Joyoti Dey

Induction of the phase II detoxification pathway suppresses neuron loss in Drosophila models of Parkinson's disease

α-Synuclein aggregates are a common feature of sporadic Parkinsons disease (PD), and mutations that increase α-synuclein abundance confer rare heritable forms of PD. Although these findings suggest that α-synuclein plays a central role in the pathogenesis of this disorder, little is known of the mechanism by which α-synuclein promotes neuron loss or the factors that regulate α-synuclein toxicity. To address these matters, we tested candidate modifiers of α-synuclein toxicity using a Drosophila model of PD. In the current work, we focused on phase II detoxification enzymes involved in glutathione metabolism. We find that the neuronal death accompanying α-synuclein expression in Drosophila is enhanced by loss-of-function mutations in genes that promote glutathione synthesis and glutathione conjugation. This neuronal loss can be overcome by genetic or pharmacological interventions that increase glutathione synthesis or glutathione conjugation activity. Moreover, these same pharmacological agents suppress neuron loss in Drosophila parkin mutants, a loss-of-function model of PD. Our results suggest that oxidative stress is a feature of α-synuclein toxicity and that induction of the phase II detoxification pathway represents a potential preventative therapy for PD.

A technology platform to assess multiple cancer agents simultaneously within a patient’s tumor

Simultaneous in vivo assessment of multiple cancer drugs and drug combinations using microinjection technology predicts systemic response in model tumors and has shown feasibility for assessment of drug efficacy in a pilot study in cancer patients. There’s no place like the human Animal models of human tumors and dish cultures of cancer cells are not sufficient to predict an individual patient’s response to therapy. In the emerging era of personalized medicine, why limit ourselves to rodent models and engineered in vitro tumor models when we can study a drug directly in the patient’s tumor? This question was answered by Klinghoffer et al. by creating a microinjection system called CIVO that delivers small doses of up to eight different drugs simultaneously, directly into the tumor. The tumors could then be removed and evaluated for various markers of cancer response; in short, the authors looked for markers of cell death and drug-related mechanisms of action. By using an injection-tracking dye, Klinghoffer and colleagues could see where the drug was deposited and then use an automated analyzer for quantitative image processing along the 6-mm injection tract. In mouse models of human lymphoma, the authors were able to correctly predict systemic responsiveness to doxorubicin or vincristine—or not, in the case of resistant lymphomas. They also uncovered unexpected drug sensitivities, which were not picked up by traditional cell culture, including to novel anticancer agents, and confirmed these in vivo. The authors pilot-tested the device in dog and human patients, demonstrating the ability of CIVO to inject and track local tumor response to chemotherapies. Ultimately, such a personalized approach to drug sensitivity testing will allow for rational selection of therapeutics while sparing patients the pain—and time—associated with ineffective treatments. A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.

Albumin-bound nanoparticle (nab) paclitaxel exhibits enhanced paclitaxel tissue distribution and tumor penetration

Purposenab-paclitaxel demonstrates improved clinical efficacy compared with conventional Cremophor EL (CrEL)-paclitaxel in multiple tumor types. This study explored the distinctions in drug distribution between nab-paclitaxel and CrEL-paclitaxel and the underlying mechanisms.MethodsUptake and transcytosis of paclitaxel were analyzed by vascular permeability assay across human endothelial cell monolayers. The tissue penetration of paclitaxel within tumors was evaluated by local injections into tumor xenografts and quantitative image analysis. The distribution profile of paclitaxel in solid-tumor patients was assessed using pharmacokinetic modeling and simulation.ResultsLive imaging demonstrated that albumin and paclitaxel were present in punctae in endothelial cells and could be observed in very close proximity, suggesting cotransport. Uptake and transport of albumin, nab-paclitaxel and paclitaxel were inhibited by clinically relevant CrEL concentrations. Further, nab-paclitaxel causes greater mitotic arrest in wider area within xenografted tumors than CrEL- or dimethyl sulfoxide-paclitaxel following local microinjection, demonstrating enhanced paclitaxel penetration and uptake by albumin within tumors. Modeling of paclitaxel distribution in patients with solid tumors indicated that nab-paclitaxel is more dependent upon transporter-mediated pathways for drug distribution into tissues than CrEL-paclitaxel. The percent dose delivered to tissue via transporter-mediated pathways is predicted to be constant with nab-paclitaxel but decrease with increasing CrEL-paclitaxel dose.ConclusionsCompared with CrEL-paclitaxel, nab-paclitaxel demonstrated more efficient transport across endothelial cells, greater penetration and cytotoxic induction in xenograft tumors, and enhanced extravascular distribution in patients that are attributed to carrier-mediated transport. These observations are consistent with the distinct clinical efficacy and toxicity profile of nab-paclitaxel.

A Distinct Smoothened Mutation Causes Severe Cerebellar Developmental Defects and Medulloblastoma in a Novel Transgenic Mouse Model

ABSTRACT Deregulated developmental processes in the cerebellum cause medulloblastoma, the most common pediatric brain malignancy. About 25 to 30% of cases are caused by mutations increasing the activity of the Sonic hedgehog (Shh) pathway, a critical mitogen in cerebellar development. The proto-oncogene Smoothened (Smo) is a key transducer of the Shh pathway. Activating mutations in Smo that lead to constitutive activity of the Shh pathway have been identified in human medulloblastoma. To understand the developmental and oncogenic effects of two closely positioned point mutations in Smo, we characterized NeuroD2-SmoA2 mice and compared them to NeuroD2-SmoA1 mice. While both SmoA1 and SmoA2 transgenes cause medulloblastoma with similar frequencies and timing, SmoA2 mice have severe aberrations in cerebellar development, whereas SmoA1 mice are largely normal during development. Intriguingly, neurologic function, as measured by specific tests, is normal in the SmoA2 mice despite extensive cerebellar dysplasia. We demonstrate how two nearly contiguous point mutations in the same domain of the encoded Smo protein can produce striking phenotypic differences in cerebellar development and organization in mice.

A Platform for Rapid, Quantitative Assessment of Multiple Drug Combinations Simultaneously in Solid Tumors In Vivo

While advances in high-throughput screening have resulted in increased ability to identify synergistic anti-cancer drug combinations, validation of drug synergy in the in vivo setting and prioritization of combinations for clinical development remain low-throughput and resource intensive. Furthermore, there is currently no viable method for prospectively assessing drug synergy directly in human patients in order to potentially tailor therapies. To address these issues we have employed the previously described CIVO platform and developed a quantitative approach for investigating multiple combination hypotheses simultaneously in single living tumors. This platform provides a rapid, quantitative and cost effective approach to compare and prioritize drug combinations based on evidence of synergistic tumor cell killing in the live tumor context. Using a gemcitabine resistant model of pancreatic cancer, we efficiently investigated nine rationally selected Abraxane-based combinations employing only 19 xenografted mice. Among the drugs tested, the BCL2/BCLxL inhibitor ABT-263 was identified as the one agent that synergized with Abraxane® to enhance acute induction of localized apoptosis in this model of human pancreatic cancer. Importantly, results obtained with CIVO accurately predicted the outcome of systemic dosing studies in the same model where superior tumor regression induced by the Abraxane/ABT-263 combination was observed compared to that induced by either single agent. This supports expanded use of CIVO as an in vivo platform for expedited in vivo drug combination validation and sets the stage for performing toxicity-sparing drug combination studies directly in cancer patients with solid malignancies.

Voruciclib, a clinical stage oral CDK9 inhibitor, represses MCL-1 and sensitizes high-risk Diffuse Large B-cell Lymphoma to BCL2 inhibition

Aberrant regulation of BCL-2 family members enables evasion of apoptosis and tumor resistance to chemotherapy. BCL-2 and functionally redundant counterpart, MCL-1, are frequently over-expressed in high-risk diffuse large B-cell lymphoma (DLBCL). While clinical inhibition of BCL-2 has been achieved with the BH3 mimetic venetoclax, anti-tumor efficacy is limited by compensatory induction of MCL-1. Voruciclib, an orally bioavailable clinical stage CDK-selective inhibitor, potently blocks CDK9, the transcriptional regulator of MCL-1. Here, we demonstrate that voruciclib represses MCL-1 protein expression in preclinical models of DLBCL. When combined with venetoclax in vivo, voruciclib leads to model-dependent tumor cell apoptosis and tumor growth inhibition. Strongest responses were observed in two models representing high-risk activated B-cell (ABC) DLBCL, while no response was observed in a third ABC model, and intermediate responses were observed in two models of germinal center B-cell like (GCB) DLBCL. Given the range of responses, we show that CIVO, a multiplexed tumor micro-dosing technology, represents a viable functional precision medicine approach for differentiating responders from non-responders to BCL-2/MCL-1 targeted therapy. These findings suggest that the combination of voruciclib and venetoclax holds promise as a novel, exclusively oral combination therapy for a subset of high-risk DLBCL patients.

Abstract 2835: Voruciclib, a clinical stage oral CDK inhibitor, sensitizes triple negative breast cancer xenografts to proteasome inhibition

Triple negative breast cancer (TNBC) is a highly heterogeneous disease, notoriously challenging to treat with standard chemotherapy options, and therefore is an area of intense focus for discovery of novel effective combination therapies. Here we used a previously described technology platform called CIVO, which enables assessment of multiple drugs and drug combinations simultaneously in living tumors, to identify drug combinations that result in synergistic anti-tumor activity in the HCC1187 model of TNBC. Our study focused on agents that combine with Voruciclib, a novel clinical stage oral CDK inhibitor with potent activity ( Citation Format: Joyoti Dey, Joseph Casalini, Sally Ditzler, Matt Biery, Angela Merrell, Derek Thirstrup, Marc Grenley, Richard Klinghoffer. Voruciclib, a clinical stage oral CDK inhibitor, sensitizes triple negative breast cancer xenografts to proteasome inhibition. [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 2835.

Abstract A39: A platform to assess multiple therapy options simultaneously in a patient's own tumor

Assessment of anti-cancer drug efficacy is an imprecise and challenging undertaking. Early candidate selection is typically based on results from systemically treated animal models and later by performance in human trials where patients are exposed to often toxic levels of drug, prior to obtaining readouts of tumor response. In both of these testing models, only one drug can be tested at a time. Using these methods, over 90% of candidate new oncology drugs fail to provide benefit for patients in human clinical trials. To improve the predictive value of preclinical candidate selection in animal models and enable a new type of pre-Phase 1 toxicity-sparing comparative drug efficacy study in humans, amenable for use in the solid tumor clinic, we have developed a technology platform called CIVO™. This platform allows for simultaneous assessment of multiple drugs or drug combinations directly in a single solid tumor to assess efficacy, resistance and drug synergies. In this study, precise, controlled delivery of classic chemotherapy drugs vincristine and doxorubicin induced spatially defined (ranging 0.3 – 2.0 mm in diameter), readily detectable, and mechanism-specific cellular changes around sites of tumor microinjection across three xenograft models of lymphoma. The extent of apoptosis induced via CIVO™ microdosing of each drug ( The data presented here generated in drug-responsive and non-responsive solid tumors in the preclinical setting sets the stage for future application of this technology to demonstrate tumor responsiveness to novel drug candidates in the context of human patients. Citation Format: Richard Klinghoffer, Alicia Moreno-Gonzalez, Michael Carleton, Marc Grenley, Beryl Hatton, Jason Frazier, William Kerwin, Ilona Tretyak, Nathan Hedin, Joyoti Dey, Joseph Casalini, Sally Ditzler, James Olson, Nathan Caffo. A platform to assess multiple therapy options simultaneously in a patient9s own tumor. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A39.

Abstract 3129: A platform to assess multiple therapy options simultaneously in a patient's own tumor

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Proper selection of anti-cancer agents at the earliest stage of patient treatment following diagnosis of disease relapse is expected to substantially impact clinical response to treatment. Currently, genomic approaches to personalized cancer treatments have been yielded mixed results, while empirical tests to assess tumor responsiveness have been limited to ex vivo systems that disrupt the native tumor microenvironment and show limited predictive value. To address the need for multiplexed in vivo chemosensitivity testing, we have developed a technology that allows simultaneous assessment of multiple cancer therapeutics directly in a patients tumor. This technology could provide a valuable decision-making tool to prioritize effective treatments in the oncology clinic. Data herein highlight how this technology enables controlled and reliable microinjection of multiple drugs simultaneously in preclinical tumor models, canine lymphoma, and human lymphoma patients. Consistent with the controlled drug delivery of this system, spatially localized, readily detectable, and mechanism-specific cellular changes were observed around sites of microinjection in response to classic chemotherapy drugs (vincristine and doxorubicin) as well as to a small molecule inhibitor of TOR kinase. Importantly, localized response (or lack thereof) to individual components of CHOP combination therapy correlated with response to long-term systemic drug administration across multiple cell line and patient-derived xenograft models of lymphoma. Underscoring the importance of assessing drug efficacy in the context of an intact in vivo system, tumor responses to vincristine were impacted by the local tumor microenvironment. Our results also emphasize the importance of selecting effective therapies early in the course of treatment, as drug resistance mechanisms induced cross-resistance to otherwise efficacious drugs. These studies set the stage for use of this platform in oncology drug development, where the ability to more rapidly assess drug efficacy using clinically relevant in vivo tumors may decrease the current reliance on in vitro cell-based models of cancer and possibly increase the likelihood of clinical success. This platform may thus be useful a clinical decision-making tool for selection of patient-specific anti-cancer therapies. Citation Format: Richard Klinghoffer, Alicia Moreno-Gonzalez, Michael Carleton, Jason Frazier, Marc Grenley, Ilona Tretyak, Nathan Hedin, Joyoti Dey, Joseph Casalini, Beryl Hatton, Sally Ditzler, James Olson, Daniel Pierce, Ellen Filvaroff, Nathan Caffo. A platform to assess multiple therapy options simultaneously in a patients own tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3129. doi:10.1158/1538-7445.AM2014-3129