HMGA1 sensitizes esophageal squamous cell carcinoma to mTOR inhibitors through the ETS1-FKBP12 axis.

Esophageal carcinoma is amongst the prevalent malignancies worldwide, characterized by unclear molecular classifications and varying clinical outcomes. The PI3K/AKT/mTOR signaling, one of the frequently perturbed dysregulated pathways in human malignancies, has instigated the development of various inhibitory agents targeting this pathway, but many ESCC patients exhibit intrinsic or adaptive resistance to these inhibitors. Here, we aim to explore the reasons for the insensitivity of ESCC patients to mTOR inhibitors. We assessed the sensitivity to rapamycin in various ESCC cell lines by determining their respective IC50 values and found that cells with a low level of HMGA1 were more tolerant to rapamycin. Subsequent experiments have supported this finding. Through a transcriptome sequencing, we identified a crucial downstream effector of HMGA1, FKBP12, and found that FKBP12 was necessary for HMGA1-induced cell sensitivity to rapamycin. HMGA1 interacted with ETS1, and facilitated the transcription of FKBP12. Finally, we validated this regulatory axis in in vivo experiments, where HMGA1 deficiency in transplanted tumors rendered them resistance to rapamycin. Therefore, we speculate that mTOR inhibitor therapy for individuals exhibiting a reduced level of HMGA1 or FKBP12 may not work. Conversely, individuals exhibiting an elevated level of HMGA1 or FKBP12 are more suitable candidates for mTOR inhibitor treatment.

Primary prophylaxis with mTOR inhibitor enhances T cell effector function and prevents heart transplant rejection during talimogene laherparepvec therapy of squamous cell carcinoma.

The application of mammalian target of rapamycin inhibition (mTORi) as primary prophylactic therapy to optimize T cell effector function while preserving allograft tolerance remains challenging. Here, we present a comprehensive two-step therapeutic approach in a male patient with metastatic cutaneous squamous cell carcinoma and heart transplantation followed with concomitant longitudinal analysis of systemic immunologic changes. In the first step, calcineurin inhibitor/ mycophenolic acid is replaced by the mTORi everolimus to achieve an improved effector T cell status with increased cytotoxic activity (perforin, granzyme), enhanced proliferation (Ki67) and upregulated activation markers (CD38, CD69). In the second step, talimogene laherparepvec (T-VEC) injection further enhances effector function by switching CD4 and CD8 cells from central memory to effector memory profiles, enhancing Th1 responses, and boosting cytotoxic and proliferative activities. In addition, cytokine release (IL-6, IL-18, sCD25, CCL-2, CCL-4) is enhanced and the frequency of circulating regulatory T cells is increased. Notably, no histologic signs of allograft rejection are observed in consecutive end-myocardial biopsies. These findings provide valuable insights into the dynamics of T cell activation and differentiation and suggest that timely initiation of mTORi-based primary prophylaxis may provide a dual benefit of revitalizing T cell function while maintaining allograft tolerance.

PI3K/mTOR inhibition induces tumour microenvironment remodelling and sensitises pS6high uterine leiomyosarcoma to PD-1 blockade.

BACKGROUND: Uterine leiomyosarcomas (uLMS) are aggressive tumours with poor prognosis and limited treatment options. Although immune checkpoint blockade (ICB) has proven effective in some 'challenging-to-treat' cancers, clinical trials showed that uLMS do not respond to ICB. Emerging evidence suggests that aberrant PI3K/mTOR signalling can drive resistance to ICB. We therefore explored the relevance of the PI3K/mTOR pathway for ICB treatment in uLMS and explored pharmacological inhibition of this pathway to sensitise these tumours to ICB. METHODS: We performed an integrated multiomics analysis based on TCGA data to explore the correlation between PI3K/mTOR dysregulation and immune infiltration in 101 LMS. We assessed response to PI3K/mTOR inhibitors in immunodeficient and humanized uLMS patient-derived xenografts (PDXs) by evaluating tumour microenvironment modulation using multiplex immunofluorescence. We explored response to single-agent and a combination of PI3K/mTOR inhibitors with PD-1 blockade in humanized uLMS PDXs. We mapped intratumoural dynamics using single-cell RNA/TCR sequencing of serially collected biopsies. RESULTS: PI3K/mTOR over-activation (pS6high) associated with lymphocyte depletion and wound healing immune landscapes in (u)LMS, suggesting it contributes to immune evasion. In contrast, PI3K/mTOR inhibition induced profound tumour microenvironment remodelling in an ICB-resistant humanized uLMS PDX model, fostering adaptive anti-tumour immune responses. Indeed, PI3K/mTOR inhibition induced macrophage repolarisation towards an anti-tumourigenic phenotype and increased antigen presentation on dendritic and tumour cells, but also promoted infiltration of PD-1+ T cells displaying an exhausted phenotype. When combined with anti-PD-1, PI3K/mTOR inhibition led to partial or complete tumour responses, whereas no response to single-agent anti-PD-1 was observed. Combination therapy reinvigorated exhausted T cells and induced clonal hyper-expansion of a cytotoxic CD8+ T-cell population supported by a CD4+ Th1 niche. CONCLUSIONS: Our findings indicate that aberrant PI3K/mTOR pathway activation contributes to immune escape in uLMS and provides a rationale for combining PI3K/mTOR inhibition with ICB for the treatment of this patient population.

Design, Synthesis, Formulation, and Bioevaluation of Trisubstituted Triazines as Highly Selective mTOR Inhibitors for the Treatment of Human Breast Cancer.

The aberrant activation of the PI3K/mTOR signaling pathway is implicated in various human cancers. Thus, the development of inhibitors targeting mTOR has attracted considerable attention. In this study, we used a structure-based drug design strategy to discover a highly potent and kinase-selective mTOR inhibitor 24 (PT-88), which demonstrated an mTOR inhibitory IC50 value of 1.2 nM without obvious inhibition against another 195 kinases from the kinase profiling screening. PT-88 displayed selective inhibition against MCF-7 cells (IC50: 0.74 µM) with high biosafety against normal cells, in which autophagy induced by mTOR inhibition was implicated. After successful encapsulation in a lipodisc formulation, PT-88 demonstrated favorable pharmacokinetic and biosafety profiles and exerted a large antitumor effect in an MCF-7 subcutaneous bearing nude mice model. Our study shows the discovery of a highly selective mTOR inhibitor using a structure-based drug discovery strategy and provides a promising antitumor candidate for future study and development.

A Novel Dual PI3K/mTOR Inhibitor, XIN-10, for the Treatment of Cancer.

An imbalance in PI3K/AKT/mTOR pathway signaling in humans often leads to cancer. Therefore, the investigation of anti-cancer medications that inhibit PI3K and mTOR has emerged as a significant area of research. The aim of this study was to explore the effect of XIN-10, a dual PI3K/mTOR inhibitor, on the growth as well as antiproliferation of tumor cells and to investigate the anti-tumor mechanism of XIN-10 by further exploration. We screened three cell lines for more in-depth exploration by MTT experiments. From the AO staining, cell cycle and apoptosis, we found that XIN-10 had a more obvious inhibitory effect on the MCF-7 breast cancer cell line and used this as a selection for more in-depth experiments. A series of in vitro and in vivo experiments showed that XIN-10 has superior antiproliferative activity compared with the positive drug GDC-0941. Meanwhile, through the results of protein blotting and PCR experiments, we concluded that XIN-10 can block the activation of the downstream pathway of mTOR by inhibiting the phosphorylation of AKT(S473) as well as having significant inhibitory effects on the gene exons of PI3K and mTOR. These results indicate that XIN-10 is a highly potent inhibitor with low toxicity and has a strong potential to be developed as a novel PI3Kα/mTOR dual inhibitor candidate for the treatment of positive breast cancer.

mTOR Inhibitors Modulate the Physical Properties of 3D Spheroids Derived from H9c2 Cells.

To establish an appropriate in vitro model for the local environment of cardiomyocytes, three-dimensional (3D) spheroids derived from H9c2 cardiomyoblasts were prepared, and their morphological, biophysical phase contrast and biochemical characteristics were evaluated. The 3D H9c2 spheroids were successfully obtained, the sizes of the spheroids decreased, and they became stiffer during 3-4 days. In contrast to the cell multiplication that occurs in conventional 2D planar cell cultures, the 3D H9c2 spheroids developed into a more mature form without any cell multiplication being detected. qPCR analyses of the 3D H9c2 spheroids indicated that the production of collagen4 (COL4) and fibronectin (FN), connexin43 (CX43), ß-catenin, N-cadherin, STAT3, and HIF1 molecules had increased and that the production of COL6 and α-smooth muscle actin (α-SMA) molecules had decreased as compared to 2D cultured cells. In addition, treatment with rapamycin (Rapa), an mTOR complex (mTORC) 1 inhibitor, and Torin 1, an mTORC1/2 inhibitor, resulted in significantly decreased cell densities of the 2D cultured H9c2 cells, but the size and stiffness of the H9c2 cells within the 3D spheroids were reduced with the gene expressions of several of the above several factors being reduced. The metabolic responses to mTOR modulators were also different between the 2D and 3D cultures. These results suggest that as unique aspects of the local environments of the 3D spheroids, the spontaneous expression of GJ-related molecules and hypoxia within the core may be associated with their maturation, suggesting that this may become a useful in vitro model that replicates the local environment of cardiomyocytes.

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.

NSCLC Cells Resistance to PI3K/mTOR Inhibitors Is Mediated by Delta-6 Fatty Acid Desaturase (FADS2).

Hyperactivation of the phosphatidylinositol-3-kinase (PI3K) pathway is one of the most common events in human cancers. Several efforts have been made toward the identification of selective PI3K pathway inhibitors. However, the success of these molecules has been partially limited due to unexpected toxicities, the selection of potentially responsive patients, and intrinsic resistance to treatments. Metabolic alterations are intimately linked to drug resistance; altered metabolic pathways can help cancer cells adapt to continuous drug exposure and develop resistant phenotypes. Here we report the metabolic alterations underlying the non-small cell lung cancer (NSCLC) cell lines resistant to the usual PI3K-mTOR inhibitor BEZ235. In this study, we identified that an increased unsaturation degree of lipid species is associated with increased plasma membrane fluidity in cells with the resistant phenotype and that fatty acid desaturase FADS2 mediates the acquisition of chemoresistance. Therefore, new studies focused on reversing drug resistance based on membrane lipid modifications should consider the contribution of desaturase activity.

Brain-restricted mTOR inhibition with binary pharmacology.

On-target-off-tissue drug engagement is an important source of adverse effects that constrains the therapeutic window of drug candidates1,2. In diseases of the central nervous system, drugs with brain-restricted pharmacology are highly desirable. Here we report a strategy to achieve inhibition of mammalian target of rapamycin (mTOR) while sparing mTOR activity elsewhere through the use of the brain-permeable mTOR inhibitor RapaLink-1 and the brain-impermeable FKBP12 ligand RapaBlock. We show that this drug combination mitigates the systemic effects of mTOR inhibitors but retains the efficacy of RapaLink-1 in glioblastoma xenografts. We further present a general method to design cell-permeable, FKBP12-dependent kinase inhibitors from known drug scaffolds. These inhibitors are sensitive to deactivation by RapaBlock, enabling the brain-restricted inhibition of their respective kinase targets.

mTOR inhibition prevents angiotensin II-induced aortic rupture and pseudoaneurysm but promotes dissection in Apoe-deficient mice.

Aortic dissection and rupture are triggered by decreased vascular wall strength and/or increased mechanical loads. We investigated the role of mTOR signaling in aortopathy using a well-described model of angiotensin II-induced dissection, aneurysm, or rupture of the suprarenal abdominal aorta in Apoe-deficient mice. Although not widely appreciated, nonlethal hemorrhagic lesions present as pseudoaneurysms without significant dissection in this model. Angiotensin II-induced aortic tears result in free rupture, contained rupture with subadventitial hematoma (forming pseudoaneurysms), dilatation, or healing, while the media invariably thickens regardless of mural tears. Medial thickening results from smooth muscle cell hypertrophy and extracellular matrix accumulation, including matricellular proteins. Angiotensin II activates mTOR signaling in vascular wall cells, and inhibition of mTOR signaling by rapamycin prevents aortic rupture but promotes dissection. Decreased aortic rupture correlates with decreased inflammation and metalloproteinase expression, whereas extensive dissection correlates with induction of matricellular proteins that modulate adhesion of vascular cells. Thus, mTOR activation in vascular wall cells determines whether aortic tears progress to dissection or rupture. Previous mechanistic studies of aortic aneurysm and dissection by angiotensin II in Apoe-deficient mice should be reinterpreted as clinically relevant to pseudoaneurysms, and mTOR inhibition for aortic disease should be explored with caution.

Turnover of the mTOR inhibitor, DEPTOR, and downstream AKT phosphorylation in multiple myeloma cells, is dependent on ERK1-mediated phosphorylation.

DEPTOR is a 48 kDa protein upregulated in multiple myeloma (MM) cells. DEPTOR inhibits mTOR and, by repressing a negative feedback loop, promotes AKT activation. We previously identified a compound that binds to DEPTOR in MM cells and induces its proteasomal degradation. To identify the mechanism of degradation, here, we screened for drug-induced posttranslational modifications and identified reduced phosphorylation of DEPTOR on serine 235 (S235). We show that an S235 phosphomimetic DEPTOR mutant was resistant to degradation, confirming the importance of this posttranslational modification. In addition, a DEPTOR mutant with a serine-to-alanine substitution at S235 could only be expressed upon concurrent proteasome inhibition. Thus, S235 phosphorylation regulates DEPTOR stability. Screening the DEPTOR interactome identified that the association of USP-7 deubiquitinase with DEPTOR was dependent upon S235 phosphorylation. Inhibition of USP-7 activity resulted in DEPTOR polyubiquitination and degradation. A scansite search suggested that ERK1 may be responsible for S235 phosphorylation, which was confirmed through the use of inhibitors, ERK1 knockdown, and an in vitro kinase assay. Inhibition of ERK1 also downregulated AKT phosphorylation. To test if DEPTOR phosphorylation mediated this crosstalk, MM cells were transfected with WT or phosphomimetic DEPTOR and exposed to ERK inhibitors. Although WT DEPTOR had no effect on the inhibition of AKT phosphorylation, the phosphomimetic DEPTOR prevented inhibition. These results indicate that ERK1 maintains AKT activity in MM cells via phosphorylation of DEPTOR. We propose that DEPTOR-dependent crosstalk provides MM cells with a viability-promoting signal (through AKT) when proliferation is stimulated (through ERK).

JAK and mTOR inhibitors prevent cytokine release while retaining T cell bispecific antibody in vivo efficacy.

BACKGROUND: T cell engaging therapies, like chimeric antigen receptor T cells and T cell bispecific antibodies (TCBs), efficiently redirect T cells towards tumor cells, facilitating the formation of a cytotoxic synapse and resulting in subsequent tumor cell killing, a process that is accompanied by the release of cytokines. Despite their promising efficacy in the clinic, treatment with TCBs is associated with a risk of cytokine release syndrome (CRS). The aim of this study was to identify small molecules able to mitigate cytokine release while retaining T cell-mediated tumor killing. METHODS: By screening a library of 52 Food and Drug Administration approved kinase inhibitors for their impact on T cell proliferation and cytokine release after CD3 stimulation, we identified mTOR, JAK and Src kinases inhibitors as potential candidates to modulate TCB-mediated cytokine release at pharmacologically active doses. Using an in vitro model of target cell killing by human peripheral blood mononuclear cells, we assessed the effects of mTOR, JAK and Src kinase inhibitors combined with 2+1 T cell bispecific antibodies (TCBs) including CEA-TCB and CD19-TCB on T cell activation, proliferation and target cell killing measured by flow cytometry and cytokine release measured by Luminex. The combination of mTOR, JAK and Src kinase inhibitors together with CD19-TCB was evaluated in vivo in non-tumor bearing stem cell humanized NSG mice in terms of B cell depletion and in a lymphoma patient-derived xenograft (PDX) model in humanized NSG mice in terms of antitumor efficacy. RESULTS: The effect of Src inhibitors differed from those of mTOR and JAK inhibitors with the suppression of CD19-TCB-induced tumor cell lysis in vitro, whereas mTOR and JAK inhibitors primarily affected TCB-mediated cytokine release. Importantly, we confirmed in vivo that Src, JAK and mTOR inhibitors strongly reduced CD19-TCB-induced cytokine release. In humanized NSG mice, continuous treatment with a Src inhibitor prevented CD19-TCB-mediated B cell depletion in contrast to mTOR and JAK inhibitors, which retained CD19-TCB efficacy. Ultimately, transient treatment with Src, mTOR and JAK inhibitors minimally interfered with antitumor efficacy in a lymphoma PDX model. CONCLUSIONS: Taken together, these data support further evaluation of the use of Src, JAK and mTOR inhibitors as prophylactic treatment to prevent occurrence of CRS.

mTOR inhibitor PP242 increases antitumor activity of sulforaphane by blocking Akt/mTOR pathway in esophageal squamous cell carcinoma.

BACKGROUND: Sulforaphane (SFN) is a kind of isothiocyanate from cruciferous vegetables with extensive anti-tumor activity. Esophageal squamous cell carcinoma (ESCC) is a popular malignancy in East Asia, East and South Africa, while the more efficient medicines and therapeutic strategies are still lack. This study aims to explore the anti-tumor activity of SFN alone and combined with Akt/mTOR pathway inhibitors as well as the potential molecular mechanism in ESCC. METHODS AND RESULTS: Cell proliferation, migration, cell cycle phase, apoptosis and protein expression were detected with MTT assay, clone formation experiment, wound healing assays, flow cytometry and Western blot, respectively, after ESCC cells ECa109 and EC9706 treated with SFN alone or combined with Akt/mTOR inhibitors. Xenograft models were used to evaluate the efficiency and mechanism of SFN combined with PP242 in vivo. The results showed that SFN significantly inhibited the viability and induced apoptosis of ECa109 and EC9706 cells by increasing expression of Cleaved-caspase 9. SFN combined with PP242, but not MK2206 and RAD001, synergetic inhibited proliferation of ESCC cells. Moreover, compared to SFN alone, combination of SFN and PP242 had stronger inhibiting efficiency on clone formation, cell migratory, cell cycle phase and growth of xenografts, as well as the more powerful apoptosis-inducing effects on ESCC. The mechanism was that PP242 abrogated the promoting effects of SFN on p-p70S6K (Thr389) and p-Akt (Ser473) in ESCC. CONCLUSIONS: Our findings demonstrate that PP242 enhances the anti-tumor activity of SFN by blocking SFN-induced activation of Akt/mTOR pathway in ESCC, which provides a rationale for treating ESCC using SFN combined with Akt/mTOR pathway inhibitors.

KDM1A inhibition augments the efficacy of rapamycin for the treatment of endometrial cancer.

Endometrial cancer (EC) often exhibit aberrant activation of PI3K/Akt/mTOR signaling and targeted therapies using mTOR inhibitors showed limited success. The epigenetic modifier, lysine-specific histone demethylase-1A (KDM1A/LSD1) is overexpressed in EC, however, the mechanistic and therapeutic implications of KDM1A in EC are poorly understood. Here, using 119 FDA-approved drugs screen, we identified that KDM1A inhibition is highly synergistic with mTOR inhibitors. Combination therapy of KDM1A and mTOR inhibitors potently reduced the cell viability, survival, and migration of EC cells. Mechanistic studies demonstrated that KDM1A inhibition attenuated the activation of mTOR signaling cascade and abolished rapamycin induced feedback activation of Akt. RNA-seq analysis identified that KDM1A inhibition downregulated the expression of genes involved in rapamycin induced activation of Akt, including the mTORC2 complex. Chromatin immunoprecipitation experiments confirmed KDM1A recruitment to the promoter regions of mTORC2 complex genes and that KDM1A inhibition promoted enrichment of repressive H3K9me2 marks at their promoters. Combination therapy of KDM1A inhibitor and rapamycin reduced the tumor growth in EC xenograft and patient derived xenograft models in vivo and patient derived tumor explants ex vivo. Importantly, in silico analysis of TCGA EC patients data sets revealed that KDM1A expression positively correlated with the levels of PI3K/Akt/mTOR genes. Collectively, our results provide compelling evidence that KDM1A inhibition potentiates the activity of mTOR inhibitors by attenuating the feedback activation of Akt survival signaling. Furthermore, the use of concurrent KDM1A and mTOR inhibitors may be an attractive targeted therapy for EC patients.

Transcriptomic Repositioning Analysis Identifies mTOR Inhibitor as Potential Therapy for Epidermolysis Bullosa Simplex.

Expression-based systematic drug repositioning has been explored to predict novel treatments for a number of skin disorders. In this study, we utilize this approach to identify, to our knowledge, previously unreported therapies for epidermolysis bullosa simplex (EBS). RNA sequencing analysis was performed on skin biopsies of acute blisters (<1 week old) (n = 9) and nonblistered epidermis (n = 11) obtained from 11 patients with EBS. Transcriptomic analysis of blistered epidermis in patients with EBS revealed a set of 1,276 genes dysregulated in EBS blisters. The IL-6, IL-8, and IL-10 pathways were upregulated in the epidermis from EBS. Consistent with this, predicted upstream regulators included TNF-α, IL-1ß, IL-2, IL-6, phosphatidylinositol 3-kinase, and mTOR. The 1,276 gene EBS blister signature was integrated with molecular signatures from cell lines treated with 2,423 drugs using the Connectivity Map CLUE platform. The mTOR inhibitors and phosphatidylinositol 3-kinase inhibitors most opposed the EBS signature. To determine whether mTOR inhibitors could be used clinically in EBS, we conducted an independent pilot study of two patients with EBS treated with topical sirolimus for painful plantar keratoderma due to chronic blistering. Both individuals experienced marked clinical improvement and a notable reduction of keratoderma. In summary, a computational drug repositioning analysis successfully identified, to our knowledge, previously unreported targets in the treatment of EBS.

Rapalogs induce non-apoptotic, autophagy-dependent cell death in HPV-negative TP53 mutant head and neck squamous cell carcinoma.

TP53 is the most frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). Patients with HPV-negative TP53 mutant HNSCC have the worst prognosis, necessitating additional agents for treatment. Since mutant p53 causes sustained activation of the PI3K/AKT/mTOR signaling pathway, we investigated the effect of rapalogs RAD001 and CCI-779 on HPV-negative mutTP53 HNSCC cell lines and xenografts. Rapalogs significantly reduced cell viability and colony formation. Interestingly, rapalogs-induced autophagy with no effect on apoptosis. Pretreatment with autophagy inhibitors, 3-methyladenine (3-MA) and ULK-101 rescued the cell viability by inhibiting rapalog-induced autophagy, suggesting that both RAD001 and CCI-779 induce non-apoptotic autophagy-dependent cell death (ADCD). Moreover, rapalogs upregulated the levels of ULK1 and pULK1 S555 with concomitant downregulation of the mTORC1 pathway. However, pretreatment of cells with rapalogs prevented the ULK-101-mediated inhibition of ULK1 to sustained autophagy, suggesting that rapalogs induce ADCD through the activation of ULK1. To further translate our in vitro studies, we investigated the effect of RAD001 in HPV-negative mutTP53 (HN31 and FaDu) tumor cell xenograft model in nude mice. Mice treated with RAD001 exhibited a significant tumor volume reduction without induction of apoptosis, and with a concomitant increase in autophagy. Further, treatment with RAD001 was associated with a considerable increase in pULK1 S555 and ULK1 levels through the inhibition of mTORC1. 3-MA reversed the effect of RAD001 on FaDu tumor growth suggesting that RAD001 promotes ACDC in HPV-negative mutTP53 xenograft. This is the first report demonstrating that rapalogs promote non-apoptotic ADCD in HPV-negative mutTP53 HNSCC via the ULK1 pathway. Further studies are required to establish the promising role of rapalogs in preventing the regrowth of HPV-negative mutTP53 HNSCC.

Jolkinolide B sensitizes bladder cancer to mTOR inhibitors via dual inhibition of Akt signaling and autophagy.

The monotherapy of mTOR inhibitors (mTORi) in cancer clinical practice has achieved limited success due to the concomitant activation of compensatory pathways, such as Akt signaling and cytoprotective autophagy. Thus, the combination of mTORi and the inhibitors of these pro-survival pathways has been considered a promising therapeutic strategy. Herein, we report the synergistic effects of a natural anti-cancer agent Jolkinolide B (JB) and mTORi (temsirolimus, rapamycin, and everolimus) for the effective treatment of bladder cancer. A mechanistic study revealed that JB induced a dual inhibition of Akt feedback activation and cytoprotective autophagy, potentiating the anti-proliferative efficacy of mTORi in both PTEN-deficient and cisplatin-resistant bladder cancer cells. Meanwhile, mTORi augmented the pro-apoptotic and pro-paraptotic effects of JB by reinforcing JB-activated endoplasmic reticulum stress and MAPK pathways. These synergistic mechanisms were related to cellular reactive oxygen species accumulation. Our study suggests that dual inhibition of Akt feedback activation and cytoprotective autophagy is an effective strategy in mTORi-based therapy, and JB + mTORi combination associated with multiple anti-cancer mechanisms and good tolerance in mouse models may serve as a promising treatment for bladder cancer.

Epithelial X-Box Binding Protein 1 Coordinates Tumor Protein p53-Driven DNA Damage Responses and Suppression of Intestinal Carcinogenesis.

BACKGROUND & AIMS: Throughout life, the intestinal epithelium undergoes constant self-renewal from intestinal stem cells. Together with genotoxic stressors and failing DNA repair, this self-renewal causes susceptibility toward malignant transformation. X-box binding protein 1 (XBP1) is a stress sensor involved in the unfolded protein response (UPR). We hypothesized that XBP1 acts as a signaling hub to regulate epithelial DNA damage responses. METHODS: Data from The Cancer Genome Atlas were analyzed for association of XBP1 with colorectal cancer (CRC) survival and molecular interactions between XBP1 and p53 pathway activity. The role of XBP1 in orchestrating p53-driven DNA damage response was tested in vitro in mouse models of chronic intestinal epithelial cell (IEC) DNA damage (Xbp1/H2bfl/fl, Xbp1ΔIEC, H2bΔIEC, H2b/Xbp1ΔIEC) and via orthotopic tumor organoid transplantation. Transcriptome analysis of intestinal organoids was performed to identify molecular targets of Xbp1-mediated DNA damage response. RESULTS: In The Cancer Genome Atlas data set of CRC, low XBP1 expression was significantly associated with poor overall survival and reduced p53 pathway activity. In vivo, H2b/Xbp1ΔIEC mice developed spontaneous intestinal carcinomas. Orthotopic tumor organoid transplantation revealed a metastatic potential of H2b/Xbp1ΔIEC-derived tumors. RNA sequencing of intestinal organoids (H2b/Xbp1fl/fl, H2bΔIEC, H2b/Xbp1ΔIEC, and H2b/p53ΔIEC) identified a transcriptional program downstream of p53, in which XBP1 directs DNA-damage-inducible transcript 4-like (Ddit4l) expression. DDIT4L inhibits mechanistic target of rapamycin-mediated phosphorylation of 4E-binding protein 1. Pharmacologic mechanistic target of rapamycin inhibition suppressed epithelial hyperproliferation via 4E-binding protein 1. CONCLUSIONS: Our data suggest a crucial role for XBP1 in coordinating epithelial DNA damage responses and stem cell function via a p53-DDIT4L-dependent feedback mechanism.

Investigation of the effects of mTOR inhibitors rapamycin and everolimus in combination with carboplatin on canine malignant melanoma cells.

BACKGROUND: Malignant melanoma in dogs is considered to be largely resistant to conventional chemotherapy, although responses to carboplatin have been documented. Invasion and early metastasis are common features of certain melanoma subtypes that contribute to tumour progression despite aggressive local and systemic therapy. Upregulation of the PI3K/AKT/mTOR pathway has been observed in canine malignant melanoma and may represent a potential target for therapy. Rapamycin (sirolimus) and everolimus are commercially available small molecule inhibitors that target mTOR and therefore may have anticancer activity in canine melanoma. It was hypothesized that there is synergism between rapamycin or everolimus and platinum chemotherapy, and that combination drug treatment would inhibit target/downstream proteins involved in cell viability/proliferation and increase cell death in canine melanoma cells. It was further hypothesized that rapamycin or everolimus would impact metabolism by reducing glycolysis in these cells. Four canine melanoma cell lines were treated in vitro with rapamycin and everolimus as sole treatment or combined with carboplatin. Cell viability, apoptosis, target modulation, and glycolytic metabolism were evaluated by crystal violet colourimetric assay, Annexin V/PI flow cytometry, western blotting, and Seahorse bioanalyzer, respectively. RESULTS: When combined with carboplatin chemotherapy, rapamycin or everolimus treatment was overall synergistic in reducing cell viability. Carboplatin-induced apoptosis was noted at 72 h after treatment compared to the vehicle control. Levels of phosphorylated mTOR were reduced by rapamycin and everolimus in all four cell lines, but activation of the downstream protein p70S6K was not consistently reduced by treatment in two of the cell lines. Both mTOR inhibitors decreased the extracellular acidification rate of canine melanoma cells, indicating reduced cancer cell glycolytic activity. CONCLUSIONS: Inhibition of mTOR by rapalogs, such as rapamycin and everolimus combined with carboplatin chemotherapy may have activity in canine melanoma. Future mechanistic investigation is warranted, including in vivo assessment of this combination therapy.

Topoisomerase 1 activity during mitotic transcription favors the transition from mitosis to G1.

As cells enter mitosis, chromatin compacts to facilitate chromosome segregation yet remains transcribed. Transcription supercoils DNA to levels that can impede further progression of RNA polymerase II (RNAPII) unless it is removed by DNA topoisomerase 1 (TOP1). Using ChIP-seq on mitotic cells, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity by RNAPII during elongation allowed RNAPII clearance from genes in prometaphase and enabled chromosomal segregation. Disruption of the TOP1-RNAPII interaction impaired RNAPII spiking at promoters and triggered defects in the post-mitotic transcription program. This program includes factors necessary for cell growth, and cells with impaired TOP1-RNAPII interaction are more sensitive to inhibitors of mTOR signaling. We conclude that TOP1 is necessary for assisting transcription during mitosis with consequences for growth and gene expression long after mitosis is completed. In this sense, TOP1 ensures that cellular memory is preserved in subsequent generations.