IL-7R is essential for leukemia-initiating cell activity of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from the dysregulation of signaling pathways that control intrathymic T-cell development. Relapse rates are still significant, and prognosis is particularly bleak for relapsed patients. Therefore, development of novel therapies specifically targeting pathways controlling leukemia-initiating cell (LIC) activity is mandatory for fighting refractory T-ALL. The interleukin-7 receptor (IL-7R) is a crucial T-cell developmental pathway that is commonly expressed in T-ALL and has been implicated in leukemia progression; however, the significance of IL-7R/IL-7 signaling in T-ALL pathogenesis and its contribution to disease relapse remain unknown. To directly explore whether IL-7R targeting may be therapeutically efficient against T-ALL relapse, we focused on a known Notch1-induced T-ALL model, because a majority of T-ALL patients harbor activating mutations in NOTCH1, which is a transcriptional regulator of IL-7R expression. Using loss-of-function approaches, we show that Il7r-deficient, but not wild-type, mouse hematopoietic progenitors transduced with constitutively active Notch1 failed to generate leukemia upon transplantation into immunodeficient mice, thus providing formal evidence that IL-7R function is essential for Notch1-induced T-cell leukemogenesis. Moreover, we demonstrate that IL-7R expression is an early functional biomarker of T-ALL cells with LIC potential and report that impaired IL-7R signaling hampers engraftment and progression of patient-derived T-ALL xenografts. Notably, we show that IL-7R-dependent LIC activity and leukemia progression can be extended to human B-cell acute lymphoblastic leukemia (B-ALL). These results have important therapeutic implications, highlighting the relevance that targeting normal IL-7R signaling may have in future therapeutic interventions, particularly for preventing T-ALL (and B-ALL) relapse.

MicroRNA214 Is Associated With Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-Associated Cancer in Mice.

BACKGROUND & AIMS: Persistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC). METHODS: We performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohn's disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214). RESULTS: A high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN. CONCLUSIONS: Interleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.

A functional microRNA library screen reveals miR-410 as a novel anti-apoptotic regulator of cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma is characterized by late diagnosis and a poor survival rate. MicroRNAs have been involved in the pathogenesis of different cancer types, including cholangiocarcinoma. Our aim was to identify novel microRNAs regulating cholangiocarcinoma cell growth in vitro and in vivo. METHODS: A functional microRNA library screen was performed in human cholangiocarcinoma cells to identify microRNAs that regulate cholangiocarcinoma cell growth. Real-time PCR analysis evaluated miR-9 and XIAP mRNA levels in cholangiocarcinoma cells and tumors. RESULTS: The screen identified 21 microRNAs that regulated >50 % cholangiocarcinoma cell growth. MiR-410 was identified as the top suppressor of growth, while its overexpression significantly inhibited the invasion and colony formation ability of cholangiocarcinoma cells. Bioinformatics analysis revealed that microRNA-410 exerts its effects through the direct regulation of the X-linked inhibitor of apoptosis protein (XIAP). Furthermore, overexpression of miR-410 significantly reduced cholangiocarcinoma tumor growth in a xenograft mouse model through induction of apoptosis. In addition, we identified an inverse relationship between miR-410 and XIAP mRNA levels in human cholangiocarcinomas. CONCLUSIONS: Taken together, our study revealed a novel microRNA signaling pathway involved in cholangiocarcinoma and suggests that manipulation of the miR-410/XIAP pathway could have a therapeutic potential for cholangiocarcinoma.

Reprogramming of miRNA networks in cancer and leukemia.

We studied miRNA profiles in 4419 human samples (3312 neoplastic, 1107 nonmalignant), corresponding to 50 normal tissues and 51 cancer types. The complexity of our database enabled us to perform a detailed analysis of microRNA (miRNA) activities. We inferred genetic networks from miRNA expression in normal tissues and cancer. We also built, for the first time, specialized miRNA networks for solid tumors and leukemias. Nonmalignant tissues and cancer networks displayed a change in hubs, the most connected miRNAs. hsa-miR-103/106 were downgraded in cancer, whereas hsa-miR-30 became most prominent. Cancer networks appeared as built from disjointed subnetworks, as opposed to normal tissues. A comparison of these nets allowed us to identify key miRNA cliques in cancer. We also investigated miRNA copy number alterations in 744 cancer samples, at a resolution of 150 kb. Members of miRNA families should be similarly deleted or amplified, since they repress the same cellular targets and are thus expected to have similar impacts on oncogenesis. We correctly identified hsa-miR-17/92 family as amplified and the hsa-miR-143/145 cluster as deleted. Other miRNAs, such as hsa-miR-30 and hsa-miR-204, were found to be physically altered at the DNA copy number level as well. By combining differential expression, genetic networks, and DNA copy number alterations, we confirmed, or discovered, miRNAs with comprehensive roles in cancer. Finally, we experimentally validated the miRNA network with acute lymphocytic leukemia originated in Mir155 transgenic mice. Most of miRNAs deregulated in these transgenic mice were located close to hsa-miR-155 in the cancer network.

23ME-00610, a genetically informed, first-in-class antibody targeting CD200R1 to enhance antitumor T cell function.

Immune checkpoint inhibition (ICI) has revolutionized cancer treatment; however, only a subset of patients benefit long term. Therefore, methods for identification of novel checkpoint targets and development of therapeutic interventions against them remain a critical challenge. Analysis of human genetics has the potential to inform more successful drug target discovery. We used genome-wide association studies of the 23andMe genetic and health survey database to identify an immuno-oncology signature in which genetic variants are associated with opposing effects on risk for cancer and immune diseases. This signature identified multiple pathway genes mapping to the immune checkpoint comprising CD200, its receptor CD200R1, and the downstream adapter protein DOK2. We confirmed that CD200R1 is elevated on tumor-infiltrating immune cells isolated from cancer patients compared to the matching peripheral blood mononuclear cells. We developed a humanized, effectorless IgG1 antibody (23ME-00610) that bound human CD200R1 with high affinity (KD <0.1 nM), blocked CD200 binding, and inhibited recruitment of DOK2. 23ME-00610 induced T-cell cytokine production and enhanced T cell-mediated tumor cell killing in vitro. Blockade of the CD200:CD200R1 immune checkpoint inhibited tumor growth and engaged immune activation pathways in an S91 tumor cell model of melanoma in mice.

MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis.

Progress in understanding the biology of multiple myeloma (MM), a plasma cell malignancy, has been slow. The discovery of microRNAs (miRNAs), a class of small noncoding RNAs targeting multiple mRNAs, has revealed a new level of gene expression regulation. To determine whether miRNAs play a role in the malignant transformation of plasma cells (PCs), we have used both miRNA microarrays and quantitative real time PCR to profile miRNA expression in MM-derived cell lines (n = 49) and CD138+ bone marrow PCs from subjects with MM (n = 16), monoclonal gammopathy of undetermined significance (MGUS) (n = 6), and normal donors (n = 6). We identified overexpression of miR-21, miR-106b approximately 25 cluster, miR-181a and b in MM and MGUS samples with respect to healthy PCs. Selective up-regulation of miR-32 and miR-17 approximately 92 cluster was identified in MM subjects and cell lines but not in MGUS subjects or healthy PCs. Furthermore, two miRNAs, miR-19a and 19b, that are part of the miR-17 approximately 92 cluster, were shown to down regulate expression of SOCS-1, a gene frequently silenced in MM that plays a critical role as inhibitor of IL-6 growth signaling. We also identified p300-CBP-associated factor, a gene involved in p53 regulation, as a bona fide target of the miR106b approximately 25 cluster, miR-181a and b, and miR-32. Xenograft studies using human MM cell lines treated with miR-19a and b, and miR-181a and b antagonists resulted in significant suppression of tumor growth in nude mice. In summary, we have described a MM miRNA signature, which includes miRNAs that modulate the expression of proteins critical to myeloma pathogenesis.

Fhit-Fdxr interaction in the mitochondria: modulation of reactive oxygen species generation and apoptosis in cancer cells.

Fhit protein is lost in cancers of most, perhaps all, cancer types; when restored, it can induce apoptosis and suppress tumorigenicity, as shown in vitro and in mouse tumor models in vivo. Following protein cross-linking and proteomics analyses, we characterized a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes the heat-shock chaperonin pair, HSP60/10, which is likely involved in importing Fhit into the mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin, in electron transport chain complex III. Overexpression of Fhit protein in Fhit-deficient cancer cells modulates the production of intracellular reactive oxygen species, causing increased ROS, following peroxide treatment, with subsequent increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape ROS overproduction and ROS-induced apoptosis, likely carrying oxidative damage. Thus, characterization of Fhit-interacting proteins has identified direct effectors of a Fhit-mediated apoptotic signal pathway that is lost in many cancers. This is of translational interest considering the very recent emphasis in a number of high-profile publications, concerning the role of oxidative phosphorylation in the treatment of human cancers, and especially cancer stem cells that rely upon oxidative phosphorylation for survival. Additionally, we have shown that cells from a Fhit-deficient lung cancer cell line, are sensitive to killing by exposure to atovaquone, thought to act as a selective oxidative phosphorylation inhibitor by targeting the CoQ10 dependence of the mitochondrial complex III, while the Fhit-expressing sister clone is resistant to this treatment.

A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia.

BACKGROUND: MicroRNA expression profiles can be used to distinguish normal B cells from malignant B cells in patients with chronic lymphocytic leukemia (CLL). We investigated whether microRNA profiles are associated with known prognostic factors in CLL. METHODS: We evaluated the microRNA expression profiles of 94 samples of CLL cells for which the level of expression of 70-kD zeta-associated protein (ZAP-70), the mutational status of the rearranged immunoglobulin heavy-chain variable-region (IgV(H) ) gene, and the time from diagnosis to initial treatment were known. We also investigated the genomic sequence of 42 microRNA genes to identify abnormalities. RESULTS: A unique microRNA expression signature composed of 13 genes (of 190 analyzed) differentiated cases of CLL with low levels of ZAP-70 expression from those with high levels and cases with unmutated IgV(H) from those with mutated IgV(H) . The same microRNA signature was also associated with the presence or absence of disease progression. We also identified a germ-line mutation in the miR-16-1-miR-15a primary precursor, which caused low levels of microRNA expression in vitro and in vivo and was associated with deletion of the normal allele. Germ-line or somatic mutations were found in 5 of 42 sequenced microRNAs in 11 of 75 patients with CLL, but no such mutations were found in 160 subjects without cancer (P<0.001). CONCLUSIONS: A unique microRNA signature is associated with prognostic factors and disease progression in CLL. Mutations in microRNA transcripts are common and may have functional importance.

Fhit tumor suppressor: guardian of the preneoplastic genome.

Environmental agents induce intragenic alterations in the FRA3B/FHIT chromosome fragile site, resulting in fragile FHIT allele loss early in cancer development. Fhit knockout mice are predisposed to tumor development and Fhit gene therapy reduces tumor burden. Repair-deficient cancers are likely to be Fhit-deficient and Fhit-deficient cells show enhanced resistance to ultraviolet C, mitomycin C, camptothecin and oxidative stress-induced cell killing. Loss of Fhit leads to alterations in the DNA damage response checkpoint and contributes to DNA instability. Hsp60/Hsp10 are Fhit interactors, suggesting a direct role for Fhit in stress responses. Fhit also interacts with and stabilizes ferrodoxin reductase (Fdxr), a mitochondrial flavoprotein that transfers electrons from NADPH to cytochrome P450, suggesting a role for Fhit in the modulation of reactive oxygen species production and of genomic damage.

Physical association with WWOX suppresses c-Jun transcriptional activity.

WWOX is a tumor suppressor that functions as a modular protein partner of transcription factors. WWOX contains two WW domains that mediate protein-protein interactions. In this report, we show that WWOX, via its first WW domain, specifically associates with the proline-rich motif of c-Jun proto-oncogene. Our data show that phosphorylation of c-Jun caused by overexpression of mitogen-activated protein kinase kinase kinase 1 (Mekk1), an upstream activator of c-Jun, enhances the interaction of c-Jun with WWOX. Furthermore, exposure of HaCaT keratinocytes to UVC radiation resulted in the association of endogenous WWOX and c-Jun. The WWOX-c-Jun complexes mainly occur in the cytoplasm. Expression of WWOX attenuates the ability of MEKK1 to increase the activity of a c-Jun-driven activating protein-1 (AP-1)-luciferase reporter plasmid. In contrast, a point mutation in the first WW domain of WWOX has no effect on transactivation of AP-1 when coexpressed with c-Jun protein. Our findings reveal a novel functional cross-talk between c-Jun transcription factor and WWOX tumor suppressor protein.

Preclinical assessment of FHIT gene replacement therapy in human leukemia using a chimeric adenovirus, Ad5/F35.

PURPOSE: Expression of the FHIT protein is lost or reduced in most solid tumors and a significant fraction of hematopoietic malignancies. Adenovirus 5 (Ad5) virus or adeno-associated viral vectors have been used to study the tumor suppressor function of FHIT in solid tumors, but these tools have not been effective in leukemias. We have generated a chimeric FHIT-containing adenovirus composed of Ad5 and the group B adenovirus called F35 with which we have been able to efficiently infect hematopoietic cells. EXPERIMENTAL DESIGN: Infection efficiency of Ad5/F35-FHIT and Ad5/F35-GFP viruses was tested in leukemia cell lines that lacked FHIT expression, and biological effects of successful infection were assessed. An acute myelogenous leukemia, a chronic myelogenous leukemia, and four acute lymphoblastic leukemia human cell lines were examined as well as two EBV-transformed B lymphoblastoid cell lines that expressed endogenous FHIT. RESULTS: Two of four acute lymphoblastic leukemia cell lines, Jurkat and MV4;11, which were efficiently infected with Ad5/F35-FHIT, underwent growth suppression and massive induction of apoptosis without apparent activation of caspase-8 or caspase-2 and late activation of caspase-3. Treatment of infected cells with caspase-9 and caspase-3 inhibitors partially blocked FHIT-induced apoptosis. The two remaining infected acute lymphoblastic leukemia cell lines, Molt-3 and RS4;11, were apparently unaffected. Restoration of FHIT expression in the chronic myelogenous leukemia K562 cell line and the acute myelogenous leukemia KG1a cell line also induced apoptosis but at later time points than seen in the acute lymphoblastic leukemia Jurkat and MV4;11 cell lines. I.v. injection of Ad5/F35-FHIT-infected Jurkat cells resulted in abrogation of tumorigenicity in the NOD/SCID xenogeneic engraftment model. CONCLUSION: FHIT restoration in some FHIT-deficient leukemia cells induces both antiproliferative and proapoptotic effects involving the intrinsic caspase apoptotic pathway.

Bruton Tyrosine Kinase Inhibition Attenuates Liver Damage in a Mouse Warm Ischemia and Reperfusion Model.

BACKGROUND: Bruton tyrosine kinase (Btk) is a central player in multiple signaling pathways of lymphoid and myeloid cells. Myeloid cells are crucial early effectors in organ ischemia-reperfusion (IR) injury. BTKB66 is a selective, irreversible inhibitor of Btk. In this study, we hypothesized that Btk inhibition would reduce hepatocellular injury in a murine model of liver warm hepatic IR. METHODS: First, BTKB66 was tested in in vitro models of lipopolysaccharide-mediated neutrophil and macrophage activation. Then, to assess its efficacy in vivo, BTKB66 was administered orally to mice for 7 days before subjecting them to 90 minutes of warm hepatic ischemia followed by reperfusion for 6 or 24 hours. Clinical and pathologic features in the livers, including AST, ALT, and a panel of cytokines and chemokines, were examined. RESULTS: BTKB66 potently inhibited lipopolysaccharide-mediated activation of bone marrow-derived neutrophils and macrophages in vitro. It also reduced the severity of IR injury as determined by AST and ALT levels, as well as immune cell infiltrates. BTKB66 significantly decreased hepatic markers of sterile inflammation, such as C-X-C motif chemokine 1, C-X-C motif chemokine 2, and C-X-C motif chemokine 10, in parallel with depression of serum markers of the myeloid cell activation, such as CCL5, CCL11, and C-X-C motif chemokine 5. CONCLUSIONS: BTKB66 treatment ameliorated hepatocellular injury in a well-established model of liver partial warm ischemia and in situ reperfusion. These findings confirm that neutrophil recruitment and activation play an essential role in IR stress, and that targeting Btk activity may provide a useful approach for preventing hepatocellular damage and improving outcomes in liver transplantation.

The eighth fibronectin type III domain of protein tyrosine phosphatase receptor J influences the formation of protein complexes and cell localization.

Regulation of receptor-type phosphatases can involve the formation of higher-order structures, but the exact role played in this process by protein domains is not well understood. In this study we show the formation of different higher-order structures of the receptor-type phosphatase PTPRJ, detected in HEK293A cells transfected with different PTPRJ expression constructs. In the plasma membrane PTPRJ forms dimers detectable by treatment with the cross-linking reagent BS(3) (bis[sulfosuccinimidyl]suberate). However, other PTPRJ complexes, dependent on the formation of disulfide bonds, are detected by treatment with the oxidant agent H(2)O(2) or by a mutation Asp872Cys, located in the eighth fibronectin type III domain of PTPRJ. A deletion in the eighth fibronectin domain of PTPRJ impairs its dimerization in the plasma membrane and increases the formation of PTPRJ complexes dependent on disulfide bonds that remain trapped in the cytoplasm. The deletion mutant maintains the catalytic activity but is unable to carry out inhibition of proliferation on HeLa cells, achieved by the wild type form, since it does not reach the plasma membrane. Therefore, the intact structure of the eighth fibronectin domain of PTPRJ is critical for its localization in plasma membrane and biological function.

MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia.

MicroRNAs (miRNAs) are small RNAs of 19 to 25 nucleotides that are negative regulators of gene expression. To determine whether miRNAs are associated with cytogenetic abnormalities and clinical features in acute myeloid leukemia (AML), we evaluated the miRNA expression of CD34(+) cells and 122 untreated adult AML cases using a microarray platform. After background subtraction and normalization using a set of housekeeping genes, data were analyzed using Significance Analysis of Microarrays. An independent set of 60 untreated AML patients was used to validate the outcome signatures using real-time polymerase chain reaction. We identified several miRNAs differentially expressed between CD34(+) normal cells and the AML samples. miRNA expression was also closely associated with selected cytogenetic and molecular abnormalities, such as t(11q23), isolated trisomy 8, and FLT3-ITD mutations. Furthermore, patients with high expression of miR-191 and miR-199a had significantly worse overall and event-free survival than AML patients with low expression (overall survival: miR-191, P = .03; and miR-199a, P = .001, Cox regression). In conclusion, miRNA expression in AML is closely associated with cytogenetics and FLT3-ITD mutations. A small subset of miRNAs is correlated with survival.

Fhit interaction with ferredoxin reductase triggers generation of reactive oxygen species and apoptosis of cancer cells.

Fhit protein is lost in most cancers, its restoration suppresses tumorigenicity, and virus-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. We have used protein cross-linking and proteomics methods to characterize a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Viral-mediated Fhit restoration increases production of intracellular reactive oxygen species, followed by increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, carrying serious oxidative DNA damage that may contribute to an increased mutation rate. Characterization of Fhit interacting proteins has identified direct effectors of the Fhit-mediated apoptotic pathway that is lost in most cancers through loss of Fhit.

MicroRNA fingerprints during human megakaryocytopoiesis.

microRNAs are a highly conserved class of noncoding RNAs with important regulatory functions in proliferation, apoptosis, development, and differentiation. To discover novel regulatory pathways during megakaryocytic differentiation, we performed microRNA expression profiling of in vitro-differentiated megakaryocytes derived from CD34(+) hematopoietic progenitors. The main finding was down-regulation of miR-10a, miR-126, miR-106, miR-10b, miR-17 and miR-20. Hypothetically, the down-regulation of microRNAs unblocks target genes involved in differentiation. We confirmed in vitro and in vivo that miR-130a targets the transcription factor MAFB, which is involved in the activation of the GPIIB promoter, a key protein for platelet physiology. In addition, we found that miR-10a expression in differentiated megakaryocytes is inverse to that of HOXA1, and we showed that HOXA1 is a direct target of miR-10a. Finally, we compared the microRNA expression of megakaryoblastic leukemic cell lines with that of in vitro differentiated megakaryocytes and CD34(+) progenitors. This analysis revealed up-regulation of miR-101, miR-126, miR-99a, miR-135, and miR-20. Our data delineate the expression of microRNAs during megakaryocytopoiesis and suggest a regulatory role of microRNAs in this process by targeting megakaryocytic transcription factors.