Peer review MOLONC-22-0375:
In this interesting manuscript, Schelch et al. describe how exploiting YB-1 affects in vitro and in vivo behavior of pleural mesothelioma cells, as well as its consequences on regulation of selected target genes. The authors build this article upon their previous work, which had already demonstrated reduced tumor growth, migration and invasion in four pleural mesothelioma cell lines (PMID: 29113949) as well as induction of apoptosis and cell cycle alterations in three pleural mesothelioma cell lines (PMID: 32823952) upon YBX1 knockdown. Despite certain similarities to their previous work (as per below), overall, this current manuscript provides novelty on several aspects and clearly furthers our knowledge about a mechanistical role of YB-1 in pleural mesothelioma. Given that pleural mesothelioma associates with poor survival and unfortunately still limited systemic treatment options, identifying and characterizing new targets, i.e. YB-1, is of clinical relevance.

This article is well written and conclusive, and its methods are appropriately used to proof and investigate the authors hypotheses. However, there are certain aspects that should be addressed prior to consideration for publication to further both novelty and potential clinical implications:

Major comments:
YB-1 is (over)expressed in pleural mesothelioma tumor cells and associates with migration and invasion, as previously demonstrated by the authors (PMID: 29113949). Some evidence suggests a tendency for particularly higher expression in sarcomatoid mesotheliomas (PMID: 24457449). In this manuscript, the authors primarily focused on mesothelioma cell line SPC212, which is of biphasic histology, and investigated the in vitro effect of YB-1 expression on migration, morphology and intravasation. They proof the effect on migration in five additional cell lines, which were either biphasic (MST-211H, VMC40, MM05) or epithelioid (Ren, VMC23). Interestingly, Ren and VMC23 seem to be among the cell lines with the lowest baseline migration potential (per Fig. 4C). Accordingly, first, it would be helpful if the authors mention in the methods the histological subtypes of the cell lines. Second, it would be of high interest and potential clinical relevance to evaluate in vitro tumor cell behavior with respect to YB-1 (over)expression in a sarcomatoid mesothelioma cell line as comparison. I assume there could be a histology-dependent effect associated with exploiting YB-1. Of note, in (PMID: 29113949), the authors previously demonstrated that YB-1 is overexpressed in pleural mesothelioma cell lines compared to the immortalized mesothelial cell line Met5a. Accordingly, it would also be interesting, as control, to consider investigating some effects of YB-1 exploitation in Met5a in a limited in vitro approach, given that mesothelioma cell lines already at baseline express high levels of YB-1, as stated by the authors in the discussion.

The authors primarily investigate the effects of YB-1 exploitation on migration, morphology, intravasation and selected target gene expressions, with overall very robust and convincing data. However, YB-1 overexpression has been showed to confer resistance to chemotherapy, particularly towards platinum agents, as recently reviewed by the authors (PMID: 31632972). In line with this, as stated in the discussion, Snail has been linked to cisplatin resistance (PMID: 34593606). Thus, to significantly further the importance of YB-1 as potential target in pleural mesothelioma and to strengthen its association with tumor cell aggressiveness, it would be interesting to consider investigating whether there is an effect of exploiting YB-1 on platinum-sensitivity in vitro.

In lung cancer, YB-1 has been showed to associate with EMT (PMID: 25645014), as demonstrated by downregulation of E-cadherin, upregulation of N-cadherin, and increased EMT response to TGF-β. In this current manuscript, Schelch et al. demonstrate and conclude that SNAI1 is a critical regulator of YB-1 mediated cell motility, in contrary to TWIST, EGFR, MMP1 or VIM. Importantly, SNAI1 and TWIST are major genes involved in EMT, and I am wondering whether other major EMT markers including ZEB1, SNAI2 and MMP2 may follow a similar pattern with regards to regulating YB-1 mediated cell motility. Furthermore, the authors selected two biphasic cell lines (SPC212 and MSTO-211H) to investigate the link between YB-1, migration and SNAI1. Biphasic tumor cell lines might be more sensitive to EMT-related changes including changes in migration. Therefore, investigating whether SNAI1 also critically regulates YB-1 mediated cell motility in an epithelioid and/or sarcomatoid cell line may provide crucial information to further our knowledge on the role of YB-1 and possibly EMT in pleural mesothelioma, and therefore strengthen the overall conclusion of this article.

Minor comments:
The authors should discuss the limited (but existing) evidence of YB-1 and its prognostic implication in MPM, as they had recently discussed it in (PMID: 31632972).

The authors should discuss the link between Snail/YB-1, and EMT in pleural mesothelioma in more detail.

The authors should consider omitting the term “malignant” as prefix for pleural mesothelioma, as recently recommended by the 2021 WHO classification update of pleural tumors (PMID: 35026477).

In the introduction, regarding nivo/ipi versus SoC chemotherapy, the authors should list “18.1 months vs 14.1 months” instead of “14.1 months vs 18.1 months”. The authors should also consider damping down the statement “… not all patients respond…”, given that the objective response rate to ipi/nivo was noted as only 40% per Baas et al. Lancet 2021.

In the last paragraph of the discussion, the reference 38 is not properly formatted.

All my comments addressed by the authors.