Beryl A. Hatton

The SmoA1 mouse model reveals that notch signaling is critical for the growth and survival of sonic hedgehog-induced medulloblastomas

To develop a genetically faithful model of medulloblastoma with increased tumor incidence compared with the current best model we activated the Sonic Hedgehog (Shh) pathway by transgenically expressing a constitutively active form of Smoothened in mouse cerebellar granule neuron precursors (ND2:SmoA1 mice). This resulted in early cerebellar granule cell hyper-proliferation and a 48% incidence of medulloblastoma formation. Gene expression studies showed an increase in the known Shh targets Gli1 and Nmyc that correlated with increasing hyperplasia and tumor formation. Notch2 and the Notch target gene, HES5, were also significantly elevated in Smoothened-induced tumors showing that Shh pathway activation is sufficient to induce Notch pathway signaling. In human medulloblastomas reverse transcription-PCR for Shh and Notch targets revealed activation of both of these pathways in most tumors when compared with normal cerebellum. Notch pathway inhibition with soluble Delta ligand or γ secretase inhibitors resulted in a marked reduction of viable cell numbers in medulloblastoma cell lines and primary tumor cultures. Treatment of mice with D283 medulloblastoma xenografts with a γ secretase inhibitor resulted in decreased proliferation and increased apoptosis, confirming that Notch signaling contributes to human medulloblastoma proliferation and survival. Medulloblastomas in ND2:SmoA1 mice and humans have concomitant increase in Shh and Notch pathway activities, both of which contribute to tumor survival.

The Smo/Smo Model: Hedgehog-Induced Medulloblastoma with 90% Incidence and Leptomeningeal Spread

Toward the goal of generating a mouse medulloblastoma model with increased tumor incidence, we developed a homozygous version of our ND2:SmoA1 model. Medulloblastomas form in 94% of homozygous Smo/Smo mice by 2 months of age. Tumor formation is, thus, predictable by age, before the symptomatic appearance of larger lesions. This high incidence and early onset of tumors is ideal for preclinical studies because mice can be enrolled before symptom onset and with a greater latency period before late-stage disease. Smo/Smo tumors also display leptomeningeal dissemination of neoplastic cells to the brain and spine, which occurs in many human cases. Despite an extended proliferation of granule neuron precursors (GNP) in the postnatal external granular layer (EGL), the internal granular layer formed normally in Smo/Smo mice and tumor formation occurred only in localized foci on the superficial surface of the molecular layer. Thus, tumor formation is not simply the result of over proliferation of GNPs within the EGL. Moreover, Smo/Smo medulloblastomas were transplantable and serially passaged in vivo, demonstrating the aggressiveness of tumor cells and their transformation beyond a hyperplastic state. The Smo/Smo model is the first mouse medulloblastoma model to show leptomeningeal spread. The adherence to human pathology, high incidence, and early onset of tumors thus make Smo/Smo mice an efficient model for preclinical studies.

N-myc Is an Essential Downstream Effector of Shh Signaling during both Normal and Neoplastic Cerebellar Growth

We examined the genetic requirements for the Myc family of oncogenes in normal Sonic hedgehog (Shh)-mediated cerebellar granule neuronal precursor (GNP) expansion and in Shh pathway-induced medulloblastoma formation. In GNP-enriched cultures derived from N-myc(Fl/Fl) and c-myc(Fl/Fl) mice, disruption of N-myc, but not c-myc, inhibited the proliferative response to Shh. Conditional deletion of c-myc revealed that, although it is necessary for the general regulation of brain growth, it is less important for cerebellar development and GNP expansion than N-myc. In vivo analysis of compound mutants carrying the conditional N-myc null and the activated Smoothened (ND2:SmoA1) alleles showed, that although granule cells expressing the ND2:SmoA1 transgene are present in the N-myc null cerebellum, no hyperproliferation or tumor formation was detected. Taken together, these findings provide in vivo evidence that N-myc acts downstream of Shh/Smo signaling during GNP proliferation and that N-myc is required for medulloblastoma genesis even in the presence of constitutively active signaling from the Shh pathway.

Hedgehog pathway inhibitor saridegib (IPI-926) increases lifespan in a mouse medulloblastoma model

The Sonic Hedgehog (Shh) pathway drives a subset of medulloblastomas, a malignant neuroectodermal brain cancer, and other cancers. Small-molecule Shh pathway inhibitors have induced tumor regression in mice and patients with medulloblastoma; however, drug resistance rapidly emerges, in some cases via de novo mutation of the drug target. Here we assess the response and resistance mechanisms to the natural product derivative saridegib in an aggressive Shh-driven mouse medulloblastoma model. In this model, saridegib treatment induced tumor reduction and significantly prolonged survival. Furthermore, the effect of saridegib on tumor-initiating capacity was demonstrated by reduced tumor incidence, slower growth, and spontaneous tumor regression that occurred in allografts generated from previously treated autochthonous medulloblastomas compared with those from untreated donors. Saridegib, a known P-glycoprotein (Pgp) substrate, induced Pgp activity in treated tumors, which likely contributed to emergence of drug resistance. Unlike other Smoothened (Smo) inhibitors, the drug resistance was neither mutation-dependent nor Gli2 amplification-dependent, and saridegib was found to be active in cells with the D473H point mutation that rendered them resistant to another Smo inhibitor, GDC-0449. The fivefold increase in lifespan in mice treated with saridegib as a single agent compares favorably with both targeted and cytotoxic therapies. The absence of genetic mutations that confer resistance distinguishes saridegib from other Smo inhibitors.

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.

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.

Notch signaling is not essential in sonic hedgehog-activated medulloblastoma.

Dysregulated signal transduction through the notch pathway has been noted in human and mouse medulloblastoma studies. Gamma secretase inhibitors (GSIs) impair notch signaling by preventing the cleavage of transmembrane notch proteins into their active intracellular domain fragments. Previous studies have shown that GSI treatment caused apoptosis and impaired medulloblastoma cell engraftment in xenograft systems. In this study, we used in vivo genetic and pharmacologic approaches to quantify the contribution of notch signaling to sonic hedgehog (shh)-activated mouse medulloblastoma models. In contrast to prior in vitro studies, pharmacologic inhibition of notch pathways did not reduce the efficiency of medulloblastoma xenotransplantation nor did systemic therapy impact tumor size, proliferation, or apoptosis in genetically engineered mouse medulloblastoma models. The incidence and pathology of medulloblastomas driven by the SmoA1 transgene was unchanged by the bi-allelic absence of Notch1, Notch2, or Hes5 genes. These data show that notch signaling is not essential for the initiation, engraftment, or maintenance of sonic hedgehog pathway-driven medulloblastomas.

Minimally invasive in tumor multidrug comparative analysis with contrast‐enhanced MRI in mice

To facilitate decision making in the oncology clinic, technologies have recently been developed to independently inject and assess multiple anticancer agents directly in a patients tumor. To increase the flexibility of this approach beyond histological readouts of response, contrast‐enhanced MRI was evaluated for the detection of cell death in living tumors after injection.

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