R lorenzo Falchi Biologia e terapia della malattia fl udarabina resistente ruolo delle mutazioni di TP53

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R lorenzo Falchi Biologia e terapia della malattia fl udarabina resistente ruolo delle mutazioni di TP53 Powered By Docstoc
					        Mediterranean School of Oncology
             Orvieto, 20-22/11/2009




 Biologia e terapia della LLC
   fludarabina-resistente:
ruolo delle mutazioni di TP53
                Lorenzo Falchi
               Marco Gunnellini

     S.C. di Oncoematologia con Autotrapianto
                A.O. S.Maria, Terni
   Incidence and Anatomy of p53 Mutations

• Somatic mutations of p53 occur in >50% of human cancers;

• missense mutations account for ~75% of TP53 mutations,
(frameshift, nonsense, silent mutations and deletions less
frequent);

• the bulk of TP53 mutations cluster within the DNA binding
domain (exons 5 to 8); there appear to be clear “hotspots”;
• unselected cohorts of untreated patients can be expected
to show TP53 mutations in <10% of cases;

• the highest incidence of TP53 mutation is seen in patients
with Fludarabine-refractory CLL (37%).
                          Genomic abnormalities in CLL are
                          independent predictors of survival

                         100
                                                                                17p deletion
                                                                                11q deletion
                                                                                12q trisomy
Patients surviving (%)




                         80
                                                                                Normal
                                                                                13q deletion as
                                                                                sole abnormality
                         60


                         40


                         20


                          0
                               0   12 24 36 48 60 72 84 96 108 120 132 144 156 168 180
                                                    Time (months)
                                                                       Döhner H et al. N Engl J Med 2000
     p53-dependent gene expression response of CLL
         cells to treatment with fludarabine in vivo




DNA microarray analysis identifies a homogeneous p53-dependent gene expression response of
CLL cells to treatment with fludarabine in vivo.
pretreatment level (black squares).
(*) Sequence-verified Lymphochip clones.
(IM) IMAGE clone numbers for unnamed genes.
(LC) Lymphochip number for unnamed genes without sequence verification.
                                                                            Rosenwald, Blood 2004
           Dissecting fludarabine resistance

Gene expression changes at 24 and        Immunohistochemical staining
48 hours for each gene relative to its   for p53: overexpression in
expression in control cultures.          30% of tumor cells.




                                                     Rosenwald, Blood 2004
A model of the p53 network and its potential
            impairment in CLL.
                                        Deleted in 22%
                                       of p53 WT cases
                                          (virtually all
                                         unmutated),
                                        associated to
                                          monoallelic
                                           mutations




                                       Zenz, Cell Cycle 2009
      Del(17p)/TP53 alterations: overview


(dominant negative)   ≈5%                                                  ≈   95%




         copy number neutral
              1. mutated allele frequency >50%, likely UPD
              2. mutated allele frequency ≤50%, both a) and b) may be present.
                                                               Zenz, Cell Cycle 2009
                TP53: a “stand-alone” player in
               del(17p)-related fluda-resistance?
      Molecular cytogenetics (FISH)   Time to treatment



                   94%




Dicker et al, Leukemia 2009
Overall survival of CLL patients according to absence
    or presence of TP53 mutation/17p deletion
                                 (from the time of mutation analysis)




                                p < 0.0001




                      n = 125



                                                     Zenz, Cell Cycle 2009
     Isolated TP53 mutations are an independent
               prognostic factor in CLL
                               Multivariate analyses
    Author             TP53 mutations                                   Del(17p)
              Hazard ratio       95% CI           p       Hazard           95% CI      p
                                                           ratio
    Cerri,     PFS 2.481                         .21
    Rossi      OS 3.202;                        .002
              CT-refr. 3.972                    <.001
    Grever3    ORR 1.082                        .809     PFS 3.428                    .0002

    Dicker4     TTT 6.461         2.409-        .001        1.733          0.816-     .153
                                  17.330                                   3.682
    Zenz5        OS 3.71        1.27-10.8        .02        11.2          5.00-24.9   .001

1 Cerri et al, Haematologica 2008;    4   Dicker et al, Leukemia 2008
2 Rossi et al. Clin Cancer Res 2009   5   Zenz et al, Blood 2008
3 Grever et al J Clin Oncol 2007;
                Viability of CLL cells after in vitro
                  administration of fludarabine

                                              no TP53 defect (n=8)

                                              monoallelic TP53 defect
                                              (n=7)
                                              biallelic TP53 defect
                                              (n=7)




*4 ATM deletion +4 wt unmut IgVH.             Malcikova, Blood prepub. Oct 2009
               Monoallelic TP53 abnormalities impair
                     survival of CLL patients
                                           Survival analysed from time of TP53 / ATM investigation




                                               (P<0.001)*
                                                (P=0.021)*
                                   (P=0.55)*




* p53-wt/Atm-wt harboring unmutated IgVH as control             Malcikova, Blood prepub. Oct 2009
 Induction of p53-downstream target genes PUMA, BAX and
       CDKN1A (p21) after fludarabine administration.

                                              PUMA   Fludarabine concentration
                                                     3.6 μg/ml for 24 h.



                                                     TYPE A PROFILE
                                                     (missense mutations):
                                              BAX    high baseline p53 levels,
                                                     failure to induce p21

                                                     TYPE B PROFILE
                                                     (frameshift mutations):
                                              p21    low baseline levels of
                                                     p53, failure to induce
                                                     both p53 and p21



biallelic TP53   monoallelic      no TP53
 inactivation       TP53        abnormality
                 inactivation                         Malcikova, Blood prepub. Oct 2009
   Model of the p53/miR-34a network and its potential
                  impairment in CLL.
50% of F-refractory cases in the absence of TP53 mutations/17p deletions




    INTACT                              INTACT



                                                         No type A or B
                                                         profile




                                                 Zenz, Blood 2009 & Cell Cycle 2009
Del17p and TP53 mutation are associated with miR-
34a low expression and defective induction after IR
                                                  17p         TP53 mutation w/o17p


                                                                           (**) wild-type TP53
                                                                           allele in 50%
                                                                           (*) wild-type TP53
                                                                           allele in 70% of cells




                                             control   5 Gy   control   5 Gy


                                                 ↓miR-34


                           Cell viability%




                                             ↑miR-34                     Zenz et al, Blood 2009
      Deregulated miR component of the p53 pathway
                    in refractory CLL
                                 Before FLUDA                       FLUDA-refractory




                                p53          p21                   p53           p21


                                                                  17p, TP53 mutation




                                                                  p53              p21




In cases with F-refractory CLL (without TP53 mutation or 17p), miR-34a levels are on average
higher, but a subset of cases show expression levels similar to TP53 mutation/17p cases. Bar
represents mean expression.                                                 Zenz et al, Blood 2009 (II)
                        to restore …..
   Stresses activate the TP53-MDM2 negative regulatory loop.

   Manipulating the MDM2-p53 complex
       Small Molecule E3-Ligase Inhibitors
       Inhibitors of HDM2-dependent p53 degradation (RITA)
       MDM2-antagonists (Nutlins)
       Restore wt and DNA-binding capacity (PRIMA-1)


 ? Toxicity of p53 activation to normal tissues
(Embrionic lethality, Mielotoxicity, Gastrointestinal toxicity)



                 …… and to overcome!!
ADDITIONAL SLIDES
                         Prognostic factors in CLL:
                         identifying high-risk patients
 Survival from start of therapy (fludarabine or pentostatin; n=50)


                   100


                    80
    Survival (%)




                    60                           No p53 deletion
                    40

                    20        p53 deletion           P < 0.001

                     0
                         0   12    24       36      48        60        72

                                            (months)
                                        TimeTime (months)


                                                         Dohner F, et al. Blood 1995;85:1580–89.

				
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