The transcription factor Hypoxia-Inducible Factor 2α (HIF2α) plays a crucial role in cancer cell adaptation to hypoxic conditions, particularly in clear cell renal cell carcinoma, promoting tumor growth and angiogenesis. Targeting HIF2α through pharmacologic inhibition offers a promising therapeutic strategy for HIF2α-driven cancers. An in silico docking study using 10,000 drug-like compounds was performed using the previously solved X-ray crystal structure of HIF2α. Select compounds predicted to bind to the Per-Arnt-Sim-A (PAS-A) and PAS-B domains of HIF2α were further evaluated for biological activity in clear cell renal cell carcinoma and normal kidney cell lines. Biochemical and cell-based assays were performed to define the mechanism of action for a lead compound. Here, we identify Compound-c2 as a selective HIF2α inhibitor that binds to the PAS-B domain of HIF2α. Notably, Compound-c2 disrupts the interaction between HIF2α and the molecular chaperone Hsp70, leading to proteasomal degradation of HIF2α and the induction of apoptosis in ccRCC. The distinctive inhibitory mechanism of the HIF2α inhibitor identified here, Compound-c2, sets it apart from previous HIF2α antagonists. This positions Compound-c2 as a promising alternative with potential applications in addressing drug resistance, providing a unique approach to inhibit HIF2α-related processes. Cancer occurs when cells grow out of control. In the most common type of kidney cancer, this is caused by abnormal activity of a specific part of cellular machinery. In this study, we used computer simulations to find drugs that can stop this growth-causing activity. By testing drugs identified from these simulations, we found one drug that kills kidney cancer cells, but not normal cells. We show that this drug works differently than another drug used to kill kidney cancer. These results show that in the future, this drug could be used to treat kidney cancer, particularly in patients where other available drugs have not worked. Heritz et al. use an orthogonal approach to identify a selective inhibitor for HIF2α that disrupts its interaction with the molecular chaperone Hsp70. This inhibitor utilizes an alternative mechanism of action to previous HIF2α antagonists, providing a promising approach in addressing kidney cancer drug resistance.

Supplementary Figures 3-5 from Targeted Cancer Gene Therapy Using a Hypoxia Inducible Factor–Dependent Oncolytic Adenovirus Armed with Interleukin-4

Supplementary Figure Legends 1-5 from Targeted Cancer Gene Therapy Using a Hypoxia Inducible Factor–Dependent Oncolytic Adenovirus Armed with Interleukin-4

Supplementary Figures 1-2 from Targeted Cancer Gene Therapy Using a Hypoxia Inducible Factor–Dependent Oncolytic Adenovirus Armed with Interleukin-4

280 Background: Prostate cancer is characterized by heterogeneity of mechanisms which are poorly understood but pointing to epithelial plasticity as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, is proposed to act as tumor suppressor in prostate cancer, but the mechanism of tumor progression due to Abi1 loss is not clear. Methods: To address Abi1’s role in prostate cancer we used CRISPR-based gene editing and retroviral expression to manipulate Abi1 levels in prostate cancer cell lines. Levels of Abi1 expression in prostate organoid tumor cell lines were evaluated by Western blotting and/or RNA sequencing. Association of Abi1 loss with tumor grade was evaluated by immunohistochemistry. Results: Abi1 expression is downregulated in tumor organoid cell lines from metastatic bone and lymph node biopsies. Moreover, low Abi1 expression is associated with high-grade prostate tumors (GG3 or higher, p < 0.001). Disruption of Abi1 gene in a benign prostate epithelial cell line RWPE-1 resulted in a gain of invasive phenotype, which is characterized by loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 Abi1 KO spheroids. Through RNA sequencing and protein expression analysis we discovered that Abi1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of Abi1. Furthermore, increase in STAT3 phosphorylation upon Abi1 inactivation and evidence for high affinity interaction of FYN-SH2 domain with Abi1 pY421 support the model that Abi1 acts as a gatekeeper of the non-canonical WNT-EMT pathway activation downstream from FZD2 receptor. The gene expression profile of Abi1-EMT-WNT pathway overlaps with the reported gene signature of high-risk prostate tumors. Conclusions: Abi1 contributes to prostate cancer progression and epithelial plasticity through regulation of EMT-WNT pathways. Understanding of Abi1’s role may provide more mechanistic understanding of prostate cancer tumor progression.

Abstract American Cancer Society predicts over 160,000 new prostate cancer cases in 2017, which amounts to about 20 percent of all cancer diagnoses in men. Despite several treatment options being available, poor prognosis of high-risk and metastasis remains a major concern for prostate cancer (PCa) patients. Therefore, it is important to understand molecular mechanisms of PCa progression to aggressive disease. Our previous research identified Abelson interactor 1 (Abi1), a member of WAVE complex, as a tumor suppressor in human prostate. Not only did we find mutations and deletions in the gene in prostate tumor patient samples, but WAVE complex levels were also found to be inversely correlated to disease recurrence and castration resistance. Moreover, Abi1 is downregulated in majority of prostate cancer cell lines, and patient organoid cultures derived from metastatic bone and lymph node biopsies. These data point to the clinical significance of defining Abi1-dependent tumor suppression mechanisms. To understand these mechanisms we generated CRISPR-mediated Abi1 KO in the RWPE-1 non-tumor human prostate cell line. When plated in 3D cultures in matrigel, we observed striking phenotypic differences in the appearance and behavior of the Abi1 KO spheroids compared to the parental cells. While the parental RWPE-1 grew into spherical organoids with tight cell-cell boundaries, the Abi1 KO cells grew as loose irregular-shaped spheres, with many cells migrating out of the organoids. Hence, the loss of Abi1 in these non-tumor cells lead to gain of an invasive phenotype. Western Blots of Abi1-KO cells demonstrated concurrent disruption of WAVE complex and deregulation of WAVE complex protein levels. Moreover, cell-cell adhesion proteins such as E-cadherin and β-catenin showed modest decrease and/or a substantial loss of membrane localization. Rescue experiments of Abi1 re-expression in the KO cells reversed the phenotype indicating that the observed phenotype is Abi1-dependent. Analyses of RNA sequencing of cells obtained from 3D cultures demonstrated differences in expression signatures characteristic of altered cell-cell and cell-matrix adhesion pathways and indicated upregulation of pathways associated with the invasive phenotype of cancer cells. We propose that Abi1 loss and WAVE complex deregulation represents key mediators of invasive phenotype in PCa. [Supported by NCI R01 CA161018 and NYS Department of Health Prostate Cancer Hypothesis Development RFA #1410200115] Citation Format: Disharee Das, Ming Chen, Claudia Mondragon, Dawn Post, Alexander Nappi, Heidi Hehnly, Anita Hryniewicz-Jankowska, Pier Paolo Pandolfi, Gennady Bratslavsky, Leszek Kotula. Modeling of Abi1 loss using spheroid cultures to investigate mechanisms of prostate tumorigenesis [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 1403.

Prostate cancer is the second most common cancer in American men, and it is the third leading cause of cancer-related deaths in American men. Distant stage and metastatic disease is often treated with androgen-deprivation therapy (ADT); however, this treatment ultimately fails, leading to castration-resistant prostate cancer (CRPC). This further emphasizes the need for better therapeutic targets. Our work focuses on understanding the role of the Abi1 gene in prostate cancer tumor progression. Our current in vitro data demonstrate an invasive phenotype with our CRISPR-mediated Abi1 knockout (KO) cell lines. In total, using CRISPR-mediated technology we have generated four Abi1 gene KO cell lines, PC-3, DU145, LnCaP, and RWPE-1. Importantly, the androgen-dependent cell line, LNCaP, demonstrated upregulation of Abi1 to the androgen receptor stimulation and the opposite response to the receptor inhibition upon treatment with enzalutamide. These data indicated that low Abi1 level could be induced by ADT therapies and are consistent with our previous observation of low Abi1/WAVE complex in prostate tumor samples. To learn more about this mechanism, we set out to investigate the role of Abi1 in castrated mice. The overall goal of the experiment is to compare the ability of cells lacking the Abi1 gene to engraft in bone under androgen deprivation condition and compare it to the control cells and examine any change in the engraftment phenotype. It is hoped that this research will lead to better understanding of the Abi1/WAVE complex role in CRPC. Supported by NCI R01 CA16101. Citation Format: Alexander Nappi, Dawn Post, Disharee Das, Ishita Joshi, Megan Oest, Gennady Bratslavsky, Leszek Kotula. Defining bone metastatic potential of Abi1 CRISPR prostate cancer cell lines [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 1088.

Abstract Prostate cancer accounts for 20-25% of cancer cases every year and is predicted at over 180,000 new cases in 2016. However, the mechanisms of prostate cancer initiation and castration resistance still remains poorly understood. Our previous research shows mutations and deletions in Abelson interactor 1 (Abi1), an integral part of WAVE complex, in prostate tumor samples. Moreover, WAVE complex levels are inversely correlated to prostate cancer recurrence and castration resistance, outlining their clinical significance. A common phenomenon found early on in most cancers, including prostate cancer, is dysregulated cell-cell adhesion. This points towards a possible tumor suppressor mechanism, since Abi1 and the WAVE complex are in fact the primary regulators of branched actin polymerization via the Arp2/3 complex, and are critical for the formation and maintenance of cell-cell adhesion complexes. Hence, to study Abi1 in prostate cancer, we generated a prostate-specific conditional Abi1 knock-out (KO) mouse. In these mice, we observed evidence of prostatic intraepithelial neoplasia (PIN), a precursor of invasive carcinoma, with changes in proliferation and cell-cell adhesion markers. The goal of this study is to elucidate the mechanism by which the loss of Abi1 leads to the development of PIN in these mice. We are leading this analysis by primary 3D cultures with cells from our Abi1 KO prostates, and 2D cultures with mouse embryonic fibroblasts (MEFs). Loss of Abi1 leads to increase in size of the cultured spheroids, which indicates increased proliferation and dysregulated cell-cell adhesion. Also, adherens junction (AJ) markers E-cadherin and β-catenin levels are lower in western blots and immunostaining, suggesting defects in cell-cell adhesion. In MEFs, we observe loss of membrane localization of AJ proteins in Abi1 KO cells. Loss of Abi1 is often associated with upregulation of Abi2, which may be a contributing factor to the phenotype. To validate our findings in a human system, we have recently developed a CRISPR-mediated KO of Abi1 in the RWPE-1 non-tumor prostate cell line. We will use this KO cell line to determine cell-cell adhesion formation and maintenance in 2D and 3D cultures. In conclusion, loss of Abi1 leads to dysregulation of cell-cell adhesion in prostate epithelial cells, which may be a key mechanism for tumor development and progression in prostate cancer. Supported by NCI R01 CA161018 (LK). Citation Format: Disharee Das, Anita Hryniewicz-Jankowska, Heidi Hehnly, Dawn Post, Gennady Bratslavsky, Leszek Kotula. The key role of Abi1 loss in dysregulating cell-cell adhesion during prostate cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4466. doi:10.1158/1538-7445.AM2017-4466

We propose to understand how the mitotic kinase PLK1 drives chromosome segregation errors, with a specific focus on Gravin, a PLK1 scaffold. In both three-dimensional primary prostate cancer cell cultures that are prone to Gravin depletion and Gravin short hairpin RNA (shRNA)-treated cells, an increase in cells containing micronuclei was noted in comparison with controls. To examine whether the loss of Gravin affected PLK1 distribution and activity, we utilized photokinetics and a PLK1 activity biosensor. Gravin depletion resulted in an increased PLK1 mobile fraction, causing the redistribution of active PLK1, which leads to increased defocusing and phosphorylation of the mitotic centrosome protein CEP215 at serine-613. Gravin depletion further led to defects in microtubule renucleation from mitotic centrosomes, decreased kinetochore-fiber integrity, increased incidence of chromosome misalignment, and subsequent formation of micronuclei following mitosis completion. Murine Gravin rescued chromosome misalignment and micronuclei formation, but a mutant Gravin that cannot bind PLK1 did not. These findings suggest that disruption of a Gravin-PLK1 interface leads to inappropriate PLK1 activity contributing to chromosome segregation errors, formation of micronuclei, and subsequent DNA damage.

The molecular chaperone Hsp90 protects deregulated signaling proteins that are vital for tumor growth and survival. Tumors generally display sensitivity and selectivity toward Hsp90 inhibitors; however, the molecular mechanism underlying this phenotype remains undefined. We report that the mitotic checkpoint kinase Mps1 phosphorylates a conserved threonine residue in the amino-domain of Hsp90. This, in turn, regulates chaperone function by reducing Hsp90 ATPase activity while fostering Hsp90 association with kinase clients, including Mps1. Phosphorylation of Hsp90 is also essential for the mitotic checkpoint because it confers Mps1 stability and activity. We identified Cdc14 as the phosphatase that dephosphorylates Hsp90 and disrupts its interaction with Mps1. This causes Mps1 degradation, thus providing a mechanism for its inactivation. Finally, Hsp90 phosphorylation sensitizes cells to its inhibitors, and elevated Mps1 levels confer renal cell carcinoma selectivity to Hsp90 drugs. Mps1 expression level can potentially serve as a predictive indicator of tumor response to Hsp90 inhibitors.

http://sro.sussex.ac.uk Woodford, Mark R, Truman, Andrew W, Dunn, Diana M, Jensen, Sandra M, Cotran, Richard, Bullard, Renee, Abouelleil, Mourad, Beebe, Kristin, Wolfgeher, Donald, Wierzbicki, Sara, Post, Dawn E, Caza, Tiffany, Tsutsumi, Shinji, Panaretou, Barry, Kron, Stephen J et al. (2016) Mps1 mediated phosphorylation of Hsp90 confers renal cell carcinoma sensitivity and selectivity to Hsp90 inhibitors. Cell Reports, 14 (4). pp. 872-884. ISSN 2211-1247

The epidermal growth factor receptor (EGFR) pathway is aberrantly activated in tumors and plays a key role in promoting tumor growth. Small molecule inhibitors which bind reversibly to EGFR have demonstrated limited clinical activity. Thus, there is a continued need to develop novel EGFR inhibitors with improved anti-tumor activity. Bay846 is a newly developed small molecule inhibitor that binds irreversibly to the tyrosine kinase domains of EGFR and Her2. The in vitro and in vivo efficacy of Bay846 was tested using a panel of nine human malignant brain tumor (glioma) models. Lapatinib, a reversible inhibitor of EGFR and Her2, was included for comparison. Six glioma cell lines were sensitive to Bay846 treatment. Bay846 strongly suppressed tumor cell growth in vitro by inducing cell lysis/death rather than cell cycle arrest. Consistent with this, Bay846 had potent anti-tumor activity which led to regressions in tumor size. The active, phosphorylated form of EGFR was reduced by Bay846 treatment in vitro and in tumors. Importantly, the efficacy of Bay846 was significantly greater than lapatinib in all assays. Bay846-sensitivity was associated with expression of a wild-type PTEN in conjunction with high levels of an oncogenic EGFR variant (A289V or EGFRvIII). These studies demonstrate that targeting the EGFR pathway with the irreversible inhibitor Bay846 has great potential to increase the efficacy of this cancer therapy.

There is a need to develop more potent oncolytic adenoviruses (Ads) that show increased antitumor activity in patients. The HYPR-Ads are targeted oncolytic Ads that specifically kill tumor cells, which express active hypoxia-inducible factor (HIF). While therapeutically efficacious, the HYPR-Ads showed attenuated replication and oncolytic activity. To overcome these deficiencies and improve antitumor efficacy, we created new HIF-activated oncolytic Ads, HIF-Ad and HIF-Ad-IL4, which have two key changes: (i) a modified HIF-responsive promoter to regulate the E1A replication gene and (ii) insertion of the E3 gene region. The HIF-Ads showed conditional activation of E1A expression under hypoxia. Importantly, the HIF-Ads show hypoxia-dependent replication, oncolytic and cellular release activities, and potent antitumor efficacy, all of which are significantly greater than that of the HYPR-Ads. Notably, HIF-Ad-IL4 treatment led to regressions in tumor size by 70% and extensive tumor infiltration by leukocytes resulting in an antitumor efficacy that is up to six-fold greater than that of the HYPR-Ads, HIF-Ad and wild-type Ad treatment. These studies show that treatment with an HIF-activated oncolytic Ad leads to a measurable therapeutic response. The novel design of the HIF-Ads represents a significant improvement compared with first-generation oncolytic Ads and has great potential to increase the efficacy of this cancer therapy.

We exploited the differential activation of hypoxia-inducible factor (HIF)-dependent gene expression in tumors versus normal tissue for the design of a targeted oncolytic herpes simplex virus type-1 (HSV-1). A gene that is essential for viral replication, infected cell polypeptide 4 (ICP4), was placed under the regulation of an HIF-responsive promoter and then introduced into the thymidine kinase locus (U L 23) of HSV d120, which contains partial deletions in the two endogenous ICP4 genes. Recombinant HIF-HSV was isolated and their derivation from d120 was verified by expression of a truncated, non-functional form of ICP4 protein. Disruption of the U L 23 locus was confirmed by loss of thymidine kinase expression and resistance to acyclovir. Unexpectedly, HIF-HSV expressed ICP4 and induced tumor cell lysis at similar levels under normoxia and hypoxia. The lack of HIF-dependent ICP4 transgene expression by HIF-HSV was due to two factors that have not previously been reported—reversion of the ICP4 gene region to its wild-type configuration and increased HIF-transcriptional activity under normoxia when cells were infected with any strain of HSV-1. The findings that an oncolytic HSV-1 is genetically unstable and can activate a tumor-related promoter in a non-specific manner have important implications for any proposed use of this virus in cancer therapy.

A clinically important target for anti-tumor therapy is hypoxia-inducible factor (HIF). HIF is a transcription factor that is expressed in a wide range of primary tumors, metastases, and cancer stem cells, whereas it is undetectable in normal healthy tissues. HIF expression in tumors is associated with (i) resistance to apoptosis, chemotherapy, and radiotherapy and (ii) increased invasion/metastasis, angiogenesis, and patient mortality. Oncolytic adenoviruses (Ad) are a cancer therapy which utilize the cytolytic replication cycle of the virus to specifically kill tumor cells (oncolysis). A wild-type Ad cannot be used for cancer therapy because it lacks tumor-specificity and is toxic to normal healthy tissues. We exploited the differential activation of HIF-dependent gene expression in tumors versus normal tissue for the design of targeted HIF-activated oncolytic Ads. In the first-generation HIF-activated oncolytic Ad called HYPR-Ad, we placed the E1A gene which is essential for Ad replication under the regulation of a HIF-inducible promoter. Our published studies with HYPR-Ad provided critical proof that a HIF-activated oncolytic Ad can specifically kill HIF-active tumor cells and inhibit tumor growth. While therapeutically efficacious, HYPR-Ad exhibited attenuated replication and oncolytic activity when compared to a wild-type Ad in vitro. To overcome these deficiencies and improve anti-tumor efficacy, we created a second-generation HIF-activated oncolytic Ad, called HIF-Ad, which has two key changes: (i) a modified HIF-responsive promoter to regulate the E1A replication gene and (ii) insertion of the E3 gene region. To augment the anti-tumor activity of HIF-Ad beyond the killing of HIF-active tumor cells, it was modified to express interleukin-4 (HIF-Ad-IL4). The IL4 cytokine was selected because it has potent multimodal anti-tumorigenic activities. Using normal and tumor cell lines in vitro, we found that the HIF-Ads have HIF-dependent E1A expression, replication, and oncolytic activity. Importantly, the replication and oncolytic efficacy of the HIF-Ads was similar to a wild-type Ad and superior to the HYPR-Ads. The HIF-Ads demonstrated strong anti-tumor activity against subcutaneous human tumor xenografts. Notably, HIF-Ad-IL4 treatment led to tumor regressions and its anti-tumor efficacy was up to 6-fold greater than HYPR-Ad-IL4, HIF-Ad, and wild-type Ad treatment. These studies demonstrate that the HIF-Ads have improved anti-tumor activity compared to the HYPR-Ads and that treatment with a HIF-activated oncolytic Ad leads to a measurable therapeutic response. The novel design of the HIF-Ads represents a significant improvement compared to first-generation oncolytic Ads and has great potential to increase the anti-tumor efficacy of this cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 586.

The replication cycle of many viruses kills the host cell, and this property is an attractive feature for the development of anticancer viral therapy. The challenge resides in modifying the viral genome so that it will direct the formation of viral particles that can preferentially replicate in and destroy tumor cells while sparing normal tissue. In this review, we will discuss the different strategies that have been developed to engineer cancer-targeted adenoviruses, both replication-deficient gene-therapy vectors and replication-competent oncolytic vectors. Their specific application to the targeting of malignant gliomas in preclinical studies and the results of the first eight clinical trials are presented. Lastly, new ongoing developments in the design, imaging, and replication potency of new generations of therapeutic viruses are discussed.