c-Myc plays a critical role in the antileukemic activity of the Mcl-1-selective inhibitor AZD5991 in acute myeloid leukemia.
Apoptosis : an international journal on programmed cell death
Acute myeloid leukemia (AML) is an aggressive disease with a low 5-year overall survival rate of 29.5%. Thus, more effective therapies are in need to prolong survival of AML patients. Mcl-1 is overexpressed in AML and is associated with poor prognosis, representing a promising therapeutic target. The oncoprotein c-Myc is also overexpressed in AML and is a significant prognostic factor. In addition, Mcl-1 is required for c-Myc induced AML, indicating that c-Myc-driven AML harbors a Mcl-1 dependency and co-targeting of Mcl-1 and c-Myc represents a promising strategy to eradicate AML. In this study, we investigated the role of c-Myc in the antileukemic activity of Mcl-1 selective inhibitor AZD5991 and the antileukemic activity of co-targeting of Mcl-1 and c-Myc in preclinical models of AML. We found that c-Myc protein levels negatively correlated with AZD5991 ECs in AML cell lines and primary patient samples. AZD5991 combined with inhibition of c-Myc synergistically induced apoptosis in AML cell lines and primary patient samples, and cooperatively targeted leukemia progenitor cells. AML cells with acquired resistance to AZD5991 were resensitized to AZD5991 when c-Myc was inhibited. The combination also showed promising and synergistic antileukemic activity in vitro against AML cell lines with acquired resistance to the main chemotherapeutic drug AraC and primary AML cells derived from a patient at relapse post chemotherapy. The oncoprotein c-Myc represents a potential biomarker of AZD5991 sensitivity and inhibition of c-Myc synergistically enhances the antileukemic activity of AZD5991 against AML.
Pharmacologic Targeting of Mcl-1 Induces Mitochondrial Dysfunction and Apoptosis in B-Cell Lymphoma Cells in a and Dependent Manner.
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Bcl-2 has been effectively targeted in lymphoid malignancies. However, resistance is inevitable, and novel approaches to target mitochondrial apoptosis are necessary. AZD5991, a selective BH3-mimetic in clinical trials, inhibits Mcl-1 with high potency. EXPERIMENTAL DESIGN:We explored the preclinical activity of AZD5991 in diffuse large B-cell lymphoma (DLBCL) and ibrutinib-resistant mantle cell lymphoma (MCL) cell lines, MCL patient samples, and mice bearing DLBCL and MCL xenografts using flow cytometry, immunoblotting, and Seahorse respirometry assay. Cas9 gene editing and functional drug screen assays helped identify mechanisms of resistance to Mcl-1 inhibition. RESULTS:Mcl-1 was expressed in DLBCL and MCL cell lines and primary tumors. Treatment with AZD5991 restricted growth of DLBCL cells independent of cell of origin and overcame ibrutinib resistance in MCL cells. Mcl-1 inhibition led to mitochondrial dysfunction as manifested by mitochondrial membrane depolarization, decreased mitochondrial mass, and induction of mitophagy. This was accompanied by impairment of oxidative phosphorylation. and were essential for sensitivity to Mcl-1, and oxidative phosphorylation was implicated in resistance to Mcl-1 inhibition. Induction of prosurvival proteins (e.g., Bcl-xL) in stromal conditions that mimic the tumor microenvironment rendered protection of primary MCL cells from Mcl-1 inhibition, while BH3-mimetics targeting Bcl-2/xL sensitized lymphoid cells to AZD5991. Treatment with AZD5991 reduced tumor growth in murine lymphoma models and prolonged survival of MCL PDX mice. CONCLUSIONS:Selective targeting Mcl-1 is a promising therapeutic approach in lymphoid malignancies. apoptotic network and metabolic reprogramming underlie susceptibility to Mcl-1 inhibition.
Combined inhibition of BCL-2 and MCL-1 overcomes BAX deficiency-mediated resistance of TP53-mutant acute myeloid leukemia to individual BH3 mimetics.
Blood cancer journal
TP53-mutant acute myeloid leukemia (AML) respond poorly to currently available treatments, including venetoclax-based drug combinations and pose a major therapeutic challenge. Analyses of RNA sequencing and reverse phase protein array datasets revealed significantly lower BAX RNA and protein levels in TP53-mutant compared to TP53-wild-type (WT) AML, a finding confirmed in isogenic CRISPR-generated TP53-knockout and -mutant AML. The response to either BCL-2 (venetoclax) or MCL-1 (AMG176) inhibition was BAX-dependent and much reduced in TP53-mutant compared to TP53-WT cells, while the combination of two BH3 mimetics effectively activated BAX, circumventing survival mechanisms in cells treated with either BH3 mimetic, and synergistically induced cell death in TP53-mutant AML and stem/progenitor cells. The BH3 mimetic-driven stress response and cell death patterns after dual inhibition were largely independent of TP53 status and affected by apoptosis induction. Co-targeting, but not individual targeting of BCL-2 and MCL-1 in mice xenografted with TP53-WT and TP53-R248W Molm13 cells suppressed both TP53-WT and TP53-mutant cell growth and significantly prolonged survival. Our results demonstrate that co-targeting BCL-2 and MCL-1 overcomes BAX deficiency-mediated resistance to individual BH3 mimetics in TP53-mutant cells, thus shifting cell fate from survival to death in TP53-deficient and -mutant AML. This concept warrants clinical evaluation.
Mechanisms of MCL-1 Protein Stability Induced by MCL-1 Antagonists in B-Cell Malignancies.
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Several MCL-1 inhibitors (MCL-1i), including AMG-176 and AZD5991, have shown promise in preclinical studies and are being tested for the treatment of hematologic malignancies. A unique feature of these agents is induction and stability of Mcl-1 protein; however, the precise mechanism is unknown. We aim to study the mechanism of MCL-1i-induced Mcl-1 protein stability. EXPERIMENTAL DESIGN:Using several B-cell leukemia and lymphoma cell lines and primary chronic lymphocytic leukemia (CLL) lymphocytes, we evaluated molecular events associated with Mcl-1 protein stability including protein half-life, reverse-phase protein array, protein-protein interaction, phosphorylation, ubiquitination, and de-ubiquitination, followed by molecular simulation and modeling. RESULTS:Using both in vivo and in vitro analysis, we demonstrate that MCL-1i-induced Mcl-1 protein stability is predominantly associated with defective Mcl-1 ubiquitination and concurrent apoptosis induction in both cell lines and primary CLL subjects. These MCL1i also induced ERK-mediated Mcl-1Thr163 phosphorylation, which partially contributed to Mcl-1 stability. Disruption of Mcl-1:Noxa interaction followed by Noxa degradation, enhanced Mcl-1 de-ubiquitination by USP9x, and Mule destabilization are the major effects of these inhibitors. However, unlike other BH3 proteins, Mule:Mcl-1 interaction was unaffected by MCL-1i. WP1130, a global deubiquitinase (DUB) inhibitor, abrogated Mcl-1 induction reaffirming a critical role of DUBs in the observed Mcl-1 protein stability. Further, in vitro ubiquitination studies of Mcl-1 showed distinct difference among these inhibitors. CONCLUSIONS:We conclude that MCL-1i blocked Mcl-1 ubiquitination via enhanced de-ubiquitination and dissociation of Mcl-1 from Noxa, Bak and Bax, and Mule de-stabilization. These are critical events associated with increased Mcl-1 protein stability with AMG-176 and AZD5991.
AMG 176, a Selective MCL1 Inhibitor, Is Effective in Hematologic Cancer Models Alone and in Combination with Established Therapies.
Caenepeel Sean,Brown Sean P,Belmontes Brian,Moody Gordon,Keegan Kathleen S,Chui Danny,Whittington Douglas A,Huang Xin,Poppe Leszek,Cheng Alan C,Cardozo Mario,Houze Jonathan,Li Yunxiao,Lucas Brian,Paras Nick A,Wang Xianghong,Taygerly Joshua P,Vimolratana Marc,Zancanella Manuel,Zhu Liusheng,Cajulis Elaina,Osgood Tao,Sun Jan,Damon Leah,Egan Regina K,Greninger Patricia,McClanaghan Joseph D,Gong Jianan,Moujalled Donia,Pomilio Giovanna,Beltran Pedro,Benes Cyril H,Roberts Andrew W,Huang David C,Wei Andrew,Canon Jude,Coxon Angela,Hughes Paul E
The prosurvival BCL2 family member MCL1 is frequently dysregulated in cancer. To overcome the significant challenges associated with inhibition of MCL1 protein-protein interactions, we rigorously applied small-molecule conformational restriction, which culminated in the discovery of AMG 176, the first selective MCL1 inhibitor to be studied in humans. We demonstrate that MCL1 inhibition induces a rapid and committed step toward apoptosis in subsets of hematologic cancer cell lines, tumor xenograft models, and primary patient samples. With the use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by reductions in B cells, monocytes, and neutrophils. Furthermore, the combination of AMG 176 and venetoclax is synergistic in acute myeloid leukemia (AML) tumor models and in primary patient samples at tolerated doses. These results highlight the therapeutic promise of AMG 176 and the potential for combinations with other BH3 mimetics. SIGNIFICANCE: AMG 176 is a potent, selective, and orally bioavailable MCL1 inhibitor that induces a rapid commitment to apoptosis in models of hematologic malignancies. The synergistic combination of AMG 176 and venetoclax demonstrates robust activity in models of AML at tolerated doses, highlighting the promise of BH3-mimetic combinations in hematologic cancers...
AMG-176, an Mcl-1 Antagonist, Shows Preclinical Efficacy in Chronic Lymphocytic Leukemia.
Yi Xue,Sarkar Aloke,Kismali Gorkem,Aslan Burcu,Ayres Mary,Iles LaKesla R,Keating Michael J,Wierda William G,Long James P,Bertilaccio Maria Teresa Sabrina,Gandhi Varsha
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Survival of CLL cells due to the presence of Bcl-2 and Mcl-1 has been established. Direct inhibition of Bcl-2 by venetoclax and indirect targeting of Mcl-1 with transcription inhibitors have been successful approaches for CLL. AMG-176 is a selective and direct antagonist of Mcl-1, which has shown efficacy in several hematologic malignancies; however, its effect on CLL is elusive. We evaluated biological and molecular effects of AMG-176 in primary CLL cells. EXPERIMENTAL DESIGN:Using samples from patients ( = 74) with CLL, we tested effects of AMG-176 on CLL and normal hematopoietic cell death and compared importance of CLL prognostic factors on this biological activity. We evaluated CLL cell apoptosis in the presence of stromal cells and identified cell death pathway including stabilization of Mcl-1 protein. Finally, we tested a couplet of AMG-176 and venetoclax in CLL lymphocytes. RESULTS:AMG-176 incubations resulted in time- and dose-dependent CLL cell death. At 100 and 300 nmol/L, there was 30% and 45% cell death at 24 hours. These concentrations did not result in significant cell death in normal hematopoietic cells. Presence of stroma did not affect AMG-176-induced CLL cell death. IGHV unmutated status, high β2M and Mcl-1 protein levels resulted in slightly lower cell death. Mcl-1, but not Bcl-2 protein levels, in CLL cells increased with AMG-176. Low concentrations of venetoclax (1-30 nmol/L) were additive or synergistic with AMG-176. CONCLUSIONS:AMG-176 is active in inducing CLL cell death while sparing normal blood cells. Combination with low-dose venetoclax was additive or synergistic.
Discovery of an Oral, Beyond-Rule-of-Five Mcl-1 Protein-Protein Interaction Modulator with the Potential of Treating Hematological Malignancies.
Journal of medicinal chemistry
Avoidance of apoptosis is critical for the development and sustained growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family of proteins which is overexpressed in many cancers. Upregulation of Mcl-1 in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Therefore, pharmacological inhibition of Mcl-1 is regarded as an attractive approach to treating relapsed or refractory malignancies. Herein, we disclose the design, synthesis, optimization, and early preclinical evaluation of a potent and selective small-molecule inhibitor of Mcl-1. Our exploratory design tactics focused on structural modifications which improve the potency and physicochemical properties of the inhibitor while minimizing the risk of functional cardiotoxicity. Despite being in the "non-Lipinski" beyond-Rule-of-Five property space, the developed compound benefits from exquisite oral bioavailability and induces potent pharmacodynamic inhibition of Mcl-1 in a mouse xenograft model.
A novel Mcl-1 inhibitor synergizes with venetoclax to induce apoptosis in cancer cells.
Molecular medicine (Cambridge, Mass.)
BACKGROUND:Evading apoptosis by overexpression of anti-apoptotic Bcl-2 family proteins is a hallmark of cancer cells and the Bcl-2 selective inhibitor venetoclax is widely used in the treatment of hematologic malignancies. Mcl-1, another anti-apoptotic Bcl-2 family member, is recognized as the primary cause of resistance to venetoclax treatment. However, there is currently no Mcl-1 inhibitor approved for clinical use. METHODS:Paired parental and Mcl-1 knockout H1299 cells were used to screen and identify a small molecule named MI-238. Immunoprecipitation (IP) and flow cytometry assay were performed to analyze the activation of pro-apoptotic protein Bak. Annexin V staining and western blot analysis of cleaved caspase 3 were employed to measure the cell apoptosis. Mouse xenograft AML model using luciferase-expressing Molm13 cells was employed to evaluate in vivo therapeutic efficacy. Bone marrow samples from newly diagnosed AML patients were collected to evaluate the therapeutic potency. RESULTS:Here, we show that MI-238, a novel and specific Mcl-1 inhibitor, can disrupt the association of Mcl-1 with BH3-only pro-apoptotic proteins, selectively leading to apoptosis in Mcl-1 proficient cells. Moreover, MI-238 treatment also potently induces apoptosis in acute myeloid leukemia (AML) cells. Notably, the combined treatment of MI-238 with venetoclax exhibited strong synergistic anti-cancer effects in AML cells in vitro, MOLM-13 xenografts mouse model and AML patient samples. CONCLUSIONS:This study identified a novel and selective Mcl-1 inhibitor MI-238 and demonstrated that the development of MI-238 provides a novel strategy to improve the outcome of venetoclax therapy in AML.
Lymphoma cells lacking pro-apoptotic BAX are highly resistant to BH3-mimetics targeting pro-survival MCL-1 but retain sensitivity to conventional DNA-damaging drugs.
Cell death and differentiation
BH3-mimetic drugs are an anti-cancer therapy that can induce apoptosis in malignant cells by directly binding and inhibiting pro-survival proteins of the BCL-2 family. The BH3-mimetic drug venetoclax, which targets BCL-2, has been approved for the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia by regulatory authorities worldwide. However, while most patients initially respond well, resistance and relapse while on this drug is an emerging and critical issue in the clinic. Though some studies have begun uncovering the factors involved in resistance to BCL-2-targeting BH3-mimetic drugs, little focus has been applied to pre-emptively tackle resistance for the next generation of BH3-mimetic drugs targeting MCL-1, which are now in clinical trials for diverse blood cancers. Therefore, using pre-clinical mouse and human models of aggressive lymphoma, we sought to predict factors likely to contribute to the development of resistance in patients receiving MCL-1-targeting BH3-mimetic drugs. First, we performed multiple whole genome CRISPR/Cas9 KO screens and identified that loss of the pro-apoptotic effector protein BAX, but not its close relative BAK, could confer resistance to MCL-1-targeting BH3-mimetic drugs in both short-term and long-term treatment regimens, even in lymphoma cells lacking the tumour suppressor TRP53. Furthermore, we found that mouse Eµ-Myc lymphoma cells selected for loss of BAX, as well as upregulation of the untargeted pro-survival BCL-2 family proteins BCL-XL and A1, when made naturally resistant to MCL-1 inhibitors by culturing them in increasing doses of drug over time, a situation mimicking the clinical application of these drugs. Finally, we identified therapeutic approaches which could overcome these two methods of resistance: the use of chemotherapeutic drugs or combined BH3-mimetic treatment, respectively. Collectively, these results uncover some key factors likely to cause resistance to MCL-1 inhibition in the clinic and suggest rational therapeutic strategies to overcome resistance that should be investigated further.
Synergistic activity and mechanism of cytarabine and MCL-1 inhibitor AZD5991 against acute myeloid leukemia.
The 5-year overall survival rate of acute myeloid leukemia (AML) is less than 30%. Improving clinical outcomes is still a clinical challenge for AML treatment. Simultaneous use of chemotherapeutic drugs and targeting of apoptosis pathways has become a first-line clinical treatment for AML. Myeloid cell leukemia 1 (MCL-1) is a candidate target for AML treatment. In this study, we demonstrated that inhibition of the anti-apoptotic protein MCL-1 by AZD5991 synergistically increased chemotherapeutic agent cytarabine (Ara-C)-induced apoptosis in AML cell lines and primary patient samples. Apoptosis induced by a combination of Ara-C and AZD5991 was partially dependent on caspase activity and Bak/Bax. The downregulation of MCL-1 by Ara-C and the enhancement of Ara-C-induced DNA damage through inhibition of MCL-1 are potential mechanisms underlying the synergistic anti-AML activity between Ara-C and AZD5991. Our data support the application of MCL-1 inhibitor in combination with the conventional chemotherapeutic agent for the clinical treatment of AML.
The Role of BCL-2 and PD-1/PD-L1 Pathway in Pathogenesis of Myelodysplastic Syndromes.
International journal of molecular sciences
Myelodysplastic syndromes (MDSs) belong to a group of clonal bone marrow malignancies. In light of the emergence of new molecules, a significant contribution to the understanding of the pathogenesis of the disease is the study of the B-cell CLL/lymphoma 2 (BCL-2) and the programmed cell death receptor 1 (PD-1) protein and its ligands. BCL-2-family proteins are involved in the regulation of the intrinsic apoptosis pathway. Disruptions in their interactions promote the progression and resistance of MDSs. They have become an important target for specific drugs. Bone marrow cytoarchitecture may prove to be a predictor of response to its use. The challenge is the observed resistance to venetoclax, for which the MCL-1 protein may be largely responsible. Molecules with the potential to break the associated resistance include S63845, S64315, chidamide and arsenic trioxide (ATO). Despite promising in vitro studies, the role of PD-1/PD-L1 pathway inhibitors has not yet been established. Knockdown of the PD-L1 gene in preclinical studies was associated with increased levels of BCL-2 and MCL-1 in lymphocytes T, which could increase their survival and promote tumor apoptosis. A trial (NCT03969446) is currently underway to combine inhibitors from both groups.
The Impact of Panobinostat on Cell Death in Combination with S63845 in Multiple Myeloma Cells.
Indian journal of hematology & blood transfusion : an official journal of Indian Society of Hematology and Blood Transfusion
Multiple myeloma is a B cell neoplasm characterized by bone marrow infiltration with malignant plasma cells. The Overexpression of histone deacetylase prevents apoptosis of myeloma cells by different mechanisms. The combination of Panobinostat with a BH3 mimetic, S63845, has demonstrated significant antitumor activity in multiple myeloma. We examined the impact of Panobinostat combined with MCL-1 inhibitor on multiple myeloma cell lines and as well as on fresh human myeloma cells. Our study shows that MCL-1 remains a major resistant factor to cell death induced by Panobinostat. Therefore, the inhibition of the MCL-1 member is considered a therapeutic strategy to kill the myeloma cells. We examined that the MCL-1 inhibitor (S63845) enhanced the cytotoxic effect of Panobinostat and decreased the viability of human cell lines and primary myeloma patient cells. Mechanistically, Panobinostat/S63845 control cell death via an intrinsic pathway. Given these data, the combination can be a promising therapeutic target for myeloma patients and should be further explored in clinical trials.
AMG176, an MCL-1 inhibitor, is active in pre-clinical models of aggressive B-cell lymphomas.
Leukemia & lymphoma
Upregulation of the anti-apoptotic protein MCL-1 has been implicated in chemotherapy resistance and poor clinical outcomes in B-cell lymphoma (BCL). We report the activity of AMG176, a direct, selective MCL-1 inhibitor, in preclinical models of BCL. A panel of cell lines representing diffuse large B-cell lymphoma (DLBCL), double-hit lymphoma (DHL) and Burkitt's lymphoma (BL) was selected. AMG176 induced apoptotic cell death in a dose- and time-dependent manner in all BCL cell lines. Baseline MCL-1 expression was not predictive of response. AMG176 exhibited impressive synergy with venetoclax and chemotherapeutic agents, less so with proteasomal inhibitors, and antagonism with anti-CD20 monoclonal antibodies. The activity of AMG176 could not be confirmed in murine models of BCL. Combination therapy targeting MCL-1 and BCL-2 may provide an alternative therapeutic approach in BCL, however optimal patient selection will remain the key to obtaining high response rates and tolerability.
Platycodin D induces proliferation inhibition and mitochondrial apoptosis in diffuse large B-cell lymphoma.
Patients with diffuse large B-cell lymphoma (DLBCL) have unsatisfactory outcomes, especially when relapse occurs after initial chemotherapy. Platycodin D (PD), a triterpenoid saponin isolated from the root of Platycodon grandiflorum (Jacq.) A. DC., has demonstrated potent anticancer activities. However, information regarding the effect of PD on malignant lymphoma remains unavailable. In the present study, we showed that PD dose dependently inhibited the viability of a serial of established DLBCL cell lines representing different molecular subtypes, and their sensitivities to PD were comparable. Mitochondrial dysfunction and subsequent intrinsic apoptosis were induced by PD, as indicated by the loss of mitochondrial membrane potential (MMP) and the increase in the percentage of Annexin Ⅴ-positive cells. Mechanistically, PD treatment downregulated the expression levels of antiapoptotic proteins including MCL-1, BCL-2, and BCL-XL, whereas the expression level of proapoptotic protein BAK was upregulated, followed by the cleavage of the DNA repair enzyme PARP. Moreover, PD synergistically enhanced the cytotoxicity of BCL-2 inhibitor venetoclax. In a SUDHL-4-derived xenograft mouse model, the PD administration significantly constrained the tumor growth without obvious side effects. Therefore, our results provide new insights into the role of PD in lymphoma therapy.
A phase 1b study of venetoclax and alvocidib in patients with relapsed/refractory acute myeloid leukemia.
Relapsed/refractory (R/R) Acute Myeloid Leukemia (AML) is a genetically complex and heterogeneous disease with a poor prognosis and limited treatment options. Thus, there is an urgent need to develop therapeutic combinations to overcome drug resistance in AML. This open-label, multicenter, international, phase 1b study evaluated the safety, efficacy, and pharmacokinetics of venetoclax in combination with alvocidib in patients with R/R AML. Patients were treated with escalating doses of venetoclax (400, 600, and 800 mg QD, orally, days 1-28) and alvocidib (45 and 60 mg/m , intravenously, days 1-3) in 28-day cycles. The combination was found to be safe and tolerable, with no maximum tolerated dose reached. Drug-related Grade ≥3 adverse events were reported in 23 (65.7%) for venetoclax and 24 (68.6%) for alvocidib. No drug-related AEs were fatal. Gastrointestinal toxicities, including diarrhea, nausea, and vomiting were notable and frequent; otherwise, the toxicities reported were consistent with the safety profile of both agents. The response rate was modest (complete remission [CR] + incomplete CR [CRi], 11.4%; CR + CRi + partial response rate + morphologic leukemia-free state, 20%). There was no change in alvocidib pharmacokinetics with increasing doses of venetoclax. However, when venetoclax was administered with alvocidib, AUC and C decreased by 18% and 19%, respectively. A recommended phase 2 dose was not established due to lack of meaningful increase in efficacy across all cohorts compared to what was previously observed with each agent alone. Future studies could consider the role of the sequence, dosing, and the use of a more selective MCL1 inhibitor for the R/R AML population.
Synergistic Action of MCL-1 Inhibitor with BCL-2/BCL-XL or MAPK Pathway Inhibitors Enhances Acute Myeloid Leukemia Cell Apoptosis and Differentiation.
International journal of molecular sciences
Acute myeloid leukemia (AML) is a hematological malignancy characterized by excessive proliferation of abnormal myeloid precursors accompanied by a differentiation block and inhibition of apoptosis. Increased expression of an anti-apoptotic MCL-1 protein was shown to be critical for the sustained survival and expansion of AML cells. Therefore, herein, we examined the pro-apoptotic and pro-differentiating effects of S63845, a specific inhibitor of MCL-1, in a single-agent treatment and in combination with BCL-2/BCL-XL inhibitor, ABT-737, in two AML cell lines: HL-60 and ML-1. Additionally, we determined whether inhibition of the MAPK pathway had an impact on the sensitivity of AML cells to S63845. To assess AML cells' apoptosis and differentiation, in vitro studies were performed using PrestoBlue assay, Coulter electrical impedance method, flow cytometry, light microscopy and Western blot techniques. S63845 caused a concentration-dependent decrease in the viability of HL-60 and ML-1 cells and increased the percentage of apoptotic cells. Combined treatment with S63845 and ABT-737 or MAPK pathway inhibitor enhanced apoptosis but also induced differentiation of tested cells, as well as altering the expression of the MCL-1 protein. Taken together, our data provide the rationale for further studies regarding the use of MCL-1 inhibitor in combination with other pro-survival protein inhibitors.
MCL-1 Inhibitor S63845 Distinctively Affects Intramedullary and Extramedullary Hematopoiesis.
Conventional chemotherapy for killing cancer cells using cytotoxic drugs suffers from low selectivity, significant toxicity, and a narrow therapeutic index. Hyper-specific targeted drugs achieve precise destruction of tumors by inhibiting molecular pathways that are critical to tumor growth. Myeloid cell leukemia 1 (MCL-1), an important pro-survival protein in the BCL-2 family, is a promising antitumor target. In this study, we chose to investigate the effects of S63845, a small-molecule inhibitor that targets MCL-1, on the normal hematopoietic system. A mouse model of hematopoietic injury was constructed, and the effects of the inhibitor on the hematopoietic system of mice were evaluated via routine blood tests and flow cytometry. The results showed that S63845 affected the hematopoiesis of various lineages in the early stage of action, causing extramedullary compensatory hematopoiesis in the myeloid and megakaryocytic lineages. The maturation of the erythroid lineage in the intramedullary and extramedullary segments was blocked to varying degrees, and both the intramedullary and extramedullary lymphoid lineages were inhibited. This study provides a complete description of the effects of MCL-1 inhibitor on the intramedullary and extramedullary hematopoietic lineages, which is important for the selection of combinations of antitumor drugs and the prevention of adverse hematopoiesis-related effects.