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Significant strides have been made in the development of precision therapeutics for cancer. Aberrantly expressed glycoproteins represent a potential avenue for therapeutic development. The MUC16/CA125 glycoprotein serves as a biomarker of disease and a driver of malignant transformation in epithelial ovarian cancer. Previously, we demonstrated a proof-of-principle approach to selectively targeting MUC16+ cells. In this report, we performed a synthetic lethal kinase screen using a human kinome RNAi library and identified key pathways preferentially targetable in MUC16+ cells using isogenic dual-fluorescence ovarian cancer cell lines. Using a separate approach, we performed high-content small-molecule screening of six different libraries of 356,982 compounds for MUC16/CA125-selective agents and identified lead candidates that showed preferential cytotoxicity in MUC16+ cells. Compounds with differential activity were selected and tested in various other ovarian cell lines or isogenic pairs to identify lead compounds for structure–activity relationship (SAR) selection. Lead siRNA and small-molecule inhibitor candidates preferentially inhibited invasion of MUC16+ cells in vitro and in vivo, and we show that this is due to decreased activation of MAPK, and non–receptor tyrosine kinases. Taken together, we present a comprehensive screening approach to the development of a novel class of MUC16-selective targeted therapeutics and identify candidates suitable for further clinical development.

Supplementary Figure from Dual Fluorescence Isogenic Synthetic Lethal Kinase Screen and High-Content Secondary Screening for MUC16/CA125-Selective Agents

Supplementary data file shows supplementary methods and figures S1 to S7. Supplementary Figure S1 shows the establishment of the in vitro sarcoma TAM model. Supplementary Figure S2 shows the effect of anti-CSF-1 and anti-CSF-1R mAb and pazopanib on in vitro sarcoma TAMs. Supplementary Figure S3 show spontaneous lung metastasis in LM8 orthotopic osteosarcoma mouse model. Supplementary Figure S4 shows CSF-1R inhibition by PLX3397 blocks LM8-luc tumor growth and lung metastasis in orthotopic osteosarcoma mouse model. Supplementary Figure S5 shows toxicity assessment by body weight and blood chemistry per treatment group at the end of therapeutic experiments (day 22). Supplementary Figure S6 show histopathological examination of orthotopic osteosarcoma mouse models. Supplementary Figure S7 shows alteration of the TME composition in PLX3397-treated mice after CSF-1R inhibition

Significant strides have been made in the development of precision therapeutics for cancer. Aberrantly expressed glycoproteins represent a potential avenue for therapeutic development. The MUC16/CA125 glycoprotein serves as a biomarker of disease and a driver of malignant transformation in epithelial ovarian cancer. Previously, we demonstrated a proof-of-principle approach to selectively targeting MUC16+ cells. In this report, we performed a synthetic lethal kinase screen using a human kinome RNAi library and identified key pathways preferentially targetable in MUC16+ cells using isogenic dual-fluorescence ovarian cancer cell lines. Using a separate approach, we performed high-content small-molecule screening of six different libraries of 356,982 compounds for MUC16/CA125-selective agents and identified lead candidates that showed preferential cytotoxicity in MUC16+ cells. Compounds with differential activity were selected and tested in various other ovarian cell lines or isogenic pairs to identify lead compounds for structure–activity relationship (SAR) selection. Lead siRNA and small-molecule inhibitor candidates preferentially inhibited invasion of MUC16+ cells in vitro and in vivo, and we show that this is due to decreased activation of MAPK, and non–receptor tyrosine kinases. Taken together, we present a comprehensive screening approach to the development of a novel class of MUC16-selective targeted therapeutics and identify candidates suitable for further clinical development.

Summary of immunogenicity experiment.

A. Tumors were undetectable at 4 weeks by IVIS imaging when b-AP15 was administered from day 1 after injecting A673 cells expressing GFP-luciferase. NSG mice bearing A673 xenografts were treated with DMSO, bortezomib or b-AP15 after they reached 80-100mm3 and weekly tumor size measurements were taken. B. b-AP15 slowed the growth of the established A673 xenografts and C. prolonged survival of A673 tumor bearing mice compared to bortezomib. D. VLX-1570 and b-AP15 treatment did not cause significant weight loss in xenograft models. (two way Anova with Tukey's multiple comparison test).

Purpose: In patients whose lung adenocarcinomas harbor epidermal growth factor receptor (EGFR) tyrosine kinase domain mutations, acquired resistance to the tyrosine kinase inhibitors (TKI) gefitinib (Iressa) and erlotinib (Tarceva) has been associated with a second-site EGFR mutation, which leads to substitution of methionine for threonine at position 790 (T790M). We aimed to elucidate the frequency and nature of secondary EGFR mutations in patients with acquired resistance to TKI monotherapy.Experimental Design: Tumor cells from patients with acquired resistance were examined for secondary EGFR kinase domain mutations by molecular analyses.Results: Eight of 16 patients (50% observed rate; 95% confidence interval, 25-75%) had tumor cells with second-site EGFR mutations. Seven mutations were T790M and one was a novel D761Y mutation found in a brain metastasis. When combined with a drug-sensitive L858R mutation, the D761Y mutation modestly reduced the sensitivity of mutant EGFR to TKIs in both surrogate kinase and cell viability assays. In an autopsy case, the T790M mutation was found in multiple visceral metastases but not in a brain lesion.Conclusions: The T790M mutation is common in patients with acquired resistance. The limited spectrum of TKI-resistant mutations in EGFR, which binds to erlotinib in the active conformation, contrasts with a wider range of second-site mutations seen with acquired resistance to imatinib, which binds to ABL and KIT, respectively, in closed conformations. Collectively, our data suggest that the type and nature of kinase inhibitor resistance mutations may be influenced by both anatomic site and mode of binding to the kinase target.

Ewing sarcoma is a primitive round cell sarcoma with a peak incidence in adolescence that is driven by a chimeric oncogene created from the fusion of the EWSR1 gene with a member of the ETS family of genes. Patients with metastatic and recurrent disease have dismal outcomes and need better therapeutic options. We screened a library of 309,989 chemical compounds for growth inhibition of Ewing sarcoma cells to provide the basis for the development of novel therapies and to discover vulnerable pathways that might broaden our understanding of the pathobiology of this aggressive sarcoma. This screening campaign identified a class of benzyl-4-piperidone compounds that selectively inhibit the growth of Ewing sarcoma cell lines by inducing apoptosis. These agents disrupt 19S proteasome function through inhibition of the deubiquitinating enzymes USP14 and UCHL5. Functional genomic data from a genome-wide shRNA screen in Ewing sarcoma cells also identified the proteasome as a node of vulnerability in Ewing sarcoma cells, providing orthologous confirmation of the chemical screen findings. Furthermore, shRNA-mediated silencing of USP14 or UCHL5 in Ewing sarcoma cells produced significant growth inhibition. Finally, treatment of a xenograft mouse model of Ewing sarcoma with VLX1570, a benzyl-4-piperidone compound derivative currently in clinical trials for relapsed multiple myeloma, significantly inhibited in vivo tumor growth. Overall, our results offer a preclinical proof of concept for the use of 19S proteasome inhibitors as a novel therapeutic strategy for Ewing sarcoma. Cancer Res; 76(15); 4525–34. ©2016 AACR.

Cell lines were treated with the indicated compounds for 96 h and then growth determined. Results represent the mean {plus minus} SD of 2-4 experiments in which each condition was assayed in triplicate.

2-step SADA-PRIT imaging by PET/CT

Results and analysis of a genome-wide functional genomic screen using the Sigma-Aldrich shRNA Human Genome Library consisting of 63,093 shRNAs targeting 11,748 human genes.

A. Pair�wise expression profile comparisons of EWS cell lines treated with the known proteasome inhibitors bortezomib and MG�262 demonstrated highly significant overlap with profiles treated with compounds EWS-P and EWS-W. B. Overlap included 13 core genes established through published studies as being upregulated by 20S inhibitors such as bortezomib and MG-262. C. EWS cell lines TC-71 and CHP100 show demonstrate an increase in polyubiquitinated proteins following treatment with proteasome inhibitors MG-262, bortezomib, EWS-P, and EWS-W.

Supplementary Tables S2-S5

Supplementary Table 2

Supplementary figure S4

2-step IgG-PRIT imaging by PET/CT (no CA)

Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5′ untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC.Significance:These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.

General Procedures for synthesis of 2,6-Bis-arylidenepiperidone scaffold

Supplementary Methods, Supplementary Figures S1-S11, Supplementary Tables S1, S6, S8-11

Ingenuity Pathway Analysis of positive hits identifies enrichment of genes involved in the ubiquitin-proteasome pathway.

Supplementary Table 4

Supplementary Data (Tables and Figure legends)

Supplementary Table S7

Supplementary Methods and Figure Legends from Novel D761Y and Common Secondary T790M Mutations in Epidermal Growth Factor Receptor–Mutant Lung Adenocarcinomas with Acquired Resistance to Kinase Inhibitors

Purpose:Many cancer treatments suffer from dose-limiting toxicities to vital organs due to poor therapeutic indices. To overcome these challenges we developed a novel multimerization platform that rapidly removes tumor-targeting proteins from the blood to substantially improve therapeutic index.Experimental Design:The platform was designed as a fusion of a self-assembling and disassembling (SADA) domain to a tandem single-chain bispecific antibody (BsAb, anti-ganglioside GD2 × anti-DOTA). SADA–BsAbs were assessed with multiple in vivo tumor models using two-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry, and antitumor responses.Results:SADA–BsAbs self-assembled into stable tetramers (220 kDa), but could also disassemble into dimers or monomers (55 kDa) that rapidly cleared via renal filtration and substantially reduced immunogenicity in mice. When used with rapidly clearing DOTA-caged PET isotopes, SADA–BsAbs demonstrated accurate tumor localization, dosimetry, and improved imaging contrast by PET/CT. When combined with therapeutic isotopes, two-step SADA-PRIT safely delivered massive doses of alpha-emitting (225Ac, 1.48 MBq/kg) or beta-emitting (177Lu, 6,660 MBq/kg) S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) payloads to tumors, ablating them without any short-term or long-term toxicities to the bone marrow, kidneys, or liver.Conclusions:The SADA–BsAb platform safely delivered large doses of radioisotopes to tumors and demonstrated no toxicities to the bone marrow, kidneys, or liver. Because of its modularity, SADA–BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve therapeutic index and maximize the delivered dose.See related commentary by Capala and Kunos, p. 377

72 hour IC50 growth inhibition levels (μM) of 23 compounds (labeled EWS-A through EWS-X) against five EWS cell lines and 11 non-EWS cell lines

Supplementary Figures 1-3 from Novel D761Y and Common Secondary T790M Mutations in Epidermal Growth Factor Receptor–Mutant Lung Adenocarcinomas with Acquired Resistance to Kinase Inhibitors

Supplementary Table 1

Supplementary Table 3

3-step IgG-PRIT imaging by PET/CT

Panel upper left: CHP100 cells exposed 3 hours to different concentrations of b-AP15. Extracts were labeled with HA-Ub-VS and subjected to western blot. Filters probed with HA antibody. Note the lack of inhibition of any of the DUBs labeled by HA-Ub-VS. Panel upper right: CHP100 cells exposed 3 hours to 1 uM b-AP15 Extracts were labeled with Ub-VS and subjected to western blot. Filters probed with USP14, UCHL5 or UCHL1 antibodies. b-AP15 treated extracts demonstrate inhibition of USP14, weak inhibition of UCHL5, and no inhibition of UCHL1. Middle Panels: Purified 26S proteasomes (5 nM) were treated with indicate concentrations for b-AP15 and DUB activity was determined by cleavage of Ub-AMC. Note the reduction in 26S associated proteasome activity following treatment with b-AP15. Lower Panels: Recombinant UCHL1 or UCHL3 was treated with b-AP15 and DUB activity was monitored using Ub-AMC cleavage as above. No inhibition of either UCHL1 or UCHL3 was observed.

Purpose:Nucleotide excision repair (NER) gene alterations constitute potential cancer therapeutic targets. We explored the prevalence of NER gene alterations across cancers and putative therapeutic strategies targeting these vulnerabilities.Experimental Design:We interrogated our institutional dataset with mutational data from more than 40,000 patients with cancer to assess the frequency of putative deleterious alterations in four key NER genes. Gene-edited isogenic pairs of wild-type and mutant ERCC2 or ERCC3 cell lines were created and used to assess response to several candidate drugs.Results:We found that putative damaging germline and somatic alterations in NER genes were present with frequencies up to 10% across multiple cancer types. Both in vitro and in vivo studies showed significantly enhanced sensitivity to the sesquiterpene irofulven in cells harboring specific clinically observed heterozygous mutations in ERCC2 or ERCC3. Sensitivity of NER mutants to irofulven was greater than to a current standard-of-care agent, cisplatin. Hypomorphic ERCC2/3-mutant cells had impaired ability to repair irofulven-induced DNA damage. Transcriptomic profiling of tumor tissues suggested codependencies between DNA repair pathways, indicating a potential benefit of combination therapies, which were confirmed by in vitro studies.Conclusions:These findings provide novel insights into a synthetic lethal relationship between clinically observed NER gene deficiencies and sensitivity to irofulven and its potential synergistic combination with other drugs.See related commentary by Jiang and Greenberg, p. 1833

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Colony-stimulating factor 1 (CSF1) is a primary regulator of the survival, proliferation, and differentiation of monocyte/macrophage that sustains the protumorigenic functions of tumor-associated macrophages (TAMs). Considering current advances in understanding the role of the inflammatory tumor microenvironment, targeting the components of the sarcoma microenvironment, such as TAMs, is a viable strategy. Here, we investigated the effect of PLX3397 (pexidartinib) as a potent inhibitor of the CSF1 receptor (CSF1R). PLX3397 was recently approved by the Food and Drug Administration (FDA) to treat tenosynovial giant cell tumor and reprogram TAMs whose infiltration correlates with unfavorable prognosis of sarcomas. First, we confirmed by cytokine arrays of tumor-conditioned media (TCM) that cytokines including CSF1 are secreted from LM8 osteosarcoma cells and NFSa fibrosarcoma cells. The TCM, like CSF1, stimulated ERK1/2 phosphorylation in bone marrow–derived macrophages (BMDMs), polarized BMDMs toward an M2 (TAM-like) phenotype, and strikingly promoted BMDM chemotaxis. In vitro administration of PLX3397 suppressed pERK1/2 stimulation by CSF1 or TCM, and reduced M2 polarization, survival, and chemotaxis in BMDMs. Systemic administration of PLX3397 to the osteosarcoma orthotopic xenograft model significantly suppressed the primary tumor growth and lung metastasis, and thus improved metastasis-free survival. PLX3397 treatment concurrently depleted TAMs and FOXP3+ regulatory T cells and, surprisingly, enhanced infiltration of CD8+ T cells into the microenvironments of both primary and metastatic osteosarcoma sites. Our preclinical results show that PLX3397 has strong macrophage- and T-cell–modulating effects that may translate into cancer immunotherapy for bone and soft-tissue sarcomas.

Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5′ untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC.Significance:These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.

Trametinib is an extremely potent allosteric inhibitor of mitogen-activated protein kinase (MAPK)/extracellular-signal–regulated kinase (ERK) (MEK) 1/2, which has been approved for treatment of metastatic melanoma and anaplastic thyroid cancer in patients with confirmed BRAF(V600E)/K mutations. Though trametinib is highly efficacious, adverse side effects, including skin, gastrointestinal, and hepatic toxicity, are dose-limiting and can lead to treatment termination. Development of a noninvasive tool to visualize and quantify the delivery and distribution of trametinib (either as a single agent or in combination with other therapeutics) to tumors and organs would be helpful in assessing therapeutic index, personalizing individual dose, and potentially predicting resistance to therapy. Methods: To address these issues, we have developed a radiolabeled trametinib and evaluated the in vitro and in vivo properties. (123)I-, (124)I-, and (131)I-trametinib, pure tracer analogs to trametinib, were synthesized in more than 95% purity, with an average yield of 69.7% and more than 100 GBq/μmol specific activity. Results: Overall, (124)I-trametinib uptake in a panel of cancer cell lines can be blocked with cold trametinib, confirming specificity of the radiotracer in vitro and in vivo. (124)I-trametinib was taken up at higher rates in KRAS and BRAF mutant cell lines than in wild-type KRAS cancer cell lines. In vivo, biodistribution revealed high uptake in the liver 2 h after injection, followed by clearance through the gastrointestinal tract over 4 d. Importantly, uptake higher than expected was observed in the lung and heart for up to 24 h. Peak uptake in the skin and gastrointestinal tract was observed between 6 and 24 h, whereas in B16F10 melanoma–bearing mice peak tumor concentrations were achieved between 24 and 48 h. Tumor uptake relative to muscle and skin was relatively low, peaking at 3.4- to 8.1-fold by 72 h, respectively. The biodistribution of (124)I-trametinib was significantly reduced in mice on trametinib therapy, providing a quantitative method to observe MEK inhibition in vivo. Conclusion: (124)I-trametinib serves as an in vivo tool to personalize the dose instead of using the current single-fixed-dose scheme and, when combined with radiomic data, to monitor the emergence of therapy resistance. In addition, the production of iodinated trametinib affords researchers the ability to measure drug distribution for improved drug delivery studies.

Clearing agents (CAs) can rapidly remove non-localized targeting biomolecules from circulation for hepatic catabolism, thereby enhancing the therapeutic index (TI), especially for blood (marrow), of the subsequently administered radioisotope in any multi-step pretargeting strategy. Herein we describe the synthesis and in vivo evaluation of a fully synthetic glycodendrimer-based CA for DOTA-based pretargeted radioimmunotherapy (DOTA-PRIT). The novel dendron-CA consists of a non-radioactive yttrium-DOTA-Bn molecule attached via a linker to a glycodendron displaying sixteen terminal α-thio-N-acetylgalactosamine (α-SGalNAc) units (CCA α-16-DOTA-Y(3+); molecular weight: 9059 Da). Pretargeting [(177)Lu]LuDOTA-Bn with CCA α-16-DOTA-Y(3+) to GPA33-expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [(177)Lu]LuDOTA-Bn for blood, tumor, liver, spleen, and kidneys of 11.7, 468, 9.97, 5.49, and 13.3 cGy/MBq, respectively. Tumor-to-normal tissues absorbed-dose ratios (i.e., TIs) ranged from 40 (e.g., for blood and kidney) to about 550 for stomach.