Targeting autophagy as a therapeutic strategy in pediatric acute lymphoblastic leukemia.

Autophagy is activated in response to a variety of stress conditions including anti-cancer therapies, and tumors cells often depend on autophagy for survival. In this study, we have evaluated inhibition of autophagy as therapeutic strategy in acute lymphoblastic leukemia (ALL) in children, both as a single treatment and in combination with glucocorticoid (GC) Dexamethasone (Dexa). Analysis of proteomics and RNA-seq of ALL cell lines and primary samples identified an upregulation of Vps34 and ATG14 proteins and autophagy and lysosomal pathway enrichment in a genetic subgroup with a recurrent t(12;21) translocation. Cells from this sugbroup were also significantly more sensitive to the selective autophagy or lysosomal inhibitors than cells with other genetic rearrangements. Further, combination of Dexa with either lysosomal or autophagy inhibitors was either synergistic or additive in killing leukemic cells across various genetic and lineage backgrounds, for both cell lines and primary samples, as assessed using viability assays and SynergyFinder as well as apoptotic caspase 3/7-based live-cell assays. Our data demonstrate that targeting autophagy represents a promising strategy for the treatment of pediatric ALL, both as a selective modality for the t(12;21) pre-B-ALL subgroup, and in combination treatments to sensitize to GC-induced cytotoxicity.

FORALL: an interactive shiny/R web portal to navigate multi-omics high-throughput data of pediatric acute lymphoblastic leukemia.

Motivation: Pediatric acute lymphoblastic leukemia (ALL) is the most common cancer among children worldwide. The availability of easily accessible multi-omics data provides unprecedented resources and opportunities for discovering and refining disease biology, cancer biomarkers, and drug mechanisms of action. This has led to exponential growth of omics data available in public repositories. However, delivering the useful information and knowledge extraction from this data is one of the bottlenecks of multi-omics. Presenting, navigating, and downloading ALL omics data in a user-friendly interface provide a valuable platform for biologists and clinicians to get most of the omics data. Our in-house data provides in-depth mass spectrometry-based protein abundance data for a large panel of commercially available ALL cell lines. Providing this data to the scientific community in the form of a user-friendly web-portal allows for easy and detailed exploration of the data. Results: We have developed the Functional Omics Resource of Acute Lymphoblastic Leukemia (FORALL) web-portal. FORALL is a shiny-based web portal designed to navigate in-depth mass spectrometry-based proteomics data of 51 cell lines. The proteomics data can be navigated and visualized along with matched RNA expression data as well as drug sensitivity data of 528 investigational and approved drugs. Availability and implementation: FORALL is available at https://proteomics.se/forall/.

Identification of mechanism of cancer-cell-specific reactivation of hTERT offers therapeutic opportunities for blocking telomerase specifically in human colorectal cancer.

Transcriptional reactivation of hTERT is the limiting step in tumorigenesis. While mutations in hTERT promoter present in 19% of cancers are recognized as key drivers of hTERT reactivation, mechanisms by which wildtype hTERT (WT-hTERT) promoter is reactivated, in majority of human cancers, remain unknown. Using primary colorectal cancers (CRC) we identified Tert INTeracting region 2 (T-INT2), the critical chromatin region essential for reactivating WT-hTERT promoter in CRCs. Elevated ß-catenin and JunD level in CRC facilitates chromatin interaction between hTERT promoter and T-INT2 that is necessary to turn on hTERTexpression. Pharmacological screens uncovered salinomycin, which inhibits JunD mediated hTERT-T-INT2 interaction that is required for the formation of a stable transcription complex on the hTERT promoter. Our results showed for the first time how known CRC alterations, such as APC, lead to WT-hTERT promoter reactivation during stepwise-tumorigenesis and provide a new perspective for developing cancer-specific drugs.

Healthy and cancer cells harbor the same DNA sequence, but reactivation of the Human Telomerase Reverse Transcriptase (hTERT) gene is observed only in cancer cells. How does that happen was not known for over three decades of research? This study identifies a specific DNA structure that forms only in cancer cells and brings the necessary molecular machinery into the correct position to activate the hTERT gene. The detailed mechanism of hTERT activation provided in this study will be instrumental in designing cancer cell-specific hTERT inhibitors, especially since all the other ways of inhibiting telomerase failed in the clinic.

Integrative multi-omics and drug response profiling of childhood acute lymphoblastic leukemia cell lines.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although standard-of-care chemotherapeutics are sufficient for most ALL cases, there are subsets of patients with poor response who relapse in disease. The biology underlying differences between subtypes and their response to therapy has only partially been explained by genetic and transcriptomic profiling. Here, we perform comprehensive multi-omic analyses of 49 readily available childhood ALL cell lines, using proteomics, transcriptomics, and pharmacoproteomic characterization. We connect the molecular phenotypes with drug responses to 528 oncology drugs, identifying drug correlations as well as lineage-dependent correlations. We also identify the diacylglycerol-analog bryostatin-1 as a therapeutic candidate in the MEF2D-HNRNPUL1 fusion high-risk subtype, for which this drug activates pro-apoptotic ERK signaling associated with molecular mediators of pre-B cell negative selection. Our data is the foundation for the interactive online Functional Omics Resource of ALL (FORALL) with navigable proteomics, transcriptomics, and drug sensitivity profiles at https://proteomics.se/forall .

TP63, SOX2, and KLF5 Establish a Core Regulatory Circuitry That Controls Epigenetic and Transcription Patterns in Esophageal Squamous Cell Carcinoma Cell Lines.

BACKGROUND & AIMS: We investigated the transcriptome of esophageal squamous cell carcinoma (ESCC) cells, activity of gene regulatory (enhancer and promoter regions), and the effects of blocking epigenetic regulatory proteins. METHODS: We performed chromatin immunoprecipitation sequencing with antibodies against H3K4me1, H3K4me3, and H3K27ac and an assay for transposase-accessible chromatin to map the enhancer regions and accessible chromatin in 8 ESCC cell lines. We used the CRC_Mapper algorithm to identify core regulatory circuitry transcription factors in ESCC cell lines, and determined genome occupancy profiles for 3 of these factors. In ESCC cell lines, expression of transcription factors was knocked down with small hairpin RNAs, promoter and enhancer regions were disrupted by CRISPR/Cas9 genome editing, or bromodomains and extraterminal (BET) family proteins and histone deacetylases (HDACs) were inhibited with ARV-771 and romidepsin, respectively. ESCC cell lines were then analyzed by whole-transcriptome sequencing, immunoprecipitation, immunoblots, immunohistochemistry, and viability assays. Interactions between distal enhancers and promoters were identified and verified with circular chromosome conformation capture sequencing. NOD-SCID mice were given injections of modified ESCC cells, some mice where given injections of HDAC or BET inhibitors, and growth of xenograft tumors was measured. RESULTS: We identified super-enhancer-regulated circuits and transcription factors TP63, SOX2, and KLF5 as core regulatory factors in ESCC cells. Super-enhancer regulation of ALDH3A1 mediated by core regulatory factors was required for ESCC viability. We observed direct interactions between the promoter region of TP63 and functional enhancers, mediated by the core regulatory circuitry transcription factors. Deletion of enhancer regions from ESCC cells decreased expression of the core regulatory circuitry transcription factors and reduced cell viability; these same results were observed with knockdown of each core regulatory circuitry transcription factor. Incubation of ESCC cells with BET and HDAC disrupted the core regulatory circuitry program and the epigenetic modifications observed in these cells; mice given injections of HDAC or BET inhibitors developed smaller xenograft tumors from the ESCC cell lines. Xenograft tumors grew more slowly in mice given the combination of ARV-771 and romidepsin than mice given either agent alone. CONCLUSIONS: In epigenetic and transcriptional analyses of ESCC cell lines, we found the transcription factors TP63, SOX2, and KLF5 to be part of a core regulatory network that determines chromatin accessibility, epigenetic modifications, and gene expression patterns in these cells. A combination of epigenetic inhibitors slowed growth of xenograft tumors derived from ESCC cells in mice.

MicroRNA-138 is a Prognostic Biomarker for Triple-Negative Breast Cancer and Promotes Tumorigenesis via TUSC2 repression.

Breast cancer manifests as a spectrum of subtypes with distinct molecular signatures, and different responses to treatment. Of these subtypes, triple-negative breast cancer (TNBC) has the worst prognoses and limited therapeutic options. Here we report aberrant expression of microRNA-138 (miR-138) in TNBC. Increased miR-138 expression is highly specific to this subtype, correlates with poor prognosis in patients, and is functionally relevant to cancer progression. Our findings establish miR-138 as a specific diagnostic and prognostic biomarker for TNBC. OncomiR-138 is pro-survival; sequence-specific miR-138 inhibition blocks proliferation, promotes apoptosis and inhibits tumour growth in-vivo. miR-138 directly targets a suite of pro-apoptotic and tumour suppressive genes, including tumour suppressor candidate 2 (TUSC2). miR-138 silences TUSC2 by binding to a unique 5'-UTR target-site, which overlaps with the translation start-site of the transcript. Over-expression of TUSC2 mimics the phenotype of miR-138 knockdown and functional rescue experiments confirm that TUSC2 is a direct downstream target of miR-138. Our report of miR-138 as an oncogenic driver in TNBC, positions it as a viable target for oligonucleotide therapeutics and we envision the potential value of using antimiR-138 as an adjuvant therapy to alleviate this therapeutically intractable cancer.

HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis.

The repression of telomerase activity during cellular differentiation promotes replicative aging and functions as a physiological barrier for tumorigenesis in long-lived mammals, including humans. However, the underlying mechanisms remain largely unclear. Here we describe how miR-615-3p represses hTERT expression. mir-615-3p is located in an intron of the HOXC5 gene, a member of the highly conserved homeobox family of transcription factors controlling embryogenesis and development. Unexpectedly, we found that HoxC5 also represses hTERT expression by disrupting the long-range interaction between hTERT promoter and its distal enhancer. The 3'UTR of hTERT and its upstream enhancer region are well conserved in long-lived primates. Both mir-615-3p and HOXC5 are activated upon differentiation, which constitute a feed-forward loop that coordinates transcriptional and post-transcriptional repression of hTERT during cellular differentiation. Deregulation of HOXC5 and mir-615-3p expression may contribute to the activation of hTERT in human cancers.

Genome and transcriptome delineation of two major oncogenic pathways governing invasive ductal breast cancer development.

Invasive ductal carcinoma (IDC) is a major histo-morphologic type of breast cancer. Histological grading (HG) of IDC is widely adopted by oncologists as a prognostic factor. However, HG evaluation is highly subjective with only 50%-85% inter-observer agreements. Specifically, the subjectivity in the assignment of the intermediate grade (histologic grade 2, HG2) breast cancers (comprising ~50% of IDC cases) results in uncertain disease outcome prediction and sub-optimal systemic therapy. Despite several attempts to identify the mechanisms underlying the HG classification, their molecular bases are poorly understood.We performed integrative bioinformatics analysis of TCGA and several other cohorts (total 1246 patients). We identified a 22-gene tumor aggressiveness grading classifier (22g-TAG) that reflects global bifurcation in the IDC transcriptomes and reclassified patients with HG2 tumors into two genetically and clinically distinct subclasses: histological grade 1-like (HG1-like) and histological grade 3-like (HG3-like). The expression profiles and clinical outcomes of these subclasses were similar to the HG1 and HG3 tumors, respectively. We further reclassified IDC into low genetic grade (LGG = HG1+HG1-like) and high genetic grade (HGG = HG3-like+HG3) subclasses. For the HG1-like and HG3-like IDCs we found subclass-specific DNA alterations, somatic mutations, oncogenic pathways, cell cycle/mitosis and stem cell-like expression signatures that discriminate between these tumors. We found similar molecular patterns in the LGG and HGG tumor classes respectively.Our results suggest the existence of two genetically-predefined IDC classes, LGG and HGG, driven by distinct oncogenic pathways. They provide novel prognostic and therapeutic biomarkers and could open unique opportunities for personalized systemic therapies of IDC patients.