Purpose: This phase I/II study sought to determine the safety and maximum tolerated dose (MTD) of a novel schedule of belinostat, a histone deacetylase inhibitor (HDAC) administered before and in combination with cisplatin (P), doxorubicin (A), and cyclophosphamide (C) in thymic epithelial tumors (TET). Antitumor activity, pharmacokinetics, and biomarkers of response were also assessed.
Experimental Design: Patients with advanced, unresectable TET received increasing doses of belinostat as a continuous intravenous infusion over 48 hours with chemotherapy in 3-week cycles. In phase II, belinostat at the MTD was used.
Results: Twenty-six patients were enrolled (thymoma, 12; thymic carcinoma, 14). Dose-limiting toxicities at 2,000 mg/m2 belinostat were grade 3 nausea and diarrhea and grade 4 neutropenia and thrombocytopenia, respectively, in two patients. Twenty-four patients were treated at the MTD of 1,000 mg/m2 with chemotherapy (P, 50 mg/m2 on day 2; A, 25 mg/m2 on days 2 and 3; C, 500 mg/m2 on day 3). Objective response rates in thymoma and thymic carcinoma were 64% (95% confidence interval, 30.8%-89.1%) and 21% (4.7%–50.8%), respectively. Modulation of pharmacodynamic markers of HDAC inhibition and declines in regulatory T cell (Treg) and exhausted CD8+ T-cell populations were observed. Decline in Tregs was associated with response (P = 0.0041) and progression-free survival (P = 0.021). Declines in TIM3+ CD8+ T cells were larger in responders than nonresponders (P = 0.049).
Purpose: Although hypoxia has been long recognized as a crucial factor impairing tumor response in many therapeutic schemes, atraumatic and reliable methods of individually quantifying tumor oxygenation are still lacking in day-to-day clinical practice. The aim of this work was to investigate the potentially quantitative properties of our recently described noninvasive magnetic resonance (MR) technique "MOBILE" (mapping of oxygen by imaging lipids relaxation enhancement) and to qualify this endogenous contrast as a tumor hypoxia marker.
Experimental Design: The "MOBILE" technique, which assesses the longitudinal MR relaxation rate, R1, of lipid protons, was benchmarked with the parent technique which assesses the global (or water) R1, in response to a hyperoxic challenge (carbogen breathing) and to a hypoxic challenge (combretastatin A4) in MDA-MB-231 xenografts and in NT2 mammary tumors. Electron paramagnetic resonance (EPR) oximetry was used to quantitatively assess the tumor pO2 in matching tumors longitudinally.
Purpose: Epstein–Barr virus (EBV) infects B cells, as well as T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoid malignancies. In various tumor cells, mTOR performs an essential function together with Akt with regard to cell growth. We investigated the effects of mTOR inhibitors on EBV-associated T- and NK-cell lymphomas.
Experimental Design: We investigated the Akt/mTOR activation pathway in EBV-positive and -negative T- and NK-cell lines (SNT13, SNT16, Jurkat, SNK6, KAI3, and KHYG1). We evaluated the antitumor effects of mTOR inhibitors (rapamycin and its analogue, CCI-779) against these cell lines in culture and in a murine xenograft model that was established by subcutaneous injection of SNK6 cells into NOG mice.
Results: All EBV-positive and -negative T- and NK-cell lines tested displayed activation of the Akt/mTOR pathway, and treatment with mTOR inhibitors suppressed mTOR activation. The inhibitors induced G1 cell-cycle arrest and inhibited cell proliferation in T- and NK-cell lines. Overall, T cell lines were more sensitive to rapamycin, but there were no significant differences between EBV-positive and -negative cell lines. Treatment with rapamycin did not affect lytic or latent EBV gene expression. Intraperitoneal treatment with CCI-779 significantly inhibited the growth of established tumors in NOG mice and reduced the EBV load in peripheral blood.
Purpose: Non–small cell lung cancers (NSCLC) that express EGF receptor with activating mutations frequently develop resistance to EGFR kinase inhibitors. The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed in NSCLC cells and confers a poor prognosis; however, the functional involvement of MUC1 in mutant EGFR signaling is not known.
Experimental Design: Targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in NSCLC cells harboring mutant EGFR was studied for effects on signaling, growth, clonogenic survival, and tumorigenicity.
Results: Stable silencing of MUC1-C in H1975/EGFR(L858R/T790M) cells resulted in downregulation of AKT signaling and inhibition of growth, colony formation, and tumorigenicity. Similar findings were obtained when MUC1-C was silenced in gefitinib-resistant PC9GR cells expressing EGFR(delE746_A750/T790M). The results further show that expression of a MUC1-C(CQC->AQA) mutant, which blocks MUC1-C homodimerization, suppresses EGFR(T790M), AKT and MEK->ERK activation, colony formation, and tumorigenicity. In concert with these results, treatment of H1975 and PC9GR cells with GO-203, a cell-penetrating peptide that blocks MUC1-C homodimerization, resulted in inhibition of EGFR, AKT, and MEK->ERK signaling and in loss of survival. Combination studies of GO-203 and afatinib, an irreversible inhibitor of EGFR, further demonstrate that these agents are synergistic in inhibiting growth of NSCLC cells harboring the activating EGFR(T790M) or EGFR(delE746-A750) mutants.
Purpose: In a recent phase II clinical trial, low-dose (100 mg/m2) gemcitabine showed promise as a radiosensitizer in bladder cancer, but underlying mechanisms lack elucidation. Here, we investigated the mechanism of radiosensitization by low-dose gemcitabine in bladder cancer cell lines.
Experimental Design: Four bladder cancer cell lines were screened for radiosensitization by low-dose gemcitabine using clonogenic assay, and gemcitabine-resistant RT112gem and CALgem cells created by exposure to increasing gemcitabine doses. Four key gemcitabine-regulatory genes were knocked down by transient siRNA. Nude mice carrying CALgem subcutaneous xenografts were exposed to 100 mg/kg gemcitabine ± ionizing radiation (IR) and response assessed by tumor growth delay.
Results: Gemcitabine was cytotoxic in the low nanomolar range (10–40 nmol/L) in four bladder cancer cell lines and radiosensitized all four lines. Sensitizer enhancement ratios at 10% survival were: RT112 1.42, CAL29 1.55, T24 1.63, and VMCUB1 1.47. Transient siRNA knockdown of deoxycytidine kinase (dCK) significantly reduced radiosensitization by gemcitabine (P = 0.02). RT112gem and CALgem cells displayed robust decreases of dCK mRNA and protein levels; reexpression of dCK restored gemcitabine sensitivity. However, CALgem xenografts responded better to combination gemcitabine/IR than either treatment alone (P < 0.001) with dCK strongly expressed in the tumor vasculature and stroma.
Purpose: High levels of BCL-2 family members in colorectal carcinoma cause resistance to treatment. Inhibition of NANOG or its paralog NANOGP8 reduces the proliferation, stemness, and tumorigenicity of colorectal carcinoma cells. Our hypothesis was that inhibition of NANOG/NANOGP8 enhances the cytotoxic effect of BH3 mimetics targeting BCL-2 family members in colorectal carcinoma cells through reducing expression of MCL-1, a prosurvival BCL-2 protein.
Experimental Design: Lentiviral vector (LV) shRNA to NANOG (shNG-1) or NANOGP8 (shNp8-1) transduced colorectal carcinoma cells that were also exposed to the BH3 mimetics ABT-737 or ABT-199 in vivo in colorectal carcinoma xenografts and in vitro where proliferation, protein and gene expression, and apoptosis were measured.
Results: Clone A and CX-1 were sensitive to ABT-737 and ABT-199 at IC50s of 2 to 9 μmol/L but LS174T was resistant with IC50s of 18 to 30 μmol/L. Resistance was associated with high MCL-1 expression in LS174T. LVshNG-1 or LVshNp8-1 decreased MCL-1 expression, increased apoptosis, and decreased replating efficiency in colorectal carcinoma cells treated with either ABT-737 or ABT-199 compared with the effects of either BH3 mimetic alone. Inhibition or overexpression of MCL-1 alone replicated the effects of LVshNG-1 or LVshNp8-1 in increasing or decreasing the apoptosis caused with the BH3 mimetic. The combination therapy inhibited the growth of LS174T xenografts in vivo compared with untreated controls or treatment with only LV shRNA or ABT-737.
Purpose: Imiquimod is a Toll-like receptor 7 agonist used topically to treat external genital warts and basal cell carcinoma. We examined the combination of topical imiquimod with intramuscular administration of CRT/E7, a therapeutic human papillomavirus (HPV) vaccine comprised of a naked DNA vector expressing calreticulin fused to HPV16 E7.
Experimental Design: Using an orthotopic HPV16 E6/E7+ syngeneic tumor, TC-1, as a model of high-grade cervical/vaginal/vulvar intraepithelial neoplasia, we assessed if combining CRT/E7 vaccination with cervicovaginal deposition of imiquimod could result in synergistic activities promoting immune-mediated tumor clearance.
Results: Imiquimod induced cervicovaginal accumulation of activated E7-specific CD8+ T cells elicited by CRT/E7 vaccination. Recruitment was not dependent upon the specificity of the activated CD8+ T cells, but was significantly reduced in mice lacking the IFN receptor. Intravaginal imiquimod deposition induced upregulation of CXCL9 and CXCL10 mRNA expression in the genital tract, which are produced in response to IFN receptor signaling and attract cells expressing their ligand, CXCR3. The T cells attracted by imiquimod to the cervicovaginal tract expressed CXCR3 as well as CD49a, an integrin involved in homing and retention of CD8+ T cells at mucosal sites. Our results indicate that intramuscular CRT/E7 vaccination in conjunction with intravaginal imiquimod deposition recruits antigen-specific CXCR3+ CD8+ T cells to the genital tract.
Purpose: Perturbations in the retinoblastoma pathway are over-represented in advanced prostate cancer; retinoblastoma loss promotes bypass of first-line hormone therapy. Conversely, preliminary studies suggested that retinoblastoma-deficient tumors may become sensitized to a subset of DNA-damaging agents. Here, the molecular and in vivo consequence of retinoblastoma status was analyzed in models of clinical relevance.
Experimental Design: Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAPC4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of retinoblastoma using shRNA). Multiple mechanisms were interrogated including cell cycle, apoptosis, and DNA damage repair. Transcriptome analysis was performed, validated, and mechanisms discerned. Cell survival was measured using clonogenic cell survival assay and in vivo analysis was performed in nude mice with human derived tumor xenografts.
Results: Loss of retinoblastoma enhanced the radioresponsiveness of both hormone-sensitive and castrate-resistant prostate cancer. Hypersensitivity to ionizing radiation was not mediated by cell cycle or p53. Retinoblastoma loss led to alteration in DNA damage repair and activation of the NF-B pathway and subsequent cellular apoptosis through PLK3. In vivo xenografts of retinoblastoma-deficient tumors exhibited diminished tumor mass, lower PSA kinetics, and decreased tumor growth after treatment with ionizing radiation (P < 0.05).
Purpose: Activating mutations in the RAS oncogene occur frequently in human leukemias. Direct targeting of RAS has proven to be challenging, although targeting of downstream RAS mediators, such as MEK, is currently being tested clinically. Given the complexity of RAS signaling, it is likely that combinations of targeted agents will be more effective than single agents.
Experimental Design: A chemical screen using RAS-dependent leukemia cells was developed to identify compounds with unanticipated activity in the presence of an MEK inhibitor and led to identification of inhibitors of IGF1R. Results were validated using cell-based proliferation, apoptosis, cell-cycle, and gene knockdown assays; immunoprecipitation and immunoblotting; and a noninvasive in vivo bioluminescence model of acute myeloid leukemia (AML).
Results: Mechanistically, IGF1R protein expression/activity was substantially increased in mutant RAS-expressing cells, and suppression of RAS led to decreases in IGF1R. Synergy between MEK and IGF1R inhibitors correlated with induction of apoptosis, inhibition of cell-cycle progression, and decreased phospho-S6 and phospho-4E-BP1. In vivo, NSG mice tail veins injected with OCI-AML3-luc+ cells showed significantly lower tumor burden following 1 week of daily oral administration of 50 mg/kg NVP-AEW541 (IGF1R inhibitor) combined with 25 mg/kg AZD6244 (MEK inhibitor), as compared with mice treated with either agent alone. Drug combination effects observed in cell-based assays were generalized to additional mutant RAS-positive neoplasms.
Purpose: Cisplatin and its platinum derivatives are first-line chemotherapeutic agents in the treatment of ovarian cancer; however, treatment is associated with tumor resistance and significant toxicity. Here we investigated the antitumoral activity of lipoplatin, one of the most promising liposomal platinum drug formulations under clinical investigation.
Experimental Design:In vitro effects of lipoplatin were tested on a panel of ovarian cancer cell lines, sensitive and resistant to cisplatin, using both two-dimensional (2D) and 3D cell models. We evaluated in vivo the lipoplatin anticancer activity using tumor xenografts.
Results: Lipoplatin exhibited a potent antitumoral activity in all ovarian cancer cell lines tested, induced apoptosis, and activated caspase-9, -8, and -3, downregulating Bcl-2 and upregulating Bax. Lipoplatin inhibited thioredoxin reductase enzymatic activity and increased reactive oxygen species accumulation and reduced EGF receptor (EGFR) expression and inhibited cell invasion. Lipoplatin demonstrated a synergistic effect when used in combination with doxorubicin, widely used in relapsed ovarian cancer treatment, and with the albumin-bound paclitaxel, Abraxane. Lipoplatin decreased both ALDH and CD133 expression, markers of ovarian cancer stem cells. Multicellular aggregates/spheroids are present in ascites of patients and most contribute to the spreading to secondary sites. Lipoplatin decreased spheroids growth, vitality, and cell migration out of preformed spheroids. Finally, lipoplatin inhibited more than 90% tumor xenograft growth with minimal systemic toxicity, and after the treatment suspension, no tumor progression was observed.
Purpose: Primary cutaneous T-cell lymphomas (CTCL) are neoplastic disorders of skin-homing T cells. Affected skin areas show morphologic similarities with alterations in other T-cell–mediated dermatoses. Furthermore, as in atopic dermatitis but in contrast with psoriasis, patients with CTCL are frequently afflicted by cutaneous bacterial infections that support the survival of lymphoma cells. Our aim was to investigate the mechanisms of elevated susceptibility to cutaneous infections in patients with CTCL.
Experimental Design: Skin samples from CTCL, psoriasis, and atopic dermatitis patients were used to illuminate the antibacterial competence status and the presence of its modulating cytokines. For substantiation of findings, 3-dimensional epidermis models, isolated and in vitro generated Th-subpopulations, were applied. Parameters were analyzed via qPCR and IHC.
Results: CTCL lesions compared with psoriatic lesions presented an impaired upregulation of antibacterial proteins (ABPs), with levels even below those in atopic dermatitis. This was associated with a relative deficiency of the ABP-inducing cytokine IL-17 and a strong presence of the ABP-downregulating cytokine IL-13. The simultaneous presence of the Th17-cell cytokine IL-26 indicated that IL-17 deficiency in CTCL lesions results from functional deviation of Th17 cells. Accordingly, IL-17 but not IL-26 production by Th17 cells in vitro was inhibited by IL-4Rα ligand. Levels of other ABP inducers were comparable between CTCL and psoriasis lesions. The same was true about IL-22/TNF-α targets, including the keratinocyte hyper-regeneration marker K16 and the matrix-degrading enzyme MMP1.
Purpose: Ameloblastoma is an odontogenic neoplasm whose overall mutational landscape has not been well characterized. We sought to characterize pathogenic mutations in ameloblastoma and their clinical and functional significance with an emphasis on the mitogen-activated protein kinase (MAPK) pathway.
Experimental Design: A total of 84 ameloblastomas and 40 non-ameloblastoma odontogenic tumors were evaluated with a combination of BRAF V600E allele–specific PCR, VE1 immunohistochemistry, the Ion AmpliSeq Cancer Hotspot Panel, and Sanger sequencing. Efficacy of a BRAF inhibitor was evaluated in an ameloblastoma-derived cell line.
Results: Somatic, activating, and mutually exclusive RAS–BRAF and FGFR2 mutations were identified in 88% of cases. Somatic mutations in SMO, CTNNB1, PIK3CA, and SMARCB1 were also identified. BRAF V600E was the most common mutation, found in 62% of ameloblastomas and in ameloblastic fibromas/fibrodentinomas but not in other odontogenic tumors. This mutation was associated with a younger age of onset, whereas BRAF wild-type cases arose more frequently in the maxilla and showed earlier recurrences. One hundred percent concordance was observed between VE1 immunohistochemistry and molecular detection of BRAF V600E mutations. Ameloblastoma cells demonstrated constitutive MAPK pathway activation in vitro. Proliferation and MAPK activation were potently inhibited by the BRAF inhibitor vemurafenib.
Purpose: Loss of function of PTEN is a frequent event in melanoma, particularly in tumors with BRAFV600 mutations. The prevalence, pathologic features, and clinical outcomes associated with PTEN loss in patients with stage IIIB/C melanoma were interrogated to improve our understanding of the clinical significance of this molecular event.
Experimental Design: Archival tissue from lymphadenectomy specimens among patients (n = 136) with stage IIIB or IIIC melanoma was assessed by DNA sequencing for activating BRAF and NRAS mutations, and by immunohistochemistry for the expression of PTEN protein. Associations of these molecular aberrations with demographics, tumor characteristics, and clinical outcomes were determined.
Results: The prevalence of BRAFV600 mutations (40% overall), NRAS mutations (10%), and PTEN loss (25%) did not vary by pathologic substage. BRAF/NRAS mutation status did not correlate with distant disease-free survival (DDFS) or overall survival (OS). Complete loss of PTEN expression correlated with shorter OS but not DDFS. When stratified by specific sites of distant metastasis, PTEN loss was associated with significantly shorter time to melanoma brain metastasis (MBM), but not to liver, lung, or bone metastasis. Analysis of PTEN in mutationally defined subsets showed that PTEN loss was significantly associated with OS and time to MBM in patients with BRAFV600 mutations.
Purpose: An improved understanding of the molecular pathogenesis of brain metastases, one of the most common and devastating complications of advanced melanoma, may identify and prioritize rational therapeutic approaches for this disease. In particular, the identification of molecular differences between brain and extracranial metastases would support the need for the development of organ-specific therapeutic approaches.
Experimental Design: Hotspot mutations, copy number variations (CNV), global mRNA expression patterns, and quantitative analysis of protein expression and activation by reverse-phase protein array (RPPA) analysis were evaluated in pairs of melanoma brain metastases and extracranial metastases from patients who had undergone surgical resection for both types of tumors.
Results: The status of 154 previously reported hotspot mutations, including driver mutations in BRAF and NRAS, were concordant in all evaluable patient-matched pairs of tumors. Overall patterns of CNV, mRNA expression, and protein expression were largely similar between the paired samples for individual patients. However, brain metastases demonstrated increased expression of several activation-specific protein markers in the PI3K/AKT pathway compared with the extracranial metastases.
Purpose: It has been recognized for almost a decade that concentrations of signaling androgens sufficient to activate the androgen receptor are present in castration-resistant prostate cancer tissue. The source of these androgens is highly controversial, with three competing models proposed. We, therefore, wished to determine the androgenic potential of human benign and malignant (hormone-naïve and treated) prostate tissue when incubated with various precursors and examine concomitant changes in enzyme expression.
Experimental Design: Freshly harvested prostate tissue [benign, hormone-naïve, and hormone-refractory prostate cancer (HRPC)] was incubated in excess concentrations of cholesterol, progesterone, DHEA, androstenedione, or testosterone for 96 hours, and steroid concentrations in the conditioned media measured by gas chromatography–mass spectroscopy. Changes in the expression of androgen synthetic and/or degradative enzymes were determined by expression microarray and qPCR. Significant changes were confirmed in an independent dataset.
Results: Of the precursor molecules tested, only incubation with androstenedione gave rise to significant concentrations of signaling androgens. Although this was observed in all tissue types, it occurred to a significantly greater degree in hormone-refractory compared with hormone-naïve cancer. Consistent with this, gene set enrichment analysis of the expression microarray data revealed significant upregulation of 17HSD17B activity, with overexpression of the canonical enzyme AKR1C3 confirmed by qPCR in the same samples and in a publicly available expression dataset. Importantly, we found no evidence to support a significant contribution from either the "backdoor" or "5-α dione" pathway.
Purpose: Radiotherapy can result in lymphopenia, which has been linked to poorer survival. Here, we test the hypothesis that radiotherapy-induced lymphopenia is mediated by a tumor-secreted factor, Galectin-1 (Gal-1), which possesses T-cell proapoptotic activities.
Experimental Design: Matched Gal-1 wild-type (WT) or null mice were implanted with Lewis lung carcinoma (LLC-1) that either expressed Gal-1 or had Gal-1 stably downregulated. Tumors were irradiated locally and circulating Gal-1 and T cells were measured. Tumor growth, lung metastasis, intratumoral T-cell apoptosis, and microvessel density count were quantified. Thiodigalactoside (TDG), a Gal-1 inhibitor, was used to inhibit Gal-1 function in another group of mice to validate the observations noted with Gal-1 downregulation. Lymphocyte counts, survival, and plasma Gal-1 were analyzed in cohorts of radiotherapy-treated lung [non–small cell lung cancer (NSCLC)] and head and neck cancer patients.
Results: LLC irradiation increased Gal-1 secretion and decreased circulating T cells in mice, regardless of host Gal-1 expression. Inhibition of tumor Gal-1 with either shRNA or thiodigalactoside ablated radiotherapy-induced lymphopenia. Irradiated shGal-1 tumors showed significantly less intratumoral CD8+ T-cell apoptosis and microvessel density, which led to marked tumor growth delay and reduced lung metastasis compared with controls. Similar observations were made after thiodigalactoside treatment. Radiotherapy-induced lymphopenia was associated with poorer overall survival in patients with NSCLC treated with hypofractionated radiotherapy. Plasma Gal-1 increased whereas T-cell decreased after radiation in another group of patients.