Biology subjects

Chang, J. W.

Publications and source records attributed to Chang, J. W..

3 recordsLinked to original sources

bioRxiv2021

Non-conserved metabolic regulation by LKB1 distinguishes human and mouse lung adenocarcinoma

KRAS is the most frequently mutated oncogene in human lung adenocarcinomas (hLUAD) and activating mutations in KRAS frequently co-occur with loss-of-function mutations in the tumor suppressor genes, TP53 or STK11/LKB1. However, mutation of all three genes is rarely observed in hLUAD, even though engineered mutations of all three genes produces a highly aggressive lung adenocarcinoma in mice (mLUAD). Here we provide an explanation of this difference between hLUAD and mLUAD by uncovering an evolutionary divergence in regulation of the glycolytic enzyme triosephosphate isomerase (TPI1). Using KRAS/TP53 mutant hLUAD cell lines, we show that TPI1 enzymatic activity can be altered via phosphorylation at Ser21 by the Salt Inducible Kinases (SIKs) in an LKB1-dependent manner; this allows modulation of glycolytic flux between completion of glycolysis and production of glycerol lipids. This metabolic flexibility appears to be critical in rapidly growing cells with KRAS and TP53 mutations, explaining why loss of LKB1 creates a metabolic liability in these tumors. In mice, the amino acid at position 21 of TPI1 is a Cys residue which can be oxidized to alter TPI1 activity, allowing regulation of glycolytic flux balance without a need for SIK kinases or LKB1. Our findings reveal an unexpected role for TPI1 in metabolic reprogramming and suggest that LKB1 and SIK family kinases are potential targets for treating KRAS/TP53 mutant hLUAD. Our data also provide a cautionary example of the limits of genetically engineered murine models as tools to study human diseases such as cancers.

cancer biology↗

bioRxiv2021

Non-Viral Engineering of CAR-NK and CAR-T cells using the Tc Buster Transposon System™

Cancer immunotherapy using T cells and NK cells modified with viral vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in treating hematological malignancies in clinical trials. However, viral vectors are limited in their cargo size capacity, and large-scale manufacturing for clinical use remains complex and cost prohibitive. As an alternative, CAR delivery via DNA transposon engineering is a superior and cost-effective production method. Engineering via transposition is accomplished using a two-component system: a plasmid containing a gene expression cassette flanked by transposon inverted terminal repeats (ITRs) paired with a transposase enzyme that binds to the ITRs, excises the transposon from the plasmid, and stably integrates the transposon into the genome. Here, we used the newly developed hyperactive Tc Buster (Bio-Techne) transposon system to deliver a transposon containing a multicistronic expression cassette (CD19-CAR, mutant DHFR, and EGFP) to primary human peripheral blood (PB) NK cells and T cells. We optimized methods to avoid DNA toxicity and maximize efficiency. Our cargo contained a mutant dihydrofolate reductase (DHFR) which allowed us to enrich for stable transposon integration using methotrexate (MTX) selection. We then tested CAR-NK and CAR-T cells in functional assays against CD19-expressing Raji cells. CAR-expressing NK and T cells produced significantly more cytokines than CAR-negative controls and efficiently killed target cells. We recognize that cryopreservation manufactured CAR-expressing cells will be necessary for clinical translation. We observed reduced cytotoxicity of CAR-NK cells immediately after thaw, but increasing the NK dose overcame this loss of function. Our work provides a platform for robust delivery of multicistronic, large cargo via transposition to primary human NK and T cells. We demonstrate that CAR-expressing cells can be enriched using MTX selection, while maintaining high viability and function. This non-viral approach represents a versatile, safe, and cost-effective option for the manufacture of CAR-NK and CAR-T cells compared to viral delivery.

bioengineering↗

bioRxiv2021

Human Astrocytes Exhibit Tumor Microenvironment-, Age-, and Sex-Related Transcriptomic Signatures

Astrocytes are dynamic cells with important roles in brain function and neurological disease. There are notable species differences between human astrocytes and commonly used animal models. However, changes of the molecular attributes of human astrocytes across disease states, sex, and age are largely unknown, which is a barrier in understanding human astrocyte biology and its potential involvement in neurological diseases. To better understand the properties of human astrocytes, we acutely purified astrocytes from the cerebral cortices of over 40 humans across various ages, sexes, and disease states. We performed RNA sequencing to generate transcriptomic profiles of these astrocytes and identified genes associated with these biological variables. Here, we identified a robust transcriptomic signature of human astrocytes in the tumor-surrounding microenvironment, including upregulation of proliferation processes, along with downregulation of genes involved in ionic homeostasis and synaptic function, suggesting involvement of peri-tumor astrocytes in tumor-associated neural circuit dysfunction. In aging, we also found downregulation of synaptic regulators and upregulation of markers of astrocyte reactivity, while in maturation we identified changes in ionic transport with implications for calcium signaling. In addition, we identified some of the first transcriptomic evidence of sexual dimorphism in human cortical astrocytes, which has implications for observed sex differences across many neurological disorders. Overall, genes involved in synaptic function exhibited dynamic changes in multiple conditions. This data provides powerful new insights into human astrocyte biology in several biologically relevant states, that will aid in generating novel testable hypotheses about homeostatic and reactive astrocytes in humans. Significance StatementAstrocytes are an abundant class of cells playing integral roles in the central nervous system. Astrocyte dysfunction is implicated in a variety of human neurological diseases. Yet our knowledge of astrocytes is largely based on mouse studies. Direct knowledge of human astrocyte biology remains limited. Here, we present transcriptomic profiles of human cortical astrocytes, and we identified molecular differences associated with age, sex, and disease state. We found changes suggesting involvement of peritumor astrocytes in tumor-associated neural circuit dysfunction, aging-associated decline in astrocyte-synapse interactions, ionic transport changes with brain maturation, and some of the first evidence of sexual dimorphism in human astrocytes. These data provide necessary insight into human astrocyte biology that will improve our understanding of human disease.

neuroscience↗