Supplemental material Mutant DNMT3A: A Marker of Poor Prognosis in Acute Myeloid Leukemia Ana Flávia Tibúrcio Ribeiro, Marta Pratcorona, Claudia

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1 Supplemental material Mutant DNMT3A: A Marker of Poor Prognosis in Acute Myeloid Leukemia Ana Flávia Tibúrcio Ribeiro, Marta Pratcorona, Claudia Erpelinck-Verschueren, Veronika Rockova, Mathijs Sanders, Saman Abbas, Maria E. Figueroa, Annelieke Zeilemaker, Ari Melnick, Bob Löwenberg, Peter J. M. Valk, Ruud Delwel.

2 Table S1. Samples numbers and respective mutation sites Sample Mutation Allele Amino acid number Type change change 1174 Missense c.2645g>a p.arg882his 1747 Missense c.2645g>a p.arg882his 1766 Missense c.2645g>a p.arg882his 2177 Missense c.2651c>t p.ala884val 2181 Missense c.2645g>a p.arg882his 2183 Missense c.2644c>t p.arg882cys 2185 Frameshift deletion c.2409_2478del70 p.arg803fs 2187 Missense c.2645g>a p.arg882his 2188 Missense c.2645g>a p.arg882his 2191 Missense c.2645g>a p.arg882his 2195 Missense c.2645g>a p.arg882his 2198 Missense c.2309c>t p.ser770leu 2199 Missense c.2245c>t p.arg749cys 2206 Missense c.2645g>a p.arg882his 2225 Missense c.2645g>a p.arg882his 2226 Missense c.2644c>t p.arg882cys 2229 Missense c.1904g>c p.arg635pro 2231 In frame deletion c.2580_2597del18 p.cys861if 2234 Missense c.2312g>a p.arg771gln 2239 Missense c.1644g>a p.met548ile 2244 Missense c.2645g>a p.arg882his 2255 Missense c.2378a>c p.tyr793ser 2256 Missense c.2645g>a p.arg882his 2257 Missense c.2644c>t p.arg882cys 2261 Missense c.2645g>a p.arg882his 2272 Missense c.1741t>c ptrp581arg 2282 Missense c.2645g>a p.arg882his 2283 Missense c.2645g>a p.arg882his 2284 Missense c.1598a>g p.tyr533cys 2289 Missense c.1640t>a p.leu547his 2291 Missense c.2605g>a p.gly869ser 2296 Missense c.2141c>g p.ser714cys 2300 Missense c.2056g>a p.asp686gly 2467 Missense c.2645g>a p.arg882his 2550 Frameshift deletion c.1711delg p.ala571fs 2669 Missense c.2645g>a p.arg882his 2672 Missense c.2645g>a p.arg882his 2679 Missense c.2644c>t p.arg882cys 2683 Missense c.2185g>t p.arg729trp 2685 Missense c.2644c>a p.arg882ser 2687 Missense c.2644c>t p.arg882cys 2689 Missense c.1627g>t p.gly543cys 2692 Missense c.2645g>a p.arg882his 2757 Missense c.2644c>t p.arg882cys 2771 Missense c.2645g>a p.arg882his 3278 Missense c.2645g>a p.arg882his 3310 Missense c.2645g>a p.arg882his 3311 Missense c.1741t>a p.trp581arg 3321 Missense c.1994t>g pval665gys 3331 # Missense c.2645g>a p.arg882his 3481 Missense c.2645g>a p.arg882his 3491 # Missense c.2206c>t p.arg736cys 4334 Frameshift insertion c.2196_2197inst p.glu733x 5285 Missense c.2645g>a p.arg882his 5286 Missense c.2512a>g p.asn838asp 5349# Missense c.2645g>a p.arg882his

3 Table S1. Samples numbers and respective mutation sites (continued) Sample Mutation Allele Amino acid number Type change change 5354 # Missense c.1627g>t p.gly543cys 5355 # Frameshift insertion and deletion c.1656_1663del8+1656_1657insa p.asn552fs 5362 Missense c.1903c>t p.arg635trp 5363 Missense c.2644c>t p.arg882cys 6239 Frameshift deletion c.1872dela p.pro624fs 6245 Missense c.2141c>g p.ser714cys 6366 Missense c.2645g>a p.arg882his 6368 Missense c.2141c>g p.ser714cys 6369 Missense c.2729c>t p.ala910val 6370 Missense c.2645g>a p.arg882his 6371 Missense c.2645g>a p.arg882his 6374 Nonsense c.2311c>t p.arg771x 6724 Missense c.2644c>t p.arg882cys 6732 Missense c.2644c>t p.arg882cys 6734 Missense c.1903c>t p.arg635trp 6952 Missense c.2645g>a p.arg882his 6979 Missense c.2645g>a p.arg882his 7057 Missense c.2645g>a p.arg882his 7065 In frame c.1713_1721deltgcccaggc p.ala571if 7067 Missense c.2645g>a p.arg882his 7074 Missense c.1627g>t p.gly543cys 7075 Missense c.2645g>a p.arg882his 7078 Missense c.2645g>a p.arg882his 7119 Missense c.2645g>a p.arg882his 7120 Frameshift deletion c.2203delt p.tyr735fs 7121 Missense c.2645g>a p.arg882his 7122 Missense c.2645g>a p.arg882his 7124 Missense c.2644c>t p.arg882cys 7131 Missense c.2645g>a p.arg882his 7144 Missense c.1628g>t p.gly543val 7145 Nonsense c.2311c>t p.arg771x 7153 Missense c.2645g>a p.arg882his 7164 Missense c.2645g>a p.arg882his # 7167 Missense c.2644c>t p.arg882cys 7168 Missense c.2645g>a p.arg882his 7172 Missense c.2644c>t p.arg882cys 7186 Missense p.1903c>t p.arg635trp 7188 Missense c.2645g>a p.arg882his 7276 Missense c.2645g>a p.arg882his 7309 Missense c.2171a>g p.tyr724cys 7311 Missense c.2645g>a p.arg882his 7313 Missense c.2644c>t p.arg882cys 7314 Missense c.2314t>a p.phe772ile 7316 Missense c.2644c>t p.arg882cys 7318 Missense c.2644c>t p.arg882cys 7319 Missense c.2645g>a p.arg882his # 7322 Missense c.2645g>a p.arg882his # 7327 Missense c.2578t>c p.trp860arg 7328 # Missense c.2645g>a p.arg882his 7407 Missense c.2644c>t p.arg882cys 7408 Missense c.2645g>a p.arg882his 7414 Missense c.2645g>a p.arg882his 2541* Missense c.2135g>a p.asp712asn 2541* Missense c.2353g>a p.val785met 3483* Missense c.1502a>g p.asn501ser 3483* Fameshift deletion c.2407_2476del70 p.arg803fs 7324* Missense c.1685g>a p.cys562tyr 7324* Missense c.2146g>a p.val716ile 7411* # Nonsense c.1776c>g p.tyr592x 7411* # Frameshift insertion c.2307_2308inst p.ile769fs

4 *One individual but with two mutations. # Patients of > 60 years of age. These patients were not within the 415 AML taken along in this study. DNMT3A mutations in AML with MDS related changes or therapy related AMLs. These AMLs were also not among the 415 cases studied.

5 References 1. Abbas S, Lugthart S, Kavelaars F, et al. Acquired Mutations in the Genes Encoding Idh1 and Idh2 Both Are Recurrent Aberrations in Acute Myeloid Leukemia (Aml): Prevalence and Prognostic Value. Haematologica-the Hematology Journal. 2010;95: Thol F, Damm F, Ludeking A, et al. Incidence and Prognostic Influence of DNMT3A Mutations in Acute Myeloid Leukemia. J Clin Oncol Prepublished on 2011/06/15 as DOI JCO [pii] /JCO Yan XJ, Xu J, Gu ZH, et al. Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia. Nat Genet Prepublished on 2011/03/15 as DOI ng.788 [pii] /ng Ley TJ, Ding L, Walter MJ, et al. DNMT3A Mutations in Acute Myeloid Leukemia. N Engl J Med Prepublished on 2010/11/12 as DOI /NEJMoa Figueroa ME, Lugthart S, Li Y, et al. DNA methylation signatures identify biologically distinct subtypes in acute myeloid leukemia. Cancer Cell. 2010;17(1): Breems DA, Lowenberg B. Acute myeloid leukemia with monosomal karyotype at the far end of the unfavorable prognostic spectrum. Haematologica. 2011;96(4): Valk PJ, Bowen DT, Frew ME, Goodeve AC, Lowenberg B, Reilly JT. Second hit mutations in the RTK/RAS signaling pathway in acute myeloid leukemia with inv(16). Haematologica. 2004;89(1):106. Prepublished on 2004/02/03 as DOI. 8. Barjesteh van Waalwijk van Doorn-Khosrovani S, Erpelinck C, Meijer J, et al. Biallelic mutations in the CEBPA gene and low CEBPA expression levels as prognostic markers in intermediate-risk AML. Hematol J. 2003;4(1): Care RS, Valk PJ, Goodeve AC, et al. Incidence and prognosis of c-kit and FLT3 mutations in core binding factor (CBF) acute myeloid leukaemias. Br J Haematol. 2003;121(5): Verhaak RG, Goudswaard CS, van Putten W, et al. Mutations in nucleophosmin (NPM1) in acute myeloid leukemia (AML): association with other gene abnormalities and previously established gene expression signatures and their favorable prognostic significance. Blood. 2005;106(12):

6 DNMT3A protein 912 amino acid residue 45 different DNMT3A mutations in 515 AML samples Frameshift deletion Frameshift insertion Missense Nonsense In frame deletion PWWP PHD MTase motifs N501S Y533C G543V G543C L547H M548I I N552fs2fs C562Y A571 A571fs W581R Y592* P624fs R635W R635P V665G D686G D712N S714C V716I Y724C R729W F732fs Y735fs R736C R749C S770L I769f s P79 9fs R771Q R771* F772I V785M Y793S G800 R882S N838D W860R G869S R882L R882H R882C W860 fs A884V A910V Figure S1: Schematic representation of the types and locations of the mutations in the DNMT3A gene. DNMT3A mutation analysis was carried out in 415 de novo AML patients of 60 years and younger, which revealed 35 mutants. Mutation analysis was also carried out on 24 APLs (1 mutant) and on 76 AML patients > 60 years (9 mutants). All mutants are shown in Table S1.

7 Figure S2. Survival analyses of patients with AML with DNMT3A wild-type or DNMT3A R882 AML mutations. (A) Overall Survival in all AML patients with DNMT3A wild-type or DNMT3A R882 AML mutations (B) Relapse Free Survival in all AML patients with DNMT3A wild-type or DNMT3A R882 AML mutations P indicated p-value follows from the Wald test in Cox regression analysis.

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9 Figure S3: Relapse-free survival within specifics genotype subgroups of AML patients with or without DNMT3A mutations. Figures A, B and C represent RFS of distinct subgroups determined within the total cohort of 415 AML patients younger than 60 and excluding APLs. (A) RFS of AML patients from which CEBPA dm patients were excluded (B) RFS in AMLs without FLT3 ITD and without mutations in NPM1. (C) RFS in AML patients without FLT3 ITD, without CEBPA dm and without mutations in NPM1. (D) RFS within the cytogenetically defined intermediate risk group. Figures E and F represent RFS of distinct subgroups determined within the cytogenetically defined intermediate risk group of 236 AML patients. (E) RFS in AMLs without FLT3 ITD and without mutations in NPM1. (F) RFS in AML patients without FLT3 ITD, without CEBPA dm and without mutations in NPM1.

10 Figure S4: Clustering of AML patient samples based on Gene Expression profiling: Distribution of DNMT3A mutant and DNMT3A wild-type AMLs. Pearson correlation view based on gene expression profiling of 285 AML cases (Valk et. al. New Engl.J.Med. 2004, 350: ). The cluster (different colours) and presence (red) or absence (green) of FLT3-ITD, NPM1 mutation and DNMT3A mutation are indicated for the individual AML cases adjacent to the correlation matrix. Cluster #5 is enriched for DNMT3 mutant AML patients.

11 Figure S5: HOXB5 expression in DNMT3A/NPM1 composite genotypes.