SAMD9 and SAMD9L
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2019 International Conference & 60th Annual Meeting of Korean Society of Hematology Roles of Monosomy 7 and SAMD9/SAMD9L mutations in myeloid leukemogenesis Hirotaka Matsui Department of Molecular Laboratory Medicine Faculty of Life Sciences, Kumamoto University 대한혈액학회 Korean Society of Hematology COI disclosure Name of author ： Hirotaka MATSUI I have no personal or financial interests to declare: I have no financial support from an industry source at the current presentation. 北海道 (Hokkaido) 本州 (Honshu) 日本 (JAPAN) 熊本 四国 (Kumamoto) 九州 (Shikoku) (Kyushu) Kumamoto Castle Amakusa Islands Areake Sea Aso volcano Monosomy 7 seen in a wide range of myeloid disorders. 1. De novo disorders: primary MDS, primary AML, infant monosomy 7 syndrome etc. 2. Secondary disorders: those following therapy for previous malignancy. 3. Constitutional disorders: Fanconi’s anemia, Kostmann’s syndrome, Familial monosomy 7 etc. Luna-Fineman, Blood 85, 1995 Interstitial deletion of chr.7q Raca G, ASH Image Bank 2016 Monosomy 7 (in adult MDS) Deletion of chr.7 is the second frequent karyotype change in adult MDS. Frequently associated with complex karyotype and predicts worse prognosis. Found in 11% of adult MDS patients Haase D. et al., Blood 110, 2007 Monosomy 7 (in pediatric MDS) In pediatric MDS, deletion of chr.7 is the most common cytogenetic abnormality. Deletion of chr.7 is often associated with germline GATA2 deficiency. Germline GATA2 deficiency is associated with monosomy 7 Hasle H, Hematol Am Soc Hematol Educ Prog, 2016 Donadieu J et al., Hematologica 2018 Isolation of candidate responsible genes Chr. 7 1964 1st report of Mono.7 1996 7q22, 7q32-34 5Mbp 220 genes 2009 MIKI, SAMD9L 7q22 Asou et al. BBRC 12Mbp 180 genes 7q32-34 Isolation of candidate responsible genes from Juvenile myelomonocytic leukemia without overt loss of chr.7. 235 (1 per 110kb) probes in 7q21.3-7q31.1 (25Mb) q21 q22 q31 7q Probe ID 1112131415161718 1.4 1 1.2 2 3 1 Patient 4 0.8 ID 5 0.6 6 candidate deleted Fluorescenceratio 0.4 7 0.2 region | | | | | | 8 0 1 50 100 150 200 Probe ID no. SAMD9 SAMD9L MIKI (=HEPACAM2) Telomere Probe #14 Probe #15 Probe #16 Asou H et al. BBRC 383: 245, 2009 Isolation of candidate responsible genes MIKI and SAMD9/9L as promising candidates for AML/MDS responsible genes MIKI SAMD9L (HEPACAM2) MIKI MIKI MIKI Golgin97 Merge Cell cycle interphase G1 S Golgi apparatus G2 M MIKI α-Tubulin Merge Mitotic phase Centrosome/spindle Ozaki Y, Matsui H et al. Mol Cell 47: 694, 2012. MIKI locates at centrosomes and spindles Pro Prometa Meta Ana Telo α-tubulin MIKI Hoechst MIKI Control MIKI siRNA MIKI inhibition causes: Decreased spindle tension MIKI Hoechst Disorganized alignment of chromosomes Ozaki Y, Matsui H et al. Mol Cell 47: 694, 2012. MIKI regulates mitosis Control cells MIKI siRNA MIKI siRNA MIKI is poly(ADP)-ribosylated No G1 G2 M synchro S M Poly(ADP)-ribosylation by Tankyrase-1 125 M.W. (KDa) 50 Isolated spindles and MASS analysis centrosomes 125 M.W. (KDa) 50 MIKI promotes centrosome maturation interphase Interphase mitosis centrosomes Mitotic centrosomes MIKI knockdown Results in inhibition of Centrosome maturation Interphase centrosomes γ-tubulin Pseudo-metaphase Hoechst Inhibit Normal K562(CML l evel ion of MIKIis associated with myelodysplasia p125 MIKI of MIKI MIKI of - BC) HeLa K562 MDS Kasumi-1 Low level MIKI of level Low KG-1a - L ( Monosomy7 L Monosomy7 ( ) F-36P Monosomy FKH-1 MDS-KZ MDS-L MONO-7 7 OHN-GM FA-AML-1 Marimo MIKI MIKI K562 parent K562 K562 MIKI shRNA shRNA SAMD9/9L Samd9l K/O aged mice：hematological disorders 60% identical SAMD9 SAMD9L MIKI (HEPACAM2) Human 7q21 Miki (Hepacam2) EG667335 Samd9L Mouse 6A1 Immunoblot Death by hematological tumors 1 (kidney) (kDa) +/++/- -/- 0.8 220 Samd9L 0.6 Samd9L -/- (n=15) 97 0.4 1.0 0.23 Samd9L +/- (n=15) 0 Samd9L +/+ (n=23) 0.2 0 0 5 10 15 20 25 Months after birth Nagamachi A, Matsui H et al. Cancer Cell 24: 305, 2013. Samd9l is located at early endosome Cytokine receptors Early endosome Samd9L Rab5 merge Fusion of early endosomes SAMD9L Late lysosome endosome Degradation of receptors Nagamachi A, Matsui H et al. Cancer Cell 24: 305, 2013. Samd9l promotes fusion of endosomes Cytokine 0min. stimulation Endocytosis Fusion of Wild type Samd9L shRNA vesicles 15min. Fused early Smaller endosomes endosomes Early endosome (fusion impaired) 2.0 Merged EEA1 PDGFR Merged EEA1 PDGFR 1.6 1.2 0.8 Wild type 0.4 Samd9L shRNA 0.0 0min 5min 15min30min Nagamachi A, Matsui H et al. Cancer Cell 24: 305, 2013. Samd9l is required for regulation of cytokine signals Wild type Samd9L shRNA Early PDGF-BB (100ng/mL) 0 5 15 30 0 5 15 30 min. Endosome Cell surface receptors PDGFRB SAMD9L Endosomal receptors phospho- AKT PDGFRB Wild type Samd9L shRNA Transient PDGF-BB (100ng/mL) 0 5 15 30 0 5 15 30 min. Signaling phospho-AKT total AKT p-AKT Sustained Signaling Nagamachi A, Matsui H et al. Cancer Cell 24: 305, 2013. Short summary Candidate monosomy 7 responsible genes MIKI (HEPACAM2) Plays a role in the maintenance of mitotic integrity. SAMD9 and SAMD9L Negatively regulate cytokine signals by promoting fusion of endosomes. Involved in myeloid leukemogenesis. But… We haven’t identified somatic SAMD9/9L mutations in patients…. MIRAGE syndrome: germline SAMD9 mutation Adrenal hypoplasia 1. Without extra-adrenal features 2. Syndromic adrenal hypoplasia AAA syndrome, IMAGe syndrome, Syndrome with MCM4 mutations 30% of patients remains genetically undiagnosed SAMD9 mutations were identified by WES. The patients commonly exhibited symptoms below. Myelodysplasia Infection Restriction of growth Adrenal hypoplasia Genital phenotypes Enteropathy MIRAGE syndrome website (written in Japanese) MIRAGE syndrome: germline SAMD9 mutation Loss of Chr.7 with SAMD9 mutation in MDS cells Narumi S. et al., Nat genet. 48(7), 792-797, 2016 MIRAGE syndrome: germline SAMD9 mutation Hematological manifestations include… Thrombocytopenia Anemia: requiring transfusions Severe infection Spontaneous remission of cytopenia mut WT Loss of mut chr.7 in blood cells mut WT WT WT WT WT MDS WT WT Narumi S. et al., Nat genet. 48(7), 792-797, 2016 MIRAGE syndrome: germline SAMD9 mutation Enlarged late endosomes in fibroblasts of affected individuals. Narumi S. et al., Nat genet. 48(7), 792-797, 2016 MIRAGE syndrome: germline SAMD9 mutation Gain of function of mutant SAMD9. Cells expressing mutant SAMD9 proliferate slowly. Narumi S. et al., Nat genet. 48(7), 792-797, 2016 Ataxia-pancytopenia syndrome (ATXPC) Cerebellar ataxia Variable hematologic cytopenias Myeloid malignancy accompanied by Mono.7 Li FP. et al., Cancer Genet Cytogenet. 4(3), 189-196, 1981 Li FP. Et al., Am J Med. 65(6), 933-940, 1978 Ataxia-pancytopenia syndrome: SAMD9L mutation Cerebellar ataxia Variable hematologic cytopenias Myeloid malignancy accompanied by Mono.7 SAMD9L was isolated as the causative gene c.2640C>A Chen DH. Et al., Am J Hum Genet. 98, 1146-1158, 2016 Ataxia-pancytopenia syndrome: SAMD9L mutation Cerebellar ataxia Variable hematologic cytopenias Myeloid malignancy accompanied by Mono.7 SAMD9L was isolated as the causative gene c.3687G>C also from the original family. Chen DH. Et al., Am J Hum Genet. 98, 1146-1158, 2016 Ataxia-pancytopenia syndrome 6mo. Copy number-neutral VAF was decreased when the transformed lymphocytes were cultured. mut WT mut WT mut WT LOH mut WT Somatic mosaicism of chromosome 7 was suspected. Chen DH. Et al., Am J Hum Genet. 98, 1146-1158, 2016 MLSM7: a syndrome overlapping with ATXPC (MLSM7: Myelodysplasia and leukemia syndrome with -7) Severe neurological Mild to no neurological phenotype phenotype SAMD9L ATXPC mutation MLSM7 MDS with Mono.7 (at least two siblings are affected) Tesi B. et al., Blood. 129, 2266-2279, 2017 Loss of mutant SAMD9L in MSLM7 and ATXPC SAMD9L mutation Unfavorable for cell growth mut WT Reversion MDS with Mono.7 mut WT WT WT WT Somatic mut. CN-LOH Mono 7 in cis Recovery from cytopenia Tesi B. et al., Blood. 129, 2266-2279, 2017 LOF germline mutation of SAMD9/9L in adult MDS SAMD9 SAMD9L missense nonsense Nagata Y et al., Blood 132, 2309, 2018 LOF germline mutation of SAMD9/9L in adult MDS MUT in children = C-terminal SAMD9 SAMD9L missense nonsense Nagata Y et al., Blood 132, 2309, 2018 LOF germline mutation of SAMD9/9L in adult MDS SAMD9 MUT in Adult = N-terminal SAMD9L missense nonsense Nagata Y et al., Blood 132, 2309, 2018 LOF germline mutation of SAMD9/9L in adult MDS Adult type (LOF) mut WT Long latency mut WT mut Additional somatic Bi-allelic LOF mutation GL mutation + Mono 7 Nagata Y et al., Blood 132, 2309, 2018 Germline mutations in adult vs pediatric MDS Pediatric type (GOF) Adult type (LOF) Unfavorable for Favorable for cell growth cell growth mut WT Selective mut WT pressure Short latency Long latency mut WT mut WT WT mut WT mut Somatic CN-LOH Mono 7 Additional somatic Bi-allelic LOF mutation mutation GL mutation + Mono 7 in cis Pediatric MDS Adult Recovery from cytopenia MDS Clonal hematopoiesis Acknowledgement Research Institute for Radiation Research Institute, National Center Biology and Medicine for Global Health and Medicine Hiroshima University Keiyo Takubo Toshiya Inaba Akiko Nagamachi IRCMS Akinori Kanai Kumamoto University Yuko Ozaki Toshio Suda Hiroya Aso Hiroaki Honda.