Description: Ribonucleotide reductase (RNR) is an attractive target for anticancer agents given its central function in DNA synthesis, growth, metastasis, and drug resistance of cancer cells. The current clinically established RNR inhibitors have the shortcomings of short half-life, drug resistance, and iron chelation. Here, we report the development of a novel class of effective RNR inhibitors addressing these issues. A novel ligand-binding pocket on the RNR small subunit (RRM2) near the C-terminal tail was proposed by computer modeling and verified by site-directed mutagenesis and nuclear magnetic resonance (NMR) techniques. A compound targeting this pocket was identified by virtual screening of the National Cancer Institute (NCI) diverse small-molecule database. By lead optimization, we developed the novel RNR inhibitor COH29 that acted as a potent inhibitor of both recombinant and cellular human RNR enzymes. COH29 overcame hydroxyurea and gemcitabine resistance in cancer cells. It effectively inhibited proliferation of most cell lines in the NCI 60 human cancer panel, most notably ovarian cancer and leukemia, but exerted little effect on normal fibroblasts or endothelial cells. In mouse xenograft models of human cancer, COH29 treatment reduced tumor growth compared with vehicle. Site-directed mutagenesis, NMR, and surface plasmon resonance biosensor studies confirmed COH29 binding to the proposed ligand-binding pocket and offered evidence for assembly blockade of the RRM1-RRM2 quaternary structure. Our findings offer preclinical validation of COH29 as a promising new class of RNR inhibitors with a new mechanism of inhibition, with broad potential for improved treatment of human cancer. Cancer Res; 73(21); 6484–93. ©2013 AACR.

Description: Angiogenesis is a major clinical feature of esophageal squamous cell carcinoma (ESCC), an aggressive disease of increasing incidence in developed countries. In ESCCs, the proangiogenic factor VEGF-C is an independent prognostic factor for ESCC, where understanding the mechanisms of VEGF-C upregulation may cue possible therapeutic insights. Here, we report that expression of the transcription factor Nkx2-8 is downregulated in ESCCs where it inversely correlates with progression and VEGF-C upregulation. Patients with ESCCs with lower Nkx2-8 expression exhibited reduced overall survival. Modulating expression of Nkx2-8 up or down inhibited or enhanced, respectively, proangiogenic activity in vitro and in vivo. Mechanistic investigations showed that Nkx2-8 repressed NF-κB activity by restraining nuclear localization of NF-κB p65 via downregulation of AKIP1, a NF-κB p65 binding partner, and also by directly targeting the AKIP1 promoter. We confirmed evidence for the importance of the Nkx2-8/AKIP1/NF-κB axis identified in ESCC cell models through an immunohistochemical analysis of a large cohort of human ESCC specimens. Taken together, our results showed that Nkx2-8 functions as a tumor suppressor in ESCCs, the downregulation of which contributes to NF-κB activation and ESCC angiogenesis. Cancer Res; 73(12); 3638–48. ©2013 AACR.

Description: Macrophages are important drivers in the development of inflammation-associated colon cancers, but the mechanistic underpinnings for their contributions are not fully understood. Furthermore, Toll-like receptors have been implicated in colon cancer, but their relevant cellular sites of action are obscure. In this study, we show that the endoplasmic reticulum chaperone gp96 is essential in tumor-associated macrophages (TAM) to license their contributions to inflammatory colon tumorigenesis. Mice where gp96 was genetically deleted in a macrophage-specific manner exhibited reduced colitis and inflammation-associated colon tumorigenesis. Attenuation of colon cancer in these mice correlated strikingly with reduced mutation rates of β-catenin, increased efficiency of the DNA repair machinery, and reduced expression of proinflammatory cytokines, including interleukin (IL)-17 and IL-23 in the tumor microenvironment. The genotoxic nature of TAM-associated inflammation was evident by increased expression of genes in the DNA repair pathway. Our work deepens understanding of how TAM promote oncogenesis by altering the molecular oncogenic program within epithelial cells, and it identifies gp96 as a lynchpin chaperone needed in TAM to license their function and impact on expression of critical inflammatory cytokines in colon tumorigenesis. Cancer Res; 74(2); 446–59. ©2013 AACR.

Description: Purpose: The oncogenic PI3K/Akt/mTOR pathway is an attractive therapeutic target in cancer. However, it is unknown whether the pathway blockade required for tumor growth inhibition is clinically achievable. Therefore, we conducted pharmacodynamic studies with GDC-0068, an ATP competitive, selective Akt1/2/3 inhibitor, in preclinical models and in patients treated with this compound. Experimental Design: We used a reverse phase protein array (RPPA) platform to identify a biomarker set indicative of Akt inhibition in cell lines and human-tumor xenografts, and correlated the degree of pathway inhibition with antitumor activity. Akt pathway activity was measured using this biomarker set in pre- and post-dose tumor biopsies from patients treated with GDC-0068 in the dose escalation clinical trial. Results: The set of biomarkers of Akt inhibition is composed of 10 phosphoproteins, including Akt and PRAS40, and is modulated in a dose-dependent fashion, both in vitro and in vivo . In human-tumor xenografts, this dose dependency significantly correlated with tumor growth inhibition. Tumor biopsies from patients treated with GDC-0068 at clinically achievable doses attained a degree of biomarker inhibition that correlated with tumor growth inhibition in preclinical models. In these clinical samples, compensatory feedback activation of ERK and HER3 was observed, consistent with preclinical observations. Conclusion: This study identified a set of biomarkers of Akt inhibition that can be used in the clinical setting to assess target engagement. Here, it was used to show that robust Akt inhibition in tumors from patients treated with GDC-0068 is achievable, supporting the clinical development of this compound in defined patient populations. Clin Cancer Res; 19(24); 6976–86. ©2013 AACR .

Description: Histone lysine methyltransferase NSD2 (WHSC1/MMSET) is overexpressed frequently in multiple myeloma due to the t(4;14) translocation associated with 15% to 20% of cases of this disease. NSD2 has been found to be involved in myelomagenesis, suggesting it may offer a novel therapeutic target. Here we show that NSD2 methyltransferase activity is crucial for clonogenicity, adherence, and proliferation of multiple myeloma cells on bone marrow stroma in vitro and that NSD2 is required for tumorigenesis of t(4;14)+ but not t(4;14)− multiple myeloma cells in vivo. The PHD domains in NSD2 were important for its cellular activity and biological function through recruiting NSD2 to its oncogenic target genes and driving their transcriptional activation. By strengthening its disease linkage and deepening insights into its mechanism of action, this study provides a strategy to therapeutically target NSD2 in multiple myeloma patients with a t(4;14) translocation. Cancer Res; 73(20); 6277–88. ©2013 AACR.

Description: Purpose: The human tumor-derived soluble MHC I-chain–related molecule (sMIC) is highly immune suppressive in cancer patients and correlates with poor prognosis. However, the therapeutic effect of targeting sMIC has not been determined, due to the limitation that mice do not express homologs of human MIC. This study is to evaluate the therapeutic effect of a monoclonal antibody (mAb) targeting sMIC in a clinically relevant transgenic animal model. Experimental Design: We treated the engineered MIC-expressing "humanized" TRAMP/MIC bitransgenic mice at advanced disease stages with a sMIC-neutralizing nonblocking anti-MIC mAb and assessed the therapeutic efficacy and associated mechanisms. Results: A sMIC-neutralizing nonblocking anti-MIC mAb effectively induced regression of primary tumors and eliminated metastasis without inducing systemic toxicity. The therapeutic effect is conferred by revamping endogenous antitumor immune responses, exemplified by restoring natural killer (NK) cell homeostasis and function, enhancing susceptibility of MIC + -tumor cells to NK cell killing, reviving and sustaining antigen-specific CD8 T-cell responses, augmenting CD4 T cells to Th1 responses, priming dendritic cells for antigen presentation, and remodeling tumor microenvironment to be more immune reactive. Conclusions: Therapy with a sMIC-neutralizing nonblocking anti-MIC mAb can effectuate antitumor immune responses against advanced MIC + tumors. Our study provided strong rationale for translating sMIC-neutralizing therapeutic mAb into clinics, either alone or in combination with current ongoing standard immunotherapies. Clin Cancer Res; 21(21); 4819–30. ©2015 AACR .

Description: Oncogenic transformation may reprogram tumor metabolism and render cancer cells addicted to extracellular nutrients. Deprivation of these nutrients may therefore represent a therapeutic opportunity, but predicting which nutrients cancer cells become addicted remains difficult. Here, we performed a nutrigenetic screen to determine the phenotypes of isogenic pairs of clear cell renal cancer cells (ccRCC), with or without VHL, upon the deprivation of individual amino acids. We found that cystine deprivation triggered rapid programmed necrosis in VHL-deficient cell lines and primary ccRCC tumor cells, but not in VHL-restored counterparts. Blocking cystine uptake significantly delayed xenograft growth of ccRCC. Importantly, cystine deprivation triggered similar metabolic changes regardless of VHL status, suggesting that metabolic responses alone are not sufficient to explain the observed distinct fates of VHL-deficient and restored cells. Instead, we found that increased levels of TNFα associated with VHL loss forced VHL-deficient cells to rely on intact RIPK1 to inhibit apoptosis. However, the preexisting elevation in TNFα expression rendered VHL-deficient cells susceptible to necrosis triggered by cystine deprivation. We further determined that reciprocal amplification of the Src–p38 (MAPK14)–Noxa (PMAIP1) signaling and TNFα–RIP1/3 (RIPK1/RIPK3)–MLKL necrosis pathways potentiated cystine-deprived necrosis. Together, our findings reveal that cystine deprivation in VHL-deficient RCCs presents an attractive therapeutic opportunity that may bypass the apoptosis-evading mechanisms characteristic of drug-resistant tumor cells. Cancer Res; 76(7); 1892–903. ©2016 AACR.

Description: Triple-negative breast cancer (TNBC) is a highly aggressive tumor subtype lacking effective prognostic indicators or therapeutic targets. Mitochondrial function is dysregulated frequently in cancer cells to allow for adaptation to a harsh tumor microenvironment. Targeting mitochondrial biogenesis and bioenergetics is, therefore, an attractive therapeutic strategy. In this study, we performed quantitative proteomic analyses in human parental and metastatic breast cancer cell lines to identify mitochondrial proteins involved in TNBC metastasis. We found that single-strand DNA-binding protein 1 (SSBP1) was downregulated in highly metastatic breast cancer cells. Moreover, SSBP1 downregulation promoted TNBC cell metastasis in vitro and in vivo. Mechanistically, SSBP1 loss decreased mitochondrial DNA copy number, thereby potentiating calcineurin-mediated mitochondrial retrograde signaling that induced c-Rel/p50 nuclear localization, activated TGFβ promoter activity, and TGFβ-driven epithelial-to-mesenchymal transition. Low SSBP1 expression correlated with tumor progression and poor prognosis in patients. Collectively, our findings identified SSBP1 as a novel metastasis suppressor and elucidated the mechanisms by which dysregulated mitochondrial signaling contributes to metastatic potential, providing potential new prognostic indicators for patients with TNBC. Cancer Res; 76(4); 952–64. ©2015 AACR.

Description: Traditional approaches to evaluating antitumor agents using human tumor xenograft models have generally used cohorts of 8 to 10 mice against a limited panel of tumor models. An alternative approach is to use fewer animals per tumor line, allowing a greater number of models that capture greater molecular/genetic heterogeneity of the cancer type. We retrospectively analyzed 67 agents evaluated by the Pediatric Preclinical Testing Program to determine whether a single mouse, chosen randomly from each group of a study, predicted the median response for groups of mice using 83 xenograft models. The individual tumor response from a randomly chosen mouse was compared with the group median response using established response criteria. A total of 2,134 comparisons were made. The single tumor response accurately predicted the group median response in 1,604 comparisons (75.16%). The mean tumor response correct prediction rate for 1,000 single mouse random samples was 78.09%. Models had a range for correct prediction (60%–87.5%). Allowing for misprediction of ± one response category, the overall mean correct single mouse prediction rate was 95.28%, and predicted overall objective response rates for group data in 66 of 67 drug studies. For molecularly targeted agents, occasional exceptional responder models were identified and the activity of that agent confirmed in additional models with the same genotype. Assuming that large treatment effects are targeted, this alternate experimental design has similar predictive value as traditional approaches, allowing for far greater numbers of models to be used that more fully encompass the heterogeneity of disease types. Cancer Res; 76(19); 5798–809. ©2016 AACR.

Description: Purpose: Somatic mutations in the tyrosine kinase domain of human epidermal growth factor receptor 2 (HER2) may be an alternative mechanism to HER2 activation and can affect the sensitivity toward HER2-targeted therapies. We aimed to investigate the prevalence, clinicopathologic characteristics, and functional relevance of novel HER2 mutations in breast cancer. Experimental Design: We performed Sanger sequencing of all exons of the HER2 gene in 1,248 primary tumors and 18 paired metastatic samples. Novel HER2 mutations were functionally characterized. Results: The total HER2 somatic mutation rate was 2.24% (28/1,248). Of the seven novel HER2 mutations, L768S and V773L were only detected in HER2-negative tumors, whereas K753E was found in HER2-positive disease. L768S and V773L mutations exhibited a significant increase in tyrosine kinase–specific activity and strongly increased the phosphorylation of signaling proteins in various cell lines. Xenograft experiments showed that NIH3T3 cells bearing the L768S and V773L mutations displayed more rapid growth. MCF10A, BT474, and MDA-MB-231 cells bearing the K753E mutation were resistant to lapatinib, but could be inhibited by neratinib. Finally, comparison of HER2 mutations in 18 pairs of primary and metastatic lesions revealed that the drug-resistant HER2 mutations (K753E and L755S) were enriched in metastatic lesions. Conclusions: HER2-negative breast cancer with activating mutations can benefit from HER2-targeted therapies. Meanwhile, mutations in the HER2 kinase domain might be a key mechanism of resistance to HER2-targeted therapy, and irreversible tyrosine kinase inhibitors such as neratinib may offer alternative treatment options. Clin Cancer Res; 22(19); 4859–69. ©2016 AACR .