p53-independent ibrutinib responses in an Eμ-TCL1 mouse model demonstrates efficacy in high-risk CLL

Abstract

Deletion of the short-arm of chromosome 17 (17p-) is one of the most critical genetic alterations used in chronic lymphocytic leukemia (CLL) risk stratification. The tumor suppressor TP53 maps to this region, and its loss or mutation accelerates CLL progression, hampers response to chemotherapy and shortens survival. Although florescent in situ hybridization analyses for 17p deletions are routinely performed during clinical diagnoses, p53 mutational status is often unexamined. Given the limited clinical data that exists for frontline treatment of patients with CLL harboring TP53 mutations, there is a need to understand the biology of CLL with TP53 mutations and identify treatment strategies for this subset of patients. Herein, we used a CLL mouse model (Eμ-TCL1) harboring one of the most common TP53 hot-spot mutations observed in CLL (p53(R172H), corresponding to p53(R175H) in humans) to evaluate its impact on disease progression, survival, response to therapy and loss of the remaining wild-type Trp53 allele following ibrutinib treatment. We show that ibrutinib was effective in increasing survival, activating cellular programs outside the p53 pathway and did not place selective pressure on the remaining wild-type Trp53 allele. These data provide evidence that ibrutinib acts as an effective treatment for aggressive forms of CLL with TP53 mutations.

Figure 1

Mutant p53 results in reduced survival, but phenotypically similar disease progression in Eμ-TCL1 mice. (a) Kaplan–Meier curves of Eμ-TCL1;p53^R172H/+ and Eμ-TCL1 mice. (b) Flow cytometry data demonstrating the CD5⁺/CD19⁺ immunophenotypes in the peripheral blood of Eμ-TCL1;p53^R172H/+ and Eμ-TCL1 mice at 4, 6 and 8 months of age. Cells were gated by their expression of CD5, CD19 and CD5/CD19. (c) Bar graph representing the percent of CD5⁺/CD19⁺ cells in the peripheral blood of Eμ-TCL1;p53^R172H/+ (n=6) and Eμ-TCL1 (n=6) mice at 4, 6 and 8 months of age. NS represents non-significant values (P>0.05) (d–f) Hematoxylin and eosin staining of tumor burdened spleens, lymph nodes, livers of Eμ-TCL1;p53^R172H/+ and Eμ-TCL1 mice. The scale bar represents 100 microns.

Figure 2

Differential gene expression profiling between B-cells from Eμ-TCL1 and Eμ-TCL1;p53^R172H/+ mice. (a) Heat map of hierarchical clustering of gene expression profiles between B-cells from Eμ-TCL1 and Eμ-TCL1;p53^R172H/+ mice. (b) Gene set enrichment analysis enrichment plots of differentially expressed gene sets from B-cells isolated from Eμ-TCL1 and Eμ-TCL1;p53^R172H/+ mice. Reactome Meiotic Recombination, normalized enrichment score (NES)=2.39 and false-discovery rate (FDR)=0.075; Amundsom Gamma Radiation Response, NES=2.014 and FDR=0.198; Kong E2F3, NES=1.99 and FDR=0.192; and Whitefield Cell Cycle Literature, NES=1.97, FDR=0.200. (c) KEGG-pathway analyses of differentially regulated biological pathways in B-cells from Eμ-TCL1 and Eμ-TCL1;p53^R172H/+ mice.

Figure 3

Ibrutinib prolongs survival and activates downstream pathways independent of p53. (a) Kaplan–Meier curves of vehicle- and ibrutinib-treated Eμ-TCL1;p53^R172H/+ mice. (b) Kaplan–Meier curves of vehicle- and ibrutinib-treated Eμ-TCL1 mice. (c) Western blot of total BTK and phospho-BTK-(Tyr223) levels in spleens from short-term ibrutinib-treated Eμ-TCL1 and Eμ-TCL1;p53^R172H/+ mice. (d) Western blot of total ERK and phospho-ERK (Thr202/Tyr204) levels in spleens from short-term ibrutinib-treated Eμ-TCL1 and Eμ-TCL1;p53^R172H/+ mice. (e) Flow cytometry data demonstrating CD5⁺/CD19⁺/AnnexinV⁺ cells in the peripheral blood of Eμ-TCL1;p53^R172H/+ and Eμ-TCL1 mice pre and post short-term ibrutinib treatment. Cells were initially gated by their expression of CD5, CD19 and CD5/CD19 and then further analyzed by their uptake of Annexin V. (f) Bar graph representing the percent of CD5⁺/CD19⁺/AnnexinV⁺ cells in the peripheral blood of Eμ-TCL1;p53^R172H/+ (n=4) and Eμ-TCL1 (n=4) mice pre- and post-ibrutinib treatment.

Figure 4

Ibrutinib does not induce LOH of the remaining wild-type Trp53 allele compared with vehicle treatment. (a) LOH at the Trp53 locus in B-cells from tumor burdened spleens and lymph nodes from vehicle- and ibrutinib-treated Eμ-TCL1;p53^R172H/+ mice. (b) Percentage of LOH at the Trp53 locus in spleens and lymph nodes from tumor burdened Eμ-TCL1;p53^R172H/+ mice treated with ibrutinib (n=29) or vehicle (n=37).