The enzyme aromatase (CYP19A1) is an important target for the discovery of new therapeutic drugs against breast cancer. A series of novel heteroaryl steroidal derivatives, C-2- and C-16-thiadiazole-substituted estranes, were synthesized and biologically evaluated as potential aromatase inhibitors. These compounds exhibited cytotoxicity against breast cancer cells with IC50 values of 5.5 µM and higher. The lead compound, 2-(2'-P(O)(NHtBu)2-1',3',4'-thiadiazole)-Δ1,3,5(10)-estratrien-17-one, was shown to be selective against MCF7 cells with IC50 (MCF7) = 5.5 µM vs IC50 (MDA-MB-231) = 26.1 µM. It acts as both a potent selective agent blocking CYP19A1 and an effective apoptosis inducer, with no effects on the hormone receptors ERα and AR. The toxicity of the lead compound against normal epithelial cells has not been revealed. Molecular docking was used to more accurately define the binding mode of the lead compound to CYP19A1. In a word, here we describe a novel heteroaryl steroidal derivative promising as a CYP19A1 inhibitor for the treatment of hormone-dependent breast cancer.

Anti-cluster-of-differentiation (anti-CD) antibody microarray is an attractive multiplex analogue to leukocyte immunocytochemistry for surface markers. The transparent plastic microarray support permits to sort leukocytes by their surface lineage-specific markers making them available for subsequent high-resolution morphology examination. The combined data on the pathologic cells’ immunophenotype, morphology and cytochemistry from the microarray is sufficient to suggest preliminary diagnosis in many leukemia types. However, in some cases additional staining for internal markers is required, i.e. in T cell acute leukemia the T cell origin of the blasts can only be proved by the presence of cytoplasmic CD3 (cytCD3). Here we describe a new protocol of immunocytochemical detection of internal proteins in mononuclear cells captured by mouse anti-CD antibodies on the microarray surface. The protocol uses primary mouse FITC-conjugated (FITC – fluorescein isothiocyanate) antibodies against the target, secondary anti-FITC antibodies conjugated with alkaline phosphatase and colorimetric alkaline phosphatase substrate, BCIP/NBT. We show on normal leukocytes and leukemic cell lines that the protocol is sensitive and specific. The percentages of cytCD3-positive cells determined by this method in bone marrow aspirates of two patients with T cell acute lymphoblastic leukemia are in excellent agreement with flow cytometry results. This method expands the diagnostic capabilities of anti-CD antibody microarray.

Modulation of the molecular chaperone HSP90 represents a promising therapeutic strategy in oncology. As a continuation of our research aimed at designing new HSP90 modulators based on the 6,7-dihydrobenzo[d]isoxazol-4(5H)-one scaffold, we established a strategy for the synthesis of a novel class of compounds: N-(4,5,6,7-tetrahydrobenzo[d]isoxazol-5-yl)carboxamides. A series of such substances was synthesized and evaluated for antiproliferative activity against breast cancer cell lines. Several compounds exhibited potent activity in the low micromolar to submicromolar range. The lead molecule, 5c, demonstrated an IC50 of 0.45 & micro;M in HER2+ HCC1954 cells, where it effectively suppressed the expression of HER2 and p-HER2, as well as p-c-MET and p-EGFR. The results suggest a dual mechanism of action, involving both HSP90 modulation and direct inhibition of oncogenic kinases, as supported by molecular docking and molecular dynamics studies. Moreover, the potential of compound 5c in combination with established antiproliferative agents was assessed in a cellular model, highlighting its translational relevance. Collectively, our results identify N-(4,5,6,7-tetrahydrobenzo[d]isoxazol-5-yl)carboxamides as a promising class of multitarget anticancer agents, providing a foundation for the development of next-generation inhibitors acting on both HSP90 and oncogenic kinases.

Novel N-(4,5,6,7-tetrahydrobenzisoxazol-4-yl)amide derivatives were designed and synthesized as potential HSP90 inhibitors. The synthetic pathway commenced with 6,7-dihydrobenzo[d]isoxazol-4(5H)-ones, utilizing the Ritter reaction as a key step. Molecular docking, molecular dynamics simulations, and MM/GBSA analysis guided the selection of compounds for synthesis and provided insights into the interaction mode of the most active compound with HSP90α. The synthesized compounds exhibited significant antiproliferative effects against breast cancer cell lines ERα+ MCF7 and HER2+ HCC1954. Lead compounds with submicromolar IC50 values, initially synthesized as racemates, were subsequently obtained and tested in their enantiopure forms. In HER2+ HCC1954 cancer cells, the molecular pathways regulated by compound (R)-8n were characterized. Treatment with compound (R)-8n resulted in the pronounced suppression of HSP90-related pathways, including key oncoreceptors (HER2, EGFR, c-MET) and mitogenic kinases (AKT, CDK4). Additionally, compound (R)-8n induced apoptosis, as evidenced by the accumulation of cleaved PARP. The inhibitory effect of compound (R)-8n on the HSP90 pathway was corroborated by molecular modeling and further validated through the observed suppression of client proteins, along with an upregulation of HSP70, a well-established marker of HSP90 inhibition. The activity of compound (R)-8n was associated with cell cycle arrest at the G2/M phases, ultimately leading to dose-dependent cell death. Notably, compound (R)-8n demonstrated substantial selectivity toward breast tumor cells. These findings suggest that N-(4,5,6,7-tetrahydrobenzisoxazol-4-yl)amides represent a promising class of HSP90 inhibitors for anticancer therapy.

Background/Objectives: Breast cancer remains one of the most prevalent and life-threatening malignancies worldwide. This study describes the design and biological evaluation of a series of secosteroid-2-pyrazoline hybrids as novel antitumor agents against ERα-positive breast cancer cell lines MCF-7 and T47D. Methods: A simple and efficient method for synthesizing secosteroid-2-pyrazoline hybrids was developed starting from 13α-hydroxy-3-methoxy-13,17-secoestra-1,3,5(10)-triene-17-oic acid hydrazide and 1,3-diketones. The resulting secosteroid derivatives were evaluated against hormone-dependent MCF-7 and T47D breast cancer cells. Furthermore, the selectivity and effects of three lead compounds on signaling pathways in MCF-7 cells were examined. Flow cytometry was used to assess the cell-cycle distribution of MCF-7 cells treated with the lead compound. Results: Among the synthesized hybrids, compounds 3f, 3j, and 3k exhibited potent antiproliferative activity with IC50 values of 0.2-0.5 μM against breast cancer cells, while demonstrating very low cytotoxicity towards normal cells (IC50 > 25 μM), indicating a favorable safety profile. The antitumor activity of lead compound 3j was additionally investigated in combination with standard chemotherapeutics, docetaxel and doxorubicin, yielding synergistic effects. The lead compounds showed a dual mechanism of action by inhibiting S6 kinase and promoting Bcl-2 phosphorylation at 0.9 μM, without significantly affecting hormonal breast cancer targets such as ERα, GREB1, and AR. Compound 3j induced apoptosis accompanied by a reduction of the G1/G0 phase in MCF-7 cells. Conclusions: These findings highlight secosteroid-2-pyrazoline hybrids as promising candidates for the development of next-generation breast cancer therapeutics targeting apoptosis and S6K signaling pathways.

Mitotic inhibitors, such as Vinca alkaloids and taxanes, are one of the most effective chemotherapeutic agents used in the clinic. Despite their advantages, there are drawbacks to their use - primarily development of resistance and a high rate of side-effects, including damage to non-proliferating tissues. A range of new inhibitors targeting mitosis, whose activity does not depend on the binding to tubulin, are currently tested in clinical trials. Among such agents, inhibitors of Eg5 kinesin are highly promising due to their high activity and specificity. Here we show that compared to other drugs that target mitosis, an Eg5 inhibitor, SB743921, preferentially eliminates TP53-mutated cells and induces irreversible senescence, even after the drug washout, regardless of the p53 status. These effects are not defined by the immediate block of mitosis where SB743921 and a clinically used mitotic inhibitor Ixabepilone induce similar rates of mitotic arrest, apoptosis and induction of p53 and p21, but rather a long-term reaction, with absence of proteins required for replication, such as Cyclin A, E2F1, pRB. While after the washout Ixabepilone-treated cells can exit senescence and resume proliferation, cells treated with SB743921 did not exit the senescent state and did not resume proliferation as based on SA-beta-galactosidase staining and EdU incorporation. The remaining senescent cells were effectively eliminated by Bcl2/Bcl-xL/Bcl-w inhibitor ABT-263, showing a potential of the combinational therapy with senolytic drugs. In total, we show the capacity of Eg5-targeting drugs for therapy of high-risk TP53-mutated tumors, which are potentially resistant to clinically approved mitotic inhibitors. ### Competing Interest Statement The authors have declared no competing interest.

CDK8 and CDK19 paralogs are regulatory kinases associated with the transcriptional Mediator complex. We have generated mice with the systemic inducible Cdk8 knockout on the background of Cdk19 constitutive knockout. Cdk8/19 double knockout (iDKO) males, but not single Cdk8 or Cdk19 KO, had an atrophic reproductive system and were infertile. The iDKO males lacked postmeiotic spermatids and spermatocytes after meiosis I pachytene. Testosterone levels were decreased whereas the amounts of the luteinizing hormone were unchanged. Single-cell RNA sequencing showed marked differences in the expression of steroidogenic genes (such as Cyp17a1, Star, and Fads ) in Leydig cells concomitant with alterations in Sertoli cells and spermatocytes, and were likely associated with an impaired synthesis of steroids. Star and Fads were also downregulated in cultured Leydig cells after iDKO. The treatment of primary Leydig cell culture with a CDK8/19 inhibitor did not induce the same changes in gene expression as iDKO, and a prolonged treatment of mice with a CDK8/19 inhibitor did not affect the size of testes. iDKO, in contrast to the single knockouts or treatment with a CDK8/19 kinase inhibitor, led to depletion of cyclin C (CCNC), the binding partner of CDK8/19 that has been implicated in CDK8/19-independent functions. This suggests that the observed phenotype was likely mediated through kinase-independent activities of CDK8/19, such as CCNC stabilization.

Imatinib mesylate (IM) and other BCR-ABL tyrosine kinase inhibitors (BCR-ABLi) are the mainstay of chronic myelogenous leukemia (CML) treatment. However, activation of circumventing signaling pathways and quiescence may limit BCR-ABLi efficacy. CDK8/19 Mediator kinases have been implicated in the emergence of non-genetic drug resistance. Dissecting the effects of pharmacological CDK8/19 inhibition on CML survival in response to BCR-ABLi, we found that a selective, non-toxic CDK8/19 inhibitor (CDK8/19i) Senexin B (SenB) and other CDK8/19i sensitized K562 cells to different BCR-ABLi via attenuation of cell cycle arrest. In particular, SenB prevented IM-induced upregulation of genes that negatively regulate cell cycle progression. SenB also antagonized IM-activated p27Kip1 elevation thereby diminishing the population of G1-arrested cells. After transient G1 arrest, cells treated with IM + SenB re-entered the S phase, where they were halted and underwent replicative stress. Consequently, the combination of IM and SenB intensified apoptotic cell death, measured by activation of caspase 9 and 3, subsequent cleavage of poly(ADPriboso)polymerase 1, positive Annexin V staining and increase of subG1 fraction. In contrast, IM-treated BCR-ABL-positive KU812 CML cells, which did not induce p27Kip1, readily died regardless of SenB treatment. Thus, CDK8/19i prevent the quiescence-mediated escape from BCR-ABLi-induced apoptosis, suggesting a strategy for avoiding the CML relapse.

Introduction: The design and development of antitumor compounds based on an isatin core led to the synthesis of 1-substituted isatin-5-sulfonamides with potent antiproliferative activity [...]

Replication stress (RS) is a characteristic state of cancer cells as they tend to exchange precision of replication for fast proliferation and increased genomic instability. To overcome the consequences of improper replication control, malignant cells frequently inactivate parts of their DNA damage response (DDR) pathways (the ATM-CHK2-p53 pathway), while relying on other pathways which help to maintain replication fork stability (ATR-CHK1). This creates a dependency on the remaining DDR pathways, vulnerability to further destabilization of replication and synthetic lethality of DDR inhibitors with common oncogenic alterations such as mutations of TP53, RB1, ATM, amplifications of MYC, CCNE1 and others. The response to RS is normally limited by coordination of cell cycle, transcription and replication. Inhibition of WEE1 and PKMYT1 kinases, which prevent unscheduled mitosis entry, leads to fragility of under-replicated sites. Recent evidence also shows that inhibition of Cyclin-dependent kinases (CDKs), such as CDK4/6, CDK2, CDK8/19 and CDK12/13 can contribute to RS through disruption of DNA repair and replication control. Here, we review the main causes of RS in cancers as well as main therapeutic targets—ATR, CHK1, PARP and their inhibitors.

In mammals, a large number of proteins are expressed as more than one isoform, resulting in the increased diversity of their proteome. Understanding the functions of isoforms is very important, since individual isoforms of the same protein can have oncogenic or pathogenic properties, or serve as disease markers. The high homology of isoforms with ubiquitous expression makes it difficult to study them. In this work, we propose a new approach for the study of protein isoforms in mammalian cells, which makes it possible to individually detect and investigate the functions of an individual isoform. The approach was developed to study the functions of isoforms of the PHF10 protein, a chromatin subunit of the PBAF remodeling complex. We demonstrated the possibility of induced simultaneous suppression of all endogenous PHF10 isoforms and the expression of a single recombinant FLAG-tagged isoform. For this purpose, we created constructs based on the pSLIK plasmid with a cloned cassette containing the recombinant gene of interest and miR30 with the corresponding shRNAs. The doxycycline-induced activation of the cassette allows on and off switching. Using this construct, we achieved the preferential expression of only one recombinant PHF10 isoform with a simultaneously reduced number of all endogenous isoforms. Our approach can be used to study the role of point mutations, the functions of individual domains and important sites, or to individually detect untagged isoforms with knockdown of all endogenous isoforms.

The Polybromo-associated BAF (BRG1- or BRM-associated factors) (PBAF) chromatin-remodeling complex is essential for transcription in mammalian cells. In this study, we describe a novel variant of the PBAF complex from differentiated neuronal cells, called dcPBAF, that differs from the canonical PBAF existing in proliferating neuroblasts. We describe that in differentiated adult neurons, a specific subunit of PBAF, PHF10, is replaced by a PHF10 isoform that lacks N- and C-terminal domains (called PHF10D). In addition, dcPBAF does not contain the canonical BRD7 subunit. dcPBAF binds promoters of the actively transcribed neuron-specific and housekeeping genes in terminally differentiated neurons of adult mice. Furthermore, in differentiated human neuronal cells, PHF10D-containing dcPBAF maintains a high transcriptional level at several neuron-specific genes.

The promising antitumor effects of progesterone derivatives have been identified in many studies. However, the specific mechanism of action of this class of compounds has not been fully described. Therefore, in this study, we investigated the antiproliferative and (anti)estrogenic activities of novel pentacyclic derivatives and benzylidenes of the progesterone series. The antiproliferative effects of the compounds were evaluated on hormone-dependent MCF7 breast cancer cells using the MTT test. Estrogen receptor α (ERα) activity was assessed by a luciferase-based reporter assay. Immunoblotting was used to evaluate the expression of signaling proteins. All benzylidenes demonstrated inhibitory effects with IC50 values below 10 µM, whereas pentacyclic derivatives were less active. These patterns may be associated with the lability of the geometry of benzylidene molecules, which contributes to an increase in the affinity of interaction with the receptor. The selected compounds showed significant anti-estrogenic potency. Benzylidene 1d ((8 S,9 S,10R,13 S,14 S,17 S)-17-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15-decahydrocyclopenta[a]phenanthren-3-one) was the most active in antiproliferative and anti-estrogenic assays. Apoptosis induced by compound 1d was accompanied by decreases in CDK4, ERα, and Cyclin D1 expression. Compounds 1d and 3d were characterized by high inhibitory potency against resistant breast cancer cells. Apoptosis induced by the leader compounds was confirmed by PARP cleavage and flow cytometry analysis. Compound 3d caused cell arrest in the G2/M phase. Further analysis of novel derivatives of the progesterone series is of great importance for medicinal chemistry, drug design, and oncology.

Опухолевой супрессор p53 является центральным звеном защиты клетки от злокачественной трансформации. Мутации кодирующего гена TP53 наблюдаются приблизительно в половине опухолей человека и способствуют не только опухолевой прогрессии, но также устойчивости или чувствительности к противоопухолевым препаратам. Получение изогенных линий, имеющих разный статус TP53, необходимо не только для исследования его роли, но и для скрининга новых противоопухолевых препаратов и их комбинаций. Получение изогенных моделей с помощью системы CRISPR/Cas9 часто связано с одноклеточным клонированием, что приводит к клональным эффектам из-за гетерогенности опухолевых культур. В данной работе описано получение новых нокаутов TP53 в линиях MCF7 и A549 с помощью системы CRISPR/Cas9 и отбора по устойчивости к нутлину-3, позволяющей отбирать нокаутные клетки без этапа клонального отбора. Фенотипически нокаут был подтвержден по полному отсутствию белка p53, снижению экспрессии p53-зависимого гена CDKN1A и изменению чувствительности к ДНК-повреждающим противоопухолевым препаратам. The tumor suppressor p53 is the central point of cellular defense against oncogenic transformation. Mutations of TP53 gene are present in approximately half of human tumors and promote not only tumor progression, but also resistance to anticancer drugs. Creation of isogenic models, differing in their TP53 status, is valuable not only for studying its role in carcinogenesis, but also for screening of anticancer drugs and their combinations. Establishment of isogenic models using the CRISPR/Cas9 system is usually done through single cell cloning, which can lead to clonal effects, because of heterogeneity of the cell cultures. In this article we present the process of creation of new knockout cell sublines of MCF7 and A549 using CRISPR/Cas9 and selection using nutlin-3, which allows selecting cell sublines without clonal selection. Phenotypically the knockout of TP53 was confirmed by total absence of p53, absence of induction of p53-dependent gene CDKN1A, and shift in sensitivity to DNA-damaging drugs.

This study aimed to investigate the activities of novel 20(R)-3,20-dihydroxy-19-norpregn-1,3,5(10)-trienes (kuz7 and kuz8b) of natural 13β- and epimeric 13α-series against triple-negative MDA-MB-231 breast cancer cells. High antiproliferative activity of synthesized compounds kuz8b and kuz7 against MDA-MB-231 triple-negative cancer cells was revealed. The steroid kuz7 of natural 13β-configuration was more active against MDA-MB-231 cells than the 13α-steroid kuz8b. Cell cycle analysis revealed common patterns for the action of both tested compounds. The number of cells in the subG1 phase increased in a dose-dependent manner, indicating induction of apoptosis, which was also verified by PARP cleavage. In contrast, the number of cells in the G0/G1 phase decreases with increasing compound concentration. Steroid kuz7 at micromolar concentrations reduced the expression of GLUT1, a glucose transporter. High efficacy of the combination of kuz7 with biguanide metformin was shown, and synergistic effects on MDA-MB-231 cell growth and expression of the anti-apoptotic protein Bcl-2 were revealed. According to the obtained results, including the high activity of kuz7 against triple-negative cancer cells, the detected induction of apoptosis, and the decrease in GLUT1 expression, 13β-steroid kuz7 is of interest for further preclinical studies both alone and in combination with the metabolic drug metformin.

Inhibitors of CDK4/6 have provided a major addition to the clinical armamentarium in estrogen receptor (ER)-positive breast cancers. Palbociclib and other FDA-approved CDK4/6 inhibitors slow down tumor growth and induce senescence (irreversible cell cycle arrest) by inhibiting downstream phosphorylation of the Rb protein by the CDK4/6-Cyclin D complex. However, long-term treatment with CDK4/6 inhibitors inevitably leads to drug resistance and an overall decline in drug efficacy, both in laboratory models and in the clinic. Therefore, identifying an approach to preventing the development of resistance to CDK4/6 inhibitors would be beneficial to improving patient outcomes.CDK8 or its isoform, CDK19, together with their binding partner Cyclin C (CCNC) and proteins MED12 and MED13, form the regulatory CDK module of the transcriptional Mediator complex. CDK8/19 are actively pursued drug targets, with highly selective pharmacological inhibitors of CDK8/19 becoming available. The primary function of CDK8/19 Mediator kinase is potentiation of several signal-responsive transcription factors, such as ER, NFκB, SMADs, STATs and HIF1α; CDK8/19 inhibition suppresses signal-induced expression of a subset of genes activated by such transcription factors. This function defines CDK8/19 as mediators of transcriptional reprogramming, a critical process for the development of drug resistance, and CDK8/19 inhibition has been found to prevent the development of resistance to several classes of anticancer drugs. We have now tested if selective CDK8/19 inhibitors, Senexin B and SNX631, could prevent the development of Palbociclib resistance in ER-positive breast cancers. Our experiments have shown that Palbociclib-treated ER-positive breast cell lines, when cultured in the presence of Palbociclib, rapidly become resistant. CDK8/19 inhibitors have only a moderate growth-inhibitory effect in these cells and do not overcome acquired Palbociclib resistance. However, when CDK8/19 inhibitors are added together with Palbociclib, resistance does not develop. We have found that CDK8/19 inhibitors counteract some of the transcriptomic effects of Palbociclib and interact with this drug in modulating Rb expression and phosphorylation and attenuating the senescent phenotype. These results suggest specific mechanisms through which CDK8/19 inhibition prevents the development of adaptive resistance to Palbociclib. These in vitro findings are currently being investigated in vivo in an ER-positive breast cancer xenograft model. Citation Format: Zachary Mack, Margarita Yastrebova, Alvina Khamidullina, Amanda Sharko, Xiaokai Ding, Stephan Bowe, Nikitha Sashi, Samantha Safa, Kaitlyn Digsby, Amanda Eckstrom, Vitali Sikirzhitski, Chang-uk Lim, Victor Tatarskiy, Igor Roninson, Eugenia Broude. Preventing adaptive therapeutic resistance to CDK4/6 inhibition with CDK8/19 inhibitors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-17.

Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ERT2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen-the active metabolites of tamoxifen-measured by LC-MS in these tissues. We also describe two cases of Cdk8floxed/floxed/Rosa-Cre-ERT2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.