Lloyd Hutchinson

Prolonged dormancy of human liposarcoma is associated with impaired tumor angiogenesis

The disease state of cancer appears late in tumor development. Before being diagnosed, a tumor can remain for prolonged periods of time in a dormant state. Dormant human cancer is commonly defined as a microscopic tumor that does not expand in size and remains asymptomatic. Dormant tumors represent an early stage in tumor development and may therefore be a potential target for nontoxic, antiangiogenic therapy that could prevent tumor recurrence. Here, we characterize an experimental model that recapitulates the clinical dormancy of human tumors in mice. We demonstrate that these microscopic dormant cancers switch to the angiogenic phenotype at a predictable time. We further show that while angiogenic liposarcomas expand rapidly after inoculation of tumor cells in mice, nonangiogenic dormant liposarcomas remain microscopic up to one‐third of the normal severe combined immune deficiency (SCID) mouse life span, although they contain proliferating tumor cells. Nonangiogenic dormant tumors follow a similar growth pattern in subcutaneous (s.c.) and orthotopic environments. Throughout the dormancy period, development of intratumoral vessels is impaired. In nonangogenic dormant tumors, small clusters of endothelial cells without lumens are observed early after tumor cell inoculation, but the nonangiogenic tumor cannot sustain these vessels, and they disappear within weeks. There is a concomitant decrease in microvessel density, and the nonangiogenic dormant tumor remains harmless to the host. In contrast, microvessel density in tumors increases rapidly after the angiogenic switch and correlates with rapid expansion of tumor mass. Both tumor types cultured in vitro contain fully transformed cells, but only cells from the nonangiogenic human liposarcoma secrete relatively high levels of the angiogenesis inhibitors thrombospondin‐1 and TIMP‐1. This model suggests that as improved blood or urine molecular biomarkers are developed, the microscopic, nonangiogenic, dormant phase of human cancer may be vulnerable to antiangiogenic therapy years before symptoms, or before anatomical location of a tumor can be detected, by conventional methods.—Almog, N., Henke, V., Flores, L., Hlatky, L., Kung, A. L., Wright, R. D., Berger, R., Hutchinson, L., Naumov, G., Bender, E., Akslen, L., Achilles, E.‐G., Folkman, J. Prolonged dormancy of human liposarcoma is associated with impaired tumor angiogenesis. FASEB J. 20, E1–E10 (2006)

The BRAF Oncoprotein Functions through the Transcriptional Repressor MAFG to Mediate the CpG Island Methylator Phenotype

Most colorectal cancers (CRCs) containing activated BRAF (BRAF[V600E]) have a CpG island methylator phenotype (CIMP) characterized by aberrant hypermethylation of many genes, including the mismatch repair gene MLH1. MLH1 silencing results in microsatellite instability and a hypermutable phenotype. Through an RNAi screen, here we identify the transcriptional repressor MAFG as the pivotal factor required for MLH1 silencing and CIMP in CRCs containing BRAF(V600E). In BRAF-positive human CRC cell lines and tumors, MAFG is bound at the promoters of MLH1 and other CIMP genes, and recruits a corepressor complex that includes its heterodimeric partner BACH1, the chromatin remodeling factor CHD8, and the DNA methyltransferase DNMT3B, resulting in hypermethylation and transcriptional silencing. BRAF(V600E) increases BRAF/MEK/ERK signaling resulting in phosphorylation and elevated levels of MAFG, which drives DNA binding. Analysis of transcriptionally silenced CIMP genes in KRAS-positive CRCs indicates that different oncoproteins direct the assembly of distinct repressor complexes on common promoters.

Human Barrett's adenocarcinoma of the esophagus, associated myofibroblasts, and endothelium can arise from bone marrow-derived cells after allogeneic stem cell transplant.

This study characterizes the contribution of bone marrow-derived cells (BMDCs) to Barretts adenocarcinoma of the esophagus using a mouse surgical model of disease and human specimens. Transplantation of bone marrow expressing beta galactosidase into a wild-type mouse, followed by surgical esophagojejunostomy, allowed tracking of BMDCs into the surgical anastomosis and resulting Barretts metaplasia. Human tissue from a male patient who had been transplanted with female bone marrow and later developed esophageal adenocarcinoma allowed us to tract donor-derived cells into the tumor. Using a combination of antibodies directed against beta-galactosidase (animal studies) and X/Y fluorescent in situ hybridization (FISH) (human studies), combined with specific lineage staining directed against epithelial, fibroblast, endothelial, and leukocyte markers, we show that bone marrow cells contribute to both the epithelial and stromal component of esophageal adenocarcinoma. These findings demonstrate that BMDCs can generate cancer-associated fibroblasts as well as contribute directly to epithelial cells in cancer of the esophagus.

A KRAS-directed transcriptional silencing pathway that mediates the CpG island methylator phenotype

Approximately 70% of KRAS-positive colorectal cancers (CRCs) have a CpG island methylator phenotype (CIMP) characterized by aberrant DNA hypermethylation and transcriptional silencing of many genes. The factors involved in, and the mechanistic basis of, CIMP is not understood. Among the CIMP genes are the tumor suppressors p14ARF, p15INK4B, and p16INK4A, encoded by the INK4-ARF locus. In this study, we perform an RNA interference screen and identify ZNF304, a zinc-finger DNA-binding protein, as the pivotal factor required for INK4-ARF silencing and CIMP in CRCs containing activated KRAS. In KRAS-positive human CRC cell lines and tumors, ZNF304 is bound at the promoters of INK4-ARF and other CIMP genes. Promoter-bound ZNF304 recruits a corepressor complex that includes the DNA methyltransferase DNMT1, resulting in DNA hypermethylation and transcriptional silencing. KRAS promotes silencing through upregulation of ZNF304, which drives DNA binding. Finally, we show that ZNF304 also directs transcriptional silencing of INK4-ARF in human embryonic stem cells. DOI: http://dx.doi.org/10.7554/eLife.02313.001

A therapeutically targetable mechanism of BCR-ABL–independent imatinib resistance in chronic myeloid leukemia

A large-scale RNA interference screen reveals a new mechanism of imatinib resistance in chronic myeloid leukemia that can be therapeutically targeted. An Irresistible Combination Unlike most cancers, which have variable mutation patterns, chronic myeloid leukemia is normally associated with a specific genetic alteration, which produces a fusion protein called BCR-ABL. Famously, this protein was targeted with the first cancer-specific drug, known as imatinib (Gleevec), which is still the standard therapy for this cancer. Unfortunately, leukemia cells can develop resistance to imatinib, which does not always require mutations in BCR-ABL. Now, Ma et al. have identified a mechanism for imatinib resistance in chronic myeloid leukemia cells that lack mutations in BCR-ABL. The authors also demonstrated that a U.S. Food and Drug Administration–approved drug called trametinib can overcome this resistance and kill leukemia stem cells without harming the nonmalignant precursors that give rise to normal blood cells. Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) poses a major problem for the treatment of chronic myeloid leukemia (CML). IM resistance often results from a secondary mutation in BCR-ABL that interferes with drug binding. However, in many instances, there is no mutation in BCR-ABL, and the basis of such BCR-ABL–independent IM resistance remains to be elucidated. To gain insight into BCR-ABL–independent IM resistance mechanisms, we performed a large-scale RNA interference screen and identified IM-sensitizing genes (IMSGs) whose knockdown renders BCR-ABL+ cells IM-resistant. In these IMSG knockdown cells, RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) signaling is sustained after IM treatment because of up-regulation of PRKCH, which encodes the protein kinase C (PKC) family member PKCη, an activator of CRAF. PRKCH is also up-regulated in samples from CML patients with BCR-ABL–independent IM resistance. Combined treatment with IM and trametinib, a U.S. Food and Drug Administration–approved MEK inhibitor, synergistically kills BCR-ABL+ IMSG knockdown cells and prolongs survival in mouse models of BCR-ABL–independent IM-resistant CML. Finally, we showed that CML stem cells contain high levels of PRKCH, and this contributes to their intrinsic IM resistance. Combined treatment with IM and trametinib synergistically kills CML stem cells with negligible effect on normal hematopoietic stem cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL–independent IM resistance in CML and CML stem cells.

C-terminal variations in ?-thymosin family members specify functional differences in actin-binding properties

Mammalian cells express several isoforms of β‐thymosin, a major actin monomer sequestering factor, including thymosins β4, β10, and β15. Differences in actin‐binding properties of different β‐thymosin family members have not been investigated. We find that thymosin β15 binds actin with a 2.4‐fold higher affinity than does thymosin β4. Mutational analysis was performed to determine the amino acid differences in thymosin β15 that specify its increased actin‐affinity. Previous work with thymosin β4 identified an α‐helical domain, as well as a conserved central motif, as crucial for actin binding. Mutational analysis confirms that these domains are also vital for actin binding in thymosin β15, but that differences in these domains are not responsible for the variation in actin‐binding properties between thymosins β4 and β15. Truncation of the unique C‐terminal residues in thymosin β15 inhibits actin binding, suggesting that this domain also has an important role in mediating actin‐binding affinity. Replacement of the 10 C‐terminal amino acids of thymosin β15 with those of thymosin β4 did, however, reduce the actin‐binding affinity of the hybrid relative to thymosin β15. Similarly, replacement of the thymosin β4 C‐terminal amino acids with those of thymosin β15 led to increased actin binding. We conclude that functional differences between closely related β‐thymosin family members are, in part, specified by the C‐terminal variability between these isoforms. Such differences may have consequences for situations where β‐thymosins are differentially expressed as in embryonic development and in cancer. J. Cell. Biochem. 77:277–287, 2000.

Systemic mastocytosis with associated clonal hematological non‐mast cell lineage disease: Clinical significance and comparison of chomosomal abnormalities in SM and AHNMD components

Some patients with systemic mastocytosis have concurrent hematological neoplasms, designated in the World Health Organization (WHO) classification as systemic mastocytosis with associated clonal hematological non‐mast cell lineage disease (SM‐AHNMD). In this study, we analyzed 29 patients with SM‐AHNMD and compared them to 40 patients with pure SM. The AHNMDs were classified as chronic myelomonocytic leukemia (CMML) (n = 10), myelodysplastic syndrome (MDS) (n = 7), myeloproliferative neoplasms (n = 4), B‐cell lymphoma/leukemia/plasma cell neoplasms (n = 7), and acute myeloid leukemia (n = 1). Patients with SM‐AHNMD were older, more frequently had constitutional symptoms and hematological abnormalities, less often had skin lesions, and had an inferior overall survival compared with pure SM patients (48 months vs. not‐reached, P < 0.001). Karyotypic abnormalities were detected in 9/28 (32%) patients with SM‐AHNMD but not in pure SM patients (P < 0.001). Combined imaging/ fluorescence‐in‐situ hybridization performed in four SM‐AHNMD cases revealed shared abnormal signals in mast cells and myeloid cells in two patients with SM‐CMML and one patient with SM‐MDS, but not in the mast cells of a case SM‐associated with chronic lymphocytic leukemia with ATM‐deletion. Quantitative mutation analysis showed higher levels of mutant KIT D816V in SM‐CMML and SM‐MDS than in pure SM (P < 0.001). Our data indicate that the SM‐AHNMD category in the WHO classification is heterogeneous, including clonally related and unrelated forms of AHNMD. The presentation, treatment, and outcome of patients with SM‐AHNMD is often dictated by the type of AHNMD. Am. J. Hematol. 88:219–224, 2013.

The Blk pathway functions as a tumor suppressor in chronic myeloid leukemia stem cells

A therapeutic strategy for treating cancer is to target and eradicate cancer stem cells (CSCs) without harming their normal stem cell counterparts. The success of this approach relies on the identification of molecular pathways that selectively regulate CSC function. Using BCR-ABL–induced chronic myeloid leukemia (CML) as a disease model for CSCs, we show that BCR-ABL downregulates the Blk gene (encoding B-lymphoid kinase) through c-Myc in leukemic stem cells (LSCs) in CML mice and that Blk functions as a tumor suppressor in LSCs but does not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Blk suppresses LSC function through a pathway involving an upstream regulator, Pax5, and a downstream effector, p27. Inhibition of this Blk pathway accelerates CML development, whereas increased activity of the Blk pathway delays CML development. Blk also suppresses the proliferation of human CML stem cells. Our results show the feasibility of selectively targeting LSCs, an approach that should be applicable to other cancers.

Expression of CD25 independently predicts early treatment failure of acute myeloid leukaemia (AML)

CD25+ CD34+ CD38 leukaemic cells have been shown to be chemotherapy-resistant and initiate acute myeloid leukaemia (AML) in xenograft models, suggesting a leukaemic stem cells (LSC) biology (Saito et al, 2010). High CD25 (also known as interleukin 2 receptor alpha, IL2RA) expression (>10%) at diagnosis in young (<60 years) AML patients in retrospective analysis correlated with a significantly shorter overall survival (OS, P = 0 0005) and relapse-free survival (RFS, P = 0 005). CD25 expression was also associated with FLT3-internal tandem duplication (ITD) mutation, and double positive patients had the poorest OS and RFS (P = 0 001 and P = 0 003, respectively; Terwijn et al, 2009).

Clinicopathological features of extramedullary recurrence/relapse of multiple myeloma

Extramedullary relapses of multiple myeloma (MM) during the course of disease are rare. We report a series of six patients with primary intramedullary MM that were treated with immunomodulatory therapy and/or stem cell transplant, and that later developed extramedullary relapses at various body sites. These six cases represent 3.9% of the 156 patients treated for MM at our institution over a 9‐yr period (1999–2007). Five (83.3%) of the six cases showed immature/high‐grade histology in the extramedullary relapses as compared with their antecedent MM. The neural cell adhesion molecule, CD56, was immunohistochemically demonstrable in 75% (three of four) of the original myelomas tested, but was absent in 83.3% (five of six) of their extramedullary relapses. The disease typically behaved aggressively and was rapidly fatal in all six patients even when therapy was administered. The median time of progression to extramedullary relapse was 29 months (range 9–64 months), and the median survival after diagnosis of the relapses was only 38 d (range 1–106 d). Our case series shows that extramedullary relapse of MM is characterized by high‐grade histology, loss of CD56 expression, frequent resistance to current therapeutic regimens, aggressive biological behavior, and very short survival.