Discovery of a CD10-Ve Fetal B Progenitor

Re-ordering human B lymphopoiesis: Discovery of a CD10-ve fetal B progenitor

Andi Roy University of Oxford Is leukaemia the same disease at any age?

FETAL LIFE BIRTH INFANCY CHILDHOOD ADULTHOOD

? = ? =

Overall Survival paediatric ALL: 91.5%

infant ALL: 53.8%

adult ALL: 39% Proportion of patients (%) patients of Proportion

Time (years) Fetal origins of infant ALL

FETAL LIFE BIRTH INFANCY CHILDHOOD

iALL

1st HIT

lymphopoiesis 2ndHIT

pALL Fetal B Fetal FETAL LIVER FETAL BM PAED BM

• What is the target (fetal) cell for leukaemic transformation in infant/ childhood ALL?

• Where do these lie in the B cell developmental hierarchy?

• Are these progenitors restricted to a particular developmental stage or site?

• Do the fetal specific characteristics of these progenitors define the biology of the leukaemia? Sites of haematopoiesis through ontogeny

FETAL LIFE BIRTH CHILDHOOD ADULTHOOD 5w 7w 12w 40w

AGM Liver

Bone marrow

FL and FBM from 6 weeks- 21 post conceptional weeks Fetal specific B progenitors

CD34

HSC MPP LMPP CLP ProB B cell

CD19-10+ CD19+10+

HSC MPP LMPP ELP PreProB ProB B cell

CD127+ CD19+10- CD19+10+ Roy et al, 2012 Sanz et al (CB), 2010 Roy et al (fetal vs Boiers et al, 2018 Roy et al (FL), 2012 postnatal), 2017

High frequency of PreProB progenitors in FBM Are fetal specific B progenitors site/ stage specific

HSC MPP LMPP ELP PreProB ProB B cell

CD19+10- CD19+10+ 40 FL (n= 49)

+ FBM (n= 27) **** 4 30

n 20

****

o 10 *

0 C P P P B B S P P L o o H M M E r r L P P re P

15 40 40

FL ELP FL PreProB FL ProB

+ +

4 FBM PrePro B

FBM ELP 4 FBM ProB

3 3

3 30 30

D C

10 C

2 2 20 2

n 20

f f

5 f o

o 10 o

% % 0 0 0 6 7 8 9 0 1 3 5 7 9 1 6 7 8 9 0 1 3 5 7 9 1 1 1 1 1 1 1 2 6 7 8 9 0 1 3 5 7 9 1 1 1 -1 -1 -1 -1 -2 - - - - - 1 1 -1 -1 -1 -1 -2 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 1 1 1 1 2 1 1 1 1 2 1 1 1 1 2 gestational age (weeks) Post conceptional weeks gestationalPost conceptional age (weeks)weeks gestationalPost conceptional age (weeks)weeks Are upstream FBM progenitors ‘lymphoid primed’?

HSC MPP LMPP ELP PreProB ProB B cell

CD19+10- CD19+10+

CMP GMP

Lineage-CD34+ MEP

viable

30,000 cells Lineage

CD34

SAMPLES 2 x Fetal liver (FL) Chromium 10 X 2 x Fetal BM (FBM) HT single cell gene expression Are upstream FBM progenitors ‘lymphoid primed’?

ERYTHROID MYELOID LYMPHOID MEGAKARYOCYTIC EPOR ELANE CD79A C6orf25 KLF1 AZU1 VPREB1 CD9 TFR2 PRTN3 VPREB3 CLU LYMPHOID TFR1 CFD IL7R PF4 MEGA CSF2RB MPO EBF1 GP9

ERYTHROID APOE CSF3R IGHM ITGA2B APOC1 CSF1R JCHAIN ITGAB3

MYELOID CNRIP1 LYZ CD22 CLEC1B MS4A3 GP6

CST7 PDGFA

CTSG FLI1

THBS1 N Ashley SELP B Psaila S Thongjuea Are upstream FBM progenitors ‘lymphoid primed’?

(matched) FL FBM ABM

ERYTHROID/Mk MYELOID LYMPHOID UNDEFINED

Erythroid affiliated HSPC compartment predominant in FL and lymphoid affiliated in FBM

The Lin-CD34+ HSPC compartment varies in its composition and lineage specification in a site- specific manner in the same fetus and is most likely, directed by cell intrinsic and/or specific microenvironmental factors Characterising human FBM B lymphopoiesis

CD34

HSC MPP LMPP ELP PreProB ProB B cell

CD19+10- CD19+10+

Immunophenotyping Functional Gene expression Epigenetics CD10

CD19

O’Byrne et al, manuscript under revision Cellular hierarchy of human fetal B lymphopoiesis

? ? HSC MPP LMPP ELP PreProB ProB B cell

CD19+10- CD19+10+ FUNCTIONAL

HSC MPP LMPP ELP Pre ProB ProB

erythroid ✔ ✔

myeloid ✔ ✔ ✔ ✔

T ✔ ✔ ✔ ✔

NK ✔ ✔ ✔ ✔

B ✔ ✔ ✔ ✔ ✔ ✔ ELP PrePreProB ProB ProBProB 100 100

80 80

+ +

5 5 4

4 60 60

D D

C C

f f

o o

40 40

% % 20 20

0 0 D0 D3 D7 D14 D0 D3 D7 D14 Day of analysis Day of analysis O’Byrne et al, manuscript under revision Cellular hierarchy by gene expression

HSC MPP LMPP ELP PreProB ProB B cell

CD19+10- CD19+10+

BULK FACS SORTING (100 cells/population) RNA SEQUENCING

BIOINFORMATICS

SINGLE CELL INDEX SORTING BIOMARK SINGLE CELL RQ-PCR

Natalina Elliott Gemma Buck 96 GENE PANEL (lineage/lymphoid/ iALL associated genes) S O’Byrne Cellular hierarchy by gene expression

HSC MPP LMPP ELP PreProB ProB B cell

CD19+10- CD19+10+

Single Cell analysis

Index sorted CD34+ cells Index sorted lymphoid populations

lymphoid

DC2 DC2

myeloid

DC1 DC1 • B lymphoid differentiation trajectory : HSC->MPP->LMPP->ELP->PPB->PB->B O’Byrne et al, manuscript under revision Cellular hierarchy by gene expression

HSC MPP LMPP ELP PreProB ProB B cell

B cell genes

IL7R CD79A VPREB1 EBF1 DNTT

PAX5 CD19 MME RAG1 RAG2

CD72 CD9 CD20 LEF1 TCF3

SC RQ-PCR analysis shows upregulation of B cell specific gene expression from ELP  PreProB  ProB Pre ProB progenitors are distinct from ProB progenitors

HSC MPP LMPP ELP PreProB ProB B cell

CD19+10- CD19+10+ RNA-SEQ

PreProB ProB HOXA5 LIN28B 40 200 KIT 25

30 20 150 20 15

M M

P * 10 P 100

T 10 * 5 50 5

0 0 0 C P P P B B C P P P B B C P P P B B L S P P L o ro S P P L o S P P o ro H M M E r P E ro r H M M E r P L P H M M P L P e L P e r re r P P P

25 RUNX2 2000 MPO 40 CSF1R

808 genes DE (FDR<0.1) 20 * 1500 30

15 M

P 1000 20 T 10 * * Lineage and leukaemia associated genes 500 10 5 CDKN2A IGKV1-33 0 0 0 TCL1A C P P P B B C P P P B B C P P P B B L L PreProB S P P L o o S P P o ro S P P o ro CD40 H M M E r r H M M E r P H M M E r P L P P L P L P IGKV1-27 e e re r r IGKV3-11 P P P IGHJ3 MME CD9 80 CD244 6 CD7 200 CD3D MZB1 B cell genescell specific B CD72 CD79A 60 IGHM 150 MPO 4

CD7 M

GP9 P 40 * 100 CSF1R T CD3D 2 * CD248 20 *

myeloid/T 50 CD244 SPINK2 BAALC 0 BLK 0 0 C P P P B B LIN28B L C P P P B B C P P P B B ProB S P P o ro S P P L o o S P P L o o PROM1 H M M E r P H M M E r r H M M E r r L P L P P L P P HOXA3 e r re re HOXA5 P P P RUNX2

Stem cell genes cell Stem S100Z NEGR1

0 0 5 0 5 (*FDR<0.1)

1 1 - log2 FC Pre ProB progenitors are distinct from ProB progenitors

Chromatin accessibility ProB PreProB ProB Lin+ (ATAC sequencing) CD19+10- CD19+10+ CD34-Lin+ PC2 PC2 (2000 cells) (2000 cells) (5000 cells)

PreProB

PC1

PreProB

ProB

Lin+ MNC

MME/CD10 DNTT RAG1

PreProB

ProB

Lin+ MNC

LIN28B PROM1 MPO S O’Byrne C Garnett Cellular hierarchy by IgH status

HSC MPP LMPP ELP PreProB ProB B cell

RAG1 and DNTT

NO REARRANGEMENT D-J? D-J? D-J?/VDJ V-D-J

IgH rearrangements

RAG1100 DNTT VDJ

e t 80 DJ

nil s 60

a 40

o 20

P P B B ls S O’Byrne P L ro ro l M E P P e L e c r B S Rice P G Wright (GOSH) Cellular hierarchy using in vivo models

HSC MPP LMPP ELP PreProB ProB B cell

CD19+10- CD19+10+

CMP GMP

Lineage-CD34+19-10- MEP

Week 2-3 bone marrow S Rice Check for engraftment and differentiation N Fordham S O’Byrne Cellular hierarchy using in vivo models

HSC MPP LMPP ELP PreProB ProB B cell

CD19+10- CD19+10+

CMP GMP

Lineage-CD34+19-10- MEP Summary

FETAL LIFE BIRTH INFANCY CHILDHOOD ADULTHOOD

CD10-

CD10+

FETAL LIVER FETAL BM PAED BM ADULT BM

• There are 2 different B cell progenitors in fetal life based on CD10 expresssion

• The CD10- Pre ProB progenitor is more abundant in fetal BM compared to FL

• It is distinct from and lies upstream of the CD10+ ProB progenitor

• It is fetal specific and virtually absent in adult BM Cellular hierarchy of fetal B lymphopoiesis

HSC MPP LMPP ELP PreProB ProB B cell Why is this important?

FETAL LIFE BIRTH INFANCY CHILDHOOD ADULTHOOD

MLLr CD10- iALL

1st HIT 2ndHIT CD10+ pALL

FETAL LIVER FETAL BM PAED BM ADULT BM

• All infant and many paediatric ALL originate in fetal life

• The CD10- Pre ProB progenitor may be the target for infant ALL

• The CD10+ ProB progenitor may be the target for paediatric ALL Acknowledgments

Roberts lab CBRG Prof Irene Roberts Supat Thongjuea Guanlin Wang Sorcha O’Byrne Siobhan Rice Single Cell Facility (WIMM) Natalina Elliott Neil Ashley Gemma Buck Nicholas Fordham C Garnett Flow facility (WIMM) Peng Hua Genome Engineering Facility Eleni Louka Biomedical Services, Oxford Lucy Field Imperial College London Prof G Hollander Prof Tassos Karadimitris V Caputo A Rotolo Milne lab D Iskander Tom Milne L Godfrey Great Ormond St Hospital Phil Ancliff J Kerry Sarah Inglott Nick Crump Gary Wright D Ladon Mead lab Menendez Lab Prof Adam Mead (Barcelona) B Povinelli Beth Psaila

Hashem Koohy