Generation of a CRISPR activation mouse that enables modelling of aggressive lymphoma and interrogation of venetoclax resistance

Abstract

CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been exploited in this context. We establish a CRISPRa mouse (dCas9a-SAMKI) for inducing gene expression in vivo and in vitro. Using dCas9a-SAMKI primary lymphocytes, we induce B cell restricted genes in T cells and vice versa, demonstrating the power of this system. There are limited models of aggressive double hit lymphoma. Therefore, we transactivate pro-survival BCL-2 in Eµ-MycT/+;dCas9a-SAMKI/+ haematopoietic stem and progenitor cells. Mice transplanted with these cells rapidly develop lymphomas expressing high BCL-2 and MYC. Unlike standard Eµ-Myc lymphomas, BCL-2 expressing lymphomas are highly sensitive to the BCL-2 inhibitor venetoclax. We perform genome-wide activation screens in these lymphoma cells and find a dominant role for the BCL-2 protein A1 in venetoclax resistance. Here we show the potential of our CRISPRa model for mimicking disease and providing insights into resistance mechanisms towards targeted therapies.

Lymphoma cells lacking pro-apoptotic BAX are highly resistant to BH3-mimetics targeting pro-survival MCL-1 but retain sensitivity to conventional DNA-damaging drugs

“…Resistance to BH3-mimetic drugs is a critical and emerging issue which threatens to limit the benefit of this class of anti-cancer therapy. While genetic changes underlying resistance to the BCL-2 inhibitor venetoclax are beginning to be uncovered [5,23,26,[35][36][37][38][39], less attention has been focused on resistance to MCL-1 inhibitors, which have entered clinical trials for diverse haematological malignancies [5]. To this end, we sought to identify factors that could confer resistance to MCL-1 inhibitors using murine and human models of highly aggressive, MYC-driven lymphoma.…”

“…The second major mechanism driving resistance we identified in our S63845-resistant cells was upregulation of the pro-survival proteins BCL-XL and A1. Many studies have reported the over-expression of pro-survival BCL-2 proteins as resistance factors for venetoclax therapy, including in patient samples and in in vitro whole genome CRISPR/Cas9 screens [ 35 , 37 , 39 , 52 , 53 ]. In our study, Eµ-Myc lymphoma cells that had achieved resistance to S63845 through upregulation of BCL-XL and A1 also showed resistance to etoposide treatment due to the over-expression of these pro-survival proteins preventing apoptosis.…”

BCL-W makes only minor contributions to MYC-driven lymphoma development

“…A frequent mechanism of resistance in malignant cells to loss/inhibition of BCL-2 pro-survival proteins by BH3-mimetic drugs is the upregulation of one/several of the non-targeted pro-survival BCL-2 family proteins [ 16 , 30 ]. If BCL-W is required to support the ongoing survival of lymphoma cells, it is possible that such compensatory changes could occur to allow MYC-driven lymphomagenesis in the absence of BCL-W. To investigate this, we performed Western blot analyses on tumour tissues from Eµ-Myc T/+ ;Bcl-w +/+ and Eµ-Myc T/+ ;Bcl-w −/− mice ( n = 12 per genotype; n (females) = 15, n (males) = 9) (Fig.…”

“…Finally, in various solid cancers, BCL-W has been detected as a potential resistance factor [ 39 ], and is upregulated in a host of these malignancies [ 40 ], but to our knowledge this is not the case in haematological cancers. We recently published the results of a CRISPR activation (CRISPRa) screen in a murine model of double-hit lymphoma (DHL), where we identified resistance factors to Venetoclax treatment [ 30 ]. Interestingly, in this screen we identified upregulation of genes expressing the pro-survival BCL-2 family members BCL-XL, MCL-1, and A1 as factors that promoted DHL resistance to Venetoclax, but not BCL-W.…”

Preclinical Anticipation of On- and Off-Target Resistance Mechanisms to Anti-Cancer Drugs: A Systematic Review

“…Resistance of MCF-7 breast cancer cells to palbociclib is linked to stimulation of cyclin E/CDK2 and Myc activity, as evidenced by CRISPRa experiments [194]. Another CRISPRa screen performed in BCL-2-expressing Eµ-Myc/dCas9a-SAM Kl/+ /sgBcl-2 lymphoma cells showed the BCL-2-related A1 protein to be associated to resistance to venetoclax [195]. A CRISPRa screening approach was also applied to activate expression of lncRNAs in order to identify putative players with a role in vemurafenib resistance in A375 melanoma cells, and loci potentially involved have been uncovered [196].…”