Developing Therapeutic Strategies for CLL with Mutant p53

Chronic lymphocytic leukemia (CLL) is a clonal disease marked by genetic heterogeneity, often resulting in varied therapeutic responses. As a consequence of these genetic variations, cytogenetic analyses are routinely employed to select the most efficacious treatment strategies. One of the most critical genetic variants used for CLL risk-stratification is deletion of the short arm of chromosome 17 (17p-). The tumor suppressor TP53 maps to this region, and its loss correlates with dismal outcomes. While FISH analyses for 17p- are routinely performed, the mutational status of TP53 is typically unknown. The importance of p53 mutations in leukemic progression have recently become apparent, as clinical studies have identified subsets of CLL patients that harbor TP53 mutations without an accompanying 17p- (Zenz et. al., JCO 2010); while other studies have suggest that p53 mutations are present in leukemic clones that expand following cytotoxic treatment (Wong et. al., Nature 2015). Thus, it is critical that we identify frontline treatment modalities that do not directly place undo selective pressure on the p53 pathway in CLL patients harboring a single TP53 mutation. In recent clinical trials, the Bruton's tyrosine kinase (BTK) inhibitor, Ibrutinib, has been shown effective in improving progression free survival in patients with CLL regardless of 17p- status (O'Brien et. al., Lancet Oncology 2014). However, little is known regarding how p53 mutations impact therapeutic responses given their known dominant-negative and gain-of-function effects. To this end, we have utilized a preclinical in vivo mouse model of B-CLL ( Eµ-TCL1 ) in the presence or absence of a TP53 hot-spot mutation ( p53R172H , corresponding to p53R175H in humans) to study its impact on therapeutic response, survival, and dynamic loss of the remaining wild-type Trp53 allele during the natural course of B-CLL following ibrutinib-based therapy. Cohorts of Eµ-TCL1; p53R172H /+ and Eµ-TCL1; p53WT mice were treated with ibrutinib 25mg/kg/day by oral gavage starting at 8 months of age. Ibrutinib significantly extended survival in both the Eµ-TCL1; p53R172H /+ and Eµ-TCL1; p53WT cohorts (Fig. 1A) and resulted in a reduction in CD5+CD19+ cells (Fig. 1B). Together, these data indicate ibrutinib's therapeutic efficacy even in the presence of mutated p53. To investigate the molecular pathways altered by ibrutinib in both the wild type and p53 mutant setting, we performed RNA-Seq analyses using malignant B-cells from treated and untreated Eµ-TCL1; p53R172H and Eµ-TCL1; p53WT mice. Critically, these analyses revealed that ibrutinib did not impact genes or cellular programs governed by p53 (Fig. 1C). Furthermore, RNA and protein analyses of tumor samples revealed that ibrutinib was directly impacted the BTK-, PLC-, MAPK-, and ERK- pathways regardless of p53 mutational status. Perhaps most important, we did not observe a significant loss of heterozygosity of the remaining wild type Trp53 allele in the ibrutinib treated Eµ-TCL1; p53R172H /+ lymphomas compared to untreated Eµ-TCL1; p53R172H /+. Together, these results directly indicate that ibrutinib does not place undo selective pressure on the remaining wild type Trp53 allele (Fig. 1D) and demonstrates that ibrutinib based treatment modalities may be effective treatment regimens in CLL patients harboring mutant p53. Even though ibrutinib effectively improved overall survival compared to vehicle treated animals, these mice eventually succumbed to the disease. Thus, it will be critical to examine mechanisms of ibrutinib-resistance in the setting of Trp53 mutations. To this end, expression analysis of ibrutinib treated Eµ-TCL1; p53R172H /+ mice revealed an interesting increase in T-cell immune regulatory pathways compared to Eµ-TCL1; p53WT treated cohorts. Currently, we are exploring the mechanism by which mutant p53 expression, exposure to ibrutinib, and regulations of immune-checkpoint genes. ![Figure][1] Disclosures No relevant conflicts of interest to declare. [1]: pending:yes