Upregulation Of Mcl-1 Research Articles

Abstract 4515: A novel obatoclax derivative is a promising agent to overcome bortezomib's resistance in acute leukemia cells

Abstract Background: Our previous work showed that bortezomib, a reverse-inhibitor of the 26S proteasome, exerts differential cytotoxic effects to acute leukemia cells through down-regulation of phosphor-Akt (AACR 2010 abstract 1575). Interestingly, some acute leukemia cells (MOLT-3 and K562) are resistant to bortezomib. One of the resistant mechanisms of bortezomib involves the abnormality in intrinsic apoptotic pathway members, the bcl-2 family. Of particular, it has been shown that bortezomib induces upregulation of Mcl-1 and that Mcl-1 accumulation may delay bortezomib-induced apoptosis. Obatoclax, a small molecule inhibitor of pro-survival BCL-2 members, including MCL-1, synergizes with bortezomib in mantle cell lymphoma (Blood 2007) and may overcome bortezomib resistance. Here, we designed a novel Mcl-1 inhibitor deriving from obatoclax, SC-2001, to overcome the resistance. Methods: MOLT-3 and K562 cell lines were used for in vitro studies. Apoptosis was examined by both flow cytometry and Western blot. Signal transduction pathways in cells were assessed by Western Blot. Gene silencing was done by small interference RNA (siRNA). Results: MOLT-3 and K562 were resistant to bortezomib up to 100nM. Upregulation of Mcl-1 expression after bortezomib treatment were evident in these cells. Interestingly, the addition of either obatoclax or SC-2001, restored the sensitivity of K562 and MOLT-3 cells to bortezomib. SC-2001, comparing to obatoclax, exerted better efficacy in sensitivity restoration of bortezomib in association with Mcl-1 downregulation in MOLT-3 cells. Moreover, down-regulation of Mcl-1 by siRNA overcame the apoptotic resistance to bortezomib in K562 cells. These data indicate that novel Mcl-1 inhibitors could restore bortezomib sensitivity in resistant acute leukemia cells. Conclusions: a novel Mcl-1 inhibitor SC-2001, is a promising agent to overcome bortezomib's resistance in acute leukemia, further elucidating the drug mechanism regulating Mcl-1 inhibition is needed. Supported by VGHTPE 99DHA0100332 and 100DHA0100591 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4515. doi:10.1158/1538-7445.AM2011-4515

Abstract 5033: Stromal-derived conditioned media activates STAT3 via JAK2 and TYK2 and results in de novo resistance to Nilotinib

Abstract Complete eradication of CML has not been possible in patients because of the presence of minimal residual disease (MRD) in sanctuary sites like the bone marrow (BM). One of the reasons for MRD is the milieu of cytokines and growth factors present in the BM microenvironment, that contribute to BCR-ABL independent drug resistance in CML. For example, in the present study, we demonstrate the importance of STAT3-mediated drug resistance in the BM microenvironment by showing that conditioned media (CM) derived from both normal and patient-derived mesenchymal stem cells and osteoblasts leads to STAT3 phosphorylation (pSTAT3-Y705) and concomitant drug resistance to Nilotinib in CML cells. Inhibiting the activity or reducing the expression of JAK2 and TYK2 was necessary for sensitizing cells to Nilotinib in CM. Moreover, increased pSTAT3-Y705 levels were associated with increased STAT3 transcriptional activity and up-regulation of Mcl-1 and cyclin D1. Our study indicates that there is an inherent redundancy in CM-mediated STAT3 activation pathway because concomitant neutralization of two potent activators of STAT3, IL-6 and G-CSF, does not reverse CM-mediated protection against Nilotinib. Furthermore, utilizing a SCID-hu bone implant in vivo model, we show that K562 cells with reduced STAT3 showed significantly lower tumor burden, which could be attributed to not only the slower proliferation rate, but also to the observation that these cells underwent cell death in BM stromal derived CM. Finally, CD34+ cells from CML patients when exposed to CM showed an increase in STAT3 phosphorylation which correlated with the development of de novo resistance to Nilotinib-mediated cell death. Taken together our data indicate that STAT3 is an important target in CML and its direct inhibition, along with BCR-ABL inhibitors, will likely be the most effective therapeutic strategy for reversing resistance within the BM microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5033. doi:10.1158/1538-7445.AM2011-5033

Abstract 5366: The small molecule Bcl-2 antagonist obatoclax interacts synergistically with the irreversible proteasome inhibitor carfilzomib in GC and ABC- sub types of DLBCL cells both in vitro and in vivo

Abstract Introduction: Based on previous studies showing synergistic interactions between proteasome inhibitors (e.g., bortezomib) and Bcl-2 antagonists, we have investigated whether the activity of the irreversible proteasome inhibitor carfilzomib (CFZ) could be enhanced in DLBCL cells by a clinically relevant Bcl-2 antagonist such as obatoclax (OBTX). Experimental Procedures: SUDHL16, SUDHL4 (GC -subtype) and OCI-LY10, OCI-LY-3 (ABC -subtype) cell lines were used as model systems. CFZ and OBTX were provided by Onyx Pharmaceuticals and GeminX Inc, respectively. Apoptosis was monitored by Annexin V/PI following drug treatment with OBTX ± CFZ. Synergism was assessed by CalcuSyn software. Effects of drug treatment on protein expression was monitored by western blotting. Beige-nude-XID (NIH-III) mice were used for in vivo studies. Results : Co-treatment (24- 48 hrs) with minimally toxic CFZ concentrations (i.e., 2-6 nM) and sub-toxic concentrations of either OBTX (0.05-2.0µM) or HA14-1 (2-10 µM) resulted in a striking increase in apoptosis in diverse DLBCL cells. Median Dose Effect analysis demonstrated that interactions were highly synergistic, with Combination Index values less than 1.0. The CFZ/OBTX regimen also strikingly induced apoptosis in 2 primary human DLBCL (1-GC and 1-ABC types) samples but exhibited minimal toxicity toward normal hematopoietic cells (i.e., CD34+ cells). Synergistic interactions were associated with a sharp increase in caspase-3 activation, PARP cleavage, induction of p-JNK, up-regulation of Noxa, and de-phosphorylation of AKT. Additionally, combined exposure diminished up-regulation of Mcl-1 induced by CFZ. Enhanced activity of this drug regimen was associated with Bax translocation, as well as Bax conformational change and dimerization. Notably, genetic interruption of JNK signaling (e.g., by shRNA knockdown) significantly attenuated CFZ/OBTX-mediated apoptosis, demonstrating a significant role for JNK activation in enhanced lethality. Additionally, shRNA knock down of Noxa or enforced activation of AKT significantly attenuated the lethality of the CFZ/ OBTX regimen in DLBCL cells. Notably, co-administration of CFZ/ OBTX resulted in sharp increases in apoptosis in 3 separate bortezomib-resistant DLBCL models. Finally, in vivo administration of CFZ and OBTX to mice inoculated with SUDHL4 cells substantially suppressed tumor growth compared to the effects of single agent treatment. Conclusions: A strategy combining the carfilzomib with obatoclax is active against parental DLBCL cells, including both GC- and ABC-subtypes, their bortezomib-resistant DLBCL counterparts, as well as primary DLBCL cells. It also displays significant in vivo activity in a xenograft DLBCL model. This approach warrants further exploration as therapeutic strategy in DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5366. doi:10.1158/1538-7445.AM2011-5366

Ets-1 mediates upregulation of Mcl-1 downstream of XBP-1 in human melanoma cells upon ER stress

Past studies have shown that upregulation of the anti-apoptotic Bcl-2 family protein Mcl-1 is a major adaptive mechanism of melanoma cells to endoplasmic reticulum (ER) stress, and has an important role in resistance of the cells to apoptosis. In this study, we show that the increase in transcription of Mcl-1 in melanoma cells triggered by pharmacological ER stress inducers is mediated by the transcription factor Ets-1. By incremental deletion analysis of the Mcl-1 promoter, we identified a DNA fragment containing an Ets-1 binding site that is transcriptionally responsive to ER stress. Mutations in the Ets-1 binding site or knockdown of Ets-1 inhibited the increase in Mcl-1, indicating that Ets-1 has a critical role in transcriptional upregulation of Mcl-1. Similar to Mcl-1, Ets-1 was transcriptionally upregulated by ER stress. This was mediated by the IRE1α/XBP-1 branch of the unfolded protein response, as upregulation of Ets-1 was inhibited in melanoma cell lines deficient in IRE1α or XBP-1 established by short hairpin RNA knockdown. Activation of the PI3k/Akt pathway downstream of XBP-1 was also involved, in that inhibition of the pathway blocked upregulation of Ets-1. Inhibition of Ets-1 enhanced ER stress-induced apoptosis in melanoma cell lines and in fresh melanoma isolates, recapitulating the effect of inhibition of Mcl-1. These results reveal a key mechanism by which Mcl-1 is transcriptionally upregulated in melanoma cells by ER stress, and identify Ets-1 as a potential target for inhibition to sensitize melanoma cells to apoptosis.

Epidermal growth factor regulates Mcl-1 expression through the MAPK-Elk-1 signalling pathway contributing to cell survival in breast cancer

Myeloid cell leukaemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. In breast tumours, increased Mcl-1 expression correlates with high tumour grade and poor patient survival. We have previously demonstrated that Her-2 levels correspond to increased Mcl-1 expression in breast tumours. Epidermal growth factor (EGF) receptor signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival. Herein, we determined the critical downstream signals responsible for the EGF mediated increase of Mcl-1 and their role in cell survival. We found that both Mcl-1 mRNA and protein levels are rapidly induced upon stimulation with EGF. Promoter analysis revealed that an Elk-1 transcription factor-binding site is critical for EGF activation of the Mcl-1 promoter. Furthermore, we found that knockdown of Elk-1or inhibition of the Erk signalling pathway was sufficient to block EGF upregulation of Mcl-1 and EGF mediated cell survival. Using chromatin immunoprecipitation and biotin labelled probes of the Mcl-1 promoter, we found that Elk-1 and serum response factor are bound to the promoter after EGF stimulation. To determine whether Mcl-1 confers a survival advantage, we found that knockdown of Mcl-1 expression increased apoptosis whereas overexpression of Mcl-1 inhibited drug induced cell death. In human breast tumours, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels. These results indicate that the EGF induced activation of Elk-1 is an important mediator of Mcl-1 expression and cell survival and therefore a potential therapeutic target in breast cancer.

NO signaling in exercise training-induced anti-apoptotic effects in human neutrophils

Short-lived neutrophils play a predominant role in innate immunity, the effects of exercise training on neutrophil survival is unclear. In this study, we investigated the underlying mechanisms of training effects on human neutrophil apoptosis. Healthy male subjects were trained on a cycling ergometer for 8 weeks and followed by 4 weeks of detraining. Blood neutrophils were collected before exercise, after training, and after detraining. Comparing with pre-exercise specimens, neutrophils collected after training showed reduced apoptosis rate, which partially returned after detraining. Various intracellular proteins, including iNOS, Mcl-1, A1, Grp78, and IL-8, were upregulated by training, and they remained high after detraining. Upregulated iNOS was closely correlated with these anti-apoptotic molecules in neutrophils. Furthermore, the possible mechanism by which iNOS suppressed apoptosis was explored. Neutrophil apoptosis was accelerated by blocking and retarded by stimulating the endogenous iNOS activity. As an anti-apoptosis mediator of NO signaling, the Mcl-1 level dropped by depletion of the major NO downstream molecule cGMP and such loss of Mcl-1 was avoidable when supplying exogenous NO. Upon activation of NO-cGMP signaling, neutrophils held increased Mcl-1 expression and delayed apoptosis. Collectively, our results suggested that exercise training may retard neutrophil apoptosis by upregulating the iNOS-NO-cGMP-Mcl-1 pathway.

Contrasting Effects of Nutlin-3 on TRAIL- and Docetaxel-Induced Apoptosis Due to Upregulation of TRAIL-R2 and Mcl-1 in Human Melanoma Cells

Wild-type p53 is commonly expressed in melanoma but does not appear to be effective in the induction of apoptosis. One explanation is that p53 is targeted for degradation by the E3 ligase MDM2. However, we found in this study that blockade of the interaction of p53 and MDM2 by the MDM2 antagonist nutlin-3 in melanoma cells did not induce apoptosis, even though it upregulated p53 and its proapoptotic targets. Nevertheless, nutlin-3 enhanced TRAIL-induced apoptosis as a result of p53-mediated upregulation of TRAIL-R2. Unexpectedly, nutlin-3 upregulated Mcl-1, which attenuated apoptotic signaling triggered by TRAIL, and inhibited apoptosis induced by the microtubule-targeting drug docetaxel. The increase in Mcl-1 was related to a p53-independent transcriptional mechanism, but stabilization of the Mcl-1 protein played a dominant role, as nutlin-3 upregulated the Mcl-1 protein to a much greater extent than the Mcl-1 mRNA, and this was associated with prolonged half-life time and reduced ubiquitination of the protein. Knockdown of p53 blocked the upregulation of the Mcl-1 protein, indicating that p53 plays a critical role in the stabilization of Mcl-1. The contrasting effects of nutlin-3 on TRAIL- and docetaxel-induced apoptosis were confirmed in fresh melanoma isolates. Collectively, these results show that nutlin-3 may be a useful agent in combination with TRAIL and, importantly, uncover a novel regulatory effect of p53 on the expression of Mcl-1 in melanoma cells on treatment with nutlin-3, which may antagonize the therapeutic efficacy of other chemotherapeutic drugs in addition to docetaxel in melanoma.

MDM2 Antagonist Nutlin-3 Enhances Bortezomib-Mediated Mitochondrial Apoptosis In TP53-Mutated Mantle Cell Lymphoma

AbstractAbstract 3920Mantle cell lymphoma (MCL) is a subtype of B-cell lymphoma and frequently resistant to standard chemotherapeutic agents, and the aggressive blastoid MCL cases often possess mutant (mt-) TP53. Although a proteasome inhibitor bortezomib demonstrates single agent efficacy in MCL, more than 50% of the relapsed or refractory MCL are not enough sensitive to bortezomib. Therefore, development of rationally designed combinations of bortezomib and other antineoplastic agents is needed. We previously reported a dose- and time- dependent inhibition of cellular proliferation in wt-TP53 MCL cells by an MDM2 inhibitor nutlin-3, and its synergistic effects when combined with bortezomib in both mt-TP53- and wt-TP53- bearing MCL cells (Tabe et al., Clin Cancer Res. 2009).In this study, we investigated the molecular mechanism of the synergistical cytotoxic effects of nutlin-3/bortezomib in the mt-TP53 MCL cells which are intrinsically resistant to bortezomib. The MCL cell lines with wild-type and mutant TP53 (wt-TP53: Z-138, Granta-519, mt-TP53: MINO) have been utilized. First, we established the IC50 of nutlin-3 and bortezomib (nutlin-3: 19.1 μM for MINO, 8.2 μM for Granta 519, 1.0 μM for Z-138, bortezomib: 21.8 nM for MINO, 3.7 nM for Granta, 5.0 nM for Z138, MTS assay at 48 hours). The combination of nutlin-3 and bortezomib synergistically induced cytotoxicity more prominently in the bortezomib resistant and mt-TP53 bearing MINO cells (combination index: 0.05 for MINO, 0.71 for Granta 519, 0.12 for Z-138, Calcusyn software). The combination of nutlin-3/bortezomib caused G0/G1 cell cycle arrest in the bortezomib sensitive wt-TP53 Granta 519 and Z138, but not in MINO. In contrast, nutlin-3/bortezomib induced marked increase in the sub-G1 fraction in MINO, but only minimal or no further increase in Granta 519 and Z138, which was confirmed by annexin V/PI staining. BH3-only protein NOXA expression was increased by bortezomib in all tested cells, and the nutlin-3/bortezomib enhanced NOXA accumulation in MINO but not in Granta 519 and Z138 (western blot). On the other hand, anti-apoptotic Mcl-1 has been upregulated by bortezomib in Granta 519 and Z138. In MINO cells, Mcl-1 upregulation was induced only by nutlin-3/bortezomib, which was coimmunoprecipitated with NOXA along with the induction of caspase-3 and -9 cleavage products. We then, assess the subsequent conformational change of proapoptotic BAX/BAK by the nutlin-3/bortezmib combination in MINO (flow cytometric analysis). Taken together, our findings indicate that mitochondrial apoptotic pathway may be an important mechanism contributing to synergistic apoptosis induction by the nutlin-3/bortezomib combination in MCL cells expressing mt-TP53 and intrinsically resistant to bortezomib. The nutlin-3/bortezomib combination may be a potential efficient therapeutic strategy for the chemorefractory MCL. Disclosures:No relevant conflicts of interest to declare.

CCR4:CCL17 Interaction Influences TLR-9 Mediated Cell Survival and Proliferation In Chronic Lymphocytic Leukemia

AbstractAbstract 3593Unlike other hematologic disorders, chronic lymphocytic leukemia(CLL) exhibits remarkable heterogeneity in the rates of disease progression among cases. CLL cells survive by receiving signals from the microenvironment via various receptors: B-cell antigen receptor (BCR), Toll-like receptors (TLRs) and cytokine and chemokine receptors. We previously reported that CLL clones with somatically mutated IGHVs and high (≥30%) percentage of CD38 expressing cells have the highest percentage of CCR4-expressing cells. To further explore the functional contribution of the CCR4:CCL17 axis in CLL, we studied CCL17-induced chemotactic behavior in 16 CLL cases. In transwell cultures we observed a bimodal migratory response to CCL17 at 2 doses in a dose range of 0.78– 25ng/ml, in ~60% of cases; the remaining cases showed maximal migration at a single dose (1.56 or 3.12ng/ml). A comparison of phenotypes of the migrated and non-migrated cell populations was undertaken in 10 cases, analyzing CXCR3, CXCR4, CCR4 and CCR7 that are involved in homing of cells to sites favoring growth, and CD31, CD38 and CD69, activation related molecules. The migrated cells consistently showed significantly higher percentages and densities of CD38 expression than the non-migrated cells suggesting a role for CD38 in the CCR4-mediated downstream pathway.CCR4 ligand, CCL17, is constitutively expressed in the thymus and is produced by dendritic cells, endothelial cells, keratinocytes and fibroblasts, whereas CCL22 is produced by tumor cells and the tumor microenvironment. Serum levels of both these ligands in untreated patients were quantified by ELISA. CCL17 levels ranged between 45-1, 229 pg/ml in U-CLL cases (n=23) and between 43-1, 418 pg/ml in M-CLL cases (n=30). CCL22 levels ranged between 121-5, 497 pg/ml in U-CLL cases (n=23) and 409-5, 502 pg/ml in M-CLL cases (n=30). The percentages of CCR4- expressing B cells directly correlated with percentages of T cells expressing CCR4 in individual cases, whereas they inversely correlated with both, serum levels of CCL17 (p< 0.01) and CCL22 (p< 0.05).CCL17 produced by DCs in peripheral organs may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells. We therefore tested if CCL17 supported BCR- and TLR-mediated proliferative responses in a cohort of 31 (16 U-CLL and 15M-CLL) CLL cases. CCL17 augmented BCR-mediated B-cell proliferation in 9/16 (56%) U-CLL cases, but only in 3/15 (20%) M-CLL cases. On the other hand, CCL17 showed an additive effect in promoting TLR-9-mediated cell proliferation in 13/15 (87%) M-CLL cases at a dose of 2ng/nl (approximating that detected in serum); it also augmented TLR-9 mediated B cell proliferation in 6/16 U-CLL cases but at a 5-fold or higher dose (10-25 ng/ml).In a subset of this cohort (8 cases) CCL17-induced modulation of molecules involved in the apoptotic process was studied. We found upregulation of anti-apoptotic proteins Mcl-1 and Bcl2 and down-regulation of pro-apoptotic molecules Bim, PUMA, and Bid in 5 of these cases. The pro-survival effects of CCL17 were partially abrogated by the blocking anti-CCR4 mAb (1G1). Taken together, these findings suggest that CCL17 plays a role in modulating TLR-9-mediated signaling and migration in CLL. Therefore, inhibition of CCR4:CCL17 interaction in vivo represents a novel therapy by preventing migration of CLL cells towards an environment that promotes their survival. Disclosures:No relevant conflicts of interest to declare.

The BH3 Mimetic, ABT-737, Overcomes Stromal-Mediated Pro-Survival Signals and Synergizes with PHA-767491, a Dual Cdc7/CDK9 Inhibitor, In Acute Myeloid Leukaemia

AbstractAbstract 1841ABT-737 is a small molecule inhibitor of Bcl-2 and Bcl-xL with reported activity in pre-clinical in-vitro and in-vivo studies of acute myeloid leukaemia(AML) but to date no data has been reported on its activity in an AML co-culture model. To address this, we examined the effects of co-culture of AML cell lines (MOLM-13, ML-1, KG-1, OCI-AML2) with HS5 cells, a human stromal cell line, on sensitivity to Ara-C and ABT-737. All cell lines cultured in the presence of HS-5 stroma demonstrated a significant reduction in Ara-C-induced apoptosis (% relative reduction - OCI-AML2:80%; ML1:65%; MOLM-13:53%; KG-1:55%) as compared to cells cultured in suspension in normal complete media, with the effect on expression of Bcl-2 family members being currently under evaluation. In contrast, in the presence of ABT-737, HS-5 co-culture did not provide any protective effect whatsoever to AML cells, with IC50 ranging from 0.1 to 0.3μM in the cell lines noted above, regardless of the presence of stroma. OCI-AML3, an AML cell line known to express high levels of Mcl-1 was resistant to ABT-737 in both normal suspension cultures and co-culture. Indeed Mcl-1, an important pro-survival protein in haematopoietic cells is thought to be a key factor promoting resistance to ABT-737 and it has recently been reported that transcriptional upregulation of Mcl-1 may follow exposure to ABT-737. Thus, the combination of ABT-737 with strategies to deplete Mcl-1 is particularly attractive. Cdk9 inhibition is such a strategy. Since Cdk9 phosphorylates RNA polymerase II affecting the rate of transcription, inhibition leads to a depletion of proteins with short half-lives, such as Mcl-1. Here we report that resistance of OCI-AML3 cells to ABT-737-induced apoptosis can be overcome by combination with PHA-767491, a novel dual Cdc7/CDK9 inhibitor. OCI-AML3 cells were treated with increasing concentrations of ABT-737, PHA-767491 or both. Co-administration resulted in a strong synergistic apoptosis-inducing effect as assessed by AnnexinV staining, with combination indices, as calculated by Chou et Talalay, for a range of doses of both drugs of <1 (range 0.3–0.9). Importantly, the sensitising effect of PHA-767491 was seen only at concentrations (≥ 2μM) that resulted in significant downregulation of Mcl-1 protein expression, implicating Mcl-1 downregulation as a possible cause of synergy. We are currently investigating the precise role of Mcl-1 in this regard. In conclusion, taken together, these studies support that ABT-737, possibly in combination with agents to deplete Mcl-1, represents a promising therapeutic strategy for AML and warrants further evaluation. Disclosures:No relevant conflicts of interest to declare.

Reduced expression of the tumor suppressor PHLPP1 enhances the antiapoptotic B-cell receptor signal in chronic lymphocytic leukemia B-cells

The PI3K/Akt pathway is activated in response to various microenvironmental stimuli that regulate the survival and proliferation of chronic lymphocytic leukemia (CLL) B-cells, including triggering of the B-cell receptor (BCR). Although this pathway is frequently targeted in cancer, no significant alterations have yet been identified in CLL. We now show that the phosphatase PH domain leucin-rich repeat protein phosphatase (PHLPP1), a recently identified tumor suppressor and negative regulator of the Akt kinase, is absent or expressed at substantially reduced levels in CLL B-cells. To determine what the consequences of PHLPP1 loss on BCR signaling are, we downregulated or re-expressed PHLPP1 in lymphoma cell lines and primary CLL B-cells, respectively. Downregulation of PHLPP1 increased BCR-induced phosphorylation and activation of the Akt, GSK3 and ERK kinases, whereas re-expression had the opposite effect. Importantly, re-expression of PHLPP1 in primary CLL cells prevented upregulation of Mcl-1 and inhibited the increase in leukemic cell viability induced by sustained BCR engagement. Enforced expression of PHLPP1 also affected the response to other microenvironmental stimuli, particularly in terms of ERK phosphorylation. Collectively, these data show that CLL cells lack an important negative regulator of the Akt and ERK pathways, which could confer them a growth advantage by facilitating the propagation of crucial microenvironment-derived stimuli.

Prokineticin-1/endocrine gland-derived vascular endothelial growth factor is a survival factor for human multiple myeloma cells

Prokineticin-1 (PK1) has been identified as a mitogen-specific protein for the endothelium of steroidogenic glands. Here we report a novel function of PK1 in the regulation of multiple myeloma (MM) cells. PK1 activates multiple signals including mitogen-activated protein kinase (MAPK), PI3K-AKT, and Jak-STAT3, sphingosine kinase-1 (SPK1) in MM cells. Treatment of MM cells with PK1 causes a time- and dose-dependent phosphorylation of MAPK, AKT and STAT3 and upregulation of SPK1 expression and cellular activity. We also show that PK1 upregulates Mcl-1 expression in a time- and dose-dependent manner in human MM cell lines and in the cells of patients with MM. Pertussis toxin, a pan-PK1 receptor inhibitor, can block PK1-induced upregulation of Mcl-1, indicating it relates to a G-protein-coupled receptor. We also show that PK1 protects MM cells against apoptosis induced by starvation for fetal calf serum (FBS), or for FBS and IL-6. Taken together, PK1 activates multiple signaling pathways and, upregulates Mcl-1 expression, leading to proliferation and survival of MM cells.

The Bcl-Xl Inhibitor, ABT-737, Efficiently Induces Apoptosis and Suppresses Growth of Hepatoma Cells in Combination with Sorafenib

Tumor cells are characterized by uncontrolled proliferation, often driven by activation of oncogenes, and apoptosis resistance. The oncogenic kinase inhibitor sorafenib can significantly prolong median survival of patients with advanced hepatocellular carcinoma (HCC), although the response is disease-stabilizing and cytostatic rather than one of tumor regression. Bcl-xL (B cell lymphoma extra large), an antiapoptotic member of the B cell lymphoma-2 (Bcl-2) family, is frequently overexpressed in HCC. Here, we present in vivo evidence that Bcl-xL overexpression is directly linked to the rapid growth of solid tumors. We also examined whether ABT-737, a small molecule that specifically inhibits Bcl-xL but not myeloid cell leukemia-1 (Mcl-1), could control HCC progression, especially when used with sorafenib. Administration of ABT-737, even at an in vivo effective dose, failed to suppress Huh7 xenograft tumors in mice. ABT-737 caused the levels of Mcl-1 expression to rapidly increase by protein stabilization. This appeared to be related to resistance to ABT-737, because decreasing Mcl-1 expression levels to the baseline by a small interfering RNA-mediated strategy made hepatoma cells sensitive to this agent. Importantly, administration of ABT-737 to Mcl-1 knockout mice induced severe liver apoptosis, suggesting that tumor-specific inhibition of Mcl-1 is required for therapeutic purposes. Sorafenib transcriptionally down-regulated Mcl-1 expression specifically in tumor cells and abolished Mcl-1 up-regulation induced by ABT-737. Sorafenib, not alone but in combination with ABT-737, efficiently induced apoptosis in hepatoma cells. This combination also led to stronger suppression of xenograft tumors than sorafenib alone. Bcl-xL inactivation by ABT-737 in combination with sorafenib was found to be safe and effective for anti-HCC therapy in preclinical models. Direct activation of the apoptosis machinery seems to unlock the antitumor potential of oncogenic kinase inhibitors and may produce durable clinical responses against HCC.

Spleen tyrosine kinase inhibition prevents chemokine- and integrin-mediated stromal protective effects in chronic lymphocytic leukemia

AbstractThe microenvironment provides essential growth and survival signals to chronic lymphocytic leukemia (CLL) cells and contributes to their resistance to cytotoxic agents. Pharmacologic inhibition of spleen tyrosine kinase (SYK), a key mediator of B-cell receptor (BCR) signaling, induces apoptosis in primary CLL cells and prevents stroma contact-mediated cell survival. This report demonstrates a role of SYK in molecularly defined pathways that mediate the CLL-microenvironmental crosstalk independent from the BCR. Chemokine and integrin stimulation induced SYK phosphorylation, SYK-dependent Akt phosphorylation, and F-actin formation in primary CLL cells. Inhibition of SYK by 2 pharmacologic inhibitors and siRNA-knockdown abrogated downstream SYK signaling and morphologic changes induced by these stimuli. CLL cell migration toward CXCL12, the major homing attractor, and CLL cell adhesion to VCAM-1, a major integrin ligand expressed on stromal cells, were markedly reduced by SYK inhibition. In combination with fludarabine, the SYK inhibitor R406 abrogated stroma-mediated drug resistance by preventing up-regulation of the antiapoptotic factor Mcl-1 in CLL cells. SYK blockade in CLL is a promising therapeutic principle not only for its inhibition of the BCR signaling pathway, but also by inhibiting protective stroma signals in a manner entirely independent of BCR signaling.

651 Apurinic/Apyrimidinic Endonuclease (APE-1) Regulates Oxidative Signaling Pathways Including RAC1 and NADPH Oxidase in H. pylori (HP) Infected Gastric Epithelial Cells (GEC)

Mcl-1 expression in gastric cells co-cultured with strain 7.13, consistent with the marked downregulation of miR-320. To define the effect of the H. pylori oncoprotein CagA on these events, cells were also co-cultured with a 7.13 cagAisogenic mutant. Loss of cagA attenuated both the reduction of miR-320 and the subsequent increase in Mcl-1 expression observed in response to wild-type strain 7.13. In summary,H. pylori strain 7.13 specifically downregulates miR-320 in a CagA-dependent manner, which relieves repression of Mcl-1. Upregulation of Mcl-1 by this carcinogenic strain ofH. pylori likely promotes cell survival through inhibition of apoptosis, which may heighten retention of mutagenized cells and increase the risk of subsequent gastric carcinogenesis.

654 Nitric Oxide Inhibits Helicobacter pylori-Induced Innate Immune Function of Gastric Epithelial Cells by a Heme Oxygenase-1-Dependent Pathway

Mcl-1 expression in gastric cells co-cultured with strain 7.13, consistent with the marked downregulation of miR-320. To define the effect of the H. pylori oncoprotein CagA on these events, cells were also co-cultured with a 7.13 cagAisogenic mutant. Loss of cagA attenuated both the reduction of miR-320 and the subsequent increase in Mcl-1 expression observed in response to wild-type strain 7.13. In summary,H. pylori strain 7.13 specifically downregulates miR-320 in a CagA-dependent manner, which relieves repression of Mcl-1. Upregulation of Mcl-1 by this carcinogenic strain ofH. pylori likely promotes cell survival through inhibition of apoptosis, which may heighten retention of mutagenized cells and increase the risk of subsequent gastric carcinogenesis.

Abstract 1198: Adaptation to ER stress as a driver of increased expression of Mcl-1 with melanoma progression

Abstract Past studies have shown that Mcl-1 expression increases with melanoma progression, which is an important mechanism of resistance of melanoma cells to apoptosis. We show here that up-regulation of Mcl-1 in melanoma is a consequence of adaptation of melanoma cells to endoplasmic reticulum (ER) stress. Exposure of cultured melanoma cells and fresh melanoma isolates to pharmacological ER stress inducers results in up-regulation of Bcl-2 and Mcl-1. Inhibition of Mcl-1 renders melanoma cells more sensitive to ER stress-induced apoptosis even when Bcl-2 is over-expressed, but inhibition of Bcl-2 does not increase the sensitivity to ER stress-induced apoptosis in melanoma cells over-expressing Mcl-1. Up-regulation of Mcl-1 by ER stress involves multiple mechanisms. The transcriptional increase is mediated by the transcription factor ETS-1 downstream of the XBP1 axis of the unfolded protein response, whereas down-regulation of GSK3α is responsible for reduced proteasomal degradation of Mcl-1 in melanoma cells under ER stress. Studies on a large cohort of melanocytic tumor tissue sections revealed that expression of GRP78, a classic marker of ER stress, and expression of LDH5, an indicator of hypoglycemia that is one of the causes of ER stress, correlate with Mcl-1 expression, indicating that the increase in Mcl-1 with melanoma progression in vivo is associated with ER stress. In addition, we demonstrate in this report that induction of apoptosis by ER stress in melanoma cells deficient in Mcl-1 is mediated by the BH3-only proteins PUMA and Noxa, whereas another BH3-only protein Bim does not appear to play a part in melanoma cells as it does in other cell types. In summary, our results indicate that adaptation to ER stress is a driver of up-regulation of Mcl-1 during progression of melanoma, and suggest that strategies aimed at inhibiting the activity of Mcl-1 may be important in the treatment of melanoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1198.

Abstract LB-342: Matrix metalloproteinase-9 is a novel pathogenic factor in B-cell chronic lymphocytic leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation in peripheral blood of CD5+ B lymphocytes, which ‘ progressively infiltrate lymphoid tissues. Several molecules, including matrix metalloproteinase-9 (MMP-9), are involved in these processes and could represent therapeutic targets. We previously showed that MMP-9 plays an essential role in B-CLL cell migration through basement membranes or endothelial cells (Redondo-Muñoz et al., Blood 108: 3143, 2006). We also recently demonstrated that alpha4beta1 integrin and a 190 kDa CD44v isoform are receptors for MMP-9 in B-CLL but not in normal B cells and that the MMP-9 hemopexin domain is essential for these interactions. Binding of MMP-9 to B-CLL cells inhibited cell migration and this required MMP-9 proteolytic activity (Redondo-Muñoz et al., Blood 112: 169, 2008). To further define the role of surface-bound MMP-9 in B-CLL we have employed several techniques including western blot, zymography, immunofluorescence, immunoprecipitation, siRNA transfection, cell adhesion/soluble binding assays, apoptosis, and cell migration assays. B-CLL cells cultured on pro- or mature MMP-9, proMMP-9 hemopexin domain or proMMP-9 catalytically-inactive mutant, showed decreased spontaneous apoptosis. This was due to induction of a survival pathway involving Lyn activation, STAT3 phosphorylation and Mcl-1 upregulation and did not require catalytic activity. This pathway was induced in all B-CLL cases and was active in B-CLL lymphoid tissues. Our results highlight the role of MMP-9 in B-CLL pathogenesis and indicate that MMP-9 hemopexin domain could constitute a therapeutic target in this malignancy. Supported by grants PI060400 and SAF2009-07035 from the Ministerio de Ciencia e Innovación and by the Fundación de Investigación Médica Mutua Madrileña. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-342.

PI3K-dependent upregulation of Mcl-1 by human cytomegalovirus is mediated by epidermal growth factor receptor and inhibits apoptosis in short-lived monocytes.

Monocytes are a primary target for human CMV (HCMV) infection and are a key cell type responsible for hematogenous dissemination of the virus. Biologically, these cells have a short lifespan of 1-3 d in the circulation, yet infected cells remain viable for weeks despite the lack of viral antiapoptotic gene expression during this period. To understand the mechanism by which HCMV inhibits the initial phase of monocyte apoptosis, we focused on the viral modulation of early prosurvival cell signaling events after infection. We demonstrate in this study that the viral upregulation of the PI3K pathway promotes an early block in apoptosis after infection. Temporal transcriptome and protein analyses revealed Mcl-1, a member of the Bcl-2 family, was transiently induced in a PI3K-dependent manner during the early stages of HCMV infection. In accord with the survival studies, virally induced levels of Mcl-1 expression dissipated to mock levels by 72 h postinfection. Through the use of Mcl-1-specific small interfering RNA, we confirmed the functional role that Mcl-1 plays as a key early regulator of apoptosis in monocytes. Lastly, we showed that HCMV engagement and activation of the epidermal growth factor receptor during viral binding triggered the upregulation of Mcl-1. Overall, our data indicates that activation of the epidermal growth factor receptor/PI3K signaling pathway, via the PI3K-dependent upregulation of Mcl-1, is required to circumvent apoptosis in naturally short-lived monocytes during the early stages of HCMV infection, thus ensuring the early steps in the viral persistence strategy.

TRAIL-activated stress kinases suppress apoptosis through transcriptional upregulation of MCL-1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potentially useful anticancer agent with exquisite selectivity for cancer cells. Unfortunately, many cancers show or acquire resistance to TRAIL. In this study we report that TRAIL activates a TGF-beta-activated kinase 1 --> mitogen-activated protein kinase (MAPK) kinase 3 (MKK3)/MKK6 --> p38 pathway in prostate cancer cells that transcriptionally upregulates expression of the antiapoptotic BCL-2 family member MCL-1. TRAIL alone triggered robust formation of the 'death-inducing signaling complex' (DISC), activation of the initiator caspase-8, and truncation of the BH3-only protein BID (tBID). Nevertheless, simultaneous disruption of the p38 MAPK pathway was required to suppress MCL-1 expression, thereby allowing tBID to activate the proapoptotic BCL-2 family member BAK and stimulate mitochondrial outer membrane permeabilization (MOMP). Release of the inhibitor-of-apoptosis (IAP) antagonist, Smac/DIABLO, from the intermembrane space was sufficient to promote TRAIL-induced apoptosis, whereas release of cytochrome c and activation of the apoptosome was dispensable. Even after MOMP, however, mitochondrial-generated reactive oxygen species (ROS) activated a secondary signaling pathway, involving c-Jun N-terminal kinases (JNKs), that similarly upregulated MCL-1 expression and partially rescued some cells from death. Thus, stress kinases activated at distinct steps, before and after mitochondrial injury, mediate TRAIL resistance through maintenance of MCL-1 expression.