Severe congenital neutropenia

Transcription

1 Severe congenital neutropenia

2 Milestones of the history of congenital neutropenias Agranulocytosis- Schultz-syndrome Preleukemic Syndrome G-CSF (Phase 1-3 clinical trials) CSF3R mutations* G6PT mutations SBDS mutations P14 mutations RUNX1 mutations* Infantile genetic agranulocytosis Kostmannsyndrome Recombinant G-CSF Establishment of the SCNIR ELANE mutations HAX1 mutations G6PC3 mutations * = acquired mutations

3 Patient 8716/27: 5 µg/kg/d Severe congenital neutropenia G-CSF Treatment Absolute Neutrophil Count [x10^3/µl] Days Absolute Neutrophil Count [x10^3/µl] Patient 8716/01: 20 µg/kg/d Absolute Neutrophil Count [x10^3/µl] Patient 8716/27: 50 µg/kg/d Days Days

4 Identification of Neutropenia causing gene defects Establishment of the SCNIR SBDS Boocock GR, Morrison JA, Popovic M et. al. (2003) P14 Bohn G, Allroth A, Brandes G et. Al. (2007) ELANE Horwitz M, Benson KF, Person RE et. al. (1999) CXCR4 Hernandez PA, Gorlin RJ, Lukens JN et. al. (2003) HAX1 Klein C, Grudzien M, Appaswamy G, et. al. (2007) G6PC3 Boztug K, Appaswamy G, Ashikov A et. al.(2009).

5 Congenital Neutropenia (CN) 189 patients, 29 AML/MDS 1 patient c. -9 A>G 3 patients 1 MDS/AML M1R 1 M1T Severe Chronic Neutropenia International Registry (SCNIR) ELANE Mutations in Cyclic and Congenital Neutropenia 29 patients 4 MDS/AML F43L A61G V45M A61V S46F V65D 1 C55S M66R G56R 1 S67W A57T C71F A57V 1 C71R I60M C71S I60T 1 C71Y 29 patients 4 MDS/AML A79fs 1 R103L R81P R103P V83D I118N L84P I120F G85E 1 I120N 1 G85R L121F V98_Q102del L121H V101M 1 Linear Localization 4 patients 1 MDS/AML IVS3-8 C>A 1 IVS C>T 63 patients 7 MDS/AML L123H W156G S126L 1 W156R S126W V174_C181del 1 A127D C181fs T128del V186I I129del V190fs 1 P139L R191S C151S R193Q C151Y 3 Q194ter L152P 1 V197fs A153P F199fs M154R 11 patients 2 MDS/AML IVS4 +1 G>A 1 IVS4 +1 G>T IVS4 +5 G>A IVS4 +6 3bp ins 1 49 patients 10 MDS/AML D201fs 1 R220Q S202fs 1 G221ter G203R C223fs L206fs 1 C223ter 1 V207D S225ter C208G G226R C208ter 1 Y228ter 1 G210V D230fs 1 G210W F232fs G214E A233fs G214R 3 Q237fs G214ter N240del V219I 5 UTR Exon 1 Exon 2 Exon 3 Intron III Exon 4 Intron IV Exon 5 Cyclic Neutropenia (CyN) 118 patients, 0 MDS/AML 10 patients F43L A61V 3 patients Q97L V101M D117V 1 patient IVS3-2 A>C 16 patients S126L S126W P139L D174ins V186_D201del Q194ter 49 patients IVS4 +1 G>A IVS4 +3 A>T IVS4 +5 G>A 39 patients L206F G214ter R220Q Y228ter W241G W241L W241ter Dale D, Welte K, et al.,curr Opin Hematol. 2015;22:3-11

6 Unfolded protein response (UPR) Transcription of new BiP Apoptosis Translational arrest Protein degradation Adapted from Dudek, J. et al., Cell. Mol. Life. Sci. 2008

7 ATF6 is upregulated in myeloid cells of CN, but not CyN patients CD33 + bone marrow cells ATF6/β-actin mrna Ratio, AU CN CN CyN ctrl ctrl G-CSF Healthy CN DAPI ATF6 Nustede R., et al., BJH 2016

8 HAX transcript variants Mutations affecting both isoforms are associated with neutropenia and a neurological phenotype: Isoform 2 is critical for neuronal functions, Mutations affecting isoform 1 only (e.g. Trp44X) are associated with neutropenia only. Klein, C., et al., Nat Gen 2007 Germeshausen M., et al., Blood 2008 Carlsson G., et al., J Intern Med 2008

9 HCLS1 is phosphorylated by Lyn and Syk upon G-CSF stimulation G-CSF G-CSFR Lyn Syk HAX1 HCLS1 LEF-1 Grb2 JAK2 STATs HCLS1 is a Hematopoietic Cell specific Lyn Substrate 1 HAX1 is a HCLS1 Associated protein X 1

10 G-CSF failed to phosphorylate HCLS1 in hematopoietic cells of CN patients harboring HAX1 mutations G-CSF 0` 30` total HCLS1 Isotype ctrl phospho-hcls1 CN patient healthy individual total HCLS1 / phospho-hcls1

11 HCLS1 is essential for myeloid differentiation Skokowa, J., et al., Nat Med 2012

12 HCLS1 is involved in the nuclear transport of LEF-1 protein LEF-1 WT LEF-1 HCLS1 binding MUT

13 LEF-1 and its target gene C/EBPα expression are downregulated in ELA2 and HAX1 mutated CN patients LEF-1/β-actin mrna Ratio, AU * * ** CN CN CyN ctrl G-CSF C/EBPα/β-actin mrna Ratio, AU * * CN G-CSF * CN CN CyN ctrl G-CSF DAPI CN CyN ctrl anti-lef-1 target genes CN: congenital neutropenia; CyN: cyclic neutropenia; Skokowa, J., et al., Nat Med 2006; 12:

14 Restoration of defective LEF-1 expression promotes granulocytic differentiation of CD34 + progenitors of CN patients LEF-1/β-actin mrna ratio, AU CD15 expression, % positive cells * * ** CD11b expression, % positive cells mock ctrl lv LEF-1 lv * * ** time, days time, days mock ctrl lv LEF-1 lv Skokowa, J., et al., Nat Med 2006; 12:

15 HCLS1 interacts with LEF-1 transcription factor inducing its nuclear translocation and activation upon G-CSF treatment f LEF-1 C/EBPα Medizinische Hochschule Hannover Skokowa J. et al., Nature Medicine, 2012

16 Glucose-6-Phosphatase Komplex Cytosol Disease Gene Expression Phenotype GSD1a G6PC1 Liver, kidney, intestine GSD GSD1b G6PT ubiquitous GSD + CN G6PC3-deficiency G6PC3 ubiquitous CN G6PC1 G6PT G6PC3 ER Lumen

17 JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia Boztug K., et al., Nature Genetics 46, (2014) JAGN1-mutant granulocytes are characterized by ultrastructural defects, absence of secretory vesicles and aberrant N-glycosylation of multiple proteins, and increased apoptosis.

18 a Ig-like domain Cytokine receptor homology region Fibronectin III-like domains Transmembrane region Intracytoplasmic region Healthy individual G-CSF CN patient c.998-2a>t * No signal transduction p.w547* b Neutrophils Monocytes Events Events isotype ctrl healthy individual isotype ctrl CN patient anti-g-csfr healthy individual anti-g-csfr CN patient G-CSFR c G-CSF (up to 110μg/kg/day) GM-CSF 6 μg/kg/day twice a week GM-CSF 3 μg/kg/day twice a week ANC, x1000/ul 4 3,5 3 2,5 2 1, , Time, months after birth Figure 1

19 S E V E R E C H R O N I C N E U T R O P E N I A I n t e r n a t i o n a l R e g i s t r y TAZ+; 6; 3% G6PT+; 22; 10% WAS+; 5; 2% P14+; 4; 2% other mutation; 8; 4% SBDS+; 44; 19% ELANE+; 91; 40% DIGENIC; 4; 2% G6PC3+; 10; 4% HAX1+; 32; 14% Distribution of gene mutations in 226 European congenital neutropenia patients

20 Neutropenia causing mutations Most of cases of SCN are attributable to ELANE mutations, but there are mutations in genes affecting G-CSF signaling (CSF3R, HAX1) genes affecting glucose homeostasis (SLC37A4, G6PC3), lysosomal function (LYST, RAB27A, ROBLD3/p14, AP3B1, VPS13B, TCIRG1), ribosomal proteins (SBDS, RMRP), mitochondrial proteins (HAX1, TAZ), immune functions (STK4, GFI1, CXCR4), and X-linked (WAS) ultrastructural defects, absence of secretory vesicles and

21 How does G-CSF induce granulopoiesis (overcome senescence) in CN, if both LEF-1 and HCLS1 are severely downregulated? Medizinische Hochschule Hannover

22 How does G-CSF induce granulopoiesis in CN? LEF-1 dependent steady-state granulopoiesis LEF-1 independent emergency granulopoiesis * C/EBPα/β-actin mrna Ratio, AU 20 * * * C/EBPß/ß-actin mrnaratio, AU CN CN CyN IN MN ctrl G-CSF CN CN CyN IN MN ctrl G-CSF G-CSF induces C/EBPß in CN!

23 Nampt triggers myeloid differentiation of CD34 + cells 55 kda Nampt 110 kda SIRT1 55 kda Nampt 55 kda 37.5 kda C/EBPβ 40 kda CEBPα 45 kda β-actin 45 kda β-actin control Nampt 20% 84% G-CSFR G-CSF ng/ml medium 0.6 ** ctrl Nampt

24 G-CSF induces Nampt/PBEF and NAD + in myeloid progenitors from CN patients G-CSF STAT3/Nampt NA NAD + SIRT1 Regulation of transcription Nampt/PBEF protein Ng/ml * bone marrow promyelocytes 0 CN CN MN ctrl ctrl G-CSF * inad +, mg/l 5 * CN MN ctrl G-CSF kDa loading control SIRT1 Skokowa, J., et al., Nat Med 2009; 15: 151-8

25 Nampt triggers myeloid differentiation of CD34 + cells % of CD16 + cells ctrl Nampt G-CSF Nampt +G-CSF Time (d) % of CD15 + cells Time (d) - NAMPT + NAMPT 10 µm 10 µm

26 Vitamin B3 treatment of patient with cyclic neutropenia Neutrophil granulocytes in peripheral bloot ( x 10 3 µl -1 ) Treatment with G-CSF Neutrophil granulocytes in peripheral bloot ( x 10 3 µl -1 ) Treatment with Vitamin B3 without G-CSF

27 G-CSF signaling pathways G-CSF G-CSFR HAX1 HCLS1 Lyn Syk LEF-1 SHP-2 Grb2 RAS STAT3,5 JAK2 MAPK Vitamin B3 (Nicotinamide) Nampt NAD + JAK2 SHP-1 SOCS3 Sirtuins, protein deacetylases HAX1 HCLS1 LEF-1 C/EBPs STAT3,5 C/EBPs Nampt: Skokowa J, et al, Nat. Med 2009

28 Risk of leukemia in CN patients First reports on leukemias in CN: Miller RW, (Period ). Pediat Res 1969 Gilman PA, et al., Blood 1970 Rosenberg, et al., BJH 2010

29 G-CSF Treatment by Neutropenia- Genotype Neutropenia Code No Leukemia (n) Median G-CSF dose (µg/kg/d) Leukemia (n) Median G-CSF dose (µg/kg/d) ELANE-CN 72 4, ,7 HAX1-CN 25 3,5 6 7,05 ELANEneg/HAX1neg 19 11,7 6 15,05 neg tested 15 4,43 1 4,86 WAS 3 3,23 2 3,09 SDS 6 1,72 1 4,3 CN not tested 44 5,69 6 5,22 GSD1B 19 3,21 1 3,0 CyC not tested 24 1, ,76 * Median G-CSF Dose for all Congenital Patients 4,85 µg/kg/d and for all Cyclic Patients 1,6 µg/kg/d

30 Congenital Neutropenia Incidence of Leukemia CI at 30 Years by Genetic Subtype Log Rank p=0,649 No ELA/HAX mut. Events/N 6/35 ELA mut. Events/N 11/74 HAX mut. Events/N 4/31

31 VAFs of CSF3R mutant clones in CN and CN/AML patients 20 Frequency of mutant allele, % 17, ,25 13,6 13,4 12,6 13,2 11,2 10,4 10 7,1 7,1 6,1 5 4,65 3,1 3 2,3 2,3 2,2 1 1, Years JS0DK G-CSFR p.q749* JS01F G-CSFR p.q754* JS0QV G-CSFR p.q749* JS0SA G-CSFR p.q741* JS018 G-CSFR p.q739* JS0QE G-CSFR p.y752* JS0QE G-CSFR p.q739* JSA24 G-CSFR p.q768* JS0PG G-CSFR p.q741* JSB15 G-CSFR p.q749* JS0QC G-CSFR p.q749* JS0SJ G-CSFR p.q743* JS0SJ G-CSFR p.q752*

32 CSF3R mutations

33 VAFs of CSF3R mutant clones in CN and CN/AML patients.

34 Leukemia-associated mutations in 31 CN/AML patients Targeted deep sequencing 23 (74 %) CSF3R 20 (64,5 %) RUNX1 2 FLT3-ITD 4 EP300 2 SUZ12 1 CREBB 1 CBL 1 NRAS!!! Neg. for: CEBPA, DNMT3A, IDH1, IDH2, NPM1, TET2

35 High frequency of cooperating RUNX1 and CSF3R mutations in 31 CN/AML patients CSF3R

36 Segregation of RUNX1 and CSF3R mutations in blasts of CN/AML patient 4 1 RUNX1 MUT + CSF3R MUT RUNX1 MUT only CSF3R MUT only 43 N=48

37 First detection of CSF3R- and Runx1 mutations in months prior to AML Patient CSF3R mut Runx1 mut # # # # # #

38 G-CSF treatment in combination with mutations in CSF3R and RUNX1 are leukemogenic Skokowa et al., EHA 2014 Presidential Symposium

39 A 1 st sequential ANC count of CyN-AML patient B 2 nd sequential ANC count of CyN-AML patient (4 months later) ANC, cells/ul Platelets, x1000/ul ANC, cells/ul Platelets, x1000/ul Neutrophils, ANC Platelets time, days Neutrophils, ANC Platelets time, days C c.697g<c c.703delg D CyN-AML CyN-AML mutant allele ELANE ß-actin CyN-AML wild type allele Mother Father ELANE, exon 5 Blood 2016 Figure 1

40 Mutated RUNX1 enhanced clonogenic capacity of lin - cells from d715 Csf3r mice BM lin - cells from d715 Csf3r mice expansion 48 h 72 h transduction with lentiviral constructs with WT or MUT RUNX1 plating FACS sorting BFP + cells 1 st replating 2 nd replating number of colonies 20 ** ** st replating number of colonies no colonies 2 nd replating number of cells 2 weeks aafter plating, x ** ** ctrl BFP WT RUNX1 R139G RUNX1 R174X RUNX1 RUNX1 MUT 1 RHD MUT 2 TAD

41 The two-hit hypothesis of leukemogenesis in CN ELANE-, HAX1- mutations, Genotoxic stress pre-leukemia HSC stem cell 1 st hit 2 nd hit leukemia stem cell leukemic blasts CSF3R mutation RUNX1 mutation Monosomy 7 Trisomy 21 diminished LEF-1, C/EBPa, HCLS1 hyperactivated NAMPT/SIRTs, Akt, STAT5a deacetylated p53, FOXO3a, LEF-1

42 Improvement of maturation arrest after genetic correction HAX1 3F5 +GFP HAX1 3F5 +HAX1 Morishima T et al. Haematologica. 2014; 99:19-27.

43 Correction of ELANE mutations in ipscs from a patient with congenital neutropenia by CRISP/Cas9 technology Nayak RC, et al. JCI 2015

44 Acknowledgement Dept. Molecular Hematopoiesis Julia Skokowa Ünalan Murat Kandabarau Sergey Klimenkova Olga Klimiankou Max Samareh Bardia SCN Registries Cornelia Zeidler David Dale Jean Donadieu and the LLP Physicians Dept. Ped Hem./Oncology MHH Dirk Reinhardt Martin Stanulla Dept. of Hem./Oncology MHH Arnold Ganser Michael Heuser Institute of Cell/Mol. Pathology MHH Doris Steinemann Brigitte Schlegelberger Dept. of Exp Hematology MHH Axel Schambach Zhixiong Li Heinrich-Pette-Institut Hamburg Carol Stocking Washington Univ. School of Medicine Dan Link Department of Pathology MHH H.-H. Kreipe Kais Hussein Erasmus University Rotterdam Ivo Touw Haunersche Kinderklinik München Christoph Klein Munich Leukemia Laboratory Susanne Schnittgers Andreas Kohlmann Medizinische Hochschule Hannover und Universitätsklinikum Tübingen

45 Abteilung und/oder Titel Abteilung und/oder Titel Datum Elternverein krebskranker Kinder Hannover e.v.

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