ABT-737 Targets Intrinsic Apoptosis during Cooperation of BCL-2 and Oncogenic NRAS in An in Vivo Progression Model of Myelodysplasia/Acute Myeloid Leukaemia

OBJECTIVES: Activating RAS mutations and over-expression of BCL-2 are prognostic features of myelodysplastic syndromes/acute myeloid leukemia (MDS/AML) transformation. Using NRASD12 and BCL-2, we created two distinct transplantable in vivo models of MDS and AML. Expression of hBCL-2 in a primitive compartment by MMTV-LTR results in a disease resembling human MDS with bone marrow blasts of 15% with increased apoptosis assayed by TUNEL on liver sections, whilst the myeloid MRP8 promoter induces a disease with characteristics of human AML with marrow blasts of up to 90% with liver apoptosis patterns similar to wild type. In MDS mice RAS and BCL-2 do not co-localize in the mitochondria, but localize to the plasma membrane, where active pro-apoptotic RAS is normally located, whereas in the AML disease RAS and BCL-2 co-localize in the mitochondria, where BCL-2 is normally found; consistent with its anti-apoptotic properties. We next examine the mouse hematopoietic FDCP-1 in vitro model with stable exogenous expression of NRASD12, hBCL-2 or NRASD12 and hBCL-2. Furthermore, we report the in vivo effects of the BH-3 mimetic inhibitor ABT-737.

RESULTS: FDCP-1 lines demonstrate that whilst hBCL-2 alone prevents staurosporine-induced mitochondrial-dependent (caspase-9-mediated intrinsic) apoptosis, both NRAS-D12 and NRASD12/hBCL-2 cell lines were pro-apoptotic. No significant difference was observed in cell-death receptor (caspase-8-mediated extrinsic) apoptosis between the cell lines following pharmacological induction with Fas-ligand or TRAIL. Annexin-V/Propidium Iodide flow cytometry and caspase activity assays on hematopoietic primary cells from the mouse models recapitulated the findings; namely the MDS-model demonstrates increased caspase-9-mediated apoptosis (within the Lin⁻/Sca-1⁺/KIT⁺ (LSK) sub-population), whilst the AML-model illustrate anti-apoptotic activity. This is concordant with the signaling profile where phosphorylated AKT is reduced in the RAS-mediated MDS mice and active-AKT is increased in the BCL-2 mediated AML disease. Expanded leukemic stem cell LSK populations had increased hBCL-2 expression in the RAS-GTP complex in both MDS/AML diseases. Thus we suggest that this complex can be a specific target for therapy. When hBCL-2 is switched off with doxycycline in the conditional MDS mice, only partial reversal of the phenotype was observed as RAS recruits endogenous mouse BCL-2 to remain active; thus demonstrating the role of the complex in the disease. In order to target both human and mouse BCL-2 the efficacy of in vivo treatment with the specific BH-3 mimetic inhibitor ABT-737 was determined. We show that the treatment significantly reduced the progression of the disease, increased the peripheral blood platelet counts, decreased the bone marrow blast and cleared the tissue invasion in the MDS mice or reduced tissue infiltration of the AML. In vivo imaging by single-photon emission computed tomography (SPECT) using ^99mTc-labelled Annexin-V shows that ABT-737 induces apoptosis of the blast cells that infiltrate the liver and the spleen of the treated mice, which was confirmed by TUNEL on liver sections. Additionally, ABT-737 can reduce the myeloid colony growth and the LSK expansion in these mice; whilst abrogating BCL-2:RAS-GTP complex formation illustrated via biochemical and confocal assays.

CONCLUSION: This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex rescued by ABT-737 via intrinsic apoptosis and thus support the case for BH-3 mimetic therapy.