Supplementary Figure 1 from Substrate Overlap between Mrp4 and Abcg2/Bcrp Affects Purine Analogue Drug Cytotoxicity and Tissue Distribution

Abstract Novel T cell–based therapies for the treatment of B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), are thought to have strong potential. Progress, however, has been hampered by low efficacy and high toxicity. Tumor targeting by Vγ9Vδ2 T cells, a conserved T-cell subset with potent intrinsic antitumor properties, mediated by a bispecific antibody represents a novel approach promising high efficacy with limited toxicity. Here, we describe the generation of a bispecific Vγ9Vδ2 T-cell engager directed against CD40, which, due to its overexpression and biological footprint in malignant B cells, represents an attractive target. The CD40-targeting moiety of the bispecific antibody was selected because it can prevent CD40L-induced prosurvival signaling and reduce CD40-mediated resistance of CLL cells to venetoclax. Selective activation of Vγ9Vδ2 T cells in the presence of CD40+ tumor cells induced potent Vγ9Vδ2 T-cell degranulation, cytotoxicity against CLL and MM cells in vitro, and in vivo control of MM in a xenograft model. The CD40-bispecific γδ T-cell engager demonstrated lysis of leukemic cells by autologous Vγ9Vδ2 T cells present in patient-derived samples. Taken together, our CD40 bispecific γδ T-cell engager increased the sensitivity of leukemic cells to apoptosis and induced a potent Vγ9Vδ2 T cell–dependent antileukemic response. It may, therefore, represent a potential candidate for the development of novel treatments for B-cell malignancies.

In autoimmune diseases like rheumatoid arthritis (RA), multidrug resistance (MDR) transporters of the ATP-binding cassette (ABC) transporter superfamily harbor dual functions by extruding pro-inflammatory mediators and exporting disease modifying anti-rheumatics drugs (DMARDs), hence contributing to diminished treatment response. Herein we determined the expression (mRNA/protein) and functional efflux activities of multiple selected ABC transporters in immune-effector cells of RA patients in relation to DMARD response. ABC transporter profiling included ABCB1 (P-glycoprotein), ABCC1-6/ABCC10-12 (multidrug resistance proteins 1-9) and ABCG2 (Breast Cancer Resistance Protein). Analyses were performed in peripheral blood lymphocytes (PBL) and monocyte-derived macrophages (MDM) obtained from 52 RA patients (DMARD-naive and DMARD (non)-responders) and HC (n = 19) using PCR, immunohistochemistry and flow cytometry. Notwithstanding the large inter-patient variabilities, PBLs from RA patients displayed significantly higher mRNA levels of ABCC1 (2.1-fold), ABCC4 (1.6-fold) and ABCC10 (1.9-fold) compared with HC. Expression levels of ABCB1, ABCC1, ABCC4 and ABCC10 were significantly and positively correlated with each other. Furthermore, significantly increased ABCG2 mRNA (2.8-fold) and protein levels (2.4-fold) were observed in MDM from RA patients compared to HC. Additional analyses revealed that a 1.8-fold increased functional activity of ABCB1 in CD3+ cells in RA patients receiving DMARD treatment versus DMARD-naive patients, was exclusively contributed by DMARD non-responders. Although up to 1.7-fold higher levels of MDR mRNA levels were noted in PBL of DMARD non-responders over DMARD responders, these differences were not statistically significant. Together, these results underscore the involvement of multiple ABC transporters in immune-competent cells in relation to RA and DMARD response.

Background The hexapeptide 4A6 (Ac-Thr(tBu)-His(Bzl)-Thr(Bzl)-Nle-Glu(OtBu)-Gly-Bza) was isolated from a peptide library constructed to identify peptide-based transport inhibitors of multidrug resistance (MDR) efflux pumps including P-glycoprotein and Multidrug Resistance-associated Protein 1. 4A6 proved to be a substrate but not an inhibitor of these MDR efflux transporters. In fact, 4A6 and related peptides displayed potent cytotoxic activity via an unknown mechanism. Objective To decipher the mode of cytotoxic activity of 4A6. Methods Screening of 4A6 activity was performed against the NCI60 panel of cancer cell lines. Possible interactions of 4A6 with the 26S proteasome were assessed via proteasome activity and affinity labeling, and cell growth inhibition studies with leukemic cells resistant to the proteasome inhibitor bortezomib (BTZ). Results The NCI60 panel COMPARE analysis revealed that 4A6 had an activity profile overlapping with BTZ. Consistently, 4A6 proved to be a selective and reversible inhibitor of β5 subunit (PSMB5)-associated chymotrypsin-like activity of the 26S proteasome. This conclusion is supported by several lines of evidence: (i) inhibition of chymotrypsin-like proteasome activity by 4A6 and related peptides correlated with their cell growth inhibition potencies; (ii) 4A6 reversibly inhibited functional β5 active site labeling with the affinity probe BodipyFL-Ahx3L3VS; and (iii) human myeloid THP1 cells with acquired BTZ resistance due to mutated PSMB5 were highly (up to 287-fold) cross-resistant to 4A6 and its related peptides. Conclusion 4A6 is a novel specific inhibitor of the β5 subunit-associated chymotrypsin-like proteasome activity. Further exploration of 4A6 as a lead compound for development as a novel proteasome-targeted drug is warranted.

Background: The fecal immunochemical test (FIT) for detecting hemoglobin is used widely for noninvasive colorectal cancer (CRC) screening, but its sensitivity leaves room for improvement. Objective: To identify novel protein biomarkers in stool that outperform or complement hemoglobin in detecting CRC and advanced adenomas. Design: Case-control study. Setting: Colonoscopy-controlled referral population from several centers. Participants: 315 stool samples from one series of 12 patients with CRC and 10 persons without colorectal neoplasia (control samples) and a second series of 81 patients with CRC, 40 with advanced adenomas, and 43 with nonadvanced adenomas, as well as 129 persons without colorectal neoplasia (control samples); 72 FIT samples from a third independent series of 14 patients with CRC, 16 with advanced adenomas, and 18 with nonadvanced adenomas, as well as 24 persons without colorectal neoplasia (control samples). Measurements: Stool samples were analyzed by mass spectrometry. Classification and regression tree (CART) analysis and logistic regression analyses were performed to identify protein combinations that differentiated CRC or advanced adenoma from control samples. Antibody-based assays for 4 selected proteins were done on FIT samples. Results: In total, 834 human proteins were identified, 29 of which were statistically significantly enriched in CRC versus con-trol stool samples in both series. Combinations of 4 proteins reached sensitivities of 80% and 45% for detecting CRC and advanced adenomas, respectively, at 95% specificity, which was higher than that of hemoglobin alone (P < 0.001 and P = 0.003, respectively). Selected proteins could be measured in small sample volumes used in FIT-based screening programs and discriminated between CRC and control samples (P < 0.001). Limitation: Lack of availability of antibodies prohibited validation of the top protein combinations in FIT samples. Conclusion: Mass spectrometry of stool samples identified novel candidate protein biomarkers for CRC screening. Several protein combinations outperformed hemoglobin in discriminating CRC or advanced adenoma from control samples. Proof of concept that such proteins can be detected with antibody-based assays in small sample volumes indicates the potential of these biomarkers to be applied in population screening.

Complex interactions between DNA herpesviruses and host factors determine the establishment of a life-long asymptomatic latent infection. The lymphotropic Epstein-Barr virus (EBV) seems to avoid recognition by innate sensors despite massive transcription of immunostimulatory small RNAs (EBV-EBERs). Here we demonstrate that in latently infected B cells, EBER1 transcripts interact with the lupus antigen (La) ribonucleoprotein, avoiding cytoplasmic RNA sensors. However, in coculture experiments we observed that latent-infected cells trigger antiviral immunity in dendritic cells (DCs) through selective release and transfer of RNA via exosomes. In ex vivo tonsillar cultures, we observed that EBER1-loaded exosomes are preferentially captured and internalized by human plasmacytoid DCs (pDCs) that express the TIM1 phosphatidylserine receptor, a known viral- and exosomal target. Using an EBER-deficient EBV strain, enzymatic removal of 5'ppp, in vitro transcripts, and coculture experiments, we established that 5'pppEBER1 transfer via exosomes drives antiviral immunity in nonpermissive DCs. Lupus erythematosus patients suffer from elevated EBV load and activated antiviral immunity, in particular in skin lesions that are infiltrated with pDCs. We detected high levels of EBER1 RNA in such skin lesions, as well as EBV-microRNAs, but no intact EBV-DNA, linking non-cell-autonomous EBER1 presence with skin inflammation in predisposed individuals. Collectively, our studies indicate that virus-modified exosomes have a physiological role in the host-pathogen stand-off and may promote inflammatory disease.

Abstract Background The fecal immunochemical test (FIT) is used in many countries for non-invasive screening for colorectal cancer (CRC), but its characteristics leave room for improvement. We aimed to identify novel stool-based protein markers in stool that outperform or complement hemoglobin in detecting CRC. Materials and methods A series of large scale proteomic discovery and validation was conducted on a total of 313 human stool specimens. A discovery set consisted of 22 stool specimens (12 CRC and 10 controls), and isolated proteins were analyzed by an in-dept mass spectrometry GeLC-MS/MS workflow. An external validation set consisted of 291 independent stool specimens (79 CRC, 40 advanced adenoma (AA), 43 nonadvanced adenoma (A) and 129 controls), and isolated proteins were analyzed by single-shot mass spectrometry (Q-Exactive). As proof of concept, commercially available meso scale discovery (MSD) assays were performed on an independent validation set of 72 FIT fluid samples set including 14 CRC, 16 AA, 18 A and 24 controls. Results Of the 468 human proteins quantified in the discovery set, 93 were significantly enriched in CRC vs controls (p<0.05). Of these, 29 were significantly found enriched in CRC vs controls in the validation set (q<0.05). CART analysis and exhaustive search revealed a combination of 4 proteins as the most optimal combination to differentiate CRC from controls. A logistic regression model of this combination of proteins detected CRC with a sensitivity of 73% as compared to 43% for hemoglobin (HBA1) alone at a fixed specificity of 95% (p = 0.00003). Another combination of 4 proteins was the most optimal combination to differentiate AA from controls, which showed a sensitivity of 48% vs 8% for hemoglobin (p = 0.0002) at fixed specificity of 95%. Evaluation of one of the markers on FIT fluid samples provided proof of concept that these proteins can be detected in FIT fluid and can significantly discriminate CRC from healthy controls (p < 0.001). Conclusion Proteome profiling on stool samples revealed 29 validated proteins significantly enriched in CRC samples compared to controls. A panel of 4 complementary protein markers outperformed hemoglobin for detection of CRC as well as AA. Proof of concept for detecting the proteins in FIT fluids confirmed the high potential of these markers for screening purposes in a non-invasive and cost-effective manner. Citation Format: Linda J. Bosch, Meike De Wit, Annemieke C. Hiemstra, Sander R. Piersma, Thang V. Pham, Gideon Oudgenoeg, Veerle Coupe, George Scheffer, Sandra Mongera, Jochim Terhaar Sive droste, Frank Oort, Sietze van Turenhout, Ilhame Ben Larbi, Chris Mulder, Beatriz Carvalho, Remond JA Fijneman, Connie R. Jimenez, Gerrit A. Meijer. Improved colorectal cancer screening by new stool-based protein markers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3176.

Aminopeptidase inhibitors are receiving attention as combination chemotherapeutic agents for the treatment of refractory acute myeloid leukemia. However, the factors determining therapeutic efficacy remain elusive. Here we identified the molecular basis of acquired resistance to CHR2863, an orally available hydrophobic aminopeptidase inhibitor prodrug with an esterase-sensitive motif, in myeloid leukemia cells. CHR2863 enters cells by diffusion and is retained therein upon esterase activity-mediated conversion to its hydrophilic active metabolite drug CHR6768, thereby exerting amino acid depletion. Carboxylesterases (CES) serve as candidate prodrug activating enzymes given CES1 expression in acute myeloid leukemia specimens. We established two novel myeloid leukemia sublines U937/CHR2863(200) and U937/CHR2863(5uM), with low (14-fold) and high level (270-fold) CHR2863 resistance. The latter drug resistant cells displayed: (i) complete loss of CES1-mediated drug activation associated with down-regulation of CES1 mRNA and protein, (ii) marked retention/sequestration of the prodrug, (iii) a substantial increase in intracellular lipid droplets, and (iv) a dominant activation of the pro-survival Akt/mTOR pathway. Remarkably, the latter feature coincided with a gain of sensitivity to the mTOR inhibitor rapamycin. These finding delineate the molecular basis of CHR2863 resistance and offer a novel modality to overcome this drug resistance in myeloid leukemia cells.

Abstract Introduction and objectives: Early detection of colorectal cancer (CRC) and its precursor lesions is an effective approach to reduce CRC mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test that detects small traces of the blood protein hemoglobin. Although beneficial in its current form, the FIT test characteristics leave room for improvement. The aim of the present study was to identify and validate novel protein biomarkers in stool that complement or outperform the current hemoglobin-based test, to improve its diagnostic accuracy. Methods: Proteins isolated from stool from 10 subjects without any signs of colorectal neoplasia (controls) at colonoscopy and from 12 CRC patients were analyzed by GeLC-MS/MS (discovery set). Data were analyzed by comparing protein abundancies, measured as spectral counts. Analysis of differential proteins was performed using the beta-binomial test. Findings were validated by mass spectrometry (Q-Exactive) in an independent series of 292 stool samples obtained from control subjects (n = 109) and subjects with adenomas (n = 55), advanced adenomas (n = 53), or CRCs (n = 75). Results and Discussion: In total 830 human proteins were identified in the discovery set, of which 134 were significantly enriched in CRC. These included 78 proteins that were significantly more enriched in FIT-negative CRC stool samples compared to controls. Preliminary analysis of the validation set indicates that more than half of these markers are significantly more abundant in CRC samples compared to controls. Conclusion: Proteome profiling of stool samples revealed novel candidate biomarkers to improve current CRC screening tests. More data analysis is currently ongoing to select most promising protein biomarkers for clinical assay development. Citation Format: Linda JW Bosch, Meike de Wit, Annemieke C. Hiemstra, Sander Piersma, Thang Pham, Gideon Oudgenoeg, George Scheffer, Sandra Mongera, Malgorzata Komor, Jochim Terhaar Sive Droste, Frank A. Oort, Sietze van Turenhout, Ilhame Ben Larbi, Chris JJ Mulder, Beatriz Carvalho, Remond JA Fijneman, Connie Jimenez, Gerrit A. Meijer. Stool proteomics reveals novel candidate biomarkers for colorectal cancer screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1563. doi:10.1158/1538-7445.AM2015-1563

Several lines of evidence predict that specific pathways must exist in metazoans for the escorted movement of heme, an essential but cytotoxic iron-containing organic ring, within and between cells and tissues, but these pathways remain obscure. In Caenorhabditis elegans, embryonic development is inextricably dependent on both maternally derived heme and environmentally acquired heme. Here, we show that the multidrug resistance protein MRP-5/ABCC5 likely acts as a heme exporter, and targeted depletion of mrp-5 in the intestine causes embryonic lethality. Transient knockdown of mrp5 in zebrafish leads to morphological defects and failure to hemoglobinize red blood cells. MRP5 resides on the plasma membrane and endosomal compartments and regulates export of cytosolic heme. Together, our genetic studies in worms, yeast, zebrafish, and mammalian cells identify a conserved, physiological role for a multidrug resistance protein in regulating systemic heme homeostasis. We envision other MRP family members may play similar unanticipated physiological roles in animal development.

We aimed to determine whether the multidrug-resistance-proteins MRP4 (ABCC4) and MRP5 (ABCC5) confer resistance to the antimetabolites cytarabine (Ara-C), gemcitabine (GEM), and the L-nucleoside analog troxacitabine. For this purpose we used HEK293 and the transfected HEK/MRP4 (59-fold increased MRP4) or HEK/MRP5i (991-fold increased MRP5) as model systems and tested the cells for drug sensitivity using a proliferation test. Drug accumulation was performed by using radioactive Ara-C, and for GEM and troxacitabine with HPLC with tandem-MS or UV detection. At 4-hr exposure HEK/MRP4 cells were 2-4-fold resistant to troxacitabine, ara-C and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and HEK/MRP5i to ara-C and PMEA, but none to GEM. The inhibitors probenecid and indomethacin reversed resistance. After 4-hr exposure ara-C-nucleotides were 2-3-fold lower in MRP4/5 cells, in which they decreased more rapidly after washing with drug-free medium (DFM). Trocacitabine accumulation was similar in the 3 cell lines, but after the DFM period troxacitabine decreased 2-4-fold faster in MRP4/5 cells. Troxacitabine-nucleotides were about 25% lower in MRP4/5 cells and decreased rapidly in MRP4, but not in MRP5 cells. Accumulation of GEM-nucleotides was higher in the MRP4/5 cells. In conclusion: MRP4 and MRP5 overexpression confer resistance to troxacitabine and ara-C, but not to GEM, which was associated with a rapid decline of the ara-C and troxacitabine-nucleotides in HEK/MRP4-5 cells.

Epstein-Barr virus (EBV) BARF1 (BamHI-A rightward frame 1) protein is a viral oncogen and immunomodulator. BARF1 mRNA is highly and selectively expressed in nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC), however, evidence for presence of BARF1 protein in situ is lacking. In vitro, the hexameric form of BARF1 is rapidly secreted (sBARF1). Secretion of BARF1 from NPC tumor cells may result in the presence of sBARF1 in sera of NPC patients. This study describes the development of new monoclonal and polyclonal antibodies that strongly and selectively bind to ‘native’ NPC-derived sBARF1 protein in its natural hexameric conformation. Using these antibodies, a capture enzyme linked immunosorbent assay (ELISA) was developed to detect sBARF1 protein in sera of NPC patients. Although a detection limit of 10 ng/ml sBARF1 in serum was reached for the ELISA, only 3 of 71 NPC patients at diagnosis showed elevated levels of sBARF1 protein. Possibly sBARF1 is complexed with serum proteins shielding antibody recognition sites. However, sample denaturation prior to ELISA testing did not affect the levels observed. A MALDI-MS/MS approach was developed to overcome this problem, yielding a specific BARF1 identifier peptide. However, the sensitivity of the current setup was less than BARF1-capture ELISA. The BARF1 identifier peptide can be used to further develop a quantitative targeted proteomics approach. Using immunohistochemical staining with various anti-BARF1 antibody reagents, BARF1 could be detected in paraffin-embedded BARF1 expressing cell lines revealing a cytoplasmic localisation. Unfortunately, in patient material, no BARF1 protein expression could be demonstrated. In conclusion, the sensitive sBARF1 capture ELISA (10 ng/mL serum) demonstrated only low levels of sBARF1 in NPC patient sera, conflicting with previously published data. A higher sensitivity of both ELISA and proteomic approach is needed for a diagnostic tool. sBARF1 remains a potential serum biomarker for NPC and GC and its diagnostic value using a targeted proteomics approach should be further evaluated. Chapter 8 EBV BARF1 detection

While multidrug resistance (MDR) in cancer is well established, little is known about the cellular pathways regulating the expression and trafficking of the MDR efflux transporter like BCRP (ABCG2). Here we evaluated the role of signalling downstream of EGFR on BCRP expression and sub-cellular localization using lung cancer cells harboring BCRP but expressing various EGFR and Ras activating mutations; A549 (K-Ras-G12S), H292 wild-type EGFR and Ras, and H1650 (EGFR-DelE747-A750). Immunocytochemistry and immunofluorescence studies demonstrated that BCRP was predominantly intracellular but its expression was found also on the plasma membrane in A549 and H1650 cells with activated Ras and EGFR. Remarkably, EGFR inhibition by erlotinib at IC₅₀ concentrations induced a differential timedependent alteration in BCRP gene and protein expression. In H1650 cells, erlotinib enhanced both the total and plasma membrane degradation of BCRP by ubiquitination within 6-24 hours, whereas BCRP expression regained the original basal levels after 48 hours. In erlotinib treated H292 cells, BCRP levels decreased at 24 hours until 72 hours, whereas in A549 cells erlotinib initially reduced BCRP expression but then induced its accumulation on the plasma membrane at 72 hours. We further found that the PI3K/Akt inhibitor LY294002 down-regulated BCRP expression, hence showing that the Akt pathway is involved in the regulation of BCRP expression but not in its localization in these lung cancer cell lines. Finally, the selective BCRP transport inhibitor Ko143 did not increase erlotinib sensitivity, but did decrease the transport activity of BCRP in A549 and H1650 cells as it induced the accumulation of its transport substrate topotecan. In conclusion, our results suggest that the EGFR and Akt pathways are involved in regulation of BCRP expression, trafficking and drug transport activity. These findings warrant future studies on the pharmacologic modulation of these pathways to enhance the efficacy of anticancer combinations of erlotinib with drugs that are BCRP transport substrates.

Abstract Background In clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence. Methods Here we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC). Results Characterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a) 10–12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive β5 proteasome subunit) introducing an amino acid substitution (Met45Ile) in the BTZ-binding pocket, (b) a significant 2–4 fold increase in the mRNA and protein levels of the constitutive β5 proteasome subunit along with unaltered immunoproteasome expression, (c) full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent) the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC. Conclusions These results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease.

Submitted 5 Inactivating PSMB5 mutations and P-glyco-protein (MDR1/ ABCB1) mediate resistance to proteasome inhibitors: ex vivo efficacy of (immuno) proteasome inhibitors in mononuclear blood cells from rheumatoid arthritis patients 106 Abstract Bortezomib (BTZ), a registered proteasome inhibitor (PI) for multiple myeloma, has also been proposed as a potential anti-rheumatic agent. Its reported side effects however, make it unappealing for long-term administration and resistance may also develop. To overcome this, second generation PIs became available. Here, we investigated whether a novel class of peptide epoxyketone-based PIs including carfilzomib, ONX 0912 and ONX 0914, might escape two established BTZ-resistance mechanisms: (a) mutation(s) in the proteasome β5 subunit (PSMB5) targeted by these PIs, and (b) drug efflux mediated by ABC transporters. THP1 myeloid sublines with acquired resistance to BTZ (54-235 fold) due to mutations in PSMB5, displayed marked cross-resistance but less pronounced to carfilzomib (9-32 fold), ONX 0912 (39-62 fold) and ONX 0914 (27-97 fold). As for ABC transporter mediated efflux: lymphoid CEM/VLB cells with P-glycoprotein (Pgp/MDR1) overexpression exhibited substantial resistance to carfilzomib (114-fold), ONX 0912 (23-fold), ONX 0914 (162-fold) whereas less resistance to BTZ (4.5-fold) was observed. Consistently, the β5 subunit-associated chymo-trypsin-like proteasome activity was significantly less inhibited in these CEM/ VLB cells. Ex vivo analysis of peripheral blood mononuclear cells (PBMCs) from therapy naive rheumatoid arthritis patients revealed that, although basal Pgp levels were low, it compromised the inhibitory effect of carfilzomib and ONX 0914. However, the use of P121, a Pgp transport inhibitor, restored parental cell inhibitory levels both in CEM/VLB cells as well as in PBMCs. These results indicate that the pharmacologic activity of these PIs may be hindered by drug resistance mechanisms involving PSMB5 mutations and PI extrusion via Pgp.

We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics.

Adipose tissue-derived stem cells (ASCs) are promising candidates for regenerative therapy, like after myocardial infarction. However, when transplanted into the infarcted heart, ASCs are jeopardized by the ischemic environment. Interestingly, it has been shown that multidrug resistance (MDR) proteins like the breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) have a protective effect in haematopoietic stem cells. In ASC, however, only expression of BCRP was shown until now. In this study, we therefore analysed the expression and functional activity of BCRP and P-gp and their putative function in ischemia in ASC. BCRP and P-gp protein expression was studied over time (passages 2–6) using western blot analysis and immunohistochemical staining. MDR activity was analysed using protein-specific substrate extrusion assays. Ischemia was induced using metabolic inhibition. All analyses demonstrated protein expression and activity of BCRP in ASCs. In contrast, only minor expression of P-gp was found, without functional activity. BCRP expression was most prominent in early passage ASCs (p2) and decreased during culture. Finally, ischemia induced expression of BCRP. In addition, when BCRP was blocked, a significant increase in dead ASCs was found already after 1 h of ischemia. In conclusion, ASCs expressed BCRP, especially in early passages. In addition, we now show for the first time that BCRP protects ASCs against ischemia-induced cell death. These data therefore indicate that for transplantation of ASCs in an ischemic environment, like myocardial infarction, the optimal stem cell protective effect of BCRP theoretically will be achieved with early culture passages ASCs.

INTRODUCTION:Bone is the most common site of breast cancer metastasis, and complications associated with bone metastases can lead to a significantly decreased patient quality of life. Thus, it is essential to gain a better understanding of the molecular mechanisms that underlie the emergence and growth of breast cancer skeletal metastases.METHODS:To search for novel molecular mediators that influence breast cancer bone metastasis, we generated gene-expression profiles from laser-capture microdissected trephine biopsies of both breast cancer bone metastases and independent primary breast tumors that metastasized to bone. Bioinformatics analysis identified genes that are differentially expressed in breast cancer bone metastases compared with primary, bone-metastatic breast tumors.RESULTS:ABCC5, an ATP-dependent transporter, was found to be overexpressed in breast cancer osseous metastases relative to primary breast tumors. In addition, ABCC5 was significantly upregulated in human and mouse breast cancer cell lines with high bone-metastatic potential. Stable knockdown of ABCC5 substantially reduced bone metastatic burden and osteolytic bone destruction in mice. The decrease in osteolysis was further associated with diminished osteoclast numbers in vivo. Finally, conditioned media from breast cancer cells with reduced ABCC5 expression failed to induce in vitro osteoclastogenesis to the same extent as conditioned media from breast cancer cells expressing ABCC5.CONCLUSIONS:Our data suggest that ABCC5 functions as a mediator of breast cancer skeletal metastasis. ABCC5 expression in breast cancer cells is important for efficient osteoclast-mediated bone resorption. Hence, ABCC5 may be a potential therapeutic target for breast cancer bone metastasis.