Interplay of mutations, alternate mechanisms, and treatment breaks in leukaemia: Understanding and implications studied with stochastic models.

Bcr-Abl1 kinase domain mutations are the most prevalent cause of treatment resistance in chronic myeloid leukaemia (CML). Alternate resistance pathways nevertheless exist, and cell line experiments show certain patterns in the gain, and loss, of some of these alternate adaptations. These adaptations have clinical consequences when the tumour develops mechanisms that are beneficial to its growth under treatment, but slow down its growth when not treated. The results of temporarily halting treatment in CML have not been widely discussed in the clinic and there is no robust theoretical model that could suggest when such a pause in therapy can be tolerated. We constructed a dynamic model of how mechanisms such as Bcr-Abl1 overexpression and drug transporter upregulation evolve to produce resistance in cell lines, and investigate its behaviour subject to different treatment schedules, in particular when the treatment is paused ('drug holiday'). Our study results suggest that the presence of additional resistance mechanisms creates an environment which favours mutations that are either preexisting or occur late during treatment. Importantly, the results suggest the existence of tumour drug addiction, where cancer cells become dependent on the drug for (optimal) survival, which could be exploited through a treatment holiday. All simulation code is available at https://github.com/Sandalmoth/dual-adaptation.

Rotating between ponatinib and imatinib temporarily increases the efficacy of imatinib as shown in a chronic myeloid leukaemia model.

Targeted therapies for chronic myeloid leukaemia (CML) are effective, but rarely curative. Patients typically require treatment indefinitely, which gives ample time for drug resistance to evolve. Drug resistance issues are one of the main causes of death owing to CML, thus any means of preventing resistance are of importance. Drug rotations, wherein treatment is switched periodically between different drugs are one such option, and have been theorized to delay the onset of resistance. In vitro testing of drug rotation therapy is a first step towards applying it in animal or human trials. We developed a method for testing drug rotation protocols in CML cell lines based around culturing cells with a moderate amount of inhibitors interspersed with washing procedures and drug swaps. Drug rotations of imatinib and ponatinib were evaluated in a CML specific cell line, KCL-22. The growth of KCL-22 cells was initially reduced by a drug rotation, but the cells eventually adapted to the protocol. Our results show that ponatinib in a drug rotation temporarily sensitizes the cells to imatinib, but the effect is short-lived and is eventually lost after a few treatment cycles. Possible explanations for this observation are discussed.

Combating drug resistance in acute myeloid leukaemia by drug rotations: the effects of quizartinib and pexidartinib.

BACKGROUND: Acute myeloid leukaemia (AML) is an aggressive blood cancer. In approximately 30% of the cases, driver mutations in the FLT3 gene are identified. FLT3 inhibitors are used in treatment of such patients together with cytotoxic drugs or (in refractory AML) as single agents. Unfortunately, resistance to FLT3 inhibitors limits their efficacy. Resistance is often due to secondary mutations in the gene encoding the molecular target. The gatekeeper mutation F691L confers resistance to specific FLT3 inhibitors such as quizartinib, but pexidartinib is much less resistance to this mutation. Pexidartinib alone is however sensitive to many other resistance mutations. In chronic myeloid leukaemia (CML), it has been suggested that rotation between drugs with a different landscape of resistance mutations might postpone the emergence of resistance. METHODS: We studied the effect of quizartinib and pexidartinib in AML cell lines that express FLT3 (MOLM-14 and MV4-11). Using a rotation protocol, we further examined whether the emergence of resistance could be postponed. Computational modelling was used to analyse the onset of resistance and suggest which mutations are most likely to occur in a quantitative fashion. RESULTS: The cells were sensitive to both inhibitors but quickly developed resistance that could be inherited, suggesting a genetic origin. Rotation protocols were not useful to postpone the emergence of resistance, which implies that such protocols, or changing from pexidartinib to quizartinib (or vice-versa) should not be used in patients. The computational modelling led to similar conclusions and suggested that F691L is the most common mutation to occur with quizartinib, and also when both drugs are used in rotation. CONCLUSIONS: AML patients are not likely to benefit from a quizartinib/pexidartinib rotation protocol. A combination of tyrosine kinase inhibitors (with different molecular targets) might be more useful in the future. Development of specific FLT3 inhibitors that are less sensitive to resistance mutations might also lead to a better outcome.

Inferring time-dependent population growth rates in cell cultures undergoing adaptation.

BACKGROUND: The population growth rate is an important characteristic of any cell culture. During sustained experiments, the growth rate may vary due to competition or adaptation. For instance, in presence of a toxin or a drug, an increasing growth rate indicates that the cells adapt and become resistant. Consequently, time-dependent growth rates are fundamental to follow on the adaptation of cells to a changing evolutionary landscape. However, as there are no tools to calculate the time-dependent growth rate directly by cell counting, it is common to use only end point measurements of growth rather than tracking the growth rate continuously. RESULTS: We present a computer program for inferring the growth rate over time in suspension cells using nothing but cell counts, which can be measured non-destructively. The program was tested on simulated and experimental data. Changes were observed in the initial and absolute growth rates, betraying resistance and adaptation. CONCLUSIONS: For experiments where adaptation is expected to occur over a longer time, our method provides a means of tracking growth rates using data that is normally collected anyhow for monitoring purposes. The program and its documentation are freely available at https://github.com/Sandalmoth/ratrack under the permissive zlib license.

The effects of combination treatments on drug resistance in chronic myeloid leukaemia: an evaluation of the tyrosine kinase inhibitors axitinib and asciminib.

BACKGROUND: Chronic myeloid leukaemia is in principle a treatable malignancy but drug resistance is lowering survival. Recent drug discoveries have opened up new options for drug combinations, which is a concept used in other areas for preventing drug resistance. Two of these are (I) Axitinib, which inhibits the T315I mutation of BCR-ABL1, a main source of drug resistance, and (II) Asciminib, which has been developed as an allosteric BCR-ABL1 inhibitor, targeting an entirely different binding site, and as such does not compete for binding with other drugs. These drugs offer new treatment options. METHODS: We measured the proliferation of KCL-22 cells exposed to imatinib-dasatinib, imatinib-asciminib and dasatinib-asciminib combinations and calculated combination index graphs for each case. Moreover, using the median-effect equation we calculated how much axitinib can reduce the growth advantage of T315I mutant clones in combination with available drugs. In addition, we calculated how much the total drug burden could be reduced by combinations using asciminib and other drugs, and evaluated which mutations such combinations might be sensitive to. RESULTS: Asciminib had synergistic interactions with imatinib or dasatinib in KCL-22 cells at high degrees of inhibition. Interestingly, some antagonism between asciminib and the other drugs was present at lower degrees on inhibition. Simulations revealed that asciminib may allow for dose reductions, and its complementary resistance profile could reduce the risk of mutation based resistance. Axitinib, however, had only a minor effect on T315I growth advantage. CONCLUSIONS: Given how asciminib combinations were synergistic in vitro, our modelling suggests that drug combinations involving asciminib should allow for lower total drug doses, and may result in a reduced spectrum of observed resistance mutations. On the other hand, a combination involving axitinib was not shown to be useful in countering drug resistance.

Stochastic modelling of tyrosine kinase inhibitor rotation therapy in chronic myeloid leukaemia.

BACKGROUND: Resistance towards targeted cancer treatments caused by single nucleotide variations is a major issue in many malignancies. Currently, there are a number of available drugs for chronic myeloid leukaemia (CML), which are overcome by different sets of mutations. The main aim of this study was to explore if it can be possible to exploit this and create a treatment protocol that outperforms each drug on its own. METHODS: We present a computer program to test different treatment protocols against CML, based on available resistance mutation growth data. The evolution of a relatively stable pool of cancer stem cells is modelled as a stochastic process, with the growth of cells expressing a tumourigenic protein (here, Abl1) and any emerging mutants determined principally by the drugs used in the therapy. RESULTS: There can be some benefit to Bosutinib-Ponatinib rotation therapy even if the mutation status is unknown, whereas Imatinib-Nilotinib rotation is unlikely to improve the outcomes. Furthermore, an interplay between growth inhibition and selection effects generates a non-linear relationship between drug doses and the risk of developing resistance. CONCLUSIONS: Drug rotation therapy might be able to delay the onset of resistance in CML patients without costly ongoing observation of mutation status. Moreover, the simulations give credence to the suggestion that lower drug concentrations may achieve better results following major molecular response in CML.

Correlation between translation efficiency and outcome of combination therapy in chronic hepatitis C genotype 3.

Combination therapy with interferon-alpha (IFN-alpha) and ribavirin (RBV) in chronic hepatitis C demonstrates the best responses against hepatitis C virus (HCV) of genotype 3. Still, it has proven to be ineffective in 20-30% of patients infected with this genotype. In the present study, we analysed the translation efficiency mediated by the internal ribosome entry site (IRES) region in HCV genotype 3 genomes isolated from sustained responders (SR) and non-responders (NR), assuming that this may influence the outcome of treatment. Pretreatment isolates of genotype 3 from 22 individuals (15 SR, seven NR) were selected for such analyses. The IRES region [nucleotide (nt) 1-407] was cloned into a dual luciferase vector and IRES activity assessed following transfection into various cell lines. Low relative translation efficiency was observed for IRES elements derived from SR patients, whereas those of NR patients showed significantly greater translation efficiency (29.7 +/- 13 vs 69.4 +/- 22; P < 0.01). Subsequently, the effect of IFN-alpha plus RBV on IRES-driven translation in vitro was determined. A greater suppressive effect was observed on IRES activity isolated from seven SR patients, when compared with seven NR patients. In conclusion, IRES efficiency in vitro correlated with treatment response for HCV genotype 3. Further studies are warranted to investigate whether IRES efficiency in vitro, or sequence motifs associated with IRES efficiency, will be worthwhile to explore as prognostic tools for other HCV genotypes in the treatment of chronic HCV infection.

The implant-supported telescopic prosthesis: a biomechanical analysis.

This in vitro project investigated load transfer through screw-retained telescopic prostheses. Three Brånemark System implants incorporating strain gauges were embedded in an aluminum block. Telescopic prostheses that included 1 mesial and 1 distal cantilever were fabricated over 1 central EsthetiCone and 2 Ti-Adapt abutments. The buffering capacity of the cement in a combined screw-retained/cemented prosthesis was studied. The degree of misfit of the prostheses could be adjusted by applying shims of various thicknesses under the EsthetiCone. Load distribution was measured while a 50-N load was applied in turn over each implant and each cantilever. The results showed that tightening the central prosthetic screw widened the load distribution. The cement accommodated misfits between the layers of the telescope, significantly reducing bending moments on some supporting implants. The system exhibited a degree of tolerance to misfit and can provide a versatile prosthodontic option.

Tilting of posterior mandibular and maxillary implants for improved prosthesis support.

Rehabilitation of atrophied edentulous arches with endosseous implants in the posterior regions is often associated with anatomic problems such as jaw shape and location of the mental loop, mandibular canal, and maxillary sinuses. The purpose of this investigation was to modify the method for implant placement in the posterior part of the jaws to extend fixed implant-connected prostheses further distally, and to reduce the length of cantilevers in complete-arch prostheses without transpositioning the mandibular nerve or performing bone grafting in the maxilla. Forty-seven consecutive patients were treated with implants (25 patients/36 mandibular implants, 22 patients/30 maxillary implants) placed in tilted positions. They were followed a mean of 40 months (mandibles) and 53 months (maxillae). In the mandible, implants close to the mental foramina were tilted posteriorly approximately 25 to 35 degrees. In the maxilla, the posterior implants were placed close to and parallel with the sinus walls and were titled anteriorly/posteriorly approximately 30 to 35 degrees. Patients gained a mean distance of 6.5 mm of prosthesis support in the mandible and 9.3 mm in the maxilla, as a result of implant tilting. There were no implant failures in mandibles. The cumulative success rates in the maxilla at 5 years were 98% for tilted implants and 93% for non-tilted implants. Paresthesias of the mental nerve were observed on 4 sides during the first 2 to 3 weeks after implant placement. Analysis of the load distribution in one mandibular case showed no significant difference between tilted and the non-tilted implants, and the improved prosthesis support was confirmed. Satisfactory medium-term results concerning osseointegration and significant extension of prosthesis support show that the method can be recommended. This technique may allow for longer implants to be placed with improved bone anchorage.

Brånemark System Wide Platform implants for single molar replacement: clinical evaluation of prospective and retrospective materials.

BACKGROUND: The use of oral implants for single tooth replacement has become a predictable treatment modality. As single tooth loss is most common in posterior areas of the jaws, the use of the protocol is of specific interest in those regions. New implant designs aimed at this purpose have also been introduced. PURPOSE: The aim of the study was to present the outcomes for wide diameter implant treatment when being used in posterior areas of the jaws. MATERIALS AND METHODS: This paper presents the 3-year results of a prospective multicenter study (three clinics; n = 38 implants) and the 1-year results from a retrospective multicenter study (two clinics; n = 20 implants) on wide diameter implants for single molar replacement. Based on the hypothesis that dense bone in posterior mandibles would benefit from careful surgery and longer remodeling time, the influences of surgical technique and healing time on implant success and bone resorption were particularly addressed. RESULTS: The outcome demonstrated a good predictability for Brånemark System Wide Platform implants, at least short term, when used as single molar support (prospective group cumulative success rate [CSR] = 92% after 3 years; retrospective group CSR = 95% after 1 year). The increased mechanical strength of the wide platform implant/abutment complex also turned out to be important for mechanical stability. CONCLUSIONS: The study indicated that it was important to carefully perform surgery in posterior mandibles in order to preserve and optimally use the existing dense bone. It was suggested that from bone healing and remodeling aspects, posterior mandibles may be more demanding to handle than corresponding areas of maxillae.

Effects of long-term treatment with methyl mercury on the developing rat brain.

Sprague-Dawley rats were exposed to low doses of methyl mercury (3.9 mg mercury/kg diet), via their dams during gestation and lactation and directly via their diet until sacrifice at 50 days postpartum, in order to study possible detrimental effects on CNS development. The methyl mercury exposure of the rats resulted in a brain concentration of 1.45 +/- 0.06 mg mercury/kg wet weight (mean +/- SEM). No general toxic effects were observed; body weight was not affected, brain weight was only slightly increased. No discernible general morphological alterations were seen in the brain as evaluated using cresyl violet histology. Furthermore, no effects on GFA-positive astrocytes in brain sections were observed and computerized morphometry of smeared astrocytes from frontal cortex, hippocampus, and cerebellum did not reveal any effects of the methyl mercury treatment. The noradrenaline (NA) and dopamine (DA) systems were also studied. In cerebellum the NA levels were increased (117% of controls, P = 0.008), whereas in other regions analyzed NA and DA levels were unchanged. Thus, long-term low-dosage exposure of methyl mercury in rats during development does not appear to exert any major effects on the morphological maturation of neurons and astrocytes. However, the results indicate that effects may occur in specific transmitter-identified systems, such as the NA input to cerebellum. The results therefore underline the need for detailed biochemical analyses to study the effects of long-term low-dosage exposure to neurotoxic compounds.

Cloning and characterization of the human ADH4 gene.

Human alcohol dehydrogenase (ADH) constitutes a set of isozymes and enzymes with different tissue and substrate specificities. The subunits are coded for by at least five gene loci, ADH1-ADH5. We now report the cloning and analysis of the human ADH4 gene coding for the class-II ADH with pi-subunits. The gene spans a region of 21 kb and is divided into nine exons and eight introns. The arrangement is the same as for all analyzed mammalian class-I genes, but the region covered is 50% larger than that in the human class-I genes. The nucleotide (nt) sequences of the exons, exon/intron boundaries and 5'- and 3'-untranslated regions were determined. The transcription start point (tsp) of the ADH4 gene was defined by primer extension and localized to a position 61 nt upstream from the ATG start codon. A TATA box and a CAAT element were identified by homology to consensus sequences for tsp. No DNA structures homologous to the glucocorticoid-responsive elements (GRE) present in the ADH2 gene were found in the upstream region of the ADH4 gene, but two structures with a 70% identity to the GRE consensus sequence were found at nonhomologous locations. The difference and the overall low degree of identity, 41%, of the upstream regions suggest different regulatory mechanisms for the class-I and class-II genes.

Effects of prenatal exposure to tributyltin and trihexyltin on behaviour in rats.

The effects of prenatal administration of tributyltin (1 and 5 mg/kg) and trihexyltin (5 mg/kg) upon the development and behavioural repertoire of rats were studied. The dose levels were selected so as not to induce maternal toxicity. No consistent delay upon occurrence of various maturation markers of the organotin-treated offspring was seen. As adults the tributyltin-treated offspring showed considerable hyperactivity following the initial habituation whereas the trihexyltin-treated offspring showed hyperactivity to a lesser degree. In the spatial learning tasks applied, the radial arm maze and the circular swim maze, tributyltin-treated rats demonstrated a clearly retarded aquisition of the radial arm maze task whereas trihexyltin-treated rats performed as well as the control rats; no differences were obtained in the swim maze task. The tributyltin-treated offspring showed a drastic potentiation of d-amphetamine-induced hyperactivity, whereas trihexyltin treatment induced only a marginal increase.

Plant-derived neurotoxic amino acids (beta-N-oxalylamino-L-alanine and beta-N-methylamino-L-alanine): effects on central monoamine neurons.

In the present study the subacute effects of beta-N-oxalylamino-L-alanine (BOAA) and beta-N-methylamino-L-alanine (BMAA) on CNS monoamine neurons in rats were investigated following intracisternal injections or local intracerebral administration into substantia nigra. In vitro effects of BOAA and BMAA on high-affinity synaptosomal uptake of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) were also examined. Intracisternal administration of BMAA decreased NA levels in hypothalamus, whereas no effects were seen on DA or 5-HT levels. Following intranigral injections of BOAA, NA levels tended to decrease in several regions, whereas the DA levels and the levels of DA metabolites were unaffected in all regions analyzed. Loss of tyrosine hydroxylase (TH) immunoreactivity in the intranigral injection sites and the presence of TH-immunoreactive pyknotic neurons near the borders of the injection sites were observed following both BOAA and BMAA treatments. Furthermore, substance P-immunoreactive terminals in substantia nigra pars reticulata were also found to have disappeared within the lesioned area following either BOAA or BMAA injections. Incubations with both BOAA and BMAA (10(-5) M) reduced high-affinity [3H]NA uptake in cortical synaptosomes to 69% and 41% of controls, respectively, whereas the striatal high-affinity [3H]DA uptake and the cortical high-affinity [3H]5-HT uptake were unaffected by BOAA or BMAA. The results demonstrate that both BOAA and BMAA can affect central monoamine neurons, although the potency and specificity of these substances on monoamine neurons when administered acutely into cerebral tissue or liquor cerebri seem to be low. However, the in vitro studies indicate selective effects of both compounds on NA neurons in synaptosomal preparations.

Purification of transfer RNA (m5U54)-methyltransferase from Escherichia coli. Association with RNA.

tRNA (m5U54)-methyltransferase (EC 2.1.1.35) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to transfer ribonucleic acid (tRNA) and thereby forming 5-methyluridine (m5U, ribosylthymine) in position 54 of tRNA. This enzyme, which is involved in the biosynthesis of all tRNA chains in Escherichia coli, was purified 5800-fold. A hybrid plasmid carrying trmA, the structural gene for tRNA (m5U54)-methyltransferase was used to amplify genetically the production of this enzyme 40-fold. The purest fraction contained three polypeptides of 42 kDa, 41 kDa and 32 kDa and a heterogeneous 48-57-kDa RNA-protein complex. All the polypeptides seem to be related to the 42/41-kDa polypeptides previously identified as the tRNA (m5U54)-methyltransferase. RNA comprises about 50% (by mass) of the complex. The RNA seems not to be essential for the methylation activity, but may increase the activity of the enzyme. The amino acid composition is presented and the N-terminal sequence of the 42-kDa polypeptide was found to be: Met-Thr-Pro-Glu-His-Leu-Pro-Thr-Glu-Gln-Tyr-Glu-Ala-Gln-Leu-Ala-Glu-Lys- . The tRNA (m5U54)-methyltransferase has a pI of 4.7 and a pH optimum of 8.0. The enzyme does not require added cations but is stimulated by Mg2+. The apparent Km for tRNA and S-adenosyl-L-methionine are 80 nM and 17 microM, respectively.

Mitochondrial aldehyde dehydrogenase from horse liver. Correlations of the same species variants for both the cytosolic and the mitochondrial forms of an enzyme.

The primary structure of the mitochondrial form of horse liver aldehyde dehydrogenase has been determined, utilizing peptide analyses and homology with other enzyme forms. The subunit exhibits N-terminal heterogeneity in size similar to that for the corresponding human mitochondrial protein, the longest form having 500 residues. Catalase was identified as a contaminant of the preparations. All four pairs within a set of aldehyde dehydrogenases can now be compared, including the same two species variants (horse and human) for both the cytosolic and mitochondrial enzyme, revealing characteristic differences although Cys-302 and other segments of presumed functional importance are unchanged. The cytosolic and mitochondrial enzymes are clearly different (172 exchanges in the horse pair; 160 exchanges in the human pair) and the mitochondrial forms are more conserved (28 exchanges of 500 residues) than the cytosolic ones (43 exchanges). Distributions of the residue substitutions also differ between the two enzyme types. These results suggest a comparatively distant separation of the cytosolic and mitochondrial enzymes into forms with separate functional constraints that are more strict on the mitochondrial than the cytosolic enzyme. Unexpectedly, positions with residues unique to one of the four enzymes are about twice as common in both of the horse proteins than in either of the human proteins. This difference may reflect a general pattern for human/non-human proteins, showing that not only functional properties of the protein, but also other factors, such as generation time (longer in man than in horse), are important for enzyme divergence.

Regulation of gene expression of class I alcohol dehydrogenase by glucocorticoids.

The effect of glucocorticoids on gene expression of rat class I alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was investigated. A cDNA clone for the beta-subunit of human ADH (ADH2) was used to analyze class I ADH mRNA levels in rat hepatoma cells, which are known to contain a functional glucocorticoid receptor. RNA gel blot analysis of total cellular RNA isolated from these cells showed hybridization of the human ADH2 cDNA probe to a single approximately equal to 1500-base RNA species. Treatment of the cells with dexamethasone (0.1 nM to 1 microM) caused a dose-dependent increase in total cellular class I ADH mRNA levels by a factor of 2-4. Maximal levels were reached within 18-24 hr of treatment. This effect was reversible following withdrawal of dexamethasone. The glucocorticoid induction of class I ADH mRNA does not seem to require ongoing protein synthesis since treatment of the cells with cycloheximide did not affect the increase in class I ADH mRNA levels by dexamethasone. The human ADH2 gene contains both upstream and within the coding region sequence motifs that display homology with response elements of genes positively regulated by glucocorticoids. These data suggest a receptor-mediated transcriptional enhancement of the ADH2 gene as the mechanism of regulation. However, analysis of RNA decay in cells treated with actinomycin D indicates that the dexamethasone-induced increase in class I ADH mRNA might, at least in part, be due to enhanced ADH mRNA stability.

Expression in Escherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation.

Human alcohol dehydrogenase (ADH, beta beta isozyme of class I) was expressed in Escherichia coli, purified to homogeneity, and characterized regarding N-terminal processing. The expression system was obtained by ligation of a cDNA fragment corresponding to the beta-subunit of human liver alcohol dehydrogenase into the vector pKK 223-3 containing the tac promoter. The enzyme, detected by Western-blot analysis and ethanol oxidizing activity, constituted up to 3% of the total amount of protein. Recombinant ADH was separated from E. coli ADH by ion-exchange chromatography and the isolated enzyme was essentially pure as judged by SDS-polyacrylamide gel electrophoresis and sequence analysis. The N-terminal sequence was identical to that of the authentic beta-subunit except that the N-terminus was non-acetylated, indicating a correct removal of the initiator methionine, but lack of further processing.