A Metabolic Reprogramming Amino Acid Polymer as an Immunosurveillance Activator and Leukemia Targeting Drug Carrier for T-Cell Acute Lymphoblastic Leukemia

Abstract

Compromised immunosurveillance leads to chemotherapy resistance and disease relapse of hematological malignancies. Amino acid metabolism regulates immune responses and cancer; however, a druggable amino acid metabolite to enhance antitumor immunosurveillance and improve leukemia targeting-therapy efficacy remains unexplored. Here, an L-phenylalanine polymer, Metabolic Reprogramming Immunosurveillance Activation Nanomedicine (MRIAN), is invented to effectively target bone marrow (BM) and activate the immune surveillance in T-cell acute lymphoblastic leukemia (T-ALL) by inhibiting myeloid-derived suppressor cells (MDSCs) in T-ALL murine model. Stable-isotope tracer and in vivo drug distribution experiments show that T-ALL cells and MDSCs have enhanced cellular uptake of L-phenylalanine and MRIANs than normal hematopoietic cells and progenitors. Therefore, MRIAN assembled Doxorubicin (MRIAN-Dox) specifically targets T-ALL cells and MDSCs but spare normal hematopoietic cells and hematopoietic stem and progenitor cells with enhanced leukemic elimination efficiency. Consequently, MRIAN-Dox has reduced cardiotoxicity and myeloablation side effects in treating T-ALL mice. Mechanistically, MRIAN degrades into L-phenylalanine, which inhibits PKM2 activity and reduces ROS levels in MDSCs to disturb their immunosuppressive function and increase their differentiation toward normal myeloid cells. Overall, a novel amino acid metabolite nanomedicine is invented to treat T-ALL through the combination of leukemic cell targeting and immunosurveillance stimulation.

Keywords: Metabolic Reprogramming Immunosurveillance Activation Nanomedicine (MRIAN); T-cell acute lymphoblastic leukemia (T-ALL); amino acid metabolism; immunosurveillance; myeloid-derived suppressor cells (MDSCs).

Scheme 1

Graphical abstract to illustrate the synthesis process and functional features of Metabolic Reprogramming Immunosurveillance Activation Nanomedicine (MRIAN). MRIAN targets T‐cell acute lymphoblastic leukemia (T‐ALL) cells and myeloid‐derived suppressor cells (MDSCs) in the bone marrow (BM). MRIAN degrades into l‐Phe to reprogram the energy metabolism of MDSCs, which drives their differentiation toward normal myeloid cells and unarms their immunosuppressive function to reinforce immune surveillance in T‐ALL.

Figure 1

MRIAN efficiently penetrates bone marrow and explicitly targets leukemic cells and MDSCs in T‐ALL mice. a) Bioluminescence images (upper) and quantification (below) of DiR dye intensity in mice treated with DiR dye, DiR@8P2 NPs, DiR@8P4 NPs, DiR@MRIAN, DiR@8L6 NPs, and DiR@PLGA NPs at the indicated time after injection (n = 5 mice). b,c) Representative images (b) and quantification (c) of DiR intensity in the indicated organs at 48 h after injection (n = 5 mice). d) Scheme for quantifying DiL@MRIAN uptake efficiency by leukemia cells and BM cells in T‐ALL mice. e,f) Representative FACS histogram (e) and quantification (f) of the fluorescent intensity of DiL@MRIAN in T‐ALL cells, MDSCs, and normal hematopoietic cells as indicated (n = 5 mice). g) Scheme for stable‐isotope tracer experiment and quantification of cellular uptake of [¹³C, ¹⁵N] l‐Phe in T‐ALL cells, MDSCs, and normal hematopoietic cells from T‐ALL mice (n = 4 mice). Liquid chromatography mass spectrometry (LC–MS). Data represent mean ± s.d. Repeated measures one‐way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons, ^ǂ p < 0.05, ^ǂǂ p < 0.01, ^ǂǂǂ p < 0.001.

Figure 2

MRIAN‐Dox has reduced cardiotoxicity and tissue damage than Dox. a) Scheme for synthesizing of MRIAN‐Dox. b) Time‐dependent stability of MRIAN‐Dox in PBS or PBS with 10% fetal bovine serum (FBS). c,d) Scheme (c) and pharmacokinetics of Dox and MRIAN‐Dox in mice (d). e) Treatment scheme for T‐cell acute lymphoblastic leukemia (T‐ALL) mice. f–i) HE, Masson staining, representative immunohistochemistry images of TUNEL, γ‐H₂AX (f), quantification of the fibrostic area (g), γ‐H₂AX+ cells frequency (h), and Tunel⁺ cells frequency (i) in the heart section of hearts from T‐ALL mice after indicated treatments (n = 5 mice). j–l) M‐mode echocardiography (j), quantification of ejection fraction (k), and fractional shortening (l) of T‐ALL mice after indicated treatments (n = 5 mice). m,n) HE of kidney or liver (m) and quantification of glomerular necrosis frequency in the kidney (left) and liver function ALT (middle) and AST (right) in mice serum after indicated treatments. Repeated measures one‐way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons, ^ǂ p < 0.05, ^ǂǂ p < 0.01, ^ǂǂǂ p < 0.001.

Figure 3

MRIAN‐Dox enhances DNA damage and apoptosis of Jurkat cells induced by Dox in vitro (a,b). Representative images (a) and quantification (b) of cellular uptake of Dox in Jurkat cells at indicated times after incubation with Dox, MRIAN‐Dox, or DoxiL (n = 3 independent experiments). c) Cell proliferation rate of Jurkat cells at the indicated time after treatments (n = 3 independent experiments). d,e) Representative FACS plots (d) and quantification (e) of Annexin V⁺ apoptotic Jurkat cells at 72 h after treatments (n = 3 independent experiments). f,g) Representative image (f) and quantification (g) of γ‐H₂AX staining in Jurkat cells at 72 h after indicated treatments (n = 50 cells). h) Representative FACS plots (left) and quantification (right) of γ‐H₂AX positive Jurkat cells at 72 h after indicated treatments (n = 3 independent experiments). i) Representative image (left) and quantification (right) of comet assay in Jurkat cells at 72 h after indicated treatments (n = 30 cells). Scale bar 50 µm (a) and 5 µm (f,i). Data represent mean ± s.d. Repeated measures one‐way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons, ^ǂ p < 0.05, ^ǂǂ p < 0.01, ^ǂǂǂ p < 0.001.

Figure 4

MRIAN‐Dox has enhanced therapeutic efficacy to treat T‐ALL in vivo. a) Treatment scheme for T‐ALL mice. b,c) The frequency of T‐ALL cells in peripheral blood (PB) (b) and bone marrow (BM) (c) at the indicated time after treatments (n = 4 mice). Leukemia cells (GFP⁺ cells). d) The body weight of T‐ALL mice 24 days after transplantation with indicated treatment (n = 4 mice). e,f) The representative image (e) and quantification (f) of γ‐H₂AX staining in T‐ALL cells from T‐ALL mice 24 days after transplantation with indicated treatment (n = 30 cells). Scale bar 5 µm (e). g,h) Quantification of apoptotic cells in GFP^– normal hematopoietic cells and GFP⁺ T‐ALL cells from T‐ALL mice 24 days after transplantation with indicated treatment (n = 4 mice). i) The blood routine analysis of normal C57B6J without transplantation (WT) and T‐ALL mice 24 days after transplantation with indicated treatment (n = 4 mice). j–k) Representative images of the PB smear (j) and the representative images (left) and weight quantification (right) of liver and spleen (k) of T‐ALL mice 24 days after transplantation with indicated treatment (n = 4 mice). l,m) Treatment scheme (l) and survival curve T‐ALL mice with indicated treatments (m) (n = 10 mice). n,o) Treatment Scheme (n) and survival curve T‐ALL mice with indicated treatments (o) (n = 10 mice). Data represent mean ± s.d. Repeated measures one‐way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons, ^ǂ p < 0.05, ^ǂǂ p < 0.01, ^ǂǂǂ p < 0.001.

Figure 5

MRIAN‐Dox improved leukemia microenvironment in T‐ALL. a) In vitro MDSC differentiation assay scheme. b,c) ROS level (b) and frequency of MDSCs, macrophages, and dendritic cells (DCs) (c) of purified MDSCs after indicated treatment (n = 3 mice). d) Treatment scheme for T‐ALL mice. e,f) The frequency of MDSCs in bone marrow (BM) (e) and spleen (f) in T‐ALL mice after indicated treatments (n = 4 mice per group). g–m) The frequency of macrophages (g), DCs (h), NK cells (i), CD4⁺ T cells (j), CD8⁺ T cells (k), CD3⁺CD8⁺IFNγ ⁺ cytotoxic T lymphocytes (CTL T cells) (l), and CD3⁺CD4⁺IFNγ ⁺ Type 1 T helper cells (Th1 T cells) (m) in BM in T‐ALL mice 24 days after transplantation with indicated treatment (n = 4 mice per group). n,o) Experiment scheme (n) and quantification of proliferating T cells after co‐culture with MDSCs with indicated ratios (o). MDSCs were isolated from T‐ALL mice with or without treatment as indicated. p) Quantification of ROS levels in MDSCs from T‐ALL 24 days after transplantation with indicated treatment (n = 4 mice per group). Data represent mean ± s.d. Repeated measures one‐way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons, ^ǂ p < 0.05, ^ǂǂ p < 0.01, ^ǂǂǂ p < 0.001.

Figure 6

MRIAN reduces MDSC without affecting the metabolic level of leukemia cells. a–c) Scheme and representative gas chromatography mass spectrometry (GC‐MS) spectrum of Phe (a) and quantification (b) for the cellular Phe levels (b) and Tyr levels (c) in T‐ALL cells and MDSCs by GC‐MS after indicated treatments (n = 3 replicates per group). d) Western blot analyses (upper) and quantification (down) of PAH protein levels in T‐ALL cells and MDSCs from T‐ALL mice (n = 4 mice). 1#, 2# indicated two representative individual T‐ALL mice. e) Scheme of glucose metabolism in MDSCs. f–m) Relative intracellular pyruvate kinase (f), relative intracellular pyruvate concentration (g), lactate production (h), LDH activity (i), mitochondrial activity (TMRM) (j), intracellular ATP concentration (k), Cell ROXDeep (ROS^high) cell frequency (l), and relative glucose uptake (m) in MDSCs 72 h after indicated treatments (n = 4 mice per group). n) Scheme of glucose metabolism in T‐ALL cells. o–v) Relative intracellular pyruvate kinase (o), relative glucose uptake (p), relative intracellular pyruvate concentration (q), lactate production (r), LDH activity (s), intracellular ATP concentration (t), mitochondrial activity (TMRM) (u), and Cell ROXDeep (ROS^high) cell frequency (v) in T‐ALL cells 72 h after indicated treatments (n = 4 mice per group). Data represent mean ± s.d. Two‐tailed Student's t‐tests were used to assess statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Repeated measures one‐way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons, ^ǂ p < 0.05, ^ǂǂ p < 0.01, ^ǂǂǂ p < 0.001.

Figure 7

PAH catalyzes Phe and regulates PKM2 activity in T‐ALL cells. a,b) Western blot analyses (a) and quantification (b) of PAH protein levels in Jurkat cells after PAH knockdown with indicated treatment (n = 3 independent experiments). c,d) Quantification of gas chromatography mass spectrometry for the cellular Phe levels (c) and Tyr levels (d) in Jurkat cells with indicated treatment (n = 3 independent experiments). e–g) Relative intracellular pyruvate kinase (e), ROS level (f), and apoptotic cells (g) in Jurkat cells with indicated treatment (n = 3 independent experiments). Data represent mean ± s.d. Two‐tailed Student's t‐tests were used to assess statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001. Repeated measures one‐way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons, ^ǂ p < 0.05, ^ǂǂ p < 0.01, ^ǂǂǂ p < 0.001.