Gene Set Enrichment Analysis of single cell RNAseq data reveals downregulation of several inflammation-regulated pathways in monocytic populations.

Changes in MIB binding ordered by p value (unpaired t-tests of log2LFQ).

Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1), encoded by the gene INPP5D, is a lipid phosphatase that negatively regulates immune receptor signaling in hematopoietic cells and microglia. Here, we describe a pyridyl-pyrazole-piperidine scaffold and the lead compound 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (32), which demonstrates SHIP1 target engagement, brain exposure, and evidence of a central pharmacodynamic response in vivo. Structure-activity relationship studies, guided by biochemical and cellular assays using multiple human and murine protein constructs and cells, identified SHIP1-active ligands. A thermal shift assay using full-length SHIP1 was used to assess compounds for cellular target engagement, while studies in IL-4 conditioned THP-1 cells was used to demonstrate changes in downstream AKT signaling. Targeted lipidomics revealed changes in the overall phosphoinositide pool consistent with SHIP1 target engagement and reduction of phospho-AKT levels. In a protein-lipid overlay assay, compound 32 induced changes in the relative association of SHIP1 with multiple phosphatidylinositols on a membrane surface. In high-content cellular imaging assays, compound 32 enhanced the uptake of myelin/membrane debris and fibrillar amyloid by primary murine microglia, phenocopying a genetic model with reduced SHIP1 expression. Finally, oral administration of compound 32 resulted in brain exposure sufficient to alter gene expression and reduce IL-1β levels as pharmacodynamic markers of microglial activation and neuroinflammation in an amyloidosis mouse model of Alzheimer's disease. Collectively, these results define a scaffold with SHIP1 target engagement, CNS exposure, and in vivo activity, providing a foundation for the optimization of brain-penetrant SHIP1 ligands suitable for further mechanistic studies and therapeutic development for the treatment of Alzheimer's disease.

Abstract Osteosarcoma (OS) in pediatric, adolescent, and young adult (AYA) patients is an aggressive malignancy with limited therapeutic progress. About 40% of OS patients develop metastases over time with 15-20% of OS patients already exhibiting metastases at initial diagnosis. Thus, there is a critical need to develop better therapeutics. Genomic analyses from our institution and others highlight recurrent alterations in CDKN2A and CDK4/6, suggesting that CDK4/6 inhibition (CDK4/6i) is a rational therapeutic vulnerability. Although RB1 proficiency (RB1+) is considered essential for CDK4/6i response, over 70% of OS tumors are RB1-deficient (RB1–), raising questions about the utility of CDK4/6i in these patients. Emerging evidence in other solid tumors and in our preliminary OS studies, suggests that CDK4/6i may retain antitumor activity in RB1– contexts. Compounding this complexity, pharmacologic CDK4/6 blockade induces compensatory PI3K/mTOR signaling, which can restore cyclin D–CDK4/6 activity and drive resistance. We hypothesized that co-targeting PI3K/mTOR would enhance CDK4/6i efficacy irrespective of RB1 status by suppressing adaptive signaling. We evaluated palbociclib, voxtalisib, and the combination across RB1+ and RB1- OS models, using cell lines, patient derived xenografts (PDXs), and an experimental metastasis model. In vitro drug interactions (Chou–Talalay, Bliss) and mechanistic assays (cell cycle, senescence, autophagy) were complemented by in vivo tumor growth kinetics and pharmacodynamic profiling through histopathology, kinome, and proteomic analyses. In RB1+ OS cells in vitro, palbociclib induced G1 arrest and senescence, accompanied by increased PI3K/AKT phosphorylation consistent with adaptive feedback. Voxtalisib suppressed this response, reinforced autophagic signaling, and maintained pathway inhibition. In vivo, combination therapy was well tolerated and produced significant tumor growth suppression in treatment-naïve and metastatic PDXs. In the RB1+ lung-colonization model, CDK4/6i alone reduced metastatic burden, with combination therapy achieving comparable control. To interrogate RB1 as a biomarker, CRISPR-engineered human and mouse RB1– OS clones (MG63.3, K7M2) were generated and characterized. As expected, voxtalisib response was RB1-independent, and palbociclib sensitivity was reduced in RB1- cells. However, RB1- cells still exhibited growth inhibition at higher palbociclib concentrations. Notably, combination therapy of palbociclib+voxtalisib produced additive-to-synergistic growth inhibition regardless of RB1 status. In vivo validation studies are ongoing. These findings identify convergent CDK4/6–PI3K/mTOR hyperactivation as a targetable axis in OS and support further evaluation of CDK4/6i-based strategies, including in RB1-deficient disease. Citation Format: Lauren K. Stevens, M. Reza Saadatzadeh, Farinaz Barghi, Erika A. Dobrota, Harlan E. Shannon, Rada Malko, Ryli Justice, Christopher Davis, Keiko Kreklau, Melissa A. Trowbridge, Kathy Coy, Felicia M. Kennedy, Anthony L. Sinn, Kyle W. Jackson, George Sandusky, L. Daniel Wurtz, Christopher D. Collier, Dana Mitchell, Ed Greenfield, Emma H. Doud, Amber L. Mosley, Steven P. Angus, Pankita H. Pandya, Karen E. Pollok. Convergent CDK4/6 and PI3K/mTOR pathway hyperactivation defines a targetable axis in osteosarcoma across RB1-proficient and RB1-deficient contexts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6483.

Data from Correlative Molecular Analysis at Various Time Points.

SRC proto-oncogene, non-receptor tyrosine kinase expression in scRNAseq.

Hyperactive RAS signaling, induced by mutations in NRAS, HRAS, or KRAS, drives tumorigenesis in most PAX3/7::FOXO1 fusion-negative rhabdomyosarcomas (FN-RMS). Despite the frequency of these mutations, indirect RAS pathway-directed therapies have been ineffective for RAS-driven RMS. Farnesyltransferase (FTase) inhibitors (FTI), such as tipifarnib, inhibit HRAS membrane localization and blunt RAS effector signaling, leading to an antitumor effect in HRAS-mutant FN-RMS preclinical models. However, the effect is not durable. In this study, we investigated the mechanisms of adaptive resistance that limit the activity of FTIs, revealing that response to FTIs was limited by adaptive feedback reactivation of ERK signaling and upregulation of wild-type RAS. The combination of HRAS suppression with FTI and MEK inhibition impaired ERK reactivation and reduced ERK transcriptional output in HRAS-mutant RMS models. Cotargeting FTase and MEK restrained tumor progression and induced terminal myogenic differentiation. These findings highlight an effective combinatorial strategy and support its preclinical translation for patients with HRAS-mutant RMS. SIGNIFICANCE:Farnesyltransferase and MEK inhibition suppresses ERK reactivation, decreases tumor growth, and promotes myogenesis in HRAS-mutant rhabdomyosarcoma.

Genetic and methylation analysis across timepoints.

Osteosarcoma (OS) in pediatric, adolescent, and young adult (AYA) patients is an aggressive bone cancer with limited treatment options. Dysregulation of the CDK4/6–cyclin D axis and the PI3K/mTOR pathway contributes to OS pathogenesis, providing a biological rationale for co-targeting these signaling nodes. However, pharmacologic CDK4/6 inhibition can trigger compensatory activation of the PI3K/mTOR pathway, restoring D-type cyclin expression and partially reactivating CDK4/6 signaling. Thus, dual inhibition of the CDK4/6 and PI3K/mTOR pathways not only addresses two parallel oncogenic drivers but may also prevent potential CDK4/6 inhibitor resistance mediated by feedback activation of PI3K/mTOR. In this study, we tested the hypothesis that coordinated targeting of these pathways would improve tumor control in preclinical OS models. In vitro sensitivity analyses using palbociclib and voxtalisib demonstrated additive to synergistic OS growth suppression, with palbociclib inducing G1 arrest and senescence, and the combination enhancing autophagy. Furthermore, the efficacy, tolerability, and mechanisms of palbociclib and voxtalisib, alone or in combination, were evaluated in molecularly defined primary treatment-naïve, and relapsed/metastatic OS models. In the relapsed/metastatic PDX77-TT2 model, short-term palbociclib exposure activated PI3K/mTOR signaling, whereas the combination of palbociclib and voxtalisib in long-term studies produced marked tumor suppression and extended survival. In the primary treatment-naïve PDX96 model, long-term palbociclib exposure generated a robust CDK4/6 pharmacodynamic response. The addition of voxtalisib reinforced autophagy, sustained CDK pathway inhibition, and improved overall tumor control. In an OS lung-colonization model, CDK4/6 inhibition alone markedly reduced OS lung nodules, with combination therapy providing comparable suppression. Dual CDK4/6–PI3K/mTOR inhibition achieves tumor control across various OS models, supporting the use of genomically guided, pathway-targeted strategies for pediatric and AYA OS.

Adverse events, grade 3+ with attribution of possible, probable, or definite.

Responders and non-responders have distinct patterns of kinase/transcript changes after exposure to trametinib.

Type 1 diabetes (T1D) is characterized by autoimmune destruction of pancreatic β-cells, insulin insufficiency, and hyperglycemia. Receptor interacting protein kinase 1 (RIPK1) is a multifunctional regulator of cell fate with kinase and scaffolding functions, and we previously identified RIPKs as regulators of β-cell cytotoxicity in vitro. Here we report that Ripk1 expression is increased in islets from aged non-obese diabetic (NOD) mice and β-cells from T1D donors, suggesting that RIPK1 may drive cytokine- and autoimmune-mediated β-cell demise in T1D. Using NIT-1 β-cells derived from NOD mice, we observed that TNFα + IFNγ increases RIPK1 phosphorylation, caspase 3/7 activity, and cell death. In contrast, this cytotoxicity was blocked with small molecule RIPK1 inhibition or in Ripk1 gene-edited (Ripk1Δ) β-cells. Small molecule caspase inhibition studies and co-labeling of caspase 3/7 activation and cell death in single cells revealed protection from caspase-dependent and -independent forms of death in Ripk1Δ cells. RNAseq uncovered differential cell death-, immune-, and identity-related gene expression, and kinome profiling identified changes in MAPK, Eph, JAK, and other kinase activity associated with protection from cell death in RIPK1 deficient β-cells. Furthermore, in vitro co-culture assays and in vivo adoptive transfer experiments revealed that NIT-1 Ripk1Δ cells are protected from autoimmune destruction by splenocytes isolated from diabetic NOD mice. Collectively, our findings indicate that RIPK1 promotes β-cell demise in response to cytokine and autoimmune stress via actions on gene expression and kinase signaling. Therapeutics targeting RIPK1 may provide novel opportunities for the prevention or treatment of autoimmune diabetes.

Representativeness of Study Participants.

Downregulation of MAPK and inflammatory signaling after trametinib treatment.

Kinases are central regulators of multiple signaling cascades, controlling processes such as cellular growth, proliferation, and differentiation. Given their vital role within the cell, dysregulated kinase activity contributes to several skin diseases, including melanoma and dermatitis. Poor disease response or resistance to targeted inhibitors can be driven by adaptive kinase responses. Genomic assays are highly informative but do not accurately capture kinase abundance and activity at the protein level. In this paper, we review 2 complementary mass spectrometry-based proteomics methods for functional kinome analysis that are readily applicable to dermatology research. Multiplexed inhibitor beads coupled with mass spectrometry (MIB-MS) uses broad-spectrum, immobilized kinase inhibitors to enrich for kinases in active conformation, providing an unbiased, pathway-level readout of kinase network dynamics, adaptive rewiring, and drug specificity. Internal standard triggered-parallel reaction monitoring (IS-PRM)-targeted proteomics, including the Thermo SureQuant acquisition method, leverages heavy peptide triggers to deliver sensitive, consistent quantification of predefined kinase peptides from limited input clinical specimens, including formalin fixed, paraffin embedded. We summarize optimized workflows, instrument set-up, sample requirements, technical considerations, and limitations. Together, MIB-MS and IS-PRM SureQuant offer orthogonal, scalable strategies to profile kinase networks in skin biology and to inform target discovery, biomarker development, and rational therapeutic strategies.

Multidrug-resistant bacteria necessitate innovative antibacterial strategies. Bacteriophages (phages) offer a promising alternative; however, bacterial immune defenses limit their effectiveness. Small-molecule inhibitors of these defenses may facilitate mechanistic studies and serve as adjuvants to enhance phage therapy. Here, we identify inhibitors targeting the bacterial cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector nuclease Cap5. Cap5 is hypothesized to degrade genomic DNA in virally infected cells, leading to cell death through abortive infection. Guided by the crystal structure of the Cap5 SAVED domain bound to its activating ligand, we performed structure-guided virtual screening to identify candidate inhibitors. Biochemical assays revealed ∼16% of the top docking hits inhibited Cap5. Cellular assays revealed one compound could enter E. coli cells and inhibit Cap5 activity. Our integrated approach—combining structure-based virtual screening with biochemical validation—provides a framework for discovering small-molecule inhibitors of bacterial immune defenses to advance adjunctive therapies and deepen our understanding of phage-bacteria interactions.

NF2 (neurofibromatosis type 2)-related schwannomatosis (NF2-SWN) is a cancer predisposition syndrome characterized by the development of bilateral vestibular (VS) and spinal schwannomas. While benign, these tumors can cause substantial morbidity, and effective pharmacological treatments remain limited. Here, we demonstrate that genetic ablation of focal adhesion kinase (Fak/Ptk2) impairs tumor formation and preserves hearing in a murine model of NF2. Mechanistically, we show that Fak deletion decreases macrophage infiltration, attenuates nucleotide-binding oligomerization domain-containing protein 2-, leucine rich repeats (LRR)- and pyrin domain-containing protein 3 inflammasome activation, and suppresses the hepatocyte growth factor-MET axis. Pharmacological inhibition of FAK with single agent VS-4718 did not significantly reduce macroscopic tumor volume; however, its use in combination with the mitogen-activated protein kinase kinase (MEK) inhibitor selumetinib resulted in both a significant reduction in tumor volume and the preservation of dorsal root ganglion architecture. Our findings establish a critical role for FAK in schwannoma development and provide rationale for evaluation of combination FAK plus MEK inhibition in future clinical trials for NF2-associated SWN.