TALEN-mediated genetic tailoring as a tool to analyze the function of acquired mutations in multiple myeloma cells.

Multiple myeloma (MM) is a clonal plasma cell malignancy that is initiated by a number of mutations and the process of disease progression is characterized by further acquisition of mutations. The identification and functional characterization of these myelomagenic mutations is necessary to better understand the underlying pathogenic mechanisms in this disease. Recent advancements in next-generation sequencing have made the identification of most of these mutations a reality. However, the functional characterization of these mutations has been hampered by the lack of proper and efficient tools to dissect these mutations. Here we explored the possible utility of transcription activator-like effector nuclease (TALEN) genome engineering technology to tailoring the genome of MM cells. To test this possibility, we targeted the HPRT1 gene and found that TALENs are a very robust and efficient genome-editing tool in MM cells. Using cotransfected green fluorescent protein as an enrichment marker, single-cell subclones with desirable TALEN modifications in the HPRT1 gene were obtained in as little as 3-4 weeks of time. We believe that TALENs will greatly facilitate the functional study of somatic mutations in MM as well as other cancers.

Molecular analysis of immunoglobulin genes reveals frequent clonal relatedness in double monoclonal gammopathies.

Monoclonal gammopathies (MGs) are hematological diseases characterized by high levels of a monoclonal immunoglobulin (Ig) or M-protein. Within this group are patients with more than one M-protein, referred to as double MGs (DMGs). The M-proteins in DMG patients may have different heavy chain (HC) isotypes that are associated with different light chains (LCs), or different HCs that are LC matched. In this study, we examined the clonal relatedness of the M-proteins in the latter type in a cohort of 14 DMG patients. By using PCR, we identified 7/14 DMG patients that expressed two Ig HC isotypes with identical Ig HC variable (IGHV), diversity (IGHD), joining (IGHJ), and complementarity determining region (HCDR3) sequences. Two additional DMG patients had two Ig transcripts using the same IGHV, IGHD and IGHJ genes but with slight differences in variable region or HCDR3 mutations. LC analysis confirmed that a single LC was expressed in 3/7 DMG patients with identical HC transcripts and in the two DMGs with highly similar transcripts. The PCR findings were confirmed by immunofluorescence for HC and LC expression. Clonally related HC-dissimilar/LC-matched DMGs may occur often and defines a new subtype of MG that may serve as a tool for studies of disease pathogenesis.

Infectious complications among individuals with clinical monoclonal B-cell lymphocytosis (MBL): a cohort study of newly diagnosed cases compared to controls.

Although the risk of progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL) has been well characterized, it is unknown whether other common complications associated with CLL, such as increased risk of infection, occurs in individuals with MBL. We used the Mayo CLL database to identify cohorts of individuals with newly diagnosed MBL (n=154) or newly diagnosed CLL (n=174) who resided within 50 miles of Mayo Clinic. A cohort of 689 adult patients seen for a general medical examination who resided within 50 miles of Mayo clinic and who enrolled in a case-control study of non-Hodgkin lymphoma (NHL) was used as a comparison cohort. Hospitalization with infection was more common among individuals with MBL (25/154; 16.2%), and CLL (32/174; 18.4%) than controls (18/689; 2.6%). On pooled multivariable Cox proportional hazards analysis of all 1017 patients (controls, MBL and CLL), male sex (hazards ratio (HR)=2.3; P=0.002), major co-morbid health problems (HR=1.7, P=0.04), the presence of CLL (HR=3.2, P<0.001), treatment for progressive CLL (HR=2.4, P=0.001) and the presence of MBL (HR=3.0, P=0.001) were independently associated with risk of hospitalization for infection. These results suggest the risk of serious infection in clinical MBL is substantially greater than the risk of progression requiring treatment.

Increased expression of extracellular matrix metalloproteinase inducer (CD147) in multiple myeloma: role in regulation of myeloma cell proliferation.

Multiple myeloma (MM) is preceded by the asymptomatic pre-malignant state, monoclonal gammopathy of undetermined significance (MGUS). Although MGUS patients may remain stable for years, they are at increased risk of progressing to MM. A better understanding of the relevant molecular changes underlying the transition from an asymptomatic to symptomatic disease state is urgently needed. Our studies show for the first time that the CD147 molecule (extracellular matrix metalloproteinase inducer) may be having an important biological role in MM. We first demonstrate that CD147 is overexpressed in MM plasma cells (PCs) vs normal and pre-malignant PCs. Next, functional studies revealed that the natural CD147 ligand, cyclophilin B, stimulates MM cell growth. Moreover, when MM patient PCs displaying bimodal CD147 expression were separated into CD147(bright) and CD147(dim) populations and analyzed for proliferation potential, we discovered that CD147(bright) PCs displayed significantly higher levels of cell proliferation than did CD147(dim) PCs. Lastly, CD147-silencing significantly attenuated MM cell proliferation. Taken together, these data suggest that the CD147 molecule has a key role in MM cell proliferation and may serve as an attractive target for reducing the proliferative compartment of this disease.

Evidence for ongoing DNA damage in multiple myeloma cells as revealed by constitutive phosphorylation of H2AX.

DNA double-strand breaks (DSBs) are deleterious lesions that can lead to chromosomal anomalies, genomic instability and cancer. The histone protein H2AX has an important role in the DNA damage response (DDR) and the presence of phospho-H2AX (γH2AX) nuclear foci is the hallmark of DSBs. We hypothesize that ongoing DNA damage provides a mechanism by which chromosomal abnormalities and intratumor heterogeneity are acquired in malignant plasma cells (PCs) in patients with multiple myeloma (MM). Therefore, we assessed PCs from patients with the premalignant condition, monoclonal gammopathy of undetermined significance (MGUS) and MM, as well as human MM cell lines (HMCLs) for evidence of DSBs. γH2AX foci were detected in 2/5 MGUS samples, 37/40 MM samples and 6/6 HMCLs. Notably, the DSB response protein 53BP1 colocalized with γH2AX in both MM patient samples and HMCLs. Treatment with wortmannin decreased phosphorylation of H2AX and suggests phosphoinositide (PI) 3-kinases and/or PI3-kinase-like family members underlie the presence of γH2AX foci in MM cells. Taken together, these data imply that ongoing DNA damage intensifies across the disease spectrum of MGUS to MM and may provide a mechanism whereby clonal evolution occurs in the monoclonal gammopathies.

Prevalence and risk of progression of light-chain monoclonal gammopathy of undetermined significance: a retrospective population-based cohort study.

BACKGROUND: Monoclonal gammopathy of undetermined significance (MGUS) is defined by expression of heavy-chain immunoglobulin (IgH) and is the precursor lesion for 80% of cases of multiple myeloma. The remaining 20% are characterised by absence of IgH expression; we aimed to assess prevalence of a corresponding precursor entity, light-chain MGUS. METHODS: We used a population-based cohort, previously assembled to estimate MGUS prevalence, of 21,463 residents of Olmsted County, MN, USA, aged 50 years and older. We did a serum free light-chain assay on all samples with sufficient serum remaining, and immunofixation electrophoresis was done for all samples with an abnormal free light-chain ratio or abnormal protein electrophoresis results from the original study. Light-chain MGUS was defined as an abnormal free light-chain ratio with no IgH expression, plus increased concentration of the involved light chain. We calculated age-specific and sex-specific prevalence and rates of progression to lymphoproliferative disorders for light-chain and conventional MGUS and assessed incidence of renal disorders in patients with light-chain MGUS. FINDINGS: 610 (3.3%) of 18,357 people tested had an abnormal free light-chain ratio, of whom 213 had IgH expression that was diagnostic of conventional MGUS. 146 of the remaining 397 individuals had an increase of at least one free light chain and met criteria for light-chain MGUS. Prevalence of light-chain MGUS was 0.8% (95% CI 0.7-0.9), contributing to an overall MGUS prevalence of 4.2% (3.9-4.5). Risk of progression to multiple myeloma in patients with light-chain MGUS was 0.3% (0.1-0.8) per 100 person-years. 30 (23%) of 129 patients with light-chain MGUS were diagnosed with renal disease. INTERPRETATION: We define a clinical entity representing the light-chain equivalent of conventional MGUS and posing a risk of progression to light-chain multiple myeloma and related disorders. FUNDING: US National Cancer Institute.

Elevation of soluble CD307 (IRTA2/FcRH5) protein in the blood and expression on malignant cells of patients with multiple myeloma, chronic lymphocytic leukemia, and mantle cell lymphoma.

CD307 is a differentiation antigen expressed in B-lineage cells. One soluble and two membrane-bound forms have been predicted and an enzyme-linked immunosorbent assay (ELISA) for soluble CD307 established. Our goal was to determine if CD307 is expressed on the surface of cells from patients with multiple myeloma (MM), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and other B-cell malignancies and if soluble CD307 levels are elevated in the blood of patients with these B-cell malignancies. Cells and blood were collected from patients. Expression of CD307 was measured by flow cytometry and blood levels of soluble CD307 by ELISA. High soluble CD307 levels were detected in 21/43 (49%) of patients with MM, 36/46 (78%) with CLL and 9/24 (38%) with MCL. Soluble CD307 levels correlated with plasma cell percentages in bone marrow aspirates in MM and total white blood cells in CLL. CD307 on the cell membrane was detected by flow cytometry in 8/8 MM, 23/29 CLL and 4/5 MCL samples. Because CD307 is present on malignant cells from patients with MM, CLL and MCL, CD307 may be a useful therapeutic target for the treatment of these diseases.

CD38 expression levels in chronic lymphocytic leukemia B cells are associated with activation marker expression and differential responses to interferon stimulation.

CD38, a surface protein whose expression increases upon normal B-cell activation, is a marker of disease aggression in B-cell chronic lymphocytic leukemia (B-CLL). Higher percentages of CD38-expressing CLL B cells may be found in lymphoid compartments compared to peripheral blood. Therefore, it is possible that although CLL B cells are resting, CD38 may be a marker of recent cell activation prior to entry into the periphery. To address this hypothesis, we examined the association of CD38 expression with other activation antigens identified in gene expression profiling experiments and include CD18, CD49d, CD20, and subunit 5 of the anaphase-promoting complex/cyclosome. We found that all these markers were more highly expressed in leukemic B cells from CD38-positive CLL patients. Lastly, because interferon is known to modulate CD38 expression, we used IFN-alpha to test the ability of CLL B cells to increase CD38 expression in vitro. Interestingly, IFN stimulation only modulated CD38 expression in CLL B cells that already expressed CD38. Taken together, these data suggest that CD38 is a marker of a more recently activated CLL B cell. This in turn may explain the biological and clinical differences between CD38-positive type B-CLL and CD38-negative type B-CLL.

ZAP-70 is expressed by a subset of normal human B-lymphocytes displaying an activated phenotype.

The Syk family tyrosine kinase ZAP-70 is essential for normal T-cell development and signaling. Recently, leukemic cells from some patients with B-cell chronic lymphocytic leukemia (B-CLL) were shown to express ZAP-70. Owing to the prognostic value of B-CLL ZAP-70 expression, this phenotype may reflect intrinsic biological differences between the two subsets of disease. However, it remains unclear whether CLL-B cells aberrantly acquire ZAP-70 expression during the transformation process or whether ZAP-70 may be expressed under certain conditions in normal human B-lymphocytes. To discriminate between these two possibilities, we assessed ZAP-70 expression in normal human B-lymphocytes. Our data demonstrate that ZAP-70 is expressed in a subpopulation of tonsillar and splenic normal B-lymphocytes that express an activated phenotype. Furthermore, ZAP-70 expression can be induced in vitro upon stimulation of blood and tonsillar B cells. Finally, we show that phosphorylation of ZAP-70 occurs in tonsillar B cells with stimulation through the B-cell receptor. These results provide new insight into normal human B-cell biology as well as provide clues about the transformed cell in B-CLL.

VEGF receptors on chronic lymphocytic leukemia (CLL) B cells interact with STAT 1 and 3: implication for apoptosis resistance.

We have previously shown that chronic lymphocytic leukemia (CLL) B cells secrete vascular endothelial growth factor (VEGF) in vitro, have constitutively active VEGF receptors R1 and R2, and respond to exogenous VEGF by specifically upregulating Mcl-1 and XIAP in association with decreased cell death. We found that epigallocatechin (EGCG) decreases VEGF receptor phosphorylation and induces apoptosis in CLL B cells. The mechanism(s) by which VEGF receptor activation increases Mcl-1 and XIAP and promotes survival remains unknown. To further define the signaling pathway mediating VEGF induction of antiapoptotic proteins in CLL B-cells, we investigated downstream effects of VEGF-VEGF receptor binding on the STAT signaling pathway. We find that CLL B cells abundantly express cytoplasmic serine phosphorylated (p)-STAT-1 and p-STAT-3, VEGF-R1/2 are physically associated with p-STAT-1 and p-STAT-3, and p-STAT-3 (but not p-STAT-1) is found in the CLL nucleus. VEGF receptor ligation selectively induces activation and perinuclear translocation of STAT 3 through receptor-mediated endocytosis. The inhibition of VEGF receptor activation with either tyrosine kinase inhibitors or VEGF neutralizing antibodies inhibit VEGF receptor phosphorylation, decrease p-STAT-3 (serine 727), Mcl-1, and induces cell death in CLL B cells. Thus, a VEGF-VEGF receptor pathway in CLL B cells can be linked to activation of STAT proteins that are able to enhance their apoptotic resistance.

Inhibition of survivin expression suppresses the growth of aggressive non-Hodgkin's lymphoma.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family and functions both as an apoptosis inhibitor and a regulator of cell division. Survivin overexpression is common in many human tumors and correlates with survival in large cell non-Hodgkin's lymphoma. To evaluate this molecule as a potential therapeutic target in large-cell lymphoma, we evaluated the effect of survivin inhibition both in vitro and in vivo. Using an antisense oligonucleotide (ASO) approach, cell growth was significantly inhibited in the DoHH2, RL and HT lymphoma cell lines. In a lymphoma xenograft model, the development of tumors as well as the growth of established tumors was inhibited in the survivin ASO-treated mice compared to controls. To assess the efficacy of the survivin ASO in combination with other biological agents, we combined the survivin ASO with an anti-CD20 monoclonal antibody, rituximab. The effect of survivin ASO and rituximab in combination was additive in vitro. In vivo, however, suppression of tumor growth with the combination was not significantly superior to controls. We conclude that inhibition of survivin expression is an attractive therapeutic strategy in aggressive non-Hodgkin's lymphomas, and that combining survivin ASO with rituximab may enhance the efficacy of this approach.

Interleukin 6 induces monocyte chemoattractant protein-1 expression in myeloma cells.

Interleukin 6 (IL-6) is known to play an important role in the biology of the malignant plasma cells in multiple myeloma. In an effort to better understand IL-6 stimulated myeloma cell growth, we have performed gene expression profiling to identify IL-6 early response genes. Using the KAS-6/1 IL-6-dependent human myeloma cell line, IL-6 stimulation dramatically induced expression of monocyte chemoattractant protein-1 (MCP-1) mRNA. To verify this result, we used reverse transcriptase PCR and RNAse protection assays and demonstrated using both assays that MCP-1 is indeed an IL-6 responsive gene in a variety of IL-6-responsive myeloma cell lines. Moreover, we also demonstrated IL-6 stimulated MCP-1 secretion by the myeloma cell lines as well as by fresh patient tumor cells. Lastly, we present evidence that fresh patient tumor cells express mRNA for the MCP-1 receptor, CCR2, as do myeloma cell lines along with a second MCP-1 receptor, CCR11. Although MM cell chemotaxis in response to MCP-1 was only minimal, we were able to demonstrate that MCP-1 stimulated activation of MAPK. Because of the important role that this chemokine plays in both angiogenesis and bone homeostasis, and the ability of MCP-1 to activate myeloma cells, these results suggest a new mechanism by which IL-6 may contribute to disease pathogenesis.

Analysis of IL-6-mediated growth control of myeloma cells using a gp130 chimeric receptor approach.

Interleukin 6 (IL-6) has been shown to be a key growth factor for myeloma cells. To study IL-6 signal transduction in multiple myeloma (MM), we employed chimeric receptors composed of the epidermal growth factor receptor (EGFR) extracellular domain, gp130 transmembrane domain, and full-length or truncated gp130 cytoplasmic domains lacking regions previously shown to be necessary for MAPK, STAT1, and STAT3 activation. The IL-6-dependent KAS-6/1 MM cell line was transfected with various chimeric receptor constructs and assayed for EGF responsiveness. EGF stimulation surprisingly stimulated DNA synthesis in all transfectants, regardless of receptor length. When cell proliferation was assayed instead, only transfectants capable of inducing high levels of STAT3 activation proliferated in response to EGF. Additional studies revealed that EGF stimulation resulted in tyrosine phosphorylation of endogenous gp130 in cells expressing the chimeric receptor. Replacing the gp130 transmembrane region with the EGFR transmembrane domain diminished but did not disrupt this interaction. This receptor interaction was also observed in the IL-6-dependent MM cell line ANBL-6. In summary, although our results suggest that STAT activation is crucial in gp130-mediated proliferation of myeloma cells, these results must be interpreted with caution given our demonstration of the interaction between chimeric and endogenous receptors in myeloma cells. Importantly, this interaction has not been noted in studies utilizing the same gp130 chimeric receptor system in non-MM cells.

B-CLL cells are capable of synthesis and secretion of both pro- and anti-angiogenic molecules.

Initial work has shown that clonal B cells from B-chronic lymphocytic leukemia (B-CLL) are able to synthesize pro-angiogenic molecules. In this study, our goal was to study the spectrum of angiogenic factors and receptors expressed in the CLL B cell. We used ELISA assays to determine the levels of basic fibroblast growth factors (bFGF), vascular endothelial growth factor (VEGF), endostatin, interferon-alpha (IFN-alpha) and thrombospondin-1 (TSP-1) secreted into culture medium by purified CLL B cells. These data demonstrated that CLL B cells spontaneously secrete a variety of pro- and anti-angiogenic factors, including bFGF (23.9 pg/ml +/- 7.9; mean +/- s.e.m.), VEGF (12.5 pg/ml +/- 2.3) and TSP-1 (1.9 ng/ml +/- 0.3). Out of these three factors, CLL B cells consistently secreted bFGF and TSP-1, while VEGF was expressed in approximately two-thirds of CLL patients. Of interest, hypoxic conditions dramatically upregulated VEGF expression at both the mRNA and protein levels. We also employed ribonuclease protection assays to assay CLL B cell expression of a variety of other angiogenesis-related molecules. These analyses revealed that CLL B cells consistently express mRNA for VEGF receptor 1 (VEGFR1), thrombin receptor, endoglin, and angiopoietin. Further analysis of VEGFR expression by RT-PCR revealed that CLL B cells expressed both VEGFR1 mRNA and VEGFR2 mRNA. In summary, these data collectively indicate that CLL B cells express both pro- and anti-angiogenic molecules and several vascular factor receptors. Because of the co-expression of angiogenic molecules and receptors for some of these molecules, these data suggest that the biology of the leukemic cells may also be directly impacted by angiogenic factors as a result of autocrine pathways of stimulation.

The effectiveness of double-stranded short inhibitory RNAs (siRNAs) may depend on the method of transfection.

RNA interference (RNAi) is a recently described powerful experimental tool that can cause sequence-specific gene silencing, thereby facilitating functional analysis of gene function. Consequently, we became interested in using RNAi to determine the function of aberrantly expressed ErbB3 in the KAS-6/1 human myeloma cell line. Despite the wealth of information available on the use of RNAi, dsRNA target design, and the transfection of dsRNA in vitro, little information is available for transfecting dsRNA into nonadherent cells from any species. In the present study, we report that gene silencing of ErbB3 was not observed in myeloma cells when dsRNA targeting ErbB3 was introduced using conventional transfection agents and protocols that have proved successful for several adherent cell lines. Silencing of ErbB3, however, was observed in T47D cells, an adherent breast carcinoma cell line, using the same transfection methods, indicating that our target sequence was functional for gene silencing of ErbB3. Interestingly, ErbB3 was silenced in myeloma cells when the dsRNA target was introduced by electroporation. Thus, our studies illustrate the striking dependence of dsRNA-mediated gene silencing in some cells on the methods of dsRNA transfection.