Purpose: BAF57, a component of the switching-defective and sucrose nonfermenting (SWI/SNF) chromatin-remodeling complex conglomerate, modulates androgen receptor activity to promote prostate cancer. However, the molecular consequences of tumor-associated BAF57 expression have remained undefined in advanced disease such as castration-resistant prostate cancer and/or metastasis.
Experimental Design: Clinical human specimens of primary and metastatic prostate cancer were immunohistochemically examined for tumor-grade association of BAF57 expression. Global gene expression analyses were conducted in models mimicking tumor-associated BAF57 expression. Aberrant BAF57-dependent gene expression changes, bypass of androgen-mediated signaling, and chromatin-specific SWI/SNF complex alterations with respect to cytoskeletal remodelers such as integrins were validated. Cell migration assays were used to profile the biologic phenotypes conferred under conditions simulating tumor-derived BAF57 expression.
Results: Immunohistochemical quantitation of primary human specimens revealed that BAF57 was significantly and aberrantly elevated as a function of tumor grade. Critically, gene expression analyses showed that BAF57 deregulation circumvented androgen-mediated signaling, elicited α2 integrin upregulation, and altered other SWI/SNF complex components at the α2 integrin locus. BAF57-dependent α2 integrin induction conferred a prometastatic migratory advantage, which was attenuated by anti-α2 integrin antibody blockade. Furthermore, BAF57 was found to be markedly upregulated in human prostate cancer metastases of the lung, lymph node, and dura.
Purpose: We queried whether comprehensive genomic profiling using a next-generation sequencing–based assay could identify novel and unanticipated targets of therapy for patients with relapsed invasive lobular carcinoma (ILC).
Experimental Design: DNA sequencing (Illumina HiSeq 2000) was conducted for 3,320 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer on indexed, adaptor-ligated, hybridization-captured libraries using DNA isolated from formalin-fixed paraffin-embedded sections from 22 histologically verified ILC.
Results: A total of 75 genomic alterations were identified with an average of 3.4 alterations per tumor (range, 1–6), of which 35 were actionable for an average of 1.59 actionable alterations per patient (range, 0–3). Nineteen of 22 (86%) of the ILC samples harbored at least one actionable alteration. Six (27%) cases featured alterations in ERRB2 including 4 (18%) with ERBB2 mutation, 1 (5%) with an ERBB2 gene fusion, and 1 (5%) with an ERBB2 copy number gain (amplification). The enrichment of ERBB2 mutations/fusion in CDH1-mutated ILC (5 of 22, 23%) compared with the 5 ERBB2 mutations in a series of 286 non-CDH1-mutated breast cancers from which the ILC cases were obtained (5 of 286, 2%) was significant (P = 0.0006).
Purpose: To analyze the antimyeloma potential of TG02, an ERK5/CDK inhibitory drug.
Experimental Design: Utilizing different multiple myeloma cell lines we determined the effect of TG02 over viability by MTT assays. The apoptotic effect over multiple myeloma patient samples was studied ex vivo by cytometry. The mechanism of action of TG02 was analyzed in the cell line MM1S, studying its effect on the cell cycle, the induction of apoptosis, and the loss of mitochondrial membrane potential by cytometry and Western blot. Two models of multiple myeloma xenograft were utilized to study the in vivo action of TG02.
Results: TG02 potently inhibited proliferation and survival of multiple myeloma cell lines, even under protective bone marrow niche conditions, and selectively induced apoptosis of primary patient-derived malignant plasma cells. TG02 displayed significant single-agent activity in two multiple myeloma xenograft models, and enhanced the in vivo activity of bortezomib and lenalidomide. Signaling analyses revealed that the drug simultaneously blocked the activity of CDKs 1, 2, and 9 as well as the MAP kinase ERK5 in MM1S cells, leading to cell-cycle arrest and rapid commitment to apoptosis. TG02 induced robust activation of both the intrinsic and extrinsic pathways of apoptosis, and depletion of XIAP and the key multiple myeloma survival protein Mcl-1.
Purpose:BRAFV600E mutations are associated with poor clinical prognosis in colorectal cancer (CRC). Although selective BRAF inhibitors are effective for treatment of melanoma, comparable efforts in CRC have been disappointing. Here, we investigated potential mechanisms underlying this resistance to BRAF inhibitors in BRAFV600E CRC.
Experimental Design: We examined phosphoinositide 3-kinase (PI3K)/mTOR signaling in BRAFV600E CRC cell lines after BRAF inhibition and cell viability and apoptosis after combined BRAF and PI3K/mTOR inhibition. We assessed the efficacy of in vivo combination treatment using a novel genetically engineered mouse model (GEMM) for BRAFV600E CRC.
Results: Western blot analysis revealed sustained PI3K/mTOR signaling upon BRAF inhibition. Our BRAFV600E GEMM presented with sessile serrated adenomas/polyps, as seen in humans. Combination treatment in vivo resulted in induction of apoptosis and tumor regression.
Purpose: An understanding of how hematopoietic cells respond to therapy that causes myelosuppression will help develop approaches to prevent this potentially life-threatening toxicity. The goal of this study was to determine how human myeloid precursor cells respond to temozolomide (TMZ)-induced DNA damage.
Experimental Design: We developed an ex vivo primary human myeloid precursor cells model system to investigate the involvement of cell-death pathways using a known myelosuppressive regimen of O6-benzylguanine (6BG) and TMZ.
Results: Exposure to 6BG/TMZ led to increases in p53, p21, -H2AX, and mitochondrial DNA damage. Increases in mitochondrial membrane depolarization correlated with increased caspase-9 and -3 activities following 6BG/TMZ treatment. These events correlated with decreases in activated AKT, downregulation of the DNA repair protein O6-methylguanine–DNA methyltransferase (MGMT), and increased cell death. During myeloid precursor cell expansion, FAS/CD95/APO1(FAS) expression increased over time and was present on approximately 100% of the cells following exposure to 6BG/TMZ. Although c-flipshort, an endogenous inhibitor of FAS-mediated signaling, was decreased in 6BG/TMZ–treated versus control, 6BG-, or TMZ alone–treated cells, there were no changes in caspase-8 activity. In addition, there were no changes in the extent of cell death in myeloid precursor cells exposed to 6BG/TMZ in the presence of neutralizing or agonistic anti-FAS antibodies, indicating that FAS-mediated signaling was not operative.
Purpose: Effective therapy for malignant melanoma, the leading cause of death from skin cancer, remains an area of significant unmet need in oncology. The elevated expression of PKC in advanced metastatic melanoma results in the increased phosphorylation of the transcription factor ATF2 on threonine 52, which causes its nuclear localization and confers its oncogenic activities. The nuclear-to-mitochondrial translocation of ATF2 following genotoxic stress promotes apoptosis, a function that is largely lost in melanoma cells, due to its confined nuclear localization. Therefore, promoting the nuclear export of ATF2, which sensitizes melanoma cells to apoptosis, represents a novel therapeutic modality.
Experimental Design: We conducted a pilot high-throughput screen of 3,800 compounds to identify small molecules that promote melanoma cell death by inducing the cytoplasmic localization of ATF2. The imaging-based ATF2 translocation assay was conducted using UACC903 melanoma cells that stably express doxycycline-inducible GFP-ATF2.
Results: We identified two compounds (SBI-0089410 and SBI-0087702) that promoted the cytoplasmic localization of ATF2, reduced cell viability, inhibited colony formation, cell motility, and anchorage-free growth, and increased mitochondrial membrane permeability. SBI-0089410 inhibited the 12-O-tetradecanoylphorbol-l3-acetate (TPA)–induced membrane translocation of protein kinase C (PKC) isoforms, whereas both compounds decreased ATF2 phosphorylation by PKC and ATF2 transcriptional activity. Overexpression of either constitutively active PKC or phosphomimic mutant ATF2T52E attenuated the cellular effects of the compounds.
Purpose: Patients with triple-negative breast cancer (TNBC) and residual disease after neoadjuvant chemotherapy generally have worse outcome; however, some patients with residual tumor after neoadjuvant chemotherapy do not relapse. We hypothesize that there are subgroups of patients with chemoresistant TNBC with different prognosis.
Experimental Design: Forty-nine chemoresistant cases from 111 patients with TNBC treated with neoadjuvant chemotherapy (M.D. Anderson Cancer Center, Houston, TX) constituted the discovery cohort, and 25 chemoresistant samples from 47 neoadjuvant chemotherapy-treated TNBC (The Methodist Hospital, Houston, TX) were chosen for validation. Extended validation was carried out in 269 operable TNBC predicted to be chemoresistant by expression pattern from published datasets.
Results: We established a seven-gene prognostic signature using dChip and gene set enrichment analyses. In the independent validation cohort, the classifier predicted correctly with positive predictive value of 75.0% and negative predictive value (i.e., relapse-free survival; RFS) of 76.9% at 3 years. Those predicted to relapse had a HR of 4.67 [95% confidence interval (CI): 1.27–17.15] for relapse in 3 years. In extended validation, patients predicted not to relapse exhibited 3-year RFS of 78.9%, whereas the 3-year RFS was 48.5% for patients predicted to relapse, with HR of 2.61 (95% CI: 1.52–4.49). The TNBC subgroup that predicted to have relatively favorable prognosis was characterized by high expression of "luminal-like" genes [androgen-receptor (AR) and GATA3], whereas the subgroup with worse prognosis was characterized by expression of cancer stem-cell markers.
Purpose: Multiple injections of oncolytic adenovirus could enhance immunologic response. In the first part of this article, the focus was on immunologic aspects. Sixty patients previously naïve to oncolytic virus and who had white blood cells available were treated. Thirty-nine of 60 were assessed after a single virus administration, whereas 21 of 60 received a "serial treatment" consisting of three injections within 10 weeks. In the second part, we focused on 115 patients treated with a granulocyte macrophage colony-stimulating factor (GM–CSF)–coding capsid chimeric adenovirus, CGTG-102.
Results: Following serial treatment, both increase and decrease in antitumor T cells in blood were seen more frequently, findings which are compatible with induction of T-cell immunity and trafficking of T cells to tumors, respectively. Safety was good in both groups. In 115 patients treated with CGTG-102 (Ad5/3-D24-GMCSF), median overall survival was 111 days following single and 277 days after serial treatment in nonrandomized comparison. Switching the virus capsid for avoiding neutralizing antibodies in a serial treatment featuring three different viruses did not impact safety or efficacy. A correlation between antiviral and antitumor T cells was seen (P = 0.001), suggesting that viral oncolysis can result in epitope spreading and breaking of tumor-associated immunologic tolerance. Alternatively, some patients may be more susceptible to induction of T-cell immunity and/or trafficking.
Purpose: We assessed adding the multikinase inhibitor sorafenib to gemcitabine or capecitabine in patients with advanced breast cancer whose disease progressed during/after bevacizumab.
Experimental Design: This double-blind, randomized, placebo-controlled phase IIb study (ClinicalTrials.gov NCT00493636) enrolled patients with locally advanced or metastatic human epidermal growth factor receptor 2 (HER2)–negative breast cancer and prior bevacizumab treatment. Patients were randomized to chemotherapy with sorafenib (400 mg, twice daily) or matching placebo. Initially, chemotherapy was gemcitabine (1,000 mg/m2 i.v., days 1, 8/21), but later, capecitabine (1,000 mg/m2 orally twice daily, days 1–14/21) was allowed as an alternative. The primary endpoint was progression-free survival (PFS).
Results: One hundred and sixty patients were randomized. More patients received gemcitabine (82.5%) than capecitabine (17.5%). Sorafenib plus gemcitabine/capecitabine was associated with a statistically significant prolongation in PFS versus placebo plus gemcitabine/capecitabine [3.4 vs. 2.7 months; HR = 0.65; 95% confidence interval (CI): 0.45–0.95; P = 0.02], time to progression was increased (median, 3.6 vs. 2.7 months; HR = 0.64; 95% CI: 0.44–0.93; P = 0.02), and overall response rate was 19.8% versus 12.7% (P = 0.23). Median survival was 13.4 versus 11.4 months for sorafenib versus placebo (HR = 1.01; 95% CI: 0.71–1.44; P = 0.95). Addition of sorafenib versus placebo increased grade 3/4 hand–foot skin reaction (39% vs. 5%), stomatitis (10% vs. 0%), fatigue (18% vs. 9%), and dose reductions that were more frequent (51.9% vs. 7.8%).
Purpose: To investigate the short-term biologic effects of neoadjuvant chemotherapy +/– zoledronic acid (ZOL) in invasive breast cancer.
Experimental Design: Forty patients were randomized to receive a single 4 mg infusion of ZOL 24 hours after the first cycle of FE100C chemotherapy, or chemotherapy alone. Randomization was stratified for tumor stage, ER, HER2, and menopausal status. All patients had repeat breast core biopsy at day 5 (D5) ± day 21 (D21). Effects on apoptotic index, proliferation (Ki67), growth index, surrogate serum markers of angiogenesis (VEGF), and serum reproductive hormones within the TGFβ family (activin-A, TGFβ1, inhibin-A, and follistatin) were evaluated and compared.
Results: Baseline clinicopathologic characteristics were well balanced. Cell growth index (increased apoptosis and reduced proliferation) fell at D5 in both groups but recovered more rapidly with chemotherapy + ZOL compared with chemotherapy alone by D21 (P = 0.006). At D5, a greater reduction in serum VEGF occurred with chemotherapy + ZOL compared with chemotherapy: median percentage change –23.8% [interquartile range (IQR): –32.9 to –15.8] versus –8.4% (IQR: –27.3 to +8.9; P = 0.02), but these effects were lost by D21. Postmenopausal women showed a decrease in follistatin levels from baseline in the chemotherapy + ZOL group at D5 and D21, compared with chemotherapy alone (Pinteraction = 0.051).
Purpose: To conduct a first-in-human phase I study to determine the dose-limiting toxicities (DLT), characterize the pharmacokinetic profile, and document the antitumor activity of IPI-926, a new chemical entity that inhibits the Hedgehog pathway (HhP).
Experimental Design: Patients with solid tumors refractory to standard therapy were given IPI-926 once daily (QD) by mouth in 28-day cycles. The starting dose was 20 mg, and an accelerated titration schedule was used until standard 3 + 3 dose-escalation cohorts were implemented. Pharmacokinetics were evaluated on day –7 and day 22 of cycle 1.
Results: Ninety-four patients (32F, 62M; ages, 39–87) received doses ranging from 20 to 210 mg QD. Dose levels up to and including 160 mg administered QD were well tolerated. Toxicities consisted of reversible elevations in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and bilirubin, fatigue, nausea, alopecia, and muscle spasms. IPI-926 was not associated with hematologic toxicity. IPI-926 pharmacokinetics were characterized by a slow absorption (Tmax = 2–8 hours) and a terminal half-life (t1/2) between 20 and 40 hours, supporting QD dosing. Of those HhP inhibitor-naïve patients with basal cell carcinoma (BCC) who received more than one dose of IPI-926 and had a follow-up clinical or Response Evaluation Criteria in Solid Tumors (RECIST) assessment, nearly a third (8 of 28 patients) showed a response to IPI-926 at doses ≥130 mg.
Purpose: Estrogen withdrawal by treatment with aromatase inhibitors is the most effective form of endocrine therapy for postmenopausal estrogen receptor–positive (ER+) breast cancer. However, response to therapy varies markedly and understanding of the precise molecular effects of aromatase inhibitors and causes of resistance is limited. We aimed to identify in clinical breast cancer those genes and pathways most associated with resistance to aromatase inhibitors by examining the global transcriptional effects of AI treatment.
Experimental Design: Baseline and 2-week posttreatment biopsies were obtained from 112 postmenopausal women with ER+ breast cancer receiving neoadjuvant anastrozole. Gene expression data were obtained from 81 baseline and 2-week paired samples. Pathway analysis identified (i) the most prevalent changes in expression and (ii) the pretreatment genes/pathways most related to poor antiproliferative response.
Results: A total of 1,327 genes were differentially expressed after 2-week treatment (false discovery rate < 0.01). Proliferation-associated genes and classical estrogen-dependent genes were strongly downregulated whereas collagens and chemokines were upregulated. Pretreatment expression of an inflammatory signature correlated with antiproliferative response to anastrozole and this observation was validated in an independent study. Higher expression of immune-related genes such as SLAMF8 and TNF as well as lymphocytic infiltration were associated with poorer response (P < 0.001) and validated in an independent cohort.