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.Granulocyte-macrophage-colony-stimulating factor (GM-CSF) hypersensitivity is a hallmark of juvenile myelomonocytic leukemia (JMML) but has not been systematically shown in the related human disease chronic myelomonocytic leukemia (CMML). We find that primary CMML samples demonstrate GM-CSF-dependent hypersensitivity by hematopoietic colony formation assays and phospho-STAT5 (pSTAT5) flow cytometry compared with healthy donors. Among CMML patients, the pSTAT5 hypersensitive response positively correlated with high-risk disease, peripheral leukocytes, monocytes, and signalingassociated mutations. When compared with IL-3 and G-CSF, GM-CSF hypersensitivity was cytokine specific and thus a possible target for intervention in CMML. To explore this possibility, we treated primary CMML cells with KB003, a novel monoclonal anti-GM-CSF antibody, and JAK2 inhibitors. We found that an elevated proportion of immature GM-CSF receptor-a(R) subunit-expressing cells were present in the bone marrow myeloid compartment of CMML. In survival assays, we found that myeloid and monocytic progenitors were sensitive to GM-CSF signal inhibition. Our data indicate that a committed myeloid precursor expressing CD38 may represent the progenitor population with enhanced GM-CSF dependence in CMML, consistent with results in JMML. These preclinical data indicate that GM-CSF signaling inhibitors merit further investigation in CMML and that GM-CSFR expression on myeloid progenitors may be a biomarker for this therapy. (Blood. 2013;121(25):5068-5077)
IntroductionChronic lymphocytic leukemia (CLL) represents 30% of adult leukemia and is an incurable B-cell malignancy. Malignant CLL cells use a limited repertoire of immunoglobulin heavy and light chain genes to manufacture their BCRs, [1][2][3] and are very responsive to in vitro anti-IgM stimulation. 4,5 Thus, Ag stimulation has been proposed to drive malignant progression of CLL.The functions of the endoplasmic reticulum (ER) and its associated molecules in CLL have not attracted extensive investigative efforts because CLL cells do not exhibit a readily prominent ER structure like professional secretory cells. Although no protein Ag has been shown to drive malignant progression of CLL in vivo, exposure to Toll-like receptor ligands activates CLL cells, allowing rapid proliferation, 6,7 a cellular process accompanied by robust production and folding of membrane receptors and secretory proteins in the ER. We hypothesize that the ER may play an important role in malignant progression of CLL. First, electron microscopy examinations of human CLL cells showed clear ER expansions and immunoglobulin staining in the ER. [8][9][10] Second, treatments that target ER-Golgi protein transport or inhibit BiP (HSP70 in the ER) and GRP94 (HSP90 in the ER) can sensitize CLL cells to drug-induced apoptosis. 9,11,12 The IRE-1/XBP-1 pathway is activated in response to stress conditions like proteotoxicity or hypoxia in the ER, but it also plays important roles in maintaining basal cellular functions. 13,14 IRE-1 is an ER-resident transmembrane protein that contains a stress sensor domain in the lumen of the ER, and a serine/threonine kinase domain linked to an RNase domain in the cytoplasm. On stress conditions, IRE-1 oligomerizes via its luminal domains in the ER, bringing together the cytoplasmic kinase domains which can undergo autophosphorylation and up-regulate IRE-1's RNase activity. The IRE-1 RNase then splices 26 nucleotides from the mature XBP-1 mRNA, allowing the spliced XBP-1 mRNA to encode the functional 54-kDa transcription factor XBP-1. [15][16][17] XBP-1 regulates a panel of important genes 18 and can crosstalk with other B-cell transcription factors, such as IRF4 and Blimp-1. 19 Overexpression of XBP-1 in B cells has been shown to cause monoclonal gammopathy of undetermined significance, a precursor condition for multiple myeloma. 20 Here, we investigate the roles of the ER stress response in the E-TCL1 CLL mouse model, in which the TCL1 gene is under the control of the immunoglobulin heavy chain promoter/enhancer driving TCL1 overexpression in B cells. 21 TCL1 is expressed in ϳ 90% human CLL patients, 22 and its overexpression is associated with strong BCR signaling, 23,24 allowing malignant CLL cells to undergo high-rate proliferation. E-TCL1 mice initially develop a preleukemic state with clear CD5 ϩ IgM ϩ B-cell characteristics in the blood, spleen, lymph nodes, and bone marrow, and slowly progress to the full-blown monoclonal CLL stage with all clinical features of aggressive human CLL. 21,25 Similar to huma...
Myleodysplastic syndromes (MDS) are premalignant diseases characterized by cytopenias, myeloid dysplasia, immune dysregulation with association to autoimmunity, and variable risk for acute myeloid leukemia (AML) transformation. Studies of Forkhead-box P3 (FoxP3)+ regulatory T-cells (Tregs) indicate that the number and/or activation state may influence cancer progression in these patients. Focusing on patients with a lower-risk for leukemia transformation, 18 (34.6%) of 52 patients studied displayed an altered Treg compartment compared to age-matched controls. Delineation of unique Treg subsets revealed that an increase in the absolute number of CD4(+)FoxP3(+)CD25(+)CD127(low)CD45RA(−)CD27(−) Tregs (effector memory Tregs; TregEM) was significantly associated with anemia (p=0.046), reduced hemoglobin (p=0.038), and blast counts ≥5% (p=0.006). In healthy donors, this TregEM population constitutes only 2% of all Tregs (1–6 Treg cells/μl) in peripheral blood, but when isolated, exhibit greater suppressive activity in vitro. With a median follow-up of 3.1 years (range-2.7 to 4.9) from sample acquisition, increased numbers of TregEM cells proved to have independent prognostic importance in survival estimates suggesting that enumeration of this Treg subset may be a more reliable indicator of immunological escape than FoxP3+ T-cells as a whole. Based on multivariate analyses, TregEM impacted survival independently from myeloblast characteristics, cytopenias, karyotype and comorbidities. Based on these findings, TregEM cell expansion may be synonymous with human Treg activation and indicate microenvironmental changes conducive to transformation in MDS.
Tumor-induced immune suppression involves the accumulation of immune-suppressive infiltrates in the microenvironment. This study demonstrates increased numbers of CD4+CD25+FoxP3+ regulatory T cells (Tregs) in the lungs of C57BL/6 mice bearing a metastatic Lewis lung carcinoma (LLC) variant. These Tregs suppressed the proliferation of endogenous CD4+CD25− cells and expressed higher levels of the chemokine receptor CCR4 than other types of T cells. LLC-bearing lungs secreted elevated levels of the CCR4-associated chemokine CCL22 compared with normal lungs. However, CCL22 was not secreted by LLC or normal epithelial controls, suggesting that CCL22 is secreted by a nonepithelial component of the microenvironment. Migration assays revealed that medium conditioned by LLC-bearing lungs selectively recruited Tregs at higher frequencies than did medium conditioned by normal lungs. Neutralization of CCL22 significantly reduced this selective recruitment toward both conditioned media. A series of immunomagnetic isolations, FACS, and flow cytometric analyses were used to isolate different cellular fractions from both normal and LLC-bearing lungs. When isolated, only the NK-containing fractions secreted CCL22, and the same fraction isolated from LLC-bearing lungs secreted higher levels. Depletion of NK cells from both normal and LLC-bearing lung tissue significantly reduced CCL22 secretion, suggesting that a large portion of secreted CCL22 is NK cell dependent. Flow cytometric analysis of the lung NK compartments revealed no significant increase in NK cell numbers across LLC-bearing lung tissue as a whole as compared with normal tissue. However, immunofluorescent staining revealed an increased frequency of NK cells at the tumor periphery that were closely associated with the elevated FoxP3+ infiltrate.
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