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Cancer testis antigens have been of interest as possible targets for cancer immunotherapies. To better understand the opportunities for the use of such immunotherapy targets, we used a chemical complementarity scoring algorithm and an original web tool to establish aspects of electrostatic complementarity of the CTAs, MAGEA3 and MAGEA6, with melanoma specimen resident, T-cell receptor (TCR) complementarity determining region 3 (CDR3) amino acid sequences. Greater electrostatic complementarity between T-cell receptor CDR3 and tumor CTAs MAGEA3/6 was associated with a greater probability of overall survival, for both the cancer genome atlas and Moffitt Cancer Center samples; and was associated with high levels of T-cell cytotoxicity-related gene expression. Most importantly, this approach allowed for the highly efficient screening of specific segments of the MAGEA3/6 antigens which indicated that certain MAGE segments would have either more or less risk of auto-reactivity. In sum, the chemical complementarity algorithm, and its efficient application via the web tool, adaptivematch.com, offers a convenient opportunity to identify likely parameters important for immunotherapy considerations and melanoma patient risk stratifications.

Transient transfection data

Details of experimental methods

The transcriptional coactivator YAP1 is the major oncogenic component of the Hippo signaling pathway and contributes to the genesis and progression of various tumors, including non–small cell lung cancer (NSCLC). YAP1 levels are regulated by the canonical Hippo kinases, MST1/2 and LATS1/2, which modulate its cytoplasmic retention and proteasomal degradation. While noncanonical regulation of YAP1 has been reported, its role in hypoxic response is not fully elucidated. The studies presented here show that YAP1 levels and function are modulated by von Hippel–Lindau (VHL) and prolyl hydroxylase 2 (PHD2). YAP1 could regulate multiple genes involved in angiogenesis through E2F1; it also associates with HIF1α in cancer cells under hypoxic conditions, inducing the VEGF-A promoter. Under normoxic conditions, PHD2 associates with and hydroxylates specific proline residues on YAP1, facilitating its interaction with VHL and promoting ubiquitination and subsequent proteasomal degradation. Exposure to hypoxia dissociates YAP1 from PHD2 and VHL, elevating YAP1 levels and enhancing its association with HIF1α. YAP1–HIF1α interaction was higher in NSCLC and renal cell carcinoma samples, indicating a role for this interaction in the genesis of these cancers. Our results thus reveal a novel mode of regulation of YAP1 by PHD2 and VHL in normoxic cells, suggesting that YAP1-mediated induction of VEGF and other genes contributes to hypoxic response in tumors.Significance:YAP1 under normoxic conditions is regulated by a novel nonclassical regulatory pathway involving PHD2-mediated prolylhydroxylation and proteasomal degradation; absence of this regulation under hypoxic conditions stabilizes YAP1, contributing to neoangiogenesis.

Supplementary Methods, radiologist's reading of CT images, supplementary figure legends, supplementary references

Supplementary Table 1: Table provides information on the siRNA sequences and/or chemical structures of the non-FDA approved inhibitors for bromodomain proteins, transcription factors and kinases described in the current study. Supplementary Table 2: Sequences of primers used for qRT-PCR analysis of various targets described in the study. Supplementary Table 3: Sequences of primers used for ChIP analysis of various targets described in the study. Supplementary Table 4: Analysis of HOXB13 target gene expression in the GSE92721 data set. Supplementary Table 5: Analysis of HOTBIN10 gene expression in the GSE92721data set. Supplementary Table 6: Analysis of HOTBIN10 gene expression in the Moffitt TCC data set. Supplementary Table 7: Clinical parameters of the primary cancers in the Moffitt TCC data set. Supplementary Table 8: Clinical parameters of the metastatic cancers in the Moffitt TCC data set. Supplementary Table 9: Analysis of HOTBIN10 genes in MSKCC gene expression data set (GSE21034). Supplementary Table 10: Analysis of HOTBIN10 gene expression in the GSE67980 CTC data set.

MiRNA expression in Malignant (N=21) versus Benign (N=21) IPMN cases, sorted by P value

Pancreatic cancer is an aggressive and deadly malignancy responsible for the death of over 37,000 Americans each year. Gemcitabine-based therapy is the standard treatment for pancreatic cancer but has limited efficacy due to chemoresistance. In this study, we evaluated the in vitro and in vivo effects of gemcitabine combined with the selective nuclear export (CRM1) inhibitor KPT-330 on pancreatic cancer growth. Human pancreatic cancer MiaPaCa-2 and metastatic pancreatic cancer L3.6pl cell lines were treated with different concentrations of KPT-330 and gemcitabine alone or in combination, and anchorage–dependent/independent growth was recorded. In addition, L3.6pl cells with luciferase were injected orthotopically into the pancreas of athymic nude mice, which were treated with (i) vehicle (PBS 1 mL/kg i.p., 2/week and povidone/pluronic F68 1 mL/kg p.o., 3/week), (ii) KPT-330 (20 mg/kg p.o., 3/week), (iii) gemcitabine (100 mg/kg i.p., 2/week), or (iv) KPT-330 (10 mg/kg) + gemcitabine (50 mg/kg) for 4 weeks. KPT-330 and gemcitabine alone dose-dependently inhibited anchorage-dependent growth in vitro and tumor volume in vivo compared with vehicle treatment. However, the combination inhibited growth synergistically. In combination, KPT-330 and gemcitabine acted synergistically to enhance pancreatic cancer cell death greater than each single-agent therapy. Mechanistically, KPT-330 and gemcitabine promoted apoptosis, induced p27, depleted survivin, and inhibited accumulation of DNA repair proteins. Together, our data suggest that KPT-330 potentiates the antitumor activity of gemcitabine in human pancreatic cancer through inhibition of tumor growth, depletion of the antiapoptotic proteins, and induction of apoptosis. Mol Cancer Ther; 14(7); 1570–81. ©2015 AACR.

This study evaluated the preclinical activity of δ-tocotrienol (DT3), a bioactive form of vitamin E, in the inhibition of colorectal cancer growth and development in vitro and in vivo. DT3 is the most bioactive isomer of vitamin E in inhibiting growth of colorectal cancer cells. However, it had little effect on the proliferation of normal colon mucosal cells NCM460. In HCT-116 and SW-620 colorectal cancer cells, DT3 (50 μmol/L) significantly inhibited malignant transformation (P < 0.02, P < 0.001), cell migration (P < 0.02, P < 0.05), and invasion (P < 0.05, P < 0.01) compared with vehicle. DT3 inhibited markers for epithelial (E-cadherin) to mesenchymal (vimentin) transition, metastasis (matrix metalloproteinase 9), angiogenesis VEGF, inflammation (NF-κB), and Wnt signaling (β-catenin) compared with vehicle in colorectal cancer cells. DT3 induced apoptosis selectively in colorectal cancer cells (SW-620 cells, HCT-116 cells, and HT-29) without affecting the normal colon cells. In the azoxymethane-induced colorectal carcinogenesis model in rats, DT3 (200 mg/kg orally twice a day) for 20 weeks significantly inhibited colorectal polyps by 70% and colorectal cancer by almost 99% compared with the vehicle treatment group (P < 0.02, P < 0.001), and the cancer inhibition effect was more potent than sulindac (50%). Taken together, these data demonstrate that DT3 is a potential chemopreventive agent in colorectal cancer, warranting further investigation into its clinical use in the prevention and treatment of colorectal cancer.

Supplemental legend for Fig. S1

This document contains the figure legends for Supplementary Figures 1-5.

miR-145-5p expression differentiates between cases and non-diseased controls. A) Box plot reveals miR-145-5p expression is higher in cases versus controls. B) ROC analysis reveals that miR-145-5p expression can differentiate between groups with an AUC=79.3 (95% CI: 68.3-90.3). Estimates of sensitivity, specificity, positive predictive value, and negative predictive value are 81%, 62.5%, 79%, and 65%, respectively.

Supplementary Fig. 1. Epigenetic landscape and the transcription cofactor recruitment at the HOXB13 enhancer region under various treatments; Supplementary Figure 2. BRD4 is a target of the dual BET-kinase inhibitors in CRPCs; Supplementary Figure 3. Sensitivity of normal and AR negative cell lines to the novel BET-kinase inhibitors; Supplementary Figure 4. HOXB13 but not FOXA1 is under the epigenetic control of BRD4 in prostate cancer cells; Supplementary Figure S5. Haploinsufficiency of HOXB13 sensitizes CRPCs to the novel BET-kinase inhibitors; Supplementary Figure S6. AURKB expression correlates with HOXB13 in human CTCs from mCRPC patients following Abiraterone therapy; Supplementary Fig. S7. AURKB is a therapeutic vulnerability of HOXB13 positive prostate cancers

This file contains Supplementary Table 1.

microCT scans

Supplementary Figure S1. Effect of KPT-330 on human normal pancreatic epithelial (HPNE) cell proliferation (MTT assay).

Please see this file for descriptions

SFigure 1: Histological examination of mouse lungs; SFigure 2: Tissue sections from human lung adenocarcinoma; SFigure 3: Cabozantinib (CBT) and vandetanib (VDT) resistant cells; SFigure 4: Comparison of in vitro inhibition of RET, RETV804L and RETV804M by ponatinib, cabozantinib, vandetanib, and lenvatinib; SFigure 5: Ponatinib treatment schedule and body weight measurements; SFigure 6: Lung section histology from Dox-induced C/KR mice and treated with vehicle or ponatinib for 1 months; SFigure 7: μCT examination of dox-induced C/KR mice with lung tumors before and after one month treatment with vehicle or ponatinib.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers worldwide, partly because methods are lacking to detect disease at an early, operable stage. Noninvasive PDAC precursors called intraductal papillary mucinous neoplasms (IPMN) exist, and strategies are needed to aid in their proper diagnosis and management. Data support the importance of miRNAs in the progression of IPMNs to malignancy, and we hypothesized that miRNAs may be shed from IPMN tissues and detected in blood. Our primary goals were to measure the abundance of miRNAs in archived preoperative plasma from individuals with pathologically confirmed IPMNs and healthy controls and discover plasma miRNAs that distinguish between IPMN patients and controls and between “malignant” and “benign” IPMNs. Using novel nCounter technology to evaluate 800 miRNAs, we showed that a 30-miRNA signature distinguished 42 IPMN cases from 24 controls [area underneath the curve (AUC) = 74.4; 95% confidence interval (CI), 62.3–86.5, P = 0.002]. The signature contained novel miRNAs and miRNAs previously implicated in pancreatic carcinogenesis that had 2- to 4-fold higher expression in cases than controls. We also generated a 5-miRNA signature that discriminated between 21 malignant (high-grade dysplasia and invasive carcinoma) and 21 benign (low- and moderate-grade dysplasia) IPMNs (AUC = 73.2; 95% CI, 57.6–88.9, P = 0.005), and showed that paired plasma and tissue samples from patients with IPMNs can have distinct miRNA expression profiles. This study suggests feasibility of using new cost-effective technology to develop a miRNA-based blood test to aid in the preoperative identification of malignant IPMNs that warrant resection while sparing individuals with benign IPMNs the morbidity associated with overtreatment. Cancer Prev Res; 8(9); 826–34. ©2015 AACR.

Figure legends for supplementary figures

Supplementary Figure S2. Distribution of mean Δβ for each experimental condition by normalization: (a) DASEN and (a) minfi-Illumina. Normal distribution of mean Δβ peaked approximately at 0 for DASEN and

Supplementary Figure Legend from Expanding Epigenomics to Archived FFPE Tissues: An Evaluation of DNA Repair Methodologies

IKBKE interacts with EGFR.

Association of IKBKE level with overall survival

Supplementary Table S2. The number and overlap of discordant loci (|Δβ|>0.3) across internal replicates of the Restore and REPLI-g Ligase methods for each patient sample.

Effects of inhibitor of IKBKE and MEK on HCC827 xenograft growth.

Purpose: This phase I study was conducted to determine the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) for the combination of bortezomib and alvocidib in patients with B-cell malignancies (multiple myeloma, indolent lymphoma, Waldenstrom macroglobulinemia, and mantle cell lymphoma).Experimental Design: Patients received bortezomib (intravenous push), followed by alvocidib (1-hour infusion), on days 1, 4, 8, and 11 of a 21-day treatment cycle. Patients experiencing responses or stable disease continued on treatment at the investigator's discretion. A standard 3+3 dose-escalation design was used to identify the MTD based on DLTs, and pharmacokinetic and pharmacodynamic studies were conducted.Results: A total of 44 patients were enrolled, with 39 patients assessed for response. The MTD was established as 1.3 mg/m2 for bortezomib and 40 mg/m2 for alvocidib. The most common hematologic toxicities included leukopenia, lymphopenia, neutropenia, and thrombocytopenia. The most common nonhematologic toxicities included diarrhea, fatigue, and sensory neuropathy. Three complete remissions (8%) and 10 partial remissions (26%) were observed for a total response rate of 33%. Pharmacokinetic findings with the current dosing regimen were consistent with the comparable literature and the hybrid dosing regimen. Pharmacodynamic study results did not correlate with clinical responses.Conclusions: The combination of bortezomib and alvocidib is tolerable, and an MTD has been established for this schedule. The regimen appears to be efficacious in patients with relapsed/refractory multiple myeloma or indolent non-Hodgkin lymphoma. As the nonhybrid regimen is less cumbersome than the previous hybrid dosing schedule regimen, the current schedule is recommended for successor studies. Clin Cancer Res; 20(22); 5652–62. ©2014 AACR.

Supplementary Figures S1-S2; Supplementary Tables S1-S3.

Identification of IKBKE tyrosine residue(s) phosphorylated by EGFR

Background: Epigenome-wide association studies are emerging in the field of cancer epidemiology with the rapid development of large-scale methylation array platforms. Until recently, these methods were only valid for DNA from flash frozen (FF) tissues. Novel techniques for repairing DNA from formalin-fixed paraffin-embedded (FFPE) tissues have emerged; however, a direct comparison of FFPE DNA repair methods before analysis on genome-wide methylation array to matched FF tissues has not been conducted.Methods: We conducted a systematic performance comparison of two DNA repair methods (REPLI-g Ligase vs. Infinium HD Restore Kit) on FFPE-DNA compared with matched FF tissues on the Infinium 450K array. A threshold of discordant methylation between FF-FFPE pairs was set at Δβ > 0.3. The correlations of β-values from FF–FFPE pairs were compared across methods and experimental conditions.Results: The Illumina Restore kit outperformed the REPLI-g ligation method with respect to reproducibility of replicates (R2 > 0.970), highly correlated β-values between FF-FFPE (R2 > 0.888), and fewest discordant loci between FF-FFPE (≤0.61%). The performance of the Restore kit was validated in an independent set of 121 FFPE tissues.Conclusions: The Restore kit outperformed RELPI-g ligation in restoring FFPE-derived DNA before analysis on the Infinium 450K methylation array. Our findings provide critical guidance that may significantly enhance the breadth of diseases that can be studied by methylomic profiling.Impact: Epigenomic studies using FFPE tissues should now be considered among cancers that have not been fully characterized from an epigenomic standpoint. These findings promote novel epigenome-wide studies focused on cancer etiology, identification of novel biomarkers, and developing targeted therapies.See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.”Cancer Epidemiol Biomarkers Prev; 23(12); 2622–31. ©2014 AACR.

The Akt activation inhibitor triciribine and the farnesyltransferase inhibitor tipifarnib have modest to little activity in clinical trials when used as single agents. In this article, preclinical data show that the combination is more effective than single agents both in cultured cells and in vivo. Combination index data analysis shows that this combination is highly synergistic at inhibiting anchorage-dependent growth of breast cancer cells. This synergistic interaction is also observed with structurally unrelated inhibitors of Akt (MK-2206) and farnesyltransferase (FTI-2153). The triciribine/tipifarnib synergistic effects are seen with several cancer cell lines including those from breast, leukemia, multiple myeloma and lung tumors with different genetic alterations such as K-Ras, B-Raf, PI3K (phosphoinositide 3-kinase), p53 and pRb mutations, PTEN, pRB and Ink4a deletions, and ErbB receptor overexpression. Furthermore, the combination is synergistic at inhibiting anchorage-independent growth and at inducing apoptosis in breast cancer cells. The combination is also more effective at inhibiting the Akt/mTOR/S6 kinase pathway. In an ErbB2-driven breast tumor transgenic mouse model, the combination, but not single agent, treatment with triciribine and tipifarnib induces significant breast tumor regression. Our findings warrant further investigation of the combination of farnesyltransferase and Akt inhibitors. Clin Cancer Res; 17(9); 2852–62. ©2011 AACR.

Supplementary Materials. Supplemental Table 1. C1D1 alvocidib pharmacokinetics by dose Supplemental Figure 1. Sample alvocidib concentration-time profile. Samples were obtained pre-infusion, immediately following infusion, and at 1, 2, 4, 8, 12, and 24 hours post-infusion.

IKBKE inhibitor amlexanox induces G1 arrest and pERK

Supplementary Figures S1-S2; Supplementary Tables S1-S2.

EGFR mutant NSCLCs express high levels of pIKBKE-Y153.

Effect of EGFR phosphorylation of IKBKE-Y153/179 on EGFR-induced cell phenotype

Supplementary Figures 1-3, Tables 1-2 from Downregulation of Notch Pathway by a γ-Secretase Inhibitor Attenuates AKT/Mammalian Target of Rapamycin Signaling and Glucose Uptake in an ERBB2 Transgenic Breast Cancer Model