In Vivo RNAi Screening Identifies HDAC4 As a Mediator Of Chemoresistance In Acute Myeloid Leukemia

The majority of AML patients still relapse, despite intensive chemotherapy. A subset of more chemoresistant leukemia cells might be responsible for this phenomenon. These relapse-initiating cells might differ epigenetically from the more chemosensitive leukemia bulk. Since interaction of leukemic cells with the niche might contribute to chemoresistance, we decided to perform an in vivo RNAi screen for modulators of chemoresistance. We constructed a focussed shRNA library with ∼400 constructs that target 142 genes involved in stem cell functions, leukemogenesis and epigenetic regulation. Myc/Bcl2 induced murine leukemias were retrovirally transduced and injected into sublethally irradiated syngeneic mice. Mice (n=24) were either treated with Cytarabine, Azacytidine, Decitabine, or vehicle. Upon signs of leukemia, DNA was extracted and the shRNA pool was PCR-amplified from bone marrow, spleen and liver of each individual mouse. Overall, 61 libraries were sequenced with more than 500-fold coverage of each construct. Suppression of HDAC4, Pik3r1, Atr, Ccna2 and HDAC1 significantly enhanced sensitivity towards Cytarabine treatment. Histone deacetylase 4 (HDAC4) was identified as a top candidate since it enhanced the efficacy of all three drugs. It was depleted more than sixteen-fold upon Cytarabine treatment. HDAC4 suppression by the specific shRNAs was confirmed by real-time RT-PCR and western blot analysis. In a competitive re-transplantation assay in vivo, HDAC4 suppression inhibited leukemic growth. Further, we analyzed inhibition of HDAC4 by LMK235, a novel and specific inhibitor of HDAC4 and 5 in human HL60 leukemia cells. Synergistic inhibition was demonstrated for LMK235 and Cytarabine in proliferation assays and in colony formation assays. These findings demonstrate that in vivo RNAi screening for therapeutic efficacy is feasible. HDAC4 might be an important target to enhance efficacy of anti-leukemic drugs.