Oncogenic alterations in the RAS/RAF/MEK/ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAF
-driven cancers, effective targeted ...therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the SHP2 phosphatase (encoded by PTPN11) with RMC-4550, a small-molecule allosteric inhibitor, is effective in human cancer models bearing RAS-GTP-dependent oncogenic BRAF (for example, class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (for example, KRAS
). SHP2 inhibitor treatment decreases oncogenic RAS/RAF/MEK/ERK signalling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS-GTP-dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.
The proteomes of blood plasma and serum represent a potential gold mine of biological and diagnostic information, but challenges such as dynamic range of protein concentration have hampered efforts ...to unlock this resource. Here we present a method to label and isolate N-terminal peptides from human plasma and serum. This process dramatically reduces the complexity of the sample by eliminating internal peptides. We identify 772 unique N-terminal peptides in 222 proteins, ranging over six orders of magnitude in abundance. This approach is highly suited for studying natural proteolysis in plasma and serum. We find internal cleavages in plasma proteins created by endo- and exopeptidases, providing information about the activities of proteolytic enzymes in blood, which may be correlated with disease states. We also find signatures of signal peptide cleavage, coagulation and complement activation, and other known proteolytic processes, in addition to a large number of cleavages that have not been reported previously, including over 200 cleavages of blood proteins by aminopeptidases. Finally, we can identify substrates from specific proteases by exogenous addition of the protease combined with N-terminal isolation and quantitative mass spectrometry. In this way we identified proteins cleaved in human plasma by membrane-type serine protease 1, an enzyme linked to cancer progression. These studies demonstrate the utility of direct N-terminal labeling by subtiligase to identify and characterize endogenous and exogenous proteolysis in human plasma and serum.
Abstract Current KRAS G12C (OFF) inhibitors that target inactive GDP-bound KRAS G12C cause responses in less than half of patients and these responses are not durable. A class of RAS G12C (ON) ...inhibitors that targets active GTP-bound KRAS G12C blocks ERK signaling more potently than the inactive-state inhibitors. Sensitivity to either class of agents is strongly correlated with inhibition of mTORC1 activity. We have previously shown that PI3K/mTOR and ERK-signaling pathways converge on key cellular processes and that inhibition of both pathways is required for inhibition of these processes and for significant antitumor activity. We find here that the combination of a KRAS G12C inhibitor with a selective mTORC1 kinase inhibitor causes synergistic inhibition of Cyclin D1 expression and cap-dependent translation. Moreover, BIM upregulation by KRAS G12C inhibition and inhibition of MCL-1 expression by the mTORC1 inhibitor are both required to induce significant cell death. In vivo, this combination causes deep, durable tumor regressions and is well tolerated. This study suggests that the ERK and PI3K/mTOR pathways each mitigate the effects of inhibition of the other and that combinatorial inhibition is a potential strategy for treating KRAS G12C -dependent lung cancer.
Shp1, encoded by the gene
, is a protein tyrosine phosphatase that transduces inhibitory signals downstream of immunoreceptors in many immune cell types. Blocking Shp1 activity represents an exciting ...potential immunotherapeutic strategy for the treatment of cancer, as Shp1 inhibition would be predicted to unleash both innate and adaptive immunity against tumor cells. Antibodies blocking the interaction between CD47 on tumor cells and SIRPα on macrophages enhance macrophage phagocytosis, show efficacy in preclinical tumor models, and are being evaluated in the clinic. Here we found that Shp1 bound to phosphorylated peptide sequences derived from SIRPα and transduced the anti-phagocytic signal, as Shp1 loss in mouse bone marrow-derived macrophages increased phagocytosis of tumor cells
. We also generated a novel mouse model to evaluate the impact of global, inducible
deletion on anti-tumor immunity. We found that inducible Shp1 loss drove an inflammatory disease in mice that was phenotypically similar to that seen when
is knocked out from birth. This indicates that acute perturbation of Shp1
could drive hyperactivation of immune cells, which could be therapeutically beneficial, though at the risk of potential toxicity. In this model, we found that Shp1 loss led to robust anti-tumor immunity against two immune-rich syngeneic tumor models that are moderately inflamed though not responsive to checkpoint inhibitors, MC38 and E0771. Shp1 loss did not promote anti-tumor activity in the non-inflamed B16F10 model. The observed activity in MC38 and E0771 tumors was likely due to effects of both innate and adaptive immune cells. Following Shp1 deletion, we observed increases in intratumoral myeloid cells in both models, which was more striking in E0771 tumors. E0771 tumors also contained an increased ratio of effector to regulatory T cells following Shp1 loss. This was not observed for MC38 tumors, though we did find increased levels of IFNγ, a cytokine produced by effector T cells, in these tumors. Overall, our preclinical data suggested that targeting Shp1 may be an attractive therapeutic strategy for boosting the immune response to cancer via a mechanism involving both innate and adaptive leukocytes.
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.
Abstract The protein tyrosine phosphatase SHP2 binds to phosphorylated signaling motifs on regulatory immunoreceptors including PD-1, but its functional role in tumor immunity is unclear. Using ...preclinical models, we show that RMC-4550, an allosteric inhibitor of SHP2, induces antitumor immunity, with effects equivalent to or greater than those resulting from checkpoint blockade. In the tumor microenvironment, inhibition of SHP2 modulated T-cell infiltrates similar to checkpoint blockade. In addition, RMC-4550 drove direct, selective depletion of protumorigenic M2 macrophages via attenuation of CSF1 receptor signaling and increased M1 macrophages via a mechanism independent of CD8+ T cells or IFNγ. These dramatic shifts in polarized macrophage populations in favor of antitumor immunity were not seen with checkpoint blockade. Consistent with a pleiotropic mechanism of action, RMC-4550 in combination with either checkpoint or CSF1R blockade caused additive antitumor activity with complete tumor regressions in some mice; tumors intrinsically sensitive to SHP2 inhibition or checkpoint blockade were particularly susceptible. Our preclinical findings demonstrate that SHP2 thus plays a multifaceted role in inducing immune suppression in the tumor microenvironment, through both targeted inhibition of RAS pathway–dependent tumor growth and liberation of antitumor immune responses. Furthermore, these data suggest that inhibition of SHP2 is a promising investigational therapeutic approach. Significance: Inhibition of SHP2 causes direct and selective depletion of protumorigenic M2 macrophages and promotes antitumor immunity, highlighting an investigational therapeutic approach for some RAS pathway–driven cancers.
Abstract Oncogenic RAS binds to multiple effector proteins to activate downstream signaling pathways, including the MAPK and PI3K axes. While RAS-mediated MAPK activation has a well-validated role in ...driving cancer progression, the contribution of PI3K activation has been more difficult to assess. Understanding the degree to which different KRAS-mutant genotypes stimulate PI3K activity may inform biomarker and combination strategies for RAS inhibitors currently undergoing clinical evaluation.RMC-7977 is a RASMULTI(ON) inhibitor that targets GTP-bound RAS(ON) and blocks effector protein binding. RMC-7977 exhibits similar potency for wild-type and oncogenic mutant RAS and is a powerful preclinical tool, representative of the investigational RASMULTI(ON) inhibitor RMC-6236, for comparing how RAS-mutant genotypes differ with respect to signal activation. MAPK signaling proceeds through a protein phosphorylation cascade that is readily detectable using standard cell biology methods, but PI3K is a lipid kinase whose activity is not amenable to protein characterization techniques. An assay of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) was developed to directly measure the activity of PI3K in the context of RAS inhibition.In isogenic LIM1215 cell lines expressing different KRASG12X mutants (KRASG12D, KRASG12V, KRASG12C and KRASG12R), RAS inhibition by RMC-7977 completely suppressed PI3K activity in cells with wild-type KRAS and KRASG12D, had a moderate inhibitory effect in KRASG12C and KRASG12V, and had no effect in KRASG12R. To confirm that this inhibition is dependent on a direct interaction between RAS(ON) and PI3K, CRISPR engineering was used to introduce mutations in PIK3CA to perturb the RAS-binding domain. In cells with disrupted RAS-PI3K interactions, the basal levels of PIP3 are significantly reduced and no longer change in response to treatment with RMC-7977.Based on the isogenic cell line results, KRASG12D and KRASG12R-mutant pancreatic cancer cell lines were profiled for their response to RAS inhibition. RMC-7977 decreased the levels of MAPK signaling in both KRASG12D and KRASG12R genotypes, but PI3K activity was only inhibited in KRASG12D lines. MEK inhibitors only inhibited MAPK signaling and had no effect on PI3K activity in either genotype. The simultaneous inhibition of MAPK and PI3K signaling by RMC-7977 led to deeper suppression of cyclin D levels and a more pronounced G1 cell cycle arrest as compared with MAPK inhibitors.We demonstrate that the coupling between RAS activation and PI3K signaling varies between oncogenic mutants, with KRASG12D showing the greatest degree of PI3K stimulation. In KRASG12D-mutant pancreatic cancer cell lines, a RASMULTI(ON) inhibitor suppresses both PI3K and MAPK signaling. These preclinical results may be used to inform potential, rational, mechanism-based combination treatment strategies for RAS(ON) inhibitors. Citation Format: Priyanka S. Bapat, Kyle Seamon, Andrea Gould, David Wildes. The RASMULTI(ON) inhibitor RMC-7977 blocks downstream MAPK and PI3K pathway activation in KRASG12X_mutant cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4709.
The clinical benefits of pan-mTOR active-site inhibitors are limited by toxicity and relief of feedback inhibition of receptor expression. To address these limitations, we designed a series of ...compounds that selectively inhibit mTORC1 and not mTORC2. These ‘bi-steric inhibitors’ comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. Structural modification of these components modulated their affinities for their binding sites on mTOR and the selectivity of the bi-steric compound. mTORC1-selective compounds potently inhibited 4EBP1 phosphorylation and caused regressions of breast cancer xenografts. Inhibition of 4EBP1 phosphorylation was sufficient to block cancer cell growth and was necessary for maximal antitumor activity. At mTORC1-selective doses, these compounds do not alter glucose tolerance, nor do they relieve AKT-dependent feedback inhibition of HER3. Thus, in preclinical models, selective inhibitors of mTORC1 potently inhibit tumor growth while causing less toxicity and receptor reactivation as compared to pan-mTOR inhibitors.Design of a bivalent inhibitor containing an ATP-competitive moiety and rapamycin-modified FRB binding ligand that selectively inhibits mTORC1 results in potent and durable inhibition of 4EBP1 phosphorylation and cell proliferation in vitro and in vivo.
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).
PDF
