scholarly journals Negative regulation of TGFβ-induced apoptosis by RAC1B enhances intestinal tumourigenesis

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Victoria Gudiño ◽  
Patrizia Cammareri ◽  
Caroline V. Billard ◽  
Kevin B. Myant
Keyword(s):  
Ex Vivo ◽  
Tumour Progression ◽  
Small Gtpase ◽  
Tumour Invasion ◽  
Cytotoxic Effects ◽  
Tgfβ Signalling ◽  
Splice Isoform ◽  
Induced Apoptosis ◽  
Tumour Initiation

AbstractRAC1B is a tumour-related alternative splice isoform of the small GTPase RAC1, found overexpressed in a large number of tumour types. Building evidence suggests it promotes tumour progression but compelling in vivo evidence, demonstrating a role in driving tumour invasion, is currently lacking. In the present study, we have overexpressed RAC1B in a colorectal cancer mouse model with potential invasive properties. Interestingly, RAC1B overexpression did not trigger tumour invasion, rather it led to an acceleration of tumour initiation and reduced mouse survival. By modelling early stages of adenoma initiation we observed a reduced apoptotic rate in RAC1B overexpressing tumours, suggesting protection from apoptosis as a mediator of this phenotype. RAC1B overexpressing tumours displayed attenuated TGFβ signalling and functional analysis in ex vivo organoid cultures demonstrated that RAC1B negatively modulates TGFβ signalling and confers resistance to TGFβ-driven cell death. This work defines a novel mechanism by which early adenoma cells can overcome the cytostatic and cytotoxic effects of TGFβ signalling and characterises a new oncogenic function of RAC1B in vivo.

Next Generation XPO1 Inhibitor KPT-8602 for the Treatment of Drug-Resistant Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1818-1818 ◽  
Author(s):  
Joel G Turner ◽  
Jana L Dawson ◽  
Christopher L Cubitt ◽  
Erkan Baluglo ◽  
Steven Grant ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Ex Vivo ◽  
Single Agent ◽  
Resistant Cell ◽  
Drug Resistant ◽  
Newly Diagnosed ◽  
Induced Apoptosis ◽  
Drug Resistant Cell

Abstract Purpose Human multiple myeloma (MM) remains an incurable disease despite relatively effective treatments, including proteasome inhibitors, immunomodulator-based therapies, and high-dose chemotherapy with autologous stem cell rescue. New agents are needed to further improve treatment outcomes. In previous studies, we have shown that inhibitors of the nuclear export receptor XPO1, in combination with bortezomib, carfilzomib, doxorubicin, or melphalan, synergistically induced apoptosis in MM cells in vitro, in vivo and ex vivo without affecting non-myeloma cells. In early clinical trials, the oral, brain penetrating XPO1 inhibitor selinexor showed clear anti-myeloma activity however adverse events have been recorded, including nausea and anorexia. Our purpose was to investigate the use of oral KPT-8602, a novel small-molecule inhibitor of XPO1 with minimal brain penetration, which has been shown to have reduced toxicities in rodents and primates while maintaining potent anti-tumor effects. Experimental Procedures To test the efficacy of KPT-8602, we treated human MM cell lines (both parental and drug-resistant) with KPT-8602 ± currently used MM drugs, including bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, and VP-16. Human MM cell lines assayed included RPMI-8226 (8226), NCI-H929 (H929), U266, and MM1.S, PI-resistant 8226-B25 and U266-PSR, doxorubicin-resistant 8226-Dox6 and 8226-Dox40, and melphalan-resistant 8226-LR5 and U266-LR6 cell lines. MM cells (2-4x106 cells/mL) were treated for 24 hours with KPT-8602 (300 nM), followed by treatment with one of the listed anti-MM agents for an additional 24 hours. MM cells were then assayed for cell viability (CellTiter-Blue, Promega). In addition, cells were treated with KPT-8602 ± anti-MM agents concurrently for 20 hours and assayed for apoptosis by flow cytometry. In vivo testing was done in NOD/SCID-g mice by intradermal injection of U266 MM cells. Treatment started 2 weeks after tumor challenge with KPT-8602 (10 mg/kg) ± melphalan (1 or 3 mg/kg) 2X/week (Tuesday, Friday) or with KPT-8602 alone 5X weekly (10 mg/kg) (Monday-Friday). A parallel experiment was run using the clinical XPO1 inhibitor KPT-330 (selinexor). Ex vivo testing was performed on MM cells from newly diagnosed/relapsed patient bone marrow aspirates with KPT-8602 ± bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, or VP16. CD138+/light-chain+ cells were assayed for apoptosis by flow cytometry. Results Viability assay showed that KPT-8602 had low IC50values (~140 nM) as a single agent and functioned synergistically with bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16. (CI values < 1.0). This synergistic effect was less pronounced in myeloma cells when KPT-8602 was used in combination with dexamethasone or lenalidomide. KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 combination therapy also induced apoptosis in all MM cell lines tested, including drug-resistant cell lines, as shown by caspase 3 cleavage and flow cytometric analyses. NOD/SCID-gamma mouse tumor growth was reduced and survival increased in KPT-8602/melphalan-treated mice when compared to single-agent controls. In addition, mice treated with KPT-8602 5X weekly had significantly reduced tumor growth and increased survival when compared to 2X weekly drug administration. No toxicity was observed in KPT-8602-treated mice as determined by weight loss in both the 2X and 5X groups. In patient bone marrow biopsies, the combination of KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 was more effective than single agents at inducing apoptosis in CD138+/LC+ MM cells in both newly diagnosed and relapsed/refractory patient samples. Conclusions We found that the novel XPO1 inhibitor KPT-8602 sensitizes MM cells to bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 as shown by apoptosis in parental and drug-resistant cell lines and by cell viability assays. Sensitization was found to be synergistic. In addition, KPT-8602 was effective in treatment of human MM tumors in mice as a single agent or in combination with melphalan and was effective when combined with several MM drugs in MM cell lines and MM patient bone marrow aspirates. KPT-8602 may be a potential candidate for future clinical trials. Disclosures Shacham: Karyopharm: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties.

scholarly journals JAK/STAT pathway inhibition sensitizes CD8 T cells to dexamethasone-induced apoptosis in hyperinflammation

Blood ◽  
2020 ◽  
Vol 136 (6) ◽  
pp. 657-668 ◽  
Author(s):  
Lauren K. Meyer ◽  
Katherine C. Verbist ◽  
Sabrin Albeituni ◽  
Brooks P. Scull ◽  
Rachel C. Bassett ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Immune System Activation ◽  
Stat Signaling ◽  
Induced Apoptosis ◽  
Stat Pathway

Abstract Cytokine storm syndromes (CSS) are severe hyperinflammatory conditions characterized by excessive immune system activation leading to organ damage and death. Hemophagocytic lymphohistiocytosis (HLH), a disease often associated with inherited defects in cell-mediated cytotoxicity, serves as a prototypical CSS for which the 5-year survival is only 60%. Frontline therapy for HLH consists of the glucocorticoid dexamethasone (DEX) and the chemotherapeutic agent etoposide. Many patients, however, are refractory to this treatment or relapse after an initial response. Notably, many cytokines that are elevated in HLH activate the JAK/STAT pathway, and the JAK1/2 inhibitor ruxolitinib (RUX) has shown efficacy in murine HLH models and humans with refractory disease. We recently reported that cytokine-induced JAK/STAT signaling mediates DEX resistance in T cell acute lymphoblastic leukemia (T-ALL) cells, and that this could be effectively reversed by RUX. On the basis of these findings, we hypothesized that cytokine-mediated JAK/STAT signaling might similarly contribute to DEX resistance in HLH, and that RUX treatment would overcome this phenomenon. Using ex vivo assays, a murine model of HLH, and primary patient samples, we demonstrate that the hypercytokinemia of HLH reduces the apoptotic potential of CD8 T cells leading to relative DEX resistance. Upon exposure to RUX, this apoptotic potential is restored, thereby sensitizing CD8 T cells to DEX-induced apoptosis in vitro and significantly reducing tissue immunopathology and HLH disease manifestations in vivo. Our findings provide rationale for combining DEX and RUX to enhance the lymphotoxic effects of DEX and thus improve the outcomes for patients with HLH and related CSS.

scholarly journals Rac1 Inhibits Apoptosis in Human Lymphoma Cells by Stimulating Bad Phosphorylation on Ser-75

2004 ◽  
Vol 24 (14) ◽  
pp. 6205-6214 ◽  
Author(s):  
Baolin Zhang ◽  
Yaqin Zhang ◽  
Emily Shacter
Keyword(s):  
Cell Survival ◽  
Phosphorylation Site ◽  
Small Gtpase ◽  
Lymphoma Cells ◽  
Drug Induced ◽  
Chemotherapy Drugs ◽  
Human Lymphoma ◽  
Induced Apoptosis

ABSTRACT The small GTPase Rac1 has emerged as an important regulator of cell survival and apoptosis, but the mechanisms involved are not completely understood. In this report, constitutively active Rac1 is shown to stimulate the phosphorylation of the Bcl-2 family member Bad, thereby suppressing drug-induced caspase activation and apoptosis in human lymphoma cells. Rac1 activation leads to human Bad phosphorylation specifically at serine-75 (corresponding to murine serine-112) both in vivo and in vitro. Inhibition of constitutive and activated Rac1-induced Bad phosphorylation by a cell-permeable competitive peptide inhibitor representing this Bad phosphorylation site sensitizes lymphoma cells to drug-induced apoptosis. The data show further that endogenous protein kinase A is a primary catalyst of cellular Bad phosphorylation in response to Rac activation, while Akt is not involved. These findings define a mechanism by which active Rac1 promotes lymphoma cell survival and inhibits apoptosis in response to cancer chemotherapy drugs.

scholarly journals PIDDosome Expression and the Role of Caspase-2 Activation for Chemotherapy-Induced Apoptosis in RCCs

2010 ◽  
Vol 32 (1-2) ◽  
pp. 29-42
Author(s):  
Sebastian Heikaus ◽  
Igor Pejin ◽  
Helmut Erich Gabbert ◽  
Uwe Ramp ◽  
Csaba Mahotka
Keyword(s):  
Tumour Progression ◽  
Chemotherapy Resistance ◽  
Clear Cell Type ◽  
Alternatively Spliced ◽  
Caspase 2 ◽  
Induced Apoptosis ◽  
Few Data

Background: The importance of caspase-2 activation for mediating apoptosis in cancer is not clear and seems to differ between different tumour types. Furthermore, only few data have been obtained concerning the expression of caspase-2, which can be alternatively spliced into caspase-2L and caspase-2S, and the other PIDDosome members PIDD and RAIDD in human tumours in vivo. We, therefore, investigated their expression in renal cell carcinomas (RCCs) of the clear cell type in vivo and analysed the role of caspase-2 in chemotherapy-induced apoptosis in RCCs in vitro.Methods: The analyses were performed by semiquantitative real-time PCR, Western Blot and Caspase-2 Assay.Results: Our in vivo results showed an overall decrease in proapoptotic caspase-2L expression during tumour progression due to an increase in the relative share of caspase-2S mRNA in total caspase-2 mRNA expression. Furthermore, an increase in the expression of PIDD and RAIDD could be observed. In contrast, antiapoptotic BCL-2 expression increased only during early tumour stages, whereas expression decreased in pT3 RCCs. In vitro, caspase-2 activation in RCC cell lines coincidenced with sensitivity of tumour cells towards Topotecan-induced apoptosis. However, inhibition of caspase-2 could not prevent Topotecan-induced apoptosis. Interestingly, Topotecan-resistance could be overcome by the apoptosis-sensitizing drug HA14-1.Conclusions: Our study confirms the concept of a shift towards a more antiapoptotic transcriptional context during tumour progression in RCCs. Furthermore, it shows that caspase-2 participates in chemotherapy-induced apoptosis in RCCs although it is not mandatory for it. Additionally, inhibition of antiapoptotic BCL-2 family members might provide a possible way to overcome chemotherapy resistance of RCCs.

Abstract 448: The Loss of Rad GTPase Protects Against Infarction-induced Myocardial Remodeling, Scar Formation, and Decompensatory Dilation

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Janet R Manning ◽  
Bryana M Levitan ◽  
Ayman Haroun ◽  
Catherine N Withers ◽  
Prabhakara R Nagareddy ◽  
...  
Keyword(s):  
Contractile Function ◽  
Ex Vivo ◽  
Neutrophil Infiltration ◽  
Left Ventricular ◽  
Small Gtpase ◽  
Calcium Handling ◽  
Myocardial Inflammation ◽  
Loss Of Function ◽  
Ventricular Dilation

Rationale: Myocardial infarction (MI) is a leading cause of death in the U.S. A non-contractile infarct compromises the overall mechanical function of the heart, reducing cardiac output and triggering decompensatory ventricular dilation. Rad GTPase, a member of the small GTPase RGK (Rem, Gem, Kir) family, is a calcium channel blocker that is endogenously expressed in the myocardium. We have previously shown that Rad deletion in mice results in increased Ca 2+ handling and a sustained non-pathological improvement in left ventricular function compared to wildtype. Hypothesis: Rad-ablation attenuates post-ischemic loss of function, resulting in reduced remodeling and improved long-term contractility. Methods and Results: We subjected Rad-deficient mice to ligation of the left anterior descending (LAD) coronary artery, and monitored cardiac function using echocardiography. We found that Rad deletion reduces both mortality and contractile dysfunction after MI, as well as ventricular dilation over five weeks. This improvement is also accompanied by preserved calcium handling in isolated myocytes. Histological and MRI examination of both ex vivo global ischemia and in vivo 24 hour LAD ligated myocardium revealed that initial infarct size is comparable between knockout and wildtype. We found that Rad loss reduced scar development and elongation independent of preserving tissue viability. Investigation of inflammatory pathways to account for this revealed increased expression of the anti-inflammatory protein thrombospondin accompanied by a reduction in neutrophil infiltration into the myocardium after MI. Conclusion: Rad deletion results in reduced cardiac remodeling, diminished myocardial inflammation, and improved contractile function after MI.

Abstract 15: Novel Functions of Small GTPase Rap1 in Regulating Endothelial Homeostasis: Control of Nitric Oxide Release, Vascular Function and Blood Pressure

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Sribalaji Lakshmikanthan ◽  
Xiaodong Zheng ◽  
Yoshinori Nishijima ◽  
Jeannette Vasquez-Vivar ◽  
David X Zhang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Shear Flow ◽  
Knockout Mice ◽  
Ex Vivo ◽  
Left Ventricular ◽  
Small Gtpase ◽  
No Production ◽  
Endothelial Homeostasis

Endothelial dysfunction, resulting from decreased nitric oxide (NO) bioavailability is a pathology linked to endothelial vasomotor dysfunction and hypertension, inflammation and atherosclerosis, perturbed endothelial barrier and progression of diabetes. In blood vessels, NO is produced by the endothelial NO synthase (eNOS), the activity of which is regulated by Ca2+/calmodulin, binding of regulatory cofactors, and posttranslational modifications, including phosphorylation events on Ser1177, which stimulate NO production. Rap1 is a ubiquitously expressed small GTPase implicated in promoting vascular barrier. We have shown that endothelial cell (EC)-specific Rap1 deletion leads to defective angiogenesis in vivo due to faulty VEGFR2 activation and signaling. Importantly, EC-specific Rap1 knockout mice developed hypertension and pathological left ventricular hypertrophy. The objective of the study was to determine the role of small G protein Rap1 in regulating endothelial NO production and endothelial-dependent vasorelaxation in vivo and ex vivo. Using ex vivo myography and tamoxifen-inducible, endothelial-specific Rap1-knockout mice (Cadh5-CreERT2+/0;Rap1f/f), we demonstrate that Rap1 deficiency completely abrogates NO-dependent vasodilation and attenuates NO production. Mechanistically, we show that Rap1 is rapidly activated in response to receptor agonists that activate eNOS via Ca2+/calmodulin- dependent pathway and in response to shear flow, which modules eNOS activity by its phosphorylation. Rap1 deletion in human ECs, in vitro, leads to deficient NO release in response to both these stimuli, and interferes with PI3K/Akt pathway and eNOS Ser1177 phosphorylation. Further, we demonstrate Rap1 is required for transducing signals from the endothelial mechanosensing complex comprising PECAM-1, VE-cadherin and VEGFR2 in response to shear flow, leading to ligand-independent VEGFR2 activation and signaling to stimulate NO production. We conclude that Rap1 in endothelium is critically required for endothelial homeostasis and NO production, thereby affecting vascular tone and regulation of blood pressure. Furthermore, this study establishes Rap1 as a novel regulator of mechanotransduction in response to shear flow.

scholarly journals TMIC-27. GLUTAMATERGIC NEURON-GLIOMA SYNAPSES DRIVE BRAIN TUMOUR PROGRESSION

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi253-vi253
Author(s):  
Varun Venkataramani ◽  
Dimitar Tanev ◽  
Christopher Strahle ◽  
Alexander Studier-Fischer ◽  
Laura Fankhauser ◽  
...  
Keyword(s):  
Glutamate Receptors ◽  
Brain Tumour ◽  
Neuronal Activity ◽  
Ex Vivo ◽  
Tumour Progression ◽  
Glioma Cells ◽  
Tumour Cells ◽  
Glioma Invasion ◽  
Synaptic Ultrastructure

Abstract A network of communicating tumour cells established by tumour microtubes (TMs) is supposed to mediate relevant aspects of progression and resistance of incurable gliomas. Moreover, neuronal activity has been shown to foster malignant behavior of glioma cells by non-synaptic paracrine and autocrine mechanisms. Here, we report an unexpected direct communication channel between neurons and glioma cells in multiple disease models as well as in astrocytomas and glioblastomas (GBs) of adult patients: functional bona fide chemical synapses formed between presynaptic neurons and postsynaptic glioma cells. These neurogliomal synapses (NGS) show a typical synaptic ultrastructure, are located on TM networks, and produce depolarizing postsynaptic currents mediated by glutamate receptors of the AMPA subtype. AMPA-type glutamate receptors (AMPAR) are expressed by a molecularly and morphologically distinct subpopulation of network-integrated glioma cells. Increased neuronal activity under epileptic conditions ex vivo or neuronal optogenetic stimulation in vivo enhanced, while general anesthesia diminished synchronized calcium transients in TM-connected glioma networks. Accordingly, anesthesia reduced invasiveness of TM-positive tumour cells in mice. Genetic perturbation of AMPAR or chronic AMPAR inhibition by perampanel decreased glioma invasion and proliferation in mice and deletion of GluRII in Drosophila glioma increased survival. These findings reveal a hitherto unappreciated direct synaptic communication between neurons and glioma cells that appears relevant for brain tumour biology, implying new avenues for glioma treatment.

scholarly journals Identification of a novel 82 kDa proMMP-9 species associated with the surface of leukaemic cells: (auto-)catalytic activation and resistance to inhibition by TIMP-1

2007 ◽  
Vol 405 (3) ◽  
pp. 547-558 ◽  
Author(s):  
Christian Ries ◽  
Thomas Pitsch ◽  
Reinhard Mentele ◽  
Stefan Zahler ◽  
Virginia Egea ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Ex Vivo ◽  
Tumour Progression ◽  
Critical Role ◽  
Active Form ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Collagen Type Iv ◽  
Leukaemic Cells ◽  
Ic50 Values

MMP-9 (matrix metalloproteinase 9) plays a critical role in tumour progression. Although the biochemical properties of the secreted form of proMMP-9 are well characterized, little is known about the function and activity of cell surface-associated proMMP-9. We purified a novel 82 kDa species of proMMP-9 from the plasma membrane of THP-1 leukaemic cells, which has substantial differences from the secreted 94 kDa proMMP-9. The 82 kDa form was not detected in the medium even upon stimulation with a phorbol ester. It is truncated by nine amino acid residues at its N-terminus, lacks O-linked oligosaccharides present in the 94 kDa proMMP-9, but retains N-linked carbohydrates. Incubation of 94 kDa proMMP-9 with MMP-3 generated the well-known 82 kDa active form, but the 82 kDa proMMP-9 was converted into an active species of 35 kDa, which was also produced by autocatalytic processing in the absence of activating enzymes. The activated 35 kDa MMP-9 efficiently degraded gelatins, native collagen type IV and fibronectin. The enzyme was less sensitive to TIMP-1 (tissue inhibitor of metalloproteinase 1) inhibition with IC50 values of 82 nM compared with 1 nM for the 82 kDa active MMP-9. The synthetic MMP inhibitor GM6001 blocked the activity of both enzymes, with similar IC50 values below 1 nM. The 82 kDa proMMP-9 is also produced in HL-60 and NB4 leukaemic cell lines as well as ex vivo leukaemic blast cells. It is, however, absent from neutrophils and mononuclear cells isolated from peripheral blood of healthy individuals. Thus, the 82 kDa proMMP-9 expressed on the surface of malignant cells may escape inhibition by natural TIMP-1, thereby facilitating cellular invasion in vivo.

scholarly journals Cell-based approaches in drug development – a concise review

2020 ◽  
Vol 8 (1) ◽  
pp. 44-49
Author(s):  
Ievgeniia Kocherova ◽  
Bartosz Kempisty ◽  
Greg Hutchings ◽  
Lisa Moncrieff ◽  
Claudia Dompe ◽  
...  
Keyword(s):  
Drug Development ◽  
Development Process ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Cytotoxic Effects ◽  
Safety Issues ◽  
Third Phase ◽  
Concise Review

AbstractIn vitro models represent an alternative technique to in vivo or ex vivo studies in the drug development process. Cell-based assays are used to measure the level of proliferation and toxicity, as well as activation of signalling pathways and changes in morphology in cultivated cells. The studies conducted in vitro are aimed to estimate the newly synthesised drugs’ ability to permeate biological barriers and exert their therapeutic or cytotoxic effects. However, more than half of all studied drugs fail in the second or third phase of clinical trials due to a lack of confirmed efficacy. About a third of drugs fail because of safety issues, such as unacceptable levels of toxicity. To reduce attrition level in drug development, it is crucial to consider the implementation of translational phenotypic assays as well as to decipher various molecular mechanisms of action for new molecular entities. In this review, we summarise the existing cell-based methods most frequently used in the studies on drugs, taking into account their advantages and drawbacks.Running title: Cell-based approaches in drug development

scholarly journals Role of RIPK1 in SMAC mimetics-induced apoptosis in primary human HIV-infected macrophages

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ramon Edwin Caballero ◽  
Simon Xin Min Dong ◽  
Niranjala Gajanayaka ◽  
Hamza Ali ◽  
Edana Cassol ◽  
...  
Keyword(s):  
Cell Death ◽  
Hiv Infection ◽  
Ex Vivo ◽  
Laboratory Strain ◽  
Interacting Protein ◽  
Heat Stable Antigen ◽  
Smac Mimetics ◽  
Induced Apoptosis

AbstractMacrophages serve as viral reservoirs due to their resistance to apoptosis and HIV-cytopathic effects. We have previously shown that inhibitor of apoptosis proteins (IAPs) confer resistance to HIV-Vpr-induced apoptosis in normal macrophages. Herein, we show that second mitochondrial activator of caspases (SMAC) mimetics (SM) induce apoptosis of monocyte-derived macrophages (MDMs) infected in vitro with a R5-tropic laboratory strain expressing heat stable antigen, chronically infected U1 cells, and ex-vivo derived MDMs from HIV-infected individuals. To understand the mechanism governing SM-induced cell death, we show that SM-induced cell death of primary HIV-infected macrophages was independent of the acquisition of M1 phenotype following HIV infection of macrophages. Instead, SM-induced cell death was found to be mediated by IAPs as downregulation of IAPs by siRNAs induced cell death of HIV-infected macrophages. Moreover, HIV infection caused receptor interacting protein kinase-1 (RIPK1) degradation which in concert with IAP1/2 downregulation following SM treatment may result in apoptosis of macrophages. Altogether, our results show that SM selectively induce apoptosis in primary human macrophages infected in vitro with HIV possibly through RIPK1. Moreover, modulation of the IAP pathways may be a potential strategy for selective killing of HIV-infected macrophages in vivo.

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