BCL-2 Inhibitor ABT-737 Effectively Targets Leukemia-Initiating Cells with Differential Regulation of Relevant Genes Leading to Extended Survival in a NRAS/BCL-2 Mouse Model of High Risk-Myelodysplastic Syndrome.

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.

GEP analysis validates high risk MDS and acute myeloid leukemia post MDS mice models and highlights novel dysregulated pathways.

BACKGROUND: In spite of the recent discovery of genetic mutations in most myelodysplasic (MDS) patients, the pathophysiology of these disorders still remains poorly understood, and only few in vivo models are available to help unravel the disease. METHODS: We performed global specific gene expression profiling and functional pathway analysis in purified Sca1+ cells of two MDS transgenic mouse models that mimic human high-risk MDS (HR-MDS) and acute myeloid leukemia (AML) post MDS, with NRASD12 and BCL2 transgenes under the control of different promoters MRP8NRASD12/tethBCL-2 or MRP8[NRASD12/hBCL-2], respectively. RESULTS: Analysis of dysregulated genes that were unique to the diseased HR-MDS and AML post MDS mice and not their founder mice pointed first to pathways that had previously been reported in MDS patients, including DNA replication/damage/repair, cell cycle, apoptosis, immune responses, and canonical Wnt pathways, further validating these models at the gene expression level. Interestingly, pathways not previously reported in MDS were discovered. These included dysregulated genes of noncanonical Wnt pathways and energy and lipid metabolisms. These dysregulated genes were not only confirmed in a different independent set of BM and spleen Sca1+ cells from the MDS mice but also in MDS CD34+ BM patient samples. CONCLUSIONS: These two MDS models may thus provide useful preclinical models to target pathways previously identified in MDS patients and to unravel novel pathways highlighted by this study.

DNA-mediated adjuvant immunotherapy extends survival in two different mouse models of myeloid malignancies.

We have previously shown that a specific promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) DNA vaccine combined with all-trans retinoic acid (ATRA) increases the number of long term survivors with enhanced immune responses in a mouse model of acute promyelocytic leukemia (APL). This study reports the efficacy of a non-specific DNA vaccine, pVAX14Flipper (pVAX14), in both APL and high risk myelodysplastic syndrome (HR-MDS) models. PVAX14 is comprised of novel immunogenic DNA sequences inserted into the pVAX1 therapeutic plasmid. APL mice treated with pVAX14 combined with ATRA had increased survival comparable to that obtained with a specific PML-RARA vaccine. Moreover, the survival advantage correlated with decreased PML-RARA transcript levels and increase in anti-RARA antibody production. In HR-MDS mice, pVAX14 significantly improved survival and reduced biomarkers of leukemic transformation such as phosphorylated mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1. In both preclinical models, pVAX14 vaccine significantly increased interferon gamma (IFNγ) production, memory T-cells (memT), reduced the number of colony forming units (CFU) and increased expression of the adapter molecule signalling to NF-κB, MyD88. These results demonstrate the adjuvant properties of pVAX14 providing thus new approaches to improve clinical outcome in two different models of myeloid malignancies, which may have potential for a broader applicability in other cancers.

BCL-2 inhibition with ABT-737 prolongs survival in an NRAS/BCL-2 mouse model of AML by targeting primitive LSK and progenitor cells.

Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1(+)/c-Kit(+), and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells.

Tracking the extramedullary PML-RARα-positive cell reservoirs in a preclinical model: biomarker of long-term drug efficacy.

Using an acute promyelocytic leukemia (APL) preclinical model, we show that oncogene-specific PCR (Polymerase Chain Reaction)-based assays allow to evaluate the efficacy of immunotherapy combining all-trans retinoic acid (ATRA) and a DNA-based vaccine targeting the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) oncogene. Kaplan-Meier survival analysis according to the peripheral blood PML-RARα normalized copy number (NCN) clearly shows that ATRA + DNA-treated mice with an NCN lower than 10 (43%) formed the group with a highly significant (p < 0.0001) survival advantage. Furthermore, a PCR assay was used to assess various tissues and organs for the presence of PML-RARα-positive cells in long-term survivors (n = 15). As expected, the majority of mice (n = 10) had no measurable tissue level of PML-RARα. However, five mice showed a weak positive signal in both the brain and spleen (n = 2), in the brain only (n = 2) and in the spleen only (n = 1). Thus tracking the oncogene-positive cells in long-term survivors reveals for the first time that extramedullary PML-RARα-positive cell reservoirs such as the brain may persist and be involved in relapses.

Localization of the NRAS:BCL-2 complex determines anti-apoptotic features associated with progressive disease in myelodysplastic syndromes.

We have previously demonstrated that two prognostic features of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML), mutant NRAS and over-expressing BCL-2, cooperate physically and functionally in vivo. Screening of MDS patient bone marrow (BM) identified NRAS:BCL-2 co-localization in 64% cases, correlating with percentage BM blasts, apoptotic features and disease status (p<0.0001). Localization of the complex at the plasma membrane or the mitochondria correlated with disease and apoptosis features in MDS patients, whilst caspase-9 mediated mechanism was elucidated in vivo and in vitro. The intensity and localization of the RAS:BCL-2 complex merits further evaluation as a novel biomarker of MDS.

DNA vaccination with all-trans retinoic acid treatment induces long-term survival and elicits specific immune responses requiring CD4+ and CD8+ T-cell activation in an acute promyelocytic leukemia mouse model.

DNA vaccination and all-trans retinoic acid (ATRA) result in a survival advantage in a mouse model of acute promyelocytic leukemia (APL). Depletion of CD4(+) or CD8(+) cells abolished this effect. CD4(+) depletions of long-term survivors resulted in relapse and death within 3 months, thus demonstrating the need of both CD4(+) and CD8(+) subsets for the generation of DNA-driven antileukemic immune responses and underscoring a crucial role of CD4(+) cells in the maintenance of durable remissions. Degranulation and cytotoxic carboxyfluorescein diacetate succinimidyl ester-based assays showed major histocompatibility complex-restricted APL-specific T cell-mediated immune responses. Sorted APL-specific CD8(+)CD107a(+) T cells showed an increase of antileukemic activity. Effectors from ATRA + DNA-treated mice were shown to secrete interferon-gamma when stimulated with either APL cells or peptides from the promyelocytic leukemia-RARalpha vaccine-derived sequences as detected by ELISpot assays. Our results demonstrate that DNA vaccination with ATRA confers the effective boosting of interferon-gamma-producing and cytotoxic T cells in the leukemic mice.

BCL-2 and mutant NRAS interact physically and functionally in a mouse model of progressive myelodysplasia.

Myelodysplastic syndromes (MDS) are clonal stem cell hematologic disorders that evolve to acute myeloid leukemia (AML) and thus model multistep leukemogenesis. Activating RAS mutations and overexpression of BCL-2 are prognostic features of MDS/AML transformation. Using NRASD12 and BCL-2, we created two distinct models of MDS and AML, where human (h)BCL-2 is conditionally or constitutively expressed. Our novel transplantable in vivo models show that expression of hBCL-2 in a primitive compartment by mouse mammary tumor virus-long terminal repeat results in a disease resembling human MDS, whereas the myeloid MRP8 promoter induces a disease with characteristics of human AML. Expanded leukemic stem cell (Lin(-)/Sca-1(+)/c-Kit(+)) populations and hBCL-2 in the increased RAS-GTP complex within the expanded Sca-1(+) compartment are described in both MDS/AML-like diseases. Furthermore, the oncogenic compartmentalizations provide the proapoptotic versus antiapoptotic mechanisms, by activating extracellular signal-regulated kinase and AKT signaling, in determination of the neoplastic phenotype. When hBCL-2 is switched off with doxycycline in the MDS mice, partial reversal of the phenotype was observed with persistence of bone marrow blasts and tissue infiltration as RAS recruits endogenous mouse (m)BCL-2 to remain active, thus demonstrating the role of the complex in the disease. This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex. The colocalization of BCL-2 and RAS in the bone marrow of MDS/AML patients offers targeting either oncogene as a therapeutic strategy.