Potent SHP2 Research Articles

Discovery of JAB-3312, a Potent SHP2 Allosteric Inhibitor for Cancer Treatment.

As an oncogenic phosphatase, SHP2 acts as a converging node in the RTK-RAS-MAPK signaling pathway in cancer cells and suppresses antitumor immunity by passing signals downstream of PD-1. Here, we utilized the extra druggable pocket outside the previously identified SHP2 allosteric tunnel site by the (6,5 fused), 6 spirocyclic system. The optimized compound, JAB-3312, exhibited a SHP2 binding Kd of 0.37 nM, SHP2 enzymatic IC50 of 1.9 nM, KYSE-520 antiproliferative IC50 of 7.4 nM and p-ERK inhibitory IC50 of 0.23 nM. For JAB-3312, an oral dose of 1.0 mg/kg QD was sufficient to achieve 95% TGI in KYSE-520 xenograft model of mouse. JAB-3312 was well-tolerated in animal models, and a close correlation was observed between the plasma concentration of JAB-3312 and the p-ERK inhibition in tumors. Currently, JAB-3312 is undergoing clinical trials as a potential anticancer agent.

Discovery of a novel SHP2 allosteric inhibitor using virtual screening, FMO calculation, and molecular dynamic simulation.

SHP2 is a non-receptor protein tyrosine phosphatase to remove tyrosine phosphorylation. Functionally, SHP2 is an essential bridge to connect numerous oncogenic cell-signaling cascades including RAS-ERK, PI3K-AKT, JAK-STAT, and PD-1/PD-L1 pathways. This study aims to discover novel and potent SHP2 inhibitors using a hierarchical structure-based virtual screening strategy that combines molecular docking and the fragment molecular orbital method (FMO) for calculating binding affinity (referred to as the Dock-FMO protocol). For the SHP2 target, the FMO method prediction has a high correlation between the binding affinity of the protein-ligand interaction and experimental values (R2 = 0.55), demonstrating a significant advantage over the MM/PBSA (R2 = 0.02) and MM/GBSA (R2 = 0.15) methods. Therefore, we employed Dock-FMO virtual screening of ChemDiv database of ∼2,990,000 compounds to identify a novel SHP2 allosteric inhibitor bearing hydroxyimino acetamide scaffold. Experimental validation demonstrated that the new compound (E)-2-(hydroxyimino)-2-phenyl-N-(piperidin-4-ylmethyl)acetamide (7188-0011) effectively inhibited SHP2 in a dose-dependent manner. Molecular dynamics (MD) simulation analysis revealed the binding stability of compound 7188-0011 and the SHP2 protein, along with the key interacting residues in the allosteric binding site. Overall, our work has identified a novel and promising allosteric inhibitor that targets SHP2, providing a new starting point for further optimization to develop more potent inhibitors. All the molecular docking studies were employed to identify potential leads with Maestro v10.1. The protein-ligand binding affinities of potential leads were further predicted by FMO calculations at MP2/6-31G* level using GAMESS v2020 system. MD simulations were carried out with AmberTools18 by applying the FF14SB force field. MD trajectories were analyzed using VMD v1.9.3. MM/GB(PB)SA binding free energy analysis was carried out with the mmpbsa.py tool of AmberTools18. The docking and MD simulation results were visualized through PyMOL v2.5.0.

Abstract 1975: Discovery of I-1000233, a potent, selective and brain permeable SHP2 allosteric inhibitor

Abstract Background: SHP2 (Src homology region 2-containing protein tyrosine phosphatase 2) is a target of interest for cancer therapy due to its key role in the regulation of RAS/MAPK signal transduction downstream of Receptor Tyrosine Kinases (RTKs). We report here the identification of I-1000233, a novel, highly potent, orally available and brain permeable SHP2 allosteric inhibitor, with potential for the treatment of tumors with dysregulated RTK/RAS/ERK signaling pathways. Material and Methods: High-throughput biological, biochemical and pharmacodynamic (PD) assays were used to inform Structure Activity Relationship studies which led to the identification of a chemical series of potent SHP2 inhibitors. Iterative optimization of physicochemical and pharmacological properties resulted in the identification of I-1000233, an orally available SHP2 inhibitor that showed excellent drug-like properties in preclinical studies. Results: I-1000233 is a potent inhibitor of the SHP2 enzyme in vitro (IC50 = 2 nM) with an exceptionally long residence time on the target. When tested in cell lines, it exhibits significant anti-proliferation activity against brain tumor cells and multiple RAS or EGFR mutant models. It strongly inhibits pERK, a downstream marker of MAPK pathway activity, in a dose dependent manner. Further, in vivo preclinical data confirmed that I-1000233 penetrates the BBB and shows anti-tumor activity, supporting its potential in the treatment of CNS and brain-metastasized tumors. Conclusions: I-1000233 is a potent, selective and orally available SHP2 inhibitor with potential for once-a-day dosing and its brain permeability in humans makes it a promising therapeutic agent against SHP2i responsive brain tumors and metastases. Citation Format: Francesca Puca, Danilo Fabbrini, Alina Ciammaichella, Antonino Missineo, Monica Bisbocci, Martina Nibbio, Annalise Di Marco, Claudia Apicella, Fabrizio Colaceci, Romina Sasso, Daniela Natale, Francesco Scalabrì, Cristina Alli, Vincenzo Pucci, Christian Montalbetti, Alessia Petrocchi, Alessandro Carugo, Carlo Toniatti. Discovery of I-1000233, a potent, selective and brain permeable SHP2 allosteric inhibitor [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 1975.

Abstract 6060: Discovery of potent SHP2 protein degraders with strong anti-tumor activities in KRAS mutant cancers

Abstract Background: SHP2 plays a pivotal role in modulating the RTK/RAS/MAPK signaling pathway, which is frequently dysregulated in various cancers. Multiple allosteric SHP2 inhibitors have been under active development for the treatment of RTK/RAS/MAPK-dependent cancers, while certain limitations have come to light, such as their inability to target the active form of SHP2 and narrow therapeutic index necessitating intermittent dosing to mitigate toxicity. To overcome these challenges, we have discovered SHP2 protein degraders as next generation SHP2 targeting agents. Here, we present compelling profiles of our SHP2 degraders. Methods: We employed a variety of cancer cell lines harboring mutations in RTK, KRAS, or SHP2 for assessing in vitro activity of SHP2 degraders. For in vivo assessment of pharmacological profile, we utilized an NCI-H358 KRAS G12C mutant NSCLC xenograft. SHP2 protein levels in cell lysates and tumors were analyzed to assess SHP2 degradation efficiency. Simultaneously, ERK phosphorylation and DUSP6 mRNA levels were analyzed to evaluate downstream effects. Immunoprecipitation assays were used to assess the impact on SHP2's scaffolding roles. Results: A series of SHP2 degraders have been identified, demonstrating highly efficient SHP2 degradation activity with DC50 values in subnanomolar ranges. Robust inhibition of ERK phosphorylation, followed by remarkable anti-cancer effects in multiple KRAS mutant cancer cell lines were observed. Interestingly, in vitro cell growth inhibition by SHP2 degraders are 10 times more potent than SHP2 inhibitors currently under clinical development. Further characterization reveals several advantages of SHP2 degraders compared to SHP2 inhibitors. First, we observed a more efficient disruption of SHP2 scaffolding function, such as the formation of the SHP2-GRB2-GAB1 complex. Second, we detected effective degradation of activating mutants of SHP2, such as E76K and T507K, which are typically not targeted by SHP2 inhibitors. Experiments using a KRAS mutant CDX model demonstrate compelling in vivo profiles. A single administration of lead compounds resulted in Dmax values exceeding 95% in tumors, accompanied by the significant reduction of downstream signaling markers, such as p-ERK and DUSP6 mRNA. Notably, the reduced levels of SHP2 and downstream markers persist for several days. This suggests that utilizing intermittent dosing could be a viable approach with SHP2 degraders. Once-weekly intravenous administration achieved strong tumor growth inhibition without causing significant body weight loss. Conclusion: We have successfully discovered novel SHP2 degraders exhibiting remarkable activities. We demonstrate that SHP2 degraders have potential to overcome the limitations associated with allosteric SHP2 inhibitors and provide a promising therapeutic option for RTK/RAS/MAPK-dependent cancer patients. Citation Format: So Hyun Kim, Jihye Lee, Jiyoung Song, Geon-Tae Park, Jieun Jung, Jungtae Na, Dong Ho Lee, Soohyun Lee, Ji Youn Park, Jiyoung Kim, Onnuri Bae, Jeonghwa Han, Ju Young Kang, Mijin Moon, Yongje Shin, Je Ho Ryu, Song Hee Lee, Sook-Kyung Park. Discovery of potent SHP2 protein degraders with strong anti-tumor activities in KRAS 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 6060.

Identification of a Novel, Potent, and Orally Bioavailable Guanidine-Based SHP2 Allosteric Inhibitor from Virtual Screening and Rational Structural Optimization for the Treatment of KRAS Mutant Cancers.

Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) is a highly attractive therapeutic target for treating Kirsten rat sarcoma viral oncogene (KRAS) mutant cancers. In this work, a series of guanidine-based SHP2 allosteric inhibitors were discovered via virtual screening and rational structural optimization. Notably, lead compound 23 with potent SHP2 inhibitory activity (IC50 = 17.7 nM) effectively inhibited the proliferation, migration, and invasion of MIA PaCa-2 pancreatic cancer cells. Furthermore, compound 23 featured great in vivo pharmacokinetic properties (AUCpo = 4320 nM·h; F = 66.3%) and exhibited significant antitumor efficacy in the MIA PaCa-2 xenograft mouse model. This demonstrates that compound 23 is a potential lead compound for the development of SHP2 allosteric inhibitors to treat KRAS mutant cancers. Moreover, these guanidine-based scaffolds may provide an opportunity to mitigate the potential safety risks of the alkyl amine motif predominately incorporated in current SHP2 allosteric inhibitors.

Discovery of a Novel Series of Potent SHP2 Allosteric Inhibitors.

Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is the first reported nonreceptor oncogenic tyrosine phosphatase connecting multiple signal transduction cascades and exerting immunoinhibitory function through the PD-1 checkpoint receptor. As part of a drug discovery program aimed at obtaining novel allosteric SHP2 inhibitors, a series of pyrazopyrazine derivatives bearing an original bicyclo[3.1.0]hexane basic moiety on the left-hand side region of the molecule were identified. We report herein the discovery process, the in vitro pharmacological profile, and the early developability features of compound 25, one of the most potent members of the series.

Discovery of a Novel Series of Imidazopyrazine Derivatives as Potent SHP2 Allosteric Inhibitors.

Protein tyrosine phosphatase SHP2 is an oncogenic protein that can regulate different cytokine receptor and receptor tyrosine kinase signaling pathways. We report here the identification of a novel series of SHP2 allosteric inhibitors having an imidazopyrazine 6,5-fused heterocyclic system as the central scaffold that displays good potency in enzymatic and cellular assays. SAR studies led to the identification of compound 8, a highly potent SHP2 allosteric inhibitor. X-ray studies showed novel stabilizing interactions with respect to known SHP2 inhibitors. Subsequent optimization allowed us to identify analogue 10, which possesses excellent potency and a promising PK profile in rodents.

99 (PB089) - Discovery of an allosteric small molecule inhibitor that can potently target SHP2 in vitro and in vivo

Background: SHP2 (Src homology region 2-containing protein tyrosine phosphatase 2) is the only known tumor-promoting phosphatase, that relays the signals from the diverse receptor tyrosine kinases to the RAS/MAPK pathway. Abnormal SHP2 activation and activating mutations are frequently observed in various cancers and accordingly genetic or pharmacological inhibition of SHP2 has been reported to suppress RTK/KRAS-mediated cancer progression. Moreover, SHP2 serves as a key component of the ERK re-activation in the resistance mechanism to KRAS pathway inhibitors. Therefore, SHP2 inhibitor has a great therapeutic potential in treating RTK/ RAS-mediated cancer as monotherapy and in combination treatment. Material and methods: Biochemical phosphatase assays were performed by detecting DiFMU which is converted by SHP2(WT or mutants) with various concentrations of compounds. For the cellular pERK analysis, compounds were treated into H358 cells followed by pERK measurement using the AlphaLISA system (PerkinElmer). Cell viability was measured by CellTiter-Glo (Promega) after 3-day treatment with compounds. For the in vivo tumor model, cancer cells were inoculated into the flank region of BALB/ c nude mice. Once the tumor size reached 100 mm3, compounds were administrated (P.O.,Q.D). Tumor size was measured twice a week. Results: Cpd1, an orally available and highly potent SHP2 inhibitor, profoundly suppressed SHP2 activity in vitro and KRAS-mutated tumor growth in mouse xenograft models, outcompeting the positive control TNO-155. In detail, Cpd1 potently inhibited SHP2(WT) and also the highly-active oncogenic SHP2 mutants. It impeded the proliferation of cancer cells encoding KRAS and/or SHP2 mutations. With an excellent PK profile, Cpd1 induced in vivo tumor regression in KRAS mutation-driven models. Conclusions: Cpd1 is a potent and selective SHP2 allosteric inhibitor, with a great anti-tumor effect in vitro and in vivo. It may provide a novel therapeutic option for diverse cancers, including KRAS-driven tumors, as a monotherapy or in combination therapies. No conflict of interest.

95 (PB085) - Preliminary results from FLAGSHP-1: A Phase I dose escalation study of ERAS-601, a potent SHP2 inhibitor, in patients with previously treated advanced or metastatic solid tumors

Background: SHP2 is an oncogenic tyrosine phosphatase that transduces receptor tyrosine kinase signaling to the RAS/MAPK pathway via its phosphatase-mediated regulation of guanine nucleotide exchange factors. ERAS-601 is a potent, selective, and orally bioavailable allosteric inhibitor of SHP2 that has demonstrated robust anti-proliferative activity in vitro and in vivo as monotherapy and in combination in preclinical models of cancer harboring EGFR, KRAS, BRAF class III and NF1LOF mutations.

The Discovery of Potent SHP2 Inhibitors with Anti-Proliferative Activity in Breast Cancer Cell Lines.

Despite available treatments, breast cancer is the leading cause of cancer-related death. Knowing that the tyrosine phosphatase SHP2 is a regulator in tumorigenesis, developing inhibitors of SHP2 in breast cells is crucial. Our study investigated the effects of new compounds, purchased from NSC, on the phosphatase activity of SHP2 and the modulation of breast cancer cell lines’ proliferation and viability. A combined ligand-based and structure-based virtual screening protocol was validated, then performed, against SHP2 active site. Top ranked compounds were tested via SHP2 enzymatic assay, followed by measuring IC50 values. Subsequently, hits were tested for their anti-breast cancer viability and proliferative activity. Our experiments identified three compounds 13030, 24198, and 57774 as SHP2 inhibitors, with IC50 values in micromolar levels and considerable selectivity over the analogous enzyme SHP1. Long MD simulations of 500 ns showed a very promising binding mode in the SHP2 catalytic pocket. Furthermore, these compounds significantly reduced MCF-7 breast cancer cells’ proliferation and viability. Interestingly, two of our hits can have acridine or phenoxazine cyclic system known to intercalate in ds DNA. Therefore, our novel approach led to the discovery of SHP2 inhibitors, which could act as a starting point in the future for clinically useful anticancer agents.

Phosphorylation of SHP2 at Tyr62 Enables Acquired Resistance to SHP2 Allosteric Inhibitors in FLT3-ITD-Driven AML.

These findings suggest that combined inhibition of SHP2 and FLT3 effectively treat FLT3-ITD-positive AML, highlighting the need for development of more potent SHP2 inhibitors and combination therapies for clinical applications.

Studies on molecular mechanism between SHP2 and pyridine derivatives by 3D-QSAR, molecular docking and MD simulations

BackgroundSrc homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) as a major phosphatase would affect the development of tumors by regulating several cellular processes, and is a significant potential target for cancer treatment. MethodsIn the present work, a series of pyridine derivatives possessing a wide range of inhibitory activity was employed to investigate the structural requirements by developing three dimensional quantitative structure–activity relationship (3D-QSAR) models using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. The results show that CoMFA (R2cv = 0.646, R2pred = 0.5587) and CoMSIA (R2cv = 0.777, R2pred = 0.7131) have excellent stability and predictability. The relationship between the inhibitory activity and structure of the inhibitors was analyzed by the derived contour maps. Furthermore, the QSAR models were validated by molecular docking and molecular dynamics simulations, which were also applied to reveal the potential molecular mechanism of these inhibitors. FindingsIt was found that Arg110, Asn216, Thr218, Thr252 and Pro490 play a crucial role in stabilizing the inhibitors. Additionally, MM/PBSA calculations provided the binding free energy were also conducted to explain the discrepancy of binding activities. Overall, the outcomes of this work could provide useful information and theoretical guidance for the development of novel and potent SHP2 inhibitors.

Allosteric inhibition reveals SHP2-mediated tumor immunosuppression in colon cancer by single-cell transcriptomics

Colorectal cancer (CRC), a malignant tumor worldwide consists of microsatellite instability (MSI) and stable (MSS) phenotypes. Although SHP2 is a hopeful target for cancer therapy, its relationship with innate immunosuppression remains elusive. To address that, single-cell RNA sequencing was performed to explore the role of SHP2 in all cell types of tumor microenvironment (TME) from murine MC38 xenografts. Intratumoral cells were found to be functionally heterogeneous and responded significantly to SHP099, a SHP2 allosteric inhibitor. The malignant evolution of tumor cells was remarkably arrested by SHP099. Mechanistically, STING–TBK1–IRF3-mediated type I interferon signaling was highly activated by SHP099 in infiltrated myeloid cells. Notably, CRC patients with MSS phenotype exhibited greater macrophage infiltration and more potent SHP2 phosphorylation in CD68+ macrophages than MSI-high phenotypes, suggesting the potential role of macrophagic SHP2 in TME. Collectively, our data reveals a mechanism of innate immunosuppression mediated by SHP2, suggesting that SHP2 is a promising target for colon cancer immunotherapy.

Abstract 1475: Discovery of ETS-001, a highly potent allosteric SHP2 inhibitor to treat RTK/RAS-driven cancers

Abstract The non-receptor protein tyrosine phosphatase (PTP) SHP2, encoded by PTPN11, functions as a convergent node downstream of multiple receptor tyrosine kinases (RTKs) and is required for full activation of the MAPK pathway. Suppression of SHP2 activity via either genetic silencing or pharmacological inhibition has been shown to inhibit RAS/MAPK signaling and cell growth in a wide spectrum of cancers, pointing to a therapeutic opportunity for targeting SHP2 in cancer therapy. Here we report the discovery of a highly potent, selective and orally bioavailable small-molecule allosteric inhibitor of SHP2, ETS-001, that is effective in various human cancer models bearing oncogenic alterations in the RTK/RAS/MAPK pathway. Treatment with ETS-001 as a single agent resulted in dramatic growth inhibition of cancer cells as well as in vivo tumor models bearing genetic alterations of EGFR, or components in the nucleotide cycling-dependent KRAS pathway. Besides, as the effectiveness of RAS pathway inhibitors are ultimately limited by rapid emergence of drug resistance via multiple mechanisms including bypass activation of alternative RTKs, SHP2 inhibition has the potential to overcome the resistance as combinational strategies associated with various RAS pathway-targeted therapies. Here we show that ETS-001 exhibited strong synergistic effect with EGFR-TKI (osimertinib), KRASG12Ci (AMG510) or CDK4/6i (ribociclib) in appropriate cancer models in vitro and in vivo. In EGFR mutant NSCLC cells, treatment with both ETS-001 and osimertinib showed combinational benefit that was coincident with sustained ERK inhibition. In a KRASG12C mutant pancreatic cancer cell xenograft model, ETS-001 greatly enhanced the efficacy of AMG510 to suppress in vivo tumor growth. In addition, combinational benefit of ETS-001 and ribociclib was observed in esophageal cancer models, which exhibited high frequency of concurrent oncogenic alterations in RTKs and cell cycle pathway. Taken together, our findings provide preclinical evidence that ETS-001 as a potent SHP2 inhibitor, alone or in combination with other targeted-therapy agents, can effectively inhibit MAPK activation and treat cancers with diverse oncogenic mutations in the RAS/MAPK pathway. Citation Format: Xi Xia, Lin Du, Hao Zhuge, Qiangang Zheng, Wenqi Cui, Jidong Zhu, Guo Li. Discovery of ETS-001, a highly potent allosteric SHP2 inhibitor to treat RTK/RAS-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1475.

Discovery of SHP2-D26 as a First, Potent, and Effective PROTAC Degrader of SHP2 Protein.

Src homology 2 domain-containing phosphatase 2 (SHP2) is an attractive therapeutic target for human cancers and other human diseases. Herein, we report our discovery of potent small-molecule SHP2 degraders whose design is based upon the proteolysis-targeting chimera (PROTAC) concept. This work has led to the discovery of potent and effective SHP2 degraders, exemplified by SHP2-D26. SHP2-D26 achieves DC50 values of 6.0 and 2.6 nM in esophageal cancer KYSE520 and acute myeloid leukemia MV4;11 cells, respectively, and is capable of reducing SHP2 protein levels by >95% in cancer cells. SHP2-D26 is >30-times more potent in inhibition of phosphorylation of extracellular signal-regulated kinase (ERK) and of cell growth than SHP099, a potent SHP2 inhibitor, in KYSE520 and MV4;11 cancer cell lines. This study demonstrates that induced SHP2 degradation is a very effective approach to inhibit the function of SHP2. Further optimization of these SHP2 degraders may lead to the development of a new class of therapies for cancers and other human diseases.

PCC0208023, a potent SHP2 allosteric inhibitor, imparts an antitumor effect against KRAS mutant colorectal cancer

The non-receptor tyrosine phosphatase SHP2, encoded by PTPN11, plays an indispensable role in tumors driven by oncogenic KRAS mutations, which frequently occur in colorectal cancer. Here, PCC0208023, a potent SHP2 allosteric inhibitor, was synthesized to evaluate its inhibitory effects against the SHP2 enzyme, and the KRAS mutant colorectal cancer in vitro and in vivo, and its impart on the RAS/MAPK pathway. Consistent with an allosteric mode of inhibition, PCC0208023 can non-competitively inhibit the activity of full-length SHP2 enzyme, but lacks activity against the free catalytic domain of SHP2. Furthermore, PCC0208023 inhibited the proliferation of KRAS mutation-driven human colorectal cancer cells by inhibiting the RAS/MAPK signaling pathway in vitro. Importantly, PCC0208023 displayed good anti-tumor efficacy against KRAS-driven LS180 and HCT116 xenograft models in nude mice with the decreased Ki67 and p-ERK level, and increased cleaved caspase-3 expression in tumors. Interestingly, PCC0208023 maintained high levels in LS180 tumors within 24 h after administration and was mainly distributed in both intestines and lungs. Molecular docking studies revealed a higher affinity of PCC0208023 with key residues in the SHP2 allosteric pocket than RMC-4550. PCC0208023 deserves further optimization to identify additional low-toxic and potent SHP2 allosteric inhibitors with novel scaffolds for the treatment of patients with KRAS mutation-positive colorectal cancer.

Abstract 1322: Maximizing the therapeutic potential of SHP2 inhibition with rational combination strategies in tumors driven by aberrant RAS-MAPK signaling

Abstract SHP2 (PTPN11) is a protein tyrosine phosphatase and scaffold protein that functions as a convergent node downstream of multiple receptor tyrosine kinases (RTKs), integrating growth factor signals to promote RAS activation. Recently we reported that a SHP2 allosteric inhibitor suppresses RAS activation and proliferation in cancers driven by nucleotide cycling oncogenic KRAS, e.g., KRASG12C and other RAS-GTP dependent downstream mutations, e.g., NF1LOF, BRAFclass3. Daily oral administration of a SHP2 allosteric inhibitor at the maximally tolerated dose achieved tumor cytostasis in cell line-derived and patient-derived xenograft models harboring these mutations, and in some instances, tumor regressions. In most models, tumor cytostasis was followed by regrowth of tumors, reminiscent of patterns observed with MEK inhibitors. In the NCI-H358 KRASG12C model of non-small cell lung cancer, the maximum daily dose of the SHP2 inhibitor resulted in ~75% inhibition (relative to control) of pERK1/2, a downstream indicator of RAS-MAPK signaling in tumor lysates. These results support the hypothesis that targeting a single node results in incomplete RAS-MAPK pathway inhibition, which may permit tumor persistence in these models. To achieve deeper suppression of oncogenic flux through the RAS-MAPK pathway in tumors driven by nucleotide cycling KRAS, NF1LOF, or BRAFclass3, we evaluated combinations of MEK and ERK inhibitors with a SHP2 inhibitor as rational “in-pathway”, anti-proliferative combinations. Additionally, we examined the combination with CDK4/6 inhibition as a strategy to impact RAS-MAPK signaling both directly and via cell cycle effects. We found that concurrent targeting of SHP2 and MEK or ERK drives greater anti-tumor efficacy than that observed with monotherapy with either agent. Comparable synergy was observed when SHP2 inhibition was combined with CDK4/6 inhibition. Given the complementary anti-tumor mechanisms triggered by MEK/ERK and CDK4/6 inhibition, we further interrogated a triple combination of SHP2, MEK, and CDK4/6 inhibition to drive complete elimination of RAS-MAPK oncogenic signals. We found that this combination regimen was tolerated, resulted in more significant responses in all tumor models we tested, and delayed the evolution of on-treatment resistance. Investigation of the downstream effects of the triple therapy on tumor cells revealed profound combinatorial effects on cell cycle progression, proliferation and induction of apoptosis. These observed synergies are likely due to a combination of RAS pathway extinction and the inhibition of parallel escape mechanisms. In summary, our findings reveal rational combination strategies to maximize the therapeutic potential of a potent SHP2 inhibitor in patients with tumors harboring nucleotide cycling oncogenic KRAS or other RAS-GTP-dependent downstream mutations. Citation Format: Grace J. Lee, Carlos Stahlhut, James Evans, Denise F. Reyes, Edward G. Lorenzana, Shaoling Li, Elena S. Koltun, Dong Lee, Zhengping Wang, Robert J. Nichols, Jacqueline A. Smith, Mallika Singh. Maximizing the therapeutic potential of SHP2 inhibition with rational combination strategies in tumors driven by aberrant RAS-MAPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1322.

The tyrosine phosphatase SHP2 controls TGF\u03b2-induced STAT3 signaling to regulate fibroblast activation and fibrosis

Uncontrolled activation of TGFβ signaling is a common denominator of fibrotic tissue remodeling. Here we characterize the tyrosine phosphatase SHP2 as a molecular checkpoint for TGFβ-induced JAK2/STAT3 signaling and as a potential target for the treatment of fibrosis. TGFβ stimulates the phosphatase activity of SHP2, although this effect is in part counterbalanced by inhibitory effects on SHP2 expression. Stimulation with TGFβ promotes recruitment of SHP2 to JAK2 in fibroblasts with subsequent dephosphorylation of JAK2 at Y570 and activation of STAT3. The effects of SHP2 on STAT3 activation translate into major regulatory effects of SHP2 on fibroblast activation and tissue fibrosis. Genetic or pharmacologic inactivation of SHP2 promotes accumulation of JAK2 phosphorylated at Y570, reduces JAK2/STAT3 signaling, inhibits TGFβ-induced fibroblast activation and ameliorates dermal and pulmonary fibrosis. Given the availability of potent SHP2 inhibitors, SHP2 might thus be a potential target for the treatment of fibrosis.

Abstract 20959: Inhibition of Shp2 Ameliorates Systemic Lupus Erythematosus-Associated Cardiac Pathogenesis

Introduction: Systemic lupus erythematous (SLE) is a devastating autoimmune disorder affecting multiple tissues, including heart. However, the molecular mechanisms that cause cardiac effects in SLE remain unclear. PTPN11 , the gene encoding the SH2 domain-containing protein tyrosine phosphatase SHP2, is critical for development and is the first tyrosine phosphatase known to have a functional role in the adult heart. Moreover, gain-of-function mutations in SHP2 have been associated with onset of SLE. Here, sought to identify the mechanistic role for SHP2 in the cardiac pathogenesis of SLE. Methods and Results: We analyzed hearts from 18-week old female MRL/lpr (SLE-prone) and MRL/MpJ (control) mice. As compared to controls, MRL/lpr hearts showed substantial interstitial fibrosis and had elevated numbers of infiltrated CD11b + leukocytes. Moreover, MRL/lpr hearts displayed decreased fractional shortening and enlarged chamber dimensions, as measured by echocardiography. MRL/lpr heart lysates revealed significantly elevated SHP2 phosphatase activity, as compared to controls. Immunoprecipitation of SHP2 from MRL/lpr hearts revealed Focal Adhesion Kinase (FAK) in complex with SHP2. FAK is required to maintain cardiac function under conditions of stress. Significantly, SHP2 is a negative regulator of FAK, suggesting SHP2 may be involved in cardiac pathogenesis through dephosphorylation of FAK. Here, MRL/lpr heart lysates had decreased pY397-FAK activity, in conjunction with decreased p-ERK levels, suggesting that SHP2 may play a role in cardiac pathogenesis of SLE through aberrant FAK -mediated regulation of ERK. To test whether higher SHP2 activity contributes to the cardiac pathogenesis in lupus, we utilized a potent SHP2 inhibitor, 11a-1, to treat MRL/lpr mice for 6 weeks starting at 12 weeks of age. Hearts from these mice displayed improved cardiac structure and function, with reduced chamber dimensions, had decreased numbers of CD11b + leukocytes and lessened cardiac fibrosis. Moreover, pY397-FAK and p-ERK activities were both nearly normalized in response to treatment with the SHP2 inhibitor. Conclusion: These data suggest that SHP2 affects cardiac pathogenesis in SLE hearts through regulation of FAK and ERK signaling.

Identification of demethylincisterol A3 as a selective inhibitor of protein tyrosine phosphatase Shp2

Shp2 is a classical non-receptor protein tyrosine phosphatase (PTP) involved in many human diseases such as Noonan syndrome and tumors, and identified as a potential therapeutic target. In order to find a potent and selective Shp2 inhibitor, we screened a diverse collection of the secondary metabolites from endophyte fungi using an in vitro enzyme assay, and finally identified a potent Shp2 inhibitor, HLP46 (demethylincisterol A3) from Pestalotiopsis sp. HLP46 was reported to have anti-tumor and anti-inflammation activity previously. We provide the first evidence that HLP46 is an inhibitor of the Shp2. HLP46 shows high selective inhibition of Shp2 over Shp1, PTP1B, Lyp, STEP, PTPRA and Cdc25b. Enzymatic kinetic analyses showed that HLP46 is a non-competitive inhibitor of Shp2. HLP46 interrupts Gab1-Shp2 association and blocked Shp2-dependent activation of the Ras/ERK signal pathway induced by EGF. Furthermore, HLP46 decreased Src activation and inhibit tumor cell migration and invasion. As expected, HLP46 has no effect on the Shp2-independent activation of ERK induced by PMA or on the activation of the PI3K/Akt pathway. We testified therapeutic efficacy targeting both Shp2 and PI3K in MCF7 cells. HLP46 does not show any synergistic inhibition with PI3K inhibitor in suppressing cell growth. Collectively, these results suggest that HLP46 is a selective Shp2 inhibitor and could inhibit Shp2-dependent cell signaling in human cells.