Dissecting the impact of bromodomain inhibitors on the Interferon Regulatory Factor 4-MYC oncogenic axis in multiple myeloma.

B-cell progenitor fate determinant interferon regulatory factor 4 (IRF4) exerts key roles in the pathogenesis and progression of multiple myeloma (MM), a currently incurable plasma cell malignancy. Aberrant expression of IRF4 and the establishment of a positive auto-regulatory loop with oncogene MYC, drives a MM specific gene-expression program leading to the abnormal expansion of malignant immature plasma cells. Targeting the IRF4-MYC oncogenic loop has the potential to provide a selective and effective therapy for MM. Here we evaluate the use of bromodomain inhibitors to target the IRF4-MYC axis through combined inhibition of their known epigenetic regulators, BRD4 and CBP/EP300. Although all inhibitors induced cell death, we found no synergistic effect of targeting both of these regulators on the viability of MM cell-lines. Importantly, for all inhibitors over a time period up to 72 h, we detected reduced IRF4 mRNA, but a limited decrease in IRF4 protein expression or mRNA levels of downstream target genes. This indicates that inhibitor-induced loss of cell viability is not mediated through reduced IRF4 protein expression, as previously proposed. Further analysis revealed a long half-life of IRF4 protein in MM cells. In support of our experimental observations, gene network modeling of MM suggests that bromodomain inhibition is exerted primarily through MYC and not IRF4. These findings suggest that despite the autofeedback positive regulatory loop between IRF4 and MYC, bromodomain inhibitors are not effective at targeting IRF4 in MM and that novel therapeutic strategies should focus on the direct inhibition or degradation of IRF4.

Thrombocytopenia: the good, the bad and the ugly.

New thrombocytopenia may be associated with a variety of conditions and diagnosis can be challenging. Presentation can vary from life-threatening bleeding or thrombosis to an incidental finding in an asymptomatic patient. New thrombocytopenia requires urgent investigation. Investigations are mainly guided by findings from the clinical history, physical examination, full blood count and blood film analysis. Aside from the actively bleeding patient, rare but life-threatening causes of thrombocytopenia must be identified early as they require urgent treatment. These include thrombotic thrombocytopenic purpura, disseminated intravascular coagulation, suspicion of new acute promyelocytic leukaemia, and vaccine-induced prothrombotic immune thrombocytopenia. Here, we discuss how to approach a patient with new thrombocytopenia, along with key differentials not to be missed.

Lymphocytosis and chronic lymphocytic leukaemia: investigation and management.

Lymphocytosis is a common blood-test finding. Establishing whether the cause of lymphocytosis is benign or malignant is key to managing patients appropriately. A lymphocytosis should always prompt clinical review including a thorough history, examination and appropriate preliminary investigations (blood tests, blood film). The majority of patients with chronic lymphocytic leukaemia (CLL) present incidentally due to a lymphocytosis found on routine blood tests. Patient outcomes vary considerably based on genetic pre-disposition and various prognostic markers (age, Binet or Rai staging, and B2-microglobulin). Although not curative, chemo-immunotherapy is an effective treatment strategy for the majority of CLL patients with progressive disease. More recently, novel oral therapies have been developed that target key signalling and apoptosis pathways and that are being used in relapse settings and as first-line treatments for certain patients.

Acute leukaemia: no reason to panic.

Acute leukaemia is characterised by uncontrolled expansion of immature leukocytes, either myeloid or lymphoid progenitors, leading to acute myeloid leukaemia (AML) and acute lymphoid leukaemia (ALL), respectively. If left untreated, it is life-threatening and can lead to death within weeks. When acute leukaemia is suspected, urgent haematology input should be sought. Appropriate investigations are needed promptly to confirm diagnosis and start treatment. A multidisciplinary approach is vital to ensure appropriate management.

Randomised open-label trial comparing intravenous iron and an erythropoiesis-stimulating agent versus oral iron to treat preoperative anaemia in cardiac surgery (INITIATE trial).

BACKGROUND: Preoperative anaemia is a risk factor for adverse postoperative outcomes after cardiac surgery. Iron deficiency is a frequent cause of low preoperative haemoglobin. An effective treatment for preoperative anaemia associated with iron deficiency has not been determined. METHODS: We conducted a single-centre, open-label, pragmatic randomised trial, enrolling 156 elective cardiac surgery patients who had low preoperative haemoglobin (100-130 g L-1) with iron deficiency (serum ferritin <100 µg L-1 or transferrin saturation <30%) to compare intravenous ferric derisomaltose 1000 mg and darbepoetin 200 µg subcutaneously (intervention group) with oral ferrous sulphate 600 mg daily (control group). The primary outcome was transfusion of at least one unit of allogeneic red cells during surgery and within the following 5 days. Secondary outcomes included the change in haemoglobin concentration between randomisation and surgery, red cell transfusion volume, postoperative blood loss, pre-specified postoperative complications, length of hospital stay, and in-hospital death. RESULTS: The odds of red cell transfusion were lower in the intervention group compared with the control group (adjusted odds ratio=0.33; 95% confidence interval [CI], 0.15-0.75; P=0.008). Of the secondary outcomes, the only significant difference was the increase in haemoglobin between randomisation and surgery, intervention vs control 9.5 g L-1 (95% CI, 6.8-12.2; P<0.001). CONCLUSIONS: In patients with a low preoperative haemoglobin and iron deficiency, preoperative treatment with a single dose of ferric derisomaltose and darbepoetin decreased the proportion of participants who received a perioperative blood transfusion as a consequence of a greater increase in haemoglobin compared with treatment with oral ferrous sulphate. CLINICAL TRIAL REGISTRATION: ISRCTN Number: 41421863; EUDRACT number: 2011-003695-36.

Management of adverse events in patients with acute myeloid leukemia in remission receiving oral azacitidine: experience from the phase 3 randomized QUAZAR AML-001 trial.

BACKGROUND: Most older patients with acute myeloid leukemia (AML) who attain morphologic remission with intensive chemotherapy (IC) will eventually relapse and post-relapse prognosis is dismal. In the pivotal QUAZAR AML-001 trial, oral azacitidine maintenance therapy significantly prolonged overall survival by 9.9 months (P < 0.001) and relapse-free survival by 5.3 months (P < 0.001) compared with placebo in patients with AML in first remission after IC who were not candidates for transplant. Currently, the QUAZAR AML-001 trial provides the most comprehensive safety information associated with oral azacitidine maintenance therapy. Reviewed here are common adverse events (AEs) during oral azacitidine treatment in QUAZAR AML-001, and practical recommendations for AE management based on guidance from international cancer consortiums, regulatory authorities, and the authors' clinical experience treating patients in the trial. METHODS: QUAZAR AML-001 is an international, placebo-controlled randomized phase 3 study. Patients aged ≥ 55 years with AML and intermediate- or poor-risk cytogenetics at diagnosis, who had attained first complete remission (CR) or CR with incomplete blood count recovery (CRi) within 4 months before study entry, were randomized 1:1 to receive oral azacitidine 300 mg or placebo once-daily for 14 days in repeated 28-day cycles. Safety was assessed in all patients who received ≥ 1 dose of study drug. RESULTS: A total of 469 patients received oral azacitidine (n = 236) or placebo (n = 233). Median age was 68 years. Patients received a median of 12 (range 1-80) oral azacitidine treatment cycles or 6 (1-73) placebo cycles. Gastrointestinal AEs were common and typically low-grade. The most frequent grade 3-4 AEs during oral azacitidine therapy were hematologic events. AEs infrequently required permanent discontinuation of oral azacitidine (13%), suggesting they were effectively managed with use of concomitant medications and oral azacitidine dosing modifications. CONCLUSION: Oral azacitidine maintenance had a generally favorable safety profile. Prophylaxis with antiemetic agents, and blood count monitoring every other week, are recommended for at least the first 2 oral azacitidine treatment cycles, and as needed thereafter. Awareness of the type, onset, and duration of common AEs, and implementation of effective AE management, may maximize treatment adherence and optimize the survival benefits of oral azacitidine AML remission maintenance therapy. Trial registration This trial is registered on clinicaltrials.gov: NCT01757535 as of December 2012.

BRD4-mediated repression of p53 is a target for combination therapy in AML.

Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report enhanced toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi's ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.

The renin-angiotensin system - a therapeutic target in COVID-19?

COVID-19, caused by infection with SARS-CoV-2, is a disease characterised by cough, fever and fatigue, which progresses to life-threatening lung injury in approximately 5% of patients. The SARS-CoV-2 virus enters the cell via ACE2. ACE2 is a component of the renin-angiotensin system (RAS) which has an important counterregulatory effect on the classical ACE-dependent pathway. Several antihypertensives increase ACE2 expression or activity, leading to concern that this may facilitate SARS-CoV-2 entry and worsen COVID-19 disease. However, ACE2 is protective against lung injury while ANG II (which is catabolised by ACE2) is associated with lung injury both in mice and humans. We propose that medications which inhibit the RAS ACE-dependent pathway may be beneficial in treating COVID-19 and should be explored in animal models and clinical trials. Here we give an overview of the RAS pathway with respect to COVID-19 and argue that strategies which manipulate this pathway might reduce the destructive lung manifestations of COVID-19 and improve patient outcomes.

Chronic Lymphocytic Leukaemia in 2020: the Future Has Arrived.

PURPOSE OF REVIEW: Chronic lymphocytic leukaemia is now recognised as a heterogenous disease with a variety of clinical outcomes. Here we summarise the way it is currently stratified according to genetic risk and patient characteristics and the treatment approaches used for these different subgroups. RECENT FINDINGS: Certain patients appear to sustain MRD negativity after combination chemoimmunotherapy, leading to the suggestion that their CLL may be cured. However, 17p-deleted, p53-mutated or IGHV-UM subgroups are generally resistant to FCR, and much better responses are seen with ibrutinib and venetoclax, frequently inducing MRD negativity that hopefully will be translated into durable remissions. Small molecule inhibitors have already revolutionised CLL treatment. Going forward, we anticipate their use in the majority of patients, early after diagnosis and with curative intent.

Dissecting the role of the CXCL12/CXCR4 axis in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is the most common adult acute leukaemia with the lowest survival rate. It is characterised by a build-up of immature myeloid cells anchored in the protective niche of the bone marrow (BM) microenvironment. The CXCL12/CXCR4 axis is central to the pathogenesis of AML as it has fundamental control over AML cell adhesion into the protective BM niche, adaptation to the hypoxic environment, cellular migration and survival. High levels of CXCR4 expression are associated with poor relapse-free and overall survival. The CXCR4 ligand, CXCL12 (SDF-1), is expressed by multiple cells types in the BM, facilitating the adhesion and survival of the malignant clone. Blocking the CXCL12/CXCR4 axis is an attractive therapeutic strategy providing a 'multi-hit' therapy that both prevents essential survival signals and releases the AML cells from the BM into the circulation. Once out of the protective niche of the BM they would be more susceptible to destruction by conventional chemotherapeutic drugs. In this review, we disentangle the diverse roles of the CXCL12/CXCR4 axis in AML. We then describe multiple CXCR4 inhibitors, including small molecules, peptides, or monoclonal antibodies, which have been developed to date and their progress in pre-clinical and clinical trials. Finally, the review leads us to the conclusion that there is a need for further investigation into the development of a 'multi-hit' therapy that targets several signalling pathways related to AML cell adhesion and maintenance in the BM.

Real-world experience of ibrutinib therapy in relapsed chronic lymphocytic leukemia: results of a single-center retrospective analysis.

BACKGROUND: Ibrutinib is a Bruton's tyrosine-kinase (BTK) inhibitor that is approved as a second-line treatment in chronic lymphocytic leukemia (CLL). While recent trials have demonstrated impressive results for ibrutinib, there remains a paucity of real-world data on its use in the clinical setting. METHODS: In this single-center study carried out at Brighton and Sussex University Hospitals, we retrospectively compared outcomes in 38 patients with relapsed CLL who received ibrutinib versus those who received conventional first- and second-line therapies. RESULTS: Our results demonstrate improved progression-free survival (PFS, p=0.022) with ibrutinib versus conventional second-line therapies and survival comparable to conventional first-line therapies. However, there was a high frequency (81.6%) of adverse events associated with ibrutinib therapy, including 2 cases of death secondary to sepsis and a further 7 cases of discontinuation of treatment due to adverse events. We also identify del13q14.3 as an adverse predictor of response to ibrutinib with respect to both overall survival (p=0.014) and PFS (p=0.008), suggesting that these patients may be better suited to receiving the BCL2 inhibitor venetoclax. CONCLUSION: Whilst there is robust evidence for improved outcomes with ibrutinib, we find that survival in patients with del13q14.3 is reduced and that the rate of adverse events and discontinuation in clinical practice is higher than anticipated from clinical trials.

Targeting SHP-1, 2 and SHIP Pathways: A Novel Strategy for Cancer Treatment?

Well-balanced levels of tyrosine phosphorylation, maintained by the reversible and coordinated actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), are critical for a wide range of cellular processes including growth, differentiation, metabolism, migration, and survival. Aberrant tyrosine phosphorylation, as a result of a perturbed balance between the activities of PTKs and PTPs, is linked to the pathogenesis of numerous human diseases, including cancer, suggesting that PTPs may be innovative molecular targets for cancer treatment. Two PTPs that have an important inhibitory role in haematopoietic cells are SHP-1 and SHP-2. SHP-1, 2 promote cell growth and act by both upregulating positive signaling pathways and by downregulating negative signaling pathways. SHIP is another inhibitory phosphatase that is specific for the inositol phospholipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). SHIP acts as a negative regulator of immune response by hydrolysing PIP3, and SHIP deficiency results in myeloproliferation and B-cell lymphoma in mice. The validation of SHP-1, 2 and SHIP as oncology targets has generated interest in the development of inhibitors as potential therapeutic agents for cancers; however, SHP-1, 2 and SHIP have proven to be an extremely difficult target for drug discovery, primarily due to the highly conserved and positively charged nature of their PTP active site, and many PTP inhibitors lack either appro-priate selectivity or membrane permeability. To overcome these caveats, novel techniques have been employed to synthesise new inhibitors that specifically attenuate the PTP-dependent signaling inside the cell and amongst them; some are already in clinical development which are discussed in this review.

BET Inhibition Suppresses S100A8 and S100A9 Expression in Acute Myeloid Leukemia Cells and Synergises with Daunorubicin in Causing Cell Death.

S100A8 and S100A9 are both members of the S100 family and have been shown to play roles in myeloid differentiation, autophagy, apoptosis, and chemotherapy resistance. In this study we demonstrate that the BET-bromodomain inhibitor JQ1 causes rapid suppression of S100A8 and S100A9 mRNA and protein in a reversible manner. In addition, we show that JQ1 synergises with daunorubicin in causing AML cell death. Daunorubicin alone causes a dose- and time-dependent increase in S100A8 and S100A9 protein levels in AML cell lines which is overcome by cotreatment with JQ1. This suggests that JQ1 synergises with daunorubicin in causing apoptosis via suppression of S100A8 and S100A9 levels.

DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers.

DIS3 (defective in sister chromatid joining) is the catalytic subunit of the exosome, a protein complex involved in the 3'-5' degradation of RNAs. DIS3 is a highly conserved exoribonuclease, also known as Rrp44. Global sequencing studies have identified DIS3 as being mutated in a range of cancers, with a considerable incidence in multiple myeloma. In this work, we have identified two protein-coding isoforms of DIS3. Both isoforms are functionally relevant and result from alternative splicing. They differ from each other in the size of their N-terminal PIN (PilT N-terminal) domain, which has been shown to have endoribonuclease activity and tether DIS3 to the exosome. Isoform 1 encodes a full-length PIN domain, whereas the PIN domain of isoform 2 is shorter and is missing a segment with conserved amino acids. We have carried out biochemical activity assays on both isoforms of full-length DIS3 and the isolated PIN domains. We find that isoform 2, despite missing part of the PIN domain, has greater endonuclease activity compared with isoform 1. Examination of the available structural information allows us to provide a hypothesis to explain this altered behaviour. Our results also show that multiple myeloma patient cells and all cancer cell lines tested have higher levels of isoform 1 compared with isoform 2, whereas acute myeloid leukaemia and chronic myelomonocytic leukaemia patient cells and samples from healthy donors have similar levels of isoforms 1 and 2. Taken together, our data indicate that significant changes in the ratios of the two isoforms could be symptomatic of haematological cancers.

Targeting Developmental Pathways: The Achilles Heel of Cancer?

Developmental pathways (e.g., Notch, Hippo, Hedgehog, Wnt, and TGF-ß/BMP/FGF) are networks of genes that act co-ordinately to establish the body plan, and disruptions of genes in one pathway can have effects in related pathways and may result in serious dysmorphogenesis or cancer. Interestingly, all developmental pathways are highly conserved cell signalling systems present in almost all multicellular organisms. In addition, they have a crucial role in cell proliferation, apoptosis, differentiation, and finally in organ development. Of note, almost all of these pathways promote oncogenesis through synergistic associations with the Hippo signalling pathway, and several lines of evidence have also indicated that these pathways (e.g., Wnt/ß-catenin) may be implicated in checkpoint inhibitor resistance (e.g., CTLA-4, PD-1, and PD-L1). Since Notch inhibition in vivo results in partial loss of its stemness features such as self-renewal, chemoresistance, invasive and migratory potential, and tumorigenesis, these highly conserved developmental pathways are regarded as being critical for regulation of self-renewal in both embryonic and adult stem cells and hence are likely to be implicated in the maintenance of cancer stem cells. Many small molecules are currently in preclinical and early clinical development, and only two compounds are approved for treatment of advanced or metastatic basal cell carcinoma (vismodegib and sonidegib). Furthermore, therapeutic targeting of cancer stem cells using drugs that disrupt activated developmental pathways may also represent an attractive strategy that is potentially relevant to many types of malignancy, notably blood cancers, where the evidence for leukaemia stem cells is well established. Future work will hopefully pave the way for the development of new strategies for targeting these pervasive oncogenic pathways.

Use of FDG PET/CT in identification of bone marrow involvement in diffuse large B cell lymphoma and follicular lymphoma: comparison with iliac crest bone marrow biopsy.

Background Non-Hodgkin's lymphoma (NHL) accounts for around 4% of new cancer cases annually. Bone marrow involvement is important for staging and management. Fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) is used increasingly to identify this, in addition to bone marrow biopsy (BMB), which is seen as "gold" reference standard. Purpose To compare determination of bone marrow involvement by FDG PET/CT against BMB in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Material and Methods This was a retrospective study of patients with histologically confirmed NHL at a single UK cancer center undergoing pre-treatment FDG PET/CT and BMB between June 2010 and February 2013. Information was collected from patient notes, cancer registry, histological and imaging reports. Diagnostic accuracy of FDG PET/CT was determined, compared to BMB as the reference standard. Results Twenty-four patients with DLBCL and 12 with FL were included. Five DLBCL patients had bone marrow involvement on PET/CT; all were confirmed on BMB. Three FL patients had marrow involvement on PET/CT but not on BMB; one FL patient had positive BMB but negative PET/CT. Using BMB as the reference standard, the sensitivity and specificity of FDG PET/CT for detecting bone marrow involvement in DLBCL were 100% and 100%, respectively, and in FL were 0% and 72.7%, respectively. Conclusion FDG PET/CT is accurate for detection of bone marrow involvement in newly diagnosed DLBCL, but not FL. In DLBCL, positive FDG PET/CT may negate the need for routine BMB, although BMB in addition or combination may be appropriate if this would influence management or prognosis.

Epigenetic Guardian: A Review of the DNA Methyltransferase DNMT3A in Acute Myeloid Leukaemia and Clonal Haematopoiesis.

Acute myeloid leukaemia (AML) is a haematological malignancy characterized by clonal stem cell proliferation and aberrant block in differentiation. Dysfunction of epigenetic modifiers contributes significantly to the pathogenesis of AML. One frequently mutated gene involved in epigenetic modification is DNMT3A (DNA methyltransferase-3-alpha), a DNA methyltransferase that alters gene expression by de novo methylation of cytosine bases at CpG dinucleotides. Approximately 22% of AML and 36% of cytogenetically normal AML cases carry DNMT3A mutations and around 60% of these mutations affect the R882 codon. These mutations have been associated with poor prognosis and adverse survival outcomes for AML patients. Advances in whole-exome sequencing techniques have recently identified a large number of DNMT3A mutations present in clonal cells in normal elderly individuals with no features of haematological malignancy. Categorically distinct from other preleukaemic conditions, this disorder has been termed clonal haematopoiesis of indeterminate potential (CHIP). Further insight into the mutational landscape of CHIP may illustrate the consequence of particular mutations found in DNMT3A and identify specific "founder" mutations responsible for clonal expansion that may contribute to leukaemogenesis. This review will focus on current research and understanding of DNMT3A mutations in both AML and CHIP.

DOT1L as a therapeutic target for the treatment of DNMT3A-mutant acute myeloid leukemia.

Mutations in DNA methyltransferase 3A (DNMT3A) are common in acute myeloid leukemia and portend a poor prognosis; thus, new therapeutic strategies are needed. The likely mechanism by which DNMT3A loss contributes to leukemogenesis is altered DNA methylation and the attendant gene expression changes; however, our current understanding is incomplete. We observed that murine hematopoietic stem cells (HSCs) in which Dnmt3a had been conditionally deleted markedly overexpress the histone 3 lysine 79 (H3K79) methyltransferase, Dot1l. We demonstrate that Dnmt3a(-/-) HSCs have increased H3K79 methylation relative to wild-type (WT) HSCs, with the greatest increases noted at DNA methylation canyons, which are regions highly enriched for genes dysregulated in leukemia and prone to DNA methylation loss with Dnmt3a deletion. These findings led us to explore DOT1L as a therapeutic target for the treatment of DNMT3A-mutant AML. We show that pharmacologic inhibition of DOT1L resulted in decreased expression of oncogenic canyon-associated genes and led to dose- and time-dependent inhibition of proliferation, induction of apoptosis, cell-cycle arrest, and terminal differentiation in DNMT3A-mutant cell lines in vitro. We show in vivo efficacy of the DOT1L inhibitor EPZ5676 in a nude rat xenograft model of DNMT3A-mutant AML. DOT1L inhibition was also effective against primary patient DNMT3A-mutant AML samples, reducing colony-forming capacity (CFC) and inducing terminal differentiation in vitro. These studies suggest that DOT1L may play a critical role in DNMT3A-mutant leukemia. With pharmacologic inhibitors of DOT1L already in clinical trials, DOT1L could be an immediately actionable therapeutic target for the treatment of this poor prognosis disease.

The 3' to 5' Exoribonuclease DIS3: From Structure and Mechanisms to Biological Functions and Role in Human Disease.

DIS3 is a conserved exoribonuclease and catalytic subunit of the exosome, a protein complex involved in the 3' to 5' degradation and processing of both nuclear and cytoplasmic RNA species. Recently, aberrant expression of DIS3 has been found to be implicated in a range of different cancers. Perhaps most striking is the finding that DIS3 is recurrently mutated in 11% of multiple myeloma patients. Much work has been done to elucidate the structural and biochemical characteristics of DIS3, including the mechanistic details of its role as an effector of RNA decay pathways. Nevertheless, we do not understand how DIS3 mutations can lead to cancer. There are a number of studies that pertain to the function of DIS3 at the organismal level. Mutant phenotypes in S. pombe, S. cerevisiae and Drosophila suggest DIS3 homologues have a common role in cell-cycle progression and microtubule assembly. DIS3 has also recently been implicated in antibody diversification of mouse B-cells. This article aims to review current knowledge of the structure, mechanisms and functions of DIS3 as well as highlighting the genetic patterns observed within myeloma patients, in order to yield insight into the putative role of DIS3 mutations in oncogenesis.