Elevated Epidermal Growth Factor Receptor (EGFR) expression is observed in most cervical cancers, and it is frequently associated with poor clinical outcomes. The limited efficacy of existing EGFR-targeted therapies in cervical cancer highlights the need for a deeper understanding of EGFR role in this cancer type. To investigate EGFR separately from its interaction with Epidermal Growth Factor (EGF), we removed the key amino acids from the ligand bindings site. We used CRISPR/Cas9 genome editing to generate a panel of EGFR mutant cell lines and then sequenced and characterized them in detail. Studying the phenotypes of mutant cell clones, we show that a pair of amino acid substitutions L14R and Y45M within Domain I of EGFR protein completely disrupts EGF binding and changes EGFR subcellular distribution. A single substitution Y45M significantly reduced EGF binding but did not lead to subcellular redistribution of EGFR. Upon editing, EGFR mRNA and protein expression were decreased in mutant clones compared to wild type cells. Genome wide profiling of different CRISPR/Cas9 clones confirmed correct editing of EGFR with no off target CRISPR/Cas9 generated mutations. At the same time, spontaneous mutations that could impact cell phenotypes were detected in mutant clones. Disruption of ligand binding domain of EGFR by sequential knock in CRISPR/Cas9 genome editing altered subcellular localization and phosphorylation of EGFR in cervical cancer cells. The results presented here provide insights that may accelerate the development of CRISPR/Cas9-based therapies for EGFR-dependent cancers and reinforce the importance of thorough evaluation of CRISPR/Cas9-generated phenotypes.

Abstract Background: To develop effective therapies for DIPG at different clinical stages, we examined efficacy and mechanisms of action of a combination therapy—abemaciclib (a CDK4/6 inhibitor), temozolomide (TMZ), and radiation (XRT)—in two patient-derived orthotopic xenograft (PDOX) models derived from treatment-naïve (IBs-9119DIPG) and autopsied (IBs-A0317DIPG) tumors. Methods: In vitro synergistic anti-tumor activities were examined in tumor organoids, and in vivo efficacy in the PDOX models treated with abemaciclib (75 mg/Kg × 14 days), TMZ (50 mg/Kg × 5 days) and XRT (2 Gy/day × 5 days) alone and in combination (n=10/group, 60 mice/model). Changes of animal survival times were analyzed with log-rank analysis. Mechanisms of treatment response and resistance were elucidated by immunohistochemistry and scRNA-seq analysis. Results: The triple therapy generated synergistic anti-tumor effects in organoids and significantly extended survival times in both PDOX models (P<0.05) despite their strong cellular state differences. scRNAseq identified reduction of oligodendrocyte-progenitor-like (OPC-like) cells in both models and astrocyte-like (AC-like) cells in IBs-A0317DIPG as response mediators; and revealed expansion of neural progenitor-like (NPC-like) cells in IBs-A0317DIPG and of mesenchymal-like (MES-like) and mitotic-like cells in IBs-9119DIPG as resistance contributors. Pseudotime trajectory analysis uncovered the exit of stemness into differentiation in oligodendrocyte-progenitor-like (OPC-like) cells as a novel mechanism of resistance in the treatment-naïve IBs-9119DIPG, in contrast to the enrichment of stem-like cells in the recurrent model IBs-A0317DIPG. A radiation-resistant subpopulation with novel candidate targets (NPAS3, TBC1D5, INPP4B) was also discovered. Conclusions: This study demonstrated strong anti-DIPG capacities of the triple therapy in both untreated and recurrent DIPG tumors by acting on distinct cellular and molecular targets, and identifies previously unrecognized mechanisms underlying DIPG therapy response and resistance. Citation Format: Zilu Huang, Tongchao Jiang, Milagros M. Suarez Palacios, Tommy Ouyang, Aalaa Abdallah, Long Niu, Jinnan Chen, Xin Zhai, Emily Ciolak, Wenan Qiang, Runxin Wu, Nitin Wadhwani, Alicia Lenzen, Michael DeCuypere, Sandi Lam, Shi-Yuan Cheng, Ching Man Wai, Brian Wray, Matthew John Schipma, Xinkun Wang, Wan-Yee Teo, Daniel J. Brat, Yuchen Du, Yunfei Xia, Xiao-Nan Li, John Kalapurakal. Combining CDK4/6 inhibitor with TMZ and radiation alters cell states for synergistic responses in orthotopic DIPG models [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 1150.

Diffuse intrinsic pontine glioma (DIPG) is a highly lethal and therapeutically refractory pediatric brain tumor, and the effects of distinct disease backgrounds on treatment response and residual cell-state plasticity remain poorly understood. We evaluated abemaciclib, an FDA-approved CDK4/6 inhibitor, combined with temozolomide (TMZ) and radiation (XRT) in two patient-derived orthotopic xenograft (PDOX) models established from a treatment-naïve biopsy (IBs-9119DIPG) and a previously treated autopsy tumor (IBs-A0317DIPG). Treatment activity was assessed in PDOX-derived 3D tumor organoids and in randomized DIPG PDOX studies, followed by survival analysis, immunohistochemistry, and endpoint single-cell RNA sequencing (scRNA-seq). The triple therapy generated synergistic antitumor effects in PDOX-derived organoids and significantly prolonged survival in both PDOX models (P < 0.05) despite their distinct baseline molecular and cell-state differences. Endpoint scRNA-seq revealed reduced oligodendrocyte-progenitor-like (OPC-like) cells in both models and decreased astrocyte-like cells in the IBs-A0317DIPG model as cell-state changes associated with treatment response. In contrast, neural progenitor-like (NPC-like) cells expanded in IBs-A0317DIPG, whereas mesenchymal-like and Mitotic populations persisted in IBs-9119DIPG as candidate therapy-tolerant states. Pseudotime trajectory analysis uncovered a resistance-associated trajectory characterized by an exit from stemness toward differentiation in OPC-like cells in treatment-naïve IBs-9119DIPG, in contrast to the enrichment of stem-like OPC-like and NPC-like cells in therapy-resistant IBs-A0317DIPG. A transcriptionally defined radiation-resistance-associated subpopulation with candidate radiosensitization target genes (NPAS3, TBC1D15, and INPP4B) was also identified. Overall, the triple therapy improved survival in clinically distinct DIPG PDOX models and revealed therapy-associated residual cell-state changes that may inform future strategies to improve durable DIPG tumor control.

2073 Background: Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, with two-year survival less than 10%. Radiation therapy (XRT) offers limited survival benefits, and effective therapies are urgently needed. This study investigates the FDA-approved CDK4/6 inhibitors-Abemaciclib, XRT, and temozolomide (TMZ) as a therapeutic regimen for DIPG using organoids and patient-derived orthotopic xenograft (PDOX) models, aiming to improve survival outcomes and gain insights into the underlying cellular and molecular mechanisms of DIPG treatment responsiveness and resistance to support rapid translation into clinical trials. Methods: The efficacy of Abemaciclib, XRT, TMZ, and their combinations was evaluated in DIPG organoids and PDOX models (IBs-A0317DIPG and IBs-9119DIPG, H3.3K27M mutation). In vitro, PDOX organoids were treated with Abemaciclib, TMZ, with/without XRT. Synergy was assessed using the Bliss Independence model. In vivo, six treatment arms were tested: (1) control, (2) XRT (2 Gy/day × 5), (3) Abemaciclib (75 mg/kg, p.o., 14 days), (4) Abemaciclib + XRT, (5) TMZ (50 mg/kg, p.o., 5 days) + XRT, (6) Abemaciclib + TMZ + XRT. Single-cell RNA sequencing and IHC were used to assess cellular subtypes responses, gene expression changes, and resistance mechanisms. Results: In DIPG organoids, the combination treatment yielded Over Bliss values > 0 (0.25 and 0.58 in A0317DIPG and 9119DIPG models, respectively) demonstrating synergistic activities. In PDOX models, the triple therapy showed improved median survival compared to other treatment arms and significant survival advantage over control ( p = 0.0157) and Abemaciclib alone ( p = 0.0461) in A0317DIPG, and control ( p < 0.0001), XRT alone ( p = 0.0032), Abemaciclib alone ( p = 0.0006), Abemaciclib + XRT ( p = 0.0046), and TMZ + XRT ( p = 0.0001) in 9119DIPG models. Single-cell RNA sequencing revealed six tumor subtypes: AC-like, NPC-like, OPC-like, MES-like, mitotic, and radiation-resistant cells. The triple therapy increased NPC-like and mitotic cell populations while decreasing AC-like and OPC-like cells in both models. Additionally, we identified a novel radiation-resistant subpopulation that expanded after XRT treatment. Dynamic gene expression analysis in different cell types identified key target genes and cell-type specific pathways that mediate therapy responsiveness and resistance. Conclusions: Our study demonstrates that the combination of Abemaciclib, TMZ, and XRT offers a novel, synergistic approach for DIPG, significantly improving survival in preclinical PDOX models. Single-cell RNA sequencing reveals the roles of different cell types and molecular changes underlying resistance, highlighting potential targets for future anti-resistance strategies in DIPG management.

Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain cancer. Here, we demonstrated that combining the CDK4/6 inhibitor abemaciclib with temozolomide and radiation (the triple therapy) yields synergistic anti-tumor effects in DIPG organoids and significantly extends survival times in patient-derived orthotopic (intra-brain stem) xenograft (PDOX) models. Median survival times increased from 42 days in the control group to 54 days (19% increase) in triple combination in IBs-9119DIPG (P < 0.001) and from 103 days to 140.5 days (30% increase) in IBs-A0317DIPG (P < 0.05). Mechanistically, we performed scRNA-seq and identified dynamic reprogramming of tumor cell states, including the expansion of NPC-like, and NPC-like cells displayed a consistent treatment-resistant profile across both DIPG models, with notable enrichment under triple therapy. In IBs-A0317DIPG, NPC-like cells rose from 39.7% in the control to 76.8% (1.9-fold) under the triple therapy. This trend was paralleled by increasing median potency scores, peaking at 0.378 with the triple combination, as well as decreasing pseudotime values across all treatment groups. In IBs-9119DIPG, although baseline NPC-like fractions were low (~1%), they also rose by ~1.5-fold (to 1.5%) following triple therapy. Concurrently, persistent MES-like and mitotic-like populations contributed significantly to therapeutic resistance, while OPC- and AC-like subpopulations were depleted, potentially enhancing therapy responsiveness. Pseudotime trajectory analysis further revealed therapy-induced cell fate transitions within NPC- and OPC-like compartments, including the enrichment of stem-like cells. More importantly, we discovered that an alternative trajectory—exit from stemness toward more differentiated states—may also contribute to therapeutic failure. Additionally, we identified a novel radiation-resistant subpopulation and found a new set of transcriptional targets indicative of therapeutic vulnerability. This study demonstrated the triple therapy as a promising DIPG regimen that can be rapidly translated into clinical trials and nominated new cellular and molecular targets to overcome residual disease.

Increased expression of a set of homeodomain transcription factors, including HoxA10, characterizes an adverse prognosis subtype of acute myeloid leukemia (AML). Examples of this subtype include AML with KMT2A or MYST3/CREBBP gene rearrangements, and an AML subset with normal cytogenetics. Previously, we identified ARIH2, the gene encoding Triad1, as a HoxA10 target gene. We determined that transcriptional activation of ARIH2 by HoxA10 was necessary to terminate emergency granulopoiesis during the innate immune response but also antagonized leukemogenesis in a murine model of KMT2A-rearranged AML. Triad1 expression progressively decreases during the latent period preceding AML in this model, and Triad1 knockdown accelerates AML development. Triad1 is an E3 ubiquitin ligase, and we found that knocking down Triad1 decreased protein ubiquitination in myeloid cells. Therefore, proteins with Triad1-dependent ubiquitination might regulate leukemogenesis and/or the innate immune response. By proteomic screen, we identified Triad1-dependent ubiquitination of a set of proteins that regulate the integrated stress response (ISR), including Gcn1. The ISR prevents metabolic exhaustion during sustained inflammation by decreasing total mRNA translation and global protein synthesis, while altering the translatome to correct metabolic deficiencies and inhibit apoptosis. In cells with Triad1-knockdown, we defined a translatome consistent with ISR-activation and reversed by co-knockdown of Gcn1. Gcn1-knockdown also delayed AML development in a KMT2A-rearranged murine model, and reversed the effects of Triad1-knockdown on leukemogenesis. These results suggest ISR-inhibition mediates Triad1-related leukemia suppression, and activation of the ISR enhances leukemogenesis in this adverse-prognosis AML subtype.

Neutrophil (PMN) tissue accumulation is an established feature of ulcerative colitis (UC) lesions and colorectal cancer (CRC). To assess the PMN phenotypic and functional diversification during the transition from inflammatory ulceration to CRC we analyzed the transcriptomic landscape of blood and tissue PMNs. Transcriptional programs effectively separated PMNs based on their proximity to peripheral blood, inflamed colon, and tumors. In silico pathway overrepresentation analysis, protein-network mapping, gene signature identification, and gene-ontology scoring revealed unique enrichment of angiogenic and vasculature development pathways in tumor-associated neutrophils (TANs). Functional studies utilizing ex vivo cultures, colitis-induced murine CRC, and patient-derived xenograft models demonstrated a critical role for TANs in promoting tumor vascularization. Spp1 (OPN) and Mmp14 (MT1-MMP) were identified by unbiased -omics and mechanistic studies to be highly induced in TANs, acting to critically regulate endothelial cell chemotaxis and branching. TCGA data set and clinical specimens confirmed enrichment of SPP1 and MMP14 in high-grade CRC but not in patients with UC. Pharmacological inhibition of TAN trafficking or MMP14 activity effectively reduced tumor vascular density, leading to CRC regression. Our findings demonstrate a niche-directed PMN functional specialization and identify TAN contributions to tumor vascularization, delineating what we believe to be a new therapeutic framework for CRC treatment focused on TAN angiogenic properties.

Supplemental Figure 2. Histological evaluation of tumor progression and confirmation of IDO-mGFP expression in brain tumors.

Abstract BACKGROUND Circulating tumor DNA (ctDNA) assays are being evaluated to inform clinical decisions in cancer care in adult patients, but the study in pediatric brain tumor patients is rare partly due to very low levels of ctDNA in blood. Currently, treatment decisions require invasive diagnostic surgical biopsies that carry risks and morbidity. The aim of this study is to utilize methylomes from plasma ctDNA for non-invasive diagnosis in pediatric brain tumor patients. METHODS Using cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq), which with low input DNA requirement, we measured methylation profiles of plasma samples from 77 pediatric brain tumor patients and 16 patients with non-neoplastic diseases like epilepsy. Binomial GLMnet classifiers of tumor or tumor subtype were built, in 2000 iterations of 60% training sets. Performance was evaluated in 40% test sets. RESULTS. These 77 pediatric brain tumor patients were a mixture of the most common pediatric brain tumors, include circumscribed astrocytic gliomas (N=23), glioneuronal tumors (N=16), embryonal tumor (N=9), ependymal tumor (N=7), pediatric type diffuse high grade glioma (N= 5), glioma NOS (N=4), mesenchymal tumor (N=4) and other pediatric brain tumors (N=9). The methylation profile differentiated brain tumors from non-neoplastic diseases with 82.9% accuracy (precision = 92.6%, sensitivity = 86.2%, specificity = 66.7 %). For subtype analysis, the methylation profile detected circumscribed astrocytic glioma from non-neoplastic diseases with 85.7% accuracy (precision = 87.5%, sensitivity = 87.5 %, specificity = 83.3 %), and glioneuronal tumors from non- neoplastic diseases with 83.3% accuracy (all precision, sensitivity, and specificity = 83.3 %). CONCLUSIONS The results suggest that methylation profiling of plasma cell-free DNA has the potential to discriminate between pediatric brain tumor patients and patients with non- neoplastic diseases. Larger population cohorts to train even more accurate classifiers will be needed.

Supplementary Table 4. Mass spectrometry parameters for detecting CFH and FHL-1 in plasma.

Supplemental Figure 1. Overall survival of syngeneic mice with intracranial IDO-/-tGBM tumors and depleted for CD4+ T, CD8+ T, and NK1.1+ immune cells.

Supplementary Table 2. Nucleic acid-based reagents utilized.

Supplementary Table 1. Demographics of patient-isolated tissue samples.

Supplementary Table 3. Differentially expressed genes that possess the strongest correlation with IDO in human GBM cells.

Suppressor of cytokine signaling-1 (SOCS1) exerts control over inflammation by targeting p65 nuclear factor-κB (NF-κB) for degradation in addition to its canonical role regulating cytokine signaling. We report here that SOCS1 does not operate on all p65 targets equally, instead localizing to a select subset of pro-inflammatory genes. Promoter-specific interactions of SOCS1 and p65 determine the subset of genes activated by NF-κB during systemic inflammation, with profound consequences for cytokine responses, immune cell mobilization, and tissue injury. Nitric oxide synthase-1 (NOS1)-derived nitric oxide (NO) is required and sufficient for the displacement of SOCS1 from chromatin, permitting full inflammatory transcription. Single-cell transcriptomic analysis of NOS1-deficient animals led to detection of a regulatory macrophage subset that exerts potent suppression on inflammatory cytokine responses and tissue remodeling. These results provide the first example of a redox-sensitive, gene-specific mechanism for converting macrophages from regulating inflammation to cells licensed to promote aggressive and potentially injurious inflammation.

description of the methods in the supplementary data

Tumor-initiating cells with reprogramming plasticity are thought to be essential for cancer development and metastatic regeneration in many cancers; however, the molecular mechanisms are not fully understood. This study reports that CD81, a tetraspanin protein marker of small extracellular vesicles (exosomes), functions as a binding partner of CD44 and facilitates self-renewal of tumor initiating cells. Using machine learning-assisted protein structure modeling, co-immunoprecipitation, and mutagenesis approaches, we further demonstrate that CD81 interacts with CD44 on the cellular membrane through their extracellular regions. In-depth global and phosphoproteomic analyses of clustering tumor cells unveils endocytosis-related signature pathways of proteins and phosphorylation patterns regulated by CD81 and CD44 specifically or shared between two. Notably, CRISPR Cas9-mediated depletion of either CD44 or CD81 results in loss of both proteins in cancer cell-secreted exosomes, a state which abolishes exosome-induced self-renewal of recipient cells for mammosphere formation. CD81 is expressed in >80% of human circulating tumor cells (CTCs) and specifically enriched in clustered CTCs along with CD44 isolated from breast cancer patients. Mimicking the phenotypes of CD44 deficiency, loss of CD81 also inhibits tumor cluster aggregation, tumorigenesis, and lung metastasis of triple negative breast cancer (TNBC), supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights the novel role of CD81 and its partnership with CD44 in cancer exosomes, self-renewal, CTC clustering, and metastasis initiation of TNBC.

Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is coexpressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.

Purpose: Glioblastoma (GBM) is an incurable primary brain tumor that has not benefited from immunotherapy to date. More than 90% of GBM expresses the tryptophan (Trp) metabolic enzyme, indoleamine 2,3-dioxygenase 1 (IDO). This observation supported the historical hypothesis that IDO suppresses the antitumor immune response solely through a mechanism that requires intratumoral Trp depletion. However, recent findings led us to investigate the alternative hypothesis that IDO suppresses the anti-GBM immune response independent of its association with Trp metabolism.Experimental Design: IDO-deficient GBM cell lines reconstituted with IDO wild-type or IDO enzyme-null cDNA were created and validated in vitro and in vivo. Microarray analysis was conducted to search for genes that IDO regulates, followed by the analysis of human GBM cell lines, patient GBM and plasma, and The Cancer Genome Atlas (TCGA) database. Ex vivo cell coculture assays, syngeneic and humanized mouse GBM models, were used to test the alternative hypothesis.Results: Nonenzymic tumor cell IDO activity decreased the survival of experimental animals and increased the expression of complement factor H (CFH) and its isoform, factor H like protein 1 (FHL-1) in human GBM. Tumor cell IDO increased CFH and FHL-1 expression independent of Trp metabolism. Increased intratumoral CFH and FHL-1 levels were associated with poorer survival among patients with glioma. Similar to IDO effects, GBM cell FHL-1 expression increased intratumoral regulatory T cells (Treg) and myeloid-derived suppressor cells while it decreased overall survival in mice with GBM.Conclusions: Our study reveals a nonmetabolic IDO-mediated enhancement of CFH expression and provides a new therapeutic target for patients with GBM.