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.

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.

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.

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.

Analysis of clonal B-cell CD38 and immunoglobulin variable region sequence status in relation to clinical outcome for B-chronic lymphocytic leukaemia.

Recent reports suggest that the expression of germline (GL) Ig variable region heavy-chain genes (VH) is a negative prognostic factor for B-cell chronic lymphocytic leukaemia (B-CLL) patients and that CLL B-cell CD38 expression may be a surrogate marker of Ig VH gene status. Currently, however, the usefulness of this surrogate marker is controversial. Therefore, our goal was to study the ability of CD38 to act as a surrogate marker for Ig VH somatic mutation (SM), and to identify differences in overall survival (OS), progression-free survival (PFS) and response in B-CLL patients based on these two markers. We first assessed the relationship between CD38 expression and Ig VH status on 131 B-CLL patients, including 66 patients enrolled in three North Central Cancer Treatment Group Trials. Although the mean percentages of CD38+ clonal B cells were significantly higher for patients classified as GL versus SM, CD38 was not a reliable marker for clonal B-cell SM. Overall, GL patients exhibited significantly shorter OS and PFS times than SM patients. Despite the inability of clonal B-cell CD38 expression to predict Ig VH mutation status, patients with < or =30% CD38+ cells did have shorter PFS and OS times than did CLL patients with < 30% CD38+ cells. Thus, the relationship between CD38 expression and Ig VH mutation status in B-CLL is not straightforward. Nevertheless, analysis in a co-operative group clinical trial setting suggests that both B-cell markers alone or in combination may have clinical usefulness. These data strongly encourage the study of these biological markers as they relate to disease heterogeneity in B-CLL.

Dissection of the signalling requirements of interleukin-6-stimulated myeloma cell growth.

The growth characteristics of myeloma cells are in striking contrast with those of normal, non-dividing end-stage plasma cells. Interleukin 6 (IL-6) has been shown to be a key growth factor for myeloma cells, however, IL-6 only acts as a differentiation factor for normal B cells. Wc have hypothesized that the differential response of myeloma cells to IL-6 may either result from altered IL-6 signal transduction and or from inappropriate IL-6-induced expression of genes whose products are key for continued tumor cell growth. To test this hypothesis, we have employed two experimental strategies. First, we are using cDNA array screening technology to identify IL-6 responsive genes in myeloma cells. Second. we are using a chimeric receptor approach to identify the regions of gp130, the signal transducing component of the IL-6 receptor, that are essential for myeloma cell proliferation. To this end, we have utilized a panel of IL-6 growth-responsive myeloma cell lines and a panel of mutant gp130 chimeric receptors. This combined approach has the potential to assess the relative importance of several signalling events in myeloma cell growth control and identify IL-6 responsive genes in this malignancy.

Differential human multiple myeloma cell line responsiveness to interferon-alpha. Analysis of transcription factor activation and interleukin 6 receptor expression.

Although IFN-alpha is commonly used as maintenance treatment for multiple myeloma patients, its effectiveness is varied. In this study, we have used a panel of IL-6 responsive myeloma cell lines that vary remarkably in responsiveness to IFN-alpha. Three cell lines were growth arrested by IFN-alpha; however, IFN-alpha significantly stimulated growth of the fourth cell line, KAS-6/1. Our studies have focused on elucidating the mechanism of differential IFN-alpha responsiveness. First, we have shown that IFN-alpha-stimulated growth of the KAS-6/1 cells did not result from induction of autocrine IL-6 expression. Second, analysis of Stats 1, 2, and 3 and IFN regulatory factor-1 (IRF-1) and IRF-2 activation failed to reveal differences between the IFN-alpha growth-arrested or growth-stimulated cells. Third, although IFN-alpha treatment of the IFN-alpha growth-inhibited cell lines reduced IL-6 receptor (IL-6R) expression, IFN-alpha also reduced KAS-6/1 IL-6R expression. Finally, although IFN-alpha treatment reduced IL-6R numbers on each cell line, analysis of Stat protein activation revealed that the receptors were still functional. We conclude that myeloma cell responsiveness to IFN-alpha is heterogeneous and that mechanisms of IFN-alpha-mediated growth inhibition other than IL-6R downregulation must exist in myeloma. Identification of these mechanisms may allow development of agents that are more universally effective than IFN-alpha.

Ethacrynic acid: outflow effects and toxicity in human trabecular meshwork in perfusion organ culture.

The effect of ethacrynic acid, a potential outflow agent for the treatment of glaucoma, was studied in a series of 25 pairs of human eyes in perfusion organ culture. Concentrations varied from 0.01 mM to 2.4 mM and were used in single or repetitive doses. Intraocular pressure was continuously recorded for up to two weeks after exposure. Eyes were then fixed and the meshwork examined histologically. Ethacrynic acid in single doses of 0.05 mM, 0.3 mM, and 0.6 mM increased facility of outflow at least 40% when compared with fellow control eyes. The duration of effect was approximately 18 hours, during which time the intraocular pressure gradually returned to baseline. Histologic examination revealed dose related effects on the trabecular cells, ranging from clumping of nuclear chromatin in some eyes to cellular swelling, disruption of cytoplasmic membranes, and cell necrosis in other eyes at concentrations of 0.1 mM and higher. No recovery or reversal of these changes was noted with time, even two weeks after a single exposure to the drug. Although ethacrynic acid is effective in temporarily lowering intraocular pressure in the human eye, a low therapeutic index may limit its clinical usefulness.

Human trabecular meshwork phagocytosis. Observations in an organ culture system.

Perfusion organ culture of the trabecular meshwork was used to study the phagocytic ability of human trabecular cells. Cultured eyes were challenged with blood, latex microspheres, or zymosan particles for periods of 1 hour to 7 days. Trabecular cells were capable of ingesting all three types of particles. The presence of a foreign particle did not necessarily induce a phagocytic response, however, as free particles were seen in the intertrabecular spaces and Schlemm's canal. In contrast to studies in animals which indicate trabecular cell migration from the eye may play a role in the removal of foreign debris, limited human trabecular cell migration was observed. The effect of the culture process on trabecular cell phagocytosis was also studied, using adult cats. One eye received a phagocytic challenge in vivo with the fellow eye later receiving the phagocytic challenge in vitro. Phagocytosis was demonstrated in each eye, although more cells were involved with phagocytosis in vivo. The additional cells involved in vivo may have been recruited by an accompanying inflammation. Organ culture of human trabecular meshwork may be useful in the study of trabecular cell phagocytosis, and it allows separation of the effects of inflammation from the potential effects of phagocytosis itself.

Trabecular meshwork cellularity. Differences between fellow eyes.

The difference in trabecular meshwork cellularity between fellow eyes was determined in a series of seven pairs of normal human eyes. Variations in cellularity between different quadrants of individual eyes also were determined. Characterization of these differences and development of appropriate sampling techniques to determine trabecular cellularity has become important in light of recent studies that find a decrease in trabecular cell numbers with age and with primary open-angle glaucoma, and the apparent stimulation of trabecular cell division by laser trabeculoplasty. The difference in absolute cell numbers (nuclei per section) between fellow eyes was 12.8% +/- 4.3% (standard error of the mean, SEM). The difference in cellularity (nuclei per solid tissue area) was 9.4% +/- 4.5% (SEM) in a subgroup of five pairs of these eyes. The coefficient of variation between different quadrants of an eye was 18.4% for absolute cell numbers and 17.3% for cellularity for the entire group of eyes. Sample size calculations indicate that for future studies, four pairs of eyes would be needed to detect a 20% difference in absolute cell numbers between fellow eyes and three pairs needed for detecting a similar difference in cellularity (alpha = 0.05, beta = 0.1). At least two quadrants per eye should be examined.

Glycosaminoglycans of human trabecular meshwork in perfusion organ culture.

The synthetic profile of glycosaminoglycans (GAGs) of human trabecular meshwork in perfusion organ culture was studied in a series of 34 human eyes. The anterior segments of these eyes were cultured for periods of two to 28 days and received medium containing 3H-glucosamine and 35S-sulfate during the final 48 hours of culture. The meshwork was then dissected and the GAGs isolated and subjected to sequential enzyme digestion. Active labelling of hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate was found in all time periods. Eyes cultured seven and 14 days had similar incorporation profiles to "fresh" eyes (cultured 48 hours to allow for labelling). Eyes cultured 21 days showed an increase in dermatan sulfate labelling and a slight decrease in keratan sulfate labelling when compared with "fresh" eyes. Light microscopic autoradiography confirmed the trabecular meshwork incorporation of the radiolabelled precursors at all time periods. Thus, the trabecular meshwork remains metabolically active and GAG synthetic profiles remain reasonably similar to fresh eyes for up to three weeks in a perfusion organ culture system. This system may serve as a model for future studies of human trabecular meshwork GAGs.

Stimulation of cell division by argon and Nd:YAG laser trabeculoplasty in cynomolgus monkeys.

Although laser treatment of the trabecular meshwork is the most common form of surgery for glaucoma, the tissue response to this therapy is still incompletely understood. We applied argon or Nd:YAG laser to the trabecular meshwork of six monkeys. Cell division was identified by injecting tritiated thymidine into the anterior chamber 24 hr after laser application. Autoradiography of tissue sections revealed significantly more labelled cells in eyes treated with laser than in the untreated controls. In addition, cells in neighboring tissues such as iris, ciliary body and sclera showed labelling in association with laser application. Furthermore, comparison of argon-induced lesions with those caused by pulsed Nd:YAG suggests that there are quantitative and qualitative differences in the response of trabecular meshwork and surrounding tissues to these two forms of laser energy.

The effect of organ culture on human trabecular meshwork.

The effect of organ culture on the trabecular meshwork was studied in a series of human eyes using a perfusion culture system. One eye of a pair was cultured while the fellow eye was immediately fixed in glutaraldehyde. Culture periods ranged from 2 to 28 days, during which a mean intraocular pressure of 27 mmHg +/- 15 was maintained. The perfused culture medium appeared to leave the eye via Schlemm's canal and collector channels, mimicking the in vivo situation. The trabecular meshwork was well maintained with this system in 21 of 25 eyes. Cells remained in position on trabecular lamellae, cellular organelles usually remained normal, and the lamellae remained intact. Culture-induced changes were noted, with some cells developing intracytoplasmic lipid vacuoles and other cells developing swollen mitochondria. In addition, scattered focal cell necrosis was observed, most often in the uveal meshwork. Meshwork cellularity (nuclei:solid tissue) was determined with an image analysis system; there was an average cell loss of 20-40% in the cultured eyes as compared to their respective fellow control eyes. Overall, organ culture appears to maintain the human trabecular meshwork for at least 28 days, allowing controlled experimental studies to be performed.