POT1 MUTATIONS DRIVE GENOMIC INSTABILITY AND CONFER SYNTHETIC LETHALITY WITH G-QUADRUPLEX STABILIZING AGENTS IN CHRONIC LYMPHOCYTIC LEUKEMIA
Author(s): ,
Saskia Rudat
Affiliations:
Cancer, Ageing and Somatic Mutation,Wellcome Sanger Institute,Hinxton,United Kingdom
,
Guillaume Guilbaud
Affiliations:
Division of Protein & Nucleic Acid Chemistry,Medical Research Council Laboratory of Molecular Biology,Cambridge,United Kingdom
,
Beiyuan Fu
Affiliations:
Molecular Cytogenetics Core Facility,Wellcome Sanger Institute,Hinxton,United Kingdom
,
Andreia Silva
Affiliations:
Cambridge Blood and Stem Cell Biobank (CBSB) and Department of Haematology,University of Cambridge,Cambridge,United Kingdom
,
Mamunur Rashid
Affiliations:
Cancer, Ageing and Somatic Mutation,Wellcome Sanger Institute,Hinxton,United Kingdom
,
Joanna Baxter
Affiliations:
Cambridge Blood and Stem Cell Biobank (CBSB) and Department of Haematology,University of Cambridge,Cambridge,United Kingdom
,
Fengtang Yang
Affiliations:
Molecular Cytogenetics Core Facility,Wellcome Sanger Institute,Cambridge,United Kingdom
,
Julian Sale
Affiliations:
Division of Protein & Nucleic Acid Chemistry,Medical Research Council Laboratory of Molecular Biology,Cambridge,United Kingdom
,
David Adams
Affiliations:
Cancer, Ageing and Somatic Mutation,Wellcome Sanger Institute,Hinxton,United Kingdom
Chi Wong
Affiliations:
Cancer, Ageing and Somatic Mutation,Wellcome Sanger Institute,Hinxton,United Kingdom
EHA Library. Rudat S. Jun 15, 2019; 267478; S895
Saskia Rudat
Saskia Rudat
Contributions
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Abstract

Abstract: S895

Type: Oral Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 16:00 - 16:15

Location: Hall 3B

Background

The fact that chronic lymphocytic leukemia (CLL) is the most common leukemia in adults has been fueling efforts to optimize clinical treatment. Indeed, recent sequencing studies have identified recurrent mutations that hold great promise for novel targeted therapies. One candidate is represented by the POT1 gene, which encodes a member of the shelterin complex involved in telomere maintenance, and whose missense mutations disrupt binding of POT1 proteins to telomeric single-stranded DNA (ssDNA). 

Aims

We focused on POT1 mutations, since they are associated with inferior CLL prognosis, and aimed to understand how mutant POT1 impacts CLL development in vivo and to test the susceptibility of CLL cells to telomere-binding agents.

Methods

We performed exome sequencing of 280 cancer driver genes, including POT1, on 123 CLL patient samples. Furthermore, we generated four knock-in mouse models to recapitulate two CLL-associated mutations (p.Y89C or p.Q94E) in the murine POT1 paralogues, Pot1a and Pot1b.

Results

We determined that POT1 was disrupted in 5.7% of CLL patients, including five unique missense mutations, of which three have never been associated with CLL while two have previously been described, and two splice site mutations. Subsequently, we found that all but one of the missense mutations prevented POT1 from binding to telomeric ssDNA in an electrophoretic mobility shift assay, and that mutant POT1-transduced cells displayed longer telomeres compared to wildtype POT1-transduced cells in a telomere length assay using the telomerase-positive fibrosarcoma cell line HT1080. Similarly, we observed defective telomeric ssDNA binding of mutant Pot1a and Pot1b proteins, however, only Pot1a-mutant murine embryonic fibroblasts demonstrated telomere elongation in fluorescence in situ hybridization experiments and increased numbers of fragile telomeres and chromatid fusions; phenotypes which were enhanced by a compound Pot1b mutation. Importantly, these phenotypes translated into greater CLL tumor burden on an Emμ-TCL1 background as determined by flow cytometric analysis of CD5-positive B cells, and significantly shorter survival times of mice harboring Pot1a and compound Pot1a/b mutations, with leukemic cells displaying gross karyotypic abnormalities. Considering that G-rich telomeric ssDNA has a propensity to form secondary structures such as G-quadruplexes, which can impede DNA replication and provoke replication stress, we evaluated the effects of the G-quadruplex stabilizing agent pyridostatin (PDS) in the presence of wildtype or mutant Pot1a/b. We observed that especially on a mutant Pot1a or Pot1a/b background, PDS treatment increased the frequency of telomeric γH2AX foci indicative of DNA damage and enhanced apoptosis as determined by Annexin-V levels, ultimately culminating in impaired cell viability as demonstrated in IC50 assays.

Conclusion

Our complementary approaches using human and mouse models expand our understanding of POT1 in human CLL. We propose that while POT1 mutations drive genomic instability to confer tumor survival advantage, they also reveal an actionable opportunity to selectively target POT1-mutated CLL.

Session topic: 5. Chronic lymphocytic leukemia and related disorders - Biology & Translational Research

Keyword(s): B cell chronic lymphocytic leukemia, Genomic instability, Targeted therapy, Telomere

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