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Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. To investigate the role of microRNAs (miRNAs) in regulating drug resistance and leukemic stem cell (LSC) fate, we performed global transcriptome profiling in treatment-naive chronic myeloid leukemia (CML) stem/progenitor cells and identified that miR-185 levels anticipate their response to ABL tyrosine kinase inhibitors (TKIs). miR-185 functions as a tumor suppressor: its restored expression impaired survival of drug-resistant cells, sensitized them to TKIs in vitro, and markedly eliminated long-term repopulating LSCs and infiltrating blast cells, conferring a survival advantage in preclinical xenotransplantation models. Integrative analysis with mRNA profiles uncovered PAK6 as a crucial target of miR-185, and pharmacological inhibition of PAK6 perturbed the RAS/MAPK pathway and mitochondrial activity, sensitizing therapy-resistant cells to TKIs. Thus, miR-185 presents as a potential predictive biomarker, and dual targeting of miR-185-mediated PAK6 activity and BCR-ABL1 may provide a valuable strategy for overcoming drug resistance in patients.

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38−) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.

Xenograft models are transforming our understanding of the output capabilities of primitive human hematopoietic cells in vivo . However, many variables that affect posttransplantation reconstitution dynamics remain poorly understood. Here, we show that an equivalent level of human chimerism can be regenerated from human CD34 + cord blood cells transplanted intravenously either with or without additional radiation-inactivated cells into 2- to 6-month-old NOD-Rag1 –/– -IL2Rγc –/– (NRG) mice given a more radioprotective conditioning regimen than is possible in conventionally used, repair-deficient NOD- Prkdc scid/scid - IL2Rγc –/– (NSG) hosts. Comparison of sublethally irradiated and non-irradiated NRG mice and W 41 /W 41 derivatives showed superior chimerism in the W 41 -deficient recipients, with some differential effects on different lineage outputs. Consistently superior outputs were observed in female recipients regardless of their genotype, age, or pretransplantation conditioning, with greater differences apparent later after transplantation. These results define key parameters for optimizing the sensitivity and minimizing the intraexperimental variability of human hematopoietic xenografts generated in increasingly supportive immunodeficient host mice.

Human B-cell precursor acute lymphoid leukemias (BCP-ALLs) comprise a group of genetically and clinically distinct disease entities with features of differentiation arrest at known stages of normal B-lineage differentiation. We previously showed that BCP-ALL cells display unique and clonally heritable, stable DNA replication timing (RT) programs (ie, programs describing the variable order of replication and subnuclear 3D architecture of megabase-scale chromosomal units of DNA in different cell types). To determine the extent to which BCP-ALL RT programs mirror or deviate from specific stages of normal human B-cell differentiation, we transplanted immunodeficient mice with quiescent normal human CD34+ cord blood cells and obtained RT signatures of the regenerating B-lineage populations. We then compared these with RT signatures for leukemic cells from a large cohort of BCP-ALL patients with varied genetic subtypes and outcomes. The results identify BCP-ALL subtype-specific features that resemble specific stages of B-cell differentiation and features that seem to be associated with relapse. These results suggest that the genesis of BCP-ALL involves alterations in RT that reflect biologically significant and potentially clinically relevant leukemia-specific epigenetic changes.

Summary Patients with chronic myeloid leukemia (CML) often require lifelong therapy with ABL1 tyrosine kinase inhibitors (TKIs) due to a persisting TKI-resistant population of leukemic stem cells (LSCs). From transcriptome profiling, we show integrin-linked kinase (ILK), a key constituent of focal adhesions, is highly expressed in TKI-nonresponsive patient cells and their LSCs. Genetic and pharmacological inhibition of ILK impaired the survival of nonresponder patient cells, sensitizing them to TKIs, even in the presence of protective niche cells. Furthermore, ILK inhibition eliminated TKI-refractory LSCs from patients, but not normal HSCs, in vitro and in vivo. RNA-sequencing and functional validation studies implicated an important role of ILK in maintaining a requisite level of mitochondrial oxidative metabolism in highly purified, quiescent LSCs. Thus, these findings point to ILK as a critical survival mediator to TKIs and quiescent stem cells, offering an attractive therapeutic target and model for curative combination therapies in stem-cell-driven cancers.

Plerixafor (P) together with granulocyte colony–stimulating factor (G) is now recognized as an important strategy for mobilizing hematopoietic cells for use in patients given myelosuppressive therapies. However, quantitative comparisons of their ability to mobilize human cells with different hematopoietic activities in vitro or in vivo (in immunodeficient mice) and their interrelationships have not been investigated. To address these questions, we collected samples from 5 normal adult volunteers before and after administering P alone and from another 5 before and after a 4-day course of G and again after a subsequent injection of P. Measurements of their blood content of CD34+ cells, in vitro myeloid colony–forming cells, 3- and 6-week long-term culture (LTC) cell outputs, and levels of circulating human platelets, as well as myeloid and lymphoid cells obtained in immunodeficient mice that received transplants, showed all activities were maximal 4 hours after P preceded by G, and 3-week LTC outputs showed the highest concordance with the 3-week circulating human neutrophil levels obtained in mice that received transplants. Thus, human cells capable of producing neutrophils rapidly in vivo were optimally mobilized by the G + P protocol, and the 3-week LTC assay appears to offer a more specific predictor of their levels than conventional CD34+ cell or colony-forming cell counts.

A 60-year-old woman with Sjögren's syndrome showed recurrence of parotid mucosa-associated lymphoid tissue (MALT) lymphoma with a simultaneous increase of serum sIL-2R antigen levels 10 years after surgical treatment. Helicobacter pylori infection had been detected in the stomach since the beginning of the lymphoma. Although H. pylori was not detected in the recurrent parotid lymphoma, antibiotic therapy contributed not only to successful eradication of gastric H. pylori but also to disappearance of the recurrent lymphoma. Further studies on the mechanisms of occurrence of extragastric MALT lymphomas are needed to establish the treatment of extragastric MALT lymphomas.

ABCG2 is a transporter preferentially expressed in a primitive subpopulation of cells and recently reported as a surviving factor for trophoblasts. To date, manner of ABCG2 expression in lymphoid tissues is not known. Immunohistochemically, strong ABCG2 expression was found in a small proportion of plasma cells mainly located in the interfollicular space of lymphoid tissues. The number of ABCG2-high plasma cells increased in interleukin-6- (IL-6) rich lesions, such as Castleman's disease of plasma cell type. Plasma cells are subjected to endoplasmic reticulum (ER) stress when excess proteins are synthesized, and IL-6 stimulates protein synthesis. Therefore, the effect of IL-6 and ER stress on ABCG2 expression in plasma cells was examined. The expression level of ABCG2 increased by treatment with either IL-6 or ER stress inducers, and further increased with both. The promoter analysis revealed that the effect of IL-6 and ER stress inducers was mediated through the site overlapping XBP-1 and HIF-1 binding sequences. Knocked-down of ABCG2 by siRNA or ABCG2 inhibitor reduced plasma cell viability under ER stress. These suggest that ABCG2 is a surviving factor for plasma cells. To our knowledge, this is the first study reporting the effect of ER stress on ABCG2 expression.

Cancer stem cells are supposed to be resistant to apoptosis, but information for this is quite limited. Cancer stem cells are usually isolated as dye-effluxing cells with Hoechst 33342 staining, called side-population (SP) cells. Because Hoechst 33342 dye itself induces apoptosis, the SP cells isolated by such method are not suitable for evaluation of apoptosis. For accurate assessment, SP cells must be isolated without Hoechst 33342. Here, we found that CD55 was highly expressed in SP cells of two mammary gland carcinoma cell lines. Then, the high expression of CD55 was used for isolation of cancer stem cells among mammary carcinoma cell lines as a surrogate character. Cells expressing high level of CD55 (CD55(hi)) were resistant to apoptosis induced by serum depletion as in the case of SP cells. In ceramide-inducing apoptosis, CD55(hi) cells showed high tolerance. Anti-apoptotic molecules such as Bcl-2 were abundantly expressed in both SP cells and CD55(hi) cells. These findings indicated that SP cells as revealed to be CD55(hi) cells were tolerant to apoptosis. The high expression of CD55 may be a useful character for SP cells in evaluating their functions.

Genome-wide DNA replication timing (RT) profiles reflect the global 3D chromosome architecture of cells. They also provide a comprehensive and unique megabase-scale picture of the cellular epigenetic state. Thus normal differentiation involves reproducible changes in RT and transformation generally perturbs these, although the potential effects of altered RT on the properties of transformed cells remain largely unknown. A major challenge to interrogating these issues in human acute lymphoid leukemia (ALL) is the low proliferative activity of most of the cells, which may be further reduced in cryopreserved samples and difficult to overcome in vitro. In contrast, the ability of many human ALL cell populations to expand when transplanted in highly immunodeficient mice is well documented. To examine the stability of DNA RT profiles of serially passaged xenografts of primary human B- and T-ALL cells, we first devised a method that circumvents the need for BrdU incorporation to distinguish early versus late S-phase cells. Using this and more standard protocols, we found consistent strong retention in xenografts of the original patient-specific RT features, for all 8 primary human ALL cases surveyed (7 B-ALLs and one T-ALL). Moreover, in a case where genomic analyses indicated changing subclonal dynamics in serial passages, the RT profiles tracked concordantly. These results show that DNA RT is a relatively stable feature of human ALLs propagated in immunodeficient mice. In addition, they suggest the power of this approach for future interrogation of the origin and consequences of altered DNA RT in these diseases.

Some thrombopoietin receptor-agonists (TPR-As) have been developed and shown to be highly effective in the treatment of immune thrombocytopenic purpura (ITP). Soluble cytotoxic T-lymphocyte-associated antigen 4 (sCTLA-4) can modulate and terminate the immune response. Several reports have shown that sCTLA-4 levels are elevated in patients with some autoimmune disorders. However, sCTLA-4 levels have not previously been investigated in TPR-A exposed patients with ITP. We investigated the levels of transforming growth factor (TGF) β1 and sCTLA-4 in ITP patients to determine the clinical association with TGFβ1 and sCTLA-4 in TPR-A-exposed patients with ITP. Thirty-seven ITP patients were divided into 2 groups (TPR-A-exposed: 13 patients; unexposed: 24 patients). Doses of eltrombopag ranging from 12.5 mg to 50 mg were administered daily, and biochemical data obtained before and after eltrombopag administration were compared. Eltrombopag therapy significantly increased sCTLA-4 and TGFβ1 levels relative to baseline in patients with ITP. In addition, plasma TGFβ1 was positively correlated with platelet counts and sCTLA-4 in patients with ITP in the eltrombopag-exposed group. However, no significant change in the detection rate for anti-glycoprotein antibody was observed before and 24 weeks after eltrombopag treatment. These results suggest that eltrombopag can partially modulate some immune responses by TGFβ1 and sCTLA-4, but it does not induce immune tolerance by 24 weeks after treatment.

We report a case of acute mixed-lineage leukemia, as seen in a 65 year-old female with MLL gene amplification and biallelic loss of wild type p53 gene. The diagnosis was based on the findings that her bone marrow (BM) blasts expressed cytoplasmic CD3 (cyCD3), B-lineage antigens and myeloid antigens accompanied by clonal rearrangements of IgH gene. The BM blasts consisted of small-sized peroxidase-negative blasts (97%) and large-sized peroxidase-positive blasts (3%). The BM blasts showed a complex "karyotype," including dic(17;20) (p11;q11), -5 and add (11q23). Add (11q23) abnormality was found in sideline karyotypes as well as the stemline abnormality of dic(17;20) (p11;q11). For the p53 gene, which is located at 17p13, fluorescence in situ hybridization analysis showed the loss of one of two p53 alleles. Furthermore, polymerase chain reaction-single-strand conformation polymorphism and following nucleotide sequencing showed that the p53 gene was mutated at codon 215, leading to an amino acid substitution from Ser to Arg. For the MLL gene, southern blot analysis showed that the MLL gene locus was amplified but not rearranged at its breakpoint cluster region, which is usually rearranged in balanced translocations with many partner genes. These findings suggest that MLL gene amplification may in this case be based on the genetic instability caused by the preceding biallelic loss of the wild type p53 gene.

Statins, which are used as cholesterol-lowering agents, have pleiotropic immunomodulatory properties. Although beneficial effects of statins have been reported in autoimmune diseases, the mechanisms of these immunomodulatory effects are still poorly understood. Type I interferons (IFNs) and plasmacytoid dendritic cells (PDCs) represent key molecular and cellular pathogenic components in autoimmune diseases such as systemic lupus erythematosus (SLE). Therefore, PDCs may be a specific target of statins in therapeutic strategies against SLE. This study was undertaken to investigate the immunomodulatory mechanisms of statins that target the IFN response in PDCs.We isolated human blood PDCs by flow cytometry and examined the effects of simvastatin and pitavastatin on PDC activation, IFNalpha production, and intracellular signaling.Statins inhibited IFNalpha production profoundly and tumor necrosis factor alpha production modestly in human PDCs in response to Toll-like receptor ligands. The inhibitory effect on IFNalpha production was reversed by geranylgeranyl pyrophosphate and was mimicked by either geranylgeranyl transferase inhibitor or Rho kinase inhibitor, suggesting that statins exert their inhibitory actions through geranylgeranylated Rho inactivation. Statins inhibited the expression of phosphorylated p38 MAPK and Akt, and the inhibitory effect on the IFN response was through the prevention of nuclear translocation of IFN regulatory factor 7. In addition, statins had an inhibitory effect on both IFNalpha production by PDCs from SLE patients and SLE serum-induced IFNalpha production.Our findings suggest a specific role of statins in controlling type I IFN production and a therapeutic potential in IFN-related autoimmune diseases such as SLE.

Purpose: Our recent study revealed that CD55-high population in breast cancer cell line was resistant to apoptosis and formed colonies in vitro more efficiently than CD55-low population. The present study was conducted to examine whether CD55-high population in breast cancer cell line possesses higher tumorigenic potential in vivo and presence of CD55-high cells in breast cancer affects clinicopathologic behavior of patients.Experimental Design: CD55-high and CD55-low population was sorted from breast cancer cell line, injected into immunodeficient mice, and the resultant tumor volume was measured. CD55 expression was immunohistochemically examined in clinical samples from 74 cases with breast cancers, and cases with >1% of tumor cells showing high level of CD55 expression were categorized as CD55 high.Results: The xenotransplanted tumor volume derived from CD55-high population was significantly larger than that from CD55-low population. Fifty (67.6%) of 74 cases of breast cancer were CD55-high. A significant correlation was observed between CD55-high character and relapse rate (P < 0.001). Univariate analysis showed that tumor size (P = 0.005) and CD55 expression (P = 0.005) were unfavorable prognostic factors. Multivariate analysis revealed that the tumor size (P = 0.013) and CD55 expression (P = 0.011) were independent prognostic factors.Conclusions: CD55 play an important role in tumorigenesis of breast cancer, and presence of small population of cells with strong CD55 expression would be sufficient to predict poor prognosis of patients.

The majority of cases of intravascular lymphomatosis (IVL) is derived from B cells. However, IVL may also arise from T cells, or more rarely NK cells. The clinicopathological findings in six cases of NK-cell IVL (NK-IVL), including one new case, were summarised and compared with B-cell IVL (B-IVL) and T-cell IVL (T-IVL). Earlier onset of disease and female predominance were found in NK-IVL. NK-IVL was typically Epstein-Barr virus (EBV)-positive, whereas EBV was rarely detected in B-IVL. Cutaneous manifestations were common in NK-IVL with constant EBV infection. B-IVL showed a more favourable prognosis than T- or NK-IVL. Irrespective of immunophenotype, however, IVL showed a less favourable prognosis than ordinary lymphomas within the same immunophenotype. In summary, IVL of the B-, T- and NK-cell phenotypes is clinicopathologically distinct and shows similarities to their more common counterparts, i.e. diffuse large B-cell lymphoma, peripheral T-cell lymphoma, unspecified and extranodal NK/T-cell lymphoma, nasal type.

A 69-year-old man was admitted to our hospital with fever and right neck lymphadenopathy. A neck lymph node biopsy was performed. In the specimens, immunoblasts were present with an admixture of small T lymphocytes and macrophagocytes. Immunohistochemcal staining of immunoblasts was positive for CD20, CD30 and EBER. Epstein-Barr virus (EBV) serology showed elevated IgG antibody to VCA, and EBV DNA was detected in the peripheral blood. Since he showed latency II without immunodeficiency disease, we diagnosed age-related EBV-associated B-cell lymphoproliferative disorder (EBV-LPD) as a result of aging and EBV infection. He was treated with 6 courses of the CHOP regimen plus rituximab, and achieved complete remission. EBV-DNA became undetectable at remission. In age-related EBV-LPD, there is some possibility that EBV-DNA in the peripheral blood is valuable as a tumor biomarker.

CDCP1, a novel stem cell marker, is expressed in hematopoietic cell line K562 but not in Jurkat. When CDCP1 promoter was transfected exogenously, Jurkat showed comparable promoter activity with K562, suggesting that the factor to enhance transcription was present but interfered to function in Jurkat. The reporter assay and si-RNA-mediated knockdown experiment revealed that zfp67, a zinc-finger protein, enhanced CDCP1 transcription. Amount of zfp67 in Jurkat was comparable with K562, but chromatin immunoprecipitation showed that zfp67 bound to CDCP1 promoter in K562 but not in Jurkat. There are CpG sequences around the promoter of CDCP1, which were heavily methylated in Jurkat but not in K562. Addition of demethylating reagent to Jurkat induced CDCP1 expression, and increased the zfp67 binding to CDCP1 promoter. Among normal hematopoietic cells such as CD34+CD38- cells, lymphocytes and granulocytes, inverse correlation between proportion of methylated CpG sequences and CDCP1 expression level was found. Demethylation of CpG sequences in lymphocytes, in which CpG sequences were heavily methylated, induced CDCP1 expression and its expression level further increased through zfp67 overexpression. The methylation of DNA appeared to regulate the cell-type-specific expression of CDCP1 through the control of interaction between chromatin DNA and transcription factors.

An adult patient suffering from G-CSF-resistant very severe aplastic anemia received a cord blood transplantation from a three-loci HLA-mismatched unrelated donor after nonmyeloablative conditioning. Cord blood was infused after conditioning with fludarabine (180 mg/m2) and cyclophosphamide (100 mg/kg). Cyclosporin A and short-term methotrexate were used for prophylaxis against acute GVHD. Engraftment was achieved on day 23, and there was no serious GVHD. A full-donor type T-cell chimerism was obtained by day 30. Normal hematopoiesis and complete chimerism have been maintained 14 months after transplantation.