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Abstract Introduction Pulmonary fibrosis is characterized by excessive fibroblast activation and alveolar remodeling, leading to progressive lung dysfunction. Osteoglycin (OGN), a small leucine-rich proteoglycan, and its functional fragment MC002 have been identified as regulators of alveolar epithelial regeneration, a process that is impaired in fibrotic lung disease. This study investigates the effects of OGN and MC002 on TGF-β-induced fibrotic responses in lung fibroblasts, organoids, and an in vivo fibrosis model. Methods Human lung fibroblasts (MRC5) were treated with OGN or MC002 for 48 hours, with or without transforming growth factor-beta (TGF-β; 2 ng/mL). Expression of the myofibroblast marker α-smooth muscle actin (ACTA2) and matrix markers collagen 1a1 (COL1A1) and fibronectin (FN1) was assessed by RT-PCR and Western blotting. Global transcriptomic changes were analyzed by RNA sequencing. Myofibroblast differentiation was evaluated by phalloidin staining and quantified using the phalloidin/DAPI ratio. Murine lung organoids were generated by co-culturing epithelial progenitor cells with fibroblasts and treated with TGF-β (2 ng/mL) in the presence or absence of OGN or MC002. Organoid number, size, and alveolar differentiation were quantified after 14 days. In vivo, pulmonary fibrosis was induced in mice using intratracheal bleomycin. MC002 was administered at different doses (6.75 µg or 20.25 µg) every other day starting from day 7 post-bleomycin, and lung weight and pressure-volume (PV) loops were assessed to evaluate fibrotic burden and lung mechanics. Osteoglycin expression in human lung tissue from 13 non-smokers, 11 ex-smokers, and 12 IPF patients was analyzed by immunostaining. Results TGF-β induced pro-fibrotic gene expression in fibroblasts, while OGN and MC002 alone had no pro-fibrotic effects. MC002 fully reversed TGF-β-induced ACTA2, COL1A1, and FN1 expression, whereas OGN selectively attenuated COL1A1. RNA-sequencing revealed broad suppression of fibrosis- and ECM-associated gene programs by MC002. At the protein level, TGF-β-induced ACTA2 expression was reversed by MC002 but not by OGN. Both compounds reduced TGF-β-induced stress fiber formation. In lung organoids, TGF-β impaired organoid formation and alveolar differentiation, effects that were restored by OGN and MC002 in a concentration-dependent manner. In the bleomycin model, MC002 significantly reduced lung weight and improved PV loop profiles, indicating attenuation of fibrotic stiffening and improved lung compliance. Osteoglycin protein expression was significantly diminished in IPF whole lung tissue and parenchyma compared to controls. Conclusion OGN, and particularly MC002, counteract TGF-β-driven fibroblast activation and myofibroblast differentiation while promoting alveolar preservation. The superior efficacy of MC002 supports its potential as a therapeutic candidate for pulmonary fibrosis. This abstract is funded by: Lung Foundation Netherlands 10.1.23.021PPS

Abstract As an intrinsically mechanosensitive organ, the lung experiences a range of mechanical forces. Chronic obstructive pulmonary disease (COPD) involves abnormal macroscopic cellular and extracellular matrix (ECM) changes that impact mechanical properties of the lung. Mechanosensitive Piezo1/2 channels are expressed in the lung including on airway smooth muscle cells (ASM) that mediate cellular responses to stretch and ECM biomechanics. The expression and roles of Piezos in COPD lung ASM are not known. We hypothesized that Piezo expression and activation are altered in COPD lung ASM influencing ECM regulation. Distribution of Piezo proteins in ASM and epithelium of small airways of COPD stage II and IV vs. non-COPD controls was assessed using immunohistochemistry and ImageJ (n=10-17/group). Isolated ASM cells from control (n=6) vs. COPD stage II and IV patients (n=3 each stage) were exposed to stretch or the Piezo1 agonist Yoda1 followed by measurement of ECM gene and protein expression. Less Piezo2 staining was observed in COPD IV patients compared to controls, with lesser area and intensity of staining in the epithelial layer, and lower intensity of staining in ASM and small airways as a whole. Fura-2-based imaging of ASM Ca 2+ showed lower influx after Yoda1 exposure in COPD II compared to control and COPD IV. Gene expression of Piezo1 increased upon stretching in controls but not in COPD ASM, while Piezo2 protein expression decreased with stretching in all groups. Yoda1 treatment resulted in decreased collagen1 , fibulin1 and periostin gene and collagen 1 and periostin protein expression in ASM. Overall, these results support a role for Piezo activation in abnormal ECM-ASM cell crosstalk in COPD.

4562 Background: Approximately 50-80% of patients (pts) with la/mUC have tumors with HER2 protein expression. As HER2-directed therapies enter development, a robust understanding of HER2 prevalence and real-world outcomes with standard-of-care (SOC) therapies across HER2 expression subgroups is needed. SGNDV-003 (NCT05902494) is a global retrospective chart review study in which HER2 status was assessed via central lab testing and outcomes were analyzed by HER2 status in pts with la/mUC. Methods: SGNDV-003, conducted 6/2023-7/2025, included pts with la/mUC who received ≥1 prior treatment (tx) (incl. 1 line of platinum-based chemotherapy [chemo]). HER2 status was centrally tested using a UC-specific investigational immunohistochemistry [IHC] and in situ hybridization (ISH) assays. HER2 status was defined as: HER2-positive (IHC3+ or IHC2+/ISH+), HER2-low (IHC2+/ISH- or IHC1+), HER2-expressing (IHC3+, 2+, 1+), and HER2-zero (IHC0). Index line of therapy (LOT) was defined as the first LOT received after platinum-based chemo. Outcomes included real-world objective response rate (rwORR) (per RECIST 1.1 or physician-assessed response) to index LOT, real-world progression-free survival (rwPFS), and overall survival (OS) from start of index LOT. Data was analyzed via descriptive statistics. Results: A total of 353 pts with la/mUC and available HER2 status were analyzed (male, 76%; median age, 70 years). 70% of pts were HER2 expressing (19% HER2-positive, 51% HER2-low); 30% were HER2-zero. Disease characteristics were well balanced by HER2 status. Index LOT tx and real-world outcomes are shown in the Table. Immuno-oncology therapies (IO) and enfortumab vedotin (EV) were the most common index LOTs. For IO, rwORRs were 21% for HER2-expressing and 17% for HER2-zero. For EV mono, rwORRs were 36% (HER2-expressing) and 38% (HER2-zero), respectively. Conclusions: This global study provides a robust estimate of HER2 distribution in la/mUC based on standardized central lab testing, showing high prevalence of HER2 expression. Real-world outcomes with current SOC therapies were broadly consistent across HER2 subgroups, establishing an important benchmark for the clinical development of HER2-directed therapies in this setting. HER2-positive (n=67) HER2-low (n=181) HER2-expressing (n=248) HER2-zero (n=105) Total (n=353) Real-world Outcomes rwORR, n (%)[95% CI] 17 (25.4)[15.5, 37.5] 50 (27.6)[21.3, 34.7] 67 (27.0)[21.6, 33.0] 22 (21.0)[13.6, 30.0] 89 (25.2)[20.8, 30.1] Median rwPFS (95% CI), mo 2.8(2.0, 4.1) 3.7(2.7, 5.1) 3.0(2.7, 4.5) 2.6(2.1, 3.4) 2.9 (2.6, 3.5) Median OS(95% CI), mo 12.2(9.3, 16.5) 11.5(9.5, 13.5) 11.9(10.3, 13.4) 7.7(5.9, 9.9) 10.8(9.1, 12.3) Index LOT, n% IO EV monoChemoOther tx 37 (55)17 (25)10 (15)3 (5) 94 (52)51 (28)24 (13)14 (8) 131 (53)68 (27)34 (14)17 (7) 65 (62)21 (20)12 (11)7 (7) 196 (56)89 (25)46 (13)24 (7)

11502 Background: To report results from the open-label alveolar soft part sarcoma (ASPS) cohort of the phase III APROMISS trial (NCT03016819) evaluating Catequentinib hydrochloride (AL3818), an oral multitargeted receptor tyrosine kinase inhibitor, in adults with advanced or metastatic ASPS. Methods: Adult patients with pathologic and molecularly confirmed advanced ASPS, either treatment-naive or previously treated (excluding cediranib), were enrolled in the open-label, non-randomized ASPS cohort of the phase III APROMISS trial conducted across the United States, Asia, and Europe. Catequentinib hydrochloride was administered orally at 12 mg once daily in 21-day cycles (14 days on/7 days off) until disease progression, toxicity, or death. The primary endpoint was objective response rate (ORR) assessed per RECIST by Blinded Independent Central Review (BICR) (predefined threshold: 31%). Secondary endpoints included duration of response (DOR) with a predefined target of =/>6 months, progression free survival (PFS) and overall survival (OS). Results: From July 2017 to August 2022, a total of 56 patients entered the trial; 56 patients were evaluable for the primary endpoint. Median (m-) age was 29.5 years (range: 18 – 66), with 24 female and 32 male. Best overall response included 1/56 complete response (1.8%), 14/56 partial responses (25.0%), 36/56 stable disease (64.3%), and 3/56 (5.4%) progressive disease. RECIST ORR by BICR was 26.8% (95% CI, 15.8–40.3); m-DOR was 22.57 months (95%CI: 10.38-NE). The study data was collected close to 8 years for all patients. mPFS was 18.33 months (95% CI, 14.19–23.13), with 6- and 12-month progression-free rates of 88.77% (95% CI, 76.68–94.79) and 65.54% (95% CI, 50.42–77.06), respectively. mOS was 59.27 months (95% CI, 44.09–NE), with 6- and 12-month overall survival rates of 94.64% (95% CI, 84.30–98.24) and 90.81% (95% CI, 79.30–96.08), respectively. Overall, 27/56 (48.2%) patients experienced grade ≥3 treatment-related adverse events, most commonly diarrhea (50.0%), hypertension (44.6%), fatigue (37.5%), nausea (35.7%), and palmar-plantar erythrodysesthesia syndrome (30.4%); one patient (1.8%) had a grade 5 event (respiratory failure). Conclusions: Catequentinib hydrochloride demonstrated meaningful antitumor activity and durable responses in adults with advanced ASPS. Despite the ORR of 26.8% slightly below the predefined 31% threshold, disease control was prolonged, with a m-DOR exceeding 22 months. Favorable PFS and OS outcomes, together with a manageable safety profile, support its clinical benefit in this ultra-rare sarcoma. Clinical trial information: NCT03016819 .

We previously reported initial results of the pivotal phase II trial of atezolizumab for patients with alveolar soft part sarcoma (ASPS; ClinicalTrials.gov identifier: NCT03141684 ). Here, we report on three additional years of observation. Fifty-three patients with ASPS received atezolizumab. Median duration of response increased to 37.0 months. Objective response rate (ORR) and median progression-free survival (mPFS) remained essentially as previously reported (35.8% [95% CI, 23.1 to 50.2] and 20.8 months [IQR, 7.6-not reached], respectively). ASPSCR1::TFE3 fusion type was determined for 47/53 patients; ORR and mPFS were higher among the 41 patients expressing type 1 (43.9% [95% CI, 28.5 to 60.2] and 28.3 months [IQR, 9.2-not reached], respectively) than the six patients expressing type 2 (0% [95% CI, 0 to 45.9] and 7.5 months [IQR, 3.9-not reached], respectively, PFS HR, 3.2 [95% CI, 1.01 to 10.2]). Eleven patients chose a per-protocol drug holiday (range, 3.5-26.4 months) after ≥2 years of treatment; two experienced disease progression during the holiday. Nine eligible patients elected to receive bevacizumab plus atezolizumab after progressing on monotherapy; ORR was 0% and mPFS was 18.5 months (IQR, 7.9-21.1) in this small cohort. Long-term results support using atezolizumab to treat ASPS, even for several years; a drug holiday with careful monitoring may be an option for some patients.

Abstract Background Primary CNS lymphoma (PCNSL) is a rare, aggressive diffuse large B-cell lymphoma confined to the CNS, with poor outcomes. Virally associated PCNSL, including AIDS-related PCNSL (AR-PCNSL) and CNS post-transplant lymphoproliferative disorder (CNS-PTLD) accounts for ~10% of cases and is strongly linked to EBV infection and poor prognosis. In our prior Blood (Gandhi et al. 2021) study, targeted sequencing showed that EBV⁺ PCNSL lacks canonical MYD88, CD79B, and PIM1 mutations and retains intact HLA class I/II, suggesting distinct immunobiology. However, questions remain regarding the immune microenvironment, broad mutational landscape, and intratumoral heterogeneity. Here, we present the first large-scale comprehensive integrated analysis of virally associated PCNSL to define subtype-specific vulnerabilities and therapeutic implications. Methods FFPE brain biopsies from 72 PCNSL patients (40 EBV- de novo PCNSL, 32 EBV+ PCNSL including 18 AR-PCNSL and 14 CNS-PTLD) were analyzed. Immune gene expression was profiled using the NanoString PanCancer Immune Panel. Whole exome sequencing (WES) defined recurrent driver mutations, tumor mutational burden (TMB), and LymphGen classification (excluding A53 subtype). Single nuclei RNA sequencing (snRNA-seq) using the 10x Genomics Chromium Flex platform characterized cellular heterogeneity. Results WES demonstrated a marked reduction in canonical mutations (MYD88L265P, CD79B, PIM1, CARD11) in CNS-PTLD (0%) and AR-PCNSL (29%) vs. de novo PCNSL (87.5%; p<0.0001). AR-PCNSL had a distinct mutational signature when compared with CNS-PTLD and de novo PCNSL. NOTCH1/2 mutations were significantly enriched in AR-PCNSL (71%) vs. CNS-PTLD (9%) and de novo PCNSL (33%) (p<0.01). NLRC5 mutations were present in 64% of AR-PCNSL cases, higher than the 25% observed in de novo PCNSL and were absent in CNS-PTLD. TMB was significantly elevated in AR-PCNSL (mean 26.27 mutations/Mb) vs. de novo PCNSL (17.22) and CNS-PTLD (11.01; p=0.0018) underscoring pronounced genomic instability and suggesting HIV may exert a mutagenic influence beyond immunosuppression. EBV driven subtypes lacked MCD features and were more often classified as ST2 or unassigned. Despite NOTCH2 mutations, AR-PCNSL cases were not classified under the BN2 subtype, further highlighting limitations in current molecular classification systems to accurately stratify virally associated PCNSL. Immune transcriptomic profiling revealed further distinctions within EBV+ PCNSL. CNS-PTLD showed increased expression of genes related to antigen presentation (p<0.0001), cytotoxicity (p=0.0019), and T cell function (p<0.0001), suggesting retained immune responsiveness. AR-PCNSL exhibited significant suppression of HLA class I/II genes (p=0.0027 and p<0.0001 respectively), despite intact genomic loci, implicating post-transcriptional or epigenetic silencing. Immune cell composition revealed reduced macrophage infiltration and suppressed CD4 expression in AR-PCNSL. CNS-PTLD showed higher expression of checkpoint molecules LAG3, TIM3, and CTLA4, consistent with T cell exhaustion. snRNA-seq profiling of AR-PCNSL identified distinct malignant and non-malignant cell populations, including a reduced CD4⁺ compartment and altered microglial/macrophage populations and cell states compared to de novo PCNSL, corroborating bulk transcriptomic data, amongst other ongoing analysis. Conclusion This study represents the largest and most comprehensive immunogenomic investigation of virally associated PCNSL. AR-PCNSL and CNS-PTLD lack canonical PCNSL driver mutations and instead harbor distinct mutational signatures, including NOTCH pathway alterations in AR-PCNSL. These findings underscore divergent pathogenic mechanisms and support tailored therapies. AR-PCNSL is marked by high TMB, suppressed antigen presentation, and reduced CD4⁺ infiltration, indicating profound immune dysfunction. CNS-PTLD retains immune functionality but shows checkpoint upregulation and T cell exhaustion. Despite both being EBV-driven and CNS-restricted, they display distinct molecular and immunological programs, suggesting context specific EBV mechanisms. These findings challenge existing classification systems and support CNS-specific molecular frameworks to guide therapeutic interventions including BTK inhibition for NOTCH-mutated lymphoma. They lay the foundation for precision approaches in EBV⁺ PCNSL, including immune restoration, antigen-specific T cell therapy, and epigenetic modulation.

Abstract BACKGROUND: DNA methyltransferase inhibitors (DNMTi) are US FDA approved for the management of CMML. These agents are associated with an overall response rate (ORR) of 40-50%, with a true complete remission (CR) rate of <20%. TET2 mutations are seen in 60% of CMML, with TET2 being a ferrous and oxoglutarate dependent DNA-dioxygenase that oxidizes 5mC to generate 5hmC, 5fC and 5caC. Ascorbic acid (AA) is critical for TET activity and can enhance TET 1/2/3 dioxygenase function by binding to the catalytic domain and when given in high doses, can result in glutathione depletion and free radical induced cell death. In a pilot study (Xie et al. Blood 2024), we demonstrated that high dose IV AA can be clinically effective and induce changes in differentially methylated regions (DMR) involving hematopoietic enhancers and improve cytopenias in TET2-mutant (mt) patients [CCUS]. We expanded this study to combine high dose IV AA with DNMTi to enhance the demethylation effect and produce durable responses in new diagnosed, treatment requiring, TET2mt CMML patients. METHODS: At Mayo Clinic, the LS1781 (NCT03418038) clinical trial was amended to include Arm E, combing high dose IV AA with standard of care decitabine in newly diagnosed TET2mt CMML patients that needed therapy. IV AA was dosed at 1 g/kg on days 1, 3 and 5 (per 28-day cycle, max dose 100 gm) through a central line, based on demonstrated safety from phase 1 studies. Standard dosing was adopted for decitabine, and patients were then asked to take oral AA, 1 gm daily from days 6-28. Key inclusion criteria included a WHO-defined diagnosis of CMML with at least one detectable TET2 mutation requiring therapy with adequate performance status (ECOG PS <2) and organ function (creatinine clearance > 55 mL/min). Response was evaluated by the MDS/MPN International Working Group Criteria (Savona et al. Blood 2015). We measured pre- and post-therapy (4 cycles) bone marrow variant allele fractions (VAF) through next generation sequencing assays, transcriptomic changes through RNA seq, and DNA methylation changes using the Illumina Methylation EPIC array. RESULTS: Between February 2024 and July 2025, a total of six patients were enrolled. The median age at enrollment was 73 years (range: 65-80); all Caucasian with 3 (50%) males. All patients had a diagnosis of CMML-1 with a median bone marrow (BM) blast percentage of 3 (0-10), median hemoglobin 13 (9.5-14.5) g/dL, median WBC count of 5.7 (2.1-12.6) x 109/L, absolute monocyte count 1.4 (0.7-2.7) x 109/L, and platelet count of 92.5 (32-123) x 109/L. TET2 (100%, 4 patients > 1 TET2 mutation at mean VAF of 36%), SRSF2 (67%), and KRAS (33%) were the most frequent molecular abnormalities. Neutropenia (without infection/sepsis) was the only treatment-associated adverse effect seen in 3 (50%, all grade 3) patients. Response was assessed in patients who completed at least 4 cycles (n=5) of therapy, with all (100%) patients meeting criteria for either a complete response (n=2, 40%), optimal marrow response (n=1, 20%) or clinical benefit (platelet response, n=2, 40%). In five patients who had NGS assessment pre-and post-therapy, there was a >50% reduction in TET2mt VAF, with one patient loosing the KRAS G12D clone in its entirety. Furthermore, we identified significant changes in DNA methylation associated with treatment with >85% of the CpGs being hypomethylated post-treatment. RNA-sequencing on patients pre- and post-treatment, identified key transcriptomic changes, with a decrease in expression of leukemogenic genes. Integration of the differential methylation data with transcriptomic changes identified several genes in which changes in DNA methylation correlated with a change in gene expression. These genes include regulators of blood cell development and inflammatory response including GATA2, BCL2, and HLA-DPA. Several leukemia-associated genes were downregulated including MEIS1, TCL1A, and SETBP1. CONCLUSION: In TET2mt CMML, high dose IV AA acid enhances TET activity and synergizes with DNMTi with encouraging clinical responses in all patients and with no new safety signals. On completion of this study, we plan a randomized phase 2 trial comparing high dose IV AA with decitabine vs decitabine alone in newly diagnosed TET2/IDHmt CMML.

Abstract BACKGROUND: DNA methyltransferase inhibition offers palliative benefit but does not alter disease biology. in chronic myelomonocytic leukemia (CMML, Merlevede J et al. Nat Comm 2016). This was conclusively demonstrated in the phase 3 DACOTA study, where decitabine did not improve event-free survival in comparison to hydroxyurea (median 12.1 versus 10.3 months, P=0.27) in advanced myeloproliferative CMML (MP-CMML). Hence, there is in urgent need to develop disease modifying therapies for this dismal condition. We and others have demonstrated a role for RAS mutations in MP-CMML through the activation of the KMT2A-PLK1 axis. Targeting of this axis using onvansertib, an inhibitor of PLK1, has demonstrated its pre-clinical efficacy in MP-CMML patient-derived organoid and xenograft models. (Carr RM et al. Nat Comm 2021). METHODS: We conducted a single center phase 1 study to assess the safety and efficacy of onvansertib in relapsed/refractory MP-CMML (NCT05549661). We used a Bayesian Optimal Interval (BOIN) study design to determine the maximum tolerated dose (MTD). The three dose cohorts included 6, 9 and 12 mg/m2 given orally from days 1 to 21 of each 28-day cycle. Key inclusion criteria included a WHO-defined diagnosis of MP-CMML with a white blood cell (WBC) count > 13,000/mm3 and adequate organ function (estimated GFR > 50 mL/min/m2). Response was evaluated per the MDS/MPN International Working Group Criteria (Savona et al. Blood 2015). Targeting efficacy was assessed by measuring phosphorylation of TCTP (a PLK1 substrate), specifically pTCTP/TCTP ratios by immunoblotting in peripheral blood mononuclear cell (PBMC) samples pre and post cycle 1 (Days 1, 7, 21) and end of cycle 3. Pharmacokinetic studies and translational correlative studies were conducted per protocol. RESULTS: Between April 2023 and July 2025, a total of 9 patients were enrolled. The median age at enrollment was 68 years (range: 60-83); all caucasian with 4 (44%) males. All patients had a diagnosis of CMML-1 with a median bone marrow (BM) blast percentage of 4 (0-9), median hemoglobin 10 (7.1-13.2) g/dL, median WBC count of 11.1 (3.1-72.7) x 109/L (4 patients had a WBC count < 13 x 109/L at enrollment due to hydroxyurea use but were confirmed to be MP-CMML at diagnosis), absolute monocyte count 2.1 (1.9-19.6) x 109/L, and platelet count of 36 (20-494) x 109/L. TET2 (89%), SRSF2 (78%), ASXL1 (56%), NRAS, KRAS and CBL (22% each) were the most frequent molecular abnormalities. Previous therapies included hydroxyurea (n=8) and decitabine (n=1). Four patients were enrolled at the 6mg/m2dose level, 2 at the 9 mg/m2 dose level and we currently have one patient at the 12 mg/m2 dose level. While there were 2 planned patients at the 6 mg/m2 dose level, due to a case of pericardial effusion that was possibly drug related, an additional two patients were enrolled at the same dose level, without any dose limiting toxicity (DLT). Targeting efficacy was confirmed by pTCTP immunoblotting in PBMC samples from 4 patients (6 and 9 mg/m2 dose levels) showing suppressed pTCTP/TCTP ratios. No confirmed DLT has been identified thus far, and no grade 3 or 4 toxicities attributable to the study drug have been reported. Possible or unrelated grade 3 adverse effects included infections (sepsis/cellulitis, n=2, 22%), acute kidney injury (n=1, 11%), pericardial effusion (n=1, 11%), and atrial fibrillation with rapid ventricular response (n=1, 11%). Median duration on therapy was 124 (range: 15-654) days with most common cause of study discontinuation being disease progression (33%). Response was assessed as per the MDS/MPN IWG criteria in patients who received at least 1 cycle (n=8) with 3 (38%) patients meeting criteria for either hematological or optimal marrow response. Specifically, there was one (14%) patient with >100% platelet response, one (14%) with normalization of WBC count (baseline WBC count >50 x 109/L), one (14%) optimal marrow response with BM blast% reduction from 8 to 2, facilitating candidacy for an allogeneic hematopoietic stem cell transplantation, and three patients (38%) remained as stable disease. CONCLUSION: In MP-CMML, PLK1 inhibition with onvansertib is relatively well tolerated and shows preliminary efficacy in approximately 40% of patients, with one patient having an optimal marrow response at the 9 mg/m2 dose. Future directions include a dose expansion phase and studying upfront combination with DNA methyltransferase inhibitors.