Dihydroartemisinin-induced ferroptosis in acute myeloid leukemia: links to iron metabolism and metallothionein.

Artemisinin is an anti-malarial drug that has shown anticancer properties. Recently, ferroptosis was reported to be induced by dihydroartemisinin (DHA) and linked to iron increase. In the current study, we determined the effect of DHA in leukemic cell lines on ferroptosis induction and iron metabolism and the cytoprotective effect triggered in leukemic cells. We found that treatment of DHA induces early ferroptosis by promoting ferritinophagy and subsequent iron increase. Furthermore, our study demonstrated that DHA activated zinc metabolism signaling, especially the upregulation of metallothionein (MT). Supportingly, we showed that inhibition MT2A and MT1M isoforms enhanced DHA-induced ferroptosis. Finally, we demonstrated that DHA-induced ferroptosis alters glutathione pool, which is highly dependent on MTs-driven antioxidant response. Taken together, our study indicated that DHA activates ferritinophagy and subsequent ferroptosis in AML and that MTs are involved in glutathione regenerating and antioxidant response.

High mTORC1 activity drives glycolysis addiction and sensitivity to G6PD inhibition in acute myeloid leukemia cells.

Alterations in metabolic activities are cancer hallmarks that offer a wide range of new therapeutic opportunities. Here we decipher the interplay between mTORC1 activity and glucose metabolism in acute myeloid leukemia (AML). We show that mTORC1 signaling that is constantly overactivated in AML cells promotes glycolysis and leads to glucose addiction. The level of mTORC1 activity determines the sensitivity of AML cells to glycolysis inhibition as switch-off mTORC1 activity leads to glucose-independent cell survival that is sustained by an increase in mitochondrial oxidative phosphorylation. Metabolic analysis identified the pentose phosphate pathway (PPP) as an important pro-survival pathway for glucose metabolism in AML cells with high mTORC1 activity and provided a clear rational for targeting glucose-6-phosphate dehydrogenase (G6PD) in AML. Indeed, our analysis of the cancer genome atlas AML database pinpointed G6PD as a new biomarker in AML, as its overexpression correlated with an adverse prognosis in this cohort. Targeting the PPP using the G6PD inhibitor 6-aminonicotinamide induces in vitro and in vivo cytotoxicity against AML cells and synergistically sensitizes leukemic cells to chemotherapy. Our results demonstrate that high mTORC1 activity creates a specific vulnerability to G6PD inhibition that may work as a new AML therapy.

Vemurafenib pharmacokinetics and its correlation with efficacy and safety in outpatients with advanced BRAF-mutated melanoma.

Vemurafenib is a BRAF kinase inhibitor approved for first-line treatment of metastatic BRAF (V600) -mutant melanoma. However, data on the pharmacokinetic/pharmacodynamic (PK/PD) relationship are lacking. The aim of this prospective, multicenter study was to explore the PK/PD relationship for vemurafenib in outpatients with advanced BRAF-mutated melanoma. Fifty-nine patients treated with single-agent vemurafenib were prospectively analyzed. Vemurafenib plasma concentration (n = 159) was measured at days 15, 30, 60, and 90 after treatment initiation. Clinical and biological determinants (including plasma vemurafenib concentration) for efficacy and safety were assessed using Cox's model and multivariate stepwise logistic regression. Median progression-free survival (PFS) and overall survival were 5.0 (95 % confidence interval [95 % CI] 2.0-6.0) and 11.0 (95% CI 7.0-16.0) months, respectively. Twenty-nine patients (49 %) experienced any grade ≥3 toxicity and the most frequent grade ≥2 toxicity was skin rash (37 %). Severe toxicities led to definitive discontinuation in seven patients (12 %). Grade ≥2 skin rash was not statistically associated with better objective response at day 60 (p = 0.06) and longer PFS (hazard ratio 0.47; 95 % CI 0.21-1.08; p = 0.075). Grade ≥2 skin rash was statistically increased in patients with ECOG ≥ 1 (odds ratio 4.67; 95 % CI 1.39-15.70; p = 0.012). Vemurafenib concentration below 40.4 mg/L at day 15 was significantly associated with a shorter PFS (1.5 [0.5-5.5] vs. 4.5 [2-undetermined] months, p = 0.029). Finally, vemurafenib concentration was significantly greater in patients developing grade ≥2 rash (61.7 ± 25.0 vs. 36.3 ± 17.9 mg/L, p < 0.0001). These results suggest that early plasma drug monitoring may help identify outpatients at high risk of non-response or grade ≥ 2 skin rash.

Flow cytometric detection of dyserythropoiesis: a sensitive and powerful diagnostic tool for myelodysplastic syndromes.

Several groups have published flow cytometry scores useful for the diagnosis or prognosis of myelodysplastic syndromes (MDS), mainly based on the detection of immunophenotypic abnormalities in the maturation of granulocytic/monocytic and lymphoid lineages. As anemia is the most frequent symptom of early MDS, the aim of this study was to identify markers of dyserythropoiesis relevant for the diagnosis of MDS analyzed by selecting erythroblasts in a whole no-lysis bone marrow strategy by using a nuclear dye. This prospective study included 163 patients, including 126 with cytopenias leading to MDS suspicion and 46 controls without MDS. In a learning cohort of 53 unequivocal MDS with specific markers, there was a significant difference between the coefficients of variation of mean fluorescence intensities of CD71 and CD36 in MDS patients compared with controls. These two parameters and the hemoglobin level were used to build a RED-score strongly suggestive of MDS if ≥ 3. Using the RED-score in the whole cohort, 80% of MDS or non-MDS patients were correctly classified. When combined with the flow score described by Ogata et al., this strategy allowed to reach a very high sensitivity of 88% of patients correctly classified.

A phase Ib GOELAMS study of the mTOR inhibitor RAD001 in association with chemotherapy for AML patients in first relapse.

The mTORC1 signaling pathway is constitutively activated in almost all acute myelogenous leukemia (AML) patients. We conducted a phase Ib trial combining RAD001 (everolimus), an allosteric inhibitor of mTORC1, and conventional chemotherapy, in AML patients under 65 years of age at first relapse (clinical trial NCT 01074086). Increasing doses of RAD001 from 10-70 mg were administrated orally on days 1 and 7 (d1 and d7) of a 3+7 daunorubicin+cytarabine conventional induction chemotherapy regimen. Twenty-eight patients were enrolled in this trial. The treatment was well tolerated with <10% toxicity, mainly involving the gastrointestinal tract and lungs. In this phase Ib trial, the RAD001 maximum tolerated dose was not reached at 70 mg. Sixty-eight percent of patients achieved CR, of which 14 received a double induction. Eight subsequently were intensified with allogeneic-stem cell transplant. Strong plasma inhibition of P-p70S6K was observed after RAD001 administration, still detectable at d7 (d7)at the 70 mg dosage. CR rates in patients with RAD001 areas under or above the curve median were 53% versus 85%. A 70 mg dose of RAD001 at d1 and d7 of an induction chemotherapy regimen for AML has acceptable toxicity and may improve treatment.

The dual mTORC1 and mTORC2 inhibitor AZD8055 has anti-tumor activity in acute myeloid leukemia.

The serine/threonine kinase mammalian target of rapamycin (mTOR) is crucial for cell growth and proliferation, and is constitutively activated in primary acute myeloid leukemia (AML) cells, therefore representing a major target for drug development in this disease. We show here that the specific mTOR kinase inhibitor AZD8055 blocked mTORC1 and mTORC2 signaling in AML. Particularly, AZD8055 fully inhibited multisite eIF4E-binding protein 1 phosphorylation, subsequently blocking protein translation, which was in contrast to the effects of rapamycin. In addition, the mTORC1-dependent PI3K/Akt feedback activation was fully abrogated in AZD8055-treated AML cells. Significantly, AZD8055 decreased AML blast cell proliferation and cell cycle progression, reduced the clonogenic growth of leukemic progenitors and induced caspase-dependent apoptosis in leukemic cells but not in normal immature CD34+ cells. Interestingly, AZD8055 strongly induced autophagy, which may be either protective or cell death inducing, depending on concentration. Finally, AZD8055 markedly increased the survival of AML transplanted mice through a significant reduction of tumor growth, without apparent toxicity. Our current results strongly suggest that AZD8055 should be tested in AML patients in clinical trials.

Perspectives on inhibiting mTOR as a future treatment strategy for hematological malignancies.

Mammalian target of rapamycin (mTOR) is a protein kinase implicated in the regulation of various cellular processes, including those required for tumor development, such as the initiation of mRNA translation, cell-cycle progression and cellular proliferation. In a wide range of hematological malignancies, the mTORC1 signaling pathway has been found to be deregulated and has been designed as a major target for tumor therapy. Given that pre-clinical studies have clearly established the therapeutic value of mTORC1 inhibition, numerous clinical trials of rapamycin and its derivates (rapalogs) are ongoing for treatment of these diseases. At this time, although disease stabilization and tumor regression have been observed, objective responses in some tumor types have been modest. Nevertheless, some of the mechanisms underlying cancer-cell resistance to rapamycin have now been described, thereby leading to the development of new strategy to efficiently target mTOR signaling in these diseases. In this review, we discuss the rationale for using mTOR inhibitors as novel therapies for a variety of hematological, malignancies with a focus on promising new perspectives for these approaches.

PI-103, a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, has antileukemic activity in AML.

The phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin complex 1 (mTORC1) signaling pathways are frequently activated in acute myelogenous leukemia (AML). mTORC1 inhibition with RAD001 induces PI3K/Akt activation and both pathways are activated independently, providing a rationale for dual inhibition of both pathways. PI-103 is a new potent PI3K/Akt and mTOR inhibitor. In human leukemic cell lines and in primary blast cells from AML patients, PI-103 inhibited constitutive and growth factor-induced PI3K/Akt and mTORC1 activation. PI-103 was essentially cytostatic for cell lines and induced cell cycle arrest in the G1 phase. In blast cells, PI-103 inhibited leukemic proliferation, the clonogenicity of leukemic progenitors and induced mitochondrial apoptosis, especially in the compartment containing leukemic stem cells. In contrast, apoptosis was not induced with RAD001 and IC87114 association, which specifically inhibits mTORC1 and p110delta activity, respectively. PI-103 had additive proapoptotic effects with etoposide in blast cells and in immature leukemic cells. Interestingly, PI-103 did not induce apoptosis in normal CD34(+) cells and had moderate effects on their clonogenic and proliferative properties. Here, we demonstrate that multitargeted therapy against PI3K/Akt and mTOR with PI-103 may be of therapeutic value in AML.

The esterase-like activity of serum albumin may be due to cholinesterase contamination.

PURPOSE: The "esterase-like activity" of human serum albumin (HSA) is described in the literature, but a contamination of commercially available HSA preparations by plasma cholinesterase is conceivable in some cases. The purpose of the present work was to examine this hypothesis. METHODS: The hydrolytic activity of HSA and its inhibition by physostigmine were measured fluorimetrically by monitoring the hydrolysis of the ester substrate moxisylyte. Affinity chromatography was used to separate cholinesterase and HSA. The cholinesterase activity in the eluted fractions was assessed using Ellman's reagent and butyrylthiocholine as substrate. RESULTS: A significant variation in the esterase-like activity of different albumin batches was observed. This activity was strongly inhibited by physostigmine, a well-known inhibitor of cholinesterase. Affinity chromatography led to a complete separation between HSA and the esterase activity, which was found exclusively in the cholinesterase fraction. CONCLUSIONS: The apparent esterase-like activity of HSA toward moxisylyte and butyrylthiocholine was due to a contamination by cholinesterase. With these substrates, HSA showed a total lack of esterase-like activity.

Estimation of passive horizontal linear whole-body displacement in humans.

1. Passive linear self-motion estimation along the X and Y axes was investigated in human subjects. 2. A target was viewed from a distance of 0.8 or 2.4 m from the starting point. Subjects were then blindfolded and transported toward the target on a distance of 3.2 m and back to the start. Acceleration was constant: +/- 0.2 or +/- 1 m/s2. The subjects pushed a button on both outward and return paths, when they passed the previously seen target. 3. The results showed anticipation of the target on the outward path, and not on the return. This was identical for both axes and both accelerations. 4. The data are in accord with a model of double integration of the otolith signal, suggesting that linear path integration is a basic sensory mechanism.

[Flexible choledochoscopy: two years experience]. / La cholédocoscopie souple: deux ans d'expérience.

This study is a retrospective review of 258 procedures on the gallbladder and bile ducts (115 without and 143 with flexible choledochoscope). There were 14 bile duct explorations for stone removal without choledochoscopy and 16 with a flexible choledochoscope. Residual stones were found in 2/14 (14.2%) cases without and 1/16 (6.3%) case with choledochoscope. A review of the literature confirms that there are fewer residual bile duct stones following flexible choledochoscopy. There were no complications attributable to the procedure.

[Simultaneous proximal and distal revascularization for arterial obstruction of the leg at two sites. Indications, techniques and results]. / Revascularisation proximale et distale simulanée pour obstruction artérielle des membres inférieurs à deux niveaux. Indications, techniques et résultats.

In chronic aortoiliac occlusive disease, 50 to 75% of the patients have further femoropopliteal lesions. The surgical treatment of these multilevel obstructions is sometimes controversial: the distal reconstruction can be performed at the time of the proximal one or deferred to a later date. It can be difficult to determine the hemodynamic importance of an iliac stenosis. Angiography is not a perfect predictor of the iliac segment hemodynamics because as it underestimates the severity of aorto-iliac stenoses when single plane views are taken. Direct measurement of the femoral pressure at angiography disclosing a gradient between brachial and femoral arteries indicates significant iliac stenosis. Intraarterial papaverine administration can also be of importance to determine critical stenoses. Despite proximal reconstruction, distal ischemia can persist if peripheral resistance is high. Distal revascularization can relieve ischemic symptoms, providing an adequate outflow bed. However some patients do not benefit from this two-level procedure because of a steal phenomenon in the intermediate vascular bed.

[In situ venous grafts. Indications, technique and results. Prospective study of 26 consecutive cases]. / Pontages veineux in situ. Indications, technique et résultats. A propos d'une étude prospective de 26 cas consécutifs.

The in situ saphenous vein bypass has been introduced in our department since 1989. A total of 26 bypasses in 22 patients have been followed prospectively. Indications for revascularisation have been severe arterial insufficiency in 73% of the cases (stage III or IV). With the exception of one postoperative death (myocardial infarction), all the patients have recovered uneventfully, with a regression to stage I. No amputation has been necessary. Morbidity has been 30%, with mainly minor local complications. The primary patency rate is 83% at one year and 78% after 2 and 3 years, whereas the secondary patency rate is 91% at one year, and remains constant thereafter up to 3 years. Considering our results and those from the literature, we believe that the in situ technique is very valuable, especially for below-knee vascular reconstruction. Technical difficulties of the method are analysed.

Effect of post-training unilateral labyrinthectomy in a spatial orientation task by guinea pigs.

The effects of unilateral labyrinthectomy in guinea pigs have been studied on an angular orientation task consisting, in an open field, of running to a hidden goal oriented at 45 degrees with respect to the cephalocaudal axis of the animal placed in a starting-box. The task was conducted in light but in an homogeneous environment, i.e. without visual, auditory or olfactory cues indicating the location of the goal. A second group of animals was submitted to a similar task running to a hidden goal but the place of the goal was indicated by a colored card. All the animals were trained before the lesion and tested in their respective task for 1 month after the lesion. In the task conducted without conspicuous cues, animals were dramatically disturbed. In contrast, animals pretrained in the visually guided task were not impaired after the lesion. These results point out the important role of vestibular information in performing spatial tasks based on angular estimation, since, even if proprioceptive and visuokinesthetic information remain available, subjects seemed not able to maintain a correct angular trajectory. The trajectories being not disturbed in the visually guided task, one can exclude the hypothesis that such deficit was due to a purely motor disturbance.

Spatial problem-solving in a wheel-shaped maze: quantitative and qualitative analyses of the behavioural changes following damage to the hippocampus in the rat.

The behaviour of sham-operated rats and rats with damage to the dorsal hippocampus was compared in a complex spatial problem-solving task using a 'hub-spoke-rim' wheel type maze. Compared to the classical Olton 8-arm radial maze and Morris water maze, this apparatus presents the animal with a series of possible alternative routes both direct and indirect to the goal (food). The task included 3 main stages: exploration, feeding and testing, as do the classic problem-solving tasks. During exploration, hippocampal rats were found to be more active than sham rats. Nevertheless, they displayed habituation and a relatively efficient circumnavigation, though, in both cases, different from those of sham rats. During test trials, hippocampal rats were characterized as being less accurate, making more errors than sham rats. Nevertheless, both groups increased their accuracy of first choices over trials. The qualitative analyses of test trial performance indicated that hippocampal rats were less accurate in terms of the initial error's deviation from the goal, and less efficient in terms of corrective behaviour than sham rats which used either the periphery or the spokes to attain economically the goal. Surprisingly, hippocampal rats were not limited to a taxon type orientation but learned to use the periphery, a tendency which developed over time. Seemingly, for sham rats, the problem-solving process took the form of updating information during transit. For hippocampal rats, the use of periphery reflected both an ability to discriminate its usefulness in reaching the goal via a taxis type behaviour, and some sparing of ability to generalize the closeness and the location of the goal. These results, especially the strategic correction patterns, are discussed in the light of Sutherland and Rudy's 'configurational association theory'.

Object permanence in cats: Analysis in locomotor space.

Stages IV and V object permanence were studied with 38-40-week-old cats. A constraining apparatus preventing animals from pursuing the bowl containing meat before it was concealed was used. Either the bowl was seen moving and disappeared from view behind a screen (stage IV trials), or after this sequence, it reappeared from behind the first screen and disappeared behind a second screen (stage V trials). In both situations cats performed significantly above chance but the paths taken to reach the food were different according to the stage. In stage V trials, cats expressed a preference for the path leading to the end of the second screen where the food was last seen disappearing.