RNA Splicing Alterations Induce a Cellular Stress Response Associated with Poor

Author Manuscript Published OnlineFirst on March 2, 2020; DOI: 10.1158/1078-0432.CCR-20-0184 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Anande et al – revised manuscript RNA splicing alterations in AML

RNA splicing alterations induce a cellular stress response associated with poor

prognosis in Acute Myeloid Leukemia

Running title: RNA splicing alterations in AML

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#* & John E. Pimanda1,10,11#*

1Adult Cancer Program, Lowy Cancer Research Centre & Prince of Wales Clinical

School, UNSW Sydney, NSW, Australia

2School of Biotechnology and Biomolecular Sciences, UNSW Sydney, NSW,

Australia

3Hematology Centre, Karolinska University Hospital and Department of Medicine,

Karolinska Institutet, Huddinge, Stockholm, Sweden

4Institute of Medical Data Processing, Biometrics and Epidemiology, Faculty of

Medicine, LMU Munich, Munich, Germany

5Data Integration for Future Medicine, LMU Munich, Munich, Germany

6Department of Medicine III, University Hospital, LMU Munich, Munich, Germany

7Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Zentrum

München, German Research Center for Environmental Health, Munich, Germany

8Department of Medical Sciences, Uppsala University, Uppsala, Sweden

9School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of

Hong Kong, Hong Kong

Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 2, 2020; DOI: 10.1158/1078-0432.CCR-20-0184 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Anande et al – revised manuscript RNA splicing alterations in AML

10Department of Pathology, School of Medical Sciences, UNSW Sydney, NSW,

Australia

11Department of Haematology, Prince of Wales Hospital, Sydney, NSW, Australia

#Joint senior authors

*correspondences to be addressed to: [email protected] (A.U.) or

[email protected] (J.P.)

Conflict of Interest Disclosures:

The authors declare no potential conflicts of interests.

Anande et al – revised manuscript RNA splicing alterations in AML

Key Points

 Widespread and recurrent alternative splicing differences exist between AML patients with good or poor prognosis

 Missplicing of RNA splicing factors leads to altered splicing of their target transcripts

 Aberrant splicing of protein translation genes triggers the induction of an integrated stress response and concomitant inflammatory response

 Alternative RNA splicing information can be used to improve the accuracy of existing prognostic algorithms in AML

 The addition of the splicing signature accurately classifies ELNInt AML patients, effectively converting a three-group classification system into a two- group one, facilitating better treatment decisions to be made.

Translational Relevance

Utilising cytogenetic and mutational information, the European LeukemiaNet (ELN)

algorithm is the clinical standard for prognosis in AML. However, there is

considerable room for improvement, especially in patients classified as intermediate-

risk for whom treatment is challenging. The 4-gene splicing signature that we have

discovered improves the accuracy of classification, converting the existing three-

group risk classification (favorable, intermediate and adverse-risk) into essentially

two groups with significantly different overall survival. This will facilitate improved

treatment decisions to be made for patients.

Our findings also reveal new molecular vulnerabilities that can be potential drug

targets for the treatment of AML, a disease that currently has poor overall outcomes

(<30% five-year survival rate). Direct pharmacological inhibition of splicing factors is

potentially challenging clinically due to the toxicity of the drugs. Our data suggests

that targeting integrated stress response or pathways stimulated as a consequence of

missplicing in leukemic cells could be an alternative approach.

Anande et al – revised manuscript RNA splicing alterations in AML

Abstract

Purpose: RNA splicing is a fundamental biological process that generates protein

diversity from a finite set of genes. Recurrent somatic mutations of splicing factor

genes are common in some hematological cancers but are relatively uncommon in

Acute Myeloid Leukemia (AML, < 20% of patients). We examined whether RNA

splicing differences exist in AML, even in the absence of splicing factor mutations.

Experimental Design: We developed a bioinformatics pipeline to study alternative

RNA splicing in RNA-seq data from large cohorts of AML patients.

Results: We have identified recurrent differential alternative splicing between

patients with poor and good prognosis. These splicing events occurred even in

patients without any discernible splicing factor mutations. Alternative splicing

recurrently occurred in genes with specific molecular functions, primarily related to

protein translation. Developing tools to predict the functional impact of alternative

splicing on the translated protein, we discovered that ~45% of the splicing events

directly affected highly conserved protein domains. Several splicing factors were

themselves misspliced and the splicing of their target transcripts were altered.

Studying differential gene expression in the same patients, we identified that

alternative splicing of protein translation genes in ELNAdv patients resulted in the

induction of an integrated stress response and up-regulation of inflammation-related

genes. Lastly, using machine learning techniques, we identified a splicing signature of

four genes which refine the accuracy of existing risk prognosis schemes and validated

it in a completely independent cohort.

Anande et al – revised manuscript RNA splicing alterations in AML

Conclusions: Our discoveries therefore identify aberrant alternative splicing as a

molecular feature of adverse AML with clinical relevance.