Arrayed CRISPR library screen to identify synthetic lethal partners of the histone methyltransferase KMT2D in diffuse large B cell lymphoma


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Elizabeth Blaikley1,Mark Cockerill1,Cath Eberlein1,Ben Hodgson1,Graeme Walker1,Caroline Springer1
1CRUK Manchester Institute

Abstract

Background

Diffuse large B cell lymphoma (DLBCL) is a disease with unmet clinical need and poor patient survival. The gene encoding the Histone 3 Lysine 4 methyltransferase KMT2D is frequently mutated in DLBCL, resulting in loss of function. Synthetic lethality occurs when loss of two genes’ functions together, but not individually, result in cell death. Our aim is to identify novel therapeutic targets for the treatment of DLBCL with KMT2D loss, by identifying synthetic lethal partners of KMT2D. This therapeutic strategy would provide tumour selectivity, a key aim of cancer therapy.

Method

The Invitrogen™ LentiArray™ Human Epigenetics CRISPR Library will be used to identify synthetic lethal partners of KMT2D. The library contains gRNA targeting 394 epigenetic genes in array format. An isogenic DLBCL cell line pair, positive or negative for KMT2D, and stably expressing CAS9 will be screened with the library.

Results

A panel of 6 DLBCL cell lines were characterised to determine their suitability for the CRISPR screen. KMT2D mutation status and protein expression levels were confirmed. As crucial cell line features; proliferation, clonogenic potential and transfection efficiency were assessed. The DLBCL cell line OCI-Ly-1, with bi-allelic KMT2D loss, was selected. Current efforts focus on stably integrating and expressing wild-type KMT2D in OCI-Ly1 cells to generate an isogenic cell line pair. CAS9 will subsequently be stably expressed in these cells prior to commencement of the screen.

Conclusion

Synthetic lethality is an attractive strategy to selectively target DLBCL with KMT2D loss. The major challenges of this project have been the large size of KMT2D coupled with the low transfection efficiency of DLBCL cell lines. Current efforts focus on the generation of the KMT2D positive and negative isogenic cell line pair.