Genetic evaluation of a combination therapy for the treatment of KRAS mutant lung adenocarcinoma

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 17-12-2019Esta tesis tiene embargado el acceso al texto completo hasta el 17-06-2021Genetic ablation of CDK4 is known to prevent tumor development of Kras-driven lung adenocarcinoma (LUAD). Yet, elimination of the protein does not mimic pharmacological inhibition. We have utilized a genetically engineered mouse model (GEMM) that closely recapitulates this pharmacological treatment to evaluate the consequences of inactivating CDK4. In the present thesis, we have used two kinase dead (KD) isoforms, K35M and D140A, to eliminate the catalytic activity of CDK4. We demonstrated that the kinase function of CDK4 is not essential for mouse embryonic development. These mutant mice recapitulate most of the features of the complete elimination of CDK4 but later in time and with less penetrance. Mice expressing these CDK4 kinase dead isoforms are smaller than their wildtype counterparts and develop diabetes due to defects in the β-pancreatic cells development. Furthermore, CDK4 inactivation impairs lung tumor initiation, although to a lesser extent than CDK4 ablation, suggesting the existence of kinase independent functions for the maintenance of cell cycle progression. Nevertheless, inactivation of CDK4 is not sufficient to eradicate aggressive KrasG12V;Trp53-null adenocarcinomas. Hence, combinatorial targets to improve therapeutic responses have been validated. Recently, it has been reported that elimination of RAF1 decreases lung tumor progression but unfortunately and similarly to CDK4 inactivation, it is insufficient to completely eliminate the disease in a Trp53 deficient background. Here, we demonstrated that concomitant inhibition of CDK4 and RAF1 leads to regression of 100% of the Kras-driven adenocarcinomas even in the most aggressive context where the tumor suppressor Trp53 is depleted. Of note, 24% were complete responders according to the RECIST criteria. Still, there is a small population of remaining non-proliferative cells that survive in the absence of both targets being able to evolve and spawn new cancer growth. In vitro characterization of those cells confirmed the heterogenous mechanisms a cell can acquire in order to sustain tumor growth independently of CDK4 and RAF1. Both, epigenetic modifications as well as activation of alternative signaling pathways such as PI3K, are found to be responsible of CDK4 and RAF1 independent cell growth. In fact, selective treatment with 5-Azacytidine or PI3K inhibitors halts proliferation of CDK4 and RAF1 resistant cells. Further studies to understand the complex crosstalk between the cell cycle and MAP Kinase signaling will shed light on future novel treatments against KRAS mutant tumors.La eliminación genética de CDK4 en un modelo murino de adenocarcinoma de pulmón inducido por el oncogén Kras impide el desarrollo tumoral. Sin embargo, la eliminación completa de la proteína no reproduce la inhibición farmacológica. Hemos utilizado un modelo de ratón genéticamente modificado que recapitula dicha inhibición farmacológica, permitiéndonos evaluar las consecuencias de la inactivación de la actividad quinasa de CDK4. En esta tesis hemos usado dos modelos, K35M y D140A, para eliminar la actividad catalítica de la proteína. Hemos demostrado que la función quinasa no es esencial para el desarrollo embrionario del ratón. Estos animales recapitulan la mayoría de las características asociadas a la eliminación completa de CDK4, pero con mayor latencia y menor penetrancia. Los ratones que expresan una forma catalíticamente inactiva de CDK4 son más pequeños que sus hermanos de camada para los que Cdk4 está intacto y, además, desarrollan diabetes debido a un defecto en el desarrollo de las células β pancreáticas. Por otra parte, la inactivación de CDK4 ralentiza la iniciación de los tumores de pulmón, aunque en menor medida que la eliminación completa de la proteína, sugiriendo que existen funciones catalíticamente independientes para el mantenimiento de la progresión celular. Sin embargo, la inactivación de CDK4 no es suficiente para erradicar los tumores de pulmón más agresivos dirigidos por el oncogén Kras donde, además, el tumor supresor Trp53 es eliminado. Por lo tanto, hemos buscado dianas terapéuticas adicionales para ser combinadas con CDK4. Recientemente, hemos reportado que la eliminación de RAF1 disminuye la progresión de los tumores de pulmón, pero desafortunadamente como la inactivación de CDK4, es insuficiente para eliminar completamente la enfermedad en un fondo sin Trp53. En esta tesis hemos demostrado que la inhibición conjunta de CDK4 y RAF1 favorece la disminución del tamaño del 100% de los tumores de pulmón dirigidos por el oncogén Kras en un contexto donde la función de Trp53 ha sido eliminada. Cabe destacar que el 24% de los tumores han desaparecido completamente de acuerdo a los criterios RECIST. Aún así, hay un pequeño porcentaje de células que sobreviven en ausencia de las dos dianas terapéuticas, siendo capaces de evolucionar y promover nuevo crecimiento tumoral. La caracterización de dichas células, confirma la existencia de diversos mecanismos heterogéneos como cambios epigenéticos o la activación de vías de señalización alternativas como la vía de PI3K. De hecho, el tratamiento selectivo con 5-Azacitidina o inhibidores de PI3K impiden la progresión de las células resistentes a la inactivación de CDK4 y RAF1. Futuros estudios para entender las complejas interacciones entre el ciclo celular y la vía de MAP quinasa arrojarán luz para favorecer el desarrollo de nuevos tratamientos contra los tumores con mutaciones en KRA

Gamificación en el aula de matemáticas a través de recursos virtuales

El trabajo que se presenta a continuación recoge el marco teórico sobre el que se sustenta la gamificación así como el uso de las llamadas Tecnologías de la Información y la Comunicación en la educación. El juego y los beneficios de su aplicación en la educación se muestran a través de autores y profesionales de la neuroeducación, así como su aplicación en la enseñanza de las matemáticas mediante distintas experiencias. A partir de la fundamentación teórica, se trata de dar respuesta ante la desmotivación y los problemas detectados en el ámbito de las matemáticas con una propuesta didáctica que comprende la gamificación en el aula de Matemáticas a través de recursos virtuales. Concretamente, se trata de una experiencia orientada a convertir en protagonista del proceso de enseñanza-aprendizaje al alumnado del curso de 4º ESO.The work presented below presents the theoretical basis on which gamification is constructed as well as the use of the so-called Information and Communication Technologies in education. The game and the benefits of its application in education are shown through authors and professionals of neuroeducation, as well as its application in the teaching of mathematics through different experiences. Based on the theoretical foundation, the aim followed is to respond to demotivation and problems detected in the field of mathematics through a didactic proposal that includes gamification in the Mathematics classroom through virtual resources. Specifically, it is an experience aimed at turning fourth year of the Secondary School students into protagonists of the teaching-learning process.Máster Universitario en Formación del Profesorado de ESO, Bachillerato, Formación Profesional y Enseñanza de Idiomas. Especialidad en Matemáticas (M088

RAF1 kinase activity is dispensable for KRAS/p53 mutant lung tumor progression.

We thank Dr. Shiva Malek and her colleagues (Genentech Inc.) for sharing their results with us before publication. We also thank M. San Roman, R. Villar, M.C. Gonzalez, A. Lopez, N. Cabrera, P. Villanueva, J. Condo, O. Dominguez, and S. Ortega for excellent technical support. This work was supported by grants from the European Research Council (ERC-2015-AdG/695566, THERACAN); the Spanish Ministry of Science, Innovation, and Universities (RTC-2017-6576-1 and RTI2018094664-B-I00) and the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM) to M.B., as well as by a grant from the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00) to M.B. and M.M. M.B. is a recipient of an Endowed Chair from the AXA Research Fund. M.S., P.N., and F.F.-G. were supported by FPU fellowships from the Spanish Ministry of Education. L.E.-B. was a recipient of an FPI fellowship from the Spanish Ministry of Economy and Competitiveness. S.G.-A. is a recipient of a postdoctoral fellowship from the Asociacion Espanola Contra el Cancer (AECC).S

Tumor regression and resistance mechanisms upon CDK4 and RAF1 inactivation in KRAS/P53 mutant lung adenocarcinomas.

KRAS mutant lung adenocarcinomas remain intractable for targeted therapies. Genetic interrogation of KRAS downstream effectors, including the MAPK pathway and the interphase CDKs, identified CDK4 and RAF1 as the only targets whose genetic inactivation induces therapeutic responses without causing unacceptable toxicities. Concomitant CDK4 inactivation and RAF1 ablation prevented tumor progression and induced complete regression in 25% of KRAS/p53-driven advanced lung tumors, yet a significant percentage of those tumors that underwent partial regression retained a population of CDK4/RAF1-resistant cells. Characterization of these cells revealed two independent resistance mechanisms implicating hypermethylation of several tumor suppressors and increased PI3K activity. Importantly, these CDK4/RAF1-resistant cells can be pharmacologically controlled. These studies open the door to new therapeutic strategies to treat KRAS mutant lung cancer, including resistant tumors.We thank S. Ortega for the generation of the Cdk4FxKD mouse model; and M. San Roman, R. Villar, M. C. Gonzalez, A. Lopez, N. Cabrera, P. Villanueva, J. Condo, J. Klett, A. Cebria, A. Otero, O. Dominguez, G. Luengo, G. Garaulet, F. Mulero, and D. Megias for excellent technical support. This work was supported by European Research Council Grant ERC-2015-AdG/695566, THERACAN, Spanish Ministry of Science, Innovation, and Universities Grant RTC-2017-6576-1, and the Autonomous Community of Madrid Grant B2017/BMD-3884 iLUNG-CM (to M.B.); Spanish Ministry of Science, Innovation, and Universities Grant RTI2018-094664B-I00 (to M.B. and M.M.); and National Natural Science Foundation of China Grant 31771469 (to H.W.). M.B. is a recipient of an Endowed Chair from the AXA Research Fund. L.E.-B. is the recipient of an FPI fellowship from the Spanish Ministry of Economy and Competitiveness. F.F.-G., M.S., and P.N. were supported by an FPU fellowships from the Spanish Ministry of Education.S

Latency reversal agents affect differently the latent reservoir present in distinct CD4+ t subpopulations

Latency reversal agents (LRAs) have proven to induce HIV-1 transcription in vivo but are ineffective at decreasing the size of the latent reservoir in antiretroviral treated patients. The capacity of the LRAs to perturb the viral reservoir present in distinct subpopulations of cells is currently unknown. Here, using a new RNA FISH/flow ex vivo viral reactivation assay, we performed a comprehensive assessment of the viral reactivation capacity of different families of LRAs, and their combinations, in different CD4 T cell subsets. We observed that a median of 16.28% of the whole HIV-reservoir induced HIV-1 transcripts after viral reactivation, but only 10.10% of these HIV-1 RNA cells produced the viral protein p24. Moreover, none of the LRAs were powerful enough to reactivate HIV-1 transcription in all CD4 T cell subpopulations. For instance, the combination of Romidepsin and Ingenol was identified as the best combination of drugs at increasing the proportion of HIV-1 RNA cells, in most, but not all, CD4 T cell subsets. Importantly, memory stem cells were identified as highly resistant to HIV-1 reactivation, and only the combination of Panobinostat and Bryostatin-1 significantly increased the number of cells transcribing HIV within this subset. Overall, our results validate the use of the RNA FISH/flow technique to assess the potency of LRAs among different CD4 T cell subsets, manifest the intrinsic differences between cells that encompass the latent HIV reservoir, and highlight the difficulty to significantly impact the latent infection with the currently available drugs. Thus, our results have important implications for the rational design of therapies aimed at reversing HIV latency from diverse cellular reservoirs

Self-Reported Health Status in Primary Health Care: The Influence of Immigration and Other Associated Factors

OBJECTIVE: The aims of this study are to compare self-reported health status between Spanish-born and Latin American-born Spanish residents, adjusted by length of residence in the host country; and additionally, to analyse sociodemographic and psychosocial variables associated with a better health status. DESIGN: This is a cross-sectional population based study of Latin American-born (n = 691) and Spanish-born (n = 903) in 15 urban primary health care centres in Madrid (Spain), carried out between 2007 and 2009. The participants provided information, through an interview, about self-reported health status, socioeconomic characteristics, psychosocial factors and migration conditions. Descriptive and multiple logistic regression analyses were conducted. RESULTS: The Spanish-born participants reported a better health status than the Latin America-born participants (79.8% versus 69.3%, p<0.001). Different patterns of self-reported health status were observed depending on the length of residence in the host country. The proportion of immigrants with a better health status is greater in those who have been in Spain for less than five years compared to those who have stayed longer. Better health status is significantly associated with being men, under 34 years old, being Spanish-born, having a monthly incomes of over 1000 euros, and having considerable social support and low stress. CONCLUSIONS: Better self-reported health status is associated with being Spanish-born, men, under 34 years old, having an uppermiddle-socioeconomic status, adequate social support, and low stress. Additionally, length of residence in the host country is seen as a related factor in the self-reported health status of immigrants

Estructura económica de Risaralda

Para conocer la historia de un país, también es indispensable conocer como han estado conformadas sus ciudades y cómo ha sido su recorrido a través del tiempo para tener una perspectiva mucho más amplia de su economía durante cada coyuntura. Es por esto, que la Historia Económica de Risaralda busca poner a disposición y resaltar una visión bastante amplia de su recorrido histórico y económico, partiendo desde los primeros habitantes y estructuras económicas del territorio, datadas desde tiempo precoloniales, y atravesando por las diferentes transformaciones que fueron afectando a Risaralda durante la colonia y en los años posteriores a esta, especialmente con acontecimientos como la colonización antioqueña, que dejó arraigada la actividad agrícola como la más importante de la economía departamental, aunque la misma con el pasar de los años en el siglo XX fue perdiendo importancia a causa del auge manufacturero, y especialmente del auge de la terciarización sustentada en la actividad comercial. Este fenómeno de terciarización económica será considerado de forma tanto histórica como actual, identificando cual es la situación económica vigente de Risaralda, y a partir de ello darse una idea de lo que le puede deparar al territorio en los próximos años, donde los objetivos de sostenibilidad y calidad de vida parecen ser las claves de toda iniciativa

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to &lt;90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], &gt;300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of &lt;15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P&lt;0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P&lt;0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Global burden and strength of evidence for 88 risk factors in 204 countries and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

Background: Understanding the health consequences associated with exposure to risk factors is necessary to inform public health policy and practice. To systematically quantify the contributions of risk factor exposures to specific health outcomes, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 aims to provide comprehensive estimates of exposure levels, relative health risks, and attributable burden of disease for 88 risk factors in 204 countries and territories and 811 subnational locations, from 1990 to 2021. Methods: The GBD 2021 risk factor analysis used data from 54 561 total distinct sources to produce epidemiological estimates for 88 risk factors and their associated health outcomes for a total of 631 risk–outcome pairs. Pairs were included on the basis of data-driven determination of a risk–outcome association. Age-sex-location-year-specific estimates were generated at global, regional, and national levels. Our approach followed the comparative risk assessment framework predicated on a causal web of hierarchically organised, potentially combinative, modifiable risks. Relative risks (RRs) of a given outcome occurring as a function of risk factor exposure were estimated separately for each risk–outcome pair, and summary exposure values (SEVs), representing risk-weighted exposure prevalence, and theoretical minimum risk exposure levels (TMRELs) were estimated for each risk factor. These estimates were used to calculate the population attributable fraction (PAF; ie, the proportional change in health risk that would occur if exposure to a risk factor were reduced to the TMREL). The product of PAFs and disease burden associated with a given outcome, measured in disability-adjusted life-years (DALYs), yielded measures of attributable burden (ie, the proportion of total disease burden attributable to a particular risk factor or combination of risk factors). Adjustments for mediation were applied to account for relationships involving risk factors that act indirectly on outcomes via intermediate risks. Attributable burden estimates were stratified by Socio-demographic Index (SDI) quintile and presented as counts, age-standardised rates, and rankings. To complement estimates of RR and attributable burden, newly developed burden of proof risk function (BPRF) methods were applied to yield supplementary, conservative interpretations of risk–outcome associations based on the consistency of underlying evidence, accounting for unexplained heterogeneity between input data from different studies. Estimates reported represent the mean value across 500 draws from the estimate's distribution, with 95% uncertainty intervals (UIs) calculated as the 2·5th and 97·5th percentile values across the draws. Findings: Among the specific risk factors analysed for this study, particulate matter air pollution was the leading contributor to the global disease burden in 2021, contributing 8·0% (95% UI 6·7–9·4) of total DALYs, followed by high systolic blood pressure (SBP; 7·8% [6·4–9·2]), smoking (5·7% [4·7–6·8]), low birthweight and short gestation (5·6% [4·8–6·3]), and high fasting plasma glucose (FPG; 5·4% [4·8–6·0]). For younger demographics (ie, those aged 0–4 years and 5–14 years), risks such as low birthweight and short gestation and unsafe water, sanitation, and handwashing (WaSH) were among the leading risk factors, while for older age groups, metabolic risks such as high SBP, high body-mass index (BMI), high FPG, and high LDL cholesterol had a greater impact. From 2000 to 2021, there was an observable shift in global health challenges, marked by a decline in the number of all-age DALYs broadly attributable to behavioural risks (decrease of 20·7% [13·9–27·7]) and environmental and occupational risks (decrease of 22·0% [15·5–28·8]), coupled with a 49·4% (42·3–56·9) increase in DALYs attributable to metabolic risks, all reflecting ageing populations and changing lifestyles on a global scale. Age-standardised global DALY rates attributable to high BMI and high FPG rose considerably (15·7% [9·9–21·7] for high BMI and 7·9% [3·3–12·9] for high FPG) over this period, with exposure to these risks increasing annually at rates of 1·8% (1·6–1·9) for high BMI and 1·3% (1·1–1·5) for high FPG. By contrast, the global risk-attributable burden and exposure to many other risk factors declined, notably for risks such as child growth failure and unsafe water source, with age-standardised attributable DALYs decreasing by 71·5% (64·4–78·8) for child growth failure and 66·3% (60·2–72·0) for unsafe water source. We separated risk factors into three groups according to trajectory over time: those with a decreasing attributable burden, due largely to declining risk exposure (eg, diet high in trans-fat and household air pollution) but also to proportionally smaller child and youth populations (eg, child and maternal malnutrition); those for which the burden increased moderately in spite of declining risk exposure, due largely to population ageing (eg, smoking); and those for which the burden increased considerably due to both increasing risk exposure and population ageing (eg, ambient particulate matter air pollution, high BMI, high FPG, and high SBP). Interpretation: Substantial progress has been made in reducing the global disease burden attributable to a range of risk factors, particularly those related to maternal and child health, WaSH, and household air pollution. Maintaining efforts to minimise the impact of these risk factors, especially in low SDI locations, is necessary to sustain progress. Successes in moderating the smoking-related burden by reducing risk exposure highlight the need to advance policies that reduce exposure to other leading risk factors such as ambient particulate matter air pollution and high SBP. Troubling increases in high FPG, high BMI, and other risk factors related to obesity and metabolic syndrome indicate an urgent need to identify and implement interventions