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Suppl Figure 1. Chemical structure and molecular data for TAK-733

Suppl Figure 3. Immunoblot of Effector Proteins and Markers of Apoptosis in Melanoma Cell Lines

Suppl Figure 2. Immunoblot Analysis of Effector Proteins in Melanoma Cell Lines

Suppl Table 1. IC50 Value Matrix

Suppl Figure 5. Immunoblot of Effector Proteins in Tumors from PDTX Mice Treated with TAK-733

Suppl Figure 4. Representative PDTX Model MB1255 Treated with Vehicle or TAK-733

Suppl Table 2. TAK-733 PD/ Conc. Summary, A375 tumor bearing nude mouse

Suppl Table 4. Antitumor Activity of Orally Administered TAK-733 Against A375 Melanoma Tumor Xenografts in Female Athymic Nude Mice

Suppl Figure 6. Genomic Analyses of PDTX MB1374 Treated to Resistance

Supplementary Figure Legends

The goal of this study was to investigate the activity of the selective MEK1/2 inhibitor TAK-733 in both melanoma cell lines and patient-derived melanoma xenograft models. In vitro cell proliferation assays using the sulforhodamine B assay were conducted to determine TAK-733 potency and melanoma responsiveness. In vivo murine modeling with eleven patient-derived melanoma explants evaluated daily dosing of TAK-733 at 25 or 10 mg/kg. Immunoblotting was performed to evaluate on-target activity and downstream inhibition by TAK-733 in both in vitro and in vivo studies. TAK-733 demonstrated broad activity in most melanoma cell lines with relative resistance observed at IC50 > 0.1 μmol/L in vitro. TAK-733 also exhibited activity in 10 out of 11 patient-derived explants with tumor growth inhibition ranging from 0% to 100% (P < 0.001–0.03). Interestingly, BRAFV600E and NRAS mutational status did not correlate with responsiveness to TAK-733. Pharmacodynamically, pERK was suppressed in sensitive cell lines and tumor explants, confirming TAK-733–mediated inhibition of MEK1/2, although the demonstration of similar effects in the relatively resistant cell lines and tumor explants suggests that escape pathways are contributing to melanoma survival and proliferation. These data demonstrate that TAK-733 exhibits robust tumor growth inhibition and regression against human melanoma cell lines and patient-derived xenograft models, suggesting that further clinical development in melanoma is of scientific interest. Particularly interesting is the activity in BRAF wild-type models, where current approved therapy such as vemurafenib has been reported not to be active. Mol Cancer Ther; 14(2); 317–25. ©2014 AACR.

Purpose: This first-in-human study assessed safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary clinical activity of single and multiple doses of TAK-441, an investigational inhibitor of the Hedgehog signaling pathway.Experimental Design: Patients with advanced, solid tumors received daily oral TAK-441 (50–1,600 mg/day); daily dose was doubled in each subsequent cohort until the maximum tolerated/feasible dose (MTD/MFD) was reached. Blood was collected to evaluate TAK-441 plasma concentrations. Skin biopsies were obtained to evaluate suppression of the Hedgehog-regulated gene Gli1.Results: Thirty-four patients were enrolled (median age 59). The most common diagnoses were colorectal cancer (26%), basal cell carcinoma (BCC, 21%), and pancreatic cancer (9%). The MFD of 1,600 mg/day (based on tablet size and strength) was considered the MTD. Dose-limiting toxicities included muscle spasms and fatigue. Grade ≥3 treatment-emergent adverse events, regardless of causality, occurred in 15 patients (44%), of which hyponatremia (n = 4) and fatigue (n = 3) were most common. Oral absorption was fairly rapid; median Tmax was 2.0 to 4.0 hours after a single dose. Mean elimination half-life was 13.5 to 22.6 hours. Systemic exposure of TAK-441 based on the area under the plasma concentration–time curve was linear across the dose range. Gli1 expression in skin biopsies was strongly inhibited at all dose levels. Best response was partial response (1 patient with BCC) and stable disease (7 patients with various solid tumors).Conclusions: TAK-441 was generally well tolerated up to MFD of 1,600 mg/day, with preliminary antitumor activity. Further study of TAK-441 may be appropriate in populations selected for tumors with ligand-dependent or independent Hedgehog signaling. Clin Cancer Res; 21(5); 1002–9. ©2014 AACR.

Supplementary Table S1. On-study patient deaths

Purpose: This first-in-human study assessed safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary clinical activity of single and multiple doses of TAK-441, an investigational inhibitor of the Hedgehog signaling pathway. Experimental Design: Patients with advanced, solid tumors received daily oral TAK-441 (50–1,600 mg/day); daily dose was doubled in each subsequent cohort until the maximum tolerated/feasible dose (MTD/MFD) was reached. Blood was collected to evaluate TAK-441 plasma concentrations. Skin biopsies were obtained to evaluate suppression of the Hedgehog-regulated gene Gli1. Results: Thirty-four patients were enrolled (median age 59). The most common diagnoses were colorectal cancer (26%), basal cell carcinoma (BCC, 21%), and pancreatic cancer (9%). The MFD of 1,600 mg/day (based on tablet size and strength) was considered the MTD. Dose-limiting toxicities included muscle spasms and fatigue. Grade ≥3 treatment-emergent adverse events, regardless of causality, occurred in 15 patients (44%), of which hyponatremia (n = 4) and fatigue (n = 3) were most common. Oral absorption was fairly rapid; median Tmax was 2.0 to 4.0 hours after a single dose. Mean elimination half-life was 13.5 to 22.6 hours. Systemic exposure of TAK-441 based on the area under the plasma concentration–time curve was linear across the dose range. Gli1 expression in skin biopsies was strongly inhibited at all dose levels. Best response was partial response (1 patient with BCC) and stable disease (7 patients with various solid tumors). Conclusions: TAK-441 was generally well tolerated up to MFD of 1,600 mg/day, with preliminary antitumor activity. Further study of TAK-441 may be appropriate in populations selected for tumors with ligand-dependent or independent Hedgehog signaling. Clin Cancer Res; 21(5); 1002–9. ©2014 AACR.

The goal of this study was to investigate the activity of the selective MEK1/2 inhibitor TAK-733 in both melanoma cell lines and patient-derived melanoma xenograft models. In vitro cell proliferation assays using the sulforhodamine B assay were conducted to determine TAK-733 potency and melanoma responsiveness. In vivo murine modeling with eleven patient-derived melanoma explants evaluated daily dosing of TAK-733 at 25 or 10 mg/kg. Immunoblotting was performed to evaluate on-target activity and downstream inhibition by TAK-733 in both in vitro and in vivo studies. TAK-733 demonstrated broad activity in most melanoma cell lines with relative resistance observed at IC50 > 0.1 μmol/L in vitro. TAK-733 also exhibited activity in 10 out of 11 patient-derived explants with tumor growth inhibition ranging from 0% to 100% (P < 0.001–0.03). Interestingly, BRAFV600E and NRAS mutational status did not correlate with responsiveness to TAK-733. Pharmacodynamically, pERK was suppressed in sensitive cell lines and tumor explants, confirming TAK-733–mediated inhibition of MEK1/2, although the demonstration of similar effects in the relatively resistant cell lines and tumor explants suggests that escape pathways are contributing to melanoma survival and proliferation. These data demonstrate that TAK-733 exhibits robust tumor growth inhibition and regression against human melanoma cell lines and patient-derived xenograft models, suggesting that further clinical development in melanoma is of scientific interest. Particularly interesting is the activity in BRAF wild-type models, where current approved therapy such as vemurafenib has been reported not to be active. Mol Cancer Ther; 14(2); 317–25. ©2014 AACR.

Background: Immunohistochemistry provides visual context to protein modulation and is relied upon to elucidate mechanism of action, marker translocation and pharmacodynamics (PD) in drug discovery. Markers of the PI3K-MTOR pathway have been monitored in this way to aid in the clinical development of various inhibitors targeting this pathway. Here, assays were developed using preclinical xenograft PD studies to explore the feasibility of automated staining of pAKT 473, pS6, p4EBP1 and pRb. In preparation for clinical assessment of these markers in human skin samples; these markers were also examined in normal mouse skin. p4EBP1 was selected to examine the reproducibility of these automated assays and to explore the correlation between pathway inhibition in PD xenograft tissues and murine skin tissues. The automated staining technique was coupled with whole slide scanning and digital image analysis for quantitative comparison. Methods: Assays were chosen for evaluation based on known biology. FFPE PTEN-null breast cancer primary human tumor xenografts with high, medium and low biomarker expression, with companion skin samples, were selected for feasibility and reproducibility testing. Whole slide scanning and reproducibility analysis was executed using Aperio Scanscope XT and Spectrum software. Statistical analysis methods of mixed effect linear models and two sample t-tests were applied. Xenograft and skin samples from NCI-H1048 tumor bearing Nude mice treated with MTOR (MLN0128)and PI3Kα (MLN1117) investigational inhibitors were immunohistochemically stained for p4EBP1. Definiens object-based imaging software was used to isolate and analyze viable tumor area and exclude necrotic and stromal components in xenografts. A customized Definiens® algorithm was used on skin to identify the epithelial layer for analysis. Results: After evaluation, baseline biomarker expression in the xenograft and skin samples determined marker selection for future steps. Baseline p4EBP1 levels were high in both xenograft and skin. The reproducibility testing yielded consistent results in both xenograft and skin, validating autostaining use for PD studies. Phosphomarker p4EBP1 expression was inhibited after MLN0128 administration, with increased reduction after combination with MLN1117. Development of a novel analysis solution using Definiens software for skin samples permitted the area of interest to be restricted to the epithelial layer and excluded other epidermal features with similar morphometric characteristics. The p4EBP1 modulation was concordant between skin and tumor though more pronounced in skin, indicating that skin is a suitable surrogate tissue for determining modulation of this PI3K-MTOR pathway marker. Conclusions: Autostaining platforms provide reproducible results and stain consistently for PI3K-MTOR phosphomarkers on both xenografts and mouse skin samples. Utilizing p4EBP1, target effect was observed after MTOR and PI3Kα administration. This development and evaluation of the phosphobiomarker using automated staining technology and advanced image analysis supports preclinical PD assessment and combination clinical trials as well as influenced timepoint selection for clinical assessment. Citation Format: Anna Kreshock, Natalia Iartchouk, Yueying Cao, Jeffrey Szwaya, Feng Gao, Christopher Simpson, Evan Luongo, Kristine Burke, Esha Gangolli, Keisuke Kuida, Rachael Brake, Stephen Tirrell, Vaishali Shinde. Automated immunohistochemistry of phosphobiomarkers: Case study of MTOR (MLN0128) and PI3Kα (MLN1117) investigational inhibitors, single agent and in combination, on xenografts and mouse skin. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A41.

Background: MLN0128 is an investigational, potent, and highly selective inhibitor of mTORC1/2, which are integral to cell proliferation, angiogenesis, and cellular metabolism. This first-in-human study (NCT01058707) aimed to determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary antitumor activity of oral MLN0128. Methods: Pts aged ≥18 years with advanced solid tumors were enrolled in a 3+3 dose-escalation design initially to receive MLN0128 once daily (QD), and then enrolled in escalating intermittent schedules of once weekly (QW), 3 days/week (QDx3d QW), or 5 days/week (QDx5d QW) dosing, in 28-day cycles. Blood samples were collected at multiple timepoints for PK analysis. PD endpoints were evaluated in skin to determine the effect on mTORC1/2-dependent biomarkers. A preclinical PK-efficacy model was generated (Phoenix NLME v1.1) with tumor xenograft efficacy data, and implemented using clinical PK parameters to simulate tumor volume-time curves for various MLN0128 doses/schedules. Response was assessed by RECIST v1.1. Results: 115 pts received MLN0128 doses in the ranges 2-7 mg QD (n=30), 7-40 mg QW (n=30), 6-20 mg QDx3d QW (n=33), and 7-13 mg QDx5d QW (n=22). Median age was 60 years (range 24-89); 40% were male. The most common tumor types were colorectal (22%), renal (9%), and ovarian (8%) cancer. The MTDs were 6 mg QD, 40 mg QW, 16 mg QDx3d QW, and 10 mg QDx5d QW. Based on the overall safety profiles, the recommended phase 2 doses were 5 mg QD, 40 mg QW, 9 mg QDx3d QW, and 7 mg QDx5d QW. Pts received a median of 2 cycles (range 1-23). The most common drug-related adverse events (AEs) were hyperglycemia (65%), nausea (60%), vomiting (44%), decreased appetite (36%), diarrhea (33%), asthenia (30%), and mucosal inflammation (30%). The most common drug-related grade ≥3 AEs were hyperglycemia (12%), asthenia (9%), and mucosal inflammation (5%). MLN0128 exhibited dose-linear PK with a plasma half-life of ∼8 h, and did not accumulate in plasma with QD dosing. Modeling of preclinical PK-efficacy data and simulation of human tumor volume-time curves using clinical PK parameters suggested potential greater antitumor effect with schedules using more frequent dosing (i.e. QD, QDx5d QW). There was a treatment-related inhibition of mTORC1/2 biomarkers in skin. Two of 10 pts (20%) with renal cancer receiving MLN0128 at 15 and 40 mg QW had a partial response; 5 pts (6%) had stable disease for ≥6 cycles. Conclusions: Based on the safety profile, PK/PD, simulated tumor volume-time curves, and responses, MLN0128 5 mg QD and 40 mg QW were selected for further evaluation in an expansion phase in pts with renal, endometrial, or urothelial cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C252. Citation Format: Jeffrey R. Infante, Josep Tabernero, Andres Cervantes, Shadia Jalal, Howard A. Burris, Teresa Macarulla, J. Alejandro Perez-Fidalgo, Rachel Neuwirth, Chirag Patel, Esha Gangolli, Rachael Brake, Jeffrey Sturm, Eric H. Westin, Michael Gordon. A phase 1, dose-escalation study of MLN0128, an investigational oral mammalian target of rapamycin complex 1/2 (mTORC1/2) catalytic inhibitor, in patients (pts) with advanced non-hematologic malignancies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C252.