Class â…  Histone Deacetylases Regulate BRCA1, CHK1, And RAD51Expression In Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is the most common form of acuteleukemia in adults and the second most common form of acute leukemiain Children. Ara-C (cytosine arabinoside) and daunorubicin (DNR) arethe two main drugs used for treating AML, and resistance to the twodrugs remains the leading cause of treatment failure in AML. Therefore,introducing new agents to enhance the activity and/or reduce the toxicityof the conventional chemotherapy drugs has theoretical and practicalsignificance.Histone deacetylase inhibitors (HDACIs) have showedanticanceractivitiesagainst numerous malignancies including AML and haveattracted significant attention of researchers in the cancer research field.Four novel HDACIs, Vorinostat (Suberoylanilide hydroxamic acid,SAHA), depsipeptide, belinostat, and panobinostat, have been approvedby the US Food and Drug Administration (US FDA) for treatinghematological malignancies. In addition, at least a dozen other HDACIsare in the early stages of clinical development for treating cancer.HDACIs have been demonstrated to induce cell cycle arrest,differentiation， and apoptosis, but less so in normal cells. Therefore,HDACIs represent a promising new class of agents for treating AML.Though single-agent efficacy of HDACIs in the clinic has been modest, HDACIs can significantly enhance the anti-tumor activities ofconventional chemotherapeutic drugs or other molecularly targeted drugs.Developing combination therapies with HDACIs has been a hot researcharea in the cancer research field.In a recent study, we demonstrated that treatment of AML cells withpanobinostat, a pan-HDACI which has recentlybeen approved by the USFDA for the treatment of multiple myeloma, resulted in decreased proteinand mRNA levels for BRCA1, CHK1，and RAD51, which are criticalfactors in the DNA damage response network. It also partially abolishedara-C-and DNR-induced S and/or G2/M cell cycle checkpoints, andenhanced ara-C-and DNR-induced DNA damage， and apoptosis.However, it remains unknown which HDAC isoforms are responsible forthe downregulation of BRCA1, CHK1，and RAD51in AML cells treatedwith panobinostat. The main focus of this study wasto identify the HDACisoforms which regulate the expression of BRCA1, CHK1， and RAD51in AML cells.First, we used class-selective HDACIs to determine the roles ofdifferent classes of HDACs in the regulation of the expression of BRCA1,CHK1, and RAD51in AML cells. We found that the class I-selectiveHDACI, MGCD0103which targets HDACs1,2， and3, candown-regulate the protein and mRNA levels for BRCA1, CHK1, andRAD51. Consistent with the results obtained with panobinostat, MGCD0103treatment also partially abolished ara-C-and DNR-inducedS and/or G2/M cell cycle arrest, accompanied by significant enhancementof ara-C-and DNR-induced DNA damage，and apoptosis in AML cells.However, treatment of AML cells with a class IIa-selective HDACI,MC1568, and a HDAC6-selective inhibitor, Tubustatin A, resulted in noalterations of the protein levels for BRCA1, CHK1, and RAD51. Theseresults demonstrate that the simultaneous inhibition of HDACs1,2， and3can cause downregulation of BRCA1, CHK1, and RAD51, leading toabolishment of ara-C-and DNR-induced S and/or G2/M cell cyclecheckpoint activation and enhancement of ara-C-and DNR-induced DNAdamage, and apoptosis in AML cells.To further confirm the results mentioned above, immunoprecipitationof class I HDACs (HDACs1,2,3, and8) was performed, followed byHDAC enzymatic assays. As expected, MGCD0103showed specificinhibition of HDACs1,2, and3, but not HDAC8, confirming thatMGCD0103selectively targets HDACs1,2, and3。Therefore,this furtherconfirms that the simultaneous inhibition of HDACs1,2, and3can causedownregulation of BRCA1, CHK1, and RAD51in AML cells. Ourexploratory studies of the molecular mechanism underlying thedownregulation of BRCA1, CHK1, and RAD51by inhibition of HDACs1,2, and3suggested that transcription factor E2F1is a mediator of thisprocess. To further determine which of HDACs1,2, and3arerequired for theregulation of the expression of BRCA1, CHK1, and RAD51in AML cells,the individual lentiviral shRNA knockdowns of HDAC1,2,and3wereperformed. Interestingly, individual knockdowns of these HDACs had noimpact on the protein or mRNA levels for BRCA1, CHK1, and RAD51.In addition, treatment of the HDAC1and HDAC2knockdown cells witha HDAC3-selective inhibitor, RGFP966, did not affect the expressionlevels of BRCA1, CHK1, and RAD51either. These results indicate thatHDAC1and HDAC2or all the three HDACs are involved in theregulation of the expression of BRCA1, CHK1, and RAD51in AML cells.However, this needs to be further determined in future studies.In summary, we have demonstrated that the simultaneous inhibitionof HDAC1,2, and3can cause downregulation of BRCA1, CHK1, andRAD51, leading to abolishment of ara-C-and DNR-induced S and/orG2/M cell cycle checkpoint activation, and enhancement of ara-C-andDNR-induced DNA damage, and apoptosis. Further studies indicated thatboth HDACs1and2are involved in the regulation of the expression ofBRCA1, CHK1, and RAD51in AML cells. However, additionalexperiments are needed to further determine the role of HDAC3in thisprocess. The success of this study will shed important light on thedesigning of more selective HDACIs and the clinical use of HDACIs forthe treatment of AML.