Therapeutic targeting of LCK tyrosine kinase and mTOR signaling in T-cell acute lymphoblastic leukemia.

Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.

JARID2 and the PRC2 complex regulate the cell cycle in skeletal muscle.

JARID2 is a noncatalytic member of the polycomb repressive complex 2 (PRC2) which methylates of histone 3 lysine 27 (H3K27). In this work, we show that JARID2 and the PRC2 complex regulate the cell cycle in skeletal muscle cells to control proliferation and mitotic exit. We found that the stable depletion of JARID2 leads to increased proliferation and cell accumulation in S phase. The regulation of the cell cycle by JARID2 is mediated by direct repression of both cyclin D1 and cyclin E1, both of which are targets of PRC2-mediated H3K27 methylation. Intriguingly, we also find that the retinoblastoma protein (RB1) is a direct target of JARID2 and the PRC2 complex. The depletion of JARID2 is not sufficient to activate RB1. However, the ectopic expression of RB1 can suppress cyclin D1 expression in JARID2-depleted cells. Transient depletion of JARID2 in skeletal muscle cells leads to a transient up-regulation of cyclin D1 that is quickly suppressed with no resulting effect on proliferation, Taken together, we show that JARID2 and the PRC2 complex regulate skeletal muscle proliferation in a precise manner that involves the repression of cyclin D1, thus restraining proliferation and repressing RB1, which is required for mitotic exit and terminal differentiation.

Integrated Constraint-Based Modeling of E. coli Cell-Free Protein Synthesis.

Cell-free protein expression has become a widely used research tool in systems and synthetic biology and a promising technology for protein biomanufacturing. Cell-free protein synthesis relies on in-vitro transcription and translation processes to produce a protein of interest. However, transcription and translation depend upon the operation of complex metabolic pathways for precursor and energy regeneration. Toward understanding the role of metabolism in a cell-free system, we developed a dynamic constraint-based simulation of protein production in the myTXTL E. coli cell-free system with and without electron transport chain inhibitors. Time-resolved absolute metabolite measurements for â"³ = 63 metabolites, along with absolute concentration measurements of the mRNA and protein abundance and measurements of enzyme activity, were integrated with kinetic and enzyme abundance information to simulate the time evolution of metabolic flux and protein production with and without inhibitors. The metabolic flux distribution estimated by the model, along with the experimental metabolite and enzyme activity data, suggested that the myTXTL cell-free system has an active central carbon metabolism with glutamate powering the TCA cycle. Further, the electron transport chain inhibitor studies suggested the presence of oxidative phosphorylation activity in the myTXTL cell-free system; the oxidative phosphorylation inhibitors provided biochemical evidence that myTXTL relied, at least partially, on oxidative phosphorylation to generate the energy required to sustain transcription and translation for a 16-hour batch reaction.

Endothelial cells metabolically regulate breast cancer invasion toward a microvessel.

Breast cancer metastasis is initiated by invasion of tumor cells into the collagen type I-rich stroma to reach adjacent blood vessels. Prior work has identified that metabolic plasticity is a key requirement of tumor cell invasion into collagen. However, it remains largely unclear how blood vessels affect this relationship. Here, we developed a microfluidic platform to analyze how tumor cells invade collagen in the presence and absence of a microvascular channel. We demonstrate that endothelial cells secrete pro-migratory factors that direct tumor cell invasion toward the microvessel. Analysis of tumor cell metabolism using metabolic imaging, metabolomics, and computational flux balance analysis revealed that these changes are accompanied by increased rates of glycolysis and oxygen consumption caused by broad alterations of glucose metabolism. Indeed, restricting glucose availability decreased endothelial cell-induced tumor cell invasion. Our results suggest that endothelial cells promote tumor invasion into the stroma due, in part, to reprogramming tumor cell metabolism.

Constructing Cell-Free Expression Systems for Low-Cost Access.

Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203-424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.

Single-cell analysis and functional characterization uncover the stem cell hierarchies and developmental origins of rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a common childhood cancer that shares features with developing skeletal muscle. Yet, the conservation of cellular hierarchy with human muscle development and the identification of molecularly defined tumor-propagating cells has not been reported. Using single-cell RNA-sequencing, DNA-barcode cell fate mapping and functional stem cell assays, we uncovered shared tumor cell hierarchies in RMS and human muscle development. We also identified common developmental stages at which tumor cells become arrested. Fusion-negative RMS cells resemble early myogenic cells found in embryonic and fetal development, while fusion-positive RMS cells express a highly specific gene program found in muscle cells transiting from embryonic to fetal development at 7-7.75 weeks of age. Fusion-positive RMS cells also have neural pathway-enriched states, suggesting less-rigid adherence to muscle-lineage hierarchies. Finally, we identified a molecularly defined tumor-propagating subpopulation in fusion-negative RMS that shares remarkable similarity to bi-potent, muscle mesenchyme progenitors that can make both muscle and osteogenic cells.

Myogenin is required for assembly of the transcription machinery on muscle genes during skeletal muscle differentiation.

Skeletal muscle gene expression is governed by the myogenic regulatory family (MRF) which includes MyoD (MYOD1) and myogenin (MYOG). MYOD1 and MYOG are known to regulate an overlapping set of muscle genes, but MYOD1 cannot compensate for the absence of MYOG in vivo. In vitro, late muscle genes have been shown to be bound by both factors, but require MYOG for activation. The molecular basis for this requirement was unclear. We show here that MYOG is required for the recruitment of TBP and RNAPII to muscle gene promoters, indicating that MYOG is essential in assembling the transcription machinery. Genes regulated by MYOD1 and MYOG include genes required for muscle fusion, myomaker and myomerger, and we show that myomaker is fully dependent on activation by MYOG. We also sought to determine the role of MYOD1 in MYOG dependent gene activation and unexpectedly found that MYOG is required to maintain Myod1 expression. However, we also found that exogenous MYOD1 was unable to compensate for the loss of Myog and activate muscle gene expression. Thus, our results show that MYOD1 and MYOG act in a feed forward loop to maintain each other's expression and also show that it is MYOG, and not MYOD1, that is required to load TBP and activate gene expression on late muscle gene promoters bound by both factors.

Proteasomal Regulation of Mammalian SPT16 in Controlling Transcription.

FACT (facilitates chromatin transcription), an essential and evolutionarily conserved heterodimer from yeast to humans, controls transcription and is found to be upregulated in various cancers. However, the basis for such upregulation is not clearly understood. Our recent results deciphering a new ubiquitin-proteasome system regulation of the FACT subunit SPT16 in orchestrating transcription in yeast hint at the involvement of the proteasome in controlling FACT in humans, with a link to cancer. To test this, we carried out experiments in human embryonic kidney (HEK293) cells, which revealed that human SPT16 undergoes ubiquitylation and that its abundance is increased following inhibition of the proteolytic activity of the proteasome, thus implying proteasomal regulation of human SPT16. Furthermore, we find that the increased abundance/expression of SPT16 in HEK293 cells alters the transcription of genes, including ones associated with cancer, and that the proteasomal degradation of SPT16 is impaired in kidney cancer (Caki-2) cells to upregulate SPT16. Like human SPT16, murine SPT16 in C2C12 cells also undergoes ubiquitylation and proteasomal degradation to regulate transcription. Collectively, our results reveal a proteasomal regulation of mammalian SPT16, with physiological relevance in controlling transcription, and implicate such proteasomal control in the upregulation of SPT16 in cancer.

The PRC2 complex directly regulates the cell cycle and controls proliferation in skeletal muscle.

The polycomb repressive complex 2 (PRC2) is an important developmental regulator responsible for the methylation of histone 3 lysine 27 (H3K27). Here, we show that the PRC2 complex regulates the cell cycle in skeletal muscle cells to control proliferation and mitotic exit. Depletions of the catalytic subunit of the PRC2 complex, EZH2, have shown that EZH2 is required for cell viability, suggesting that EZH2 promotes proliferation. We found that EZH2 directly represses both positive and negative cell cycle genes, thus enabling the PRC2 complex to tightly control the cell cycle. We show that modest inhibition or depletion of EZH2 leads to enhanced proliferation and an accumulation of cells in S phase. This effect is mediated by direct repression of cyclin D1 (Ccnd1) and cyclin E1 (Ccne1) by the PRC2 complex. Our results show that PRC2 has pleiotropic effects on proliferation as it serves to restrain cell growth, yet clearly has a function required for cell viability as well. Intriguingly, we also find that the retinoblastoma protein gene (Rb1) is a direct target of the PRC2 complex. However, modest depletion of EZH2 is not sufficient to maintain Rb1 expression, indicating that the PRC2 dependent upregulation of cyclin D1 is sufficient to inhibit Rb1 expression. Taken together, our results show that the PRC2 complex regulates skeletal muscle proliferation in a complex manner that involves the repression of Ccnd1 and Ccne1, thus restraining proliferation, and the repression of Rb1, which is required for mitotic exit and terminal differentiation.

The transcription elongation factor TCEA3 induces apoptosis in rhabdomyosarcoma.

TCEA3 is one of three genes representing the transcription elongation factor TFIIS family in vertebrates. TCEA3 is upregulated during skeletal muscle differentiation and acts to promote muscle specific gene expression during myogenesis. Rhabdomyosarcoma (RMS) is a pediatric cancer derived from the muscle lineage, but the expression or function of TCEA3 in RMS was uncharacterized. We found that TCEA3 expression was strongly inhibited in RMS cell lines representing both ERMS and ARMS subtypes of RMS. TCEA3 expression correlates with DNA methylation and we show that TBX2 is also involved in the repression of TCEA3 in RMS cell lines. Ectopic expression of TCEA3 inhibited proliferation of RMS cell lines and initiated apoptosis through both the intrinsic and extrinsic pathways. We found that only pan-caspase inhibitors could block apoptosis in the presence of TCEA3. While expression of TCEA3 is highest in skeletal muscle, expression has been detected in other tissues as well, including breast, ovarian and prostate. We found that ectopic expression of TCEA3 also promotes apoptosis in HeLa, MCF7, MDA-231, and PC3 cell lines, representing cervical, breast, and prostate cancer, respectively. Restoration of TCEA3 expression in RMS cell lines enhanced sensitivity to chemotherapeutic drugs, including TRAIL. Thus, TCEA3 presents a novel target for therapeutic strategies to promote apoptosis and enhance sensitivity to current chemotherapeutic drugs.

Effective Biophysical Modeling of Cell Free Transcription and Translation Processes.

Transcription and translation are at the heart of metabolism and signal transduction. In this study, we developed an effective biophysical modeling approach to simulate transcription and translation processes. The model, composed of coupled ordinary differential equations, was tested by comparing simulations of two cell free synthetic circuits with experimental measurements generated in this study. First, we considered a simple circuit in which sigma factor 70 induced the expression of green fluorescent protein. This relatively simple case was then followed by a more complex negative feedback circuit in which two control genes were coupled to the expression of a third reporter gene, green fluorescent protein. Many of the model parameters were estimated from previous biophysical studies in the literature, while the remaining unknown model parameters for each circuit were estimated by minimizing the difference between model simulations and messenger RNA (mRNA) and protein measurements generated in this study. In particular, either parameter estimates from published studies were used directly, or characteristic values found in the literature were used to establish feasible ranges for the parameter estimation problem. In order to perform a detailed analysis of the influence of individual model parameters on the expression dynamics of each circuit, global sensitivity analysis was used. Taken together, the effective biophysical modeling approach captured the expression dynamics, including the transcription dynamics, for the two synthetic cell free circuits. While, we considered only two circuits here, this approach could potentially be extended to simulate other genetic circuits in both cell free and whole cell biomolecular applications as the equations governing the regulatory control functions are modular and easily modifiable. The model code, parameters, and analysis scripts are available for download under an MIT software license from the Varnerlab GitHub repository.

IFN-γ and CIITA modulate IL-6 expression in skeletal muscle.

Interleukin 6 (IL-6) is a secreted cytokine that is an important mediator of the immune response in numerous tissues, including skeletal muscle. IL-6 is considered a myokine as it can be secreted by muscle. IL-6 is secreted following exercise, where it exerts both pro-myogenic effects as well as anti-myogenic effects such as promoting atrophy and muscle wasting. The regulation of IL-6 in skeletal muscle is not well understood. The purpose of this study was to determine if IFN-γ and TNF-ɑ stimulate IL-6 in skeletal muscle. We found that both IFN-γ and TNF-α stimulate IL-6 in skeletal muscle, but the stimulation is not cooperative as seen in monocytes. We have previously shown that the IFN-γ stimulated class II major histocompatibility complex transactivator (CIITA) mediates many of the effects of IFN-γ in skeletal muscle and we show here that CIITA directly stimulates IL-6. The regulation of IL-6 by CIITA is clearly complex, as we found that CIITA both stimulates and restrains IL-6 expression. To show that these effects could be observed in a physiological setting, mice were treated with IFN-γ and we found that both CIITA and IL-6 were upregulated in skeletal muscle.

The transcription elongation factor TCEA3 promotes the activity of the myogenic regulatory factors.

The transcription elongation factor TFIIS is encoded by a three member gene family in vertebrates. Here we show that one member of this family, TCEA3, is upregulated during skeletal muscle differentiation and acts to promote gene activation by the myogenic regulatory family of transcription factors, which includes MyoD and myogenin. We show that myogenin is a direct regulator of Tcea3. Myogenin binds to the Tcea3 promoter and is required to recruit RNA polymerase II. TCEA3 can bind to both myogenin and MyoD and is co-recruited with the MRFs to promoters dependent on the MRFs. Depletion of myogenin inhibits the recruitment of TCEA3, suggesting that the interaction of TCEA3 with the MRFs serves to aid in recruitment to target promoters. Like TFIIS, we show that TCEA3 interacts with RNA polymerase II. TCEA3 travels with the elongating RNA polymerase II in the coding region of genes and depletions of TCEA3 inhibit the recruitment of RNA polymerase II to promoters. In proliferating cells, TCEA3 expressed at low levels and is present in both the nucleus and cytoplasm. However, upon differentiation, TCEA3 is upregulated and transported exclusively to the nucleus. Thus, our data show that TCEA3 is a required co-factor for MRF driven gene expression during myogenesis.

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry.

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

TBX3 represses TBX2 under the control of the PRC2 complex in skeletal muscle and rhabdomyosarcoma.

TBX2 and TBX3 function as repressors and are frequently implicated in oncogenesis. We have shown that TBX2 represses p21, p14/19, and PTEN in rhabdomyosarcoma (RMS) and skeletal muscle but the function and regulation of TBX3 were unclear. We show that TBX3 directly represses TBX2 in RMS and skeletal muscle. TBX3 overexpression impairs cell growth and migration and we show that TBX3 is directly repressed by the polycomb repressive complex 2 (PRC2), which methylates histone H3 lysine 27 (H3K27me). We found that TBX3 promotes differentiation only in the presence of early growth response factor 1 (EGR1), which is differentially expressed in RMS and is also a target of the PRC2 complex. The potent regulation axis revealed in this work provides novel insight into the effects of the PRC2 complex in normal cells and RMS and further supports the therapeutic value of targeting of PRC2 in RMS.

JARID2 and the PRC2 complex regulate skeletal muscle differentiation through regulation of canonical Wnt signaling.

BACKGROUND: JARID2 is a non-catalytic member of the polycomb repressive complex 2 (PRC2), which is known to regulate developmental target genes in embryonic stem cells. Here, we provide mechanistic insight into the modulation of Wnt signaling by JARID2 during murine skeletal muscle differentiation. RESULTS: We show that JARID2 is expressed in proliferating myoblasts, but downregulated upon muscle differentiation. Unexpectedly, depletion of JARID2 or the catalytic subunit of the PRC2 complex, EZH2, inhibited differentiation, suggesting that JARID2 and the PRC2 complex are required to initiate this process. Expression of the myogenic regulatory factors required to promote differentiation, MYOD and MYOG, was downregulated in the absence of JARID2, even though decreases in the methylation of histone H3 lysine 27 (H3K27me3) were observed on both promoters. We found that activation of the Wnt signaling pathway upregulated MYOD and restored differentiation. Activation of the Wnt pathway in JARID2 depleted cells caused ß-catenin to translocate to the nucleus, where it bound to and activated the Myod1 promoter. We show that the Wnt antagonist SFRP1 is highly upregulated in the absence of JARID2 and is a direct target of JARID2 and the PRC2 complex. Ectopic expression of SFRP1 blocked MYOD and late muscle gene expression and inhibited the translocation of ß-catenin to the nucleus. Finally, we show that JARID2 and SFRP1 are inversely correlated in melanoma, confirming that the JARID2-mediated repression of SFRP1 extends beyond skeletal muscle and has important implications in many cellular systems, including cancer. CONCLUSIONS: We show that JARID2 and the PRC2 complex regulate muscle differentiation by modulating Wnt signaling through the direct repression of Wnt antagonists.

EGR1 interacts with TBX2 and functions as a tumor suppressor in rhabdomyosarcoma.

EGR1, one of the immediate-early response genes, can function as a tumor suppressor gene or as an oncogene in cancer. The function of EGR1 has not been fully characterized in rhabdomyosarcoma (RMS), a pediatric cancer derived from the muscle linage. We found that EGR1 is downregulated in the alveolar RMS (ARMS) subtype but expressed at levels comparable to normal skeletal muscle in embryonal RMS (ERMS). We found that overexpression of EGR1 in ARMS significantly decreased cell proliferation, mobility, and anchorage-independent growth while also promoting differentiation. We found that EGR1 interacts with TBX2, which we have shown functions as an oncogene in RMS. The interaction inhibits EGR1 dependent gene expression, which includes the cell cycle regulators p21 and PTEN as well as other important cell growth drivers such as NDRG1 and CST6. We also found that EGR1 induced apoptosis by triggering the intrinsic apoptosis pathway. EGR1 also activated two pro-apoptotic factors, BAX and dephosphorylated BAD, which are both located upstream of the caspase cascades in the intrinsic pathway. EGR1 also sensitized RMS cells to chemotherapeutic agents, suggesting that activating EGR1 may improve therapeutic targeting by inducing apoptosis. Our results establish the important role of EGR1 in understanding RMS pathology.