ACK1 Inhibitors Research Articles

Epigenetic reprogramming of cell cycle genes by ACK1 promotes breast cancer resistance to CDK4/6 inhibitor

Hormone receptor-positive, HER2-negative advanced breast cancers exhibit high sensitivity to CDK4/6 inhibitors such as palbociclib. However, most patients inevitably develop resistance, thus identification of new actionable therapeutic targets to overcome the recurrent disease is an urgent need. Immunohistochemical studies of tissue microarray revealed increased activation of non-receptor tyrosine kinase, ACK1 (also known as TNK2) in most of the breast cancer subtypes, independent of their hormone receptor status. Chromatin immunoprecipitation studies demonstrated that the nuclear target of activated ACK1, pY88-H4 epigenetic marks, were deposited at cell cycle genes, CCNB1, CCNB2 and CDC20, which in turn initiated their efficient transcription. Pharmacological inhibition of ACK1 using its inhibitor, (R)-9b dampened CCNB1, CCNB2 and CDC20 expression, caused G2/M arrest, culminating in regression of palbociclib-resistant breast tumor growth. Further, (R)-9b suppressed expression of CXCR4 receptor, which resulted in significant impairment of metastasis of breast cancer cells to lung. Overall, our pre-clinical data identifies activated ACK1 as an oncogene that epigenetically controls the cell cycle genes governing the G2/M transition in breast cancer cells. ACK1 inhibitor, (R)-9b could be a novel therapeutic option for the breast cancer patients that have developed resistance to CDK4/6 inhibitors.

Design, Synthesis, and Antitumor Activity of Potent and Selective EGFR L858R/T790M Inhibitors and Identification of a Combination Therapy to Overcome Acquired Resistance in Models of Non-small-cell Lung Cancer.

Epidermal growth factor receptor (EGFR) is one of the most studied drug targets for the treatment of non-small-cell lung cancer (NSCLC). Here, we report the identification, structure optimization, and structure-activity relationship studies of quinazoline derivatives as novel selective EGFR L858R/T790M inhibitors. The most promising compound, 28f, exhibited strong inhibitory activity against EGFR L858R/T790M (IC50 = 3.5 nM) and greater than 368-fold selectivity over EGFR WT (IC50 = 1290 nM), a 6.7-fold improvement over osimertinib. Furthermore, 28f effectively inhibited downstream signaling pathways and induced apoptosis in mutant cells. In the H1975 xenograft in vivo model, 28f exhibited a good tumor suppressive effect. Furthermore, the combination of 28f with the ACK1 inhibitor dasatinib produced synergistic antiproliferative efficacy with 28f in 28f-resistant cells and in vivo. In conclusion,28f could become a candidate drug for the treatment of NSCLC, and the combination of 28f and dasatinib is expected to overcome EGFR resistance.

Identification of Activated Cdc42-Associated Kinase Inhibitors as Potential Anticancer Agents Using Pharmacoinformatic Approaches.

Activated Cdc42-associated kinase (ACK1) is essential for numerous cellular functions, such as growth, proliferation, and migration. ACK1 signaling occurs through multiple receptor tyrosine kinases; therefore, its inhibition can provide effective antiproliferative effects against multiple human cancers. A number of ACK1-specific inhibitors were designed and discovered in the previous decade, but none have reached the clinic. Potent and selective ACK1 inhibitors are urgently needed. In the present investigation, the pharmacophore model (PM) was rationally built utilizing two distinct inhibitors coupled with ACK1 crystal structures. The generated PM was utilized to screen the drug-like database generated from the four chemical databases. The binding mode of pharmacophore-mapped compounds was predicted using a molecular docking (MD) study. The selected hit-protein complexes from MD were studied under all-atom molecular dynamics simulations (MDS) for 500 ns. The obtained trajectories were ranked using binding free energy calculations (ΔG kJ/mol) and Gibb's free energy landscape. Our results indicate that the three hit compounds displayed higher binding affinity toward ACK1 when compared with the known multi-kinase inhibitor dasatinib. The inter-molecular interactions of Hit1 and Hit3 reveal that compounds form desirable hydrogen bond interactions with gatekeeper T205, hinge region A208, and DFG motif D270. As a result, we anticipate that the proposed scaffolds might help in the design of promising selective ACK1 inhibitors.

Improvement of ACK1-targeted therapy efficacy in lung adenocarcinoma using chloroquine or bafilomycin A1

BackgroundActivated Cdc42-associated kinase 1 (ACK1) is a promising druggable target for cancer, but its inhibitors only showed moderate effects in clinical trials. The study aimed to investigate the underlying mechanisms and improve the antitumor efficacy of ACK1 inhibitors.MethodsRNA-seq was performed to determine the downstream pathways of ACK. Using Lasso Cox regression analysis, we built a risk signature with ACK1-related autophagy genes in the lung adenocarcinoma (LUAD) patients from The Cancer Genome Atlas (TCGA) project. The performance of the signature in predicting the tumor immune environment and response to immunotherapy and chemotherapy were assessed in LUAD. CCK8, mRFP-GFP-LC3 assay, western blot, colony formation, wound healing, and transwell migration assays were conducted to evaluate the effects of the ACK1 inhibitor on lung cancer cells. A subcutaneous NSCLC xenograft model was used for in vivo study.ResultsRNA-seq revealed the regulatory role of ACK1 in autophagy. Furthermore, the risk signature separated LUAD patients into low- and high-risk groups with significantly different prognoses. The two groups displayed different tumor immune environments regarding 28 immune cell subsets. The low-risk groups showed high immune scores, high CTLA4 expression levels, high immunophenoscore, and low DNA mismatch repair capacity, suggesting a better response to immunotherapy. This signature also predicted sensitivity to commonly used chemotherapy and targeted drugs. In vitro, the ACK1 inhibitors (AIM-100 and Dasatinib) appeared to trigger adaptive autophagy-like response to protect lung cancer cells from apoptosis and activated the AMPK/mTOR signaling pathway, partially explaining its moderate antitumor efficacy. However, blocking lysosomal degradation with chloroquine/Bafilamycine A1 or inhibiting AMPK signaling with compound C/shPRKAA1 enhanced the ACK1 inhibitor’s cytotoxic effects on lung cancer cells. The efficacy of the combined therapy was also verified using a mouse xenograft model.ConclusionsThe resulting signature from ACK1-related autophagy genes robustly predicted survival and drug sensitivity in LUAD. The lysosomal degradation inhibition improved the therapeutic effects of the ACK1 inhibitor, suggesting a potential role for autophagy in therapy evasion.

ACK1 Contributes to the Pathogenesis of Inflammation and Autoimmunity by Promoting the Activation of TLR Signaling Pathways.

Toll-like receptors (TLRs) are the first line of defense in the immune system, whose activation plays a key role in the pathogenesis of inflammation and autoimmunity. TLRs can activate a variety of immune cells such as macrophages and dendritic cells, which produce proinflammatory cytokines, chemokines, and co-stimulatory molecules that lead to the development of inflammation and autoimmune diseases. As a nonreceptor tyrosine kinase, ACK1 is involved in multiple signaling pathways and physiological processes. However, the roles of ACK1 in the activation of TLR pathways and in the pathogenesis of inflammation and autoimmune diseases have not yet been reported. We found that the expression of ACK1 could be upregulated by TLR pathways in vivo and in vitro. Intriguingly, overexpression of ACK1 significantly promoted the activation of TLR4, TLR7, and TLR9 pathways, while knockdown of ACK1 or the use of the ACK1 inhibitor AIM-100 significantly inhibited the activation of TLR4, TLR7, and TLR9 pathways. In vivo studies showed that the inhibition of ACK1 activity by AIM-100 could significantly protect mice from the TLR4 agonist lipopolysaccharide (LPS)-mediated endotoxin shock and alleviate the condition of imiquimod-mediated lupus-prone mice and MRL/lpr mice. In summary, ACK1 participates in TLR-mediated inflammation and autoimmunity and has great potential in controlling inflammation and alleviating autoimmune diseases.

A mechanism of ACK1 activation in cancer via C‐terminal UBA domain truncation

We recently developed a machine learning (ML) based program to predict cancer hotspots. Here, we applied the ML program to 32 non‐receptor tyrosine kinases (NRTKs) and identified 36 potential cancer driver mutations, with high probability mutations in 10 genes, including ABL1, ABL2, JAK1, JAK3, and ACK1. Interestingly among all the NRTKs, ACK1 is the only kinase that, when altered, shows a significant drop in overall survival, supporting the idea that ACK1 is an oncogenic tyrosine kinase. ACK1 is a member of the poorly understood ACK family of NRTKs that also includes TNK1. Although ACK1 is an established oncogene and high‐interest therapeutic target, the exact mechanism of ACK1 regulation is largely unknown and there is still no ACK1 inhibitor in clinical use. The ACK kinase family has a unique domain arrangement with, most notably, a predicted ubiquitin association (UBA) domain at its C‐terminus. The presence of a functional UBA domain on a kinase is unique to the ACK family, but the role of the UBA domain on ACK1 is unknown. Interestingly, the ML program identified the ACK1 UBA‐truncating mutation p633fs* as a cancer hotspot. Our preliminary data suggest that the ACK1 UBA domain helps activate full‐length ACK1 through induced proximity, but also acts as a mechanism of negative feedback by tethering ACK1 to ubiquitinated cargo that is ultimately degraded. Indeed, our preliminary data suggest that truncation of the ACK1 UBA stabilizes ACK1 protein levels, which results in spontaneous ACK1 oligomerization and activation. Thus, our data provide a model to explain how human mutations in ACK1 convert the kinase into an oncogenic driver.

Small Molecules Targeting Activated Cdc42-Associated Kinase 1 (ACK1/TNK2) for the Treatment of Cancers.

Activated Cdc42-associated kinase 1 (ACK1/TNK2) is a nonreceptor tyrosine kinase with a unique structure. It not only can act as an activated transmembrane effector of receptor tyrosine kinases (RTKs) to transmit various RTK signals but also can play a corresponding role in epigenetic regulation. A number of studies have shown that ACK1 is a carcinogenic factor. Blockage of ACK1 has been proven to be able to inhibit cancer cell survival, proliferation, migration, and radiation resistance. Thus, ACK1 is a promising potential antitumor target. To date, despite many efforts to develop ACK1 inhibitors, no specific small molecule inhibitors have entered clinical trials. This Perspective provides an overview of the structural features, biological functions, and association with diseases of ACK1 and in vitro and in vivo activities, selectivity, and therapeutic potential of small molecule ACK1 inhibitors with different chemotypes.

Identification of ACK1 inhibitors as anticancer agents by using computer-aided drug designing

ACK1, an intracellular non-receptor tyrosine kinase when abnormally activated and amplified causes numerous sorts of human cancers, therefore, act as a major target of cancer drug development. Currently, there are very small numbers of inhibitors reported for ACK1 inhibition and none of them are in clinical trials. In this study, to identify potential ACK1 inhibitors, a small dataset was prepared from already known inhibitors as a training set for ligand-based pharmacophore model generation using the Hip-Hop algorithm available in the Discovery Studio. Selected pharmacophore hypothesis, Hypo1 displayed the highest rank and consists of five features including two hydrogen bond acceptors (HBA), two-ring aromatic (RA) and one hydrophobic (HYP). Hypo1 was further validated by Receiver Operating Characteristic (ROC) curve and Güner-Henry (GH) approach which give an ROC value of 0.92 and highest GH score of 0.78. Drug-like database was generated from ZINC, ANX, NCI and Princeton database using Lipinski's rule of five and ADMET descriptors. Validated Hypo1 was used for searching new potential hit compounds from the generated drug-like database. From molecular docking analysis, ten potential inhibitor compounds were selected on the basis of Goldscore >67.72, the score of reference inhibitor Dasatinib. Furthermore, molecular dynamics simulation study was performed to study the stability of docking conformations. Subsequently, molecular dynamics simulation analyses revealed that the Hit1 and Hit2 compounds have desirable molecular interaction with key residues Thr205 and Ala208 in the hinge region of ACK1 with better binding affinity. Finally, various pharmacokinetic properties over the Hit1 and Hit2 compounds were analyzed using pkCSM tool and it was found that our hit compounds have shown comparable results with reference. Therefore, we propose the Hit1 and Hit2 compounds may be crucial against ACK1 as potential anticancer agents subjected to experimental validation.

Inhibition of ACK1 delays and overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib

ObjectivesThe emergence of acquired resistance to the third generation EGFR inhibitor, osimertinib (AZD9291 or TAGRISSO™), is an unavoidable huge clinical challenge. The involvement of ACK1, a non-receptor tyrosine kinase with an oncogenic function, in regulating cell response to osimertinib has not been investigated and thus is the focus of this study. Material and MethodsDrug effects on cell growth were evaluated by measuring cell numbers and colony formation. Apoptosis was monitored with flow cytometry for annexin V-positive cells and Western blotting for protein cleavage. Intracellular protein and mRNA alterations were detected with Western blotting and qRT-PCR, respectively. Drug effects on delaying osimertinib acquired resistance were determined using colony formation in vitro and xenografts in nude mice in vivo, respectively. Cell senescence was assayed by β-galactosidase staining. ResultsInhibition of ACK1 with the novel ACK1 inhibitor, (R)-9b synergized with osimertinib in inhibiting the growth of EGFR mutant NSCLC cell lines. Similar results were also generated with ACK1 gene knockdown. The combination of osimertinib and (R)-9b enhanced induction of apoptosis. In both in vitro and in vivo long-term resistance delay assays, the combination of (R)-9b and osimertinib clearly delayed the emergence of osimertinib-resistance. Further, the (R)-9b and osimertinib combination was also effective in inhibiting the growth of EGFR mutant NSCLC cell lines with acquired resistance to osimertinib, which possess elevated levels of ACK1, and the growth of osimertinib-resistant tumors in vivo. In some resistant cell lines, the combinations induced senescence in addition to induction of apoptosis. ConclusionsThese novel findings suggest that ACK1 inhibition might be a potential and innovative strategy for delaying and overcoming osimertinb acquired resistance.

Abstract 73: Inhibition of ACK1 delays and overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib

Abstract The approval of osimertinib (AZD9291) as a third generation EGFR inhibitor for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (first-line) or those who have become resistant to 1st generation EGFR inhibitors through the T790M mutation (second-line) have generated tremendous benefits to these NSLCL patients. However, the unavoidable development of acquired resistance to osimertinib limits the long-term benefits of patients. Thus, effective treatment options are urgently needed. Activated Cdc42-associated kinase 1 or ACK1 (also named as TNK2) is a non-receptor tyrosine kinase with oncogenic function. Although ACK1 gene is amplified in lung squamous cell carcinoma, its involvement in lung cancer including regulation of drug resistance has largely not been investigated. We found that inhibition of ACK1 with (R)-9bMS, a specific ACK1 inhibitor, synergized with AZD9291 in inhibiting the growth of EGFR mutant NSCLC cell lines. Similar results were also generated when ACK1 is genetically inhibited through transient shRNA gene knockdown. The combination of AZD9291 and (R)-9bMS enhanced induction of apoptosis as evaluated by detecting annexin V-positive cells with flow cytometry and PARP cleavage using Western blotting. In an in vitro long-term resistance delay assay, the combination of (R)-9bMS and AZD9291 clearly prevented the emergence of AZD9291-resistance as evaluating resistant cell colony formation and growth. Beyond the combination was also effective in inhibiting the growth of EGFR mutant NSCLC cell lines with acquired resistance to AZD9291. In some resistant cell lines, the combinations induced senescence in addition to induction of apoptosis. These preliminary findings suggest that ACK1 inhibition might be a potential and innovative strategy for preventing and overcoming AZD9291 acquired resistance. Hence further in vivo and mechanistic studies are warranted. (This work was supported by the NIH/NCI R01 CA223220 and Winship lung cancer pilot award to SYS; NIH/NCI R01 CA208258, Bankhead Coley Award-6BC08 and Prostate Cancer Foundation (PCF) Challenge award-17CHAL06 to NPM). Citation Format: Jiajia Gu, Xia He, Nupam P. Mahajan, Taofeek K. Owonikoko, Suresh S. Ramalingam, Shi-Yong Sun. Inhibition of ACK1 delays and overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 73.

Effect of set up protocols on the accuracy of alchemical free energy calculation over a set of ACK1 inhibitors.

Hit-to-lead virtual screening frequently relies on a cascade of computational methods that starts with rapid calculations applied to a large number of compounds and ends with more expensive computations restricted to a subset of compounds that passed initial filters. This work focuses on set up protocols for alchemical free energy (AFE) scoring in the context of a Docking–MM/PBSA–AFE cascade. A dataset of 15 congeneric inhibitors of the ACK1 protein was used to evaluate the performance of AFE set up protocols that varied in the steps taken to prepare input files (using previously docked and best scored poses, manual selection of poses, manual placement of binding site water molecules). The main finding is that use of knowledge derived from X-ray structures to model binding modes, together with the manual placement of a bridging water molecule, improves the R2 from 0.45 ± 0.06 to 0.76 ± 0.02 and decreases the mean unsigned error from 2.11 ± 0.08 to 1.24 ± 0.04 kcal mol-1. By contrast a brute force automated protocol that increased the sampling time ten-fold lead to little improvements in accuracy. Besides, it is shown that for the present dataset hysteresis can be used to flag poses that need further attention even without prior knowledge of experimental binding affinities.

First SAR Study for Overriding NRAS Mutant Driven Acute Myeloid Leukemia.

GNF-7, a multitargeted kinase inhibitor, served as a dual kinase inhibitor of ACK1 and GCK, which provided a novel therapeutic strategy for overriding AML expressing NRAS mutation. This SAR study with GNF-7 derivatives, designed to target NRAS mutant-driven AML, led to identification of the extremely potent inhibitors, 10d, 10g, and 11i, which possess single-digit nanomolar inhibitory activity against both ACK1 and GCK. These substances strongly suppress proliferation of mutant NRAS expressing AML cells via apoptosis and AKT/mTOR signaling blockade. Compound 11i is superior to GNF-7 in terms of kinase inhibitory activity, cellular activity, and differential cytotoxicity. Moreover, 10k possessing a favorable mouse pharmacokinetic profile prolonged life-span of Ba/F3-NRAS-G12D injected mice and significantly delayed tumor growth of OCI-AML3 xenograft model without causing the prominent level of toxicity found with GNF-7. Taken together, this study provides insight into the design of novel ACK1 and GCK dual inhibitors for overriding NRAS mutant-driven AML.

Abstract A084: Reprogramming of androgen receptor gene expression by ACK1/TNK2 mediated histone H4 Tyr88-phosphorylation promotes castration resistance

Abstract Aberrant androgen receptor (AR) transcriptional co-activator activity underlies the onset and progression of prostate cancer (PC) to castration-resistant (CRPC) state. While anti-androgens provide immediate palliative benefits by negating AR transcriptional activity, resistance inevitably develops—either by AR gene amplification, mutation, or expression of splice variants, such as AR-V7. To achieve complete remission, ablation of AR expression appears to be the key. However, targeted inhibition of AR and AR-V7 gene expression with small-molecule inhibitors has not yet been accomplished. Recently, we uncovered a novel chromatin-regulated mechanism of AR transcriptional regulation mediated by the tyrosine kinase ACK1 (also known as TNK2). We observed that ACK1 phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site. The WDR5/MLL2 complex is read by H4-Y88-phosphorylation and in turn deposits the transcriptionally activating H3K4-trimethyl marks, promoting AR transcription in androgen-deficient environment. Reversal of the pY88-H4 epigenetic marks by the ACK1 inhibitor (R)-9bMS sensitized naive and enzalutamide-resistant prostate cancer cells and reduced AR and AR-V7 levels to mitigate CRPC tumor growth. Interestingly, we observed that AR itself was modified by acetylation at Lys609 residue in enzalutamide-resistant PC cells, revealing an additional mechanisms underlying castration resistance. Overall, these data reveal previously undocumented epigenetic circuitry that not only sustains AR and AR-V7 expression, but also provides mechanistic insight into rapid acquisition of resistance to antiandrogens. Citation Format: Nupam Mahajan, Kiran Mahajan, Harshani Lawrence, Nicholas Lawrence. Reprogramming of androgen receptor gene expression by ACK1/TNK2 mediated histone H4 Tyr88-phosphorylation promotes castration resistance [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A084.

Abstract 2378: Histone H4 tyr88-phosphorylation mediated by ACK1 promotes castration-resistant prostate cancer by epigenetic reprogramming of androgen receptor

Abstract Androgen receptor (AR) plays a paramount role in the onset and progression of prostate cancer (PC). This very facet underlies androgen deprivation therapy (ADT), a preferred treatment to negate AR transcriptional activity. While ADT provides immediate palliative benefits, it is ineffective long term, as the recalcitrant disease recurs within 2-3 years and progresses to a lethal stage, referred to as the metastatic castration-resistant prostate cancer (mCRPC). The AR gene (AR) is amplified or mutated in >50% of mCRPCs, suggesting that PC cells may have reinvigorated AR transcription as a response to the loss of existing AR activity by ADT. Consequently, resistance to ADT has become one of the most vexing problems in PC therapy. AR antagonists are often ineffective due to their inability to repress the expression of AR or its splice variant, AR-V7. Here, we report that the tyrosine kinase ACK1 (TNK2) phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site. The WDR5/MLL2 complex reads the H4-Y88-phosphorylation marks and deposits the transcriptionally activating H3K4-trimethyl marks promoting AR transcription. We developed a new class of ACK1 kinase-specific small molecule inhibitor (R)-9bMS that suppresses ACK1 kinase activity at IC50= 48 nM. Reversal of the pY88-H4 epigenetic marks by the ACK1 inhibitor (R)-9bMS-sensitized naive and enzalutamide-resistant prostate cancer cells and reduced AR and AR-V7 levels to mitigate CRPC tumor growth. Thus, a feedforward ACK1/pY88-H4/WDR5/MLL2/AR epigenetic circuit drives CRPC and is necessary for maintenance of the malignant state. Citation Format: Nupam P. Mahajan, Kiran Mahajan, Nicholas Lawrence, Harshani Lawrence. Histone H4 tyr88-phosphorylation mediated by ACK1 promotes castration-resistant prostate cancer by epigenetic reprogramming of androgen receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2378.

Blockade of ACK1/TNK2 To Squelch the Survival of Prostate Cancer Stem-like Cells

Prostate cancer stem-like cells (PCSCs) are not only enriched in the CD44+PSA−/lo subpopulation but also employ androgen-independent signaling mechanisms for survival. CD44+ PCSCs defy androgen deprivation, resist chemo- and radiotherapy and are highly tumorigenic. Human prostate tissue microarray (TMA) staining revealed an increased membranous staining of CD44 in the luminal compartment in higher grade G7-G9 tumors versus staining of the basal layer in benign hyperplasia. To uncover tyrosine kinase/s critical for the survival of the CD44+PSA−/lo subpopulation, we performed an unbiased screen targeting 87 tyrosine kinases with gene specific siRNAs. Among a subset of tyrosine kinases crucial for PCSC survival, was a non-receptor tyrosine kinase, ACK1/TNK2, a critical regulator of castration resistant prostate cancer (CRPC) growth. Consistently, activated ACK1 as measured by phosphorylation at Tyr284 was significant in the CD44+PSA−/lo population. Conversely, pharmacological inhibition by ACK1 inhibitor, (R)-9bMS mitigated CD44+PSA−/lo sphere formation, overcame resistance to radiation-induced cell death, induced significant apoptosis in PCSCs and inhibited CD44+PSA−/lo xenograft tumor growth in castrated mice suggesting dependency of PCSCs on ACK1 for survival. Thus, blockade of ACK1/TNK2 could be a new therapeutic modality to target recalcitrant PCSCs.

ACK1/TNK2 Regulates Histone H4 Tyr88-phosphorylation and AR Gene Expression in Castration-Resistant Prostate Cancer

The androgen receptor (AR) is critical for the progression of prostate cancer to a castration-resistant (CRPC) state. AR antagonists are ineffective due to their inability to repress the expression of AR or its splice variant, AR-V7. Here, we report that the tyrosine kinase ACK1 (TNK2) phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site. The WDR5/MLL2 complex reads the H4-Y88-phosphorylation marks and deposits the transcriptionally activating H3K4-trimethyl marks promoting AR transcription. Reversal of the pY88-H4 epigenetic marks by the ACK1 inhibitor (R)-9bMS-sensitized naive and enzalutamide-resistant prostate cancer cells and reduced AR and AR-V7 levels to mitigate CRPC tumor growth. Thus, a feedforward ACK1/pY88-H4/WDR5/MLL2/AR epigenetic circuit drives CRPC and is necessary for maintenance of the malignant state.

Histone deacetylase inhibitors induce proteolysis of activated CDC42-associated kinase-1 in leukemic cells.

Activated CDC42-associated kinase-1 (ACK1/TNK2) and epigenetic regulators of the histone deacetylase (HDAC) family regulate the proliferation and survival of leukemic cells. 18 HDACs fall into four classes (I-IV). We tested the impact of clinically relevant histone deacetylase inhibitors (HDACi) on ACK1 and if such drugs combine favorably with the therapeutically used ACK1 inhibitor Dasatinib. We applied the broad-range HDACi Panobinostat/LBH589 and the class I HDAC-specific inhibitor Entinostat/MS-275 to various acute and chronic myeloid leukemia cells (AML/CML). We also used the replicative stress inducer Hydroxyurea (HU), a standard drug for leukemic patients, and the apoptosis inducer Staurosporine (STS). To assess cytotoxic effects of HDACi, we measured cell cycle profiles and DNA fragmentation by flow cytometry. Western blot was employed to analyze protein expression and phosphorylation. LBH589 and MS-275 induce proteolysis of ACK1 in CML and AML cells. Panobinostat more strongly induces apoptosis than Entinostat, and this correlates with a significantly pronounced loss of ACK1. STS and HU also propel the degradation of ACK1 in leukemic cells. Moreover, the caspase inhibitor z-VAD-FMK reduces ACK1 degradation in the presence of HDACi. Concomitant with the attenuation of ACK1, we noticed decreased phosphorylation of STAT3. Direct inhibition of ACK1 with Dasatinib also suppresses STAT3 phosphorylation. Furthermore, Dasatinib and HDACi combinations are effective against CML cells. HDACs sustain the ACK1-STAT3 signaling node and leukemic cell growth. Consistent with their different effects on ACK1 stability or auto-phosphorylation, Dasatinib and HDACi combinations produce beneficial antileukemic effects.

ACK1/TNK2 tyrosine kinase: molecular signaling and evolving role in cancers.

Deregulated tyrosine kinase signaling alters cellular homeostasis to drive cancer progression. The emergence of a non-receptor tyrosine kinase, ACK1 as an oncogenic kinase, has uncovered novel mechanisms by which tyrosine kinase signaling promotes cancer progression. While early studies focused on ACK1 (also known as activated Cdc42-associated kinase 1 or TNK2) as a cytosolic effecter of activated transmembrane receptor tyrosine kinases (RTKs), wherein it shuttles between the cytosol and the nucleus to rapidly transduce extracellular signals from the RTKs to the intracellular effectors, recent data unfold a new aspect of its functionality as an epigenetic regulator. ACK1 interacts with the Estrogen Receptor (ER)/histone demethylase KDM3A (JHDM2a) complex, modifies KDM3A by tyrosine phosphorylation to regulate transcriptional outcome at HOXA1 locus to promote the growth of tamoxifen-resistant breast cancer. It is also well established that ACK1 regulates the activity of Androgen Receptor (AR) by tyrosine phosphorylation to fuel the growth of hormone-refractory prostate cancers. Further, recent explosion in genomic sequencing has revealed recurrent ACK1 gene amplification and somatic mutations in a variety of human malignancies, providing a molecular basis for its role in neoplastic transformation. In this review, we will discuss the various facets of ACK1 signaling, including its newly uncovered epigenetic regulator function, which enables cells to bypass the blockade to major survival pathways to promote resistance to standard cancer treatments. Not surprisingly, cancer cells appear to acquire an `addiction’ to ACK1 mediated survival, particularly under stress conditions, such as growth factor deprivation or genotoxic insults or hormone deprivation. With the accelerated development of potent and selective ACK1 inhibitors, targeted treatment for cancers harboring aberrant ACK1 activity may soon become a clinical reality.

Abstract 2511: New inhibitors the tyrosine kinase ACK1/TNK2 active in prostate, breast and pancreatic cancer

Abstract ACK1, is a non-receptor tyrosine kinase that is aberrantly activated, over expressed or mutated in many cancer cell types. It interacts with several important ligand-activated receptor tyrosine kinases (RTKs), for example, EGFR, MerTK, HER2, PDGFR and insulin receptor to initiate intracellular signaling cascades. ACK1 activation has been reported in various cancers including prostate, breast and pancreatic tumors. We have shown that the androgen receptor (AR) and AKT are two major downstream effectors of ACK1. ACK1 directly phosphorylates AKT at Tyr176 resulting in AKT membrane localization and activation. We have also found that in prostate cancer cells ACK1 phosphorylates AR at Tyr-267 in an androgen-independent manner. Tyr284-phosphorylated-ACK1, Tyr176-phosphorylated-AKT and Tyr267-phosphorylated-AR levels are positively correlated with the severity of disease progression, and inversely correlated with the survival of patients with prostate cancer. Similarly, ACK1 mediated AKT tyrosine phosphorylation was found to correlate positively with breast cancer progression. Further, we have shown that a 5,6-biaryl-furo[2,3-d]pyrimidine inhibitor of ACK1 (AIM-100) inhibits ACK1 activation and suppresses pTyr267-AR phosphorylation and AKT Tyr176-phosphorylation, inhibiting AR and AKT activity. These findings indicate that Ack1 is prognostic of progression of prostate, breast and pancreatic cancer and inhibitors of ACK1 activity have potential as novel cancer therapeutic agents. We will discuss the development of new potent ACK1 inhibitors by fragment-recombination and structure-based design by incorporating fragments of key ACK1 inhibitors. Focused chemical libraries were developed and structure activity relationships will be described. The work described has produced compounds capable of inhibiting ACK1 in vitro at low nanomolar concentrations. We have used an ELISA based assay that measures the ability of ACK1 to phosphorylate a peptide derived from AKT, as a primary screening assay. ACK1 inhibition of hits was confirmed in the 33P ATP “Hotspot” assay platform. Selected compounds from the library have been shown to inhibit ACK1 autophosphorylation and the phosphorylation of AR at Tyr267, a surrogate for ACK1 inhibition in vivo. Furthermore we will show that ACK1 is a promising drug target for cancer therapy. Citation Format: Harshani R. Lawrence, Yunting Luo, Daniel Zhang, Nathan Tindall, Sevil Ozcan, Miles Huseyin, Sakib Kazi, Sayantani Bandyopadhyay, Kiran Mahajan, Nupam P. Mahajan, Nicholas J. Lawrence. New inhibitors the tyrosine kinase ACK1/TNK2 active in prostate, breast and pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2511. doi:10.1158/1538-7445.AM2014-2511

Abstract 2599: KDM3A tyrosine phosphorylation by Ack1 promotes tamoxifen-resistance in breast cancer

Abstract Hormone therapy with the selective estrogen-receptor modulator tamoxifen (TAM) provides palliative benefit for patients with breast cancer. However, estrogen receptor α (ER hereafter) positive breast tumors with high expression of HER2 receptor tyrosine kinase often develop significant resistance to tamoxifen therapy. Thus, precise understanding of activation of Estrogen (E2)-independent ER regulated gene transcription in tamoxifen-resistant breast tumors could open new therapeutic avenues to target drug-resistance and ameliorate poor prognosis. The non-receptor tyrosine kinase, Ack1 (also known as TNK2) has emerged as a major integrator of signaling from various receptor tyrosine kinases including HER2. Here we demonstrate that heregulin promoted ER activity at Estrogen Response Element (ERE) in the presence of tamoxifen, which was significantly downregulated upon Ack1 knockdown. We observed that Ack1 phosphorylated the ER co-activator, KDM3A, a H3K9 demethylase, at evolutionary conserved tyrosine 1114 in heregulin dependent manner in the presence of tamoxifen. KDM3A demethylates H3 mono- and dimethyl-K9 in vitro and in vivo. Heregulin mediated Ack1 activation resulted in significant decrease in the level of monomethyl-H3K9, while, monomethyl-H3K27 levels remained unchanged. Further, inhibition of Ack1 by small molecule inhibitor AIM-100 or Dasatinib resulted in significant increase in the level of monomethyl-H3K9. Therefore by its ability to regulate the activity of ER co-activator such as KDM3A, activated Ack1 may modulate ER target gene expression in the absence of estrogen and confer TAM-resistance to cancer cells. Thus, Ack1 inhibitors hold the potential as novel therapeutics to treat a subset of tamoxifen resistant breast cancers. Citation Format: Kiran Mahajan, Sayantani Bandyopadhyay, Nupam Mahajan. KDM3A tyrosine phosphorylation by Ack1 promotes tamoxifen-resistance in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2599. doi:10.1158/1538-7445.AM2014-2599