Immunosuppression in Isocitrate Dehydrogenase Mutated Acute Myeloid Leukemia

Introduction: Mutations in the isocitrate dehydrogenase (IDHmut) enzymes 1 and 2 are prevalent in a diverse array of human malignancies, including melanoma, glioma, acute immunoblastic T cell lymphoma (AITL) and acute myeloid leukemia (AML,15-20% of patients). IDHmut enzymes produce R-2-hydroxyglutarate (R-2-HG), an oncometabolite with immunomodulatory capability (Notarangelo et al, Science 2022). Immunotherapies in AML outside of allogeneic stem cell transplantation have proven to be largely ineffective, possibly due to immune evasion mechanisms intrinsic to myeloid malignancies. Herein, we aim to characterize the immune repertoire in IDHmut AML patients at the time of diagnosis, as well as the mechanisms by which R-2-HG may suppress immune cell function.

Methods: The immune repertoire in cryopreserved peripheral blood mononuclear cells (PBMCs, n=10) and bone marrow mononuclear cells (BMMCs, n=8) of IDHmut AML patients were characterized using full spectrum flow cytometry to evaluate 30 different immune cell markers. T cell activation (CD25, CD69, CD40L, CD134), exhaustion (PD-1, CTLA-4, Lag-3, Tim-3), differentiation (Foxp3, CXCR3, CCR4, CCR6), and memory (CD45RA, CCR7, CD28, CD95) markers were assessed. IDH wild-type (IDHwt) AML PBMCs (n=4), BMMCs (n=12) and healthy volunteer PBMCs (n=6) and BMMCs (n=3) were used as controls.

To investigate the effects of R-2-HG on CD4 ⁺ T cell activation, primary human naïve CD4 ⁺ T cells (CD4 ⁺, CD45RA ⁺) were isolated from healthy donors and activated (anti-CD3/CD28) in the presence of R-2-HG (20 mM) or PBS control. Activation, proliferation and cytokine production were assessed with flow cytometry, intracellular cytokine staining (ICS) and LEGENDplex at 4, 8, 24 and 72 hrs. The OT-II transgenic mouse model was used to assess antigen-specific T cell activation.

T cell metabolism at rest and after activation in the presence of R-2-HG was evaluated using SCENITH (Argüello et al, Cell Metab 2021) and ¹³C ₆-glucose isotope labeling coupled with mass spectrometry (MS) performed by the UCLA Metabolomics Center.

Results: Full-spectrum flow cytometry analysis revealed a trend for higher CD4:CD8T cell ratio in AML BMMCs (fold=1.95, p=0.229) as well as increased CD4 ⁺ T cell activation and exhaustion markers compared to healthy donor BMMCs. No appreciable differences in CD4 ⁺ T cell markers were found between IDHwt AML and IDHmut AML, likely due to AML heterogeneity and small sample size. Nevertheless, our preliminary results suggest CD4 ⁺ T cell engagement in AML; thus, we chose to investigate how CD4 ⁺ T cells are affected by R-2-HG.

Primary human naïve CD4 ⁺ T cells activated in the presence of R-2-HG exhibited a defect in early activation demonstrated by decreased CD69 expression (n=7, fold=0.773, p=0.001) and concomitant decreased production of IL-2 measured by ICS (n=7, fold=0.403, p=0.0001) and LEGENDplex (n=3, fold=0.557, p=0.046). Proliferation in the presence of R-2-HG was inhibited in a dose dependent manner (n=6, Division Index fold change=0.768, p=0.006 at 20mM). Antigen-specific OT-II CD4 ⁺ T cells showed a decrease in proliferation in response to OVA _323-339 (n=3, Division Index fold change=0.620, p=0.004).

Given R-2-HG's structural similarity to α-ketoglutarate (αKG), we hypothesized that disruption in CD4 ⁺ T cell activation when cultured with R-2-HG may be due to metabolic dysfunction. Indeed, we found a decrease in global metabolism in CD4 ⁺ T cells cultured with R-2-HG (fold = 0.678, p = 0.009). MS revealed decreased levels of succinate and malate downstream and accumulation of αKG upstream of α-ketoglutarate dehydrogenase (KGDH) in the tricyclic acid (TCA) cycle, suggesting a defect in TCA flux (fold succinate / αKG ratio =0.134 unactivated, p = 0.029; 0.231 4h activation, p = 0.012; 0.328 24h activation, p = 0.004; Figure 1). Moreover, MS also showed increased amounts of essential amino acids (Ile, Leu, Met, Phe, Thr, Trp, Val, His; Figure 2), suggesting a compensatory reliance on amino acid import and metabolism.

Conclusions: By characterizing primary AML samples at diagnosis, we found trending increases in CD4:CD8 ratio and expression of CD4 ⁺ T cell activation and exhaustion markers compared to healthy controls. We also demonstrate R-2-HG suppression of CD4 ⁺ T cells, potentially via inhibition of KGDH in the TCA cycle. Our studies show R-2-HG in IDHmut tumors can modulate CD4 ⁺ T cell function and contribute to immune escape in human AML.