Abstract A014 INO80-complex N-terminal module resolves replication stress in PAX3-FOXO1 positive rhabdomyosarcoma and exposes therapeutic vulnerability
Bibliographic record
Abstract
Abstract PAX3-FOXO1 fusion positive rhabdomyosarcoma (FP-RMS) is an aggressive form of pediatric cancer with limited therapeutic options. The fusion oncoprotein PAX3-FOXO1 binds to chromatin and rewires gene expression to stop terminal differentiation and promote cancer cell proliferation. In this study, we aimed to find genetic dependencies downstream of PAX3-FOXO1 to discover the new therapeutic targets and identified selective dependency for the INO80 complex (INO80c) N-terminal subunits UCHL5 and NFRKB. We conducted a CRISPRi screen in the FP-RMS Rh30 cell line, transduced with either a control shRNA or one targeting the PAX3-FOXO1 fusion boundary which suppressed fusion expression. Loss of UCHL5 in this screen led to selective depletion of PAX3-FOXO1+ cells compared to PAX3-FOXO1KD cells. We validated the selective effects of UCHL5 loss in a set of two FP-RMS and two fusion negative (FN)-RMS cell lines. Data from the Dependancy Map project supported our finding of UCHL5 depednence in FP-RMS, not FN-RMS, and revealed that the top UCHL5 co-dependents genes are components of the INO80c. UCHL5 is a part of the N-terminal module of INO80c that stabilizes another subunit, NFRKB, by deubiquitination. We observed that NFRKB knockdown phenocopies the growth defects seen in UCHL5 loss in FP-RMS. The data suggests that PAX3-FOXO1 selectivity resides in the INO80c N-terminal subunits. INO80c functions as a chromatin remodeler with roles in in DNA replication and repair. We found that γH2AX, signaling DNA damage, is increased upon UCHL5 loss in FP-RMS compared to FN-RMS. Further, pulse labeling of replicating DNA fibers confirmed that loss of UCHL5 slowed replication fork speed in FP-RMS. Loss of NFRKB again phenocopied this induction of replication stress in FP-RMS cells, whereas FN-RMS cells didn’t demonstrate slowed replication forks upon UCHL5 or NFRKB loss. We tested the impact of PAX3-FOXO1 loss in replication; unexpectedly, we found that PAX3-FOXO1 loss reduces the replication fork speed. Our biochemical assays showed lack of physical interaction between PAX3-FOXO1 and UCHL5. But we found PAX3-FOXO1 interaction with TOP2A that increases upon UCHL5 loss. Combination of UCHL5 loss with chemotherapeutics that induce replication stress by TOP2A inhibition (doxorubicin) highlighted the therapeutic impact of this finding as the FP-RMS cells showed hypersensitivity to doxorubicin compared to FN-RMS cells. Finally, we performed in vivo experiments using cell line xenografts to confirm that loss of UCHL5 slows the growth of FP-RMS (Rh30), but not FN-RMS (RD) in vivo. This study identifies UCHL5 and NFRKB as a selective dependency in PAX3-FOXO1 positive RMS. Loss of UCHL5 increases replication stress in FP-RMS and may also increase the efficacy of replication stress inducing drugs, suggesting a combination therapeutic approach to improve in vivo selectivity of these toxic therapies. Together, our study suggests that UCHL5-NFRKB can serve as potential precision medicine target for the treatment of PAX3-FOXO1 positive RMS. Citation Format: Pushpendra Kumar Sahu, Yue Pan, Kristen Kwong, Amit J. Sabnis. INO80-complex N-terminal module resolves replication stress in PAX3-FOXO1 positive rhabdomyosarcoma and exposes therapeutic vulnerability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A014.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".