Cancer is a complex disease, and its treatment is a big challenge, with variable efficacy of conventional anticancer drugs. A two-drug cocktail hybrid approach is a potential strategy in recent drug discovery that involves the combination of two drug pharmacophores into a single molecule. The hybrid molecule acts through distinct modes of action on several targets at a given time with more efficacy and less susceptibility to resistance. Thus, there is a huge scope for using hybrid compounds to tackle the present difficulties in cancer medicine. Recent work has applied this technique to uncover some interesting molecules with substantial anticancer properties. In this study, we report data on numerous promising hybrid anti-proliferative/anti-tumor agents developed over the previous 10 years (2011–2021). It includes quinazoline, indole, carbazole, pyrimidine, quinoline, quinone, imidazole, selenium, platinum, hydroxamic acid, ferrocene, curcumin, triazole, benzimidazole, isatin, pyrrolo benzodiazepine (PBD), chalcone, coumarin, nitrogen mustard, pyrazole, and pyridine-based anticancer hybrids produced via molecular hybridization techniques. Overall, this review offers a clear indication of the potential benefits of merging pharmacophoric subunits from multiple different known chemical prototypes to produce more potent and precise hybrid compounds. This provides valuable knowledge for researchers working on complex diseases such as cancer. Cancer is a complex group of multiple diseases characterized by inappropriately controlled cell proliferation and replication eventually resulting in disruption of normal physiology, metabolism, or structure. Benign tumors are self-limited and do not invade or metastasize, but in complex stages groups of cells display uncontrolled growth, invasion and metastasis . In the Cancer is a major global health care problem that continues to remain a leading cause of morbidity and mortality, with >277 different cancer types. According to estimates from the World Health Organization (WHO) in 2019, in 112 of 183 countries, cancer is the first or second major cause of death before the age of 70 and ranks third or fourth in a further 23 countries. In 2020, for all cancers, 19,292,789 new cases were estimated globally, with a total of 9,958,133 cancer deaths . AccordiOver the past 20 years, cancer treatments have improved significantly, and more potent medications have better safety profiles and more precise molecular targeting. Drug resistance is a major challenge with cancer treatment. During clinical usage, almost all targeted anticancer medications encounter resistance. Numerous processes have been related to drug resistance, including genetic and/or epigenetic mutation, amplification, cancer stem cells (CSCs), efflux transporters, apoptotic dysregulation, and autophagy, among others ,7,8,9.(a) two or more medicines that operate on distinct sites simultaneously or concurrently; (b) multi-targeting or promiscuous drugs treatment; and (c) hybridization drugs ..36 . Mechanistically, compound 36(a) induced appototic cell death via caspase activation with an IC50 of 7.8 µM quinoline (DIMIQ) at IC50 values 1.14, 2.19, 1.50, 9.70, and 5.70 µM, respectively. Furthermore, compound 38a showed excellent topo II inhibitory activity at concentration of 0.05 µM with control DIMIQ (Conc. 0.5 µM), m-AMSA (Conc. 0.05 µM), and daunorubicind (Conc. 0.5 µM) and cell cycle inhibitory activity at a concentration of 0.10 µM with control DIMIQ (Conc. 1.02 µM). Additionally, it showed positive inhibitory results in the G2M phase of the cell cycle. The structure of quinoline-indole based hybrid derivatives is given in 38(b–e) against human cancer cell lines is shown in Luniewski et al. (2012) synthesized quinoline based indole hybrids and evaluated their anticancer activity. Most of the synthesized compounds showed potent anticancer activity. Among them, compound 39(a) showed excellent activity against MDA-MB-231, HeLa, SMMC-7721, and QSG-7701 (hepatocyte cell line) at IC50 0.12, 0.08, 0.34, and 10.76 µM with control Etoposide with IC50 values of 5.26, 2.98, 3.48, and 28.75 µM, respectively. Furthermore, compound 39(a) showed excellent activity apoptosis-inducing activity in on HeLa cell lines (for 48 h). The structure of quinolinebased ursolic acid hybrids derivatives is shown in 39(b–e) against human cancer cell lines is shown in Jin et al. (2019) synthesized quinoline-based ursolic acid hybrids and evaluated their anticancer activity. Most of the synthesized compounds showed potent anticancer activity. Among them, compound 40(a) showed excellent activity against MB231, MB468, MCF7, 184B5 , and MCF10A cell lines, having GI50 values of 3.4, 0.7, 2.3, 9.0, and 12.3 µM. Control chloroquine, had GI50 values of 22.5, 28.6, 38.4, 76.1, and 81.26 µM and Cisplatin having GI50 value 23.7, 31.0, 25.8, 25.5, and 51.51µM, respectively. Furthermore, compound 40(a) showed cell cycle interruption at the meta phase, and also increased the liposamal volume in cancerous cells which led to cell death. The structure of quinoline based piperazine hybrids is given in 40(b–e) against human cancer cell lines is shown in Solomon et al. (2019) synthesized quinoline based sulphonamide/piperazine hybrids and evaluated their anticancer activity. Most of the synthesized compounds showed potent anticancer activity. Among them, compound 41 showed potent activity against HCT-116, Caco-2 (human colon cancer cell), HT-29, and CCD-18C (colonic fibroblasts), having IC50 values of 14.26, 19.56, 19.66, and 28.28 µM, respectively. Control etoposide had comparative values of 38.10, 32.90, 35.10, and 58.90 µM, respectively. Furthermore, hybrid 41 was evaluated for anti-malarial activity, showing more potent activity than lumefantrine on the 3D7 strain of Plasmodium falciparum. A quinoline-based gallium(III) hybrid is depicted in Kumar et al. (2014) synthesized quinoline-based gallium(III) hybrids and evaluated their anticancer activity. Synthesized hybrid Quinoline containing drugs that have received FDA approval include lenvatinib, cabozantinib, and bosutinib which are protein kinase inhibitors and are used to cure medullary thyroid cancer and chronic myelogenous leukemia accordingly. FDA approved drugs with quinoline hybrids are depicted in Quinones are found in all living beings; particularly animals, plant and microbes. Through serving as crucial links in the cell nucleus respiratory cycle, they play a vital role in the power generation of such species. Numerous distinct medicinal uses of quinones have been noted, such as antiviral, antithrombotic, antiplatelet, antibacterial, antifungal, anti-inflammatory, and antiallergic properties ,102.42(a) showed excellent activity against HeLa, LS174 , A549, and MRC-5 (multipotent stem cells) with IC50 values of 2.73, 6.44, 28.84, and 48.76, respectively. Furthermore, compound 42(a) showed caspase based apoptosis in G2/M and S phase of cell division. The structure of quinone-based chalcone hybrids derivatives is given in 42(b–e) against human cancer cell lines is shown in Markovic et al. (2015) synthesized quinone based chalcone hybrids and evaluated their anticancer activity. Most of the synthesized compounds showed potent anticancer activity. Among them, compound 43(a) showed excellent activity against KB (throat cancer cells), KB/VCR (colon cancer cells), A549, and HL60, with IC50 values 4.05, 1.28, 0.62, and 1.73 µg/mL. A control vincristine had IC50 values of 0.46, 0.26, and 12.09 µg/mL , respectively, and adriamycin had an IC50 value 0.02 µg/mL (HL60). The structure of quinone-based pyran hybrid derivatives is given in 43(b–e) against human cancer cell lines is shown in Jiang et al. (2015) synthesized quinone based pyran hybrids and evaluated their anticancer activity. Most of the synthesized compounds showed potent anticancer activity. Among them, compound Doxorubicin, daunorubicin, and mitoxantrone drugs are used to treat several types of cancer. These drugs are shown in The imidazole derivatives have recently gained a lot of interest and are already present in many existing treatments. Imidazole was first synthesized in 1858 using glyoxal and formaldehyde by Heinrich Debus . Various44(a–e)  are listed in Xiao-Dong Yang et al. (2012) synthesized different derivatives of novel hybrid compounds between 2-phenylbenzofuran and imidazole. Myeloid liver carcinoma (SMMC-7721), colon carcinoma (SW480), breast carcinoma (MCF-7), lung carcinoma (A549) and leukemia (HL-60) cell lines were used for in vitro study of the cytotoxic effects of synthesized hybrids with cisplatin (DPP) as the standard drug. Five derivatives showed more potent cytotoxic activity than standard DPP. The structure of imidazole based benzofuran hybrid derivatives are given in 2-phenyl-3-alkylbenzofuran and imidazole compounds. Myeloid liver carcinoma (SMMC-7721), colon carcinoma (SW480), breast carcinoma (MCF-7), lung carcinoma (A549) and leukemia (HL-60) cell lines were used for in vitro cytotoxic testing of synthesized hybrids, with DPP as the standard drug. They found that five derivatives showed more potent cytotoxic activity than standard DPP. The structure of imidazole based benzofuran hybrid derivatives is given in 45(a–e)  are listed in Wen Chen et al. (2013) synthesized numerous derivatives of novel hybrid 46(a–e) are listed in Al-blewi et al. (2013) synthesized numerous derivatives of novel imidazole-1,2,3-triazole hybrids. The synthesized compounds were screened for their anticancer activity against three different types of cancer, namely human colon carcinoma (Caco2 and HCT116), human cervical carcinoma (HeLa) and human breast adenocarcinoma (MCF-7) cancer cells-, using doxorubicin as a standard drug. They found that these derivatives showed potent more cytotoxic activity than standard doxorubicin. The structure of imidazole based triazole hybrid derivatives is given in 47(a–e) are listed in Yanping Hu et al. (2019) designed and synthesized a series of artemisinin-imidazole hybrids derivatives with multidrug resistance (MDR) reversal activity. All hybrids were screened in vitro for anticancer activities against four human cancer cell lines, human breast cancer (MCF-7), human non-small-cell lung (A549), (HEPG-2), breast cancer (MDA-MB-231) and normal human hepatic cells (L02). Adriamycin was used as reference drug. They found that most of the synthesized compounds showed higher anticancer activities than artemisinin. The structure of imidazole based artemisinin hybrid derivatives is given in 48(a–e) are listed in Wen-Jian Song et al. (2012) designed and synthesized a series of novel hybrid compounds of 2-substituted benzofuran and imidazole. In vitro anticancer activity of synthesized hybrids against a panel of human tumor cell lines i.e., ovarian carcinoma cell line (Skov-3), leukemia (HL-60) and breast carcinoma (MCF-7) was evaluated. They found that the hybrid compounds were more selective than standard DPP. The structure of imidazole based benzofuran hybrid derivatives is given in Different imidazole-based anticancer hybrid drugs that are FDA approved/or under clinical trials are shown in , and theSelenium (Se) is a unique trace element. Se directly or indirectly exerts antioxidant functions in the human body. However, during recent years, researchers reported that Se containing compounds exhibit superior anticancer effects, with high efficacy and selectivity . It has Organic Se compounds have lower systemic effects, fewer side effects, and strong anti-tumor action. They also have a greater ability to inhibit metastasis. Several novel organic Se compounds have been synthesized in order to further improve the selectivity, specificity and efficacy and to lower the toxicity .3) derivatives as potential anticancer agents. The anticancer activity of the synthesized compounds was assessed using human cancer cell lines, human colon adenocarcinoma cells (SW480), human cervical cancer cells (HeLa), human lung carcinoma cells (A549), human breast adenocarcinoma cells (MCF-7). The in vitro biological evaluation was conducted at 24, 48 and 72 h intervals, and it was found that the organoselenium hybrid compounds were more selective than standard Fluorouracil (5FU). The structure of organoselenium hybrid derivatives is given in 49(a–c) are listed in Xianran He et al. (2020) synthesized organoselenium (SeCF50(a–e) are listed in Guilherme A. Jardim et al. (2017) synthesized selenium-containing quinone-based 1,2,3-triazoles with potential antitumor activity via rhodium-catalyzed C-H bond activation and click reactions. All compounds were evaluated against five types of cancer cell lines: human promyelocytic leukemia cells (HL-60), human colon carcinoma cells (HCT-116), human glioblastoma cells (SF295), human lung cells (NCIH-460) and human prostate cancer cells (PC3), using paclitaxel as a positive control. L929 cells were also used to test the cytotoxic potential of the naphthoquinoidal derivatives in non-tumor cells. Overall, these compounds represented promising new lead derivatives with potential antitumor activity. The structure of selenium based quinone triazole hybrids is given in 51(a–e) are listed in An B et al. (2018) synthesized selenium-containing 4-anilinoquinazoline hybrids and evaluated them as tubulin polymerization inhibitors. All the synthesized compounds were screened against a panel of six human tumor cell lines, human colon cancer cells (RKO), HEPG2, breast adenocarcinoma (MCF-7), human epithelial cervical cancer cells (HeLa), human colon cancer cells (HCT116), and human gastric cancer cells (MGC803). An antiproliferative activity assay showed that most of compounds inhibited human cancer cells at low nano-molar concentrations. These compounds disturbed microtubule dynamics, lowered mitochondrial membrane potential, and stopped Hela cells in the G2/M phase, ultimately leading to apoptosis. The structure of selenium based anilino quinazoline hybrids is given in 51(a–d) are listed in Hairong Tang et al. (2021) synthesized novel selenium-containing chiral 1,4-diarylazetidin-2-ones and biologically evaluated for antitumor activities. All the newly synthesized selenium-containing compounds were screened for their antiproliferative activity against four human cancer cell lines, human epithelial cervical cancer cells (HeLa), human hepatoma cells (HUH-7), ovarian carcinoma cells (Skov-3) and human ovarian cancer cells (A2780), using paclitaxel as the positive control. The structure of Selenium based arylazetidin hybrids is shown in 53 displayed more than two-fold higher cytotoxicity to A2780 cells than to normal ovarian epithelial cells (IOSE80). Further research demonstrated that these inhibitors generated reactive oxygen species (ROS), harmed mitochondrial membrane potential (MMP), stopped cells from entering G0/G1 phase, and ultimately promoted apoptosis in the A2780 cells. The structure of selenium-based diarylimidazole hybrids is shown in 53(a–e) are listed in Sheng Huang et al. (2021) designed and synthesized fourteen novel selenium N-heterocyclic carbene (Se-NHC) compounds derived from 4,5-diarylimidazole and evaluated their antiproliferative activity towards ovarian cancer cells (A2780) and normal ovarian epithelial cells (IOSE80). Most of them were more effective towards ovarian cancer cells (A2780) than HepG2 hepatocellular carcinoma (HCC) cells. In addition, compound Different selenium-based potent anticancer drugs their chemical structure , mode ofPlatinum (Pt) medicines are still among the most often used anticancer treatments after more than 40 years of use. It is not unexpected that new research into changes in DNA repair pathways provides a reasonable explanation for Pt medicines’ efficacy since they primarily target DNA . The fir54(a–e) are listed in A. Graham et al. (2012) synthesized platinum−acridine hybrid anticancer agents. In vitro cytotoxicity activity was evaluated on different cell lines, ovarian cancer (OVCAR-3), breast cancer , pancreatic (PANC1) and non-small cell lung cancer cells (NCI-H460) using cisplatin as the positive control. The structure of Platinum−acridine hybrids is given in 55–60) are listed in Jian Zhao et al. (2012) designed and synthesized six novel platinum (II) complexes 1−6 bearing different furoxan moieties as nitric oxide (NO) donors. The furoxan groups were introduced to the platinum complexes to release NO, which may have synergisticaction with the platinum-based moieties on the tumor cells. It was found that all compounds exhibited higher cytotoxicity against human cancer cell lines HCT-116 and SGC-7901 compared to standard carboplatin, and oxaliplatin. The structure of platinum hybrids is shown in 50 values. The structure of Platinum (iv) dihydro-2-quinolone hybrids is given in 61(a–e) are listed in Liu Z et al. (2021) prepared dihydro-2-quinolone (DHQLO) platinum (IV) compounds. Cytotoxic profiles of DHQLO platinum (IV) complexes were tested against five carcinoma cell lines including, ovarian cancer (SKOV-3), human cervical cancer cell (HeLa), human lung cancer (A549), murine colon cancer (CT-26), a cisplatin resistant cell line (A549R), and one normal human embryonic kidney cell line (293T). The antitumor activities of DHQLO platinum (IV) compounds were tested using the MTT assay with cisplatin and oxaliplatin as reference drugs. Cells were treated with drugs at different concentrations for 48 h, and the results were given as IC62−66, 62−66) were tested against four tumor cell lines including human lung cancer (A549), human ovarian cancer (SKOV-3), murine colon cancer (CT-26) and a cisplatin resistant cell line (A549R).A human normal liver cell LO-2 was also evaluated. The results are given in 62–66 displayed moderate to effective antitumor activities against the tested tumor cell lines   benzodiazepine-3,11-dione (PBD) derivatives and evaluated their HDAC6 inhibitory effect. When R = H, the activity of the linker with the benzene ring was moderate. However, when the linker was extended by one methylene, the compound’s activity drastically dropped (107a vs. 107b). The structure of pyrrolo-benzodiazepine-dione hybrids is given in 50 of compounds  against HDAC6 enzyme is shown in Li et al. (2021) synthesized new pyrrolo  against cancer cell lines is shown in Chen et al. (2013) synthesized a new series of PBD-triazole hybrids and their cytotoxicity was investigated on various mouse and human cells, taking tubastatin A as a positive control. Cis-hybrids , has been shown to have strong anticancer effects on a variety of cancer types.One of the most significant bioactive substances with a chalcone structure (isoliquiritigenin) was isolated from liquorice roots. Butein, a physiologically active flavonoid found in Chalcone-based hybrid compounds have the potential to improve selectivity and anticancer activity while also overcoming drug resistance. Therefore, combining the chalcone moiety with additional anticancer pharmacophores is a promising strategy for creating new anticancer drugs. In recent times, a number of chalcone hybrids have been prepared and evaluated for their cytotoxic activity; some of them are found to have remarkable activity both in vitro and in vivo .111(a) and 111(b) were the most effective compounds with IC50 values of 7.14 µg/mL and 7.6 g/mL, respectively. The structure of chalcone based phenothiazine hybrids is shown in 111(a–d) against cancer cell lines is shown in Zahrani et al. (2020) synthesized chalcone-based phenothiazine derivatives and evaluated their cytotoxic activity against two carcinoma cell lines (human breast cancer cell line MCF-7 and human hepatocellular carcinoma HEPG-2 cells) compared with anticancer standard drugs cisplatin and doxorubicin under the similar conditions following the MTT (methylthiazol-tetrazolium) colorimetric assay. 112(a–d) against cancer cell lines is shown in Shivapriya et al. (2021) produced chalcone benzoxadiazole hybrids and evaluated their cytotoxic activity against the human epidermal carcinoma (KB) cell line using MTT assay. Chalcone-benzoxadiazole hybrids were prepared through the Claisen–Schmidt condensation reaction. The structure of chalcone-based benzoxadiazole hybrids is given in 113a–d, and evaluated their cytotoxic activity against three target cell lines: human breast adenocarcinoma (MCF-7), human colon carcinoma (HCT-116) and human hepatocellular carcinoma (HEPG-2) using the MTT assay method with doxorubicin as a reference drug. The initial results showed that some of the chalcones exhibited significant antiproliferative effects against most of the cell lines, with selective or non-selective behavior, with IC50 values found to be in the 1.65 to 34.28 µM range. Compound 113(a) showed maximum cytotoxic activity towards given cell lines. The structure of chalcone-based triazolo-quinoxaline hybrids is given in Alswah (2017) et al., designed and synthesized a series of triazolo-quinoxaline-chalcone derivatives 114(a) maximum activity. The structure of chalcone based melatonin hybrids is given in 114(a–d) against cancer cell lines is shown in Yepes et al. (2021) synthesized a new series of hybrid molecules by inclusion of two scaffolds: chalcone and melatonin. To achieve this goal, biologically active chalcone was attached via a non-hydrolizable thioalkyloxy linker to the corresponding melatonin bioisostere scaffold. The anticancer activity of the synthesized hybrids was evaluated against an in vitro model of colorectal cancer. In this study, two cell lines were used, i.e., the non-malignant (CHO-K1) and human colon adenocarcinoma cells (SW480). The reference drug used was 5-FU. Compound 115a–e) were tested in vitro for their anticancer activities against, PC12, BPH-1 and MCF-7 cells using the MTT assay. With IC50 values in the micro molar range (9.1–98.7 mM) against all tested cancer cells, all synthesized compounds demonstrated moderate to good antiproliferative activity and compound 115(e) showed maximum cytotoxic activity. The structure of chalcone-based quinoxalin hybrids is given in 115(a–e) against cancer cell lines is shown in Ma et al. (2021) synthesized chalcone-based quinoxalin as anti-cancer hybrids. All of the synthesized compounds (The coumarin scaffold (2H-1-benzopyran-2-one) is extensive in nature, and it’s derivatives exhibit various antibacterial, antifungal, antimalarial, and anticancer pharmacological properties.1R2) were the most potent with inhibition of most of the cell lines. The compound with ethanolamine as a substituent (NR1R2) at position 7 of the coumarin-benzimidazole scaffold, was the most potent synthesized compound. The structure of coumarin-benzimidazole hybrids is given in 116(a–e) against cancer cell lines is shown in Kamaldeep Paul et al. (2013) synthesized coumarin-benzimidazole hybrids. The synthesized compounds showed potent activity against leukemia cancer cells , K-562, RPMI-8226), colon cancer cells , melanoma cancer cells  and breast cancer cells . The compounds containing substitution (NR50 of compounds 117 (a–e) against cancer cell lines are shown in R. An et al. synthesized amide containing 1,2,3-triazole hybrids, which showed moderate to excellent activity against MDB-MB 231 cell lines under both normoxic and hypoxic conditions. The structure of coumarin-triazole hybrids is given in 118(a) was more potent than 3,4-dimethoxyphenyl chalcone 118(b), which showed higher activity than chalcone with 3,4,5-trimethoxyphenyl moiety 118(c).H.A. Elshemy et al., (2017) synthesized coumarin-chalcone hybrids having anticancer activity against HEPG2, leukemia, and WI-38 cell lines. All coumarin-chalcone hybrids had potent activity against HEPG2 and K562, and weak activity against WI-38 cell. Among the coumarin-chalcone hybrids, 4-methoxyphenyl chalcone 119(c) showed the highest activity against a leukemia cell line (k562) while compound (119a and 119b) showed potent activity against the HEPG2 cell line and moderate activity against the WI-38 cell line. The structure of coumarin containing chalcone hybrids is shown in 50 (µM) of compounds  against cancer cell lines is shown in In the coumarin-acrylohydrazide series, compound 50 = 1.55µM) contained A with fluorine atom as R with two carbon chain lengths between triazole and coumarin moieties. The structure of coumarin containing uracil hybrids is given in 50 (µM) of compounds 120(a–e) against cancer cell lines is shown in Mohit Sanduja et al. (2020) synthesized uracil-coumarin-based compounds as potent anticancer hybrid compounds. The uracil-coumarin hybrid compounds inhibited the MCF-7 cancer cell proliferation more effectively compared to standard 5-FU. The most potent compound 120b (GI121(e) had the highest antiproliferative activity and was more potent than standard, doxorubicin. The structure of coumarin containing furoxin hybrids is given in 50 of compounds 121(a–e) against cancer cell lines is shown in Zhuo zing et al., (2018) synthesized novel coumarin-based furoxin hybrids which had potent activity against Hela cell proliferation. The compound 122(b) induced apoptosis and affected cell cycle progression in human hepatoma (Bel-7402) cells. The structure of nitrogen mustard contain oridonin hybrids is given in 50 of compounds 122(a–d) against the cancer cell line (Bel-7402) is shown in Shengtao Xu et al. (2014) synthesized natural oridonin bearing nitrogen mustard hybrid as potential anticancer compound. The hybrid showed activity against the multidrug resistant cell lines SW620/AD300 and NCL-H460/MX20. Compound 123(a–e) was most potent and exhibited activity against human leukemia (MV4-11) cells. The compounds 123(b) and 123(e) had potent activity against MCF-7 and HCT116. The structure of nitrogen mustard contain thiazole hybrids is given in 50 of compounds 123(a–e) against cancer cell lines is shown in Laczkowski et al. (2016) synthesized nitrogen mustard-based thiazole hybrid drugs that showed antiproliferative activity against human cancer cell lines MV4-11, A549, MCF-7 and HCT-116. Among the all derivatives, b) and 124(d) were the most potent. The structure of nitrogen mustard-containing triazine hybrids is shown in 50 of compounds 124(a–d) against cancer cell lines is shown in Kolesinska et al. (2012) synthesized hybrids containing a nitrogen mustard-triazine scaffold as potent anticancer compounds. These were prepared by rearrangement of mono, bis and tris--1,4-diazacyclo [2.2.2] octanium chlorides leading to the formation of 2-chloroethylamino fragments attached to 1,3,5-triazine via one, two or three piperazine, respectively. Their cytotoxicity and alkylating activity depended on substituents on the triazine ring and nitrogen mustard. Among the above mentioned compounds, 124(125(a) showed the highest growth inhibition on IGROV (ovarian cancer cells) with a GI50 of 0.937 µg/mL. The structure of androstene oxime-nitrogen mustard hybrids is shown in 50 (µg/mL) of compounds (125(a-b),126(a-b)) against cancer cell lines is shown in Acharya et al. (2017) synthesized androstene oxime-nitrogen mustard hybrids and nitrogen mustard conjugates of various steroidal oximes. The conjugation was achieved by oxime-ester linkage. The 17-E-steroidal oxime benzoic acid mustard ester, 3β acetoxy-17E-[p-amino]benzoyloxiamino-androst-5ene 126a and 126b showed excellent anticancer activity against the HepG2 cancer cell line with an IC50 of 6.1 and 7.9 µM, respectively. The structure of the pyrazole-based indole hybrid is given in 50 of compounds 127(a–e) against cancer cell lines are shown in Hassan et al. (2021) synthesized indole-pyrazole hybrids as anticancer agents. The newly synthesized hybrids were screened for their cytotoxicity activities in vitro against four human cancer types, i.e., colorectal (HCT-116), breast carcinoma (MCF-7), liver carcinoma (HepG2) and lung carcinoma (A549). Among all indole-pyrazole hybrids, 128c showed good src inhibition activity at 10 µM and was found to be most potent compound. The structure of benzofuran-pyrazole hybrids is given in 50 of compounds 128(a–d) against cancer cell lines is shown in Somaia et al. (2015) synthesized benzofuran-pyrazole-based hybrids as anticancer drugs. The newly synthesized compounds showed remarkable growth inhibitory activity against leukemia (CCRF-CEM), A549 (lung carcinoma), and HCT-116  cells. Compound 50 of compounds (129–133) against cancer cell lines is shown in Abdelaal et al. (2021) synthesized quinazoline-pyrazole hybrids as potential antiproliferative agents. The newly synthesized compounds showed activity against hepatocellular carcinoma (liver) HEPG2, mammary gland (breast) MCF-7 and colon cancer HCT-116 cells. The structure of quinazoline-pyrazole hybrids is given in 134 (b) was found to active against A549, SiHa, COLO205 and HePG2 cell lines with IC50 values of 4.94, 4.54, 4.98 and 2.09 µM, respectively, and was non-toxic against normal cells. The structure of pyrazoline-pyrazole hybrids is given in 50 of compounds 134(a–e) against cancer cell lines is shown in Washim Akhtar et al. (2021) synthesized 15 novel pyrazoline-pyrazole hybrids, all of which showed activity in the MTT growth inhibition assay against five cancer cells lines: MCF-7, A549, SiHa, COLO205 and HePG2 cells. Compound a–c) demonstrated promising results against all tested cell lines. The non-cyclized compounds (amide derivatives) favored anticancer activity. The structure of pyrazole acrylic acid based oxadiazole hybrids is shown in 50 of compounds 135(a–c) against cancer cell lines is shown in Garima Verma et al. (2018) synthesized pyrazole acrylic acid-based oxadiazole and amide hybrids as anticancer and antimalarial agents. The anticancer activity of newly synthesized compounds was measured using the sulforhodamine B assay. Compounds 135(136(d) was found to be most potent compound. The structure of biquinoline-pyridine hybrids is given in 50 of compounds 136(a–e) against cancer cell lines is shown in Chetan B. Sangani et al. (2014) synthesized biquinoline-pyridine hybrids as potential EGFR and HER-2 kinase inhibitors by base catalyzed cyclocondensation through one potent multicomponent reaction. All the synthesized compounds were tested against A549  and Hep G2 (liver cancer) cell lines. Enzyme inhibitory activity was measured against HER-2. Compound 137 showed the most potent activity against HEPG2 with an IC50 of 2.5 µM, while compound 138(c) was the most potent compound against A549 and MCF-7 cell line with IC50 values of 10.8 and 6.3 µM, respectively. The structure of isatin-pyridine hybrids is shown in 50 of compounds  against cancer cell lines is shown in W.M. Eldehna et al. (2015) synthesized isatin-pyridine hybrids as potential antiproliferative agents. They showed antiproliferative activity against hepatocellular carcinoma (HEPG2), lung cancer (A549) and breast cancer (MCF-7) cell lines. Compound 139(a–d) showed activity against HCT116 and compounds 140(a–d) showed activity against MCF-7 cancer cell lines. The structure of pyrazolo  against cancer cell lines is shown in E.K. Hamza et al. (2020) synthesized pyrazolo ,4 pyridiFor a very long time, medicinal chemists from all over the world have been trying to develop new and effective cancer treatments. Combination therapy and hybrid chemotherapeutics haves become more common, because a complex disease such as cancer cannot be properly treated with a single drug. This study presents rational approaches behind the design of anticancer agents employing molecular hybridization. This method has potential because it combines two moieties to create new molecular scaffolds. Molecular hybridization offers a wide range of applications since it can produce compounds with distinct and/or multiple modes of action and minimal side effects. The few examples included in this article are not intended to be an exhaustive collection of anticancer hybrids, but to provide a quick explanation of the idea and its potential uses for researchers working in this field.


Knowledge of the transport systems in the BBB and motor neurons will help to deliver drugs to the brain and develop the therapeutic strategy for treating CNS and neurological diseases.Defective solute carrier (SLC) transporters are responsible for neurotransmitter dysregulation, resulting in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). We provided the role and kinetic parameters of transporters such as ASCTs, Taut, LAT1, CAT1, MCTs, OCTNs, CHT, and CTL1, which are mainly responsible for the transport of essential nutrients, acidic, and basic drugs in blood–brain barrier (BBB) and motor neuron disease. The affinity for LAT1 was higher in the BBB than in the ALS model cell line, whereas the capacity was higher in the NSC-34 cell lines than in the BBB. Affinity for MCTs was lower in the BBB than in the NSC-34 cell lines. CHT in BBB showed two affinity sites, whereas no expression was observed in ALS cell lines. CTL1 was the main transporter for choline in ALS cell lines. The half maximal inhibitory concentration (IC The solute carrier (SLC) superfamily constitutes more than 65 families and over 400 genes responsible for the influx and efflux of a wide range of molecules such as organic and inorganic ions, sugars, and amino acids across membranes . These tAmong motor neuron diseases, ALS is considered to be the most prevalent disorder resulting in muscle paralysis . ALS casG93A, a mutant cell line (MT), has an overexpression of human SOD1 gene mutation due to the substitution at the 93 position of glycine with alanine d-serine was markedly higher in the MT cells, whereas [3H]l-serine was higher in the WT cells. The kinetics parameters also elucidate the altered affinity and capacity. In case of [3H]d-serine, the affinity was higher in the MT cells as compared to WT; however, the affinity for [3H]l-serine in WT cells were two times lower in MT cells as compared to WT cells  gene . For a cWT cells . From thd-serine . The altd-serine .3H]taurine is transported by TauT/Slc6a6 in rat brain capillary endothelial cells (TR-BBB13) [3H]taurine uptake has shown the activity of transporters at both membranes luminal and antiluminal of BCECs. Saturable taurine transport showed high affinity and low capacity systems, Michaelis-Menten constant ; 12.1 ± 0.5 μM and velocity (Vmax); 4.32 ± 0.05 nmol/30 min/mg protein, for the luminal uptake, whereas, Km; 13.6 ± 2.4 μM and Vmax; 2.81 ± 0.02 nmol/30 min/mg protein, for antiluminal uptake of [3H]taurine taurine .3H]taurine, where the uptake was markedly higher in the MT cell line as compared to the WT cell  expressing H1 high strain mice) in a pattern similar to that of the motor neurons of ALS patients . Due to  WT cell . FurtherHeat shock factor-1 (HSF1) mediated expression of TauT showed a compensatory effect against oxidative stress, which is considered the key factor in ALS pathogenesis. It has been concluded that TauT is one of the key markers for diagnosing stress in motor neurons, and the regulation of Slc6a6 may slow the process of neurodegeneration . Additiol-arginine [14C]citrulline through the BBB was carried by Slc7a5/LAT1 [14C]citrulline was mediated by Slc7a5/LAT1 transporter In a similar manner, the roles of essential amino acids such as tryptophan in both the BBB and ALS model cell lines have been studied, and the findings suggested that LAT1 was also involved in the transport of [3H]tryptophan citrulline transport in the BBB showed that two saturable processes are involved in the transport, and the results showed that at high affinity site, higher affinity and capacity, whereas at a low affinity site, there was a lower affinity and capacity in the TR-BBB cell lines [m values 1–10 μM for high affinity and 10–100 μM for low affinity [14C]citrulline in ALS have also been studied. The data showed that the high affinity and low capacity transport systems were involved in the MT compared to the WT [A previous study on [ll lines . Anotheraffinity . Similaro the WT . These r14C]l-citrulline in a concentration-dependent manner. A previous study reported no inhibition at 0.2 mM, whereas a significant inhibition was observed at 0.5 mM in both NSC-34 cell lines l-arginine was found to be concentration-dependent in ALS model cell lines, and the kinetics revealed that in the MT cell line, the affinity was lower and capacity was higher at a high affinity site, whereas at a low affinity site there was no significant difference between WT and MT cell lines, as shown in 3H]arginine in ALS cell lines [The SLC7 family is subdivided as LATs and cationic amino acid transporters (CATs) . For theell line  and alsoell line . L-Argins of ALS  and has s of ALS . The uptll lines . Similarll lines . Furtherll lines .14C]PBA transport to the brain across the BBB [14C]PBA transport characteristics and transporters involved have been studied in ALS cell lines, and the results indicated that sodium-coupled MCT1 (SMCT1/Slc5a8) and MCT1 both help [14C]PBA transport to NSC-34 cell lines [MCTs play a vital role in cellular metabolism and energy pathways in several tissues . The SLC the BBB . Additioll lines .14C]PBA transport by TR-BBB showed that the transport was concentration-dependent, and the Michaelis–Menten constant demonstrated that the carrier-mediated transport of PBA was pH-dependent, with the Km four times higher at pH 7.4 than the Km at pH 6.0. In contrast, Vmax was five times lower at pH 7.4 than pH 6.0  analysis in MT revealed that high edaravone concentration is required to reach 50% inhibition [3H]VPA uptake.Conversely, our previous study on valproic acid (VPA) has shown the neuroprotective effects of VPA in the ALS disease model. The study data suggested that the transporter SMCT1 was commonly involved in mediating the transport of VPA in NSC-34 cell lines . Transpo6–0.8 mM , that isll lines . IbuprofT and MT . In addill lines  (Table 2hibition . These rl-carnitine (ALCAR) in the BBB [3H]ALCAR transport in TR-BBB cells was carried by OCTN2. Expression of OCTN2 in the cells confirm the involvement of OCTN2 in TR-BBB cells. Another study has reported that OCTN2 in the brain and astrocytes are responsible for the transport of ALCAR and l-carnitine [l-carnitine has shown the involvement of both OCTN1 and OCTN2 in the transport of carnitine in motor neuron NSC-34 cell lines [l-carnitine to neuronal cells in human has increased neuronal mitochondrial functions and thus has a role in preventing the pathological conditions related to ALS disease [3H]l-carnitine in ALS model cell lines exhibits saturable processes and two affinity sites. The data shown in l-carnitine possesses two affinity sites in ALS model cell lines. Additionally, various pharmacological compounds such as quinidine, pyrilamine, diphenhydramine (DPH) and metformin have shown the significant inhibitory effect of the uptake of carnitine in ALS model cell lines. The organic cationic compounds showed competitive inhibition with l-carnitine, showing they compete for the same binding sites [l-carnitine showed significant inhibition on the uptake of [3H]paeonol in ALS model cell lines. The OCTN1 and PMAT transporters showed altered behavior in the disease model of ALS in the uptake of paeonol ..93].Further, in our previous study on TR-BBB cell lines, CHT1 was expressed in the brain, whereas OCT2 was expressed in the brain and TR-BBB B 50]. C. C50]. C3H]choline transport in the BBB were retrieved from our previously published article. The data presented in 3H]choline uptake was concentration-dependent, and the Eadie–Hofstee plot showed a straight line, indicating a single saturable process with Km (26.2 µM) and Vmax (397 pmol/mg protein/min) choline uptake by half in the ALS disease model cell lines. Amiloride was the most sensitive drug in the ALS model cell line to achieve the maximal half concentration, whereas choline inhibition showed the most sensitivity in the TR-BBB cell line. A previous study on muscle cell lines involving the H1 receptor antagonist, DPH, showed the inhibition of histamine receptors, and the estimated IC50 value, 1.01 µM, helped control the intracellular calcium taurine was higher in the disease model as compared to the control. It was concluded that increase in the levels of taurine might be due to increase in the TauT levels in the disease model cell line. TauT modulation might delay the neurodegeneration and is considered to be a novel biomarker for ALS. PBA was transported across the BBB by MCT1; however, in addition to MCT1, PBA transport in ALS cell lines was mediated by SMCT. L-Carnitine was transported by OCTN1/2 in ALS cell lines whereas its acetylated form ALCAR transport was mediated by OCTN2 in TR-BBB cells. CHT1 was the main transport for choline across the BBB; however, no CHT1 expression was observed in ALS cell lines. CTL1 is responsible for choline transport in NSC-34 cell lines. In addition, IC50 evaluation of basic drugs showed that choline in BBB and amiloride were the most sensitive in ALS cell lines. Additionally, it is shown that the therapeutics for ALS such as riluzole and edaravone can be transported by LAT1 or SMCT1, respectively, in ALS cell lines. In conclusion, ALS is a devastating neuronal disease and need of hour is to find the possibilities to cure and alleviate the symptoms related to ALS. Hence, adequate knowledge of transporter involvement will be beneficial in delivering novel drugs in ALS. Comprehending the concept of SLC transporters involved in the transport of compounds will aid the development of new drugs and their delivery in brain and motor neuron diseases.Overall, in this review, the representative transporters, which are related with the transport of amino acids including small amino acid, large neutral amino acids, basic amino acids, as well as acidic drugs including PBA and VPA and essential nutrients such as choline and carnitine in BBB and ALS model cell lines. Affinity and capacity plays important role in understanding the transport ability of compounds into the cells. Therefore, we compared the kinetic parameters obtained in BBB and ALS cell lines. In the BBB, affinity of LAT1 is very high, but capacity is very lower than ALS cell lines.


However, most studies that make use of EOC cell lines fail to recognize the importance of subtype. Furthermore, the similarity of cell lines to their cognate primary tumors is often ignored. Identification of cell lines with high molecular similarity to primary tumors is needed in order to better guide pre-clinical EOC research and to improve development of targeted therapeutics and diagnostics for each distinctive subtype. This study aims to generate a reference dataset of cell lines representative of the major EOC subtypes. We found that non-negative matrix factorization (NMF) optimally clustered fifty-six cell lines into five groups, putatively corresponding to each of the five EOC subtypes. These clusters validated previous histological groupings, while also classifying other previously unannotated cell lines. We analysed the mutational and copy number landscapes of these lines to investigate whether they harboured the characteristic genomic alterations of each subtype. Finally we compared the gene expression profiles of cell lines with 93 primary tumor samples stratified by subtype, to identify lines with the highest molecular similarity to HGSOC, CCOC, ENOC, and MOC. In summary, we examined the molecular features of both EOC cell lines and primary tumors of multiple subtypes. We recommend a reference set of cell lines most suited to represent four different subtypes of EOC for both in silico and in vitro studies. We also identify lines displaying poor overall molecular similarity to EOC tumors, which we argue should be avoided in pre-clinical studies. Ultimately, our work emphasizes the importance of choosing suitable cell line models to maximise clinical relevance of experiments.Epithelial ovarian cancer (EOC) is the most fatal gynaecological malignancy, accounting for over 200,000 deaths worldwide per year. EOC is a highly heterogeneous disease, classified into five major histological subtypes–high-grade serous (HGSOC), clear cell (CCOC), endometrioid (ENOC), mucinous (MOC) and low-grade serous (LGSOC) ovarian carcinomas. Classification of EOCs is clinically beneficial, as the various subtypes respond differently to chemotherapy and have distinct prognoses. Cell lines are often used as  BRCA1/2 mutations and hormone receptor (HR) deficiencies in non-HGSOC/high grade ENOC subtypes limits the use of poly (ADP-ribose) polymerase (PARP) inhibitors in disease management (Ovarian cancer (OC) is the most deadly cancer of gynaecological origin, with over 300,000 new cases and 200,000 deaths occurring in 2020 . In the nagement . Targetenagement  but haveCell lines are often utilized to study cancer biology, as a cheaper and less time-consuming alternative to patient derived explants or animal models. There exists over 100 OC cell lines, with around 70 commercially available through the American Type Culture Collection (ATCC), European Collection of Authenticated Cell Cultures (ECACC), Rikagaku Kenkyusho , Leibniz Institute DSMZ, CellBank Australia (CBA) and/or the Japanese Collection of Research Biosources Cell Bank (JCRB) . Cancer Since cell lines do not possess the morphological traits necessary for histological subtyping, current studies utilize molecular characteristics in order to determine the subtypes of these EOC models. A seminal study by in vitro models to study different subtypes of ovarian cancer. However, there exists no study that compares the molecular profiles of EOC cell lines to primary tumor tissue of multiple EOC subtypes. Here, we integrate and analyse EOC cell line molecular data from multiple different sources, with the aim of generating a reference dataset of lines representative of the major EOC subtypes. We apply non-negative matrix factorisation (NMF) to a panel of 56 EOC cell lines. This resulted in five stable clusters that largely validated results obtained in previous studies, while also suggesting new subtype annotations for five previously unannotated cell lines. Additionally, genetic profiles of EOC cell lines were compared to 93 EOC primary tumor samples, from multiple independent datasets, stratified by subtype. We investigate the molecular similarity of cell lines to not only HGSOC primary tumors, but also CCOC, ENOC, and MOC. Ultimately, we identify the most and least representative cell lines to represent these EOC subtypes, generating a reference dataset for future in silico and in vitro studies. Consequently, our work highlights the need for the generation of additional, subtype specific datasets, in particular for the LGSOC and ENOC subtypes.These studies have made remarkable progress in identifying the most suitable An overview of the methodology followed is displayed in Ovarian cancer cell line RNA sequencing (RNA-seq) data was obtained from three sources: n = 11, TP53 mutational status and treatment for each cell line and is detailed in OC cell lines that were misclassified, engineered or non-epithelial in origin were removed from analysis n = 11, . Two celn = 5), HGSOC (n = 5), ENOC (n = 4) and MOC (n = 5). RNA-seq data for these 19 primary tumors were analyzed in this study. n = 11), HGSOC (n = 8) and ENOC (n = 4). There is no publicly available RNA-seq data for LGSOC primary tumors . In total, transcriptomic data for 16 CCOCs, 64 HGSOCs, 8 ENOCs and 5 MOCs were used.Ovarian primary tumor tissue data was obtained from three independent datasets . Corona Quality control checks were performed on all FASTQ files using the FastQC package . Forwardr) was carried out by running NMF for values of r from 2 to 8, using 50 random initiation points. Quality measures including the cophenetic correlation coefficient, residual sum of squares and dispersion were used to select the most suitable value of r. In the presented data, clustering for r values of 2 and 5 displayed high quality metrics . A variance stabilizing transform was applied to read counts using the DESeq2 package . In orde metrics . After sFor cell lines present in the CCLE study, mutational and copy number landscape was originally determined by The uniformly processed EOC cell line and tumor tissue counts were upper quartile normalized. ComBat  was thenW and H. The basis matrix W delineates the contribution of a small subset of genes to what are termed ‘metagenes’. This is essentially a decomposition of genes into those whose co-expression influences cluster assignment . Seeding is used to initialize the starting point for the NMF algorithm . The NMF package contains a number of built in seeding methods. Here the ‘random’ method is used, whereby these initial values are obtained from a uniform distribution with the same range as the input matrix. For reproducibility, a numerical value is passed into the seed function to seed the random number generator. In order to achieve a stable result from this random seeding method, multiple runs of NMF are required.Non-negative matrix factorization is a method of unsupervised learning often applied to gene expression data to extract biologically relevant information . It funcsignment . The coer), a critical parameter that is selected by the user-ideally, r should be small enough to reduce noise but large enough to preserve biologically relevant information. r for which the cophenetic correlation coefficient starts decreasing. The cophenetic correlation coefficient indicates the robustness of clusters for a given choice of r. r is only relevant if the information captured by factorization is greater than that derived from permuted data, otherwise this increase will lead to overfitting. They suggest using the smallest value of r where the decrease in residual sum of squares (RSS) is lower than the decrease observed in randomized data. By comparing the residual error of NMF from the original data to that of permuted data, a solution is identified that is more information than noise, while preventing overfitting.The number of metagenes and sample clusters are defined by the factorization rank  and ENOC/CCOC/MOC (ARID1A)  have not been investigated in terms of EOC subtype since their creation, with original annotation data unavailable for four of these lines . Additionally, these cell lines are rarely cited in literature and as such, mutational and copy number data was largely unavailable . TYKNUCP(ARID1A) . Ultimatin vitro models. Some progress has been made in identifying suitable cell lines to utilize as HGSOC models. Both As discussed previously, subtype assignment of certain cell lines can prove difficult to resolve using transcriptional data, especially when mutational or copy number data is unavailable. Comparison of cell lines to tumor tissue is required to determine the suitability of these Publicly available transcriptomic data was available for primary tumors of HGSOC, CCOC, ENOC and MOC origins  were within the top 20 most correlated cell lines to HGSOC primary tumors, with Caov4, COV413A, OVCA432, OVCAR3 and OVKATE representing the most highly correlated cell lines . This stBRCA2, MYC amplifications), as well as those that are generally not observed in HGSOC   . This hiin vitro EOC studies should be avoided, and importantly, there is a striking need to develop additional ENOC cell lines. We observe a similar pattern for other lines classified as ENOC. Cell lines classified as HSGOC and CCOC may represent the best available models of this subtype, as they possess the highest median correlations to ENOC primary tumors (None of the six lines classified as ENOC ranked within the top 20 most correlated lines to ENOC tumors . In facty tumors . Indeed y tumors  and CCOCy tumors .All lines classified as MOC (apart from JHOM2B) fell within the top 20 most highly correlated lines to MOC primary tumors . COV644,Most cell lines classified as LGSOC  were poorly correlated to primary tumors of HGSOC, CCOC, ENOC, and MOC origin –5. AdditOverall, we show that correlation of gene expression patterns between EOC cell lines and primary tumors can identify models with high molecular similarity to specific subtypes and aid in subtype identification, with the use of techniques such as NMF. In summary, we generated a reference dataset of cell lines most representative of HGSOC, CCOC, and MOC . We alsoin vitro models, at least for gene expression-based studies. Potential cell line models of LGSOC were also reported. Finally, we highlight the need for generation of additional subtype-specific datasets to help identify suitable models of less studied EOC subtypes, including LGSOC.In this study, we utilized publicly available transcriptomic, mutational and copy number data to investigate the molecular characteristics of 56 EOC cell lines. We demonstrated that cell lines optimally clustered into five stable groups that likely represent the five histological subtypes of EOC. We also compared the gene expression profiles of these cell lines to primary tumors of four EOC subtypes, generally observing that HGSOC, CCOC and MOC cell lines were highly correlated to their respective primary tumors. We noted that ENOC cell lines, especially A2780, were poorly correlated overall to not only ENOC tumors but EOC overall. We identified cell lines that feature the major genomic and transcriptomic features of each particular subtype and are therefore the most suitable in silico investigation that lacks experimental validation. However, we note that our results are in agreement with other studies of this nature , genes involved in homologous repair , or genes found to be associated with an increased risk of developing EOC overall   .in vitro models such as spheroids, multicellular organoid systems and tumor-on-a-chip models (It is possible that the use of two-dimensional, monocultures of individual cell lines may become greatly reduced or outdated in cancer research, due to the rising popularity of three dimensional p models . Cell lip models . In any In conclusion, although major technological progress and healthcare improvements have facilitated more effective cancer treatments, the last 20 years have only seen modest improvements in OC survival rates . Indeed,


To determine whether the width of the shoulder and the size of the bystander effect arecorrelated using clonal lineages derived from a cultured cell line.HCT 116 (p53 wildtype) cells were grown at cloning density and individual viablecolonies were picked off and grown to establish a series of cell lines from bothunirradiated and irradiated progenitors. These cell lines were then irradiated togenerate full survival curves. Highly variant clones were then tested to determine thelevel of the bystander effect using a medium transfer protocol.n is assessed in terms of radiosensitivity. The parent cell line hasan n value of 1.1 while the most variant clones have nvalues of 0.88 (Clone G) and 5.5 (Clone A). Clonal lines subject to irradiation prior toisolation differed in bystander signal strength in comparison to clonal lines which werenot initially irradiated (P = .055).The multi-target model gave the best fit in these experiments and size of the shoulderBased on these experiments we suggest there may be a link between shoulder size of amammalian cell line and the strength of a bystander effect produced in vitro. This mayhave implications for radiotherapy related to out-of-field effects. Recently, the effects of low dose IR have gained attention due both to the increasinguse of IR in medical diagnostics, the use of novel protocols in radiotherapy such as FLASHand MRT, and the interest in small modular reactors as energy sources in remoteenvironments.2 High dose direct IRgenerally leads to significant cell death through processes such as reproductive death orapoptosis35 while non-targeted and low dose radiationappears to involve other mechanisms.7 Radiation-induced bystander effects (RIBE)are of particular interest since they involve cell killing, transformation and initiation ofcell signaling pathways in cells that have not been directly exposed to IR but have receivedsignals from directly exposed cells.814 RIBE have been widely studied both in vivo and in vitro and they appearto be associated with low dose radiosensitivity1518 with somesuggestion that they require wildtype p53 to be expressed. This is relevant since many tumors have compromised p53 function,2022 meaning that additional killing due to RIBE would predominantly affectnormal cells around the tumor rather than the tumor itself. However, the research in thisarea is quite controversial with contradictory reports about RIBE even in laboratories usingthe same protocols and cells. A possible explanation for this is “drift” within cultured cell lines leading toclonal heterogeneity in populations of genetically identical cells. To test whether thismight be a factor, we decided to revisit clonal heterogeneity with respect to clonalsensitivity. Through investigation of clonal populations we aim to approach in a moresystematic way the often heterogenous nature of malignancies.25 The literature often refers to clonalheterogeneity within a tumor as a “fuel for resistance” and studying this key challenge inoptimizing individual therapies is necessary to advance cancer treatment.25 Since radiotherapy can lead to secondmalignancies,2628 some cell lines were derived fromcultures of cells exposed to 1 Gy to determine whether there was greater variability interms of radiosensitivity in these lines. With a better understanding of the heterogeneityof response in clonal sub-populations we may gain a new perspective which could improveradiation treatment.The impacts of ionizing radiation (IR) on human cells are important for radiationprotection, environmental risk assessment, and radiation therapy.2 Falcontissue culture flasks at 37°C and 5% CO2. Subcultures were conducted using0.25% phenol red-free trypsin solution with 0.192 mM EDTA every 6-7 days. Trypsinizedcells were neutralized using a greater volume of growth media. Cell cultures were 70–80%confluent upon culture. Cell concentrations were determined using Bio-Rad TC20 automatedcell counter . All reagents were purchasedfrom Gibco, ThermoFisher Scientific.The immortalized human epithelial HCT116 (p53 wildtype) cell line derived from a largeintestine/colon carcinoma was used in this study. Clonal cell lines were isolated fromthis parent cell line. These cells were routinely cultured in Roswell Park MemorialInstitute (RPMI) 1640 growth medium supplemented with 10% fetal bovine serum (FBS), 100U/mL penicillin, 100 ug/mL streptomycin, and 2.05 mM L-Glutamine. This growth medium wasalso used in the bystander effect assays. Cells were grown in 75 cmPetri dishes were seeded with 200–300 cells. These were allowed to form viable coloniesof at least 50 cells. After 7 days, individual clones of various sizes were chosen forclonal expansion. These colonies were scraped off the dish and resuspended in smallmultiwall plates . The cloneswere passaged into T25 flasks when confluent and grown to produce a sufficient supply ofcells for the experiments. Some clones were isolated from plates where cells had beenexposed to 1 Gy radiation after cells had adhered to the culture plate in order to examinethe effect of this dose on subsequent clonal heterogeneity .Figure 1All irradiations were performed using a Cesium-137 gamma-emitting source with a dose rateof 198.4 mGy/min and flasks were placed 30 cm away from the source . Direct irradiations for survival curve data generation wereconducted 15–20 hours post seeding. Irradiations to generate medium for bystander effectassays were also completed in this manner. Cell seeding densities were determined using the plating efficiencies (PE)determined for each clonal cell line. Clonogenic assays were conducted to develop fullsurvival curves upon irradiation of cells at the following dose points: 0, 0.5, 1.0, 3.0,5.0, 7.0, 10.0, and 15.0 Gy. This wide range in dose points provided an overall assessmentat cell survival across doses. Flasks were irradiated at the appropriate dose and returnedto the incubator immediately following irradiation and grown for nine days at 37°C in anatmosphere of 5% CO2 in air. On day nine, all flasks were stained with 15%Carbol Fuchsin solution . Colonies were counted manuallyto determine the surviving fraction for each dose. The data were entered into GraphPadPrism 8 software , to generate survival curvegraphs.Flasks containing approximately 70% confluent cultures were used for clonogenic survivalassays. Cells were removed from the flask using a Trypsin-EDTA working solution describedabove. Detached cells were neutralized with growth medium and mixed to form a single cellsuspension. These cells were counted and plated to perform a clonogenic assay using themethod described by Puck and Marcus.2) were seeded with cells in 5 mL growthmedia for the following treatments and incubated for 6 hours at 37°C and 5%CO2: plating efficiency, direct irradiation (2.0 Gy), bystander effect donorand recipient, and sham donor and recipient . Least square error linear regression analyses were performed ondata to produce the multi-target and linear-quadratic models using GraphPad Prism 8. Datafor bystander effect assays were also collected as mean of three replicates in threeindependent trials. Standard error of the mean error bars are used in all figures. Todetermine variance between groups in bystander effect experiments, t-tests were conductedbetween the sham and bystander groups and the sham and direct irradiation groups for eachcell line. These post hoc analyses were performed using Welch’s t-test. Significance wasdetermined at the 95% confidence interval.+/+ line alongside all clonal cell lines derived from either anirradiated or non-irradiated population prior to clone isolation. Data were not fitted toany established model for this figure. Clonogenic survival over a dose range of 0–15 Gyafter direct exposure to a cesium-137 gamma source shows variation between each clonalline. Plating efficiency variations were also observed between clonal lines and arepresented in n and D0 areobserved where n is a common indicator for low dose radiosensitivity orthe size of the cell survival curve shoulder. In comparison to the parent cell line, CloneA had the largest n value of 5.5 while Clone G had the smallestn value of 0.88 . There is a trendfor n value to correlate with bystander induced reduction in survivalhowever other additional factors could be involved. Also, while to our knowledge no previousexperiments were set up to examine this relationship, there is anecdotal evidence in theliterature that shoulder size and bystander signal strength are related inversely. The paperby Mothersill et al (2002) examined parent cell lines and radiosensitive lines with variousDNA repair defects derived from these parents. Irrespective of the nature of the repair defect, all radiosensitive lines were moreradiosensitive than their parent line. Also many radioresistant cell lines such as PC3 donot show bystander associated cell death while radiosensitive lines such as SW48 do showstrong bystander effects.When all clonal populations were tested for the presence of bystander signals following thebystander medium transfer assay, it was demonstrated that most clonal populations regardlessof their origin from a non-irradiated or irradiated population, displayed a significantreduction in cell survival following receipt of ICCM . Howeverder size . Unfortuder size  does sugIn certain clonal populations, bystander signals were not produced even though they wereradiosensitive. Consistent with previous findings this could suggest the presence of a lowdose hyperradiosensitivity with increased radioresistance as the dose increased (HRS/IRR)mechanism. In instances of hyperradiosensitivity a generally greater than expected responseto radiation is observed. However, various studies have shown that certain cell lines onlyrespond to bystander signals in the lower dose region where HRS is seen.Apart from the findings in relation to RIBE, the data in this paper suggest that themathematical expressions based on classical target theory predictions do not provide goodfits to these results. This is important to note because many of the classical experimentswere done using a few cell lines such as CHO or V79 cells. These have high platingefficiencies of the order of 80-90% but limited expression of tissue of origincharacteristics. Most modern radiobiology is done using lines which express importantparameters related to epithelial cell or tumor function, but which have plating efficienciesbelow 50%. High plating efficiencies are necessary to derive meaningful target theory basedconclusions. This is because of the statistical probability that radiation is the cause of acell not forming a colony if the PE is high. With a low plating efficiency, the cause of notforming a colony need not be the radiation effect. Nowadays the focus is on moleculareffects so that plating efficiency is not such an issue.In conclusion, the data presented show marked clonal heterogeneity in cell lines derivedfrom both irradiated and unirradiated progenitors. This manifests as differences in doublingtime, plating efficiency and radiosensitivity. The data also reveal a weak correlationbetween shoulder size (a surrogate for low dose radioresistence) and the ability of ICCM toreduce the plating efficiency of unirradiated cells. The data also suggest that the commonlyused mathematical expressions traditionally used to fit survival curve data provide poorfits to the data in this paper, possibly due to the low plating efficiency of the celllines. In conclusion, the results may have implications for tumor radiotherapy where clonalheterogeneity is an important limitation for treatment.


B-cell lymphomas represent the majority of non-Hodgkin lymphomas and are the most common lymphoid malignancies in the Western world. Genetic alterations or epigenetic modulations can lead to tumor initiation and tumor progression. Aside from standard care, targeted, individualized therapies can be highly effective. Here, we evaluated the impact of simultaneous specific inhibition of two key regulators involved in B lymphoid tumor progression. Spleen tyrosine kinase (SYK) is a B-cell receptor-associated kinase acting as a proto-oncogene in B-cell malignancies, while bromodomain and extra-terminal domain (BET) proteins are epigenetic reader proteins involved in histone recognition and transcription regulation. The simultaneous inhibition of SYK and BET showed enhanced anti-proliferative effects, as well as inducing a distinct combination-specific gene expression profile, suggesting SYK and BET inhibition as a promising combination in the treatment of B-cell lymphoma.Background: Both bromodomain and extra-terminal domain (BET) proteins and spleen tyrosine kinase (SYK) represent promising targets in diffuse large B-cell (DLBCL) and Burkitt’s lymphoma (BL). We evaluated the anti-lymphoma activity of the isoform-specific bivalent BET inhibitor AZD5153 (AZD) and the pan-BET inhibitor I-BET151 (I-BET) as single agents and in combination with SYK inhibitor Entospletinib (Ento) in vitro. Methods: The effect of the single agents on cell proliferation and metabolic activity was evaluated in two DLBCL and two BL cell lines. Proliferation, metabolic activity, apoptosis, cell cycle and morphology were further investigated after a combined treatment of AZD or I-BET and Ento. RNAseq profiling of combined AZD+Ento treatment was performed in SU-DHL-4 cells. Results: Both BET inhibitors reduced cell proliferation and metabolic activity in a dose- and time-dependent manner. Combined BET and SYK inhibition enhanced the anti-proliferative effect and induced a G0/G1 cell cycle arrest. SU-DHL-4 demonstrated a pronounced modulation of gene expression by AZD, which was markedly increased by additional SYK inhibition. Functional enrichment analyses identified combination-specific GO terms related to DNA replication and cell division. Genes such as ADGRA2, MYB, TNFRSF11A, S100A10, PLEKHH3, DHRS2 and FOXP1-AS1 were identified as possible key regulators. Conclusion: Simultaneous inhibition of BET and SYK enhanced the anti-proliferative effects, and induced a combination-specific gene expression signature. Targeted therapies by small molecule inhibitors and their combinations have been shown to efficiently affect tumor growth and survival in hematological neoplasias. B-cell receptor-associated kinases (BAKs) like spleen tyrosine kinase (SYK), as well as the bromodomain (BRD)-containing proteins such as the bromodomain and extra-terminal domain (BET) proteins play a critical role in signaling pathway transduction. The specific inhibition of BET and SYK represents a promising tool in hematological neoplasias.BET co-activators represent a group of epigenetic readers involved in histone recognition and transcription regulation ,2. BET pEarly BET inhibitors JQ1 and I-BET762 (GSK525762A) were designed to target the BRD4-NUT (nuclear protein in testis) fusion protein in aggressive NUT midline carcinoma. The specific binding of these inhibitors to BRD4, induces the displacement of BRD4 fusion from chromatin and leads to tumor regression in vivo ,11. FollBased on the promising results of targeting BET proteins in hematological and solid tumors, several BET inhibitors have been developed and are under clinical investigation, although currently without FDA approval .Most prominent BET inhibitors are pan-BET inhibitors, with specificity to more than one representative BET family protein. Representative pan-BET inhibitors are I-BET151 GSK1210151A) , ABBV-07151A , ABHere, we aimed to compare the anti-tumorigenic effect of a simultaneous SYK inhibition by Entospletinib with pan-BET inhibition by I-BET151 (I-BET) or isoform-specific bivalent BET inhibition by AZD5153 (AZD) in a DLBCL and Burkitt’s lymphoma in vitro approach. We investigated the synergistic potential of additional specific SYK inhibition and the resulting specific gene expression modulation patterns in both B-lymphoma subtypes. Inhibitor-based transcriptome modulation was evaluated by RNA sequencing and supported by microarray analyses.We uncovered additional anti-tumorigenic effects in B-lymphoma cell lines induced by simultaneous SYK and BET inhibition, which was investigated by analyzing proliferation, metabolic activity, apoptosis and cell cycle analyses. Cellular processes such as DNA replication and cell cycle were identified as key modulating pathways by high-throughput RNA sequencing.Human Burkitt’s lymphoma (BL) cell lines DG-75 and RAJI and diffuse large B-cell lymphoma (DLBCL) cell lines SU-DHL-4 (GCB) and U-2946 (ABC) were purchased from the German Collection of Microorganisms and Cell Cultures , cultured as recommended by the manufacturer in RPMI 1640 media  supplemented with 10% heat-inactivated FCS  and 100 µg/mL penicillin and streptomycin . Cultured cells were regularly tested for mycoplasma contamination and authenticity (cell surface markers by flow cytometry).AZD5153 (AZD), I-BET 151 (GSK1210151A) (I-BET) and Entospletinib (GS-9973) (Ento) were obtained from Selleck Chemicals  and prepared according to the manufacturer’s instructions to a 10 mM stock and stored at −80 °C until ready for use.5 cells/mL) were exposed to AZD, I-BET, Ento or DMSO (control) as mono substance (0.001 µM–10 µM) or in combination for up to 72 h Cell lines . Viable cells were determined by trypan blue staining and cell count with a hemocy-tometer . Metabolic activity was determined via tetrazolium compound WST-1  according to the manufacturer’s protocol and previously described in .TM  and BD FACS Suite Software .Early and late apoptosis was analyzed by Annexin V FITC  and Propidium Iodide (PI)  staining according to the manufacturer’s protocol and previously described in . MeasureTM, BD Biosciences, Heidelberg, Germany). Cell cycle distribution was examined by FlowJo Software .Protocol was previously described in . Briefly4 cells per slide  in a Cytospin 3 centrifuge . Slides were stained for 6 min with May–Grunwald working solution , rinsed with microscopy buffer according to WEISE (pH 7.2) , stained 20 min in Giemsa working solution  and rinsed thoroughly with microscopy buffer again. The slides were air dried at room temperature before analysis. Morphological changes were analyzed by the EVOS® XL Core Imaging System .Coated cytoslides  were prepared with 5 × 10Cell pellets were resuspended in QIAzol Lysis Reagent  and RNA isolation was performed with miRNeasy Mini Kit  according to the manufacturer’s instructions and previously described in .Poly-A RNA was enriched from 1 µg total RNA using the NEBNext Poly (A) mRNA Magnetic Isolation Module  and sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit . Sequencing was conducted on an Illumina NextSeq500  as single reads with 75 bp length with a total of 480 million reads.Gene expression analyses were also performed by microarray, using the Human Clariom D Array  according to manufacturer’s instructions and previously described in . In brieaffycoretools with probe mappings drawn from clariomhumantranscriptcluster.db. and a linear model was fitted of the form:ijY is the intensity for gene j in sample I, iTreatment is the treatment which sample i was subjected to, iLine is the cell line from which sample i was drawn, Batchi is the batch which sample i belongs to and ij is the error term, with the coefficients β0, β1 and β2 estimated by least squares. All contrasts were tested using the eBayes method of limma to perform a moderated t-test for each gene and the results were corrected for multiplicity using the Benjamini–Hochberg correction.Microarray data were RMA normalized using limma and probes were mapped to genes using Rsubread [edgeR was used to filter low read counts using the filterByExpr function, normalized using the TMM method, and a differential expression analysis was performed using the GLM method to fit a generalized linear model, specifying no intercept and with treatment group and batch as covariates. All contrasts were tested using the glmQLFTest function of edgeR, which performs an empirical Bayes quasi-likelihood F-test for each gene [p-value of <0.05 and a fold-change > 2.The RNAseq data were processed as follows: The raw reads from different lanes were concatenated and then trimming and quality control were performed using CutAdapt via TrimGalore! . TrimmedRsubread . edgeR wach gene , and thePrincipal component analysis (PCA) of the microarray data  and the RNAseq data  was performed to identify trends in the data. For the PCA plots, the cell batch effect was controlled by first using the removeBatchEffect function of limma.p-value of <0.05 being taken as significant. When considering the importance of GO terms, terms were ranked by Enrichr’s combined score. This is calculated by multiplying the log of the p-value for a given term by the z-score of the deviation of that term from its expected rank, given the size of the gene set. In this way the combined score aims to better account for biases introduced into enrichment analysis by variation in gene set size.Gene ontology (GO) enrichment analysis of biological process terms was performed using Enrichr  with a BTo compare the two platforms, the overlaps between all like gene lists were obtained and can be seen in p < 0.05, ** p < 0.01, *** p < 0.001.Statistical data are presented as mean  ±  standard deviation (SD) from at least three biologically independent samples. Statistical differences between multiple comparisons were analyzed using one-way ANOVA followed by Tukey’s test as post-hoc analysis for normally-distributed data. The Kruskal–Wallis test was applied to non-parametric data. Differences were considered statistically significant for * Synergy was evaluated by a mathematical Bliss independence model . Bliss sBurkitt’s lymphoma (BL) cell lines DG-75 and RAJI and diffuse large B-cell lymphoma (DLBCL) cell lines SU-DHL-4 and U-2946 were exposed to dose ranges of isoform-specific bivalent BET inhibitor AZD5153 (AZD) or pan-BET inhibitor I-BET 151 (I-BET) (0.001–10 µM) for 24 h, 48 h and 72 h . Both inp < 0.05) and 72 h (p < 0.01) Entospletinib (Ento) exposure. Metabolic activity was significantly reduced within 48 h but not after 72 h of substance application .Both combinations  induced a significant reduction of cell proliferation after 72 h exposure compared to the DMSO control in DG-75, SU-DHL-4 and U-2946 a,b. CellHowever, compared to the Ento single agent, the combinations induced an additional anti-proliferative effect in DG-75, RAJI and U-2946. Further, in SU-DHL-4 the combination induced a significant reduction of proliferation and metabolic activity compared to both BET inhibitors as single agents.Furthermore, both combinations reduced metabolic activity significantly in the DLBCL cell line SU-DHL-4 compared to the DMSO control and AZD or I-BET single agents, respectively b. HoweveThe synergistic potential of BET and SYK inhibition in BL and DLBCL cell lines was evaluated by a Bliss independence model  staining at 72 h a,b. The Moreover, we investigated if simultaneous inhibition of BET and SYK is able to boost the effect on cell cycle blockade after 72 h exposure, compared to the single agents.AZD as a mono application significantly increased the percentage of cells in G0/G1 phase in the BL cell line DG-75 and the DLBCL cell line SU-DHL-4 a,b. The Application of I-BET as a single agent did not induce cell cycle changes, while the I-BET+Ento combination significantly intensified the G0/G1 blockade compared to control as well as to the single agents in DG-75, SU-DHL-4.However, the cell cycle of BL cell line RAJI was not significantly affected a. RepresTo evaluate if Ento, AZD, I-BET as single agent or in combination induce apoptosis in BL or DLBCL cell lines after 72 h exposure, Annexin V/PI double staining was performed and analyzed by flow cytometry. During the tested conditions, no significant apoptosis induction could be observed for any tested lymphoma cell lines after 72 h c,d.RNA sequencing was performed in order to evaluate the underlying molecular mechanism of simultaneous BET and SYK inhibition in the DLBCL cell line SU-DHL-4. Principal component analysis (PCA) of exposed SU-DHL-4 cells (after batch correction) showed a distinct clustering of the treatment groups a.Volcano plots of the differential gene expression analysis highlight the significant deregulated genes by each respective treatment. In particular, Ento+AZD combined exposure .Ento and AZD single agent exposures vs. DMSO control . The comFurthermore, comparing Ento+AZD combined exposure to the single agents Ento –j.To validate the RNAseq data, Human Clariom D Array analyses  were carried out for the DLBCL cell line SU-DHL-4 and the BL cell line DG-75  affirminFOXP1-AS1) and solute carrier family 30 member 4 (SLC30A4) highly overexpressed by both procedures.Within the Ento+AZD combination, we found the genes of the long non-coding RNA (lncRNA) FOXP1 antisense RNA 1, E2F transcription factor 8 (E2F8) and chromatin assembly factor 1 subunit B (CHAF1B) were identified as highly downregulated by both tools and in both cell lines.Furthermore, MYB proto-oncogene, transcription factor AICDA) and BTB domain and CNC homolog 2 (BACH2) genes as significantly overexpressed, affirmed by both the RNAseq and microarray analyses. In DG-75, AICDA was also significantly upregulated by Ento single agent exposure. However, SPARC-like 1 (SPARCL1) was identified as significantly downregulated in SU-DHL-4.Ento single agent exposure revealed activation-induced cytidine deaminase (COBLL1), Ras-association domain family member 6 (RASSF6) and Rhotekin 2 (RTKN2) were identified as significantly downregulated, while COBLL1 and RTKN2 were also found to be significantly downregulated in DG-75.AZD single agent only showed overlaps in downregulated genes. Here, Cordon-Bleu WH2 repeat protein-like 1  were unique to this treatment.Gene ontology (GO) enrichment analyses by Enrichr identified several GO terms for the respective condition in the DLBCL cell line SU-DHL-4.The combination of Ento and AZD induced a significant downregulation of several genes, allocated to GO terms, which are mostly related to DNA replication and cell cycle processes . Howeverp < 0.05 level. The Ento+AZD combination revealed no enriched terms for the overexpressed differentially expressed genes but a total of 120 enriched terms for the underexpressed genes , centromer (CEN), cell division cycle (CDC), kinesin family (KIF), GINS complex subunit (GINS), having more than one gene allocated to the top 10 GO terms. Furthermore, GO term intersections between the conditions are summarized in A further approach was to identify which of the most strongly downregulated DEGs were allocated to the top 10 significant GO terms . This anFurthermore, GO terms were condensed using REVIGO and subsequently visualized by CirGO identifying GO clusters. Three big clusters were identified revealing DNA replication, kinetochore organization and cell cycle as the most affected biological processes . Most GOEpigenetic changes and deregulated BCR signaling by molecular lesions are able to promote B-lymphomagenesis ,38,39. THere, we comparatively evaluated the anti-tumorigenic effect of isoform-specific bivalent BET inhibitor AZD5153 and pan-BET inhibitor I-BET151 in a DLBCL and Burkitt’s lymphoma in vitro approach and further investigated the cell- and molecular effect of simultaneous SYK inhibition by Entospletinib.Both, AZD5153 and I-BET151 as single agents efficiently reduced proliferation and metabolic activity at low dosage in all tested BL and DLBCL cell lines, with slightly lower IC50 values for AZD compared to I-BET. Compared to Rhyasen et al., our evaluated IC50 values were slightly higher for AZD . FurtherFollowing on from this, the simultaneous exposure of BET inhibitor AZD or I-BET with SYK inhibitor Entospletinib to the BL and DLBCL cell lines further affects proliferation. A pronounced effect was observed in DLBCL cell line SU-DHL-4 by Ento addition, revealing additionally reduced proliferation and metabolic activity. However, the corresponding Bliss values of both combinations showed no synergistic effect, due to the good response to Ento single agent exposure by SU-DHL-4 cells. Ento addition in all other tested cell lines improved anti-proliferative activity moderately, while respective positive Bliss values indicate slight synergy due to the low response to Ento single agent exposure. In a recent study by Kim et al., it was shown that BET inhibition by GS-5829 in combination with Ibrutinib (BTK inhibitor) or Entospletinib synergistically increased anti-leukemia activity and induced apoptosis in primary co-cultured CLL cells . CombinaMoreover, we identified a significant G0/G1 cell cycle arrest induced by both combinations in BL cell lines DG-75 and DLBCL cell line SU-DHL-4 compared to control. In DLBCL U-2946 only Ento+AZD induced a significant cell cycle arrest. However, the comparison of both combinations to the single agents only slightly increased the cell cycle blockade.Similar findings were observed for BET inhibition as a single agent with BRD4 inhibitor JQ1, inducing a G1 cell cycle arrest in DLBCL cell lines . JQ1 wasIn order to clarify the underlying molecular mechanism, we further investigated the gene expression modulation and functional enrichment of single agents Ento and AZD and the specific gene set modulation by the Ento+AZD combination.By RNAseq analyses we identified that both single agents induced gene expression changes, while the combination was capable of highly increasing the gene expression modulation compared to control, as well as to both single agents. The fold change range of the combination likewise increased compared to the single agents. Rhyasen et al. investigated the effect of AZD5153 in xenograft leukemia and lymphoma models and also performed RNAseq on a hematologic cell line panel revealing 174 differentially expressed genes (DEGs) across this panel . CompareADGRA2) as the most significantly underexpressed gene in SU-DHL-4 cells. ADGRA2 or TEM5/GPR124  was identified as an orphan G protein-coupled receptor in endothelial cells with roles in brain angiogenesis and Wnt signaling [ADGRA2 had a worse overall survival [ADGRA2 in glioblastoma significantly decreased the proliferation of the cancer cells by disrupting the mitotic progression and chromosome segregation [ADGRA2 and its role in lymphoma is unknown. However, the DLBCL cell line SU-DHL-4 exhibits an ADGRA2 missense mutation inducing a protein change p.A765T [ADGRA2 expression remains to be clarified.Within the Ento+AZD combination, we identified the adhesion G protein-coupled receptor A2 . This receptor regulates various biological processes such as apoptosis, cell survival and differentiation through the activation of several signal transduction pathways such as nuclear factor κB (NF-κB), Jun N-terminal kinase (JNK), p38, extracellular signal-related kinase (ERK) and phosphoinositide 3-kinase (PI3K) [TNFRSF11A (RANK) expression has been linked to several cancers. Its overexpression is correlated to breast cancer and prostate cancer with high metastatic potential and to gliomas [TNFRSF11A induces cancer cell proliferation, migration and invasiveness in cervical cancer [KLF5 was not significantly affected by any of the treatments. Finally, TNFRSF11A induces survival and proliferation pathways in B cells in vitro with progression to B-cell malignancies [TNFRSF11A could be one reason for the effective anti-proliferative effect by the single agents and the intensification by Ento+AZD combination in SU-DHL-4 cells.A further significant downregulation has been observed in the gene TNF receptor superfamily member 11a (e (PI3K) ,52. Aber gliomas ,54,55. I gliomas ,57,58. Tgnancies . The sigS100A10). S100A10 is involved in various cellular processes, regulates plasminogen activation [S100A10 expression is correlated with tumor development, invasion, poor prognosis and further can act as a biomarker due to its expression in various tumor cells [The Ento+AZD combination was exclusively able to significantly downregulate the S100 calcium binding protein A10 (tivation  and has tivation ). Furtheor cells ,62,63,64MYB  as highly underexpressed. The transcription regulator is known to be aberrantly expressed or rearranged in leukemias, lymphomas and also solid tumors [MYB [MYB downregulation inhibits cell proliferation and invasion in solid tumors [The combined exposure additionally revealed d tumors ,66. SU-Dors [MYB , which wd tumors ,69.PLEKHH3) were also identified as significantly downregulated. The function of PLEKHH3 is not yet understood, while pleckstrin homology (PH) domains are known to be part of several proteins, e.g., cytoskeletal proteins, function in signal transduction, mediating protein–protein interactions and membrane association [PLEKHH3 was identified in blood immune cells associated with obstructive sleep apnea [PLEKHH3 in cancer has not been described until now.Pleckstrin homology, MyTH4 and FERM domain containing H3  was highly and significantly upregulated. High DHRS2 expression was observed in BET inhibitor resistant liver cancer cell lines. The specific BET inhibition in liver cancer cells induced an increase of DHRS2 expression in sensitive (Hep3B) and resistant cells (HepG2). This could be assumed to be related to drug escape mechanisms [DHRS2 was further related to invasion, lymph node metastasis and is associated with worse prognosis in esophageal squamous cell carcinoma. The overexpression of DHRS2 leads to decreased tumor proliferation and tumor volume both in vitro and in vivo, while the DHRS2 knockdown increases it [DHRS2 levels, which was linked to HDACi sensitivity [DHRS2 needs to be clarified in DLBCL.However, within the Ento+AZD combination we identified that the gene dehydrogenase/reductase 2  was significantly upregulated after combined Ento+AZD exposure in SU-DHL-4 cells. Currently, detailed knowledge about the actual function of COLCA2 is limited. However, it is assumed to have tumor suppressor function and low expression has been correlated to colorectal cancer [Furthermore, the colorectal cancer-associated 2 gene  enrichment analyses identified the main biological processes which were affected after combined or single agent exposure in DLBCL cell line SU-DHL-4. For the Ento+AZD combination, GO term enrichment analysis identified combination-specific GO terms compared to the single agents. Furthermore, only the underexpressed results of Ento+AZD combined exposure revealed several highly significantly modified GO terms. All other conditions showed no significance, but sometimes a high combined score.CDK2, CDCA5, CDC7, WEE1, E2F8, CCNA2, CDC6, CDC25A and CDC25C.The identified downregulation of general biological processes is related to DNA replication and cell division processes which may be caused by cell death induction due to combined BET and SYK inhibition. This assumption can be affirmed by the observed significant downregulation of cell cycle-related genes such as MCM), centromer (CEN), cell division cycle (CDC), kinesin family (KIF), GINS complex subunit (GINS). Several top GO terms contain significantly modulated genes related to the minichromosome maintenance complex (MCM). MCM proteins are DNA helicases regulating and initiating DNA replication processes by unwinding duplex DNA, thus playing a pivotal role in cell division [MCMs have been shown to be involved in tumorigenesis. MCM3 for example, has been identified being overexpressed in several cancers including leukemia and lymphoma [MCM2 expression was also suggested as a negative prognostic marker in DLBCL [MCM5 by JQ1 BET inhibitor was already identified in thyroid cancer cells [MCM genes in our study. Further, the cell division cycle gene CDC7 is highly expressed in a variety of tumors, suggesting a role as a biomarker [CDC7 and MCM2 have been also suggested as prognostic markers in DLBCL [Furthermore, the relation of the top 10 GO terms  and significant DEGs identified various gene sets with crucial roles in DNA replication processes such as mini chromosome maintenance . CDC7 ain DLBCL .GINS1 and GINS2 are likewise involved in DNA replication initiation and are upregulated in a variety of tumors, indicating potential as a prognostic marker [c marker ,91. CDC4c marker ,93. The c marker ). The siSeveral of the identified genes related to the top 10 GO terms are involved in DNA replication processes. We further identified their functions in tumorigenesis or high expression in cancers, suggesting biomarker and prognostic marker potential.Furthermore, GO terms were condensed using REVIGO, summarizing the GO terms based on semantic similarities and they were then visualized using CirGO, which highlighted changes in GO clusters. In particular, the arising GO clusters enriched in the underexpressed DEGs revealed that more than 50% of identified GO terms are related to DNA replication and cell cycle processes, as with the top GO terms when simply sorted by combined score. The emerging GO clusters match with the biological data of the cell cycle blockade as well as the mode of action of BET inhibition by AZD and its interference in histone recognition.The intervention in the biological processes is suggested to be mainly caused by the BET inhibition process, due to its major effects on biological and gene expression levels. However, the Ento addition leads to the specific gene and pathway deregulation caused by the combined exposure.The combination-specific emerging GO terms as well as gene modulation are, for the most part, exclusive and therefore they provide an indication for a combination-specific gene set modulation by Entospletinib and AZD5153 combined exposure. Both the enhanced biological response as well as the strongly enhanced gene modulation by the combination suggest an advantageous intervention for DLBCL subtypes, which requires further investigation.Here, we identified a moderate synergy of simultaneous BET and SYK inhibition in a BL and DLBCL in vitro approach affecting cell cycle modulation but not apoptosis. Expression analysis characterized a distinct gene expression modulation, while the combination strongly increased this modulation. The combination-specific emerging DEGs and GO terms are mostly exclusive and therefore provide an indication for a combination-specific gene set modulation. Although the changes in the main processes are mainly provoked by AZD exposure, Ento addition leads to specifically modified gene and pathway deregulation. The main affected biological processes can be related to DNA replication and cell division.ADGRA2, MYB, TNFRSF11A, S100A10, PLEKHH3, DHRS2 and FOXP1-AS1 presumably play a pivotal role of the Ento+AZD acting mode in SU-DHL-4 cells.The significantly differentially expressed genes Future work remains to fully understand the underlying mechanism of the Ento+AZD combination. Prospectively, it remains to clarify which modulated genes are key regulators by including functional analyses and extending the work to other B-lymphoma cell lines. Proteomic analyses as well as STRING analyses could further validate the affected biological processes investigated by RNAseq. A full understanding of the Ento+AZD combination-induced effects will also need to involve 3D and in vivo models. This is especially important for a better transferability and investigation of clinical value.