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Background: This study set out to investigate the antitumor effects of a treatment strategy combining the mTOR inhibitor CCI-779 with cisplatin in vitro and in a human melanoma SCID mouse model. Methods: In vitro 518A2, Mel-JUSO or 607B cell lines were incubated with CCI-779, cisplatin and CCI-779 plus cisplatin. Based on these results, a 4-group, parallel, controlled experimental study design was initiated in vivo. SCID mice were injected with melanoma cells, and after the development of tumors the mice received daily injections of CCI-779 or solvent. On treatment days 2 and 6 cisplatin or mock solution were administered. Results: In vitro a synergistic antitumor effect was observed for the treatment regimen of CCI-779 plus cisplatin. In SCID mice after 2 weeks of therapy with CCI-779 plus cisplatin 4 of 6 tumors of the 518A2 cell line were completely eradicated. In the two remaining 518A2 xenografts this treatment strategy reduced the tumor weight by 94 ± 9% compared to solvent. CCI-779 plus cisplatin also exerted a significant antitumor effect in Mel-JUSO and 607B xenografts compared to mock-treated animals. Conclusion: We provide circumstantial evidence that the use of CCI-779 plus cisplatin might qualify as a promising strategy in the treatment of human melanoma. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The epidermal growth factor receptor (EGFR) is a validated target in squamous cell carcinoma (SCC) of the head and neck. Most patients, however, do not respond or develop resistance to this agent. Mammalian target of rapamycin (mTOR) is involved in the pathogenesis of SCC of the head and neck (SCCHN). This study aimed to determine if targeting mTOR in combination with EGFR is effective in SCC, and to develop early pharmacodynamic markers of efficacy. Two SCC cell lines, one resistant (HEP2) and one of intermediate susceptibility (Detroit 562) to EGFR inhibitors, were xenografted in vivo and treated with an mTOR inhibitor (temsirolimus), an EGFR inhibitor (erlotinib) or a combination of both. Temsirolimus exerted superior growth arrest in both cell lines than erlotinib. The combined treatment resulted in synergistic antitumor effects in the Detroit 562 cell line. Immunohistochemical assessment of pharmacodynamic effects in fine-needle aspiration (FNA) biopsies early after treatment using phospho MAPK, Phospho-P70 and Ki67 as end points demonstrated pathway abrogation in the Detroit 562 tumours treated with the combination, the only group where regressions were seen. In conclusion, an mTOR inhibitor showed antitumor activity in EGFR-resistant SCC cell lines. Marked antitumor effects were associated with dual pathway inhibition, which were detected by early FNA biopsies. [ABSTRACT FROM AUTHOR]

Sirolimus is a mammalian target of rapamycin (mTOR) inhibitor that inhibits cell cycle progression and has proven to be a potent immunosuppressive agent for use in solid organ transplant recipients. The drug was initially studied as an adjunct to ciclosporin (cyclosporine) to prevent acute rejection in kidney transplant recipients. Subsequent studies have shown efficacy when combined with a variety of other immunosuppressive agents. The most common adverse effects of sirolimus are hyperlipidaemia and myelosuppression. The drug has unique antiatherogenic and antineoplastic properties, and may promote immunological tolerance and reduce the incidence of chronic allograft nephropathy. Although sirolimus is relatively non-nephrotoxic when administered as monotherapy, it pharmacodynamically enhances the toxicity of calcineurin inhibitors. Ironically, the drug has been used to facilitate calcineurin inhibitor-free protocols designed to preserve renal function after solid organ transplantation. Whether sirolimus can be used safely over the long term with low doses of calcineurin inhibitors requires further study. The use of sirolimus as a corticosteroid-sparing agent also remains to be proven in controlled trials. Postmarketing studies have revealed a number of unforeseen adverse effects including impaired wound healing and possibly proteinuria, oedema, pneumonitis and thrombotic microangiopathy. Overall, sirolimus is a powerful agent when used judiciously with other available immunosuppressants. As is true for all immunosuppressive drugs available for treatment of solid organ transplant recipients, the efficacy of the drug must be balanced against its considerable adverse effects. [ABSTRACT FROM AUTHOR]

Immunosuppressive calcineurin inhibitors, like cyclosporine (CsA), can be used for the clinical management of severe ulcerative colitis. However, patients treated with CsA are at a risk for developing kidney failure and may be more susceptible to colon cancer. Furthermore, severe neurotoxicity and hypertension are common problems. To avoid the side effects of CsA, new immunosuppressive drugs to treat colitis are needed. The aim of the present study was to test the immunosuppressive mammalian target of rapamycin inhibitor rapamycin in an experimental model of chronic colitis and to compare its effectiveness with CsA. Chronic colitis was established in Balb/c mice after four feeding cycles of dextran sodium sulfate. Because leukocyte recruitment to sites of intestinal inflammation is crucial for the development of chronic colitis, intravital microscopy was used to study the effect of rapamycin and CsA on leukocyte–endothelium interactions and leukocyte extravasation. To assess the degree of colitis, histological sections were evaluated. Both rapamycin and cyclosporine effectively reduced leukocyte sticking (>60%) in submucosal venules, as compared to controls. Furthermore, rapamycin, but not CsA, reduced (>35%) leukocyte extravasation in the mucosa. Both rapamycin and CsA treatments significantly improved the histologic inflammation score. Our in vivo results demonstrate that rapamycin reduces leukocyte sticking and extravasation during chronic colitis induction and proves to be as effective as CsA at reducing experimental chronic colitis. These results support the use of rapamycin in clinical trials to avoid serious side effects of CsA therapy in chronic colitis patients. [ABSTRACT FROM AUTHOR]

Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), inhibits tumor cell motility. However, the underlying mechanism is poorly understood. Here, we show that rapamycin inhibited type I insulin-like growth factor (IGF-I)-stimulated motility of a panel of cell lines. Expression of a rapamycin-resistant mutant of mTOR (mTORrr) prevented rapamycin inhibition of cell motility. However, cells expressing a kinase-dead mTORrr remained sensitive to rapamycin. Downregulation of raptor or rictor by RNA interference (RNAi) decreased cell motility. However, only downregulation of raptor mimicked the effect of rapamycin, inhibiting phosphorylation of S6 kinase 1 (S6K1) and 4E-BP1. Cells infected with an adenovirus expressing constitutively active and rapamycin-resistant mutant of p70 S6K1, but not with an adenovirus expressing wild-type S6K1, or a control virus, conferred to resistance to rapamycin. Further, IGF-I failed to stimulate motility of the cells, in which S6K1 was downregulated by RNAi. Moreover, downregulation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) by RNAi-attenuated rapamycin inhibition of cell motility. In contrast, expression of constitutively active 4E-BP1 dramatically inhibited IGF-I-stimulated cell motility. The results indicate that both S6K1 and 4E-BP1 pathways, regulated by TORC1, are required for cell motility. Rapamycin inhibits IGF-I-stimulated cell motility, through suppression of both S6K1 and 4E-BP1/eIF4E-signaling pathways, as a consequence of inhibition of mTOR kinase activity.Oncogene (2006) 25, 7029–7040. doi:10.1038/sj.onc.1209691; published online 22 May 2006 [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma (KS) is a recognised complication following kidney transplantation, but the incidence varies according to the geographical area. Although it is a rare tumour, its incidence increases dramatically after solid-organ transplantation. The immunosuppressive medications reactivate human herpes virus 8, which has been proposed as the offending agent. The usual treatment of KS is to reduce immunosuppression, chemotherapy and radiotherapy. Nevertheless, the mortality still remains considerably high and has been reported between 8 and 14%. Sirolimus (SRL) has properties which may be useful in the management of some post-transplant tumours such as KS. We report a renal transplant patient with KS, who had multiple relapses after radiotherapy but responded well to the change of immunosuppression from cyclosporine to SRL. [ABSTRACT FROM AUTHOR]

The serine/threonine kinase, mTOR (mammalian Target of Rapamycin) has become a focus for cancer drug development. Rapamycins are highly specific inhibitors of mTOR and potently suppress tumour cell growth by retarding cells in G1 phase or potentially inducing apoptosis. Currently, both rapamycin and several analogues are being evaluated as anticancer agents in clinical trials. Results indicate that many human cancers have intrinsic resistance and tumours initially sensitive to rapamycins become refractory, demonstrating acquired resistance. Here, we consider mechanisms of resistance to inhibitors of mTOR.British Journal of Cancer (2006) 95, 955–960. doi:10.1038/sj.bjc.6603353 www.bjcancer.com Published online 5 September 2006 [ABSTRACT FROM AUTHOR]

Proteins regulating the mammalian target of rapamycin (mTOR), as well as some of the targets of the mTOR kinase, are overexpressed or mutated in cancer. Rapamycin, the naturally occurring inhibitor of mTOR, along with a number of recently developed rapamycin analogs (rapalogs) consisting of synthetically derived compounds containing minor chemical modifications to the parent structure, inhibit the growth of cell lines derived from multiple tumor types in vitro, and tumor models in vivo. Results from clinical trials indicate that the rapalogs may be useful for the treatment of subsets of certain types of cancer. The sporadic responses from the initial clinical trials, based on the hypothesis of general translation inhibition of cancer cells are now beginning to be understood owing to a more complete understanding of the dynamics of mTOR regulation and the function of mTOR in the tumor microenvironment. This review will summarize the preclinical and clinical data and recent discoveries of the function of mTOR in cancer and growth regulation.Oncogene (2006) 25, 6436–6446. doi:10.1038/sj.onc.1209886 [ABSTRACT FROM AUTHOR]

The Edmonton Protocol for islet transplantation has provided hope for type 1 diabetic patients. However, this protocol requires lifelong immunosuppression, specifically sirolimus, a cellular antiproliferate. The effect of sirolimus on human pancreatic ductal cells (HDCs) is not known. This may be important since HDCs are believed to be islet precursors. Since neonatal porcine islets (NPIs), which contain many ductal precursor cells, could be a potential clinical source of islets, we also tested the effects of sirolimus on this tissue. HDCs ( n=4), NPIs ( n=9) and human islets ( n=5) were cultured with and without sirolimus (20 ng/ml) for 6 days. HDCs and NPIs cultured with sirolimus showed a 50 and 28% decrease, respectively, in cell number relative to control ( p<0.05). Control cultures expanded 1.65- and 2.44-fold relative to time 0. Decreases in cell number of sirolimus-treated HDCs were not due to apoptosis as measured by TUNEL staining. No functional effects on human islets or NPIs were observed following static incubation with high glucose. Treatment of syngeneically transplanted and naïve BALC/c mice with sirolimus resulted in altered OGTT profiles with prolonged elevation of hyperglycaemia and weight gain. There was no difference in graft and organ insulin content between treatment groups. Our results indicate that sirolimus decreases ductal cell numbers in culture and alters glucose-stimulated insulin secretion in vivo. The administration of sirolimus to islet transplant recipients is likely to impair graft function as a result of decreasing ductal neogenesis and induction of insulin resistance. [ABSTRACT FROM AUTHOR]

Background and purpose:Rapamycin is a widely used drug with antifungal, immunosuppressant and antiangiogenic effects. Herein, we studied whether immunosuppressive doses of rapamycin are capable of influencing endometriotic lesions.Experimental approach:We tested in vitro the potential of rapamycin to inhibit endothelial cell sprouting using the aortic ring assay and we further studied its effect on the expression of proliferating cell nuclear antigen (PCNA), apoptotic cell death-associated activated caspase-3 and vascular endothelial growth factor (VEGF) in cultured endometrial tissue fragments. In addition, we analyzed the drug in vivo after induction of endometriotic lesions by transplanting isolated endometrial fragments into the dorsal skinfold chamber of Syrian golden hamsters. Using intravital fluorescence microscopy, we repetitively analyzed angiogenesis, neovascularization and microcirculatory parameters over a time period of 14 days in rapamycin-treated animals and DMSO-treated controls.Key results:Administration of rapamycin significantly reduced the size of the endometriotic lesions. This was associated by inhibition of VEGF-mediated angiogenesis as indicated by a suppression of endothelial cell sprouting in vitro and a reduction of microvessel density in endometriotic lesions in vivo. Moreover, rapamycin directly inhibited cell proliferation within endometrial tissue, while manifestation of apoptotic cell death remained unaffected.Conclusions and implications:Our data indicate that administration of rapamycin may represent a novel therapeutic approach for an antiangiogenic treatment of endometriosis.British Journal of Pharmacology (2006) 149, 137–144. doi:10.1038/sj.bjp.0706857; published online 7 August 2006 [ABSTRACT FROM AUTHOR]

mTOR (mammalian target of rapamycin) has come a long way since its humble beginnings as a kinase of unknown function. As part of the mTORC1 and mTORC2 complexes mTOR has key roles in several pathways that are involved in human cancer, stimulating interest in mTOR inhibitors and placing it on the radar of the pharmaceutical industry. Here, I discuss the rationale for the use of drugs that target mTOR, the unexpectedly complex mechanism of action of existing mTOR inhibitors and the potential benefits of developing drugs that function through different mechanisms. The purpose is not to cover all aspects of mTOR history and signalling, but rather to foster discussion by presenting some occasionally provocative ideas. [ABSTRACT FROM AUTHOR]

Mammalian target of rapamycin (mTOR) is a kinase that functions as a master switch between catabolic and anabolic metabolism and as such is a target for the design of anticancer agents. The most established mTOR inhibitors--rapamycin and its derivatives--showed long-lasting objective tumour responses in clinical trials, with CCI-779 being a first-in-class mTOR inhibitor that improved the survival of patients with advanced renal cell carcinoma. This heralded the beginning of extensive clinical programmes to further evaluate mTOR inhibitors in several tumour types. Here we review the clinical development of this drug class and look at future prospects for incorporating these agents into multitarget or multimodality strategies against cancer. [ABSTRACT FROM AUTHOR]

Kaposi’s sarcoma (KS) is an angioproliferative neoplasia associated with human herpesvirus 8 (HHV-8) infection. HHV-8 generates KS by means of the secretion of vascular endothelial growth factor (VEGF) andup-regulation of VEGF receptor, KDR, in endothelial cells. We report a case of KS in a 72-year-old male with a renal transplant who had received immunosuppressant drugs including sirolimus, mycophenolate mofetil, tacrolimus and steroids. KS developed 11 months after transplantation, in relation to deep venous thrombosis and withdrawal of sirolimus due to toxicity. Multiple purple papules and nodules were observed exclusively in the limb affected by thrombosis. Diagnosis of KS was confirmed by biopsy. Progressive withdrawal of prednisone was accompanied by full remission of the tumour. The thrombosis and withdrawal of sirolimus may have acted as cofactors in the development of KS, favouring the activation of the VEGF/KDR autocrine loop. Our experience contributes to further evidence that sirolimus may protect against KS. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The aim of this investigation is to determine whether rapamycin treatment has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days in culture, attached cells were stimulated with rapamycin (in a series of final concentrations: 0.1, 1.0, 2.0 and 5.0 g/ml) for 6, 12, 24 and 48 h. EPCs were characterized as adherent cells, double positive for DiLDL uptake and lectin binding by direct fluorescence staining. EPC proliferation and migration were determined using the MTT assay and a modified version of the Boyden chamber assay, respectively. An EPC adhesion assay was performed by replating the cells on fibronectin-coated dishes; adherent cells were then counted. Tube formation activity was assayed by using a tube formation assay kit and endothelial nitric oxide synthase (eNOS) was assayed by Western blot analysis. Incubation of isolated human MNCs with rapamycin decreased the number of EPCs present; rapamycin also decreased EPCs proliferative, migratory, adhesive, tube formation capacity and eNOS production in a concentration- and time-dependent manner. Rapamycin was found to decrease the number, proliferative, migratory, adhesive and tube formation capacities of the EPCs, and also was found to decreases eNOS in the EPCs. [ABSTRACT FROM AUTHOR]

Inhibitors of the kinase mammalian target of rapamycin (mTOR) have shown sporadic activity in cancer trials, leading to confusion about the appropriate clinical setting for their use. Here we show that loss of the Von Hippel-Lindau tumor suppressor gene (VHL) sensitizes kidney cancer cells to the mTOR inhibitor CCI-779 in vitro and in mouse models. Growth arrest caused by CCI-779 correlates with a block in translation of mRNA encoding hypoxia-inducible factor (HIF1A), and is rescued by expression of a VHL-resistant HIF1A cDNA lacking the 5′ untranslated region. VHL-deficient tumors show increased uptake of the positron emission tomography (PET) tracer fluorodeoxyglucose (FDG) in an mTOR-dependent manner. Our findings provide preclinical rationale for prospective, biomarker-driven clinical studies of mTOR inhibitors in kidney cancer and suggest that FDG-PET scans may have use as a pharmacodynamic marker in this setting. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the synergistic antiangiogenic effect of low dose of vinblastine (VBL) and rapamycin (RAP) in neuroblastoma (NB). Both in vitro (endothelial cells proliferation assay; TUNEL assay; phosphatidylserine exposure and cell cycle analysis) and in vivo (chick embryo chorioallantoic membrane, CAM) assays were used. Each compound alone was able to induce a significant dose- and time-response inhibition of in vitro endothelial cells (EC) growth. Interaction index evaluation indicates that a synergistic effect was found when both drugs were combined at very low doses. Comparable effects were obtained when EC were preincubated with conditioned medium (CM) derived from the human NB cell line HTLA-230. Morphological changes were induced by each drug, and their combination resulted in a clear and stronger effect. Apoptosis was demonstrated by the TUNEL assay and confirmed by Annexin V-FITC staining of EC treated with VBL, showing an increase in the percentage of cells with a G2–M and sub-G1 DNA content, whereas in those treated with RAP a block in the G1 cell fraction and inhibition of progression to the S phase were observed. Here too, the combination resulted in a synergistic cell cycle arrest and induction of apoptosis. Similar results were obtained in vivo with the CAM assay. The angiogenic responses induced by HTLA-230-derived CM, NB tumor xenografts, and human NB biopsy specimens were inhibited by each drug and more significantly by their combination. The observation that these well-known drugs display synergistic effects as antiangiogenics when administered frequently at very low dose may be of significance in the designing of new ways of treating NB.Oncogene (2005) 24, 6785–6795. doi:10.1038/sj.onc.1208829; published online 11 July 2005 [ABSTRACT FROM AUTHOR]

It is known that radiation activates the phosphoinositol-3 kinase (PI3K)/Akt pathway and that inhibition of PI3K or Akt sensitizes tumor vasculature to radiotherapy. Mammalian target of rapamycin (mTOR) is a downstream target of Akt, and we hypothesized that irradiation activates mTOR signaling in both glioma and endothelial cells (ECs) and that radiosensitization results from inhibiting mTOR signaling. mTOR inhibitors, rapamycin and RAD001 (everolimus) were found to radiosensitize vascular ECs, but failed to sensitize glioma cells as determined by clonogenic assay. Therefore, we investigated the anti-angiogenic effects of mTOR inhibitors. Increased phospho-mTOR protein was detected in irradiated human umbilical vein endothelial cells (HUVEC), but not in GL261 glioma cells. Phospho-S6, a biomarker for mTOR signaling, was also found to be induced following irradiation in HUVEC and this effect was inhibited by PI3K or mTOR inhibitors. Significant increase in cleaved caspase 3 was detected when Rad001 was combined with radiation. Endothelial tube formation was significantly diminished following treatment with rapamycin and 3 Gy of radiation. Histological sections of GL261 tumors from mice showed a greatly reduced vascular density when treated with RAD001 and radiation. Power Weighted Doppler of glioma xenografts in mice showed a significant reduction in vasculature and blood flow compared with mice treated with 3 Gy or RAD001 alone. We conclude that irradiation activates mTOR signaling in vascular endothelium and that rapamycin and RAD001 increased apoptosis of ECs in response to radiation. To the authors' best knowledge this is the first study which demonstrates that mTOR inhibitors may be a way to target the vasculature by radiosensitizing the vascular endothelium resulting in better tumor control as seen in experiments demonstrating increased tumor growth delay in mice treated with rapamycin with radiation compared with mice treat with either treatment alone. We conclude that mTOR inhibitors have increased efficacy as antiangiogenics when combined with radiation.Oncogene (2005) 24, 5414–5422. doi:10.1038/sj.onc.1208715; published online 23 May 2005 [ABSTRACT FROM AUTHOR]

Non-myeloablative conditioning has extended the use of allogeneic haematopoietic transplant to many previously ineligible patients. We added the immunosuppressive and antitumour agent sirolimus (rapamycin) to an established transplant regimen of fludarabine 25 mg/m2 days −7 through −3 and cyclophosphamide 1000 mg/m2 days −7 and −6, with tacrolimus and methotrexate immunoprophyllaxis. A total of 23 patients with acute myelogenous leukaemia (AML) were treated, with a median age of 59 years (range: 28–72) at transplant. Only seven patients in total were in complete remission prior to transplantation. Nine patients were in chemotherapy-refractory progression and seven were primarily refractory to induction therapy. Six patients received matched sibling, 11 unrelated donor, 1–5/6 matched and five haploidentical (haplo – three of six or four of six matched) transplants. The haplo-recipients also received antithymocyte globulin, all patients engrafted. Only two, both recipients of haploidentical cells, have died from transplant-related causes. Twelve of 23 patients survived at 198–1162-d post-transplant (median 578). Four of 12 survivors relapsed at 83, 88, 243 and 508 d and three were in remission after chemotherapy and donor lymphocyte infusion. Although follow up is short, this data suggests that non-myeloablative haematopoietic cell transplantation with sirolimus (rapamycin)-based immunosuppression may provide disease control over several years in some patients with advanced and poor prognosis AML. [ABSTRACT FROM AUTHOR]

Rapamycin, a complex macrolide and potent fungicide, immunosuppressant and anticancer agent, is a highly specific inhibitor of mammalian target of rapamycin (mTOR). Rapamycin has been shown to induce G1-phase cell cycle arrest in diverse tumor cell types, and its derivatives RAD001 and CCI-779 are currently in phase I and phase II clinical trials, respectively, as anticancer agents. In this study, we show that rapamycin induced the apoptotic death of JN-DSRCT-1 cells, the only available in vitro model for Desmoplastic Small Round Cell Tumors (DSRCT), while having only minor effects on their cell cycle. Rapamycin induced apoptosis by increasing the Bax?:?Bcl-xL ratio as a consequence of the concomitant downregulation of Bcl-xL and upregulation of Bax, both at the post-transcriptional level. Rapamycin also downregulated the levels of EWS/WT1, the fusion protein characteristic of DSRCT. Transient transfection studies using kinase-dead and rapamycin-resistant forms of mTOR demonstrated that only the downregulation of Bcl-xL was caused by the mTOR inhibitory action of rapamycin, which prevented cap-dependent translation initiation, whereas Bax upregulation was induced by rapamycin through a mechanism independent of its mTOR inhibitory activity. Moreover, rapamycin treatment downregulated the mRNA and protein levels of the 26S p44.5 proteasome subunit, suggesting the involvement of the proteasome complex in the mechanisms of rapamycin-induced apoptosis. Treatment of JN-DSRCT-1 cells with MG-132, a proteasome specific inhibitor, also resulted in the induction of apoptosis through a similar increase in the Bax?:?Bcl-xL ratio specifically caused by inhibiting Bax degradation and turnover. These results suggested that rapamycin induces apoptosis by preventing the degradation of the Bax protein by the proteasome, and that this process is independent of mTOR inhibition. Furthermore, these results strongly support the introduction of the use of rapamycin as a cytotoxic agent for the treatment of DSRCT.Oncogene (2005) 24, 3348-3357. doi:10.1038/sj.onc.1208471 Published online 14 March 2005 [ABSTRACT FROM AUTHOR]

The pathophysiology of the myelodysplastic syndromes (MDS) involves disturbed regulation of angiogenesis, apoptosis, proliferation and differentiation as well as immune surveillance. Increasing data suggest that sirolimus might affect these pathways positively, thus being of possible therapeutic benefit in patients with this disease. Nineteen patients (n = 19) with a median age of 72 years (range 54–80 years) diagnosed with MDS received sirolimus orally with a target blood concentration of 3–12 ng/ml. Sirolimus was administered for a median of 3·7 months (range 0·3–11 months). Three patients [1 × refractory anaemia with excess blasts (RAEB)-2, 1 × RAEB-1, 1 × refractory cytopenia with multilineage dysplasia] showed either a major (1 × platelet, 1 × neutrophil) or a minor (1 × erythroid, 2 × platelet) haematological response according to International Working Group criteria. Major side-effects were hyperlipidaemia (n = 4), stomatitis (n = 3), thrombocytopenia (n = 2) and urinary tract infection (n = 1). These data suggest that sirolimus has activity in a subset of patients with more advanced MDS. [ABSTRACT FROM AUTHOR]

Sirolimus (rapamycin) is a macrocyclic lactone isolated from a strain of Streptomyces hygroscopicus that inhibits the mammalian target of rapamycin (mTOR)-mediated signal-transduction pathways, resulting in the arrest of cell cycle of various cell types, including T- and B-lymphocytes. Sirolimus has been demonstrated to prolong graft survival in various animal models of transplantation, ranging from rodents to primates for both heterotopic, as well as orthotopic organ grafting, bone marrow transplantation and islet cell grafting. In human clinical renal transplantation, sirolimus in combination with ciclo-sporin (cyclosporine) efficiently reduces the incidence of acute allograft rejection. Because of the synergistic effect of sirolimus on ciclosporin-induced nephrotoxicity, a prolonged combination of the two drugs inevitably leads to progressive irreversible renal allograft damage. Early elimination of calcineurin inhibitor therapy or complete avoidance of the latter by using sirolimus therapy is the optimal strategy for this drug. Prospective randomised phase II and III clinical studies have confirmed this approach, at least for recipients with a low to moderate immunological risk. For patients with a high immunological risk or recipients exposed to delayed graft function, sirolimus might not constitute the best therapeutic choice – despite its ability to enable calcineurin inhibitor sparing in the latter situation – because of its anti-proliferative effects on recovering renal tubular cells. Whether lower doses of sirolimus or a combination with a reduced dose of tacrolimus would be advantageous in these high risk situations remains to be determined. Clinically relevant adverse effects of sirolimus that require a specific therapeutic response or can potentially influence short- and long-term patient morbidity and mortality as well as graft survival include hypercholesterolaemia, hypertriglyceridaemia, infectious and non-infectious pneumonia, anaemia, lymphocele formation and impaired wound healing. These drug-related adverse effects are important determinants in the choice of a tailor-made immunosuppressive drug regimen that complies with the individual patient risk profile. Equally important in the latter decision is the lack of severe intrinsic nephrotoxicity associated with sirolimus and its advantageous effects on arterial hypertension, post-transplantation diabetes mellitus and esthetic changes induced by calcineurin inhibitors. Mild and transient thrombocytopenia, leukopenia, gastrointestinal adverse effects and mucosal ulcerations are all minor complications of sirolimus therapy that have less impact on the decision for choosing this drug as the basis for tailor-made immunosuppressive therapy. It is clear that sirolimus has gained a proper place in the present-day immunosuppressive armament used in renal transplantation and will contribute to the development of a tailor-made immunosuppressive therapy aimed at fulfilling the requirements outlined by the individual patient profile. [ABSTRACT FROM AUTHOR]

Rapamycin has antiangiogenic activity against tumors. This has been discussed while addressing the problem of cancer in organ transplantation. Here we investigated effective dosing schedules against tumors and angiogenesis. Growth of established CT-26 colon adenocarcinoma tumors was measured in Balb/c mice treated with total equivalent rapamycin doses (1.5 mg/kg/day) given once a day, once every 3 days, or by continuous infusion. Tumors were most inhibited with continuous rapamycin infusion, and less by bolus dosing. Interestingly, however, continuous dosing produced the lowest rapamycin blood levels (15 ng/ml). As rapamycin-sensitive p70S6-kinase intracellular signaling is critical for angiogenesis, p70S6-kinase activation was measured in endothelial cells by Western blotting. Maximal p70S6-kinase inhibition occurred from 1–5 ng/ml rapamycin. These same rapamycin concentrations optimally blocked vessel-sprouting from cultured aortic rings. Therefore, low-level rapamycin dosing most effectively controls tumors in mice. Importantly, antiangiogenic rapamycin levels are compatible with immunosuppressive doses, supporting its potential use in transplant patients with cancer. [ABSTRACT FROM AUTHOR]

Chronic lymphocytic B-cell leukemia (B-CLL) is an incurable disease characterized by the accumulation of monoclonal mature B cells, although disease progression relies upon cycling B-CLL cells in proliferation centers in central lymph organs. Rapamycin and its analogs are immunosuppressant drugs that exert their activity by specific inhibition of the mammalian target of rapamycin (mTOR). mTOR inhibition induces cell cycle arrest not only in normal lymphocytes but also in malignant cells. Therefore, rapamycins have recently entered the field of cancer treatment. In the present review we discuss how progression through the cell cycle is regulated in B-CLL cells and how rapamycin and its analogs can be used as target therapies against proliferating B-CLL cells. We also focus on additional effects of rapamycin, such as targeting the interaction between malignant B cells and the microenvironment. [ABSTRACT FROM AUTHOR]

Rapamycin has previously been shown to be efficacious against intracerebral glioma xenografts and to act in a cytostatic manner against gliomas. However, very little is known about the mechanism of action of rapamycin. The purpose of our study was to further investigate the in vitro and in vivo mechanisms of action of rapamycin, to elucidate molecular end points that may be applicable for investigation in a clinical trial, and to examine potential mechanisms of treatment failure. In the phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-null glioma cell lines U-87 and D-54, but not the oligodendroglioma cell line HOG (PTEN null), doses of rapamycin at the IC50 resulted in accumulation of cells in G1, with a corresponding decrease in the fraction of cells traversing the S phase as early as 24 h after dosing. All glioma cell lines tested had markedly diminished production of vascular endothelial growth factor (VEGF) when cultured with rapamycin, even at doses below the IC50. After 48 h of exposure to rapamycin, the glioma cell lines (but not HOG cells) showed downregulation of the membrane type-1 matrix metalloproteinase (MMP) invasion molecule. In U-87 cells, MMP-2 was downregulated, and in D-54 cells, both MMP-2 and MMP-9 were downregulated after treatment with rapamycin. Treatment of established subcutaneous U-87 xenografts in vivo resulted in marked tumor regression (P < 0.05). Immunohistochemical studies of subcutaneous U-87 tumors demonstrated diminished production of VEGF in mice treated with rapamycin. Gelatin zymography showed marked reduction of MMP-2 in the mice with subcutaneous U-87 xenografts that were treated with rapamycin as compared with controls treated with phosphate-buffered saline. In contrast, treatment of established intracerebral U-87 xenografts did not result in increased median survival despite inhibition of the Akt pathway within the tumors. Also, in contrast with our findings for subcutaneous tumors, immunohistochemistry and quantitative Western blot analysis results for intracerebral U-87 xenografts indicated that there is not significant VEGF production, which suggests possible deferential regulation of the hypoxia-inducible factor 1α in the intracerebral compartment. These findings demonstrate that the complex operational mechanisms of rapamycin against gliomas include cytostasis, anti-VEGF, and anti-invasion activity, but these are dependent on the in vivo location of the tumor and have implications for the design of a clinical trial. [ABSTRACT FROM AUTHOR]

Mathew T, Kreis H, Friend P. Two-year incidence of malignancy in sirolimus-treated renal transplant recipients: results from five multicenter studies. Clin Transplant 2004 DOI: 10.1111/j.1399-0012.2004.00188.x © Blackwell Munksgaard, 2004 We examined the rates of malignancy at 2 yr after transplantation in renal allograft patients receiving sirolimus (SRL) in continuous combination with cyclosporine (CsA), SRL as base therapy or SRL maintenance therapy after early withdrawal of CsA. A total of 1295 patients were enrolled in two double-blind studies comparing SRL with azathioprine (AZA) or placebo administered in continuous regimens with CsA. In two other trials (n = 161), SRL given as base therapy was compared with CsA. In the fifth trial, patients were randomly assigned at 3 months to either remain on CsA + SRL therapy (n = 215) or to have CsA eliminated with SRL being continued in concentration-controlled doses (n = 215). At 2 yr after transplantation, patients receiving SRL in continuous combination with CsA had a significantly lower incidence of skin cancer compared with patients receiving placebo. Patients receiving SRL as base therapy had no malignancies compared with a 5% incidence in those receiving CsA. The incidence of malignancy was significantly lower in patients receiving concentration-controlled SRL with elimination of CsA compared with those who remained on CsA + SRL. Based on the currently available data, patients receiving SRL-based therapy without CsA or SRL maintenance therapy after early CsA withdrawal have lower rates of malignancy in the first 2 yr after renal transplantation. SRL immunotherapy may be beneficial in protecting renal transplant patients from skin cancer even when given in combination with CsA. [ABSTRACT FROM AUTHOR]

Jiménez-Rivera C, Avitzur Y, Fecteau AH, Jones N, Grant D, Ng VL. Sirolimus for pediatric liver transplant recipients with post-transplant lymphoproliferative disease and hepatoblastoma. Pediatr Transplantation 2004: 8: 243–248. © 2004 Blackwell Munksgaard Sirolimus is a promising immune suppressive agent, with the potential to reduce calcineurin inhibitor associated nephrotoxicity, halt progression of chronic rejection and prevent tumor proliferation. The aim of this study was to review the experience using sirolimus in pediatric liver transplant recipients at a single center. Database and medical charts of all pediatric liver transplant recipients receiving sirolimus at the Hospital for Sick Children in Toronto were reviewed. Eight patients received sirolimus between October, 2000 and September, 2002. Indications for using sirolimus were post-transplant lymphoproliferative disease (PTLD) (n = 6) and hepatoblastoma (n = 2). Two patients with PTLD concurrently had renal impairment and chronic rejection. Sirolimus dosages ranged between 1.5 and 5 mg once daily. Median duration of follow-up was 17 months. Persistently elevated liver transaminase levels in the two children with chronic rejection decreased during sirolimus therapy. Recurrence of PTLD occurred in one patient. Two patients were diagnosed with acute cellular rejection after transition to maintenance sirolimus monotherapy. Resolution of adverse effects including mouth sores (n = 3), leg swelling (n = 2) and hyperlipidemia (n = 3) occurred either spontaneously or with dose reduction. Sirolimus was discontinued in four patients because of persisting bone marrow suppression, interstitial pneumonitis, life-threatening sepsis and refractory diarrhea. Children with PTLD or hepatoblastoma may benefit from immune suppression with sirolimus after liver transplantation. Further multi-center, prospective, randomized controlled trials will be instrumental to further the knowledge of long-term efficacy, safety and tolerability of sirolimus for selected children following liver transplantation. [ABSTRACT FROM AUTHOR]

Rapamycin, a macrocyclic lactone, is a highly specific inhibitor of the serine/threonine protein kinase target of rapamycin (TOR). Although it is clear that TOR controls initiation of protein translation, recent results indicate that TOR is a central controller, integrating a plethora of signalling pathways that respond to growth factors and nutritional status. In addition to the role of rapamycin as an immune suppressant, emerging data indicate that genetic and metabolic changes accompanying malignant transformation might cause hypersensitivity to TOR inhibition. [ABSTRACT FROM AUTHOR]

Conventional immunosuppressive drugs have been used effectively to prevent immunologic rejection in organ transplantation. Individuals taking these drugs are at risk, however, for the development and recurrence of cancer. In the present study we show that the new immunosuppressive drug rapamycin (RAPA) may reduce the risk of cancer development while simultaneously providing effective immunosuppression. Experimentally, RAPA inhibited metastatic tumor growth and angiogenesis in in vivo mouse models. In addition, normal immunosuppressive doses of RAPA effectively controlled the growth of established tumors. In contrast, the most widely recognized immunosuppressive drug, cyclosporine, promoted tumor growth. From a mechanistic perspective, RAPA showed antiangiogenic activities linked to a decrease in production of vascular endothelial growth factor (VEGF) and to a markedly inhibited response of vascular endothelial cells to stimulation by VEGF. Thus, the use of RAPA, instead of cyclosporine, may reduce the chance of recurrent or de novo cancer in high-risk transplant patients. [ABSTRACT FROM AUTHOR]

Summary Background Recent studies suggest that patients on mammalian target of rapamycin (mTOR) inhibitors experience a reduction in cutaneous carcinogenesis by an estimated 50% or more compared with calcineurin inhibitors. While randomized trials are running, organ transplant recipients are frequently switched from calcineurin inhibitors to mTOR inhibitors when cutaneous carcinogenesis increases. Objectives To slow carcinogenesis in our patient, a heart transplant recipient with a neuropathic diabetic foot syndrome who had developed cutaneous carcinogenesis at a rate of more than 20 squamous cell carcinomas (SCC) annually. Methods The patient's immunosuppression was switched from the calcineurin inhibitor ciclosporin to the mTOR inhibitor everolimus. Results Carcinogenesis slowed to six SCC annually; however, he developed recalcitrant diabetic foot ulcers which were purely neuropathic and nonangiopathic, and a limb-threatening fistulating necrotic erysipelas of the right leg. Both sites responded poorly to antibiotic therapy, offloading and debridement. This skin fistula became chronic and some toes were at risk for minor amputation. In view of the propensity for mTOR inhibitors to impair would healing, immunosuppression was switched back to ciclosporin. All wounds healed rapidly, but skin carcinogenesis rose to former levels. Conclusions This case impressively illustrates the clinical dilemma for mTOR inhibitor use where benefit in carcinogenesis is counterbalanced by impairment in wound healing. Changes in immunosuppressive regimens should thus be made on an individual basis with careful consideration of the relative risks. [ABSTRACT FROM AUTHOR]

Targeting mTOR (mammalian target of rapamycin) is an effective approach in the treatment of advanced RCC (renal cell carcinoma). Rapamycin-like drugs (rapalogues) have shown clinical activities and have been approved for the treatment of RCC. Recently, with the development of ATP-competitive inhibitors of mTOR, therapies targeting mTOR have entered a new era. Here, we discuss the biological relevance of blocking mTOR in RCC and review the mechanisms of action of rapalogues in RCC. We also advance some perspectives on the use of ATP-competitive inhibitors of mTOR in RCC. [ABSTRACT FROM AUTHOR]

The article discusses the target of rapamycin (TOR) inhibitor rapamycin and its effect on the life span of transplant recipients. It cites the experiments conducted by D. E. Harrison and colleagues via The National Institute on Aging and Intervention Testing Program. The program employs thorough testing techniques in genetically heterogeneous mice. Results showed that continuous rapamycin treatment has resulted in the increase in both the median and maximal lifespans of the mice.

Renal transplant recipients are susceptible to Kaposi's sarcoma (KS) because of treatment with immunosuppressive drugs. Sirolimus, a new immunosuppressive agent, has been successfully used for immune-suppression in kidney transplant recipients. Several studies have shown the potential role of sirolimus to inhibit progression of KS in kidney-transplant recipients. This report details a kidney-transplant recipient with cutaneous KS who had a complete remission in response to sirolimus therapy. [ABSTRACT FROM AUTHOR]

Radiotherapy is routinely used as a neoadjuvant, adjuvant or palliative treatment in various cancers. There is significant variation in clinical response to radiotherapy with or without traditional chemotherapy. Patients with a good response to radiotherapy demonstrate better clinical outcomes universally across different cancers. The PI3K/AKT/mTOR pathway upregulation has been linked to radiotherapy resistance. We reviewed the current literature exploring the role of inhibiting targets along this pathway, in enhancing radiotherapy response. We identified several studies using in vitro cancer cell lines, in vivo tumour xenografts and a few Phase I/II clinical trials. Most of the current evidence in this area comes from glioblastoma multiforme, non-small cell lung cancer, head and neck cancer, colorectal cancer, and prostate cancer. The biological basis for radiosensitivity following pathway inhibition was through inhibited DNA double strand break repair, inhibited cell proliferation, enhanced apoptosis and autophagy as well as tumour microenvironment changes. Dual PI3K/mTOR inhibition consistently demonstrated radiosensitisation of all types of cancer cells. Single pathway component inhibitors and other inhibitor combinations yielded variable outcomes especially within early clinical trials. There is ample evidence from preclinical studies to suggest that direct pharmacological inhibition of the PI3K/AKT/mTOR pathway components can radiosensitise different types of cancer cells. We recommend that future in vitro and in vivo research in this field should focus on dual PI3K/mTOR inhibitors. Early clinical trials are needed to assess the feasibility and efficacy of these dual inhibitors in combination with radiotherapy in brain, lung, head and neck, breast, prostate and rectal cancer patients. [ABSTRACT FROM AUTHOR]

The article points out that sirolimus is a very attractive candidate drug for treating several skin diseases. The drug is able to accomplish two things because of its unique combination of anticancer and immunosuppressant abilities. The absence of nephrotoxicity, its immunosuppresant and antitumoral characteristics makes sirolimus makes its an attractive drug for the dermatologist.

Colorectal cancer (CRC) occurs with more aggressiveness in kidney transplant recipients compared to the general population. Immunosuppressive therapy plays a crucial role in the development of post-transplant malignancy. Concretely, cyclosporine A (CsA) has intrinsic pro-oncologic properties, while several studies report a regression of cancer after the introduction of rapamycin (RAPA). However, their effect on the extracellular vesicle (EV) content from CRC cell lines and their relevance in the pre-metastatic niche have not yet been studied. Here, we investigated the effect of RAPA and CsA in EV-miRNAs from metastatic and non-metastatic CRC cell lines and the role of relevant miRNAs transferred into a pre-metastatic niche model. EV-miRNA profiles showed a significant upregulation of miR-6127, miR-6746-5p, and miR-6787-5p under RAPA treatment compared to CsA and untreated conditions in metastatic cell lines that were not observed in non-metastatic cells. From gene expression analysis of transfected lung fibroblasts, we identified 22 shared downregulated genes mostly represented by the histone family involved in chromatin organization, DNA packaging, and cell cycle. These results suggest that EV-miR-6127, miR-6746-5p and miR-6787-5p could be a potential epigenetic mechanism induced by RAPA therapy in the regulation of the pre-metastatic niche of post-transplant colorectal cancer. [ABSTRACT FROM AUTHOR]

Maffucci syndrome is characterized by multiple benign vascular anomalies and enchondromas present on the distal extremities. Effective treatment options are currently not available for Maffucci syndrome‐associated vascular lesions. Sirolimus is a mTOR pathway inhibitor, and has been tried successfully in the treatment of various vascular anomalies. We treated a 23‐year‐old female with Maffucci syndrome‐associated spindle cell hemangiomas with oral sirolimus (2mg/day, 0.04mg/kg/day). There was improvement in pain, but no change in colour or size of the vascular nodules. In view of unsatisfactory response and treatment‐related adverse effects (oral aphthae, mild transaminitis), sirolimus was stopped after 6 months. [ABSTRACT FROM AUTHOR]

While calcineurin inhibitors (CNIs) are effective for preventing acute rejection in kidney transplant recipients, long‐term use may cause chronic kidney injury and is associated with increased risks of cardiovascular events, cancer, and infection‐associated death. Immunosuppression strategies are needed to balance risks of acute and subclinical rejection with long‐term benefits of improved kidney function. Sirolimus, an inhibitor of mammalian target of rapamycin, is used for immunosuppression in kidney transplantation. Its clinical utility has evolved, over more than 15 years, including de novo sirolimus with and without concomitant CNIs and conversion from CNI‐based regimens to sirolimus. Sirolimus‐containing regimens are associated with preservation of good renal function, with promising characteristics for improving long‐term graft and patient survival, including antiviral and anticancer effects. Based on clinical evidence, use of low‐dose sirolimus in a de novo approach with tacrolimus/steroids in the immediate posttransplantation period is appropriate. A feasible alternative is a long term, CNI‐free combination with mycophenolate mofetil (following CNI‐to‐sirolimus conversion at 3‐6 months). These strategies are appropriate for a broad range of patients with various levels of immunologic risk, including those receiving expanded criteria donor kidneys or at increased risk of delayed graft function, particular challenges in Latin America and other global regions. [ABSTRACT FROM AUTHOR]

Aim: Nanotheranostics consisting of highly-fluorescent quantum dots coupled with gelatin/chondroitin layer-by-layer assembled nanocapsules were developed. Materials & methods: The hydrophobic drugs celecoxib (CXB) and rapamycin (RAP) were co-loaded into the oily core of nanocapsules (NCs) to enable synergistic growth inhibition of breast cancer cells. To overcome the nonspecific binding of actively targeted CS-NCs with normal cells, a matrix metalloproteinase (MMP-2)-degradable cationic gelatin layer was electrostatically deposited onto the surface of the negatively-charged CS-NCs. Results: The prepared nanocarriers displayed strong fluorescence which enabled tracing their internalization into cancer cells. An enhanced cytotoxicity of the NCs against breast cancer cells was demonstrated. In vivo, the nanoplatforms displayed superior antitumor efficacy as well as nonimmunogenic response. Conclusion: Therefore, these multifunctional nanoplatforms could be used as potential cancer theranostics. [ABSTRACT FROM AUTHOR]

The article discusses research being done on the treatment for port-wine stains (PWS) capillary vascular abnormalities. It references the study "Topical Axitinib Suppresses Angiogenesis Pathways Induced by Pulsed Dye Laser," by L. Gao et al. published in the 2015 issue. Several issues are noted in this study including the antiangiogenic properties of rapamycin, its impact on vascular endothelial growth factor (VEGF), and the ability of axitinib to inhibit VEGF receptor tyrosine kinases.

An introduction to the journal is presented in which the editors discuss research on the growth of pediatric renal transplant recipients on sirolimus (SRL) treatment.

The article presents a study on the use of sirolimus to prevent leukemias and lymphomas in T-cell replete transplantations from haplotype-mismatched donors. Seventeen patients are tracked, who all underwent transplants and lists survival rates and causes of death. The authors claim that sirolimus should be a primary immunosuppressive to prevent tumors in these cases.

A letter to the editor is presented on the role of sirolimus in the regression of Kaposi's sarcoma (KS) in a 32-year-old patient that underwent renal transplantation.

Summary Early conversion to a calcineurin-inhibitor (CNI)-free maintenance immunosuppression with sirolimus (SRL), mycophenolate mofetil (MMF) and steroids was associated with an improved 1-year renal function as compared with a cyclosporine (CsA)-based regimen (SMART core-study). This observational follow-up describes 132 patients followed up within the SMART study framework for 36 months. At 36 months, renal function continued to be superior in SRL-treated patients [ITT-eGFR@36m: 60.88 vs. 53.72 (CsA) ml/min/1.73 m2, P = 0.031]. However, significantly more patients discontinued therapy in the SRL group 59.4% vs.42.3% (CsA). Patient [99% (SRL) vs.97% (CsA) and graft 96% (SRL) vs.94% (CsA)] survival at 36 months was excellent in both arms. There was no difference in late rejection episodes. Late infections and adverse events were similar in both arms except of a higher rate of hyperlipidemia in SRL and a higher incidence of malignancy in CsA-treated patients. In a multivariate analysis, donor age >60 years, S-creatinine at conversion >2 mg/dl, CMV naïve(-) recipients and immunosuppression with CsA were predictive of an impaired renal function at 36 months. Early conversion to a CNI-free SRL-based immunosuppression is associated with a sustained improvement of renal function up to 36 months after transplantation. Patient selection will be key to derive long-term benefit and avoid treatment failure using this mTOR-inhibitor-based immunosuppressive regimen. [ABSTRACT FROM AUTHOR]

Background. The specific mTor inhibitor sirolimus has been implicated in the pathogenesis of renal glomerular lesions and nephrotic syndrome appearance after transplantation. Podocyte injury and focal segmental glomerulosclerosis have been related to sirolimus therapy in some patients but the pathways underlying these lesions remain hypothetical. Methods. To go further in the comprehension of these mechanisms, primary cultures of human podocytes were exposed to therapeutic-range concentrations of sirolimus. Results. Cell viability was not affected after 2 days’ exposure to the drug but changes in cell phenotype and cytoskeleton reorganization were observed. We also evidenced that vascular endothelial growth factor (VEGF) synthesis and Akt phosphorylation were decreased by sirolimus addition. We did not observe any loss of podocyte differentiation markers with the notable exception of WT1, a transcription factor essential for maintaining podocyte integrity. WT1 gene and protein expression in podocytes were decreased in a dose-dependent manner after incubation with sirolimus. Conclusion. Taken together, these data suggest that sirolimus could impair pathways essential for podocyte integrity and therefore predisposes to glomerular injury. [ABSTRACT FROM AUTHOR]

Reports on the findings of researchers that immunosuppressant rapamycin can inhibit primary and metastatic tumor growth in organ transplantation. Ability to block interleukin-2 stimulation of lymphocytes and prevent graft rejection; Mechanism of action.

BACKGROUND. Idiopathic multicentric Castleman disease (iMCD) is a hematologic illness involving cytokine-induced lymphoproliferation, systemic inflammation, cytopenias, and life-threatening multi-organ dysfunction. The molecular underpinnings of interleukin-6 (IL-6) blockade-refractory patients remain unknown; no targeted therapies exist. In this study, we searched for therapeutic targets in IL-6 blockade-refractory iMCD patients with the thrombocytopenia, anasarca, fever/elevated C-reactive protein, reticulin myelofibrosis, renal dysfunction, organomegaly (TAFRO) clinical subtype. METHODS. We analyzed tissues and blood samples from 3 IL-6 blockade-refractory iMCD-TAFRO patients. Cytokine panels, quantitative serum proteomics, flow cytometry of PBMCs, and pathway analyses were employed to identify novel therapeutic targets. To confirm elevated mTOR signaling, a candidate therapeutic target from the above assays, immunohistochemistry was performed for phosphorylated S6, a read-out of mTOR activation, in 3 iMCD lymph node tissue samples and controls. Proteomic, immunophenotypic, and clinical response assessments were performed to quantify the effects of administration of the mTOR inhibitor sirolimus. RESULTS. Studies of 3 IL-6 blockade-refractory iMCD cases revealed increased [CD8.sup.+] T cell activation, VEGF-A, and PI3K/Akt/mTOR pathway activity. Administration of sirolimus substantially attenuated [CD8.sup.+] T cell activation and decreased VEGF-A levels. Sirolimus induced clinical benefit responses in all 3 patients with durable and ongoing remissions of 66, 19, and 19 months. CONCLUSION. This precision medicine approach identifies PI3K/Akt/mTOR signaling as the first pharmacologically targetable pathogenic process in IL-6 blockade-refractory iMCD. Prospective evaluation of sirolimus in treatment- refractory iMCD is planned (NCT03933904). FUNDING. This study was supported by the Castleman's Awareness & Research Effort/Castleman Disease Collaborative Network, Penn Center for Precision Medicine, University Research Foundation, Intramural NIH funding, and the National Heart Lung and Blood Institute., Introduction Multicentric Castleman disease (MCD) is characterized by polyclonal lymphoproliferation, hypervascularized lymph nodes containing dysmorphic germinal centers, cytokine-driven systemic inflammation, cytopenias, and multi-organ dysfunction. The estimated annual incidence of MCD [...]