Different Immunotherapeutic Combinations Enhance Specific T Cell Immune Responses Against Leukemic Cells, as well as Leukemic Progenitor Cells in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematologic malignancy typified by uncontrolled proliferation and impaired differentiation of myeloid progenitor cells in the bone marrow. It primarily affects older adults, many of whom cannot tolerate intensive chemotherapy. Standard chemotherapy alone remains insufficient, as relapse rates are high and the 5-year survival rate remains low, highlighting the urgent need for new therapeutic approaches  (1,2). Growing evidence supports the role of immunotherapy for the treatment of AML, particularly approaches that target leukemic cells and their precursors. Strategic combinations of immunotherapies may help prevent immune escape while minimizing toxicity (3,4,5). Although immune-based approaches such as stem cell transplantation have shown efficacy for nearly 50 years, relapse remains a major challenge, underscoring the need for improved strategies.

Combination therapies are already shaping AML management, especially for patients ineligible for intensive treatment (6). Regimens such as 5’-azacytidine (AZA) with venetoclax, or ivosidenib with AZA, are now standard, while agents including FLT3 inhibitors, menin inhibitors, and immunomodulatory drugs are showing promise in double or triple combinations. Immunotherapy may complement these regimens. Immune checkpoint inhibitors, chimeric antigen receptor (CAR) T-cell therapies, and bispecific T-cell engagers (BiTEs) are gaining traction, while epigenetic immunomodulators in combination with programmed death-1 (PD-1) blockade remain under investigation. Leukemia associated antigens (LAAs) represent key immunogenic targets for cytotoxic T lymphocytes (CTLs) and thus provide an avenue for antigen-specific immunotherapy (7, 8, 9,10).

The role of immune checkpoint inhibition in AML is still under debate. While checkpoint monotherapy yields modest benefits, combinations with hypomethylating agents (HMA) or chemotherapy have shown encouraging results (11). Single-agent PD-1 blockade has limited benefits as an AML treatment, but its use with HMA has shown potential for relapsed/refractory disease and as frontline therapy for older patients (12,13). The immunomodulatory drug Lenalidomide (Len) exhibits anti-inflammatory, anti-proliferative, pro-apoptotic, and anti-angiogenic properties, thereby enhancing anti-tumor immunity. Unlike other agents used in the treatment of AML, Len’s unique profile, it’s direct anti-tumor effects, makes it a promising candidate for both monotherapy and combination approaches (8).

Nucleophosmin1 (NPM1) is one of the most commonly mutated (mut) genes in AML and is often associated with a favorable prognosis. Immune responses play an increasing role in AML treatment decisions; however, the role of immune checkpoint inhibition is still not clear. To address this, we investigated specific immune responses against NPM1, and three other LAAs - PRAME, Wilms' tumor 1 and RHAMM in AML patients (10). We investigated T cell destruction of leukemic progenitor/colony forming cells (LPC/CFC) using colony-forming immunoassays (CFI) and flow cytometry. We examined whether anti-PD1 antibodies could enhance the immune response against stem cell-like cells, comparing cells from patients with NPM1^mut and NPM1 wild-type (WT) AML. We found that the anti-PD-1 antibody, nivolumab, increased the number of LAA stimulated CTLs and their cytotoxic effects against LPC/CFC. These effects were most notable against NPM1^mut cells when the immunogenic epitope was derived from the NPM1^mut peptide and were further enhanced through the addition of anti-PD-1. These data suggest that NPM1^mut AML patients could be treated with anti-PD-1 and that this treatment combined with NPM1^mut directed immunotherapy could be even more effective for AML NPM^mut patients (10).

In 2025 (14), we published the effect of anti-PD-1 (nivolumab) alone or in combination with one of four other immunotherapeutics, ipilimumab (anti-CTLA4), lenalidomide (Len), all-trans retinoic acid (ATRA) or AZA. We examined the inhibition of LPC/CFC in mixed lymphocyte peptide cultures (MPLC) which were created through the incubation of mononuclear cells (MNC) from healthy donors with MNCs from 20 AML patients (Figure 1, Co-Culture).

Figure 1

The prevention of AML NPM1^mut LPC/CFC formation by LAA-stimulated T cell populations was most effective in the presence of NPM1^mut peptide, anti-PD-1 and AZA leading to a 64% reduction in CFC compared to the incubation of the MPLC with NPM^mut peptide alone. However, it was also notable when anti-PD-1 and Len were used together with NPM1^mut peptide, that there was a 46% reduction in CFC, compared to incubation of the MPLC with NPM^mut peptide alone. Samples from patients with NPM1^WT showed similar responses to the therapies, with an average reduction in CFCs of 32% when MPLCs were treated with NPM1^WT peptide and AZA, compared with a 39% reduction when MPLCs were treated with NPM1^WT peptide and Len. When LPC/CFC from AML NPM1^WT patients were treated with NPM1^WT peptide, anti-PD-1 and Len there was a 42% reduction in CFC. MPLCs in which the incubation only included the peptide of choice served as a growth control (Figure 2). 

Figure 2

 We were able to demonstrate a significant reduction in LPC/CFC numbers when MPLC involved primary AML NPM1^mut patient samples. This likely reflected the stimulation of the immune system by non-native NPM1^mut protein, which was exacerbated by the use of anti-PD-1 antibodies in combination with AZA (14, 15). This is predicted to have enhanced anti-leukemia responses against NPM^mut target proteins compared with NPM^WT proteins, which are unlikely to have primed the immune system during early AML development.   

In an extended analysis in which MNCs from NPM1^mut patients served as a negative control, the addition of allogeneic MNCs treated with NPM1 peptide and anti-PD-1 and/or AZA, were assessed in CFIs. We showed a significant reduction in the number of LPC/CFC following the addition of NPM1^mut (34%), NPM1 ^mut and anti-PD-1 (66%), NPM1 ^mut and AZA (79%) and NPM1 ^mut and anti-PD-1/AZA (86%) to the MPLCs.

A key challenge in targeted therapy is overcoming resistance mechanisms. Due to clonal evolution, the surviving cancer cells appear to be able to adapt rapidly. In AML, somatic mutations accumulate in hematopoietic stem and progenitor cells, driving uncontrolled proliferation. This process fosters resistance, immune evasion, and sustained leukemic growth (16).

In our ex vivo studies (14), we have observed that combining the checkpoint-inhibitor, anti-PD-1, with AZA produced the most notable impact, enhancing T-cell activation and suppressing LPC/leukemic stem cells growth. This gives an indication that immune checkpoint therapy can help counteract immune evasion in myeloid malignancies.

References

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