ACK1-CSK and ACK1-AR SIGNALING

Solid tumors are highly refractory to immune checkpoint blockade (ICB) therapies due to the functional impairment of effector T cells and their inefficient trafficking to tumors. T-cell activation is negatively regulated by C-terminal Src kinase (CSK); however, the exact mechanism remains unknown. We uncovered that ACK1 phosphorylate CSK at Tyrosine 18 (pY18), which enhances CSK function, constraining T-cell activation. Mice deficient in the ACK1/Tnk2 exhibit spontaneous activation of CD8+ and CD4+ T cells, resulting in inhibited growth of transplanted ICB-resistant tumors. Furthermore, ICB treatment of castration-resistant prostate cancer (CRPC) patients results in re-activation of ACK1/pY18-CSK signaling, confirming the involvement of this pathway in ICB insensitivity. An ACK1 small-molecule inhibitor, (R)-9b, recapitulates inhibition of ICB-resistant tumors, which provides evidence for ACK1 playing a pivotal role in generating ICB resistance (Figure 1). Nature Communications, 2022.

ACK1-AR SIGNALING IN PROSTATE CANCER

ACK1 (TNK2) interacts with androgen receptor or AR in an androgen-independent manner. Expression of activated ACK1 correlates positively with the progression of disease to CRPC stage, and PC patients whose tumors display moderate to strong staining of activated ACK1 have poor prognosis.

AR plays a paramount role in the onset and progression of prostate cancer (PC). A majority of the PC patients progress to a lethal stage of the disease, referred to as the metastatic Castration Resistant Prostate Cancer (mCRPC). There is increasing expression of AR as disease progress to later stages (Figure 2). CRPC remains an incurable malignancy with limited treatment options. 

Need to venture beyond Anti-Androgen Therapies

Reliance of CRPCs on AR despite androgen-depletion, has led to development of AR antagonists, Enzalutamide & Abiraterone. Unfortunately, in spite of early response, most patients relapsed within 2 years, exhibiting renewed AR activity.   

ACK1, a new target in CRPCs

ACK1 is upregulated in ~40% of prostate adenocarcinomas. Importantly, 10 out of 13 CRPCs exhibited ACK1 overexpression. Further, LNCaP cells that are poorly tumorigenic in castrated mice, formed robust CRPC tumors following expression of activated ACK1.

ACK1, a histone kinase

We uncovered that ACK1 interacts with AR and deposit novel epigenetic marks, histone H4 Tyr88-phosphorylation (Figure 3). Inhibition of ACK1 by (R)-9b suppressed AR & AR-V7 transcription, inhibiting proliferation of enzalutamide-resistant CRPC tumors.

  • Mahajan K, Malla P, Kim J, Coppola D, Lawrence N and Mahajan NP*. ACK1 regulates histone Tyr-phosphorylation and AR gene expression in castration resistant prostate cancer. Cancer Cell 2017. Pubmed ID:  28609657
Figure 1. a HEK293T cells were co-transfected with FLAG-tagged CSK and HA-tagged ACK1 or cACK1. Post-MG132 treatment, lysates were IP with FLAG beads, followed by IB with p-Tyr antibodies (top panel). b Docking model of CSK (green) bound to ACK1 (gray) with ATPγS (yellow)/Mg2+ (green) modelled into the active site of ACK1. The SH3 domain of CSK docks against the activation loop of ACK1 (red). c Lysates from spleen of WT and Ack1 KO mice were immunoblotted with the indicated antibodies. d HEK293T cells were co-transfected with or without ACK1, LCK and CSK or CSK-Y18F mutant expressing constructs. Lysates were immunoblotted with indicated antibodies. e X-ray co-crystal structure of ACK1 kinase domain bound to (R)-9b. f Jurkat cells were treated with (R)-9b (3 μM) and immunoblotted with indicated antibodies. g RNA was prepared from Jurkat cells treated with vehicle or (R)-9b, followed by real time PCR of NF- κB.
Figure 2. (Top) Prostate TMA stained with AR antibody. (Bottom) Boxplot summarize distributions of staining intensity for AR in prostate biopsies
Figure 3. ACK1 phosphorylates histone H4 at Y88 upstream of the AR transcription start site, leading to theWDR5/MLL2 complex mediated increase of AR transcription. Inhibition of ACK1 reverses the pY88-H4 marks and reduces AR and AR-V7 levels to mitigate castration-resistant prostate tumor growth.