Denise K. Walters

CD147 regulates the expression of MCT1 and lactate export in multiple myeloma cells.

Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis, tumor cells must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells.

Induction of Malignant Plasma Cell Proliferation by Eosinophils

The biology of the malignant plasma cells (PCs) in multiple myeloma (MM) is highly influenced by the bone marrow (BM) microenvironment in which they reside. More specifically, BM stromal cells (SCs) are known to interact with MM cells to promote MM cell survival and proliferation. By contrast, it is unclear if innate immune cells within this same space also actively participate in the pathology of MM. Our study shows for the first time that eosinophils (Eos) can contribute to the biology of MM by enhancing the proliferation of some malignant PCs. We first demonstrate that PCs and Eos can be found in close proximity in the BM. In culture, Eos were found to augment MM cell proliferation that is predominantly mediated through a soluble factor(s). Fractionation of cell-free supernatants and neutralization studies demonstrated that this activity is independent of Eos-derived microparticles and a proliferation-inducing ligand (APRIL), respectively. Using a multicellular in vitro system designed to resemble the native MM niche, SCs and Eos were shown to have non-redundant roles in their support of MM cell growth. Whereas SCs induce MM cell proliferation predominantly through the secretion of IL-6, Eos stimulate growth of these malignant cells via an IL-6-independent mechanism. Taken together, our study demonstrates for the first time a role for Eos in the pathology of MM and suggests that therapeutic strategies targeting these cells may be beneficial.

Atypical expression of ErbB3 in myeloma cells: cross-talk between ErbB3 and the interferon-alpha signaling complex.

We have previously demonstrated that the responsiveness of multiple myeloma (MM) cells to interferon-alpha (IFN-α) stimulation is variable, with an atypical growth response displayed by some cells. Here we report the ability of IFN-α to induce tyrosine phosphorylation of a 180 kDa band in the KAS-6/1 MM cell line, which is growth responsive to IFN-α. Further characterization demonstrated that this band corresponds to ErbB3. To our knowledge, this is the first report of ErbB3 expression in a cell type of the hematopoietic lineage. Although ErbB receptors have been shown to crosscommunicate with various other receptors, our results show for the first time that the IFN-α receptor can crosscommunicate with ErbB3. To address the significance of these observations, we transfected ErbB3-negative DP-6 MM cells with ErbB3 and used siRNA to silence ErbB3 in the KAS-6/1 cell line. Although IFN-α transactivated ErbB3 in the DP-6 transfectants, it did not confer growth responsiveness to IFN-α. Interestingly, silencing ErbB3 expression in the KAS-6/1 cells decreased the overall growth response to IFN-α and to interleukin-6. These results suggest that ErbB3 expression alone does not uniquely confer IFN-α growth responsiveness, but instead may amplify proliferation rates in MM cells that have acquired atypical expression of this receptor.

Transactivation of gp130 in myeloma cells

Receptor transactivation, i.e., interaction between unrelated receptor systems, is a growing theme in cytokine and growth factor signaling. In this study we reveal for the first time the ability of IFN-α to transactivate gp130 in myeloma cells. An epidermal growth factor receptor/gp130 chimeric receptor previously shown by us to transactivate endogenous gp130, provided a complementary tool to study the underlying mechanisms of receptor cross-talk. Further analysis revealed that transactivation of gp130 by IFN-α did not require the extracellular or trans-membrane domain of gp130. Moreover, transactivation of gp130 was critically dependent upon Janus kinase activation by the initiating receptor and correlated with rapid and sustained Janus kinase 1 and tyrosine kinase (Tyk) 2 tyrosine phosphorylation. Finally, transactivation of gp130 may be a common theme in myeloma cells, perhaps providing a mechanism for enhanced or qualitatively distinct cellular responses to specific stimuli.

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 CD147bright and CD147dim populations and analyzed for proliferation potential, we discovered that CD147bright PCs displayed significantly higher levels of cell proliferation than did CD147dim 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.

A role for Janus kinases in crosstalk between ErbB3 and the interferon-alpha signaling complex in myeloma cells.

Receptor crosstalk is an emerging and recurrent theme in cytokine and growth factor signaling; however, insight into the mechanism(s) underlying these interactions remains limited. Recently, we reported that crosstalk occurs between ErbB3 and the interferon alpha (IFN-α) signaling complex in the myeloma cell line KAS-6/1 and that this crosstalk contributes to the regulation of cell proliferation. In this study, we examined the mechanism underlying the transactivation of ErbB3 in the IFN-α growth-responsive KAS-6/1 cells. The examination of IFN-α receptor 1 and 2 (IFNAR1 and IFNAR2) levels revealed that the KAS-6/1 cell line overexpresses IFNAR1 relative to other myeloma cell lines that are growth arrested by IFN-α. Subsequent investigation of Tyk2, which is constitutively associated with IFNAR1, demonstrated that Tyk2 activation is uniquely sustained in the KAS-6/1 cell line following IFN-α stimulation. Interestingly, silencing of Tyk2 expression via siRNA resulted in attenuation of ErbB3 transactivation. However, inhibition of Jak1 expression also decreased IFN-α-induced tyrosine phosphorylation of ErbB3. Finally, siRNA downregulation of Tyk2 and Jak1 was found to decrease IFN-α-stimulated proliferation. These findings validate our previous report of ErbB3 involvement in IFN-α-induced proliferation and further suggest that both Janus kinase members, Tyk2 and Jak1, play a role in the transactivation of ErbB3 in this model system.

Responsiveness of cytogenetically discrete human myeloma cell lines to lenalidomide: Lack of correlation with cereblon and interferon regulatory factor 4 expression levels

The introduction of novel immunomodulatory drugs (IMiDs) has dramatically improved the survival of patients with multiple myeloma (MM). While it has been shown that patients with specific cytogenetic subtypes, namely t(4;14), have the best outcomes when treated with bortezomib‐based regimens, the relationship between cytogenetic subtypes and response to IMiDs remains unclear. Using DNA synthesis assays, we investigated the relationship between cytogenetic subtype and lenalidomide response in a representative panel of human myeloma cell lines (HMCLs). We examined HMCL protein expression levels of the lenalidomide target cereblon (CRBN) and its downstream target interferon regulatory factor‐4 (IRF4), which have previously been shown to be predictive of lenalidomide response in HMCLs. Our results reveal that lenalidomide response did not correlate with specific cytogenetic translocations. There were distinct groups of lenalidomide‐responsive and non‐responsive HMCLs, as defined by inhibition of cellular proliferation; notably, all of the hyperdiploid HMCLs fell into the latter category. Repeated dosing of lenalidomide significantly lowered the IC50 of the responsive HMCL ALMC‐1 (IC50 = 2.6 μm vs. 0.005 μm, P < 0.0001), but did not have an effect on the IC50 of the non‐responsive DP‐6 HMCL (P > 0.05). Moreover, no association was found between lenalidomide responsiveness and CRBN and IRF4 expression. Our data indicate that lenalidomide sensitivity is independent of cytogenetic subtype in HMCLs. While CRBN and IRF4 have been shown to be associated with response to lenalidomide in patients, these findings do not translate back to HMCLs, which could be attributable to factors present in the bone marrow microenvironment.

Biological Evaluation of CpG Stimulation of Normal Human B-Cells: Implications for B-Cell Biology and Cytogenetic Analysis of CLL B-Cells

CpG oligodeoxynucleotides (CpG) are potent normal B-cell immune modulating agents and have been used therapeutically as adjuvants in immunotherapy and vaccines (Klinman, 2004). CpG are also used in clinical laboratories as a mitogenic tool to reveal chromosomal abnormalities in malignant B-cells from chronic lymphocytic leukaemia (CLL) patients (Dicker et al., 2006; Haferlach et al., 2007; Mayr et al., 2006). The latter utility has greatly advanced the cytogenetic analysis of CLL cells beyond what can be achieved by interphase fluorescence in situ hybridization (FISH) analysis. However, it remains unclear whether the stimulatory effects of CpG could also induce de novo genomic abnormalities in CLL B-cells in addition to revealing pre-existing genetic lesions. The answer to this question would also have fundamental B lineage tumourigenesis implications that are relevant to the therapeutic use of CpG in the clinic. To validate the role of CpG in revealing but not inducing cytogenetic abnormalities in CLL B-cells, we stimulated peripheral blood B-cells from 24 healthy adults with CpG and assessed the incidence of chromosomal abnormalities. These cells are considered to have no or very few pre-existing abnormalities. All 24 B-cell samples lacked CLL FISH panel-specific pre-existing abnormalities (Fink et al., 2005). Following CpG stimulation under the conditions identical to those used by the clinical cytogenetic laboratory, we performed karyotype analysis (Table 1). Of note, none of the 24 samples showed evidence of clonal abnormalities as defined in clinical laboratories as having ≥2 cells with the same cytogenetic abnormality(s). Thus, our data confirm that CpG remains a powerful tool for revealing pre-existing CLL B-cell clonal chromosomal abnormalities. Table 1 Effect of CpG stimulation on the cytogenetics of normal B-cells However, 9 of 24 samples (37.5%) stimulated with CpG did show ≥1 cell(s) with nonclonal (single appearance / 20 metaphase cells) karyotypic abnormalities. The type and location of chromosomal abnormalities appeared to be random. Among them, 3 of 24 samples (12.5%) had greater than 10% of the cells (equivalent to 2 abnormal cells/20 cells) displaying nonclonal chromosomal abnormalities. In a smaller subset of normal controls (n=8), B-cells were also stimulated with CD40 ligand (CD40L), a known B-cell activator, and 4 out 8 samples tested (50%) showed nonclonal chromosomal abnormalities. These data suggest that both CpG and CD40L may induce chromosomal abnormalities when B-cells are stimulated in vitro. Of interest, it has also been shown that some CpG-activated CLL cells also exhibit nonclonal cytogenetic abnormalities, which are normally not scored clinically (Put et al., 2009). To address the remote possibility that CpG-induced cytogenetic abnormalities in healthy donor B-cells reflected pre-existing genetic lesions, we further fractionated B-cells from 6 healthy donors into naive and memory subsets. Here, we reasoned that memory B-cells were more likely than naive B-cells to harbour certain types of pathological translocations, albeit at very low frequencies (Roulland et al., 2006). However, after CpG stimulation, we observed that both memory and naive B-cells (3/6 samples in both cases) exhibited nonclonal abnormalities (data not shown) at comparable frequencies. These observations strongly suggest that the nonclonal abnormalities did not pre-exist but instead were induced de novo by CpG stimulation. Given that the mutagenic enzyme activation-induced cytidine deaminase (AICDA), which induces mutations in the variable and switch regions of immunoglobulin genes, also induces chromosomal translocations in mouse models (Ramiro et al., 2006; Robbiani et al., 2009), we questioned if CpG stimulation could induce AICDA expression, which in turn may play a role in induction of chromosomal abnormalities. Therefore, we performed polymerase chain reaction (PCR) analysis of AICDA expression on freshly isolated as well as 3-day CpG stimulated B-cells from 5 healthy donors. While freshly isolated normal B-cells expressed little or no detectable AICDA, CpG stimulation resulted in robust induction of AICDA transcripts (Figure 1A). Additional quantitative PCR analyses showed that CpG and CD40L stimulation induced about 15-fold and 25-fold higher levels of AICDA expression, respectively (Figure 1B). These data suggest that CpG activated normal B-cells indeed express significant levels of mutagenic AICDA. Figure 1 Analysis of CpG activated normal or malignant B-cells Finally, we investigated whether CpG or CD40L-stimulated B-cells exhibit evidence of double stranded DNA breaks (DSBs), known intermediates of chromosomal abnormalities (Ramiro et al, 2006). To that end, we evaluated CpG and CD40L-stimulated B-cells for phospho-histone 2AX (γH2AX) foci, the hallmark of DSBs. As presented in Figure 1C, although freshly isolated B-cells did not display intranuclear γH2AX foci, CD40L-activated B-cells exhibited a number of large and coarse γH2AX foci and CpG-stimulated B-cells exhibited a number of γH2AX foci, albeit more diffuse in appearance. Therefore, B-cells activated in vitro with CpG as well as CD40L do indeed show evidence of DNA damage, in addition to the induction of the DNA mutating enzyme AICDA. These observations are consistent with the explanation that CpG activation has the potential to induce genetic instability in B-cells through the mutagenic activity of AICDA. Recently, in a separate study, we found that the activation of B-cells with CpG or CD40L induced a robust induction of AICDA expression, echoing the data presented in this study. More importantly, while AICDA expression is induced, CpG stimulation does not induce expression of various DNA repair activities that we believe are necessary to offset AICDA mutagenic activity (unpublished observations). Given that CpG stimulation induces robust expression of AICDA and nonclonal cytogenetic abnormalities in vitro, it is reasonable to question what would happen to these B-cells if they were activated in vivo when CpG is used as a therapeutic agent. Similarly, what is the role of natural bacterial CpG in the development and progression of B-cell malignancies? Indeed, a recent report showing that CpG could induce aberrant class switch recombination in mature or immature B-cells is consistent with our findings (Edry et al., 2008). Although most CpG-induced chromosomal abnormalities may be incompatible with cell survival, it is possible that some rarely occurring abnormalities could indeed be pathogenic and play a role in lymphomagenesis. Thus, CpG activation of malignant B-cells could also play a role in clonal evolution and further progression of B-cell malignancies such as CLL. Therefore, our study suggests the need for more in-depth studies to address these important issues.

Characterization and use of the novel human multiple myeloma cell line MC-B11/14 to study biological consequences of CRISPR-mediated loss of immunoglobulin A heavy chain

The genetic abnormalities underlying multiple myeloma (MM) are notoriously complex and intraclonal heterogeneity is a common disease feature. In the current study, we describe the establishment of a monoclonal immunoglobulin A (IgA) kappa (κ) MM cell line designated MC-B11/14. Cytogenetic and fluorescence in situ hybridization analyses of the original and relapse patient samples revealed that the MM clone was nonhyperdiploid and possessed an 11;14 chromosomal translocation. The MC-B11/14 cell line, established from the relapse sample, is tetraploid and houses the t(11;14) abnormality. Given our long-standing interest in Ig function and secretion, we next used CRISPR technology to knock out IgA heavy-chain expression in the MC-B11/14 cells to assess the biological consequences of converting this cell line to one only expressing κ light chains. As expected, secretion of intact IgA was undetectable from MC-B11/14IgA- cells. Sensitivity to pomalidomide treatment was similar between the MC-B11/14WT and MC-B11/14IgA- cells; however, MC-B11/14IgA- cells were found to be significantly more resistant to bortezomib treatment. This study describes the establishment of a new human MM cell line tool with which to study disease biology and the use of CRISPR technology to create a potentially useful model with which to study MM light-chain escape.