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Supplementary Figure S1: Mesoporous silica-based nanoconstruct for targeted delivery of siRNA; Supplementary Figure S2: Screening of siPLK1 by mRNA knockdown and viability; Supplementary Figure S3: ROS intensity and NOX1-5 mRNA levels of TNBC cell lines; Supplementary Figure S4: HER2 signaling in LM2-4luc+/H2N cells; Supplementary Figure S5: Representative whole body bioluminescent IVIS imaging of a mouse injected with cancer cells; Supplementary Figure S6: Representative images of lung nodes of mice from short-term study; Supplementary Figure S7: Representative images of the whole lung, H&E, and anti-human vimentin staining of mouse lungs from the long-term study.

PDF file - 379K, Dose response curves of human PDA cell lines treated with either MEK inhibitor GSK1120212, AKT inhibitor GSK690693, or both together

XLS file - 41K, Cell lines used in this study and their genotype

PDF file - 194K, RAF and MEK co inhibition

Detailed description of the methods used in the manuscript.

XLS file - 65K, Interaction indicies of MEK inhibitor GSK1120212, AKT inhibitor GSK690693 in PDA cell lines

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PDF file - 331K, MEK inhibitor PD325901 inhibits MEK in vivo

PDF file - 578K, Characterization of a new, syngeneic, orthotopic mouse model of PDA

Cytokines are potent stimulators of the immune system and have long been investigated and used as therapeutics to treat cancer. However, free cytokines have short half-life and the lack of tumor targeting often results in systemic toxicity. IL-15 is a stimulatory cytokine that shares the common beta and gamma receptors with IL-2. In contrast to IL-2, IL-15 is more selective for NK and effector memory T cells, with less proliferative effect on Tregs, making it a more preferred therapeutic candidate for cancer. QL415 is an anti-PD-L1 x IL-15 fusion protein designed to enhance tumor accumulation and prolong blood circulation, thereby widening the therapeutic window. IL-15 and IL-15 receptor alpha sushi domain are covalently linked to the C-terminus of our proprietary PD-L1 antibody. Fc-mediated effector functions were ablated using mutations in the CH2 domain, to prevent depletion of effector immune cells. In vitro studies showed QL415 to be highly potent in promoting proliferation and pSTAT5 signaling of IL-15 -responding NK92 and M07e cells. QL415 selectively induced proliferation of NK and CD8+ T cells from human PBMC in vitro and mouse CD8+ and NK1.1 cells in vivo. In a MC38 mouse colon cancer model expressing human PD-L1, QL401 was highly effective in suppressing tumor growth, in contrast to PD-L1 monoclonal antibody or a non-targeted IL-15 control. Follow up tumor rechallenge study showed protective memory against cognate MC38 but not B16F10. In a separate MC38 tumor model using a mouse surrogate molecule of QL415, immunophenotyping of antigen presenting cells offered preliminary evidence of enrichment of conventional dendritic cells 1 in the tumor draining lymph nodes. Therefore, QL415 not only directly stimulates effector immune cells, but also indirectly through antigen presenting cells, promotes robust anti-tumor response. In non-human primates, Q415 was tolerated up to 1.5 mg/kg, well above the effective therapeutic dose. In light of these favorable preclinical efficacy and safety results, a phase 1 dose escalation and expansion study has been initiated to evaluate the safety, tolerability, and early efficacy of QL415. Citation Format: Irene Tang, Lauren Schwimmer, Monica Jin, Shenda Gu, Wei Wei Prior, Arianne Capacio, Hieu V. Tran, Allan Chan, Anna McClain, Shihao Chen, Chuanzeng Cao, Xiaoran Wu, Yanling Xu, Dongmei Guan, Xi Chen, Xianli Su. QL415, a tumor targeted IL-15 fusion protein stimulating both lymphoid and myeloid immune cells for optimal anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3504.

4-1BB (CD137, TNFRSF9) is a potent co-stimulatory receptor found on T and NK cells. Activation of 4-1BB requires receptor clustering, which is naturally mediated by the endogenous trimeric 4-1BB ligand. Cross-linking via agnostic monoclonal antibodies can also activate 4-1BB but has generally resulted in unwanted side effects, mainly liver toxicity. To address the shortcomings of 4-1BB agonists, we have developed QL301, a PD-L1 x 4-1BB bispecific antibody that conditionally activates 4-1BB only when concurrently engaged to PD-L1, an immune checkpoint mediator elevated in the immuno-suppressive tumor microenvironment. The IgG1 backbone of QL301 was mutated to eliminate Fc gamma receptor-dependent functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), thereby preventing depletion of effector immune cells and non-specific activation of 4-1BB through Fc gamma receptor binding. In addition, QL301 blocks the suppressive interaction of PD-1 with PD-L1. Therefore, through a combination of 4-1BB agonism and PD-L1 blockade, QL301 provides both a co-stimulatory signal and the release of a suppressive signal to reinvigorate anti-tumor immune response. In vitro, QL301 induced robust cytokine release and expansion of CD8+ T cells conditional on the presence of tumor or antigen presenting cells expressing PD-L1. In xenograft and transgenic mice models, QL301 had potent anti-tumor effect that was superior to PD-L1 monoclonal antibodies. Tumor regression correlated with the expansion of CD8+ T cells. In addition, QL301 potentiated tumor cell killing mediated by an EGFR targeted CD3 bispecific antibody, providing a basis for combination therapy. In non-human primates, QL301 was well tolerated up to 30 mg/kg. There was no chronic elevation of serum AST and ALT levels, and histopathology analysis showed minimal inflammation of liver tissue. A phase 1 clinical trial to evaluate the safety, tolerability, and early efficacy of QL301 is currently ongoing. Citation Format: Shenda Gu, Irene Tang, Shirley Mihardja, Wei Wei Prior, Hieu V. Tran, Allan Chan, Anna McClain, Aaron Kurtzman, Shihao Chen, Youguang Luo, Xiaoyan Kang, Xiaoran Wu, Qingmei Zheng, Guodong Jia. QL301, a PD-L1 dependent 4-1BB agonist with enhanced preclinical anti-tumor efficacy and minimal liver toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4245.

Upregulation of CD47, a “Do Not Eat Me” signal, is observed in nearly all solid and hematological malignancies. Engagement of CD47 on tumor cells with SIRPα on macrophages inhibits phagocytosis of tumor cells. Anti-CD47 antibodies block the CD47 - SIRPα engagement and reactivate phagocytosis of tumor cells by macrophages. Yet the ubiquitous expression of CD47 on normal cells, including red blood cells, presents a therapeutic challenge. Systemic targeting of CD47, by either anti-CD47 monoclonal antibodies or SIRPα-Fc fusion proteins, yielded only moderate clinical benefit due to severe adverse side effects, mainly anemia. QL401 is PD-L1 x CD47 bispecific antibody engineered to reduce binding to red blood cells while retaining potent phagocytic activation of macrophages in vitro and delayed tumor growth in vivo. QL401 comprises three functional components: a PD-L1 binding Fab arm, a CD47 binding scFv arm, and a human IgG4 backbone. The PD-L1 binding arm provides both tumor targeting and blocking of PD-1 for reactivating T cells. The CD47 arm blocks the binding of SIRPα, the “Do Not Eat Me” signal, while the IgG4 Fc retains Fc gamma receptor binding to provide a phagocytic “Eat Me” signal. Therefore, QL401 is multi-functional antibody that re-actives both the innate and adaptive immune response. In preclinical efficacy studies, QL401 potently blocked SIRPα to promote phagocytosis of tumor cells with sub-nanomolar potency. In vivo efficacy studies in mouse models of solid and hematological tumors showed QL401 to be comparable or superior to PD-L1 or CD47 monoclonal antibodies alone and in combination. In vitro safety evaluation of Q401 showed significantly reduced binding and phagocytosis of red blood cells, in contrast to CD47 monoclonal antibodies. In addition, Q401 did not induce hemagglutination. In non-human primates, QL401 was well tolerated up to 100 mg/kg without reduction of red blood cells below normal range. A phase 1 dose escalation and expansion study is expected to start Q1 2022 to evaluate the safety, tolerability, and early efficacy of QL401. Citation Format: Irene Tang, Lauren Schwimmer, Shenda Gu, Wei Wei Prior, Hieu V. Tran, Allan Chan, Anna McClain, Shihao Chen, Chuanzeng Cao, Chunyan Sun, Meimei Si, Guijiang Wang. QL401, a dual PD-L1/CD47 blocker effective against both solid and hematological malignancies with improved blood safety [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2894.

Radiation sensitizers that can selectively act on cancer cells hold great promise to patients who receive radiation therapy. We developed a novel targeted therapy and radiation sensitizer for non-small cell lung cancer (NSCLC) based on cetuximab conjugated nanoparticle that targets epidermal growth factor receptor (EGFR) and delivers small interfering RNA (siRNA) against polo-like kinase 1 (PLK1). EGFR is overexpressed in 50% of lung cancer patients and a mediator of DNA repair, while PLK1 is a key mitotic regulator whose inhibition enhances radiation sensitivity. The nanoparticle construct (C-siPLK1-NP) effectively targets EGFR + NSCLC cells and reduces PLK1 expression, leading to G2/M arrest and cell death. Furthermore, we show a synergistic combination between C-siPLK1-NP and radiation, which was confirmed in vivo in A549 flank tumors. We also demonstrate the translational potential of C-siPLK1-NP as a systemic therapeutic in an orthotopic lung tumor model, where administration of C-siPLK1-NP reduced tumor growth and led to prolonged survival. Our findings demonstrate that C-siPLK1-NP is effective as a targeted therapy and as a potent radiation sensitizer for NSCLC. Potential application to other EGFR + cancer types such as colorectal and breast cancer is also demonstrated.

Multiparametric and high-content protein analysis of single cells or tissues cannot be accomplished with the currently available flow cytometry or imaging techniques utilizing fluorophore-labelled antibodies, because the number of spectrally resolvable fluorochromes is limited. In contrast, mass cytometry can resolve more signals by exploiting lanthanide-tagged antibodies; however, only about 100 metal reporters can be attached to an antibody molecule. This makes the sensitivity of lanthanide-tagged antibodies substantially lower than fluorescent reporters. A new probe that can carry more lanthanide molecules per antibody is a desirable way to enhance the sensitivity needed for the detection of protein with low cellular abundance. Herein, we report on the development of new probes utilizing mesoporous silica nanoparticles (MSNPs) with hydroxyl, amine, or phosphonate functional groups. The phosphonated MSNPs proved to be best at loading lanthanides for up to 1.4 × 106 molecules per particle, and could be loaded with various lanthanide elements (Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb, and Lu) at relatively similar molar extents. The modified MSNPs can also load a fluorescent dye, allowing bimodal mass and fluorescence-based detection. We achieved specificity of antibody-conjugated nanoparticles (at 1.4 × 103 antibodies per nanoparticle) for targeting proteins on the cell surface. The new materials can potentially be used as mass cytometry probes and provide a method for simultaneous monitoring of a large host of factors comprising the tumor microenvironment (e.g., extracellular matrix, cancer cells, and immune cells). These novel probes may also benefit personalized medicine by allowing for high-throughput analysis of multiple proteins in the same specimen.

This Review discusses the various types of non-coding oligonucleotides, which have garnered extensive interest as new alternatives for targeted cancer therapies over small molecule inhibitors and monoclonal antibodies. These oligonucleotides can target any hallmark of cancer, no longer limited to so-called “druggable” targets. Thus, any identified gene that plays a key role in cancer progression or drug resistance can be exploited with oligonucleotides. Among them, small-interfering RNAs (siRNAs) are frequently utilized for gene silencing due to the robust and well established mechanism of RNA interference. Despite promising advantages, clinical translation of siRNAs is hindered by the lack of effective delivery platforms. This Review provides general criteria and consideration of nanoparticle development for systemic siRNA delivery. Different classes of nanoparticle candidates for siRNA delivery are discussed, and the progress in clinical trials for systemic cancer treatment is reviewed. Lastly, this Review presents HER2 (human epidermal growth factor receptor type 2)-positive breast cancer as one example that could benefit significantly from siRNA technology. How siRNA-based therapeutics can overcome cancer resistance to such therapies is discussed.

HER2 is overexpressed in about 20% of breast cancers and contributes to poor prognosis. Unfortunately, a large fraction of patients have primary or acquired resistance to the HER2-targeted therapy trastuzumab, thus a multi-drug combination is utilized in the clinic, putting significant burden on patients. We systematically identified an optimal HER2 siRNA from 76 potential sequences and demonstrated its utility in overcoming intrinsic and acquired resistance to trastuzumab and lapatinib in 18 HER2-positive cancer cell lines. We provided evidence that the drug-resistant cancer maintains dependence on HER2 for survival. Importantly, cell lines did not readily develop resistance following extended treatment with HER2 siRNA. Using our recently developed nanoparticle platform, systemic delivery of HER2 siRNA to trastuzumab-resistant tumors resulted in significant growth inhibition. Moreover, the optimal HER2 siRNA could also silence an exon 16 skipped HER2 splice variant reported to be highly oncogenic and linked to trastuzumab resistance.

Leucine-rich repeat containing 15 (LRRC15) antigen is highly expressed on both stromal fibroblasts of multiple solid tumors and on cancer cells of mesenchymal origin. As its expression is very low in normal tissues, targeting LRRC15 has shown therapeutic potential as an antibody-drug conjugate (Demetri, ASCO 2019), which could lead to significant tumor inhibition by solely targeting stromal LRRC15 expression. In this study, we have developed and rank ordered an array of IgG-based CD3 bispecific molecules based on biophysical and biologic attributes and anti-cancer efficiency through T-cell engagement and redirection/activation against LRRC15-expressing tumors. The selected lead, QL315, robustly and conditionally induced T cell activation and proliferation only in the presence of LRRC15-expressing cancer cells with an EC50 in a picomolar range in vitro but not in the coculture with LRRC15-negative cancer cells. Moreover, QL315 strongly induced tumor cell lysis in vitro and led to significant tumor regression and T cell expansion in a mouse xenograft model. Importantly, toxicologic evaluation of QL315 in cynomolgus monkeys showed antibody-like stability and half-life, and good tolerability with no changes of circulating blood cell homeostasis and organ damage. At highest levels, liver enzymes, as well as cytokine release, were transiently elevated but returned to normal within one week of treatment. In conclusion, these data demonstrate drug-like properties, promising anti-tumor capability and good tolerability of the anti-LRRC15/CD3 bispecific antibody, QL315, indicating its potential as a therapeutic option in the treatment of solid tumors of diverse origin by targeting either tumor stromal micro environment or patient cancers expressing LRRC15. Citation Format: Monica Jin, Aaron Kurtzman, Weiwei W. Prior, Allan Chan, Anna McClain, Shenda Gu, Irene Tang, Zijie Cai, Lauren Schwimmer, Hieu Tran, Shihao Chen. QL315, A CD3 bispecific antibody redirecting T cell activation to the tumor stroma antigen LRRC15 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4541.

Worapol Ngamcherdtrakul,1,2,* Thanapon Sangvanich,1,* Moataz Reda,1 Shenda Gu,1 Daniel Bejan,2 Wassana Yantasee1,2 1Department of Biomedical Engineering, Oregon Health and Science University, Portland, OR, USA; 2Nanomedicine Research Unit, PDX Pharmaceuticals, LLC, Portland, OR, USA *These authors contributed equally to this work Introduction: Long-term stability of therapeutic candidates is necessary toward their clinical applications. For most nanoparticle systems formulated in aqueous solutions, lyophilization or freeze-drying is a common method to ensure long-term stability. While lyophilization of lipid, polymeric, or inorganic nanoparticles have been studied, little has been reported on lyophilization and stability of hybrid nanoparticle systems, consisting of polymers, inorganic particles, and antibody. Lyophilization of complex nanoparticle systems can be challenging with respect to preserving physicochemical properties and the biological activities of the materials. We recently reported an effective small-interfering RNA (siRNA) nanoparticle carrier consisting of 50-nm mesoporous silica nanoparticles decorated with a copolymer of polyethylenimine and polyethyleneglycol, and antibody. Materials and methods: Toward future personalized medicine, the nanoparticle carriers were lyophilized alone and loaded with siRNA upon reconstitution by a few minutes of simple mixing in phosphate-buffered saline. Herein, we optimize the lyophilization of the nanoparticles in terms of buffers, lyoprotectants, reconstitution, and time and temperature of freezing and drying steps, and monitor the physical and chemical properties (reconstitution, hydrodynamic size, charge, and siRNA loading) and biological activities (gene silencing, cancer cell killing) of the materials after storing at various temperatures and times. Results: The material was best formulated in Tris-HCl buffer with 5% w/w trehalose. Freezing step was performed at -55°C for 3 h, followed by a primary drying step at -40°C (100 μBar) for 24 h and a secondary drying step at 20°C (20 μBar) for 12 h. The lyophilized material can be stored stably for 2 months at 4°C and at least 6 months at -20°C. Conclusion: We successfully developed the lyophilization process that should be applicable to other similar nanoparticle systems consisting of inorganic nanoparticle cores modified with cationic polymers, PEG, and antibodies. Keywords: nanoparticles, lyophilization, cancer, mesoporous silica, antibody, siRNA

Fibrotic diseases such as scleroderma have been linked to increased oxidative stress and upregulation of pro-fibrotic genes. Recent work suggests a role of NADPH oxidase 4 (NOX4) and heat shock protein 47 (HSP47) in inducing excessive collagen synthesis, leading to fibrotic diseases. Herein, we elucidate the relationship between NOX4 and HSP47 in fibrogenesis and propose to modulate them altogether as a new strategy to treat fibrosis. We developed a nanoparticle platform consisting of polyethylenimine (PEI) and polyethylene glycol (PEG) coating on a 50-nm mesoporous silica nanoparticle (MSNP) core. The nanoparticles effectively delivered small interfering RNA (siRNA) targeting HSP47 (siHSP47) in an in vitro model of fibrosis based on TGF-β stimulated fibroblasts. The MSNP core also imparted an antioxidant property by scavenging reactive oxygen species (ROS) and subsequently reducing NOX4 levels in the in vitro fibrogenesis model. The nanoparticle was far superior to n-acetyl cysteine (NAC) at modulating pro-fibrotic markers. In vivo evaluation was performed in a bleomycin-induced scleroderma mouse model, which shares many similarities to human scleroderma disease. Intradermal administration of siHSP47-nanoparticles effectively reduced HSP47 protein expression in skin to normal level. In addition, the antioxidant MSNP also played a prominent role in reducing the pro-fibrotic markers, NOX4, alpha smooth muscle actin (α-SMA), and collagen type I (COL I), as well as skin thickness of the mice.

Background High mitotic activity is associated with the genesis and progression of many cancers. Small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents. With clinical trials of several of these experimental compounds underway, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that carry molecular aberrations that confer high mitotic activity, and to develop molecular markers that distinguish which tumors will be most responsive to mitotic apparatus inhibitors. Methods We identified a coordinately regulated mitotic apparatus network by analyzing gene expression profiles for 53 malignant and non-malignant human breast cancer cell lines and two separate primary breast tumor datasets. We defined the mitotic network activity index (MNAI) as the sum of the transcriptional levels of the 54 coordinately regulated mitotic apparatus genes. The effect of those genes on cell growth was evaluated by small interfering RNA (siRNA). Results High MNAI was enriched in basal-like breast tumors and was associated with reduced survival duration and preferential sensitivity to inhibitors of the mitotic apparatus proteins, polo-like kinase, centromere associated protein E and aurora kinase designated GSK462364, GSK923295 and GSK1070916, respectively. Co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25 was associated with the transcriptional upregulation of this network of 54 mitotic apparatus genes, and we identify transcription factors that localize to these regions and putatively regulate mitotic activity. Knockdown of the mitotic network by siRNA identified 22 genes that might be considered as additional therapeutic targets for this clinically relevant patient subgroup. Conclusions We define a molecular signature which may guide therapeutic approaches for tumors with high mitotic network activity. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0728-y) contains supplementary material, which is available to authorized users.

RNA interference (RNAi) is an endogenous post-transcriptional gene regulatory mechanism, where non-coding, double-stranded RNA molecules interfere with the expression of certain genes in order to silence it. Since its discovery, this phenomenon has evolved as powerful technology to diagnose and treat diseases at cellular and molecular levels. With a lot of attention, short interfering RNA (siRNA) therapeutics has brought a great hope for treatment of various undruggable diseases, including genetic diseases, cancer, and resistant viral infections. However, the challenge of their systemic delivery and on how they are integrated to exhibit the desired properties and functions remains a key bottleneck for realizing its full potential. Nanoparticles are currently well known to exhibit a number of unique properties that could be strategically tailored into new advanced siRNA delivery systems. This review summarizes the various nanoparticulate systems developed so far in the literature for systemic delivery of siRNA, which include silica and silicon-based nanoparticles, metal and metal oxides nanoparticles, carbon nanotubes, graphene, dendrimers, polymers, cyclodextrins, lipids, hydrogels, and semiconductor nanocrystals. Challenges and barriers to the delivery of siRNA and the role of different nanoparticles to surmount these challenges are also included in the review.

Metastatic breast cancer is developed in about 20% to 30% of newly diagnosed patients with early-stage breast cancer despite treatments. Herein, we report a novel nanoparticle platform with intrinsic antimetastatic properties for the targeted delivery of Polo-like kinase 1 siRNA (siPLK1). We first evaluated it in a triple-negative breast cancer (TNBC) model, which shows high metastatic potential. PLK1 was identified as the top therapeutic target for TNBC cells and tumor-initiating cells in a kinome-wide screen. The platform consists of a 50-nm mesoporous silica nanoparticle (MSNP) core coated layer-by-layer with bioreducible cross-linked PEI and PEG polymers, conjugated with an antibody for selective uptake into cancer cells. siRNA is loaded last and fully protected under the PEG layer from blood enzymatic degradation. The material has net neutral charge and low nonspecific cytotoxicity. We have also shown for the first time that the MSNP itself inhibited cancer migration and invasion in TNBC cells owing to its ROS- and NOX4-modulating properties. In vivo, siPLK1 nanoconstructs (six doses of 0.5 mg/kg) knocked down about 80% of human PLK1 mRNA expression in metastatic breast cancer cells residing in mouse lungs and reduced tumor incidence and burden in lungs and other organs of an experimental metastasis mouse model. Long-term treatment significantly delayed the onset of death in mice and improved the overall survival. The platform capable of simultaneously inhibiting the proliferative and metastatic hallmarks of cancer progression is unique and has great therapeutic potential to also target other metastatic cancers beyond TNBC. Mol Cancer Ther; 16(4); 763–72. ©2017 AACR.

Manhattan plots for each mitotic network gene illustrating the strength of association (-log10 p value) between the expression of the gene and genome-wide somatic copy number alterations (see Fig S6). (PDF 8516 kb)

Table S1. List of genes that are significantly correlated with either PLK1, CENPE or AURKB in breast cancer cell lines and used for constructing gene network. Table S2. Gene ontology (GO) analysis of genes significantly correlated with PLK1, CENPE or AURKB in human breast cancer cell lines. Table S3. Mitotic network gene list. Table S4. Pearson coefficients for correlation (and significance) between cell line responses across 53 breast cell lines derived from tumor and normal tissues. The number of lines used to establish the correlation is listed below each correlation in parentheses. (DOC 282 kb)

KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDA) but remains an intractable pharmacologic target. Consequently, defining RAS effector pathway(s) required for PDA initiation and maintenance is critical to improve treatment of this disease. Here, we show that expression of BRAFV600E, but not PIK3CAH1047R, in the mouse pancreas leads to pancreatic intraepithelial neoplasia (PanIN) lesions. Moreover, concomitant expression of BRAFV600E and TP53R270H result in lethal PDA. We tested pharmacologic inhibitors of RAS effectors against multiple human PDA cell lines. Mitogen-activated protein (MAP)/extracellular signal–regulated (ERK) kinase (MEK) inhibition was highly effective both in vivo and in vitro and was synergistic with AKT inhibition in most cell lines tested. We show that RAF→MEK→ERK signaling is central to the initiation and maintenance of PDA and to rational combination strategies in this disease. These results emphasize the value of leveraging multiple complementary experimental systems to prioritize pathways for effective intervention strategies in PDA. Significance: PDA is difficult to treat, in large part, due to recurrent mutations in the KRAS gene. Here, we define rational treatment approaches for the disease achievable today with existing drug combinations by thorough genetic and pharmacologic dissection of the major KRAS effector pathways, RAF→MEK→ERK and phosphoinositide 3′-kinase (PI3′K)→AKT. Cancer Discov; 2(8); 685–93. ©2012 AACR. Read the Commentary on this article by Hanrahan et al., p. 666. This article is highlighted in the In This Issue feature, p. 653.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease. Overall survival is typically 6 months from diagnosis(1). Numerous phase 3 trials of agents effective in other malignancies have failed to benefit unselected PDA populations, although patients do occasionally respond. Studies in other solid tumors have shown that heterogeneity in response is determined, in part, by molecular differences between tumors. Furthermore, treatment outcomes are improved by targeting drugs to tumor subtypes in which they are selectively effective, with breast(2) and lung(3) cancers providing recent examples. Identification of PDA molecular subtypes has been frustrated by a paucity of tumor specimens available for study. We have overcome this problem by combined analysis of transcriptional profiles of primary PDA samples from several studies, along with human and mouse PDA cell lines. We define three PDA subtypes: classical, quasimesenchymal and exocrine-like, and we present evidence for clinical outcome and therapeutic response differences between them. We further define gene signatures for these subtypes that may have utility in stratifying patients for treatment and present preclinical model systems that may be used to identify new subtype specific therapies.

© 2016 The Author(s). Background: High mitotic activity is associated with the genesis and progression of many cancers. Small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents. With clinical trials of several of these experimental compounds underway, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that carry molecular aberrations that confer high mitotic activity, and to develop molecular markers that distinguish which tumors will be most responsive to mitotic apparatus inhibitors. Methods: We identified a coordinately regulated mitotic apparatus network by analyzing gene expression profiles for 53 malignant and non-malignant human breast cancer cell lines and two separate primary breast tumor datasets. We defined the mitotic network activity index (MNAI) as the sum of the transcriptional levels of the 54 coordinately regulated mitotic apparatus genes. The effect of those genes on cell growth was evaluated by small interfering RNA (siRNA). Results: High MNAI was enriched in basal-like breast tumors and was associated with reduced survival duration and preferential sensitivity to inhibitors of the mitotic apparatus proteins, polo-like kinase, centromere associated protein E and aurora kinase designated GSK462364, GSK923295 and GSK1070916, respectively. Co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25 was associated with the transcriptional upregulation of this network of 54 mitotic apparatus genes, and we identify transcription factors that localize to these regions and putatively regulate mitotic activity. Knockdown of the mitotic network by siRNA identified 22 genes that might be considered as additional therapeutic targets for this clinically relevant patient subgroup. Conclusions: We define a molecular signature which may guide therapeutic approaches for tumors with high mitotic network activity.

In vivo delivery of siRNAs designed to inhibit genes important in cancer and other diseases continues to be an important biomedical goal. We now describe a new nanoparticle construct that has been engineered for efficient delivery of siRNA to tumors. The construct is comprised of a 47-nm mesoporous silica nanoparticle (MSNP) core coated with a cross-linked PEI-PEG copolymer, carrying siRNA against the HER2 oncogene, and coupled to the anti-HER2 monoclonal antibody (trastuzumab). The construct has been engineered to increase siRNA blood half-life, enhance tumor-specific cellular uptake, and maximize siRNA knockdown efficacy. The optimized anti-HER2-nanoparticles produced apoptotic death in HER2 positive (HER2+) breast cancer cells grown in vitro, but not in HER2 negative (HER2-) cells. One dose of the siHER2-nanoparticles reduced HER2 protein levels by 60% in trastuzumab-resistant HCC1954 xenografts. Multiple doses administered intravenously over 3 weeks significantly inhibited tumor growth (p < 0.004). The siHER2-nanoparticles have an excellent safety profile in terms of blood compatibility and low cytokine induction, when exposed to human peripheral blood mononuclear cells. The construct can be produced with high batch-to-batch reproducibility and the production methods are suitable for large-scale production. These results suggest that this siHER2-nanoparticle is ready for clinical evaluation.

HJURP (Holliday Junction Recognition Protein) is a newly discovered gene reported to function at centromeres and to interact with CENPA. However its role in tumor development remains largely unknown. The goal of this study was to investigate the clinical significance of HJURP in breast cancer and its correlation with radiotherapeutic outcome. We measured HJURP expression level in human breast cancer cell lines and primary breast cancers by Western blot and/or by Affymetrix Microarray; and determined its associations with clinical variables using standard statistical methods. Validation was performed with the use of published microarray data. We assessed cell growth and apoptosis of breast cancer cells after radiation using high-content image analysis. HJURP was expressed at higher level in breast cancer than in normal breast tissue. HJURP mRNA levels were significantly associated with estrogen receptor (ER), progesterone receptor (PR), Scarff-Bloom-Richardson (SBR) grade, age and Ki67 proliferation indices, but not with pathologic stage, ERBB2, tumor size, or lymph node status. Higher HJURP mRNA levels significantly decreased disease-free and overall survival. HJURP mRNA levels predicted the prognosis better than Ki67 proliferation indices. In a multivariate Cox proportional-hazard regression, including clinical variables as covariates, HJURP mRNA levels remained an independent prognostic factor for disease-free and overall survival. In addition HJURP mRNA levels were an independent prognostic factor over molecular subtypes (normal like, luminal, Erbb2 and basal). Poor clinical outcomes among patients with high HJURP expression were validated in five additional breast cancer cohorts. Furthermore, the patients with high HJURP levels were much more sensitive to radiotherapy. In vitro studies in breast cancer cell lines showed that cells with high HJURP levels were more sensitive to radiation treatment and had a higher rate of apoptosis than those with low levels. Knock down of HJURP in human breast cancer cells using shRNA reduced the sensitivity to radiation treatment. HJURP mRNA levels were significantly correlated with CENPA mRNA levels. HJURP mRNA level is a prognostic factor for disease-free and overall survival in patients with breast cancer and is a predictive biomarker for sensitivity to radiotherapy.

Post-genomic analyses of major transcription factor families, in both malignant and non-malignant cell types, have resulted in a broader revision of understanding the biology of transcription factor families. A number of oncogenic transcription factors, such as activator protein (AP)-1, estrogen receptor(ER) and signal transducer and activator of transcription (STAT)-3/STAT-5, are overactivated in human carcinomas. Up to now, three main groups of transcription factors are known to be important in human cancer. Transcription factor-directed anticancer drug development has focused on membrane or cytosolic targeting of molecules acting as ligand receptors. Recent technologies, such as small interfering RNA (siRNA), have shifted transcription factor targeting toward a more sophisticated rationale to elucidate the mechanisms that govern gene expression by analyzing the functions of proteins commonly over-expressed in transformed and malignant cells. In this study, we've applied a cell spot microarray (CSMA) method which provides reproducible multi-parametric phenotypic readouts for large-scale gene knockdown genetic screens. A human transcription factor siRNA library including 350 transcription factor genes which were significantly overexpressed in human breast cancer was screened against 6 breast cancer cell lines including (Triple negative breast cancer, HER2 positive ER positive) SUM149, BT20, HCC1569, SKBR3, MDAMB361 and MCF7 for measuring cell proliferation using EdU incorporation, cell apoptosis with c-PARP staining. We ranked the candidate gene list by the change of the rate of EdU or level of c-PARP or the combination of both. Our data showed that the silencing of LHX1, BCL6, GABPA or SMAD1 increased the c-PARP levels in most of cell lines. The Knockdown of ZNF215, NFATC1, HOXD4 or ZNF169 reduced the levels of EdU staining. Selected siRNAs were then tested on more different breast cancer cell lines to confirm the spectrum of activity. In the near future, based on this pilot screen, a complete siRNA library of transcription factors including 1000 gene list will be tested on our panel of 50 breast cancer cell lines. Our screen will provide comprehensive information on the central role of transcription factor in breast cancer, and will lead to the identity of novel therapeutics selectively against transcription factors. Citation Format: Zhi Hu, Shenda Gu, Joe Gray, Juha Rantala. Small interfering RNA library screen to identify therapeutic transcription factor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-208. doi:10.1158/1538-7445.AM2013-LB-208

Over-expression of oncogene HER2 was found in 20-30% primary breast cancers which involved in tumor initiation and progression. Breast cancer patients with high levels of HER2 (HER2 positive) showed poor clinical outcome. Increasing evidence indicated that the majority of patients with HER2 positive did not respond well to anti-HER2 drugs Trastuzumab (Herceptin) and Lapatinib. Even those who showed initial response ultimately became resistant to the treatment. The molecular mechanism of resistance includes the mutation of HER2 gene and the blocking of antigen of HER2 protein. The discovery of small interfering RNA (siRNA) raises the possibility of exploring new approach for cancer treatment. The siRNA against HER2 (siHER2) can reduce HER2 protein level by degrading mRNA expression. In our study, seventy-six siRNA sequences targeting the coding region of human HER2 gene were designed and synthesized. We quantified the RNA levels of HER2 and cell viability after HER2+ breast cancer cells (BT474, SkBr3 HCC1954) were transfected with siHER2 and scramble control siRNAs. Ten siRNA sequences were selected with greater than 50% knockdown efficiency and 40% inhibition of cell viability. We also measured the dose of 50% inhibition of cell growth (GI50) of top 5 siHER2 sequences in 20 HER2+ cell lines. The GI50 data showed that most of cell lines which were resistant to Trastuzumab were still sensitive to siHER2 treatment. Therefore, our data indicated that the siRNA against HER2 can potentially be used to overcome resistance of HER2 drugs, making it an alternative cancer therapeutic for HER2 positive patients. Currently, in vivo delivery of optimized siHER2 using nanoparticle is under evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-258. doi:1538-7445.AM2012-LB-258

Deregulation of aspects of the mitotic apparatus leads to increased genome instability, carcinogenesis and aggressive tumor behavior in human and rodent model systems1. This knowledge has stimulated development of inhibitors of elements of the mitotic apparatus as anticancer agents including PLK1, CENPE, and AURKB and several are now being tested for efficacy clincially2-6. These trials and eventual clinical use will benefit from molecular markers that predict response. In order to identify such markers, we assessed quantitative responses to the agents GSK461364, GSK923295 and GSK1070916 that target PLK1, CENPE and AURKB; respectively, in a panel of 50 breast cancer cell lines. This analysis showed that basal subtype cell lines were preferentially sensitive to all three agents and that responses among the lines to the three agents were strongly correlated. This may be explained by our discovery that components of the mitotic apparatus including PLK1, CENPE and AURKB form a transcriptionally co-regulated network comprised of more than 50 genes that is preferentially active in basal subtype of breast cell lines and primary tumors. Remarkably, this network also is activate in subsets of cancers of the lung, ovarian, prostate and brain, Wilms tumor, human blood malignancies and selected normal tissues. We then defined a mitotic apparatus network index (MANI) and showed that high MANI was associated with poor outcome clinically and with preferential responsive to GSK461364, GSK923295 and GSK1070916 in preclinical models. This suggests that measures of the MANI will identify poor outcome tumors that will likely respond well to mitotic apparatus network gene inhibitors as well as potential dose limiting normal tissues.Reference1. Quigley, D.A. et al. Nature 458, 505-8 (2009).2. Strebhardt, K. & Ullrich, A. Nat. Rev. Cancer 6, 321-330 (2006).3. Toyoshima-Morimoto, F., Taniguchi, E., Shinya, N., Iwamatsu, A. & Nishida, E. Nature 410, 215-20 (2001).4. Barr, F.A., Sillje, H.H. & Nigg, E.A. Nat. Rev. Mol. Cell. Biol. 5, 429–440 (2004).5. McInnes, C. et al. Nat. Chem. Biol. 2, 608–617 (2006).6. Yamada, S. et al. Oncogene 23, 5901-5911(2004). Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2020.

Figure S1. The mitotic network is conserved across human malignancies including lung cancer [GEO:GSE3141] (Bild et al., 2006); ovarian cancer [GEO:GSE3149] (Bild et al., 2006); Wilms’ tumor [GEO:GSE10320] (Huang et al., 2009); prostate cancer [GEO:GSE8218]; glioblastomas [GEO:GSE13041] (Lee et al., 2008); acute lymphoblastic leukemia and acute myelogenous leukemia [GEO:GSE12417] (Metzeler et al., 2008); and lymphoblast cell lines [GEO:GSE11582] (Choy et al., 2008). Figure S2. Mitotic network activity is significantly higher in intraductal breast carcinoma (IDC) [GEO:GSE7307] as compared to normal tissues. Figure S3. A The activity of the mitotic network (MNAI) is significantly associated with pathological mitotic counts (p

Figure S1. The mitotic network is conserved across human malignancies including lung cancer [GEO:GSE3141] (Bild et al., 2006); ovarian cancer [GEO:GSE3149] (Bild et al., 2006); Wilms’ tumor [GEO:GSE10320] (Huang et al., 2009); prostate cancer [GEO:GSE8218]; glioblastomas [GEO:GSE13041] (Lee et al., 2008); acute lymphoblastic leukemia and acute myelogenous leukemia [GEO:GSE12417] (Metzeler et al., 2008); and lymphoblast cell lines [GEO:GSE11582] (Choy et al., 2008). Figure S2. Mitotic network activity is significantly higher in intraductal breast carcinoma (IDC) [GEO:GSE7307] as compared to normal tissues. Figure S3. A The activity of the mitotic network (MNAI) is significantly associated with pathological mitotic counts (p