Impaired immunomodulatory function of chronic myeloid leukemia cancer stem cells and the possible mechanism involved in it.

BACKGROUND: Cancer stem cells (CSCs) are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Development of specific therapies targeted at CSCs holds hope for the improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease. This is particularly true in chronic myeloid leukemia (CML). METHODS: In this study, we isolated fetal liver kinase-1-positive (Flk1(+)) cells carrying the BCR/ABL fusion gene from the bone marrow of Philadelphia chromosome-positive (Ph(+)) patients with stem cells property. We examined their biological characteristics as well as immunological function and further detected the possible molecular mechanism involved in the leukemia genesis. RESULTS: We showed that CML patient-derived Flk1(+)CD31(-)CD34(-) MSCs had normal morphology, phenotype and karyotype but appeared impaired immunomodulatory function. The capacity of Flk1(+)CD31(-)CD34(-) MSCs from CML patients to inhibit T lymphocyte activation and proliferation was impaired in vitro. CML patient-derived MSCs have dampening immunomodulatory functions, suggesting that the dysregulation of hematopoiesis and immune response might originate from MSCs rather than HSCs. These Ph(+) putative CML hemangioblast upregulated TGF-ß1 and resultantly activated matrix metalloproteinase-9 (MMP-9) to enhance s-KitL and s-ICAM-1 secretion, which activated c-kit(+) HSCs from the quiescent state to proliferative state. Further studies showed that phosphatidylinositol-3 kinase (PI3K)/Akt/nuclear factor (NF)-κB signaling pathway was involved in CML pathogenesis. CONCLUSIONS: Flk1(+)CD31(-)CD34(-) MSCs that express BCR/ABL leukemia oncogene are CSCs of CML and they play a critical role in the progression of CML through PI3K/Akt/NF-κB/MMP-9/s-ICAM-1/s-KitL signaling pathway beyond HSCs.

Graft-versus-leukemia (GVL) against mouse blast-crisis chronic myelogenous leukemia (BC-CML) and chronic-phase chronic myelogenous leukemia (CP-CML): shared mechanisms of T cell killing, but programmed death ligands render CP-CML and not BC-CML GVL resistant.

Graft-versus-leukemia (GVL) against chronic-phase chronic myelogenous leukemia (CP-CML) is potent, but it is less efficacious against acute leukemias and blast-crisis chronic myelogenous leukemia (BC-CML). The mechanisms underlying GVL resistance are unknown. Previously, we found that alloreactive T cell targeting of GVL-sensitive bcr-abl-induced mouse CP-CML (mCP-CML) required TCR-MHC interactions and that multiple and redundant killing mechanisms were in play. To better understand why BC-CML is resistant to GVL, we performed a comprehensive analysis of GVL against mouse BC-CML (mBC-CML) induced by the retroviral transfer of the bcr-abl and NUP98/HOXA9 fusion cDNAs. Like human BC-CML, mBC-CML was GVL resistant, and this was not due to accelerated kinetics or a greater leukemia burden. To study T cell recognition and killing mechanisms, we generated a panel of gene-deficient leukemias by transducing bone marrow from gene-deficient mice. T cell target recognition absolutely required that mBC-CML cells express MHC molecules. GVL against both mCP-CML and mBC-CML required leukemia expression of ICAM-1. We hypothesized that mBC-CML would be resistant to some of the killing mechanisms sufficient to eliminate mCP-CML, but we found instead that the same mechanisms were effective against both types of leukemia, because GVL was similar against wild-type or mBC-CML genetically lacking Fas, TRAIL-R, Fas/TRAIL-R, or TNFR1/R2 or when donor T cells were perforin(-/-). However, mCP-CML, but not mBC-CML, relied on expression of programmed death-1 ligands 1 and 2 (PD-L1/L2) to resist T cell killing, because only GVL against mCP-CML was augmented when leukemias lacked PD-L1/L2. Thus, mBC-CML cells have cell-intrinsic mechanisms, distinct from mCP-CML cells, which protect them from T cell killing.

ICSBP-mediated immune protection against BCR-ABL-induced leukemia requires the CCL6 and CCL9 chemokines.

Interferon (IFN) is effective at inducing complete remissions in patients with chronic myelogenous leukemia (CML), and evidence supports an immune mechanism. Here we show that the type I IFNs (alpha and beta) regulate expression of the IFN consensus sequence-binding protein (ICSBP) in BCR-ABL-transformed cells and as shown previously for ICSBP, induce a vaccine-like immunoprotective effect in a murine model of BCR-ABL-induced leukemia. We identify the chemokines CCL6 and CCL9 as genes prominently induced by the type I IFNs and ICSBP, and demonstrate that these immunomodulators are required for the immunoprotective effect of ICSBP expression. Insights into the role of these chemokines in the antileukemic response of IFNs suggest new strategies for immunotherapy of CML.

Induced dendritic cell differentiation of chronic myeloid leukemia blasts is associated with down-regulation of BCR-ABL.

Although differentiation of leukemic blasts to dendritic cells (DC) has promise in vaccine strategies, the mechanisms underlying this differentiation and the differences between leukemia and normal progenitor-derived DC are largely undescribed. In the case of chronic myeloid leukemia (CML), understanding the relationship between the induction of DC differentiation and the expression of the BCR-ABL oncogene has direct relevance to CML biology as well as the development of new therapeutic approaches. We now report that direct activation of protein kinase C (PKC) by the phorbol ester PMA in the BCR-ABL(+) CML cell line K562 and primary CML blasts induced nonterminal differentiation into cells with typical DC morphology (cytoplasmic dendrites), characteristic surface markers (MHC class I, MHC class II, CD86, CD40), chemokine and transcription factor expression, and ability to stimulate T cell proliferation (equivalent to normal monocyte-derived DC). PKC-induced differentiation was associated with down-regulation of BCR-ABL mRNA expression, protein levels, and kinase activity. This down-regulation appeared to be signaled through the mitogen-activated protein kinase pathway. Therefore, PKC-driven differentiation of CML blasts into DC-like cells suggests a potentially novel strategy to down-regulate BCR-ABL activity, yet raises the possibility that CML-derived DC vaccines will be less effective in presenting leukemia-specific Ags.

The BCR/ABL transgene causes abnormal NK cell differentiation and can be found in circulating NK cells of advanced phase chronic myelogenous leukemia patients.

NK cells from the blood of chronic myelogenous leukemia (CML) patients are progressively decreased in number as the disease progresses from chronic phase to blast crisis. We hypothesize that BCR/ABL may be directly responsible by interfering with NK cell differentiation. CD34(+)HLA-DR(+) cells from CML patients were studied for their capacity to differentiate into NK cells. The NK cell cloning frequency was significantly decreased from CML CD34(+)HLA-DR(+) cells compared with cells from normal donors, yet CD34(+)HLA-DR(+) cells gave rise to BCR/ABL(+) NK cells in some patients. This finding prompted us to further investigate circulating NK cells from the blood of CML patients. CD56(+)CD3(-) NK cells were sorted from CML patients and examined by fluorescence in situ hybridization (FISH). In contrast to chronic phase CML, significant numbers of NK cells from advanced phase CML patients were BCR/ABL(+), whereas T cells were always BCR/ABL(-) regardless of the disease stage. To test the effects of BCR/ABL as the sole genetic abnormality, BCR/ABL was transduced into umbilical cord blood CD34(+) cells, and NK development was studied. p210-enhanced green fluorescence protein-transduced cells gave rise to significantly decreased numbers of NK cells compared with enhanced green fluorescence protein transduction alone. In addition, the extrinsic addition of BCR/ABL-transduced autologous CD34(+) cells suppressed the NK cell differentiation of normal umbilical cord blood CD34(+)CD38(-) cells. This study provides the first evidence that BCR/ABL is responsible for the altered differentiation of NK cells and that the NK cell lineage can be involved with the malignant clone in advanced stage CML.

bcl2 and v-abl oncogenes cooperate to immortalize murine B cells that secrete antigen specific antibodies.

In this manuscript, a general strategy was designed and used to rapidly test whether any combination(s) of p53, v-abl, bcl2 and ras oncogenes could act cooperatively to immortalize B cells. Here we report that only the combination of v-abl and bcl2 was successful. Splenic B cells from beta galactosidase-immunized mice were stimulated in vitro with lipopolysaccharide and dextran sulphate for 48 h and co-infected with ecotropic A-MuLV (v-abl) and amphotropic pZip-bcl2 (human bcl2) viruses. When inoculated i.p. into naive pristane-primed mice, these B cells generated mesenteric lymphadenopathy, intraperitoneal lymph nodules and ascites in 100% (8/8) of the mice within 36-53 days. The ascites fluid contained 69.5-122 microg/ml IgG and 2.5-13 microg/ml IgM against the immunogen. The ascites cells were passed intraperitoneally up to three times. In all passages, ascites tumors were generated, and the ascites fluid contained beta galactosidase-specific IgG and IgM, indicating that some immunoglobulin secreting B cells had been immortalized. Neither ascites nor tumors were produced when B cells infected with only one of the viruses was injected into the mice. The presence of each oncogene in ascites cells was verified by immunohistochemistry or RT-PCR. This study provides evidence for the cooperativity of an unexpected pair of oncogenes in B cell immortalization.

Distinct tumorigenic potential of abl and raf in B cell neoplasia: abl activates the IL-6 signaling pathway.

The development of murine plasma cell tumors induced by raf/myc containing retroviruses is facilitated by T cells and completely dependent on IL-6. To determine whether kinases with differing specificities reflect alternative biochemical pathways in B cell tumorigenesis, we have employed an abl/myc containing retrovirus to assess neoplastic development. In contrast with raf/myc, abl/myc disease is T cell and IL-6 independent. An examination of the IL-6 signal transduction pathway reveals that this pathway, as defined by activation of Stat3, is inducible by IL-6 in raf/myc tumors but constitutively activated in abl/myc tumors. These findings provide a mechanism for the derivation of cytokine-independent plasma cell tumors and suggest that both IL-6-dependent and independent tumors may arise in vivo depending on the particular mutational events incurred during tumorigenesis.