Description: The Hsp90 facilitates proper folding of signaling proteins associated with cancer progression, gaining attention as a target for therapeutic intervention. The natural rotenoid deguelin was identified as an Hsp90 inhibitor, but concerns about neurotoxicity have limited prospects for clinical development. In this study, we report progress on deguelin analogues that address this limitation, focusing on the novel analogue SH-1242 as a candidate to broadly target human lung cancer cells, including those that are chemoresistant or harboring KRAS mutations. In a KRAS-driven mouse model of lung cancer, SH-1242 administration reduced tumor multiplicity, volume, and load. Similarly, in human cell line–based or patient-derived tumor xenograft models, SH-1242 induced apoptosis and reduced tumor vasculature in the absence of detectable toxicity. In contrast to deguelin, SH-1242 toxicity was greatly reduced in normal cells and when administered to rats did not produce obvious histopathologic features in the brain. Mechanistic studies revealed that SH-1242 bound to the C-terminal ATP-binding pocket of Hsp90, disrupting the ability to interact with its co-chaperones and clients and triggering a degradation of client proteins without affecting Hsp70 expression. Taken together, our findings illustrate the superior properties of SH-1242 as an Hsp90 inhibitor and as an effective antitumor and minimally toxic agent, providing a foundation for advancing further preclinical and clinical studies. Cancer Res; 76(3); 686–99. ©2015 AACR.

Description: Purpose: The intratumoral heterogeneity (ITH) and the evolution of genomic architectures associated with the development of distant metastases are not well understood in colorectal cancers. Experimental Design: We performed multiregion biopsies of primary and liver metastatic regions from five colorectal cancers with whole-exome sequencing and copy number profiling. Results: In addition to a substantial level of genetic ITH, multiregion genetic profiling identifies the subclonal mutational architecture, leading to the region-based or spatial categorization of somatic mutations and the inference of intratumoral evolutionary history of cancers. The universal mutations (those observed in all the regional biopsies) are enriched in known cancer genes such as APC and TP53 with distinct mutational spectra compared with biopsy- or region-specific mutations, suggesting that major operative mutational mechanisms and their selective pressures are not constant across the metastatic progression. The phylogenies inferred from genomic data show branching evolutionary patterns where some primary biopsies are often segregated with metastastic lesions. Our analyses also revealed that copy number changes such as the chromosomal gains of c-MYC and chromothripsis can be region specific and the potential source of genetic ITH. Conclusions: Our data show that the genetic ITH is prevalent in colorectal cancer serving as a potential driving force to generate metastasis-initiating clones and also as a means to infer the intratumoral evolutionary history of cancers. The paucity of recurrent metastasis-clonal events suggests that colorectal cancer distant metastases may not follow a uniform course of genomic evolution, which should be considered in the genetic diagnosis and the selection of therapeutic targets for the advanced colorectal cancer. Clin Cancer Res; 21(19); 4461–72. ©2015 AACR .

Description: Purpose: Gastric adenoma (GA) is a premalignant lesion that precedes intestinal-type gastric carcinoma (GC). However, genetic progression mechanisms from GA to GC have not been clarified. Experimental Design: We performed whole-exome sequencing–based mutational analyses for 15 synchronous pairs of attached GAs and GCs. Results: There was no significant difference in the number of driver mutations or copy-number alterations between GAs and GCs. Well-known mutations of TP53, APC, RNF43, and RPL22 were recurrently detected in synchronous GA/GC pairs. In addition, we discovered novel KDM6A, PREX2, FAT1, KMT2C, GLI3, and RPL22 mutations and hypermutation in GAs, but did not identify recurrent drivers for GA-to-GC progression. Clonal structure analyses revealed that most GA/GC pairs exhibit parallel evolution with early divergence rather than stepwise evolution during GA-to-GC progression. Of note, three cases were identified as clonally nonrelated GA/GC pairs despite the lack of histologic differences. We found differences in dominant mutational signatures 1, 6, 15, and 17 in GA/GC trunks, GA branches, and GC branches. Compared with our previous work on synchronous colon adenoma/carcinoma genome structures, where most drivers were in the trunk with parallel evolution, synchronous GA/GC genomes showed a different model of parallel evolution, with many drivers in the branches. Conclusions: The preferred sequence of mutational events during GA-to-GC progression might be more context-dependent than colon adenoma progression. Our results show that nonclonal synchronous GA/GC is common and that GA genomes have already acquired distinct genomic alterations, suggesting caution in the diagnosis of synchronous GA and GC, especially in residual or recurrent cases. Clin Cancer Res; 24(19); 4715–25. ©2018 AACR .

Description: The generation of reactive oxygen species (ROS) is required for proper cell signaling, but must be tightly regulated to minimize deleterious oxidizing effects. Activation of the NADPH oxidases (Nox) triggers ROS production and, thus, regulatory mechanisms exist to properly control Nox activity. In this study, we report a novel mechanism in which Nox1 activity is regulated through the proteasomal degradation of Nox organizer 1 (NoxO1). We found that through the interaction between NoxO1 and growth receptor–bound protein 2 (Grb2), the Casitas B–lineage lymphoma (Cbl) E3 ligase was recruited, leading to decreased NoxO1 stability and a subsequent reduction in ROS generation upon epidermal growth factor (EGF) stimulation. Additionally, we show that EGF-mediated phosphorylation of NoxO1 induced its release from Grb2 and facilitated its association with Nox activator 1 (NoxA1) to stimulate ROS production. Consistently, overexpression of Grb2 resulted in decreased Nox1 activity, whereas knockdown of Grb2 led to increased Nox1 activity in response to EGF. CRISPR/Cas9-mediated NoxO1 knockout in human colon cancer cells abrogated anchorage-independent growth on soft agar and tumor-forming ability in athymic nude mice. Moreover, the expression and stability of NoxO1 were significantly increased in human colon cancer tissues compared with normal colon. Taken together, these results support a model whereby Nox1 activity and ROS generation are regulated by Grb2/Cbl-mediated proteolysis of NoxO1 in response to EGF, providing new insight into the processes by which excessive ROS production may promote oncogenic signaling to drive colorectal tumorigenesis. Cancer Res; 76(4); 855–65. ©2016 AACR.

Description: Purpose: This study was aimed at developing and validating a quantitative multigene assay for predicting tumor recurrence after gastric cancer surgery. Experimental Design: Gene expression data were generated from tumor tissues of patients who underwent surgery for gastric cancer ( n = 267, training cohort). Genes whose expression was significantly associated with activation of YAP1 (a frequently activated oncogene in gastrointestinal cancer), 5-year recurrence-free survival, and 5-year overall survival were first identified as candidates for prognostic genes (156 genes, P 〈 0.001). We developed the recurrence risk score (RRS) by using quantitative RT-PCR to identify genes whose expression levels were significantly associated with YAP1 activation and patient survival in the training cohort. Results: We based the RRS assay on 6 genes, IGFBP4, SFRP4, SPOCK1, SULF1, THBS , and GADD45B , whose expression levels were significantly associated with YAP1 activation and prognosis in the training cohort. The RRS assay was further validated in an independent cohort of 317 patients. In multivariate analysis, the RRS was an independent predictor of recurrence [HR, 1.6; 95% confidence interval (CI), 1.02–2.4; P = 0.03]. In patients with stage II disease, the RRS had an HR of 2.9 (95% CI, 1.1–7.9; P = 0.03) and was the only significant independent predictor of recurrence. Conclusions: The RRS assay was a valid predictor of recurrence in the two cohorts of patients with gastric cancer. Independent prospective studies to assess the clinical utility of this assay are warranted. Clin Cancer Res; 22(24); 6228–35. ©2016 AACR .

Description: Purpose: This multicenter randomized trial was designed to evaluate whether melanoma helper peptides augment cytotoxic T lymphocyte (CTL) responses to a melanoma vaccine and improve clinical outcome in patients with advanced melanoma. Experimental Design: One hundred seventy-five patients with measurable stage IV melanoma were enrolled into 4 treatment groups, vaccinated with 12 MHC class I-restricted melanoma peptides to stimulate CTL (12MP, group A), plus a tetanus peptide (group B), or a mixture of 6 melanoma helper peptides (6MHP, group C) to stimulate helper T lymphocytes (HTL), or with 6 melanoma helper peptide (6MHP) alone (group D), in incomplete Freund's adjuvant plus granulocyte macrophage colony-stimulating factor. CTL responses were assessed using an in vitro -stimulated IFN- ELIspot assay, and HTL responses were assessed using a proliferation assay. Results: In groups A to D, respectively, CTL response rates to 12 melanoma peptides were 43%, 47%, 28%, and 5%, and HTL response rates to 6MHP were in 3%, 0%, 40%, and 41%. Best clinical response was partial response in 7 of 148 evaluable patients (4.7%) without significant difference among study arms. Median overall survival (OS) was 11.8 months. Immune response to 6 MHP was significantly associated with both clinical response ( P = 0.036) and OS ( P = 0.004). Conclusion: Each vaccine regimen was immunogenic, but MHPs did not augment CTL responses to 12 melanoma peptides. The association of survival and immune response to 6MHP supports further investigation of helper peptide vaccines. For patients with advanced melanoma, multipeptide vaccines should be studied in combination with other potentially synergistic active therapies. Clin Cancer Res; 19(15); 4228–38. ©2013 AACR .

Description: Purpose: Common treatment modalities for non–small cell lung cancer (NSCLC) involve the EGF receptor-tyrosine kinase inhibitors (EGFR-TKIs) like gefitinib and erlotinib. However, the vast majority of treated patients acquire resistance to EGFR-TKIs, due, in large part, to secondary mutations in EGFR or amplification of the MET gene. Our purpose was to test ubiquitin-specific peptidase 8 (USP8) as a potential therapeutic target for gefitinib-resistant and -sensitive non–small cell lung cancer (NSCLC). Experimental Design: Testing the effect of knockdown of USP8 and use of a synthetic USP8 inhibitor to selectively kill gefitinib-resistant (or -sensitive) NSCLCs with little effect on normal cells in cell culture and a xenograft mouse model. Results: Knockdown of ubiquitin-specific peptidase 8 (USP8) selectively kills gefitinib-resistant NSCLCs while having little toxicity toward normal cells. Genetic silencing of USP8 led to the downregulation of several receptor tyrosine kinases (RTK) including EGFR, ERBB2, ERBB3, and MET. We also determined that a synthetic USP8 inhibitor markedly decreased the viability of gefitinib-resistant and -sensitive NSCLC cells by decreasing RTK expression while having no effect on normal cells. Moreover, treatment with a USP8 inhibitor led to significant reductions in tumor size in a mouse xenograft model using gefitinib-resistant and -sensitive NSCLC cells. Conclusions: Our results show for the first time that the inhibition of USP8 activity or reduction in USP8 expression can selectively kill NSCLC cells. We propose USP8 as a potential therapeutic target for gefitinib-resistant and -sensitive NSCLC cells. Clin Cancer Res; 19(14); 3894–904. ©2013 AACR .

Description: Purpose: The goals of this study were to investigate the clinical activity, safety, and biomarkers of dacomitinib, an irreversible tyrosine kinase inhibitor of EGFR, HER2, and HER4, in recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M-SCCHN). Experimental Design: Patients were eligible if the diseases were not amenable to curative treatment and had progressed on platinum-based chemotherapy, and were treated with dacomitinib 45 mg/day. The primary endpoint was objective response rate by RECISTv1.1. Exploratory analysis included the characterization of somatic mutation, gene copy number, gene expression, p16 INK4A expression by IHC, and investigation of their relationship with clinical outcomes. Results: Forty-eight patients were evaluable for efficacy and toxicity. Ten patients (20.8%) had partial responses and 31 patients (65%) had stable diseases. The median progression-free survival (PFS) and overall survival (OS) were 3.9 months [95% confidence interval (CI), 2.9–5.0] and 6.6 months (95% CI, 5.4–10.3). Adverse events were mostly grade 1–2. Mutations in the PI3K pathway ( PIK3CA , PTEN ) and high expression of inflammatory cytokines ( IL6 , IL8 , IL1A , IL1B , IL4 , and TNF ) were significantly associated with shorter PFS (2.9 vs. 4.9 months without mutations, P = 0.013; 2.8 vs. 9.9 months with low expression, P = 0.004). Those harboring PI3K pathway mutations or high inflammatory cytokine expression had shorter median OS (6.1 vs. 12.5 months lacking PI3K pathway mutations and with low inflammatory cytokine expression, P = 0.005). Conclusions: Dacomitinib demonstrated clinical efficacy with manageable toxicity in platinum-failed R/M-SCCHN patients. Screening of PI3K pathway mutation and inflammatory cytokine expression may help identify which R/M-SCCHN patients are likely to gain benefit from dacomitinib. Clin Cancer Res; 21(3); 544–52. ©2014 AACR.

Description: Purpose: Activation of YAP1, a novel oncogene in the Hippo pathway, has been observed in many cancers, including colorectal cancer. We investigated whether activation of YAP1 is significantly associated with prognosis or treatment outcomes in colorectal cancer. Experimental Design: A gene expression signature reflecting YAP1 activation was identified in colorectal cancer cells, and patients with colorectal cancer were stratified into two groups according to this signature: activated YAP1 colorectal cancer (AYCC) or inactivated YAP1 colorectal cancer (IYCC). Stratified patients in five test cohorts were evaluated to determine the effect of the signature on colorectal cancer prognosis and response to cetuximab treatment. Results: The activated YAP1 signature was associated with poor prognosis for colorectal cancer in four independent patient cohorts with stage I–III disease (total n = 1,028). In a multivariate analysis, the impact of the YAP1 signature on disease-free survival was independent of other clinical variables [hazard ratio (HR), 1.63; 95% confidence interval (CI), 1.25–2.13; P 〈 0.001]. In patients with stage IV colorectal cancer and wild-type KRAS, IYCC patients had a better disease control rate and progression-free survival (PFS) after cetuximab monotherapy than did AYCC patients; however, in patients with KRAS mutations, PFS duration after cetuximab monotherapy was not different between IYCC and AYCC patients. In multivariate analysis, the effect of YAP1 activation on PFS was independent of KRAS mutation status and other clinical variables (HR, 1.82; 95% CI, 1.05–3.16; P = 0.03). Conclusions: Activation of YAP1 is highly associated with poor prognosis for colorectal cancer and may be useful in identifying patients with metastatic colorectal cancer resistant to cetuximab. Clin Cancer Res; 21(2); 357–64. ©2014 AACR .

Description: Purpose: Malignancy relapse remains a major obstacle for successful allogeneic hematopoietic cell transplantation (HCT). Chronic graft-versus-host disease (cGVHD) is associated with fewer relapses. However, when studying effects of cGVHD on relapse, it is difficult to separate from acute GVHD effects as most cases of cGVHD occur within the first year after transplant at the time when acute GVHD is still active. Experimental Design: This study based on CIBMTR registry data investigated cGVHD and its association with the incidence of late relapse and survival in 7,489 patients with acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and myelodysplastic syndromes (MDS), who were leukemia free at 12 months after myeloablative allogeneic HCT. Results: Forty-seven percent of the study population was diagnosed with cGVHD at 12 months after transplant. The protective effect of cGVHD on late relapse was present only in patients with CML [RR, 0.47; 95% confidence interval (CI), 0.37–0.59; P 〈 0.0001). cGVHD was significantly associated with higher risk of treatment-related mortality (TRM; RR, 2.43; 95% CI, 2.09–2.82; P 〈 0.0001) and inferior overall survival (RR, 1.56; 95% CI, 1.41–1.73; P 〈 0.0001) for all diseases. In patients with CML, all organ sites and presentation types of cGVHD were equally associated with lower risk of late relapse. Conclusions: These results indicate that clinically relevant antileukemia effects of cGVHD on late relapses are present only in CML but not in AML, ALL, or MDS. Chronic GVHD in patients who are 1-year survivors after myeloablative allogeneic HCT is primarily associated with higher TRM and inferior survival. Clin Cancer Res; 21(9); 2020–8. ©2014 AACR . See related commentary by Gill, p. 1981