEHD MEHD MEHD MEHD MEHD 342 L

14 B Samples ID pegfr tegfr pher3 ther3 TCK CT CT CT CT MEHD MEHD MEHD MEHD GDC GDC GDC GDC GDC GDC GDC GDC GDC68 + MEHD GDC68 + MEHD GDC68 + MEHD GDC68 + MEHD GDC941 + MEHD GDC941 + MEHD GDC941 + MEHD GDC941 + MEHD

15 C Samples ID pegfr tegfr pher3 ther3 CK CT CT CT 257 RP CT CT CT 257 RA CT Cetux 261 L Cetux 24 L Cetux 258 L Cetux 255 L Cetux 227 L Cetux 233 L MEHD MEHD MEHD MEHD MEHD MEHD 217 L MEHD 22 L L GDC L GDC L GDC L GDC L GDC GDC L GDC L

16 GDC Cetux + GDC L Cetux + GDC L Cetux + GDC L Cetux + GDC L Cetux + GDC L Cetux + GDC L MEHD + GDC L MEHD + GDC L MEHD + GDC L L L L L MEHD + GDC MEHD + GDC L MEHD + GDC L

17 Table S2. Patient clinicopathological information. Pre-Th= pre-treatment; post-th=post treatment. Statistical analysis was performed using Microsoft Excel and Statistics Epidemiology Medicine (SEM). The increase in HER3 abundance in panitumumab and cetuximab-treated patient samples (pooled) was statistically significant (p=.15). Sample ID PANITUMUMAB TRIAL CASES WHO DID NOT ACHIEVE pcr Treatment Response EGFR score HER3 score PTEN status pre- postpre-th post-th pre-th post-th Th Th 9H242 P-FEC-TAX non-pcr 24 6 neg neg 9H2679 P-FEC-TAX non-pcr pos pos 9H2587 P-FEC-TAX non-pcr neg neg 1F192 P-FEC-TAX non-pcr 4 uninterpretable uninterpretable nd nd 1H283 P-FEC-TAX non-pcr pos pos 1H54 P-FEC-TAX non-pcr pos neg 12F13 P-FEC-TAX non-pcr 1 6 neg pos 1H1565 P-FEC-TAX non-pcr neg nd 1F32 P-FEC-TAX non-pcr 3 3 neg pos 1F226 P-FEC-TAX non-pcr neg nd 1F383 P-FEC-TAX non-pcr neg neg 1F352 P-FEC-TAX non-pcr neg pos 1F542 P-FEC-TAX non-pcr 5 15 neg neg 1F621 P-FEC-TAX non-pcr neg neg 11F48 P-FEC-TAX non-pcr neg neg 11F47 P-FEC-TAX non-pcr neg neg 12F12 P-FEC-TAX non-pcr 13 2 pos pos 11F41 P-FEC-TAX non-pcr pos pos 1F712 P-FEC-TAX non-pcr nd nd 11H283 P-FEC-TAX non-pcr nd (exhausted) nd (exhausted) pos nd 1H153 P-FEC-TAX non-pcr pos pos

18 1F539 P-FEC-TAX non-pcr 8 8 neg neg 1F626 P-FEC-TAX non-pcr neg neg 11F651 P-FEC-TAX non-pcr neg neg PANITUMUMAB TRIAL CASES WHO ACHIEVED pcr Sample ID Treatment Response EGFR score HER3 score PTEN status pre- pre- postpost-th pre-th post-th Th Th Th 1H824 P-FEC-TAX pcr 3 nd (no tumor cells) 5 nd (no tumor cells) pos nd 1H1168 P-FEC-TAX pcr 13 nd (no tumor cells) 5 nd (no tumor cells) pos nd 1F548 P-FEC-TAX pcr 3 nd (no tumor cells) 2 nd (no tumor cells) pos nd 1F225 P-FEC-TAX pcr nd (no tumor cells) 25 nd (no tumor cells) neg nd 1H1621 P-FEC-TAX pcr 19 nd (no tumor cells) 15 nd (no tumor cells) neg nd 11F131 P-FEC-TAX pcr nd (no tumor cells) 5 nd (no tumor cells) pos nd 1F722 P-FEC-TAX pcr 11 nd (no tumor cells) nd (no tumor cells) neg nd 11F34 P-FEC-TAX pcr 2 nd (no tumor cells) nd (no tumor cells) neg nd 1F547 P-FEC-TAX pcr 11 nd (no tumor cells) 1 nd (no tumor cells) nd nd 1F622 P-FEC-TAX pcr 19 nd (no tumor cells) nd (no tumor cells) neg nd 1F549 P-FEC-TAX pcr 19 nd (no tumor cells) 5 nd (no tumor cells) nd nd 1F538 P-FEC-TAX pcr 5 nd (no tumor cells) 1 nd (no tumor cells) neg nd 1H1873 P-FEC-TAX pcr 14 nd (no tumor cells) nd (no tumor cells) neg nd 1H757 P-FEC-TAX pcr 14 nd (no tumor cells) 5 nd (no tumor cells) neg nd 1F224 P-FEC-TAX pcr nd (no tumor cells) 5 nd (no tumor cells) neg nd 9H2631 P-FEC-TAX pcr 26 nd (no tumor cells) 5 nd (no tumor cells) pos nd

19 CETUXIMAB TRIAL CASES WHO DID NOT ACHIEVE pcr Sample ID Treatment Response EGFR score HER3 score PTEN status pre-th post-th pre-th post-th pre- Th post-th 9H557 CTX-TAX non-pcr pos pos 9H627 CTX-TAX non-pcr neg neg 12F514 CTX-TAX non-pcr 19 neg neg 9H211 CTX-TAX non-pcr neg neg 9H2262 CTX-TAX non-pcr pos pos 9H2432 CTX-TAX non-pcr nd nd 9H2358 CTX-TAX non-pcr neg neg 11H1178 CTX-TAX non-pcr pos pos 11H1512 CTX-TAX non-pcr pos neg 12F155A CTX-TAX non-pcr neg neg 12F157 CTX-TAX non-pcr pos pos 12F154 CTX-TAX non-pcr 8 15 pos neg 12R19 CTX-TAX non-pcr uninterpretable pos pos 12H8 CTX-TAX non-pcr neg pos 12H143 CTX-TAX non-pcr neg pos 12H1116 CTX-TAX non-pcr neg neg 12F156A CTX-TAX non-pcr pos neg 12F24 CTX-TAX non-pcr neg neg 12F36 CTX-TAX non-pcr neg neg 12H145 CTX-TAX non-pcr pos pos

20 CETUXIMAB TRIAL CASES WHO ACHIEVED pcr Sample ID preth Treatment Response EGFR score HER3 score PTEN status pre- pre- postpost-th pre-th post-th Th Th Th 12F327 CTX-TAX pcr nd (no tumor cells) 5 nd (no tumor cells) pos nd 12F515 CTX-TAX pcr 9 nd (no tumor cells) nd (no tumor cells) neg nd 12F513 CTX-TAX pcr nd (no tumor cells) nd (no tumor cells) neg nd 11H923 CTX-TAX pcr 9 nd (no tumor cells) 8 nd (no tumor cells) pos nd 12F12 CTX-TAX pcr 8 nd (no tumor cells) 2 nd (no tumor cells) pos nd 12F142 CTX-TAX pcr 12 nd (no tumor cells) 1 nd (no tumor cells) pos nd