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Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, our understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, we have identified binding regions for the Hox TF Deformed (Dfd) in the Drosophila genome. Our analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. We also find that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, our results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo.

In this Editorial, our Chief Editor and members of our Advisory Editorial Board discuss recent breakthroughs, current challenges, and emerging opportunities in single-cell biology and share their vision of "where the field is headed."

Integrins are heterodimeric cell surface glycoproteins that bind cells to extracellular matrix (ECM) and regulate tumour cell proliferation, migration and survival. We have previously observed that de novo expression of integrin αVβ3 in Cal27-derived cell clone 2B1 conferred resistance to several anticancer drugs (cisplatin, mitomycin C and doxorubicin) through the mechanism which involves downregulation of pSrc(Y418) and increased cell migration and invasion. In addition, in 2B1 cells we observed increased expression of integrin αVβ5 whose expression was unrelated to drug resistance (Stojanović et al., BBA-MCR, 2016). Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) are formed, creating links to the cell cytoskeleton. Functional and morphological analyses have defined major forms of IACs, including focal adhesions, fibrillar adhesions, hemidesmosomes and reticular adhesions. Several protocols have been developed to purify ventral IACs from 2D cell cultures, but most preparations have been isolated from cells seeded on fibronectin which led to the definition of a FN- induced adhesion components. In order to further analyse the adhesion components whose differential expression contributes to the observed changes in Cal27 and 2B1 cell adhesion and anticancer drug resistance, we isolated IACs from both cell lines cultured on dishes without prior coating with ECM proteins enabling us to detect adhesion components and various ECM proteins secreted by the cells themselves. Mass spectrometry (MS)-based proteomics analysis showed that both cell lines preferentially use integrin α6β4 for adhesion. Anticancer drug resistant cell clone 2B1 show increased level of α6β4 accompanied with increased deposition of laminin 332 but decreased level of keratins 5 and 14 (KRT-5/14). Validation of MS data using western blot, immunofluorescence and electron microscopy experiments demonstrated formation of type II hemidesmosomes which are not linked to KRT-5/14. Keratins 5/14 are very likely part of desmosomes whose levels are reduced in 2B1 cells. The downregulation of integrin α6β4 heterodimer achieved by β4 subunit knockdown conferred resistance to anticancer drugs of both, Cal27 and 2B1 cells, revealing another mechanism of drug resistance. Taken together, our results show multiple integrin crosstalk events triggered by αVβ3 expression i.e. upregulation of αVβ5 and α6β4, and the role of α6β4 in regulating anticancer drug sensitivity.

INTRODUCTION: Lack of differentiation is a hallmark of acute myeloid leukemia (AML), making differentiation therapy a promising treatment strategy. Several novel differentiation targets have been recently identified, including dihydroorotate dehydrogenase (DHODH), one of the key enzymes in de novo pyrimidine synthesis. Our recent studies demonstrated that pyrimidine synthesis inhibitors 5- aminoimidazole-4- carboxamide ribonucleoside (AICAr), a UMP synthase inhibitor, and brequinar, a DHODH inhibitor, induce differentiation of leukemia cells by activating the ataxia telangiectasia and RAD3related (ATR)/checkpoint kinase 1 (Chk1) 29 Systems approches in cacer DNA-damage signaling pathway via pyrimidine depletion. Cytarabine (cytosine arabinoside or AraC) has been the mainstay of the standard first-line induction therapy in adults with AML for the last several decades, as well as one of the main medications used in low-dose regimens administered to patients unfit for intensive chemotherapy. Apart from its cytotoxic effects, monocytic or granulocytic differentiation in response to cytarabine have been confirmed in several AML cell lines. However, the mechanism responsible for cytarabine-induced differentiation remained incompletely understood. Cytarabine interference with the process of DNA synthesis leads to DNA damage by staling replication forks, and ATR/Chk1 pathway is known to promote survival by signaling replication stress. In this study, we tested the role of Chk1 in differentiation of AML cells induced by cytarabine and compared the effects of cytarabine with the effects of AICAr and brequinar. MATERIALS AND METHODS: Human AML cell lines U937 and THP-1 were purchased from ECACC (UK) and DSMZ (Germany). Non adherent mononuclear cells from bone marrow of five AML patients were obtained by density gradient separation, purified by overnight adherence to plastic, and seeded in liquid medium containing 50 ng/mL interleukin-3, interleukin-6, stem cell factor and FLT3-ligand. Cells were collected after obtaining patients’ written informed consent in accordance with the Declaration of Helsinki and approval obtained by the University Hospital Centre Zagreb Institutional ethic committee. Cells were incubated in the presence of cytarabine (10, 100, 1000 nM), AICAr (0.2 mM), brequinar (0.5 µM), and pharmacological inhibitors of ATR/Chk1, Torin2 (10 nM, 100 nM) or VE-821 (2 µM, 10 µM). The number of viable cells was determined by trypan blue exclusion. The expression of differentiation markers, as well as DNA content for cell cycle analysis were determined by flow cytometry (Attune, Life Technologies) and FlowJo software. siRNA transfections were performed using siRNA targeting Chk1 (Dharmacon) and NeonTM transfection system (Invitrogen). Total cell lysates were analyzed for the level of Chk1, Ser-345-p-Chk1, Tyr-15-p-CDC2 and β actin by Western blot. Statistical analysis was performed using Student’s t-test (p < 0.05). RESULTS: The effects of cytarabine on cell viability and the expression of differentiation markers in monocytic leukemia cells are similar to the effects of AICAr and brequinar. Cytarabine dose-dependently decreases cell number and induces expression of differentiation markers CD11b and CD64 in AML cell lines. Western blot analysis revealed the increase in the level of Ser-345-phosphorylated Chk1, a marker of Chk1 activation, in response to cytarabine. Cytarabine, AICAr and brequinar increased the level of inhibitory Tyr-15- phosphorylation of cyclin-dependent kinase 1 (Cdk1, CDC2), suggesting that CDC2 inactivation is an important downstream target of Chk1 in cell cycle restriction. Pharmacological inhibition of ATR/Chk1 pathway by Torin2 and VE821 reduced differentiative effects and cell cycle arrest in response to cytarabine, AICAr and brequinar. Moreover, transfection of U937 cells with siRNA targeting Chk1 reduced differentiative effects of all agents tested. In addition, cytarabine dose- dependently induced differentiation in two primary AML samples that were responsive to pyrimidine synthesis inhibitors. CONCLUSION: Low dose cytarabine-mediated differentiation of AML cells occurs through the activation of DNA damage checkpoint kinase Chk1.

Cancer whole-genome sequencing provided new insights into the mutational landscape of various tumors, processes that drive their development, and how to exploit these genetic instabilities for a personalized therapeutic approach. The most common and widely accepted way for designing a specific therapy for each patient involves identifying certain somatic or germline mutations in genes like DNA-repair genes. Such treatments include the best-known example of poly ( ADP- ribose) polymerase (PARP) inhibitors in patients with deficient homologous recombination (HR) pathways. In the clinics, several PARP inhibitors have demonstrated improvement in progression-free survival in patients harboring mutations in certain HRrelated genes. Nowadays, there are four FDA-approved PARP inhibitors for treatments of patients with pathogenic BRCA1/2 somatic or germline mutation. Despite the success of selecting and treating HRdeficient patients based on testing for pathogenic BRCA1/2 mutations, there are patients sensitive to PARP inhibitors with a similar phenotype to HR-deficient tumors but are lacking mutations in BRCA genes. An increasing number of patients that can benefit from PARP inhibitor therapy can now be discovered by the latest methods based on the WGS analysis. Different genomic scars and damages, from mutational signatures to large-scale transition, have been associated in cancers with HR-deficient tumors harboring BRCA1/2 mutations . Two tools, CHORD and HRDetect, based on machine-learning approaches utilize these different genomic features of HR- deficient cancers to estimate the probability of a certain tumor to be HR-deficient. Both of these tools have a high accuracy of above 90% for detecting HR-deficient tumors, however, their application in clinical diagnostics needs to be validated further across a variety of tumors. Here we have benchmarked CHORD and HRDetect prediction for breast, pancreatic, ovarian, or prostate cancer patients from previous studies. The goal was to assess the performance of these classifiers and to investigate new alterations in genes related to HR deficiency. For each patient, we looked into the biallelic inactivation of HR- related genes alongside the promoter hypermethylation of those genes to evaluate the performance of the two tools. The average sensitivity across these four tumor types was above 98% (area under the curve (AUC) = 0.98) with both tools. The majority of HR-deficient samples contained BRCA1/2 germline mutations followed by BRCA1/2 somatic mutations. The promoter methylation status of BRCA1 genes in breast and ovarian cancer patients with HR deficiency was shown as a valuable resource in determining a tumor's HR status. These samples do not contain clear pathogenic variants in HR-related genes and therefore would not receive the appropriate therapy using standard targeted panel sequencing in clinical diagnostics. Using 10-fold nested cross-validation we estimated the optimal threshold values for each cancer type in both tools since the threshold for determining HR status has not been optimized for specific cancer types. Although some optimal thresholds may significantly vary from the default ones, the accuracy in majority tumors was the same or higher for about 2-3% except for the optimal HRDetect threshold for pancreatic cancer where an increase was 9%. Alongside using these WGS approaches in improving clinical diagnostics, they can expand existing knowledge of gene repertoire associated with HR defects tumors in those samples declared to be HR deficient by these tools.