Ibrutinib therapy downregulates AID enzyme and proliferative fractions in chronic lymphocytic leukemia.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination of the immunoglobulin genes. As a trade-off for its physiological function, AID also contributes to tumor development through its mutagenic activity. In chronic lymphocytic leukemia (CLL), AID is overexpressed in the proliferative fractions (PFs) of the malignant B lymphocytes, and its anomalous expression has been associated with a clinical poor outcome. Recent preclinical data suggested that ibrutinib and idelalisib, 2 clinically approved kinase inhibitors, increase AID expression and genomic instability in normal and neoplastic B cells. These results raise concerns about a potential mutagenic risk in patients receiving long-term therapy. To corroborate these findings in the clinical setting, we analyzed AID expression and PFs in a CLL cohort before and during ibrutinib treatment. We found that ibrutinib decreases the CLL PFs and, interestingly, also reduces AID expression, which correlates with dampened AKT and Janus Kinase 1 signaling. Moreover, although ibrutinib increases AID expression in a CLL cell line, it is unable to do so in primary CLL samples. Our results uncover a differential response to ibrutinib between cell lines and the CLL clone and imply that ibrutinib could differ from idelalisib in their potential to induce AID in treated patients. Possible reasons for the discrepancy between preclinical and clinical findings, and their effect on treatment safety, are discussed.

LPL protein in Chronic Lymphocytic Leukaemia have different origins in Mutated and Unmutated patients. Advances for a new prognostic marker in CLL.

Lipoprotein lipase (LPL) mRNA expression in chronic lymphocytic leukaemia (CLL) is associated with an unmutated immunoglobulin profile and poor clinical outcome. We evaluated the subcellular localization of LPL protein in CLL cells that did or did not express LPL mRNA. Our results show that LPL protein is differently located in CLL cells depending on whether it is incorporated from the extracellular medium in mutated CLL or generated de novo by leukaemic cells of unmutated patients. The specific quantification of endogenous LPL protein correlates with mRNA expression levels and mutational IGHV status, suggesting LPL protein as a possible reliable prognostic marker in CLL.

Regulatory T cells (Tregs) in lymphoid malignancies and the impact of novel therapies.

Regulatory T cells (Tregs) are responsible for maintaining immune homeostasis by controlling immune responses. They can be characterized by concomitant expression of FoxP3, CD25 and inhibitory receptors such as PD-1 and CTLA-4. Tregs are key players in preventing autoimmunity and are dysregulated in cancer, where they facilitate tumor immune escape. B-cell lymphoid malignancies are a group of diseases with heterogenous molecular characteristics and clinical course. Treg levels are increased in patients with B-cell lymphoid malignancies and correlate with clinical outcomes. In this review, we discuss studies investigating Treg immunobiology in B-cell lymphoid malignancies, focusing on clinical correlations, mechanisms of accumulation, phenotype, and function. Overarching trends suggest that Tregs can be induced directly by tumor cells and recruited to the tumor microenvironment where they suppress antitumor immunity to facilitate disease progression. Further, we highlight studies showing that Tregs can be modulated by novel therapeutic agents such as immune checkpoint blockade and targeted therapies. Treg disruption by novel therapeutics may beneficially restore immune competence but has been associated with occurrence of adverse events. Strategies to achieve balance between these two outcomes will be paramount in the future to improve therapeutic efficacy and safety.

TGF-ß/SMAD Pathway Is Modulated by miR-26b-5p: Another Piece in the Puzzle of Chronic Lymphocytic Leukemia Progression.

Clinical and molecular heterogeneity are hallmarks of chronic lymphocytic leukemia (CLL), a neoplasm characterized by accumulation of mature and clonal long-lived CD5 + B-lymphocytes. Mutational status of the IgHV gene of leukemic clones is a powerful prognostic tool in CLL, and it is well established that unmutated CLLs (U-CLLs) have worse evolution than mutated cases. Nevertheless, progression and treatment requirement of patients can evolve independently from the mutational status. Microenvironment signaling or epigenetic changes partially explain this different behavior. Thus, we think that detailed characterization of the miRNAs landscape from patients with different clinical evolution could facilitate the understanding of this heterogeneity. Since miRNAs are key players in leukemia pathogenesis and evolution, we aim to better characterize different CLL behaviors by comparing the miRNome of clinically progressive U-CLLs vs. stable U-CLLs. Our data show up-regulation of miR-26b-5p, miR-106b-5p, and miR-142-5p in progressive cases and indicate a key role for miR-26b-5p during CLL progression. Specifically, up-regulation of miR-26b-5p in CLL cells blocks TGF-ß/SMAD pathway by down-modulation of SMAD-4, resulting in lower expression of p21-Cip1 kinase inhibitor and higher expression of c-Myc oncogene. This work describes a new molecular mechanism linking CLL progression with TGF-ß modulation and proposes an alternative strategy to explore in CLL therapy.

Childhood Vaccine Acceptance and Refusal among Warao Amerindian Caregivers in Venezuela; A Qualitative Approach.

OBJECTIVES: Acceptance of childhood vaccination varies between societies, affecting worldwide vaccination coverage. Low coverage rates are common in indigenous populations where parents often choose not to vaccinate their children. We aimed to gain insight into reasons for vaccine acceptance or rejection among Warao Amerindians in Venezuela. METHODS: Based on records of vaccine acceptance or refusal, in-depth interviews with 20 vaccine-accepting and 11 vaccine-declining caregivers were performed. Parents' attitudes were explored using a qualitative approach. RESULTS: Although Warao caregivers were generally in favor of vaccination, fear of side effects and the idea that young and sick children are too vulnerable to be vaccinated negatively affected vaccine acceptance. The importance assigned to side effects was related to the perception that these resembled symptoms/diseases of another origin and could thus harm the child. Religious beliefs or traditional healers did not influence the decision-making process. CONCLUSIONS: Parental vaccine acceptance requires educational programs on the preventive nature of vaccines in relation to local beliefs about health and disease. Attention needs to be directed at population-specific concerns, including explanation on the nature of and therapeutic options for side effects.

Estudio de mecanismos moleculares asociados a la proliferación y sobrevida del clon tumoral en la Leucemia Linfoide Crónica: hacia la identificación de un posible blanco terapéutico / Study of molecular mechanisms associated with the proliferation and survival of the tumor clone in Chronic Lymphoid Leukemia: towards the identification of a possible therapeutic target

La Leucemia Linfoide Crónica (LLC) es una neoplasia caracterizada por la acumulación de Linfocitos B (LB) maduros en sangre periférica, médula ósea y órganos linfoides secundarios (OLS). Es la leucemia más frecuente en adultos de países occidentales, y afecta principalmente a individuos mayores de 50 años. Esta enfermedad presenta una evolución clínica heterogénea, donde si bien hay un grupo de pacientes que se mantienen con una LLC estable desde su diagnóstico sin requerir tratamiento, existen otros que debutan o progresan con una enfermedad más agresiva, observándose con frecuencia resistencia o recaídas posterior a la terapia. Por lo que, en la actualidad la LLC sigue considerándose una patología incurable. El estudio de los mecanismos moleculares que conllevan a la progresión de la enfermedad permite identificar nuevos blancos terapéuticos que podrían redundar en el hallazgo de un mejor tratamiento que cure definitivamente esta leucemia y/o mejore la calidad de vida de los pacientes. El microambiente tumoral juega un rol fundamental en la fisiopatología de la LLC. La interacción de células no B con el linfocito leucémico provee señales que conlleva a la sobrevida y/o proliferación del clon tumoral. En el año 2005, Messmer y col. describieron que en la LLC existe una subpoblación celular con alta capacidad proliferativa. Acorde con esto, nuestro grupo identificó en la sangre periférica de pacientes con pronóstico desfavorable la presencia de una fracción proliferante (FP) con un activo cambio de clase y expresión de la enzima deaminasa de citidina inducida por activación (AID). Posteriormente, otras FPs expresando la enzima AID como una señal de activación linfocitaria también han sido descritas en pacientes con mala evolución clínica. Y dado a su actividad mutagénica, AID ha sido asociada a la generación de mutaciones puntuales en el ADN y translocaciones cromosómicas en linfomas de células B. La presencia de estas FP con una expresión anómala de AID en la sangre periférica de pacientes con una enfermedad progresiva nos lleva a hipotetizar que esta enzima podría estar relacionada con el origen de las aberraciones cromosómicas de mal pronóstico en la LLC. En este trabajo demostramos que la FP IgMpos/IgGpos se asocia al desarrollo de una enfermedad más agresiva y que esta subpoblación pareciera ser la primera en adquirir la del11q que conlleva a la pérdida del gen ATM, siendo dicha alteración cromosómica asociada a la refractariedad terapéutica en la LLC. De igual manera, nuestros resultados muestran que las FP y la expresión de AID se ven afectados por el tratamiento con Ibrutinib, un inhibidor de la enzima tirosina quinasa de Brutón que ha sido recientemente aprobado por la FDA para el tratamiento de la LLC. Además, los resultados de esta tesis proveen datos originales sobre un mecanismo alternativo al previamente descrito para esta droga sobre el clon leucémico. Demostrando así que la disminución de la enzima AID luego del tratamiento con ibrutinib estaría originada en la desactivación de la vía IL-4/JAK/STAT6. En una segunda parte de la tesis nuestros esfuerzos fueron dirigidos al estudio del proceso inflamatorio en los pacientes progresores con LLC y como moléculas implicadas en la activación del inflamasoma podrían convertirse en nuevos blancos terapéuticos. Si bien, la presencia de un microambiente tumoral activado favorece el desarrollo de una LLC más agresiva. También ha sido descrito una paradójica regulación del sistema inmune en esta leucemia, donde una modulación negativa de la respuesta inflamatoria puede promover el crecimiento tumoral y tolerancia inmunológica. En este sentido nuestros resultados muestran que la sobreexpresión de la proteína TMEM176A conlleva a menos actividad del inflamasoma y resistencia a la piroptosis en la LLC, postulando la hipótesis de que las células tumorales de pacientes con un desarrollo agresivo de LLC podrían aumentar la expresión de TMEM176A para escapar de la muerte celular programada. Finalmente, este trabajo propone que la modulación farmacológica de TMEM176A podría ser utilizada como un complemento junto con drogas como Venetoclax o Ibrutinib en la terapia para esta leucemia. Los resultados en células humanas leucémicas, así como en un modelo murino de LLC, mostrados en este trabajo de tesis proveen un aporte racional a esta hipótesis y dejan planteada una nueva perspectiva terapéutica para esta leucemia