ALBERT

Description: Characterization of RING finger E3-Ubiquitin Ligase Cereblon in Hematopoietic Regulation Cereblon is a RING-domain E3 ubiquitin ligase (UbL) that is the direct protein target for thalidomide and lenalidomide. Inhibition of this molecule mediates antiproliferative activity in myeloma cells and in activated B-cell-like subtype diffuse large B-cell lymphoma. In addition to its anticancer properties, lenalidomide potentiates T-cell effector function through the engagement of the CD28-mediated co-stimulatory pathway by a CRBL-dependent mechanism that is poorly characterized. Human and animal studies show CRBL to be ubiquitously expressed in the hematopoietic compartment and in neurons, where a nonsense mutation causes a mild autosomal recessive non-syndromic intellectual disability. Ubiquitin ligase activity underlies suppression of growth factor signaling and maintains homeostasis within the hematopoietic compartment. C-Cbl, cbl-b, GRAIL, and ITCH represent four partially overlapping E3-UbL regulators of cytokine signaling and hematopoietic regulation. To understand the role of CRBL in hematopoetic development and function, we studied a CRBL-deficient mouse strain. All mice used in this study were bred and maintained under specific pathogen-free conditions according to institutional guidelines. The strategy used to create germline crbn deletion (crbl-/-) was described previously, although these mice were not investigated for aberrations in hematopoietic development. First, crbl-/- mice were viable, fertile and normal in appearance without limb malformations. Consistent with a role for CRBL in the hematopoietic development, crbl-/- mice exhibited marked changes in their hematopoietic profiles, including lymphoid hyperplasia, a markedly expanded peripheral white blood cell count and neutrophil expansion. In the T-cell compartment, splenic lymphocytes numbers were increased with demargination of the T-cells into B-cell zones by immunohistochemical staining. Double-negative thymocytes ratio were altered with an accumulation of DN1 and DN3 cells and lower DN4. In contrast, differentiated cell lineages such as mature T-cells, including CD4 and CD8 single-positive (SP) thymocytes, SP peripheral T-cells and naïve and memory T-cells were similar in number to wild-type littermates. The mature T-cells, similar to Cbl-b KO mice showed superior proliferation and IL-2 production in the absence of CD28 co-ligation. Collectively, these histological changes are indicative of a role for crbl in negatively regulating cytokine and receptor signaling events that control cell proliferation or survival. Interestingly, this phenotype overlaps partially with c-cbl and cbl-b KO mice suggesting that they could be coordinately regulating similar pathways. In summary, our findings demonstrate a novel role for crbl, the molecular target of thalidomide and lenalidomide in hematopoiesis and suggest that this molecule may act by an unknown mechanism in concert with other E3 UbLs involved in cytokine receptor signaling. This study provides the first characterization of crbl genetic deficiency in a murine model and demonstrates a unique regulatory relationship between CRBL and other known E3 UbLs. Disclosures: McDaniel: Celgene: Employment.

Description: Abstract 1703 Myleodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic diseases marked by various cytopenias including neutropenia and anemia. Hematologic improvement in response to T-cell depleting agents and immunosuppressants in a subset of MDS patients suggests that hematopoietic impairment may result from autoreactive T-cell activation. Conversely, suppression of the T-cell compartment may be permissible to the development of malignant myeloid clones. The suspicion that T-cell homeostatic balance may be disrupted in MDS has spawned numerous studies investigating mechanisms of immune suppression and autoimmune regulation. T-cell autoimmunity is regulated in part by regulatory T-cells (Tregs), which express T cell receptors (TCRs) that recognize self-antigens and function to maintain immune homeostasis. Indeed, higher numbers of Tregs have been found in higher risk MDS patients, and those MDS subtypes with higher blast percentages. However, the role of Tregs in lower risk patients is less clear, but may still represent an important component of pathogenesis in early disease. Because of the autoimmune nature of low risk MDS, we hypothesized that alterations in the Treg compartment may play a role in lower risk MDS disease progression. To address this, we analyzed Treg phenotype and numbers in MDS patients largely classified as low risk by the International Prognostic Scoring System (IPSS) and 41 age-matched controls using cell surface markers as analyzed by flow cytometry. Tregs were identified as CD3+CD4+CD25+CD127dim. The use of CD127 as a Treg marker was validated by intracellular staining with FoxP3. Naïve and memory phenotypes within the Treg compartment were analyzed using CD45RA and CD27 expression. Naïve Tregs (TregN) were characterized as CD45RA+CD27+, central memory Tregs (TregCM) as CD45RA−CD27+, and effector memory Tregs (TregEM) as CD45RA−CD27−. Two distinct subgroups of MDS patients were identified: one with elevated total numbers of Tregs, and a second with a marked shift in Treg memory phenotype away from the common TregCM phenotype and toward the rare TregEM phenotype. These two subgroups of patients were distinct from each other, and appeared to be independent of MDS WHO subtype, IPSS score, karyotype, neutropenic state, and thrombocytopenic state. MDS patients with a shift toward TregEMwere associated with anemia (p=0.0433). This association was not found in MDS patients with total increases in Treg numbers. Next, we sought to determine if there are differences in overall survival (OS) of MDS patients with either increased total numbers of Tregs or in patients with a shift toward TregEM phenotype. Patients with greater than 50 Tregs/μl did have reduced OS compared to patients with less Tregs, but this difference was not statistically significant (p=0.1387). In contrast, a highly significant difference was observed in MDS patients with a shift toward the TregEM phenotype (p=0.0200), with patients having greater than 2.5TregEM/μl displaying the worst OS (Figure 1). Figure 1 Overall Survival in lower risk MDS patients with Low, Intermediate, or High absolute numbers of TregEM cells Figure 1. Overall Survival in lower risk MDS patients with Low, Intermediate, or High absolute numbers of TregEM cells Lastly, because an increased TregEM compartment is associated with reduced OS in MDS patients, we sought to compare the suppressive capacities of TregN, TregCM, and TregEM cells to better understand the functional consequences of Treg memory shift. CD4+ T-cells were isolated by negative selection, and then TregN, TregCM, and TregEM cells were sorted using CD45RA and CD27 expression. Conventional CD4+ responder cells were then labeled with Carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with increasing ratios of each isolated Treg population. Cellular division in response to CD3/CD28 stimulation was measured 5 days later by analyzing CFSE dilution. TregCM appear to be the least suppressive, while TregN and TregEM cells were more potently suppressive. These results indicate that a shift in Treg phenotype toward TregEM may result in a more suppressive Treg compartment without obvious increases in total Treg numbers. Taken together, this data suggests that in lower risk MDS, the phenotypic makeup of the Treg compartment may be more important than total Treg numbers. In addition, this data suggests that increased TregEM cells may be a novel prognostic indicator in low risk MDS. Disclosures: Komrokji: Celgene: Honoraria, Research Funding, Speakers Bureau. List:Celgene: Consultancy.

Description: Background Large Granular Lymphocyte (LGL) leukemia is associated with chronic lymphoproliferation in association with unexplained cytopenias. Discovery of somatic mutations in STAT3 provides new insights into the molecular basis of LGL leukemia, but the precise mechanism leading to cytopenias remains unresolved. To gain insight into this mechanism, bone marrow fibrosis (Fig. 1A) was found to occur in 88% of cases and is significantly associated with splenomegaly, the presence of cytopenias, and co- existence of autoimmune diseases. In this study, we are reporting the identification of the fibrosis initiating cell population. Methods Primary mesenchymal stromal cultures (MSCs) from LGL leukemia BM were isolated and grown under reduced oxygen conditions using undifferentiated and differentiation-specific culture conditions (Fig. 1B). These cultured cells were confirmed to deposit greater amounts of fibrillar Type I, III and V collagen matrix consistent with the heavy reticulin and trichrome positive stains observed in bone marrow biopsies (Fig. 1C). This collagen-producing cell population failed to support normal hematopoietic progenitor proliferation and colony formation in co-culture assays. Microarray analysis of the collagen-producing mesenchymal cell population was conducted and pathway analysis was performed using the GeneGo analysis platform from MetaCore™. Each list of genes was individually grouped and evaluated for membership on the set of GeneGo® pathway maps under the functional ontology enrichment tool. Calculation of p-values using hypergeometric distribution was used to determine pathway statistical significance. Flow cytometry was conducted to further evaluate the mesenchymal lineage. Results Supervised hierarchical clustering was performed to determine the overall similarity of LGL MSCs to publicly available, platform-compatible, microarrays from cells of mesenchymal lineage. Clustering showed that both patient and control MSCs display expression patterns distinct from committed mesenchymal lineages including osteoblasts, chondrocytes, and adipocytes. Mesenchymal-derived fibroblast differentiation can be distinguished by down-regulated CD29, CD44, CD105, CD106, CD117, bone morphogenetic protein receptor, and Sca-1 expression, and by up-regulated collagen type I, collagen type III, tenacin C, fibronectin, matrix metalloproteinase 1, fibroblast-specific protein 1, and vimentin. By microarray analysis and flow cytometry, we find no evidence that the cells are differentiated cells aside from collagen over expression. In order to define their pluripotent potential, sub-confluent cultures were grown in three types of differentiating media. Primary MSCs from both LGL leukemia patients and healthy controls were similar in their capacity to undergo trilineage differentiation into osteoblasts (stained with Alizarin Red S), adipocytes (Oil red O), and chondrocytes (Alican blue) (Fig. 1D). Next, pathway analyses revealed significant re-programming of the pluripotent MSCs. ECM production pathways were increased while proliferation pathways were down-regulated. Proliferation of MSCs is governed by production of autocrine growth factors. Both leukemia inhibitor factor (LIF) and basic fibroblast growth factor (FGFb) were significantly reduced compared to control MSCs (p

Description: Abstract 932 Large Granular Lymphocyte Leukemia (LGLL) is a chronic lymphoproliferative syndrome of clonal mature T or NK cells frequently associated with peripheral cytopenias including neutropenia and anemia. The ability of LGLL cells to lyse pro-erythrocytes, the association with secondary autoimmune disorders, and the chronically activated state of LGLL clones suggest that the disease is secondary to a systemic reactive process. Yet, the mechanism causing severe and chronic cytopenias remains unresolved. Because LGLL cells heavily reside in the bone marrow and the bone marrow microenvironment plays such a large role in supporting hematopoiesis, we hypothesized that constituents of the bone marrow microenvironment may be dysfunctional in LGLL patients, and contribute toward the pathogenesis of LGLL and the devolvement of cytopenias. To address this hypothesis, bone marrow core biopsies, aspirates, and peripheral smears taken from 24 patients diagnosed with LGLL were analyzed retrospectively by three independent pathology reviews. Reticulin and trichrome stains revealed clinically relevant myelofibrosis in 21 patients at the time of diagnosis. Of these, 15 had severe myelofibrosis (MF2-3, semi-quantitative grading). The severity of myelofibrosis correlated with neutropenia, anemia, splenomegaly, secondary autoimmune disorders, and the degree of LGLL bone marrow infiltration. The severity of fibrosis also correlated with novel disease aspects reported here for the first time, including increased pseudo-Pelger-Huet and immature neutrophils in peripheral smears, and increased monocytes in the bone marrow and periphery. Because myelofibrosis is so strongly associated with disease severity, we sought to understand the pathogenesis of medullary fibrosis using primary mesenchymal stem cell (MSC) cultures isolated from bone marrow aspirates of LGLL patients or healthy controls. Patient MSC cultures displayed abnormal morphologies, impaired growth kinetics, and reduced growth potential compared to controls, suggesting that patient MSCs may be prematurely senescent. Microarray analysis showed global gene expression changes as healthy MSCs expand in culture including increased expression of pro-hematopoietic cytokines and autocrine growth factors vital to MSC self-renewal such as basic fibroblast growth factor (FGFb) and leukemia inhibitory factor (LIF). Patient MSC cultures appeared incapable of these gene expression changes, instead maintaining low levels of pro-hematopoietic cytokines, FGFb, and LIF expression, while maintaining heightened collagen expression. Cytokine secretion was confirmed using ELISA, and immunofluorescent staining of collagen on MSC cultures verified abnormally elevated collagen deposition from patient MSCs. In particular, collagen I and collagen III matrices were heavily deposited over patient MSCs reiterating the increased trichrome and reticulin staining seen in patient bone marrow biopsies. In an effort to rescue senescence, exogenous FGFb and LIF were added to patient MSCs. FGFb, but not LIF, completely restored the growth kinetics, growth potential, and morphology of these cells. Moreover, collagen deposition of patient MSCs was comparable to healthy donors after exposure to FGFb. We also investigated the ability of MSC cultures to support the growth of hematopoietic progenitors. Healthy bone marrow mononuclear cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE) and placed in co-culture with patient or control MSCs. Four days later, cellular division of CD34+ cells was analyzed by CFSE dilution. Control MSCs were able to support high levels of CD34+ cell division after expansion in culture. Conversely, patient MSCs supported only minimal growth of CD34+ cells. This suggests that gene expression changes seen in healthy MSC cultures during expansion may be required to support hematopoiesis, and that these changes are blocked in senescent MSCs in LGLL patients. Collectively, this data implicates aberrant MSCs and collagen production in the pathogenesis of LGLL, and suggests that autocrine growth factors such as FGFb, may represent a novel therapeutic option to rescue pro-hematopoietic behavior and reduce excessive buildup of collagen fibers in the bone marrow microenvironment.Figure 1MSCs from Patient Bone Marrow Deposite increase collagen matrix compared to healthy controls.Figure 1. MSCs from Patient Bone Marrow Deposite increase collagen matrix compared to healthy controls. Disclosures: No relevant conflicts of interest to declare.

Description: Background Durable responses to lenalidomide (LEN, an immunomodulatory drug, IMiDTM) occur in both non-del5q and del5q Myelodysplastic Syndrome (MDS). In non-del5q MDS and multiple myeloma, modulation of the bone marrow microenvironment by an unknown mechanism has been theorized. IMiDs initiate numerous effects on the immune system, some of which may induce an anti-leukemic and anti-inflammatory response. The connection between the drug's immune modulating activity and its hematopoietic stimulatory activity in MDS is currently untested. We and others have shown that LEN expands T-cells and increases IL-2 (p

Description: Immunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only molecular target for this drug class, where drug-induced, ubiquitin-dependent degradation of known “neosubstrates,” such as IKAROS, AIOLOS, and CK1α, accounts for their biological activity. Far less clear is whether these CRBN E3 ligase-modulating compounds disrupt the endogenous functions of CRBN. We report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T cells augments their central metabolism manifested as elevated bioenergetics, with supraphysiological levels of polyamines, secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Treatment with CRBN-modulating compounds similarly augments central metabolism of human CD8+ T cells. Notably, the metabolic control of CD8+ T cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn-deficient T cells have augmented antigen-specific cytolytic activity vs melanoma tumor cells, ex vivo and in vivo, and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of CD8+ T cells, and this phenotype can be exploited by treatment with drugs.

Description: Chronic lymphocytic leukemia (CLL) represents 30% of adult leukemia. TCL1 is expressed in ∼ 90% of human CLL. Transgenic expression of TCL1 in murine B cells (Eμ-TCL1) results in mouse CLL. Here we show for the first time that the previously unexplored endoplasmic reticulum (ER) stress response is aberrantly activated in Eμ-TCL1 mouse and human CLL. This includes activation of the IRE-1/XBP-1 pathway and the transcriptionally up-regulated expression of Derlin-1, Derlin-2, BiP, GRP94, and PDI. TCL1 associates with the XBP-1 transcription factor, and causes the dysregulated expression of the transcription factors, Pax5, IRF4, and Blimp-1, and of the activation-induced cytidine deaminase. In addition, TCL1-overexpressing CLL cells manufacture a distinctly different BCR, as we detected increased expression of membrane-bound IgM and altered N-linked glycosylation of Igα and Igβ, which account for the hyperactive BCR in malignant CLL. To demonstrate that the ER stress-response pathway is a novel molecular target for the treatment of CLL, we blocked the IRE-1/XBP-1 pathway using a novel inhibitor, and observed apoptosis and significantly stalled growth of CLL cells in vitro and in mice. These studies reveal an important role of TCL1 in activating the ER stress response in support for malignant progression of CLL.

Description: Key Points GM-CSF–dependent STAT5 hypersensitivity is detected in 90% of CMML samples and is enhanced by signaling mutations. Treatment with a GM-CSF–neutralizing antibody and JAK2 inhibitors reveals therapeutic potential.

Description: Background: Immunomodulatory drugs (IMiDs), including thalidomide and its analogs, are highly effective for the treatment of both non-del5q and del5q Myelodysplastic Syndrome (MDS) and other hematological cancers. IMiDs have also been shown to play an additional role as a potent T-cell stimulant for cancer therapy. The first identified target of IMiDs is cereblon, an E3 ubiquitin ligase substrate receptor. From crystal structure analysis of human and murine crbn/ddb1 bound to lenalidomide or thalidomide, IMiDs bind to a conserved hydrophobic pocket of crbn called the thalidomide-binding domain. To explore possible mechanisms of crbn regulation of immune response, we studied immune regulation in cereblondeficient mice (crbn-/-), which have increased T-cell proliferation and cytokine production. Results: We have previously shown that purified T-cells from crbn deficient mice have greater immune activation with increased proliferation and pro-inflammatory cytokines, including IL-2, IFNγ, and TNFα, when compared to wild-type T-cells. Additionally crbn-/- T-cells mount a greater alloimmune response in vivo. To determine the molecular effects of crbn deficiency on T-cell response following activation, we analyzed gene expression by Affymetrix microarray in wild-type and crbn-/- purified T-cells before and 12 hours following activation with anti-CD3 and anti-CD28 antibodies. Based on hierarchal clustering, the majority of gene expression changes were driven by T-cell activation. To explore the effect of crbn deficiency on T-cell activation, we compared genes from wild type and crbn-/- T-cells that showed a 2-fold (FDR

Description: Abstract 2595 Large Granular Lymphocyte (LGL) leukemia is a chronic lymphoproliferative syndrome that can be broadly classified into two groups depending on whether the expanded cells are T-cells or NK-cells. The clinical characteristics of the disease include lymphocytosis, neutropenia, anemia, that can be associated with rheumatoid arthritis and pulmonary arterial hypertension (PAH). Hematologic improvement with immunosuppressive agents such as cyclosporine and low-dose methotrexate has lead to the widely accepted theory that cytopenias are mediated by autoimmune destruction of the hematopoietic stem cell (HSC) compartment or lysis of mature myeloid cells in circulation. We found, however, that autologous HSCs and mature granulocyte populations fail to be recognized or lysed ex vivo by T-LGL leukemia cells suggesting that an alternate mechanism may be involved. In contrast to research done on the T-LGL cells themselves, the role of the bone marrow microenvironment and HSC compartment in T-LGL leukemia patients is completely unexplored. Therefore, bone marrow core biopsies, aspirates, and peripheral blood smears were obtained from 22 patients with LGL leukemia and 14 patients with non-hematological malignancies to serve as controls. Morphology and extracellular matrix composition were examined by H&E and reticulin stains, respectively. Utilizing the European consensus guidelines, grading scale (0 – 3), we determined that bone marrow reticulin fibrosis is present in patients with T-LGL leukemia (Figure 1). The mean fibrosis grade for the LGL group was 2.32 (median of 2.5), whereas, the mean fibrosis grade for the control group was 1.46 (median of 1.50), p-value of 0.01. Our analysis revealed that reticulin fibrosis in LGL leukemia bone marrow was particularly associated with the interstitial stroma and lymphoid aggregates. The degree of fibrosis in T-LGL bone marrow showed no relation to absolute neutrophil counts. However, an in depth analysis of neutrophil morphology revealed several dysplastic features within the neutrophil compartment in T-LGL patients. These features include decreased segmentation, increased numbers of pseudo-Pelger-Huet forms, and an increase percentage of immature neutrophils. Of these, the proportion of immature neutrophils positively correlated with fibrosis grade in T-LGL patients (Spearman r=0.7302; p=0.0002), indicating a possible link between reticulin fibrosis and the quality of hematopoiesis. To explore the pathogenesis of medullary fibrosis, mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates of 6 LGL leukemia patients and 5 healthy controls and then expanded ex vivo under non-differentiating conditions. During expansion, healthy MSCs produce cytokines and growth factors necessary for self renewal and for the support of hematopoiesis. However, MSCs from T-LGL patients displayed severely reduced self-renewal potential, reaching a mean of 7 population doublings compared to a mean of 23 for normal MSCs, and were unable to support the proliferation of healthy HSCs in a co-culture proliferation assay. Microarray analysis (H6 V133 plus 2.0) was performed on the MSCs from both control and T-LGL patients with analysis focused on genes regulating basement membrane composition. For normal MSCs, significant reductions in the expression of numerous collagen genes occured as the cells underwent expansion in self-renewal conditions. However, MSCs from T-LGL patients failed to downregulate these genes despite months of culture. The most prominent collagen genes following this pattern were types I (α1, α2), III (reticulin), IV (α1, α2), and V (α1, α2). A combination of qRT-PCR and immunflourescent staining (Figure 2) were utilized to confirm these gene expression changes. Collectively, these results implicate aberrant MSC self-renewal capacity and skewed basement membrane protein expression in the pathogenesis of T-LGL leukemia and suggest that these abnormalities may represent novel targets for future drug discovery. Figure 1: T-LGL patients have increase reticulin fibrosis in bone marrow stroma. Paraffin embeddeb from control (a) or T-LGL patient (b) bone marrow biopsies with reticulin. Figure 1:. T-LGL patients have increase reticulin fibrosis in bone marrow stroma. Paraffin embeddeb from control (a) or T-LGL patient (b) bone marrow biopsies with reticulin. Figure 2: MSCs from T-LGL patients display altered basement membrane composition following ex vivo expansion. Immunofluorescent staining for collagen type I (green), collagen type II (red), and DAPI (blue) was performed on primary MSCs isolated from the bone marrow of normal donors at early passage (a), late passage (b), or T-LGL patients at late passage. Figure 2:. MSCs from T-LGL patients display altered basement membrane composition following ex vivo expansion. Immunofluorescent staining for collagen type I (green), collagen type II (red), and DAPI (blue) was performed on primary MSCs isolated from the bone marrow of normal donors at early passage (a), late passage (b), or T-LGL patients at late passage. Disclosures: No relevant conflicts of interest to declare.