Mice Receiving Combination Therapy Research Articles

HDAC inhibitor SAHA enhances antitumor immunity via the HDAC1/JAK1/FGL1 axis in lung adenocarcinoma

BackgroundHistone deacetylase (HDAC), a kind of protease that regulates gene expression by modifying protein acetylation levels, is usually aberrantly activated in tumors. The approved pan-HDAC inhibitors (HDACi) have exhibited clinical...

Potential effectiveness of parenteral nemonoxacin in the treatment of Clostridioides difficile infections: in vitro, ex vivo, and mouse studies.

Antimicrobial therapy plays a crucial role in the management of CDI patients. However, the standard agent for treating CDIs is limited to oral fidaxomicin or vancomycin. For patients made nil by mouth, there is a clinically urgent and essential need to develop an intravenous antibiotic. For C. difficile with the lowest MIC of nemonoxacin and vancomycin, the inhibitory effects were tested using the kinetic time-kill assay and ex vivo co-culture model. The effectiveness of nemonoxacin and vancomycin in inhibiting spore germination, the sporicidal activity, and the treatment of mice with CDIs were compared. For clinical isolates and laboratory strains, lower MICs of nemonoxacin against C. difficile than levofloxacin and ciprofloxacin were observed, even in those harboring point mutations in the quinolone-resistance determining region. Although nemonoxacin failed to suppress spore outgrowth and germination in C. difficile, it exhibited an effective inhibitory effect against C. difficile in the kinetic time-kill assay and the ex vivo co-culture model. Mice receiving intraperitoneal nemonoxacin had less weight loss, higher cecum weight, a longer colon length, and lower expression of the tcdB gene, compared with untreated mice. Notably, there were no significant differences observed in weight loss, cecum weight, colon length, or tcdB gene expression between mice treated with vancomycin and those treated with any dose of nemonoxacin. Similarly, no significant differences were found between mice receiving combination therapy of intraperitoneal nemonoxacin plus oral vancomycin and those treated with intraperitoneal nemonoxacin or oral vancomycin alone. The potential role of nemonoxacin, which can be administered parenterally, for treating CDIs was evidenced through the in vitro, ex vivo, and mouse models.

AAV mediated repression of Neat1 lncRNA combined with F8 gene augmentation mitigates pathological mediators of joint disease in haemophilia.

Haemophilic arthropathy (HA), a common comorbidity in haemophilic patients leads to joint pain, deformity and reduced quality of life. We have recently demonstrated that a long non-coding RNA, Neat1 as a primary regulator of matrix metalloproteinase (MMP) 3 and MMP13 activity, and its induction in the target joint has a deteriorating effect on articular cartilage. In the present study, we administered an Adeno-associated virus (AAV) 5 vector carrying an short hairpin (sh)RNA to Neat1 via intra-articular injection alone or in conjunction with systemic administration of a capsid-modified AAV8 (K31Q) vector carrying F8 gene (F8-BDD-V3) to study its impact on HA. AAV8K31Q-F8 vector administration at low dose, led to an increase in FVIII activity (16%-28%) in treated mice. We further observed a significant knockdown of Neat1 (~40 fold vs. untreated injured joint, p = 0.005) in joint tissue of treated mice and a downregulation of chondrodegenerative enzymes, MMP3, MMP13 and the inflammatory mediator- cPLA2, in mice receiving combination therapy. These data demonstrate that AAV mediated Neat1 knockdown in combination with F8 gene augmentation can potentially impact mediators of haemophilic joint disease.

Combination of Thymoquinone and Intermittent Fasting as a Treatment for Breast Cancer Implanted in Mice.

Breast cancer stands out as a particularly challenging form of cancer to treat among various types. Traditional treatment methods have been longstanding approaches, yet their efficacy has diminished over time owing to heightened toxicity, adverse effects, and the emergence of multi-drug resistance. Nevertheless, a viable solution has emerged through the adoption of a complementary treatment strategy utilizing natural substances and the incorporation of intermittent fasting to enhance therapeutic outcomes. This study aimed to assess the anticancer activity of thymoquinone (TQ), intermittent fasting, and their combination using in vivo and in vitro methods. The anti-proliferative activity of TQ and fasting (glucose/serum restriction) were evaluated against the T47D, MDA-MB-231, and EMT6 cell lines and compared to normal cell lines (Vero) using the MTT colorimetric assay method. Additionally, this study aimed to determine the half-maximal inhibitory concentration (IC50) of TQ. For the in vivo experiment, the antitumor activity of TQ and intermittent fasting (IF) was assessed by measuring the tumor sizes using a digital caliper to determine the change in the tumor size and survival rates. At the molecular level, the serum levels of glucose, β-hydroxybutyrate (β-HB), leptin, and insulin growth factor-1 (IGF-1) were measured using standard kits. Additionally, the aspartate transaminase (AST), alanine transaminase (ALT), and creatinine serum levels were measured. The inhibition of the breast cancer cell lines was achieved by TQ. TQ and intermittent fasting both had an additional anticancer effect against breast tumors inoculated in mice. The combination therapy was evaluated and found to significantly reduce the tumor size, with a change in tumor size of -57.7%. Additionally, the combination of TQ and IF led to a decrease in the serum levels of glucose, IGF-1 (24.49 ng/mL) and leptin (1.77 ng/mL) while increasing β-hydroxybutyrate in the mice given combination therapy (200.86 nM) with no toxicity on the liver or kidneys. In the mice receiving combination therapy, TQ and IF treated breast cancer in an additive way without causing liver or kidney toxicity due to decreased levels of glucose, IGF-1, and leptin and increased levels of β-hydroxybutyrate. Further investigation is required to optimize the doses and determine the other possible mechanisms exhibited by the novel combination.

Direct, Potent, and TP53-Independent Activity of Sting Agonists Against Acute Myeloid Leukaemia Enhances Venetoclax Efficacy In Vivo

Introduction TP53 mutation occurs in approximately 20% of patients with newly diagnosed acute myeloid leukaemia (AML). Treatment outcomes are universally poor and discovery of new therapies with TP53 independent activity represents an area of very high unmet need. Cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) sense cytoplasmic double-stranded DNA to elicit type I interferon production. Direct pharmacologic activation of STING has been investigated in clinical trials with the goal of enhancing immunogenicity. During apoptosis, activated caspases have been shown to suppress activation of the cGAS/STING pathway to render cell death immunologically silent. Whilst the canonical role of STING is stimulation of an inflammatory response, we now show that direct activation of STING by potent and clinically relevant agonists can induce apoptosis directly in cancerous cells by leveraging the high levels of expressed STING in a variety of blood cancers. Importantly, apoptosis induced by STING agonists may be enhanced through combination with diverse BH3-mimetic drugs in a TP53 independent manner (Diepstraten et al, submitted). In this abstract, we highlight the activity of STING agonists in AML and its potential for clinical testing, especially among patients with TP53 mutated disease. Methods We tested the clinically relevant small molecule STING agonist diABZI (purchased from SYNthesis Med Chem), which is currently in early phase clinical trials (NCT03843359, NCT05424380). diABZI was tested alone or in combination with the BCL-2 specific BH3 mimetic drug venetoclax (purchased from Chemgood). Drug sensitivity assays were performed using human AML derived cell lines and primary bone marrow or peripheral blood mononuclear cells from patients with AML or myelodysplastic syndrome with excess blasts 2 (MDS-EB2). Cells were cultured for 48 hours, and cell death assessed by PI or DAPI staining. In vivo experiments were conducted by transplanting NOD- scidIL2Rgamma null (NSG) mice with MOLM-13 TP53 wildtype (WT) or TP53 knockout(KO) cells. Mice then received either no treatment (control), diABZI alone (1.5 mg/kg intravenously twice a week), venetoclax alone (50 mg/kg orally Monday-Friday), or combination diABZI and venetoclax for two weeks ( n=5 per treatment arm). Survival analysis was performed using the log-rank (Mantel-Cox) test with Bonferroni correction. Results Single agent diABZI was active in 5/6 human AML cell lines tested, including cell lines with TP53 mutation. Isogenic MOLM-13 TP53 KO cells demonstrated the same sensitivity as the TP53 WT parental cells. BAX/BAK KO and STING KO cell lines were resistant to diABZI, confirming that cell death occurred by apoptosis being dependent on BAX/BAK as well as STING. Combination of diABZI and venetoclax resulted in additive killing in MOLM-13 and OCI-AML3 cells (Bliss scores of 7.62 and 8.06, respectively). Ex vivo testing was performed on 18 different primary specimens (16 AML and 2 MDS-EB2; 12/18 cases had high risk genetic or clinical features such TP53 mutation, complex karyotype, secondary disease, or relapse after prior therapy) (Figure 1). Strikingly, diABZI was active as a single agent in 16/18 cases, with marked efficacy in 11 cases (IC 50 <100 nM). The addition of venetoclax enhanced killing of AML cells, especially in cases resistant to STING agonist alone. In vivo, diABZI monotherapy and combination therapy with venetoclax was well tolerated without significant weight loss or hematologic toxicity. Treatment with diABZI prolonged survival in the highly aggressive MOLM-13 xenograft model (Figure 2). In both the TP53 WT and TP53 KO context, single agent diABZI was superior to single agent venetoclax ( p=0.012 and p=0.011, respectively), but survival was most prolonged in mice receiving combination therapy ( p=0.013and p=0.012, respectively). Conclusion Activation of STING can exert direct anti-tumour effects in blood cancers via induction of apoptosis. Human AML cells are highly sensitive to the pro-apoptotic effects of STING agonists. The combination of venetoclax and STING agonist shows marked efficacy in ex vivo and in vivo studies, including in models of TP53 defective AML. Combining STING agonists with venetoclax represents a highly promising and novel therapeutic approach for AML and should be considered for accelerated early phase clinical trials.

Can Ganciclovir and Quercetin-P188 Ameliorate Cytomegalovirus Induced Hearing Loss?

Determine whether combination therapy with ganciclovir (GCV) and a Quercetin-P188 solution improves hearing outcomes in a murine cytomegalovirus (CMV) model. BALB/c mice were infected with murine CMV on postnatal day 3 (p3). Quercetin was solubilized in saline using P188 (QP188). Treatment groups received either GCV, QP188, GCV and QP188, or P188 delivery vehicle BID at 12-hour intervals via intraperitoneal injection. All treatment groups were treated for 14 days starting at p3. Uninfected controls were treated with the combined regimen, saline or P188 delivery vehicle. Auditory thresholds were assessed using distortion product otoacoustic emission (DPOAE) and auditory brainstem response (ABR) testing at 4, 6, and 8 weeks of age. Temporal bones from separate CMV-infected groups were harvested at p10, and viral load was determined by quantitative polymerase chain reaction. CMV-infected mice receiving combination therapy GCV+QP188 demonstrated statistically significant lower ABR (p < 0.001) and DPOAE thresholds (p < 0.001) compared with mice treated with GCV monotherapy, QP188 monotherapy, and P188 delivery vehicle at 4, 6, and 8 weeks of age. GCV+QP188 combination therapy, GCV monotherapy, and QP188 monotherapy resulted in a nonsignificant reduction in mean viral titers compared to P188 monotherapy (p = 0.08). Combining GCV with the excipients quercetin and P188 effectively ameliorated CMV-induced sensorineural hearing loss in a murine model. This result may be partially explained by a reduction in viral titers in mouse temporal bones that correlate with in vitro studies demonstrating additive antiviral effect in cell culture. NA Laryngoscope, 134:1457-1463, 2024.

Combining idebenone and rosuvastatin prevents atherosclerosis by suppressing oxidative stress and NLRP3 inflammasome activation

Atherosclerosis is a progressive inflammatory disease activated by excessive oxidized low-density lipoprotein (ox-LDL). Statins are the first-line choice to reduce the risk of cardiovascular disease. However, statin-associated side effects prompt dose reduction or discontinuation. Idebenone could protect against atherosclerosis by scavenging reactive oxygen species (ROS). Although both idebenone and statins have certain efficacy, neither of them can achieve a completely satisfactory effect. Here, we aim to investigate the anti-atherosclerotic effect of the combination of idebenone and statins. Apolipoprotein E knockout (ApoE−/−) mice were given idebenone (400 mg/kg/d), rosuvastatin (10 mg/kg/d) or a combination of idebenone and rosuvastatin. Histological and immunohistochemical staining were used to analyze the size and composition of the plaque. In vivo and in vitro experiments were conducted to explore the possible mechanism. Idebenone and rosuvastatin both reduced plaque burden and increased the stability of atherosclerotic plaques in the ApoE−/− mice. Mice receiving the combination therapy had even reduced and more stable atherosclerotic plaques than mice treated with idebenone or rosuvastatin alone. NLRP3 and IL-1β were further downregulated in mice receiving combination therapy compared with mice treated with monotherapy. The combination treatment also markedly reduced oxidative stress and cell apoptosis in vivo and in vitro. In conclusion, our data demonstrate that the combination of idebenone and rosuvastatin works synergistically to inhibit atherosclerosis, and that the use of both substances together is more effective than using either substance alone. From a therapeutic point, combining idebenone and rosuvastatin appears to be a promising strategy to further prevent atherosclerosis.

Oncolytic therapy with recombinant vaccinia viruses targeting the interleukin-15 pathway elicits a synergistic response

We developed recombinant variants of oncolytic vaccinia virusLIVP strain expressing interleukin-15 (IL-15) or its receptor subunit alpha (IL-15Rα) to stimulate IL-15-dependent immune cells. We evaluated their oncolytic activity either alone or in combination with each other invitro and invivo using the murine CT26 colon carcinoma and 4T1 breast carcinoma models. We demonstrated that the admixture of these recombinant variants could promote the generation of the IL-15/IL-15Rα complex. Invitro studies indicated that 4T1 breast cancer cells were more susceptible to the developed recombinant viruses. Invivo studies showed significant survival benefits and tumor regression in 4T1 breast cancer syngeneic mice that received a combination of LIVP-IL15-RFP with LIVP-IL15Ra-RFP. Histological analysis showed recruited lymphocytes at the tumor region, while no harmful effects to the liver or spleen of the animals were detected. Evaluating tumor-infiltrated lymphocytes represented profound activation of cytotoxic Tcells and macrophages in mice receiving combination therapy. Thus, our experiments showed superior oncolytic effectiveness of simultaneous injection of LIVP-IL15-RFP and LIVP-IL15Ra-RFP in breast cancer-bearing mice.The combined therapy by these recombinant variants represents a potent and versatile approach for developing new immunotherapies for breast cancer.

IFNα and 5-Aza-2'-deoxycytidine combined with a dendritic-cell targeting DNA vaccine alter tumor immune cell infiltration in the B16F10 melanoma model.

DNA vaccines containing a fusion of the gene encoding chemokine MIP-3α (CCL20), the ligand for CCR6 on immature dendritic cells (DCs), to melanoma-associated antigen genes have enhanced anti-tumor immunity and efficacy compared to those lacking the chemokine gene. Previous work has shown that type-I interferon (IFNα or IFN) and 5-Aza-2'-deoxycytidine (5Aza) significantly enhance the therapeutic benefit of DNA vaccines as measured by reduced tumor burden and improved mouse survival. Here, we explored mouse intratumoral immune correlates underlying the therapeutic benefit of this combination regimen (vaccine, IFN, and 5Aza) as compared to vaccine alone and IFN and 5Aza without vaccine, focusing on chemokine mRNA expression by qRT-PCR and inflammatory cellular infiltration into the tumor microenvironment (TME) by flow cytometry and immunohistochemistry (IHC). The combination group significantly upregulated intratumoral mRNA expression of key immune infiltration chemokines XCL1 and CXCL10. Flow cytometric analyses of tumor suspensions exhibited greater tumor infiltration of CD8+ DCs, CCR7+ DCs, and NK cells in the combination group, as well as reduced levels of myeloid-derived suppressor cells (MDSCs) in vaccinated groups. The mice receiving combination therapy also had greater proportions of effector/memory T-cells (Tem), in addition to showing an enhanced infiltration of Tem and central memory CD8+ T-cells, (Tcm). Tem and Tcm populations both correlated with smaller tumor size. Immunohistochemical analysis of tumors confirmed that CD8+ cells were more abundant overall and especially in the tumor parenchyma with combination therapy. Efficient targeting of antigen to immature DCs with a chemokine-fusion vaccine offers a potential alternative approach to classic and dendritic cell-based vaccines. Combining this approach with IFNα and 5Aza treatments significantly improved vaccine efficacy. This treatment creates an environment of increased inflammatory chemokines that facilitates the trafficking of CD8+ DCs, NK cells, and CD8+ T-cells, especially memory cells, while reducing the number of MDSCs. Importantly, in the combination group, CD8+ cells were more able to penetrate the tumor mass in addition to being more numerous. Further analysis of the pathways engaged by our combination therapy is expected to provide additional insights into melanoma pathogenesis and facilitate the development of novel treatment strategies.

The Multi-CDK Inhibitor Dinaciclib Shows Synergistic Activity with the BET Inhibitor PLX51107 and Reverses BET Resistance through Inhibition of Canonical Wnt Signaling in Acute Myeloid Leukemia (AML)

PLX51107 is a novel BET inhibitor (BETi) with antineoplastic activity in pre-clinical models of both aggressive B-cell malignancies and AML. Differentiating it from other BET inhibitors currently under investigation, PLX51107 has a unique binding site in the acetylated lysine binding pocket of BRD4. Through interaction with pTEFb, which is comprised of CDK9 and several cyclin T1 heterodimers, BRD4 promotes phosphorylation of RNA polymerase II, ultimately influencing gene transcription. Dinaciclib is a potent inhibitor of multiple cyclin dependent kinases including CDK9 and has been shown to have pre-clinical activity in AML. Therefore, we hypothesized that PLX51107 would have synergistic activity when combined with dinaciclib. We found synergistic effects on proliferation in primary AML cells when PLX51107 was combined with dinaciclib in stromal co-cultures across a range of dose combinations below the IC50 of either compound alone. Synergistic activity was also present in cytokine support culture conditions, colony forming assays, and synergy assays utilizing COMbenefit software. Interestingly, the highly conserved canonical β-catenin dependent Wnt signaling pathway is a known BETi resistance mechanism in AML. Therefore, destruction of β-catenin and inhibition of Wnt signaling could represent a novel therapeutic target to circumvent development of BET-mediated resistance. We found dinaciclib treatment reduced protein levels of total β-catenin in primary AML cells and AML cell lines (Figure 1A). Phosphorylated β-catenin and non-phosphorylated "active" β-catenin protein levels were also reduced following dinaciclib treatment (Figure 1A). In addition, mRNA expression of multiple components of the Wnt pathway, including the Wnt co-receptor LRP6, AXIN2, FZD1, and many Wnt target genes were significantly decreased following dinaciclib and combination treatment. Moreover, in a luciferase based Wnt reporter cell line, we found dinaciclib significantly reduced Wnt3a induced luciferase activity, suggesting a direct effect on β-catenin mediated transcription of Wnt target genes. Collectively, this data suggests dinaciclib has multiple inhibitory effects on canonical β-catenin dependent Wnt signaling. We found combination treatment of PLX51107 and dinaciclib produced synergistic reduction in expression of both Wnt signaling components and Wnt target genes. Conversely, β-catenin and Wnt target gene expression levels were either not significantly changed or even increased when treated with PLX51107 alone. In order to examine the effects of dinaciclib in the setting of BET resistance, we developed PLX51107 resistant MOLM-13 and MV4-11 AML cell lines via serial drugging. These resistant cell lines remained sensitive to dinaciclib treatment and to cytotoxic chemotherapy drugs including cytarabine and etoposide while demonstrating resistance to PLX51107 via Annexin PI and MTS assays. We found sensitivity to dinaciclib treatment in these resistant cell lines remained unaffected by BETi resistance and continued to demonstrate inhibition of Wnt signaling, similar to the effects seen in parental cells. Therefore, dinaciclib represents a novel strategy to overcome potential therapy emergent BETi resistance. We next examined the efficacy of PLX51107 in combination with dinaciclib in vivo, utilizing luciferase labeled MOLM-13 AML cells xenotransplanted into NOD / SCID / IL2rgnull (NSG) immunodeficient mice. Daily oral dosing with 20 mg/kg PLX51107 resulted in prolonged survival (median 42 days) compared to vehicle treated control animals (median 28 days, p< 0.001). Weekly intraperitoneal 10 mg/kg dinaciclib did not significantly improve survival (median 30 days, p=0.09) in MOLM-13 xenograft mice. However, mice receiving combination therapy survived a median of 56.5 days, significantly longer than with either drug alone (p<0.05) (Figure 1B). We also found decreased disease burden by bioluminescence and by histopathology in combination treated animals, compared to both vehicle and single agent controls. In conclusion, we have demonstrated in vitro and in vivo synergistic activity of the BET inhibitor PLX51107 in combination with dinaciclib in pre-clinical models of AML and have identified novel inhibition of Wnt / β-catenin signaling by dinaciclib, thereby providing a strategy to circumvent resistance to BET inhibitors in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Protective Effect of Selenium against Methotrexate Induced Hepatotoxicity

Introduction: The liver is the largest internal organ by percent weight in the human body and has crucial functions, including cholesterol production, intermediary metabolism, hormone synthesis, bile and urea production and drug detoxification. Objectives: The main objective of the study is to analyse the Protective effect of selenium against methotrexate induced hepatotoxicity in mice. Material and methods: This descriptive study was conducted in Jinnah Hospital Lahore during 2020. Selenium and all chemical reagents of analytical grades were purchased from Sigma Chemical Co. (St. Louis, Mo, USA). The dose of MTX diluted in water (50% 1ml/kg of body weight of Mice were applied twice a week for six weeks to induced hepatocellular injury determined by liver function tests. The mice were maintained under pathogen-free conditions with air conditioning, a 12-hr light/12-hr dark cycle, and %55 humidity. Results: Results shows that a significant increase in serum enzymes (ALT, AST, ALP) and significant decrease in total protein was observed in all groups receiving MTX for the induction of hepatic injury. Mice receiving combination therapy of selenium along with MTX shows a time course recovery towards normalcy. The highest value of ALT (94.83 IU/L), AST (73.21 IU/L) and ALP (157 IU/L) were recorded in Mice receiving MTX for hepatic injury. The lowest values (63.67, 51.49 and 139.95 IU/L) was recorded in group treated with MTX+ Se (200mg/kg b.w) but both the groups differed non significantly and shows the same trend but a decreasing trend in serum enzymes (ALT 32.85%), (AST 29.67%) and (ALP 11.40%). Conclusion: It is concluded that selenium supplementation in MTX treated rats elicited a reduction in the toxic effects of the pesticide by improving the studied parameters, which was confirmed by the biochemical analysis of serum.

Adjunctive S100A8/A9 Immunomodulation Hinders Ciprofloxacin Resistance in Pseudomonas aeruginosa in a Murine Biofilm Wound Model.

Objective Pseudomonas aeruginosa is known to contribute to the pathogenesis of chronic wounds by biofilm-establishment with increased tolerance to host response and antibiotics. The neutrophil-factor S100A8/A9 has a promising adjuvant effect when combined with ciprofloxacin, measured by quantitative bacteriology, and increased anti- and lowered pro-inflammatory proteins. We speculated whether a S100A8/A9 supplement could prevent ciprofloxacin resistance in infected wounds.MethodFull-thickness 2.9cm2-necrosis was inflicted on 32 mice. On day 4, P.aeruginosa in seaweed alginate was injected sub-eschar to mimic a mono-pathogenic biofilm. Mice were randomized to receive ciprofloxacin and S100A8/A9 (n=14), ciprofloxacin (n=12) or saline (n=6). Half of the mice in each group were euthanized day 6 and the remaining day 10 post-infection. Mice were treated until sacrifice. Primary endpoint was the appearance of ciprofloxacin resistant P.aeruginosa. The study was further evaluated by genetic characterization of resistance, means of quantitative bacteriology, wound-size and cytokine-production.ResultsThree mice receiving ciprofloxacin monotherapy developed resistance after 14 days. None of the mice receiving combination therapy changed resistance pattern. Sequencing of fluoroquinolone-resistance determining regions in the ciprofloxacin resistant isolates identified two high-resistant strains mutated in gyrA C248T (MIC>32µg/ml) and a gyr B mutation was found in the sample with low level resistance (MIC=3µg/ml). Bacterial densities in wounds were lower in the dual treated group compared to the placebo group on both termination days.ConclusionThis study supports the ciprofloxacin augmenting effect and indicates a protective effect in terms of hindered ciprofloxacin resistance of adjuvant S100A8/A9 in P.aeruginosa biofilm infected chronic wounds.

Combined treatment with a gastric inhibitory polypeptide receptor antagonist and a peptidyl peptidase-4 inhibitor improves metabolic abnormalities in diabetic mice.

ObjectivesDipeptidyl peptidase-4 inhibition and gastric inhibitory polypeptide (GIP) receptor antagonism have therapeutic effects in type 2 diabetes mellitus. We assessed the effects of sitagliptin and Pro3(GIP) in a mouse model of diabetes.MethodsDiabetes was induced in C57BL/6J mice by a high-fat diet and intraperitoneal injection of streptozocin. Blood glucose was assessed weekly. Six weeks later, serum triglycerides, total cholesterol and glucose tolerance were assessed and pancreatic and adipose tissues were collected.ResultsCombination therapy with sitagliptin and Pro3(GIP) resulted in significantly greater reductions of blood glucose and triglycerides than either monotherapy. Combination therapy also improved insulin sensitivity and glucose tolerance. β-cell mass and insulin-positive cell percentage in the pancreas was higher in mice receiving combination therapy compared with either monotherapy. Crown-like structures, inflammatory markers in adipose tissue, and serum leptin concentrations were decreased in mice receiving combination therapy compared with either monotherapy.ConclusionsCombination therapy with Pro3(GIP) and sitagliptin improved metabolic abnormalities in diabetic mice. Changes in serum leptins and reduced inflammatory cell infiltration in adipose tissue might account for the observed effects.

Rebalancing Protein Homeostasis Enhances Tumor Antigen Presentation.

Despite the accumulation of extensive genomic alterations, many cancers fail to be recognized as "foreign" and escape destruction by the host immune system. Immunotherapies designed to address this problem by directly stimulating immune effector cells have led to some remarkable clinical outcomes, but unfortunately, most cancers fail to respond, prompting the need to identify additional immunomodulatory treatment options.Experimental Design: We elucidated the effect of a novel treatment paradigm using sustained, low-dose HSP90 inhibition in vitro and in syngeneic mouse models using genetic and pharmacologic tools. Profiling of treatment-associated tumor cell antigens was performed using immunoprecipitation followed by peptide mass spectrometry. We show that sustained, low-level inhibition of HSP90 both amplifies and diversifies the antigenic repertoire presented by tumor cells on MHC-I molecules through an IFNγ-independent mechanism. In stark contrast, we find that acute, high-dose exposure to HSP90 inhibitors, the only approach studied in the clinic to date, is broadly immunosuppressive in cell culture and in patients with cancer. In mice, chronic non-heat shock-inducing HSP90 inhibition slowed progression of colon cancer implants, but only in syngeneic animals with intact immune function. Addition of a single dose of nonspecific immune adjuvant to the regimen dramatically increased efficacy, curing a subset of mice receiving combination therapy. These highly translatable observations support reconsideration of the most effective strategy for targeting HSP90 to treat cancers and suggest a practical approach to repurposing current orally bioavailable HSP90 inhibitors as a new immunotherapeutic strategy.See related commentary by Srivastava and Callahan, p. 6277.

Abstract 583: DNA demethylating agents enhance susceptibility of lung cancer cells to γδ T cell-based immunotherapy via MHC-independent mechanisms

Abstract Immune-based therapy is revolutionizing lung cancer management in the clinic. In addition to rejuvenating T cells through immune checkpoint blockade, enhancing the immunogenicity of cancer cells and their interactions with immune cells may also be key to boosting the clinical efficacy of immunotherapy. Previous transcriptomic studies have shown that DNA demethylating agents may alter antigen processing/presentation machinery, upregulate cancer-testis antigens, reactivate endogenous retroviruses to facilitate efficacy of immunotherapy. Nevertheless, how DNA demethylating agents modulate surface immune synapse proteins and affect interactions between cancer and immune cells remain unclear. We utilized stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative membrane proteomics to identify surface proteins altered in decitabine-treated human lung cancer cells. The results showed that decitabine upregulated multiple immune synapse proteins. Gene enrichment analyses discovered that these surface proteins are predominantly involved in γδT cell-mediated cytotoxicity. γδT cells are a distinct subgroup of T cells that bridge between the innate and the adaptive immune systems and do not require the major histocompatibility complexes for antigen recognition. Co-culture of decitabine-pretreated human lung cancer cells with allogenic γδT cells in vitro confirmed sensitization effects of decitabine for the killing of γδT cells. A similar effect was also observed in immunocompromised mice bearing xenograft tumors in vivo. Moreover, the comparison of decitabine-induced surface proteomes between sensitizable and unsensitizable lung cancer cells revealed that intercellular adhesion molecule 1 (ICAM1), was among the top differentially expressed proteins. Imaging analyses confirmed that ICAM1 participated in the formation of immune synapses between cancer cells and γδT cells. Increased expression of ICAM1 was also observed in xenograft tumors from mice receiving combination therapy of decitabine and γδT cells. Overall, our data characterized decitabine-induced surface proteomes and uncovered an immunomodulatory effect of decitabine for enhancing MHC-independent killing by γδT cells. These findings have great clinical implications and provide a novel molecular basis for coupling DNA demethylation agents with γδT cell-based immunotherapy in the treatment of lung cancer patients. Citation Format: Rueyhung Weng, Chien-Ting Lin, Tai-Chung Huang, Hsuan-Hsuan Lu, Yi-Chieh Wu, Xuan-Hui Lin, Rong-Shan Lin, Chong-Jen Yu, Hsing-Chen Tsai. DNA demethylating agents enhance susceptibility of lung cancer cells to γδ T cell-based immunotherapy via MHC-independent mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 583.

The Combination of PARP Inhibitors and DNMT Inhibitors Modulates Immune Activity and Suggests a Role for Immune Therapy in AML

Acute myeloid leukemia (AML) is a genetically heterogeneous disease where malignant clonal proliferation of immature myeloid cells in the bone marrow leads to disruption of normal hematopoiesis and bone marrow failure. Most treatment improvements have consisted of limited chemotherapeutic interventions, and morbidity and mortality remain unacceptably high. While patients unfit for intensive chemotherapy have the options of epigenetic treatments with DNA methyltransferase inhibitors (DNMTis), and the preclinical research underpinning these treatments is robust and promising, clinical response rates have been poor. Therefore, we have derived a unique treatment strategy combining poly-ADP ribose polymerase inhibitors (PARPis) with DNMTis, and we have previously demonstrated efficacy of this combination in AML in both in vitroand in vivomodels. This combination increases the amount and retention time of PARP1 at laser-induced DNA damage sites, increases DNA DSBs and tumor cytotoxicity, blunts cell self-renewal, and leads to strong anti-tumor responses. These results form the basis for our ongoing Phase I/II clinical trial of this combination therapy in patients with relapsed/refractory AML.To identify molecular pathways underpinning this combination, genome-wide expression microarray analysis was performed on RNA extracted from multiple BRCA-intact sporadic AML cell lines treated with single agents or drug combination. Interestingly, combination drug treatment, more than either single treatment alone, up regulated genes in immune response pathways, including interferons, natural killer cell-mediated cytotoxicity and Toll-like receptor signaling pathway genes. In both AML cell lines (N=10) and primary samples (N=8), we used qPCR to validate these expression increases, with particular focus on interferon type 1-inducible genes, including IFI6, IFI27, IFI30, IRF7, ISG15, and OASL. These interferon-inducible genes were similarly increased in selected AML clinical trial samples post combination treatment. Interferon stimulation studies showed that treatment of multiple cell lines with IFN-β similarly up regulates our genes of interest, and suggests that the immune system plays a pivotal role in mediating the effects of our combination treatment.To functionally validate this immune involvement in vivo, we use an immune competent mouse model that spontaneously develops AML. The bone marrow cells collected from leukemic FLT3/ITD;NHD13 mice are transplanted into sublethally irradiated syngeneic recipients where they undergo clonal leukemic expansion and expression of specific surface markers (CD45.1 vs CD45.2) allow for the tracking of both host and transplanted blood cells. After validating that our combination treatment increases survival and reduces tumor burden in this in vivo model, we isolated mononuclear cells from spleens and livers and found that mice in the combination therapy group had increased T cell populations with increased PD1, CD28 and 4-1BB expression. Tal single agent and combination therapy treated mice also had decreased myeloid-derived suppressor cells and increased antigen presenting cells, specifically dendritic cells, monocytes and macrophages. Finally, upon ex vivo stimulation, T cells from mice receiving combination therapy produced significantly more IFN-γ and GM-CSF than mice from other treatment groups.These results indicate that our combination therapy results in immune activation, and suggest the potential to target the immune system in order to induce anti-tumor effects, enhance efficacy of our combination treatment and improve clinical outcomes in AML. DisclosuresLapidus:KinaRx, LLC: Other: Founder and Scientific Advisor.

Abstract 3778: Combination immunotherapy intercepts murine pancreatic cancer progression

Abstract Immunotherapy agents that activate T cells are changing clinical practice for some metastatic cancer patients. Vaccines targeting viral oncogenes prevent the development of hepatocellular and cervical cancers. We therefore tested the hypothesis that a vaccine targeting the oncogene that initiates pancreatic cancer (PDA) given in combination with immunotherapy agents that reprogram the earliest procarcinogenic inflammatory changes will slow (intercept) PDA development. The KRAS proto-oncogene is a driver mutation expressed in &amp;gt;90% of pancreatic intraepithelial neoplasms (PanINs) and an ideal candidate target for early vaccination against PDA. However, even early PanINs have immunosuppressive microenvironments highly expressing inhibitory immune checkpoints like CTLA-4 and infiltrating immunosuppressive cells such as regulatory T cells (Tregs). The overall goal of this research is to reprogram the immunosuppressive environment while specifically activating immunity against early premalignancies to prevent PDA development. We previously reported that early dosing of KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice, which endogenously recapitulate human PDA tumorigenesis, with the Treg depleting and inactivating agents cyclophosphamide (cy) and PC61 antibody in combination with an attenuated Listeria monocytogenes vaccine expressing mutant KrasG12D (LM-Kras) can slow tumorigenesis in 40% of mice. In this study we attempted to improve the efficacy of a vaccine approach by specifically inhibiting the immunosuppressive effects of the CTLA-4 immune checkpoint on Tregs with a CTLA-4 blocking antibody. KPC mice at 4-5 weeks with early PanINs received 2 doses of LM-Kras and cy/PC61 and 8 doses of anti-CTLA-4. Control mice received isotype antibody with LM-Kras and cy/PC61 or no treatment. At 10-11 weeks, pancreata were pathologically evaluated and assigned grades normal, low (1) and high-grade (2 and 3) PanINs, and PDA. Mice receiving LM-Kras + anti-CTLA-4 had a significantly lower incidence of low- and high-grade PanINs and PDA compared to controls. Furthermore, mice receiving LM-Kras, cy/PC61, and anti-CTLA-4 did not develop lesions beyond PanIN 1, indicating that the combination therapy may halt PDA development at early stages. To investigate the immune profiles of mice receiving treatment, flow cytometry was performed on tumor-infiltrating lymphocytes (TILs) isolated from pancreata to assess abundance of Tregs, infiltration of effector and memory T cell populations, and expression of inhibitory immune checkpoint molecules. Mice receiving combination therapy had decreased numbers of Tregs, increased numbers of effector and memory T cells, and increased CD8+ T cells to Tregs ratio in the microenvironment compared to controls. Further studies using immunohistochemistry and RNA expression profiles are under way to further characterize the immune changes in the microenvironment following treatment. Citation Format: Aleksandra Popovic, Nina J. Chu, Todd Armstrong, Elizabeth M. Jaffee. Combination immunotherapy intercepts murine pancreatic cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3778.

Abstract 2981: Clonal deletion of tumor-specific T cells by combination checkpoint blockade compromises antitumor efficacy in low tumor burden states

Abstract A myriad of combination immunotherapies is being developed as cancer treatments with the goal of enhancing antitumor immunity. We found that depending on the disease setting, combined immune checkpoint blockade may not always lead to synergistic antitumor effects. While combined anti-CTLA-4 and anti-PD-1 improves control of established tumors, this combination can paradoxically compromise antitumor immunity in the low tumor burden state in preclinical models as well as in melanoma patients. This paradoxical outcome results from treatment-induced apoptosis of tumor-specific T cells. These changes further alter the overall T-cell receptor repertoire. Activated tumor-specific T cells express higher levels of IFN-γ receptor and are more susceptible to apoptosis. Deficiency of IFN-γ receptor on immune cells rescues this phenotype and restores antitumor activity. Additionally, tumor-specific T cells lacking the IFN-γ receptor demonstrate a significant survival advantage compared to their wild-type counterparts in tumor-bearing mice receiving combination therapy. Finally, combination therapy induces significantly higher levels of IFN-γ in the low versus high tumor burden state on a per cell basis, reflecting their less exhausted immune status. Thus, the optimal immunotherapy strategy may depend on disease context, and may not always favor more potent combination immunotherapies. Citation Format: Chien-Chun Steven Pai, Xiaoqing Lu, Donald Simons, Michel DuPage, Kole T. Roybal, Mingyi Chen, Serena Kwek, Amy-Jo Casbon, Gillian A. Kinsbury, Lawrence Fong. Clonal deletion of tumor-specific T cells by combination checkpoint blockade compromises antitumor efficacy in low tumor burden states [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2981.

Pharmacological Inhibition of NOS Activates ASK1/JNK Pathway Augmenting Docetaxel-Mediated Apoptosis in Triple-Negative Breast Cancer.

Purpose: Chemoresistance in triple-negative breast cancer (TNBC) is associated with the activation of a survival mechanism orchestrated by the endoplasmic reticulum (EnR) stress response and by inducible nitric oxide synthase (iNOS). Our aim was to determine the effects of pharmacologic NOS inhibition on TNBC.Experimental Design: TNBC cell lines, SUM-159PT, MDA-MB-436, and MDA-MB-468, were treated with docetaxel and NOS inhibitor (L-NMMA) for 24, 48, and 72 hours. Apoptosis was assessed by flow cytometry using Annexin-V and propidium iodide. Western blot was used to assess ER stress and apoptosis, and rtPCR was used to evaluate s-XBP1. TNBC patient-derived xenografts (PDX) were treated either with vehicle, docetaxel, or combination therapy (NOS inhibition + docetaxel). Mouse weight and tumor volumes were recorded twice weekly. Docetaxel concentration was determined using mass spectrometry. To quantify proliferation and apoptosis, PDX tumor samples were stained using Ki67 and TUNEL assay.Results:In vitro, L-NMMA ameliorated the iNOS upregulation associated with docetaxel. Apoptosis increased when TNBC cells were treated with combination therapy. In TNBC PDXs, combination therapy significantly reduced tumor volume growth and increased survival proportions. In the BCM-5998 PDX model, intratumoral docetaxel concentration was higher in mice receiving combination therapy. Coupling docetaxel with NOS inhibition increased EnR-stress response via coactivation of ATF4 and CHOP, which triggered the pASK1/JNK proapoptotic pathway, promoting cleavage of caspases 3 and 9.Conclusions: iNOS is a critical target for docetaxel resistance in TNBC. Pharmacologic inhibition of NOS enhanced chemotherapy response in TNBC PDX models. Combination therapy may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. Clin Cancer Res; 24(5); 1152-62. ©2018 AACR.

Combination therapy with octyl gallate and ferulic acid improves cognition and neurodegeneration in a transgenic mouse model of Alzheimer's disease

To date, there is no effective Alzheimer's disease (AD)-modifying therapy. Nonetheless, combination therapy holds promise, and nutraceuticals (natural dietary compounds with therapeutic properties) and their synthetic derivatives are well-tolerated candidates. We tested whether combination therapy with octyl gallate (OG) and ferulic acid (FA) improves cognition and mitigates AD-like pathology in the presenilin-amyloid β-protein precursor (PSAPP) transgenic mouse model of cerebral amyloidosis. One-year-old mice with established β-amyloid plaques received daily doses of OG and FA alone or in combination for 3 months. PSAPP mice receiving combination therapy had statistically significant improved cognitive function versus OG or FA single treatment on some (but not all) measures. We also observed additional statistically significant reductions in brain parenchymal and cerebral vascular β-amyloid deposits as well as brain amyloid β-protein abundance in OG- plus FA-treated versus singly-treated PSAPP mice. These effects coincided with enhanced nonamyloidogenic amyloid β-protein precursor (APP) cleavage, increased α-secretase activity, and β-secretase inhibition. We detected elevated expression of nonamyloidogenic soluble APP-α and the α-secretase candidate, a disintegrin and metalloproteinase domain-containing protein 10. Correspondingly, amyloidogenic β-carboxyl-terminal APP fragment and β-site APP-cleaving enzyme 1 expression levels were reduced. In parallel, the ratio of β- to α-carboxyl-terminal APP fragment was decreased. OG and FA combination therapy strikingly attenuated neuroinflammation, oxidative stress, and synaptotoxicity. Co-treatment afforded additional statistically significant benefits on some, but not all, of these outcome measures. Taken together, these data provide preclinical proof-of-concept for AD combination therapy.