dasatinib is an effective treatment for angioimmunoblastic ......1 supplemental information...

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Supplemental information

Dasatinib Is An Effective Treatment For Angioimmunoblastic T-Cell

Lymphoma

Tran B. Nguyen1+, Mamiko Sakata-Yanagimoto1,2+*, Manabu Fujisawa3, Sharna Tanzima Nuhat3, Hiroaki Miyoshi4, Yasuhito Nannya5, Koichi Hashimoto6, Kota Fukumoto3, Olivier A. Bernard7, Yusuke Kiyoki2, Kantaro Ishitsuka2, Haruka Momose2, Shinichiro Sukegawa2, Atsushi Shinagawa8, Takuya Suyama8, Yuji Sato9, Hidekazu Nishikii1,2, Naoshi Obara1,2, Manabu Kusakabe1,2, Shintaro Yanagimoto10, Seishi Ogawa5, Koichi Ohshima4, and Shigeru Chiba1,2,11* 1. Department of Hematology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba,

Ibaraki 305-8575, Japan. 2. Department of Hematology, University of Tsukuba Hospital, 2-1-1 Amakubo, Tsukuba, Ibaraki

305-8576, Japan. 3. Department of Hematology, Graduate School of Comprehensive Human Sciences, University of

Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. 4. Department of Pathology, Kurume University, School of Medicine, 67 Asahi, Kurume, Fukuoka

830-0011, Japan. 5. Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University,

Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. 6. Tsukuba Clinical Research and Development Organization (TCReDo), University of Tsukuba, 1-

1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. 7. INSERM U1170, Gustave Roussy, Université Paris-Saclay, Equipe Labellisée Ligue Nationale

Contre le Cancer, Villejuif, France. 8. Department of Hematology, Hitachi General Hospital, 2-1-1 Jonan-cho, Hitachi, Ibaraki 317-0077,

Japan. 9. Department of Hematology and Oncology, Tsukuba Memorial Hospital, 1187-299 Kaname,

Tsukuba, Ibaraki 300-2622, Japan. 10. Division for Health Service Promotion, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-

0033, Japan. 11. Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance, University of

Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. + These authors contributed equally. * Corresponding authors Supplemental Methods

Supplemental Results

Supplemental Figures 1-12

Supplemental Tables 1-8

Supplemental References

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Supplemental Methods

Southern blot assay

DNA from tails of mice was extracted using Phenol-Chloroform method and 15 μg of DNA

was digested with EcoRI. The DNA was precipitated by ethanol and sodium acetate, and then

subjected to electrophoresis through 1% agarose gel. After electrophoresis, the gel was

transferred into a nylon membrane (Hybond-N; Amersham Biosciences, Buckinghamshire,

UK). The probe detecting human RHOA cDNA was made by PCR with primers listed in

Supplemental Table 1 and was labeled with deoxycytidine triphosphate, labeled on the alpha

phosphate group with 32P [dCTP, (!- 32P); PerkinElmer Inc., MA, USA]. Then, the labeled

probe was hybridized with the membrane to detect the human RHOA cDNA at the site of 600

base pairs (bp). Pictures were taken by Typhoon FLA7000 (GE health care, Chicago, USA).

Quantitative PCR of genomic DNA to check copy number of human RHOA (hRHOA)

transgene

Mouse tail DNA was extracted by QIAmp DNA Blood Mini kit (QIAGEN, Hilden, Germany).

Quantitative PCR was performed in duplicate with 12.5 ng of DNA using SYBER Green

Universal Master Mix (Applied Biosystems, CA, USA). Primers used to detect hRHOA

transgene were listed in Supplemental Table 1. VA vector harboring hRHOA transgene

(hRHOA-VA) was used for making standard curve and mouse Actin was used as a house-

keeping gene. The number of hRHOA transgene copies was calculated from their respective

cycle threshold using the linear equation from the respective hRHOA-VA standard curve.

hRHOA copy number per diploid cell was calculated by dividing the copy number in the DNA

samples by the number of cells from which the DNA was isolated. Given that a C57BL/6 mouse

cell yields 6 pg of DNA, 12.5 ng of DNA is equal with 2.08 x 103 diploid cells.

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T-cell receptor rearrangement analysis

One hundred ng of CD4+ splenocytes DNA was extracted using QIAmp DNA Blood Mini kit

(QIAGEN, Hilden, Germany). PCR was performed using Takara Ex-Taq (Takara Bio Inc.,

Shiga, Japan) using the primers listed in Supplemental Table 1 with the following conditions:

(94oC: 3 min; [94oC: 45s, 65oC: 1 min 30s, 72oC: 2 min 30s] ×35 cycles; 72oC: 10 min]). Direct

sequencing was performed with the dominant PCR products. Then, the sequences were

analyzed using IGMT/V Quest tool (1,2).

Relative quantitative reverse transcript PCR (qRT-PCR) to check gene expression

Relative qRT-PCR using SYBER Green Universal Master Mix (Applied Biosystems, CA,

USA) and 18S rRNA as a housekeeping gene was performed (3). The primers used to detect

human RHOA, mouse RhoA, and mouse Tet2 mRNA are listed in Supplemental Table 1.

Immunohistochemistry of phosphorylation of Vav1 (pVav1) and cell count of pVav1

positive cells

Sections were incubated with primary antibodies against mouse pVav1 Tyr-174 (Supplemental

Table 2, 1:100) for 60 minutes at RT. Then, the sections were incubated with HRP-conjugated

anti rabbit antibody (Supplemental Table 7, 1:100) for 60 minutes at room temperature. After

washing with PBS(-), sections were incubated with liquid DAB (Dako liquid DAB+Subtrate

Chromogen System, K3468, Dako, USA) for 15 to 20 minutes at RT. Pictures were taken using

a Keyence BZ X710 microscope (Keyence Corporation, Osaka, Japan). pVav1 positive cells

were manually counted in a total of 10 fields at 40× magnification for each slide.

Validation of VAV1 tandem duplication mutation in patient 5

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The primers detecting the junction site of VAV1 tandem duplication mutation are listed in

Supplemental Table 1. Twenty ng of genomic DNA extracted from the swollen lymph node of

patient 5 was analyzed with PCR using KOD plus neo taq (Toyobo, Osaka, Japan) with these

primers under following conditions: 94oC: 2 min; (98oC: 10s, 50oC: 30s, 68oC: 30s) × 40 cycles.

Direct sequencing was performed with the PCR product. The sequence was confirmed using

UCSC Genome Browser Blat tool (4).

Supplemental Results

Mouse generation

To generate transgenic mice expressing G17V RHOA in T cells , human G17V RHOA cDNA

was inserted into the VA CD2 cassette (5) (Supplemental Figure 1a). Then, the construct was

injected into fertilized eggs to get G17V RHOA transgenic mice. Two lines of G17V RHOA

transgenic mice (A and B) were obtained. Southern blotting of genomic DNA was performed

to confirm transgene integration using tail DNA from each line. The EcoRI fragment containing

the human G17V RHOA cDNA was 600 bp, and both lines exhibited bands of that size

(Supplemental Figure 1b). Genomic quantitative real-time polymerase chain reaction (qPCR)

analysis of tail DNA also showed that these lines harbored similar copy numbers of RHOA

transgenes: line A and B mice showed 2.07±0.86 and 3.17±1.41 copies on average, respectively

(Supplemental Figure 1c).

To recapitulate the human AITL genome, both lines of G17V RHOA transgenic mice were

crossed with Tet2flox/flox mice (6) and Mx-Cre (7) mice to establish Mx-Cre x Tet2flox/flox × G17V

RHOA transgenic mice, in which Tet2 was deleted in all lineages of blood cells following

intraperitoneal injection of polyinosinic:polycytidylic acid (pIpC) (Tet2-/-G17VRHOA mice).

Both integration of the G17V RHOA transgene and Tet2 deletion were confirmed using

genomic PCR of CD4+ splenocytes purified from 6-8 week-old Tet2-/-G17VRHOA mice

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(Supplemental Figure 2a). We also performed qRT-PCR to check expression of G17V RHOA

transgene-derived mRNA (representing the human RHOA sequence) and Tet2 mRNA in CD4+

splenocytes purified from either Tet2-/-G17VRHOA or wild-type (WT) mice. Expression levels

of human RHOA mRNA in CD4+ cells of Tet2-/-G17VRHOA mice were significantly higher

than those in WT mice but lower than those in Jurkat cells. Meanwhile, expression levels of

mouse RhoA mRNA were comparable in WT and transgenic mice (Supplemental Figure 2b).

Tet2 mRNA expression levels in CD4+ splenocytes of Tet2-/-G17VRHOA mice were much

lower than those detected in WT mice (Supplemental Figure 2c).

Clinical trial results in details

Dasatinib has been used in clinical practices for the treatment of Ph-positive leukemias for

many years, but its safety evaluation in relapsed/refractory AITL patients was encouraged.

Therefore, a single center phase I trial was conducted at the University of Tsukuba Hospital.

Given the rareness of this disease, enrollment of five patients was planned in the trial. Firstly,

the dasatinib therapy was planned for 30 days for all five patients, because it was thought to be

a minimal period to make an evaluation. The committee of the hospital, however, let us extend

the therapy period after the first patient responded to the drug well, and the protocol was

changed to give dasatinib for 90 days.

VAV1 mutations were identified in two patients (PAT2 and PAT5). PAT5 had a tandem

duplication mutation (Supplemental Table 8, Supplemental Figures 12a-d). It was predicted

that exon 6 was fused in frame to 3’ of exon 20. Consequently, the autoinhibitory SH3-SH2-

SH3 module was disrupted at E638 in the N-terminal SH3 domain and flanked by the DH

domain (Supplemental Figure 12d). In the PAT2, three missense mutations and an in-frame

two amino acids deletion mutation in the N-terminal SH3 domain were found (Supplemental

Table 8, Supplemental Figure 11a-b). We previously reported another missense mutation,

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c.C1844T, p.Pro615Leu in the N-terminal SH3 domain in an AITL patient and experimentally

demonstrated that this mutation actually confers enhanced functions on VAV1 (10). All these

findings together indicate that both the missense/deletion mutations and the structural variation

found in the current two patients should be activating mutations.

Patient 1 previously had AITL refractory to 4 different regimens and presented with

persistent high fever, parotid swelling, and generalized lymphadenopathy. Those symptoms

were ameliorated within a week of dasatinib initiation. The detailed clinical course of this

patient was described in the Result section.

Prior to this study, Patient 3 had had a biopsy-proven relapse three years after autologous

transplantation and had presented with generalized lymphadenopathy. After enrollment in our

study, the patient's CT scan on day 27 after treatment initiation indicated fulfillment of PR

(Supplemental Figure 10b). However, dasatinib treatment was stopped on day 55, as disease

progression became apparent and was manifested by high fever and abnormal lung shadow

(data not shown). Tumor infiltration was confirmed by bone marrow biopsy, which was not

observed prior to dasatinib prescription.

Prior to this study, Patient 4 had had an 11-year history of AITL, and had relapsed with

tumor cells appearing in peripheral blood and multiple lung regions, generalized

lymphadenopathy, and pleural effusion. High-dose dexamethasone treatment rapidly decreased

the number of peripheral blood tumor cells and pleural effusion, but lymphadenopathy

persisted. After enrollment in this study, the patient's swollen lymph nodes gradually decreased

in size, and abnormal lymph node swelling was not detectable by CT scan on day 31 after

treatment initiation, although multiple lung lesions persisted (Supplemental Figure 10b).

Dasatinib treatment in this patient was intermittent due to QTc elongation and anemia and

terminated on day 59 due to disease progression.

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Prior to this study, Patient 5 experienced biopsy-proven relapse two and a half years after

achieving the first remission. At the start of this study, generalized lymphadenopathy was

evident. CT scans performed on days 30 and 60 after treatment initiation demonstrated

markedly decreased lymph node swelling, with an estimation of PR (Supplemental Figure 10b).

Dasatinib treatment was interrupted from days 17 to 32 due to QTc elongation, and the study

was stopped on day 79 due to severe and concomitant adverse events: coronary heart disease,

found incidentally during the course of treatment, and grade 3 neuropathy, potentially

attributable to drug (Table 2).

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Supplemental Figure 1

Generation of G17VRHOA mice. a. Graph shows the construct harboring the human G17V RHOA transgene. b. Southern blot analysis of tail DNA shows G17V RHOA gene integration in 2 lines (A and

B) of transgenic mice. WT, wild-type. c. Average number of copies of human G17V RHOA transgenes as detected via analysis of tail

DNA of lines A and B (upper). Average cycle quantification value of mouse Actin, which served as a housekeeping gene (lower).

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Generation of Tet2-/-G17VRHOA mice. a. Genomic polymerase chain reaction (PCR) analysis shows human G17V RHOA transgene

(upper blot) and Tet2-deleted band (lower blot) in CD4+ splenocytes of young (6-8 week-old) wild-type (WT) and Tet2-/-G17VRHOA mice, n=3 each. PC, postive control; Flox, floxed Tet2; DW, distilled water control; Deleted, deleted Tet2.

b. Expression of human RHOA mRNA (left side) in CD4+ splenocytes from young (6-8 week- old) WT or Tet2-/-G17VRHOA mice (lines A and B), and Jurkat cells; and expression of mouse RhoA mRNA (right side) in CD4+ splenocytes from WT and Tet2-/-G17VRHOA mice, n=3 each.

c. Expression of mouse Tet2 mRNA in CD4+ splenocytes from WT and Tet2-/-G17VRHOA mice, n=3 each.

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Pathology of tumors in Tet2-/-G17VRHOA mice. a. Macroscopy (i) and weights (ii) of spleens from mice of indicated genotypes (n=7, WT;

n=10, Tet2-/-G17VRHOA). (iii) Swollen lymph nodes of Tet2-/-G17VRHOA mice. b. Low (×4, above) and high (×100 for spleen and lymph nodes, and ×40 for lung and liver;

below) magnification images showing haematoxyllin and eosin staining of indicated tissues from WT and Tet2-/-G17VRHOA mice. Arrowheads and arrows show immunoblasts and eosinophils, respectively.

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Cellular analysis of tumor-bearing Tet2-/-G17VRHOA mice. a. Flow cytometric analysis of cells from spleen tissues of WT and Tet2-/-G17VRHOA mice.

Gr1, Mac1: myeloid cells; CD19, B220: B-lymphocytes; CD4, CD8: T-lymphocytes; PD1, ICOS: markers of T follicular helper (TFH).

b. Percentage of cells in various myeloid and lymphoid fractions, as indicated, from spleens of WT and Tet2-/-G17VRHOA mice, as analyzed using flow cytometry. n=7 (WT) and n=10 (Tet2-/-G17VRHOA mice).

c. Immunofluorescence staining of CD4, PD1 and ICOS in spleen tissues from mice of indicated genotypes. Images are at x100 magnification.

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T-cell receptor beta arrangement in CD4+ splenocytes from Tet2-/-G17VRHOA tumor-bearing mice. a, T-cell receptor (TCR) rearrangement in CD4+ splenocytes of Tet2-/-

G17VRHOA tumor bearing mice. Arrows show dominant PCR products in each numbered mouse. DW, distilled water control. b, Pattern of rearrangement and amino acid sequence of junction of TCR beta in three different Tet2-/-G17VRHOA tumor bearing mice established from the B line. c, Sequence chromatogram of DNA representing rearranged TCR beta in CD4+ splenocytes. The dominant PCR bands of Tet2-/-G17VRHOA tumor bearing mice were sequenced. Arrows show V, D, and J regions of TCR beta.

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Characteristics of tumors seen in Tet2-/-G17VRHOA mice. a, Plasma concentrations of indicated inflammatory cytokines in wild-type (WT) and Tet2-/-

G17VRHOA tumor-bearing mice. n=6, each. *, p<0.05; **, p<0.01. b, Gene set enrichment analysis (GSEA) of nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) in CD4+ splenocytes from Tet2-/-G17VRHOA tumor-bearing mice. Graph shows the enrichment score plot for NF-κB pathway gene set (upper). Heat map shows the expression of genes in the pathway (lower). c, As in (b), enrichment of T follicular helper-(TFH-) related genes. d, Validation of relatively increased expression of BCL6, ICOS, and IL-21 mRNAs in CD4+ splenocytes from WT and Tet2-/-G17VRHOA mice. n=3, each.

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Characteristics of tumor-engrafted nude mice. a. Macroscopic view of spleens of recipient nude mice that were not transplanted (non-

transplanted) or transplanted with tumor cells from Tet2-/-G17VRHOA mice (engrafted). b. Low (×4, above) and high (×100, below) magnification images showing HE staining of

spleen from indicated recipients. c. Flow cytometric analysis of lymph nodes indicating engraftment of donor tumor cells in

lymph nodes of a recipient nude mouse. H2Kd and H2Kb, markers of recipient nude and donor C57BL/6 mice, respectively; CD4, ICOS, and PD1, markers of T follicular helper cells.

d. PCR analysis showing upper G17V RHOA amplicon and lower Tet2-deleted band in CD4+ splenocytes from tumor-engrafted nude mice (N4, N8). Flox, floxed Tet2; PC, positive control; deleted, deleted Tet2.

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Anti-tumor effect of dasatinib in tumor-engrafted recipient mice. a. Effect of vehicle or dasatinib treatment on plasma levels of indicated cytokines at treatment

day 0 (pre) and 15 days later (post). n=9 (vehicle) and n=8 (dasatinib). b. Effect of dasatinib on Vav1 phosphorylation (pVav1). (i) Low (×10, above) and high

(×100, below) magnification images show staining with a pVav1 antibody of spleen tissues treated as indicated. Staining was performed 6 days after vehicle or dasatinib treatment. Brown dots show the pVav1+ cells. (ii) Quantification of pVav1+ splenocytes, which were counted in 10 fields of ×40 magnification, 6 days after treatment with vehicle (black) and dasatinib (red). n=4, each.

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Responses of patient 3, 4 and 5 to dasatinib. a. Serum soluble IL-2 receptor levels before (Baseline) and on first assessment day (PAT3:

day 27, PAT4: day 31, PAT5: day 30) after initiation of dasatinib treatment (After). b. CT scan performed before and on first assessment day. Arrows show lymph nodes. Stars

indicate spleens.

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Digital droplet PCR (ddPCR) analysis of G17V RHOA mutations. a. G17V RHOA mutation status in indicated samples obtained from all five patients enrolled

in clinical trial. LN, lymph node; BM, bone marrow mononuclear cells; PB, peripheral blood mononuclear cells; FFPE, formalin-fixed paraffin embedded.

b. 2D images of ddPCR assay testing G17V RHOA mutations in tumor DNA of Patient 3 (PAT3, upper) and Patient 4 (PAT4, lower). Pink lines indicate FAM (horizontal) and HEX (vertical) threshold. Red dots indicate G17V RHOA positive events.

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Analysis of the VAV1 mutations in Patient 2. a. Integrative Genomics Viewer (IGV) image of the VAV1 mutations in Patient 2 (PAT2). b. Locations of the VAV1 mutations. Stars indicate mutations. CH, calponin homology

domain; DH, Dbl homology domain; PH, pleckstrin homology domain; C1, protein kinase C-like phorbol ester/diacylglycerol-binding domain; and SH, Src homology domain. Stars indicate the mutations.

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Analysis of the VAV1 tandem duplication mutation in Patient 5. a, (i) Schema showing VAV1 tandem duplication mutation in Patient 5 (PAT5). F, forward primer; R, reverse primer. (ii) PCR analysis shows the product amplified using F and R primers shown in (i). Healthy, healthy control; DW, distilled water control. b, Sequence chromatogram of the amplified PCR product (above) at the junction site. c, Integrative Genomics Viewer (IGV) image of the VAV1 tandem duplication mutation in PAT5. d, Predicted protein domain structure of the VAV1 tandem duplication mutation. CH, calponin homology domain; DH, Dbl homology domain; PH, pleckstrin homology domain; C1, protein kinase C-like phorbol ester/diacylglycerol-binding domain; and SH, Src homology domain.

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Supplemental Table 1: List of used prim

ers

Primer

Forward (5’ to 3’)

Reverse (5’ to 3’)

Genotyping and detection of hum

an RHOA

transgene in G17V

RHO

A m

ouse A

ATG

ATG

AG

CACA

CAA

GG

CG

AG

CAG

CTCTCGTA

GCCA

TTT

Hum

an RHOA cD

NA

probe for southern blot assay

ACTG

GTG

ATTG

TTGG

TGA

TGG

A

CTTCATCTTG

GCTA

GCTCCCG

TCR beta arrangement

GCG

CTTCTCACCTCA

GTCTTCA

G

TGA

GA

GCTG

TCTCCTACTA

TCGA

T

Hum

an RHOA m

RNA

for qRT-PCR A

ATG

ATG

AG

CACA

CAA

GG

CG

AG

CAG

CTCTCGTA

GCCA

TTT

Mouse RhoA m

RNA

for qRT-PCR G

CAG

GTA

GA

TTGG

CTTTATG

G

CTTGTG

TGCCA

TCATTCCG

A

Mouse Tet2 m

RNA

for qRT-PCR CG

GTTG

TGCTG

TCATTTG

TT G

TCGA

AA

GCG

TTCCTCTCTG

Validation of VAV1 m

utation of patient 5 A

CCGA

TCATCTCTCTG

CCTATTTTT

TGTCA

GTG

TACTTCTCCTCCG

TCT

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Supplemental Table 2: List of used antibodies

Assay Antibody Conjugati-on Company Clone Order

number

Flow- cytometry

H2Kb biotin Biolegend AF6-88.5

116503

B220 FITC eBio- science

RA3-6B2

11-0452-82

H2Kd FITC Biolegend SF1-1.1 116605

ICOS FITC eBio- science

C398.4A 11-9949-82

Gr1 FITC eBio-science RB6-8C5

11-5931-82

CD19 PE eBio-science eBio1D3 12-0193-81

CD8a PE eBio-science 53-6.7 17-5921-82

Mac1 PE Biolegend M1/70 101207

PD1 PE eBio-science RMP1-30

12-9981-81

CD4 PE-Cy7 eBio-science RM4-5 25-0042-82

7ADD PerCP eBio-science

00-6993-50

Immuno- staining

CD4 eBio-science 14-0041-81

PD1 R&D system AF1021

ICOS FITC eBio-science C398.4A 11-9949-82

pVav1 (Tyr-174)

Introvigen PA5-36699

pPLCƔ1 CST 2821

anti goat Alexa 594 Introvigen A11058

anti rat Alexa 488 Introvigen A11006

anti rat Alexa 594 Introvigen A21209

anti rabbit Alexa 488 Introvigen A11034

anti rabbit Alexa 594 Introvigen A11072

anti rabbit HRP Dako P4050

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Supplemental Table 3: Lists of genes analyzed by target sequencing

Gene symbol Entrez gene ID

Gene name

ABL1 25 ABL proto-oncogene 1, non-receptor tyrosine kinase ACTB 60 actin beta ADNP 23394 activity dependent neuroprotector homeobox AKT1 207 AKT serine/threonine kinase 1 AKT2 208 AKT serine/threonine kinase 2 AKT3 10000 AKT serine/threonine kinase 3 ALK 238 anaplastic lymphoma receptor tyrosine kinase AR 367 androgen receptor ARHGEF1 9138 Rho guanine nucleotide exchange factor 1 ARID1A 8289 AT-rich interaction domain 1A ARID1B 57492 AT-rich interaction domain 1B ARID2 196528 AT-rich interaction domain 2 ARID5B 84159 AT-rich interaction domain 5B ASXL2 55252 ASXL transcriptional regulator 2 ASXL3 80816 additional sex combs like 3, transcriptional regulator ATM 472 ATM serine/threonine kinase ATP6AP1 537 ATPase H+ transporting accessory protein 1 ATP6V1B2 526 ATPase H+ transporting V1 subunit B2 ATR 545 ATR serine/threonine kinase ATXN1 6310 ataxin 1 ATXN7L1 222255 ataxin 7 like 1 AURKA 6790 aurora kinase A B2M 567 beta-2-microglobulin BAP1 8314 BRCA1 associated protein 1 BCL10 8915 B-cell CLL/lymphoma 10 BCL11A 53335 B-cell CLL/lymphoma 11A BCL11B 64919 B-cell CLL/lymphoma 11B BCL2 596 BCL2, apoptosis regulator BCL6 604 B-cell CLL/lymphoma 6 BCL7A 605 BCL tumor suppressor 7A BCOR 54880 corepressor BCR 613 BCR, RhoGEF and GTPase activating protein BIRC3 330 baculoviral IAP repeat containing 3 BRAF 673 B-Raf proto-oncogene, serine/threonine kinase BRCA1 672 BRCA1, DNA repair associated BRCA2 675 BRCA2, DNA repair associated BRD4 23476 bromodomain containing 4

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BRSK1 84446 BR serine/threonine kinase 1 BTG1 694 BTG anti-proliferation factor 1 BTG2 7832 BTG anti-proliferation factor 2 BTK 695 Bruton tyrosine kinase

CAD 790 carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase

CARD11 84433 caspase recruitment domain family member 11 CASP8 841 caspase 8 CBLB 868 Cbl proto-oncogene B CCND1 595 cyclin D1 CCND2 894 cyclin D2 CCND3 896 cyclin D3 CCR4 1233 C-C motif chemokine receptor 4 CCR7 1236 C-C motif chemokine receptor 7 CCT6B 10693 chaperonin containing TCP1 subunit 6B CD22 933 CD22 molecule CD28 940 CD28 molecule CD36 948 CD36 molecule CD58 965 CD58 molecule CD70 970 CD70 molecule CD79A 973 CD79a molecule CD79B 974 CD79b molecule CD80 941 CD80 molecule CD83 9308 CD83 molecule CDC73 79577 cell division cycle 73 CDK4 1019 cyclin dependent kinase 4 CDK6 1021 cyclin dependent kinase 6 CDKN1B 1027 cyclin dependent kinase inhibitor 1B CDKN2A 1029 cyclin dependent kinase inhibitor 2A CDKN2B 1030 cyclin dependent kinase inhibitor 2B CDKN2C 1031 cyclin dependent kinase inhibitor 2C CEBPA 1050 CCAAT/enhancer binding protein alpha CHAF1A 10036 chromatin assembly factor 1 subunit A CHD2 1106 chromodomain helicase DNA binding protein 2 CHUK 1147 conserved helix-loop-helix ubiquitous kinase CIITA 4261 class II major histocompatibility complex transactivator CNOT2 4848 CCR4-NOT transcription complex subunit 2 CPS1 1373 carbamoyl-phosphate synthase 1 CR2 1380 complement C3d receptor 2 CREBBP 1387 CREB binding protein

CSF2RB 12983 colony stimulating factor 2 receptor, beta, low-affinity (granulocyte-macrophage)

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CSMD3 114788 CUB and Sushi multiple domains 3 CSNK1A1 1452 casein kinase 1 alpha 1 CSNK2A1 1457 casein kinase 2 alpha 1 CSNK2B 1460 casein kinase 2 beta CTCF 10664 CCCTC-binding factor CTLA4 1493 cytotoxic T-lymphocyte associated protein 4 CTNNA2 1496 catenin alpha 2 CTNNA3 29119 catenin alpha 3 CTNNB1 1499 catenin beta 1 CXCR4 7852 C-X-C motif chemokine receptor 4 DDR2 4921 discoidin domain receptor tyrosine kinase 2 DDX3X 1654 DEAD-box helicase 3, X-linked DDX60 55601 DExD/H-box helicase 60 DDX60L 91351 DExD/H-box 60 like DNAH14 127602 dynein axonemal heavy chain 14 DNAH5 1767 dynein axonemal heavy chain 5 DNMT3A 1788 DNA methyltransferase 3 alpha DROSHA 29102 drosha ribonuclease III DSCAM 1826 DS cell adhesion molecule DSP 1832 desmoplakin DST 667 dystonin DTX1 1840 deltex E3 ubiquitin ligase 1 DUSP2 1844 dual specificity phosphatase 2 DUSP9 1852 dual specificity phosphatase 9 EBF1 1879 early B-cell factor 1 EEF1A1 1915 eukaryotic translation elongation factor 1 alpha 1 EGFR 1956 epidermal growth factor receptor EGR1 1958 early growth response 1 EIF4A1 1973 eukaryotic translation initiation factor 4A1 EIF4A2 1974 eukaryotic translation initiation factor 4A2 EIF4G1 1981 eukaryotic translation initiation factor 4 gamma 1 EP300 2033 E1A binding protein p300 EPHA7 2045 EPH receptor A7 EPPK1 83481 epiplakin 1 ERBB2 2064 erb-b2 receptor tyrosine kinase 2 ERBB3 2065 erb-b2 receptor tyrosine kinase 3 ERBB4 2066 erb-b2 receptor tyrosine kinase 4 ETS1 2113 ETS proto-oncogene 1, transcription factor ETV6 2120 ETS variant 6 EZH2 2146 enhancer of zeste 2 polycomb repressive complex 2 subunit FAS 355 Fas cell surface death receptor FAT1 2195 FAT atypical cadherin 1

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FAT2 2196 FAT atypical cadherin 2 FAT3 120114 FAT atypical cadherin 3 FAT4 79633 FAT atypical cadherin 4 FBXO11 80204 F-box protein 11 FBXO31 79791 F-box protein 31 FBXW7 55294 F-box and WD repeat domain containing 7 FGFR1 2260 fibroblast growth factor receptor 1 FGFR2 2263 fibroblast growth factor receptor 2 FGFR3 2261 fibroblast growth factor receptor 3 FGFR4 2264 fibroblast growth factor receptor 4 FGFRL1 53834 fibroblast growth factor receptor like 1 FHIT 2272 fragile histidine triad FLT3 2322 fms related tyrosine kinase 3 FOXC1 2296 forkhead box C1 FOXO1 2308 forkhead box O1 FOXP1 27086 forkhead box P1 FRMPD1 22844 FERM and PDZ domain containing 1 FUBP1 8880 far upstream element binding protein 1 FYN 2534 FYN proto-oncogene, Src family tyrosine kinase GADD45B 4616 growth arrest and DNA damage inducible beta GATA3 2625 GATA binding protein 3 GNA11 2767 G protein subunit alpha 11 GNA12 2768 G protein subunit alpha 12 GNA13 10672 G protein subunit alpha 13 GNAI2 2771 G protein subunit alpha i2 GNAQ 2776 G protein subunit alpha q GNAS 2778 GNAS complex locus GPR183 1880 G protein-coupled receptor 183 GRB2 2885 growth factor receptor bound protein 2 GRHPR 9380 glyoxylate and hydroxypyruvate reductase GSG2 83903 germ cell associated 2, haspin HDAC4 9759 histone deacetylase 4 HDAC7 51564 histone deacetylase 7

HERC1 8925 HECT and RLD domain containing E3 ubiquitin protein ligase family member 1

HERC2 8924 HECT and RLD domain containing E3 ubiquitin protein ligase 2 HIST1H1B 3009 histone cluster 1 H1 family member b HIST1H1C 3006 histone cluster 1 H1 family member c HIST1H1D 3007 histone cluster 1 H1 family member d HIST1H1E 3008 histone cluster 1 H1 family member e HIST1H2AC 8334 histone cluster 1 H2A family member c HIST1H2AD 3013 histone cluster 1 H2A family member d

26

HIST1H2AE 3012 histone cluster 1 H2A family member e HIST1H2AG 8969 histone cluster 1 H2A family member g HIST1H2AK 8330 histone cluster 1 H2A family member k HIST1H2AL 8332 histone cluster 1 H2A family member l HIST1H2AM 8336 histone cluster 1 H2A family member m HIST1H2BC 8347 histone cluster 1 H2B family member c HIST1H2BF 8343 histone cluster 1 H2B family member f HIST1H2BJ 8970 histone cluster 1 H2B family member j HIST1H2BK 85236 histone cluster 1 H2B family member k HIST1H2BO 8348 histone cluster 1 H2B family member o HIST1H3B 8358 histone cluster 1 H3 family member b HIST1H3C 8352 histone cluster 1 H3 family member c HIST1H3D 8351 histone cluster 1 H3 family member d HIST1H3F 8968 histone cluster 1 H3 family member f HIST1H3G 8355 histone cluster 1 H3 family member g HIST1H3I 8354 histone cluster 1 H3 family member i HIST2H3D 653604 histone cluster 2 H3 family member d HIST4H4 121504 histone cluster 4 H4 HLA-A 3105 major histocompatibility complex, class I, A HLA-B 3106 major histocompatibility complex, class I, B HLA-C 3107 major histocompatibility complex, class I, C HLA-DMB 3109 major histocompatibility complex, class II, DM beta HLA-DPA1 3113 major histocompatibility complex, class II, DP alpha 1 HLA-DPB1 3115 major histocompatibility complex, class II, DP beta 1 HLA-DQA1 3117 major histocompatibility complex, class II, DQ alpha 1 HLA-DQB1 3119 major histocompatibility complex, class II, DQ beta 1 HLA-DRB1 3123 major histocompatibility complex, class II, DR beta 1 HLA-DRB5 3127 major histocompatibility complex, class II, DR beta 5 HNRNPA2B1 3181 heterogeneous nuclear ribonucleoprotein A2/B1 HNRNPD 3184 heterogeneous nuclear ribonucleoprotein D HNRNPU 3192 heterogeneous nuclear ribonucleoprotein U HRAS 3265 HRas proto-oncogene, GTPase ICK 22858 intestinal cell kinase ID2 3398 inhibitor of DNA binding 2, HLH protein ID3 3399 inhibitor of DNA binding 3, HLH protein IDH1 3417 isocitrate dehydrogenase (NADP(+)) 1, cytosolic IDH2 3418 isocitrate dehydrogenase (NADP(+)) 2, mitochondrial IGF1R 3480 insulin like growth factor 1 receptor

IGLL5 100423062

immunoglobulin lambda like polypeptide 5

IKBKB 3551 inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta

27

IKBKE 9641 inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon

IKZF1 10320 IKAROS family zinc finger 1 IKZF2 22807 IKAROS family zinc finger 2 IKZF3 22806 IKAROS family zinc finger 3 IL10 3586 interleukin 10 IL16 3603 interleukin 16 IL22RA1 58985 interleukin 22 receptor subunit alpha 1 IL2RB 3560 interleukin 2 receptor subunit beta IMMP2L 83943 inner mitochondrial membrane peptidase subunit 2 IRAK1 3654 interleukin 1 receptor associated kinase 1 IRF2BP2 359948 interferon regulatory factor 2 binding protein 2 IRF4 3662 interferon regulatory factor 4 IRF8 3394 interferon regulatory factor 8 ITGB1 3688 integrin subunit beta 1 ITPKB 3707 inositol-trisphosphate 3-kinase B JAK1 3716 Janus kinase 1 JAK2 3717 Janus kinase 2 JAK3 3718 Janus kinase 3 JUNB 3726 JunB proto-oncogene, AP-1 transcription factor subunit KCNA3 3738 potassium voltage-gated channel subfamily A member 3 KDM1A 23028 lysine demethylase 1A KDM2B 84678 lysine demethylase 2B KDM4C 23081 lysine demethylase 4C KDM6A 7403 lysine demethylase 6A KDR 3791 kinase insert domain receptor KIT 3815 KIT proto-oncogene, receptor tyrosine kinase KLF2 10365 Kruppel like factor 2 KLHL14 57565 kelch like family member 14 KLHL6 89857 kelch like family member 6 KMT2C 58508 lysine methyltransferase 2C KMT2D 8085 lysine methyltransferase 2D KRAS 3845 KRAS proto-oncogene, GTPase LEF1 51176 lymphoid enhancer binding factor 1 LRCH1 23143 leucine rich repeats and calponin homology domain containing 1 LRP1B 53353 LDL receptor related protein 1B LTB 4050 lymphotoxin beta LTBP2 4053 latent transforming growth factor beta binding protein 2 LTBP3 4054 latent transforming growth factor beta binding protein 3 LYN 4067 LYN proto-oncogene, Src family tyrosine kinase LYST 1130 lysosomal trafficking regulator MAGT1 84061 magnesium transporter 1

28

MALT1 10892 MALT1 paracaspase MAP2K1 5604 mitogen-activated protein kinase kinase 1 MAP2K2 5605 mitogen-activated protein kinase kinase 2 MAP3K14 9020 mitogen-activated protein kinase kinase kinase 14 MAP3K6 9064 mitogen-activated protein kinase kinase kinase 6 MAP3K7 6885 mitogen-activated protein kinase kinase kinase 7 MCL1 4170 BCL2 family apoptosis regulator MDM2 4193 MDM2 proto-oncogene MDM4 4194 MDM4, p53 regulator MED12 9968 mediator complex subunit 12

MEF2B 100271849

myocyte enhancer factor 2B

MEF2C 4208 myocyte enhancer factor 2C MET 4233 MET proto-oncogene, receptor tyrosine kinase MFHAS1 9258 malignant fibrous histiocytoma amplified sequence 1 MGA 23269 MGA, MAX dimerization protein MGAM 8972 maltase-glucoamylase MKI67 4288 marker of proliferation Ki-67 MPEG1 219972 macrophage expressed 1 MSH3 4437 mutS homolog 3 MSH6 2956 mutS homolog 6 MSN 4478 moesin MTF2 22823 metal response element binding transcription factor 2 MTOR 2475 mechanistic target of rapamycin MYC 4609 v-myc avian myelocytomatosis viral oncogene homolog MYD88 4615 myeloid differentiation primary response 88 MYL1 4632 myosin light chain 1 MYOM2 9172 myomesin 2 NCOR1 9611 nuclear receptor corepressor 1 NCOR2 9612 nuclear receptor corepressor 2 NF1 4763 neurofibromin 1 NF2 4771 neurofibromin 2 NFATC1 4772 nuclear factor of activated T-cells 1 NFKB1 4790 nuclear factor kappa B subunit 1 NFKB2 4791 nuclear factor kappa B subunit 2 NFKBIA 4792 NFKB inhibitor alpha NFKBIE 4794 NFKB inhibitor epsilon NFKBIZ 64332 NFKB inhibitor zeta NOD1 10392 nucleotide binding oligomerization domain containing 1 NOL9 79707 nucleolar protein 9 NOTCH1 4851 notch receptor 1 NOTCH2 4853 notch receptor 2

29

NOTCH3 4854 notch receptor 3 NPM1 4869 nucleophosmin NRAS 4893 neuroblastoma RAS viral oncogene homolog NRP1 8829 neuropilin 1 NRP2 8828 neuropilin 2 NRXN3 9369 neurexin 3 NTRK1 4914 neurotrophic receptor tyrosine kinase 1 NTRK2 4915 neurotrophic receptor tyrosine kinase 2 NTRK3 4916 neurotrophic receptor tyrosine kinase 3 OSBPL10 114884 oxysterol binding protein like 10 P2RY8 286530 purinergic receptor P2Y8 PARD3 56288 par-3 family cell polarity regulato PASK 23178 PAS domain containing serine/threonine kinase PAX5 5079 paired box 5 PC 5091 pyruvate carboxylase PCBP1 5093 poly(rC) binding protein 1 PCLO 27445 piccolo presynaptic cytomatrix protein PDCD1 5133 programmed cell death 1 PDE7B 27115 phosphodiesterase 7B PDGFA 5154 platelet derived growth factor subunit A PDGFC 56034 platelet derived growth factor C PDGFRA 5156 platelet derived growth factor receptor alpha PDGFRB 5159 platelet derived growth factor receptor beta PDGFRL 5157 platelet derived growth factor receptor like PIK3C3 5289 phosphatidylinositol 3-kinase catalytic subunit type 3 PIK3CA 5290 phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha PIK3CB 5291 phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta PIK3CD 5293 phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta

PIK3CG 5294 phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma

PIK3R1 5295 phosphoinositide-3-kinase regulatory subunit 1 PIM1 5292 Pim-1 proto-oncogene, serine/threonine kinase PIM2 11040 Pim-2 proto-oncogene, serine/threonine kinase PLCE1 51196 phospholipase C epsilon 1 PLCG1 5335 phospholipase C gamma 1 PLCG2 5336 phospholipase C gamma 2 PMS2 5395 PMS1 homolog 2, mismatch repair system component POT1 25913 protection of telomeres 1 POU2F2 5452 POU class 2 homeobox 2 PRDM1 639 PR/SET domain 1 PRF1 5551 perforin 1 PRKCB 5579 protein kinase C beta

30

PRKCD 5580 protein kinase C delta PRKCQ 5588 protein kinase C theta PRKD1 5587 protein kinase D1 PTCH1 5727 patched 1 PTEN 5728 phosphatase and tensin homolog PTPN1 5770 protein tyrosine phosphatase, non-receptor type 1 PTPN11 5781 protein tyrosine phosphatase, non-receptor type 11 PTPN6 5777 protein tyrosine phosphatase, non-receptor type 6 PTPRB 5787 protein tyrosine phosphatase receptor type B PTPRC 5788 protein tyrosine phosphatase, receptor type C PTPRD 5789 protein tyrosine phosphatase, receptor type D PTPRK 5796 protein tyrosine phosphatase, receptor type K RAC2 5880 Rac family small GTPase 2 RAF1 5894 Raf-1 proto-oncogene, serine/threonine kinase RASAL2 9462 RAS protein activator like 2 RASGEF1A 221002 RasGEF domain family member 1A RB1 5925 RB transcriptional corepressor 1 REL 5966 REL proto-oncogene, NF-kB subunit RELA 5970 RELA proto-oncogene, NF-kB subunit RELB 5971 RELB proto-oncogene, NF-kB subunit RELN 5649 reelin RET 5979 ret proto-oncogene RFX7 64864 regulatory factor X7 RHOA 387 ras homolog family member A RHOH 399 ras homolog family member H ROBO1 6091 roundabout guidance receptor 1 ROBO2 6092 roundabout guidance receptor 2 ROS1 6098 ROS proto-oncogene 1, receptor tyrosine kinase RPS6KA1 6195 ribosomal protein S6 kinase A1 RRAGC 64121 Ras related GTP binding C S1PR1 1901 sphingosine-1-phosphate receptor 1 S1PR2 9294 sphingosine-1-phosphate receptor 2 SEMA3C 10512 semaphorin 3C SETD1B 23067 SET domain containing 1B SETD2 29072 SET domain containing 2 SF3B1 23451 splicing factor 3b subunit 1 SGK1 6446 serum/glucocorticoid regulated kinase 1 SH2D1A 4068 SH2 domain containing 1A SIN3A 25942 SIN3 transcription regulator family member A SKI 6497 SKI proto-oncogene

SMARCA1 6594 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 1

31

SMARCA4 6597 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4

SMARCB1 6598 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1

SMO 6608 smoothened, frizzled class receptor SOCS1 8651 suppressor of cytokine signaling 1 SOX5 6660 SRY-box 5 SPEN 23013 spen family transcriptional repressor SPIB 6689 Spi-B transcription factor SPTA1 6708 spectrin alpha, erythrocytic 1 SPTBN2 6712 spectrin beta, non-erythrocytic 2 STAB2 55576 stabilin 2 STAT3 6774 signal transducer and activator of transcription 3 STAT5B 6777 signal transducer and activator of transcription 5B STAT6 6778 signal transducer and activator of transcription 6 STK11 6794 serine/threonine kinase 11 STXBP2 6813 syntaxin binding protein 2 SYK 6850 spleen associated tyrosine kinase SYNCRIP 10492 synaptotagmin binding cytoplasmic RNA interacting protein TAF1 6872 TATA-box binding protein associated factor 1 TAP1 6890 transporter 1, ATP binding cassette subfamily B member TBL1XR1 79718 transducin beta like 1 X-linked receptor 1 TBX3 6926 T-box 3 TCF3 6929 transcription factor 3 TCF4 6925 transcription factor 4 TCL1A 8115 T-cell leukemia/lymphoma 1A TERT 7015 telomerase reverse transcriptase TET2 54790 tet methylcytosine dioxygenase 2 TFDP3 51270 transcription factor Dp family member 3 TGFB2 7042 transforming growth factor beta 2 TGFBI 7045 transforming growth factor beta induced TLL2 7093 tolloid like 2 TLR2 7097 toll like receptor 2 TMEM30A 55754 transmembrane protein 30A TMSB4X 7114 thymosin beta 4, X-linked TNFAIP3 7128 TNF alpha induced protein 3 TNFRSF11A 8792 TNF receptor superfamily member 11a TNFRSF13C 115650 TNF receptor superfamily member 13C TNFRSF14 8764 TNF receptor superfamily member 14 TNFRSF1B 7133 TNF receptor superfamily member 1B TOX 9760 thymocyte selection associated high mobility group box TP63 8626 tumor protein p63

32

TP73 7161 tumor protein p73 TRAF1 7185 TNF receptor associated factor 1 TRAF2 7186 TNF receptor associated factor 2 TRAF3 7187 TNF receptor associated factor 3 TRAF5 7188 TNF receptor associated factor 5 TSC1 7248 tuberous sclerosis 1 TSC2 7249 tuberous sclerosis 2 UBE2A 7319 ubiquitin conjugating enzyme E2 A UBR5 51366 ubiquitin protein ligase E3 component n-recognin 5 UNC5C 8633 unc-5 netrin receptor C UNC5D 137970 unc-5 netrin receptor D USH2A 7399 usherin USP7 7874 ubiquitin specific peptidase 7 VAV1 7409 vav guanine nucleotide exchange factor 1 VEGFA 7422 vascular endothelial growth factor A VEGFC 7424 vascular endothelial growth factor C VMP1 81671 vacuole membrane protein 1 WHSC1 7468 Wolf-Hirschhorn syndrome candidate 1 WIF1 11197 WNT inhibitory factor 1 WNT1 7471 Wnt family member 1 XBP1 7494 X-box binding protein 1 XPO1 7514 exportin 1 YTHDF2 51441 YTH N6-methyladenosine RNA binding protein 2 ZC3H12A 80149 zinc finger CCCH-type containing 12A ZEB1 6935 zinc finger E-box binding homeobox 1 ZFP36L1 677 ZFP36 ring finger protein like 1 ZFP36L2 678 ZFP36 ring finger protein like 2 ZNF296 162979 zinc finger protein 296 ZNF608 57507 zinc finger protein 608 ZNF638 27332 zinc finger protein 638

33

Supplemental T

able 4: Enriched pathw

ays in CD

4+ splenocytes as analyzed by G

SEA

with hallm

ark matrix

NA

ME

SIZE ES

NES

NO

M p-val

FDR

q-val FW

ER p-val

HA

LLMA

RK

_IL2_STAT5_SIG

NA

LING

186

0.56 2.14

0 0

0 H

ALLM

AR

K_O

XID

ATIV

E_PHO

SPHO

RY

LATIO

N

189 0.52

2.02 0

0 0

HA

LLMA

RK

_GLY

CO

LYSIS

164 0.48

1.84 0

0 0

HA

LLMA

RK

_AN

GIO

GEN

ESIS 27

0.62 1.76

0 0

0.01 H

ALLM

AR

K_TN

FA_SIG

NA

LING

_VIA

_NFK

B

183 0.47

1.81 0

0 0

HA

LLMA

RK

_HY

POX

IA

154 0.47

1.77 0

0 0.01

HA

LLMA

RK

_MY

C_TA

RG

ETS_V1

197 0.43

1.67 0

0.01 0.05

HA

LLMA

RK

_EPITHELIA

L_MESEN

CH

YM

AL_TR

AN

SITION

143

0.45 1.67

0 0.01

0.04 H

ALLM

AR

K_D

NA

_REPA

IR

131 0.42

1.56 0

0.02 0.15

HA

LLMA

RK

_AD

IPOG

ENESIS

179 0.39

1.5 0

0.03 0.27

HA

LLMA

RK

_CO

MPLEM

ENT

151 0.4

1.51 0.01

0.03 0.26

HA

LLMA

RK

_P53_PATH

WA

Y

180 0.39

1.49 0.01

0.03 0.31

HA

LLMA

RK

_INTER

FERO

N_A

LPHA

_RESPO

NSE

86 0.43

1.51 0.01

0.03 0.25

HA

LLMA

RK

_IL6_JAK

_STAT3_SIG

NA

LING

77

0.44 1.52

0.01 0.03

0.24 H

ALLM

AR

K_IN

TERFER

ON

_GA

MM

A_R

ESPON

SE 177

0.39 1.52

0 0.03

0.23 H

ALLM

AR

K_K

RA

S_SIGN

ALIN

G_U

P 149

0.37 1.42

0.01 0.05

0.51 H

ALLM

AR

K_PR

OTEIN

_SECR

ETION

88

0.4 1.4

0.02 0.06

0.57 H

ALLM

AR

K_IN

FLAM

MA

TOR

Y_R

ESPON

SE 167

0.37 1.41

0.01 0.06

0.56 H

ALLM

AR

K_A

POPTO

SIS 141

0.36 1.37

0.02 0.07

0.7 H

ALLM

AR

K_U

V_R

ESPON

SE_UP

130 0.36

1.34 0.03

0.09 0.8

HA

LLMA

RK

_MTO

RC

1_SIGN

ALIN

G

185 0.34

1.33 0.03

0.1 0.82

HA

LLMA

RK

_ALLO

GR

AFT_R

EJECTIO

N

166 0.34

1.3 0.04

0.11 0.89

HA

LLMA

RK

_E2F_TAR

GETS

193 0.33

1.3 0.03

0.11 0.9

HA

LLMA

RK

_FATTY

_AC

ID_M

ETAB

OLISM

126

0.35 1.29

0.05 0.12

0.91

34

Supplemental T

able 5: Enriched pathw

ays in CD

4+ splenocytes as analyzed by G

SEA

with K

yoto Encyclopedia of G

enes and Genom

es (K

EG

G) m

atrix

NA

ME

SIZ

E

ES

NE

S N

OM

p-val F

DR

q-val F

WE

R p-val

KEG

G_PA

RK

INSO

NS_D

ISEASE

97 0.63

2.23 0

0 0

KEG

G_A

LZHEIM

ERS_D

ISEASE

139 0.57

2.15 0

0 0

KEG

G_O

XID

ATIV

E_PHO

SPHO

RY

LATIO

N

99 0.61

2.14 0

0 0

KEG

G_PR

OTEA

SOM

E 42

0.63 1.98

0 0

0 K

EGG

_HU

NTIN

GTO

NS_D

ISEASE

148 0.54

2.01 0

0 0

KEG

G_C

YTO

KIN

E_CY

TOK

INE_R

ECEPTO

R_IN

TERA

CTIO

N

171 0.48

1.85 0

0.01 0.03

KEG

G_PR

ION

_DISEA

SES 26

0.65 1.83

0 0.01

0.05 K

EGG

_CY

TOSO

LIC_D

NA

_SENSIN

G_PA

THW

AY

39

0.59 1.81

0 0.01

0.06 K

EGG

_CA

RD

IAC

_MU

SCLE_C

ON

TRA

CTIO

N

50 0.55

1.75 0

0.02 0.14

KEG

G_PR

OTEIN

_EXPO

RT

22 0.63

1.68 0.01

0.03 0.29

KEG

G_PEN

TOSE_PH

OSPH

ATE_PA

THW

AY

19

0.63 1.62

0.01 0.06

0.52 K

EGG

_GLY

CO

LYSIS_G

LUC

ON

EOG

ENESIS

38 0.53

1.62 0

0.07 0.51

KEG

G_TY

PE_I_DIA

BETES_M

ELLITUS

18 0.62

1.57 0.04

0.1 0.7

KEG

G_FR

UC

TOSE_A

ND

_MA

NN

OSE_M

ETAB

OLISM

29

0.55 1.56

0.02 0.1

0.75 K

EGG

_RETIN

OL_M

ETAB

OLISM

16

0.62 1.52

0.05 0.13

0.86 K

EGG

_ECM

_REC

EPTOR

_INTER

AC

TION

63

0.46 1.52

0.01 0.13

0.85 K

EGG

_SPLICEO

SOM

E 119

0.41 1.5

0 0.15

0.9 K

EGG

_GA

LAC

TOSE_M

ETAB

OLISM

22

0.55 1.46

0.05 0.19

0.96 K

EGG

_SNA

RE_IN

TERA

CTIO

NS_IN

_VESIC

ULA

R_TR

AN

SPOR

T 31

0.49 1.45

0.05 0.21

0.98 K

EGG

_LON

G_TER

M_D

EPRESSIO

N

47 0.45

1.44 0.04

0.21 0.98

35

Supplemental Table 6: Mortality frequency of dasatinib- and vehicle-treated mice at

day 30 after treatment.

Supplemental Table 7: Lists of tumor samples

Patient Sample type Sample status Time of collection

PAT1 lymph node FFPE diagnosis

PAT2 lymph node FFPE diagnosis

PAT3 bone marrow MNCs fresh relapse*

PAT4 peripheral blood

MNCs fresh relapse

PAT5 lymph node frozen relapse

FFPE, formalin-fixed paraffin embedded; MNCs, mononuclear cells.

* The sample was obtained at the relapse of dasatinib treatment.

Alive (n, %) Dead (n, %) Total (n, %)

Dasatinib 12 (66.67%) 6 (33.33%) 18 (100%)

Vehicle 4 (22.22%) 14 (77.78%) 18 (100%)

36

Supplemental Table 8: List of m

utations identified in AITL patients

Sample ID

A

nnotated G

enes R

efSeq Exonic Function

Nucleotide

Change

Am

ino Acid

Change

VA

F D

epth

PAT1

TET2 N

M_001127208

frameshift insertion

c.3187dupA

p.V1062fs

0.25 523

PAT1

TET2 N

M_001127208

nonsilent SNV

c.G

3619A

p.E1207K

0.28 374

PAT1

KM

T2C N

M_170606

nonsilent SNV

c.C2633T

p.A878V

0.04

579

PAT1

EPPK1

NM

_031308 nonsilent SN

V

c.C6773T p.T2258M

0.08

2792

PAT1

GN

AQ

N

M_002072

nonsilent SNV

c.A

175C p.M

59L 0.05

504

PAT2

JAK

1 N

M_002227

nonsilent SNV

c.G

3290A

p.G1097D

0.15

400

PAT2

TET2 N

M_001127208

frameshift insertion

c.3015dupG

p.K1005fs

0.03 480

PAT2

TET2 N

M_001127208

nonsense c.G

5440T p.G

1814X

0.14 432

PAT2

SEMA

3C N

M_006379

nonsilent SNV

c.C2069T

p.P690L 0.06

422

PAT2

KM

T2C N

M_170606

nonsilent SNV

c.C2410G

p.L804V

0.09

1225

PAT2

EPPK1

NM


V

c.G7069A

p.V

2357M

0.06 5059

PAT2

ARID

2 N

M_152641

nonsilent SNV

c.G

1768A

p.G590R

0.07 440

PAT2

FOX

O1

NM

_002015 nonfram

eshift

insertion c.299_300insCG

C p.A

100delinsAA

0.28

61

PAT2

VA

V1

NM


V

c.G1916A

p.G

639D

0.04 390

PAT2

VA

V1

NM

_001258206 nonfram

eshift

deletion c.1918_1923del

p.640_641del 0.03

647

37

PAT2

VA

V1

NM


V

c.A1925T

p.Q642L

0.04 384

PAT2

VA

V1

NM


V

c.C1934G

p.S645C 0.04

380

PAT2

PLCG1

NM


V

c.G2120A

p.R707Q

0.07

376

PAT2

EP300 N

M_001429

nonsilent SNV

c.G

3227C p.R1076P

0.09 561

PAT2

DD

X3X

N

M_001193417

nonsilent SNV

c.A

1487C p.H

496P 0.18

219

PAT3

ATX

N1

NM

_001128164 nonfram

eshift

deletion c.627_644del

p.209_215del 0.20

86

PAT3

FBXO

31 N

M_024735

nonsilent SNV

c.A

1217G

p.Q406R

0.03 219

PAT3

RHO

A

NM


V

c.G50T

p.G17V

0.01

#

PAT4

RHO

A

NM


V

c.G50T

p.G17V

0.06

492

PAT4

TET2 N

M_001127208

nonsense c.C1061G

p.S354X

0.18

709

PAT4

TET2 N

M_001127208

frameshift deletion

c.2498delC p.S833fs

0.10 704

PAT4

FAT1

NM


V

c.C4409T p.A

1470V

0.04 453

PAT4

ATX

N1

NM


V

c.G651T

p.Q217H

0.14

74

PAT4

HIST1H

2BC N

M_003526

nonsilent SNV

c.A

208G

p.I70V

0.06 797

PAT4

ATM

N

M_000051

nonsilent SNV

c.A

3854G

p.D1285G

0.04

472

PAT5

TET2 N

M_001127208

frameshift deletion

c.2873delA

p.Q958fs

0.10 1112

PAT5

TET2 N

M_001127208

frameshift deletion

c.3533delA

p.E1178fs 0.09

412

PAT5

VA

V1

NM

_001258206 tandem

_duplication

0.05

212

RefSeq, Reference Sequence; VA

F, variant allele frequency; #, defined by digital droplet PCR.

38

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dasatinib is an effective treatment for angioimmunoblastic ......1 supplemental information...

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