Relapsed childhood acute lymphoblastic leukemia (ALL) carries a poor prognosis despite intensive retreatment, due to intrinsic drug resistance1-2. The biological pathways that mediate resistance are unknown. Here we report the transcriptome profiles of matched diagnosis and relapse bone marrow specimens from ten pediatric B lymphoblastic leukemia patients using RNA-sequencing. Transcriptome sequencing identified 20 newly acquired novel non-synonymous mutations not present at initial diagnosis, of which two patients harbored relapse specific mutations in the same gene, NT5C2, a 5′-nucleotidase. Full exon sequencing of NT5C2 was completed in 61 additional relapse specimens, identifying five additional cases. Enzymatic analysis of mutant proteins revealed that base substitutions conferred increased enzymatic activity and resistance to treatment with nucleoside analogue therapies. Clinically, all patients who harbored NT5C2 mutations relapsed early, or within 36 months of initial diagnosis (p=0.03). These results suggest that mutations in NT5C2 are associated with the outgrowth of drug resistant clones in ALL.

• Genetic or pharmacologic inhibition of MEK4 and MEK2 enhances prednisoloneinduced cell death in ALL models.• MAPK signaling cascades are activated at relapse compared to diagnosis in ALL samples and have enhanced response to MEK inhibition.The outcome for pediatric acute lymphoblastic leukemia (ALL) patients who relapse is dismal. A hallmark of relapsed disease is acquired resistance to multiple chemotherapeutic agents, particularly glucocorticoids. In this study, we performed a genome-scale short hairpin RNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of these data with an integrated analysis of relapse-specific genetic and epigenetic changes allowed us to identify the mitogen-activated protein kinase (MAPK) pathway as a mediator of prednisolone resistance in pediatric ALL. We show that knockdown of the specific MAPK pathway members MEK2 and MEK4 increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to prednisolone by increasing the levels of the glucocorticoid receptor. MEK2 knockdown increased sensitivity to all chemotherapy agents tested by increasing the levels of p53. Furthermore, we demonstrate that inhibition of MEK1/2 with trametinib increased sensitivity of ALL cells and primary samples to chemotherapy in vitro and in vivo. To confirm a role for MAPK signaling in patients with relapsed ALL, we measured the activation of the MEK1/2 target ERK in matched diagnosis-relapse primary samples and observed increased phosphorylated ERK levels at relapse. Furthermore, relapse samples have an enhanced response to MEK inhibition compared to matched diagnosis samples in xenograft models. Together, our data indicate that inhibition of the MAPK pathway increases chemosensitivity to glucocorticoids and possibly other agents and that the MAPK pathway is an attractive target for prevention and/or treatment of relapsed disease. (Blood. 2015;126(19):2202-2212

Dandekar S, Privitera C, Carney T, Klein SA. Neural saccadic response estimation during natural viewing. J Neurophysiol 107: 1776-1790, 2012. First published December 14, 2011 doi:10.1152/jn.00237.2011Studying neural activity during natural viewing conditions is not often attempted. Isolating the neural response of a single saccade is necessary to study neural activity during natural viewing; however, the close temporal spacing of saccades that occurs during natural viewing makes it difficult to determine the response to a single saccade. Herein, a general linear model (GLM) approach is applied to estimate the EEG neural saccadic response for different segments of the saccadic main sequence separately. It is determined that, in visual search conditions, neural responses estimated by conventional eventrelated averaging are significantly and systematically distorted relative to GLM estimates due to the close temporal spacing of saccades during visual search. Before the GLM is applied, analyses are applied that demonstrate that saccades during visual search with intersaccadic spacings as low as 100 -150 ms do not exhibit significant refractory effects. Therefore, saccades displaying different intersaccadic spacings during visual search can be modeled using the same regressor in a GLM. With the use of the GLM approach, neural responses were separately estimated for five different ranges of saccade amplitudes during visual search. Occipital responses time locked to the onsets of saccades during visual search were found to account for, on average, 79 percent of the variance of EEG activity in a window 90 -200 ms after the onsets of saccades for all five saccade amplitude ranges that spanned a range of 0.2-6.0 degrees. A GLM approach was also used to examine the lateralized ocular artifacts associated with saccades. Possible extensions of the methods presented here to account for the superposition of microsaccades in event-related EEG studies conducted in nominal fixation conditions are discussed. saccades; EEG; visual search; microsaccades; artifact removal EEG IS WELL SUITED FOR THE study of fast neural dynamics, and a variety of EEG studies have successfully explored saccadic planning (Everling et al. 1997;Richards 2003) and postsaccadic neural responses (Bellebaum et al. 2005;Ossandón et al. 2010). However, most EEG studies of saccadic eye movements are conducted with sparse synthetic visual targets and/or temporally well separated saccades in conditions that do not resemble those that are involved in natural viewing conditions. A variety of fMRI studies of saccadic eye movements, most of which were conducted in more controlled conditions than those associated with natural viewing, have identified various sites of neural activation likely involved in saccadic eye movements (Connolly et al. 2002;Corbetta et al. 1998). However, the low temporal resolution of fMRI makes the study of fast temporal neural dynamics during natural viewing conditions impractical. A general linear least squares or general linear model (GLM...

Summary While childhood acute lymphoblastic leukemia (ALL) is now highly curable, the dismal prognosis for children who relapse warrants novel therapeutic approaches. Previously, using an integrated genomic analysis of matched diagnosis - relapse paired samples, we identified overactivation of the Wnt pathway as a possible mechanism of recurrence. To validate these findings and document whether Wnt inhibition may sensitize cells to chemotherapy, we analyzed the expression of Activated β-catenin (and its downstream target BIRC5) using multiparameter phosphoflow cytometry and tested the efficacy of a recently developed Wnt inhibitor, iCRT14, in ALL cell lines and patient samples. We observed increased activation of β-catenin at relapse in 6 /10 patients. Furthermore, treatment of leukemic cell lines with iCRT14 led to significant downregulation of Wnt target genes and combination with traditional chemotherapeutic drugs resulted in a synergistic decrease in viability as well as a significant increase in apoptotic cell death. Finally, pre-treatment of purified blasts from patients with relapsed leukemia with the Wnt inhibitor followed by exposure to prednisolone, restored chemosensitivity in these cells. Our results demonstrate that overactivation of the Wnt pathway may contribute to chemoresistance in relapsed childhood ALL and that Wnt-inhibition may be a promising therapeutic approach.

Attending to a task-relevant location changes how neural activity oscillates in the alpha band (8–13Hz) in posterior visual cortical areas. However, a clear understanding of the relationships between top-down attention, changes in alpha oscillations in visual cortex, and attention performance are still poorly understood. Here, we tested the degree to which the posterior alpha power tracked the locus of attention, the distribution of attention, and how well the topography of alpha could predict the locus of attention. We recorded magnetoencephalographic (MEG) data while subjects performed an attention demanding visual discrimination task that dissociated the direction of attention from the direction of a saccade to indicate choice. On some trials, an endogenous cue predicted the target’s location, while on others it contained no spatial information. When the target’s location was cued, alpha power decreased in sensors over occipital cortex contralateral to the attended visual field. When the cue did not predict the target’s location, alpha power again decreased in sensors over occipital cortex, but bilaterally, and increased in sensors over frontal cortex. Thus, the distribution and the topography of alpha reliably indicated the locus of covert attention. Together, these results suggest that alpha synchronization reflects changes in the excitability of populations of neurons whose receptive fields match the locus of attention. This is consistent with the hypothesis that alpha oscillations reflect the neural mechanisms by which top-down control of attention biases information processing and modulate the activity of neurons in visual cortex.