Oncogene (2013) 32, 3111–3118 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc

REVIEW Role of the adaptor LNK in normal and malignant hematopoiesis

S Gery1 and HP Koeffler1,2

The signal transduction pathways, orchestrating the differentiation of hematopoietic stem and progenitor cells in response to stimulation, are strictly controlled by networks of feedback loops, highly selective protein interactions and finely tuned on/off switches. In hematological malignancies, the aberrant activation of signaling pathways is usually associated with mutations in tyrosine kinases. Recently, the role of negative signaling regulators is increasingly being recognized as an alternative mechanism involved in diseases such as leukemias and myeloproliferative neoplasms (MPNs). The adaptor protein LNK (Src homology 2 (SH2)B3) is a negative regulator of cytokine signaling that has an essential, nonredundant role in normal hematopoiesis. Indeed, LNK-deficient mice show marked expansion of early hematopoietic precursors, more mature myeloid and B-lineage lymphoid cells, as well as enhanced hematopoietic reconstitution. Murine models show that loss of LNK enhances the development of MPNs and may have a role in additional pathologies. LNK mutations were recently identified in patients with MPNs, and studies in animal models and hematopoietic cell lines suggest that LNK controls the aberrant signaling pathways induced by activated oncogenic kinases. In addition, genome-wide studies show that LNK is associated with autoimmune and cardiovascular disorders. These findings have implications for the future study of hematopoiesis, as well as for the development of novel stem cell and disease-specific therapies.

Oncogene (2013) 32, 3111–3118; doi:10.1038/onc.2012.435; published online 8 October 2012 Keywords: cytokine signaling; LNK; hematopoiesis

INTRODUCTION structure (Figure 1) with a N-terminal dimerization domain Throughout life, hematopoiesis is a continuing process, in which containing a phenylalanine zipper motif, which mediates homo- 14 hematopoietic stem cells (HSCs) replenish multilineage progeni- and heterodimerization of SH2B family members; proline-rich tors that further differentiate into all lineages of mature blood regions; a pleckstrin homology (PH) domain believed to recognize cells. and their cognate receptors are key factors phosphoinositides and control localization; influencing the function and survival of hematopoietic cells. The a SH2 domain essential for binding phosphotyrosine in target magnitude and kinetics of cytokine receptor activation is tightly ; and a conserved C-terminal CBL recognition motif. SH2B regulated at multiple levels to ensure a proper cellular response adaptor proteins are implicated in signal transduction pathways and prevent leukemic transformation. Dysregulation of signaling downstream of various RTKs, including receptors for insulin, by receptor tyrosine kinases (RTKs), as well as non-RTKs (i.e., Janus insulin-like I, nerve growth factor, -derived kinases (JAKs)) is, a frequent event in cancer, particularly in growth factor (PDGF), fibroblast growth factor, stem cell hematopoietic malignancies.1,2 Adaptor proteins that bind RTKs factor (SCF)/c-KIT and macrophage colony-stimulating factor 15,16 and JAKs have an important role in the control of cytokine (M-CSF)/FMS, as well as JAKs. signaling pathways. These adaptor proteins usually lack enzymatic Deletion of SH2B1 in mice results in severe , leptin 17 activity and instead serve as molecular platforms coordinating and insulin resistance, type 2 diabetes and infertility. Genetic signaling events. The Src homology 2 (SH2)B family is a group of studies have also indicated that SH2B1 has a key role in controlling adaptor proteins implicated in a broad range of signaling body weight and glucose homeostasis in humans. Deletion of APS pathways. Among mice deficient for SH2B family members, does not alter adiposity, energy balance or glucose metabolism, those who are LNK-deficient have the most prominent but may negatively regulate leptin and insulin sensitivity under 18 aberrations in hematopoiesis.3,4 Moreover, LNK mutations have certain conditions. LNK was originally cloned from a lymph node 19 recently been identified in myeloproliferative neoplasms (MPNs) cDNA library and shown to participate in T-cell signaling. Shortly and some leukemias,5–13 demonstrating the importance of LNK after, two groups generated LNK-deficient mice providing 3,4 not only for normal but also for aberrant hematopoiesis, thus important insights into LNK function in hematopoiesis. forming the rationale for this review. Although high levels of LNK are found in non-hematopoietic tissues such as the testis, brain and muscle, the only noticeable phenotypes of Lnk À / À mice are in the hematopoietic SH2B FAMILY compartments. Further studies established LNK as a key hub The SH2B family of adaptor proteins consists of SH2B1, APS integrating multiple signaling pathways in the hematopoietic/ (SH2B2) and LNK (SH2B3). These proteins share a common domain immunologic system.

1Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA, USA and 2Cancer Science Institute, National Cancer Institute, National University of Singapore, Singapore. Correspondence: Dr S Gery, Hematology/Oncology, Cedars-Sinai Medical Center, 8700 Beverly boulevard, Los Angeles, CA 90048, USA. E-mail: [email protected] Received 13 June 2012; revised 3 August 2012; accepted 4 August 2012; published online 8 October 2012 Adaptor protein LNK S Gery and HP Koeffler 3112 SH2B1 (PSM) Y HSC

DD PH SH2 Tpo/JAK2 1756Myeloid Lymphoid progenitor progenitor APS (SH2B2) Y LNK DDPH SH2 1632MEP GMP ProB ProT IL-3, G-CSF, Lnk (SH2B3) Y FLT3, PDGF DDPH SH2 erythrocytes granulocytes macrophages B cells T cells 1575 14-3-3 14-3-3 FLNA KIT CBL* (S13) (S129) FMS (Y572) PDGFRA Epo Tpo/JAK2 SCF, IL-7 PDGFRB M-CSF FLT3 JAK2 Figure 2. JAK3 LNK is a broad inhibitor of cytokine signaling pathways in LCK hematopoietic lineages. The major lineage-specific cytokine signal- TRKA ing pathways targeted by LNK and additional cytokine signaling MPL TCRζ pathways under LNK control are shown in red. Epo, erythropoietin; GMP, granulocyte macrophage progenitor; HSC, hematopoietic stem Figure 1. Schematic representation of SH2B family members. LNK- cells; M-CSF, macrophage colony-stimulating factor; MEP, mega- binding partners are shown below the LNK protein relative to their karyocyte erythroid progenitor; Tpo, thrombopoietin. main binding region. Interacting proteins for which the LNK-binding domain is unknown are not shown. *CBL-binding site is based on .71 A full colour version of this figure is available at the Oncogene journal online.

LNK IN NORMAL HEMATOPOIESIS BM. In addition, production of reactive oxygen species is increased À / À LNK in hematopoietic lineages in Lnk macrophages, and LNK inhibits M-CSF-induced LNK is highly expressed in multipotent hematopoietic cells and migration of macrophages. their precursors. Furthermore, expression levels of LNK are regulated during normal hematopoiesis, and enhanced by cytokine stimulation. Although LNK functions as a negative LNK in non-hematopoietic BM cells regulator of cytokine signaling in various lineages, the specific Non-hematopoietic (stromal) BM cells consist of a heterogeneous pathways targeted by LNK appear to differ between the lineages cell population, including fibroblasts, endothelial cells, osteoblasts (Figure 2). The most striking features of Lnk À / À mice are profound and osteoclasts, providing the structural microenvironment that splenomegaly with extramedullary hematopoiesis.3,4 The enlarged facilitates hematopoiesis. In addition to its strong expression in spleens of these mice had marked expansion of B lineage hematopoietic cells, LNK is also expressed in non-hematopoietic lymphocytes resulting in part from hypersensitivity of B cells BM cells. The aorta-gonad-mesonephros region is the first intra- precursors to SCF. LNK was originally reported to be involved in embryonic site for definitive hematopoiesis and LNK was found to T-cell receptor signal transduction; yet, T-cell development is be a negative regulator of hematopoiesis in this region.31 LNK was unaffected in LNK-deficient mice. detected in aorta-gonad-mesonephros endothelial cells and its Several studies demonstrated a critical role of LNK in controlling expression pattern overlapped with that of CD34, suggesting that self-renewal and quiescence of HSCs.20–24 LNK-deficient mice LNK might be involved in hematopoietic cell development from have a 10–15-fold increase in HSC numbers with enhanced endothelial precursors. In addition in endothelial cells, the multi-lineage repopulation after bone marrow (BM) transplanta- proinflammatory cytokine tumor necrosis factor-a, through a tion. LNK constrains quiescence and self-renewal of HSCs/ phosphatidylinositol 3-kinase-dependent signaling pathway, hematopoietic progenitor cell (HPCs), predominantly through rapidly phosphorylates and subsequently upregulates LNK.32 thrombopoietin/JAK2 signaling. LNK-deficient mice also display a In turn, LNK negatively regulates tumor necrosis factor signaling. 3–5-fold increase in circulating white blood cells and platelets, as Adhesion and migration of endothelial cells is also affected well as accumulation of erythroid and megakaryocytes cells by LNK. Following b1- activation, LNK is rapidly phospho- in the BM. These changes are due to hypersensitivity to signaling rylated leading to a signaling cascade resulting in increased cell by erythropoietin and thrombopoietin.25,26 Hematopoietic pro- adhesion and reduced cell motility.33 LNK also has a pivotal role in genitors in Lnk À / À mice show hypersensitivity to several other specific modulation of cell growth, endothelial commitment and cytokines such as interleukin (IL)-3 and IL-7. mobilization of endothelial progenitor cells (EPCs) from BM into In murine platelets, LNK regulates integrin aIIbb3 outside-in the peripheral blood.34 Loss of LNK in mice increases EPC kinetics signaling, leading to stabilization of thrombus development in response to ischemia-related cytokines (vascular endothelial in vivo.27 Involvement of LNK in integrin-mediated signaling growth factor, stromal cell-derived factor-1, granulocyte CSF and pathways was also reported the in maturation process of SCF) enhancing neovascularization. In addition, LNK deficiency megakaryocytes, where LNK functions during cell adhesion and increases the commitment of c-KIT-positive, Sca-1-positive and is involved in a crosstalk between integrin- and cytokine-mediated lineage marker-negative (KSL) cells to EPCs.35 Likewise, vasculo- signaling.28 LNK also has a role in mast cell development genesis/angiogenesis and osteogenesis are augmented in LNK- by attenuating signaling of c- KIT.29 Compared with wild-type deficient mice by the mobilization and recruitment of HSCs/EPCs (WT) cells, Lnk À / À BM mast cells display increased SCF- also via activation of the SCF/c-KIT signaling pathway.36 In dependent proliferation and migration. In macrophages, LNK addition, osteoblasts from LNK-deficient mice show a greater regulates signaling by M-CSF.30 Clonogenic assays demonstrated potential for terminal differentiation in response to signaling by elevated number of macrophage colony-forming cells in Lnk À / À SCF/c-KIT in vitro.

Oncogene (2013) 3111 – 3118 & 2013 Macmillan Publishers Limited Adaptor protein LNK S Gery and HP Koeffler 3113 Activated TKs target by LNK associates with the juxtamembrane region of homodimeric LNK interacts with several TK as well as other signaling molecules receptors including erythropoietin R, myeloproliferative leukemia (Figure 1, Table 1). The interaction between LNK and JAK2 is well- (MPL) (thrombopoietin R), G-CSF R and IL-3R. Ligand binding to characterized. JAK2 is a cytoplasmic non-receptor TK that has a the receptor results in JAK phosphorylation, recruitment of STAT crucial role in cytokine signaling during hematopoiesis. JAK2 proteins and activation of downstream signaling pathways. LNK, as well as the other SHB family members (SH2B1 and APS), via their SH2 domain directly bind the phosphorylated tyrosine 15,24,37 Table 1. Binding partners of LNK residue 813 of activated JAK2 (Figure 3a). Interestingly, while LNK is a negative regulator of JAK2, SH2B1 and at least in Binding partner Binding Main LNK-binding Reference some contexts, APS act as potent activators of JAK2 signaling. group partner domain Similar to other negative regulators, such as CIS and SOCS proteins, LNK mRNA and protein expression is induced following 3,29,40 RTK class III c-KIT SH2 JAK2 activation.9,38 LNK, therefore, is part of a negative feedback RTK class III PDGFRA SH2 41 41 loop that controls the recptor JAK2/STAT signaling in RTK class III PDGFRB SH2 hematopoiesis. In HSCs, the LNK–JAK2 interaction is further RTK class III FLT3 SH2 42 RTK class III c-FMS SH2 30 negatively regulated by 14-3-3 adaptor proteins; LNK binds to 9,24,38 14-3-3, and this abrogates the LNK–JAK2 interaction, thereby Kinase JAK2 SH2 39 Kinase JAK3 SH2 47 elevating the inhibitory function of LNK in JAK2 signaling. Kinase PI3K NK 19 Although earlier studies showed that tyrosine phosphorylations, Kinase LCK SH2 72 particularly the conserved tyrosine in the C-terminal domain of LNK, Kinase ILK NK 33 are important for LNK inhibitory functions, binding of 14-3-3 requires Kinase TRKA SH2 43 two serine phosphorylation sites in LNK, which are phosphorylated Adaptor GRB2 NK 19 39 by glycogen synthase kinase 3 and protein kinase A. Together, Adaptor 14-3-3 Ser13 Ser129 these finding suggest that LNK is a central signaling node allowing Adaptor CBL NK 50 Other MPL SH2 24,46 the integration of multiple signaling pathways. Other TCRz SH2 72 c-KIT was the first cytokine receptor identified as a direct target Other PLCg NK 72 of LNK. Studies by us and others pinpointed the LNK–c-KIT Other FLNA PH/SH2 73 interaction to the phosphorylated tyrosine 568 in the juxtamem- brane domain of c-KIT and the SH2 domain of LNK (Figure 3b).29,40 Abbreviations: FLT3, FMS-like tyrosine kinase 3; ILK, integrin-linked kinase; LNK negatively modulates SCF-dependent signaling pathways JAK, Janus kinases; MPL, myeloproliferative leukemia; NK, not known; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PI3K, involved in proliferation and migration of mast and primary phosphatidylinositol 3-kinase; PLCg, phospholipase Cg; RTK, receptor TKs; hematopoietic cells. c-KIT is a member of the RTK subclass III SH2, Src homology 2; TCRz, T-cell receptor z. family, which includes FMS, FMS-like TK 3 (FLT3) and the receptors for PDGF A and B (PDGFRA and PDGFRB). The Y568 amino acid

Figure 3. LNK through its SH2 domain binds to phosphorylated tyrosines in JAK2 and class III RTKs. (a) LNK binds to tyrosine 813 in JAK2 (Y813). (b) LNK binds to tyrosine 568 in c-KIT (Y568). (b) Sequence alignment of the juxtamembrane region of class III RTKs. c-KIT conserved tyrosine 568 is highlighted. (a, b) are adapted from Toffalini and Demoulin.1

& 2013 Macmillan Publishers Limited Oncogene (2013) 3111 – 3118 Adaptor protein LNK S Gery and HP Koeffler 3114 Growth factor/cytokine receptor Integrin Table 2. LNK binding to oncogenic alleles Oncogenic allele Main LNK-binding domain Reference

KITD816V KITD816H SH2 48 FIP1L1-PDGFRA SH2 41 TEL-PDGFRB SH2 41 FLT3ITD SH2 42 MPLW515L SH2 47 JAK2V617F JAK2K539L SH2 9,24,38 JAK3A572V SH2 47 Proliferation Migration Abbreviations: FLT3, FMS-like tyrosine kinase 3; JAK, Janus kinases; MPL, Apoptosis Adhesion myeloproliferative leukemia; PDGF, platelet-derived growth factor; SH2, Src homology 2 domain. Self renewal

Figure 4. LNK is part of negative feedback loops in signaling 45 pathways that control various cellular processes critical for chronic myelomonocytic leukemia and unclassifiable MPNs). The hematopoiesis. A full colour version of this figure is available at activating JAK2V617F mutation is the most prevalent mutation in the Oncogene journal online. BCR-ABL1-negative MPNs, detected in virtually all PV cases and in about half of the ET and PMF cases. After the discovery of JAK2V617F, investigators began searching for other mutations found in c-KIT is conserved among RTK III family members relevant to JAK signaling that might contribute to MPN (Figure 3c), and we hypothesized that LNK might similarly bind pathogenesis. Indeed, the identification of rare MPL receptor and regulate additional RTK III members. Indeed, LNK binds and JAK2 exon 12 mutations were soon discovered. Genome-wide to FMS and inhibits M-CSF-induced signaling and migration studies identified a number of novel MPN-associated 30 in macrophages. LNK also binds PDGFRA and PDGFRB, and including IKZF1, CBL, IDH1/2, EZH2, ASXL1, TET2, as well as LNK. LNK overexpression inhibits PDGF-dependent proliferation of hematopoietic Ba/F3 cells.41 LNK interacts with FLT3, and FLT3 activation and downstream-induced signaling are suppressed LNK regulation of oncogenic TKs in myeloid cells by LNK in 32D hematopoietic cells.42 In addition, loss of LNK Although LNK mutations occur at a low frequency in MPNs, LNK enhances expansion of HPCs stimulated by FL ligand. The SH2 may regulate the abnormal signaling pathways induced by domain is the main LNK domain involved in the LNK interactions oncogenic TKs (Table 2). LNK binds to and negatively regulates with RTK III, as well as JAK2, and is required for the inhibitory MPN-associated mutant MPL and JAK2 alleles, including effects of LNK. MPLW515L, JAK2V617F and JAK2K539L.9,38,46 We found that LNK Recently, LNK was reported to interact with two additional TKs. also interacted with and was phosphorylated by JAK3, as well as In endothelial cells, LNK interacts with the integrin-linked by JAK3A572V, an activating JAK3 mutation associated with acute kinase, involved in integrin-mediated signal transduction megakaryoblastic leukemia.47 LNK mRNA is overexpressed in MPN pathways.33 Through its interaction with integrin-linked kinase, patients and positively correlates with the JAK2V617F allele LNK controls cell adhesion and migration. In neuronal cells, burden.9,48 Studies in hematopoietic cell lines showed that LNK LNK binds to phosphorylated nerve growth factor receptor, together with MPL and JAK2 are involved in a finely controlled TRKA.43 Overexpression of LNK inhibits nerve growth factor- feedback mechanism implicated in megakaryopoiesis, where induced differentiation of PC12 neural cells and reduces neurite changes in LNK expression modulate JAK2/JAK2V617F-mediated outgrowth of primary cortical neurons. signaling. In a murine model, loss of LNK accelerates and Taken together, LNK is emerging as an important negative exacerbates oncogenic JAK2 (either JAK2V617F or the TEL/JAK2 regulator involved in restraining multiple signaling pathways critical fusion)-induced myeloproliferative diseases.49 to hematopoietic homeostasis in various lineages (Figure 4). The BCR/ABL1 fusion is the hallmark of CML. Although LNK does not interact with BCR/ABL1, it does inhibit proliferation of BCR/ ABL1-positive hematopoietic cells.48 LNK-growth inhibition activity is associated with downregulation of STAT5, a key mediator of the LNK IN HEMATOPOIETIC NEOPLASMS pathogenesis of CML. Furthermore, in a murine model for CML, LNK The World Health Organization classification of myeloid malig- deficiency cooperates with the BCR/ABL1 oncogene.49 Thus, LNK nancies includes five major subgroups: 1. MPNs, 2. myelodysplas- regulates aberrant signal transduction pathways in both BCR/ABL1- tic syndromes (MDS), 3. MDS/MPNs, 4. myeloid and lymphoid negative and -positive MPNs. Notably, elevated levels of LNK mRNA neoplasms with eosinophilia and abnormalities of PDGFRA, are also found in MDS and AML cells, suggesting that LNK may PDGFRB or fibroblast growth factor R1 and 5. acute myeloid influence not only the development of MPNs but also that of other leukemia (AML).44 TK signal transduction pathways are often hematopoietic malignancies.48 altered in these diseases, and LNK has been suspected to As discussed above, we have shown that LNK associates with have a role in their pathogeneses. Interestingly, a number and negatively regulates RTK III family members. We found similar of features displayed by LNK-deficient mice are reminiscent of interactions between LNK and a number of mutant RTK III alleles the myeloproliferative abnormalities found in MPN patients, i.e., present in hematopoietic disorders (Table 2). FIP1L1/PDGFRA hypersensitivity to cytokines, splenomegaly together with fibrosis, fusion protein is found in chronic eosinophilic leukemia and thrombocytosis and extramedullary hematopoiesis, as well as systemic mastocytosis patients; TEL/PDGFRB is associated with increased number of multilineage, erythroid and megakaryocytic chronic myelomonocytic leukemia. The growth of hematopoietic clonogenic cells. 32D cells transformed by either FIP1L1/PDGFRA or TEL/PDGFRB MPN includes chronic myelogenous leukemia (CML), also was inhibited by overexpression of LNK, and this effect was known as BCR/ABL1-positive MPNs, classic BCR/ABL1-negative dependent on the SH2 domain of LNK.41 Interestingly, LNK can MPNs: polycythemia vera (PV), essential thrombocythemia (ET) inhibit growth of cells transformed by either FIP1L1/PDGFRA or and primary myelofibrosis (PMF), as well as nonclassic MPNs TEL/PDGFRB; nevertheless in co-expression experiments, LNK only (including systemic mastocytosis, chronic eosinophilic leukemia, interacts with and is phosphorylated by FIP1L1/PDGFRA but not

Oncogene (2013) 3111 – 3118 & 2013 Macmillan Publishers Limited Adaptor protein LNK S Gery and HP Koeffler 3115 TEL/PDGFRB. We speculate that while LNK may not directly inhibit W262R (SNP) some oncogenic kinases, such as BCR/ABL1 and TEL/PDGFRB, it Y can still attenuate their induced growth stimulation by dampening DD PH SH2 downstream signaling pathways in the transformed cells. 1 575 Somatic mutations of FLT3 involving internal tandem duplica- E208 D234 V65A

tion of the juxtamembrane domain or point mutations in the TK I257T I568T V465I F287S P129S R308X S360C Q423X R551W G152R E208Q domain occur in AML, acute lymphoblastic leukemia (ALL) and E208X occasionally MPN patients. LNK can bind and decrease signaling A215V 42 A215V induced by WT or mutant FLT3-TK domain. Activating mutations A215V of c-KIT occur in patients with gastrointestinal stromal tumors and G220R G220V systemic mastocytosis, as well as several other hematological A223V malignancies. LNK binds the systemic mastocytosis-associated G229S c-KIT mutant alleles, KITD816V and KITD816H.48 Given its ability to D234N interact with oncogenic c-KIT, and modulate c-KIT activity, we Figure 5. LNK point mutations are shown below the LNK protein. In hypothesize that LNK may be occasionally mutated in mast cell MPNs, LNK point mutations mostly target a hot spot in the PH disorders. domain spanning glutamic acid 208 (E208) to aspartic acid 234 (D234). See Table 3 for more details. LNK nonsynonymous W262R single-nucleotide polymorphism is located in the PH domain and is LNK mutations in myeloid neoplasms associated with autoimmune and cardiovascular diseases. A full colour version of this figure is available at the Oncogene journal The relevance of LNK to human disease is highlighted by the online. recent identification of LNK mutations in patients (Figure 5, Table 3). A study by Oh et al.5 was the first to report LNK mutations in MPNs. A screen of 33 ET and PMF patients identified two early T-cell precursor T-ALL cases.13 This was the first report mutations in exon 2 of LNK, which encodes part of the PH domain. describing LNK mutations in lymphoid leukemia. We have Cell lines and primary patient samples expressing these mutant sequenced the PH domain of LNK in 147 lymphoma samples forms of LNK exhibited aberrant cytokine-dependent JAK/STAT (mantle cell lymphoma, peripheral T-cell lymphoma (PTCL) and signaling and proliferation. A follow-up study found that 11 of adult T-cell leukemia/lymphoma cases) but could not detect any 341 (3%) samples from MPN patients had LNK mutations, mutations. Based on the newly discovered mutations outside the including 3/61 (5%) ET, 3/75 (4%) PMF and 5/71 (7%) chronic PH domain, additional sequencing of the full-length LNK gene may myelomonocytic leukemia patients.6 All the mutations localized to be warranted. In rare cases LNK mutations were also detected in a the PH domain of LNK. In a subsequent study of 61 patients with number of solid tumors.51–53 leukemia (MPN-blast phase), 9 heterozygous LNK mutations were In addition to mutations, a nonsynonymous polymorphism identified in 8 patients (13%); of which 8 affected the PH domain.7 (rs3184504, R262W ) in the LNK PH domain is associated with LNK mutations were not detected in 78 additional patients with various autoimmune and cardiovascular disorders including type 1 chronic phase MPNs, suggesting that LNK mutations may be diabetes,54,55 celiac disease,55,56 systemic lupus erythematous,57 involved in leukemic transformation. In addition, some samples rheumatoid arthritis,58 multiple sclerosis,59 hypothyroidism,60 had either a JAK2V617F or IDH mutation concurrent with LNK generalized vitiligo61 elevated blood pressure,62,63 increased risk mutations. In a study of eight patients with idiopathic of hypertension62,63 and myocardial infarction.64 The R262W LNK erythrocytosis (IE), two cases (25%) were found to have LNK polymorphism has also been associated with elevated peripheral PH domain mutations.8 These patients do not have a predilection blood white cells, eosinophils, lymphocytes and platelets,64 as well to develop leukemia, suggesting that Lnk mutation are not as increased proliferation of monocytes in diabetic patients.65 A necessarily leukemogenic. More recently, several studies described recent functional study found that LNK R262W is associated with LNK mutations outside the PH domain in MPN samples.9,11,12 higher cytokine production and stronger activation of the NOD2 Most of the LNK mutations identified in MPNs are missense pathway (a critical pathway for mediating immune defense), mutations targeting a ‘hot spot’ spanning residues Glu208-Asp234 suggesting that LNK might have a role in protection against in the PH domain. The PH domain is probably involved in bacterial infection.66 In addition to LNK R262W, a nonsynonymous membrane localization of LNK; and these mutations may lead to polymorphism (rs72650673, E400K) in the SH2 domain of LNK is mislocalization of LNK to the cytoplasm. The mutations are associated with IE.67 predominantly heterozygous, raising the question of whether they In summary, abnormal signaling in leukemia is often linked to contribute to MPN pathogenesis via haploinsufficiency and/or a activated TKs such as BCR/ABL1, JAK2 and FLT3. In recent years, dominant-negative effect. Lnk þ / À mice exhibit an intermediate the importance of aberrant negative feedback regulation is MPN phenotype, consistent with a haploinsufficiency model. increasingly recognized as an alternative mechanism leading to Alternatively, as the majority of LNK PH mutations leave the deregulated TK activity in transformed cells. Although infrequently N-terminal dimerization domain intact, mutant LNK may bind and mutated, LNK, CBL and SOCSs, all negative regulators of cytokine sequester WT LNK, resulting in a dominant-negative effect. signaling pathways, appear to have impotent roles in hemato- Another question is whether specific mutations may contribute poietic malignancies. to different MPN disease phenotypes or responses to therapy. To begin dissecting the molecular mechanisms of LNK PH mutations, we analyzed the effect of different mutations on signaling pathways downstream of either WT or oncogenic TKs.50 We LNK AS A THERAPEUTIC TARGET found that LNK PH domain mutants have mild, variable, partial Given its role as a broad spectrum regulator of cytokine signaling loss of function compared with WT LNK and did not have a in normal and pathological hematopoietic cells, LNK may be a dominant-negative effect. In addition, the mutants retain binding promising therapeutic target for hematopoietic diseases, as well capacity for JAK2, as well as to several other known LNK targets. as a number of other disorders (Figure 6). In addition, inhibiting Early T-cell precursor ALL is a subgroup of T-ALL, which is a LNK may augment expansion and engraftment of HSCs/HPCs. As neoplasm of early hematopoietic cells. Deep sequencing of 64 more data become available from whole-genome sequencing early T-cell precursor ALL patients identified 4 (6%) samples with studies, further associations between LNK and MPNs and possibly LNK mutations, whereas no LNK mutations were found in non- other malignancies may emerge. Moreover, as resistance to

& 2013 Macmillan Publishers Limited Oncogene (2013) 3111 – 3118 Adaptor protein LNK S Gery and HP Koeffler 3116 Table 3. Published LNK mutations

Diagnosis Mutation Mutation type location JAK2V617F Reference

PV C-terminal Misssense/I568T Positive 12 PV PH domain Misssense/F287S Positive 11 ET PH domain Misssense/E208Q Negative 5 ET SH2 domain Nonsense/Q423X Positive 12 ET N-terminal Misssense/G152R Negative 9 ET C-terminal Frameshift Negative 11 PMF PH domain Frameshift Negative 5 PMF C-terminal Misssense/R551W Positive 12 PMF/AML PH domain Misssense/G220R Positive 7 PMF/AML PH domain Misssense/A215V Negative 7 PMF/AML PH domain Misssense/A215V NA 7 PMF/AML PH domain Misssense/G220V Negative 7 PMF/AML PH domain Misssense/G229S Positive 7 PMF/AML PH domain Misssense/A223V, D234N Negative 7 PMF/AML PH domain Frameshift Positive 7 IE PH domain Nonsense/E208X Negative 8 IE PH domain Misssense/A215V Negative 8 ETP ALL PH domain E3_exon 13 ETP ALL PH/SH2 Nonsense/R308X 13 ETP ALL DD domain Misssense/V65A 13 ETP ALL PH domain Misssense/I257T 13 Colon cancer C-terminal Misssense/V465I 51 Ovarian cancer PH/SH2 Misssense/S360C 52 Breast cancer N-terminal Misssense/P129S 53 Abbreviations: AML, acute myeloblastic leukemia; DD, dimerization domain; ET, essential thrombocythemia; ETP ALL, early T-cell precursor acute lymphoblastic leukemia; IE, idiopathic erythrocytosis; JAK, Janus kinases; NA, not available; PH, pleckstrin homology; PMF, primary myelofibrosis; PV, polycythemia vera; SH2, Src homology 2 domain.

21 Myeloproliferative mice. In addition, human embryonic stem cells may provide an neoplasms alternative source for transplantation. Knockdown of LNK increased the hematopoietic progenitors generated from human Cardiovascular Leukemia diseases embryonic stem cells, suggesting that targeting LNK maybe a beneficial strategy for generation of clinically transplantable HSCs from human embryonic stem cells.69 Together, these studies Ex vivo expansion of suggest that LNK is an excellent candidate for the genetic Bone diseases HSC/HPC manipulation of HSC/HPC self-renewal in vivo and in vitro. Murine models place Lnk as a potential therapeutic target in several other diseases. For example, LNK regulates integrin aIIbb3- Autoimmune mediated signaling in platelets and stabilizes developing thrombi In vivo engraftment in vivo.27 LNK might therefore represent a safe therapeutic target diseases of HSC/HPC Stem cell therapy for the treatment and/or prevention of cardiovascular disease. Another example is featured in a murine hindlimb ischemia Figure 6. LNK as a therapeutic target. HSC/HPC, hematopoietic model; Lnk-deficient EPCs more potently produced hindlimb stem/progenitor cells. A full colour version of this figure is available perfusion recovery and ischemic neovascularization compared at the Oncogene journal online. with Lnk-intact cells.34 Similarly, Lnk–/– mice have augmented retinal neovascularization without an increase of pathogenic angiogenesis in an in vivo model of retinopathy. Further, the TK-targeted therapies continues to be a vexing problem, under- KSL fraction of murine BM represents a population of HSCs and standing the role of downstream signaling molecules such as LNK EPCs. Administration of KSL cells to a murine spinal cord injury have in TK pathways may aid in the development of novel model promotes angiogenesis, astrogliosis, axon growth and therapeutic approaches to overcome drug resistance. If indeed functional recovery following injury. LNK-deficient KSL cells were LNK functions as a tumor suppressor in leukemia, perhaps it can even more effective in promoting these regenerative events.35 In be a therapeutic target. In vitro studies demonstrated that addition, in a murine bone fracture model, loss of LNK enhances retroviral expression of LNK or octa-arginine-mediated delivery the regenerative response during healing of a fracture by the of LNK suppresses hematopoietic leukemic cell proliferation.48,68 mobilization and recruitment of HSCs/EPCs.36 Moreover, LNK is Nevertheless, gene therapy is still a daunting technical challenge also expressed in adult brain and is a negative regulator of brain and a more practical method to activate LNK might be through neural stem cell proliferation after stroke induction in a murine discovery of small molecules that can modulate its activity. model; raising the possibility that LNK may be a therapeutic target Expansion of HSCs/HPCs and enhancement of engrafting in the postischemic brain.70 potential is a therapeutic goal aimed to treat hematological LNK is associated with an array of pathogenic conditions, as well malignancies or genetic diseases by transplantation. Loss of LNK as control of self-renewal of HSCs; thus Lnk-targeted interventions enhances both cytokine-dependent ex vivo expansion and in vivo may be of considerable clinical value. However, whether LNK itself engraftment of HSCs/HPCs.20–23 Notably, even transient inhibition can be effectively targeted needs to be established; and if so, of LNK facilitated engraftment of HSCs/HPCs under nonmyeloabl- whether this will be more useful therapeutically than targeting of ative conditions, leading to full reconstitution in immunodeficient upstream TKs.

Oncogene (2013) 3111 – 3118 & 2013 Macmillan Publishers Limited Adaptor protein LNK S Gery and HP Koeffler 3117 CONCLUSION 18 Li M, Ren D, Iseki M, Takaki S, Rui L. Differential role of SH2-B and APS in Research over the last decade clearly points to a critical role for regulating energy and glucose homeostasis. Endocrinology 2006; 147: LNK in hematopoiesis including the control of cytokine signaling 2163–2170. and cell proliferation. Also, a growing body of evidence suggests 19 Huang X, Li Y, Tanaka K, Moore KG, Hayashi JI. Cloning and characterization of that LNK is involved in mitigating the effects of hematopoietic Lnk, a signal transduction protein that links T-cell receptor activation signal to transformation. Mutation or deregulation of LNK activity occur in phospholipase C gamma 1, Grb2, and phosphatidylinositol 3-kinase. Proc Natl several hematopoietic malignances boosting the notion that LNK Acad Sci USA 1995; 92: 11618–11622. 20 Ema H, Sudo K, Seita J, Matsubara A, Morita Y, Osawa M et al. Quantification of is a tumor suppressor. Better understanding of this important self-renewal capacity in single hematopoietic stem cells from normal and adaptor protein will likely pave the way for novel therapeutic Lnk-deficient mice. Dev Cell 2005; 8: 907–914. strategies to modulate critical signaling pathways in cancer and 21 Takizawa H, Kubo-Akashi C, Nobuhisa I, Kwon SM, Iseki M, Taga T et al. Enhanced other disorders. engraftment of hematopoietic stem/progenitor cells by the transient inhibition of an adaptor protein, Lnk. Blood 2006; 107: 2968–2975. 22 Buza-Vidas N, Antonchuk J, Qian H, Månsson R, Luc S, Zandi S et al. Cytokines CONFLICT OF INTEREST regulate postnatal hematopoietic stem cell expansion: opposing roles of throm- The authors declare no conflict of interest. bopoietin and LNK. 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Oncogene (2013) 3111 – 3118 & 2013 Macmillan Publishers Limited