SHP2 as a primordial epigenetic enzyme expunges histone H3 pTyr-54 to amend androgen receptor homeostasis.

Mutations that decrease or increase the activity of the tyrosine phosphatase, SHP2 (encoded by PTPN11), promotes developmental disorders and several malignancies by varying phosphatase activity. We uncovered that SHP2 is a distinct class of an epigenetic enzyme; upon phosphorylation by the kinase ACK1/TNK2, pSHP2 was escorted by androgen receptor (AR) to chromatin, erasing hitherto unidentified pY54-H3 (phosphorylation of histones H3 at Tyr54) epigenetic marks to trigger a transcriptional program of AR. Noonan Syndrome with Multiple Lentigines (NSML) patients, SHP2 knock-in mice, and ACK1 knockout mice presented dramatic increase in pY54-H3, leading to loss of AR transcriptome. In contrast, prostate tumors with high pSHP2 and pACK1 activity exhibited progressive downregulation of pY54-H3 levels and higher AR expression that correlated with disease severity. Overall, pSHP2/pY54-H3 signaling acts as a sentinel of AR homeostasis, explaining not only growth retardation, genital abnormalities and infertility among NSML patients, but also significant AR upregulation in prostate cancer patients.

Prostate-Specific Membrane Antigen-Targeted Imaging and Its Correlation with HOXB13 Expression.

Homeobox 13 (HOXB13) is an oncogenic transcription factor that directly regulates expression of folate hydrolase 1, which encodes prostate-specific membrane antigen (PSMA). HOXB13 is expressed in primary and metastatic prostate cancers (PCs) and promotes androgen-independent PC growth. Since HOXB13 promotes resistance to androgen receptor (AR)-targeted therapies and regulates the expression of folate hydrolase 1, we investigated whether SUVs on PSMA PET would correlate with HOXB13 expression. Methods: We analyzed 2 independent PC patient cohorts who underwent PSMA PET/CT for initial staging or for biochemical recurrence. In the discovery cohort, we examined the relationship between HOXB13, PSMA, and AR messenger RNA (mRNA) expression in prostate biopsy specimens from 179 patients who underwent PSMA PET/CT with 18F-piflufolastat. In the validation cohort, we confirmed the relationship between HOXB13, PSMA, and AR by comparing protein expression in prostatectomy and lymph node (LN) sections from 19 patients enrolled in 18F-rhPSMA-7.3 PET clinical trials. Correlation and association analyses were also used to confirm the relationship between the markers, LN positivity, and PSMA PET SUVs. Results: We observed a significant correlation between PSMA and HOXB13 mRNA (P < 0.01). The association between HOXB13 and 18F-piflufolastat SUVs was also significant (SUVmax, P = 0.0005; SUVpeak, P = 0.0006). Likewise, the PSMA SUVmax was significantly associated with the expression of HOXB13 protein in the 18F-rhPSMA-7.3 PET cohort (P = 0.008). Treatment-naïve patients with LN metastases demonstrated elevated HOXB13 and PSMA levels in their tumors as well as higher PSMA tracer uptake and low AR expression. Conclusion: Our findings demonstrate that HOXB13 correlates with PSMA expression and PSMA PET SUVs at the mRNA and protein levels. Our study suggests that the PSMA PET findings may reflect oncogenic HOXB13 transcriptional activity in PC, thus potentially serving as an imaging biomarker for more aggressive disease.

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.

Histone H2A Lys130 acetylation epigenetically regulates androgen production in prostate cancer.

The testicular androgen biosynthesis is well understood, however, how cancer cells gauge dwindling androgen to dexterously initiate its de novo synthesis remained elusive. We uncover dual-phosphorylated form of sterol regulatory element-binding protein 1 (SREBF1), pY673/951-SREBF1 that acts as an androgen sensor, and dissociates from androgen receptor (AR) in androgen deficient environment, followed by nuclear translocation. SREBF1 recruits KAT2A/GCN5 to deposit epigenetic marks, histone H2A Lys130-acetylation (H2A-K130ac) in SREBF1, reigniting de novo lipogenesis & steroidogenesis. Androgen prevents SREBF1 nuclear translocation, promoting T cell exhaustion. Nuclear SREBF1 and H2A-K130ac levels are significantly increased and directly correlated with late-stage prostate cancer, reversal of which sensitizes castration-resistant prostate cancer (CRPC) to androgen synthesis inhibitor, Abiraterone. Further, we identify a distinct CRPC lipid signature resembling lipid profile of prostate cancer in African American (AA) men. Overall, pY-SREBF1/H2A-K130ac signaling explains cancer sex bias and reveal synchronous inhibition of KAT2A and Tyr-kinases as an effective therapeutic strategy.

Epigenetic and transcriptomic characterization reveals progression markers and essential pathways in clear cell renal cell carcinoma.

Identifying tumor-cell-specific markers and elucidating their epigenetic regulation and spatial heterogeneity provides mechanistic insights into cancer etiology. Here, we perform snRNA-seq and snATAC-seq in 34 and 28 human clear cell renal cell carcinoma (ccRCC) specimens, respectively, with matched bulk proteogenomics data. By identifying 20 tumor-specific markers through a multi-omics tiered approach, we reveal an association between higher ceruloplasmin (CP) expression and reduced survival. CP knockdown, combined with spatial transcriptomics, suggests a role for CP in regulating hyalinized stroma and tumor-stroma interactions in ccRCC. Intratumoral heterogeneity analysis portrays tumor cell-intrinsic inflammation and epithelial-mesenchymal transition (EMT) as two distinguishing features of tumor subpopulations. Finally, BAP1 mutations are associated with widespread reduction of chromatin accessibility, while PBRM1 mutations generally increase accessibility, with the former affecting five times more accessible peaks than the latter. These integrated analyses reveal the cellular architecture of ccRCC, providing insights into key markers and pathways in ccRCC tumorigenesis.

Cytomorphology of follicular dendritic cell sarcoma: a report of 7 cases with an emphasis on the diagnostic challenges.

BACKGROUND: Follicular dendritic cell sarcoma is a malignant neoplasm derived from germinal center follicular dendritic cells, which both share a characteristic immunophenotype (namely CD21, CD23, and CD35). Cytomorphologic descriptions are few, consisting of only 26 prior cases from 24 publications. Identification by cytologic means appears challenging as the majority of previous reports disclose an erroneous or indeterminate initial cytologic diagnosis. Herein, we present the largest cytology series to date with the aim of expanding upon this small body of literature and discuss possible factors resulting in misinterpretation. MATERIALS AND METHODS: A retrospective search was conducted from 2 academic medical centers to identify histologically confirmed cases of follicular dendritic cell sarcoma with an associated cytologic component. Clinicopathologic data were tabulated and a comparative analysis of cytomorphologic and immunohistochemical features was performed. RESULTS: Seven separate cases were identified. All cases showed cohesive tumor cells with a characteristic voluminous, ill-defined cytoplasm with interconnecting fibrillary processes and intimately admixed mature lymphocytes. Features were maintained across various cytologic preparations, including conventional smear, liquid-based cytology, and touch imprint. Unusual immunohistochemical profiles were noted in a subset of cases. CONCLUSIONS: Cytomorphology is highly conserved across cases and preparations; however, a propensity for aberrant immunoexpression may contribute to diagnostic errors. Cytomorphologic features, supported by immunohistochemistry, suggest fine-needle aspiration as a reasonable diagnostic modality. Tumors with these features should include CD21, CD23, and/or CD35 in the workup.

TNK2/ACK1-mediated phosphorylation of ATP5F1A (ATP synthase F1 subunit alpha) selectively augments survival of prostate cancer while engendering mitochondrial vulnerability.

The challenge of rapid macromolecular synthesis enforces the energy-hungry cancer cell mitochondria to switch their metabolic phenotypes, accomplished by activation of oncogenic tyrosine kinases. Precisely how kinase activity is directly exploited by cancer cell mitochondria to meet high-energy demand, remains to be deciphered. Here we show that a non-receptor tyrosine kinase, TNK2/ACK1 (tyrosine kinase non receptor 2), phosphorylated ATP5F1A (ATP synthase F1 subunit alpha) at Tyr243 and Tyr246 (Tyr200 and 203 in the mature protein, respectively) that not only increased the stability of complex V, but also increased mitochondrial energy output in cancer cells. Further, phospho-ATP5F1A (p-Y-ATP5F1A) prevented its binding to its physiological inhibitor, ATP5IF1 (ATP synthase inhibitory factor subunit 1), causing sustained mitochondrial activity to promote cancer cell growth. TNK2 inhibitor, (R)-9b reversed this process and induced mitophagy-based autophagy to mitigate prostate tumor growth while sparing normal prostate cells. Further, depletion of p-Y-ATP5F1A was needed for (R)-9b-mediated mitophagic response and tumor growth. Moreover, Tnk2 transgenic mice displayed increased p-Y-ATP5F1A and loss of mitophagy and exhibited formation of prostatic intraepithelial neoplasia (PINs). Consistent with these data, a marked increase in p-Y-ATP5F1A was seen as prostate cancer progressed to the malignant stage. Overall, this study uncovered the molecular intricacy of tyrosine kinase-mediated mitochondrial energy regulation as a distinct cancer cell mitochondrial vulnerability and provided evidence that TNK2 inhibitors can act as "mitocans" to induce cancer-specific mitophagy.Abbreviations: ATP5F1A: ATP synthase F1 subunit alpha; ATP5IF1: ATP synthase inhibitory factor subunit 1; CRPC: castration-resistant prostate cancer; DNM1L: dynamin 1 like; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; Mdivi-1: mitochondrial division inhibitor 1; Mut-ATP5F1A: Y243,246A mutant of ATP5F1A; OXPHOS: oxidative phosphorylation; PC: prostate cancer; PINK1: PTEN induced kinase 1; p-Y-ATP5F1A: phosphorylated tyrosine 243 and 246 on ATP5F1A; TNK2/ACK1: tyrosine kinase non receptor 2; Ub: ubiquitin; WT: wild type.

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

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.

Acetylated HOXB13 Regulated Super Enhancer Genes Define Therapeutic Vulnerabilities of Castration-Resistant Prostate Cancer.

PURPOSE: Androgen receptor (AR) antagonism is exacerbated by HOXB13 in castration-resistant prostate cancers (CRPC). However, it is unclear when and how HOXB13 primes CRPCs for AR antagonism. By mass-spectrometry analysis of CRPC extract, we uncovered a novel lysine 13 (K13) acetylation in HOXB13 mediated by CBP/p300. To determine whether acetylated K13-HOXB13 is a clinical biomarker of CRPC development, we characterized its role in prostate cancer biology. EXPERIMENTAL DESIGN: We identified tumor-specific acK13-HOXB13 signal enriched super enhancer (SE)-regulated targets. We analyzed the effect of loss of HOXB13K13-acetylation on chromatin binding, SE proximal target gene expression, self-renewal, enzalutamide sensitivity, and CRPC tumor growth by employing isogenic parental and HOXB13K13A mutants. Finally, using primary human prostate organoids, we evaluated whether inhibiting an acK13-HOXB13 target, ACK1, with a selective inhibitor (R)-9b is superior to AR antagonists in inhibiting CRPC growth. RESULTS: acK13-HOXB13 promotes increased expression of lineage (AR, HOXB13), prostate cancer diagnostic (FOLH1), CRPC-promoting (ACK1), and angiogenesis (VEGFA, Angiopoietins) genes early in prostate cancer development by establishing tumor-specific SEs. acK13-HOXB13 recruitment to key SE-regulated targets is insensitive to enzalutamide. ACK1 expression is significantly reduced in the loss of function HOXB13K13A mutant CRPCs. Consequently, HOXB13K13A mutants display reduced self-renewal, increased sensitivity to enzalutamide, and impaired xenograft tumor growth. Primary human prostate tumor organoids expressing HOXB13 are significantly resistant to AR antagonists but sensitive to (R)-9b. CONCLUSIONS: In summary, acetylated HOXB13 is a biomarker of clinically significant prostate cancer. Importantly, PSMA-targeting agents and (R)-9b could be new therapeutic modalities to target HOXB13-ACK1 axis regulated prostate cancers.

Pilot Study: PARP1 Imaging in Advanced Prostate Cancer.

PURPOSE: PARP inhibitor (PARPi) therapy is approved for patients with metastatic castration-resistant prostate cancer (mCRPC) and homologous recombination repair (HRR) genomic aberrations. However, only a fraction of patients with BRCA1/2 mutations respond to PARPi therapy. In this pilot study, we assess PARP-1 expression in prostate cancer patients with and without HRR genomic alternations using a novel PARP-based imaging agent. PROCEDURES: Nine advanced prostate cancer patients were studied with PET/CT and [18F]FluorThanatrace (FTT), an analogue of the PARPi rucaparib. Images were analyzed using maximum standardized uptake values (SUVmax). PARP expression was assessed by immunohistochemistry (IHC) when feasible (n = 4). RESULTS: We found great variability in FTT uptake (SUVmax range: 2.3-15.4). Patients with HRR mutations had a significantly higher SUVmax (p = 0.0379) than patients with non-HRR mutations although there was an overlap in FTT uptake between groups. Three patients without HRR and one with HRR mutations had similarly high PARP1 IHC expression. CONCLUSIONS: FTT-PET/CT may serve as an alternate biomarker for PARP1 expression and a potential method for PARPi treatment selection.

Chronologically modified androgen receptor in recurrent castration-resistant prostate cancer and its therapeutic targeting.

Resistance to second-generation androgen receptor (AR) antagonists such as enzalutamide is an inevitable consequence in patients with castration-resistant prostate cancer (CRPC). There are no effective therapeutic options for this recurrent disease. The expression of truncated AR variant 7 (AR-V7) has been suggested to be one mechanism of resistance; however, its low frequency in patients with CRPC does not explain the almost universal acquisition of resistance. We noted that the ability of AR to translocate to nucleus in an enzalutamide-rich environment opens up the possibility of a posttranslational modification in AR that is refractory to enzalutamide binding. Chemical proteomics in enzalutamide-resistant CRPC cells revealed acetylation at Lys609 in the zinc finger DNA binding domain of AR (acK609-AR) that not only allowed AR translocation but also galvanized a distinct global transcription program, conferring enzalutamide insensitivity. Mechanistically, acK609-AR was recruited to the AR and ACK1/TNK2 enhancers, up-regulating their transcription. ACK1 kinase-mediated AR Y267 phosphorylation was a prerequisite for AR K609 acetylation, which spawned positive feedback loops at both the transcriptional and posttranslational level that regenerated and sustained high AR and ACK1 expression. Consistent with these findings, oral and subcutaneous treatment with ACK1 small-molecule inhibitor, (R)-9b, not only curbed AR Y267 phosphorylation and subsequent K609 acetylation but also compromised enzalutamide-resistant CRPC xenograft tumor growth in mice. Overall, these data uncover chronological modification events in AR that allows prostate cancer to evolve through progressive stages to reach the resilient recurrent CRPC stage, opening up a therapeutic vulnerability.

Pilot Study of the Performance of 19-G Needle in Endobronchial Ultrasound-guided Transbronchial Aspiration for the Diagnosis and Testing of Molecular Markers in Lung Cancer.

BACKGROUND: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has become the standard for diagnosis and staging of lung cancer. Historically, 21- and 22-G needles have been paired with EBUS. We evaluated the performance of EBUS-TBNA using a larger 19-G needle in the assessment of tumor tissue obtained and success of testing for molecular markers. METHODS: We prospectively enrolled adult patients with lymphadenopathy concerning for metastatic lung cancer. Patients underwent diagnostic EBUS-TBNA utilizing 19-G needles. Cases of non-small cell lung cancer (NSCLC) were evaluated for programmed cell death receptor ligand (PD-L1) expression. Cases of adenocarcinoma or undifferentiated NSCLC were further evaluated for 3 molecular markers for driver mutations: epidermal growth factor receptor (EGFR), c-ros oncogene 1 (ROS-1), and anaplastic lymphoma kinase (ALK). RESULTS: Fifty patients were enrolled and underwent EBUS-TBNA using 19-G needles. PD-L1 assay was successfully performed in 90% of NSCLC cases. In adenocarcinoma or undifferentiated NSCLC cases, the success rate in testing was 90% for EGFR and 86% for ALK. ROS-1 testing had a success rate of 67%; 24% of these specimens had adequate tumor cells but there was technical difficulty with the assay. Block quality was judged by total number of tumor cells per hematoxylin and eosin-stained slide of each cell block (58% of specimens had >500 cells and 22% had 200 to 500 cells). There were no adverse events. CONCLUSION: EBUS-TBNA using 19-G needles can obtain a high number of tumor cells and has a high rate of success in performing assays for PD-L1, EGFR, and ALK in NSCLC patients without an increase in adverse events. The success rate of ROS-1 testing was lower.