Long-Term Follow-Up Defines the Population That Benefits from Early Interception in a High-Risk Smoldering Multiple Myeloma Clinical Trial Using the Combination of Ixazomib, Lenalidomide, and Dexamethasone.

Background: Early therapeutic intervention in high-risk SMM (HR-SMM) has demonstrated benefit in previous studies of lenalidomide with or without dexamethasone. Triplets and quadruplet studies have been examined in this same population. However, to date, none of these studies examined the impact of depth of response on long-term outcomes of participants treated with lenalidomide-based therapy, and whether the use of the 20/2/20 model or the addition of genomic alterations can further define the population that would benefit the most from early therapeutic intervention. Here, we present the results of the phase II study of the combination of ixazomib, lenalidomide, and dexamethasone in patients with HR-SMM with long-term follow-up and baseline single-cell tumor and immune sequencing that help refine the population to be treated for early intervention studies. Methods: This is a phase II trial of ixazomib, lenalidomide, and dexamethasone (IRD) in HR-SMM. Patients received 9 cycles of induction therapy with ixazomib 4mg on days 1, 8, and 15; lenalidomide 25mg on days 1-21; and dexamethasone 40mg on days 1, 8, 15, and 22. The induction phase was followed by maintenance with ixazomib 4mg on days 1, 8, and 15; and lenalidomide 15mg d1-21 for 15 cycles for 24 months of treatment. The primary endpoint was progression-free survival after 2 years of therapy. Secondary endpoints included depth of response, biochemical progression, and correlative studies included single-cell RNA sequencing and/or whole-genome sequencing of the tumor and single-cell sequencing of immune cells at baseline. Results: Fifty-five patients, with a median age of 64, were enrolled in the study. The overall response rate was 93%, with 31% of patients achieving a complete response and 45% achieving a very good partial response or better. The most common grade 3 or greater treatment-related hematologic toxicities were neutropenia (16 patients; 29%), leukopenia (10 patients; 18%), lymphocytopenia (8 patients; 15%), and thrombocytopenia (4 patients; 7%). Non-hematologic grade 3 or greater toxicities included hypophosphatemia (7 patients; 13%), rash (5 patients; 9%), and hypokalemia (4 patients; 7%). After a median follow-up of 50 months, the median progression-free survival (PFS) was 48.6 months (95% CI: 39.9 - not reached; NR) and median overall survival has not been reached. Patients achieving VGPR or better had a significantly better progression-free survival (p<0.001) compared to those who did not achieve VGPR (median PFS 58.2 months vs. 31.3 months). Biochemical progression preceded or was concurrent with the development of SLiM-CRAB criteria in eight patients during follow-up, indicating that biochemical progression is a meaningful endpoint that correlates with the development of end-organ damage. High-risk 20/2/20 participants had the worst PFS compared to low- and intermediate-risk participants. The use of whole genome or single-cell sequencing of tumor cells identified high-risk aberrations that were not identified by FISH alone and aided in the identification of participants at risk of progression. scRNA-seq analysis revealed a positive correlation between MHC class I expression and response to proteasome inhibition and at the same time a decreased proportion of GZMB+ T cells within the clonally expanded CD8+ T cell population correlated with suboptimal response. Conclusions: Ixazomib, lenalidomide and dexamethasone in HR-SMM demonstrates significant clinical activity with an overall favorable safety profile. Achievement of VGPR or greater led to significant improvement in time to progression, suggesting that achieving deep response is beneficial in HR-SMM. Biochemical progression correlates with end-organ damage. Patients with high-risk FISH and lack of deep response had poor outcomes. ClinicalTrials.gov identifier: (NCT02916771).

Assay Validation of Cell-Free DNA Shallow Whole-Genome Sequencing to Determine Tumor Fraction in Advanced Cancers.

Blood-based liquid biopsy is increasingly used in clinical care of patients with cancer, and fraction of tumor-derived DNA in circulation (tumor fraction; TFx) has demonstrated clinical validity across multiple cancer types. To determine TFx, shallow whole-genome sequencing of cell-free DNA (cfDNA) can be performed from a single blood sample, using an established computational pipeline (ichorCNA), without prior knowledge of tumor mutations, in a highly cost-effective manner. We describe assay validation of this approach to facilitate broad clinical application, including evaluation of assay sensitivity, precision, repeatability, reproducibility, pre-analytic factors, and DNA quality/quantity. Sensitivity to detect TFx of 3% (lower limit of detection) was 97.2% to 100% at 1× and 0.1× mean sequencing depth, respectively. Precision was demonstrated on distinct sequencing instruments (HiSeqX and NovaSeq) with no observable differences. The assay achieved prespecified 95% agreement of TFx across replicates of the same specimen (repeatability) and duplicate samples in different batches (reproducibility). Comparison of samples collected in EDTA and Streck tubes from single venipuncture in 23 patients demonstrated that EDTA or Streck tubes were comparable if processed within 8 hours. On the basis of a range of DNA inputs (1 to 50 ng), 20 ng cfDNA is the preferred input, with 5 ng minimum acceptable. Overall, this shallow whole-genome sequencing of cfDNA and ichorCNA approach offers sensitive, precise, and reproducible quantitation of TFx, facilitating assay application in clinical cancer care.

TSC2 inactivation, low mutation burden and high macrophage infiltration characterise hepatic angiomyolipomas.

AIMS: Although TSC1 or TSC2 inactivating mutations that lead to mTORC1 hyperactivation have been reported in hepatic angiomyolipomas (hAML), the role of other somatic genetic events that may contribute to hAML development is unknown. There are also limited data regarding the tumour microenvironment (TME) of hAML. The aim of the present study was to identify other somatic events in genomic level and changes in TME that contribute to tumorigenesis in hAML. METHODS AND RESULTS: In this study, we performed exome sequencing in nine sporadic hAML tumours and deep-coverage targeted sequencing for TSC2 in three additional hAML. Immunohistochemistry and multiplex immunofluorescence were carried out for 15 proteins to characterise the tumour microenvironment and assess immune cell infiltration. Inactivating somatic variants in TSC2 were identified in 10 of 12 (83%) cases, with a median allele frequency of 13.6%. Five to 18 somatic variants (median number: nine, median allele frequency 21%) not in TSC1 or TSC2 were also identified, mostly of uncertain clinical significance. Copy number changes were rare, but detection was impaired by low tumour purity. Immunohistochemistry demonstrated numerous CD68+ macrophages of distinct appearance from Küpffer cells. Multiplex immunofluorescence revealed low numbers of exhausted PD-1+/PD-L1+, FOXP3+ and CD8+ T cells. CONCLUSION: hAML tumours have consistent inactivating mutations in TSC2 and have a low somatic mutation rate, similar to other TSC-associated tumours. Careful histological review, standard IHC and multiplex immunofluorescence demonstrated marked infiltration by non-neoplastic inflammatory cells, mostly macrophages.

Systematic characterization of therapeutic vulnerabilities in Multiple Myeloma with Amp1q reveals increased sensitivity to the combination of MCL1 and PI3K inhibitors.

The development of targeted therapy for patients with Multiple Myeloma (MM) is hampered by the low frequency of actionable genetic abnormalities. Gain or amplification of chr1q (Amp1q) is the most frequent arm-level copy number gain in patients with MM, and it is associated with higher risk of progression and death despite recent advances in therapeutics. Thus, developing targeted therapy for patients with MM and Amp1q stands to benefit a large portion of patients in need of more effective management. Here, we employed large-scale dependency screens and drug screens to systematically characterize the therapeutic vulnerabilities of MM with Amp1q and showed increased sensitivity to the combination of MCL1 and PI3K inhibitors. Using single-cell RNA sequencing, we compared subclones with and without Amp1q within the same patient tumors and showed that Amp1q is associated with higher levels of MCL1 and the PI3K pathway. Furthermore, by isolating isogenic clones with different copy number for part of the chr1q arm, we showed increased sensitivity to MCL1 and PI3K inhibitors with arm-level gain. Lastly, we demonstrated synergy between MCL1 and PI3K inhibitors and dissected their mechanism of action in MM with Amp1q.

Circulating Tumor DNA Is Prognostic in Intermediate-Risk Rhabdomyosarcoma: A Report From the Children's Oncology Group.

PURPOSE: Novel biomarkers are needed to differentiate outcomes in intermediate-risk rhabdomyosarcoma (IR RMS). We sought to evaluate strategies for identifying circulating tumor DNA (ctDNA) in IR RMS and to determine whether ctDNA detection before therapy is associated with outcome. PATIENTS AND METHODS: Pretreatment serum and tumor samples were available from 124 patients with newly diagnosed IR RMS from the Children's Oncology Group biorepository, including 75 patients with fusion-negative rhabdomyosarcoma (FN-RMS) and 49 with fusion-positive rhabdomyosarcoma (FP-RMS) disease. We used ultralow passage whole-genome sequencing to detect copy number alterations and a new custom sequencing assay, Rhabdo-Seq, to detect rearrangements and single-nucleotide variants. RESULTS: We found that ultralow passage whole-genome sequencing was a method applicable to ctDNA detection in all patients with FN-RMS and that ctDNA was detectable in 13 of 75 serum samples (17%). However, the use of Rhabdo-Seq in FN-RMS samples also identified single-nucleotide variants, such as MYOD1L122R, previously associated with prognosis. Identification of pathognomonic translocations between PAX3 or PAX7 and FOXO1 by Rhabdo-Seq was the best method for measuring ctDNA in FP-RMS and detected ctDNA in 27 of 49 cases (55%). Patients with FN-RMS with detectable ctDNA at diagnosis had significantly worse outcomes than patients without detectable ctDNA (event-free survival, 33.3% v 68.9%; P = .0028; overall survival, 33.3% v 83.2%; P < .0001) as did patients with FP-RMS (event-free survival, 37% v 70%; P = .045; overall survival, 39.2% v 75%; P = .023). In multivariable analysis, ctDNA was independently associated with worse prognosis in FN-RMS but not in the smaller FP-RMS cohort. CONCLUSION: Our study demonstrates that baseline ctDNA detection is feasible and is prognostic in IR RMS.

Clinical Efficacy and Whole-Exome Sequencing of Liquid Biopsies in a Phase IB/II Study of Bazedoxifene and Palbociclib in Advanced Hormone Receptor-Positive Breast Cancer.

PURPOSE: Sensitivity to endocrine therapy (ET) is critical for the clinical benefit from the combination of palbociclib plus ET in hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer. Bazedoxifene is a third-generation selective estrogen receptor (ER) modulator and selective ER degrader with activity in preclinical models of endocrine-resistant breast cancer, including models harboring ESR1 mutations. Clinical trials in healthy women showed that bazedoxifene is well tolerated. PATIENTS AND METHODS: We conducted a phase Ib/II study of bazedoxifene plus palbociclib in patients with HR+/HER2- advanced breast cancer who progressed on prior ET (N = 36; NCT02448771). RESULTS: The study met its primary endpoint, with a clinical benefit rate of 33.3%, and the safety profile was consistent with what has previously been seen with palbociclib monotherapy. The median progression-free survival (PFS) was 3.6 months [95% confidence interval (CI), 2.0-7.2]. An activating PIK3CA mutation at baseline was associated with a shorter PFS (HR = 4.4; 95% CI, 1.5-13; P = 0.0026), but activating ESR1 mutations did not impact the PFS. Longitudinal plasma circulating tumor DNA whole-exome sequencing (WES; N = 68 plasma samples) provided an overview of the tumor heterogeneity and the subclonal genetic evolution, and identified actionable mutations acquired during treatment. CONCLUSIONS: The combination of palbociclib and bazedoxifene has clinical efficacy and an acceptable safety profile in a heavily pretreated patient population with advanced HR+/HER2- breast cancer. These results merit continued investigation of bazedoxifene in breast cancer.

Durvalumab plus tremelimumab alone or in combination with low-dose or hypofractionated radiotherapy in metastatic non-small-cell lung cancer refractory to previous PD(L)-1 therapy: an open-label, multicentre, randomised, phase 2 trial.

BACKGROUND: Patients with non-small-cell lung cancer (NSCLC) that is resistant to PD-1 and PD-L1 (PD[L]-1)-targeted therapy have poor outcomes. Studies suggest that radiotherapy could enhance antitumour immunity. Therefore, we investigated the potential benefit of PD-L1 (durvalumab) and CTLA-4 (tremelimumab) inhibition alone or combined with radiotherapy. METHODS: This open-label, multicentre, randomised, phase 2 trial was done by the National Cancer Institute Experimental Therapeutics Clinical Trials Network at 18 US sites. Patients aged 18 years or older with metastatic NSCLC, an Eastern Cooperative Oncology Group performance status of 0 or 1, and progression during previous PD(L)-1 therapy were eligible. They were randomly assigned (1:1:1) in a web-based system by the study statistician using a permuted block scheme (block sizes of three or six) without stratification to receive either durvalumab (1500 mg intravenously every 4 weeks for a maximum of 13 cycles) plus tremelimumab (75 mg intravenously every 4 weeks for a maximum of four cycles) alone or with low-dose (0·5 Gy delivered twice per day, repeated for 2 days during each of the first four cycles of therapy) or hypofractionated radiotherapy (24 Gy total delivered over three 8-Gy fractions during the first cycle only), 1 week after initial durvalumab-tremelimumab administration. Study treatment was continued until 1 year or until progression. The primary endpoint was overall response rate (best locally assessed confirmed response of a partial or complete response) and, along with safety, was analysed in patients who received at least one dose of study therapy. The trial is registered with ClinicalTrials.gov, NCT02888743, and is now complete. FINDINGS: Between Aug 24, 2017, and March 29, 2019, 90 patients were enrolled and randomly assigned, of whom 78 (26 per group) were treated. This trial was stopped due to futility assessed in an interim analysis. At a median follow-up of 12·4 months (IQR 7·8-15·1), there were no differences in overall response rates between the durvalumab-tremelimumab alone group (three [11·5%, 90% CI 1·2-21·8] of 26 patients) and the low-dose radiotherapy group (two [7·7%, 0·0-16·3] of 26 patients; p=0·64) or the hypofractionated radiotherapy group (three [11·5%, 1·2-21·8] of 26 patients; p=0·99). The most common grade 3-4 adverse events were dyspnoea (two [8%] in the durvalumab-tremelimumab alone group; three [12%] in the low-dose radiotherapy group; and three [12%] in the hypofractionated radiotherapy group) and hyponatraemia (one [4%] in the durvalumab-tremelimumab alone group vs two [8%] in the low-dose radiotherapy group vs three [12%] in the hypofractionated radiotherapy group). Treatment-related serious adverse events occurred in one (4%) patient in the durvalumab-tremelimumab alone group (maculopapular rash), five (19%) patients in the low-dose radiotherapy group (abdominal pain, diarrhoea, dyspnoea, hypokalemia, and respiratory failure), and four (15%) patients in the hypofractionated group (adrenal insufficiency, colitis, diarrhoea, and hyponatremia). In the low-dose radiotherapy group, there was one death from respiratory failure potentially related to study therapy. INTERPRETATION: Radiotherapy did not increase responses to combined PD-L1 plus CTLA-4 inhibition in patients with NSCLC resistant to PD(L)-1 therapy. However, PD-L1 plus CTLA-4 therapy could be a treatment option for some patients. Future studies should refine predictive biomarkers in this setting. FUNDING: The US National Institutes of Health and the Dana-Farber Cancer Institute.

Donor Clonal Hematopoiesis and Recipient Outcomes After Transplantation.

PURPOSE: Clonal hematopoiesis (CH) can be transmitted from a donor to a recipient during allogeneic hematopoietic cell transplantation. Exclusion of candidate donors with CH is controversial since its impact on recipient outcomes and graft alloimmune function is uncertain. PATIENTS AND METHODS: We performed targeted error-corrected sequencing on samples from 1,727 donors age 40 years or older and assessed the effect of donor CH on recipient clinical outcomes. We measured long-term engraftment of 102 donor clones and cytokine levels in 256 recipients at 3 and 12 months after transplant. RESULTS: CH was present in 22.5% of donors, with DNMT3A (14.6%) and TET2 (5.2%) mutations being most common; 85% of donor clones showed long-term engraftment in recipients after transplantation, including clones with a variant allele fraction < 0.01. DNMT3A-CH with a variant allele fraction ≥ 0.01, but not smaller clones, was associated with improved recipient overall (hazard ratio [HR], 0.79; P = .042) and progression-free survival (HR, 0.72; P = .003) after adjustment for significant clinical variables. In patients who received calcineurin-based graft-versus-host disease prophylaxis, donor DNMT3A-CH was associated with reduced relapse (subdistribution HR, 0.59; P = .014), increased chronic graft-versus-host disease (subdistribution HR, 1.36; P = .042), and higher interleukin-12p70 levels in recipients. No recipient of sole DNMT3A or TET2-CH developed donor cell leukemia (DCL). In seven of eight cases, DCL evolved from donor CH with rare TP53 or splicing factor mutations or from donors carrying germline DDX41 mutations. CONCLUSION: Donor CH is closely associated with clinical outcomes in transplant recipients, with differential impact on graft alloimmune function and potential for leukemic transformation related to mutated gene and somatic clonal abundance. Donor DNMT3A-CH is associated with improved recipient survival because of reduced relapse risk and with an augmented network of inflammatory cytokines in recipients. Risk of DCL in allogeneic hematopoietic cell transplantation is driven by somatic myelodysplastic syndrome-associated mutations or germline predisposition in donors.

Phase II Clinical Trial of Everolimus in a Pan-Cancer Cohort of Patients with mTOR Pathway Alterations.

PURPOSE: This was a multicenter, histology-agnostic, single-arm prospective phase II trial of therapeutic activity of everolimus, an oral mTORC1 inhibitor, in patients with advanced solid tumors that harbored TSC1/TSC2 or MTOR mutations. PATIENTS AND METHODS: Patients with tumors with inactivating TSC1/TSC2 or activating MTOR mutations identified in any Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory were eligible. Patients were treated with everolimus 10 mg once daily until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR). Whole-exome sequencing was performed to identify co-occurring genomic alterations. RESULTS: Between November 2015 and October 2018, 30 patients were enrolled at Dana-Farber Cancer Institute and Memorial Sloan Kettering Cancer Center. Tumors harbored TSC1 (13/30), TSC2 (15/30), concurrent TSC1 and TSC2 (1/30), or MTOR (1/30) mutations. The most common treatment-related adverse event of any grade was mucositis (8/30, 27%); 1 patient had fatal pneumonitis. Partial responses were seen in 2 patients [7%; 95% confidence interval (CI), 1%-22%]. Median progression-free survival was 2.3 months (95% CI, 1.8-3.7 months) and median overall survival (OS) was 7.3 months (95% CI, 4.5-12.7 months). There was no clear association between other genomic alterations and response. Of the 2 patients with objective response, 1 had upper tract urothelial carcinoma with biallelic inactivation of TSC1 and high tumor mutation burden, and the other had uterine carcinoma with biallelic TSC2-inactivating mutations and PEComa-like pathologic features. CONCLUSIONS: Everolimus therapy had a disappointing ORR (7%) in this pan-cancer, mutation-selected, basket study.See related commentary by Kato and Cohen, p. 3807.

Annotation of chromatin states in 66 complete mouse epigenomes during development.

The morphologically and functionally distinct cell types of a multicellular organism are maintained by their unique epigenomes and gene expression programs. Phase III of the ENCODE Project profiled 66 mouse epigenomes across twelve tissues at daily intervals from embryonic day 11.5 to birth. Applying the ChromHMM algorithm to these epigenomes, we annotated eighteen chromatin states with characteristics of promoters, enhancers, transcribed regions, repressed regions, and quiescent regions. Our integrative analyses delineate the tissue specificity and developmental trajectory of the loci in these chromatin states. Approximately 0.3% of each epigenome is assigned to a bivalent chromatin state, which harbors both active marks and the repressive mark H3K27me3. Highly evolutionarily conserved, these loci are enriched in silencers bound by polycomb repressive complex proteins, and the transcription start sites of their silenced target genes. This collection of chromatin state assignments provides a useful resource for studying mammalian development.

Somatic piRNAs and Transposons are Differentially Expressed Coincident with Skeletal Muscle Atrophy and Programmed Cell Death.

PIWI-interacting RNAs (piRNAs) are small single-stranded RNAs that can repress transposon expression via epigenetic silencing and transcript degradation. They have been identified predominantly in the ovary and testis, where they serve essential roles in transposon silencing in order to protect the integrity of the genome in the germline. The potential expression of piRNAs in somatic cells has been controversial. In the present study we demonstrate the expression of piRNAs derived from both genic and transposon RNAs in the intersegmental muscles (ISMs) from the tobacco hawkmoth Manduca sexta. These piRNAs are abundantly expressed, ∼27 nt long, map antisense to transposons, are oxidation resistant, exhibit a 5' uridine bias, and amplify via the canonical ping-pong pathway. An RNA-seq analysis demonstrated that 19 piRNA pathway genes are expressed in the ISMs and are developmentally regulated. The abundance of piRNAs does not change when the muscles initiate developmentally-regulated atrophy, but are repressed coincident with the commitment of the muscles undergo programmed cell death at the end of metamorphosis. This change in piRNA expression is correlated with the repression of several retrotransposons and the induction of specific DNA transposons. The developmentally-regulated changes in the expression of piRNAs, piRNA pathway genes, and transposons are all regulated by 20-hydroxyecdysone, the steroid hormone that controls the timing of ISM death. Taken together, these data provide compelling evidence for the existence of piRNA in somatic tissues and suggest that they may play roles in developmental processes such as programmed cell death.

Cross-Site Concordance Evaluation of Tumor DNA and RNA Sequencing Platforms for the CIMAC-CIDC Network.

PURPOSE: Whole-exome (WES) and RNA sequencing (RNA-seq) are key components of cancer immunogenomic analyses. To evaluate the consistency of tumor WES and RNA-seq profiling platforms across different centers, the Cancer Immune Monitoring and Analysis Centers (CIMAC) and the Cancer Immunologic Data Commons (CIDC) conducted a systematic harmonization study. EXPERIMENTAL DESIGN: DNA and RNA were centrally extracted from fresh frozen and formalin-fixed paraffin-embedded non-small cell lung carcinoma tumors and distributed to three centers for WES and RNA-seq profiling. In addition, two 10-plex HapMap cell line pools with known mutations were used to evaluate the accuracy of the WES platforms. RESULTS: The WES platforms achieved high precision (> 0.98) and recall (> 0.87) on the HapMap pools when evaluated on loci using > 50× common coverage. Nonsynonymous mutations clustered by tumor sample, achieving an index of specific agreement above 0.67 among replicates, centers, and sample processing. A DV200 > 24% for RNA, as a putative presequencing RNA quality control (QC) metric, was found to be a reliable threshold for generating consistent expression readouts in RNA-seq and NanoString data. MedTIN > 30 was likewise assessed as a reliable RNA-seq QC metric, above which samples from the same tumor across replicates, centers, and sample processing runs could be robustly clustered and HLA typing, immune infiltration, and immune repertoire inference could be performed. CONCLUSIONS: The CIMAC collaborating laboratory platforms effectively generated consistent WES and RNA-seq data and enable robust cross-trial comparisons and meta-analyses of highly complex immuno-oncology biomarker data across the NCI CIMAC-CIDC Network.

High-resolution analysis of differential gene expression during skeletal muscle atrophy and programmed cell death.

Skeletal muscles can undergo atrophy and/or programmed cell death (PCD) during development or in response to a wide range of insults, including immobility, cachexia, and spinal cord injury. However, the protracted nature of atrophy and the presence of multiple cell types within the tissue complicate molecular analyses. One model that does not suffer from these limitations is the intersegmental muscle (ISM) of the tobacco hawkmoth Manduca sexta. Three days before the adult eclosion (emergence) at the end of metamorphosis, the ISMs initiate a nonpathological program of atrophy that results in a 40% loss of mass. The ISMs then generate the eclosion behavior and initiate a nonapoptotic PCD during the next 30 h. We have performed a comprehensive transcriptomics analysis of all mRNAs and microRNAs throughout ISM development to better understand the molecular mechanisms that mediate atrophy and death. Atrophy involves enhanced protein catabolism and reduced expression of the genes involved in respiration, adhesion, and the contractile apparatus. In contrast, PCD involves the induction of numerous proteases, DNA methylases, membrane transporters, ribosomes, and anaerobic metabolism. These changes in gene expression are largely repressed when insects are injected with the insect steroid hormone 20-hydroxyecdysone, which delays death. The expression of the death-associated proteins may be greatly enhanced by reductions in specific microRNAs that function to repress translation. This study not only provides fundamental new insights into basic developmental processes, it may also represent a powerful resource for identifying potential diagnostic markers and molecular targets for therapeutic intervention.

Integrated miRNA-/mRNA-Seq of the Habenulo-Interpeduncular Circuit During Acute Nicotine Withdrawal.

Tobacco use is the leading preventable cause of mortality in the world. The limited number of smoking cessation aids currently available are minimally effective, highlighting the need for novel therapeutic interventions. We describe a genome-wide approach to identify potential candidates for such interventions. Next-generation sequencing was performed using RNA isolated from the habenulo-interpeduncular circuit of male mice withdrawn from chronic nicotine treatment. This circuit plays a central role in the nicotine withdrawal response. Differentially expressed miRNAs and mRNAs were validated using RT-qPCR. Many of the differentially expressed mRNAs are predicted targets of reciprocally expressed miRNAs. We illustrate the utility of the dataset by demonstrating that knockdown in the interpeduncular nucleus of a differentially expressed mRNA, that encoding profilin 2, is sufficient to induce anxiety-related behavior. Importantly, profilin 2 knockdown in the ventral tegmental area did not affect anxiety behavior. Our data reveal wide-spread changes in gene expression within the habenulo-interpeduncular circuit during nicotine withdrawal. This dataset should prove to be a valuable resource leading to the identification of substrates for the design of innovative smoking cessation aids.

Predicting human splicing branchpoints by combining sequence-derived features and multi-label learning methods.

BACKGROUND: Alternative splicing is the critical process in a single gene coding, which removes introns and joins exons, and splicing branchpoints are indicators for the alternative splicing. Wet experiments have identified a great number of human splicing branchpoints, but many branchpoints are still unknown. In order to guide wet experiments, we develop computational methods to predict human splicing branchpoints. RESULTS: Considering the fact that an intron may have multiple branchpoints, we transform the branchpoint prediction as the multi-label learning problem, and attempt to predict branchpoint sites from intron sequences. First, we investigate a variety of intron sequence-derived features, such as sparse profile, dinucleotide profile, position weight matrix profile, Markov motif profile and polypyrimidine tract profile. Second, we consider several multi-label learning methods: partial least squares regression, canonical correlation analysis and regularized canonical correlation analysis, and use them as the basic classification engines. Third, we propose two ensemble learning schemes which integrate different features and different classifiers to build ensemble learning systems for the branchpoint prediction. One is the genetic algorithm-based weighted average ensemble method; the other is the logistic regression-based ensemble method. CONCLUSIONS: In the computational experiments, two ensemble learning methods outperform benchmark branchpoint prediction methods, and can produce high-accuracy results on the benchmark dataset.

DNApi: A De Novo Adapter Prediction Algorithm for Small RNA Sequencing Data.

With the rapid accumulation of publicly available small RNA sequencing datasets, third-party meta-analysis across many datasets is becoming increasingly powerful. Although removing the 3´ adapter is an essential step for small RNA sequencing analysis, the adapter sequence information is not always available in the metadata. The information can be also erroneous even when it is available. In this study, we developed DNApi, a lightweight Python software package that predicts the 3´ adapter sequence de novo and provides the user with cleansed small RNA sequences ready for down stream analysis. Tested on 539 publicly available small RNA libraries accompanied with 3´ adapter sequences in their metadata, DNApi shows near-perfect accuracy (98.5%) with fast runtime (~2.85 seconds per library) and efficient memory usage (~43 MB on average). In addition to 3´ adapter prediction, it is also important to classify whether the input small RNA libraries were already processed, i.e. the 3´ adapters were removed. DNApi perfectly judged that given another batch of datasets, 192 publicly available processed libraries were "ready-to-map" small RNA sequence. DNApi is compatible with Python 2 and 3, and is available at https://github.com/jnktsj/DNApi. The 731 small RNA libraries used for DNApi evaluation were from human tissues and were carefully and manually collected. This study also provides readers with the curated datasets that can be integrated into their studies.

Evaluation of preprocessing, mapping and postprocessing algorithms for analyzing whole genome bisulfite sequencing data.

Cytosine methylation regulates many biological processes such as gene expression, chromatin structure and chromosome stability. The whole genome bisulfite sequencing (WGBS) technique measures the methylation level at each cytosine throughout the genome. There are an increasing number of publicly available pipelines for analyzing WGBS data, reflecting many choices of read mapping algorithms as well as preprocessing and postprocessing methods. We simulated single-end and paired-end reads based on three experimental data sets, and comprehensively evaluated 192 combinations of three preprocessing, five postprocessing and five widely used read mapping algorithms. We also compared paired-end data with single-end data at the same sequencing depth for performance of read mapping and methylation level estimation. Bismark and LAST were the most robust mapping algorithms. We found that Mott trimming and quality filtering individually improved the performance of both read mapping and methylation level estimation, but combining them did not lead to further improvement. Furthermore, we confirmed that paired-end sequencing reduced error rate and enhanced sensitivity for both read mapping and methylation level estimation, especially for short reads and in repetitive regions of the human genome.

The Role of H3K4me3 in Transcriptional Regulation Is Altered in Huntington's Disease.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder resulting from expansion of CAG repeats in the Huntingtin (HTT) gene. Previous studies have shown mutant HTT can alter expression of genes associated with dysregulated epigenetic modifications. One of the most widely studied chromatin modifications is trimethylated lysine 4 of histone 3 (H3K4me3). Here, we conducted the first comprehensive study of H3K4me3 ChIP-sequencing in neuronal chromatin from the prefrontal cortex of six HD cases and six non-neurologic controls, and its association with gene expression measured by RNA-sequencing. We detected 2,830 differentially enriched H3K4me3 peaks between HD and controls, with 55% of them down-regulated in HD. Although H3K4me3 signals are expected to be associated with mRNA levels, we found an unexpected discordance between altered H3K4me3 peaks and mRNA levels. Gene ontology (GO) term enrichment analysis of the genes with differential H3K4me3 peaks, revealed statistically significantly enriched GO terms only in the genes with down-regulated signals in HD. The most frequently implicated biological process terms are organ morphogenesis and positive regulation of gene expression. More than 9,000 H3K4me3 peaks were located not near any recognized transcription start sites and approximately 36% of these "distal" peaks co-localized to known enhancer sites. Six transcription factors and chromatin remodelers are differentially enriched in HD H3K4me3 distal peaks, including EZH2 and SUZ12, two core subunits of the polycomb repressive complex 2 (PRC2). Moreover, PRC2 repressive state was significantly depleted in HD-enriched peaks, suggesting the epigenetic role of PRC2 inhibition associated with up-regulated H3K4me3 in Huntington's disease. In summary, our study provides new insights into transcriptional dysregulation of Huntington's disease by analyzing the differentiation of H3K4me3 enrichment.

MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites.

Mitochondria provide numerous essential functions for cells and their dysfunction leads to a variety of diseases. Thus, obtaining a complete mitochondrial proteome should be a crucial step toward understanding the roles of mitochondria. Many mitochondrial proteins have been identified experimentally but a complete list is not yet available. To fill this gap, methods to computationally predict mitochondrial proteins from amino acid sequence have been developed and are widely used, but unfortunately, their accuracy is far from perfect. Here we describe MitoFates, an improved prediction method for cleavable N-terminal mitochondrial targeting signals (presequences) and their cleavage sites. MitoFates introduces novel sequence features including positively charged amphiphilicity, presequence motifs, and position weight matrices modeling the presequence cleavage sites. These features are combined with classical ones such as amino acid composition and physico-chemical properties as input to a standard support vector machine classifier. On independent test data, MitoFates attains better performance than existing predictors in both detection of presequences and in predicting their cleavage sites. We used MitoFates to look for undiscovered mitochondrial proteins from 42,217 human proteins (including isoforms such as alternative splicing or translation initiation variants). MitoFates predicts 1167 genes to have at least one isoform with a presequence. Five-hundred and eighty of these genes were not annotated as mitochondrial in either UniProt or Gene Ontology. Interestingly, these include candidate regulators of parkin translocation to damaged mitochondria, and also many genes with known disease mutations, suggesting that careful investigation of MitoFates predictions may be helpful in elucidating the role of mitochondria in health and disease. MitoFates is open source with a convenient web server publicly available.

Bisulfighter: accurate detection of methylated cytosines and differentially methylated regions.

Analysis of bisulfite sequencing data usually requires two tasks: to call methylated cytosines (mCs) in a sample, and to detect differentially methylated regions (DMRs) between paired samples. Although numerous tools have been proposed for mC calling, methods for DMR detection have been largely limited. Here, we present Bisulfighter, a new software package for detecting mCs and DMRs from bisulfite sequencing data. Bisulfighter combines the LAST alignment tool for mC calling, and a novel framework for DMR detection based on hidden Markov models (HMMs). Unlike previous attempts that depend on empirical parameters, Bisulfighter can use the expectation-maximization algorithm for HMMs to adjust parameters for each data set. We conduct extensive experiments in which accuracy of mC calling and DMR detection is evaluated on simulated data with various mC contexts, read qualities, sequencing depths and DMR lengths, as well as on real data from a wide range of biological processes. We demonstrate that Bisulfighter consistently achieves better accuracy than other published tools, providing greater sensitivity for mCs with fewer false positives, more precise estimates of mC levels, more exact locations of DMRs and better agreement of DMRs with gene expression and DNase I hypersensitivity. The source code is available at http://epigenome.cbrc.jp/bisulfighter.