Selective Inhibition Of mTORC1 Research Articles

Abstract C020: First-in-human phase 1/1b trial of the first-in-class bi-steric mTORC1-selective inhibitor RMC-5552 in patients with advanced solid tumors

Abstract Activation of the mTOR pathway plays a role in the oncogenesis and progression of many cancer types, and is frequently co-altered in RAS-driven tumors. First-generation mTORC1-selective inhibitors were limited by incomplete inhibition of 4EBP1 phosphorylation, while second-generation pan-mTOR inhibitors had a low therapeutic index due to mTORC2-mediated toxicity such as hyperglycemia. RMC-5552 is a first-in-class third-generation bi-steric mTOR inhibitor that is mTORC1-selective via its FKBP12-binding moiety and potently inhibits phosphorylation of the 4EBP1 tumor suppressor via a linked ATP-competitive moiety, addressing these key limitations of prior mTOR inhibitors in preclinical models. RMC-5552 is being investigated in a Phase I/Ib study (NCT04774952). Eligible patients (pts) were ≥18 yrs old with ECOG 0-1 and had advanced solid tumors that had failed or were ineligible for standard-of-care treatments. Pts received RMC-5552 as a weekly infusion. To address mucositis, a common toxicity in pts treated with mTOR inhibitors, pts prophylactically used dexamethasone mouthwash either alone or with tacrolimus mouthwash (TM), which binds to the RMC-5552 presenter protein FKBP12. Objectives included assessment of safety/tolerability (CTCAEv5), defining the maximum tolerated and recommended Phase 2 doses, pharmacokinetics, and preliminary efficacy (RECIST v1.1). As of June 12, 2023, 47 pts have been evaluated at 6 dose levels from 1.6 mg to 12 mg. The most common (> 20%) treatment-related adverse events (TRAE, any grade) were stomatitis/mucositis (45%), fatigue (40%), nausea (38%), decreased appetite (32%), and vomiting (23%). Treatment-related events of hyperglycemia occurred in 4% of pts (both grade 2) and were not dose limiting. The most common grade 3 TRAE at dose levels ≥6 mg was mucositis/stomatitis, which was dose-dependent and limited tolerability in the absence of TM. Between 8 and 12 mg, the rate of treatment related stomatitis/mucositis was 65% (15% grade 3) in pts treated without TM (N = 20) versus 30% (no grade 3) in pts treated with TM (N = 10). TM prophylaxis enabled dose escalation beyond dose levels previously considered intolerable due to mucositis (10-12 mg; escalation ongoing), and unbound plasma exposures at 8-12 mg were within the range that induced significant anti-tumor activity in preclinical models. The disease control rate was 67% among efficacy evaluable pts (N = 39). One pt with a salivary gland tumor harboring a PTEN mutation had a confirmed partial response (PR) with 78% reduction and 16 months duration of response. In addition, molecular responses of circulating tumor DNA were observed in pts with mTOR pathway variants. Updated efficacy results will be presented. These early data indicate that RMC-5552 monotherapy is tolerated at doses predicted to be efficacious, and that selective inhibition of mTORC1 alleviates mTORC2-mediated toxicity. Dose escalation is ongoing, and mucositis prophylaxis has enabled further dose intensification for future combination studies with RAS(ON) inhibitors. Citation Format: Alison M Schram, Abdul Rafeh Naqash, Eric B Haura, Jonathan W Riess, Susanna V Ulahannan, Sai-Hong I Ou, Anna Capasso, Pamela N Munster, Michael L Cheng, W Clay Gustafson, Bojena Bitman, Sumit Kar, Zhican Wang, Lin Tao, Justin G Meyerowitz, Howard A Burris. First-in-human phase 1/1b trial of the first-in-class bi-steric mTORC1-selective inhibitor RMC-5552 in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C020.

Blazing a trail for the clinical use of rapamycin as a geroprotecTOR.

Treatment with rapamycin, an inhibitor of the mechanistic Target Of Rapamycin Complex One (mTORC1) protein kinase, has been repeatedly demonstrated to extend lifespan and prevent or delay age-related diseases in diverse model systems. Concerns over the risk of potentially serious side effects in humans, including immunosuppression and metabolic disruptions, have cautiously limited the translation of rapamycin and its analogs as a treatment for aging associated conditions. During the last decade, we and others have developed a working model that suggests that while inhibition of mTORC1 promotes healthy aging, many of the negative side effects of rapamycin are associated with "off-target" inhibition of a second mTOR complex, mTORC2. Differences in the kinetics and molecular mechanisms by which rapamycin inhibits mTORC1 and mTORC2 suggest that a therapeutic window for rapamycin could be exploited using intermittent dosing schedules or alternative rapalogs that may enable more selective inhibition of mTORC1. However, the optimal dosing schedules and the long-term efficacy of such interventions in humans are unknown. Here, we highlight ongoing or upcoming clinical trials that will address outstanding questions regarding the safety, pharmacokinetics, pharmacodynamics, and efficacy of rapamycin and rapalogs on several clinically oriented outcomes. Results from these early phase studies will help guide the design of phase 3 clinical trials to determine whether rapamycin can be used safely to inhibit mTORC1 for the treatment and prevention of age-related diseases in humans.

Discovery of RMC-5552, a Selective Bi-Steric Inhibitor of mTORC1, for the Treatment of mTORC1-Activated Tumors.

Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of "bi-steric inhibitors" that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRASG12C shows increased antitumor activity in a preclinical model of KRASG12C mutant NSCLC that exhibits resistance to KRASG12C inhibitor monotherapy.

Abstract P244: Tubular Sites Of Mtorc2 Inhibition Of Sodium Transport In Dahl Salt-sensitive Rats.

Background: There is evidence that the mTOR signaling pathway plays an important role in salt induced hypertension in the Dahl salt-sensitive (SS) rat via regulation of Na + excretion. PP242 inhibition of mTORC1-2 prevented hypertension in SS rats and intrarenal PP242 produced a 4-fold increase of Na + excretion while selective inhibition of mTORC1 with rapamycin had no effect. The goal of the present study was to identify the renal tubular Na + transporters regulated by the mTORC2 pathway. Methods: Natriuretic effectors of mTORC2 were deduced from the differences of Na + excreted with PP242 treatment beyond that observed by the natriuretic actions of benzamil (B; ENaC inhibitor), furosemide (F; NKCC2 inhibitor), hydrochlorothiazide (H; NCC inhibitor) and in combination (B+F+H). Unanesthetized SS rats were first administered B, F, H or B+F+H (i.p.) and 2 days later with co-administration of PP242 with these same reagents. Rats were studied when fed 0.4% NaCl (LS) and then following 3 weeks of a 4.0% NaCl (HS) diet. The effect of PP242 on ENaC activity in freshly isolated cortical collecting ducts was also examined using cell-attached patch clamp electrophysiology. Results: Urine Na + excretion rates determined at 20 min. intervals over 80 minutes increased nearly 2-fold with administration of PP242 + B, compared to B alone [0.10 + 0.02 vs. 0.04 + 0.01 mmol/100gbw (n=8; P<0.05)] and similarly in the F and H comparisons [F: 0.20 + 0.02 vs. 0.10 + 0.02 mmol/100gbw (n=8; P<0.05); H: 0.05 + 0.01 vs. 0.03 + 0.01 mmol/100gbw (n=8; P=0.06)]. PP242 given in the presence of F+B+H did not further enhance the overall natriuretic response. Rats fed HS showed little enhancement of natriuresis when PP242 was administered with any of the drugs. Minimum changes in K + excretion were observed with PP242 in LS or HS studies. Patch clamp studies found that PP242 reduced ENaC activity through reduction of the open channel probability (Po) in both LS and HS fed rats. Western blot data indicated that PP242 reduced pNCC T53 and phosphorylation of SGK1. Conclusion: Together, these studies found evidence that the natriuretic actions of PP242 are a consequence of inhibitory actions on the ENaC and NCC transporters in the distal nephron segments.

Further Insights Into the Impact of Bariatric Surgery on the Progression of Nonalcoholic Fatty Liver Disease

Seeberg KA, Borgeraas H, Hofsø H, et al. Gastric bypass versus sleeve gastrectomy in type 2 diabetes: effects on hepatic steatosis and fibrosis: a randomized controlled trial. Ann Intern Med 2022;175:74–83. Bariatric surgery has an increasingly recognized role in treating metabolic syndrome, and benefits nonalcoholic fatty liver disease (NAFLD) specifically (Gastroenterology 2020;159:1290–1301). However, the extent to which bariatric surgery might impact NAFLD progression and whether various bariatric approaches affect this differently remain relatively unexplored. Seeberg et al undertook a single-center randomized trial in which 100 participants with type 2 diabetes mellitus (T2DM) and obesity were randomized 1:1 to either Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG) and followed up to 1 year postoperatively. Although the primary trial outcomes related to T2DM remission have been published (Lancet Diabetes Endocrinol 2019;7:912–924), this present study described predefined outcomes related to hepatic steatosis (assessed via liver fat fraction on magnetic resonance imaging), enhanced liver fibrosis test (as a proxy of hepatic fibrosis), and other noninvasive NAFLD markers. At 1-year follow-up, there was a 20% reduction (95% CI, 23% to 17%) in liver fat fraction in the SG group and 22% (95% CI, 24% to 19%) observed in the RYGB group, with no significant difference between groups. Almost all patients—94% of the SG group and 100% of the RYGB group—had no or low-grade steatosis at 1 year (compared with ∼25% of participants at baseline). The enhanced liver fibrosis score category remained stable in 77% of patients, but 18% experienced worsening of fibrosis at 1 year, with no substantial between-group difference. Weight loss, improvements in lipid profile, and remission rates for T2DM favored RYGB compared with SG. Collectively, these data demonstrate that both evaluated forms of bariatric surgery are of comparable high efficacy in improving hepatic steatosis, although this may not translate into short-term improvements in hepatic fibrosis. Study limitations include it being a single-center trial, possible underpowering for NAFLD-related outcomes (given the primary outcome relating to T2DM), and the reliance on a noninvasive marker (enhanced liver fibrosis) as the main measure of hepatic fibrosis, with no liver biopsies obtained. Given that steatohepatitis typically progresses to fibrosis over the course of years rather than months in NAFLD, results of the already planned longer-term follow-up will be of great pertinence for gauging the full impact of bariatric surgery. Splitting Hairs: Folliculin Parts the Good From the Bad in mTORC1 Control Over Lipid MetabolismGastroenterologyVol. 163Issue 2PreviewGosis BS, Wada S, Thorsheim C, et al. Inhibition of nonalcoholic fatty liver disease in mice by selective inhibition of mTORC1. Science 2022;376(6590):eabf8271. Full-Text PDF

Splitting Hairs: Folliculin Parts the Good From the Bad in mTORC1 Control Over Lipid Metabolism

Gosis BS, Wada S, Thorsheim C, et al. Inhibition of nonalcoholic fatty liver disease in mice by selective inhibition of mTORC1. Science 2022;376(6590):eabf8271. The mechanistic target of rapamycin complex 1 (mTORC1) is a signaling kinase complex that mediates several fundamental processes, including de novo lipogenesis. Therefore, mTORC1 is an attractive target to treat nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). However, a barrier to effectively target mTORC1 is an incomplete understanding of how mTORC1 regulates liver fat metabolism. To illustrate this point, both hyperactivating and inactivating mTORC1 signaling attenuates de novo lipogenesis and hepatic fat accumulation in mice. What is known is that mTORC1 activates de novo lipogenesis by phosphorylating 2 primary downstream effectors—p70 S6 kinase (P70S6K) and transcription factor E3 (TFE3)—which both promote de novo lipogenesis. At the same time, these de novo lipogenesis–promoting actions of mTORC1 are counterbalanced by an important negative feedback loop; mTORC1-P70S6K signaling inhibits the insulin receptor complex to attenuate further anabolic insulin signaling. Therefore, selective targeting of mTORC1 through TFE3, which prevents de novo lipogenesis but does not break the P70S6K anabolic insulin signaling feedback loop, could be an ideal target for NAFLD. Gosis and colleagues provided exciting new data to unify prior disparate observations and identify a potential therapeutic target for NAFLD. They identified the GTPase-activating protein folliculin (FLCN) as a factor that confers mTORC1 substrate specificity for TFE3, but not p70S6K. Using a variety of diet-induced models for NAFLD/NASH, they showed that liver-specific FLCN deletion blocked mTORC1-mediated TFE3 phosphorylation, thereby promoting lipid catabolic gene transcription. However, FLCN deletion preserved the insulin-mediated P70S6K-negative feedback loop, thereby reducing further anabolic insulin signaling. In these diet-induced NAFLD/NASH models, FLCN deletion promoted fat oxidation and prevented fat accumulation and fibrosis in the liver. Furthermore, in established diet-induced NASH, inhibiting FLCN reversed fat accumulation and fibrosis marker gene expression, but not fibrosis scores. The numerous strengths of this study include the detailed mechanistic in vivo genetic models used to demonstrate the cell biology, physiology, and translational significance of this pathway. Equally exciting, the clinical and translational impact is that these data nominate FLCN as a novel and tractable target to precisely inhibit mTORC1 signaling to treat NAFLD, NASH, and their complications, albeit a specific mechanism to target FLCN in patients will need to be developed. Although both of these studies (Science 2022;376(6590):eabf8271; Ann Intern Med 2022;175:74–83) offered insights into promising therapeutic options for NAFLD, the challenge of preventing the ensuing liver fibrosis remains, and additional studies are needed to determine whether either of these options will “crack that nut.” Further Insights Into the Impact of Bariatric Surgery on the Progression of Nonalcoholic Fatty Liver DiseaseGastroenterologyVol. 163Issue 2PreviewSeeberg KA, Borgeraas H, Hofsø H, et al. Gastric bypass versus sleeve gastrectomy in type 2 diabetes: effects on hepatic steatosis and fibrosis: a randomized controlled trial. Ann Intern Med 2022;175:74–83. Full-Text PDF

Inhibition of nonalcoholic fatty liver disease in mice by selective inhibition of mTORC1.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) remain without effective therapies. The mechanistic target of rapamycin complex 1 (mTORC1) pathway is a potential therapeutic target, but conflicting interpretations have been proposed for how mTORC1 controls lipid homeostasis. We show that selective inhibition of mTORC1 signaling in mice, through deletion of the RagC/D guanosine triphosphatase-activating protein folliculin (FLCN), promotes activation of transcription factor E3 (TFE3) in the liver without affecting other mTORC1 targets and protects against NAFLD and NASH. Disease protection is mediated by TFE3, which both induces lipid consumption and suppresses anabolic lipogenesis. TFE3 inhibits lipogenesis by suppressing proteolytic processing and activation of sterol regulatory element-binding protein-1c (SREBP-1c) and by interacting with SREBP-1c on chromatin. Our data reconcile previously conflicting studies and identify selective inhibition of mTORC1 as a potential approach to treat NASH and NAFLD.

Targeting eIF4F translation complex sensitizes B-ALL cells to tyrosine kinase inhibition

The mechanistic target of rapamycin (mTOR) is a kinase whose activation is associated with poor prognosis in pre-B cell acute lymphoblastic leukemia (B-ALL). These and other findings have prompted diverse strategies for targeting mTOR signaling in B-ALL and other B-cell malignancies. In cellular models of Philadelphia Chromosome-positive (Ph+) B-ALL, mTOR kinase inhibitors (TOR-KIs) that inhibit both mTOR-complex-1 (mTORC1) and mTOR-complex-2 (mTORC2) enhance the cytotoxicity of tyrosine kinase inhibitors (TKIs) such as dasatinib. However, TOR-KIs have not shown substantial efficacy at tolerated doses in blood cancer clinical trials. Selective inhibition of mTORC1 or downstream effectors provides alternative strategies that may improve selectivity towards leukemia cells. Of particular interest is the eukaryotic initiation factor 4F (eIF4F) complex that mediates cap-dependent translation. Here we use novel chemical and genetic approaches to show that selective targeting of either mTORC1 kinase activity or components of the eIF4F complex sensitizes murine BCR-ABL-dependent pre-B leukemia cells to dasatinib. SBI-756, a small molecule inhibitor of eIF4F assembly, sensitizes human Ph+ and Ph-like B-ALL cells to dasatinib cytotoxicity without affecting survival of T lymphocytes or natural killer cells. These findings support the further evaluation of eIF4F-targeted molecules in combination therapies with TKIs in B-ALL and other blood cancers.

Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17β signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression.

Abstract PR04: 4EBP1 reactivation by potent and selective bi-steric inhibitors of mTORC1

Abstract Aberrant activation of the PI3K/AKT/mTOR pathway is implicated in human cancer. The protein kinase mechanistic target of rapamycin (mTOR) is a key node in this pathway and participates in two complexes: mTORC1 and mTORC2. mTORC1 regulates cap-dependent protein translation, both by activating downstream effectors, including p70S6 kinase 1 (S6K1), and by deactivating the translation-initiation repressor eIF4E binding protein 1 (4EBP1). mTORC2 regulates cellular proliferation via AKT phosphorylation. mTOR inhibitors have diverse pharmacologic effects on the activity of these complexes. Rapamycin and its analogs (rapalogs) cause selective but incomplete inhibition of mTORC1, reducing phosphorylation of some substrates, including S6K1, but not 4EBP1. In contrast, ATP-competitive inhibitors, such as MLN0128, inhibit mTORC1, mTORC2, and various lipid kinases. The suboptimal antitumor activity of rapalogs has been attributed, at least in part, to their limited spectrum of inhibitory activity against mTORC1. Conversely, the broad inhibitory profile of the ATP competitive inhibitors may be the basis for observed toxicities in the clinic and lack of optimal inhibition of tumor mTORC1 at tolerated doses. Recently, a new class of mTOR inhibitor that exploits a bivalent interaction with both the ATP- and FRB-binding sites of mTOR has been reported, which we have termed “bi-steric” to reflect the interaction with both allosteric and orthosteric sites. The prototype bi-steric inhibitor, RapaLink-1, blocks phosphorylation of many mTORC1 substrates, including 4EBP1, but exhibits only modest differentiation between inhibition of mTORC1 and mTORC2 in vitro. Using our Revblocks® “modular synthesis” platform, we have generated novel bi-steric mTOR inhibitors that exhibit potent and selective inhibition of mTORC1 in vitro. By independently tuning the affinities of the rapamycin-derived and ATP-competitive moieties, we have designed bi-steric inhibitors that exhibit distinct mTORC1/2 selectivity profiles as well as different durations of action. Following intraperitoneal administration, mTORC1-selective bi-steric inhibitors produced sustained inhibition of tumor 4EBP1 phosphorylation in vivo in MCF7, an estrogen receptor-positive and PI3KCA mutant xenograft model of mammary carcinoma in nude mice, at doses that induced rapid and sustained tumor regression. In contrast, daily oral administration of everolimus at a clinically relevant dose did not alter tumor 4EBP1 phosphorylation and was less efficacious in comparison with bi-steric mTOR inhibitors. Naïve mice treated with mTORC1-selective inhibitors showed a reduced spike in blood glucose in a glucose tolerance test compared to nonselective mTORC1 inhibitors, indicative of reduced insulin resistance driven by mTORC2 inhibition. We will compare and contrast the biologic profiles of different levels of mTORC1/2 selectivity that can be achieved with selected examples from our collection of diverse bi-steric mTOR inhibitors. This abstract is also being presented as Poster A19. Citation Format: Nidhi Tibrewal, James B. Aggen, Abraham I. Bassan, G. Leslie Burnett, Jim Evans, Micah J. Gliedt, Daphne Hsieh, Gert Kiss, Bianca J. Lee, Dong Lee, Ed Lorenzana, Abby Marquez, Arun Thottumkara, Zhengping Wang, Stacy Wilson, Frances Zhao, Mark Goldsmith, Mallika Singh, Pete Wildes, Adrian L. Gill, Jacqueline A.M. Smith. 4EBP1 reactivation by potent and selective bi-steric inhibitors of mTORC1 [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr PR04.

Abstract IA17: Strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma

Abstract mTORC1 signaling is elevated in most lymphoid malignancies and frequently is associated with poor prognosis. Targeting mTORC1 with rapalogs or mTOR kinase inhibitors (TOR-KIs) are two strategies that both have limitations. Rapalogs are weak inducers of apoptosis, mainly due to incomplete mTORC1 inhibition, resulting in reduced phosphorylation of some substrates such as S6K1, but not 4E-BP1. TOR-KIs inhibit mTORC1 more completely, but also inhibit mTORC2, which likely contributes to dose-limiting toxicities in TOR-KI clinical trials. We have evaluated alternative strategies to target mTORC1 and downstream survival pathways in B-cell leukemia and lymphoma. In one approach, we have used novel bi-steric inhibitors of mTORC1, generated using our RevBlocksTM modular synthesis platform. These inhibitors engage both the FRB- and ATP-binding sites of mTOR and exhibit potent and selective inhibition of mTORC1. We found that RM-001, a proprietary bi-steric compound ~13-fold-selective for mTORC1 over mTORC2 in vitro, enhances the efficacy of the BCR-ABL1 inhibitor dasatinib in Philadelphia chromosome-positive pre-B cell acute lymphoblastic leukemia (Ph+ B-ALL) cell lines, causing profound and sustained phosphorylation inhibition of 4E-BP1 and S6, cell growth inhibition, and cell cycle arrest. Once-weekly dosing with RM-001 (10 mg/kg, IP) in a Ph+ B-ALL mouse model was well tolerated and effectively reduced leukemic burden as a single agent. A corresponding inhibition of phosphorylation of 4E-BP1 and S6 was observed in bone marrow. A key downstream effector pathway of mTORC1 is formation of the eIF4F translation initiation complex, which consists of eIF4E, eIF4G, and eIF4A. To assess targeting eIF4F as an alternative approach, we evaluated the function of eIF4F in both tumors and normal cells of the B lymphocyte lineage. In both B-ALL and diffuse large B-cell lymphoma (DLBCL) cells, disruption of eIF4F with a constitutively active 4E-BP1 mutant phenocopied the effect of TOR-KIs and bi-steric inhibitors. Notably, reduced gene dosage of Eif4e (eIF4E+/-) impaired the initiation and maintenance of B-cell transformation without affecting normal B-cell development and function. Most existing chemical inhibitors of eIF4F target either the cap-binding protein eIF4E or the RNA helicase eIF4A. We tested a novel eIF4G-binding molecule, SBI-756, shown previously to overcome resistance to BRAF inhibitors in melanoma models. SBI-756 (250 – 500 nM) disrupted eIF4F formation and caused marked cytotoxic effects in B-ALL, DLBCL, and normal B lymphocytes, while sparing T cells and NK cells. SBI-756 also synergized with targeted agents including dasatinib in Ph+ B-ALL and venetoclax in DLBCL. The combination of SBI-756 with venetoclax was more effective than single agents in a DLBCL xenograft model. These findings establish proof of concept for molecules that selectively bind to the scaffolding protein eIF4G. Collectively, these data highlight distinct strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma. Citation Format: Lee-or Herzog, Bianca J. Lee, Thanh-Trang Vo, Honyin Chiu, Sharmila Mallya, Amos Fung, Mallika Singh, James Aggen, Nidhi Tibrewal, Jacqueline A.M. Smith, David Wildes, Ze'ev Ronai, Davide Ruggero, David A. Fruman. Strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA17.

Regulation of RLR-Mediated Antiviral Responses of Human Dendritic Cells by mTOR.

To detect replicating viruses, dendritic cells (DCs) utilize cytoplasmic retinoic acid inducible gene-(RIG) I-like receptors (RLRs), which play an essential role in the subsequent activation of antiviral immune responses. In this study, we aimed to explore the role of the mammalian target of rapamycin (mTOR) in the regulation of RLR-triggered effector functions of human monocyte-derived DCs (moDCs) and plasmacytoid DCs (pDCs). Our results show that RLR stimulation increased the phosphorylation of the mTOR complex (mTORC) 1 and mTORC2 downstream targets p70S6 kinase and Akt, respectively, and this process was prevented by the mTORC1 inhibitor rapamycin as well as the dual mTORC1/C2 kinase inhibitor AZD8055 in both DC subtypes. Furthermore, inhibition of mTOR in moDCs impaired the RLR stimulation-triggered glycolytic switch, which was reflected by the inhibition of lactate production and downregulation of key glycolytic genes. Blockade of mTOR diminished the ability of RLR-stimulated moDCs and pDCs to secret type I interferons (IFNs) and pro-inflammatory cytokines, while it did not affect the phenotype of DCs. We also found that mTOR blockade decreased the phosphorylation of Tank-binding kinase 1 (TBK1), which mediates RLR-driven cytokine production. In addition, rapamycin abrogated the ability of both DC subtypes to promote the proliferation and differentiation of IFN-y and Granzyme B producing CD8 + T cells. Interestingly, AZD8055 was much weaker in its ability to decrease the T cell proliferation capacity of DCs and was unable to inhibit the DC-triggered production of IFN-y and Granyzme B by CD8 + T cells. Here we demonstrated for the first time that mTOR positively regulates the RLR-mediated antiviral activity of human DCs. Further, we show that only selective inhibition of mTORC1 but not dual mTORC1/C2 blockade suppresses effectively the T cell stimulatory capacity of DCs that should be considered in the development of new generation mTOR inhibitors and in the improvement of DC-based vaccines.

Selective inhibition of mTORC1 in tumor vessels increases antitumor immunity.

A tumor blood vessel is a key regulator of tissue perfusion, immune cell trafficking, cancer metastasis, and therapeutic responsiveness. mTORC1 is a signaling node downstream of multiple angiogenic factors in the endothelium. However, mTORC1 inhibitors have limited efficacy in most solid tumors, in part due to inhibition of immune function at high doses used in oncology patients and compensatory PI3K signaling triggered by mTORC1 inhibition in tumor cells. Here we show that low-dose RAD001/everolimus, an mTORC1 inhibitor, selectively targets mTORC1 signaling in endothelial cells (ECs) without affecting tumor cells or immune cells, resulting in tumor vessel normalization and increased antitumor immunity. Notably, this phenotype was recapitulated upon targeted inducible gene ablation of the mTORC1 component Raptor in tumor ECs (RaptorECKO). Tumors grown in RaptorECKO mice displayed a robust increase in tumor-infiltrating lymphocytes due to GM-CSF–mediated activation of CD103+ dendritic cells and displayed decreased tumor growth and metastasis. GM-CSF neutralization restored tumor growth and metastasis, as did T cell depletion. Importantly, analyses of human tumor data sets support our animal studies. Collectively, these findings demonstrate that endothelial mTORC1 is an actionable target for tumor vessel normalization, which could be leveraged to enhance antitumor immune therapies.

Preclinical efficacy of dual mTORC1/2 inhibitor AZD8055 in renal cell carcinoma harboring a TFE3 gene fusion

BackgroundRenal cell carcinomas (RCC) harboring a TFE3 gene fusion (TfRCC) represent an aggressive subset of kidney tumors. Key signaling pathways of TfRCC are unknown and preclinical in vivo data are lacking. We investigated Akt/mTOR pathway activation and the preclinical efficacy of dual mTORC1/2 versus selective mTORC1 inhibition in TfRCC.MethodsLevels of phosphorylated Akt/mTOR pathway proteins were compared by immunoblot in TfRCC and clear cell RCC (ccRCC) cell lines. Effects of the mTORC1 inhibitor, sirolimus, and the dual mTORC1/2 inhibitor, AZD8055, on Akt/mTOR activation, cell cycle progression, cell viability and cytotoxicity were compared in TfRCC cells. TfRCC xenograft tumor growth in mice was evaluated after 3-week treatment with oral AZD8055, intraperitoneal sirolimus and respective vehicle controls.ResultsThe Akt/mTOR pathway was activated to a similar or greater degree in TfRCC than ccRCC cell lines and persisted partly during growth factor starvation, suggesting constitutive activation. Dual mTORC1/2 inhibition with AZD8055 potently inhibited TfRCC viability (IC50 = 20-50 nM) due at least in part to cell cycle arrest, while benign renal epithelial cells were relatively resistant (IC50 = 400 nM). Maximal viability reduction was greater with AZD8055 than sirolimus (80–90% versus 30–50%), as was the extent of Akt/mTOR pathway inhibition, based on significantly greater suppression of P-Akt (Ser473), P-4EBP1, P-mTOR and HIF1α. In mouse xenograft models, AZD8055 achieved significantly better tumor growth inhibition and prolonged mouse survival compared to sirolimus or vehicle controls.ConclusionsAkt/mTOR activation is common in TfRCC and a promising therapeutic target. Dual mTORC1/2 inhibition suppresses Akt/mTOR signaling more effectively than selective mTORC1 inhibition and demonstrates in vivo preclinical efficacy against TFE3-fusion renal cell carcinoma.

A novel rapamycin analog is highly selective for mTORC1 in vivo

Rapamycin, an inhibitor of mechanistic Target Of Rapamycin Complex 1 (mTORC1), extends lifespan and shows strong potential for the treatment of age-related diseases. However, rapamycin exerts metabolic and immunological side effects mediated by off-target inhibition of a second mTOR-containing complex, mTOR complex 2. Here, we report the identification of DL001, a FKBP12-dependent rapamycin analog 40x more selective for mTORC1 than rapamycin. DL001 inhibits mTORC1 in cell culture lines and in vivo in C57BL/6J mice, in which DL001 inhibits mTORC1 signaling without impairing glucose homeostasis and with substantially reduced or no side effects on lipid metabolism and the immune system. In cells, DL001 efficiently represses elevated mTORC1 activity and restores normal gene expression to cells lacking a functional tuberous sclerosis complex. Our results demonstrate that highly selective pharmacological inhibition of mTORC1 can be achieved in vivo, and that selective inhibition of mTORC1 significantly reduces the side effects associated with conventional rapalogs.

Discovery of Small-Molecule Selective mTORC1 Inhibitors via Direct Inhibition of Glucose Transporters

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular metabolic processes. Dysregulation of this kinase complex can result in a variety of human diseases. Rapamycin and its analogs target mTORC1 directly; however, chronic treatment in certain cell types and invivo results in the inhibition of both mTORC1 and mTORC2. We have developed a high-throughput cell-based screen for the detection of phosphorylated forms of the mTORC1 (4E-BP1, S6K1) and mTORC2 (Akt) substrates and have identified and characterized a chemical scaffold that demonstrates a profile consistent with the selective inhibition of mTORC1. Stable isotope labeling of amino acids in cell culture-based proteomic target identification revealed that class I glucose transporters were the primary target for these compounds yielding potent inhibition of glucose uptake and, as a result, selective inhibition of mTORC1. The link between the glucose uptake and selective mTORC1 inhibition are discussed in the context of a yet-to-be discovered glucose sensor.

Next Generation Strategies for Geroprotection via mTORC1 Inhibition

Inhibition of mTORC1 (mechanistic Target Of Rapamycin Complex 1) with the pharmaceutical rapamycin prolongs the lifespan and healthspan of model organisms including rodents, with evidence now emerging that rapamycin and its analogs may also have rejuvenative effects in dogs and humans. However, the side effects associated with long-term rapamycin treatment, many of which are due to inhibition of a second mTOR complex, mTORC2, have seemed to preclude the routine use of rapamycin as a therapy for age-related diseases. Here, we discuss recent findings suggesting that strong, chronic inhibition of both mTOR complexes may not be necessary to realize the geroprotective effects of rapamycin. Instead, modestly but specifically inhibiting mTORC1 via a variety of emerging techniques, including intermittent or transient treatment with rapamycin derivatives, or specific dietary regimens, may be sufficient to promote health and longevity with reduced side effects. We will also discuss prospects for the development of new molecules that, by harnessing the detailed molecular understanding of mTORC1 signaling developed over the last decade, will provide new routes to the selective inhibition of mTORC1. We conclude that therapies based on the selective inhibition of mTORC1 may soon permit the safer treatment of diseases of aging.

Abstract 3452: TAK228, a kinase inhibitor of mTORC1 and mTORC2, is highly effective in ER+ breast cancer model

Abstract Background: mTOR is an atypical Ser/Thr kinase involved in regulating major cellular functions, such as nutrients sensing, growth, proliferation and angiogenesis. Current FDA approved clinical mTOR inhibitors only inhibit mTORC1 and have several limitations. First, inhibiting mTORC1 releases the negative feedback of PI3K-AKT signaling and therefore activates upstream AKT signaling. Secondly, allosteric inhibitors of rapalogs block the phosphorylation of some but not all mTORC1 downstream substrates. In particular phosphorylation of 4EBP1 is largely insensitive to rapalogs. In order to address these issues, “second generation” ATP competitive catalytic inhibitors including TAK228 against mTORC1/C2 have also been developed and are now in active in various stages of clinical trials. Purpose: Resistance of breast cancers (BC) to targeted hormone receptor inhibitors often occurs through deregulation of the PI3K-AKT-mTOR pathway. Mutations or loss of function of upstream regulators such as TSC1/2, LKB1, or components of the PI3K pathway such as PIK3CA, AKT, or PTEN have been reported in most types of human tumors including BC. Here, we investigated the effects of TAK228 alone or in conjunction with letrozole in ER+ BC cell lines. Methodology: Anti-proliferative, apoptotic, cell cycle, and intracellular signaling effects of TAK228 alone and in combination with letrozole were evaluated in ER +/PIK3CA mutated and ER+/PTEN mutated BC cell lines. Results: 1) TAK228 caused a strong differential growth inhibition in ER+/PIK3CA or ER+/PTEN mutated or ER+ (WT of PI3K pathway molecules) BC cell lines by 3D-ON-TOP clonogenic assay and real-time monitoring in an IncuCyte Zoom. Inhibition is greater when TAK228 was combined with letrozole. 2) Administration of TAK228 induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependent manner. 3) qRT-PCR data showed that pro-apoptotic transcript, PUMA and cell survival transcripts SURVIVIN & STATHMIN mRNA expressions were significantly increased and decreased respectively following the treatment of TAK228. 4) CYCLIN D1 mRNA expression was significantly decreased in both 24 & 48 hrs following the treatment of TAK228. 5) TAK228 is mechanistically distinct from everolimus by a more potent and comprehensive inhibition of the PI3K-AKT-mTOR signaling network: a) avoided S6K inhibition-mediated feedback activation of PI3K-AKT and b) abrogated 4EBP1 phosphorylation. Conclusion: Data showed that TAK228 effectively inhibited proliferation and enhanced apoptosis via inhibition of the PI3K-AKT-mTORC1/C2 signaling pathway and dual TORC1/2 inhibition also mitigated the feedback activation of AKT caused by selective inhibition of mTORC1, and because of this, TAK228 may yield greater therapeutic benefit in BC patients. Citation Format: Pradip De, Jennifer H. Carlson, Tyler Jepperson, Casey Williams, Nandini Dey, Brian Leyland Jones. TAK228, a kinase inhibitor of mTORC1 and mTORC2, is highly effective in ER+ breast cancer model [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 3452.

Discovery of novel leucyladenylate sulfamate surrogates as leucyl-tRNA synthetase (LRS)-targeted mammalian target of rapamycin complex 1 (mTORC1) inhibitors

According to recent studies, leucyl-tRNA synthetase (LRS) acts as a leucine sensor and modulates the activation of the mammalian target of rapamycin complex 1 (mTORC1) activation. Because overactive mTORC1 is associated with several diseases, including colon cancer, LRS-targeted mTORC1 inhibitors represent a potential option for anti-cancer therapy. In this work, we developed a series of simplified leucyladenylate sulfamate analogues that contain the N-(3-chloro-4-fluorophenyl)quinazolin-4-amine moiety to replace the adenine group. We identified several compounds with comparable activity to previously reported inhibitors and exhibited selective mTORC1 inhibition and anti-cancer activity. This study further supports the hypothesis that LRS is a promising target to modulate the mTORC1 pathway.

A small molecule inhibitor of Rheb selectively targets mTORC1 signaling

The small G-protein Rheb activates the mechanistic target of rapamycin complex 1 (mTORC1) in response to growth factor signals. mTORC1 is a master regulator of cellular growth and metabolism; aberrant mTORC1 signaling is associated with fibrotic, metabolic, and neurodegenerative diseases, cancers, and rare disorders. Point mutations in the Rheb switch II domain impair its ability to activate mTORC1. Here, we report the discovery of a small molecule (NR1) that binds Rheb in the switch II domain and selectively blocks mTORC1 signaling. NR1 potently inhibits mTORC1 driven phosphorylation of ribosomal protein S6 kinase beta-1 (S6K1) but does not inhibit phosphorylation of AKT or ERK. In contrast to rapamycin, NR1 does not cause inhibition of mTORC2 upon prolonged treatment. Furthermore, NR1 potently and selectively inhibits mTORC1 in mouse kidney and muscle in vivo. The data presented herein suggest that pharmacological inhibition of Rheb is an effective approach for selective inhibition of mTORC1 with therapeutic potential.