Pharmacological Targeting of STK19 Inhibits Oncogenic NRAS-Driven Melanomagenesis.

Activating mutations in NRAS account for 20%-30% of melanoma, but despite decades of research and in contrast to BRAF, no effective anti-NRAS therapies have been forthcoming. Here, we identify a previously uncharacterized serine/threonine kinase STK19 as a novel NRAS activator. STK19 phosphorylates NRAS to enhance its binding to its downstream effectors and promotes oncogenic NRAS-mediated melanocyte malignant transformation. A recurrent D89N substitution in STK19 whose alterations were identified in 25% of human melanomas represents a gain-of-function mutation that interacts better with NRAS to enhance melanocyte transformation. STK19D89N knockin leads to skin hyperpigmentation and promotes NRASQ61R-driven melanomagenesis in vivo. Finally, we developed ZT-12-037-01 (1a) as a specific STK19-targeted inhibitor and showed that it effectively blocks oncogenic NRAS-driven melanocyte malignant transformation and melanoma growth in vitro and in vivo. Together, our findings provide a new and viable therapeutic strategy for melanomas harboring NRAS mutations.

Mutational Landscape of Secondary Glioblastoma Guides MET-Targeted Trial in Brain Tumor.

Low-grade gliomas almost invariably progress into secondary glioblastoma (sGBM) with limited therapeutic option and poorly understood mechanism. By studying the mutational landscape of 188 sGBMs, we find significant enrichment of TP53 mutations, somatic hypermutation, MET-exon-14-skipping (METex14), PTPRZ1-MET (ZM) fusions, and MET amplification. Strikingly, METex14 frequently co-occurs with ZM fusion and is present in ∼14% of cases with significantly worse prognosis. Subsequent studies show that METex14 promotes glioma progression by prolonging MET activity. Furthermore, we describe a MET kinase inhibitor, PLB-1001, that demonstrates remarkable potency in selectively inhibiting MET-altered tumor cells in preclinical models. Importantly, this compound also shows blood-brain barrier permeability and is subsequently applied in a phase I clinical trial that enrolls MET-altered chemo-resistant glioma patients. Encouragingly, PLB-1001 achieves partial response in at least two advanced sGBM patients with rarely significant side effects, underscoring the clinical potential for precisely treating gliomas using this therapy.

ARAF protein kinase activates RAS by antagonizing its binding to RASGAP NF1.

RAF protein kinases are effectors of the GTP-bound form of small guanosine triphosphatase RAS and function by phosphorylating MEK. We showed here that the expression of ARAF activated RAS in a kinase-independent manner. Binding of ARAF to RAS displaced the GTPase-activating protein NF1 and antagonized NF1-mediated inhibition of RAS. This reduced ERK-dependent inhibition of RAS and increased RAS-GTP. By this mechanism, ARAF regulated the duration and consequences of RTK-induced RAS activation and supported the RAS output of RTK-dependent tumor cells. In human lung cancers with EGFR mutation, amplification of ARAF was associated with acquired resistance to EGFR inhibitors, which was overcome by combining EGFR inhibitors with an inhibitor of the protein tyrosine phosphatase SHP2 to enhance inhibition of nucleotide exchange and RAS activation.

Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription.

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.

Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

A designed ankyrin-repeat protein that targets Parkinson's disease-associated LRRK2.

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

A drug-resistant mutation in plant target of rapamycin validates the specificity of ATP-competitive TOR inhibitors in vivo.

Kinases are major components of cellular signaling pathways, regulating key cellular activities through phosphorylation. Kinase inhibitors are efficient tools for studying kinase targets and functions, however assessing their kinase specificity in vivo is essential. The identification of resistant kinase mutants has been proposed to be the most convincing approach to achieve this goal. Here, we address this issue in plants via a pharmacogenetic screen for mutants resistant to the ATP-competitive TOR inhibitor AZD-8055. The eukaryotic TOR (Target of Rapamycin) kinase is emerging as a major hub controlling growth responses in plants largely thanks to the use of ATP-competitive inhibitors. We identified a dominant mutation in the DFG motif of the Arabidopsis TOR kinase domain that leads to very strong resistance to AZD-8055. This resistance was characterized by measuring root growth, photosystem II (PSII) activity in leaves and phosphorylation of YAK1 (Yet Another Kinase 1) and RPS6 (Ribosomal protein S6), a direct and an indirect target of TOR respectively. Using other ATP-competitive TOR inhibitors, we also show that the dominant mutation is particularly efficient for resistance to drugs structurally related to AZD-8055. Altogether, this proof-of-concept study demonstrates that a pharmacogenetic screen in Arabidopsis can be used to successfully identify the target of a kinase inhibitor in vivo and therefore to demonstrate inhibitor specificity. Thanks to the conservation of kinase families in eukaryotes, and the possibility of creating amino acid substitutions by genome editing, this work has great potential for extending studies on the evolution of signaling pathways in eukaryotes.

Evolution of the Cdk4/6-Cdkn2 system in invertebrates.

The cell cycle is driven by cyclin-dependent kinases (Cdks). The decision whether the cell cycle proceeds is made during G1 phase, when Cdk4/6 functions. Cyclin-dependent kinase inhibitor 2 (Cdkn2) is a specific inhibitor of Cdk4/6, and their interaction depends on D84 in Cdkn2 and R24/31 in Cdk4/6. This knowledge is based mainly on studies in mammalian cells. Here, we comprehensively analyzed Cdk4/6 and Cdkn2 in invertebrates and found that Cdk4/6 was present in most of the investigated phyla, but the distribution of Cdkn2 was rather uneven among and within the phyla. The positive charge of R24/R31 in Cdk4/6 was conserved in all analyzed species in phyla with Cdkn2. The presence of Cdkn2 and the conservation of the positive charge were statistically correlated. We also found that Cdkn2 has been tightly linked to Fas associated factor 1 (Faf1) during evolution. We discuss potential interactions between Cdkn2 and Cdk4/6 in evolution and the possible cause of the strong conservation of the microsynteny.

KinScan: AI-based rapid profiling of activity across the kinome.

Kinases play a vital role in regulating essential cellular processes, including cell cycle progression, growth, apoptosis, and metabolism, by catalyzing the transfer of phosphate groups from adenosing triphosphate to substrates. Their dysregulation has been closely associated with numerous diseases, including cancer development, making them attractive targets for drug discovery. However, accurately predicting the binding affinity between chemical compounds and kinase targets remains challenging due to the highly conserved structural similarities across the kinome. To address this limitation, we present KinScan, a novel computational approach that leverages large-scale bioactivity data and integrates the Multi-Scale Context Aware Transformer framework to construct a virtual profiling model encompassing 391 protein kinases. The developed model demonstrates exceptional prediction capability, distinguishing between kinases by utilizing structurally aligned kinase binding site features derived from multiple sequence alignment for fast and accurate predictions. Through extensive validation and benchmarking, KinScan demonstrated its robust predictive power and generalizability for large-scale kinome-wide profiling and selectivity, uncovering associations with specific diseases and providing valuable insights into kinase activity profiles of compounds. Furthermore, we deployed a web platform for end-to-end profiling and selectivity analysis, accessible at https://kinscan.drugonix.com/softwares/kinscan.

Illuminating phenotypic drug responses of sarcoma cells to kinase inhibitors by phosphoproteomics.

Kinase inhibitors (KIs) are important cancer drugs but often feature polypharmacology that is molecularly not understood. This disconnect is particularly apparent in cancer entities such as sarcomas for which the oncogenic drivers are often not clear. To investigate more systematically how the cellular proteotypes of sarcoma cells shape their response to molecularly targeted drugs, we profiled the proteomes and phosphoproteomes of 17 sarcoma cell lines and screened the same against 150 cancer drugs. The resulting 2550 phenotypic profiles revealed distinct drug responses and the cellular activity landscapes derived from deep (phospho)proteomes (9-10,000 proteins and 10-27,000 phosphorylation sites per cell line) enabled several lines of analysis. For instance, connecting the (phospho)proteomic data with drug responses revealed known and novel mechanisms of action (MoAs) of KIs and identified markers of drug sensitivity or resistance. All data is publicly accessible via an interactive web application that enables exploration of this rich molecular resource for a better understanding of active signalling pathways in sarcoma cells, identifying treatment response predictors and revealing novel MoA of clinical KIs.

TRKing down drug resistance in NTRK fusion-positive cancers†.

In a recent issue of The Journal of Pathology, Chen and colleagues established novel patient-derived ex vivo models of NTRK fusion-positive soft tissue sarcoma to characterize resistance mechanisms against targeted therapy with tyrosine kinase inhibitors. Prolonged exposure to escalating concentrations of the tyrosine kinase inhibitor, entrectinib, ultimately led to the occurrence of resistant clones that harbored an inactivating mutation in the NF2 gene, not previously described in this context, accompanied by increased PI3K/AKT/mTOR and Ras/Raf/MEK/ERK signaling. Finally, an inhibitor screen identified, among others, MEK and mTOR inhibitors as potential combination agents. © 2024 The Pathological Society of Great Britain and Ireland.

EGFR-T790M Mutation-Derived Interactome Rerouted EGFR Translocation Contributing to Gefitinib Resistance in Non-Small Cell Lung Cancer.

Secondary mutation, T790M, conferring tyrosine kinase inhibitors (TKIs) resistance beyond oncogenic epidermal growth factor receptor (EGFR) mutations presents a challenging unmet need. Although TKI-resistant mechanisms are intensively investigated, the underlying responses of cancer cells adapting drug perturbation are largely unknown. To illuminate the molecular basis linking acquired mutation to TKI resistance, affinity purification coupled mass spectrometry was adopted to dissect EGFR interactome in TKI-sensitive and TKI-resistant non-small cell lung cancer cells. The analysis revealed TKI-resistant EGFR-mutant interactome allocated in diverse subcellular distribution and enriched in endocytic trafficking, in which gefitinib intervention activated autophagy-mediated EGFR degradation and thus autophagy inhibition elevated gefitinib susceptibility. Alternatively, gefitinib prompted TKI-sensitive EGFR translocating toward cell periphery through Rab7 ubiquitination which may favor efficacy to TKIs suppression. This study revealed that T790M mutation rewired EGFR interactome that guided EGFR to autophagy-mediated degradation to escape treatment, suggesting that combination therapy with TKI and autophagy inhibitor may overcome acquired resistance in non-small cell lung cancer.

Structure-based design of selective, orally available salt-inducible kinase inhibitors that stimulate bone formation in mice.

Osteoporosis is a major public health problem. Currently, there are no orally available therapies that increase bone formation. Intermittent parathyroid hormone (PTH) stimulates bone formation through a signal transduction pathway that involves inhibition of salt-inducible kinase isoforms 2 and 3 (SIK2 and SIK3). Here, we further validate SIK2/SIK3 as osteoporosis drug targets by demonstrating that ubiquitous deletion of these genes in adult mice increases bone formation without extraskeletal toxicities. Previous efforts to target these kinases to stimulate bone formation have been limited by lack of pharmacologically acceptable, specific, orally available SIK2/SIK3 inhibitors. Here, we used structure-based drug design followed by iterative medicinal chemistry to identify SK-124 as a lead compound that potently inhibits SIK2 and SIK3. SK-124 inhibits SIK2 and SIK3 with single-digit nanomolar potency in vitro and in cell-based target engagement assays and shows acceptable kinome selectivity and oral bioavailability. SK-124 reduces SIK2/SIK3 substrate phosphorylation levels in human and mouse cultured bone cells and regulates gene expression patterns in a PTH-like manner. Once-daily oral SK-124 treatment for 3 wk in mice led to PTH-like effects on mineral metabolism including increased blood levels of calcium and 1,25-vitamin D and suppressed endogenous PTH levels. Furthermore, SK-124 treatment increased bone formation by osteoblasts and boosted trabecular bone mass without evidence of short-term toxicity. Taken together, these findings demonstrate PTH-like effects in bone and mineral metabolism upon in vivo treatment with orally available SIK2/SIK3 inhibitor SK-124.

Tissue-restricted inhibition of mTOR using chemical genetics.

Mammalian target of rapamycin (mTOR) is a highly conserved eukaryotic protein kinase that coordinates cell growth and metabolism, and plays a critical role in cancer, immunity, and aging. It remains unclear how mTOR signaling in individual tissues contributes to whole-organism processes because mTOR inhibitors, like the natural product rapamycin, are administered systemically and target multiple tissues simultaneously. We developed a chemical-genetic system, termed selecTOR, that restricts the activity of a rapamycin analog to specific cell populations through targeted expression of a mutant FKBP12 protein. This analog has reduced affinity for its obligate binding partner FKBP12, which reduces its ability to inhibit mTOR in wild-type cells and tissues. Expression of the mutant FKBP12, which contains an expanded binding pocket, rescues the activity of this rapamycin analog. Using this system, we show that selective mTOR inhibition can be achieved in Saccharomyces cerevisiae and human cells, and we validate the utility of our system in an intact metazoan model organism by identifying the tissues responsible for a rapamycin-induced developmental delay in Drosophila.

Remibrutinib demonstrates favorable safety profile and sustained efficacy in chronic spontaneous urticaria over 52 weeks.

BACKGROUND: Remibrutinib (LOU064), an oral, highly selective Bruton tyrosine kinase inhibitor, offers fast disease control in patients with chronic spontaneous urticaria (CSU) who remain symptomatic despite treatment with second-generation H1 antihistamines. It is currently in phase 3 development for CSU. OBJECTIVE: We sought to evaluate long-term safety and efficacy of remibrutinib in patients with CSU inadequately controlled with H1 antihistamines. METHODS: In this phase 2b extension study, patients who completed the core study and had a weekly Urticaria Activity Score (UAS7) ≥16 at the beginning of the extension study received remibrutinib 100 mg twice daily for 52 weeks. The primary objective was to assess long-term safety and tolerability. Key efficacy end points included change from baseline in UAS7 and proportion of patients with complete response to treatment (UAS7 = 0) and well-controlled disease (UAS7 ≤6) at week 4 and over 52 weeks. RESULTS: Overall, 84.3% (194/230) of patients entered the treatment period and received ≥1 doses of remibrutinib. The overall safety profile of remibrutinib was comparable between the extension and core studies. Most treatment-emergent adverse events were mild to moderate and considered unrelated to remibrutinib by investigators. The 3 most common treatment-emergent adverse events by system organ class were infections (30.9%), skin and subcutaneous tissue (26.8%), and gastrointestinal disorders (16.5%). At week 4 and 52, mean ± SD change from baseline in UAS7 was -17.6 ± 13.40 and -21.8 ± 10.70; UAS7 = 0 (as observed) was achieved in 28.2% and 55.8% and UAS7 ≤6 (as observed) was achieved in 52.7% and 68.0% of patients, respectively. CONCLUSIONS: Remibrutinib demonstrated a consistent favorable safety profile with fast and sustained efficacy for up to 52 weeks in patients with CSU.

BTK signaling-a crucial link in the pathophysiology of chronic spontaneous urticaria.

Chronic spontaneous urticaria (CSU) is an inflammatory skin disorder that manifests with itchy wheals, angioedema, or both for more than 6 weeks. Mast cells and basophils are the key pathogenic drivers of CSU; their activation results in histamine and cytokine release with subsequent dermal inflammation. Two overlapping mechanisms of mast cell and basophil activation have been proposed in CSU: type I autoimmunity, also called autoallergy, which is mediated via IgE against various autoallergens, and type IIb autoimmunity, which is mediated predominantly via IgG directed against the IgE receptor FcεRI or FcεRI-bound IgE. Both mechanisms involve cross-linking of FcεRI and activation of downstream signaling pathways, and they may co-occur in the same patient. In addition, B-cell receptor signaling has been postulated to play a key role in CSU by generating autoreactive B cells and autoantibody production. A cornerstone of FcεRI and B-cell receptor signaling is Bruton tyrosine kinase (BTK), making BTK inhibition a clear therapeutic target in CSU. The potential application of early-generation BTK inhibitors, including ibrutinib, in allergic and autoimmune diseases is limited owing to their unfavorable benefit-risk profile. However, novel BTK inhibitors with improved selectivity and safety profiles have been developed and are under clinical investigation in autoimmune diseases, including CSU. In phase 2 trials, the BTK inhibitors remibrutinib and fenebrutinib have demonstrated rapid and sustained improvements in CSU disease activity. With phase 3 studies of remibrutinib ongoing, it is hoped that BTK inhibitors will present an effective, well-tolerated option for patients with antihistamine-refractory CSU, a phenotype that presents a considerable clinical challenge.

Prevention of allergic reactions during oxaliplatin desensitization through inhibition of Bruton tyrosine kinase.

BACKGROUND: Acute infusion reactions to oxaliplatin, a chemotherapeutic used to treat gastrointestinal cancers, are observed in about 20% of patients. Rapid drug desensitization (RDD) protocols often allow the continuation of oxaliplatin in patients with no alternative options. Breakthrough symptoms, including anaphylaxis, can still occur during RDD. OBJECTIVE: Our aim was to evaluate whether pretreatment with acalabrutinib, a Bruton tyrosine kinase inhibitor, can prevent anaphylaxis during RDD in a patient sensitized to oxaliplatin. METHODS: A 52-year-old male with locally advanced gastric carcinoma developed anaphylaxis during his fifth cycle of oxaliplatin. As he required 6 additional cycles to complete his curative-intent treatment regimen, he underwent RDD to oxaliplatin but still developed severe acute reactions. The risks and benefits of adding acalabrutinib before and during RDD were reviewed, and the patient elected to proceed. RESULTS: With acalabrutinib taken before and during the RDD, the patient was able to tolerate oxaliplatin RDD without complication. Consistent with its mechanism of action, acalabrutinib completely blocked the patient's positive skin prick response to oxaliplatin. Acalabrutinib did not alter the percentage of circulating basophils (1.24% vs 0.98%) before the RDD but did protect against basopenia (0.74% vs 0.09%) after the RDD. Acalabrutinib was associated with a drastic reduction in the ability of basophils to upregulate CD63 in vitro following incubation with oxaliplatin (0.11% vs 2.38%) or polyclonal anti-human IgE antibody (0.08% vs 44.2%). CONCLUSIONS: Five doses of acalabrutinib, 100 mg, orally twice daily starting during the evening 2 days before and continuing through RDD allowed a sensitized patient to receive oxaliplatin successfully and safely.

Mimicry of Short Linear Motifs by Bacterial Pathogens: A Drugging Opportunity.

Bacterial pathogens have developed complex strategies to successfully survive and proliferate within their hosts. Throughout the infection cycle, direct interaction with host cells occurs. Many bacteria have been found to secrete proteins, such as effectors and toxins, directly into the host cell with the potential to interfere with cell regulatory processes, either enzymatically or through protein-protein interactions (PPIs). Short linear motifs (SLiMs) are abundant peptide modules in cell signaling proteins. Here, we cover the reported examples of eukaryotic-like SLiM mimicry being used by pathogenic bacteria to hijack host cell machinery and discuss how drugs targeting SLiM-regulated cell signaling networks are being evaluated for interference with bacterial infections. This emerging anti-infective opportunity may become an essential contributor to antibiotic replacement strategies.

CDK inhibitors from past to present: A new wave of cancer therapy.

Deregulation of the cell cycle machinery, which has been linked to dysregulation of cyclin-dependent kinases (CDKs), is a defining characteristic of cancer, eventually promoting abnormal proliferation that feeds tumorigenesis and disease development. In this regard, several CDK inhibitors (CDKIs) have been developed during the last few decades (1st, 2nd, and 3rd generation CDKIs) to inhibit cancer cell proliferation. 1st and 2nd generation CDKIs have not received much clinical attention for the treatment of cancer patients because of their limited specificity and high toxicity. However, the recent development of combination strategies allowed us to reduce the toxicity and side effects of these CDKIs, paving the way for their potential application in clinical settings. The 3rd generation CDKIs have yielded the most promising results at the preclinical and clinical levels, propelling them into the advanced stages of clinical trials against multiple malignancies, especially breast cancer, and revolutionizing traditional treatment strategies. In this review, we discuss the most-investigated candidates from the 1st, 2nd, and 3rd generations of CDKIs, their basic mechanisms of action, the reasons for their failure in the past, and their current clinical development for the treatment of different malignancies. Additionally, we briefly highlighted the most recent clinical trial results and advances in the development of 3rd generation FDA-approved selective CDK4/6 inhibitors that combat the most prevalent cancer. Overall, this review will provide a thorough knowledge of CDKIs from the past to the present, allowing researchers to rethink and develop innovative cancer therapeutic regimens.

A Rapid One-Pot Workflow for Sensitive Microscale Phosphoproteomics.

Compared to advancements in single-cell proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We present a simple and rapid one-pot phosphoproteomics workflow (SOP-Phos) integrated with data-independent acquisition mass spectrometry (DIA-MS) for microscale phosphoproteomic analysis. SOP-Phos adapts sodium deoxycholate based one-step lysis, reduction/alkylation, direct trypsinization, and phosphopeptide enrichment by TiO2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n-dodecyl ß-d-maltoside precoated tubes and shortening the digestion time, SOP-Phos is completed within 3-4 h with a 1.4-fold higher identification coverage. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved reproducibility (8%-10% CV). With a sample size-comparable spectral library, SOP-Phos-DIA identified 33,787 ± 670 to 22,070 ± 861 phosphopeptides from 5 to 0.5 µg cell lysate and 30,433 ± 284 to 6,548 ± 21 phosphopeptides from 50,000 to 2,500 cells. Such sensitivity enabled mapping key lung cancer signaling sites, such as EGFR autophosphorylation sites Y1197/Y1172 and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on EGFR-TKI sensitive and resistant cells, revealing the interplay of multipathway Hippo-EGFR-ERBB signaling cascades underlying the mechanistic insight into EGFR-TKI resistance. Overall, SOP-Phos-DIA is an efficient and robust protocol that can be easily adapted in the community for microscale phosphoproteomic analysis.