ALBERT

Description: Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase with important roles in hematopoietic progenitor cell survival and proliferation. It is mutated in approximately one-third of AML patients, mostly by internal tandem duplications (ITDs). Adaptor protein Lnk is a negative regulator of hematopoietic cytokine signaling. In the present study, we show that Lnk interacts physically with both wild-type FLT3 (FLT3-WT) and FLT3-ITD through the SH2 domains. We have identified the tyrosine residues 572, 591, and 919 of FLT3 as phosphorylation sites involved in direct binding to Lnk. Lnk itself was tyrosine phosphorylated by both FLT3 ligand (FL)–activated FLT3-WT and constitutively activated FLT3-ITD. Both shRNA-mediated depletion and forced overexpression of Lnk demonstrated that activation signals emanating from both forms of FLT3 are under negative regulation by Lnk. Moreover, Lnk inhibited 32D cell proliferation driven by different FLT3 variants. Analysis of primary BM cells from Lnk-knockout mice showed that Lnk suppresses the expansion of FL-stimulated hematopoietic progenitors, including lymphoid-primed multipotent progenitors. The results of the present study show that through direct binding to FLT3, Lnk suppresses FLT3-WT/ITD–dependent signaling pathways involved in the proliferation of hematopoietic cells. Therefore, modulation of Lnk expression levels may provide a unique therapeutic approach for FLT3-ITD–associated hematopoietic disease.

Description: Multiple myeloma (MM) is a neoplasm of plasma cells that often remains fatal despite the use of high-dose chemotherapy with hematopoietic stem cell transplantation. In the clinical setting, the introduction of novel agents, such as proteasome inhibitors and immunomodulatory drugs, has improved the clinical outcomes of both patients with newly diagnosed MM and patients with advanced MM. However, most patients eventually relapse and develop drug resistance. T-LAK cell-originated protein kinase (TOPK), also known as PDZ-binding kinase (PBK), is a mitogen-activated protein kinase kinase (MAPKK)-like serine/threonine kinase that plays a critical role in many cellular functions, such as cell proliferation, apoptotic cell death, and inflammation, in normal tissues. Because the expression of TOPK is up-regulated during mitosis and is activated by the Cdk1/cyclin B1 complex, TOPK is thought to have a role in cytokinesis. While the expression of TOPK is very low in most normal human tissues except for testis and placenta, it is overexpressed in various malignant neoplasms, indicating its crucial role in tumorigenesis. Phosphorylation of TOPK leads to the activation of the MAPK signaling pathway including p38 and Ras extracellular signal-regulated kinase (ERK). Moreover, TOPK interacts with p53 tumor suppressor protein and inhibits its function. Ribosomal protein S6 kinase (RSK2) is a downstream target of the ERK/MAPK signaling cascade and it has a pivotal role in cell survival and proliferation. Recent studies suggest that RSK2 inhibition induces apoptotic cell death and sensitizes MM cells to lenalidomide. Suppression of p53 function is also involved in MM progression. Taken together, these data suggest that TOPK might be an attractive target for new therapeutic agents against this incurable hematological malignancy. HI-TOPK-032, which is a potent and specific inhibitor of TOPK, occupies the ATP-binding site of TOPK and thereby suppresses TOPK kinase activity. In the present study, we investigated the role of TOPK/PBK in MM as a potential therapeutic target by using HI-TOPK-032. MTSand trypan blue dye exclusion assays showed that HI-TOPK-032 inhibited the proliferation of various MM cell lines, including U266, RPMI8226, MM1.S, OPM-2, and KMS-11, in a dose- (0 to 10 mM) and time- (0 to 72 h) dependent manner. To examine the mechanisms behind the growth inhibition effect induced by HI-TOPK-032, assays for apoptotic cell death were performed; these assays demonstrated that HI-TOPK-032 induced both early and late apoptosis in MM cells. To investigate the molecular mechanisms of HI-TOPK-032-induced cell death in MM cells, the expression of various cell death-associated proteins and down-stream molecules of TOPK was examined. Western blotting analysis showed that HI-TOPK-032 arrested cell growth and induced apoptotic cell death in MM cells in a dose-dependent manner by reducing t he phosphorylation of ERK and RSK2, thereby reducing the expression of the target molecules of RSK2, i.e., MCL1 and c-Myc. Moreover, HI-TOPK-032 induced p53 expression in a dose-dependent manner. We next examined the effects of HI-TOPK-032 on bortezomib (BTZ)-resistant MM cells, which represent an urgent issue in clinics and for which a therapeutic solution is important. Interestingly, HI-TOPK-032 inhibited the proliferation of both BTZ-sensitive wild-type KMS cells and BTZ-resistant KMS cells, suggesting that BTZ resistance can be overcome by targeting TOPK. Because our results showed that HI-TOPK-032 reduced the phosphorylation of RSK2, and previous studies have suggested that RSK2 inhibition sensitized MM cells to lenalidomide, we next studied the effects of HI-TOPK-032 in combination with lenalidomide on MM cell growth. HI-TOPK-032 and lenalidomide synergisticallyinduced growth arrest in not only lenalidomide-sensitive MM cells, but also in lenalidomide-resistant cells. To determine whether HI-TOPK-032 can re-sensitize BTZ-resistant cells to the anti-MM activity of BTZ, the effects of the combination of HI-TOPK-032 and BTZ were tested using an MTS assay. Interestingly, HI-TOPK-032 was able to re-sensitize BTZ-resistant MM cells to BTZ. These results indicate that the inhibition of TOPK may serve as an attractive therapeutic option for both patients with BTZ- or lenalidomide-resistant MM. In conclusion, these data suggest that TOPK/PBK can be a promising molecular target for the treatment of MM. Disclosures Kizaki: Nippon Shinyaku Co., Ltd.: Research Funding; Ono Phranacutical Co., Ltd.: Consultancy; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Phrarmaceutical Co., Ltd.: Research Funding.

Description: Abstract 3406 Adaptor protein Lnk negatively regulates not only several hematopoietic cytokine receptors including MPL, EpoR and c-Kit, but also non-receptor tyrosine kinases such as JAK2 and Src. Our previous studies demonstrated that Lnk, when expressed in hematopoietic cell lines, binds and regulates the mutant proteins, JAK2V617F and MPLW515L. Recent in vivo studies have shown that Lnk has an important role in the development of myeloproliferative neoplasms. These data suggest that Lnk may have the ability to inhibit constitutively activated signaling pathways in hematopoietic malignancies. However, how Lnk can attenuate the activity of Bcr-Abl is unclear. In the present study, we tested the hypothesis that Lnk might play a role in regulating Bcr-Abl function. In order to assess if Lnk can inhibit the proliferation of Bcr-Abl-positive hematopoietic cells, Bcr-Abl-expressing BaF3 cells were stably transfected with either Lnk (BaF3/Bcr-Abl/Lnk) or vector only (BaF3/ Bcr-Abl). Colony-formation assays revealed that Lnk significantly inhibited the proliferation of Bcr-Abl-expressing BaF3 cells. Similarly, overexpression of Lnk inhibited growth in the human CML cell line, K562. To determine the cause of growth inhibition by Lnk, assays for apoptosis were performed. Annexin V staining demonstrated that Lnk overexpression induced apoptosis in Bcr-Abl-expressing BaF3 cells. Western blotting analysis of protein lysates from BaF3/ Bcr-Abl /Lnk cells and BaF3/ Bcr-Abl cells found that Lnk-mediated growth inhibition was associated with downregulation of the Stat5 signaling pathway, but not associated with MAPK and PI3K signaling pathways. In addition, experiments in 293T cells expressing Bcr-Abl and Stat5 with either wild-type Lnk or SH2 mutant Lnk revealed that wild-type Lnk, but not SH2 mutant Lnk, inhibited phosphorylation of Stat5. Interestingly, Lnk inhibited Bcr-Abl-induced Stat5 phosphorylation in a dose-dependent manner. These data suggest that the SH2 domain of Lnk is essential for Lnk–mediated downregulation of the Stat5 signaling pathway in Bcr-Abl-positive cells. Taken together, our data suggest that Lnk inhibits Bcr-Abl-induced cell proliferation by attenuating the Stat5 signal transduction and may become a therapeutic target for Bcr-Abl-positive leukemias such as chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Substantial efficacy has been demonstrated with bortezomib (Bor)-containing regimens for the treatment of refractory/relapsed (RR) patients with transplant-ineligible (TI) multiple myeloma (MM). However, the dosing schedules for induction therapy and the significance of maintenance therapy using Bor in clinical settings have yet to be standardized. Discontinuations and dose reduction of Bor owing to severe adverse events (AEs) such as peripheral neuropathy (PN) and infection are frequent causes of adjustments to the standard 9 cycles of Bor/dexamethasone (BD) therapy used for induction in clinical practice. We have previously demonstrated that a change in Bor administration to a schedule of weekly or longer intervals had similar or greater efficacy for patients with RR-MM including transplant-eligible (Tokuhira M, et al. Leuk Res. 35:591-7, 2011). To investigate the efficacy of this modified Bor dosing schedule in the treatment focusing of TI-RR-MM, we retrospectively analyzed 22 patients receiving weekly-BD induction followed by a modified schedule of biweekly or longer intervals using Bor as maintenance therapy in a single institution. Methods: Data on the 22 TI-RR-MM patients treated with BD therapy in our institution were retrospectively analyzed. The induction therapy consisted of intravenous injections of Bor (1.3 mg/m2) on days 1, 8, 15, and 22 in combination with dexamethasone (10–20 mg) on days 1 and 2 after Bor infusion, every 5 weeks. After BD induction, Bor maintenance was initiated with a schedule of biweekly or longer intervals. The dose of BD could be reduced based on AEs or other social aspects at the discretion of the treating physician. Overall survival (OS) was defined as the period between BD initiation of treatment to the last follow-up. Results: The median age of patients at the time of BD induction was 72.6 years (range, 49–84 years), and the subtypes of MM were the IgG type in 14 patients, IgA type in 6 patients, and BJP in 2 patients. Twelve patients were men and 10 were women. On the basis of the International Staging System classification, the clinical stage was I in 3 patients, II in 16 patients, and III in 3 patients. The median number of prior treatment regimens was 2.0 (range, 1–6). The median follow-up duration was 4.3 years (inter-quartile range, 3.0–7.8 years). Ten patients were alive, and 12 patients had died at the time of reporting. The median duration from the start of the first line treatment to BD initiation was 1.8 years (range, 0.1–14.8 years). The median number of BD induction cycles was 2.0 (range, 1–6), and 8 patients (36%) achieved a partial response (PR), although the other 14 patients (64%) showed stable disease or only a minimal response. Although AEs such as PN and gastrointestinal tract symptoms developed in 10 of 22 patients during BD induction, all 22 patients could move on to the maintenance phase. The median duration of Bor maintenance was 14.5 months (range, 1.8–76.5 months). Three patients were still receiving maintenance therapy at the time of reporting. Six patients (27.2%) received maintenance therapy for over 2 years, and 3 (13.6%) received it for over 4 years. The median OS was 3.6 years (range, 0.4–6.5 years), and the duration of Bor therapy from induction to the last administration was 2.5 years. The median progression free survival was 1.6 years (range, 0.3–6.5 years). During maintenance therapy, 5 patients achieved a PR. The reasons for cessation of Bor maintenance therapy were AEs in 8 patients and progressive disease in 11 patients. No patient died during Bor administration in this study. Discussion: We retrospectively analyzed 22 TI-RR-MM patients receiving modified BD therapy. Pantani L et al. reported the results of using BD therapy for 85 RR-MM patients, and demonstrated that twice-weekly Bor administration resulted in a clinical response rate of 19% and a median OS of 22 months (Ann Hematol, 2014). Although the clinical response rate in our study was inferior to that of Pantani L et al., the median OS in our study was superior (3.6 years) than those of previous reports. Because the majority of patients in this study were frail or elderly, the early decision to change to maintenance therapy before they developed severe AEs resulted in a good clinical outcome and a longer OS. Continuing BD therapy without a satisfactory molecular response might be an attractive therapeutic approach for TI-RR-MM patients. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2939 Multiple myeloma (MM) remains incurable despite the use of conventional and high-dose chemotherapy with stem cell transplantation. However, novel agents such as thalidomide, lenalidomide, and bortezomib have already achieved promising clinical results. In particular, bortezomib, a proteasome inhibitor, is widely used in the treatment of MM and has remarkable clinical effects in both refractory/relapsed and newly diagnosed patients. However, acquired drug resistance and serious toxicities such as peripheral neuropathy are often triggered by bortezomib in the clinical settings. Therefore, novel targeted agents with less toxicity are necessary to overcome bortezomib-resistance and improve clinical outcomes in MM. We have previously reported that 1'-acetoxychavicol acetate (ACA), a component of a traditional Asian condiment obtained from the rhizomes of the commonly used ethno-medicinal plant Languas galanga, has an inhibitory effect on NF-κB, and induces cell death in MM cells both in vitro and in vivo (Cancer Res, 2005; 65: 4417). With the aim of discovering more potent NF-κB inhibitors, we subsequently developed several ACA analogs based on quantitative structure-activity relationship (QSAR) analysis. This showed that a novel benzhydrol-type analog of ACA (TM-233) exhibited greater inhibition of NF-κB activity in MM cells when compared with ACA. In the present study, we examined the effects of TM-233 on various MM cells, including those resistant to bortezomib and we investigated the molecular mechanism of TM-233-induced cell death in these cells. TM-233 inhibited cell proliferation and induced cell death in various MM cell lines (U266, RPMI8226, and MM-1S cells) in a time (0–48 hours)- and dose (0–5 μM)- dependent manner. Similarly potent activity was observed against primary MM cells. Exposure of bone marrow mononuclear cells from 3 patients with MM to TM-233 revealed selective activity under the same conditions as for cell lines. Moreover, TM-233 did not inhibit colony formation in CD34-positive cells from human umbilical cord blood; therefore, TM-233 did not influence the normal blood cells. We next investigated the effects of TM-233 on the various signaling pathways including JAK/STAT and NF-κB in MM cells. TM-233 activated apoptosis-related caspase-3, -8, -9, and PARP, suggesting that cell death related to TM-233 is mediated by both mitochondrial- and Fas-dependent pathways. Furthermore, treatment with TM-233 inhibited constitutive activation of JAK2 and STAT3, and then downregulated the expression of anti-apoptotic Mcl-1 protein, but not that of Bcl-2 and Bcl-xL proteins. TM-233 directly bound and activated the transcription of the Mcl-1 gene promoter, suggesting that TM-233-induced cell death in MM cells down-regulated Mcl-1 via modulation of the JAK/STAT pathway. In addition, the DNA-binding activity of NF-κB in MM cells was inhibited by the treatment of TM-233. Further, TM-233 rapidly decreased the nuclear expression of NF-κB, but increased the accumulation of cytosol NF-κB, suggesting that TM-233 inhibits the translocation of NF-κB from the cytosol to the nucleus. These results indicate that TM-233 induced cell death in MM cells occurs via both JAK/STAT and NF-κB pathways. We finally examined the effects of TM-233 on bortezomib-resistant MM cells. We have recently established the bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ (Leukemia 2010; 24: 1506). We found that these cells have a unique point mutation, G322A, in the gene encoding the proteasome b5 subunit, resulting in bortezomib-resistance mediated through prevention of the accumulation of unfolded proteins and fatal ER stress. TM-233, but not bortezomib, inhibited cellular proliferation and induced cell death in KMS-11/BTZ and OPM-2/BTZ cells in a time- and dose-dependent manner. Interestingly, the combination of TM-233 and bortezomib significantly induced cell death in these bortezomib-resistant MM cells via inhibition of NF-κB activity. These results indicate that TM-233 could overcome bortezomib resistance in MM cells by acting according to a different mechanism from that of bortezomib, possibly inhibition of the JAK/STAT pathway. In conclusion, TM-233, a novel analog of ACA, might be a more potent NF-κB inhibitor than ACA and could overcome bortezomib-resistance in MM cells. Further studies investigating clinical applications are necessary and may yield interesting results. Disclosures: Iida: Janssen Pharmaceutical K.K.: Honoraria. Kizaki:Janssen Pharmaceutical K.K.: Honoraria.

Description: Abstract 1312 Background The production and lineage commitment of hematopoietic cells is controlled by the actions of a complex network of signaling pathways. Mutations and translocations of tyrosine kinases within these pathways lead to constitutive signaling and enhanced proliferation. Classic examples are BCR-ABL in CML, Janus kinase 2 (JAK2) mutations in MPN, Fms-like tyrosine kinase 3 (FLT3) and c-KIT mutations in AML. FLT3 is a receptor tyrosine kinase with important roles in hematopoietic progenitor cell survival and proliferation. It is mutated in about 1/3 of AML patients, mostly by internal tandem duplications (ITD). Adaptor protein Lnk is expressed in hematopoietic cells and is an important negative regulator in cytokine signaling and hematopoiesis. Previously, we and others have shown that Lnk interacts with the JXM domain of c-KIT, PDGFRA, PDGFRB and FMS, all of which share a similar sequence in this domain. The fact that FLT3 harbors a conserved JXM domain prompted us to investigate whether Lnk interacts with FLT3. Methods and Results Co-immunoprecipitation and GST-pulldown assay showed that Lnk physically interacts with both wild-type FLT3 (FLT3-WT) and FLT3-ITD through its SH2 domain in multiple types of hematopoietic cells. Through affinity fishing assay with immobilized peptides, we identified the tyrosine residues 572, 591 and 919 of FLT3 as phosphorylation sites involved in direct binding to Lnk. Importantly, Lnk itself was tyrosine-phosphorylated by both FLT3 ligand (FL)-activated FLT3-WT and constitutively activated FLT3-ITD. Functionally, both shRNA-mediated depletion and ectopic expression of Lnk demonstrated that activation signals emanating from both forms of FLT3 are under negative regulation by Lnk. Consequently, Lnk inhibited 32D cell proliferation driven by different FLT3 oncogenic variants. Moreover, analysis of primary bone marrow cells from Lnk−/−mice showed that Lnk suppresses the expansion of FL-stimulated hematopoietic progenitors, including lymphoid-primed multipotent progenitors, mainly through inhibiting MAPK-ERK activation by FL. Conclusions This study reveals that through direct binding to FLT3-WT and FLT3-ITD, Lnk constrains FLT3-WT/ITD-dependent signaling pathways involved in the proliferation and expansion of hematopoietic cells as well as related leukemic cells. Modulation of Lnk expression levels may provide a unique therapeutic approach for FLT3-ITD-associated hematopoietic diseases. Disclosures: No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) is a hematological malignancy characterized by abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents such as proteasome inhibitors, immunomodulatory drugs, and antibodies that have significantly improved clinical outcomes of the patients of MM, most patients eventually relapse and develop drug resistance. In particular, the prognosis of patients harboring either chromosome TP53 deletion or mutations remains very poor, suggesting the prevalence of TP53 abnormalities increases with disease progression. Therefore, novel therapeutic strategies to overcome this unfavorable feature are urgently needed in clinical settings. WEE1 is a cell-cycle checkpoint kinase and a key regulator of DNA damage surveillance pathways. In response to extrinsically induced DNA damage, WEE1 kinase induces cell cycle arrest, allowing damaged DNA to be repaired before the cell undergoes DNA replication in S phase, and preventing cells harboring unrepaired, damaged DNA from mitotic lethality. Furthermore, WEE1 overexpression has been observed in many types of cancers. In addition, our previous studies revealed that monotherapy with AZD1775, a potent and highly selective inhibitor of WEE1, inhibited the proliferation of various MM cell lines irrespective of TP53 status. (Blood 2016; 128: 3256). On the other hand, one of the defining features of MM cells is the production of large amounts of protein, such as immunoglobulin, that must be processed within the endoplasmic reticulum (ER). Due to the accumulation of abundant immunoglobulin in ER, MM cells are constitutively under conditions of ER stress. The unfolded protein response (UPR)-signaling pathway is a cytoprotective mechanism against ER stress, and is therefore activated in MM cells to survive these conditions. Activation of the UPR has been observed in many types of cancers, and loss of TP53 has shown to enhance the UPR. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is one of three ER transmembrane protein kinases implicated as primary effectors of the UPR. Recent studies have suggested that PERK inhibition resulted in dose-dependent inhibition of tumor growth both in vitro and in vivo. In addition, more recent studies have proposed that PERK induces resistance to cell death elicited by chemotherapy. The combination of WEE1 and PERK inhibitors might thus offer an attractive therapeutic option against this incurable hematological malignancy. Here, we investigated the therapeutic utility of AZD1775 and GSK2606414, a highly selective inhibitor of PERK kinase, alone and in combination in various MM cells including TP53 wild-type (MM1.S) as well as TP53-deficient (KMS-11) and TP53-mutated (U266, RPMI8226, OPM-2) cell lines. AZD1775 and GSK2606414 alone induced dose-dependent cell growth inhibition in all investigated MM cells irrespective of TP53 status. Interestingly, GSK2606414 in combination with AZD1775 inhibited proliferation of all MM cells more effectively than either single agent. Assays for apoptotic cell death demonstrated that AZD1775 in combination with GSK2606414 induced significant and marked apoptotic cell death in MM cells used in this study compared to monotherapy alone. Next, western blotting analysis was performed to address the mechanisms of apoptotic cell death by the treatment of WEE1 and PERK inhibitors in MM cells. GSK2606414 inhibited PERK activation and decreased its downstream substrates (phospho-eIF2a, ATF4, and CHOP). Combination treatment with WEE1 inhibitor and various doses of PERK inhibitor significantly increased PARP and caspase 3 cleavage, CDK1 phosphorylation, and histone H2AX expression. Taken together, these data suggest that combining AZD1775 and GSK2606414 synergistically induced DNA damage and promoted premature mitotic entry, resulting in apoptotic cell death of TP53-deleted or -mutated MM cells. In conclusion, dual targeting of WEE1 and PERK might be a promising therapeutic approach for MM irrespective of TP53 status. Disclosures Tokuhira: Bristol-Myers Squibb: Speakers Bureau; AYUMI Pharmaceutical Corporation: Speakers Bureau; Mitsubishi Tanabe Pharma Corporation: Speakers Bureau; Chugai: Speakers Bureau. Kizaki:Novartis: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Nippon Shinyaku,: Research Funding, Speakers Bureau.

Description: Multiple myeloma (MM) is a hematological malignancy that derives from the proliferation of unregulated plasma cells. Dramatic improvement in the clinical outcomes of both newly diagnosed and relapsed/refractory patients with MM has been achieved using many clinical approaches, including use of high-dose chemotherapy followed by hematopoietic stem cell transplantation, and new drugs, such as proteasome inhibitors, immunomodulatory drugs, and histone deacetylase inhibitors. However, most patients eventually relapse and develop drug resistance. Moreover, the prognosis of patients with bortezomib (BTZ) and/or lenalidomide (LEN)-resistant MM (key drugs in the treatment of MM) is very poor. Therefore, novel therapeutic approaches to overcome BTZ and LEN resistance are urgently needed in clinical settings. WEE1 is a cell-cycle checkpoint kinase and a key regulator of DNA damage surveillance pathways. In response to extrinsically induced DNA damage, WEE1 catalyzes inhibitory phosphorylation of both cyclin-dependent kinase1 and 2 (CDK1 and CDK2), leading to CDK1- and CDK2-induced cell cycle arrest at the G1, S, or G2-M phases. This cell-cycle arrest, in turn, allows for the damaged DNA to be repaired before the cell undergoes DNA replication, and prevents cells harboring unrepaired damaged DNA from mitotic lethality. Furthermore, recent research has shown that knockdown of WEE1 leads to DNA double-strand breaks specifically in S-phase cells undergoing DNA replication, and that WEE1 is most active in the S-phase, suggesting that WEE1 is involved in DNA synthesis. Overexpression of WEE1 has been observed in many types of cancers, including hepatic cancer, breast cancer, glioblastoma and gastric cancer, and high expression of WEE1 has been shown to correlate with poor prognosis. In addition, research has shown that inhibition of checkpoint kinase 1 (Chk1), a critical transducer of the DNA damage response, potentiates the cytotoxicity of chemotherapy on p53-deficient MM cells, which are regarded as chemotherapy-resistant, suggesting that inhibition of cell-cycle checkpoint kinase is involved in re-sensitization of refractory MM cells to anticancer drugs. These data suggest that WEE1 might be an attractive target for novel therapeutic agents against this incurable hematological malignancy. MK-1775 is a potent and highly-selective small-molecule inhibitor of WEE1. In the present study, we investigated the role of WEE1 in MM as a potential therapeutic target using MK-1775. MTSassays showed that single agent MK-1775 inhibited the proliferation of various MM cell lines, including the intrinsically LEN-resistant cell line, RPMI-8226, in a dose- (0 to 10 mM) and time- (0 to 72 h) dependent manner. Furthermore, the growth inhibition effect is irrespective of p53 status. To examine the mechanisms behind the growth inhibition effect induced by MK-1775, assays for apoptotic cell death were performed. These assays demonstrated that MK-1775 induces both early and late apoptosis in MM cells. To investigate the molecular mechanisms of MK-1775-induced cell death in MM cells, the expression of various cell death-associated proteins and downstream molecules of WEE1 were examined. Western blotting analysis showed that MK-1775 arrested cell growth and induced apoptotic cell death in MM cells in a dose-dependent manner by inhibiting both, the expression of the target molecules of Bcl-2 and MCL1, and the cleavage of PARP and Caspase 3. Similarly, there was a substantial inhibition of CDK1 phosphorylation downstream of WEE1. Moreover, an increased expression of histone H2AX was observed following administration of MK-1775, suggesting that MK-1775 results in cytotoxicity by direct DNA damage. Next, we examined the effects of MK-1775 on BTZ-resistant MM cells. Interestingly, MK-1775 inhibited the proliferation of both BTZ-sensitive wild-type MM cells and BTZ-resistant MM cells, suggesting that BTZ resistance can be overcome by targeting WEE1. Furthermore, in combination with BTZ, MK-1775 was able to re-sensitize BTZ-resistant MM cells to BTZ. These results indicate that inhibition of WEE1 might serve as an attractive therapeutic option for patients with both BTZ-resistant and LEN-resistant MM. In conclusion, our data suggest that WEE1 might be a promising molecular target for the treatment of MM. Disclosures Tokuhira: Bristol Myers Squibb Co., Ltd: Honoraria; Pfizer Co., Ltd: Honoraria; Eizai Co., Ltd: Honoraria.

Description: 【Background】Despite numerous attempts to elucidate the mechanism underlying other iatrogenic immunodeficiency-associated lymphoproliferative diseases (OIIA-LPDs), the mechanism remains poorly understood. Methotrexate-associated lymphoproliferative disorder (MTX-LPD) is one of the disease entities included in OIIA-LPDs, which is especially developed in rheumatoid arthritis (RA) as we demonstrated (Tokuhira et al, Leuk Lymphoma. 2012;53:616-23). Spontaneous regression of LPD after MTX withdrawal is one of the distinct characteristics of MTX-LPD, and recent articles suggest that the increase in peripheral blood (PB) lymphocytes affect this phenomenon. However, the population of lymphocytes necessary to induce MTX-LPD regression is still unknown. 【Methods】We prospectively analyzed 10 patients with RA developing confirmed LPD during MTX administration, in our institutions between January 2014 to October 2015. All patients ceased MTX after developing lymphoadenopathies. To investigate the factors involved in spontaneous regression of LPD following MTX withdrawal, we performed flowcytometric analysis of whole blood including lymphocytes from PB for CD3, 4, 8, 14, 15, 20, 56, 127, and 45RO, HLA-DR, CCR3, CCR4, CCR6, CCR7, and EBV-tetramer expression, for all patients at three time points during the clinical course of treatment; at the time of MTX cessation (w0), and 4 weeks (w4) and 12 weeks (w12) after MTX cessation. We selected 10 age-, sex-, MTX dose-, and RA duration-matched RA patients treated with MTX for more than 6 months as controls, randomly selected from among the RA patients at our institutions. The patients with MTX-LPD were divided into two groups based on the status of LPD after MTX cessation, regressive LPD (R group) and persistent LPD (P group), and clinical parameters were compared. 【Results】In the 10 RA patients with MTX-LPD, the median age was 62.4 years (40-74), and the ratio of male:female patients was 2:8. The median duration from the time of RA diagnosis to the time of MTX development was 7.2 years (1.2-20.4), and the median duration of MTX administration was 4.3 years (0.7-9.8). The pathological diagnosis of LPDs was diffuse large B cell lymphoma (DLBCL, n=5), Classical Hodgkin lymphoma (cHL, n = 3), and non-specific LPD (LPD-nos, n=2). The averages values for LDH, CRP, and sIL-2R in the serum were 220 IU/mL (176-554), 0.85 mg/dL (0.06-2.53), and 579 IU/L(206-1210), respectively. The number of patients in the R group and the P group was 7 and 3, respectively. According to LPD phenotypes, all 5 cases of DLBCL belonged to the R group, whereas 1 of 3 HL and 1 of 2 LPD-nos belonged to the R group. At w0, the median WBC count and absolute lymphocyte number in 10 MTX-LPD patients were 5810/µL (2700-11400) and 964/µL (220-2070), respectively. On the other hand, the median WBC count and absolute lymphocyte number in the 10 control patients were 5930/µL (3900-9000) and 1512/µL (755-2379), respectively. The absolute lymphocyte count in the P group was significantly higher than that in the R group. In addition, a significant increase in the lymphocyte number following MTX withdrawal was observed only in the R group, but not in the P group. There were several observations from the flowcytometric analysis. First, the proportions of effector memory CD8+ T cells (EM CD8+), EBV specific CD8+ T cells, and T helper 1 (Th1) cells were significantly decreased both in the R group and in the P group compared to the control group at w0, and this subset was significantly increased in the R group at w4 and w12 compared to the P group. Second, the proportion of CD14-CD15+ cells in the lymphocyte fraction, which includes myeloid-derived suppressor cells (MDSC), was significantly increased in LPD group at w0, and significantly decreased following MTX cessation only in the R group, but not in the P group. Third, the proportion of CD14-CD15+ cells negatively and significantly correlated with the proportion of EM CD8+T cells (r2 = 0.47, p 〈 0.0001). 【Conclusions】Although the mechanism of MTX-LPD regression following MTX withdrawal is still unknown, this study demonstrates that reconstitution with specific lymphocytes in the PB is definitely correlated with LPD-regression. The proportion of Th1 cells, EM CD 8+, EBV specific CD8+ along with CD14-CD15+ cells dramatically indicated restoration and transition following MTX cessation, suggesting their potent effect toward the progression and regression of MTX-LPD. Disclosures Tokuhira: Bristol Myers Squibb Co., Ltd: Honoraria; Pfizer Co., Ltd: Honoraria; Eizai Co., Ltd: Honoraria. Okamoto:Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Shionogi & Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Teijin Pharma Limited: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Astellas Pharma Inc.: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

Description: Abstract 2785 The adaptor protein LNK has an important role during the development and maturation of cells in the hematopoietic system. LNK KO mice have an expansion of hematopoietic stem cells (HSCS) and an increase in circulating neutrophils, platelets, as well as immature B cells. On the other hand, overexpression of WT LNK under the control of a lymphocyte-specific expression vector results in an impaired expansion of lymphoid precursor cells and altered mature B cell subpopulations. Recently, LNK PH domain mutations were described in myeloproliferative neoplasms (MPN). The non-receptor tyrosine kinase JAK3 is expressed mainly in hematopoietic cells and together with JAK1, transmits signals from cytokine receptors of the IL-2 family. While JAK3 loss of function causes severe combined immune deficiency (SCID), JAK3 activating mutations were described in AML, particularly in acute megakaryocytic leukemia (AMKL). The importance of both LNK and JAK3 in lymphopoiesis, coupled with the finding that LNK binds to another JAK family member (JAK2), has led us to study whether LNK and JAK3 interact. LNK and JAK3 co- immunoprecipitated (co-IP) in the following leukemic cell lines- HEL, NALM6 (containing both LNK and WT JAK3), CMK (containing both LNK and an activating JAK3 mutant) and not in K562 (no JAK3). WT-JAK3 and WT LNK also co-IP when overexpressed in 293T cells, and this binding was associated with phosphorylation of LNK. To determine which domain of LNK is responsible for JAK3 binding, WT JAK3 was overexpressed in 293T cells with the following LNK mutants: 1. point mutation in the SH2 domain (R392E); 2. Deletion of the SH2 domain (del SH2); 3. Deletion of the PH and SH2 (del SH2/PH) domains; 4. Point mutation in the PH domain, recently found in a patient with a JAK2 V617F negative MPN (E208Q); 5. LNK SNP (rs3184504) described in GWAS to associate with an increased risk of autoimmune disease and which also causes an amino acid change in the LNK PH domain (W262R). LNK binding was retained in the E208Q and W262R mutants, markedly reduced in the R392E and del SH2 mutants and completely abolished with the del SH2/PH mutant, suggesting that LNK SH2 domain is important for the binding of LNK and WT-JAK3, although some binding may occur even in its absence. The effect of the LNK mutants R392E, E208Q and W262R on WT-JAK3 and LNK phosphorylation was also studied in 293T cells. Notably, WT-JAK3 was phosphorylated in the presence of WT-LNK and the E208Q and W262R mutants and not in the presence of the R392E mutant or an empty vector; also, the WT-LNK, LNK E208Q and W262R were phophorylated and the LNK R392E was not. These results suggest that WT-JAK3 phosphorylates LNK and that the LNK SH2 domain is important for this to occur. Interestingly, we found that the LNK PH domain mutant (E208Q) was much more phosphorylated than the WT- LNK when co-expressed with WT-JAK3. A similar result was obtained when LNK E208Q was co-expressed in 293T cells with vectors expressing an activating JAK3 mutant- A572V and the JAK2 V617F mutant but not when it was co-expressed with a BCR-ABL vector. This, again, supports the fact that direct binding underlines LNK phosphorylation by JAK3, as was previously shown for JAK2, while the BCR-ABL kinase does not bind LNK. The LNK PH domain mutation E208Q may cause a conformational change in LNK, allowing better access to tyrosine residues, or a change in LNK localization. The finding in 293T cells that WT-JAK3 is phophorylated only in the presence of LNK that retains a PH domain is intriguing. The other LNK family members- SH2B1 and APS were previously shown to homo- and heterodimerize. LNK contains a dimerization domain and we speculate that LNK homodimerization may serve as a scaffold for WT-JAK3 in the absence of a cytokine receptor, enabling JAK3 autophosphorylation. Since LNK PH domain mutations were recently described in MPN and may involve activation of the JAK2-STAT pathway we hypothesized that similar mutations might be found in lymphoma cases where JAK3 is reported to be activated. We, therefore, sequenced LNK PH domain in 147 lymphoma samples including- mantle cell lymphoma, peripheral T cell lymphoma (PTCL) and adult T cell leukemia/lymphoma cases but could not detect any mutations. In summary- we found that JAK3 binds and phosphorylates both WT-LNK and to a greater extent, the LNK PH domain mutant E208Q and in the absence of a cytokine receptor can lead to JAK3 phosphorylation. Taken together, LNK may likely control JAK3 activity. Disclosures: No relevant conflicts of interest to declare.