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

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

Ozaki Y, Matsui H et al. Mol Cell 47: 694, 2012. MIKI regulates

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 Inhibition of MIKI is associated with myelodysplasia

Monosomy 7 1 1 shRNA - 7 GM KZ L 1 - - - - 1a - AML - - 36P MIKI - MONO - OHN HeLa K562 Kasumi KG F FKH MDS MDS FA Marimo K562parent p125 MIKI

K562 (CML-BC) MDS-L ( Monosomy7 ) K562 Normal level of MIKI Low level of MIKI MIKI 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 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 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