FOXM1 inhibitor, RCM‑1, enhances venetoclax mediated apoptosis through downregulation of ATP2B4 in rhabdomyosarcoma.
Provisional — an automated or curator check raised a specific concern and points reviewers here. This is NOT a final assessment and not a determination about the authors.
The main result did not reproduce in this reproduction attempt. Where our recomputation produced values that differ from the published ones, those discrepancies are listed below. This is a single automated attempt — not peer review and not a finding of error or misconduct — and differences can also arise from data access, undocumented parameters or the computing environment. The verdict can be contested via “report an error”.
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Described well enough to attempt a faithful 1:1 reproduction, and the pipeline reproduced cleanly END-TO-END on «our HPC» -- but the endpoint gene claim is a MISMATCH. Brief correction: code=EnhancedVolcano pairs with the authors' OWN mouse RNA-seq SRA PRJNA1204196 (12 samples, 4 groups x 3 reps), NOT the brief's GSE143704 (external DeMicheli human-muscle scRNA atlas used only as a Seurat reference). Pipeline: ENA fastq -> Kallisto 0.52.0 (Ensembl GRCm38 r102 cDNA) -> tximport gene-level -> DESeq2 (Wald/BH/alpha=0.1, design ~condition, ref=Venetoclax) -> EnhancedVolcano. Fig 3C is explicitly the Combo-vs-Venetoclax volcano (confirmed from PMC full text + legend 'unique genetic profile'). In that exact contrast Atp2b4 is log2FC -0.213, padj 0.735 -- non-significant, rank 2439 of all genes, NOT among the 2078 sig-down genes -- so the paper's 'Atp2b4 was one of the most downregulated genes in the combination treatment' does NOT reproduce for the figure's stated comparison. Atp2b4 IS significantly down vs Control/RCM1 (and venetoclax alone also lowers it), i.e. not unique to the combination and not driven by adding RCM-1 to venetoclax. Flagged as POSSIBLE-OVERCLAIM (not fabrication) for human audit. NOTE: this is the re-queued run; requeue was triggered by a tooling artifact (stale bogus-folder ROOM_RESULT), not by any problem with the science. Re-confirmation «job» in progress; numbers will be refreshed on completion.
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
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v1 current initial assessment Score 10assessed: 2026-06-19 ⛓ 052932feb377
✎ I am an author of this paper
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-22
- Rubric version
- not recorded
- Assessed by
- —
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetThe study tested whether combining the FOXM1 inhibitor RCM-1 with the Bcl2 inhibitor venetoclax exerts synergistic anti-tumor, pro-apoptotic effects in rhabdomyosarcoma (RMS), potentially offering a more effective and less toxic therapy than either agent alone.
- ★ Combination therapy of RCM-1 and venetoclax inhibits RMS tumor growth more efficiently than venetoclax alone in an animal model by decreasing proliferation and inducing apoptosis finding
- ★ RNA-sequencing showed the combination therapy uniquely decreased expression of ATP2B4, a plasma membrane calcium channel highly expressed in RMS compared with normal muscle cells finding
- ★ RCM-1, but not venetoclax, inhibits ATP2B4 and enhances RMS cell sensitivity to apoptosis mechanism
- ★ Knockdown of ATP2B4 decreases RMS tumor cell proliferation, migration and colony formation in vitro finding
- ★ Knockdown of ATP2B4 increases tumor cell apoptosis, while overexpression of ATP2B4 decreases apoptosis in vitro finding
- ★ Depletion of ATP2B4 decreases tumor growth in the animal model of RMS finding
- ★ ATP2B4 is a promising therapeutic target for RMS, providing rationale for early-stage clinical trials of RCM-1/venetoclax combination finding
- Venetoclax, though FDA-approved and effective in hematologic malignancies, is not effective as a monotherapy in solid tumors finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| CCK-8 dose-response / IC50 determination | 76-9 (mouse) and RD (human) RMS cell lines | RCM-1 and venetoclax, single agent | cell viability, IC50 | CCK8 kit, microplate reader (450 nm) |
| Caspase 3/7 activation assay | RMS cell lines (76-9, RD) | RCM-1 and venetoclax, sequential combination | apoptosis via caspase 3/7 luminescence | Caspase-Glo 3/7 Assay (Promega) |
| Subcutaneous syngeneic mouse tumor model | 76-9 cells injected into C57BL/6 mice | RCM-1 nanoparticle (tail vein) and/or venetoclax (oral gavage) | tumor volume, proliferation (Ki-67), apoptosis (Cleaved-Caspase3, BAX) | caliper measurement, immunofluorescence |
| RNA-sequencing | 76-9 RMS cells, control vs. treated | RCM-1/venetoclax treatment | differential gene expression | Illumina NovaSeq6000, Kallisto, DESeq2 |
| siRNA/shRNA knockdown and overexpression assays | RMS cell lines (76-9, RD, RH30) | ATP2B4 knockdown (siRNA/shRNA) or overexpression (ATP2B4a/b), FOXM1 knockdown (shRNA) | gene/protein expression, downstream phenotypes | Dharmafect, lentiviral transduction, TransIT-X2 |
| Clonogenic growth assay | 76-9 cells (control vs. siATP2B4) | ATP2B4 knockdown | colony formation (crystal violet staining) | ImageJ quantification |
| Wound healing (migration) assay | 76-9 cells (control vs. siATP2B4) | ATP2B4 knockdown | cell migration/wound closure | Ibidi inserts, EVOS FL Auto 2, TScratch software |
| Single-cell RNA-seq analysis of public datasets | Human muscle tissue (GSE143704) and RMS patient samples (GSE195709) | none | ATP2B4 expression across cell clusters (violin plot) | Seurat (v4.3.1), NextSeq 500 |
- – IC50 of RCM-1 was lower than venetoclax in both cell lines (RCM-1: 2.5 μM RD, 1.37 μM 76-9; venetoclax: 9.4 μM RD, 6.3 μM 76-9)
- ▼ Combination therapy inhibited RMS tumor growth more effectively than venetoclax alone in vivo
- – Combination therapy decreased tumor cell proliferation and increased apoptosis in vivo
- ▼ RNA-seq identified ATP2B4 as uniquely downregulated by combination therapy
- ▼ RCM-1 (not venetoclax) inhibited ATP2B4 expression and enhanced apoptosis sensitivity in RMS cells
- ▼ ATP2B4 knockdown reduced RMS proliferation, migration and colony formation in vitro
- – ATP2B4 knockdown increased apoptosis; ATP2B4 overexpression decreased apoptosis in vitro
- ▼ ATP2B4 depletion decreased tumor growth in the RMS animal model
- other IC50 RCM-1 = 2.5 μM (RD cells dose-response)
- other IC50 RCM-1 = 1.37 μM (76-9 cells dose-response)
- other IC50 venetoclax = 9.4 μM (RD cells dose-response)
- other IC50 venetoclax = 6.3 μM (76-9 cells dose-response)
- count n=19 control, n=12 RCM-1, n=12 venetoclax, n=12 combination (mouse tumor study group sizes)
- mean RNA Quality Number = 9.87 (RNA-seq sample quality)
- count ~30 million reads per sample (RNA-sequencing depth)
- other DESeq2 FDR alpha = 0.1 (default); significance threshold P<0.05 (differential expression and general statistical analysis)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study used a combination of in vitro functional assays (cell viability by CCK-8, apoptosis by caspase 3/7, migration, colony formation) and a syngeneic murine subcutaneous model (n=55 mice, four groups) to evaluate RCM-1 plus venetoclax in rhabdomyosarcoma. Bulk RNA-seq in the 76-9 cell line was analyzed with DESeq2 (negative binomial model, Wald test) with Benjamini-Hochberg FDR correction; public scRNA-seq datasets were integrated and visualized via Seurat/UMAP. For all other comparisons, unpaired Student's t-tests or one-way/two-way ANOVA with Tukey's post hoc correction were applied as appropriate, using GraphPad Prism 9.5.1. All continuous data were reported as mean ± SD, with P<0.05 as the significance threshold.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Unpaired Student's t-test | Pairwise comparisons throughout in vitro and in vivo experiments, as appropriate per figure | In vitro: triplicate experiments; in vivo: n=12–19 per group | not stated |
| One-way ANOVA with Tukey's multiple comparisons post hoc | Single-factor multi-group in vitro or in vivo comparisons, as appropriate | In vitro: triplicate experiments; in vivo: n=12–19 per group | not stated |
| Two-way ANOVA with Tukey's multiple comparisons post hoc | Factorial comparisons (e.g., dose × treatment interactions), as appropriate | In vitro: triplicate experiments; in vivo: n=12–19 per group | not stated |
| DESeq2 Wald test (negative binomial model) | Bulk RNA-seq differential gene expression between treatment conditions in 76-9 cells | Number of biological replicates per RNA-seq condition not stated | not stated |
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In vitro experiments were performed in triplicate; the text does not specify whether these are biological replicates (independent cell passages) or technical replicates (repeated measurements of the same passage)↳ Could also: Explicitly distinguish biological from technical replicates and base inferential statistics on biological replicates — Biological replicates support generalizability beyond a single passage; their distinction from technical replicates is standard practice recommended by ARRIVE and Nature guidelines for in vitro studies
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Continuous data throughout are reported as mean ± SD↳ Could also: Report 95% confidence intervals alongside or instead of SD, particularly for small n — Confidence intervals convey precision of the group mean estimate; for triplicate experiments (n=3), they communicate uncertainty in a way that SD, which reflects spread of individual observations, does not
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Both unpaired t-tests and ANOVA with Tukey's correction were used across different comparisons; no correction across the full study-wide family of t-tests is described↳ Could also: Apply a Benjamini-Hochberg or Bonferroni correction across the complete set of t-tests, or frame all multi-group questions within a single ANOVA — A unified multiplicity framework explicitly controls the false-discovery or family-wise error rate across all comparisons simultaneously, which is particularly relevant when many contrasts are conducted across multiple assays
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The in vivo experiment was described as a pilot study with no formal power calculation↳ Could also: Include a prospective power calculation based on published effect sizes for similar RMS models, or provide a post hoc power estimate — Even in a pilot context, citing the effect size and variance assumptions used to arrive at n=12 per group helps readers interpret the study's ability to detect the observed differences and plan future confirmatory experiments
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The number of biological replicates submitted per condition for bulk RNA-seq was not stated↳ Could also: State the number of independent biological replicates per RNA-seq condition explicitly in the methods — DESeq2 requires at least n≥2 per group and variance estimation improves substantially with n≥3; reporting this is a MINSEQE/MIAME best practice and allows readers to assess statistical power of the differential expression analysis
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IC50 values were derived from dose-response curves, but the curve-fitting method was not described↳ Could also: Specify the model used for IC50 derivation (e.g., four-parameter logistic/Hill equation nonlinear regression) and report confidence intervals on the IC50 estimates — The four-parameter logistic is the standard for sigmoidal dose-response data; reporting the fit model and its uncertainty allows reproduction of the IC50 calculation and comparison across studies
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Reproduction scope — pmid-41789627
Paper: Merjaneh et al. (2026) FOXM1 inhibitor, RCM‑1, enhances venetoclax mediated apoptosis through downregulation of ATP2B4 in rhabdomyosarcoma. Int J Oncol. PMID 41789627 · PMCID PMC12987556 · DOI 10.3892/ijo.2026.5865
Key artifact discrepancy (documented up front)
The room brief lists code = EnhancedVolcano and data = GSE143704. Reading the paper shows these two do not pair with each other:
- EnhancedVolcano (Fig 3C volcano plot) is applied to the authors' own
bulk RNA-seq, deposited in SRA
PRJNA1204196(mouse, GRCm38, Kallisto → DESeq2). This is the genuine pipeline-derived result tied to EnhancedVolcano. - GSE143704 is an external dataset (DeMicheli "Single-cell transcriptomic atlas of human donor muscle tissue", 10 healthy donors). The paper re-uses it only as a normal-muscle reference for a separate Seurat scRNA integration with patient RMS samples (GSE195709). It is NOT the input to EnhancedVolcano.
We therefore reproduce the EnhancedVolcano / DESeq2 pipeline on the paper's own
data (PRJNA1204196) — the result the named tool actually produces — and note
the brief's data accession was the external reference dataset, not the DEG input.
Pipeline (from Methods + Data Availability)
- Reads aligned/quantified to GRCm38 with Kallisto, standard settings.
- Raw counts normalized with DESeq2; DESeq2 for differential expression (negative binomial, Wald test, Benjamini–Hochberg, FDR α = 0.1).
- Volcano plot via EnhancedVolcano = Fig 3C, comparison combination therapy (RCM-1 + venetoclax) vs venetoclax monotherapy.
Data: PRJNA1204196 (mouse RNA-seq, 12 samples = 4 groups × 3 reps, paired-end)
- Control: SRR31850352 (D1), SRR31850351 (D2), SRR31850348 (D3)
- RCM1: SRR31850347 (R1), SRR31850346 (R2), SRR31850345 (R3)
- Venetoclax: SRR31850344 (V1), SRR31850343 (V2), SRR31850342 (V3)
- Combo: SRR31850341 (C1), SRR31850350 (C2), SRR31850349 (C3)
IN SCOPE (attempted) — pipeline-derived
RU-1 (Fig 3C): Kallisto(GRCm38, Ensembl r102 cDNA) → tximport(gene-level) → DESeq2 contrast Combo vs Venetoclax (Wald, BH) → EnhancedVolcano.
- Reproduced claim: "the combination therapy uniquely decreased expression of ATP2B4 … Atp2b4 was one of the most downregulated genes" (Results; Fig 3C).
- Verification = sign(log2FC) < 0, padj significant, and rank of Atp2b4 among significantly downregulated genes. (Paper gives no exact log2FC/padj for Atp2b4 in main text, so the comparison is the direction + significance + top-rank claim; reproduced numbers are reported for human audit.)
OUT OF SCOPE (the hard ~20%, not attempted — with reasons)
- Seurat scRNA integration of GSE143704 + GSE195709 (normal muscle vs RMS): integration parameters not specified in Methods; output is a UMAP/feature plot not reducible to a single checkable number → poor 1:1 target. Skipped per 80/20.
- Pathway/GSEA enrichment mentioned for Fig 3: enrichment tool/parameters not named → not cleanly reproducible. Skipped.
- All wet-lab quantities (qPCR knockdown %, Annexin V / DEVD apoptosis %, luciferase activity, in-vivo tumor knockdown, Figs 4–8): not pipeline-derived.
Compute plan
All on «our HPC» (SLURM, partition=std). Data + intermediates on «infra» under
«path».
Only small results (Atp2b4 stats, top-DEG table, volcano PNG, versions) come back
to the «host» dataset folder.
Assessments & scoring basis
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Reproduction footprint
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