Supplementary Table S4 shows the most negatively enriched pathways in non-cycling persister cells by GSEA

Supplementary Figure S6 shows the increase in NF-kB activity following chemotherapy and its role in survival which is enhanced by specific TAMs and CAFs

Abstract It was recently shown that inhibition of polo-like kinase 4 (PLK4) induces synthetic lethality in cancers with chromosome 17q-encoded TRIM37 copy number gain due to cooperative regulation of centriole duplication and mitotic spindle nucleation. We show here that chromosome 17q/TRIM37 gain is a defining feature of high-risk neuroblastoma and renders patient-derived cell lines hypersensitive to the novel PLK4 inhibitor RP-1664. We demonstrate that centriole amplification at low doses of RP-1664 contributes to this sensitivity in a TRIM37 -independent fashion. CRISPR screens and live cell imaging reveal that upon centriole amplification, neuroblastoma cells succumb to multipolar mitoses due to an inability to cluster or inactivate supernumerary centrosomes. RP-1664 monotherapy showed robust anti-tumor activity in 14/15 human neuroblastoma-derived xenograft models, and significantly extended survival in a transgenic MYCN -driven murine model of neuroblastoma. RP-1664 combined with GD2-directed chemoimmunotherapy resulted in maintained complete responses in 6/9 mice with established MYCN -driven murine neuroblastomas. These data support clinical development of PLK4 inhibitors for high-risk neuroblastoma and other cancers with somatically acquired TRIM37 overexpression.

Abstract Developing effective chimeric antigen receptor (CAR) T cell therapies for pediatric solid tumors requires discovery of highly tumor-selective cell surface molecules. Using a multimodal immunotherapeutic target discovery platform, we identified a receptor composed of the nicotinic acetylcholine receptor subunits α3 and β4 as a new previously unrecognized immunotherapeutic target in neuroblastoma. CHRNA3 (α3) and CHRNB4 (β4) are robustly differentially overexpressed in neuroblastoma compared with normal tissues, driven by a super-enhancer upstream of the CHRNA3/B4 locus on chromosome 15q25 that is occupied by neuroblastoma core regulatory transcription factors and physically engages both promoters. Neuroblastoma cells exhibit abundant α3 and β4 protein, which bind and assemble into a stabilized α3β4 complex displayed at high levels on the neuroblastoma cell surface. Functionally, genetic depletion of α3 also reduced β4 levels, induced mesenchymal cell state-associated marker expression, and increased proliferation and invasion in neuroblastoma cell line models. To therapeutically exploit this tumor-restricted α3β4 receptor expression, we engineered Dual Interacting Subunit COmplex (DISCO)-specific single chain variable fragments (scFvs) designed to recognize an epitope requiring simultaneous engagement of both α3 and β4 subunits of the α3β4 cell surface receptor. DISCO CAR T cells generated from these scFvs bound selectively to and were potently activated by the α3β4 receptor, but not either subunit individually. α3β4-targeted DISCO CAR T cells demonstrated potent and selective cytotoxicity across diverse neuroblastoma cell lines and three patient-derived xenograft (PDXs) models, significantly extending the survival of mice harboring PDXs with a range of α3 and β4 expression (P < 0.05). Beyond neuroblastoma, CHRNA3 and CHRNB4 are also highly expressed in small cell lung cancer (SCLC) and retinoblastoma, among several other tumors. The identical super-enhancer is associated with high levels of CHRNA3 and CHRNB4 in SCLC, suggesting similar mechanisms of tumor-driven overexpression. In an intraocular retinoblastoma xenograft model, a single intravitreal dose of α3β4-redirected DISCO CAR T cells achieved marked tumor control and significantly extended ocular survival (P = 0.0015), collectively showing disease-relevance and targetability beyond neuroblastoma. Together, these findings broaden the repertoire of actionable CAR T cell targets in pediatric and adult solid tumors by validating the α3β4 receptor, and more broadly the nicotinic acetylcholine receptor family, as bona fide immunotherapeutic candidates. Moreover, these studies demonstrate that CARs can be designed to recognize disease-relevant subunit configurations within multidomain surface receptors. Citation Format: Patrick M. Schürch, Vincent P. Zecchino, Anna M. Giudice, Rebecca S. Kaufman, Evan Cresswell-Clay, Guillem Pascual-Pasto, Sydney L. Roth, Brendan C. McIntyre, Rawan Shraim, Amber K. Hamilton, Kush Parikh, Karina L. Conkrite, Khanh B. Trang, Grant P. Grothusen, David Groff, Pamela Mishra, Simona Lombardi, Tyler Skinner, Andrew D. Wells, Struan F. Grant, Daniel Martinez, Wei Li, Sharon J. Diskin, Kristopher R. Bosse. Selective targeting of the α3β4 nicotinic acetylcholine receptor by DISCO (dual interacting subunit complex) CAR T cells [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 1332.

Supplementary Table S2 links the CN number to the HTAN biospecimen numbers

While CD19- and BCMA-directed immunotherapies have improved outcomes for B-lymphoid and plasma cell malignancies, frequent relapses with antigen loss/downregulation highlight the need for new targets. Here, using transcriptomic datasets and newly-developed monoclonal antibodies, we show that P2RX5 , long considered a pseudogene in humans, encodes a stable protein in 80% of individuals of African descent carrying the ancestral haplotype. Like CD19, P2RX5 displays B-cell lineage-restricted expression in normal tissues. Unlike CD19, P2RX5 is expressed not only in B-cell neoplasms, but also in T-cell leukemia (T-ALL) and multiple myeloma (MM). We developed P2RX5-directed bispecific T-cell engagers and CAR T cells, which killed T-ALL cells with no evidence of T-cell fratricide. These agents were non-inferior to FDA-approved CD19- and BCMA-directed immunotherapeutics in cell culture and xenograft models of Burkitt lymphoma and MM, while maintaining potency against CD19- and BCMA-negative variants. Hence, P2RX5 is a unique multi-lineage target for frontline or salvage immunotherapy.

Background Solid tumors present unique barriers to treatment with CAR T cells, including poor tumor infiltration into a highly immunosuppressive and metabolically challenging tumor microenvironment (TME). Objectives To enhance both CAR T cell efficacy and the overall immune response against solid tumors, this study explored the therapeutic potential of combining chimeric antigen receptor (CAR) T cells with CD40 stimulation via an agonistic CD40 antibody (αCD40). We hypothesized that CAR T cells could serve as targeted vaccines, promoting antigen release and cooperating with αCD40 to activate and mobilize the endogenous immune cells, thus "heating up" the TME and potentially rendering it more receptive to subsequent therapies. Methods We used a syngeneic mouse model of pancreatic ductal adenocarcinoma (PDAC) and further validated our findings in a triple-negative breast cancer (TNBC) mouse model Results This combined strategy was associated with enhanced anti-tumor activity over CAR T cells alone. This included rapid and sustained tumor necrosis, increased immune cell activation both systemically and within the TME, as well as an overall improvement in survival rates. Comprehensive immune profiling at early timepoints revealed mechanistic insights into the enhanced anti-tumor effects of CAR T cell therapy and αCD40 treatment. Conclusions These findings set the stage for future clinical applications of CAR T cells in combination with CD40 agonists for the treatment of challenging solid tumors.

Supplementary Table S1 shows the patient clinical characteristics and genetic alterations at diagnosis

Abstract Effective cellular therapies for solid tumors are limited by the lack of tumor-specific antigens. We previously showed that non-mutated self-peptides from essential intracellular neuroblastoma (NB) oncoproteins are presented by common HLA allotypes, enabling selective targeting (Nature 2023). Here, we report IND-enabling studies of second-generation PHOX2B PC-CAR T cells incorporating a 4-1BB costimulatory domain. A GMP-grade lentiviral vector encoding the PHOX2B peptide-HLA-specific CAR was produced at CHOP and used to transduce healthy donor and patient T cells. PHOX2B expression and epitope heterogeneity were analyzed across solid tumors, NB xenografts, and cell lines by RNA-seq, IHC, and a PHOX2B scFv assembled with klickmer detecting the QYNPIRTTF/HLA complex. Safety was evaluated using the X-scan and sCRAP cross-reactivity algorithms integrated with experimental testing across 25 normal HLA-A24/23 primary cell lines and HLA-matched PHOX2B negative cancers. Functional activity was assessed via IncuCyte based cytotoxicity, multiplex cytokine profiling, and T-cell activation/proliferation by flow cytometry. Efficacy was tested in four HLA-A24/23 NB xenografts (including chemotherapy-resistant models) and one non HLA-A*24:02/23:01 NB control in NSG MHC-I/II deficient mice receiving a single ∼7×106 PC-CAR T-cell infusion. Longitudinal expansion, immunophenotype, and transcriptional states were characterized by flow cytometry and single-cell RNA-seq. PHOX2B was highly expressed in all neuroblastomas and pheochromocytomas and not expressed in other cancers. Klickmer staining revealed variable epitope density correlating with PC-CAR T cell activation. PHOX2B PC-CAR T cells mediated potent, dose-dependent killing of HLA-A24/23 neuroblastomas, sparing antigen-negative non-HLA-A24/23 cancer cells unless peptide-pulsed. Antigen engagement triggered proliferation, effector cytokine release, and IFN-γ dependent upregulation of HLA-I, significantly increasing antigen density. No cross-reactivity was detected in co-culture assays. A single dose of PC-CAR T cells induced complete regression (>50 days) across all xenografts. One relapse in each of two models either regressed spontaneously with re-emergence of PC-CAR T cells or after re-treatment. Responses were durable >120 days, whereas PHOX2B+ non-HLA-A24:02/23:01 controls showed progressive disease. Expansion peaked at day 14 with CD8+ effector-memory predominance transitioning to central-memory persistence. PHOX2B-directed PC-CAR T cells show stringent specificity, potent cytotoxicity, and curative efficacy in preclinical patient-derived NB models. These IND-enabling data supported the ongoing first-in-human/child Phase 1 trial (NCT07007117), validating PHOX2B as a lineage-restricted immunotherapeutic target in high-risk neuroblastoma. Citation Format: Muzamil Y. Want, Richa Kapoor, David Groff, Keelan O’Reilly, Liron Grossmann, Alvin Farrel, Rebeca A. Ventura, Peiyao Li, Quinlen Marshall, Jenny pogoriler, Daniel Martinez, Matt Beasley, Ben Kiefel, Mark Yarmarkovich, John Maris. Durable remission of high-risk and refractory neuroblastoma by PHOX2B derived peptide-HLA directed CAR T cells: An IND enabling study [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 3707.

Supplementary Figure S2 shows the details of the pathway-based clustering algorithm including the autocorrelation pathway scores, cell cycle annotation, pathway enrichment analysis and region analysis.

Supplementary Figure S4 differences in persister cell composition between diagnosis and definitive surgery as well as decrease in MYC/N protein level in human samples, PDX and cell lines following chemotherapy

Supplementary Figure S3 shows the WGS analysis of the diagnostic and definitive surgery including clonal evolution of ALK mutation from diagnosis to definitive surgery in one tumor.

Supplementary Table S3 provides the genes of each persister subtype signature

Supplementary Figure S5 shows the connection between NF-kB/stemness subtype and mesenchymal transcriptional state.

Supplementary Figure S1 shows cell type markers and inferred copy number of tumors as well as characterization of malignant, Schwann cells, tumor associated macrophages and cancer associated fibroblasts at time of definitive surgery

GPC2 on SCLC cells activate D3-GPC2 CAR T cells.

High-risk neuroblastoma, a leading cause of pediatric cancer mortality, exhibits substantial intratumoral heterogeneity, contributing to therapeutic resistance. To understand tumor microenvironment evolution during therapy, we longitudinally profiled 22 patients with high-risk neuroblastoma before and after induction chemotherapy using single-nucleus RNA and ATAC sequencing and whole-genome sequencing. This revealed profound shifts in tumor and immune cell subpopulations after therapy and identified enhancer-driven transcriptional regulators of neuroblastoma neoplastic states. Poor outcome correlated with proliferative and metabolically active neoplastic states, whereas more differentiated neuronal-like states predicted better prognosis. Proportions of mesenchymal neoplastic cells increased after therapy and a high proportion correlated with a poorer chemotherapy response. Macrophages significantly expanded towards pro-angiogenic, immunosuppressive and metabolic phenotypes. We identified paracrine signaling networks and validated the HB-EGF-ERBB4 axis between macrophage and neoplastic subsets, which promoted tumor growth through the induction of ERK signaling. These findings collectively reveal intrinsic and extrinsic regulators of therapy response in high-risk neuroblastoma.

D3-GPC2 CAR-BBz T cells from a second T cell donor are robustly and selectively activated by GPC2-high neuroblastoma cell lines.

D3-GPC2 CAR-28z T cells are robustly and selectively cytotoxic to GPC2-expressing neuroblastoma cells.

To overcome the paucity of known tumor-specific surface antigens in pediatric high-grade glioma (pHGG), we contrasted splicing patterns in pHGGs and normal brain samples. Among alternative splicing events affecting extracellular protein domains, the most pervasive alteration was the skipping of ≤30-nt-long exons. Several of these skipped microexons mapped to L1-immunoglobulin cell adhesion molecule (IgCAM) family members, such as neuronal CAM (NRCAM). Bulk and single-nuclei short- and long-read RNA-seq revealed uniform skipping of NRCAM microexons 5 and 19 in virtually every pHGG sample. Importantly, the Δex5Δex19 (but not the full-length) NRCAM proteoform was essential for pHGG cell migration and invasion in vitro and tumor growth in vivo. We developed a monoclonal antibody selective for Δex5Δex19 NRCAM and demonstrated that "painting" pHGG cells with this antibody enables killing by T cells armed with an FcRI-based universal immune receptor. Thus, pHGG-specific NRCAM and possibly other L1-IgCAM proteoforms are promising and highly selective targets for adoptive immunotherapies.