Publications by Year: 2021

Short hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting > 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs.

The architecture of chromatin regulates eukaryotic cell states by controlling transcription factor access to sites of gene regulation. Here we describe a dual transposase-peroxidase approach, integrative DNA and protein tagging (iDAPT), which detects both DNA (iDAPT-seq) and protein (iDAPT-MS) associated with accessible regions of chromatin. In addition to direct identification of bound transcription factors, iDAPT enables the inference of their gene regulatory networks, protein interactors and regulation of chromatin accessibility. We applied iDAPT to profile the epigenomic consequences of granulocytic differentiation of acute promyelocytic leukemia, yielding previously undescribed mechanistic insights. Our findings demonstrate the power of iDAPT as a platform for studying the dynamic epigenomic landscapes and their transcription factor components associated with biological phenomena and disease.

A major barrier to the successful application of nanotechnology for cancer treatment is the suboptimal delivery of therapeutic payloads to metastatic tumor deposits. We previously discovered that cabozantinib, a tyrosine kinase inhibitor, triggers neutrophil-mediated anticancer innate immunity, resulting in tumor regression in an aggressive PTEN/p53-deficient genetically engineered murine model of advanced prostate cancer. Here, we specifically investigated the potential of cabozantinib-induced neutrophil activation and recruitment to enhance delivery of BSA-coated polymeric nanoparticles (BSA-NPs) into murine PTEN/p53-deficient prostate tumors. On the basis of the observation that BSA coating of NPs enhanced association and internalization by activated neutrophils by approximately 6-fold in vitro, relative to uncoated NPs, we systemically injected BSA-coated, dye-loaded NPs into prostate-specific PTEN/p53-deficient mice that were pretreated with cabozantinib. Flow cytometric analysis revealed an approximately 4-fold increase of neutrophil-associated BSA-NPs and an approximately 32-fold increase in mean fluorescent dye uptake following 3 days of cabozantinib/BSA-NP administration, relative to BSA-NP alone. Strikingly, neutrophil depletion with Ly6G antibody abolished dye-loaded BSA-NP accumulation within tumors to baseline levels, demonstrating targeted neutrophil-mediated intratumoral NP delivery. Furthermore, we observed an approximately 13-fold decrease in accumulation of BSA-NPs in the liver, relative to uncoated NPs, post-cabozantinib treatment, suggesting that BSA coating of NPs can significantly enhance cabozantinib-induced, neutrophil-mediated targeted intratumoral drug delivery, while mitigating off-target toxicity. Collectively, we demonstrate a novel targeted nano-immunotherapeutic strategy for enhanced intratumoral delivery of BSA-NPs, with translational potential to significantly augment therapeutic indices of cancer medicines, thereby overcoming current pharmacologic barriers commonly encountered in preclinical/early-phase drug development.

Loss of the tumor suppressor gene Pten in murine prostate recapitulates human carcinogenesis and causes stromal proliferation surrounding murine prostate intraepithelial neoplasia (mPIN), which is reactive to microinvasion. In turn, invasion has been shown to be regulated in part by de novo fatty acid synthesis in prostate cancer. We therefore investigated the effects of genetic ablation of Fasn on invasive potential in prostate-specific Pten knockout mice. Combined genetic ablation of Fasn and Pten reduced the weight and volume of all the prostate lobes when compared to single knockouts. The stromal reaction to microinvasion and the cell proliferation that typically occurs in Pten knockout were largely abolished by Fasn knockout. To verify that Fasn knockout indeed results in decreased invasive potential, we show that genetic ablation and pharmacologic inhibition of FASN in prostate cancer cells significantly inhibit cellular motility and invasion. Finally, combined loss of PTEN with FASN overexpression was associated with lethality as assessed in 660 prostate cancer patients with 14.2 years of median follow-up. Taken together, these findings show that de novo lipogenesis contributes to the aggressive phenotype induced by Pten loss in murine prostate and targeting Fasn may reduce the invasive potential of prostate cancer driven by Pten loss. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencing in aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas.

NF-κB activation has been linked to prostate cancer progression and is commonly observed in castrate-resistant disease. It has been suggested that NF-κB-driven resistance to androgen-deprivation therapy (ADT) in prostate cancer cells may be mediated by aberrant androgen receptor (AR) activation and AR splice variant production. Preventing resistance to ADT may therefore be achieved by using NF-κB inhibitors. However, low oral bioavailability and high toxicity of NF-κB inhibitors is a major challenge for clinical translation. Dimethylaminoparthenolide (DMAPT) is an oral NF-κB inhibitor in clinical development and has already shown favorable pharmacokinetic and pharmacodyanamic data in patients with heme malignancies, including decrease of NF-κB in circulating leuchemic blasts. Here, we report that activation of NF-κB/p65 by castration in mouse and human prostate cancer models resulted in a significant increase in AR variant-7 (AR-V7) expression and modest upregulation of AR. In vivo castration of VCaP-CR tumors resulted in significant upregulation of phosphorylated-p65 and AR-V7, which was attenuated by combination with DMAPT and DMAPT increased the efficacy of AR inhibition. We further demonstrate that the effects of DMAPT-sensitizing prostate cancer cells to castration were dependent on the ability of DMAPT to inhibit phosphorylated-p65 function. IMPLICATIONS: Our study shows that DMAPT, an oral NF-κB inhibitor in clinical development, inhibits phosphorylated-p65 upregulation of AR-V7 and delays prostate cancer castration resistance. This provides rationale for the development of DMAPT as a novel therapeutic strategy to increase durable response in patients receiving AR-targeted therapy.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.

Activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is a pervasive event in tumorigenesis due to PI3K mutation and dysfunction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Pharmacological inhibition of PI3K has resulted in variable clinical outcomes, however, raising questions regarding the possible mechanisms of unresponsiveness and resistance to treatment. WWP1 is an oncogenic HECT-type ubiquitin E3 ligase frequently amplified and mutated in multiple cancers, as well as in the germ lines of patients predisposed to cancer, and was recently found to activate PI3K signaling through PTEN inactivation. Here, we demonstrate that PTEN dissociated from the plasma membrane upon treatment with PI3K inhibitors through WWP1 activation, whereas WWP1 genetic or pharmacological inhibition restored PTEN membrane localization, synergizing with PI3K inhibitors to suppress tumor growth both in vitro and in vivo. Furthermore, we demonstrate that WWP1 inhibition attenuated hyperglycemia and the consequent insulin feedback, which is a major tumor-promoting side effect of PI3K inhibitors. Mechanistically, we found that AMPKα2 was ubiquitinated and, in turn, inhibited in its activatory phosphorylation by WWP1, whereas WWP1 inhibition facilitated AMPKα2 activity in the muscle to compensate for the reduction in glucose uptake observed upon PI3K inhibition. Thus, our identification of the cell-autonomous and systemic roles of WWP1 inhibition expands the therapeutic potential of PI3K inhibitors and reveals new avenues of combination cancer therapy.