1. Supplementary Figures and Tables
Entrectinib, a Pan-TRK, ROS1 and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications
Elena Ardini, Maria Menichincheri, Patrizia Banfi, Roberta Bosotti, Cristina De Ponti, Romana Pulci, Dario Ballinari, Marina Ciomei, Gemma Texido, Anna Degrassi, Nilla Avanzi, Nadia Amboldi, Maria Beatrice Saccardo, Daniele Casero, Paolo Orsini, Tiziano Bandiera, Luca Mologni, David Anderson, Ge Wei, Jason Harris, Jean-Michel Vernier, Gang Li, Eduard Felder, Daniele Donati, Antonella Isacchi, Enrico Pesenti, Paola Magnaghi and Arturo Galvani
Supplementary Figures and Tables

2. Supplementary Figure 1
days after tumor cell injection
Figure S1. In vivo efficacy of entrectinib in KM12 xenograft model
Nu/Nu mice bearing established KM12 xenografts were administered entrectinib per os bid at the indicated doses or vehicle daily for 4 consecutive days followed by 3 days off treatment for a total of 3 cycles. Tumor volume for each group was measured. Data are expressed as mean ± SD (n = 7).

3. Supplementary Figure 2
Figure S2. Mechanism of Action of entrectinib in ALK-transformed Ba/F3 cells
Ba/F3 cells expressing TEL-ALK were treated with the indicated concentrations of entrectinib or crizotinib for 2 h. Levels of ALK and phospho-ALK were detected by immunoblot analysis using specific antibodies.

4. Supplementary Figure 3 A B
Figure S3. Analysis of cell cycle distribution
(A and B) Karpas-299 (A) and SR786 (B) ALCL cells were treated with different doses of entrectinib for 24 h, 48 h or 72 h. Cell cycle distribution was evaluated by PI staining and cytofluorimetric analysis.

5. A B Supplementary Figure 4
Figure S4. Mechanism of action of entrectinib in ALK-driven ALCL cell lines
(A and B) SUP-M2 (A) and SU-DHL-1 cells (B) were treated with entrectinib at different concentrations for 2 h. Levels of proteins and phospho-proteins were detected by immunoblot analysis using specific antibodies.

6. Supplementary Figure 5
days after tumor cell injection
Figure S5. Activity of entrectinib against NCI-H2228 NSCLC tumors
Nu/Nu mice bearing established NCI-H2228 xenografts were administered entrectinib per os bid at the indicated doses or vehicle daily for 4 consecutive days followed by 3 days off treatment for a total of 4 cycles. Tumor volume for each group was measured. Data are expressed as mean ± SD (n = 8).

7. Supplementary Figure 6 A B C D
PRE-TREATMENT
POST-TREATMENT
VEHICLE
ENTRECTINIB 120 mg/Kg
ENTRECTINIB 60 mg/Kg
CRIZOTINIB 100 mg/Kg A D C B
Figure S6. Activity of entrectinib against an intracranial growth model
Mice bearing intracranial tumors were administered (A) vehicle, (B and C) entrectinib per os bid or (D) crizotinib per os die at the indicated doses for 10 consecutive days. Representative magnetic resonance images of vehicle- or drug-treated mice are shown.

8. Supplementary Table 1. Antiproliferative activity of entrectinib on human tumor cell lines

9. Supplementary Table 1. Antiproliferative activity of entrectinib on human tumor cell lines
(con’t)

10. Supplementary Table 1. Antiproliferative activity of entrectinib on human tumor cell lines
(con’t)

11. Supplementary Table 1. Antiproliferative activity of entrectinib on human tumor cell lines
(con’t)

12. Supplementary Table 2. Pharmacokinetic parameters of entrectinib in CD1 mice
§ All values cited are those measured following a single administration of entrectinib, except for brain/plasma ratio, which was measured following 10 daily repeated administrations.