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Deregulated expression of the HSP40 family members Auxilin-1 and -2 is indicative of proteostasis imbalance and predicts patient outcome in Ph+ leukemia
© Vieri et al. 2016
- Received: 10 November 2015
- Accepted: 2 February 2016
- Published: 9 February 2016
Proteostasis is defined by the orchestrated control of anabolic and catabolic protein pathways. Disruption of proteostasis results in cell stress and adaptation to proteostasis imbalance is mediated by adaptive pathways such as the Heat Shock Response (including heat-shock proteins) or the unfolded protein response (UPR). The BCR-ABL1 kinase (Philadelphia chromosome) is the hallmark of chronic myeloid leukemia (CML) and defines a historically poor subset in acute lymphoblastic leukemia (Ph+ ALL). We previously demonstrated the importance of the UPR and particularly of the IRE1/XBP1 signaling axis in Ph+ ALL, while others demonstrated the therapeutic relevance of HSP70 in ALL. In this regard, HSP70 is regulated by smaller HSP40 s, whose function is so far poorly characterized.
Herein, we characterize the expression of HSP40 s in Ph+ ALL and CML. We show that these genes are not regulated in a pan-class manner and identify a homologous gene pair, namely Auxilin-1 (DNAJC6) and Auxilin-2 (GAK) with a unique expression profile. Overexpression of Auxilin-2, the ubiquitously expressed homologue of Auxilin-1 correlated with superior clinical outcome in ALL and was tightly linked to both IRE1 RNase and BCR-ABL1 kinase activities.
Our findings suggest that HSP40 gens are uniquely regulated and provide a rationale for further studies between BCR-ABL1/IRE1-based therapies in combination with HSP40 inhibitors, thus opening potentially novel therapeutic avenues.
- Unfolded protein response
- Acute lymphoblastic leukemia
- Chronic myeloid leukemia
Control of protein homeostasis (proteostasis) is an essential driver of cell survival. In certain hematological malignancies such as multiple myeloma, the concept of disrupting proteostasis by weakening cancer cells’ adaptation to oncogene-induced increased metabolic demand and/or to challenging microenvironment has shown clinical success . Under challenging conditions, proteostasis maintenance is achieved through the activation of selective adaptive machineries such as the heat shock response (HSR) or the unfolded protein response (UPR) [2, 3]. The HSR is mainly mediated by Heat Shock Factor-1 (HSF1) which is responsible for the induction of the heat shock proteins (HSPs), that were initially classified based on their molecular weight [4, 5] and are generally regulated by proteotoxic stress [6, 7]. For instance, the HSP40 (DNAJ) family of proteins is important for protein translation, folding, translocation and degradation, mainly by modulating HSP70 ATPase activity. While the role of HSP70 is well characterized, that of HSP40 proteins, specifically in hematological malignancies remains unclear . Moreover, we have recently identified the IRE1/XBP1 axis of the UPR as an important survival cue in B-ALL [2, 9]. The IRE1/XBP1 axis is known to control the expression of several HSP40 family members , which suggests potential functional links between HSR and UPR.
Auxilin-1 is considered as the neuron-specific homologue of Auxilin-2  with many overlapping functions, and genetic loss of Auxilin-1 causes upregulation of Auxilin-2 . Since one caveat in gene-expression approaches is that a probe-set only detects changes between two conditions, even if the overall expression is marginal, we hypothesized that (a) while Auxilin-1 expression might be increased in comparison to pre-B cells, the overall expression might be negligible in baseline due to its neuron-specificity and (b) Auxilin-1 upregulation could cause a compensatory downregulation of Auxilin-2, thus biasing our studies. Our hypothesis was supported by the fact that mRNA levels of Auxilin-1 were low, while that of Auxilin-2 were detectable in primary cases of myeloproliferative neoplasms (MPNs), B-ALL and B cell Non-Hodgkin Lymphoma (B-NHL) and also established leukemia cell lines (Fig. 1d, e, Additional file 3: Table S1). These primary cases (Additional file 3: Table S1) were obtained in compliance with the institutional review board of the University of Aachen, ethical vote number EK 206/09. The potential upregulation of Auxilin-1 outside the nervous system is supported by its upregulation in hepatocellular carcinoma patients  leading to enhanced HCC proliferation and invasion, and suggesting neuron-independent and oncogenic functions. Following this notion, while Auxilin-1 expression was very low in baseline, treatment of Ph+ leukemia cell lines with the ER stresser Thapsigargin caused significant upregulation of both Auxilin-1 and -2 (Additional file 2: Figure S2c, d) suggesting that Auxilin-1 can be upregulated outside the nervous system. A similar pattern of regulation was previously observed for instance for the oncogene BCL6 which is non-detectable at baseline but can strikingly be upregulated upon Imatinib (IM) treatment emitting its oncogenic function [13, 14] by allowing leukemia cells to escape TKI-mediated cell death.
The recent finding that Auxilin-2 knockout in MEFs does not cause the upregulation of Auxilin-1 suggests that the genes might act independently under certain conditions , even though Auxilin-1 knockout mice showed compensatory upregulation Auxilin-2 . Taken together, the comparison of the studied HSP40 family members in leukemia suggested quite unique regulation (Additional file 1: Figure S1b). Since several HSP40 family members were upregulated in CML and Ph+ ALL, both driven by the BCR-ABL1 oncogene, we wanted to further understand if the expression of those genes might be linked to BCR-ABL1 kinase activity. We first studied their expression in a microarray of BCR-ABL1 transformed ALL cells treated with the tyrosine kinase inhibitor (TKI) Imatinib (Additional file 2 : Figure S2a). Several members which were previously downregulated in Ph+ ALL cases (Additional file 1: Figure S1a) were now upregulated upon TKI treatment including Jdd1 (Dnajc9), Mdg1 (Dnajb9), Tpr2 (Dnajc7), Erdj1 (Dnajc1), Mcj (Dnajc15) and Auxilin-2 (Gak), while others, previously upregulated in Ph+ ALL cases such as Auxilin-1 (Dnajc6), Nedd7 (Dnaja1), Rdj2 (Dnaja2), Tid1 (Dnaja3) and Dnajc16 were downregulated upon TKI treatment (Additional file 1: Figure S2a). Due to the very low baseline expression of Auxilin-1 mRNA levels, we focused our studies on Auxilin-2 and verified the upregulation of Auxilin-2 mRNA levels in Ph+ ALL and CML cell lines treated with either Imatinib, or the 2nd generation TKI, Nilotinib (Fig. 2f, Additional file 2: Figure 2b). The differential regulation of individual HSP40 s upon TKI treatment suggest rather specific than a pan-class function. For instance NEDD7 (DNAJA1) which was downregulated by TKI-treatment has been described to be important for activation-induced cytidine deaminase (AID) function, a known oncogene in ALL [18, 19], whereas TKI treatment causes downregulation of AID . Our finding might be the missing link between TKI treatment and AID downregulation through potential downregulation of NEDD7 (DNAJA1) and thereby negatively affecting AID function. Other important HSP40 members included MDG1 (DNAJB9) and TPR2 (DNAJC7), both upregulated by TKI-treatment and known to positively regulate the tumor suppressor p53 [21, 22]. We had previously shown that TKI treatment leads to downregulation of XBP1 s, while inhibition of XBP1 s activation by pharmacological inhibitors of IRE1 RNase activity caused apoptosis in Ph+ ALL cells . As such the IRE1 RNase inhibitor MKC-8866 caused upregulation of Auxilin-2 in Ph+ ALL and CML cells (Fig. 2g). This suggests that BCR-ABL1 kinase activity might cause potentially proteostasis imbalance leading to activation of the UPR, as previously identified by us and downregulation of individual HSP40 family members, which might hold the key to novel therapeutic approaches targeting the proteostatic network.
Taken together, our study provides for the first time, a more comprehensive overview of the expression and regulation of this understudied gene family in leukemias and more importantly shows that HSP40 family members can act as predictors of clinical outcome and are rather regulated by the BCR-ABL1 kinase activity specifically than in a pan-class manner.
BKM conceptually designed the study, performed experiments, analyzed the results and wrote the manuscript. MV designed and performed experiments and analyzed the results. HG performed experiments and analyzed results. AS, EC and JBP supported analysis. JP and SW treated patients and collected samples. All authors discussed the results and commented on the manuscript. All authors read and approved the final manuscript.
This work is supported by grants from the Ernst Jung Foundation, German Cancer Aid, RWTH START and RWTH START UP to BKM; by Belgian grants (Interuniversity Attraction Poles, IAP 7/32) and Health Research Board (Grant no. HRA_HSR/2010/24) to AS; Institut National du Cancer (INCa) to EC.
The authors declare that they have no competing interests.
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