Inhibition of MCL‐1 by obatoclax sensitizes Sp2/0‐Ag14 hybridoma cells to glutamine deprivation‐induced apoptosis

For several cancer cell types, the lack of an adequate supply of the amino acidl ‐glutamine (Gln) triggers apoptosis, a phenomenon termed Gln addiction. In this report, we examined the role of the anti‐apoptotic proteins of the B‐cell lymphoma 2 (BCL‐2) protein family in the survival of Sp2/0‐Ag14 (Sp2/0) mouse hybridoma cells, a cell line that undergoes apoptosis within minutes of Gln deprivation. Western blot analysis revealed that myeloid cell leukaemia 1 (MCL‐1) was expressed at much higher levels than BCL‐2, B‐cell lymphoma extra‐large and BCL‐2‐like protein 2 making it the prominent pro‐survival BCL‐2 family member in this hybridoma. Gln deprivation triggered a progressive decrease in MCL‐1 protein levels, which coincided with the decrease in Sp2/0 cell survival. Moreover, Sp2/0 cells were much more sensitive to the broad Bcl‐2 homology domain‐3 (BH3) mimetic obatoclax (which targets MCL‐1) than to the more selective drug ABT‐737 (which does not target MCL‐1). Finally, we show that obatoclax sensitizes Sp2/0 cells to apoptosis following Gln starvation. All together, the data presented here reveal that modulation of the pro‐survival protein MCL‐1 is an important step in the sequence of events leading to the initiation of apoptosis in Gln‐starved Sp2/0 cells. Cancer cells require an adequate supply ofl ‐glutamine for their survival. Using a mouse hybridoma cell line that is exquisitely sensitive to glutamine starvation, we show that the levels of the pro‐survival BCL‐2 family protein MCL‐1 decrease upon glutamine starvation in a manner that correlates with the loss of cell viability. Moreover, inhibiting MCL‐1 with the drug obatoclax sensitizes hybridoma cells to glutamine starvation. Thus, in some cancer cells, glutamine starvation triggers the inactivation of pro‐survival proteins. Our data suggest that the combined inhibition of glutamine biosynthesis pathways and BCL‐2 proteins may prove effective against some cancers. Copyright © 2015 John Wiley & Sons, Ltd.     

WIN55,212-2 induces cytoplasmic vacuolation in apoptosis-resistant MCL cells

Cannabinoid receptors 1 (CB1) and/or 2 (CB2) are overexpressed in many types of human malignancies including mantle cell lymphoma (MCL). Agonists to CB1 and CB2 promote ceramide de novo synthesis, p38–mitogen-activated protein kinase-dependent activation of caspase-3 and apoptotic cell death in most MCLs. However, in this report we describe that in some MCLs the response to treatment with cannabinoids decreased cell viability as assessed by metabolic activity but did not involve the caspase-3 cascade or loss of plasma membrane integrity. Both primary cells from one MCL patient and the MCL cell line Granta519 responded to treatment with cannabinoids by formation of cycloheximide-sensitive cytoplasmic vacuoles, but did not enter apoptosis. The persistent expression of mammalian homolog of Atg8 with microtubule-associated protein-1 light chain-3 II (LC3 II) and p62, as well as the lack of protection from chloroquine, indicates that lysosomal degradation is not involved in this cytoplasmic vacuolation process, distinguishing from classical autophagy. Transmission electron microscopy images and immunofluorescence staining of endoplasmic reticulum (ER) chaperone calreticulin showed that the vacuoles were of ER origin and that chromatin remained normal. These features resemble paraptosis-like cell death—a third type of a programmed cell death not previously described in response to cannabinoids.     

Toll-Like Receptor 1/2 and 5 Ligands Enhance the Expression of Cyclin D1 and D3 and Induce Proliferation in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin’s lymphoma with a still undefined etiology. Several lines of evidence are consistent with the possible involvement of peculiar microenvironmental stimuli sustaining tumor cell growth and survival, as the activation of Toll-like receptors (TLR) 4 and 9. However, little is known about the contribution of other TLRs of pathogenic relevance in the development of MCL. This study reports evidence that MCL cell lines and primary MCL cells express different levels of TLR2 and TLR5, and that their triggering is able to further activate the Akt, MAPK, and NF-κB signaling cascades, known to be altered in MCL cells. This leads to the enhancement of cyclin D1 and D3 over-expression, occurring at post-translational level through a mechanism that likely involves the Akt/GSK-3α/β pathway. Interestingly, in primary B cells, TLR1/2 or TLR5 ligands increase protein level of cyclin D1, which is not usually expressed in normal B cells, and cyclin D3 when associated with CD40 ligand (CD40L), IL-4, and anti-human-IgM co-stimulus. Finally, the activation of TLR1/2 and TLR5 results in an increased proliferation of MCL cell lines and, in the presence of co-stimulation with CD40L, IL-4, and anti-human-IgM also of primary MCL cells and normal B lymphocytes. These effects befall together with an enhanced IL-6 production in primary cultures. Overall, our findings suggest that ligands for TLR1/2 or TLR5 may provide critical stimuli able to sustain the growth and the malignant phenotype of MCL cells. Further studies aimed at identifying the natural source of these TLR ligands and their possible pathogenic association with MCL are warranted in order to better understand MCL development, but also to define new therapeutic targets for counteracting the tumor promoting effects of lymphoma microenvironment.     

Combination bortezomib and rituximab treatment affects multiple survival and death pathways to promote apoptosis in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a distinct histologic subtype of B cell non-Hodgkins lymphoma (NHL) associated with an aggressive clinical course. Inhibition of the ubiquitin-proteasome pathway modulates survival and proliferation signals in MCL and has shown clinical benefit in this disease. This has provided rationale for exploring combination regimens with B-cell selective immunotherapies such as rituximab. In this study, we examined the effects of combined treatment with bortezomib and rituximab on patient-derived MCL cell lines (Jeko, Mino, SP53) and tumor samples from patients with MCL where we validate reversible proteasome inhibition concurrent with cell cycle arrest and additive induction of apoptosis. When MCL cells were exposed to single agent bortezomib or combination bortezomib/rituximab, caspase dependent and independent apoptosis was observed. Single agent bortezomib or rituximab treatment of Mino and Jeko cell lines and patient samples resulted in decreased levels of nuclear NFκB complex(es) capable of binding p65 consensus oligonucleotides, and this decrease was enhanced by the combination. Constitutive activation of the Akt pathway was also diminished with bortezomib alone or in combination with rituximab. On the basis of in vitro data demonstrating additive apoptosis and enhanced NFκB and phosphorylated Akt depletion in MCL with combination bortezomib plus rituximab, a phase II trial of bortezomib-rituximab in patients with relapsed/refractory MCL is underway.Key words: mantle cell lymphoma, proteasome inhibition, CD20, survival and death pathways, apoptosis     

The role of B cell antigen receptors in mantle cell lymphoma

Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and secondary resistance to currently available therapies in most cases. Therefore, despite recent advances in the treatment of this disease, it is still considered to be incurable in the majority of cases. MCL B cells retain their B cell antigen receptor (BCR) expression during and after neoplastic transformation. BCRs in MCL show distinct patterns of antigen selection and ongoing BCR signaling. However, little is known about the involved antigens and the mechanisms leading to lymphomagenesis and lymphoma progression in MCL. Recent preclinical and clinical studies have established a crucial role of the BCR and the potential of inhibiting its signaling in this disease. This has established the B cell antigen receptor signaling cascade as a very promising therapeutic target to improve outcome in MCL alone or in combination with chemo-immunotherapy in recent years.     

Cannabinoid receptor ligands mediate growth inhibition and cell death in mantle cell lymphoma

We have earlier reported overexpression of the central and peripheral cannabinoid receptors CB1 and CB2 in mantle cell lymphoma (MCL), a B cell non-Hodgkin lymphoma. In this study, treatment with cannabinoid receptor ligands caused a decrease in viability of MCL cells, while control cells lacking CB1 were not affected. Interestingly, equipotent doses of the CB1 antagonist SR141716A and the CB1/CB2 agonist anandamide inflicted additive negative effects on viability. Moreover, treatment with the CB1/CB2 agonist Win-55,212-2 caused a decrease in long-term growth of MCL cells in culture. Induction of apoptosis, as measured by FACS/Annexin V-FITC, contributed to the growth suppressive effect of Win-55,212-2. Our data suggest that cannabinoid receptors may be considered as potential therapeutic targets in MCL.     

Combination bortezomib and rituximab treatment affects multiple survival and death pathways to promote apoptosis in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a distinct histologic subtype of B cell non-Hodgkins lymphoma (NHL) associated with an aggressive clinical course. Inhibition of the ubiquitin-proteasome pathway modulates survival and proliferation signals in MCL and has shown clinical benefit in this disease. This has provided rationale for exploring combination regimens with B-cell selective immunotherapies such as rituximab. In this study, we examined the effects of combined treatment with bortezomib and rituximab on patient-derived MCL cell lines (Jeko, Mino, SP53) and tumor samples from patients with MCL where we validate reversible proteasome inhibition concurrent with cell cycle arrest and additive induction of apoptosis. When MCL cells were exposed to single agent bortezomib or combination bortezomib/rituximab, caspase dependent and independent apoptosis was observed. Single agent bortezomib or rituximab treatment of Mino and Jeko cell lines and patient samples resulted in decreased levels of nuclear NF-kB complex(es) capable of binding p65 consensus oligonucleotides, and this decrease was enhanced by the combination. Constitutive activation of the AKT pathway was also diminished with bortezomib alone or in combination with rituximab. On the basis of in vitro data demonstrating additive apoptosis and enhanced NF-kB and phosphorylated AKT depletion in MCL with combination bortezomib plus rituximab, a phase II trial of bortezomib-rituximab in patients with relapsed/refractory MCL is underway.     

Autophagy controls everolimus (RAD001) activity in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive neoplasm, which lacks effective therapy. The mechanistic target of rapamycin (MTOR) kinase inhibitor everolimus (RAD001) has shown activity in preclinical and clinical models of MCL, despite the fact that its mechanism of action has not been fully elucidated. We found that everolimus activity in MCL cells is closely linked to AKT phosphorylation status, and that the prevention of AKT rephosphorylation upon everolimus treatment by means of a selective AKT inhibitor, greatly enhances everolimus activity. Furthermore, our data show that an accumulation of autophagic vacuoles correlates with a lack of efficacy of dual AKT-MTOR targeting and that the complete therapeutic potential of this strategy can be restored by ATG gene selective knockdown or secondary inhibition of autolysosome formation by hydroxychloroquine. We thus demonstrated for the first time that the use of an autophagy inhibitor can overcome resistance to the combination of MTOR and AKT inhibitors in MCL cell lines and primary samples, demonstrating the prosurvival role of autophagy in AKT-MTOR compromised cells, and pointing out some potential opportunities using this triple combinational strategy in hematological malignancies.     

BCL2 and related prosurvival proteins require BAK1 and BAX to affect autophagy

It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.     

Inhibition of constitutive NF-kappa B activation in mantle cell lymphoma B cells leads to induction of cell cycle arrest and apoptosis

Constitutive activation of the NF-kappaB has been documented to be involved in the pathogenesis of many human malignancies, including hemopoietic neoplasms. In this study, we examined the status of NF-kappaB in two non-Hodgkin's lymphoma cell lines derived from mantle cell lymphoma (MCL) samples and in patient MCL biopsy specimens by EMSA and confocal microscopic analysis. We observed that NF-kappaB is constitutively activated in both the MCL cell lines and in the MCL patient biopsy cells. Since NF-kappaB has been shown to play an important role in a variety of cellular processes, including cell cycle regulation and apoptosis, targeting the NF-kappaB pathways for therapy may represent a rational approach in this malignancy. In the MCL cell lines, inhibition of constitutive NF-kappaB by the proteasome inhibitor PS-341 or a specific pIkappaBalpha inhibitor, BAY 11-7082, led to cell cycle arrest in G(1) and rapid induction of apoptosis. Apoptosis was associated with the down-regulation of bcl-2 family members bcl-x(L) and bfl/A1, and the activation of caspase 3, that mediates bcl-2 cleavage, resulting in the release of cytochrome c from the mitochondria. PS-341or BAY 11-induced G(1) cell cycle arrest was associated with the inhibition of cyclin D1 expression, a molecular genetic marker of MCL. These studies suggest that constitutive NF-kappaB expression plays a key role in the growth and survival of MCL cells, and that PS-341 and BAY 11 may be useful therapeutic agents for MCL, a lymphoma that is refractory to most current chemotherapy regimens.     

BCL2 and related prosurvival proteins require BAK1 and BAX to affect autophagy

It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.     

Design,synthesis and biological evaluation of novel 2,3-indolinedione derivatives against mantle cell lymphoma

2,3-Indolinedione derivatives have been identified as a novel class of promising agents for cancer treatment. In this study, eighteen 2,3-indolinedione derivatives were designed and synthesized, and their anticancer activities against mantle cell lymphoma (MCL) cells were evaluated. Most of them exhibited significant antiproliferative activity against the tested cell lines, and compound K5 was the most potent (MCL cellular IC₅₀ = 0.4–0.7 μM). Further, compound K5 could induce cell apoptosis and cell cycle arrest in G2/M phase. Additionally, the results of drug-likeness analysis demonstrated that these novel 2,3-indolinedione derivatives could have potential as novel treatment strategies for MCL.     

Ki-67 as a prognostic marker in mantle cell lymphoma��consensus guidelines of the pathology panel of the European MCL Network

Mantle cell lymphoma (MCL) has a heterogeneous clinical course and is mainly an aggressive B cell non-Hodgkin lymphoma; however, there are some indolent cases The Ki-67 index, defined by the percentage of Ki-67-positive lymphoma cells on histopathological slides, has been shown to be a very powerful prognostic biomarker. The pathology panel of the European MCL Network evaluated methods to assess the Ki-67 index including stringent counting, digital image analysis, and estimation by eyeballing. Counting of 2 × 500 lymphoma cells is the gold standard to assess the Ki-67 index since this value has been shown to predict survival in prospective randomized trials of the European MCL Network. Estimation by eyeballing and digital image analysis showed a poor concordance with the gold standard (concordance correlation coefficients [CCC] between 0.29 and 0.61 for eyeballing and CCC of 0.24 and 0.37 for two methods of digital image analysis, respectively). Counting a reduced number of lymphoma cells (2 × 100 cells) showed high interobserver agreement (CCC = 0.74). Pitfalls of the Ki-67 index are discussed and guidelines and recommendations for assessing the Ki-67 index in MCL are given.     

Peritumoral monocytes induce cancer cell autophagy to facilitate the progression of human hepatocellular carcinoma

Macroautophagy/autophagy is an important catabolic process mediating cellular homeostasis and plays critical roles in cancer development. Whereas autophagy has been widely studied in various pathological models, little is known about the distribution, clinical significance and regulatory mechanism of this process in human hepatocellular carcinoma (HCC). In the present study, we found that tumor tissues exhibited significantly increased levels of autophagy compared with non-tumor tissues, and cancer cells with higher levels of autophagy were predominantly enriched in the invading edge regions of human HCC. Increased MAP1LC3B/LC3B expression in the invading edge regions was significantly correlated with a higher density of closely located monocytes, and TNF and IL1B derived from tumor-activated monocytes synergistically induced cancer cell autophagy in the invading edge regions of HCC. Monocyte-elicited autophagy induced the epithelial-mesenchymal transition (EMT) of cancer cells and promoted tumor metastasis by activating the NFKB-SNAI1 signaling pathway. Moreover, the increase of LC3B⁺ cancer cells in the invading edge areas was associated with high mortality and reduced survival of patients with HCC. These findings indicated that cancer cell autophagy is regulated by a collaborative interaction between tumor and immune cell components in distinct HCC microenvironments, thus allowing the inflammatory monocytes to be rerouted in a tumor-promoting direction.     

Placental autophagy regulation by the BOK-MCL1 rheostat

Autophagy is the catabolic degradation of cellular cytoplasmic constituents via the lysosomal pathway that physiologically elicits a primarily cytoprotective function, but can rapidly be upregulated in response to stressors thereby inducing cell death. We have reported that the balance between the BCL2 family proteins BOK and MCL1 regulates human trophoblast cell fate and its alteration toward cell death typifies preeclampsia. Here we demonstrate that BOK is a potent inducer of autophagy as shown by increased LC3B-II production, autophagosomal formation and lysosomal activation in HEK 293. In contrast, using JEG3 cells we showed that prosurvival MCL1 acts as a repressor of autophagy via an interaction with BECN1, which is abrogated by BOK. We found that MCL1-cleaved products, specifically MCL1c157, trigger autophagy while the splicing variant MCL1S has no effect. Treatment of JEG3 cells with nitric oxide donor SNP resulted in BOK-MCL1 rheostat dysregulation, favoring BOK accumulation, thereby inducing autophagy. Overexpression of MCL1 rescued oxidative stress-induced autophagy. Of clinical relevance, we report aberrant autophagy levels in the preeclamptic placenta due to impaired recruitment of BECN1 to MCL1. Our data provided the first evidence for a key role of the BOK-MCL1 system in regulating autophagy in the human placenta, whereby an adverse environment as seen in preeclampsia tilts the BOK-MCL1 balance toward the build-up of isoforms that triggers placental autophagy.     

Inhibition of autophagosome-lysosome fusion by ginsenoside Ro via the ESR2-NCF1-ROS pathway sensitizes esophageal cancer cells to 5-fluorouracil-induced cell death via the CHEK1-mediated DNA damage checkpoint

Modulation of autophagy has been increasingly regarded as a promising cancer therapeutic approach. In this study, we screened several ginsenosides extracted from Panax ginseng and identified ginsenoside Ro (Ro) as a novel autophagy inhibitor. Ro blocked the autophagosome-lysosome fusion process by raising lysosomal pH and attenuating lysosomal cathepsin activity, resulting in the accumulation of the autophagosome marker MAP1LC3B/LC3B and SQSTM1/p62 (sequestosome 1) in various esophageal cancer cell lines. More detailed studies demonstrated that Ro activated ESR2 (estrogen receptor 2), which led to the activation of NCF1/p47^PHOX (neutrophil cytosolic factor 1), a subunit of NADPH oxidase, and subsequent reactive oxygen species (ROS) production. Treatment with siRNAs or inhibitors of the ESR2-NCF1-ROS axis, such as N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), apocynin (ACN), Tiron, and Fulvestrant apparently decreased Ro-induced LC3B-II, GFP-LC3B puncta, and SQSTM1, indicating that ROS instigates autophagic flux inhibition triggered by Ro. More importantly, suppression of autophagy by Ro sensitized 5-fluorouracil (5-Fu)-induced cell death in chemoresistant esophageal cancer cells. 5-Fu induced prosurvival autophagy, and by inhibiting such autophagy, siRNAs against BECN1/beclin 1, ATG5, ATG7, and LC3B enhanced 5-Fu-induced autophagy-associated and apoptosis-independent cell death. We observed that Ro potentiates 5-Fu cytotoxicity via delaying CHEK1 (checkpoint kinase 1) degradation and downregulating DNA replication process, resulting in the delayed DNA repair and the accumulation of DNA damage. In summary, these data suggest that Ro is a novel autophagy inhibitor and could function as a potent anticancer agent in combination therapy to overcome chemoresistance.     

MCL1 is critical for mitochondrial function and autophagy in the heart

MCL1 (myeloid cell leukemia sequence 1 [BCL2-related]) is an anti-apoptotic BCL2 family protein that is upregulated in several human cancers. In malignancies, overexpression of MCL1 promotes cell survival and confers chemotherapeutic resistance. MCL1 is also highly expressed in normal myocardium, but the functional importance of MCL1 in myocytes has not been explored. We recently discovered that MCL1 plays an essential role in myocardial homeostasis and autophagy. Here, we discuss how loss of MCL1 in the adult mouse heart leads to mitochondrial dysfunction, impaired autophagy and development of heart failure.