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PDF file - 106K, Immunoblot analysis of the indicated proteins in U-266 and LP-1 pre-treated with 10 muM CQ, 10 muM zVAD.fmk or 4 muM MG132 (for 4h) followed by 10 mu M Sor for 24h

PDF file - 26K, The indicated myeloma cell lines were treated with 10 muM Sor for 24h and cell cycle distribution was analysed by NucleoCounter NC-3000 (Chemometec).

PDF file - 36K, A, Quantitative analysis of Annexin V/PI positive murine 5T33MMvitro cells treated the indicated concentrations of sorafenib for 24h and 48h; B, Immunoblot analysis of the indicated proteins from the 5T33MMvitro cell line treated with 10 muM Sor for the indicated time points. C, Immunoblot analysis of the indicated proteins from murine 5T33MMvivo treated ex-vivo with 10 muM Sor for 24h

We present the generation and validation of an updated version of the TanDEM-X digital elevation model (DEM) of Antarctica: the TanDEM-X PolarDEM 90 m of Antarctica. Improvements compared to the global TanDEM-X DEM version comprise filling gaps with newer bistatic synthetic aperture radar (SAR) acquisitions of the TerraSAR-X and TanDEM-X satellites, interpolation of smaller voids, smoothing of noisy areas, and replacement of frozen or open sea areas with geoid undulations. For the latter, a new semi-automatic editing approach allowed for the delineation of the coastline from DEM and amplitude data. Finally, the DEM was transformed into the cartographic Antarctic Polar Stereographic projection with a homogeneous metric spacing in northing and easting of 90 m. As X-band SAR penetrates the snow and ice pack by several meters, a new concept for absolute height adjustment was set up that relies on areas with stable penetration conditions and on ICESat (Ice, Cloud, and land Elevation Satellite) elevations. After DEM generation and editing, a sophisticated height error characterization of the whole Antarctic continent with ICESat data was carried out, and a validation over blue ice achieved a mean vertical height error of just −0.3 m ± 2.5 m standard deviation. The filled and edited Antarctic TanDEM-X PolarDEM 90 m is outstanding due to its accuracy, homogeneity, and coverage completeness. It is freely available for scientific purposes and provides a high-resolution data set as basis for polar research, such as ice velocity, mass balance estimation, or orthorectification.

Lenalidomide (Len) plus dexamethasone (Dex) is approved for the treatment of relapsed or refractory multiple myeloma (RRMM). It is possible that single‐agent Len may be effective as prolonged treatment regimen in RRMM once patients demonstrate an initial response to Len+Dex induction. Patients with RRMM who responded to first‐line Len+Dex in an observational study (NCT01430546) received up to 24 cycles of either Len (25 mg/day) or Len+Dex (25 mg/day and 40 mg/week) as prolonged treatment in a subsequent phase 2 clinical trial (NCT01450215). In the observational study (N = 133), median time to response was 1.7 (range 0.6–9.6) months. A complete response to all treatments received in both studies was observed in 11% of patients; very good partial response and partial response rates were 31% and 38%, respectively. Corresponding response rates in the subgroup of patients who did not enter the phase 2 trial (n = 71) were 3%, 18%, and 39%, respectively. Rates of disease progression at 2 years in the phase 2 trial were 47% versus 31% for Len versus Len+Dex (P = 0.14). After 36 months median follow‐up in surviving patients, median time to progression was not reached with Len+Dex and was 24.9 months (95% confidence interval 12.5–not calculable, P < 0.001) with Len. Three‐year OS among the total observational study population was 61% (95% CI, 52–69%). The corresponding rate among patients who entered the phase 2 clinical trial was 73% (95% CI, 60–83%) and was significantly lower among those patients who achieved ≥PR but did not proceed into the phase 2 trial (55%; P = 0.01). In the phase 2 trial, OS was 73% in both treatment arms (P = 0.70). Neutropenia and thrombocytopenia were more common with prolonged (phase 2 trial) versus short‐term (observational study) Len administration but remained manageable. Prolonged treatment with Len with or without Dex provides sustained, clinically relevant responses and demonstrates an acceptable safety profile. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Purpose Despite therapeutic advances, patients with multiple myeloma (MM) continue to experience disease relapse and treatment resistance. The gene ABCB1 encodes the drug transporter P-glycoprotein, which confers resistance through drug extrusion across the cell membrane. Lenalidomide (Len) is excreted mainly via the kidneys, and, given the expression of P-gp in the renal tubuli, single-nucleotide polymorphisms (SNPs) in the ABCB1 gene may influence Len plasma concentrations and, subsequently, the outcome of treatment. We, therefore, investigated the influence of ABCB1 genetic variants on Len treatment outcomes and adverse events (AEs). Methods Ninety patients with relapsed or refractory MM, who received the second-line Len plus dexamethasone in the Rev II trial, were genotyped for the ABCB1 SNPs 1199G amp;gt; A (Ser400Asn, rs2229109), 1236C amp;gt; T (silent, rs1128503), 2677G amp;gt; T/A (Ala893Ser, rs2032582), and 3435C amp;gt; T (silent, rs1045642) using pyrosequencing, and correlations to response parameters, outcomes, and AEs were investigated. Results No significant associations were found between genotype and either best response rates or hematological AEs, and 1236C amp;gt; T, 2677G amp;gt; T or 3435C amp;gt; T genotypes had no impact on survival. There was a trend towards increased time to progression (TTP) in patients carrying the 1199A variant, and a significant difference in TTP between genotypes in patients with standard-risk cytogenetics. Conclusions Our findings show a limited influence of ABCB1 genotype on lenalidomide treatment efficacy and safety. The results suggest that 1199G amp;gt; A may be a marker of TTP following Len treatment in standard-risk patients; however, larger studies are needed to validate and clarify the relationship. Funding Agencies|Swedish Cancer Society; Swedish Research Council; AFA Insurance; Linkoping University; ALF Grants, Region Ostergotland; Celgene Corporation

Treatment with Daratumumab (Dara), a monoclonal anti-CD38 antibody of IgG1 subtype, is effective in patients with multiple myeloma (MM). However, Dara also impairs the cellular immunity, which in turn may lead to higher susceptibility to infections. The exact link between immune impairment and infectious complications is unclear. In this study, we report that nine out of 23 patients (39%) with progressive MM had infectious complications after Dara treatment. Five of these patients had viral infections, two developed with bacterial infections and two with both bacterial and viral infections. Two of the viral infections were exogenous, i.e. acute respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), while five consisted of reactivations, i.e. one herpes simplex (HSV), 1 varicella-zoster (VZV) and three cytomegalovirus (CMV). Infections were solely seen in patients with partial response or worse. Assessment of circulating lymphocytes indicated a selective depletion of NK cells and viral reactivation after Dara treatment, however this finding does not exclude the multiple components of viral immune-surveillance that may get disabled during this monoclonal treatment in this patient cohort. These results suggest that the use of antiviral and antibacterial prophylaxis and screening of the patients should be considered.

Background: Novel efficacious treatments with long-term tolerability are needed for transplant-ineligible, newly diagnosed multiple myeloma (NDMM) patients. This phase 2 study evaluated the safety and efficacy of all-oral ixazomib-cyclophosphamide-dexamethasone (ICd) followed by single-agent ixazomib maintenance. Patients and methods: Patients were randomised (1:1) to receive 4.0 mg of ixazomib, 300 (Arm A) or 400 (Arm B) mg/m2 of cyclophosphamide (days 1, 8, and 15), and 40 mg of dexamethasone (days 1, 8, 15, and 22) as induction (up to 13 × 28-day cycles), followed by single-agent ixazomib maintenance (28-day cycles) until progressive disease, death, or unacceptable toxicity. Primary end-point was complete response (CR) + very good partial response (VGPR) rate for ICd induction. Results: Seventy patients were enrolled (n = 36 Arm A; n = 34 Arm B); median age was 73 years (range, 61–87). At data cut-off, 66% of patients had completed 13 induction cycles followed by ixazomib maintenance. Median overall treatment duration was 19 cycles (range, 1–29); 21% of patients discontinued treatment during induction and 3% during maintenance due to adverse events (AEs). During induction, among 67 response-evaluable patients, CR+VGPR rate was 25%, and overall response rate (ORR) was 73%. Including the maintenance phase, CR+VGPR rate was 33%, and ORR was 76%. Median progression-free survival was 23.5 months (median follow-up: 26.1 months). The most common all-grade AE was neutropenia (31%). Grade ≥3 AEs were reported by 73% of patients. Five on-study deaths occurred (not treatment-related). Conclusions: ICd treatment followed by ixazomib maintenance is tolerable and active in elderly, transplant-ineligible NDMM patients. Trial registration number: NCT02046070.

Since 2010, TanDEM-X and its twin satellite TerraSAR-X fly in a close orbit formation and form a single-pass synthetic aperture radar (SAR) interferometer. The formation was established to acquire a global high-precision digital elevation model (DEM) using SAR interferometry (InSAR). In order to achieve the required height accuracy of the TanDEM-X DEM, at least two global coverages have to be acquired. However, in difficult and mountainous terrain, up to five coverages are present. Here, acquisitions from ascending and descending orbits are needed to fill gaps and to overcome geometric limitations. Therefore, a strategy to properly combine the available height estimates is mandatory. The objective of this paper is the presentation of the operational TanDEM-X DEM mosaicking approach. In general, multiple InSAR DEM heights are combined by means of a weighted average with the height error as weight. Apart from this widely used mosaicking approach, one big challenge remains with the handling of larger height discrepancies between the input data, which are mainly caused by phase unwrapping errors, but also by temporal changes between acquisitions. In the case of inconsistencies, the TanDEM-X mosaicking approach performs a grouping into height levels. A priority concept is set up to evaluate the different groups of heights considering the number of DEMs and several InSAR quality parameters: the height error, the phase unwrapping method, and the height of ambiguity. This allows the identification of the most reliable height level for mosaicking. This fusion concept is verified on different test areas affected by phase unwrapping errors in flat and mountainous terrain as well as by height discrepancies in forests. The results show that the quality of the final TanDEM-X DEM mosaic benefits a lot from this mosaicking approach.

Multiple Myeloma patients refractory to both proteasome inhibitors and immuno-modulatory drugs have poor prognoses, with median overall survival estimates of 9. We present data from two patients with MM in disease progression on ongoing treatment; both had received Daratumumab (Dara) as monotherapy earlier and were re-challenged with Dara on relapse. Bone Marrow and blood samples were collected before start of treatment and the later before administration of each dose. Detailed analyses of circulating cell populations were performed. We demonstrate that Dara had durable single-agent activity on re-treatment in both patients while both patients had no effective treatment options. As expected, we show that following initial Dara treatment, MM cells at relapse retained high levels of CD38 expression. Dara treatment depletes circulating NK cells almost immediately after the start of the treatment. Furthermore, both NK cells and CD8+ populations were partially increased at treatment interruption. Anti-inflammatory myelomonocytic cells showed a sharp decrease at the same time. These data strongly suggest that continuous treatment with Dara decreases ADCC mediated antitumor activity using two independent mechanisms, which can both be recovered by a short treatment interruption. In line with this, maintenance treatment should include an intermittent treatment regime with appropriate time for recovery. Figure 1. Figure 1. Disclosures Off Label Use: In this study Daratumumab was used in a compassionate use program provided by Janssen.. Ahmadi:Janssen: Employment. Khan:Janssen: Employment. Nahi:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

In this paper we present for the first time the new digital elevation model (DEM) for Greenland produced by the TanDEM-X (TerraSAR add-on for digital elevation measurement) mission. The new, full coverage DEM of Greenland has a resolution of 0.4 arc seconds corresponding to 12 m. It is composed of more than 7.000 interferometric synthetic aperture radar (InSAR) DEM scenes. X-Band SAR penetrates the snow and ice pack by several meters depending on the structures within the snow, the acquisition parameters, and the dielectricity constant of the medium. Hence, the resulting SAR measurements do not represent the surface but the elevation of the mean phase center of the backscattered signal. Special adaptations on the nominal TanDEM-X DEM generation are conducted to maintain these characteristics and not to raise or even deform the DEM to surface reference data. For the block adjustment, only on the outer coastal regions ICESat (Ice, Cloud, and land Elevation Satellite) elevations as ground control points (GCPs) are used where mostly rock and surface scattering predominates. Comparisons with ICESat data and snow facies are performed. In the inner ice and snow pack, the final X-Band InSAR DEM of Greenland lies up to 10 m below the ICESat measurements. At the outer coastal regions it corresponds well with the GCPs. The resulting DEM is outstanding due to its resolution, accuracy and full coverage. It provides a high resolution dataset as basis for research on climate change in the arctic.

The TanDEM-X mission will derive a global digital elevation model (DEM) with satellite SAR interferometry. Two radar satellites (TerraSAR-X and TanDEM-X) will map the Earth in a resolution and accuracy with an absolute height error of 10m and a relative height error of 2m for 90% of the data. In order to fulfill the height requirements in general two global coverages are acquired and processed. Besides the final TanDEM-X DEM, an intermediate DEM with reduced accuracy is produced after the first coverage is completed. The last step in the whole workflow for generating the TanDEM-X DEM is the calibration of remaining systematic height errors and the merge of single acquisitions to 1°x1° DEM tiles. In this paper the current status of generating the intermediate DEM and first validation results based on GPS tracks, laser scanning DEMs, SRTM data and ICESat points are shown for different test sites.

Leukemic cells isolated from most patients with acute myelogenous leukemia (AML) have higher low density lipoprotein (LDL) uptake than normal mononuclear blood cells. Little is known, however, about the mechanism behind the elevated LDL uptake. We investigated if AML cells secrete factors that stimulate cellular LDL uptake. Mononuclear blood cells were isolated from peripheral blood from 42 patients with AML at diagnosis. Cellular LDL uptake was determined from the degradation rate of 125I-labelled LDL. Conditioned media from AML cells stimulated the LDL degradation in the leukemic cell lines KG1 and HL60, and in isolated AML cells. The stimulatory effect correlated with the LDL degradation in the AML cells directly after isolation from blood. Conditioned media also autostimulated LDL degradation in the AML cells themselves. Concentrations of IL-6 and IL-8 in AML cell conditioned media correlated with the LDL degradation in AML cells directly after isolation from blood. Addition of R-TNF-α, but not IL-6 or IL-8, stimulated LDL degradation in HL60, KG1, and AML cells. The LDL degradation in AML cells could be inhibited by a LDL receptor blocking antibody. AML cells secrete factors that stimulate LDL uptake in a paracrine and autocrine pattern which open up therapeutic possibilities to inhibit the uptake of LDL by administration of antibodies to these factors.

The outcome for multiple myeloma patients has improved since the introduction of bortezomib, thalidomide and lenalidomide. However, studies comparing new and conventional treatment include selected patient groups. We investigated consecutive patients (n = 1638) diagnosed in a defined period and compared survival with a gender- and age-matched cohort Swedish population (n = 9 340 682). Median overall survival for non-high-dose treated patients was 2·8 years. The use of bortezomib, thalidomide or lenalidomide in first line therapy predicted a significantly longer overall survival (median 4·9 years) compared to conventional treatment (2·3 years). Among non-high-dose treated patients receiving at least 2 lines with bortezomib, thalidomide or lenalidomide, 69% and 63% have survived at 3 and 5 years as compared to 48% and 22% with conventional drugs and 88% and 79% in the matched cohort populations, respectively. The median overall survival in high-dose treated patients was 6·9 years. Of these patients, 84% survived at 3 years and 70% at 5 years as compared to 98% and 95% in the matched cohort population. Overall survival in the best non-high-dose treated outcome group is closing the gap with the matched cohort. Upfront use of new drugs is clearly better than waiting until later lines of treatment.

The combination of melphalan, prednisone, and thalidomide (MPT) is considered standard therapy for newly diagnosed patients with multiple myeloma who are ineligible for stem cell transplantation. Long-term treatment with thalidomide is hampered by neurotoxicity. Melphalan, prednisone, and lenalidomide, followed by lenalidomide maintenance therapy, showed promising results without severe neuropathy emerging. We randomly assigned 668 patients between nine 4-week cycles of MPT followed by thalidomide maintenance until disease progression or unacceptable toxicity (MPT-T) and the same MP regimen with thalidomide being replaced by lenalidomide (MPR-R). This multicenter, open-label, randomized phase 3 trial was undertaken by Dutch-Belgium Cooperative Trial Group for Hematology Oncology and the Nordic Myeloma Study Group (the HOVON87/NMSG18 trial). The primary end point was progression-free survival (PFS). A total of 318 patients were randomly assigned to receive MPT-T, and 319 received MPR-R. After a median follow-up of 36 months, PFS with MPT-T was 20 months (95% confidence interval [CI], 18-23 months) vs 23 months (95% CI, 19-27 months) with MPR-R (hazard ratio, 0.87; 95% CI, 0.72-1.04; P = .12). Response rates were similar, with at least a very good partial response of 47% and 45%, respectively. Hematologic toxicity was more pronounced with MPR-R, especially grades 3 and 4 neutropenia: 64% vs 27%. Neuropathy of at least grade 3 was significantly higher in the MPT-T arm: 16% vs 2% in MPR-R, resulting in a significant shorter duration of maintenance therapy (5 vs 17 months in MPR-R), irrespective of age. MPR-R has no advantage over MPT-T concerning efficacy. The toxicity profile differed with clinically significant neuropathy during thalidomide maintenance vs myelosuppression with MPR.

center dot In vitro studies show that daunorubicin (DNR) cytotoxicity decreases with increasing cell density because of a high cellular uptake and depletion of drug in the medium. center dot It i ...

Anthracyclines like daunorubicin (DNR) are important drugs in the treatment of acute myeloid leukaemia (AML). In vitro studies have shown that cellular metabolism of antrhacyclines could play a role in drug resistance. Currently, it is not known what enzyme is responsible for anthracycline metabolism in leukemic cells. To study C-13 reduction of DNR to daunorubicinol (DOL) in leukemic cells isolated from patients with AML and to determine the most important enzyme involved. Mononuclear blood cells from 25 AML patients were isolated at diagnosis and used in a metabolic assay to determine the % DOL formed. MRNA and western blot analysis were performed on the 2 most likely candidates for anthracycline metabolism; carbonyl reductase 1 (CR1) and aldoketoreductase 1A1 (AKR1A1). DNR and DOL concentrations were determined by HPLC. We found a large interindividual variation (up to 47-fold) in leukemic cell DNR metabolism. The specific CR1 inhibitor zeraleone analogue 5 significantly inhibited DNR metabolism with a mean inhibitory effect of 68 %. No correlation between mRNA levels of the enzymes and metabolism were found. Cellular DNR metabolism correlated significantly with CR1 protein expression, determined by western blot, (p < 0.05, R2 = 0,229) while no significant correlation was found with AKR1A1 protein expression. DNR metabolism in AML cells shows a pronounced interindividual variability. Our results support that CR1 is the most important enzyme for conversion of DNR to DOL in AML cells. This information could in the future be used to genotype CR1 and possibly help to individualise dosing.

In mammals, excess energy is stored in the form of triacylglycerol primarily in lipid droplets of white adipose tissue. The first step of lipolysis (i.e. the mobilization of fat stores) is catalyzed by adipose triglyceride lipase (ATGL). The enzymatic activity of ATGL is strongly enhanced by CGI-58 (comparative gene identification-58), and the loss of either ATGL or CGI-58 function causes systemic triglyceride accumulation in humans and mice. However, the mechanism by which CGI-58 stimulates ATGL activity is unknown. To gain insight into CGI-58 function using structural features of the protein, we generated a three-dimensional homology model based on sequence similarity with other proteins. Interestingly, the model of CGI-58 revealed that the N terminus forms an extension of the otherwise compact structure of the protein. This N-terminal region (amino acids 1-30) harbors a lipophilic tryptophan-rich stretch, which affects the localization of the protein. (1)H NMR experiments revealed strong interaction between the N-terminal peptide and dodecylphosphocholine micelles as a lipid droplet-mimicking system. A role for this N-terminal region of CGI-58 in lipid droplet binding was further strengthened by localization studies in cultured cells. Although wild-type CGI-58 localizes to the lipid droplet, the N-terminally truncated fragments of CGI-58 are dispersed in the cytoplasm. Moreover, CGI-58 lacking the N-terminal extension loses the ability to stimulate ATGL, implying that the ability of CGI-58 to activate ATGL is linked to correct localization. In summary, our study shows that the N-terminal, Trp-rich region of CGI-58 is essential for correct localization and ATGL-activating function of CGI-58.

The Myc/Max/Mad network transcription factors are known to govern target gene expression through recruiting histone acetyltransferases or deacetylases. In the present study, we show that Mad1 recruits the histone demethylase RBP2 to the Myc target telomerase reverse transcriptase (hTERT) gene promoter to repress transcription. With differentiation of leukemic HL60 cells, Mad1 and RBP2 were both up-regulated, interacted, and cooccupied the hTERT promoter accompanied by histone H3-K4 demethylation. In immortalized p493-6 B cells, shutting down c-Myc led to the accumulation of Mad1 and RBP2 at hTERT promoter and diminished hTERT mRNA expression. When RBP2 was depleted, hTERT expression was significantly enhanced, coupled with dissociation of RBP2 with and increased H3-K4 methylation at the hTERT promoter in p493-6 cells. Moreover, RBP2 and Mad1 were present on the hTERT promoter in human fibroblasts having a silent hTERT gene, and RBP2 depletion resulted in gene derepression. Taken together, Mad1 recruits RBP2 to the hTERT promoter that, in turn, demethylates H3-K4, thereby contributing to a stable repression of the hTERT gene in normal or differentiated malignant cells. Our findings reveal a novel mechanism through which the Myc/Max/Mad network proteins control their target gene transcription and provide insights into mechanisms underlying telomerase silencing and activation.

Complex-binding of anthracyclines to DNA may increase their therapeutic efficacy. In a previous randomized trial patients with acute myelocytic leukaemia (AML) receiving combination chemotherapy including a DNA-bound doxorubicin preparation had a longer duration of first complete remission (CR) and survival than patients receiving free doxorubicin. In a parallel phase I/II study a combination of mitoxantrone, activity. In this randomized study of AML patients (15-60 years) induction treatment with MEA was compared to a combination of doxorubicin/DNA conjugate ara-C, thioguanine, vincristine and prednisolone (POCAL-DNA). The study was closed after an interim analysis of 86 patients. Thirty-five/42 (83%) and 20/44 (45%) patients entered CR in the MEA and POCAL-DNA groups, respectively (p < 0.001). With rescue therapy the corresponding figures were 88 and 64% (p < 0.02). Median survival was 27.8 and 13.1 months for MEA and POCAL-DNA patients, respectively (p < 0.03). In conclusion, the MEA regimen has a very high antileukaemic activity in good accordance with our previous experience. Since we could not reproduce our earlier clinical results using DNA-bound anthracyclines, the source and preparation of DNA seem to be of major importance.

Cellular accumulation of vincristine (VCR) and doxorubicin (DOX) was studied in 15 patients with low-grade B-cell malignancies. Their leukemic lymphocytes were isolated and the effect of verapamil on drug uptake was studied. The immunophenotype was characterized using anti-IgM, -IgD, -B 1, monoclonal antibodies. The more that cells bound the IgD or B1 antibodies, the more vincristine and doxorubicin did they accumulate. The fewer the cells that bound IgM antibodies, the more drug did they accumulate.

Cytosine arabinoside (ara-C) and etoposide are often used in combination in the treatment of acute myelocytic leukemia (AML). The intracellular phosphorylation of ara-C to its 5'-triphosphate (ara-CTP) is a prerequisite for its cytotoxic effects. It has been shown in vitro that etoposide can impair the formation of ara-CTP in leukemia cells. The present study was undertaken in order to elucidate whether this interaction may be of clinical importance. Leukemia cells were isolated from 3 patients with acute myelocytic leukemia and incubated in medium (RPMI-1640) with or without 10% fetal calf serum or in human plasma. When the cells were incubated in RPMI-1640 with ara-C (10 mumol/l) and etoposide during 2 h, the formation of ara-CTP was decreased to 71 +/- 18 (mean +/- S.D.) and 30 +/- 15% of control at 1 and 10 micrograms/ml etoposide, respectively. When the cells were incubated in human plasma, the formation of ara-CTP was not influenced by the presence of etoposide (101 +/- 6 and 103 +/- 20% at 1 and 10 micrograms/ml etoposide). When incubated in RPMI supplemented with 10% fetal calf serum, the corresponding figures were 81 +/- 8 and 70 +/- 20%. Six patients with AML were therefore treated with ara-C 0.5 or 1.0 g/m2 as a 2-h infusion every 12 h and, during 1 h before the second ara-C infusion, 100 or 200 mg/m2 etoposide was administered. The median change in the AUC of cellular ara-CTP between the first and second ara-C dose was 0% (-37 to +21%). The corresponding median change in rate of accumulation of ara-CTP in leukemia cells was 12% (-26 to +110%). The concentration of etoposide in plasma during the ara-C infusion was 18.7 +/- 5.1 micrograms/ml while the non-protein bound etoposide was 0.73 +/- 0.34 micrograms/ml. Thus, despite exposure to higher etoposide concentrations in vivo than in vitro, no impairment of ara-CTP formation was seen in the patients. This corresponds to the results obtained when leukemic cells were incubated in plasma. It is concluded that the inhibition of ara-CTP formation by etoposide seen in vitro is offset by the high protein binding of etoposide in plasma (96%) and that etoposide does not impair the formation of ara-CTP in leukemia cells in vivo during treatment with standard-dose etoposide.

Activation and expansion of T helper (Th) cells followed by regulation of activation are essential to the generation of immune responses while limiting concomitant autoreactivity. In order to characterize T cells reactive towards myeloma-derived monoclonal immunoglobulin (mIg), an autologous coculture assay for single-cell analysis of mIg-responding cells was developed. When cultured with dendritic cells loaded with mIg, CD4+ Th cells from patients with progressing multiple myeloma (MM) showed a proliferative MHC class II–dependent response. CD8+ T-cell reactivity and Th1 activation were consistently low or absent, and Th2 and regulatory cytokines were expressed. The presence of such non-Th1 CD4+ T cells in peripheral blood was independent of treatment status, while the frequencies of responding cells varied between patients and reached the same order of magnitude as those measured for tetanus toxoid–specific Th memory cells. Furthermore, investigations of T-cell subpopulations indicated a possible regulatory role on the mIg responsiveness mediated by suppressive CD25highFOXP3+CD4+ T cells. It is proposed from the present results that a predominant in vivo activation of non-Th1 mIg-reactive CD4+ T cells constitute an Ig-dependent autoregulatory mechanism in human MM, with possible tumor growth supporting or permissive effects.

Recent evidence has accumulated that the dynamic histone methylation mediated by histone methyltransferases and demethylases plays key roles in regulation of chromatin structure and transcription. In the present study, we show that SET and MYND domain-containing protein 3 (SMYD3), a histone methyltransferase implicated in oncogenesis, directly trans-activates the telomerase reverse transcriptase (hTERT) gene that is essential for cellular immortalization and transformation. SMYD3 occupies its binding motifs on the hTERT promoter and is required for maintenance of histone H3-K4 trimethylation, thereby contributing to inducible and constitutive hTERT expression in normal and malignant human cells. Knocking down SMYD3 in tumor cells abolished trimethylation of H3-K4, attenuated the occupancy by the trans-activators c-MYC and Sp1, and led to diminished histone H3 acetylation in the hTERT promoter region, which was coupled with down-regulation of hTERT mRNA and telomerase activity. These results suggest that SMYD3-mediated trimethylation of H3-K4 functions as a licensing element for subsequent transcription factor binding to the hTERT promoter. The present findings provide significant insights into regulatory mechanisms of hTERT/telomerase expression; moreover, identification of the hTERT gene as a direct target of SMYD3 contributes to a better understanding of SMYD3-mediated cellular transformation. [Cancer Res 2007;67(6):2626–31]

Alternative splicing of telomerase reverse transcriptase (hTERT) mRNA is known to contribute to regulation of telomerase activity in normal and cancerous cells, however, previous studies indicated that normal human T and B cells exhibited constitutive expression of full-length hTERT mRNA without splicing variants and that activation of telomerase upon stimulation of the cells was due to the shuttling of hTERT protein from cytoplasm to nucleus [Proc. Natl. Acad. Sci. USA 96 (1999) 5147; J. Immunol. 166 (2001) 4826]. We found that typical variants of hTERT mRNA were widespread in human lymphocyte-derived cell lines and normal stimulated T cells. In activated T cells, induction of the full-length hTERT mRNA was coupled with increased hTERT protein expression and telomerase activity. Collectively, human normal and malignant lymphocytes, like other human cells, express splicing variants of hTERT mRNA and require transcriptional activation of the hTERT gene to acquire telomerase activity.

Telomerase activity and telomerase reverse transcriptase (hTERT), the key component of the telomerase complex, are tightly proliferation regulated in normal and malignant cells both in vitro and in vivo; however, underlying mechanisms are unclear. In the present study, we identified mitogen-activated protein kinase (MAPK) cascade-mediated histone H3 ser10 phosphorylation to be a molecular link between proliferation and induction of hTERT/telomerase activity. In normal human T lymphocytes and fibroblasts, growth or stress stimuli known to drive H3 phosphorylation through the MAPK signaling induce hTERT expression and/or telomerase activity that was preceded by phosphorylated histone H3 (ser10) at the hTERT promoter. Blockade of the MAPK-triggered H3 phosphorylation significantly abrogates hTERT induction and ser10 phosphorylation at this promoter. However, H3 ser10 phosphorylation alone resulted in low, transient hTERT induction, as seen in fibroblasts, whereas H3 phosphorylation followed by its acetylation at lys14 robustly trans-activated the hTERT gene accompanying constitutive telomerase activity in normal and malignant T cells. H3 acetylation without phosphorylation similarly exerted weak effects on hTERT expression. These results define H3 phosphorylation as a key to hTERT transactivation induced by proliferation and reveal a fundamental mechanism for telomerase regulation in both normal human cells and transformed T cells.

Activation of telomerase, a key event during immortalization and malignant transformation, requires expression of the telomerase reverse transcriptase (hTERT). Consistently, lack of telomerase activity and hTERT expression occurs in most normal human somatic cells. However, it has been observed that both normal and cancerous renal tissues express hTERT whereas only the latter exhibits telomerase activity. The mechanism underlying the dissociation between hTERT expression and telomerase activity is unclear. In the present study, we examined telomerase activity and alternative splicing of hTERT transcripts in renal cell carcinoma (RCC) specimens and adjacent normal tissues from 33 patients with RCC. Telomerase activity was detectable in 27 of 33 (82%) RCC samples but none in their normal counterparts. Thirty-two of 33 tumors expressed overall hTERT mRNA and 27 of them contained full-length hTERT transcripts, all with telomerase activity. Although 42% (14 of 33) of normal renal samples expressed hTERT mRNA, none of them had full-length hTERT transcripts, coinciding with lack of telomerase activity. The presence of full-length hTERT mRNA and telomerase activity was significantly associated with c-MYC induction. In tumors, absence of full-length hTERT mRNA or telomerase activity defines a subgroup of nonmetastatic, early-stage RCCs. Taken together, telomerase repression in normal renal tissues is attributed to the absence of full-length hTERT transcripts, whereas telomerase activation is achieved via induction of or switch to expression of full-length hTERT mRNA during the oncogenic process of kidneys, and associated with aggressive RCCs.

In a majority of malignant human tumors telomerase activity can be detected, suggesting an immortal phenotype. Expression of the reverse transcriptase subunit, hTERT, in the human telomerase complex is required for telomerase activity. The regulation of hTERT, from gene level to a fully functional protein, is still a poorly understood process. Increased copy number of the hTERT gene has been demonstrated in a significant portion of established cell lines and tumors of different origin but its relevance for telomerase activity levels is unclear. In the present study, we examined the hTERT gene copy number using fluorescence in situ hybridization (FISH) in samples from 64 colorectal carcinomas and an increased copy number (or = 3 hTERT gene copies/nucleus) was observed in 31 cases (48%). No statistical association existed between hTERT gene copy number and hTERT RNA expression or telomerase activity. However, a significant relationship was found between an increase in hTERT gene copy number and p53 protein accumulation (p = 0.002) and aneuploidy (p = 0.036). Only 4 tumors showed microsatellite instability, 3 of which had a normal hTERT gene copy number. The data indicated that the increased copy number of the hTERT gene in colorectal carcinoma was a result of genomic instability with no obvious consequence for telomerase activity levels.

Although human papillomavirus (HPV) has been defined as the pathogen for cervical carcinomas, molecular events underlying the oncogenic process are unclear. As telomere dysfunction-mediated chromosomal instability and telomerase activation have been suggested as key events in carcinogenesis, we dissected the dynamic changes in telomere length, checkpoint response, and temporal profile of telomerase expression during the evolution from precursor lesions (cervical intraepithelial neoplasia, CINs) to invasive cancers of the uterine cervix in sequential samples from 16 patients. Telomeres were significantly shortened in all CIN samples and no further substantial attritions occurred in most cases with the acquisition of malignant phenotype. Very short telomeres were coupled with constitutive activation of the DNA damage response pathway (Chk2 phosphorylation) and increased cellular proliferation in those cervical specimens. Telomerase reverse transcriptase (hTERT) expression was preferably induced at advanced CINs or invasive cancers. The present finding demonstrates that excessive telomere shortening predominantly occurs in the early carcinogenesis of the uterine cervix largely prior to telomerase activation. Widespread over-erosion of telomeres or telomere dysfunction in very early stages of cervical tumorigenesis might fuel transformation processes by driving chromosomal instability.

Multiple myeloma (MM) is considered to be an essentially incurable haematological malignant disease, probably because of the existence of resistant clonal precursor cell with self-renewal capacity. Recent data have indicated that the myeloma cell hierarchy includes circulating clonal memory B cells, which differ considerably from the classical end-stage plasma cells, infiltrating the bone marrow. The pathophysiological significance of these cells is unknown, but hypothetically they may serve as ‘sleeping’ myeloma stem cells responsible for and ‘feeding’ post-treatment relapse and progression. The present study evaluates the toxicity and feasibility of fludarabine, added to the VAD-induction regimen in MM, and investigates the effect on the myeloma cell hierarchy. Nineteen patients were randomised to receive either four cycles of VAD (n = 9) or two cycles of VAD, followed by two cycles of VAD combined with 5 days fludarabine 25 mg/m2/day i.v. (n = 10). Toxicity evaluation showed more profound neutropenia in the fludarabine-treated patients and two infectious episodes in each study arm: three were fever of unknown origin while one, in the fludarabine-arm, was a local skin infection at the insertion site of the central venous line. Nine of the fludarabine-treated patients responded to treatment (two complete remission, seven partial remission), compared with five responders (all PR) in the control-arm. The effects on the blood circulating myeloma compartments identified an increased reduction of CD19+ B cells and myeloma plasma cells in the fludarabine-arm. In conclusion, adding fludarabine to VAD induction in multiple myeloma is feasible and may be clinically effective by reducing the myeloma clone.

Cri du chat syndrome (CdCS) results from loss of the distal portion of chromosome 5p, where the telomerase reverse transcriptase (hTERT) gene is localized (5p15.33). hTERT is the rate-limiting component for telomerase activity that is essential for telomere-length maintenance and sustained cell proliferation. Here, we show that a concomitant deletion of the hTERT allele occurs in all 10 patients with CdCS whom we examined. Induction of hTERT mRNA in proliferating lymphocytes derived from five of seven patients was lower than that in unaffected control individuals (P

The objective of this study was to determine the role of transport proteins in daunorubicin (Dnr) accumulation and efflux in leukemia cells from 36 patients with acute myeloid leukemia (AML). Mononuclear cells were isolated and incubated with 1 microM Dnr with/without addition of 3 microM cyclosporin A (CyA) or metabolic inhibitors (MI). Cellular Dnr concentration in leukemia blast cells was measured with flow cytometry. After washing and reincubation of the cells in drug-free medium, Dnr efflux was followed with/without addition of CyA or MI. Levels of mRNA expression for mdr1, multidrug resistance associated protein (mrp) and lung resistance protein (lrp) were determined with reverse transcriptase-polymerase chain reaction (RT-PCR). MI enhanced cellular Dnr accumulation to a higher extent than CyA whereas CyA reduced Dnr efflux more efficiently than MI (P0.001). There was a significant difference in Dnr accumulation between samples with low and high mdr1 mRNA levels but only in the presence of MI or CyA. Our results imply that other factors than P-glycoprotein (Pgp) are of major importance for in vitro Dnr accumulation in AML blasts and that the role of Pgp as a drug efflux pump is not conclusive.

It is still controversial how to treat elderly patients with acute myeloid leukaemia (AML), and results have been poor with most regimens. We report the long-term results of a randomised study performed by the Leukaemia Group of Middle Sweden during 1984-88 comparing two intensive chemotherapeutic drug combinations. Ninety patients >or=60-yr old with untreated AML were randomly allocated to treatment with daunorubicin, cytosine arabinoside (ara-C), and thioguanine (TAD) (43 patients) or a combination in which aclarubicin was substituted for daunorubicin (TAA) (47 patients). Forty-four patients (49%) entered complete remission (CR), 22/43 (51%) in the TAD group and 22/47 (47%) in the TAA group (ns). The CR rate in patients 70 yr 14/48 (29%) (P 70 yr than in patients or=10 yr after inclusion of the last patient, 5/90 patients (one in the TAD group and four in the TAA group, respectively) were still alive, four in continuous complete remission and one in second complete remission. Thus, both treatment regimens appear to have similar efficacy, with a relatively high complete remission rate, and a reasonable survival as compared to other studies including some long-term survivors. However, early deaths are still numerous, particularly in patients above 70 yr of age, and the relapse rate is substantial.

Activation of telomerase, essential for cellular immortalization and transformation, requires the induction of its catalytic component, telomerase reverse transcriptase (hTERT). However, biochemical and genetic mechanisms for the control of hTERT expression remain undefined. In the present study, we demonstrate that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) induces hyperacetylation of histones at the hTERT proximal promoter, directly transactivates the hTERT gene in normal human telomerase-negative cells, and upregulates hTERT expression in telomerase-positive tumor cells. Overexpression of HDAC1 leads to repression of the hTERT promoter activity. TSA-mediated activation of the hTERT promoter is abolished by the mutation of Sp1 sites at the proximal promoter, suggesting that the effect of TSA is regulated through Sp1 motifs. We also show a physical interaction of Sp1 with HDAC1 and the presence of HDAC1 at the hTERT promoter region. Moreover, hyperacetylation of histones at the hTERT promoter is associated with the natural up-regulation of hTERT expression that occurs in activated T lymphocytes. Taken together, histone acetylation/deacetylation may be a common underlying feature to hTERT transactivation/repression in human normal and malignant cells.

The expression of telomerase reverse transcriptase (hTERT), the catalytic component of the telomerase complex, is required for activation of telomerase during immortalization and transformation of human cells. However, the biochemical and genetic mechanisms governing hTERT expression remain to be elucidated. In the present study, we examined hTERT amplification as a potential genetic event contributing to telomerase activation in cervical carcinomas. An amplification of the hTERT gene was found in 1/4 cervical cancer cell lines and 21/88 primary tumor samples derived from the patients with cervical carcinomas. An increase in the hTERT copy number was significantly correlated with higher levels of hTERT protein expression. Moreover, the hTERT alterations with the enhanced hTERT expression were exclusively observed in those tumors with high-risk human papillomavirus infection. Taken together, the hTERT gene amplification, directly or indirectly targeted by human papillomavirus, may be one of the driving forces responsible for upregulation of hTERT expression and activation of telomerase in cervical cancers. © 2002 Wiley-Liss, Inc.

Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy with high mortality rate. Treatment outcomes have improved since the introduction of new drugs such as the IMiD lenalidomide, but relapse rates and resistance is still a problem. The gene ABCB1 encodes the drug transporter p-glycoprotein (p-gp) which confers resistance through extrusion of drugs over the cell membrane. Lenalidomide is subject to limited metabolism and excreted mainly via the kidneys. In vitro studies have shown lenalidomide to be an ABCB1 substrate, and single nucleotide polymorphisms (SNPs) affecting gene expression, transporter function and/or activity may affect drug distribution and the subsequent outcome and risk of adverse events. However, in vivo studies of the effect of ABCB1 on lenalidomide pharmacokinetics are contradictory. Our aim was to investigate the influence of ABCB1 SNPs on lenalidomide treatment outcome and adverse events (AE). Materials & Methods: In the observational part of two connected studies, 133 Lenalidomide naïve patients at 1st relapse/refractory MM were treated with lenalidomide and dexamethasone for up to 9 cycles of 4 weeks. In the prospective 2nd part, 62 patients that had achieved at least partial response according to IMWG-criteria followed by at least two additional treatment cycles were randomized to either lenalidomide/dexamethasone or lenalidomide as a single drug. 90 patients (of which 47 was further randomized to the 2nd part) had samples available for genotyping of the ABCB1 SNPs 1199G>A (Ser400Asn, rs2229109), 1236C>T (rs1128503), 2677G>T/A (Ala893Ser, rs2032582) and 3435C>T (rs1045642) using Pyrosequencing. Correlations to overall survival, time to progression (TTP), response parameters and AE were investigated, and a p-value of 0.05 was considered significant. Results: No significant correlations to hematological AE or response rates were found, and no impact on survival for 1236C>T, 2677G>T/A or 3435C>T, neither in the whole population nor in patients randomized to the 2nd part. The results were similar also when risk (according to FISH) was considered. There was a trend towards improved TTP for patients carrying the 1199A variant; mean TTP 3.2 years (95%CI 2.3-4.1) vs 2.2 years (95%CI 1.8-2.6) for G/A and G/G, respectively (p=0.076). This trend was confirmed in the multivariable cox regression analysis; HR=0.280 (95%CI 0.74-1.054), p=0.06. The difference in TTP was significant in the non-high risk subgroup; mean TTP 4.3 years (95%CI 3.7-4.9) vs 2.3 years (95%CI 1.8-2.8), p=0.034, for G/A and G/G, respectively. Conclusion: No evidence was found for a large impact of 1236C>T, 2677G>T/A or 3435C>T on lenalidomide treatment outcome or risk of hematological AE. 1199G>A may be a potential marker of TTP in non-high risk MM but further studies in a larger cohort is needed to clarify the relationship and whether this is due to altered drug transport or efflux independent mechanisms. Citation Format: Ingrid Jakobsen Falk, Johan Lund, Henrik Gréen, Astrid Gruber, Evren Alici, Birgitta Lauri, Cecilie Blimark, Ulf-Henrik Mellqvist, Agneta Swedin, Karin Forsberg, Conny Carlsson, Mats Hardling, Lucia Ahlberg, Hareth Nahi, Kourosh Lotfi. The impact of ABCB1 single nucleotide polymorphisms on the outcome in lenalidomide treated multiple myeloma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5030. doi:10.1158/1538-7445.AM2017-5030