Src Homology Region 2-containing Protein Research Articles

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.

SHP2 deneddylation mediates tumor immunosuppression in colon cancer via the CD47/SIRPα axis.

SIPRα on macrophages binds with CD47 to resist proengulfment signals, but how the downstream signal of SIPRα controls tumor-infiltrating macrophages (TIMs) is still poorly clarified. Here, we report that the CD47/signal regulatory protein α (SIRPα) axis requires the deneddylation of tyrosine phosphatase SHP2. Mechanistically, Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) was constitutively neddylated on K358 and K364 sites; thus, its autoinhibited conformation was maintained. In response to CD47-liganded SIRPα, SHP2 was deneddylated by sentrin-specific protease 8 (SENP8), which led to the dephosphorylation of relevant substrates at the phagocytic cup and subsequent inhibition of macrophage phagocytosis. Furthermore, neddylation inactivated myeloid-SHP2 and greatly boosted the efficacy of colorectal cancer (CRC) immunotherapy. Importantly, we observed that supplementation with SHP2 allosteric inhibitors sensitized immune treatment-resistant CRC to immunotherapy. Our results emphasize that the CRC subtype that is unresponsive to immunotherapy relies on SIRPαhiSHP2hiNEDD8lo TIMs and highlight the need to further explore the strategy of SHP2 targeting in CRC therapy.

Waking immune-resistant tumors with neddylation.

The CD47/signal regulatory protein α (SIRPα) axis, which functions as an inhibitory phagocytosis checkpoint, also serves as a key mediator in cancer immune evasion. Many cancers, including colorectal cancer (CRC), exploit the expression of CD47 to escape phagocytic clearance and activate the innate immune system. Previous work has indicated that distinct paradigms of posttranslational modifications mediate the regulatory mechanisms of the CD47/SIRPα axis. In this issue of the JCI, Li et al. show that neddylation, a ubiquitin-like modification, inactivates Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2), a downstream target of this pathway. They further show that inhibition of SHP2 sensitizes CRC cells to immunotherapies to which they were previously resistant. Collectively, the results underscore the need for cotargeting SHP2 and immune checkpoints (e.g., programmed death 1 [PD1]) in CRC and possibly other immunotherapy-resistant tumors.

Aberrant Cortical Layer Development of Brain Organoids Derived from Noonan Syndrome-iPSCs.

Noonan syndrome (NS) is a genetic disorder mainly caused by gain-of-function mutations in Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2). Although diverse neurological manifestations are commonly diagnosed in NS patients, the mechanisms as to how SHP2 mutations induce the neurodevelopmental defects associated with NS remain elusive. Here, we report that cortical organoids (NS-COs) derived from NS-induced pluripotent stem cells (iPSCs) exhibit developmental abnormalities, especially in excitatory neurons (ENs). Although NS-COs develop normally in their appearance, single-cell transcriptomic analysis revealed an increase in the EN population and overexpression of cortical layer markers in NS-COs. Surprisingly, the EN subpopulation co-expressing the upper layer marker SATB2 and the deep layer maker CTIP2 was enriched in NS-COs during cortical development. In parallel with the developmental disruptions, NS-COs also exhibited reduced synaptic connectivity. Collectively, our findings suggest that perturbed cortical layer identity and impeded neuronal connectivity contribute to the neurological manifestations of NS.

Potential Therapeutic Effects of Thiazolidinedione on Malignant Glioma.

Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant tumor of the central nervous system. GBM has a very low 5-year survival rate and reaching merely a median of ~15 months even with aggressive treatments. PPARγ (Peroxisome proliferator- activated receptor gamma) agonists (ciglitazone), while being widely used on patients of type 2 diabetes mellitus, also have approved anticancer effects. Their action mechanisms on malignant glioma are not fully understood. The aim of this study is to investigate the potential therapeutic effect of PPARγ agonists on maligant glioma. Glioma cell line and in-vivo/ex-vivo animal model intervened by ciglitazone were used to assess the associated mechanism and therapeutic effect. Our results from in vivo and ex vivo experiments showed that ciglitazone not only inhibited tumor growth and its associated angiogenesis, but it also reduced colony formation and migration of tumors. Ciglitazone inhibited the phosphorylation of STAT3 (signal transducer and activator of transcription 3) (at the point of tyrosine 705 by increasing both the amount and activity of SHP-2 (Src homology region 2-containing protein tyrosine phosphatase 2) proteins, based on evidence obtained from immunoprecipitation and immunohistochemistry. Furthermore, ciglitazone activated proteasomes and lysosomes to degrade cell-cycle-related proteins like Cyclin D1, Cyclin E, CDK2 (Cyclin-dependent kinase 2), and CDK4 (Cyclin-dependent kinase 4). Ciglitazone triggered expressions of LC3 (Microtubule-associated protein 1A/1B-light chain 3) and formation of acidic vesicular organelles (AVOs), both of which were implicated in the autophagy pathway. In conclusion, ciglitazone showed the multiple actions to regulate the growth of glioma, which appeared to be a potential candidate for treating malignant glioma.

77 (PB067) - Discovery of I-0436650, a potent and selective SHP2 allosteric inhibitor for the treatment of RAS driven solid tumors

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-0436650, a novel, highly potent, orally available SHP2 allosteric inhibitor, with potential for the treatment of tumors with dysregulated RTK/RAS/ERK signaling pathways.

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.

159 (PB039) - A combination vertical inhibition approach with inhibitors of SHP2 and ERK provides improved activity in KRAS-mutant pancreatic and colorectal cancer models

Background: Inhibitors of the MAPK pathway are approved therapeutic agents with a high initial response rate, however single agent use of MAPK inhibitors in the clinic is often limited by resistance. Resistance commonly occurs through mechanisms which result in reactivation of MAPK signaling, such as activation of receptor tyrosine kinases (RTKs). Vertical pathway combination strategies are therefore of interest for addressing resistance through pathway reactivation. Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) is a key regulator of the MAPK pathway downstream of RTKs and upstream of RAS, whilst ERK1/2 (ERK) is the final node of the kinase cascade.

SHP2 regulates adipose maintenance and adipocyte-pancreatic cancer cell crosstalk via PDHA1.

Adipocytes are the most abundant cell type in the adipose tissue, and their dysfunction is a significant driver of obesity-related pathologies, such as cancer. The mechanisms that (1) drive the maintenance and secretory activity of adipocytes and (2) mediate the cancer cellular response to the adipocyte-derived factors are not fully understood. To address that gap of knowledge, we investigated how alterations in Src homology region 2-containing protein (SHP2) activity affect adipocyte function and tumor crosstalk. We found that phospho-SHP2 levels are elevated in adipose tissue of obese mice, obese patients, and differentiating adipocytes. Immunofluorescence and immunoprecipitation analyses as well as in-silico protein-protein interaction modeling demonstrated that SHP2 associates with PDHA1, and that a positive association promotes a reactive oxygen species (ROS)-driven adipogenic program. Accordingly, this SHP2-PDHA1-ROS regulatory axis was crucial for adipocyte maintenance and secretion of interleukin-6 (IL-6), a key cancer-promoting cytokine. Mature adipocytes treated with an inhibitor for SHP2, PDHA1, or ROS exhibited an increased level of pro-lipolytic and thermogenic proteins, corresponding to an increased glycerol release, but a suppression of secreted IL-6. A functional analysis of adipocyte-cancer cell crosstalk demonstrated a decreased migration, invasion, and a slight suppression of cell cycling, corresponding to a reduced growth of pancreatic cancer cells exposed to conditioned media (CM) from mature adipocytes previously treated with inhibitors for SHP2/PDHA1/ROS. Importantly, PDAC cell growth stimulation in response to adipocyte CM correlated with PDHA1 induction but was suppressed by a PDHA1 inhibitor. The data point to a novel role for (1) SHP2-PDHA1-ROS in adipocyte maintenance and secretory activity and (2) PDHA1 as a regulator of the pancreatic cancer cells response to adipocyte-derived factors.

Abstract 1041: HBI-2376, HUYABIO clinical stage SHP2 inhibitor, possess robust in vitro potency and in vivo efficacy in several preclinical tumor models carrying KrasG12C or EGFR mutations

Abstract SHP2 (Src homology region 2-containing protein tyrosine phosphatase 2) is an attractive target in oncology drug development due to its dual roles on the direct growth and proliferation of cancer cells and also acting as an immune checkpoint molecule to suppress tumor immunity. HBI-2376 is an orally bioavailable selective SHP2 inhibitor that is being developed for the treatment of solid tumors harboring KRAS or EGFR mutations, such as non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). Additionally, HBI-2376 has the potential to suppress tumor growth via induction of the activity of immune infiltrating cells in the TME (tumor microenvironment). In a head-to-head comparison, in vitro studies showed greater potency of HBI-2376 to inhibit cell proliferation in a variety of cancer cell lines compared to other SHP2 inhibitors such as TNO-155 (Novartis AG) and RMC-4550 (Revolution Medicines). Consistently, HBI-2376 found to be more efficacious in tumor growth inhibition than TNO-155 and RMC-4550 in NCI-H1975L858R_T790M_C797S osimertinib-resistant cell line and other tumor models. In addition to xenograft models, we tested efficacy of HBI-2376 as single agent or in combination with anti-PD1 mAb in MC38 CRC syngeneic model. Consistent with xenograft data, HBI-2376 showed greater efficacy in tumor growth inhibition vs. TNO-155 and RMC-4550 both as single agent and in combination therapy. IHC data showed reduction in infiltration of M2 macrophages (F4/80+Arg1+) in the HBI-2376 treated tumors suggesting a MoA for its greater efficacy in MC38 model. Furthermore, Western blotting showed inhibition of p-ERK and DUSP6 in HBI-2376 treated cells indicative of a biomarker of response to the therapy. These findings, along with a positive safety profile led to HBI-2376 IND clearance by FDA and suggested promising responses in NSCLC or CRC patients in the forthcoming clinical studies. Citation Format: Farbod Shojaei, Farbod Shojaei, Jill M. Ricono, Che Fang, Fairooz Kabbinavar, Bob Goodenow, Mireille Gillings. HBI-2376, HUYABIO clinical stage SHP2 inhibitor, possess robust in vitro potency and in vivo efficacy in several preclinical tumor models carrying KrasG12C or EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1041.

Hexachlorophene, a selective SHP2 inhibitor, suppresses proliferation and metastasis of KRAS-mutant NSCLC cells by inhibiting RAS/MEK/ERK and PI3K/AKT signaling pathways

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations account for 35% of the genetic alterations in non-small cell lung cancer (NSCLC). The Src-homology region 2-containing protein tyrosine phosphatase 2 (SHP2), encoded by PTPN11, is closely involved in RAS downstream pathways and development of many tumors by affecting cell proliferation, differentiation, and immunity. Targeting SHP2 with small molecules may be a promising avenue for the treatment of KRAS-mutant (mut) NSCLC. Herein, hexachlorophene (HCP) was identified as a SHP2 inhibitor with an IC50 value of 5.63 ± 0.75 μM through screening of the FDA-approved drug library. HCP specifically inhibited SHP2 rather than other phosphatases. Molecular docking showed that HCP displayed an orientation favorable for nucleophilic attack in the catalytic domain of SHP2. HCP suppressed viability of multiple KRAS-mut and KRAS-wild type cells and induced senescence and apoptosis in KRAS-mut cells. Moreover, HCP reversed epithelial-mesenchymal transition to suppress metastasis in KRAS-mut cells, and inhibited the RAS/MEK/ERK and PI3K/AKT signaling pathways by suppression of SHP2 phosphorylation and formation SHP2/Grb2/Gab1/SOS1 complex. In summary, HCP can act as a specific SHP2 inhibitor to inhibit KRAS-mut NSCLC cell proliferation and metastasis and induce senescence through suppression of the RAF/MEK/ERK and PI3K/AKT pathways. HCP warrants further investigation as a new compound skeleton for the development of selective SHP2 inhibitors for the treatment of NSCLC.

Retinal Src homology region 2-containing protein tyrosine phosphatase 2 silencing alleviates diabetic retinopathy via suppressing inflammatory response and oxidative stress by regulating Yes-associated protein 1 activity.

Diabetic retinopathy (DR) is the prevalent microvascular complication of diabetes mellitus (DM), and it may lead to permanent blindness. The previous publication has indicated that both inflammatory response and oxidative stress are critical factors involved in DR progression, however, the accurate regulatory mechanism remains to be revealed. Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), a member of the protein tyrosine phosphatase family, was reported to play a role in diabetic nephropathy, whereas its function in DR was unknown and required further exploration. The level of phosphorylated, not the total, SHP2 increased in the retinas of rats with streptozotocin injection-induced DM. Further, the intravitreal injection of SHP2 shRNA lentivirus alleviated retinal pathological changes, and inhibited inflammatory response and oxidative stress, which were accompanied with Yes-associated protein 1 (YAP1) deactivation in DR rats. Additional co-immunoprecipitation results confirmed the interaction of SHP2 and YAP1. Collectively, our data preliminarily show that DR amelioration-induced by SHP2 inhibition in rats may attribute to the deactivation of YAP1 pathway.

Polyphyllin D Shows Anticancer Effect through a Selective Inhibition of Src Homology Region 2-Containing Protein Tyrosine Phosphatase-2 (SHP2).

Natural products have continued to offer tremendous opportunities for drug development, as they have long been used in traditional medicinal systems. SHP2 has served as an anticancer target. To identify novel SHP2 inhibitors with potential anticancer activity, we screened a library containing 658 natural products. Polyphyllin D was found to selectively inhibit SHP2 over SHP1, whereas two other identified compounds (echinocystic acid and oleanolic acid) demonstrated dual SHP1 and SHP2 inhibition. In a cell-based assay, polyphyllin D exhibited cytotoxicity in Jurkat cells, an acute lymphoma leukemia cell line, whereas the other two compounds were ineffective. Polyphyllin D also decreased the level of phosphorylated extracellular signal-regulated kinase (p-ERK), a proliferation marker in Jurkat cells. Furthermore, knockdown of protein tyrosine phosphatase (PTP)N6 (SHP1) or PTPN11 (SHP2) decreased p-ERK levels. However, concurrent knockdown of PTPN6 and PTPN11 in Jurkat cells recovered p-ERK levels. These results demonstrated that polyphyllin D has potential anticancer activity, which can be attributed to its selective inhibition of SHP2 over SHP1.

The BTLA and PD-1 signaling pathways independently regulate the proliferation and cytotoxicity of human peripheral blood γδ T cells.

BackgroundB‐ and T‐lymphocyte attenuator (BTLA) and programmed cell death‐1 (PD‐1) inhibit γδ T cell homeostasis and activation. This study aimed to determine whether BTLA and PD‐1 signaling pathways were convergent or independent in human peripheral blood γδ T cells. Herein we demonstrate that the signalings of BTLA and PD‐1 regulated proliferation and cytotoxicity of human γδ T cells, respectively.MethodsHuman peripheral blood γδ T cells were cultured with inactivated Jurkat cells in the presence of interleukin‐2 and zoledronate (Zol) for 14 days. Flow cytometry was performed to evaluate the phenotypes and functions of γδ T cells.ResultsThe proliferation of the γδ T cells was increased when PBMCs were cocultured with inactivated herpes virus entry mediator (HVEM)low Jurkat cells. The cytotoxicity of the expanded γδ T cells was not affected by coculture with inactivated HVEMlow Jurkat cells and was further increased in the presence of anti‐PD‐L1 mAb. These results suggest that the inactivation of the BTLA signaling pathway during expansion could help produce more γδ T cells without compromising γδ T cell function. The inhibition of BTLA or PD‐1 signaling repressed phosphorylation of the src homology region 2‐containing protein tyrosine phosphatase 2 and increased the phosphorylation of protein kinase B in γδ T cells. However, there were no synergistic or additive effects by a combination of BTLA and PD‐1 blockade.ConclusionThese results suggest that BTLA signaling is crucial in regulating γδ T cell proliferation and function and that the BTLA and PD‐1 signaling pathways act independently on the proliferation and cytotoxicity of human peripheral γδ T cells.

Therapeutic effect of the injectable thermosensitive hydrogel loaded with SHP099 on intervertebral disc degeneration

AimsIntervertebral disc (IVD) degeneration (IDD), a common musculoskeletal disease with limited self-healing ability, is challenging to treat. The development of innovative therapies to reverse IDD depends on the elucidation of its regulatory mechanisms. Therefore, the role of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) in the pathogenesis of IDD and the therapeutic effect of its small-molecule inhibitor, SHP099, were investigated. Materials and methodsThe expression of SHP2 by nucleus pulposus (NP) cells in IVD was investigated in vitro and in vivo, and its molecular mechanism in IDD was explored using transfection technology. Injectable N-isopropylacrylamide-based thermosensitive hydrogels were synthesized for SHP099 delivery. Key findingsSHP2 was highly expressed in degenerated IVDs, where its overexpression in NP cells inhibited the expression of Sry-related HMG box-9 (Sox9), leading to the decreased expression of key proteins (collagen II and aggrecan) and consequently to IDD. SHP099 reversed the degeneration of NP cells in vitro. Moreover, its administration in rats via the injectable thermosensitive hydrogel had a therapeutic effect on IDD. SignificanceOur results suggest that SHP2 is a key factor in IDD progression, and SHP099 inhibits both its expression and NP cell degeneration. Therefore, SHP099 delivery via injectable thermosensitive hydrogels is a potential treatment strategy for IDD.

Abstract 2173: CRISPR-Cas9 gene editing of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma

Abstract Early clinical trials have demonstrated the therapeutic potential of chimeric antigen receptor (CAR) T cells against glioblastoma (GBM), but also highlighted the obstacles that need to be addressed to improve their anti-tumor activity. GBM is highly immunosuppressive and thus necessitates endowing adoptively transferred CAR T cells with resistance mechanisms to achieve sustained treatment responses. This is, in part, due to the effect of immune-inhibitory receptors, including PD1, CTLA4, LAG3 and BTLA, that antagonize the tumor-directed T cells. A common mechanism of T cell inhibition involves the recruitment of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) to the immune synapse upon engagement of multiple co-inhibitory receptors. SHP2, in turn, de-phosphorylates key signaling proteins involved in T cell activation. We hypothesized that disrupting SHP2 expression will offset multiple co-inhibitory pathways, thereby improving the anti-tumor activity of CAR T cells. Using CRISPR/Cas9 gene editing, we disrupted the SHP2-encoding gene locus, PTPN11, in primary human T cells by generating and testing a library of single-guide RNA (sgRNA). We selected the top candidate that reproducibly reduced the SHP2 protein expression to <10%. A HER2-targeting second-generation CAR (HER2-CAR) was then expressed in SHP2 knock-out (SHP2KO) T cells via gamma retroviral vector transduction. SHP2KO CAR T cell manufacturing following a single round of stimulation was optimized to minimize ex vivo manipulation of the cells. The phenotypic profiles of SHP2KO and wild-type HER2-CAR T cells were comparable at baseline. Loss of SHP2 did not alter the CAR T cell viability, expansion or proliferative capacity in comparison to the wild-type CAR T cells. Mass cytometry (CyTOF)-based analysis of SHP2KO CAR T cells after encountering GBM cells demonstrated the preservation of the central memory (CCR7+CD45RA-) T cell subset. The majority of PD1+ SHP2KO CAR T cells co-expressed functional activation markers such as ICOS and CD107a. The PD1+ T cell subset was comprised of mostly T-betHi progenitor population and only a smaller percentage of EomesHi terminally exhausted population. Functionally, HER2-specific SHP2KO CAR T cells from different donors (n=4) exhibited improved anti-tumor activity that was sustained in long-term cytotoxicity assays. In an experimental orthotopic LN229-GBM mouse model, SHP2KO HER2-CAR T cells better controlled the growth of established xenografts and improved survival in comparison to wild-type HER2-CAR T cells. In conclusion, our results show that disrupting SHP2 expression using a CRISPR/Cas9 gene editing strategy could improve the CAR T cell function against GBM and warrants further clinical testing. Citation Format: Khaled Sanber, Zeid Nawas, Vita Salsman, Ahmed Gad, Pretty Rose Matthew, Daniel Landi, Ciaran Lee, Amel Sengal, Rikhia Chakraborty, Sujith Joseph, Ahmed Nabil, Meenakshi Hegde. CRISPR-Cas9 gene editing of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2173.

Modulation of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma

Background & Aim Early clinical trials have demonstrated the safety of chimeric antigen receptor (CAR) T cells targeting glioblastoma (GBM), however, their efficacy remains limited by multiple obstacles including the highly immunosuppressive tumor microenvironment. These adoptively transferred CAR T cells are susceptible to inhibition via the engagement of inhibitory receptors on their surface including PD1, CTLA4, LAG3 and BTLA. The recruitment of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) to the immune synapse upon engagement of these co-inhibitory receptors may represent a common mechanism of T cell inhibition. Upon its recruitment and activation, SHP2 dephosphorylates key stimulatory signaling molecules, thus limiting T cell activation. We hypothesized that disrupting SHP2 expression will simultaneously offset multiple co-inhibitory pathways, thereby improving the anti-tumor activity of CAR T cells. Methods, Results & Conclusion CRISPR/Cas9 technology was used to knockout (KO) SHP2 from human T cells. Retroviral vector transduction was used to express a clinically-utilized second generation CAR (with a CD28 signaling endodomain) targeting HER2. The phenotype of wild-type and edited CAR T cells was investigated using mass cytometry and confirmed with flow cytometry. Their function was tested in vitro using xcelligence (an electrical impedance-based long-term cytotoxicity assay) and in vivo in an orthotopic xenograft mouse model of GBM. Efficient SHP2 KO in human T cells was verified using western blotting. The Inference of CRISPR Efficiency (ICE) Assay confirmed efficient editing of the PTPN11 gene encoding SHP2. An anti-HER2 CAR was efficiently expressed in the SHP2KO T cells. SHP2 deletion did not significantly affect T cell expansion, proliferation or phenotypic profile. However, SHP2KO CAR T cells eliminated LN229 cells (a GBM cell line) in vitro more efficiently compared to wild-type CAR T cells. In vivo, SHP2KO CAR T cells showed better control of engrafted LN229 tumor cells and improved the survival of treated mice in comparison to wild-type CAR T cells. In conclusion, these results suggest that SHP2 may limit the function of CAR T cells and that SHP2 deletion may improve their anti-GBM activity.

Loss of Shp2 Rescues BDNF/TrkB Signaling and Contributes to Improved Retinal Ganglion Cell Neuroprotection.

Glaucoma is characterized by the loss of retinal ganglion cells (RGC), and accordingly the preservation of RGCs and their axons has recently attracted significant attention to improve therapeutic outcomes in the disease. Here, we report that Src homology region 2-containing protein tyrosine phosphatase 2 (Shp2) undergoes activation in the RGCs, in animal model of glaucoma as well as in the human glaucoma tissues and that Shp2 dephosphorylates tropomyosin receptor kinase B (TrkB) receptor, leading to reduced BDNF/TrkB neuroprotective survival signaling. This was elucidated by specifically modulating Shp2 expression in the RGCs invivo, using adeno-associated virus serotype 2 (AAV2) constructs. Shp2 upregulation promoted endoplasmic reticulum (ER) stress and apoptosis, along with functional and structural deficits in the inner retina. In contrast, loss of Shp2 decelerated the loss of RGCs, preserved their function, and suppressed ER stress and apoptosis in glaucoma. This report constitutes the first identification of Shp2-mediated TrkB regulatory mechanisms in the RGCs that can become a potential therapeutic target in both glaucoma and other neurodegenerative disorders.

Clinicopathological and prognostic significance of SHP2 and Hook1 expression in patients with thyroid carcinoma

Some thyroid carcinomas (TCs) have an aggressive biological behavior and poor prognosis, and lacking of effective molecular markers is still the main obstacle for clinical stratified diagnosis and treatment of TC. The aim of the study was to discover the clinicopathological and prognostic implications of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) and Hook microtubule tethering protein 1 (Hook1) expression in TC. The expression of SHP2 and Hook1 was detected by immunohistochemistry on tissue microarrays from 313 primary TCs who underwent surgery in January 2006 and January 2010 in Zhejiang Cancer Hospital. The χ2 test, Kaplan-Meier method, and Cox proportional-hazards regression models were used to analyze the associations between their expressions and clinicopathological features and prognosis. The expression rates of SHP2 and Hook1 in TC were 57.5% (180/313) and 22.0% (69/313), respectively. SHP2 was positively correlated with Hook1 in TC. SHP2 expression differed significantly by age, histologic variants, maximal tumor diameter, intrathyroidal dissemination, metastases, and disease stage (P < .05). Moreover, patients with high SHP2 expression had reduced risk for death of disease compared with those with low SHP2 expression (hazard ratio, 0.267; 95% confidence interval, 0.105-0.684; P = .006) in univariate analysis, but that multivariate analysis failed to suggest that SHP2 was an independent prognostic factor. Hook1 expression differed significantly by histologic variants, maximal tumor diameter, and intrathyroidal dissemination (P < .05). However, there was no significant correlation between Hook1 expression and outcome in TC (P > .05). Our results suggested that SHP2 may be a favorable indicator of prognosis in TC.

SHP2 negatively regulates HLA-ABC and PD-L1 expression via STAT1 phosphorylation in prostate cancer cells.

Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) is a ubiquitous protein tyrosine phosphatase that activates the signal transduction pathways of several growth factors and cytokines. In our study, SHP2 expression was very high in prostate cancer (PCa) cell lines, and the expression of phospho-signal transducer and activator of transcription 1 (p-STAT1) and STAT1 was very low. SHP2 knockdown upregulated the expression of p-STAT1 and downregulated phospho-extracellular signal regulated kinase (p-ERK). SHP2 depletion also increased the expression of human leukocyte antigen (HLA)-ABC and programmed death ligand 1 (PD-L1). When tumor cells were pretreated with Janus kinase 2 (JAK2) inhibitor, SHP2 depletion failed to induce HLA-ABC and PD-L1 expression. Furthermore, treating tumor cells with the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitor PD0325901 did not upregulate HLA-ABC and PD-L1. SHP2 depletion was associated with increased T-cell activation (CD25 MFI of CD8+) by coculture of allogeneic healthy donor peripheral blood monocytes (PBMC) with SHP2 siRNA pretreated PCa cell lines. These results show that SHP2 targeting upregulates HLA-ABC and PD-L1 expression via STAT1 phosphorylation in PCa cells and SHP2 depletion could increase T-cell activation.