Cytokine-regulated expression of survivin in myeloid leukemia

: Survivin, a member of the inhibitors-of-apoptosis gene family, is expressed in a cell-cycle-dependent manner in all the most common cancers but not in normal differentiated adult tissues. Survivin expression and regulation were examined in acute myeloid leukemia (AML). Survivin was detected by Western blot analysis in all myeloid leukemia cell lines and in 16 of 18 primary AML samples tested. In contrast, normal CD34(+) cells and normal peripheral blood mononuclear cells expressed no or very low levels of survivin. Cytokine stimulation increased survivin expression in leukemic cell lines and in primary AML samples. In cultured primary samples, single-cytokine stimulation substantially increased survivin expression in comparison with control cells, and the combination of G-CSF, GM-CSF, and SCF increased survivin levels even further. Conversely, all-trans retinoic acid significantly decreased survivin protein levels in HL-60, OCI-AML3, and NB-4 cells within 96 hours, parallel to the induction of myelomonocytic differentiation. Using selective pharmacologic inhibitors, the differential involvement of mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol-3 kinase (PI3K) pathways were demonstrated in the regulation of survivin expression. The MEK inhibitor PD98059 down-regulated survivin expression in both resting and GM-CSF-stimulated OCI-AML3 cells, whereas the PI3K inhibitor LY294002 inhibited survivin expression only on GM-CSF stimulation. In conclusion, these results demonstrate that survivin is highly expressed and cytokine-regulated in myeloid leukemias and suggest that hematopoietic cytokines exert their antiapoptotic and mitogenic effects, at least in part, by increasing survivin levels

Inhibition of phosphatidylinositol 3-kinase dephosphorylates BAD and promotes apoptosis in myeloid leukemias

: The phosphatidylinositol 3-kinase (PI3K)/AKT protein kinase pathway is involved in cell growth, proliferation, and apoptosis. The functional activation of PI3K/AKT provides survival signals and blockade of this pathway may facilitate cell death. Downstream targets of PI3K-AKT include the proapoptotic protein BAD, caspase-9, NF-kappaB, and Forkhead. We have previously reported that BAD is constitutively phosphorylated in primary acute myeloid leukemia (AML) cells, a post-transcriptional modification, which inactivates its proapoptotic function. In this study, we tested the hypothesis that the inhibition of PI3K by LY294002 results in the dephosphorylation of AKT and BAD, and thus promote leukemia cell apoptosis. We investigated the effects of LY294002 in megakaryocytic leukemia-derived MO7E cells, primary AML and normal bone marrow progenitor cells. In MO7E cells, LY294002 reduced AKT kinase activity, induced dephosphorylation of AKT and BAD, and increased apoptosis. Concomitant inhibition of mitogen-activated protein kinase signaling or combination with all-trans retinoic acid further enhanced apoptosis of leukemic cells. In primary AML samples, clonogenic cell growth was significantly reduced. Normal hematopoietic progenitors were less affected, suggesting preferential targeting of leukemia cells. In conclusion, the data suggest that the inhibition of the PI3K/AKT signaling pathway restores apoptosis in AML and may be explored as a novel target for molecular therapeutics in AML

At the bedside:Profiling and treating patients with CXCR4-expressing cancers

The chemokine receptor, C-X-C chemokine receptor type 4 (CXCR4) and its ligand, C-X-C motif chemokine 12, are key mediators of hematopoietic cell trafficking. Their roles in the proliferation and metastasis of tumor cells, induction of angiogenesis, and invasive tumor growth have been recognized for over 2 decades. CXCR4 is a promising target for imaging and therapy of both hematologic and solid tumors. To date, Sanofi Genzyme's plerixafor is the only marketed CXCR4 inhibitor (i.e., Food and Drug Administration-approved in 2008 for stem cell mobilization). However, several new CXCR4 inhibitors are now being investigated as potential therapies for a variety of fluid and solid tumors. These small molecules, peptides, and Abs include balixafortide (POL6326, Polyphor), mavorixafor (X4P-001, X4 Pharmaceuticals), motixafortide (BL-8040, BioLineRx), LY2510924 (Eli Lilly), and ulocuplumab (Bristol-Myers Squibb). Early clinical evidence has been encouraging, for example, with motixafortide and balixafortide, and the CXCR4 inhibitors appear to be generally safe and well tolerated. Molecular imaging is increasingly being used for effective patient selection before, or early during CXCR4 inhibitor treatment. The use of radiolabeled theranostics that combine diagnostics and therapeutics is an additional intriguing approach. The current status and future directions for radioimaging and treating patients with CXCR4-expressing hematologic and solid malignancies are reviewed. See related review - At the Bench: Pre-Clinical Evidence for Multiple Functions of CXCR4 in Cancer. J. Leukoc. Biol. xx: xx-xx; 2020

Quantitative single cell determination of ERK phosphorylation and regulation in relapsed and refractory primary acute myeloid leukemia

: We investigated the constitutive activation of the MEK/ERK pathway in acute myelogenous leukemia (AML) via a flow cytometric technique to quantitate expression of phosphorylated ERK (p-ERK). A total of 42 AML samples (16 newly diagnosed, 26 relapsed/refractory) were analyzed. Normal bone marrow CD34+ cells (n = 10) had little or no expression of p-ERK, while G-CSF-mobilized CD34+ cells exhibited enhanced p-ERK levels. Markedly elevated p-ERK levels were found in 83.3% of the AML samples, with no differences observed between the newly diagnosed and relapsed/refractory samples. Treatment with a MEK inhibitor resulted in significantly decreased p-ERK levels in both the newly diagnosed and relapsed/refractory samples, which was associated with growth arrest, but not apoptosis induction. In summary, we defined conditions for the analysis of MAPK signaling in primary AML samples. Normal CD34+ cells expressed very low levels of p-ERK, and increased p-ERK levels were found in normal G-CSF-stimulated circulating CD34+ cells. Constitutively high p-ERK levels observed in the majority of AML samples suggest deregulation of this pathway that appears to be independent of disease status. The ability of ERK inhibition to promote growth arrest rather than apoptosis suggests that clinical trials of MEK/ERK inhibitors may be more effective when combined with chemotherapy

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

Regulation and targeting of antiapoptotic XIAP in acute myeloid leukemia

: XIAP is a member of the inhibitors-of-apoptosis family of proteins, which inhibit caspases and block cell death, with prognostic importance in AML. Here we demonstrate that cytokines regulate the expression of XIAP in leukemic cell lines and primary AML blasts. Inhibition of phosphatidylinositol-3 kinase (PI3K) with LY294002 and of the mitogen-activated protein kinase (MAPK) cascade by PD98059 resulted in decreased XIAP levels (34+/-8.7 and 23+/-5.7%, respectively). We then generated OCI-AML3 cells with constitutively phosphorylated Akt (p473-Akt) by retroviral gene transfer. Neither these nor Akt inhibitor-treated OCI-AML3 cells showed changes in XIAP levels, suggesting that XIAP expression is regulated by PI3K downstream effectors other than Akt. The induction of XIAP expression by cytokines through PI3K/MAPK pathways is consistent with its role in cell survival. Exposure of leukemic cells to chemotherapeutic agents decreased XIAP protein levels by caspase-dependent XIAP cleavage. Targeting XIAP by XIAP antisense oligonucleotide resulted in downregulation of XIAP, activation of caspases and cell death, and sensitized HL-60 cells to Ara-C. Our results suggest that XIAP is regulated by cytokines through PI3K, and to a lesser degree through MAPK pathways. Selective downregulation of XIAP expression might be of therapeutic benefit to leukemic patients

The organic arsenic derivative GMZ27 induces PML-RARα-independent apoptosis in myeloid leukemia cells

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is very effective against acute promyelocytic leukemia. However, organic arsenic derivatives (OAD) have a more favorable toxicity profile than ATO. We herein characterized dipropil-S-glycerol arsenic (GMZ27), a novel OAD. GMZ27 had potent antiproliferative activity against human acute myeloid leukemia (AML) cell lines that was higher than that of ATO. In contrast to ATO, GMZ27 only marginally induced maturation of leukemia cells and had no effect on the cell cycle. The anti-leukemia activity of GMZ27 against AML cells was independent of the presence of the PML-RARα fusion protein. GMZ27 dissipates mitochondrial transmembrane potential, and induces cleavage of caspase 9 and activation of caspase 3 without altering the expression levels of (BCL-2), BAX and BCL-xl. GMZ27 induces the formation of intracellular superoxide, a reactive oxygen species (ROS) which plays a major role in the antileukemia activity of this OAD. In addition to ROS generation, GMZ27 concomitantly reduces intracellular glutathione which markedly weakens the cellular antioxidant capacity, thus enhancing the detrimental intracellular effects of ROS production. These results indicate that GMZ27 induces apoptosis in AML cells in a PML-RARα-independent fashion, through the induction of ROS production. This activity provides the rationale for the testing of GMZ27 in patients with AML

Synergistic induction of apoptosis by simultaneous disruption of the Bcl-2 and MEK/MAPK pathways in acute myelogenous leukemia

: Recent studies suggest that the Bcl-2 and mitogen-activated protein kinase (MAPK) pathways together confer an aggressive, apoptosis-resistant phenotype on acute myelogenous leukemia (AML) cells. In this study, we analyzed the effects of simultaneous inhibition of these 2 pathways. In AML cell lines with constitutively activated MAPK, MAPK kinase (MEK) blockade by PD184352 strikingly potentiated the apoptosis induced by the small-molecule Bcl-2 inhibitor HA14-1 or by Bcl-2 antisense oligonucleotides. Isobologram analysis confirmed the synergistic nature of this interaction. Moreover, MEK blockade overcame Bcl-2 overexpression-mediated resistance to the proapoptotic effects of HA14-1. Most importantly, simultaneous exposure to PD184352 significantly (P =.01) potentiated HA14-1-mediated inhibition of clonogenic growth in all primary AML samples tested. These findings show that the Bcl-2 and MAPK pathways are relevant molecular targets in AML and that their concurrent inhibition could be developed into a new therapeutic strategy for this disease

MEK blockade converts AML differentiating response to retinoids into extensive apoptosis

: The aberrant function of transcription factors and/or kinase-based signaling pathways that regulate the ability of hematopoietic cells to proliferate, differentiate, and escape apoptosis accounts for the leukemic transformation of myeloid progenitors. Here, we demonstrate that simultaneous retinoid receptor ligation and blockade of the MEK/ERK signaling module, using the small-molecule inhibitor CI-1040, result in a strikingly synergistic induction of apoptosis in both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells with constitutive ERK activation. This proapoptotic synergism requires functional RAR and RXR retinoid receptors, as demonstrated using RAR- and RXR-selective ligands and RAR-defective cells. In the presence of MEK inhibitors, however, retinoid-induced chromatin remodeling, target-gene transcription, and granulocytic differentiation are strikingly inhibited and apoptosis induction becomes independent of death-inducing ligand/receptor pairs; this suggests that apoptosis induction by combined retinoids and MEK inhibitors is entirely distinct from the classical "postmaturation" apoptosis induced by retinoids alone. Finally, we identify disruption of Bcl-2-dependent mitochondrial homeostasis as a possible point of convergence for the proapoptotic synergism observed with retinoids and MEK inhibitors. Taken together, these results indicate that combined retinoid treatment and MEK blockade exert powerful antileukemic effects and could be developed into a novel therapeutic strategy for both AML and APL