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Patients with acute myeloid leukemia (AML) often achieve remission after allogeneic hematopoietic cell transplantation (allo-HCT) but subsequently die of relapse driven by leukemia cells resistant to elimination by allogeneic T cells based on decreased major histocompatibility complex II (MHC-II) expression and apoptosis resistance. Here we demonstrate that mouse-double-minute-2 (MDM2) inhibition can counteract immune evasion of AML. MDM2 inhibition induced MHC class I and II expression in murine and human AML cells. Using xenografts of human AML and syngeneic mouse models of leukemia, we show that MDM2 inhibition enhanced cytotoxicity against leukemia cells and improved survival. MDM2 inhibition also led to increases in tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2 (TRAIL-R1/2) on leukemia cells and higher frequencies of CD8+CD27lowPD-1lowTIM-3low T cells, with features of cytotoxicity (perforin+CD107a+TRAIL+) and longevity (bcl-2+IL-7R+). CD8+ T cells isolated from leukemia-bearing MDM2 inhibitor-treated allo-HCT recipients exhibited higher glycolytic activity and enrichment for nucleotides and their precursors compared with vehicle control subjects. T cells isolated from MDM2 inhibitor-treated AML-bearing mice eradicated leukemia in secondary AML-bearing recipients. Mechanistically, the MDM2 inhibitor-mediated effects were p53-dependent because p53 knockdown abolished TRAIL-R1/2 and MHC-II upregulation, whereas p53 binding to TRAILR1/2 promotors increased upon MDM2 inhibition. The observations in the mouse models were complemented by data from human individuals. Patient-derived AML cells exhibited increased TRAIL-R1/2 and MHC-II expression on MDM2 inhibition. In summary, we identified a targetable vulnerability of AML cells to allogeneic T-cell-mediated cytotoxicity through the restoration of p53-dependent TRAIL-R1/2 and MHC-II production via MDM2

MicroRNAs (miR) are small noncoding RNAs that regulate gene expression, posttranscription, and manipulate immune responses in different types of cancers. In this study, we identify miR-146a as a negative regulator of immune activation, comparable to immune-checkpoint molecules. miR-146a levels were increased in melanoma microenvironmental tissue, and miR-146a−/− mice survived longer and developed less metastases in comparison with wild-type melanoma-bearing mice. T cells isolated from miR-146a−/− mice revealed higher expression levels of the miR-146a target gene Stat1 and the Stat1-regulated cytokine IFNγ. Neutralization of IFNγ in miR-146a−/− mice decreased survival and increased melanoma metastasis patterns to those of wild-type mice. In vitro, IFNγ reduced melanoma cell migration, cell-cycle activity, and basal metabolic rate. Conversely, IFNγ also increased PD-L1 levels on the melanoma cells, which may counterbalance some of the beneficial effects increasing immune escape in vivo. Combined treatment with a miR-146a antagomiR and anti–PD-1 resulted in improved survival over isotype control or anti–PD-1 treatment alone. In summary, these data show that miR-146a plays a central role within the STAT1/IFNγ axis in the melanoma microenvironment, affecting melanoma migration, proliferation, and mitochondrial fitness as well as PD-L1 levels. Additionally, combined inhibition of PD-1 and miR-146a could be a novel strategy to enhance antitumor immune response elicited by checkpoint therapy. Significance: These findings identify a microRNA–based mechanism by which melanoma cells escape the immune system, providing a new therapeutic strategy to improve the current management of patients with melanoma.

Acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT) has a dismal prognosis. We found that T cells of patients relapsing with AML after allo-HCT exhibited reduced glycolysis and interferon-γ production. Functional studies in multiple mouse models of leukemia showed that leukemia-derived lactic acid (LA) interfered with T cell glycolysis and proliferation. Mechanistically, LA reduced intracellular pH in T cells, led to lower transcription of glycolysis-related enzymes, and decreased activity of essential metabolic pathways. Metabolic reprogramming by sodium bicarbonate (NaBi) reversed the LA-induced low intracellular pH, restored metabolite concentrations, led to incorporation of LA into the tricarboxylic acid cycle as an additional energy source, and enhanced graft-versus-leukemia activity of murine and human T cells. NaBi treatment of post–allo-HCT patients with relapsed AML improved metabolic fitness and interferon-γ production in T cells. Overall, we show that metabolic reprogramming of donor T cells is a pharmacological strategy for patients with relapsed AML after allo-HCT.

The bone marrow microenvironment (BMME) is key player in regulation and maintenance of hematopoiesis. Oncogenic

Immune checkpoint inhibitor (ICI) therapy has shown a significant benefit in the treatment of a variety of cancer entities. However, immune-related adverse events (irAEs) occur frequently and can lead to ICI treatment termination. MicroRNA-146a (miR-146a) has regulatory functions in immune cells. We observed that mice lacking miR-146a developed markedly more severe irAEs compared with WT mice in several irAE target organs in 2 different murine models. miR-146a(–/–) mice exhibited increased T cell activation and effector function upon ICI treatment. Moreover, neutrophil numbers in the spleen and the inflamed intestine were highly increased in ICI-treated miR-146a(–/–) mice. Therapeutic administration of a miR-146a mimic reduced irAE severity. To validate our preclinical findings in patients, we analyzed the effect of a SNP in the MIR146A gene on irAE severity in 167 patients treated with ICIs. We found that the SNP rs2910164 leading to reduced miR-146a expression was associated with an increased risk of developing severe irAEs, reduced progression-free survival, and increased neutrophil counts both at baseline and during ICI therapy. In conclusion, we characterized miR-146a as a molecular target for preventing ICI-mediated autoimmune dysregulation. Furthermore, we identified the MIR146A SNP rs2910164 as a biomarker to predict severe irAE development in ICI-treated patients.

Oncogenic Ras mutations occur in various leukemias. It was unclear if, besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation-related effect of KRAS contributes to the disease. Here, we identify a functional link between oncogenic KrasG12D and NLRP3 inflammasome activation in murine and human cells. Mice expressing active KrasG12D in the hematopoietic system developed myeloproliferation and cytopenia, which is reversed in KrasG12D mice lacking NLRP3 in the hematopoietic system. Therapeutic IL-1-receptor blockade or NLRP3-inhibition reduces myeloproliferation and improves hematopoiesis. Mechanistically, KrasG12D-RAC1 activation induces reactive oxygen species (ROS) production causing NLRP3 inflammasome-activation. In agreement with our observations in mice, patient-derived myeloid leukemia cells exhibit KRAS/RAC1/ROS/NLRP3/IL-1β axis activity. Our findings indicate that oncogenic KRAS not only act via its canonical oncogenic driver function, but also enhances the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1β axis. This paves the way for a therapeutic approach based on immune modulation via NLRP3 blockade in KRAS-mutant myeloid malignancies., Oncogenic Ras mutations are common drivers in myeloid leukemia. Here, the authors show in patient cells and in mice that oncogenic K-Ras activates NLRP3 inflammasome to drive myeloproliferation, which can be reversed by genetic or pharmacologic NLRP3 blockade.

Contains fulltext : 190745.pdf (Publisher’s version ) (Closed access) Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD(+) leukemia cells. This synergized with the allogeneic CD8(+) T cell response, leading to long-term survival in six mouse models of FLT3-ITD(+) AML. Sorafenib-related IL-15 production caused an increase in CD8(+)CD107a(+)IFN-gamma(+) T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD(+) AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8(+) T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.

Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive PD-L1 expression may help in developing new therapeutic strategies. Here, we show that oncogenic JAK2 activity caused STAT3 and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2(V617F)-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2(V617F)-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2(V617F)-myeloproliferative neoplasms (MPN) compared to healthy individuals and declined upon JAK2 inhibition. JAK2(V617F) mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient-derived monocytes, megakaryocytes, and platelets. PD-1 inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2(V617F)-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1-mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2(V617F)-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.