Tractable Therapeutic Target Research Articles

Tales of Cancer-Induced Bone Disease from the Senescent Osteocyte Crypt

Cancer-induced bone disease greatly diminishes the quality of life for patients with bone metastatic breast cancer, resulting in painful skeletal-related events including bone loss and fracture. Improved understanding of the roles of osteoblasts and osteoclasts, and how tumors alter their biology, has led to blockbuster therapies that significantly reduce skeletal-related events, but the disease remains incurable. However, emerging technologies and tools for studying the role of other stromal and immune components in controlling tumor–host interactions have begun to reveal new insights that may yield tractable therapeutic targets to further mitigate the painful effects of bone metastases. In this issue of Cancer Research, Kaur and colleagues study osteocytes, which are terminally differentiated osteoblasts and entombed within the bone matrix, from established bone metastatic breast cancer and report how the disease ages them as characterized by a senescence-associated secretory phenotype. This premature development of osteocyte senescence in turn enhances bone destruction and osteoclastogenic potential. Targeting senescent cells using senolytics suppressed bone resorption and preserved bone mass. Collectively, these findings underscore osteocyte involvement in the “vicious cycle” of bone metastasis, and targeting senescent osteocytes represents a new avenue for managing cancer-induced bone disease.See related article by Kaur et al., p. 3936

Guanine nucleotide exchange factors and colon neoplasia.

Despite many diagnostic and therapeutic advances, colorectal cancer (CRC) remains the second leading cause of cancer death for men and women in the United States. Alarmingly, for reasons currently unknown, the demographics of this disease have shifted towards a younger population. Hence, understanding the molecular mechanisms underlying CRC initiation and progression and leveraging these findings for therapeutic purposes remains a priority. Here, we review critically the evidence that canonical and noncanonical actions of guanine nucleotide exchange factors (GEFs) play important roles in CRC evolution. Rho GEF GTPases, which switch between inactive GDP-bound and active GTP-bound states, are commonly overexpressed and activated in a variety of cancers, including CRC, and may be tractable therapeutic targets. In addition to comprehensively reviewing this field, we focus on Rho/Rac GEFs that are involved in regulating key functions of normal and neoplastic cells like cell polarity, vesicle trafficking, cell cycle regulation, and transcriptional dynamics. Prime examples of such Rho/Rac GEFs include βPak-interacting exchange factor (βPix), a Rho family GEF for Cdc42/Rac1, Tiam1, GEF-H1, RGNEF, and other GEFs implicated in CRC development and progression. Throughout this analysis, we explore how these findings fill key gaps in knowledge regarding the molecular basis of colon carcinogenesis and how they may be leveraged to treat advanced CRC. Lastly, we address potential future directions for research into the role of GEFs as CRC biomarkers and therapeutic targets. In this regard, leveraging the noncanonical actions of GEFs appears to provide a relatively unexplored opportunity requiring further investigation.

Mitochondrial complex III-derived ROS amplify immunometabolic changes in astrocytes and promote dementia pathology.

Neurodegenerative disorders alter mitochondrial functions, including the production of reactive oxygen species (ROS). Mitochondrial complex III (CIII) generates ROS implicated in redox signaling, but its triggers, targets, and disease relevance are not clear. Using site-selective suppressors and genetic manipulations together with mitochondrial ROS imaging and multiomic profiling, we found that CIII is the dominant source of ROS production in astrocytes exposed to neuropathology-related stimuli. Astrocytic CIII-ROS production was dependent on nuclear factor-κB (NF-κB) and the mitochondrial sodium-calcium exchanger (NCLX) and caused oxidation of select cysteines within immune and metabolism-associated proteins linked to neurological disease. CIII-ROS amplified metabolomic and pathology-associated transcriptional changes in astrocytes, with STAT3 activity as a major mediator, and facilitated neuronal toxicity in a non-cell-autonomous manner. As proof-of-concept, suppression of CIII-ROS in mice decreased dementia-linked tauopathy and neuroimmune cascades and extended lifespan. Our findings establish CIII-ROS as an important immunometabolic signal transducer and tractable therapeutic target in neurodegenerative disease.

P-217 ULK3-mediated autophagy is a tractable therapeutic target for the treatment of multiple myeloma

P-217 ULK3-mediated autophagy is a tractable therapeutic target for the treatment of multiple myeloma

How Molecular Discoveries Have Changed Liver Tumor Pathology: A Brief Review.

Recent molecular discoveries have led to improved understanding of tumor biology and the development of new diagnostic assays. To review primarily 3 examples of liver tumors and to briefly illustrate how recent molecular discoveries have altered clinical liver pathology practice. First, we will discuss fibrolamellar carcinoma, which will be the main focus of discussion, as an example for new diagnostic tests that have been developed as a result of molecular discoveries. Additional information on the role of molecular diagnostics in hepatocellular adenoma and hepatocellular carcinoma will be provided. Second, we will use the example of epithelioid hemangioendothelioma as an example of how new diagnostic tools, based on molecular discoveries, may support improved prognostication. Finally, we will use the example of intrahepatic cholangiocarcinoma as an example of a liver tumor where new molecular discoveries have identified tractable therapeutic targets and led to new effective therapies. This portion of the manuscript will also include a description of the anatomic and molecular differences between intrahepatic, hilar, and extrahepatic cholangiocarcinoma. Fueled by molecular discoveries, new and better diagnostic tests and therapeutic targets have improved clinical care in patients affected by liver tumors.

Detection of early Alzheimer's disease-like molecular alterations in a mouse model expressing human ApoE4.

The E4 allele of apolipoprotein E (ApoE4) is a key genetic risk factor for late-onset Alzheimer's disease (AD), increasing the risk of developing the disease by up to three-fold. However, the mechanisms by which ApoE4 contributes to AD pathogenesis are poorly understood. Here, we utilize a mouse model expressing either human ApoE3 or human ApoE4 to examine the effects of the E4 allele on a wide range of genetic and molecular pathways that are altered in early AD pathology. We demonstrate that ApoE4-expressing mice begin to show early differential expression of multiple genes, leading to alterations in downstream pathways related to neural cell maintenance, insulin signaling, amyloid processing and clearance, and synaptic plasticity. These alterations may result in the earlier accumulation of pathological proteins such as β-amyloid that may build up within cells, leading to the accelerated degeneration of neurons and astrocytes as observed in ApoE4-positive individuals. We also examine the metabolic effects associated with a high-fat diet (HFD) in male ApoE4-expressing mice compared with regular chow diet (RD) fed mice at different ages. We found that young ApoE4-expressing mice fed HFD developed metabolic disturbances, such as elevated weight gain, blood glucose, and plasma insulin levels that cumulatively have been observed to increase the risk of AD in humans. Taken together, our results reveal early pathways that could mediate ApoE4-related AD risk and may help identify more tractable therapeutic targets for treating ApoE4-associated AD.

Dependency of Tamoxifen Sensitive and Resistant ER+ Breast Cancer Cells on Semaphorin 3C (SEMA3C) for Growth.

Estrogen receptor positive (ER+) breast cancer (BCa) accounts for the highest proportion of breast cancer-related deaths. While endocrine therapy is highly effective for this subpopulation, endocrine resistance remains a major challenge and the identification of novel targets is urgently needed. Previously, we have shown that Semaphorin 3C (SEMA3C) is an autocrine growth factor that drives the growth and treatment resistance of various cancers, but its role in breast cancer progression and endocrine resistance is poorly understood. Here, we report that SEMA3C plays a role in maintaining the growth of ER+ BCa cells and is a novel, tractable therapeutic target for the treatment of ER+ BCa patients. Analyses of publicly available clinical datasets indicate that ER+ BCa patients express significantly higher levels of SEMA3C mRNA than other subtypes. Furthermore, SEMA3C mRNA expression was positively correlated with ESR1 mRNA expression. ER+ BCa cell lines (MCF7 and T47D) expressed higher levels of SEMA3C mRNA and protein than a normal mammary epithelial MCF10A cell line. ER siRNA knockdown was suppressed, while dose-dependent beta-estradiol treatment induced SEMA3C expression in both MCF7 and T47D cells, suggesting that SEMA3C is an ER-regulated gene. The stimulation of ER+ BCa cells with recombinant SEMA3C activated MAPK and AKT signaling in a dose-dependent manner. Conversely, SEMA3C silencing inhibited Estrogen Receptor (ER) expression, MAPK and AKT signaling pathways while simultaneously inducing apoptosis, as monitored by flow cytometry and Western blot analyses. SEMA3C silencing significantly inhibited the growth of ER+ BCa cells, implicating a growth dependency of ER+ BCa cells on SEMA3C. Moreover, the analysis of tamoxifen resistant (TamR) cell models (TamC3 and TamR3) showed that SEMA3C levels remain high despite treatment with tamoxifen. Tamoxifen-resistant cells remained dependent on SEMA3C for growth and survival. Treatment with B1SP Fc fusion protein, a SEMA3C pathway inhibitor, attenuated SEMA3C-induced signaling and growth across a panel of tamoxifen sensitive and resistant ER+ breast cancer cells. Furthermore, SEMA3C silencing and B1SP treatment were associated with decreased EGFR signaling in TamR cells. Here, our study implicates SEMA3C in a functional role in ER+ breast cancer signaling and growth that suggests ER+ BCa patients may benefit from SEMA3C-targeted therapy.

Functional roles of reactive astrocytes in neuroinflammation and neurodegeneration.

Despite advances in uncovering the mechanisms that underlie neuroinflammation and neurodegenerative disease, therapies that prevent neuronal loss remain elusive. Targeting of disease-defining markers in conditions such as Alzheimer disease (amyloid-β and tau) or Parkinson disease (α-synuclein) has been met with limited success, suggesting that these proteins do not act in isolation but form part of a pathological network. This network could involve phenotypic alteration of multiple cell types in the CNS, including astrocytes, which have a major neurosupportive, homeostatic role in the healthy CNS but adopt reactive states under acute or chronic adverse conditions. Transcriptomic studies in human patients and disease models have revealed the co-existence of many putative reactive sub-states of astrocytes. Inter-disease and even intra-disease heterogeneity of reactive astrocytic sub-states are well established, but the extent to which specific sub-states are shared across different diseases is unclear. In this Review, we highlight how single-cell and single-nuclei RNA sequencing and other 'omics' technologies can enable the functional characterization of defined reactive astrocyte states in various pathological scenarios. We provide an integrated perspective, advocating cross-modal validation of key findings to define functionally important sub-states of astrocytes and their triggers as tractable therapeutic targets with cross-disease relevance.

PRSet: Pathway-based polygenic risk score analyses and software.

Polygenic risk scores (PRSs) have been among the leading advances in biomedicine in recent years. As a proxy of genetic liability, PRSs are utilised across multiple fields and applications. While numerous statistical and machine learning methods have been developed to optimise their predictive accuracy, these typically distil genetic liability to a single number based on aggregation of an individual's genome-wide risk alleles. This results in a key loss of information about an individual's genetic profile, which could be critical given the functional sub-structure of the genome and the heterogeneity of complex disease. In this manuscript, we introduce a 'pathway polygenic' paradigm of disease risk, in which multiple genetic liabilities underlie complex diseases, rather than a single genome-wide liability. We describe a method and accompanying software, PRSet, for computing and analysing pathway-based PRSs, in which polygenic scores are calculated across genomic pathways for each individual. We evaluate the potential of pathway PRSs in two distinct ways, creating two major sections: (1) In the first section, we benchmark PRSet as a pathway enrichment tool, evaluating its capacity to capture GWAS signal in pathways. We find that for target sample sizes of >10,000 individuals, pathway PRSs have similar power for evaluating pathway enrichment as leading methods MAGMA and LD score regression, with the distinct advantage of providing individual-level estimates of genetic liability for each pathway -opening up a range of pathway-based PRS applications, (2) In the second section, we evaluate the performance of pathway PRSs for disease stratification. We show that using a supervised disease stratification approach, pathway PRSs (computed by PRSet) outperform two standard genome-wide PRSs (computed by C+T and lassosum) for classifying disease subtypes in 20 of 21 scenarios tested. As the definition and functional annotation of pathways becomes increasingly refined, we expect pathway PRSs to offer key insights into the heterogeneity of complex disease and treatment response, to generate biologically tractable therapeutic targets from polygenic signal, and, ultimately, to provide a powerful path to precision medicine.

Nicotinamide Adenosine Dinucleotide Phosphate Oxidase-Mediated Signaling in Cardiac Remodeling.

Significance: Reactive oxygen species (ROS) play a key role in the pathogenesis of cardiac remodeling and the subsequent progression to heart failure (HF). Nicotinamide adenosine dinucleotide phosphate (NADPH) oxidases (NOXs) are one of the major sources of ROS and are expressed in different heart cell types, including cardiomyocytes, endothelial cells, fibroblasts, and inflammatory cells. Recent Advances: NOX-derived ROS are usually produced in a regulated and spatially confined fashion and typically linked to specific signaling. The two main cardiac isoforms, namely nicotinamide adenine dinucleotide phosphate oxidase isoform 2 (NOX2) and nicotinamide adenine dinucleotide phosphate oxidase isoform 4 (NOX4), possess different biochemical and (patho)physiological properties and exert distinct effects on the cardiac phenotype in many settings. Recent work has defined important cell-specific effects of NOX2 that contribute to pathological cardiac remodeling and dysfunction. NOX4, on the other hand, may exert protective effects by stimulating adaptive stress responses, with recent data showing that NOX4-mediated signaling regulates transcription and metabolism in the heart. Critical Issues: The inhibition of NOX2 appears to be a very promising therapeutic target to ameliorate pathological cardiac remodeling. If the beneficial effects of NOX4 can be enhanced, this might be a unique approach to boosting adaptive responses and thereby impact cell survival, activation, contractility, and growth. Future Directions: Increasing knowledge regarding the intricacies of NOX-mediated signaling may yield tractable therapeutic targets, in contrast to the non-specific targeting of oxidative stress. Antioxid. Redox Signal. 38, 371-387.

Abstract B017: MICAL2 promotes pancreatic cancer growth and metastasis independent of MRTF-A signaling

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in large part due to its propensity for early metastasis. We used ChIP-seq to identify differentially tagged super-enhancer associated genes in PDAC and identified MICAL2 as a gene of interest. MICAL (molecule interacting with CasL) proteins are flavin monooyxgenases that induce actin depolymerization. MICAL2 canonically modulates nuclear actin to regulate SRF/MRTF-A (myocardin related transcription factor) mediated transcription to drive EMT (epithelial-mesenchymal transition) and fibrosis. In this study, we sought to determine the impact of MICAL2 on PDAC progression and its dependence on SRF/MRTF-A signaling. Methods: Studies were performed in human and murine PDAC models. MICAL2 and MRTF-A were knocked down with shRNA and gene expression quantitated with qPCR. In vitro cell viability was assessed by MTT colorimetric assay. Cell migration was tested with in vitro wound scratch assays. Cell cycle analysis was done with flow cytometry-based DNA content measurement. Subcutaneous, orthotopic, and intrasplenic injections of PDAC cells were performed to assess in vivo growth and metastasis. Protein expression was measured with Western blotting. Student’s t-test was used to confirm statistical significance of experimental results. Results: In human PDAC cell lines, MICAL2 knockdown decreased cell viability, reduced cell migration and wound healing, and induced cell cycle arrest at G0/G1. MICAL2 inhibition was associated with decreased p-AKT, c-Myc, and increased p27 by Western blot. Orthotopic and subcutaneous injections of KPC46 shMICAL2 cells were performed in syngeneic mice; orthotopic injections of shMICAL2 cells led to decreased tumor growth compared to control (0.26 gm vs. 1 gm, p = 0.008), not observed with shMRTF-A (1.032 gm vs. 1 gm, p = 0.95). Subcutaneous injections of MICAL2 knockdown cells led to no tumor growth; injections of MRTF-A knockdown cells again led to comparable tumor growth as control (3.02 gm vs. 1.72 gm, p = 0.24). GSEA (Gene Set Enrichment Analysis) of RNAseq data demonstrated a correlation between MICAL2 knockdown and downregulation of Hallmark EMT pathways. Intrasplenic injection studies in syngeneic mice revealed grossly decreased liver metastases after intrasplenic injection of shMRTF-A cells, with total abrogation of metastases after injection of shMICAL2 cells. Conclusions: MICAL2 promotes pancreatic cancer cell growth and metastasis. While MICAL2’s effect on metastasis appears largely dependent on SRF/MRTF-A, tumor growth appears to be MRTF-A independent, suggesting putative novel functions of MICAL2. Given its potentially targetable enzymatic domain and important role in promoting cell growth in vitro and tumor progression and metastasis in vivo, MICAL2 holds promise as a novel tractable therapeutic target in pancreatic cancer. Citation Format: Sohini Khan, Shweta Sharma, Divya Sood, Bharti Garg, Dawn Jaquish, Evangeline Mose, Edgar Esparza, Herve Tiriac, Andrew M. Lowy. MICAL2 promotes pancreatic cancer growth and metastasis independent of MRTF-A signaling [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B017.

Thrombopoietin Receptor Signaling Promotes the Competitive Advantage of TET2-Mutated Hematopoietic Stem and Progenitor Cells in Clonal Hematopoiesis

Clonal Hematopoiesis of Indeterminate Potential (CHIP) is an age-related condition that predisposes individuals to blood cancers. The time interval from acquisition of CHIP to overt neoplasia can span many years, providing an opportunity to delay or prevent malignant transformation. A key to achieving this goal is identifying differential dependencies between mutant and normal hematopoietic stem and progenitor cells (HSPCs). Targeting such dependencies may prevent against CHIP-related health risks. One of the most commonly mutated genes in CHIP is TET2, which encodes a methylcytosine dioxygenase. In mouse models, Tet2 knockout (KO) HSPCs have a competitive advantage over wild-type (WT) HSPCs. In this study, we aimed to identify differential dependencies between Tet2KO and Tet2WT HSPCs. We first generated isogenic murine cell lines by overexpressing HOXB4, a transcription factor involved in self-renewal, in HSPCs derived from a Tet2KO mouse and Tet2WT littermate. Using the isogenic cell lines termed HPCHOXB4, we conducted an epigenetic drug screen and found SGC0946, a selective inhibitor of H3K79 methyltransferase DOT1L, reduced the advantage of Tet2KO HPCHOXB4 cells in competition assays by preferentially reducing their proliferation and viability. SGC0946 also reduced the competitive advantage of unmodified Tet2KO HSPCs in competition assays, indicating that the dependency was not a consequence of HOXB4 overexpression. To decipher the mechanism behind this differential dependency, we performed RNA-seq and ChIP-seq for H3K79me2 on Tet2KO and Tet2WT HPCHOXB4 cells at baseline and after treatment with SGC0946. Expression of Mpl, which encodes the thrombopoietin receptor TPOR, was 20-fold higher in Tet2KO HPCHOXB4 than in Tet2WT cells at baseline and decreased after drug treatment. Mpl expression at the mRNA and protein level was also elevated in unmodified Tet2KO HSPCs compared with Tet2WT cells. Importantly, ChIP-seq analysis revealed that the level of H3K79me2 which associates with active transcription at the Mpl gene locus was higher in Tet2KO than in Tet2WT HPCHOXB4 cells at baseline, a difference that was erased with SGC0946 treatment. Based on the above findings, we hypothesized that Tet2KO HSPCs are more dependent on TPOR signaling than Tet2WT cells. Consistent with our hypothesis, a higher concentration of TPO in the culture medium increased the competitive advantage of Tet2KO HPCHOXB4 cells. Moreover, overexpression of Mpl in Tet2KO HPCHOXB4 cells rescued the effects of DOT1L inhibition, and shRNA-mediated suppression of Mpl expression was sufficient to decrease the competitive advantage of Tet2KO HPCHOXB4 cells and unmodified Tet2KO HSPCs over their corresponding Tet2WT counterparts. To explore the translational relevance of our findings, we tested if inhibition of JAK2, a critical mediator of TPOR intracellular signaling, would also reduce the competitive advantage of Tet2KO HSPCs. In line with our hypothesis, treatment with JAK2 inhibitors (fedratinib and AZ960), but not JAK1 inhibitors, suppressed the competitive advantage of Tet2KO HPCHOXB4 cells in vitro. Importantly, competitive repopulation studies showed that treatment with ruxolitinib, a clinically approved JAK1/2 inhibitor, also reduced the competitive advantage of untransformed Tet2KO HSPCs in vivo. To determine if our findings apply to humans, we generated two TET2KO cell pools from cord blood HSPCs using CRISPR/Cas9. The TET2KO cells expressed higher levels of TPOR and were more sensitive to treatment with SGC0946 and ruxolitinib compared with control cells. In addition, we expanded HSPCs from TET2-mutated mobilized peripheral blood samples in short-term cultures and monitored the size of the mutant clones by measuring the allele frequency of the sample-specific TET2 variants. In untreated cultures, the relative size of the mutant clones increased over time, an effect that was diminished by treatment with either SGC0946 or ruxolitinib. In summary, our findings demonstrate that TET2-mutated HSPCs are more dependent on TPOR signaling than TET2WT HSPCs. This differential dependency is mediated through increased H3K79 methylation at the MPL gene locus and enhanced expression of MPL. Furthermore, inhibition of the TPOR/JAK2 signaling axis is effective in suppressing the competitive advantage of TET2-mutated HSPCs, thus identifying a tractable therapeutic target for TET2-mutated CHIP.

SFPQ promotes RAS-mutant cancer cell growth by modulating 5'-UTR mediated translational control of CK1α.

Oncogenic mutations in the RAS family of small GTPases are commonly found in human cancers and they promote tumorigenesis by altering gene expression networks. We previously demonstrated that Casein Kinase 1α (CK1α), a member of the CK1 family of serine/threonine kinases, is post-transcriptionally upregulated by oncogenic RAS signaling. Here, we report that the CK1α mRNA contains an exceptionally long 5′-untranslated region (UTR) harbouring several translational control elements, implicating its involvement in translational regulation. We demonstrate that the CK1α 5′-UTR functions as an IRES element in HCT-116 colon cancer cells to promote cap-independent translation. Using tobramycin-affinity RNA-pulldown assays coupled with identification via mass spectrometry, we identified several CK1α 5′-UTR-binding proteins, including SFPQ. We show that RNA interference targeting SFPQ reduced CK1α protein abundance and partially blocked RAS-mutant colon cancer cell growth. Importantly, transcript and protein levels of SFPQ and other CK1α 5′-UTR-associated RNA-binding proteins (RBPs) are found to be elevated in early stages of RAS-mutant cancers, including colorectal and lung adenocarcinoma. Taken together, our study uncovers a previously unappreciated role of RBPs in promoting RAS-mutant cancer cell growth and their potential to serve as promising biomarkers as well as tractable therapeutic targets in cancers driven by oncogenic RAS.

LncExACT1 and DCHS2 Regulate Physiological and Pathological Cardiac Growth.

The heart grows in response to pathological and physiological stimuli. The former often precedes cardiomyocyte loss and heart failure; the latter paradoxically protects the heart and enhances cardiomyogenesis. The mechanisms underlying these differences remain incompletely understood. Although long noncoding RNAs (lncRNAs) are important in cardiac development and disease, less is known about their roles in physiological hypertrophy or cardiomyogenesis. RNA sequencing was applied to hearts from mice after 8 weeks of voluntary exercise-induced physiological hypertrophy and cardiomyogenesis or transverse aortic constriction for 2 or 8 weeks to induce pathological hypertrophy or heart failure. The top lncRNA candidate was overexpressed in hearts with adeno-associated virus vectors and inhibited with antisense locked nucleic acid-GapmeRs to examine its function. Downstream effectors were identified through promoter analyses and binding assays. The functional roles of a novel downstream effector, dachsous cadherin-related 2 (DCHS2), were examined through transgenic overexpression in zebrafish and cardiac-specific deletion in Cas9-knockin mice. We identified exercise-regulated cardiac lncRNAs, called lncExACTs. lncExACT1 was evolutionarily conserved and decreased in exercised hearts but increased in human and experimental heart failure. Cardiac lncExACT1 overexpression caused pathological hypertrophy and heart failure; lncExACT1 inhibition induced physiological hypertrophy and cardiomyogenesis, protecting against cardiac fibrosis and dysfunction. lncExACT1 functioned by regulating microRNA-222, calcineurin signaling, and Hippo/Yap1 signaling through DCHS2. Cardiomyocyte DCHS2 overexpression in zebrafish induced pathological hypertrophy and impaired cardiac regeneration, promoting scarring after injury. In contrast, murine DCHS2 deletion induced physiological hypertrophy and promoted cardiomyogenesis. These studies identify lncExACT1-DCHS2 as a novel pathway regulating cardiac hypertrophy and cardiomyogenesis. lncExACT1-DCHS2 acts as a master switch toggling the heart between physiological and pathological growth to determine functional outcomes, providing a potentially tractable therapeutic target for harnessing the beneficial effects of exercise.

Nascent chains can form co-translational folding intermediates that promote post-translational folding outcomes in a disease-causing protein

During biosynthesis, proteins can begin folding co-translationally to acquire their biologically-active structures. Folding, however, is an imperfect process and in many cases misfolding results in disease. Less is understood of how misfolding begins during biosynthesis. The human protein, alpha-1-antitrypsin (AAT) folds under kinetic control via a folding intermediate; its pathological variants readily form self-associated polymers at the site of synthesis, leading to alpha-1-antitrypsin deficiency. We observe that AAT nascent polypeptides stall during their biosynthesis, resulting in full-length nascent chains that remain bound to ribosome, forming a persistent ribosome-nascent chain complex (RNC) prior to release. We analyse the structure of these RNCs, which reveals compacted, partially-folded co-translational folding intermediates possessing molten-globule characteristics. We find that the highly-polymerogenic mutant, Z AAT, forms a distinct co-translational folding intermediate relative to wild-type. Its very modest structural differences suggests that the ribosome uniquely tempers the impact of deleterious mutations during nascent chain emergence. Following nascent chain release however, these co-translational folding intermediates guide post-translational folding outcomes thus suggesting that Z’s misfolding is initiated from co-translational structure. Our findings demonstrate that co-translational folding intermediates drive how some proteins fold under kinetic control, and may thus also serve as tractable therapeutic targets for human disease.

A JAK/STAT-mediated inflammatory signaling cascade drives oncogenesis in AF10-rearranged AML

AbstractLeukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins (FPs) are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of acute myeloid leukemia (AML) driven by the most common AF10 FPs, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a detailed map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent antioncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias.

Germline ALK variations are associated with a poor prognosis in glioma and IDH-wildtype glioblastoma.

Glioma is the most common primary brain tumor. Clear classification is crucial for accurate diagnosis and individualized treatment. Histopathological characteristics and genetic alterations have shown to be related to prognosis and treatment response. Germline variants are important components of genetic alterations. However, the distribution of germline variations in glioma patients and their association with survival remain unknown. We carried out whole-exome sequencing on 99 cases to explore germline variants in glioma. We also analyzed the association of germline variants with clinicopathological features and other prognostic indicators. All the glioma cases harbored rare germline variants. Germline ALK variants (gALK-Mut) were identified in 12/99 (12.12%) patients. The gALK-Mut patients had significantly shorter overall survival than germline ALK wildtype (gALK-WT) patients in the all glioma group (99 cases) and the subset of patients with IDH-wildtype glioblastoma (IDH-WT-GBM, 39 cases) (P = 0.013 and 0.027, respectively). The gALK-Mut patients also had higher frequency of BIRC5, PIK3CA and RPN1 somatic mutations than the gALK-WT patients in IDH-WT-GBM. Other confounding factors appeared to contribute to patient survival. The subgroup of patients in IDH-WT-GBM with gALK-Mut/TP53-Mut had worse prognosis than the gALK-WT/TP53-Mut subgroup (P = 0.031); The gALK-Mut/TERT-WT and gALK-Mut/TERT-Mut subgroups both had a worse prognosis than the gALK-WT/TERT-Mut subgroup (P = 0.031 and 0.018, respectively). Our study revealed ALK variation was an independent indicator of poor prognosis in glioma and IDH-WT-GBM. It could be a promising biomarker and tractable therapeutic target for this deadly disease.

New small molecule fluorescent probes for G protein-coupled receptors: valuable tools for drug discovery.

G protein-coupled receptors (GPCRs) are essential signaling proteins and tractable therapeutic targets. To develop new drug candidates, GPCR drug discovery programs require versatile, sensitive pharmacological tools for ligand binding and compound screening. With the availability of new imaging modalities and proximity-based ligand binding technologies, fluorescent ligands offer many advantages and are increasingly being used, yet labeling small molecules remains considerably more challenging relative to peptides. Focusing on recent fluorescent small molecule studies for family A GPCRs, this review addresses some of the key challenges, synthesis approaches and structure-activity relationship considerations, and discussesadvantages of using high-resolution GPCR structures to inform conjugation strategies. While no single approach guarantees successful labeling without loss of affinity or selectivity, the choice of fluorophore, linker type and site of attachment have proved to be critical factors that can significantly affect their utility in drug discovery programs, and as discussed, can sometimes lead to very unexpected results.

Abstract 14620: LncExACT1 Acts as a Pivotal Switch Between Physiological and Pathological Cardiac Growth

Rationale: Pathological hypertrophy commonly leads to heart failure (HF) and loss of cardiomyocytes, while physiological hypertrophy protects the heart and enhances cardiomyogenesis. The mechanisms underlying these differences remain unclear. While long noncoding RNAs (lncRNAs) are important in cardiac development and disease, their role in physiological hypertrophy is unknown. Objective: To investigate the role of lncRNAs in physiological hypertrophy. Methods and Results: Mice underwent voluntary wheel running for eight weeks or transverse aortic constriction (TAC) for two or ten weeks. RNAseq identified a novel set of lncRNAs altered in exercised hearts, which we termed l ong n on c oding Ex ercise- A ssociated C ardiac T ranscripts (lncExACTs). lncExACT1 was highly conserved and uniquely downregulated in exercised hearts but upregulated in pathological animal models and hearts from HF patients vs controls (1.8-fold; p <0.001, N=24) as well as plasma from HF patients with reduced (2.9-fold; p =0.032, N=16) and preserved ejection fraction (3.4-fold; p =0.006, N=18). In mice, AAV9 lncExACT1 overexpression increased cardiac lncExACT1 7-fold at 16 weeks and increased heart (HW) and lung (LW) weight relative to tibial length (TL), reduced fractional shortening (FS) and increased relative wall thickness (RWT) ( p <0.05 for all). These changes were associated with a pathological gene expression pattern. In contrast, antisense lncExACT1 inhibition reduced cardiac expression 2-fold at 2 weeks and increased HW/TL without an increase in LW/TL, improved cardiac function, and increased RWT ( p <0.05 for all). LncExACT1 inhibition induced a physiological gene expression pattern and increased markers of cardiomyogenesis. LncExACT1 inhibition reduced TAC-induced HW/TL and fibrosis, while increasing FS ( p <0.05 for all). Mechanistic studies revealed that lncExACT1 works by binding miR-222 and as a novel regulator of Hippo/Yap1 signaling through modulation of dachsous cadherin-related 2. Conclusions: lncExACT1 acts as a master switch toggling the heart between physiological and pathological growth and provides a potentially tractable therapeutic target for harnessing the beneficial effects of exercise.

Identifying collagen VI as a target of fibrotic diseases regulated by CREBBP/EP300

Fibrotic diseases remain a major cause of morbidity and mortality, yet there are few effective therapies. The underlying pathology of all fibrotic conditions is the activity of myofibroblasts. Using cells from freshly excised disease tissue from patients with Dupuytren's disease (DD), a localized fibrotic disorder of the palm, we sought to identify new therapeutic targets for fibrotic disease. We hypothesized that the persistent activity of myofibroblasts in fibrotic diseases might involve epigenetic modifications. Using a validated genetics-led target prioritization algorithm (Pi) of genome wide association studies (GWAS) data and a broad screen of epigenetic inhibitors, we found that the acetyltransferase CREBBP/EP300 is a major regulator of contractility and extracellular matrix production via control of H3K27 acetylation at the profibrotic genes, ACTA2 and COL1A1 Genomic analysis revealed that EP300 is highly enriched at enhancers associated with genes involved in multiple profibrotic pathways, and broad transcriptomic and proteomic profiling of CREBBP/EP300 inhibition by the chemical probe SGC-CBP30 identified collagen VI (Col VI) as a prominent downstream regulator of myofibroblast activity. Targeted Col VI knockdown results in significant decrease in profibrotic functions, including myofibroblast contractile force, extracellular matrix (ECM) production, chemotaxis, and wound healing. Further evidence for Col VI as a major determinant of fibrosis is its abundant expression within Dupuytren's nodules and also in the fibrotic foci of idiopathic pulmonary fibrosis (IPF). Thus, Col VI may represent a tractable therapeutic target across a range of fibrotic disorders.