Synthesis and evaluation of derivatives of the proteasome deubiquitinase inhibitor b-AP15.

The ubiquitin-proteasome system (UPS) is increasingly recognized as a therapeutic target for the development of anticancer therapies. The success of the 20S proteasome core particle (20S CP) inhibitor bortezomib in the clinical management of multiple myeloma has raised the possibility of identifying other UPS components for therapeutic intervention. We previously identified the small molecule b-AP15 as an inhibitor of 19S proteasome deubiquitinase (DUB) activity.

Immunophenotyping of Waldenströms macroglobulinemia cell lines reveals distinct patterns of surface antigen expression: potential biological and therapeutic implications.

Waldenströms macroglobulinemia (WM) is a subtype of Non-Hodgkin's lymphoma in which the tumor cell population is markedly heterogeneous, consisting of immunoglobulin-M secreting B-lymphocytes, plasmacytoid lymphocytes and plasma cells. Due to rarity of disease and scarcity of reliable preclinical models, many facets of WM molecular and phenotypic architecture remain incompletely understood. Currently, there are 3 human WM cell lines that are routinely used in experimental studies, namely, BCWM.

Targeted inhibition of the deubiquitinating enzymes, USP14 and UCHL5, induces proteotoxic stress and apoptosis in Waldenström macroglobulinaemia tumour cells.

Deubiquitinase enzymes (DUBs) of the proteasomal 19S regulatory particle are emerging as important therapeutic targets in several malignancies. Here we demonstrate that inhibition of two proteasome-associated DUBs (USP14 and UCHL5) with the small molecule DUB inhibitor b-AP15, results in apoptosis of human Waldenström macroglobulinaemia (WM) cell lines and primary patient-derived WM tumour cells. Importantly, b-AP15 produced proteotoxic stress and apoptosis in WM cells that have acquired resistance to the proteasome inhibitor bortezomib.

The investigational agent MLN2238 induces apoptosis and is cytotoxic to CLL cells in vitro, as a single agent and in combination with other drugs.

Chronic lymphocytic leukaemia (CLL) is the most common haematological malignancy in the U.S. The course of the disease has been shown to be negatively impacted by increased levels of BCL2.

AT-101 downregulates BCL2 and MCL1 and potentiates the cytotoxic effects of lenalidomide and dexamethasone in preclinical models of multiple myeloma and Waldenström macroglobulinaemia.

Multiple myeloma, the second most common haematological malignancy in the U.S., is currently incurable.

Neem leaf extract induces cell death by apoptosis and autophagy in B-chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and is currently incurable. To expand the therapeutic armamentarium, we investigated neem leaf extract (NLE) after a patient with CLL demonstrated disease regression upon taking oral NLE. NLE-mediated apoptosis was examined in peripheral blood mononuclear cells (PBMCs) from 41 patients with CLL.

Development and characterization of a novel human Waldenström macroglobulinemia cell line: RPCI-WM1, Roswell Park Cancer Institute - Waldenström Macroglobulinemia 1.

Understanding the biology of Waldenström macroglobulinemia is hindered by a lack of preclinical models. We report a novel cell line, RPCI-WM1, from a patient treated for WM. The cell line secretes human immunoglobulin M (h-IgM) with κ-light chain restriction identical to the primary tumor.

Downregulation of BCL2 by AT-101 enhances the antileukaemic effect of lenalidomide both by an immune dependant and independent manner.

Over-expression of anti-apoptotic BCL2 has been reported in chronic lymphocytic leukaemia (CLL), but targeting BCL2 alone did not yield appreciable clinical results. However, it was demonstrated that BCL2 inhibitors enhanced the clinical efficacy of chemo and immunotherapeutics. Lenalidomide, an immunomodulator, is clinically effective in CLL and can enhance the anti-CLL effects of CD20 targeting monoclonal antibody, rituximab.

Targeted treatment for chronic lymphocytic leukemia.

The treatment of chronic lymphocytic leukemia (CLL) has evolved over the last few decades. Recognition has increased of several key components of CLL biology currently manipulated for therapeutics. A milestone in the treatment of CLL was reached with the incorporation of immunotherapy with conventional chemotherapy.

Biological effects and clinical significance of lenalidomide-induced tumour flare reaction in patients with chronic lymphocytic leukaemia: in vivo evidence of immune activation and antitumour response.

Lenalidomide has demonstrated impressive antileukaemic effects in patients with chronic lymphocytic leukaemia (CLL). The mechanism(s) by which it mediates these effects remain unclear. Clinically, CLL patients treated with lenalidomide demonstrate an acute inflammatory reaction, the tumour flare reaction that is suggestive of an immune activation phenomenon.

Phosphorylation of the SSBP2 and ABL proteins by the ZNF198-FGFR1 fusion kinase seen in atypical myeloproliferative disorders as revealed by phosphopeptide-specific MS.

The ZNF198-fibroblast growth factor receptor-1 (FGFR1) fusion kinase is a constitutively activated tyrosine kinase associated with a specific atypical myeloproliferative disease. The chimeric protein localizes to the cytoplasm, unlike the wild type FGFR1 receptor kinase, and presumably inappropriately phosphorylates specific targets as part of the oncogenic signaling cascade. Other than known targets of the FGFR1 kinase itself, few specific targets of ZNF198-FGFR1 have been identified.

Expression characteristics of prostate-derived Ets factor support a role in breast and prostate cancer progression.

The purpose of this study was to understand the characteristics of prostate-derived Ets factor (PDEF) protein expression in breast and prostate cancer progression. A polyclonal antibody specific to PDEF was raised and reacted with tissue microarrays consisting of benign breast, in situ ductal, invasive ductal, and invasive lobular breast carcinomas. The antibody was also reacted with tissue microarrays, including benign prostate, prostate intraepithelial neoplasias (PINs), and prostate carcinomas.

HSPA1A is an important regulator of the stability and function of ZNF198 and its oncogenic derivative, ZNF198-FGFR1.

Mass spectroscopy analysis demonstrated that the HSPA1A protein is found in complex with the ZNF198 protein which is involved in a chromosome rearrangement with the FGFR1 gene in an atypical myeloproliferative disease. HSPA1A is a member of the HSP70 family of genes which has been shown to be inducible in a variety of circumstances. Exogenous expression of the ZNF198-FGFR1 fusion kinase gene as well as ZNF198 in a model cell system results in a large (>650-fold) increase in HSP70 mRNA levels.

ZNF198, a zinc finger protein rearranged in myeloproliferative disease, localizes to the PML nuclear bodies and interacts with SUMO-1 and PML.

The ZNF198/FGFR1 fusion gene in atypical myeloproliferative disease produces a constitutively active cytoplasmic tyrosine kinase, unlike ZNF198 which is normally a nuclear protein. We have now shown that the ZNF198/FGFR1 fusion kinase interacts with the endogenous ZNF198 protein suggesting that the function of ZNF198 may be compromised in cells expressing it. Little is currently known about the endogenous function of ZNF198 and to investigate this further we performed a yeast two-hybrid analysis and identified SUMO-1 as a binding partner of ZNF198.

Induction of the plasminogen activator inhibitor-2 in cells expressing the ZNF198/FGFR1 fusion kinase that is involved in atypical myeloproliferative disease.

The ZNF198/FGFR1 fusion kinase associated with an atypical myeloproliferative disease is constitutively activated and regulates several STAT transcription factors. We used oligonucleotide microarrays to compare the gene-expression profiles between HEK-293 cells that stably express either the ZNF198/FGFR1 chimeric protein or the wild-type ZNF198 gene. Expression of the plasminogen activator inhibitor-2 (PAI-2/SERPINB2) was highly increased in cells expressing the fusion gene.

Mass spectroscopy identifies the splicing-associated proteins, PSF, hnRNP H3, hnRNP A2/B1, and TLS/FUS as interacting partners of the ZNF198 protein associated with rearrangement in myeloproliferative disease.

ZNF198 is fused with FGFR1 in an atypical myeloproliferative disease that results in constitutive activation of the kinase domain and mislocalization to the cytoplasm. We have used immunoprecipitation of a GFP-tagged ZNF198 combined with MALDI-TOF mass spectroscopy to identify interacting proteins. P splicing factor (PSF) was identified as one of the proteins and this interaction was confirmed by Western blotting.

LGI1, a putative tumor metastasis suppressor gene, controls in vitro invasiveness and expression of matrix metalloproteinases in glioma cells through the ERK1/2 pathway.

Gliomas take a number of different genetic routes in the progression to glioblastoma multiforme, a highly invasive variant that is mostly unresponsive to current therapies. Gliomas express elevated levels of matrix metalloproteinases (MMPs), which have been implicated in the control of proliferation and invasion as well as neovascularization. Progressive loss of LGI1 expression has been associated with the development of high grade gliomas.

Suppression of the cell proliferation and invasion phenotypes in glioma cells by the LGI1 gene.

The leucine-rich, glioma-inactivated (LGI1) gene, located in 10q24, was originally identified because it was interrupted and inactivated by a reciprocal chromosome translocation in the T98G glioma cell line. Loss of LGI1 expression in high-grade brain tumors is correlated with the frequent loss of chromosome 10 during progression of gliomas. To investigate whether this gene can suppress the malignant phenotype in glioma cells, we introduced the LGI1 gene into cells that do (U87) and do not (T98G and A172) express LGI1 endogenously.

Tumor antigens and markers for breast and ovarian cancers.

Use of vaccines to prevent and treat breast and ovarian cancer is a highly attractive approach because of the expected minimal side effects and the potential to predict individuals likely to benefit from vaccination. To fully harness the capacity of the immune system for this purpose, it is necessary to characterize tumor antigens for these cancers so that purified antigens can be tested for their immunogenicity in individual patients and for their suitability as targets of vaccine-induced immunity. Discovery of novel breast and ovarian tumor antigens is also necessary for developing multi-antigen vaccines composed of multiple tumor antigens.