A Novel BCMA Immunohistochemistry Assay Reveals a Heterogenous and Dynamic BCMA Expression Profile in Multiple Myeloma.

B-cell maturation antigen (BCMA) is a promising target for the treatment of multiple myeloma (MM) because the expression of this protein is largely limited to B-cell sets, plasma cells, MM, and other B-cell malignancies. Early studies assessing BCMA protein expression and localization have used insufficiently qualified immunohistochemistry assays, which have reported broad ranges of BCMA expression. As a result, our understanding of BCMA tissue expression derived from these data is limited, specifically the prevalence of BCMA expression on the cell surface/membrane, which has mechanistic relevance to the antimyeloma activity of several novel biotherapeutics. Here, we report on the qualification and application of a novel anti-BCMA immunohistochemistry antibody, 805G12. This antibody shows robust detection of BCMA in formalin-fixed, decalcified bone marrow tissue and provides key insights into membrane BCMA expression. The clone 805G12, which was raised against an intracellular C-terminal domain peptide of membrane BCMA, exhibited increased sensitivity and superior specificity across healthy and diseased tissue compared with the frequently referenced commercial reagent AF193. The new clone also demonstrated a broad range of expression of BCMA in MM and diffuse large B-cell lymphoma specimens. Additionally, cross-reactivity with closely related tumor necrosis factor receptor family members was observed with AF193 but not with 805G12. Furthermore, via established 805G12 and other independent BCMA assays, it was concluded that proteolytic processing by γ-secretase contributes to the levels of BCMA localized to the plasma membrane. As BCMA-directed therapeutics emerge to address the need for more effective treatment in the relapsed or refractory MM disease setting, the implementation of a qualified assay would ensure that reliable and consistent data on BCMA surface expression are used to inform clinical trial decisions and patient responses.

A Potent Isoprenylcysteine Carboxylmethyltransferase (ICMT) Inhibitor Improves Survival in Ras-Driven Acute Myeloid Leukemia.

Blockade of Ras activity by inhibiting its post-translational methylation catalyzed by isoprenylcysteine carboxylmethyltransferase (ICMT) has been suggested as a promising antitumor strategy. However, the paucity of inhibitors has precluded the clinical validation of this approach. In this work we report a potent ICMT inhibitor, compound 3 [UCM-1336, IC50 = 2 µM], which is selective against the other enzymes involved in the post-translational modifications of Ras. Compound 3 significantly impairs the membrane association of the four Ras isoforms, leading to a decrease of Ras activity and to inhibition of Ras downstream signaling pathways. In addition, it induces cell death in a variety of Ras-mutated tumor cell lines and increases survival in an in vivo model of acute myeloid leukemia. Because ICMT inhibition impairs the activity of the four Ras isoforms regardless of its activating mutation, compound 3 surmounts many of the common limitations of available Ras inhibitors described so far. In addition, these results validate ICMT as a valuable target for the treatment of Ras-driven tumors.

Control of RhoA methylation by carboxylesterase I.

A number of proteins that play key roles in cell signaling are post-translationally modified by the prenylation pathway. The final step in this pathway is methylation of the carboxyl terminus of the prenylated protein by isoprenylcysteine carboxylmethyltransferase. Due to the impact of methylation on Rho function, we sought to determine if the process was reversible and hence could control Rho function in a dynamic fashion. Elevating isoprenylcysteine carboxylmethyltransferase activity in cells has profound effects on MDA-MB-231 cell morphology, implying the presence of a pool of unmethylated prenyl proteins in these cells under normal conditions. Using a knockdown approach, we identified a specific esterase, carboxylesterase 1, whose function had a clear impact not only on the methylation status of RhoA but also RhoA activation and cell morphology. These data provide compelling evidence that C-terminal modification of prenyl proteins, rather than being purely a constitutive process, can serve as a point of regulation of function for this important class of protein.

Dual inhibition of αV integrins and Src kinase activity as a combination therapy strategy for colorectal cancer.

Both Src and αV integrins are important for tumor growth and angiogenesis. They are interconnected and responsible for important features of the tumor phenotype including invasiveness, metastasis, angiogenesis, and resistance to apoptosis. This study examines whether combinational inhibition of both integrin and Src pathways would exert greater antiangiogenesis and antitumor effects than either pathway alone. Using in-vitro cell culture systems, the activity of CNTO95 (Intetumumab), an αV integrin inhibitor, and dasatinib, an Src inhibitor, on proliferation, adhesion, and migration was evaluated in colon cancer cell lines, HCT-116 and RKO, as well as HUVEC cells. The antiangiogenic effect of this combinatory regimen was also tested using an in-vitro tubular network formation assay. The effects of CNTO95 and dasatinib on the activation of Src and integrin pathway signal transduction were also determined by western blotting. The combination of CNTO95 plus dasatinib inhibited adhesion, migration, and paxillin phosphorylation in both HCT-116 and RKO cells. CNTO95 and dasatinib also led to increased apoptosis of HCT-116 cells; however, similar effects were not observed in RKO cells. In addition, dual treatment of CNTO95 and dasatinib exerted enhanced effects on HUVEC cell proliferation, invasion, tubular network formation, and paxillin phosphorylation. In conclusion, our results suggest that concurrent inhibition of both the integrin and the Src pathways exert more pronounced antiangiogenic and antitumor effects than with either pathway being inhibited alone.

G12 signaling through c-Jun NH2-terminal kinase promotes breast cancer cell invasion.

Signaling through the heterotrimeric G protein, G12, via Rho induces a striking increase in breast cancer cell invasion. In this study, evidence is provided that the c-Jun NH(2)-terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively-active Gα12 or activation of G12 signaling by thrombin leads to increased JNK and c-Jun phosphorylation. Pharmacologic inhibition of JNK or knockdown of JNK expression by siRNA significantly decreases G12-induced JNK activation as well as the ability of breast cancer cells to invade a reconstituted basement membrane. Furthermore, expression of dominant-negative Rho or treatment of cells with an inhibitor of the Rho kinase, ROCK, reduces G12-induced JNK and c-Jun activation, and ROCK inhibitor treatment also inhibits G12-induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. Taken together, our data indicate that JNK activation is required for G12-induced invasion of breast cancer cells and that JNK is downstream of Rho and ROCK on this pathway. This study implicates a G12-stimulated mitogen-activated protein kinase cascade in cancer cell invasion, and supports a role for JNK in cancer progression.

Prenylated c17orf37 induces filopodia formation to promote cell migration and metastasis.

Post-translational modification by covalent attachment of isoprenoid lipids (prenylation) regulates the functions and biological activities of several proteins implicated in the oncogenic transformation and metastatic progression of cancer. The largest group of prenylated proteins contains a CAAX motif at the C-terminal that serves as a substrate for a series of post-translational modifications that convert these otherwise hydrophilic proteins to lipidated proteins, thus facilitating membrane association. C17orf37 (chromosome 17 open reading frame 37), also known as C35/Rdx12/MGC14832, located in the 17q12 amplicon, is overexpressed in human cancer, and its expression correlates with the migratory and invasive phenotype of cancer cells. Here we show that C17orf37 contains a functional CAAX motif and is post-translationally modified by protein geranylgeranyltransferase-I (GGTase-I). Geranylgeranylation of C17orf37 at the CAAX motif facilitates association of the protein to the inner leaflet of plasma membrane, enhances migratory phenotype of cells by inducing increased filopodia formation, and potentiates directional migration. A prenylation-deficient mutant of C17orf37 is functionally inactive and fails to trigger dissemination of tail vein-injected cells in a mouse model of metastasis. These findings demonstrate that prenylation is required for the function of the C17orf37 protein in cancer cells and imply that the post-translational modification may functionally regulate metastatic progression of disease.

RHO methylation matters: a role for isoprenylcysteine carboxylmethyltransferase in cell migration and adhesion.

Numerous proteins involved in diverse aspects of cell biology undergo a process of post-translational modification termed prenylation. The prenylation pathway consists of three enzymatic steps, the final of which is methylation of the carboxyl-terminal prenylcysteine formed in the first two steps by the enzyme isoprenylcysteine carboxylmethyltransferase (Icmt). Due to the prevalence of prenylated proteins in cancer biology, and the findings that several of the proteins are involved in processes controlling cell migration and adhesion, we sought to examine the role of Icmt - mediated methylation on cell behavior associated with metastasis. We found that inhibition of methylation reduces migration of the highly metastatic MDA-MB-231 breast cancer cell line. In addition, cell adhesion and cell spreading were also impaired by Icmt inhibition. Further investigation revealed that inhibition of Icmt significantly decreased the activation of both RhoA and Rac1, which are both prenylated proteins. The data obtained were consistent with the decreased activation being due to an increase in Rho GDP-dissociation inhibitor (GDI) binding by both proteins in the absence of their methylation. Importantly, the addition of exogenous RhoA or Rac1 to cells in which Icmt was inhibited was able to partially, but selectively, rescue directed and random migration, respectively. These results establish a role for Icmt-mediated methylation in cell migration, and point to specific prenylated proteins involved in this biology. The prenylation pathway has been targeted for oncogenic therapies, but the role of methylation in cell motility had been largely unexplored until now. The finding that methylation of Rho family members impacts on a specific component of their function provides an additional avenue through which to interrogate the biology of this important class of regulatory proteins.

CaM kinase kinase beta-mediated activation of the growth regulatory kinase AMPK is required for androgen-dependent migration of prostate cancer cells.

While patients with advanced prostate cancer initially respond favorably to androgen ablation therapy, most experience a relapse of the disease within 1-2 years. Although hormone-refractory disease is unresponsive to androgen-deprivation, androgen receptor (AR)-regulated signaling pathways remain active and are necessary for cancer progression. Thus, both AR itself and the processes downstream of the receptor remain viable targets for therapeutic intervention. Microarray analysis of multiple clinical cohorts showed that the serine/threonine kinase Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß) is both highly expressed in the prostate and further elevated in prostate cancers. Using cellular models of prostate cancer, we have determined that androgens (a) directly increase the expression of a CaMKKß splice variant and (b) increase functional CaMKKß protein levels as determined by the phosphorylation of both CaMKI and AMP-activated protein kinase (AMPK), two of CaMKKß's primary substrates. Importantly, inhibition of the CaMKKß-AMPK, but not CaMKI, signaling axis in prostate cancer cells by pharmacological inhibitors or siRNA-mediated knockdown blocks androgen-mediated migration and invasion. Conversely, overexpression of CaMKKß alone leads to both increased AMPK phosphorylation and cell migration. Given the key roles of CaMKKß and AMPK in the biology of prostate cancer cells, we propose that these enzymes are potential therapeutic targets in prostate cancer.

Role of isoprenylcysteine carboxylmethyltransferase-catalyzed methylation in Rho function and migration.

A number of proteins that play key roles in biological regulatory events undergo a process of post-translational modifications termed prenylation. The prenylation pathway consists of three enzymatic steps; the final processed protein is isoprenoid-modified and methylated on the C-terminal cysteine. This protein modification pathway plays a significant role in cancer biology because many oncogenic proteins undergo prenylation. Methylation of the C terminus by isoprenylcysteine carboxylmethyltransferase (Icmt) is the final step in the prenylation pathway. Cysmethynil, a specific Icmt inhibitor discovered in our laboratory, is able to inhibit Ras-mediated signaling, cell growth, and oncogenesis. We sought to examine the role of Icmt-mediated methylation on the behaviors of cancer cells associated with metastatic potential. Our results indicate that inhibition of methylation reduces migration of the highly metastatic MDA-MB-231 breast cancer cell line. In addition, cell adhesion and cell spreading are also significantly impacted by cysmethynil. To examine the mechanism of Icmt-dependent migration we focused on RhoA and Rac1, prenylated proteins that are important mediators of cell migration through their control of the actin cytoskeleton. Inhibition of Icmt significantly decreases the activation of both RhoA and Rac1; an increase in Rho GDP-dissociation inhibitor (RhoGDI) binding in the absence of methylation appears to contribute to this effect. Furthermore, in the absence of Icmt activity the addition of exogenous RhoA or Rac1 is able to partially rescue directed and random migration, respectively. These findings establish a role for Icmt-mediated methylation in cell migration and advance our understanding of the biological consequences of Rho methylation.

Utilizing peptide SPOT arrays to identify protein interactions.

SPOT arrays consist of synthesized peptides 12- to 18-amino acids long, with overlapping sequences that cover the entire sequence of a protein, covalently linked to a solid support. This unit describes how to construct peptide SPOT arrays, biotinylate recombinant proteins, and conduct overlay assays to identify binding interactions. In addition, directions describing how to analyze results to determine single amino acid binding contributions are included. The two techniques in this unit describe how to scan protein sequences to find binding motifs and how to conduct site-directed mutagenesis studies.

Using peptide arrays to define nuclear carrier binding sites on nucleoporins.

In the peptide SPOT array technique, an array of different peptides are synthesized on, and covalently linked to, cellulose membranes. In one usage of this technique, these peptides are screened in an overlay assay to determine which short sequence(s) contains a binding site for an interacting protein. By preparing overlapping peptides that cover the entire sequence of a protein, all of the binding domains on the protein for a second protein can be identified. We have utilized the peptide SPOT array technique to identify the short amino acid sequences within nuclear pore complex proteins (also known as nucleoporins or Nups) that bind the nuclear carrier importin-beta. Crystallization studies by others have indicated that nuclear carriers such as importin-beta bind to phenylalanine-glycine (FG) repeats present in numerous copies in the sequences of a family of nucleoporins. Consistent with this, we found that most (but not all) of the Nup binding sites for importin-beta identified by this technique contain Fx, FG, FxFG, FxFx, or GLFG sequences, although not all such sequences bound importin-beta. Peptide SPOT array substitution studies confirmed a crucial role for the phenylalanine in FG repeats and identified a lysine residue flanking some repeats that is crucial for importin-beta binding to those repeats. In addition to these expected binding sequences for importin-beta, we found multiple instances of a peptide lacking a canonical FG repeat that strongly bound importin-beta, indicating that additional Nup sequences may form binding sites for importin-beta.

The fission yeast Schizosaccharomyces pombe has two importin-alpha proteins, Imp1p and Cut15p, which have common and unique functions in nucleocytoplasmic transport and cell cycle progression.

The nuclear import of classical nuclear localization signal-containing proteins depends on importin-alpha transport receptors. In budding yeast there is a single importin-alpha gene and in higher eukaryotes there are multiple importin-alpha-like genes, but in fission yeast there are two: the previously characterized cut15 and the more recently identified imp1. Like other importin-alpha family members, Imp1p supports nuclear protein import in vitro. In contrast to cut15, imp1 is not essential for viability, but imp1delta mutant cells exhibit a telophase delay and mild temperature-sensitive lethality. Differences in the cellular functions that depend on Imp1p and Cut15p indicate that they each have unique physiological roles. They also have common roles because the imp1delta and the cut15-85 temperature-sensitive mutations are synthetically lethal; overexpression of cut15 partially suppresses the temperature sensitivity, but not the mitotic delay in imp1delta cells; and overexpression of imp1 partially suppresses the mitotic defect in cut15-85 cells but not the loss of viability. Both Imp1p and Cut15p are required for the efficient nuclear import of both an SV40 nuclear localization signal-containing reporter protein and the Pap1p component of the stress response MAP kinase pathway. Imp1p and Cut15p are essential for efficient nuclear protein import in S. pombe.

Computational and biochemical identification of a nuclear pore complex binding site on the nuclear transport carrier NTF2.

Nuclear transport carriers interact with proteins of the nuclear pore complex (NPC) to transport their cargo across the nuclear envelope. One such carrier is nuclear transport factor 2 (NTF2), whose import cargo is the small GTPase Ran. A domain highly homologous to the small NTF2 protein (14kDa) is also found in a number of additional proteins, which together make up the NTF2 domain containing superfamily of proteins. Using structural, computational and biochemical analysis we have identified a functional site that is present throughout this superfamily, and our results indicate that this site functions as an NPC binding site in NTF2. Previously we showed that a D23A mutant of NTF2 exhibits increased affinity for the NPC. The mechanism of this mutation, however, was unknown as this region of NTF2 had not been implicated in binding to NPC proteins. Here we show that the D23A mutation in NTF2 does not result in gross structural changes affecting other known NPC binding sites. Instead, the D23 residue is located in an evolutionarily important region in the NTF2 domain containing superfamily, that in NTF2, is involved in binding to the NPC.

The dynamic association of RCC1 with chromatin is modulated by Ran-dependent nuclear transport.

Regulator of chromosome condensation (RCC1) binding to chromatin is highly dynamic, as determined by fluorescence recovery after photobleaching analysis of GFP-RCC1 in stably transfected tsBN2 cells. Microinjection of wild-type or Q69L Ran markedly slowed the mobility of GFP-RCC1, whereas T24N Ran (defective in nucleotide loading) decreased it further still. We found significant alterations in the mobility of intranuclear GFP-RCC1 after treatment with agents that disrupt different Ran-dependent nuclear export pathways. Leptomycin B, which inhibits Crm1/RanGTP-dependent nuclear export, significantly increased the mobility of RCC1 as did high levels of actinomycin D (to inhibit RNA polymerases I, II, and III) or alpha-amanitin (to inhibit RNA polymerases II and III) as well as energy depletion. Inhibition of just mRNA transcription, however, had no affect on GFP-RCC1 mobility consistent with mRNA export being a Ran-independent process. In permeabilized cells, cytosol and GTP were required for the efficient release of GFP-RCC1 from chromatin. Recombinant Ran would not substitute for cytosol, and high levels of supplemental Ran inhibited the cytosol-stimulated release. Thus, RCC1 release from chromatin in vitro requires a factor(s) distinct from, or in addition to, Ran and seems linked in vivo to the availability of Ran-dependent transport cargo.