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

aLunenfeld-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) The Lnk (Sh2b3) adaptor dampens the response of hema- exhibit hyperactivation of the Jak–Stat and Erk MAP kinase − − topoietic stem cells and progenitors (HSPCs) to a variety of pathways. Previous studies have shown that Lnk / hematopietic − − by inhibiting JAK2 signaling. As a consequence, Lnk / mice develop stem cells (HSCs) have increased self-renewal and engraftment − − hematopoietic hyperplasia, which progresses to a phenotype resem- capacities and that Lnk / animals have an expanded HSC − − bling the nonacute phase of myeloproliferative neoplasm. In addition, compartment (10, 11). Indeed, aged Lnk / mice exhibit a mye- Lnk mutations have been identified in human myeloproliferative loproliferative neoplasm (MPN)-like phenotype (12), consistent neoplasms and acute leukemia. We find that Lnk suppresses the with the discovery of mutations in the human LNK in some development of radiation-induced acute B-cell malignancies in cases of essential thrombocythemia, primary myelofibrosis, and mice. Lnk-deficient HSPCs recover more effectively from irradia- −/− erythrocytosis (13, 14). Interestingly, LNK mutations have also tion than their wild-type counterparts, and this resistance of Lnk been described in patients with acute T-cell leukemia (15, 16), HSPCs to radiation underlies the subsequent emergence of leuke- implicating LNK deficiency as being involved in leukemias of mia. A search for the mechanism responsible for radiation resis- both myeloid and lymphoid origin. tance identified the IL-11 as being critical for the ability of −/− − − Based on the fact that Lnk mice exhibit excessive cytokine Lnk / HSPCs to recover from irradiation and subsequently be- signaling and a hyperproliferative disease, we considered the pos- come leukemic. In IL-11 signaling, wild-type Lnk suppresses tyrosine −/− phosphorylation of the Src homology region 2 domain-containing sibility that Lnk mice might provide a useful model in which to phosphatase-2/protein tyrosine phosphatase nonreceptor type study the role of cytokines in radio resistance and radiotherapy- 11 and its association with the receptor-bound related leukemia. protein 2, as well as activation of the Erk MAP kinase pathway. Results Indeed, Src homology region 2 domain-containing phosphatase-2 fi has a binding motif for the Lnk Src Homology 2 domain that is Accelerated Acute B-Cell Lymphoma in Lnk-De cient Mice After fi −/− phosphorylated in response to IL-11 stimulation. IL-11 therefore Ionizing Radiation. The nding that Lnk mice do not usually drives a pathway that enhances HSPC radioresistance and radia- develop acute malignancy despite their hyperproliferative disorder tion-induced B-cell malignancies, but is normally attenuated by the inhibitory adaptor Lnk. Significance

cancer | lymphoma | survival | gp130 Recurrence of cancer in patients treated with radiation therapy infers that tumor cells have the capacity to escape the lethal adiation therapy is used to treat a significant proportion of effects of irradiation. Surviving tumor cells rely on signaling Rpatients with cancers such as lymphoma but can also have pathways triggered by hematopoietic interleukins in a mecha- undesirable consequences. For example, some tumor cells may nism that is poorly understood. We find that the adaptor escape the initial lethal effects of irradiation, resulting in radio protein Lnk is key in this process, acting as a negative regulator resistance and disease recurrence. In addition, a second ma- of interleukin 11 survival signaling. In Lnk knockout derived lignant neoplasm (SMN) can develop years or decades after mice, hematopoietic stem cells have a marked level of irradia- CELL BIOLOGY treatment (1). In this context, there is increasing evidence that tion resistance, causing B-cell malignancies. Such observations cytokines can influence cell malignancy (2) and that mutations are consistent with the physiology seen in a subset of human affecting involved in cytokine signaling can influence leukemia patients that have Lnk gene mutations. Inhibition of cancer development (3). However, little is known about the interleukin 11 signaling in the mouse model circumvented this potential roles of the cytokine network in radio resistance and irradiation-resistance phenomenon and is suggestive of a po- radiotherapy-related leukemia. tential for therapeutic intervention. Lnk and the closely related proteins PH domain-containing Author contributions: I.L.-H. and T.P. designed research; I.L.-H., C.F., J.R., G.G., J.-P.L., and adapter protein and Src homology 2 B (SH2-B) form a subfamily M.M. performed research; I.L.-H., J.J., M.M.K., H.A.A., and R.H. contributed new reagents/ of SH2 domain-containing proteins. The Lnk adaptor protein, 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. which is primarily expressed in the hematopoietic system, con- and T.P. wrote the paper. tains an N-terminal proline-rich region, a pleckstrin homology The authors declare no conflict of interest. (PH) domain, an SH2 domain, and a C-terminal sequence with This article is a PNAS Direct Submission. potential tyrosine phosphorylation sites (4, 5). The hematopoietic 1To whom correspondence should be addressed. E-mail: [email protected]. −/− cells of Lnk mice (6) are hypersensitive to a range of cytokines, 2Deceased August 7, 2013. including interleukin (IL)-3, IL-7, erythropoietin (Epo), stem cell This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. factor (SCF), and thrombopoietin (TPO) (6–9), and consequently 1073/pnas.1319665110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1319665110 PNAS | December 17, 2013 | vol. 110 | no. 51 | 20599–20604 Downloaded by guest on October 4, 2021 − − (6) suggests that additional genetic aberrations are required To explore whether Lnk / HSCs are protected from irra- for disease progression, which might be provided by irradiation. diation treatment in vivo, we used a competitive repopulation − − − − To test this possibility, we subjected Lnk / mice with hemato- assay in which donor bone marrow (BM) cells from Lnk / or poietic hyperplasia (Fig. 1A and Fig. S1) to a single sublethal WT mice (expressing the marker CD45.2) were mixed with WT dose of 500 rad (5 Gy) gamma irradiation. Eight to 10 mo after competitor cells (CD45.1) and then transplanted into lethally − − gamma irradiation, 45% of Lnk / mice developed acute lym- irradiated WT mice (CD45.1) and measured for their relative phoma (P < 0.0005) (Fig. 1 B and C). Histological analysis abilities to compete for BM repopulation (Fig. 2 A–D). Before revealed that the normal architecture of the thymus, lymph nodes, transplantation, the donor animals were either untreated or −/− and spleen was replaced by a diffuse sheet of large (25–35 μm) whole body irradiated. Lnk HSCs have a 10-fold higher fi repopulation capability than WT HSCs (9, 10). To compensate neoplastic lymphocytes. The neoplastic lymphocytes also in l- −/− trated the liver and lung, and further immunostaining revealed for this relative advantage of the mutant cells, we mixed Lnk and competitor cells in a 1:1 ratio, but WT and competitor cells that the neoplastic cells were B-cell derived (Fig. 1C). This − − − − B / fi phenotype was not found in Lnk / mice that had not been ir- in a 10:1 ratio (Fig. 2 ). Strikingly, Lnk HSCs were signi - radiated nor in irradiated wild-type (WT) mice, with the excep- cantly more resistant to irradiation than WT HSCs, as assessed B in the competitive repopulation assay (Fig. 2 C and D). In ad- tion of one lymphoma in a WT irradiated animal (Fig. 1 ), which −/− was T cell in nature (P < 0.0005). Spectral karyotyping and array dition, for each irradiated Lnk marrow transplanted, 1 out of 10 recipient mice developed early B-cell precursor acute lym- comparative genomic hybridization analysis of three gamma- − − phoblastic leukemia 3 to 8 mo after bone marrow transplantation irradiation–induced B-cell lymphomas from Lnk / mice revealed (BMT) (Fig. 2 E and F and Fig. S3). These results identify Lnk as genomic aberrations and chromosomal translocations, including a negative regulator of HSC recovery after gamma irradiation t (5, 17) translocations, indicating that the tumors were geneti- treatment and support the hypothesis that irradiation induces the D − − cally unstable and clonal in origin (Fig. 1 ). These data suggest transformation of a rare Lnk / hematopoietic stem cells and that murine Lnk normally suppresses the emergence of B-cell progenitors (HSPCs) population, which leads to radiation-related malignancy following irradiation. B-cell malignancies. − − / − − Superior Recovery of Lnk Hematopoietic Stem Cells and Progen- Lnk / HSPCs Are Protected from Gamma Irradiation Treatment due itors After Gamma Irradiation Leads to Early B-Cell Precursor Acute to Enhanced IL-11 Signaling. Because Lnk regulates cellular Lymphoblastic Leukemia in Recipient Mice. Our finding that irradi- −/− responses to a variety of cytokines (6), we hypothesized that in ated Lnk mice developed B-cell lymphoma prompted us to the absence of Lnk, hyperactive cytokine signaling might pro- − − explore the cellular basis for this phenotype. No differences in mote the survival of Lnk / HSPCs and thereby allow them to −/− survival were detected when WT or Lnk mice were lethally escape the effect of irradiation and develop the potential to irradiated (10 Gy); however, sublethal irradiation (5 Gy) revealed become cancerous. To identify such cytokines, we tested the −/− an enhanced recovery of the blood system in Lnk mice com- ability of a single HSC to create a colony under different cyto- + + − pared with WT animals, as assessed by the numbers of myeloid kine conditions Single, purified Rho123loKit Sca1 Lin CD49blo progenitor colonies and endogenous spleen colony-forming units (RLSKa2lo) long-term HSC (LT-HSC) (18) were cultured in (S-CFUs) (Fig. S2 A–C). Moreover, under these circumstances serum-free medium with cytokines, or following the withdrawal − + + we detected a higher rate of recovery of the Lin Sca1 cKit (LSK) of SCF, IL-11, or Fms-like tyrosine kinase 3 (Flt3) ligand, which − − population in Lnk / mice compared with their WT counter- were shown to protect HSPCs from irradiation. Strikingly, the − − parts (Fig. S2D). absence of IL-11 profoundly reduced the ability of Lnk / HSC

Lnk-/- Lnk-/- A WT hyperplasia1 hyperplasia2 B

BM

Spleen 60 80 100 C H&E CD3 B220 LN 40

% tumor free Fig. 1. Accelerated acute B-cell lymphoma in irra- − − diated Lnk / mice with hyperplasia. Hyperplasia in Thymus WT −/− KO Lnk nontreated Lnk mice. (A) Hematopoietic hyper- − − WT+IR plasia in Lnk / BM and spleen as analyzed by He- KO Lnk+IR 020 matoxylin/eosin staining. (B) Kaplan–Meier analysis Lung −/− 0 5 10 15 20 of lymphoma incidence. WT and Lnk mice (n = 20) months were exposed to 5 Gy of whole body irradiation. D The percentage of lymphoid tumor-free animals is Liver plotted against time (in months). Log-rank test P = 0.0003. (C) Hematoxylin/eosin staining of represen- tative Lnk−/− lymphoma and B220 and CD3 immuno- spleen histochemistry on a lymphoma section is shown. − − Representative lymph nodes and thymus from Lnk / Lymph nodes lymphoma are shown in the picture. (D) Spectral − − Karyotyping (SKY) of Lnk / gamma-irradiated/ induced B-cell Lymphoma. Classification-colored Thymus Lung chromosoms are shown. Tumor display t (5, 18).

20600 | www.pnas.org/cgi/doi/10.1073/pnas.1319665110 Louria-Hayon et al. Downloaded by guest on October 4, 2021 A B WT CD45.1 WT/ Lnk-deficient CD45.2 Not treated competitor NT, 3Gy or 5Gy irradiated Not treated 100

80

60

BM 40 WT

20 KO Lnk BMT % donor derived WT recipient CD45.1 0 Lethal irradiated 8 16 24 32 Weeks

C 3Gy IR D 5Gy IR 100 100 WT 80 80 KO Lnk

60 WT 60 − − Fig. 2. Radio resistance of Lnk / HSPCs leads to 40 KO Lnk 40 acute pre–B-cell leukemia in recipient mice. (A) Schematic illustration shows reconstitution compe- 20 20 % donor derived % donor derived tition assay before and after irradiation. BMs from 0 0 not treated (B) and whole body irradiated WT or − − 8 16 24 32 8 16 24 32 Lnk / donor mice (C and D) expressing the CD45.2 Weeks Weeks cell surface marker were mixed with WT competitor cells expressing the CD45.1 cell surface marker, and were transplanted into lethally irradiated WT re- E F − − 200 cipient mice. Lnk / and competitor cells were mixed BM 180 in ratio of 1:1 (1 million cells of each), and WT and 160 competitor cells were mixed in ratio of 10:1 (10 140 million WT cells and 1 million competitor cells). At Leukemia 8–32 wk posttransplantation, peripheral blood was 120 withdrawn from recipients and analyzed for the 100 percentage of CD45.2 cells in total leukocytes. + − 10^9/L 10^9/L 80 Liver Results show means ± / SD from at least six animals 60 (C and D). P < 0.001. (E) Differential blood counts (109/L) from three leukemic recipients of 5 Gy–irra- 40 − − diated Lnk / donor mice. (*) P < 0.001. (F) Lym- 20 phoblast cells in BM and infiltration of lymphoblast 0 cells into the liver of leukemic recipient mice ana- WBC NE Ly MO Ba Eo lyzed by Hematoxylin/eosin staining.

to create colonies (Fig. 3A). To elucidate the effects of Lnk on this treatment, the BM was flushed and subjected to a myeloid IL-11 signaling, colonies derived from single LT-HSCs were colony assay. Interestingly, anti–IL-11 antibody significantly re- − − stimulated with IL-11 and examined for activating phosphory- duced the capacity of BM cells derived from irradiated Lnk / lation events on the Stat3 transcription factor and the Erk MAP mice to form colonies compared with those from irradiated − − − − kinase. Lnk / colonies exhibited enhanced IL-11 signaling as Lnk / mice that were not injected or were treated with anti- evaluated by higher Stat3 and Erk phosphorylation in compari- angiopoietin-1 receptor (Tie2) antibody (Fig. 3C). These data son with the WT colonies (Fig. 3B). suggest that blocking IL-11 signaling debilitates the recovery of − − − − To understand how IL-11 influences the survival of Lnk / Lnk / HSPC after irradiation. HSPCs after irradiation, we examined the sensitivity of LSK Based on these results, we explored the long-term effects of anti– − − populations to apoptosis using an annexin-V assay (17). By this IL-11 treatment on the HSPC population of Lnk / mice (Fig. S5).

− − CELL BIOLOGY measure, Lnk / LSK cells showed enhanced resistance to irra- Strikingly, we found that a single dose of anti–IL-11 after irradia- − − diation in the presence of IL-11 compared with the WT popula- tion reduced the recovery of Lnk / LSK cells and HSCs even when − − tion; however, this resistance of Lnk / cells strikingly collapsed measured after 5 mo (Fig. S5 B and C). Most interestingly, there in the absence of IL-11 (Fig. S4). Together, these results indi- was a 50% reduction in the number of animals that developed − − cate that Lnk / HSPCs are hypersensitive to IL-11 stimulation, acute B-cell lymphoma when the irradiated mice were injected with suggesting that Lnk is a negative regulator of IL-11 signaling. anti–IL-11 and a 3-mo delay in the appearance of the disease (Fig. Most interestingly, our experiments indicate that the resistance 3D). These results indicate that anti–IL-11 treatment suppresses − − − − of Lnk / HSPC to irradiation is IL-11 dependent. the recovery of Lnk / HSPCs after irradiation and thus reduces the incidence of radiation-related malignancies. Inhibition of IL-11 Signaling Reduces HSPC Recovery After Radiation − − Treatment and Delays Radiation-Induced B-Cell Lymphoma in Lnk / Selection for Enhanced Src Homology Region 2 Domain-Containing Mice. Based on the observation that IL-11 signaling is enhanced Phosphatase-2–Erk Signaling in Radiation-Induced Lnk−/− B-Cell − − in Lnk / HSPCs and is critical for their survival after irradiation, Malignancies. IL-11 belongs to the gp130 cytokine family, which we examined whether in vivo treatment with a neutralizing an- includes IL-11, IL-6, leukemia inhibitory factor (LIF), oncostatin − − tibody to IL-11 could affect HSPC recovery of irradiated Lnk / M (OSM), Cardiotrophin-1 (CT-1), Ciliary neurotrophic factor − − mice. We therefore sublethally irradiated Lnk / mice, and then (CNTF) and IL-27 (19). The binding of cytokines such as IL-11 injected them, or not, with anti–IL-11 antibody. Nine days after and IL-6 to the gp130 receptor results in its homodimerization

Louria-Hayon et al. PNAS | December 17, 2013 | vol. 110 | no. 51 | 20601 Downloaded by guest on October 4, 2021 A B WT KO Lnk IL-11 - + - + 100 wt pSTAT3 80

60 KO Lnk STAT3 40 pERK

% of maximum 20

0 − − Fig. 3. Enhanced IL-11 signaling in Lnk / HSPCs pro- All -IL-11 -SCF -Flt3 ERK ligand motes resistance to irradiation and lymphomas. (A) fi lo + + − lo C D Single puri ed LT-HSC (Rho123 Kit Sca1 Lin CD49b ) KO Lnk BM-CFU-GM 9 days post treatment were plated in serum-free media with the cytokines 120 IL-11, SCF, and Flt3 ligand or without the indicated 100 cytokine. Seven days after plating the ability to create acolonywasindicated.n = 5; (*) P < 0.001. (B)Western 80 blot analysis for the level of pStat3 and pErk in LT-HSC– 60 derived colonies with/without stimulation of IL-11. (C) Lnk−/− mice were treated with IR (5Gy) or together 40 with anti-IL-11 (R&D SYSTEMS 100 μg per mouse) or Colony numbers 20 anti-Tie2 (R&D SYSTEMS 100 μg per mouse). Nine days % tumor free fl 0 posttreatment, the BM was ashed and the recovery NT IR IR IR was determined by number of myeloid colonies. n = 5, +anti-IL-11 +anti-Tie2 (*) 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). months Log-rank test P = 0.45.

(20, 21), leading to activation of the Jak2 tyrosine kinase and additional targets for Lnk regulation in IL-11 signaling, we syn- phosphorylation of the Stat3 transcription factor. A distinct thesized a panel of phosphotyrosine-containing peptides (33), signaling pathway downstream of the gp130/Jak2 complex in- derived from 13 proteins involved in gp130 signaling, and tested volves the recruitment and phosphorylation of Src homology them for binding to the Lnk SH2 domain (list of peptides in Table region 2 domain-containing phosphatase-2/protein tyrosine S1). Far Western blot analysis using a bacterially expressed Lnk phosphatase nonreceptor type 11 (Shp2/PTPN11), resulting in SH2 domain revealed direct interactions with phosphotyrosine- the stimulation of both Shp2 ty