Search

Background: Ziftomenib (KO-539) is an oral selective menin inhibitor with known preclinical activity in menin-dependent acute myeloid leukaemia models. The primary objective of this study was to determine the recommended phase 2 dose in patients with relapsed or refractory acute myeloid leukaemia based on safety, pharmacokinetics, pharmacodynamics, and preliminary activity., Methods: KOMET-001 is a multicentre, open-label, multi-cohort, phase 1/2 clinical trial of ziftomenib in adults with relapsed or refractory acute myeloid leukaemia. Results of the phase 1 study, conducted at 22 hospitals in France, Italy, Spain, and the USA, are presented here and comprise the dose-escalation (phase 1a) and dose-validation and expansion (phase 1b) phases. Eligible patients were aged 18 years or older, had relapsed or refractory acute myeloid leukaemia, and had an Eastern Cooperative Oncology Group performance status of 2 or less. For phase 1a, patients (all molecular subtypes) received ziftomenib (50-1000 mg) orally once daily in 28-day cycles. For phase 1b, patients with NPM1 mutations or with KMT2A rearrangements were randomly assigned (1:1) using third-party interactive response technology to two parallel dose cohorts (200 mg and 600 mg ziftomenib). Primary endpoints were maximum tolerated dose or recommended phase 2 dose in phase 1a, and safety, remission rates, and pharmacokinetics supporting recommended phase 2 dose determination in phase 1b. Analyses were performed in all patients who received at least one dose of ziftomenib (modified intention-to-treat population). Phase 1a/1b is complete. This trial is registered with ClinicalTrials.gov, NCT04067336, and the EU Clinical Trials register, EudraCT 2019-001545-41., Findings: From Sept 12, 2019, to Aug 19, 2022, 83 patients received 50-1000 mg ziftomenib (39 [47%] were male and 44 [53%] were female). Median follow-up was 22·3 months (IQR 15·4-30·2). Of 83 patients, the most common grade 3 or worse treatment-emergent adverse events were anaemia (20 [24%]), febrile neutropenia (18 [22%]), pneumonia (16 [19%]), differentiation syndrome (12 [15%]), thrombocytopenia (11 [13%]), and sepsis (ten [12%]). Overall, 68 of 83 patients had serious adverse events, with two reported treatment-related deaths (one differentiation syndrome and one cardiac arrest). Differentiation syndrome rate and severity influenced the decision to halt enrolment of patients with KMT2A rearrangements. In Phase 1b, no responses were reported in patients treated at the 200 mg dose level. At the recommended phase 2 dose of 600 mg, nine (25%) of 36 patients with KMT2A rearrangement or NPM1 mutation had complete remission or complete remission with partial haematologic recovery. Seven (35%) of 20 patients with NPM1 mutation treated at the recommended phase 2 dose had a complete remission., Interpretation: Ziftomenib showed promising clinical activity with manageable toxicity in heavily pretreated patients with relapsed or refractory acute myeloid leukaemia. Phase 2 assessment of ziftomenib combination therapy in the upfront and relapsed or refractory setting is ongoing., Funding: Kura Oncology., Competing Interests: Declaration of interests ESW declares honoraria for educational talks from Aptitude, Astellas, Bioascend, CEA, CCO, Curio Sciences, Dava Oncology, Medscape, MD Education, OncLive, PER, Peerview, RTP, and Pfizer; participation on Data Safety Monitoring Committees and research grant committees for AbbVie and Gilead; advisory board participation for Abbvie, Astellas, Blueprint, Bristol Myers Squibb, Daiichi Sankyo, Gilead, GlaxoSmithKline, Immunogen, Janssen, Jazz, Kite, Novartis, NuProbe, PharmaEssentia, Pfizer, Qiagen, Rigel, Schrodinger, Sumitomo, Syndax, and Takeda; other financial and non-financial interests with UptoDate; and medical writing support for this work from Kura Oncology. GCI declares research funding to their institution from Astex, Cullinan Oncology, Kura Oncology, Merck, Novartis, and Syndax Pharmaceuticals; consultancy fees from AbbVie, Kura Oncology, Novartis, and Syndax Pharmaceuticals; support for attending meetings and travel from Kura Oncology; steering committee role with Kura Oncology and Novartis; receipt of material for sample analysis from NuProbe; and medical writing support for this work from Kura Oncology. HPE declares a leadership role with AbbVie (Chair, Independent Review Committee for VIALE A and VIALE C), Bristol Myers Squibb (Chair, AML Registry Steering Committee), and Glycomimetics (Scientific Steering Committee); speakers bureau for AbbVie, Bristol Myers Squibb, Incyte, Jazz, Novartis, and Servier; contracted research from AbbVie, ALX Oncology, Amgen, Aptose, Ascentage, Daiichi Sankyo, Forma, Gilead, Glycomimetics, Immunogen, Jazz, Kura Oncology, MacroGenics, Novartis, PTC, and Sumitomo Pharma; consultancy fees from AbbVie, Astellas, Bristol Myers Squibb, Daiichi Sankyo, Glycomimetics, Incyte, Jazz, Kura Oncology, Novartis, Pfizer, Servier, Stemline, and Sumitomo Pharma; and medical writing support for this work from Kura Oncology. JKA declares a leadership role with the American Society of Hematology (guidelines panel), National Comprehensive Cancer Network (acute myeloid leukaemia panel vice-chair), and National Cancer Institute (co-chair on Leukemia Steering Committee); honoraria from Astellas, HMP Education, MD Education, and VJ HemOnc; advisory board role with AbbVie, Aptitude Health, Astellas, BlueBird Bio, Curio, Daiichi Sankyo, Dark Blue Therapeutics, Gilead, Kura Oncology, Kymera, Rigel, Stemline Therapeutics, Syros, and Treadwell Therapeutics; meeting attendance and travel expenses from Astellas, Daiichi Sankyo, HMP Education, MD Education, and VJ HemOnc; medical writing support for this work from Kura Oncology; and participation on a Data Safety Committee for Glycomimetics. PM declares consultancy fees from Kura Oncology and Syndax Pharmaceuticals and medical writing support for this work from Kura Oncology. SDB declares honoraria from Astellas, Bristol Myers Squibb, Menarini, and Servier; consultancy fees from AbbVie, Bristol Myers Squibb, Forma, Remix, Rigel, and Servier; travel expenses from Janssen, Pfizer, Rigel, and Servier; and medical writing support for this work from Kura Oncology. RBW declares clinical trial support from Kura Oncology and medical writing support for this work from Kura Oncology. KP declares honoraria from Merck (investigator meeting lecture); advisory board role with AbbVie, Incyte, PharmaEssentia, Protagonist, and Sobi; and medical writing support for this work from Kura Oncology. MRS declares research funding to institution from ALX Oncology, Astex, Incyte, Takeda, and TG Therapeutics; consultancy fees from AbbVie, Bristol Myers Squibb, CTI BioPharma, Forma, Geron, GlaxoSmithKline, Karyopharm, Rigel, Ryvu, Taiho, and Treadwell; stock or stock options in Empath Bioscience, Karyopharm, and Ryvu; and medical writing support for this work from Kura Oncology. MVS declares research grant to their institution from AbbVie, Astellas, Celgene, Kura Oncology, and MRKR Therapeutics; travel expenses from Dava Oncology; and medical writing support for this work from Kura Oncology. MK declares consultancy fees from AbbVie, CTI Biopharma, Incyte, and Protagonist; advisory board role with CTI BioPharma, Incyte, Kura Oncology, and Morphosys; travel expenses from Protagonist; and medical writing support for this work from Kura Oncology. MRB declares research funding to their institution from AbbVie, Ascentage, Astellas, Gilead, Kura Oncology, and Takeda and medical writing support for this work from Kura Oncology. JMF declares leadership role with National Cancer Institute Leukemia Steering Committee and the National Heart, Lung, and Blood Institute national Myelodysplastic Syndrome Study Steering Committee; stock or stock options with Aurinia; honoraria from AmerisourceBergen/IntrinsiQ Specialty Solutions, Aptitude Health, and MJH LifeSciences; consultancy fees from Autolus, Bristol Myers Squibb, Remix, and Syndax; grants to institution for clinical trial support from Actinium, Astellas, Celgene, Chordia, Kura Oncology, Novartis, Pfizer, Roivant, Sellas, and Servier; and medical writing support for this work from Kura Oncology. GS declares contracts through their institution from AbbVie, Actinium, Actuate, Agios, Arog, Astellas, AlloVir, Amgen, Aptevo, AltruBio, AVM Bio, Bristol Myers Squibb/Celgene; BioMea, Biopath, Biosight, Cellularity, Celator, Constellation, Cogent, Cellectis, Cullinan, Daiichi Sankyo, Deciphera, Delta-Fly, Fate, Forma, FujiFilm, Gamida, Genentech-Roche, Rigel Glycomimetics, Geron, Gilead, Incyte, Janssen, Jazz, Karyopharm, Kite/Gilead, Kronos Bio, Kura Oncology, Immunogene, ImmuneOnc, Loxo, Marker, Mateon, Novartis, Onconova, Ono-UK, Orca, Pfizer, PrECOG, Regimmune, Samus, Sangamo, Sellas, Stemline, Syros, Takeda, Tolero, and Trovagene; consultancy fees from Bristol Myers Squibb, Curios, Daiichi, and Novartis; speakers bureau role for AbbVie, Agios, Amgen, Astellas, Blueprint Medicine, Bristol Myers Squibb, Celgene, Karyopharm, GlaxoSmithKline, Kite (Gilead), Jazz, Rigel, Seattle genetics, and Stemline; board or advisory committee membership for Agios, Autolus, AVM Biotech, Bristol Myers Squibb, Gamida, Gilead, GlaxoSmithKline, Incyte, Novartis, Orca, Rigel, and Stemline; board of trustees membership for Leukemia Lymphoma Society Los Angeles; secretary or treasurer membership for the American Society of Hematology Research Collaborative Board of Directors; and holds stock with Amgen, Bristol Myers Squibb, and Janssen/Johnson & Johnson. LA declares research funding to institution from AbbVie, Bristol Myers Squibb, Jazz Pharmaceuticals, and Novartis; consultancy fees from AbbVie, Jazz Pharmaceuticals, and Novartis; and medical writing support for this work from Kura Oncology. ER declares consulting fees from Astellas and Laboratories Delbert; honoraria from AbbVie, Astellas, Eurocept, and Jazz Pharmaceuticals; support for attending meetings and travel from AbbVie, Gilead, and Jazz Pharmaceuticals; and medical writing support for this work from Kura Oncology. MMP declares research funding to institution from Epigenetix, Kura Oncology, Polaris, Solutherapeutics, and Stem Line Pharma; medical writing support for this work from Kura Oncology; and Data Safety Monitoring Boards with CTI Biopharma. CP declares honoraria from AbbVie, Amgen, Astellas, Blueprint, Bristol Myers Squibb, Celgene, GlaxoSmithKline, Incyte, Janssen, Jazz Pharmaceuticals, Laboratories Delbert, Menarini-Stemline, Novartis, Paladin Labs, Pfizer, and Servier; travel expenses from AbbVie, Amgen and Pfizer; medical writing support for this work from Kura Oncology; and Data Safety Monitoring Board or advisory board membership for AbbVie, Amgen, Astellas, Blueprint, Bristol Myers Squibb, Celgene, GlaxoSmithKline, Incyte, Janssen, Jazz Pharmaceuticals, Laboratories Delbert, Paladin Labs, and Pfizer. JG declares research support to institution, consultancy fees, royalties, patents, and stock with Kura Oncology, and medical writing support for this work from Kura Oncology. TC declares research support to institution and royalties from, and patents and owns stock with Kura Oncology; and medical writing support for this work from Kura Oncology. BC, JR, BML, MH, PP, and OS declare medical writing support for this work from Kura Oncology. KN declares employment with Kura Oncology; received stock options from Kura Oncology; and medical writing support for this work from Kura Oncology. AM declares employment with Kura Oncology; owns stock with Kura Oncology; patents and patent applications with Kura Oncology; and medical writing support for this work from Kura Oncology. JMA declares employment and stock or stock options with and travel expenses from Kura Oncology, and medical writing support for this work from Kura Oncology. MT declares employment with Kura Oncology; stock and restricted stock units with Kura Oncology; reimbursement for conference fees, hotels and travel expenses from Kura Oncology; and medical writing support for this work from Kura Oncology. HSS declares employment, patents and patent applications, and owns stock with, Kura Oncology, and medical writing support for this work from Kura Oncology. DC declares employment with Kura Oncology; stock or stock options with Kura Oncology; and medical writing support for this work from Kura Oncology. ML declares employment, patents and patent applications, stock or stock options, support for attending meetings and travel, and other financial or non-financial interests with Kura Oncology; and medical writing support for this work from Kura Oncology. SD declares employment, patents and patent applications, stock or stock options, and other financial or non-financial interests with Kura Oncology; and medical writing support for this work from Kura Oncology. ATF declares consultancy fees from AbbVie, Amgen, Astellas, AstraZeneca, Autolus, Bristol Myers Squibb/Celgene, Daiichi Sankyo, EnClear, Forma, Genentech, Gilead, Immunogen, Ipsen, Kite, Mablytics, Menarini, Novartis, Orum, Pfizer, PureTech, Remix, Rigel, Servier, and Takeda; clinical trial support from AbbVie, Bristol Myers Squibb, and Servier; and medical writing support for this work from Kura Oncology. CB declares no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Outcomes for patients with recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) are poor, with median overall survival (OS) ranging from 6 to 18 months. For those who progress on standard-of-care (chemo)immunotherapy, treatment options are limited, necessitating the development of rational therapeutic strategies. Toward this end, we targeted the key HNSCC drivers PI3K-mTOR and HRAS via the combination of tipifarnib, a farnesyltransferase (FTase) inhibitor, and alpelisib, a PI3Kα inhibitor, in multiple molecularly defined subsets of HNSCC. Tipifarnib synergized with alpelisib at the level of mTOR in PI3Kα- or HRAS-dependent HNSCCs, leading to marked cytotoxicity in vitro and tumor regression in vivo. On the basis of these findings, the KURRENT-HN trial was launched to evaluate the effectiveness of this combination in PIK3CA-mutant/amplified and/or HRAS-overexpressing R/M HNSCC. Preliminary evidence supports the clinical activity of this molecular biomarker-driven combination therapy. Combined alpelisib and tipifarnib has potential to benefit >45% of patients with R/M HNSCC. By blocking feedback reactivation of mTORC1, tipifarnib may prevent adaptive resistance to additional targeted therapies, enhancing their clinical utility., Significance: The mechanistically designed, biomarker-matched strategy of combining alpelisib and tipifarnib is efficacious in PIK3CA- and HRAS-dysregulated head and neck squamous carcinoma and could improve outcomes for many patients with recurrent, metastatic disease. See related commentary by Lee et al., p. 3162., (©2023 The Authors; Published by the American Association for Cancer Research.)

Purpose: FGFR genomic alterations (amplification, mutations, and/or fusions) occur in ∼8% of gliomas, particularly FGFR1 and FGFR3. We conducted a multicenter open-label, single-arm, phase II study of a selective FGFR1-3 inhibitor, infigratinib (BGJ398), in patients with FGFR-altered recurrent gliomas., Patients and Methods: Adults with recurrent/progressive gliomas harboring FGFR alterations received oral infigratinib 125 mg on days 1 to 21 of 28-day cycles. The primary endpoint was investigator-assessed 6-month progression-free survival (PFS) rate by Response Assessment in Neuro-Oncology criteria. Comprehensive genomic profiling was performed on available pretreatment archival tissue to explore additional molecular correlations with efficacy., Results: Among 26 patients, the 6-month PFS rate was 16.0% [95% confidence interval (CI), 5.0-32.5], median PFS was 1.7 months (95% CI, 1.1-2.8), and objective response rate was 3.8%. However, 4 patients had durable disease control lasting longer than 1 year. Among these, 3 had tumors harboring activating point mutations at analogous positions of FGFR1 (K656E; n = 2) or FGFR3 (K650E; n = 1) in pretreatment tissue; an FGFR3-TACC3 fusion was detected in the other. Hyperphosphatemia was the most frequently reported treatment-related adverse event (all-grade, 76.9%; grade 3, 3.8%) and is a known on-target toxicity of FGFR inhibitors., Conclusions: FGFR inhibitor monotherapy with infigratinib had limited efficacy in a population of patients with recurrent gliomas and different FGFR genetic alterations, but durable disease control lasting more than 1 year was observed in patients with tumors harboring FGFR1 or FGFR3 point mutations or FGFR3-TACC3 fusions. A follow-up study with refined biomarker inclusion criteria and centralized FGFR testing is warranted., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: Treatment options are sparse for patients with advanced cholangiocarcinoma after progression on first-line gemcitabine-based therapy. FGFR2 fusions or rearrangements occur in 10-16% of patients with intrahepatic cholangiocarcinoma. Infigratinib is a selective, ATP-competitive inhibitor of fibroblast growth factor receptors. We aimed to evaluate the antitumour activity of infigratinib in patients with locally advanced or metastatic cholangiocarcinoma, FGFR2 alterations, and previous gemcitabine-based treatment., Methods: This multicentre, open-label, single-arm, phase 2 study recruited patients from 18 academic centres and hospitals in the USA, Belgium, Spain, Germany, Singapore, Taiwan, and Thailand. Eligible participants were aged 18 years or older, had histologically or cytologically confirmed, locally advanced or metastatic cholangiocarcinoma and FGFR2 fusions or rearrangements, and were previously treated with at least one gemcitabine-containing regimen. Patients received 125 mg of oral infigratinib once daily for 21 days of 28-day cycles until disease progression, intolerance, withdrawal of consent, or death. Radiological tumour evaluation was done at baseline and every 8 weeks until disease progression via CT or MRI of the chest, abdomen, and pelvis. The primary endpoint was objective response rate, defined as the proportion of patients with a best overall response of a confirmed complete or partial response, as assessed by blinded independent central review (BICR) according to Response Evaluation Criteria in Solid Tumors, version 1.1. The primary outcome and safety were analysed in the full analysis set, which comprised all patients who received at least one dose of infigratinib. This trial is registered with ClinicalTrials.gov, NCT02150967, and is ongoing., Findings: Between June 23, 2014, and March 31, 2020, 122 patients were enrolled into our study, of whom 108 with FGFR2 fusions or rearrangements received at least one dose of infigratinib and comprised the full analysis set. After a median follow-up of 10·6 months (IQR 6·2-15·6), the BICR-assessed objective response rate was 23·1% (95% CI 15·6-32·2; 25 of 108 patients), with one confirmed complete response in a patient who only had non-target lesions identified at baseline and 24 partial responses. The most common treatment-emergent adverse events of any grade were hyperphosphataemia (n=83), stomatitis (n=59), fatigue (n=43), and alopecia (n=41). The most common ocular toxicity was dry eyes (n=37). Central serous retinopathy-like and retinal pigment epithelial detachment-like events occurred in 18 (17%) patients, of which ten (9%) were grade 1, seven (6%) were grade 2, and one (1%) was grade 3. There were no treatment-related deaths., Interpretation: Infigratinib has promising clinical activity and a manageable adverse event profile in previously treated patients with locally advanced or metastatic cholangiocarcinoma harbouring FGFR2 gene fusions or rearrangements, and so represents a potential new therapeutic option in this setting., Funding: QED Therapeutics and Novartis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Although recent advances in immunotherapy have transformed the treatment landscape for many anatomically defined cancers, these therapies are currently not approved for patients diagnosed with cancer of unknown primary (CUP). Molecular cancer classification using gene expression profiling (GEP) assays has the potential to identify tumor type and putative primary cancers and thereby may allow consideration of immune checkpoint inhibitor (ICI) therapy options for a subset of patients with CUP. Herein, we evaluated and characterized the ability of a 92-gene assay (CancerTYPE ID) to provide a molecular diagnosis and identify putative tumor types that are known to be sensitive to ICI therapies in patients with CUP or uncertain diagnosis., Findings: A total of 24,426 cases from a large-scale research database of 92-gene assay clinical cases were classified, of which 9,350 (38%) were predicted to have an ICI-eligible tumor type. All ICIs with approved indications as of March 2020 were included in the analysis. Non-small cell lung cancer (NSCLC) was the most frequent molecular diagnosis and accounted for 33% of the ICI-eligible tumor types identified and 13% of the overall reportable results. In addition to NSCLC, the assay also frequently identified urothelial carcinomas, gastric cancer, and head and neck squamous cell carcinoma. The distributions of identified tumor types with indications for ICI therapy were similar across age and gender., Conclusions: Results suggest that molecular profiling with the 92-gene assay identifies a subset of ICI-eligible putative primary cancers in patients with CUP. We propose a treatment strategy based on available tests, including clinicopathologic features, GEP, and ICI biomarkers of response., (© 2020 The Authors. The Oncologist published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Background: Infigratinib (BGJ398) is a potent and selective fibroblast grown factor receptor 1 to 3 (FGFR1-3) inhibitor with significant activity in patients with advanced or metastatic urothelial carcinoma bearing FGFR3 alterations. Given the distinct biologic characteristics of upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB), the authors examined whether infigratinib had varying activity in these settings., Methods: Eligible patients had metastatic urothelial carcinoma with activating FGFR3 mutations and/or fusions. Comprehensive genomic profiling was performed on formalin-fixed, paraffin-embedded tissues. Blood was collected for cell-free DNA analysis using a 600-gene panel. Patients received infigratinib at a dose of 125 mg orally daily (3 weeks on/1 week off) until disease progression or intolerable toxicity occurred. The overall response rate (ORR; partial response [PR] plus complete response [CR]) and disease control rate (DCR; CR plus PR plus stable disease [SD]) were characterized., Results: A total of 67 patients were enrolled; the majority (70.1%) had received ≥2 prior antineoplastic therapies. In 8 patients with UTUC, 1 CR and 3 PRs were observed (ORR, 50%); the remaining patients achieved a best response of SD (DCR, 100%). In patients with UCB, 13 PRs were observed (ORR, 22%), and 22 patients had a best response of SD (DCR, 59.3%). Notable differences in genomic alterations between patients with UTUC and those with UCB included higher frequencies of FGFR3-TACC3 fusions (12.5% vs 6.8%) and FGFR3 R248C mutations (50% vs 11.9%), and a lower frequency of FGFR3 S249C mutations (37.5% vs 59.3%)., Conclusions: Differences in the cumulative genomic profile were observed between patients with UTUC and those with UCB in the current FGFR3-restricted experience, underscoring the distinct biology of these diseases. These results support a planned phase 3 adjuvant study predominantly performed in this population., (© 2020 American Cancer Society.)

Background: Ampullary adenocarcinoma is a rare gastrointestinal cancer associated with diverse outcomes due to clinical and pathological heterogeneity. Standardized methods to better prognosticate and inform therapeutic selection for ampullary adenocarcinoma are needed. This study explored the novel use and potential prognostic utility of a 92-gene cancer classifier in ampullary adenocarcinomas., Methods: In this prospectively-defined, blinded study of ampullary adenocarcinoma [N =54; stage T3 or higher (57 %); Grade III (44 %); Node positive (55 %)], the performance of a 92-gene classifier was examined to predict the ampullary subtype that was derived from histomorphological examination of resected ampullary samples. Outcome data for relapse-free survival (RFS) and overall survival (OS) were plotted to compare the prognostic utility of histological subtyping, histomolecular phenotyping, and the 92-gene classifier. Multivariate analysis was used to determine clinicopathological variables that were independently associated with overall survival., Results: The 92-gene classifier demonstrated sensitivities and specificities of 85 % [95 % CI, 66-94] and 68 % [95 % CI, 48-84] and 64 % [95 % CI, 46-79] and 88 % [95 % CI, 70-98] for the pancreaticobiliary and intestinal histological subtypes, respectively. For the 92-gene classifier, improved outcomes were observed for the intestine versus the pancreaticobiliary prediction (median OS 108.1 v 36.4 months; HR, 2.17; 95 % CI, 0.98 to 4.79; P = 0.05). Similar results were seen for ampullary adenocarcinoma stratification by histological subtype (P = 0.04) and histomolecular phenotype (P = 0.02). Within poorly differentiated ampullary adenocarcinomas only the 92-gene classifier demonstrated statistically significant differences in RFS and OS (P < 0.05)., Conclusions: Prognostic stratification of ampullary adenocarcinoma was similar for the 92-gene classifier, histological subtype, and histomolecular phenotype. The 92-gene classifier provides an unbiased standardized molecular-based approach to stratify ampullary tumors.

Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

TOK-001 and abiraterone are potent 17-heteroarylsteroid (17-HAS) inhibitors of Cyp17, one of the rate-limiting enzymes in the biosynthesis of testosterone from cholesterol in prostate cancer cells. Nevertheless, the molecular mechanism underlying the prevention of prostate cell growth by 17-HASs still remains elusive. Here, we assess the effects of 17-HASs on androgen receptor (AR) activity in LNCaP and LAPC-4 cells. We demonstrate that both TOK-001 and abiraterone reduced AR protein and mRNA expression, and antagonized AR-dependent promoter activation induced by androgen. TOK-001, but not abiraterone, is an effective apparent competitor of the radioligand [(3)H]R1881 for binding to the wild type and various mutant AR (W741C, W741L) proteins. In agreement with these data, TOK-001 is a consistently superior inhibitor than abiraterone of R1881-induced transcriptional activity of both wild type and mutant AR. However, neither agent was able to trans-activate the AR in the absence of R1881. Our data demonstrate that phospho-4EBP1 levels are significantly reduced by TOK-001 and to a lesser extent by abiraterone alcohol, and suggest a mechanism by which cap-dependent translation is suppressed by blocking assembly of the eIF4F and eIF4G complex to the mRNA 5' cap. Thus, the effects of these 17-HASs on AR signaling are complex, ranging from a decrease in testosterone production through the inhibition of Cyp17 as previously described, to directly reducing both AR protein expression and R1881-induced AR trans-activation.

Antisense oligodeoxyribonucleotides have been used for decades to achieve sequence-specific silencing of gene expression. However, all early generation oligonucleotides (e.g., those with no other modifications than the phosphorothioate backbone) are inactive in vitro unless administered using a delivery vehicle. These delivery vehicles are usually lipidic but can also be polyamines or some other particulate reagent. We have found that by employing locked nucleic acid (LNA) phosphorothioate gap-mer nucleic acids of 16 mer or less in length, and by carefully controlling the plating conditions of the target cells and duration of the experiment, sequence-specific gene silencing can be achieved at low micromolar concentrations in vitro in the absence of any delivery vehicle. This process of naked oligonucleotide delivery to achieve gene silencing in vivo, which we have termed gymnosis, has been observed in many both adherent and nonadherent cell lines against several different targets genes.

For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

Efficient delivery of small interfering (si)RNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We show that siRNA synthetically linked to a CpG oligonucleotide agonist of toll-like receptor (TLR)9 targets and silences genes in TLR9(+) myeloid cells and B cells, both of which are key components of the tumor microenvironment. When a CpG-conjugated siRNA that targets the immune suppressor gene Stat3 is injected in mice either locally at the tumor site or intravenously, it enters tumor-associated dendritic cells, macrophages and B cells. Silencing of Stat3 leads to activation of tumor-associated immune cells and ultimately to potent antitumor immune responses. Our findings demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment.

In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures.

Dicer-substrate small interfering RNAs (DsiRNAs) are synthetic RNA duplexes that are processed by Dicer into 21-mer species and show improved potency as triggers of RNA interference, particularly when used at low dose. Chemical modification patterns that are compatible with high potency 21-mer small interfering RNAs have been reported by several groups. However, modification patterns have not been studied for Dicer-substrate duplexes. We therefore synthesized a series of chemically modified 27-mer DsiRNAs and correlated modification patterns with functional potency. Some modification patterns profoundly reduced function although other patterns maintained high potency. Effects of sequence context were observed, where the relative potency of modification patterns varied between sites. A modification pattern involving alternating 2'-O-methyl RNA bases was developed that generally retains high potency when tested in different sites in different genes, evades activation of the innate immune system, and improves stability in serum.

MicroRNAs (miRNAs) are 21-24 nucleotide (nt) duplex RNAs that are created from precursor transcripts by subsequent processing steps mediated by members of the RNAseIII family, Drosha and Dicer. One of the two strands is incorporated into the active sites of the Argonaute family of proteins, where it serves as a guide for Watson-Crick base pairing with complementary sequences in target messenger RNAs (mRNAs). In mammals, the majority of miRNAs guide the RNA-induced silencing complex (RISC) to the 3' untranslated regions (UTRs) of mRNA targets, with the consequence that translation of the target mRNAs is inhibited. The importance of miRNAs in normal cellular development and metabolism is only now being realized. miRNA deficiencies or excesses have been correlated with a number of clinically important diseases ranging from myocardial infarction to cancers. The loss or gain of miRNA function can be caused by a single point mutation in either the miRNA or its target or by epigenetic silencing of primary miRNA transcription units. This review summarizes miRNA biogenesis and biology, explores the potential roles miRNAs can play in a variety of diseases, and suggests some therapeutic applications for restoring or inhibiting miRNA function.

Whether long interspersed element-1 (L1 or LINE-1) retrotransposition can occur in quiescent, nondividing, and/or terminally differentiated somatic cells has remained an unanswered fundamental question in human genetics. Here, we used a ubiquitously active phosphoglycerate kinase-1 promoter to drive the expression of a highly active human L1 element from an adenovirus-L1 hybrid vector. This vector system achieved retrotransposition in up to 91% of actively growing immortalized cells, and we demonstrated that L1 retrotransposition can be suppressed by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine. This adenovirus vector enabled efficient delivery of the L1 element into differentiated primary human somatic cells and G1/S-arrested cells, resulting in retrotransposition in both cases; however, it was not detected in G0-arrested cells. Thus, these data indicate that L1 retrotransposition can occur in nondividing somatic cells.

The crystal structure based model of the catalytic center of Ago2 revealed that the siRNA and the mRNA must be able to form an A-helix for correct positing of the scissile phosphate bond for cleavage in RNAi. This suggests that base pairing of the target mRNA with itself, i.e. secondary structure, must be removed before cleavage. Early on in the siRNA design, GC-rich target sites were avoided because of their potential to be involved in strong secondary structure. It is still unclear how important a factor mRNA secondary structure is in RNAi. However, it has been established that a difference in the thermostability of the ends of an siRNA duplex dictate which strand is loaded into the RNA-induced silencing complex. Here, we use a novel secondary structure prediction method and duplex-end differential calculations to investigate the importance of a secondary structure in the siRNA design. We found that the differential duplex-end stabilities alone account for functional prediction of 60% of the 80 siRNA sites examined, and that secondary structure predictions improve the prediction of site efficacy. A total of 80% of the non-functional sites can be eliminated using secondary structure predictions and duplex-end differential.

Long interspersed nuclear elements (LINE-1 or L1) comprise 17% of the human genome, although only 80-100 L1s are considered retrotransposition-competent (RC-L1). Despite their small number, RC-L1s are still potential hazards to genome integrity through insertional mutagenesis, unequal recombination and chromosome rearrangements. In this study, we provide several lines of evidence that the LINE-1 retrotransposon is susceptible to RNA interference (RNAi). First, double-stranded RNA (dsRNA) generated in vitro from an L1 template is converted into functional short interfering RNA (siRNA) by DICER, the RNase III enzyme that initiates RNAi in human cells. Second, pooled siRNA from in vitro cleavage of L1 dsRNA, as well as synthetic L1 siRNA, targeting the 5'-UTR leads to sequence-specific mRNA degradation of an L1 fusion transcript. Finally, both synthetic and pooled siRNA suppressed retrotransposition from a highly active RC-L1 clone in cell culture assay. Our report is the first to demonstrate that a human transposable element is subjected to RNAi.

It has long been recognized that the mechanisms mediating retrotransposition might be adapted for genomic integration and long-term expression of foreign genes. In particular, long interspersed nuclear elements (LINEs), an abundant class of retrotransposons that are the most active mobile genetic elements in the human genome, have been largely ignored as candidates for development as an integrating vector system because there has been no suitable method for efficiently introducing them into target cells. We have recently developed a LINE-based retrotransposon-adenovirus hybrid vector, in which a helper-dependent adenovirus (HDAd) is utilized as the platform for delivery of a human L1 element and its linked heterologous transgene cassette into the host cell nuclei. While a major drawback to the use of HDAd vectors has been their lack of specific mechanisms to achieve permanent integration into the host genome, the inserted retrotransposon sequences overcome this limitation. The L1-HDAd hybrid thus represents a single vector capable of mediating long-term gene expression by a two-stage mechanism: in the first (adenovirus) stage, the helper-dependent adenovirus serves as a carrier for efficient delivery and transient expression of its encoded L1/transgene cassette, and in the second (retrotransposon) stage, the L1 retro-element and its associated transgene then permanently integrate into the genome of the adenovirus-transduced cells. We propose that this novel retrotransposon-adenovirus hybrid vector system will be useful both as a vehicle for efficient delivery and long-term stable transduction of therapeutic genes, as well as a tool to elucidate aspects of retrotransposon biology that have previously been difficult to study.