Cooperation of germ line JAK2 mutations E846D and R1063H leads to erythroid hyperplasia with megakaryocytic atypia

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1 Cooperation of germ line JAK2 mutations E846D and R1063H leads to erythroid hyperplasia with megakaryocytic atypia Katarina Kapralova, Ph.D. Department of Biology Faculty of Medicine and Dentistry Palacký University Olomouc Czech Republic Co-authors: Monika Horvathova, Christian Pecquet, Jana Fialova Kucerova, Dagmar Pospisilova, Emilie Leroy, BarboraKralova, Jelena D. Milosevic Feenstra, Fiorella Schischlik, Robert Kralovics, Stefan N. Constantinescu and Vladimir Divoky Blood. 2016;128(10): Erythrocytosis / Polycythemia increased production of erythrocytes Erythrocytosis increased of red blood cell mass (>25% above mean normal predicted value) elevated hematocrit (>49% in men and >48% in women) congenital or acquired mutations -> affect proteins and pathways crucial in the process of erythropoiesis increased hemoglobin level (>165 g/l in men and >160 g/l in women) Hematopoietic stem cell HSC Burst forming unit - erythroid BFU-E Colony forming unit - erythroid CFU-E Erythroblasts Reticulocytes Erythrocytes RBCs 1

2 EPO/EPOR signaling pathway MPN JAK2 V617F JAK2 exon 12 mut Thrombocytosis JAK2 V617I JAK2 R564Q JAK2 R867Q JAK2 S755R JAK2 R938Q PFCP Sensitivity of erythroid progenitors to growth factors blood centrifugation mononuclear cells BFU-E BFU-E Histopaque after 14 days normal growth 0.12U/mL EPO hypersensitivity 100% BFU-E (% of maximum) 80% 60% 40% 20% JAK2 V617F EPOR Q434X VHL R200W controls 0% EPO mu/ml 2

3 Case presentation We analyzed a young Czech patient with congenital erythrocytosis. Age RBC (x10 12 /L) Hb (g/l) Hct (%) Red cell mass (ml/kg) EPO (IU/L) Clinical signs Patient (Normal values) ± 0.1 ( ) 172 ± 5 ( ) 49 ± 1.0 (37-49) 32.1 ( ) 8.9 ± 1.8 (4.3-29) plethora, splenomegaly Mother (Normal values) ( ) 149 ( ) 45 (37-47) ND 8.7 (4.3-29) - Father (Normal values) ( ) 162 ( ) 47 (42-52) ND 4.7 (4.3-29) - ND not done Case presentation A. hypercellularity C. abnormal megakaryocytes B. erythroid hyperplasia glycophorin C 3

4 In vitro sensitivity assay of erythroid progenitors Mutational analysis Whole Exome Sequencing: (CEMM, Vienna, Austria) JAK2 R1063H JAK2 E846D JAK2 R1063H JAK2 E846D Occurrence in healthy population: R1063H: 0.18 % in 1000 Genomes database E846D: 0.05 % in ESP6500 database 4

5 Functional analysis of JAK2 mutations Ba/F3 - wt hepor IL3/EPO dependent stable transfection Ba/F3-EPOR JAK2_wt Ba/F3-EPOR JAK2_V617F = positive control Ba/F3-EPOR JAK2_E846D Ba/F3-EPOR JAK2_R1063H Ba/F3-EPOR JAK2_vector EPO [U/mL] E846D P value R1063H P value V617F P value NS NS NS NS 4.37E-04 P value < 0.05 = significant NS = not significant In vitro signaling of JAK2 E846D and R1063H Exponentially growing IL-3 dependent Ba/F3-EPOR transfectants Starvation from IL-3 for 12h Stimulation with low EPO concentration for 15 min 5

6 Increased STAT5 activation via EPOR γ2a cells transient transfection Increased STAT5 phosphorylation in patient s BM BM aspirates - brown nuclear staining of phospho-stat5 erythroid positive cells negative cells of granulocytic lineage 6

7 In vitro colony assay on murine bone marrow cells JAK2 wt and mutant retroviral particles preparation transduction of C57BL/6 mouse bone marrow in vitro 48h cultivation in vitro test of erythroid colony growth In silico modeling of JAK2 mutations E846D reinforces the active conformation facilitates the active conformation R1063H E. Leroy, S.N. Constantinescu de Duve Institute, Brusel 7

8 Prolonged activation of JAK2signal pathway without EPO Exponentially growing IL-3 dependent Ba/F3-EPOR transfectants Starvation from IL-3 for 12h Stimulation with high dose EPO concentration for 15 min EPO withdrawal + JAK2 inhibitors Screening of the cohort of patients with different etiology Diagnosis Total number Number of JAK2 E846D positive Number of JAK2 R1063H positive Healthy controls n = 27 n = 0 n = 0 MPN JAK2 V617F+ n = 15 n = 1* n = 1 MPN CALR mutation+ n = 31 n = 1* n = 0 Pediatric essential thrombocythemia Triple negative MPN Pediatric congenital erythrocytosis n = 10 n = 0 n = 0 CEMM, Vienna, Austria (unpublished data) n = 0 n = 1 n = 17 n = 0 n = 0 Adult erythrocytosiscases n = 6 n = 1 n = 0 * JAK2 V617F+ / CALR+ pacient Occurrence in MPN patients 3.6% Occurrence in erythrocytosis patients 4.3% 8

9 Summary of published data The first case of two germ line JAK2 mutations associated with erythrocytosis and abnormal megakaryopoiesis - partially resembling polycythemia vera cases with JAK2 exon 12 mutations E846D and R1063H JAK2 mutations led to abnormal increased STAT5 activation in the specific context of EPO receptor Cooperation of two weak germ line mutations is necessary to augment JAK2 activity above a threshold level necessary for the induction of a pathological phenotype New diagnosed erythrocytosis patient JAK2 E846D found in 53 years old erythrocytosis patient: Hb 182 g/l (normal values: g/l) Hct 53 % (normal values: %) In vitro sensitivity assay hypersensitivity of patient erythroid progenitors to EPO 9

10 Mutational screening two mutations in EPOR pathway: 1. JAK2 E846D => increased and prolonged activation of EPOR pathway 2. in JAK2 negative regulator - LNK R262W => previously associated with elevated hematological parameters The possible combinatory effect is now under investigation Conclusion Unexplained idiopathic erythrocytosis cooperative effects of weak germ line mutations affecting the critical pathways in erythroid cells need further investigations 10

11 Acknowledgment Prof. Stefan N. Constantinescu, M.D. Prof. Robert Kralovics, Ph.D. Prof. Dagmar Pospíšilová, M.D. Prof. Vladimír Divoký, Ph.D. Monika Horváthová, Ph.D. Barbora Kráľová Department of Biology Grant support: GACR S (PI Divoky) IGA_LF_2017_015 Czech Republic Program KONTAKT II (LH15223) MEXP31C1(PI Constantinescu) Thank you for your attention 11