MYD88 and Beyond: Novel Opportunities for Diagnosis, Prognosis and Treatment in Waldenstro¨M’S Macroglobulinemia

CONCISE REVIEW MYD88 and beyond: novel opportunities for diagnosis, prognosis and treatment in Waldenstro¨m’s Macroglobulinemia

O Landgren and N Tageja

Waldenstro¨m’s Macroglobulinemia (WM) is a rare disease of the elderly with a median age of 63–68 years at diagnosis. Despite recent progress in biological insights and therapeutics, WM remains clinically challenging to diagnose and is difficult to manage with significant morbidity and lack of established curative therapies. Recently, the use of whole-genome sequencing has helped to identify a highly recurrent somatic mutation, myeloid differentiation factor 88 [MYD88] L265P in WM. This has fueled major interest in the field and as newer evidence accumulates, it is clear that that discovery of MYD88 L265P mutation may represent an important breakthrough in understanding the pathogenesis of WM and lymphoproliferative disorders. Recent scientific work in this field has also guided the identification of new targets such as CXCR4 and PI3K-delta that may have major implications in the future treatment of WM. This review discusses the role of MYD88 L265P mutations as well as targets beyond MYD88 in the setting of pathogenesis and development of future rational therapeutic trials focusing on patients diagnosed with WM.

Leukemia (2014) 28, 1799–1803; doi:10.1038/leu.2014.88

INTRODUCTION transduces signals to the NF-kB transcription factors in response to Waldenstro¨m’s Macroglobulinemia (WM) is a rare hematological IL-1R1 signaling. MYD88 has a modular structure with a Toll/IL-1R malignancy with a reported age-adjusted incidence rate of 3.4 per (TIR) domain at its COOH terminus and a death domain at its 12 11 million among men and 1.7 per million among women in the NH2 terminus. Wesche et al. and Muzio et al. used United States of America that increases geometrically with age.1 coimmunoprecipitation experiments to show that the TIR Clinically and pathologically, WM represents an intermediate stage domain of MYD88 interacts with the TIR domains of IL-1R1 and between lymphoproliferative disorders and plasma cell dyscrasias IL-1RAP. MYD88 binds the serine–threonine kinases IRAK1 and with characteristics that may lie anywhere between the two IRAK2, mammalian homologs of Drosophila Pelle in the Toll spectrums.2 While the molecular pathogenesis of WM is not fully pathway, via a heterotypic death domain-mediated interaction understood,2,3 the crucial role of nuclear factor-kappa B (NF-kB) and thus acts as a pure adaptor linking the IL-1R1 to downstream 13 activation in tumorigenesis has been reported for at least half a IRAK kinases. Subsequent work by Adachi et al. found MYD88 to decade.4 More recently, whole-genome sequencing (WGS) in WM be essential for induction of NF-kB and MAPK signaling in allowed the identification of a somatic variant (T-C) at position response to IL-18, a cytokine structurally related to IL-1beta. 38182641 in chromosome 3p22.2 that results in an amino-acid Deficiency of MYD88 in mice causes defects in T-cell proliferation change from leucine to proline (L265P) in the MYD88 gene.5 as well as induction of acute phase proteins and cytokines in 13 Myeloid differentiation factor 88 (MYD88) is a crucial adaptor response to IL-1. Increases in interferon-gamma production and protein in the interleukin (IL)-1, IL-18 and Toll-like receptors (TLRs) natural killer cell activity in response to IL-18 are also abrogated. signaling pathway that directs the assemblage of a multisubunit Absence of MYD88 in humans causes life-threatening pyogenic 14 15 signaling complex that includes IL-1R-associated kinase (IRAK) 1 bacterial infections. Finally, Medzhitov et al. using over- 16 and IRAK4, leading up to the activation of Janus kinase–signal expression studies and Kawai et al. using MYD88-deficient transducer and activator of transcription (JAK–STAT) 3 signaling mice showed that MYD88 is critical for TLR signaling. Taken and NF-kB pathways.6,7 This paper summarizes recent evidence together, these studies defined MYD88 as a crucial adaptor for that has provided useful insights into the role of MYD88 and its signaling through the IL-1/IL-18/Toll receptor superfamily. We now alterations in the pathogenesis of WM and the potential know that following stimulation by IL-1R or TLR, MYD88 is applicability in diagnosis, prognosis, therapeutics as well as recruited to the activated receptor complex as a homodimer that 17,18 response assessment. It also addresses recent discovery work complexes with IRAK 4 to activate IRAK 1. This activates the showing a role for CXC chemokine receptor 4 (CXCR4) and PI3K- tumor necrosis factor-associated factor 6 leading to NF-kB 17,18 delta as potential new therapeutic targets in WM.8,9 activation via IkB-alpha phosphorylation (Figure 1).

THE MYD88 PATHWAY AND FUNCTIONAL ROLE MYD88 IN LYMPHOMA, WM AND IGM MGUS MYD88 was first identified as a myeloid differentiation primary Ngo et al.19 implicated TLR signaling deregulation in lympho- response gene in 1990.10 Subsequent work by Muzio et al.11 and magenesis by discovering MYD88 mutations in 39% of cases with Wesche et al.12 identified MYD88 as a proximal adaptor of the activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). IL-1R signaling pathway and went on to show that MYD88 Using high-throughput mRNA resequencing of ABC cell lines

Multiple Myeloma Section, Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. Correspondence: Dr O Landgren, Multiple Myeloma Section, Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 10/Room 13N240, Bethesda 20892, MD, USA. E-mail: [email protected] Received 16 December 2013; revised 13 January 2014; accepted 15 January 2014; accpted article preview online 27 February 2014; advance online publication, 16 May 2014 MYD88 and beyond: opportunities for WM O Landgren and N Tageja 1800 use of inhibitors that block MYD88 and IRAK activity, Yang et al.21 observed that MYD88 signaling was supportive of WM growth and survival. Overexpression of MYD88 L265P promoted enhanced survival of WM cells versus WT MYD88, consistent with a gain-of- function mutation as reported by Ngo et al.19 in ABC DLBCL cells. MYD88 was preferentially complexed to phosphorylated BTK in both L265P-expressing WM cell lines, whereas little complexing was observed in MYD88 WT cells. Importantly, knockdown of MYD88 or the use of a MYD88 inhibitor abrogated BTK activity in L265P-expressing cell lines, whereas overexpression of MYD88 L265P showed more robust BTK activity. The use of ibrutinib, an inhibitor of BTK kinase activity, resulted in decreased MYD88-BTK complexing in MYD88 L265P-expressing cells.21 These studies by Ngo et al.19 and Yang et al.21 show that IRAK and BTK independently direct downstream NF-kB activation, and combined use of IRAK and BTK inhibitors leads to synergistic tumor cell killing in MYD88 L265P-expressing WM cells. These studies would suggest a model wherein MYD88 L265P triggers Figure 1. Stimulation of Toll-like receptor 4 facilitates the activation NF-kB via dual but independent pathways which signal through BTK and/or IRAK1/IRAK4. of two pathways: the MYD88 (myeloid differentiation primary- 5 response protein 88)-dependent and MYD88-independent Translating the preclinical work to bedside, Treon et al. pathways. The MYD88-dependent pathway involves the early phase conducted an important study where they reported MYD88 of nuclear factor-B (NF-B) activation, which leads to the production L265P to be a commonly recurring mutation in patients with WM. of inflammatory cytokines. The MYD88-independent pathway Forty nine of 54 patients (91%) with WM and 3 of 3 patients activates interferon (IFN)-regulatory factor (IRF3) and involves the (100%) with non–IgM-secreting LPL had MYD88 L265P expres- late phase of NF-B activation, both of which lead to the production sion by whole-genome sequencing, and confirmed on Sanger of IFN and the expression of IFN-inducible genes. ask1, apoptosis sequencing. This high frequency of the MYD88 L265P somatic signal-regulating kinase 1; ERK, extracellular signal-regulated kinase; Fks, glucan synthase; IKK, inhibitor of k-B kinase; IRAK, interleukin-1 mutation in patients with WM and IgM MGUS has been receptor-associated kinase; IRF, interferon regulatory transcription confirmed with subsequent studies using Sanger, polymerase factor; JNK, c-Jun N-terminal kinase; MEKK3, mitogen- chain reaction (PCR), and allele-specific PCR (AS-PCR) assays activated protein kinase kinase kinase 3; NF-kB, nuclear factor-kB; (Table 1).22–27 TLR, Toll-like receptor; Tpl2, tumor progression Locus 2; TRAF, TNF Whereas the knowledge on this subject is constantly evolving, receptor-associated factor; TRIF, TIR-domain-containing adapter- the present evidence points to MYD88 L265P as an early inducing interferon-b. oncogenic event for the development of WM. Our group used Sanger sequencing to assess the status of MYD88 L265P expression in nine patients with IgM MGUS.25 These patients and sequence analyses of MYD88-coding region in 382 biopsied were extensively worked up with immunohistochemical analyses, tumor samples, a single amino-acid substitution, L265P, was flow cytometry, molecular (immunoglobulin gene rearrangement) observed in 29% of the biopsies. This mutation was lacking in analysis, imaging and laboratory and clinical examinations to rule other subtypes of DLBCL. Subsequent copy number variation out WM and confirm the diagnosis of IgM MGUS. MYD88 L265P analysis implied that the tumor cells might be preferentially expression was found in five of them (56%);25 all five patients had selecting this allele, indicating the biological potency of the L265P clonal plasma cells and clonal lymphocytes in bone marrow mutant. An RNA interference screen followed by high-throughput (lymphoplasmacytic precursor neoplasm). In contrast, the four resequencing and comparative genomic hybridization were patients negative for MYD88 L265P expression had either clonal employed to authenticate these findings. During IL-1 signaling, plasma cells or clonal lymphocytes in bone marrow (plasma cell or IRAK1 became hyperphosphorylated by IRAK4, resulting in slowly lymphoid precursor neoplasm), suggesting that the cases of migrating IRAK1 isoforms. In cells bearing the MYD88 L265P, a MYD88 L265P IgM MGUS may represent a precursor to WM rather prominent, slow-migrating IRAK1 species co-immunoprecipitated that a transformation from IgM-MGUS to WM. These findings were with MYD88. By contrast, wild-type MYD88 did not associate subsequently confirmed by the Treon group.23 Varettoni et al.24 strongly with these IRAK1 isoforms nor did the other MYD88 performed a retrospective case–control study in IgM MGUS mutants tested in the study. Phosphorylation of endogenous patients and found that MYD88 L265P in patients with IgM MGUS IRAK1 was observed in an ABC DLBCL line with L265P but not significantly increases the risk of malignant evolution. Whereas in a GCB DLBCL line. The ABC cell-line survival was jeopardized these results need to be validated prospectively, the authors’ by IRAK4 knockdown, and introducing wild type but not series of six MYD88 L265P IgM MGUS patients who progressed to kinase-inactivated IRAK4 reversed this suggesting tumor WM certainly had higher levels of IgM, lower levels of IgGand IgA dependence on the kinase activity of IRAK4. Moreover, an and a higher incidence of Bence–Jones proteinuria at diagnosis, inhibitor of the kinase activity of IRAK1 and IRAK4 killed suggesting MYD88 (L265P) as a genetic event associated with ABC cell lines, but spared GC or myeloma cell lines. These disease progression. An important aspect to consider when observations may be seen as a potential promise for the interpreting results from studies focusing on IgM MGUS is the pharmacological inhibition of IRAK4 in lymphomas bearing fact that the current definition of IgM MGUS does not take oncogenic MYD88 mutations or other mutations that into account whether the infiltrating tumor cells are of plasma enhanceTLR–NFkB signaling.19,20 The decreases in STAT3 cell, lymphoid or mixed cellular origin. The cellular origin has signaling noted after MYD88 knockout also pointed to increased not been described in full detail in published studies. Thus, JAK–STAT3 signaling and interferon-b production as important biological heterogeneity may account for the observed variances downstream effects of MYD88 L265P, which could serve as in the rates of MYD88 L265P detection across prior investigations. potential targets in future. Furthermore, the discovery of MYD88 L265P mutation in Along the same lines, WM cell survival is enhanced by MYD88 individuals with IgM MGUS may also reflect differences in L265P overexpression.21 By lentiviral knockdown of MYD88 and/or technology and primers used.

Leukemia (2014) 1799 – 1803 & 2014 Macmillan Publishers Limited MYD88 and beyond: opportunities for WM O Landgren and N Tageja 1801 Table 1. MYD88 L265P expression in WM and IgM MGUS

Authors Reference Tissue Method MYD88 L265P positive/total MYD88 L265P positive/total number of WM patients tested, number of IgM MGUS patients n/N (%) tested, n/N (%)

Treon et al. 5 BM CD19 þ WGS/Sanger 49/54 (91) 2/21 (10) Gachard et al. 22 BM PCR 21/31 (67) ND Xu et al. 23 BM CD19 þ AS-PCR 97/104 (93) 13/24 (54) Varettoni et al. 24 BM AS-PCR 58/58 (100) 36/77 (47) Landgren and Staudt 25 BM Sanger 5/9 (56) Jimenez et al. 26 BM AS-PCR 100/117 (86) 27/31 (87) Ansell et al. 27 NA WGS/Sanger/AS-PCR 38/39 (97) ND Poulain et al. 37 BM CD19 þ PCR 54/67 (80) ND Abbreviations: AS-PCR, allele-speciﬁc PCR; BM, bone marrow; MYD88, myeloid differentiation factor 88; not applicable; ND, not determined; PCR, polymerase chain reaction; WGS, whole genome sequencing; WM, Waldenstro¨ m’s Macroglobulinemia.

As suggested before,24,28 a few hypotheses could seem evident: MYD88 AS A THERAPEUTIC TOOL (1) The MYD88 (L265P) mutation is a critical component of disease Preclinical work has shown that knockout of MYD88 pathogenesis and may be a universal progression event. The and/or downstream targets such as BTK, IRAK 1 and IRAK4 can interpretation of MYD88-negative IgM-MGUS patients implies a suppress NF-kB signaling and thus induce WM cell killing.21,27 clinical challenge and may be partially attributable to the present Ibrutinib, a BTK kinase inhibitor, has shown promising activity in detection methods that are unable to detect the mutation in the WM in a phase 1 study34 (responses seen in three of the four presence of very small B-cell clones. While the use of AS-PCR with included WM patients) as well as in an ongoing phase 2 clinical CD19 þ sorting may increase the diagnostic yield, the process is trial.31 As discussed previously, Yang et al.21 had shown that cumbersome and will not be broadly applicable. (2) There is ibrutinib-mediated inhibition of BTK activity did not affect heterogeneity in the biological behavior of IgM MGUS with activation state or protein levels of IRAK 1 and vice versa, multiple possible subtypes: it is possible that only a subset of IgM supporting that MYD88 signaling through these pathways is MGUS patients have MYD88 mutation as founder mutation and likely independent of each other. Supporting this hypothesis, the are thus at a higher risk of progression from onset. Alternatively, combined use of IRAK and BTK inhibitors indeed resulted in patients may have other, still unrecognized, founding mutations augmented inhibition of NF-kB signaling and more robust WM cell that drive the initial pathogenesis of disease with MYD88 L265P as killing. The development of small molecule MYD88 inhibitors a second hit that accelerates the malignant clones leading to could act proximally to block both BTK and IRAK signaling smoldering WM or full-blown WM. While the search for these pathways. On the other hand, the use of a combination of BTK and founding mutations continues, researchers at Dana-Farber Cancer IRAK inhibitors could be explored in patients with WM patients. Institute have used paired normal/WM lymphoplasmacytic cell- The detection of MYD88 L265P could help to identify those paired tissues and WGS to identify somatic mutations in the patients who are more suitable for treatments targeting MYD88 29 CXCR4 gene in 16/55 (29%) WM patients. They have also found L265P signaling. highly recurrent copy number alternations in WM that include genes with critical regulatory roles in lymphocytic growth and survival signaling.30 Mutations in genes related to NF-kB have also been reported; however, they are relatively uncommon and MYD88 FOR RESPONSE ASSESSMENT deletions of the long arm of chromosome 6 are seen in only half of The potential of using real-time AS-PCR for MYD88 L265P in the cases.31 The multiple hit hypothesis32 could explain why response assessment for patients with WM was explored by Xu 23 26 26 Jimenez et al.26 were unable to demonstrate any differences in et al. and Jimenez et al. Jimenez et al. evaluated six patients time to first therapy, response to treatment or progression-free or who responded very well to therapy (490% M-component overall survival based on the MYD88 mutational status in patients reduction) and found that AS-PCR persisted in five of them with 23 with WM and IgM MGUS. comparable results to those yielded by flow cytometry. Xu et al. found a strong correlation between the percentage changes of BM involvement and levels of MYD88 L265P (r ¼ 0.89, P ¼ 0.008) MYD88 AS A DIAGNOSTIC TOOL that was independent of treatment received. In one case, MYD88 mutational status may help to discriminate WM/LPL from undetectable MYD88 L265P levels accompanied the attainment marginal zone lymphomas, IgM-secreting myeloma or chronic of complete response to therapy. While these results would need lymphocytic leukemia with plasmacytic differentiation that have to be validated in larger prospective series using multiple overlapping clinicopathologic features presenting a diagnostic treatment regimens, one could envision that future studies dilemma.25,33 Jimenez et al.26 found that none of the non-IgM would explore the role of measuring the quantitative burden of MGUS or lymphoplasmacytic lymphomas harbored the MYD88 MYD88 L265P at baseline as a prognostic marker for the mutation, nor did the chronic lymphocytic leukemia or multiple attainment of deep molecular remissions as well as correlation myeloma cases, including those with an IgM monoclonal with progression-free and overall survival. The Treon group at component. By contrast, three of 14 splenic marginal zone DFCI recently presented results of an elegant study that examined lymphomas (all with an IgM monoclonal component and BM the feasibility of testing MYD88 in the peripheral blood using real- infiltration, but without a clear lymphoplasmacytic pattern) and time AS-PCR.35 Simultaneously analyzed PB and BM samples from nine of 48 ABC DLBCLs (non-GC type) displayed the mutation, 65 untreated WM patients demonstrated positivity for MYD88 suggesting that MYD88 should not be considered exclusively L265P in 58/65 (89.2%) and 55/65 (84.6%) of CD19-selected BM diagnostic of WM/LPL. In further support of these findings above, and PB samples, respectively. The detection of MYD88 L265P by all of our IgM MGUS patients with an IgM spike and MYD88 L265P CD19-selected PB AS-PCR examination will provide a convenient expression in malignant cells had both clonal B cells and plasma and suitable alternative to repeated bone marrow biopsies in cells in the BM.25 guiding the treatment of patients with WM.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1799 – 1803 MYD88 and beyond: opportunities for WM O Landgren and N Tageja 1802 TARGETS BEYOND MYD88 AUTHOR CONTRIBUTIONS Scientific work published by Ngo et al.36 in 2008 showed that WM NT and OL equally contributed towards writing the manuscript. Both authors cells express high levels of chemokine and adhesion receptors, approved the final version of the manuscript. including CXCR4 and VLA-4. The CXCR4/SDF-1 axis interacts with VLA-4 in regulating migration and adhesion of WM cells in the bone marrow microenvironment.36 More recently, REFERENCES researchers at DFCI led by Dr Hunter used paired normal/WM 1 Groves FD, Travis LB, Devesa SS, Ries LA, Fraumeni Jr JF. Waldenstrom’s lymphoplasmacytic cell-paired tissues and WGS to identify macroglobulinemia: incidence patterns in the United States 1988-1994. Cancer somatic mutations in the CXCR4 gene that were present in 1998; 82: 1078–1081. 16/55 (29%) WM patients.29 In results presented at the 2013 2 Braggio E, Philipsborn C, Novak A, Hodge L, Ansell S, Fonseca R. Molecular American Society of Hematology meeting, the DFCI group found pathogenesis of Waldenstrom’s macroglobulinemia. Haematologica 2012; 97: that CXCR4 mutations are associated with resistance to ibrutinib- 1281–1290. 3 Buske C, Leblond V. How to manage Waldenstrom’s macroglobulinemia. Leukemia mediated ERK1/2 and AKT signaling, as well as growth 2013; 27: 762–772. suppression in the presence of the CXCR4 ligand, CXCL12, in 4 Leleu X, Eeckhoute J, Jia X, Roccaro AM, Moreau AS, Farag M et al. Targeting 8 WM cells. In addition to its potential use in predicting outcome NF-kappaB in Waldenstrom macroglobulinemia. Blood 2008; 111: 5068–5077. for patients undergoing ibrutinib therapy, based on data 5 Treon SP, Xu L, Yang G, Zhou Y, Liu X, Cao Y et al. MYD88 L265P somatic mutation obtained from WM cell lines, selective targeting of CXCR4 may in Waldenstrom’s macroglobulinemia. N Engl J Med 2012; 367: 826–833. be a strategy to overcome resistance to BTK inhibitors.8 Future 6 Kawai T, Akira S. Toll-like receptor and RIG-I-like receptor signaling. Ann N Y Acad clinical trials are needed to explore these mechanisms in patients Sci 2008; 1143: 1–20. diagnosed with WM. 7 Warner N, Nunez G. MyD88: a critical adaptor protein in innate immunity signal Researchers are also investigating the downstream signaling transduction. J Immunol 2013; 190: 3–4. associated with MYD88 L265P in WM cells to identify new 8 Cao Y, Hunter ZR, Liu X, Yang G, Tripsas CK, Manning R et al. Somatic 9 Activating Mutations In CXCR4 Are Common In Patients With Waldenstrom’s therapeutic targets. It has recently been shown that in addition Macroglobulinemia, and Their Expression In WM Cells Promotes Resistance To to activation of NF-kB through IRAK and BTK signaling, Ibrutinib Blood (ASH Annual Meeting Abstracts), December 2013; 4424 2013. MYD88 L265P promotes survival of WM cells by activation of 9 Yang G, Xu L, Zhou Y, Xu L, Cao Y, Manning R et al. PI3K/AKT Pathway Is Activated PI3K/AKT signaling. Inhibition of PI3K-delta with CAL-101 and By MYD88 L265P and Use Of PI3K-Delta Inhibitors Induces Robust Tumor Cell PIK-294 was associated with robust killing of WM cells and Killing In Waldenstrom’s Macroglobulinemia Blood (ASH Annual Meeting provide the preclinical rationale for studying PI3K-delta Abstracts), December 2013; 4255. inhibitors in WM.9 10 Lord KA, Hoffman-Liebermann B, Liebermann DA. Nucleotide sequence and expression of a cDNA encoding MyD88, a novel myeloid differentiation primary response gene induced by IL6. Oncogene 1990; 5: 1095–1097. 11 Muzio M, Ni J, Feng P, Dixit VM. IRAK (Pelle) family member IRAK-2 and MyD88 as SUMMARY AND FUTURE DIRECTIONS proximal mediators of IL-1 signaling. Science 1997; 278: 1612–1615. 12 Wesche H, Henzel WJ, Shillinglaw W, Li S, Cao Z. MyD88: an adapter that recruits The discovery of MYD88 L265P in WM presents multiple exciting IRAK to the IL-1 receptor complex. Immunity 1997; 7: 837–847. avenues with the impact on clinical management as well as 13 Adachi O, Kawai T, Takeda K, Matsumoto M, Tsutsui H, Sakagami M et al. Targeted future research. In addition to allowing an accurate diagnosis of disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. WM and exclusion of other lymphoproliferative disorders with Immunity 1998; 9: 143–150. overlapping features, the MYD88 L265P mutation in IgM-MGUS 14 von Bernuth H, Picard C, Jin Z, Pankla R, Xiao H, Ku CL et al. Pyogenic bacterial has been associated with higher risk of progression and may infections in humans with MyD88 deficiency. Science 2008; 321: 691–696. therefore prove useful as a prognostic marker. Carefully 15 Medzhitov R, Preston-Hurlburt P, Janeway Jr CA. A human homologue of the conducted studies published over the past year also provide Drosophila Toll protein signals activation of adaptive immunity. Nature 1997; 388: 394–397. the framework for the investigation of inhibitors targeting 16 Kawai T, Adachi O, Ogawa T, Takeda K, Akira S. Unresponsiveness of MyD88- MYD88 and its downstream pathways for the treatment deficient mice to endotoxin. Immunity 1999; 11: 115–122. of WM and/or other lymphoproliferative disorders harboring 17 Suzuki N, Chen NJ, Millar DG, Suzuki S, Horacek T, Hara H et al. IL-1 receptor- this mutation. Quantitative assessment of the mutation associated kinase 4 is essential for IL-18-mediated NK and Th1 cell responses. in bone marrow and/or peripheral blood may be an opportunity, J Immunol 2003; 170: 4031–4035. if successful, to provide a sensitive and inexpensive method 18 Suzuki N, Suzuki S, Duncan GS, Millar DG, Wada T, Mirtsos C et al. Severe to assess minimal residual disease in WM post treatment. impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK- Recent discovery work supports a role for CXCR4 and PI3K-delta 4. Nature 2002; 416: 750–756. as therapeutic targets in WM (8.9). Interestingly, preclinical 19 Ngo VN, Young RM, Schmitz R, Jhavar S, Xiao W, Lim KH et al. Oncogenically active investigations show that WM cell lines harboring CXCR4 MYD88 mutations in human lymphoma. Nature 2011; 470: 115–119. 20 Jeelall YS, Horikawa K. Oncogenic MYD88 mutation drives Toll pathway to mutations were resistant to BTK inhibition and the addition lymphoma. Immunol Cell Biol 2011; 89: 659–660. of AMD3100 (Plerixafor, trade name Mozobil) was able to 21 Yang G, Zhou Y, Liu X, Xu L, Cao Y, Manning RJ et al. A mutation in MYD88 8 overcome the resistance. Future studies are needed to expand (L265P) supports the survival of lymphoplasmacytic cells by activation of our knowledge on pathogenesis of WM and to develop Bruton tyrosine kinase in Waldenstrom macroglobulinemia. Blood 2013; 122: translational clinical trials for WM patients based on these 1222–1232. insights. 22 Gachard N, Parrens M, Soubeyran I, Petit B, Marfak A, Rizzo D et al. IGHV gene features and MYD88 L265P mutation separate the three marginal zone lymphoma entities and Waldenstrom macroglobulinemia/lymphoplasmacytic lymphomas. Leukemia 2013; 27: 183–189. CONFLICT OF INTEREST 23 Xu L, Hunter ZR, Yang G, Zhou Y, Cao Y, Liu X et al. MYD88 L265P in Waldenstrom macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other The authors declare no conflict of interest. B-cell lymphoproliferative disorders using conventional and quantitative allele- specific polymerase chain reaction. Blood 2013; 121: 2051–2058. 24 Varettoni M, Arcaini L, Zibellini S, Boveri E, Rattotti S, Riboni R et al. Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenstrom’s ACKNOWLEDGEMENTS macroglobulinemia and related lymphoid neoplasms. Blood 2013; 121: 2522–2528. The Intramural Research Program of the National Cancer Institute of the National 25 Landgren O, Staudt L. MYD88 L265P somatic mutation in IgM MGUS. N Engl J Med Institutes of Health supported this work. 2012; 367: 2255–2256; author reply 6-7.

Leukemia (2014) 1799 – 1803 & 2014 Macmillan Publishers Limited MYD88 and beyond: opportunities for WM O Landgren and N Tageja 1803 26 Jimenez C, Sebastian E, Chillon MC, Giraldo P, Mariano Hernandez J, Escalante F 32 Treon SP, Hunter ZR. A new era for Waldenstrom macroglobulinemia: MYD88 et al. MYD88 L265P is a marker highly characteristic of, but not restricted to, L265P. Blood 2013; 121: 4434–4436. Waldenstrom’s macroglobulinemia. Leukemia 2013; 27: 1722–1728. 33 Pangalis GA, Kyrtsonis MC, Kontopidou FN, Vassilakopoulos TP, Siakantaris MP, 27 Ansell SM HL, Secreto F, Manske M, Braggio E, Price-Troska T et al. Activation Dimopoulou MN et al. Differential diagnosis of Waldenstrom’s macroglobulinemia Of TAK1 By MYD88 L265P Drives Malignant B Cell Growth In Non-Hodgkin from other low-grade B-cell lymphoproliferative disorders. Semin Oncol 2003; 30: Lymphoma Blood (ASH Annual Meeting Abstracts) 2013; 120: Abstract 245. 201–205. 28 Fonseca R, Braggio E. The MYDas touch of next-gen sequencing. Blood 2013; 121: 34 Advani RH, Buggy JJ, Sharman JP, Smith SM, Boyd TE, Grant B et al. Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant activity 2373–2374. in patients with relapsed/refractory B-cell malignancies. J Clin Oncol 2013; 31: 29 Cao Y, Xu L, Liu X, Zhou Y, Yang G, Patterson CJ et al. Whole Genome Sequencing 88–94. Identifies Recurring Somatic Mutations in the C-Terminal Domain of CXCR4, 35 Xu L, Hunter Z, Yang G, Zhou Y, Liu X, Cao Y et al. Detection Of MYD88 L265P In Including a Gain of Function Mutation in Waldenstrom’s Macroglobinemia Blood Peripheral Blood Of Patients With Waldenstro¨m’s Macroglobulinemia and IgM (ASH Annual Meeting Abstracts) 2012; 120: Abstract 27152012. Monoclonal Gammopathy Of Undetermined Significance Blood (ASH Annual 30 Hunter Z, Xu L, Yang G, Zhou Y, Liu X, Cao Y et al. The genomic landscape of Meeting Abstracts), December 2013; 3024 2013. Waldenstom’s Macroglobulinemia is characterized by highly recurring MYD88 36 Ngo HT, Leleu X, Lee J, Jia X, Melhem M, Runnels J et al. SDF-1/CXCR4 and VLA-4 and WHIM-like CXCR4 mutations, and small somatic deletions associated with interaction regulates homing in Waldenstrom macroglobulinemia. Blood 2008; B-cell lymphomagenesis. Blood 2014; 123: 1637–1646. 112: 150–158. 31 Schop RF, Kuehl WM, Van Wier SA, Ahmann GJ, Price-Troska T, Bailey RJ et al. 37 Poulain S, Roumier C, Decambron A, Renneville A, Herbaux C, Bertrand E et al. Waldenstrom macroglobulinemia neoplastic cells lack immunoglobulin heavy chain MYD88 L265P mutation in Waldenstrom macroglobulinemia. Blood 2013; 121: locus translocations but have frequent 6q deletions. Blood 2002; 100: 2996–3001. 4504–4511.

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