Search

Pathologically activated neutrophils (PMN) with immunosuppressive activity, which are termed myeloid-derived suppressor cells (PMN-MDSC), play a critical role in regulating tumor progression. These cells have been implicated in promoting tumor metastases by contributing to premetastatic niche formation. This effect was facilitated by enhanced spontaneous migration of PMN from bone marrow to the premetastatic niches during the early-stage of cancer development. The molecular mechanisms underpinning this phenomenon remained unclear. In this study, we found that syntaphilin (SNPH), a cytoskeletal protein previously known for anchoring mitochondria to the microtubule in neurons and tumor cells, could regulate migration of PMN. Expression of SNPH was decreased in PMN from tumor-bearing mice and patients with cancer as compared with PMN from tumor-free mice and healthy donors, respectively. In Snph-knockout (SNPH-KO) mice, spontaneous migration of PMN was increased and the mice showed increased metastasis. Mechanistically, in SNPH-KO mice, the speed and distance travelled by mitochondria in PMN was increased, rates of oxidative phosphorylation and glycolysis were elevated, and generation of adenosine was increased. Thus, our study reveals a molecular mechanism regulating increased migratory activity of PMN during cancer progression and suggests a novel therapeutic targeting opportunity.

Supplemental Video

Supplemental Figures, legends and tables

Supplementary Figure 2

Supplementary Figure Legends for S1-4

Supplementary Figure 4

Antibodies targeting CTLA-4 induce durable responses in some patients with melanoma and are being tested in a variety of human cancers. However, these therapies are ineffective for a majority of patients across tumor types. Further understanding the immune alterations induced by these therapies may enable the development of novel strategies to enhance tumor control and biomarkers to identify patients most likely to respond. In several murine models, including colon26, MC38, CT26, and B16 tumors cotreated with GVAX, anti–CTLA-4 efficacy depends on interactions between the Fc region of CTLA-4 antibodies and Fc receptors (FcR). Anti–CTLA-4 binding to FcRs has been linked to depletion of intratumoral T regulatory cells (Treg). In agreement with previous studies, we found that Tregs infiltrating CT26, B16-F1, and autochthonous BrafV600EPten−/− melanoma tumors had higher expression of surface CTLA-4 (sCTLA-4) than other T-cell subsets, and anti–CTLA-4 treatment led to FcR-dependent depletion of Tregs infiltrating CT26 tumors. This Treg depletion coincided with activation and degranulation of intratumoral natural killer cells. Similarly, in non–small cell lung cancer (NSCLC) and melanoma patient-derived tumor tissue, Tregs had higher sCTLA-4 expression than other intratumoral T-cell subsets, and Tregs infiltrating NSCLC expressed more sCTLA-4 than circulating Tregs. Patients with cutaneous melanoma who benefited from ipilimumab, a mAb targeting CTLA-4, had higher intratumoral CD56 expression, compared with patients who received little to no benefit from this therapy. Furthermore, using the murine CT26 model we found that combination therapy with anti–CTLA-4 plus IL15/IL15Rα complexes enhanced tumor control compared with either monotherapy.

Supplementary Figure 1

PDF containing all supplementary figures. Supplementary Figure Legends. Supplemental Figure 1. A, graph indicating the number of patients for which best response data to pembrolizumab was obtained, and from which institution. B, various response groups of patients treated with pembrolizumab, separated by gender, and using 62 years of age as the most statistically significant cut-off. Supplemental Figure 2. A, B Change in mouse weight on final day of experiment from start in female and male mice bearing BSC9AJ2 tumors, after treatment with 3 doses of 300µg rat IgG2AK (N=5) or 4 doses of 300µg anti-PD1 (N=5) every 5 days, starting on day 0. Supplemental Figure 3. A, CD45+ cells within Yumm1.7 and B. BSC9AJ2 tumors. Err=95%CI. B, CD45+ cells within in tumors of Yumm1.7 and Yumm2.1 tumors. C, CD8b+ cells following 5 hour incubation with PMA and ionomycin analyzed by FACS analysis. Err=SD. D, E CD45+CD8b+ cells expressing TNFα and IFNγ within spleens from mice harboring Yumm1.7 or BSC9AJ2 tumors. Err=95%CI. Significance was determined using individual two-tailed student's t-test assuming unequal variance for 1-2 factors and using a 2-way ANOVA for 3 or more factors. Supplemental Figure 4. A 10X view with 40X zoom of FOXP3+ foci staining in melanoma patient biopsies. B, FOXP3+ staining from patient tumors across various age groups. C, CD8+ staining from patient tumors across various age groups. D, percentages of patients in the indicated age groups, whose CD8:FOXP3 ratio is low (< median - 1), med (median +/- 1), or high (> median + 1). Supplemental Figure 5. A. Mouse weight on final day of experiment from start in mice treated with anti-CD25 or an IgG1A isotype 5 days prior to sub-dermal of BSC9AJ2 cells with treatments continuing every 5 days until sacrifice. Anti-PD1 was administered every 3-4 days, starting on day 9 post-tumoral injection. Err=95%CI. Statistical significance determined by 2 way ANOVA. Err bars = SEM. B. Tumor growth for individual mice, per condition in A. Supplemental Figure 6. A. Ratios of CD3+CD8a+ to CD3+CD4+FoxP3+ in BSC9AJ2 tumors from young female mice 4 days following cyclophosphamide intraperitoneal injection at indicated doses. 5 mice in PBS group, and 3 mice per dose. Err=SEM. B, intra-tumoral CD3+CD4+FoxP3+ populations. Err=SD. C, tumor growth in young female mice bearing BSC9AJ2 tumors were injected 6 days post tumor implantation with either 100µL PBS control, or 100µL PBS containing 25 mgs/kg cyclophosphamide intra-peritoneally. Four days later, mice were also given either 300µg anti-PD1 or vehicle control. A total of 4 doses of PD1 was given on day 0, 5, 10, and 14. Statistical significance was determined using a linear mixed-effect model. Err.=SEM. D, fold change in mouse weight from C. Err=SD. E. Intra-tumoral ratios of CD3+CD8a+ to CD3+CD4+FoxP3+ at the end of the experiment. Err.=SEM.

Figure Legends for Supplemental Data

Purpose: We have shown that the aged microenvironment increases melanoma metastasis, and decreases response to targeted therapy, and here we queried response to anti-PD1.Experimental Design: We analyzed the relationship between age, response to anti-PD1, and prior therapy in 538 patients. We used mouse models of melanoma, to analyze the intratumoral immune microenvironment in young versus aged mice and confirmed our findings in human melanoma biopsies.Results: Patients over the age of 60 responded more efficiently to anti-PD-1, and likelihood of response to anti-PD-1 increased with age, even when we controlled for prior MAPKi therapy. Placing genetically identical tumors in aged mice (52 weeks) significantly increased their response to anti-PD1 as compared with the same tumors in young mice (8 weeks). These data suggest that this increased response in aged patients occurs even in the absence of a more complex mutational landscape. Next, we found that young mice had a significantly higher population of regulatory T cells (Tregs), skewing the CD8+:Treg ratio. FOXP3 staining of human melanoma biopsies revealed similar increases in Tregs in young patients. Depletion of Tregs using anti-CD25 increased the response to anti-PD1 in young mice.Conclusions: While there are obvious limitations to our study, including our inability to conduct a meta-analysis due to a lack of available data, and our inability to control for mutational burden, there is a remarkable consistency in these data from over 500 patients across 8 different institutes worldwide. These results stress the importance of considering age as a factor for immunotherapy response. Clin Cancer Res; 24(21); 5347–56. ©2018 AACR.See related commentary by Pawelec, p. 5193

Pathologically activated neutrophils (PMN) with immunosuppressive activity, which are termed myeloid-derived suppressor cells (PMN-MDSC), play a critical role in regulating tumor progression. These cells have been implicated in promoting tumor metastases by contributing to premetastatic niche formation. This effect was facilitated by enhanced spontaneous migration of PMN from bone marrow to the premetastatic niches during the early-stage of cancer development. The molecular mechanisms underpinning this phenomenon remained unclear. In this study, we found that syntaphilin (SNPH), a cytoskeletal protein previously known for anchoring mitochondria to the microtubule in neurons and tumor cells, could regulate migration of PMN. Expression of SNPH was decreased in PMN from tumor-bearing mice and patients with cancer as compared with PMN from tumor-free mice and healthy donors, respectively. In Snph-knockout (SNPH-KO) mice, spontaneous migration of PMN was increased and the mice showed increased metastasis. Mechanistically, in SNPH-KO mice, the speed and distance travelled by mitochondria in PMN was increased, rates of oxidative phosphorylation and glycolysis were elevated, and generation of adenosine was increased. Thus, our study reveals a molecular mechanism regulating increased migratory activity of PMN during cancer progression and suggests a novel therapeutic targeting opportunity.

Anti-CTLA-4 antibodies such as ipilimumab were among the first immuno-oncology agents to show significantly improved outcomes for patients. However, existing anti-CTLA-4 therapies fail to induce a response in a majority of patients and can induce severe, immune-related adverse events. It has been assumed that checkpoint inhibition, i.e., blocking the interaction between CTLA-4 and its ligands, is the primary mechanism of action for ipilimumab. Here we present evidence that checkpoint inhibition may not be the primary mechanism of action for efficacy of anti-CTLA-4 antibodies. Instead, the primary mechanism for efficacy may be FcR-mediated Treg depletion in the tumor microenvironment. First, we identified a monoclonal antibody (mAb), GIGA-564, that binds to CTLA-4 at an epitope that differs from ipilimumab’s by only a few amino acids, yet has limited checkpoint inhibitor activity. Surprisingly, GIGA-564 has superior anti-tumor activity compared to ipilimumab in a murine model. GIGA-564 also induces less Treg proliferation and has increased ability to induce in vitro FcR signaling and in vivo depletion of intratumoral Tregs. Further experiments showed that the enhanced FcR activity of GIGA-564 likely contributes to its enhanced anti-tumor activity. Importantly, we also showed that GIGA-564 was associated with lower toxicity in murine models. Our work suggests that new anti-CTLA-4 drugs should be optimized for Treg depletion rather than checkpoint inhibition. Citation Format: Erica L Stone, Kyle P Carter, Ellen K Wagner, Michael A Asensio, Emily Benzie, Yao Yuan Chiang, Garry L Coles, Chelsea Edgar, Bishal K Gautam, Ashley Gras, Jackson Leong, Renee Leong, Vishal A Manickam, Rena A Mizrahi, Ariel R Niedecken, Jasmeen Saini, Savreet K Sandhu, Jan Fredrick Simons, Kacy Stadtmiller, Brendan Tinsley, LaRee Tracy, Nicholas P Wayham, Yoong Wearn Lim, Adam S Adler, David S Johnson. GIGA-564, a third generation anti-CTLA-4 with minimal ability to block CTLA-4 binding to B7 ligands, has enhanced efficacy but reduced toxicity compared to ipilimumab in pre-clinical models [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A32.

Peer-to-peer learning in a prelicensure nursing program allows students to critique one another with decreased stress and anxiety. The use of video recording technology affords students the opportunity to engage in reflective practices of physical assessment and psychomotor skills checkoffs outside of a traditional instructor proctored evaluation session. Students reported improved learning associated with peer-to-peer feedback. Instructor-proctored evaluation, although more authentic, produced more stress and anxiety.

Potent, selective antitumour AhR ligands 5F 203 and GW 610 are bioactivated by CYPs 1A1 and 2W1. Herein we reason that DNA adducts' generation resulting in lethal DNA double strand breaks (DSBs) underlies benzothiazoles' activity. Treatment of sensitive carcinoma cell lines with GW 610 generated co-eluting DNA adducts (R(2)>0.7). Time-dependent appearance of γ-H2AX foci revealed subsequent DNA double strand breaks. Propensity for systemic toxicity of benzothiazoles steered development of prodrugs' hydrogels for localised delivery. Clinical applications of targeted therapies include prevention or treatment of recurrent disease after surgical resection of solid tumours. In vitro evaluation of 5F 203 prodrugs' activity demonstrated nanomolar potency against MCF-7 breast and IGROV-1 ovarian carcinoma cell lines.

Summary Foxo transcription factors integrate extrinsic signals to regulate cell division, differentiation and survival, and specific functions of lymphoid and myeloid cells. Here, we showed the absence of Foxo1 severely curtailed the development of Foxp3 + regulatory T (Treg) cells and those that developed were nonfunctional in vivo. The loss of function included diminished CTLA-4 receptor expression as the Ctla4 gene was a direct target of Foxo1. T cell-specific loss of Foxo1 resulted in exocrine pancreatitis, hind limb paralysis, multiorgan lymphocyte infiltration, anti-nuclear antibodies and expanded germinal centers. Foxo-mediated control over Treg cell specification was further revealed by the inability of TGF-β cytokine to suppress T-bet transcription factor in the absence of Foxo1, resulting in IFN-γ secretion. In addition, the absence of Foxo3 exacerbated the effects of the loss of Foxo1. Thus, Foxo transcription factors guide the contingencies of T cell differentiation and the specific functions of effector cell populations.

New fluorinated 2-aryl-benzothiazoles, -benzoxazoles, and -chromen-4-ones have been synthesized and their activity against MCF-7 and MDA 468 breast cancer cell lines compared with the potent antitumor benzothiazole 5. Analogues such as 9a, b and 12a, d yielded submicromolar GI50 values in both cell lines; however, none of the new compounds approached 5 in terms of antitumor potency. For 5, binding to the aryl hydrocarbon receptor appeared to be necessary but not sufficient for growth inhibition.

A series of new 2-phenylbenzothiazoles has been synthesized on the basis of the discovery of the potent and selective in vitro antitumor properties of 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (8n; GW 610, NSC 721648). Synthesis of analogues substituted in the benzothiazole ring was achieved via the reaction of o-aminothiophenol disulfides with substituted benzaldehydes under reducing conditions. Compounds were evaluated in vitro in four human cancer cell lines, and compound 8n was found to possess exquisitely potent antiproliferative activity (GI(50) < 0.1 nM for MCF-7 and MDA 468). Potent and selective activity was also observed in the NCI 60 human cancer cell line panel. Structure-activity relationships established that the compound 8n stands on a pinnacle of potent activity, with most structural variations having a deactivating in vitro effect. Mechanistically, this new series of agents contrasts with the previously reported 2-(4-aminophenyl)benzothiazoles; compound 8n is not reliant on induction of CYP1A1 expression for antitumor activity.

Compounds within the 2-(4-aminophenyl)benzothiazole class represent extremely potent and selective experimental antitumour agents. The lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole is undergoing phase I clinical evaluation. Extensive studies to elucidate mechanisms underlying the stark selectivity demonstrated potent cytosolic AhR ligand binding and cytochrome P450 1A1-catalysed bioactivation. Two human derived breast cell lines, initially exquisitely sensitive to this class of agent (GI505 nM) have been derived displaying acquired resistance to 2-(4-amino-3-methylphenyl)benzothiazole (DF 203; GI5050 microM). Cross resistance to 2-(4-amino-3-iodophenyl)benzothiazole and 2-(4-amino-3-cyanophenyl)benzothiazole is observed (GI5030 microM) as is100-fold reduced sensitivity of the two variant lines to 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203). In contrast, cell lines possessing acquired resistance to DF 203 (203R) retain sensitivity to benzo[a]pyrene and doxorubicin. Examination of DF 203-treated cells by confocal microscopy and HPLC analyses of nutrient media concur revealing diminished depletion of DF 203 from medium and impaired intracellular DF 203 retention. In contrast to cytosolic arylhydrocarbon (AhR) receptors of wild type cells, AhR appears constitutively localised within nuclei of 203R cells; consequently, DF 203 fails to drive transcription of cyp1a1. DF 203- and 5F 203-derived DNA adducts fall significantly in 203R cells. Reduced number and intensity of gamma H2AX foci report protection against DF 203-evoked DNA double strand breaks. In conclusion, aberrant AhR signalling underlies at least in part acquired resistance to DF 203. Intriguingly, comparisons of gene transcription profiles between sensitive and resistant paired lines reveal5-fold up-regulation of cyp1b1 expression, a protein implicated in resistance to therapeutic agents.