A novel MDM2-p53 antagonist APG115 exerts synergistic effect with ibrutinib via targeting MCL-1 in chronic lymphocytic leukemia

doi:10.21203/rs.3.rs-1983828/v1

Download PDF

Article

A novel MDM2-p53 antagonist APG115 exerts synergistic effect with ibrutinib via targeting MCL-1 in chronic lymphocytic leukemia

https://doi.org/10.21203/rs.3.rs-1983828/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

APG115 is a highly selective small-molecule inhibitor of MDM2-p53 interaction with oral activity, which restores p53 activation in patients with solid tumors in clinical trials. The Bruton tyrosine kinase inhibitor (BTKi) ibrutinib exhibits significant efficacy in chronic lymphocytic leukemia (CLL) patients including high-risk patients. However, the chemoresistance of ibrutinib still needs to be addressed urgently. Herein, we first demonstrated that the APG115 exerted apoptogenic and antiproliferative effects, and induced G0/G1 cell cycle arrest in CLL. As an agent used either alone or in combination with ibrutinib together, APG115 provided remarkable antitumor activity and overall survival extension in vivo. Mechanistically, the activation of p53 positively regulates the p53/p21 pathway, prompting MCL-1 degradation via inducing its ubiquitination. On basis of the upregulation of MCL-1 in CLL cells with ibrutinib resistance, these evidences explain how APG115 reduces the resistance of ibrutinib in CLL. This study offers promising prospects to constitute effective regimens of APG115 combined with ibrutinib for the CLL treatment.

Chronic lymphocytic leukemia

APG115

Ibrutinib

p53

MCL-1

Chronic lymphocytic leukemia (CLL) is a clonal malignant disease with slow accumulation of monoclonal mature B lymphocytes, commonly occurring in the elderly ^1–3. Del17p/ TP53 mutation, unmutated IGHV status, NOTCH1 mutation and complex karyotype have been reported to predict chemoresistance and a faster progression⁴. The emergence of novel targeted therapies^{5, 6}, based on B-cell lymphoma 2 (BCL-2) inhibitors and Bruton’s tyrosine kinase (BTK) inhibitors, has undoubtedly reshaped the traditional treatment patterns for CLL ^{7, 8}. Whereas, the recurrence and chemoresistance in CLL still needs to be addressed urgently⁹.

The BTK molecule is responsible for linking B-cell antigen receptor signaling ¹⁰. The first irreversible BTK inhibitor, ibrutinib, has shown significant efficacy in high-risk CLL patients with del17p/ TP53 mutation ¹¹. Ibrutinib, however, has some limitations, such as resistance development (mainly due to C481 BTK mutations), and toxicity (mainly due to off-target activity) ^{12, 13}. It is suggested that MCL-1 protein levels are essential to CLL survival, and increased in CLL patients with ibrutinib resistance ¹⁴. Through the introduction of more precise targeted therapies and novel combination treatments, CLL patients will benefit from more individualized and effective treatment strategies.

MDM2 is considered to be the most closely associated functional target of p53¹⁵. Combined with the transcriptional activation domain of p53, the N-terminal domain of MDM2 blocks the interaction of p53 with its co-transcriptional activators, which obstructs the function of p53 target genes. On the other hand, the C-terminal RING domain of MDM2 has E3 ubiquitin ligase activity, which can be ubiquitinated to degrade p53 and MDMX¹⁶. p53 has been reported to negatively regulate MCL-1 ¹⁷. We hypothesized that ibrutinib resistance could be reduced by inhibiting MCL-1 expression. APG115 is a highly selective small-molecule inhibitor with oral activity, which destabilizes MDM2-p53 interaction and restores p53 activation ¹⁸. However, as reported that APG115 promoted cell apoptosis in multiple cancers, its role and mechanism in the treatment of CLL remains unclear.

The aim of this study was to investigate the antitumor effect of APG115 as either or combined with the ibrutinib and further to explore its mechanism of resistance to ibrutinib in CLL. Therefore, we hope that this study will supply sufficient theoretical basis for improving treatment regimen of CLL.

Cell line and cultures

The p53 wild type CLL cell line EHEB and the p53 mutant CLL cell line MEC-1 from American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured with IMDM and RPMI-1640 (Hyclone, UT, USA) include 10% fetal bovine serum (Hyclone, UT, USA) respectively. All cells were tested for mycoplasma infection and placed in an incubator with 5% CO₂, at 37 ℃.

Patient specimens

This study has been approved by the Medical Ethics Committee of Shandong Provincial Hospital and meets the requirements of medical ethics. On basis of International Workshop on Chronic Lymphocytic revised criterion, all CLL patients newly diagnosed have signed informed consent prior to sample collection according to the Declaration of Helsinki. Peripheral blood mononuclear cells were extracted from newly diagnosed CLL patients by Ficoll-Hypaque solution (TBD Science, Tianjin, China).

Reagent and antibodies

APG115 was provided by Ascentage Pharma Group Inc. (Ascentage, Suzhou, China). Ibrutinib was originated from MedChemExpress Inc. (MCE, Shanghai, China). Antibodies against p53, MDM2, PUMA, p21, MCL-1 were purchased from Proteintech Group Inc. (PTG, Chicago, USA). And all other antibodies including β-actin, and GAPDH were obtained from Zhongshan Goldenbridge (ZG, Beijing, China).

RNA isolation and quantitative real-time PCR

Total RNA was extracted from the collected cells by RNAiso Plus (TaKaRa, Dalian, China) and the concentration was determined by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, WALTHAM, MA). The RNA was reversely transcribed into cDNA according to the instructions of complementary DNA reverse transcription kit (TaKaRa, Dalian, China). Quantitative real time polymerase chain reaction (qRT-PCR) was performed with the Power SYBR™ Green PCR Master Mix (TaKaRa, Dalian, China) through the 7400 real-time PCR system. Finally, 2^{− ΔΔCT} was used to calculate the fold change to represent the relative mRNA expression level of the gene. Primer sequences were as follows: GAPDH-F: 5’-GCACCGTCAAGGCTGAGAAC-3’; GAPDH-R: 5’-TGGTGAAGACGCCAGTGGA-3’;

Western blotting

Proteins were lysed by RIPA protein lysate (Beyotime, Shanghai, China) supplemented with 1% protease inhibitor (Beyotime, Shanghai, China) and 1% phosphatase inhibitor (Beyotime, Shanghai, China) and placed on ice for 30 min. After centrifugation (Shenergy Biocolor, Shanghai, China), the protein concentration was determined by bicinchoninic acid assay (BCA) method. The same amounts of proteins were added into the polyacrylamide gel well and separated at 200 V for 30 min. Then the protein was transferred to the polyvinylidene fluoride membrane at 10 V for 30 min. The membrane was sealed in 5% skim milk at room temperature for 1 h and soaked in primary antibody at 4 ℃ overnight. The next day, the membranes were washed in Tris buffered saline containing tween (TBST) for three times. After incubated with appropriate secondary antibodies at room temperature for 1 h, another three washes in TBST were needed. Covering with the chemiluminescent reagent (Pierce), the membranes were placed in at Bio-Rad Image Lab ™ (Bio-Rad, Hercules, CA) for visualization. The Image J software was taken to analyze the protein gray density while GAPDH was used as internal references.

Cell proliferation assays

The density of cells in new culture medium to be detected during logarithmic growth period was adjusted to 1×10⁵cells/ml. Inoculate 100ul cell suspension in each well of 96-well plate. After incubation for 24, 48, and 72 h, add 10 μl cell counting kit-8 (Beyotime, Shanghai, China) solution to each well and wait for 2 h to measure the absorbance at 450 nm via a SpectraMax M2 microplate reader (Molecular Devices, CA, USA).

Immunofluorescence assays

PBS was added to cells at logarithmic growth stage to adjust the density to 10⁵cells/ml. The cells were thrown onto the slide and fixed with 4% paraformaldehyde for 15 min. Then 0.1% Triton X 100 was added to the slide for 10 min. After being sealed with 5% goat serum for 1 h, the slides were incubated with primary antibody at 4 °C overnight. The next day, Dylight 488 labeled goat anti-rat IgG antibody (Abbkine, Beijing, China) was dropped at room temperature for 1 h. The slides were washed by PBS and stained with DAPI. Microscopic analysis was performed under a Nikon C2 confocal microscope.

Analysis of cell apoptosis and cell cycle

Cells with cell cycle to be measured were collected and added 70% ethanol at 4 °C overnight. After washing with PBS, stain the cells by PI/RNase staining buffer (BD Biosciences, Bedford, MA, USA) at 37 ℃ for 15 min. Before detecting the cells by Navios flow cytometer (Beckman Coulter Inc. USA), 500 μl 1× binding buffer was added to adjust the density to 10⁶ cells/ml. The cell cycle was analyzed by Modfit software. Cells were collected to be washed with PBS and adjust the density to 10⁶cells/ml by binding buffer. After incubated for 30 min with Annexin V-PE/7AAD antibody, the apoptosis assays were performed by A Navios flow cytometer from Beckman Coulter. The United States).

SiRNA transfection

The RNA interference sequences were designed by Gene Pharma (Shanghai, China): sip53#1, 5′-GGAAATGGAGTCCATTGATCA-3′; sip53#2, 5′-GCACCAAGAAGCTGAGAATGC-3′. 1ug plasmid and 1 μl lipo2000 (Invitrogen, California, USA) were diluted in 50 μl opti-MEM respectively for 5 min. Then gently mix the two tubes of diluted suspension and add the mixture into cell culture roles after 25 min. 48 h later, the cell culture medium was changed for drug intervention and further testing.

Co-Immunoprecipitation (Co-IP) assay

Cell proteins were extracted with NP-40 lysis buffer (Beyotime, Shanghai, China) on ice for 30 min and then centrifuged to discard the precipitation. Fully incubated 1-3 μg primary antibody with supernatant cracking buffer at 4 ℃ overnight. Then 30ul protein A/G PLUS agarose (Santa Cruz Biotechnology, USA) was joined the suspension at 4 °C for 2 h incubation. Wash the protein agarose with PBS for 3 times and add 2× loading buffer of the same volume at 100 °C for 10 min. The proteins were then detected by Western blotting.

Xenograft in vivo

5 ×10⁶ EHEB cells suspended in 100 μL PBS was injected into tail vein of each female severe combined immunodeficiency (NSG) mouse (6 mice in each group, 5 weeks old). All mice in this study were randomly selected. On fifth day, flow cytometry was used to verify the establishment of CLL xenograft model. From seventh day, mice in 4 groups were injected intraperitoneally. Meanwhile, their weights were measured. After 2 weeks, the invasion ranges of CLL in mice were observed by imaging in vivo. All animal experiments were carried out in accordance with the procedures approved by the Institutional Animal Care and Research Advisory Committee of Shandong Provincial Hospital Affiliated to Shandong University (SPHASU).

Statistical analyses

Mean ± standard deviation (SD) was used to represent the results of three independent experiments. Student T test and Mann-Whitney U test were utilized to compare the differences between the two groups. Univariate ANOVA or two-factor ANOVA were performed for multi-group comparison. All data were analyzed using SPSS 23.0 software (Chicago, IL, USA) and Graphpad Prism 9.0 statistical software (San Diego, CA, USA). *p<0.05 was considered statistically significant.

APG115 promotes cell apoptosis by increasing p53 expression in CLL

To study the role of APG115, we added APG115 into CLL cell line and CLL primary cells selected appropriate concentration according to IC₅₀ (Fig.1A and 1B). The IC₅₀ of MEC-1 and EHEB were 50.44μM and 11.37μM respectively (Fig.1C). Appropriate concentration of APG115 was selected to intervene in MEC-1, EHEB and CLL primary cells. Under the same concentration, the intervention effect of APG115 on cells increased over time, and the inhibition effect of APG115 on cells increased with the increase of concentration at the same time (Fig. 1D), which suggested that CLL cells were time-dose-dependent on APG115. In addition, the protein levels of MDM2, p53, PUMA and p21 increased under the treatment of APG115, indicating that APG115 relieved the inhibition of MDM2 on p53 and activated downstream apoptotic pathways (Fig. 1E). The cell viability of CLL cells gradually decreased along with the extension of time or concentration augment (Fig. 1F). Meanwhile, the APG115 induced cell apoptotic elevation and cell cycle arrest in G0/G1 phase (Fig. 1G and 1H).

APG115 enhances ibrutinib sensitivity via promoting MCL-1 degradation under the activity of p53

Subsequently, aimed to explore whether APG115 functions depending on p53, small interference plasmids (si p53) were transfected into cells after verification of p53 knockdown efficiency with sequence 2 selected for subsequent experiments (Fig. 2A). While p53 was silencing, both the apoptotic rate and cell viability in knockdown group were significantly lower than control group (Fig. 2B, 2C and 2D).The addition of APG115 activated the increasement of p53 in the nucleus and reduction in the cytoplasm (Fig. 2E).

The combination of APG115 and ibrutinib exhibits a better effect than single agent

Furthermore, in order to observe the synergistic effect of APG115 on ibrutinib, the cell viability of combination group under both APG115 and ibrutinib was obviously suppressed than groups with single drug intervention (Fig. 3A). Likewise, the APG115 elevated the cell apoptosis rate under ibrutinib treatment. Compared with single drug group, apoptosis rate was increased in combination group (Fig. 3B and 3C). Finally, immunofluorescence images revealed that p53 promoted MCL-1 into cytoplasm under the action of APG115 (Fig. 3E, 3F and 3G).

APG115 enhances ibrutinib sensitivity via promoting MCL-1 degradation under the activity of p53

Ibrutinib-resistant cells were generated by long-term exposure of CLL primary cells to ibrutinib, and the verified resistance index RI was 10.35 (Fig. 4A). Compared with the primary cells and the corresponding drug-resistant strains, the expression of MCL-1 in ibrutinib drug-resistant strains was detected by PCR (Fig. 4B). After adding APG115, it was found that the survival rate of drug-resistant strain was lower than that of sensitive strain (Fig. 4C). It was proved that p53 induced the ubiquitination process when combined with MCL-1 to reduce the inhibitory effect of apoptosis via CO-IP (Fig. 4D, E, F). After APG115 was added, the apoptosis rate of drug-resistant strains was higher than that of sensitive strains (Fig. 4G and 4H). When APG115 was added on the basis of ibrutinib, the difference in apoptosis rate of drug-resistant strains was greater than that of sensitive strains (Fig. 4I and 4J).

APG115 suppressed CLL development and enhanced ibrutinib sensibility in a xenograft mouse model

Each mouse was injected with 5×10⁶ EHEB cells suspension into tail vein. The xenograft mouse model was successfully established by flow cytometry on the 5th day, and intraperitoneal injection was started in 4 groups from the 7th day. Body weight was recorded by injection of vehicle, APG115 5mg/kg, Ibrutinib 3mg/kg, and APG115 5mg/kg + Ibrutinib 3mg/kg. In vivo imaging was performed after 2 weeks of injection. Spleen bone marrow was dissected after the death of mice for immunohistochemistry (Fig. 5A). After injection, each group of mice gradually lost weight. Both the monotherapy group and the combination group were lower than the control group, but there was no significant difference between the monotherapy group and the combination group (Fig. 5B). In vivo imaging showed that the invasion range of CLL cells in the combination group was smaller than that in the single agent group, and the invasion range of CLL cells in the single agent group was lower than that in the control group 2 weeks after injection (Fig. 5C). The survival curve of mice was obtained by recording the time of death (Fig. 5D). The survival rate of agent combination group was higher than that of single agent group, and the survival rate of single agent group was higher than that of control group. Immunohistochemistry of the dissected spleens of mice showed that the malignancy of the cells in the single agent group was lower than that in the control group, while that in the combination group was lower than that in the monotherapy group (Fig. 5E).

The mechanism of APG115 exerts synergistic effect of ibrutinib against CLL.

As shown in Figure 6, APG115 could block the inhibitory effect of MDM2 on p53 and promote p53 expression. After entering the nucleus, p53 promoted transcription of PUMA and NOXA to release BAX and BAK. Meanwhile, ibrutinib motivated cell apoptosis by inhibiting BTK to block transcription of BCL-2 and BCL-XL under NK-κB. Nevertheless, the expression of MCL-1 was increased in the resistant cells treated with long-term ibrutinib. Then p53 bond to MCL-1 and accelerated its ubiquitination and proteasome degradation. The decreased expression of MCL-1 promoted BAK, BAX and other downstream apoptotic molecules, thus promoting cell apoptosis.

Our study first elucidated that the APG115, a potent MDM2-p53 antagonist ¹⁸, exhibited significant apoptogenic and antiproliferative effects, and induced G0/G1 cell cycle arrest in CLL. Alone or in combination with ibrutinib together, APG115 provided remarkable antitumor activity and overall survival extension in vivo. Mechanistically, the suppression of MCL-1 targeted by p53 played a key role in APG115's mitigation of ibrutinib resistance.

As it turns out, APG115 diminished cells multiplication in a time-dose dependent patent among CLL cell lines and CLL primary cells. Compared with p53^MUT mutant cells, p53^WT cells were more sensitive to APG115, which was consistent with subsequent verification that APG115 exert effects in a p53-dependent way. Similar to the research in acute myeloid leukemia (AML) ^{19, 20}, APG-115 not only exhibited anti-proliferative activity, but also induced cell apoptosis ^{21, 22} and G0 /G1 cell cycle arrest.

Although ibrutinib has shown excellent efficacy in CLL patients with untreated and relapsed/refractory, its resistance and side effects still needs to be addressed ^{23, 24}. In order to identify the role that APG115 plays in chemotherapy resistance, CLL cells viability was measured under several chemotherapy regimens. Obviously, APG-115 was synergistic with ibrutinib, reducing chronic lymphocyte invasion and prolonging overall survival in vivo. Similarly, it has been reported that APG115 enhanced the radiotherapy sensitivity in gastric cancer ²⁵. Combined with SOC agents, it has activated the p53/p21 pathway and upregulated DNA damage in AML cell ¹⁹. At present, APG-115 or other MDM2 inhibitors in combination with SOC drugs are also underway in AML and MDS patients in several clinical trials ²⁶.

Furthermore, BTK and the anti-apoptotic proteins of the BCL-2 family, especially BCL-2 and MCL-1, were involved in the proliferation and development in CLL^27,28. As the top molecule for cell multiplication in cancers ^29–34, the upregulation of MCL-1 is a vital factor for the poor therapeutic effect of CLL. Currently, the inhibitors of MCL-1 have also been developed such as AZD-5991 ³⁵, AMG-176 ^{36, 37}, S63845 ³⁸ and S64315 ³⁹. Mechanically, the activation of p53 can regulate the phosphorylation of MCL-1 via negatively regulating Ras/Raf/MEK/ERK pathway ⁴⁰. Domoste et al. showed that p53 could induce MCL-1 polyubiquitination after interfering with RiBi⁴¹. Ubiquitinated MCL-1 is subsequently degraded by the proteasome, resulting in apoptotic cell death ⁴¹. As previously reported, dual inhibitors of mTOR and p-AKT impaired MCL-1 expression, which leaded to BAX activation and cell death. PI3Kδ inhibitor idelalisib suppressed MCL-1 and sensitized ABT199-resitant cells by BAX reaction ⁴². Whether MCL-1 protein can be targeted by direct antagonists (such as APG115) to sensitize CLL cells during ibrutinib treatment remains unclear.

Herein, we found that MCL-1 expression was upregulated in long-term ibrutinib-treated drug-resistant cells. P53 binds to MCL-1 and promotes ubiquitination, which is then transferred to proteasome degradation. The decreased expression of MCL-1 promotes apoptosis by up-regulating downstream apoptotic molecules PUMA and NOXA^43–45. Additionally, the PUMA and NOXA further enhances the functions of the pro-apoptotic proteins BAX and BAK. BAX/BAK oligomerization leads to changes in permeability of mitochondrial outer membrane, resulting in cytochrome C release and apoptosis ⁴⁶. Similarly, APG115 can synergistically promote apoptosis of ibrutinib by promoting MCL-1 degradation ^{45, 47, 48}. The current potential application direction of APG115 is cooperating with cancer immunotherapy. The anti-PD-1 antibody Pembrolizumab (Keytruda) is being tested with APG-115 in patients with advanced solid tumors and metastatic melanoma (NCT03611868) ³⁸. Moreover, APG-115 enhances MDM2 in T cells to stabilize STAT5, improving T cell immunity. The evaluation of the feasibility in appropriate CLL complementary immunotherapy regimens becomes a part of future exploration ⁴⁹.

In conclusion, our data affords new insights into anti-cancer effects of the novel MDM2-p53 inhibitor APG115 in CLL. The activation of p53 under APG115 positively regulates the p53/p21 pathway, prompting MCL-1 ubiquitination for degradation, which revealed the molecular mechanism of the synergistic effects with ibrutinib. This study offers promising prospects of APG115 combined with ibrutinib for new therapy regimens of CLL patients.

Funding

This study was funded by National Natural Science Foundation (No.82000195, No.82070203 and No.81770210); Key Research and Development Program of Shandong Province (No.2018CXGC1213); Translational Research Grant of NCRCH (No.2021WWB02, No.2020ZKMB01); Taishan Scholars Program of Shandong Province; Shandong Provincial Natural Science Foundation (No.ZR2020QH094); Shandong Provincial Engineering Research Center of Lymphoma; Technology Development Project of Jinan City (No.202019182); Academic Promotion Programme of Shandong First Medical University (No.2019QL018, No.2020RC007); Shandong Provincial Hospital Youth Talent Plan.

Author Contributions

X.W., Y.Z. and Y.H. conceived and designed the experiments. Y.H., X.H. and H.W. performed the experiments. Z.T., X.Z. and S.H. assisted with figures drawing. Y.H. and Y.Z. analyzed the data and wrote the paper. All authors read and approved the final manuscript.

Data Availability Statement

The data analyzed during the current study is available from the corresponding author on reasonable request.

Competing Interests

The authors declare no competing interests.

Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Dohner H, et al. iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL. Blood 2018 Jun 21; 131(25): 2745–2760.
Eichhorst B, Robak T, Montserrat E, Ghia P, Niemann CU, Kater AP, et al. Chronic lymphocytic leukaemia: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of oncology: official journal of the European Society for Medical Oncology 2021 Jan; 32(1): 23–33.
Tian Z, Liu M, Fang X, Zhou X, Li P, Li Y, et al. Distinct Age-Related Clinical Features and Risk Assessment in Chinese With Chronic Lymphocytic Leukemia. Frontiers in oncology 2022; 12: 885150.
Yun X, Zhang Y, Wang X. Recent progress of prognostic biomarkers and risk scoring systems in chronic lymphocytic leukemia. Biomarker research 2020; 8: 40.
Zhang Y, Zhou X, Li Y, Xu Y, Lu K, Li P, et al. Inhibition of maternal embryonic leucine zipper kinase with OTSSP167 displays potent anti-leukemic effects in chronic lymphocytic leukemia. Oncogene 2018 Oct; 37(41): 5520–5533.
Mamidi MK, Mahmud H, Maiti GP, Mendez MT, Fernandes SM, Vesely SK, et al. Idelalisib activates AKT via increased recruitment of PI3Kdelta/PI3Kbeta to BCR signalosome while reducing PDK1 in post-therapy CLL cells. Leukemia 2022 Jul; 36(7): 1806–1817.
Weis TM, Gutierrez J, Kabel CC, King AC, Daley RJ, Stump SE. Real-world management of targeted therapies in chronic lymphocytic leukemia. Journal of oncology pharmacy practice: official publication of the International Society of Oncology Pharmacy Practitioners 2022 Mar 29: 10781552221090869.
Noy A, de Vos S, Thieblemont C, Martin P, Flowers CR, Morschhauser F, et al. Targeting Bruton tyrosine kinase with ibrutinib in relapsed/refractory marginal zone lymphoma. Blood 2017 Apr 20; 129(16): 2224–2232.
Bond DA, Huang Y, Fisher JL, Ruppert AS, Owen DH, Bertino EM, et al. Second cancer incidence in CLL patients receiving BTK inhibitors. Leukemia 2020 Dec; 34(12): 3197–3205.
Brullo C, Villa C, Tasso B, Russo E, Spallarossa A. Btk Inhibitors: A Medicinal Chemistry and Drug Delivery Perspective. International journal of molecular sciences 2021 Jul 16; 22(14).
Robak T, Witkowska M, Smolewski P. The Role of Bruton's Kinase Inhibitors in Chronic Lymphocytic Leukemia: Current Status and Future Directions. Cancers 2022 Feb 2; 14(3).
O'Brien S, Furman RR, Coutre S, Flinn IW, Burger JA, Blum K, et al. Single-agent ibrutinib in treatment-naive and relapsed/refractory chronic lymphocytic leukemia: a 5-year experience. Blood 2018 Apr 26; 131(17): 1910–1919.
Zhou H, Hu P, Yan X, Zhang Y, Shi W. Ibrutinib in Chronic Lymphocytic Leukemia: Clinical Applications, Drug Resistance, and Prospects. OncoTargets and therapy 2020; 13: 4877–4892.
Bauvois B, Pramil E, Jondreville L, Quiney C, Nguyen-Khac F, Susin SA. Activation of Interferon Signaling in Chronic Lymphocytic Leukemia Cells Contributes to Apoptosis Resistance via a JAK-Src/STAT3/Mcl-1 Signaling Pathway. Biomedicines 2021 Feb 13; 9(2).
Konopleva M, Martinelli G, Daver N, Papayannidis C, Wei A, Higgins B, et al. MDM2 inhibition: an important step forward in cancer therapy. Leukemia 2020 Nov; 34(11): 2858–2874.
Kowalczyk D, Nakasone MA, Smith BO, Huang DT. Bivalent binding of p14ARF to MDM2 RING and acidic domains inhibits E3 ligase function. Life science alliance 2022 Dec; 5(12).
Liu J, Chen G, Feng L, Zhang W, Pelicano H, Wang F, et al. Loss of p53 and altered miR15-a/16-1short right arrowMCL-1 pathway in CLL: insights from TCL1-Tg:p53(-/-) mouse model and primary human leukemia cells. Leukemia 2014 Jan; 28(1): 118–128.
Aguilar A, Lu J, Liu L, Du D, Bernard D, McEachern D, et al. Discovery of 4-((3'R,4'S,5'R)-6''-Chloro-4'-(3-chloro-2-fluorophenyl)-1'-ethyl-2''-oxodispiro[ cyclohexane-1,2'-pyrrolidine-3',3''-indoline]-5'-carboxamido)bicyclo[2.2.2]octane – 1-carboxylic Acid (AA-115/APG-115): A Potent and Orally Active Murine Double Minute 2 (MDM2) Inhibitor in Clinical Development. Journal of medicinal chemistry 2017 Apr 13; 60(7): 2819–2839.
Fang DD, Tang Q, Kong Y, Rong T, Wang Q, Li N, et al. MDM2 inhibitor APG-115 exerts potent antitumor activity and synergizes with standard-of-care agents in preclinical acute myeloid leukemia models. Cell death discovery 2021 May 3; 7(1): 90.
Werner LR, Huang S, Francis DM, Armstrong EA, Ma F, Li C, et al. Small Molecule Inhibition of MDM2-p53 Interaction Augments Radiation Response in Human Tumors. Molecular cancer therapeutics 2015 Sep; 14(9): 1994–2003.
Chen H, Luo D, Zhang L, Lin X, Luo Q, Yi H, et al. Restoration of p53 using the novel MDM2-p53 antagonist APG115 suppresses dedifferentiated papillary thyroid cancer cells. Oncotarget 2017 Jun 27; 8(26): 43008–43022.
Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004 Feb 6; 303(5659): 844–848.
Hillmen P, Diels J, Healy N, Iraqi W, Aschan J, Wildgust M. Ibrutinib for chronic lymphocytic leukemia: international experience from a named patient program. Haematologica 2018 May; 103(5): e204-e206.
Furman RR, Cheng S, Lu P, Setty M, Perez AR, Guo A, et al. Ibrutinib resistance in chronic lymphocytic leukemia. The New England journal of medicine 2014 Jun 12; 370(24): 2352–2354.
Yi H, Yan X, Luo Q, Yuan L, Li B, Pan W, et al. A novel small molecule inhibitor of MDM2-p53 (APG-115) enhances radiosensitivity of gastric adenocarcinoma. Journal of experimental & clinical cancer research: CR 2018 May 2; 37(1): 97.
Tisato V, Voltan R, Gonelli A, Secchiero P, Zauli G. MDM2/X inhibitors under clinical evaluation: perspectives for the management of hematological malignancies and pediatric cancer. Journal of hematology & oncology 2017 Jul 3; 10(1): 133.
Jayappa KD, Portell CA, Gordon VL, Capaldo BJ, Bekiranov S, Axelrod MJ, et al. Microenvironmental agonists generate de novo phenotypic resistance to combined ibrutinib plus venetoclax in CLL and MCL. Blood advances 2017 Jun 13; 1(14): 933–946.
Yi X, Jain N, Iles LR, Ayres ML, Wierda WG, Gandhi V. Targeting Mcl-1 by AMG-176 During Ibrutinib and Venetoclax Therapy in Chronic Lymphocytic Leukemia. Frontiers in oncology 2022; 12: 833714.
Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J, et al. The landscape of somatic copy-number alteration across human cancers. Nature 2010 Feb 18; 463(7283): 899–905.
Cervantes-Gomez F, Lamothe B, Woyach JA, Wierda WG, Keating MJ, Balakrishnan K, et al. Pharmacological and Protein Profiling Suggests Venetoclax (ABT-199) as Optimal Partner with Ibrutinib in Chronic Lymphocytic Leukemia. Clinical cancer research: an official journal of the American Association for Cancer Research 2015 Aug 15; 21(16): 3705–3715.
Haselager MV, Kielbassa K, Ter Burg J, Bax DJC, Fernandes SM, Borst J, et al. Changes in Bcl-2 members after ibrutinib or venetoclax uncover functional hierarchy in determining resistance to venetoclax in CLL. Blood 2020 Dec 17; 136(25): 2918–2926.
Guieze R, Liu VM, Rosebrock D, Jourdain AA, Hernandez-Sanchez M, Martinez Zurita A, et al. Mitochondrial Reprogramming Underlies Resistance to BCL-2 Inhibition in Lymphoid Malignancies. Cancer cell 2019 Oct 14; 36(4): 369–384 e313.
Kapoor I, Bodo J, Hill BT, Hsi ED, Almasan A. Targeting BCL-2 in B-cell malignancies and overcoming therapeutic resistance. Cell death & disease 2020 Nov 2; 11(11): 941
Jayappa KD, Gordon VL, Morris CG, Wilson B, Shetty BD, Cios KJ, et al. Extrinsic interactions in the microenvironment in vivo activate an antiapoptotic multidrug-resistant phenotype in CLL. Blood advances 2021 Sep 14; 5(17): 3497–3510.
Tron AE, Belmonte MA, Adam A, Aquila BM, Boise LH, Chiarparin E, et al. Discovery of Mcl-1-specific inhibitor AZD5991 and preclinical activity in multiple myeloma and acute myeloid leukemia. Nature communications 2018 Dec 17; 9(1): 5341.
Caenepeel S, Brown SP, Belmontes B, Moody G, Keegan KS, Chui D, et al. AMG 176, a Selective MCL1 Inhibitor, Is Effective in Hematologic Cancer Models Alone and in Combination with Established Therapies. Cancer discovery 2018 Dec; 8(12): 1582–1597.
Yi X, Sarkar A, Kismali G, Aslan B, Ayres M, Iles LR, et al. AMG-176, an Mcl-1 Antagonist, Shows Preclinical Efficacy in Chronic Lymphocytic Leukemia. Clinical cancer research: an official journal of the American Association for Cancer Research 2020 Jul 15; 26(14): 3856–3867.
Fang DD, Tang Q, Kong Y, Wang Q, Gu J, Fang X, et al. MDM2 inhibitor APG-115 synergizes with PD-1 blockade through enhancing antitumor immunity in the tumor microenvironment. Journal for immunotherapy of cancer 2019 Nov 28; 7(1): 327.
Kotschy A, Szlavik Z, Murray J, Davidson J, Maragno AL, Le Toumelin-Braizat G, et al. The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models. Nature 2016 Oct 27; 538(7626): 477–482.
Pan R, Ruvolo V, Mu H, Leverson JD, Nichols G, Reed JC, et al. Synthetic Lethality of Combined Bcl-2 Inhibition and p53 Activation in AML: Mechanisms and Superior Antileukemic Efficacy. Cancer cell 2017 Dec 11; 32(6): 748–760 e746.
Domostegui A, Peddigari S, Mercer CA, Iannizzotto F, Rodriguez ML, Garcia-Cajide M, et al. Impaired ribosome biogenesis checkpoint activation induces p53-dependent MCL-1 degradation and MYC-driven lymphoma death. Blood 2021 Jun 17; 137(24): 3351–3364.
Choudhary GS, Al-Harbi S, Mazumder S, Hill BT, Smith MR, Bodo J, et al. MCL-1 and BCL-xL-dependent resistance to the BCL-2 inhibitor ABT-199 can be overcome by preventing PI3K/AKT/mTOR activation in lymphoid malignancies. Cell death & disease 2015 Jan 15; 6: e1593.
Townsend PA, Kozhevnikova MV, Cexus ONF, Zamyatnin AA, Jr., Soond SM. BH3-mimetics: recent developments in cancer therapy. Journal of experimental & clinical cancer research: CR 2021 Nov 9; 40(1): 355.
Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI, et al. Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes & development 2005 Jun 1; 19(11): 1294–1305.
Senichkin VV, Pervushin NV, Zamaraev AV, Sazonova EV, Zuev AP, Streletskaia AY, et al. Bak and Bcl-xL Participate in Regulating Sensitivity of Solid Tumor Derived Cell Lines to Mcl-1 Inhibitors. Cancers 2021 Dec 30; 14(1).
Juin P, Geneste O, Gautier F, Depil S, Campone M. Decoding and unlocking the BCL-2 dependency of cancer cells. Nature reviews Cancer 2013 Jul; 13(7): 455–465.
Suksamai D, Racha S, Sriratanasak N, Chaotham C, Aphicho K, Lin ACK, et al. 5-O-(N-Boc-l-Alanine)-Renieramycin T Induces Cancer Stem Cell Apoptosis via Targeting Akt Signaling. Marine drugs 2022 Mar 29; 20(4).
Weller S, Toenniessen A, Schaefer B, Beigl T, Muenchow A, Bopple K, et al. The BCL-2 inhibitor ABT-199/venetoclax synergizes with proteasome inhibition via transactivation of the MCL-1 antagonist NOXA. Cell death discovery 2022 Apr 20; 8(1): 215.
Zhou J, Kryczek I, Li S, Li X, Aguilar A, Wei S, et al. The ubiquitin ligase MDM2 sustains STAT5 stability to control T cell-mediated antitumor immunity. Nature immunology 2021 Apr; 22(4): 460–470.

There is NO conflict of interest to disclose.

Download PDF

Version 1

posted

You are reading this latest preprint version

A novel MDM2-p53 antagonist APG115 exerts synergistic effect with ibrutinib via targeting MCL-1 in chronic lymphocytic leukemia

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Declarations

References

Additional Declarations

Status:

Version 1