Reversible Suppression Research Articles

Contextual Reprogramming of CAR-T Cells for Treatment of HER2+ Cancers

Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities. To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS. The conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.

Irradiated mesenchymal stem cells support stemness maintenance of hepatocellular carcinoma stem cells through Wnt/\u03b2-catenin signaling pathway

BackgroundCancer stem cells are the main reason of relapse, metastasis and resistance to anti-cancer therapies of Hepatocellular carcinoma (HCC). Mesenchymal stem cells (MSCs) are an important part of the tumor microenvironment. MSCs have been demonstrated to be involved in drug resistance in tumor. How MSCs contribute to radiotherapy resistance of HCC is still indistinct.MethodsFlow cytometry analysis was performed to isolate CD133+ cells from HCC cell lines Huh7 and PLC. The stemness of Huh7-CD133 and PLC-CD133 those were co-cultured with IR-MSCs were investigated by Colony formation assay. Tumor formation in nude mice was used to explore the tumorigenicity of CD133+ cancer cells. The activating Wnt/β-catenin signaling pathway in CSCs were also detected by RT-PCR and Western blotting.ResultsWe report that irradiated MSCs (IR-MSCs) could increase the ratio of CD133+ cells in hepatocellular carcinoma cells. IR-MSCs could promote stemness maintenance of HCC stem cells. After co-cultured with IR-MSCs, liver cancer stem cells (CSCs) presented increased colony formation ability and tumor formation ability. We also found IR-MSCs promoted Wnt expression of CSCs. Reverse suppression experiment showed that when Wnt inhibitor was added into the culture medium, the effect of IR-MSCs on stemness maintenance was counteracted.ConclusionsThese data showed that IR-MSCs could support stemness maintenance of CSCs by activating Wnt/β-catenin signaling pathway.

Dose-escalation trial of lobaplatin weekly plus concurrent radiotherapy for local-regionally advanced nasopharyngeal carcinoma

Objective To define the maximum-tolerated dose (MTD) of lobaplatin (LBP) in a weekly regimen combined with concurrent radiotherapy in the treatment of locally advanced nasopharyngeal carcinoma (NPC). Methods A total of 18 cases with stage Ⅲ/IV A NPC were enrolled. Concurrent chemoradiotherapy was given to all the patients with a dose escalation of LBP. The initial dose of LBP was 15 mg/m2 with an escalating dose of 5 mg/m2. At least 3 patients were assigned into each group. Patients were proceeded into the next dose group if no dose-limiting toxicity (DLT) occurred until the MTD was achieved. Efficacy and toxicity were evaluated regularly. Results Three patients were assigned into the 10 mg/m2, 3 into the 15 mg/m2, and 6 into the 20 mg/m2 and 25 mg/m2 groups, respectively. Two patients experienced DLT in the 25 mg/m2 group. Hence, the MTD was determined as 20 mg/m2. At 3 months after corresponding treatment, the remission rate of nasopharyngeal tumors and neck-positive lymph nodes of the patients was 100%. The most common toxicity was reversible bone marrow suppression. Conclusions The MTD of weekly lobaplatin plus concurrent IMRT is 20 mg/m2 for locally advanced NPC. This regimen is reliable and safe, which is worthy of further clinical study. Key words: Nasopharyngeal neoplasm/concurrent chemoradiotherapy; Lobaplatin; Dose-limiting toxicity; Maximum tolerated dose

Extracellular Vesicles From Human Urine-Derived Stem Cells Inhibit Glucocorticoid-Induced Osteonecrosis of the Femoral Head by Transferring Pro-Angiogenic DMBT1 and Anti-Apoptotic TIMP1

Osteonecrosis of the femoral head (ONFH) frequently occurs after glucocorticoid (GC) treatment. Extracellular vesicles (EVs) are important nano-sized paracrine mediators of intercellular crosstalk. Herein, we found that intravenous administration of EVs from human urine-derived stem cells (USC-EVs) at the early stage of GC exposure rescues angiogenesis impairment, reduces apoptosis of bone marrow and trabecular bone cells, prevent trabecular bone destruction and improves bone microarchitecture in the femoral heads of rats. In vitro, USC-EVs reverse GC-induced suppression of endothelial angiogenesis and activation of cellular apoptosis. Deleted in malignant brain tumors 1 (DMBT1) and tissue inhibitor of metalloproteinases 1 (TIMP1) are enriched in USC-EVs and essential for USC-EVs-induced pro-angiogenic and anti-apoptotic effects in GC-treated cells, respectively. Knockdown of TIMP1 in USC-EVs attenuates their protective effects against GC-induced ONFH. Our study suggests that USC-EVs are a novel promising nano-sized agent for the prevention of GC-induced ONFH by delivering pro-angiogenic DMBT1 and anti-apoptotic TIMP1.

The re‐emerging pathophysiological role of the cystathionine‐β‐synthase ‐ hydrogen sulfide system in Down syndrome

Down syndrome (DS) is associated with significant perturbances in many morphological and biochemical features. Cystathionine-β-synthase (CBS) is one of the key mammalian enzymes that is responsible for the biological production of the gaseous transmitter hydrogen sulfide (H2 S). When H2 S is overproduced, it can exert detrimental cellular effects, in part due to inhibition of mitochondrial Complex IV activity. An increased expression of CBS and the consequent overproduction of H2 S are well documented in individuals with DS. Two decades ago, it has been proposed that a toxic overproduction of H2 S importantly contributes to the metabolic and neurological deficits associated with DS. However, until recently, this hypothesis has not yet been tested experimentally. Recent data generated in human dermal fibroblasts show that DS cells overproduce H2 S, which, in turn, suppresses mitochondrial Complex IV activity and impairs mitochondrial oxygen consumption and ATP generation. Therapeutic CBS inhibition lifts the tonic (and reversible) suppression of Complex IV: This results in the normalization of mitochondrial function in DS cells. H2 S may also contribute to the cellular dysfunction via several other molecular mechanisms through interactions with various mitochondrial and extramitochondrial molecular targets. The current article provides a historical background of the field, summarizes the recently published data and their potential implications, and outlines potential translational approaches (such as CBS inhibition and H2 S neutralization) and future experimental studies in this re-emerging field of pathobiochemistry.

Elagolix for Heavy Menstrual Bleeding in Women with Uterine Fibroids

BackgroundUterine fibroids are hormone-responsive neoplasms that are associated with heavy menstrual bleeding. Elagolix, an oral gonadotropin-releasing hormone antagonist resulting in rapid, reversible suppression of ovarian sex hormones, may reduce fibroid-associated bleeding.MethodsWe conducted two identical, double-blind, randomized, placebo-controlled, 6-month phase 3 trials (Elaris Uterine Fibroids 1 and 2 [UF-1 and UF-2]) to evaluate the efficacy and safety of elagolix at a dose of 300 mg twice daily with hormonal “add-back” therapy (to replace reduced levels of endogenous hormones; in this case, estradiol, 1 mg, and norethindrone acetate, 0.5 mg, once daily) in women with fibroid-associated bleeding. An elagolix-alone group was included to assess the impact of add-back therapy on the hypoestrogenic effects of elagolix. The primary end point was menstrual blood loss of less than 80 ml during the final month of treatment and at least a 50% reduction in menstrual blood loss from baseline to the final month; missing data were imputed with the use of multiple imputation.ResultsA total of 412 women in UF-1 and 378 women in UF-2 underwent randomization, received elagolix or placebo, and were included in the analyses. Criteria for the primary end point were met in 68.5% of 206 women in UF-1 and in 76.5% of 189 women in UF-2 who received elagolix plus add-back therapy, as compared with 8.7% of 102 women and 10% of 94 women, respectively, who received placebo (P<0.001 for both trials). Among the women who received elagolix alone, the primary end point was met in 84.1% of 104 women in UF-1 and in 77% of 95 women in UF-2. Hot flushes (in both trials) and metrorrhagia (in UF-1) occurred significantly more commonly with elagolix plus add-back therapy than with placebo. Hypoestrogenic effects of elagolix, especially decreases in bone mineral density, were attenuated with add-back therapy.ConclusionsElagolix with add-back therapy was effective in reducing heavy menstrual bleeding in women with uterine fibroids. (Funded by AbbVie; Elaris UF-1 and Elaris UF-2 ClinicalTrials.gov numbers, NCT02654054 and NCT02691494.)

&lt;i&gt;Leishmania donovani&lt;/i&gt;-Induced Immune Dysregulation among Sudanese Patients with Visceral and Post Kala-Azar Dermal Leishmaniases: Possible Roles in Pathogenesis

L. donovani infections (visceral and post kala-azar dermal leishmaniases) are characterized by infection-induced reversible immune suppression. Autoimmunity is a well-documented phenomenon among patients with primary immune deficiencies. This study aimed to study auto-immune phenomena accompanying L. donovani infections. In a prospective case-controlled study and following informed consent, 155 individuals with visceral leishmaniasis (VL; n = 62), post kala-azar dermal leishmaniasis (PKDL; n = 31) and apparently healthy volunteers (n = 62) were recruited. Sera antinuclear (ANA), anti-dsDNA, anti-thyroid peroxidase (TPO), anti-smooth muscles (ASMA) and F-actin auto-antibodies were measured using ELISA and indirect immune-fluorescence assay. The mean ages of VL, PDKL patients and apparently healthy volunteers were: 17.5 ± 12.5, 15.0 ± 7.0 and 17.5 ± 9.5 years with Male:Female ratios of 2:0, 1:2 and 1:5 respectively. Significantly high frequencies of F-actin (74.2%; 46/62) and ASMA (50%; 31/62) auto-antibodies were seen among VL patients (p = 0.003, p = 0.001) compared to apparently healthy volunteers. Likewise, significantly high frequencies of F-actin (64.5%; 20/31; p = 0.001), ASMA (42%; 13/31; p = 0.003), ANA (36%; 11/31; p = 0.001) and anti-dsDNA (16%; 5/31; p = 0.01) auto-antibodies were seen among PKDL patients. Development of tissue-based autoantibodies in L. donovani infections probably indicates loss of peripheral tolerance with activation of circulating auto-reactive T and B cells probably contributing to disease pathogenesis (increased bilirubin/liver enzymes, prolonged QT interval/arrythmias and blood cytopenias). In conclusion, L. donovani infection-induced immune suppression with development of tissue-based auto-antibodies is prevalent among Sudanese patients with VL and PKDL leishmaniases and contributes to some aspects of the disease pathogenesis.

SM09419, a Novel, Small-Molecule CDC-like Kinase (CLK) Inhibitor, Demonstrates Strong Inhibition of the Wnt Signaling Pathway and Antitumor Effects in Mantle Cell Lymphoma Models

Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) that accounts for ~7% of all NHL in the U.S. MCL is associated with aberrant activation of the Wnt signaling pathway, which plays a key role in the survival and maintenance of MCL-initiating cells. Many MCL patients experience relapse and subsequent disease progression due to chemoresistance following initial therapy; hence, novel therapies are needed. CLKs regulate the activity of serine/arginine-rich splicing factors (SRSFs) that modulate spliceosome assembly, mRNA splicing, and gene expression. SM09419 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt pathway. The purpose of these studies was to examine the antitumor activity of SM09419 in preclinical models of MCL. SM09419 potently inhibited both CLK1-CLK4 (IC50 for all &lt;0.02 µM) and Wnt signaling pathway (average EC50=0.068 µM) activities. In REC-1 and GRANTA-519 MCL cells, SM09419 dose-dependently inhibited SRSF6 phosphorylation and potently suppressed expression of Wnt-related genes (CCND1, LEF1, TCF7) and proteins vs. vehicle. In tests on 5 MCL cell lines, cell proliferation was strongly impaired by SM09419 across all lines (average EC50=0.102 µM [0.021-0.236]). SM09419 also induced apoptosis in REC-1 and GRANTA-519 cells, increasing caspase 3/7 activation and PARP cleavage while reducing survivin and MCL-1 expression vs. vehicle. In vivo antitumor effects and tolerability of oral SM09419 (QD 20-21 days) were assessed in mice bearing REC-1 and JeKo-1 flank xenografts (n=5/group). In REC-1 xenografts, strong tumor growth inhibition (TGI) vs. vehicle occurred in mice treated with 12.5, 25, and 50 mg/kg SM09419 (TGI 88% [p&lt;0.01], 100%, and 100% [p&lt;0.001], respectively), and the two highest doses induced complete tumor regression in all mice from D14. Similarly, in JeKo-1 xenografts, SM09419 (12.5 and 25 mg/kg) induced significant TGI vs. vehicle (71% and 100%, respectively; p&lt;0.0001) with complete tumor regression at 25 mg/kg, whereas acalabrutinib (50 mg/kg BID) was not efficacious (27% TGI) when tested in parallel. SM09419 25mg/kg induced reversible suppression of phospho-SRSF6 protein and inhibited Wnt pathway-related gene expression (TCF7 and DVL2) in JeKo-1 tumors in a single-dose PD study, demonstrating downstream target engagement in vivo. SM09419 was also assessed in 2 patient-derived xenograft (PDX) mouse models of MCL. PDX cells were injected intravenously and treatment was initiated upon 8-12% engraftment of human CD45+CD19+ cells in peripheral blood. In the first model, derived from a patient who was progressive after 8 modalities including ibrutinib, SM09419 (25 mg/kg QD) increased survival vs. vehicle (100% through D26 vs. 0% by D12, respectively; n=6/group) and suppressed MCL engraftment in the blood (12% at D26 vs. 69% at D8 and D12, respectively; p=0.002) and bone marrow (30% at D26 vs. 91% at D8 and D12, respectively; p=0.002). In the second model, derived from a patient refractory after ibrutinib and anti-PDL1 treatment, SM09419 (25 mg/kg QD) significantly suppressed MCL engraftment vs. vehicle in the blood (8% vs. 72%), bone marrow (20% vs. 57%), and spleen (15% vs. 96%) at D28 (study end; p&lt;0.001 for all; n=4/group). In addition, SM09419 greatly inhibited splenomegaly vs. vehicle (0.04 g vs. 0.4 g, respectively; p&lt;0.001). In a subsequent experiment in the same model, mice (n=7/group) were treated with 12.5 or 25 mg/kg SM09419 or vehicle for 12 weeks (to D85). Blood MCL engraftment at D41 was significantly lower in mice treated with SM09419 (40% at 12.5 mg/kg and 23% at 25 mg/kg) vs. vehicle (88%; p&lt;0.01 and p&lt;0.001, respectively). SM09419 dose-dependently increased survival (28.6% at 12.5 mg/kg and 85.7% at 25 mg/kg at D85) vs. vehicle (0% at D63); survival was maintained in both dose groups during post-treatment monitoring (to D99). SM09419 was well tolerated in all tested mouse models based on body weight measurements. In summary, SM09419 potently inhibited SRSF6 phosphorylation Wnt signaling pathway activity, and cell proliferation and induced apoptosis in MCL cell lines. The strong in vivo antitumor effects observed as a single agent suggest that SM09419 may provide a clinical benefit for patients with treatment-resistant or refractory MCL. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies is being initiated. Disclosures Chung: Samumed, LLC: Employment, Equity Ownership. Creger:Samumed, LLC: Employment, Equity Ownership. Sitts:Samumed, LLC: Employment, Equity Ownership. Chiu:Samumed, LLC: Employment, Equity Ownership. Mak:Samumed, LLC: Employment, Equity Ownership. KC:Samumed, LLC: Employment, Equity Ownership. Tam:Samumed, LLC: Employment, Equity Ownership. Bucci:Samumed, LLC: Employment, Equity Ownership. Stewart:Samumed, LLC: Employment, Equity Ownership. Phalen:Samumed, LLC: Employment, Equity Ownership. Cha:Samumed, LLC: Employment, Equity Ownership.

TMIC-59. INVESTIGATING PHYSIOLOGY OF THERAPY RESISTANT, TUMOR-INITIATING GBM CELLS IN HYPOXIC NICHES USING SPATIALLY-SENSITIVE HYPOXIA REPORTER SYSTEM AND SINGLE-CELL RNA SEQUENCING

Abstract Glioblastoma (GBM) is the most common and lethal brain cancer that invariably recurs after therapy due to presence of resistant GBM cells within hypoxic and peri-necrotic regions. Eradicating such GBM cells, which constitute a major source of tumor recurrence, is important to curb disease relapse. An endogenously expressed, spatially sensitive hypoxia reporter would therefore be a valuable tool to evaluate hypoxic zones in GBM in detail, and to measure the efficacy of hypoxia-activated drugs. For this purpose, we engineered a lentiviral vector that carries a hypoxia reporter, consisting of HIF response elements (HRE) that drive expression of UnaG fluorescent protein, which fluoresces independent of oxidative maturation. We validated the sensitivity of our reporter in vitro using U87MG, GBM2, and patient-derived GBM stem cell lines, and we performed intracranial transplantations of GBM cells in SCID mice to identify cells undergoing hypoxic stress in in vivo microenvironment. In addition, GL261 murine GBM cells with hypoxia reporter were intracranially implanted in C57BL/6 mice as syngeneic model for studies on immune responses. Brains from our transplant studies were dissociated and single-cell RNA sequencing (Drop-Seq) was performed to investigate heterogeneity in response to hypoxia within GBM cells and the cellular composition of microenvironment. We will also apply a hypoxia-activated prodrug, Evofosfamide (Evo), in our ongoing studies that can potentially eradicate hypoxic tumor cells and increase T cell infiltration and reverse immune suppression. As hypoxic niches are thought to confer resistance to radiation therapy (XRT), combining XRT with Evo could thus improve therapy efficacy. Our hypoxia gene reporter, combined with single-cell transcriptomics, could therefore serve as an effective tool to enable fundamental investigation of GBM microenvironment and could be used to evaluate therapies targeting tumor microenvironment to enhance GBM patient survival.

Male Hormonal Contraception.

The economic and public health burdens of unplanned pregnancies are evident globally. Since the introduction of the condom >300 years ago, assumptions about male willingness to participate in contraception, as well as concerns about failure rates and side effects, have stagnated the development of additional reversible male contraceptives. However, changing attitudes and recent research advances have generated renewed interest in developing reversible male contraceptives. To achieve effective and reversible suppression of spermatogenesis, male hormonal contraception relies on suppression of testicular testosterone and sperm production using an androgen-progestin combination. While these may be associated with side effects-changes in libido, weight, hematocrit, and cholesterol-recently, novel androgens and progestins have shown promise for a "male pill" with reduced side effects. Here we summarize landmark studies in male contraceptive development, showcase the most recent advances, and look into the future of this field, which has the potential to greatly impact global public health.

Chilli leaf curl virus infection downregulates the expression of the genes encoding chloroplast proteins and stress-related proteins.

Virus infection alters the expression of several host genes involved in various cellular and biological processes in plants. Most of the studies performed till now have mainly focused on genes which are up-regulated and later projected them as probable stress tolerant/susceptible genes. Nevertheless, genes which are down-regulated during plant-virus interaction could also play a critical role on disease development as well as in combating the virus infection. Hence, to identify such down-regulated genes and pathway, we performed reverse suppression subtractive hybridization in Capsicum annuum var. Punjab Lal following Chilli leaf curl virus (ChiLCV) infection. The screening and further processing suggested that majority of the genes (approximately 35% ESTs) showed homology with the genes encoding chloroplast proteins and 16% genes involved in the biotic and abiotic stress response. Additionally, we identified several genes, functionally known to be involved in metabolic processes, protein synthesis and degradation, ribosomal proteins, energy production, DNA replication and transcription, and transporters. We also found 3% transcripts which did not show homology with any known genes. The redundancy analysis revealed the maximum percentage of chlorophyll a-b binding protein (15/96) and auxin-binding proteins (13/96). We developed a protein interactome network to characterise the relationships between proteins and pathway involved during the ChiLCV infection. We identified that the most of the interaction occurs either among the chloroplast proteins (Arabidopsis proteins interactive map) or biotic and abiotic stress responsive proteins (Solanum lycopersicum interactome). Taken together, our study provides the first transcriptome and protein interactome of the down-regulated genes during C. annuum-ChiLCV interaction. These resources could be exploited in deciphering the steps involved in the process of virus infection.

A Low-cost Continuous-flow Gas Interface for Coupling an Elemental Analyzer with a Micadas AMS: gas flow Mathematical Model and first results

ABSTRACTA fully automatic continuous-flow gas injection interface was built to couple an elemental analyzer with a MICADAS accelerator mass spectrometer (AMS) as a low-cost option that does not require an absorber trap for CO2 injection. The complication of the variable ion current during gas injection can be overcome by understanding and controlling the mass flow-dependent ionization yield. The time-varying CO2 concentrations and carbon mass flows are estimated with a mathematical model in order to investigate their relationship with the abundant isotope (12C–) signal. This model is based on a complete CO2 diffusion equation and instantaneous mass flow. It shows a good agreement between model calculations and the measurements. A reversible suppression of the formation of ions occurs, if the carbon mass flow exceeds 2.0–2.3 µg C/min. This result repeats for different injection capillaries and for different carrier volumetric flow rates.

Novel conjugates with dual suppression of glutathione S-transferases and tryptophan-2,3-dioxygenase activities for improving hepatocellular carcinoma therapy

Tryptophan-2,3-dioxygenase (TDO) is an immune checkpoint enzyme expressed in human tumors and involved in immune evasion and tumor tolerance. While glutathione S-transferases (GSTs) are pharmacological targets for several cancer. Here we demonstrated the utility of NBDHEX (GSTs inhibitor) and TDO inhibitor by the combinatorial linker design. Two novel conjugates with different linkers were prepared to reverse tumor immune suppression. The conjugates displayed significant antitumor activity against TDO and GSTs expression of HepG2 cancer cells. Further study indicated that compound 4 could induce higher apoptotic effect than its mother compounds via a mitochondrial-dependent pathway, simultaneously more effective to inhibit TDO and GSTs protein expression. Further study indicated that 4 could decrease the production of kynurenine and deactivate aryl hydrocarbon receptor (AHR), leading to CD3+T-cell activation and proliferation to involve in antitumor immune response.

Reversible neuroinhibition by focused ultrasound is mediated by a thermal mechanism

BackgroundTranscranial focused ultrasound (tFUS) at low intensities has been reported to directly evoke responses and reversibly inhibit function in the central nervous system. While some doubt has been cast on the ability of ultrasound to directly evoke neuronal responses, spatially-restricted transcranial ultrasound has demonstrated consistent, inhibitory effects, but the underlying mechanism of reversible suppression in the central nervous system is not well understood. Objective/hypothesisIn this study, we sought to characterize the effect of transcranial, low-intensity, focused ultrasound on the thalamus during somatosensory evoked potentials (SSEP) and investigate the mechanism by modulating the parameters of ultrasound. MethodsTFUS was applied to the ventral posterolateral nucleus of the thalamus of a rodent while electrically stimulating the tibial nerve to induce an SSEP. Thermal changes were also induced through an optical fiber that was image-guided to the same target. ResultsFocused ultrasound reversibly suppressed SSEPs in a spatially and intensity-dependent manner while remaining independent of duty cycle, peak pressure, or modulation frequency. Suppression was highly correlated and temporally consistent with in vivo temperature changes while producing no pathological changes on histology. Furthermore, stereotactically-guided delivery of thermal energy through an optical fiber produced similar thermal effects and suppression. ConclusionWe confirm that tFUS predominantly causes neuroinhibition and conclude that the most primary biophysical mechanism is the thermal effect of focused ultrasound.

Neoadjuvant Treatment With Müllerian-Inhibiting Substance Synchronizes Follicles and Enhances Superovulation Yield.

Müllerian-inhibiting substance (MIS), also known as anti-Müllerian hormone, is thought to be a negative regulator of primordial follicle activation. We have previously reported that treatment with exogenous MIS can induce complete ovarian suppression within 5 weeks of treatment in mice. To investigate the kinetics of the return of folliculogenesis following the reversal of suppression, we treated animals with recombinant human MIS (rhMIS) protein for 40 days in adult female Nu/Nu mice and monitored the recovery of each follicle type over time. Following cessation of MIS therapy, secondary, and antral follicles returned within 30 days, along with the normalization of reproductive hormones, including LH, FSH, MIS, and Inhibin B. Furthermore, 30 days following MIS pretreatment, the number of antral follicles were significantly higher than controls, and superovulation with timed pregnant mare serum gonadotropin and human chorionic gonadotropin stimulation at this time point resulted in an approximately threefold increased yield of eggs. Use of the combined rhMIS-gonadotropin superovulation regimen in a diminished ovarian reserve (DOR) mouse model, created by 4-vinylcyclohexene dioxide treatment, also resulted in a twofold improvement in the yield of eggs. In conclusion, treatment with rhMIS can induce a reversible ovarian suppression, following which a rapid and synchronized large initial wave of growing follicles can be harnessed to enhance the response to superovulation. Therapies modulating MIS signaling may therefore augment the response to current ovarian stimulation protocols and could be particularly useful to women with DOR or poor responders to controlled ovarian hyperstimulation during in vitro fertilization.

Abstract 1207: Reversing lactate-driven immunosuppression using the novel, potent and selective MCT4 inhibitor AZD0095

Abstract Metabolic adaptation to a Warburg phenotype is a hallmark of cancer, where tumors rely on glucose to produce ATP and building blocks required to support rapid tumor growth. Glycolytic metabolism requires continuous export of lactate from cells by monocarboxylate transporters (MCTs) resulting in accumulation of lactic acid in the tumor microenvironment. Tumor-derived lactate is immune-suppressive and has been shown to modify immune cell function. Inhibition of the MCT4 lactate transporter has the potential to reduce lactate in the tumor microenvironment and reverse immune suppression. AZD0095 is a highly selective, potent inhibitor of MCT4 with a cellular activity of 1-3 nM with a &amp;gt;1000-fold selectivity over MCT1. We confirm that lactic acid inhibits T cell proliferation and viability. AZD0095 does not inhibit T cell proliferation or survival (IC50 &amp;gt; 10 μM). Oral administration of AZD0095 (10-100 mg/kg BID) modulates lactate transport in mouse syngeneic models which do not express the MCT1 lactate transporter. AZD0095 treatment causes an increase in tumor-infiltrating lymphocytes and an increase in cytotoxic T cells in combination with α-PD1. AZD0095 treatment suppresses myeloid infiltration as a monotherapy and in combination with checkpoint inhibitor. Consistent with the observed changes in immune cell infiltration, the combination of AZD0095 with checkpoint inhibitors α-PD1 or α-CTLA4 (10 mg/kg 3x week) enhances the anti-tumor activity of checkpoint inhibitor monotherapy in MC-38 and EMT6 (MCT1 knock-out) mouse syngeneic models. These data demonstrate that AZD0095 can reverse lactate-driven immunosuppression and enhance response to checkpoint inhibition in pre-clinical models. Citation Format: Susan E. Critchlow, Gareth Hughes, Anna Staniszewska, Matt King, Filippos Michopoulos, Lorna Hopcroft, Martin R. Brown, Jenna Bradley, Beverley Hammond, Pablo MorentinGutierrez, Larissa Carnevalli, Elizabeth Hardaker, Frederick W. Goldberg. Reversing lactate-driven immunosuppression using the novel, potent and selective MCT4 inhibitor AZD0095 [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 1207.

Abstract 1140: A new twist on an old strategy: Can the lymph node environment help cancers escape immune surveillance

Abstract Cancer cells are selected for strategies to proliferate, invade, and metastasize within an organism. One such strategy is an ability to create an acidic microenvironment. This acidity stems from an increase in H+ production associated with elevated glucose metabolism. This, combined with poor perfusion, results in an acidic extracellular pH (pHe) of 6.5 - 6.9 in malignant tumors. The resulting acidic environment is toxic to normal cells, giving tumor cells an adaptive advantage. Further, it inhibits immune surveillance, although the mechanism by which this occurs is not entirely clear. However, it is well known that an acidic pH profoundly inhibits T cell effector function with minimal effect on T cell viability. Lymph nodes are the primary site for naïve T cell activation yet are the most common site of metastasis. Reversible suppression of T cell effector function naturally occurs within the lymph node, creating a site of immune privilege. As a result of these findings we hypothesized that cancer cells exploit this normal physiological function, which may be occurring in the lymph nodes. While lymph nodes are known to be hypoxic, nothing to date is known regarding their pH. Here, we show using four different approaches that the pH of lymph nodes is acidic. All experiments were performed using a murine model under normal, inflammatory and tumor bearing conditions. We interrogated lymph node pH using microelectrodes, chemical exchange saturation transfer (CEST) MRI, imaging with pH-low inserting peptide, pHLIP, and intravital imaging (IVI) of pH sensitive fluorescent dyes; all of which reported an acidic pH between 6.2-6.8. With the aid of a novel lymph node window chamber technique we were able to both image and measure the in vivo pH of a murine lymph node. This novel technique granted us the unique opportunity to acquire enough data to accurately map the pH of the lymph node. These IVI images together with pHLIP immunostaining showed that this acidity was restricted to subcortical regions known to contain antigen presenting dendritic cells and lymphocytes. Because this acidity was so extreme and was not affected by NaHCO3, we hypothesize that its maintenance must be an active process. Collective these data demonstrate that murine lymph nodes sustain an acidic pH under altering physiologic conditions. We hypothesize that cancer cells may be adopting the normal physiological conditions found in the lymph nodes to escape immune surveillance. Citation Format: Veronica Estrella, Pawel Swietach, Kim Luddy, Dominique Abrahams, Pedro Enriquez-Navas, Yana Reshetnyak, Mehdi Damaghi, Robert J. Gillies. A new twist on an old strategy: Can the lymph node environment help cancers escape immune surveillance [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 1140.

Mitochondrial Mechanisms Underlying Tolerance to Fluctuating Oxygen Conditions: Lessons from Hypoxia-Tolerant Organisms.

Oxygen (O2) is essential for most metazoan life due to its central role in mitochondrial oxidative phosphorylation (OXPHOS), which generates >90% of the cellular adenosine triphosphate. O2 fluctuations are an ultimate mitochondrial stressor resulting in mitochondrial damage, energy deficiency, and cell death. This work provides an overview of the known and putative mechanisms involved in mitochondrial tolerance to fluctuating O2 conditions in hypoxia-tolerant organisms including aquatic and terrestrial vertebrates and invertebrates. Mechanisms of regulation of the mitochondrial OXPHOS and electron transport system (ETS) (including alternative oxidases), sulphide tolerance, regulation of redox status and mitochondrial quality control, and the potential role of hypoxia-inducible factor (HIF) in mitochondrial tolerance to hypoxia are discussed. Mitochondrial phenotypes of distantly related animal species reveal common features including conservation and/or anticipatory upregulation of ETS capacity, suppression of reactive oxygen species (ROS)-producing electron flux through ubiquinone, reversible suppression of OXPHOS activity, and investment into the mitochondrial quality control mechanisms. Despite the putative importance of oxidative stress in adaptations to hypoxia, establishing the link between hypoxia tolerance and mitochondrial redox mechanisms is complicated by the difficulties of establishing the species-specific concentration thresholds above which the damaging effects of ROS outweigh their potentially adaptive signaling function. The key gaps in our knowledge about the potential mechanisms of mitochondrial tolerance to hypoxia include regulation of mitochondrial biogenesis and fusion/fission dynamics, and HIF-dependent metabolic regulation that require further investigation in hypoxia-tolerant species. Future physiological, molecular and genetic studies of mitochondrial responses to hypoxia, and reoxygenation in phylogenetically diverse hypoxia-tolerant species could reveal novel solutions to the ubiquitous and metabolically severe problem of O2 deficiency and would have important implications for understanding the evolution of hypoxia tolerance and the potential mitigation of pathological states caused by O2 fluctuations.

Engineering Adenylate Cyclase Activated by Near-Infrared Window Light for Mammalian Optogenetic Applications.

Light in the near-infrared optical window (NIRW) penetrates deep through mammalian tissues, including the skull and brain tissue. Here we engineered an adenylate cyclase (AC) activated by NIRW light (NIRW-AC) and suitable for mammalian applications. To accomplish this goal, we constructed fusions of several bacteriophytochrome photosensory and bacterial AC modules using guidelines for designing chimeric homodimeric bacteriophytochromes. One engineered NIRW-AC, designated IlaM5, has significantly higher activity at 37 °C, is better expressed in mammalian cells, and can mediate cAMP-dependent photoactivation of gene expression in mammalian cells, in favorable contrast to the NIRW-ACs engineered earlier. The ilaM5 gene expressed from an AAV vector was delivered into the ventral basal thalamus region of the mouse brain, resulting in the light-controlled suppression of the cAMP-dependent wave pattern of the sleeping brain known as spindle oscillations. Reversible spindle oscillation suppression was observed in sleeping mice exposed to light from an external light source. This study confirms the robustness of principles of homodimeric bacteriophytochrome engineering, describes a NIRW-AC suitable for mammalian optogenetic applications, and demonstrates the feasibility of controlling brain activity via NIRW-ACs using transcranial irradiation.

Differential posttranslational modification of mitochondrial enzymes corresponds with metabolic suppression during hibernation.

During hibernation, small mammals, including the 13-lined ground squirrel (Ictidomys tridecemlineatus), cycle between two distinct metabolic states: torpor, where metabolic rate is suppressed by >95% and body temperature falls to ~5°C, and interbout euthermia (IBE), where both metabolic rate and body temperature rapidly increase to euthermic levels. Suppression of whole animal metabolism during torpor is paralleled by rapid, reversible suppression of mitochondrial respiration. We hypothesized that these changes in mitochondrial metabolism are regulated by posttranslational modifications to mitochondrial proteins. Differential two-dimensional gel electrophoresis and two-dimensional blue-native PAGE revealed differences in the isoelectric point of several liver mitochondrial proteins between torpor and IBE. Quadrupole time-of-flight LC/MS and matrix-assisted laser desorption/ionization MS identified these as proteins involved in β-oxidation, the tricarboxylic acid cycle, reactive oxygen species detoxification, and the electron transport system (ETS). Immunoblots revealed that subunit 1 of ETS complex IV was acetylated during torpor but not IBE. Phosphoprotein staining revealed significantly greater phosphorylation of succinyl-CoA ligase and the flavoprotein subunit of ETS complex II in IBE than torpor. In addition, the 75-kDa subunit of ETS complex I was 1.5-fold more phosphorylated in torpor. In vitro treatment with alkaline phosphatase increased the maximal activity of complex I from liver mitochondria isolated from torpid, but not IBE, animals. By contrast, phosphatase treatment decreased complex II activity in IBE but not torpor. These findings suggest that the rapid changes in mitochondrial metabolism in hibernators are mediated by posttranslational modifications of key metabolic enzymes, perhaps by intramitochondrial kinases and deacetylases.