Transcriptional regulation is dependent upon the interactions between the RNA pol II holoenzyme complex and chromatin. RNA pol II is part of a highly conserved multiprotein complex that includes the core mediator and CDK8 subcomplex. In Saccharomyces cerevisiae, the CDK8 subcomplex, composed of Ssn2p, Ssn3p, Ssn8p, and Srb8p, is thought to play important roles in mediating transcriptional control of stress-responsive genes. Also central to transcriptional control are histone post-translational modifications. Lysine methylation, dynamically balanced by lysine methyltransferases and demethylases, has been intensively studied, uncovering significant functions in transcriptional control. A key question remains in understanding how these enzymes are targeted during stress response. To determine the relationship between lysine methylation, the CDK8 complex, and transcriptional control, we performed phenotype analyses of yeast lacking known lysine methyltransferases or demethylases in isolation or in tandem with SSN8 deletions. We show that the RNA pol II CDK8 submodule components SSN8/SSN3 and the histone demethylase JHD2 are required to inhibit pseudohyphal growth-a differentiation pathway induced during nutrient limitation-under rich conditions. Yeast lacking both SSN8 and JHD2 constitutively express FLO11, a major regulator of pseudohyphal growth. Interestingly, deleting known FLO11 activators including FLO8, MSS11, MFG1, TEC1, SNF1, KSS1, and GCN4 results in a range of phenotypic suppression. Using chromatin immunoprecipitation, we found that SSN8 inhibits H3 Lys4 trimethylation independently of JHD2 at the FLO11 locus, suggesting that H3 Lys4 hypermethylation is locking FLO11 into a transcriptionally active state. These studies implicate the CDK8 subcomplex in finetuning H3 Lys4 methylation levels during pseudohyphal differentiation.?
Meiosis-specific chromatin structures, guided by histone modifications, are critical mediators of a meiotic transient transcription program and progression through prophase I. Histone H3K4 can be methylated up to three times by the Set1-containing COMPASS complex and each methylation mark corresponds to a different chromatin conformation. The level of H3K4 modification is directed by the activity of additional COMPASS components. In this study, we characterized the role of the COMPASS subunits during meiosis in S. cerevisiae. In vegetative cells, previous studies revealed a role for subunits Swd2, Sdc1, and Bre2 for H3K4me2 while Spp1 supported trimethylation. However, we found that Bre2 and Sdc1 are required for H3K4me3 as yeast prepare to enter meiosis while Spp1 is not. Interestingly, we identified distinct meiotic functions for the core COMPASS complex members that required for all H3K4me, Set1, Swd1, and Swd3. While Set1 and Swd1 are required for progression through early meiosis, Swd3 is critical for late meiosis and spore morphogenesis. Furthermore, the meiotic requirement for Set1 is independent of H3K4 methylation, suggesting the presence of non-histone substrates. Finally, checkpoint suppression analyses indicate that Set1 and Swd1 are required for both homologous recombination and chromosome segregation. These data suggest that COMPASS has important new roles for meiosis that are independent of its well-characterized functions during mitotic divisions.
Background Despite significant success in treating hematological malignancies, adoptive cell therapies have yielded limited efficacy in solid tumors. 1 Macrophages are myeloid cells of the innate immune system and are naturally recruited to solid tumors, 2 where they have the potential to phagocytose tumor cells, activate the tumor microenvironment (TME), and prime a broad anti-tumor adaptive immune response via T cell recruitment and activation. We have previously developed chimeric antigen receptor macrophages (CAR-M) targeting HER2 and showed efficacy in a variety of pre-clinical models, 3 with a Phase I clinical trial ongoing. Mesothelin is overexpressed in a variety of solid tumors, including mesothelioma, lung, pancreatic, and ovarian cancers. 4 Here, we present preclinical data summarizing the development of CT-1119, a mesothelin targeted CAR-M for solid tumors. Methods Using the chimeric adenoviral vector Ad5f35, we engineered primary human macrophages to express a CAR comprising a human scFv targeted against human mesothelin. To assess the activity of CT-1119, in vitro cell based assays and in vivo murine xenograft models were utilized. Donormatched untransduced (UTD) macrophages served as controls.Results Primary human CAR-M engineered with an Ad5f35 vector demonstrated high CAR expression, high viability, upregulated M1 (anti-tumor) macrophage markers, and downregulated M2 (pro-tumor) macrophage markers. CT-1119 demonstrated increased resistance to repolarization by M2 (pro-tumor) polarizing cytokines as compared to donor matched UTD macrophages. CT-1119 specifically bound mesothelin and binding was not impacted by mesothelin shedding. CT-1119 specifically phagocytosed multiple mesothelin expressing tumor cell lines in a CAR-dependent and antigen-dependent manner. CT-1119 demonstrated robust in vitro killing of the relevant tumor cell lines A549 and MES-OV expressing mesothelin. CAR engagement also induced the release of proinflammatory cytokines such as TNFa following stimulation with mesothelin in both cell-free and cell-based contexts in a dose-dependent manner. In vivo, CT-1119 significantly reduced tumor burden in a murine xenograft model of lung cancer. Similarly, human monocytes targeting mesothelin were successfully generated using the same Ad5f35 vector and demonstrated specific activity against mesothelin positive tumor cells. Conclusions The presented results demonstrate that CT-1119, an autologous human anti-mesothelin CAR-M, can cause phagocytosis, tumor cell killing, and pro-inflammatory cytokine release in response to stimulation with mesothelin. These results show that CAR-M is a feasible approach for the treatment of mesothelin expressing sold tumors via the potential for induction of a systemic anti-tumor response.
Histones are proteins that can be post‐translationally modified and these modifications will determine the expression of genes. Many studies have shown that it is very common for cancers to have mutations in their histone genes. The concept of synthetic lethality states that if a cell has a single mutation it will be able to survive. But if a second mutation is applied, the cell will become synthetically sick and/or die. In our study, we are seeking to determine synthetically lethal interactions between the histone H3 gene and RPD3 histone deacetylase enzyme. In order to discover potential genetic targets to develop therapies against cancers. To achieve this we used a genetically engineered yeast strain that has the histone H3 gene and RPD3 gene deleted. The H3 gene deletion is covered by a wild type H3 plasmid. These yeast were then transformed with plasmids from the SHIMA library. The yeast were then grown on plates lacking uracil and tryptophan. The individual colonies were then patched and replica plated onto tryptophan deficient media. Finally, they were replica plated onto agar plate media containing 5‐ FOA. The DNA from yeast deemed synthetically lethal or sick were then put through “Library Prep” which is a high throughput method used to isolate the H3 gene within the plasmid with low error rate. The lethals were then put into Next Generation sequencing, aligned, and transferred in the IGV. Finally, we were able to estimate with high accuracy the frequency of H3 mutants in our lethal pool. The top three frequencies discovered were F67A, F84A, and Q93A. They represented 35%, 12% and 10% of our lethal pool respectively. In total we discovered ten new potential synthetically lethal interactions between histone H3 and RPD3. We also used this information in combination with previous knowledge to generate a genetic interaction map for RPD3 from our findings. Although still in the preliminary stages when verified with duplication studies our new potential synthetically lethal interactions can be targets to develop cancer therapies. Also, the knowledge gained from our study can be used to understand genetic interaction between histones and histone modification enzymes.Support or Funding InformationSummer Medical Research Fellowship at Rowan University School of Osteopathic MedicineThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
While adoptive cell therapies have seen significant success in the treatment of hematological malignancies, solid tumors remain challenging for the field. A significant obstacle is the exclusion of T cells from the tumor microenvironment (TME). In contrast, monocytes/macrophages are naturally recruited to the TME. These cells then have the potential to phagocytose tumor cells, activate the TME, and prime a broad anti-tumor adaptive immune response via T cell recruitment and activation. We have previously developed CT-0508, a chimeric antigen receptor macrophage (CAR-M) targeting HER2 which showed efficacy in a variety of pre-clinical models and is currently in a Phase I clinical trial for patients with HER2+ solid tumors. Mesothelin is overexpressed in a variety of solid tumors, including mesothelioma, lung, pancreatic, and ovarian cancers. To leverage tumor biology with myeloid cells, we engineered primary human macrophages using the chimeric adenoviral vector Ad5f35 to express a CAR containing a human scFv against human mesothelin. We used both in vitro cell based assays and in vivo xenograft models to assess the activity of CT-1119. CAR-M engineered with an Ad5f35 vector demonstrated high CAR expression, high viability, upregulated M1 (anti-tumor) macrophage markers, and downregulated M2 (pro-tumor) macrophage markers. CT-1119 specifically phagocytosed multiple mesothelin expressing tumor cell lines in a CAR-dependent and antigen-dependent manner. CT-1119 demonstrated robust in vitro killing of the relevant tumor cell lines A549 and MES-OV expressing mesothelin. CAR engagement also induced the release of pro-inflammatory cytokines such as TNFα following stimulation with mesothelin in both cell-free and cell-based contexts in a dose-dependent manner. In vivo, CT-1119 significantly reduced tumor burden in a murine xenograft model of lung cancer. Similarly, human monocytes targeting mesothelin were successfully generated using the same Ad5f35 vector and demonstrated specific activity against mesothelin positive tumor cells. The presented results demonstrate that CT-1119, an autologous human anti-mesothelin CAR-M, can cause phagocytosis, tumor cell killing, and pro-inflammatory cytokine release in response to stimulation with mesothelin. These results show that CAR-M is a feasible approach for the treatment of mesothelin expressing sold tumors via the potential for induction of a systemic anti-tumor response. Citation Format: Nicholas R. Anderson, Brinda Shah, Alison Worth, Rashid Gabbasov, Brett Menchel, Kerri Ciccaglione, Daniel Blumenthal, Stefano Pierini, Sabrina Ceeraz DeLong, Sascha Abramson, Thomas Condamine, Michael Klichinsky. A mesothelin targeting chimeric antigen receptor macrophage (CAR-M) for solid tumor immunotherapy: pre-clinical development of CT-1119. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4053.
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