Chronic loss of STAG2 leads to altered chromatin structure contributing to de-regulated transcription in AML.

BACKGROUND: The cohesin complex plays a major role in folding the human genome into 3D structural domains. Mutations in members of the cohesin complex are known early drivers of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), with STAG2 the most frequently mutated complex member. METHODS: Here we use functional genomics (RNA-seq, ChIP-seq and HiChIP) to investigate the impact of chronic STAG2 loss on three-dimensional genome structure and transcriptional programming in a clinically relevant model of chronic STAG2 loss. RESULTS: The chronic loss of STAG2 led to loss of smaller loop domains and the maintenance/formation of large domains that, in turn, led to altered genome compartmentalisation. These changes in genome structure resulted in altered gene expression, including deregulation of the HOXA locus and the MAPK signalling pathway, resulting in increased sensitivity to MEK inhibition. CONCLUSIONS: The altered genomic architecture driven by the chronic loss of STAG2 results in altered gene expression that may contribute to leukaemogenesis and may be therapeutically targeted.

Post-diagnostic antipsychotic use and cancer mortality: a population based cohort study.

BACKGROUND: Many antipsychotics elevate prolactin, a hormone implicated in breast cancer aetiology however no studies have investigated antipsychotic use in patients with breast cancer. This study investigated if antipsychotic use is associated with an increased risk of cancer-specific mortality among breast cancer patients. METHODS: A cohort of 23,695 women newly diagnosed with a primary breast cancer between 1st January 1998 and 31st December 2012 was identified from the UK Clinical Practice Research Datalink linked to English cancer-registries and followed for until 30th September 2015. Time-dependent Cox proportional hazards models were used to calculate adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) of breast cancer-specific mortality comparing use of antipsychotics with non-use, overall, and by prolactin elevating activitiy. Analyses were repeated restricting to patients with a history of severe mental illness to control for potential confounding by indication. RESULTS: In total 848 patients were prescribed an antipsychotic and of which 162 died due to their breast cancer. Compared with non-use, antipsychotic use was associated with an increased risk of breast-cancer specific mortality (HR 2.25, 95%CI 1.90-2.67), but this did not follow a dose response relation. Restricting the cohort to patients with severe mental illness attenuated the association between antipsychotic use and breast cancer-specific mortality (HR 1.11, 95%CI 0.58-2.14). CONCLUSIONS: In this population-based cohort of breast cancer patients, while the use of antipsychotics was associated with increased breast cancer-specific mortality, there was a lack of a dose response, and importantly null associations were observed in patients with severe mental illness, suggesting the observed association is likely a result of confounding by indication. This study provides an exemplar of confounding by indication, highlighting the importance of consideration of this important bias in studies of drug effects in cancer patients.

The RNA processing factors THRAP3 and BCLAF1 promote the DNA damage response through selective mRNA splicing and nuclear export.

mRNA splicing and export plays a key role in the regulation of gene expression, with recent evidence suggesting an additional layer of regulation of gene expression and cellular function through the selective splicing and export of genes within specific pathways. Here we describe a role for the RNA processing factors THRAP3 and BCLAF1 in the regulation of the cellular DNA damage response (DDR) pathway, a key pathway involved in the maintenance of genomic stability and the prevention of oncogenic transformation. We show that loss of THRAP3 and/or BCLAF1 leads to sensitivity to DNA damaging agents, defective DNA repair and genomic instability. Additionally, we demonstrate that this phenotype can be at least partially explained by the role of THRAP3 and BCLAF1 in the selective mRNA splicing and export of transcripts encoding key DDR proteins, including the ATM kinase. Moreover, we show that cancer associated mutations within THRAP3 result in deregulated processing of THRAP3/BCLAF1-regulated transcripts and consequently defective DNA repair. Taken together, these results suggest that THRAP3 and BCLAF1 mutant tumors may be promising targets for DNA damaging chemotherapy.

Selective serotonin reuptake inhibitor use and breast cancer survival: a population-based cohort study.

BACKGROUND: Nearly 50% of breast cancer patients suffer from depression or anxiety. Selective serotonin reuptake inhibitors (SSRIs), the first-line pharmacological treatment for depression, have been implicated in breast cancer development through increased prolactin levels and tamoxifen metabolism inhibition. Previous studies of breast cancer progression have focused on tamoxifen users, or have been limited by their small sample size and methodology. Therefore, we used UK population-based data to more robustly investigate the association between SSRI use and cancer-specific mortality. METHODS: A cohort of patients with newly-diagnosed breast cancer between 1998 and 2012 was selected from English cancer registries and linked to prescription records from the Clinical Practice Research Datalink, and to death records from the Office for National Statistics. We used Cox regression models to calculate hazard ratios (HRs) comparing mortality between post-diagnostic SSRI users and non-users (using time-dependant covariates), after adjusting for demographics, comorbidities and pre-diagnosis use of hormone replacement therapy or oral contraceptives. We conducted several additional analyses to assess causality. RESULTS: Our cohort included 23,669 breast cancer patients, of which 2672 used SSRIs and 3053 died due to their breast cancer during follow-up. After adjustment, SSRI users had higher breast cancer-specific mortality than non-users (HR = 1.27; 95% confidence interval (CI) 1.16, 1.40). However, this association was attenuated when restricting to patients with a prior history of depression (HR = 1.14; 95% CI 0.98, 1.33), and when comparing to users of other antidepressant medications (HR = 1.06; 95% CI 0.93, 1.20). There was some evidence of higher mortality among long-term SSRI users, even when restricting to patients with prior depression (HR = 1.54; 95% CI 1.03, 2.29). CONCLUSIONS: In this large breast cancer cohort, SSRI use was associated with a 27% increase in breast cancer mortality. The cause of this is unknown; however, confounding by indication seems likely as it was largely attenuated when restricting to patients with prior depression, or when comparing SSRIs to other antidepressant medications. Clinicians should not be unduly concerned when prescribing SSRIs to breast cancer patients, but the increase in mortality among long-term SSRI users warrants further investigation.

Postdiagnostic Calcium Channel Blocker Use and Breast Cancer Mortality: A Population-based Cohort Study.

BACKGROUND: There have long been concerns that calcium channel blockers (CCBs), widely used to treat hypertension, may contribute to malignant growth through the evasion of apoptosis and proliferation of cancer cells. Worryingly, a recent cohort study found breast cancer patients who used CCBs had higher death rates, but interpreting these results was difficult as they were based on all-cause mortality and medication use before cancer diagnosis. We used UK population-based data to more robustly investigate the association between CCB use and cancer-specific mortality. METHODS: We selected a cohort of patients with breast cancer diagnosed between 1998 and 2012 from English cancer registries. We linked to prescription and clinical records from the Clinical Practice Research Datalink, and to death records from the Office for National Statistics. We used adjusted, time-dependent Cox regression models to calculate hazard ratios (HRs) comparing breast cancer-specific and all-cause mortality between postdiagnostic CCB users and nonusers. RESULTS: Our cohort included 23,669 breast cancer patients, of whom 5,141 used CCBs and 3,053 died due to their breast cancer during follow-up. After adjustment, CCB users had similar breast cancer-specific mortality to nonusers (HR = 0.98, 95% confidence interval [CI] = 0.88, 1.08). There was no evidence of a dose-response relationship. We found similar associations for specific CCBs, and for all-cause mortality. CONCLUSIONS: In this large population-based breast cancer cohort, we did not find any evidence that CCB use is associated with increased mortality.

A combined connectivity mapping and pharmacoepidemiology approach to identify existing medications with breast cancer causing or preventing properties.

PURPOSE: We applied a novel combined connectivity mapping and pharmacoepidemiological approach to identify medications that alter breast cancer risk. METHODS: The connectivity mapping process identified 6 potentially cancer-causing (meloxicam, azithromycin, rizatriptan, citalopram, rosiglitazone, and verapamil) and 4 potentially cancer-preventing (bendroflumethiazide, sertraline, fluvastatin, and budesonide) medications that were suitable for pharmacoepidemiological investigation. Within the UK Clinical Practice Research Datalink, we matched 45,147 breast cancer cases to 45,147 controls based on age, year, and general practice. Medication use was determined from electronic prescribing records. We used conditional logistic regression to calculate odds ratios (ORs) for the association between medication use and cancer risk after adjustment for comorbidities, lifestyle factors, deprivation, and other medication use. RESULTS: Bendroflumethiazide was associated with increased breast cancer risk (OR: 1.11; 95% CI: 1.06, 1.15); however the connectivity mapping exercise predicted that this medication would reduce risk. There were no statistically significant associations for any of the other candidate medications, with ever use ORs ranging from 0.93 (95% CI: 0.78, 1.11) for azithromycin to 1.16 (95% CI: 0.99, 1.37) for verapamil. CONCLUSIONS: In this instance, our combined connectivity mapping and pharmacoepidemiological approach did not identify any additional medications that were substantially associated with breast cancer risk. This could be due to limitations in the connectivity mapping, such as implausible dosage requirements, or the pharmacoepidemiology, such as residual confounding.

An integrated meta-analysis approach to identifying medications with potential to alter breast cancer risk through connectivity mapping.

BACKGROUND: Gene expression connectivity mapping has gained much popularity in recent years with a number of successful applications in biomedical research testifying its utility and promise. A major application of connectivity mapping is the identification of small molecule compounds capable of inhibiting a disease state. In this study, we are additionally interested in small molecule compounds that may enhance a disease state or increase the risk of developing that disease. Using breast cancer as a case study, we aim to develop and test a methodology for identifying commonly prescribed drugs that may have a suppressing or inducing effect on the target disease (breast cancer). RESULTS: We obtained from public data repositories a collection of breast cancer gene expression datasets with over 7000 patients. An integrated meta-analysis approach to gene expression connectivity mapping was developed, which involved unified processing and normalization of raw gene expression data, systematic removal of batch effects, and multiple runs of balanced sampling for differential expression analysis. Differentially expressed genes stringently selected were used to construct multiple non-joint gene signatures representing the same biological state. Remarkably these non-joint gene signatures retrieved from connectivity mapping separate lists of candidate drugs with significant overlaps, providing high confidence in their predicted effects on breast cancers. Of particular note, among the top 26 compounds identified as inversely connected to the breast cancer gene signatures, 14 of them are known anti-cancer drugs. CONCLUSIONS: A few candidate drugs with potential to enhance breast cancer or increase the risk of the disease were also identified; further investigation on a large population is required to firmly establish their effects on breast cancer risks. This work thus provides a novel approach and an applicable example for identifying medications with potential to alter cancer risks through gene expression connectivity mapping.

Repurposing medicinal compounds for blood cancer treatment.

Drug development is being continuously scrutinised for its lack of productivity. Novel drug development is associated with high costs, high failure rates and lengthy development process. These downfalls combined with a huge demand in blood cancer for new therapeutic treatments have led many to consider the method of drug repurposing. Finding new therapeutic indications for already established drug substances is known as redirecting, repositioning, reprofiling, or repurposing of drugs. Off-patent and on-patent drugs can be screened for additional targets and new indications thus bringing them to clinical trials at a faster pace. This approach offers smaller research groups, such as those that are academic based, into the drug development industry. Drug repurposing can make use of previously published data concerning dosage, toxicology and mechanism of activity.

HOXA/PBX3 knockdown impairs growth and sensitizes cytogenetically normal acute myeloid leukemia cells to chemotherapy.

The cytogenetically normal subtype of acute myeloid leukemia is associated with an intermediate risk which complicates therapeutic options. Lower overall HOX/TALE expression appears to correlate with more favorable prognosis/better response to treatment in some leukemias and solid cancer. The functional significance of the associated gene expression and response to chemotherapy is not known. Three independent microarray datasets obtained from large cohorts of patients along with quantitative polymerase chain reaction validation were used to identify a four-gene HOXA/TALE signature capable of prognostic stratification. Biochemical analysis was used to identify interactions between the four encoded proteins and targeted knockdown used to examine the functional importance of sustained expression of the signature in leukemia maintenance and response to chemotherapy. An 11 HOXA/TALE code identified in an intermediate-risk group of patients (n=315) compared to a group with a favorable risk (n=105) was reduced to a four-gene signature of HOXA6, HOXA9, PBX3 and MEIS1 by iterative analysis of independent platforms. This signature maintained the favorable/intermediate risk partition and where applicable, correlated with overall survival in cytogenetically normal acute myeloid leukemia. We further showed that cell growth and function are dependent on maintained levels of these core genes and that direct targeting of HOXA/PBX3 sensitizes cytogenetically normal acute myeloid leukemia cells to standard chemotherapy. Together the data support a key role for HOXA/TALE in cytogenetically normal acute myeloid leukemia and demonstrate that targeting of clinically significant HOXA/PBX3 elements may provide therapeutic benefit to patients with this subtype of leukemia.

Prediction of prime editing insertion efficiencies using sequence features and DNA repair determinants.

Most short sequences can be precisely written into a selected genomic target using prime editing; however, it remains unclear what factors govern insertion. We design a library of 3,604 sequences of various lengths and measure the frequency of their insertion into four genomic sites in three human cell lines, using different prime editor systems in varying DNA repair contexts. We find that length, nucleotide composition and secondary structure of the insertion sequence all affect insertion rates. We also discover that the 3' flap nucleases TREX1 and TREX2 suppress the insertion of longer sequences. Combining the sequence and repair features into a machine learning model, we can predict relative frequency of insertions into a site with R = 0.70. Finally, we demonstrate how our accurate prediction and user-friendly software help choose codon variants of common fusion tags that insert at high efficiency, and provide a catalog of empirically determined insertion rates for over a hundred useful sequences.

Cancer-Associated SF3B1 Mutations Confer a BRCA-Like Cellular Phenotype and Synthetic Lethality to PARP Inhibitors.

Mutations in SF3B1 have been identified across several cancer types. This key spliceosome component promotes the efficient mRNA splicing of thousands of genes including those with crucial roles in the cellular response to DNA damage. Here, we demonstrate that depletion of SF3B1 specifically compromises homologous recombination (HR) and is epistatic with loss of BRCA1. More importantly, the most prevalent cancer-associated mutation in SF3B1, K700E, also affects HR efficiency and as a consequence, increases the cellular sensitivity to ionizing radiation and a variety of chemotherapeutic agents, including PARP inhibitors. In addition, the SF3B1 K700E mutation induced unscheduled R-loop formation, replication fork stalling, increased fork degradation, and defective replication fork restart. Taken together, these data suggest that tumor-associated mutations in SF3B1 induce a BRCA-like cellular phenotype that confers synthetic lethality to DNA-damaging agents and PARP inhibitors, which can be exploited therapeutically. SIGNIFICANCE: The cancer-associated SF3B1K700E mutation induces DNA damage via generation of genotoxic R-loops and stalled replication forks, defective homologous recombination, and increased replication fork degradation, which can be targeted with PARP inhibitors.

Biomolecular condensation of NUP98 fusion proteins drives leukemogenic gene expression.

NUP98 fusion proteins cause leukemia via unknown molecular mechanisms. All NUP98 fusion proteins share an intrinsically disordered region (IDR) in the NUP98 N terminus, featuring repeats of phenylalanine-glycine (FG), and C-terminal fusion partners often function in gene control. We investigated whether mechanisms of oncogenic transformation by NUP98 fusion proteins are hardwired in their protein interactomes. Affinity purification coupled to mass spectrometry (MS) and confocal imaging of five NUP98 fusion proteins expressed in human leukemia cells revealed that shared interactors were enriched for proteins involved in biomolecular condensation and that they colocalized with NUP98 fusion proteins in nuclear puncta. We developed biotinylated isoxazole-mediated condensome MS (biCon-MS) to show that NUP98 fusion proteins alter the global composition of biomolecular condensates. An artificial FG-repeat-containing fusion protein phenocopied the nuclear localization patterns of NUP98 fusion proteins and their capability to drive oncogenic gene expression programs. Thus, we propose that IDR-containing fusion proteins combine biomolecular condensation with transcriptional control to induce cancer.

Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8.

BACKGROUND: Activation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded. METHODS: We carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation. RESULTS: Here we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced. CONCLUSIONS: Expression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway.

The histone deacetylase inhibitor Romidepsin induces as a cascade of differential gene expression and altered histone H3K9 marks in myeloid leukaemia cells.

Myelodysplastic syndromes (MDS) are a heterogeneous, clonal haematopoietic disorder, with ~1/3 of patients progressing to acute myeloid leukaemia (AML). Many elderly MDS patients do not tolerate intensive therapeutic regimens, and therefore have an unmet need for better tolerated therapies. Epigenetics is important in the pathogenesis of MDS/AML with DNA methylation, and histone acetylation the most widely studied modifications. Epigenetic therapeutic agents have targeted the reversible nature of these modifications with some clinical success. The aim of this study was to characterise the molecular consequences of treatment of MDS and AML cells with the histone deacetylase inhibitor (HDACi) Romidepsin. Romidepsin as a single agent induced cell death with an increasing dose and time profile associated with increased acetylation of histone H3 lysine 9 (H3K9) and decreased HDAC activity. Gene expression profiling, qPCR, network and pathway analysis recognised that oxidation-reduction was involved in response to Romidepsin. ROS was implicated as being involved post-treatment with the involvement of TSPO and MPO. Genomic analysis uncoupled the differences in protein-DNA interactions and gene regulation. The spatial and temporal transcriptional differences associated with acetylated, mono- and tri-methylated H3K9, representative of two activation and a repression mark respectively, were identified. Bioinformatic analysis uncovered positional enrichment and transcriptional differences between these marks; a degree of overlap with increased/decreased gene expression that correlates to increased/decreased histone modification. Overall, this study has unveiled a number of underlying mechanisms of the HDACi Romidepsin that could identify potential drug combinations for use in the clinic.

Altered splicing and cytoplasmic levels of tRNA synthetases in SF3B1-mutant myelodysplastic syndromes as a therapeutic vulnerability.

Myelodysplastic syndromes (MDS) are haematopoietic malignancies that are characterised by a heterogeneous clinical course. In recent years, sequencing efforts have uncovered recurrent somatic mutations within RNA splicing factors, including SF3B1, SRSF2, U2AF1 and ZRSR2. The most frequently mutated gene is SF3B1, mutated in 17% of MDS patients. While SF3B1 mutations and their effects on splicing have been well characterised, much remains to be explored about their more far-reaching effects on cellular homeostasis. Given that mRNA splicing and nuclear export are coordinated processes, we hypothesised that SF3B1 mutation might also affect export of certain mRNAs and that this may represent a targetable pathway for the treatment of SF3B1-mutant MDS. We used CRISPR/Cas9-genome editing to create isogenic cellular models. Comprehensive transcriptome and proteome profiling of these cells identified alterations in the splicing and export of components of the translational machinery, primarily tRNA synthetases, in response to the SF3B1 K700E mutation. While steady-state protein synthesis was unaffected, SF3B1 mutant cells were more sensitive to the clinically-relevant purine analogue, 8-azaguanine. In this study, we also demonstrated that 8-azaguanine affects splicing. Our results suggest that the simultaneous targeting of RNA metabolism and splicing by 8-azaguanine represents a therapeutic opportunity for SF3B1-mutant myelodysplastic syndromes.

Integrated analysis of the molecular action of Vorinostat identifies epi-sensitised targets for combination therapy.

Several histone deacetylase inhibitors including Vorinostat have received FDA approval for the treatment of haematological malignancies. However, data from these trials indicate that Vorinostat has limited efficacy as a monotherapy, prompting the need for rational design of combination therapies. A number of epi-sensitised pathways, including sonic hedgehog (SHH), were identified in AML cells by integration of global patterns of histone H3 lysine 9 (H3K9) acetylation with transcriptomic analysis following Vorinostat-treatment. Direct targeting of the SHH pathway with SANT-1, following Vorinostat induced epi-sensitisation, resulted in synergistic cell death of AML cells. In addition, xenograft studies demonstrated that combination therapy induced a marked reduction in leukemic burden compared to control or single agents. Together, the data supports epi-sensitisation as a potential component of the strategy for the rational development of combination therapies in AML.

Identification and validation of the dopamine agonist bromocriptine as a novel therapy for high-risk myelodysplastic syndromes and secondary acute myeloid leukemia.

Myelodysplastic syndromes (MDS) represent a broad spectrum of diseases characterized by their clinical manifestation as one or more cytopenias, or a reduction in circulating blood cells. MDS is predominantly a disease of the elderly, with a median age in the UK of around 75. Approximately one third of MDS patients will develop secondary acute myeloid leukemia (sAML) that has a very poor prognosis. Unfortunately, most standard cytotoxic agents are often too toxic for older patients. This means there is a pressing unmet need for novel therapies that have fewer side effects to assist this vulnerable group. This challenge was tackled using bioinformatic analysis of available transcriptomic data to establish a gene-based signature of the development and progression of MDS. This signature was then used to identify novel therapeutic compounds via statistically-significant connectivity mapping. This approach suggested re-purposing an existing and widely-prescribed drug, bromocriptine as a novel potential therapy in these disease settings. This drug has shown selectivity for leukemic cells as well as synergy with current therapies.

Rational drug repurposing using sscMap analysis in a HOX-TALE model of leukemia.

Drug discovery and development are often hampered by lack of target identification and clinical tractability. Repurposing of approved drugs to life-threatening diseases such as leukemia is emerging as a promising alternative approach. Connectivity mapping systems link approved drugs with disease-related gene signatures. Relevant preclinical models provide essential tools for system validation and proof-of-concept studies. Herein we describe procedures aimed at generating disease-based gene signatures and applying them to established cross-referencing databases of potential candidate drugs. As a proof of principle, we present the identification of Entinostat as a candidate drug for the treatment of HOX-TALE-related leukemia.