CRISPR/Cas9-Generated Models Uncover Therapeutic Vulnerabilities of Del(11q) Chronic Lymphocytic Leukemia Cells to Dual BCR and PARP Inhibition

Chromosome 11q22.3 deletion (del(11q)) is one of the most common cytogenetic alterations in CLL and usually involves both ATM and BIRC3 genes. Concomitant mutations in ATM and/or BIRC3 in the remaining allele have been associated with poor survival. Despite the encouraging efficacy of novel agents targeting BCR and BCL2 pathways, del(11q) patients still have an inferior outcome and the development of resistance to these drugs has been increasingly reported. We therefore investigated the functional impact of del(11q) together with loss-of-function mutations in ATM and/or BIRC3 and whether CLLs harboring these alterations could benefit from novel combinatorial therapies.

To address these questions, we used the CRISPR/Cas9 system to generate an isogenic CLL cell line to model del(11q) derived from HG3 cells by introducing two guide RNAs targeting the 11q22.1 and 11q23.3 regions. The presence of a monoallelic deletion (size ~17 Mb) was confirmed in 100% of the cells by FISH. Truncating mutations in ATM and/or BIRC3 were introduced in the remaining allele, generating HG3 del(11q) ATM^KO, del(11q) BIRC3^KO and del(11q) ATM^KOBIRC3^KO (three clones per condition). In addition, single ATM^KO and BIRC3^KO mutations, or the combination of both, were introduced into both HG3 and MEC1 CLL-derived cells (three clones per condition).

Functional in vitro studies revealed that del(11q) BIRC3^KO cells had increased growth rates compared to del(11q) BIRC3^WT clones (P<0.01). Similar results were observed in HG3 and MEC1 BIRC3^KO cells (P<0.01; P<0.05). Moreover, biallelic inactivation of BIRC3 in del(11q) cells resulted in cytoplasmic stabilization of NF-kB-inducing kinase (NIK), leading to higher nuclear NF-kB2 (p52) activation (P<0.01) as measured by ELISA. In parallel, we analyzed the DNA damage response (DDR) of these cells, and showed that del(11q) ATM^KO cells displayed reduced pH2AX levels (P<0.001) and an accumulation of unrepaired double strand breaks (DSB) (P<0.001) after irradiation, as determined by comet assays. Consistently, in vivo subcutaneous xenografts showed that HG3 ATM^KOBIRC3^KO tumors presented proliferative advantage, higher p52 levels and greater genomic and mitotic instability than HG3^WT tumors, indicating a more aggressive phenotype.

We next assessed the response of these CRISPR/Cas9-edited CLL cell lines to therapy. Of note, only TP53^KO clones (also generated by CRISPR/Cas9), and not del(11q) BIRC3^KO cells, showed resistance to fludarabine (mean IC₅₀ 16.9 uM vs. 4.1 uM; mean apoptotic cells (5 uM) 5.5% vs. 22.5%; P<0.05). Moreover, del(11q) cells were slightly more resistant to ibrutinib (IBRU) treatment compared to WT cells (mean IC₅₀ 10 uM vs. 3.7 uM; P<0.05). Interestingly, exploiting the DDR deficiencies underlying del(11q) by targeting the single strand break repair pathway with the PARP inhibitor olaparib (OLA), del(11q) ATM^KO cells were not able to proliferate even 12 days after treatment (3 uM), independently of the mutational status of BIRC3 (P<0.01). In vivo intravenous HG3-derived xenografts (N=20) showed that OLA (100 mg/kg) reduced hCD45+ cells in the peripheral blood (P<0.01) and significantly improved survival of del(11q) ATM^KOBIRC3^KO xenografted mice (P<0.01).

Moreover, IBRU potentiated the effects of OLA in cell viability (72h) in all the del(11q) clones (combination indexes 0.69-0.85), leading to an increased necrotic cell death, as shown by annexin V/PI staining (P<0.001) and HMGB1 release. Remarkably, we found that IBRU caused downregulation of the DNA repair protein RAD51, leading to impaired RAD51 foci formation in DSB lesions (P<0.01). Consistently, IBRU (1 uM) reduced the homologous recombination (HR) repair efficiency in HG3 cells (P=0.001), as determined by an HR-reporter construct. This IBRU-dependent impairment of HR repair could explain the synergistic effects with OLA by synthetic lethality.

In conclusion, we demonstrate that del(11q) CLL cells with biallelic inactivation of BIRC3 and ATM show enhanced proliferation through activation of the non-canonical NF-kB pathway, and accumulation of DNA damage contributing to genomic instability. We show that these defects on the DDR can be therapeutically targeted by synthetic lethal approaches using PARP inhibitors either alone or in combination with BCR inhibitors, providing a rationale for the study of this combination in relapsed del(11q) CLL patients.

PI15/01471 SA085U16 JCyL-MQ FEHH-MH