Panobinostat (LBH589) a Promising New Partner for Combination with Doxorubicin in Acute Myeloid Leukemia.
Authors: Maiso Patricia , Colado Enrique , Ocio Enrique M , Garayoa Mercedes , Atadja Peter , Pandiella Atanasio , Miguel J. F. San 
Published: April 2020

Abstract
Background and Aims: The combination of cytarabine with an anthracyclin has been the gold standard for the induction treatment of acute myeloid leukaemia (AML) for the last three decades. Nevertheless, 20–50% of patients fail to respond to this scheme and among those who achieve complete response (CR) the relapse rate come close to 50%. In the present study, we have investigated the potential value of panobinostat (LBH589) for the treatment of AML. This drug has already demonstrated antileukemic activity. Nevertheless, since there is a large body of evidence indicating that AML treatment requires drug combination, we analysed the potential synergism of panobinostat with other well established anti-AML agents.
Material and Methods: The efficacy of panobinostat and of its combination with each one of three other agents (cytarabine, doxorubicin and fludarabine) was analyzed both in vitro, (in four AML cell lines: HEL, HL-60, KG-1 and MV4.11, by MTT) and ex vivo (in freshly isolated cells from 6 AML patients by flow cytometry). In addition the toxicity in normal hematopoietic cells was analyzed. The mechanism of action was investigated by Annexin V, cell cycle profile, DioC6 staining, Western Blot and gene expression profile (GEP) analysis by microarrays.
Results: Panobinostat potently suppressed the viability of AML cells. IC50 values were 9 nM (HEL), 7 nM (HL-60), 17 nM (KG1), and 6 nM (MV4-11). Comparison of the IC50 of panobinostat with other drugs commonly used in AML, indicated that panobinostat was more potent than cytarabine, fludarabine, and doxorubicin (IC50 = 740 nM, 362 nM and 220 nM respectively, for HEL cells). Panobinostat increased the anti-AML effect of cytarabine and fludarabine. Nevertheless, the most significant effect was observed for the combination with doxorubicin. The CI range values were 0.05–0.41 in all cell lines, and accordingly, remaining experiments focused on this combination. This efficacy was also confirmed in ex vivo experiments. Using quadruple staining (annexin V-FITC/CD33-PE/CD45-PerCP/CD34-APC), we identify and distinguish the blast cell population (CD34−/+, CD33−/+, CD45dim) from the normal residual lymphocytes (CD45+, SSClo) and quantify the number of apoptotic cells in each cell population. In all six cases a potentiation was observed with P+D. Interestingly no effect was observed in terms of toxicity on non leukemic residual hematopoietic cells from the same patients’ samples. An important question to be asked, upon using drug combinations, is whether the genes deregulated by the combination just represent the sum of those targeted by each of the drugs or if the drug combination is inducting new targeting pathways. In order to answer this question we compared the GEP of HEL cells exposed to the P+D with those as single agents. While there were 285 genes deregulated with panobinostat and 43 with doxorubicin after 24 hours with each drug; 12 hours of treatment with P+D resulted in the deregulation of 841 genes. Accordingly, 588 genes were exclusively deregulated after P+D treatment, indicating that panobinostat and doxorubicin affect different groups of genes and pathways. The two most significantly deregulated functional categories were genes involved in the control of cell cycle and apoptosis. Treatment with P+D down-regulated Cyclin B1 (−3.04), AVEN (−2.58), Bcl-X (−2.96), TNFRSF25 (−3.48) or HSPA5 (−3.87), and up-regulated the levels of Cyclin G2 (5.49), BTG1 (5.47), or BNIP3L (2.17). c-jun was upregulated after treatment with panobinostat (3.77) and particularly with doxorubicin (26.30), and the upregulation was even higher upon treatment the AML cells with the combination of both drugs (58.38). Mechanistic experiments showed that P+D activated apoptosis (Annexin V staining and PARP- and caspases-cleavage by Western Blot) at concentrations that did not induce any cytotoxic effect when panobinostat and doxorubicin were used as single agents. P+D activated the intrinsic pathway of apoptosis with loss of mitochondrial membrane potential and subsequent release of Cytochrome C to the cytoplasm. A decrease in MCL-1 and BCL-X cleavage was also observed with the combination while not with the single agents. Interestingly, P+D also induced cell cycle arrest.
Conclusion: Panobinostat + doxorubicin show a marked synergistic activity against AML cells, with unique mechanism of action, and represent a most attractive combination for clinical investigation.