The T-cell leukemia-associated ribosomal RPL10 R98S mutation enhances JAK-STAT signaling.

Several somatic ribosome defects have recently been discovered in cancer, yet their oncogenic mechanisms remain poorly understood. Here we investigated the pathogenic role of the recurrent R98S mutation in ribosomal protein L10 (RPL10 R98S) found in T-cell acute lymphoblastic leukemia (T-ALL). The JAK-STAT signaling pathway is a critical controller of cellular proliferation and survival. A proteome screen revealed overexpression of several Jak-Stat signaling proteins in engineered RPL10 R98S mouse lymphoid cells, which we confirmed in hematopoietic cells from transgenic Rpl10 R98S mice and T-ALL xenograft samples. RPL10 R98S expressing cells displayed JAK-STAT pathway hyper-activation upon cytokine stimulation, as well as increased sensitivity to clinically used JAK-STAT inhibitors like pimozide. A mutually exclusive mutation pattern between RPL10 R98S and JAK-STAT mutations in T-ALL patients further suggests that RPL10 R98S functionally mimics JAK-STAT activation. Mechanistically, besides transcriptional changes, RPL10 R98S caused reduction of apparent programmed ribosomal frameshifting at several ribosomal frameshift signals in mouse and human Jak-Stat genes, as well as decreased Jak1 degradation. Of further medical interest, RPL10 R98S cells showed reduced proteasome activity and enhanced sensitivity to clinical proteasome inhibitors. Collectively, we describe modulation of the JAK-STAT cascade as a novel cancer-promoting activity of a ribosomal mutation, and expand the relevance of this cascade in leukemia.

RPL5 on 1p22.1 is recurrently deleted in multiple myeloma and its expression is linked to bortezomib response.

Chromosomal region 1p22 is deleted in ⩾20% of multiple myeloma (MM) patients, suggesting the presence of an unidentified tumor suppressor. Using high-resolution genomic profiling, we delimit a 58 kb minimal deleted region (MDR) on 1p22.1 encompassing two genes: ectopic viral integration site 5 (EVI5) and ribosomal protein L5 (RPL5). Low mRNA expression of EVI5 and RPL5 was associated with worse survival in diagnostic cases. Patients with 1p22 deletion had lower mRNA expression of EVI5 and RPL5, however, 1p22 deletion status is a bad predictor of RPL5 expression in some cases, suggesting that other mechanisms downregulate RPL5 expression. Interestingly, RPL5 but not EVI5 mRNA levels were significantly lower in relapsed patients responding to bortezomib and; both in newly diagnosed and relapsed patients, bortezomib treatment could overcome their bad prognosis by raising their progression-free survival to equal that of patients with high RPL5 expression. In conclusion, our genetic data restrict the MDR on 1p22 to EVI5 and RPL5 and although the role of these genes in promoting MM progression remains to be determined, we identify RPL5 mRNA expression as a biomarker for initial response to bortezomib in relapsed patients and subsequent survival benefit after long-term treatment in newly diagnosed and relapsed patients.

Heterogeneous patterns of amplification of the NUP214-ABL1 fusion gene in T-cell acute lymphoblastic leukemia.

Episomes with the NUP214-ABL1 fusion gene have been observed in 6% of T-ALL. In this multicentric study we collected 27 cases of NUP214-ABL1-positive T-ALL. Median age was 15 years with male predominance. Outcome was poor in 12 patients. An associated abnormality involving TLX1 or TLX3 was found in all investigated cases. Fluorescent in situ hybridization revealed a heterogeneous pattern of NUP214-ABL1 amplification. Multiple episomes carrying the fusion were detected in 24 patients. Episomes were observed in a significant number of nuclei in 18 cases, but in only 1-5% of nuclei in 6. In addition, intrachromosomal amplification (small hsr) was identified either as the only change or in association with episomes in four cases and two T-ALL cell lines (PEER and ALL-SIL). One case showed insertion of apparently non-amplified NUP214-ABL1 sequences at 14q12. The amplified sequences were analyzed using array-based CGH.These findings confirm that the NUP214-ABL1 gene requires amplification for oncogenicity; it is part of a multistep process of leukemogenesis; and it can be a late event present only in subpopulations. Data also provide in vivo evidence for a model of episome formation, amplification and optional reintegration into the genome. Implications for the use of kinase inhibitors are discussed.

Intrinsic differences between the catalytic properties of the oncogenic NUP214-ABL1 and BCR-ABL1 fusion protein kinases.

The NUP214-ABL1 fusion kinase has recently been identified in 6% of patients with T-cell acute lymphoblastic leukemia. In contrast to the more common oncogenic ABL1 fusion BCR-ABL1, NUP214-ABL1 localizes to the nuclear pore complexes and has attenuated transforming properties in hematopoietic cells and in mouse bone marrow transplant models. We have performed a thorough biochemical comparative analysis of NUP214-ABL1 and BCR-ABL1 and show that, despite their common tyrosine kinase domain, the two fusion proteins differ in many critical catalytic properties. NUP214-ABL1 has lower in vitro tyrosine kinase activity, which is in agreement with the absence of phosphorylation on its activation loop. NUP214-ABL1 was more sensitive to imatinib (Glivec) than BCR-ABL1 in vitro and in cells, indicating a different activation state and conformation of the two ABL1 fusion kinases. Using a peptide array, we identified differences in the spectrum and efficiency of substrate peptide phosphorylation and a differential involvement of Src kinases in downstream signaling. These results clearly indicate that different fusion partners of the same kinase can determine not only localization, but also critical functional properties of the enzyme such as inhibitor sensitivity and substrate preference, with subsequent differences in downstream signaling effectors and likely consequences in disease pathogenesis.

ABL1 fusions in T-cell acute lymphoblastic leukemia.

Mutant tyrosine kinases are common in cancer and can be therapeutically targeted with kinase inhibitors. To obtain insight in the contribution of activated kinases to the pathogenesis ofT-cell acute lymphoblastic leukemia (T-ALL), we studied the NUP214-ABL1 fusion gene that is found in 6% of T-ALL and EML1-ABL1 that we identified in one T-ALL patient. NUP214-ABL1 and EML1-ABL1 display constitutive kinase activity and are sensitive to the kinase inhibitor imatinib. Both proteins transform hematopoietic cells and we established mouse models of EML1-ABL1 and NUP214-ABL1 induced T-ALL. Interestingly, whereas EML1-ABL1 activity requires homo-oligomerization via its coiled coil domain, activity of NUP214-ABL1 depends on its cellular localization to the nuclear pore complexes. These results for NUP214-ABL1 delineate a novel mechanism for fusion kinase activation in cancer and provide new options to interfere with NUP214-ABL1 activity. T-ALL development requires accumulation of different mutations. Little is known about the cooperation between the mutations and about their sequence of accumulation. We provide evidence that tyrosine kinase mutations occur late in T-ALL development, suggesting limited potential for kinase inhibitor monotherapy. We therefore combined NUP214-ABL1 inhibition with other therapies and show that inhibition of NUP214-ABL1 and NOTCH1 in vitro can result in synergistic effects. Further validation of these and other combination therapies requires animal models containing several of the mutations in T-ALL and thus reflecting the multistep oncogenic process of human T-ALL genesis. The models for ABL1 fusion induced T-ALL will serve as starting point here.

Chronic myeloproliferative disorders: a tyrosine kinase tale.

Chronic myeloproliferative diseases (CMPDs) are characterized by the abnormal proliferation and survival of one or more myeloid cell types. The archetype of this class of hematological diseases is chronic myeloid leukemia (CML), characterized by the presence of the Philadelphia (Ph) chromosome, the result of t(9;22)(q34;q11), and the associated BCR-ABL1 oncogene. Some of the Ph-negative myeloproliferative diseases are characterized by other chromosomal translocations involving a variety of tyrosine kinase genes, including ABL1, ABL2, PDGFRA, PDGFRB, FGFR1, and JAK2. The majority of Ph-negative CMPDs, however, such as chronic eosinophilic leukemia, polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis are not characterized by the presence of recurrent chromosomal abnormalities. Recent studies have identified the FIP1L1-PDGFRA fusion gene, generated due to a small cryptic deletion on chromosome 4q12, and the activating V617F mutation in JAK2 in a significant fraction of Ph-negative CMPDs. These results show that abnormalities in tyrosine kinase genes are central to the molecular pathogenesis of CMPDs. Genome-wide screenings to identify novel tyrosine kinase abnormalities in CMPDs may contribute to further improvement of the diagnosis and the treatment of these diseases.

Implications of the use of neuromuscular transmission monitoring on immediate postoperative extubation in off-pump coronary artery bypass surgery.

BACKGROUND AND OBJECTIVE: When continuous infusions of neuromuscular blocking drugs are administered during lengthy interventions and no routine antagonism of their effects is applied, there is a dramatic incidence of residual curarization. We have examined whether the use of neuromuscular transmission monitoring results in differences in the incidence of postoperative residual curarization, the use of antagonist agents, and the endotracheal extubation rate and outcome after continuous infusion of rocuronium in patients undergoing off-pump coronary artery bypass surgery. METHODS: Twenty patients were assigned to group 1 (n = 10, non-blinded neuromuscular transmission monitoring) or group 2 (n = 10, blinded neuromuscular transmission monitoring). In group 1, patients were given rocuronium at an infusion rate of 6 microg kg(-1) min(-1). The rate was manually adjusted in order to maintain T1/T0 at 10%. In group 2, a rocuronium infusion was started 30 min after induction of anaesthesia, at a rate of 6 microg kg(-1) min(-1); this rate was left unchanged during surgery. The rocuronium infusion was discontinued on completion of all vascular anastomoses; propofol was stopped at the beginning of closure of the subcutis and pirinitramide (piritramide) 15 mg was administered intravenously. Remifentanil was discontinued at the beginning of skin closure and neostigmine (50 microg kg(-1)) administered at the end of surgery when the train-of-four ratio was < 0.9 in group 1, and routinely in group 2. A 20 min test period for spontaneous ventilation was allowed once surgery had been accomplished. When the train-of-four ratio was > or = 0.9 (group 1), patients were extubated if also breathing spontaneously, fully awake and able to follow commands. When they met the clinical criteria for normal neuromuscular function after induced blockade, patients in group 2 were extubated when fully awake and able to follow commands. RESULTS: In group 1, the rate of rocuronium infusion required to keep T1/T0 at 10% was 5 +/- 1.9 microg kg(-1) min(-1); this was not significantly different from the fixed rate in group 2 (P = 0.15). One patient in group 2 was excluded. Eight out of 10 and eight out of nine patients in groups 1 and 2, respectively, reached the extubation criteria. Three out of eight, and five out of eight, patients from groups 1 and 2, respectively, were extubated in the operating room. At that time of endotracheal extubation, all three patients from group 1, but only four of the five patients from group 2 had a train-of-four ratio > or = 0.9. In group 2, one patient was reintubated in the intensive care unit. The incidence of pharmacological reversal was high in group 1. CONCLUSIONS: Although we found no additional benefit of using neuromuscular transmission monitoring, it seems an absolute necessity for safety reasons. Pharmacological antagonism was mandatory. However, in our opinion, it is not wise routinely to perform immediate postoperative extubation in off-pump coronary artery bypass surgery.