KT-474 is a first-in-class IRAK4 heterobifunctional degrader that utilizes cereblon (CRBN) for E3 ligase recruitment and was rationally designed to be devoid of immunomodulatory imide drug (IMiD)-related neosubstrate degradation. Like KT-474, most degraders in clinical trials to date rely on CRBN for E3 ligase recruitment to harness the ubiquitin-proteasome system to selectively degrade disease-associated proteins. Structural similarities of the CRBN-binding portion of these degraders to IMiDs (e.g., thalidomide) have raised safety concerns due to potential degradation of CRBN neosubstrates implicated in teratogenicity, such as SALL4. To address this theoretical concern, the potential of KT-474 to degrade CRBN neosubstrates in vitro and cause developmental toxicity in vivo was evaluated. Proteomic analyses across 3 human cell systems (peripheral blood mononuclear cells [PBMCs], induced pluripotent stem cells, and SK-N-DZ cells) demonstrated that KT-474 selectively degraded IRAK4 without affecting SALL4 or other detected CRBN neosubstrates. In embryo-fetal development studies, no KT-474-related malformations or embryo-fetal toxicity were observed in rats or rabbits at the highest doses tested. Associated exposures (AUC) provided 23- to 9-fold multiples, respectively, over exposures at the clinical dose of KT-474 associated with robust degradation of IRAK4 and early signals of efficacy. Deep IRAK4 degradation by KT-474 in primary rat cells, rabbit PBMCs, and a range of tissues provides confidence in the appropriateness of the animal species tested. Taken together, these data clearly differentiate KT-474 from IMiDs, support that CRBN-mediated teratogenicity seen with IMiD drugs is neosubstrate-driven, and demonstrate that structure-based design can generate highly selective degraders devoid of teratogenic risk.

Multiple p38 MAP kinase inhibitors have been developed for the treatment of inflammatory diseases such as rheumatoid arthritis, but their effectiveness has been limited due to toxicity and tachyphylaxis, leading to a lack of clinical benefit. Efforts have been made to circumvent this limitation by targeting individual substrates downstream of p38, including MK2 and MK5. This approach has failed to yield clinical benefit despite preclinical evidence of a therapeutic effect. We hypothesized that there is redundancy in the MAPK activating kinase family that would necessitate blocking multiple kinases to sufficiently impact inflammatory processes. We used heterobifunctional protein degraders that either specifically degraded MK2 selectively or degraded MK2/3/5 simultaneously to test the hypothesis, in addition to genetic approaches to enable knockdown. In human PBMCs, elimination of MK2/3/5 with heterobifunctional degraders resulted in full reduction of TLR4 or TLR7/8 induced TNFα, whereas MK2-specific degradation only attenuated TNFα biosynthesis. In contrast, both specific MK2 degradation and broad MK2/3/5 degradation inhibited TGF-β-induced collagen production in human fibroblasts. This observation was consistent with genetic deletions of MK2, MK3 and MK5 (singly and in combination) whereby single deletion of MK2, MK3 or MK5 attenuated lipopolysaccharide (LPS) induced TNFα production and had no effect on R848-induced TNFα production. Double deletion of MK2 and MK3 or MK2 and MK5 or MK2/3/5 triple deletion had a significantly greater effect on TNFα production regardless of stimulus. The combined data suggest cooperativity between MK2 and either MK3 or MK5 for efficient, cell context-dependent modulation of inflammatory responses.

Gut microbiota can regulate host brain functions and influence various physiological and pathological processes through the brain-gut axis. To systematically elucidate the intervention of different gut environments on different brain regions, we implemented an integrated approach that combines 11-plex DiLeu isobaric tags with a "BRIDGE" normalization strategy to comparatively analyze the proteome of six brain regions in germ-free (GF)- and conventionally raised (ConvR)-mice. A total of 5945 proteins were identified and 5656 were quantifiable, while 1906 of them were significantly changed between GF- and ConvR-mice; 281 proteins were filtered with FC greater than 1.2 in at least one brain region, of which heatmap analysis showed clear protein profile disparities, both between brain regions and gut microbiome conditions. Gut microbiome impact is most overt in the hypothalamus and the least in the thalamus region. Collectively, this approach allows an in-depth investigation of the induced protein changes by multiple gut microbiome environments in a brain region-specific manner. This comprehensive proteomic work improves the understanding of the brain region protein association networks impacted by the gut microbiome and highlights the critical roles of the brain-gut axis.

Histone citrullination is an essential epigenetic post-translational modification (PTM) that affects many important physiological and pathological processes, but effective tools to study histone citrullination are greatly limited due to several challenges, including the small mass shift caused by this PTM and its low abundance in biological systems. Although previous studies have reported frequent occurrences of histone citrullination, these methods failed to provide a high-throughput and site-specific strategy to detect histone citrullination. Recently, we developed a biotin thiol tag that enabled precise identification of protein citrullination coupled with mass spectrometry. However, very few histone citrullination sites were identified, likely due to the highly basic nature of these proteins. In this study, we develop a novel method utilizing limited digestion and biotin derivative tag enrichment to facilitate direct in vivo identification of citrullination sites on histones. We achieve improved coverage of histone identification via partial enzymatic digestion and lysine block by dimethylation. With biotin tag-assisted chemical derivatization and enrichment, we also achieve precise annotation of histone citrullination sites with high confidence. We further compare different fragmentation methods and find that the electron-transfer-dissociation-based approach enables the most in-depth analysis and characterization. In total, we unambiguously identify 18 unique citrullination sites on histones in human astrocytoma U87 cells, including 15 citrullinated sites being detected for the first time. Some of these citrullination sites are observed to exhibit noticeable alterations in response to DNA damage, which demonstrates the superiority of our strategy in understanding the roles of histone citrullination in critical biological processes.

Interleukin-1 receptor associated kinase 4 (IRAK4) is an essential mediator of the IL-1R and TLR signaling pathways, both of which have been implicated in multiple autoimmune conditions. Hence, blocking the activity of IRAK4 represents an attractive approach for the treatment of autoimmune diseases. The activity of this serine/threonine kinase is dependent on its kinase and scaffolding activities; thus, degradation represents a potentially superior approach to inhibition. Herein, we detail the exploration of structure-activity relationships that ultimately led to the identification of KT-474, a potent, selective, and orally bioavailable heterobifunctional IRAK4 degrader. This represents the first heterobifunctional degrader evaluated in a nononcology indication and dosed to healthy human volunteers. This molecule successfully completed phase I studies in healthy adult volunteers and patients with atopic dermatitis or hidradenitis suppurativa. Phase II clinical trials in both of these indications have been initiated.

Developing therapies for the activated B-cell like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL) remains an area of unmet medical need. A subset of ABC DLBCL tumors is driven by activating mutations in myeloid differentiation primary response protein 88 (MYD88), which lead to constitutive activation of interleukin-1 receptor associated kinase 4 (IRAK4) and cellular proliferation. IRAK4 signaling is driven by its catalytic and scaffolding functions, necessitating complete removal of this protein and its escape mechanisms for complete therapeutic suppression. Herein, we describe the identification and characterization of a dual-functioning molecule, KT-413 and show it efficiently degrades IRAK4 and the transcription factors Ikaros and Aiolos. KT-413 achieves concurrent degradation of these proteins by functioning as both a heterobifunctional degrader and a molecular glue. Based on the demonstrated activity and safety of KT-413 in preclinical studies, a phase 1 clinical trial in B-cell lymphomas, including MYD88 mutant ABC DLBCL, is currently underway.

Abstract To elucidate the underlying mechanisms of STAT3 degradation and the suitability of VHL as an E3 ligase partner for targeting STAT3 in cancer using our clinical degrader, KT-333. Signal transducer and activator of transcription 3 (STAT3) is an undrugged oncogenic transcription factor and its role as cancer driver and tumor microenvironment modulator has been validated in a multitude of studies. Based on it’s potential as a target for cancer therapeutics and limitations of prior approaches, we developed KT-333, a first-in-class, potent, highly selective, heterobifunctional STAT3 degrader currently in Phase 1 clinical trials. Here, based on STAT3 degradation by multiple E3s based degraders, structure of STAT3-KT333-VHL and a lysine site-resolved target ubiquitination model, we provide evidence for VHL as the ideal partner E3 for targeting STAT3 in cancer. We successfully identified potent and selective STAT3 degraders using either CRBN or VHL as the E3 ligase, but ultimately preferred VHL based degraders for their increased potency, and consistent STAT3 degradation across multiple cancer lines. KT-333, our VHL based clinical STAT3 degrader, induces a strong ternary complex between STAT3 and VHL. We present a high resolution cryo-electron microscopy based ternary complex structure of STAT3-KT333-VHL which provides mechanistic insights further validating VHL as the E3 of choice for deep, selective and fast STAT3 degradation. Specifically, KT-333 enables favorable protein-protein interactions between STAT3 and VHL, burying a large protein interface in the ternary complex to the extent typically observed only in native protein complexes. Additionally, the residues forming the STAT3-VHL interface are not conserved in other STAT family members corroborating with degradation selectivity observed with KT-333. Next, we leveraged this structure and generated a ubiquitination super-complex model by deploying a combination of biochemical and proteomics techniques and reveal that KT-333-induced ubiquitination occurs by precise targeting of specific lysine resides on STAT3. Furthermore, to decode the mechanism for KT-333 activity against SUDHL1 cell line both in vitro and in a mouse xenograft model, we measured temporal changes at protein level by discovery proteomics. Reduced expression of canonical STAT3 targets and down-regulation of cytokine-mediated signaling and cell cycle signature genes indicated that cell cycle arrest and subsequent apoptosis are the main drivers of efficacy in vitro and in vivo. In summary, we show that our clinical STAT3 degrader, KT-333, is designed and optimally paired with VHL resulting in a very stable ternary complex exhibiting properties of native protein complexes. Our data provides precise structural and molecular mechanisms behind potent, selective, consistent, and fast degradation of STAT3, and downstream mechanism of action observed in vitro and in vivo. Citation Format: Kirti Sharma, Xue Fei, Yatao Shi, Christopher Browne, Dirk Walther, Caroline Daigle, Anand Ramanathan, Richard Miller, Karen Yuan, Kiran Mahasenan, Sean Zhu, Xin Huang, Bin Yang. E3 pairing and structural mechanisms underlying anti-tumor activity of clinical STAT3 degrader KT-333 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB037.

TAM receptors (TYRO3, AXL, and MERTK) comprise a family of homologous receptor tyrosine kinases (RTK) that are expressed across a range of liquid and solid tumors where they contribute to both oncogenic signaling to promote tumor proliferation and survival, as well as expressed on myeloid and immune cells where they function to suppress host anti-tumor immunity. In recent years, several strategies have been employed to inhibit TAM kinases, most notably small molecule tyrosine kinase inhibitors and inhibitory neutralizing monoclonal antibodies (mAbs) that block receptor dimerization. Targeted protein degraders (TPD) use the ubiquitin proteasome pathway to redirect E3 ubiquitin ligase activity and target specific proteins for degradation. Here we employ first-in-class TPDs specific for MERTK/TAMs that consist of a cereblon E3 ligase binder linked to a tyrosine kinase inhibitor targeting MERTK and/or AXL and TYRO3. A series of MERTK TPDs were designed and investigated for their capacity to selectively degrade MERTK chimeric receptors, reduce surface expression on primary efferocytic bone marrow-derived macrophages, and impact on functional reduction in efferocytosis (clearance of apoptotic cells). We demonstrate proof-of-concept and establish that TPDs can be tailored to either selectivity degrades MERTK or concurrently degrade multiple TAMs and modulate receptor expression in vitro and in vivo. This work demonstrates the utility of proteome editing, enabled by tool degraders developed here towards dissecting the therapeutically relevant pathway biology in preclinical models, and the ability for TPDs to degrade transmembrane proteins. These data also provide proof of concept that TPDs may serve as a viable therapeutic strategy for targeting MERTK and other TAMs and that this technology could be expanded to other therapeutically relevant transmembrane proteins.

Citrullination is a key post-translational modification (PTM) that affects protein structures and functions. Although it has been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize this PTM. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of citrullination via mass spectrometry. We perform global mapping of the citrullination proteome of mouse tissues. In total, we identify 691 citrullination sites from 432 proteins which represents the largest data set to date. We discover novel distribution and functions of this PTM. This study depicts a landscape of protein citrullination and lays the foundation for further deciphering their physiological and pathological roles.

Protein citrullination is a key post-translational modification (PTM) that leads to the loss of positive charge on arginine and consequent protein structural and functional changes. Though it has been indicated to play critical roles in various physiological and pathological processes, effective analytical tools are largely limited due to a few challenges such as the small mass shift induced by this PTM and its low-abundance nature. Recently, we developed a biotin thiol tag, which enabled large-scale profiling of protein citrullination from complex biological samples via mass spectrometry. However, a high-throughput quantitative approach is still in great need to further improve the understanding of this PTM. In this study, we report an efficient pipeline using our custom-developed N,N-dimethyl leucine isobaric tags to achieve a multiplexed quantitative analysis of citrullination from up to 12 samples for the first time. We then apply this strategy to investigating citrullination alterations in response to DNA damage stress using human cell lines. We unveil important biological functions regulated by protein citrullination and observe hypercitrullination on RNA-binding proteins and DNA repair proteins, respectively. Our results reveal the involvement of citrullination in DNA damage pathways and may provide new insights into DNA-damage-related disease pathogenesis.

Background: The murine double minute 2 (MDM2) oncoprotein is a key E3 ubiquitin ligase that degrades the tumor-suppressor p53. Reversible SMIs of the MDM2/p53 interaction have been developed to stabilize p53 and to induce apoptosis in wildtype p53 tumors. However, MDM2 SMIs induce a p53/MDM2 feedback loop, resulting in upregulation of MDM2 protein levels and p53 pathway inhibition, thus drastically limiting their biological activity and clinical application. MDM2 targeted protein degradation suppresses p53-dependent MDM2 protein feedback upregulation and is therefore expected to lead to a superior response compared to SMIs. Objectives: We discovered KT-253, a novel, highly potent heterobifunctional MDM2 degrader. We characterized the activity and molecular mechanism of KT-253 in wildtype p53 cell lines and xenograft models and demonstrated that KT-253 has superior activity compared to SMIs in these models. Methods: We characterized KT-253 in MDM2-dependent cell lines in vitro, as well as in in vivo mouse AML and ALL xenograft models. Methods included in vitro cell proliferation and apoptosis assays, gene expression profiling, and in vivo pharmacological studies. Results: KT-253 selectively depletes MDM2 protein levels with sub-nanomolar cellular potency. We demonstrated in the Acute Lymphoblastic Leukemia (ALL) cell line RS4;11 that KT-253 can overcome the p53-dependent upregulation of MDM2 protein levels as seen for reversible SMIs. In addition, even brief exposures to KT-253 in these cells could more potently stabilize p53 compared to SMIs, leading to apoptosis mediated through p53 target genes. The superior MDM2/p53 pathway inhibition and induction of apoptosis by KT-253 translates into a >200-fold stronger cell growth inhibition, compared to SMIs, across a panel of solid and hematological tumor cell lines. Using mouse xenograft models, we established an intermittent dosing schedule that drives anti-tumor efficacy. Importantly, a single dose of KT-253 at 1 mg/kg resulted in sustained tumor regression in the RS4;11 mouse xenograft model. In this model, KT-253 exposures correlated with induction of apoptotic p53 target genes and tumor growth inhibition. In addition, weekly administration of KT-253 significantly prolonged the survival in a disseminated Acute Myeloid Leukemia (AML) model when compared to vehicle treated animals. Conclusions: In summary, KT-253, our development candidate, has significantly improved potency relative to reversible SMIs leading to potent in vitro and in vivo efficacy that is superior to all clinically active agents. In addition, an intermittent dosing schedule of the highly potent KT-253 degrader can induce rapid apoptosis in MDM2-dependent cancer cells potentially leading to improved efficacy and safety profile. Citation Format: Yogesh Chutake, Michele Mayo, Dapeng Chen, Bradley Enerson, Patricia Cho, Jessica Filiatrault, Crystal Brown, Michael Placke, Madison Adams, Rahul Karnik, James Shaw, Yatao Shi, Dirk Walther, Alice McDonald, Frank Qi, Phillip Liu, Joseph D. Growney, Kirti Sharma, Duncan Walker, Stefanie Schalm, Juliet Williams, Matthew Weiss. KT-253, a highly potent and selective heterobifunctional MDM2 degrader for the treatment of wildtype p53 tumors with superior potency and differentiated biological activity compared to small molecule inhibitors (SMI) [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 3934.

Signal Transducer and Activator of Transcription 3 (STAT3) plays important roles in the transduction of signals from growth factors and cytokines in both normal and malignant cells. Upon activation, STAT3 controls expression of genes that regulate cell growth, survival, differentiation, stemness and cell-cell interactions. Aberrant activation of STAT3 has been observed in many cancers including lymphoma and leukemias through activating mutations, hyper-signaling through upstream regulators or loss of negative feedback regulation. Additionally, STAT3-mediated cross-talk in the tumor microenvironment results in suppression of immune surveillance compromising anti-tumor immunity.

The extracellular matrix (ECM) is unique to each tissue and capable of guiding cell differentiation, migration, morphology, and function. The ECM proteome of different developmental stages has not been systematically studied in the human pancreas. In this study, we apply mass spectrometry-based quantitative proteomics strategies using N,N-dimethyl leucine isobaric tags to delineate proteome-wide and ECM-specific alterations in four age groups: fetal (18-20 weeks gestation), juvenile (5-16 years old), young adults (21-29 years old) and older adults (50-61 years old). We identify 3,523 proteins including 185 ECM proteins and quantify 117 of them. We detect previously unknown proteome and matrisome features during pancreas development and maturation. We also visualize specific ECM proteins of interest using immunofluorescent staining and investigate changes in ECM localization within islet or acinar compartments. This comprehensive proteomics analysis contributes to an improved understanding of the critical roles that ECM plays throughout human pancreas development and maturation.

High‐resolution, noninvasive and nondestructive imaging of the subepithelial structures of the larynx would enhance microanatomic tissue assessment and clinical decision making; similarly, in situ molecular profiling of laryngeal tissue would enhance biomarker discovery and pathology readout. Towards these goals, we assessed the capabilities of high‐resolution magnetic resonance imaging (MRI) and matrix‐assisted laser desorption/ionisation‐mass spectrometry (MALDI‐MS) imaging of rarely reported paediatric and adult cadaveric larynges that contained pathologies. The donors were a 13‐month‐old male, a 10‐year‐old female with an infraglottic mucus retention cyst and a 74‐year‐old female with advanced polypoid degeneration and a mucus retention cyst. MR and molecular imaging data were corroborated using whole‐organ histology. Our MR protocols imaged the larynges at 45–117 μm 2 in‐plane resolution and capably resolved microanatomic structures that have not been previously reported radiographically—such as the vocal fold superficial lamina propria, vocal ligament and macula flavae; age‐related tissue features—such as intramuscular fat deposition and cartilage ossification; and the lesions. Diffusion tensor imaging characterised differences in water diffusivity, primary tissue fibre orientation, and fractional anisotropy between the intrinsic laryngeal muscles, mucosae and lesions. MALDI‐MS imaging revealed peptide signatures and putative protein assignments for the polypoid degeneration lesion and the N‐glycan constituents of one mucus retention cyst. These imaging approaches have immediate application in experimental research and, with ongoing technology development, potential for future clinical application.

Gut microbiota can regulate host physiological and pathological status through gut-brain communications or pathways. However, the impact of the gut microbiome on neuropeptides and proteins involved in regulating brain functions and behaviors is still not clearly understood. To address the problem, integrated label-free and 10-plex DiLeu isobaric tag-based quantitative methods were implemented to compare the profiling of neuropeptides and proteins in the hypothalamus of germ-free (GF)- vs conventionally raised (ConvR)-mice. A total of 2943 endogenous peptides from 63 neuropeptide precursors and 3971 proteins in the mouse hypothalamus were identified. Among these 368 significantly changed peptides (fold changes over 1.5 and a p-value of <0.05), 73.6% of the peptides showed higher levels in GF-mice than in ConvR-mice, and 26.4% of the peptides had higher levels in ConvR-mice than in GF-mice. These peptides were mainly from secretogranin-2, phosphatidylethanolamine-binding protein-1, ProSAAS, and proenkephalin-A. A quantitative proteomic analysis employing DiLeu isobaric tags revealed that 282 proteins were significantly up- or down-regulated (fold changes over 1.2 and a p-value of <0.05) among the 3277 quantified proteins. These neuropeptides and proteins were mainly involved in regulating behaviors, transmitter release, signaling pathways, and synapses. Interestingly, pathways including long-term potentiation, long-term depression, and circadian entrainment were involved. In the present study, a combined label-free and 10-plex DiLeu-based quantitative method enabled a comprehensive profiling of gut microbiome-induced dynamic changes of neuropeptides and proteins in the hypothalamus, suggesting that the gut microbiome might mediate a range of behavioral changes, brain development, and learning and memory through these neuropeptides and proteins.

STAT3 (signal transducers and activators of transcription 3) is a transcription factor and a member of the STAT protein family that is activated through a variety of different cytokine and growth factor receptors via JAKs, as well as through oncogenic fusion proteins and gain-of-function (GoF) mutations in STAT3 itself. STAT3 hyperactivation and GoF mutations are found in numerous cancers, including clinically aggressive hematologic malignancies with high unmet medical need, such as peripheral T cell lymphomas (PTCLs) (Andersson et al., 2020). We have previously shown that a potent and selective STAT3 heterobifunctional degrader, KTX-201, strongly represses cell growth in models of STAT3-dependent heme malignancies (Csibi et al., 2019). Herein, we report on the cellular mechanisms underlying the anti-tumor effect of STAT3 degradation in PTCL and provide a model for the relationship between pharmacokinetics/ pharmacodynamics (PK/PD) and activity of KTX-201 in vivo. The relationship between STAT3 degradation by KTX-201, anti-tumor mechanism of action and in vivo activity were investigated in anaplastic large T cell lymphoma (ALCL) models, a subset of PTCLs. In vitro, a decrease of STAT3 by 90% for 48hr was required for ALCL cells to commit to death. To identify anti-tumor mechanism(s) of KTX-201 at the systems level, we performed a time-resolved analysis of the proteomic changes of SU-DHL-1 cells undergoing growth inhibition mediated by KTX-201 at GI95. We measured the abundance of 10,000 proteins and confirmed selective degradation of STAT3 by KTX-201 after 8h of treatment. Significant changes in several marker proteins known to be involved in STAT3-mediated proximal signaling in ALCL including SOCS3, Myc and Granzyme B were observed after 16h. Functional annotation analysis of proteins identified pathways that were significantly enriched in at least one time point. Using unsupervised hierarchical clustering of annotations, we found that proteins that increased in abundance over 48h of exposure to KTX-201 were associated with markers of apoptosis and those that decreased in abundance by 24h and 48h were associated with cytokine signaling and cell cycle, respectively. Based on these data, this study identifies inhibition of cytokine signaling, G1 cell cycle arrest and induction of apoptosis as key anti-tumor mechanisms associated with KTX-201 consistent with observed cell phenotypes. STAT3 degradation in tumor was characterized in mice bearing SU-DHL-1 tumors following single dose IV administration. The STAT3 PD response in tumor was correlated with exposures in tumor. At the dose of 25 mg/kg weekly where complete tumor regression was achieved, KTX-201 achieves >90% STAT3 degradation at 24h post dosing in SUDHL1 xenografts. STAT3 degradation was maintained at 90% at 4 days post dosing. The results from the PK/PD study suggests that STAT3 degradation in tumor of >90% is necessary for anti-tumor efficacy in vivo of KTX-201, but only for a limited duration, such as 4 days out of a weekly dosing cycle. Collectively, our data demonstrate that significant STAT3 degradation for a limited time during dosing interval with KTX-201 in ALCL promotes early changes in key signaling nodes involved with proliferation and cytokine stimulation, followed by profound changes in apoptotic proteins. By integrating mechanistic biology with a deep understanding of PK/PD and efficacy, this study provides a foundation for the clinical development of STAT3 degraders using intermittent dosing regimen for treatment of PTCL and other STAT3-dependent heme malignancies. Disclosures Rong: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Csibi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company, Ended employment in the past 24 months. Yang:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rusin:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Shi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayo:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yuan:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chutake:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonald:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhu:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ji:Kymera Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Liu:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Li:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Walker:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollob:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mainolfi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Desavi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections. The results demonstrated that the signal-to-noise ratios for target saccharides were notably improved after chemical derivatization, in which signals were enhanced by 230-fold for glucose and over 28-fold for maltooctaose. Improved glycome coverage was obtained for N-glycans derived from glycoproteins and tissue samples after chemical derivatization. Furthermore, on-tissue derivatization was applied for MALDI-MSI of N-glycans from human laryngeal cancer and ovarian cancer tissues. Differentially expressed N-glycans among the tumor region, adjacent normal tissue region, and tumor proximal collagen stroma region were imaged, revealing that high-mannose type N-glycans were predominantly expressed in the tumor region. Overall, our results indicate that the on-tissue labeling strategy coupled with MALDI-MSI shows great potential to spatially characterize N-glycan expression within heterogeneous tissue samples with enhanced sensitivity. This study provides a promising approach to better understand the pathogenesis of cancer related aberrant glycosylation, which is beneficial to the design of improved clinical diagnosis and therapeutic strategies.

The Spatiotemporal Dynamics of Low-abundance Bioactive Lipids in Arteries Undergoing Restenosis Observed and Identified at High Spatial Resolving Power with Multi-modal Mass Spectrometry Imaging - Volume 26 Issue S2