Interlinked switch circuits of biological intelligence.

Eukaryotic cells learn and adapt via unknown network architectures. Recent work demonstrated a circuit of two GTPases used by cells to overcome growth factor scarcity, encouraging our view that artificial and biological intelligence share strikingly similar design principles and that cells function as deep reinforcement learning (RL) agents in uncertain environments.

Diverse gut pathogens exploit the host engulfment pathway via a conserved mechanism.

Macrophages clear infections by engulfing and digesting pathogens within phagolysosomes. Pathogens escape this fate by engaging in a molecular arms race; they use WxxxE motif-containing "effector" proteins to subvert the host cells they invade and seek refuge within protective vacuoles. Here, we define the host component of the molecular arms race as an evolutionarily conserved polar "hot spot" on the PH domain of ELMO1 (Engulfment and Cell Motility protein 1), which is targeted by diverse WxxxE effectors. Using homology modeling and site-directed mutagenesis, we show that a lysine triad within the "patch" directly binds all WxxxE effectors tested: SifA (Salmonella), IpgB1 and IpgB2 (Shigella), and Map (enteropathogenic Escherichia coli). Using an integrated SifA-host protein-protein interaction network, in silico network perturbation, and functional studies, we show that the major consequences of preventing SifA-ELMO1 interaction are reduced Rac1 activity and microbial invasion. That multiple effectors of diverse structure, function, and sequence bind the same hot spot on ELMO1 suggests that the WxxxE effector(s)-ELMO1 interface is a convergence point of intrusion detection and/or host vulnerability. We conclude that the interface may represent the fault line in coevolved molecular adaptations between pathogens and the host, and its disruption may serve as a therapeutic strategy.

A circuit for secretion-coupled cellular autonomy in multicellular eukaryotic cells.

Cancers represent complex autonomous systems, displaying self-sufficiency in growth signaling. Autonomous growth is fueled by a cancer cell's ability to "secrete-and-sense" growth factors (GFs): a poorly understood phenomenon. Using an integrated computational and experimental approach, here we dissect the impact of a feedback-coupled GTPase circuit within the secretory pathway that imparts secretion-coupled autonomy. The circuit is assembled when the Ras-superfamily monomeric GTPase Arf1, and the heterotrimeric GTPase Giαßγ and their corresponding GAPs and GEFs are coupled by GIV/Girdin, a protein that is known to fuel aggressive traits in diverse cancers. One forward and two key negative feedback loops within the circuit create closed-loop control, allow the two GTPases to coregulate each other, and convert the expected switch-like behavior of Arf1-dependent secretion into an unexpected dose-response alignment behavior of sensing and secretion. Such behavior translates into cell survival that is self-sustained by stimulus-proportionate secretion. Proteomic studies and protein-protein interaction network analyses pinpoint GFs (e.g., the epidermal GF) as key stimuli for such self-sustenance. Findings highlight how the enhanced coupling of two biological switches in cancer cells is critical for multiscale feedback control to achieve secretion-coupled autonomy of growth factors.

Olaparib enhances the Resveratrol-mediated apoptosis in breast cancer cells by inhibiting the homologous recombination repair pathway.

Although sensitization of BRCA-mutated, homologous recombination (HR)-deficient breast cancer cells through PARP inhibitor is widely studied, not much is known about the treatment of BRCA-wild-type, HR-proficient breast cancer. Here, we aim to investigate whether a bioactive compound, Resveratrol (RES), can induce DNA double-strand breaks in HR-proficient breast cancer cells and Olaparib (OLA), a PARP inhibitor, can enhance the RES-mediated apoptosis by deregulating the HR repair pathway. The detailed mechanism of anti-cancer action of RES + OLA combination in breast cancer has been evaluated using in vitro, ex vivo, and in vivo preclinical model systems. OLA increased RES-mediated DNA damage, downregulated the HR pathway proteins, caused a late S/G2 cell cycle arrest, enhanced apoptosis and cell death in RES pre-treated breast cancer cells at much lower concentrations than their individual treatments. Direct measurement of HR pathway activity using a GFP plasmid-based assay demonstrated reduced HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. Moreover, RES + OLA treatment also caused significant reduction in PARP1-mediated PARylation and efficiently trapped PARP1 at the DNA damage site. Upon RES treatment, PARylated PARP1 was found to interact with BRCA1, which then activated other HR pathway proteins. But after addition of OLA in RES pre-treated cells, PARP1 could not interact with BRCA1 due to inhibition of PARylation. This resulted in deregulation of HR pathway. To further confirm the role of BRCA1 in PARP1-mediated HR pathway activation, BRCA1 was knocked down that caused complete inhibition of HR pathway activity, and further enhanced apoptosis after RES + OLA treatment in BRCA1-silenced cells. In agreement with in vitro data, similar experimental results were obtained in ex vivo patient-derived breast cancer cells and in vivo xenograft mice. Thus, RES + OLA combination treatment enhanced breast cancer cell death by causing excessive DNA damage and also simultaneously inhibiting the HR pathway.

Network approach to mutagenesis sheds insight on phage resistance in mycobacteria.

MOTIVATION: A rigorous yet general mathematical approach to mutagenesis, especially one capable of delivering systems-level perspectives would be invaluable. Such systems-level understanding of phage resistance is also highly desirable for phage-bacteria interactions and phage therapy research. Independently, the ability to distinguish between two graphs with a set of common or identical nodes and identify the implications thereof, is important in network science. RESULTS: Herein, we propose a measure called shortest path alteration fraction (SPAF) to compare any two networks by shortest paths, using sets. When SPAF is one, it can identify node pairs connected by at least one shortest path, which are present in either network but not both. Similarly, SPAF equalling zero identifies identical shortest paths, which are simultaneously present between a node pair in both networks. We study the utility of our measure theoretically in five diverse microbial species, to capture reported effects of well-studied mutations and predict new ones. We also scrutinize the effectiveness of our procedure through theoretical and experimental tests on Mycobacterium smegmatis mc2155 and by generating a mutant of mc2155, which is resistant to mycobacteriophage D29. This mutant of mc2155, which is resistant to D29 exhibits significant phenotypic alterations. Whole-genome sequencing identifies mutations, which cannot readily explain the observed phenotypes. Exhaustive analyses of protein-protein interaction network of the mutant and wild-type, using the machinery of topological metrics and differential networks does not yield a clear picture. However, SPAF coherently identifies pairs of proteins at the end of a subset of shortest paths, from amongst hundreds of thousands of viable shortest paths in the networks. The altered functions associated with the protein pairs are strongly correlated with the observed phenotypes. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Targeting cancer stem cells in the tumor microenvironment: An emerging role of PARP inhibitors.

Cancer stem cells (CSCs) constitute a small population of cancer cells in the tumor microenvironment (TME), which are responsible for metastasis, angiogenesis, drug resistance, and cancer relapse. Understanding the key signatures and resistance mechanisms of CSCs may help in the development of novel chemotherapeutic strategies to specifically target CSCs in the TME. PARP inhibitors (PARPi) are known to enhance the chemosensitivity of cancer cells to other chemotherapeutic agents by inhibiting the DNA repair pathways and chromatin modulation. But their effects on CSCs are still unknown. Few studies have reported that PARPi can stall replication fork progression in CSCs. PARPi also have the potential to overcome chemoresistance in CSCs and anti-angiogenic potentiality as well. Previous reports have suggested that epigenetic drugs can synergistically ameliorate the anti-cancer activities of PARPi through epigenetic modulations. In this review, we have systematically discussed the effects of PARPi on different DNA repair pathways with respect to CSCs and also how CSCs can be targeted either as monotherapy or as a part of combination therapy. We have also talked about how PARPi can help in reversal of chemoresistance of CSCs and the role of PARPi in epigenetic modifications to hinder cancer progression. We have also elaborated on the aspects of research that need to be investigated for development of successful therapeutic interventions using PARPi to specifically target CSCs in the TME.

NIR irradiation enhances the apoptotic potentiality of quinacrine-gold hybrid nanoparticles by modulation of HSP-70 in oral cancer stem cells.

Cancer stem cells (CSCs) are the tumor cell subpopulations that can self-renew, differentiate, initiate and maintain tumor growth. CSCs are frequently drug-resistant, resulting in tumor recurrence, metastasis, and angiogenesis. Herein, using in vitro oral squamous cell carcinoma (OSCC) CSCs and in vivo xenograft mice model, we have systematically studied the apoptotic potentiality of quinacrine-gold hybrid nanoparticle (QAuNP) and its underlying mechanism after NIR irradiation. QAuNP + NIR caused DNA damage and induced apoptosis in SCC-9-CSCs by deregulating mitochondrial membrane potential (ΔΨm) and activation of ROS. Upregulation of CASPASE-3 and DR-5/DR-4 and reduction of heat shock protein (HSP-70) up to 5-fold were also noticed upon the treatment. The increased expression of DR-5 and CASPASE-3 and decreased expression of HSP-70, CD-44 and Ki-67 were also noted in the xenograft mice treated with QAuNP + NIR + TRAIL. Thus, data suggest that the combined treatment enhances apoptosis in OSCC-CSCs by modulating HSP-70 in the DISC.

Modeling Bacteria-Phage Interactions and Its Implications for Phage Therapy.

Bacteriophages are more abundant than any other organism on our planet. The interaction of bacteriophages and bacteria and their coevolution is well known. In this chapter, we describe various aspects of modeling such systems and their dynamics. We explore their interaction in: (i) liquid media, which leads to well-mixed populations and (ii) solid media, where their interaction is spatially restricted. Such modeling, when used in conjunction with experiments would not only shed deep insight into the underlying dynamics but also provide useful clues toward potential therapeutic applications.

Dynamics of Mycobacteriophage-Mycobacterial Host Interaction: Evidence for Secondary Mechanisms for Host Lethality.

Mycobacteriophages infect mycobacteria, resulting in their death. Therefore, the possibility of using them as therapeutic agents against the deadly mycobacterial disease tuberculosis (TB) is of great interest. To obtain better insight into the dynamics of mycobacterial inactivation by mycobacteriophages, this study was initiated using mycobacteriophage D29 and Mycobacterium smegmatis as the phage-host system. Here, we implemented a goal-oriented iterative cycle of experiments on one hand and mathematical modeling combined with Monte Carlo simulations on the other. This integrative approach lends valuable insight into the detailed kinetics of bacterium-phage interactions. We measured time-dependent changes in host viability during the growth of phage D29 in M. smegmatis at different multiplicities of infection (MOI). The predictions emerging out of theoretical analyses were further examined using biochemical and cell biological assays. In a phage-host interaction system where multiple rounds of infection are allowed to take place, cell counts drop more rapidly than expected if cell lysis is considered the only mechanism for cell death. The phenomenon could be explained by considering a secondary factor for cell death in addition to lysis. Further investigations reveal that phage infection leads to the increased production of superoxide radicals, which appears to be the secondary factor. Therefore, mycobacteriophage D29 can function as an effective antimycobacterial agent, the killing potential of which may be amplified through secondary mechanisms.

Microtubule-stabilizing properties of the avocado-derived toxins (+)-(R)-persin and (+)-(R)-tetrahydropersin in cancer cells and activity of related synthetic analogs.

The avocado toxin (+)-R-persin (persin) is active at low micromolar concentrations against breast cancer cells and synergizes with the estrogen receptor modulator 4-hydroxytamoxifen. Previous studies in the estrogen receptor-positive breast cancer cell line MCF-7 indicate that persin acts as a microtubule-stabilizing agent. In the present study, we further characterize the properties of persin and several new synthetic analogues in human ovarian cancer cells. Persin and tetrahydropersin cause G2M cell cycle arrest and increase intracellular microtubule polymerization. One analog (4-nitrophenyl)-deshydroxypersin prevents cell proliferation and blocks cells in G1 of the cell cycle rather than G2M, suggesting an additional mode of action of these compounds independent of microtubules. Persin can synergize with other microtubule-stabilizing agents, and is active against cancer cells that overexpress the P-glycoprotein drug efflux pump. Evidence from Flutax-1 competition experiments suggests that while the persin binding site on ß-tubulin overlaps the classical taxoid site where paclitaxel and epothilone bind, persin retains activity in cell lines with single amino acid mutations that affect these other taxoid site ligands. This implies the existence of a unique binding location for persin at the taxoid site.

Targeting signaling pathways in cancer stem cells: A potential approach for developing novel anti-cancer therapeutics.

Cancer stem cells (CSCs) have emerged as prime players in the intricate landscape of cancer development, progression, and resistance to traditional treatments. These unique cellular subpopulations own the remarkable capability of self-renewal and differentiation, giving rise to the diverse cellular makeup of tumors and fostering their recurrence following conventional therapies. In the quest for developing more effective cancer therapeutics, the focus has now shifted toward targeting the signaling pathways that govern CSCs behavior. This chapter underscores the significance of these signaling pathways in CSC biology and their potential as pivotal targets for the development of novel chemotherapy approaches. We delve into several key signaling pathways essential for maintaining the defining characteristics of CSCs, including the Wnt, Hedgehog, Notch, JAK-STAT, NF-κB pathways, among others, shedding light on their potential crosstalk. Furthermore, we highlight the latest advancements in CSC-targeted therapies, spanning from promising preclinical models to ongoing clinical trials. A comprehensive understanding of the intricate molecular aspects of CSC signaling pathways and their manipulation holds the prospective to revolutionize cancer treatment paradigms. This, in turn, could lead to more efficacious and personalized therapies with the ultimate goal of eradicating CSCs and enhancing overall patient outcomes. The exploration of CSC signaling pathways represents a key step towards a brighter future in the battle against cancer.

FORWARD: A Learning Framework for Logical Network Perturbations to Prioritize Targets for Drug Development.

Despite advances in artificial intelligence (AI), target-based drug development remains a costly, complex and imprecise process. We introduce F.O.R.W.A.R.D [ Framework for Outcome-based Research and Drug Development ], a network-based target prioritization approach and test its utility in the challenging therapeutic area of Inflammatory Bowel Diseases (IBD), which is a chronic condition of multifactorial origin. F.O.R.W.A.R.D leverages real-world outcomes, using a machine-learning classifier trained on transcriptomic data from seven prospective randomized clinical trials involving four drugs. It establishes a molecular signature of remission as the therapeutic goal and computes, by integrating principles of network connectivity, the likelihood that a drug's action on its target(s) will induce the remission-associated genes. Benchmarking F.O.R.W.A.R.D against 210 completed clinical trials on 52 targets showed a perfect predictive accuracy of 100%. The success of F.O.R.W.A.R.D was achieved despite differences in targets, mechanisms, and trial designs. F.O.R.W.A.R.D-driven in-silico phase '0' trials revealed its potential to inform trial design, justify re-trialing failed drugs, and guide early terminations. With its extendable applications to other therapeutic areas and its iterative refinement with emerging trials, F.O.R.W.A.R.D holds the promise to transform drug discovery by generating foresight from hindsight and impacting research and development as well as human-in-the-loop clinical decision-making.

Growth factor-dependent phosphorylation of Gαi shapes canonical signaling by G protein-coupled receptors.

A long-standing question in the field of signal transduction is how distinct signaling pathways interact with each other to control cell behavior. Growth factor receptors and G protein-coupled receptors (GPCRs) are the two major signaling hubs in eukaryotes. Given that the mechanisms by which they signal independently have been extensively characterized, we investigated how they may cross-talk with each other. Using linear ion trap mass spectrometry and cell-based biophysical, biochemical, and phenotypic assays, we found at least three distinct ways in which epidermal growth factor affected canonical G protein signaling by the Gi-coupled GPCR CXCR4 through the phosphorylation of Gαi. Phosphomimicking mutations in two residues in the αE helix of Gαi (tyrosine-154/tyrosine-155) suppressed agonist-induced Gαi activation while promoting constitutive Gßγ signaling. Phosphomimicking mutations in the P loop (serine-44, serine-47, and threonine-48) suppressed Gi activation entirely, thus completely segregating growth factor and GPCR pathways. As expected, most of the phosphorylation events appeared to affect intrinsic properties of Gαi proteins, including conformational stability, nucleotide binding, and the ability to associate with and to release Gßγ. However, one phosphomimicking mutation, targeting the carboxyl-terminal residue tyrosine-320, promoted mislocalization of Gαi from the plasma membrane, a previously uncharacterized mechanism of suppressing GPCR signaling through G protein subcellular compartmentalization. Together, these findings elucidate not only how growth factor and chemokine signals cross-talk through the phosphorylation-dependent modulation of Gαi but also how such cross-talk may generate signal diversity.

Human-specific elimination of epithelial Siglec-XII suppresses the risk of inflammation driven colorectal cancers.

Carcinomas are common in humans but rare among closely related "great apes". Plausible explanations, including human-specific genomic alterations affecting the biology of sialic acids are proposed, but causality remains unproven. Here, an integrated evolutionary genetics-phenome-transcriptome approach studied the role of SIGLEC12 gene (encodes Siglec-XII) on epithelial transformation and cancer. Exogenous expression of the protein in cell lines and genetically engineered mice recapitulated ~30% of the human population in whom the protein is expressed in a form that cannot bind ligand due to a fixed, homozygous, human-universal missense mutation. Siglec-XII null cells/mice recapitulated the remaining ~70% of the human population in whom an additional polymorphic frameshift mutation eliminates the entire protein. Siglec-XII expression drove several pro-oncogenic phenotypes in cell lines, and increased tumor burden in mice challenged with chemical carcinogen and inflammation. Transcriptomic studies yielded a 29-gene signature of Siglec-XII-positive disease and when used as a computational tool for navigating human datasets, pinpointed with surprising precision that SIGLEC12 expression (model) recapitulates a very specific type of colorectal carcinomas (disease) that is associated with mismatch-repair defects and inflammation, disproportionately affects European-Americans, and carries a better prognosis. They revealed a hitherto unknown evolutionary genetic mechanism for an ethnic/environmental predisposition of carcinogenesis.

Resveratrol nanoparticles induce apoptosis in oral cancer stem cells by disrupting the interaction between ß-catenin and GLI-1 through p53-independent activation of p21.

Cancer stem cells (CSCs) are mainly responsible for tumorigenesis, chemoresistance, and cancer recurrence. CSCs growth and progression are regulated by multiple signaling cascades including Wnt/ß-catenin and Hh/GLI-1, which acts independently or via crosstalk. Targeting the crosstalk of signaling pathways would be an effective approach to control the CSC population. Both Wnt/ß-catenin and Hh/GLI-1 signaling cascades are known to be regulated by p53/p21-dependent mechanism. However, it is interesting to delineate whether p21 can induce apoptosis in a p53-independent manner. Therefore, utilizing various subtypes of oral CSCs (SCC9-PEMT p53+/+p21+/+, SCC9-PEMT p53-/-p21+/+, SCC9-PEMT p53+/+p21-/- and SCC9-PEMT p53-/-p21-/-), we have examined the distinct roles of p53 and p21 in Resveratrol nanoparticle (Res-Nano)-mediated apoptosis. It is interesting to see that, besides the p53/p21-mediated mechanism, Res-Nano exposure also significantly induced apoptosis in oral CSCs through a p53-independent activation of p21. Additionally, Res-Nano-induced p21-activation deregulated the ß-catenin-GLI-1 complex and consequently reduced the TCF/LEF and GLI-1 reporter activities. In agreement with in vitro data, similar experimental results were obtained in in vivo mice xenograft model.

Nano formulated Resveratrol inhibits PD-L1 in oral cancer cells by deregulating the association between tumor associated macrophages and cancer associated fibroblasts through IL-6/JAK2/STAT3 signaling axis.

Tumor associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) in the tumor microenvironment secrete several cytokines, which involved in tumor initiation, progression, metastatic outgrowth and angiogenesis. However, the association between TAMs and CAFs in the context of tumor development remain unclear. Here, we studied the relationship between TAMs and CAFs along with the involvement of cytokines in the production of cancer-stem-like-cells (CSCs) in oral cancer cells and explored the potential anticancer effects of Nano-formulated Resveratrol (Res-NP) using an activated macrophage-M1 (AM-M1) and activated fibroblast cells as the model system. IL-6 secretion was found to be enhanced in the conditioned-medium (CM) when AM-M1 cells + CAFs-like cells were cocultured together. CSCs-enriched population was developed after addition of CM of AM-M1 +CAFs in H-357 cells and patient-derived-primary-oral-cancer cells. AM-M1 cells+ CAFs-like cells secreted IL-6 enhanced CSCs growth, proliferation, metastasis, and angiogenesis. IL-6 was found to promote PD-L1 expression in CSCs-enriched cells via JAK2/STAT3 pathway, as evident from the enhanced expression of p-JAK2 and p-STAT3. Nevertheless, Res-NP inhibited CSCs proliferation and reduced the expression of metastatic and angiogenic markers, in ovo blood vascularization, NO production and MMPs expression. Res-NP delinked the association between AM-M1 and CAFs by blocking IL-6 production and also disrupted the potential connection between IL-6 and PD-L1 with considerable decrease in p-JAK2 and p-STAT3 expressions. IL-6 depletion inhibited stemness and angiogenesis in oral CSCs by downregulating PD-L1 via JAK2/STAT3 cascade. Similar observations were also observed in Res-NP treated xenograft mice. Thus, data demonstrate that CSCs growth is dependent on IL-6/PD-L1 axis. Res-NP deregulates the association between AM-M1 and CAFs along-with attenuates carcinogenesis in in vitro, in ovo, ex vivo and in vivo model systems by inhibiting PD-L1 via IL-6/JAK2/STAT3 axis.

Growth signaling autonomy in circulating tumor cells aids metastatic seeding.

Self-sufficiency (autonomy) in growth signaling, the earliest recognized hallmark of cancer, is fueled by the tumor cell's ability to "secrete-and-sense" growth factors (GFs); this translates into cell survival and proliferation that is self-sustained by autocrine/paracrine secretion. A Golgi-localized circuitry comprised of two GTPase switches has recently been implicated in the orchestration of growth signaling autonomy. Using breast cancer cells that are either endowed or impaired (by gene editing) in their ability to assemble the circuitry for growth signaling autonomy, here we define the transcriptome, proteome, and phenome of such an autonomous state, and unravel its role during cancer progression. We show that autonomy is associated with enhanced molecular programs for stemness, proliferation, and epithelial-mesenchymal plasticity. Autonomy is both necessary and sufficient for anchorage-independent GF-restricted proliferation and resistance to anticancer drugs and is required for metastatic progression. Transcriptomic and proteomic studies show that autonomy is associated, with a surprising degree of specificity, with self-sustained epidermal growth factor receptor (EGFR)/ErbB signaling. Derivation of a gene expression signature for autonomy revealed that growth signaling autonomy is uniquely induced in circulating tumor cells (CTCs), the harshest phase in the life of tumor cells when it is deprived of biologically available epidermal growth factor (EGF). We also show that autonomy in CTCs tracks therapeutic response and prognosticates outcome. These data support a role for growth signaling autonomy in multiple processes essential for the blood-borne dissemination of human breast cancer.

Near infrared-responsive quinacrine-gold hybrid nanoparticles deregulate HSP-70/P300-mediated H3K14 acetylation in ER/PR+ breast cancer stem cells.

Aim: This study aimed to determine if quinacrine-gold hybrid nanoparticles (QAuNPs) + near-infrared (NIR) deregulate HSP-70/P300 complex-mediated H3K14 acetylation in estrogen receptor/progesterone receptor (ER/PR+) breast cancer stem cells (CSCs). Materials & methods: Various cells and mouse-based systems were used as models. Results: QAuNP + NIR treatment reduced the nuclear translocation of HSP-70, affected the histone acetyltransferase activity of P300 and specifically decreased H3K14 acetylation in ER/PR+ breast CSCs. Finally, HSP-70 knockdown showed a reduction in P300 histone acetyltransferase activity, decreased H3K14 acetylation and inhibited activation of the TGF-ß gene. Conclusion: This study revealed that QAuNP + NIR irradiation inhibits oncogenic activation of the TGF-ß gene by decreasing H3K14 acetylation mediated through the HSP-70/P300 nuclear complex in ER/PR+ breast CSCs.

Combination of Resveratrol and PARP inhibitor Olaparib efficiently deregulates homologous recombination repair pathway in breast cancer cells through inhibition of TIP60-mediated chromatin relaxation.

Recently, we reported that a combination of a natural, bioactive compound Resveratrol (RES) and a PARP inhibitor Olaparib (OLA) deregulated the homologous recombination (HR) pathway, and enhanced apoptosis in BRCA1-wild-type, HR-proficient breast cancer cells. Upon DNA damage, chromatin relaxation takes place, which allows the DNA repair proteins to access the DNA lesion. But whether chromatin remodeling has any role in RES + OLA-mediated HR inhibition is not known. By using in vitro and ex vivo model systems of breast cancer, we have investigated whether RES + OLA inhibits chromatin relaxation and thereby blocks the HR pathway. It was found that RES + OLA inhibited PARP1 activity, terminated PARP1-BRCA1 interaction, and deregulated the HR pathway only in the chromatin fraction of MCF-7 cells. DR-GFP reporter plasmid-based HR assay demonstrated marked reduction in HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. RES + OLA efficiently trapped PARP1 at the DNA damage site in the chromatin of MCF-7 cells. Unaltered expressions of HR proteins were found in the chromatin of PARP1-silenced MCF-7 cells, which confirmed that RES + OLA-mediated DNA damage response was PARP1-dependent. Histone Acetyltransferase (HAT) activity and histone H4 acetylation assays showed reduction in HAT activity and H4 acetylation in RES + OLA-treated chromatin fraction of cells. Western blot analysis showed that the HAT enzyme TIP60, P400 and acetylated H4 were downregulated after RES + OLA exposure. In the co-immunoprecipitation assay, it was observed that RES + OLA caused abolition of PARP1-TIP60-BRCA1 interaction, which suggested the PARP1-dependent TIP60-BRCA1 association. Unaltered expressions of PAR, BRCA1, P400, and acetylated H4 in the chromatin of TIP60-silenced MCF-7 cells further confirmed the role of TIP60 in PARP1-mediated HR activation in the chromatin. Similar results were obtained in ex vivo patient-derived primary breast cancer cells. Thus, the present study revealed that RES + OLA treatment inhibited PARP1 activity in the chromatin, and blocked TIP60-mediated chromatin relaxation, which, in turn, affected PARP1-dependent TIP60-BRCA1 association, resulting in deregulation of HR pathway in breast cancer cells.

Machine-Learning Identifies a Strategy for Differentiation Therapy in Solid Tumors.

BACKGROUND: Although differentiation therapy can cure some hematologic malignancies, its curative potential remains unrealized in solid tumors. This is because conventional computational approaches succumb to the thunderous noise of inter-/intratumoral heterogeneity. Using colorectal cancers (CRCs) as an example, here we outline a machine learning(ML)-based approach to track, differentiate, and selectively target cancer stem cells (CSCs). METHODS: A transcriptomic network was built and validated using healthy colon and CRC tissues in diverse gene expression datasets (~5,000 human and >300 mouse samples). Therapeutic targets and perturbation strategies were prioritized using ML, with the goal of reinstating the expression of a transcriptional identifier of the differentiated colonocyte, CDX2, whose loss in poorly differentiated (CSC-enriched) CRCs doubles the risk of relapse/death. The top candidate target was then engaged with a clinical-grade drug and tested on 3 models: CRC lines in vitro, xenografts in mice, and in a prospective cohort of healthy (n = 3) and CRC (n = 23) patient-derived organoids (PDOs). RESULTS: The drug shifts the network predictably, induces CDX2 and crypt differentiation, and shows cytotoxicity in all 3 models, with a high degree of selectivity towards all CDX2-negative cell lines, xenotransplants, and PDOs. The potential for effective pairing of therapeutic efficacy (IC50) and biomarker (CDX2-low state) is confirmed in PDOs using multivariate analyses. A 50-gene signature of therapeutic response is derived and tested on 9 independent cohorts (~1700 CRCs), revealing the impact of CDX2-reinstatement therapy could translate into a ~50% reduction in the risk of mortality/recurrence. CONCLUSIONS: Findings not only validate the precision of the ML approach in targeting CSCs, and objectively assess its impact on clinical outcome, but also exemplify the use of ML in yielding clinical directive information for enhancing personalized medicine.