Selective mTORC1 Inhibitor Research Articles

MTOR Regulation of N-Myc Downstream Regulated 1 (NDRG1) Phosphorylation in Clear Cell Renal Cell Carcinoma.

The mechanistic target of rapamycin (mTOR) kinase is a component of two signaling complexes that are known as mTOR complex 1 (mTORC1) and mTORC2. We sought to identify mTOR-phosphorylated proteins that are differently expressed in clinically resected clear cell renal cell carcinoma (ccRCC) relative to pair-matched normal renal tissue. Using a proteomic array, we found N-Myc Downstream Regulated 1 (NDRG1) showed the greatest increase (3.3-fold) in phosphorylation (on Thr346) in ccRCC. This was associated with an increase in total NDRG1. RICTOR is a required subunit in mTORC2, and its knockdown decreased total and phospho-NDRG1 (Thr346) but not NDRG1 mRNA. The dual mTORC1/2 inhibitor, Torin 2, significantly reduced (by ~100%) phospho-NDRG1 (Thr346). Rapamycin is a selective mTORC1 inhibitor that had no effect on the levels of total NDRG1 or phospho-NDRG1 (Thr346). The reduction in phospho-NDRG1 (Thr346) due to the inhibition of mTORC2 corresponded with a decrease in the percentage of live cells, which was correlated with an increase in apoptosis. Rapamycin had no effect on ccRCC cell viability. Collectively, these data show that mTORC2 mediates the phosphorylation of NDRG1 (Thr346) in ccRCC. We hypothesize that RICTOR and mTORC2-mediated phosphorylation of NDRG1 (Thr346) promotes the viability of ccRCC cells.

Abstract 4859: Bi-steric mTORC1 inhibitors are superior to rapamycin and induce apoptotic cell death in tumor models with hyperactivated mTORC1

Abstract The PI3K-mTOR pathway is one of the most commonly dysregulated pathways in human tumors. Rapalogs have been used extensively in human clinical trials but exhibit modest clinical benefit for the most part, possibly due to their lack of effect on 4E-BP1, a key target downstream of mTORC1. ATP-competitive mTOR inhibitors can fully inhibit mTORC1 but are poorly tolerated possibly due to their inhibition of mTORC2. A new class of selective mTORC1 inhibitors has been developed and termed ‘bi-steric’, which comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. We report that bi-steric mTORC1-selective inhibitors (RMC-4627 and RMC-6272) have greater activity than rapalogs for tumors with mTORC1 hyperactivation. RMC-4627 and RMC-6272 showed more effective growth inhibition in multiple tumor cell lines with mTORC1 dysfunction compared to rapamycin. Both RMC-4627 and RMC-6272 at ~1 nM showed near complete inhibition of p4E-BP1T37/46, which was not seen with rapamycin treatment. Rapamycin and both bi-steric compounds markedly reduced kidney tumor burden and human bladder cancer PDX tumor size after four weeks of treatment. Tumor regrowth assessed after two-month treatment cessation was significantly reduced in the RMC-6272 group as compared to the rapamycin group. Finally, TUNEL staining showed RMC-6272 treatment led to a greater induction of apoptosis in kidney tumor cells and human bladder cancer PDX tumors relative to rapamycin after a single dose of each compound. This is the first time that clear evidence for cell death after mTORC1 inhibition has been seen. Integrative multi-omic analysis reveals differential global rewiring induced by RMC-6272 in comparison to rapamycin. Genes involved in cell cycle transition were downregulated by RMC-6272 vs. rapamycin. Proteins downregulated by RMC-6272 treatment relative to rapamycin were enriched for translation and ribosome-related proteins, consistent with effects of non-phosphorylated 4E-BP1 on synthesis of those proteins. Multiple purine metabolites were decreased to a greater extent in RMC-6272 treated cells as compared to rapamycin treatment, including deoxyadenosine, AICAR, AMP, IMP, and GMP. Notably, there was no significant change in R5P, suggesting that downregulation of the purine de novo synthesis pathway was the cause of these changes. Strong tumor cell apoptosis was induced by the suppression of de novo purine synthesis through mTORC1-PRPS1 axis. PRPS1, the rate-limit enzyme involving in purine synthesis was regulated by mTORC1 activity. In summary, RMC-4627 and RMC-6272 demonstrate improved in vitro and in vivo inhibition of mTORC1 in comparison to rapamycin, and induced more cell death in preclinical models, indicating the potential of bi-steric mTORC1-selective inhibitors as a novel therapeutic strategy to treat tumors with mTORC1 dysregulation. Citation Format: Heng Du, Yu Chi Yang, Heng-Jia Liu, Min Yuan, John Asara, Kwok-Kin Wong, Mallika Singh, David Kwiatkowski. Bi-steric mTORC1 inhibitors are superior to rapamycin and induce apoptotic cell death in tumor models with hyperactivated mTORC1. [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 4859.

Selective mTORC1 Inhibitors Block Cystogenesis in a Human Kidney Organoid Model of Polycystic Kidney Disease

Selective mTORC1 Inhibitors Block Cystogenesis in a Human Kidney Organoid Model of Polycystic Kidney Disease

Abstract LB089: Effective in vivo treatment of endometrial tumor models with coexistent mutant PI3K and PTEN inactivation with a selective bi-steric mTORC1 kinase inhibitor

Abstract Dysregulation of PI3K signaling is a common event in human cancer, but feedback inhibition of receptor kinase signaling and/or induction of PTEN translation by activated PI3K/AKT/mTOR signaling reduces pathway output and tumor dependency. In some cancers, including the majority of endometrial carcinomas, these lesions coexist and cause synergistic hyperactivation of pathway output, particularly mTORC1 kinase signaling. Although this suggests that PI3K signaling is a driver of these endometrial cancers, standard inhibitors have had only marginal activity in patients with advanced disease. We here show that, in experimental isogenic models and PDX models with PI3K and PTEN alterations, PI3K inhibitors and rapalogs are weak inhibitors of TORC1 targets and slow but do not suppress the in vivo growth of these tumors. Revolution Medicines has developed a series of bi-steric selective and potent mTORC1 inhibitors that inhibit the phosphorylation of 4EBP-1, S6K and other mTORC1 substrates at concentrations that do not affect TORC2 substrates and thus do not induce the hyperglycemia caused by downregulation of AKT signaling by pan-mTOR kinase inhibitors. We have explored the biologic and anti-tumor effects of these inhibitors in models of endometrial cancer with coexistent PI3K mutation and PTEN inactivation. The mTORC1 kinase selective bi-steric inhibitor RMC-6272 can potently suppress both mTORC1 activity and the growth of these tumor cells in vitro. Moreover, in contrast to PI3K inhibition, RMC-6272 effectively inhibited the phosphorylation of mTORC1 kinase substrates and completely suppressed the growth in vivo of multiple PDXs with this genotype without inducing hyperglycemia or causing weight loss in the mice. The results suggest that dysregulation of TORC1 activity is an important driver of endometrial carcinomas with coexistent PTEN inactivation and PI3K mutations and that selective TORC1 kinase inhibitors may prove to be useful therapeutics in this context. The bi-steric mTORC1 kinase inhibitor RMC-5552 is the first clinical candidate of this class and clinical testing is ongoing (NCT04774952). Citation Format: Hilla Solomon, Radha Mukherjee, Xiaoping Chen, HuiYong Zhao, Elisa de Stanchina, Britta Weigelt, Alison M. Schram, Carol Aghajanian, Mallika Singh, Jan Smith, Neal Rosen. Effective in vivo treatment of endometrial tumor models with coexistent mutant PI3K and PTEN inactivation with a selective bi-steric mTORC1 kinase inhibitor [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 LB089.

Abstract 2662: Bi-steric mTORC1 inhibitor RMC-6272 synergizes with immune checkpoint inhibitors to induce sustained regression of MYC-driven hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths in the world. Current treatments for HCC have limited efficacy. First-line HCC therapy atezolizumab with bevacizumab increased patient 6-month PFS from 37% to 55% relative to sorafenib, a multi-kinase inhibitor currently serving as a standard-of-care treatment. Although immunotherapies provide survival benefits, reported patient response and disease control rates were 27% and 74%, respectively, highlighting the need for novel therapeutic agents that can re-establish responsiveness to immunotherapies and enhance their therapeutic effects. Revolution Medicines has developed a class of selective mTORC1 inhibitors, termed ‘bi-steric’, which comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. RMC-6272 is a representative bi-steric inhibitor that exhibits potent and selective (>30-fold) inhibition of mTORC1 over mTORC2. Several lines of evidence suggest that inhibition of the mTORC1 pathway may result in synthetic lethality of MYC-driven HCC. Thus, we hypothesized that MYC-driven HCC tumors are sensitive to mTORC1 inhibition and p-4EBP1 suppression. A single dose of RMC-6272 induced HCC regression in 80% of the Lap-tTA/Tet-O-MYC mouse model of MYC-driven HCC, whereas sorafenib did not cause tumor regression. Mechanistically, RMC-6272 inhibited the phosphorylation of mTORC1 downstream substrates S6k and 4EBP1 and depleted MYC protein levels. Given that the MYC oncogene enables immune evasion in HCC, we hypothesized that by reducing MYC, RMC-6272 can re-establish anti-tumor immune surveillance and responsiveness to α-PD-1 immunotherapy. Indeed, RMC-6272 induced tumor recruitment of dendritic cells, T cells, B cells, and natural killer (NK) cells, producing similar anti-tumor immunity as observed upon MYC genetic inactivation. Moreover, combining RMC-6272 with α-PD-1 resulted in deeper tumor regression and a more durable response as compared to either RMC-6272 or α-PD-1 alone in the MYC-driven HCC mouse model. By immunohistochemical staining and analysis, RMC-6272 in combination with α-PD-1, but not either agent alone, promoted tumor recruitment of CD4+ T cells and NK cells, immune cell degranulation, and the release of perforins and granzymes in MYC-driven HCC tumors. Our results show that the bi-steric mTORC1 inhibitor RMC-6272 synergizes with α-PD-1 to induce immune activation and the release of perforins and granzymes, resulting in sustained tumor regression in a MYC-driven HCC mouse model. The bi-steric mTORC1 inhibitor RMC-5552 is the first clinical candidate of this class and clinical testing is ongoing (NCT04774952). These preclinical data highlight a prospective anti-cancer therapeutic opportunity for mTORC1 selective inhibitors in combination with immune checkpoint inhibitors in MYC-driven cancers. Citation Format: Wadie D. Mahauad-Fernandez, Yu C. Yang, Ian Lai, Jangho Park, Lilian Yao, James W. Evans, Jacqueline A. Smith, Mallika Singh, Dean W. Felsher. Bi-steric mTORC1 inhibitor RMC-6272 synergizes with immune checkpoint inhibitors to induce sustained regression of MYC-driven hepatocellular carcinoma [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 2662.

Investigating the therapeutic efficacy of a novel mTORC1 inhibitor, RMC‐6272, on liver tumors with b‐catenin activation

Hepatocellular cancers (HCC) are a challenge to treat and lack precision‐based therapies. Gain‐of‐function (GOF) CTNNB1 mutations are evident in around 25‐35% of all HCCs. Co‐expression of mutant CTNNB1 (S45Y) along with proto‐oncogene Met or constitutively active Nrf2, recapitulates 11% and 10% of clinical HCC, respectively. GOF CTNNB1 mutations are also observed in >90% of hepatoblastomas (HBs). Co‐expression of active β‐catenin and Yap in mice leads to the development of HB, representing 80% of all cases. Aberrant activation of the mTOR complex 1 (mTORC1) is observed in both HCC and HB models. In HCC, mutant‐ β‐catenin causes glutamine synthetase‐dependent increase in glutamine, leading to increased S2448‐pmTOR, indicative of mTORC1 activation. In Yap/Ctnnb1 model, mTORC1 activation occurs due to Yap‐regulated increase in glutamine transporter. Here we examine the therapeutic effects of a novel mTORC1 inhibitor RMC‐6272 on the progression of β‐catenin‐driven HCCs and HB. RMC‐6272 belongs to a class of selective mTORC1 inhibitors, termed ‘bi‐steric’, which comprise a rapamycin‐like core moiety covalently linked to an mTOR active‐site inhibitor.MethodsHydrodynamic tail‐vein injection (HDTVI) of S127A‐YAP1 and ΔN90‐CTNNB1 plasmids, together with SB transposase, was used to induce HB development in FVB mice. HCC was induced by co‐expression of S45YCTNNB1 with either Met or G31A‐NFE2L2. Weekly IP administration of RMC‐6272 (Revolution Medicines, 10 mg/kg) was started at 1 week post HDTVI in HB, at 3 weeks in Met‐β‐catenin, and 5 weeks in Nrf2‐β‐catenin models. Animals were euthanized after 7 and 11 weeks of treatment for HB; at 2.5 and 5 weeks for Met‐β‐catenin HCC; and 5 weeks for Nrf2‐ β‐catenin HCC. Liver weight to body weight ratio (LW/BW) was used to address tumor burden. Durability of the RMC‐6272‐elicited response was studied in Yap/β‐catenin and in Nrf2/β‐catenin models by first administering the drug as described above, and then switching to the control treatment for 3 additional weeks, prior to sacrificing them 15 and 13 weeks, respectively, after HDTVI.ResultsTreatment with RMC‐6272 significantly reduced tumor burden in all 3 models. Although both early‐ and late‐end point groups showed some malignant growth, the size and quantity of tumors was diminished by the treatment. The tumor inhibitory effect of RMC‐6272 was more pronounced at late time points by both significantly reduced LW/BW as well as by histology. Moreover, tumor suppressive effects were sustained in both a HB and in a Nrf2‐β‐catenin‐driven HCC, as evident by the significantly reduced LW/BW, even if the treatment had been terminated 3 weeks prior to analysis.ConclusionsOur study demonstrates that RMC‐6272, a novel bi‐steric mTORC1 inhibitor, had a potent multifactorial effect on diminishing HB and HCC growth in relevant murine models. These observations further solidify the potential use of mTORC1 inhibitors as therapeutic agents for CTNNB1‐mutated HCCs and in most HB cases. The bi‐steric mTORC1 inhibitor RMC‐5552 is the first clinical candidate of this class and clinical testing is ongoing (NCT04774952)

Abstract ND10: Discovery of RMC-5552: A selective bi-steric inhibitor of mTORC1 that suppresses 4EBP1 phosphorylation, for the treatment of mTORC1-activated tumors including RAS pathway escape

Abstract The PI3K/mTOR and RAS signaling pathways are hyperactivated in a wide range of human cancers. Several points of cross-talk and intersecting feedback mechanisms have been described and activation of mTOR signaling frequently mediates escape from RAS pathway inhibition. Revolution Medicines has developed a novel class of selective mTORC1 inhibitors, termed ‘bi-steric inhibitors', that interact with both the ATP- and FKBP12/FRB-binding sites of mTORC1. These compounds potently and selectively inhibit mTORC1 over mTORC2 and other lipid kinases. Unlike rapalogs, such as everolimus, these bi-steric inhibitors suppress 4EBP1 phosphorylation in vivo and induce growth suppression and apoptosis in various genetically-defined preclinical models of human cancer. We will describe our SAR investigations and findings for our preclinical tool compound, RMC-6272 (also known as RM-006) and for RMC-5552, our clinical candidate. We will demonstrate that mTORC1-selective bi-steric inhibitors show combinatorial activity with both KRASG12C(OFF) and KRASG12C(ON) inhibitors, in KRASG12C STK11deficient NSCLC models and can forestall resistance to KRASG12C inhibitor monotherapy. In addition, we will show single agent activity of bi-steric mTORC1 inhibitors in preclinical models of tumors bearing PI3K/mTOR pathway alterations as primary drivers, including PTEN-deficient glioblastoma multiforme, TSC1/2-deficient renal cystadenoma cancer, and hepatocellular cancers harboring mTOR-activating β-catenin mutations. RMC-5552, through reactivation of 4EBP1 and inhibition of cap-dependent translation, has the potential to benefit cancer patients with genetically-defined tumors with activating mutations in the RAS and PI3K/mTOR pathways both as a single agent or in combination with RAS inhibitors. Citation Format: G. Leslie Burnett. Discovery of RMC-5552: A selective bi-steric inhibitor of mTORC1 that suppresses 4EBP1 phosphorylation, for the treatment of mTORC1-activated tumors including RAS pathway escape [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 ND10.

Abstract 1002: A bi-steric mTORC1 inhibitor that selectively reactivates 4EBP1 and induces regression of MYC-driven hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) accounts for ~90% of all liver cancers and is the third leading cause of cancer deaths in the world. HCC patients have a 5-year survival rate of 31%. Current treatments for HCC are scarce and have limited efficacy. These treatments include first-line therapy Sorafenib and second-line combination therapy nivolumab (anti-PD-1) with ipilimumab (anti-CTLA-4). The former therapy increased survival by less than 3 months and the latter increased survival from 37% to 68%. However, patient responses are variable and limited. Thus, there is a need to identify new therapies for HCC with sustained and efficacious therapeutic effects. Revolution Medicines has developed a class of selective mTORC1 inhibitors, termed ‘bi-steric', which comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. RMC-5552 is the first clinical candidate of this class and clinical testing is planned in 2021. Bi-steric mTORC1 inhibitors exhibit potent and selective (>10-fold) inhibition of mTORC1 over mTORC2 and durably suppress p-S6K and p-4EBP1. Several lines of evidence suggest that inhibition of the mTORC1 pathway may result in synthetic lethality of MYC-driven HCC. Thus, we hypothesize that MYC-driven HCC tumors are sensitive to mTORC1 inhibition and p-4EBP1 suppression (4EBP1 reactivation). To test this hypothesis, we first evaluated the short-term in vivo effect of two representative bi-steric tool compounds, RM-001 and RM-006 (also known as RMC-6272) in the LAP-tTA/TRE-MYC mouse model of MYC-driven HCC. We compared their anti-tumor activity to first-line HCC therapy Sorafenib. Our results show that a single dose of RM-006 induced HCC regression in 80% of mice while RM-001 reduced HCC growth in 68% of mice and Sorafenib reduced tumor growth in 50% of mice. Mechanistically, we found that RM-006 potently and durably inhibits the phosphorylation of mTORC1 downstream effectors 4EBP1 and S6 (a direct substrate of the mTORC1 substrate S6K), and decreased protein expression levels of MYC. Our results show for the first time that inhibition of mTORC1 with concomitant suppression of p-4EBP1 drove regression in MYC-driven HCC tumors, and the significant anti-tumor activity of RM-006 may result from sustained reduction of MYC protein levels due to reduced protein translation upon 4EBP1 reactivation. Future directions will aim at defining whether RM-006 affects global MYC-regulated cell-intrinsic and host immune pathways. These preclinical data highlight a potential therapeutic opportunity for the investigational bi-steric mTORC1 inhibitor RMC-5552 in combination with immune checkpoint inhibitors to overcome MYC-mediated immune suppression. Under this potential therapeutic paradigm HCC patient survival may be extended via a dual mechanism that reduces MYC levels via 4EBP1 reactivation and that rescues anti-tumor immune surveillance. Citation Format: Wadie D. Mahauad-Fernandez, Yu C. Yang, Ian Lai, Jangho Park, James W. Evans, Mallika Singh, Jacqueline A. Smith, Dean W. Felsher. A bi-steric mTORC1 inhibitor that selectively reactivates 4EBP1 and induces regression of MYC-driven hepatocellular carcinoma [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 1002.

Abstract 1026: Bi-steric mTORC1-selective inhibitors demonstrate improved potency and efficacy in tumors with mTORC1 hyperactivation

Abstract The PI3K-mTOR pathway is one of the most commonly dysregulated pathways in human tumors. Rapalogs have been used extensively in human clinical trials but exhibit modest clinical benefit for the most part, possibly due to their lack of effect on 4E-BP1, a key target downstream of mTORC1. ATP-competitive mTOR inhibitors can fully inhibit mTORC1 but are poorly tolerated possibly due to their inhibition of mTORC2. A new class of selective mTORC1 inhibitors has been developed and termed ‘bi-steric', which comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. RMC-5552 is the first clinical candidate of this class and clinical testing is planned in 2021. RM-001 and RM-006 (also known as RMC-6272) are representative bi-steric tool compounds that exhibit potent and selective (> 10-fold) inhibition of mTORC1 over mTORC2. We report that bi-steric mTORC1-selective inhibitors have greater activity than rapalogs for tumors with mTORC1 hyperactivation secondary to TSC1/2 loss. RM-001 and RM-006 showed more effective growth inhibition in multiple TSC1 or TSC2 mutant tumor cell lines compared to rapamycin. RM-001 and RM-006 caused a more profound growth inhibition in the TSC1 or TSC2 mutant cells than the wild type cells. Two-week low dilution clonogenic assays in TSC1 or TSC2 mutant cells showed clone numbers were significantly reduced by rapamycin, RM-001, and RM-006, whereas in the wild type cells, Rapamycin and RM compounds had less reduction in clone numbers, indicating a differential sensitivity in TSC-null cells. Both RM-001 and RM-006 at ~1 nM showed near complete inhibition of p4E-BP1T37/46, which was not seen with rapamycin treatment, while inhibition of pS6S240/244 levels was similar for rapamycin and RM compounds. Further, the RM compounds had prolonged anti-proliferative activity in TSC-null cells, as shown by washout studies. Rapamycin, MLN0128 and both RM compounds markedly reduced kidney tumor burden in Tsc2+/- A/J mice after four weeks of treatment. Tumor regrowth was assessed two months after treatment cessation, tumor burden was significantly reduced in the RM-006 group as compared to the rapamycin and MLN0128 groups. Finally, TUNEL staining showed RM-006 treatment led to a greater induction of apoptosis in kidney tumor cells relative to rapamycin and MLN0128 after a single dose of each compound in Tsc2+/- A/J mice. This is the first time that clear evidence for cell death after mTORC1 inhibition has been seen in TSC rodent models. In summary, RM-001 and RM-006 demonstrate improved in vitro and in vivo inhibition of mTORC1 in comparison to rapamycin, and induced more cell death in TSC2 null tumors in vivo, indicating the potential of bi-steric mTORC1-selective inhibitors as a novel therapeutic strategy to treat tumors with mTORC1 dysregulation. Citation Format: Heng Du, Yu Chi Yang, Mallika Singh, Heng-jia Liu, David Kwiatkowski. Bi-steric mTORC1-selective inhibitors demonstrate improved potency and efficacy in tumors with mTORC1 hyperactivation [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 1026.

A low-dose combination of ketamine and guanosine counteracts corticosterone-induced depressive-like behavior and hippocampal synaptic impairments via mTORC1 signaling

Ketamine exhibits rapid and sustained antidepressant responses, but its repeated use may cause adverse effects. Augmentation strategies have been postulated to be useful for the management/reduction of ketamine's dose and its adverse effects. Based on the studies that have suggested that ketamine and guanosine may share overlapping mechanisms of action, the present study investigated the antidepressant-like effect of subthreshold doses of ketamine and guanosine in mice subjected to repeated administration of corticosterone (CORT) and the role of mTORC1 signaling for this effect. The ability of the treatment with ketamine (0.1 mg/kg, i.p.) plus guanosine (0.01 mg/kg, p.o.) to counteract the depressive-like behavior induced by CORT (20 mg/kg, p.o., for 21 days) in mice, was paralleled with the prevention of the CORT-induced reduction on BDNF levels, Akt (Ser473) and GSK-3β (Ser9) phosphorylation, and PSD-95, GluA1, and synapsin immunocontent in the hippocampus. No changes on mTORC1 and p70S6K immunocontent were found in the hippocampus and prefrontal cortex of any experimental group. No alterations on BDNF, Akt/GSK-3β, mTORC1/p70S6K, and synaptic proteins were observed in the prefrontal cortex of mice. The antidepressant-like and pro-synaptogenic effects elicited by ketamine plus guanosine were abolished by the pretreatment with rapamycin (0.2 nmol/site, i.c.v., a selective mTORC1 inhibitor). Our results showed that the combined administration of ketamine and guanosine at low doses counteracted CORT-induced depressive-like behavior and synaptogenic disturbances by activating mTORC1 signaling. This study supports the notion that the combined administration of guanosine and ketamine may be a useful therapeutic strategy for the management of MDD.

Physical exercise stimulates hippocampal mTORC1 and FNDC5/irisin signaling pathway in mice: Possible implication for its antidepressant effect

Several lines of evidence have consistently indicated that physical exercise has antidepressant effects by improving hippocampal function, although the signaling pathways underpinning these responses are not well established. Therefore, this study investigated the role of mechanistic target of rapamycin complex 1 (mTORC1) and fibronectin type III domain-containing protein 5 (FNDC5)/irisin signaling in the antidepressant-like effect of physical exercise. We showed that physical exercise (treadmill running – 45 min/day/5 days/week for 4 weeks) produced an antidepressant-like effect as indicated by a reduction on the immobility time in mice subjected to the forced swimming test (FST) without altering locomotor activity in the open field test (OFT). Rapamycin (a selective mTORC1 inhibitor, 0.2 nmol/site, i.c.v.) administration completely abolished the antidepressant-like effect of physical exercise in the FST, suggesting that mTORC1 activation plays a role for its behavioral effect. Accordingly, physical exercise increased the number of phosphorylated mTORC1 (Ser2448)-positive cells in the entire and ventral subgranular zone of the hippocampal dentate gyrus. Physical exercise was also effective in augmenting the hippocampal FNDC5/irisin immunocontent, but rapamycin administration did not alter this effect. Our results reinforce the notion that physical exercise exerts an antidepressant-like effect and identifies the mTORC1-mediated signaling pathway as a target for its behavioral effects. This study provides additional evidence that physical exercise increases hippocampal FNDC5/irisin immunocontent, but this effect seems to be independent on hippocampal mTORC1 activation. Altogether the results contribute to elucidate possible molecular targets implicated in the antidepressant effects of physical exercise and highlight the role of mTORC1 signaling for its behavioral response.

Abstract LB-113: Positioning a selective, bi-steric inhibitor of mTORC1 as a combination partner in RAS-driven cancers

Abstract The RAS and PI3K/mTOR signaling pathways function downstream of receptor tyrosine kinases (RTK), and are hyperactivated in a wide range of human cancers. Several points of cross-talk and intersecting feedback mechanisms have also been described, in which inhibition of one pathway node leads to activation of the parallel signaling pathway, thereby decreasing the effectiveness of single-pathway targeted therapies. Concomitant inhibition of both pathways is clearly effective in RAS-driven cancers as demonstrated via genetic means and by the combination of pharmacologic inhibitors of MEK and PI3 kinase in a variety of preclinical models (Engelman 2008, Collisson 2012, Eser 2013) However, the clinical translation of this combination has been largely unsuccessful, primarily due to development of dose-limiting toxicities that prohibit the achievement of optimal therapeutic drug concentrations. Recent advances in the treatment paradigm for RAS mutant non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) include several first-in-class covalent inhibitors of KRASG12C (e.g., AMG510, MRTX849) which have demonstrated a favorable safety profile along with early promising anti-tumor activity in patients with advanced cancers harboring a KRASG12C mutation. Drug-anchored CRISPR screens and combination experiments have identified mTOR signaling as a key mediator that limits therapeutic response to KRASG12C inhibition (Molina-Arcas 2019, Hallin 2019). We have developed a novel class of selective mTORC1 inhibitors, termed ‘bi-steric', that covalently links rapamycin analogs with a mTOR kinase active-site inhibitor, and interacts with both the ATP- and FKBP12/FRB-binding sites of mTORC1. These compounds exhibit potent and selective (>10-fold) inhibition of mTORC1 over mTORC2, durably suppress S6K and 4EBP1 phosphorylation, and induce growth suppression and apoptosis in multiple cancer cell lines. In preclinical tests, these mTORC-1 selective inhibitors do not induce hyperglycemia and are predicted to have reduced mTORC2-mediated toxicities. We tested the combinatorial activity of these mTOR inhibitors and covalent KRASG12C inhibitors in preclinical models of two KRASG12C mutant tumor types predicted to have limited sensitivity to KRASG12C inhibitor monotherapy: a) STK11 deficient NSCLC wherein activation of mTORC1 signaling is also an oncogenic driver (~20% co-occurrence in KRAS-mutant NSCLC, Scheffler 2018) and b) colorectal cancer wherein RTK activation is thought to co-activate PI3 kinase signaling (Ebi, 2011). In each case, we showed combinatorial activity manifest as decreased tumor cell viability in vitro and tumor regressions in vivo. In contrast, single agents exhibited cytostatic effects in vitro and modest effects on tumor growth in vivo. Our data demonstrate that the combination of KRASG12C and bi-steric mTORC1 selective inhibitors elicits marked anti-tumor activity at well tolerated doses in vivo in models of cancer patient populations with current unmet medical need. The mechanistic bases for these combinatorial effects are currently under investigation. Our preclinical results reinforce the importance of mTORC1 as a concurrent target in genetically defined RAS-mutant cancers, and showcase the therapeutic potential of a novel and selective bi-steric mTORC1 inhibitor, currently in IND-enabling studies. Citation Format: Yu Chi Yang, Christopher Schulze, Jingjing Jiang, Denise F. Reyes, Tiffany Choy, Tram Nguyen, Zhican Wang, Dong Lee, Robert J. Nichols, Zhengping Wang, Jacqueline A. Smith, Steve M. Kelsey, Mallika Singh. Positioning a selective, bi-steric inhibitor of mTORC1 as a combination partner in RAS-driven cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-113.

MTORC1 pathway is involved in the kappa opioid receptor activation-induced increase in excessive alcohol drinking in mice

KOP-r agonist U50,488H produces strong aversion and anxiety/depression-like behaviors that enhance alcohol intake and promote alcohol seeking and relapse-like drinking in rodents. Mammalian target of rapamycin complex 1 (mTORC1) pathway in mouse striatum is highly involved in excessive alcohol intake and seeking, and in the U50,488H-induced conditioned place aversion. Therefore, we hypothesized that KOP-r activation increases alcohol consumption through the mTORC1 activation. This study focuses on: (1) how chronic excessive alcohol drinking (4-day drinking-in-the-dark paradigm followed by 3-week chronic intermittent access drinking paradigm [two-bottle choice, 24-h access every other day]) affected nuclear transcript levels of the mTORC1 pathway genes in mouse nucleus accumbens shell (NAcs), using transcriptome-wide RNA sequencing analysis; and (2) whether selective mTORC1 inhibitor rapamycin could alter excessive alcohol drinking and prevent U50,488H-promoted alcohol intake. Thirteen nuclear transcripts of mTORC1 pathway genes showed significant up-regulation in the NAcs, with two genes down-regulated, after excessive alcohol drinking, suggesting the mTORC1 pathway was profoundly disrupted. Single administration of rapamycin decreased alcohol drinking in a dose-dependent manner. U50,488H increased alcohol drinking, and pretreatment with rapamycin, at a dose lower than effective doses, blocked the U50,488H-promoted alcohol intake in a dose-dependent manner, indicating a mTORC1-mediated mechanism. Our results provide supportive and direct evidence relevant to the transcriptional profiling of the critical mTORC1 genes in mouse NAc shell: with functional and pharmacological effects of rapamycin, altered nuclear transcripts in the mTORC1 signaling pathway after excessive alcohol drinking may contribute to increased alcohol intake triggered by KOP-r activation.

Abstract B108: 4EBP3 mRNA as a biomarker of therapeutic response to treatment with mTORC1 inhibitors

Abstract Aberrantly activated PI3K/mTOR signaling is frequently implicated as an oncogenic driver in human cancer. In response to nutrient and growth factor stimuli, mTOR complex 1 (mTORC1) regulates cell growth and cap-dependent protein translation through phosphorylation and activation of S6K, and through phosphorylation and inactivation of the translational repressor 4EBP1. While suppression of mTORC1 activity causes rapid dephosphorylation of its substrates S6K and 4EBP1, it has also been reported that the translational repressor 4EBP3 is transcriptionally induced during prolonged mTORC1 inhibition.1 We have designed a series of complete and selective bi-steric inhibitors of mTORC1 by covalently linking rapamycin, a selective yet incomplete mTORC1 inhibitor, with mTOR kinase active-site inhibitors. These compounds potently and durably suppress phosphorylation of S6K and 4EBP1, induce growth suppression and apoptosis in multiple cancer cell lines, and cause tumor growth inhibition in xenograft models. In the present study we evaluated the utility of 4EBP3 mRNA expression as a pharmacodynamic (PD) biomarker of mTORC1 inhibition by our bi-steric inhibitors. We further interrogated whether tumor mTORC1 inhibition and anti-tumor efficacy could be predicted by monitoring this PD marker in surrogate tissue. First, we demonstrated that 4EBP3 mRNA and protein levels increased in response to mTORC1 inhibition in a concentration- and time-dependent manner in MDA-MB-468, MCF-7, and HCC1954 breast carcinoma cells. Kinetic studies in which we compared the response of bi-steric inhibitors to that of short duration-of-action active-site inhibitors revealed that 4EBP3 induction requires sustained mTORC1 inhibition, as loss of mTORC1 inhibition correlated with a rapid return to baseline 4EBP3 expression. Treatment with inhibitors of varying potency against mTORC1 also demonstrated a strong correlation between magnitude of cell growth inhibition and 4EBP3 mRNA induction. Additionally, suppression of tumor 4EBP1 phosphorylation and induction of 4EBP3 mRNA was observed in MCF-7 and HCC9154 tumor xenografts following in vivo administration of bi-steric inhibitors with a concomitant induction of murine 4ebp3 in peripheral blood mononuclear cells (PBMCs). Initial studies indicate that ex vivo exposure of human PBMCs to a mTORC1 inhibitor also promotes 4EBP3 mRNA expression. Together, these data suggest that 4EBP3 could provide a sensitive mRNA-based in vivo biomarker of prolonged mTORC1 inhibition, as is the case following administration of a single dose of a bi-steric mTORC1 inhibitor. The concordance between the responses in tumor and PBMC suggests it may be possible to monitor tumor mTORC1 inhibition by monitoring 4EBP3 in surrogate tissue. In summary, 4EBP3 mRNA represents a potential PD biomarker of mTORC1 inhibitor response as an alternative to measurement of the phosphorylation status of mTORC1 substrates. 1. Tsukumo, T., et al. Translation control during prolonged mTORC1 inhibition mediated by 4E-BP3. Nat Commun 7:11776. doi: 10.1038/ncomms11776 (2016). Citation Format: Bianca J. Lee, Nuntana Dinglasan, Tram Nguyen, Ed Lorenzana, Stacy Wilson, G. Leslie Burnett, James B. Aggen, Robert J. Nichols, Mallika Singh, David Wildes, Jaqueline A.M. Smith. 4EBP3 mRNA as a biomarker of therapeutic response to treatment with mTORC1 inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B108. doi:10.1158/1535-7163.TARG-19-B108

DDIS-33. SELECTIVE AND COMPLETE INHIBITION OF mTORC1 BY BI-STERIC mTOR INHIBITORS DRIVES THERAPEUTIC RESPONSE IN GLIOBLASTOMA

Abstract Activation of phosphatidylinositol 3-kinase (PI3K)/PTEN pathway and oncogenic signaling via the mechanistic target of rapamycin (mTOR) occur in a majority of high-grade glial brain tumors. Allosteric mTOR inhibitors, such as rapamycin and other rapalogs, incompletely block mTORC1 by reducing phosphorylation of some substrates, including S6K1, but not 4EBP1. In contrast, ATP-competitive inhibitors, such as sapanisertib, fully inhibit mTORC1. However these inhibitors are also active against mTORC2 and lipid kinases, likely enhancing toxicity. A new class of selective mTORC1 inhibitor that interacts with both the ATP- and FKBP12/FRB-binding sites has been developed, which we term ‘bi-steric’. The prototype bi-steric inhibitor, RapaLink-1, blocks phosphorylation of many mTORC1 substrates, including 4EBP1. Importantly, RapaLink-1 showed improved efficacy in glioblastoma models in vivo as compared to rapamycin or sapanisertib (Fan et al., Cancer Cell 2017). Revolution Medicines has developed novel next-generation bi-steric mTORC1-selective inhibitors that exhibit potent and selective (>10-fold) inhibition of mTORC1 over mTORC2 in vitro in cell line models. Two of these compounds, RM-001 and RM-006, showed sustained blockade of mTORC1 signaling, including dephosphorylation of 4EBP1, following weekly ip dosing in an orthotopic U87MG-Luc model of glioblastoma. Repeated weekly administration of these agents resulted in significantly greater anti-tumor efficacy, as assessed via tumor burden (bioluminescence imaging) and overall survival in comparison to daily sapanisertib and the rapalog everolimus, and weekly dosing of the first generation bi-steric RapaLink-1, all at maximally tolerated doses. In summary, our data demonstrate that bi-steric mTORC1 selective inhibitors elict marked anti-tumor efficacy at doses that are well tolerated in a preclinical model of glioblastoma. Our study emphasizes the importance of mTOR as a central target in glioblastoma, and showcases the therapeutic potential of novel and selective clinical bi-steric mTORC1 inhibitors under development as investigational new drugs.

N-Methyl-D-aspartate receptor antagonist d-methadone produces rapid, mTORC1-dependent antidepressant effects.

Currently available antidepressants have a delayed onset and limited efficacy, highlighting the need for new, rapid and more efficacious agents. Ketamine, an NMDA receptor antagonist, has emerged as a new rapid-acting antidepressant, effective even in treatment resistant patients. However, ketamine induces undesired psychotomimetic and dissociative side effects that limit its clinical use. The d-stereoisomer of methadone (dextromethadone; REL-1017) is a noncompetitive NMDA receptor antagonist with an apparently favorable safety and tolerability profile. The current study examined the rapid and sustained antidepressant actions of d-methadone in several behavioral paradigms, as well as on mTORC1 signaling and synaptic changes in the medial prefrontal cortex (mPFC). A single dose of d-methadone promoted rapid and sustained antidepressant responses in the novelty-suppressed feeding test (NSFT), a measure of anxiety, and in the female urine sniffing test (FUST), a measure of motivation and reward. D-methadone also produced a rapid reversal of the sucrose preference deficit, a measure of anhedonia, in rats exposed to chronic unpredictable stress. D-methadone increased phospho-p70S6 kinase, a downstream target of mTORC1 in the mPFC, and intra-mPFC infusion of the selective mTORC1 inhibitor rapamycin blocked the antidepressant actions of d-methadone in the FUST and NSFT. D-methadone administration also increased levels of the synaptic proteins, PSD95, GluA1, and Synapsin 1 and enhanced synaptic function in the mPFC. Studies in primary cortical cultures show that d-methadone also increases BDNF release, as well as phospho-p70S6 kinase. These findings indicate that d-methadone induces rapid antidepressant actions through mTORC1-mediated synaptic plasticity in the mPFC similar to ketamine.

Mammalian target of rapamycin complex 1 and its downstream effector collapsin response mediator protein-2 drive reinstatement of alcohol reward seeking.

Alcohol use disorder is a chronic relapsing disease. Maintaining abstinence represents a major challenge for alcohol-dependent patients. Yet the molecular underpinnings of alcohol relapse remain poorly understood. In the present study, we investigated the potential role of the mammalian target of rapamycin complex 1 (mTORC1) in relapse to alcohol-seeking behavior by using the reinstatement of a previously extinguished alcohol conditioned place preference (CPP) response as a surrogate relapse paradigm. We found that mTORC1 is activated in the nucleus accumbens shell following alcohol priming-induced reinstatement of alcohol place preference. We further report that the selective mTORC1 inhibitor, rapamycin, abolishes reinstatement of alcohol place preference. Activation of mTORC1 initiates the translation of synaptic proteins, and we observed that reinstatement of alcohol CPP is associated with increased protein levels of one of mTORC1's downstream targets, collapsin response mediator protein-2 (CRMP2), in the nucleus accumbens. Importantly, the level of mTORC1 activation and CRMP2 expression positively correlate with the CPP score during reinstatement. Finally, we found that systemic administration of the CRMP2 inhibitor, lacosamide, attenuates alcohol priming-induced reinstatement of CPP. Together, our results reveal that mTORC1 and its downstream target, CRMP2, contribute to mechanisms underlying reinstatement of alcohol reward seeking. Our results could have important implications for the treatment of relapse to alcohol use and position the Food and Drug Administration approved drugs, rapamycin and lacosamide, for the treatment of alcohol use disorder.

MTORC1 controls long-term memory retrieval

Understanding how stored information emerges is a main question in the neurobiology of memory that is now increasingly gaining attention. However, molecular events underlying this memory stage, including involvement of protein synthesis, are not well defined. Mammalian target of rapamycin complex 1 (mTORC1), a central regulator of protein synthesis, has been implicated in synaptic plasticity and is required for memory formation. Using inhibitory avoidance (IA), we evaluated the role of mTORC1 in memory retrieval. Infusion of a selective mTORC1 inhibitor, rapamycin, into the dorsal hippocampus 15 or 40 min but not 3 h before testing at 24 h reversibly disrupted memory expression even in animals that had already expressed IA memory. Emetine, a general protein synthesis inhibitor, provoked a similar impairment. mTORC1 inhibition did not interfere with short-term memory retrieval. When infused before test at 7 or 14 but not at 28 days after training, rapamycin impaired memory expression. mTORC1 blockade in retrosplenial cortex, another structure required for IA memory, also impaired memory retention. In addition, pretest intrahippocampal rapamycin infusion impaired object location memory retrieval. Our results support the idea that ongoing protein synthesis mediated by activation of mTORC1 pathway is necessary for long but not for short term memory.

Abstract 137: FT-1518, a new generation selective and potent mTORC1 and mTORC2 inhibitor: an in vitro and in vivo profile

Abstract Mammalian target of rapamycin (mTOR) is a clinically validated target in the treatment of cancer. mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2 which regulate cell growth, metabolism, proliferation, and survival. Rapamycin analogues target only the mTORC1 complex but do not affect the mTORC2 complex, which is an important driver for cancer cell growth and survival. The new generation of “Selective” mTOR inhibitors, blocking both mTORC1 and mTORC2 signaling might increase the efficacy and safety while expanding the therapeutic potential of these anticancer agents. Herein we describe FT-1518, a low nanomolar potent, kinase and PI3K sub family selective mTOR inhibitor. FT-1518 not only exhibited high oral bioavailability in preclinical species but has demonstrated excellent microsomal stability with no inhibitory activity towards undesired CYPs. FT-1518 showed high sustained tumor exposure and target Inhibition in a single oral dose xenograft model. FT-1518 depicted very good growth inhibitory activity across a large panel of hematologic and solid tumor cell lines with most activities falling into low nanomolar range. mTOR kinase inhibition in cells, by FT-1518, resulted in more potent inhibition of the mTOR pathway biomarkers (mTORC 1 & 2 biomarkers [pAkt(S473) and pS6(S240/244) or p70 S6K), no inhibition of PI3K biomarker [pAkt(T308)], and improved anti-proliferative activity as compared with rapamycin. FT-1518 exhibited dose-dependent and higher tumor growth inhibition (TGI) in multiple solid tumor xenografts compared with rapalogs and is poised to enter the clinic with a favorable toxicology profile. Citation Format: Alain C. Mita, Monica M. Mita, Anthony D. William, Khalid Pasha, Chandra Siddamadappa, Kevin Zikaras, Felix T. Garzon. FT-1518, a new generation selective and potent mTORC1 and mTORC2 inhibitor: an in vitro and in vivo profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 137. doi:10.1158/1538-7445.AM2017-137

Ketamine accelerates fear extinction via mTORC1 signaling

Impaired fear extinction contributes to the persistence of post-traumatic stress disorder (PTSD), and can be utilized for the study of novel therapeutic agents. Glutamate plays an important role in the formation of traumatic memories, and in the pathophysiology and treatment of PTSD, highlighting several possible drug targets. Recent clinical studies demonstrate that infusion of ketamine, a glutamate NMDA receptor antagonist, rapidly and significantly reduces symptom severity in PTSD patients. In the present study, we examine the mechanisms underlying the actions of ketamine in a rodent model of fear conditioning, extinction, and renewal. Rats received ketamine or saline 24h after fear conditioning and were then subjected to extinction-training on each of the following three days. Ketamine administration enhanced extinction on the second day of training (i.e., reduced freezing behavior to cue) and produced a long-lasting reduction in freezing on exposure to cue plus context 8days later. Additionally, ketamine and extinction exposure increased levels of mTORC1 in the medial prefrontal cortex (mPFC), a region involved in the acquisition and retrieval of extinction, and infusion of the selective mTORC1 inhibitor rapamycin into the mPFC blocked the effects of ketamine on extinction. Ketamine plus extinction also increased cFos in the mPFC and administration of a glutamate-AMPA receptor antagonist blocked the effects of ketamine. These results support the hypothesis that ketamine produces long-lasting mTORC1/protein synthesis and activity dependent effects on neuronal circuits that enhance the expression of extinction and could represent a novel approach for the treatment of PTSD.