Behind the Paper

Turning leukemia cell death into an immune alarm

How OT-55 and Bcl-xL inhibition made apoptosis immunogenic in myelomonocytic AML and opened a route to PD-1/Tim-3 blockade
YEJIN LEE Jul 06, 2026

The question that started the work

Many anticancer drugs are judged by a simple question: do they kill the cancer cell? In acute myeloid leukemia (AML), that question is necessary but not sufficient. AML remains an aggressive blood cancer, and immune escape is one reason durable responses to immunotherapy remain difficult to achieve 1 2. In our laboratory at Seoul National University (SNU), we framed this study around a second question: when a leukemia cell dies, can its death provide instructive signals to the immune system?

This matters because apoptosis can be quiet. BH3 mimetics have changed the way we think about drugging the BCL-2 family in AML 3, 4, but mitochondrial apoptosis is not automatically an immune alarm. In some settings, it may even blunt interferon signaling and anticancer immunity 4, 5. We began with the concern that a leukemia cell could be eliminated without leaving the antigenic and adjuvant cues needed for durable adaptive immunity.

The conceptual bridge was immunogenic cell death (ICD). ICD is not simply cell death plus inflammation; it depends on the coordinated appearance and function of damage-associated molecular patterns, including surface calreticulin, extracellular ATP, and HMGB1, in an appropriate redox and immunological context 6, 7. We were especially interested in the hydroxycoumarin OT-55, which had previously been associated with ER stress, DAMP release, and leukemia-cell phagocytosis 8. A related compound, OT52, had also shown synergy with BH3 mimetics in lung cancer 9. These observations led to the central experimental question pursued in the SNU lab: could OT-55 convert BH3-mimetic-induced apoptosis from a largely tolerogenic event into an immunogenic one?

Although the project was conceived and experimentally driven in our Seoul National University laboratory, it was also enabled by important international collaborations. OT-55 itself was originally designed and synthesized by Prof. Artur Silva at the University of Aveiro, Portugal, years before this study began. We are grateful to Prof. Artur Silva for providing the chemical starting point that enabled the present work.

The project was further sharpened by a 2025 discussion with Prof. Dmitri V. Krysko at Ghent University, Belgium, whose contributions have strongly influenced the field of cancer cell death immunology. That exchange helped us refine the distinction between DAMP release and DAMP function, a point that became central to our interpretation of HMGB1 in this study. Prof. Krysko sadly passed away later in 2025. We dedicate this Behind the Paper reflection to his memory with deep respect and gratitude.

 

Turning AML apoptosis into an immune signal. OT-55 induces ER stress and danger signaling in C1498 myelomonocytic AML cells, whereas A-1331852 promotes Bim release by targeting Bcl-xL. Together, the drugs drive caspase-3-dependent immunogenic apoptosis, marked by calreticulin exposure and ATP release. HMGB1 is released, oxidized, and functionally muted. The resulting immune priming activates dendritic cells, expands antigen-specific CD8+ T cells, and cooperates with PD-1/Tim-3 blockade to promote local and distant tumor control.

Image credit: Created in BioRender. Lee, Y., and Diederich, M. (2026).

 

What the data told us

We first asked whether an ICD signature had clinical meaning in AML. By screening 34 ICD-associated genes across TARGET-AML, BEAT-AML, and GSE37642, we derived a nine-gene ICD score associated with a favorable prognosis 10. The signal was not only prognostic. ICD-high AML showed greater immune activity, with increased CD8+ T cells, activated dendritic cells, and Tim-3 expression. The score was highest in myelomonocytic AML, which led us to use C1498, a myelomonocytic AML model, as a mechanistic and immunological test system.

These patient-cohort analyses changed the way we framed the project. Rather than evaluating OT-55/A-1331852 solely as a cytotoxic combination, we treated it as a means to probe an immune state: could we induce a form of AML cell death that dendritic cells could read, process, and convert into T-cell priming?

This part of the study was developed through sustained discussions within the SNU lab, particularly with Dr. Eun-Ji Kwon, whose expertise in bioinformatics helped us move from a mechanistic observation to a patient-cohort question. Together, we asked whether ICD could be evaluated directly in AML datasets before returning to the bench. The resulting ICD scoring system enabled us to stratify patients into ICD-high and ICD-low groups and to compare their prognoses and immune landscapes. In this way, the bioinformatic analyses did more than complement the experiments; they connected the SNU laboratory findings to a broader clinical and translational context.

What surprised us

The combination that emerged from the SNU experiments was OT-55 with A-1331852, a selective Bcl-xL inhibitor. Mechanistically, the two drugs induced apoptosis via complementary pathways. OT-55 induced ER stress, reduced Mcl-1, and increased NOXA, thereby promoting Bim displacement from Mcl-1. A-1331852 promoted Bcl-xL phosphorylation and Bim release from Bcl-xL. Together, these events converged on caspase-3 cleavage and apoptotic death.

The key point, however, was not only that more cells died. The dying C1498 cells displayed increased surface calreticulin and secreted more ATP, two signals that help dendritic cells engulf and interpret tumor material. HMGB1 was also released, but in the oxidative context induced by OT-55, HMGB1 appeared functionally inert. For us, this was one of the most useful surprises of the paper. It made the study less of a checklist of ICD markers and more of a functional dissection of which signals actually carried the immune effect.

The vaccination experiments were the decisive test. AML cells treated with OT-55/A-1331852 protected immunocompetent mice in a CD8+ T-cell-dependent and antigen-specific manner. When calreticulin was neutralized or ATP was degraded, vaccine efficacy was markedly reduced; blocking HMGB1 did not have the same effect. The immune system was not responding to cell death in general. It was responding to a particular configuration of danger signals.

The mechanistic interpretation of the BCL-2 family pathway also benefited from international training and discussion. During Dr. Yejin Lee’s internship at the Laboratoire de Biologie Moléculaire et Cellulaire du Cancer (LBMCC) in Luxembourg, exchanges with Dr. Claudia Cerella, whose expertise in the BCL-2 family was highly valuable, helped refine our analysis of OT-55/A-1331852 synergy. They contributed to our conceptual framework for interpreting experiments conducted in the SNU lab.

Why checkpoint blockade mattered

The final experimental step in the SNU study was the administration of checkpoint blockade. Because ICD-high AML showed PD-1/Tim-3 biology and single-cell analysis revealed exhausted CD8+ T-cell states, we tested whether immunogenic vaccination could be combined with anti-PD-1 and anti-Tim-3 antibodies. The combination amplified IFN-gamma and granzyme B CD8+ T-cell responses, improved tumor rejection, and, in a bilateral model, reduced distant tumor growth, consistent with a systemic antitumor response.

Taken together, the study led by our SNU lab describes that apoptosis is not a single immunological outcome. It can be silent, tolerogenic, or immunogenic, depending on the stress pathways and DAMP functions that accompany it. OT-55 did not merely add cytotoxicity to a BH3 mimetic; it changed the immunological meaning of the death process.

This remains a preclinical proof-of-concept, not a clinical regimen. Its value lies in defining a therapeutic logic for AML: combine a cell-death trigger with a signal-shaping agent that makes dying leukemia cells visible and interpretable to dendritic cells, and then release exhausted T cells through rational checkpoint blockade. The goal is not only to remove leukemia cells but to convert their death into a form of immune instruction.

Topics

Acute myeloid leukemia; cancer immunotherapy; immunogenic cell death; apoptosis; BH3 mimetics; checkpoint blockade; translational oncology; hematology; tumor immunology.

References

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