The integrated stress response promotes immune evasion through lipocalin 2

Nature News · Feb 18, 2026 · Collected from RSS

Full Article

MainImmune checkpoint inhibitors (ICIs) have transformed anti-cancer therapy and become a first-line treatment for several cancers4,5. However, many solid tumours do not respond to standard-of-care ICIs6,7,8. Gaining a better understanding of the mechanisms that underlie immune evasion of ICI-refractory cancers is therefore essential to improve anti-cancer treatments8,9.Solid tumours progressively reshape their immediate tumour microenvironment (TME), producing features that affect the fitness and characteristics of cells within the TME, such as hypoxia, nutrient scarcity and waste accumulation1,10,11,12,13. In response, cancer cells activate the integrated stress response (ISR), an evolutionarily conserved cellular defence system that is triggered by diverse stressors, including misfolded proteins, amino acid starvation and mitochondrial dysfunction1,3,11,14,15,16,17,18,19. The central driver of ISR activation is the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which reduces global translation while selectively promoting the translation of activating transcription factor 4 (ATF4), a central regulator of transcriptional programs that support adaptation and survival1. The ISR and its master transcriptional effector, ATF4, are emerging as key players that respond to several intrinsic stressors during tumorigenesis and contribute to therapy resistance1,2,3,15,17,18,20.Although these TME features also profoundly influence anti-tumour immunity, the cancer-cell-extrinsic contribution of the ISR and the ATF4 axis to this process remains mainly unknown10,11,16,21. So far, most studies investigating the role of ATF4 in cancer cells have been performed in vitro or in immunodeficient animals, and consequently its potential role in adaptation and resistance to host immune responses during tumorigenesis has not been defined.Loss of ATF4 impedes tumour growth in miceTo evaluate the importance of ATF4 in tumorigenesis, we used a Kras and p53 (also known as Trp53) mutant (KrasLSL-G12D/+;p53fl/fl; hereafter, KP) genetically engineered mouse model (GEMM) of lung adenocarcinoma (LUAD), with conditional CRISPR–Cas9-based loss of Atf4 (sgAtf4) or control (sgTom) (Fig. 1a,b). Loss of Atf4 decreased the tumour burden, as compared with Atf4 wild-type (WT) tumours (Fig. 1c). To dissect the importance of ATF4 in tumour progression, we generated isogenic Atf4 knockout (Atf4KO) and WT (Atf4WT) lung cancer KP primary cell lines (Extended Data Fig. 1a) and transplanted them subcutaneously (s.c.) into syngeneic immunocompetent C57BL/6J mice (Fig. 1d and Extended Data Fig. 1b,c). In agreement with the GEMM experiment (Fig. 1c) and previous studies2,3,15, ATF4-deficient tumours grew significantly more slowly than control tumours did, despite showing no differences in markers of cellular proliferation or apoptosis (Extended Data Fig. 1d) and no differences in proliferation in vitro (Extended Data Fig. 1a), excluding the possibility that the intrinsic properties of the ATF4-deficient cells caused their growth delay in immunocompetent mice. Notably, Atf4KO tumours grew as well as Atf4WT tumours did in immunodeficient NOD scid IL2Rγnull (NSG) mice (Fig. 1e and Extended Data Fig. 1c). Consistent with these findings, ATF4 was required for tumour growth in two additional syngeneic models—Lewis lung carcinoma (LLC) (Fig. 1f and Extended Data Fig. 1e) and B16F10 melanoma (Fig. 1g and Extended Data Fig. 1f). By contrast, loss of ATF4 in B16F10 tumours had no effect on tumour growth in immunodeficient NU/J (nude) mice (Fig. 1h and Extended Data Fig. 1g). Furthermore, pharmacological inhibition of ATF4 with integrated stress response inhibitor (ISRIB), which allosterically antagonizes phosphorylated eIF2α22 (Fig. 1i), resulted in a significant reduction in tumour growth and prolonged survival in immunocompetent mice (Fig. 1j,k and Extended Data Fig. 1h,i), but not in immunodeficient NSG mice (Fig. 1l and Extended Data Fig. 1j,k). Collectively, these results show that ATF4 is required for tumour progression by curtailing anti-tumour immunity in several cancers.Fig. 1: ATF4 is essential for tumour growth in immunocompetent mice.a, Scheme of the GEMM KrasLSL-G12D/+;p53fl/fl;Rosa26LSL-Cas-P2A-GFP model, in which we infected mice with bifunctional lentiviruses (pUSEC), which express Cre recombinase and sgRNAs for conditional CRISPR–Cas9-based loss of Atf4 (sgAtf4) or control (sgTom). b, Representative IHC of lung tumours stained for ATF4. Arrowheads indicate ATF4-positive cells. Scale bars, 50 μm. c, Tumour burden 14 weeks after infection with lentiviruses carrying guides against control sgTom (n = 7) and sgAtf4 (n = 8). Two-tailed t-test. d,e, Tumour growth in C57BL/6J (Atf4WT n = 4, Atf4KO n = 6) (d) and NSG (Atf4WT n = 10, Atf4KO n = 11) (e) mice injected s.c. with KP primary LUAD cells (Atf4WT or Atf4KO). Data were analysed by a repeated-measures two-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test. NS, not significant. f, Tumour growth of LLC s.c. injected cell lines with Atf4WT (n = 13) or Atf4KO (n = 15) status in C57BL/6J mice. Data were analysed by a repeated-measures two-way ANOVA with Fisher’s LSD test. g,h, Tumour growth of B16F10 s.c. transplanted tumours with Atf4WT (n = 17) or Atf4KO (n = 8) status in C57BL/6J mice (g) and nude mice (h) (Atf4WT n = 8, Atf4KO n = 8). Data were analysed by a repeated-measures two-way ANOVA with Šidák’s multiple-comparisons test. i, Mechanism of ISR pharmacological inhibition by ISRIB, which disrupts the dimerization of phospho (p)-eIF2α and the subsequent accumulation of ATF4. j, Luminescence of orthotopically transplanted KP tumours in the lungs of C57BL/6J mice treated daily with 2.5 mg per kg ISRIB (n = 7) or vehicle (n = 5). Data were analysed by a repeated-measures two-way ANOVA with Šidák’s multiple-comparisons test. k, Survival of C57BL/6J mice with orthotopic KP tumours (vehicle n = 11, ISRIB n = 12). Data were analysed by a log-rank (Mantel–Cox) test. l, Luminescence of orthotopically transplanted KP tumours in the lungs of NSG mice treated with ISRIB (n = 4) or vehicle (n = 3). Data were analysed by a repeated-measures two-way ANOVA with Šidák’s multiple-comparisons test. Data are mean ± s.e.m. The illustrations in a,i were created in BioRender. Bossowski, J. (2025) https://BioRender.com/wryu8nk.Source dataFull size imageLCN2 mediates ATF4-dependent immune evasionTo identify ATF4-driven immunosuppressive factors, we performed a pooled negative-selection CRISPR–Cas9 screen in a syngeneic C57BL/6J mouse using a custom library of 477 putative ATF4 targets23 (Fig. 2a and Supplementary Table 1). We identified candidate genes whose single guide RNAs (sgRNAs) were preferentially depleted in the immunocompetent hosts—including Atf4, consistent with our previous data (Fig. 2b, Extended Data Fig. 2a and Supplementary Table 2). Moreover, we identified lipocalin 2 (Lcn2) as one of the most significantly depleted genes (Fig. 2b and Extended Data Fig. 2a).Fig. 2: LCN2 loss slows tumour progression in immunocompetent mice.a, Schematic of the CRISPR library experimental set-up to screen ATF4-regulated genes in vivo and in vitro. b, Differential score of gene dropout frequency between KP tumours implanted into immunodeficient NSG versus immunocompetent C57BL/6J mice and isolated 12 days after s.c. transplantation. c,d, Growth of Lcn2WT and Lcn2KO KP tumours transplanted into C57BL/6J mice (n = 8 in Lcn2WT, n = 10 in Lcn2KO group) (c) and NSG immunodeficient mice (n = 24 in Lcn2WT, n = 13 in Lcn2KO group) (d). Data were analysed by a repeated-measures two-way ANOVA with Šidák’s multiple-comparisons test. e, Normalized luminescence signal from orthotopic KP tumours expressing Lcn2WT (n = 6), Lcn2KO (n = 5), or Lcn2KO KP cells with ectopic overexpression of mouse Lcn2 (mLcn2, n = 6) in C57BL/6J mice. Data were analysed by a repeated-measures two-way ANOVA with Fisher’s LSD test. f, Relative tumour growth in C57BL/6J mice injected s.c. with KP cells with Atf4 KO or WT status, with ectopic overexpression of control (Ctr) vector or Lcn2 (Lcn2OE) (Atf4WTCtr n = 7, Atf4WTLcn2OE n = 7, Atf4KOCtr n = 7, Atf4KOLcn2OE n = 6). Data were analysed by a repeated-measures two-way ANOVA with Šidák’s multiple-comparisons test. g, Growth, in C57BL/6J mice, of s.c. implanted Lcn2KO KP tumours ectopically expressing empty vector (n = 9), Lcn2WT (n = 9), Lcn2 lacking the secretion signal peptide sequence (sec_del, n = 14) and two independent triple mutant Lcn2 constructs expressing Lcn2 with substituted amino acids responsible for binding to iron (Lcn2Y127A/K147A/K156A, n = 6; Lcn2R103G/K147A/K156A, n = 7). Data were analysed by a repeated-measures two-way ANOVA with Fisher’s LSD test. h, Scheme of the KPC GEMM KrasLSL-G12D/+;p53fl/fl;Rosa26LSL-Cas-P2A-GFP model, in which we infected mice with bifunctional lentiviruses (pUSEC), which express Cre recombinase and double-guides for conditional CRISPR–Cas9-based loss of Lcn2 (sgLcn2) or control (sgNeo). i, Representative magnetic resonance imaging (MRI) images 14 and 21 weeks after infection of KPC mice with sgLcn2 or sgNeo lentivirus. j, Histological tumour burden (LUAD) 24 weeks after tumour initiation (sgNeo n = 7, sgLcn2 n = 6). Data were analysed by a two-tailed t-test. k, Scheme of the orthotopic PDAC transplant mouse model. l, Tumour weight of PDAC (KPC7) tumours five weeks after orthotopic transplantation with Lcn2WT (n = 6) or Lcn2KO (n = 7) cells. Data were analysed by a two-tailed t-test. Data are mean ± s.e.m. The illustrations in a,h,k were created in BioRender. Bossowski, J. (2025) https://BioRender.com/wryu8nk.Source dataFull size imageLCN2 is a secreted glycoprotein that has been implicated in inflammatory responses24,25. We performed a series of in vivo experiments using Lcn2 knockout (Lcn2KO) and wild-type (Lcn2WT) KP cells. Similar to our findings for ATF4 (Fig. 1d–h), loss of LCN2 resulted in a reduction in tumo