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Dasatinib Is an Effective Treatment for Angioimmunoblastic T-Cell

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Dasatinib Is an Effective Treatment for Angioimmunoblastic T-Cell 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 1 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. 2 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 3 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 4 (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, 5 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.
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