Nature - USA (2019-07-18)

Article reSeArcH

MF01 MF02 MF03 MF04

HSPC WT

HSPC MUT

MkP WT

MkP MUT

HSPC

MkP

MEP

Log

(Cell cycle module) 2

3

4

5

6

P = 0.66

3

4

5

6

P = 0.047

2

3

4

5

P = 0.39

2

3

4

5

P = 0.23

WT

(n=27)

MUT

(n=23)

WT

(n=102)

MUT

(n=554)

WT

(n=51)

MUT

(n=332)

WT

(n=173)

MUT

(n=339)

3

4

5

6

7

P = 3.2 x 10-8

2

4

6

8

P =1.5 x 10-8

0

2

4

6

8

P = 0.15

0

2

4

6

P = 0.019

WT

(n=11)

MUT

(n=16)

WT

(n=56)

MUT

(n=418)

WT

(n=79)

MUT

(n=737)

WT

(n=184)

MUT

(n=569)

4

5

6

7

8

P = 1.5 x 10-9

3

4

5

6

7

8

P = 2.2 x 10-6

3

4

5

6

7

8

P = 6.8 x 10-6

2

3

4

5

6

P = 0.32

WT

(n=30)

MUT

(n=40)

WT

(n=40)

MUT

(n=375)

WT

(n=43)

MUT

(n=424)

WT

(n=118)

MUT

(n=503)

Log

(Cell cycle module) 2

Log

(Cell cycle module) 2

MEP WT

MEP MUT

4

5

6

7

4

5

6

7

4

5

6

7

3

4

5

P = 0.14 P = 2.7 x 10-4 P = 4.2 x 10-7 P = 5.9 x 10-15

WT

(n=29)

MUT

(n=36)

WT

(n=44)

MUT

(n=273)

WT

(n=128)

MUT

(n=892)

WT

(n=180)

MUT

(n=533)

EP

Log

(Cell cycle module) 2

EP WT

EP MUT

e

HSP90AA1

LIMS1

HMGN2

H2AFZ

CCND3

TYMS

SMC2

TMSB4X

CKS1B

RAN

0

5

10

15

20

-2 02

Log 2 (Fold Change)

-Log

(adjusted 10

Pvalue)

CD74

CYTL1

PRSS57

CRHBP

CPPED1

ACTB

PDLIM1

SLC44A1

HBD

Higher in WT MkPs

with High Cell Cycle

Higher in WT MkPs

with Low Cell Cycle

Cell cycle-related genes

Unfolded protein binding genes

d

0

0.1

0.2

0.3

0.4

MUTWT

Pseudotime

P = 0.08

c

Patient ID

MF01

MF02

MF03

MF04

t-SNE2

t-SNE1

t-SNE2

t-SNE1

Pseudotime

Early

Late

t-SNE2

t-SNE1

a b

0

5

10

15

MUT TGFb module / WT TGFb module

MF-2/3

MF-3/3

Fibrosis Score (MF)

P = 4.7 x 10-4

P = 0.0058

P = 0.0091

P = 0.062

MF02MF04MF01MF03

# cells = 11,093

Extended Data Fig. 8 | CALR-mutated haematopoietic progenitor cells
from myelofibrosis show upregulation of the IRE1-mediated UPR.
a, t-SNE projection of CD34+ progenitor cells from samples MF01–MF04,
after integration and batch correction using the Seurat package (Methods)
(n = 11,093). b, Left, t-SNE projection of CD34+ progenitor cells from
samples MF01–MF04 labelled with pseuodotime^21 (n = 11,093). Right,
pseudotime comparison between wild-type (n = 2,221) and mutant
(n = 7,483) cells. P values from likelihood ratio tests of linear mixed model
with genotype as fixed effect and individual patient samples as random
effect, against the model without the genotype effect (Methods). c, Cell-
cycle module score comparison between wild-type and mutant cells in

patients with myelofibrosis (two-sided Wilcoxon rank-sum test). d, Ratio

of TGFβ-signalling-pathway gene expression of mutant and wild-type

MkPs. One mutant and one wild-type MkP were randomly sampled for

each round of analysis (n = 100 iterations; two-sided Wilcoxon-rank sum

test). e, Differentially expressed genes between wild-type MkPs with high

cell-cycle expression (n = 220) and wild-type MkPs with low cell-cycle

expression (n = 110), common across samples MF02–MF04. P values

were combined using Fisher’s combined test with Benjamini–Hochberg

adjustments. Weighted average of fold change (expressed in log 2 ) based on

cell number across samples is shown (Methods).