High-Throughput Drug Screen for Potential Combinations With Venetoclax Guides the Treatment of Transformed Follicular Lymphoma

Abstract

Transformed follicular lymphoma (t-FL) is an aggressive malignancy that is refractory and rapidly progressing with poor prognosis. There is currently no effective treatment. High-throughput screening (HTS) platforms are used to profile the sensitivity or toxicity of hundreds of drug molecules, and this approach is applied to identify potential effective treatments for t-FL. We randomly selected a compound panel from the School of Pharmaceutical Sciences Xiamen University, tested the effects of the panel on the activity of t-FL cell lines using HTS and the CCK-8 assay, and identified compounds showing synergistic anti-proliferative activity with the Bcl-2 inhibitor venetoclax (ABT-199). Bioinformatics tools were used to analyze the potential synergistic mechanisms. The single-concentration compound library demonstrated varying degrees of activity across the t-FL cell lines evaluated, of which the Karpas422 cells were the most sensitive, but it was the cell line with the least synergy with ABT-199. We computationally identified 30 drugs with synergistic effects in all cell lines. Molecularly, we found that the targets of these 30 drugs didn’t directly regulate Bcl-2 and identified 13 medications with high evidence value above .9 of coordination with ABT-199, further confirming TP53 may play the largest role in the synergistic effect. Collectively, these findings identified the combined regimens of ABT-199 and further suggested that the mechanism is far from directly targeting Bcl-2, but rather through the regulation and synergistic action of p53 and Bcl-2. This study intended to reveal the best synergistic scheme of ABT-199 through HTS to more quickly inform the treatment of t-FL.

Keywords

t-FL HTS ABT-199 TP53 synergistic effect

Introduction

Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma (NHL) and accounts for 20% of all NHL cases.^1,2 Translocation t (14; 18) (q32; q21) places the antiapoptotic Bcl-2 oncogene under the control of the immunoglobulin (Ig) heavy-chain enhancer and is a genetic hallmark of FL.^3,4 Although the disease follows a progression-free course over a long period, progression and transformation still occur.^2,5 Among them, the transformation into more aggressive forms is called transformed follicular lymphoma (t-FL), and the incidence of this aggressive form of lymphoma is 2-3% per year.⁴ Patients with t-FL have rapid progression, treatment resistance, poor prognosis, and outcomes that are unfortunate and far inferior to those of FL patients whose disease did not undergo transformation. Despite the improvement in the efficacy of chemoimmunotherapy regimens, most patients remain incurable due to the genetic diversity and the high heterogeneity in the clinical presentation of t-FL.^4–7 Therefore, further updated therapy regimens are urgently needed to impede progression, ameliorate prognosis, and achieve personalized treatment.

Bcl-2 is a member of the Bcl-2 family, which regulates cell apoptosis; it was first discovered by its involvement in the t (14; 18) chromosomal translocations commonly found in FL.^8,9 Bcl-2, classified as an oncogene, has been identified as a cause for the onset of lymphomas, confers a survival advantage on B cells by inhibiting apoptosis, and more generally may block a common cell death pathway induced by chemotherapy, conferring clinical drug resistance on cancer cells overexpressing Bcl-2 protein.^10,11 Therefore, many strategies have been developed to block or modulate the expression of Bcl-2. The first genuine BH3 mimetic and a specific antagonist of the antiapoptotic proteins Bcl-2, Bcl-xL, and Bcl-w, ABT-737 has attracted the attention of numerous biologists. However, the obvious targeted thrombocytopenia (through Bcl-xL inhibition) observed in clinical trials of this agent limited its clinical utility.^12,13

Venetoclax (ABT-199), a highly selective Bcl-2 inhibitor, is currently approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with relapsed chronic lymphocytic leukemia (CLL) with a 17p deletion.¹⁴ Research has demonstrated that venetoclax has potent activity against FL, diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL) cell lines, as well as in a t (14; 18)-carrying xenograft model.^15,16 Although venetoclax is active as a single agent in NHL, the clinical benefit varies depending on the histologic subtype. The combination of venetoclax with chemotherapy, monoclonal antibodies, and B-cell receptor signaling inhibitors suggests that venetoclax would be a logical partner in a range of combination regimens.^16,17 Given the high heterogeneity of t-FL, the search for the optimal combination of venetoclax will enable targeted inhibition of Bcl-2 to have a greater clinical impact.

Currently, multitargeted drug combinations are evaluated in vitro and preclinically to establish anticancer efficacy and potential standards of care for different types of malignancies.¹⁸ The further clinical development of venetoclax in B-cell malignancies, especially t-FL, will be aided by establishing venetoclax combination treatments with maximum synergistic effects. High-throughput screening (HTS) is often used to profile the sensitivity or toxicity of hundreds of drug molecules to identify a few hit compounds with desired response profiles for further development.^19,20 We designed a drug HTS platform to systematically screen for combinations of venetoclax with approved therapeutics, emerging drugs, and well-characterized molecular probes targeting different aspects of the MAPK and PI3K signaling pathways known to be relevant in the pathogenesis of cancer. Compared to target-based drug discovery, which starts from the individual disease-related protein Bcl-2 in lymphoma, phenotype-based drug discovery assays profile the complex cellular system as a whole. The goal of the present study was to identify a venetoclax combination regimen that causes apoptosis in t-FL cell lines.

Material and Methods

Cell Culture

The t-FL cell line RL was obtained from the Institute of Hematology, Xiamen University School of Medicine (Xiamen, Fujian, China). The t-FL cell lines SU-DHL4 (CBP60628) and Karpas422 (CBP60629) were purchased from Nanjing Cobioer Biotechnology Co., Ltd. (Nanjing, Jiangsu, China), and SC-1 cells were generously donated by the Department of Hematology, Jiangsu Province Hospital (Nanjing, Jiangsu, China). All these cell lines were cultured in RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, CA, USA), 100 units/ml penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). The cells were maintained in a 37°C incubator with a 5% CO₂ environment.

The Automated Drug Screening System

The plate and handler automated screening system of PerkinElmer Co., Ltd. (PerkinElmer, Waltham, MA, USA) was used to conduct experiments through the Core Facility of Biomedical Sciences, Xiamen University (Xiamen, Fujian, China). The system is based on the plate and handler Flex multiaxis robotic arms, integrates various equipment such as a JANUS liquid workstation, a Nivo multifunction microplate reader, and an incubator, and has the function of high-throughput automated drug screening.

Compound Library

Venetoclax (ABT-199; GDC-0199) was provided by Topscience Co. Ltd. (Shanghai, China) and dissolved in sterile DMSO (Sigma, MO, USA) to produce a 5 mM stock solution stored at −20°C. Venetoclax was combined with a compound library obtained from the School of Pharmaceutical Sciences Xiamen University (Xiamen, Fujian, China). All drugs were approved by the FDA and dissolved in sterile DMSO to produce a 2.5 mM stock solution stored at −80°C. The layout of the compound library 384-well plate in the experiment can be seen in Supplementary Table 1, and the details of the bioactive compound library can be confirmed in Supplementary Table 2. All compounds are currently approved therapies for malignancies.

Anti-Proliferation Assay

All t-FL cell lines were seeded in 384-well plates (Nest Life Technology Co., Ltd., 761 001, Wuxi, Jiangsu, China) at 3000 cells per well in 50 μL of the respective medium using a Multidrop Combi automatic dispenser (Thermo Fisher Scientific, Waltham, MA, USA) with or without venetoclax (20 nM). Then, single-dose drugs of the test compound plates were thawed completely, and .1 μL of the respective drug volume (5 μM) was added to each well using a plate and handler automated instrument (PerkinElmer). Cells were treated for 24 h followed by an assessment of cell viability utilizing the Cell Counting Kit-8 (CCK-8, Zeta Life, CA, USA) assay and incubated for an additional 2 h, after which absorbance at 450 nm (OD value) was detected on a Nivo multifunction microplate reader (PerkinElmer). Cell viability × (%) = [OD value (drug)-OD value (blank)]/[ OD value (DMSO)-OD value (blank)] ×100. Experiments were conducted in triplicate.

Synergy Assessment

Due to there being only one concentration of all drugs in the compound library, we chose a method with higher specificity rather than sensitivity in this synergistic drug screening. To identify antiproliferative combination effects at on-target concentrations, the single-agent dose axis for ABT-199 was limited to a concentration of 20 nM. With this method, a cell viability of less than 80% compared to ABT-199 (which is considered a control) was considered an effect. If the medication is effective, and the value of medication monotherapy subtracted from the value after the combined ABT-199 is greater than 0, we defined the drug to be synergistic with ABT-199.

Network Construction and Pathway Analysis

The interaction network of proteins and the involved pathways were analyzed through the STRING database (https://string-db.org/). Information on drug targets was provided by Topscience Co., Ltd. (Supplementary Table 2). All the interactive relationships were constructed for the network by Cytoscape (Version 3.9.0, MD, USA).

Results

t-FL Cell Lines Exhibited Individual Sensitivities to 320 Drugs

To determine the sensitivity of transformed follicular lymphoma to various drugs, a compound library plate was randomly selected from the School of Pharmaceutical Sciences Xiamen University, which contained 320 compounds in a 384-well plate. Four t-FL cell lines, SU-DHL4, Karpas422, RL, and SC-1, were treated with this compound library (the final concentration was 5 μM) under the drug screening platform. As shown in Figure 1, the four cell lines exhibited completely different sensitivities to the 320 drugs at 24 h. In addition, Karpas422 cells were determined as the most sensitive cell line under the action of most agents. To obtain a more effective treatment strategy for t-FL, this compound plate was combined with the Bcl-2 targeting drug ABT-199, attempting to better gain the coordinated lethality of these 320 drugs and ABT-199.

Figure 1.

Cell viability assay in t-FL cell lines treated with a compound library. The cell viability percentage of (A) SU-DHL4, (B) Karpas422, (C) RL, and (D) SC-1 cells was measured after treatment with 320 drugs compared to equal volume DMSO. Heat maps were drawn by the GraphPad Prism software according to cell viability percentage.

ABT-199 Alters the Effect of the Compound Library on t-FL Cell Lines

To investigate the synergistic drugs for ABT-199, the concentration of ABT-199 was fixed at 20 nM and cotreated for four cell lines with the compound library for 24 h. Then the value of cell viability with a combined treatment regimen was divided by the cell viability with ABT-199 treatment alone to accurately define drugs with an efficacious effect on t-FL cells. Drug percentages above 80% would be deemed to lack cytotoxicity of the t-FL cell line. As expected, it was found that many drugs showed different effects than the original single drugs by combination therapy (Figure 2), suggesting that their target molecules may coordinate with Bcl-2, which is the target molecule of ABT-199.

Figure 2.

Cell viability of t-FL cell lines treated with ABT-199 combined with compound plates. The combination of ABT-199 (20 nM) with special drugs was applied (A-D) to four t-FL cell lines to obtain the percentage of cell viability relative to ABT-199 treatment alone. The blue color of some squares means the percentage was more than 100%.

In cases where these combination treatments were effectively lethal to t-FL cell lines, the combined percentages were subtracted from the original percentages of these drugs to define the drugs for which ABT-199 contributed to their effects. As shown in Figure 3, despite being sensitive to most drugs in the library, Karpas422 cells showed the least promotion of drug effects by ABT-199.

Figure 3.

The effects of ABT-199 on this compound library. (A-D) The difference values of the combined regimen from the original percentage in four t-FL cell lines, in which the value of treatment with ABT-199 alone was subtracted. The blue color in some squares means the value is less than 0.

Certain Drugs Show Synergism With ABT-199 in t-FL Cell Lines

To eliminate random errors, a percentage of less than 80% was interpreted to mean that the drug was effective for t-FL. If a drug combined with ABT-199 was effective and ABT-199 promoted the efficacy of this agent, it was judged that the drug and ABT-199 have a synergistic effect on the treatment of t-FL. This approach avoids most combinatorial strategies that exert no cooperation or an antagonistic effect.

As shown in Figure 4, certain drugs were demonstrated to synergize with ABT-199, and in Karpas422 cells, there were only 43 different types of medications that work in synergy with ABT-199. All drugs were subsequently statistically analyzed that synergized with ABT-199 in all four cell lines, and the results are shown in Figure 5. Ignoring less synergistic or antagonistic drugs, 30 drugs were interpreted to be synergistic with ABT-199 in four cell lines (Table 1).

Figure 4.

Coordination of compound library plates with ABT-199 in t-FL. The association of 320 drugs with ABT-199 demonstrated in (A) SU-DHL4, (B) Karpas422, (C) RL, and (D) SC-1 cells. The yellow color in the square means that the specific drugs are synergistic with ABT-199. The whiteness or darkness indicates that the drug combined with ABT-199 has no effect or antagonism.

Figure 5.

The compound library exhibited synergism with ABT-199 in all cell lines. The color changes from blue to white to pink, which means that the drug’s synergism with ABT-199 ranges from a single cell line to all cell lines in t-FL.

Table 1.

Details of Drugs That Synergize With ABT-199 in t-FL Cell Lines.

Number	ID	MOLENAME	CAS	SMILES	Formula	MolWt	Bioactivity	Pathways	Receptor
1	T3104	LY2608204	1234703-40-2	C1CCC(CC1)[C@@H]1C[C@]1(C(=O)Nc1ncc(s1)SCCN1CCCC1)c1cc c(cc1)S(=O)(=O)C1CC1	C28H37N3O3 S3	559.81	LY2608204 has been used in trials studying the treatment of Diabetes Mellitus, Type 2.	Metabolism	Glucokinase
2	T6233	Entinostat	209783-80-2	c1ccc(c(c1)N)NC(=O)c1ccc(cc1)CN C(=O)OCc1cnccc1	C21H20N4O3	376.41	Entinostat (MS-275) is an inhibitor of HDACs that inhibits HDAC1 and HDAC3 (IC50s: 0.18/0.74 μM).	Chromatin/Epigenetic; DNA Damage/DNA Repair	HDAC1; HDAC3
3	T1849	Momelotinib	1056634-68-4	O=C(NCC#N)c1ccc(cc1)c1nc(Nc2c cc(cc2)N2CCOCC2)ncc1	C23H22N6O2	414.46	Momelotinib is an orally bioavailable small-molecule inhibitor of Janus kinases 1 and 2 (JAK1/2) with IC50 of 11 nM/18 nM. JAK1/2 inhibitor CYT387 competes with JAK1/2 for ATP binding, which may result in inhibition of JAK1/2 activation, inhibition of the JAK-STAT signaling pathway, and so the induction of apoptosis and a reduction of tumor cell proliferation in JAK1/2-expressing tumor cells.	Angiogenesis; Chromatin/Epigenetic; JAK/STAT signaling; Stem Cells	JAK1; JAK2; JAK3
4	T1784	Everolimus	159351-69-6	C(O)CO[C@H]1[C@@H](C[C@@H](CC1)C[C@H]([C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=[C @]2([C@@H](CC[C@H](O2)C[C @@H](/C(=C/C=C/C=C/[C@H](C[ C@H](C(=O)[C@@H]([C@@H](/C(=C/[C@H](C(=O)C1)C)/C)O)OC)C)C)/C)OC)C)O)C)OC	C53H83NO14	958.22	Everolimus is a potent mTOR inhibitor that binds to FKBP-12. It is used alone or in combination with calcineurin inhibitors.	PI3K/Akt/mTOR signaling	mTOR (FKBP12)
5	T2668	JNK-IN-8	1410880-22-6	Cc1c(ccc(c1)NC(=O)c1cc(ccc1)NC(=O)/C=C/CN(C)C)Nc1nccc(n1)c1cn ccc1	C29H29N7O2	507.59	JNK-IN-8 is an irreversible JNK1/2/4 inhibitor (IC50: 4.7/18.7/1 nM). The selectivity is higher 10-fold than MNK2, Fms and no inhibition of Met, c-Kit, PDGFRβ in A375 cell line.	MAPK; Tyrosine Kinase/Adaptors	Kit (V559D); Kit (V559D,T670I); JNK1; JNK2; JNK3
6	T2037	TH588	1609960-31-7	Nc1nc(cc(NC2CC2)n1)c1c(Cl)c(Cl) ccc1	C24H27N7O	429.52	TH588 is nudix hydrolase family inhibitor that effectively and selectively engages and inhibits the MTH1(IC50: 5 nM) in cells.	DNA Damage/DNA Repair	MTH1
7	T0968	Paclitaxel	33069-62-4	CC(=O)O[C@@H]1C2=C(C)[C@H](C[C@@](O)([C@H](OC(=O)c3ccccc3)[C@H]3[C@@]4(CO[C@@H]4C[C@H](O)[C@@]3(C)C1=O)OC(C)=O)C2(C)C)OC(=O)[C@H](O)[C@@H](NC(=O)c1ccccc1)c1ccccc 1	C47H51NO14	853.92	Paclitaxel is a cyclodecane isolated from the bark of the Pacific yew tree. It stabilizes microtubules in their polymerized form leading to cell death.	Cytoskeletal Signaling	Microtubule
8	T1986L	Atopaxar Hydrobromide	474550-69-1	c1(c(c(cc2c1C(=N)N(C2)CC(=O)c1 cc(c(c(c1)N1CCOCC1)OC)C(C)(C) C)OCC)OCC)F.Br	C29H39BrFN3 O5	608.54	Atopaxar, a reversible PAR-1 thrombin receptor antagonist, interferes with platelet signaling. Atopaxar has been investigated for the treatment of Coronary Artery Disease and Acute Coronary syndrome	GPCR/G Protein	PAR-1
9	T6045	Torin 1	1222998-36-8	CCC(=O)N1CCN(CC1)c1c(cc(cc1) n1c(=O)ccc2cnc3ccc(cc3c12)c1cc2c cccc2nc1)C(F)(F)F	C35H28F3N5 O2	607.62	Torin 1 is an effective inhibitor of mTORC1/2 with (IC50: 2 nM/10 nM); has 1000-fold selectivity for mTOR than PI3K.	DNA Damage/DNA Repair; PI3K/Akt/mTOR signaling	DNA-PK; mTOR; mTORC1; mTORC2; p110γ
10	T2217	Cephalomannine	71610-00-9	O1[C@H]2[C@](OC(=O)C)([C@@H]3[C@@]([C@@H](O)C2)(C(=O)[C@H](OC(=O)C)C2=C([C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)/C(=C/C)/C)c4ccccc4)C[C@@]( O)([C@H]3OC(=O)c3ccccc3)C2(C) C)C)C)C1	C45H53NO14	831.91	Cephalomannine is a taxol derivative obtained from Taxus yunnanensis with antitumor activity.	Cytoskeletal Signaling	Microtubule/Tubul in
11	T1758	ABT-751	141430-65-1	COc1ccc(cc1)S(=O)(=O)Nc1c(Nc2c cc(O)cc2)nccc1	C18H17N3O4 S	371.41	ABT-751 has been investigated for the treatment of Lung Cancer, Non-Small Cell Lung Cancer, and Non-Small-Cell Lung Cancer.	Cytoskeletal Signaling	Microtubule
12	T3108	CUDC-101	1012054-59-9	n1cnc(c2c1cc(c(c2)OCCCCCCC(= O)NO)OC)Nc1cc(ccc1)C#C	C24H26N4O4	434.49	CUDC-101 is a potent inhibitor of HDAC, EGFR and HER2 with IC50s of 4.4, 2.4 and 15.7 nM, respectively.	Chromatin/Epigenetic; Angiogenesis; DNA Damage/DNA Repair; JAK/STAT signaling; NF-Κb; Tyrosine Kinase/Adaptors	EGFR; HDAC; HDAC1; HDAC10; HDAC2; HDAC3; HDAC4; HDAC5; HDAC6; HDAC7; HDAC8; HDAC9; HER2
13	T6173	BI 2536	755038-02-9	CC[C@@H]1C(=O)N(c2cnc(nc2N1C1CCCC1)Nc1c(cc(cc1)C(=O)NC1 CCN(CC1)C)OC)C	C28H39N7O3	521.66	BI2536 is an effective Plk1 inhibitor (IC50: 0.83 nM). It has 4- and 11-fold greater selectivity than Plk2 and Plk3.	Cell Cycle/Checkpoint; Chromatin/Epigenetic	BRD4; PLK1; PLK2; PLK3
14	T1917	6-BIO	667463-62-9	O/N=C/1\c2ccccc2N\C1=C\1/C(=O) Nc2c1ccc(Br)c2	C16H10BrN3O 2	356.17	BIO is a specific inhibitor of GSK-3.	PI3K/Akt/mTOR signaling; Cell Cycle/Checkpoint; Tyrosine Kinase/Adaptors; Stem Cells	CDK1/CyclinB; CDK2/CyclinA; CDK5/p35; GSK-3; Tyk2
15	T2677	Crenolanib	670220-88-9	CC1(COC2=CC3=C(C=C2)N(C=N 3)C2=NC3=C(C=CC=C3N3CCC(N)CC3)C=C2)COC1	C26H29N5O2	443.54	Crenolanib is an orally bioavailable type III tyrosine kinases inhibitor of PDGFRα/β and FLT3 (IC50s: 11, 3.2, and 4 nM).	Angiogenesis; Tyrosine Kinase/Adaptors	PDGFRα; PDGFRβ; FLT3
16	T2005	TP0903	1341200-45-0	CN(S(=O)(=O)c1ccccc1Nc1nc(Nc2 ccc(cc2)CN2CCN(CC2)C)ncc1Cl)C	C24H30ClN7O 2S	516.06	TP-0903 is a potent and selective Axl kinase inhibitor.	Tyrosine Kinase/Adaptors	AXL
17	T1846	Y320	288250-47-5	Cc1c(cnn1c1ccc(Cl)cc1)C(=O)Nc1c c(C#N)c(cc1)N1CCC(CC1)N1CCOCC1	C27H29ClN6O 2	505.01	Y-320 is a new phenylpyrazoleanilide immunomodulator.	Metabolism	ATGL
18	T1999	Taselisib	1282512-48-4	CC(C)n1nc(C)nc1- c1cn2CCOc3cc(ccc3-c2n1)- c1cnn(c1)C(C)(C)C(N)=O	C24H28N8O2	460.53	Taselisib is an orally bioavailable inhibitor of the class I phosphatidylinositol 3-kinase (PI3K) alpha isoform (PIK3CA), with potential antineoplastic activity.	PI3K/Akt/mTOR signaling	PI3Kα; PI3Kβ; PI3Kγ; PI3Kδ
19	T1818	Tenovin-6	1011557-82-6	CC(C)(C)c1ccc(cc1)C(=O)NC(=S)N c1ccc(cc1)NC(=O)CCCCN(C)C	C25H34N4O2 S	454.63	Tenovin-6 is a p53 transcriptional activity agonist.	Chromatin/Epigenetic; DNA Damage/DNA Repair	SIRT1; SIRT2; SIRT3
20	T1811	WH-4-023	837422-57-8	COc1ccc(N(C(=O)Oc2c(C)cccc2C)c 2nc(Nc3ccc(N4CCN(C)CC4)cc3)ncc2)c(OC)c1	C32H36N6O4	568.67	WH-4-023 is a potent and orally active Lck/Src inhibitor. It also potently inhibits SIK.	Angiogenesis	Lck; Src
21	T1827	Buparlisib	944396-07-0	Nc1ncc(c2cc(nc(n2)N2CCOCC2)N2 CCOCC2)c(c1)C(F)(F)F	C18H21F3N6 O2	410.39	Buparlisib is an orally bioavailable specific oral inhibitor of the pan-class I PI3K (IC50s: 52/166/116/nM for p110α, p110β, and p110δ).	PI3K/Akt/mTOR signaling	p110α; p110β; p110γ; p110δ; Vps34
22	T1916	Apitolisib	1032754-93-0	c12sc(CN3CCN(C(=O)[C@@H](O)C)CC3)c(C)c1nc(c1cnc(N)nc1)nc2N 1CCOCC1	C23H30N8O3 S	498.6	Apitolisib, an effective, class I PI3K inhibitor for P13Kα (IC50=5 nM), P13Kβ (IC50=27 nM), P13Kδ (IC50=7 nM), P13Kγ (IC50=14 nM), is used in trials study of solid cancers, breast cancer, prostate cancer, renal cell carcinoma, and endometrial carcinoma, among others.	PI3K/Akt/mTOR signaling	mTOR; p110α; p110β; p110γ; p110δ
23	T1844	KPT-330	1421923-86-5	FC(F)(F)c1cc(cc(c1)c1nn(/C=C/C(= O)NNc2nccnc2)cn1)C(F)(F)F	C17H11F6N7 O	443.31	KPT-330 is a CRM1-selective inhibitor of nuclear export. It inhibits protein trafficking from the nucleus and induces cell cycle arrest and apoptosis in mesothelioma cells.	Membrane transporter/Ion channel; Others	CRM1; XPO1
24	T3111	abemaciclib mesylate	1231930-82-7	CCN1CCN(Cc2ccc(Nc3nc(c4cc(F)c 5nc(C)n(C(C)C)c5c4)c(F)cn3)nc2)CC1.CS(=O)(=O)O	C27H32F2N8· CH4O3S	602.7	LY2835219 is a specific and effective inhibitor of CDK4(IC50=2 nM) and CDK6(IC50=10 nM).	Cell Cycle/Checkpoint	CDK4; CDK6
25	T2220	2-Methoxyestradiol	362-07-2	c1c(c(cc2CC[C@H]3[C@@H]4CC[ C@@H]([C@]4(CC[C@@H]3c12) C)O)O)OC	C19H26O3	302.41	2-Methoxyestradiol is an orally bioavailable estradiol metabolite with potential antineoplastic activity. 2-Methoxyestradiol inhibits angiogenesis by reducing endothelial cell proliferation and inducing endothelial cell apoptosis. This agent also inhibits tumor cell growth by binding to tubulin, resulting in antimitotic activity, and by inducing caspase activation, resulting in cell cycle arrest in the G2 phase, DNA fragmentation, and apoptosis.	Angiogenesis; Cytoskeletal Signaling; Chromatin/Epigenetic	HIF-1α; HIF-2α; Microtubule depolymerization
26	T1852	Belinostat	414864-00-9	ONC(=O)/C=C/c1cc(ccc1)S(=O)(= O)Nc1ccccc1	C15H14N2O4 S	318.35	Belinostat is a novel hydroxamic acid-type histone deacetylase (HDAC) inhibitor with antineoplastic activity. Belinostat targets HDAC enzymes, thereby inhibiting tumor cell proliferation, inducing apoptosis, promoting cellular differentiation, and inhibiting angiogenesis. This agent may sensitize drug-resistant tumor cells to other antineoplastic agents, possibly through a mechanism involving the down-regulation of thymidylate synthase.	Chromatin/Epigenetic; DNA Damage/DNA Repair; NF-Κb	HDAC
27	T1912	Dinaciclib	779353-01-4	c1(cc(nc2n1ncc2CC)N1[C@@H](C CCC1)CCO)NCc1c[n+](ccc1)[O-]	C21H28N6O2	396.49	Dinaciclib is a new-type and effective CDK inhibitor for CDK2/5/1/9 (IC50: 1 nM/1 nM/3 nM/4 nM) with potential antineoplastic activity.	Cell Cycle/Checkpoint	CDK1; CDK2; CDK5; CDK9
28	T6226	Pemetrexed Disodium Hydrate	357166-30-4	[Na+].[Na+].Nc1nc(=O)c2c(CCc3cc c(cc3)C(=O)N[C@@H](CCC([O-]) =O)C([O-])=O)c[nH]c2[nH]1	C20H21N5O6· 5/2H2O·2Na	516.42	Pemetrexed Disodium Hydrate is a new-type antifolate and antimetabolite for TS, DHFR, and GARFT. The Ki of Pemetrexed Disodium Hydrate for TS, DHFR and GARFT is 1.3 nM, 7.2 nM and 65 nM, respectively.	DNA Damage/DNA Repair; Metabolism	DHFR; DHFR; GARFT; Thymidylate synthase
29	T1773	Afatinib Dimaleate	850140-73-7	c12cc(c(cc1c(ncn2)Nc1cc(c(cc1)F)C l)NC(=O)/C=C/CN(C)C)O[C@H]1 CCOC1.C(=C\C(=O)O)\C(=O)O.C(=C\C(=O)O)\C(=O)O	C32H33ClFN5 O11	718.08	Afatinib is an orally bioavailable anilino-quinazoline derivative and inhibitor of the receptor tyrosine kinase (RTK) epidermal growth factor receptor (ErbB; EGFR) family, with antineoplastic activity.	Angiogenesis; JAK/STAT signaling; Tyrosine Kinase/Adaptors	EGFR (L858R); EGFR (L858R/T790M); EGFR (wt); HER2
30	T1911	Afuresertib	1047644-62-1	c1(cnn(c1c1c(sc(c1)C(=O)N[C@@ H](Cc1cc(ccc1)F)CN)Cl)C)Cl	C18H17Cl2FN 4OS	427.32	Afuresertib is an orally bioavailable inhibitor of the serine/threonine protein kinase Akt (protein kinase B) with potential antineoplastic activity.	Cytoskeletal Signaling; PI3K/Akt/mTOR signaling	Akt1; Akt2; Akt3

Drugs Synergistic With ABT-199 Target Various Molecules

Due to the noticeable results discovered above, the potential mechanism of synergism was further investigated. Considering that Bcl-2 is the most effective target of ABT-199, how many proteins may be affected by ABT-199 were first identified. Through calculations on the STRING database (https://cn.string-db.org/), proteins that are directly regulated by Bcl-2 were searched for. The 10 proteins most directly regulated by Bcl-2 were identified, including BID, BIK, and TP53 (Figure 6A).

Figure 6.

Proteins directly affected by Bcl-2 and targets of the compound library plate. (A) Bcl-2 directly affects 10 proteins. (B) The targets of 30 drugs synergistic with ABT-199 are plated in rounds filled with red color, and the drugs in the green square.

However, these 10 proteins were not present in the target library of a defined set of 30 drugs that synergized with ABT-199. Therefore, all direct targets of the 30 defined agents were found (Figure 6B), including CDK2, JAK, AKT. These results indicate that the mechanism of combination therapy is more complex than the direct interaction with Bcl-2.

The Synergistic Effect of ABT-199 With Each Drug Arises Through a Complex Interaction Network and Pathway

To describe the interaction between ABT-199 and 30 defined drugs, their target molecules were entered into the STRING database to complete their interaction network (Figure 7). Medications with evidence values of less than .9 were discarded to obtain more specific results, resulting in 13 medications with high evidence of coordination with ABT-199 (Table 2). Notably, TP53 is at the center of this network crosslinking map.

Figure 7.

Interaction regulation of Bcl-2-related networks with 13 drug targets. The interaction network around Bcl-2 shows the drugs taking part in Bcl-2 regulation.

Table 2.

Details of 13 Drugs With High Evidence of Coordination With ABT-199.

Number	ID	MOLENAME	CAS	SMILES	Formula	MolWt	Bioactivity	Pathways	Receptor
1	T6233	Entinostat	209783-80-2	c1ccc(c(c1)N)NC(=O)c1ccc(cc1)CN C(=O)OCc1cnccc1	C21H20N4O3	376.41	Entinostat (MS-275) is an inhibitor of HDACs that inhibits HDAC1 and HDAC3 (IC50s: 0.18/0.74 μM).	Chromatin/Epigenetic; DNA Damage/DNA Repair	HDAC1; HDAC3
2	T1849	Momelotinib	1056634-68-4	O=C(NCC#N)c1ccc(cc1)c1nc(Nc2c cc(cc2)N2CCOCC2)ncc1	C23H22N6O2	414.46	Momelotinib is an orally bioavailable small-molecule inhibitor of Janus kinases 1 and 2 (JAK1/2) with IC50 of 11 nM/18 nM. JAK1/2 inhibitor CYT387 competes with JAK1/2 for ATP binding, which may result in inhibition of JAK1/2 activation, inhibition of the JAK-STAT signaling pathway, and so the induction of apoptosis and a reduction of tumor cell proliferation in JAK1/2-expressing tumor cells.	Angiogenesis; Chromatin/Epigenetic; JAK/STAT signaling; Stem Cells	JAK1; JAK2; JAK3
3	T2668	JNK-IN-8	1410880-22-6	Cc1c(ccc(c1)NC(=O)c1cc(ccc1)NC(=O)/C=C/CN(C)C)Nc1nccc(n1)c1cn ccc1	C29H29N7O2	507.59	JNK-IN-8 is an irreversible JNK1/2/4 inhibitor (IC50: 4.7/18.7/1 nM). The selectivity is higher 10-fold than MNK2, Fms and no inhibition of Met, c-Kit, PDGFRβ in A375 cell line.	MAPK; Tyrosine Kinase/Adaptors	Kit (V559D); Kit (V559D,T670I); JNK1; JNK2; JNK3
4	T1986L	Atopaxar Hydrobromide	474550-69-1	c1(c(c(cc2c1C(=N)N(C2)CC(=O)c1 cc(c(c(c1)N1CCOCC1)OC)C(C)(C) C)OCC)OCC)F.Br	C29H39BrFN3 O5	608.54	Atopaxar, a reversible PAR-1 thrombin receptor antagonist, interferes with platelet signaling. Atopaxar has been investigated for the treatment of Coronary Artery Disease and Acute Coronary Syndrome.	GPCR/G Protein	PAR-1
5	T3108	CUDC-101	1012054-59-9	n1cnc(c2c1cc(c(c2)OCCCCCCC(= O)NO)OC)Nc1cc(ccc1)C#C	C24H26N4O4	434.49	CUDC-101 is a potent inhibitor of HDAC, EGFR and HER2 with IC50s of 4.4, 2.4 and 15.7 nM, respectively.	Chromatin/Epigenetic; Angiogenesis; DNA Damage/DNA Repair; JAK/STAT signaling; NF-Κb; Tyrosine Kinase/Adaptors	EGFR; HDAC; HDAC1; HDAC10; HDAC2; HDAC3; HDAC4; HDAC5; HDAC6; HDAC7; HDAC8; HDAC9; HER2
6	T6173	BI 2536	755038-02-9	CC[C@@H]1C(=O)N(c2cnc(nc2N1 C1CCCC1)Nc1c(cc(cc1)C(=O)NC1CCN(CC1)C)OC)C	C28H39N7O3	521.66	BI2536 is an effective Plk1 inhibitor (IC50: 0.83 nM). It has 4- and 11-fold greater selectivity than Plk2 and Plk3.	Cell Cycle/Checkpoint; Chromatin/Epigenetic	BRD4; PLK1; PLK2; PLK3
7	T2005	TP0903	1341200-45-0	CN(S(=O)(=O)c1ccccc1Nc1nc(Nc2ccc(cc2)CN2CCN(CC2)C)ncc1Cl)C	C24H30ClN7O2S	516.06	TP-0903 is a potent and selective Axl kinase inhibitor.	Tyrosine Kinase/Adaptors	AXL
8	T1818	Tenovin-6	1011557-82-6	CC(C)(C)c1ccc(cc1)C(=O)NC(=S)N c1ccc(cc1)NC(=O)CCCCN(C)C	C25H34N4O2 S	454.63	Tenovin-6 is a p53 transcriptional activity agonist.	Chromatin/Epigenetic; DNA Damage/DNA Repair	SIRT1; SIRT2; SIRT3
9	T1844	KPT-330	1421923-86-5	FC(F)(F)c1cc(cc(c1)c1nn(/C=C/C(= O)NNc2nccnc2)cn1)C(F)(F)F	C17H11F6N7 O	443.31	KPT-330 is a CRM1-selective inhibitor of nuclear export. It inhibits protein trafficking from the nucleus and induces cell cycle arrest and apoptosis in mesothelioma cells.	Membrane transporter/Ion channel; Others	CRM1; XPO1
10	T3111	abemaciclib mesylate	1231930-82-7	CCN1CCN(Cc2ccc(Nc3nc(c4cc(F)c5nc(C)n(C(C)C)c5c4)c(F)cn3)nc2)C C1.CS(=O)(=O)O	C27H32F2N8· CH4O3S	602.7	LY2835219 is a specific and effective inhibitor of CDK4(IC50=2 nM) and CDK6(IC50=10 nM).	Cell Cycle/Checkpoint	CDK4; CDK6
11	T1852	Belinostat	414864-00-9	ONC(=O)/C=C/c1cc(ccc1)S(=O)(=O)Nc1ccccc1	C15H14N2O4 S	318.35	Belinostat is a novel hydroxamic acid-type histone deacetylase (HDAC) inhibitor with antineoplastic activity. Belinostat targets HDAC enzymes, thereby inhibiting tumor cell proliferation, inducing apoptosis, promoting cellular differentiation, and inhibiting angiogenesis. This agent may sensitize drug-resistant tumor cells to other antineoplastic agents, possibly through a mechanism involving the down-regulation of thymidylate synthase.	Chromatin/Epigenetic; DNA Damage/DNA Repair; NF-Κb	HDAC
12	T1912	Dinaciclib	779353-01-4	c1(cc(nc2n1ncc2CC)N1[C@@H](C CCC1)CCO)NCc1c[n+](ccc1)[O-]	C21H28N6O2	396.49	Dinaciclib is a new-type and effective CDKinhibitor for CDK2/5/1/9 (IC50: 1 nM/1 nM/3 nM/4 nM) with potential antineoplastic activity.	Cell Cycle/Checkpoint	CDK1; CDK2; CDK5; CDK9
13	T1773	Afatinib Dimaleate	850140-73-7	c12cc(c(cc1c(ncn2)Nc1cc(c(cc1)F)C l)NC(=O)/C=C/CN(C)C)O[C@H]1 CCOC1.C(=C\C(=O)O)\C(=O)O.C(=C\C(=O)O)\C(=O)O	C32H33ClFN5 O11	718.08	Afatinib is an orally bioavailable anilino-quinazoline derivative and inhibitor of the receptor tyrosine kinase (RTK) epidermal growth factor receptor (ErbB; EGFR) family, with antineoplastic activity.	Angiogenesis; JAK/STAT signaling; Tyrosine Kinase/Adaptors	EGFR (L858R); EGFR (L858R/T790M); EGFR (wt); HER2

Bcl-2 was placed at the center of the network for a better scan of molecules that interact with ABT-199 targets (Figure 7). None of the drug targets were found to directly interact with Bcl-2, again implying that the mechanism of synergism is more complex. Some proteins present in the network that coordinate directly with Bcl-2 mediate the interactions between some drugs and Bcl-2, such as TP53, BIK, and BID. These proteins may be significant for the synergism of ABT-199 in some pathways.

Finally, the top 20 GO-MF and KEGG pathways were used to describe the processes in which the 30 drugs may be involved (Figure 8). Notably, among them, pathways such as “phosphatidylinositol 3-kinase (PI3K) binding”, “JUN kinase activity”, “p53 signaling pathway” and “EGFR tyrosine kinase inhibitor resistance” have been extensively studied in malignancy, which also implies that their changes may be instructive for a collaborative regimen with ABT-199.

Figure 8.

Functional analysis based on 30 drug targets. The top 20 GO-MF and KEGG pathways involve targets of 30 drugs synergistic with ABT-199.

Discussion

t-FL is a devastating malignancy because of its association with treatment-refractory disease and dismal outcomes.⁵ This study, for the first time, determined the feasibility of HTS as a precision medicine tool to identify effective targeted drug combinations for the preclinical treatment of patients with t-FL. We randomly selected a compound panel containing 320 U.S. FDA-approved drugs and found that they exhibited different degrees of cytotoxicity on four t-FL cell lines, of which Karpas422 was the most sensitive. Monotherapy is often unable to control the progression of transformed diseases. Some studies have reported that ABT-199 alone rarely exhibits notable efficacy in other B-cell malignancies except chronic lymphocytic leukemia (CLL),^21,22 and significant drug resistance has also emerged.²³ This study aimed to identify an effective combination regimen with ABT-199.

Unexpectedly, the most effective cell line during compound library screening, Karpas422, proved to be the least synergistic cells following ABT-199 was administered. We used a computational approach to screen 30 drugs in four cell lines and revealed the specific molecular targets of those 30 drugs, most of which involve HDAC inhibitors, the MAPK/JNK signaling pathway, the PI3K/AKT/mTOR signaling pathway, the cell cycle, and death regulation, which are consistent with the results of combination therapies reported by most of the current studies.^24–26 However, the underlying cellular response patterns and target mechanisms of the screened drugs often remain unknown in HTS experiments. Thus, we analyzed whether the targets of these 30 agents have some effect on the ABT-199 target Bcl-2 but found that they are not directly related, indicating that the potential mechanism of the combined regimen is far more than the direct effect on the Bcl-2 protein, and is largely involved in the intersection of multiple pathways or histone modification of intermediate proteins.

Resistance to ABT-199 is a potential concern, as with other targeted therapeutics. Although the initial response might be promising, resistance is often inevitable due to various compensatory mechanisms mediated by complex cancer signaling networks.^27,28 We further sought the cross-linking network between the targets of these 30 drugs and Bcl-2 and found that TP53 was located in the middle of this network. Evasion of apoptosis is a hallmark of cancer, and Bcl-2 and p53 represent two important nodes in apoptosis signaling pathways,²⁹ implying that the combined effect of these drugs lies mainly in modulating their relationship.

Mutations in TP53 are frequent in cancer and hematologic malignancies, where they correlate with unfavorable prognosis and chemotherapy resistance.^30,31 TP53^mut is generally not detected at diagnosis in t-FL, but mutations may be detected at follow-up and are usually associated with transformation.^32,33 Through the Cellosaurus database, we found that in addition to the SC-1-cell line, the other three cell lines have been confirmed to have TP53 mutations; we further verified that transformation is closely related to TP53 mutations. Preclinical studies have suggested that TP53^mut confers intrinsic resistance to ABT-199 through the perturbation of mitochondrial homeostasis and cellular metabolism, including increased oxidative phosphorylation.³⁴ Among the 30 drugs screened in this study, the p53 transcriptional activity agonist Tenovin-6 exerts an antitumor effect by inhibiting the protein deacetylation activity of SirT1 and SirT2.³⁵ In addition, the activation of p53 also leads to proteasomal degradation of Mcl-1 through activation of GSK3,²⁹ perhaps inhibiting any Mcl-1 upregulation that may occur as a consequence of exposure to ABT-199 and may further confer the synergy seen in these experiments.

Ultimately, our drug screening results also confirmed the synergistic effect of inhibitors of the PI3K/AKT/mTOR signaling pathway with ABT-199. Considering that most targeted compounds focus on upstream nodes of cancer signaling pathways, we mainly discussed the PI3K kinase here. Studies have confirmed that the delta subunit of class IA PI3K is mainly expressed in hematopoietic cells and is involved in signal transduction, development, and survival of B cells.³⁶ Excessive activation of PI3Kδ promotes the development of B-cell malignancies.³⁷ Downstream of PI3Kδ lie several effector molecules, including AKT. This is especially relevant for the study of p53, as AKT phosphorylates MDM2, enhancing its ability to ubiquitinate p53 and target it for proteasomal degradation.³⁸ Thus, in addition to blocking the upregulation of the antiapoptotic protein Mcl-1, inhibition of PI3Kδ may partly restore p53 activity by stabilizing p53.³⁹ This is also consistent with our findings.

Conclusion

In conclusion, the goal of this study was to evaluate the cytotoxicity of a combined regimen containing ABT-199 on t-FL utilizing high-throughput drug screening and to further reveal that TP53 plays a role in the combination regimen based on ABT-199. However, there are some limitations to our experiment. First, the HTS analysis is limited by the utilization of a single concentration of the compound library plate. Second, the study was performed on tumor cell lines; further efficacy evaluation and toxicity testing on patient samples are required in the future. Our data complement ABT-199 monotherapy and highlight the need for ABT-199-based combination therapy to improve outcomes in patients with TP53^mut t-FL. This approach may uncover new drug combinations that have not been evaluated before, which could motivate the design of future clinical trials.

Supplemental Material

Supplemental Material - High-Throughput Drug Screen for Potential Combinations With Venetoclax Guides the Treatment of Transformed Follicular Lymphoma

Supplemental Material for High-Throughput Drug Screen for Potential Combinations With Venetoclax Guides the Treatment of Transformed Follicular Lymphoma by Zhifeng Li, Guangchao Pan, Mengya Zhong, Li Zhang, Xingxing Yu, Jie Zha, and Bing Xu in International Journal of Toxicology.

Footnotes

Author Contributions

Zhifeng Li contributed to conception and design, contributed to acquisition, and critically revised manuscript; Guangchao Pan contributed to conception and design, contributed to analysis, and drafted manuscript; Mengya Zhong contributed to acquisition, analysis, and interpretation and critically revised manuscript; Li Zhang contributed to acquisition and critically revised manuscript; Xingxing Yu contributed to interpretation and drafted manuscript; Jie Zha contributed to conception and design, contributed to acquisition, and critically revised manuscript; Bing Xu contributed to conception and design, contributed to acquisition, and critically revised manuscript. All authors gave final approval and agree to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (U22A20290, 82170180, 82100204), the Natural Science Foundation of Fujian Province (2020J011246, 2021J011359), the Backbone Talent Training Project of Fujian Provincial Health(2020GGB054), and the Xiamen Municipal Bureau of Science and Technology (3502Z20209003, 3502Z20209008).

Data Availability Statement

The data used to support the findings of this study are available from the corresponding author upon request.

ORCID iD

Jie Zha

Supplemental Material

Supplemental material for this article is available online.

Appendix

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