Therapeutic Strategy For Acute Myeloid Leukemia Research Articles

Mechanisms of Antileukemic Activity of the Novel Bcl-2 Homology Domain-3 Mimetic GX15-070 (Obatoclax)

In this study, we investigated the mechanism of apoptosis induction of obatoclax (GX15-070), a novel Bcl-2 homology domain-3 (BH3) mimetic, in acute myeloid leukemia (AML) cell lines and primary AML samples. Obatoclax inhibited cell growth of HL-60, U937, OCI-AML3, and KG-1 cell lines. Apoptosis induction contributed to the observed antiproliferative effects at concentrations of this agent that mirror its affinity for antiapoptotic Bcl-2 proteins. We show that obatoclax can promote the release of cytochrome c from isolated leukemia cell mitochondria and that apoptosis induced by this agent is preceded by the release of Bak from Mcl-1, liberation of Bim from both Bcl-2 and Mcl-1, and the formation of an active Bak/Bax complex. Notably, apoptosis was diminished, but not fully prevented, in the absence of Bak/Bax or Bim, suggesting that obatoclax has additional targets that contribute to its cytotoxicity. At growth inhibitory doses that did not induce apoptosis or decrease viability, obatoclax induced an S-G(2) cell-cycle block. Obatoclax induced apoptosis in AML CD34+ progenitor cells with an average IC(50) of 3.59 +/- 1.23 micromol/L although clonogenicity was inhibited at concentrations of 75 to 100 nmol/L. Obatoclax synergized with the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells and synergistically induced apoptosis in combination with AraC in leukemic cell lines and in primary AML samples. In conclusion, we show that obatoclax potently induces apoptosis and decreases leukemia cell proliferation and may be used in a novel therapeutic strategy for AML alone and in combination with other targeted agents and chemotherapeutics.

MTOR Inhibitor Rapamycin Interacts Synergistically with Farnesyltransferase Inhibitor FTI-277 To Induce Growth Inhibition in Human Leukemia Cells.

Proteins regulating the mammalian target of rapamycin (mTOR), as well as some of the targets of the mTOR kinase, are overexpressed or mutated in cancer. Rapamycin inhibits the growth of cell lines derived from multiple tumor types in vitro, and tumor models in vivo. However, it has been suggested that substantial proportion of acute myeloid leukemia (AML) cells showed resistance to rapamycin-induced growth inhibition.Aim: We aim to investigate the effects of the farnesyltransferase inhibitor (FTI)-277 on the rapamycin-induced growth inhibition of human leukemia cells.Patients and methods: Flow cytometric evaluation and Western blot analysis for mTOR and Ras-like GTPase Rheb expression in the leukemia cell lines (HL60,NB4,THP1,KG1,U937) and primary leukemia cells obtained from AML patients were performed. We also observed the inhibition of cell growth and the changes in expression of mTOR and up- or down-streams of mTOR after mTOR inhibitor rapamycin treatment with or without FTI-277.Results: Both flow cytometric evaluation and Western blot analysis demonstrated that mTOR expression in the leukemia cell lines (HL60, NB4, THP1, KG1, U937) and primary leukemia cells obtained from AML patients were significantly higher compared to normal bone marrow mononuclear cells (p<0.001). Expression of Ras-like GTPase Rheb, a mTOR upstream, was also significantly increased in the leukemia cell lines and primary AML cells compared to normal bone marrow mononuclear (p<0.001 and p<0.005, respectively). We observed the inhibition rate of leukemia cell growth after treatment of cells with mTOR inhibitor rapamycin (100mM) in the absence or presence of farnesyltransferase inhibitor FTI-277 (10mM). Clonogenic cell growth in the leukemia cell lines was 69.3 ± 5.3% in the rapamycin group and 78.7 ± 4.4% in the FTI-277 group compared to that of the control group. Cotreatment of THP1 and HL-60 leukemia cells with rapamycin and FTI-277 exerted synergistic decrease in the clonogenic cell growth, as well as arrest at the G2/M phase of cell cycle, in a dose-and time-dependent manner (p<0.01). This was associated with marked attenuation of protein levels of Rheb, phospho-mTOR, and mTOR downstreams phospho-p70S6 kinase, phospho-4E-BP1. Interestingly decreased expression of mTOR upstreams Akt/PKB activity, Akt/PKB phosphorylation and PTEN phosphorylation was also observed in these leukemia cells after cotreatment with FTI-277 and rapamycin. These findings were also observed in the primary leukemia cells obtained from untreated patients with AML.Conclusions: Taken together, these findings indicate that farnesyltransferase inhibitor FTI-277 potentially enhance the growth-inhibitory property of rapamycin, with inducing multiple perturbations in PI3K - Akt/PKB - mTOR signaling pathway in human leukemia cells. Combined rapamycin and FTI blockade can exert powerful anti-leukemia effects and could be developed into a novel therapeutic strategy for AML.

Transcriptional dysregulation during myeloid transformation in AML

The current paradigm on leukemogenesis indicates that leukemias are propagated by leukemic stem cells. The genomic events and pathways involved in the transformation of hematopoietic precursors into leukemic stem cells are increasingly understood. This concept is based on genomic mutations or functional dysregulation of transcription factors in malignant cells of patients with acute myeloid leukemia (AML). Loss of the CCAAT/enhancer binding protein-alpha (CEBPA) function in myeloid cells in vitro and in vivo leads to a differentiation block, similar to that observed in blasts from AML patients. CEBPA alterations in specific subgroups of AML comprise genomic mutations leading to dominant-negative mutant proteins, transcriptional suppression by leukemic fusion proteins, translational inhibition by activated RNA-binding proteins, and functional inhibition by phosphorylation or increased proteasomal-dependent degradation. The PU.1 gene can be mutated or its expression or function can be blocked by leukemogenic fusion proteins in AML. Point mutations in the RUNX1/AML1 gene are also observed in specific subtypes of AML, in addition to RUNX1 being the most frequent target for chromosomal translocation in AML. These data are persuasive evidence that impaired function of particular transcription factors contributes directly to the development of human AML, and restoring their function represents a promising target for novel therapeutic strategies in AML.

Synergistic Cytotoxic Activity of Recombinant TRAIL Plus the Non-Genotoxic Activator of the p53 Pathway Nutlin-3 in Acute Myeloid Leukemia Cells

To potentiate the response of acute myeloid leukemia (AML) to TRAIL cytotoxicity, we have adopted a strategy of combining nutlin-3, a potent non-genotoxic activator of the p53 pathway, with recombinant TRAIL. The rationale for using such a combination was that deletions and/or mutations of the p53 gene occur in only 5-10% of AML and that TRAIL and nutlin-3 activate the extrinsic and intrinsic pathways of apoptosis, respectively. TRAIL induced a rapid increase of apoptosis when added to OCI M4-type and MOLM M5-type AML cells, carrying a wild-type p53, as well as to NB4 M3-type AML, carrying a mutated p53. On the other hand, the small molecule activator of the p53 pathway nutlin-3 induced p53 accumulation, cell cycle arrest and a slow progressive increase of apoptosis in OCI and MOLM but not in NB4. Of note, nutlin-3 up-regulated the surface expression of TRAIL-R2 and synergized with TRAIL in inducing apoptosis in OCI and MOLM as well as in primary M4-type and M5-type AML blasts, but not in NB4 cells. Moreover, while nutlin-3 up-regulated the expression of cyclin dependent kinase inhibitor p21, a p53-target gene mediating cell cycle block and showing anti-apoptotic activity, the simultaneous addition of TRAIL plus nutlin-3 induced the caspase-dependent cleavage of p21. The relevance of p21 down-regulation for sensitizing AML cells to apoptosis was underscored in knocking-down experiments with small interfering RNAs. Our data suggest that the combined treatment of nutlin-3 plus TRAIL might offer a novel therapeutic strategy for AML.

Inhibition of Bcl-2 Signaling by Small Molecule BH3 Inhhibitor GX15-070 as a Novel Therapeutic Strategy in AML.

GX15-070 is a novel cycloprodigiosin derived small molecule BH3 inhibitor that binds with moderate affinity to all antiapoptotic Bcl-2 family members, including Mcl-1, and is currently undergoing Phase I clinical trials in leukemias. In this study, we investigated the activity of GX15-070 in acute myeloid leukemia (AML) cell lines and primary AML samples. GX15-070 inhibited cell growth of HL-60, U937, OCI-AML3 and KG-1 cell lines at IC50's of 0.1, 0.5, 0.5 and 2.5μM, respectively, at 72 hours. Neither overexpression of Bcl-2 or Bcl-XL nor loss of expression of Bax conferred resistance to GX15-070. GX15-070 inhibited Bim/Bcl-2 heterodimerization and induced association of activated Bak with Bax in OCI-AML3 cells, as demonstrated by co-immunoprecipitation studies using CHAPS buffer. This was associated with cytosolic release of cytochrome c followed by an increase in annexin positivity, caspase activation and a decrease in mitochondrial inner membrane potential. Notably, GX15-070 induced cytochrome c release from isolated mitochondria of leukemic cells. GX15-070 synergized with both AraC and the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells, a notoriously chemoresistant cell line (GX15-070 and ABT-737 average CI value 0.3; GX15-070 and AraC average CI value 0.36). In 6/7 primary AML samples, GX15-070 induced apoptosis in CD34+ progenitor cells at an average IC50 of 3.6±1.2μM at 24 hours. GX15-070 potently inhibited clonogenic ability of AML blasts at sub-micromolar doses (58.5±10.6% CFU-Blast at 0.1μM and 38.1±10.5% at 0.25μM, n=7). In summary, BH3 inhibitor GX15-070 induces apoptosis in AML cells via inhibition of association of pro-survival Bcl-2 family proteins and BH3-only proteins, followed by Bax/Bak activation and initiation of the intrinsic apoptotic pathway. Hence, GX15-070 alone or in combination with chemotherapeutic agents may have utility in AML therapy.

Inhibition of Bcl-2 Signaling by Small Molecule BH3 Inhibitor GX15-070 as a Novel Therapeutic Strategy in AML.

GX15-070 is a novel cycloprodigiosin derived small molecule BH3 inhibitor that binds with moderate affinity to all antiapoptotic Bcl-2 family members, including Mcl-1, and is currently undergoing Phase I clinical trials in CLL. In this study, we investigated anti-leukemic activity of GX15-070 in acute myeloid leukemia (AML) cell lines and primary AML samples. GX15-070 inhibited cell growth of HL-60, U937, OCI-AML3 and KG-1 at IC50's of 0.1, 0.5, 0.5 and 2.5μM, respectively as determined by trypan blue exclusion method at 72 hours. Overexpression of Bcl-2 or Bcl-XL did not confer resistance to GX15-070 (IC50's of HL-60/neo, HL-60/Bcl-2 and HL-60/Bcl-XL cells 0.25μM). MDR-overexpressing HL-60/Dox cells were similarly sensitive compared to their non-MDR counterpart, and specific MDR inhibitor (PSC 833) did not enhance apoptosis caused by GX15-070. GX15-070 (250nM) inhibited Mcl-1/Bak heterodimerization in HL-60 cells starting at 3 hours as evidenced by IP/Western blot analysis, however it did not affect Bax/Bcl-2 heterodimerization. This was associated with cytosolic release of cytochrome c followed by an increase in annexin positivity (21±2.5% annexin + cells with 250nM) and a decrease in mitochondrial inner membrane potential with 64±4.8% of cells losing membrane potential at 72 hours. GX15-070 did not affect cell cycle distribution of HL-60 cells. In 6/7 primary AML samples, GX15-070 induced apoptosis in CD34+ progenitor cells at an average IC50 of 3.6±1.2μM at 24 hours. In conclusion, the small molecule BH3 inhibitor GX15-070 potently induces apoptosis in myeloid leukemia cells via disruption of Mcl-1/Bak dimerization and activation of the intrinsic apoptotic cascade. Our findings provide rationale for the further development of this BH3 inhibitor as a novel therapeutic strategy for AML.

MDM2 antagonists induce p53-dependent apoptosis in AML: implications for leukemia therapy

AbstractAlthough TP53 mutations are rare in acute myeloid leukemia (AML), inactivation of wild-type p53 protein frequently occurs through overexpression of its negative regulator MDM2 (murine double minute 2). Recently, small-molecule antagonists of MDM2, Nutlins, have been developed that inhibit the p53-MDM2 interaction and activate p53 signaling. Here, we study the effects of p53 activation by Nutlin-3 in AML cells. Treatment with MDM2 inhibitor triggered several molecular events consistent with induction of apoptosis: loss of mitochondrial membrane potential, caspase activation, phosphatidylserine externalization, and DNA fragmentation. There was a positive correlation in primary AML samples with wild-type p53 between baseline MDM2 protein levels and apoptosis induced by MDM2 inhibition. No induction of apoptosis was observed in AML samples harboring mutant p53. Colony formation of AML progenitors was inhibited in a dose-dependent fashion, whereas normal CD34+ progenitor cells were less affected. Mechanistic studies suggested that Nutlin-induced apoptosis was mediated by both transcriptional activation of proapoptotic Bcl-2 family proteins, and transcription-independent mitochondrial permeabilization resulting from mitochondrial p53 translocation. MDM2 inhibition synergistically enhanced cytotoxicity of cytosine arabinoside and doxorubicin in AML blasts but not in normal hematopoietic progenitor cells. p53 activation by targeting the p53-MDM2 interaction might offer a novel therapeutic strategy for AML that retain wild-type p53.

Inhibition of Bcl-2 Signaling at Nanomolar Concentrations by Small Molecule BH3 Inhibitor, ABT-737 as a Novel Therapeutic Strategy in Acute Myeloid Leukemia (AML).

In this study, we investigated the anti-leukemic activity of a novel small molecule BH3 inhibitor (ABT-737) that binds with high affinity to Bcl-2, Bcl-xL and Bcl-w (Ki < 1nM). ABT-737 inhibited 50% of cell growth of HL-60, NB4 and KG-1 cells at IC50's below 100nM, while its less active enantiomer did not affect cell growth at concentrations of 1μM. The dose-response curve was shifted to the right in cells over-expressing the drug targets, Bcl-2 or Bcl-XL (IC50 for HL-60/neo - 1μM, HL-60/BclXL - 4μM and HL-60/Bcl-2 - 5μM). ABT-737 inhibited Bcl-2/Bax heterodimerization starting at 1 hour as evidenced by IP/Western blot analysis. Furthermore, Bax cleavage (at 3 hours) resulted in pro-apoptotic Bax-p18 and later a change to “death conformation” of Bax. This resulted in mitochondrial depolarization and cleavage of caspases-9, -3 and -8. Bax-knockout Hct116 cells were largely protected from cytolytic activity of ABT-737 demonstrating the critical role of Bax in apoptosis induction. In primary AML samples (n=9), 100 nM ABT-737 induced apoptosis in 70±7% of CD34+ progenitor cells (DMSO-control, 28±4%, p<0.001) at 24 hrs. ABT-737 inhibited clonogenic growth of AML with IC50 of 25nM (n=3) but spared normal progenitors n=3 (no inhibition at 100nM). To investigate the role of Bcl-2 phosphorylation in the sensitivity to BH3 inhibitor, we used IL-3 dependent NSF.N1/H7 mouse myeloid cells modified by site-directed mutagenesis to produce various Bcl-2 phospho-mutants. NSF.N1/H7 cells stably transfected with phosphomimetic T69E/S70E/S87E (EEE) Bcl-2 mutants were resistant to ABT-737 (IC50>500nM) as compared to cells expressing wt-Bcl-2 or the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutants (IC50s of 50 and 25nM). This suggests that phosphorylation-dependent conformational change of Bcl-2 affects the ability of BH3 inhibitors to disrupt Bcl-2/Bax dimers and induce apoptosis. Since the S70E phosphorylation site of Bcl-2 is a known ERK substrate, we examined combined effects of ABT-737 and MEK inhibitor PD98059 in OCI-AML3 cells resistant to ABT-737 alone. The combined activity of PD98059 and ABT-737, evaluated by isobologram analysis, revealed a striking synergistic interaction between the MEK and BH3 inhibitors, with combination indices (CI) of 0.09 to 0.56. The hypothesis that phosphorylation-dependent conformational change of Bcl-2 affects the ability of BH3 inhibitors to disrupt Bcl-2/Bax dimers and induce apoptosis is presently under investigation. Inhibition of MEK signaling may therefore enhance the efficacy of BH3 inhibitors in AML and possibly other malignancies with phosphorylated Bcl-2. In conclusion, small molecule BH3 inhibitors are potent inducers of apoptosis in myeloid leukemias via disruption of Bcl-2/Bax dimerization and activation of the downstream apoptotic cascade. Differential inhibition of AML but not normal progenitor cells at low nanomolar concentrations advocates further development of BH3 inhibitors as a novel therapeutic strategy for AML.

Therapeutic targeting of the MEK/MAPK signal transduction module in acute myeloid leukemia.

The mitogen-activated protein kinase (MAPK) pathway regulates growth and survival of many cell types, and its constitutive activation has been implicated in the pathogenesis of a variety of malignancies. In this study we demonstrate that small-molecule MEK inhibitors (PD98059 and PD184352) profoundly impair cell growth and survival of acute myeloid leukemia (AML) cell lines and primary samples with constitutive MAPK activation. These agents abrogate the clonogenicity of leukemic cells but have minimal effects on normal hematopoietic progenitors. MEK blockade also results in sensitization to spontaneous and drug-induced apoptosis. At a molecular level, these effects correlate with modulation of the expression of cyclin-dependent kinase inhibitors (p27(Kip1) and p21(Waf1/CIP1)) and antiapoptotic proteins of the inhibitor of apoptosis proteins (IAP) and Bcl-2 families. Interruption of constitutive MEK/MAPK signaling therefore represents a promising therapeutic strategy in AML.