CDKN1A is a target for phagocytosis-mediated cellular immunotherapy in acute leukemia

Abstract

Targeting the reprogramming and phagocytic capacities of tumor-associated macrophages (TAMs) has emerged as a therapeutic opportunity for cancer treatment. Here, we demonstrate that tumor cell phagocytosis drives the pro-inflammatory activation of TAMs and identify a key role for the cyclin-dependent kinase inhibitor CDKN1A (p21). Through the transcriptional repression of Signal-Regularity Protein α (SIRPα), p21 promotes leukemia cell phagocytosis and, subsequently, the pro-inflammatory reprogramming of phagocytic macrophages that extends to surrounding macrophages through Interferon γ. In mouse models of human T-cell acute lymphoblastic leukemia (T-ALL), infusion of human monocytes (Mos) engineered to overexpress p21 (p21TD-Mos) leads to Mo differentiation into phagocytosis-proficient TAMs that, after leukemia cell engulfment, undergo pro-inflammatory activation and trigger the reprogramming of bystander TAMs, reducing the leukemic burden and substantially prolonging survival in mice. These results reveal p21 as a trigger of phagocytosis-guided pro-inflammatory TAM reprogramming and highlight the potential for p21TD-Mo-based cellular therapy as a cancer immunotherapy.

Fig. 1. Tumor cell phagocytosis triggers the proinflammatory activation of macrophages.

a Schematic representation of the coculture of CMFDA-labeled MDMs with CMTMR-labeled malignant hematologic cells. b Confocal micrograph of MDMs and Jurkat cells after 8 h of coculture. c, d Percentage of phagocytosis of leukemia cells or PBLs (c) or CD34⁺ AML cells (d) in control (Co.)- or ZVAD (100 µM)-treated cocultures. e FACS dot plot of Phago⁺ MDMs or Phago^- MDMs sorted after 2 h of coculture with MOLT4 cells. f, g Confocal micrographs and percentages of CMTMR⁺CMFDA⁺ MDMs at 2 h (**p = 0.0079) (f, g) and 96 h (h, i) after Phago⁺ MDMs and Phago^- MDMs sorting. j–n Phago⁺ MDMs were analyzed in comparison to Phago^- MDMs to characterize modulated genes by a microarray (**p = 0.0022, *p = 0.0152) (j), CD163 membrane expression by FACS (**p = 0.0039) (k), and IRF5 expression by western blot (WB) analysis (l) and the supernatant (SN) was evaluated for indicated proinflammatory cytokines by WB analysis (l) or for IFNγ by a cytokine microarray (*p = 0.0286) (m) or ELISA (***p = 0.0008) (n) at 96 h (j, k, m) and 7 d (l, n) after FACS sorting. o, p Transwell coculture model of Phago⁺ MDMs and Phago^- MDMs at 2 h after FACS sorting (o), and iNOS expression identified by WB analysis of Phago^- MDMs cocultured in the bottom chambers for 15 d (p). In b, l, p and e, f, h, the data are representative of n = 3 and n = 5 donors. In c, n, d, and g, i, the data are presented as the mean±SEM from n = 3, n = 6, and n = 5 donors. In d, the CD34⁺ cells were from n = 4 AML patients. In j, box plots show centre line as median, box limits as upper and lower quartiles, and whiskers as a minimum to maximum values, from n = 3 donors. In k and m, the data are donor matched from n = 9 and n = 4 donors. Exact p-values are indicated and determined with two- (g) or one-tailed (m) unpaired Mann-Whitney test, the Kolmogorov–Smirnov test (j), a two-tailed paired Wilcoxon test (k), and a two-tailed unpaired t test (n). Source data are provided as a Source Data file.

Fig. 2. Macrophage-expressed p21 governs the phagocytosis of leukemia cells through the repression of SIRPα transcription.

a p21 expression in control (siCo.) or p21-silenced (sip21) MDMs after 24 h silencing. b Confocal micrograph of siCo. or sip21 CMFDA⁺ MDMs cocultured with CMTMR⁺ Jurkat cells. c–e Percentages of Jurkat (c), MOLT4 (d) or patient CD34⁺ AML (e) phagocytosis by siCo. or sip21 MDMs (**p = 0.0078, *p = 0.0312, ****p = 6.1e-5). f Phagocytosis (Fluorescence Units (FU)) of pHrodo Green E. coli bioparticle by siCo. or sip21 MDMs. g Percentages of CrCD47MOLT4 or CrCo.MOLT4 phagocytosis by MDMs transduced with control (Co.TD) or p21-expressing (p21TD) lentiviral vectors (LVs) after 72 h of transduction (**p = 0.0061, **p = 0.0053, **p = 0.0048). h–m SIRPα and p21 proteins (h, k) or mRNAs (i, j, l, m) in siCo. or sip21 MDMs (**p = 0.0024, **p = 0.0020) (h–j) or in Co.TD-MDMs or p21TD-MDMs (**p = 0.0063, *p = 0.0291) (k–m). n ChIP-qPCR assays performed with control MDMs, Co.TD-MDMs (**p = 0.0016) or p21TD-MDMs (**p = 0.0017, *p = 0.0194) immunoprecipitated with control IgG or anti-p21 antibodies and analyzed by qPCR at SIRPα promoter. o, p Luciferase activities in siCo. or sip21 MDMs (o) or in Co.TD-MDMs or p21TD-MDMs (p) transduced with a lentiviral vector expressing a luciferase reporter gene under the control of the SIRPα promoter (*p = 0.0431, *p = 0.0458). q–s MDMs derived from monocytes transduced with Co.TD and/or p21TD LVs and/or SIRPα-expressing LVs (SIRPαTD) assessed for the indicated proteins (q) and for phagocytosis of mCherry⁺ MOLT4 cells (**p = 0.0079, *p = 0.0472, ***p = 0.0002, ***p = 0.0004) (r, s). In a, b, h, k, q and r, the data are representative of n = 3 donors. In c, d, e and o, p, the data are donor matched from n = 8, n = 6, n = 9, n = 4 and n = 3 donors. In e, the CD34⁺ cells were from n = 6 AML patients. In i, j and g, l, m, n, s, the data are presented as the mean±SEM from n = 5 and n = 3 donors. Exact p-values are indicated and determined with two-tailed paired Wilcoxon (c, d, e), two- (i, j, l, n) or one-tailed (m) ratio-paired t, and one-tailed paired t (o, p) tests and two-way ANOVA with Sidak’s (g) or one-way ANOVA Tukey’s (s) multiple comparison tests. Source data are provided as a Source Data file.

Fig. 3. Prophylactic adoptive transfer of p21TD-Mos decreases the leukemic burden and prolongs mouse survival in a model of human T-ALL.

a Schematic representation showing prophylactic adoptive transfer of CFSE-labeled control (Co.TD), p21 (p21TD) and/or SIRPα (SIRPαTD) genetically engineered human monocytes (Mos) into NSG mice previously treated with total body irradiation (TBI), which were then engrafted with mCherry⁺ MOLT4 cells 7 days later. b–k Engrafted mice that received the indicated monocytes were assessed at 21 d to measure body (**p = 0.0079) (b, c) and spleen (*p = 0.00317) weights (d, e); the leukemic burdens in the blood, bone marrow (BM) and spleen by FACS (**p = 0.0079) (f, g, h) and in liver tissues by confocal microscopy (**p = 0.0079) (i, j); and survival (***p = 0.0003) (k). l Survival of engrafted mice that received indicated monocytes and were treated 21 d after monocyte transfer with control (Co.) or clodronate (Clodro)-containing liposomes (****p = 4.8e-9, ****p = 0.1.1e-5). m Survival of engrafted mice that received the indicated monocytes (****p = 3.2e-5, ****p = 7e-10, ****p = 2.1e-7, ****p = 1e-6). n Survival of engrafted mice that received Co.TD-Mos, p21TD-Mos, SIRPαTD-Mos or p21+SIRPαTD-Mos and were treated on d 15 after Mo transfer for 14 d with daily injections of isotype control (IgG) or anti-CD47 blocking antibodies (100 μg/mouse) (****p = 9.4e-12, p = 0.0942 (n.s.), ****p = 2.9e-7, ****p = 2.7e-6, ****p < 1e-15, ****p = 4.5e-14, ****p = 9e-10). In b, d and i, the data are representative of n = 5 mice/group. In c, e, f, g, h and j, the data are presented as the mean±SEM from n = 5 mice/group. In k, m, n and l, the survival data are from n = 5 and n = 6 mice/group. Exact p-values are indicated and determined with two-tailed unpaired Mann-Whitney (c, e, f, g, h, j) and log-rank Mantel–Cox (k, l, m, n), tests. Source data are provided as a Source Data file.

Fig. 4. Prophylactic adoptive transfer of p21TD-Mos triggers the proinflammatory activation of TAMs and prolongs the survival of human T-ALL-engrafted mice in an IFNγ-dependent manner.

a, b Confocal micrographs of phagocytic (mCherry⁺CFSE⁺) macrophages in the spleen (a) and CD163 membrane expression identified by FACS on sorted single-positive CFSE⁺ (Phago^-) MDMs or phagocytic mCherry⁺CFSE⁺ (Phago⁺) MDMs from the spleen and BM (*p = 0.033) (b) of mCherry⁺ MOLT4 cell-engrafted NSG mice that received Co.TD-Mos or p21TD-Mos, as shown in Fig. 3a, on d 21 after Mo transfer. c–e Percentages of iNOS-expressing (iNOS⁺) cells, determined by immunofluorescence staining, among Phago⁺ MDMs (c), MDMs (d) or Phago^- MDMs (****p = 4.6e-5, ****p = 5e-5, ***p = 0.0004) (e) sorted from tumor-free (d) or mCherry⁺ MOLT4 cell-engrafted (c, e) NSG mice that received Co.TD-Mos or p21TD-Mos; cells collected on d 21 and 35 after Mo transfer. f Percentages of iNOS⁺ cells among human TAMs (hCD14⁺hCD11b⁺hCD163⁺CFSE⁺), sorted from the BM and spleen of mCherry⁺ MOLT4 cell-engrafted NSG mice adoptively transferred with CFSE⁺ human Mo; cells collected on d 35 after Mo transfer. These cells were cocultured, as shown in Supplementary Fig. 16, in the bottom chambers of Transwell devices with Phago^- MDMs or Phago⁺ MDMs sorted from the spleen and BM of mCherry⁺ MOLT4 cell-engrafted NSG mice adoptively transferred with Co.TD-Mos or p21TD-Mos (d 35 after Mo transfer) or with control MDMs differentiated in vitro from Co.TD-Mos or p21TD-Mos (****p = 1.4e-8, ****p = 4.2e-5, ****p = 3.8e-10). g, h Schematic representation (g) and survival Fig. 4h (****p = 1.2e-7, ***p = 0.0002) (h) of mCherry⁺ MOLT4 cell-engrafted NSG mice that received Co.TD-Mos or p21TD-Mos and were treated 15 d after Mo transfer with isotype control (IgG) or anti-IFNγ blocking antibodies. In (a), the data are representative of n = 5 mice/group. In b, the data are mouse matched from n = 3 mice/group. In c–f, the data are presented as the mean±SEM from n = 3 mice/group. In h, the survival data are from n = 7 mice/group. Exact p-values are indicated and determined with one-way ANOVA with Friedman’s (b) and Sidak’s (f) or two-way ANOVA with Tukey’s (e) multiple comparison tests or determined with log-rank Mantel-Cox (h), test. Source data are provided as a Source Data file.

Fig. 5. Curative adoptive transfer of p21TD-Mos prolongs mouse survival in diagnosed or relapsed T-ALL-derived PDX models.

a Schematic representation showing curative adoptive transfer of Co.TD-Mos or p21TD-Mos into NSG mice engrafted with T-ALL PDXs. b–d Survival of mice engrafted with T-ALL PDX#1 (****p = 1.1e-5) (b), PDX#2 (****p = 1.9e-6) (c) or PDX#3 (**p = 0.003) (d) and treated curatively, as shown in a, with Co.TD-Mos or p21TD-Mos. PDX#1 and PDX#2 were from the same patient but isolated at the diagnosis and relapse stages, respectively. In b–d, the survival data are from n = 5 mice/group. Exact p-values are indicated and determined with the log-rank Mantel-Cox (b–d) test. Source data are provided as a Source Data file.