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COMMENTARY JAK-STAT1:2,77–79;April/May/June2012;G2012LandesBioscience The interplay between inhibition of JAK2 and HSP90 Jordan S. Fridman and Nicholas J. Sarlis* IncyteCorporation;Wilmington,DEUSA A recent article by Weigert et al. is overcome by HSP90 inhibition,” published in The Journal of described the in vitro generation of three Experimental Medicine described the in synthetic mutations in the JAK2 kinase vitro generation of synthetic mutations domain—G935R, Y931C and E864K— in Janus kinase 2 (JAK 2) that decreased that decreased the potency of multiple the potency of JAK2 (or JAK1/JAK2) JAK2 inhibitors in cellular assay systems. inhibitors in artificial systems. The The authors go on to report an ability of authors found that heat shock protein heat shock protein 90 (HSP90) inhibitors 90 (HSP90) inhibitors circumvented the to circumvent the potency shift observed potency shift and suggested that HSP90 with JAK inhibition.4 JAK2 inhibitors inhibition may abrogate JAK inhibitor were less potent when these in vitro- resistance in these experimental systems. generated synthetic residue substitutions However, the clinical relevance of these were present in cis with clinically relevant laboratory-generated JAK2 mutations, somatic-activating JAK2 mutations, i.e., which have not been identified to-date JAK2 V617F, which is characteristic of in patients treated with JAK inhibitors, MPNs,5 and JAK2 R683G, which is and the therapeutic potential of found in a subset of individuals with HSP90 inhibitors in diseases involving B-cell acute lymphoblastic leukemia aberrant JAK-STAT signaling remain to (B-ALL) with rearrangements of cytokine be determined. receptor-likefactor2(CRLF2).6Structural modeling studies determined that the synthetic G935R, Y931C and E864K Alterations in Janus kinase-signal trans- amino acid changes were located near the ducer and activator of transcription (JAK- JAK2 ATP binding site, which led to the STAT) signaling have been identified in hypothesis that they would interfere with several disease states, including inflam- JAK2 inhibitor binding.4 Keywords: Janus kinase, JAK inhibitor, matory conditions (rheumatoid arthritis, The in vitro experimental process HSP90, resistance, mutation psoriasis) and some cancers [myelopro- yielded G935R, Y931C and E864K by Abbreviations: JAK, Janus kinase; liferative neoplasms (MPNs), other hema- exposure of CRLF2-expressing murine HSP, heat shock protein; STAT, signal tologic cancers, pancreatic cancer]. Thus, Ba/F3 cells transduced with synthetically transducer and activator of transcription JAK inhibitors are under very active altered human JAK2 R683G cDNA to investigation as novel therapeutic agents high concentrations of the JAK2 inhibitor Submitted: 03/22/12 with the potential for broad application. NVP-BVB808. These JAK2 variants also Accepted: 04/07/12 ThefirstsuchinhibitortoreceiveUSFood reducedtheresponsiveness oferythropoie- and Drug Administration (FDA) approval tin receptor(EpoR)-expressingBa/F3 cells http://dx.doi.org/10.4161/jkst.20293 is ruxolitinib (Incyte Corporation) for the to this JAK inhibitor. Using similar in *Correspondenceto:NicholasJ.Sarlis; treatment of patients with intermediate vitro methods, others have also identified Email:[email protected] or high-risk myelofibrosis (MF).1,2 This these JAK2 alterations,7-9 though it is oral, small-molecule, JAK1 and JAK2 noteworthy that they have not been Commentaryto:WeigertO,LaneAA,BirdL, inhibitorisalsobeinginvestigatedinother reported in either JAK2 V617F-driven KoppN,ChapuyB,vanBodegomD,etal.Genetic resistancetoJAK2enzymaticinhibitorsisover- malignancies.3 mouse models of MPN-like diseases comebyHSP90inhibition.JExpMed2012; ArecentarticlepublishedinTheJournal following treatment with JAK inhibitors 209:259–73;PMID:22271575;http://dx.doi.org/10. of Experimental Medicine, “Genetic resis- or in patients. Testing of a panel of 1084/jem.20111694 tance to JAK2 enzymatic inhibitors JAK2 inhibitors against the mutant www.landesbioscience.com JAK-STAT 77 EpoR-expressing Ba/F3 cells transduced inhibitors with signaling pathways not due to the emergence of JAK2 secondary with mouse JAK2 V617F revealed that involving JAK2 contributed to cell kill. mutations in patients treated with JAK G935RandY931C decreased thepotency In nude mice transplanted with Ba/F3 inhibitors. While this may be the result of ruxolitinib in this system. Of note, the cells containing the Y931C mutation, of a less pronounced oncogenic role for concentration of ruxolitinib required treatment with the HSP90 inhibitor JAK2 signaling in MF patients, it is to inhibit cell growth by 50% (GI50) NVP-AUY922 improved overall survival intriguing, yet perhaps coincidental, that increased approximately 3-fold in the compared with vehicle; however, the all three publications describing JAK2 presence of the G935R mutation and effects of NVP-BVB808 were not evalu- variants with reduced sensitivity to 9-fold with the Y931C mutation.4 ated in this setting and the tolerability JAK inhibitors utilized the expression of Because JAK2 is an HSP90 client,10 to NVP-AUY922 was not described. unnatural secondary JAK2 mutations and inhibition of HSP90 results in wild- In CRLF2-rearranged B-ALL xenografts rather than relying on natural selection. type and mutant JAK2 depletion,11 established from the bone marrow of Quantitatively, the mutations described HSP90 inhibitors were also evaluated in B-ALL patients and implanted into mice, by Weigert et al. conveyed a modest these in vitro-generated JAK2 mutant NVP-AUY922 was more efficacious than degree (, 4-fold to 9-fold) of resistance clones. In this report by Weigert et al., NVP-BVB808 at suppressing JAK-STAT, relative to the wild-type protein. This is addition of HSP90 inhibitors led to frank MAP kinase and AKT signaling and was in contrast to those mutations in BCR- cytotoxicity rather than growth inhibition associated with prolonged survival com- ABL identified by Ray et al. in a similar because of cell cycle accumulation in G pared with NVP-BVB808. However, one model system. In that study, five clini- 1 or G , which is typically seen in other should recognize that these xenografts cally occurring BCR-ABL mutations 2 experimental settings with HSP90 inhibi- lacked any secondary JAK2 mutations conveyed a degree of resistance to the tors.12Thiscytotoxiceffectledtheauthors that would confer resistance to JAK second-generation inhibitor nilotinib to suggest that HSP90 inhibition may inhibition.4 Moreover, the dose of NVP- greater than 20-fold with at least two be mechanistically relevant in overcoming BVB808 used in this experiment was BCR-ABL mutants conveying greater JAK2 inhibitor resistance (Fig.1). How- insufficient to produce any noteworthy than 100-fold resistance over wild-type ever, these findings also suggest that pharmacodynamic suppression of either BCR-ABL.13 HSP90 inhibition is likely a less selective pJAK2 or pSTAT. What are the implications of this study approach than direct inhibition of JAK2. Although the resistance observed with for patients being treated with ruxolitinib? Indeed, HSP90 has numerous client pro- JAK inhibitors in this study is intriguing, While the findings are of interest, their teins in addition to JAK2, and HSP90 there are qualitative and quantitative dif- clinical relevance remains to be deter- inhibitorshaveshowncytotoxicactivityin ferences between the resistance described mined. It is not known whether patients a great variety of in vitro malignancy- by Weigert et al. and that described for treated with JAK2 inhibitors develop derived cell lines in addition to strictly BCR-ABL inhibitors, such as imatinib, or G935R,Y931CorE864Kmutationsdur- JAK2-dependent cell lines. Given the themorepotentsecond-generationinhibi- ing treatment, as these variants have not above, the authors acknowledged the tor nilotinib. Most significant is the lack been identified in the clinic. Additionally, possibility that interference of HSP90 (to date) of documented clinical resistance a clinical definition of resistance to JAK Figure1.AmodelbasedonthefindingsofthestudybyWeigertetal.wouldimplythatinthepresenceofcertainJAK2mutations,JAK2inhibitorsmay beunabletomaximallyinhibitJAK-STATsignaling(A).Inthismodel,theco-administrationofanHSP90inhibitorcouldsupplementtheeffectofaJAK2 inhibitortoovercomecompromisedJAK2inhibitorpotency(B). 78 JAK-STAT Volume1Issue2 inhibitors in the setting of MPNs, and JAK2 mutations is higher (e.g., acute exploring the combination of HSP90 specifically MF, has not been published. leukemia), true “resistance mutations” inhibitors and a JAK inhibitor may be Indeed, long-term follow-up of patients may be more readily selected. Lastly, the the most efficient way to translate the treatedwithJAK2(orJAK1/JAK2)inhibi- lack of specificity of HSP90 inhibitors preclinical work of Weigert et al., it will tors to determine whether they develop withregardtomolecular moietiesorpath- beequallyimportanttoexplorethesingle- resistancetosuchagents,andifsoelucida- ways whose activity would secondarily be agent antineoplastic efficacy of HSP90 tion of the mechanisms of resistance, will influenced by these agents may result in inhibitors, interrogate whether HSP90 be necessary to understand whether the tolerability issues. The clinical utility of system-related biology underlies differ- G935R, Y931C and E864K amino acid HSP90 inhibitors in diseases in which ences in the magnitude of clinical benefit changes are clinically meaningful. Perhaps JAK-STAT inhibition is (or may become) of JAK inhibitors and characterize emerg- in clinical situations where the selective of therapeutic value will require further ing mechanisms of resistance to the latter pressure for spontaneous emergence of study.10 Indeed, while clinical studies class of agents. References 7. Hornakova T, Springuel L, Devreux J, Dusa A, 11. ProiaDA,FoleyKP,KorbutT,SangJ,SmithD,Bates Constantinescu SN, Knoops L, et al. Oncogenic RC, et al. Multifaceted intervention by the Hsp90 1. Pressrelease.FDAapprovesIncyte’sJakafiTM(ruxoli- JAK1 and JAK2-activating mutations resistant to inhibitor ganetespib (STA-9090) in cancer cells with tinib)forpatientswithmyelofibrosis.11/16/11. ATP-competitive inhibitors. Haematologica 2011; activated JAK/STAT signaling. PLoS One 2011; 6: 2. 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