Fabio Liberante

Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1 -mutant myelodysplastic syndromes

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3′ splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3′ splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

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

BackgroundActivation 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.MethodsWe 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.ResultsHere 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.ConclusionsExpression 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.

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.

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

Abstract 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.

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.

High and low, but not intermediate, PRAME expression levels are poor prognostic markers in myelodysplastic syndrome at disease presentation

This work was supported in part by Wessex Medical Research and a Wellcome Value In People award (BG), Northern Ireland Leukaemia Research Fund (NILRF) and the NI Department of Education and Learning (FL) and Leukaemia and Lymphoma Research (AP and JB).

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.

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

BackgroundGene 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).ResultsWe 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.ConclusionsA 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.

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

We applied a novel combined connectivity mapping and pharmacoepidemiological approach to identify medications that alter breast cancer risk.

Post-diagnostic calcium channel blocker use and breast cancer mortality: a population-based cohort study: 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.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.