Published OnlineFirst March 2, 2020; DOI: 10.1158/1078-0432.CCR-20-0184 CLINICAL CANCER RESEARCH | PRECISION MEDICINE AND IMAGING RNA Splicing Alterations Induce a Cellular Stress Response Associated with Poor Prognosis in Acute Myeloid Leukemia Govardhan Anande1, Nandan P. Deshpande2, Sylvain Mareschal3, Aarif M. N. Batcha4,5, Henry R. Hampton1, Tobias Herold6,7, Soren Lehmann3,8, Marc R. Wilkins2, Jason W.H. Wong1,9, Ashwin Unnikrishnan1, and John E. Pimanda1,10,11 ABSTRACT ◥ Purpose: RNA splicing is a fundamental biological process that predict the functional impact of alternative splicing on the generates protein diversity from a finite set of genes. Recurrent translated protein, we discovered that approximately 45% somatic mutations of splicing factor genes are common in some of the splicing events directly affected highly conserved protein hematologic cancers but are relatively uncommon in acute myeloid domains. Several splicing factors were themselves misspliced leukemia (AML, < 20% of patients). We examined whether RNA and the splicing of their target transcripts were altered. Studying splicing differences exist in AML, even in the absence of splicing differential gene expression in the same patients, we identified factor mutations. that alternative splicing of protein translation genes in ELNAdv Experimental Design: We developed a bioinformatics pipeline patients resulted in the induction of an integrated stress to study alternative RNA splicing in RNA-sequencing data from response and upregulation of inflammation-related genes. Final- large cohorts of patients with AML. ly, using machine learning techniques, we identified a splicing Results: We have identified recurrent differential alternative signature of four genes which refine the accuracy of existing risk splicing between patients with poor and good prognosis. prognosis schemes and validated it in a completely independent These splicing events occurred even in patients without any cohort. discernible splicing factor mutations. Alternative splicing recur- Conclusions: Our discoveries therefore identify aberrant alter- rently occurred in genes with specific molecular functions, native splicing as a molecular feature of adverse AML with clinical primarily related to protein translation. Developing tools to relevance. Introduction Acute myeloid leukemia (AML) is a hematologic malignancy associated with a poor prognosis and a <30% 5-year survival rate (1). 1Adult Cancer Program, Lowy Cancer Research Centre & Prince of Wales Clinical With an incidence rate of 4 per 100,000 adults per year (2) and a 5-fold School, University of New South Wales Sydney, New South Wales, Australia. higher rate in people over the age of 65, AML represents approximately 2School of Biotechnology and Biomolecular Sciences, University of New South 40% of all new adult-onset leukemias in developed societies (3). AML is Wales Sydney, New South Wales, Australia. 3Hematology Centre, Karolinska characterized by the clonal proliferation of undifferentiated myeloid University Hospital and Department of Medicine, Karolinska Institutet, Hud- precursor cells in the bone marrow and impaired hematopoiesis (4). 4 dinge, Stockholm, Sweden. Institute of Medical Data Processing, Biometrics Patients with AML have recurrent somatic driver mutations (5–7) in and Epidemiology, Faculty of Medicine, LMU Munich, Munich, Germany. 5Data Integration for Future Medicine, LMU Munich, Munich, Germany. 6Department of addition to characteristic cytogenetic and chromosomal abnormali- fi Medicine III, University Hospital, LMU Munich, Munich, Germany. 7Research Unit ties. These alterations have prognostic signi cance and are used to Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum Munchen,€ German classify AML (5). However, not all of these mutations are exclusive to Research Center for Environmental Health, Munich, Germany. 8Department of AML, with many also being detected in myelodysplastic syndrome 9 Medical Sciences, Uppsala University, Uppsala, Sweden. School of Biomedical (MDS; ref. 8) as well as in healthy individuals with age-related clonal Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong hematopoiesis (9). Kong. 10Department of Pathology, School of Medical Sciences, University of New South Wales Sydney, New South Wales, Australia. 11Department of Haematol- The standard-of-care treatment for AML is intensive induction ogy, Prince of Wales Hospital, Sydney, New South Wales, Australia. chemotherapy. However, despite complete remission (CR) rates of >50%, long-term disease-free survival remains poor at <10% and a Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). median overall survival of less than 12 months in patients aged over 60 years (10). In addition, because of significant comorbidities, inten- A. Unnikrishnan and J.E. Pimanda contributed equally as co-senior authors of sive chemotherapy may not suit older patients (11). Alternate therapies this article. for these individuals may include lower intensity treatments, DNA Corresponding Authors: Ashwin Unnikrishnan, UNSW Sydney, Room 217, Level hypomethylating agents (HMA; ref. 12), or targeted therapies. How- 2, C25 Lowy Cancer Research Centre, UNSW Sydney, Sydney, NSW 2052, fi Australia. Phone: 612-9385-8045; E-mail: [email protected]; ever, response rates and survival bene ts still remain poor (13), and John E. Pimanda, [email protected] highlighting an important need to develop new therapeutic options for the management of AML. Clin Cancer Res 2020;XX:XX–XX To develop more effective drugs for AML, it is necessary to better doi: 10.1158/1078-0432.CCR-20-0184 understand the molecular aberrations present in leukemic cells. Aber- Ó2020 American Association for Cancer Research. rations in RNA splicing, a fundamental and highly conserved process AACRJournals.org | OF1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 2, 2020; DOI: 10.1158/1078-0432.CCR-20-0184 Anande et al. RNA-sequencing data analyses Translational Relevance RNA-sequencing (RNA-seq) data were analyzed for multiple types Utilizing cytogenetic and mutational information, the European of alternative splicing using a custom in-house bioinformatics pipeline LeukemiaNet (ELN) algorithm is the clinical standard for prog- incorporating available tools, including Mixture of Isoforms (MISO) to nosis in AML. However, there is considerable room for improve- determine Percent Spliced In (PSI) values in each sample and rMATS ment, especially in patients classified as intermediate-risk for for differential splicing analyses. Differential gene expression analyses whom treatment is challenging. The 4-gene splicing signature that were performed using DESeq2. A custom in-house pipeline was we have discovered improves the accuracy of classification, con- developed to identify possible changes in well-annotated protein verting the existing three-group risk classification (favorable, domains due to differentially spliced events. Full details are provided intermediate, and adverse risk) into essentially two groups with as Supplementary Data. significantly different overall survival. This will facilitate improved treatment decisions to be made for patients. Our findings also Transcript motif analyses reveal new molecular vulnerabilities that can be potential drug Predictions of differential binding of RNA-binding proteins were targets for the treatment of AML, a disease that currently has poor made using rMAPS (29). Maximum entropy modeling was done with overall outcomes (<30% 5-year survival rate). Direct pharmaco- MaxEntScan (30). Full details are provided as Supplementary Data. logic inhibition of splicing factors is potentially challenging clin- ically due to the toxicity of the drugs. Our data suggest that Prognostic model generation targeting integrated stress response or pathways stimulated as a The splicing signature was generated using LASSO Cox Regression consequence of missplicing in leukemic cells could be an alternative with 10-fold cross-validation implemented in glmnet (R package v 2.0- approach. 16). The splicing risk score for each patient was calculated from the regression coefficients. Performances of prognostic models were assessed by Harrel C index. Risk contributions and variable impor- tance of all prognostic models were estimated as described previous- occurring in >95% of multi-exon human genes (14), are increasingly ly (31). Full details are provided as Supplementary Data. being described in many cancers. Pan-cancer studies have begun to reveal that tumors have an average of approximately 20% more alternative splicing events than matched healthy tissues (15, 16). Results Splicing is a cotranscriptional event, orchestrated by cis-acting regu- Identification of differential alternative splicing related to latory elements as well as trans-acting factors of the spliceosomal outcome in patients with AML complex. Dysregulation of the expression of splicing factors (17) and To determine whether RNA splicing alterations might be a factor in upstream signaling pathways (18), as well as genomic mutations in cis- adverse outcomes in AML, we developed a bioinformatics pipeline to splice sites (19) have all been reported in cancers. In addition, in quantify differential alternative splicing in RNA-seq data. We first hematologic malignancies such as MDS and chronic myelomonocytic analyzed AML transcriptomes