hsp90molecularchaperoneinhibitorsarewethereyet.pdf

1、<p>　　Hsp90 molecular chaperone inhibitors: Are we there yet?</p><p>　　Len Neckers1 and Paul Workman2</p><p>　　Urologic Oncology Branch, Center for Cancer Research, National Cancer Institut

2、e, 9000</p><p>　　Rockville Pike Bldg. 10/CRC, Room 1-5940, Bethesda，MD 20892-1107 USA</p><p>　　Signal Transduction and Molecular Pharmacology Team，Cancer Research UK Cancer</p><p>　

3、　Therapeutics Unit, Division of Cancer Therapeutics, The Institute of Cancer Research, Haddow</p><p>　　Laboratories，15 Cotswold Road, Sutton SM2 5NG UK</p><p><b>　　Abstract</b></p

4、><p>　　Heat shock protein (Hsp) 90 is an ATP-dependent molecular chaperone exploited by malignant cells to support activated oncoproteins，including many cancer-associated kinases and transcription factors，and i

5、s essential for oncogenic transformation. Originally viewed with skepticism, Hsp90 inhibitors are now actively pursued by the pharmaceutical industry, with 17 agents having entered clinical trials. Hsp90’s druggability w

6、as established using the natural products geldanamycin and radicicol which mi</p><p>　　Introduction</p><p>　　Heat shock protein (Hsp) 90 is an ATP-dependent molecular chaperone that regulates la

7、te stage maturation，activation，and stability of a diverse range of 'client’ proteins (defined as proteins with demonstrated binding to Hsp90 whose steady-state level declines upon Hsp90 inhibitor treatment, usually a

8、s a result of proteasome-mediated degradation; see http://www.picard.ch/downloads for a curated list) many which are involved in signal transduction and other key pathways that are especially importa</p><p>

9、　　Corresponding authors: Lcn Ncckcrs PhD, ncckcrsl@mail.nih.gov Paul Workman PhD, paul.workman@icr.ac.uk .</p><p>　　Conflict of interest Paul Workman is an employee of The Institute of Cancer Research, which

10、 has a commercial interest in Hsp90 inhibitors and which operates a reward (o inventors scheme. Intellectual property on Hsp90 inhibitors developed at The Institute of’ Cancer Research was licensed to Vcrnalis and Novart

11、is. Paul Workman has also been a consultant to Novartis，and is a scientific founder of Chroma Therapeutics and current Chairman of Chroma Therapeutics Scientific Advisory Board.</p><p>　　activated or metasta

12、ble (e.g.，labile) forms of oncoproteins, including many kinases and transcription factors, that are mutated，translocated, amplified or overexpressed in malignancy; and 2) to buffer cellular stresses induced by the malign

13、ant lifestyle (Figure 1) (2, 3). Hsp90 is itself often overexpressed (4) and present in an activated multichaperone complex in cancer cells ⑶，and it is now regarded as essential for malignant transformation and progressi

14、on (2, 3).</p><p>　　When the concept of targeting Hsp90 in cancer was first promulgated in the early 1990s，it was viewed with considerable skepticism by the pharmaceutical industry. This was primarily becaus

15、e it was unprecedented to propose targeting a housekeeping protein that is abundantly expressed in normal cells and there was perceived risk that Hsp90 inhibition might therefore generate unacceptable toxicity. Thus，the

16、early clinical development of Hsp90 inhibitors was undertaken by the U. S. National Cancer In</p><p>　　In parallel with recent preclinical and clinical therapeutic developments，there has been considerable pr

17、ogress in understanding the molecular，cellular and organismal contributions of Hsp90 (1-3). Experience gained over the last several years in both the basic biology and the translational drug development around Hsp90, enh

18、anced by the use of Hsp90 inhibitors as chemical probes, has helped us to understand how best to achieve clinical success through inhibition of the molecular chaperone. As part</p><p>　　Target validation, ch

19、emical tools, drug discovery and development</p><p>　　The druggability of Hsp90 was established using the natural products radicicol and geldanamycin (Figure 2). These were isolated in 1953 and 1970, respect

20、ively, and were shown to have biologic activity by a then unknown mechanism (11). A critical observation for the Hsp90 field was the demonstration in 1994 that Hsp90 was the molecular target of geldanamycin and that Hsp9

21、0 inhibition by this agent prevented formation of the complex between Hsp90 and its client protein SRC, resulting in SRC destab</p><p>　　selective inhibition of ATP binding and hydrolysis (15, 16) and in tur

22、n to the depletion of oncogenic clients through ubiquitin-mediated proteasomal degradation (17, 18).</p><p>　　The natural products provided important chemical probes that proved invaluable to the Hsp90 field

23、, enabling its function to be queried in detail and validating Hsp90 as a druggable target (19). Moreover, although geldanamycin and radicicol proved too toxic and unstable/reactive for clinical use, they each provided t

24、he chemical basis for drugs that subsequently entered the clinic. The first to progress to clinical trials was the better tolerated geldanamycin analog 17-aIlylamino-17-demethoxygelda</p><p>　　In Phase I stu

25、dies, tanespimycin showed proof-of-mechanism for target inhibition using a validated pharmacodynamic biomarker signature of client protein depletion and HSF1- dependent Hsp induction (20, 21). It also exhibited evidence

26、of clinical activity in various malignancies, most impressively to date in Phase I and II studies in HER2+，trastuzumab- refractory breast cancer where objective RECIST responses were seen on a weekly schedule of 450 mg/m

27、2 (22-24). This activity is attributed to the </p><p>　　However, despite the promising results seen in HER2+ breast cancer and also in multiple myeloma, where encouraging activity was seen in combination wit

28、h the proteasome inhibitor bortezomib (25, 26)，tanespimycin’s development was subsequently discontinued. It has been speculated that termination by the company supplying tanespimycin (Bristol- Myers Squibb) may have been

29、 related to production/formulation and patent expiry concerns (24, 27)</p><p>　　(http://www.myelomabeaconxom/news/2010/07/22/tanespimycin-development-halted/). Although prolonged disease stabilization was ac

30、hieved in Phase I studies of tanespimycin in various additional tumor types expressing particular Hsp90 client proteins, neither complete nor partial tumor responses were seen (28, 29). This limited activity has been att

31、ributed to suboptimal inhibition of the target client proteins, most likely owing to insufficient drug dose or frequency of administration. In fact，consi</p><p>　　Another geldanamycin analog (17-DMAG，17-dime

32、thylaminoethylamino-17- demethoxygeldanamycin，alvespimycin; Figure 2) is much less sensitive to, and dependent on，NQOl and also has improved formulation and pharmacokinetic properties. Of interest，a recent study with alv

33、espimycin reported one complete response in castrate-refractory prostate cancer (CRPC), seen by prostate specific antigen levels and confirmed on CT, as well as stable disease in other patients with CRPC，chondrosarcoma，a

34、nd renal cancer </p><p>　　Activity in CRPC was likely related to depletion of the androgen receptor, an Hsp90 client protein，as seen in preclinical models (33). There appears to be no further development of

35、either 17-DMAG or of IPI-493 (the 17-amino analog and metabolite of tanespimycin) which is similarly less dependent on NQOl (39) (Figure 2). Still in clinical development，however， is the soluble stabilized hydroquinone f

36、orm of 17-A AG, IPI-504 (retaspimycin hydrochloride. Figure 2). Its evaluation in gastrointestinal </p><p>　　The trailblazing proof-of-concept work with geldanamycin analogues stimulated the race to discover

37、 synthetic small molecule Hsp90 inhibitors that would overcome some or all of the limitations of this class，including the ability to use doses and schedules that would deliver sufficiently sustained client depletion whil

38、e sparing the liver toxicity，hypothesized to be caused by quinone metabolism (41，42)，as well as avoiding P-glycoprotein-mediated efflux seen with tanespimycin (30>. A large number of</p><p>　　Success in d

39、iscovering these new Hsp90 drug candidates has benefited greatly from structure-based design using available X-ray crystal structures of Hsp90, with initial chemical matter frequently emerging from high-throughput, fragm

40、ent or virtual screening (39, 43). The two initial examples of success were with: 1) the purine scaffold series, based initially on PU3 (44,45)，leading to the clinical candidates BBIIB021 (CNF-2024) and BTIB028 (structur

41、e not disclosed), as well as PU-H71，now in phase</p><p>　　Importantly, where co-crystal structures of drug bound to the N-terminal domain of Hsp90 have been determined these inhibitors all seem to exploit th

42、e same core network of water- mediated hydrogen bonding interactions exploited by geldanamycin and radicicol (as well as ATP/ADP) to anchor the drugs into the base of the N-terminal nucleotide-binding pocket of the chape

43、rone (16, 57, 58). This is illustrated in Figure 3 for the current clinical drug NVP- AUY922. The various new agents have the poten</p><p>　　earlier). Encouraging early clinical data have been reported conce

44、rning these agents pharmacodynamic and antitumor activity in diverse malignancies, again with the expected client protein and genetic profiles，including breast，non-small cell lung and rectal cancer，as well as melanoma an

45、d leukemia (40, 59-65).</p><p>　　Biological insights and therapeutic implications</p><p>　　As more is learned about the role of Hsp90 in modulating signaling networks and about the sensitivity o

46、f various client proteins to Hsp90 inhibition, a better understanding of Hsp90 biology has educated and will continue to inform the ongoing clinical development of Hsp90 inhibitor-based therapy，in part by supporting the

47、correct choice of tumor types and revealing additional molecular targets whose inhibition synergizes with Hsp90 inhibition, as discussed below.</p><p>　　Hsp90 helps to coordinate cellular and organismal resp

48、onses to environmental stresses. For the fruit fly D. melanogaster, this may involve the ability to survive in a habitat where temperature fluctuations are common. For the protozoan parasite P. falciparum, this certainly

49、 involves successful adaptation to a life cycle that includes more than one host，as well as dramatically different local environments and temperatures. For cancer cells， unavoidable environmental perturbations include nu

50、trient</p><p>　　While specific Hsp90-dependent pathways that promote unlimited growth，survival in low oxygen conditions, escape from apoptosis，and overcoming nutrient deprivation by fostering angiogenesis ha

51、ve been known for some time, data have been reported more recently to suggest that Hsp90 inhibition promotes enhanced host natural killer cell-mediated tumor killing (66). Further, the receptor tyrosine kinase ephrin rec

52、eptor A2 (EphA2) was recently identified to be an Hsp90 client (67). EphA2 is abundantly </p><p>　　Cancer cells are inherently genetically unstable and aneuploidy is a hallmark of cancer. Recently，data were

53、reported identifying the Hsp90 inhibitor tanespimycin to be among a small group of compounds demonstrating enhanced efficacy toward aneuploid cancer cell lines in vitro (69). Thus, several colorectal cancer cell lines wi

54、th high-grade aneuploid karyotypes were significantly more sensitive to the Hsp90 inhibitor than were colorectal cell lines with near euploid karyotypes. Similar differenti</p><p>　　The enhanced sensitivity

55、of aneuploid cancer cells to Hsp90 inhibition may reflect enhanced proteotoxic stress associated with aneuploidy, in part as a consequence of accumulating abundant misfolded proteins (69>. Thus，it is not surprising th

56、at Hsp90 inhibitors synergize with proteasome inhibitors in multiple myeloma，a cancer in which the proteasome</p><p>　　degradation machinery is taxed to the utmost. As described earlier in this review， combi

57、nation of tanespimycin and the proteasome inhibitor bortezomib has been associated with durable responses in heavily pretreated multiple myeloma patients，including those with bortezomib-refractory disease (25, 26, 71，72)

58、. Additional Hsp90 inhibitors are also being evaluated in this setting (50, 73, 74).</p><p>　　Some Hsp90 clients are known tumor driver proteins. Would tumors ‘a(chǎn)ddicted’ to (i.e.， dependent on) these clients

59、 be most likely to show clinical responses to Hsp90 inhibitors? The strong relationship between client driver protein dependence on Hsp90 and potential clinical efficacy of Hsp90 inhibition is demonstrated by two powerfu

60、l examples. The first is the tyrosine kinase receptor HER2, which was reported 15 years ago to be an Hsp90 client that is extremely sensitive (compared to most other c</p><p>　　The second example is the muta

61、ted (rearranged) tyrosine kinase ALK，perhaps the only Hsp90 client that is as sensitive or more sensitive to chaperone inhibition than is HER2 (77). ALK rearrangements, particularly the EML4-ALK fusion protein, are found

62、 in a small subset (approximately 4%) of NSCLC patients. In a recent, non-randomized phase II study of the Hsp90 inhibitor retaspimycin in patients with molecularly defined NSCLC, an overall response rate of 7% was obser

63、ved. However, of the three pat</p><p>　　These two examples suggest that client protein sensitivity to Hsp90 inhibition may be a key contributor to Hsp90 inhibitor clinical efficacy, but only in cases where t

64、he tumor is ?addicted to’ the client. Hsp90 inhibition may also represent an effective strategy to overcome or prevent tyrosine kinase inhibitor (TKI) resistance (78-81). A recent study reported acquired resistance to th

65、e ALK inhibitor crizotinib in an NSCLC patient whose cancer relapsed after five months of treatment. Molecular an</p><p>　　Chronic myelogenous leukemia in patients treated with the Abl TKI imatinib eventuall

66、y becomes resistant to the drug due to development or outgrowth of Bcr-Abl drug resistant mutations，the most common being T315I. This Bcr-Abl mutant is resistant to all first- and second-line TKIs tested but remains sens

67、itive to Hsp90 inhibition (Bcr-Abl is a highly dependent Hsp90 client) (83)- These examples suggest that, at least in certain circumstances, combination of an Hsp90 inhibitor with a TKI may suppre</p><p>　　I

68、n addition to the relevance of amplification of - and dependence on - client proteins such as HER2 or rearranged ALK as predictors of tumor response to Hsp90 inhibitors, overexpression of Hsp90 itself has been identified

69、 as an independent prognostic factor in breast cancer (4). This may represent a clinical example of non-oncogene addiction to stress protection pathways and it may explain the Hsp90 inhibitor efficacy of triple negative

70、breast cancer in preclinical models (46). Hsp90 expression </p><p>　　Future prospects: What next?</p><p>　　Several approaches are being investigated to enhance cancer cell sensitivity to Hsp90 i

71、nhibitors, including targeting Hsp90 co-chaperone proteins，HSF1 and its transcriptional targets，and post-translational modifiers of Hsp90. Modulating co-chaperone expression affects cancer cell sensitivity to Hsp90 inhib

72、itors. For example, deletion of p23 in yeast results in hypersensitivity to the Hsp90 inhibitors geldanamcyin and radicicol，while p23 over-expression protects yeast from these agents (85). As t</p><p>　　Acti

73、vation of the heat shock transcription factor HSF1 occurs uniformly in response to the Hsp90 inhibitors currently under clinical evaluation. Although useful to provide pharmacodynamic biomarkers，when this occurs in tumor

74、 cells it is generally considered to limit the activity of Hsp90 inhibitors because HSF1-dependent transcriptional induction of Hsp70, Hsp27, and to some degree Hsp90 itself, protects cancer cells from apoptosis.</p&g

75、t;<p>　　Indeed，cells in which HSF1 has been knocked out are much more sensitive to Hsp90 inhibitors than are their wild type counterparts (92). Likewise, silencing of either Hsp70 or Hsp27 has been shown to dramat

76、ically increase cancer cell sensitivity to Hsp90 inhibition and to induction of apoptosis (93, 94). Efforts are underway to identify and validate inhibitors of HSF1，Hsp70, and Hsp27, and to explore their combination with

77、 Hsp90 inhibitors (95-99).</p><p>　　Hsp90 is subject to several post-translational modifications that affect chaperone function. For example, the tyrosine kinase WEE 1，an Hsp90 client, directly phosphorylate

78、s the chaperone on a conserved tyrosine residue in Hsp90’s N-terminal domain (100). Phosphorylation at this site positively affects the ability of Hsp90 to chaperone a number of cancer-related kinases, including HER2, SR

79、C, CRAF，CDK4, and WEE1 itself. Importantly， WEE1 phosphorylation of Hsp90 negatively affects tanespimycin bin</p><p>　　Although to date the vast majority of drug development efforts have focused on targeting

80、 the N-domain ATP binding site of Hsp90, a second druggable site has been identified in the C- domain of the protein (102, 103)- Coumarin antibiotics, such as novobiocin，are the prototypic inhibitors that interact at thi

81、s site. Recently，significant advances have been made in improving the affinity of these compounds for Hsp90, and their ability to induce apoptosis</p><p>　　in cancer cells，in some cases with superior efficac

82、y to tanespimycin，has been demonstrated (104, 105). One potential benefit of these drugs is that some of the C-terminal inhibitors appear to be associated with significantly less robust HSF1 activation than is characteri

83、stic of N-terminal inhibitors (106). Existing data strongly support further medicinal chemistry optimization and pre-clinical evaluation of C-terminal Hsp90 inhibitors.</p><p>　　The relative importance in ca

84、ncer of the four Hsp90 isoforms (Hsp90a and Hsp90p in the cytoplasm and the nucleus, GRP94 in the endoplasmic reticulum, and TRAP 1 in the mitochondria) remains poorly understood. Although current inhibitors most potentl

85、y interact with Hsp90a and P isoforms (e.g. see ref 41)，the impact of isoform selectivity on the therapeutic index and toxic efYects of these inhibitors should be explored in greater detail.</p><p>　　The cri

86、tical role of Hsp90 in promoting cellular and organismal survival in response to environmental stress cannot be disputed. Thus although the protein has been validated as a bona fide molecular target in cancer and the inh

87、ibitors are generally well tolerated in the clinic, it should nonetheless be remembered that this chaperone also helps to maintain normal cellular homeostasis and has certain functions in specific cellular contexts that

88、one would not want to inhibit, at least on a long-ter</p><p>　　Hsp90 also positively regulates at least one transcription factor with tumor suppressor activity，namely interferon regulatory factor 1 (IRF1). L

89、oss of IRF1 cooperates with Ras mutation to transform cells, and IRFI is deleted in certain cancers (110). Hsp90 inhibition is associated with inhibition of IRFI transcriptional activity and IRFI protein degradation</

90、p><p><b>　　(HD-</b></p><p>　　LATS1 and LATS2 kinases are both clients of Hsp90 and are degraded upon Hsp90 inhibition (112). Both kinases positively regulate the Hippo tumor suppressor

91、pathway，and LATS-deficient mice are prone to develop ovarian cancers and sarcomas (113, 114). LATS1 activity was disrupted in Hsp90 inhibitor-treated ovarian cancer cell lines in vitro, and in ovarian cancer xenograft tu

92、mors obtained from mice treated with the Hsp90 inhibitor tanespimycin (112).</p><p>　　Finally，low-penetrant mutations of the retinoblastoma (RB) tumor suppressor gene give rise to RB proteins that retain app

93、roximately 50% of wild type tumor suppressor activity. However, these mutant RB proteins interact with and depend on Hsp90 for stability, and they are inactivated and destabilized in cells exposed to the Hsp90 inhibitor

94、geldanamycin (115). Thus, Hsp90 inhibitor treatment of patients harboring these low-penetrant RB mutant alleles, which normally are characterized by reduced or</p><p>　　Taken together，these data demonstrate

95、that several tumor suppressor pathways may be deregulated following Hsp90 inhibition，and they emphasize that the cumulative impact of an Hsp90 inhibitor on both the individual and the cancer cell is multi-factorial，and w

96、ill almost certainly be influenced by the duration of treatment，the disparate sensitivity to Hsp90 inhibition of the various client proteins present in normal and cancer cells，the dependence of the particular cancer on t