Correction: Mutant JAK3 phosphoproteomic profiling predicts synergism between JAK3 inhibitors and MEK/BCL2 inhibitors for the treatment of T-cell acute lymphoblastic leukemia.

Following the publication of this article the authors noted that data describing precisely where phosphorylation sites in proteins modulated following JAK1 or JAK3 inhibition in mutant T-ALL samples was not clearly annotated. Therefore an additional sheet has been added to Supplementary Table 2.

Mutant JAK3 phosphoproteomic profiling predicts synergism between JAK3 inhibitors and MEK/BCL2 inhibitors for the treatment of T-cell acute lymphoblastic leukemia.

Mutations in the interleukin-7 receptor (IL7R) or the Janus kinase 3 (JAK3) kinase occur frequently in T-cell acute lymphoblastic leukemia (T-ALL) and both are able to drive cellular transformation and the development of T-ALL in mouse models. However, the signal transduction pathways downstream of JAK3 mutations remain poorly characterized. Here we describe the phosphoproteome downstream of the JAK3(L857Q)/(M511I) activating mutations in transformed Ba/F3 lymphocyte cells. Signaling pathways regulated by JAK3 mutants were assessed following acute inhibition of JAK1/JAK3 using the JAK kinase inhibitors ruxolitinib or tofacitinib. Comprehensive network interrogation using the phosphoproteomic signatures identified significant changes in pathways regulating cell cycle, translation initiation, mitogen-activated protein kinase and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signaling, RNA metabolism, as well as epigenetic and apoptotic processes. Key regulatory proteins within pathways that showed altered phosphorylation following JAK inhibition were targeted using selumetinib and trametinib (MEK), buparlisib (PI3K) and ABT-199 (BCL2), and found to be synergistic in combination with JAK kinase inhibitors in primary T-ALL samples harboring JAK3 mutations. These data provide the first detailed molecular characterization of the downstream signaling pathways regulated by JAK3 mutations and provide further understanding into the oncogenic processes regulated by constitutive kinase activation aiding in the development of improved combinatorial treatment regimens.

Defining the molecular basis of oncogenic cooperation between TAL1 expression and Pten deletion in T-ALL using a novel pro-T-cell model system.

T-cell acute lymphoblastic leukemia (T-ALL) is caused by the accumulation of multiple mutations combined with the ectopic expression of transcription factors in developing T cells. However, the molecular basis underlying cooperation between transcription factor expression and additional oncogenic mutations in driving T-ALL has been difficult to assess due to limited robust T-cell model systems. Here we utilize a new ex vivo pro-T-cell model to study oncogenic cooperation. Using a systems biological approach we first dissect the pro-T-cell signaling network driven by interleukin-7, stem cell factor and Notch1 and identify key downstream Akt, Stat, E2f and Myc genetic signaling networks. Next, this pro-T-cell system was used to demonstrate that ectopic expression of the TAL1 transcription factor and Pten deletion are bona-fide cooperating events resulting in an increased stem cell signature, upregulation of a specific E2f signaling network and metabolic reprogramming with higher influx of glucose carbons into the tricarboxylic acid cycle. This ex vivo pro-T-cell system thereby provides a powerful new model system to investigate how normal T-cell signaling networks are perturbed and/or hijacked by different oncogenic events found in T-ALL.

Characterization of the genome-wide TLX1 binding profile in T-cell acute lymphoblastic leukemia.

The TLX1 transcription factor is critically involved in the multi-step pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) and often cooperates with NOTCH1 activation during malignant T-cell transformation. However, the exact molecular mechanism by which these T-cell specific oncogenes cooperate during transformation remains to be established. Here, we used chromatin immunoprecipitation followed by sequencing to establish the genome-wide binding pattern of TLX1 in human T-ALL. This integrative genomics approach showed that ectopic TLX1 expression drives repression of T cell-specific enhancers and mediates an unexpected transcriptional antagonism with NOTCH1 at critical target genes, including IL7R and NOTCH3. These phenomena coordinately trigger a TLX1-driven pre-leukemic phenotype in human thymic precursor cells, reminiscent of the thymus regression observed in murine TLX1 tumor models, and create a strong genetic pressure for acquiring activating NOTCH1 mutations as a prerequisite for full leukemic transformation. In conclusion, our results uncover a functional antagonism between cooperative oncogenes during the earliest phases of tumor development and provide novel insights in the multi-step pathogenesis of TLX1-driven human leukemia.

The Fat1 cadherin is overexpressed and an independent prognostic factor for survival in paired diagnosis-relapse samples of precursor B-cell acute lymphoblastic leukemia.

Improved survival of patients with acute lymphoblastic leukemia (ALL) has emerged from identifying new prognostic markers; however, 20% of children still suffer recurrence. Previously, the altered expression of Fat1 cadherin has been implicated in a number of solid tumors. In this report, in vitro analysis shows that Fat1 protein is expressed by a range of leukemia cell lines, but not by normal peripheral blood (PB) and bone marrow (BM) cells from healthy donors. In silico analysis of expression of array data from clinical leukemias found significant levels of Fat1 transcript in 11% of acute myeloid leukemia, 29% and 63% of ALL of B and T lineages, respectively, and little or no transcript present in normal PB or BM. Furthermore, in two independent studies of matched diagnosis-relapse of precursor B-cell (preB) ALL pediatric samples (n=32 and n=27), the level of Fat1 mRNA expression was prognostic at the time of diagnosis. High Fat1 mRNA expression was predictive of shorter relapse-free and overall survival, independent of other traditional prognostic markers, including white blood cell count, sex and age. The data presented demonstrate that Fat1 expression in preB-ALL has a role in the emergence of relapse and could provide a suitable therapeutic target in high-risk preB-ALL.

Ets-1 mediates upregulation of Mcl-1 downstream of XBP-1 in human melanoma cells upon ER stress.

Past studies have shown that upregulation of the anti-apoptotic Bcl-2 family protein Mcl-1 is a major adaptive mechanism of melanoma cells to endoplasmic reticulum (ER) stress, and has an important role in resistance of the cells to apoptosis. In this study, we show that the increase in transcription of Mcl-1 in melanoma cells triggered by pharmacological ER stress inducers is mediated by the transcription factor Ets-1. By incremental deletion analysis of the Mcl-1 promoter, we identified a DNA fragment containing an Ets-1 binding site that is transcriptionally responsive to ER stress. Mutations in the Ets-1 binding site or knockdown of Ets-1 inhibited the increase in Mcl-1, indicating that Ets-1 has a critical role in transcriptional upregulation of Mcl-1. Similar to Mcl-1, Ets-1 was transcriptionally upregulated by ER stress. This was mediated by the IRE1α/XBP-1 branch of the unfolded protein response, as upregulation of Ets-1 was inhibited in melanoma cell lines deficient in IRE1α or XBP-1 established by short hairpin RNA knockdown. Activation of the PI3k/Akt pathway downstream of XBP-1 was also involved, in that inhibition of the pathway blocked upregulation of Ets-1. Inhibition of Ets-1 enhanced ER stress-induced apoptosis in melanoma cell lines and in fresh melanoma isolates, recapitulating the effect of inhibition of Mcl-1. These results reveal a key mechanism by which Mcl-1 is transcriptionally upregulated in melanoma cells by ER stress, and identify Ets-1 as a potential target for inhibition to sensitize melanoma cells to apoptosis.

Association of thymidylate synthase enhancer region polymorphisms with thymidylate synthase activity in vivo.

Two known polymorphisms in the 5' enhancer region (ER) of the thymidylate synthase (TS) gene, a variable number of tandem repeats of a 28 bp sequence (2R/3R) and a further G>C single nucleotide substitution within the repeats, result in genotypes with 0-5 functional upstream stimulatory factor (USF) E-box consensus elements. However, the relationship between these polymorphisms, regulation of TS expression and patient response to fluoropyrimidine treatment has been inconsistent. In this study, seven possible TSER allele configurations showed similar patterns of luciferase gene expression regardless of cell type or USF-1 content, with no significant difference in promoter activity between the wild-type 2RGC and 3RGGC (1.40±0.37 vs 1.43±0.32, P=0.90), whereas the minor alleles, 2RCC and 3RGCC, were significantly reduced (0.84±0.17, P=0.01) and increased (3.19±0.72, P=0.001) respectively. Patient plasma levels of 2'-deoxyuridine, a surrogate marker of TS activity, were significantly different between genotypes (P<0.001) and inversely related to luciferase activity (P=0.02) but not to the absolute number of functional repeated elements (P=0.16), suggesting that the position, rather than the number of functional USF E-box repeats in the TSER, is responsible for determining gene expression in vitro and TS activity in vivo.

Spatial variation in diurnal surface temperatures and the distribution and abundance of an alpine grasshopper.

We studied the influence of spatial differences in diurnal surface temperatures due to topography on an alpine grasshopper (Aeropedellus clavatus). Temperature measurements on east-and west-facing alpine dry meadow slopes on Niwot Ridge in Colorado demonstrated a significant difference in diurnal surface temperatures between the two slopes throughout the growing season. A. clavatus body temperature was found to be highly correlated with nearby shaded surface temperature, and individual grasshoppers enclosed on the two slopes had significantly higher relative growth rates on the warmer east slope. Temperature effects were manifest at the population level as well. A. clavatus was significantly more abundant on the east-facing than on the west-facing slope, despite similar vegetation in both areas. This study contributes to our understanding of the myriad of factors governing insect distribution and abundance by attempting to integrate the physiological and ecological, abiotic and biotic, influences on both individuals and populations.

Reptilian and avian ovarian cycles and the evolutionary origin of volant birds.

The first birds probably evolved from a line of theropod dinosaurs in the late Jurassic or early Cretaceous. The "trees down" theory proposes that avian ancestors were arboreal, whereas the "ground-up" (cursorial) theory suggests that they were terrestrial, and ran and jumped for prey. We present suggestive evidence from reptilian and avian female reproductive biology that supports the arboreal theory, although neontological evidence can never authenticate a paleontological event. The "law of follicular constancy" is an empirical observation that the number of ovulations per female per ovulatory cycle (instantaneous fecundity, or IF) is the same regardless of the amount of ovarian tissue present. In vertebrates with an IF of two or more, surgical removal of one of the paired ovaries (unilateral ovariectomy, or ULO) leads to a doubling of ovulations from the remaining ovary (compensatory ovarian hypertrophy, or COH), this ovary cycling at the same frequency as it did before surgery. In vertebrates that produce one egg alternately from each ovary (an IF of one), however, ULO leads to a form of COH in which the remaining ovary still ovulates one egg at a time but twice as frequently. In most birds, only the left ovary is present; it ovulates a single egg every 1-2 days until the species-specific clutch size is reached. Inasmuch as this avian ovulatory pattern is similar to an accelerated version of that occurring after ULO in a species that alternates ovulation, we propose that birds evolved from dinosaurs with such a pattern. A great majority of extant reptiles with an IF of one are either anoline or gekkonid lizards, and many of these ovulate several times a year. Furthermore, most species in these tropical groups are arboreal. Even considering phylogenetic constraints within anoline and gekkonid lizards, we propose that correlations of arboreality and an IF of one in these groups are implied as adaptive relationships and represent ecological parallelisms. Therefore, we propose that the ancestors of birds were arboreal. Furthermore, they probably were of small size, as are all lizards with an IF of one.

Field experimental evidence for diffuse competition among Southwestern riparian birds.

In a 6-yr study of 70 generally insectivorous bird species in Arizona riparian woodlands, abundances of cavity-nesting species increased on 50 experimental plots compared with 49 control plots, following addition of artificial nest boxes. Open-nesting birds increased in abundance on control plots during the study but avoided experimental plots relative to control plots after box addition. Multivariate analysis revealed that a negative response of open nesters to the experiment was widespread among a large and ecologically diverse group of species. Results of this field experiment suggest that communitywide (diffuse) competition was an important factor influencing the distribution and abundance of birds in these riparian ecosystems.

Social plasticity in the acorn woodpecker.

Acorn woodpeckers (Melanerpes formicivorus) in southeastern Arizona exhibited two different types of social organization: one of highly cooperative and resident groups and another of birds that migrated and formed only temporary male-female pairs during reproduction. The occurrence of both patterns in the same population indicates a high degree of social flexibility in this species.