Many acute myeloid leukemia (AML) patients exhibit hallmarks of immune exhaustion, such as increased myeloid-derived suppressor cells, suppressive regulatory T cells and dysfunctional T cells. Similarly, we have identified the same immune-related features, including exhausted CD8+ T cells (TEx) in a mouse model of AML. Here we show that inhibitors that target bromodomain and extra-terminal domain (BET) proteins affect tumor-intrinsic factors but also rescue T cell exhaustion and ICB resistance. Ex vivo treatment of cells from AML mice and AML patients with BET inhibitors (BETi) reversed CD8+ T cell exhaustion by restoring proliferative capacity and expansion of the more functional precursor-exhausted T cells. This reversal was enhanced by combined BETi and anti-PD1 treatment. BETi synergized with anti-PD1 in vivo, resulting in the reduction of circulating leukemia cells, enrichment of CD8+ T cells in the bone marrow, and increase in expression of Tcf7, Slamf6, and Cxcr5 in CD8+ T cells. Finally, we profiled the epigenomes of in vivo JQ1-treated AML-derived CD8+ T cells by single-cell ATAC-seq and found that JQ1 increases Tcf7 accessibility specifically in Tex cells, suggesting that BETi likely acts mechanistically by relieving repression of progenitor programs in Tex CD8+ T cells and maintaining a pool of anti-PD1 responsive CD8+ T cells.
Acute Myeloid Leukemia (AML) is the most common adult leukemia and has a 5-year survival of under 30%. AML is caused by uncontrolled proliferation of myeloid cells resulting from a combination of mutations that affect proliferation, differentiation and epigenetic state. For this reason, drugs targeting epigenetic modifications are being studied in AML. AML cells avoid immune recognition though inhibiting the function of multiple cell types, especially T cells and therefore immune checkpoint blockade presents a promising therapy; however, clinical trials to date have shown very modest efficacy. T cell exhaustion has been shown to be a regulated process involving transcriptional and epigenetic changes. BET proteins, which are chromatin readers, have been implicated in maintaining this exhaustion state. In these studies, we investigated the effects of the BET inhibitor (BETi) JQ1 on T cell exhaustion and checkpoint responsiveness in a murine model of AML and AML patient samples. The AML mouse model bears FLT3-ITD and deletion of TET2 restricted to myeloid lineages and is resistant to anti-PD1 therapy. This mouse model of AML expanded terminally exhausted T cells and impaired proliferative capacity after TCR stimulation. Ex vivo treatment with BETi and anti-PD1 reverses CD8+ T cell exhaustion via rescue of proliferative dysfunction and expansion of more functional precursor exhausted T cells in patient samples and AML splenocytes. Finally, we show that BETi rescues anti-PD1 resistance in vivo and reduces tumor burden in multiple organ sites and enriches CD8+ T cells in the bone marrow. In total, we demonstrate that combining BETi and anti-PD1 therapy in the treatment of AML is a rational strategy to overcome anti-PD1 resistance.
Acute myeloid leukemia (AML) is a hematological cancer with a very poor prognosis. FLT3-ITD mutations that cause constitutive FLT3 signaling are commonly seen in patients. Our lab and others have shown that AML patient samples exhibit hallmarks of immune exhaustion such as T cell dysfunction, increased MDSCs, and increased Tregs that associate with worse survival. In solid tumor models DCs have been shown to be important for anti-tumor T cell activity and we aim to further describe the role of DCs in the immune response to leukemia. FLT3 signaling is critical for DC development but the effects of FLT3-ITD on DCs and how it contributes to leukemogenesis remains unclear. In a novel FLT3-ITD driven AML mouse model we found that DCs from these mice have elevated pFLT3, indicating constitutive FLT3 signaling. Moreover, significantly expanded DC progenitors and DCs in the bone marrow and spleen are observed. Analysis of serum cytokines identified increased levels of IL-27, IL-10, and IL-17a in leukemia mice compared to healthy controls, suggesting that the expansion of DCs in these leukemia mice may be driving pathogenic expansion of T helper subsets. Furthermore, we measured higher levels of circulating Th2, Th17, and Treg phenotypes in the blood compared to healthy controls. Thus, we hypothesize that FLT3-ITD DCs are a significant contributor to T cell skewing in AML resulting in poor anti-tumor responses. We are using scRNA-seq methods to identify transcriptional changes in FLT3-ITD DCs that lead to the phenotypes we identified. Understanding how FLT3-ITD DCs contribute to AML immune suppression is critical to interpreting leukemia etiology and the development of targeted therapies such as immune checkpoint blockade or small molecule inhibitors.
Acute myeloid leukemia (AML) is an aggressive myeloid lineage white blood cell cancer with an extremely poor prognosis that occurs most frequently above 60 years of age. The current standard of care consists of an intensive induction chemotherapy regimen. Many patients cannot withstand this intensive treatment regimen and rely on other targeted therapies such as small-molecule inhibitors, which inhibit specific oncogenic proteins, but these are not durable. Immunotherapies, which block stimuli that inhibit T cell functions, have had great success in achieving durable remissions in metastatic melanoma. Studies have shown that AML patients exhibit markers of immune suppression by the tumor microenvironment such as increased levels of PD1/LAG3+ exhausted T cells as well as regulatory T cells and myeloid-derived suppressor cells. In addition, inhibitors targeting the BET proteins family, which are histone readers, potently target leukemia cells by reducing myc but also increase IFNy and IL-2 production in T cells by inhibiting BATF, a negative regulator of TCF1. TCF1 is a critical factor in effector memory T cells and TCF1+ T cells are the primary expanders with aPD1. We therefore hypothesized that BETi and aPD1 would synergize in treating AML by modulating both tumor intrinsic and extrinsic factors (enhanced T cell activity). T cells derived from an immune competent FLT3-ITD/TET2/Lys-Cre AML mouse model show significant proliferative dysfunction and enhanced exhaustion markers, but are rescued by ex vivo treatment with BETi + aPD1. Further, In vivo treatment with this novel drug combination showed enhanced reduction of leukemic blasts, increased T cell proliferation, and in vitro expansion of TCF1+ effector memory CD8 T cells.
BackgroundAcute Myeloid Leukemia (AML) is the most common adult leukemia and has a very poor prognosis. With a 5-year survival of under 30% (seer.cancer.gov), most people diagnosed with AML will die from the disease. AML is caused by an uncontrolled proliferation of poorly differentiated myeloid precursor cells which results from a combination of three classes of mutations that affect proliferation, differentiation and epigenetic state. For this reason, drugs targeting epigenetic modifications are being actively studied in AML. AML has been shown to avoid immune recognition though inhibiting the function of multiple cell types, especially T cells1 2 and therefore immune checkpoint blockade presents a promising therapy for any immune-targeted strategy; however, clinical trials to date have shown very modest efficacy.3–5 T cell exhaustion in cancer has been shown to be a regulated process involving transcriptional and epigenetic changes.6–9 BRD4 has been shown to be important for maintaining this exhaustion state.10 11 It stands to reason that drugs designed to target epigenetic pathways in tumors will have effects on T cell populations present in the tumor microenvironment. In these studies, we investigated the effects of the BET inhibitor (BETi) JQ1 on T cell exhaustion and checkpoint responsiveness in a murine model of AML.MethodsThe AML mouse model bears FLT3-ITD and deletion of TET2 restricted to the myeloid lineage. For in vitro studies, splenocytes were stimulated with anti-CD3 and either JQ1, anti-PD1 or both and proliferation and differentiation status were assessed by flow cytometry. For in vivo studies, treatment consisted of 2 weeks with JQ1, anti-PD1 or both.ResultsThis mouse model of AML exhibits an expansion of terminally exhausted T cells and impaired proliferative capacity after stimulation through the TCR (figure 1). Ex vivo treatment with BETi and anti-PD1 reverses CD8+ T cell exhaustion via rescue of proliferative dysfunction and expansion of more functional precursor exhausted T cells (TPEx-CD8, PD1+, TCF1+, TIM3-) (figure 2). Finally, we show that BETi synergizes with anti-PD1 in vivo leading to a reduction of tumor cells in multiple organ sites, and enrichment of CD8+ T cells in the bone marrow (figure 3).ConclusionsUsing an AML mouse model that exhibits leukemia-induced immune exhaustion, we demonstrate the pre-clinical efficacy of combining BETi and anti-PD1 therapy in the treatment of AML.ReferencesLamble AJ, Lind EF. Targeting the immune microenvironment in acute myeloid leukemia: a focus on T Cell immunity. Front Oncol 2018;8:213.Lamble AJ, Kosaka Y, Laderas T, Maffit A, Kaempf A, Brady LK, et al. Reversible suppression of T cell function in the bone marrow microenvironment of acute myeloid leukemia. Proc Natl Acad Sci U S A. 2020;117(25):14331–41.Boddu P, Kantarjian H, Garcia-Manero G, Allison J, Sharma P, Daver N. The emerging role of immune checkpoint based approaches in AML and MDS. Leuk Lymphoma 2018;59(4):790–802.Bewersdorf JP, Shallis RM, Zeidan AM. Immune checkpoint inhibition in myeloid malignancies: moving beyond the PD-1/PD-L1 and CTLA-4 pathways. Blood Rev 2020:100709.Daver N, Garcia-Manero G, Basu S, Boddu PC, Alfayez M, Cortes JE, et al. Efficacy, safety, and biomarkers of response to azacitidine and nivolumab in relapsed/Refractory acute myeloid leukemia: a nonrandomized, Open-Label, Phase II Study. Cancer Discov 2019;9(3):370–83.Beltra JC, Manne S, Abdel-Hakeem MS, Kurachi M, Giles JR, Chen Z, et al. Developmental Relationships of Four Exhausted CD8(+) T Cell subsets reveals underlying transcriptional and epigenetic landscape control mechanisms. Immunity 2020;52(5):825–41 e8.Khan O, Giles JR, McDonald S, Manne S, Ngiow SF, Patel KP, et al. TOX transcriptionally and epigenetically programs CD8(+) T cell exhaustion. Nature 2019;571(7764):211–8.Pauken KE, Sammons MA, Odorizzi PM, Manne S, Godec J, Khan O, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354(6316):1160–5.Abdel-Hakeem MS, Manne S, Beltra JC, Stelekati E, Chen Z, Nzingha K, et al. Epigenetic scarring of exhausted T cells hinders memory differentiation upon eliminating chronic antigenic stimulation. Nat Immunol 2021;22(8):1008–19.Milner JJ, Toma C, Quon S, Omilusik K, Scharping NE, Dey A, et al. Bromodomain protein BRD4 directs and sustains CD8 T cell differentiation during infection. J Exp Med 2021;218(8).Kagoya Y, Nakatsugawa M, Yamashita Y, Ochi T, Guo T, Anczurowski M, et al. BET bromodomain inhibition enhances T cell persistence and function in adoptive immunotherapy models. J Clin Invest 2016;126(9):3479–94.Ethics ApprovalThis study has been approved by the OHSU IACUC committee protocol IP00000907 “Immune-based therapeutic approaches for acute myeloid leukemia” Evan Lind PI.Abstract 734 Figure 1T cell exhaustion in the AML mouse model. (A) Cytotoxic T cells show an exhausted phenotype in mice with AML. Spleen cultures from mice with AML or WT controls were stained with antibodies to CD3, CD8, TIM3, PD1, and TCF1. Left shows percent of TPEX CD8 T cells. Right panel shows TEX CD8 T cells. N = 12 animals per group. (B) Proliferative defect in T cells in mice with AML. Splenocytes were labeled with the proliferation dye CFSE. Whole spleen suspensions were stimulated with anti-CD3 or anti-CD3 and anti-CD28 for 3 days. FACs plots show proliferation of T cells in each conditionAbstract 734 Figure 2Treatment with JQ1 results in expansion of T cells with TPEX. (A) Example of proliferation (CFSE dilution) vs TCF-1 expression showing unstimulated, CD3 or CD3+JQ1 120 nM in in vitro 3-day culture. Results gated on CD8 T cells. (B) Summary of T cell proliferation from 4 independent experiments showing the percent proliferation of CD8 T cells with TPEX (PD1+ Tim-3- TCF-1+) (black line) or TEX (PD1+Tim-3+TCF1-) phenotype (red line). Statistics are unpaired T-Test for each treatment condition.Abstract 734 Figure 3In vivo treatment of FTL mice with the BETi JQ1. (A) Schematic overview of treatment protocol. (B) White blood cell counts at pre-treatment, 1 week and 2 weeks after JQ1, PD1 blockade or both. (C) Percent of CD8+ T cells of all CD3-gated T cells in the BM of treated animals. (D) A-C Percent of precursor-exhausted CD8+ T cells as a percent of all T cells in the spleen of treated animals. Results combined from 2 separate experiments n=7. D One experiment n=3.
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