Apoptosis Repressor Research Articles

MicroRNA-532-3p regulates mitochondrial fission through targeting apoptosis repressor with caspase recruitment domain in doxorubicin cardiotoxicity.

Doxorubicin (DOX) is a wide-spectrum antitumor drug, but its clinical application is limited by its cardiotoxicity. However, the mechanisms underlying DOX-induced cardiomyopathy remain mostly unclear. Here we observed that apoptosis repressor with caspase recruitment domain (ARC) was downregulated in mouse heart and cardiomyocytes upon DOX treatment. Furthermore, enforced expression of ARC attenuated DOX-induced cardiomyocyte mitochondrial fission and apoptosis. ARC transgenic mice demonstrated reduced cardiotoxicity upon DOX administration. DOX-induced mitochondrial fission required the activity of dynamin-related protein 1 (Drp1). In elucidating the molecular mechanism by which ARC was downregulated upon DOX treatment, miR-532-3p was found to directly target ARC and participated in DOX-induced mitochondrial fission and apoptosis. MiR-532-3p was not involved in DOX-induced apoptosis in cancer cells. Taken together, these findings provide novel evidence that miR-532-3p and ARC constitute an antiapoptotic pathway that regulates DOX cardiotoxicity. Therefore, the development of new therapeutic strategies based on ARC and miR-532-3p is promising for overcoming the cardiotoxicity of chemotherapy for cancer therapy.

Regrowth after skeletal muscle atrophy is impaired in aged rats, despite similar responses in signaling pathways

Skeletal muscle regrowth after atrophy is impaired in the aged and in this study we hypothesized that this can be explained by a blunted response of signaling pathways and cellular processes during reloading after hind limb suspension in muscles from old rats. Male Brown Norway Fisher 344 rats at 6 (young) and 32 (old) months of age were subjected to normal ambulatory conditions (amb), hind limb suspension for 14days (HS), and HS followed by reloading through normal ambulation for 14days (RE); soleus muscles were used for analysis of intracellular signaling pathways and cellular processes. Soleus muscle regrowth was blunted in old compared to young rats which coincided with a recovery of serum IGF-1 and IGFBP-3 levels in young but not old. However, the response to reloading for p-Akt, p-p70s6k and p-GSK3β protein abundance was similar between muscles from young and old rats, even though main effects for age indicate an increase in activation of this protein synthesis pathway in the aged. Similarly, MAFbx mRNA levels in soleus muscle from old rats recovered to the same extent as in the young, while Murf-1 was unchanged. mRNA abundance of autophagy markers Atg5 and Atg7 showed an identical response in muscle from old compared to young rats, but beclin did not. Autophagic flux was not changed at either age at the measured time point. Apoptosis was elevated in soleus muscle from old rats particularly with HS, but recovered in HSRE and these changes were not associated with differences in caspase-3, -8 or -9 activity in any group. Protein abundance of apoptosis repressor with caspase-recruitment domain (ARC), cytosolic EndoG, as well as cytosolic and nuclear apoptosis inducing factor (AIF) were lower in muscle from old rats, and there was no age-related difference in the response to atrophy or regrowth. Soleus muscles from old rats had a higher number of ED2 positive macrophages in all groups and these decreased with HS, but recovered in HSRE in the old, while no changes were observed in the young. Pro-inflammatory cytokines in serum did not show a differential response with age to different loading conditions. Results indicate that at the measured time point the impaired skeletal muscle regrowth after atrophy in aged animals is not associated with a general lack of responsiveness to changes in loading conditions.

Functional, morphological, and apoptotic alterations in skeletal muscle of ARC deficient mice.

Apoptotic signaling plays an important role in the development and maintenance of healthy skeletal muscle. However, dysregulation of apoptotic signals in skeletal muscle is associated with atrophy and loss of function. Apoptosis repressor with caspase recruitment domain (ARC) is a potent anti-apoptotic protein that is highly expressed in skeletal muscle; however, its role in this tissue has yet to be elucidated. To investigate whether ARC deficiency has morphological, functional, and apoptotic consequences, skeletal muscle from 18 week-old wild-type and ARC knockout (KO) mice was studied. In red muscle (soleus), we found lower maximum tetanic force, as well as a shift towards a greater proportion of type II fibers in ARC KO mice. Furthermore, the soleus of ARC KO mice exhibited lower total, as well as fiber type-specific cross sectional area in type I and IIA fibers. Interestingly, these changes in ARC KO mice corresponded with increased DNA fragmentation, albeit independent of caspase or calpain activation. However, cytosolic fractions of red muscle from ARC KO mice had higher apoptosis inducing factor content, suggesting increased mitochondrial-mediated, caspase-independent apoptotic signaling. This was confirmed in isolated mitochondrial preparations, as mitochondria from skeletal muscle of ARC KO mice were more susceptible to calcium stress. Interestingly, white muscle from ARC KO mice showed no signs of altered apoptotic signaling or detrimental morphological differences. Results from this study suggest that even under basal conditions ARC influences muscle apoptotic signaling, phenotype, and function, particularly in slow and/or oxidative muscle.

Crystallization and preliminary X-ray crystallographic analysis of the CARD domain of apoptosis repressor with CARD (ARC).

Apoptosis repressor with caspase-recruiting domain (ARC) is an apoptosis repressor that inhibits both intrinsic and extrinsic apoptosis signalling. Human ARC contains an N-terminal caspase-recruiting domain (CARD domain) and a C-terminal proline- and glutamic acid-rich (P/E-rich) domain. The CARD domain in ARC is the domain that is directly involved in inhibition of the extrinsic pathway. In this study, the N-terminal CARD domain of ARC was overexpressed, purified and crystallized. X-ray diffraction data were collected to a resolution of 2.1 Å and the crystals were found to belong to space group P6(1) or P65, with unit-cell parameters a=98.28, b=98.28, c=51.86 Å, α=90, β=90, γ=120°.

Aβ induces PUMA activation: a new mechanism for Aβ-mediated neuronal apoptosis

p53 upregulated modulator of apoptosis (PUMA) is a promising tumor therapy target because it elicits apoptosis and profound sensitivity to radiation and chemotherapy. However, inhibition of PUMA may be beneficial for curbing excessive apoptosis associated with neurodegenerative disorders. Alzheimer's disease (AD) is a representative neurodegenerative disease in which amyloid-β (Aβ) deposition causes neurotoxicity. The regulation of PUMA during Aβ-induced neuronal apoptosis remains poorly understood. Here, we reported that PUMA expression was significantly increased in the hippocampus of transgenic mice models of AD and hippocampal neurons in response to Aβ. PUMA knockdown protected the neurons against Aβ-induced apoptosis. Furthermore, besides p53, PUMA transactivation was also regulated by forkhead box O3a through p53-independent manner following Aβ treatment. Notably, PUMA contributed to neuronal apoptosis through competitive binding of apoptosis repressor with caspase recruitment domain to activate caspase-8 that cleaved Bid into tBid to accelerate Bax mitochondrial translocation, revealing a novel pathway of Bax activation by PUMA to mediate Aβ-induced neuronal apoptosis. Together, we demonstrated that PUMA activation involved in Aβ-induced apoptosis, representing a drug target to antagonize AD progression.

SNX13 reduction mediates heart failure through degradative sorting of apoptosis repressor with caspase recruitment domain.

Heart failure (HF) is associated with complicated molecular remodelling within cardiomyocytes; however, the mechanisms underlying this process remain unclear. Here we show that sorting nexin-13 (SNX13), a member of both the sorting nexin and the regulator of G protein signalling (RGS) protein families, is a potent mediator of HF. Decreased levels of SNX13 are observed in failing hearts of humans and of experimental animals. SNX13-deficient zebrafish recapitulate HF with striking cardiomyocyte apoptosis. Mechanistically, a reduction in SNX13 expression facilitates the degradative sorting of apoptosis repressor with caspase recruitment domain (ARC), which is a multifunctional inhibitor of apoptosis. Consequently, the apoptotic pathway is activated, resulting in the loss of cardiac cells and the dampening of cardiac function. The N-terminal PXA structure of SNX13 is responsible for mediating the endosomal trafficking of ARC. Thus, this study reveals that SNX13 profoundly affects cardiac performance through the SNX13-PXA-ARC-caspase signalling pathway.

MicroRNA-30d regulates cardiomyocyte pyroptosis by directly targeting foxo3a in diabetic cardiomyopathy.

Diabetic cardiomyopathy is a common cardiac condition in patients with diabetes mellitus, which can result in cardiac hypertrophy and subsequent heart failure, associated with pyroptosis, the pro-inflammatory programmed cell death. MicroRNAs (miRNAs), small endogenous non-coding RNAs, have been shown to be involved in diabetic cardiomyopathy. However, whether miRNAs regulate pyroptosis in diabetic cardiomyopathy remains unknown. Our study revealed that mir-30d expression was substantially increased in streptozotocin (STZ)-induced diabetic rats and in high-glucose-treated cardiomyocytes as well. Upregulation of mir-30d promoted cardiomyocyte pyroptosis in diabetic cardiomyopathy; conversely, knockdown of mir-30d attenuated it. In an effort to understand the signaling mechanisms underlying the pro-pyroptotic property of mir-30d, we found that forced expression of mir-30d upregulated caspase-1 and pro-inflammatory cytokines IL-1β and IL-18. Moreover, mir-30d directly repressed foxo3a expression and its downstream protein, apoptosis repressor with caspase recruitment domain (ARC). Furthermore, silencing ARC by siRNA mimicked the action of mir-30d: upregulating caspase-1 and inducing pyroptosis. These findings promoted us to propose a new signaling pathway leading to cardiomyocyte pyroptosis under hyperglycemic conditions: mir-30d↑→foxo3a↓→ ARC↓→caspase-1↑→IL-1β, IL-18↑→pyroptosis↑. Therefore, mir-30d may be a promising therapeutic target for the management of diabetic cardiomyopathy.

Abstract 1181: The Apoptosis repressor with a CARD domain (ARC) is a direct HIF1 target gene and promotes survival and proliferation of VHL deficient renal cancer cells

Abstract The induction of hypoxia inducible factors (HIFs) is essential for the adaptation of tumor cells to a low oxygen environment. We found that the expression of the apoptosis inhibitor ARC was induced by hypoxia in a variety of cancer cell types and its induction is primarily HIF1 dependent. Chromatin immunoprecipitation (ChIP) and reporter assays also indicate that the ARC gene is regulated by direct binding of HIF1 to a hypoxia response element (HRE) located at -190 bp upstream of the transcription start site. HIFs play an essential role in the pathogenesis of renal cell carcinoma (RCC) under normoxic conditions, through the loss of the Von Hippel Lindau (VHL) gene. Accordingly, our results show that ARC is not expressed in normal renal tissue, but is highly expressed in 65% of RCC tumors, which also express high levels of Carbonic Anhydrase IX (CAIX), a HIF1-dependent gene. Compared to controls, ARC-deficient RCCs exhibited decreased colony formation and increased apoptosis in vitro. In addition, loss of ARC resulted in a dramatic reduction of RCC tumor growth in SCID mice in vivo. Thus, HIF-mediated increased expression of ARC in RCC can explain how loss of VHL can promote survival early in tumor formation. Citation Format: Olga V. Razorenova, Laura Castellini, Renata Colavitti, Laura E. Edgington, Monica Nicolau, Xin Huang, Barbara Bedogni, Edward M. Mills, Matthew Bogyo, Amato J. Giaccia. The Apoptosis repressor with a CARD domain (ARC) is a direct HIF1 target gene and promotes survival and proliferation of VHL deficient renal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1181. doi:10.1158/1538-7445.AM2014-1181

Abstract 2266: Investigating the role of the anti-apoptotic protein ARC in breast cancer cell DNA repair

Abstract According to the American Cancer Society, more than 90% of breast cancer patients who succumb to the condition die as a result of the cancer metastasizing to other areas of the body. Our lab has previously shown that the presence of a chemoresistant invasive subpopulation of breast carcinoma cells is correlated with the prevalence of metastasis. Additionally, we have hypothesized that chemoresistance and survival of these cancer cells which eventually leads to relapse and metastasis following initial treatment with chemotherapeutic agents is achieved by enhanced DNA repair capability in this population. We reported that an anti-apoptotic protein ARC (apoptosis repressor with caspase recruitment domain) is over expressed in the invasive mammary cancer cells that are resistant to standard chemotherapeutic agents. Further, we have shown that ARC over expressing cells are resistant to chemotherapy both in vivo and in vitro. In this study, we examined cellular DNA repair signaling pathways in order to delineate the role of ARC in the pathogenesis of breast cancer. Utilizing cell lines where ARC was either knocked down or overexpressed, we investigated potential correlations of ARC with DNA repair capability by means of the single cell gel electrophoresis assay (comet assay). The results of these experiments indicated that over expression of ARC leads to increased cellular DNA repair while knocking it down leads to decreased cellular DNA repair. In order to determine the molecular basis of the role of ARC in the DNA repair pathways, we have performed qRT-PCR based gene expression analysis on ARC modified cell lines using techniques previously established in our lab. The results of these experiments indicated that increased levels of ARC are correlated with upregulation of key genes in single stranded DNA repair pathways such as the base excision and nucleotide excision repair pathways. We are working to identify the specific domain of ARC that serves as its effector in the DNA repair pathways. In particular, we are looking at the caspase recruitment domain (CARD) of ARC which is known to be key for ARC`s function in the apoptotic pathways. Once this domain is identified, it may serve as a potential drug target, the silencing of which could result in the disruption of key pathways required for invasive breast cancer cell survival. Such therapy may facilitate the ablation of chemoresistant breast cancer cell populations, prevent metastasis and tumor relapse, and increase long term patient survival. Citation Format: Eli Grunblatt, Evanka Madan, Sweta Roy, Sumanta Goswami. Investigating the role of the anti-apoptotic protein ARC in breast cancer cell DNA repair. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2266. doi:10.1158/1538-7445.AM2014-2266

Apoptosis repressor with caspase recruitment domain modulates second mitochondrial-derived activator of caspases mimetic-induced cell death through BIRC2/MAP3K14 signalling in acute myeloid leukaemia.

Overexpression of the apoptosis repressor with caspase recruitment domain (ARC, also termed NOL3) protein predicts adverse outcome in patients with acute myeloid leukaemia (AML) and confers drug resistance to AML cells. The second mitochondrial-derived activator of caspases (SMAC, also termed DIABLO) mimetic, birinapant, promotes extrinsic apoptosis in AML cells. SMAC mimetics induce cleavage of cellular inhibitor of apoptosis (cIAP) proteins, leading to stabilization of the nuclear factor-κB (NF-κB)-inducing kinase (MAP3K14, also termed NIK) and activation of non-canonical NF-κB signalling. To enhance the therapeutic potential of SMAC mimetics in AML, we investigated the regulation and role of ARC in birinapant-induced apoptosis. We showed that birinapant increases ARC in AML and bone marrow-derived mesenchymal stromal cells (MSCs). Downregulation of MAP3K14 by siRNA decreased ARC levels and suppressed birinapant-induced ARC increase. Reverse-phase protein array analysis of 511 samples from newly diagnosed AML patients showed that BIRC2 (also termed cIAP1) and ARC were inversely correlated. Knockdown of ARC sensitized, while overexpression attenuated, birinapant-induced apoptosis. Furthermore, ARC knockdown in MSCs sensitized co-cultured AML cells to birinapant-induced apoptosis. Our data demonstrate that ARC is regulated via BIRC2/MAP3K14 signalling and its overexpression in AML or MSCs can function as a resistant factor to birinapant-induced leukaemia cell death, suggesting that strategies to inhibit ARC will improve the therapeutic potential of SMAC mimetics.

MicroRNA-185 regulates chemotherapeutic sensitivity in gastric cancer by targeting apoptosis repressor with caspase recruitment domain.

Gastric cancer remains the second leading cause of cancer deaths worldwide. Resistance to chemotherapy is a significant barrier for effective cancer treatment. Here, we identified miR-185 to be a contributor to chemosensitivity in gastric cancer. We observed low levels of miR-185 in gastric cancer cell lines and clinical tissues, compared with gastric epithelium cell line and noncancerous tissues. Furthermore, enforced expression of miR-185 increased the sensitivity of gastric cancer cells to low-dose chemotherapeutic agents, which alone cannot trigger significant apoptosis. Conversely, knockdown of endogenous miR-185 prevented high-dose chemotherapy-induced apoptosis. In elucidating the molecular mechanism by which miR-185 participated in the regulation of chemosensitivity in gastric cancer, we discovered that apoptosis repressor with caspase recruitment domain (ARC) is a direct target of miR-185. The role of miR-185 was confirmed in gastric tumor xenograft model. The growth of established tumors was suppressed by a combination therapy using enforced miR-185 expression and a low dose of anticancer drugs. Finally, we found that RUNX3 (Runt-related transcription factor) was involved in the activation of miR-185 at the transcriptional level. Taken together, our results reveal that RUNX3, miR-185 and ARC regulate the sensitivity of gastric cancer cells to chemotherapy.

The Apoptosis Repressor with a CARD Domain (ARC) Gene Is a Direct Hypoxia-Inducible Factor 1 Target Gene and Promotes Survival and Proliferation of VHL-Deficient Renal Cancer Cells

The induction of hypoxia-inducible factors (HIFs) is essential for the adaptation of tumor cells to a low-oxygen environment. We found that the expression of the apoptosis inhibitor ARC (apoptosis repressor with a CARD domain) was induced by hypoxia in a variety of cancer cell types, and its induction is primarily HIF1 dependent. Chromatin immunoprecipitation (ChIP) and reporter assays also indicate that the ARC gene is regulated by direct binding of HIF1 to a hypoxia response element (HRE) located at bp -190 upstream of the transcription start site. HIFs play an essential role in the pathogenesis of renal cell carcinoma (RCC) under normoxic conditions, through the loss of the Von Hippel-Lindau gene (VHL). Accordingly, our results show that ARC is not expressed in normal renal tissue but is highly expressed in 65% of RCC tumors, which also express high levels of carbonic anhydrase IX (CAIX), a HIF1-dependent protein. Compared to controls, ARC-deficient RCCs exhibited decreased colony formation and increased apoptosis in vitro. In addition, loss of ARC resulted in a dramatic reduction of RCC tumor growth in SCID mice in vivo. Thus, HIF-mediated increased expression of ARC in RCC can explain how loss of VHL can promote survival early in tumor formation.

A novel role for the apoptosis inhibitor ARC in suppressing TNFα-induced regulated necrosis.

TNFα signaling can promote apoptosis or a regulated form of necrosis. ARC (apoptosis repressor with CARD (caspase recruitment domain)) is an endogenous inhibitor of apoptosis that antagonizes both the extrinsic (death receptor) and intrinsic (mitochondrial/ER) apoptosis pathways. We discovered that ARC blocks not only apoptosis but also necrosis. TNFα-induced necrosis was abrogated by overexpression of wild-type ARC but not by a CARD mutant that is also defective for inhibition of apoptosis. Conversely, knockdown of ARC exacerbated TNFα-induced necrosis, an effect that was rescued by reconstitution with wild-type, but not CARD-defective, ARC. Similarly, depletion of ARC in vivo exacerbated necrosis caused by infection with vaccinia virus, which elicits severe tissue damage through this pathway, and sensitized mice to TNFα-induced systemic inflammatory response syndrome. The mechanism underlying these effects is an interaction of ARC with TNF receptor 1 that interferes with recruitment of RIP1, a critical mediator of TNFα-induced regulated necrosis. These findings extend the role of ARC from an apoptosis inhibitor to a regulator of the TNFα pathway and an inhibitor of TNFα-mediated regulated necrosis.

Apoptosis repressor with caspase recruitment domain is regulated by MAPK/PI3K and confers drug resistance and survival advantage to AML

The apoptosis repressor with caspase recruitment domain (ARC) protein is known to suppress both intrinsic and extrinsic apoptosis. We previously reported that ARC expression is a strong, independent adverse prognostic factor in acute myeloid leukemia (AML). Here, we investigated the regulation and role of ARC in AML. ARC expression is upregulated in AML cells co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and suppressed by inhibition of MAPK and PI3K signaling. AML patient samples with RAS mutations (N=64) expressed significantly higher levels of ARC than samples without RAS mutations (N=371) (P=0.016). ARC overexpression protected and ARC knockdown sensitized AML cells to cytarabine and to agents that selectively induce intrinsic (ABT-737) or extrinsic (TNF-related apoptosis inducing ligand) apoptosis. NOD-SCID mice harboring ARC-overexpressing KG-1 cells had significantly shorter survival than mice injected with control cells (median 84 vs 111days) and significantly fewer leukemia cells were present when NOD/SCID IL2Rγ null mice were injected with ARC knockdown as compared to control Molm13 cells (P=0.005 and 0.03 at 2 and 3weeks, respectively). Together, these findings demonstrate that MSCs regulate ARC in AML through activation of MAPK and PI3K signaling pathways. ARC confers drug resistance and survival advantage to AML in vitro and in vivo, suggesting ARC as a novel target in AML therapy.

Apoptosis Repressor with a CARD Domain (ARC) Restrains Bax-Mediated Pathogenesis in Dystrophic Skeletal Muscle

Myofiber wasting in muscular dystrophy has largely been ascribed to necrotic cell death, despite reports identifying apoptotic markers in dystrophic muscle. Here we set out to identify the contribution of canonical apoptotic pathways to skeletal muscle degeneration in muscular dystrophy by genetically deleting a known inhibitor of apoptosis, apoptosis repressor with a card domain (Arc), in dystrophic mouse models. Nol3 (Arc protein) genetic deletion in the dystrophic Sgcd or Lama2 null backgrounds showed exacerbated skeletal muscle pathology with decreased muscle performance compared with single null dystrophic littermate controls. The enhanced severity of the dystrophic phenotype associated with Nol3 deletion was caspase independent but dependent on the mitochondria permeability transition pore (MPTP), as the inhibitor Debio-025 partially rescued skeletal muscle pathology in Nol3 -/- Sgcd -/- double targeted mice. Mechanistically, Nol3 -/- Sgcd -/- mice showed elevated total and mitochondrial Bax protein levels, as well as greater mitochondrial swelling, suggesting that Arc normally restrains the cell death effects of Bax in skeletal muscle. Indeed, knockdown of Arc in mouse embryonic fibroblasts caused an increased sensitivity to cell death that was fully blocked in Bax Bak1 (genes encoding Bax and Bak) double null fibroblasts. Thus Arc deficiency in dystrophic muscle exacerbates disease pathogenesis due to a Bax-mediated sensitization of mitochondria-dependent death mechanisms.

ARC Supports Leukemia-Stromal Interactions By Regulating Multiple Receptor-Chemokine Axes In AML

We have reported that “apoptosis repressor with caspase recruitment domain” (ARC), an antiapoptotic protein promotes leukemia-stromal interactions in part by increasing the expression of CXCR4 in AML cells and of CXCL12 (SDF-1) in mesenchymal stromal cells (MSCs) (Carter et al., ASH 2012). To further understand the mechanisms of action of ARC in mediating leukemia-stromal interactions, we examined the expression of multiple chemokines by quantitative cytokine PCR array in ARC knockdown (KD) and control MSCs. We found that among 6 C-X-C motif and 22 C-C motif chemokines, CXCL12, CCL2 and CCL4 are highly expressed and upregulated by ARC in MSCs.We then determined the protein levels of CCR2 and CCR5, the respective receptors for CCL2 and CCL4, in bone marrow samples from 8 AML patients and found that CCR2 was highly expressed in all samples and CCR5 was detectable in 75 % suggesting that the CCR2/CCL2 and CCR5/CCL4 axes may also contribute to leukemia-stromal interactions in AML. Trans-well migration assays showed that CCL2 and CCL4 induced the migration of AML cells which was antagonized by blocking antibodies or small molecule inhibitors. AML cells migrated less to ARC KD than to control MSCs. Primary leukemia cells from AML patient samples also migrated towards CCL2 and CCL4, and this migration correlated with the expression of the respective receptors in the leukemia cells.Co-culture of MSCs with leukemia cells greatly increased the levels of CXCL12, CCL2, and CCL4 in MSCs and the increase was diminished when co-cultured with ARC KD and increased when co-cultured with ARC overexpressing (OE) cells was compared to their respective controls, suggesting that chemokine expression in MSCs is in part regulated by ARC-mediated signaling from AML cells. Cytokine PCR array revealed that IL1β expression is increased in ARC OE and decreased in ARC KD cells. Conversely, the level of the IL1 receptor antagonist IL1RN is lower in ARC OE and higher in ARC KD. ARC OE cells also secreted more IL1β while ARC KD cells secreted less. Furthermore, co-cultures with MSCs increased the expression of IL1β in AML cells. IL1β greatly increased the levels of CCL2, CCL4, and CXCL12 in MSCs and the increase was largely diminished by co-treating the MSCs with IL1β antagonist IL1βRA. Similarly, treating MSCs with conditioned media from AML cells increased the expression of the chemokines in MSCs, and the increase was reduced with conditioned medium from ARC KD cells; this increase was diminished by IL1βRA. Furthermore, IL1βRA blocked the migration of these cells toward CCL2 and CCL4.Using a 3-D model in which cancellous bone was covered with MSCs, we found fewer OCI-AML3 cells attached to MSCs when ARC was knocked down in these cells further supporting the role of ARC in leukemia-stromal interactions.ARC is a member of caspase recruitment domain (CARD) containing proteins that have diverse functions such as antiapoptosis and regulation of NFκB activity. ARC participation in cell death suppression and leukemia-stromal interactions suggests that ARC may interact not only with apoptosis regulators but also with signaling proteins to regulate multiple cellular functions in AML. ConclusionsOur findings suggest that multiple ARC-regulated receptor/ligand pairs play important roles in leukemia-stromal interactions, and that there is reciprocal crosstalk between malignant cells and microenvironment cells that is in part mediated through ARC-regulated inflammatory cytokine IL1β. ARC is therefore a novel target not only for direct apoptosis induction in leukemia cells but also for disruption of protective leukemia-stromal interactions to further sensitize leukemia cells to their elimination by chemotherapy. Disclosures:No relevant conflicts of interest to declare.

ARC Suppresses Cell Death By Antagonizing p53 Function and Suppressing TRAIL In AML Cells

The apoptosis repressor with caspase recruitment domain (ARC) protein is a unique antiapoptotic protein as it can suppress the activation of both the intrinsic and extrinsic apoptosis pathways. ARC levels are prognostic in AML (Carter BZ et al., Blood 2011) and inhibition of ARC sensitizes leukemic cells to chemotherapy-, to the Bcl-2 inhibitor ABT-737-, and to TRAIL-induced apoptosis in AML (Mak PY et al., ASH 2012 abstract #893). Here we investigated the connection between p53 and TRAIL in the antiapoptotic function of ARC in AML.Confocal microscopic analysis of wild type p53 OCI-AML3 and Molm13 AML cells showed co-localization of p53 and ARC, suggesting possible functional interactions between the two proteins. Contrary to a previous report (Li YZ, Mol Cell Biol, 2008), the activation of p53 by nutlin3a did not decrease ARC levels in AML cells. This was confirmed by Western blot and RT-PCR analysis.ARC knockdown (KD) in OCI-AML3 and Molm13 cells resulted in increased expression of basal and nultin3a-induced p53 and p53 target proteins, suggesting that ARC inhibits p53 expression and antagonizes its function. Consistent with this finding, ARC KD in AML cells sensitized these cells to nutlin3a-induced cell death (EC50 for ARC KD and control OCI-AML3 cells was 2.06 ± 0.50 µM and 4.86 ± 0.05 µM, respectively, and for ARC KD and control Molm13 cells 0.59 ± 0.03 µM and 1.09 ± 0.05 µM, respectively, at 48 hours) (p<0.05).CRM1 is known to transport p53 from the nucleus into the cytoplasm and decrease p53 transcriptional activity (Kojima K et al., Blood 2013). DR5, the TRAIL receptor is also a known transcriptional target of p53. ARC also regulated the expression of CRM1 and TRAIL. CRM1 protein levels are lower in ARC KD cells than in their controls. Conversely, TRAIL mRNA levels were lower in ARC overexpressing (OE) and higher in ARC KD cells. The combination of nutlin3a and TRAIL synergistically induced cell death in OCI-AML3 and Molm13 cells cultured alone or co-cultured with mesenchymal stromal cells (MSCs) (CI < 0.1), and this combination induced statistically significantly (P < 0.05) more cell death in ARC KD cells than in controls even when cells were co-cultured with MSCs. Similarly, combinations of nutlin3a and ABT-737 or TRAIL and ABT-737 synergistically induced cell death in AML cells cultured alone or co-cultured with MSCs (CI < 0.1). Cell death was more pronounced in ARC KD cells than in their respective controls (P < 0.05). Furthermore, the combination of TRAIL and ABT-737 synergistically induced cell death in KG-1 cells, and the cell death was significantly reduced in ARC OE KG-1 than the control cells. ConclusionsThis study identifies novel mechanisms by which ARC suppresses cell death in AML cells. Specifically, ARC antagonizes p53 function by suppressing its expression and that of its target proteins and by increasing CRM1 levels, and ARC inhibits TRAIL expression. These findings suggest that combined p53 activation and ARC inhibition can augment apoptosis induction and sensitize AML cells to chemotherapeutic agents. Disclosures:No relevant conflicts of interest to declare.

Humoral control of cardiac remodeling in fish: Role of Angiotensin II

Angiotensin II (AngII), the principal effector of the Renin-Angiotensin-System (RAS), is a multipotent hormone whose biological actions include short-term modulation as well as long-term adjustments. In the eel heart, AngII elicits short-term inotropic and chronotropic effects. However, information regarding the influence of AngII on cardiac remodeling, expressed as morphological and hemodynamic changes, is lacking. To clarify the putative actions of AngII on eel cardiac remodeling, we used freshwater eels (Anguilla anguilla) intraperitoneally injected for 4weeks with saline or AngII (0.4 or 1.2nmolgBW−1) or AngII (1.2nmolgBW−1) plus the AT2 receptor antagonist CGP42112. Using an in vitro working heart preparation, the cardiac response (stroke volume changes) to preload and afterload increases has been evaluated. Hearts of all groups showed similar Frank–Starling responses. However, in response to afterload increases, stroke volume rapidly decreased in control hearts, while it was better maintained in AngII-treated counterparts. These effects were abolished by an antagonist of the AT2 receptor, whose cardiac expression was revealed by western blotting analysis. We also found by immunolocalization and immunoblotting that AngII influences both expression and localization of molecules which regulate cell growth [such as c-kit, heat shock protein 90 (Hsp-90), endothelial Nitric Oxide Synthase “(eNOS)-like” isoform] and apoptosis [i.e. apoptosis repressor with CARD domain (ARC)], thus playing a role in cardiac long-term adjustments. These results point to a role of AngII in eel heart remodeling, providing new insights regarding the modulation of cardiac plasticity in fish.

ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in vitro and in vivo

Abstract Abstract 893 ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival. We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors. To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively. Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively. In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. Download : Download high-res image (24KB) Download : Download full-size image Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures: No relevant conflicts of interest to declare.

HOX antisense lincRNA HOXA-AS2 is an apoptosis repressor in allTransretinoic acid treated NB4 promyelocytic leukemia cells

HOXA cluster antisense RNA 2 (HOXA-AS2) is a long non-coding RNA located between the HOXA3 and HOXA4 genes in the HOXA cluster. Its transcript is expressed in NB4 promyelocytic leukemia cells and human peripheral blood neutrophils, and expression is increased in NB4 cells treated with all trans retinoic acid (ATRA). Knockdown of HOXA-AS2 expression by transduced shRNA decreases the number of viable cells and increases the proportion of apoptotic cells, measured by annexin V binding and by activity and cleavage of caspases-3, -8, and -9. The increase in death of HOXA-AS2 knockdown cells was accompanied by an elevated TNF-related apoptosis-inducing ligand (TRAIL) levels, but ATRA-induced NB4 cells treated with TRAIL did show an increase in HOXA-AS2 expression. These results demonstrate that ATRA induction of HOXA-AS2 suppresses ATRA-induced apoptosis, possibly through a TRAIL-mediated pathway. HOXA-AS2-mediated negative regulation thus contributes to the fine-tuning of apoptosis during ATRA-induced myeloid differentiation in NB4 cells.