Recurrent RARB Translocations in Acute

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Published OnlineFirst June 19, 2018; DOI: 10.1158/0008-5472.CAN-18-0840

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Recurrent RARB Translocations in Acute Promyelocytic Leukemia Lacking RARA Translocation Tomoo Osumi1,2, Shin-ichi Tsujimoto2,3, Moe Tamura4, Meri Uchiyama1, Kazuhiko Nakabayashi5, Kohji Okamura6, Masanori Yoshida1,3, Daisuke Tomizawa2, Akihiro Watanabe7, Hiroyuki Takahashi8, Tsukasa Hori9, Shohei Yamamoto10, Kazuko Hamamoto11, Masahiro Migita12, Hiroko Ogata-Kawata5, Toru Uchiyama13, Hiroe Kizawa14, Hitomi Ueno-Yokohata1, Ryota Shirai1,3, Masafumi Seki15, Kentaro Ohki1, Junko Takita15, Takeshi Inukai16, Seishi Ogawa17, Toshio Kitamura4, Kimikazu Matsumoto2, Kenichiro Hata5, Nobutaka Kiyokawa1, Susumu Goyama4, and Motohiro Kato1,2

Abstract

Translocations of retinoic acid receptor-a (RARA), typically and inhibited myeloid maturation of human cord blood cells PML–RARA, are a genetic hallmark of acute promyelocytic as PML–RARA did. However, the response of APL with RARB leukemia (APL). However, because a small fraction of APL lack translocation to retinoids was attenuated compared with that translocations of RARA, we focused here on APL cases without of PML–RARA, an observation in line with the clinical resis- RARA translocation to elucidate the molecular etiology of tance of RARB-positive APL to ATRA. Our results demonstrate RARA-negative APL. We performed whole-genome sequenc- that the majority of RARA-negative APL have RARB transloca- ing, PCR, and FISH for five APL cases without RARA translocations, thereby forming a novel, distinct subgroup of APL. tions. Four of five RARA-negative APL cases had translocations TBL1XR1–RARB as an oncogenic protein exerts effects similar involving retinoic acid receptor-b (RARB) translocations, and to those of PML–RARA, underpinning the importance of TBL1XR1–RARB was identified as an in-frame fusion in three retinoic acid pathway alterations in the pathogenesis of APL. cases; one case had an RARB rearrangement detected by FISH, although the partner gene could not be identified. When Significance: These findings report a novel and distinct transduced in cell lines, TBL1XR1–RARB homodimerized and genetic subtype of acute promyelocytic leukemia (APL) by diminished transcriptional activity for the retinoic acid recep- illustrating that the majority of APL without RARA trans- tor pathway in a dominant-negative manner. TBL1XR1–RARB locations harbor RARB translocations. Cancer Res; 78(16); enhanced the replating capacity of mouse bone marrow cells 4452–8. 2018 AACR.

Introduction location, which induces a fusion of promyelocytic leukemia Acute promyelocytic leukemia (APL) accounts for 5% to 10% (PML) and retinoic acid receptor-a (RARA;ref.2).PML–RARA of acute myeloid leukemia (AML; ref. 1), which is characterized is a potent oncoprotein that deregulates transcription and by unique morphological features typically including abnormal transforms cells ex vivo and in vivo (3). APL cells with PML– promyelocytes containing azurophilic granules. APL was once RARA respond well to all-trans retinoic acid (ATRA; ref. 4), considered as the most intractable form of AML because of life- providing one of the most successful examples of targeted threatening coagulopathy and high relapse rate. The genomic therapy for cancer. Furthermore, arsenic trioxide (ATO) also basis of APL is characterized by the t(15;17)(q22;q21) trans- exerts extraordinarily potent anti-APL effects (4), and

1Department of Pediatric Hematology and Oncology Research, National and Development, Tokyo, Japan. 14Department of Clinical Laboratory Medicine, Research Institute for Child Health and Development, Tokyo, Japan. 2Children's National Center for Child Health and Development, Tokyo, Japan. 15Department Cancer Center, National Center for Child Health and Development, Tokyo, Japan. of Pediatrics, The University of Tokyo, Tokyo, Japan. 16Department of Pediatrics, 3Department of Pediatrics, Yokohama City University, Yokohama, Japan. 4Divi- University of Yamanashi, Chuo, Japan. 17Department of Pathology and Tumor sion of Cellular Therapy, the Institute of Medical Science, The University of Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan. Tokyo, Tokyo, Japan. 5Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Tokyo, Japan. 6Department of Note: Supplementary data for this article are available at Cancer Research Systems BioMedicine, National Research Institute for Child Health and Devel- Online (http://cancerres.aacrjournals.org/). 7 opment, Tokyo, Japan. Department of Pediatrics, Niigata Cancer Center Hos- T. Osumi and S.-i. Tsujimoto contributed equally to this study. pital, Niigata, Japan. 8Department of Pediatrics, Toho University, Tokyo, Japan. 9Department of Pediatrics, Sapporo Medical University School of Medicine, Corresponding Author: Motohiro Kato, National Center for Child Health and Sapporo, Japan. 10Department of Pediatrics, Showa University Fujigaoka Hos- Development, Tokyo 1578535, Japan. Phone: 81-03-3416-0181; E-mail: pital, Yokohama, Japan. 11Department of Pediatrics, Hiroshima Red Cross Hos- [email protected] pital & Atomic-Bomb Survivors Hospital, Hiroshima, Japan. 12Department of doi: 10.1158/0008-5472.CAN-18-0840 Pediatrics, Japanese Red Cross Kumamoto Hospital, Kumamoto, Japan. 13Department of Human Genetics, National Research Institute for Child Health 2018 American Association for Cancer Research.

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reportedly cures up to 70% of patients when used alone (5). Patients and Methods Recent clinical trials demonstrated that a combination of ATRA Patients and ATO achieved excellent outcomes, with more than 90% A 2-year-old boy presented with leukocytosis and coagulopathy event-free survival for typical APL with PML–RARA,evenwith- (unique patient identifier, UPN1). Bone marrow aspiration out conventional cytotoxic chemotherapy (4). revealed the presence of blasts with azurophilic granules, and Indeed, translocated RARA genes are a hallmark of APL. APL was morphologically diagnosed (Fig. 1A), but treatment with Although a minority of APL cases (<5%) lacks canonical PML– ATRA was ineffective. PCR for PML–RARA was negative, and RARA (6), most had a translocation between RARA and other break-apart FISH analysis for RARA did not show a split signal partners including ZBTB16 (PLZF; ref. 6), NPM1 (7), NUMA1 (8), (Fig. 1B). STAT5B (9), FNDC3B (10), PRAR1A (11), BCOR (12), FIP1L1 Furthermore, we collected four additional cases morpholog- (13), NABP1 (14), GTF2I (15), IRF2BP2 (16), and TBL1XR1 (17). ically diagnosed as APL lacking a RARA translocation. As Almost all known, APL-associated translocations involve RARA, shown in Supplementary Fig. S1, all cases had proliferation which accounts for 99% of APL (6). A study showed whole of promyelocytes with azurophilic granules, which could not genome sequencing could detect a cryptic PML–RARA, which be distinguished from APL with PML–RARA,althoughfew were missed by conventional cytogenetics or FISH (18); however, faggot cells were observed in all RARA-negative APL cases. genomic analyses focusing on RARA translocation are incapable Most of the cases had a similar pattern of surface markers of detecting RARA rearrangement in the remainder of APL cases with typical APL, such as positivity for CD13 and CD33, and (6). A recent study with whole exome sequencing revealed negativity for CD34 and HLA-DR. As in the first case, all the detailed genomic landscape of APL with PML–RARA (19), but additional cases were refractory to ATRA; hence AML-based few studies have been performed to identify novel fusions in multiagent chemotherapy was given. However, in total, four of RARA-negative APL due to the rarity. Previous case reports iden- five cases suffered a relapse or failed to achieve remission tified single cases with RARG translocations in APL (20, 21); (Supplementary Fig. S2). The details of the characteristics and however, the molecular mechanisms underlying RARA-negative clinical course of the cases are shown in Supplementary Table APL await clarification. S1. The clinical course of one case (UPN5) has previously been We herein focused on APL cases without RARA transloca- reported elsewhere (22). tion, performed a high-throughput sequencing analysis, identified a novel recurrent fusion of retinoic acid receptor-b (RARB) in the majority of patients with RARA-negative APL, Genomic analysis and elucidated the features of this novel fusion in the path- For whole genome sequencing, DNA sample from leukemic ogenesis of APL. cells was sonicated to produce random short fragments. Library

Figure 1. Morphologic and cytogenetic features of the APL case with TBL1XR1–RARB translocation (UPN1) and the model of the fusion gene. A, Morphologic features of RARB-positive APL blasts in the bone marrow (May-Giemsa staining). Hypergranular promyelocytic blast cells with irregular nuclear membranes and prominent, heavily stained granules lacking Auer rods. B, Break-apart FISH analysis for RARB. Arrows, split signal for one allele; arrowhead, fusion signal of the wild- type allele. C and D, Reverse transcription PCR (C) and Sanger sequencing (D)conﬁrming the in-frame TBL1XR1–RARB fusion.

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construction was performed with NEBNext Ultra II DNA Library required written informed consent was obtained from the patients Prep Kit according to the manufacturer's protocol. All the libraries or guardians. This study was conducted in according to the were sequenced using the Illumina HiSeq 2500. In the use of Declaration of Helsinki. Integrative Genomics Viewer 2.3.66, coloring by chromosome on which their mates were mapped can visualize the candidate Results mutations. Custom Perl and C programs, which were used in this analysis, are available upon request. Genomic analysis of RARA-negative APL fi For whole exome sequencing, library construction was per- We rst performed whole-genome sequencing to identify the formed with SureSelect Human All Exon Kit V5 or V6, Agilent potential genomic alterations in the initial case (UPN1). Intrigu- fi Technology, according to the manufacturer's protocol. Enriched ingly, we successfully identi ed an in-frame fusion between TBL1XR1 RARB fi fragment libraries were then sequenced on an Illumina HiSeq and , which was con rmed by FISH and PCR RARB 2500 in 101-bp paired-end mode. Image analyses and base calling (Fig. 1C and D; Supplementary Tables S2 and S3). encodes RARB RARA were performed using CASAVA 1.8.4 with default parameters. To , a paralog of , both of which are members of the RARB validate the mutations detected by whole-exome sequencing, nuclear receptor superfamily. We searched further for RARA Sanger sequencing of each genes was performed. alterations in the four additional cases of -negative APL, using whole-genome sequencing, RT-PCR and FISH according to Luciferase assay sample availability (Supplementary Table S1). Strikingly, two Transcriptional activity of TBL1XR1–RARB was analyzed by additional cases had the TBL1XR1–RARB fusion with a break luciferase assay. Retinoic acid-responsive element (RARE) signal point identical to that of UPN1, whereas one case had a RARB reporter (Qiagen) including transcription factor responsive con- rearrangement detected by FISH although the partner gene struct and constitutively expressing Renilla luciferase construct, could not be identified. No structural variant in RARA, RARB, and respective EX-B0004-M07 expression vectors (Mock, RARB, and RARG was observed in UPN5 by whole-genome sequencing. RARA, TBL1XR1–RARB, PML–RARA) were transiently cotrans- In total, four of five (80%) RARA-negative APL cases had RARB fected into HEK293 cells. Different concentration of ATRA were translocations. added after 24 hours and incubated 24 hours. Previous reports showed that PML–RARA alone was insufficient to develop leukemia, requiring additional molecular events such Coimmunoprecipotation and immunoblotting analysis as internal tandem duplication (ITD) of FLT3 (19). We thus For coimmunoprecipitation, expression vectors with MYC- or performed whole-exome sequencing for UPN1 and UPN2, for HA-tag proteins were transiently transfected into HEK293 cells. which diagnostic and remission samples were available, to inves- After 24 hours, ATRA, tamibarotene, and ATO were added and tigate additional somatic mutations other than the RARB trans- incubated. For immunoblotting analysis, extracted proteins from location. Although we failed to find recurrent mutations in our the cell lines were separated in polyacrylamide gel electrophoresis cohort (Supplementary Table S4), it should be noted that one case gels and transferred to nitrocellulose membrane. The proteins had a somatic mutation of TBL1XR1 in a remaining allele that had were analyzed by immunoblotting using each antibody. not fused to RARB (Supplementary Fig. S3). Flow cytometry and morphologic analysis U937, which is a cell line widely used for the biological studies Effects of TBL1XR1–RARB on retinoic acid pathway and cell including the preceding study for TBL1XR1-RARA, were infected differentiation in vitro with prepared retrovirus (Mock, RARB, TBL1XR1–RARB, PML– The recurrent TBL1XR1–RARB fusion had a construct similar to RARA). ATRA-treated or nontreated selected U937 cell lines were that of TBL1XR1-RARA (17) and PML–RARA (Supplementary incubated with FITC-antihuman CD11b antibody (BioLegend) Fig. S4). To investigate the functional effect of this novel fusion and examined by flow cytometer. For morphologic analysis, on the etiology of APL, we transduced the HEK293 cell lines with cytospin slides of 0 and 5 days after ATRA-treated U937 cells TBL1XR1–RARB, PML–RARA, RARB, and RARA. The luciferase were stained with Wright-Giemsa staining solution. assay demonstrated that the transcriptional activity of TBL1XR– RARB for a RARE was reduced. The fusion also suppressed trans- Human cord blood cell culture activation of the reporter induce by RARA or RARB in response to Human umbilical cord blood (CB) cells were obtained from ATRA (Fig. 2A and B), suggesting a dominant negative effect Riken BRC or the Japanese Red Cross Kanto-Koshinetsu Cord against both RARA and RARB. þ Blood Bank (Tokyo, Japan). CD34 cells were separated and those All reported partners of RARA fusion in APL shared the ability to transduced with vector, TBL1XR1–RARB, PML–RARA,orRUNX1- form homodimers, and the lissencephaly type-1-like homology RUNX1T1 were cultured. motif (LisH) domain of TBL1XR1 was capable of homodimerization. We therefore assessed the dimerization ability of the Colony replating assay TBL1XR1–RARB fusion protein. As with PML–RARA and þ Mousebonemarrowprogenitors(c-Kit cells) were separat- TBL1XR1-RARA (17), MYC-tagged TBL1XR1–RARB was able to þ ed. The c-Kit cells transduced with vector, TBL1XR1–RARB, coimmunoprecipitate with the HA-tagged fusion protein whereas PML–RARA,orRUNX1-RUNX1T1 were plated in M3234 meth- wild-type RARB was not (Fig. 2C). The homodimerization of ylcellulose. Colony counting and replating were performed TBL1XR1–RARB was weakened by the therapeutic dose of ATRA every 5 days. but the effect remained partial. The impact of tamibarotene, a synthetic retinoic acid with enhanced biological functions, was Institutional Review Board approval also insufficient, even though both retinoids had a potent effect This study was approved by the institutional ethics board of the on PML–RARA. ATO had also no effect on TBL1XR1–RARB National Center for Child Health and Development (#1035), and transduced cell lines.

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Figure 2. Effects of TBL1XR1–RARB on transcriptional activity, differentiation, and homodimerization. A and B, HEK293 cells were transiently transfected with a RARE reporter and each expression plasmid. The transfected cells were then incubated with different dosages of ATRA and luciferase activity was assayed. The transcriptional activity of TBL1XR1–RARB was signiﬁcantly lower than that of wild-type RARB or RARA. TBL1XR1–RARB dominant-negatively inhibited the transcriptional activity of wild-type RARB and wild-type RARA. C, HEK293 cells were transiently transfected with MYC- and HA-tagged expression plasmids. Coimmunoprecipitation demonstrated that both TBL1XR1–RARB and PML–RARA were able to homodimerize, whereas wild-type RARB was not. The homodimerization of PML–RARA was canceled in the presence of ATRA, tamibarotene, and ATO. In contrast, for TBL1XR1–RARB, the effect of ATRA was partial, and no effect of tamibarotene or ATO was observed. D, U937 cells were infected with a retrovirus that expressed control retrovirus, wild-type RARB, TBL1XR1–RARB,andPML–RARA. These cells were incubated, and the percentage of CD11b-positive cells was measured.

TBL1XR1–RARB also failed to differentiate the U937 cell line passage, whereas TBL1XR1–RARB-, PML–RARA-, and RUNX1- whereas wild-type RARB did with increased expression of CD11b RUNX1T1-transduced cells acquired extensive serial replating (Fig. 2D). ATRA was able to differentiate TBL1XR1–RARB-trans- capacity. TBL1XR1–RARB-expressing colonies contained many þ duced U937 although the effect was much smaller than the Gr-1 c-Kitlow/ myeloblasts and resembled PML–RARA-expres- PML–RARA-transduced effect. The blockage of differentiation by sing colonies but were immunophenotypically distinct from TBL1XR1–RARB and the partial neutrophil differentiation in RUNX1-RUNX1T1-expressing colonies (Fig. 3C; Supplementary response to ATRA was conﬁrmed by morphologic changes Fig. S6B). þ (Supplementary Fig. S5). We next transduced these fusions into human CB CD34 cells and cultured them with cytokines designed to induce myeloid Effects of TBL1XR1–RARB on cell differentiation and differentiation (Supplementary Fig. S7A). TBL1XR1–RARB and proliferation ex vivo PML–RARA both showed increased frequency of myeloblasts and þ To assess the leukemogenic activity of TBL1XR1–RARB ex vivo, inhibited myeloid differentiation towards CD66 granulocytes þ we ﬁrst performed a colony replating assay using mouse bone and CD14 monocytes in the culture (Fig. 4A and B). Unlike marrow (BM) cells and the results were compared with those RUNX1-RUNX1T1, TBL1XR1–RARB did not increase the frequen- þ obtained for PML–RARA and RUNX1-RUNX1T1 (ETO), a t(8;21)- cy of primitive CD34 cells under these culture conditions (Sup- derived fusion protein shown to promote self-renewal of hemato- plementary Fig. S7B). Thus, TBL1XR1–RARB enhances the replat- poietic progenitor cells (Fig. 3A and B; Supplementary Fig. S6A). ing capacity of mouse BM cells and inhibits myeloid maturation Vector-transduced cells did not produce colonies beyond the third of human CB cells as PML–RARA does.

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Figure 3. Effects of TBL1XR1–RARB on differentiation and proliferation in mouse bone marrow. A, Colony numbers of vector, PML–RARA, TBL1XR1–RARB,orRUNX1-RUNX1T1- transduced mouse bone marrow c-Kitþ cells. Shown are weekly colony counts per 5 104 replated cells. B and C, Wright-Giemsa staining (B) and Gr-1 and c-Kit expression (C) in cells at the third round. All vector- transduced cells were c-Kitþ mastocytes, whereas oncogene-transduced colonies contained myeloblasts. The percentages of Gr-1þ and c-Kitþ cells are indicated.

When PML–RARA- and TBL1XR1–RARB-transduced CB cells ity for the retinoic acid pathway with a dominant negative effect were cultured in the presence of ATRA, ATRA inhibited the growth on both RARA and RARB, blocked cell differentiation, and of TBL1XR1–RARB- and PML–RARA-expressing CB cells as evi- induced proliferation. Our findings underscored the fact that þ denced by the decrease of GFP cells in the culture (Fig. 4C). A alterations in the RAR pathway play a central role in APL path- therapeutic dose of ATRA induced myeloid differentiation of ogenesis, as repeatedly observed for PML–RARA-positive APL. PML–RARA-expressing CB cells to the same extent as they did in The effect of therapeutic retinoids on TBL1XR1–RARB was vector-transduced CB cells. In contrast, ATRA did not fully induce significantly smaller than that observed in PML–RARA, which differentiation of TBL1XR1–RARB-expressing CB cells, indicating agreed with the finding of clinical resistance to ATRA in RARB- their attenuated response to ATRA (Fig. 4D; Supplementary Fig. positive cases. Recent studies of the combination of ATRA and S7B). Surprisingly, tamibarotene showed only modest effects on ATO reported excellent outcomes for PML–RARA positive APL, inhibiting the growth and promoting the differentiation of but the RARB-positive cases in our cohort showed resistance not TBL1XR1–RARB cells while showing potent effects on PML–RARA only to ATRA but also to multiagent chemotherapy. There is no cells (Fig. 4D; Supplementary Fig. S7C). case treated with ATO, but ATO had no effect on TBL1XR1–RARB The attenuated response of TBL1XR1–RARB cells to tamibar- transduced cell lines in vitro because the ATO targets the PML otene was also confirmed in the murine colony-forming cell assay moiety of PML–RARA by preventing multimerization and induc- (Supplementary Fig. S8). ing degradation. Of note, two of the five RARA-negative APL cases had an extramedullary relapse, an extremely rare event in classical APL Discussion (1). Furthermore, our results suggested that tamibarotene, which We found that the majority of RARA-negative APL had a RARB is effective against relapsed APL (23), was shown to be less translocation, and that TBL1XR1–RARB was the first recurrent effective to RARB-transduce cells in vitro and ex vivo. Considering fusion observed in APL other than the RARA fusions. RARB is the dramatic success of precision medicine for APL with PML– located at 3p24 and TBL1XR1 at 3q26, thus fusion would involve RARA, a novel, small molecules/compounds capable of differen- a t(3;3) or an inv(3) and might therefore be very hard to recognize tiating APL with the RARB translocation is required to improve by standard karyotype analysis. outcomes in this resistant subgroup. RARB has a function similar to that of RARA as a member of We demonstrated that TBL1XR1 is the recurrent partner gene of retinoic acid receptors (RAR) family of proteins. RARB forms RARB. TBL1XR1 is reportedly a partner of RARA translocation in heterodimers with retinoid X receptors (RXR), binds to RAREs, APL (17). The other known fusion partners of RARA, such as PML, and recruits corepressors and histone deacetylases to target genes NPM1, and NUMA1, share the characteristic of having a dimer- in resting cells. The RARs/RXR heterodimers are activated by ization/oligomerization domain. The dimerization activity of the physiologic concentration of retinoic acid, which mediates cellu- LisH domain in TBL1XR1 contributes to homodimerization and lar signaling in embryonic morphogenesis, cell growth, and the dominant negative effect of RARs. Interestingly, the deletion differentiation. Our study demonstrated that TBL1XR1–RARB and mutations in TBL1XR1 have been reported in various malig- was an oncogenic protein having effects similar to those of nancies (24), and a previous study showed that TBL1XR1 was a PML–RARA; it homodimerized, diminished transcriptional activ- component of the protein complex regulating the retinoic acid

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Figure 4. Effects of TBL1XR1–RARB on differentiation and proliferation in human cord blood. A and B, Human CB CD34þ cells were transduced with vector, PML–RARA, TBL1XR1–RARB,orRUNX1-RUNX1T1 and were cultured with cytokines (SCF, TPO, FLT3L, IL3, IL6, each 10 ng/mL) to induce myeloid differentiation. The frequency of myeloblasts and mature myeloid cells (A) and the frequency of CD66þ and CD14þ cells (B) in human CB cells transduced with vector, PML–RARA or TBL1XR1–RARB at 10 days (A) and 24 days (B) of culture, respectively. TBL1XR1–RARB and PML–RARA increased the frequency of myeloblasts and decreased CD66/CD14 expression compared with the vector control, indicating their effects on inhibiting myeloid maturation. C, Human CB cells transduced with vector, PML–RARA,orTBL1XR1–RARB were cultured in cytokine-containing media. The mixed transduction culture containing both transduced GFP(þ)and untransduced GFP() cells were passaged to score the frequency of GFP(þ) cells by ﬂow cytometric analysis as a measure of the impact of the transduced gene on the cellular proliferation rate. The initial frequency of GFP(þ) cells immediately after transduction was set at 1. Both ATRA and tamibarotene showed a substantial growth-inhibitory effect on PML–RARA cells. TBL1XR1–RARB cells were relatively resistant to the retinoid treatments, especially to tamibarotene. D, CD66 and CD14 expression in cultures described in 10 days after addition of ATRA or tamibarotene. The retinoids induced myeloid differentiation of PML–RARA cells efﬁciently but showed only limited effects on TBL1XR1–RARB cells.

pathway (25). Considering an interesting result that one case had retinoic acid pathway, and have oncogenic potency. Our study a mutation of TBL1XR1, alteration of TBL1XR1 may simulta- confirmed that the dysregulation of the retinoic acid pathway neously contribute to the pathogenesis of APL. was a hallmark of APL; however, the response of APL with Our study included only five pediatric cases of RARA-nega- RARB translocation to retinoids was partial and was in line tive APL, and notably, most of our cases were young child with with the clinical finding of the resistance of RARA-negative APL median of 2.6 years of age. In terms of additional mutations, no to ATRA. Thus, in the context of precision medicine, RARB cases in our cohort had FLT3-ITD, which is detected commonly alteration should be screened for in RARA-negative APL cases, in adult APL. APL in young child might have distinct genetic and further investigation is required to improve outcomes for landscape, and RARB-translocation is a candidate for genetic RARB-positive APL. hallmark of APL in younger age. Further investigation in larger cohorts including older age and adult cases is required to assess Disclosure of Potential Conflicts of Interest frequency, clinical characteristics, and outcomes of RARB- No potential conflicts of interest were disclosed. translocation in APL. Further molecular investigation, such as transcriptome comparison between RARA-positive and RARB- Authors' Contributions positive APLs, would be informative to elucidate underlying Conception and design: T. Osumi, H. Takahashi, T. Uchiyama, M. Kato mechanism of APLs. Development of methodology: T. Osumi, M. Uchiyama, K. Hata, M. Kato Acquisition of data (provided animals, acquired and managed patients, In conclusion, we found that the majority of RARA-negative RARB provided facilities, etc.): T. Osumi, M. Tamura, M. Uchiyama, K. Nakabayashi, APL had translocation, and thereby formed a novel, distinct D. Tomizawa, A. Watanabe, T. Hori, S. Yamamoto, K. Hamamoto, M. Migita, subgroup of APL. Unlike wild-type RARB, both the TBL1XR1– H. Kizawa, R. Shirai, T. Inukai, S. Ogawa, T. Kitamura, K. Matsumoto, RARB and PML–RARA proteins homodimerize, suppress the N. Kiyokawa, S. Goyama, M. Kato

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Analysis and interpretation of data (e.g., statistical analysis, biostatistics, proofreading and editing this manuscript. The authors are responsible for all computational analysis): T. Osumi, S.-i. Tsujimoto, M. Tamura, M. Uchiyama, final modifications to the manuscript prior to submission. K. Nakabayashi, K. Okamura, M. Yoshida, H. Ogata-Kawata, H. Ueno-Yokohata, This study was supported in part by the Japan Society for the Promotion of M. Seki, K. Ohki, J. Takita, T. Inukai, T. Kitamura, K. Hata, S. Goyama, M. Kato Science (JSPS) through a Grant-in-Aid for Scientific Research (grant number Writing, review, and/or revision of the manuscript: T. Osumi, M. Yoshida, 17H04234 to M. Kato), by a grant from the National Center for Child Health D. Tomizawa, K. Hamamoto, M. Migita, H. Ogata-Kawata, H. Kizawa, T. Inukai, and Development (grant numbers 28-5 to M. Kato and 27-4 to D. Tomizawa), a S. Goyama, M. Kato grant from the Japan Foundation for Pediatric Research (to M. Kato), a grant Administrative, technical, or material support (i.e., reporting or organizing from the Kawano Memorial Foundation for the Promotion of Pediatrics of data, constructing databases): H. Takahashi, T. Uchiyama, K. Ohki, Japan (to S. Goyama), and a grant from The Cell Science Research Foundation N. Kiyokawa, M. Kato (to S. Goyama). Study supervision: H. Takahashi, K. Matsumoto The costs of publication of this article were defrayed in part by the payment of Acknowledgments page charges. This article must therefore be hereby marked advertisement in The authors would like to thank Ms. Natsuko Ariizumi, Ms. Shinobu accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Kobayashi, and Ms. Yoshie Fukumasa for their technical assistance. The authors thank Mr. James R. Valera of the Department of Education for Clinical Research Received March 26, 2018; revised May 17, 2018; accepted June 14, 2018; of the National Center for Child Health and Development for his assistance in published first June 19, 2018.

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4458 Cancer Res; 78(16) August 15, 2018 Cancer Research

Recurrent RARB Translocations in Acute Promyelocytic Leukemia Lacking RARA Translocation

Tomoo Osumi, Shin-ichi Tsujimoto, Moe Tamura, et al.

Cancer Res 2018;78:4452-4458. Published OnlineFirst June 19, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-0840

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