Editorial

The promise and challengesof targeting RSK for thetreatment of cancerAnna L Stratford† & Sandra E DunnUniversity of British Columbia -- Pediatrics, Vancouver, British Columbia, Canada

Expert Opin. Ther. Targets (2011) 15(1):1-4

The p90 ribosomal S6 kinases (RSKs) are a family of AGC serine/threonine kinasesof which there are four members (RSK 1 -- 4). RSK1 and RSK2 are expressed ina wide range of cancers including breast [1], prostate [2], multiple myelomas [3,4],T-cell lymphoma [4,5] and melanoma [6] as well as head and neck cancers [7]. Fur-thermore, RSK2 has been shown to be critical to the early events of cancer develop-ment. In particular, it has been demonstrated that RSK2 is essential for celltransformation induced by constitutively activated Ras [8] and fibroblast growth fac-tor receptor 1 (FGFR1) [9]. RSK3 and RSK4 however have been described as tumorsuppressor genes [10,11]. Given the role of RSK1 and RSK2 in cancer, isoform-specific inhibitors could prove to be therapeutically beneficial. The review byChen et al. describes the specific promise of RSK2 targeted agents [12].

RSK1 and RSK2 lie downstream of the PI3K and MAPK pathways [13]. Follow-ing their activation they phosphorylate a wide range of substrates. The list ofdownstream substrates of RSK is continually growing and includes, amongstothers, transcription factors, anti-apoptotic proteins and cell cycle regulators(reviewed in [14]). More recently, the transcription/translation factor Y-box bindingprotein-1 (YB-1) has been described as a RSK substrate [15]. YB-1 is a particularlynotable substrate because it is a developmental gene that is silenced in adult tissuesyet preferentially expressed in tumors. There have been no other tumor-specificsubstrates reported for RSK1 or RSK2. In breast cancer, RSK and YB-1 partnerto promote tumor cell growth [15] and drug resistance [16]. RSK also phosphorylatesimportant nuclear hormone receptors such as the estrogen and androgen recep-tors [17,18]. RSK2 cooperates with estrogen to maximally promote the activation ofthe estrogen receptor [17]. Thus, RSK2 inhibitors combined with anti-estrogenicagents could improve the treatment of breast cancer.

RSKs are likely to serve a fundamental role in sustaining tumor growth as they areconvergence points for many receptor tyrosine kinases (RTKs) linked to can-cer [14,19]. For example, RSKs are activated by the EGFR [15] and human EGF recep-tor 2 (Her-2), IGF-1R [20] as well as FGFR as noted by Kang et al. [4]. For this veryreason, targeting RSK may be more advantageous than inhibiting individual RTKs.For example, trastuzumab inactivates the Her-2 receptor; however, the expression ofIGF-1R renders the cells resistant [21]. Yet, IGF-1R inhibitors have not proven to beas effective as expected, perhaps due to an uncharacterized role for RSK. Indeed,RSK is in fact highly activated in breast cancer cells that have acquired trastuzumabresistance, prompting the idea that it may serve as an important therapeutic target inHer-2-positive tumors [16]. As a testament to this idea, BI-D1870 inhibited thegrowth of trastuzumab-resistant breast cancer cell lines [16]. This could be the tipof the iceberg, and RSK inhibitors will likely overcome resistance to other RTKinhibitors given its central role in cancer cell signaling.

As described in the upcoming review, there are numerous RSK inhibitors(BI-D1870, SL0101 and fluoromethyl ketone (FMK)) that have demonstratedspecificity in vitro [22] but have not, as yet, advanced to being tested inclinical trials. The current inhibitors are not isoform-specific; however, with the

10.1517/14728222.2011.537656 © 2011 Informa UK, Ltd. ISSN 1472-8222 1All rights reserved: reproduction in whole or in part not permitted

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elucidation of the crystal structure of the RSK2 N-terminalkinase domain [23] isoform-specific inhibitors may become areality. With the crystal structure described, one approachto identifying novel RSK2 inhibitors would be to docklibraries of known drugs (Prestwick collection, Microsourceand Library of Pharmacologically Active Compounds(LOPAC) to name a few) to the kinase domain using bio-informatics (Figure 1). Chemicals that produce informativedocking scores can then be screened for inhibitory effectsin vitro using recombinant RSK where kinase activity wouldbe monitored in the presence of radioactive ATP and com-parisons can be made with the other RSK isoforms. Thelead agents would then be assessed for their ability toinhibit cancer cell growth and to block signal transductionin vitro and in vivo (Figure 1). This process of drug reposi-tioning has many advantages including saving time and thecost of drug development. Since these drugs have alreadybeen approved for use in humans their safety profiles arealso largely known. Alternatively, novel compounds thatinhibit the individual RSK isoforms specifically can be syn-thesized or may be identified by screening natural productlibraries. For example, the natural product kaempferol, aplant flavonoid, was originally identified as having RSKinhibitory activity. Further, SL0101 was identified byscreening botanical extracts for RSK2 inhibitors [1].

SL0101 was identified in the flower extract from Forsteroniarefracta, belonging to the Apocynum cannabinum family(commonly called dogbane, Indian hemp or wild cotton)that is found in the rainforests of South America, southernCanada and the USA. It was structurally classified as akaempferol glycoside. Given that kaempferol was previouslyreported to inhibit RSK the finding was rather serendipi-tous. While they originally identified SL0101 in a RSK2kinase screen they also reported that it blocked RSK1 aswell. Thus as yet, isoform-specific inhibitors of RSK2have not been reported. The development of selective kinaseinhibitors is challenging because the ATP binding pocketsof many kinases are highly conserved. BI-D1870 for exam-ple, inhibits RSK1 -- 4 more then other kinases [22] how-ever, it does have activity against polo-like kinase 1(PLK-1) and aurora B [24] at higher concentrations. IfRSK2 inhibitors were developed they could potentiallyeliminate such off-target effects.

Another benefit to targeting RSK is the apparent safety/therapeutic window. Since RSK is activated by the MAPKpathway, and therefore by many receptor tyrosine kinasesthat have been linked to cancers, such as IGF-1R [20],FGFR [4] and EGFR [15], it can be instrumental in promotinggrowth and inhibiting apoptosis. Immortalized normal celllines express RSK; however, they do not depend on it

PrestwickLOPAC

Microsource

Dock

RSK2 kinase assay

Validateagainst RSK

targets

GSK3βERαYB-1

Clinical trials

RSK2 N-terminal kinase domain

Figure 1. RSK2-specific inhibitors would be identified by molecular docking given that the crystal structure of its

N-terminal kinase domain was recently reported. The structure is particularly useful as it was resolved in its active state that is

bound to ATP [23]. Small-molecule libraries of off-patent drugs could be evaluated in order to expedite the lead-time to

discovery and validation. Such compounds could be assessed in vitro using RSK2 kinase assays and comparisons could be made

against RSK1, RSK3 and RSK4 to assess isoform specificity. Next the agents of interest could be assessed in cancer cells lines

that express RSK2 for anti-neoplastic activity (changes in proliferation, invasion and/or drug sensitivity). The identification of

RSK2 agents could then be used to tailor treatment for patients that developed tumors that specifically express this isoform.

The promise and challenges of targeting RSK for the treatment of cancer

2 Expert Opin. Ther. Targets (2011) 15(1)

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and do not respond to RSK inhibitors ([1,25] and personalobservation). Further evidence for the safety of RSK inhibi-tors is the fact that RSK2 knockout mice are viable, with min-imal phenotypic changes; they were 10% smaller than thewild-type littermates [26,27] and the knockout had a very mod-est effect on insulin sensitivity [27]. The RSK2 knockout micealso form normal levels of T- and B- cells however there maybe a delay in the activation of the T-lymphocytes [28] and thiscould perturb host response times to infection. Further,RSK1 and RSK2 are reportedly expressed in the blood [29];it is therefore conceivable that inhibiting it may have deleteri-ous side-effects. However, aspirin suppresses RSK2 kinaseactivity but does not lead to related toxicities [29]. Further-more, loss of RSK2 via knockout also had no affect on thehematopoietic stem cell subpopulation [4]. Likewise, inhibit-ing RSK with the small molecule FMK [30] does not suppresscolony-forming units as compared with wild-type bone mar-row cells [5] or delay the growth of primary myeloid cellsfrom healthy individuals [4]. Thus, RSK2 is expressed in awide range of cancers and importantly many tumors continueto depend on it for sustaining growth. Based on what isknown thus far it is likely that RSK2 inhibitors will not behighly toxic to normal cells however additional studiesare needed to firmly conclude this definitively. With theelucidation of the crystal structure for RSK2 it is only a matter

of time before potent, selective inhibitors are identified for thetreatment of cancer.

Expert opinion

The recent interest in RSK as a potential therapeutictarget for cancer treatment is well-founded considering thegrowing number of tumors that overexpress this kinase.The inhibition of RSK2 is rationalized by pre-clinicalstudies, however, it is unclear how frequently this isoformis expressed in human tumors in general. Assuming thatRSK2 is commonly expressed it is likely to be activatedgiven the fact that so many RTKs found in cancer activateit. The role of RSK1 and RSK2 in tumor cell growth hasbrought them to the forefront of target identificationfor the pursuit of chemotherapeutic agents that could indi-vidualize therapy and may in some cases also be used toovercome drug resistance.

Declaration of interest

The authors have been sponsered by the Canadian BreastCancer Foundation (CBCF), the Canadian Institutesof Health Research (CIHR) and the Michael CuccioneFoundation. The authors state no conflict of interest.

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AffiliationAnna L Stratford† & Sandra E Dunn†Author for correspondence

University of British Columbia -- Pediatrics,

Vancouver,

British Columbia V5Z 4H4, Canada

E-mail: anna.stratford@gmail.com

The promise and challenges of targeting RSK for the treatment of cancer

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