Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is an aggressive non-Hodgkin lymphoma of T/null cell immunophenotype [1–3]. This lymphoma primarily presents in children, adolescents, and young adults where it accounts for 10–20% of childhood non-Hodgkin lymphomas . ALK+ ALCL is characterized by the presence of chromosomal translocations involving the ALK gene, which encodes for a receptor tyrosine kinase belonging to the insulin receptor super-family. These translocations result in the expression of ALK fusion proteins that are critical for the pathogenesis of ALK+ ALCL [2, 3]. Moreover, ALK fusion proteins have been implicated in the pathogenesis of a subset of non-small cell lung carcinomas (ALK+ NSCLC) [4–7] and inflammatory myofibroblastic tumours (ALK+ IMT) [8–10]. In ALK+ ALCL several different ALK translocations have been described [2, 3]; however, the most common (~80%) is the t(2;5)(p23;q35) translocation involving the nucleophosmin (NPM) gene which generates the NPM-ALK oncogene [1–3].
NPM-ALK consists of the N-terminal region of NPM and the C-terminal kinase and intracellular domains of ALK [11, 12]. The NPM portion of this fusion protein possesses a dimerization domain required for the tyrosine kinase activity and transforming ability of NPM-ALK [13, 14]. The activity of the NPM-ALK oncoprotein is also critically dependent on the molecular chaperone, heat shock protein-90 (Hsp90) [15–18]. Hsp90 is a ubiquitously expressed protein that assists in the proper folding and activity of numerous cellular proteins [19, 20]. Hsp90 promotes the stability of NPM-ALK [15–18], as treatment of cell lines with the Hsp90 inhibitor, 17-Allylamino-Demethoxygeldanamycin (17-AAG), resulted in the proteasomal degradation of NPM-ALK . The treatment of ALK+ ALCL cell lines with 17-AAG resulted in cell cycle arrest and the induction of apoptosis [15, 18]; however, these effects are likely due to more than just decreased NPM-ALK levels. Hsp90 inhibition also decreased levels of the pro-survival serine/threonine kinase Akt, the cell cycle-associated proteins cyclin D1, cyclin-dependent kinase 4 (cdk4), and cdk6, as well as several other proteins in ALK+ ALCL [15, 18, 21]. The treatment of ALK+ ALCL cell lines with 17-AAG resulted in decreased phosphorylation of the serine/threonine kinase Erk without affecting Erk levels . Moreover, the treatment of ALK+ NSCLC with Hsp90 inhibitors resulted in Erk dephosphorylation as well as the degradation of Akt and the EML4-ALK oncoprotein in these tumours [22–24].
Hsp90 inhibitors are also effective at inhibiting EML4-ALK-driven tumourigenesis in vivo in the mouse [22, 23], and the treatment of three ALK+ NSCLC patients with the Hsp90 inhibitor, IPI-504, resulted in a partial response in two of the patients and stable disease in the other . Importantly, Hsp90 inhibitors are effective against tumour cells expressing ALK fusion proteins that possess mutations that render them resistant to the ALK inhibitor, Crizotinib [24, 26]. Thus, Hsp90 inhibitors may be useful in treating patients that develop resistance to ALK inhibitors.
One aspect of Hsp90 biology that is largely unstudied in ALK-expressing tumours is the role of Hsp90 co-chaperones. Many functions of Hsp90 are dependent on its association with co-chaperone proteins [19, 20]. Co-chaperones mediate various aspects of Hsp90 function, including the association of Hsp90 with client proteins and the regulation of Hsp90 ATPase activity [19, 20]. Cyclophilin 40 (Cyp40), FK506-binding protein (FKBP) 51, and FKBP52 are members of the immunophilin family of Hsp90 co-chaperones. This family is best characterized for its association with Hsp90-steroid hormone receptor complexes containing client proteins such as the glucocorticoid, estrogen, progesterone, and androgen receptors [27–30]. The individual immunophilin family members show some preference for specific hormone receptors, and they can both antagonize and promote the transcription mediated by these receptors. For example, FKBP51 inhibits the transcriptional activity of the glucocorticoid receptor [31–33], while FKBP52 is important for promoting the transcriptional activity of this receptor [32–35]. In addition to steroid hormone receptors, immunophilin co-chaperones have been found to complex with the Lck  and Fes  tyrosine kinases. As well, the expression and activity of ectopically expressed v-Src oncoprotein in Saccharomyces cerevisiae is dependent on the Cyp40 homolog, Cpr7 . Immunophilin co-chaperones are important in cancer, as Cyp40 and FKBP51 have been shown to promote the proliferation of androgen-dependent and androgen-independent prostate cancer cell lines .
We identified Cyp40 in a mass spectrometry screen designed to identify proteins regulated by the JunB transcription factor in ALK+ ALCL (R.J.I and J.D.P; unpublished observation). JunB is an AP-1 family transcription factor that is highly expressed in ALK+ ALCL [40–42], and has been shown to promote the proliferation of the Karpas 299 ALK+ ALCL cell line . This transcription factor also promotes the expression of CD30 [44, 45] and the cytotoxic protein, Granzyme B , in ALK+ ALCL, which are phenotypic characteristics of this lymphoma [1, 47]. Since co-chaperone proteins are important for Hsp90 function, and Hsp90 activity is critical in ALK+ ALCL, we were intrigued by our observation that JunB might promote the expression of Cyp40 in ALK+ ALCL. In this study, we examined whether the expression of the immunophilin co-chaperones was regulated by oncogenic signalling in ALK+ ALCL. We also investigated whether the immunophilin co-chaperone proteins were important for the viability of ALK+ ALCL cell lines. We found that NPM-ALK induced the transcription of two immunophilin family co-chaperones, Cyp40 and FKBP52, but that only Cyp40 transcription was promoted by JunB. In addition, knocking-down the expression of Cyp40, but not FKBP51 or FKBP52, reduced the viability of ALK+ ALCL cell lines. However, knock-down of the immunophilin proteins did not appear to regulate NPM-ALK stability or activation. In conclusion, we demonstrate that some members of the immunophilin family of Hsp90 co-chaperone proteins are targets of NPM-ALK signalling, and that Cyp40 plays an important role(s) in ALK+ ALCL that is not shared by other immunophilin family co-chaperones.