the spectrum and clinical impact of epigenetic modifier...

The spectrum and clinical impact of epigenetic modifier mutations in myelomaRunning title: Epigenetic modifier mutations in myeloma

Authors Charlotte Pawlyn1, Martin F Kaiser1, Christoph Heuck2, Lorenzo Melchor1, Christopher P Wardell1, Alex Murison1, Shweta Chavan2, David C Johnson1, Dil Begum1, Nasrin Dahir1, Paula Proszek1#, David A Cairns3, Eileen M Boyle1, John R Jones1, Gordon Cook4, Mark T Drayson5, Roger G Owen6, Walter M Gregory3, Graham H Jackson7, Bart Barlogie2, Faith E Davies1,2, Brian A Walker1,2, Gareth J Morgan1,2

Affiliations1Centre for Myeloma Research, The Institute of Cancer Research, London, United Kingdom2Myeloma Institute, University of Arkansas for Medical Sciences, Little Rock, AR, United States3Clinical Trials Research Unit, University of Leeds, Leeds, United Kingdom4University of Leeds, Leeds, 5Clinical Immunology, School of Immunity & Infection, University of Birmingham, Birmingham6St James’s University Hospital, Leeds7Northern Centre for Bone Marrow Transplantation, Newcastle upon Tyne Hospitals, Newcastle upon Tyne, United Kingdom

Supplementary Information

Supplementary Tables:

Supplementary table 1: Demographics of the 463 patients included in the Myeloma XI analysis

Supplementary table 2: List of epigenetic modifier genes used to interrogate the list of mutations in the Myeloma XI dataset

Supplementary table 3: Demographics of the 156 patients included in the UAMS dataset

Supplementary table 4: List of epigenetic modifier genes sequenced in UAMS dataset

Supplementary table 5: SIFT analysis of Histone 1 family gene mutations

Supplementary table 6: Multivariate cox-regression model for overall survival in Myeloma XI patients

Supplementary table 7: Percentage of patients with mutations in genes encoding epigenetic modifiers sequenced in both the MYXI and UAMS datasets (as shown in Supplementary table 4)

Supplementary Figures:

Supplementary Figure 1: Epigenetic genes mutated in ≥5/463 patients (>1%) in Myeloma XI dataset – location of mutation with respect to protein functional domains

Supplementary Figure 2: Translocation subgroup distribution by presence or absence of epigenetic modifier mutation in the Myeloma XI dataset

Supplementary Figure 3: Survival curves for Histone 1 mutations in Myeloma XI dataset

Supplementary Figure 4: A comparison of the variant allele frequency between the MyXI and UAMS mutations

Supplementary Figure 5: The distribution of mutations in epigenetic modifiers in previously treated patients by GEP70 risk score

Supplementary Figure 6: A comparison of the UAMS molecular subgroup distribution by presence of epigenetic modifier mutations in previously treated patients

Supplementary table 1: Demographics of the 463 patients included in the Myeloma XI analysis

(Reproduced from Walker BA et al, Nat Comm 2015)

Variable n=463

Median age (range) 68 (31-89)Sex ratio (Male:Female) 1.4:1Race

CaucasianNon-caucasian

93% (n=449)3% (n=14)

PathwayIntensiveNon-intensive

57% (n=262)43% (n=201)

IsotypeIgAIgDIgGLight chain onlyNon-SecretorsData missing

26% (n=121)1.7% (n=8)57% (n=263)12% (n=55)1% (n=2)3% (n=14)

ISS stageIIIIIIData missing

30% (n=140)29% (n=135)34% (n=159)6% (n=29)

Beta-2-microglobulin< 3.53.5≤-<5.5≥5.5Data missing

33% (n=153)26% (n=119)35% (n=162)6% (n=29)

Creatinine≥ 104 µM≥ 150 µM

35% (n=162)13% (n=57)

Bone disease 70% (n=324)Hypercalcemia ≥ 2.65 mM 12.5% (n=58)Copy number abnormality (gene)

del(1p33) (FAF1/CDNK2C)del(1p12) (FAM46C)gain(1q21.2) (CKS1B)del(13q) (RB1)del(17p) (TP53)Hyperdiploidy

8.5% (n=39)24% (n=111)36% (n=166)42% (n=195)9.5% (n=44)52% (n=239)

Translocationst(4;14)t(6;14)t(11;14)t(14;16)t(14;20)

12.7% (n=59)1% (n=5)18.6% (n=86)4% (n=17)1% (n=4)

Ig = immunoglobulin

Supplementary table 2List of epigenetic modifier genes used to interrogate the list of mutations in the Myeloma XI dataset

Core/linker histones

Histone demethylases

Histone methyl-transferases

Histone deacetylases

Histone acetyl-transferases

DNA methylation modifiers

Chromatin assembly and remodelling

Readers

Histone 1:HIST1H1AHIST1H1BHIST1H1CHIST1H1DHIST1H1EHIST1H1TH1FOOH1FNTH1F0H1FX

Histone 2A:HIST1H2AAHIST1H2ABHIST1H2ACHIST1H2ADHIST1H2AEHIST1H2AGHIST1H2AHHIST1H2AJHIST1H2AKHIST1H2ALHIST1H2AMHIST2H2AB

KDM1AKDM1BKDM2AKDM2BKDM3AKDM3BKDM4AKDM4BKDM4CKDM4DKDM5AKDM5BKDM5CKDM5DKDM6AKDM6BKDM7A/JHDM1DKDM8/JMJD5UTYPHF2PHF8

ASH1LCARM1

DOT1LEEDEHMT1EHMT2EZH1EZH2MEN1NSD1PRDM2PRMT1PRMT2PRMT5PRMT6PRMT7SETD1ASETD1BSETD2SETD3SETD7SETD8SETDB1

BAZ2AHDAC1HDAC10HDAC11HDAC2HDAC3HDAC4HDAC5HDAC6HDAC7HDAC8HDAC9SIRT1SIRT2SIRT3SIRT4SIRT5SIRT6SIRT7ZBTB33

BRPF1CLOCK

CREBBPELP3EP300EP400GNAT1GNAT2GNAT3GTF3C4HAT1KAT2AKAT2BKAT5MYST3/KAT6AMYST4/KAT6BMYST2/KAT7MYST1/KAT8MORF4L1NCOA1NCOA2

DNMT1DNMT3ADNMT3BIDH1IDH2TET1TET2TET3

ACTL6A

ACTL6B

ARID1A

ARID1B

ARID2

ARID3A

ARID3B

ARID3C

ARID4A

ARID4B

ARID5A

ARID5B

ASXL1

ATRX

BPTFBRD2BRD4BRD8CBX5CBX7CTCFMBD1MBD2MBD3MBD4MBD5MBD6MECP2RAG2TDGTP53BP1

HIST2H2ACHIST3H2AH2AFB3H2AFJH2AFVH2AFXH2AFYH2AFY2H2AFZ

Histone 2B:HIST1H2BAHIST1H2BBHIST1H2BCHIST1H2BDHIST1H2BEHIST1H2BFHIST1H2BGHIST1H2BHHIST1H2BIHIST1H2BJHIST1H2BKHIST1H2BLHIST1H2BMHIST1H2BNHIST1H2BOHIST2H2BEHIST2H2BFHIST3H2BBH2BFMH2BFWT

Histone 3:

SETDB2SETMARSMYD1SMYD2SMYD3SUV39H1SUV39H2SUV420H1SUV420H2SUZ12WHSC1/MMSETWHSC1L1

MLL family:MLLMLL2MLL3MLL4MLL5

NCOA3TAF1TAF1LTAF3

BRD7

CHD1

CHD2

CHD3

CHD4

CHD5

CHD6

CHD8

CHD9

DPF1

DPF2

INO80

KLF1

MLF1IP

PBRM1

PHF10

RBBP4

SET

HIST1H3AHIST1H3BHIST1H3CHIST1H3DHIST1H3EHIST1H3FHIST1H3GHIST1H3HHIST1H3IHIST1H3JHIST2H3DHIST3H3H3F3AH3F3BH3F3C

Histone 4:HIST1H4AHIST1H4BHIST1H4CHIST1H4DHIST1H4EHIST1H4FHIST1H4GHIST1H4HHIST1H4IHIST1H4JHIST1H4KHIST1H4LHIST4H4

SMARCA1

SMARCA2

SMARCA4

SMARCA5

SMARCAD1

SMARCAL1

SMARCB1

SMARCC1

SMARCC2

SMARCD1

SMARCD2

SMARCD3

SMARCE1

SRCAP

Supplementary Table 3Demographics of the 156 patients included in the UAMS dataset

Variable N=156

Median age (range) 67 (34-84)Sex ratio (Male:Female) 1.9:1 (103:53)Race

CaucasianNon-caucasianData missing

85% (133)13% (21)1.2% (2)

ISS stageIIIIIIData missing

29% (46)36% (56)24% (37)11% (17)

Beta-2-microglobulin< 3.5 mg/L3.5≤-<5.5 mg/L≥5.5 mg/LData missing

40% (62)24% (38)25% (39)11% (17)

Creatinine<2 mg/dL>=2 mg/dL

90% (141)10% (15)

Hypercalcaemia <12 mg/dL >=12 mg/dL

99% (155)0.6% (1)

UAMS GEP70 risk group Low High

66% (103)34% (53)

UAMS molecular subgroup CD1 CD2 MF MS PR LB HY

5.8% (9)22% (34)2.6% (4)11% (17)29% (45)10% (15)21% (32)

Supplementary table 4List of epigenetic modifier genes sequenced in UAMS dataset.

Core/linker histones

Histone demethylases

Histone methyl-transferases

Histone deacetylases

Histone acetyl-transferases

DNAmethylationmodifiers

Chromatin assembly and remodelling

Readers

Histone 1:HIST1H1CHIST1H1DHIST1H1E

Histone 2A:HIST1H2ACHIST1H2AGHIST1H2ALHIST1H2AM

Histone 2B:HIST1H2BCHIST1H2BJHIST1H2BKHIST1H2BO

Histone 3:HIST1H3B

KDM2BKDM4CKDM5AKDM5CKDM6A

DOT1LEEDEZH2MEN1NSD1WHSC1SETD2SUZ12

MLL family:MLLMLL2MLL3

HDAC1HDAC4HDAC7

CREBBPEP300KAT6ATAF1

DNMT3AIDH1IDH2TET2

ARID1AARID2ASXL1ATRXCHD2PBRM1SMARCA1SMARCA4SMARCB1

BRD4CTCF

Supplementary table 5SIFT analysis of Histone 1 family gene mutations

Chromosome, position, residue change (input)

PROTEIN_ID CODONCHANGE

POS RESIDUEREF

RESIDUEALT

TYPE SCORE

PREDICTION (cutoff=0.05)

MEDIANINFO

#SEQ

1 6,27835056,C,G ENSP00000330074

ATT AA[G/C] CTG

84 K N Single AA Change

0.022 Damaging 2.79 385

2 6,26056617,G,A ENSP00000339566

CCT [C/T]CT GCG

14 P S Single AA Change

0.152 Tolerated 3.01 174

3 6,26056233,C,T ENSP00000339566

GCG [G/A]CT GGC

142 A T Single AA Change

0.535 Tolerated 3.51 40

4 6,26056421,C,T ENSP00000339566

AGC C[G/A]T ATC

79 R H Single AA Change

0.017 Damaging 2.86 395

5 6,26056643,G,A ENSP00000339566

ACT G[C/T]T CCT

5 A V Single AA Change

0.5 Tolerated 3.01 188

6 6,26056440,C,G ENSP00000339566

GAT [G/C]TG GAG

73 V L Single AA Change

0.008 Damaging 2.86 394

7 6,26056356,C,T ENSP00000339566

GGT [G/A]CT TCT


0.008 Damaging 2.88 392

8 6,26056478,G,C ENSP00000339566

CTG G[C/G]T GCT

60 A G Single AA Change

0 Damaging 2.86 393

9 6,26056475,G,A ENSP00000339566

GCT G[C/T]T CTG


0.001 Damaging 2.86 393

10 6,26056463,G,T ENSP00000339566

AAA G[C/A]G TTG

65 A E Single AA Change

0.039 Damaging 2.87 387

11 6,26056310,G,C ENSP00000339566

GAA G[C/G]C

116 A G Single AA Change

0.073 Tolerated 3.01 88

AAG12 6,26056394,A,A

CENSP00000339566

Frameshift NA NA NA NA

13 6,26056305,G,A ENSP00000339566

AAG [C/T]CC AAG


0.607 Tolerated 3.01 84

14 6,26234968,TTCTT,T

ENSP00000244534

Frameshift NA NA NA NA

15 6,26234977,G,A ENSP00000244534

GCC G[C/T]G CTT


0.002 Damaging 2.92 394

16 6,26234840,G,A ENSP00000244534

AAA [C/T]TC AAC

108 L F Single AA Change

0.001 Damaging 2.93 384

17 6,26156849,C,G ENSP00000307705

AAC AA[C/G] AGC

77 N K Single AA Change

0.038 Damaging 2.88 359

18 6,26156802,C,T ENSP00000307705

GCT [C/T]TC AAG

62 L F Single AA Change

0.007 Damaging 2.87 359

19 6,26156812,C,T ENSP00000307705

AAA G[C/T]G CTG


0.082 Tolerated 2.87 350

20 6,26156811,G,C ENSP00000307705

AAA [G/C]CG CTG

65 A P Single AA Change

0.019 Damaging 2.87 350

21 6,26156911,G,A ENSP00000307705

AAG G[G/A]C ACC

98 G D Single AA Change

0 Damaging 2.88 359

22 6,26156799,G,C ENSP00000307705

GCC [G/C]CT CTC


0.001 Damaging 2.87 359

23 6,26156861,G,C ENSP00000307705

ATC AA[G/C] CTG

81 K N Single AA Change

0.017 Damaging 2.88 359

24 6,26156932,T,A ENSP00000307705

TCC T[T/A]C AAA

105 F Y Single AA Change

0.025 Damaging 2.88 358

25 6,26156757,G,C ENSP00000307705

AAA [G/C]CT GTT


0.001 Damaging 2.87 353

26 6,26156791,C,T ENSP00000307705

GTA T[C/T]T TTG

58 S F Single AA Change

0 Damaging 2.87 359

27 6,26157204,C,T ENSP00000307705

AAA [C/T]CC AAG


0.158 Tolerated 3.16 33

28 6,26156925,G,A ENSP00000307705

TCG [G/A]GT TCC

103 G S Single AA Change

0.001 Damaging 2.87 356

29 6,26156752,C,T ENSP00000307705

ATT A[C/T]T AAA

45 T I Single AA Change

0.23 Tolerated 2.87 354

30 6,26157213,G,A ENSP00000307705

GCG [G/A]CT AAA


0.559 Tolerated 3.18 31

31 6,26156926,G,C ENSP00000307705

TCG G[G/C]T TCC

103 G A Single AA Change

0.035 Damaging 2.87 356

Supplementary table 6Multivariate cox-regression model for overall survival in Myeloma XI patients

P Hazard ratio 95% CI for Hazard ratio

Lower UpperAny adverse translocation

0.165 1.418 0.867 2.321

Del17p 0.000 3.611 2.244 5.812Age > 70 0.122 1.377 0.918 2.067KDM6A mut/del

0.130 1.832 0.836 4.013

DNA meth mut

0.023 2.470 1.134 5.380

ISS 0.003ISS 2 0.409 1.277 0.714 2.284ISS 3 0.002 2.299 1.358 3.8921q+ 0.0391q+ gain 0.327 1.262 0.792 2.010 1q+ amplification

0.011 2.127 1.189 3.802

DNA meth mut = Mutation in any DNA methylation modifier, KDM6A mut/del = Mutation or deletion in KDM6A

Supplementary table 7 Percentage of patients with mutations in genes encoding epigenetic modifiers sequenced in both the MYXI and UAMS datasets (as shown in Supplementary table 4)

MyXI(n=463)

UAMS(n=156)

n % n %

Any epigenetic modifier 116 25 69 44

Core/linker histonesAny 38 8.21 12 7.69

Any Histone 1: 27 5.83 8 5.13HIST1H1C 12 2.59 3 1.92HIST1H1D 3 0.65 2 1.28HIST1H1E 13 2.81 3 1.92

Any Histone 2A: 7 1.51 5 3.21HIST1H2AC 2 0.43 0 0.00HIST1H2AG 4 0.86 1 0.64HIST1H2AL 0 0.00 2 1.28HIST1H2AM 1 0.22 2 1.28

Any Histone 2B: 2 0.43 1 0.64HIST1H2BC 1 0.22 1 0.64HIST1H2BJ 1 0.22 0 0.00HIST1H2BK 0 0.00 0 0.00HIST1H2BO 0 0.00 0 0.00

Any Histone 3: 2 0.43 0 0.00HIST1H3B 2 0.43 0 0.00

Histone methyltransferasesAny 32 6.91 26 16.67DOT1L 3 0.65 1 0.64EED 0 0.00 0 0.00EZH2 0 0.00 0 0.00MEN1 0 0.00 0 0.00MLL (any) 21 4.54 18 11.54MLL 8 1.73 2 1.28MLL2 6 1.30 6 3.85MLL3 7 1.51 10 6.41NSD1 0 0.00 4 2.56WHSC1 2 0.43 0 0.00SETD2 6 1.30 4 2.56SUZ12 1 0.22 0 0.00

Histone demethylasesAny 14 3.02 3 1.92KDM2B 1 0.22 0 0.00KDM4C 3 0.65 0 0.00KDM5A 3 0.65 1 0.64KDM5C 2 0.43 1 0.64KDM6A 6 1.30 1 0.64

Histone deacetylasesAny 3 0.65 2 1.28HDAC1 0 0.00 0 0.00HDAC4 2 0.43 2 1.28HDAC7 1 0.22 0 0.00

Histone acetyltransferasesAny 11 2.38 11 7.05CREBBP 3 0.65 6 3.85EP300 6 1.30 3 1.92

KAT6A/MYST3 2 0.43 1 0.64TAF1 0 0.00 1 0.64

DNA modifiersAny 9 1.94 13 8.33DNMT3A 2 0.43 8 5.13IDH1 1 0.22 2 1.28IDH2 1 0.22 0 0.00TET2 5 1.08 4 2.56

Chromatin Assembly and RemodellingAny 25 5.40 11 7.05ARID1A 6 1.30 2 1.28ARID2 6 1.30 2 1.28ASXL1 0 0.00 0 0.00ATRX 2 0.43 1 0.64

CHD2 7 1.51 1 0.64PBRM1 2 0.43 1 0.64SMARCA1 0 0.00 0 0.00SMARCA4 1 0.22 4 2.56SMARCB1 1 0.22 0 0.00

ReadersAny 2 0.43 1 0.64BRD4 0 0.00 1 0.64CTCF 2 0.43 0 0.00

Supplementary Figure 1Epigenetic genes mutated in ≥5/463 patients (>1%) in Myeloma XI dataset – location of mutation with respect to protein functional domains. (except Histone 1 genes which are shown in figure 1)

The sites of mutation are indicated by the markers with red dots indicating missense mutations, blue dots nonsense mutations, yellow dots splice site mutations and purple dots frameshift mutations.

Supplementary Figure 2Translocation subgroup distribution by presence or absence of epigenetic modifier mutation in the Myeloma XI dataset

Patien

ts with

out any e

pigeneti

c modifier

mutations

Patien

ts with

any e

pigeneti

c modifier

mutations

0

20

40

60

80

100

None/UTD16 or 201164%

of p

atien

ts

There was a slightly higher proportion of patients with a MAF translocation (t(14;16) or t(14;20)) in the patients with any epigenetic modifier mutation 15/246 (6.1%) vs 6/216 (2.8%). This did not reach statistical significance in this dataset (z test of proportions). UTD: Unable to determine, 4: t(4;14), 6: t(6;14), 11: t(11;14), 16 or 20:t(14;16) or t(14;20)

Supplementary Figure 3Survival curves for Histone 1 mutations in Myeloma XI dataset

Kaplan-Meier curves showing MyXI patients with a mutation in a gene encoding one of the Histone 1 protein variants, (HIST1H1B-E). (n=28, dashed) and those without (n=435, solid). Progression-free survival median mutated 21.2 months vs wild type 26.6 months (logrank p = 0.609). Overall survival medians not reached, (logrank p = 0.684)

Supplementary Figure 4A comparison of the variant allele frequency between the MyXI and UAMS mutations

The variant allele frequency was compared for all genes sequenced in the UAMS dataset (those in supplementary table 3), by class of epigenetic modifier and then by individual genes (where there were at least 2 mutated samples in each dataset). Myeloma XI samples are shown in blue and UAMS in red. The bars shows the mean and standard error of the mean of the variant allele frequency (%). Unpaired t-tests with multiple test correction (Holm-Sidak method) were performed to compare the mean values between the MyXI and UAMS samples. No statistically significant differences were found.

Supplementary Figure 5The distribution of mutations in epigenetic modifiers in previously treated patients by GEP70 risk score.

Core/linker h

istones

Histone m

ethyltransfe

rases

Histone demethyla

ses

Histone deace

tylase

s

Histone ace

tyltra

nsferase

s

Readers

Chromatin Assembly

and Remodelling

DNA methyla

tion modifiers

Any epigenetic m

odifier0

10

20

30

40

50

60

GEP70 high risk

GEP70 low risk

% o

f pati

ents

with

in e

ach

risk

grou

p w

ith a

mut

ation

in e

ach

class

of e

pige

entic

mod

ifier

Bar chart showing the percentage of patients within each risk score with a mutation in a gene encoding the indicated type of epigenetic modifier, in the UAMS F1 dataset. Total number of patients with a high risk GEP70 score = 53/156, low risk GEP70 score = 103/156. The number of patients within each risk group with a mutation in each epigenetic modifier is expressed as a percentage of the total number of patients within each risk group. Z score test of proportions did not show any statistically significant differences between groups.

Supplementary Figure 6A comparison of the UAMS molecular subgroup distribution by presence of epigenetic modifier mutations in previously treated patients

Patients with no epigenetic mod-ifier mutations (n=87)

LBHYCD2CD1PRMFMS

Patients with any epigenetic modifier mutation (n=69)

LBHYCD2CD1PRMFMS

A comparison of the UAMS molecular subgroup distribution for patients without (left) and with (right) epigenetic modifier mutations. Comparison of proportions between the groups demonstrated a difference in the proportion of patients in the PR subgroup depending on presence or absence of mutation (No mut 45/87, 29%, With mut 26/69, 38%, Z-score P=0.03) which does not remain significant following multiple test correction (Bonferroni method).

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