Inhibition of bromodomain and extra‐terminal proteins increases sensitivity to venetoclax in chronic lymphocytic leukaemia

The development of drugs able to target BTK, PI3k‐delta and BCL2 has dramatically improved chronic lymphocytic leukaemia (CLL) therapies. However, drug resistance to these therapies has already been reported due to non‐recurrent changes in oncogenic pathways and genes expression signatures. In this study, we investigated the cooperative role of the BCL2 inhibitor venetoclax and the BRD4 inhibitor JQ1. In particular, we found that JQ1 shows additional activity with venetoclax, in CLL cell lines and in ex vivo isolated primary CD19⁺ lymphocytes, arguing in favour of combination strategies. Lastly, JQ1 is also effective in venetoclax‐resistant CLL cell lines. Together, our findings indicated that the BET inhibitor JQ1 could be a promising therapy in CLL, both as first‐line therapy in combination with venetoclax and as second‐line therapy, after the emergence of venetoclax‐resistant clones.     

Copy number aberrations of BCL2 and CDKN2A/B identified by array-CGH in thymic epithelial tumors

The molecular pathology of thymic epithelial tumors (TETs) is largely unknown. Using array comparative genomic hybridization (CGH), we evaluated 59 TETs and identified recurrent patterns of copy number (CN) aberrations in different histotypes. GISTIC algorithm revealed the presence of 126 significant peaks of CN aberration, which included 13 cancer-related genes. Among these peaks, CN gain of BCL2 and CN loss of CDKN2A/B were the only genes in the respective regions of CN aberration and were associated with poor outcome. TET cell lines were sensitive to siRNA knockdown of the anti-apoptotic molecules BCL2 and MCL1. Gx15-070, a pan-BCL2 inhibitor, induced autophagy-dependent necroptosis in TET cells via a mechanism involving mTOR pathways, and inhibited TET xenograft growth. ABT263, an inhibitor of BCL2/BCL-XL/BCL-W, reduced proliferation in TET cells when administered in combination with sorafenib, a tyrosine kinase inhibitor able to downregulate MCL1. Immunohistochemistry on 132 TETs demonstrated that CN loss of CDKN2A correlated with lack of expression of its related protein p16^INK4 and identified tumors with poor prognosis. The molecular markers BCL2 and CDKN2A may be of potential value in diagnosis and prognosis of TETs. Our study provides the first preclinical evidence that deregulated anti-apoptotic BCL2 family proteins may represent suitable targets for TET treatment.     

The putative BH3 mimetic S1 sensitizes leukemia to ABT-737 by increasing reactive oxygen species, inducing endoplasmic reticulum stress, and upregulating the BH3-only protein NOXA

S1 is a putative BH3 mimetic proposed to inhibit BCL2 and MCL1 based on cell-free assays. However, we previously demonstrated that it failed to inhibit BCL2 or induce apoptosis in chronic lymphocytic leukemia (CLL) cells, which are dependent on BCL2 for survival. In contrast, we show here that S1 rapidly increases reactive oxygen species, initiates endoplasmic reticulum stress, and upregulates the BH3-only protein NOXA. The BCL2 inhibitors, ABT-737, ABT-263, and ABT-199, have demonstrated pro-apoptotic efficacy in cell lines, while ABT-263 and ABT-199 have demonstrated efficacy in early clinical trials. Resistance to these inhibitors arises from the upregulation of anti-apoptotic factors, such as MCL1, BFL1, and BCLXL. This resistance can be induced by co-culturing CLL cells on a stromal cell line that mimics the microenvironment found in patients. Since NOXA can inhibit MCL1, BFL1, and BCLXL, we hypothesized that S1 may overcome resistance to ABT-737. Here we demonstrate that S1 induces NOXA-dependent sensitization to ABT-737 in a human promyelocytic leukemia cell line (NB4). Furthermore, S1 sensitized CLL cells to ABT-737 ex vivo, and overcame resistance to ABT-737 induced by co-culturing CLL cells with stroma.     

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

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

Endogenous neurotrophins and Trk signaling in diffuse large B cell lymphoma cell lines are involved in sensitivity to rituximab-induced apoptosis

Background

Diffuse large B-cell lymphoma (DLBCL) is a common and often fatal malignancy. Immunochemotherapy, a combination of rituximab to standard chemotherapy, has resulted in improved survival. However a substantial proportion of patients still fail to reach sustained remission. We have previously demonstrated that autocrine brain-derived neurotrophic factor (BDNF) production plays a function in human B cell survival, at least partly via sortilin expression. As neurotrophin receptor (Trks) signaling involved activation of survival pathways that are inhibited by rituximab, we speculated that neurotrophins may provide additional support for tumour cell survival and therapeutic resistance in DLBCL.

Methodology/Principal Findings

In the present study, we used two DLBCL cell lines, SUDHL4 and SUDHL6, known to be respectively less and more sensitive to rituximab. We found by RT-PCR, western blotting, cytometry and confocal microscopy that both cell lines expressed, in normal culture conditions, BDNF and to a lesser extent NGF, as well as truncated TrkB and p75^NTR/sortilin death neurotrophin receptors. Furthermore, BDNF secretion was detected in cell supernatants. NGF and BDNF production and Trk receptor expression, including TrkA, are regulated by apoptotic conditions (serum deprivation or rituximab exposure). Indeed, we show for the first time that rituximab exposure of DLBCL cell lines induces NGF secretion and that differences in rituximab sensitivity are associated with differential expression patterns of neurotrophins and their receptors (TrkA). Finally, these cells are sensitive to the Trk-inhibitor, K252a, as shown by the induction of apoptosis. Furthermore, K252a exhibits additive cytotoxic effects with rituximab.

Conclusions/Significance

Collectively, these data strongly suggest that a neurotrophin axis, such NGF/TrkA pathway, may contribute to malignant cell survival and rituximab resistance in DLBCL.     

Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.     

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.     

BAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia

BCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance.     

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.     

Inducer-and cell type-specific regulation of antiapoptotic MCL1 in myeloid leukemia and multiple myeloma cells exposed to differentiation-inducing or microtubule-disrupting agents

The antiapoptotic BCL2 family member MCL1 is rapidly upregulated upon exposure of ML-1 myeloid leukemia cells to either differentiation-inducing phorbol 12′-myristate 13′-acetate (PMA) or chemotherapeutic microtubule disrupting agents (MTDAs). This report examined how signaling for MCL1 upregulation is coupled to these two different phenotypic changes, and tested for upregulation in other hematopoietic cancers. With PMA, ERK stimulated MCL1 mRNA expression and ML-1 cell differentiation, and ERK additionally stabilized expression of the MCL1 protein. However, with MTDAs, transient ERK and ensuing JNK activation contributed to initial MCL1 upregulation and viability-retention, but sustained JNK activation eventually resulted in cell death. MCL1 was upregulated by PMA in THP-1 and U937 myeloid leukemia cells, but by MTDAs only in THP-1 cells. MCL1 expression was constitutively elevated in multiple myeloma cell lines, and was not affected by PMA/ERK or MTDAs. Thus, MCL1 expression level and sensitivity to regulation are important considerations in selecting approaches for targeting this antiapoptotic gene product to kill cancer cells.     

N-terminal truncation of antiapoptotic MCL1, but not G2/M-induced phosphorylation, is associated with stabilization and abundant expression in tumor cells

The antiapoptotic BCL2 family member MCL1 is normally up- and down-modulated in response to environmental signals and conditions, but is constitutively expressed in cancer where it promotes cell survival and drug resistance. A post-translational modification identified here, truncation at the N terminus, was found to act along with previously described ERK- and GSK3-induced phosphorylation events to regulate the turnover of the MCL1 protein and thus its availability for antiapoptotic effects. Although both N-terminally truncated and full-length MCL1 contain sequences enriched in proline, glutamic acid, serine, and threonine and were susceptible to proteasomal degradation, the truncated form decayed less rapidly and was maintained for an extended period in the presence of ERK activation. This was associated with extended cell survival because the truncated form of MCL1 (unlike those of BCL2 and BCLX) retained antiapoptotic activity. N-terminal truncation slightly increased the electrophoretic mobility of MCL1 and differed from the phosphorylation/band shift to decreased mobility, which occurs in the G2/M phase and was not found to affect MCL1 turnover. The N-terminally truncated form of MCL1 was expressed to varying extents in normal lymphoid tissues and was the predominant form present in lymphomas from transgenic mice and human tumor lines of B-lymphoid origin. The degradation versus stabilized expression of antiapoptotic MCL1 is thus controlled by N-terminal truncation as well as by ERK- and GSK3 (but not G2/M)-induced phosphorylation. These modifications may contribute to dysregulated MCL1 expression in cancer and represent targets for promoting its degradation to enhance tumor cell death.     

Multiple BH3 mimetics antagonize antiapoptotic MCL1 protein by inducing the endoplasmic reticulum stress response and up-regulating BH3-only protein NOXA

BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-X(L), or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.     

Ran GTPase-Activating Protein 1 Is a Therapeutic Target in Diffuse Large B-Cell Lymphoma

Lymphoma-specific biomarkers contribute to therapeutic strategies and the study of tumorigenesis. Diffuse large B-cell lymphoma (DLBCL) is the most common type of malignant lymphoma. However, only 50% of patients experience long-term survival after current treatment; therefore, developing novel therapeutic strategies is warranted. Comparative proteomic analysis of two DLBCL lines with a B-lymphoblastoid cell line (LCL) showed differential expression of Ran GTPase-activating protein 1 (RanGAP1) between them, which was confirmed using immunoblotting. Immunostaining showed that the majority of DLBCLs (92%, 46/50) were RanGAP1⁺, while reactive lymphoid hyperplasia (n = 12) was RanGAP1⁺ predominantly in germinal centers. RanGAP1 was also highly expressed in other B-cell lymphomas (BCL, n = 180) with brisk mitotic activity (B-lymphoblastic lymphoma/leukemia: 93%, and Burkitt lymphoma: 95%) or cell-cycle dysregulation (mantle cell lymphoma: 83%, and Hodgkin’s lymphoma 91%). Interestingly, serum RanGAP1 level was higher in patients with high-grade BCL (1.71 ± 2.28 ng/mL, n = 62) than in low-grade BCL (0.75 ± 2.12 ng/mL, n = 52) and healthy controls (0.55 ± 1.58 ng/mL, n = 75) (high-grade BCL vs. low-grade BCL, p = 0.002; high-grade BCL vs. control, p < 0.001, Mann-Whitney U test). In vitro, RNA interference of RanGAP1 showed no effect on LCL but enhanced DLBCL cell death (41% vs. 60%; p = 0.035) and cell-cycle arrest (G0/G1: 39% vs. 49%, G2/M: 19.0% vs. 7.5%; p = 0.030) along with decreased expression of TPX2 and Aurora kinases, the central regulators of mitotic cell division. Furthermore, ON 01910.Na (Estybon), a multikinase inhibitor induced cell death, mitotic cell arrest, and hyperphosphorylation of RanGAP1 in DLBCL cell lines but no effects in normal B and T cells. Therefore, RanGAP1 is a promising marker and therapeutic target for aggressive B-cell lymphoma, especially DLBCL.     

Gossypol Increases Expression of the Pro-apoptotic BH3-only Protein NOXA through a Novel Mechanism Involving Phospholipase A2, Cytoplasmic Calcium,and Endoplasmic Reticulum Stress

Gossypol is a putative BH3 mimetic proposed to inhibit BCL2 and BCLXL based on cell-free assays. We demonstrated previously that gossypol failed to directly inhibit BCL2 in cells or induce apoptosis in chronic lymphocytic leukemia (CLL) cells or platelets, which require BCL2 or BCLXL, respectively, for survival. Here, we demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA. Pretreatment with the PLA2 inhibitor, aristolochic acid, abrogated the increase in calcium, ER stress, and NOXA. Calcium chelation also abrogated the gossypol-induced increase in calcium, ER stress, and NOXA, but not the increase in PLA2 activity, indicating that PLA2 is upstream of these events. In addition, incubating cells with the two products of PLA2 (lysophosphatidic acid and arachidonic acid) mimicked treatment with gossypol. NOXA is a pro-apoptotic protein that functions by binding the BCL2 family proteins MCL1 and BFL1. The BCL2 inhibitor ABT-199 is currently in clinical trials for CLL. Resistance to ABT-199 can occur from up-regulation of other BCL2 family proteins, and this resistance can be mimicked by culturing CLL cells on CD154⁺ stroma cells. We report here that AT-101, a derivative of gossypol in clinical trials, overcomes stroma-mediated resistance to ABT-199 in primary CLL cells, suggesting that a combination of these drugs may be efficacious in the clinic.     

Inhibition of Polo-like kinase 4 induces mitotic defects and DNA damage in diffuse large B-cell lymphoma

Polo-like kinase 4 (PLK4), a key regulator of centriole biogenesis, has recently been shown to play key roles in tumorigenesis. Blocking PLK4 expression by interference or targeted drugs exhibits attractive potential in improving the efficacy of chemotherapy. Nevertheless, the role of PLK4 in diffuse large B-cell lymphoma (DLBCL) is still undefined. In this study, we discover that PLK4 is a potential target for the treatment of DLBCL, and demonstrate the efficacy of a PLK4 inhibitor when used in combination with doxorubicin. Pharmaceutical inhibition of PLK4 with CFI-400945 inhibited DLBCL cell proliferation and induced apoptotic cell death. The anti-tumor effects were accompanied by mitotic defects, including polyploidy and cytokinesis failure. Activation of p53 and Hippo/YAP tumor suppressor signaling pathway was identified as the potential mechanisms driving CFI-400945 activity. Moreover, CFI-400945 treatment resulted in activation of DNA damage response. Combining CFI-400945 with doxorubicin markedly delayed tumor progression in DLBCL xenografts. Finally, PLK4 was increased in primary DLBCL tissues and cell lines. High levels of PLK4 expression were associated with poor survival in the patients receiving CHOP-based treatment, implicating PLK4 as a predictive biomarker of DLBCL chemosensitivity. These results provide the therapeutic potential of CFI-400945 both as monotherapy or in combination with doxorubicin for the treatment of DLBCL.Subject terms: B-cell lymphoma, Cytokinesis, DNA damage response