Dissecting the role of TP53 alterations in del(11q) chronic lymphocytic leukemia

Abstract

Background: Several genetic alterations have been identified as driver events in chronic lymphocytic leukemia (CLL) pathogenesis and oncogenic evolution. Concurrent driver alterations usually coexist within the same tumoral clone, but how the cooperation of multiple genomic abnormalities contributes to disease progression remains poorly understood. Specifically, the biological and clinical consequences of concurrent high-risk alterations such as del(11q)/ATM-mutations and del(17p)/TP53-mutations have not been established.

Methods: We integrated next-generation sequencing (NGS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 techniques to characterize the in vitro and in vivo effects of concurrent monoallelic or biallelic ATM and/or TP53 alterations in CLL prognosis, clonal evolution, and therapy response.

Results: Targeted sequencing analysis of the co-occurrence of high-risk alterations in 271 CLLs revealed that biallelic inactivation of both ATM and TP53 was mutually exclusive, whereas monoallelic del(11q) and TP53 alterations significantly co-occurred in a subset of CLL patients with a highly adverse clinical outcome. We determined the biological effects of combined del(11q), ATM and/or TP53 mutations in CRISPR/Cas9-edited CLL cell lines. Our results showed that the combination of monoallelic del(11q) and TP53 mutations in CLL cells led to a clonal advantage in vitro and in in vivo clonal competition experiments, whereas CLL cells harboring biallelic ATM and TP53 loss failed to compete in in vivo xenotransplants. Furthermore, we demonstrated that CLL cell lines harboring del(11q) and TP53 mutations show only partial responses to B cell receptor signaling inhibitors, but may potentially benefit from ATR inhibition.

Conclusions: Our work highlights that combined monoallelic del(11q) and TP53 alterations coordinately contribute to clonal advantage and shorter overall survival in CLL.

Keywords: CRISPR/Cas9 system; TP53 gene; biomarkers; chromosomal abnormality; chronic lymphocytic leukemia; next-generation sequencing.

FIGURE 1

Mutational analysis and overall survival (OS) of del(11q) patients. (A) Mutational landscape of del(11q) patients; each column represents a patient and each row corresponds to a genetic alteration. Mutation or cytogenetic events are indicated in red, IGHV unmutated status in dark yellow, and IGHV mutated status in light yellow. White indicates missing data. (B) Significantly high and low co‐occurrences of ATM and TP53 alterations in CLL patients (chi‐square test). Each column corresponds to one patient, and the presence of mutations or deletions is clustered according to the type of ATM and TP53 alterations, shown in blue. Left red rectangle indicates the presence of high co‐occurrence between monoallelic del(11q) and TP53 alterations (** p < 0.01). Right table indicates the grades of low co‐occurrences between the indicated conditions. (C) Impact of TP53 mutations in the survival of del(11q) CLL patients. (D) OS of del(11q) patients according to the presence of monoallelic or biallelic TP53 alterations (deletion and/or mutation). (E) Clinical impact of the presence of del(11q) in patients with TP53 alterations (deletion and/or mutations). (F) Mutational analysis and (G) overall survival (OS) of del(11q) patients in the validation cohort from the UK LRF CLL4 trial

FIGURE 2

Generation of CRISPR/Cas9‐edited CLL cell lines harboring del(11q), TP53 and/or ATM mutations and phenotypical analysis of edited cells. (A) Upper panel: experimental design for the introduction of del(11q), TP53, and ATM mutations in the HG3 CLL derived cell line using the CRISPR/Cas9 system. sgRNAs targeting 11q22.1 and 11q23.3 were nucleofected for transitory expression in HG3‐Cas9 cells. Nucleofected single‐cell sorted clones were screened for the presence of del(11q), and the presence of this deletion was validated by Sanger sequencing and FISH. The resulting HG3‐del(11q) isogenic cell line, as well as parental HG3‐Cas9 cells, was further transduced with sgRNAs targeting TP53 and/or ATM genes for the generation of truncating mutations. In total, 3 HG3^WT, 3 HG3 TP53 ^MUT, 3 HG3‐del(11q), 5 HG3‐del(11q) TP53 ^MUT, and 5 HG3‐del(11q) ATM ^MUT TP53 ^MUT clones were generated. Lower panel: number of alleles affected by mutations and deletions in the CRISPR/Cas9‐generated cell lines. (B) Representative FISH images of HG3^WT and HG3‐del(11q) cells. Green signals correspond to 11q22/ATM probe and the control red signals correspond to 17p13/TP53 probe. (C) Western blot analysis of isogenic HG3‐edited clones with TP53 mutations. Upper panel shows TP53 protein expression of 2 HG3^WT clones and 3 HG3 TP53 ^MUT clones. Lower panel shows GAPDH, which was used as loading control. (D) Western blot analysis of HG3‐edited single‐cell clones. Upper and middle panels show ATM and TP53 expression, respectively, of 2 HG3^WT clones, 5 HG3‐del(11q) TP53 ^MUT clones, 5 HG3‐del(11q) TP53 ^MUT ATM ^MUT clones, and 2 HG3‐del(11q) clones. β‐actin was used as loading control. (E) Bright field and Giemsa stained representative images of HG3‐edited cell lines

FIGURE 3

Effects of biallelic loss of TP53 and ATM on viability, cell growth, apoptosis, and cell cycle control of CRISPR/Cas9‐edited cell lines. (A) Effects of del(11q), TP53, and/or and ATM mutations on proliferation of HG3 cells after 72 hours. MTT absorbance values are normalized with the HG3^WT clones. Data are summarized as the mean ± SD. (B) HG3‐edited cell lines were seeded at a concentration of 3 × 10⁴ cells/mL, and cell growth was assessed at five time points every 24 hours by Trypan Blue exclusion. Data were fitted in an exponential growth equation, and time point values are presented as the mean ± SEM. (C) Representative immunoblot analyses of HG3^WT, HG3 TP53 ^MUT, HG3‐del(11q), HG3‐del(11q) TP53 ^MUT, and HG3‐del(11q) ATM ^MUT TP53 ^MUT whole cell lysates. ATM, PARP1, and Caspase‐3 protein expression and/or cleavage were analyzed. β‐actin was used as loading control. (D) Cell cycle phase distribution of HG3‐edited cell lines upon exposure to irradiation at the indicated time points. Data represent the mean values ± SD of at least three independent experiments. p < 0.05 (*), p < 0.01 (**). (E) Representative cell cycle profiles of CRISPR/Cas9‐edited clones after 72 hours irradiation exposure (2 Gy). All the events placed right from the G2/M peak at 400 DNA content units were considered >4n population

FIGURE 4

In vivo clonal competition analysis of xenotransplanted NSG mice. (A) HG3^WT GFP‐tagged and HG3 TP53 ^MUT RFP‐tagged cells were mixed at a ratio 1:1 and injected into NSG mice (n = 8). Spleen and bone marrow infiltration were assessed by flow cytometry 7 (n = 4) and 14 (n = 4) days post‐injection. (B) HG3‐del(11q) RFP‐tagged, HG3‐del(11q) TP53 ^MUT GFP‐tagged, and HG3‐del(11q) ATM ^MUT TP53 ^MUT GFP, and RFP‐tagged cells were mixed at a ratio 1:1:1 and injected into NSG mice (n = 8). Spleen and bone marrow infiltration were assessed by flow cytometry 7 (n = 4) and 14 (n = 4) days post‐injection. Data are represented as the mean ± SD. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). (C) Quantification of GFP+ and/or RFP+ cell populations in the peripheral blood of HG3^WT and HG3 TP53 ^MUT (left), and HG3‐del(11q), HG3‐del(11q) TP53 ^MUT, and HG3‐del(11q) ATM ^MUT TP53 ^MUT (right) xenografted mice 17 days after intravenous injection. Data are shown as mean ± SEM. p < 0.001 (***). (D) Kaplan‐Meier overall survival curve of HG3^WT (n = 5) and HG3 TP53 ^MUT (n = 5) xenografted mice (left panel) and HG3‐del(11q) (n = 5), HG3‐del(11q) TP53 ^MUT (n = 5), and HG3‐del(11q) ATM ^MUT TP53 ^MUT (n = 5) xenotransplants (right panel). The reported p value was calculated by using the Log‐rank test

FIGURE 5

Cell viability studies of HG3‐edited clones in response to different drug treatments. (A and B) Tables indicating the IC₅₀ ± SEM values of each CRISPR/Cas9‐edited HG3 cell line in response to fludarabine, ibrutinib, idelalisib or AZD6738, as well as the p‐values of the comparison between HG3^WT IC₅₀ mean concentrations with the mean IC₅₀ values from the rest of the conditions. (C‐J) HG3‐edited clones were treated with escalating doses of fludarabine (C and D), ibrutinib (E and F), idelalisib (G and H), and AZD6738 (I and J), and cell viability was assessed by MTT assay after the indicated treatment times. Surviving fraction is expressed relative to untreated controls. Data are summarized as the mean ± SD of three independent experiments