Division of Hematology/Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Seoul National University Hospital and.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea.
Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center.
Vanderbilt Center for Immunobiology.
Vanderbilt-Ingram Cancer Center, and.
Program in Cancer Biology, Vanderbilt University, Nashville, Tennessee, USA.
Veterans Affairs Tennessee Valley Healthcare, Nashville, Tennessee, USA.
Abstract
Effective eradication of leukemic stem cells (LSCs) remains the greatest challenge in treating acute myeloid leukemia (AML). The immune receptor LAIR-1 has been shown to regulate LSC survival; however, the therapeutic potential of this pathway remains unexplored. We developed a therapeutic LAIR-1 agonist antibody, NC525, that induced cell death of LSCs, but not healthy hematopoietic stem cells in vitro, and killed LSCs and AML blasts in both cell- and patient-derived xenograft models. We showed that LAIR-1 agonism drives a unique apoptotic signaling program in leukemic cells that was enhanced in the presence of collagen. NC525 also significantly improved the activity of azacitidine and venetoclax to establish LAIR-1 targeting as a therapeutic strategy for AML that may synergize with standard-of-care therapies.
Keywords
LeukemiasOncology
Figures
Figure 1 LAIR-1 is a target for AML therapy. (A and B) LAIR-1 transcript levels, as measured by RNA sequencing and quantified by RNA Sequencing by Expectation and Maximization (RSEM) software, in AML patient samples clustered by French-American-British (FAB) classification (A) or by molecular mutation (B). UD, undiagnosed (non-AML). n = 1–47 patient samples per group. (C) Illustration of leukemopoiesis from LSC precursors into granulocyte-macrophage progenitor–like (GMP-like) LSCs, lymphoid-primed multipotent progenitor–like (LMPP-like) LSCs, or multipotential progenitor–like (MPP-like) LSCs. (D) Mean fluorescence intensity (MFI) of LAIR-1 cell surface expression on the indicated LSC subpopulations. Each symbol/line represents a unique patient. n = 25 AML patients. (E) Illustration of normal hematopoiesis from hematopoietic stem cells (HSCs) into multipotential progenitors (MPPs), common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), or granulocyte-monocyte progenitors (GMPs). (F) MFI of LAIR-1 cell surface expression on the indicated HSC subpopulations. Each symbol/line represents a unique donor. n = 7 non-AML healthy donors. (G) Comparison of LAIR-1 cell surface expression on cells from comparable compartments from AML patients or healthy donors (H). Each dot represents a unique donor/patient. n = 7 healthy donors or 25 AML patients. P value determined by Student’s t test. Data are shown as the mean ± SEM.Figure 2 LAIR-1 agonist mAb NC525 inhibits colony formation in LSCs but not healthy stem cells. (A) Representative images of ex vivo colony formation of LSCs from AML patient 19-0029 when plated at the indicated cell number and treated with 5 μg /mL of NC525 or isotype control. (B) Colony-forming units (CFUs) formed by ex vivo plating of (left to right) 50,000, 20,000, or 12,000 LSCs per well from the indicated AML patient during titrated treatment with anti–LAIR-1 agonist mAb NC525. n = 3 technical replicates per group. P values determined by 1-way ANOVA with multiple comparisons. (C) CFU formation from healthy donor BM or AML patient BM treated with 5 μg /mL NC525. Values normalized to isotype control. n = 14 AML biological replicates or 5 healthy biological replicates. P value determined by Student’s t test.Figure 3 LAIR-1 engagement eradicates primary and secondary AML in patient-derived xenograft models. (A) Schematic of the AML patient–derived xenograft (PDX) model and representative scatterplots of human (H) CD33+CD45+ leukemic cells in circulation at the indicated time after engraftment. Subgated population represents percent of parent. (B) Leukemic growth, as measured by the percentage of circulating HCD33+HCD45+ cells, in PDX mice engrafted with BM from donors with normal-karyotype AML (n = 4–5 mice per group), monocytic AML (n = 4–5 mice per group), acute myelomonocytic leukemia (AMML) (n = 4 mice per group), FLT3-ITD+ AML (n = 8 mice per group), or uncharacterized AML (n = 5 mice per group). Engrafted mice were treated with 5 mg/kg isotype control (gray) or NC525 (red). (C) Schematic of PDX secondary transplant model, where BM from PDX mice engrafted and treated as above was harvested and secondarily transplanted into naive recipient mice. Graphs show leukemic growth in secondary recipient mice after receiving BM from AMML PDX animals or normal-karyotype AML PDX animals that had been treated with 5 mg/kg isotype control (gray) or NC525 (red). n = 3 mice per group. P values calculated by 2-way ANOVA. Data are shown as the mean ± SEM.Figure 4 NC525 agonism of LAIR-1 induces AML cell death. (A–C) Blood leukocytes after ex vivo treatment with 10 μg/mL isotype (gray) or NC525 (red) with (A) or without (B) exogenous collagen or CD45loSSClo blasts with collagen (C). n = 3 technical replicates. (D) Percentage dead leukocytes or blasts treated as in A and B relative to LAIR-1 surface expression. Each dot represents an individual patient. Red or blue shading indicates LAIR-1 greater or less than 20,000 arbitrary units, respectively. Line represents linear regression. (E) Total live cells or blasts from healthy or AML donors after treatment as indicated in A and B. n = 4–7 donors. (F) AlphaLISA of phosphorylated SHP-1 relative to total SHP-1 in AML PBMCs, normalized to isotype treatment. n = 3 technical replicates. (G and H) Duplicate dot blots from phospho-arrays of AML PBMCs quantified for MAPK (G) or mTOR and NF-κB activity (H). (I) Schematic of LAIR-1 (blue) clustering using anti-IgG (green) and NC525 (red). In vitro growth of MV4-11-LAIR-1overexpressing cells treated with isotype (gray) or NC525 (red) in the absence or presence of anti-IgG. (J) Annexin V staining of cells treated as in I, with representative scatterplot of apoptotic annexin V+ Live-Dead Aqua– cells at day 3 of culture. (K and L) 4E-BP1 expression as measured by Lumit assay (K) or cleaved caspase-7 as measured by Western blot (L) of cells treated as shown in I. Mean pixel density normalized to histone-3 for triplicate samples. (M) Percentage TUNEL+ MV4-11-LAIR-1overexpressing cells at day 3 of treatment plus DMSO vehicle, 50 μM 220509-74-0 (caspase-3/7 inhibitor), or 50 μM MHY1485 (mTOR activator). Values normalized to isotype for each respective condition. n = 3–4 technical replicates. P values calculated by Student’s t test. Error bars represent SEM.Figure 5 NC525-mediated AML killing in vivo is dependent on LAIR-1 expression level. (A) Schematic of the MV4-11–luciferase or THP-1–luciferase cell-derived xenograft (CDX) model of AML. (B) In vivo leukemic growth as measured by whole-body luminescence of MV4-11–luciferase cells (left) or THP-1–luciferase cells (right) in CDX mice treated with 10 mg/kg isotype control (gray) or NC525 (red). n = 8 mice per group. P values determined by 2-way ANOVA. (C and D) MV4-11 cell counts (C) or percent dead MV4-11 cells (D) in the blood, spleen, or BM of CDX mice treated with vehicle control (gray) or 10 μg/mL NC525 (red). (E) Total cell counts or mouse (M) CD45+ cell counts in the BM of CDX mice treated with vehicle control (gray) or 10 μg/mL NC525 (red). n = 9–10 mice per group. P values determined by Student’s t test. Data are shown as the mean ± SEM. (F) Representative histograms of LAIR-1 cell surface expression on the indicated cell lines. (G) Schematic of CDX model systems to test the inhibition of leukemic growth as a function of LAIR-1 expression. (H) Percent inhibition of MV4-11-LAIR-1–knockout (green), MV4-11-LAIR-1–wild-type (purple), or MV4-11-LAIR-1–overexpression cell growth in vivo (normalized to the mean of the respective isotype controls) after treatment with 10 mg/kg NC525 (left) and plotted against LAIR-1 geometric mean fluorescence intensity (gMFI) (right). (I) Percent inhibition of MV4-11 growth fit to a logarithmic regression curve of LAIR-1 expression. n = 9 mice per group.Figure 6 LAIR-1 signaling restricts AML survival signaling pathways in vivo. (A) Model schematic (left) and MV4-11-luciferase growth (right) in CDX mice used for digital spatial imaging and target protein quantification. n = 6 mice per group. (B) Representative images of CDX mouse bones stained with DAPI (blue) and anti–human CD45 (red) used to quantify the number of MV4-11 cells in the indicated region of interest (ROI) (highlighted). ROI area is equal between samples. (C) Protein reads of human CD45, BCL-XL, or uncleaved PARP from bone or spleen harvested from CDX mice at day 22 after engraftment. Read counts are normalized to histone H3 and ribosomal protein S6. n = 3–11 quantified tissue regions across 2 isotype-treated or 2 NC525-treated mice. Data are shown as the mean ± SEM. P values determined by Student’s t test.Figure 7 NC525 synergizes with AML standard-of-care therapy. Ex vivo killing of VEN/AZA–treated AML patient leukemic cells (A) or T cells or NK cells (B). Values normalized to vehicle control. Lines represent linear regression of NC525 concentration versus normalized cell killing. (C) LAIR-1 surface expression of VEN/AZA–treated AML patient leukemic cells, T cells, or NK cells. (D and E) In vivo leukemic growth as measured by whole-body luminescence of MV4-11–luciferase cells (D) or survival (E) of CDX mice treated with 10 mg/kg isotype control (gray) or NC525 (red) or 100 mg/kg VEN (pink) or 0.5 mg/kg AZA (blue) or combination therapy (green or yellow, respectively). BLoD, below limit of detection. n = 9 mice per group. P values determined by 2-way ANOVA or log-rank (Mantel-Cox) test, respectively. (F and G) Leukemic growth in the blood (F) and in the spleen and BM (G) at week 8 after transplant of AML PDX mice treated with vehicle (gray), VEN/AZA (blue), NC525 (red), or VEN/AZA plus NC525 (green). n = 5–10 mice per group. AML cells in spleen and BM compared from 4–5 mice per group. Data are shown as the mean ± SEM. P values determined by 2-way ANOVA or 1-way ANOVA with multiple comparisons.Figure 8 NC525 induces AML apoptosis through enhanced LAIR-1 clustering and SHP-1 phosphorylation. Schematic of LAIR-1–induced cell death in leukemic cells. Engagement of LAIR-1 on AML cells by agonist mAb NC525 induces enhanced SHP-1 phosphorylation that blocks aberrant PI3K/mTOR activity, leading to the suppression of constitutively active MAPK signaling and the self-renewal mechanisms promoted by AKT and NF-κB. This loss of proliferative signaling induces the deactivation of BCL-XL, which releases an apoptotic cascade through caspase-7 and PARP, culminating in programmed cell death.
그림 출처: PubMed Central 공개 논문 (PMC10650974)
그 밖의 정보
출판 형태
Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't
저널
The Journal of clinical investigation · eISSN 1558-8238
