Biologia e terapia della LLC fludarabina-resistente: ruolo delle mutazioni di TP53

1. 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

2. 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%).

3. 17p deletion 11q deletion 12q trisomy Normal 13q deletion assole abnormality Genomic abnormalities in CLL are independent predictors of survival 100 80 60 Patients surviving (%) 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

4. 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

5. Dissecting fludarabine resistance Gene expression changes at 24 and 48 hours for each gene relative to its expression in control cultures. Immunohistochemical staining for p53: overexpression in 30% of tumor cells. Rosenwald, Blood 2004

6. Deleted in 22% of p53 WT cases (virtually all unmutated), associated to monoallelic mutations A model of the p53 network and its potential impairment in CLL. Zenz, Cell Cycle 2009

7. copy number neutral Del(17p)/TP53 alterations: overview ≈5% ≈95% (dominant negative) • mutated allele frequency >50%, likely UPD • mutated allele frequency ≤50%, both a) and b) may be present. Zenz, Cell Cycle 2009

8. TP53: a “stand-alone” player in del(17p)-related fluda-resistance? Time to treatment Molecular cytogenetics (FISH) 94% Dicker et al, Leukemia 2009

9. 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

10. Isolated TP53 mutations are an independent prognostic factor in CLL Multivariate analyses 1 Cerri et al, Haematologica 2008; 2 Rossi et al. Clin Cancer Res 2009 3 Grever et al J Clin Oncol 2007; 4 Dicker et al, Leukemia 2008 5 Zenz et al, Blood 2008

11. no TP53 defect (n=8) monoallelic TP53 defect (n=7) biallelic TP53 defect (n=7) Viability of CLL cells after in vitro administration of fludarabine *4 ATM deletion +4 wt unmut IgVH. Malcikova, Blood prepub. Oct 2009

12. Monoallelic TP53 abnormalities impair survival of CLL patients (P<0.001)* (P=0.021)* (P=0.55)* Survival analysed from time of TP53 / ATM investigation * p53-wt/Atm-wt harboring unmutated IgVH as control Malcikova, Blood prepub. Oct 2009

13. Induction of p53-downstream target genes PUMA, BAX and CDKN1A (p21) after fludarabine administration. Fludarabine concentration 3.6 μg/ml for 24 h. PUMA TYPE A PROFILE (missense mutations): high baseline p53 levels, failure to induce p21 TYPE B PROFILE (frameshift mutations): low baseline levels of p53, failure to induce both p53 and p21 BAX p21 monoallelic TP53 inactivation no TP53 abnormality biallelic TP53 inactivation Malcikova, Blood prepub. Oct 2009

14. Model of the p53/miR-34a network and its potential impairment in CLL. INTACT INTACT 50% of F-refractory cases in the absence of TP53 mutations/17p deletions No type A or B profile Zenz, Blood 2009 & Cell Cycle 2009

15. 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 in70% of cells 5 Gy control 5 Gy control ↓miR-34 Cell viability% ↑miR-34 Zenz et al, Blood 2009

16. Before FLUDA FLUDA-refractory p53 p21 p53 p21 17p, TP53 mutation p53 p21 Deregulated miR component of the p53 pathway in refractory CLL 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)

17. …… and to overcome!! 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)

19. ADDITIONAL SLIDES

20. Prognostic factors in CLL: identifying high-risk patients • Survival from start of therapy (fludarabine or pentostatin; n=50) 100 80 No p53 deletion 60 Survival (%) 40 p53 deletion 20 P < 0.001 0 0 12 24 36 48 60 72 Time (months) Time (months) Dohner F, et al. Blood 1995;85:1580–89.