Monica Bartucci

The Hippo transducers TAZ and YAP in breast cancer: oncogenic activities and clinical implications.

The Hippo signalling is emerging as a tumour suppressor pathway whose function is regulated by an intricate network of intracellular and extracellular cues. Defects in the signal cascade lead to the activation of the Hippo transducers TAZ and YAP. Compelling preclinical evidence showed that TAZ/YAP are often aberrantly engaged in breast cancer (BC), where their hyperactivation culminates into a variety of tumour-promoting functions such as epithelial-to-mesenchymal transition, cancer stem cell generation and therapeutic resistance. Having acquired a more thorough understanding in the biology of TAZ/YAP, and the molecular outputs they elicit, has prompted a first wave of exploratory, clinically-focused analyses aimed at providing initial hints on the prognostic/predictive significance of their expression. In this review, we discuss oncogenic activities linked with TAZ/YAP in BC, and we propose clinical strategies for investigating their role as biomarkers in the clinical setting. Finally, we address the therapeutic potential of TAZ/YAP targeting and the modalities that, in our opinion, should be pursued in order to further study the biological and clinical consequences of their inhibition.

Personalized Medicine Approaches in Prostate Cancer Employing Patient Derived 3D Organoids and Humanized Mice

Prostate cancer (PCa) is the most common malignancy and the second most common cause of cancer death in Western men. Despite its prevalence, PCa has proven very difficult to propagate in vitro. PCa represents a complex organ-like multicellular structure maintained by the dynamic interaction of tumoral cells with parenchymal stroma, endothelial and immune cells, and components of the extracellular matrix (ECM). The lack of PCa models that recapitulate this intricate system has hampered progress toward understanding disease progression and lackluster therapeutic responses. Tissue slices, monolayer cultures and genetically engineered mouse models (GEMM) fail to mimic the complexities of the PCa microenvironment or reproduce the diverse mechanisms of therapy resistance. Moreover, patient derived xenografts (PDXs) are expensive, time consuming, difficult to establish for prostate cancer, lack immune cell-tumor regulation, and often tumors undergo selective engraftments. Here, we describe an interdisciplinary approach using primary PCa and tumor initiating cells (TICs), three-dimensional (3D) tissue engineering, genetic and morphometric profiling, and humanized mice to generate patient-derived organoids for examining personalized therapeutic responses in vitro and in mice co-engrafted with a human immune system (HIS), employing adaptive T-cell- and chimeric antigen receptor- (CAR) immunotherapy. The development of patient specific therapies targeting the vulnerabilities of cancer, when combined with antiproliferative and immunotherapy approaches could help to achieve the full transformative power of cancer precision medicine.

BMI-1 targeting interferes with patient-derived tumor-initiating cell survival and tumor growth in prostate cancer

Purpose: Current prostate cancer management calls for identifying novel and more effective therapies. Self-renewing tumor-initiating cells (TICs) hold intrinsic therapy resistance and account for tumor relapse and progression. As BMI-1 regulates stem cell self-renewal, impairing BMI-1 function for TIC-tailored therapies appears to be a promising approach. Experimental Design: We have previously developed a combined immunophenotypic and time-of-adherence assay to identify CD49bhiCD29hiCD44hi cells as human prostate TICs. We utilized this assay with patient-derived prostate cancer cells and xenograft models to characterize the effects of pharmacologic inhibitors of BMI-1. Results: We demonstrate that in cell lines and patient-derived TICs, BMI-1 expression is upregulated and associated with stem cell–like traits. From a screened library, we identified a number of post-transcriptional small molecules that target BMI-1 in prostate TICs. Pharmacologic inhibition of BMI-1 in patient-derived cells significantly decreased colony formation in vitro and attenuated tumor initiation in vivo, thereby functionally diminishing the frequency of TICs, particularly in cells resistant to proliferation- and androgen receptor–directed therapies, without toxic effects on normal tissues. Conclusions: Our data offer a paradigm for targeting TICs and support the development of BMI-1–targeting therapy for a more effective prostate cancer treatment. Clin Cancer Res; 22(24); 6176–91. ©2016 AACR.

DNA Damage and Repair Biomarkers in Cervical Cancer Patients Treated with Neoadjuvant Chemotherapy: An Exploratory Analysis

Cervical cancer cells commonly harbour a defective G1/S checkpoint owing to the interaction of viral oncoproteins with p53 and retinoblastoma protein. The activation of the G2/M checkpoint may thus become essential for protecting cancer cells from genotoxic insults, such as chemotherapy. In 52 cervical cancer patients treated with neoadjuvant chemotherapy, we investigated whether the levels of phosphorylated Wee1 (pWee1), a key G2/M checkpoint kinase, and γ-H2AX, a marker of DNA double-strand breaks, discriminated between patients with a pathological complete response (pCR) and those with residual disease. We also tested the association between pWee1 and phosphorylated Chk1 (pChk1), a kinase acting upstream Wee1 in the G2/M checkpoint pathway. pWee1, γ-H2AX and pChk1 were retrospectively assessed in diagnostic biopsies by immunohistochemistry. The degrees of pWee1 and pChk1 expression were defined using three different classification methods, i.e., staining intensity, Allred score, and a multiplicative score. γ-H2AX was analyzed both as continuous and categorical variable. Irrespective of the classification used, elevated levels of pWee1 and γ-H2AX were significantly associated with a lower rate of pCR. In univariate and multivariate analyses, pWee1 and γ-H2AX were both associated with reduced pCR. Internal validation conducted through a re-sampling without replacement procedure confirmed the robustness of the multivariate model. Finally, we found a significant association between pWee1 and pChk1. The message conveyed by the present analysis is that biomarkers of DNA damage and repair may predict the efficacy of neoadjuvant chemotherapy in cervical cancer. Further studies are warranted to prospectively validate these encouraging findings.

Utility of Glioblastoma Patient-Derived Orthotopic Xenografts in Drug Discovery and Personalized Therapy

Despite substantial effort and resources dedicated to drug discovery and development, new anticancer agents often fail in clinical trials. Among many reasons, the lack of reliable predictive preclinical cancer models is a fundamental one. For decades, immortalized cancer cell cultures have been used to lay the groundwork for cancer biology and the quest for therapeutic responses. However, cell lines do not usually recapitulate cancer heterogeneity or reveal therapeutic resistance cues. With the rapidly evolving exploration of cancer “omics,” the scientific community is increasingly investigating whether the employment of short-term patient-derived tumor cell cultures (two- and three-dimensional) and/or patient-derived xenograft models might provide a more representative delineation of the cancer core and its therapeutic response. Patient-derived cancer models allow the integration of genomic with drug sensitivity data on a personalized basis and currently represent the ultimate approach for preclinical drug development and biomarker discovery. The proper use of these patient-derived cancer models might soon influence clinical outcomes and allow the implementation of tailored personalized therapy. When assessing drug efficacy for the treatment of glioblastoma multiforme (GBM), currently, the most reliable models are generated through direct injection of patient-derived cells or more frequently the isolation of glioblastoma cells endowed with stem-like features and orthotopically injecting these cells into the cerebrum of immunodeficient mice. Herein, we present the key strengths, weaknesses, and potential applications of cell- and animal-based models of GBM, highlighting our experience with the glioblastoma stem-like patient cell-derived xenograft model and its utility in drug discovery.

Synthesis and Characterization of Novel BMI1 Inhibitors Targeting Cellular Self-Renewal in Hepatocellular Carcinoma.

BackgroundHepatocellular carcinoma (HCC) represents one of the most lethal cancers worldwide due to therapy resistance and disease recurrence. Tumor relapse following treatment could be driven by the persistence of liver cancer stem-like cells (CSCs). The protein BMI1 is a member of the polycomb epigenetic factors governing cellular self-renewal, proliferation, and stemness maintenance. BMI1 expression also correlates with poor patient survival in various cancer types.ObjectiveWe aimed to elucidate the extent to which BMI1 can be used as a potential therapeutic target for CSC eradication in HCC.MethodsWe have recently participated in characterizing the first known pharmacological small molecule inhibitor of BMI1. Here, we synthesized a panel of novel BMI1 inhibitors and examined their ability to alter cellular growth and eliminate cancer progenitor/stem-like cells in HCC with different p53 backgrounds.ResultsAmong various molecules examined, RU-A1 particularly downregulated BMI1 expression, impaired cell viability, reduced cell migration, and sensitized HCC cells to 5-fluorouracil (5-FU) in vitro. Notably, long-term analysis of HCC survival showed that, unlike chemotherapy, RU-A1 effectively reduced CSC content, even as monotherapy. BMI1 inhibition with RU-A1 diminished the number of stem-like cells in vitro more efficiently than the model compound C-209, as demonstrated by clonogenic assays and impairment of CSC marker expression. Furthermore, xenograft assays in zebrafish showed that RU-A1 abrogated tumor growth in vivo.ConclusionsThis study demonstrates the ability to identify agents with the propensity for targeting CSCs in HCC that could be explored as novel treatments in the clinical setting.

Abstract 1264: Conditional mouse and zebrafish models of INK4-mediated tumor suppression reveal ARF-independent regulation of cellular senescence

The INK4b-ARF-INK4a locus on human chromosome 9p21 is a hot spot genomic region that undergoes frequent inactivation or deletion in a wide spectrum of human cancers. The locus encodes three cell cycle inhibitory proteins: p15INK4b encoded by CDKN2b, p16INK4a encoded by CDKN2a and p14ARF (p19Arf in mice) encoded by an alternative reading frame (ARF) of CDKN2a. We have recently identified a homologous INK4 genomic locus in zebrafish that is surprisingly devoid of ARF sequences, and encodes a single zebrafish ink4ab gene that functions to activate stress-induced senescence. Therefore, evolution of the mammalian INK4 locus to include multiple tumor suppressors sharing regulatory features could attribute to stronger mechanisms of tumor suppression in these longer-lived vertebrates. Oxidative and/or oncogenic stress provokes cellular senescence involving the retinoblastoma (Rb) and ARF/p53 pathways, leading to silencing of growth-promoting genes by methylation of histone H3 lysine 9 (H3K9me) with the histone methyltransferase Suv39h1. We found that ink4ab deficient zebrafish had deregulated Rb signaling and senescence responses, and reduced suv39h1 expression levels that led to poor overall survival. Ink4ab deficient zebrafish had increased lymphocyte proliferation, splenomegaly, and developed multiple spontaneous tumors including metastatic melanoma, osteosarcoma, hepatocellular adenoma, leukemias and myelodysplastic disorders. Moreover, zebrafish heterozygous for both p53 and ink4ab mutations displayed significantly higher tumor incidence compared to p53 heterozygotes with wild-type ink4ab, indicating that ink4ab haploinsufficiency promotes tumorigenesis. To this end, the combined deficiency of ink4ab and p53 accelerates tumor latency, likely by reversing cellular senescence responses. To further examine ARF-independent senescence regulatory roles of INK4 products in a mammalian model, we have generated conditional mice deficient for all three INK4 open reading frames in the adult hematopoietic system. Within seven months, homozygous Ink4a/Ink4b/ARF-/- mice exhibited marked splenomegaly and developed spontaneous tumors, including leukemias and soft tissue sarcomas. Peripheral blood, splenic and bone marrow analyses of homozygous mutant mice showed deregulated lymphocyte development, uncontrolled proliferation, apoptosis and Suv39h1-dependent senescence when compared to heterozygous and wild type mice. These results identify ARF-independent and Suv39h1-dependent senescence regulatory and tumor suppressor mechanisms whose inactivation permits tumor formation in response to oncogenic and/or oxidative stress. Our models offer the opportunity to reveal novel paradigms for promoting cellular senescence as an alternative strategy for cancer prevention and therapy. Citation Format: Kathleen Flaherty, Daniel Jones, Shamila Yussuf, Stephani Davis, Eric Huselid, Wei Wang, Monica Bartucci, Hatem E. Sabaawy. Conditional mouse and zebrafish models of INK4-mediated tumor suppression reveal ARF-independent regulation of cellular senescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1264. doi:10.1158/1538-7445.AM2015-1264

Abstract 223: Generation of single cell-derived normal, benign and cancer mini-prostates from primary patient-derived tissues

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnClinically effective next generation androgen deprivation therapy (ADT) and androgen receptor (AR)-directed therapies have been developed to treat castration-resistant prostate cancer (CRPC). However, disease progression inevitably occurs, and majority of relapsed tumors continue to overexpress AR. AR mutations and constitutively active AR splice variants (AR-Vs) are detected during ADT and may lead to drug resistance. Therefore, there is dire need to develop models that provide a better understanding of AR signaling and determine markers of ADT resistance in prostate cancer patient cells. Mouse models and existing human prostate cancer cell lines have inherent limitations for deciphering mechanisms of therapy resistance. Unlike the recently reported prostate tissue organoids that were derived at a low efficiency from 7/49 metastatic prostate cancer samples, we have developed a novel and exciting technology to generate normal, benign prostatic hyperplasia (BPH) and cancer “mini-prostates” from both early-stage and advanced prostate cancer patient samples. We have successfully rederived mini-prostates from 17 out of 19 primary prostatectomy tissues. We incorporated developmental epithelial and mesenchymal signals, stem-like cells and growth requirements in 3D cultures for maintaining primary normal, BPH and prostate cancer patient-derived cells as stem-like cell-derived organoids. Our preliminary data show the establishment of patient- single cell-derived normal, BPH and cancer mini-prostates comprised of organoids with glandular tissues that have a thick basement membrane, an outer mesenchymal and inner epithelial cells conveying AR signaling, expressing cytokeratins and secreting PSA. RNA sequencing, expression profiling and ADT revealed that mini-prostate cells maintain the genetic, chemoresistance, and growth rate features of the primary prostate. To model tumor heterogeneity, prostate cells from surgical tissues were isolated upon digital imaging and tissue mapping in mirror sections for AMACR, AR, AR-Vs, p63, Ki67, ERG expression and FISH analysis for TMPRSS2-ERG fusion. Living cells from mapped BPH and cancer clones are being utilized to derive single cells into mini-prostates. Our studies generated novel therapy sensitivity and drug discovery models that would allow for precision medicine and coclinical approaches to be enlisted to reveal chemo- or hormonal-resistance profiles in genetically defined mini-prostates, apply combinatory-targeted therapies and study therapy-induced clonal selection processes to ultimately guide future clinical trials.nnCitation Format: Monica Bartucci, Michele Patrizii, Eric Huselid, Shamila Yussuf, Nitu Bansal, Kathleen Flaherty, Denis Tolkunov, Hua Zhong, Mark N. Stein, Joseph Bertino, Robert DiPaola, Isaac Kim, Hatem E. Sabaawy. Generation of single cell-derived normal, benign and cancer mini-prostates from primary patient-derived tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 223. doi:10.1158/1538-7445.AM2015-223

Patient stem cell (SC)-derived prostate cancer (PC) organoids (Org) to recreate clonal heterogeneity of PC foci and measure therapeutic response potential.

252 Background: In spite of significant outcome gains in recurrent and de novo metastatic PC, resistance inevitably develops to androgen deprivation (ADT) and chemotherapy. Resistance is supported by clonal heterogeneity due to complex genomic and epigenetic alterations within PC foci in the primary and metastases (mets) and by the enrichment of residual tumor foci with PC SCs that survive therapy. PCSCs maintain self-renewal capacity, adapt over time to different microenvironments and retain tumor propagating activity leading to castration resistant (CR) PC progression. To study these processes, we generated patient SC-derived PC Org from primary and mets specimens. Methods: We isolated cells from normal epithelium and PC foci of 4 histopathologically mapped radical prostatectomy specimens and 2 CRPC mets. We developed a 3D culture system utilizing single tumor initiating cells expressing CD29hi/CD49bhi/CD44hiand co-cultured with epithelial/mesenchymal growth factors. Org growth peaked at 2 weeks, and Or...

Abstract C36: Patient-derived organoids from high-risk prostate cancer identify different biological subtypes and responsiveness to therapy

Clonal heterogeneity in primary prostate cancer (PC) is a well-established phenomenon from which rare subclones develop metastatic potential that seed distant sites. It is also well established that androgen deprivation increases clonal heterogeneity of PC foci, seeding between sites and progression to castration resistant (CR) disease that is poorly controlled by subsequent therapies. Genomic sequencing and expression profiling have revealed the complexity and variation of the molecular changes within foci and across patients. In spite of the heterogeneity, a number of gene/pathway alterations have been identified that discriminate molecular subtype and potential differences in response to therapies. To study the heterogeneity of locally advanced PC in high-risk patients, identify foci with metastatic potential and differences in biologic behavior, we generated patient-derived organoids. To this end, we isolated cells from PC foci and normal epithelial counterparts from histopathologically mapped radical prostatectomy specimens and developed a 3D culture system utilizing single cell-derived tumor initiating cells (TICs) and epithelial/mesenchymal growth factors. Organoid growth peaks at 2 weeks and serially passaged organoids can be maintained in culture for up to 6 months with no significant deviation from early passages. The organoids mirror the morphologic features (well-organized glandular structures with central E-Cad+ cells, peripheral Vimentin+ cells and a surrounding thick basement membrane), biomarker expression (AR and PSA) and molecular subtypes of the foci from which they were derived, including alterations in TMRSS2-Ets fusions, loss of PTEN, RB and CHD1, overexpression of Erg, BMI-1, AR and AR-Vs. To study the biology and responsiveness of these organoids to established therapies in the androgen sensitive and castration resistant stages of PC progression, we measured single-cell-derived organoid cell proliferation, viability and secondary organoid- forming potential in response to androgen deprivation, docetaxel, abiraterone, enzalutamide, BEZ325, BMS-345541 IKB/IKK inhibitor and a novel BMI-1 inhibitor. The results show that androgen deprivation decreases cellular growth compared to control untreated organoids, although they can be passaged for up to 4 additional weeks. Monotherapy with docetaxel, abiraterone or BEZ325 significantly reduced tumor cell viability in the absence of DHT but only abiraterone and docetaxel reduced the organoid-forming potential. The BMI-1 inhibitor selectively inhibited self-renewal, and combinations of the BMI-1 inhibitor with docetaxel rescued resistance to docetaxel in CRPC-derived cells. In conclusion, we developed a personalized and genetically defined patient-derived PC organoid model that can be utilized to identify clones with metastatic potential which may help planning systemic therapeutic interventions. Citation Format: Hatem E. Sabaawy, Monica Bartucci, Mark N. Stein, Isaac Yi Kim, Joseph R. Bertino, Robert S. DiPaola, Anna Ferrari. Patient-derived organoids from high-risk prostate cancer identify different biological subtypes and responsiveness to therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C36.