Involvement of p53 in the cytotoxic activity of the NAMPT inhibitor FK866 in myeloid leukemic cells

FK866 is a specific inhibitor of NAMPT and induces apoptosis of leukemic cells by depletion of intracellular NAD⁺. Since up‐regulation of NAMPT is associated with several cases of cancers, including leukemias, we asked whether in leukemic cells inhibition of NAMPT involves p53 pathway. We observed that FK866 induced apoptosis and reduced cell proliferation in NB‐4, OCI‐AML3 and MOLM‐13 cell lines. In contrast, the leukemia cell lines, K‐562 and Kasumi, containing nonfunctional p53 were relatively unaffected by FK866 treatment. Importantly, direct inhibition of sirtuins significantly reduced the viability of NB‐4, OCI‐AML3 and MOLM‐13 cell lines. Activation of p53 by FK866 involved increased acetylation of p53 at lysine 382 with subsequent increase in the expression of p21 and BAX. Further, knockdown of p53 attenuated the effects of FK866 on apoptosis and cell cycle arrest, which was partly associated with decreased expression of p21 and BAX. Our results suggest the role of p53 acetylation pathway in the anti‐leukemic effect of FK866.     

B‐cell receptor configuration and mutational analysis of patients with chronic lymphocytic leukaemia and trisomy 12 reveal recurrent molecular abnormalities

Trisomy 12 (+12) is the third most frequent cytogenetic aberration in chronic lymphocytic leukaemia (CLL) retrievable both as the sole chromosomal abnormality or in association with additional alterations. NOTCH1 mutations are known to be more prevalent among +12 patients, whereas mutations of FBXW7, a gene involved in NOTCH1 degradation, that lead to the constitutional activation of NOTCH1 have not been investigated in this setting. We analyzed a unicentric cohort of 44 +12 patients with CLL for mutations of TP53, NOTCH1 and FBXW7 genes, and we correlated them with B‐cell receptor (BCR) configurations. FBXW7, TP53 and NOTCH1 mutations were identified in 4.5%, 6.8% and 18.2% of patients, respectively. FBXW7 and NOTCH1 mutations appeared in a mutually exclusive fashion, suggesting that both aberrations might affect the same biological pathway. We found that 44.1% of +12 CLL patients had stereotyped B‐cell receptors, which is significantly higher than that observed in patients with CLL and no +12 (27%, p = 0.01). Subsets #1, #8, #10, #28 and #59 were the most represented stereotyped patterns, and IGHV4‐39*01 was the gene configuration most commonly used. There was a significantly higher risk for Richter's syndrome (RS) transformation in patients with NOTCH1 or FBXW7 mutations, with four of the seven (57%) patients developing RS and characterized at least by one of the two abnormalities. These observations suggest that, similarly to the aberrations of NOTCH1, FBXW7 gene mutations may also result in cell proliferation and evasion from apoptosis in patients with +12 CLL. Together with the extremely high frequency of stereotyped BCRs and RS transformation, these abnormalities appear to cluster in these CLL patients with additional chromosome 12, suggesting a connection with the prognosis of the disease. Copyright © 2013 John Wiley & Sons, Ltd.     

Loss of cooperativity of secreted CD40L and increased dose–response to IL4 on CLL cell viability correlates with enhanced activation of NF‐kB and STAT6

Chronic lymphocytic leukemia (CLL) cells fail to enter apoptosis in vivo as opposed to their non‐malignant B‐lymphocyte counterparts. The ability of CLL cells to escape apoptosis is highly dependent on their microenvironment. Compared to non‐malignant B cells, CLL cells are more responsive to complex stimuli that can be reproduced in vitro by the addition of cytokines. To understand the molecular mechanism of the environment‐dependent anti‐apoptotic signaling circuitry of CLL cells, we quantified the effect of the SDF‐1, BAFF, APRIL, anti‐IgM, interleukin‐4 (IL4) and secreted CD40L (sCD40L) on the survival of in vitro cultured CLL cells and found IL4 and sCD40L to be most efficient in rescuing CLL cells from apoptosis. In quantitative dose–response experiments using cell survival as readout, the binding affinity of IL4 to its receptor was similar between malignant and non‐malignant cells. However, the downstream signaling in terms of the amount of STAT6 and its degree of phosphorylation was highly stimulated in CLL cells. In contrast, the response to sCD40L showed a loss of cooperative binding in CLL cells but displayed a largely increased ligand binding affinity. Although a high‐throughput microscopy analysis did not reveal a significant difference in the spatial CD40 receptor organization, the downstream signaling showed an enhanced activation of the NF‐kB pathway in the malignant cells. Thus, we propose that the anti‐apoptotic phenotype of CLL involves a sensitized response for IL4 dependent STAT6 phosphorylation, and an activation of NF‐kB signaling due to an increased affinity of sCD40L to its receptor.     

The phosphatidylinositol‐3 kinase I inhibitor BKM120 induces cell death in B‐chronic lymphocytic leukemia cells in vitro

BKM120, a pan class I PI3K inhibitor, was cytotoxic in the majority of primary B‐chronic lymphocytic leukemia (CLL) lymphocytes, including samples from patients who have a high‐risk for poor response to treatment (patient with del11 and del17) at clinically obtainable concentrations. The PI3Kδ inhibitor Cal‐101 is cytotoxic in B‐CLL lymphocytes in vitro and is active in the treatment of CLL in vivo. Interestingly, we demonstrated that BKM120 is 3.6 fold more toxic than Cal‐101 in malignant B‐CLL lymphocytes in vitro. BKM120 cytotoxicity correlated with the basal expression of proteins involved in the PI3K/Akt pathway. A protein signature of PI3K pathway proteins predicts the response to BKM120 treatment. In the primary B‐CLL lymphocytes tested in vitro, BKM120 decreased the phosphorylation status of molecular biomarkers used as indicators of PI3K pathway inhibition in vivo. Also, BKM120 induced apoptosis in primary B‐CLL cells culture in the presence and absence of stromal cell support. Our findings suggest that BKM120 should be tested clinically in CLL.     

Decreased sensitivity of 17p-deleted chronic lymphocytic leukemia cells to a small molecule BCL-2 antagonist ABT-737

BACKGROUND:

Despite the high complete response rates achieved with fludarabine‐based regimens, relapse is inevitable in chronic lymphocytic leukemia (CLL). Relapsed patients often acquire deletions of the short arm of chromosome 17 (del[17p]), which are closely associated with tumor protein 53 (TP53) mutations. Wild‐type p53 up‐regulates and activates B‐cell CLL/lymphoma 2 (BCL‐2)‐associated X protein (BAX), and it down‐regulates and inactivates BCL‐2. The small‐molecule BCL‐2 inhibitor ABT‐737 induces apoptosis in a BAX‐dependent and BCL‐2 homologous antagonist‐killer (BAK)‐dependent manner. The role of p53 in sensitivity of CLL cells to BCL‐2 inhibition has not been extensively investigated.

METHODS:

The authors investigated the association of del(17p) with ABT‐737 sensitivity in CLL cells from 50 patients. Stable p53 and BAX knockdown cells were used for mechanistic studies.

RESULTS:

CLL cells with del(17p) were less sensitive to ABT‐737‐induced BAX activation and apoptosis than CLL cells without del(17p) (39% ± 7.3% vs 63.7% ± 2.9% [specific annexin V induction]; P < .01). A positive correlation between the degrees of apoptosis induced by ABT‐737 and by the p53‐activating binding protein homolog murine double minute (MDM2) antagonist nutlin‐3a (correlation coefficient [r] = 0.75; P < .0001) was observed. CLL cells with del(17p) expressed lower levels of BAX than those without del(17p) (0.67 ± 0.12 vs 1.27 ± 0.10 in relative protein expression levels; P < .01). Knockdown of p53 or BAX in leukemia cells resulted in decreased apoptosis induced by ABT‐737.

CONCLUSIONS:

The current data indicated that p53 dysfunction may lead to decreased apoptosis induction by ABT‐737. Cancer 2012;. © 2011 American Cancer Society.     

HSP70/HSF1 axis,regulated via a PI3K/AKT pathway,is a druggable target in chronic lymphocytic leukemia

Considering the role played by the heat shock protein of 70 kDa (HSP70) in cancer, we characterized this protein and its major regulator, the heat shock factor 1 (HSF1), in chronic lymphocytic leukemia (CLL). We found both HSP70 and HSF1 overexpressed in CLL patients, correlated to poor prognosis and abnormally localized in the nucleus of leukemic B cells. The two proteins were strictly correlated each other and their levels decreased consensually in those patients responding to in vivo therapeutic regimens. HSP70 and HSF1 inhibition was proved to be effective in inducing a dose-dependent in vitro apoptosis of CLL B cells. Considering that HSF1 is finely regulated by kinases belonging to pathways triggered by rat sarcoma (RAS), we benefited from a previous proteomic study performed in CLL patients aiming to assess the activation/expression of key signaling proteins. We found that patients showing high levels of HSP70 also expressed high Akt-Ser473, thus activating HSF1. Inhibition of PI3K, which activates AKT, reduced the expression of HSF1 and HSP70. By contrast, HSP70-low patients displayed high activation of MEK1/2 and ERK1/2, known to negatively regulate HSF1. These data demonstrate that the HSP70 expression is regulated by the modulation of HSF1 activity through the activation of RAS-regulated pathways and suggest the HSP70/HSF1 interplay as an interesting target for antileukemic therapies. Finally, inhibition of PI3K, that activates AKT, reduced the expression of HSF1 and HSP70.     

A NOTCH1 gene copy number gain is a prognostic indicator of worse survival and a predictive biomarker to a Notch1 targeting antibody in colorectal cancer

Dysregulation of the Notch1 receptor has been shown to facilitate the development and progression of colorectal cancer (CRC) and has been identified as an independent predictor of disease progression and worse survival. Although mutations in the NOTCH1 receptor have not been described in CRC, we have previously discovered a NOTCH1 gene copy number gain in a portion of CRC tumor samples. Here, we demonstrated that a NOTCH1 gene copy number gain is significantly associated with worse survival and a high percentage of gene duplication in a cohort of patients with advanced CRC. In our CRC patient‐derived tumor xenograft (PDTX) model, tumors harboring a NOTCH1 gain exhibited significant elevation of the Notch1 receptor, JAG1 ligand and cleaved Notch1 activity. In addition, a significant association was identified between a gain in NOTCH1 gene copy number and sensitivity to a Notch1‐targeting antibody. These findings suggest that patients with metastatic CRC that harbor a gain in NOTCH1 gene copy number have worse survival and that targeting this patient population with a Notch1 antibody may yield improved outcomes.     

Role of beta2 integrins in the prevention of apoptosis induction in chronic lymphocytic leukemia B cells.

Immunologically committed lymphocytes, especially mature, leukemic B cells, proliferate then accumulate without further cell division in chronic lymphocytic leukemia patients (CLL). These mature, leukemic B cells often produce autoantibodies. Under normal circumstances, immunologically committed lymphocytes that are autoreactive are deleted by a programmed cell death mechanism. In CLL cells, these mechanisms appear to be inhibited; therefore, cells accumulate rather than be destroyed. To understand the mechanism by which cell survival is selected over death in CLL cells, we studied the role of beta2 integrins and their ligands in the regulation of apoptosis. CLL cells were treated with monoclonal antibodies directed against beta2 integrins. Antibodies directed against the I-domain of the alpha chain of CD11b/CD18 inhibited apoptosis. The identity of the physiological ligand or counter-receptor for beta2 integrins that was required for the inhibition of apoptosis induction was sought. The ligand iC3b, but not ICAM-1 or fibrinogen, was identified as a ligand that could prevent apoptosis of CLL B cells. Free iC3b levels were elevated in CLL patients indicating that this ligand is available in vivowhere it may interact with beta2 integrins on CLL B cells and sustain their viability by preventing activation of the programmed cell death pathway. Leukemia (2000) 14, 34-39.     

The mechanism of fucoidan‐induced apoptosis in leukemic cells: Involvement of ERK1/2, JNK,glutathione, and nitric oxide

Fucoidan, a sulfated polysaccharide in brown seaweed, has various biological activities including anti‐tumor activity. We investigated the effects of fucoidan on the apoptosis of human promyeloid leukemic cells and fucoidan‐mediated signaling pathways. Fucoidan induced apoptosis of HL‐60, NB4, and THP‐1 cells, but not K562 cells. Fucoidan treatment of HL‐60 cells induced activation of caspases‐8, ‐9, and ‐3, the cleavage of Bid, and changed mitochondrial membrane permeability. Fucoidan‐induced apoptosis, cleavage of procaspases, and changes in the mitochondrial membrane permeability were efficiently blocked by depletion of mitogen‐activated protein kinase (MAPK) kinase kinase 1 (MEKK1), and inhibitors of MAPK kinase 1 (MEK1) and c Jun NH₂‐terminal kinase (JNK). The phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and JNK was increased in fucoidan‐treated HL‐60, NB4, and THP‐1 cells, but not K562 cells. ERK1/2 activation occurred at earlier times than JNK activation and JNK activation was blocked by MEK1 inhibitor. In addition, fucoidan‐induced apoptosis was inhibited by addition of glutathione and/or L ‐NAME, and fucoidan decreased intracellular glutathione concentrations and stimulated nitric oxide (NO) production. Buthionine‐[R,S]‐sulfoximine rendered HL‐60 cells more sensitive to fucoidan. Depletion of MEKK1 and inhibition of MEK1 restored the intracellular glutathione content and abrogated NO production, whereas inhibition of JNK activation by SP600125 restored intracellular glutathione content but failed to inhibit NO production in fucoidan‐treated HL‐60 cells. These results suggest that activation of MEKK1, MEK1, ERK1/2, and JNK, depletion of glutathione, and production of NO are important mediators in fucoidan‐induced apoptosis of human leukemic cells. © 2010 Wiley‐Liss, Inc.     

Mitogen‐activated protein kinase (MEK/ERK) inhibition sensitizes cancer cells to centromere‐associated protein E inhibition

Inhibition of centromere‐associated protein‐E (CENP‐E) has demonstrated preclinical anti‐tumor activity in a number of tumor types including neuroblastoma. A potent small molecule inhibitor of the kinesin motor activity of CENP‐E has recently been developed (GSK923295). To identify an effective drug combination strategy for GSK923295 in neuroblastoma, we performed a screen of siRNAs targeting a prioritized set of genes that function in therapeutically tractable signaling pathways. We found that siRNAs targeted to extracellular signal‐related kinase 1 (ERK1) significantly sensitized neuroblastoma cells to GSK923295‐induced growth inhibition (p = 0.01). Inhibition of ERK1 activity using pharmacologic inhibitors of mitogen‐activated ERK kinase (MEK1/2) showed significant synergistic growth inhibitory activity when combined with GSK923295 in neuroblastoma, lung, pancreatic and colon carcinoma cell lines. Synergistic growth inhibitory activity of combined MEK/ERK and CENP‐E inhibition was a result of increased mitotic arrest and apoptosis. There was a significant correlation between ERK1/2 phosphorylation status in neuroblastoma cell lines and GSK923295 growth inhibitory activity (r = 0.823, p = 0.0006). Consistent with this result we found that lung cancer cell lines harboring RAS mutations, which leads to oncogenic activation of MEK/ERK signaling, were significantly more resistant than cell lines with wild‐type RAS to GSK923295‐induced growth inhibition (p = 0.047). Here we have identified (MEK/ERK) activity as a potential biomarker of relative GSK923295 sensitivity and have shown the synergistic effect of combinatorial MEK/ERK pathway and CENP‐E inhibition across different cancer cell types including neuroblastoma.     

γ‐Secretase inhibitor I induces apoptosis in chronic lymphocytic leukemia cells by proteasome inhibition,endoplasmic reticulum stress increase and notch down‐regulation

γ‐Secretase inhibitors (GSIs) have been proposed for combined therapies of malignancies with a dysregulated Notch signaling. GSI I (Z‐Leu‐Leu‐Nle‐CHO) induces apoptosis of some tumor cells by inhibiting proteasome and Notch activity. Alterations in these two cell survival regulators contribute to apoptosis resistance of chronic lymphocytic leukemia (CLL) cells. Here, we investigated the mechanisms whereby GSI I increases apoptosis of primary CLL cells. Time‐course studies indicate that initial apoptotic events are inhibition of proteasome activity, concomitant with an increased endoplasmic reticulum (ER) stress apoptotic signaling, and a consistent Noxa protein up‐regulation. These events precede, and some of them contribute to, mitochondrial alterations, which occur notwithstanding Mcl‐1 accumulation induced by GSI I. In CLL cells, GSI I inhibits Notch1 and Notch2 activation only in the late apoptotic phases, suggesting that this event does not initiate CLL cell apoptosis. However, Notch inhibition may contribute to amplify GSI I‐induced CLL cell apoptosis, given that Notch activation sustains the survival of these cells, as demonstrated by the evidence that both Notch1 and Notch2 down‐regulation by small‐interfering RNA accelerates spontaneous CLL cell apoptosis. Overall, our results show that GSI I triggers CLL cell apoptosis by inhibiting proteasome activity and enhancing ER stress, and amplifies it by blocking Notch activation. These findings suggest the potential relevance of simultaneously targeting these three important apoptosis regulators as a novel therapeutic strategy for CLL.     

The effect of cantharidins on leukemic stem cells

To identify an agent with specific activity against leukemic stem cells (LSCs), we evaluated compounds that targeted hepatic leukemia factor (HLF), a gene implicated in hematopoietic stem cell (HSCs) regulation, that we found overexpressed in LSCs. Cantharidin, a natural toxin from blister beetles, used as medicinal agent since antiquity, has been described to modulate the HLF competitor NFIL3 and is under clinical evaluation as an antitumor and antimetastatic agent. The molecule is not a substrate for multidrug resistant pumps and does not cause myelosuppression, and therefore it represents a promising compound for selective ablation of LSCs. Cantharidin and norcantharidin, a derivative with reduced toxicity, decreased HLF protein levels and induced apoptosis in the AML cell line MV4‐11 by modulating the expression of several molecules that govern survival pathway, including HLF, SLUG, NFIL3 and c‐myc, thereby inducing p53 and the mitochondrial caspase cascade. In vitro, cantharidin readily targeted primary AML stem and progenitor cells in contrast to conventional chemotherapeutic agents, such as Ara‐C and daunorubicin, that mainly targeted more differentiated leukemic cells. In vitro the compound did not exhibit a therapeutic window, being equally toxic to normal HSCs and LSCs. In vivo cantharidin did not produce myelosuppression. Because of dose‐limiting toxicity in vivo, neither cantharidin nor norcantharidin proved therapeutical benefit in AML xenograft models as a single agent. However, its potent in vitro LSC activity and pathway targeting may still be exploited clinically with a new generation of cantharidin derivatives or formulations and with appropriate drug combinations. © 2008 Wiley‐Liss, Inc.     

靶向抑制TROP2基因表达对胰腺癌细胞生物学特性的影响研究

目的 探讨抑制胰腺癌组织中肿瘤相关钙信号传导因子2(TROP2)表达对胰腺癌细胞增殖和凋亡的影响及机制.方法 选取胰腺癌组织和相应的癌旁组织各46例.采用逆转录-聚合酶链反应(RT-PCR)法检测并分析胰腺癌组织和癌旁组织中TROP2基因的mRNA表达情况;将人胰腺癌PANC-1细胞分为对照组、NC-siR-NA组和TROP2-siRNA组,培养48 h后,采用蛋白质印迹法检测并分析TROP2、cleaved caspase 3、NOTCH1和HES1蛋白表达情况;采用CCK8法和流式细胞仪分别检测并分析细胞的增殖和凋亡情况.结果 在胰腺癌组织中TROP2基因的mRNA表达明显高于癌旁组织,差异有统计学意义(P﹤0.01).对照组、NC-siRNA组和TROP2-siRNA组中,TROP2蛋白表达、细胞存活率、细胞凋亡率及cleaved caspase 3、NOTCH1、HES1蛋白表达比较,差异均有明显统计学意义(P﹤0.01);TROP2-siRNA组TROP2蛋白表达、细胞存活率及NOTCH1、HES1蛋白表达均明显低于对照组,细胞凋亡率和cleaved caspase 3蛋白表达均明显高于对照组,差异均有统计学意义(P﹤0.01),而NC-siRNA组TROP2蛋白表达、细胞存活率、细胞凋亡率及cleaved caspase 3、NOTCH1、HES1蛋白表达与对照组比较,差异均无统计学意义(P﹥0.05).结论 抑制胰腺癌组织中TROP2基因表达,可以明显降低肿瘤细胞的增殖能力,促进细胞凋亡,其机制与阻断NOTCH1信号通路有关.     

TLX1 and NOTCH coregulate transcription in T cell acute lymphoblastic leukemia cells

Background  

The homeobox gene TLX1 (for T-cell leukemia homeobox 1, previously known as HOX11) is inappropriately expressed in a major subgroup of T cell acute lymphoblastic leukemia (T-ALL) where it is strongly associated with activating NOTCH1 mutations. Despite the recognition that these genetic lesions cooperate in leukemogenesis, there have been no mechanistic studies addressing how TLX1 and NOTCH1 functionally interact to promote the leukemic phenotype.