Osteopontin (OPN) is the most upregulated gene in primary central nervous system lymphoma (PCNSL) compared to non-CNS diffuse large B cell lymphoma (DLBCL). We show here that OPN is a key mediator of intracerebral tumor growth, invasion, and dissemination in CNS lymphoma, and that these effects depend upon activation of NF-κB. We further show that activation of NF-κB by OPN occurs through a unique mechanism in which intracellular OPN (iOPN) causes transcriptional downregulation of the NF-κB inhibitors, A20/TNFAIP3 and ABIN1/TNIP1, and secretory OPN (sOPN) promotes receptor-mediated activation of NF-κB. We also identify NF-κB-mediated induction of matrix metalloproteinase-8 (MMP-8) as a specific feature of OPN-mediated tissue invasion. These results implicate OPN as a candidate for development of targeted therapy for patients with PCNSL.

Ovarian cancer represents the most lethal tumor type among malignancies of the female reproductive system. Overall survival rates remain low. In this study, we identify the serine protease inhibitor Kazal type 1 (SPINK1) as a potential therapeutic target for a subset of ovarian cancers. We show that SPINK1 drives ovarian cancer cell proliferation through activation of epidermal growth factor receptor (EGFR) signaling, and that SPINK1 promotes resistance to anoikis through a distinct mechanism involving protease inhibition. In analyses of ovarian tumor specimens from a Mayo Clinic cohort of 490 patients, we further find that SPINK1 immunostaining represents an independent prognostic factor for poor survival, with the strongest association in patients with nonserous histological tumor subtypes (endometrioid, clear cell, and mucinous). This study provides novel insight into the fundamental processes underlying ovarian cancer progression, and also suggests new avenues for development of molecularly targeted therapies.

Systemic treatment is necessary for one third of patients with renal cell carcinoma. No valid biomarker is currently available to tailor personalized therapy. In this study we established a representative panel of patient derived xenograft (PDX) mouse models from patients with renal cell carcinomas and determined serum levels of high mobility group B1 (HMGB1) protein under treatment with sunitinib, pazopanib, sorafenib, axitinib, temsirolimus and bevacizumab. Serum HMGB1 levels were significantly higher in a subset of the PDX collection, which exhibited slower tumor growth during subsequent passages than tumors with low HMGB1 serum levels. Pre-treatment PDX serum HMGB1 levels also correlated with response to systemic treatment: PDX models with high HMGB1 levels predicted response to bevacizumab. Taken together, we provide for the first time evidence that the damage associated molecular pattern biomarker HMGB1 can predict response to systemic treatment with bevacizumab. Our data support the future evaluation of HMGB1 as a predictive biomarker for bevacizumab sensitivity in patients with renal cell carcinoma.

There is a clear unmet need for novel therapeutic agents for management of primary and secondary brain tumors. Novel therapeutic agents with excellent central nervous system (CNS) penetration and therapeutic activity are urgently needed. EDO-S101 is a novel alkylating and histone deacetylase inhibiting agent created by covalent fusion of bendamustine and vorinostat. We used murine models to perform CNS pharmacokinetic analysis and preclinical therapeutic evaluation of EDO-S101 for CNS lymphoma, metastatic triple-negative breast cancer of the brain, and glioblastoma multiforme. EDO-S101 has excellent CNS penetration of 13.8% and 16.5% by intravenous infusion and bolus administration respectively. It shows promising therapeutic activity against CNS lymphoma, metastatic triple-negative breast cancer of the brain, and glioblastoma multiforme with significant prolongation of survival compared to no-treatment controls. Therapeutic activity was higher with IV infusion compared to IV bolus. It should be evaluated further for therapeutic use in brain tumors.

Here we present an innovative computational-based drug discovery strategy, coupled with machine-based learning and functional assessment, for the rational design of novel small molecule inhibitors of the lipogenic enzyme stearoyl-CoA desaturase 1 (SCD1). Our methods resulted in the discovery of several unique molecules, of which our lead compound SSI-4 demonstrates potent anti-tumor activity, with an excellent pharmacokinetic and toxicology profile. We improve upon key characteristics, including chemoinformatics and absorption/distribution/metabolism/excretion (ADME) toxicity, while driving the IC50 to 0.6 nM in some instances. This approach to drug design can be executed in smaller research settings, applied to a wealth of other targets, and paves a path forward for bringing small-batch based drug programs into the Clinic.

Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies that currently has no effective therapy. We performed quantitative high-throughput screening (qHTS) in three ATC cell lines using 3,282 clinically approved drugs and drug candidates, and identified 100 active agents. Enrichment analysis of active compounds showed that inhibitors of EGFR and histone deacetylase (HDAC) were most active. Of these, the first-in-class dual inhibitor of EGFR, HER2 and HDACs, CUDC-101, had the highest efficacy and lower IC50 than established drugs. We validated that CUDC-101 inhibited cellular proliferation and resulted in cell death by inducing cell cycle arrest and caspase-dependent apoptosis. CUDC-101 also inhibited cellular migration in vitro. Mechanistically, CUDC-101 inhibited MAPK signaling and histone deacetylation in ATC cell lines with multiple driver mutations present in human ATC. The anticancer effect of CUDC-101 was associated with increased expression of p21 and E-cadherin, and reduced expression of survivin, XIAP, β-catenin, N-cadherin, and Vimentin. In an in vivo mouse model of metastatic ATC, CUDC-101 inhibited tumor growth and metastases, and significantly prolonged survival. Response to CUDC-101 treatment in vivo was associated with increased histone 3 acetylation and reduced survivin expression. Our findings provide a preclinical basis to evaluate CUDC-101 therapy in ATC.

Currently there is a lack of targeted therapies that lead to long-term attenuation or regression of disease in patients with advanced clear cell renal cell carcinoma (ccRCC). Our group has implemented a high-throughput genetic analysis coupled with a high-throughput proliferative screen in order to investigate the genetic contributions of a large cohort of overexpressed genes at the functional level in an effort to better understand factors involved in tumor initiation and progression. Patient gene array analysis identified transcripts that are consistently elevated in patient ccRCC as compared to matched normal renal tissues. This was followed by a high-throughput lentivirus screen, independently targeting 195 overexpressed transcripts identified in the gene array in four ccRCC cell lines. This revealed 31 'hits' that contribute to ccRCC cell proliferation. Many of the hits identified are not only presented in the context of ccRCC for the first time, but several have not been previously linked to cancer. We further characterize the function of a group of hits in tumor cell invasion. Taken together these findings reveal pathways that may be critical in ccRCC tumorigenicity, and identifies novel candidate factors that could serve as targets for therapeutic intervention or diagnostic/prognostic biomarkers for patients with advanced ccRCC.

The aim of this study was to compare and contrast the expression of all members of the Kallikrein (KLK) family of genes across 15 cancer types and to evaluate their utility as diagnostic and prognostic biomarkers.

Matrix metalloproteinases (MMPs) have been implicated in diverse roles in breast cancer development and progression. While many of the different MMPs expressed in breast cancer are produced by stromal cells MMP-9 is produced mainly by the tumor cells themselves. To date, the functional role of tumor cell-produced MMP-9 has remained unclear. Here, we show that human breast cancer cell-produced MMP-9 is specifically required for invasion in cell culture and for pulmonary metastasis in a mouse orthotopic model of basal-like breast cancer. We also find that tumor cell-produced MMP-9 promotes tumor vascularization with only modest impact on primary tumor growth, and that silencing of MMP-9 expression in tumor cells leads to an altered transcriptional program consistent with reversion to a less malignant phenotype. MMP-9 is most highly expressed in human basal-like and triple negative tumors, where our data suggest that it contributes to metastatic progression. Our results suggest that MMP9 may offer a target for anti-metastatic therapies for basal-like triple negative breast cancers, a poor prognosis subtype with few available molecularly targeted therapeutic options.

MISIIR is a potential target for ovarian cancer (OC) therapy due to its tissue-specific pattern of expression. 3C23K is a novel therapeutic monoclonal anti-MISIIR antibody designed to recruit effector cells and promote cell death through ADCC (antibody dependent cell-mediated cytotoxicity). Our objective was to determine the tolerability and efficacy of 3C23K in OC patient-derived xenografts (PDX) and to identify factors affecting efficacy. Quantitative RT-PCR, immunohistochemistry (IHC), and flow cytometry were used to categorize MISIIR expression in established PDX models derived from primary OC patients. We selected two high expressing models and two low expressing models for in vivo testing. One xenograft model using an MISIIR over-expressing SKOV3ip cell line (Z3) was a positive control. The primary endpoint was change in tumor size. The secondary endpoint was final tumor mass. We observed no statistically significant differences between control and treated animals. The lack of response could be secondary to a number of variables including the lack of known biomarkers of response, the low membrane expression of MISIIR, and a limited ability of 3C23K to induce ADCC in PDX models. Further study is needed to determine the magnitude of ovarian cancer response to 3C23K and also if there is a threshold surface expression to predict response.

Patient-derived tumor xenograft (PDTX) mouse models were used to discover new therapies for naïve and drug resistant BRAFV600E -mutant melanoma. Tumor histology, oncogenic protein expression, and antitumor activity were comparable between patient and PDTX-matched models thereby validating PDTXs as predictive preclinical models of therapeutic response in patients. PDTX models responsive and non-responsive to BRAF/MEK standard of care (SOC) therapy were used to identify efficacious combination therapies. One such combination includes a CDK4/6 inhibitor that blocks cell cycle progression. The rationale for this is that the retinoblastoma protein (pRb) is 95% wildtype in BRAF mutant melanoma. We discovered that 77/77 stage IV metastatic melanoma tissues were positive for inactive phosphorylated pRb (pRb-Ser780). Rb is hyperphosphorylated and inactivated by CDK4/6:cyclin D1 and when restored to its hypophosphorylated active form blocks cell cycle progression. The addition of a CDK4/6 inhibitor to SOC therapy was superior to SOC. Importantly, triple therapy in an upfront treatment and salvage therapy setting provided sustained durable response. We also showed that CDK4/6 blockade resensitized drug resistant melanoma to SOC therapy. Durable response was associated with sustained suppression of pRb-Ser780. Thus, reactivation of pRb may prove to be a clinical biomarker of response and the mechanism responsible for durable response. In light of recent clinical trial data using this triple therapy against BRAFV600E -mutant melanoma, our findings demonstrating superior and prolonged durable response in PDTX models portend use of this therapeutic strategy against naïve and SOC resistant BRAFV600E -mutant metastatic melanoma coupled with pRB-Ser780 as a biomarker of response.

Gene fusions play a critical role in some cancers and can serve as important clinical targets. In epithelial ovarian cancer (EOC), the contribution of fusions, especially by histological type, is unclear. We therefore screened for recurrent fusions in a histologically diverse panel of 220 EOCs using RNA sequencing. The Pipeline for RNA-Sequencing Data Analysis (PRADA) was used to identify fusions and allow for comparison with The Cancer Genome Atlas (TCGA) tumors. Associations between fusions and clinical prognosis were evaluated using Cox proportional hazards regression models. Nine recurrent fusions, defined as occurring in two or more tumors, were observed. CRHR1-KANSL1 was the most frequently identified fusion, identified in 6 tumors (2.7% of all tumors). This fusion was not associated with survival; other recurrent fusions were too rare to warrant survival analyses. One recurrent in-frame fusion, UBAP1-TGM7, was unique to clear cell (CC) EOC tumors (in 10%, or 2 of 20 CC tumors). We found some evidence that CC tumors harbor more fusions on average than any other EOC histological type, including high-grade serous (HGS) tumors. CC tumors harbored a mean of 7.4 fusions (standard deviation [sd] = 7.4, N = 20), compared to HGS EOC tumors mean of 2.0 fusions (sd = 3.3, N = 141). Few fusion genes were detected in endometrioid tumors (mean = 0.24, sd = 0.74, N = 55) or mucinous tumors (mean = 0.25, sd = 0.5, N = 4) tumors. To conclude, we identify one fusion at 10% frequency in the CC EOC subtype, but find little evidence for common (> 5% frequency) recurrent fusion genes in EOC overall, or in HGS subtype-specific EOC tumors.

Developing selective inhibitors for proteolytic enzymes that share high sequence homology and structural similarity is important for achieving high target affinity and functional specificity. Here, we used a combination of yeast surface display and dual-color selective library screening to obtain selective inhibitors for each of the matrix metalloproteinases (MMPs) MMP14 and MMP9 by modifying the non-specific N-terminal domain of the tissue inhibitor of metalloproteinase-2 (N-TIMP2). We generated inhibitor variants with 30- to 1175-fold improved specificity to each of the proteases, respectively, relative to wild type N-TIMP2. These biochemical results accurately predicted the selectivity and specificity obtained in cell-based assays. In U87MG cells, the activation of MMP2 by MMP14 was inhibited by MMP14-selective blockers but not MMP9-specific inhibitors. Target specificity was also demonstrated in MCF-7 cells stably expressing either MMP14 or MMP9, with only the MMP14-specific inhibitors preventing the mobility of MMP14-expressing cells. Similarly, the mobility of MMP9-expressing cells was inhibited by the MMP9-specific inhibitors, yet was not altered by the MMP14-specific inhibitors. The strategy developed in this study for improving the specificity of an otherwise broad-spectrum inhibitor will likely enhance our understanding of the basis for target specificity of inhibitors to proteolytic enzymes, in general, and to MMPs, in particular. We, moreover, envision that this study could serve as a platform for the development of next-generation, target-specific therapeutic agents. Finally, our methodology can be extended to other classes of proteolytic enzymes and other important target proteins.