Real world data on outcomes of anti-CD38 antibody treated, including triple class refractory, patients with multiple myeloma: a multi-institutional report from the Canadian Myeloma Research Group (CMRG) Database.

Multiple myeloma (MM) remains incurable despite the availability of novel agents. This multi-center retrospective cohort study used the Canadian Myeloma Research Group Database to describe real-world outcomes of patients withanti-CD38 monoclonal antibody (mAb) refractory MM subsequently treated with standard of care (SoC) regimens. Patients with triple class refractory (TCR) disease (refractory to a proteasome inhibitor, immunomodulatory drug, and anti-CD38 mAb) were examined as a distinct cohort. Overall, 663 patients had disease progression on anti-CD38 mAb therapy, 466 received further treatment (346 with SoC regimens were included, 120 with investigational agents on clinical trial and were excluded). The median age at initiation of subsequent SoC therapy of 67.9 (range 39.6-89.6) years with a median of 3 prior lines (range 1-9). The median PFS and OS from the start of subsequent therapy was 4.6 (95% CI 4.1-5.6) months and 13.3 (95% CI 10.6-16.6) months, respectively. The median PFS and OS of patients with TCR disease (n = 199) was 4.4 (95% CI 3.6-5.3) months and 10.5 (95% CI 8.5-13.8) months. Our results reinforce that real-world patients with relapsed MM, particularly those with TCR disease, have dismal outcomes. There remains an urgent unmet need for the development of and access to effective therapeutics for these patients.

An accessible pharmacodynamic transcriptional biomarker for notch target engagement.

γ-Secretase mediates amyloid production in Alzheimer's disease (AD) and oncogenic activity of Notch. γ-Secretase inhibitors (GSIs) are thus of interest for AD and oncology. A peripheral biomarker of Notch activity would aid determination of the therapeutic window and dosing regimen for GSIs, given toxicities associated with chronic Notch inhibition. This study examined the effects of GSI MK-0752 on blood and hair follicle transcriptomes in healthy volunteers. The effects of a structurally diverse GSI on rhesus blood and hair follicles were also compared. Significant dose-related effects of MK-0752 on transcription were observed in hair follicles, but not blood. The GSI biomarker identified in follicles exhibited 100% accuracy in a clinical test cohort, and was regulated in rhesus by a structurally diverse GSI. This study identified a translatable, accessible pharmacodynamic biomarker of GSI target engagement and provides proof of concept of hair follicle RNA as a translatable biomarker source.

A randomized phase III trial on maintenance treatment with bevacizumab alone or in combination with erlotinib after chemotherapy and bevacizumab in metastatic colorectal cancer: the Nordic ACT Trial.

BACKGROUND: The main objective was to study the effect on progression-free survival (PFS) of adding erlotinib to bevacizumab as maintenance treatment following chemotherapy and bevacizumab as first-line treatment of metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: Patients with untreated mCRC received doublet chemotherapy + bevacizumab during 18 weeks and those without tumor progression were eligible for randomization to bevacizumab + erlotinib (arm A) or bevacizumab alone (arm B), until progression or unacceptable toxic effect. RESULTS: Of the 249 patients enrolled, 80 started maintenance treatment in arm A and 79 in arm B. The rate of any grade 3/4 toxic effect was 53% in arm A and 13% in arm B. Median PFS was 5.7 months in arm A and 4.2 months in arm B (HR = 0.79; 95% confidence interval 0.55-1.12; P = 0.19). Overall survival (OS) from start of induction chemotherapy was 26.7 months in the randomized population, with no difference between the two arms. CONCLUSIONS: The addition of erlotinib to bevacizumab as maintenance treatment after first-line chemotherapy in mCRC did not improve PFS significantly. On-going clinical and translational studies focus on identifying subgroups of patients that may benefit from erlotinib in the maintenance setting. CLINICAL TRIALS NUMBER: NCT00598156.

MK-0457, an Aurora kinase and BCR-ABL inhibitor, is active in patients with BCR-ABL T315I leukemia.

MK-0457, an Aurora kinase and BCR-ABL inhibitor, was studied on a Phase I/II study in 77 patients with refractory hematologic malignancies. The average number of cycles per patient was 3 (range 1-21). Maximum tolerated doses for a 5-day short infusion and continuous infusion regimens were 40 mg/m(2)/h and 144 mg/m(2)/h, respectively. Drug-related adverse events (AEs) included transient mucositis and alopecia. Eight of 18 patients with BCR-ABL T315I-mutated chronic myelogenous leukemia (44%) had hematologic responses and one of three patients (33%) with Philadelphia chromosome-positive acute lymphoblastic leukemia obtained complete remission. MK-0457 has important activity in patients with leukemias expressing the highly resistant T315I BCR-ABL mutation.

Paclitaxel, Epirubicin and Capecitabine (TEX) as First-Line Treatment for Metastatic Breast Cancer: a Pilot Phase I/II Feasibility Study.

Thirteen patients with untreated metastatic breast cancer received epirubicin 60 mg/m(2), paclitaxel 155 mg/m(2) (both day 1) and capecitabine 665 mg/m(2) twice daily (days 1-14) every 21 days, with intra-patient dose escalation/reduction. Grade 3/4 events were infrequent. Nine patients (69%) achieved an objective response. Median time to progression and overall survival were 6.6 and 23.5 months, respectively.

Palladium-mediated C5 substitution of pyrimidine nucleosides.

One of the most efficient ways to link a reporter group to oligonucleotides is through the incorporation of a modified nucleoside during automated oligonucleotide synthesis. To be useful, it is important that the reporter group not interfere in hybridization reactions. This unit describes two linkers that can be used for the incorporation of a reporter group at the C5 position of deoxyuridine: a flexible aminoethylthioether linker, and a rigid amidopropynyl linker. The latter is sufficiently long and positioned so that the reporter group lies outside the major groove of the DNA duplex.

Fibrosis in undifferentiated (anaplastic) thyroid carcinomas: evidence for a dual action of tumour cells in collagen type I synthesis.

The mechanisms involved in stromal reactions and fibrosis in solid malignant tumours are incompletely understood. In the present study, collagen type I production was investigated in tissues and cell lines derived from human undifferentiated (anaplastic) thyroid carcinomas, a highly aggressive, often fibrotic malignancy with mesenchymal phenotype. In situ hybridization showed the expression of pro-alpha1(I) collagen mRNA throughout the stromal part of the tumours. However, immunofluorescence staining using an anti-pro-collagen type I antibody revealed the synthesis of pro-collagen type I protein mainly in stromal cells juxtaposed to nests of tumour cells. In one out of five tissue samples from human undifferentiated thyroid carcinomas, pro-alpha1(I) collagen mRNA expression was also found in a small number of tumour cells. Several well-characterized cell lines established from undifferentiated thyroid carcinomas, two from tumours included in the present study, expressed both pro-alpha1(I) collagen and prolyl 4-hydroxylase mRNA, and three of these cell lines also synthesized native triple-helical collagen type I. Taken together, these data suggest that stromal fibroblasts are the main producers of collagen type I in anaplastic thyroid tumours. The carcinoma cells seem to play a regulatory role, stimulating the synthesis of collagen type I protein in the surrounding stroma by increasing pro-alpha1(I) collagen mRNA translation. However, collagen type I production by the carcinoma cells might also contribute to the marked desmoplasia commonly seen in these tumours.

A conserved motif N-terminal to the DNA-binding domains of myogenic bHLH transcription factors mediates cooperative DNA binding with pbx-Meis1/Prep1.

The t(1;19) chromosomal translocation of pediatric pre-B cell leukemia produces chimeric oncoprotein E2a-Pbx1, which contains the N-terminal transactivation domain of the basic helix-loop-helix (bHLH) transcription factor, E2a, joined to the majority of the homeodomain protein, Pbx1. There are three Pbx family members, which bind DNA as heterodimers with both broadly expressed Meis/Prep1 homeo-domain proteins and specifically expressed Hox homeodomain proteins. These Pbx heterodimers can augment the function of transcriptional activators bound to adjacent elements. In heterodimers, a conserved tryptophan motif in Hox proteins binds a pocket on the surface of the Pbx homeodomain, while Meis/Prep1 proteins bind an N-terminal Pbx domain, raising the possibility that the tryptophan-interaction pocket of the Pbx component of a Pbx-Meis/Prep1 complex is still available to bind trypto-phan motifs of other transcription factors bound to flanking elements. Here, we report that Pbx-Meis1/Prep1 binds DNA cooperatively with heterodimers of E2a and MyoD, myogenin, Mrf-4 or Myf-5. As with Hox proteins, a highly conserved tryptophan motif N-terminal to the DNA-binding domains of each myogenic bHLH family protein is required for cooperative DNA binding with Pbx-Meis1/Prep1. In vivo, MyoD requires this tryptophan motif to evoke chromatin remodeling in the Myogenin promoter and to activate Myogenin transcription. Pbx-Meis/Prep1 complexes, therefore, have the potential to cooperate with the myogenic bHLH proteins in regulating gene transcription.

Lack of responsiveness to TGF-beta1 in a thyroid carcinoma cell line with functional type I and type II TGF-beta receptors and Smad proteins, suggests a novel mechanism for TGF-beta insensitivity in carcinoma cells.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine. In the present study we have investigated the expression of TGF-beta receptors (TbetaR's) and SMAD proteins in non-neoplastic and neoplastic thyroid follicle cells. We found expression of all TbetaR's (type I, II and III) and SMAD proteins analysed (Smad2, Smad3, Smad4, Smad6 and Smad7). Five out of six human anaplastic thyroid carcinoma cell lines were growth inhibited by addition of TGF-beta1, and therefore considered to be TGF-responsive. One cell line however, HTh 7, did not respond to TGF-beta1 with growth inhibition, induction of the extracellular matrix protein fibronectin or immediate early genes junB, Smad6 and Smad7 mRNA. Analysis of the TGF-beta intracellular signalling pathway in HTh 7 cells showed that receptors were capable of signalling, e.g. Smad2 phosphorylation and SMAD nuclear translocation. In summary, our data shows abundant expression of TGF-beta signalling components in thyroid follicle cells, and the escape from TGF-beta sensitivity in one anaplastic thyroid carcinoma despite an apparently functional TGF-beta/SMAD-signalling pathway, indicating a novel mechanism for TGF-beta insensitivity.

Dopamine agonist-mediated rotation in rats with unilateral nigrostriatal lesions is not dependent on net inhibitions of rate in basal ganglia output nuclei.

Current models of basal ganglia function predict that dopamine agonist-induced motor activation is mediated by decreases in basal ganglia output. This study examines the relationship between dopamine agonist effects on firing rate in basal ganglia output nuclei and rotational behavior in rats with nigrostriatal lesions. Extracellular single-unit activity ipsilateral to the lesion was recorded in awake, locally-anesthetized rats. Separate rats were used for behavioral experiments. Low i.v. doses of D1 agonists (SKF 38393, SKF 81297, SKF 82958) were effective in producing rotation, yet did not change average firing rate in the substantia nigra pars reticulata or entopeduncular nucleus. At these doses, firing rate effects differed from neuron to neuron, and included increases, decreases, and no change. Higher i.v. doses of D1 agonists were effective in causing both rotation and a net decrease in rate of substantia nigra pars reticulata neurons. A low s.c. dose of the D1/D2 agonist apomorphine (0.05 mg/kg) produced both rotation and a robust average decrease in firing rate in the substantia nigra pars reticulata, yet the onset of the net firing rate decrease (at 13-16 min) was greatly delayed compared to the onset of rotation (at 3 min). Immunostaining for the immediate-early gene Fos indicated that a low i.v. dose of SKF 38393 (that produced rotation but not a net decrease in firing rate in basal ganglia output nuclei) induced Fos-like immunoreactivity in the striatum and subthalamic nucleus, suggesting an activation of both inhibitory and excitatory afferents to the substantia nigra and entopeduncular nucleus. In addition, D1 agonist-induced Fos expression in the striatum and subthalamic nucleus was equivalent in freely-moving and awake, locally-anesthetized rats. The results show that decreases in firing rate in basal ganglia output nuclei are not necessary for dopamine agonist-induced motor activation. Motor-activating actions of dopamine agonists may be mediated by firing rate decreases in a small subpopulation of output nucleus neurons, or may be mediated by other features of firing activity besides rate in these nuclei such as oscillatory firing pattern or interneuronal firing synchrony. Also, the results suggest that dopamine receptors in both the striatum and at extrastriatal sites (especially the subthalamic nucleus) are likely to be involved in dopamine agonist influences on firing rates in the substantia nigra pars reticulata and entopeduncular nucleus.

The effect of hydrogen peroxide upon beta-adrenoceptor density and function in C6 rat glioma cells.

Hydrogen peroxide has been suggested to play an important role in the pathogenesis of Alzheimer's disease. In the present study, the effects of hydrogen peroxide upon the functional integrity of beta-adrenoceptors have been investigated in C6 glioma cells. Treatment of cells for 24 h with hydrogen peroxide in serum-free medium produced a concentration-dependent cell toxicity, seen both using cell counting and LDH release into medium as end point. There were no large nor consistent changes in either the density of cell surface beta 1, or beta 2-adrenoceptors, measured using the hydrophilic ligand [3H](-)-CGP 12177, nor in either basal, forskolin and isoprenaline-stimulated cAMP responses, following hydrogen peroxide treatment. It is concluded that the decreased adenylyl cyclase activity and responsiveness to Gs stimulation found in post-mortem brain samples from Alzheimer's disease autopsy cases is unlikely to be mediated by hydrogen peroxide.

Nucleotide analogs and new buffers improve a generalized method to enrich for low abundance mutations.

A high sensitivity method for detecting low level mutations is under development. A PCR reaction is performed in which a restriction site is introduced in wild-type DNA by alteration of specific bases. Digestion of wild-type DNA by the cognate restriction endonuclease (RE) enriches for products with mutations within the recognition site. After reamplification, mutations are identified by a ligation detection reaction (LDR). This PCR/RE/LDR assay was initially used to detect PCR error in known wild-type samples. PCR error was measured in low |Deltap K a| buffers containing tricine, EPPS and citrate, as well as otherwise identical buffers containing Tris. PCR conditions were optimized to minimize PCR error using perfect match primers at the Msp I site in the p53 tumor suppressor gene at codon 248. However, since mutations do not always occur within pre-existing restriction sites, a generalized PCR/RE/LDR method requires the introduction of a new restriction site. In principle, PCR with mismatch primers can alter specific bases in a sequence and generate a new restriction site. However, extension from 3' mismatch primers may generate misextension products. We tested conversion of the Msp I (CCGG) site to a Taq I site (TCGA). Conversion was unsuccessful using a natural base T mismatch primer set. Conversion was successful when modified primers containing the 6 H,8 H -3, 4-dihydropyrimido[4,5- c ][1,2]oxazine-7-one (Q6) base at 3'-ends were used in three cycles of preconversion PCR prior to conversion PCR using the 3' natural base T primers. The ability of the pyrimidine analog Q6 to access both a T-like and C-like tautomer appears to greatly facilitate the conversion.

Triple helix formation: binding avidity of acridine-conjugated AG motif third strands containing natural, modified and surrogate bases opposed to pyrimidine interruptions in a polypurine target.

A critical issue for the general application of triple-helix-forming oligonucleotides (TFOs) as modulators of gene expression is the dramatically reduced binding of short TFOs to targets that contain one or two pyrimidines within an otherwise homopurine sequence. Such targets are often found in gene regulatory regions, which represent desirable sites for triple helix formation. Using intercalator-conjugated AG motif TFOs, we compared the efficacy and base selectivity of 13 different bases or base surrogates in opposition to pyrimidines and purines substituted into selected positions within a paradigm 15-base polypurine target sequence. We found that substitutions closer to the intercalator end of the TFO (positions 4-6) had a more deleterious effect on the dissociation constant (K d) than those farther away (position 11). Opposite T residues at position 11, 3-nitropyrrole or cytosine in the TFO provided adequate binding avidity for useful triplex formation (K ds of 55 and 110 nM, respectively). However, 3-nitropyrrole was more base selective than cytosine, binding to T >/=4 times better than to A, G or C. None of the TFOs tested showed avid binding when C residues were in position 11, although the 3-nitropyrrole-containing TFO bound with a K d of 200 nM, significantly better than the other designs. Molecular modeling showed that the 3-nitropyrrole.T:A triad is isomorphous with the A.A:T triad, and suggests novel parameters for evaluating new base triad designs.

Two domains of MyoD mediate transcriptional activation of genes in repressive chromatin: a mechanism for lineage determination in myogenesis.

Genetic studies have demonstrated that MyoD and Myf5 establish the skeletal muscle lineage, whereas myogenin mediates terminal differentiation, yet the molecular basis for this distinction is not understood. We show that MyoD can remodel chromatin at binding sites in muscle gene enhancers and activate transcription at previously silent loci. TGF-beta, basic-FGF, and sodium butyrate blocked MyoD-mediated chromatin reorganization and the initiation of transcription. In contrast, TGF-beta and sodium butyrate did not block transcription when added after chromatin remodeling had occurred. MyoD and Myf-5 were 10-fold more efficient than myogenin at activating genes in regions of transcriptionally silent chromatin. Deletion mutagenesis of the MyoD protein demonstrated that the ability to activate endogenous genes depended on two regions: a region rich in cysteine and histidine residues between the acidic activation domain and the bHLH domain, and a second region in the carboxyl terminus of the protein. Neither region has been shown previously to regulate gene transcription and both have domains that are conserved in the Myf5 protein. Our results establish a mechanism for chromatin modeling in the skeletal muscle lineage and define domains of MyoD, independent of the activation domain, that participate in chromatin reorganization.

Participation of cyclin A in Myc-induced apoptosis.

The involvement of c-Myc in cellular proliferation or apoptosis has been linked to differential cyclin gene expression. We observed that in both proliferating cells and cells undergoing apoptosis, cyclin A (but not B, C, D1, and E) mRNA level was elevated in unsynchronized Myc-overexpressing cells when compared with parental Rat1a fibroblasts. We further demonstrated that Zn(2+)-inducible cyclin A expression was sufficient to cause apoptosis. When Myc-induced apoptosis was blocked by coexpression of Bcl-2, the levels of cyclin C, D1, and E mRNAs were also elevated. Thus, while apoptosis induced by c-Myc is associated with an elevated cyclin A mRNA level, protection from apoptosis by coexpressed Bcl-2 is associated with a complementary increase in cyclin C, D1, and E mRNAs.

3',3'-Difluoro-3'-deoxythymidine: comparison of anti-HIV activity to 3'-fluoro-3'-deoxythymidine.

3',3'-Difluoro-3'-deoxythymidine (3) has been synthesized in four steps from thymidine, and characterized by 1H NMR and NOE experiments. The JHF coupling constants support a conformation in solution that is predominantly 2'-endo (S). Although conformationally and sterically nucleoside 3 may resemble other thymidine analogs which are active against HIV-1, 3 is virtually inactive.

Nucleoside and nucleotide transport through a model liquid membrane. Periodic-catastrophic transport of a novel amantadine phosphoramidate conjugate of 5'-AMP.

Adenosine 5'-phosphor(adamantyl)amidate (5), an analog derived by linking the antiviral drug amantadine to 5'-AMP is transported through a model membrane system in a discontinuous periodic-catastrophic fashion. The system was composed of a glass cell containing two aqueous buffer phases separated by a chloroform layer. A more lipophilic, but structurally related derivative, adenosine 5'-phosphor(n-decyl)amidate (3) showed linear transport in the same system. Less lipophilic substances, including 5'-AMP and adenosine 5'-phosphor(morpholidyl)amidate (2), did not show transport. It is hypothesized that the periodic-catastrophic transport is a result of the collective activity of amidate 5 at the interface between the first aqueous interface and the chloroform layer. The time between catastrophic events is thought to be a reflection of the time necessary for molecular organization at the interface. The phenomenon is a new example of molecular organization in a system far from equilibrium leading to a repetitive dynamic process.

In vitro and in vivo studies of a promising antileukemic thymidine analogue, 5-hydroxymethyl-2' deoxyuridine.

The toxicity and metabolism of a thymidine analogue, 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd) were studied with human leukemia cells (HL-60) and with human platelets. 3 X 10(-5) M 5HmdUrd caused a 50% inhibition in the proliferation of HL-60 cells. The compound was hydrolyzed to 5-hydroxymethyluracil (5HmUra) by the enzyme thymidine phosphorylase (EC 2.4.2.4) present in leukemia cells; this catabolic product was non-toxic. The catabolism of 5HmdUrd by human platelet thymidine phosphorylase could be inhibited by 6-aminothymine. The toxicity of 5HmdUrd was effectively reversed by deoxycytidine and 5HmdUrd increased the incorporation of deoxycytidine into dCTP and DNA several fold. The two latter phenomena are explicable in terms of a feedback action to ribonucleotide reductase, resulting in deoxycytidylate starvation, which is a known effect of excess thymidine. We report here also our preliminary observations that 5HmdUrd is active against mouse leukemia in vivo.

Antirhinovirus activity of purine nucleoside analogs.

A wide variety of purine nucleoside (mainly tubercidin and adenosine) analogs, which had previously been shown to inhibit the replication of a broad spectrum of RNA viruses, were evaluated for their antirhinovirus activity in human diploid (WI-38) fibroblasts. Tubercidin, 5-(1-hydroxyethyl)tubercidin, 5-(2-buten-1-yl)tubercidin, toyocamycin, and sangivamycin emerged as the most potent inhibitors. These compounds inhibited the replication of rhinovirus types 1A, 1B, and 9 at an MIC well below 1 microgram/ml. However, these compounds proved cytotoxic for the uninfected host cells at concentrations which were only slightly higher (3- to 10-fold, on the average) than those required for inhibition of rhinovirus replication. The most selective inhibitor of rhinovirus replication was 3-deazaguanine, with a selectivity index of 50. None of the carbocyclic and acyclic analogs of adenosine tested exhibited a potent or selective antirhinovirus activity.

5-Hydroxymethyl-2'-deoxyuridine. Cytotoxicity and DNA incorporation studied by using a novel [2-14C]-derivative with normal and leukemic human hematopoietic cells.

5-Hydroxymethyl-2'-deoxyuridine is a biologically active thymidine analogue. This investigation was aimed at characterizing the cytotoxicity of 5-hydroxymethyl-2'-deoxyuridine and its incorporation into DNA. Fifty percent inhibition of cellular proliferation, assessed by incorporation of [U-14C]-L-leucine in vitro, was caused by 1.7-5.8 X 10(-5) incorporation of [U-14C]-L-leucine in vitro, was caused by 1.7-5.8 X 10(-5) M 5-hydroxymethyl-2'-deoxyuridine in seven human leukemia cell lines. Higher concentrations of 5-hydroxymethyl-2'-deoxyuridine, i.e. 6-8 X 10(-5) M, were required for a comparable inhibition in human PHA-stimulated peripheral blood lymphocytes. A new synthesis procedure for [2-14C]5-hydroxymethyl-2'-deoxyuridine was developed. The net incorporation of [2-14C]5-hydroxymethyl-2'-deoxyuridine into DNA of hematopoietic cells was low. The possibility of a repair mechanism for 5-hydroxymethyluracil bound to DNA is discussed.