Mechanisms underlying apoptosis induced by concomitant interruption of the mitogen-activated protein kinase kinase/ extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways were investigated in human leukemia cells. Inhibition of these pathways using the MEK inhibitor PD184352 or U0126 and the PI3K/ Akt inhibitor perifosine strikingly induced apoptosis in multiple malignant human hematopoietic cells, and substantially reduced the colony-forming capacity of primary acute myeloblastic leukemia, but not normal CD34 ؉ cells. These events

Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 (IL-24), a member of the IL-10 cytokine gene family, preferentially induces cell death in neoplastic epithelial cells types while sparing their normal counterparts. The effects of mda-7/ IL-24 in acute myeloid leukemia (AML) cells have not been extensively characterized. Treatment with recombinant GST-MDA-7/ IL-24 potently induced apoptosis in diverse myeloid leukemia cell types including U937, HL60, MV4-11, EOL-1, and MLL/ENL cells. MDA-7/IL-24 also markedly induced apoptosis in and suppressed the colony-forming capacity of primary AML blasts but exerted minimal toxicity toward normal CD34 ϩ hematopoietic progenitor cells. MDA-7/IL-24 lethality was associated with pronounced endoplasmic reticulum (ER) stress induction in leukemia cell lines and primary AML blasts, manifested by the accumulation of growth arrest and DNA damage-inducible protein 34 (GADD34), 78-kDa glucose-regulated protein (GRP78)/BiP, inositol-requiring enzyme 1␣ (IRE1␣), and eukaryotic initiation factor 2␣ phosphorylation. It is noteworthy that short hairpin RNA (shRNA) knockdown of IRE1␣, GADD34, or GRP78/BiP significantly enhanced MDA-7/IL-24-mediated apoptosis, indicating a protective role for these molecules against MDA-7/IL-24 lethality. MDA-7/IL-24 also down-regulated the antiapoptotic protein Mcl-1 and sharply increased expression of the proapoptotic proteins Bim and Noxa. Ectopic Mcl-1 expression or shRNA knockdown of Bim or Noxa significantly attenuated MDA-7/IL-24-mediated leukemia cell death. Finally, knockdown of Bax or Bak significantly reduced MDA-7/IL-24 lethality. Together, these findings indicate that MDA-7/IL-24 potently induces apoptosis in human myeloid leukemia cells through a process regulated by ER stress induction, Mcl-1 down-regulation, and Bim and Noxa up-regulation. They also suggest that MDA-7/IL-24 warrants further investigation in myeloid leukemia.

mda-7/IL-24, a member of the IL-10 cytokine gene family, has been shown to induce cell death in various epithelial cancer cell types while exhibiting relatively little toxicity toward normal cells. Currently, the effects of mda7/IL-24 on myeloid leukemia cells (AML) have not been extensively characterized. In the present study, we report that treatment with recombinant GST-mda7/IL24 strikingly induces apoptosis in diverse myeloid leukemia cell lines including U937, MV4-11, EOL-1 (both FLT3 mutant), and MLL/ENL cells, which display certain leukemia stem cell-like properties. In addition, mda7/IL24 potently induces apoptosis in and suppresses the colony formation capacity (L-CFU) of primary blasts isolated from multiple patients with AML. In contrast, mda-7/IL-24 exerted only minimal effects on normal CD34+ progenitor cells. These effects were associated with profound mitochondrial injury manifested by cytochrome c and AIF release into the cytosol, caspase-3 activation, and PARP cleavage. The apoptotic effects of mda7/IL24 were associated with a potent induction of ER stress in leukemia cell lines as well as in primary AML blasts, manifested by accumulation of GADD153/CHOP, GADD34, GRP78, and IRE1α. Notably molecular studies with shRNA directed against IRE1α revealed that knockdown of IRE1α significantly enhanced mda7/IL24-mediated apoptosis in U937 cells, suggesting that induction of IRE1α plays a protective role against mda7/IL24 lethality. In addition treatment with mda7/IL24 resulted in down-regulation of the anti-apoptotic protein Mcl-1, and marked accumulation of the pro-apoptotic proteins Bim and Noxa. Notably, ectopic expression of Mcl-1 or knock-down of Bim or Noxa with shRNA markedly decreased mda7/IL24-mediated cell death in leukemia cells. Collectively, these findings indicate that mda7/IL24 strikingly induces apoptosis in human myeloid leukemia through a process that involves ER stress induction, Mcl-1 downregulation, and Bim and Noxa upregulation. They also suggest that mda7/IL24 warrants attention as a potential addition to the therapeutic armamentarium in leukemia and possibly other hematologic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1511.

Previously, we demonstrated that the multikinase inhibitor sorafenib diminishes Mcl-1 protein expression in human leukemia cells by inhibiting translation through a MEK1/2/ERK1/2 signaling-independent mechanism, and that this phenomenon plays a key functional role in sorafenib lethality. However, recent evidence suggests that Bcl-2 and Bcl-xL may also limit the apoptotic activity of sorafenib. In efforts to potentiate the lethality of pharmacologically achievable concentrations of sorafenib (e.g., 5-7.5 μM) in human myeloid leukemia cells, we have examined the effects of the small molecule inhibitor obatoclax (GX15-070), which has been reported to disrupt the function of Bcl-2, Bcl-xL, and Mcl-1, on sorafenib anti-leukemic activity. Here we report that obatoclax strikingly enhances sorafenib-mediated mitochondrial dysfunction and cell death in U937 and other myeloid leukemia cell types. These events were manifested by cytochrome c and AIF release into the cytosol, caspase-3 and −9 activation, PARP cleavage, apoptosis, and a marked decrease in cell growth. Similar effects were observed in KG1 and HL-60 cells. These events were associated with the rapid and pronounced downregulation of Mcl-1. Notably, genetic studies using shRNA directed against Bim, a major Mcl-1 binding partner, revealed that knockdown of Bim strikingly attenuated obatoclax/sorafenib-induced apoptosis. Immunoprecipitation studies revealed that down-regulation of Mcl-1 in sorafenib-treated cells was associated with enhanced binding of Bim to Bcl-xL and Bcl-2. However, co-administration of obatoclax effectively prevented the increased association between Bim and Bcl-xL/Bcl-2. In addition, knockdown of Bak or Bax also markedly diminished obatoclax/sorafenib-mediated lethality. In contrast, knockdown of Bad or Noxa had no significant effect on obatoclax/sorafenib-mediated lethality. Together, these findings suggest that activation of Bim, but not Bad or Noxa, plays a key role in the pronounced antineoplastic activity of obatoclax/sorafenib toward human leukemia cells. They also raise the possibility that obatoclax may enhance sorafenib lethality by diminishing associations between Bcl-xL/Bcl-2 and Bim. Finally, in vivo studies in a murine xenograft U937 cell model revealed that co-treatment with sorafenib and obatoclax strikingly reduces in vivo tumor growth, whereas the agents administered individually had only modest effects. Combined treatment also reduced Mcl-1 expression in vivo. Collectively, these findings raise the possibility that combining sorafenib, which induces Mcl-1 down-regulation, with agents such as obatoclax that inhibit other Bcl-2 family antiapoptotic members (e.g., Bcl-2, Bcl-xL) may represent a novel and effective strategy against myeloid leukemia and possibly other hematologic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4523.