Depletion of the TP53 Family Member p73 Sensitizes Cancer Cells towards Cytarabine.

Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. 50–70 % of adults with AML achieve complete remission with induction of chemotherapy - only 20–30 % of patients enjoy long-term disease-free survival. Many AML-patients still die of their disease, most frequently because of drug resistance. We intended to study drug resistance in cancer cells by using a synthetic lethal RNAi screen with the S phase targeting drug cytarabine (Ara-C). Our goal was the identification of siRNAs that overcome drug resistance triggered by cytarabine.

For the chemosensitizer RNAi screen we used lipid-based reverse transfection for transient siRNA-mediated gene silencing in the human osteosarcoma cell line (U2OS). The human osteosarcoma cell line is a good model to study cytarabine resistance: the cells are highly refractory to Ara-C treatment, while activating DNA damage response (indicated by activation of different DNA checkpoint markers (phospho-Chk1, phospho-p53, p21)). Gene silencing was done by using a custom library comprising 437 siRNA probes specific for DNA repair and DNA damage response genes (consisting of a pool of four different siRNA-sequences for one gene). Synthetic lethality of siRNAs with a sub lethal dose of cytarabine (IC20) was measured after 72 hours of incubation with a luminescence-based cell viability assay. Hits were identified after z-score normalization of each plate and by evaluating the cytarabine-sensitivity of siRNAs reducing cell viability in combination with Ara-C vs. cell viability of untreated siRNA transfected cells. Screens were done in duplicate and high confidence hits (with z-scores greater than 2 STD) were validated. For secondary validation screens and further validation experiments we used two different single siRNA-sequences for each gene.

We reproducible identified and validated candidate genes which sensitize cells to cytarabine-treatment when silenced. Our top hits p73 and components of ubiquitine ligase complexes have been implicated in drug resistance and some are also “druggable”. The positively screened p73, a member of the p53 tumor suppressor protein family, is frequently overexpressed in cancer patients and correlates with increased tumor aggressiveness and therapy resistance. Apoptosis of p73-depleted U2OS cells is dramatically increased in the presence of cytarabine (> 50 %) compared to untreated p73-depleted cancer cells. We provide data that p73 and other screen hits enhance the PCNA-coupled post-replicative DNA repair pathway as the basis for cytarabine resistance. Analyzing the genome-wide gene expression profiles of 286 AML-patients with different karyotypes showed that our top screen hits in human osteosarcoma cells have a prognostic impact in AML-patients. Kaplan-Meier survival analysis showed that AML-patients with high-level expression of p73 displayed a significantly reduced overall survival compared to p73 low-expressing AML-patients. Together, these data may lead to a more individualized AML therapy, resulting in better treatment outcome.

Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.