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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.

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.

Intrinsic and acquired resistance to current HER2 targeted therapies remains a challenge in clinics. We have developed a therapeutic HER2 siRNA delivered using mesoporous silica nanoparticles modified with polymers and conjugated with HER2 targeting antibodies. Our previous studies have shown that our HER2 siRNA nanoparticles could overcome intrinsic and acquired resistance to trastuzumab and lapatinib in HER2-positive breast cancers. In this study, we investigated the effect of long-term (7 months) treatment using our therapeutic HER2 siRNA. Even after the removal of HER2 siRNA, the long-term treated cells grew much slower (67% increase in doubling time) than cells that have not received any treatment. The treated cells did not undergo epithelial-mesenchymal transition or showed enrichment of tumor initiating cells. Unlike trastuzumab and lapatinib, which induced resistance in BT474 cells after 6 months of treatment, HER2 siRNA did not induce resistance to HER2 siRNA, trastuzumab, or lapatinib. HER2 ablation with HER2 siRNA prevented reactivation of HER2 signaling that was observed in cells resistant to lapatinib. Altogether, our results indicate that a HER2 siRNA based therapeutic provides a more durable inhibition of HER2 signaling in vitro and can potentially be more effective than the existing therapeutic monoclonal antibodies and small molecule inhibitors.

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.

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.

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.