Downregulation Of Mcl-1 Expression Research Articles

The Synergistic Effect of Venetoclax Combined with a CDK9 Inhibitor in Primary Diffuse Large B Cell Lymphoma and Follicular Lymphoma Cells

The early phase studies have shown the high response rates in chronic lymphocytic leukemia (CLL) patients treated with the BH3 mimetic venetoclax (ABT-199). It indicated that inhibition of BCL-2 is a viable strategy for the treatment of lymphoid malignancies. Objective anti-tumor responses were also observed in patients with other common B-cell non-Hodgkin lymphomas (NHLs) such as follicular lymphoma (FL) or diffuse large B-cell lymphoma (DLBCL), however the overall response rates are not as high as those in CLL patients. Targeting only one anti-apoptotic protein may lead to or uncover resistance owing to activity of other anti-apoptotic BCL2-family members in these settings. MCL-1 is associated with both intrinsic and acquired resistance to venetoclax and thus inhibition of MCL-1 is being explored through either direct inhibition or indirect targeting. Expression of MCL-1 is maintained via p-TEFb-mediated transcription, of which CDK9 plays a critical role. Here we aimed to investigate the combined effects of CDK9 inhibitor and venetoclax in primary DLBCL and FL cells.Inhibition of CDK9 via a small molecule A-1467729.0 (AbbVie) caused rapid loss in phosphorylation (Serine 2) of RNA polymerase II and MCL-1 expression in all tested primary cells of DLBCL and FL patients, confirming the intended effect of CDK9 inhibition. Primary samples from 12 NHL cases (6 DLBCL including 3 GCB/3 non-GCB and 6 FL) were tested for their ex vivo response to A-1467729.0 or venetoclax alone or in combination. Apoptosis assays showed negligible effects (<10% induction) of A-1467729.0 at concentrations of 1, 10, 50 and 100 nM in 11 of 12 samples, while venetoclax at same dose range induced dose-dependent apoptosis in all samples. At 10 nM, the range of venetoclax-induced apoptosis was 16.8% - 55.3% (median 27.9%). Co-treatment with venetoclax and A-1467729.0 demonstrated synergistic effects at multiple doses in all 12 samples as evidenced by flow cytometry based apoptotic assay and PARP cleavage. Quantitative flow cytometry (QFC) studies (molecules/cell) showed that DLBCL and FL patient samples had comparable levels of anti-apoptotic proteins including BCL-2, MCL-1 and BCL-xl. However, BIM levels were higher in DLBCL in comparison to FL samples. Immunohistochemical staining of BIM in formalin-fixed paraffin embedded tissues confirmed this trend. Interestingly, venetoclax was more potent in inducing apoptosis in DLBCL patient samples than FL patient samples ex vivo. QFC data revealed a correlation between 1) BCL-2/BIM ratio and IC50 of venetoclax; 2) BCL-2/(MCL-1+BCL-xl) ratio and IC50 of venetoclax in FL. Importantly, venetoclax and CDK9 inhibitor combination demonstrated superior anti-tumor efficacy in xenograft mouse model of B-cell NHL than either agent alone.In summary, small molecule inhibition of CDK9 in primary NHL cells results in rapid down regulation of MCL1 expression. A-1467729.0, in combination with venetoclax, demonstrates synergistic activity as shown by apoptosis induction in primary DLBCL and FL cells. QFC of BCL-2 family proteins may be a useful biomarker for predicting response to BCL-2 inhibition in FL. The in vitro synergy between CDK9 inhibitor and venetoclax was also seen in vivo xenograft studies. These data support further investigation for combination therapy with CDK9 inhibitor and venetoclax in B-cell NHL. DisclosuresPhillips:AbbVie: Employment, Other: Shareholder, Patents & Royalties. Souers:AbbVie: Employment, Equity Ownership. Tapang:AbbVie: Employment, Equity Ownership. Albert:AbbVie: Employment, Equity Ownership.

Alvocidib Potentiates the Activity of Cytarabine and Mitoxantrone through the Targeting of MCL-1 When Administered in a Time Sequential Regimen in AML

Alvocidib has demonstrated a significant improvement in the complete response rates of newly diagnosed acute myeloid leukemia (AML) patients when administered before cytarabine and mitoxantrone (FLAM regimen) in a randomized Phase 2 study compared to 7+3, the current standard of care. Although the mechanism of action of alvocidib as a single agent is documented, the mechanism underlying synergy found in the FLAM regimen is still not fully understood. The FLAM regimen was originally developed based on the perceived benefit of time-sequential cell cycle arrest (alvocidib) followed by release of the cells from cell cycle arrest and inhibition of DNA replication (cytarabine) during S-phase. However, recent reports suggest that the transcriptional repression of key anti-apoptotic proteins (eg., MCL-1) mediated by alvocidib's CDK9 inhibition, drive the activity in the FLAM regimen. We, therefore, hypothesized that MCL-1 transcriptional repression constitutes the primary mechanism for the synergism observed with the treatment of the FLAM regimen. Here, we demonstrate that treatment with alvocidib, followed by treatment with cytarabine and mitoxantrone, is synergistic in vitro and correlates with the downregulation of MCL-1 expression. The FLAM regimen results in significant increases in caspase activity in comparison to any single agent within the combination. As has been previously reported, we also observe that increased activity of cytarabine in alvocidib-treated cells corresponds with progression into the S-phase of the cell cycle, following the washout of alvocidib. However, this observation accounts for only a small portion of the inhibition of cell proliferation. This is further confirmed by the observation that CDK4/6 (cell cycle) specific inhibitors, such as palbociclib, do not show synergistic increases in caspase activity following treatment in the same setting. In various AML cell lines treated with MCL-1 siRNA, followed by cytarabine and mitoxantrone treatment, we also observe a synergistic increase in the inhibition of cell proliferation. Therefore, considering our earlier work showing that MCL-1 dependence predicts AML patient response to the FLAM regimen, we propose that MCL-1 repression is the primary mechanism of alvocidib's biological activity and also a primary mechanism conferring resistance to cytarabine. We also conclude that the FLAM regimen is an effective regimen, clinically, in treating patients with high-risk AML, as a consequence of its inhibition of transcription via CDK9. DisclosuresKim:Tolero Pharmaceuticals: Employment. Bahr:Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Lee:Tolero Pharmaceuticals: Employment. Peterson:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment.

Loss in MCL-1 function sensitizes non-Hodgkin's lymphoma cell lines to the BCL-2-selective inhibitor venetoclax (ABT-199).

As a population, non-Hodgkin's lymphoma (NHL) cell lines positive for the t(14;18) translocation and/or possessing elevated BCL2 copy number (CN; BCL2High) are exquisitely sensitive to navitoclax or the B-cell lymphoma protein-2 (BCL-2)-selective inhibitor venetoclax. Despite this, some BCL2High cell lines remain resistant to either agent. Here we show that the MCL-1-specific inhibitor A-1210477 sensitizes these cell lines to navitoclax. Chemical segregation of this synergy with the BCL-2-selective inhibitor venetoclax or BCL-XL-selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Similarly, the CDK inhibitor flavopiridol downregulated MCL-1 expression and synergized with venetoclax in BCL2High NHL cell lines to a similar extent as A-1210477. A-1210477 also synergized with navitoclax in the majority of BCL2Low NHL cell lines. However, chemical segregation with venetoclax or A-1155463 revealed that synergy was driven by BCL-XL inhibition in this population. Collectively these data emphasize that BCL2 status is predictive of venetoclax potency in NHL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that inhibit MCL-1 function. These studies also potentially identify a patient population (BCL2Low) that could benefit from BCL-XL (navitoclax)-driven combination therapy.

Imperatorin acts as a cisplatin sensitizer via downregulating Mcl-1 expression in HCC chemotherapy.

Acquisition of cisplatin resistance is the common and critical limitation for hepatocellular carcinoma (HCC) therapy. Our study was aimed to determine whether there were conditions under which the addition of imperatorin would reverse the resistance of HCC cells to cisplatin-based therapy. In this study, we found that addition of imperatorin significantly enhanced the cytotoxicity of cisplatin to HCC cells. Since the Mcl-1 was overexpressed in HCC cell lines (HepG2, HepG3B, PLC, Huh7) compared with normal liver cell line (L-O2), we found that the Mcl-1 expression was downregulated by imperatorin but not influenced by cisplatin in HCC cells. In addition, our results showed the combination of imperatorin and cisplatin induced apoptosis and ∆Ψm collapse more significantly compared with treatment of imperatorin or cisplatin alone. Furthermore, the imperatorin-induced sensitization for cisplatin-cytotoxicity to HCC cells was abolished by the transfection of Mcl-1 expression plasmid. Finally, we found that the addition of imperatorin significantly reversed the resistance to cisplatin in cisplatin-resistant HCC cells, which was Mcl-1 dependent. In summary, our study revealed that combination with imperatorin could enhance the antitumor activity of cisplatin via targeting Mcl-1 and reverse the resistance to cisplatin in HCC.

ψ-Bufarenogin, a novel anti-tumor compound, suppresses liver cancer growth by inhibiting receptor tyrosine kinase-mediated signaling.

Resistance of hepatocellular carcinoma (HCC) to existing chemotherapeutic agents largely contributes to the poor prognosis of patients, and discovery of novel anti-HCC drug is in an urgent need. Herein we report ψ-Bufarenogin, a novel active compound that we isolated from the extract of toad skin, exhibited potent therapeutic effect in xenografted human hepatoma without notable side effects. In vitro, ψ-Bufarenogin suppressed HCC cells proliferation through impeding cell cycle progression, and it facilitated cell apoptosis by downregulating Mcl-1 expression. Moreover, ψ-Bufarenogin decreased the number of hepatoma stem cells through Sox2 depression and exhibited synergistic effect with conventional chemotherapeutics. Mechanistic study revealed that ψ-Bufarenogin impaired the activation of MEK/ERK pathway, which is essential in the proliferation of hepatoma cells. ψ-Bufarenogin notably suppressed PI3-K/Akt cascade, which was required in ψ-Bufarenogin-mediated reduction of Mcl-1 and Sox2. ψ-Bufarenogin inhibited the auto-phosphorylation and activation of epithelial growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met), thereafter suppressed their primary downstream cascades Raf/MEK/ERK and PI3-K/Akt signaling. Taken together, ψ-Bufarenogin suppressed HCC growth via inhibiting, at least partially, receptor tyrosine kinases-regulated signaling, suggesting that ψ-Bufarenogin could be a novel lead compound for anti-HCC drug.

BCR-ABL+ Bp-ALL Exhibits Heightened Sensitivity to Simultaneous Induction of Energy and ER Stress Via Downregulation of Mcl-1 Expression

Tumor metabolism has emerged as a hallmark of cancer by which the oncogenic profile of cancer cells pairs energy availability with growth and survival. BCR-ABL+ acute lymphoblastic leukemia (BCR-ABL+ ALL) accounts for 25% of ALL and the BCR-ABL fusion protein has been correlated with alterations in glucose metabolism. The glucose analogue 2-deoxy-D-glucose (2DG), induces simultaneous energy and ER-stress by inhibiting glycolysis (energy stress) via blocking HK and PGI enzymes, and interfering with N-linked glycosylation through its incorporation in place of mannose into the lipid-linked oligosaccharide (LLO) chain leading to premature termination of LLO synthesis (ER stress). We recently demonstrated that inhibition of both aerobic glycolysis and N-linked glycosylation by 2DG led to apoptosis in ALL (Mol Cancer Res 10:969, 2012). Among ALL subtypes, BCR-ABL+ ALL cell lines SupB15 and TOM1 exhibited the highest sensitivity to 2DG suggesting that in ALL cells BCR-ABL expression may be linked to heightened sensitivity to energy and/or ER stress. To test this premise, we constructed NALM6 (Bp-ALL) stable cell lines expressing the BCR-ABL p190 fusion and found that expression of the BCR-ABL p190 fusion in NALM6 cells significantly increased 2DG-induced apoptosis compared to mock transfected controls. To investigate the contribution of each form of stress (energy and ER stress) in the increased sensitivity of BCR-ABL+ cells to 2DG, we examined the effects of agents known to preferentially induce energy stress (lowering ATP) such as 2-fluoro-2-deoxy-D-glucose and oxamate, and ER stress such as tunicamycin. We found no differences in cell death or ATP levels between BCR-ABL+ and control ALL cells treated with agents solely inducing energy or ER stress, indicating the increased susceptibility of BCR-ABL+ ALL cells to 2DG results from the simultaneous induction of both types of stress. Consistent with these data, Fluorophore-Assisted Carbohydrate Electrophoresis and immunoblotting demonstrated that 2DG induced equivalent inhibition of N-linked glycosylation and upregulation of the UPR signaling in BCR-ABL+ and control ALL cells. Similar findings were seen in primary ALL patient samples. Consequently, simultaneous energy and ER stress are required for the heightened vulnerability to these forms of stress conferred by BCR-ABL in ALL. Based on the known function of Mcl-1 promoting survival in BCR-ABL+ ALL, we investigated its role in the mechanism of cell death induced by energy and/or ER stress agents. We uncovered that BCR-ABL+ ALL cell lines exhibited the lowest levels of Mcl-1 expression compared to control BCR-ABL- ALL and CML cells, and this basal level of Mcl-1 expression directly correlated with their resistance to energy and/or ER stressing agents. Further, we found that Mcl-1 knock-down by shRNA sensitized ALL and CML cells to the dual induction of energy and ER stress by 2DG. We also found that Mcl-1 expression was further downregulated in BCR-ABL+ and - ALL cells treated with energy and/or ER stressors which correlated with the level of apoptosis. Using qRT-PCR or co-treatment with the pancaspase inhibitor qVD-OPH or the proteasome inhibitor bortezomib, we showed that Mcl-1 downregulation by energy and/or ER stressors is neither due to transcriptional, caspase dependent cleavage, or post-translational regulation mechanisms but likely due to translational control of the Mcl-1 protein via AMPK/mTOR and the UPR PERK/p-eIF2α. Finally, because TKIs lead to decrease ATP (energy stress) in BCR-ABL+ cells, we co-treated BCR-ABL+ ALL cells with TKIs plus ER stressors and found that the simultaneous induction of these forms of stress by these combinations significantly increased cell death compared to single drug. Further, we found that TKIs + ER stressors further downregulated Mcl-1 and GRP78 expression compared to single agent treatment. Taken together, our data demonstrate that simultaneous induction of energy and ER stress leads to significant induction of apoptosis in BCR-ABL+ ALL cells, and these heightened vulnerability to these forms of stress is likely mediated by downregulation of Mcl-1 expression via AMPK activation/mTOR inhibition and PERK/p-eIF2α. Our study demonstrates that strategies with agents that induce energy and/or ER stress plus BCR-ABL TKIs warrant further consideration as a potential approach for future clinical translation into trials for BCR-ABL+ ALL patients. DisclosuresNo relevant conflicts of interest to declare.

Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression.

Although ABT-737, a small-molecule Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic agent, ABT-737-induced apoptosis is often blocked in several types of cancer cells with elevated expression of Mcl-1. Cafestol, one of the major compounds in coffee beans, has been reported to have anti-carcinogenic activity and tumor cell growth-inhibitory activity, and we examined whether cafestol could overcome resistance against ABT-737 in Mcl-1-overexpressed human renal carcinoma Caki cells. ABT-737 alone had no effect on apoptosis, but cafestol markedly enhanced ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells, human glioma U251MG cells, and human breast carcinoma MDA-MB231 cells. By contrast, co-treatment with ABT-737 and cafestol did not induce apoptosis in normal human skin fibroblast. Furthermore, combined treatment with cafestol and ABT-737 markedly reduced tumor growth compared with either drug alone in xenograft models. We found that cafestol inhibited Mcl-1 protein expression, which is important for ABT-737 resistance, through promotion of protein degradation. Moreover, cafestol increased Bim expression, and siRNA-mediated suppression of Bim expression reduced the apoptosis induced by cafestol plus ABT-737. Taken together, cafestol may be effectively used to enhance ABT-737 sensitivity in cancer therapy via downregulation of Mcl-1 expression and upregulation of Bim expression.

Abstract 342: Telomerase-dependent oncolytic adenovirus sensitizes human osteosarcoma cells to chemotherapy through Mcl-1 downregulation

Abstract Osteosarcoma is the most common malignant primary bone tumor. Despite advances in multi-agent chemotherapy, poor response to chemotherapy is one of the critical prognostic factors in osteosarcoma. Therefore, enhancement of chemosensitivity is one approach to improve the survival of osteosarcoma patients. We recently developed a telomerase-specific replication-competent oncolytic adenovirus, Telomelysin (OBP-301). A Phase I clinical trial in the U.S. showed the safety of OBP-301 in advanced cancer patients, and a Phase I/II clinical trial for malignant thoracic tumors is currently undergoing. We recently confirmed the antitumor effect of OBP-301 monotherapy in human osteosarcoma cells. In this study, we investigated the chemosensitizing effect of OBP-301 in human osteosarcoma cells and the molecular mechanism in the OBP-301-mediated enhancement of cell death. We used four human osteosarcoma cell lines, HOS, MNNG/HOS, 143B and SaOS-2. OBP-301 is an attenuated adenovirus, in which the hTERT promoter drives the expression of E1 gene, and causes tumor-selective lysis in a variety of human malignant tumor cells with telomerase activity. OBP-301 infection enhanced the cytotoxic effect of chemotherapeutic agents, cisplatin and doxorubicin, that are commonly used for the treatment of osteosarcomas. The calculation of combination index revealed the synergistic effects in all human osteosarcoma cell lines. Combination of OBP-301 increased apoptosis in the chemotherapeutic agents-treated cells. To clarify the molecular mechanism for the chemosensitizing effect of OBP-301, the expression of Bcl-2 family proteins, which are critical regulators of apoptosis, were investigated. In OBP-301-infected MNNG/HOS and SaOS-2 cells, the expression level of Mcl-1 was markedly decreased by OBP-301 infection. Downregulation of Mcl-1 expression by siRNA enhanced the chemotherapeutic agents-induced apoptosis. Moreover, combination therapy of chemotherapeutic agents with OBP-301 significantly suppressed tumor growth in a subcutaneous xenograft tumor model compared to monotherapy with chemotherapeutic agents or OBP-301. Anti-apoptotic Mcl-1 protein has been shown to be frequently overexpressed in human sarcomas. Overexpression of anti-apoptotic proteins is an important factor to prevent chemotherapy-induced apoptosis in cancer. We demonstrated the involvement of Mcl-1 suppression in the chemosensitizing effect of OBP-301 in human osteosarcoma cells. These results suggest that Mcl-1 is a critical target for improvement of the chemosensitivity in human osteosarcoma cells and the combination of chemotherapy with OBP-301 may be a novel therapeutic strategy for osteosarcoma patients. Citation Format: Shuhei Osaki, Toshinori Omori, Hiroshi Tazawa, Joe Hasei, Yasuaki Yamakawa, Tsuyoshi Sasaki, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Telomerase-dependent oncolytic adenovirus sensitizes human osteosarcoma cells to chemotherapy through Mcl-1 downregulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2014-342

Abstract 2933: Assessment of ABT-263 activity across a comprehensive cancer cell line collection leads to a novel, potent combination therapy for small cell lung cancer

Abstract BH3 mimetics like ABT-263 induce apoptosis in subsets of cancer. However, they have shown limited clinical efficacy as single agents, and rational combination strategies remain underexplored. Toward the development of a novel combined therapy, we examined efficacy of ABT-263 across 331 cancer cell lines. Cell lines with high expression levels of the pro-apoptotic gene BIM were more sensitive to ABT-263 activity. This was particularly evident in small cell lung cancer (SCLC), which tends to have higher BIM levels and is among the most sensitive to ABT-263. However, MCL-1 expression mitigated the impact of ABT-263-induced apoptosis by sequestering BIM released from BCL-2 and BCL-XL. Since MCL-1 expression is dependent on TORC1 activity in some cancers, we attempted to overcome the apoptotic block in SCLC by combining ABT-263 with the TORC1/2 inhibitor AZD8055. AZD8055 treatment alone downregulated MCL-1 expression; combination AZD8055 and ABT-263 induced marked apoptosis and growth arrest in vitro, as well as tumor regressions in multiple SCLC xenograft models. Strikingly, low-dose AZD8055 was sufficient to promote tumor regressions in the combination strategy. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve single-agent ABT-263 activity in SCLC. Citation Format: Anthony C. Faber, Carlotta Costa, Anahita Dastur, Cyril Benes, Jeffrey Engelman. Assessment of ABT-263 activity across a comprehensive cancer cell line collection leads to a novel, potent combination therapy for small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2933. doi:10.1158/1538-7445.AM2014-2933

Targeting CDK9 by wogonin and related natural flavones potentiates the anti-cancer efficacy of the Bcl-2 family inhibitor ABT-263.

Tumor initiation, progression and resistance to therapies are tightly associated with over-expression of anti-apoptotic proteins Bcl-2, Bcl-x(L), Bcl-w and Mcl-1. ABT-263 (Navitoclax), an orally bio-available small-molecule mimetic of the Bcl-2 homology domain 3, inhibits Bcl-2, Bcl-x(L), and Bcl-w and has shown anti-cancer effects mainly on lymphomas and lymphocytic leukemia. Despite promising results obtained from the clinical trials, the use of ABT-263 in patients is dose-limited due to causing thrombocytopenia via inhibition of Bcl-x(L) in platelets. ABT-199 specifically inhibits Bcl-2; however, its use is limited to tumors over-expressing only Bcl-2. Besides, many tumors resist treatment due to high levels of Mcl-1 expression or develop resistance via up-regulation of Mcl-1 during long-term exposure. These obstacles highlight the demand to improve the ABT-263-based therapy. In this study, we show that anti-cancer flavones, e.g., wogonin, baicalein, apigenin, chrysin and luteolin enhance ABT-263-induced apoptosis in different cancer cell lines and in primary AML and ALL cells by down-regulation of Mcl-1 expression. Importantly, wogonin does not enhance the toxicity of ABT-263 to proliferating normal T cells and thrombocytes. Wogonin also potentiates the lethality of ABT-263 in cancer cells which have acquired resistance to ABT-263. Furthermore, we show that combination of wogonin with ABT-263 promotes in vivo tumor regression in a human T-cell leukemia xenograft mouse model. Our study demonstrates that wogonin (and related flavones) reduce the effective dose of ABT-263 thereby possibly decreasing the risk of adverse side effects.

Downregulation of Mcl-1 through inhibition of translation contributes to benzyl isothiocyanate-induced cell cycle arrest and apoptosis in human leukemia cells

Benzyl isothiocyanate (BITC) is one of the compounds of ITCs' family that has attracted a great deal of interest because of its ability to exhibit anticancer activity. In this study, we investigated the effects of BITC on cell cycle arrest and apoptosis in human leukemia cell lines, primary leukemia cells, and nude mice Jurkat xenograft. Exposure of Jurkat cells to BITC resulted in dose- and time-dependent increase in apoptosis, caspase activation, cytochrome c release, nuclear apoptosis-inducing factor (AIF) accumulation, Bcl2-associated X protein (Bax) translocation, and myeloid cell leukemia-1 (Mcl-1) downregulation. Treatment with these cells also resulted in cell cycle arrest at the G2/M phase. The G2/M-arrested cells are more sensitive to undergoing Mcl-1 downregulation and apoptosis mediated by BITC. BITC downregulates Mcl-1 expression through inhibition of translation, rather than through a transcriptional, post-translational, or caspase-dependent mechanism. Dephosphorylation of eukaryotic initiation factor 4G could contribute to the inhibition of Mcl-1 translation mediated by BITC. Furthermore, ectopic expression of Mcl-1 substantially attenuates BITC-mediated lethality in these cells, whereas knockdown of Mcl-1 through small interfering RNA significantly enhances BITC-mediated lethality. Finally, administration of BITC markedly inhibited tumor growth and induced apoptosis in Jurkat xenograft model in association with the downregulation of Mcl-1. Taken together, these findings represent a novel mechanism by which agents targeting Mcl-1 potentiate BITC lethality in transformed and primary human leukemia cells and inhibitory activity of tumor growth of Jurkat xenograft model.

Oxaliplatin Sensitizes OS Cells to TRAIL-induced Apoptosis Via Down-regulation of Mcl1

To investigate the killing effect on OS cells of a combination of oxaliplatin and TRAIL and related molecular mechanisms. TRAIL and oxaliplatin were applied to OS732 cells singly or jointly and survival inhibition rates were measured by MTT assay, changes of cellular shape being assessed with inverted phase contrast and fluorescence microscopy. Apoptotic rates were analyzed by flow cytometry (FCM) and immunocytochemistry was used to examine Mcl1 expression of OS732 cells. The survival inhibition rate of combined application of 100 μg/ml TRAIL and 1 μg/ml oxaliplatin on OS-732 cells was significantly higher than that of either agent singly (p<0.01). Changes of cellular shape and apoptotic rates also indicated apoptosis-inducing effects of combined application to be much stronger than those of individual application. Oxaliplatin had the effect of down-regulating Mcl1 expression and sensitizing OS cells to TRAIL-induced apoptosis. A combination of TRAIL and oxaliplatin exerts strong killing effects on OS-732 cells which might be related to down-regulation of Mcl1 expression.

The HSP90 Inhibitor XL888 Overcomes BRAF Inhibitor Resistance Mediated through Diverse Mechanisms

The clinical use of BRAF inhibitors is being hampered by the acquisition of drug resistance. This study shows the potential therapeutic use of the HSP90 inhibitor (XL888) in six different models of vemurafenib resistance. The ability of XL888 to inhibit growth and to induce apoptosis and tumor regression of vemurafenib-resistant melanoma cell lines was shown in vitro and in vivo. A novel mass spectrometry-based pharmacodynamic assay was developed to measure intratumoral HSP70 levels following HSP90 inhibition in melanoma cell lines, xenografts, and melanoma biopsies. Mechanistic studies were carried out to determine the mechanism of XL888-induced apoptosis. XL888 potently inhibited cell growth, induced apoptosis, and prevented the growth of vemurafenib-resistant melanoma cell lines in 3-dimensional cell culture, long-term colony formation assays, and human melanoma mouse xenografts. The reversal of the resistance phenotype was associated with the degradation of PDGFRβ, COT, IGFR1, CRAF, ARAF, S6, cyclin D1, and AKT, which in turn led to the nuclear accumulation of FOXO3a, an increase in BIM (Bcl-2 interacting mediator of cell death) expression, and the downregulation of Mcl-1. In most resistance models, XL888 treatment increased BIM expression, decreased Mcl-1 expression, and induced apoptosis more effectively than dual mitogen-activated protein-extracellular signal-regulated kinase/phosphoinositide 3-kinase (MEK/PI3K) inhibition. HSP90 inhibition may be a highly effective strategy at managing the diverse array of resistance mechanisms being reported to BRAF inhibitors and appears to be more effective at restoring BIM expression and downregulating Mcl-1 expression than combined MEK/PI3K inhibitor therapy.

Abstract 4724: Anti-tumor effects of SC-2001, a novel STAT3 inhibitor, in breast cancer cells

Abstract Background: STAT3 has been shown to play an important role in breast carcinogenesis and is often overexpressed in primary breast tumors. STAT3 has emerged as a novel potential anti-cancer target. Previously, we have designed SC-2001 ((Z)-2-((3-methoxy-2H-pyrrol-2-ylidene)methyl)-1H-pyrrole) and reported its efficacy in HCC cells (AACR 2011, abstract#4516). SC-2001 is structurally related to the Mcl-1 inhibitor obatoclax, and showed better antitumor effects than obatoclax in HCC cells. Here, we examined the efficacy and drug mechanism of SC-2001 in breast cancer cells. Methods: breast cancer cell lines were used for in vitro studies. Apoptosis was examined by both flow cytometry and Western blot. Signal transduction pathways in cells were assessed by Western Blot. Tyrosine Phosphatase Assay Kit was used for SHP-1 activity assay. Gene silencing was done by small interference RNA (siRNA). Results: SC-2001 inhibited tumor cell growth (by MTT assay) and induced apoptosis in several breast cancer cell lines (MDA MB-468, MDA MB-231, MDA MB-453, MCF-7). SC-2001 inhibited the protein-protein interactions between Mcl-1 and Bak similar to obatoclax. In addition, SC-2001 downregulated Mcl-1 expression by reducing its transcription, whereas obatoclax had no such effect. SC-2001 downregulated the phosphorylation of STAT3 (Tyr 705) and subsequently inhibited its transcriptional activities in a dose-dependent manner. Moreover, STAT3-regulated proteins, including Mcl-1, survivin and cyclin D1, were also repressed by SC-2001. Over-expression of STAT3 in MDA MB-468 cells protected cells from SC-2001-induced apoptosis. Importantly, SC-2001 enhanced the expression of SHP-1, a negative regulator of STAT3. Inhibition of SHP-1 by either specific inhibitor or siRNA reduced the apoptotic effects of SC-2001, indicating that SHP-1 plays a key role in mediating SC-2001-induced cell death. Conclusions: our results suggest that SC-2001 induced apoptosis in breast cancer cells, and that this effect is mediated through SHP-1-dependent STAT3 inactivation. (Supported by NSC100-2325-B-002-036, NSC98-2320-B-010-005-My3 and NSC-100-2325-B-010-007, VGHTPE V100D-005-4, 101DHA0100318, and 101DHA0100230.) Keyword: obatoclax, breast cancer, STAT3, SHP-1, apoptosis Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4724. doi:1538-7445.AM2012-4724

Abstract 2016: A subset of small cell lung cancer (SCLC) cell lines is Mcl-1-dependent and responds to cyclin-dependent kinase (cdk)9 inhibition in vitro and in vivo

Abstract Small cell lung cancer (SCLC) accounts for approximately 13% of lung cancer cases, and current treatment options are limited to radiation and/or chemotherapy, which frequently lead to drug resistance. Recently, multiple SCLC cell lines have been found to be responsive to the Bcl-2/Bcl-xL inhibitor, ABT-737, both in vitro and in vivo. Clinical trials of the related orally-bioavailable compound ABT-263 have shown promising activity in SCLC patients. However, a subset of SCLC cell lines that express Mcl-1, another anti-apoptotic protein, are resistant to ABT-737. Therefore, drugs targeting Mcl-1 may be particularly useful in this disease. Here, we have investigated dependence of a panel of SCLC cell lines on Mcl-1 for survival. A subset of Mcl-1-expressing SCLC cell lines, including NCI-H82, NCI-N417 and SHP-77, demonstrate apoptosis upon Mcl-1 knockdown. However, other SCLC cell lines, including NCI-H69, NCI-H196 and SW1271, are not responsive to Mcl-1 knockdown. In addition, the dependence of SCLC cell lines on Mcl-1 for survival can be predicted by BH3 profiling, a methodology that measures dependence of a cell on a variety of anti-apoptotic proteins. We have also investigated the efficacy of Flavopiridol, a potent inhibitor of the transcriptional cyclin-dependent kinase (cdk)9, against the panel of SCLC cell lines. Flavopiridol acutely down-regulates Mcl-1 expression in all SCLC cell lines tested; however, only Mcl-1-dependent cells, including NCI-H82, NCI-N417 and SHP-77, undergo abrupt apoptosis. In contrast, cells that were not found to be Mcl-1 dependent are not effectively killed by Flavopiridol. We have further investigated the activity of Flavopiridol against Mcl-1-dependent SCLC in vivo utilizing nude mice bearing NCI-H82 xenografts. Flavopiridol treatment results in substantial tumor growth inhibition in this aggressive and chemotherapy-refractory model. To potentially stratify patients who would be predicted to be responsive to Flavopiridol treatment, we have performed MCL-1 gene fluorescence in situ hybridization in SCLC cell lines and deidentified patient specimens. MCL-1 gene copy number gains exist in both patient and cell line samples, and copy number gain of MCL-1 or MYC in cell lines correlates with Mcl-1 dependence. Strategies for targeting MCL-1 and MYC copy number gains will be discussed further. Finally, we have demonstrated that the combination of Flavopiridol and ABT-737 significantly reduces viability in Mcl-1-independent SW1271 cells compared to treatment with either drug alone. Taken together, our studies suggest that Flavopiridol-mediated cdk9 inhibition may effectively target Mcl-1-dependent SCLCs, including those resistant to Bcl-2/Bcl-xL inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2016. doi:1538-7445.AM2012-2016

Ran Is a Potential Therapeutic Target for Cancer Cells with Molecular Changes Associated with Activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK Pathways

Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities. Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. K-Ras-mutated, c-Met-amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways.

PDGF-RTK Inhibition Restores CD95/Fas-Mediated Apoptosis in Leukemic T-LGL Via Promoting ASM Translocation, Ceramide Production and CD95 Signal Amplification

Abstract 1374Human memory T cells are protected from Fas/CD95-induced apoptosis. The mechanisms involved in blocking of CD95 signal transduction revealed the presences of anti-apoptotic factors. T-LGL leukemic cells are terminal effector memory T cells (Temra [CD45RA+/CD62L−]) featuring over-expression of Fas/Fas ligand, constitutive activation of PDGF-RTK and its downstream Src family kinase (SFK), PI3K/PKB/AKT/ERK1/2 signal cascade, as well as high expression levels of c-FLIP and Mcl-1. Inhibition of the downstream targets of PDGF-RTK pathway, such as SFK and PI3K induce significant level of apoptosis in leukemic LGL. However, direct targeting of PDGF-RTK showed a limited efficacy in induction of cell death in vitro. Loss of Fas sensitivity is the key pathological aberration in leukemic LGL, and restoration of CD95/Fas-mediated apoptosis in LGL leukemic cells remains a challenge. Therefore, reprogramming LGL leukemic cells to become sensitive to the already existing over-expressed Fas/Fas L in patients can be a potential therapeutic strategy. In the current study, we observed that inhibition of PDGF-RTK activity with inhibitors, such as AG1296 or imatinib myselate (Gleevec) restored CD95/Fas sensitivity in T-LGL leukemic cells. Our data suggest that the mechanism of PDGF-RTK inhibition restores Fas sensitivity in T-LGL leukemic cells via induction of acid sphingomyelinase (ASM) cell membrane translocation and activation, promoting ceramide production and Fas receptor aggregation in the cell membrane lipid rafts of leukemic cells. We also observed that PDGF-RTK inhibition significantly downregulates Mcl-1 expression in T-LGL leukemic cells. These observations suggest a possible role for future clinical application of TKIs in clinical management of this disease. Disclosures:No relevant conflicts of interest to declare.

Abstract LB-22: A subset of small cell lung cancer (SCLC) cell lines is Mcl-1-dependent and responds to cyclin-dependent kinase (cdk)9 inhibition in vitro and in vivo

Abstract Small cell lung cancer (SCLC) accounts for approximately 13% of lung cancer cases, and current treatment options are limited to radiation and/or chemotherapy, which frequently lead to drug resistance. Recently, multiple SCLC cell lines have been found to be responsive to the Bcl-2/Bcl-xL inhibitor, ABT-737, both in vitro and in vivo. Clinical trials of the related orally-bioavailable compound ABT-263 have shown promising activity in SCLC patients. However, a subset of SCLC cell lines that express Mcl-1, another anti-apoptotic protein, are resistant to ABT-737. Therefore, drugs targeting Mcl-1 may be particularly useful in this disease. Here, we have investigated dependence of a panel of SCLC cell lines on Mcl-1 for survival. A subset of Mcl-1-expressing SCLC cell lines, including NCI-H82 and SHP-77, demonstrate apoptosis upon Mcl-1 knockdown. However, other SCLC cell lines, including NCI-H69 and SW1271, are not responsive to Mcl-1 knockdown. In addition, the dependence of SCLC cell lines on Mcl-1 for survival can be predicted by BH3 profiling, a methodology that measures dependence of a cell on a variety of anti-apoptotic proteins. We have also investigated the efficacy of Flavopiridol, a potent inhibitor of the transcriptional cyclin-dependent kinase (cdk)9, against the panel of SCLC cell lines. Flavopiridol acutely down-regulates Mcl-1 expression in all cell lines tested; however, only Mcl-1-dependent cells undergo abrupt apoptosis. In contrast, cells that were not found to be Mcl-1 dependent, including H69 and SW1271 cells, are not effectively killed by Flavopiridol. We have further investigated the activity of Flavopiridol against Mcl-1-dependent SCLC in vivo utilizing nude mice bearing H82 xenografts. Flavopiridol treatment results in substantial tumor growth inhibition in this aggressive and chemotherapy-refractory model. To potentially stratify patients who would be predicted to be responsive to Flavopiridol treatment, we have performed MCL-1 fluorescent in situ hybridization in SCLC cell lines and deidentified patient specimens. MCL-1 gene copy number gains exist in both patient and cell line samples, and copy number gain in cell lines correlates with Mcl-1 dependence. Finally, we have demonstrated that the combination of Flavopiridol and ABT-737 can synergistically kill both Mcl-1-dependent and – independent SCLC cells, with significantly reduced viability in SHP-77 and SW1271 cells compared to treatment with either drug alone. Taken together, our studies suggest that Flavopiridol-mediated cdk9 inhibition may effectively target Mcl-1-dependent SCLCs, including those resistant to Bcl-2/Bcl-xL inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-22. doi:10.1158/1538-7445.AM2011-LB-22

Modulation of Multiple Myeloma Apoptosis and Adhesion by Seliciclib, A Selective Inhibitor of Cyclin Dependent Kinase (CDK)

AbstractAbstract 3971Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cells that eventually develop resistance to chemotherapy. Novel agents such as Thalidomid, Bortezomib and Lenalidomide improve response rate and prolong progression free and overall survival. Drug resistance, differentiation block and increased survival of the MM tumor cells result from genomic alterations including high levels of cyclin D. In addition, MM displays a high genomic instability. The most common translocations in MM lead to dysregulation of cyclin D (CCND), a regulatory protein that governs the activation of a key cell cycle regulator – cyclin dependent kinase (CDK). Cell-proliferation is tightly regulated by activity of CDKs at checkpoints located at the boundaries of cell cycle stages. The association of CDK4/6 with CCND is critical for G1 phase progression, whereas CDK2-cycllin E is necessary for S phase initiation. Deviation from the orderly control may lead to malignant transformation. Furthermore, genomic instability was reported to be affected by over expression of cyclin E. Cyclin E deregulation induces genomic instability as an early event in tumorgenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore, sought to investigate the role of cyclin E in MM. We first examined the expression profile of CCNE1 in various MM cell lines (hMMCLs) including ArP-1, ARH77, CAG, NCIH929, RPMI8226 and U266 by immunoblotting utilizing CCNE1 Abs and by qRT-PCR. CCNE1 expression was found to be heterogeneous being high in U266 and NCIH292, intermediate in ARH77, CAG and PRMI8226 and low in ArP-1 MM cell lines, respectively. A direct correlation between the level of protein and the transcript was observed. We then evaluated the effect of Seliciclib (Roscovitine, CYC202), a selective inhibitor of CDK's on hMMCLs viability, using the MTT assay as hMMCLs have been reported to be sensitive to CDK inhibition. The various hMMCLs were incubated with increasing concentration of Seliciclib (0-100 μM) for 3 days. Seliciclib inhibition of CDK resulted in dose-dependent cytotoxicity with IC50 of 20–80 μM with PRMI8226 being the most sensitive, while ARH77 hMMCL the most resistant. Cell cycle study demonstrated increased apoptosis (based on sub G1 and annexin V analysis) in the sensitive hMMCLs, with no effect on the resistant cell lines. Annexin V binding was observed within 5h and reached maximal level 12h flowing Seliciclib (50 μM) exposure. We next assessed the effect Seliciclib on MCL1, an anti-apoptotic BCL2 family member that is consistitutively expressed in MM cells. Seliciclib significantly down-regulated MCL1 expression in a time and dose dependant manner. Assessing the effect of Seliciclib on cell cycle regulators including CCND, CCNE and P27kip1 indicated a very significant reduction in CCND2 and P27 expression levels in most of the hMMCLs in a dose and time dependant manner, while CCNE1 expression levels increased. Transfection of the low expressing hMMCL RPMI8226 with CCNE1, inducing over expression of cyclin E, resulted in reduced sensitivity of the MM tumor cells to the Seliciclib induced cell death in comparison to the paternal cell line. Lastly, we investigated the effect of Seliciclib on MM cell adhesion to fibronectin (FN), as cell-adhesion-mediated-drug-resistance (CAM-DR) is a major importance protecting MM tumor cells from cytotoxic drugs induced apoptosis. hMMCLs were pre-incubated with increasing concentration of Seliciclib for 1h, plated onto 40 μg/ml FN coated plates for 1h, washed and stained by crystal violet. Seliciclib resulted in a direct dose dependant inhibition of adhesion. The adhesive protective property was evalued by CAM-DR assay, whereas FN-adhesion protected the MM tumor cells against doxorubicin mediated cytotoxicity. There was no difference between the cytotoxic effects of Seliciclib on FN-MM adherent vs. suspended tumor cells. In summary incubation of MM tumor cells with Seliciclib, a selective inhibitor of CDK's, results in apoptosis which is mediated by down regulation of MCL1 and is accompanied by reduced adhesion to FN. Its ability to prevent adhesion – mediated – drug resistance of MM tumor cells may turn Seliciclib as essential component of modern anti MM drug combination therapy. Further experiments should test its synergism with anti MM novel agents. Disclosures:No relevant conflicts of interest to declare.

Actinomycin D synergistically enhances the efficacy of the BH3 mimetic ABT-737 by downregulating Mcl-1 expression

Many types of cancer cells possess the ability to evade apoptosis, leading to their rapid and uncontrolled proliferation. As major regulators of apoptosis, Bcl-2 proteins serve as emerging targets for novel chemotherapeutic strategies. In this study, we examined the involvement of Bcl-2 proteins in apoptosis induced by the chemotherapeutic agent actinomycin D. A dramatic decrease in anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) mRNA and protein expression was detected upon actinomycin D treatment. Further, Mcl-l over-expression caused resistance to cell death upon treatment with actinomycin D, implicating a role for the down-regulation of Mcl-1 in actinomycin D-induced apoptosis. We also explored the therapeutic potential of actinomycin D in combination with ABT-737, an experimental agent that inhibits anti-apoptotic Bcl-2 proteins. Actinomycin D sensitized cells to ABT-737 treatment in a Bak- or Bax-dependent manner. Importantly, low concentrations of actinomycin D and ABT-737 were more effective in inducing cell death in transformed cells than their untransformed counterparts. A synergistic effect of actinomycin D and ABT-737 on cell death was observed in several human tumor cell lines. Like actinomycin D treatment, knocking down Mcl-1 expression greatly sensitized tumor cells to ABT-737, and Mcl-1 over-expression abrogated the cytotoxic effect induced by ABT-737 and actinomycin D. These results suggest that the down-regulation of Mcl-1 by actinomycin D is likely responsible for the observed synergistic effect between the two drugs. Overall, our studies provide compelling evidence that the combination of actinomycin D and ABT-737 may lead to an effective cancer treatment strategy.