Aristotle said it best: “ We are what we repeatedly do. Excellence, then, is not an act, but a habit. ” This insight is profound. Let us just zoom in to our brain for a moment. Even though it has no will of its own, we know what it is repeatedly doing. Processing information. Our brain processes information all the time. Sensory information, auditory information

JAK3 is a cytosolic tyrosine kinase that associates with the common gamma chain in different cytokine receptors, in which the JAK1 kinase is another essential signaling protein. Large scale sequencing efforts recently identified mutations in the IL7R, JAK1 or JAK3 genes in about 25% of T-cell acute lymphoblastic leukemia (T-ALL) cases, with JAK3 being the most frequently (15% of T-ALL cases) mutated gene in this pathway. To determine if all mutations in JAK3 are true oncogenic mutations, we generated expression plasmids for 16 JAK3 mutants (M511I, A572T, A573V, R657Q, R657W, V674A, V678M, V678L, R775C, L857Q, Q865E, L875H, P906S, R925S, E958K, E1106G) and determined the in vitro and in vivo transforming properties, as well as their sensitivity to JAK kinase inhibitors. For 12 of 16 mutants expression in the IL3-dependent Ba/F3 cell line resulted in transformation to IL3 independent growth. Similarly, expression of the transforming mutants in IL7-receptor reconstituted 293T cells confirmed their ligand independent activation, while this was not observed for the non-transforming mutants. These 4 non-transforming mutants are likely to be passenger mutations, illustrating that results from sequencing always need to be confirmed by functional assays to distinguish driver mutations from passenger mutations. Most JAK3 mutants, except JAK3 L857Q and JAK3 L875H, were dependent on JAK1 kinase activity for their transforming capacities. In agreement with this, we observed that Ba/F3 cells transformed by the JAK1 dependent JAK3 mutants could be inhibited by ruxolitinib, a JAK1/JAK2 selective inhibitor, while the JAK3 L857Q and JAK3 L875H transformed cells were significantly less sensitive to ruxolitinib treatment. As expected, all JAK3 mutants were sensitive to the JAK3 selective inhibitor tofacitinib, except for JAK3 L875H, which showed resistance to all inhibitors tested. To determine the in vivo oncogenic properties of the JAK3 mutants, we expressed selected JAK3 mutants (M511I, A573V, L857Q, V674A and R657Q) in mouse hematopoietic cells through viral transduction. Mice transplanted with cells expressing JAK3 M511I, A573V or V674A showed a gradual increase of the WBC count and developed a T-ALL like disease within 120 to 200 days. In contrast, mice transplanted with cells expressing JAK3 L857Q or R657Q showed a lower increase in WBC count, and did present with severe splenomegaly and lymphadenopathy. Expression of JAK3 L857Q caused severe thymus hyperplasia, while the JAK3 R657Q mutant caused B-cell leukemia, illustrating that different JAK3 mutants seem to have variable oncogenic characteristics. Mice transplanted with cells expressing JAK3 M511I were treated with the JAK3 selective inhibitor tofacitinib and disease progression was followed by white blood cell count measurements. Treatment of the animals for 5 weeks with tofacitinib (oral gavage, 40 mg/kg/day) significantly decreased the disease progression compared to placebo treated mice. Moreover, we observed severe apoptosis of the leukemia cells in spleen and thymus in tofacitinib treated animals and not in placebo treated mice. However, tofacitnib treatment could not eradicate all leukemia cells, and the mice progressed when treatment was stopped. In conclusion, JAK3 is recurrently mutated in T-ALL patients, and we demonstrate that most JAK3 mutants are transforming proteins using In vitro and in vivo experiments. Our results show that JAK1 is an essential kinase for most JAK3 mutants, and that the majority of JAK3 mutants are sensitive to JAK3 and JAK1 specific inhibitors such as tofacitinib and ruxolitinib. However, some JAK3 mutants do show resistance to these inhibitors, which will need to be taken into account when trials are initiated for the treatment of ALL patients with JAK specific inhibitors. Disclosures No relevant conflicts of interest to declare.

The NUP214-ABL1 fusion protein is a constitutively active protein tyrosine kinase that is found in 6% of patients with T-cell acute lymphoblastic leukemia and that promotes proliferation and survival of T-lymphoblasts. Although NUP214-ABL1 is sensitive to ABL1 kinase inhibitors, development of resistance to these compounds is a major clinical problem, underlining the need for additional drug targets in the sparsely studied NUP214-ABL1 signaling network. In this work, we identify and validate the SRC family kinase LCK as a protein whose activity is absolutely required for the proliferation and survival of T-cell acute lymphoblastic leukemia cells that depend on NUP214-ABL1 activity. These findings underscore the potential of SRC kinase inhibitors and of the dual ABL1/SRC kinase inhibitors dasatinib and bosutinib for the treatment of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia. In addition, we used mass spectrometry to identify protein interaction partners of NUP214-ABL1. Our results strongly support that the signaling network of NUP214-ABL1 is distinct from that previously reported for BCR-ABL1. Moreover, we found that three NUP214-ABL1-interacting proteins, MAD2L1, NUP155, and SMC4, are strictly required for the proliferation and survival of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia cells. In conclusion, this work identifies LCK, MAD2L1, NUP155 and SMC4 as four new potential drug targets in NUP214-ABL1-positive T-cell acute lymphoblastic leukemia.

Activated forms of the platelet derived growth factor receptor alpha (PDGFRα) have been described in various tumors, including FIP1L1-PDGFRα in patients with myeloproliferative diseases associated with hypereosinophilia and the PDGFRα(D842V) mutant in gastrointestinal stromal tumors and inflammatory fibroid polyps. To gain a better insight into the signal transduction mechanisms of PDGFRα oncogenes, we mutated twelve potentially phosphorylated tyrosine residues of FIP1L1-PDGFRα and identified three mutations that affected cell proliferation. In particular, mutation of tyrosine 720 in FIP1L1-PDGFRα or PDGFRα(D842V) inhibited cell growth and blocked ERK signaling in Ba/F3 cells. This mutation also decreased myeloproliferation in transplanted mice and the proliferation of human CD34(+) hematopoietic progenitors transduced with FIP1L1-PDGFRα. We showed that the non-receptor protein tyrosine phosphatase SHP2 bound directly to tyrosine 720 of FIP1L1-PDGFRα. SHP2 knock-down decreased proliferation of Ba/F3 cells transformed with FIP1L1-PDGFRα and PDGFRα(D842V) and affected ERK signaling, but not STAT5 phosphorylation. Remarkably, SHP2 was not essential for cell proliferation and ERK phosphorylation induced by the wild-type PDGF receptor in response to ligand stimulation, suggesting a shift in the function of SHP2 downstream of oncogenic receptors. In conclusion, our results indicate that SHP2 is required for cell transformation and ERK activation by mutant PDGF receptors.

RNA interference (RNAi) is one of the processes in the cell that regulates mRNA expression levels. RNAi can be exploited to experimentally knockdown the expression of one or more genes in cell lines or even in cells in vivo and also became an interesting tool to develop new therapeutic approaches. One of the major challenges of using RNAi is selecting effective shRNAs or siRNAs that sufficiently down-regulate the expression of the target gene. Here, we describe a system to select functional shRNAs or siRNAs that makes use of the leukemia cell line Ba/F3 that is dependent on the expression of a mutant form of the PDGFRα kinase for its proliferation and survival. The basis of this system is the generation of an expression construct, where part of the open reading frame of the gene of interest is linked to the mutant PDGFRα. Thus, shRNAs or siRNAs that effectively target the gene of interest also result in a reduction of the expression of the mutant PDGFRα protein, which can be detected by a reduction of the proliferation of the cells. We demonstrate that this validation system can be used for the selection of effective siRNAs as well as shRNAs. Unlike other systems, the system described here is not dependent on obtaining high-transduction efficiencies, and nonspecific effects of the siRNAs or shRNAs can be detected by comparing the effects in the presence or absence of the growth factor interleukin-3.

Janus kinases are important signaling proteins implicated in cytokine signaling. In particular, Janus kinase 3 (JAK3) has gained attention as a target for inhibition of the immune system, due to its importance for T and B cell development and function. In this issue however, Haan et al. (2011) show that inhibition of JAK3 activity may not be sufficient for this purpose.

Janus kinases are important signaling proteins implicated in cytokine signaling. In particular, Janus kinase 3 (JAK3) has gained attention as a target for inhibition of the immune system, due to its importance for T and B cell development and function. In this issue however, Haan et al. (2011) show that inhibition of JAK3 activity may not be sufficient for this purpose.

Signaling by the many ligands of the TGFβ family strongly converges towards only five receptor-activated, intracellular Smad proteins, which fall into two classes i.e. Smad2/3 and Smad1/5/8, respectively. These Smads bind to a surprisingly high number of Smad-interacting proteins (SIPs), many of which are transcription factors (TFs) that co-operate in Smad-controlled target gene transcription in a cell type and context specific manner. A combination of functional analyses in vivo as well as in cell cultures and biochemical studies has revealed the enormous versatility of the Smad proteins. Smads and their SIPs regulate diverse molecular and cellular processes and are also directly relevant to development and disease. In this survey, we selected appropriate examples on the BMP-Smads, with emphasis on Smad1 and Smad5, and on a number of SIPs, i.e. the CPSF subunit Smicl, Ttrap (Tdp2) and Sip1 (Zeb2, Zfhx1b) from our own research carried out in three different vertebrate models.

The genetics of classical Hodgkin lymphoma (cHL) is poorly understood. The finding of a JAK2-involving t(4;9)(q21;p24) in 1 case of cHL prompted us to characterize this translocation on a molecular level and to determine the prevalence of JAK2 rearrangements in cHL. We showed that the t(4;9)(q21;p24) leads to a novel SEC31A-JAK2 fusion. Screening of 131 cHL cases identified 1 additional case with SEC31A-JAK2 and 2 additional cases with rearrangements involving JAK2. We demonstrated that SEC31A-JAK2 is oncogenic in vitro and acts as a constitutively activated tyrosine kinase that is sensitive to JAK inhibitors. In vivo, SEC31A-JAK2 was found to induce a T-lymphoblastic lymphoma or myeloid phenotype in a murine bone marrow transplantation model. Altogether, we identified SEC31A-JAK2 as a chromosomal aberration characteristic for cHL and provide evidence that JAK2 rearrangements occur in a minority of cHL cases. Given the proven oncogenic potential of this novel fusion, our studies provide new insights into the pathogenesis of cHL and indicate that in at least some cases, constitutive activation of the JAK/STAT pathway is caused by JAK2 rearrangements. The finding that SEC31A-JAK2 responds to JAK inhibitors indicates that patients with cHL and JAK2 rearrangements may benefit from targeted therapies.

With every heartbeat the heart must contract and relax. This seemingly trivial process critically needs tight control of contraction and relaxation phases, and extremely efficient coordination between these two phases to control blood flow and maintain cardiac homeostasis. To achieve this, specialized sensors are required to detect the inherent repeatedly changing environment and needs. One sensor is a stretch-sensor that monitors the filling of the ventricles. Its molecular identity and localization are only partly understood. Here we give a synopsis of the genetic models that leap into our understanding of stretch-sensors. We focus on the widely acknowledged sarcomeric sensor at the Z-disc and the costamere sensor at the sarcolemma. Recently, several novel components of both sensors were discovered. Given that these two sensors seem physically connected, it is likely that these two models are not mutually exclusive and might even communicate. We describe briefly how candidate and known proteins within these sensors receive and transduce mechanical signals in the cardiomyocyte that lead to changes in gene expression underlying homeostasis and its restoration in the heart. Emphasis is placed on the putative link between altered stretch-sensor function and heart failure observed in different genetic mouse models of stretch-sensor components.

Smads are intracellular signaling proteins that transduce signals elicited by members of the transforming growth factor (TGF)-beta superfamily. Smad5 and Smad1 are highly homologous, and they mediate primarily bone morphogenetic protein (Bmp) signals. We used the Cre-loxP system and Sm22-Cre and Tie-1-Cre mice to study the function of Smad5 in the developing blood vessel wall. Analysis of embryos demonstrated that deletion of Smad5 in endothelial or smooth muscle cells resulted in a normal organization of embryonic and extra-embryonic vasculature. Angiogenic assays performed in adult mice revealed that mutant mice display a comparable angiogenic and vascular remodeling response to control mice. In Sm22-Cre; Smad5(fl/-) mice, Smad5 is also deleted in cardiomyocytes. Echocardiographic analysis on those 9-month-old female mice demonstrated larger left ventricle internal diameters and decreased fractional shortening compared with control littermates without signs of cardiac hypertrophy. The decreased cardiac contractility was associated with a decreased performance in a treadmill experiment. In isolated cardiomyocytes, fractional shortening was significantly reduced compared with control cells. These data demonstrate that restricted deletion of Smad5 in the blood vessel wall results in viable mice. However, loss of Smad5 in cardiomyocytes leads to a mild heart defect.

The mouse mutant Ozzy, originating from an ENU-mutagenesis programme, displays a head bobbing phenotype. We report here that Ozzy mice show a clear deficit in vestibulo-ocular reflex (VOR). Micro-CT scanning of the inner ears showed narrowing and truncations of at least one of the semicircular canals and loss of the ampullae. Frequency-specific auditory-evoked brainstem response (ABR) tests revealed a slight threshold increase in the middle frequency range compared to wild-type littermates. Linkage analysis localised the gene in a 5.5-cM region on chromosome 2. Subsequently, a 499 T→A missense mutation was identified in Jag1, leading to a substitution of an evolutionary conserved tryptophane (W167R). Mutations in the human homologue of Jag1 cause Alagille syndrome (AGS), an autosomal dominant disorder associated with liver, heart, eye and skeletal abnormalities, accompanied by a characteristic facies. In human patients, it occasionally affects other organ systems like the kidney or the inner ear. Liver disease is the main diagnostic factor for AGS. Ozzy mice showed significantly less intrahepatic bile ducts than wild-type littermates. Thirty-seven percent of Ozzy mice showed heart defects. No eye or vertebral abnormalities could be detected. In conclusion, Ozzy mice show two of the major and one minor characteristic of AGS.