Pb1775: transposable element participates in composing stat3::rara::ltr40a-rc trinary fusion in variant acute promyelocytic leukemia and confers atra resistance via ligand binding domain truncation

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Attribute to the application of all-trans retinoic acid (ATRA) and arsenic, acute promyelocytic leukemia (APL) positive for PML::RARA fusion gene (FG) has achieved a high cure rate. There are still cases manifest like APL but negative for PML::RARA, that is, variant APL (APLv). So far, fifteen RARA fusion partners have been reported in APLv, and all RARA-FGs were reported as X::RARA (X stands for 5’ partner genes). Unlike PML::RARA-positive APL, most other X::RARA cases were resistant to ATRA, suggesting molecular mechanisms remain to be revealed. Here we report the finding of transposable element participates in composing STAT3::RARA::LTR40a-RC (RC, abb. of reverse complement) trinary fusion in APLv and confer ATRA resistance via RARA ligand binding domain (LBD) truncation. Aims: To analyze the structural traits of the RARA-FG in APLv and to explore its sensitivity to ATRA treatment. Methods: The FG was analyzed by transcriptome sequencing (RNA-seq), whole genome sequencing (WGS), and optical genome mapping (OGM). The biological effects and responsiveness to ATRA of fusion proteins were evaluated in vitro. Results: A 35-year-old female came with 2 months of skin ecchymosis and 2 days of lower abdominal pain and diarrhea. Laboratory tests showed moderate anemia and abnormal coagulation function. Morphological examination showed 79% myeloblasts and immunophenotype analysis showed APL phenotype. RT-PCR screening for PML::RARA was negative. The karyotype showed inv(17)(p12q21). The patient received ATRA and arsenic combination therapy, but the response was poor. Unfortunately, the patient died from non-medical reasons 20 days after starting treatment. We identified a special form of STAT3::RARA::LTR40a trinary fusion, which is spliced in tandem with 3 fragments of STAT3 exon 1_21, RARA exon 3 to truncated exon 9, and LTR40a-RC sequence together with its 5’ adjacent 151bp sequence. Both long-read WGS, OGM, and RT-PCR confirmed that these 3 fragments spliced within the same cistron. RNA-seq data showed the gene expression pattern of this case was like classic APL, and the aberrant activation expression of the transposon element LTR40a. The 3’ fusion sequence confers a short derived 3 amino acids coding sequence with a swift termination. Importantly, LTR40a-RC confers a poly(A) signal sequence, which is necessary for a qualified mRNA. The RARA exon 9 truncation led to the deletion of helix 12 (H12) of its LBD, which plays a pivotal role in response to ATRA [Structure.2019;27:1270-1285.e6]. In principle, the RARA protein with LBD-H12 truncation will lose responsiveness to ATRA. Our in vitro experiments confirmed that cells transfected with chimeric protein with intact RARA LBD responded well to ATRA. In contrast, chimeric proteins with RARA LBD-H12 truncation were unresponsive to ATRA, even at high concentrations. Summary/Conclusion: This is the first report of pathological trinary fusion, the first report of transposable element participates in composing pathological fusion gene, and the first report of the RARA LBD truncation confers ATRA resistance in APLv. Moreover, in the two previously reported STAT3::RARA cases, the authors also observed breakpoints of exon 9 at the genomic level but did not conduct further analysis [Blood. 2018;131(8):935- 939.]. It strongly suggests that RARA LBD truncation might be the recurrent event in STAT3::RARA fusion or even other APLv cases. It may be due to the inertial only focusing on RARA 5’ splicing and the complexity of transposon sequence analysis, so RARA 3’ splicing was previously unobserved.Keywords: AML, Retinoic acid receptor (RAR), APL, Gene fusion