Experimental & Molecular Medicine

Angiogenesis, the process by which new blood vessels form from existing vasculature, is fundamental to tissue repair and regeneration but also underlies pathological conditions such as cancer progression. Targeting angiogenesis has thus become a promising approach for developing novel cancer therapeutics. While various phytochemicals have demonstrated anti-angiogenic effects, the role of 2-5(H)-Furanone, a naturally occurring lactone found in various plants and marine sources with diverse biological activities, remains insufficiently explored. In this study, we systematically evaluate the anti-angiogenic potential of 2-5(H)-Furanone using Human Umbilical Vein Endothelial Cells (HUVECs) as an in vitro model and zebrafish embryos as an in vivo model. Experimental findings demonstrated that treatment of HUVECs with increasing concentrations of 2-5(H)-Furanone led to significant, dose-dependent reductions in proliferation, invasion, migration, and tube formation. Analyses of gene expression revealed marked downregulation of key pro-angiogenic mediators, VEGF, and HIF-1. Complementing these in vitro results, in vivo studies in zebrafish embryos showed robust, dose-dependent inhibition of intersegmental vessel (ISV) formation, accompanied by suppression of critical angiogenesis-related genes. Molecular docking further supported these observations by indicating stable binding of 2-5(H)-Furanone to major angiogenic targets, including VEGFR2, MMP2, HIF-1, and PIK3CA. Collectively, our data demonstrate that 2-5(H)-Furanone potently inhibits angiogenesis, as evidenced in both HUVEC and zebrafish models, through functional and molecular mechanisms. These findings support the further development of 2-5(H)-Furanone as a promising anti-angiogenic therapy candidate.

The evolutionary trajectory of lung squamous cell carcinoma (LUSC) remains poorly defined, hindering the development of effective therapies. By integrating genomic and transcriptomic sequencing from human LUSC specimens, we delineated a polyclonal-to-monoclonal evolutionary trajectory during LUSC progression. This evolutionary pattern was corroborated by single-cell RNA sequencing, which revealed consistent tumor cell heterogeneity. Specifically, the SBS5 mutational signature was enriched and correlated with poor prognosis independent of tumor stage. By further utilizing the spontaneous LUSC mouse model to identify key genomic and genetic events in LUSC progression, we observed that the JNK pathway was inhibited and that cytoskeleton-related pathways were dysregulated during LUSC development, and identified the mutations in the JNK pathway (e.g., DACT1) and cytoskeletal regulators (e.g., KIF26A). Collectively, these findings established a polyclonal-monoclonal evolution paradigm for LUSC, potentially regulated by JNK pathways, which could benefit LUSC precision therapeutics.

Single-cell expression quantitative trait loci (sc-eQTL) analyses are powerful in identifying context-specific susceptibility genes from genome-wide association studies (GWAS) loci. However, few studies have comprehensively investigated cells of lung cancer origin in non-European populations. Here, we built a lung sc-eQTL dataset from 129 Korean women never-smokers with epithelial cell enrichment. eQTL mapping identified 2,229 genes with an eQTL in 33 cell types, including East Asian-specific findings when compared to predominantly European datasets. Integration with single-cell chromatin accessibility data demonstrated an enrichment of cell-type specific eQTLs in cell-type matched candidate enhancers, while shared eQTLs were more frequently found near promoters. Colocalization and transcriptome-wide association study unveiled 36 susceptibility genes from 22 cell types in 22 lung cancer loci, including 10 loci not achieving genome-wide significance in prior GWAS. Around 47% of these genes were from cells of the alveoli, underscoring their importance, especially in lung adenocarcinoma (LUAD) susceptibility. Focusing on the trajectory of alveolar epithelial cell regeneration, we detected 785 cell-state-interacting QTLs, which overlapped with 28% (10) of the identified susceptibility genes. Finally, we experimentally validated East Asian-and alveolar type 2 cell-specific eQTL of TCF7L2 underlying East Asian LUAD locus, 10q25.2. Consistent with its role as a Wnt/{beta}-catenin effector, TCF7L2 displayed significant effect on lung adenocarcinoma cell growth. Our data highlighted context-specific susceptibility genes, especially from alveolar cells of lung, contributing to lung cancer etiology.

Nitrogen mustard (NM)-caused severe cutaneous damage lacks effective targeted therapies. Vitamin D3 (VD3) shows promise as a therapy for NM-induced dermal toxicity; however, the underlying mechanisms remain elusive. Herein, we initially confirmed that NM induced gut flora dysbiosis, characterized by a decrease of Akkermansia muciniphila (AKK) abundance, thereby leading to butyrate reduction. Antibiotics (ABX) significantly promoted NM-induced skin injury, whereas fecal microbiota transplantation of the controls feces (HC-FMT) or AKK administration attenuated NM-induced dermal toxicity. HC-FMT or AKK significantly increased butyrate levels in feces and serum of NM-treated mice. Butyrate notably attenuated ABX-caused acceleration of NM-induced skin injury. Meanwhile, NM markedly decreased the expression of -defensins, MMP7, and VDR. NM failed to further decrease AKK abundance and BA contents in intestinal MMP7-deficient mice, which was abolished by human alpha defensin 5 (HD5) overexpression. And intestinal MMP7 deficiency enhanced NM-caused skin injury, which was markedly attenuated by HD5 overexpression, AKK transplantation, or BA supplementation. Moreover, NM also failed to further reduce MMP7 and -defensin expression, AKK abundance, and butyrate levels in intestinal VDR-silenced mice. Finally, VD3 remodeled the gut microbiome particularly enriching AKK, increased butyrate contents and promoted the expression of -defensins, MMP7, and VDR, thereby attenuating NM-induced skin damage. The protective effect of VD3 against NM-caused dermal toxicity was abolished by either ABX or intestinal-specific knockdown of MMP7 or VDR in mice; however, this impairment was reversed by butyrate or AKK. In conclusion, VD3 attenuated NM-caused dermal toxicity by promoting BA production via remodeling the gut microbiota, and this effect was partially mediated by the intestinal VDR--defensin signaling pathway. These highlight that targeting the gut flora or supplementing with BA could be potential therapies for NM-induced dermal toxicity.

Anemia is one of the most debilitating and frequent complications of inflammatory bowel diseases (IBD) and is often treated with iron supplementation, which has limited efficacy. Damaged intestinal barrier function is a hallmark of IBD and causes the translocation of endotoxins from gut bacteria into the bloodstream. In a previous study in mice, we reported that endotoxin suppresses erythropoiesis by reprogramming erythroblastic island macrophages (EBI M{varphi}). Here, we show that IBD patients and mice with acute colitis developed endotoxemia associated with anemia. Endotoxemia in IBD patients was negatively correlated with blood erythrocyte counts. In line with this, mice with acute colitis caused by drinking water containing dextrin sodium sulphate (DSS) had endotoxemia together with anemia characterized by reduced red blood cell counts, hemoglobin content and hematocrit., and reduced medullary erythropoiesis which was in part compensated by increased extramedullary erythropoiesis. As the endotoxin receptor TLR4 is expressed by CD169+ gut-resident macrophages and erythroid island macrophages in the bone marrow, we tested the hypothesis that TLR4 expressed by these CD169+ macrophages mediate both inflammatory colitis and anemia. Indeed, mice with conditional deletion of the Tlr4 gene specifically in CD169+ tissue-resident macrophages were protected from DSS-induced anemia and colitis. In addition, treatment of DSS mice with the TLR4 inhibitor C34 abated inflammation and anemia. These results suggest that endotoxins leaking from the inflamed gut may play a crucial role in IBD and associated anemia and that drugs targeting TLR4 may protect against IBD-associated anemia. Key pointsO_LIPatients with IBD and mice with acute colitis are anemic with increased endotoxemia and inflammation. C_LIO_LIEndotoxemia is inversely correlated with blood erythrocyte counts in IBD patients. C_LIO_LIConditional deletion of endotoxin receptor gene Tlr4 specifically in CD169+ tissue-resident macrophages or administration of synthetic TLR4 inhibitor significantly reduced colitis-induced anemia in mice. C_LI

Despite decades of research, current understanding of the spectrum of targets bound by kinase inhibitors remains incomplete. This complicates mechanism of action studies, drug repurposing, and understanding of adverse responses. Here, we describe kinome-wide profiling of an optimal kinase library (OKL) comprising 192 small molecules selected based on stage of clinical development, chemical diversity, and target coverage. Our results show that polypharmacology is widespread among kinase inhibitors independent of regulatory approval. The generally understood ("assigned") targets of approved molecules are not necessarily the most potently inhibited and off targets include multiple understudied kinases. Moreover, median selectivity has not increased over time. We illustrate the use of synoptic OKL-kinome profiles in identifying potential toxicity targets, repurposing anti-inflammatory drugs for neurodegenerative and infectious diseases, and performing chemical genetic studies. Our studies illustrate how much remains to be discovered about the chemistry and biology of one of the largest classes of human therapeutics.

Spinal muscular atrophy (SMA) is caused by loss of SMN protein and is increasingly recognized as a multisystem disorder involving molecular pathology beyond motor neurons. Recently, we identified NRF2-KEAP1 signaling as dysregulated in SMA mice. Because NRF2 coordinates transcriptional programs that maintain cellular redox homeostasis and adaptive stress responses, we investigated whether NRF2 signaling is similarly altered in SMA type I patient-derived fibroblasts and whether it can be pharmacologically engaged. Compared with control fibroblasts, SMA fibroblasts displayed reduced basal expression of NRF2 target proteins, including NQO1 and xCT (SLC7A11), along with decreased levels of PGC1. Omaveloxolone (OMAV), a pharmacological NRF2 activator approved for the treatment of Friedreichs ataxia, increased cell viability and upregulated NRF2 target proteins in both control and SMA fibroblasts. Notably, OMAV produced a modest increase in SMN protein abundance and PGC1 levels selectively in SMA cells. Together, these findings support diminished NRF2 pathway output as a feature of SMA fibroblasts and demonstrate that OMAV induces NRF2 target proteins in this human SMA cellular model, consistent with enhanced cytoprotective signaling. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/712434v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1904bfeorg.highwire.dtl.DTLVardef@6d20e2org.highwire.dtl.DTLVardef@89f365org.highwire.dtl.DTLVardef@ca9638_HPS_FORMAT_FIGEXP M_FIG C_FIG

Osteoporosis affects millions of women globally. In this study, we applied bioinformatics methods to screen for novel diagnostic biomarkers of osteoporosis in women using the GSE62402 and GSE56814 datasets. PCSK5, ZNF225, and H1FX were used to construct a diagnostic model. ROC, calibration, and decision curve analyses were performed to assess the diagnostic performance on the training (GSE56814) and external (GSE56815) datasets. The expression level of model genes was validated in GEO datasets. Furthermore, five transcription factors (ETS1, NOTCH1, MAZ, ERG, and FLI1) were identified as common upstream regulators of model genes. PCSK5, ZNF225, and H1FX serve as novel diagnostic biomarkers, providing new insights into the pathogenesis of and treatment strategies for osteoporosis in women.

Hyperuricemia, a major risk factor for gout and kidney disease, arises from the evolutionary loss of human uricase and remains a significant medical challenge due to its high prevalence. However, limited therapeutic options are available for refractory hyperuricemia that typically require long-term treatment. Here we developed a circRNA-based uricase replacement strategy and evaluated its efficacy in uricase-knockout mice as a model for severe hyperuricemia. Lipid nanoparticle-mediated delivery of circRNA enabled efficient in vivo expression of an engineered human-like uricase, which rapidly reduced serum urate levels after a single injection and maintained the urate-lowering effect for up to 10 days. Repeated administration led to sustained urate reduction for 10 weeks, mitigated renal injury, and exhibited favorable biosafety. These findings highlight the therapeutic potential of circRNA-based uricase replacement for the long-term treatment of hyperuricemia and its associated complications.

Mutations in human LIS1 cause lissencephaly, a severe developmental brain malformation. Although most stud-ies focus on development, LIS1 is also expressed in adult mouse tissues. We previously induced LIS1 knockout (iKO) in adult mice using a Cre-Lox approach with an actin promoter driving CreERT2 expression. This proved to be rapidly lethal, with evidence pointing toward nervous system dysfunction. CreERT2 activity was observed in astrocytes, brainstem and spinal motor neurons, and axons and Schwann cells in the sciatic and phrenic nerves, suggesting dysfunctional cardiorespiratory and motor circuits. However, it is unclear how LIS1 knockout in these different cell types contributes to the lethal phenotype. We now report that LIS1 depletion from astro-cytes is not lethal to mice (male or female), although glial fibrillary protein (GFAP) expression is increased in all LIS1-depleted astrocytes. In contrast, LIS1 depletion from projection neurons causes motor deficits and rapid lethality in both males and females. This is accompanied by progressive, widespread axonal degeneration along the entire length of both motor and sensory axons. Interestingly, sensory neurons harvested from iKO mice ini-tially extend axons in culture but soon develop axonal swellings and fragmentation, indicating axonal degenera-tion. LIS1 is a prominent regulator of cytoplasmic dynein 1 (dynein, hereafter), a microtubule motor whose dis-ruption can cause both cortical malformations and later-onset neurodegenerative diseases, such as Charcot-Marie-Tooth disease. Our results raise the possibility that LIS1 depletion, through disruption of dynein function in mature axons, may lead to Wallerian-like axon degeneration without traumatic nerve injury.

Motor performance and coordination deficits are hallmarks of spinocerebellar ataxias, yet effective disease-modifying therapies remain limited. Here, we investigate the expression of the interleukin 17 receptor subunit A (IL-17RA) in cerebellum and assess the therapeutic potential of its ligand in a mouse model for Spinocerebellar Ataxia Type 2 (SCA2). We found that IL-17RA is highly enriched in cerebellar molecular layer interneurons (MLIs), which provide inhibitory input to Purkinje neurons. In-vitro electrophysiological recordings revealed that symptomatic SCA2 mice exhibited increased spontaneous inhibitory synaptic input onto Purkinje neurons, which was normalized by IL-17A application to control levels. Concomitantly, IL-17A application restored Purkinje neuron firing, a parameter characteristically reduced in SCA2 mice. Behaviorally, intranasal administration of IL-17A restored motor performance of symptomatic SCA2 mice to control levels in both rotarod and beam-crossing assays. Collectively, our results indicate that IL-17A rescues Purkinje neuron dysfunction and motor deficits in SCA2 mice, highlighting IL-17A signaling as a promising therapeutic target for spinocerebellar ataxia.

Chronic pain affects billions globally, yet safe, long-lasting, and non-addictive analgesics remain lacking. Nav1.7 is a genetically validated pain target, but traditional small molecules have repeatedly failed. Therapeutic oligonucleotides-antisense oligonucleotides (ASOs) and siRNAs-offer selective, durable silencing. We developed N02C0702, an ASO-siRNA conjugate (ASC), achieving robust Nav1.7 knockdown and sustained analgesia without additional delivery vehicles. N02C0702 outperformed individual ASO (N02A114) and siRNA (N02S154) moieties at mRNA and protein levels and in pain relief. In CFA-induced inflammatory pain, a single intrathecal dose exceeded naproxen and suzetrigine, while in SNL neuropathic pain, efficacy persisted up to 56 days, comparable to or surpassing pregabalin. Genome-wide RNA sequencing confirmed minimal off-target effects. N02C0702 highlights Nav1.7 as a key analgesic target and demonstrates the ASC platforms potential for chronic pain and other CNS-related pathologies, offering durable, selective, and safe therapeutic effects.

Myocardial ischemia-reperfusion injury significantly exacerbates cardiac damage and worsens clinical outcomes, with oxidative stress in cardiomyocytes representing a central pathological mechanism. In this study, we reveal that APEX1, a key redox regulator, is markedly downregulated in cardiomyocytes under oxidative stress conditions. Functional analyses demonstrate that APEX1 knockdown intensifies oxidative stress-induced cardiomyocyte injury, whereas APEX1 overexpression confers robust protection against hypoxia reoxygenation mediated damage. Mechanistically, APEX1 exerts its cardioprotective effects by stabilizing the p53 protein and modulating its ubiquitination status. These findings establish APEX1 as a critical defender against oxidative injury in cardiomyocytes through direct regulation of p53 protein stability, highlighting its potential as a therapeutic target for ischemia-reperfusion related heart disease.

Major depressive disorders (MDD) are predicted to become the first cause of burden of disease worldwide in 2030, but 30% of patients still do not respond to antidepressants. Current rodent models of MDD mainly result either from one genetic or one environmental risk factor exposure, not recapitulating the multifactorial and polygenic nature of MDD. We recently generated a polygenic mouse model of MDD from selective breeding after mild stress in the Tail Suspension Test (TST), named H-TST. Here, we selected animals exhibiting high immobility during the Forced Swim Test (FST) to generate a new stable polygenic model of MDD, called H-FST. Unlike our previous H-TST model, H-FST mice did not exhibit any anxiety-or anhedonia-like behaviors, nor did they display any sleep disturbances. Moreover, H-TST and H-FST mice showed opposite response after administration of various antidepressant treatments. The gene expression level in the prefrontal cortex of H-TST and H-FST mice revealed little overlap in genes and biological pathways associated with depressive-like behaviors and opposite dysregulation of excitatory/inhibitory synaptic imbalance. Finally, these two models allowed in humans the identification biomarkers of treatment response specific of clinical subgroup of patients.

Localized rupture of the nuclear envelope has recently been reported in various pathologies, including cancer 1,2, neurodegenerative disease 3-5, myocardial infarction 6, as well as dilated cardiomyopathy caused by Lamin A/C gene mutations (LMNA-DCM) 7. Whether and how nuclear rupture contributes to disease remains unknown. Here, we report that nuclear rupture causes global transcriptional deficiency in a mouse model of LMNA-DCM. We observed that ruptured nuclei lost RNA polymerase II, leading to downregulation of numerous genes essential for cardiomyocyte structure and function. We identified endogenous resealing of nuclear rupture as a cardioprotective mechanism in LMNA-DCM mouse hearts. Resealing involved the ESCRT-III membrane remodeling complex recruited to nuclear rupture sites. Resealed nuclei restored transcription while inhibiting ESCRT-III activity accelerated cardiomyopathy. However, resealed nuclei were short-lived: they re-ruptured at twice the rate of resealing. A kinetic model predicted progressive accumulation of ruptured nuclei despite ongoing resealing. Consistently, a human LMNA-DCM heart contained numerous ruptured nuclei at disease presentation. These findings linked nuclear rupture to organ deterioration through global transcriptional deficiency and suggested rupture resealing as a critical modifier of nuclear rupture-associated conditions.

SEZ6L2 autoantibodies have been identified in patients with subacute cerebellar ataxia, but the underlying immune mechanisms and pathogenic pathways remain poorly understood. We previously established a C57BL/6 mouse model of SEZ6L2 autoimmunity that recapitulates key features of the disease. Here, we evaluated whether genetic background influences the magnitude and organization of SEZ6L2-directed immune responses. Pilot screening of autoimmune-prone strains identified SJL mice as exhibiting accelerated and enhanced antibody responses following SEZ6L2 immunization. In a large-cohort study, SEZ6L2-immunized SJL mice developed robust and sustained antibody responses, along with antigen-specific CD4 and CD8 T-cell activation. Expanded immune profiling revealed increased CNS infiltration of multiple lymphocyte populations, including CD4 T cells, CD8 T cells, B cells, and dendritic cells, as well as the presence of SEZ6L2-specific B cells within the brain. In addition, SJL mice exhibited strain-specific immunodominant T-cell epitopes distinct from those observed in C57BL/6 mice. Functionally, SEZ6L2-immunized SJL mice developed motor deficits consistent with cerebellar dysfunction. Integration of behavioral outcomes demonstrated a consistent overall impairment, and multivariate analysis revealed that coordinated humoral and cellular immune responses were associated with behavioral deficits. Together, these findings demonstrate that SEZ6L2-directed immune responses produce coordinated adaptive immune activation linked to neurological dysfunction and establish the SJL strain as an enhanced model for studying SEZ6L2 autoimmunity. This model also provides a platform for investigating disease mechanisms and therapeutic strategies.

Head and neck squamous cell carcinoma (HNSCC) remains associated with substantial morbidity and a 5-year overall survival rate of approximately 60%, reflecting persistent radio- and chemo-resistance and the lack of effective precision medicine strategies. Patient-Derived Tumor Organoids (PDTO) constitute promising functional models that may predict individual treatment response. In this study, we generated PDTO from surgically resected HNSCC of the oral cavity, oropharynx, larynx, and hypopharynx. A total of 20 long-term PDTO lines were established, maintaining growth over seven passages and successfully cryopreserved, capturing the molecular and clinical diversity of the patient cohort. These PDTO faithfully recapitulated histological features, major tumor marker expression, and the genomic and transcriptomic landscapes of their tumors of origin, with stability over time. Functional assays revealed heterogeneous responses to cisplatin and X-rays. Importantly, in vitro sensitivity of PDTO was associated with clinical outcome of patients at 24 months. Cisplatin response of PDTO predicted prognosis with 66.7% sensitivity and 100% specificity, while X-ray response showed 91.7% sensitivity and 75% specificity. Notably, all patients whose PDTO were classified as resistant to both cisplatin and X-rays experienced relapse and/or death within 24 months. Collectively, the successful long-term expansion and cryopreservation of HNSCC PDTO establish a stable and scalable preclinical resource that captures the molecular and clinical heterogeneity of the disease. This biobank provides a valuable platform for mechanistic studies and for the evaluation of innovative therapeutic strategies. This cohort represents one of the largest clinically annotated HNSCC PDTO collections to date, demonstrating a robust association between PDTO response to cisplatin and X-rays and patient prognosis. These findings support the predictive potential of PDTO-based functional assays and argue for their integration into standardized, rapid, and miniaturized precision oncology workflows for HNSCC.

Tuberculosis, often considered the worlds deadliest infectious disease, is associated with over one million deaths annually. The emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) makes anti-tuberculosis drug development a critical priority. Griselimycin (GM) is a cyclic peptide that targets the essential DNA sliding clamp of Mtb. While GM is a promising Mtb antibiotic, its poorly understood structure-activity relationship has stalled derivatization. To investigate the contribution of each amino acid towards its activity, we assessed the antibiotic activity of an alanine scan library in M. tuberculosis and M. smegmatis. Residues essential for activity and tolerable to modification were identified, and the impact of backbone N-methylation at each position was determined. Edits to cyclization chemistry, unnatural amino acid incorporation, and replacing the acetylated N-terminus with a free amine were also investigated. Lastly, incorporation of an N-terminal fluorophore enabled visualization of GM accumulation inside of mycobacteria both in and outside of macrophage cells, where Mtb natively resides. These findings present the first comprehensive structure-activity investigation into GM and can be used to rationally design future analogues.

Non-syndromic cleft lip and palate (NSCLP) represents the most prevalent and clinically severe subtype within non-syndromic orofacial cleft (NSOFC), and 2p24.2 is the most significant reported risk locus for NSCLP. However, the causal variant at 2p24.2 and the underlying pathogenic mechanism remain unclear, limiting clinical translation. Here, we defined a 104-kb linkage disequilibrium (LD) block tagged by the lead SNP rs7552 at 2p24.2. Through a two-stage genetic screen within this block, including targeted sequencing and replication involving 2,437 Chinese NSCLP patients and 2,391 unaffected individuals, we identified a common non-coding single-nucleotide polymorphism, rs4263114, at 2p24.2 as the causal variant that confers susceptibility to NSCLP by residing within a previously unrecognized enhancer. Mechanistically, this enhancer physically bridges to the MYCN promoter through distal spatial contact, implicating MYCN as the pathogenic gene at this locus. Specifically, the rs4263114 risk variant reduces the recruitment of FOXP2 to the enhancer and disrupts liquid-liquid phase separation (LLPS)-driven droplet assembly. This biophysical defect impairs MYCN transcriptional activation and subsequently suppresses cranial neural crest cell (cNCC) differentiation. Notably, MYCN expression in cNCCs carrying homozygous risk alleles were partially restored by promoting FOXP2 LLPS. Collectively, our study functionally annotates the 2p24.2 locus and identifies a mechanism by which a non-coding variant disrupts transcription factor phase separation to increase susceptibility to NSCLP, providing a basis for future clinical translation.

Reliable, minimally invasive biomarkers for predicting immunotherapy response in head and neck squamous cell carcinoma (HNSCC) remain an unmet clinical need. Here, using patients from a prospective, multi-institutional phase II clinical trial (NCT02641093), we performed whole genome sequencing of 185 plasma cell-free DNA (cfDNA) samples collected longitudinally from 68 patients with locally advanced, surgically resectable HNSCC undergoing neoadjuvant and adjuvant pembrolizumab treatment. We developed the regional motif diversity score (rMDS), a novel fragmentomic metric quantifying the entropy of cfDNA 5' end motifs across genomic regions. Remarkably, unsupervised analysis revealed that rMDS robustly distinguished immunotherapy responders from non-responders, outperforming established cfDNA fragmentomic metrics and copy number alterations, while demonstrating independence from technical confounders. Longitudinal analysis revealed dynamic rMDS changes in genomic regions enriched for immune, lectin, and keratinization-related genes, hallmarks of squamous cell carcinoma, reflecting the interplay between tumor and peripheral immunity during the immunotherapy treatment. Interestingly, the regions with the most dynamic rMDS changes were highly enriched in telomere proximal loci, suggesting a novel link between telomere biology and cfDNA fragmentation. A machine learning classifier based on rMDS achieved robust predictive performance across multiple validation settings (AUC 0.89-0.99), with the highest accuracy at post-treatment timepoints and superior to PD-L1 expression and tumor fraction in the same sample. Predicted responders demonstrated significant trends toward improved disease-free survival (log rank test p=0.035, hazard ratio: 2.67, 95% confidence interval: 1.03-6.92), underscoring the clinical utility of rMDS-based stratification. These findings position rMDS as a biologically meaningful and clinically actionable biomarker for immunotherapy response in HNSCC, supporting its integration into future risk assessment frameworks and broader cancer care.