From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
on p21WAF1/Cip1, a negative regulator of cell cycle progression. This protein is upregulated in CLL cells on lenalidomide treatment in vitro and in patients, and its depletion abrogates the antiproliferative effect. Depletion of cereblon, the only known molecular target of lenalidomide, prevents lenalidomide’s activity. Cereblon is part of the E3 ubiquitin ligase complex that consists of cullin 4A and damaged DNA binding protein 1 and ubiquitinates specific target proteins that are subsequently degraded by proteasome activity. In this complex, cereblon is responsible for target protein recognition and binding (see figure). Lenalidomide and related compounds like thalidomide directly bind to cereblon and thereby inhibit the autoubiquitination activity of the E3 ligase, which is the basis for the teratogenic activity of thalidomide.5 Recently, 2 groups independently showed that lenalidomide enhances the binding of cereblon to the 2 transcription factors Ikaros (IKZF1) and Aiolos (IKZF3).6,7 These transcription factors are essential for B- and T-cell development and are highly expressed in B-cell malignancies like CLL. Lenalidomide treatment results in ubiquitination and degradation of IKZF1 and 3, which is necessary for the antiproliferative effect of the drug in multiple myeloma. Together, these findings show that lenalidomide can either act as an inhibitor of E3 ubiquitin ligase activity, which results in a stabilization and accumulation of cereblon target proteins, or regulate the degradation of proteins by altering cereblon’s substrate specificity. As in multiple myeloma, lenalidomide treatment results in increased levels of p21WAF1/Cip1 in CLL, which is dependent on cereblon activity. Thus far, the molecular mechanism of this induction is not known. Data by Fecteau et al suggest a transcriptional upregulation of p21WAF1/Cip1. Future studies need to analyze whether IKZF1 and 3 or another transcription factor that is directly or indirectly regulated by cereblon are involved in p21WAF1/Cip1 expression. IKZF1 and 3 are known to act as repressors or activators of gene expression in different settings. Of interest, the gene coding for p21WAF1/Cip1 contains a potential binding site for IKZF1 in the promoter region, and down-regulation of IKZF1 in acute lymphocytic leukemia cells, resulted in increased p21WAF1/Cip1 levels.8 Alternatively, a posttranslational regulation of p21WAF1/Cip1 by lenalidomide impacting on the stability or
1546
degradation of the protein is also conceivable. In this respect, it is of interest that many regulatory proteins of the cell cycle, including p21WAF1/Cip1, are targets of cullin 4–based E3 ubiquitin ligase complexes.9 With the advances in understanding the molecular mechanism of lenalidomide, several questions concerning its therapeutic activity in CLL arise. (1) Are the transcription factors IKZF1 and 3 involved in the antiproliferative effect of lenalidomide in CLL cells? (2) Are cereblon and the transcription factors also responsible for the activity within the CLL microenvironment? An IKZF1- and 3-mediated activation of T cells on lenalidomide treatment has been described. Here, the degradation of both transcription factors results in a derepression of the interleukin-2 gene.10 (3) Why does only a subset of CLL patients respond to lenalidomide treatment? A discrimination of responders and nonresponders might become possible by analyzing the expression and activity of cereblon and its target proteins. On this basis, the rational design of drug combinations including lenalidomide should be advanced. Within recent years, enormous progress in elucidating the pathomechanisms of CLL and in developing novel targeted compounds has been made. This knowledge now has to be translated into novel treatment protocols. By selecting therapeutics according to the molecular and genetic setup of every patient, we are getting closer to an individualized treatment of CLL with a maximal therapy success.
Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Fecteau J-F, Corral LG, Ghia EM, et al. Lenalidomide inhibits the proliferation of CLL cells via a cereblon/ p21WAF1/Cip1-dependent mechanism independent of functional p53. Blood. 2014;124(10):1637-1644. 2. Giannopoulos K, Mertens D, Stilgenbauer S. Treating chronic lymphocytic leukemia with thalidomide and lenalidomide. Expert Opin Pharmacother. 2011;12(18): 2857-2864. 3. Ramsay AG, Johnson AJ, Lee AM, et al. Chronic lymphocytic leukemia T cells show impaired immunological synapse formation that can be reversed with an immunomodulating drug. J Clin Invest. 2008;118(7): 2427-2437. 4. Schulz A, D¨urr C, Zenz T, et al. Lenalidomide reduces survival of chronic lymphocytic leukemia cells in primary cocultures by altering the myeloid microenvironment. Blood. 2013;121(13):2503-2511. 5. Ito T, Ando H, Suzuki T, et al. Identification of a primary target of thalidomide teratogenicity. Science. 2010;327(5971):1345-1350. 6. Kr¨onke J, Udeshi ND, Narla A, et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science. 2014;343(6168):301-305. 7. Lu G, Middleton RE, Sun H, et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science. 2014;343(6168):305-309. 8. Iacobucci I, Iraci N, Messina M, et al. IKAROS deletions dictate a unique gene expression signature in patients with adult B-cell acute lymphoblastic leukemia. PLoS ONE. 2012;7(7):e40934. 9. Abbas T, Sivaprasad U, Terai K, Amador V, Pagano M, Dutta A. PCNA-dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex. Genes Dev. 2008;22(18):2496-2506. 10. Gandhi AK, Kang J, Havens CG, et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.). Br J Haematol. 2014;164(6):811-821. © 2014 by The American Society of Hematology
l l l MYELOID NEOPLASIA
Comment on Brown et al, page 1655
Driving toward targeted therapy for LCH ----------------------------------------------------------------------------------------------------Robert A. Baiocchi
THE OHIO STATE UNIVERSITY
In this issue of Blood, Brown et al identify somatic mutations of MAP2K1 capable of driving the RAS-RAF-MEK-ERK pathway in Langerhans cell histiocytosis (LCH). Their findings lend important insight into the pathogenesis of this disease and provide the rationale for exploring targeted approaches in clinical trials.1
L
CH is a rare, often misdiagnosed histiocytic proliferative disorder afflicting pediatric and adult patients. The clonal (CD1a1, S1001,
and CD2071) proliferating cells have morphologic features most consistent with bone marrow–derived Langerhans cells that
BLOOD, 4 SEPTEMBER 2014 x VOLUME 124, NUMBER 10
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
The RAS-RAF-MEK-ERK pathway represents a driver in the pathogenesis of LCH while offering the potential for targeted therapeutic approaches. As much as 60% of LCH patients carry mutated forms of ARAF or BRAF (V600E) capable of constitutive phosphorylation of the downstream effectors MEK1 and MEK2 that ultimately drive ERK activity. Up to 50% BRAF-V600E–negative LCH cases (27% total) harbor a mutation in MAP2K1 that leads to constitutive MEK1 and consequent ERK activation, promoting downstream proliferation and survival networks to drive LCH. The mutually exclusive presence of BRAF and MAP2K1 mutations presents an ideal opportunity to intervene with selective agents like vemurafenib or trametinib to target 2 critical enzymes essential to LCH pathogenesis.
represent an antigen presenting dendritic cell commonly located in the skin and mucosa. The clinical course of LCH is diverse, ranging from solitary skin or bone lesions that may spontaneously resolve to widely disseminated, multisystem disease associated with high morbidity and mortality.2 The broad spectrum of disease activity, inflammatory nature of the lesions, normal karyotype, and associated immune dysregulation suggested that LCH represented an immunoreactive disorder. However, in 2010 Badalian-Very et al reported 57% of archived LCH lesions harbored a recurrent somatic, activating genetic mutation of the BRAF gene (BRAF-V600E).3 Furthermore, they found evidence of constitutive activation of the RAS-RAF-MAPK-ERK pathway regardless of whether BRAF was mutated or wild-type in LCH lesions. The fact that BRAF-V600E mutations were present as single alleles suggested that this might reflect a driver mutation for this disease. These data presented the first convincing genetic evidence supporting the characterization of LCH as a myeloid
neoplastic disorder and provided rationale for considering a targeted approach to treat patients with this disease. Several years later, Haroche et al4 reported impressive clinical responses in 3 patients with BRAF-V600E–mutated LCH treated with the single-agent BRAF inhibitor vemurafenib. Constitutive ERK activity in all LCH lesions led others to explore additional genetic drivers of ERK activity. Nelson et al5 used whole-exome sequencing of DNA isolated from purified LCH cells from 3 patients with wild-type BRAF. Interestingly, their studies documented the first somatic, activating mutations (F351L and Q347_A3438del) within the kinase-encoded domain of ARAF, leading to both ARAF and MEK constitutive kinase activity. Transfection of mouse fibroblast cells with the ARAF double-mutant clone led to acquired contact-independent growth in soft agar, a hallmark feature of cellular transformation, and further evidence that ERK activation may represent a driver of LCH growth. In this issue, Brown and colleagues1 provide additional convincing evidence that
BLOOD, 4 SEPTEMBER 2014 x VOLUME 124, NUMBER 10
links constitutive MAPK-ERK signaling to the pathogenesis of LCH. They used targeted next-generation sequencing to evaluate 8 LCH cases and found BRAF-V600E in 3 cases, consistent with prior work, and an E102_I103del mutation in the MAP2K1 gene that encodes for the MEK1 protein kinase, an enzyme directly upstream of extracellular signal-regulated kinases ERK1/2. Interestingly, this MAP2K1 mutation occurred in an LCH case that was BRAF wild-type, which then led the investigators to examine an additional 32 cases by BRAF-V600E allele–specific polymerase chain reaction and Sanger sequencing of exons 2 and 3 of MAP2K1. Surprisingly, 11 of 40 (27%) cases showed somatic MAP2K1 mutations that occurred mutually exclusive to BRAF mutations, with 50% of wild-type BRAF cases showing MAP2K1 mutation. Most mutations identified in this study were deletions within exons 2 and 3 and were previously shown to encode for markedly enhanced MEK1 kinase activity (see Figure 1 in Brown et al).6 Collectively, these results, when taken in the context of studies led by Badalian-Very et al, suggest that the majority of LCH patients harbor a somatic, activating mutation in critical signaling elements of the RAS-RAF-MEK-ERK pathway (see figure). Do activating mutations of components of the RAS-RAF-MEK-ERK pathway represent true drivers of LCH pathogenesis? Recent work by Berres et al7 examines the biological relevance of recurrent BRAF-V600E mutations in the pathogenesis of LCH. Sixty-four lesions from 100 LCH patients showed the presence of the BRAF-V600E mutation, an event that was linked to increased risk of relapse. In patients with high-risk disease, the BRAF-V600E mutation could be detected in bone marrow CD341 hematopoietic progenitor cells, whereas patients with low-risk LCH showed presence of the mutation restricted to mature CD2071 dendritic cells (DCs) within primary lesions. The authors went on to develop a novel in vivo mouse model, allowing for conditional expression of BRAF-V600E in various subsets of DCs. Mice with conditional expression of BRAF-V600E in DC progenitors (under CD11c promoter) rapidly developed an aggressive, multisystem LCH-like disease. Interestingly, granulomatous lesions in these mice showed inflammatory infiltrates with accumulation of regulatory T cells and abundant expression of
1547
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
inflammatory cytokines that are observed in the lesions of humans with aggressive LCH. In stark contrast, mice with conditional expression of BRAF-V600E in mature DCs (under langerin promoter) developed a low-grade, localized LCH-like disease. By definition, driver mutations contribute toward the initiation of cellular transformation and the progression of malignant disease, 2 features clearly illustrated in this interesting preclinical model of LCH. Elegant models such as this will certainly improve our understanding of the complex pathogenesis of LCH and provide a useful setting to test novel targeted therapies exploiting this pathway. Additional studies supporting the driver nature of these mutated signaling components have illustrated the capacity of activating ARAF mutations to drive fibroblast transformation,5 MAP2K1 mutations driving constitutive ERK phosphorylation in melanomas6 and hairy cell leukemia,8 and MAP kinase activity to drive transformation of mammalian cells.9 Perhaps the most intriguing implications reported by Brown et al1 and others3,5 lie within the potential for pursuing targeted therapeutic strategies. Current up-front therapeutic modalities to manage patients with multisystem LCH have traditionally used a risk-stratified approach (single vs multisystem LCH), often using intensive cytotoxic chemotherapy.10 Patients with relapsed or refractory disease have benefitted from single-agent modalities delivering cytarabine or cladribine. Although these regimens have generally improved outcome, toxicity remains a challenge and patients with high-risk, multisystem disease still face a high mortality rate. The identification of activating mutations in A/BRAF and MAP2K1, genes encoding 2 critical signaling enzymes in the ERK pathway, allows for strategic approaches using novel targeted agents. Promising Food and Drug Administration–approved, selective agents like vemurafenib and trametinib to target mutant A/BRAF and MAP2K1/2, respectively, provide an ideal opportunity to develop well-designed, multicenter, genetically stratified (using targeted sequencing approaches) clinical trials. The collective findings reported by Brown et al1 and others3,5 identifying activating somatic mutations in the majority of LCH patients have shed light on the complex pathogenesis of LCH while offering patients with LCH hope for improved treatment strategies in the near future.
1548
Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Brown NA, Furtado LV, Betz BL, et al. High prevalence of somatic MAP2K1 mutations in BRAF V600E negative Langerhans cell histiocytosis. Blood. 2014;124(10): 1655-1658. 2. Delprat C, Aric`o M. Blood spotlight on Langerhans cell histiocytosis. Blood. 2014;124(6):867-872. 3. Badalian-Very G, Vergilio JA, Degar BA, et al. Recurrent BRAF mutations in Langerhans cell histiocytosis. Blood. 2010;116(11):1919-1923. 4. Haroche J, Cohen-Aubart F, Emile JF, et al. Dramatic efficacy of vemurafenib in both multisystemic and refractory Erdheim-Chester disease and Langerhans cell histiocytosis harboring the BRAF V600E mutation. Blood. 2013;121(9):1495-1500. 5. Nelson DS, Quispel W, Badalian-Very G, et al. Somatic activating ARAF mutations in Langerhans cell histiocytosis. Blood. 2014;123(20):3152-3155.
6. Nikolaev SI, Rimoldi D, Iseli C, et al. Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma. Nat Genet. 2012;44(2): 133-139. 7. Berres ML, Lim KP, Peters T, et al. BRAF-V600E expression in precursor versus differentiated dendritic cells defines clinically distinct LCH risk groups. J Exp Med. 2014;211(4):669-683. 8. Waterfall JJ, Arons E, Walker RL, et al. High prevalence of MAP2K1 mutations in variant and IGHV434-expressing hairy-cell leukemias. Nat Genet. 2014; 46(1):8-10. 9. Mansour SJ, Matten WT, Hermann AS, et al. Transformation of mammalian cells by constitutively active MAP kinase kinase. Science. 1994;265(5174):966-970. 10. Gadner H, Minkov M, Grois N, et al; Histiocyte Society. Therapy prolongation improves outcome in multisystem Langerhans cell histiocytosis. Blood. 2013; 121(25):5006-5014. © 2014 by The American Society of Hematology
l l l THROMBOSIS & HEMOSTASIS
Comment on Sherman et al, page 1659
Taking a leaf from the book of oral tolerance ----------------------------------------------------------------------------------------------------David W. Scott
UNIFORMED SERVICES UNIVERSITY OF THE HEALTH SCIENCES
In this issue of Blood, Sherman et al demonstrate that mucosal delivery of engineered tobacco leaves can lead to tolerance to factor VIII (FVIII) inhibitor production. Eat your veggies for tolerance!1
Proposed clinical application: FVIII domains expressed in transgenic plants can be ingested and processed by the mucosal immune system to induce tolerance to FVIII.
BLOOD, 4 SEPTEMBER 2014 x VOLUME 124, NUMBER 10
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
2014 124: 1546-1548 doi:10.1182/blood-2014-07-587378
Driving toward targeted therapy for LCH Robert A. Baiocchi
Updated information and services can be found at: http://www.bloodjournal.org/content/124/10/1546.full.html Articles on similar topics can be found in the following Blood collections Free Research Articles (4041 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
on p21WAF1/Cip1, a negative regulator of cell cycle progression. This protein is upregulated in CLL cells on lenalidomide treatment in vitro and in patients, and its depletion abrogates the antiproliferative effect. Depletion of cereblon, the only known molecular target of lenalidomide, prevents lenalidomide’s activity. Cereblon is part of the E3 ubiquitin ligase complex that consists of cullin 4A and damaged DNA binding protein 1 and ubiquitinates specific target proteins that are subsequently degraded by proteasome activity. In this complex, cereblon is responsible for target protein recognition and binding (see figure). Lenalidomide and related compounds like thalidomide directly bind to cereblon and thereby inhibit the autoubiquitination activity of the E3 ligase, which is the basis for the teratogenic activity of thalidomide.5 Recently, 2 groups independently showed that lenalidomide enhances the binding of cereblon to the 2 transcription factors Ikaros (IKZF1) and Aiolos (IKZF3).6,7 These transcription factors are essential for B- and T-cell development and are highly expressed in B-cell malignancies like CLL. Lenalidomide treatment results in ubiquitination and degradation of IKZF1 and 3, which is necessary for the antiproliferative effect of the drug in multiple myeloma. Together, these findings show that lenalidomide can either act as an inhibitor of E3 ubiquitin ligase activity, which results in a stabilization and accumulation of cereblon target proteins, or regulate the degradation of proteins by altering cereblon’s substrate specificity. As in multiple myeloma, lenalidomide treatment results in increased levels of p21WAF1/Cip1 in CLL, which is dependent on cereblon activity. Thus far, the molecular mechanism of this induction is not known. Data by Fecteau et al suggest a transcriptional upregulation of p21WAF1/Cip1. Future studies need to analyze whether IKZF1 and 3 or another transcription factor that is directly or indirectly regulated by cereblon are involved in p21WAF1/Cip1 expression. IKZF1 and 3 are known to act as repressors or activators of gene expression in different settings. Of interest, the gene coding for p21WAF1/Cip1 contains a potential binding site for IKZF1 in the promoter region, and down-regulation of IKZF1 in acute lymphocytic leukemia cells, resulted in increased p21WAF1/Cip1 levels.8 Alternatively, a posttranslational regulation of p21WAF1/Cip1 by lenalidomide impacting on the stability or
1546
degradation of the protein is also conceivable. In this respect, it is of interest that many regulatory proteins of the cell cycle, including p21WAF1/Cip1, are targets of cullin 4–based E3 ubiquitin ligase complexes.9 With the advances in understanding the molecular mechanism of lenalidomide, several questions concerning its therapeutic activity in CLL arise. (1) Are the transcription factors IKZF1 and 3 involved in the antiproliferative effect of lenalidomide in CLL cells? (2) Are cereblon and the transcription factors also responsible for the activity within the CLL microenvironment? An IKZF1- and 3-mediated activation of T cells on lenalidomide treatment has been described. Here, the degradation of both transcription factors results in a derepression of the interleukin-2 gene.10 (3) Why does only a subset of CLL patients respond to lenalidomide treatment? A discrimination of responders and nonresponders might become possible by analyzing the expression and activity of cereblon and its target proteins. On this basis, the rational design of drug combinations including lenalidomide should be advanced. Within recent years, enormous progress in elucidating the pathomechanisms of CLL and in developing novel targeted compounds has been made. This knowledge now has to be translated into novel treatment protocols. By selecting therapeutics according to the molecular and genetic setup of every patient, we are getting closer to an individualized treatment of CLL with a maximal therapy success.
Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Fecteau J-F, Corral LG, Ghia EM, et al. Lenalidomide inhibits the proliferation of CLL cells via a cereblon/ p21WAF1/Cip1-dependent mechanism independent of functional p53. Blood. 2014;124(10):1637-1644. 2. Giannopoulos K, Mertens D, Stilgenbauer S. Treating chronic lymphocytic leukemia with thalidomide and lenalidomide. Expert Opin Pharmacother. 2011;12(18): 2857-2864. 3. Ramsay AG, Johnson AJ, Lee AM, et al. Chronic lymphocytic leukemia T cells show impaired immunological synapse formation that can be reversed with an immunomodulating drug. J Clin Invest. 2008;118(7): 2427-2437. 4. Schulz A, D¨urr C, Zenz T, et al. Lenalidomide reduces survival of chronic lymphocytic leukemia cells in primary cocultures by altering the myeloid microenvironment. Blood. 2013;121(13):2503-2511. 5. Ito T, Ando H, Suzuki T, et al. Identification of a primary target of thalidomide teratogenicity. Science. 2010;327(5971):1345-1350. 6. Kr¨onke J, Udeshi ND, Narla A, et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science. 2014;343(6168):301-305. 7. Lu G, Middleton RE, Sun H, et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science. 2014;343(6168):305-309. 8. Iacobucci I, Iraci N, Messina M, et al. IKAROS deletions dictate a unique gene expression signature in patients with adult B-cell acute lymphoblastic leukemia. PLoS ONE. 2012;7(7):e40934. 9. Abbas T, Sivaprasad U, Terai K, Amador V, Pagano M, Dutta A. PCNA-dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex. Genes Dev. 2008;22(18):2496-2506. 10. Gandhi AK, Kang J, Havens CG, et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.). Br J Haematol. 2014;164(6):811-821. © 2014 by The American Society of Hematology
l l l MYELOID NEOPLASIA
Comment on Brown et al, page 1655
Driving toward targeted therapy for LCH ----------------------------------------------------------------------------------------------------Robert A. Baiocchi
THE OHIO STATE UNIVERSITY
In this issue of Blood, Brown et al identify somatic mutations of MAP2K1 capable of driving the RAS-RAF-MEK-ERK pathway in Langerhans cell histiocytosis (LCH). Their findings lend important insight into the pathogenesis of this disease and provide the rationale for exploring targeted approaches in clinical trials.1
L
CH is a rare, often misdiagnosed histiocytic proliferative disorder afflicting pediatric and adult patients. The clonal (CD1a1, S1001,
and CD2071) proliferating cells have morphologic features most consistent with bone marrow–derived Langerhans cells that
BLOOD, 4 SEPTEMBER 2014 x VOLUME 124, NUMBER 10
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
The RAS-RAF-MEK-ERK pathway represents a driver in the pathogenesis of LCH while offering the potential for targeted therapeutic approaches. As much as 60% of LCH patients carry mutated forms of ARAF or BRAF (V600E) capable of constitutive phosphorylation of the downstream effectors MEK1 and MEK2 that ultimately drive ERK activity. Up to 50% BRAF-V600E–negative LCH cases (27% total) harbor a mutation in MAP2K1 that leads to constitutive MEK1 and consequent ERK activation, promoting downstream proliferation and survival networks to drive LCH. The mutually exclusive presence of BRAF and MAP2K1 mutations presents an ideal opportunity to intervene with selective agents like vemurafenib or trametinib to target 2 critical enzymes essential to LCH pathogenesis.
represent an antigen presenting dendritic cell commonly located in the skin and mucosa. The clinical course of LCH is diverse, ranging from solitary skin or bone lesions that may spontaneously resolve to widely disseminated, multisystem disease associated with high morbidity and mortality.2 The broad spectrum of disease activity, inflammatory nature of the lesions, normal karyotype, and associated immune dysregulation suggested that LCH represented an immunoreactive disorder. However, in 2010 Badalian-Very et al reported 57% of archived LCH lesions harbored a recurrent somatic, activating genetic mutation of the BRAF gene (BRAF-V600E).3 Furthermore, they found evidence of constitutive activation of the RAS-RAF-MAPK-ERK pathway regardless of whether BRAF was mutated or wild-type in LCH lesions. The fact that BRAF-V600E mutations were present as single alleles suggested that this might reflect a driver mutation for this disease. These data presented the first convincing genetic evidence supporting the characterization of LCH as a myeloid
neoplastic disorder and provided rationale for considering a targeted approach to treat patients with this disease. Several years later, Haroche et al4 reported impressive clinical responses in 3 patients with BRAF-V600E–mutated LCH treated with the single-agent BRAF inhibitor vemurafenib. Constitutive ERK activity in all LCH lesions led others to explore additional genetic drivers of ERK activity. Nelson et al5 used whole-exome sequencing of DNA isolated from purified LCH cells from 3 patients with wild-type BRAF. Interestingly, their studies documented the first somatic, activating mutations (F351L and Q347_A3438del) within the kinase-encoded domain of ARAF, leading to both ARAF and MEK constitutive kinase activity. Transfection of mouse fibroblast cells with the ARAF double-mutant clone led to acquired contact-independent growth in soft agar, a hallmark feature of cellular transformation, and further evidence that ERK activation may represent a driver of LCH growth. In this issue, Brown and colleagues1 provide additional convincing evidence that
BLOOD, 4 SEPTEMBER 2014 x VOLUME 124, NUMBER 10
links constitutive MAPK-ERK signaling to the pathogenesis of LCH. They used targeted next-generation sequencing to evaluate 8 LCH cases and found BRAF-V600E in 3 cases, consistent with prior work, and an E102_I103del mutation in the MAP2K1 gene that encodes for the MEK1 protein kinase, an enzyme directly upstream of extracellular signal-regulated kinases ERK1/2. Interestingly, this MAP2K1 mutation occurred in an LCH case that was BRAF wild-type, which then led the investigators to examine an additional 32 cases by BRAF-V600E allele–specific polymerase chain reaction and Sanger sequencing of exons 2 and 3 of MAP2K1. Surprisingly, 11 of 40 (27%) cases showed somatic MAP2K1 mutations that occurred mutually exclusive to BRAF mutations, with 50% of wild-type BRAF cases showing MAP2K1 mutation. Most mutations identified in this study were deletions within exons 2 and 3 and were previously shown to encode for markedly enhanced MEK1 kinase activity (see Figure 1 in Brown et al).6 Collectively, these results, when taken in the context of studies led by Badalian-Very et al, suggest that the majority of LCH patients harbor a somatic, activating mutation in critical signaling elements of the RAS-RAF-MEK-ERK pathway (see figure). Do activating mutations of components of the RAS-RAF-MEK-ERK pathway represent true drivers of LCH pathogenesis? Recent work by Berres et al7 examines the biological relevance of recurrent BRAF-V600E mutations in the pathogenesis of LCH. Sixty-four lesions from 100 LCH patients showed the presence of the BRAF-V600E mutation, an event that was linked to increased risk of relapse. In patients with high-risk disease, the BRAF-V600E mutation could be detected in bone marrow CD341 hematopoietic progenitor cells, whereas patients with low-risk LCH showed presence of the mutation restricted to mature CD2071 dendritic cells (DCs) within primary lesions. The authors went on to develop a novel in vivo mouse model, allowing for conditional expression of BRAF-V600E in various subsets of DCs. Mice with conditional expression of BRAF-V600E in DC progenitors (under CD11c promoter) rapidly developed an aggressive, multisystem LCH-like disease. Interestingly, granulomatous lesions in these mice showed inflammatory infiltrates with accumulation of regulatory T cells and abundant expression of
1547
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
inflammatory cytokines that are observed in the lesions of humans with aggressive LCH. In stark contrast, mice with conditional expression of BRAF-V600E in mature DCs (under langerin promoter) developed a low-grade, localized LCH-like disease. By definition, driver mutations contribute toward the initiation of cellular transformation and the progression of malignant disease, 2 features clearly illustrated in this interesting preclinical model of LCH. Elegant models such as this will certainly improve our understanding of the complex pathogenesis of LCH and provide a useful setting to test novel targeted therapies exploiting this pathway. Additional studies supporting the driver nature of these mutated signaling components have illustrated the capacity of activating ARAF mutations to drive fibroblast transformation,5 MAP2K1 mutations driving constitutive ERK phosphorylation in melanomas6 and hairy cell leukemia,8 and MAP kinase activity to drive transformation of mammalian cells.9 Perhaps the most intriguing implications reported by Brown et al1 and others3,5 lie within the potential for pursuing targeted therapeutic strategies. Current up-front therapeutic modalities to manage patients with multisystem LCH have traditionally used a risk-stratified approach (single vs multisystem LCH), often using intensive cytotoxic chemotherapy.10 Patients with relapsed or refractory disease have benefitted from single-agent modalities delivering cytarabine or cladribine. Although these regimens have generally improved outcome, toxicity remains a challenge and patients with high-risk, multisystem disease still face a high mortality rate. The identification of activating mutations in A/BRAF and MAP2K1, genes encoding 2 critical signaling enzymes in the ERK pathway, allows for strategic approaches using novel targeted agents. Promising Food and Drug Administration–approved, selective agents like vemurafenib and trametinib to target mutant A/BRAF and MAP2K1/2, respectively, provide an ideal opportunity to develop well-designed, multicenter, genetically stratified (using targeted sequencing approaches) clinical trials. The collective findings reported by Brown et al1 and others3,5 identifying activating somatic mutations in the majority of LCH patients have shed light on the complex pathogenesis of LCH while offering patients with LCH hope for improved treatment strategies in the near future.
1548
Conflict-of-interest disclosure: The author declares no competing financial interests. n REFERENCES 1. Brown NA, Furtado LV, Betz BL, et al. High prevalence of somatic MAP2K1 mutations in BRAF V600E negative Langerhans cell histiocytosis. Blood. 2014;124(10): 1655-1658. 2. Delprat C, Aric`o M. Blood spotlight on Langerhans cell histiocytosis. Blood. 2014;124(6):867-872. 3. Badalian-Very G, Vergilio JA, Degar BA, et al. Recurrent BRAF mutations in Langerhans cell histiocytosis. Blood. 2010;116(11):1919-1923. 4. Haroche J, Cohen-Aubart F, Emile JF, et al. Dramatic efficacy of vemurafenib in both multisystemic and refractory Erdheim-Chester disease and Langerhans cell histiocytosis harboring the BRAF V600E mutation. Blood. 2013;121(9):1495-1500. 5. Nelson DS, Quispel W, Badalian-Very G, et al. Somatic activating ARAF mutations in Langerhans cell histiocytosis. Blood. 2014;123(20):3152-3155.
6. Nikolaev SI, Rimoldi D, Iseli C, et al. Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma. Nat Genet. 2012;44(2): 133-139. 7. Berres ML, Lim KP, Peters T, et al. BRAF-V600E expression in precursor versus differentiated dendritic cells defines clinically distinct LCH risk groups. J Exp Med. 2014;211(4):669-683. 8. Waterfall JJ, Arons E, Walker RL, et al. High prevalence of MAP2K1 mutations in variant and IGHV434-expressing hairy-cell leukemias. Nat Genet. 2014; 46(1):8-10. 9. Mansour SJ, Matten WT, Hermann AS, et al. Transformation of mammalian cells by constitutively active MAP kinase kinase. Science. 1994;265(5174):966-970. 10. Gadner H, Minkov M, Grois N, et al; Histiocyte Society. Therapy prolongation improves outcome in multisystem Langerhans cell histiocytosis. Blood. 2013; 121(25):5006-5014. © 2014 by The American Society of Hematology
l l l THROMBOSIS & HEMOSTASIS
Comment on Sherman et al, page 1659
Taking a leaf from the book of oral tolerance ----------------------------------------------------------------------------------------------------David W. Scott
UNIFORMED SERVICES UNIVERSITY OF THE HEALTH SCIENCES
In this issue of Blood, Sherman et al demonstrate that mucosal delivery of engineered tobacco leaves can lead to tolerance to factor VIII (FVIII) inhibitor production. Eat your veggies for tolerance!1
Proposed clinical application: FVIII domains expressed in transgenic plants can be ingested and processed by the mucosal immune system to induce tolerance to FVIII.
BLOOD, 4 SEPTEMBER 2014 x VOLUME 124, NUMBER 10
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
2014 124: 1546-1548 doi:10.1182/blood-2014-07-587378
Driving toward targeted therapy for LCH Robert A. Baiocchi
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