ALBERT

Description: We investigated the antileukemic activity and molecular mechanisms of action of a newly synthesized ring-substituted diindolylmethane (DIM) derivative, named, 1,1-bis [3′-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane (DIM #34), in myeloid leukemic cells. DIM #34 inhibited leukemic cell growth via induction of apoptosis. DIM #34 inhibited clonogenic growth and induced apoptosis of AML CD34+ progenitor cells but spared normal progenitors. DIM #34 induced loss of mitochondrial membrane potential, which was accompanied by the release of cytochrome c into the cytosol and early cleavage of caspase-9 followed by the cleavage of caspases -8, and -3. Bcl-2 overexpression and caspase-9-deficient cells were partially protected against DIM #34-induced apoptosis, suggesting activation of the intrinsic apoptotic pathway. DIM #34 induced Bax cleavage, and Bax knockout cells were partially resistant to cell death. Furthermore, DIM #34 transiently inhibited the phosphorylation and the activity of the extracellular-regulated kinase (ERK) and abrogated Bcl-2 phosphorylation. Because other methylene substituted DIM analogs transactivate the nuclear receptor PPARγ, we studied the role of PPARγ in apoptosis induction. Although the co-treatment of cells with a selective PPARγ antagonist T007, and a low dose of DIM #34 partially diminished apoptosis, apoptosis was not inhibited at higher concentrations of DIM #34, suggesting the involvement of both, receptor-dependent and independent mechanisms. Co-treatment with RXR- and RAR-ligands enhanced DIM #34-induced cell death. Together, these findings showed that substituted DIMs represent a new class of compounds that selectively induce apoptosis in AML cells through interference with ERK and activation of PPARγ signaling pathways.

Description: Outcome results in AML are a continued challenge for the development of novel therapeutic strategies. C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid, CDDO-Me, a novel triterpenoid, induces apoptosis in myeloid and lymphoid leukemic cell lines and in primary AML samples in sub-micromolar concentrations. We reported previously that CDDO-Me inhibits the activation of ERK1/2, blocks Bcl-2 phosphorylation, and promoted apoptosis in AML-derived U937 cells (Blood 2002, 99(1):326–35). Here, we examined the effects of CDDO-Me on CD34+ AML progenitor cells in vitro. Exposure to 1μM CDDO-Me induced apoptosis in all but one AML sample (46±4% annexin(+) CD34+ cells, n=20). To assess the anti-leukemia activity of CDDO-Me in vivo, scid mice intravenously injected with U937 cells were treated with liposomally-delivered CDDO-Me (20mg/kg/day IV every other day, starting at day 7, for a total of 9 injections). While CDDO-Me was not toxic to the mice, pathological and flow cytometry analysis revealed reduced (55–86%) leukemia burden in the bone marrow, liver, and spleen of mice. Since we had shown that CDDO-Me inhibits phosphorylation of pERK in U937 cells, a further goal of this study was to assess the role of ERK in CDDO-Me-induced cell death in primary AML samples. ERK was expressed and phosphorylated in all (n=15) patients’ samples studied. CDDO-Me inhibited ERK phosphorylation in 9 of 15 patient samples and promoted apoptosis. However, CDDO-Me still induced apoptosis in 5/6 samples that displayed no significant changes in pERK levels in response to the drug. This finding suggests that ERK is not the sole target of the compound. The stress-activated protein kinases JNK and p38 are related to ERK but in general activate pathways that are opposed to ERK. By Western blot analysis, CDDO-Me induced early (30 min) phosphorylation of JNK and p38, which preceded induction of cell death. Pre-treatment of U937 cells with JNK and p38 inhibitors SP600125 and SB203580 partially abrogated induction of apoptosis, while MEK inhibitor PD-98059 significantly enhanced cytotoxicity. CDDO-Me induced p38 phosphorylation in 5 of 6 primary AML samples tested. Collectively, these finding indicate that CDDO-Me markedly shifts signaling toward the JNK and p38 MAPK stress-related pathways and away from the cytoprotective MAPK cascade. In summary, the triterpenoid CDDO-Me is a potent inducer of apoptosis in primary AML including CD34+ AML progenitor cells. Induction of apoptosis is in part mediated via inhibition of ERK signaling combined with JNK/p38 activation. These studies in primary AML samples and the anti-leukemia activity of the compound in vivo suggest potential utility of CDDO-Me for the treatment of AML patients.

Description: Background: Hyperglycemia represents a common side effect of steroid therapy and is not uncommon during treatment of patients with acute lymphocytic leukemia (ALL). Our recent retrospective study in 278 adult patients with ALL demonstrated that hyperglycemia during induction chemotherapy occurred in up to 37% of patients and was associated with a shorter median complete remission duration and a shorter median survival (Cancer 2004 Mar 15;100(6):1179–85). The serine/threonine AKT, a major downstream PI3K target, promotes continued cell growth and metabolism by increasing glucose uptake, stimulating glycolysis and ATP production. Recent data in transgenic models demonstrated that AKT activation resulted in transcriptional upregulation of enzymes essential for glycolysis including hexokinase (HK) and Glut-1 (Nature Med., 2004, 10:594). The purpose of this study was to determine whether high AKT activity in leukemic blasts in the setting of hyperglycemia promotes survival and chemoresistance of leukemic cells by stimulation of glycolysis. Methods and Results: A 24-hour culture of REH cells in serum-free medium supplemented with 14mM glucose resulted in induction of AKT phosphorylation (by Western blot analysis) and increase in mRNA levels of Glut-1 and HK-2 (by quantitative TaqMan PCR). PI3K inhibitor LY294002 (15 μM) prevented AKT phosphorylation and decreased mRNA expression of Glut-1 and HK-2 in high-glucose conditions and enhanced doxorubicin- and dexamethasone-induced killing. We next investigated expression of AKT 1, Glut1, and HK2 mRNA in bone marrow samples from 14 adult patients with ALL (9 pre-B-, 2 B-, 3 pre-T-ALL). The results demonstrated elevated (〉 2-fold compared with normal bone marrow or peripheral blood) levels of AKT in 7/14 patients, 6 of those showed increased levels of Glut1 and 5 of HK2. In addition, in samples from 2 patients with low baseline AKT expression high HK-2 mRNA levels were observed. Conclusions: High expression of AKT, glucose transporter Glut-1 and the glycolytic enzyme HK-2 is prevalent in primary ALL blast cells. Our results suggest that high glucose further stimulates AKT signaling in ALL blast cells resulting in upregulation of glucose transporter Glut-1 and of the glycolytic enzyme HK-2. This effect was abrogated by inhibition of PI3K/AKT signaling, resulting in chemosensitization. These data provide rationale for use of PI3K/AKT inhibitors in the therapy of ALL. The hypothesis that tight blood glucose control improves outcome in ALL patients is currently being tested in a Phase III clinical trial at M.D. Anderson Cancer Center.

Description: The multidrug resistance 1 (MDR1) gene product P-glycoprotein (P-gp) is frequently implicated in cross-resistance of tumors to chemotherapeutic drugs. In contrast, acute promyelocytic leukemia (APL) cells do not express MDR1 and are highly sensitive to anthracyclines. The combination of ATRA and the novel histone deacetylase inhibitor (HDACI) depsipeptide (FK228) induced P-gp expression and prevented growth inhibition and apoptosis in NB4 APL cells subsequently exposed to doxorubicin (DOX). ATRA/FK228 treatment after exposure to DOX, however, enhanced apoptosis. Both agents, ATRA or FK228, induced MDR1 mRNA. This effect was significantly enhanced by ATRA/FK228 administered in combination, due in part to increased H4 and H3-Lys9 acetylation of the MDR1 promoter and recruitment of the nuclear transcription factor Y alpha (NFYA) transcription activator to the CCAAT box. Cotreatment with specific P-gp inhibitor PSC833 reversed cytoprotective effects of ATRA/FK228. G1 cell-cycle arrest and p21 mRNA induction were also observed in response to ATRA/FK228, which may restrict DOX-induced apoptosis of cells in G2 phase. These results indicate that epigenetic mechanisms involving NF-YA transcription factor recruitment and histone acetylation are activated by ATRA and HDACI, induce MDR1 in APL cells, and point to the critical importance of mechanism-based sequential therapy in future clinical trials that combine HDAC inhibitors, ATRA, and anthracyclines.

Description: 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.

Description: 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%, p500nM) 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.

Description: Annexin A1 (ANX-A1) is a calcium-dependent membrane-binding protein involved in the modulation of apoptosis and phagocytosis (FASEB J.2003;17:1544). We have previously reported that HDAC inhibitor depsipeptide (FK228) caused marked growth inhibition and apoptosis in t(8;21) Kasumi-1 AML cells with up-regulation of 123 genes (by cDNA array) including ANX-A1 (3.5 fold; Tabe, Blood 2004). By chromatin immunoprecipitation (ChIP) assay, FK228 induced H4 and H3-K9 acetylation in the ANX-A1 promoter with corresponding induction of ANX-A1 mRNA (7.2±1.7 fold, TaqMan RT-PCR) and protein (western blot analysis). The markedly increased ANX-A1 protein localized on the cell membrane of Kasumi-1 cells exposed to FK228 was confirmed by immunofluorecence analysis using confocal microscopy. ANX-A1 membrane localization was diminished by treatment with anti-ANX-A1 mAb. To investigate the contribution of ANX-A1 to FK228-induced apoptosis, we neutralized ANX-A1 by anti-ANX-A1 mAb. This moderately decreased FK228 induced apoptosis (36.0±4.1 vs 26.5±3.7% AnnexinV(+)/PI(+) cells, p=0.01). Similarly, Kasumi-1 cells transfected with siRNA/ANX-A1 were less sensitive to FK228-induced cell death compared with nonsense (N) siRNA transfected cells (siRNA 31.2±3.1% vs NsiRNA 39.5±2.9% annexin(+) cells, p=0.03). These data indicate that the upregulation of endogeneous ANX-A1 (either membrane-binding or secreted form) promotes cell apoptosis in an autocrine fashion. Next, we investigated the functional role of ANX-A1 on leukemia cell phagocytosis. The engulfment of Kasumi-1 cells by cocultured human THP-1 monocyte-derived macrophages was evaluated by cell adherence assay. Compared with untreated cells, the exposure to FK228 induced a dramatic increase in Kasumi-1 cells attachment to macrophages (untreated vs FK228 treated; 57 ± 9 cells vs 196 ± 33 cells/ microscopic fields (0.08 mm2/field), n = 5; p=0.01). FK228-induced cell attachment was completely abrogated in the siRNA/ANX-A1 transfected Kasumi-1 cells (60.5% ± 10.5% decrease; n = 5; p

Description: 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.

Description: Background: The serine/threonine kinase AKT, a major downstream PI3K target, promotes continued cell growth and metabolism by increasing glucose uptake, stimulating glycolysis and ATP production. It has been proposed that cancer cells with a high level of constitutively active AKT depend on glucose for survival. Recent data in transgenic models demonstrated that AKT activation results in mTOR dependent transcriptional upregulation of the glycolytic enzyne HK2 and glucose transporter Glut1 via induction of HIF1-α (Nature Med2004;10:594). The purpose of this study was to determine the frequency and prognostic significance of increased expression of HK2 in patients with ALL, to determine if proglycolytic conditions, high glucose (HG) or hypoxia, promote chemoresistance in ALL blast cells, and if chemoresistance is affected by mTOR inhibition. Methods: HK2 mRNA expression was measured by quantitative Taqman PCR in 28 primary ALL samples (14 newly diagnosed and 14 relapsed). Primary bone marrow samples (N=5) were co-cultured on either MS-5 or HS-5 stroma supplemented with 4 or 14 mM glucose for 24–48 hours. Jurkat cells were cultured in serum-free medium supplemented with 4 or 14 mM glucose under hypoxic (3% O2) or normoxic (21% O2) conditions. All samples were treated with doxorubicin (DOX - 25–50 ng/mL) +/− RAD001 (10–20 nM) or CCI779 (2.5–5 mg/mL). Results: Increased expression of HK2 (≥ 2-fold relative to normal samples) occurred in 5/28 samples (18%). Patients with HK2 levels above the median (0.43) had a worse failure free survival (FFS) (HR 2.95; CI 0.89–9.81; P=0.08) and a significantly worse overall survival (OS) (HR 5.01; CI 1.21–20.7; P=0.03). In 4/5 (80%) of primary ALL samples, exposure to HG (14 mM) resulted in decreased apoptosis with DOX compared to normal glucose (4 mM) (mean % Ann+ 32.0 vs 42.0; P=0.047). This effect was reversed with addition of RAD001 or CCI779 (mean % Ann+ 49.36 vs 54.02; P=0.11). Jurkat cells cultured under hypoxic conditions and treated with DOX demonstrated decreased apoptosis and increased cell viability compared to normoxic conditions [mean % Ann (+) cells 39.85 (Hypoxic) vs 55.00 (Normoxic); P=0.016). This effect was reversed with addition of RAD001 under hypoxic conditions [mean % Ann (+) cells 39.85 (DOX) vs 51.92 (DOX+RAD); P=0.009] in association with decreased expression of HK2 mRNA (RE to DMSO mean 62 vs 47; P=0.26). Conclusions: Overexpression of HK2 is associated with worse OS in pts with newly diagnosed or relapsed ALL. Exposure of ALL blast cells to high glucose or hypoxic environment results in chemoresistance to DOX. This effect is abrogated by mTOR inhibition in association with decreased expression of HK2 mRNA, suggesting that upregulation of glycolysis via the AKT/mTOR/HIF1a pathway may be an important mechanism of chemoresistance in ALL and that mTOR inhibition or glucose normalization may play an important role in chemosensitization and improved outcomes in ALL.

Description: First-line therapy of acute myeloid leukemia (AML) consists of combinations of cytarabine and an anthracycline. While initial complete remissions are frequent, most patients succumb to resistant disease underlining the need for novel, more effective agents. The most striking progress in AML therapy was achieved by targeting the nuclear receptor RARα with ATRA. Research in our laboratory has demonstrated that the novel synthetic triterpenoid CDDO (2-cyano-3,12- dioxooleana-1,9-dien-28-oic acid) and its more active C28 methyl ester derivative CDDO-Me inhibit growth and induce apoptosis in a variety of cancers including AML, CLL and blast crisis CML. CDDO and to a much higher degree CDDO-Me are potent activators of the nuclear transcription factor Peroxisome Proliferator-Activated Receptor gamma (PPARγ). In a mammalian two-hybrid assay, the CDDO and CDDO-Me induced activation of PPARγ was associated with a marked increase in multiple coactivator recruitment (SRC-1, SRC-2, SRC-3, TRAP220/DRIP205, CARM-1 and PGC-1) that is qualitatively different from that induced by other PPARγ ligands. CDDO induced a higher degree of myelo-monocytic differentiation in DRIP205-overexpressing HL-60 cells suggesting that high cellular levels of DRIP205 co-activator modulate differentiation response to PPARγ ligation. CDDO induced p21 mRNA and protein in leukemic cells and transactivation of the p21 promoter in a p53-independent fashion. We have recently identified the PPARγ-independent depletion of mitochondrial glutathione (GSHm) as a novel mechanism of action resulting in redox disbalance and mitochondrial damage as mechanisms of pro-apoptotic effects of CDDO and CDDO-Me. Gene expression studies using cDNA arrays demonstrate that CDDO induces genes involved in the antioxidant response (AR) including phase II detoxifying enzymes (glutamate cysteine ligase, GSH transferase, etc.) and antioxidant enzymes (heme oxygenase 1, thioredoxin reductase). Cotreatment with the GSH precursor, n-acetyl cystein prevented apoptosis and loss of viability induced by CDDOs, whereas alkylation of intracellular thiols by diethylmaleate decreased the accumulation of a biotinylated derivative of CDDO, TP-301, in U937 leukemic cells suggesting that intracellular reduced thiols are functional targets of CDDO and its derivatives. The in vivo studies using liposomal CDDO-Me in a conditional leukemia model demonstrated significant reduction of leukemia burden as measured by bioluminescence imaging and prolongation of survival. Based on the ample pre-clinical evidence of anti-leukemia effects and on the favorable PK/toxicity profile of the parental compound, CDDO will enter Phase I clinical trials in hematologic malignancies in 3Q 2005 and CDDO-Me in 1Q 2006.