Downregulation Of Mcl-1 Research Articles

BDA-366, a putative Bcl-2 BH4 domain antagonist, induces apoptosis independently of Bcl-2 in a variety of cancer cell models

Several cancer cell types, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) upregulate antiapoptotic Bcl-2 to cope with oncogenic stress. BH3 mimetics targeting Bcl-2’s hydrophobic cleft have been developed, including venetoclax as a promising anticancer precision medicine for treating CLL patients. Recently, BDA-366 was identified as a small molecule BH4-domain antagonist that could kill lung cancer and multiple myeloma cells. BDA-366 was proposed to switch Bcl-2 from an antiapoptotic into a proapoptotic protein, thereby activating Bax and inducing apoptosis. Here, we scrutinized the therapeutic potential and mechanism of action of BDA-366 in CLL and DLBCL. Although BDA-366 displayed selective toxicity against both cell types, the BDA-366-induced cell death did not correlate with Bcl-2-protein levels and also occurred in the absence of Bcl-2. Moreover, although BDA-366 provoked Bax activation, it did neither directly activate Bax nor switch Bcl-2 into a Bax-activating protein in in vitro Bax/liposome assays. Instead, in primary CLL cells and DLBCL cell lines, BDA-366 inhibited the activity of the PI3K/AKT pathway, resulted in Bcl-2 dephosphorylation and reduced Mcl-1-protein levels without affecting the levels of Bcl-2 or Bcl-xL. Hence, our work challenges the current view that BDA-366 is a BH4-domain antagonist of Bcl-2 that turns Bcl-2 into a pro-apoptotic protein. Rather, our results indicate that other mechanisms beyond switching Bcl-2 conformation underlie BDA-366’s cell-death properties that may implicate Mcl-1 downregulation and/or Bcl-2 dephosphorylation.

Abstract 608: The MPS1 inhibitor S81694 is active in acute myeloid leukemia (AML)

Abstract Background: AML is a genetically heterogenous disease with poor prognosis and new treatments are needed. MPS1 is the main kinase of the spindle assembly checkpoint (SAC), critical for segregation of chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. MPS1 inhibition has been investigated in several solid tumors but not yet in AML. Here, we demonstrate the efficacy of the small molecule MPS1 inhibitor (MPS1i) S81694 in AML cell lines, primary cells and mouse models. Materials and Methods: Cell viability, cell stress and apoptosis were assessed by MTS assays, DiOC2(3) or annexin-V modifications and PI incorporation after exposure to S81694. Cell-cycle and polyploidy were determined by cytofluorometry and FISH. Phosphorylation of MPS1 was detected in synchronized cells by immunofluorescence detecting phosphorylated Thr33/Ser37 residues. Protein modifications were studied by WB. Time-lapse microscopy was used to determine mitosis duration using cells stained with the SiR-DNA live cell probe. Bone marrow cells from patients were obtained after informed consent and incubated on a MSC feeder or in methylcellulose for colony formation assays. All animal studies were performed in accordance with the animal ethics committee's guidelines. NSG mice were sub-lethally-irradiated and injected with AML-NS8 or AML-PS patient derived xenografts. Mice were treated intravenously with S81694 or with vehicle. Results: IC50 for viability after S81694 exposure alone was <1000nM in a panel of 8 AML cell lines. Three cell lines had IC50 >1000nM: the NPM1cmut cell line OCI-AML3, OCI-AML3-TP53mut and K562. In the most sensitive cell lines, including OCI-AML2, S81694 led to inhibition of MPS1 auto-phosphorylation, induced significant cell stress and apoptosis as detected by mitochondrial membrane potential loss, phosphatidylserine exposure and PI incorporation. In these cell lines, the cell cycle was strongly affected by treatment with S81694 showing aberrant 2n/4n ploidy distribution due to SAC abrogation as well as polyploidy (8n and 16n). S81694 exposure triggered mitotic exit as shown by cyclin B1 downregulation and significantly accelerated mitosis. For treated OCI-AML2 and OCI-AML3 cells we observed induction of gamma-H2AX (ser139), p53 upregulation and downstream caspase-3 and PARP cleavage indicating that S81694 induces p53 dependent apoptosis. Furthermore, MPS1i induced downregulation of anti-apoptotic proteins MCL-1 and BCLXL. Finally, for AML patient derived blast cells cells we observed apoptosis after exposure to S81694 and significant reduction of colony number. In two AML PDX models, intravenous administration of S81694 was shown to improve significantly median survival time, as compared to control, by 28 to 41 days and by 33 to 46 days respectively. Conclusion: The MPS1i S81694 shows significant preclinical activity in vitro and in vivo in AML. Combinations with different drugs active in AML are ongoing. Citation Format: Anna Kaci, Emilie Adicéam, Etienne De Braekeleer, Marine Garrido, Jeannig Berrou, Mélanie Dupont, Hanane Djamai, Virginie Eclache, Baruchel André, Claude Gardin, Hervé Dombret, Mike Burbridge, Thorsten Braun. The MPS1 inhibitor S81694 is active in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 608.

Abstract LB-177: BET and CDK9 protein inhibitors: Novel epigenetic therapy to synergistically kill human melanoma cells

Abstract Background: Dysregulation of epigenetic modifiers is a frequent event in melanoma and underlies many aspects of melanoma biology including resistance to targeted and immunotherapies. Here we report that dual targeting of BET and CDK9 proteins have synergistic effect in melanoma cells in vitro and in vivo. Methods: Cell viability was measured by Cell titre glow and colony forming assays. Annexin V/PI staining was performed to assess apoptosis. Apoptotic, anti-apoptotic, pro-survival signalling proteins were probed by immunoblotting. Genome wide expression analysis were performed by RNA-seq analysis of sensitive cell lines. Live-dead staining and apoptosis were performed in 3D spheroid models. In vivo studies were carried out on the YUMM1.1 mouse model in C57BL/6 syngeneic mice. Results: We report that BET and CDK9 inhibitor iBET151 and CDKI73 synergistically killed melanoma cells independent of their genetic background compared to single inhibitors. Combination index (CI) was calculated by calcusyn software also showed strong synergism (<0.3). However, these drugs did not kill human dermal fibroblast suggestive of a cancer cell specific effect. There was a time dependent increase in cell death that was associated with downregulation of anti-apoptotic proteins BCL2 and MCL1 and the oncogene c-MYC. These findings in 2D models were corroborated in 3D spheroid models. Gene Set Enrichment Analysis (GSEA) revealed that HALLMARK G2M CHECKPOINT, HALLMARK E2F TARGETS, HALLMARK MYC TARGETS were negatively enriched in the combination treatment (Normalized enrichment score =<-2.5). The in-vivo studies on the anti-PD1 resistant mouse melanoma cell line YUMM 1.1. showed that iBET151 and CDKI73 combination had marked effects on tumour growth compared to vehicle control and the individual drugs (p<0.05). Conclusion: Our findings have revealed that this novel combination of two epigenetic inhibitors targets multiple downstream genes associated with cell cycle, oncogenesis and, apoptotic pathways which augments cell death compared to effects of the single drugs. Overall, the findings warrant further investigation of key targets involved in the responses and the extent to which effects of CDK9 inhibition is due to effects on chromatin modelling. The combination would appear promising to consider for future clinical trial in melanoma patients not responding to current therapies. Citation Format: Abdullah Al Emran, Hsin-Yi Tseng, Dilini Gunatilake, Stuart Cook, Jessamy Tiffen, Stuart Gallagher, Peter Hersey. BET and CDK9 protein inhibitors: Novel epigenetic therapy to synergistically kill human melanoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-177.

Abstract 4849: YPN-005, an oral CDK7 inhibitor, shows a significant antitumor activity in Myc amplified solid tumors and MCL-1 overexpressed blood cancer

Abstract Background: Cyclin dependent kinase 7 (CDK7) has been recognized as one of potential therapeutic targets in Myc-driven cancers. Previously, we synthesized selective irreversible CDK7 inhibitor and we demonstrated a significant inhibition of the proliferation of TNBC and HCC cells by down-regulation of c-Myc and MCL-1. We further report YPN-005 of the preclinical data with biological characterization. Methods: Antiproliferative activity of YPN-005 was measured in solid tumor cell lines with Myc amplification and in hematologic cancer cells with MCL-1overexpression. To investigate the mechanism of action of YPN-005, the expression levels of phospho-RNA polymerase II, phospho-CDK2 and -CDK4, BClXL, cleaved-PARP, c-Myc and MCL-1 were analyzed by Western Blot analyses. CDK7 occupancy was determined by using a biotinylated small molecule probe following incubating of TNBC, HCC, AML cells and hPBMC with YPN-005. The therapeutic efficacy of YPN005 was evaluated in TNBC and HCC xenograft mouse model. Results: YPN-005 treatment induced apoptosis and cell cycle arrest. Selectivity of YPN-005 on CDK7 was confirmed in the 468 kinases panel assay. YPN-005 significantly inhibited the proliferation of TNBC, ER+ breast cancer, HCC, ovarian cancer, small cell lung cancer (SCLC), pancreas, prostate and various types of blood cancer cells and IC50's were determined in 1-20 nM range. Inhibition of cell proliferation was accompanied by down-regulation of RNA PolII phosphorylation, c-Myc and MCL-1 expression. Washout assay by Western Blot demonstrated that YPN-005 irreversibly blocked RNA PolII phosphorylation. In addition, YPN-005 downregulated the adaptive immune checkpoint PD-L1 by the depletion of c-Myc in TNBCs. CDK7 occupancy over 90% was observed in TNBC, HCC, and AML cells with 100nM of YPN-005. In vivo xenograft models of TNBC and HCC showed that daily oral administration of YPN-005 for 21 days (once, and 10~20mg/kg) efficiently inhibited the growth of tumor. All the mice survived and there was no significant changes in their hematologic profiles. Conclusion: Our findings suggest that YPN-005 is an orally available, potent, and selective CDK7 inhibitor which can be used for the treatment of Myc and MCL-1 overexpressing cancer patients. These data support the rationale for advancing YPN-005 towards IND-enabling studies. Citation Format: Jakyung Yoo, Jun Hee Lee, Mi Jung Lee, Kang Woo Lee, Ji Eun Min, Jinhwan Kim, Ki-Nam Min, Da-Hye Jeon, Dae Seong Lim, Kwang-Ok Lee. YPN-005, an oral CDK7 inhibitor, shows a significant antitumor activity in Myc amplified solid tumors and MCL-1 overexpressed blood cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4849.

Regulation of human Mcl-1 by a divergently-expressed antisense transcript

Mcl-1 is a member of the Bcl-2 anti-apoptotic protein family with important roles in the development, lifespan and metabolism of lymphocytes, as well as oncogenesis. Mcl-1 displays the shortest half-life of all Bcl-2 family members, with miRNA interference and proteasomal degradation being major pathways for Mcl-1 downregulation. In this study, we have identified a previously undescribed control mechanism active at the RNA level. A divergently transcribed lncRNA LOC107985203 (named here mcl1-AS1) negatively modulated Mcl-1 expression resulting in downregulation of Mcl-1 at both mRNA and protein level in a time-dependent manner. Using reporter assays, we confirmed that the mcl1-AS1 lncRNA promoter was located within Mcl-1 coding region. We next placed mcl1-AS1 under tetracycline-inducible control and demonstrated decreased viability in HEK293 cells upon doxycycline induction. Inhibition of mcl1-AS1 with shRNA reversed drug sensitivity. Bioinformatics surveys predicted direct mcl1-AS1 lncRNA binding to Mcl-1 transcripts, suggesting its mechanism in Mcl-1 expression is at the transcriptional level, consistent with a common role for anti-sense transcripts. The identification of a bi-directional promoter and lncRNA controlling Mcl-1 expression will have implications for controlling Mcl-1 activity in cancer cells, or for the purpose of enhancing the lifespan and quality of anti-cancer T lymphocytes.

Phosphodiesterase Type 5 Inhibitors Synergize Vincristine in Killing Castration-Resistant Prostate Cancer Through Amplifying Mitotic Arrest Signaling.

Combination therapies that display cancer-killing activities through either coexistent targeting of several cellular factors or more efficient suppression of a specific pathway are generally used in cancer treatment. Sildenafil, a specific phosphodiesterase type 5 (PDE5) inhibitor, has been suggested to display both cardioprotective and neuroprotective activities that provide a rationale for the combination with vincristine on the treatment against castration-resistant prostate cancer (CRPC). In the present work, vincristine arrested cells in the metaphase stage of mitosis. Vincristine-induced mitotic arrest was identified by Cdk1 activation (i.e., increased Cdk1Thr161 phosphorylation and decreased Cdk1Tyr15 phosphorylation), cyclin B1 upregulation, and increased phosphorylation of multiple mitotic proteins and stathmin. Sildenafil synergistically potentiated vincristine-induced mitotic arrest and a dramatic increase of mitotic index. Furthermore, sildenafil potentiated vincristine-induced mitochondrial damage, including Mcl-1 downregulation, Bcl-2 phosphorylation and downregulation, Bak upregulation and loss of mitochondrial membrane potential, and sensitized caspase-dependent apoptotic cell death. Sildenafil-mediated synergistic effects were mimicked by other PDE5 inhibitors including vardenafil and tadalafil, and also by PDE5A knockdown in cells, suggesting PDE5-involved mechanism. Notably, sildenafil amplified vincristine-induced phosphorylation and cleavage of BUBR1, a protein kinase in spindle assembly checkpoint (SAC) function and chromosome segregation. Sildenafil also significantly decreased kinetochore tension during SAC activation. Moreover, sildenafil synergized with vincristine on suppressing tumor growth in an in vivo model. In conclusion, the data suggest that sildenafil, in a PDE5-dependent manner, potentiates vincristine-induced mitotic arrest signaling, and sensitizes mitochondria damage–involved apoptosis in CRPC. Both in vitro and in vivo data suggest the combination potential of PDE5 inhibitors and vincristine on CRPC treatment.

The Histone Lysine-specific Demethylase 1 Inhibitor, SP2509 Exerts Cytotoxic Effects against Renal Cancer Cells through Downregulation of Bcl-2 and Mcl-1.

Lysine-specific histone demethylase 1 (LSD1), also known as KDM1A, can remove the methyl group from lysine 4 and 9 at histone H3, which regulates transcriptional suppression and activation. Recently, high expression of LSD1 in tumors has been shown to be involved in cancer cell proliferation, metastasis, and poor prognosis. We found that SP2509, a potent and reversible inhibitor of LSD1, induced apoptosis in human renal carcinoma (Caki and ACHN) and glioma (U87MG) cells. Pharmacological inhibition and siRNA-mediated silencing of LSD1 expression effectively downregulated anti-apoptotic proteins such as Bcl-2 and Mcl-1. Ectopic expression of these proteins markedly attenuated SP2509-induced apoptosis. At a mechanistic level, we found that inhibition of LSD1 downregulated Bcl-2 at a transcriptional level. Interestingly, protein expression of Mcl-1 was modulated at a post-translation level. Our results reveal that LSD1 could induce apoptotic cell death in renal carcinoma cells through downregulation of Bcl-2 and Mcl-1.

Melatonin inhibits high glucose-induced ox-LDL/LDL expression and apoptosis in human umbilical endothelial cells

Cardiovascular disease (CVD) is one of the major cause of mortality in diabetic patients. Evidence suggests that hyperglycemia in diabetic patients contributes to increased risk of CVD. This study is to investigate the therapeutic effects of melatonin on glucose-treated human umbilical vein endothelial cells (HUVEC) and provide insights on the underlying mechanisms. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Reactive oxygen species (ROS) and membrane potential was detected using 2',7'-dichlorofluorescein diacetate and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) dye staining, respectively. While, cell apoptosis was determined by Annexin-V staining and protein expression was measured using Western blot. Our results suggested that melatonin inhibited glucose-induced ROS elevation, mitochondria dysfunction and apoptosis on HUVEC. Melatonin inhibited glucose-induced HUVEC apoptosis via PI3K/Akt signaling pathway. Activation of Akt further activated BcL-2 pathway through upregulation of Mcl-1 expression and downregulation Bax expression in order to inhibit glucose-induced HUVEC apoptosis. Besides that, melatonin promoted downregulation of oxLDL/LOX-1 in order to inhibit glucose-induced HUVEC apoptosis. In conclusion, our results suggested that melatonin exerted vasculoprotective effects against glucose-induced apoptosis in HUVEC through PI3K/Akt, Bcl-2 and oxLDL/LOX-1 signaling pathways.

ROR1 Is Expressed in Diffuse Large B-Cell Lymphoma (DLBCL) and a Small Molecule Inhibitor of ROR1 (KAN0441571C) Induced Apoptosis of Lymphoma Cells.

The receptor tyrosine kinase ROR1 is absent in most normal adult tissues, but overexpressed in several malignancies. In this study, we explored clinical and functional inhibitory aspects of ROR1 in diffuse large B-cell lymphoma (DLBCL). ROR1 expression in tumor cells was more often observed in primary refractory DLBCL, Richter’s syndrome and transformed follicular lymphoma than in relapsed and non-relapsed DLBCL patients (p < 0.001). A survival effect of ROR1 expression was preliminarily observed in relapsed/refractory patients independent of gender and stage but not of age, cell of origin and international prognostic index. A second generation small molecule ROR1 inhibitor (KAN0441571C) induced apoptosis of ROR1+ DLBCL cell lines, similar to venetoclax (BCL-2 inhibitor) but superior to ibrutinib (BTK inhibitor). The combination of KAN0441571C and venetoclax at EC50 concentrations induced almost complete killing of DLBCL cell lines. Apoptosis was accompanied by the downregulation of BCL-2 and MCL-1 and confirmed by the cleavage of PARP and caspases 3, 8, 9. PI3Kδ/AKT/mTOR (non-canonical Wnt pathway) as well as β-catenin and CK1δ (canonical pathway) were inactivated. In zebra fishes transplanted with a ROR1+ DLBCL cell line, KAN0441571C induced a significant tumor reduction. New drugs with mechanisms of action other than those available for DLBCL are warranted. ROR1 inhibitors might represent a novel promising approach.

GSK3β suppression inhibits MCL1 protein synthesis in human acute myeloid leukemia cells.

Previous studies have shown that glycogen synthase kinase 3β (GSK3β) suppression is a potential strategy for human acute myeloid leukemia (AML) therapy. However, the cytotoxic mechanism associated with GSK3β suppression remains unresolved. Thus, the underlying mechanism of N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418)-elicited GSK3β suppression in the induction of AML U937 and HL-60 cell death was investigated in this study. Our study revealed that AR-A014418-induced MCL1 downregulation remarkably elicited apoptosis of U937 cells. Furthermore, the AR-A014418 treatment increased p38 MAPK phosphorylation and decreased the phosphorylated Akt and ERK levels. Activation of p38 MAPK subsequently evoked autophagic degradation of 4EBP1, while Akt inactivation suppressed mTOR-mediated 4EBP1 phosphorylation. Furthermore, AR-A014418-elicited ERK inactivation inhibited Mnk1-mediated eIF4E phosphorylation, which inhibited MCL1 mRNA translation in U937 cells. In contrast to GSK3α, GSK3β downregulation recapitulated the effect of AR-A014418 in U937 cells. Transfection of constitutively active GSK3β or cotransfection of constitutively activated MEK1 and Akt suppressed AR-A014418-induced MCL1 downregulation. Moreover, AR-A014418 sensitized U937 cells to ABT-263 (BCL2/BCL2L1 inhibitor) cytotoxicity owing to MCL1 suppression. Collectively, these results indicate that AR-A014418-induced GSK3β suppression inhibits ERK-Mnk1-eIF4E axis-modulated de novo MCL1 protein synthesis and thereby results in U937 cell apoptosis. Our findings also indicate a similar pathway underlying AR-A014418-induced death in human AML HL-60 cells.

Aspirin Induced Glioma Apoptosis through Noxa Upregulation.

Clinically, high cyclooxygenase-2 expression in malignant glioma correlates well with poor prognosis and the use of aspirin is associated with a reduced risk of glioma. To extend the current understanding of the apoptotic potential of aspirin in most cell types, this study provides evidence showing that aspirin induced glioma cell apoptosis and inhibited tumor growth, in vitro and in vivo. We found that the human H4 glioma cell-killing effects of aspirin involved mitochondria-mediated apoptosis accompanied by endoplasmic reticulum (ER) stress, Noxa upregulation, Mcl-1 downregulation, Bax mitochondrial distribution and oligomerization, and caspase 3/caspase 8/caspase 9 activation. Genetic silencing of Noxa or Bax attenuated aspirin-induced viability loss and apoptosis, while silencing Mcl-1 augmented the effects of aspirin. Data from genetic and pharmacological studies revealed that the axis of ER stress comprised an apoptotic cascade leading to Noxa upregulation and apoptosis. The apoptotic programs and mediators triggered by aspirin in H4 cells were duplicated in human U87 glioma cell line as well as in tumor-bearing BALB/c nude mice. The involvement of ER stress in indomethacin-induced Mcl-1 downregulation was reported in our previous study on glioma cells. Therefore, the aforementioned phenomena indicate that ER stress may be a valuable target for intervention in glioma apoptosis.

Ginsenoside panaxatriol reverses TNBC paclitaxel resistance by inhibiting the IRAK1/NF-κB and ERK pathways.

BackgroundPaclitaxel (PTX) resistance is a major obstacle in the treatment of triple-negative breast cancer (TNBC). Previously, we have reported that interleukin-1 receptor-associated kinase 1 (IRAK1) and its downstream pathways are associated with PTX resistance in TNBC cells. In this study, we sought to investigate the combination treatment of ginsenoside panaxatriol (GPT), one of the main active components in Panax ginseng, with PTX on viability and apoptosis of TNBC PTX resistant cells, and explore the role of IRAK1 mediated signaling pathways in the therapeutic effects.MethodsCellTiter-Glo and colony formation assays were used to assess cell viability. Flow cytometry was used to analyze subG1 and apoptosis. Western blot was used to detect expressions of proteins involved in apoptosis and the IRAK1/NF-κB and ERK pathways. The mRNA expression of inflammatory cytokines, S100A7/8/9 and cancer stem cell (CSC)-related genes were examined by qPCR. Stem cells were identified by tumor sphere assay. Cell invasion ability was examined by transwell assay.ResultsWe show that GPT inhibits MDA-MB-231 PTX resistant (MB231-PR) cell viability in a dose-dependent manner. When combined with PTX, GPT synergistically causes more cell death, induces subG1 accumulation and cell apoptosis. Besides, up-regulation of BAX/BCL-2 ratio, and down-regulation of MCL-1 are also observed. Moreover, this combination inhibits IRAK1, NF-κB and ERK1/2 activation, and leads to down-regulation of inflammatory cytokines (IL6, IL8, CXCL1, CCL2), S100A7/9 and CSC-related genes (OCT4, SOX2, NANOG, ALDH1, CD44) expression. In addition, the combination treatment suppresses MB231-PR cell invasion ability, and impairs tumor sphere growth both in MB231-PR and SUM159 PTX resistant (SUM159-PR) cells.ConclusionOur study demonstrates that GPT can resensitize TNBC PTX resistant cells to PTX by inhibiting the IRAK1/NF-κB and ERK pathways and reducing stem cell characteristics.

HSP90 overexpression potentiates the B-cell receptor and fibroblast growth factor receptor survival signals in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is still an incurable disease despite aggressive chemotherapies including the B-cell receptor (BCR) targeted-inhibitors. Therefore, we assessed the expression status of key signal mediators of the BCR pathway in CLL cells. Indeed, we detected aberrantly elevated levels of CD79a, B-cell adaptor for PI3K (BCAP) and phospholipase C (PLC)γ2, key mediators of BCR signal, in CLL cells. As HSP90 is also overexpressed in CLL cells, we hypothesized that HSP90 could potentiate the BCR signal via stabilization of multiple key components of the BCR-signalosome. We found that HSP90 formed a multi-molecular complex with CD79a, BCAP, PLCγ2, LYN, SYK, Bruton tyrosine kinase (BTK) and AKT and that, pharmacologic inhibition or partial depletion of HSP90 reduced the expression of these signal mediators in CLL cells. In addition, our findings also demonstrated that HSP90 could stabilize the tyrosine phosphatase, PTPN22 which positively regulates AKT phosphorylation, and the constitutively active fibroblast growth factor receptor 3 (FGFR3) in CLL cells. Finally, HSP90 inhibition induced apoptosis in CLL cells in a dose-dependent manner likely via downregulation of anti-apoptotic proteins MCL-1 and XIAP, but not BCL2, reported to be overexpressed in CLL cells. In total, our findings suggest that HSP90-inhibition may sensitize the leukemic B-cells to BCR-targeted agents, particularly those become resistant to these therapies.

Role of mutant KRAS as a biomarker in FL118-induced apoptosis, reactive oxygen species (ROS) production and inhibition of survivin, Mcl-1 and XIAP in human bladder cancer.

e15651 Background: Effective treatment of bladder cancer patients has not been realized over the past two decades due to heterogeneity with key oncogenic and tumor suppressor gene mutations, which is a major hurdle for effective treatment of the disease. Methods: Cancer cell growth/viability was determined using MTT assays; protein gene expression profiles were determined by both Western blots and proteomics technologies; PARP cleavage were determined by Western blots; genetic modulation of protein gene expression was realized by shRNA silencing or forced expression of the gene; reactive oxygen species (ROS) production was measured using a ROS assay kit; and human bladder xenograft tumor growth was determined using animal models. Results: Our study indicated that FL118, an innovative small molecule, exhibits differential anticancer efficacy in high-grade bladder cancer cell lines (HT1376, T24, UMUC-3). FL118 is highly effective at inhibiting the viability of T24 and UMUC-3 bladder cancer cells, which have Hras and Kras mutations, respectively. In contrast, HT1376 with wild-type Ras exhibits insensitivity to FL118-mediated inhibition of cell viability. Consistently, FL118 strongly induced PARP cleavage (hallmark of apoptosis) and inhibited the expression of survivin, XIAP and/or Mcl-1 in both T24 and UMUC-3 cells but not in the HT1376 cells. Our studies further indicated that the constitutive AKT and ERK1/2 signaling does not play a major role in these FL118-mediated effects. Instead, mutant Kras appeared to be involved in FL118 efficacy and played a positive role in mediating FL118 effects. Silencing of mutant Kras via shRNA reduced both FL118-induced PARP cleavage and downregulation of survivin, XIAP and Mcl-1 in UMUC-3 cells, suggesting mutant Kras is required for FL118 to exhibit high efficacy. FL118 increases ROS production in T24 and UMUC-3 cells but not in HT1376 cells. Silencing of mutant Kras with shRNA in UMUC-3 cells significantly reduced FL118-mediated ROS generation. Additionally, proteomics analyses of UMUC3 and HT1376 cell protein profiles after FL118 treatment support the obtained results described above. Consistent with these in vitro results, in vivo studies indicated that UMUC3 is highly sensitive and HT1376 is highly resistant to FL118 treatment. Conclusions: Our studies suggest that mutant Kras is a favorable biomarker for FL118 targeted treatment in human bladder cancer.

Arsenic trioxide-induced p38 MAPK and Akt mediated MCL1 downregulation causes apoptosis of BCR-ABL1-positive leukemia cells

In this study, we investigated the mechanisms underlying arsenic trioxide (ATO)-induced death of human BCR-ABL1-positive K562 and MEG-01 cells. ATO-induced apoptotic death in K562 cells was characterized by ROS-mediated mitochondrial depolarization, MCL1 downregulation, p38 MAPK activation, and Akt inactivation. ATO-induced BCR-ABL1 downregulation caused Akt inactivation but not p38 MAPK activation. Akt inactivation increased GSK3β-mediated MCL1 degradation, while p38 MAPK-mediated NFκB activation coordinated with HDAC1 suppressed MCL1 transcription. Inhibition of p38 MAPK activation or overexpression of constitutively active Akt increased MCL1 expression and promoted the survival of ATO-treated cells. Overexpression of MCL1 alleviated mitochondrial depolarization and cell death induced by ATO. The same pathway was found to be involved in ATO-induced death in MEG-01 cells. Remarkably, YM155 synergistically enhanced the cytotoxicity of ATO on K562 and MEG-01 cells through suppression of MCL1 and survivin. Collectively, our data indicate that ATO-induced p38 MAPK- and Akt-mediated MCL1 downregulation triggers apoptosis in K562 and MEG-01 cells, and that p38 MAPK activation is independent of ATO-induced BCR-ABL1 suppression.

Formononetin inhibits tumor growth by suppression of EGFR-Akt-Mcl-1 axis in non-small cell lung cancer

BackgroundEpidermal growth factor receptor (EGFR) activating mutations play crucial roles in the tumorigenesis of human non-small cell lung cancer (NSCLC). The mechanism regarding how EGFR signaling regulates myeloid cell leukemia sequence 1 (Mcl-1) protein stability and ubiquitination remains undefined.MethodsMTS assay was used for natural product library screening. The effect of formononetin (Formo) on NSCLC cells was determined by MTS assay and soft agar assay. Molecular modeling was performed to analyze the potential different binding modes between Formo and EGFR WT or mutants. Mcl-1 protein level and the inhibitory effect of Formo on EGFR signaling were examined by immunoblot, in vitro kinase assay, in vitro pulldown and ATP competition assays, co-immunoprecipitation assay, ubiquitination analysis, in vivo xenograft model, and immunohistochemical staining.ResultsFormo was identified as an EGFR inhibitor by a 98 commercially available natural product screening. Formo suppresses WT and mutant EGFR kinases activity in vitro, ex vivo, and in vivo. Molecular modeling indicates that Formo docks into the ATP-binding pocket of both WT and mutant EGFR. Formo inhibits EGFR-Akt signaling, which in turn activates GSK3β and promotes Mcl-1 phosphorylation in NSCLC cells. Treatment with Formo enhances the interaction between Mcl-1 and SCFFbw7, which eventually promotes Mcl-1 ubiquitination and degradation. Depletion of either GSK3β or SCFFbw7 compromised Formo-induced Mcl-1 downregulation. Finally, Formo inhibits the in vivo tumor growth in a xenograft mouse model.ConclusionThis study highlights the importance of promoting ubiquitination-dependent Mcl-1 turnover might be an alternative strategy to enhance the anti-tumor efficacy of EGFR-TKI.

The HDAC and PI3K dual inhibitor CUDC-907 synergistically enhances the antileukemic activity of venetoclax in preclinical models of acute myeloid leukemia.

Venetoclax is a promising agent in the treatment of acute myeloid leukemia (AML), though its antileukemic activity is limited to combination therapies. Mcl-1 downregulation, Bim upregulation, and DNA damage have been identified as potential ways to enhance venetoclax activity. In this study, we combine venetoclax with the dual PI3K and histone deacetylase inhibitor CUDC-907, which can downregulate Mcl-1, upregulate Bim, and induce DNA damage, as well as downregulate c-Myc. We establish that CUDC-907 and venetoclax synergistically induce apoptosis in AML cell lines and primary AML patient samples ex vivo. CUDC-907 downregulates CHK1, Wee1, RRM1, and c-Myc, which were found to play a role in venetoclax-induced apoptosis. Interestingly, we find that venetoclax treatment enhances CUDC-907-induced DNA damage potentially through inhibition of DNA repair. In vivo results show that CUDC-907 enhances venetoclax efficacy in an AML cell line derived xenograft mouse model, supporting the development of CUDC-907 in combination with venetoclax for the treatment of AML.

Inhibition of CDK9 by voruciclib synergistically enhances cell death induced by the Bcl-2 selective inhibitor venetoclax in preclinical models of acute myeloid leukemia

Venetoclax, an FDA-approved Bcl-2 selective inhibitor for the treatment of chronic lymphocytic leukemia and acute myeloid leukemia (AML), is tolerated well in elderly patients with AML and has good overall response rates; however, resistance remains a concern. In this study, we show that targeting CDK9 with voruciclib in combination with venetoclax results in synergistic antileukemic activity against AML cell lines and primary patient samples. CDK9 inhibition enhances venetoclax activity through downregulation of Mcl-1 and c-Myc. However, downregulation of Mcl-1 is transient, which necessitates an intermittent treatment schedule to allow for repeated downregulation of Mcl-1. Accordingly, an every other day schedule of the CDK9 inhibitor is effective in vitro and in vivo in enhancing the efficacy of venetoclax. Our preclinical data provide a rationale for an intermittent drug administration schedule for the clinical evaluation of the combination treatment for AML.

α‐Mangostin attenuates stemness and enhances cisplatin‐induced cell death in cervical cancer stem‐like cells through induction of mitochondrial‐mediated apoptosis

Cancer stem cells (CSCs) exhibit specific characteristics includingdecontrolled self-renewal, tumor-initiating, promoting, and metastatic potential, abnormal stemness signaling, and chemotherapy resistance. Thus, targeting CSC is becoming an emerging cancer treatment. α-Mangostin has been shown to have potent and multiple anticancer activities. Accordingly, we hypothesized that α-mangostin may diminish the stemness and proliferation of CSC-like cervical cancer cells. In our results, comparing to the parent cells, CSC-like SiHa and HeLa cells highly expressed CSC marker Sox2, Oct4, Nanog, CK-17, and CD49f. α-Mangostin significantly reduced the cell viability, sphere-forming ability, and expression of the CSC stemness makers of CSC-like cervical cancer cells. Further investigation showed that α-mangostin induced mitochondrial depolarization and mitochondrial apoptosis signaling, including upregulation of Bax, downregulation of Mcl-1 and Bcl-2, and activation of caspase-9/3. Moreover, α-mangostin synergically enhanced the cytotoxicity of cisplatin on CSC-like SiHa cells by promoting mitochondrial apoptosis and inhibiting the expression of CSC markers. Consistent with in vitro findings, in vivo tumor growth assay revealed that α-mangostin administration significantly inhibited the growth of inoculated CSC-like SiHa cells and synergically enhanced the antitumor effect of cisplatin. Our findings indicate that α-mangostin can reduce the stemness and proliferation of CSC-like SiHa and HeLa cells and promote the cytotoxicity of cisplatin, which may attribute to the mitochondrial apoptosis activation. Thus, it suggests that α-mangostin may have clinical potential to improve chemotherapy for cervical cancer by targeting cervical CSC.

Formononetin Inhibits Tumor Growth by Suppression of EGFR Signaling and Destabilization of Mcl-1 Protein in Non-Small Cell Lung Cancer

Background: Epidermal growth factor receptor (EGFR) activating mutations play crucial roles in the tumorigenesis of human non-small cell lung cancer (NSCLC). The mechanism regarding how EGFR signaling regulates myeloid cell leukemia sequence 1 (Mcl-1) protein stability and ubiquitination remains undefined. Methods: MTS assay was used for natural product library screening. The effect of formononetin on NSCLC cells was determined by MTS assay and soft agar assay. Molecular modeling was performed to analyze the potential different binding modes between formononetin and EGFR WT or mutants. The inhibitory effect of formononetin on EGFR signaling and Mcl-1 expression were examined by immunoblot, in vitro kinase assay, in vitro pulldown and ATP competition assays, co-immunoprecipitation assay, ubiquitination analysis, in vivo xenograft model, and immunohistochemical staining. Findings: Formononetin was identified as an EGFR inhibitor by a 98 commercially available natural product screening. Formononetin suppresses WT and mutant EGFR kinases activity in vitro, ex vivo, and in vivo. Molecular modeling indicates that formononetin docks into the ATP-binding pocket of both WT and mutant EGFR. Formononetin inhibits EGFR-Akt signaling, which in turn activates GSK3β and promotes Mcl-1 phosphorylation in NSCLC cells. Treatment with formononetin enhances the interaction between Mcl-1 and SCFFbw7, which eventually promotes Mcl-1 ubiquitination and degradation. Depletion of either GSK3β or SCFFbw7 compromised formononetin-induced Mcl-1 downregulation. Finally, formononetin inhibits the in vivo tumor growth in a xenograft mouse model. Interpretation: This study highlights the importance of promoting ubiquitination-dependent Mcl-1 turnover might be an alternative strategy to enhance the anti-tumor efficacy of EGFR-TKI. Funding Statement: This work was supported by the National Natural Science Foundation of China (No.81904262, and No.81972837), the Natural Science Foundation of Hunan Province (2018JJ3787, 2018JJ2604, 2019JJ50682). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: All mice experiments were performed according to strict guidelines established by the Medical Research Animal Ethics Committee, Central South University, China.