Preclinical Activity of LBH589 Alone or in Combination with Chemotherapy in a Xenogeneic Mouse Model of Human Acute Lymphoblastic Leukemia

Abstract 1520

Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here we analyzed the therapeutic effect of LBH589 or panobinostat, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL).

In vitro, LBH589 induced a significant dose-dependent increase in cell apoptosis and a markedly inhibition of cell proliferation, which were associated with increased H3 and H4 histone acetylation. While apoptosis of ALL cells was detected between 12 and 24 hours after treatment with LBH589, changes in acetylated H3 and H4 were detected as early as 2 hours. Phosphorylation of H2AX, as an early marker of DNA damaged, was detected 12 to 24 hours after in vitro treatment with LBH589. These results suggest that H3 and H4 acetylation precede DNA damaged and induction of apoptosis indicating that inhibition of HDAC is likely to be responsible at least in part for LBH589 induced apoptosis and inhibition of cell proliferation.

The in vivo activity of LBH589 was initially examined in a subcutaneous ALL mouse model. The ALL cell lines TOM-1 and MOLT-4 were transplanted (1×10⁶ cell per animal) subcutaneously into the left flanks of 6-week-old female BALB/cA-Rag2^−/−γc^−/−. These cell lines develop into a rapidly growing tumor. Treatment with 5mg/kg of LBH589 was initiated 24 hours after injection of the leukemic cells, included 3 cycles of 5 consecutive days of LBH589 with two days rest between cycles and animals were monitored for 24 days. A significant inhibition of tumor growth was demonstrated in animals treated with LBH589 compared with control animals (P <0.01). Inhibition of leukemia cell growth was associated with an increase in the levels of acetylated H3 and H4 and an increase in phosphorylated H2AX in the leukemic cells obtained after sacrifice of mice. These results suggest that LBH589 has a powerful antileukemic effect not only in vitro but also in vivo.

Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2^−/−γc^−/− mice was established, allowing continuous passages of transplanted cells to several mouse generations. A total of 10 million cells from a patient with T-ALL (ALL-T1) and a patient with B-ALL (ALL-B1) were administered intravenously into the tail vein of 6-week-old immunodeficient female BALB/cA-Rag2^−/−γc^−/− mice. Kinetics of engraftment of leukemic cells was monitored in PB and BM by phenotyping while organ infiltration was analyzed by immunohistochemistry. There were no significant differences in the genome, methylome or transcriptome between the original sample and the samples obtained after multiple generations on mice.

To determine the efficacy of LBH589 alone or in combination with drugs currently used for treatment of ALL, BALB/cA-RAG2^−/−γc^−/− mice engrafted with ALL-T1 and ALL-B1 cells were treated with LBH589, Vincristine and Dexamethasone or a combination of LBH589 with Vincristine and Dexamethasone. Treatment was initiated when disease could be detected in PB by FACS (24 hours after injection of cells for ALL-T1 and between day 17 and 21 after injection for ALL-B1). LBH589 was administered i.p. on days 1–5, 8–12 and 15–19, Vincristine i.v. on days 1, 8 and 21 and Dexamethasone daily until day 21 i.p. and survival was analyzed. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving Vincristine and Dexametasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with Vincristine and Dexametasone.

Our results demonstrate the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL.