Highlights on globin gene switch: new target for therapeutics

Renzo Galanello Pediatric Clinic 2 and Thalassemia Unit–

University of Cagliari (Italy) Ospedale Regionale Microcitemie ASL8

6th International Scientific Meeting New biologci and analytic issue on Hemoglobin A2 and other minor Hemoglobins

ORGANIZATION AND DEVELOPMENTAL EXPRESSION OF GLOBIN GENES

birth

α β

γ

γ

β ζ ε

1 year 4 weeks

δ

LCR

ε β δ Gγ Aγ CHROMOSOME 11

CHROMOSOME 16 ζ α α

HS 40

Moi P.

3

Type And Distribution Of β-Thalassemia Mutations

Thein et al. 2004.

Promoter

RNA Processing

5’ & 3’ UTR

ATG Nonsense codons Frameshifts

Β+

Β+

Β+, βº

βº

βº

βº

1 2 3

CCAAT

CACCC

CACCC

ATAAA ATAA

3’ 3’ UTR 105 146

AG TAA

IVS 2

GT AG GT CATG

IVS 1 5’ UTR 30 104 30 1

5’

Globin chain synthesis with β-thalassemia mutations

Brith

α

γ

γ

β 1year 4 weeks

α

HbA

HbF

Moi P.

Pathophysiology of β-thalassemia

Normal production alpha chains

Absent or decreased beta chains

Excess unstable alpha tetramers

Absent or decreased haemoglobin A

Ineffective erythropoiesis

Hemolysis

ANEMIA

HbA alpha 2/beta 2

6

a globin

β + γ globin

Mild Phenotype

α thalassemia Mild/silent β alleles Increased γ

Severe Phenotype

a globin

β + γ globin

Mechanisms Of β-Thalassemia Intermedia

7

HOMOZYGOUS SARDINIAN δβ°-THALASSEMIA

MCV fl 72.7 MCH pg 24.2 F 18.8 A2 % 2.8

Hb g/dl 11.9 MCV pg 63.4 MCH pg 21.6 F % 99.7 A2% 0.3 Aγ % 72.0

δβ°-thalassemia

DNA analysis: β39 C→T homozygous Aγ -196 C→T homozygous

Age 3.5 y

Galanello et al,Blood 2002

Why interest in fetal hemoglobin and hemoglobin switching?

ü  Hb F is a strong modifier of hemoglobinopathies’ severity

ü  level of HbF is a variable and inducible quantitative trait in humans

ü  to understand the mechanisms of thalassemia intermedia

ü  to understand the general mechanisms of gene expression and of developmental gene regulation

ü  to develop targeted therapeutic approaches for ameliorating the severity of the beta-hemoglobinopathies

.

.

Population distribution of fetal hemoglobin

Thein SL,2009

•  interindividual HbF variation is highly heritable

•  genetic investigation is expected to identify genetic factors that controls HbF production

11346–11351 PNAS July 3, 2007 vol. 104 no. 27

QTLs map to HBS1L-MYB and BCL11A

Copyright ©2008 by the National Academy of Sciences

Uda M.,Galanello R et al, Proc. Natl. Acad. Sci. USA 105, 1620-1625 (2008)

GWAS results for HbF

Hb F variation associated SNPs

Chr 2

2p16 BCL11A

rs766432

rs1427407

rs11886868

rs4671393

Chr 6

6q23 MYB

rs9399137

rs4895441

Chr 11

11p15 HBB

rs4910742

rs7482144

p= 9.14e-08

p= 0.018 p= 0.446

Bcl11A and HbF v genetic association detected by G-WAS

v SNPs in IVS2 described in different populations, in HPFH, beta thalassemia, HbE, SCD

v High HbF is associated with low Bcl11A expression

v Bcl11A expression varies at different developmental stages

v Bcl11A is down-regulated by KLF1 gene

VOLUME 42 | NUMBER 9 | SEPTEMBER 2010 Nature Genetics

Schematic mechanism of KLF1 action

KLF1 direct binding direct binding

β-promoter BCL11A promoter

induces β-globin gene expression

inhibition of γ globin gene expression

 γ-­‐globin  gene  expression  in  β-­‐talassemia  and  in    HPFH  

Birth

α

γ

γ

β 1 year 4 weeks

δ

α

γ-HPFH

Moi  P.  

0

0.2

0.4

0.6

0.8

1

0 1 2 3 4

Tal. Major

Tal. Inter

Numero di alleli positivi

Pro

porz

ione

paz

ient

i

CONTRIBUTO DI ALCUNI ALLELI NEL DETERMINARE IL FENOTIPO DI TALASSEMIA MAJOR E INTERMEDIA

Blood,2009

Locus   p   Hazards  Ra<o   Harrell’s  C-­‐index   Predictor  for  later  transfusion  start  

HBG2:g.-­‐58C>T   <0.001   0.081   0.54   +/-­‐  α  gene  defects   <0.001   0.514   0.61   class  2    

BCL11A  rs1427407   <0.001   2.391  

0.63  T  allele  

rs10189857   0.005   1.312   G  allele  

HBS1L/MYB  rs4895441   <0.001   1.979  

0.57  G  allele  

rs6904897   0.020   0.697   TT  genotype  Gender   0.016   0.738   0.52   Male  

First  quar*le  (pa*ents  with  more  posi*ve  predictors  combina*ons)  

Second  quar*le  

Third  quar*le  

Fourth  quar*le  (pa*ents  with  more  nega*ve  predictors  combina*ons)  

Survival  curves  for    316  pa<ents  with  different  combina<ons  of  predictors  for  later  and  earlier  <me  to  transfusion  

Danjou F et al.,2012

Clinically relevant fetal hemoglobin modifiers

•  Genetic: - in cis variants: - γ globin genes - β globin cluster - LCR

- in trans variants: - BCL11A - HBS1L-MYB - KLF1 mutations

- GATA1 - trisomy 13 •  Epigenetic:

- DNA methylation

- histone modification (acetylation, phosphorylation, methylation)

- erythropoiesis expansion (perturbation of erythroid kinetics)

Cao A Moi P. Galanello R.: Pediatric Reports 2011

Current hemoglobin switching model

Variance components of the F-cell trait

Age ad sex 2% Chr.11p15.4 – b globin locus 10%

Chr.6q23.3 – HMIP 19%

Chr.2q16.1 – BCL11A 15%

Other, unknown, genes

Environment 9%

(MenzelS, 2009)

Rationale for HbF induction!

ü Gamma globin genes are intact"

ü HbF will functionally compensate for the absence of HbA (HPFH homozygotes, genetic compounds (β-thal/HPFH)"

"

ü HbF in post-natal life is influenced by physiological and genetic factors (pregnancy, acute erythroid expansion, haematological diseases, haemoglobinopathies)"

ü High cost of conventional treatment"

ü  Problems wit blood safety and availability in several countries" HbA = adult haemoglobin; HbF = fetal haemoglobin.

HbF inducers

Class Compound

Hypomethylating/cytotoxic agents 5-azacytidine, decitabine

Cytotoxic agents hydroxyurea

Histone Deacetylase Inhibitors butyrate analogs, short chain fatty acids, adipicin, scriptaid, trichostatin A, valproic acid,

Antioxidants resveratrol, angelicin, curcumin………

Others

Erythropoietin, thalidomide

Atweh g eta l Hematol Incol Clin N Am 2010

•  histone hyperacetylation

•  p38-MAPK phosphorylation

•  ERK MAPK dephosphorylation

•  sGC/cGMP pathway activation

•  increased γmRNA translation

• sGC/cGMP pathway activation

•  increase of CAMP

• hypomethylation by inhibition of DNA

• hyperacetylation (?)

thalidomide

Mechanisms for γ-globin induction by pharmacologic agents

Summary of clinical trials with HU in β-thalassemia syndromes

ü  18 trials

ü  7 thalassemia major, 8 intermedia, 3 HbE/β-thal

ü  treated patients 573

ü  number of patients/trial: 2 to 163

ü  variable doses 3 to 30 mg/kg/day

ü  responding patients: 25 to 100%

ü  total hemoglobin increase: <1 g up to 4g/dl

ü  best response in thalassemia intermedia

ü  response correlates with XmnI polymorphism

Thalidomide therapy in patients with thalassemia major.

A genotype –28A→C/cd39

B genotype IVS1-6/cd44C

100 mg/kg/d HbF=∼100%

75 mg/kg/d HbF=50 → 73%

Aguilar-Lopez et al Blood Cell Mol Dis 2008

Pharmacologic induction of HbF:concerns and limitations

Ø  potential toxicity→ safety an long term

Ø  unpredictable and variable response

Ø  effectiveness and long term (loss of efficacy overtime)

Ø  compliance

Future directions

Ø  other safer and more effective compounds

Ø  better understanding of molecular mechanisms

Ø  combination therapies

Ø  correlation with individual genetic determinants

Ø  personalized therapies

Ø  impact on natural history

Bianchi N et al

Rapamycin

Induction of HbF by rapamycin

Conclusions v second generation DNA technologies have improved the knowledge of

globin gene modifiers

v well phenotyped cohorts of patients are essential for genetic research:

v gene modifiers are useful in the follow-up of disease -related complications and in drug selection and dosing

v genetic studies of disease modifiers can identify new potential targets for therapy

v an integrated approach by clinicians, geneticists, clinical researchers and basic scientists is needed to further improve the knowledge of variability and the treatment of patients with hemoglobinopathies

your genetics is only as good as your phenotype