Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer. Despite recent advances in treating BRAFV600E-driven ATC, therapy resistance remains a significant challenge, often resulting in disease progression and death. Leveraging a focused CRISPR/KO screen in parallel with a CRISPR/activation screen, both tailored on response to BRAFV600E inhibitor treatment, we identified TAZ (encoded by WWTR1 gene) deficiency as synthetically lethal with BRAF inhibitor in ATC. TAZ is overexpressed in ATC compared to well-differentiated thyroid tumors. We demonstrate that TAZ-deficient ATC cells display heightened sensitivity to BRAF inhibitors. Using gene essentiality score across cancer cell lines, we found that BRAFV600E-driven cancers are highly sensitive to TAZ loss, unlike their counterparts with wild-type BRAF and non-BRAFV600E. Mechanistically, we demonstrate that dabrafenib triggers the Unfolded Protein Response (UPR) under ER stress and suppresses protein synthesis. TAZ loss represses the UPR, reverses the inhibition of protein synthesis, and triggers increased cell death by ferroptosis in dabrafenib-treated ATC. Collectively, our findings unveil TAZ as a new target to overcome resistance to BRAF inhibitors in undifferentiated thyroid cancer.

X-linked severe combined immunodeficiency (SCID-X1) presents as T-B+NK- SCID, due to mutations in the common γ chain (IL2RG/CD132), a shared component of several cytokine receptors. Without definitive therapy, SCID-X1 is fatal within two years due to severe recurrent infections. Atypical SCID-X1 can arise from hypomorphic variants that preserve partial immunity and delay diagnosis, sometimes into adulthood.IL2RG is produced as a precursor with a 22-amino acid cleavable N-terminal signal peptide (SP), recognized by the signal recognition particle (SRP) during translation initiation. SRP engagement is essential for mRNA stability; when the SP fails to bind SRP efficiently, the corresponding mRNA is degraded through the regulation of aberrant protein production (RAPP) pathway. Because the SP is critical for proper protein expression, variants in this region have been linked to human disease. To date, however, none have been reported in association with SCID.We studied a patient with a novel 25 bp deletion in exon 1 of the IL2RG (c.26_50del; p.Ser10Cysfs*6). Although this frameshift would be expected to generate a null allele and typical SCID-X1, he instead presented at age 17 with severe T cell lymphopenia (110 cells/uL), arthritis, disseminated zoster, and at age 22 developed progressive multifocal leukoencephalopathy due to the JC virus. We found that this mutation produces an IL2RG mRNA that enables in-frame translation from an upstream translation initiation site, generating a protein nearly identical to the wild-type except for the N-terminal 17 amino acids of the SP. Patient cells showed markedly reduced and variable expression of functional wild-type IL2RG, and proportionally defective cytokine response. The altered SP exhibited reduced hydrophobicity and an extremely low Bowman index, predicting impaired SP–SRP interaction and suggesting RAPP-mediated mRNA degradation as the cause of diminished and fluctuating protein levels. Assessment of mRNA turnover in patient versus healthy control EBV-transformed cells confirmed preferential degradation of the patient’s IL2RG mRNA. We thus believe inefficient SP-SRP binding triggered IL2RG mRNA degradation, in turn causing low, variable IL2RG expression and atypical XSCID-X1.These findings identify a novel SP-region mutation that disrupts mRNA stability and causes SCID-X1, underscoring the pathogenic impact of SP mutations in immunodeficiencies.Figure 1.Schematic representation of the mechanism of disease pathogenesis in the patient is shown. Normally, the signal recognition particle (SRP) recognizes the signal peptide upon exiting from the ribosome exit tunnel, which halts translation of the protein until the ribosome–nascent chain complex (RNC) is translocated to the ER membrane. At the ER membrane, SRP binds to the SRP-receptor, and the RNC complex is transferred to the translocon, resuming translation. The translated protein is processed (signal peptide cleavage, protein folding, and post-translational modifications), and the protein is transported to the plasma membrane via the Golgi apparatus. In the patient, the mutation in the signal peptide results in inefficient SRP recognition during translation. SRP recognition protects the mRNA, and failure of signal peptide recognition by SRP triggers regulation of aberrant protein production (RAPP)-mediated mRNA degradation, resulting in reduced and variable IL2RG protein expression in the patient.

CD40L-deficient X-linked hyper IgM (XHIGM) syndrome is characterized by defective B cell class switching, deficiency of immunoglobulins G, A, and E, plus other immune abnormalities due to disrupted CD40L–CD40 interaction. Allogeneic hematopoietic stem cell (HSC) transplant can cure XHIGM but requires myeloablative conditioning and risks graft versus host disease (GVHD), posing a high mortality risk. We developed ex vivo base editing (BE) of autologous HSC and T cells to treat a 37-year-old man (P1) diagnosed late in life with a CD40LG c.C>T p.Q220X mutation in a single-patient Investigational New Drug application targeting his mutation (IND32000; NIH protocol 002385). Preclinical studies achieved >90% gene correction of P1 HSC and T cells with base editor ABE8e SpWT/A5 guide, devoid of significant unintended off-target edits. BE-HSC was intended as a definitive transplant to achieve long-term hematopoietic and immune reconstitution, whereas the BE-T cells were designed to serve as a transient bridge therapy of functional T cells during the period preceding differentiation of T cells from the BE-HSC graft.P1 presented pre-gene therapy with sclerosing cholangitis, cryptosporidium infection, nodular regenerative hyperplasia, portal hypertension, bronchiectasis, and lacked HLA-matched donors. Autologous BE-HSC transplant was given after low-dose busulfan conditioning together with prophylactic defibrotide to mitigate chemo-related veno-occlusive disease.The original treatment schema was alemtuzumab (days −21, −20, −19), busulfan 6mg/kg total (days −3, −2) with prophylactic defibrotide, BE-HSC (day 0), and BE-T on day +14 and as indicated for lymphopenia and/or infection. Following alemtuzumab, P1’s baseline abnormal liver function tests (LFTs) worsened (transaminases approximately doubled, bilirubin increased ∼18-fold) with persistent detection of previously undetectable cryptosporidium. We paused busulfan and BE-HSC and administered BE-T products, which corrected LFTs sufficiently to allow uneventful busulfan conditioning and BE-HSC product infusion. Post-treatment at months 1 and 2, blood studies confirmed robust engraftment with normal wild-type alleles in myeloid cells (>90%) and increasing levels in natural killer (>60%), B (>40%), and T (27%) cells and with first-time detection of class-switched IgG+ B cells.This innovative, dual-product strategy provides immediate, on-demand immune protection through infusion of functional BE-T cells, effectively bridging patients through the critical pre-engraftment period and until BE-HSC achieves durable, multilineage reconstitution.

The variegated expression of the KIR family of class I MHC receptors generates specialized natural killer (NK) cells capable of allele-specific HLA recognition. Understanding the mechanism of KIR gene activation will lead to improved methods for the generation of fully functional NK cells. A central RUNX-binding site in the KIR proximal promoter is required for gene activation. RUNX proteins recruit ten-eleven translocation (TET) proteins that generate 5-hydroxymethylcytosine (5hmC) and drive DNA demethylation. Assessment of 5-methylcytosine (5mC) and 5hmC residues at four stages of NK cell development reveals deposition of 5hmC primarily in a CREB site next to the RUNX site at the CD56Bright stage but not the subsequent CD56Dim stage representing fully mature NK cells. KIR promoter demethylation is delayed relative to other lineage-associated genes, indicating a high threshold for KIR gene demethylation in developing NK cells, and a window of opportunity for RUNX/TET-dependent KIR gene activation in CD56Bright NK cells.

HIV-1 cores enter the nucleus and undergo capsid disassembly (uncoating) near their integration site. Although most viral cores are localized to nuclear speckles (NSs), the spatial relationship between the uncoating site and integration site remains unclear. Here, using fluorescently labeled HIV-1 cores and NS markers, we show that uncoating predominantly occurs within NSs. Treatment of infected cells with capsid inhibitors PF-3450074 (PF74) or lenacapavir (LEN) after nuclear entry induced rapid disruption of interactions between capsid and cleavage and polyadenylation specificity factor 6 (CPSF6) followed by exit of HIV-1 cores from NSs, indicating that CPSF6 binding is required to retain the viral cores in the NSs. Treatment with PF74 or LEN led to core disruption and appearance of transcriptionally active proviruses further from the NSs compared to viral cores that uncoated in the NSs in untreated cells. This spatial shift correlated with reduction in integration into gene-rich, transcriptionally active speckle-associated chromatin domains, the preferred sites of HIV-1 integration, and increased integration into gene-sparse lamina-associated domains located away from the nuclear envelope. These findings demonstrate that the HIV-1 uncoating site is a key determinant of integration targeting, and that capsid inhibitors can misdirect integration by relocalizing uncoating to outside of NSs.

BACKGROUND & AIMS:Gallbladder cancer (GBC) is a rare and highly lethal biliary tract cancer with limited treatment options and lack of diagnostic and prognostic noninvasive biomarkers. Circulating cell-free DNA (cfDNA) offers a noninvasive means to capture fragmentomics alterations that may assist with diagnosis and biological characterization. This pilot study aimed to identify cfDNA-based features that differentiate GBC from individuals with gallstones and healthy controls. METHODS:cfDNA was extracted from archived plasma samples from 67 individuals in two case-control studies from China and Chile, followed by low coverage whole genome sequencing to evaluate fragmentomics and related features. cfDNA computational packages were leveraged to generate features and create a classification model. External cfDNA datasets including other hepatopancreatobiliary disease groups were processed and compared to the current study. RESULTS:In this pilot, individuals with GBC displayed significantly altered cfDNA features compared to healthy controls and individuals with gallstones (P<0.05). Key differences were observed in fragment lengths, end motif patterns, estimated tumor fractions, detectable copy number alterations, and transcription factor binding site accessibility, all of which discriminated GBC from a combined non-cancer group (AUC: 0.852). Many of these cfDNA alterations were consistent with the results from paired and unpaired tissue genomics datasets and other related cancer groups (liver cancer, pancreatic cancer, and non-GBC biliary tract cancer). CONCLUSIONS:This proof-of-concept study demonstrates that archived plasma samples can be successfully used for cfDNA sequencing. These methods capture biologically meaningful alterations in GBC that are consistent with tissue-based genomics data. Collectively, these findings highlight the potential of cfDNA profiling for biological characterization and as a promising noninvasive diagnostic tool for GBC. IMPACT AND IMPLICATIONS:This pilot study demonstrates that archived plasma EDTA samples can be used for cfDNA sequencing and reinforces the utility of cfDNA fragmentomics analyses for studying gallbladder disease. By assessing biologically relevant cfDNA features across related hepatopancreatobiliary cancers, we identified common and distinct features that may be used for classification and risk stratification. In high-risk settings for GBC, cfDNA fragmentomics might offer complementary information that could be used to guide clinical decision-making or help optimize waitlists for cholecystectomy.

Detection of the off-target effects of base editors is important for identifying their safety risks but current methods for understanding their global activities have limitations in terms of sensitivity or bias by computationally selecting a subset of sites for experimental analysis. We present CHANGE-seq-BE, a method to assess the guide RNA-dependent off-target profile of both adenine and cytosine base editors that is simultaneously sensitive and unbiased. CHANGE-seq-BE relies on selective sequencing of base-editor-modified genomic DNA in vitro and provides comprehensive identification of genome-wide off-target mutations. We found that 98.8% of validated off-target sites were unique to ABE8e adenine base editors compared to Cas9 nuclease, suggesting substantially higher off-target activity of the former. We further applied CHANGE-seq-BE to support genotoxicity studies in an emergency investigational new drug application for customized adenine base editor treatment for a person with CD40L-deficient X-linked hyper IgM syndrome. Our results emphasize the importance of using a base-editor-specific method for identifying off-target activity.

Abstract Background Kaposi sarcoma (KS) is an angioproliferative tumor caused by Kaposi sarcoma herpesvirus (KSHV) that occurs in people with HIV. Concurrent KSHV-associated diseases (KAD), including multicentric Castleman disease, primary effusion lymphoma, and KSHV-associated inflammatory cytokine syndrome may modify KS biology and impact clinical outcomes. Transcriptomic profiling of archival KS tissue enables investigation of molecular heterogeneity associated with these overlapping disease states. Methods Archival formalin-fixed paraffin-embedded (FFPE) KS skin biopsies from 42 patients with HIV-associated KS between 2017 and 2022 were analyzed based on confirmed histopathologic diagnosis, tissue adequacy for RNA profiling, and availability of linked clinical data. Bulk transcriptomic analyses were conducted using Nanostring nCounter PanCancer ImmunoOncology panel supplemented with KSHV-specific probes. Spatial RNA profiling was performed on four tissues from participants with KS and concurrent KAD (KS+KAD) using GeoMx digital spatial profiling (DSP) platform. Regions of interest were selected using LANA-1, CD45 and CD31 staining to characterize tumor (LANA-1+, CD31+), vessel (LANA-1-negative, CD31+) and immune cells (CD45+) areas. For bulk transcriptomic analyses and spatial transcriptomic analyses, p-values were adjusted for multiple comparisons using the Benjamin-Hochberg FDR approach, and adjusted p-values (padj) are reported. Results KS samples were obtained from 42 men with HIV (median age 40 years). Median HIV viral load of 27 copies/mL and median CD4+ T-cell count was 211 cells/µL. Forty-eight percent had KS alone and 52% had KS+KAD. Patients with KS+KAD had worse survival compared to those with KS alone. Transcriptomic analyses identified increased expression of STC1 (log2FC = 2.02, padj = 0.001), a secreted glycoprotein, and MKI67 (log2FC = 1.11, padj = 0.02), a common proliferation marker, in KS+KAD lesions, along with lower expression of cytokine-associated pathways. Spatial RNA profiling from 4 KS samples from patients with KS+KAD identified increased abundance of lymphatic endothelial cells, elevated LYVE1 expression in LANA-1+ tumor areas as compared to LANA-negative areas. Conclusions Bulk and spatial transcriptomic profiling of archival HIV-associated KS lesions revealed disease-specific molecular programs associated with concurrent KAD that altered tumor and microenvironment features. These findings demonstrate the heterogeneity of HIV-associated KS lesions that may guide future studies on KS pathogenesis and potential therapeutic targets.

8118 Background: Development of circulating cell-free (cf) DNA assays for cancer detection and monitoring is an area of active research. Limited data are available on the utility of cfDNA for thymic epithelial tumors (TETs). Evaluation of cfDNA fragmentation patterns (fragmentomics) offers advantages over conventional cfDNA analysis. Studies have shown that concurrent generation of fragmentomics and methylomics features via enzymatic methyl-sequencing (EM-Seq) are highly predictive at classifying cancer vs. non-cancer and capture key cancer-related biological features. We conducted a study to determine the feasibility of fragmentomics and methylomics for evaluation of TETs. Methods: cfDNA was extracted from 1-2 mL of plasma EDTA derived from patients with advanced TETs using the QIAamp Circulating Nucleic Acid Kit, underwent enzymatic conversion via the NEBNext Enzymatic Methyl-Seq v2 Kit, and sequenced on a NovaSeq X 10B flowcell. Estimated tumor fractions were generated using ichorCNA with low tumor settings and via fragle . Recurrent copy number alterations (CNAs) were plotted and compared against external TET copy number datasets. Short fragment percentage (90-150 bps) was calculated and average DELFI scores (DNA evaluation of fragments for early interception) were generated via FinaleToolkit . Results: Plasma samples from 47 of 50 participants (thymic carcinoma: 26, thymoma: 24; median age: 55 years (range 23-77); females 22; stage distributions: I/II/III/IV = 1/0/1/48) yielded high-quality cfDNA which was sufficient for sequencing. Individuals with thymic carcinoma displayed higher estimated tumor fractions via ichorCNA (P<0.05) and Fragle (P=0.05). Moreover, although not statistically significant, individuals with thymic carcinoma exhibited a general trend of higher short fragment percentage and higher average DELFI scores. When evaluating recurrent CNAs, individuals with thymic carcinoma displayed greater enrichments in CNAs in general with noted amplifications/gains at chr 1, 5, 12, and 17 and deletions at chr 16, whereas individuals with thymoma showed deletions at chr 3. When grouped and compared to external thymoma tissue CNAs, recurrent CNAs detected in plasma showed partial concordance. Large alterations in fragmentation profiles were also identified across both groups, which showed increased variability near recurrent CNA regions. Conclusions: This proof-of-concept study demonstrates that plasma cfDNA is detectable and fragmentomics is feasible among individuals with TETs, thus providing a non-invasive means of capturing cancer-related aberrant signals. Validation of this result in larger studies would establish cfDNA fragmentomics as a novel non-invasive means for disease monitoring and biomarker identification in patients with TETs.

Using qualitative and quantitative magnetic resonance imaging (MRI) features, this study aimed to distinguish between isocitrate dehydrogenase (IDH)-mutant astrocytomas (IDH-mA) and IDH-wildtype glioblastomas (IDH-wG) based on the fifth edition of the World Health Organization’s (WHO’s) classification of central nervous system (CNS) tumors (WHO CNS5), published in 2021. We enrolled 87 IDH-mA and 102 IDH-wG patients with pathologically confirmed disease according to the WHO CNS5 standard. Pretreatment brain MRI images and genetic information were obtained for each patient. Qualitative imaging features were assessed, including the side of lesion center, multifocality/multicentricity, hemorrhage, pial invasion, ependymal invasion, cortical involvement, midline location invasion, and enhancement mode. The quantitative imaging features assessed included tumor volume-related metrics and the relative apparent diffusion coefficient (rADC)-related metrics based on tumor segmentation. Contrast-enhanced and non-enhanced areas of the tumors were analyzed separately. univariable analysis and logistic regression were used to select the candidate predictors. The discrimination performance of the logistic regression model was evaluated using the area under the receiver operating characteristic curve (AUC). Internal validation was performed using the bootstrap approach. In terms of the qualitative features, IDH-mA exhibited less multifocality/ multicentricity (p = 0.032), more hemorrhage (p = 0.009), and more cortical involvement (p = 0.009) than IDH-wG. Regarding the quantitative imaging features, IDH-mA demonstrated higher values in Vall (p = 0.001), Vne (p < 0.001), rmaxADCce (p < 0.001), rminADCne (p = 0.015), rmaxADCne (p = 0.004), and rmeanADCall (p = 0.001) than IDH-wG. In the multivariable analysis of all patients, multifocality/multicentricity (odds ratio [OR] = 2.87, p = 0.033), Vne (OR = 1.02, p = 0.049), and rmaxADCce (OR = 2.82, p < 0.001) were independent predictive factors for distinguishing IDH-mA from IDH-wG. A combination of multifocality/multicentricity, Vne, and rmaxADCce (model) had a superior performance in distinguishing IDH-mA from IDH-wG, with an AUC, accuracy, sensitivity, and specificity of 0.849 (95

11530 Background: Kaposi sarcoma (KS) is an angioproliferative tumor caused by Kasposi sarcoma herpesvirus (KSHV) that typically manifests as skin lesions. Other KSHV-associated disease (KAD) that can occur with KS include multicentric Castleman disease (MCD), primary effusion lymphoma (PEL) and KSHV-associated inflammatory cytokine syndrome (KICS). KS with concurrent KAD, which occurs frequently in people with HIV (PWH), contributes to morbidity and mortality. Novel sequencing technologies that evaluate archival KS may further our understanding of HIV-associated KS pathogenesis. Methods: Gene expression profiling of archival KS skin samples of 42 PWH was performed with the custom nCounter PanCancer ImmunoOncology panel with the addition of KSHV probes. Spatial RNA profiling was performed using GeoMx digital spatial profiling (DSP) platform on 4 formalin fixed paraffin-embedded tissue sections randomly selected from patients (pts) with concurrent KS and KAD. LANA, CD45, and CD31 expression in samples identified KS (LANA + , CD31 +) ) and other areas of interest (AOIs) including vessels (LANA - , CD31 + ) and immune cells (CD45 + ) on tissue sections. Gene Set Enrichment analysis was performed using R package ClusterProfiler. Results: Samples were taken from 42 men with HIV with a median age of 40 years. The median CD4 T cell count was 211 cells/ µl and a median HIV viral load of 27 copies/ml. Fifty-two percent of pts with KS had a concurrent KAD, most commonly KICS with KS (30%) followed by MCD with KS (19%). In nCounter analyses, samples from pts with KS alone demonstrated upregulation of STC1, a secreted glycoprotein, (log2FC=2.02, padj=0.001) and MKI67, a proliferation marker, (log2FC=1.11, padj=0.02) as compared to pts with KS and concurrent KAD. Pathway analyses highlighted reduced enrichment in specific cytokine activity profiles (padj = 0.01), natural killer cell activation markers (padj=0.02), and B cell proliferation (padj=0.004), in KS with concurrent KAD specimens. Cell deconvolution analyses showed increased abundance of CD8 T cells and regulatory T cells in KS alone specimens as compared to those with KS and other KAD. DSP of 4 samples of pts with KS and concurrent KAD (2 pts with MCD+ KS, 2 pts with KICS+ KS) identified higher expression of TSPAN (log2FC=1.32, padj=0.04) and LYVE1 (log2FC=1.82, padj=9.17e-5) in LANA+ tumor regions than vessel and immune AOIs, and lower ICAM1 (log2FC=-1.08, padj=3.95e-6), highlighting the role of virus-infected areas in oncogenesis and modulating immune activity. Conclusions: Sequencing data of archival HIV-associated KS samples highlighted distinct gene expression profiles by concurrent KAD, particularly in MCD or KICS, demonstrating disruptions in immune activity and increased cell proliferation thus shedding light on the molecular pathways driving KS pathogenesis and avenues for future targeted study.

Extent of IMPACT CpG island promoter methylation in mouse cell lines (KPC, 4964-POP, and 4964-HOP), and five human pancreatic liver metastases compared to three primary PDACs, and two normal pancreas and liver tissue.

BACKGROUNDNaive cells comprise 90% of the CD4+ T cell population in neonates and exhibit distinct age-specific capacities for proliferation and activation. We hypothesized that HIV-infected naive CD4+ T cell populations in children on long-term antiretroviral therapy (ART) would thus be distinct from infected memory cells.METHODSPeripheral blood naive and memory CD4+ T cells from 8 children with perinatal HIV on ART initiated at age 1.7-17 months were isolated by FACS. DNA was extracted from sorted cells, and HIV proviruses were counted, evaluated for intactness, and subjected to integration site analysis (ISA).RESULTSNaive CD4+ T cells containing HIV proviruses were detected in children with 95% statistical confidence. A median 4.7% of long terminal repeat-containing naive CD4+ T cells also contained HIV genetic elements consistent with intactness. Full-length proviral sequencing confirmed intactness of 1 provirus. In the participant with the greatest degree of naive cell infection, ISA revealed infected expanded cell clones in both naive and memory T cells, with no common HIV integration sites detected between subsets. Divergent integration site profiles reflected differential gene expression patterns of naive and memory T cells.CONCLUSIONThese results demonstrate that HIV persisted in both naive and memory CD4+ T cells that underwent clonal expansion and harbored intact proviruses, and suggest that infected memory T cell clones do not frequently arise from naive cell differentiation in children with perinatal HIV on long-term ART.FUNDINGCenter for Cancer Research, NCI; Office of AIDS Research; NCI FLEX; Children's and Emory Junior Faculty Focused Award.

Introduction: Current modalities for minimal residual disease (MRD) detection in B-ALL include flow cytometry and next-generation sequencing (NGS). While highly sensitive and specific, they require the presence of intact leukemia cells. Cell-free DNA (cfDNA) offers the potential advantage of subclinical disease detection in the absence of circulating blasts, but its use in B-ALL has been understudied. As an exploratory objective on “Pilot Prospective Study for PET-CT Imaging in Participants with Relapsed/Refractory Acute Leukemias” (NCT05969002), the use of cfDNA as an adjunctive marker for disease monitoring was evaluated in patients with relapsed/refractory (RR) B-ALL proceeding to Chimeric Antigen Receptor T-cells (CAR-T). Methods: Plasma samples were prospectively collected on day 0 (pre-infusion), +14, and +28 post-CAR-T infusion in patients with RR B-ALL enrolled on this study. cfDNA was isolated using the QIAmp Circulating Nucleic Acid kit (Qiagen). Libraries were constructed using the KAPA HyperPrep kit (Roche) and xGen UDI-UMI Adapters (IDT) and uniformly generated with 8 PCR cycles. Shallow whole-genome NGS was performed to a mean depth of 6x on a NovaSeq S4 platform (lllumina). cfDNA was assessed for genomic instability (copy number alteration (CNA) derived tumor fraction (TF) using ichorCNA) and fragmentomics (cfDNA fragment end motifs, non-negative matrix factorization (NMF) of fragment sizes, NMF end motifs (F-profiles), genome wide short/long fragment binned ratios (binwise)). Statistical analysis was performed in R, calculating features' area under the curve (AUC) from receiver operating characteristic curves with Youden's index. Dominant B-ALL VDJ sequences were identified by the Adaptive clonoSEQ® platform, based on NGS-MRD analysis from each patient. cfDNA libraries were queried using NCBI's Basic Local Alignment Search Tool (BLAST) for dominant VDJ sequences. Personalized droplet digital PCR (ddPCR) assays were designed based on a priori knowledge of each patient's VDJ clonotype(s). VDJ clonotype quantifications of cfDNA by ddPCR were normalized to housekeeping gene MRTFB. Active leukemia was defined as detectable disease in the peripheral blood (PB) or bone marrow (BM) via flow cytometry or NGS, or as extramedullary disease seen on PET scan. Results: Across 13 patients enrolled, the median age was 21.4 years (range: 5.1-38.1) and 92% were male. Libraries were successfully generated for 8 patients across 1-3 timepoints (20 total samples), all of whom had active leukemia in at least 1 timepoint. These were compared to 21 healthy control libraries for fragmentomic analysis. End-motif analysis modestly differentiated samples with active leukemia from healthy (F-profile 2 AUC 0.77, F-profile 4 AUC 0.75, F-profile 5 AUC 0.74). This improved when F-profiles 4 and 5 were combined into a polynomial support vector machine (AUC 0.77). NMF (AUC 0.95) and binwise (AUC 0.94) distinguished well between B-ALL and healthy states. Fragmentomic analyses and TF were combined into a meta-classifier, boosting performance with accurate predictions in 38/41 samples (100% specificity, 77% sensitivity). Due to lack of a dominant VDJ sequence, 3 samples from 1 patient were excluded from further testing. VDJ sequences were detected in PB cfDNA WGS libraries using BLAST for 2/10 samples (2/7 patients) with active leukemia. One of the 2 positive samples had negative PB flow cytometry and NGS, negative BM flow cytometry, and only 4-7 clones/million on BM NGS. No sequences were detected using BLAST for the 7 samples from 4 patients without active leukemia. Detection using BLAST was not consistent, likely due to depth of coverage. To improve sensitivity, we transitioned to ddPCR testing of PB cfDNA. ddPCR was positive in 9/10 samples (6/7 patients) with active leukemia. Of the 10 samples, only 1/10 had positive PB flow cytometry, and 7/9 had positive PB NGS (1 PB sample was not tested for NGS). The false negative sample was negative in PB flow, BM flow, and PB NGS, with <1 clone detected on BM NGS. ddPCR was negative in 6/7 samples (4/4 patients) without active leukemia. The false positive sample was negative on PB flow and NGS, but BM status was unknown. Conclusions: cfDNA shows promise as a non-invasive adjunctive assay for disease detection in B-ALL, even in the absence of circulating blasts. Further validation of our ddPCR testing is required to determine whether it can be used merely for detection or for quantitation of disease.

GCN1 cDNA sequence in 4964-POP and 4964-HOP cells.

Genomic alterations driving tumorigenesis in sinonasal malignancies remain largely unexplored. Here, we perform an in vivo loss-of-function screen using a pooled custom single-guide library delivered to the sinonasal cavity by adeno-associated virus vector to identify cancer driver genes across diverse sinonasal malignancies. This approach yielded sinonasal malignancies with diverse histologies, including sinonasal squamous cell carcinoma, adenocarcinoma, poorly differentiated sinonasal carcinoma, and sinonasal neuroendocrine tumors characteristic of olfactory neuroblastoma. Surprisingly, rather than observing distinct sgRNA profiles across sinonasal tumor subtypes, common recurrent mutations were identified in Nf1 (79%), Rasa1 (74%), and Trp53 (68%) across malignancies with distinct histologies. Utilizing an orthogonal approach, we confirmed that Nf1/Trp53 were required for sinonasal tumorigenesis. Given that loss-of-function in NF1 and RASA1 may lead to increased Ras activity and downstream MEK signaling, we tested small molecule targeting of the RAS-MAPK pathway in sinonasal malignancies. Indeed, both tumor cell lines derived from our loss-of-function approach as well as from human sinonasal malignancies displayed significant sensitivity to MEK inhibition in standard in vitro culture and organoid models. These findings demonstrate that loss of NF1 and RASA1-mediated Ras-GAP activity leads to Ras activation and downstream MEK signaling which is a potential common target throughout major sinonasal tumor subtypes.

Live microbial contamination poses high risks to cell and gene therapies, threatening manufacturing processes and patient safety. Rapid, sensitive detection of live microbes in complex environments, such as CAR-T cell cultures, remains an urgent need. Here, an innovative sample-to-result workflow is introduced using digital loop-mediated isothermal amplification (dLAMP), enhanced by Electrostatic Microfiltration (EM)-based enrichment, for rapid sterility testing. By rationally designing primers targeting 16S and 18S rRNA, dLAMP assay enables both universal detection (covering >80% of known species) and strain-specific identification of bacterial and fungal contaminants in CAR-T cell spent medium and final products, directly from microorganism lysates. Enhanced by EM-based enrichment of low-abundance live microbes, the workflow achieves unparalleled sensitivity and speed, detecting contamination levels as low as 1 CFU/mL in complex CAR-T cell cultures within 6 h. Compared to qPCR and 14-day compendial methods, the approach demonstrates superior accuracy and significantly faster turnaround times. This workflow holds transformative potential for real-time monitoring in cell therapy manufacturing and rapid safety assessments of CAR-T cell products prior to patient infusion. Beyond cell therapy, the method is broadly applicable to infectious disease diagnostics, biomanufacturing monitoring, food safety, and environmental surveillance.

Figure S6. Nuclear GCN1 stabilizes HNRNPK leading to immune escape

Figure S3. IMPACT limits ATF4 activation through competitive inhibition of GCN1