The mechanistic target of rapamycin (mTOR) is a kinase whose activity is elevated in hematological malignancies. mTOR-complex-1 (mTORC1) phosphorylates numerous substrates to promote cell ...proliferation and survival. Eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) are mTORC1 substrates with an integral role in oncogenic protein translation. Current pharmacological approaches to inhibit mTORC1 activity and 4E-BP phosphorylation have drawbacks. Recently we described a series of bi-steric compounds that are potent and selective inhibitors of mTORC1, inhibiting 4E-BP phosphorylation at lower concentrations than mTOR kinase inhibitors (TOR-KIs). Here we report the activity of the mTORC1-selective bi-steric inhibitor, RMC-4627, in BCR-ABL-driven models of B-cell acute lymphoblastic leukemia (B-ALL). RMC-4627 exhibited potent and selective inhibition of 4E-BP1 phosphorylation in B-ALL cell lines without inhibiting mTOR-complex-2 (mTORC2) activity. RMC-4627 suppressed cell cycle progression, reduced survival, and enhanced dasatinib cytotoxicity. Compared to a TOR-KI compound, RMC-4627 was more potent, and its effects on cell viability were sustained after washout
in vitro
. Notably, a once-weekly, well tolerated dose reduced leukemic burden in a B-ALL xenograft model and enhanced the activity of dasatinib. These preclinical studies suggest that intermittent dosing of a bi-steric mTORC1-selective inhibitor has therapeutic potential as a component of leukemia regimens, and further study is warranted.
Mesenchymal stem/stromal cells (MSCs) play crucial roles in maintaining tissue homeostasis during physiological turnovers and injuries. Very little is known about the phenotype, distribution and ...molecular nature of MSCs in freshly isolated human salivary glands (SGs) as most reports have focused on the analysis of cultured MSCs. Our results demonstrate that the cell adhesion molecule CD34 was widely expressed by the MSCs of human major SGs, namely parotid (PAG), sublingual (SLG) and submandibular (SMG) glands. Further, gene expression analysis of CD34
cells derived from fetal SMGs showed significant upregulation of genes involved in cellular adhesion, proliferation, branching, extracellular matrix remodeling and organ development. Moreover, CD34
SMG cells exhibited elevated expression of genes encoding extracellular matrix, basement membrane proteins, and members of ERK, FGF and PDGF signaling pathways, which play key roles in glandular development, branching and homeostasis. In vitro CD34
cell derived SG-MSCs revealed multilineage differentiation potential. Intraglandular transplantation of cultured MSCs in immunodeficient mice led to their engraftment in the injected and uninjected contralateral and ipsilateral glands. Engrafted cells could be localized to the stroma surrounding acini and ducts. In summary, our data show that CD34
derived SG-MSCs could be a promising cell source for adoptive cell-based SG therapies, and bioengineering of artificial SGs.
Chikungunya virus (CHIKV) is the most common alphavirus infecting humans worldwide, causing acute and chronically debilitating arthralgia at a great economic expense.
To facilitate our study of ...CHIKV, we generated a mCherry tagged replication-competent chimeric virus, CHIKV 37997-mCherry. Single particle cryoEM demonstrated icosahedral organization of the chimeric virus and the display of mCherry proteins on virus surface. CHIKV 37997-mCherry is attenuated in both IFNαR knockout and wild-type mice. Strong anti-CHIKV and anti-mCherry antibody responses were induced in CHIKV 37997-mCherry infected mice.
Our work suggests that chimeric alphaviruses displaying foreign antigen can serve as vaccines against both aphaviruses and other pathogens and diseases.
Membranes surrounding the fetus play a crucial role in providing a physical and immunological barrier between a semiallogeneic fetus and mother during pregnancy. In this study, we tested whether ...cotransplantation of fetal membranes (FMs) and allogeneic donor cells would improve the retention and function of allografts in mice.
Intact and enzyme-digested membranes obtained from E18-E19 pregnant mice were subcutaneously cotransplanted with 10F7MN hybridoma cells that are of BALB/cByJ (Balb) origin and secrete anti-human CD235a antibody. Cells were transplanted into C57BL/6J (B6, allogeneic), Balb (syngeneic), and FVB/NJ (third-party) mice. Serum was collected after 1 and 3 weeks of cell transplantation and tested using flow cytometry for the presence of anti-human CD235a antibody. Immunosuppressive functions of membranes were further investigated by analyzing the cytokine profile of supernatants collected from allo-reactive mixed lymphocyte reactions (MLRs) using a multiplex cytokine assay.
B6 mice transplanted with 10F7MN cells along with membranes syngeneic to the host had significantly higher levels of CD235a antibody when compared to B6 mice that received cells without membranes, allogenic membranes, or third-party membranes. Syngeneic membranes significantly inhibited T-cell proliferation in the presence of allogeneic stimuli and suppressed the release of Th1-cytokines such as IFNγ, TNFα, and IL-2 in MLRs. Additionally, increases in the levels of Th2-cytokines were found in MLRs containing membrane-derived cells.
Our study highlights the potential use of syngeneic FMs to act as potent cell-carriers that could improve graft retention as well as graft-specific immunoprotection during allograft transplantation.
The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for ...therapeutic targeting.
KRAS
is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product–inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRAS
G12C
(in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRAS
G12C
tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRAS
G12C
or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).
Editor’s summary
KRAS is one of the most common oncogenes, but unfortunately it is also commonly thought of as “undruggable” because it lacks a suitable binding pocket for small-molecule drug candidates. To get around this limitation, Schulze
et al
. built on observations from natural product–derived drugs to go after oncogenic KRAS indirectly (see the Perspective by Liu). The authors identified a naturally occurring compound that binds cyclophilin A, a type of cellular chaperone, and then modified this compound to also bind oncogenic mutant KRAS in a three-way complex. The authors used this approach to design multiple small molecules that effectively bound mutant KRAS in complex with cyclophilin A. These molecules were very effective at inhibiting the downstream pathways involved in cell proliferation and at suppressing tumor growth in multiple models. —Yevgeniya Nusinovich
Small molecules recruit cyclophilin A to the active state of mutant KRAS to disrupt oncogenic signaling and tumor growth.
RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 61
. Small molecule ...inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer
. Nevertheless, KRAS
mutations account for only around 15% of KRAS-mutated cancers
, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRAS
). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRAS
cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).
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
Abstract only
591
Background: RAS proteins (such as KRAS, NRAS, HRAS) are small GTPases that drive cell proliferation and survival when bound to GTP. Mutant RAS proteins exist predominantly in the ...GTP-bound (RAS(ON)) state, leading to excessive downstream signaling via interaction with effectors such as RAF kinases. Oncogenic KRAS is required for the initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) (Hezel et al, 2006, Ying et al 2012). Although extinction of KRAS expression as well as pharmacological inhibition of RAS effectors clearly abrogate the growth of human PDAC models, clinical trials of drugs targeting key components of the RAS pathway have remained largely unsuccessful. Several factors contribute to these failures including redundancy in signaling surrogates downstream of KRAS and/or tumor complexity driven by co-occurring genomic alterations and intra-tumoral heterogeneity. Methods: RMC-6236 is a small molecule that binds to an intracellular chaperone protein, Cyclophilin A (CypA), resulting in an inhibitory binary complex that binds active, GTP-bound RAS to form a tri-complex and suppresses RAS signaling by disrupting interactions with effectors such as RAF kinases. Results: Here, we demonstrate that single agent RMC-6236, a first-in-class, orally bioavailable, RAS-selective tri-complex inhibitor of multiple RAS mutations and wild-type RAS (RASMULTI inhibitor) is highly efficacious in preclinical models of KRAS mutant PDAC (with marked activity in RAS-mutant colorectal cancer models described in Koltun et al, AACR 2021). RMC-6236 suppresses phosphorylation of ERK kinases, downstream effectors of RAS involved in cell proliferation, and induces growth suppression and apoptosis in multiple human cancer cell lines in vitro. Oral administration of RMC-6236 produces deep, durable, and dose-dependent suppression of tumor RAS pathway activation in vivo. An extended duration of tumor pharmacodynamic activity, relative to plasma exposure, is observed that likely reflects retention of RMC-6236 in tumor tissue due to high affinity binding to CypA. Daily dosing of RMC-6236 drives profound and durable tumor regressions in multiple cell line derived (CDX) and patient derived (PDX) xenograft models of KRAS mutant PDAC at doses that are well-tolerated. Conclusions: These results indicate that direct targeting of mutant and possibly wild-type RAS in PDAC, without inhibition of signaling nodes outside the canonical RAS pathway, has the potential to translate into clinical benefit for patients with pancreatic cancer harboring mutations in KRAS that may be superior to therapies aimed at upstream or downstream signaling elements within the RAS pathway. Our preclinical data strongly support the inclusion of PDAC patients in our planned clinical trial of RMC-6236 in patients with advanced solid tumors.
Objective
The phenotype of the cells present in the ductal region of salivary glands has been well characterized. However, it is imperative to identify novel biomarkers that can identify different ...cell types present in other glandular components for the development of therapeutic strategies and diagnostics of salivary gland disorders and malignancies. Our study aimed at the characterization of the expression and distribution of various cell surface markers, especially with a focus on CD29 in human fetal as well as adult glands.
Materials and methods
Paired human midgestation fetal and adult parotid, sublingual, and submandibular glands were collected. Phenotypic expression of various lineage‐specific cell surface markers including CD29 was investigated in freshly collected glands. The findings were further corroborated by immunohistochemistry.
Results
Enriched expression of CD29 was found on acinar and ductal epithelial, mesenchymal stromal, and myoepithelial cells; CD29+ cells co‐expressed epithelial (CD324, CD326, NKCC1, and CD44), mesenchymal (CD73, CD90, vimentin, and CD34), and myoepithelial (α‐SMA) cell‐specific progenitor markers in both fetal as well as adult salivary glands.
Conclusion
CD29 is widely expressed in human salivary glands, and it could serve as a potential biomarker for devising novel cellular therapeutic and diagnostic strategies for salivary gland disorders and malignancies.
Abstract
The mammalian target of rapamycin (mTOR) pathway is frequently involved in the pathogenesis of human tumorigenesis. mTOR forms two protein complexes: mTORC1 which responds to growth and ...stress factors resulting in the phosphorylation of 4EBP1 and S6K for their deactivation and activation, respectively; and mTORC2 which responds to growth factors resulting in pAKT activation. Through 4EBP1 and S6K, the mTORC1 complex regulates translation of oncogenes including MYC. The MYC oncogene is causally involved in the pathogenesis of many human cancers but remains as a challenging therapeutic target. Experimentally, inhibiting MYC results in tumor regression associated with immune reactivation. Several lines of evidence suggest that inhibition of the mTORC1 pathway may result in synthetic lethality of MYC-driven cancers. However, to date, existing mTOR pathway inhibitors such as everolimus or sapanisertib fail to sufficiently decrease MYC protein levels in vivo because of incomplete inhibition of p4EBP1 or toxicities that limit the activity, respectively. Revolution Medicines has developed third-generation bi-steric selective mTORC1 inhibitors with a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. RMC-5552, a representative of the bi-steric class of selective mTORC1 inhibitor, is currently in Phase 1 clinical trials (NCT04774952). Here, we show this new class of inhibitors suppresses both S6K and 4EBP1 phosphorylation and depletes MYC protein expression in vivo in an autochthonous conditional transgenic mouse model of MYC-driven HCC and across several human patient-derived xenograft models with MYC amplification. Furthermore, bi-steric mTORC1-selective inhibitors restore anti-tumor immune surveillance and synergize with α-PD-1 immune checkpoint therapy in the MYC-driven HCC mouse model. Overall, we provide proof-of-principle that selective mTORC1 pharmacological inhibition can effectively target MYC-driven cancers by reducing MYC levels, combine with immune checkpoint inhibition, re-establish anti-tumor immunity, and induce sustained tumor regression in the preclinical setting. These preclinical findings provide a rationale for clinical trials with selective mTORC1 inhibitors in patients with MYC-driven cancers and moreover, support clinical testing of combination with immune checkpoint inhibition.
Citation Format: Wadie D. Mahauad-Fernandez, Yu Chi Yang, Ian Lai, Jangho Park, Lilian Yao, James W. Evans, Danielle F. Atibalentja, Xinyu Chen, Zihui Zhao, G. Leslie Burnett, Bianca J. Lee, Nuntana Dinglasan, Nataliya Shifrin, Ethan Ahler, Elsa Quintana, Adrian Gill, Jacqueline A. Smith, Mallika Singh, Dean W. Felsher. Bi-steric mTORC1-selective inhibitors activate 4EBP1, suppress MYC, restore anti-tumor immunity, and cooperate with immune checkpoint inhibition to elicit tumor regression abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB015.
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