Neem Leaf Glycoprotein (NLGP) is a natural immunomodulator, have shown sustained tumor growth restriction as well as angiogenic normalization chiefly by activating CD8+ T cells. Here, we have investigated the direct role of NLGP as a regulator of tumor microenvironmental hypoxia and associated vascular endothelial growth factor (VEGF) production. We observed a significant reduction in VEGF level in both in vivo murine tumor and in vitro cancer cells (B16Mel, LLC) and macrophages after NLGP treatment. Interestingly, NLGP mediated VEGF downregulation in tumor cells or macrophages within hypoxic chamber was found at an early 4 h and again at late 24 h in mRNA level. Our data suggested that NLGP prevented hypoxia-induced strong binding of HIF1α with its co-factors, CBP/p300 and Sp3, but not with Sp1, which eventually limit the binding of HIF1α-transcriptional complex to hypoxia responsive element of VEGF promoter and results in restricted early VEGF transcription. On the otherhand, suppressed phosphorylation of Stat3 by NLGP results reduction of HIF1α at 24 h of hypoxia that further support sustained VEGF down-regulation. However, NLGP fails to regulate VHL activity as observed by both in vivo and in vitro studies. Therefore, this study for the first time reveals a mechanistic insight of NLGP mediated inhibition of angiogenesis by suppressing VEGF, which might help in vascular normalization to influence better drug delivery.
Abstract Background A dynamic interaction between tumor cells and its surrounding stroma promotes the initiation, progression, metastasis, and chemoresistance of solid tumors. Emerging evidences suggest that targeting the stromal events could improve the efficacies of current therapeutics. Within tumor microenvironment (TME), stromal progenitor cells, i.e., MSCs, interact and eventually modulate the biology and functions of cancer and immune cells. Our recent finding disclosed a novel mechanism stating that tumor-associated MSCs inhibit the T cell proliferation and effector functions by blocking cysteine transport to T cells by dendritic cells (DCs), which makes MSCs as a compelling candidate as a therapeutic target. Immunomodulation by nontoxic neem leaf glycoprotein (NLGP) on dysfunctional cancer immunity offers significant therapeutic benefits to murine tumor host; however, its modulation on MSCs and its impact on T cell functions need to be elucidated. Methods Bone marrow-derived primary MSCs or murine 10 T1/2 MSCs were tumor-conditioned (TC-MSCs) and co-cultured with B16 melanoma antigen-specific DCs and MACS purified CD4 + and CD8 + T cells. T cell proliferation of T cells was checked by Ki67-based flow-cytometric and thymidine-incorporation assays. Cytokine secretion was measured by ELISA. The expression of cystathionase in DCs was assessed by RT-PCR. The STAT3/pSTAT3 levels in DCs were assessed by western blot, and STAT3 function was confirmed using specific SiRNA. Solid B16 melanoma tumor growth was monitored following adoptive transfer of conditioned CD8 + T cells. Results NLGP possesses an ability to restore anti-tumor T cell functions by modulating TC-MSCs. Supplementation of NLGP in DC-T cell co-culture significantly restored the inhibition in T cell proliferation and IFNγ secretion almost towards normal in the presence of TC-MSCs. Adoptive transfer of NLGP-treated TC-MSC supernatant educated CD8 + T cells in solid B16 melanoma bearing mice resulted in better tumor growth restriction than TC-MSC conditioned CD8 + T cells. NLGP downregulates IL-10 secretion by TC-MSCs, and concomitantly, pSTAT3 expression was downregulated in DCs in the presence of NLGP-treated TC-MSC supernatant. As pSTAT3 negatively regulates cystathionase expression in DCs, NLGP indirectly helps to maintain an almost normal level of cystathionase gene expression in DCs making them able to export sufficient amount of cysteine required for optimum T cell proliferation and effector functions within TME. Conclusions NLGP could be a prospective immunotherapeutic agent to control the functions and behavior of highly immunosuppressive TC-MSCs providing optimum CD8 + T cell functions to showcase an important new approach that might be effective in overall cancer treatment.
Abstract Immune evasion within the tumor microenvironment supports malignant growth and is also a major obstacle for successful immunotherapy. Multiple cellular components and soluble factors coordinate to disrupt protective immune responses. Although stromal cells are well-known for their parenchymal supportive roles in cancer establishment and progression, we demonstrate for the first time, to our knowledge, that tumor-derived vascular pericytes negatively influence CD4+ T cell activation and proliferation, and promote anergy in recall response to Ag by CD4+CD44+ T cells via regulator of G protein signaling 5– and IL-6–dependent pathways. Our data support a new specific role for tumor-derived pericytes in the immune evasion paradigm within the tumor microenvironment and suggest the targeting of these cell populations in the context of successful immunotherapeutics for the treatment of cancer.
Abstract Purpose: Overexpression of Cyclin-Dependent Kinase 7 (CDK7) is linked to poor clinical outcomes in a variety of cancer types including AML, breast, ovarian, pancreatic, and lung cancers. CDK7 plays a dual role in tumor progression by regulating the cell cycle via phosphorylation of members of the CDK family and stimulation of transcription via phosphorylation of RNA Polymerase II. CDK7 has also been shown to be associated with super-enhancers which can lead to overexpression of oncogenic driver genes such as BCL2, GFI1, and MYC. The development of a selective inhibitor of CDK7 is a promising strategy to control expression of these oncogenic genes in patients’ tumors. Previously, we described the design and synthesis of a potent and selective small molecule inhibitor of CDK7, TGN-1062, which was designed to bind to the catalytic ATP pocket. We showed efficacy in both AML and ovarian in vitro and in vivo models with correlative biomarker data. Here, we have generated further data on the effect and safety of TGN-1062 in pancreatic cancer cell line (CDX) and patient derived xenograft (PDX) models, both as a single agent and in combination with standard of care (SOC) therapies. Methods: In vivo efficacy studies were conducted in mice engrafted with pancreatic CDX and PDX tumors and treated with TGN-1062 with and without SOC therapies. Tumors were measured to determine tumor growth inhibition (TGI) and correlating drug accumulation levels were measured in both tumors and plasma using mass spectrometry. Immunohistochemistry (IHC) was performed on tumors to determine changes in cleaved Caspase-3, MYC, Ki67, and RNA Pol II. Rats were dosed with TGN-1062 to assess plasma exposures and preliminary tolerability. Results: In a pancreatic CDX model (MIA PaCa-2), TGN-1062 showed 38% TGI as a single agent and 121% TGI when in combination with gemcitabine and nab-paclitaxel, which on its own showed 37% TGI. In a pancreatic PDX model, TGN-1062 did not exhibit single agent effect, but combination with gemcitabine and nab-paclitaxel showed 95% TGI compared to 79% TGI with gemcitabine/nab-paclitaxel alone. Drug accumulation, biomarker analysis, and IHC from CDX and PDX tumors, showed that TGN-1062 treatment decreased RNA Pol II phosphorylation and MYC expression, and induced apoptosis. Rat pharmacokinetics indicated 100% oral bioavailability. Conclusion: Our potent and selective small molecule inhibitor of CDK7, TGN-1062, shows promise in pancreatic cancer models as a single or in combination with SOC. Preliminary data suggests that TGN-1062 can be developed further in multiple cancer indications. Citation Format: Alexis Weston, Trason Thode, Ryan Rodriguez del Villar, Serina Ng, Samuel Sampson, Shelby Rheinschmidt, Tithi Ghosh Halder, Raffaella Soldi, Mohan Kaadige, Srinivas Kasibhatla, Haiyong Han, Justin Moser, Vincent Chung, Joseph Chao, Victoria Villaflor, Daniel Von Hoff, Sunil Sharma. TGN-1062 inhibits CDK7 and augments the effects of gemcitabine and nab-paclitaxel in pancreatic cancer models [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 3931.
Neem leaf glycoprotein (NLGP), a natural immunomodulator, attenuates murine carcinoma and melanoma metastasis, independent of primary tumor growth and alterations in basic cellular properties (cell proliferation, cytokine secretion, etc.). Colonization event of invasion-metastasis cascade was primarily inhibited by NLGP, with no effect on metastasis-related invasion, migration, and extravasation. High infiltration of interferon γ (IFN-γ)-secreting cytotoxic CD8+ T cells [CD44+, CD69+, GranB+, IFN-γ+, and interleukin 2+] was documented in the metastatic site of NLGP-treated mice. Systemic CD8+ T cell depletion abolished NLGP-mediated metastasis inhibition and reappeared upon adoptive transfer of NLGP-activated CD8+ T cells. Interferon γ-secreting from CD8+ T cells inhibit the expression of angiogenesis regulatory vascular endothelial growth factor and transforming growth factor β and have an impact on the prevention of colonization. Neem leaf glycoprotein modulates dendritic cells (DCs) for proper antigen presentation by its DC surface binding and upregulation of MHC-I/II, CD86, and CCR7. Neem leaf glycoprotein-treated DCs specifically imprint CXCR3 and CCR4 homing receptors on activated CD8+ T cells, which helps to infiltrate into metastatic sites to restrain colonization. Such NLGP's effect on DCs is translation dependent and transcription independent. Studies using ovalbumin, OVA257-264, and crude B16F10 antigen indicate MHC-I upregulation depends on the quantity of proteasome degradable peptide and only stimulates CD8+ T cells in the presence of antigen. Overall data suggest NLGP inhibits metastasis, in conjunction with tumor growth restriction, and thus might appear as a promising next-generation cancer immunotherapeutic.
Post-surgical tumor recurrence is a common problem in cancer treatment. In the present study, the role of neem leaf glycoprotein (NLGP), a novel immunomodulator, in prevention of post-surgical recurrence of solid sarcoma was examined. Data suggest that NLGP prevents tumor recurrence after surgical removal of sarcoma in Swiss mice and increases their tumor-free survival time. In NLGP-treated tumor-free mice, increased cytotoxic CD8+ T cells and a decreased population of suppressor cells, especially myeloid-derived suppressor cells (MDSCs) was observed. NLGP-treated CD8+ T cells showed greater cytotoxicity towards tumor-derived MDSCs and supernatants from the same CD8+ T cell culture caused upregulation of FasR and downregulation of cFLIP in MDSCs. To elucidate the role of CD8+ T cells, specifically in association with the downregulation in MDSCs, CD8+ T cells were depleted in vivo before NLGP immunization in surgically tumor removed mice and tumor recurrence was noted. These mice also exhibited increased MDSCs along with decreased levels of Caspase 3, Caspase 8 and increased cFLIP expression. In conclusion, it can be stated that NLGP, by activating CD8+ T cells, down regulates the proportion of MDSCs. Accordingly, suppressive effects of MDSCs on CD8+ T cells are minimized and optimum immune surveillance in tumor hosts is maintained to eliminate the residual tumor mass appearing during recurrence.
We have reported that prophylactic as well as therapeutic administration of neem leaf glycoprotein (NLGP) induces significant restriction of solid tumor growth in mice. Here, we investigate whether the effect of such pretreatment (25µg/mice; weekly, 4 times) benefits regulation of tumor angiogenesis, an obligate factor for tumor progression. We show that NLGP pretreatment results in vascular normalization in melanoma and carcinoma bearing mice along with downregulation of CD31, VEGF and VEGFR2. NLGP pretreatment facilitates profound infiltration of CD8+ T cells within tumor parenchyma, which subsequently regulates VEGF-VEGFR2 signaling in CD31+ vascular endothelial cells to prevent aberrant neovascularization. Pericyte stabilization, VEGF dependent inhibition of VEC proliferation and subsequent vascular normalization are also experienced. Studies in immune compromised mice confirmed that these vascular and intratumoral changes in angiogenic profile are dependent upon active adoptive immunity particularly those mediated by CD8+ T cells. Accumulated evidences suggest that NLGP regulated immunomodulation is active in tumor growth restriction and normalization of tumor angiogenesis as well, thereby, signifying its clinical translation.
Introduction Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated. Methods 4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After three consecutive treatments with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c. 25μg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status. Results Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8 + , IFNγ + , granzyme B + , CD11c + cells in comparison to 4NQO treated mice with a decrease in Ki67 + and CD4 + FoxP3 + cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP. Discussion Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8 + T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.
Mesenchymal stem cells (MSCs) represent an important cellular constituent of the tumor microenvironment, which along with tumor cells themselves, serve to regulate protective immune responses in support of progressive disease. We report that tumor MSCs prevent the ability of dendritic cells (DC) to promote naïve CD4 + and CD8 + T cell expansion, interferon gamma secretion and cytotoxicity against tumor cells, which are critical to immune‐mediated tumor eradication. Notably, tumor MSCs fail to prevent DC‐mediated early T cell activation events or the ability of responder T cells to produce IL‐2. The immunoregulatory activity of tumor MSCs is IL‐10‐ and STAT3‐dependent, with STAT3 repressing DC expression of cystathionase, a critical enzyme that converts methionine‐to‐cysteine. Under cysteine‐deficient priming conditions, naïve T cells exhibit defective cellular metabolism and proliferation. Bioinformatics analyses as well as in vitro observations suggest that STAT3 may directly bind to a GAS‐like motif within the cystathionase promoter (−269 to −261) leading to IL‐10‐STAT3 mediated repression of cystathionase gene transcription. Our collective results provide evidence for a novel mechanism of tumor MSC‐mediated T cell inhibition within tumor microenvironment.
Purpose of review Bromodomain and extraterminal domain (BET) proteins are evolutionarily conserved, multifunctional super-regulators that specifically recognize acetyl-lysine on histones and other proteins controlling gene transcription. Several studies show that small molecules targeting these regulators preferentially suppress the transcription of cancer-promoting genes. Consequently, several BET inhibitors reached clinical trials and are in various stages for different kind of malignancies. In this review, we provide a concise summary of the molecular basis and preliminary clinical outcomes of BET inhibitors as anticancer therapeutics. Recent findings Results from early clinical trials with BET inhibitors confirmed their antitumor potential in both hematologic and solid tumours, but the evidence does not support the application of BET inhibitors as a monotherapy for cancer treatment. Treatment-emergent toxicities such as thrombocytopenia and gastrointestinal disorders are also reported. Preclinical data suggest that BET inhibitors may have a promising future in combination with other anticancer agents. Summary Despite of various challenges, BET inhibitors have high potential in combinatorial therapy and the future development of next-generation inhibitors could be promising. Further studies are needed to determine the predictive biomarkers for therapeutic response, which would translate into the long-term success of BET inhibitors as personalized medicines in cancer treatment.
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