The Ingenious Multiple Efficacies of the Integrated Gene Fragment of Torque Teno Mini Virus in Orchestrating Promyelocytic Leukemogenesis Via Hijacking Rara

Introduction
Torque teno mini virus (TTMV) is a ubiquitous virus which symbiotic with humans, but it has recently been reported to cause acute promyelocytic leukemia (APL) through a unique form of genome fragment integration and forming the TTMV::RARA fusion gene, with 4 cases reported till now. However, the leukemogenesis mechanism of TTMV::RARA-APL has not been explored.

Methods
We recruited transcriptome sequencing (RNA-seq) data of 7 TTMV::RARA-APL cases, 3 of them also with whole genome sequencing (WGS) data, to analyze the properties of the TTMV integrated sequences. Clinical information was analyzed. In vitro cell experiments were performed to verify the functions of the conserved regulatory sequences and the fusion protein.

Results
These cases were collected from 4 centers, 5 males and 2 females, aged 3 to 35 (median 9) years, with 6 pediatric cases. Of which 3 pediatric cases were identified from a continuous acute myeloid leukemia (AML) cohort. In this cohort, 3 TTMV::RARA-APL were identified in 205 AML cases under 14 years, but no cases in another 930 AML cases, showing a strong tendency towards pediatrics (p=0.00022). Moreover, we totally identified 85 PML::RARA-APL and 3 TTMV::RARA-APL, and no other X::RAR cases in this cohort, indicating TTMV::RARA is the most common X::RAR fusion besides PML::RARA.

All cases manifested typical APL morphological features, and RNA-seq expression analysis showed clustering with PML::RARA-APL cases. Initial effect and remission can be achieved with chemotherapy regimens containing all-trans retinoic acid (ATRA), but early recurrence is prone to occur. RARA-I387T mutation was observed in case #TR4, who relapsed after treatment with ATRA, suggesting that the leukemic cells were responsive to ATRA. However, primary resistance to ATRA and extensive resistance to various agents were observed in case #TR5, who was initially diagnosed with myeloid sarcoma and transformed into APL at relapse.

The TTMV integrated sequences of 3 cases with WGS data were 1164bp, 2113bp, and 2859bp (Fig. 1A), respectively, and the common feature was the involvement of the transcription regulatory region and the open reading frame 2 (ORF2). Alignment of TTMV transcription regulatory sequences (Fig. 1B) revealed a highly conserved basal transcription factor region, including the canonical TATA box and GC box. While in the proximal promoter region showing sequence diversity, all 3 cases carried 4 RUNX1 binding elements and 1 octamer motif. In vitro experiments (Fig. 1C) showed that TTMV basal transcription element can initiate basal expression in 293T cells, while RUNX1 can significantly enhance the expression in response to the RUNX1 binding elements. RUNX1 is an important transcription factor protein in the differentiation of myeloid cells, and the 4 RUNX1 binding elements might explain the myelotropic of TTMV and confer an important basis in promyelocytic leukemogenesis.

The TTMV-ORF2 coding sequences of all 7 cases were analyzed, with the common region being the N-terminus 58 amino acids, of which 20 were highly conserved (Fig. 1D, 1E). Co-Immunoprecipitation analysis confirmed that the TTMV::RARA protein forms homopolymers and heteropolymers with RXRA (Fig.1F), which is the property shared by all X::RARA proteins. Serine/Threonine protein phosphatase (S/T PPase) assays showed that the fusion protein exhibits strong S/T PPase activity (Fig. 1G). UAS/GAL4 reporter experiment showed that TTMV::RARA protein exhibited a constrained but concentration-dependent response to ATRA compared to RARA (Fig. 1H). However, fusion proteins with ORF2-C32A/C34A mutation and impaired phosphatase activity were significantly more responsive to ATRA (Fig. 1H). This suggests that the S/T PPase activity of the ORF2 coding region plays a role in retarded ATRA response.

Conclusions
This is the first systematic report on this novelty disease. Data suggests that TTMV::RARA-APL may be more common in children, which may be related to the ubiquity of TTMV and its acquisition early in life. The clinical manifestations and treatment responses are diverse, with overall dismal prognosis. Molecular investigation indicated the multifunctional TTMV integrated molecular machinery in orchestrating RUNX1 enforced myelotropic, aberrant priming expression, homodimer formation, and S/T PPase activity, and confer promyelocytic leukemogenesis via hijacking RARA.