FDA-approved Anticancer Agents Research Articles

Advances in Cancer Therapy: A Comprehensive Review of CDK and EGFR Inhibitors.

Protein kinases have essential responsibilities in controlling several cellular processes, and their abnormal regulation is strongly related to the development of cancer. The implementation of protein kinase inhibitors has significantly transformed cancer therapy by modifying treatment strategies. These inhibitors have received substantial FDA clearance in recent decades. Protein kinases have emerged as primary objectives for therapeutic interventions, particularly in the context of cancer treatment. At present, 69 therapeutics have been approved by the FDA that target approximately 24 protein kinases, which are specifically prescribed for the treatment of neoplastic illnesses. These novel agents specifically inhibit certain protein kinases, such as receptor protein-tyrosine kinases, protein-serine/threonine kinases, dual-specificity kinases, nonreceptor protein-tyrosine kinases, and receptor protein-tyrosine kinases. This review presents a comprehensive overview of novel targets of kinase inhibitors, with a specific focus on cyclin-dependent kinases (CDKs) and epidermal growth factor receptor (EGFR). The majority of the reviewed studies commenced with an assessment of cancer cell lines and concluded with a comprehensive biological evaluation of individual kinase targets. The reviewed articles provide detailed information on the structural features of potent anticancer agents and their specific activity, which refers to their ability to selectively inhibit cancer-promoting kinases including CDKs and EGFR. Additionally, the latest FDA-approved anticancer agents targeting these enzymes were highlighted accordingly.

C-4 analogues of podophyllotoxin as tubulin inhibitors: synthesis, biological evaluation, and structure-activity relationship

The diversity of lignan small molecules derived from podophyllotoxin, a non-covalent tubulin inhibitor isolated from the Podophyllum family, has led to the clinical development of FDA-approved anticancer agents etoposide and teniposide. While these two compounds share the same tetracyclic core as podophyllotoxin, two subtle structural changes—4’ demethylation on the aromatic ring and stereospecific glycosylation at the C-4 hydroxyl—result in an alternate biological mechanism. Given the immense pharmacological importance of altering the C-4 position, we synthesised and evaluated a systematic library of diversified esters to establish a structure-activity relationship regarding modification at C-4 on the properties of podophyllotoxin. We determined the biological activity of these esters through cell viability assays, computer docking models, tubulin polymerisation assays, and cell cycle analysis. Altogether, we demonstrate that increasing steric hindrance at C-4 leads to a loss in potency against human cancer cells but has a significantly lesser impact on cell-free tubulin inhibition.

MODL-15. CHARACTERIZATION OF SPONTANEOUS CANINE HIGH-GRADE GLIOMA MODELS AS CLINICALLY-RELEVANT SURROGATES OF PEDIATRIC HIGH-GRADE GLIOMA

Abstract BACKGROUND High-grade glioma (HGG) occurring spontaneously in canines shows histological and genetic resemblance to pediatric high-grade glioma (PHGG). Both canine HGG and PHGG are treated with combinations of surgical resection, radiation therapy, and/or chemotherapy, but outcomes for both groups are poor. Studying canine HGG both serves companion dogs and provides an orthogonal preclinical model of PHGG. METHODS Canine HGG lines J3T, SDT3G, and G06A were characterized and compared with H3K27M-mutant PHGG lines DIPG7, DIPG13, and BT245 using neurosphere dilution assay and drug screening with all FDA-approved anti-cancer agents. J3T and SDT3G were orthotopically implanted into mice and monitored using MRI and bioluminescence. RESULTS Like PHGG, the canine lines J3T and SDT3G were highly sensitive to proteasome and XPO1 inhibition; compared to PHGG, the canine lines were most sensitive to protein translation inhibitors and DNA intercalators. Canine HGG lines were not as sensitive to other targeted inhibitors effective in PHGG. Compared to PHGG cell lines, canine cell lines had greater variance in self-renewal capability. J3T and SDT3G were also injected into the cortex of immunodeficient mice. Bioluminescent signal confirmed J3T engraftment one week post-surgery with rapid tumor growth, while SDT3G presented initial engraftment by MRI two weeks post-surgery but did not exhibit sustained growth. DISCUSSION Canine HGG and PHGG showed similar sensitivity to proteasome and XPO1 inhibition; they differed in self-renewal capability and susceptibility to other anti-cancer agents. This could be because canine target proteins often differ from their human counterparts and thus may respond differently to targeted medications designed for humans. Overall, canine HGG provides a novel, primary, immunocompetent surrogate that resembles PHGG and may provide a step for clinical investigation between xenograft models and PHGG trials and/or opportunities for co-clinical trials.

P10.23.A A PATIENT-DERIVED FUNCTIONAL GBM DRUG SCREEN OFFERS A TOOL TO PREDICT TMZ RESPONSE AND ASSESS SENSITIVITY TO CANDIDATE TREATMENTS ON A PER PATIENT BASIS

Abstract BACKGROUND Major obstacles that have impeded the development of effective new therapies for GBM include inter- and intratumoral heterogeneity, the blood-brain-barrier and use of sub-optimal cell line-based preclinical models. Taking these hurdles into account, we have set up a patient-derived GBM drug-screening platform. Molecular similarities and dissimilarities between tumors and derived cultures were investigated and the predictive power of our assay for patient response to TMZ was assessed. A large panel of GBM cells was screened for sensitivity to available oncological agents with the aim of deriving a set of drugs with favorable physicochemical properties for BBB crossing and potent activity in (a subset of) GBM cultures. Finally, we determined the success rate of performing a small-scale screen with 20 selected agents within 4 weeks post-surgery. MATERIAL AND METHODS Tissue samples as well as ultrasonic surgical aspirates (USA) were received from GBM surgeries and utilized to establish glioma stem cell (GSC) cultures. Correspondence of GSCs to parental tumors was assessed by DNA/RNA-sequencing. In vitro sensitivity to temozolomide was screened across 55 GSC cultures. Cell viability readouts were related to clinical parameters of corresponding patients. FDA-approved anticancer agents (n=107) were screened on 45 GBM cultures by dose-response testing and viability was assessed using Cell Titer-Glo. Twenty selected agents were screened on 24 fresh tumor samples within 4 weeks of receiving the tissue. RESULTS By combining both USA and tissue piece-derived dissociation protocols, culture success increased to 95%, ensuring representation of the near-complete spectrum of GBM subtypes. Tumor DNA and RNA sequences revealed strong similarities to corresponding GSC cultures although differences related to neuronal signaling, immune interactions, and cell cycle were observed. Tumor expression profiles of hallmark genes of GBM were faithfully retained in derived cell cultures. In vitro screening of TMZ on a large cohort identified 3 response categories (responders/intermediates/non-responders) for which Cox regression analysis revealed significantly different overall survival curves of corresponding patients. Screening of approved anticancer agents on 45 GBM cultures underscored the tremendous intertumoral heterogeneity in drug sensitivities. We identified 20 potent agents each effective at clinically-achievable concentrations in (a subset of) GBM cultures with favorable BBB penetration properties (CNS-MPO score). Screening of these agents on a per patient basis within 4 weeks of receiving tissue was successful in 18 out of 24 (75%) tested tumors. CONCLUSION Patient-derived functional drug screening offers a tool to predict TMZ response in GBM patients and assess tumor sensitivity to candidate treatments, either for GBM subsets or on a per patient basis.

DDDR-27. A PATIENT-DERIVED DRUG SCREENING PLATFORM PREDICTS RESPONSE TO TEMOZOLOMIDE AND IDENTIFIES POTENTIAL NEW TREATMENTS FOR GLIOBLASTOMA

Abstract BACKGROUND Major obstacles that have impeded the development of effective new therapies for GBM include inter- and intratumoral heterogeneity, the blood-brain-barrier and use of sub-optimal cell line-based preclinical models. Taking these hurdles into account, we have set up a patient-derived GBM drug-screening platform. We optimized protocols to improve cell culture success rate and retrospectively assessed the predictive power of our assay for patient response to TMZ. A large panel of GBM cells was screened for sensitivity to available oncological agents. Drugs of interest were selected based on favorable physicochemical properties for BBB crossing and potent activity in (a subset of) GBM cultures. Finally, we determined the success rate of performing a small-scale screen with 20 selected agents within 4 weeks of receiving tumor tissue. RESULTS By combining both CUSA and tissue piece-derived dissociation protocols, culture success increased to 95%, ensuring representation of the near-complete spectrum of GBM subtypes. Single-cell sequencing studies confirmed heterogeneity in our low-passage cell cultures. In vitro screening of TMZ on a large cohort (n = 55) identified 3 response categories (responders/intermediates/non-responders) for which Cox regression analysis revealed significantly different overall survival curves of corresponding patients. Screening of 107 FDA-approved anticancer agents on 45 GBM cultures underscored the tremendous intertumoral heterogeneity in drug sensitivities. We identified 20 potent agents each effective at clinically-achievable concentrations in (a subset of) GBM cultures and having favorable BBB penetration properties (CNS-MPO score). Screening of these agents on a per patient basis within 4 weeks of receiving tissue was successful in 18 out of 24 (75%) tested tumors. In the remaining cases the tumor cells grew very slowly and longer culture times were required. CONCLUSION Our drug screening platform offers a tool to predict TMZ response and assess sensitivity to candidate treatments, either for GBM subsets or on a per patient basis.

Preclinical evaluation of the ETS inhibitor TK216 against relapsed and refractory childhood leukemia.

10033 Background: Although survival rates have improved in the recent past, relapse and refractory disease remain a significant cause of death in children with leukemia. This calls for an urgent need for the development of novel therapies that could effectively treat leukemias in children. The E26 transformation specific (ETS) family of transcription factors regulate various normal cellular functions but are abnormally expressed in various cancers, including leukemia. TK216 is an ETS inhibitor, that has shown pre-clinical activity and clinical efficacy in solid tumors. In this study, we explore the feasibility of using TK216 as a therapeutic agent for the treatment of high risk refractory pediatric leukemia. Methods: A panel of pediatric leukemia derived cell lines and primary blast cells representing a spectrum of molecular abnormalities seen in pediatric leukemia were treated in vitro with TK216 to determine cytotoxicity. Normal lymphocytes were used as controls and cell viability was determined 72 hours post-treatment by Alamar blue assay. The induction of tumor cell apoptosis and target modulation were detected by Western blotting. Alterations in the cell cycle were assessed by FACS analysis with PI staining. Drug combination studies were carried out with established anti-leukemic agents to identify synergy for greater therapeutic efficiency. Results: TK216 decreased cell viability in leukemia cells compared to normal lymphocyte controls in a dose-dependent manner with variations in sensitivity noted with inherent molecular abnormalities. The IC50 values observed ranged from 0.22 µM for the most sensitive cell line, MV4-11 to 0.95 µM for least sensitive cell line, SUP-B15. Apoptosis induction upon TK216 treatment was confirmed by PARP cleavage and caspase 3 activation. Cell cycle analysis demonstrated increased sub-G1 population of cells after TK216 treatment. A strong correlation between sub-G1 population and sensitivity of the cell line towards TK216 (47% in MV4-11 vs 3.72% in SUP-B15) was observed. Screening of a panel of 200 FDA approved anti-cancer agents in drug combination studies identified potential agents for drug synergy. Significant drug synergy was noted with TK216 in combination with the epigenetic modifier 5-azacytidine and the Bcl-2 inhibitor, Venetoclax. [Combination Index for Venetoclax and TK216, mean = 0.65 for MV4-11 and 0.33 for SUP-B15]. Conclusions: Data from our study demonstrate that the ETS inhibitor TK216 induces apoptosis and cell cycle arrest in pediatric leukemia cells at physiologically relevant concentrations. Our combination studies identified distinct anti-cancer agents that could be used for developing effective drug combination regimens with TK216. Overall, our findings provide essential preclinical data for the consideration of TK216 in early phase clinical trials for the treatment of selected high-risk and refractory childhood leukemia.

A pilot clinical trial of genomic-based therapy assignment with co-expression extrapolation (COXEN) in advanced/metastatic urothelial carcinoma.

452 Background: We investigated the Co-eXpression ExtrapolatioN (COXEN) algorithm to select “next best therapy” for patients with advanced/metastatic urothelial cancer based on gene expression profiles. Methods: This is a single-arm, open label pilot study, that investigates the COXEN algorithm’s ability to use patients’ tumor gene expression models to predict sensitivities to 75 FDA-approved anticancer agents. The COXEN results were reviewed and discussed in a multi-disciplinary molecular tumor board. From that a treatment plan was chosen based on monotherapy clinical efficacy data in urothelial carcinoma, combination safety data (combinations of agents were allowed provided phase I data were available), side effect profile/tolerability, response or resistance to prior therapies, therapy contraindications, and feasibility of therapy. The objective of this study was to determine the feasibility of using COXEN as a clinical decision algorithm to make a real-time treatment decision within 21 days of specimen collection in patients with advanced/metastatic urothelial carcinoma. Results: A total of 8 patients enrolled and underwent tumor biopsy for COXEN analysis. Five out of 8 patients received COXEN therapy within 21 days of biopsy with various regimens including: vorinostat and etoposide; doxorubicin; sunitinib; doxorubicin and paclitaxel; erlotinib and nab-paclitaxel. There were no objective responses. The 5 patients treated with COXEN therapy had a median potential follow up of 36 months, median overall survival of 8.4 months (95% CI: 5.2 months – not estimable), and median progression free survival of 2.2 months (95% CI: 1.4 – 3.9 months). Grade 3 or 4 adverse events included: febrile neutropenia (n=2), hypophosphatemia (n=1) and hyponatremia (n=1). This trial was terminated early due to a lack of treatment responses. Conclusions: The COXEN algorithm is a unique personalized method for selecting therapy for patients with advanced urothelial carcinoma. Although the COXEN method was feasible, it did not demonstrate clinical efficacy in this small cohort of patients. Clinical trial information: NCT02788201.

Drug screening with a novel tumor-derived cell line identified alternative therapeutic options for patients with atypical teratoid/rhabdoid tumor.

Atypical teratoid/rhabdoid tumor (AT/RT) is a rare intracranial tumor occurring predominantly in young children. The prognosis is poor, and no effective treatment is currently available. To develop novel effective therapies, there is a need for experimental models for AT/RT. In this research, we established a cell line from a patient's AT/RT tissue (designated ATRT_OCGH) and performed drug screening using 164 FDA-approved anti-cancer agents, to identify candidates for therapeutic options. We found that bortezomib, a proteasome inhibitor, was among the agents for which the cell line showed high sensitivity, along with tyrosine kinase inhibitors, topoisomerase inhibitors, and histone deacetylase inhibitors, which are known to exert anti-AT/RT effects. Concomitant use of panobinostat potentiated the inhibitory effect of bortezomib on AT/RT cell proliferation. Our findings may provide a rationale for considering combination therapy of panobinostat and bortezomib for treatment of AT/RT.

Abstract 3913: Evaluation of patient-derived cell lines and cancer organoids for the prediction of drug responses in patient-derived xenograft models

Abstract Cancer organoids are heterogeneous 3D cellular clusters with complexities that mimic some characteristics of tumors in situ. Thus, assays performed with cancer organoids might enable better predictions of in vivo drug responses than those performed with cell monolayers. The National Cancer Institute (NCI) is developing a national repository of Patient-Derived (PD) models comprised of clinically annotated and molecularly characterized PD xenografts (PDXs), PD tumor cell lines (PDCs), and PD cancer organoids (PDOrgs) (https://pdmr.cancer.gov/). We evaluated the therapeutic activity of a panel of FDA-approved and investigational anticancer agents, including carboplatin, gemcitabine, paclitaxel, SN38, 5-FU, adavosertib, erlotinib, trametinib, and vemurafenib, against a cohort of PDCs, PDOrgs, and PDXs from solid tumors including colon, gastroesophageal, head and neck, NSCLC, pancreatic, bladder, and uterine cancers. Our goal was to investigate whether drug sensitivities determined using PDCs and PDOrgs correlate with responses observed in the matching PDXs. Cultures were exposed to anticancer agents at concentrations ranging from 1 pM to 100 µM for periods of 4 or 6 days. The data indicated that the GI50 values for PDOrgs were in overall agreement with in vivo PDX drug responses measured as relative median to event free survival (RMEFS), where an event is the median time (days) from treatment initiation to tumor volume quadrupling, calculated as median time to tumor volume quadrupling for treated animals/median time to tumor volume quadrupling for control animals. For both paclitaxel and trametinib, responses in PDOrgs, from most sensitive to most resistant, were similar to the corresponding PDXs. Drug sensitivities determined in PDC monolayers were less clearly related to in vivo PDX responses; particularly for PDCs treated with carboplatin, gemcitabine, and SN-38. This work is part of a larger effort to provide a rigorous comparison between fully characterized and annotated PDCs-PDOrgs-PDXs to assess the value of different in vitro model systems for the prediction of PDX drug responses. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Petreena Campbell, Curtis Hose, Lara El Touny, Erik Harris, John Connelly, Carrie Bonomi, Kelly Dougherty, Savanna Styers, Abigail Walke, Jenna Moyer, Mariah Baldwin, Anna Wade, Michael Mullendore, Kaitlyn Arthur, Matthew Murphy, Kevin Plater, Marion Gibson, Joseph Geraghty, Michelle Gottholm-Ahalt, Tara Grinnage-Pulley, Tiffanie Chase, John Carter, Howard Stotler, Debbie Trail, Luke Stockwin, Dianne Newton, Yvonne Evrard, Melinda Hollingshead, Ralph E. Parchment, Nathan P. Coussens, Beverly A. Teicher, James H. Doroshow, Annamaria Rapisarda. Evaluation of patient-derived cell lines and cancer organoids for the prediction of drug responses in patient-derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3913.

Exportin 1 Inhibition Induces Nerve Growth Factor Receptor Expression to Inhibit the NF-κB Pathway in Preclinical Models of Pediatric High-Grade Glioma.

High-grade glioma (HGG) is the leading cause of cancer-related death among children. Selinexor, an orally bioavailable, reversible inhibitor of the nuclear export protein, exportin 1, is in clinical trials for a range of cancers, including HGG. It inhibits the NF-κB pathway and strongly induces the expression of nerve growth factor receptor (NGFR) in preclinical cancer models. We hypothesized that selinexor inhibits NF-κB via upregulation of NGFR. In HGG cells, sensitivity to selinexor correlated with increased induction of cell surface NGFR expression. Knocking down NGFR in HGG cells increased proliferation, anchorage-independent growth, stemness markers, and levels of transcriptionally available nuclear NF-κB not bound to IκB-α, while decreasing apoptosis and sensitivity to selinexor. Increasing IκB-α levels in NGFR knockdown cells restored sensitivity to selinexor. Overexpression of NGFR using cDNA reduced levels of free nuclear NF-κB, decreased stemness markers, and increased markers of cellular differentiation. In all HGG lines tested, selinexor decreased phosphorylation of NF-κB at serine 536 (a site associated with increased transcription of proliferative and inflammatory genes). Because resistance to selinexor monotherapy occurred in our in vivo model, we screened selinexor with a panel of FDA-approved anticancer agents. Bortezomib, a proteasome inhibitor that inhibits the NF-κB pathway through a different mechanism than selinexor, showed synergy with selinexor against HGG in vitro Our results help elucidate selinexor's mechanism of action and identify NGFR as a potential biomarker of its effect in HGG and in addition suggest a combination therapy strategy for these challenging tumors.

Abstract 290: Formulation of tolerable protocols for combination regimens to control triple-negative breast cancer cell-derived xenograft tumor development in animals

Abstract Cumulative research and clinical results suggest an advantage to use a multiple drug combination over a single drug to control tumor growth and metastasis. In general, combination therapy may result in lower treatment failure rates, lower case fatality ratios, slower development of resistance, and fewer side effects than monotherapy, as well as reducing cost. Triple-negative breast cancer (TNBC) is aggressive, lethal, and heterogeneous. The molecular heterogeneity may account for the differential responses of TNBC to chemotherapies. TNBC is sensitive to conventional chemotherapies with initially high response rates; however, the prognosis is not optimal due to cancer recurrence and metastasis. Although recent advancements in drug development of targeted therapeutics and immunotherapy were somewhat effective in treating TNBC, these therapeutics still need extensive study. Although combination regimens are thought to have better therapeutic outcomes than single drug treatments, drug combinations may also come with overlapped toxicities. Thus, it is important to identify combination regimens that are safe and effectively treat cancers. Based on our in vitro results of combination regiments effective in the synergistic induction of TNBC cell death, we systemically investigated the toxicity and efficacy of combined triple and double agents at various doses and administration protocols to assess the treatment of mice bearing TNBC cell-derived xenograft tumors. We analyzed the adverse effects of combination regimens on the bone marrows, livers, and kidneys, as well as the blood cell profiles in immune-competent and -deficient mice. Our studies successfully identified tolerable protocols for triple and double combination regimens of gemcitabine, romidepsin, and cisplatin, which are FDA-approved anticancer agents, as well as the efficacy of these combination regimens in controlling TNBC xenograft tumors in immune-deficient mice. Our results indicated that the triple combination of gemcitabine plus romidepsin and cisplatin regimen was more effective than double combination regimens in the control of TNBC tumor development in vivo. In conclusion, our studies suggested that the triple combination regimens should be promptly considered as an advanced treatment of TNBC over the conventional gemcitabine and cisplatin regimen or the clinical trial romidepsin plus cisplatin regimen. Citation Format: Pawat Pattarawat, Shelby Wallace, Agricola Odoi, Bianca Pfisterer, Hwa-Chain R. Wang. Formulation of tolerable protocols for combination regimens to control triple-negative breast cancer cell-derived xenograft tumor development in animals [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 290.

Abstract 2076: Targeting vulnerable aberrant cancer-associated pathways by rationalized triple combination regimens to effectively control urothelial carcinoma cells in vitro and in vivo

Abstract Urinary bladder cancer (UBC) is the fifth-most common cancer in the United States, and urothelial carcinomas (UCs) account for more than 90% of UBCs. Although current therapies are effective in the short-term treatment of UCs, the high tendency (>50%) for them to recur and progress to life threatening, advanced muscle-invasive UCs requires life-long surveillance with extended medical care. Thus, it still requires extensive therapeutic development to achieve an effective control of UC recurrence and improve the response rate and overall survival for UC patients. To develop effective and safe regimens to control the development and recurrence of advanced UCs, we investigated the efficacy of rationalized combination regimens in controlling UC cells in vitro and UC cell-derived xenografts (CDXs) in vivo. Our in vitro studies identified a triple combination of gemcitabine, romidepsin, and cisplatin, which are all FDA-approved anticancer agents, effective in synergistically inducing death and reducing drug resistance of various UC cell lines. Our studies also revealed that UC-associated aberrant pathways, including mitochondrial reactive oxygen species (ROS) and the ERK-Nox pathway, which were already induced in UC cells, were enhanced by the triple combination regimen to play essential roles for inducing apoptosis and reducing resistance. We also detected that the unfolded protein response played a role in modulating cell death induced by the regimen. Our in vivo studies used a tolerable protocol for administering combination regimens into animals. Our studies verified the efficacy of the triple combination gemcitabine plus romidepsin and cisplatin regimen, versus double combination regimens, in controlling UC CDXs in immune-deficient mice. In conclusion, our studies indicated that a rationalized triple combination of gemcitabine plus romidepsin and cisplatin regimen was more effective than double combination regimens in modulating UC-associated vulnerable aberrant pathways and controlling UC tumor development and recurrence in animals with low tolerable/ reversible adverse effects. Thus, the triple combination gemcitabine plus romidepsin and cisplatin regimen should be promptly considered as an advanced treatment over the conventional double combination gemcitabine plus cisplatin regimen in clinical trials in order to control advanced UCs. Citation Format: Hwa-Chain R. Wang, Pawat Pattarawat, Jinquan Wang, Robert Donnell. Targeting vulnerable aberrant cancer-associated pathways by rationalized triple combination regimens to effectively control urothelial carcinoma cells in vitro and in vivo [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 2076.

Abstract 5016: High-throughput patient-derived 3-dimensional organoid cultures as personalized models to assess drug response and post-treatment residual disease

Abstract Cell lines grown in 2-dimensional (2D) plastic surfaces have historically been the main models to identify cancer cell vulnerabilities. In contrast, malignant tumors in vivo grow as 3-dimensional (3D) cellular masses and manifest remarkably different drug-response patterns compared to cell lines. We have shown that breast cancer (BrCa) cells in 3D extracellular matrices acquire distinct phenotypic properties, compared to the 2D counterparts. Notably, serum-free culture medium supplemented with defined cocktails of developmental morphogens was sufficient to support the growth of BrCa cell lines in 3D, but not in 2D conditions; indicating a key role of 3D architecture in in vivo growth regulation. CRISPR/Cas9-based gene editing screens in BrCa cells indicated that distinct gene subset govern the growth in 3D vs. 2D conditions. Using similar culture conditions, we expanded several molecularly annotated BrCa patient-derived 3D Organoids (3D PDOs), adapted them into a high-throughput miniaturized drug screening platform, and interrogated them against a panel of ~500 target-annotated kinase inhibitors and FDA-approved anti-cancer agents. This pharmacological screen revealed high sensitivity to distinct classes of agents (eg. PI3K inhibitors), but also variable responses to others (eg. EGFR inhibitors); and provided insight into potentially driver vs. passenger mutations in the respective patients. Co-culture of PDOs with non-malignant stromal cells from bone metastatic niche reduced sensitivity to some classes of drugs (eg. PLK inhibitors). Importantly, long-term (up to 3-4 weeks) treatment of 3D organoids did not eradicate the cells in culture (in contrast to conventional 2D assays), but generated a cell subpopulation which remained viable for the duration of the experiment, reminiscent of the residual disease after initial clinical partial responses to a drug. This residual disease phenotype was also confirmed in vivo, when the respective PD-Xenografts were treated with the same agents. The majority of transcripts with differential expression in residual PDX tumor cells were also concordantly up- or down-regulated in the respective 3D PDO model. These transcripts included canonical regulators of cell cycle, chromatin signaling and epigenetic mechanisms, cytoskeletal function and metabolic pathways; as well as other transcripts not previously linked to drug resistance in conventional 2D models. To our knowledge this is the first description of an in vitro personalized cancer model that simulates both molecularly and phenotypically the post-treatment residual disease in vivo. Our high-throughput functional platform can potentially complement genomic approaches in personalized medicine and help discover novel drugs that overcome tumor drug resistance. Citation Format: Eugen Dhimolea, Ricardo De Matos Simoes, Yoko DeRose, Pallavi Awate, Xiang Weng, Huihui Tang, Aedin Culhane, Alana Welm, Constantine Mitsiades. High-throughput patient-derived 3-dimensional organoid cultures as personalized models to assess drug response and post-treatment residual disease [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 5016. doi:10.1158/1538-7445.AM2017-5016

A Comprehensive Review of US FDA-Approved Immune Checkpoint Inhibitors in Urothelial Carcinoma.

Few effective treatment options are available for patients with advanced or metastatic urothelial carcinoma (UC) after unsuccessful first-line platinum-based chemotherapy. To date, immune checkpoint inhibitors are novel therapeutic agents for UC treatment. From May 2016 to May 2017, five anti-PD-1/PD-L1 monoclonal antibodies received accelerated or regular approval from the US Food and Drug Administration (FDA) for the treatment of patients with locally advanced or metastatic UC. The present comprehensive review presents the background information of these five US FDA-approved anticancer agents to provide a basic but concise understanding of these agents for advanced studies. We summarize their immune checkpoint mechanisms, clinical efficacy, recommended usage protocols, adverse events, and the limitations of the PD-L1 biomarker assays.

Abstract 267: Preclinical investigation of the HSP90 inhibitor, ganetespib, in combination with FDA-approved cytotoxic agents for their potential anti-tumor effects on human neuroendocrine tumors

Abstract The gastroenteropancreatic neuroendocrine tumor (GEP-NET) system is comprised of a heterogeneous group of tumors with increasing incidence. Current standard of care cytotoxic agents have limited efficacy, which necessitates the need for innovative therapeutic approaches. Heat shock protein 90 (HSP90) is overexpressed in a wide range of tumor types including human pancreatic neuroendocrine tumors (PanNETs). Ganetespib is a second-generation HSP90 inhibitor that is well tolerated in cancer patients (dosed into >1500 patients) and is currently being evaluated in several investigator sponsored clinical trials including acute myeloid leukemia (AML), ovarian cancer, breast cancer, and other tumor types. Here, we show that ganetespib inhibits the proliferation of NET cells and induces apoptosis in vitro with potency comparable to another second-generation HSP90 inhibitor (NVP-AUY922), but with potency two- to seven-fold more than first generation inhibitors (17-AAG, IPI-504) in BON-1, CM and H727 cell lines, and thirty-fold more in QGP-1 cell line. In mice bearing PanNET tumor xenografts, single agent ganetespib reduced the growth of tumors without signs of toxicity. Tumors from ganetespib treated animals demonstrated reduced phospho-AKT and phospho-ERK expression, and elevated HSP70 expression, supportive of exposures necessary for functional activity. In an effort to identify clinically meaningful agents that could further potentiate the antitumor activity of ganetespib, we performed in vitro combination screens evaluating proliferation in four NET cell lines with ganetespib and forty FDA approved anticancer agents. Ganetespib showed synergistic effects when combined with inhibitors of mTOR, topoisomerase I or II, or DNA synthesis. Results from our ongoing in vivo combination studies with ganetespib and these three classes of anticancer agents will be presented. Citation Format: Chung Wong, Evan Vosburgh, Arnold Levine, David Proia, Eugenia Xu. Preclinical investigation of the HSP90 inhibitor, ganetespib, in combination with FDA-approved cytotoxic agents for their potential anti-tumor effects on human neuroendocrine tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 267.

Abstract 3555: Targeting the BCL-2 family in small cell lung caner

Abstract Small cell lung cancer (SCLC) represents 13% of all lung cancer cases, affecting approximately 30,000 people annually in the United States. The prognosis for patients with SCLC is poor, and cancer-specific mortality of this malignancy is 95% at five years. This dismal prognosis has been further marred by the absence of major improvements in its treatment: there have been no substantial changes in the standard of care for advanced SCLC over the last three decades. Interestingly, small molecule inhibitors of anti-apoptotic BCL-2 family members BCL-2 and BCL-XL, ABT-737 and its orally bioavailable analog ABT-263, were shown to effectively kill some SCLC cell lines in preclinical studies, suggesting that targeting the BCL-2 family proteins may hold promise for the treatment of this dreadful cancer. However, these inhibitors do not inhibit MCL-1, another anti-apoptotic BCL-2 family member, which may explain why multiple clinical trials have not led to meaningful results. Because there are no effective MCL-1 inhibitors developed yet, we propose to identify the best strategy that enhances the apoptotic effect of ABT-737/263. To this end, we have performed a high-throughput screen in ABT-737/263 resistant H196 cell lines using two libraries, a library of FDA-approved anti-cancer agents and a pathway inhibitor library including ∼1000 compounds. As a result, doxorubicin and dinaciclib were identified as synergizers of ABT-737/263 in triggering apoptosis. These two drugs enhanced the death inducing activity of ABT-737/263 through downregulation of MCL-1 mRNA. Interestingly, some of the ABT-737/263 resistant cell lines rely on MCL-1 for survival and thereby were killed by dinaciclib or doxorubicin as a single agent, indicating that predictive biomarkers of cellular addiction to anti-apoptotic BCL-2 family proteins (BCL-2s) can guide treatment decisions and reduce unwanted toxicity. Importantly, we revealed that the expression ratio between BCL-2, BCL-XL, and MCL-1 could predict cellular addiction to BCL-2s. Surprisingly, we demonstrated that BCL-XL overexpression is an another potential mechanism behind ABT-263 resistance. ABT-263 failed to inhibit the interaction between BCL-XL and BAX/BAK in the absence of activator BH3-only molecules and thereby failed to kill one BCL-XL-addicted SCLC cell line with low expression of activator BH3-only molecules. Finally, we showed that BCL-2 selective inhibitor ABT-199 can also cooperate with doxorubicin or dinaciclib to kill ABT-263-resistant cell lines. The in vivo efficacy of combination therapy including ABT-199 and doxorubicin was demonstrated in a SCLC xenograft model. These data have direct translational implications for the treatment of SCLC. Citation Format: Akane Inoue-Yamauchi, Paul Jeng, Kwanghee Kim, Hui-Chen Chen, Song Han, Yogesh Tengarai Ganesan, Yiyu Dong, Sylvia Jebiwott, James J. Hsieh, Emily H. Cheng. Targeting the BCL-2 family in small cell lung caner. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3555.

Combination of Hedgehog inhibitors and standard anticancer agents synergistically prevent osteosarcoma growth.

High-dose chemotherapy and surgical intervention have improved long-term prognosis for non-metastatic osteosarcoma to 50-80%. However, metastatic osteosarcoma exhibits resistance to standard chemotherapy. We and others have investigated the function of Hedgehog pathway in osteosarcoma. To apply our previous findings in clinical settings, we examined the effects of Hedgehog inhibitors including arsenic trioxide (ATO) and vismodegib combined with standard anticancer agents. We performed WST-1 assays using ATO, cisplatin (CDDP), ifosfamide (IFO), doxorubicin (DOX), and vismodegib. Combination-index (CI) was used to examine synergism using CalcuSyn software. Xenograft models were used to examine the synergism invivo. WST-1 assays showed that 143B and Saos2 cell proliferation was inhibited by ATO combined with CDDP, IFO, DOX, and vismodegib. Combination of ATO and CDDP, IFO, DOX or vismodegib was synergistic when the two compounds were used on proliferating 143B and Saos2 human osteosarcoma cells. An osteosarcoma xenograft model showed that treatment with ATO and CDDP, IFO, or vismodegib significantly prevented osteosarcoma growth invivo compared with vehicle treatment. Our findings indicate that combination of Hedgehog pathway inhibitors and standard FDA-approved anticancer agents with established safety for human use may be an attractive therapeutic method for treating osteosarcoma.

Impact of a Biomarker-Based Strategy on Oncology Drug Development: A Meta-analysis of Clinical Trials Leading to FDA Approval.

In order to ascertain the impact of a biomarker-based (personalized) strategy, we compared outcomes between US Food and Drug Administration (FDA)-approved cancer treatments that were studied with and without such a selection rationale. Anticancer agents newly approved (September 1998 to June 2013) were identified at the Drugs@FDA website. Efficacy, treatment-related mortality, and hazard ratios (HRs) for time-to-event endpoints were analyzed and compared in registration trials for these agents. All statistical tests were two-sided. Fifty-eight drugs were included (leading to 57 randomized [32% personalized] and 55 nonrandomized trials [47% personalized], n = 38 104 patients). Trials adopting a personalized strategy more often included targeted (100% vs 65%, P < .001), oral (68% vs 35%, P = .001), and single agents (89% vs 71%, P = .04) and more frequently permitted crossover to experimental treatment (67% vs 28%, P = .009). In randomized registration trials (using a random-effects meta-analysis), personalized therapy arms were associated with higher relative response rate ratios (RRRs, compared with their corresponding control arms) (RRRs = 3.82, 95% confidence interval [CI] = 2.51 to 5.82, vs RRRs = 2.08, 95% CI = 1.76 to 2.47, adjusted P = .03), longer PFS (hazard ratio [HR] = 0.41, 95% CI = 0.33 to 0.51, vs HR = 0.59, 95% CI = 0.53 to 0.65, adjusted P < .001) and a non-statistically significantly longer OS (HR = 0.71, 95% CI = 0.61 to 0.83, vs HR = 0.81, 95% CI = 0.77 to 0.85, adjusted P = .07) compared with nonpersonalized trials. Analysis of experimental arms in all 112 registration trials (randomized and nonrandomized) demonstrated that personalized therapy was associated with higher response rate (48%, 95% CI = 42% to 55%, vs 23%, 95% CI = 20% to 27%, P < .001) and longer PFS (median = 8.3, interquartile range [IQR] = 5 vs 5.5 months, IQR = 5, adjusted P = .002) and OS (median = 19.3, IQR = 17 vs 13.5 months, IQR = 8, Adjusted P = .04). A personalized strategy was an independent predictor of better RR, PFS, and OS, as demonstrated by multilinear regression analysis. Treatment-related mortality rate was similar for personalized and nonpersonalized trials. A biomarker-based approach was safe and associated with improved efficacy outcomes in FDA-approved anticancer agents.

Abstract 5590: Screening of pediatric and adult sarcoma cell lines reveals novel patterns of sensitivity toward approved oncology drugs and investigational agents.

Abstract Sarcomas represent a heterogeneous group of neglected cancers with significant unmet medical need. To date, large-scale screening of compounds for activity against sarcomas has not been carried out in any systematic fashion to enable drug discovery. Our laboratory has compiled a panel of &amp;gt; 85 human pediatric and adult sarcoma cell lines, a tumor type not represented in the NCI60, and has carried out a high throughput screen of approximately 60 of these lines against the Approved Oncology Drugs (AOD) and Investigational Agents libraries, using inhibition of proliferation as an endpoint. The AOD library is comprised of 93 FDA-approved anticancer agents, and is available from The NCI/DTP Open Chemical Repository. The Investigational Agents library includes about 350 small molecules of interest. The compounds were screened for inhibition of cell proliferation in 9-point concentration response, starting at 10 uM and diluting by half-logs. The cells were cultured in 384-well plates for 24 hrs, at which time compounds were added with a final DMSO concentration of 0.25%. Viability was determined after 96 hrs of exposure using Alamar Blue. The % viability was determined relative to a vehicle-treated control, and this value was plotted relative to compound concentration to determine the EC50. The Ewing sarcoma lines (19) were unexpectedly more sensitive to pemetrexed, cabazitaxel, and floxuridine, and were, as expected, sensitive to topoisomerase 1 inhibitors relative to other sarcomas and to historical NCI60 data. Synovial sarcoma lines were sensitive to vinorelbine, vandetinib, and gemcitabine relative to other sarcomas. Further analyses and findings from the data will be presented. Funded by NCI Contract no. HHSN261200800001E. Citation Format: E. Michael August, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Michael Selby, Thomas Silvers, Joel Morris, Beverly A. Teicher. Screening of pediatric and adult sarcoma cell lines reveals novel patterns of sensitivity toward approved oncology drugs and investigational agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5590. doi:10.1158/1538-7445.AM2013-5590

The Akt Inhibitor ISC-4 Synergizes with Cetuximab in 5-FU-Resistant Colon Cancer

Phenylbutyl isoselenocyanate (ISC-4) is an Akt inhibitor with demonstrated preclinical efficacy against melanoma and colon cancer. In this study, we sought to improve the clinical utility of ISC-4 by identifying a synergistic combination with FDA-approved anti-cancer therapies, a relevant and appropriate disease setting for testing, and biomarkers of response. We tested the activity of ISC-4 and 19 FDA-approved anticancer agents, alone or in combination, against the SW480 and RKO human colon cancer cell lines. A synergistic interaction with cetuximab was identified and validated in a panel of additional colon cancer cell lines, as well as the kinetics of synergy. ISC-4 in combination with cetuximab synergistically reduced the viability of human colon cancer cells with wild-type but not mutant KRAS genes. Further analysis revealed that the combination therapy cooperatively decreased cell cycle progression, increased caspase-dependent apoptosis, and decreased phospho-Akt in responsive tumor cells. The synergism between ISC-4 and cetuximab was retained independently of acquired resistance to 5-FU in human colon cancer cells. The combination demonstrated synergistic anti-tumor effects in vivo without toxicity and in the face of resistance to 5-FU. These results suggest that combining ISC-4 and cetuximab should be explored in patients with 5-FU-resistant colon cancer harboring wild-type KRAS.