TP53 Gain-of-Function Mutants Display Unique Transcriptional Profiles in Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia (CLL) is the most common hematological malignancy in the United States, accounting for approximately 25% to 35% of all leukemias. Targeted therapies such as Bruton's Tyrosine Kinase (BTK) and B-cell lymphoma-2 (BCL-2) inhibitors ibrutinib and venetoclax have produced durable responses in CLL however, a subset of patients experience relapse and poor outcomes. This group with a poor prognosis is in urgent need of therapies that address the unique biology of treatment resistant CLL.

To investigate mutant TP53's contribution to disease progression, we collected samples at baseline from patients entering ibrutinib treatment for front-line and relapsed/refractory CLL. Large-scale exome sequencing was performed on 270 patient samples revealing that 106 patients harbored some TP53 aberration, mostly in the DNA binding domain (DBD). Mutational analysis revealed missense mutations R175H and R248Q were among the most frequently occurring. These variants are known gain of function (GOF) mutations in solid tumors contributing to tumor progression and therapeutic resistance. Eighty-two percent of TP53 mutations in this cohort had a simultaneous deletion of the short arm of chromosome 17 (del17p) shown by fluorescence in situ hybridization (FISH). Del17p serves as an independent predictor of progression on ibrutinib as these patients had an inferior response to ibrutinib. These data lead us to hypothesize that the presence of TP53 mutations in CLL supports a shortened time to initial therapy and progression and relapse compared to patients without mutations in TP53.

Wild type (WT) p53 is an essential transcription factor functioning as a tumor suppressor responsible for DNA damage repair, regulating apoptosis, and maintaining genomic stability. To describe mutant p53 activity, DNA damage was induced by ionizing radiation (IR) administered at 3Gy for 1 hour. WT but not mutant TP53 samples responded to DNA damage by stabilizing p53 protein expression. After stabilization, WT p53 strongly induced p21 protein, whereas p53 mutant expressing patient samples did not induce p21 protein in response to IR indicating a disrupted DNA repair pathway ex vivo. However, these studies did not show which genes are being targeted transcriptionally by mutant p53 nor how mutant p53 binds to and regulates these targets allowing these affected cells to subvert apoptosis.

To further investigate this, we independently modeled this system in two cell lines. The HG3 and OSU-CLL lines are patient-derived lines created using CRISPR/Cas9 gene editing to introduce specific mutations at R175H and R248Q into WT p53 CLL. Homozygous knock-in of R175H and R248Q mutations on p53 was confirmed through Sanger sequencing on expanded single-cell clones. A profile of differentially expressed genes was revealed during RNA-seq analysis of mutant TP53 and WT TP53 cell lines. Genes overexpressed in relation to WT include transcription factors such as AFF3 and BCL11b and known tumor suppressors like KLHL14. Interestingly, a strong downregulation was seen in CYP4F3 across mutants whose upregulation is shown to be associated with resistance to chemotherapeutic agents such as fludarabine and targeted agents such as ibrutinib in the mutant TP53 setting. To determine whether p53 mutants in CLL exhibit a unique transcriptional signature like what is seen in solid tumors, we are conducting chromatin immunoprecipitation (ChIP) for p53 in these cell lines.

In summary, our study reveals that TP53 mutations take advantage of mechanisms of resistance towards apoptosis-inducing therapies and impair the normal tumor-suppressive function of WT p53 which remains to be addressed in the clinic. These significantly worsen patient prognosis and accelerate relapse. Ongoing analysis will identify and characterize the action of these differentially expressed targets in CLL and highlight their role in facilitating survival in TP53 mutant CLL cells during therapy.

Rogers:AstraZeneca Pharmaceuticals LP.: Membership on an entity's Board of Directors or advisory committees; AbbVie Inc, Genentech, a member of the Roche Group, Novartis: Research Funding; AbbVie Inc, BeiGene Ltd, Genentech, a member of the Roche Group, Janssen Biotech Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company, Pharmacyclics LLC, an AbbVie Company: Consultancy; Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees. Bhat:AstraZeneca: Consultancy, Research Funding; Abbvie: Consultancy; Aptitude Health: Honoraria. Woyach:Genentech, Inc.: Consultancy; BeiGene: Consultancy; Janssen: Research Funding; Loxo Lilly: Consultancy; Merck: Consultancy; Newave: Consultancy; Pharmacyclics: Consultancy, Research Funding; Schrodinger: Research Funding; AstraZeneca: Consultancy; AbbVie: Research Funding; Morphosys: Research Funding.