"Next, there’s still much work to do. The most important thing is to gather more cases to further validate the conclusions of this study. Only then will there be hope for overcoming leukemia caused by viruses like TTMV."

This was the advice given by the 100-year-old Academician Zhenyi Wang after we presented the findings recently published in Leukemia.

The story of viruses and cancer dates back to 1910, long before the discovery of DNA or the central dogma of molecular biology. At that time, scientists believed that identifying pathogens responsible for cancer would make its prevention and treatment possible. The discovery of HPV is a notable example. In 2008, Professor Harald zur Hausen was awarded the Nobel Prize for establishing the link between HPV and cervical cancer.

In 2005, another type of virus caught the attention of Professor zur Hausen and his wife, Professor Ethel-Michele de Villiers. This was the Anellovirus family—small, single-stranded circular DNA viruses that coexist quietly within the human body from birth. These viruses usually remain at very low levels in individuals with normal immune function but can replicate explosively in immunocompromised conditions (e.g., newborns, transplant recipients, or HIV patients). Although traces of anelloviruses were found in lymphoma and leukemia samples, the potential link was largely overlooked.

It wasn't until 2021 that Dr. Annalisa Astolfi from Italy reported the detection of a subclass of anelloviruses, Torque teno mini virus (TTMV), in acute promyelocytic leukemia (APL) cells. TTMV was found fused with the retinoic acid receptor alpha (RARA) gene. Inspired by the fusion characteristics, we developed a custom screening tool and identified three cases with the TTMV::RARA fusion from RNA sequencing data of leukemia patients. Remarkably, one of these cases—showing TTMV::RARA in both primary and relapse samples—matched a challenging case discussed by Academician Wang in September 2020 during what he referred to as an "open-book exam." At that time, he raised three critical questions:

1. Why are other variant fusions, including PML::RARA, not detected in APL?
2. Why is RARA abnormally overexpressed in APL?
3. Is the combination therapy of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) still effective?

With these questions in mind, we collected data from seven published cases containing either fusion sequences or RNA-seq data, including the three cases we initially identified. Using bioinformatics approaches, we sought to address these questions.

By combining third-generation sequencing and Sanger PCR, we confirmed that TTMV integrates into the second intron of the RARA gene, creating a TTMV::RARA fusion protein. Structural analysis revealed that this fusion protein retains the DNA-binding domain and the ligand-binding domain for ATRA from RARA. Transcriptomic analysis of leukemia samples showed that TTMV::RARA-positive cases exhibit expression profiles highly similar to APL but also display unique features, including activation of infection and immune pathways. Interestingly, the expression profile of TTMV::RARA-positive cases closely resembles the transcriptional response to ATO treatment.

To investigate whether ATO is effective against TTMV::RARA, we analyzed the fusion protein sequence and identified a conserved cysteine residue at positions 55–59 on the TTMV side. In 2023, Professor Hugues de Thé from France demonstrated that arsenic binds to a pocket formed by three cysteine residues. Structural analysis of the TTMV::RARA fusion protein revealed that variations in the virus can alter the size of this binding pocket. Clinically, patients with smaller binding pockets showed better treatment responses (e.g., survival of 11 years compared to 10 months for relapse cases). GSVA analysis further validated the correlation between pocket size and ATO response. We cloned TTMV::RARA sequences with strong and weak ATO responses and tested them in U937 cell lines, confirming these findings.

How does TTMV::RARA form, and how does it drive leukemia development? Our preliminary exploration revealed two key factors. First, RARA is highly expressed in TTMV::RARA-positive samples. Second, TTMV DNA is detected in these patients’ genomes but absent in other leukemia types, indicating a high viral load during TTMV::RARA formation. The viral insertion site coincides with an open chromatin region in the second intron of RARA, a stage when the gene is transcriptionally active and DNA becomes more accessible. We propose a model where TTMV-driven leukemia arises during immune suppression, which allows a surge in TTMV replication in the bone marrow. To combat infection, immune pathways are activated, directing bone marrow cells toward differentiation. When differentiation reaches the promyelocyte stage—when RARA expression peaks—TTMV inserts itself into RARA, forming the TTMV::RARA fusion protein and driving leukemia development.

"Many patients remain undiagnosed clinically. By applying translational medicine to basic research data, we can use pathological and physiological concepts to improve the diagnosis and treatment of clinical cases. I find this work increasingly valuable and meaningful."
— Academician Zhenyi Wang, from Masters of Science Interview

Call for Collaboration

If you are a clinician and encounter cases with the following characteristics:

1. Myeloid sarcoma without abnormalities in the hematological system;
2. Promyelocyte increase on morphology or flow cytometry but negative for PML::RARA;
3. Confirmed TTMV fusion cases.

Please contact us at loujiacheng1986@foxmail.com with your phone number or WeChat ID. We will reach out to you. If your case meets the inclusion criteria, TTMV screening will be provided free of charge.

If you are a researcher interested in TTMV-driven leukemia, we welcome discussion, collaboration, and joint efforts to advance this research.