Lnk adaptor suppresses radiation resistance and radiation-induced B-cell malignancies by inhibiting IL-11 signaling Igal Louria-Hayona,1, Catherine Frelinb, Julie Rustona, Gerald Gisha, Jing Jina, Michael M. Koflera, Jean-Philippe Lamberta, Hibret A. Adissuc, Michael Milyavskyd,e, Robert Herringtonb, Mark D. Mindenf, John E. Dickd,e, Anne-Claude Gingrasa, Norman N. Iscoveb, and Tony Pawsona,e,2 a Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; bOntario Cancer Institute/University Health Network, Toronto, ON, Canada M5G; cCentre for Modeling Human Disease, Toronto Centre for Phenogenomics, Toronto, ON, Canada M5T 3H7; dPrincess Margaret Cancer Centre, Toronto, ON, Canada M5G 2M9; eDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; and fCampbell Family Cancer Research Institute, Princess Margaret Hospital, Ontario Cancer Institute, Toronto, ON, Canada M5G 2M9
Edited by Arthur Weiss, University of California, San Francisco, CA, and approved November 11, 2013 (received for review October 22, 2013)
cancer
exhibit hyperactivation of the Jak–Stat and Erk MAP kinase pathways. Previous studies have shown that Lnk−/− hematopietic stem cells (HSCs) have increased self-renewal and engraftment capacities and that Lnk−/− animals have an expanded HSC compartment (10, 11). Indeed, aged Lnk−/− mice exhibit a myeloproliferative neoplasm (MPN)-like phenotype (12), consistent with the discovery of mutations in the human LNK gene in some cases of essential thrombocythemia, primary myelofibrosis, and erythrocytosis (13, 14). Interestingly, LNK mutations have also been described in patients with acute T-cell leukemia (15, 16), implicating LNK deficiency as being involved in leukemias of both myeloid and lymphoid origin. Based on the fact that Lnk−/− mice exhibit excessive cytokine signaling and a hyperproliferative disease, we considered the possibility that Lnk−/− mice might provide a useful model in which to study the role of cytokines in radio resistance and radiotherapyrelated leukemia. Results Accelerated Acute B-Cell Lymphoma in Lnk-Deficient Mice After Ionizing Radiation. The finding that Lnk−/− mice do not usually
develop acute malignancy despite their hyperproliferative disorder Significance
| lymphoma | survival | gp130
Recurrence of cancer in patients treated with radiation therapy infers that tumor cells have the capacity to escape the lethal effects of irradiation. Surviving tumor cells rely on signaling pathways triggered by hematopoietic interleukins in a mechanism that is poorly understood. We find that the adaptor protein Lnk is key in this process, acting as a negative regulator of interleukin 11 survival signaling. In Lnk knockout derived mice, hematopoietic stem cells have a marked level of irradiation resistance, causing B-cell malignancies. Such observations are consistent with the physiology seen in a subset of human leukemia patients that have Lnk gene mutations. Inhibition of interleukin 11 signaling in the mouse model circumvented this irradiation-resistance phenomenon and is suggestive of a potential for therapeutic intervention.
R
adiation therapy is used to treat a significant proportion of patients with cancers such as lymphoma but can also have undesirable consequences. For example, some tumor cells may escape the initial lethal effects of irradiation, resulting in radio resistance and disease recurrence. In addition, a second malignant neoplasm (SMN) can develop years or decades after treatment (1). In this context, there is increasing evidence that cytokines can influence cell malignancy (2) and that mutations affecting proteins involved in cytokine signaling can influence cancer development (3). However, little is known about the potential roles of the cytokine network in radio resistance and radiotherapy-related leukemia. Lnk and the closely related proteins PH domain-containing adapter protein and Src homology 2 B (SH2-B) form a subfamily of SH2 domain-containing proteins. The Lnk adaptor protein, which is primarily expressed in the hematopoietic system, contains an N-terminal proline-rich region, a pleckstrin homology (PH) domain, an SH2 domain, and a C-terminal sequence with potential tyrosine phosphorylation sites (4, 5). The hematopoietic cells of Lnk−/− mice (6) are hypersensitive to a range of cytokines, including interleukin (IL)-3, IL-7, erythropoietin (Epo), stem cell factor (SCF), and thrombopoietin (TPO) (6–9), and consequently www.pnas.org/cgi/doi/10.1073/pnas.1319665110
Author contributions: I.L.-H. and T.P. designed research; I.L.-H., C.F., J.R., G.G., J.-P.L., and M.M. performed research; I.L.-H., J.J., M.M.K., H.A.A., and R.H. contributed new reagents/ analytic tools; I.L.-H., C.F., M.D.M., J.E.D., A.-C.G., N.N.I., and T.P. analyzed data; and I.L.-H. and T.P. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
To whom correspondence should be addressed. E-mail: [email protected].
2
Deceased August 7, 2013.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1319665110/-/DCSupplemental.
PNAS | December 17, 2013 | vol. 110 | no. 51 | 20599–20604
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The Lnk (Sh2b3) adaptor protein dampens the response of hematopoietic stem cells and progenitors (HSPCs) to a variety of cytokines by inhibiting JAK2 signaling. As a consequence, Lnk−/− mice develop hematopoietic hyperplasia, which progresses to a phenotype resembling the nonacute phase of myeloproliferative neoplasm. In addition, Lnk mutations have been identified in human myeloproliferative neoplasms and acute leukemia. We find that Lnk suppresses the development of radiation-induced acute B-cell malignancies in mice. Lnk-deficient HSPCs recover more effectively from irradiation than their wild-type counterparts, and this resistance of Lnk−/− HSPCs to radiation underlies the subsequent emergence of leukemia. A search for the mechanism responsible for radiation resistance identified the cytokine IL-11 as being critical for the ability of Lnk−/− HSPCs to recover from irradiation and subsequently become leukemic. In IL-11 signaling, wild-type Lnk suppresses tyrosine phosphorylation of the Src homology region 2 domain-containing phosphatase-2/protein tyrosine phosphatase nonreceptor type 11 and its association with the growth factor receptor-bound protein 2, as well as activation of the Erk MAP kinase pathway. Indeed, Src homology region 2 domain-containing phosphatase-2 has a binding motif for the Lnk Src Homology 2 domain that is phosphorylated in response to IL-11 stimulation. IL-11 therefore drives a pathway that enhances HSPC radioresistance and radiation-induced B-cell malignancies, but is normally attenuated by the inhibitory adaptor Lnk.
(6) suggests that additional genetic aberrations are required for disease progression, which might be provided by irradiation. To test this possibility, we subjected Lnk−/− mice with hematopoietic hyperplasia (Fig. 1A and Fig. S1) to a single sublethal dose of 500 rad (5 Gy) gamma irradiation. Eight to 10 mo after gamma irradiation, 45% of Lnk−/− mice developed acute lymphoma (P < 0.0005) (Fig. 1 B and C). Histological analysis revealed that the normal architecture of the thymus, lymph nodes, and spleen was replaced by a diffuse sheet of large (25–35 μm) neoplastic lymphocytes. The neoplastic lymphocytes also infiltrated the liver and lung, and further immunostaining revealed that the neoplastic cells were B-cell derived (Fig. 1C). This phenotype was not found in Lnk−/− mice that had not been irradiated nor in irradiated wild-type (WT) mice, with the exception of one lymphoma in a WT irradiated animal (Fig. 1B), which was T cell in nature (P < 0.0005). Spectral karyotyping and array comparative genomic hybridization analysis of three gammairradiation–induced B-cell lymphomas from Lnk−/− mice revealed genomic aberrations and chromosomal translocations, including t (5, 17) translocations, indicating that the tumors were genetically unstable and clonal in origin (Fig. 1D). These data suggest that murine Lnk normally suppresses the emergence of B-cell malignancy following irradiation.
To explore whether Lnk−/− HSCs are protected from irradiation treatment in vivo, we used a competitive repopulation assay in which donor bone marrow (BM) cells from Lnk−/− or WT mice (expressing the marker CD45.2) were mixed with WT competitor cells (CD45.1) and then transplanted into lethally irradiated WT mice (CD45.1) and measured for their relative abilities to compete for BM repopulation (Fig. 2 A–D). Before transplantation, the donor animals were either untreated or whole body irradiated. Lnk−/− HSCs have a 10-fold higher repopulation capability than WT HSCs (9, 10). To compensate for this relative advantage of the mutant cells, we mixed Lnk−/− and competitor cells in a 1:1 ratio, but WT and competitor cells in a 10:1 ratio (Fig. 2B). Strikingly, Lnk−/− HSCs were significantly more resistant to irradiation than WT HSCs, as assessed in the competitive repopulation assay (Fig. 2 C and D). In addition, for each irradiated Lnk−/− marrow transplanted, 1 out of 10 recipient mice developed early B-cell precursor acute lymphoblastic leukemia 3 to 8 mo after bone marrow transplantation (BMT) (Fig. 2 E and F and Fig. S3). These results identify Lnk as a negative regulator of HSC recovery after gamma irradiation treatment and support the hypothesis that irradiation induces the transformation of a rare Lnk−/− hematopoietic stem cells and progenitors (HSPCs) population, which leads to radiation-related B-cell malignancies.
Superior Recovery of Lnk−/− Hematopoietic Stem Cells and Progenitors After Gamma Irradiation Leads to Early B-Cell Precursor Acute Lymphoblastic Leukemia in Recipient Mice. Our finding that irradi-
Lnk−/− HSPCs Are Protected from Gamma Irradiation Treatment due to Enhanced IL-11 Signaling. Because Lnk regulates cellular
ated Lnk−/− mice developed B-cell lymphoma prompted us to explore the cellular basis for this phenotype. No differences in survival were detected when WT or Lnk−/− mice were lethally irradiated (10 Gy); however, sublethal irradiation (5 Gy) revealed an enhanced recovery of the blood system in Lnk−/− mice compared with WT animals, as assessed by the numbers of myeloid progenitor colonies and endogenous spleen colony-forming units (S-CFUs) (Fig. S2 A–C). Moreover, under these circumstances we detected a higher rate of recovery of the Lin−Sca1+cKit+ (LSK) population in Lnk−/− mice compared with their WT counterparts (Fig. S2D).
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responses to a variety of cytokines (6), we hypothesized that in the absence of Lnk, hyperactive cytokine signaling might promote the survival of Lnk−/− HSPCs and thereby allow them to escape the effect of irradiation and develop the potential to become cancerous. To identify such cytokines, we tested the ability of a single HSC to create a colony under different cytokine conditions Single, purified Rho123loKit+Sca1+Lin−CD49blo (RLSKa2lo) long-term HSC (LT-HSC) (18) were cultured in serum-free medium with cytokines, or following the withdrawal of SCF, IL-11, or Fms-like tyrosine kinase 3 (Flt3) ligand, which were shown to protect HSPCs from irradiation. Strikingly, the absence of IL-11 profoundly reduced the ability of Lnk−/− HSC
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Fig. 1. Accelerated acute B-cell lymphoma in irradiated Lnk−/− mice with hyperplasia. Hyperplasia in nontreated Lnk−/− mice. (A) Hematopoietic hyperplasia in Lnk−/− BM and spleen as analyzed by Hematoxylin/eosin staining. (B) Kaplan–Meier analysis of lymphoma incidence. WT and Lnk−/− mice (n = 20) were exposed to 5 Gy of whole body irradiation. The percentage of lymphoid tumor-free animals is plotted against time (in months). Log-rank test P = 0.0003. (C) Hematoxylin/eosin staining of representative Lnk−/− lymphoma and B220 and CD3 immunohistochemistry on a lymphoma section is shown. Representative lymph nodes and thymus from Lnk−/− lymphoma are shown in the picture. (D) Spectral Karyotyping (SKY) of Lnk−/− gamma-irradiated/ induced B-cell Lymphoma. Classification-colored chromosoms are shown. Tumor display t (5, 18).
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to create colonies (Fig. 3A). To elucidate the effects of Lnk on IL-11 signaling, colonies derived from single LT-HSCs were stimulated with IL-11 and examined for activating phosphorylation events on the Stat3 transcription factor and the Erk MAP kinase. Lnk−/− colonies exhibited enhanced IL-11 signaling as evaluated by higher Stat3 and Erk phosphorylation in comparison with the WT colonies (Fig. 3B). To understand how IL-11 influences the survival of Lnk−/− HSPCs after irradiation, we examined the sensitivity of LSK populations to apoptosis using an annexin-V assay (17). By this measure, Lnk−/− LSK cells showed enhanced resistance to irradiation in the presence of IL-11 compared with the WT population; however, this resistance of Lnk−/− cells strikingly collapsed in the absence of IL-11 (Fig. S4). Together, these results indicate that Lnk−/− HSPCs are hypersensitive to IL-11 stimulation, suggesting that Lnk is a negative regulator of IL-11 signaling. Most interestingly, our experiments indicate that the resistance of Lnk−/− HSPC to irradiation is IL-11 dependent. Inhibition of IL-11 Signaling Reduces HSPC Recovery After Radiation Treatment and Delays Radiation-Induced B-Cell Lymphoma in Lnk−/− Mice. Based on the observation that IL-11 signaling is enhanced
in Lnk−/− HSPCs and is critical for their survival after irradiation, we examined whether in vivo treatment with a neutralizing antibody to IL-11 could affect HSPC recovery of irradiated Lnk−/− mice. We therefore sublethally irradiated Lnk−/− mice, and then injected them, or not, with anti–IL-11 antibody. Nine days after Louria-Hayon et al.
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Fig. 2. Radio resistance of Lnk−/− HSPCs leads to acute pre–B-cell leukemia in recipient mice. (A) Schematic illustration shows reconstitution competition assay before and after irradiation. BMs from not treated (B) and whole body irradiated WT or Lnk−/− donor mice (C and D) expressing the CD45.2 cell surface marker were mixed with WT competitor cells expressing the CD45.1 cell surface marker, and were transplanted into lethally irradiated WT recipient mice. Lnk−/− and competitor cells were mixed in ratio of 1:1 (1 million cells of each), and WT and competitor cells were mixed in ratio of 10:1 (10 million WT cells and 1 million competitor cells). At 8–32 wk posttransplantation, peripheral blood was withdrawn from recipients and analyzed for the percentage of CD45.2 cells in total leukocytes. Results show means ±+/− SD from at least six animals (C and D). P < 0.001. (E) Differential blood counts (109/L) from three leukemic recipients of 5 Gy–irradiated Lnk−/− donor mice. (*) P < 0.001. (F) Lymphoblast cells in BM and infiltration of lymphoblast cells into the liver of leukemic recipient mice analyzed by Hematoxylin/eosin staining.
this treatment, the BM was flushed and subjected to a myeloid colony assay. Interestingly, anti–IL-11 antibody significantly reduced the capacity of BM cells derived from irradiated Lnk−/− mice to form colonies compared with those from irradiated Lnk−/− mice that were not injected or were treated with antiangiopoietin-1 receptor (Tie2) antibody (Fig. 3C). These data suggest that blocking IL-11 signaling debilitates the recovery of Lnk−/− HSPC after irradiation. Based on these results, we explored the long-term effects of anti– IL-11 treatment on the HSPC population of Lnk−/− mice (Fig. S5). Strikingly, we found that a single dose of anti–IL-11 after irradiation reduced the recovery of Lnk−/− LSK cells and HSCs even when measured after 5 mo (Fig. S5 B and C). Most interestingly, there was a 50% reduction in the number of animals that developed acute B-cell lymphoma when the irradiated mice were injected with anti–IL-11 and a 3-mo delay in the appearance of the disease (Fig. 3D). These results indicate that anti–IL-11 treatment suppresses the recovery of Lnk−/− HSPCs after irradiation and thus reduces the incidence of radiation-related malignancies. Selection for Enhanced Src Homology Region 2 Domain-Containing Phosphatase-2–Erk Signaling in Radiation-Induced Lnk−/− B-Cell Malignancies. IL-11 belongs to the gp130 cytokine family, which
includes IL-11, IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), Cardiotrophin-1 (CT-1), Ciliary neurotrophic factor (CNTF) and IL-27 (19). The binding of cytokines such as IL-11 and IL-6 to the gp130 receptor results in its homodimerization PNAS | December 17, 2013 | vol. 110 | no. 51 | 20601
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(20, 21), leading to activation of the Jak2 tyrosine kinase and phosphorylation of the Stat3 transcription factor. A distinct signaling pathway downstream of the gp130/Jak2 complex involves the recruitment and phosphorylation of Src homology region 2 domain-containing phosphatase-2/protein tyrosine phosphatase nonreceptor type 11 (Shp2/PTPN11), resulting in the stimulation of both Shp2 tyrosine phosphatase activity and its ability to bind the growth factor receptor-bound protein 2 (Grb2) adaptor, and activation of the Erk MAP kinase pathway (22–24). Shp2 is indispensable for embryonic development and hematopoiesis (25, 26) and is also required for maintenance of HSCs and progenitors in adult animals (27). Mutations in Shp2 are found in the developmental disorders Noonan syndrome (28) and leopard syndrome (29), and activating mutations in Shp2 have been implicated in leukemias (30). The aberrant activation of the Erk pathway induced by constitutively active Shp2 mutants is a potential mechanism underlying MPN leukemogenesis (31, 32). The ability of Shp2 to activate the Erk pathway typically requires its phosphatase activity and is potentially enhanced by C-terminal phosphotyrosine sites that bind the Grb2 adaptor in response to gp130 activation (22–24). Because we found that IL11 stimulation of Lnk−/− HSPC resulted in enhanced Erk phosphorylation (Fig. 3), we analyzed the state of the Shp2–Erk pathway in Lnk−/− radiation-related malignancies using splenocytes derived from WT or Lnk−/− mice. Strikingly, splenocytes derived from irradiated Lnk−/− mice that had developed B-cell lymphoma exhibited strongly enhanced Shp2 tyrosine phosphorylation, and association with Grb2 and gp130, compared with splenocytes from irradiated WT mice or cancer-free Lnk−/− mice (Fig. 4A). Consistent with these results, splenocytes and BM cells derived from Lnk−/− mice with radiation-related malignancies exhibited high levels of phospho-Erk (Fig. 4B). These data suggest that Lnk normally inhibits Shp2 signaling downstream of the IL-11–gp130 complex, and indicate that enhanced Shp2/Grb2–pErk signaling is selected during the development of radiation-related malignancies in Lnk−/− mice. Lnk Attenuates Shp2-Based Signaling in Response to IL-11 Stimulation.
Lnk binds and inhibits the Jak2 tyrosine kinase, providing one mechanism by which it may block cytokine signaling. To search for 20602 | www.pnas.org/cgi/doi/10.1073/pnas.1319665110
Fig. 3. Enhanced IL-11 signaling in Lnk−/− HSPCs promotes resistance to irradiation and lymphomas. (A) Single purified LT-HSC (Rho123loKit+Sca1+Lin−CD49blo) were plated in serum-free media with the cytokines IL-11, SCF, and Flt3 ligand or without the indicated cytokine. Seven days after plating the ability to create a colony was indicated. n = 5; (*) P < 0.001. (B) Western blot analysis for the level of pStat3 and pErk in LT-HSC– derived colonies with/without stimulation of IL-11. (C) Lnk−/− mice were treated with IR (5Gy) or together with anti-IL-11 (R&D SYSTEMS 100 μg per mouse) or anti-Tie2 (R&D SYSTEMS 100 μg per mouse). Nine days posttreatment, the BM was flashed and the recovery was determined by number of myeloid colonies. n = 5, (*) P < 0.001. (D) Kaplan–Meier analysis of lymphoma incidence. Lnk−/− mice (n = 20) were exposed to the indicated treatments. The percentage of lymphoid tumor-free animals is plotted against time (in months). Log-rank test P = 0.45.
additional targets for Lnk regulation in IL-11 signaling, we synthesized a panel of phosphotyrosine-containing peptides (33), derived from 13 proteins involved in gp130 signaling, and tested them for binding to the Lnk SH2 domain (list of peptides in Table S1). Far Western blot analysis using a bacterially expressed Lnk SH2 domain revealed direct interactions with phosphotyrosinecontaining peptides derived from the proteins Jak2, Jak3, and Shp2/Ptpn11 (centered on pY62) (Fig. S6 A and B). These three phosphopeptides have a similar sequence motif surrounding the key phosphotyrosine (Fig. S6B). These data confirm the previously reported interaction between Lnk and Jak2 (9) and suggest that Lnk has the potential to interact with Jak3. Interestingly, this strategy revealed the Shp2/Ptpn11 phosphatase as a potential binding partner for Lnk. To validate these findings in a cellular context, we derived an Lnk−/− cell line from lineagedepleted Lnk−/− BM cells, which were transformed by the Philadelphia chromosome BCR-ABL oncogene. We then transfected these Lnk−/− cells with Flag-tagged WT Lnk or with Lnk variants in which the ligand-binding properties of the SH2 domain (R364E) or PH domain (W191A) were inactivated. The pY62 peptide of Shp2 pulled down WT full-length Lnk and the PH-mutated Lnk, but not the SH2-mutated protein, indicating that the ability of full-length Lnk to bind phosphotyrosine-containing peptides is mediated by the SH2 domain (Fig. S6C). Using these cells, we also found that WT and PH domain-mutated (W191A) Lnk proteins immunoprecipitated (IP) with endogenous Shp2, whereas the Lnk SH2 domain mutant (R364E) did not (Fig. 4C). To further investigate the influence of IL-11 signaling on the Lnk–Shp2 interaction, the Lnk−/− cell line was transfected with a WT Lnk cDNA fused to an estrogen receptor (ER) promoter inducible by hydroxytamoxifen (HTO). Upon HTO treatment, Lnk was inducibly expressed and found to coprecipitate with endogenous Shp2. Stimulation of these cells with IL-11 increased the Lnk–Shp2 interaction, which reached a maximum binding level 15–20 min after cytokine treatment (Fig. 4D). Mass spectrometry analysis of Shp2 from Lnk−/− cells revealed that Y62 of Shp2 is indeed phosphorylated after IL-11 stimulation (Fig. S7). Intriguingly, Y62 is mutated to aspartate in rare cases of Noonan syndrome (34), and the adjacent D61 is mutated to glycine in Noonan syndrome and somatically mutated to tyrosine in Noonan-related leukemia. Louria-Hayon et al.
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These residues are therefore implicated in the regulation of Shp2 function. The preceding experiments reveal a consensus phosphopeptide motif recognized by the Lnk SH2 domain, and indicate that Lnk has two potential targets in the gp130–Jak2–Shp2 complex induced by IL-11, where it acts to suppress Shp2 phosphorylation, Grb2 binding, and Erk activation. Hence in the absence of Lnk, activation of both the Stat3 and Erk pathways may contribute to the radioresistance phenotype (Fig. 4E). Discussion Alterations in cytokine signaling and resulting survival pathways are known to participate in cancer development, but the roles of cytokines in resistance to therapy are less certain. To address this issue, we have used a mouse model that lacks the inhibitor Lnk, which has allowed us to investigate the phenotypic consequence of enhanced cytokine signaling in the context of irradiation. As Lnk is most abundantly expressed in HSPCs (10), this strategy was designed to investigate the physiological effects of irradiation on hematopoietic stem cells exposed to aberrantly high levels of cytokine signaling, and conversely to identify cytokines that must be kept in check to prevent an adverse hematopoietic response to irradiation. Our results indicate that in the absence of Lnk, hyperactive signaling is elicited by IL-11, and that this in turn causes resistance to irradiation and the development of radiation-related B-cell malignancies. The relative radio resistance of Lnk−/− HSPC was suppressed when this pathway was blocked by a neutralizing Louria-Hayon et al.
Fig. 4. A selection for enhanced Shp2–Erk pathway in Lnk−/− malignancies. (A) Interactions between IP Shp2 with Grb2 and gp130 in splenocytes derived from irradiated WT or lymphoma-free irradiated Lnk −/− or Lnk −/− lymphoma. (B) Western blot analysis of pERK in splenocytes derived from irradiated WT or KO Lnk lymphoma (B, Left), or of BM derived from recipient of irradiated WT or recipient of irradiated KO Lnk, which developed B-cell acute lymphoblastic leukemia (BALL) (B, Right). (C) Interaction between IP Flag-Lnk and endogenous Shp2. (D) Interaction between IP Flag-Lnk and Shp2 after IL-11 stimulation. (E) Model for Lnk regulation of IL-11 signaling. In response to IL-11 stimulation, both Jak2 and Shp2 are phsphorylated to increased Stat3 and ERK activity. Lnk–SH2 domain interacts with pYJAK2 and pY62Shp2 to down-regulate their activity. Inactivation of Lnk leads to enhanced Jak–Stat and Shp2–Grb2 pathways, which increase resistance to irradiation treatment and thus enlarge the tumor incidents.
antibody against IL-11 in vivo (Fig. 3C), or by IL-11 deprivation ex vivo (Fig. S4). Collectively, these data argue that Lnk−/− cells acquire radio resistance, at least in part due to enhanced IL11 signaling. IL-11 signals through the gp130 cytokine receptor and has been reported to increase the survival of irradiated mice (35). This activity has been primarily attributed to the Jak–Stat pathway. Lnk is known to inhibit Jak2, and our present data suggest that it also suppresses tyrosine phosphorylation of Shp2 and downstream signaling to Erk induced by IL-11. Lnk potentially exerts its inhibitory effect by binding through its SH2 domain to related pTyr sites on both Jak2 and Shp2. Our data are consistent with a scheme in which IL-11 stimulation of HSPCs induces hyperactivation of a Jak2–Shp2 pathway leading to enhanced Erk activation, which together with up-regulated Jak2–pStat3 signaling (Fig. 3B) provides a mechanism by which Lnk deficiency gives a selective survival advantage after irradiation treatment and may thus promote the development of radiation-resistant cancer (Fig. 3D). Lnk is most strongly expressed in HSPCs, which potentially provide a source for leukemic stem cells, as noted in cases of human acute myeloid leukemia and in a variety of leukemia mouse models (36, 37). It will be of interest to explore whether the resistance mechanism we have uncovered for irradiationinduced death of HSPCs may also apply to the ability of leukemic stem cells to survive therapeutic treatments. In this vein, it will be important to explore the relevance of IL-11 PNAS | December 17, 2013 | vol. 110 | no. 51 | 20603
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were plated in methylcellulose (MethoCult M3434) containing cytokines, including Epo (Stem Cell Technologies Inc.), and incubated at 37 °C, 5% CO2, 100% humidity.
Experimental Procedures
Blood Counts. A single 60-μl blood sample was collected in a capillary pipette flushed with EDTA. Samples were analyzed with Roche Hitachi 917 and Hemavet 950FS (Drew Scientific).
Mice. Lnk−/− mice (6) were backcrossed 10 generations into C57BL/6J-Ly5.2Gpi1b/b. Mice were housed and manipulated according to the guidelines of the Canadian Council on Animal Care and experiments were approved by the Toronto Centre for Phenogenomics animal care committee (AUP #0011a-H). Flow Cytometry. Staining and enrichment procedure for stem and progenitor population sorting and analysis were performed as previously described (17) and in SI Experimental Procedures. Cell Culture. Cells were incubated at 37 °C in 5% CO2 in U-bottom microtiter wells (Nunclon) in 100 μL Iscove’s modified Dulbecco’s medium containing 7.5 × 10–5 M a-thioglycerol, 4% FBS, 0.1% BSA, 5 μg/mL transferrin, 5 μg/mL insulin, 50 ng/mL c-kit ligand (KL), 50 ng/mL Flt3 ligand (FL), 10 ng/mL IL-11, and 10 ng/mL IL-7 conditioned medium all as detailed in ref. 17. For culture of single cells, sorted cells were plated at limiting dilution, and wells containing exactly one cell were identified visually after 18 h culture. Lnk−/− cell lines were cultured in RPMI medium supplemented with 10% FBS in the presence or absence of IL-11 or IL-6. The conditional ER system was established according to a previously described procedure (38).
Pathology. Mice were killed using a combination of CO2 and O2 and tissues collected and fixed in 10% neutral buffered formalin or Bouin’s fixative. Tissue sections (4 μm) were prepared and stained with Hematoxylin and Eosin. Protein Expression and Purification. Lnk–SH2 proteins were purified according to a previously described procedure (39). Peptide Synthesis. Synthetic peptides were prepared using Fmoc (9-fluorenyl methoxycarbonyl) solid-phase chemistry. Phosphotyrosine was incorporated using the N-fluorenylmethyloxycarbonyl-O-phospho-L-tyrosine derivative. Peptides were purified using reverse-phase HPLC, and the authenticity was confirmed by mass spectrometry.
In Vitro Hematopoietic Progenitor Colony Assays. Single cell suspensions were prepared from BM aspirates under sterile conditions. Triplicate samples of 104 or 105 cells (for CFU-granulocyte, erythrocyte, monocyte, megakaryocyte)
ACKNOWLEDGMENTS. We thank Adrian Pasculescu for statistical analysis. We thank Pier-Andreé Penttilä and Annie Bang for assistance with flow cytometry, and Zorana Berberovic for help with blood count procedures. We thank Cristina Virage, Geraldine Mbamalu, Mary Barbara, and Oleg Leikin for excellent technical assistance. This research was supported by grants from the Canadian Institutes of Health Research (CIHR MOP-6849) and the Terry Fox Foundation (Team Grant TFF 10).
1. Newhauser WD, Durante M (2011) Assessing the risk of second malignancies after modern radiotherapy. Nat Rev Cancer 11(6):438–448. 2. Grivennikov SI, Greten FR, Karin M (2010) Immunity, inflammation, and cancer. Cell 140(6):883–899. 3. Vainchenker W, Constantinescu SN (2013) JAK/STAT signaling in hematological malignancies. Oncogene 32(21):2601–2613. 4. Li Y, He X, Schembri-King J, Jakes S, Hayashi J (2000) Cloning and characterization of human Lnk, an adaptor protein with pleckstrin homology and Src homology 2 domains that can inhibit T cell activation. J Immunol 164(10):5199–5206. 5. Takaki S, et al. (2000) Control of B cell production by the adaptor protein lnk. Definition of a conserved family of signal-modulating proteins. Immunity 13(5):599–609. 6. Velazquez L, et al. (2002) Cytokine signaling and hematopoietic homeostasis are disrupted in Lnk-deficient mice. J Exp Med 195(12):1599–1611. 7. Tong W, Zhang J, Lodish HF (2005) Lnk inhibits erythropoiesis and Epo-dependent JAK2 activation and downstream signaling pathways. Blood 105(12):4604–4612. 8. Buza-Vidas N, et al. (2006) Cytokines regulate postnatal hematopoietic stem cell expansion: Opposing roles of thrombopoietin and LNK. Genes Dev 20(15):2018–2023. 9. Bersenev A, Wu C, Balcerek J, Tong W (2008) Lnk controls mouse hematopoietic stem cell self-renewal and quiescence through direct interactions with JAK2. J Clin Invest 118(8):2832–2844. 10. Ema H, et al. (2005) Quantification of self-renewal capacity in single hematopoietic stem cells from normal and Lnk-deficient mice. Dev Cell 8(6):907–914. 11. Seita J, et al. (2007) Lnk negatively regulates self-renewal of hematopoietic stem cells by modifying thrombopoietin-mediated signal transduction. Proc Natl Acad Sci USA 104(7):2349–2354. 12. Bersenev A, et al. (2010) Lnk constrains myeloproliferative diseases in mice. J Clin Invest 120(6):2058–2069. 13. Oh ST, et al. (2010) Novel mutations in the inhibitory adaptor protein LNK drive JAKSTAT signaling in patients with myeloproliferative neoplasms. Blood 116(6):988–992. 14. Pardanani A, et al. (2010) LNK mutation studies in blast-phase myeloproliferative neoplasms, and in chronic-phase disease with TET2, IDH, JAK2 or MPL mutations. Leukemia 24(10):1713–1718. 15. Roberts KG, et al. (2012) Genetic alterations activating kinase and cytokine receptor signaling in high-risk acute lymphoblastic leukemia. Cancer Cell 22(2):153–166. 16. Zhang J, et al. (2012) The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 481(7380):157–163. 17. Milyavsky M, et al. (2010) A distinctive DNA damage response in human hematopoietic stem cells reveals an apoptosis-independent role for p53 in self-renewal. Cell Stem Cell 7(2):186–197. 18. Benveniste P, et al. (2010) Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential. Cell Stem Cell 6(1):48–58. 19. Taga T, Kishimoto T (1997) Gp130 and the interleukin-6 family of cytokines. Annu Rev Immunol 15:797–819. 20. Neddermann P, Graziani R, Ciliberto G, Paonessa G (1996) Functional expression of soluble human interleukin-11 (IL-11) receptor alpha and stoichiometry of in vitro IL-11 receptor complexes with gp130. J Biol Chem 271(48):30986–30991. 21. Murakami M, et al. (1993) IL-6-induced homodimerization of gp130 and associated activation of a tyrosine kinase. Science 260(5115):1808–1810.
22. Yin T, Shen R, Feng GS, Yang YC (1997) Molecular characterization of specific interactions between SHP-2 phosphatase and JAK tyrosine kinases. J Biol Chem 272(2): 1032–1037. 23. Vogel W, Ullrich A (1996) Multiple in vivo phosphorylated tyrosine phosphatase SHP-2 engages binding to Grb2 via tyrosine 584. Cell Growth Differ 7(12): 1589–1597. 24. Kim H, Baumann H (1999) Dual signaling role of the protein tyrosine phosphatase SHP-2 in regulating expression of acute-phase plasma proteins by interleukin-6 cytokine receptors in hepatic cells. Mol Cell Biol 19(8):5326–5338. 25. Saxton TM, et al. (2000) The SH2 tyrosine phosphatase shp2 is required for mammalian limb development. Nat Genet 24(4):420–423. 26. Qu CK, et al. (1998) Biased suppression of hematopoiesis and multiple developmental defects in chimeric mice containing Shp-2 mutant cells. Mol Cell Biol 18(10): 6075–6082. 27. Chan G, et al. (2011) Essential role for Ptpn11 in survival of hematopoietic stem and progenitor cells. Blood 117(16):4253–4261. 28. Tartaglia M, et al. (2001) Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 29(4):465–468. 29. Legius E, et al. (2002) PTPN11 mutations in LEOPARD syndrome. J Med Genet 39(8): 571–574. 30. Chan RJ, Feng GS (2007) PTPN11 is the first identified proto-oncogene that encodes a tyrosine phosphatase. Blood 109(3):862–867. 31. Chan G, et al. (2009) Leukemogenic Ptpn11 causes fatal myeloproliferative disorder via cell-autonomous effects on multiple stages of hematopoiesis. Blood 113(18): 4414–4424. 32. Xu D, et al. (2010) A germline gain-of-function mutation in Ptpn11 (Shp-2) phosphatase induces myeloproliferative disease by aberrant activation of hematopoietic stem cells. Blood 116(18):3611–3621. 33. Wiesner S, et al. (2006) A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases. EMBO J 25(19):4686–4696. 34. Martinelli S, et al. (2012) Counteracting effects operating on Src homology 2 domaincontaining protein-tyrosine phosphatase 2 (SHP2) function drive selection of the recurrent Y62D and Y63C substitutions in Noonan syndrome. J Biol Chem 287(32): 27066–27077. 35. Van der Meeren A, Mouthon MA, Gaugler MH, Vandamme M, Gourmelon P (2002) Administration of recombinant human IL11 after supralethal radiation exposure promotes survival in mice: Interactive effect with thrombopoietin. Radiat Res 157(6): 642–649. 36. Lapidot T, et al. (1994) A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367(6464):645–648. 37. Eppert K, et al. (2011) Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med 17(9):1086–1093. 38. Jin J, Woodgett JR (2005) Chronic activation of protein kinase Bbeta/Akt2 leads to multinucleation and cell fusion in human epithelial kidney cells: Events associated with tumorigenesis. Oncogene 24(35):5459–5470. 39. Filippakopoulos P, et al. (2008) Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation. Cell 134(5):793–803.
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Louria-Hayon et al.
Edited by Arthur Weiss, University of California, San Francisco, CA, and approved November 11, 2013 (received for review October 22, 2013)
cancer
exhibit hyperactivation of the Jak–Stat and Erk MAP kinase pathways. Previous studies have shown that Lnk−/− hematopietic stem cells (HSCs) have increased self-renewal and engraftment capacities and that Lnk−/− animals have an expanded HSC compartment (10, 11). Indeed, aged Lnk−/− mice exhibit a myeloproliferative neoplasm (MPN)-like phenotype (12), consistent with the discovery of mutations in the human LNK gene in some cases of essential thrombocythemia, primary myelofibrosis, and erythrocytosis (13, 14). Interestingly, LNK mutations have also been described in patients with acute T-cell leukemia (15, 16), implicating LNK deficiency as being involved in leukemias of both myeloid and lymphoid origin. Based on the fact that Lnk−/− mice exhibit excessive cytokine signaling and a hyperproliferative disease, we considered the possibility that Lnk−/− mice might provide a useful model in which to study the role of cytokines in radio resistance and radiotherapyrelated leukemia. Results Accelerated Acute B-Cell Lymphoma in Lnk-Deficient Mice After Ionizing Radiation. The finding that Lnk−/− mice do not usually
develop acute malignancy despite their hyperproliferative disorder Significance
| lymphoma | survival | gp130
Recurrence of cancer in patients treated with radiation therapy infers that tumor cells have the capacity to escape the lethal effects of irradiation. Surviving tumor cells rely on signaling pathways triggered by hematopoietic interleukins in a mechanism that is poorly understood. We find that the adaptor protein Lnk is key in this process, acting as a negative regulator of interleukin 11 survival signaling. In Lnk knockout derived mice, hematopoietic stem cells have a marked level of irradiation resistance, causing B-cell malignancies. Such observations are consistent with the physiology seen in a subset of human leukemia patients that have Lnk gene mutations. Inhibition of interleukin 11 signaling in the mouse model circumvented this irradiation-resistance phenomenon and is suggestive of a potential for therapeutic intervention.
R
adiation therapy is used to treat a significant proportion of patients with cancers such as lymphoma but can also have undesirable consequences. For example, some tumor cells may escape the initial lethal effects of irradiation, resulting in radio resistance and disease recurrence. In addition, a second malignant neoplasm (SMN) can develop years or decades after treatment (1). In this context, there is increasing evidence that cytokines can influence cell malignancy (2) and that mutations affecting proteins involved in cytokine signaling can influence cancer development (3). However, little is known about the potential roles of the cytokine network in radio resistance and radiotherapy-related leukemia. Lnk and the closely related proteins PH domain-containing adapter protein and Src homology 2 B (SH2-B) form a subfamily of SH2 domain-containing proteins. The Lnk adaptor protein, which is primarily expressed in the hematopoietic system, contains an N-terminal proline-rich region, a pleckstrin homology (PH) domain, an SH2 domain, and a C-terminal sequence with potential tyrosine phosphorylation sites (4, 5). The hematopoietic cells of Lnk−/− mice (6) are hypersensitive to a range of cytokines, including interleukin (IL)-3, IL-7, erythropoietin (Epo), stem cell factor (SCF), and thrombopoietin (TPO) (6–9), and consequently www.pnas.org/cgi/doi/10.1073/pnas.1319665110
Author contributions: I.L.-H. and T.P. designed research; I.L.-H., C.F., J.R., G.G., J.-P.L., and M.M. performed research; I.L.-H., J.J., M.M.K., H.A.A., and R.H. contributed new reagents/ analytic tools; I.L.-H., C.F., M.D.M., J.E.D., A.-C.G., N.N.I., and T.P. analyzed data; and I.L.-H. and T.P. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
To whom correspondence should be addressed. E-mail: [email protected].
2
Deceased August 7, 2013.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1319665110/-/DCSupplemental.
PNAS | December 17, 2013 | vol. 110 | no. 51 | 20599–20604
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The Lnk (Sh2b3) adaptor protein dampens the response of hematopoietic stem cells and progenitors (HSPCs) to a variety of cytokines by inhibiting JAK2 signaling. As a consequence, Lnk−/− mice develop hematopoietic hyperplasia, which progresses to a phenotype resembling the nonacute phase of myeloproliferative neoplasm. In addition, Lnk mutations have been identified in human myeloproliferative neoplasms and acute leukemia. We find that Lnk suppresses the development of radiation-induced acute B-cell malignancies in mice. Lnk-deficient HSPCs recover more effectively from irradiation than their wild-type counterparts, and this resistance of Lnk−/− HSPCs to radiation underlies the subsequent emergence of leukemia. A search for the mechanism responsible for radiation resistance identified the cytokine IL-11 as being critical for the ability of Lnk−/− HSPCs to recover from irradiation and subsequently become leukemic. In IL-11 signaling, wild-type Lnk suppresses tyrosine phosphorylation of the Src homology region 2 domain-containing phosphatase-2/protein tyrosine phosphatase nonreceptor type 11 and its association with the growth factor receptor-bound protein 2, as well as activation of the Erk MAP kinase pathway. Indeed, Src homology region 2 domain-containing phosphatase-2 has a binding motif for the Lnk Src Homology 2 domain that is phosphorylated in response to IL-11 stimulation. IL-11 therefore drives a pathway that enhances HSPC radioresistance and radiation-induced B-cell malignancies, but is normally attenuated by the inhibitory adaptor Lnk.
(6) suggests that additional genetic aberrations are required for disease progression, which might be provided by irradiation. To test this possibility, we subjected Lnk−/− mice with hematopoietic hyperplasia (Fig. 1A and Fig. S1) to a single sublethal dose of 500 rad (5 Gy) gamma irradiation. Eight to 10 mo after gamma irradiation, 45% of Lnk−/− mice developed acute lymphoma (P < 0.0005) (Fig. 1 B and C). Histological analysis revealed that the normal architecture of the thymus, lymph nodes, and spleen was replaced by a diffuse sheet of large (25–35 μm) neoplastic lymphocytes. The neoplastic lymphocytes also infiltrated the liver and lung, and further immunostaining revealed that the neoplastic cells were B-cell derived (Fig. 1C). This phenotype was not found in Lnk−/− mice that had not been irradiated nor in irradiated wild-type (WT) mice, with the exception of one lymphoma in a WT irradiated animal (Fig. 1B), which was T cell in nature (P < 0.0005). Spectral karyotyping and array comparative genomic hybridization analysis of three gammairradiation–induced B-cell lymphomas from Lnk−/− mice revealed genomic aberrations and chromosomal translocations, including t (5, 17) translocations, indicating that the tumors were genetically unstable and clonal in origin (Fig. 1D). These data suggest that murine Lnk normally suppresses the emergence of B-cell malignancy following irradiation.
To explore whether Lnk−/− HSCs are protected from irradiation treatment in vivo, we used a competitive repopulation assay in which donor bone marrow (BM) cells from Lnk−/− or WT mice (expressing the marker CD45.2) were mixed with WT competitor cells (CD45.1) and then transplanted into lethally irradiated WT mice (CD45.1) and measured for their relative abilities to compete for BM repopulation (Fig. 2 A–D). Before transplantation, the donor animals were either untreated or whole body irradiated. Lnk−/− HSCs have a 10-fold higher repopulation capability than WT HSCs (9, 10). To compensate for this relative advantage of the mutant cells, we mixed Lnk−/− and competitor cells in a 1:1 ratio, but WT and competitor cells in a 10:1 ratio (Fig. 2B). Strikingly, Lnk−/− HSCs were significantly more resistant to irradiation than WT HSCs, as assessed in the competitive repopulation assay (Fig. 2 C and D). In addition, for each irradiated Lnk−/− marrow transplanted, 1 out of 10 recipient mice developed early B-cell precursor acute lymphoblastic leukemia 3 to 8 mo after bone marrow transplantation (BMT) (Fig. 2 E and F and Fig. S3). These results identify Lnk as a negative regulator of HSC recovery after gamma irradiation treatment and support the hypothesis that irradiation induces the transformation of a rare Lnk−/− hematopoietic stem cells and progenitors (HSPCs) population, which leads to radiation-related B-cell malignancies.
Superior Recovery of Lnk−/− Hematopoietic Stem Cells and Progenitors After Gamma Irradiation Leads to Early B-Cell Precursor Acute Lymphoblastic Leukemia in Recipient Mice. Our finding that irradi-
Lnk−/− HSPCs Are Protected from Gamma Irradiation Treatment due to Enhanced IL-11 Signaling. Because Lnk regulates cellular
ated Lnk−/− mice developed B-cell lymphoma prompted us to explore the cellular basis for this phenotype. No differences in survival were detected when WT or Lnk−/− mice were lethally irradiated (10 Gy); however, sublethal irradiation (5 Gy) revealed an enhanced recovery of the blood system in Lnk−/− mice compared with WT animals, as assessed by the numbers of myeloid progenitor colonies and endogenous spleen colony-forming units (S-CFUs) (Fig. S2 A–C). Moreover, under these circumstances we detected a higher rate of recovery of the Lin−Sca1+cKit+ (LSK) population in Lnk−/− mice compared with their WT counterparts (Fig. S2D).
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responses to a variety of cytokines (6), we hypothesized that in the absence of Lnk, hyperactive cytokine signaling might promote the survival of Lnk−/− HSPCs and thereby allow them to escape the effect of irradiation and develop the potential to become cancerous. To identify such cytokines, we tested the ability of a single HSC to create a colony under different cytokine conditions Single, purified Rho123loKit+Sca1+Lin−CD49blo (RLSKa2lo) long-term HSC (LT-HSC) (18) were cultured in serum-free medium with cytokines, or following the withdrawal of SCF, IL-11, or Fms-like tyrosine kinase 3 (Flt3) ligand, which were shown to protect HSPCs from irradiation. Strikingly, the absence of IL-11 profoundly reduced the ability of Lnk−/− HSC
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Fig. 1. Accelerated acute B-cell lymphoma in irradiated Lnk−/− mice with hyperplasia. Hyperplasia in nontreated Lnk−/− mice. (A) Hematopoietic hyperplasia in Lnk−/− BM and spleen as analyzed by Hematoxylin/eosin staining. (B) Kaplan–Meier analysis of lymphoma incidence. WT and Lnk−/− mice (n = 20) were exposed to 5 Gy of whole body irradiation. The percentage of lymphoid tumor-free animals is plotted against time (in months). Log-rank test P = 0.0003. (C) Hematoxylin/eosin staining of representative Lnk−/− lymphoma and B220 and CD3 immunohistochemistry on a lymphoma section is shown. Representative lymph nodes and thymus from Lnk−/− lymphoma are shown in the picture. (D) Spectral Karyotyping (SKY) of Lnk−/− gamma-irradiated/ induced B-cell Lymphoma. Classification-colored chromosoms are shown. Tumor display t (5, 18).
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to create colonies (Fig. 3A). To elucidate the effects of Lnk on IL-11 signaling, colonies derived from single LT-HSCs were stimulated with IL-11 and examined for activating phosphorylation events on the Stat3 transcription factor and the Erk MAP kinase. Lnk−/− colonies exhibited enhanced IL-11 signaling as evaluated by higher Stat3 and Erk phosphorylation in comparison with the WT colonies (Fig. 3B). To understand how IL-11 influences the survival of Lnk−/− HSPCs after irradiation, we examined the sensitivity of LSK populations to apoptosis using an annexin-V assay (17). By this measure, Lnk−/− LSK cells showed enhanced resistance to irradiation in the presence of IL-11 compared with the WT population; however, this resistance of Lnk−/− cells strikingly collapsed in the absence of IL-11 (Fig. S4). Together, these results indicate that Lnk−/− HSPCs are hypersensitive to IL-11 stimulation, suggesting that Lnk is a negative regulator of IL-11 signaling. Most interestingly, our experiments indicate that the resistance of Lnk−/− HSPC to irradiation is IL-11 dependent. Inhibition of IL-11 Signaling Reduces HSPC Recovery After Radiation Treatment and Delays Radiation-Induced B-Cell Lymphoma in Lnk−/− Mice. Based on the observation that IL-11 signaling is enhanced
in Lnk−/− HSPCs and is critical for their survival after irradiation, we examined whether in vivo treatment with a neutralizing antibody to IL-11 could affect HSPC recovery of irradiated Lnk−/− mice. We therefore sublethally irradiated Lnk−/− mice, and then injected them, or not, with anti–IL-11 antibody. Nine days after Louria-Hayon et al.
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Fig. 2. Radio resistance of Lnk−/− HSPCs leads to acute pre–B-cell leukemia in recipient mice. (A) Schematic illustration shows reconstitution competition assay before and after irradiation. BMs from not treated (B) and whole body irradiated WT or Lnk−/− donor mice (C and D) expressing the CD45.2 cell surface marker were mixed with WT competitor cells expressing the CD45.1 cell surface marker, and were transplanted into lethally irradiated WT recipient mice. Lnk−/− and competitor cells were mixed in ratio of 1:1 (1 million cells of each), and WT and competitor cells were mixed in ratio of 10:1 (10 million WT cells and 1 million competitor cells). At 8–32 wk posttransplantation, peripheral blood was withdrawn from recipients and analyzed for the percentage of CD45.2 cells in total leukocytes. Results show means ±+/− SD from at least six animals (C and D). P < 0.001. (E) Differential blood counts (109/L) from three leukemic recipients of 5 Gy–irradiated Lnk−/− donor mice. (*) P < 0.001. (F) Lymphoblast cells in BM and infiltration of lymphoblast cells into the liver of leukemic recipient mice analyzed by Hematoxylin/eosin staining.
this treatment, the BM was flushed and subjected to a myeloid colony assay. Interestingly, anti–IL-11 antibody significantly reduced the capacity of BM cells derived from irradiated Lnk−/− mice to form colonies compared with those from irradiated Lnk−/− mice that were not injected or were treated with antiangiopoietin-1 receptor (Tie2) antibody (Fig. 3C). These data suggest that blocking IL-11 signaling debilitates the recovery of Lnk−/− HSPC after irradiation. Based on these results, we explored the long-term effects of anti– IL-11 treatment on the HSPC population of Lnk−/− mice (Fig. S5). Strikingly, we found that a single dose of anti–IL-11 after irradiation reduced the recovery of Lnk−/− LSK cells and HSCs even when measured after 5 mo (Fig. S5 B and C). Most interestingly, there was a 50% reduction in the number of animals that developed acute B-cell lymphoma when the irradiated mice were injected with anti–IL-11 and a 3-mo delay in the appearance of the disease (Fig. 3D). These results indicate that anti–IL-11 treatment suppresses the recovery of Lnk−/− HSPCs after irradiation and thus reduces the incidence of radiation-related malignancies. Selection for Enhanced Src Homology Region 2 Domain-Containing Phosphatase-2–Erk Signaling in Radiation-Induced Lnk−/− B-Cell Malignancies. IL-11 belongs to the gp130 cytokine family, which
includes IL-11, IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), Cardiotrophin-1 (CT-1), Ciliary neurotrophic factor (CNTF) and IL-27 (19). The binding of cytokines such as IL-11 and IL-6 to the gp130 receptor results in its homodimerization PNAS | December 17, 2013 | vol. 110 | no. 51 | 20601
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(20, 21), leading to activation of the Jak2 tyrosine kinase and phosphorylation of the Stat3 transcription factor. A distinct signaling pathway downstream of the gp130/Jak2 complex involves the recruitment and phosphorylation of Src homology region 2 domain-containing phosphatase-2/protein tyrosine phosphatase nonreceptor type 11 (Shp2/PTPN11), resulting in the stimulation of both Shp2 tyrosine phosphatase activity and its ability to bind the growth factor receptor-bound protein 2 (Grb2) adaptor, and activation of the Erk MAP kinase pathway (22–24). Shp2 is indispensable for embryonic development and hematopoiesis (25, 26) and is also required for maintenance of HSCs and progenitors in adult animals (27). Mutations in Shp2 are found in the developmental disorders Noonan syndrome (28) and leopard syndrome (29), and activating mutations in Shp2 have been implicated in leukemias (30). The aberrant activation of the Erk pathway induced by constitutively active Shp2 mutants is a potential mechanism underlying MPN leukemogenesis (31, 32). The ability of Shp2 to activate the Erk pathway typically requires its phosphatase activity and is potentially enhanced by C-terminal phosphotyrosine sites that bind the Grb2 adaptor in response to gp130 activation (22–24). Because we found that IL11 stimulation of Lnk−/− HSPC resulted in enhanced Erk phosphorylation (Fig. 3), we analyzed the state of the Shp2–Erk pathway in Lnk−/− radiation-related malignancies using splenocytes derived from WT or Lnk−/− mice. Strikingly, splenocytes derived from irradiated Lnk−/− mice that had developed B-cell lymphoma exhibited strongly enhanced Shp2 tyrosine phosphorylation, and association with Grb2 and gp130, compared with splenocytes from irradiated WT mice or cancer-free Lnk−/− mice (Fig. 4A). Consistent with these results, splenocytes and BM cells derived from Lnk−/− mice with radiation-related malignancies exhibited high levels of phospho-Erk (Fig. 4B). These data suggest that Lnk normally inhibits Shp2 signaling downstream of the IL-11–gp130 complex, and indicate that enhanced Shp2/Grb2–pErk signaling is selected during the development of radiation-related malignancies in Lnk−/− mice. Lnk Attenuates Shp2-Based Signaling in Response to IL-11 Stimulation.
Lnk binds and inhibits the Jak2 tyrosine kinase, providing one mechanism by which it may block cytokine signaling. To search for 20602 | www.pnas.org/cgi/doi/10.1073/pnas.1319665110
Fig. 3. Enhanced IL-11 signaling in Lnk−/− HSPCs promotes resistance to irradiation and lymphomas. (A) Single purified LT-HSC (Rho123loKit+Sca1+Lin−CD49blo) were plated in serum-free media with the cytokines IL-11, SCF, and Flt3 ligand or without the indicated cytokine. Seven days after plating the ability to create a colony was indicated. n = 5; (*) P < 0.001. (B) Western blot analysis for the level of pStat3 and pErk in LT-HSC– derived colonies with/without stimulation of IL-11. (C) Lnk−/− mice were treated with IR (5Gy) or together with anti-IL-11 (R&D SYSTEMS 100 μg per mouse) or anti-Tie2 (R&D SYSTEMS 100 μg per mouse). Nine days posttreatment, the BM was flashed and the recovery was determined by number of myeloid colonies. n = 5, (*) P < 0.001. (D) Kaplan–Meier analysis of lymphoma incidence. Lnk−/− mice (n = 20) were exposed to the indicated treatments. The percentage of lymphoid tumor-free animals is plotted against time (in months). Log-rank test P = 0.45.
additional targets for Lnk regulation in IL-11 signaling, we synthesized a panel of phosphotyrosine-containing peptides (33), derived from 13 proteins involved in gp130 signaling, and tested them for binding to the Lnk SH2 domain (list of peptides in Table S1). Far Western blot analysis using a bacterially expressed Lnk SH2 domain revealed direct interactions with phosphotyrosinecontaining peptides derived from the proteins Jak2, Jak3, and Shp2/Ptpn11 (centered on pY62) (Fig. S6 A and B). These three phosphopeptides have a similar sequence motif surrounding the key phosphotyrosine (Fig. S6B). These data confirm the previously reported interaction between Lnk and Jak2 (9) and suggest that Lnk has the potential to interact with Jak3. Interestingly, this strategy revealed the Shp2/Ptpn11 phosphatase as a potential binding partner for Lnk. To validate these findings in a cellular context, we derived an Lnk−/− cell line from lineagedepleted Lnk−/− BM cells, which were transformed by the Philadelphia chromosome BCR-ABL oncogene. We then transfected these Lnk−/− cells with Flag-tagged WT Lnk or with Lnk variants in which the ligand-binding properties of the SH2 domain (R364E) or PH domain (W191A) were inactivated. The pY62 peptide of Shp2 pulled down WT full-length Lnk and the PH-mutated Lnk, but not the SH2-mutated protein, indicating that the ability of full-length Lnk to bind phosphotyrosine-containing peptides is mediated by the SH2 domain (Fig. S6C). Using these cells, we also found that WT and PH domain-mutated (W191A) Lnk proteins immunoprecipitated (IP) with endogenous Shp2, whereas the Lnk SH2 domain mutant (R364E) did not (Fig. 4C). To further investigate the influence of IL-11 signaling on the Lnk–Shp2 interaction, the Lnk−/− cell line was transfected with a WT Lnk cDNA fused to an estrogen receptor (ER) promoter inducible by hydroxytamoxifen (HTO). Upon HTO treatment, Lnk was inducibly expressed and found to coprecipitate with endogenous Shp2. Stimulation of these cells with IL-11 increased the Lnk–Shp2 interaction, which reached a maximum binding level 15–20 min after cytokine treatment (Fig. 4D). Mass spectrometry analysis of Shp2 from Lnk−/− cells revealed that Y62 of Shp2 is indeed phosphorylated after IL-11 stimulation (Fig. S7). Intriguingly, Y62 is mutated to aspartate in rare cases of Noonan syndrome (34), and the adjacent D61 is mutated to glycine in Noonan syndrome and somatically mutated to tyrosine in Noonan-related leukemia. Louria-Hayon et al.
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These residues are therefore implicated in the regulation of Shp2 function. The preceding experiments reveal a consensus phosphopeptide motif recognized by the Lnk SH2 domain, and indicate that Lnk has two potential targets in the gp130–Jak2–Shp2 complex induced by IL-11, where it acts to suppress Shp2 phosphorylation, Grb2 binding, and Erk activation. Hence in the absence of Lnk, activation of both the Stat3 and Erk pathways may contribute to the radioresistance phenotype (Fig. 4E). Discussion Alterations in cytokine signaling and resulting survival pathways are known to participate in cancer development, but the roles of cytokines in resistance to therapy are less certain. To address this issue, we have used a mouse model that lacks the inhibitor Lnk, which has allowed us to investigate the phenotypic consequence of enhanced cytokine signaling in the context of irradiation. As Lnk is most abundantly expressed in HSPCs (10), this strategy was designed to investigate the physiological effects of irradiation on hematopoietic stem cells exposed to aberrantly high levels of cytokine signaling, and conversely to identify cytokines that must be kept in check to prevent an adverse hematopoietic response to irradiation. Our results indicate that in the absence of Lnk, hyperactive signaling is elicited by IL-11, and that this in turn causes resistance to irradiation and the development of radiation-related B-cell malignancies. The relative radio resistance of Lnk−/− HSPC was suppressed when this pathway was blocked by a neutralizing Louria-Hayon et al.
Fig. 4. A selection for enhanced Shp2–Erk pathway in Lnk−/− malignancies. (A) Interactions between IP Shp2 with Grb2 and gp130 in splenocytes derived from irradiated WT or lymphoma-free irradiated Lnk −/− or Lnk −/− lymphoma. (B) Western blot analysis of pERK in splenocytes derived from irradiated WT or KO Lnk lymphoma (B, Left), or of BM derived from recipient of irradiated WT or recipient of irradiated KO Lnk, which developed B-cell acute lymphoblastic leukemia (BALL) (B, Right). (C) Interaction between IP Flag-Lnk and endogenous Shp2. (D) Interaction between IP Flag-Lnk and Shp2 after IL-11 stimulation. (E) Model for Lnk regulation of IL-11 signaling. In response to IL-11 stimulation, both Jak2 and Shp2 are phsphorylated to increased Stat3 and ERK activity. Lnk–SH2 domain interacts with pYJAK2 and pY62Shp2 to down-regulate their activity. Inactivation of Lnk leads to enhanced Jak–Stat and Shp2–Grb2 pathways, which increase resistance to irradiation treatment and thus enlarge the tumor incidents.
antibody against IL-11 in vivo (Fig. 3C), or by IL-11 deprivation ex vivo (Fig. S4). Collectively, these data argue that Lnk−/− cells acquire radio resistance, at least in part due to enhanced IL11 signaling. IL-11 signals through the gp130 cytokine receptor and has been reported to increase the survival of irradiated mice (35). This activity has been primarily attributed to the Jak–Stat pathway. Lnk is known to inhibit Jak2, and our present data suggest that it also suppresses tyrosine phosphorylation of Shp2 and downstream signaling to Erk induced by IL-11. Lnk potentially exerts its inhibitory effect by binding through its SH2 domain to related pTyr sites on both Jak2 and Shp2. Our data are consistent with a scheme in which IL-11 stimulation of HSPCs induces hyperactivation of a Jak2–Shp2 pathway leading to enhanced Erk activation, which together with up-regulated Jak2–pStat3 signaling (Fig. 3B) provides a mechanism by which Lnk deficiency gives a selective survival advantage after irradiation treatment and may thus promote the development of radiation-resistant cancer (Fig. 3D). Lnk is most strongly expressed in HSPCs, which potentially provide a source for leukemic stem cells, as noted in cases of human acute myeloid leukemia and in a variety of leukemia mouse models (36, 37). It will be of interest to explore whether the resistance mechanism we have uncovered for irradiationinduced death of HSPCs may also apply to the ability of leukemic stem cells to survive therapeutic treatments. In this vein, it will be important to explore the relevance of IL-11 PNAS | December 17, 2013 | vol. 110 | no. 51 | 20603
CELL BIOLOGY
WCL
Shp2
IP: anti Flag
IP: anti Shp2
pERK
signaling for survival of human leukemic populations and to understand the effects of mutations in human Lnk on responses to radiotherapy.
were plated in methylcellulose (MethoCult M3434) containing cytokines, including Epo (Stem Cell Technologies Inc.), and incubated at 37 °C, 5% CO2, 100% humidity.
Experimental Procedures
Blood Counts. A single 60-μl blood sample was collected in a capillary pipette flushed with EDTA. Samples were analyzed with Roche Hitachi 917 and Hemavet 950FS (Drew Scientific).
Mice. Lnk−/− mice (6) were backcrossed 10 generations into C57BL/6J-Ly5.2Gpi1b/b. Mice were housed and manipulated according to the guidelines of the Canadian Council on Animal Care and experiments were approved by the Toronto Centre for Phenogenomics animal care committee (AUP #0011a-H). Flow Cytometry. Staining and enrichment procedure for stem and progenitor population sorting and analysis were performed as previously described (17) and in SI Experimental Procedures. Cell Culture. Cells were incubated at 37 °C in 5% CO2 in U-bottom microtiter wells (Nunclon) in 100 μL Iscove’s modified Dulbecco’s medium containing 7.5 × 10–5 M a-thioglycerol, 4% FBS, 0.1% BSA, 5 μg/mL transferrin, 5 μg/mL insulin, 50 ng/mL c-kit ligand (KL), 50 ng/mL Flt3 ligand (FL), 10 ng/mL IL-11, and 10 ng/mL IL-7 conditioned medium all as detailed in ref. 17. For culture of single cells, sorted cells were plated at limiting dilution, and wells containing exactly one cell were identified visually after 18 h culture. Lnk−/− cell lines were cultured in RPMI medium supplemented with 10% FBS in the presence or absence of IL-11 or IL-6. The conditional ER system was established according to a previously described procedure (38).
Pathology. Mice were killed using a combination of CO2 and O2 and tissues collected and fixed in 10% neutral buffered formalin or Bouin’s fixative. Tissue sections (4 μm) were prepared and stained with Hematoxylin and Eosin. Protein Expression and Purification. Lnk–SH2 proteins were purified according to a previously described procedure (39). Peptide Synthesis. Synthetic peptides were prepared using Fmoc (9-fluorenyl methoxycarbonyl) solid-phase chemistry. Phosphotyrosine was incorporated using the N-fluorenylmethyloxycarbonyl-O-phospho-L-tyrosine derivative. Peptides were purified using reverse-phase HPLC, and the authenticity was confirmed by mass spectrometry.
In Vitro Hematopoietic Progenitor Colony Assays. Single cell suspensions were prepared from BM aspirates under sterile conditions. Triplicate samples of 104 or 105 cells (for CFU-granulocyte, erythrocyte, monocyte, megakaryocyte)
ACKNOWLEDGMENTS. We thank Adrian Pasculescu for statistical analysis. We thank Pier-Andreé Penttilä and Annie Bang for assistance with flow cytometry, and Zorana Berberovic for help with blood count procedures. We thank Cristina Virage, Geraldine Mbamalu, Mary Barbara, and Oleg Leikin for excellent technical assistance. This research was supported by grants from the Canadian Institutes of Health Research (CIHR MOP-6849) and the Terry Fox Foundation (Team Grant TFF 10).
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Louria-Hayon et al.
