Targeting autophagy overcomes cancer-intrinsic resistance to CAR-T immunotherapy in B-cell malignancies

Abstract

Background: Chimeric antigen receptor T (CAR-T) therapy has substantially revolutionized the clinical outcomes of patients with hematologic malignancies, but the cancer-intrinsic mechanisms underlying resistance to CAR-T cells remain yet to be fully understood. This study aims to explore the molecular determinants of cancer cell sensitivity to CAR-T cell-mediated killing and to provide a better understanding of the underlying mechanisms and potential modulation to improve clinical efficacy.

Methods: The human whole-genome CRISPR/Cas9-based knockout screening was conducted to identify key genes that enable cancer cells to evade CD19 CAR-T-cell-mediated killing. The in vitro cytotoxicity assays and evaluation of tumor tissue and bone marrow specimens were further conducted to confirm the role of the key genes in cancer cell susceptibility to CAR-T cells. In addition, the specific mechanisms influencing CAR-T cell-mediated cancer clearance were elucidated in mouse and cellular models.

Results: The CRISPR/Cas9-based knockout screening showed that the enrichment of autophagy-related genes (ATG3, BECN1, and RB1CC1) provided protection of cancer cells from CD19 CAR-T cell-mediated cytotoxicity. These findings were further validated by in vitro cytotoxicity assays in cells with genetic and pharmacological inhibition of autophagy. Notably, higher expression of the three autophagy-related proteins in tumor samples was correlated with poorer responsiveness and worse survival in patients with relapsed/refractory B-cell lymphoma after CD19 CAR-T therapy. Bulk RNA sequencing analysis of bone marrow samples from B-cell leukemia patients also suggested the clinical relevance of autophagy to the therapeutic response and relapse after CD19 CAR-T cell therapy. Pharmacological inhibition of autophagy and knockout of RB1CC1 could dramatically sensitize tumor cells to CD19 CAR-T cell-mediated killing in mouse models of both B-cell leukemia and lymphoma. Moreover, our study revealed that cancer-intrinsic autophagy mediates evasion of CAR-T cells via the TNF-α-TNFR1 axis-mediated apoptosis and STAT1/IRF1-induced chemokine signaling activation.

Conclusions: These findings confirm that autophagy signaling in B-cell malignancies is essential for the effective cytotoxic function of CAR-T cells and thereby pave the way for the development of autophagy-targeting strategies to improve the clinical efficacy of CAR-T cell immunotherapy.

Keywords

CAR-T resistance / Autophagy / Immune evasion / Apoptosis / Chemotaxis

References

[1] Salter AI, Pont MJ, Riddell SR. Chimeric antigen receptor-modified T cells: CD19 and the road beyond. Blood. 2018;131(24):2621-9.

[2] Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med. 2018;378(5):439-48.

[3] Park JH, Rivière I, Gonen M, Wang X, Sénéchal B, Curran KJ, et al. Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia. N Engl J Med. 2018;378(5):449-59.

[4] Larson RC, Maus MV. Recent advances and discoveries in the mechanisms and functions of CAR T cells. Nat Rev Cancer. 2021;21(3):145-61.

[5] Fraietta JA, Lacey SF, Orlando EJ, Pruteanu-Malinici I, Gohil M, Lundh S, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med. 2018;24(5):563-71.

[6] Orlando EJ, Han X, Tribouley C, Wood PA, Leary RJ, Riester M, et al. Genetic mechanisms of target antigen loss in CAR19 therapy of acute lymphoblastic leukemia. Nat Med. 2018;24(10):1504-6.

[7] Sotillo E, Barrett DM, Black KL, Bagashev A, Oldridge D, Wu G, et al. Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy. Cancer Discov. 2015;5(12):1282-95.

[8] Ledererova A, Dostalova L, Kozlova V, Peschelova H, Ladungova A, Culen M, et al. Hypermethylation of CD19 promoter enables antigen-negative escape to CART-19 in vivo and in vitro. J Immunother Cancer. 2021;9(8):e002352.

[9] Chen J, López-Moyado IF, Seo H, Lio CJ, Hempleman LJ, Sekiya T, et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature. 2019;567(7749):530-4.

[10] Cherkassky L, Morello A, Villena-Vargas J, Feng Y, Dimitrov DS, Jones DR, et al. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition. J Clin Invest. 2016;126(8):3130-44.

[11] Gulati P, Rühl J, Kannan A, Pircher M, Schuberth P, Nytko KJ, et al. Aberrant Lck Signal via CD28 Costimulation Augments Antigen-Specific Functionality and Tumor Control by Redirected T Cells with PD-1 Blockade in Humanized Mice. Clin Cancer Res. 2018;24(16):3981-93.

[12] Rafiq S, Yeku OO, Jackson HJ, Purdon TJ, van Leeuwen DG, Drakes DJ, et al. Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo. Nat Biotechnol. 2018;36(9):847-56.

[13] Shah NN, Fry TJ. Mechanisms of resistance to CAR T cell therapy. Nat Rev Clin Oncol. 2019;16(6):372-85.

[14] Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc. 2017;12(4):828-63.

[15] Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343(6166):84-7.

[16] Neff EP. CRISPR takes genetic screens forward. Lab Anim (NY). 2020;49(1):13-6.

[17] Lawson KA, Sousa CM, Zhang X, Kim E, Akthar R, Caumanns JJ, et al. Functional genomic landscape of cancer-intrinsic evasion of killing by T cells. Nature. 2020;586(7827):120-6.

[18] Manguso RT, Pope HW, Zimmer MD, Brown FD, Yates KB, Miller BC, et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature. 2017;547(7664):413-8.

[19] Hart T, Chandrashekhar M, Aregger M, Steinhart Z, Brown KR, MacLeod G, et al. High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities. Cell. 2015;163(6):1515-26.

[20] Liu D, Zhao X, Tang A, Xu X, Liu S, Zha L, et al. CRISPR screen in mechanism and target discovery for cancer immunotherapy. Biochim Biophys Acta Rev Cancer. 2020;1874(1):188378.

[21] Patel SJ, Sanjana NE, Kishton RJ, Eidizadeh A, Vodnala SK, Cam M, et al. Identification of essential genes for cancer immunotherapy. Nature. 2017;548(7669):537-42.

[22] Larson RC, Kann MC, Bailey SR, Haradhvala NJ, Llopis PM, Bouffard AA, et al. CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours. Nature. 2022;604(7906):563-70.

[23] Singh N, Lee YG, Shestova O, Ravikumar P, Hayer KE, Hong SJ, et al. Impaired Death Receptor Signaling in Leukemia Causes Antigen-Independent Resistance by Inducing CAR T-cell Dysfunction. Cancer Discov. 2020;10(4):552-67.

[24] Dufva O, Koski J, Maliniemi P, Ianevski A, Klievink J, Leitner J, et al. Integrated drug profiling and CRISPR screening identify essential pathways for CAR T-cell cytotoxicity. Blood. 2020;135(9):597-609.

[25] Li W, Xu H, Xiao T, Cong L, Love MI, Zhang F, et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 2014;15(12):554.

[26] Rajabzadeh A, Hamidieh AA, Rahbarizadeh F. Spinoculation and retronectin highly enhance the gene transduction efficiency of Mucin-1-specific chimeric antigen receptor (CAR) in human primary T cells. BMC Mol Cell Biol. 2021;22(1):57.

[27] Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545-50.

[28] Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611-22.

[29] Galluzzi L, Green DR. Autophagy-Independent Functions of the Autophagy Machinery. Cell. 2019;177(7):1682-99.

[30] Gatica D, Lahiri V, Klionsky DJ. Cargo recognition and degradation by selective autophagy. Nat Cell Biol. 2018;20(3):233-42.

[31] Xia H, Green DR, Zou W. Autophagy in tumour immunity and therapy. Nat Rev Cancer. 2021;21(5):281-97.

[32] Kearney CJ, Vervoort SJ, Hogg SJ, Ramsbottom KM, Freeman AJ, Lalaoui N, et al. Tumor immune evasion arises through loss of TNF sensitivity. Sci Immunol. 2018;3(23):eaar3451.

[33] Vredevoogd DW, Kuilman T, Ligtenberg MA, Boshuizen J, Stecker KE, de Bruijn B, et al. Augmenting Immunotherapy Impact by Lowering Tumor TNF Cytotoxicity Threshold. Cell. 2019;178(3):585-99.e15.

[34] Mariño G, Niso-Santano M, Baehrecke EH, Kroemer G. Self-consumption: the interplay of autophagy and apoptosis. Nat Rev Mol Cell Biol. 2014;15(2):81-94.

[35] Martínez-Lostao L, Anel A, Pardo J. How Do Cytotoxic Lymphocytes Kill Cancer Cells? Clin Cancer Res. 2015;21(22):5047-56.

[36] Bertheloot D, Latz E, Franklin BS. Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cell Mol Immunol. 2021;18(5):1106-21.

[37] Sikorski K, Wesoly J, Bluyssen HA. Data mining of atherosclerotic plaque transcriptomes predicts STAT1-dependent inflammatory signal integration in vascular disease. Int J Mol Sci. 2014;15(8):14313-31.

[38] Yamazaki T, Kirchmair A, Sato A, Buqué A, Rybstein M, Petroni G, et al. Mitochondrial DNA drives abscopal responses to radiation that are inhibited by autophagy. Nat Immunol. 2020;21(10):1160-71.

[39] González-Navajas JM, Lee J, David M, Raz E. Immunomodulatory functions of type I interferons. Nat Rev Immunol. 2012;12(2):125-35.

[40] Ivashkiv LB, Donlin LT. Regulation of type I interferon responses. Nat Rev Immunol. 2014;14(1):36-49.

[41] Hu X, Ivashkiv LB. Cross-regulation of signaling pathways by interferon-gamma: implications for immune responses and autoimmune diseases. Immunity. 2009;31(4):539-50.

[42] Debnath J, Gammoh N, Ryan KM. Autophagy and autophagy-related pathways in cancer. Nat Rev Mol Cell Biol. 2023:1-16.

[43] Jiang T, Chen X, Ren X, Yang JM, Cheng Y. Emerging role of autophagy in anti-tumor immunity: Implications for the modulation of immunotherapy resistance. Drug Resist Updat. 2021;56:100752.

[44] Jin Z, Sun X, Wang Y, Zhou C, Yang H, Zhou S. Regulation of autophagy fires up the cold tumor microenvironment to improve cancer immunotherapy. Front Immunol. 2022;13:1018903.

[45] Poillet-Perez L, Sarry JE, Joffre C. Autophagy is a major metabolic regulator involved in cancer therapy resistance. Cell Rep. 2021;36(7):109528.

[46] Katheder NS, Khezri R, O'Farrell F, Schultz SW, Jain A, Rahman MM, et al. Microenvironmental autophagy promotes tumour growth. Nature. 2017;541(7637):417-20.

[47] Yamamoto K, Venida A, Yano J, Biancur DE, Kakiuchi M, Gupta S, et al. Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I. Nature. 2020;581(7806):100-5.

[48] Amir M, Zhao E, Fontana L, Rosenberg H, Tanaka K, Gao G, et al. Inhibition of hepatocyte autophagy increases tumor necrosis factor-dependent liver injury by promoting caspase-8 activation. Cell Death Differ. 2013;20(7):878-87.

[49] Young TM, Reyes C, Pasnikowski E, Castanaro C, Wong C, Decker CE, et al. Autophagy protects tumors from T cell-mediated cytotoxicity via inhibition of TNFα-induced apoptosis. Sci Immunol. 2020;5(54):eabb9561.

[50] Tsai TH, Lieu AS, Huang TY, Kwan AL, Lin CL, Hsu YC. RTA404, an Activator of Nrf2, Activates the Checkpoint Kinases and Induces Apoptosis through Intrinsic Apoptotic Pathway in Malignant Glioma. J Clin Med. 2021;10(21):4805.

[51] Towers CG, Wodetzki D, Thorburn A. Autophagy and cancer: Modulation of cell death pathways and cancer cell adaptations. J Cell Biol. 2020;219(1):e201909033.

[52] Mgrditchian T, Arakelian T, Paggetti J, Noman MZ, Viry E, Moussay E, et al. Targeting autophagy inhibits melanoma growth by enhancing NK cells infiltration in a CCL5-dependent manner. Proc Natl Acad Sci U S A. 2017;114(44):E9271-E9279.

[53] Harlin H, Meng Y, Peterson AC, Zha Y, Tretiakova M, Slingluff C, et al. Chemokine expression in melanoma metastases associated with CD8+ T-cell recruitment. Cancer Res. 2009;69(7):3077-85.

[54] Clancy-Thompson E, Perekslis TJ, Croteau W, Alexander MP, Chabanet TB, Turk MJ, et al. Melanoma Induces, and Adenosine Suppresses, CXCR3-Cognate Chemokine Production and T-cell Infiltration of Lungs Bearing Metastatic-like Disease. Cancer Immunol Res. 2015;3(8):956-67.

[55] Wei H, Guan JL. Blocking tumor growth by targeting autophagy and SQSTM1 in vivo. Autophagy. 2015;11(5):854-5.

[56] Poillet-Perez L, Xie X, Zhan L, Yang Y, Sharp DW, Hu ZS, et al. Autophagy maintains tumour growth through circulating arginine. Nature. 2018;563(7732):569-573.

[57] Amaravadi RK, Kimmelman AC, Debnath J. Targeting Autophagy in Cancer: Recent Advances and Future Directions. Cancer Discov. 2019;9(9):1167-1181.

[58] Arnold R, Vehns E, Randl H, Djabali K. Baricitinib, a JAK-STAT Inhibitor, Reduces the Cellular Toxicity of the Farnesyltransferase Inhibitor Lonafarnib in Progeria Cells. Int J Mol Sci. 2021;22(14):7474.

[59] Pua HH, Dzhagalov I, Chuck M, Mizushima N, He YW. A critical role for the autophagy gene Atg5 in T cell survival and proliferation. J Exp Med. 2007;204(1):25-31.

[60] DeVorkin L, Pavey N, Carleton G, Comber A, Ho C, Lim J, et al. Autophagy Regulation of Metabolism Is Required for CD8(+) T Cell Anti-tumor Immunity. Cell Rep. 2019;27(2):502-513.e5.

[61] Starobinets H, Ye J, Broz M, Barry K, Goldsmith J, Marsh T, et al. Anti-tumor adaptive immunity remains intact following inhibition of autophagy and antimalarial treatment. J Clin Invest. 2016;126(12):4417-4429.

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