Stemness of B-cell progenitors in multiple myeloma bone marrow.

PURPOSE: In myeloma, B cells and plasma cells show a clonal relationship. Clonotypic B cells may represent a tumor-initiating compartment or cancer stem cell responsible for minimal residual disease in myeloma. EXPERIMENTAL DESIGN: We report a study of 58 patients with myeloma at time of diagnosis or relapse. B cells in bone marrow were evaluated by multicolor flow cytometry and sorting. Clonality was determined by light chain and/or immunoglobulin chain gene rearrangement PCR. We also determined aldehyde dehydrogenase activity and colony formation growth. Drug sensitivity was tested with conventional and novel agents. RESULTS: Marrow CD19+ cells express a light chain identical to plasma cells and are therefore termed light chain restricted (LCR). The LCR B-cell mass is small in both newly diagnosed and relapsed patients (≤ 1%). Few marrow LCR B cells (~10%) are CD19+/CD34+, with the rest being more differentiated CD19+/CD34- B cells. Marrow LCR CD19+ B cells exhibit enhanced aldehyde dehydrogenase activity versus healthy controls. Both CD19+/CD34+ and CD19+/CD34- cells showed colony formation activity, with colony growth efficiency optimized when stroma-conditioned medium was used. B-cell progenitors showed resistance to melphalan, lenalidomide, and bortezomib. Panobinostat, a histone deacetylase inhibitor, induced apoptosis of LCR B cells and CD138+ cells. LCR B cells are CD117, survivin, and Notch positive. CONCLUSIONS: We propose that antigen-independent B-cell differentiation stages are involved in disease origination and progression in myeloma. Furthermore, investigations of myeloma putative stem cell progenitors may lead to novel treatments to eradicate the potential reservoir of minimal residual disease.

Monitoring a nuclear factor-κB signature of drug resistance in multiple myeloma.

The emergence of acquired drug resistance results from multiple compensatory mechanisms acting to prevent cell death. Simultaneous monitoring of proteins involved in drug resistance is a major challenge for both elucidation of the underlying biology and development of candidate biomarkers for assessment of personalized cancer therapy. Here, we have utilized an integrated analytical platform based on SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring mass spectrometry, a versatile and powerful tool for targeted quantification of proteins in complex matrices, to evaluate a well-characterized model system of melphalan resistance in multiple myeloma (MM). Quantitative assays were developed to measure protein expression related to signaling events and biological processes relevant to melphalan resistance in multiple myeloma, specifically: nuclear factor-κB subunits, members of the Bcl-2 family of apoptosis-regulating proteins, and Fanconi Anemia DNA repair components. SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring methods were developed for quantification of these selected target proteins in amounts of material compatible with direct translation to clinical specimens (i.e. less than 50,000 cells). As proof of principle, both relative and absolute quantification were performed on cell line models of MM to compare protein expression before and after drug treatment in naïve cells and in drug resistant cells; these liquid chromatography-multiple reaction monitoring results are compared with existing literature and Western blots. The initial stage of a systems biology platform for examining drug resistance in MM has been implemented in cell line models and has been translated to MM cells isolated from a patient. The ultimate application of this platform could assist in clinical decision-making for individualized patient treatment. Although these specific assays have been developed to monitor MM, these techniques are expected to have broad applicability in cancer and other types of disease.

Ezetimibe is an inhibitor of tumor angiogenesis.

Epidemiological and preclinical observations have suggested a role for one or more products of the mevalonate/cholesterol biosynthesis pathway in the progression of prostate cancer. In this study, we used ezetimibe (Zetia), a specific, FDA-approved, cholesterol uptake-blocking drug, in combination with either a hyper- or hypocholesterolemic diet, to show that elevated circulating cholesterol levels promote, whereas a reduction in circulating cholesterol levels retard, the growth of human prostate cancer xenograft tumors in mice. Circulating cholesterol levels also modified tumor angiogenesis; higher cholesterol levels increased microvessel density and other indicators of vascularity. Consistent with these data, the reduction of cholesterol levels also increased the levels of the angiogenesis inhibitor thrombospondin-1 in the xenografts. Our results thus suggest that hypercholesterolemia directly accelerates the growth of prostate carcinomas, and that the pharmacological reduction of serum cholesterol levels may retard prostate cancer growth by inhibiting tumor angiogenesis.

Cholesterol sensitivity of endogenous and myristoylated Akt.

The serine-threonine kinase, Akt, has been linked to cholesterol-sensitive signaling mechanisms, suggesting a possible means whereby cholesterol might affect tumor cell growth and survival. However, it has not been shown whether Akt itself, as distinct from upstream components of the pathway (e.g., membrane phosphoinositides), can be directly responsible for cholesterol-mediated effects. Consistent with this possibility, we identified an Akt1 subpopulation in cholesterol-rich lipid raft fractions prepared from LNCaP human prostate cancer cells. Phosphorylation of this Akt subspecies was ablated with methyl-beta-cyclodextrin, a cholesterol-binding compound, under conditions where nonlipid raft-resident Akt was unaffected. A myristoylated Akt1 (MyrAkt1) fusion protein expressed in LNCaP cells was found to be highly enriched in lipid rafts, indicating that oncogenic Akt is overrepresented in cholesterol-rich membranes compared with wild-type Akt. Notably, lipid raft-resident MyrAkt1 exhibited a markedly distinct substrate preference compared with MyrAkt1 immunoprecipitated from cytosol and nonraft membrane fractions, suggesting a redirection of signal transduction when the protein is present in cholesterol-rich membranes. Expression of MyrAkt1 in LNCaP cells overcame their characteristic dependence on constitutive signaling through the phosphoinositide 3'-kinase pathway. This protective effect was substantially diminished with cyclodextrin treatment. Phosphorylation of Akt substrates in lipid raft fractions, but not in cytosol/nonraft membrane fractions, was ablated with cyclodextrin. In addition, in control (LacZ transfected) cells, lipid raft fractions were relatively enriched in phosphorylated Akt substrates. Collectively, these data show that a subpopulation of Akt is cholesterol sensitive and that the oncogenic effects conferred by myristoylation arise, in part, from the tendency of the membrane-targeted form of the protein to reside in cholesterol-rich membrane microdomains.

HMG-CoA reductase inhibitors induce apoptosis in pericytes.

Pericytes, which surround endothelial cells in precapillary arterioles, capillaries, and postcapillary venules, are important for the development, maturation, and maintenance of the vascular system. Pericytes are also pluripotent cells that can differentiate into a variety of mesenchymal cells including smooth muscle cells and osteoblasts. Possibly because of their vasculature regulating activities and ability to differentiate in situ, pericytes are implicated in several diseases with vascular complications, including diabetic retinopathy, as well as Reynaud's Syndrome, central nervous system dementias, and vascular calcification among others. Statin drugs, which block the conversion of HMG-CoA to mevalonate in the cholesterol synthesis pathway, are known to have apoptotic and growth inhibitory effects on cells in vitro and complex pleiotropic effects on cells and tissues in vivo. Recently, evidence has emerged that statin drug use in human patients results in a significant 20% reduction in cancer incidence. It is not known whether these results are due to direct statin action on normal tissue, growth inhibitory/pro-apoptotic effects on tumor cells, and/or effects on angiogenesis. Because of the role of pericytes in angiogenesis and the effects of statins on cancer incidence, we tested the direct effects of statins on pericytes. Specifically, we demonstrate that 3 statins, simvastatin, lovastatin, and mevastatin induce dose-dependent apoptosis in the TR-PCT1 pericyte cell line, that simvastatin (empirically shown to be the most potent of the 3 statins) induces similar levels of apoptosis in freshly isolated pericytes, and that simvastatin-induced apoptosis in pericytes is cholesterol, caspase-3, and caspase-7 mediated.