Multiplexed 3D atlas of state transitions and immune interaction in colorectal cancer Jia-Ren Lin*, Shu Wang*, Shannon Coy*, Yu-An Chen, Clarence Yapp, Madison Tyler, Maulik K. Nariya, Cody N. Heiser, Ken S. Lau, Sandro Santagata†, and Peter K. Sorger† *These (first) authors contributed equally †These (senior) authors contributed equally Associated publication DOI: 10.1016/J.CELL.2022.12.028 Learn more: tissue-atlas.org/atlas-datasets/lin-wang-coy-2021/ ----- SUMMARY Advanced solid cancers are complex assemblies of tumor, immune, and stromal cells characterized by high intratumoral variation. We use highly multiplexed tissue imaging, 3D reconstruction, spatial statistics, and machine learning to identify cell types and states underlying morphological features of known diagnostic and prognostic significance in colorectal cancer. Quantitation of these features in high-plex marker space reveals recurrent transitions from one tumor morphology to the next, some of which are coincident with long-range gradients in the expression of oncogenes and epigenetic regulators. At the tumor invasive margin, where tumor, normal, and immune cells compete, T-cell suppression involves multiple cell types and 3D imaging shows that seemingly localized 2D features such as tertiary lymphoid structures are commonly interconnected and have graded molecular properties. Thus, while cancer genetics emphasizes the importance of discrete changes in tumor state, whole-specimen imaging reveals large-scale morphological and molecular gradients analogous to those in developing tissues. ----- VIEW IMAGE DATA ONLINE Some data is available as narrated data explorations (with text and audio narration) for anonymous on-line browsing using MINERVA software (Rashid et al., 2022), which allows users to pan and zoom through the images without requiring any software installation. To view the Minerva stories, please visit tissue-atlas.org/atlas-datasets/lin-wang-coy-2021/#data-explorations. ---- ACCESS THE DATA All images at full resolution, derived image data (e.g., segmentation masks), and cell count tables have been released via the NCI-sponsored repository for Human Tumor Atlas Network (HTAN; humantumoratlas.org/explore). The dataset, consist of 47 CRC1 images (2.1 TB) and CRC2-17 images (4.4 TB), is available through Amazon Web Services S3 at the following locations:
Meningiomas are the most common primary tumor of the central nervous system, with ~28,000 new diagnoses annually in the United States1. Currently, there are no approved systemic therapies for meningiomas that recur following local treatment: chemotherapy and hormonal agents have demonstrated minimal benefit in numerous clinical trials2–4.
Meningioma comprises a heterogeneous group of neoplasms driven by mutations in a wide array of tumor suppressor genes and oncogenes5–17. Characterization of these mutations has revealed opportunities for rational therapy18–20. For example, a durable therapeutic response has been reported for a metastatic AKT1(E17K)-mutant meningioma treated with a pan-AKT inhibitor.21
Studies also suggest the potential for treating meningioma with immune checkpoint modulators22–24: programmed death receptor 1 ligand (PD-L1) is expressed in a subset of meningiomas and the tumor microenvironment is immunosuppressive22–28. Higher-grade meningiomas also harbor mutations predicted to generate neoantigens, which may foster susceptibility to immunotherapies29.
Based on these data, we initiated a phase II study of nivolumab, a humanized IgG4 PD-1 blocking monoclonal antibody, in patients with higher-grade meningiomas that recurred following surgery and radiotherapy ({type:clinical-trial,attrs:{text:NCT02648997,term_id:NCT02648997}}NCT02648997). We report here a patient with an atypical meningioma that was not controlled by repeated surgery and radiation but which was highly response to nivolumab.
ABSTRACT Cutaneous melanoma is a highly immunogenic malignancy, surgically curable at early stages, but life- threatening when metastatic. Here we integrate high-plex imaging, 3D high-resolution microscopy, and spatially-resolved micro-region transcriptomics to study immune evasion and immunoediting in primary melanoma. We find that recurrent cellular neighborhoods involving tumor, immune, and stromal cells change significantly along a progression axis involving precursor states, melanoma in situ, and invasive tumor. Hallmarks of immunosuppression are already detectable in precursor regions. When tumors become locally invasive, a consolidated and spatially restricted suppressive environment forms along the tumor-stromal boundary. This environment is established by cytokine gradients that promote expression of MHC-II and IDO1, and by PD1-PDL1 mediated cell contacts involving macrophages, dendritic cells, and T cells. A few millimeters away, cytotoxic T cells synapse with melanoma cells in fields of tumor regression. Thus, invasion and immunoediting can co-exist within a few millimeters of each other in a single specimen. STATEMENT OF SIGNIFICANCE The reorganization of the tumor ecosystem in primary melanoma is an excellent setting in which to study immunoediting and immune evasion. Guided by classical histopathology, spatial profiling of proteins and mRNA reveals recurrent morphological and molecular features of tumor evolution that involve localized paracrine cytokine signaling and direct cell-cell contact.
ABSTRACT In this data descriptor, we document a dataset of multiplexed immunofluorescence images and derived single-cell measurements of immune lineage and other markers in formaldehyde-fixed and paraffin-embedded (FFPE) human tonsil and lung cancer tissue. We used tissue cyclic immunofluorescence (t-CyCIF) to generate fluorescence images which we artifact corrected using the BaSiC tool, stitched and registered using the ASHLAR algorithm, and segmented using ilastik software and MATLAB. We extracted single-cell features from these images using HistoCAT software. The resulting dataset can be visualized using image browsers and analyzed using high-dimensional, single-cell methods. This dataset is a valuable resource for biological discovery of the immune system in normal and diseased states as well as for the development of multiplexed image analysis and viewing tools. METADATA SUMMARY
Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response. Introduction SHP2 is a non-receptor tyrosine phosphatase that is essential for mammalian development (Saxton et al., 1997). In humans, germline mutations of PTPN11 cause the developmental disorders Noonan and LEOPARD syndromes (Tartaglia and Gelb, 2005). Somatic PTPN11 activating mutations are also found frequently in juvenile myelomonocytic leukemia and, to a lesser extent, in solid tumors (Bentires-Alj et al., 2004). Numerous studies have shown that SHP2 acts as a positive effector of receptor tyrosine kinase (RTK) signaling (Bennett et al., 1994; Easton et al., 2006; Tang et al., 1995). SHP2 facilitates the full induction of Ras-dependent extracellular signal-regulated kinase (ERK) proteins following stimulation of cells with epidermal growth factor (EGF) or other receptor tyrosine kinase ligands. SHP2 also serves as a positive regulator of numerous other signaling systems, including cytokine (Xu and Qu, 2008), programmed cell death (Yokosuka et al., 2012), and immune checkpoint pathways (Gavrieli et al., 2003). The SHP2 protein consists of two SH2 domains (N-SH2, C-SH2), followed by a phosphatase (PTP) domain, with a natively disordered C-terminal tail that contains tyrosine residues known to become phosphorylated (Bennett et al., 1994; Keegan and Cooper, 1996). X-ray structures of the SHP2 protein core, encompassing the two SH2 domains and the PTP domain, show that the wild-type protein normally adopts an autoinhibited conformation in which the N-SH2 domain occludes the active site of the PTP domain (Hof et al., 1998). Activation of the enzyme requires disengagement of the N-SH2 domain from the PTP domain and subsequent recruitment of SHP2 to phosphotyrosine docking sites on substrate proteins. Recruitment can rely on the N-SH2 domain, C-SH2 domain, or both domains. Most oncogenic mutations of SHP2 lie at the interface between the N-SH2 and PTP domains, thereby increasing the propensity of the enzyme to adopt an open, active conformation (LaRochelle et al., 2018; LaRochelle et al., 2016). Numerous studies in a range of cell types have identified phosphotyrosine (pY) residues on adaptor proteins (e.g. GAB1 and GAB2 [Arnaud et al., 2004; Cunnick et al., 2001]) or on RTKs themselves (e.g. PDGFβR [Rönnstrand et al., 1999]) thought to recruit SHP2 to sites of active signaling following RTK activation (such sites include multiprotein complexes formed on RTK intracellular tails and IRS-1 adapters). Genetic and mutational studies have implicated specific pY residues on RTKs as putative SHP2 substrates (Agazie and Hayman, 2003; Bunda et al., 2015; Klinghoffer and Kazlauskas, 1995). The availability of a potent and highly selective allosteric inhibitor of SHP2 now allows us to capture the whole proteome dynamics of the SHP2 dependence of the EGF response. Thus, we use here the allosteric inhibitor SHP099 (Garcia Fortanet et al., 2016) to study the role of SHP2 on phosphoprotein dynamics at the whole proteome level following EGF stimulation of a breast cancer cell line carrying an EGFR amplification. Global analysis of time-resolved changes in pY abundance at over 400 tyrosine residues reveals several distinguishable response signatures following SHP099 exposure and washout. Putative substrate sites fall into two classes, with increased pY abundance at early and/or late timepoints, respectively. Proteins in this category include previously unidentified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ. Yet another class, which contains the largest number of dynamic pY sites, exhibits decreased pY abundance. These latter sites are enriched on proteins with two nearby pY residues, some of which are directly protected from dephosphorylation by the SH2 domains of SHP2. These data emphasize the two distinct and interrelated biochemical activities of SHP2 – dephosphorylation and phosphosite protection – and identify specific sites relevant to the activity of SHP099 and similar molecules such as TNO155 as therapeutic lead compounds (LaMarche et al., 2020). Results Dynamic regulation of the EGF-responsive phosphoproteome by SHP2 We investigated the role of SHP2 in the responsiveness to EGF by using the EGFR-amplified cell line MDA-MB-468, derived from a patient with triple-negative breast cancer. To identify timepoints for in-depth proteomic analysis, we monitored the influence of SHP2 on the response to EGF stimulation using ERK1/2 phosphorylation as a readout. Cell extracts were prepared at a series of timepoints from three treatment conditions: (1) following pretreatment with dimethyl sulfoxide (DMSO) and then stimulation with 10 nM EGF, (2) following pretreatment with the SHP2 inhibitor SHP099 for 2 hr and then stimulation with EGF, and (3) following pretreatment with SHP099 and then stimulation with EGF for 10 min, after which drug was washed out and medium containing EGF replenished. Because the response of the EGF receptor to EGF stimulation occurs in the 1–2 min time period (Jadwin et al., 2016), we focused attention on the 5–30 min time window to ensure the observation of SHP2-dependent events downstream of EGF receptor stimulation. Immunoblot analysis revealed the expected effect of EGF stimulation: a dramatic increase in phospho-ERK1/2 (p-ERK1/2) levels followed by a decline toward basal levels by 30 min. The induction of p-ERK1/2 was greatly attenuated by pretreatment of cells with 10 μM SHP099. SHP099 washout in the continued presence of EGF (condition 3) revealed p-ERK1/2 induction with a kinetic profile similar to that of EGF stimulation in the absence of drug (condition 1), showing that SHP099 inhibition of SHP2 is rapidly reversible in MDA-MB-468 cells (Figure 1A, Figure 1—figure supplement 1A). Figure 1 with 3 supplements see all Download asset Open asset Phosphoproteomic studies in MDA-MB-468 cells. (A) Left: Western blot showing the phosphoERK1/2 abundance after EGF stimulation alone, EGF stimulation in the presence of SHP099, and EGF stimulation in the presence of SHP099, followed by drug washout 10 min after EGF stimulation. Right: quantification of the Western blot data, calculated from the ratio of [pERK1/2]/[ERK1/2] band intensities. (B) Schematic illustration of treatment conditions and mass spectrometry workflow. Western blot and phosphoproteomic data are both representative of two independent biological replicates (n = 2). (C) Venn diagram showing the overlap of phosphopeptides identified in biological replicates 1 and 2. Figure 1—source data 1 Table showing Phospho-ERK1/2 levels normalized to total-ERK1/2 levels (as quantified by measuring band intensities using ImageJ) in MDA-MB-468 cells treated with SHP099 and EGF as indicated. https://cdn.elifesciences.org/articles/64251/elife-64251-fig1-data1-v2.xlsx Download elife-64251-fig1-data1-v2.xlsx To obtain an in-depth view of the effects of SHP2 inhibition on EGFR signaling, we performed quantitative phosphoproteomics, monitoring dynamic changes in pY abundance as a function of time under DMSO, SHP099, and washout conditions (Figure 1B). Tryptic peptides from DMSO, SHP099, and SHP099-washout groups were enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC) prior to labeling with 11-plex isobaric tandem mass tags (TMT). Tyrosine phosphorylated (pY) peptides were then immunoprecitiated from the TMT-labeled, pooled samples using an anti-pY antibody, and the recovered pY-containing peptides were analyzed by LC-MS3 mass spectrometry (Supplementary files 1 and 2 and Figure 1—figure supplement 1B,C). Principal component analysis revealed a high degree of consistency between matched samples from the two biological replicates (Figure 1—figure supplement 2), which yielded relative quantification for several hundred pY-containing peptides, with good concordance between biological replicates (Figure 1C and Supplementary files 1 and 2). The Western blot (Figure 1A) and mass spectrometry data for the dynamics of pY204 of ERK1 (Figure 1—figure supplement 1B) and pY187 of ERK2 (Supplementary files 1 and 2) show the same pattern of response, confirming that key pY marks associated with EGF-induced signaling events were accurately determined in the TMT experiment. The phosphoproteomic data revealed a spectrum of dependencies on EGF and SHP2 (Figure 1—figure supplement 3A). These were classified on the basis of both direction of change (increase or decrease) and timing. EGF dependencies were established based on responses to EGF addition in the absence of drug and were classified into five categories: fast, medium, or slow increases, neutral (no significant change), or decrease. The dependence of each phosphosite on SHP2 activity was established by assessing how phosphorylation levels were altered when SHP2 was inhibited: negative (more phosphorylation with inhibition; Figure 1—figure supplement 3A, top two rows), positive (less phosphorylation with inhibition; Figure 1—figure supplement 3A, bottom two rows), or neutral (Figure 1—figure supplement 3A, middle row). Positive and negative dependencies were further subdivided based on whether the change was present before EGF stimulation (Figure 1—figure supplement 3A, first and fourth rows) or only after (Figure 1—figure supplement 3A, second and fifth rows). As anticipated, SHP2 inhibition resulted in increased abundance of pY marks at a number of sites (Figure 1—figure supplement 3A, top two rows), the expected effect of blocking a tyrosine phosphatase. Remarkably, however, we found an even larger family of pY sites that showed a decreased abundance when SHP2 is inhibited even at early time points (Figure 1—figure supplement 3A, bottom two rows), indicating that SHP2 inhibition had direct and/or indirect activities other than direct dephosphorylation of substrates. Additionally, SHP2 exhibited EGFR-independent regulation of a number of pY sites: a subset of sites was regulated (either positively or negatively) by SHP2 prior to EGF addition (Figure 1—figure supplement 3A, first and fourth rows), showing that SHP2 had a role in shaping the basal signaling state of these cells. Motif analysis of phosphosites associated with different classes of SHP2 responsiveness did not reveal enrichment for specific motifs (residues flanking the modified tyrosine residue; Figure 1—figure supplement 3B). Remarkably, sites from different regulatory classes could even occur together in the same protein (see below), highlighting the importance of considering specific sites of phosphorylation when evaluating the impact of a phosphatase on a protein substrate. Hierarchical clustering was performed to identify groups of pY sites with similar kinetics in DMSO, SHP099, and washout conditions. Among the six kinetic profiles that emerged (Figure 2—figure supplement 1), we highlight three clusters of sites implicated in EGFR signaling and that display distinct responses to the drug (Figure 2A). The first class of responses, which includes the regulatory subunits of PI3K, occludin, Syntaxin 4 (Figure 2B), the adapter proteins CRK and GRB2, and ARHGAP35 (also known as p190RhoGAP), among other proteins, shows quantitatively increased tyrosine phosphorylation in the presence of SHP099 that rapidly disappears upon drug washout (Figure 2A), as predicted for a SHP2 substrate. The second class of sites accumulates pY marks slowly under SHP099 inhibition (at 30 min after EGF treatment), and not when SHP099 is omitted or washed out. This pattern, observed for pY sites on CBL E3 ligase, RAB10, hnRNPs, PLCγ proteins, and CAPRIN1 (Figure 2C), also matches the response predicted for a SHP2 substrate, but with a time delay, suggesting that the action of SHP2 on these proteins might require an intervening event, such as relocalization (e.g. after endocytosis of active EGFR signaling complexes), or alternatively, that the effect is indirect. The large number of potential substrates identified in this work suggests that SHP2 may catalyze dephosphorylation of many different proteins on different time scales. Figure 2 with 2 supplements see all Download asset Open asset Quantitative phosphoproteomics reveals distinct dynamic responses to SHP2 inhibition. (A) Heatmap showing three classes of dynamic response in which inhibition of SHP2 modulates the effect of EGF stimulation on pY abundance. Specific examples from each cluster are indicated to the left of the heatmap. (B) Plot of pY abundance as a function of treatment condition for Y251 of STX4, an example of an early substrate-like response pattern to SHP2 inhibition. (C) Plot of pY abundance as a function of treatment condition for Y818 of PLCG2, an example of a late substrate-like response pattern to SHP2 inhibition. (D) Plot of pY abundance as a function of treatment condition for Y1100 of ARHGEF5, an example of a site where the abundance of the mark decreases when SHP2 is inhibited, and rebounds after compound washout. Figure 2—source data 1 Table showing TMT relative abundance values for Y251 of STX4. https://cdn.elifesciences.org/articles/64251/elife-64251-fig2-data1-v2.xlsx Download elife-64251-fig2-data1-v2.xlsx Figure 2—source data 2 Table showing TMT relative abundance values for Y78 of CAPRIN1. https://cdn.elifesciences.org/articles/64251/elife-64251-fig2-data2-v2.xlsx Download elife-64251-fig2-data2-v2.xlsx Figure 2—source data 3 Table showing TMT relative abundance values for Y1100 of ARHGEF5. https://cdn.elifesciences.org/articles/64251/elife-64251-fig2-data3-v2.xlsx Download elife-64251-fig2-data3-v2.xlsx The third pattern of response observed is one in which accumulation of pY in response to EGF depends on the release of SHP2 from inhibition by SHP099. Examples of sites that fall into this category include Y1100 of ARHGEF5 (Figure 2D), Y659 of GAB1, Y643 of GAB2, as well as Y546 and Y584 of SHP2 itself. Functional classification of EGF-responsive pY sites (by GSEA and Reactome) differentially regulated by SHP099 (Figure 2—figure supplement 2A–C) reveals enrichment of six major cellular processes (Figure 2—figure supplement 2D). As expected, proteins implicated in MAPK and PI3K signaling, including EGFR and SHP2 itself, contain numerous EGF-responsive phosphosites differentially regulated by SHP099 treatment. The largest functional group includes proteins implicated in adhesion and migration, including tight junction proteins, catenins, Rho-GEF, and Rho-GAP proteins. These findings, along with data on the abundance of cytoskeletal proteins differentially affected by SHP099 (Figure 2—figure supplement 2D), are consistent with earlier results, suggesting that SHP2 promotes migration in MDA-MB-468 cells by regulating EGF-induced lamellipodia persistence (Hartman et al., 2013). Proteins implicated in transcription comprise another functional class with a number of differentially regulated phosphosites (Figure 2—figure supplement 2D). Two additional functional categories enriched in the analysis comprise endocytosis and mRNA processing, neither of which have previously been linked to SHP2 activity. Most of the sites in these two categories display a substrate-like response with SHP099 treatment. In addition, SHP099 also affected phosphorylation of CBL and CBLB, E3 ligases that ubiquitinate EGFR and facilitate its recruitment to clathrin-coated pits for endocytosis. Together, these results highlight the diversity of influence of SHP2, both in the function of proteins that it affects and in the manner in which it affects their phosphorylation patterns. Identification of new substrates of SHP2 Among proteins that show a substrate-like pattern, only one of the phosphosites observed has previously been reported to be dephosphorylated by SHP2 (GRB2 pY209; ARHGAP35 has also been reported to be a substrate, but its pY site has not been reported) (Bregeon et al., 2009; Chardin et al., 1993). Therefore, we carried out follow-up studies to determine whether any of the proteins with sites newly identified in this proteome-wide analysis are indeed directly dephosphorylated by SHP2. Of the potential substrates identified from the mass spectrometry data, we identified 20 proteins with sites that (1) showed an increase upon EGF stimulation, (2) showed an increase of greater than twofold in the presence of SHP099 when compared to the DMSO control for at least one timepoint, (3) were not previously reported as SHP2 substrates, and (4) had commercial antibodies available for immunoprecipitation studies. Of these twenty proteins screened (Supplementary file 3), three that were reliably immunoprecipiated at endogenous abundance showed phosphosite enrichment after EGF stimulation in the presence of SHP099 as judged by Western blot with an anti-pY antibody. These three proteins are occludin, PLCγ2, and ARHGAGP35 (Figure 3A–C). Immunoprecipitation of pY-modified proteins with an anti-pY antibody in EGF-stimulated cells treated with SHP099 likewise showed that PLCγ2, occludin, and GRB2 accumulate pY in the presence of SHP099, as judged by Western blot (Figure 3—figure supplement 1). Because it was not possible to immunoprecipitate ARHGAP35 with either the anti-pY antibody or an antibody that specifically reognizes the pY1105 phosphosite, we immunoblotted anti-ARHGAP35 immunoprecipitates with the anti-pY1105 antibody to confirm that accumulation of this specific mark occurs when SHP2 is inhibited by SHP099 (Figure 3D,E). In addition, enzyme assays using purified SHP2 and the pY-containing peptides that contain the SHP099-enriched phsophosites identified in the proteomic studies confirm that SHP2 can robustly remove the phosphate mark from pY209 of GRB2, pY443 of occludin, pY1105 of ARHGAP35, and pY818 of PLCγ2, whereas neither a control peptide containing pY708 from CSF-1R nor a Class III peptide from TMEM134 that shows a protection-type pattern are robust SHP2 substrates (Figure 3F). Together, these data suggest that ARHGAP35, PLCγ2, and occludin are newly identified substrates for SHP2 in EGF-stimulated MDA-MB-468 cells. Figure 3 with 1 supplement see all Download asset Open asset Evaluation of occludin, ARHGAP35, and PLCγ2 as SHP2 substrates. MDA-MB-468 cells pre-treated with DMSO carrier or SHP099 (10 µM) for 2 hr were mock treated or stimulated with EGF (10 nM) for 10 min (A) or 30 min (B, C, and E). Total cell lysates were immunoprecipitated with occludin (A), PLCγ2 (B), or ARHGAP35 (C, E) antibodies and the eluted samples were subjected to Western blotting with the phosphotyrosine antibody pY1000 (A–C), pY1105-ARHGAP35 (E), and anti-protein antibodies as indicated (A–C, E). Input samples represent 1% of total cell lysate. Non-specific IgG was used as a negative control. (D) Plot of pY abundance as a function of treatment condition for Y1105 of ARHGAP35. (F) Dephosphorylation activity of full-length wild-type SHP2, activated with 6 µM bisphosphorylated IRS-1 peptide [SLNY(p)IDLDLVKdPEG8-LSTY(p)ASINFQK], toward synthetic phosphopeptides (OCLN_pY443, PLCG2_pY818, GRB2_pY209, ARHGAP35_1105, TMEM134_pY57, CSF-1R_pY708). Immunoprecipitation – Western blot assays are representative of at least two independent biological replicates. Figure 3—source data 1 Table showing TMT relative abundance values for Y1105 of ARHGAP35. https://cdn.elifesciences.org/articles/64251/elife-64251-fig3-data1-v2.xlsx Download elife-64251-fig3-data1-v2.xlsx Figure 3—source data 2 Table showing micromoles of phosphate released per minute for all phosphopeptides tested for dephosphorylation by IRS1-activated wild-type SHP2. https://cdn.elifesciences.org/articles/64251/elife-64251-fig3-data2-v2.xlsx Download elife-64251-fig3-data2-v2.xlsx Allosteric inhibition of SHP2 results in reduced pY abundance at its interaction motifs SHP099 allosterically stabilizes the autoinhibited conformation of SHP2 (Chen et al., 2016), thereby not only inhibiting the catalytic activity of the enzyme, but also suppressing binding of its two SH2 domains to pY-containing motifs. Because the third pattern of response shows accumulation of pY after SHP2 is released from inhibition, we performed a dynamic analysis of whether SHP2 directly protects sites in this class from dephosphorylation and whether this protection is lost upon SHP099 binding by Western blot. Y659 of GAB1 and Y643 of GAB2 both show dramatic reductions in pY abundance upon SHP099 treatment and recover their pY marks upon compound washout (Figure 4), as judged by the phosphoproteomic data and confirmed across an extended time course by Western blot. These data are fully consistent with prior work indicating that the SH2 domains of SHP2 can bind to the bisphosphotyrosine-containing motifs of GAB1 (pY627/pY659) and GAB2 (pY614/pY643) to relieve SHP2 autoinhibition (Arnaud et al., 2004; Cunnick et al., 2001). Figure 4 with 1 supplement see all Download asset Open asset SHP2 inhibition results in reduced pY abundance at its interaction motifs. (A, B) TMT signal-to-noise intensities of GAB1 pY659 (A) and GAB2 pY643 (B) peptides showing dynamic changes in phosphorylation under DMSO- (solid line) and SHP099-treated (dotted line) conditions. (C) MDA-MB-468 cells pre-treated with DMSO carrier or SHP099 (10 µM) for 2 hr were mock treated or stimulated with EGF (10 nM). Total GAB1, total GAB2, GAB1 pY659, and GAB2 pY643 were analyzed by Western blot with both anti-protein and phosphospecific antibodies as a function of time after EGF addition. Western blot results are representative of at least two independent biological replicates. Figure 4—source data 1 Table showing TMT relative abundance values for Y659 of GAB1. https://cdn.elifesciences.org/articles/64251/elife-64251-fig4-data1-v2.xlsx Download elife-64251-fig4-data1-v2.xlsx Figure 4—source data 2 Table showing TMT relative abundance values for Y643 of GAB2. https://cdn.elifesciences.org/articles/64251/elife-64251-fig4-data2-v2.xlsx Download elife-64251-fig4-data2-v2.xlsx To determine whether pY sites of GAB1 and GAB2 exhibit the same pattern of response in other cellular contexts, we also treated other EGFR-driven cancer cell lines with SHP099. KYSE520, an EGFR-amplified esophageal cancer cell line, showed loss of GAB1 Y659 phosphorylation with SHP099 treatment under conditions of EGF stimulation (Figure 4—figure supplement 1A). H1975 cells, which carry an EGFR activating mutation, displayed high levels of constitutive GAB2-Y643 phosphorylation. SHP099 treatment eliminated phosphorylation of this site, and washout of the drug restores the mark within 5 min (Figure 4—figure supplement 1B). In addition, we tested whether deletion of the SHP2 gene recapitulated the effects of chemical inhibition on the phosphorylation of Y659 of GAB1. For these studies, we used a PTPN11 knockout U2OS cell line prepared by CRISPR/Cas9 mediated genome editing (LaRochelle et al., 2018). When stimulated with EGF, SHP2-null U2OS cells showed a reduction in GAB1 pY659 levels when compared to parental cells, and reintroduction of SHP2 fully rescued the level of accumulated pY659 (Figure 4—figure supplement 1C). Stimulation of U2OS cells with PDGFββ also showed an induction of the GAB1 pY659 mark that is lost upon treatment with SHP099 (Figure 4—figure supplement 1D). Similarly, stimulation of the T cell receptor on Jurkat cells with an anti-CD3 antibody induced GAB1 and GAB2 phosphorylation, and this induction was attenuated by SHP099 treatment (Figure 4—figure supplement 1E). These findings show that the effect of SHP2 on the abundance of GAB1 and GAB2 pY marks is broadly shared among a range of growth factor and antigen receptor signaling systems. To determine how inhibition of SHP2 reduced pY abundance at these sites, we generated a set of site-specific SHP2 mutants and asked whether they could rescue GAB1-pY659 phosphorylation in U2OS PTPN11 null cells. We constructed point mutations that abolish autoinhibition (E76K), eliminate catalytic activity (C459E), or disable both autoinhibition and catalytic activity (E76K/C459E). We also created protein truncations that contain only the catalytic domain (PTP) or only the SH2 domains (SH2) (Figure 5A). When stimulated with EGF, C459E, E76K, and the E76K/C459E double mutant, all restored GAB1-pY659 phosphorylation to a level similar to that of cells rescued with wild-type SHP2 (Figure 5B). When the SH2 or PTP constructs were introduced into U2OS cells lacking endogenous SHP2, the PTP fragment failed to rescue, whereas the SH2 fragment was as active as wild-type SHP2 (Figure 5C). Figure 5 with 3 supplements see all Download asset Open asset The SH2 domains of SHP2, but not its PTP domain, protect specific GAB1 and GAB2 pY marks. (A) Schematic representation of wild-type, point mutants, and deletion constructs of SHP2 tested. (B, C) Parental-, SHP2 knockout-, or SHP2 knockout U2OS cells stably expressing various SHP2 ‘rescue’ constructs were cultured with or without EGF stimulation (10 nM) for 10 min. Cells were lysed and subjected to Western blotting using anti-pY659-GAB1 and anti-GAB1 antibodies. (D) MDA-MB-468 cells stably expressing wild-type, PTP, or SH2 domains pre-treated with SHP099 were stimulated with EGF and immunoblotted after lysis using the specified antibodies. Western blot results are representative of at least n = 2 independent biological replicates. Since MDA-MB-468 cells cannot survive without SHP2 (see also the gene essentiality data from avana_public_18Q2 library, Project Achilles; Meyers et al., 2017), we stably expressed the SH2 or PTP fragments in parental MDA-MB-468 cells and chemically inhibited endogenous SHP2. These cell lines were stimulated with EGF and probed for phospho-GAB1 and phospho-GAB2. As expected, SHP099 treatment significantly reduced the abundance of pY659 on GAB1, as well as that of pY643 on GAB2. GAB1 pY627, which has been reported to bind to the N-SH2 domain of SHP2 (Cunnick et al., 2001), also showed reduced phosphorylation. Consistent with the results from U2OS cells, the SH2 fragment, but not the PTP fragment was active in increasing pY abundance at these positions of GAB1 and GAB2 (Figure 5D). Indeed, the SH2 fragment stabilizes these GAB1 and GAB2 phospho marks even in the absence of EGF stimulation (Figure 5D), consistent with a dominant protective function that is independent of the catalytic activity of the enzyme. In addition, the SHP099-resistant variant of SHP2 (T253M/Q257L) rescues protection of the GAB1 and GAB2 pY sites (Figure 6A,B), and point mutation of the key arginine residue of each SH2 domain (R32M of the N-SH2 domain or R138M of the C-SH2 domain) suppresses phosphosite protection of these sites, as does the R32M/R138M double mutant (Figure 6C,D). Together, these findings show that the catalytic activity of SHP2 is not required for the protection of the pY659 and pY643 marks on GAB1 and GAB2, respectively, and argue that the presence of tandem SH2 domains in SHP2 is needed to bind and protect pYs on GAB1 and GAB2 from phosphatase-mediated dephosphorylation. Figure 6 Download asset Open asset Effects of SHP099-resistance and pY binding-site mutations on pY abundance at protected sites on GAB1 and GAB2. (A) Schematic illustrating wild-type and SHP099-resistant forms of SHP2. (B) Parental MDA-MB-468 cells or MDA-MB-468 cells stably expressing wild-type or T253M/Q257L SHP2 proteins were pre-treated with SHP099 (10 µM). The cells were then mock treated or stimulated with EGF (10 nM) for 10 min and immunoblotted after lysis using the specified antibodies. (C) Schematic illustrating wild-type and mutated forms of SH2-only tandem fragments of SHP2. (D) Parental MDA-MB-468 cells or MDA-MB-468 cells stably expressing wild-type or mutated tandem SH2 fragments of SHP2 (SH2) as indicated (R32M, R138M, and R32M/R138M, denoted DM here) were pre-treated with SHP099 (10 µM). The cells were then mock treated or stimulated with EGF (10 nM) for 10 min and immunoblotted after lysis using the specified antibodies. Western blot results are representative of at least n = 2 independent biological replicates. To assess whether this protection mechanism applies to other SHP2 interacting proteins that show reduced pY abundance in the presence of SHP099, we studied MPZL1, a cell surface receptor involved in extracellular matrix-induced signaling (Beigbeder et al., 2017; Zhao et al., 2002). MPZL1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when doubly phosphorylated at Y241 and Y263, can bind the tandem SH2 domains of SHP2 (Zhao and Zhao, 2000). Our phosphoproteomic data show that b
Abstract High-Grade Serous Ovarian Cancer (HGSOC) originates from fallopian tube (FT) precursors. However, the molecular changes that occur as precancerous lesions progress to HGSOC are not well understood. To address this, we integrated high-plex imaging and spatial transcriptomics to analyze human tissue samples at different stages of HGSOC development, including p53 signatures, serous tubal intraepithelial carcinomas (STIC), and invasive HGSOC. Our findings reveal immune modulating mechanisms within precursor epithelium, characterized by chromosomal instability, persistent interferon (IFN) signaling, and dysregulated innate and adaptive immunity. FT precursors display elevated expression of MHC-class I, including HLA-E, and IFN-stimulated genes, typically linked to later-stage tumorigenesis. These molecular alterations coincide with progressive shifts in the tumor microenvironment, transitioning from immune surveillance in early STICs to immune suppression in advanced STICs and cancer. These insights identify potential biomarkers and therapeutic targets for HGSOC interception and clarify the molecular transitions from precancer to cancer.
SUMMARY Anti-cancer drugs commonly target signal transduction proteins activated by mutation. In patients with BRAF V600E melanoma, small molecule RAF and MEK kinase inhibitors cause dramatic but often transient tumor regression. Emerging evidence suggests that cancer cells adapting by non-genetic mechanisms constitute a reservoir for the development of drug-resistant tumors. Here, we show that few hours after exposure to RAF/MEK inhibitors, BRAF V600E melanomas undergo adaptive changes involving disruption of negative feedback and sporadic pulsatile reactivation of the MAPK pathway, so that MAPK activity is transiently high enough in some cells to drive proliferation. Quantitative proteomics and computational modeling show that pulsatile MAPK reactivation is possible due to the co-existence in cells of two MAPK cascades: one driven by BRAF V600E that is drug-sensitive and a second driven by receptors that is drug-resistant. Paradoxically, this may account both for the frequent emergence of drug resistance and for the tolerability of RAF/MEK therapy in patients.
