Indiana University-Purdue University Indianapolis (IUPUI) / Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm that lacks effective chemotherapies. Most commonly, patients have gain-of-function (GOF) oncogenic mutations in SHP2, leading to hyperactivation of ERK and AKT and hyperproliferation of cells in response to granulocyte macrophage-colony stimulating factor (GM-CSF). Our lab previously showed that p110δ, the hematopoietic-specific catalytic subunit of phosphoinositide 3-kinase, is a crucial mediator of mutant Shp2-induced GM-CSF hypersensitivity in vitro. We treated oncogenic Shp2-expressing mice with a p110δ inhibitor and showed that the strong effect our lab observed in vitro translated into reduced splenomegaly and prolonged survival in vivo. We investigated molecules potentially cooperating with p110δ signaling and discovered that Bruton’s tyrosine kinase (BTK) is hyperphosphorylated in GOF Shp2 myeloid cells. We used specific BTK and p110δ inhibitors to demonstrate that BTK cooperates with p110δ to hyperactivate Akt/Erk and to promote hyperproliferation. GOF Shp2-expressing mice treated in vivo with the drug combination targeting p110δ and BTK have significantly decreased splenomegaly and WBC counts. We also explored the mechanism of BTK signaling and hypothesized that B cell adaptor for PI3K (BCAP) mediated BTK upregulation of PI3K activity. In mutant Shp2 macrophages, we observed BCAP phosphorylation specifically in the larger isoforms needed for PI3K activation, and BTK inhibition led to a dose-dependent reduction in this phosphorylation. We also demonstrated reduced interaction between BCAP and the PI3K regulatory p85α subunit bearing mutated SH2 domains. Finally, we investigated the effects of mutated DNA methyltransferase 3A (Dnmt3a) in conjunction with GOF Shp2. Double mutant mice quickly became moribund with pronounced splenomegaly and leukocytosis. There was an expansion of mature myeloid cells in the periphery and myeloid progenitors in the bone marrow, plus anemia with evidence of compensatory erythropoiesis in the spleen. Our findings show that the myeloproliferative neoplasm caused by GOF Shp2 is due to hyperactive p110δ, and this is further promoted by BTK, which forms a positive feedback loop with PI3K and BCAP, thus leading to more Akt/Erk hyperphosphorylation and more hyperproliferation in response to GM-CSF. The dual inhibition of p110δ and BTK represents a novel effective treatment strategy for JMML and other diseases induced by oncogenic Shp2.

No description available.

<p>The Signalosome is a term used to define a putative signalling complex, which assembles near the plasma membrane in response to external signals received at cell surface receptors and then migrates towards downstream effectors. It is proposed to regulate the level of intracellular Ca2+ and subsequent downstream signalling events. To date it has been defined to consist of BTK, BLNK, BCAP, VAV, PLCγ2 and PI3K1-4 in B-Cells.</p><p>This work entailed initiating a new proteomic approach to investigate the nature and extent of Bruton’s tyrosine kinase, Btk, involvement in the signalosome – inherently, the aim was to study multiple interactions of Btk with other molecules. By transfecting host cells with a Btk gene-transfer plasmid, virus particles were produced that were used to up-regulate and analyse the expression of Btk in three haematopoietic cell lines: B-cells, Pre-B-cells and a myeloid cancer cell. The construction of a new gene-transfer vector was successfully carried out by plasmid sub-cloning and it was subsequently found to effectively transfect the host cells and produce virus particles. The recombinant virus particles were employed with success in transducing three haematopoietic cell lines and with immunopurification and subsequent gel separation protein signalosome complexes were obtained ready for analysis by mass spectrometrical fingerprinting (to be carried out as a joint effort in Mount Sinai Hospital in Toronto, Canada).</p>

The Signalosome is a term used to define a putative signalling complex, which assembles near the plasma membrane in response to external signals received at cell surface receptors and then migrates towards downstream effectors. It is proposed to regulate the level of intracellular Ca2+ and subsequent downstream signalling events. To date it has been defined to consist of BTK, BLNK, BCAP, VAV, PLCγ2 and PI3K1-4 in B-Cells. This work entailed initiating a new proteomic approach to investigate the nature and extent of Bruton’s tyrosine kinase, Btk, involvement in the signalosome – inherently, the aim was to study multiple interactions of Btk with other molecules. By transfecting host cells with a Btk gene-transfer plasmid, virus particles were produced that were used to up-regulate and analyse the expression of Btk in three haematopoietic cell lines: B-cells, Pre-B-cells and a myeloid cancer cell. The construction of a new gene-transfer vector was successfully carried out by plasmid sub-cloning and it was subsequently found to effectively transfect the host cells and produce virus particles. The recombinant virus particles were employed with success in transducing three haematopoietic cell lines and with immunopurification and subsequent gel separation protein signalosome complexes were obtained ready for analysis by mass spectrometrical fingerprinting (to be carried out as a joint effort in Mount Sinai Hospital in Toronto, Canada).

La Inmunodeficiencia Variable Común (IDVC) es la inmunodeficiencia primaria (IDP) sintomática más frecuente. Los pacientes con IDVC presentan un fenotipo clínico (infecciones recurrentes,etc) y un fenotipo inmunológico (hipogammaglobulinemia y respuesta nula de anticuerpos). El diagnóstico de IDVC se realiza por exclusión de otras causas de hipogammaglobulinemia. La existencia de casos atípicos, el solapamiento de fenotipos con otras IDPs y la ausencia de pruebas de laboratorio específicas de IDVC dificulta realizar un diagnóstico diferencial correcto. Recientemente, se han descrito algunos defectos monogénicos (ICOS, CD19, TACI y BAFFR) relacionados con IDVC (~10% pacientes IDVC). En el presente trabajo hemos estudiado: un método de cribaje (expresión de Btk, Lck y SAP), la asociación entre las células B de memoria y la gravedad/evolución clínica y el papel del ICOS como causa genética de la IDVC. El estudio de la expresión de Btk, lck y SAP se realizó mediante western blot (n=55 pacientes-IDVC,37 varones y 18 mujeres, media: 36,6a). El extracto de proteínas se obtuvo a partir de PBMC con NP-40 en condiciones reductoras, la electroforesis se realizó en geles de poliacrilamida de 4-12%, la transferencia en sistema semi-seco en membrana de nitrocelulosa, los anticuerpos primarios utilizados (anti-Btk: ac.mono. BD-Transduction laboratories, anti-lck: ac.mono. BD-Pharmingen, anti- SAP: ac. policlonal de conejo (K. Nichols) y la inmunodetección con un substrato Quimioluminiscente (SuperSignalR West Pico). Del total de los pacientes estudiados sólo en un paciente la expresión de la banda Btk fue negativa, confirmado por estudio de la Btk intracelular en monocitos por citometria de flujo y por análisis genético (P597L). La expresión de Lck y SAP fue normal en todos los pacientes.Se estudiaron las subpoblaciones de linfocitos B de memoria (CD19+CD27+) mediante citometria de flujo (FACSCalibur) en 41 pacientes adultos (21 varones y 20 mujeres, media: 37a) y se clasificaron en 3 grupos: 1) Grupo BM2 incluye pacientes con un número de LB de memoria con (CD19+CD27+IgD-) y sin cambio de isotipo (CD19+CD27+IgD+) dentro de los valores de referencia, n=7, 2) Grupo BM1 incluye pacientes con una reducción significativa de CD19+CD27+IgD- <6%, n=16 y 3) Grupo BM0 incluye pacientes que presentan una reducción significativa de los dos tipos de LB de memoria (CD19+CD27+ <13%), n=18. Se Observó una asociación entre los defectos de LB de memoria y la gravedad fenotípica clínica de los pacientes. En el grupo BM0, el 55% de pacientes con infecciones respiratorias recurrentes, desarrolló bronquiectasias con expectoración habitual, comparado con el 27% de los pacientes del grupo BM1 y el 0% del grupo BM2 (p<0.05). En el grupo BM0, el 50% de los pacientes con síntomas gastrointestinales, desarrolló el síndrome de malabsorción, comparado con el 19% del grupo BM1 y el 0% del BM2 (p<0.05). Los signos de linfoproliferación fueron más frecuentes en el grupo BM0 (50%) que en el grupo BM1 (16%) y BM2 (14%), (p<0.05).Se analizó la expresión de ICOS en la membrana de linfocitos T activados (48h con PMA+ionomicina, anti-ICOS-FITC C398.4A (J.Rojo), FACSCalibur) en pacientes IDVC con historia familiar de IDPs y/o enfermedades autoinmunes (4 familias, n=13). No encontramos ninguna alteración en la expresión de esta molécula en los pacientes estudiados.En conclusión, aunque el error diagnóstico encontrado en nuestro estudio es pequeño y la frecuencia del defecto ICOS es muy baja creemos que se debería incluir en el algoritmo de trabajo de soporte al diagnóstico un sistema de cribaje que incluya el estudio de proteínas asociadas a IDP con fenotipo de IDVC. La asociación entre la alteración inmunofenotípica B (BM0, BM1, BM2) y la gravedad fenotípica clínica justifica la inclusión de la determinación de los LB de memoria en el estudio inmunológico rutinario de los pacientes con IDVC. / Common Variable immunodeficiency (CVID) is a primary immunodeficiency (PID) with symptoms more frequently. The patients with CVID have a clinical phenotype (recurrent infections, etc.) and an immunologic phenotype (hypogammaglobulinemia and poor response an antibody's). The diagnosis of CVID is to make in order to rule out other causes of hypogammaglobulinemia. The existence of atypical cases, flapping of phenotypes with other PIDs and the absence of test specifics of CVID to hinder make a differential diagnosis correct. Recently, was described some monogenic defects (ICOS; CD19, TACI y BAFFR) related with CVID (~ 10% CVID patients).In this work, we are studied: a screening method (expression of btk, lck and SAP), the association between B cells memory and the severity/ clinical evolution and the role of ICOS how genetic cause of CVID. The study of expression of btk, lck, and SAP was making with western blot (n=55 patients-CVID, 37 man and 18 women, media: 36,6y). The extract of proteins was getting of PBMC with NP-40 in reduce conditions the electrophoresis was making in poliacrylamida gels of 4-12%, the transference in semi-dry system in nitrocellulose membrane, the primary antibody were (anti-btk: monoab. -Transduction laboratories, BD, anti-lck: monoab. Pharmingen, BD, anti-SAP: polyclonal ab of rabbit (K. Nichols) and the immunodetection with quimioluminiscente substrate (SuperSignal R West Pico). Of the total patients studied only one, the expression of the btk gang was negative, confirmed for studied of intracellular btk in monocytes for flow cytometry a genetic study (P597L). The expression of lck and SAP was normal in all the patients. The B cells subpopulations of memory (CD19+CD27+) was studied for flow cytometry (FACSCalibur) in 41 adults patients (21 man and 20 women, media 37y) and has been classified in 3 groups: 1) BM2 group include patients with number of memory B cells with (CD19+CD27+IgD-) and without isotype switched (CD19+CD27+IgD+) inside the reference levels, n=7, 2) BM1 group include patients with a significance reduction of CD19+CD27+IgD- <6%, n=16 y 3) BM0 group include patients with significance reduction of two types of memory BL (CD19+CD27+ <13%) n=18. We observed an association between defects of memory B cells and the severity clinical phenotype of the patients. In the group BM0, the 55% of patients with recurrent respiratory infections, development bronchiectasias with usual expectoration compared with the 27% of patients of the BM1 group and 0% of BM2 group (p<0.05). In the group BM0, the 50% of patients with symptoms gastrointestinal development the malabsortion syndrome, compared with the 19% of the group BM1 and the 0% of BM2 (p<0.05). The signs of lymphoproliferation were more frequent in the BM0 group (50%) than BM1 group (16%) and BM2 (14%), (p<0.05).Was analysed the expression of ICOS in the membrane of lymphocytes activated (48h with PMA+ionomicin), anti-ICOS-FITC C398.4A (J.Rojo), FACSCalibur in patients with CVID with familiar history of PIDs and/o autoimmune diseases (4 families, n=13). No find any alteration of the expression of this molecule in the patients studied.In conclusion, even though the error diagnosis fined in our studied is small and the frequency of the ICOS defect is low, we believe that should be include in the algorithm of work support al diagnosis, a system screening that include the protein studied associate a PID with phenotype of CVID. The association between the alteration immunophenotypic B (BM0, BM1, and BM2) and the severity clinical phenotype justify the inclusion of the determination of the memory B cell in the immunologic study routine of the patients with CVID.

No description available.

Agamaglobulinemia ligada ao X (XLA) é uma imunodeficiência primária caracterizada por ausência ou número reduzido de células B maduras em sangue periférico, de todos os isotipos de imunoglobulina e um aumento da susceptibilidade a infecções bacterianas e enterovirais graves. XLA é causada por mutações no gene Bruton\'s tirosino quinase, que codifica um membro da proteína da família das tirosino quinases citoplasmáticas que tem papel vital na modulação de muitos processos celulares. Neste estudo foram analisados trinta e três pacientes quanto à presença de mutações de BTK, por SSCP/HA e seqüenciamento. A análise da expressão foi realizada pela técnica por PCR em tempo real. Foram encontradas mutações do tipo stop codons, substituições de aminoácido, defeitos de splicing, pequenas deleções/inserções e frameshift nestes pacientes afetando os domínios PH, SH3, SH2 e o domínio da quinase da proteína. A análise da expressão mostrou níveis baixos nos pacientes com mutação do tipo stop codon, e nas outras mutações, os níveis de expressão foram de aproximadamente 15% e se correlacionaram com os tipos de mutação. / X-linked Agammaglobulinemia (XLA) is a primary immunodeficiency characterized by the absence or decreased numbers of mature B cells in peripheral blood, and by a lack of all immunoglobulin isotypes, leading to an increased susceptibility to severe bacterial and enteroviral infections. XLA is caused by mutations in the gene encoding for Bruton\'s tyrosine kinase (BTK), a protein member of the Tec family of cytoplasmic tyrosine kinases and plays a vital modulation role in many cellular processes. In this study thirty-three patients were analyzed for the presence of BTK mutations by SSCP/HA and sequencing. The expression analysis was carried out by the technique of Real-Time PCR. It was found mutations of the stop codons type, amino acid substitutions, splice defects, small deletions/insertions and frameshift in these patients affecting the PH, SH3, SH2 and tyrosine kinase domains of protein. The expression levels were very low in the patients with stop codon mutations, and in the other mutations, the expression levels were about 15% and were correlated with the mutation types.

Agamaglobulinemia ligada ao X (XLA) é uma imunodeficiência primária caracterizada por ausência ou número reduzido de células B maduras em sangue periférico, de todos os isotipos de imunoglobulina e um aumento da susceptibilidade a infecções bacterianas e enterovirais graves. XLA é causada por mutações no gene Bruton\'s tirosino quinase, que codifica um membro da proteína da família das tirosino quinases citoplasmáticas que tem papel vital na modulação de muitos processos celulares. Neste estudo foram analisados trinta e três pacientes quanto à presença de mutações de BTK, por SSCP/HA e seqüenciamento. A análise da expressão foi realizada pela técnica por PCR em tempo real. Foram encontradas mutações do tipo stop codons, substituições de aminoácido, defeitos de splicing, pequenas deleções/inserções e frameshift nestes pacientes afetando os domínios PH, SH3, SH2 e o domínio da quinase da proteína. A análise da expressão mostrou níveis baixos nos pacientes com mutação do tipo stop codon, e nas outras mutações, os níveis de expressão foram de aproximadamente 15% e se correlacionaram com os tipos de mutação. / X-linked Agammaglobulinemia (XLA) is a primary immunodeficiency characterized by the absence or decreased numbers of mature B cells in peripheral blood, and by a lack of all immunoglobulin isotypes, leading to an increased susceptibility to severe bacterial and enteroviral infections. XLA is caused by mutations in the gene encoding for Bruton\'s tyrosine kinase (BTK), a protein member of the Tec family of cytoplasmic tyrosine kinases and plays a vital modulation role in many cellular processes. In this study thirty-three patients were analyzed for the presence of BTK mutations by SSCP/HA and sequencing. The expression analysis was carried out by the technique of Real-Time PCR. It was found mutations of the stop codons type, amino acid substitutions, splice defects, small deletions/insertions and frameshift in these patients affecting the PH, SH3, SH2 and tyrosine kinase domains of protein. The expression levels were very low in the patients with stop codon mutations, and in the other mutations, the expression levels were about 15% and were correlated with the mutation types.

La voie PI3Kinase/AKT/mTOR, est une cible thérapeutique du temsirolimus, un inhibiteur de mTORC1. Dans le but d'obtenir une inhibition plus importante de cette voie j’utilise dans ce projet deux nouvelles molécules :- le NVP-BEZ 235 (BEZ) qui inhibe à la fois mTORC1 et la PI3kinase- l'AZD8055 (AZD), un inhibiteur des complexes mTORC1 et mTORC2. En utilisant différentes lignées de LCM, j’ai démontré que l'effet de ces nouveaux inhibiteurs sur la survie cellulaire est plus important que celui du temsirolimus. Cela est probablement dû à l'inhibition de la phosphorylation de l'AKT et la 4EBP. La deuxième partie de ce projet étudie la synergie entre les inhibiteurs de m-TOR kinase et l'aracytine. Un effet additif important a été démontré. J’ai trouvé en western blot que l’aracytine inhibe la phosphorylation des substrats de la voie Akt –mTOR notamment le 4EBP. L’ibrutinib (un inhibiteur de la voie Btk) a un effet modeste mais j’ai pu démontrer qu'il est capable à induire une inhibition plus importante de la survie cellulaire lorsqu'il est associé à l’aracytine. Cependant il s'est révélé antagoniste aux inhibiteurs de la voie PI3K-AKT-mTOR, cela reste difficile à décortiquer. Enfin, j’ai trouvé un effet additif de l’ibrutinib en combinaison avec la doxorubicine. Cependant les inhibiteurs de m-TOR n'ont pas le même effet. Afin d’expliquer ces résultats, j’ai étudié l’effet de ces molécules sur l’expression de GSTPi, enzyme de détoxification connue pour avoir un rôle important dans la résistance de LCM à l’anthracycline. J’ai mis en évidence une diminution de l’expression de cet enzyme par l’Ibrutinib. En revanche, les inhibiteurs de mTOR n’ont pas un effet sur l’expression de GSTPi. L’ibrutinib pourrait donc sensibiliser le LCM à l’anthracycline en diminuant l’expression de GSTPi. / The PI3K / AKT / mTOR pathway is the target of Temsirolimus. However, important resistance is observed. We tried to obtain a more important inhibition of PI3K / AKT pathway using two new molecules :- NVP-BEZ 235 (BEZ) which inhibits both mTORC1 and PI3K- AZD8055 (AZD) an inhibitor of mTORC1 and mTORC2 complexes. Using different cell lines of MCL, we have shown that the effect of these new inhibitors on cell survival was more important than that of Temsirolimus. This is probably because contrary to Temsirolimus, the two new molecules can inhibit AKT and 4EBP phosphorylation. In the second part of this project we studied the synergy between the m-TOR kinase inhibitors and aracytine (conventional treatment of MCL). We revealed a significant additive effect in MCL cell lines. We demonstrated by Western blot analysis that aracytine inhibits S6 and 4EBP phosphorylation. This may explain the results obtained from this drug association. We then showed that Ibrutinib (an inhibitor of Btk pathway) can induce a significant inhibition of cell survival when combined with aracytine. In this study, Ibrutinib proved antagonist effect to PI3K-AKT-mTOR inhibitors. The mechanisms of these results remain unclear. Finally, we demonstrated an additive effect of Ibrutinib in combination with doxorubicin. We did not obtain the same results when we combined m-TOR inhibitors with doxorubicin. To explain these data, we studied the effect of these drugs on the expression of GSTPi by western blot. This enzyme is known to have an important role in MCL resistance to anthracycline. Importantly, Ibrutinib induced a decrease in the expression of GSTPi but AZD8055, Temsirolimus and NVP-BEZ235 had no effect.

No description available.