The Histone Deacetylase Inhibitor Panobinostat (LBH-589) Exerts Anti-Leukaemic Activity in a MLL-Rearranged ALL Xenograft Mouse Model

BACKGROUND: Infant acute lymphoblastic leukaemia (ALL) is a rare but aggressive malignancy, mainly presenting with chromosomal rearrangements of the MLL (Mixed Lineage Leukaemia) gene locus on 11q23. The majority of these MLL rearrangements involve the translocation partners AF4, AF9 or ENL within the translocation events t(4;11)(q21;q23), t(9;11)(p22;q23) and t(11;19)(q23;p13.3), respectively. The resulting fusion genes, MLL-AF4, MLL-AF9 and MLL-ENL, code for chimeric transcription regulators acting as strong oncogenic drivers, rewriting the epigenetic landscape of the cell and profoundly altering gene expression. Consequently, these cytogenetic lesions define an ALL subtype both biologically and clinically distinct from other subtypes, strongly associated with drug resistance to first-line chemotherapeutics, high relapse rates and a dismal prognosis. Hence, novel treatment strategies which specifically target the underlying molecular pathobiology of this disease are urgently needed.

AIMS: Previously, our group performed extensive patient cohort profiling on both transcript and epigenetic level in order to understand the molecular events underlying the disease, and identified histone deacetylase inhibitors (HDACi) as effective therapeutic drugs both in silico and in vitro. The aim of the current study was to elucidate potential molecular mechanisms by which the candidate HDACi Panobinostat is able to target MLL-rearranged ALL (MLLr-ALL) cells, and to confirm its efficacy in vivo using pre-clinical MLLr-ALL xenograft mouse models able to recapitulate the disease phenotype observed in humans.

METHODS: Immunodeficient NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice were injected intrafemurally with a MLL-AF4+ B-ALL cell line (SEM) genetically modified to express a luciferase reporter. These mice were subsequently either treated with low-dose (1mg/kg) or high-dose (5mg/kg) Panobinostat using a continuous 5-day-on-2-day-off regimen for a period of up to 12 weeks, or they were assigned to a control group and left untreated. Disease onset and progression was monitored using in vivo bioluminescence imaging, and systemic human ALL cell infiltration was determined by multi-colour flow cytometry and histochemistry. In addition, molecular changes induced by Panobinostat exposure in MLLr-ALL and non-MLLr-ALL cell lines were assessed in vitro using immunoblotting and cell death assays.

RESULTS: High-dose Panobinostat resulted in a significantly and substantially delayed MLLr-ALL disease onset and progression in NSG mice when compared to controls; this was accompanied by a reduction of the systemic disease burden, as evidenced by significantly lower whole-body luminescence signals and substantially decreased splenomegaly. Furthermore, immunohistochemical and flow cytometric data showed hypocellularity and increased cell death in the BM of xenografted NSG mice treated with Panobinostat when compared to untreated control xenografts. This finding correlated well with in vitro results, where exposure with 5 nM Panobinostat induced cell death in MLLr-ALL cells, but not in non-MLLr ALL cells, as determined by both ANNEXINV/7AAD flow cytometry assays and immunoblotting. In addition, on a molecular level, in vitro exposure with Panobinostat induced histone H3 hyperacetylation in all leukaemic cell lines, but did not affect other histone modification marks investigated such as, i.e., histone H3K4 methylation or histone H3K79 methylation. A notable exception was observed in MLLr-ALL cell lines, where Panobinostat exposure correlated with a reduction in histone H2B ubiquitination, a histone modification recently reported to be pivotal for MLLr leukaemogenesis. Concomitantly, Panobinostat - or more generally - HDACi-mediated loss of H2B ubiquitination might play a role in the observed sensitivity of MLLr-ALL cell towards this drug class.

CONCLUSIONS: Both the in vivo and the molecular in vitro results show the HDACi Panobinostat to have promising therapeutic potential against MLLr-ALL. Currently, we are investigating Panobinostat in combination with other epigenetic drugs in xenograft models with primary MLLr-ALL patient material in order to consolidate these observations, and to confirm HDACi as a novel powerful treatment strategy in MLLr-ALL.