Precursor T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogenous hematologic malignancy resulting from accumulation of molecular lesions in a multistep process. Although survival rates have improved considerably, event free survival for patients with T-ALL are generally inferior compared to B-cell ALL. Genetic alterations are important determinants of responsiveness to therapy and serve as targets for molecularly tailored therapies. More than 75 chimeric fusion genes have been reported in T-ALL, the majority of which encode factors involved in transcriptional regulation, while only a smaller percentage codes for tyrosine kinases. We report a case of relapsed T-ALL, despite low risk stratification at the time of diagnosis, harboring a novel fusion protein SPTAN1-ABL1. Primary bone marrow specimen collected at diagnosis was transplanted in NSG-B2m mice and propagated as a patient-derived xenograft (PDX) line. For transcriptomic characterization, RNA isolated from primary and PDX samples was subjected to error-corrected targeted next-generation sequencing using ArcherDX FusionPlex HemeV2 kit. Bioinformatics analysis identified the novel SPTAN1-ABL1 gene fusion in which exon 2 of SPTAN1 was fused with exon 4 of ABL1. This fusion was confirmed by Sanger sequencing. Translation of the fusion product sequence showed in-frame fusion leading to the generation of a chimeric protein containing N-terminal SPTAN1 and C-terminal ABL1 with intact kinase domain. SPTAN1 encodes non-erythryocytic-1-spectrin-alpha protein, an actin-binding protein, with N-terminal domain possessing oligomerization activity. Because oligomerization of ABL1 promotes its kinase activity, it is possible that SPTAN1-ABL1 possesses constitutive kinase activity. The full-length SPTAN1-ABL1 fusion protein was cloned in a mammalian expression vector and expressed in BaF3 cells. SPTAN1-ABL1 fusion was detected at similar allelic frequencies in primary and PDX samples indicating the concordance between the two. Furthermore, treatment of engrafted mice with dasatinib (Qd10, 5 mg/Kg, p.o.) significantly prolonged survival compared to untreated mice (n=5 each, P<0.005). Taken together, these data suggest the possibility that the presence of SPTAN1-ABL1 fusion gene may confer a higher risk disease thereby leading to early recurrence, similar to the treatment failures observed in B-ALL patients later found to harbor BCR-ABL1 fusion gene. This study also indicates a potential therapeutic role for tyrosine kinase inhibitors in the treatment of T-ALL patients with ABL1 fusion. Citation Format: Anilkumar Gopalakrisnapillai, Erin Crowgey, Demetria Ruhl, Darcy Hamill, Nitin Mahajan, Todd Druley, E. Anders Kolb, Sonali P. Barwe. Identification of a novel fusion protein SPTAN1-ABL1 in a child with T-cell acute lymphoblastic leukemia: Functional characterization and therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-322.
Background We showed earlier that prolonged treatment with a combination of azacitidine (DNMT1 inhibitor) and panobinostat (HDAC inhibitor) induced complete remission in a disseminated xenograft model of KMT2A rearranged AML (Gopalakrishnapillai et al., 2017, Leuk Res). Pretreatment with epigenetic drugs has been shown to induce chemo-sensitivity in some malignancies. However, studies on the use of epigenetic drugs in overcoming chemoresistance mediated by the bone marrow microenvironment are limited. The aim of this study was to determine the merit of incorporating epigenetic therapy with chemotherapy in AML treatment regimen and to identify the mechanism by which epigenetic drugs can chemosensitize AML. Methods AML cell lines and PDX lines were co-cultured with HS5 bone marrow stromal cells. Cell viability following drug treatment was assessed by flow cytometry. For determination of cell adhesion, violet proliferation dye stained AML cells were co-cultured with HS5 cells. Following treatment, non-adherent cells were removed by washes with phosphate buffered saline. The number of adherent AML cells was determined by flow cytometry. NSG-B2m mice were engrafted with MV4;11 cells and bled regularly to evaluate disease progression. Once engraftment was confirmed mice were assigned to treatment groups. Epigenetic therapy constituted azacitidine and panobinostat (2.5 mg/Kg each; Qd5). Chemotherapy constituted cytarabine (50 mg/Kg; Qd5) and daunorubicin (1.5 mg/Kg; Qd3). Mice were euthanized when they reached predetermined experimental endpoints. All animal studies were approved by the Institutional Animal Care and Use Committee. Results AML cells in co-culture with HS5 bone marrow stromal cells were less sensitive to chemotherapeutics cytarabine and daunorubicin compared to AML cells in monoculture. This chemoprotection was not achieved when AML cells were cultured in HS5 conditioned media or in Transwell inserts suspended over HS5 monolayers, suggesting that direct cell-to-cell contact was required. MV4;11 cells exposed to epigenetic drugs or cytarabine alone retained 70% viability. Pre-treatment with the epigenetic drug combination of azacitidine and panobinostat prior to cytarabine exposure greatly reduced cell viability in MV4;11 cells harboring KMT2A fusion (Fig. 1A). Similar chemo-sensitization was observed when four distinct KMT2A rearranged PDX lines were used ex vivo. This sensitization was accompanied by reduced binding of AML cells to HS5 cells following treatment with epigenetic drugs (Fig. 1B). We evaluated the efficacy of the epigenetic therapy and chemotherapy combination in disseminated xenograft models of pediatric AML. NSG-B2m mice transplanted with MV4;11 cells via the tail vein were randomly assigned to four groups - 1) vehicle, 2) epigenetic therapy 3) chemotherapy (cytarabine + daunorubicin), and 4) epigenetic therapy followed by chemotherapy. The mice receiving the epigenetic therapy and chemotherapy combination survived significantly longer than mice treated with any other condition (Fig. 1C). To assess the leukemic cell distribution in peripheral blood, bone marrow and spleen following treatment, a cohort of mice receiving epigenetic therapy alone or chemotherapy alone were euthanized a day after treatment concluded. The percentage of leukemic cells in bone marrow and spleen was lower in mice treated with epigenetic therapy than those treated with chemotherapy, consistent with the survival data. Surprisingly, the peripheral blood counts were significantly higher in mice receiving epigenetic drug combination. These results together with our in vitro data indicate that epigenetic therapy induces mobilization of AML cells to the blood stream. This increased availability of AML cells may promote enhanced sensitivity to chemotherapy. Conclusion Our data suggest that direct cell-to-cell contact plays a major role in mediating chemoprotective effects of the bone marrow microenvironment. These chemoprotective effects can be overcome by pretreatment with epigenetic drug combination azacitidine and panobinostat. Epigenetic drugs interfere with AML cell interactions with the microenvironment and dislodge AML cells from their protective niche. Thus, mobilization of AML cells to peripheral blood is a potential mechanism of chemo-sensitization mediated by epigenetic drugs. Disclosures Kolb: Servier: Membership on an entity's Board of Directors or advisory committees; Roche- Genentech: Membership on an entity's Board of Directors or advisory committees.
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