Targeting ATAD3A-PINK1-mitophagy axis overcomes chemoimmunotherapy resistance by redirecting PD-L1 to mitochondria

Abstract

Only a small proportion of patients with triple-negative breast cancer benefit from immune checkpoint inhibitor (ICI) targeting PD-1/PD-L1 signaling in combination with chemotherapy. Here, we discovered that therapeutic response to ICI plus paclitaxel was associated with subcellular redistribution of PD-L1. In our immunotherapy cohort of ICI in combination with nab-paclitaxel, tumor samples from responders showed significant distribution of PD-L1 at mitochondria, while non-responders showed increased accumulation of PD-L1 on tumor cell membrane instead of mitochondria. Our results also revealed that the distribution pattern of PD-L1 was regulated by an ATAD3A-PINK1 axis. Mechanistically, PINK1 recruited PD-L1 to mitochondria for degradation via a mitophagy pathway. Importantly, paclitaxel increased ATAD3A expression to disrupt proteostasis of PD-L1 by restraining PINK1-dependent mitophagy. Clinically, patients with tumors exhibiting high expression of ATAD3A detected before the treatment with ICI in combination with paclitaxel had markedly shorter progression-free survival compared with those with ATAD3A-low tumors. Preclinical results further demonstrated that targeting ATAD3A reset a favorable antitumor immune microenvironment and increased the efficacy of combination therapy of ICI plus paclitaxel. In summary, our results indicate that ATAD3A serves not only as a resistant factor for the combination therapy of ICI plus paclitaxel through preventing PD-L1 mitochondrial distribution, but also as a promising target for increasing the therapeutic responses to chemoimmunotherapy.

Fig. 1. Mitochondrial distribution of PD-L1 is associated with therapeutic response to ICI.

a Quantitative analysis of pre- and post-chemotherapy IHC score of PD-L1 in human TNBC. IHC score of PD-L1 was determined by IOD value/area. Biopsies were taken as pre-chemotherapy samples at their first to doctor and intraoperative specimens were taken as post-chemotherapy samples after neoadjuvant treatment. Neoadjuvant chemotherapy included TE regimen (paclitaxel/docetaxel, epirubicin) or TEC regimen (docetaxel, epirubicin and cyclophosphamide) (n = 15, paired two-sided t-test). b Left, representative IHC staining for different subcellular patterns of PD-L1 in TNBC samples pre- or post-chemotherapy. Patient 15 represented the cases with increased cell membranal localization of PD-L1 (the CM pattern). Patient 7 represented the cases with cytoplasmic localization of PD-L1 (the CYTO pattern). Right, the percentage of patients with different PD-L1 subcellular distribution. Scale bars, 50 μm. c, d Left, co-localization of PD-L1 (green) and TOM20-labeled mitochondria (red) with or without 20 nM paclitaxel treatment for 12 h in MDA-MB-231 (c) and BT549 cells (d). Arrowheads, co-localization. Scale bars, 20 μm and 2 μm (inset). Right, the percentage of PD-L1 co-localized with TOM20 (n = 5 fields, t-test). e EM images of BT549 cells stably expressing PD-L1-APEX2 or control cells. Arrowheads, positive signals of PD-L1-APEX2 on outer mitochondrial membrane. Scale bars, 500 nm. f, g Left, co-localization of PD-L1 (green) and TOM20-labeled mitochondria (red) in TNBC specimens from a non-responder (f) or a responder (g) pre- or post-chemotherapy in the immunotherapy cohort from arm C of the FUTURE trial. These patients with refractory metastatic TNBC had received standard chemotherapy including taxol for previous antitumor regimens and underwent anti-PD-1 antibodies plus nab-paclitaxel. Intraoperative specimens and progressive disease biopsies were taken as pre-chemotherapy samples and post-chemotherapy samples respectively. Arrowheads, examples of co-localization. Scale bars, 20 μm and 2 μm (inset). Right, the percentage of PD-L1 co-localized with TOM20 (n = 9 fields, t-test). h The percentages of patients with CM or MITO signature in four different groups on the basis of their best responses to anti-PD-1 plus nab-paclitaxel combination therapy. PD, progressive disease; SD, stable disease; PR, partial response; CR, complete response (n = 30, Fisher’s exact test). i Kaplan-Meier analysis of the progression-free survival of TNBC patients with CM or MITO signature who received combination therapy (n = 16). Data were derived from the immunotherapy cohort of this study (Kaplan-Meier method and log-rank test). Data are representative of at least two independent experiments and are shown as means ± SD. See also Supplementary information, Fig. S1.

Fig. 2. ATAD3A prevents PD-L1 distribution to mitochondria.

a Venn diagram depicting the overlapped common genes in the mitochondrion component set (GOCC 0005739), mitochondrion organization set (GOBP 0007005), and immune response set (GOBP 0006955). b Kaplan-Meier analysis of the OS of glioblastoma patients who received anti-PD-1 antibody treatment with high or low ATAD3A mRNA expression (n = 29). Data were derived from GSE121810 cohort. Cutoff of ATAD3A expression was determined by the median (Kaplan-Meier method and log-rank test). c Kaplan-Meier analysis of the OS of TNBC patients with high or low ATAD3A transcriptional level. Data were derived from TCGA database. Cutoff of ATAD3A expression was determined by the median (Kaplan-Meier method and log-rank test). d Kaplan-Meier analysis of the OS (left) and PFS (right) of TNBC patients with high or low ATAD3A expression from the multicentric clinical cohort (Kaplan-Meier method and log-rank test). These patients underwent radical mastectomy or modified mastectomy and received the standard chemotherapy. e, f Left, co-localization of PD-L1 (green) and TOM20-labeled mitochondria (red) in TNBC specimens with high (e) or low (f) ATAD3A expression pre- or post-chemotherapy including taxol in immunotherapy cohort. Arrowheads, co-localization. Scale bars, 20 μm and 2 μm (inset). Right, the percentage of PD-L1 co-localized with TOM20 (n = 9 fields, t-test). g The correlation between ATAD3A levels and the number of tumor-infiltrating CD8⁺ T cells in TNBC tumors (n = 67). IHC score was quantified by Image-Pro Plus software 6.0, three to five fields per section (Pearson correlation analysis). h Representative images (left) and IHC score (right) of ATAD3A expression in tumors from non-responders and responders before anti-PD-1 antibody plus nab-paclitaxel therapy in immunotherapy cohort. Scale bars, 50 μm (n = 30, Mann-Whitney U test). i Patients with different responses in the immunotherapy cohort. Subjects were colored by their responses to combination therapy. CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. y-axis was determined by IHC score of ATAD3A by logarithm to the base 2. Cutoff of ATAD3A expression was determined by the median (n = 30, Fisher’s exact test). j Kaplan-Meier analysis of the progression-free survival of TNBC patients with high or low ATAD3A expression receiving combination therapy (n = 16). Data are derived from the immunotherapy cohort (Kaplan-Meier method and log-rank test). See also Supplementary information, Fig. S2.

Fig. 3. Involvement of ATAD3A-PINK1 mitophagy in the distribution and degradation of PD-L1.

a Left, immunostaining of PD-L1 (green) and TOM20-labeled mitochondria (red) in BT549 human TNBC cells with or without ATAD3A-knockdown (shATAD3A#1 and shATAD3A#2). Scale bars, 20 μm and 2 μm (inset). Right, the percentage of PD-L1 co-localized with TOM20 (n = 5 fields, t-test). b Immunoblot of PD-L1 in the cytoplasm and mitochondria of control and ATAD3A-knockdown BT549 cells. TOM20 and Tubulin were used as mitochondria and cytoplasm protein controls, respectively. Cyto, cytoplasm; mito, mitochondria. c Flow cytometry (left) and quantification (right) of surface PD-L1 in control and ATAD3A-knockdown BT549 cells (n = 3, one-way ANOVA). d Venn diagram depicting overlapped genes for the interaction protein of ATAD3A set (BioGRID, RP5-832C2.1), the protein localization to mitochondrion set (GOBP 0070585) and the intrinsic component of mitochondrial membrane (GOCC 0098573). e Immunoblot of PINK1 in control and ATAD3A-knockdown BT549 cells. f Immunoblot of PD-L1 and PINK1 in HEK293T cells overexpressing PD-L1 (OE-PD-L1) and control cells (OE-Control), assessed after immunoprecipitation with immunoglobulin G (IgG) or antibody to PINK1. g Protein direct interaction analysis of the intracellular domain of PD-L1 (ICD) and PINK1 in vitro. Purified Flag-labeled full-length PINK1 was incubated with Biotin-labeled PD-L1 ICD domain, followed by streptavidin pull-down and immunoblot. h Schematic diagram of Flag-labeled full-length (FL) and truncated mutants with indicated domains (amino acids 1–155, amino acids 156–320, amino acids 321–509, amino acids 510–581) of PINK1. MTS, mitochondrial targeting sequence; N-lobe, kinase domain N; C-lobe, kinase domain C; CTD, C-terminal domain. i Protein direct interaction analysis of the intracellular domain of PD-L1 (ICD) and truncated PINK1 mutants in vitro. Purified Flag-labeled full-length and truncated PINK1 were incubated with Biotin-labeled PD-L1 ICD domain, followed by streptavidin pull-down and immunoblot. The estimated size of PINK1-4 (amino acids 510–581) which did not express in HEK293T cells was labeled with asterisk. j Immunoblot of PD-L1 in the cytoplasm and mitochondria of MDA-MB-231 cells with or without PINK1-knockdown (shPINK1#1 and shPINK1#2). TOM20 and Tubulin were used as mitochondria and cytoplasm protein controls. Cyto, cytoplasm; mito, mitochondria. k Left, co-localization of PD-L1 (green) and TOM20 (red) in control, ATAD3A knockdown, PINK1 knockdown or ATAD3A and PINK1 double knockdown BT549 cells. Scale bars, 20 μm and 2 μm (inset). Right, the percentage of PD-L1 co-localized with TOM20 (n = 5 fields, one-way ANOVA). l Immunoblot of PD-L1 in the cytoplasm and mitochondria of control, ATAD3A-knockdown, PINK1-knockdown or ATAD3A and PINK1 double knockdown BT549 cells. m Immunoblot of indicated proteins in PD-L1-transfected HEK293T cells with or without PINK1 overexpression. n Immunoblot of PD-L1 in control and PINK1-knockdown (shPINK1#1 and shPINK1#2) BT549 cells. o Immunoblot of PD-L1 in BT549 cells transfected with control shRNA or shATAD3A (shATAD3A#1 and shATAD3A#2). p Immunoblot of total PD-L1 in control, ATAD3A-knockdown, PINK1-knockdown or ATAD3A and PINK1 double knockdown BT549 cells. q Immunoblot of PD-L1 in control and ATAD3A-knockdown BT549 cells treated with 20 μM CHX for indicated times. h, hours. r Quantification of PD-L1 intensity in immunoblot in control and ATAD3A-knockdown BT549 cells. s Immunoblot of PD-L1 in control and ATAD3A-knockdown MDA-MB-231 cells incubated with 20 nM BafA1 for indicated times. h, hours. Data are representative of at least two independent experiments and are shown as means ± SD. See also Supplementary information, Figs. S3 and S4.

Fig. 4. Paclitaxel upregulates ATAD3A to suppress PINK1-dependent mitophagy.

a Left, qRT-PCR of ATAD3A mRNA in BT549 cells in the presence of paclitaxel (10 nM or 20 nM) for 12 h (n = 3, one-way ANOVA). Right, immunoblot of ATAD3A in BT549 cells incubated with 20 nM paclitaxel for 12 h and 24 h. b Immunoblot of PINK1 after treatment with 20 nM paclitaxel for 12 h and 24 h in TNBC cell lines. c, d Quantification of mitochondrial mass (c) and the percentage of dysfunctional mitochondria (d) in TNBC cells in the presence of paclitaxel (20 nM or 50 nM) for 16 h. DMSO was used as a control (n = 3, one-way ANOVA). e Left, EM images of mitochondria in BT549 cells with 20 nM paclitaxel or DMSO treatment for 16 h. Scale bars, 5 μm and 500 nm (inset). Right, quantification of the number of dysfunctional mitochondria per cell (n = 10, t-test). f Left, co-localization of PD-L1 (green), mitochondria (white) and LAMP1-labeled lysosomes (red) with or without 20 nM paclitaxel treatment for 12 h in MDA-MB-231 cells. Mitochondria were labeled with MitoTracker Deep Red dye. Arrowheads indicate LAMP1-labeled lysosomes containing co-localization of PD-L1 and mitochondria. Airyscan images were shown. Scale bars, 20 μm and 2 μm (inset). Right, the percentages of lysosomes containing mitochondria or containing co-localization of PD-L1 and mitochondria per field (n = 15 fields, t-test). g Immunoblot of PD-L1 in BT549 cells with 20 nM paclitaxel treatment for indicated times. h, hours. h Flow cytometry (left) and quantification (right) of surface PD-L1 in BT549 cells treated with 20 nM paclitaxel for indicated times. h, hours (n = 3, one-way ANOVA). i Immunoblot of PD-L1 in TNBC cells treated with 20 nM BafA1 alone, 20 nM paclitaxel alone or both for 12 h. j Immunoblot of PD-L1 in control and PINK1-overexpressing BT549 cells treated with or without 20 nM paclitaxel for 24 h. k Immunoblot of PD-L1 in control and ATAD3A-knockdown BT549 cells treated with 20 nM paclitaxel for indicated times. h, hours. l, m Left, co-localization of PD-L1 (green), TOM20-labeled mitochondria (red) and LAMP1-labeled lysosomes (white) in tumor specimens from a patient with complete response (l) or a patient with progressive disease (m) pre- or post-chemotherapy in the immunotherapy cohort. Arrowheads, co-localization. Scale bars, 20 μm and 2 μm (inset). Right, the percentage of tumor cells containing co-localization among PD-L1, TOM20 and LAMP1 per field (n = 9 fields, t-test). Data are representative of at least two independent experiments and are shown as means ± SD. See also Supplementary information, Figs. S5 and S6.

Fig. 5. Depletion of Atad3a evokes a favorable tumor immune microenvironment and improves the efficacy of ICI.

a–h BALB/c mice were inoculated orthotopically with 5 × 10⁴ 4T1 cells transfected with control shRNA (shControl) or shRNA for Atad3a (shAtad3a#1 and shAtad3a#2). a, b The endpoint tumor images (a) and volume (b) of tumors formed by control and Atad3a-knockdown cells in BALB/c mice (n = 6, one-way ANOVA). c Left, IHC staining of Atad3a and PD-L1 on serial sections of tumors formed by control and Atad3a-knockdown cells. Scale bars, 50 μm. Right, IHC score of Atad3a in control and Atad3a-knockdown tumors (n = 6 fields, t-test). d IHC score of PD-L1 in control and Atad3a-knockdown tumors (n = 6 fields, t-test). e Quantification of the percentage of tumor-infiltrating CD8⁺ T cells in tumors formed by control and Atad3a-knockdown cells by flow cytometry (n = 5, t-test). f Quantification of the percentages of tumor-infiltrating IFNγ⁺CD8⁺ T cells and IFNγ⁺CD4⁺ T cells by flow cytometry (n = 5, t-test). g Ratio of CD8⁺ cytotoxic T lymphocytes to CD4⁺CD25⁺Foxp3⁺ T_reg cells (n = 5, t-test). h Quantification of the percentages of PD-1⁺TIM-3⁺CD8⁺ T cells (left) and PD-1⁺TIM-3⁺CD4⁺ T cells (right) by flow cytometry (n = 5, t-test). i–m BALB/c mice were inoculated orthotopically with 5 × 10⁴ 4T1 cells transfected with control shRNA (shControl) or shRNA specific for Atad3a (shAtad3a), Pink1 (shPink1) or both (shAtad3a + shPink1). i, Left, the endpoint images of tumors formed by control, Atad3a-knockdown, Pink1-knockdown or Atad3a and Pink1 double knockdown 4T1 cells in BALB/c mice. Right, immunoblot of Atad3a and Pink1 in these 4T1 cells. j The volume of tumors mentioned above (n = 6, one-way ANOVA). k Quantification of the percentage of tumor-infiltrating CD8⁺ T cells by flow cytometry (n = 5, one-way ANOVA). l Quantification of the percentage of tumor-infiltrating IFNγ⁺CD8⁺ T cells by flow cytometry (n = 5, one-way ANOVA). m Quantification of the percentage of PD-1⁺TIM-3⁺CD8⁺ T cells by flow cytometry (n = 5, one-way ANOVA). n–s 4T1 tumors formed by control and Atad3a-knockdown cells were established orthotopically in BALB/c mice and received vehicle, anti-PD-L1 antibody (PD-L1 mAb), paclitaxel (PTX) or combined anti-PD-L1 antibody with paclitaxel treatment (PD-L1 mAb + PTX). IgG2b and saline were used as controls. n Experimental protocol. o, p The endpoint tumor images (o) and the volume (p) of tumors (n = 6, one-way ANOVA). q–s Quantification of the percentages of tumor-infiltrating CD8⁺ T cells (q), IFNγ⁺CD8⁺ T cells (r) and PD-1⁺TIM-3⁺CD8⁺ T cells (s) in 4T1 tumors formed by control and Atad3a-knockdown cells received treatments as described above, determined by flow cytometry (n = 5, one-way ANOVA). t Schematic model. Patients with PD-L1-positive TNBC could be divided into two groups based on ATAD3A expression. Patients with ATAD3A-high tumors might respond more poorly to ICIs plus paclitaxel therapy, and inhibition of ATAD3A is required to improve clinical outcome. Patients with ATAD3A-low tumors might benefit significantly from ICIs plus paclitaxel combination therapy. See also Supplementary information, Figs. S7–S9.