Supplemental Figure S3. A. Tumors harvested on treatment d 7 were assessed by IHC for Arg-1 and FoxP3. Representative images are shown. Original magnification, 400X. N = 5. B-C. RT-qPCR gene expression analysis of Ripk1 and Ripk3 (B) and Gas6 (C) from tumors treated with vehicle or lapatinib on treatment day 1, followed by 6 daily treatments with vehicle or BMS-777607. Tumors were harvested 24 h after final treatment. Individual data points represent the average value of 5 experimental replicates from RNA harvested from a single tumor. Midlines are the average ({plus minus} S.D.) of the biological replicates (N = 3-4 per group). P values calculated using Student's T-test.

4T1 mouse mammary tumors were grown orthotopically in athymic Balb/C (nu/nu) female mice. Tumor-bearing mice were randomized into treatment groups for intratumoral delivery of SLR20 (or OH-SLR20, or saline) on treatment days 1, 5, and 9. Tumors were measured throughout treatment (days 1-10) and for 5 days after treatment ceased (days 10-15). N = 7 per group.

Immunohistochemistry was used to measure CD45, F4,80, CD4, and CD8 in tumors harvested at day 5. Representative Images are shown. N = 5

4T1 cells were transfected with SLR20 or OH-SLR20. At 4 hours after transfection, 4T1 cells were washed 5 times, then cultured in serum-free media for 16 hours. 4T1-cultured supernatant was collected, passed through a 0.2ï­m filter, then added neat to cultures of Raw264.7 cells for 30 minutes. Western analysis of RAW264.7 whole cell lysates was used to measure P-STAT1. Cells were transfected, and after 16 h, total RNA was assessed by RT-qPCR to measure expression of the indicated gene.. Each point represents the average of three experimental replicates, N = 3. Midlines are average {plus minus} S.D. Student's T-test.

The effect of U46619 and CPI211 on migration and invasion of 4T1 and MDA-MB-231 tumor cells through Matrigel-coated transwells was assessed. Tumor cells were seeded in the upper chambers of a Matrigel-coated transwell insert in serum-free media. Cell migration towards 1% serum in the lower chamber was assessed by crystal violet staining of the lower side of the transwell filter. Digital images of stained transwell filters were used to count the number of migrated cells. Representative images are shown (A). Each data point shown (B) represents the number of cells migrating to the lower side of the filter. Midlines are the average {plus minus} S.D., N = 4 (4T1) and 5 (MDA-MB-231), each assessed in triplicate.

PDF file - 105K, Tables describing the somatic SNPs and indels identified by WGS, SV validation results, PCR primers used for validation, and tumor measurements from the patient treated with TAK-733

Supplemental Figure S6. Tumors harvested on treatment d 7 were assessed by IHC for FoxP3 and Arg-1. Representative images are shown. Original magnification = 400X. Asterisks represent areas of acellular debris. Black arrows indicate tumor infiltrating lymphocytes (TILs). Yellow arrows indicate hyper-condensed nuclei characteristic of apoptotic bodies / apoptotic debris.

A. Schematic of treatment strategy for intra-tumoral nanoparticle delivery of SLR20 (or OH-SLR20) to WT Balb/C mice harboring 4T1 mammary tumors. Saline was delivered intratumorally as a control. Tumors were measured throughout treatment . B. Tumor volume was measured beginning at treatment day 0. N = 7-8 per group.

S3. Representative images of cells grown in 3D Matrigel for 14D and treated with ABT-263 (1.0 uM). S4. Whole cell lysates from cells treated with ABT-263 (1.0uM) for 0=24 hours.

Chemical structure of CPI211 is shown in Panel A. Western analysis of 293T cells expressing lentiviral empty vector, TPr-WT, or TPr-T399A s shown in Panel B. Antibodies used are shown to the left of each panel.

Supplemental Figure S1. Kaplan meier analysis and oncoprint mapping of Luminal A/B (A) and Basal-like (B) TCGA-curated breast cancers, showing the tumors in each set with upregulation of Akt1/2/3 mRNA (red boxes) or phospho-S473 (black arrows), and the survival of these patients (red Kaplan-Meier line) versus other patients (blue Kaplan-Meier line). Log-rank test shows insignificance. Supplemental Figure S2. Rictor expression in human breast cancer. Supplemental Figure S3. Oncoprint of TCGA-curated invasive breast cancers (TCGA, Cell 2015, N = 971) assessed for RICTOR genomic alterations. Analysis shows 8% of cases (76/971) with gene amplification, overexpression, missense mutation, or deep deletion. Supplemental Figure S4. Rictor mRNA expression was measured by qRT-PCR of total RNA from mammary glands harvested from virgin female 12-week old mice. Supplemental Figure S5. Genetic ablation of Rictor decreases growth and survival of HER2-positive breast cancer cells. Supplemental Figure S6. A. Cells were cultured 6 hours with FITC-labeled Annexin V, then imaged. B. MCF10-HER2 cells (parental, Rictor, SFN, and Rictor ZFN + exogenous expression of myc-tagged Rictor) were cultured 6 h with An-nexin V-FITC. C. The percentage of cells in panel B that were labeled with Annexin V-FITC was quantitated. Averages (midlines) ± S.D. were assessed for significance using Student€™s two-tailed unpaired T-test. Supplemental Figure S7. Rictor-mediated Akt activity is necessary and sufficient for breast cancer cell survival. Supplemental Figure S8. Cells were treated with lapatinib for the time course indicated. Total cell RNA was used for qRT-PCR to measure relative RPTOR mRNA expression. Supplemental Figure S9. Cells were cultured 7 days in 10% serum plus lapatinib (1 μM) or PP242 (1 μM) or the combination of lapatinib + PP242. Cells were stained with crystal violet. Crystal violet fluorescence was scanned using the odyssey. Representative images are shown. Supplemental Figure S10. Loss of Rictor/mTORC2 sensitizes parental and resistant cells to lapatinib in vitro Supplemental Figure S11. Cells were treated 6h with PP242 (1μM) ± lapatinib (1μM) and assessed by luminescent caspase-3/7 assay.

Supplemental Figure S1. Tumors harvested on treatment d 1 and d 7 were assessed by IHC for P-Neu Y1248. Representative images are shown. Original magnification, 400X. N = 5. Histological analysis of H&E-stained tumor sections. Tumors were collected on treatment day 2 (i.e., 1 day after treatment {plus minus} lapatinib). Representative images are shown. N = 5. Original magnification was 400X. Tumors harvested on treatment d 2 were assessed by IHC for CD3 and FoxP3. Representative images are shown. Original magnification, 400X. N = 5. Tumors harvested on treatment d 2 were assessed by IHC for CD3 and FoxP3. Representative images are shown. Original magnification, 400X. N = 5. RT-qPCR analysis of tumor RNA harvested at treatment d 7 measuring relative levels of indicated gene transcripts. Values were calculated using the ddCT method. Each data point represents the average value of 5 technical replicates, N = 5 tumors. For each transcript, values were corrected for the average value measured in vehicle-treated samples. Student's T-test.

Supplemental Figure S5. Decreased tumor cellularity of tumors treated with the combination of lapatinib + BMS-777607 + epacadostat. Low power images of H&E-stained sections of tumors collected at treatment day 7. Arrows point to tumor cells, which stain darker purple, and which are evident as solid sheets in vehicle-treated samples, but only as pockets in the samples treated with lapatinib + BMS-777607 + Epacadostat. Asterisks represent areas of acellular debris.

Supplemental Figure S7. MMTV-Neu tumors were treated lapatinib on days 1 and 7, and with BMS-777607 on days 14. Average tumor volume is shown.

PDF file - 198K, Breast cancer cell lines and receptor expression (S1); Correlations among optical metabolic measurements (S2).

S1. CCLE-curated cancer cells were grouped according to tumor type and assessed for miRNA expression for BCL2 and BCL2L1 (Bcl-xL) using cBio portal (www.cbio.org). S2. Whole cell lysates form a panel of breast cancer cell lines were assessed by western analysis (BL = Basal-like).

Supplemental Figure Legends and Supplemental tables (Table 1 and table 2)

Abstract Kinase inhibitors are accepted treatment for metastatic melanomas that harbor specific driver mutations in BRAF or KIT, but only 40% to 50% of cases are positive. To uncover other potential targetable mutations, we conducted whole-genome sequencing of a highly aggressive BRAF (V600) and KIT (W557, V559, L576, K642, and D816) wild-type melanoma. Surprisingly, we found a somatic BRAFL597R mutation in exon 15. Analysis of BRAF exon 15 in 49 tumors negative for BRAFV600 mutations as well as driver mutations in KIT, NRAS, GNAQ, and GNA11, showed that two (4%) harbored L597 mutations and another two involved BRAF D594 and K601 mutations. In vitro signaling induced by L597R/S/Q mutants was suppressed by mitogen-activated protein (MAP)/extracellular signal–regulated kinase (ERK) kinase (MEK) inhibition. A patient with BRAFL597S mutant metastatic melanoma responded significantly to treatment with the MEK inhibitor, TAK-733. Collectively, these data show clinical significance to BRAFL597 mutations in melanoma. Significance: This study shows that cells harboring BRAFL597 mutants are sensitive to MEK inhibitor treatment, providing a rationale for routine screening and therapy of BRAFL597-mutant melanoma. Cancer Discov; 2(9); 791–7. ©2012 AACR. Read the Commentary on this article by Garraway and Baselga, p. 766. This article is highlighted in the In This Issue feature, p. 753.

Supplemental Figure S2. Schematic representation of treatment groups and experimental timeline to measure the impact of PtdSer liposomes on the TME of 4T1 mouse mammary tumors IHC to detect CD3, and FoxP3 in tumors collected on treatment day 7. Quantitation of the number of positive cells per 200X field is shown. Each data point is the average of 5 random fields per tumor, midlines are the average of N = 3 samples, {plus minus}S.D. P values, Student's unpaired 2-tailed T-test.