Role of Bone Marrow Injury in Promoting Tet2-Mediated Clonal Hematopoiesis of Indeterminate Potential Zhonghe Ke, PhD; Yanmin He, MS; Eric M. Pietras, PhD Division of Hematology, University of Colorado Anschutz Medical Campus

Introduction 2 Tet2-deficient HSPC preferentially expanded in hematopoietic 4 Screening drugs that inhibit Tet2-deficient HSPC expansion • Clonal Hematopoiesis of Indeterminate Potential (CHIP) is an age-associated and non-hematopoietic organs A Tet2+/+ -IL1 ko vs wt OCR Tet2 ko vs wt ECAR 500 Rotenone & 60 B OXPHOS/Glycolysis inhibitor condition, associated with increased risks of cardiovascular disease, +/+ 500 60 Venetoclax YC1 Oligomycin A * Tet2+/+Tet2 -0GyIR D antimycin A 2-DG 400 Oligomycin 120 120 150 8080 *** ** 150 ** 400 120 120 150 +/+ +/+ hematological malignancies, et al. TetTet22-/--/- 0Gy-IR 2020 * 1515 150 Tet2 +/+ Tet2 Tet2 /min) /min) ** 40 -/- -/- 40 +/+ +/+ -/- Tet2 Tet2 ** 300 FCCP Tet2 -IL1 +/+Tet2 -IL1 90 90 Tet2 Tet2 +/++/+ 2.5Gy 300 TetTet22+/+ -IL1-IL1 90 90 Tet2 +IR -/- -/- 100 Tet2 -IL1 -/- Tet2 -IL1 100 6060 15 mpH -/- 15 pmol Glucose TetTet22 -IL1-IL1 • CHIP is characterized by selective outgrowth of hematopoietic stem/progenitor *** -/--/- Oligomyci Tet2 +/+ +IL1 Tet2 +/+ +IL1 Tet2Tet2 2.5Gy+IR 1010 100100 200200 TetTet22+/++/+ +IL1+IL1 6060 6060 20 -/- -/- OCR (pmol/min) n Tet2 +IL1 -/- Tet2 +IL1 ECAR (mpH/min) -/- * 20 TetTet22 +IL1+IL1 +/+ cells (HSPCs), frequently harboring oncogenic mutations, such as DNA *** 5050 Tet2Tet2 +/+ 4040 *** *** 100 30 1010 OCR ( 30 *** 100 30 30 -/- *** ECAR ( Tet2 -/- * Tet2 methylation regulator TET2. (%) cells derived * * 50 - 55 50 0 0 0 0 0 Inhibition of proliferation (%) Inhibition of proliferation (%) Inhibition of proliferation (%)

0 (%) viability Relative 0 0 20 40 60 80 0 20 40 60 80 0 0 0

20 0 50 500 0 8 0 0 0.05 0.1 20 5 0 8 5 0 40 0 20 Time40 (min) 60 80 0 20 40 60 80 200

Time (min) 40

50 10005000 0.1 200 500 Time (min) 0.05 Time (min) 1000 5000 • TET2 deficiency results in aberrant expansion of hyperinflammatory immune Donor Donor-derived cells (%)

Fold change of LSK number 0 Fold change of LSK number 0 0 Venetoclax (nM) YC-1 (nM) Oligomycin (ng/ml) Fold change of LSK number cells, which contribute to cardiovascular disease and other comorbidities. 0 number LSK of change Fold 00 0 BMBM PBPB SpleenSpleen LungLung LiverLiver GATGAT Lung Liver GAT 0Gy 0Gy 0Gy 0Gy 0Gy-/- 0Gy -/- -/- +/+ +/+ 2.5Gy 2.5Gy +/+ 2.5Gy 2.5Gy 2.5Gy-/- 2.5Gy -/- -/- B ** +/+ +/+ +/+ KDM inhibitors LSK Tet2 Tet2 Tet2 Tet2 C D E • TET2 mutations are ubiquitous during aging. While mutant allele frequency is Tet2 Tet2 Tet2 Tet2 Tet2 -/--/- Tet2+/++/+ Daminozide ATRA A, PercentageTet2 Tet2 of donorTet2-derived cells Tet2in BM, PB, Tet2 Tet2 IOX1 6060 *** +/+ -/- *** P4HA2 spleen, lung, liver and gonadal adipose tissue (GAT) Tet2 / Tet2 P4HA1 120 120 PLOD2 120 EGLN3 120 120 +/+ +/+ *** PTGS2 120 low in healthy individuals, some individuals experience selective expansion of KDM3A +/+ Tet2 Tet2 D EGLN1 Tet2 KDM4B HSPCs KDM6B in adoptively transferred recipient mice at 12 weeks PIR P3H3 ALKBH5 -/- -/- KDM7A -/- KDM5B Tet2 Tet2 P4HB KDMs 90 Tet2 90 +/+ PHYH KDMs 90 +/+ *** PLOD1 90 90 Tet2 TET2-deficient HSPCs, often associated with bone marrow (BM) disruption. *** * post treatment, tissue resident nucleated cells were TET1 90 Tet2 KDM2A *** HAAO ASPH JMJD6 ADI1 -/- 40 KDM5A Tet2 -/- 40 isolated after whole body PBS perfusion; B, Donor KDM4C *** KDM4A Tet2 POR *** 20% / 1% O ALKBH6 60 60 2 PHF8 60 *** *** KDM5C - + + P3H1 60 60 60 TET3 JMJD1C LSK (Lin Sca1 cKit ) chimerism; C, Representative PTGS1 PHF2 KDM3B * HIF1AN • However, causative role of BM disruption in selective expansion of TET2- ALOX5 KDM6A P3H2 flow cytometry analysis of tissue resident LSK KDM5D 30 30 PLOD3 30 TMLHE 30 30 OGFOD3 30 TET2 ETHE1 derived cells (%) cells derived 12hrs OGFOD2 UTY KDM8 - 2020 chimerism at 12 weeks post treatment in lung, liver, deficient HSPCs has not been identified. RSBN1 ALKBH8 ALKBH4 ALKBH3 EGLN2 0 0

FTO Inhibition of proliferation (%) Inhibition of proliferation (%) Relative viability (%) viability Relative

KDM2B 0 (%) viability Relative Inhibition of proliferation (%) GAT; D, fold change of tissue resident LSK number ADO (%) viability Relative 0 0 RNA-seq RIOX1 0

TYW5 0 00 0 OGFOD1

ALKBH1 00 4 4 12 60 40 HSPBAP1

20 40 12 60 300 200 JMJD4 0.8 RIOX2 20 15007500 1000 5000 in lung, liver and GAT. Two-way ANOVA was used to ALKBH7 10005000 200 ALKBH2 0.8 300

Donor P4HTM ALOX12 1000 5000 1500 7500 Donor-derived cells (%) 00 calculate p value, *p<0.05, **p<0.01, ***p<0.001. 1000 5000 IOX1 (nM) Daminozide (nM) BM PB SpleenSpleen LungLung Liver GATGAT ATRA (nM) Results Lung Liver GAT C F DNA hydroxymethylation modulation G 97 88 88 Aza VitC Aza-LSK %inhibition VitC-LSK %inhibition 1.7 4.7 Tet2 +/+ 0.74 120 120120 300 200 1 120 +/+ 300 +/+ 200 +/+ +/+ Tet2 Tet2 Tet2 Tet2 Tet2-deficient HSPC expanded more rapidly than wild-type -IR Tet2 +/++/+ -/- -/- -/-Tet2 -/- Tet2 Tet2 150 Tet2 Tet2 9090 9090 150 -/- -/- HSPCs in non-conditioned adoptively transferred recipient mice 3 1.6 6 200200 Tet2Tet2 6060 6060 100100 (%) 50 (%) 75 61 100100 BM transplantation Check 2.9 1.9 3030 3030 5050 A -IR Tet2 -/- 0.73 (10x106 cells/injection) chimerism 0 0 0 0 Inhibition of proliferation (%) Inhibition of proliferation (%) Inhibition of proliferation (%) -IR Inhibition of proliferation (%) Relative viability (%) viability Relative 0 (%) viability Relative 0 0 0 Relative LSK number LSK Relative

12 weeks number LSK Relative 0 0 0 2 0 0 2 0 2 0 28 20 10 50 20 0 2 10 50

20 100 500 10 50 250 100 500 250 35 2500 1250 20 2500 10 50 1250 Check 17 12500 12500 100 500 250 100 500 250 2500 1250 2500 chimerism 1250 12500 12500 4 weeks 88 76 88 Azacytidine (nM) Ascorbate (ug/ml) Azacytidine (nM) Ascorbate (ug/ml) 1.7 1.2 0.81 +IR Check Tet2 +/+ chimerism A, Oxygen consumption (OCR) and extracellular acidification rate (ECAR) measurement on Tet2+/+ and Tet2-/- HSPCs (cKit+) Non-conditioned +IR by seahorse; B, Small molecules inhibiting OXPHOS/Glycolysis were used in proliferation assay, including Venetoclax and recipient 12 weeks 6 10 6 Oligomycin, HIF inhibitor YC-1; C, Experimental scheme for RNA-seq; D, Histone lysine demethylase (KDM) family members upregulation was found; E, Small molecules modulating histone methylation and stem cell differentiation, including 69 B 57 54 KDM inhibitors IOX1 and Daminozide, and all-trans retinoic acid (ATRA) were tested in proliferation assay; F, Molecules that 0.87 2.1 1.3 -IR +IR Tet2 -/- reverse Tet2-deficiency induced aberrant DNA methylation were tested in proliferation assay, including Azacytidine and - - + + +IR 26 26 26 Ascorbate; G, Relative LSK (Mac1 Gr1 Sca1 cKit ) cell number were ananlyzed in proliferation assay treated with 93 25 90 41 Azacytidine and Ascorbate. 10 16 35 23 Tet2 +/+ cKit cKit CD45.1 CD45.1 CD3 CD45.1 Mac1 CD45.1 Mac1 cKit CD3 CD45.1 CD45.2 Sca1 CD45.2 Sca1 CD45.2 6 74 87 8 58 81 Sca1 CD45.2 Gr1 B220 CD45.2 Gr1 B220 Conclusions 73 35 64 28 3 7 9 What microenvironmental factors induced by radiation -/- Tet2 -/- contributed to preferential expansion of Tet2-deficient HSPC? • Tet2-deficient HSPCs preferentially expanded in non- Tet2 HSPC CD45.1 CD45.1 Mac1 Mac1 CD3 CD3 24 63 84 32 70 85 conditioned adoptively transferred mice, radiation Low O A Radiation Inflammation 2 CD45.2 Gr1 B220 CD45.2 Gr1 B220 promoted Tet2-deficient HSPCs preferential expansion; Tet2+/+ 0Gy ? Preferential expansion of O2 ↑ Tet2+/+ 2.5Gy Tet2-defieicnt HSPCs • Tet2-deficient HSPCs resided and expanded in non- BM injury C D Slope SlopeTet2-/- 0Gy Others hematopoietic organs, such as lung, liver and GAT; High O *** 1.818 -/- -/- 2 *** 30 1.818Tet2Tet2 2.5Gy 2.5Gy Proliferation assay 5 3030 30 B C 5 -IL1 8×108 +IL1

+ 2.0×2010 ) • High O tension was important for Tet2-deficient HSPC ) +/++/+ +/++/+ cKit cells 5 2 **** TetTet22 -0GyIR TetTet22 0Gy-IR 4 30 *** **** *** *** 1.273 -/- *** 5 *** Tet2 0Gy 5 6×10 +/++/+ preferential expansion; 2020 TetTet22+/++/+ +2.5GyIR 2020 1.273TetTet22+/++/+ 2.5Gy+IR 1.5×1510 6 TetTet22 11%%O2 **** **** Lung Liver GAT **** *** --/-/- -/--/- 20% O2 1% O2 --/-/- 20 TetTet22 -0GyIR TetTet22 0Gy-IR 5 TetTet22 1%1%O2 • Azacytidine and Ascorbate showed promising potential in 1.0×10105 4×104

derived cells (%)cells derived +/+ derived cells (%) cells derived +/++/+

- 10 10 --/-/- - 0.301 -/-Tet2 2.5Gy *** 10 TetTet22 +2.5GyIR 10 0.301TetTet22-/- 2.5Gy+IR TetTet22 2120%%O2 selectively inhibiting Tet2-deficient HSPC expansion. ± ± Cell number SCF IL1 SCF IL1 Cell number 5 +/+ 5.0×104 2×102 --/-/- 10 0.312 Tet2 0Gy 5 * TetTet22 2120%%O2

Donor-derived cells (%) 0.312 Donor-derived cells (%) 12d 12d *** Cell number (x10 Cellnumber Donor Donor 00 00 (x10 Cellnumber 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0.00 00

Donor-derived cells (%) 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 Cell count Cell count 0 4 8 12 00 44 88 1212 Weeks post treatment Weeks post treatment 0 4 8 12 0 Weeks4 post treatment8 12 Weeks post treatment DaysDays in cultureculture DaysDays in cultureculture Future Directions Weeks post treatment D Competition culture E 1) Use non-genotoxic approaches to increase O2 tension in the BM, such as +/++/+ +/+-/- +/+-/- -/--/- + 8080 -IL1 +IL1 E Tet2Tet2 - IR0Gy Tet2Tet2 +IR0Gy Tet2Tet2 -2.5GyIR Tet2Tet2 2.5Gy+IR cKit cells (1:9) 6060 -/- Tet2 +/+/Tet2-/- : BoyJ *** vasodilators, and confirm its impact on Tet2 HSPCs preferential expansion; 100100 100 100 100 6060 *** *** 4040 2) Identify pathways that harness high O2 tension to confer the proliferative 80080 80 80 80 *** -/- 20% O2 1% O2 4040 *** advantage of Tet2 HSPCs using RNA-seq and CRSPR/shRNA knock out/down T lym ** T lym CD45.2 %

* CD45.2 %

6060 60 60 60 % CD45.2 20 CD45.2 % CD45.2 20 approaches, and screen molecules that block such pathways; BB lymlym SCF SCF 2020 4040 40 40 40 derived cells (%)cells derived Myeloid 12d 12d - Myeloid 3) Test therapeutic potential of inhibitors that are screened in vitro in preventing 00 00 2020 20 20 20 FACS FACS 0 4 88 1212 0 4 88 1212 Tet2-/- HSPCs preferential expansion and/or eradicating Tet2-/- HSPCs in our non- Days in culture Donor-derived cells (%) Donor-derived cells (%) Donor-derived cells (%) Donor-derived cells (%) Days in culture Donor analysis analysis 00 0 0 0 Days in culture Days in culture 00 2 44 88 1212 0 2 44 88 1212 0 2 44 8 1212 0 2 4 8 1212 conditioned CHIP model mice. -IL1 +IL1 Weeks post treatment Weeks post Weekstreatment post treatmentWeeks post treatment Weeks post treatment F Tet2-/- selective 21% 1% (1) (2) -/- +/+ (3) 21% 1% A, Potential radiation induced BM Vasodilators Tet2 ::Tet2 HSPCs (1::9) inhibitors 78 79 62 74 damages contributed to preferential Prevent Tet2-CHIP CHIP A, Experimental scheme for non-conditioned adoptive BM transfer, radiation was applied at 4 weeks post transplantation; B, +/+ expansion of Tet2-deficient HSPCs; B, In Tet2-CHIP CHIP Tet2 progression Representative flow cytometry analysis for periphery blood (PB) chimerism at 12 weeks post ionizing radiation (IR) treatment; C, vitro proliferation assay scheme; C, cell mice + small mice progression BM O Percentage of donor-derived cells in PB in adoptively transferred recipient mice was checked at week 2, 4, 8, 12 post radiation number during 12 days culture ±IL1 2↑ molecules HTS flow 21 19 32 20 treatment; D, Percentages of donor-derived cells from (C) were fitted in linear regression and the slope was shown; E, Donor under 21% and 1% O2; D, Competition cytometry myeloid (Mac1+), B lymphoid (B220+) and T lymphoid (CD3+) lineage distribution in PB over time. Two-way ANOVA was used to culture scheme; E, Percentage of calculate p value, ***p<0.001. 63 77 46 75 CD45.2+ cells; F, Representative FACS Tet2 -/- plot at day 8 competition culture. Two-way Funding: R01 DK119394, Golfers Against Cancer Pilot Award University of Colorado Department of Medicine ANOVA was used to calculate p value, Outstanding Early Career Scholar Award (to E.M.P.) We thank James Chavez, Taylor Mills, Rachel Gessner, Ian CD45.1 *p<0.05, ***p<0.001. 36 22 48 20 Cartwright, Haobin Ye, Kelly Higa , Sean Colgan, James DeGregori, Craig Jordan for assistance and advices on CD45.2 this project.