PROGETTI RICERCA FINALIZZATA 2008

MOLECULAR IDENTIFICATION OF RISK-SPECIFIC PATIENT SUBSETS IN CHRONIC LYMPHOPROLIFERATIVE DISORDERS OF B-CELL TYPE
Caligaris-Cappio Federico

Specific Aim 1: clinical validation of prognostic and predictive markers in CLL and MM (first year).
Through proteomic and genomic analysis, we have identified three kinases proteins endowed with a strong prognostic and predictive value. Specifically, we found that in CLL phoshorylation of hematopoietic lineage cell-specific protein 1 (HS1) and of extracellular signal-regulated kinase-1/2 (ERK 1/2) are associated with a poor and a good clinical prognosis, respectively. In addition, in MM, we have recently identified a WNT receptor, RYK, that when overexpressed in MM patients, is strongly associated with a good prognosis (P=0.0016, log-rank). Finally, expression of another WNT receptor, ROR2, is associated with reduced bone lytic lesions. In this aim, we will confirm at the protein level in a large cohort of well defined patients the prognostic and predictive significance of the phosphorylation and overexpression of these markers. Immunohistochemistry analysis will be performed using commercially available antibodies. We anticipate that the studies performed in this specific aim will conclusively demonstrate the prognostic and predictive significance of such markers, and lead the way for their thorough clinical evaluation in prospective clinical trials.

Specific Aim 2: analysis of the kinome of CLL and MM and of the prognostic/predictive significance of alterations of the kinome/translational machinery (first – second year).
It has been shown that genetic alterations of oncogenes acting on the kinome repertoire can be useful in predicting the clinical course of cancer. Furthermore, in this context alterations of the kinome controlling translation, and the translational apparatus itself, are predictive of the sensitivity of cancer cells to drug treatments, such as, e.g. rapalogues. In this specific aim we will analyze on carefully selected samples of patients affected by CLL and MM the kinome complexity. The task will be performed by purification of phosphotyrosine-containing peptides and subsequent Mass spectrometry analysis. A selected focus will be also applied to the translational kinome, by analysis of phosphorylation patterns using in-house validated antibodies and functional assays. We anticipate that the studies performed will generate new targets to be further validated in specific aim 3, and provide useful information for patient stratification.

Specific Aim 3: clinical validation of the markers emerging from Specific Aim 2, and generation of clinical-grade antibodies (second year and beyond).
Based on our previous experience, we anticipate that between 4 and 6 kinases will emerge from the large-scale kinome analysis described in SA2. We will comprehensively examine their prognostic and predictive potential in a large series of well-studied CLL and MM cases that we have collected through the years, using immunohistochemistry. In case adequate and robust antibodies are not available, polyclonal and/or monoclonal antibodies will be generated. For the markers that will survive this screen, clinical-grade antibodies will be generated, to facilitate the implementation of ad-hoc prospective clinical trials.



LEFT VENTRICULAR HYPERTROPHY (LVH) AND OTHER CARDIOVASCULAR MANIFESTATIONS IN ADPKD: A TRANSLATIONAL STUDY
Alessandra Boletta

Left ventricular hypertrophy (LVH) is a very common condition that profoundly affects morbidity and mortality of the population. While several factors are known to cause LVH, the factors identify to date fail to fully justify all cases of LVH reported in the clinical practice. An emerging role for genetic factors able to influence LVH is being recognized, although their precise nature remains largely elusive. Monogenic disorders can be very helpful in identifying genetic factors that might be relevant for the clinics of non-genetic diseases as well.
The major objective of the current proposal is to investigate the relationship between LVH and Autosomal Dominant Polycystic Kidney Disease (ADPKD). ADPKD is one of the most common genetic diseases affecting 1:400-1:1000, whose main clinical manifestation is bilateral renal cyst formation. Of interest, ADPKD is characterized by a variety of extrarenal manifestations of which the cardiovascular defects are by far the most common and the most severe, life-threatening complication. LVH was described to be present in a high proportion of ADPKD patients, although it has been studied mainly in relation to hypertension. Recent studies, however, have reported that a high percentage of ADPKD patients have LVH in the absence of hypertension.
Our goal is to identify genetic determinants related to LVH. To this end we have designed a series of highly completmentary studies that will move from simple in vitro cellular systems, to mouse models and ultimately to a large coort of ADPKD patients that will be characterized for their cardiovascular phenotype as well as for their genetic determinants.
We have identified several advantages in the approach that we propose. Our hypothesis stems from preliminary data generated in mouse models of ADPKD carrying mutations in the Pkd1 gene. The group of Dr. Boletta has recently reported that absence of the Pkd1 gene in cells results in cellular hypertrophy. Interestingly, preliminary data seem to suggest that haploinsufficiency is sufficient to cause this effect. Taking advantage of the simple cellular and animal models available in the laboratory, the group of Dr. Boletta was able to link this particular phenotype to disregulation of the mTOR pathway, known to cause hypertrophy when misregulated. This lead to the hypothesis that cellular hypertrophy might be at the basis of LVH observed in ADPKD patients.
To test this hypothesis a new collaboration was initiated with the group of Dr. Latini, an expert of cardiovascular studies in mice mdels. The preliminary data generated suggest that cardiac hypetrophy can be found in a subset of Pkd1+/- mice. Based on these evidences we are proposing to undertake detailed studies on this aspect, which was never investigated in mice models before. Furthermore, the search for molecular candidates able to regulate this phenotype brings promise to be able to identify therapeutical targets for this clinical manifestation.
Finally, we will take advantage of the expertise that has been build at San Raffaele on ADPKD both at the clinical and at the genetic level. Dr. Manunta and collaborators have recently opened a center for ADPKD that was able to recruit a large cohort of ADPKD patients which will be further expanded within the next two years. These patients will be characterized for their hypertensive phenoptype and cardiovascular defects in collaboration with Dr. Fragasso, a cardiologist of our Institution. Most importantly, all of the patients collected will be analyzed for their genotype by full sequencing of the PKD1 and 2 genes. This is a demanding task that will be feasible by our team thanks to the fact that Dr. Carrera and Dr. Ferrari have established a semi-automated system.
In sum, we believe that we have assembled a strong team that will characterize at the clinical, molecular and genetic level LVH in ADPKD patients with several potential benefits for the italian health system at large.



GENETIC BIOMARKERS FOR A PERSONALIZED THERAPY PREVENTING CARDIOVASCULAR COMPLICATIONS IN DIABETIC NEPHROPATHY
Paolo Manunta

The main objective of this project is to identify those genetic polymorphisms that are responsible for cardiovascular event in patients with diabetic nephropathy. We will use a new candidate genes approach: "de novo" genes selected with our recent Genome Wide Association (GWA) studies in both salt sensitive hypertension and in a large population study (HYPERGENES) will be used. A number of interesting genes involved in cardiovascular, glomerular renal functions, transport systems have been selected. A cohort of 1600 DN patients with a follow-up > 4 years, where all the cardiovascular events carefully registered is already available (BENEDICT and DEMAND prospective clinical trials) for a post GWA study. A custom microchip with 1500 SNPs of genes of interest will be designed. With this study we will identify the gene or combination of genes responsible of the individual variability of cardiovascular event and therapeutic effect.

Secondary objectives will involve incident DN patients with elevated cardiovascular risk (blood pressure> 140/90 with microalbuminuria) where the following issues will be addressed:
1. the role of gene polymorphisms selected on humoral and urinary biomarkers (endogenous ouabain, peptide C, fosfoproteinomic).
2. with an intervention study will evaluate the farmacogenomics effects, on blood pressure control (systolic/diastolic <130/80 mmHg), of dual renin-angiotensin-system (RAS) blockade by benazepril and valsartan combination therapy as compared to single RAS blockade by benazepril or valsartan alone on microalbuminuria and cardiovascular events in high-risk patients with type 2 diabetes with DN. The relationships between albuminuria and cardiovascular outcomes will also be evaluated. Moreover using the the new biomarkers selected other secondary end points are:
a. To evaluate the effects of the three treatments on the incidence of fatal and non-fatal cardiovascular events, regression to low-normal albuminuria (urinary albumin excretion <10 ug/min), albuminuria (considered as a continuous variable), serum creatinine, lipid profile, and GFR (in a representative subgroup);
b. To assess the relationships, in the study group as a whole and within each treatment group, between renal outcome variables (including microalbuminuria and time-dependent changes in albuminuria) and fatal and non-fatal cardiovascular events, between achieved blood pressure or metabolic control and renal and/or cardiovascular outcome variables and between achieved albuminuria reduction or residual follow-up albuminuria and renal and/or cardiovascular outcome variables.
c. To assess the relationships, in the study group as a whole and within each treatment group, between renal outcome variables (ESRD, doubling serum creatinine, GFR decline, proteinuria) and fatal and non-fatal cardiovascular events, between achieved blood pressure or metabolic control and renal and/or cardiovascular outcome variables and between achieved proteinuria reduction or residual follow-up proteinuria and renal and/or cardiovascular outcome variables.
3. The practice relapse of this project, for the Italian health system is the possibility of design a bio-informatic predictive model that, considering the genomic informations, humoral and urinary new biomarkers, will lead to understanding the individual response to therapy to prevent cardiovascular complications. Moreover, bioinformatic analysis will gave the predictive values of all genes studied on renal failure progression, cardiac events, and blood pressure response according to the kind of molecular and cellular mechanism that are at work in each patients with DN.