Supplementary Chemical Synthesis Methods

Supplementary Figure S2. Classification of cancer cells according to WD/DD liposarcoma signatures.

Heatmaps of expression of 29 key gene signatures in (A) SARC028 and (B) Stanford RNA sequencing data

TPS11586 Background: The Wnt-signaling pathway and its downstream transcriptional effector, β-catenin, are dysregulated in osteosarcoma (OS), promoting tumor growth and metastatic spread. When the Wnt pathway is activated, transduction beta-like protein 1 (TBL1) binds nuclear β-catenin, forming a complex that is necessary to dock on the promoters of Wnt-driven genes. Tegavivint is a first-in-class small molecule inhibitor that selectively binds TBL1, inhibiting TBL1-β-catenin complex formation, and resulting in the nuclear degradation of β-catenin and inhibition of oncogenic transcriptional activation. Preclinical testing of in vivo OS models demonstrates that tegavivint reduces tumor volume and suppresses lung metastases, and has additive effects when combined with gemcitabine. In a phase 1 trial conducted by the Children’s Oncology Group, the recommended phase 2 dose (RP2D) of tegavivint was determined to be 6.5 mg/kg administered intravenously (IV) on a 3 weeks on/1 week off schedule, with no maximum tolerated dose (MTD) determined. The primary objective of this phase 1b study is to define the MTD and/or RP2D of tegavivint combined with gemcitabine, including assessment of toxicities and preliminary efficacy, in patients with relapsed or refractory (r/r) OS. Methods: TIGER is a phase 1b, multi-center, dose escalation trial assessing tegavivint combined with gemcitabine in patients with r/r OS. Eligible patients will be 1-30 years of age with either measurable or evaluable disease per RECIST v1.1 who have fully recovered from the clinically significant acute effects of prior therapy and have adequate organ function. Prior treatment with gemcitabine is allowed. Patients with active CNS disease, recent bisphosphonate treatment, metabolic bone disease or disorder, uncorrected hypocalcemia or low vitamin D, and prior receipt of tegavivint are excluded. Patients will receive tegavivint IV at the dose level assigned at study entry on days 1, 8, and 15 and gemcitabine at a fixed dose of 1000 mg/m 2 IV on days 1 and 8 of a 21-day cycle. Patients may receive up to 17 cycles provided they do not experience disease progression or unacceptable toxicity. Tegavivint will be dose escalated using a rolling six design to test two planned dose levels, 5 mg/kg [dose level (DL) 1] and 6.5 mg/kg (DL 2), with dose de-escalation to 3 mg/kg (DL 0) if needed. An additional 6 patients will be enrolled to a dose expansion cohort at the MTD/RP2D to obtain additional pharmacokinetic (PK), safety, and preliminary efficacy data. Peripheral blood for correlative studies will be obtained at serial timepoints to conduct PK studies, pharmacodynamic testing of serum protein biomarkers associated with β-catenin inhibition, and analysis of ctDNA and circulating tumor cells. Tumor tissue will be analyzed for TBL1 and Wnt/β-catenin signaling (sequencing and protein expression). Clinical trial information: NCT07144254 .

Supplementary Table 1. Cell line and growth media information.

TPS11594 Background: Dedifferentiated liposarcoma (DDLPS) is a rare adipocytic malignancy characterized by frequent amplification of MDM2 and CDK4 and associated with poor outcomes, with a median overall survival of approximately 15 months. A phase II trial at Memorial Sloan Kettering Cancer Center demonstrated activity of the CDK4/6 inhibitor palbociclib in advanced WD/DDLPS, with a 12-week progression-free survival (PFS) rate of 57%, exceeding historical chemotherapy benchmarks (<35%) and leading to its inclusion in the NCCN Guidelines. Despite this, durability of benefit remains limited, and outcomes following progression are poor. Preclinical work from our group demonstrated that CDK4/6 inhibition in DDLPS induces cellular geroconversion from quiescence to irreversible senescence, accompanied by MDM2 downregulation. This process requires suppression of HRAS signaling and inhibition of the MAPK pathway and is associated with activation of the senescence-associated secretory phenotype (SASP). Preclinical models further suggest that dual CDK4/6 and MEK inhibition enhances SASP activation, providing a mechanistic rationale for combination therapy. Based on these findings, we initiated a phase Ib/II clinical trial of mirdametinib, a MEK1/2 inhibitor, in combination with palbociclib in patients with DDLPS (NCT06843967). Methods: This is an ongoing phase Ib/II, single-arm, open-label, single-center study evaluating the safety, tolerability, and efficacy of mirdametinib plus palbociclib in patients with unresectable, recurrent, or metastatic DDLPS (NCT06843967). Patients may enroll at any line of therapy, including first line, consistent with contemporary palbociclib use; patients with prior CDK4/6 inhibitor exposure are eligible for the phase Ib portion. Key eligibility criteria include ECOG performance status 0–2 and RECIST v1.1–measurable disease. Phase Ib uses a Bayesian optimal interval (BOIN) design with three dose levels to determine dose-limiting toxicities, maximum tolerated dose, and recommended phase II dose (RP2D), enrolling up to 24 patients. Phase II will assess efficacy at the RP2D, with the primary endpoint of PFS at 18 weeks by RECIST v1.1 in 30 patients. Enrollment began February 19, 2025. As of January 2026, accrual is ongoing at dose level 2. Enrollment to dose level 3 is anticipated in February 2026. Clinical trial information: NCT06843967 .

Histologic distribution by immunotype and response

T cell subpopulations associated with shorter survival after accounting for histologic subtype and treatment

Gnathic osteosarcoma may vary from appendicular osteosarcoma in terms of the general systemic therapy approach. Consensus guidelines highlighting literature‐based recommendations allow for care recommendations and set a research direction as the field starts to approach methods to reveal osteosarcoma subtypes and improve outcomes.

Detailed histologic diagnoses according to TME subtype

Supplementary Figure S3. T cell state contour plots.

PURPOSE Irinotecan and temozolomide (iT) have antitumor activity in relapsed Ewing sarcoma (ES). The purpose of this phase II trial was to evaluate whether iT added to an alkylator-intensified five-drug regimen (Ewing Family of Tumors [EFT]) improved the 4-year event free survival (EFS) for patients with newly diagnosed localized ES. METHODS Patients with localized ES received six cycles of iT consolidation following completion of 21 weeks of EFT chemotherapy; patients with metastatic disease received 10 intercalated cycles of iT during EFT. The primary end point was 4-year EFS for patients with localized disease, with 85% and 70% of patients without events deemed promising and not promising, respectively. Secondary end points included EFS for metastatic patients, overall survival (OS), toxicity, and exploratory circulating tumor DNA analyses. RESULTS Eighty-three patients were enrolled (46 localized, 37 metastatic; median age 16 years). The 4-year EFS for patients with localized disease was 76% (95% CI, 64% to 90%), which did not exceed the predefined threshold for unpromising efficacy. Among patients with metastatic disease, 4-year EFS was 56% (95% CI, 37% to 84%) for those with lung-only metastases and 43% (95% CI, 25% to 74%) for those with extrapulmonary metastases. Febrile neutropenia occurred in 96% of patients, most frequently following alkylator-containing cycles. Secondary malignancies developed in four patients. No unexpected toxicities were observed. CONCLUSION The addition of iT to high-dose, alkylator-based up-front chemotherapy did not exceed the historical EFS benchmark in patients with radiographically localized ES.

Supplementary Table S3. Representativeness of Study Participants

PURPOSE:Mouse models demonstrate a role for RANK and its ligand, RANKL, in osteosarcoma. The primary objective of this single-arm, open-label phase 2 trial was to determine whether denosumab, a RANKL mAb, improved disease control in recurrent osteosarcoma relative to benchmarks derived from historic Children's Oncology Group clinical trial data. PATIENTS AND METHODS:Skeletally mature patients ages 11 to 49 years old with measurable disease were eligible for cohort 1, and those with complete surgical resection of all sites of disease were eligible for cohort 2. Patients received denosumab 120 mg subcutaneously every 4 weeks with calcium and vitamin D supplementation. The primary endpoints were RECIST response and remaining event-free for 4 months for cohort 1 and remaining event-free for 12 months for cohort 2. Toxicity, pharmacokinetics, and pharmacodynamic effects were additional endpoints. RESULTS:Fifteen patients in cohort 1 and 38 in cohort 2 were eligible and evaluable for the primary endpoint. One of 15 cohort 1 patients remained event-free at 4 months. There were no objective responses. Ten of 38 cohort 2 patients were event-free at 12 months. The predefined efficacy criteria were not met in either cohort. The most common ≥ grade 3 adverse events were hypocalcemia and hypophosphatemia (8% and 11%, respectively). At steady state, mean serum denosumab trough concentrations were 23.7 to 31 μg/mL in cycles 2 to 7. Serum c-telopeptide and urine n-telopeptide decreased on treatment. CONCLUSIONS:Denosumab was well-tolerated with anticipated side effects and pharmacokinetic and pharmacodynamic parameters but had insufficient activity for further development in osteosarcoma.