High Mcl-1 Expression Research Articles

Abstract 605: Apogossypolone derivative -BI97D6 effectively targets MCL1 overexpressing Acute Myeloid Leukemia cells.

Abstract Aberrant expression of anti-apoptotic proteins such as BCL2, MCL1, and BCL-XL is observed in many tumors including Acute Myeloid Leukemia (AML). An obvious strategy for AML therapy would be to target the anti-apoptotic BCL2 family members. While compounds such as ABT-737 have shown promise for treatment of some malignancies, the compound binds poorly to MCL1 and thus cells that overexpress MCL1 usually display high resistance to the drug. Furthermore, ABT-737 activates ERK signaling resulting in MCL1 overexpression in surviving cells (Konopleva, et al, Leukemia 2011). The apogossypolone derivative -BI97D6 binds MCL1 and BCL2, BCL-XL, BFL1 with high affinities (Wei, et al, J Med Chem 2010). We recently reported that -BI97D6 (a) efficiently kills AML cell lines even in the presence of protective mesenchymal stroma cells, (b) interferes with the association between MCL1/BIM and BCL2/BAX, (c) stimulates BAX conformational change, (d) requires BAX or BAK for cell killing, and (e) does not induce ER stress. These data suggests that -BI97D6, unlike other gossypol or apogossypol compounds, acts mainly as a BH3 mimetic. In the current study, we first examined the role of MCL1 in -BI97D6-mediated killing. OCI-AML3 cells are extremely resistant to ABT-737 due to its high expression of MCL1. MCL1 expression was suppressed by lenti-viral shRNA in OCI-AML3 cells and knockdown of MCL1 protein by 80% was achieved as determined by quantitative western blot. Compared to cells with non-specific control shRNA, cells with MCL1 shRNA were greatly sensitized to ABT-737, and significantly sensitized to AraC, while -BI97D6, which binds MCL1 with high affinity, is almost equipotent in knockdown and control cells. Since results with AML cell lines have been promising, we examined the efficacy of -BI97D6 on primary AML samples. Flow cytometry analysis revealed that -BI97D6 potently induced apoptosis in AML blast cells at low nM concentration (EC50<50nM). -BI97D6 also effectively killed putative CD34+CD38- leukemia stem cells (LSC). Importantly, primary AML with FLT3-ITD and FLT3-D835 mutations showed similar sensitivity as those without FLT3 mutations (EC50<50nM). As FLT3 mutations are associated with poor prognosis in AML, these findings suggests that -BI97D6 can target AML LSC and could benefit even high risk patients. In summary, compound -BI97D6 may be a promising drug candidate for MCL1-targeted AML therapy. Citation Format: Rongqing Pan, Vivian Ruvolo, Maurizio Pellecchia, Jun Wei, Marina Konopleva, John C. Reed, Peter Ruvolo, Michael Andreeff. Apogossypolone derivative -BI97D6 effectively targets MCL1 overexpressing Acute Myeloid Leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 605. doi:10.1158/1538-7445.AM2013-605

Abstract 5482: Combining the anti-mesothelin immunotoxin SS1P with the BH3-mimetic ABT-737 induces cell death in SS1P-resistant pancreatic cancer cell lines.

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) responds poorly to most chemotherapeutic agents, and alternative treatments are needed. Our laboratory focuses on the development of recombinant immunotoxins (RITs) for cancer treatment. RITs are antibody-toxin fusion proteins composed of an antigen-binding Fv fused to a 38-kDa portion of Pseudomonas exotoxin A (PE38). SS1P (SS1(dsFv)-PE38) is a mesothelin-targeting RIT that is in clinical trials for mesothelioma. Mesothelin is also highly expressed in PDAC. We previously reported that mesothelin-expressing PDAC cell lines were resistant to SS1P. This finding fuelled efforts to combine RITs with drugs that can tip the balance in favor of cell death. ABT-737, a BH3-mimitic, has a high affinity for Bcl-2, Bcl-xl and Blc-w, but little affinity for Mcl-1. High Mcl-1 expression therefore is a source of resistance to ABT-737. Since RITs inhibit protein synthesis, thereby downregulating Mcl-1, we examined whether combining SS1P and ABT-737 could induce cell death in PDAC. Methods PDAC cell lines KLM-1, BxPc-3 and Panc 3.014 were studied. Mesothelin expression was evaluated with FACS. Cell proliferation was evaluated with Cell Counting Kit-8 WST-8 assay, and cell death with 7-AAD and Annexin V-PE staining. Protein synthesis inhibition was quantified via 3H-leucine incorporation. Results KLM-1 had 92.6 x 103 mesothelin-binding sites/cell, respectively about 4- and 42-fold higher than Panc 3.014 (24.9 x 103) and BxPc-3 (2.2 x 103). In all three cell lines, 100 ng/ml SS1P and 10 uM ABT-737 separately did not induce substantial apoptosis as compared to the untreated controls. Combining both compounds, however, led to a significant increase in cell death. After 48hrs of treatment, apoptosis was observed in 92% of the KLM-1 cells, 55% of the BxPc-3 cells, and 23% of the Panc 3.014 cells. Combining 100 ng/ml SS1P and 10 uM ABT-737 also significantly enhanced the protein synthesis inhibition after 8hrs of treatment. Again, this increase was most pronounced in KLM-1 (42% inhibition versus 18% by SS1P), followed by BxPc-3 (30% inhibition versus 12% by SS1P), and Panc 3.014 (7% inhibition versus 3% by SS1P). Conclusion Combining SS1P with ABT-737 induced cell death in PDAC cell lines that are resistant to both compounds alone. The efficacy of this combo varied greatly among PDAC cell lines, and correlated with the extent to which protein synthesis was inhibited, but not with the amount of cell-surface mesothelin expression. These findings further support the therapeutic potential of combining a RIT and a BH3-mimetic. Further study is needed to elucidate differences in sensitivity among cell lines. This can improve the efficacy of this approach, discover novel targets, and eventually identify patients most likely to benefit from such a combination therapy. Citation Format: Kevin Hollevoet, Antonella Antignani, David J. Fitzgerald, Ira Pastan. Combining the anti-mesothelin immunotoxin SS1P with the BH3-mimetic ABT-737 induces cell death in SS1P-resistant pancreatic cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5482. doi:10.1158/1538-7445.AM2013-5482

Mcl-1 is essential for the survival of plasma cells

The long-term survival of plasma cells is entirely dependent on signals derived from their environment. These extrinsic factors presumably induce and sustain the expression of antiapoptotic proteins of the Bcl-2 family. It is uncertain whether there is specificity among Bcl-2 family members in the survival of plasma cells and whether their expression is linked to specific extrinsic factors. We found here that deletion of the gene encoding the antiapoptotic protein Mcl-1 in plasma cells resulted in rapid depletion of this population in vivo. Furthermore, we found that the receptor BCMA was needed to establish high expression of Mcl-1 in bone marrow but not spleen plasma cells and that establishing this survival pathway preceded the component of plasma cell differentiation that depends on the transcriptional repressor Blimp-1. Our results identify a critical role for Mcl-1 in the maintenance of plasma cells.

Invasive pneumococcal disease among HIV-1 seropositive individuals in the era of highly active antiretroviral therapy: does HIV-1 impair the macrophage host response to pneumococci?

BackgroundIn the era of highly active antiretroviral therapy, incidence of bacterial pneumonia and invasive pneumococcal disease among HIV-1 seropositive individuals is still more than 30 times higher than for seronegative individuals. Risk remains elevated in those with CD4 cell counts in the normal range. A programme of host-mediated macrophage (Mϕ) apoptosis ensures killing of Streptococcus pneumoniae when canonical phagolysosomal killing capacity is exhausted. HIV-1 infection is associated with resistance of Mϕ to apoptosis. We hypothesised that HIV-1-mediated Mϕ apoptotic resistance impairs S pneumoniae killing. MethodsThe following Mϕ models were used: healthy donor monocyte derived Mϕ (MDM) infected with HIV-1BaL (HMDM) or sham infected (SMDM), with infection confirmed with anti-p24 immunohistochemistry; and MDM treated with gp120 10–100 ng/mL (gpMDM) or vehicle (cMDM). Mϕ were challenged with opsonised type 2 S pneumonia multiplicity of infection=10 or mock-infected and apoptosis up to 20 h by observing morphological changes (counting condensed or fragmented nuclei), counting rates of positivity with the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) assay, measuring cells with hypodiploid DNA, and measuring caspase-3/7 activity. Bacterial internalisation and killing were measured by gentamicin protection assay. Data are mean and SD, and analysed by paired t test, or are median and IQR with Wilcoxon matched pairs signed rank test or Kruskal-Wallis test if non parametric. FindingsAfter S pneumoniae challenge, rates of initial S pneumoniae internalisation were similar for all MDM conditions. HMDM showed fewer apoptotic nuclei than did SMDM (19% [IQR 18–21] vs 33% [26–43], n=7, p<0·05), and smaller increases in caspase-3/7 activity and higher Mcl-1 expression on western blot after S pneumoniae challenge. Compared with cMDM, fewer gpMDM developed apoptotic nuclei after S pneumoniae challenge, which was dose dependent (cMDM 36% [IQR 30–48] vs gpMDM 20% [12–28] at 10 ng/mL and 18% [14–22] at 100 ng/mL, n=12, p<0·01, confirmed with TUNEL), and gpMDM exhibited significantly less caspase 3/7 activity (p<0·05). In gpMDM, reduced apoptosis was associated with bacterial survival. InterpretationHIV-1 infection reduces Mϕ apoptosis in response to S pneumoniae, and gp120 may be sufficient to mediate this. Apoptosis resistance may impair killing of S pneumoniae after phagocytosis and increase susceptibility to invasive pneumococcal disease in HIV-1 seropositive individuals. FundingUK Medical Research Council.

The expression of Mcl-1 in human cervical cancer and its clinical significance

Recently, the role of anti-apoptotic Mcl-1 in human carcinogenesis has become an area of great interest as overexpression of the protein has been reported in association with various types of cancer. The aim of this study was to investigate the expression profile of Mcl-1 in cervical cancer and to assess its clinical significance. Immunohistochemistry was used in the detection of Mcl-1 expression as well as the proliferation index of Ki-67, both in cervical cancer and in corresponding normal tissue. Western blotting analysis was also used for the detection of Mcl-1. The data was correlated with clinicopathological features. Survival analysis was performed to assess prognostic significance. Mcl-1 was overexpressed in cervical cancer tissue when compared with corresponding normal tissue. High expression of Mcl-1 was significantly associated with histological grade (P = 0.039), tumor size (P = 0.024) and lymph node involvement (P = 0.002). Overexpression of Ki-67 was associated with lymph node involvement (P = 0.015) and disease stage (P = 0.012). Spearman rank correlation test demonstrated a positive correlation between Mcl-1 and Ki-67 (P < 0.05). Using Kaplan-Meier analysis, a comparison of survival curves of low versus high expressers of Mcl-1 and Ki-67 revealed a highly significant difference in human cervical cancer tissue (P < 0.05), which suggests that overexpression of Mcl-1 and Ki-67 is associated with a poorer prognosis. Our results suggest that Mcl-1 may play an important role in cervical cancer and that it may have potential as a biomarker and therapeutic target. Its evaluation with Ki-67 may provide reliable prognostic information on cervical cancer.

Highlights of This Issue

Kinesin spindle protein (KSP) is a mitotic kinesin, and inhibition of this motor protein results in mitotic arrest and cell death. Basso and colleagues identified SCH 2047069, a novel KSP inhibitor with oral bioavailability and the ability to cross the blood brain barrier. The ability to dose the compound orally provides convenience of dosing as well as the ability to achieve more continuous exposure via the use of dose-dense administration. SCH 2047069 demonstrates potent pharmacodymanic marker activity in vitro and in vivo and antitumor activity in a variety of preclinical models as a single agent and in combination with paclitaxel, gemcitabine, or vincristine.ABT-263 is a first-in-class BCL2 family inhibitor that restores the ability of cancer cells to undergo apoptosis. However, many cancer cells are resistant to ABT-263 due to high expression of a BCL2 family member MCL1. MicroRNAs are small noncoding RNAs that have been shown to abnormally regulate in cancer cells. Lam and colleagues screened a library of 810 human microRNAmimics to explore its role in ABT-263 sensitivity. They identified microRNAs that restore apoptosis in the presence of ABT-263 by reducing MCL1 protein expression. Their data can facilitate the design of microRNA replacement therapies to increase sensitivity to BCL2 antagonists.Microtubule-associated protein Tau has been reported to be a predictive factor for clinical response to taxanes in metastatic breast cancer. Spicakova and colleagues studied the expression and function of Tau in several breast cancer cell lines. They derived three independent T-47D clones stably overexpressing Tau-3R and Tau-4R isoforms, and showed no change in taxane sensitivity compared to parental cells transfected with vector alone. Knock down or silencing of Tau expression in three cell lines that expressed Tau constitutively did not result in increased taxane sensitivity. Thus, neither overexpression nor depletion of Tau modulate cellular sensitivity to taxanes. Tau overexpression is not a mechanism of taxane resistance in breast cancer.

PCNA and anti-apoptotic Mcl-1 proteins predict disease-free survival in oral cancer patients treated with definitive radiotherapy

At our laboratory, we recently observed cell cycle and apoptosis-related proteins Myeloid Cell Leukemia-1 (Mcl-1) and Proliferating Cell Nuclear Antigen (PCNA) to be altered in oral tumours/cell lines. The present study aimed to evaluate the above proteins for predicting response and outcome in oral cancer patients treated with definitive radiotherapy. Pre-treatment oral cancer biopsy samples from 39 patients were examined for Mcl-1 and PCNA proteins using immunohistochemistry and correlated with clinico-pathological variables using disease-free survival (DFS) as the primary endpoint. We observed high expression of Mcl-1 in older versus younger patients (p=0.013) and in tobacco chewers+/-alcohol versus smokers+/-alcohol (p=0.037); and PCNA in node-positive versus node-negative tumours (p=0.037). On univariate analysis, high PCNA (p=0.007), Mcl-1 (p=0.050), node positivity (p=0.040) and co-expression of PCNA and Mcl-1 (p=0.008), had a significant impact on DFS. On multivariate analysis, low PCNA/Mcl-1 (p=0.006) co-expressing tumours were associated with improved DFS. Thus our study suggests that in patients undergoing primary radiotherapy, PCNA could be of potential predictive value to identify patients with risk of nodal metastases and in combination with Mcl-1 may have potential prognostic value to differentiate patients with poor DFS. These markers may be used for future trial patients requiring radiotherapy for their treatment.

Highlights of This Issue

ABT-263 is a Bcl-2 family member inhibitor with single-agent activity in numerous small cell lung carcinoma (SCLC) and leukemia/lymphoma cell lines in vitro and in vivo and is currently in ongoing clinical trials. Tahir and colleagues show that higher Mcl-1 expression was associated with resistance, and higher Bcl-2 and Noxa expression was associated with sensitivity to ABT-263 in both SCLC and leukemia/lymphoma cells lines. They also identified gene expression signature sets that are predictive of response to ABT-263. These markers and signatures are beneficial for the clinical development of ABT-263, providing guidance in the selection of patients in future clinical trials.EGFR and K-Ras mutations are associated with sensitivity/resistance to EGFR tyrosine kinase inhibitors (TKI) in non–small cell lung cancer (NSCLC), but do not explain all clinical outcomes. Similarly, the large interindividual variability in toxicity makes identification of novel pharmacogenetic markers attractive. This is the first study to show that the AKT1-SNP- A/A genotype is associated with shorter overall survival in NSCLC patients treated with gefitinib. Furthermore, the EGFR-191C/A, 216G/T, and R497K polymorphisms were associated with gastrointestinal toxicity. These polymorphisms can be assessed with a simple blood test and might enhance the selection of patients for EGFR-TKI treatment by identifying a genetically high-risk subgroup for drug-resistance or toxicity.Prostate cancer stem/progenitor cells can provide critical functions for tumor growth, metastases, resistance to current therapies, and disease relapse. Mimeault and colleagues describe the inhibition of hedgehog and EGFR signaling cascades, using gefitinib and cyclopamine, which improves the cytotoxic effects induced by the current clinical chemotherapeutic drug, docetaxel, on CD133+/CD44high/AR−/low side population (SP) cells with stem cell-like properties and the CD133−/CD44low/AR+ non-SP cell fraction from PC cells. These findings suggest that co-targeting of hedgehog and EGFR might constitute a novel therapeutic strategy for improving the efficacy of docetaxel-based therapies and, thereby, eradicate the total PC cell mass and prevent disease relapse.Akt and phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1) are components of the PI-3-kinase cell survival signal pathway. PDPK1 phosphorylates and activates AKT but also signals independently. The study reports the in vivo antitumor activity of PHT-427, a computationally designed small molecule that binds to the PI-3-phosphate binding pleckstrin homology (PH) domain of Akt and PDPK1, inhibiting their activity in cells and in animal tumors. The greatest antitumor activity is observed in tumors with activated PI-3-kinase signaling and less activity in tumors with mutant K-RAS. PHT-427 is an orally active antitumor agent and exhibits low toxicity.

The Anti-apoptotic Protein BCL2L1/Bcl-xL Is Neutralized by Pro-apoptotic PMAIP1/Noxa in Neuroblastoma, Thereby Determining Bortezomib Sensitivity Independent of Prosurvival MCL1 Expression

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.

Targeting Cdc7 Kinase in Multiple Myeloma.

Abstract 3847Poster Board III-783 IntroductionMyeloma remains an incurable cancer and the development of new therapeutic approaches is of fundamental importance. A key feature of progressive myeloma is deregulation of the mechanisms that control progression through the cell cycle, in particular, the increase in the activity of S-phase promoting factors and the loss of cell cycle brakes such as the tumour suppressor protein p53. Moreover, high expression of Mcl-1, a potent anti-apoptotic Bcl2 family member, and defects in the p53 pathway are now known to be instrumental in inhibiting responsiveness to current and emerging myeloma therapeutics. The Cell division cyle 7 (Cdc7) kinase is an essential protein for the initiation of DNA replication and for cell cycle progression. Previous studies have indicated that Cdc7 inhibition can cause p53 independent apoptosis in several cancer models both in vitro and in vivo and currently Cdc7 inhibitors are entering clinical trials as experimental drugs. In this work we study the effects of the prototype Cdc7 protein kinase inhibitor, PHA-767491, in myeloma cells. In addition to targeting Cdc7, PHA-767491 also targets cyclin dependent kinase 9 (CDK9) and can downregulate Mcl-1 in a variety of cancer cell lines. Hypothesis: Given the molecular characteristics of progressive myeloma and the spectrum of activity of PHA-767491, we hypothesized that Cdc7 inhibition could be an effective treatment strategy in myeloma. Methods The Multiple Myeloma Research Consortium Validation-Core cell lines were treated with increasing doses of PHA-767491 as a single agent and in combination with drugs that currently constitute standards of care in myeloma treatment. These lines included lines resistant to conventional anti-myeloma agents (e.g. melphalan, dexamethasone, and doxorubicin) as well as novel anti-MM agents (e.g. immunomodulatory thalidomide derivatives). A number of lines were TP53 mutants. Similarly, CD138+ purified primary cells with clincal resistance to lenalidomide and bortezomib were treated with increasing doses of PHA-767491. Cell viability was measured by Cell TitreGlo 72 hours post treatment allowing IC50 calcuations. Combination indices were calculated as described by Chou et Talalay. Apoptosis was assessed by PI and Annexin V staining using flow cytometry and immunoblotting with relevant antibodies was performed for cell cycle and apoptotic proteins of interest. ResultsWe found that PHA-767491 blocks proliferation and induces apoptosis with an IC50 in the low micromolar range in six out of seven MMRC cell lines and in primary cells from patient (s) with clinical resistance to bortezomib and lenalidomide. The averaqe and median IC50 value of MMRC cell lines was 2.25μM and 2μM respectively. TP53 mutation did not alter sensitivity to PHA-767491. Following treatment of myeloma cells with PHA-767491 there was potent inhibition of phosphorylation of the replicative helicase Mcm2 at serine 40/41 (a specific biomarker of Cdc7 activity) and complete downregulation of Mcl-1 by 6 hours, with concomitant caspase 3 cleavage and apoptosis as assessed by accumulation of sub-G1 cells. Annexin V/PI staining confirmed these cells to be apoptotic. When used in combination, PHA-767491 showed synergistic activity with melphalan and doxorubicin (Combination Index IC50 – 0.3, 0.7 respectively), even in the highly doxorubicin-resistant(p170/MDR1 mediated) line RPMI-8226-Dox-40 (Combination IndexIC50 – 0.3). ConclusionWe suggest that Cdc7 inhibition with PHA-767491 or with small molecules that have similar properties could be an effective strategy in myeloma treatment and merits further evaluation. Disclosures:Montagnoli:Nerviano Medical Sciences Oncology: Employment.

Prognostic role of myeloid cell leukemia‐1 protein (Mcl‐1) expression in human gastric cancer

Myeloid cell leukemia-1 protein (Mcl-1), an anti-apoptotic member of Bcl-2 family, has been reported to be correlated with tumor progression. The purpose of this study was to establish the prognostic value of Mcl-1 expression in human gastric cancer. Western blot assay was performed to detect the expression of Mcl-1 protein in human gastric cancer cell lines and tissues, while Mcl-1 expression in resected gastric cancer tissue samples was analysed by immunohistochemistry. The level of Mcl-1 protein expression in gastric cancer cell lines was significantly higher than that in gastric mucosa epithelial cell line. Moreover, the expression level of Mcl-1 protein was significantly higher in gastric cancer tissues than corresponding noncancerous tissues. Expression was correlated with T classification (P = 0.003), metastasis (P = 0.001), clinical stage (P = 0.010), and venous invasion (P = 0.004). Patients with high Mcl-1 expression showed poorer 5-year overall survival than patients with low Mcl-1 expression (P = 0.012; log-rank test). Further univariate and multivariate analysis suggested that high Mcl-1 expression was an independent prognostic marker for the disease. Mcl-1 is highly upregulated in gastric cancer and high Mcl-1 expression is correlated with a poor prognosis in gastric cancer patients. Thus, Mcl-1 can be utilized as an independent prognostic factor.

Mcl-1 expression predicts progression-free survival in chronic lymphocytic leukemia patients treated with pentostatin, cyclophosphamide, and rituximab

AbstractMyeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 protein family. Increased Mcl-1 expression is associated with failure to achieve remission after treatment with fludarabine and chlorambucil in patients with chronic lymphocytic leukemia (CLL). However, the influence of Mcl-1 expression has not been examined in CLL trials using chemoimmunotherapy. We investigated Mcl-1 protein expression prospectively as part of a phase 2 study evaluating the efficacy of pentostatin, cyclophosphamide, and rituximab in patients with untreated CLL. No significant difference by Mcl-1 expression was noted in pretreatment or response parameters. However, in patients with higher Mcl-1 expression, both minimal residual disease-negative status and progression-free survival was found to be significantly reduced (57% vs 19%, P = .01; 50.8 vs 18.7 months; P = .02; respectively). Mcl-1 expression may therefore be useful in predicting poor response to chemoimmunotherapy. These findings further support pursuing treatment strategies targeting this important antiapoptotic protein. (Because the trials described were conducted before the requirement to register them was implemented, they are not registered in a clinical trial database.)

Role of Myeloid Cell Leukemia-1 in Cell Growth of Squamous Cell Carcinoma

Myeloid cell leukemia-1 (Mcl-1), a member of the B-cell lymphoma-2 (Bcl-2) family, has been reported to be a critical survival factor in hematopoietic cells, yet little data exists for a role of Mcl-1 in human oral squamous cell carcinoma (SCC). A high level expression of Mcl-1 was observed in tumor cells of human primary SCC, lymph node metastasis tissues, and SCC cell lines. We manipulated expression of Mcl-1 protein in SCC cells by small interfering RNA (siRNA) for Mcl-1 and observed that Mcl-1 siRNA inhibited the growth of SCCs accompanied with apoptosis. Combination therapy of Mcl-1 siRNA and anti-tumor drug drastically inhibited the cell growth in comparison to that in each single treatment. In addition, phosphorylation of focal adhesion kinase (FAK) was decreased by treatment with Mcl-1 siRNA, resulting in decreases in phosphorylation of MEK1/2 and MAPK. The cell growth inhibition caused by knockdown of Mcl-1 was suggested to be mainly a result of suppression of proliferation via the inhibition of intracellular FAK/MAPK signaling pathways. These results imply a potentially important and novel role of the inhibition of Mcl-1 function by the use of specific siRNA in the treatment of SCC.

Apoptosis-based treatment of glioblastomas with ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins

Defects in the apoptotic signaling cascades contribute to the poor therapeutic response of malignant gliomas. As glioblastomas are characterized by high expression levels of anti-apoptotic Bcl-2 family proteins, we studied the effects of the novel Bcl-2 inhibitor, ABT-737, on malignant glioma cells. ABT-737 treatment released the pro-apoptotic Bax protein from its binding partner Bcl-2 and potently induced apoptotic cell death in glioblastoma cells in vitro and in vivo. The local administration of ABT-737 prolonged the survival in an intracranial glioma xenograft model. Downregulation of Mcl-1 and overexpression of Bcl-2 sensitized the cells to ABT-737-mediated apoptosis. Moreover, ABT-737 potentiated the cytotoxicity of the chemotherapeutic drugs vincristine and etoposide, and of the death ligand TRAIL. As glioma stem cells may play a crucial role for the tumor progression and the resistance to treatment in glioblastomas, we investigated the effects of ABT-737 on the subpopulation of glioma cells exhibiting stem cell characteristics. Inhibition of proliferation and induction of apoptosis by ABT-737 were less efficient in glioma stem cells than in non-stem cell-like glioma cells. As the resistance of glioma stem cells was associated with high Mcl-1 expression levels, ABT-737 treatment combined with downregulation of Mcl-1 could represent a promising novel approach in glioblastoma treatment.

Expression of VEGF-A/C, VEGF-R2, PDGF-α/Β, c-kit, EGFR, Her-2/Neu, Mcl-1 and Bmi-1 in Merkel cell carcinoma

Merkel cell carcinoma is a rare but very aggressive tumor of the skin. With current treatment options, Merkel cell carcinoma is associated with a high incidence of recurrence and metastasis. Targeted anticancer therapies such as receptor tyrosine kinase inhibitors and antisense oligonucleotides have been found to be a promising new type of treatment for various types of cancer. To evaluate whether the use of targeted therapies is a possible treatment option in Merkel cell carcinoma, we determined the expression of the target molecules c-kit, Mcl-1, Bmi-1, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-receptor 2 (VEGF-R2), platelet-derived growth factor (PDGF)-α, PDGF-Β, epidermal growth factor receptor (EGFR) and Her-2/Neu in a tissue microarray of 32 samples of 29 patients with Merkel cell carcinoma. C-kit-positive samples were analyzed for mutations in exons 9 and 11. The tissue microarray was stained immunohistochemically with antibodies directed against the above-mentioned proteins, and an immunoreactivity score was calculated. DNA was extracted from c-kit-positive samples and was analyzed for exon 9 and 11 mutations using direct DNA sequencing. We found that c-kit (7%), Mcl-1 (88%), Bmi-1 (78%), VEGF-A (91%), VEGF-C (75%) VEGF-R2 (88%), PDGF-α (72%) and PDGF-Β (13%) were expressed in Merkel cell carcinomas. All samples showed a lack of EGFR and Her-2/Neu expression. Analysis of c-kit revealed no mutations. As VEGF-A, VEGF-C, VEGF-R2, PDGFs and c-kit are targets of new cytostatic agents used in the treatment of other cancers, inhibition by a multitargeted chemotherapy could be a very promising treatment option. High expression of Bmi-1 and Mcl-1 warrants further studies on the use of antisense oligonucleotides in Merkel cell carcinoma.

Anti-Apoptotic MCL-1 Plays a Critical Role in Maintenance of Normal and Malignant Human Hematopoietic Stem Cells through FLT3 Signaling.

Mcl-1 (myeloid cell leukaemia-1) is an anti-apoptotic member of the Bcl-2 family protein, originally identified through the analysis of differentiating myeloid cells. We have recently reported that disruption of murine MCL-1 in adult murine hematopoiesis resulted in a complete deficiency of hematopoietic cells in the bone marrow, and that MCL-1 expression was activated in response to activation of c-Kit tyrosine kinase receptor signaling (Science 307;1101, 2005). Here we propose that human MCL-1 also plays a critical role in maintenance of normal and malignant human hematopoiesis. The analysis of FACS-sorted human HSCs and myeloid progenitors demonstrated that, as in case of murine hematopoiesis, HSCs expressed hMCL-1 at the highest level, and that its expression gradually downregulated in committed myeloid progenitors such as CMPs and GMPs. We also found that unlike murine long-term HSCs that express c-Kit but not FLT3 receptor tyrosine kinase, human long-term HSCs express both of these receptors. Ligation of FLT3 receptor in purified human HSCs immediately induced the expression of MCL-1, suggesting that FLT3 supports human HSC survival through stimulating MCL-1 expression. We then evaluated whether hMCL-1 is expressed in leukemic stem cells. Thirty patients with acute myeloid leukemia (AML) were enrolled in this study. Within the immature CD34+ blast population, the CD34+CD38− fraction expressed higher levels of MCL-1 than those in more differentiated CD34+CD38+ fraction. In most cases, CD34+CD38− AML cells expressed 2 to 10-fold higher levels of MCL-1 transcripts as compared to normal HSCs. Interestingly, the group of samples expressed highest levels of MCL-1 constituted mostly of FLT3/internal tandem duplications (ITDs) positive AML, suggesting that constitutive activation of FLT3 signaling by FLT3 mutations might induce overexpression of MCL-1. A high level expression of MCL-1 was also found in MOLM-13 and MV4-11, FLT3/ITD positive AML cell lines. Treatment of MOLM-13 and MV4-11 with the small molecule tyrosine kinase inhibitor PKC 412, resulted in induction of apoptosis, which was associated with decreased expression of MCL-1 transcripts and proteins. Based on these data, MCL-1 might play a critical role in maintenance of normal and malignant HSCs in human at least through FLT3 signaling. Our data also suggest that constitutive activation of FLT3 signaling by FLT3 mutation might contribute to leukemic transformation through enforcing survival of AML stem cells.

AT 101, an Inhibitor of Bcl-2 Family Members Is Cytotoxic to a Heterogeneous Group of CLL Samples and Synergistic with Rituximab.

Bcl-2 family proteins include both pro-survival and pro-apoptotic factors. The balance of these regulatory proteins determines a cell's threshold for death. Bcl-2, Bcl-XL, and Mcl-1 are structurally related anti-apoptotic proteins of the Bcl-2 family. Constitutive expression of Bcl-2 is characteristic of CLL and has been implicated in the leukemia's pro-survival tendency. In addition, high level expression of Mcl-1 and Bcl-2 have been shown to correlate with important clinical outcomes in CLL such as poor response to chemotherapy and decreased overall survival. Bcl-2 prevents mitochondrial cytochrome c release and inhibits apoptosis through direct regulation of caspases. The BH3-only proteins dimerize and neutralize their pro-survival counterparts via interaction with the BH3 binding grove on the anti-apoptotic proteins. This interaction results in the release of cytochrome c, subsequent caspase activation, and resultant cell death. Gossypol is a naturally occurring product found in cottonseed oil. AT 101, an orally bio-available derivative of gossypol is currently being evaluated in cancer clinical trials. AT 101 is a small molecule that mimics the inhibitory BH3 domain of endogenous antagonists of Bcl-2 negating its cytoprotective role. AT 101 induces apoptosis via caspase activation in cancer cells that over-express Bcl-2 or Bcl-XL, but had no significant cytotoxic effect on normal blood mononuclear cells (Mohammad, 2005). Fluorescence polarization assays demonstrate that AT 101 binds to Bcl-2, BcL-XL, and Mcl-1 with greater affinity than gossypol (Prada,ASH 2005). We hypothesized that inhibiting Bcl-2 and Mcl-1 by AT 101 may allow the CLL cells to enter apoptosis more readily and render them more sensitive to standard CLL therapeutics. We tested 10 different primary CLL patient samples in vitro and found gossypol to be cytotoxic in a dose and time dependent manner. We observed 50% reduction in CLL cell viability at a concentration of 2μM after 48 hours of treatment. Significant cytotoxicity was observed irrespective of ZAP-70 expression or IgVH mutational status. Increased ratios of Bcl-2/Bax and Mcl-1/Bax have been demonstrated in CLL patients who do not respond to rituximab (Bannerji 2003). To examine the ability of gossypol to enhance the cytotoxic effects of rituximab in CLL we evaluated samples from 6 different patients affected with the leukemia in vitro. CLL cells treated with rituximab at 10μg/ml showed no significant decrease in viability compared to untreated cells. When AT 101 was added at 5μM concentrations in combination with rituximab 10μg/ml for twelve hours the average CLL viability was decreased by 67.2% over that of cells treated with rituximab alone (P<0.001 by Bonferroni multiple comparison test) and by 45.3% over cells treated with AT 101 alone (P<0.001). The combined effect of AT 101 and rituximab appears synergistic and is displayed throughout CLL subtypes. In our studies we were unable to reach the IC50 of rituximab on CLL using doses up to 100μg/ml. However, the addition of AT 101 to rituximab 10μg/ml surpassed the IC50 and demonstrated similar cytotoxicity to that of high dose rituximab100μg/ml when used in conjunction with AT 101. Together our results suggest that AT 101 may have therapeutic potential in CLL as a single agent or in concert with other known CLL therapeutics. In particular, AT 101 was found to have synergistic cytotoxicity when combined with rituximab. Currently a phase I clinical trial is underway to evaluate the activity of AT 101 in previously untreated patients with CLL.

High expression of Mcl-1 in ALK positive and negative anaplastic large cell lymphoma

Aim: To gain more insight into the genes involved in the aetiology and pathogenesis of anaplastic large cell lymphoma (ALCL). Methods: Serial analysis of gene expression (SAGE) was undertaken on...

Mcl-1 and Bcl-2/Bax ratio are associated with treatment response but not with Rai stage in B-cell chronic lymphocytic leukemia.

Although both Bcl-2/Bax ratio and Mcl-1 have been identified to be of clinical relevance in B-cell chronic lymphocytic leukemia (CLL), there is controversy regarding their role; further, their relative importance is not well delineated. Expression of Bcl-2, Bax, the Bcl-2/Bax ratio, and Mcl-1 in 51 consecutive previously untreated CLL patients and 16 controls was determined by Western blotting. Only 37 patients were treated, all with chlorambucil and prednisone initially. Six patients achieved complete response (CR), 14 were non-responders (NR), and 17 had a partial response (PR), as defined by NCI criteria. There was considerable inter-patient variability in protein expression and overlap with healthy volunteers (P > 0.05). All patients with CR had low Mcl-1 levels compared to the PR + NR group (0.07 +/- 0.02 vs. 0.14 +/- 0.07, P = 0.043). Higher Mcl-1 expression as determined by dichotomizing the data was associated with a failure to achieve CR (P = 0.021). The Bcl-2/Bax ratio was significantly associated with treatment response only when CR and PR were considered together (0.89 +/- 0.53 [CR + PR] vs. 3.38 +/- 4.47 [NR], P = 0.0118). There was no association with Rai stage. Low Mcl-1 appears to be a requirement for CR, while low Bcl-2/Bax ratio is indicative of some response to conventional treatment.

IL-6-independent expression of Mcl-1 in human multiple myeloma.

Mcl-1 is a critical antiapoptotic survival factor for human multiple myeloma (MM). We examined the importance of IL-6 for Mcl-1 expression in five MM cell lines and in primary MM cells from 14 patients. While culture of MM.1S cells in IL-6 did induce Mcl-1 expression, four other MM cell lines exhibited high levels of Mcl-1 expression that were unaffected by IL-6. Similarly, Mcl-1 expression in 10 of 14 primary MM isolates was found to be IL-6-independent. An analysis of the mechanisms responsible for IL-6-independent Mcl-1 expression was undertaken. ERK1/2 activity did not influence Mcl-1 expression, distinct from Mcl-1 regulation that occurs during myeloid differentiation from hematopoietic progenitor cells. Inhibition of the PI3K pathway led to growth inhibition of 8226 and ANBL-6 cells without reduction of Mcl-1 levels, and high level Mcl-1 expression was maintained in the absence of activated STAT3. Analysis of the transcriptional activity of 5'-regulatory sequences from the human Mcl-1 gene in MM cells demonstrated high levels of IL-6-independent indicator gene activation as predicted. These data demonstrate that the mechanisms regulating Mcl-1 levels in MM cells are heterogeneous, and are often independent from IL-6 signaling pathways.