曲格鲁唑与抗PD-1联合应用于自发性黑色素瘤小鼠的模型的临床前研究文献综述

 2022-12-29 14:13:47

开题报告内容Content：（包括拟研究或解决的问题、采用的研究手段及文献综述，不少于2000字）Including objectives, methods adopted and literature review, no less than 2000 words

Aim, Rationale, Preliminary Results and Approach:

Rationale and Preliminary Results: In the past five years, immune checkpoint inhibitors have become the cornerstone of treatment for melanoma. PD-1 antibodies including pembrolizumab and nivolumab have response rates in the range of 30%-40% and improved median overall survival [26]. Resistance to anti-PD-1 therapy may be due to lack of inflammation within the tumor and/or lack of PD-L1 expression in the tumor microenvironment (TME). To assess the effects of glutamatergic signaling inhibitor (riluzole, trigriluzole) on immune cells in the tumor microenvironment, we took advantage of four available paired pre- and post-treatment specimens from a completed Phase II single-agent riluzole trial and examined for changes in MAPK and PI3K signaling and leukocyte infiltration using CD45 staining. Two patients with stable disease, showed a decrease in both pERK and pAKT by westerns and an increase in CD45 leukocytes in the tumor /stromal interphase only in post-treatment samples (Figure 3). In contrast, two patients with progressive disease where no reduction in pERK and pAKT by western and no increase in CD45 staining in intratumoral leukocytes in post-treatment samples were observed (Figure 4) [20] . These correlative studies suggest that an increase in leukocytes at the active edge of the tumor, tumor/stromal interface, may prime the tumor to be more receptive to immune checkpoint blockade. The relative efficacy of immunotherapy with small molecule inhibitor strategies targeting glutamatergic pathways are not known, we propose that combinatorial strategies using inhibitors of non-redundant independent pathways may yield synergistic activity.

Figure 3. CD45 staining on fresh paired tumor biopsies from 2 patients with stable disease (SD). CD45 from patients 1 (a) and 11 (b), before (left) and after (right) riluzole treatment, showing an increase in CD45 cells in the tumor/stroma interface.

Figure 4. CD45 staining on fresh paired tumor biopsies from 2 patients with progressive disease (PD). a) CD45 from patients 6 (a) and 8 (b), before (left) and after (right) riluzole, showing a decrease in intratumorally CD45 cells after treatment.

It is vital to use a model system that can mimic a given disease. Transplantation of human tumors into immunocompromised mice (xenograft) has been the standard approach to test candidate drug efficacy, however, with the emergent immunotherapy, an immune-competent model system is necessary. There are several tumor cell lines derived from syngeneic mice enabling one to perform such studies however the inability to perform long-term therapeutic studies, beyond a few weeks, makes it difficult to learn longitudinal consequences, such as the development of resistance, side effects, and relapse rates. Genetically engineered mouse models (GEMs) that recapitulate the progression of a given disease are valuable tools which gives one the opportunity to investigate long-term consequences of drug(s) in a natural multi-mutational physiologically relevant system [24, 25]. Our genetically driven GRM1-expressing mouse model, TGS, develops metastatic melanoma spontaneously and its tumor progression mirrors disease stages seen in patients [24, 25]. My proposed studies may uncover biomarkers that will allow for a better prediction to treatment response in melanoma patients. To assess if our working hypothesis is valid, we performed a mouse-allograft study using GRM-1-transformed mouse melanocytes (derived from C57BL/6), MASS20, which is wild type for both B-RAF and N-RAS and has used in our earlier studies [27].

Figure 1. Trigriluzole (1.7mg/kg) exhibits an increase in potency than riluzole (7.5mg/kg) in a mouse xenograft model of melanoma. C8161 human melanoma cells (106) were inoculated in the flanks of nude mice and when the tumor volume reached 100mm3 the mice were divided randomly into groups with similar tumor volumes: No treatment (NT), vehicle (Veh, DMSO), riluzole (Ril, 7.5 mg/kg), FC (trigriluzole) at 0.56 mg/kg, 1.7 mg/kg and 5mg/kg. Mice were treated daily by oral gavage. Tumor volume (mm3) mean plusmn;SD of 12 mice /group. Plt;0.001, for riluzole or trigriluzole groups (regardless of dosage) compared to NT/Veh.

Preliminary Results: We initiated a set of preliminary MASS20 allograft studies in C57BL/6 (BL6) mice and when tumor volumes reached about 40 mm3, we randomly divided the tumor-bearing mice into vehicle 1 (DMSO), vehicle 2 (rat IgG), riluzole (10 mg/kg) alone, anti-PD1 (100 ug/mouse/injection) alone, trigriluzole (1.7mg/kg) alone, combination of riluzole and anti-PD1, and combination of trigriluzole and anti-PD-1. Anti-PD1 or control IgG was administered three times the first week then once a week similar to studies described for B16 allografts [28]. Riluzole or trigriluzole was given by oral gavage daily [15]. Tumor volumes were measured twice a week. The experiment was terminated at day 25 when the tumor volumes for some of the groups reached permitted tumor size per Rutgers IACUC (about 1200mm3). Using MASS20 cells in immunocompetent syngeneic BL6 mice the combination approaches with either riluzole or riluzole prodrug, trigriluzole, and anti-PD-1 appears to produce a slight advantage in reducing allografted tumor progression (2.3 fold) over single agent therapy (Figure 5). These preliminary results are very encouraging and strongly support our working hypothesis that combining inhibitors of two distinct pathways offers great advantage in reducing tumor progression with very little if any toxicity.

Figure 5. Combination GRM1 inhibition plus PD-1 blockade results in decreased tumor growth compared to either treatment alone. One hundred thousand MASS20 cells were inoculated into flanks of C57BL/6 mice. When the tumor volumes reached 40 mm3, we randomly divided the tumor-bearing mice into vehicle 1 (DMSO), vehicle 2 (control rat IgG), riluzole (10 mg/kg) alone, anti-PD1 (100 g/mouse/injection) alone, trigriluzole (1.7mg/kg) alone, combination of riluzole and anti-PD1, and combination of trigriluzole and anti-PD-1. The treatment was terminated at 25 days after start of the treatment as day 1.

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