Cell Signaling-Based Classifier Predicts Response to Induction Therapy in Elderly Patients with Acute Myeloid Leukemia

Single-cell network profiling (SCNP) data generated from multi-parametric flow cytometry analysis of bone marrow (BM) and peripheral blood (PB) samples collected from patients >55 years old with non-M3 AML were used to train and validate a diagnostic classifier (DX_SCNP) for predicting response to standard induction chemotherapy (complete response [CR] or CR with incomplete hematologic recovery [CRi] versus resistant disease [RD]). SCNP-evaluable patients from four SWOG AML trials were randomized between Training (N = 74 patients with CR, CRi or RD; BM set = 43; PB set = 57) and Validation Analysis Sets (N = 71; BM set = 42, PB set = 53). Cell survival, differentiation, and apoptosis pathway signaling were used as potential inputs for DX_SCNP. Five DX_SCNP classifiers were developed on the SWOG Training set and tested for prediction accuracy in an independent BM verification sample set (N = 24) from ECOG AML trials to select the final classifier, which was a significant predictor of CR/CRi (area under the receiver operating characteristic curve AUROC = 0.76, p = 0.01). The selected classifier was then validated in the SWOG BM Validation Set (AUROC = 0.72, p = 0.02). Importantly, a classifier developed using only clinical and molecular inputs from the same sample set (DX_CLINICAL2) lacked prediction accuracy: AUROC = 0.61 (p = 0.18) in the BM Verification Set and 0.53 (p = 0.38) in the BM Validation Set. Notably, the DX_SCNP classifier was still significant in predicting response in the BM Validation Analysis Set after controlling for DX_CLINICAL2 (p = 0.03), showing that DX_SCNP provides information that is independent from that provided by currently used prognostic markers. Taken together, these data show that the proteomic classifier may provide prognostic information relevant to treatment planning beyond genetic mutations and traditional prognostic factors in elderly AML.     

Concordant phylogeography and cryptic speciation in two Western Palaearctic oak gall parasitoid species complexes

Little is known about the evolutionary history of most complex multi‐trophic insect communities. Widespread species from different trophic levels might evolve in parallel, showing similar spatial patterns and either congruent temporal patterns (Contemporary Host‐tracking) or later divergence in higher trophic levels (Delayed Host‐tracking). Alternatively, host shifts by natural enemies among communities centred on different host resources could disrupt any common community phylogeographic pattern. We examined these alternative models using two Megastigmus parasitoid morphospecies associated with oak cynipid galls sampled throughout their Western Palaearctic distributions. Based on existing host cynipid data, a parallel evolution model predicts that eastern regions of the Western Palaearctic should contain ancestral populations with range expansions across Europe about 1.6 million years ago and deeper species‐level divergence at both 8–9 and 4–5 million years ago. Sequence data from mitochondrial cytochrome b and multiple nuclear genes showed similar phylogenetic patterns and revealed cryptic genetic species within both morphospecies, indicating greater diversity in these communities than previously thought. Phylogeographic divergence was apparent in most cryptic species between relatively stable, diverse, putatively ancestral populations in Asia Minor and the Middle East, and genetically depauperate, rapidly expanding populations in Europe, paralleling patterns in host gallwasp species. Mitochondrial and nuclear data also suggested that Europe may have been colonized multiple times from eastern source populations since the late Miocene. Temporal patterns of lineage divergence were congruent within and across trophic levels, supporting the Contemporary Host‐tracking Hypothesis for community evolution.     

Activation of the mitogen- and stress-activated kinase 1 by arsenic trioxide

Arsenic trioxide (As2O3) is a potent inducer of apoptosis of leukemic cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As2O3 induces activation of the mitogen- and stress-activated kinase 1 (MSK1) and downstream phosphorylation of its substrate, histone H3, in leukemia cell lines. Such activation requires upstream engagement of p38 MAPK, as demonstrated by experiments using pharmacological inhibitors of p38 or p38alpha knock-out cells. Arsenic-induced apoptosis was enhanced in cells in which MSK1 expression was decreased using small interfering RNA and in Msk1 knock-out mouse embryonic fibroblasts, suggesting that this kinase is activated in a negative feedback regulatory manner to regulate As2O3 responses. Consistent with this, pharmacological inhibition of MSK1 enhanced the suppressive effects of As2O3 on the growth of primary leukemic progenitors from chronic myelogenous leukemia patients. Altogether, these findings indicate an important role for MSK1 downstream of p38 in the regulation of As2O3 responses.     

Gene flow and effective population size in two life-history types of broad whitefish Coregonus nasus from the Canadian Arctic

In this study, the magnitude and direction of gene flow and estimates of effective population sizes (N(e) ) were quantified among two life-history types (lacustrine and anadromous) of broad whitefish Coregonus nasus in the lower Mackenzie River system. The data suggest that dispersal and subsequent gene flow occurs between these groups, with the former appearing to be asymmetrical. Gene flow may potentially be directionally biased as well, a result attributed to source-sink population dynamics and the ongoing process of post-glacial colonization and contemporary range expansion. Additionally, average N(e) estimates were consistently lower for lacustrine populations of C. nasus although confidence intervals for both contemporary and historical estimates broadly overlapped. The lower average estimates of N(e) for lacustrine populations was suggested to be the result of more recent founding events following post-glacial dispersal. This study provides one of the first assessments of gene flow and N(e) in an Arctic coregonine, results that may be relevant to other freshwater and anadromous Arctic species persisting in systems near the periphery of their range.     

Species loss of stoneflies (Plecoptera) in the Czech Republic during the 20th century

1. Rapid expansion and intensification of anthropogenic activities in the 20th century has caused profound changes in freshwater assemblages. Unfortunately, knowledge of the extent and causes of species loss (SL) is limited due to the lack of reliable historical data. An unusual data set allows us to compare changes in the most sensitive of aquatic insect orders, the Plecoptera, at some 170 locations in the Czech Republic between two time periods, 1955–1960 and 2006–2010. Historical data (1890–1911) on assemblages of six lowland rivers allow us to infer even earlier changes. 2. Regional stonefly diversity decreased in the first half of the 20th century. Streams at lower altitudes lost a substantial number of species, which were never recovered. In the second half of the century, large‐scale anthropogenic pressure caused SL in all habitats, leading to a dissimilarity of contemporary and previous assemblages. The greatest changes were found at sites affected by organic pollution and a mixture of organic pollution and channelisation or impoundment. Colonisation of new habitats was observed in only three of the 80 species evaluated. 3. Species of moderate habitat specialisation and tolerance to organic pollution were most likely to be lost. Those with narrow specialisations in protected habitats were present in both historical and contemporary collections. 4. Contemporary assemblages are the consequence of more than a 100 years of anthropogenic impacts. In particular, streams at lower altitude and draining intensively exploited landscapes host a mere fragment of the original species complement. Most stonefly species are less frequently present than before, although their assemblages remain almost intact in near‐natural mountain streams. Our analyses demonstrate dramatic restriction of species ranges and, in some cases, apparent changes in altitudinal preference throughout the area.     

Regulatory effects of mammalian target of rapamycin-mediated signals in the generation of arsenic trioxide responses

Arsenic trioxide (As(2)O(3)) is a potent inducer of apoptosis of leukemic cells in vitro and in vivo, but the mechanisms that mediate such effects are not well understood. We provide evidence that the Akt kinase is phosphorylated/activated during treatment of leukemia cells with As(2)O(3), to regulate downstream engagement of mammalian target of rapamycin (mTOR) and its effectors. Using cells with targeted disruption of both the Akt1 and Akt2 genes, we found that induction of arsenic trioxide-dependent apoptosis is strongly enhanced in the absence of these kinases, suggesting that Akt1/Akt2 are activated in a negative feedback regulatory manner, to control generation of As(2)O(3) responses. Consistent with this, As(2)O(3)-dependent pro-apoptotic effects are enhanced in double knock-out cells for both isoforms of the p70 S6 kinase (S6k1/S6k2), a downstream effector of Akt and mTOR. On the other hand, As(2)O(3)-dependent induction of apoptosis is diminished in cells with targeted disruption of TSC2, a negative upstream effector of mTOR. In studies using primary hematopoietic progenitors from patients with acute myeloid leukemia, we found that pharmacological inhibition of mTOR enhances the suppressive effects of arsenic trioxide on leukemic progenitor colony formation. Moreover, short interfering RNA-mediated inhibition of expression of the negative downstream effector, translational repressor 4E-BP1, partially reverses the effects of As(2)O(3). Altogether, these data provide evidence for a key regulatory role of the Akt/mTOR pathway in the generation of the effects of As(2)O(3), and suggest that targeting this signaling cascade may provide a novel therapeutic approach to enhance the anti-leukemic properties of As(2)O(3).     

Regulation of arsenic trioxide-induced cellular responses by Mnk1 and Mnk2

Arsenic trioxide (As(2)O(3)) is a potent inducer of apoptosis of malignant cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As(2)O(3) induces phosphorylation/activation of the MAPK signal-integrating kinases (Mnks) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38alpha MAPK gene, indicating that it requires upstream engagement of the p38 MAPK pathway. Studies using Mnk1(-/-) or Mnk2(-/-), or double Mnk1(-/-)Mnk2(-/-) knock-out cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E at Ser-209. Importantly, arsenic-induced apoptosis is enhanced in cells with targeted disruption of the Mnk1 and/or Mnk2 genes, suggesting that these kinases are activated in a negative-feedback regulatory manner, to control generation of arsenic trioxide responses. Consistent with this, pharmacological inhibition of Mnk activity enhances the suppressive effects of arsenic trioxide on primary leukemic progenitors from patients with acute leukemias. Taken together, these findings indicate an important role for Mnk kinases, acting as negative regulators for signals that control generation of arsenic trioxide-dependent apoptosis and antileukemic responses.     

Induction of CAM in Mesembryanthemum crystallinum Abolishes the Stomatal Response to Blue Light and Light-Dependent Zeaxanthin Formation in Guard Cell Chloroplasts

Facultative CAM plants such as Mesembryanthemum crystallinum(ice plant) possess C3 metabolism when unstressed but developCAM under water or salt stress. When ice plants shift from C3metabolism to CAM, their stomata remain closed during the dayand open at night. Recent studies have shown that the stomatalresponse of ice plants in the C3 mode depends solely on theguard cell response to blue light. Recent evidence for a possiblerole of the xanthophyll, zeaxanthin in blue light photoreceptionof guard cells led to the question of whether changes in theregulation of the xanthophyll cycle in guard cells parallelthe shift from diurnal to nocturnal stomatal opening associatedwith CAM induction. In the present study, light-dependent stomatalopening and the operation of the xanthophyll cycle were characterizedin guard cells isolated from ice plants shifting from C3 metabolismto CAM. Stomata in epidermis detached from leaves with C3 metabolismopened in response to white light and blue light, but they didnot open in response to red light. Guard cells from these leavesshowed light-dependent conversion of violaxan-thin to zeaxanthin.Induction of CAM by NaCI abolished both white light- and bluelight-stimulated stomatal opening and light-dependent zeaxanthinformation. When guard cells isolated from leaves with CAM weretreated with 100 mM ascorbate, pH 5.0 for 1 h in darkness, guardcell zeaxanthin content increased at rates equal to or higherthan those stimulated by light in guard cells from leaves inthe C3 mode. The ascorbate effect indicates that chloroplastsin guard cells from leaves with CAM retain their competenceto operate the xanthophyll cycle, but that zeaxanthin formationdoes not take place in the light. The data suggest that inhibitionof light-dependent zeaxanthin formation in guard cells mightbe one of the regulatory steps mediating the shift from diurnalto nocturnal stomatal opening typical of plants with CAM. (Received July 5, 1996; Accepted December 12, 1996)