Research Publications
Research – bioRxiv – PIC recruitment by synthetic reader-actuators to polycomb-silenced genes blocks triple-negative breast cancer invasion
PIC recruitment by synthetic reader-actuators to polycomb-silenced genes blocks triple-negative breast cancer invasion
Williams NL, Hong L, Jaffe M, Shields CE, Haynes KA. (2023) bioRxiv. https://www.biorxiv.org/content/10.1101/2023.01.23.525196v1
An exciting new approach for cancer therapy is to turn on genes within the cancer cells that can stop them from growing and spreading. This approach, called epigenetic therapy, uses small molecules (inhibitors) to block chromatin-modifying proteins that play a role in silencing anti-cancer genes. However, this approach has shown disappointing results in clinical trials for solid cancers, perhaps due to biological limitations. For example, inhibitors can accidentally activate the proteins they are supposed to block (e.g. EZH inhibitors promote EZH2/FOXM1 complexes, Mahara et al 2016), and inhibitors can’t turn on important gene-regulating proteins that are damaged in many cancers. To overcome these limitations we have developed a new tool, synthetic reader-actuators (SRAs), that directly targets chromatin-silenced regions, activates the pre-initiation complex (PIC), and turns on gene transcription. In this report we tested SRAs in triple negative breast cancer cells (BT-549) and identified 122 activated genes. SRA-expressing BT-549 cells showed reduced spheroid size over time, loss of invasion, and activation of apoptosis. While epigenetic drugs have not been successful in many clinical trials, by using synthetic proteins we showed that robust epigenetic reprogramming is possible in cells from solid cancers.
Research – Frontiers – Adipocyte-mediated Epigenomic Instability in Human T-ALL cells is Cytotoxic and Phenocopied by Epigenetic Modifying Drugs
Adipocyte-mediated Epigenomic Instability in Human T-ALL cells is Cytotoxic and Phenocopied by Epigenetic Modifying Drugs
Lee M, Geitgey D, Hamilton JAG, Boss J, Scharer CD, Haynes KA, Henry CJ. (2022) Front Cell Dev Biol.
PMID: 36060800 | PMCID: PMC9438935
An alarming trend in public health is the increase in obesity, with a projection of 1.5 billion people with obesity by 2030. This trend is expected to impact personalized medicine because the progression and treatment of cell development-related diseases, such as cancer, differ in patients with obesity versus without. On one hand, acute myeloid leukemia (AML) and B-cell acute lymphoblastic leukemia (B-ALL) are positively associated with obesity. On the other hand, epidemiological studies show the the opposite for patients with T-cell acute lymphoblastic leukemia (T-ALL). To investigate the negative relationship between obesity and T-ALL development, we analyzed T-ALL cells from the diet-induced obese (DIO) mice and human T-ALL cells cultured in adipocyte-conditioned media (ACM). Lean mice transplanted with T-ALL cells showed worse survival (~20%) than obese mice (~50-80%). ACM-treated human T-ALL cells showed accelerated cell cycle progression, DNA damage, and cell death. We determined epigenetic changes that accompanied these changes in cell behavior. Transcription of genes involved in cellular responses to stimuli, cell cycle, DNA replication and repair, metabolism of RNA, and transcription were elevated. Acetylation and methylation marks on histone 3 were elevated when human T-ALL cells were cultured in ACM, indicating a loss of histone demethylase and deacetylase activity or gain of histone acetyltransferase activity. Treatment of T-ALL cells with inhibitors of histone demethylase and histone deacetylase reproduced the changes in chromatin and cell viability observed for ACM treatment. Our data support epidemiological studies demonstrating that adiposity suppresses T-ALL pathogenesis, and suggest a pharmacological option (epigenetic drug treatment) may benefit lean patients with T-ALL.
Research – Scientific Reports – B-cell acute lymphoblastic leukemia promotes an immune suppressive microenvironment that can be overcome by IL-12
B-cell acute lymphoblastic leukemia promotes an immune suppressive microenvironment that can be overcome by IL-12
Hunter R, Imbach K, Dougan J, Do P, Townsel A, Zhou C, Gibson G, Dreaden E, Haynes K, Henry CJ, Porter CC. (2022) Sci Reports.
PMID: 35831470 | PMCID: PMC9279427
Immunotherapy is an exciting and important new approach for cancer treatment. As with any emerging cancer therapy, a critical challenge is to optimize the therapy to be effective for an array of diverse cancer cell types, i.e. blood cancer versus solid tumors and cancer cells from diverse organs, and the chemically diverse spaces where they reside within the body (microenvironments). The duration of responses of B-cell acute lymphoblastic leukemia (B-ALL) to immunotherapy remains sub-optimal. To find out what it is that blocks immunotherapy in patients with B-ALL, we analyzed plasma collected from children with B-ALL and found decreased cytokine levels (particularly IL-7), increased expression from immune exhaustion genes, and factors that suppressed T-cell function. To determine how to restore the immune response, we used a B-ALL mouse model to show that treatment with IL-12 proteins restored the levels of several pro-inflammatory cytokines and chemokines, increased the number of splenic and bone marrow resident T-cells and dendritic cells, and blocked the activation of immune exhaustion genes. These results demonstrate that treatment with IL-12 proteins can be used to alter the leukemia microenvironment so that immunotherapy can be more effective.
Research – RETM – Differential epigenetic effects of BMI1 inhibitor PTC-028 on fusion-positive rhabdomyosarcoma cell lines from distinct metastatic sites
Differential epigenetic effects of BMI1 inhibitor PTC-028 on fusion-positive rhabdomyosarcoma cell lines from distinct metastatic sites
Shields C, Schnepp RW, Haynes KA. (2022) Regen Eng Transl Med. https://doi.org/10.1007/s40883-021-00244-9
Previously, we reported in “Epigenetic regulator BMI1 promotes alveolar rhabdomyosarcoma proliferation and constitutes a novel therapeutic target” that genetic and pharmacologic inhibition of BMI1 reduces the viability of alveolar rhabdomyosarcoma (ARMS) cells, suggesting a new treatment option for patients with the rare pediatric cancer fusion-positive rhabdomyosarcoma (FP-RMS). In a follow-on study, we identified gene expression changes that underlie the loss of cell viability and proliferation, and addressed some open questions about the affect of BMI1 inhibition on ARMS. For instance, in our previous study, BMI1 inhibition affected phosphorylation of LATS1/2, but BMI1 is a transcriptional regulator, not a kinase or phosphatase. In the new study, through RNA-sequencing we found that the mRNA levels of two kinases EPHA2 and PDGFRA were affected during BMI1 inhibition, revealing a possible gene regulation link between BMI1 and kinases that target LATS1/2. RNA-seq also revealed transcription profile differences between ARMS cells derived from two different sites (metastases to an axillary lymph node and bone marrow). Despite this heterogeneity, epigenetic intervention (BMI1 inhibition) induces an anti-cancer response.
Research – bioRxiv – Targeted regulation of episomal plasmid DNA expression in eukaryotic cells with a methylated-DNA-binding activator
Targeted regulation of episomal plasmid DNA expression in eukaryotic cells with a methylated-DNA-binding activator
Enwerem-Lackland I, Warga E, Dugoni M, Elmer J, Haynes KA. (2021) bioRxiv. https://www.biorxiv.org/content/10.1101/2021.11.01.466616v1
This work was inspired by our team’s interest in making DNA delivery into human cells easier and more more reliable. Currently, synthetic viruses are the most efficient method, but only up to ~1000 bp of DNA can be reliably packaged in these vectors, and current DNA-delivering viruses can cause dangerous and lethal immunogenic reactions. Non-viral plasmid DNA may be a safer alternative, but delivery and expression tends to be poor and unreliable. Our work and research from other labs has implicated epigenetic blockades as one culprit. Therefore, we developed a small protein, inspired by work from the van Steensel lab and Khalil lab, that carries a gene expression activator to newly-delivered methylated plasmid DNA to boost its expression. The “landing pad” for the activator is the smallest of its kind (4 base pair unit, GAmeTC), and therefore should be easy to use in any plasmid DNA. We named this system The Dpn Adaptor Linked Effector (DAL-E) to honor the pioneering biochemist Marie M. Daly, the first scientist to determine that a central epigenetic protein (histone) is lysine-rich, and the first black woman to earn a PhD in Chemistry in the U.S.
Research – Molecular Oncology – Epigenetic regulator BMI1 promotes alveolar rhabdomyosarcoma proliferation and constitutes a novel therapeutic target
Epigenetic regulator BMI1 promotes alveolar rhabdomyosarcoma proliferation and constitutes a novel therapeutic target
Shields CE, Potlapalli S, Cuya-Smith SM, Chappell SK, Chen D, Martinez D, Pogoriler J, Rathi KS, Patel SA, Oristian KM, Linardic CM, Maris JM, Haynes KA, Schnepp RW. (2021) Molecular Oncology.
PMID: 33523558
Abnormal expression and behavior of chromatin proteins occurs in many types of cancer, so scientists have investigated these proteins as possible drug targets. Treatment of alveolar rhabdomyosarcoma (ARMS) remains a major therapeutic challenge in pediatric oncology. In this collaboration of the Schnepp and Haynes labs, we demonstrate that genetic and pharmacologic inhibition of BMI1 reduces ARMS viability. We show that BMI1 inhibits the tumor suppressive Hippo pathway and, conversely, that BMI1 disruption upregulates Hippo signaling. Collectively, these findings provide an initial framework for targeting BMI1 in ARMS and additional sarcomas.
Research – bioRxiv – Delivery of cell-penetrating chromatin sensor-actuators to human osteosarcoma cells
Delivery of cell-penetrating chromatin sensor-actuators to human osteosarcoma cells
Tekel SJ, Brookhouser N, Haynes KA. (2020) bioRxiv. https://doi.org/10.1101/2020.02.28.969907
Recent research has revealed that a key vulnerability in hard-to-treat cancer cells (e.g. triple negative breast cancer) may be chromatin, a system of proteins and nucleic acids that controls chromosome organization and gene expression. Small molecule drugs called epigenetic inhibitors can easily get into the nucleus to disrupt chromatin in cancer, but these molecules do not carry enough biological information to direct specific changes in gene expression. This unmet need inspired us to build artificial transcription factors that could be delivered to cells in a similar manner as soluble drugs, bind to aberrant chromatin, and induce gene activation. In this report, we describe how the addition of short cell-penetrating signal to the chromatin sensor-actuator PcTF enabled 100% uptake in cultured cells (monolayers), and up to 50% uptake by cells grown as spheroids (used to represent tumors). For gene activation, these cell-penetrating PcTF proteins did not appear to be as effective as the PcTF protein we had expressed from synthetic DNA in past experiments. Further technical development is needed to deliver functional PcTF regulators into cancer cell nuclei.
Research – IJMS – Components from the human c-myb transcriptional regulation system reactivate epigenetically repressed transgenes
Components from the human c-myb transcriptional regulation system reactivate epigenetically repressed transgenes
Barrett CM, McCracken R, Elmer J, Haynes KA. (2020) Int. J. Mol. Sci. 21: 530.
PMID: 31947658 | PMCID: PMC7014047
This work was inspired by a problem faced by many bioengineers who are interested in adding new, synthetic genes to human cells to fight disease, produce therapeutic proteins, and regenerate tissues and organs. New genetic material that is delivered into cells generally starts out working quite well: it is transcribed and translated like, or even better than, a “normal” gene. But eventually the synthetic gene is often shut down (silenced) by a DNA packaging and regulation system called chromatin. In our paper, we report the results of a project where we tested different DNA and protein components for their ability to protect synthetic genes from silencing. We use a priori knowledge of chromatin features at the synthetic gene to understand the mechanism of reactivation.
Related:
- Pre-print: Use of MYB as a new synthetic activator to enhance transgene expression from within repressed Polycomb chromatin. Barrett CM, McCracken R, Elmer J, Haynes KA. (2018) bioRxiv. https://doi.org/10.1101/487736
Research – APL BioE – Site-directed targeting of transcriptional activation-associated proteins to repressed chromatin restores CRISPR activity
Site-directed targeting of transcriptional activation-associated proteins to repressed chromatin restores CRISPR activity
Daer R, Hamna F, Barrett CM, and Haynes KA. (2020) APL Bioengineering. 4: 016102.
PMID: 31967103 | PMCID: PMC6960031
CRISPR is a powerful and popular tool for editing DNA in living cells. Scientists are becoming more interested in using CRISPR to correct mistakes in DNA that lead to diseases, to artificially generate mutations to research the origins of diseases, and for other important applications. However, CRISPR originated in bacteria and has probably not evolved to function very well in genomes that are packed in configurations (open and closed chromatin) as complex as those found in human cells. In a recent report (Daer et al. 2017), we demonstrated that CRISPR activity was inhibited at a DNA sequence that became artificially condensed into closed chromatin. Our new study shows that targeted re-opening of closed chromatin leads to enhanced CRISPR activity in the same region. The epigenetic drug we tested (UNC1999) was not sufficient to generate a transcriptionally active or CRISPR-accessible state. In contrast, strong direct transcriptional activation of the target gene with a DNA-binding p65 protein did enhance CRISPR accessibility. We also identified four other fusion proteins that did not activate transcription but still enhanced CRISPR efficiency, showing that CRISPR can be improved without activating expression of the editing target.
Related:
- Histone modifications and active gene expression are associated with enhanced CRISPR activity in de-silenced chromatin. Daer R, Barrett C, Haynes KA. (2017) bioRxiv. https://www.biorxiv.org/content/early/2018/03/11/228601
- Press release – Opening up DNA to delete disease: Custom-built molecules enable editing of genes previously obscured by DNA’s innately protective structure. EurekAlert! https://www.eurekalert.org/pub_releases/2020-01/aiop-oud011320.php
Research – Frontiers – Engineered orthogonal quorum sensing systems for synthetic gene regulation
Engineered orthogonal quorum sensing systems for synthetic gene regulation
Tekel SJ, Smith CL, Lopez B, Mani A, Connot C, Livingstone X, Haynes KA. (2019) Front Bioeng Biotech. 7:80.
PMID: 31058147 | PMCID: PMC6478669
For the past two years, the undergraduate ASU International Genetically Engineered Machines (iGEM) Competition teams have been investigating the use of quorum sensing to build bacteria (E. coli) that can carry out chemical signaling with each other, without signaling to a different group of bacteria. In a continuation of the quorum sensing project from the 2016 team, the 2017 ASU iGEM team built and tested six new quorum sensors (Receivers) in addition to the previous sensor, LuxR. After exposing a total of seven Receivers to HSLs from ten different synthases (Senders), the team identified two sets of signaling pathways that exhibited orthogonal behavior. These results expand the toolbox of characterized components for engineering microbial communities. The plasmid constructs featured in this paper were contributed to public collections for use by the scientific community.
Related:
- Pre-print: Engineered orthogonal quorum sensing systems for synthetic gene regulation. bioRxiv. doi: https://doi.org/10.1101/499681
- Arizona_State 2017 iGEM Team Project: “EVR-QST – Engineering Variable Regulators for a Quorum Sensing Toolbox.” http://2017.igem.org/Team:Arizona_State
Haynes Lab | Emory 