Citations

PURPOSE Tumor treating fields (TTFields) use alternating electric fields to disrupt cancer cell proliferation. Feasibility of TTFields therapy with gemcitabine/nab-paclitaxel was previously demonstrated in patients with advanced pancreatic adenocarcinoma. PANOVA-3 was designed to confirm safety and efficacy of TTFields in patients with unresectable locally advanced pancreatic adenocarcinoma (LA-PAC). METHODS In this global phase III trial, 571 patients with newly diagnosed LA-PAC were randomly assigned to receive gemcitabine 1,000 mg/m 2 and nab-paclitaxel 125 mg/m 2 by intravenous infusion once a day on days 1, 8, and 15 of a 28-day cycle with or without TTFields. The primary end point was overall survival (OS). Secondary end points included progression-free survival (PFS), local PFS, pain-free survival, and overall response rate (ORR). Distant PFS was analyzed post hoc. RESULTS OS was significantly prolonged using TTFields with gemcitabine/nab-paclitaxel versus gemcitabine/nab-paclitaxel (median, 16.2 months [95% CI, 15.0 to 18.0] v 14.2 months [95% CI, 12.8 to 15.4]; hazard ratio [HR], 0.82 [95% CI, 0.68 to 0.99]; P = .039). PFS, local PFS, and ORR were not improved. Pain-free survival was significantly prolonged with TTFields with gemcitabine/nab-paclitaxel (median, 15.2 months [95% CI, 10.3 to 22.8] v 9.1 months [95% CI, 7.4 to 12.7]; HR, 0.74 [95% CI, 0.56 to 0.97]; P = .027), as was distant PFS (median, 13.9 months [95% CI, 12.2 to 16.8] v 11.5 months [95% CI, 10.4 to 12.9]; HR, 0.74 [95% CI, 0.57 to 0.96]; P = .022). Device-related skin adverse events (AEs) were experienced by 76.3% of patients. Most device-related skin AEs were mild to moderate, with 7.7% of patients reporting a grade 3 AE. CONCLUSION This study demonstrated significant OS, pain-free survival, and distant PFS benefits for TTFields with gemcitabine/nab-paclitaxel versus gemcitabine/nab-paclitaxel in patients with unresectable LA-PAC, with no additive systemic toxicity.

7049 Background: Odronextamab, an investigational off-the-shelf CD20×CD3 bispecific antibody, demonstrated compelling efficacy and a generally manageable safety profile in heavily pretreated pts with R/R FL in the primary analysis of the Phase 2 ELM-2 study (NCT03888105; Kim TM, et al. Ann Oncol 2024). We present updated efficacy and safety data for odronextamab in pts with R/R FL from ELM-2 after >2 yrs follow-up. Methods: Odronextamab was administered intravenously until disease progression/unacceptable toxicity, with Cycle (C) 1 step-up dosing to help mitigate cytokine release syndrome (CRS) risk, as reported previously. Pts with a complete response (CR) for ≥9 months (mo) switched from maintenance dosing Q2W to Q4W. Primary endpoint: objective response rate (ORR) per Lugano criteria by independent central review (ICR); secondary endpoints: CR rate, duration of response (DOR), progression-free survival (PFS), overall survival (OS). Results: At the updated data cutoff (Aug 15, 2024), 157 pts with centrally confirmed R/R FL Grade (Gr) 1–3a were enrolled. Median no. of treatment cycles: 19.0 (range 0.1–117.3); 96.2% (n = 151) and 82.8% (n = 130) of pts completed C1 and C4, respectively. The global cohort comprised 128 pts evaluable for efficacy. At a median efficacy follow-up of 28.3 mo, ORR was 80.5% (n = 103) by ICR and CR rate was 74.2% (n = 95); 92.2% of responders achieved CR. Responses were durable (median DOR, 26.0 mo; median duration of CR, 32.2 mo). ORR was consistent across high-risk subgroups (Follicular Lymphoma International Prognostic Index score 3–5, 78.4%; progression of disease within 2 yrs of frontline therapy, 81.0%). Median PFS was 23.0 mo (estimated 36-mo PFS rate, 37.5%), and median OS was 54.2 mo (estimated 36-mo OS rate, 62.6%). Median PFS was longer in pts who were minimal residual disease (MRD) negative (42.4 mo) versus MRD positive (21.6 mo) at Week 12. Of 47/128 pts who switched to Q4W dosing, 32 remained in CR. The odronextamab long-term safety profile was consistent with the primary analysis. All 157 pts had TEAEs (Gr ≥3, 86.0%), and 15.3% discontinued treatment due to TEAEs (most common: COVID-19 infection, 2.5%). With 0.7/4/20 mg step-up dosing (n = 89), CRS events were mostly low grade (Gr 1, 46.1%; Gr 2, 13.5%; Gr 3, n = 1; Gr ≥4, n = 0), occurred mostly in C1, and resolved in a median of 8.4 hrs. Immune effector cell-associated neurotoxicity syndrome was reported in one pt (Gr 2). Infections were reported in 79.0% of pts (124/157; Gr ≥3, 42.0%). COVID-19-related infections were reported in 38.2% of pts (Gr 5, 5.7%). Conclusions: With longer follow-up, odronextamab demonstrated durable responses in heavily pretreated pts with R/R FL from ELM-2, with robust efficacy in those with high-risk features, and a generally manageable safety profile. Overall, these compelling results support odronextamab as a potential off-the-shelf treatment option for pts with R/R FL. Clinical trial information: NCT03888105 .

Abstract RATIONALE: Alveolar Type 2 (AT2) cells play a critical role in maintaining alveolar function and facilitating repair processes in the lung. Despite recent advancement on transcriptomic mapping of cell identities and AT fate selection, the mechanisms governing AT2 fate plasticity remain elusive. Notably, in AT2-derived pulmonary diseases such as COPD and IPF, disruptions in AT2 differentiation are observed, suggesting alterations may exist in the regulation of fate plasticity. Therefore, a refined model describing the regulation of AT2 fate plasticity is needed, along with an assessment of the model's conservation across murine and human AT2s. METHODS: We performed scRNA-sequencing, ATAC-seq, and bioinformatic analysis on samples across murine lung development and validating findings by immunostaining of embryonic lungs. We conducted organotypic cultures as well as genetically engineered mouse model experiments to assess changes in AT2 fate plasticity in. Further, we established a Mayo Clinic patient lung cell biorepository from patient tissue derived AT2s. In parallel, we performed long read scRNA-sequencing for isoform discovery, organotypic culture and immunostaining in human AT2 organoids, to characterize the presence of similar regulatory mechanisms. RESULTS: Throughout the late embryonic and early perinatal period, we found a window of fate plasticity exists in AT2s whereby they retain the capacity to give rise to AT1s, and that its being closed over time by the bZIP transcription factor C/EBPα, as its genetic deletion in AT2s results in regaining fate plasticity. By ATAC-seq and ChIP-seq, we found C/EBPα binds closely to the Dlk1 locus, a Notch signaling regulator implicated in AT2-to-AT1 conversion that is upregulated following Cebpa deletion. Following adult lung injury, we found C/EBPα downregulation is required for re-accessing AT2 fate plasticity and is likely mediated by the dominant negative C/EBP family member CHOP. Finally, we observe Cebpa loss also activates a “defender” AT2 state enriched in interferon stimulated antipathogenic gene expression that is distinct from its reparative state and propose AT2s can toggle between these states following infection to robustly protect and repair alveoli.Further, we validated the presence of C/EBPα and absence of Dlk1 in human AT2s isolated from healthy adult patients, which persists following in vitro expansion. We conclude that AT2 cells window of fate plasticity is dictated by a molecular circuit involving C/EBPα suppressing and Dlk1. Future works aim to remove C/EBPα in human AT2s to reopen their fate plasticity for cell therapy.

Abstract RATIONALE: The alveolar epithelium is primarily comprised of two cell types – the flat alveolar type I (AT1) cell responsible for gas exchange, and the cuboidal AT2 cell responsible for surfactant secretion, alveolar defense and acts as facultative stem cells in injury repair. Both cell types emerge from a population of bipotent distal progenitors (DPs). While the FGF signaling involved in directing AT2 fate has been identified, the mechanism by which AT2 differentiation is precisely timed is not well understood. In embryonic stem cells, it has been demonstrated that differentiation is regulated by a biophysical parameter, cell membrane tension (CMT), that acts upstream of FGF signaling by suppressing receptor endocytosis. Given this, we sought to investigate whether and how CMT plays a role in timing the alveolar epithelial differentiation, particularly focusing on how CMT might regulate FGF signaling. METHODS: DPs were isolated from E15.5 mice and plated on top of or embedded in Matrigel. DPs were treated with small molecules, to rise CMT (CHIR22901 and Methyl Beta cyclodextrin; MBCD), inhibiting either endocytosis (Dynasore) or pathways downstream of FGF signaling (FR180204, Akt VIII, KRpep-2d, NSC 74859). CMT is measured both with optical trap technology and FlipTR dye, which uses fluorescence lifetime imaging microscopy. Tissues and cells were immunostained and imaged using markers for AT1 and AT2 cells, Fgfr2, and the endocytosis marker EEA1. The transgenic mice for this project are in a C57B6 strain background and include a pan-lung epithelial Cre (TgNkx2.1-Cre) bred to Cre-dependent ERK reporter (Rosa26SAE). RESULTS: We found that CMT reduces immediately prior to alveolar differentiation, and that increasing CMT with CHIR and MBCD blocks differentiation of DPs in culture. As CMT reduces, we observe an increase in endocytosis and internalization of Fgfr2 in both cultured cells and in vivo at E17.5. Further, scRNAseq analysis showed an upregulation of ERK target genes and we have validated higher ERK activity in nAT2s in the reporter line. Further, we validated that CMT regulates FGF signaling, as decrease in CMT results in more Fgfr2 endocytosis that likely activates ERK and Akt signaling. Our small molecule inhibition data suggests that ERK and Akt signaling are required for AT2 fate selection and we propose that they are the downstream targets of FGF signaling that are activated following lowered CMT and increased Fgfr2 endocytosis. Our findings are of fundamental importance for understanding how cell mechanics regulates biochemical signaling and fate selection during lung development.

Abstract RATIONALE: Alveoli form late in development and, for first breath, alveolar type 1 (AT1) as well as AT2 epithelial cell types must be functional. While prior studies have mapped the cellular and transcriptional programs underlying this process and identified AT fate inductive cues, questions remain as to the timing and regulation of their emergence. Thus, we sought to identify when, where, and how AT2 cells first arise and motivated by recent work in embryonic stem cells, we investigated whether the biophysical parameter cell membrane tension (CMT) plays a role. METHODS: Single-cell RNA-sequencing (scRNAseq) datasets sampling mouse lung development were processed using cluster correlation, velocity, and gene ontology analysis. Marker expression and AT2 morphology was assessed by immunostaining, and their migration confirmed by time-lapse confocal microscopy on embryonic precision-cut lung slices (PCLS). CMT was measured via optical tweezers (OT) on isolated cells as well as fluorescence lifetime (FLIM) on organoids and PCLS. Progenitors (E15.5) and AT2s (E16.5- >PN50) were isolated from distal region of the mouse lung, as well as human AT2s recently isolated, expanded, and banked in Mayo Clinic's recently established patient lung cell biorepository. CMT was raised using methyl-β-cyclodextran (MBCD) or polyethylene glycol (PEG). Small molecules CHIR and ICG-001 were used in culture to raise and lower β-catenin levels, respectively. Fluorescently labeled mesenchymal cells were isolated, expanded, and added to organoids in suspension for co-association experiments. RESULTS: No evidence of distal progenitors selecting either AT fate was found until E16.5, at which time we identified a distinct nascent AT2 (nAT2) state that ontology analysis detects upregulation of single-cell migration genes. Resistin-like molecule alpha (RELM-α), a type 2 inflammation regulator, labels nAT2s – which first individually emerged around E16.5 (although instances exist at E15.5) at intermediate zones between the branch tips and stalks of the distal epithelium. Following molecular emergence, we observed nAT2s extruded basally and can subsequently interact with nearby but anatomically distinct lumens, a behavior we provisionally name interlumenal junctioning and whose dynamics we validated by timelapse microscopy. Regarding CMT, we found its reduction in distal progenitors occurs prior to, and is required for, differentiation. We observed CMT reduction is sensitive to the manner of organoid culture, with on top culture resulting in more reduction and differentiation. Finally, we observed CMT is regulated cell-intrinsically via the adaptor protein β-catenin, as well as -extrinsically through osmotic (via PEG) or mesenchymal cell interactions, both of which reduce membrane fluidity.

Abstract RATIONALE: The alveolar epithelium consists of two cell types (AT2 and AT1) derived from a common Distal Progenitor (DP). Understanding how the epithelium of alveoli functions and becomes differentiated is essential for treatment of lung diseases. One possible regulator of cell fate is cell membrane tension (CMT), a biophysical property measured by the energy required to deform the membrane and known to be an important regulator of cell movement and endocytic processes. AT1 fate selection is largely driven and reinforced by YAP (Yes-associated protein), a mechano-sensitive transcription factor gated from entering the nucleus by the size of nuclear pores, which increases upon stretch. Here, we investigated whether and how CMT regulates the transduction of stretch-dependent YAP signaling and the remodeling into AT1 cells. METHODS: DPs were isolated from E15.5 mice and cultured on Matrigel for use in differentiation experiments. Immunostaining for AT1 and AT2 markers, Phalloidin, DAPI and YAP were used to assess YAP localization and architectural changes. We generated transgenic mice containing a pan-epithelial Cre (TgNkx2.1-Cre), a Cre-dependent driver of ltta (TigreAi140), and a TetOff regulated construct expressing YAP1 (Rosa26teto-Yap1), resulting in constitutive Yap1 expression in Cre-expressing DPs. CMT is measured both with optical trap technology and with the use of FlipTR dye, which uses Fluorescence Lifetime Imaging Microscopy (FLIM) to determine CMT. RESULTS: To demonstrate the role of CMT in timing AT1 fate, we found that CMT lowers in DPs concomitantly with differentiation, and that flattening cells (likely AT1s) have even lower CMT than round cells (likely AT2s). Using organotypic culture, we found that raising CMT with MBCD, PEG, or Matrigel embedding largely stops differentiation of DPs. Surprisingly, even when YAP is overdriven in mice in E15.5 distal progenitors, early precocious AT1 differentiation is not observed, suggesting YAP signaling is not alone sufficient to drive AT1 fate selection in DPs with higher CMT. We reproduced this finding when culturing DPs under high CMT conditions-promoting high YAP/TAZ levels via LATS inhibition was also not sufficient to select for AT1 fate. Finally, we found that high CMT disrupts nuclear YAP localization, nuclear-actin attachment, and cytoskeletal remodeling that is typically when DPs remodel towards AT1 fate. We propose that high baseline CMT in DPs prevents cellular remodeling, specifically that it suppresses the formation of actin-nuclear associations that imbue the nucleus with stretch sensitivity, thereby preventing an increased sensitivity to stretch and thus higher YAP signaling.

Abstract Background: Leukocyte immunoglobulin-like receptor B2 (LILRB2) suppresses immune cell activation, particularly within myeloid cells in the tumor microenvironment (TME), while the PD-1/PD-L1 axis serves as a key immune checkpoint that inhibits T-cell function. SPX-303 is a novel bispecific antibody designed to simultaneously block LILRB2 and PD-L1, thereby revitalizing antitumor immune responses. Here, we present initial findings from the ongoing first-in-human Phase 1 trial of SPX-303 in patients with advanced solid tumors, including safety, pharmacokinetics (PK), and receptor occupancy (RO). Methods: This is an open-label, multicenter Phase 1 study for patients with advanced solid malignancies with 3+3 dose-escalation design, followed by an expansion cohort at the recommended Phase 2 dose (RP2D). SPX-303 is administered intravenously every three weeks and safety is assessed through CTCAE v5.0 criteria. Primary Objectives of this study are to determine the safety, tolerability, and RP2D of SPX-303. Secondary endpoints include antitumor activity as well as characterization of the PK and PD of SPX-303. This study is currently open and enrolling in escalation cohorts. Citation Format: Justin Moser, Hao Xie, Yujie Zhao, Yousef Zakharia, Dongdong Li, Brandon William, Dinh-Duc Nguyen, Jack Hovey, Tiffany Li, Robert Cai, Jichun Ma, Jingdong Qin, Martin Siekierzycki, Anthony Haight, Hua Jin, Weiwei Mao, Kevin Bin Li, Gui-Dong Zhu. A first-in-human phase 1 study on the safety, PK, receptor occupancy, and pharmacodynamic markers of SPX-303, a first-in-class LILRB2×PD-L1 bispecific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT116.

Background: Multicellular programs in the tumor microenvironment (TME) drive cancer pathogenesis and response to therapy but remain challenging to identify and profile clinically. Here, we present Spatial EcoTyper, a multimodal machine learning framework for large-scale profiling of spatially dependent cell states and multicellular ecosystems, termed spatial ecotypes (SEs). Methods and Findings: By integrating five million single-cell and spot-level spatial transcriptomes from 10 diverse human neoplasms, including carcinomas and melanomas, we identified a rich tapestry of conserved regional plasticity across nine TME cell types. We then applied Spatial EcoTyper to deeply dissect these data, revealing nine novel SEs with broad conservation - each with unique biology, TME cell states, geospatial features, and clinical outcome associations. SEs were linked to specific biogeographic domains, including the tumor core, margin, and adjacent stroma, and were remarkably well-validated in 235 held-out tumor samples profiled by either single-cell RNA sequencing, sequencing-based spatial transcriptomics (10x Visium), or high-resolution imaging-based spatial transcriptomics (Vizgen MERSCOPE and NanoString CosMx). By deconvolving SEs from over 1,200 bulk tumor RNA-seq profiles, we discovered striking associations between SE levels and response to immune checkpoint inhibitors (ICIs), outperforming nearly 40 potential correlates of ICI response, including previously described signatures of tertiary lymphoid structures and tumor tissue PD-L1 expression. Notably, baseline levels of a proinflammatory SE - localized to the tumor core and defined by a hub of unique T cell, macrophage, fibroblast, and endothelial cell states - strongly forecasted ICI benefit, while another SE - localized to the tumor margin and defined by myofibroblast and hypoxia-associated endothelial cell states - portended ICI resistance. Conclusion: Our data reveal fundamental units of TME organization and demonstrate a platform for large-scale profiling of spatial cellular ecosystems in any tissue, with implications for improved forecasting of immunotherapy response and the development of novel therapeutic strategies. Citation Format: Wubing Zhang,Erin L. Brown,Abul Usmani,Noah Earland,Hyun Soo Jeon,Irfan Alahi,Nicholas P. Semenkovich,Janella C. Schwab,Chloé B. Steen,Chloe M. Sachs,Bilge Gungoren,Faridi Qaium,Peter K. Harris,Jacqueline Mudd,Qingyuan Cai,Antonella Bacchiocchi,Ryan C. Fields,Mario Sznol,Ruth Halaban,David Y. Chen,Aadel A. Chaudhuri,Aaron M. Newman. High-resolution profiling of the tumor microenvironment with spatial ecotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 153.

Abstract Background The mechanisms underlying metastatic latency in renal cell carcinoma (RCC) remain poorly understood. Methods This study evaluated 2 large independent cohorts for differences in tumor biology between patients who developed metastases early (≤1 year after nephrectomy) and those with late onset (>3 years). Results In the discovery cohort (n = 161), late metastatic RCC was associated with clear cell histology (88.9% vs 78.7%), lower pathological stage (pT1-2; 40.3% vs 18.0%), and favorable histopathological features including low grade (40.0% vs 2.3%), less sarcomatoid (5.6% vs 21.8%), and reduced necrosis (37.7% vs 78.3%; all P < .02). Late metastatic RCC tumors exhibited increased angiogenesis (63.5% vs 19.4%) and reduced inflammation (78.8% vs 50.0%; all P < .02) profiles. Genomic driver analyses revealed comparable rates of PBRM1 and SETD2 loss in late and early metastatic RCC, while BAP1 loss was significantly less common in late metastatic RCC (7.5% vs 27.1%; P < .02). In multivariable models, BAP1/PBRM1/SETD2 status and tumor necrosis emerged as key discriminators of late metastatic RCCs. These findings were confirmed in the second cohort (n = 307). Late metastatic RCC was enriched for fatty acid oxidation and angiogenesis pathways, supporting a less aggressive phenotype. This was further evidenced by a lower engraftment rate in murine models (0% vs 36.5%; P < .001) and significantly longer overall survival from the time of metastasis (median survival doubled, P < .001). Interestingly, late metastatic RCC shared genomic and phenotypic features with RCC that metastasizes to the pancreas, suggesting a common underlying biology influencing both metastatic latency and pancreatic tropism. Conclusions Overall, these findings advocate for recognition of late metastatic RCC because of its distinct biology and improved prognosis.