Design, synthesis, and bioevaluation of SOS1 PROTACs derived from pyrido[2,3-d]pyrimidin-7-one-based SOS1 inhibitor.

Oncogenic KRAS mutations drive an approximately 25 % of all human cancers. Son of Sevenless 1 (SOS1), a critical guanine nucleotide exchange factor, catalyzes the activation of KRAS. Targeting SOS1 degradation has engaged as a promising therapeutic strategy for KRAS-mutant cancers. Herein, we designed and synthesized a series of novel CRBN-recruiting SOS1 PROTACs using the pyrido[2,3-d]pyrimidin-7-one-based SOS1 inhibitor as the warhead. One representative compound 11o effectively induced the degradation of SOS1 in three different KRAS-mutant cancer cell lines with DC50 values ranging from 1.85 to 7.53 nM. Mechanism studies demonstrated that 11o-induced SOS1 degradation was dependent on CRBN and proteasome. Moreover, 11o inhibited the phosphorylation of ERK and displayed potent anti-proliferative activities against SW620, A549 and DLD-1 cells. Further optimization of 11o may provide us promising SOS1 degraders with favorable drug-like properties for developing new chemotherapies targeting KRAS-driven cancers.

Polyphyllin VII Promotes Apoptosis in Breast Cancer by Inhibiting MAPK/ERK Signaling Pathway through Downregulation of SOS1.

Polyphyllin VII is a biologically active herbal monomer extracted from the traditional Chinese herbal medicine Chonglou. Many studies have demonstrated the anticancer activity of polyphyllin VII against various types of cancers, such as colon, liver, and lung cancer, but its effect on breast cancer has not been elucidated. In this study, we demonstrate that polyphyllin VII inhibited proliferation, increased production of intracellular reactive oxygen species, and decreased mitochondrial membrane potential in breast cancer cells. Notably, polyphyllin VII also induced apoptosis via the mitochondrial pathway. Transcriptome sequencing was used to analyze the targets of PPVII in regulating breast cancer cells. Mechanistic studies showed that polyphyllin VII downregulated Son of Sevenless1 (SOS1) and inhibited the MAPK/ERK pathway. Furthermore, PPVII exerted strong antitumor effects in vivo in nude mice injected with breast cancer cells. Our results suggest that PPVII may promote apoptosis through regulating the SOS1/MAPK/ERK pathway, making it a possible candidate target for the treatment of breast cancer.

Reshaping the Tumor Microenvironment of KRASG12D Pancreatic Ductal Adenocarcinoma with Combined SOS1 and MEK Inhibition for Improved Immunotherapy Response.

KRAS inhibitors have demonstrated exciting preclinical and clinical responses, although resistance occurs rapidly. Here, we investigate the effects of KRAS-targeting therapies on the tumor microenvironment using a library of KrasG12D, p53-mutant, murine pancreatic ductal adenocarcinoma-derived cell lines (KPCY) to leverage immune-oncology combination strategies for long-term tumor efficacy. Our findings show that SOS1 and MEK inhibitors (SOS1i+MEKi) suppressed tumor growth in syngeneic models and increased intratumoral CD8+ T cells without durable responses. Single-cell RNA sequencing revealed an increase in inflammatory cancer-associated fibroblasts (iCAF), M2 macrophages, and a decreased dendritic cell (DC) quality that ultimately resulted in a highly immunosuppressive microenvironment driven by IL6+ iCAFs. Agonist CD40 treatment was effective to revert macrophage polarization and overcome the lack of mature antigen-presenting DCs after SOS1i+MEKi therapy. Treatment increased the overall survival of KPCY tumor-bearing mice. The addition of checkpoint blockade to SOS1i+MEKi combination resulted in tumor-free mice with established immune memory. Our data suggest that KRAS inhibition affects myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong antitumor effects. SIGNIFICANCE: Combination of SOS1 and MEK inhibitors increase T cell infiltration while blunting pro-immune myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong anti-tumor effects.

Discovery of a Potent Dual Son of Sevenless 1 (SOS1) and Epidermal Growth Factor Receptor (EGFR) Inhibitor for the Treatment of Prostate Cancer.

Multitarget medications represent an appealing therapy against the disease with multifactorial abnormalities─cancer. Therefore, simultaneously targeting son of sevenless 1 (SOS1) and epidermal growth factor receptor (EGFR), two aberrantly expressed proteins crucial for the oncogenesis and progression of prostate cancer, may achieve active antitumor effects. Here, we discovered dual SOS1/EGFR-targeting compounds via pharmacophore-based docking screening. The most prominent compound SE-9 exhibited nanomolar inhibition activity against both SOS1 and EGFR and efficiently suppressed the phosphorylation of ERK and AKT in prostate cancer cells PC-3. Cellular assays also revealed that SE-9 displayed strong antiproliferative activities through diverse mechanisms, such as induction of cell apoptosis and G1 phase cell cycle arrest, as well as reduction of angiogenesis and migration. Further in vivo findings showed that SE-9 potently inhibited tumor growth in PC-3 xenografts without obvious toxicity. Overall, SE-9 is a novel dual-targeting SOS1/EGFR inhibitor that represents a promising treatment strategy for prostate cancer.

Identifying Potential SOS1 Inhibitors via Virtual Screening of Multiple Small Molecule Libraries against KRAS-SOS1 Interaction.

The RAS-MAPK signaling pathway, crucial for cell proliferation and differentiation, involves key proteins KRAS and SOS1. Mutations in the KRAS and SOS1 genes are implicated in various cancer types, including pancreatic, lung, and juvenile myelomonocytic leukemia. There is considerable interest in identifying inhibitors targeting KRAS and SOS1 to explore potential therapeutic strategies for cancer treatment. In this study, advanced in silico techniques were employed to screen small molecule libraries at this interface, leading to the identification of promising lead compounds as potential SOS1 inhibitors. Comparative analysis of the average binding free energies of these predicted potent compounds with known SOS1 small molecule inhibitors revealed that the identified compounds display similar or even superior predicted binding affinities compared to the known inhibitors. These findings offer valuable insights into the potential of these compounds as candidates for further development as effective anti-cancer agents.

Discovery of Potent SOS1 PROTACs with Effective Antitumor Activities against NCI-H358 Tumor Cells In Vitro/In Vivo.

Directly targeted KRAS inhibitors are now facing resistance problems, which might be partially solved by the combination of SOS1 inhibitors with KRAS inhibitors. However, this combination may still have some resistance mitigation potential. Comparatively, SOS1 PROTAC may have promising applications in addressing the drug resistance problem by degrading the SOS1 protein. Herein, we report the discovery of novel SOS1 PROTACs and their antitumor activity both in vitro and in vivo. In vitro studies demonstrated that degrader 4 had strong inhibitory effects on the proliferation of NCI-H358 cells with IC50 of 5 nM, together with significant degradation of SOS1 protein with DC50 of 13 nM. In the NCI-H358 xenograft model, degrader 4 exhibited significant antitumor activities with TGITV values of 58.8% at 30 mg/kg bid. The PK and safety profiles also supported degrader 4 for further studies as an effective tool compound.

Discovery of Five SOS2 Fragment Hits with Binding Modes Determined by SOS2 X-Ray Cocrystallography.

SOS1 and SOS2 are guanine nucleotide exchange factors that mediate RTK-stimulated RAS activation. Selective SOS1:KRAS PPI inhibitors are currently under clinical investigation, whereas there are no reports to date of SOS2:KRAS PPI inhibitors. SOS2 activity is implicated in MAPK rebound when divergent SOS1 mutant cell lines are treated with the SOS1 inhibitor BI-3406; therefore, SOS2:KRAS inhibitors are of therapeutic interest. In this report, we detail a fragment-based screening strategy to identify X-ray cocrystal structures of five diverse fragment hits bound to SOS2.

Inhibition of Son of Sevenless Homologue 1 (SOS1): Promising therapeutic treatment for KRAS-mutant cancers.

Kristen rat sarcoma (KRAS) is one of the most common oncogenes in human cancers. As a guanine nucleotide exchange factor, Son of Sevenless Homologue 1 (SOS1) represents a potential therapeutic concept for the treatment of KRAS-mutant cancers because of its activation on KRAS and downstream signaling pathways. In this review, we provide a comprehensive overview of the structure, biological function, and regulation of SOS1. We also focus on the recent advances in SOS1 inhibitors and emphasize their binding modes, structure-activity relationships and pharmacological activities. We hope that this publication can provide a comprehensive compendium on the rational design of SOS1 inhibitors.

Translational relevance of SOS1 targeting for KRAS-mutant colorectal cancer.

It has been challenging to target mutant KRAS (mKRAS) in colorectal cancer (CRC) and other malignancies. Recent efforts have focused on developing inhibitors blocking molecules essential for KRAS activity. In this regard, SOS1 inhibition has arisen as an attractive approach for mKRAS CRC given its essential role as a guanine nucleotide exchange factor for this GTPase. Here, we demonstrated the translational value of SOS1 blockade in mKRAS CRC. We used CRC patient-derived organoids (PDOs) as preclinical models to evaluate their sensitivity to SOS1 inhibitor BI3406. A combination of in silico analyses and wet lab techniques was utilized to define potential predictive markers for SOS1 sensitivity and potential mechanisms of resistance in CRC. RNA-seq analysis of CRC PDOs revealed two groups of CRC PDOs with differential sensitivities to SOS1 inhibitor BI3406. The resistant group was enriched in gene sets involving cholesterol homeostasis, epithelial-mesenchymal transition, and TNF-α/NFκB signaling. Expression analysis identified a significant correlation between SOS1 and SOS2 mRNA levels (Spearman's ρ 0.56, p < 0.001). SOS1/2 protein expression was universally present with heterogeneous patterns in CRC cells but only minimal to none in surrounding nonmalignant cells. Only SOS1 protein expression was associated with worse survival in patients with RAS/RAF mutant CRC (p = 0.04). We also found that SOS1/SOS2 protein expression ratio >1 by immunohistochemistry (p = 0.03) instead of KRAS mutation (p = 1) was a better predictive marker to BI3406 sensitivity of CRC PDOs, concordant with the significant positive correlation between SOS1/SOS2 protein expression ratio and SOS1 dependency. Finally, we showed that GTP-bound RAS level underwent rebound even in BI3406-sensitive PDOs with no change of KRAS downstream effector genes, thus suggesting upregulation of guanine nucleotide exchange factor as potential cellular adaptation mechanisms to SOS1 inhibition. Taken together, our results show that high SOS1/SOS2 protein expression ratio predicts sensitivity to SOS1 inhibition and support further clinical development of SOS1-targeting agents in CRC.

SOS1-inspired hydrocarbon-stapled peptide as a pan-Ras inhibitor.

Blocking the interaction between Ras and Son of Sevenless homolog 1 (SOS1) has been an attractive therapeutic strategy for treating cancers involving oncogenic Ras mutations. K-Ras mutation is the most common in Ras-driven cancers, accounting for 86%, with N-Ras mutation and H-Ras mutation accounting for 11% and 3%, respectively. Here, we report the design and synthesis of a series of hydrocarbon-stapled peptides to mimic the alpha-helix of SOS1 as pan-Ras inhibitors. Among these stapled peptides, SSOSH-5 was identified to maintain a well-constrained alpha-helical structure and bind to H-Ras with high affinity. SSOSH-5 was furthermore validated to bind with Ras similarly to the parent linear peptide through structural modeling analysis. This optimized stapled peptide was proven to be capable of effectively inhibiting the proliferation of pan-Ras-mutated cancer cells and inducing apoptosis in a dose-dependent manner by modulating downstream kinase signaling. Of note, SSOSH-5 exhibited a high capability of crossing cell membranes and strong proteolytic resistance. We demonstrated that the peptide stapling strategy is a feasible approach for developing peptide-based pan-Ras inhibitors. Furthermore, we expect that SSOSH-5 can be further characterized and optimized for the treatment of Ras-driven cancers.

Targeting of SOS1: from SOS1 Activators to Proteolysis Targeting Chimeras.

The most frequent mutated oncogene KRAS in lung cancer is targeted by KRAS G12C-directed drugs, such as Sotorasib and Adagrasib. Still, other alleles frequently expressed in pancreatic and colon cancer may be attacked indirectly by hitting the guanine nucleotide exchange factor (GEF) SOS1 that loads and activates KRAS. The first modulators of SOS1 were found to act as agonists and defined a hydrophobic pocket at the catalytic site. High throughput screenings resulted in the detection of SOS1 inhibitors Bay-293 and BI-3406 comprising amino quinazoline scaffolds optimized for binding to the pocket by various substituents. The first inhibitor, BI-1701963, is in clinical studies alone or in combination with a KRAS inhibitor, a MAPK inhibitor or chemotherapeutics. An optimized agonist, VUBI-1, shows activity against tumor cells by destructive overactivation of cellular signaling. This agonist was used to formulate a proteolysis targeting chimera (PROTAC), that labels SOS1 for degradation by proteasomal degradation through a linked VHL E3 ligase ligand. This PROTAC exhibited the highest SOS1-directed activity due to target destruction, recycling and removal of SOS1 as a scaffolding protein. Although other first PROTACs have entered clinical trials, each conjugate must be meticulously adapted as an efficient clinical drug.

Design of Orally-bioavailable Tetra-cyclic phthalazine SOS1 inhibitors with high selectivity against EGFR.

KRAS mutations (G12C, G12D, etc.) are implicated in the oncogenesis and progression of many deadliest cancers. Son of sevenless homolog 1 (SOS1) is a crucial regulator of KRAS to modulate KRAS from inactive to active states. We previously discovered tetra-cyclic quinazolines as an improved scaffold for inhibiting SOS1-KRAS interaction. In this work, we report the design of tetra-cyclic phthalazine derivatives for selectively inhibiting SOS1 against EGFR. The lead compound 6c displayed remarkable activity to inhibit the proliferation of KRAS(G12C)-mutant pancreas cells. 6c showed a favorable pharmacokinetic profile in vivo, with a bioavailability of 65.8% and exhibited potent tumor suppression in pancreas tumor xenograft models. These intriguing results suggested that 6c has the potential to be developed as a drug candidate for KRAS-driven tumors.

Development of Son of Sevenless Homologue 1 (SOS1) Modulators To Treat Cancers by Regulating RAS Signaling.

Son of sevenless homologue 1 (SOS1) protein is universally expressed in cells and plays an important role in the RAS signaling pathway. Specifically, this protein interacts with RAS in response to upstream stimuli to promote guanine nucleotide exchange in RAS and activates the downstream signaling pathways. Thus, targeting SOS1 is a new approach for treating RAS-driven cancers. In this Perspective, we briefly summarize the structural and functional aspects of SOS1 and focus on recent advances in the discovery of activators, inhibitors, and PROTACs that target SOS1. This review aims to provide a timely and updated overview on the strategies for targeting SOS1 in cancer therapy.

Combining SOS1 and MEK Inhibitors in a Murine Model of Plexiform Neurofibroma Results in Tumor Shrinkage.

Individuals with neurofibromatosis type 1 develop rat sarcoma virus (RAS)-mitogen-activated protein kinase-mitogen-activated and extracellular signal-regulated kinase (RAS-MAPK-MEK)-driven nerve tumors called neurofibromas. Although MEK inhibitors transiently reduce volumes of most plexiform neurofibromas in mouse models and in neurofibromatosis type 1 (NF1) patients, therapies that increase the efficacy of MEK inhibitors are needed. BI-3406 is a small molecule that prevents Son of Sevenless (SOS)1 interaction with Kirsten rat sarcoma viral oncoprotein (KRAS)-GDP, interfering with the RAS-MAPK cascade upstream of MEK. Single agent SOS1 inhibition had no significant effect in the DhhCre;Nf1 fl/fl mouse model of plexiform neurofibroma, but pharmacokinetics (PK)-driven combination of selumetinib with BI-3406 significantly improved tumor parameters. Tumor volumes and neurofibroma cell proliferation, reduced by MEK inhibition, were further reduced by the combination. Neurofibromas are rich in ionized calcium binding adaptor molecule 1 (Iba1)+ macrophages; combination treatment resulted in small and round macrophages, with altered cytokine expression indicative of altered activation. The significant effects of MEK inhibitor plus SOS1 inhibition in this preclinical study suggest potential clinical benefit of dual targeting of the RAS-MAPK pathway in neurofibromas. SIGNIFICANCE STATEMENT: Interfering with the RAS-mitogen-activated protein kinase (RAS-MAPK) cascade upstream of mitogen activated protein kinase kinase (MEK), together with MEK inhibition, augment effects of MEK inhibition on neurofibroma volume and tumor macrophages in a preclinical model system. This study emphasizes the critical role of the RAS-MAPK pathway in controlling tumor cell proliferation and the tumor microenvironment in benign neurofibromas.

Son of sevenless 1 (SOS1), the RasGEF, interacts with ERα and STAT3 during embryo implantation.

Estrogen accounts for several biological processes in the body; embryo implantation and pregnancy being one of the vital events. This manuscript aims to unearth the nuclear role of Son of sevenless1 (SOS1), its interaction with estrogen receptor alpha (ERα), and signal transducer and activator of transcription 3 (STAT3) in the uterine nucleus during embryo implantation. SOS1, a critical cytoplasmic linker between receptor tyrosine kinase and rat sarcoma virus signaling, translocates into the nucleus via its bipartite nuclear localization signal (NLS) during the 'window of implantation' in pregnant mice. SOS1 associates with chromatin, interacts with histones, and shows intrinsic histone acetyltransferase (HAT) activity specifically acetylating lysine 16 (K16) residue of histone H4. SOS1 is a coactivator of STAT3 and a co-repressor of ERα. SOS1 creates a partial mesenchymal-epithelial transition by acting as a transcriptional modulator. Finally, our phylogenetic tree reveals that the two bipartite NLS surface in reptiles and the second acetyl coenzymeA (CoA) (RDNGPG) important for HAT activity emerges in mammals. Thus, SOS1 has evolved into a moonlighting protein, the special class of multi-tasking proteins, by virtue of its newly identified nuclear functions in addition to its previously known cytoplasmic function.

PPDPF Promotes the Development of Mutant KRAS-Driven Pancreatic Ductal Adenocarcinoma by Regulating the GEF Activity of SOS1.

The guanine nucleotide exchange factor (GEF) SOS1 catalyzes the exchange of GDP for GTP on RAS. However, regulation of the GEF activity remains elusive. Here, the authors report that PPDPF functions as an important regulator of SOS1. The expression of PPDPF is significantly increased in pancreatic ductal adenocarcinoma (PDAC), associated with poor prognosis and recurrence of PDAC patients. Overexpression of PPDPF promotes PDAC cell growth in vitro and in vivo, while PPDPF knockout exerts opposite effects. Pancreatic-specific deletion of PPDPF profoundly inhibits tumor development in KRASG12D -driven genetic mouse models of PDAC. PPDPF can bind GTP and transfer GTP to SOS1. Mutations of the GTP-binding sites severely impair the tumor-promoting effect of PPDPF. Consistently, mutations of the critical amino acids mediating SOS1-PPDPF interaction significantly impair the GEF activity of SOS1. Therefore, this study demonstrates a novel model of KRAS activation via PPDPF-SOS1 axis, and provides a promising therapeutic target for PDAC.

New insights on Noonan syndrome's clinical phenotype: a single center retrospective study.

BACKGROUND: Noonan syndrome (NS) is a clinically and genetically heterogeneous disorder. Since its clinical phenotype is often mild and difficult to differentiate from other syndromes, its diagnosis can be challenging and its prevalence in the pediatric population is most certainly underestimated. The difficulty in identifying Noonan syndrome is also increased by the fact that genetic tests are currently not able to detect an underlying mutation in around 10% of the cases. METHODS: This is a retrospective, observational study conducted at the Institute for Maternal and Child "Burlo Garofolo" in Trieste, Italy. We recruited all the patients with clinical and/or genetic diagnosis of NS who were evaluated at the Department of Pediatrics between October 2015 and October 2020. Statistical analyses were performed with IBM SPSS Statistics software. The association between discrete variables has been evaluated through chi-squared test, indicating statistically significant p with Pearson test or Fischer test for variables less than 5. RESULTS: We recruited a total of 35 patients affected by Noonan syndrome. In 24 patients (75%) we identified an underlying genetic substrate: 17 patients had a mutation on PTPN11 (61%), 2 in SOS1, KRAS and SHOC2 (7% each) and only 1 in RAF1 (4%). 25% of the subjects did not receive a genetic confirm. As for the phenotype of the syndrome, our study identified the presence of some clinical features which were previously unrelated or poorly related to NS. For example, renal and central nervous system abnormalities were found at a higher rate compared to the current literature. On the contrary, some features that are considered very suggestive of NS (such as lymphatic abnormalities and the classical facial features) were not frequently found in our population. CONCLUSIONS: In our analysis, we focused on the main phenotypic features of NS, identifying various clinical manifestation that were not associated with this genetic condition before. This could be helpful in raising the knowledge of NS's clinical spectrum, facilitating its diagnosis.

Design and Structural Optimization of Orally Bioavailable SOS1 Inhibitors for the Treatment of KRAS-Driven Carcinoma.

KRAS mutations (G12C, G12D, etc.) are implicated in the oncogenesis and progression of many refractory cancers. Son of sevenless homolog 1 (SOS1) is a key regulator of KRAS to modulate KRAS from inactive to active states. Herein, we disclosed efficacy-improving tetra-cyclic quinazoline derivatives as an enhanced scaffold for inhibiting the SOS1-KRAS interaction. Compound 37, which conjugated 1-carbonitrile-cyclopropane to tetra-cyclic quinazoline, showed a twofold higher oral drug exposure and 2.5-fold longer half-life than BI-3406 in CD-1 mouse plasma. In a Mia-paca-2 xenograft model, 37 administrated alone inhibited tumor growth by 71%. Preclinical investigations demonstrated that 37 had a limited inhibition of CYP and hERG. Overall, our studies showed that 37 was a promising drug candidate for treatment of KRAS-driven cancer.

SOS1 regulates HCC cell epithelial-mesenchymal transition via the PI3K/AKT/mTOR pathway.

The influence of son of sevenless homolog 1 (SOS1) on invasion and metastasis of hepatocellular carcinoma (HCC) cells was investigated. HCC cells were transfected with siRNA and lentivirus to achieve SOS1 knock down/overexpression and changes in RNA and protein levels analyzed by q-PCR and Western blotting (WB). Transwell assay was utilized to assess variations in cell invasion and migration in vitro and by a lung metastasis model of liver cancer in vivo. High expression of SOS1 was observed in most human liver cancers, which indicated a worse prognosis. SOS1 knockout in HepG2 cells significantly decreased cell invasion and migration. SOS1 knockout also reduced the number of metastatic foci in a lung metastasis model of HCC established in nude mice. SOS1 knockout inhibited the epithelial-mesenchymal transition (EMT) in HepG2 cells as well as the PI3K/AKT/mTOR pathway. Overexpression of SOS1 in Huh7 cells had the opposite effect. To conclude, SOS1 may induce the EMT by the activation of the PI3K/AKT/mTOR pathway, thereby enhancing invasion, migration and metastasis of HCC cells. These findings may expose SOS1 as a new HCC therapeutic target.

Lack of embryonic homozygous or adult heterozygous lymphatic phenotypes for a Sos1 mutation and lack of lymphatic embryonic phenotypes for a homozygous Cx47 mutation in mice.

We have studied the lymphatic phenotypes of 2 mutations, known to cause abnormalities of lymphatics in humans, in mice. The Cx47 R260C mutation (variably penetrant in humans heterozygous for it and causing limb lymphedema) had an adult mouse phenotype of hyperplasia and increased lymph nodes only in homozygous condition but we did not find any anatomical phenotype in day 16.5 homozygous embryos. Mice harboring the Sos1 mutation E846K (causing Noonan's in man which occasionally shows lymphatic dysplasia) had no adult heterozygous phenotype in lymphatic vessel appearance and drainage (homozygotes are early embryonic lethals) while day 16.5 heterozygous embryos also had no detectable anatomical phenotype.