Inhibiting ACK1-mediated phosphorylation of C-terminal Src kinase counteracts prostate cancer immune checkpoint blockade resistance

Abstract

Solid tumours are highly refractory to immune checkpoint blockade (ICB) therapies due to the functional impairment of effector T cells and their inefficient trafficking to tumours. T-cell activation is negatively regulated by C-terminal Src kinase (CSK); however, the exact mechanism remains unknown. Here we show that the conserved oncogenic tyrosine kinase Activated CDC42 kinase 1 (ACK1) is able to phosphorylate CSK at Tyrosine 18 (pY18), which enhances CSK function, constraining T-cell activation. Mice deficient in the Tnk2 gene encoding Ack1, are characterized by diminished CSK Y18-phosphorylation and spontaneous activation of CD8⁺ and CD4⁺ T cells, resulting in inhibited growth of transplanted ICB-resistant tumours. Furthermore, ICB treatment of castration-resistant prostate cancer (CRPC) patients results in re-activation of ACK1/pY18-CSK signalling, confirming the involvement of this pathway in ICB insensitivity. An ACK1 small-molecule inhibitor, (R)-9b, recapitulates inhibition of ICB-resistant tumours, which provides evidence for ACK1 enzymatic activity playing a pivotal role in generating ICB resistance. Overall, our study identifies an important mechanism of ICB resistance and holds potential for expanding the scope of ICB therapy to tumours that are currently unresponsive.

Introduction

The initiation of T-cell antigen receptor (TCR) signalling is dependent on the coordinated activation of a series of SRC-family tyrosine kinases, including CSK^1,2,3. The target of CSK is LCK, a key kinase; LCK activity is precisely regulated by two distinct tyrosine phosphorylation events with opposite consequences. LCK Y394 phosphorylation in the activation loop is needed to acquire full kinase activity; in contrast, Y505 phosphorylation in the C-terminal tail, which interacts with its own SH2 domain and promotes a closed autoinhibition conformation, causes loss of kinase activity. The crucial role played by CSK was determined using transgenic mice expressing a mutant form of CSK (CskAS), which showed reduced CSK activation and led to enhanced immune responses to very weak antigens, including antigens that might not have activated T cells under normal circumstances⁴. These data suggest that targeting CSK activation may be a promising strategy for reactivating the immune response. Despite the importance of CSK activation, our understanding of the regulation of CSK activity during TCR signal initiation remains inadequate.

Metastatic castration-resistant prostate cancer (CRPC) is a malignancy that poses a major challenge due to its immunosuppressive tumour microenvironment. As the second leading cause of cancer deaths in American men⁵, CRPCs have attracted considerable attention by researchers seeking to address resistance mechanisms. T-cell exhaustion due to chronic tumour antigen stimulation can be successfully overcome via immune checkpoint blockade (ICB) targeting cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed cell death-1 (PD-1), or programmed cell death ligand-1 (PD-L1)^6,7. Although ICBs have emerged as major successes against certain cancers⁷, prostate cancer has been found to be highly refractory to ICBs⁸, exhibiting marginal efficacy in clinical trials when administered either as a single agent or in combination with other agents⁹. Metastatic CRPC patients with a high intratumoral CD8⁺ T-cell density and an IFN-γ response gene signature exhibited favourable responses to CTLA-4-targeting antibodies¹⁰, suggesting that therapeutic strategies that increase CD8⁺ T-cell activation may show clinical benefits in CRPCs. However, targeted activation of CD8⁺ T cells in advanced prostate tumours using a therapeutically amenable small-molecule compound has not yet been achieved.

We previously revealed that a nonreceptor tyrosine kinase, ACK1 (also known TNK2)^11,12, acts as an epigenetic kinase and deposits novel pY88-H4 epigenetic marks on the androgen receptor (AR) enhancer¹³. ACK1 is a multidomain protein (Supplementary Fig. 1a) that is highly expressed in the spleen and thymus^14,15. Multiple malignancies favour ACK1 overexpression; ACK1 gene amplification, mutations, and aberrant kinase activation have been reported in various malignancies, including lung, breast, ovarian and prostate cancer^{12,13,16,17,18,19,20,21,22,23,24}. ACK1 levels are tightly regulated in cells; seven in absentia homologue (SIAH) ubiquitin ligases bind the degron motif located in the C-terminus of ACK1, driving its proteasomal turnover²⁵.

The rationale of this study was to examine the role of ACK1 in immune regulation. We observe ACK1 activation in two antagonizing cell types, immune cells and cancer cells, suggesting a dual mode of ACK1 action. T-cell priming and T-cell trafficking to the tumour microenvironment are dampened by activated ACK1. Consistently, genetic ablation or pharmacological inhibition of ACK1 impairs tumour growth by activating T cells and promoting persistent immune surveillance. The implication of ACK1 inhibition is evident in reactivating an immune response in ICB-resistant tumours, opening a new therapeutic modality for ‘cold’ tumors.

Results

Genetic ablation of Tnk2 lowers the TCR response threshold and amplifies T-cell responsiveness..