Rapamycin

 

• The most effective anti-aging intervention in mice.

• mTOR inhibitor + STAT3 inhibitor.

• Potent inducer of autophagy.

• Antimetastasis & anti-cancer in RCC.

• Effective against myeloproliferative neoplasms. 

• Cream improves aged human skin. 

Rapamycin is an effective inhibitor of human renal cancer metastasis, 2003

Rapamycin reduced the number of pulmonary metastases. Circulating levels of VEGF-A and TGF-beta1 were lower in the rapamycin-treated mice compared to untreated or cyclosporine-treated mice. Rapamycin may be of value for patients with RCC and that its antitumor efficacy is realized by cell cycle arrest and targeted reduction of VEGF-A and TGF-beta1. 

Newly Approved mTOR Inhibitors for the Treatment of Metastatic Renal Cell Carcinoma (2009)

The mammalian target of rapamycin (mtor) is an important target in the treatment of RCC. In first-line treatment for patients with disease having poor prognostic features, temsirolimus, an mtor inhibitor, has demonstrated benefit over interferon alfa in both overall and progression-free survival. Everolimus, a second mtor inhibitor that has showed activity in rcc, led to improved progression-free survival in a comparison with placebo in patients whose rcc progressed after treatment with vascular endothelial growth factor receptor tyrosine kinase inhibitors (sunitinib, sorafenib, or both). 

mTOR Inhibitors Alone and in Combination with JAK2 Inhibitors Effectively Inhibit Cells of Myeloproliferative Neoplasms, 2013

Background

Dysregulated signaling of the JAK/STAT pathway is a common feature of chronic myeloproliferative neoplasms (MPN), usually associated with JAK2V617F mutation. Recent clinical trials with JAK2 inhibitors showed significant improvements in splenomegaly and constitutional symptoms in patients with myelofibrosis but meaningful molecular responses were not documented. Accordingly, there remains a need for exploring new treatment strategies of MPN. A potential additional target for treatment is represented by the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway that has been found constitutively activated in MPN cells; proof-of-evidence of efficacy of the mTOR inhibitor RAD001 has been obtained recently in a Phase I/II trial in patients with myelofibrosis. The aim of the study was to characterize the effects in vitro of mTOR inhibitors, used alone and in combination with JAK2 inhibitors, against MPN cells.

Findings

Mouse and human JAK2V617F mutated cell lines and primary hematopoietic progenitors from MPN patients were challenged with an allosteric (RAD001) and an ATP-competitive (PP242) mTOR inhibitor and two JAK2 inhibitors (AZD1480 and ruxolitinib). mTOR inhibitors effectively reduced proliferation and colony formation of cell lines through a slowed cell division mediated by changes in cell cycle transition to the S-phase. mTOR inhibitors also impaired the proliferation and prevented colony formation from MPN hematopoietic progenitors at doses significantly lower than healthy controls. JAK2 inhibitors produced similar antiproliferative effects in MPN cell lines and primary cells but were more potent inducers of apoptosis, as also supported by differential effects on cyclinD1, PIM1 and BcLxL expression levels. Co-treatment of mTOR inhibitor with JAK2 inhibitor resulted in synergistic activity against the proliferation of JAK2V617F mutated cell lines and significantly reduced erythropoietin-independent colony growth in patients with polycythemia vera.

Conclusions/Significance

These findings support mTOR inhibitors as novel potential drugs for the treatment of MPN and advocate for clinical trials exploiting the combination of mTOR and JAK2 inhibitor.

Rapamycin inhibits STAT3 independent of mTor inhibition (link) 

In this study, the researchers showed that rapamycin prevents STAT3’s translocation to the nucleus (where it can affect expression of other genes) by binding to one of its domains that was previously considered undruggable because of its flat surface area. Moreover, in an unrelated process, rapamycin also directly downregulates c-Myc. Experiments on genetically modified cells with mTOR knocked out showed that rapamycin does all this independently of mTOR, proving that the scientists had indeed discovered a new mechanism of action.

Image: Rapamycin downregulates STAT3, which leads to lower expression of STAT3’s downstream target, c-Myc. Rapamycin also directly downregulates c-Myc in addition to its more widely known anti-mTOR activity. Together, these result in slower cell proliferation, inhibiting tumor growth.

The researchers also ran a few experiments in vivo. In one of them, in mice with induced hepatic tumors, rapamycin significantly decreased tumor size. Levels of mTOR, STAT3 and c-Myc were reduced as well.

Safety and efficacy of everolimus, a mTOR inhibitor, as single agent in a phase 1/2 study in patients with myelofibrosis, 2011 

In addition to dysregulated JAK/STAT signaling, activation of the AKT/mTOR pathway occurs in myelofibrosis, a myeloproliferative neoplasm with no approved therapies. We conducted a phase 1/2 study with everolimus, an mTOR inhibitor, in 39 high- or intermediate-risk primary or postpolycythemia vera/postessential thrombocythemia myelofibrosis subjects. Responses were evaluated in 30 patients of phase 2. No dose-limiting toxicity was observed in phase 1 up to 10 mg/d. When this dose was used in phase 2, grade ≥ 3 toxicities were infrequent; the commonest toxicity was grade 1-2 stomatitis. Rapid and sustained splenomegaly reduction of > 50% and > 30% occurred in 20% and 44% of subjects, respectively. A total of 69% and 80% experienced complete resolution of systemic symptoms and pruritus. Response in leukocytosis, anemia, and thrombocytosis occurred in 15%-25%. Clinical responses were not associated with reduced JAK2V617F burden, circulating CD34+ cells, or cytokine levels, whereas CCDN1 mRNA and phospho-p70S6K level, known targets of mTOR, and WT1 mRNA were identified as possible biomarkers associated with response. Response rate was 60% when European Network for Myelofibrosis criteria were used (8 major, 7 moderate, 3 minor responses) or 23% when IWG-MRT criteria (1 partial response, 6 clinical improvements) were used. These results provide proof-of-concept that targeting mTOR pathway in myelofibrosis may be clinically relevant.

Leukemia:

• Rapamycin, the mTOR kinase inhibitor, sensitizes acute myeloid leukemia cells, HL-60 cells, to the cytotoxic effect of arabinozide cytarabine, 2009

• Antileukemic activity of rapamycin in acute myeloid leukemia, 2005

• Rapamycin enhanced the antitumor effects of doxorubicin in myelogenous leukemia K562 cells by downregulating the mTOR/p70S6K pathway (link)

mTOR for longevity: Interview with Dr Green, second one.

Rapalogs (link)

• Genetically heterogeneous mice given rapamycin at a dose of 42 mg/kg mice saw median lifespans increase 23% in males, 26% in females. [3]

• Intermittent rapamycin (2 mg/kg i.p. every five days) starting with 20-month-old mice increases mean lifespan by 13% and median lifespan by 7%, without affecting glucose or insulin tolerance.[8]

• Healthy elderly volunteers given everolimus (a rapalog) at 0.5 mg daily, 5 mg weekly, or 20 mg weekly, for six weeks, had no serious adverse effects associated with the drug. [19]

[NEGATIVE] Effect of sirolimus on malignancy and survival after kidney transplantation: systematic review and meta-analysis of individual patient data, 2014

Patient level data were available from 5876 patients from 21 randomized trials. Sirolimus was associated with a 40% reduction in the risk of malignancy (adjusted hazard ratio 0.60, 95% confidence interval 0.39 to 0.93) and a 56% reduction in the risk of non-melanoma skin cancer (0.44, 0.30 to 0.63) compared with controls. Sirolimus was associated with an increased risk of death (1.43, 1.21 to 1.71) compared with controls.

Studies on transplant patients:

• Target of rapamycin inhibitors (TOR-I; sirolimus and everolimus) for primary immunosuppression in kidney transplant recipients (Review), 2019

• Target of rapamycin inhibitors (sirolimus and everolimus) for primary immunosuppression of kidney transplant recipients: a systematic review and meta-analysis of randomized trials 2006

• Mammalian Target of Rapamycin Inhibitors and Clinical Outcomes in Adult Kidney Transplant Recipients 2016

• This study shows the opposite for heart transplant patients. Those on sirolimus live longer! And they catch cancer less often. (link, link2)

• For lung transplant patients, sirolimus users again live longer than controls. (link) 

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A Randomized Control Trial to Establish the Feasibility and Safety of Rapamycin Treatment in an Older Human Cohort: Immunological, Physical Performance, and Cognitive Effects, 2019

Placebo-controlled pilot study in 25 generally healthy older adults (aged 70–95 years); subjects were randomized to receive either 1 mg RAPA or placebo daily. Although three subjects withdrew, 11 RAPA and 14 controls completed at least 8 weeks of treatment and were included in the analysis.

TL;DR Not much happens, which is a good thing from safety point of view.

Topical rapamycin reduces markers of senescence and aging in human skin: an exploratory, prospective, randomized trial, 2019

Aging is a major risk factor for the majority of human diseases, and the development of interventions to reduce the intrinsic rate of aging is expected to reduce the risk for age-related diseases including cardiovascular disease, cancer, and dementia. In the skin, aging manifests itself in photodamage and dermal atrophy, with underlying tissue reduction and impaired barrier function. To determine whether rapamycin, an FDA-approved drug targeting the mechanistic target of rapamycin (mTOR) complex, can reduce senescence and markers of aging in human skin, an exploratory, placebo-controlled, interventional trial was conducted in a clinical dermatology setting. Participants were greater than 40 years of age with evidence of age-related photoaging and dermal volume loss and no major morbidities. Thirty-six participants were enrolled in the study, and nineteen discontinued or were lost to follow-up. A significant (P = 0.008) reduction in p16INK4A protein levels and an increase in collagen VII protein levels (P = 0.0077) were observed among participants at the end of the study. Clinical improvement in skin appearance was noted in multiple participants, and immunohistochemical analysis revealed improvement in histological appearance of skin tissue. Topical rapamycin reduced the expression of the p16INK4A protein consistent with a reduction in cellular senescence. This change was accompanied by relative improvement in clinical appearance of the skin and histological markers of aging and by an increase in collagen VII, which is critical to the integrity of the basement membrane. These results indicate that rapamycin treatment is a potential anti-aging therapy with efficacy in humans.

A randomized controlled trial to establish effects of short-term rapamycin treatment in 24 middle-aged companion dogs, 2017

Age is the single greatest risk factor for most causes of morbidity and mortality in humans and their companion animals. As opposed to other model organisms used to study aging, dogs share the human environment, are subject to similar risk factors, receive comparable medical care, and develop many of the same age-related diseases humans do. In this study, 24 middle-aged healthy dogs received either placebo or a non-immunosuppressive dose of rapamycin for 10 weeks. All dogs received clinical and hematological exams before, during, and after the trial and echocardiography before and after the trial. Our results showed no clinical side effects in the rapamycin-treated group compared to dogs receiving the placebo. Echocardiography suggested improvement in both diastolic and systolic age-related measures of heart function (E/A ratio, fractional shortening, and ejection fraction) in the rapamycin-treated dogs. Hematological values remained within the normal range for all parameters studied; however, the mean corpuscular volume (MCV) was decreased in rapamycin-treated dogs. Based on these results, we will test rapamycin on a larger dog cohort for a longer period of time in order to validate its effects on cardiac function and to determine whether it can significantly improve healthspan and reduce mortality in companion dogs.

[NEGATIVE] Interferon α-induced Apoptosis in Tumor Cells Is Mediated through the Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Signaling Pathway* (link)

Interferon (IFN) α induces a caspase-dependent apoptosis that is associated with activation of the proapoptotic Bak and Bax, loss of mitochondrial membrane potential, and release of cytochrome c. In addition to the onset of the classical Jak-STAT pathway, IFNα also induced phosphoinositide 3-kinase (PI3K) activity. Pharmacological inhibition of PI3K activity by Ly294002 disrupted IFN-induced apoptosis upstream of mitochondria. Inhibition of mTOR by rapamycin or by overexpression of a kinase dead mutant of mTOR, efficiently blocked IFNα-induced apoptosis. A PI3K and mTOR-dependent phosphorylation of p70S6 kinase and 4E-BP1 repressor was induced by IFNα treatment of cells and was strongly inhibited by Ly294002 or rapamycin. The activation of Jak-STAT signaling upon IFNα stimulation was not affected by abrogating PI3K/mTOR pathway. Neither was the expression of several IFNα target genes affected, nor the ability of IFNα to protect against virus-induced cell death affected by inhibition of the PI3K/mTOR pathway. These data demonstrate that an intact PI3K/mTOR pathway is necessary for the ability of IFNα to induce apoptosis, whereas activation of the Jak-STAT pathway alone appears to be insufficient for this specific IFNα-induced effect.

[NEGATIVE] Pegylated IFN-α suppresses hepatitis C virus by promoting the DAPK-mTOR pathway

Significance

Death-associated protein kinase (DAPK) regulates several important biological functions through a diverse range of signal transduction pathways, including cell growth, the immune response, apoptosis, and autophagy, but its antiviral activity has not been explored. Our data provide evidence that DAPK is one of the major regulators for the pegylated IFN-α–induced antiviral activity against hepatitis C virus, suggesting that DAPK may serve as a novel antiviral target and also a new biomarker to predict the therapeutic efficacy of pegylated IFN-α and other antiviral agents.

Abstract

Death-associated protein kinase (DAPK) has been found to be induced by IFN, but its antiviral activity remains elusive. Therefore, we investigated whether DAPK plays a role in the pegylated IFN-α (peg-IFN-α)–induced antiviral activity against hepatitis C virus (HCV) replication. Primary human hepatocytes, Huh-7, and infectious HCV cell culture were used to study the relationship between peg-IFN-α and the DAPK-mammalian target of rapamycin (mTOR) pathways. The activation of DAPK and signaling pathways were determined using immunoblotting. By silencing DAPK and mTOR, we further assessed the role of DAPK and mTOR in the peg-IFN-α–induced suppression of HCV replication. Peg-IFN-α up-regulated the expression of DAPK and mTOR, which was associated with the suppression of HCV replication. Overexpression of DAPK enhanced mTOR expression and then inhibited HCV replication. In addition, knockdown of DAPK reduced the expression of mTOR in peg-IFN-α–treated cells, whereas silencing of mTOR had no effect on DAPK expression, suggesting mTOR may be a downstream effector of DAPK. More importantly, knockdown of DAPK or mTOR significantly mitigated the inhibitory effects of peg-IFN-α on HCV replication. In conclusion, our data suggest that the DAPK-mTOR pathway is critical for anti-HCV effects of peg-IFN-α.

Interferon-α enhances the susceptibility of renal cell carcinoma to rapamycin by suppressing mTOR activity, 2014

Combination of valproic acid, everolimus and low dosed interferon alpha provides amplified antineoplastic effects on prostate cancer cells, 2015