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Supplementary Table 1. Linkage disequilibrium patterns of the tag-SNPs used for genotyping the 6q23.3 locus. Supplementary Table 2. Cytogenetic and molecular information on AML patients used for functional studies (n=12). Supplementary Table 3. French-American-British (FAB) classification35 and molecular information on AML patients used to determine miR-3662's abundance (n=8). Supplementary Table 4. Canonical pathway analysis of the miR-3662-associated gene expression signature. Supplementary Figure 1. Transcription factor binding according to the transcription factor chip data from ENCODE. Supplementary Figure 2. Electrophoretic mobility shift assay comparing the binding affinity of the alleles of rs66650371 and rs9483788. Supplementary Figure 3. Endogenous miR-3662 expression levels of hematopoietic progenitor (HP) cells during differentiation, total bone marrow aspirate of three non-leukemic donors (total BM 1-3), different populations of differentiated peripheral blood cells, and three AML cell lines. Supplementary Figure 4. Top panel, Macroscopic pictures of the spleens of three mice of the scramble and miR-3662-infected groups (organs harvested post-mortem). All mice had a massive splenomegaly compared to the un-injected, sacrificed control mouse. Bottom panel, images of spleen histologies (40x enlargement). Slides were stained for CD45 to proof MV4-11 origin of the leukemia. Supplementary Figure 5. Endogenous abundance of miR-3662 and IKBKB in patient samples and cell lines. Supplementary Figure 6. Comparison of the relative miR-3662 abundance of AML patient blasts and AML cell lines before (black) and after (red) forced miR-3662 expression with the lentiviral expression construct.

Purpose:EGF-like domain 7 (EGFL7) is a secreted protein and recently has been shown to play an important role in acute myeloid leukemia (AML); however, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown.Experimental Design:Using an antibody interaction array, we measured the ability of EGFL7 to bind directly approximately 400 proteins expressed by primary AML blasts. Primary patient samples were stimulated in vitro with recombinant EGFL7 (rEGFL7) or anti-EGFL7 blocking antibody to assess alterations in downstream signaling and the ability to effect blast differentiation and survival. We treated three independent AML models with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival in vivo.Results:We found EGFL7 significantly binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH. Stimulation of AML blasts with rEGFL7 reduced NOTCH intracellular domain and NOTCH target gene expression while treatment with an anti-EGFL7 blocking antibody resulted in reactivation of NOTCH signaling, increased differentiation, and apoptosis. Competitive ligand-binding assays showed rEGFL7 inhibits DELTA-like (DLL) 4-mediated NOTCH activation while anti-EGFL7 combined with DLL4 significantly increased NOTCH activation and induced apoptosis. Using three different AML mouse models, we demonstrated that in vivo treatment with anti-EGFL7 alone results in increased survival.Conclusions:Our data demonstrate that EGFL7 contributes to NOTCH silencing in AML by antagonizing canonical NOTCH ligand binding. Reactivation of NOTCH signaling in vivo using anti-EGFL7 results in prolonged survival of leukemic mice, supporting the use of EGFL7 as a novel therapeutic target in AML.

Table S1: IC50 values of Topo IIa inhibitors (idarubicin, etoposide, mitoxantrone, daunorubicin) and selinexor at 48hrs as measured by WST-1 assay for AML cell lines MV4-11 and MOLM-13. Table S2: Combinatorial Index (CI) values of concomitant treatment of selinexor with Topo IIa inhibitors (idarubicin and daunorubicin) in AML cell lines MV4-11 and MOLM-13. Table S3: Combinatorial Index (CI) values of concomitant treatment of selinexor with Topo IIa inhibitors (idarubicin and daunorbicin) in AML patient blasts. Table S4: Combinatorial Index (CI) values of concomitant treatment of selinexor with individual Topo IIa inhibitors (etoposide, mitoxantrone) in AML cell lines MV4-11 and MOLM-13. Table S1: IC50 values of Topo IIα inhibitors and selinexor in AML cell lines Table S2: Combinatorial Index (CI) values of selinexor with idarubicin and daunorubicin in AML cell lines Table S3: Combinatorial Index (CI) values of selinexor with idarubicin/daunorubicin in AML patient cells Table S4: Combinatorial Index (CI) values of selinexor with Topo IIα inhibitors (etoposide and mitoxantrone) with selinexor in AML cell lines

Additional Supplementary Data

Purpose: Selinexor, a selective inhibitor of XPO1, is currently being tested as single agent in clinical trials in acute myeloid leukemia (AML). However, considering the molecular complexity of AML, it is unlikely that AML can be cured with monotherapy. Therefore, we asked whether adding already established effective drugs such as topoisomerase (Topo) II inhibitors to selinexor will enhance its anti-leukemic effects in AML.Experimental Design: The efficacy of combinatorial drug treatment using Topo II inhibitors (idarubicin, daunorubicin, mitoxantrone, etoposide) and selinexor was evaluated in established cellular and animal models of AML.Results: Concomitant treatment with selinexor and Topo II inhibitors resulted in therapeutic synergy in AML cell lines and patient samples. Using a xenograft MV4-11 AML mouse model, we show that treatment with selinexor and idarubicin significantly prolongs survival of leukemic mice compared with each single therapy.Conclusions: Aberrant nuclear export and cytoplasmic localization of Topo IIα has been identified as one of the mechanisms leading to drug resistance in cancer. Here, we show that in a subset of patients with AML that express cytoplasmic Topo IIα, selinexor treatment results in nuclear retention of Topo IIα protein, resulting in increased sensitivity to idarubicin. Selinexor treatment of AML cells resulted in a c-MYC–dependent reduction of DNA damage repair genes (Rad51 and Chk1) mRNA and protein expression and subsequent inhibition of homologous recombination repair and increased sensitivity to Topo II inhibitors. The preclinical data reported here support further clinical studies using selinexor and Topo II inhibitors in combination to treat AML. Clin Cancer Res; 22(24); 6142–52. ©2016 AACR.

Acute graft-versus-host disease (GVHD) is the leading cause of non-relapse mortality following allogeneic hematopoietic cell transplantation. The majority of patients non-responsive to front line treatment with steroids have an estimated overall 2-year survival rate of only 10%. Bromodomain and extra-terminal domain (BET) proteins influence inflammatory gene transcription, and therefore represent a potential target to mitigate inflammation central to acute GVHD pathogenesis. Using potent and selective BET inhibitors Plexxikon-51107 and -2853 ({"type":"entrez-protein","attrs":{"text":"PLX51107","term_id":"1321741095","term_text":"PLX51107"}}PLX51107 and PLX2853), we show that BET inhibition significantly improves survival and reduces disease progression in murine models of acute GVHD without sacrificing the beneficial graft-versus-leukemia response. BET inhibition reduces T cell alloreactive proliferation, decreases inflammatory cytokine production, and impairs dendritic cell maturation both in vitro and in vivo. RNA sequencing studies in human T cells revealed that BET inhibition impacts inflammatory IL-17 and IL-12 gene expression signatures, and Chromatin Immunoprecipitation (ChIP)-sequencing revealed that BRD4 binds directly to the IL-23R gene locus. BET inhibition results in decreased IL-23R expression and function as demonstrated by decreased phosphorylation of STAT3 in response to IL-23 stimulation in human T cells in vitro as well as in mouse donor T cells in vivo. Furthermore, PLX2853 significantly reduced IL-23R+ and pathogenic CD4+ IFNγ+ IL-17+ double positive T cell infiltration in gastrointestinal tissues in an acute GVHD murine model. Our findings identify a role for BET proteins in regulating the IL-23R/STAT3/IL-17 pathway. Based on our preclinical data presented here, {"type":"entrez-protein","attrs":{"text":"PLX51107","term_id":"1321741095","term_text":"PLX51107"}}PLX51107 will enter clinical trial for refractory acute GVHD in a Phase 1 safety, biological efficacy trial.

Acute graft versus host (aGVHD) is the second cause of death after allogeneic-hematopoietic stem cell transplant (allo-HSCT) underscoring the need for novel therapies. Based on previous work that endothelial cell dysfunction is present in aGVHD and that epidermal growth factor-like domain 7 (EGFL7) plays a significant role in decreasing inflammation by repressing endothelial cell activation and T cell migration, we hypothesized that increasing EGFL7 levels after allo-HSCT will diminish the severity of aGVHD. Here, we show that treatment with recombinant EGFL7 (rEGFL7) decreases aGVHD severity and improves survival in recipient mice after allogeneic transplantation with respect to controls without affecting graft versus leukemia effect. Histopathology analysis revealed higher amount of leukocyte infiltration in both large intestine and liver of PBS group compared to rEGFL7-treated mice. Furthermore, damage to the gut was reduced in EGFL7 treated mice. Finally, we showed that rEGFL7 treatment results in higher thymocytes, T, B and dendritic cells in recipient mice after allo-HSCT. This study constitutes a proof of concept of the ability of rEGFL7 therapy to reduce GHVD severity and mortality after allo-HSCT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Current treatment options for older and relapsed or refractory (R/R) acute myeloid leukemia (AML) patients are limited and represent an unmet need. Based on preclinical studies showing strong anti-leukemic effects in vivo, this phase I dose-escalation study assessed the safety and preliminary clinical activity of the oral exportin-1 inhibitor, selinexor, in combination with the hypomethylating agent, decitabine 20 mg/m(2), in adults with R/R AML and in older (age ≥60) untreated AML patients. There were no protocol-defined dose limiting toxicities. The recommended phase 2 dose of selinexor was 60mg (~35 mg/m(2)) given twice-weekly. Notable grade ≥3 toxicities included asymptomatic hyponatremia (68%), febrile neutropenia (44%), sepsis (44%), hypophosphatemia (36%), and pneumonia (28%). In 25 patients, the overall response rate was 40%. Modification of selinexor to a flat dose of 60mg, twice-weekly for two weeks after decitabine, improved tolerability of the regimen and demonstrated preliminary clinical activity in poor-risk patients with AML.

Background Notch intercellular communication instructs tissue-specific T-cell development and function. In this study, we explored the roles of dendritic cell (DC)-expressed Notch ligands in the regulation of T-cell effector function. Methods We generated mice with CD11c lineage-specific deletion of Notch Delta-like ligand (Dll)1 and Jagged (Jag)2. Using these genetically-ablated mice and engineered pharmacological Notch ligand constructs, the roles of various Delta-like and Jagged ligands in the regulation of T-cell-mediated immunity were investigated. We assessed tumor growth, mouse survival, cytokine production, immunophenotyping of myeloid and lymphoid populations infiltrating the tumors, expression of checkpoint molecules and T-cell function in the experimental settings of murine lung and pancreatic tumors and cardiac allograft rejection. Correlative studies were also performed for the expression of NOTCH ligands, NOTCH receptors and PD-1 on various subsets of myeloid and lymphoid cells in tumor-infiltrating immune cells analyzed from primary human lung cancers. Results Mice with CD11c lineage-specific deletion of Notch ligand gene Dll1, but not Jag2, exhibited accelerated growth of lung and pancreatic tumors concomitant with decreased antigen-specific CD8+T-cell functions and effector-memory (Tem) differentiation. Increased IL-4 but decreased IFN-γ production and elevated populations of T-regulatory and myeloid-derived suppressor cells were observed in Dll1-ablated mice. Multivalent clustered DLL1-triggered Notch signaling overcame DC Dll1 deficiency and improved anti-tumor T-cell responses, whereas the pharmacological interference by monomeric soluble DLL1 construct suppressed the rejection of mouse tumors and cardiac allograft. Moreover, monomeric soluble JAG1 treatment reduced T-regulatory cells and improved anti-tumor immune responses by decreasing the expression of PD-1 on CD8+Tem cells. A significant correlation was observed between DC-expressed Jagged and Delta-like ligands with Tem-expressed PD-1 and Notch receptors, respectively, in human lung tumor-infiltrates. Conclusion Our data show the importance of specific expression of Notch ligands on DCs in the regulation of T-cell effector function. Thus, strategies incorporating selectively engineered Notch ligands could provide a novel approach of therapeutics for modulating immunity in various immunosuppressive conditions including cancer. Electronic supplementary material The online version of this article (10.1186/s40425-019-0566-4) contains supplementary material, which is available to authorized users.

Introduction: The use of allogeneic hematopoietic cell transplantation to treat acute myeloid leukemia (AML) has risen in recent years. However, relapse remains the major cause of mortality post-transplant; while graft-versus-host disease (GVHD) - a T cell mediated immunological disorder is the major cause of non-relapse mortality. Dihydroorotate dehydrogenase (DHODH) supports T cell proliferation by playing a critical role in de novo pyrimidine synthesis and oxidative phosphorylation. We hypothesized that alloreactive T cells may rely on increased levels of pyrimidine pools and ATP to support rapid cell proliferation, making DHODH an interesting target to prevent GVHD. Additionally, DHODH inhibition is currently being pursued as a therapeutic option for AML. Therefore, we hypothesize that DHODH inhibition post-transplant will serve a dual purpose - i) target T cell metabolism to reduce GVHD and ii) prevent relapse due to direct anti-leukemic effects, thereby resulting in superior post-transplant outcomes. Methods: We tested the efficacy of a novel DHODH inhibitor (Cmpd 41), a lead clinical candidate, in preventing GVHD and retaining graft-versus-leukemia (GVL) effect. Human T cells isolated from PBMCs were activated with CD3/CD28 Dynabeads ± Cmpd 41. Cell proliferation (flow cytometry) and ATP production (Agilent Seahorse) was assessed. GVHD and GVL was assessedevaluated in a xenogeneic model where irradiated NSG mice received human PBMCS (~17x106 cells) and treated with vehicle or Cmpd 41 (10mg/kg, 2x/week) with addition of MOLM-13 cells (~1x104) for GVL. Splenocytes were harvested for analysis of cytokine production (intracellular flow cytometry). Results: DHODH inhibition with Cmpd 41 significantly reduced T cell proliferation and ATP production from both glycolysis and OXPHOS (fold change: Cmpd 41 vs. control- 0.56 and 0.68 respectively, p Conclusion: DHODH inhibition is a novel approach to prevent and mitigate GVHD while retaining GVL effects. Combined with direct anti-leukemic effects, we propose that Cmpd 41 treatment in post-transplant relapse in the setting of past or active graft versus host disease will provide dual treatment of AML and GVHD thereby leading to improved patient outcomes. Citation Format: Kara M. Braunreiter, Lotus Neidemire-Colley, Natalie Sell, Yandi Gao, Sandip Vibhute, Chad Bennett, Ola A. Elgamal, Thomas Goodwin, Erin K. Hertlein, John C. Byrd, Parvathi Ranganathan. DHODH inhibition modulates T cell metabolism reducing GVHD and prevents relapse following allogeneic HCT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2335.

Acute graft-versus-host disease (aGVHD) is a T cell–mediated immunological disorder and the leading cause of nonrelapse mortality in patients who receive allogeneic hematopoietic cell transplants. Based on recent observations that protein arginine methyltransferase 5 (PRMT5) and arginine methylation are upregulated in activated memory T cells, we hypothesized that PRMT5 is involved in the pathogenesis of aGVHD. Here, we show that PRMT5 expression and enzymatic activity were upregulated in activated T cells in vitro and in T cells from mice developing aGVHD after allogeneic transplant. PRMT5 expression was also upregulated in T cells of patients who developed aGVHD after allogeneic hematopoietic cell transplant compared with those who did not develop aGVHD. PRMT5 inhibition using a selective small-molecule inhibitor (C220) substantially reduced mouse and human allogeneic T cell proliferation and inflammatory IFN-γ and IL-17 cytokine production. Administration of PRMT5 small-molecule inhibitors substantially improves survival, reducing disease incidence and clinical severity in mouse models of aGVHD without adversely affecting engraftment. Importantly, we show that PRMT5 inhibition retained the beneficial graft-versus-leukemia effect by maintaining cytotoxic CD8(+) T cell responses. Mechanistically, we show that PRMT5 inhibition potently reduced STAT1 phosphorylation as well as transcription of proinflammatory genes, including interferon-stimulated genes and IL-17. Additionally, PRMT5 inhibition deregulates the cell cycle in activated T cells and disrupts signaling by affecting ERK1/2 phosphorylation. Thus, we have identified PRMT5 as a regulator of T cell responses and as a therapeutic target in aGVHD.

MicroRNAs (miRNAs) have been extensively reported to be associated with hematological malignancies. The loss of miR-15a/16–1 at chromosome 13q14 is a hallmark of most of human chronic lymphocytic leukemia (CLL). Deletion of murine miR-15a/16–1 and miR-15b/16–2 has been demonstrated to promote B cell malignancies. Here, we evaluate the biological role of miR-15/16 clusters, crossbreeding miR-15a/16–1 and miR-15b/16–2 knockout mice. Unexpectedly, the complete deletion of both clusters promoted myeloproliferative disorders in the majority of the mice by the age of 5 months with a penetrance of 70%. These mice showed a significant enlargement of spleen and abnormal swelling of lymph nodes. Flow cytometry characterization demonstrated an expanded CD11b/Gr-1 double-positive myeloid population both in spleen and in bone marrow. The transplantation of splenocytes harvested from double-KO mice into wild-type recipient mice resulted in the development of myeloproliferative disorders, as observed in the donors. In vivo, miR-15/16 cluster deletion up-regulated the expression of Cyclin D1, Cyclin D2, and Bcl-2. Taken together, our findings identify a driver oncogenic role for miR-15/16 cluster deletion in different leukocytic cell lineages.

Transplant-associated thrombotic microangiopathy (TA-TMA), a complication of hematopoietic cell transplant (HCT), is associated with significant morbidity and mortality. The pathophysiology and overlap of TA-TMA with other posttransplant complications such as graft-versus-host disease (GVHD) is poorly understood. We retrospectively identified cases of TA-TMA among patients with grade 3/4 gastrointestinal (GI) GVHD, reviewed intestinal biopsy specimens, and performed correlative testing of biomarkers associated with TA-TMA. TA-TMA was more common in patients with steroid-refractory GVHD compared with steroid-responsive GVHD (79.3% vs 42.1%; P = .001). Among patients surviving 100 days post-HCT, 1-year survival from day 100 was significantly better for patients who had not developed TA-TMA in the first 100 days (69.5% vs 36.7%; P < .001). Only 1 of 7 proposed TA-TMA histology criteria (mucosal hemorrhage) differed significantly based on GVHD steroid response. In multivariable modeling, steroid-refractory GVHD was a risk factor for development of TA-TMA (hazard ratio, 3.09; 95% confidence interval, 1.68-5.67; P < .001). There were no differences in complement activation at GVHD onset; however, 2 to 6 weeks later, patients with TA-TMA had higher levels of BBPlus and C5b-9, markers of alternative and terminal pathway activation (BBPlus: median, 600 vs 209.3 ng/mL; P = .0045) (C5b-9: median, 425.9 vs 258.4 ng/mL; P = .029). TA-TMA is associated with poor overall survival (OS) following HCT and may be detected early by histologic findings and may be differentiated from GVHD by measurement of alternative and terminal complement pathway activation. It is unknown whether treatment of TA-TMA will improve survival in steroid-refractory GVHD.

MicroRNA-155 (miR-155) is a small noncoding RNA critical for the regulation of inflammation as well as innate and adaptive immune responses. MiR-155 has been shown to be dysregulated in both donor and recipient immune cells during acute graft-versus-host disease (aGVHD). We previously reported that miR-155 is upregulated in donor T cells of mice and humans with aGVHD and that mice receiving miR-155–deficient (miR155−/−) splenocytes had markedly reduced aGVHD. However, molecular mechanisms by which miR-155 modulates T cell function in aGVHD have not been fully investigated. We identify that miR-155 expression in both donor CD8+ T cells and conventional CD4+ CD25− T cells is pivotal for aGVHD pathogenesis. Using murine aGVHD transplant experiments, we show that miR-155 strongly impacts alloreactive T cell expansion through multiple distinct mechanisms, modulating proliferation in CD8+ donor T cells and promoting exhaustion in donor CD4+ T cells in both the spleen and colon. Additionally, miR-155 drives a proinflammatory Th1 phenotype in donor T cells in these two sites, and miR-155−/− donor T cells are polarized toward an IL-4–producing Th2 phenotype. We further demonstrate that miR-155 expression in donor T cells regulates CCR5 and CXCR4 chemokine-dependent migration. Notably, we show that miR-155 expression is crucial for donor T cell infiltration into multiple target organs. These findings provide further understanding of the role of miR-155 in modulating aGVHD through T cell expansion, effector cytokine production, and migration.

Acute graft-versus-host disease (aGVHD) continues to be a frequent and devastating complication of allogeneic hematopoietic stem cell transplantation (HSCT), posing as a significant barrier against the widespread use of HSCTs as a curative modality. Recent studies suggested serum/plasma microRNAs (miRs) may predict aGVHD onset. However, little is known about the functional role of circulating miRs in aGVHD. In this article, we show in two independent cohorts that miR-29a expression is significantly upregulated in the serum of allogeneic HSCT patients at aGVHD onset compared with non-aGVHD patients. Serum miR-29a is also elevated as early as 2 wk before time of diagnosis of aGVHD compared with time-matched control subjects. We demonstrate novel functional significance of serum miR-29a by showing that miR-29a binds and activates dendritic cells via TLR7 and TLR8, resulting in the activation of the NF-κB pathway and secretion of proinflammatory cytokines TNF-α and IL-6. Treatment with locked nucleic acid anti–miR-29a significantly improved survival in a mouse model of aGVHD while retaining graft-versus-leukemia effects, unveiling a novel therapeutic target in aGVHD treatment or prevention.

Purpose: Selinexor, a selective inhibitor of XPO1, is currently being tested as single agent in clinical trials in acute myeloid leukemia (AML). However, considering the molecular complexity of AML, it is unlikely that AML can be cured with monotherapy. Therefore, we asked whether adding already established effective drugs such as topoisomerase (Topo) II inhibitors to selinexor will enhance its anti-leukemic effects in AML. Experimental Design: The efficacy of combinatorial drug treatment using Topo II inhibitors (idarubicin, daunorubicin, mitoxantrone, etoposide) and selinexor was evaluated in established cellular and animal models of AML. Results: Concomitant treatment with selinexor and Topo II inhibitors resulted in therapeutic synergy in AML cell lines and patient samples. Using a xenograft MV4-11 AML mouse model, we show that treatment with selinexor and idarubicin significantly prolongs survival of leukemic mice compared with each single therapy. Conclusions: Aberrant nuclear export and cytoplasmic localization of Topo IIα has been identified as one of the mechanisms leading to drug resistance in cancer. Here, we show that in a subset of patients with AML that express cytoplasmic Topo IIα, selinexor treatment results in nuclear retention of Topo IIα protein, resulting in increased sensitivity to idarubicin. Selinexor treatment of AML cells resulted in a c-MYC–dependent reduction of DNA damage repair genes (Rad51 and Chk1) mRNA and protein expression and subsequent inhibition of homologous recombination repair and increased sensitivity to Topo II inhibitors. The preclinical data reported here support further clinical studies using selinexor and Topo II inhibitors in combination to treat AML. Clin Cancer Res; 22(24); 6142–52. ©2016 AACR.

Purpose: EGF-like domain 7 (EGFL7) is a secreted protein and recently has been shown to play an important role in acute myeloid leukemia (AML); however, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown. Experimental Design: Using an antibody interaction array, we measured the ability of EGFL7 to bind directly approximately 400 proteins expressed by primary AML blasts. Primary patient samples were stimulated in vitro with recombinant EGFL7 (rEGFL7) or anti-EGFL7 blocking antibody to assess alterations in downstream signaling and the ability to effect blast differentiation and survival. We treated three independent AML models with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival in vivo. Results: We found EGFL7 significantly binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH. Stimulation of AML blasts with rEGFL7 reduced NOTCH intracellular domain and NOTCH target gene expression while treatment with an anti-EGFL7 blocking antibody resulted in reactivation of NOTCH signaling, increased differentiation, and apoptosis. Competitive ligand-binding assays showed rEGFL7 inhibits DELTA-like (DLL) 4-mediated NOTCH activation while anti-EGFL7 combined with DLL4 significantly increased NOTCH activation and induced apoptosis. Using three different AML mouse models, we demonstrated that in vivo treatment with anti-EGFL7 alone results in increased survival. Conclusions: Our data demonstrate that EGFL7 contributes to NOTCH silencing in AML by antagonizing canonical NOTCH ligand binding. Reactivation of NOTCH signaling in vivo using anti-EGFL7 results in prolonged survival of leukemic mice, supporting the use of EGFL7 as a novel therapeutic target in AML.

Figure S3. T-cell expressed PD-1 and NOTCH receptors correlate with DC-expressed NOTCH ligands in human lung tumor-infiltrate. Heatmap shows Pearson’s correlation between the indicated populations. P-values were corrected by Benjamani-Hochberg procedure. Color code indicates the strength of correlation and direction; * p

Figure S1. Clustered DLL1 and soluble JAG1 constructs modulate the differentiation of memory T-cells in vitro. Purified T cells were stimulated in vitro in a T:DC (3:1)Â stimulation co-culture with allogeneic bone marrow-derived dendritic cells in the presence of CD3/CD28 beads (1â Îźg/mL) for four days with or without treatment with the indicated concentrations of clustered DLL1 or monovalent soluble JAG1 constructs. Expression of CD62L and CD44 was assessed on gated CD8 population as indicated by flow cytometry. Dot plots from a representative experiment out of two independent experiments with duplicates are shown. (PPTX 4553 kb)

Chromosomal aberrations and multiple genome-wide association studies (GWAS) have established a major hematopoietic quantitative trait locus in chromosome 6q23.3. The locus comprises an active enhancer region, in which some of the associated SNPs alter transcription factor binding. We now identify miR-3662 as a new functional driver contributing to the associated phenotypes. The GWAS SNPs are strongly associated with higher miR-3662 expression. Genome editing of rs66650371, a three-base-pair deletion, suggests a functional link between the SNP genotype and the abundance of miR-3662. Increasing miR-3662′s abundance increases colony formation in hematopoietic progenitor cells, particularly the erythroid lineage. In contrast, miR-3662 is not expressed in acute myeloid leukemia cells, and its overexpression has potent antileukemic effects in vitro and in vivo. Mechanistically, miR-3662 directly targets NF-κB–mediated transcription. Thus, miR-3662 is a new player of the hematopoietic 6q23.3 locus. Significance: The characterization of miR-3662 has identified a new actor in the prominent hematopoietic quantitative trait locus in chromosome 6q23.3. The mechanistic insights into miR-3662′s function may reveal novel or only partially known pathways for normal and malignant hematopoietic cell proliferation. Cancer Discov; 6(9); 1036–51. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 932

We previously reported that microRNA (miR)-29b is down-regulated and has a tumor suppressor role in acute myeloid leukemia (AML). However, little is known about the mechanisms responsible for miR-29b expression downregulation in AML. In this work we screened for mutations that could affect miR-29b expression. Using Sanger sequencing, we identified a germline thymidine (T) base deletion within the miR-29b-1/miR-29a cluster precursor in 16% of AML patients. Remarkably we found a significant enrichment for the presence of the miR-29 polymorphism in core binding factor (CBF) newly diagnosed AML patients (n = 61/303; 20%) with respect to age, sex and race matched controls (n = 43/402:11%, P < 0.01). Mechanistically, this polymorphism affects the expression ratio of mature miR-29b and miR-29a by dampening the processing of miR-29a. RNA immunoprecipitation assays showed reduced DROSHA binding capacity to the polymorphism with respect to the controls. Finally, we showed that this polymorphism negatively impacts the ability of miR-29b-1/miR-29a cluster to target MCL-1 and CDK6, both known miR-29 targets.

Epidermal growth factor-like domain 7 (EGFL7) is a secreted protein. We previously found that EGFL7 is up-regulated in primary AML blasts and that a high mRNA expression correlates with shorter event-free and overall survival in AML patients (Papaioannou et al. PNAS 2017;114(23)). However, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown. To identify EGFL7 binding partners, we took an unbiased high-throughput approach by using an antibody interaction-array to measure the ability of EGFL7 to bind directly ~400 proteins expressed by primary AML blasts. Using this strategy, we found in cell lysates of 3 AML patients that EGFL7 binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH (in all patients P Next, we stimulated AML blasts from 3 patients with recombinant EGFL7 (rEGFL7) and found that it reduced NOTCH intracellular domain (NICD1/2, Figure 1A), indicating that EGFL7 inhibits NOTCH activation. In line with this, we found that stimulation of AML patients samples' with rEGFL7 resulted in a decreased expression of its well-known downstream target gene HES1 (Figure 1B; in all patients P On the other hand, treatment with an anti-EGFL7 blocking antibody (AB) caused increased levels of NICD1/2 (Figure 1C) and expression of HES1 (Figure 1D, in all patients P To determine whether blocking EGFL7 could provide a new targeted therapy for patients with AML, we treated 3 independent AML models (i.e. an AML cell line based xenotransplant model (n=4 mice per group), a primary murine AML model (n=7 mice per group), and a patient derived xenotransplant model (n=4 mice per group)) with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival in vivo. In all models, we demonstrated that in vivo treatment with anti-EGFL7 results in prolonged overall survival (cell line model: P In conclusion, our data demonstrate that EGFL7 contributes to NOTCH silencing in AML by antagonizing canonical NOTCH ligand binding. Reactivation of NOTCH signaling in vivo using anti-EGFL7 results in prolonged survival of leukemic mice, supporting EGFL7 might be a novel therapeutic target in AML. Figure 1 A AML patient blasts were cultured in the presence or absence (Unstim) of rEGFL7. Total proteins were extracted for immunoblotting with β-ACTIN as loading control. B Total RNA was extracted for quantitative real time (qRT-PCR) analysis of HES1 mRNA normalized to β-ACTIN control (*P Disclosures No relevant conflicts of interest to declare.

Introduction: Acute Graft-versus-Host Disease (aGVHD) affects 30-70% of all allogeneic stem cell transplant (alloSCT) recipients, contributing to high non-relapse mortality. aGVHD is due to donor T cell cytotoxic inflammatory effects stimulated by exposure to foreign host antigens. Epigenetic modulation by BET inhibition boasts anti-inflammatory effects. BET proteins are a class of epigenetic "reader molecules," functioning as key mediators of transcription. BET proteins regulate inflammatory gene expression and are targetable. BET inhibition decreases NF-kB dependent cytokine expression and Th1/Th17 differentiation without affecting regulatory T cell differentiation. BET inhibition disrupts BRD4 interaction with the acetylated component of NF-kB, RelA. Therefore, we hypothesize that BET inhibition is a feasible and effective strategy to mitigate T cell mediated aGVHD inflammation. In this study, we have used PLX51107 and PLX2853 - novel BET inhibitors developed by Plexxikon Inc that possess a unique binding mode and high bioavailability ideal for preclinical evaluation. PLX2853 (second generation) was designed to increase potency and improve tolerability. Materials and Methods: aGVHD allogeneic mouse model: Lethally irradiated (1200 cGy) B6D2F1 recipients received T cell depleted bone marrow cells (TCD-BM, 10x106) and CD45.1+ B6 splenocytes (15x106) intravenously via tail vein injection. Recipients were treated by oral gavage, 3x weekly with PLX51107 (10 mg/kg), PLX2853 (4 mg/kg) or vehicle. We evaluated two dosing schedules - starting at day +1 or day +7 after transplant in the PLX51107 studies and day +1 only after infusion of allogeneic CD45.1+ B6 splenocytes in the PLX2853 studies. Survival and clinical aGVHD scores were assessed. Xenogeneic mouse model: NSG mice were conditioned with 50 cGy X-ray irradiation and injected with 15-20x106 human PBMCs and treated with PLX2853 (4 mg/kg) or vehicle starting day +1. Survival was assessed. Graft-versus-Tumor model: In vivo: Lethally irradiated F1 mice were intravenously injected with firefly luciferase-transduced murine mastocytoma P815 cells on day 0 with B6 TCD-BM ± allogeneic B6 splenocytes (n=8 per cohort). Recipients were treated with PLX2853 (4 mg/kg) or vehicle (oral gavage, 3x weekly) starting at day +1 post-transplant. Survival was assessed. In vitro: Murine CD45.1 B6 CD8+ T cells were stimulated in vitro with PMA/Ionomycin ± PLX2853 (10nM) for 5 days and then degranulation was analyzed in response to P815 tumor challenge to evaluate CTL capacity via flow cytometry of intracellular CD107a expression. Study designs are illustrated in the Figure (Panel A). Results: PLX51107 demonstrates potent biological activity and improves survival in a murine model of aGVHD. Administration of PLX51107 dramatically improved survival of recipient mice (B) and reduced aGVHD clinical scores (C). PLX2853 significantly improves survival in multiple mouse models of aGVHD. Our in-vitro data show that PLX2853 (IC50 ~10nM) is more potent than PLX51107 (IC50 ~500nM). Therefore, we validated the biological activity and efficacy of PLX2853 in mouse models of aGVHD. PLX2853 significantly prolonged survival of allogeneic transplanted recipient mice (D) and resulted in reduced clinical scores (E). Recipient mice in the xenogeneic mouse model showed improved survival with PLX2853 treatment (F). PLX2853 maintains Graft Versus Tumor response in vivo and does not abrogate CD8+ cytotoxic T lymphocyte (CTL) responses in vitro. BET inhibition retained beneficial GVT effects as seen by improvement in survival comparable to vehicle group (G). PLX2853 treated CD8+ T cells showed comparable degranulation to control as measured by CD107a mobilization (H, I). These results suggest that BET inhibition does not abrogate CD8+ CTL capacity, correlating to retention of GVT effects observed in vivo. Conclusions: PLX51107 was well tolerated at both day +1 and +7 initiation, demonstrating the future feasibility of BET inhibition as a prophylactic or therapeutic strategy for aGVHD. PLX2853 given at lower doses demonstrated similar improvements in survival and GVHD scoring, consistent with increased potency. Preliminary in vivo and in vitro studies of PLX2853 on GVT and on CD8+ CTLs show that PLX2853 retains GVT effects. Additional mechanistic in vivo and in vitro studies are ongoing. Figure Disclosures Powell: Plexxikon Inc.: Employment. Bollag:Plexxikon Inc.: Employment.

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1-/- donors. PD-L1–deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1-/- donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell–mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.

CD4(+)CD25(+)FoxP3(+) thymic-derived regulatory T cells (tTregs) are indispensable for maintaining immune system equilibrium. Adoptive transfer of tTregs is an effective means of suppressing graft-versus-host disease (GVHD) in murine models and in early human clinical trials. Tumor necrosis factor receptor-associated factor 6 (TRAF6), an ubiquitin-conjugating enzyme that mediates nuclear factor κB (NF-κB) activation, plays an essential role in modulating regulatory T cell survival and function. MicroRNAs (miRNAs) are noncoding RNAs, which mediate RNA silencing and posttranscriptional gene repression. By performing comprehensive TaqMan Low Density Array miRNA assays, we identified 10 miRNAs differentially regulated in human tTreg compared with control T cells. One candidate, miR-146b, is preferentially and highly expressed in human naive tTregs compared with naive CD4 T cells. miRNA prediction software revealed that TRAF6 was the one of the top 10 scored mRNAs involved tTreg function with the highest probability as a potential miR-146b target. Antagomir-mediated knockdown of miRNA-146b, but not another miRNA-146 family member (miRNA-146a), enhanced TRAF6 expression. TRAF6, in turn, increases NF-κB activation, which is essential for tTreg function as well as Foxp3 protein and antiapoptotic gene expression, and downregulates proapoptotic gene expression. miR-146b knockdown increased the nuclear localization and expression of genes regulated by NF-κB, which was associated with enhanced tTreg survival, proliferation, and suppressive function measured in vitro and in vivo. TRAF6 inhibition had the opposite effects. We conclude that an miR-146b-TRAF6-NF-κB-FoxP3 signaling pathway restrains regulatory T cell survival, proliferation, and suppressor function. In vitro exposure of human tTregs to miR-146b antagomirs can be exploited to improve the clinical efficacy of human adoptive tTreg transfer in a GVHD setting.

In a recent publication we demonstrated that somatic hypermutation occurs in the V region of the TCR γ gene of the sandbar shark ( Carcharhinus plumbeus ). We hypothesize that similar mechanisms are used to generate somatic mutations in both immunoglobulin and TCR γ genes of the sharks. Two distinct patterns of mutation occur, single nucleotide mutations (point mutations) and mutations comprising 2–5 consecutive bases (tandem mutations). Our data indicates that point mutations occur by a mechanism similar to that of somatic hypermutation in immunoglobulin genes of mammals, whereas tandem mutations may be generated by an error-prone DNA polymerase with terminal deoxynucleotidyl transferase (TdT)-like activity. Shark hotspot motifs identical to those of higher vertebrates were identified. We confirm that, as in immunoglobulin of sharks and higher vertebrates, highly significant targeting of AID activity to the classical D G YW/WR C H motif occurs in somatic hypermutation of sandbar shark TCR γ V genes. Our analysis suggests that the purpose of somatic mutations in shark TCR γ V-regions is to generate a more diverse repertoire in γ/δ receptors, rather than receptors with higher affinity.

The nuclear export receptor, Exportin 1 (XPO1), mediates transport of growth-regulatory proteins, including tumor suppressors, and is overactive in many cancers, including chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and aggressive lymphomas. Oral selective inhibitor of nuclear export (SINE) compounds that block XPO1 function were recently identified and hold promise as a new therapeutic paradigm in many neoplasms. One of these compounds, KPT-330 (selinexor), has made progress in Phase I/II clinical trials, but systemic toxicities limit its administration to twice-per-week and requiring supportive care. We designed a new generation SINE compound, KPT-8602, with a similar mechanism of XPO1 inhibition and potency but considerably improved tolerability. Efficacy of KPT-8602 was evaluated in preclinical animal models of hematological malignancies, including CLL and AML. KPT-8602 shows similar in vitro potency compared with KPT-330 but lower central nervous system penetration, which resulted in enhanced tolerability, even when dosed daily, and improved survival in CLL and AML murine models compared with KPT-330. KPT-8602 is a promising compound for further development in hematological malignancies and other cancers in which upregulation of XPO1 is seen. The wider therapeutic window of KPT-8602 may also allow increased on-target efficacy leading to even more efficacious combinations with other targeted anticancer therapies.

In this issue of Blood , [Du et al][1][1][2] show that pirfenidone has remarkable therapeutic efficacy in bronchiolitis obliterans (BO) models of chronic graft-versus-host disease (cGVHD). Their data support the use of pirfenidone as a new agent in early-phase clinical trials for cGVHD treatment.

Introduction: Acute graft-versus-host disease (aGVHD), a T cell-mediated immunological disorder is the leading cause of non-relapse mortality in patients receiving allogeneic bone marrow transplants. Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetric dimethylation (me2s) of arginine (R) residues on histones (primarily H3R8 and H3R4) and other proteins. PRMT5 is overexpressed in many leukemias and lymphomas, and epigenetic changes driven by PRMT5 lead to repression of tumor suppressors and promote growth and survival of cancer cells. Recently it was shown that T cells are sensitive to R-methylation and PRMT5 promotes activation of memory T helper cells. Here we investigate: 1) mechanisms by which PRMT5 regulates T cell function; and 2) PRMT5 inhibition as a therapeutic strategy for aGVHD. Materials and Methods: Splenic T cells were isolated from lethally irradiated B6D2F1 mice that received either T cell depleted bone marrow (TCD-BM) or TCD-BM with C57/BL6 (B6) allogeneic splenocytes on day 21 post-transplant. In vitro activation of B6 T cells was achieved with CD3/CD28 Dynabeads or co-culture with allogeneic BM-derived dendritic cells. PRMT5 expression (RT-PCR, western blot) and function (H3R8me2s western blot) were evaluated. PRT220, a novel inhibitor of PRMT5, was used to evaluate PRMT5 inhibition on T cell function in vitro and in vivo. We assessed T cell proliferation (Cell Trace Violet, Ki67), apoptosis (Annexin V), cytokine secretion (ELISA, flow cytometry), cell cycle (PI incorporation), and cell signaling (western blot). Lethally irradiated F1 recipients received TCD-BM only (10x106 cells) or TCD-BM + B6 splenocytes (20 x 106). Recipients of allogeneic splenocytes were treated with PRT220 (2mg/kg) or vehicle by oral gavage once weekly starting day 7 post-transplant. Mice were monitored for survival and clinical aGVHD scores. Results: PRMT5 expression and function is upregulated following T cell activation. Inhibition of PRMT5 reduces T cell proliferation and IFN-g secretion. PRMT5 inhibition in CD3/CD28 stimulated T cells results in disruption of multiple histone epigenetic marks, cell-cycle progression (via G1 arrest) and perturbation of ERK-MAPK signaling cascades. Finally, administration of PRT220 resulted in significantly prolonging the survival of allo-transplanted recipient mice (median survival, PRT220 vs. vehicle, 36.5 vs. 26 days, p=0.01). PRT220-treated recipients also exhibited significant lower aGVHD clinical (p Conclusions: PRMT5 expression and function are upregulated in activated T cells. Inhibition of PRMT5 function using a novel and specific small-molecule inhibitor, PRT220, down-regulates T cells proliferative and effector response, induces cell-cycle arrest and perturbs signaling pathways. PRT220 shows potent biological activity in vivo by reducing aGVHD clinical severity and significantly prolonging survival in mouse models of aGVHD. Therefore, PRMT5 is a novel and druggable target for aGVHD. Disclosures No relevant conflicts of interest to declare.

The prognosis of acute myeloid leukemia (AML) is poor, highlighting the need for novel treatments. Hypomethylating agents, including decitabine are used to treat elderly AML patients with relative success. Targeting nuclear export receptor (exportin 1 [XPO1]) is a novel approach to restore tumor suppressor (TS) function in AML. Here, we show that sequential treatment of AML blasts with decitabine followed by selinexor (XPO1 inhibitor) enhances the antileukemic effects of selinexor. These effects could be mediated by the re-expression of a subset of TSs (CDKN1A and FOXO3A) that are epigenetically silenced via DNA methylation, and cytoplasmic-nuclear trafficking is regulated by XPO1. We observed a significant upregulation of CDKN1A and FOXO3A in decitabine- versus control-treated cells. Sequential treatment of decitabine followed by selinexor in an MV4-11 xenograft model significantly improved survival compared with selinexor alone. On the basis of these preclinical results, a phase 1 clinical trial of decitabine followed by selinexor in elderly patients with AML has been initiated.

Nucleophosmin-mutated acute myeloid leukemia (NPM1mut-AML) patients have a high rate of complete remission (CR) to induction chemotherapy. However, the mechanisms responsible for such effects are unknown. Because miR-10 family members are expressed at high levels in NPM1mut-AML, we evaluated whether these microRNAs could predict chemotherapy response in AML. We found that high baseline miR-10 family expression in 54 untreated cytogenetically heterogeneous AML patients was associated with achieving CR. However, when we included NPM1 mutation status in the multivariable model, there was a significant interaction effect between miR-10a-5p expression and NPM1 mutation status. Similar results were observed when using a second cohort of 183 cytogenetically normal older (age ≥ 60 years) AML patients. Loss- and gain-of-function experiments using miR-10a-5p in cell lines and primary blasts did not demonstrate any effect in apoptosis or cell proliferation at baseline or after chemotherapy. These data support a bystander role for the miR-10 family in NPM1mut-AML.

Introduction: Exportin-1 (XPO1), a nuclear transport protein critical for the export of tumor suppressor proteins (TSPs) and select mRNAs to the cytoplasm, is highly expressed in acute myeloid leukemia (AML) and correlates with poor survival. Selinexor, an oral, first-in-class, selective inhibitor of nuclear export, blocks XPO1 function. We previously reported that sequential treatment of AML blasts using the hypomethylating agent decitabine followed by selinexor exhibited strong anti-leukemic effects in vivo by inducing the expression of silenced TSPs that are kept in the nucleus by XPO1 inhibition (Ranganathan, Blood 2015). Methods: Based on these findings, a phase I dose-escalation study was initiated to evaluate the safety, feasibility, maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), and preliminary clinical activity of selinexor in combination with decitabine in poor-risk AML pts (NCT02093403). Adults with relapsed or refractory (R/R) AML and older (age ≥60) unfit pts with untreated AML were eligible. Pts received 10-day decitabine induction(s) at 20mg/m2 on days 1-10 for up to four 28-day cycles in combination with selinexor once daily, twice weekly beginning on day 11. Pts with Results: Twenty-four pts were enrolled (see Table 1 for baseline pt characteristics). Nineteen pts had R/R AML (median of 3 prior chemotherapy regimens, range 1-4); five pts had previously untreated AML. Seventeen pts were treated on the dose escalation phase receiving selinexor doses ranging from 23mg/m2 to 55mg/m2. After two patients in DL 1 declined continuation of study therapy beyond cycle 1 due to grade Conclusions: The combination of selinexor plus decitabine is an active regimen in poor-risk AML pts, with CR/CRi/mCR rate of 80% in older untreated and 21% in R/R AML pts. Modification of selinexor exposure (flat dose of 60mg given twice weekly for 2 weeks after decitabine) improved tolerability of the regimen. Correlative studies, including targeted mutation analysis to assess whether specific mutations are associated with response, are ongoing and will be presented. Disclosures Bhatnagar: Karyopharm: Research Funding.

Chromosome maintenance protein 1 (CRM1) is a nuclear export receptor involved in the active transport of tumor suppressors (eg, p53 and nucleophosmin) whose function is altered in cancer because of increased expression and overactive transport. Blocking CRM1-mediated nuclear export of such proteins is a novel therapeutic strategy to restore tumor suppressor function. Orally bioavailable selective inhibitors of nuclear export (SINE) that irreversibly bind to CRM1 and block the function of this protein have been recently developed. Here we investigated the antileukemic activity of KPT-SINE (KPT-185 and KPT-276) in vitro and in vivo in acute myeloid leukemia (AML). KPT-185 displayed potent antiproliferative properties at submicromolar concentrations (IC50 values; 100-500nM), induced apoptosis (average 5-fold increase), cell-cycle arrest, and myeloid differentiation in AML cell lines and patient blasts. A strong down-regulation of the oncogene FLT3 after KPT treatment in both FLT3-ITD and wild-type cell lines was observed. Finally, using the FLT3-ITD–positive MV4-11 xenograft murine model, we show that treatment of mice with oral KPT-276 (analog of KPT-185 for in vivo studies) significantly prolongs survival of leukemic mice (P < .01). In summary, KPT-SINE are highly potent in vitro and in vivo in AML. The preclinical results reported here support clinical trials of KPT-SINE in AML.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic stem cell transplant (alloHSCT), underscoring the need to further elucidate its mechanisms and develop novel treatments. Based on recent observations that microRNA-155 (miR-155) is up-regulated during T-cell activation, we hypothesized that miR-155 is involved in the modulation of aGVHD. Here we show that miR-155 expression was up-regulated in T cells from mice developing aGVHD after alloHSCT. Mice receiving miR-155–deficient donor lymphocytes had markedly reduced lethal aGVHD, whereas lethal aGVHD developed rapidly in mice recipients of miR-155 overexpressing T cells. Blocking miR-155 expression using a synthetic anti–miR-155 after alloHSCT decreased aGVHD severity and prolonged survival in mice. Finally, miR-155 up-regulation was shown in specimens from patients with pathologic evidence of intestinal aGVHD. Altogether, our data indicate a role for miR-155 in the regulation of GVHD and point to miR-155 as a novel target for therapeutic intervention in this disease.

Introduction MicroRNAs (miRs) are deregulated in AML and play a key role in leukemogenesis. MiR-155 is one of the most frequently overexpressed miRs in AML. Higher expression of miR-155 is associated with FLT3 internal tandem duplication (FLT3-ITD) and is associated with worse outcome, independent of FLT3-ITD status. Preliminary data shows that silencing of miR-155 induces strong antileukemic effects in AML cell lines. Altogether these data support a therapeutic role for miR-155 antagonism in AML. Here, we show the in vitro and in vivo activity of MRG-106, a novel LNA antimiR-155 compound that we are developing as a potential treatment for hematological malignancies. Methods Unconjugated, LNA-modified oligonucleotide against miR-155 (MRG-106) was developed by miRagen Therapeutics, Inc. MRG-106 was evaluated in FLT3-ITD+ AML cell lines and primary FLT3-ITD+ AML samples for impact on apoptosis and cellular proliferation using Annexin V and MTS assays. Predicted and validated targets of miR-155 were measured by qPCR and Western Blotting to assess the efficacy of miR-155 silencing. The in vivo antileukemic effect of MRG-106 was evaluated in NOD/SCID gamma mice engrafted with MV4-11 AML cells that have elevated miR-155 expression. One week after leukemic cell inoculation, the mice were separated in 3 cohorts and received either MRG-106 (n=12); LNA-scramble control (n=12); or saline (n=6). Results Inhibition of miR-155 decreased cell proliferation in MV-4-11 and MOLM-13 cells at 48hrs (Absorbance 450 nM (A450nM): 0.5 and 0.4 vs controls 2.4 and 2.5, respectively, p Conclusions Inhibition of miR-155 in AML cell lines and primary AML samples in vitro and in vivo induces significant antileukemic effects. These studies validate miR-155 as a therapeutic target in AML and support the testing of MRG-106 in AML patients in the context of a phase 1 clinical trial. Disclosures Dickinson: miRagen Therapeutics: Employment, Equity Ownership. Jackson:miRagen Therapeutics: Employment, Equity Ownership. Rodman:miRagen Therapeutics: Employment, Equity Ownership.

Introduction: We reported that microRNA-155 (miR-155) expression is upregulated in donor T cells during aGVHD and mice receiving miR-155 knock-out (KO) donor splenocytes do not exhibit lethal GVHD and have improved survival as compared to mice receiving wild type (WT) splenocytes.1 While we showed that miR-155 does not affect the allo-reactive proliferative potential of T cells, a significant decrease in the expression of the homing receptors CCR5, CXCR4, and S1P1 was found on miR-155-KO T cells, suggesting that the loss of miR-155 could impair the migration of donor T cells to aGVHD target organs resulting in less lethality. Here, we further investigate the impact of miR-155 expression in T cell migration. Materials and Methods: Lethally irradiated BALB/c or B6D2F1 recipients were infused with T cell depleted WT bone marrow (BM) cells (5x10^6) and GFP expressing miR-155 KO or GFP-B6 WT T cells (1x10^6). Recipients were sacrificed at day 7, 14 and 21 post-transplant, organs harvested and donor T cell infiltration evaluated via confocal microscopy. Transwell migration assays towards CCR5 ligands macrophage inflammatory protein-1a (MIP-1a) (100ng/mL) and RANTES (100ng/mL) was performed utilizing WT or miR-155-KO T cells activated using irradiated BALB/c splenocytes as allogeneic stimulators at a stimulator: responder ratio of 1:5. Lower chambers with medium only served as a control for spontaneous migration. CCR5 ligand-dependent migration was calculated according to the formula: Migration Index (MI) = number of cells CCR5 ligands / number of cells medium only. Results: On days 7, 14 and 21 post transplant, recipient mice were sacrificed, and tissues harvested in order to study the kinetics of miR-155 KO T cell migration following allogeneic hematopoietic stem cell transplant. There was a dramatic decrease in T cell infiltration of peripheral organs (PeyerÕs patches, liver, lung and skin) in recipients of miR-155-KO T cells as compared to WT T cells as evidenced by confocal microscopy of GFP labeled donor cells, Figure 1. We reasoned that these effects could be due to the modulation of CCR5 and other chemokine receptors by miR-155. There was a significant decrease in CCR5 mRNA and protein expression in miR-155-KO versus WT donor T cells obtained from recipient mice at the time of aGVHD. To demonstrate the functional significance of decreased CCR5 expression in miR-155 KO donor T cells, we performed in vitro transwell migration assays to CCR5 ligands RANTES and MIP-1a. To our knowledge, we are the first to show that allo-activated miR-155 KO T cells show significantly reduced migration towards CCR5 ligands, as demonstrated by the average MI of 1.08, when compared to the average MI of WT T cells of 4.79, p=0.004, Figure 2. There were lower percentages of CCR5 positive T cells and decreased mean fluorescent intensity in the miR-155 KO T cells after allogeneic stimulation when compared to WT T cells, both in the CD4+ and CD8+ populations, confirming lower CCR5 expression in miR-155 KO T cells after in vitro allogeneic stimulation. To further elucidate the mechanism of miR-155 mediated modulation of CCR5 expression, we focused on long non-coding RNA (lncRNA) LincR-Ccr2-5′AS located in the vicinity of several chemokine receptor encoding genes including CCR1, CCR2 and CCR5, known to be important for migration of Th2 cells. We found that LincR-Ccr2-5′AS has 3 potential miR-155 binding sites and so set out to determine if miR-155 negatively regulates the expression of this lncRNA, thereby influencing chemokine receptor expression as well as T cell migration. We isolated T cells from B6D2F1 recipients 21 days post-transplant, and showed a significant decrease in CCR5 mRNA expression in miR-155 KO versus WT donor T cells but no significant difference in the levels of LincR-Ccr2-5′AS. However, this result does not exclude the possibility that miR-155 might influence the activity rather than the levels of LincR-Ccr2-5′AS, which we hope to determine in future experiments. Conclusion: Our data suggest that miR-155 may exert its modulating effects in aGVHD by affecting T cell migration. Experiments are currently underway to determine the role of miR-155 in modulating T cell migration through other chemokine receptors such as CXCR4, as well as S1P1 and ATP receptor P2X7R. Reference 1. Ranganathan P, Heaphy CE, Costinean S, et al. Regulation of acute graft-versus-host disease by microRNA-155. Blood. 2012 May 17;119(20):4786-97. Disclosures No relevant conflicts of interest to declare.