BAG1 down‐regulation increases chemo‐sensitivity of acute lymphoblastic leukaemia cells

Abstract BCL2‐associated athanogene‐1 (BAG1) is a multi‐functional protein that is found deregulated in several solid cancers and in paediatric acute myeloid leukaemia. The investigation of BAG1 isoforms expression and intracellular localization in B‐cell acute lymphoblastic leukaemia (B‐ALL) patient‐derived specimens revealed that BAG1 levels decrease during disease remission, compared to diagnosis, but drastically increase at relapse. In particular, at diagnosis both BAG1‐L and BAG1‐M isoforms are mainly nuclear, while during remission the localization pattern changes, having BAG1‐M almost exclusively in the cytosol indicating its potential cytoprotective role in B‐ALL. In addition, knockdown of BAG1/BAG3 induces cell apoptosis and G1‐phase cell cycle arrest and, more intriguingly, shapes cell response to chemotherapy. BAG1‐depleted cells show an increased sensitivity to the common chemotherapeutic agents, dexamethasone or daunorubicin, and to the BCL2 inhibitor ABT‐737. Moreover, the BAG1 inhibitor Thio‐2 induces a cytotoxic effect on RS4;11 cells both in vitro and in a zebrafish xenograft model and strongly synergizes with pan‐BCL inhibitors. Collectively, these data sustain BAG1 deregulation as a critical event in assuring survival advantage to B‐ALL cells.

the role of BAG1 in protecting the cells from pro-apoptotic stimuli induced by drug or radiation therapies is emerging 8 . The same function was described for paediatric acute myeloid leukaemia (AML), where BAG1 over-expression sustained leukaemic phenotype 9 .
However, in these cells, a compensatory mechanism between BAG1 and BAG3 was reported 10 . Hence, a concomitant down-regulation of BAG1 and BAG3 was necessary for triggering AML cell death. The involvement of BAG1 in paediatric B-ALL is poorly defined, whereas other pro-apoptotic BCL2 family proteins have been examined extensively as molecular therapeutic targets. At the same time, BAG3 protein has been confirmed as highly expressed in primary ALL specimens and cell lines, regulating their survival 11 .

| RE SULTS AND D ISCUSS I ON
To better characterize the role of BAG1 in childhood ALL, we assessed BAG1's protein profiles in the bone marrow (BM) aspirates collected at different stages of the disease (Table S1). As shown in Figure 1A, BAG1-L and BAG1-M isoforms are predominantly concentrated in the nuclear protein fraction. We detected a substantial reduction in BAG1 protein levels within the total protein fraction, particularly of BAG1-L and BAG1-M isoforms, in the samples collected during disease remission ( Figure 1B), resembling previously reported expression profiles in healthy BM 9 . Since the three BAG1 isoforms can be found within diverse sub-cellular portions, the expression of BAG1 protein isoforms was investigated in cytosol and nucleus. At diagnosis, BAG1-L and BAG1-M were almost exclusively nuclear ( Figure 1A) while during remission BAG1-M was excluded from the nucleus ( Figure 1B). These findings sustain that even in B-ALL, like in other solid cancers, either over-expression or shuttling of BAG1-L and BAG1-M isoforms within the cell might sustain disease progression 12,13 . These data were further corroborated by examining BAG1-L and BAG1-M isoforms in matched B-ALL specimens collected at diagnosis, remission and relapse. A significant reduction in BAG1-L and BAG1-M isoforms at the remission stage was followed by a marked increase at relapse ( Figure 1C and Figure S1). Hence, it is plausible that either protein abundance or the localization of specific BAG1 isoforms may determine drug resistance in B-ALL cells sustaining tumour recurrence. This might justify why BAG1 has been neglected so far by comprehensive B-ALL transcriptomic analysis using probes mapping the BAG1 transcript, which is common to all BAG1 isoforms ( Figure 1D). Collectively, these data further highlight the importance of studying BAG1 protein localization.
To gain insight into the mechanism by which BAG1 fulfils its cytoprotective role in B-ALL, we adopted a small interfering RNA (siRNA) approach using the RS4;11 leukaemia cell line. Of note, following our previous experience, we used a combination of BAG1/BAG3specific siRNAs to prevent the functional rescue of BAG3 10  p < 0.001; Figure S2). In addition, BAG1/BAG3 silencing induced the G1-phase cell cycle blockade ( Figure 2B). Together, these data suggest that, as previously reported for AML, even ALL cells benefit from the BAG1 over-expression that assures their survival advantage over normal BM cell compartments.
BAG1 alone was able to define the susceptibility of B-ALL cells to common chemotherapeutic drugs used in leukaemia therapy protocols. The knockdown of BAG1 was sufficient to sensitize RS4;11 cells to dexamethasone (DEX) and daunorubicin (DAUNO) by increasing significantly the proportion of apoptotic cells ( Figure 2C and Figure S2). However, the strongest cytotoxic effect was observed upon treating BAG1-depleted cells with the pan-BCL inhibitor ABT-737, pointing out the pivotal role of BAG1 in protecting BCL-dependent regulation of the pro-survival pathway in B-ALL ( Figure 2C). These observations highlight that BAG1 knockdown sensitizes B-ALL cells to each of the drugs tested, strengthening the relevance of BAG1 protein in sustaining leukaemia cell survival.
Moreover, those results underlined BAG1 targeting as a promising approach for the improvement of current B-ALL treatment protocols. Nowadays, only a few compounds have been proposed as BAG1 inhibitors. Among them, the thioflavin S derivative Thio-2 disrupts the interaction between BAG1 and HSP70 14,15 , but also F I G U R E 1 BAG1 expression levels. Western blot analysis of BAG1 protein expression levels in paediatric B-ALL patients (pt) at first diagnosis (A) and remission stage (B) in the total protein lysate, cytoplasmic and nuclear fraction. While the total protein shows that the longest BAG1 isoforms (BAG1-M and BAG1-L) are highly expressed at diagnosis and predominantly localized in the nuclear fraction, their expression is almost undetectable at remission in the total protein fraction. The smaller BAG1-S isoform expression is consistent in both stages. (C) Representative Western blot analysis of BAG1-M and BAG1-L isoform in paired B-ALL specimens collected at disease onset, remission and relapse. Majority of analysed specimens show high BAG1-L level at diagnosis and during relapse. Expression of BAG1-S is included in these results in Figure S1 negatively modulates BAG1 and BCL2 protein levels in AML 9 and B-ALL ( Figure S3A). The treatment with Thio-2 alone was sufficient to cause a significant cytotoxic effect in NALM6 and RS4;11 ( Figure 2D), and it strongly enhanced the cytotoxic effect of Lasparaginase (L-ASP), vincristine (VCR), DEX and DAUNO in NALM6 ( Figure 2D). Instead, RS4;11 cells were generally less responsive to the same drug combinations ( Figure 2D). RS4;11 cells expressed higher levels of BAG1-L isoform, BCL2 and MCL1 proteins compared to NALM6 (Figure 2E), suggesting an addiction to anti-apoptotic mechanisms. Accordingly, RS4;11 cells were highly sensitive to pan-BCL inhibitors (ABT-737, navitoclax and venetoclax) and agents selective for BCL2 (S55746) and BCL XL (A-1155463), whereas NALM6 were resistant at the same dosages ( Figure S3B; Table S2). More importantly, the combination of Thio-2 with BCL inhibitors resulted in a synergistic effect, especially for S55746 and A-1155463 ( Figure 2F and Figure S3C). However, no synergistic effect has been observed for UMI-77, a selective MCL1 inhibitor, confirming a strong BCL dependence for this B-ALL cell line ( Figure 2F).
To sustain additionally the efficacy of Thio-2 against the growth of RS4;11 in vivo, we adopted a xenograft zebrafish model. Prelabelled RS4;11 cells were transplanted into the circulatory system of transgenic zebrafish embryos treated with 25µM Thio-2 or vehicle. Notably, Thio-2-treated embryos showed a significantly reduced number of circulating RS4;11 cells when compared to controls ( Figure 2G), highlighting the cytotoxic effect of Thio-2 versus leukaemia cells in vivo.

| CON CLUS IONS
In the present study, we reported the correlation between the BAG1 protein levels and B-ALL disease stage. We confirmed an over-expression of BAG1-L and BAG1-M isoforms at ALL diagnosis and relapse with their concomitant decrease during the remission stage, where only the physiological BAG1-S isoform remained detectable. Moreover, biological or chemical inhibition of endogenous BAG1 was sufficient to impair the survival of leukaemic cells while sensitizing them to chemotherapy. Yet, a large-scale prospective cohort study will be mandatory to address whether BAG1-L could be considered as a potential prognostic biomarker in B-ALL or proposed for targeting in advanced leukaemia therapy protocols. Lastly, we place out a critical remark on the unmet needs for more effective BAG1 inhibitors that could implement therapeutic options for other malignancies with confirmed BAG1 deregulation.

ACK N OWLED G EM ENTS
This study was supported by the following grants:

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.