In a Perspective, Pieter Van Vlierberghe and Steven Goossens discuss Meijerink and colleagues' findings on steroid resistance in pediatric T cell acute lymphoblastic leukemia

Goossens, Steven

Van Vlierberghe, Pieter

PLoS Medicine

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Steven Goossens

Pieter Van Vlierberghe

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Overcoming Steroid Resistance in T Cell Acute Lymphoblastic Leukemia.

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Overcoming Steroid Resistance in T Cell Acute Lymphoblastic Leukemia

Ghent University Academic Bibliography

PERSPECTIVEOvercoming Steroid Resistance in T Cell AcuteLymphoblastic LeukemiaSteven Goossens1,2,3, Pieter Van Vlierberghe1,3*1 Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium, 2 Department for BiomedicalMolecular Biology, Ghent University, Ghent, Belgium, 3 Cancer Research Institute Ghent (CRIG), Ghent,Belgium* Pieter.vanvlierberghe@ugent.beT cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer that ismainly diagnosed in children and arises from the malignant transformation of T cell progeni-tors [1]. Current treatment protocols consist of high-dose, multi-agent combination chemo-therapy and result in cure rates of greater than 85% in children. However, despite their clinicalsuccess, the toxic nature of these aggressive treatment regimens causes severe side effects suchas reduced intellectual capacity, osteonecrosis and growth deficiencies, infertility, and anincreased risk of secondary tumor development later in life. Therefore, integration of noveltargeted therapies into contemporary T-ALL treatment protocols will be required to increasethe quality of life for pediatric T-ALL survivors [2].Glucocorticoids, such as prednisone or dexamethasone, are core components of the high-dose chemotherapy schedules used for the treatment of T-ALL [3]. The apoptotic responseinduced by these glucocorticoids is mediated by their interaction with the glucocorticoidreceptor NR3C1 (Fig 1). Upon ligand binding, NR3C1 undergoes dimerization and translo-cates to the nucleus in order to regulate target gene expression through direct interaction witha specific DNA binding motif (glucocorticoid response element) [3]. One important glucocor-ticoid target gene is the pro-apoptotic BCL2 family member BCL2L11 (encoding the BIM pro-tein), which counteracts the anti-apoptotic activity of BCL2, BCL-XL, and MCL1 and willtrigger a steroid-induced apoptotic response in steroid-sensitive leukemic blasts (Fig 1). Nev-ertheless, glucocorticoid response is variable amongst T cell leukemia patients, and steroidresistance has been associated with an increased risk of relapse and poor clinical outcome [3].Therefore, increased understanding of the molecular mechanisms that drive glucocorticoidresistance in human T-ALL could trigger the development of novel strategies that restore ste-roid sensitivity in this disease. Ultimately, these efforts should improve clinical outcome inhuman T-ALL and reduce the detrimental toxic side effects associated with high-dosechemotherapy.Genetics Meet Drug Sensitivity in Human T-ALLIn this issue of PLOS Medicine, Jules Meijerink and colleagues [4] integrated next-generationsequencing data and genome-wide copy number profiles with ex vivo drug sensitivity and clin-ical outcome in a large panel of primary T-ALL specimens obtained at diagnosis. Interestingly,the authors found that mutations in the interleukin 7 receptor (IL7R) signaling pathway,including JAK1 and KRAS mutations, were significantly associated with prednisolone resis-tance and reduced survival in an initial discovery cohort of 69 primary T-ALL samples. Ofnote, these results are in line with the poor prognostic implications of JAK1 [5] and N/K-RASPLOS Medicine | DOI:10.1371/journal.pmed.1002208 December 20, 2016 1 / 6a11111OPENACCESSCitation: Goossens S, Van Vlierberghe P (2016)Overcoming Steroid Resistance in T Cell AcuteLymphoblastic Leukemia. PLoS Med 13(12):e1002208. doi:10.1371/journal.pmed.1002208Published: December 20, 2016Copyright: © 2016 Goossens, Van Vlierberghe.This is an open access article distributed under theterms of the Creative Commons AttributionLicense, which permits unrestricted use,distribution, and reproduction in any medium,provided the original author and source arecredited.Funding: The authors received no funding for thiswork.Competing Interests: The authors have declaredthat no competing interests exist.Abbreviations: B-ALL, B cell acute lymphoblasticleukemia; IL7R, interleukin 7 receptor; T-ALL, T cellacute lymphoblastic leukemia.Provenance: Commissioned; not externally peerreviewed.[6] mutations that have previously been reported in adult T-ALL. In addition, previous studieshave also shown an association between RAS mutations and steroid resistance in the contextof precursor B-lineage ALL (B cell acute lymphoblastic leukemia [B-ALL]) [7,8]. Given thatvarious mutations affecting the IL7R signaling cascade have also been reported in precursorB-ALL, it is tempting to speculate that the relation between IL7R signaling and steroid resis-tance could be a general feature of both T- and B-lineage ALL.Next, Meijerink and colleagues extended their analyses to 146 leukemia samples, for whichthey determined the mutational status of a selected panel of genes required for IL7R–RAS–MAPK–AKT signaling (IL7Ra, JAK1, JAK3, NF1, NRAS, KRAS, and AKT) by PCR-basedSanger sequencing. This analysis revealed that T-ALL specimens mutated for any of thesegenes were significantly associated with steroid resistance and poor clinical outcome. Interest-ingly, T-ALLs that harbor mutations in IL7R–RAS–MAPK–AKT signaling were associatedwith specific molecular genetic subtypes, including immature, TLX1, TLX3-rearranged, orHOXA-activated leukemias. However, it should be noted that Meijerink and colleagues are thefirst to establish that these specific genetic alterations are truly associated with steroid resis-tance in primary T-ALL specimens.Fig 1. The glucocorticoid response in T-ALL. In steroid-sensitive T-ALL cells, steroid-mediated activation, dimerization, and subsequentnuclear translocation of the steroid receptor NR3C1 results in transactivation of the pro-apoptotic BIM and PUMA, which will activate theapoptosome complex after cytochrome C release from the mitochondria. The anti-apoptotic protein MCL1 in steroid-sensitive T-ALL cells isactively targeted for proteasomal degradation via GSK3β. βP, phosphorylation; Ub, ubiquination; GRE, glucocorticoid response element.doi:10.1371/journal.pmed.1002208.g001PLOS Medicine | DOI:10.1371/journal.pmed.1002208 December 20, 2016 2 / 6From Association to Functional Implications: Genetic Drivers ofSteroid ResistanceTo functionally validate the putative contribution of each of these mutations to steroid resis-tance in T-ALL, Meijerink and colleagues generated a panel of isogenic tumor lines withinducible expression of IL7R–RAS–MAPK–AKT pathway mutations. Using these elegant invitro model systems, the authors confirmed that IL7R, JAK1, and NRAS mutations, as well asoverexpression of wild-type NRAS or AKT, could drive steroid resistance in human T-ALLcell lines (Fig 2). In contrast, no effect was observed for mutant JAK3 or expression of wild-type JAK1, JAK3, or IL7R.Notably, no differences in NR3C1 nuclear translocation or target gene transactivation uponglucocorticoid exposure were observed between steroid-resistant and steroid-sensitive isogenictumor cell lines. These data suggest that, at least in these in vitro model systems, the molecularmechanisms controlling steroid resistance are downstream or independent of the NR3C1transactivation response. Strikingly, these results are in sharp contrast with other work inwhich glucocorticoid resistance in ALL was associated with the inability to induce the pro-apoptotic NR3C1 target gene BCL2L11 upon dexamethasone treatment of patient-derivedFig 2. Molecular mechanisms that drive glucocorticoid resistance in T-ALL. In steroid-resistant T-ALL cells, the JAK/STAT, RAS/pERK,and PI3K/AKT1/mTOR signaling pathways result in: (1) up-regulation of the anti-apoptotic proteins BCL2 and BCL-XL; (2) inactivation ofGSK3β, leading to increased concentrations of the anti-apoptotic protein MCL1; and (3) direct inhibition of BIM, which collectively counteractthe NR3C1-mediated activation of the apoptosome complex. P, phosphorylation; GRE, glucocorticoid response element.doi:10.1371/journal.pmed.1002208.g002PLOS Medicine | DOI:10.1371/journal.pmed.1002208 December 20, 2016 3 / 6xenografts [9,10]. These contradictory results may be related to a differential epigenetic stateof the BCL2L11 (BIM) locus in the T-ALL cell lines versus patient-derived xenografts. In addi-tion, these results suggest that steroid resistance can be gained via various means at differentessential nodes in these converging signaling pathways.Instead of a dysfunctional glucocorticoid-induced transcriptional response, Meijerink andcolleagues identified aberrant activation of specific signaling pathways downstream of mutantIL7R, JAK1 or NRAS, or wild-type AKT as major determinants of steroid resistance in the iso-genic tumor lines (Fig 2). Indeed, expression of IL7R, JAK1, or NRAS mutations provoked astrong activation of MEK–ERK and AKT signaling with concomitant phosphorylation ofp70-S6K and an increased expression of anti-apoptotic BCL-XL. In addition, activated MEK–ERK and AKT signaling also triggered a robust inactivation of GSK3β, which would lead tostabilization of anti-apoptotic MCL1 (Fig 2). Interestingly, the association between IL7R–JAKsignaling and steroid resistance described in this study is in line with the interleukin 7-inducedanti-apoptotic rescue of primary T-ALLs against dexamethasone, as reported more than adecade ago [11]. However, in these studies, the inhibition of apoptosis by interleukin 7 wasshown to correlate with up-regulation of BCL2 [12]. In addition, recurrent mutations in theIL7R and RAS signaling pathways mainly occur in early immature T-ALLs, which are alsomore often characterized by increased concentrations of BCL2 [13,14].Small Molecules with a Future in Steroid-Resistant T-ALLMeijerink and colleagues’ study has unraveled specific pathways that influence glucocorticoidsensitivity in T-ALL by affecting the balance between pro- and anti-apoptotic factors. There-fore, specific targeting of these signaling cascades could provide a unique opportunity to over-come glucocorticoid resistance in human T-ALL. Indeed, the JAK1 inhibitor ruxolitinib, theMEK inhibitor CI1040, and the AKT inhibitor MK2206 were able to reverse steroid resistancein specific isogenic tumor lines (Fig 3). Interestingly, the authors also showed that the GSK3βinhibitor IX provoked strong steroid resistance in sensitive T-ALL tumor lines, further con-firming the pivotal role of this molecule in the regulation of glucocorticoid resistance. More-over, given the central role for the balance between pro- and anti-apoptotic factors in thisprocess, one could hypothesize that specific targeting of the apoptotic machinery using BCL2[13] or MCL1 [15] inhibitors could also serve as a useful strategy to alter steroid resistance inthe context of malignant T cell transformation.In a more clinically relevant approach, the authors subsequently evaluated the putative drugsynergism between prednisolone and seven small molecule inhibitors targeting JAK1, MEK1/2, or PI3K/AKT/mTOR in a panel of 11 genetically well-characterized primary T-ALL samplesat diagnosis. Notably, small molecule inhibitors targeting MEK–ERK or PI3K/AKT/mTORsignaling enhanced the steroid response in most primary T-ALL samples (Fig 3). In line withthis notion, recent work showed that the dual PI3K/AKT/mTOR inhibitor BEZ235 enhanceddexamethasone sensitivity in vivo using a patient-derived T-ALL xenograft [16]. Surprisingly,and in contrast with the cell line data, ruxolitinib had a synergistic effect on prednisolone treat-ment in only 1 out of 11 primary T-ALL samples. However, this might be related to the lack ofproliferative capacity of T-ALL cells in this system, given that previous work convincinglyshowed preclinical in vivo efficacy of ruxolitinib in patient-derived T-ALL xenografts [14].Nevertheless, these results could suggest that ruxolitinib might not be suitable for therapeutictargeting of dormant leukemic stem cells.In conclusion, Meijerink and colleagues’ study nicely illustrates how somatic alterationsimplicated in T-ALL disease biology influence the differential steroid response observed in theclinic. These novel insights strongly suggest that inhibition of MEK–ERK or PI3K/AKT/mTORPLOS Medicine | DOI:10.1371/journal.pmed.1002208 December 20, 2016 4 / 6signaling could enhance steroid sensitivity in T-ALL and potentially improve patient treatmentoutcome, a notion that warrants further investigation in future prospective clinical trials.References1. Durinck K, Goossens S, Peirs S, Wallaert A, Van Loocke W, Matthijssens F, et al. Novel biologicalinsights in T cell acute lymphoblastic leukemia. Exp Hematol. 2015; 43(8):625–39. doi: 10.1016/j.exphem.2015.05.017 PMID: 261233662. Hunger SP, Mullighan CG. Acute Lymphoblastic Leukemia in Children. N Engl J Med. 2015; 373(16):1541–52. doi: 10.1056/NEJMra1400972 PMID: 264659873. Inaba H, Pui CH. Glucocorticoid use in acute lymphoblastic leukaemia. Lancet Oncol. 2010; 11(11):1096–106. doi: 10.1016/S1470-2045(10)70114-5 PMID: 209474304. Li Y, Buijs-Gladdines JG, Cante´-Barrett K, Stubbs AP, Vroegindeweij EM, et al. 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Overcoming steroid resistance in T cell acute lymphoblastic leukemia

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Overcoming steroid resistance in T cell acute lymphoblastic leukemia

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