Forward and Reverse Genetics of B Cell Malignancies: From Insertional Mutagenesis to CRISPR-Cas.

Anthony G. Uren,Joanna C. Dawes

doi:10.3389/fimmu.2021.670280

Anthony G. Uren, Joanna C. Dawes

Open Access

https://doi.org/10.3389/fimmu.2021.670280

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Abstract

Cancer genome sequencing has identified dozens of mutations with a putative role in lymphomagenesis and leukemogenesis. Validation of driver mutations responsible for B cell neoplasms is complicated by the volume of mutations worthy of investigation and by the complex ways that multiple mutations arising from different stages of B cell development can cooperate. Forward and reverse genetic strategies in mice can provide complementary validation of human driver genes and in some cases comparative genomics of these models with human tumors has directed the identification of new drivers in human malignancies. We review a collection of forward genetic screens performed using insertional mutagenesis, chemical mutagenesis and exome sequencing and discuss how the high coverage of subclonal mutations in insertional mutagenesis screens can identify cooperating mutations at rates not possible using human tumor genomes. We also compare a set of independently conducted screens from Pax5 mutant mice that converge upon a common set of mutations observed in human acute lymphoblastic leukemia (ALL). We also discuss reverse genetic models and screens that use CRISPR-Cas, ORFs and shRNAs to provide high throughput in vivo proof of oncogenic function, with an emphasis on models using adoptive transfer of ex vivo cultured cells. Finally, we summarize mouse models that offer temporal regulation of candidate genes in an in vivo setting to demonstrate the potential of their encoded proteins as therapeutic targets.

Highlights

B cell neoplasms can be categorized by their cell of origin, each subtype being representative of a discrete stage in differentiation with characteristic phenotypes and genetic lesions (Table 1, Figure 1) [reviewed in [1, 2]]
Germinal Center B cell Pre B cell, Pro B cell, Mature B cell Mature B cell or Post–germinal center B cells Activated B cell or Germinal Center B cell Germinal center B cell Marginal zone/memory B cells Germinal Center B cell Plasma cells Mantle B cell Post Germinal Center B cells Marginal zone B cells Germinal Center B cell Plasma cells Pro B cells cells are characterized by remodeling of the immunoglobulin loci by recombinase activating gene (RAG) mediated V(D)J recombination of immunoglobulin variable regions and by activation induced cytidine deaminase (AID) mediated class switch recombination
Human populations are highly polymorphic and this, in combination with the mutator phenotype generated by many tumours, can make it difficult to subtract the background noise of unselected passenger mutations from the evidence for driver mutations

Summary

INTRODUCTION

B cell neoplasms can be categorized by their cell of origin, each subtype being representative of a discrete stage in differentiation with characteristic phenotypes and genetic lesions (Table 1, Figure 1) [reviewed in [1, 2]]. PAX5 is frequently mutated by translocation events creating fusion proteins in human tumors and some of these have been shown to cause B ALL in mice [58, 116] In one of these studies exome sequencing of five leukemic mice expressing a PAX5/ENL fusion identified mutations in Ptpn, Kras, Pax, and Jak genes [58]. Of these genes PAX5, PTPN11, KRAS and its homologue NRAS were found recurrently mutated across a panel of human B ALL samples from diverse subtypes These six independent studies using complementary experimental designs all identify mutations in Jak by exome sequencing and in some cases by insertional mutagenesis. We summarize recently developed methods for rapid generation of multiallelic strains and several approaches to temporal control of gene expression

B Cell Malignancy Models From Transplantation of Virus Transduced Cells

Findings

CONCLUSIONS AND FUTURE DIRECTIONS