Combination Of mTOR Inhibitors Research Articles

Impact of co-mutations and transcriptional signatures in non-small cell lung cancer patients treated with adagrasib in the KRYSTAL-1 trial.

KRAS inhibitors are revolutionizing the treatment of NSCLC, but clinico-genomic determinants of treatment efficacy warrant continued exploration. Patients with advanced KRASG12C-mutant NSCLC treated with adagrasib (KRYSTAL-1-NCT03785249) were included in the analysis. Pre-treatment NGS data were collected per protocol. HTG EdgeSeq Transcriptome Panel was used for gene expression profiling. Clinical endpoints included objective response, progression-free and overall survival. KRASG12C-mutant NSCLC cell lines and xenograft models were used for sensitivity analyses and combination drug screens. KEAP1MUT and STK11MUT were associated with shorter survival to adagrasib (KEAP1: PFS 4.1m vs 9.9m, HR 2.7, p<0.01; OS 5.4m vs 19.0m, HR 3.6, p<0.01; STK11: PFS 4.2m vs 11.0m, HR 2.2, p<0.01; OS 9.8m vs NR, HR 2.6, p<0.01). KEAP1WT/STK11WT status identified adagrasib-treated patients with significantly longer PFS (16.9m) and OS (NR). Pre-clinical analyses further validate the association between KEAP1 loss-of-function and adagrasib resistance. Adagrasib and mTOR inhibitor combinations produced higher treatment efficacy in NSCLC models harboring STK11 and KEAP1 co-mutations. NRF2HIGH signaling was associated with shorter survival to adagrasib (PFS: 4.2m vs 8.4m, HR 2.0, p=0.02; OS: 6.5m vs 19.0m, HR 2.8, p<0.01) even in KEAP1WTNSCLC patients. KEAP1WT/STK11WT/NRF2LOW status identified patients - 32% - with longer survival to adagrasib (PFS 12.0m vs 4.2m, HR 0.2, p<0.01; OS NR vs 8.0m, HR 0.1, p<0.01). KEAP1, STK11 and NRF2 status define KRASG12C-mutant NSCLC patients with markedly distinct outcomes to adagrasib. These results further support the use of genomic features - mutational and non-mutational - for treatment selection of KRASG12C-mutant NSCLC patients.

A Dual-Target and Dual-Mechanism Design Strategy by Combining Inhibition and Degradation Together.

Glioblastoma, a highly aggressive brain tumor, lacks effective treatment with low 5 year survival rates. Urgency for new therapies is evident. Mammalian targets of rapamycin (mTOR) and G1 to S phase transition 1 gene (GSPT1) are overexpressed in glioblastoma, regulating vital cellular functions. Current mTOR inhibitors face challenges in clinical efficacy and drug resistance. Similarly, GSPT1-targeting therapies have not progressed of glioblastoma in clinical trials. Research studies suggested that combining mTOR inhibition with GSPT1 degradation may overcome resistance and enhance efficacy. We propose the concept of jointly implementing inhibition and degradation on different proteins, integrating the properties of inhibitors and degraders into the same molecule. Introducing YB-3-17, a novel bifunctional molecule, robustly inhibits mTOR and selectively degrades GSPT1. As a tool compound for proof-of-concept studies, YB-3-17 sharpens selectivity, avoiding off-target effects, and selectively induces GSPT1 degradation and mTOR inhibition, showing superior efficacy in tumor cell lines compared to that of standalone therapies. RNA-seq analysis highlights the advantages of YB-3-17 over mTOR inhibitor treatment. YB-3-17 can safely and effectively inhibit tumor growth in mice, offering a promising direction for precision treatment of glioblastoma, representing the first attempt to combine mTOR inhibition with GSPT1 degradation. This work also demonstrates that it is conceptually possible to successfully combine the properties of small molecule inhibitors and degraders into a single molecule, killing two birds with one stone.

Treatment of Melanoma Cells with Chloroquine and Everolimus Activates the Apoptosis Process and Alters Lipid Redistribution.

The balance between apoptosis and autophagy plays a key role in cancer biology and treatment strategies. The aim of this study was to assess the effect of the mTOR kinase inhibitor everolimus and chloroquine on the regulation of proliferation, caspase-3 activation, and apoptosis in melanoma cells. We studied the activity of caspase-3 and the levels of caspase-3 and -9 using the Western blot technique. Cellular apoptosis was examined using a DNA fragmentation assay, and changes in the cell nucleus and cytoskeleton were examined using fluorescence microscopy DAPI, OA/IP. We also studied the rearrangement of lipid structures using fluorescent dyes: Nile Red and Nile Blue. A low nanomolar concentration of the mTOR kinase inhibitor everolimus in combination with chloroquine activated the apoptosis process and decreased cell proliferation. These changes were accompanied by an obvious change in cell morphology and rearrangement of lipid structures. Alterations in lipid redistribution accompanying the process of apoptosis and autophagy are among the first to occur in the cell and can be easily monitored in in vitro studies. The combination of mTOR inhibitors and chloroquine represents a promising area of research in cancer therapy. It has the potential to enhance treatment efficacy through complementary mechanisms.

Inhibitors of the mTOR signaling pathway can play an important role in breast cancer immunopathogenesis.

This study explores the critical role of inhibitors targeting the mammalian target of rapamycin (mTOR) signaling pathway in breast cancer research and treatment. The mTOR pathway, a central regulator of cellular processes, has been identified as a crucial factor in the development and progression of breast cancer. The essay explains the complex molecular mechanisms through which mTOR inhibitors, such as rapamycin and its analogs, exert their anticancer effects. These inhibitors can stop cell growth, proliferation, and survival in breast cancer cells by blocking critical signaling pathways within the mTOR pathway. Furthermore, the essay discusses the implications of using mTOR inhibitors as a comprehensive therapeutic strategy. It emphasizes the potential benefits of combining mTOR inhibitors with other treatment approaches to enhance the effectiveness of breast cancer treatment. The evolving landscape of breast cancer research underscores the significance of mTOR as a therapeutic target and highlights ongoing efforts to improve and optimize mTOR inhibitors for clinical use. In conclusion, the essay asserts that inhibitors of the mTOR signaling pathway offer a promising approach in the fight against breast cancer. These inhibitors provide a focused and effective intervention targeting specific dysregulations within the mTOR pathway. As research advances, the integration of mTOR inhibitors into customized combination therapies holds excellent potential for shaping a more effective and personalized approach to breast cancer treatment, ultimately leading to improved outcomes for individuals affected by this complex and diverse disease.

Enhancing immunotherapy through PD-L1 upregulation: the promising combination of anti-PD-L1 plus mTOR inhibitors.

Immune checkpoint inhibitors (ICIs) targeting the programmed cell death protein 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) pathway have transformed urothelial cancer (UC) therapy. The correlation between PD-L1 expression and ICI effectiveness is uncertain, leaving the role of PD-L1 as a predictive marker for ICI efficacy unclear. Among several ways to enhance the efficacy of ICI, trials are exploring combining ICIs with serine/threonine-protein kinase mTOR (mTOR) inhibitors in different tumor types. The potential interaction between mTOR inhibitors and PD-L1 expression in UC has not been well characterized. In our study, we investigated how phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway inhibitors (TAK-228, everolimus and TAK-117) affect PD-L1 expression and function in preclinical bladder cancer cell models. TAK-228 increased cell surface levels of glycosylated PD-L1 in all but one of the seven cell lines, regardless of baseline levels. TAK-228 promoted the secretion of epidermal growth factor (EGF) and interferon-β (IFNβ), both linked to PD-L1 protein induction. Blocking EGF and IFNβ receptors reversed the TAK-228-induced PD-L1 increase. Additionally, TAK-228 enhanced IFN-γ-induced PD-L1 expression and intracellular HLA-I levels in some cells. TAK-228-treated bladder cancer cells exhibited resistance to the cytotoxic effects of peripheral blood mononuclear cells (PBMCs) and cluster of differentiation 8 (CD8)+ Tcells. The addition of an anti-PD-L1 antibody diminished this resistance in T24 cells. Increased expression of PD-L1 under TAK-228 exposure was confirmed in patient-derived explants (PDEs) treated ex vivo. These preclinical findings suggest that mTOR inhibition with TAK-228 can increase PD-L1 levels, potentially impacting the specific immune response against UC cells. This highlights the rationale for exploring the combination of mTOR inhibitors with ICIs in patients with advanced UC.

Kidney Biopsy Findings After Lung Transplantation

The early diagnosis of histological kidney damage after lung transplantation (LT) is of paramount importance given the negative prognostic implications of kidney disease. Three pathologists analyzed all kidney biopsies (KBs) (N= 100) performed from 2010 to 2021 on lung transplant patients in 4 Paris transplantation centers. The main indication for biopsy was chronic renal dysfunction (72% of patients). Biopsies were performed at a median of 26.3 months after transplantation and 15 months after a decline in estimated glomerular filtration rate (eGFR) or the onset of proteinuria. Biopsies revealed a wide spectrum of chronic lesions involving the glomerular, vascular, and tubulointerstitial compartments. The 4 most frequent final diagnoses, observed in 18% to 49% of biopsies, were arteriosclerosis, acute calcineurin inhibitor (CNI) toxicity, thrombotic microangiopathy (TMA) and acute tubular necrosis (ATN). TMA was significantly associated with a combination of mTOR inhibitors (mTORi) or CNIs with biological signs present in only 50% of patients. The eGFR was poorly correlated with most lesions, particularly percent glomerulosclerosis, and with the risk of end-stage renal disease (ESRD). Thirty-four patients progressed to ESRD at an average of 20.1 months after biopsy. Three factors were independently associated with the risk of ESRD: postoperative dialysis, proteinuria >3 g/g and percent glomerulosclerosis >4%. Given the great diversity of renal lesions observed in lung transplant recipients, early referral to nephrologists for KB should be considered for these patients when they present with signs of kidney disease.

HMGA1 regulates trabectedin sensitivity in advanced soft-tissue sarcoma (STS): A Spanish Group for Research on Sarcomas (GEIS) study

HMGA1 is a structural epigenetic chromatin factor that has been associated with tumor progression and drug resistance. Here, we reported the prognostic/predictive value of HMGA1 for trabectedin in advanced soft-tissue sarcoma (STS) and the effect of inhibiting HMGA1 or the mTOR downstream pathway in trabectedin activity. The prognostic/predictive value of HMGA1 expression was assessed in a cohort of 301 STS patients at mRNA (n = 133) and protein level (n = 272), by HTG EdgeSeq transcriptomics and immunohistochemistry, respectively. The effect of HMGA1 silencing on trabectedin activity and gene expression profiling was measured in leiomyosarcoma cells. The effect of combining mTOR inhibitors with trabectedin was assessed on cell viability in vitro studies, whereas in vivo studies tested the activity of this combination. HMGA1 mRNA and protein expression were significantly associated with worse progression-free survival of trabectedin and worse overall survival in STS. HMGA1 silencing sensitized leiomyosarcoma cells for trabectedin treatment, reducing the spheroid area and increasing cell death. The downregulation of HGMA1 significantly decreased the enrichment of some specific gene sets, including the PI3K/AKT/mTOR pathway. The inhibition of mTOR, sensitized leiomyosarcoma cultures for trabectedin treatment, increasing cell death. In in vivo studies, the combination of rapamycin with trabectedin downregulated HMGA1 expression and stabilized tumor growth of 3-methylcholantrene-induced sarcoma-like models. HMGA1 is an adverse prognostic factor for trabectedin treatment in advanced STS. HMGA1 silencing increases trabectedin efficacy, in part by modulating the mTOR signaling pathway. Trabectedin plus mTOR inhibitors are active in preclinical models of sarcoma, downregulating HMGA1 expression levels and stabilizing tumor growth.

Abstract A117: Evaluation of nab-sirolimus in combination with PI3K pathway inhibitors to overcome PI3K/mTOR resistance in PI3K-mutant breast cancer cell lines

Abstract Background: The PI3K-AKT-mTOR pathway is frequently activated in many cancer types and plays a central role in tumorigenesis. However, clinical use of therapies targeting this pathway is limited by compensatory vertical and horizontal feedback activation loops, which limit antitumor efficacy and lead to drug resistance. Combination therapy strategies employing simultaneous inhibition of multiple PI3K-AKT-mTOR pathway elements may overcome these resistance mechanisms and improve antitumor activity. nab-Sirolimus is a novel albumin-bound nanoparticle form of the mTOR inhibitor sirolimus that has significantly greater tumor accumulation, mTOR target suppression, and improved antitumor effects compared with conventional first generation mTOR inhibitors in preclinical models. Here, we analyzed the effects of nab-sirolimus combinations in PI3K-mutant breast cancer (BrCa) cells. Methods: PI3K mutated BrCa cell lines (MDA-MD-453, hormone receptor [HR]-; and MDA-MB-361, HR+) were incubated for 5 days with increasing concentrations of nab-sirolimus, the PI3K pathway inhibitors gedatolisib and alpelisib, or the AKT inhibitors miransertinib and capivasertib. The antiproliferative and cytotoxic effects of single agent and combination treatment were assessed using cell viability and cell death assays. Cell signaling was analyzed by western blot. Results: The antiproliferative effects of gedatolisib at low doses (2.5-10 nM) were enhanced by 61-71% in combination with nab-sirolimus (20 or 80 nM) in MDA-MB-453 cells. Limited cell death (4.2-9.6%) was observed in HR- MDA-MB-453 cells with low doses (2.5, 5, and 10 nM) of gedatolisib alone. In contrast, and despite the well-established cytostatic action of mTOR inhibitors, the addition of 20 and 80 nM nab-sirolimus led to high levels of cell death (up to 48.9%). Similar results were observed with alpelisib at 1 µM in combination with nab-sirolimus (20 or 80 nM), and also with capivasertib (1 µM) or miransertib (250 nM) combined with 80 nM nab-sirolimus. Western blot analysis demonstrated that gedatolisib or alpelisib alone each triggered upregulation of p4EBP1 whereas nab-sirolimus treatment alone activated a negative feedback loop through increased pAKT. Importantly, these compensatory feedback activation loops were both reversed by the combination of PI3K inhibitors and nab-sirolimus. Similar cell death and cell viability results were observed with nab-sirolimus plus PI3K inhibitors (gedatolisib or alpelisib) and AKT inhibitors (capivasertib or miransertib) in HR+ MDA-MB-361 cells. Conclusions: The antitumor effects of PI3K and AKT inhibitors were enhanced by the addition of the novel mTOR inhibitor nab-sirolimus. The improved effectiveness of the PI3K and mTOR inhibitor combination was due to the reciprocal overcoming of resistance mechanisms induced by single agent treatment. These data support a vertical PI3K pathway inhibition strategy using nab-sirolimus and PI3K/AKT inhibitors in PIK3CA-mutated BrCa, regardless of HR status. Citation Format: Sean Wallace, Khine Nyein Myint, Shihe Hou, Maria Zalath, Andrew Kwon, Brian McMorran, Igor Vivanco. Evaluation of nab-sirolimus in combination with PI3K pathway inhibitors to overcome PI3K/mTOR resistance in PI3K-mutant breast cancer cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A117.

TP53/mTORC1-mediated bidirectional regulation of PD-L1 modulates immune evasion in hepatocellular carcinoma

BackgroundImmunotherapy has facilitated great breakthroughs in the treatment of hepatocellular carcinoma (HCC). However, the efficacy and response rate of immunotherapy are limited and vary among different patients with HCC. TP53...

#6310 ANALYSIS OF THERAPEUTIC ATTITUDES TOWARDS BK VIRUS INFECTION IN RENAL TRANSPLANTATION: A RETROSPECTIVE COHORT STUDY

Abstract Background and Aims BK virus infection continues to be one of the most common clinical issues encountered by transplants providers, which can lead to BK virus nephropathy and, ultimately, graft loss. Despite its importance, there are still many uncertainties surrounding effective prevention and treatment strategies. The aim of this study is to evaluate the BK virus prevalence disease in a cohort of kidney transplant patients, examine consequences of immunosuppression regimens readjustments and assess the progression of viremia and viruria. Method This study analyzed a cohort of patients who received kidney transplants from January 2011 to January 2020 and had a minimum graft survival of three months. In accordance with our established protocol, viremia was measured on a quarterly basis during the first year after transplantation and annually thereafter. BK virus infection (BKVi) was defined as a plasma viral load, determined by real-time PCR, of 10,000 copies/ml or greater, or a viral load of 5,000 copies/ml or greater if accompanied by viruria of greater than 10 million copies/ml. Out of the 490 patients initially selected, 42 were excluded from the study due to inadequate data. Results The maximum levels of viremia during the course of the study were classified as follows: viremia ≥ 10,000 (N = 78; 17.4%), between 5,000-10,000 (N = 11; 2.4%), between 500-1,000 (N = 17; 3.8%) and undetectable (N = 342; 76.3%). The maximum levels of viruria were classified as: viruria ≥ 1 million (N = 98, 21.9%), between 999,999-100,000 (N = 15; 3.3%), between 99,999-10,000 (N = 20, 4.5%) and undetectable (N = 315; 70.3%). In the group diagnosed with BKVi, viremia ≥ 10,000 appeared at a median post-transplant time of 5.8 (2.9-11.7) months. The treatment approach involved replacing tacrolimus or mycophenolate with mTOR inhibitor, based on the patient's immunological risk. In some cases of high immunological risk or intolerance to mTOR inhibitor during the first month, treatment with tacrolimus and mycophenolate was continued. If the previous regimens were not effective, treatment with cyclosporine and mTOR inhibitor was initiated in some cases. The key results are summarized in the Table 1. Conclusion The combination of tacrolimus and mTOR inhibitor appears to be an effective option for BK virus infection, while the combination of mTOR inhibitor and mycophenolate has a high discontinuation rate, which can result in subsequent increase in viremia. In cases where there is no favorable response, the combination of cyclosporine and mTOR inhibitor can be effective. The changes in immunosuppression in patients with BKVi did not result in significant differences in renal function at one year or five years after transplantation, although the sample size is limited and more research is needed to confirm this. Keeping tacrolimus and mycophenolate may lead to a longer time until viremia becomes negative, which could have long-term implications for renal function.

#4364 LEUCOPENIA AFTER KIDNEY TRANSPLANTATION: SWITCHING MYCOPHENOLATE TO MTOR INHIBITOR APPEARS SAFE AND FEASIBLE TO REDUCE THE RISK OF ALLOSENSITIZATION

Abstract Background and Aims Mycophenolate acid (MPA) is a standard component of maintenance immunosuppression after kidney transplantation. MPA, however, increases the risk of leucopenia. In the past, mTOR inhibitors (mTOR-I) in combination with calcineurin inhibitors (CNI), were considered immunologically safe but showed higher mortality in the long term. Method We conducted a retrospective single-center study analyzing 98 kidney transplant recipients (KTRs) under standard triple maintenance immunosuppression between 2020 and 2022 who developed leucopenia. We compared patient and kidney allograft outcomes between 31 KTRs who were temporarily switched from MPA to mTOR-I and 67 KTRs who underwent a reduction of MPA dose. Results A switch to mTOR-I was performed more often in case of a CMV high-risk status (50% vs. 21%, p = 0.009), CMV primary infection (68.2% vs. 22%, p&amp;lt;0.001), and more severe leucopenia (median nadir of neutropenia: 0.52 G/L vs. 0.86 G/L, p = 0.023). The median duration of leucopenia was comparable between the two groups (69 vs. 80 days). 9 of 67 KTRs (13%) developed de novo donor-specific antibodies (dnDSA) under reduced MPA compared to 1 of 31 KTRs (3%) under mTOR-I (p = 0.17). Biopsy-proven rejections were comparable between the two groups (1 in each). There was a slight but insignificant improvement of eGFR under mTOR-I compared to MPA (ΔeGFR +17 vs. +7 ml/min/1.73m2, p = 0.13) and no difference in proteinuria. Conclusion A temporary switch from MPA to mTOR-I is a feasible and safe strategy to overcome leucopenia, especially in cases of CMV high-risk status and CMV primary infection. No significant adverse effects on kidney allograft outcomes were observed. Development of dnDSA tended to be lower in the mTOR-I group, suggesting that this strategy may have the potential to reduce the risk of allosensitization.

Copper deprivation enhances the chemosensitivity of pancreatic cancer to rapamycin by mTORC1/2 inhibition

Cuproplasia, or copper-dependent cell proliferation, has been observed in varieties of solid tumors along with aberrant copper homeostasis. Several studies reported good response of patients to copper chelator assisted neoadjuvant chemotherapy, however, the internal target molecules are still undetermined. Unravel copper-associated tumor signaling would be valuable to forge new links to translate biology of copper into clinical cancer therapies. We evaluated the significance of high-affinity copper transporter-1 (CTR1) by bioinformatic analysis, and in 19 pairs of clinical specimens. Then, with the help of gene interference and chelating agent, enriched signaling pathways were identified by KEGG analysis and immunoblotting. Accompanying biological capability of pancreatic carcinoma-associated proliferation, cell cycle, apoptosis, and angiogenesis were investigated. Furthermore, a combination of mTOR inhibitor and CTR1 suppressor has been assessed in xenografted tumor mouse models. Hyperactive CTR1 was investigated in pancreatic cancer tissues and proven to as the key point of cancer copper homeostasis. Intracellular copper deprivation induced by CTR1 gene knock-down or systematic copper chelation by tetrathiomolybdate suppressed proliferation and angiogenesis of pancreatic cancer cell. PI3K/AKT/mTOR signaling pathway was suppressed by inhibiting the activation of p70(S6)K and p-AKT, and finally inhibited mTORC1 and mTORC2 after copper deprivation. Additionally, CTR1 gene silencing successfully improved the anti-cancer effect of mTOR inhibitor rapamycin. Our study reveals that CTR1 contributes to pancreatic tumorigenesis and progression, by up-regulating the phosphorylation of AKT/mTOR signaling molecules. Recovering copper balance by copper deprivation addresses as promising strategy for improved cancer chemotherapy.

Calcineurin-inhibitor free immunosuppression after lung transplantation - a single center case-control study in 51 patients converted to Mechanistic Target of Rapamycin (mTOR) inhibitors.

Data on calcineurin-inhibitor (CNI) free immunosuppression after lung transplantation (LTx) are limited. Aim of this study was to investigate CNI-free immunosuppression using mechanistic target of rapamycin (mTOR) inhibitors. This retrospective analysis was performed at a single center. Adult patients after LTx without CNI during the follow-up period were included. Outcome was compared to those LTx patients with malignancy who continued CNI. Among 2,099 patients in follow-up, fifty-one (2.4%) were converted median 6.2 years after LTx to a CNI-free regimen combining mTOR inhibitors with prednisolone and an antimetabolite, two patients were switched to mTOR inhibitors with prednisolone only. In 25 patients, malignancies without curative treatment options were the reason of the conversion, with a 1-year survival of 36%. The remaining patients had a 1-year survival of 100%. Most common non-malignant indication was neurological complications (n = 9). Fifteen patients were re-converted to a CNI-based regimen. The median duration of CNI-free immunosuppression was 338 days. No acute rejections were detected in 7 patients with follow-up biopsies. In multivariate analysis, CNI-free immunosuppression were not associated with improved survival after malignancy. The majority of patients with neurological diseases improved 12 months after conversion. Glomerular filtration rate increased by median 5 (25 and 75% percentiles -6; +18) ml/min/1.73 m2. mTOR inhibitor based CNI-free immunosuppression may be safely performed in selected patients after LTx. This approach was not associated with improved survival in patients with malignancy. Significant functional improvements were observed in patients with neurological diseases.

Three generations of mTOR kinase inhibitors in the activation of the apoptosis process in melanoma cells

Many signaling pathways are involved in the mammalian target of rapamycin (mTOR), and this serine/threonine kinase regulates the most important cellular processes such as cell proliferation, autophagy, and apoptosis. The subject of this research was the effect of protein kinase inhibitors involved in the AKT, MEK, and mTOR kinase signaling pathways on the expression of pro-survival proteins, activity of caspase-3, proliferation, and induction of apoptosis in melanoma cells. The following inhibitors were used: protein kinase inhibitors such as AKT—MK-2206, MEK—AS-703026, mTOR—everolimus and Torkinib, as well as dual PI3K and mTOR inhibitor—BEZ-235 and Omipalisib, and mTOR1/2—OSI-027 inhibitor in single-mode and their combinations with MEK1/2 kinase inhibitor AS-703026. The obtained results confirm the synergistic effect of nanomolar concentrations of mTOR inhibitors, especially the dual PI3K and mTOR inhibitors (Omipalisib, BEZ-235) in combination with the MAP kinase inhibitor (AS-703026) in the activation of caspase 3, induction of apoptosis, and inhibition of proliferation in melanoma cell lines. Our previous and current studies confirm the importance of the mTOR signal transduction pathway in the neoplastic transformation process. Melanoma is a case of a very heterogeneous neoplasm, which causes great difficulties in treating this neoplasm in an advanced stage, and the standard approach to this topic does not bring the expected results. There is a need for research on the search for new therapeutic strategies aimed at particular groups of patients.Graphical abstractEffect of three generations of mTOR kinase inhibitors on caspase-3 activity, apoptosis and proliferation in melanoma cell lines.

The activation of mTOR signalling modulates DNA methylation by enhancing DNMT1 translation in hepatocellular carcinoma

BackgroundBoth dysregulation of mechanistic target of rapamycin (mTOR) signalling and DNA methylation patterns have been shown to be closely associated with tumor progression and serve as promising targets for hepatocellular carcinoma (HCC) therapy. Although their respective roles in HCC have been extensively revealed, the existence of molecular interactions between them remains largely unknown.MethodsThe association of DNA methylation and mTOR signalling in HCC tissues and cell lines was assessed. A Kaplan‒Meier analysis was applied to estimate the overall survival (OS) and recurrence-free survival (RFS) of HCC patients. The modulation of DNMT1 by mTOR in HCC cell lines was determined. The effect of the drug combination in cell lines and mouse models was examined.ResultsThe results showed that the DNA methylation level was positively associated with the activation of mTOR signalling in HCC tissues and cell lines. Moreover, HCC patients with higher DNA methylation levels and enhanced activation of mTOR signalling exhibited the worst prognosis. Then, we screened methylation-related enzymes and found that the activation of mTOR signalling increased DNMT1 expression and activity. In addition, mTOR enhanced the translational efficiency of DNMT1 in a 4E-BP1-dependent manner, which is based on the pyrimidine rich translational element (PRTE)-containing 5′UTR of DNMT1. Moreover, we demonstrated that the combined inhibition of mTOR and DNMT synergistically inhibited HCC growth in vitro and in vivo.ConclusionsIn addition to some already identified pro-cancer downstream molecules, the activation of mTOR signalling was found to promote DNA methylation by increasing the translation of DNMT1. Furthermore, combined targeting of mTOR and DNMT1 has been demonstrated to have a more effective tumor suppressive function in HCC.

MTOR Inhibition Impairs the Activation and Function of Belatacept-Resistant CD4+CD57+ T Cells In Vivo and In Vitro.

Calcineurin inhibitors have improved graft survival in solid-organ transplantation but their use is limited by toxicity, requiring a switch to another immunosuppressor in some cases. Belatacept is one option that has been shown to improve graft and patient survival despite being associated with a higher risk of acute cellular rejection. This risk of acute cellular rejection is correlated with the presence of belatacept-resistant T cells. We performed a transcriptomic analysis of in vitro-activated cells to identify pathways affected by belatacept in belatacept-sensitive cells (CD4+CD57-) but not in belatacept-resistant CD4+CD57+ T cells. mTOR was significantly downregulated in belatacept-sensitive but not belatacept-resistant T cells. The inhibition of mTOR strongly decreases the activation and cytotoxicity of CD4+CD57+ cells. In humans, the use of a combination of mTOR inhibitor and belatacept prevents graft rejection and decreases the expression of activation markers on CD4 and CD8 T cells. mTOR inhibition decreases the functioning of belatacept-resistant CD4+CD57+ T cells in vitro and in vivo. It could potentially be used in association with belatacept to prevent acute cellular rejection in cases of calcineurin intolerance.

MTOR inhibition overcomes RSK3-mediated resistance to BET inhibitors in small cell lung cancer.

Small cell lung cancer (SCLC) is a recalcitrant malignancy with limited treatment options. Bromodomain and extraterminal domain inhibitors (BETis) have shown promising preclinical activity in SCLC, but the broad sensitivity spectrum limits their clinical prospects. Here, we performed unbiased high-throughput drug combination screens to identify therapeutics that could augment the antitumor activities of BETis in SCLC. We found that multiple drugs targeting the PI-3K-AKT-mTOR pathway synergize with BETis, among which mTOR inhibitors (mTORis) show the highest synergy. Using various molecular subtypes of the xenograft models derived from patients with SCLC, we confirmed that mTOR inhibition potentiates the antitumor activities of BETis in vivo without substantially increasing toxicity. Furthermore, BETis induce apoptosis in both in vitro and in vivo SCLC models, and this antitumor effect is further amplified by combining mTOR inhibition. Mechanistically, BETis induce apoptosis in SCLC by activating the intrinsic apoptotic pathway. However, BET inhibition leads to RSK3 upregulation, which promotes survival by activating the TSC2-mTOR-p70S6K1-BAD cascade. mTORis block this protective signaling and augment the apoptosis induced by BET inhibition. Our findings reveal a critical role of RSK3 induction in tumor survival upon BET inhibition and warrant further evaluation of the combination of mTORis and BETis in patients with SCLC.

Co-targeting BCL-XL and MCL-1 with DT2216 and AZD8055 synergistically inhibit small-cell lung cancer growth without causing on-target toxicities in mice

Small-cell lung cancer (SCLC) is an aggressive malignancy with limited therapeutic options. The dismal prognosis in SCLC is in part associated with an upregulation of BCL-2 family anti-apoptotic proteins, including BCL-XL and MCL-1. Unfortunately, the currently available inhibitors of BCL-2 family anti-apoptotic proteins, except BCL-2 inhibitors, are not clinically relevant because of various on-target toxicities. We, therefore, aimed to develop an effective and safe strategy targeting these anti-apoptotic proteins with DT2216 (our platelet-sparing BCL-XL degrader) and AZD8055 (an mTOR inhibitor) to avoid associated on-target toxicities while synergistically optimizing tumor response. Through BH3 mimetic screening, we identified a subset of SCLC cell lines that is co-dependent on BCL-XL and MCL-1. After screening inhibitors of selected tumorigenic pathways, we found that AZD8055 selectively downregulates MCL-1 in SCLC cells and its combination with DT2216 synergistically killed BCL-XL/MCL-1 co-dependent SCLC cells, but not normal cells. Mechanistically, the combination caused BCL-XL degradation and suppression of MCL-1 expression, and thus disrupted MCL-1 interaction with BIM leading to an enhanced apoptotic induction. In vivo, the DT2216 + AZD8055 combination significantly inhibited the growth of cell line-derived and patient-derived xenografts and reduced tumor burden accompanied by increased survival in a genetically engineered mouse model of SCLC without causing appreciable thrombocytopenia or other normal tissue injuries. Thus, these preclinical findings lay a strong foundation for future clinical studies to test DT2216 + mTOR inhibitor combinations in a subset of SCLC patients whose tumors are co-driven by BCL-XL and MCL-1.

Functional characterization of the PI3K/AKT/MTOR signaling pathway for targeted therapy in B-precursor acute lymphoblastic leukemia

B-cell precursor acute lymphoblastic leukemias (B-ALL) are characterized by the activation of signaling pathways, which are involved in survival and proliferation of leukemia cells. Using an unbiased shRNA library screen enriched for targeting signaling pathways, we identified MTOR as the key gene on which human B-ALL E2A-PBX1+ RCH-ACV cells are dependent. Using genetic and pharmacologic approaches, we investigated whether B-ALL cells depend on MTOR upstream signaling pathways including PI3K/AKT and the complexes MTORC1 or MTORC2 for proliferation and survival in vitro and in vivo. Notably, the combined inhibition of MTOR and AKT shows a synergistic effect on decreased cell proliferation in B-ALL with different karyotypes. Hence, B-ALL cells were more dependent on MTORC2 rather than MTORC1 complex in genetic assays. Using cell metabolomics, we identified changes in mitochondrial fuel oxidation after shRNA-mediated knockdown or pharmacological inhibition of MTOR. Dependence of the cells on fatty acid metabolism for their energy production was increased upon inhibition of MTOR and associated upstream signaling pathways, disclosing a possible target for a combination therapy. In conclusion, B-ALL are dependent on the PI3K/AKT/MTOR signaling pathway and the combination of specific small molecules targeting this pathway appears to be promising for the treatment of B-ALL patients.

Abstract 3232: Combined inhibition of mTOR and HSP90 potentiates cisplatin antitumor effect and reverts cisplatin resistance in vitro and in vivo models of epithelial ovarian cancer by modulating HSF1-dependent transactivation

Abstract INTRODUCTION: Epithelial ovarian cancer (EOC) represents the most lethal gynecological disease, with a 5-year relative survival rate of 46% after the diagnosis. The standard treatment of advanced EOC is based on surgery, followed by platinum (Pt)-based chemotherapy. However, the development of platinum resistant disease could occur and strongly impact on the survival of EOC patients for whom we still do not have valid therapeutic options. By using a proteomic approach followed by a bioinformatic analysis, we previously validated the role of HSP90 in the mechanism of platinum-resistance. Here, we propose a novel therapeutic strategy based on the combined pharmacologic inhibition of HSP90 and mTOR to further potentiate Pt-based chemotherapy and to revert Pt-resistance in EOC and non-small-cell lung cancer (NSCLC) models. METHODS: TOV-112D parental and Pt-resistant cells were characterized by phosphoproteomics followed by functional analysis and proteins validation by western blot. Synergistic anti-tumor effect was evaluated in Pt-sensitive and Pt-resistant EOC and NSCLC cell lines by calculating combination indexes (CI), colony formation assay, apoptosis and DNA damage, as well as on 3D in vitro microtissues obtained by co-culturing cancer cells with normal fibroblasts and in vivo EOC and NSCLC xenograft models. RESULTS: 542 differentially phosphorylated expressed proteins were identified in Pt-resistant TOV-112D compared with parental cells, and mTOR and the transcription factor HSF1 emerged as the most enriched pathways. The up-regulation of the phosphorylated form of PDK1, AKT, mTOR and rpS6 was observed in Pt-resistant compared to parental cells. Moreover, we also demonstrated the up-regulation of the activity of HSF1 along with the elevation of its targets such as heat shock proteins HSP90, HSP70 and HSP40, crucial components of chaperone complex machinery. Interestingly, among the differentially expressed proteins, we identified the kinase DYRK2 able to phosphorylate HSF1, supporting its transactivation. Accordingly, the combination of HSP90 inhibitor ganetespib and the mTOR inhibitor temsirolimus plus cisplatin, synergistically reduced colony formation, cancer cells and microtissues cell growth in vitro by increasing DNA-damage and apoptosis and in vivo by enhancing mouse survival. Mechanistically, the triple combination treatment, impaired the proteins involved in mTOR signalling and HSF1 transactivation. Notably, all these data were confirmed in Pt-resistant NSCLC models, supporting the possibility that the same mechanism is present in different tumor types. CONCLUSIONS: Our findings identify a promising new antitumor strategy based on the combination of HSP90 and mTOR inhibitors to revert Pt-resistance that that warrant further clinical evaluation. Citation Format: Rita Lombardi, Laura Addi, Biagio Pucci, Maura Sonego, Maria Serena Roca, Francesca Capone, Federica Iannelli, Francesca Bruzzese, Gustavo Baldassarre, Alfredo Budillon. Combined inhibition of mTOR and HSP90 potentiates cisplatin antitumor effect and reverts cisplatin resistance in vitro and in vivo models of epithelial ovarian cancer by modulating HSF1-dependent transactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3232.