Targeted disruption of a highly conserved distal enhancer reduces expression of the PU.1 transcription factor by 80% and leads to acute myeloid leukemia (AML) with frequent cytogenetic aberrations in mice. Here we identify a SNP within this element in humans that is more frequent in AML with a complex karyotype, leads to decreased enhancer activity, and reduces PU.1 expression in myeloid progenitors in a development-dependent manner. This SNP inhibits binding of the chromatin-remodeling transcriptional regulator special AT-rich sequence binding protein 1 (SATB1). Overexpression of SATB1 increased PU.1 expression, and siRNA inhibition of SATB1 downregulated PU.1 expression. Targeted disruption of the distal enhancer led to a loss of regulation of PU.1 by SATB1. Interestingly, disruption of SATB1 in mice led to a selective decrease of PU.1 RNA in specific progenitor types (granulocyte-macrophage and megakaryocyte-erythrocyte progenitors) and a similar effect was observed in AML samples harboring this SNP. Thus we have identified a SNP within a distal enhancer that is associated with a subtype of leukemia and exerts a deleterious effect through remote transcriptional dysregulation in specific progenitor subtypes.
Ulrich Steidl, Christian Steidl, Alexander Ebralidze, Björn Chapuy, Hye-Jung Han, Britta Will, Frank Rosenbauer, Annegret Becker, Katharina Wagner, Steffen Koschmieder, Susumu Kobayashi, Daniel B. Costa, Thomas Schulz, Karen B. O’Brien, Roel G.W. Verhaak, Ruud Delwel, Detlef Haase, Lorenz Trümper, Jürgen Krauter, Terumi Kohwi-Shigematsu, Frank Griesinger, Daniel G. Tenen
Vα24-invariant natural killer T (NKT) cells are potentially important for antitumor immunity. We and others have previously demonstrated positive associations between NKT cell presence in primary tumors and long-term survival in distinct human cancers. However, the mechanism by which aggressive tumors avoid infiltration with NKT and other T cells remains poorly understood. Here, we report that the v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN), the hallmark of aggressive neuroblastoma, repressed expression of monocyte chemoattractant protein–1/CC chemokine ligand 2 (MCP-1/CCL2), a chemokine required for NKT cell chemoattraction. MYCN knockdown in MYCN-amplified neuroblastoma cell lines restored CCL2 production and NKT cell chemoattraction. Unlike other oncogenes, MYCN repressed chemokine expression in a STAT3-independent manner, requiring an E-box element in the CCL2 promoter to mediate transcriptional repression. MYCN overexpression in neuroblastoma xenografts in NOD/SCID mice severely inhibited their ability to attract human NKT cells, T cells, and monocytes. Patients with MYCN-amplified neuroblastoma metastatic to bone marrow had 4-fold fewer NKT cells in their bone marrow than did their nonamplified counterparts, indicating that the MYCN-mediated immune escape mechanism, which we believe to be novel, is operative in metastatic cancer and should be considered in tumor immunobiology and for the development of new therapeutic strategies.
Liping Song, Tasnim Ara, Hong-Wei Wu, Chan-Wook Woo, C. Patrick Reynolds, Robert C. Seeger, Yves A. DeClerck, Carol J. Thiele, Richard Sposto, Leonid S. Metelitsa
The presumed involvement of paired box gene 5 (PAX5) in B-lymphomagenesis is based largely on the discovery of Pax5-specific translocations and somatic hypermutations in non-Hodgkin lymphomas. Yet mechanistically, the contribution of Pax5 to neoplastic growth remains undeciphered. Here we used 2 Myc-induced mouse B lymphoma cell lines, Myc5-M5 and Myc5-M12, which spontaneously silence Pax5. Reconstitution of these cells with Pax5–tamoxifen receptor fusion protein (Pax5ERTAM) increased neoplastic growth in a hormone-dependent manner. Conversely, expression of dominant-negative Pax5 in murine lymphomas and Pax5 knockdown in human lymphomas negatively affected cell expansion. Expression profiling revealed that Pax5 was required to maintain mRNA levels of several crucial components of B cell receptor (BCR) signaling, including CD79a, a protein with the immunoreceptor tyrosine-based activation motif (ITAM). In contrast, expression of 2 known ITAM antagonists, CD22 and PIR-B, was suppressed. The key role of BCR/ITAM signaling in Pax5-dependent lymphomagenesis was corroborated in Syk, an ITAM-associated tyrosine kinase. Moreover, we observed consistent expression of phosphorylated BLNK, an activated BCR adaptor protein, in human B cell lymphomas. Thus, stimulation of neoplastic growth by Pax5 occurs through BCR and is sensitive to genetic and pharmacological inhibitors of this pathway.
Diana Cozma, Duonan Yu, Suchita Hodawadekar, Anna Azvolinsky, Shannon Grande, John W. Tobias, Michele H. Metzgar, Jennifer Paterson, Jan Erikson, Teresa Marafioti, John G. Monroe, Michael L. Atchison, Andrei Thomas-Tikhonenko
Molecularly targeted kinase inhibitor cancer therapies are currently administered sequentially rather than simultaneously. We addressed the potential long-term impact of this strategy in patients with chronic myelogenous leukemia (CML), which is driven by the fusion oncogene BCR-ABL. Analysis of BCR-ABL genotypes in CML patients who relapsed after sequential treatment with the ABL inhibitors imatinib and dasatinib revealed evolving resistant BCR-ABL kinase domain mutations in all cases. Twelve patients relapsed with the pan-resistant T315I mutation, whereas 6 patients developed novel BCR-ABL mutations predicted to retain sensitivity to imatinib based on in vitro studies. Three of these patients were retreated with imatinib (or the chemically related compound nilotinib) and responded; however, selection for compound mutants (2 or 3 BCR-ABL mutations in the same molecule) can substantially limit the potential effectiveness of retreating patients with inhibitors that have previously failed. Furthermore, drug-resistant mutations, when compounded, can increase oncogenic potency relative to the component mutants in transformation assays. The Aurora kinase inhibitor VX-680, currently under clinical evaluation based on its activity against the T315I mutation, is also effective against the other commonly detected dasatinib-resistant mutation in our analysis, V299L. Our findings demonstrate the potential hazards of sequential kinase inhibitor therapy and suggest a role for a combination of ABL kinase inhibitors, perhaps including VX-680, to prevent the outgrowth of cells harboring drug-resistant BCR-ABL mutations.
Neil P. Shah, Brian J. Skaggs, Susan Branford, Timothy P. Hughes, John M. Nicoll, Ronald L. Paquette, Charles L. Sawyers
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, accounting for an estimated 600,000 deaths annually. Aberrant methylation, consisting of DNA hypomethylation and/or promoter gene CpG hypermethylation, is implicated in the development of a variety of solid tumors, including HCC. We analyzed the global levels of DNA methylation as well as the methylation status of 105 putative tumor suppressor genes and found that the extent of genome-wide hypomethylation and CpG hypermethylation correlates with biological features and clinical outcome of HCC patients. We identified activation of Ras and downstream Ras effectors (ERK, AKT, and RAL) due to epigenetic silencing of inhibitors of the Ras pathway in all HCC. Further, selective inactivation of SPRY1 and -2, DAB2, and SOCS4 and -5 genes and inhibitors of angiogenesis (BNIP3, BNIP3L, IGFBP3, and EGLN2) was associated with poor prognosis. Importantly, several epigenetically silenced putative tumor suppressor genes found in HCC were also inactivated in the nontumorous liver. Our results assign both therapeutic and chemopreventive significance to methylation patterns in human HCC and open the possibility of using molecular targets, including those identified in this study, to effectively inhibit HCC development and progression.
Diego F. Calvisi, Sara Ladu, Alexis Gorden, Miriam Farina, Ju-Seog Lee, Elizabeth A. Conner, Insa Schroeder, Valentina M. Factor, Snorri S. Thorgeirsson
A small population of plasmacytoid DCs (pDCs) in mouse tumor-draining LNs can express the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). We show that these IDO+ pDCs directly activate resting CD4+CD25+Foxp3+ Tregs for potent suppressor activity. In vivo, Tregs isolated from tumor-draining LNs were constitutively activated and suppressed antigen-specific T cells immediately ex vivo. In vitro, IDO+ pDCs from tumor-draining LNs rapidly activated resting Tregs from non–tumor-bearing hosts without the need for mitogen or exogenous anti-CD3 crosslinking. Treg activation by IDO+ pDCs was MHC restricted, required an intact amino acid–responsive GCN2 pathway in the Tregs, and was prevented by CTLA4 blockade. Tregs activated by IDO markedly upregulated programmed cell death 1 ligand 1 (PD-L1) and PD-L2 expression on target DCs, and the ability of Tregs to suppress target T cell proliferation was abrogated by antibodies against the programmed cell death 1/PD-L (PD-1/PD-L) pathway. In contrast, Tregs activated by anti-CD3 crosslinking did not cause upregulation of PD-Ls, and suppression by these cells was unaffected by blocking the PD-1/PD-L pathway. Tregs isolated from tumor-draining LNs in vivo showed potent PD-1/PD-L–mediated suppression, which was selectively lost when tumors were grown in IDO-deficient hosts. We hypothesize that IDO+ pDCs create a profoundly suppressive microenvironment within tumor-draining LNs via constitutive activation of Tregs.
Madhav D. Sharma, Babak Baban, Phillip Chandler, De-Yan Hou, Nagendra Singh, Hideo Yagita, Miyuki Azuma, Bruce R. Blazar, Andrew L. Mellor, David H. Munn
The long plasma half-life of IgG, while allowing for enhanced tumor uptake of tumor-targeted IgG conjugates, also results in increased background activity and normal-tissue toxicity. Therefore, successful therapeutic uses of conjugated antibodies have been limited to the highly sensitive and readily accessible hematopoietic tumors. We report a therapeutic strategy to beneficially alter the pharmacokinetics of IgG antibodies via pharmacological inhibition of the neonatal Fc receptor (FcRn) using high-dose IgG therapy. IgG-treated mice displayed enhanced blood and whole-body clearance of radioactivity, resulting in better tumor-to-blood image contrast and protection of normal tissue from radiation. Tumor uptake and the resultant therapeutic response was unaltered. Furthermore, we demonstrated the use of this approach for imaging of tumors in humans and discuss its potential applications in cancer imaging and therapy. The ability to reduce the serum persistence of conjugated IgG antibodies after their infusion can enhance their therapeutic index, resulting in improved therapeutic and diagnostic efficacy.
Jaspreet Singh Jaggi, Jorge A. Carrasquillo, Surya V. Seshan, Pat Zanzonico, Erik Henke, Andrew Nagel, Jazmin Schwartz, Brad Beattie, Barry J. Kappel, Debjit Chattopadhyay, Jing Xiao, George Sgouros, Steven M. Larson, David A. Scheinberg
We hypothesized that overexpression of PDGF-BB in colorectal cancer (CRC) and pancreatic cancer cells would result in increased pericyte coverage of ECs in vivo, rendering the tumor vasculature more resistant to antiangiogenic therapy. We stably transfected the cDNA for the PDGF-B into HT-29 human CRC and FG human pancreatic cancer cells. Surprisingly, when HT-29 or FG parental and transfected cells were injected into mice (subcutaneously and orthotopically), we observed marked inhibition of tumor growth in the PDGF-BB–overexpressing clones. In the PDGF-BB–overexpressing tumors, we observed an increase in pericyte coverage of ECs. Treatment of PDGF-BB–overexpressing tumors with imatinib mesylate (PDGFR inhibitor) resulted in increased growth and decreased total pericyte content compared with those in untreated PDGF-BB–overexpressing tumors. In vitro studies demonstrated the ability of VSMCs to inhibit EC proliferation by approximately 50%. These data show that increasing the pericyte content of the tumor microenvironment inhibits the growth of angiogenesis-dependent tumors. Single-agent therapy targeting PDGF receptor must be used with caution in tumors when PDGFR is not the target on the tumor cell itself.
Marya F. McCarty, Ray J. Somcio, Oliver Stoeltzing, Jane Wey, Fan Fan, Wenbiao Liu, Corazon Bucana, Lee M. Ellis
Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8+ T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8+ T cells in mice genetically deficient in all lymphocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed commensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflammatory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand–containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8+ T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy.
Chrystal M. Paulos, Claudia Wrzesinski, Andrew Kaiser,, Christian S. Hinrichs, Marcello Chieppa, Lydie Cassard, Douglas C. Palmer, Andrea Boni, Pawel Muranski, Zhiya Yu, Luca Gattinoni, Paul A. Antony, Steven A. Rosenberg, Nicholas P. Restifo
Tumor radioresponsiveness depends on endothelial cell death, which leads in turn to tumor hypoxia. Radiation-induced hypoxia was recently shown to trigger tumor radioresistance by activating angiogenesis through hypoxia-inducible factor 1–regulated (HIF-1–regulated) cytokines. We show here that combining targeted radioiodide therapy with angiogenic inhibitors, such as canstatin, enhances direct tumor cell apoptosis, thereby overcoming radio-induced HIF-1–dependent tumor survival pathways in vitro and in vivo. We found that following dual therapy, HIF-1α increases the activity of the canstatin-induced αvβ5 signaling tumor apoptotic pathway and concomitantly abrogates mitotic checkpoint and tetraploidy triggered by radiation. Apoptosis in conjunction with mitotic catastrophe leads to lethal tumor damage. We discovered that HIF-1 displays a radiosensitizing activity that is highly dependent on treatment modalities by regulating key apoptotic molecular pathways. Our findings therefore support a crucial role for angiogenesis inhibitors in shifting the fate of radiation-induced HIF-1α activity from hypoxia-induced tumor radioresistance to hypoxia-induced tumor apoptosis. This study provides a basis for developing new biology-based clinically relevant strategies to improve the efficacy of radiation oncology, using HIF-1 as an ally for cancer therapy.
Claire Magnon, Paule Opolon, Marcel Ricard, Elisabeth Connault, Patrice Ardouin, Ariane Galaup, Didier Métivier, Jean-Michel Bidart, Stéphane Germain, Michel Perricaudet, Martin Schlumberger