Everolimus Resistance Research Articles

Methuosis Inducer SGI-1027 Cooperates with Everolimus to Promote Apoptosis and Pyroptosis by Triggering Lysosomal Membrane Permeability in Renal Cancer.

The mTOR inhibitor everolimus has been approved as a sequential or second-line therapy for renal cell carcinoma (RCC). However, the development of drug resistance limits its clinical applications. This study aims to address the challenge of everolimus resistance and provide new insights into the treatment of advanced RCC. Here, the cytotoxicity of the DNA methyltransferase 1 (DNMT1) inhibitor SGI-1027 in inducing cell vacuolation and methuosis is discovered and demonstrated for the first time. Additionally, SGI-1027 exerts synergistic effects with everolimus, as their combination suppresses the growth, migration, and invasion of renal cancer cells. Mechanistically, apoptosis and GSDME-dependent pyroptosis triggered by lysosomal membrane permeability (LMP) are observed. The upregulation of GSDME expression and increased lysosomal activity in renal cancer cells provide a therapeutic window for the combination of these two drugs to treat renal cancer. The combination treatment exhibits effective anti-tumor activity and is well tolerated in a subcutaneous tumor model. Overall, this study validates and reveals the specific cytotoxicity property of SGI-1027 and its potent synergistic effect with everolimus, offering new insights into advanced RCC therapy and everolimus-resistance overcoming.

Abstract 7655: The CXCR4/CXCR7/CXCL12 axis and PBRM1 are involved in everolimus resistance in human renal cancer cell lines

Abstract Purpose: Renal cell carcinoma (RCC) is one of the most common malignancies in the genitourinary system. Combinations of Immunocheckpoints inhibitors plus TKIs represents the actual standard of therapy for metastatic RCC patients, while mTOR inhibitors play a role in the second-line treatment of mRCC in combination with lenvatinib. Thus characterizing resistance mechanism to mTOR inhibitors is unmet need. Experimental Design: A498 RAD-resistant renal cell carcinoma (RCC) cells were generated through increasing doses of RAD001 (RAD1-5-10 μM). Gene expression was assessed by qRT-PCR. pERK and transwell migration were evaluated in A498, RAD10 and RAD10shP. PBRM1sh was obtained in RAD10 with lentiviral particles while CXCR4 transfection in RAD10 was realized using pCMV6-AC-h-CXCR4-GFP. ChIP assay was assessed using Chromata ChIP kit. Results: Gene expression analysis revealed reduction in CXCR4/CXCR7 expression in A498 RAD 1-5-10-resistant cells (RAD1-5-10) (CXCR4:32.8, 37, 40.7-fold and CXCR7:2.3, 2.3, 2-fold, respectively), while PBRM1 and CXCL12 were overexpressed as compared to A498 (PBRM1: 2.2, 2.34 and 3.32-fold, and CXCL12: 7.2-, 5.6-, and 10-fold, respectively). The axis CXCL12-CXCR4 was functionally inhibited in RAD10 as demonstrated by inhibition in CXCL12- pERK and migration. YY1, already described as CXCR4 and CXCR7 negative regulator, was upregulated in RAD10 cells. ChIP confirmed higher binding of YY1 on CXCR4 and CXCR7 promoters in RAD10 as compared to A498. To address CXCR4 role, human CXCR4 was transfected in RAD10 cells (RAD10 CXCR4+) and RAD001 cytotoxicity was evaluated; interestingly, RAD10 CXCR4+ entirely restored RAD001 sensitivity in RAD10 cells. PBRM1 silencing in RAD10 (RAD10shP) downregulated mTOR and 4EBP1 (1.54 and 3-fold, compared to RAD10). As described, CXCR4 decreased in RAD10 and PBRM1 silencing did not further affect it, while CXCR7 increased (20-fold) in RAD10shP, indicating that PBRM1 regulates CXCR7 expression rather than CXCR4 in RAD001 resistant cells. RAD10shP migrated more towards CXCL11 than CXCL12 according to the overexpression of CXCR7 in RAD10shP cells. YY1 expression in RAD10 was higher than WT (2.6-fold) and was reduced in RAD10shP compared to RAD10 (1.6-fold) displaying a possible role of PBRM1 in regulating CXCR7 in RAD001-resistant RCC through YY1. CXCR4/CXCL12 signaling in RAD10shP cells more actively induced pERK and pAKT following CXCL12 or CXCL11-mediated as compared to RAD10 demonstrating that PBRM1 silencing restored a sensitive-like phenotype; however, PBRM1 silencing partially restored RAD001 sensitivity in RAD10 (RAD10 were 48-fold more resistant than A498, RAD10shP were 8.8-fold more resistant than A498). Conclusion: Taken together, these results demonstrate that both PBRM1 and CXCR4 are responsible for RAD001 resistance with PBRM1 regulating CXCR7 and mTOR shedding light on the new mechanistic roles of PBRM1 in RCC Citation Format: Federica Auletta, Caterina Ieranò, Giuseppina Rea, Maria Napolitano, Francesca Galdiero, Anna Maria Bello, Pierfrancesco Tassone, Maria Teresa Di Martino, Stefania Scala. The CXCR4/CXCR7/CXCL12 axis and PBRM1 are involved in everolimus resistance in human renal cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7655.

Abstract 6236: Systems biology modeling identifies SMARCD3 as a master regulator facilitating everolimus resistance in ER+ breast cancer

Abstract Breast cancer is the most common malignancy in women and estrogen receptor positive (ER+) breast cancers represent nearly 75% of all breast tumors. Everolimus, an mTORC1 inhibitor, in combination with exemestane has been approved for patients with metastatic ER+ breast cancer. However, many patients eventually develop resistance to everolimus and leads to poor survival outcomes. Characterizing everolimus-resistant cells can reveal targetable molecular phenotypes that can eventually lead to new therapeutic targets. In this study, we elucidate systems-level phenotypic distinctions between everolimus sensitive and refractory cells. We generated several isogenic ER+ breast cancer cell lines resistant or sensitive to everolimus. We then leveraged transcriptomic profiles from sensitive and resistant cells before or after everolimus treatment to decipher the underlying mechanisms essential to the resistant state. Using linear mixed effect models, we identified several meta-phenotypes that were distinct between sensitive and resistant cell states. These included growth-factor signaling, cell cycle, metabolism, stemness, and apoptosis meta-phenotypes. Our analyses also identified strong activation of growth-factor receptors including IGF1R/INSR and ESR1 in the resistant cells, which were maintained despite everolimus treatment. Next, we adopted a transcriptional regulatory network reconstruction framework to identify the master regulators responsible for the activation of resistance meta-phenotypes. We identified SMARCD3 as the key master regulator controlling expression of genes across diverse resistance meta-phenotypes, including growth-factor signaling. Further, using transcriptomic profiles from a neoadjuvant trial of 23 post-menopausal ER+ breast cancers treated with everolimus, we found that SMARCD3 expression was elevated in resistant tumors. To confirm the role of SMARCD3 in everolimus resistance, we repressed transcription of the SMARCD3 gene using CRISPR interference to re-sensitize resistant cells to everolimus treatment. In conclusion, we used context-specific cell transcriptomic profiles and integrative systems-biology approaches to elucidate mechanisms of resistance in response to everolimus treatment in ER+ breast cancer. Our findings suggest SMARCD3 upregulation promotes resistance to everolimus treatment and may serve as a novel therapeutic target in ER+ breast cancer. Citation Format: Eric F. Medina, Patrick A. Cosgrove, Eleni Farmaki, Vince K. Grolmusz, Feng Chi, Jason I. Griffiths, Andrea H. Bild, Aritro Nath. Systems biology modeling identifies SMARCD3 as a master regulator facilitating everolimus resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6236.

Impact of the PI3K-alpha inhibitor alpelisib on everolimus resistance and somatostatin receptor expression in an orthotopic pancreatic NEC xenograft mouse model.

The mechanistic target of rapamycin complex 1 (mTORC1) inhibitor everolimus is one of the few approved therapies for locally advanced and metastatic neuroendocrine tumours (NETs). However, after initial disease stabilisation, most patients develop resistance within 1 year. Our aim was to overcome resistance to everolimus by additional treatment with the PI3K-alpha inhibitor alpelisib in an everolimus-resistant orthotopic pancreatic neuroendocrine carcinoma xenograft mouse model. Female SCID mice underwent laparoscopic pancreatic transplantation of everolimus-sensitive (BON1KDMSO) or everolimus-resistant (BON1RR2) NET cells. Both groups were further divided into four treatment groups: placebo, everolimus, alpelisib, and everolimus + alpelisib (combination). Oral treatment was started at a tumour volume of approximately 140 mm3 and continued until 1900-2000 mm3, validated by weekly MRI. Somatostatin receptor expression and tumour viability were analysed by 68Ga-DOTATOC and 18F-FDG PET/CT. Everolimus resistance of the BON1RR2 tumours was confirmed. In the everolimus-sensitive group, everolimus alone, alpelisib alone, and combination treatment significantly prolonged survival, compared to placebo, while in the BON1RR2 group, only combination treatment significantly prolonged survival compared to placebo, but neither everolimus nor alpelisib alone. Placebo-treated everolimus-sensitive tumours grew more rapidly (median survival 45 days), compared to placebo-treated everolimus-resistant tumours (60 days). Within the everolimus-sensitive group, the combination-treated mice showed the longest median survival (52 days). Of all groups, the everolimus-resistant combination-treated group survived longest (69 days). Combination treatment with everolimus and alpelisib seems promising to overcome everolimus resistance in neuroendocrine neoplasms, and should be further examined in a clinical trial.

Molecular Characteristics of Everolimus-resistant Renal Cell Carcinoma Cells Generated by Continuous Exposure to Everolimus.

Mammalian target of rapamycin (mTOR) inhibitors represent the standard of care for metastatic renal cell carcinoma (RCC). However, treatment outcomes are relatively poor, suggesting a potential problem with tolerating mTOR inhibitors. The aim of this study was to establish everolimus-resistant sublines and to compare their molecular characteristics with those of their counterparts. Human-derived RCC, Caki-2, and 786-O cells were continuously exposed to everolimus at 1 μM, and the established resistant sublines were designated as Caki/EV and 786/EV, respectively. Cellular characteristics were compared between both cells. Caki/EV and 786/EV cells showed a decrease in sensitivity to everolimus as well as other mTOR inhibitors. Expression of mTOR and its effectors exhibited no alteration in resistant sublines and their counterparts. However, phosphorylation of S6K, an index of mTOR activity, decreased in resistant sublines. PCR array analysis of mTOR signaling pathway-related factors indicated that the expression of INSR, TP53, and IGFBP3 increased in Caki/EV cells, whereas that of TELO2, HRAS, and SGK1 was up-regulated in 786/EV cells. The levels of DDIT4, DEPTOR, HIF1A, and PLD1 mRNAs decreased in both cell lines. The novel everolimus-resistant Caki/EV and 786/EV cells exhibited cross-resistance to other mTOR inhibitors and decreased mTOR activity. Furthermore, down-regulation of DDIT4, DEPTOR, HIF1A, and PLD1 may contribute to everolimus resistance.

Abstract 388: Acquired sensitivity to ONC201/TIC10 in ER+ breast cancer progressing on the mTOR inhibitor everolimus

Abstract The mTOR inhibitor everolimus has become an important component for clinical management of metastatic ER+ breast cancer. Despite the improved patient outcomes, progression and resistance will still develop. Therefore, it is important to research strategies that can increase response durability and effectiveness of this therapy. Here we developed everolimus resistance models and performed pre-clinical evaluation of the small molecule ONC201/TIC10, a drug candidate showing promising results in clinical trials with advanced cancer patients. ONC201/TIC10 targets mitochondrial function and metabolism and activates stress response, however its efficacy has not been studied in everolimus resistant breast cancer. Transcriptomic analysis in everolimus resistant tumors identified sustained mitochondrial oxidative phosphorylation activity suggesting increased sensitivity to ONC201/TIC10. Our results show that combination treatment of ONC201/TIC10 with everolimus inhibits the growth of sensitive as well as everolimus resistant cells in 2D and 3D in vitro studies. The increased response to combination therapy was confirmed in primary cells from patients progressing on endocrine and mTOR therapy, supporting clinical relevance. We further show that the mechanism of ONC201/TIC10 anti-proliferative activity involves disruption of mitochondrial function and increased levels of the pro-apoptotic transcription factors CHOP and ATF4. Importantly, several resistant cell lines demonstrated increased dependency on mitochondrial respiration compared to sensitive cells which contributes to a vulnerability to ONC201/TIC10. In conclusion, we used pre-clinical models to characterize acquired sensitivity to mTOR inhibition. Combining patient derived data with resistant cell line driven experiments, our study provides validated findings in various settings strengthening the potential for clinical applications of ONC201/TIC10. We propose that ONC201/TIC10 and modulation of mitochondrial function is effective in drug resistant cancer cells and can enhance the anti-tumor activity of standard of care therapy in progressing patients. Citation Format: Eleni Farmaki, Rena Emond, Aritro Nath, Vince K. Grolmusz, Patrick A. Cosgrove, Andrea H. Bild. Acquired sensitivity to ONC201/TIC10 in ER+ breast cancer progressing on the mTOR inhibitor everolimus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 388.

Abstract PD5-10: PD5-10 Dual therapeutic targeting of CDK8/19 and mTOR in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is the most aggressive subtype of all breast cancers. However, unlike other breast cancer subtypes, current treatments for TNBC are restricted and this scarcity of viable options is the key contributor to the poorer prognosis. Despite early response, almost all the targeted drugs tested in TNBC eventually fail due to the development of resistance. Patients’ data have shown that the expression level of Mediator kinases CDK8 and CDK19 are elevated in TNBC, and that higher expression of CDK8/19 is correlated with more advanced diseases and worse prognosis. Selective CDK8/19 inhibitor SNX631, when used as a single agent, inhibited the growth of several TNBC cell lines in vitro, cell-derived xenografts (CDXs) as well as patient derived xenografts (PDXs) in vivo, suggesting the potential of targeting CDK8/19 in treating TNBC. We also analyzed the effect of CDK8/19 inhibition on the outcome of treatment with mTORC1 inhibitor everolimus (RAD001), an approved drug for several cancers with mutations of PTEN or PI3KCA. SNX631 exhibited a synergistic effect in combination with everolimus on suppressing TNBC cell growth in vitro. In vivo treatment with everolimus alone achieved a strong tumor growth inhibition in TNBC xenograft models but all the tumors eventually resumed growth, indicated the development of resistance. Significantly, the addition of a CDK8/19 inhibitor prevented the emergence of in vivo everolimus resistance both in CDX and PDX tumors upon treatment for up to 150 days, suggesting a potential for extending remission or even achieving cures in TNBC. Transcriptomic analysis demonstrated that this effect was due to the prevention of transcriptional reprogramming associated with everolimus resistance in tumor cells. Citation Format: Xiaokai Ding, Hao Ji, Amanda C. Sharko, Juergen Loskutov, Zachary Mack, Jadyn Myers, Mengqian Chen, Elena Pugacheva, Igor Roninson, Eugenia Broude. PD5-10 Dual therapeutic targeting of CDK8/19 and mTOR in triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-10.

Norcantharidin counteracts acquired everolimus resistance in renal cell carcinoma by dual inhibition of mammalian target of rapamycin complex 1 and complex 2 pathways in Vitro.

Everolimus, an oral mammalian target of rapamycin complex 1 (mTORC1) inhibitor, presents a therapeutic option in metastatic renal cell carcinoma (RCC) patients who were intolerant to, or previously failed, immune- and vascular endothelial growth factor-targeted therapies. However, the onset of drug resistance limits its clinical use. One possible mechanism underpinning the resistance is that inhibiting mTORC1 by everolimus results in mTORC2-dependent activation of v-Akt murine thymoma viral oncogene (AKT) and upregulation of hypoxia-inducible transcription factors (HIF). Norcantharidin (NCTD) is a demethylated derivative of cantharidin with antitumor properties which is an active ingredient of the traditional Chinese medicine Mylabris. In this study, everolimus-resistant RCC cells (786-O-R) obtained by chronic everolimus treatment revealed higher level of HIF2α and over-activated mTORC2 pathway and NCTD inhibits cell proliferation in both everolimus-resistant and -sensitive RCC cells by arresting cell cycle in G0/G1 phase and reducing cell cycle-related proteins of C-Myc and cyclin D. Furthermore, NCTD shows synergistic anticancer effects combined with everolimus in everolimus-resistant 786-O-R cells. Mechanically, NCTD repressed both mTORC1 and mTORC2 signaling pathways as well as downstream molecular signaling pathways, such as p-4EBP1, p-AKT, HIF1α and HIF2α. Our findings provide sound evidence that combination of NCTD and everolimus is a potential therapeutic strategy for treating RCC and overcoming everolimus resistance by dual inhibition of mTORC1 and mTORC2.

PAK4-NAMPT Dual Inhibition Sensitizes Pancreatic Neuroendocrine Tumors to Everolimus.

Metastatic pancreatic neuroendocrine tumors (PNET) remain an unmet clinical problem. Chronologic treatment in PNETs includes observation (watchful protocol), surgery, targeted therapy, and chemotherapy. However, increasing evidence illustrates that the outcomes of targeted therapeutic options for the treatment of advanced PNETs show minimal response. The FDA-approved mTOR inhibitor everolimus does not shrink these tumors. It only delays disease progression in a subset of patients, while a significant fraction acquires resistance and shows disease progression. Thus, there is a need for more effective targeted approaches to sensitize PNETs to everolimus for better treatment outcomes. Previously, we showed that mTOR regulator p21 activated kinase 4 (PAK4) and nicotinamide adenine dinucleotide biosynthesis enzyme nicotinamide phosphoribosyl transferase (NAMPT) were aberrantly expressed in PNET tissue and promoted everolimus resistance. In this report, we demonstrate that PAK4-NAMPT dual inhibitor KPT-9274 can synergize with everolimus (growth inhibition, colony suppression, and glucose uptake assays). KPT-9274-everolimus disrupted spheroid formation in multiple PNET models. Molecular analysis showed alteration of mTORC2 through downregulation of RICTOR as a mechanism supporting synergy with everolimus in vitro KPT-9274 suppressed β-catenin activity via inhibition of PAK4, highlighting the cross-talk between Rho GTPases and Wnt signaling in PNETs. KPT-9274, given at 150 mg/kg in combination with sub-MTD everolimus (2.5 mg/kg), significantly suppressed two PNET-derived xenografts. These studies bring forward a well-grounded strategy for advanced PNETs that fail to respond to single-agent everolimus.

Abstract 1122: Novel targets for therapy resistant pancreatic neuroendocrine tumors

Abstract Pancreatic Neuroendocrine Tumors (PNETs) remains an unmet clinical problem and epidemiologic studies indicate that their incidence has significantly increased over the years. Surgery remains the only curative option in patients with localized tumors. However, there is no effective therapy in patients with advanced or metastatic disease. Thus, unfortunately, 65% of PNET patients with advanced/unresectable disease die within 5 years after diagnosis. Current therapeutic approaches for advanced PNET patients include chemotherapy (Capecitabine, Temolozomide, 5FU etc), targeted therapies (everolimus, and sunitinib), hormonal therapies [somatostatin analogs (octreotide or lanreotide)] and the novel peptide receptor radionuclide therapy. Nevertheless, all these therapeutic modalities show only minimal response in patients with PNETs in the clinic. Therefore, novel targets need to be identified that could improve the dismal outcome of advanced PNETs. An analysis of PNET tissue identified p21-activated Kinase 4 (PAK4) and nicotinamide Phosphoribosyltransferase (NAMPT) as two new therapeutic targets. PAK4 is the downstream effector of Rac1 (members of the Rho family of GTPases) and is involved in critical cellular processes such as cell motility, proliferation, and survival. More importantly, PAK4 protein has been implicated in the activation of Ras/Raf/Mek/Erk and PI3K/Akt/mTOR signaling in cancers. Similarly, NAMPT is an enzyme that catalyzes the rate-limiting step in the principal salvage pathway of NAD biosynthesis in mammals. Tumor cells have highly active glycolytic, pentose and fatty acid synthesis pathways that require persistent high levels of NAD. Consequently, most cancers rely more heavily on NAMPT for rapid NAD biosynthesis. Earlier we demonstrated that biological or chemical inhibition of PAK4-NAMPT by RNAi or by a dual inhibitor (KPT-9274 a Phase I drug and its analogs) can suppress PNET proliferation and reduce the growth of subcutaneous xenografts. Our new studies show that PAK4-NAMPT dual inhibition can suppress PNET anchorage independent growth and metabolomic analysis of KPT-9274 treated PNET cells revealed significant alterations in a series of metabolites related to NAD signaling. KPT-9274 synergistically enhanced the anti-tumor activity of everolimus (combination index <1). Molecular analysis of combination treatment showed down-regulation of known everolimus resistance drivers including mTORC1, mTORC2, PI3K, ERK, FAK, RICTOR, ß-catenin. Importantly, combination treatment of KPT-9274 (150mg/kg) and everolimus (2.5 mg/kg used at sub-optimal dose) caused reduction of PNET xenografts. Our investigations demonstrate that PAK4 and NAMPT are two viable therapeutic targets in the difficult to treat PNETs that warrant further clinical investigations. Citation Format: Gabriel B. Mpilla, Amro Aboukameel, Md Hafiz Uddin, Mohammed N. Al-Hallak, Bayan Al-Share, Yosef Landesman, Yiwei Li, Steve Kim, Rafic Beydoun, Ramzi M. Mohammad, Philip A. Philip, Bassel El-Rayes, Asfar S. Azmi. Novel targets for therapy resistant pancreatic neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1122.

Reversal effect of Vitamin D (VitD) on exosome-mediated everolimus resistance (EveR) in hepatocellular carcinoma (HCC) cell line: A molecular study

Reversal effect of Vitamin D (VitD) on exosome-mediated everolimus resistance (EveR) in hepatocellular carcinoma (HCC) cell line: A molecular study

MEK inhibition overcomes everolimus resistance in gastric cancer.

Although substantial evidence has shown that the mammalian target of rapamycin (mTOR) pathway is an important therapeutic target in gastric cancer, the overall response rates in patients to mTOR inhibitor everolimus have been less than initially expected. We hypothesized that the limited efficacy of everolimus in gastric cancer is due to the activation of extracellular signal-regulated kinase (ERK). ERK activation was investigated using western blot. The effects of dual inhibition of ERK and mTOR via genetic and pharmacological approaches were determined using cellular assays and xenograft mouse model. We observed the decreased phosphorylation of mTOR, rS6, and 4EBP1 and increased phosphorylation of ERK and p90RSK in gastric cancer cells exposed to everolimus at clinically relevant concentration. Using both in vitro cell culture assays and in vivo xenograft mouse model, we found that trametinib overcame everolimus resistance by either effectively targeting resistant cells or further enhancing everolimus' efficacy in sensitive cells. Mechanism studies confirmed that trametinib overcame everolimus resistance via specifically inhibiting ERK and regulating ERK-mediated Bcl-2 family proteins in gastric cancer cells. Inhibition of mTOR pathway can induce "paradoxical" activation of ERK in gastric cancer, and this activation can be reversed by trametinib. Since both drugs are clinically available, our findings might accelerate the initiation of clinical trials on gastric cancer using everolimus and trametinib combination.

PAK4-NAMPT Dual Inhibition as a Novel Strategy for Therapy Resistant Pancreatic Neuroendocrine Tumors.

Pancreatic neuroendocrine tumors (PNET) remain an unmet clinical need. In this study, we show that targeting both nicotinamide phosphoribosyltransferase (NAMPT) and p21-activated kinase 4 (PAK4) could become a synthetic lethal strategy for PNET. The expression of PAK4 and NAMPT was found to be higher in PNET tissue compared to normal cells. PAK4-NAMPT dual RNAi suppressed proliferation of PNET cell lines. Treatment with KPT-9274 (currently in a Phase I trial or analogs, PF3758309 (the PAK4 selective inhibitor) or FK866 (the NAMPT inhibitor)) suppressed the growth of PNET cell lines and synergized with the mammalian target of rapamycin (mTOR) inhibitors everolimus and INK-128. Molecular analysis of the combination treatment showed down-regulation of known everolimus resistance drivers. KPT-9274 suppressed NAD pool and ATP levels in PNET cell lines. Metabolomic profiling showed a statistically significant alteration in cellular energetic pathways. KPT-9274 given orally at 150 mg/kg 5 days/week for 4 weeks dramatically reduced PNET sub-cutaneous tumor growth. Residual tumor analysis demonstrated target engagement in vivo and recapitulated in vitro results. Our investigations demonstrate that PAK4 and NAMPT are two viable therapeutic targets in the difficult to treat PNET that warrant further clinical investigation.

Everolimus resistance in clear cell renal cell carcinoma: miRNA-101 and HIF-2α as molecular triggers?

Aim: The majority of clear cell renal cell carcinoma patients develop resistance to mTOR inhibitors. Materials & methods: As an in vitro model four cell lines were used: HKC-8, 786- O, RCC-FG-2 and an everolimus-resistant cell line (786-OR) established during this study. The quantification of miRNA-101 and HIF-2α mRNA levels was assessed by real-time PCR. Results: We observed a significant decrease of miRNA-101 intracellular levels in 786-OR. However, this miRNA presented higher extracellular levels. Additionally, we found a significant increase of HIF-2αin 786-OR. Conclusion: The circulating levels of miRNA-101 may be a potential biomarker of anti-mTOR therapy response and resistance prediction. Moreover, the resistance to mTOR inhibitors seems to be related with the overexpression of HIF-2α.

Dual-Inhibition of mTOR and Bcl-2 Enhances the Anti-tumor Effect of Everolimus against Renal Cell Carcinoma In Vitro and In Vivo.

Renal cell carcinoma (RCC) is the predominant type of kidney cancer. Mammalian target of rapamycin (mTOR) inhibitor everolimus is currently used as a second-line therapy for sorafenib or sunitinib-refractory metastatic RCC patients. The clinical limitation confronted during everolimus therapy is the onset of drug resistance that decreases the efficacy of the drug. Elevated level of anti-apoptotic Bcl-2 protein is proposed to be an emerging feedback loop for the acquired drug-resistance in various cancer types. In this study, the Bcl-2 inhibitor ABT-737 was used in combination with everolimus to enhance its anti-tumor effectiveness in everolimus-resistant RCC cell lines. Everolimus and ABT-737 combination synergistically led to a decrease in the proliferation of primary site A-498 and metastatic site Caki-1 RCC cell lines, which was accompanied by a reduction in protein levels of cell cycle and mTOR pathway proteins. In both RCC cell lines, everolimus-ABT-737 combination not only induced apoptosis, caspase and PARP-1 cleavage but also a decrease in Bcl-2 protein levels in parallel with a concomitant increase in Bim and Noxa levels.In order to confirm our in vitro findings, we have generated everolimus-resistant RenCa cell line (RenCares) to establish a RCC mouse xenograft model. Animals co-treated with everolimus and ABT-737 exhibited a complete suppression of tumor growth without any notable toxicity. This study thus proposes the everolimus-ABT-737 combination as a novel therapeutic strategy for the treatment of RCC to overcome the current clinical problem of everolimus resistance.

Identification of mTOR inhibitor-resistant genes in cutaneous squamous cell carcinoma.

PurposeThe PI3K/AKT/mTOR pathway is frequently activated in various squamous cell carcinomas (SCCs). Although mTOR inhibitors are suggested as effective treatments in immunosuppressed patients with metastatic SCC, they are still not proven to be favorable in treating skin SCC patients not undergoing immunosuppressive therapy. Moreover, the exact mechanism of the mTOR signaling pathway in SCC has not yet been identified. In this study, we aimed to determine the genes associated with mTOR inhibitors in skin SCC.Materials and methodsThe identification of cell viability according to concentration of everolimus and Western blot was done. To analyze the global gene expression profiles, A431 and HSC-1 cells were treated with dimethyl sulfoxide (DMSO) or 100 nM of everolimus for 72 hours. Furthermore, differentially expressed genes (DEGs) were identified using Affymetrix analysis. To identify the gene network associated with everolimus resistance in SCC cells, pathway analysis was performed using the Ingenuity Pathway Analysis (IPA) tool.ResultsThe effects of cell death with respect to the mTOR inhibitor concentration were observed in the HSC-1 cell line; however, the mTOR inhibitor did not show effective cytotoxic activity in the A431 cell line. p-mTOR concentration also diminished with respect to everolimus concentrations in the HSC-1 cell line. Moreover, the microarray results showed that the MYC/CCND1/TP73/NUPR1/SBD/ERBB2/CDKN2B genes were related to mTOR inhibitor resistance. However, CCND1 gene overexpression was most closely related to mTOR inhibitor resistance.ConclusionWe identified mTOR inhibitor resistance genes, and our findings may help select therapeutic targets in skin SCC.

Abstract 4368: PAK4-NAMPT dual inhibition as a feasible strategy for treatment of resistant pancreatic neuroendocrine tumors

Abstract Our studies in pancreatic neuroendocrine tumor (pNET) cell lines demonstrate hyper-activation of the Rho GTPase effector p21 activated kinase 4 (PAK4) and the nicotinamide adenine dinucleotide (NAD) salvage pathway rate limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT). The PAK4 protein is known to regulate a myriad of signaling proteins in the mTOR pathway including mTORC1, mTORC2, PI3K, and IGF-1. Similarly, NAMPT is recognized to regulate mTOR through energy sensor protein, AMPK. In this study, pNET cell lines QGP-1 and Bon-1 were subjected to either PAK4 RNA interference (RNAi), PAK4-NAMPT dual inhibitors (the clinical compound KPT-9274 or an analog KPT-7523) or the PAK4 specific inhibitor, PF-3738309 and NAMPT specific inhibitor FK866 in the presence or absence of mTOR inhibitors (everolimus or INK128). Gene expression profiling and phospho-proteomic analyses were performed to capture molecular changes post single or combination treatments. The anti-tumor activity of KPT-9274-everolimus was evaluated in a subcutaneous xenograft mouse model derived from QGP-1 and Bon-1 cells. PAK4 RNAi suppressed proliferation and restored everolimus sensitivity in pNET cell lines. The dual inhibitors were effective in reducing proliferation and inducing apoptosis. KPT-9274 or KPT-7523 could synergistically enhance the anti-tumor activity of everolimus or INK128 in pNET cell lines [CI <1]. Molecular analysis of the combination treatment showed down-regulation of known everolimus resistance drivers such as mTORC1, mTORC2, PI3K, ERK, FAK, RICTOR, β-catenin and IGF-1. In addition, KPT-9274 and analogs suppressed the steady state level of NAD and ATP. Importantly, KPT-9274 given i.v. or orally at the maximum tolerated dose (140 mg/kg, once/day for 5 days/week for 4 weeks) dramatically inhibited the growth of QGP-1 and Bon-1 tumors. This is the first study demonstrating the role of PAK4 and NAMPT in pNETs. KPT-9274 is currently in a Phase I trial of patients with advance solid malignancies or NHL (NCT02702492). Our pre-clinical work establishes a solid rationale for a Phase II clinical study of KPT-9274 and an mTOR inhibitor combination for the treatment of difficult to treat pNETs. Citation Format: Gabriel Mpilla, Irfana Muqbil, Amro Aboukameel, Philip A. Philip, William Senapedis, Erkan Baloglu, Yosef Landesman, Michael Kauffman, Sharon Shacham, Ramzi M. Mohammad, Asfar S. Azmi. PAK4-NAMPT dual inhibition as a feasible strategy for treatment of resistant pancreatic neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4368.

The role of GSK3 and its reversal with GSK3 antagonism in everolimus resistance.

Pancreatic neuroendocrine tumors (panNETs) are often inoperable at diagnosis. The mTORC1 inhibitor everolimus has been approved for the treatment of advanced NETs. However, the regular development of resistance to everolimus limits its clinical efficacy. We established two independent everolimus-resistant panNET (BON1) cell lines (BON1 RR1, BON1 RR2) to find potential mechanisms of resistance. After 24 weeks of permanent exposure to 10 nM everolimus, BON1 RR1 and BON1 RR2 showed stable resistance with cellular survival rates of 96.70% (IC50 = 5200 nM) and 92.30% (IC50 = 2500 nM), respectively. The control cell line showed sensitivity to 10 nM everolimus with cellular survival declining to 54.70% (IC50 = 34 nM). Both resistant cell lines did not regain sensitivity over time and showed persistent stable resistance after a drug holiday of 13 weeks. The mechanisms of resistance in our cell line model included morphological adaptations, G1 cell cycle arrest associated with reduced CDK1(cdc2) expression and decreased autophagy. Cellular migration potential was increased and indirectly linked to c-Met activation. GSK3 was over-activated in association with reduced baseline IRS-1 protein levels. Specific GSK3 inhibition strongly decreased BON1 RR1/RR2 cell survival. The combination of everolimus with the PI3Kα inhibitor BYL719 re-established everolimus sensitivity through GSK3 inhibition and restoration of autophagy. We suggest that GSK3 over-activation combined with decreased baseline IRS-1 protein levels and decreased autophagy may be a crucial feature of everolimus resistance, and hence, a possible therapeutic target.

Silencing lnc-ASAH2B-2 Inhibits Breast Cancer Cell Growth via the mTOR Pathway.

Persistent activation of the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (mTOR) pathway is an important mechanism in resistance of breast cancer to endocrine therapy. Although everolimus has potent inhibitory effects on the mTOR pathway, it has demonstrated only modest clinical activity as a single agent. Whether long noncoding (lnc) RNA is involved in everolimus resistance is unknown. Cell viability, colony formation and cell proliferation experiments were used to measure the effects of long noncoding RNA N-acylsphingosine amidohydrolase 2B-2 (lnc-ASAH2B-2) knockdown in BT474 and MCF7 breast cancer cells. lnc-ASAH2B-2 was up-regulated by everolimus in cells with and without serum, and reduction of lnc-ASAH2B-2 expression was able to inhibit proliferation of BT474 and MCF7 cells. lnc-ASAH2B-2 was up-regulated after everolimus exposure and efficiently regulated breast cancer cell growth by activating the mTOR pathway, which may reduce the effect of everolimus, providing evidence that lnc-ASAH2B-2 might be a new therapeutic target for breast cancer.

Acquired resistance to everolimus in aromatase inhibitor-resistant breast cancer.

We previously reported the establishment of several types of long-term estrogen-depleted-resistant (EDR) cell lines from MCF-7 breast cancer cells. Type 1 EDR cells exhibited the best-studied mechanism of aromatase inhibitor (AI) resistance, in which estrogen receptor (ER) expression remained positive and PI3K signaling was upregulated. Type 2 EDR cells showed reduced ER activity and upregulated JNK-related signaling. The mTOR inhibitor everolimus reduced growth in cells similar to Type 1 EDR cells. The present study generated everolimus-resistant (EvR) cells from Types 1 and 2 EDR cells following long-term exposure to everolimus in vitro. These EvR cells modeled resistance to AI and everolimus combination therapies following first-line AI treatment failure. In Type 1 EvR cells, everolimus resistance was dependent on MAPK signaling; single agents were not effective, but hormonal therapy combined with a kinase inhibitor effectively reduced cell growth. In Type 2 EvR cells, ER expression remained negative and a JNK inhibitor was ineffective, but a Src inhibitor reduced cell growth. The mechanism of acquired everolimus resistance appears to vary depending on the mechanism of AI resistance. Strategies targeting resistant tumors should be tailored based on the resistance mechanisms, as these mechanisms impact therapeutic efficacy.