Wetmore, Cynthia

Abstract Background: NL-201 is a selective and long-acting computationally designed alpha-independent agonist of the IL-2 and IL-15 receptors, which share beta and gamma signaling subunits. NL-201 is being developed as a potent activator of CD8+ T cells and NK cells for cancer immunotherapy. Binding to the beta and gamma subunits stimulates dose-dependent expansion and tumor infiltration of cytotoxic CD8+ T cells and NK cells, thereby enhancing the immune response in the tumor. The absence of binding to the IL-2 alpha subunit reduces the undesirable effects of traditional IL-2 therapies, such as vascular leak syndrome and expansion of immunosuppressive regulatory T cells. As such, NL-201 is designed to promote the desired immunomodulatory anti-tumor effects of IL-2 with an improved safety profile. Methods: NL201-101 is a Phase 1 first-in-human, open-label, dose-escalation and cohort expansion study consisting of two parts. Part 1 is a monotherapy dose-escalation study in up to 60 adult patients with advanced and/or refractory solid tumors to define the safety profile and the recommended Phase 2 dose (RP2D) and schedule of NL-201. During dose escalation, two different schedules will be evaluated: dosing every 21 days (Schedule A), or on days 1 and 8 of each 21-day cycle (Schedule B). Tumor response will be assessed by Response Evaluation Criteria in Solid Tumours (RECIST) 1.1 and/or RECIST for use in cancer immunotherapy trials (iRECIST). In Part 2, patients with pathologically proven diagnosis of melanoma and renal cell carcinoma (up to N=30/cohort), who have advanced and/or refractory measurable disease and have failed at least one line of treatment, which may include checkpoint inhibitors, will be enrolled. Key exclusion criteria include history of brain cancer or active brain metastases, carcinomatous meningitis, neurologic autoimmune disease; patients previously receiving CAR-T or IL-2-based therapies are not eligible. Recruitment of Part 1 began in April 2021, and the trial is actively enrolling. Clinicaltrials.gov identifier: NCT04659629. Citation Format: Aung Naing, Margaret Callahan, Brian A. Costello, Brendan Curti, Evan Hall, Aaron Hansen, Georgina V. Long, Anthony M. Joshua, Cynthia Wetmore, Andrew Weickhardt. A first-in-human phase 1 study of NL-201 in patients with relapsed or refractory cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT250.

AbstractBackgroundPrexasertib (LY2606368) is a novel, second‐generation, selective dual inhibitor of checkpoint kinase proteins 1 (CHK1) and 2 (CHK2). We conducted a phase 1 trial of prexasertib to estimate the maximum‐tolerated dose (MTD) and/or recommended phase 2 dose (RP2D), to define and describe the toxicities, and to characterize the pharmacokinetics (PK) of prexasertib in pediatric patients with recurrent or refractory solid and central nervous system (CNS) tumors.MethodsPrexasertib was administered intravenously (i.v.) on days 1 and 15 of a 28‐day cycle. Four dose levels, 80, 100, 125, and 150 mg/m2, were evaluated using a rolling‐six design. PK analysis was performed during cycle 1. Tumor tissue was examined for biomarkers (CHK1 and TP53) of prexasertib activity.ResultsThirty patients were enrolled; 25 were evaluable. The median age was 9.5 years (range: 2‐20) and 21 (70%) were male. Twelve patients (40%) had solid tumors and 18 patients (60%) had CNS tumors. There were no cycle 1 or later dose‐limiting toxicities. Common cycle 1, drug‐related grade 3/4 toxicities (> 10% of patients) included neutropenia (100%), leukopenia (68%), thrombocytopenia (24%), lymphopenia (24%), and anemia (12%). There were no objective responses; best overall response was stable disease in three patients for five cycles (hepatocellular carcinoma), three cycles (ependymoma), and five cycles (undifferentiated sarcoma). The PK appeared dose proportional across the 80‐150 mg/m2 dose range.ConclusionsAlthough the MTD of prexasertib was not defined by this study, 150 mg/m2 administered i.v. on days 1 and 15 of a 28‐day cycle was determined to be the RP2D.

Abstract Background Diffuse midline glioma, formerly DIPG (diffuse intrinsic pontine glioma), is the deadliest pediatric brainstem tumor with median survival of less than one year. Here, we investigated (i) whether direct delivery of adenovirus-expressing cluster of differentiation (CD)40 ligand (Ad-CD40L) to brainstem tumors would induce immune-mediated tumor clearance and (ii) if so, whether therapy would be associated with a manageable toxicity due to immune-mediated inflammation in the brainstem. Methods Syngeneic gliomas in the brainstems of immunocompetent mice were treated with Ad-CD40L and survival, toxicity, and immune profiles determined. A clinically translatable vector, whose replication would be tightly restricted to tumor cells, rAd-Δ24-CD40L, was tested in human patient–derived diffuse midline gliomas and immunocompetent models. Results Expression of Ad-CD40L restricted to brainstem gliomas by pre-infection induced complete rejection, associated with immune cell infiltration, of which CD4+ T cells were critical for therapy. Direct intratumoral injection of Ad-CD40L into established brainstem tumors improved survival and induced some complete cures but with some acute toxicity. RNA-sequencing analysis showed that Ad-CD40L therapy induced neuroinflammatory immune responses associated with interleukin (IL)-6, IL-1β, and tumor necrosis factor α. Therefore, to generate a vector whose replication, and transgene expression, would be tightly restricted to tumor cells, we constructed rAd-Δ24-CD40L, the backbone of which has already entered clinical trials for diffuse midline gliomas. Direct intratumoral injection of rAd-Δ24-CD40L, with systemic blockade of IL-6 and IL-1β, generated significant numbers of cures with readily manageable toxicity. Conclusions Virus-mediated delivery of CD40L has the potential to be effective in treating diffuse midline gliomas without obligatory neuroinflammation-associated toxicity.

Abstract Activation of the Sonic hedgehog (Shh) pathway and increased expression of Gli1 play an important role in proliferation and transformation of granule cell progenitors (GCP) in the developing cerebellum. Medulloblastomas arising from cerebellar GCPs are frequently driven by Shh pathway–activating mutations; however, molecular mechanisms of Shh pathway dysregulation and transformation of neural progenitors remain poorly defined. We report that the transcription factor and oncogene Snail1 (Sna1) is directly induced by Shh pathway activity in GCPs, murine medulloblastomas, and human medulloblastoma cells. Enforced expression of Sna1 was sufficient to induce GCPs and medulloblastoma cell proliferation in the absence of Shh/Gli1 exposure. In addition, enforced expression of Sna1 increased transformation of medulloblastoma cells in vitro and in vivo. Analysis of potential Sna1 targets in neural cells revealed a novel Sna1 target, N-Myc, a transcription factor known to play a role in Shh-mediated GCP proliferation and medulloblastoma formation. We found that Sna1 directly induced transcription of N-Myc in human medulloblastoma cells and that depletion of N-Myc ablated the Sna1-induced proliferation and transformation. Taken together, these results provide further insight into the mechanism of Shh-induced transformation of neural progenitor cells and suggest that induction of Sna1 may serve to amplify the oncogenic potential of Shh pathway activation through N-Myc induction. Cancer Res; 71(15); 5336–45. ©2011 AACR.

Abstract Activation of the Sonic Hedgehog (SHH) pathway and increased expression of the Gli1 transcription factor have been implicated in the tumorigenesis of both sporadic and syndromic medulloblastoma, a malignant pediatric brain tumor that arises from cerebellar progenitor cells. While SHH pathway activity is required for normal neural stem cell proliferation, molecular mechanisms of SHH pathway dysregulation and transformation of neural precursor cells remain poorly defined. In epithelial cells, Snail1 was found to be induced by Gli1 expression and is believed to promote Gli1-mediated transformation (Li et al. 2006), however the role of Snail1 in neural cells is not well understood. We have identified Snail1 as a putative target of Gli1 in neural stem cells. We hypothesize that Snail1 participates in transduction of SHH/Gli1 signaling in the nervous system and plays a role in proliferation of neuronal progenitor cells in the cerebellum. Mice haploinsufficient for Patched (Ptc) have constitutive activation of the SHH pathway and a subset develop medulloblastoma. Quantitative RT-PCR analyses of mRNA isolated from early postnatal untransformed cerebella show that Snail1 mRNA is increased in vivo in Ptc+/- mice versus wild-type (WT) siblings.. Enforced expression of Gli1 in cultured WT murine granule precursor cells (GPCs) induced Snail1 mRNA.. Enforced expression of Snail1 was also sufficient to induce proliferation of GCPs in the absence of SHH/Gli1 exposure. Given that activation of SHH/Gli1-pathway induces proliferation of GPCs and is associated with medulloblastoma formation, we hypothesized that Snail1 may play a role in the proliferation and transformation of neural progenitor cells. We have found that the stem-like cells generated from medulloblastomas in Ptc+/- mice express higher levels of Snail mRNA than tumor parenchyma. Quantitative RT-PCR analysis demonstrates that Snail1 mRNA is expressed at baseline and is dramatically induced in human medulloblastoma cell lines after exposure to SHH. Enforced expression of Snail1 further increased proliferation of medulloblastoma cells as demonstrated by EdU incorporation. Analysis of potential Snail1 targets in human medulloblastoma cells reveals that β-catenin and N-Myc protein are induced by Snail1 overexpression. N-Myc is induced by SHH in GPCs (Kenney et al. 2003) and is known to induce proliferation in GPCs during both normal cerebellar development and medulloblastoma formation. Together these results give further insight into the mechanism of SHH-induced proliferation of neural progenitor and suggest that Snail1 may contribute to malignant transformation of GCPs through N-Myc. These data establish Snail1 as a target of SHH/Gli1 pathway activity in both normal and neoplastic cells derived from cerebellum. Ongoing experiments will further elucidate how this novel proliferative process affects transformation of neural cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 7.

The Sonic Hedgehog (Shh) pathway plays important roles in embryogenesis, stem cell maintenance, tissue repair, and tumorigenesis. Haploinsufficiency of Patched-1, a gene that encodes a repressor of the Shh pathway, dysregulates the Shh pathway and increases genomic instability and the development of spontaneous and ionizing radiation (IR)–induced tumors by an unknown mechanism. Here we show that Ptc1+/− mice have a defect in the IR-induced activation of the ATR–Chk1 checkpoint signaling pathway. Likewise, transient expression of Gli1, a downstream target of Shh signaling, disrupts Chk1 activation in human cells by preventing the interaction of Chk1 with Claspin, a Chk1 adaptor protein that is required for Chk1 activation. These results suggest that inappropriate Shh pathway activation promotes tumorigenesis by disabling a key signaling pathway that helps maintain genomic stability and inhibits tumorigenesis.

Abstract Sonic hedgehog (Shh) is necessary for sustaining the proliferation of neural stem cells (NSCs), yet little is known about its mechanisms. Whereas Gli1, Gli2, and Gli3, the primary mediators of Shh signaling, were all expressed in hippocampal neural progenitors, Shh treatment of NSCs induced only Gli1 expression. Acute depletion of Gli1 in postnatal NSCs by short-hairpin RNA decreased proliferation, whereas germline deletion of Gli1 did not affect NSC proliferation, suggesting a difference in mechanisms of Gli1 compensation that may be developmentally dependent. To determine whether Gli1 was sufficient to enhance NSC proliferation, we overexpressed this mitogen and were surprised to find that Gli1 resulted in decreased proliferation, accumulation of NSCs in the G2/M phase of cell cycle, and apoptosis. In contrast, Gli1-expressing lineage-restricted neural precursors demonstrated a 4.5-fold proliferation enhancement. Expression analyses of Gli1-expressing NSCs identified significant induction of Gadd45a and decreased cyclin A2 and Stag1 mRNA, genes involved in the G2-M transition and apoptosis. Furthermore, Gadd45a overexpression was sufficient to partially recapitulate the Gli1-induced G2/M accumulation and cell death of NSCs. In contrast to normal stem cells, tumor-derived stem cells had markedly higher basal Gli1 expression and did not undergo apoptosis with further elevation of Gli1. Our data suggest that Gli1-induced apoptosis may serve as a protective mechanism against premature mitosis and may give insight into mechanisms by which nonmalignant stem cells restrain hyperproliferation in the context of potentially transforming mitogenic signals. Tumor-derived stem cells apparently lack these mechanisms, which may contribute to their unrestrained proliferation and malignant potential. Disclosure of potential conflicts of interest is found at the end of this article.