Targeting Myeloid Leukemia-1 in Cancer Therapy: Advances and Directions.

As a tripartite cell death switch, B-cell lymphoma protein 2 (Bcl-2) family members precisely regulate the endogenous apoptosis pathway in response to various cell signal stresses through protein-protein interactions. Myeloid leukemia-1 (Mcl-1), a key anti-apoptotic Bcl-2 family member, is positioned downstream in the endogenous apoptotic pathway and plays a central role in regulating mitochondrial function. Mcl-1 is highly expressed in a variety of hematological malignancies and solid tumors, contributing to tumorigenesis, poor prognosis, and chemoresistance, making it an attractive target for cancer treatment. This Perspective aims to discuss the mechanism by which Mcl-1 regulates apoptosis and non-apoptotic functions in cancer cells and to outline the discovery and optimization process of potent Mcl-1 modulators. In addition, we summarize the structural characteristics of potent inhibitors that bind to Mcl-1 through multiple co-crystal structures and analyze the cardiotoxicity caused by current Mcl-1 inhibitors, providing prospects for rational targeting of Mcl-1.

AMPK inhibition sensitizes acute leukemia cells to BH3 mimetic-induced cell death.

BH3 mimetics, including the BCL2/BCLXL/BCLw inhibitor navitoclax and MCL1 inhibitors S64315 and tapotoclax, have undergone clinical testing for a variety of neoplasms. Because of toxicities, including thrombocytopenia after BCLXL inhibition as well as hematopoietic, hepatic and possible cardiac toxicities after MCL1 inhibition, there is substantial interest in finding agents that can safely sensitize neoplastic cells to these BH3 mimetics. Building on the observation that BH3 mimetic monotherapy induces AMP kinase (AMPK) activation in multiple acute leukemia cell lines, we report that the AMPK inhibitors (AMPKis) dorsomorphin and BAY-3827 sensitize these cells to navitoclax or MCL1 inhibitors. Cell fractionation and phosphoproteomic analyses suggest that sensitization by dorsomorphin involves dephosphorylation of the proapoptotic BCL2 family member BAD at Ser75 and Ser99, leading BAD to translocate to mitochondria and inhibit BCLXL. Consistent with these results, BAD knockout or mutation to BAD S75E/S99E abolishes the sensitizing effects of dorsomorphin. Conversely, dorsomorphin synergizes with navitoclax or the MCL1 inhibitor S63845 to induce cell death in primary acute leukemia samples ex vivo and increases the antitumor effects of navitoclax or S63845 in several xenograft models in vivo with little or no increase in toxicity in normal tissues. These results suggest that AMPK inhibition can sensitize acute leukemia to multiple BH3 mimetics, potentially allowing administration of lower doses while inducing similar antineoplastic effects.

Dual targeting of Mcl-1 and Bcl-2 to overcome chemoresistance in cervical and colon cancer.

After an initial positive response to chemotherapy, cancer patients often become resistant and experience relapse. Our previous research identified eukaryotic translation initiation factor 4E (eIF4E) as a crucial target to overcome chemoresistance. In this study, we delved further into the role and therapeutic potential of myeloid cell leukemia 1 (Mcl-1), an eIF4E-mediated target, in chemoresistance. We showed that the levels of phosphor and total eIF4E, as well as Mcl-1, were elevated in chemoresistant cervical but not colon cancer cells. Mcl-1 inhibitor S64315 decreased Mcl-1 levels in chemoresistant cancer cells, regardless of Mcl-1 upregulation, decreased viability in chemoresistant cancer cells and acted synergistically with chemotherapy drugs. The combined inhibition of Mcl-1 and B-cell lymphoma 2 (Bcl-2), employing both genetic and pharmacological approaches, led to a markedly more substantial decrease in viability compared with the inhibition of either target individually. The combination of S64315 and Bcl-2 inhibitors reduced tumor growth in chemoresistant cervical and colon cancer models without causing general toxicity in mice. This combination also prolonged overall survival compared with using S64315 or venetoclax alone. Our research highlights the therapeutic potential of inhibiting Mcl-1 and Bcl-2 simultaneously in chemoresistant cancers and provides a rationale for initiating clinical trials to investigate the combination of S64315 and venetoclax for the treatment of advanced colon and cervical cancer.

Enabling, Decagram-Scale Synthesis of Macrocyclic MCL-1 Inhibitor ABBV-467.

ABBV-467 is a highly potent and selective MCL-1 inhibitor that was advanced to a phase I clinical trial for the treatment of multiple myeloma. Due to its large size and structural complexity, ABBV-467 is a challenging synthetic target. Herein, we describe the synthesis of ABBV-467 on a decagram scale, which enabled preclinical characterization. The strategy is convergent and stereoselective, featuring a hindered biaryl cross coupling, enantioselective hydrogenation, and conformationally preorganized macrocyclization by C-O bond formation as key steps.

Combination of MCL-1 and BCL-2 inhibitors is a promising approach for a host-directed therapy for tuberculosis.

Tuberculosis (TB) accounts for 1.6 million deaths annually and over 25% of deaths due to antimicrobial resistance. Mycobacterium tuberculosis (M.tb) drives MCL-1 expression (family member of anti-apoptotic BCL-2 proteins) to limit apoptosis and grow intracellularly in human macrophages. The feasibility of re-purposing specific MCL-1 and BCL-2 inhibitors to limit M.tb growth, using inhibitors that are in clinical trials and FDA-approved for cancer treatment has not be tested previously. We show that specifically inhibiting MCL-1 and BCL-2 induces apoptosis of M.tb-infected macrophages, and markedly reduces M.tb growth in human and murine macrophages, and in a pre-clinical model of human granulomas. MCL-1 and BCL-2 inhibitors limit growth of drug resistant and susceptible M.tb in macrophages and act in additive fashion with the antibiotics isoniazid and rifampicin. This exciting work uncovers targeting the intrinsic apoptosis pathway as a promising approach for TB host-directed therapy. Since safety and activity studies are underway in cancer clinics for MCL-1 and BCL-2 inhibitors, we expect that re-purposing them for TB treatment should translate more readily and rapidly to the clinic. Thus, the work supports further development of this host-directed therapy approach to augment current TB treatment.

Pre-clinical Formulation Development of an in situ Meglumine Salt of AZD5991: A Novel Macrocyclic Mcl-1 Inhibitor.

PURPOSE: AZD5991 is a potent and selective macrocyclic inhibitor of Mcl-1 in clinical development. Developing an intravenous solution formulation for AZD5991 proved to be challenging primarily due to the poor intrinsic solubility of AZD5991. In this article are described studies performed to select a suitable crystalline form and to assess physicochemical properties of AZD5991 to aid in the design of a solution formulation for preclinical studies. METHODS: It is preferable that the preclinical formulation has a line of sight for clinical formulation. For AZD5991, a concentration of at least 20 mg/ml was required for toxicology studies. Toward this goal, extensive pre-formulation characterization of AZD5991 including solid form analysis, pH-solubility profiling and solubility determination in cosolvents and other solubilizing media were carried out. RESULTS & DISCUSSION: Crystalline Form A, which is more stable in aqueous solution and possesses acceptable thermal stability, was selected for preclinical and clinical development of AZD5991. Extensive solubility evaluation revealed an interesting pH-solubility profile that significantly enhances solubilization at pH > 8.5 to allow solution concentrations of at least 30 mg/ml by in situ meglumine salt formation. CONCLUSION: Developing pre-clinical formulations to support in vivo studies requires a good understanding of the physicochemical properties of the drug candidates. Candidates with challenging pharmaceutic properties like the novel macrocycle molecule AZD5991, demand extensive characterization in its polymorph landscape, solubility profile and suitability evaluation of the excipients. Meglumine, a pH-adjusting and solubilizing agent, was found to be the best choice for formulating AZD5991 into an intravenous product to support preclinical studies.

The Role of BCL-2 and PD-1/PD-L1 Pathway in Pathogenesis of Myelodysplastic Syndromes.

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.

Combined inhibition of aurora kinases and Bcl-xL induces apoptosis through select BH3-only proteins.

Aurora kinases (AURKs) are mitotic kinases important for regulating cell cycle progression. Small-molecule inhibitors of AURK have shown promising antitumor effects in multiple cancers; however, the utility of these inhibitors as inducers of cancer cell death has thus far been limited. Here, we examined the role of the Bcl-2 family proteins in AURK inhibition-induced apoptosis in colon cancer cells. We found that alisertib and danusertib, two small-molecule inhibitors of AURK, are inefficient inducers of apoptosis in HCT116 and DLD-1 colon cancer cells, the survival of which requires at least one of the two antiapoptotic Bcl-2 family proteins, Bcl-xL and Mcl-1. We further identified Bcl-xL as a major suppressor of alisertib- or danusertib-induced apoptosis in HCT116 cells. We demonstrate that combination of a Bcl-2 homology (BH)3-mimetic inhibitor (ABT-737), a selective inhibitor of Bcl-xL, Bcl-2, and Bcl-w, with alisertib or danusertib potently induces apoptosis through the Bcl-2 family effector protein Bax. In addition, we identified Bid, Puma, and Noxa, three BH3-only proteins of the Bcl-2 family, as mediators of alisertib-ABT-737-induced apoptosis. We show while Noxa promotes apoptosis by constitutively sequestering Mcl-1, Puma becomes associated with Mcl-1 upon alisertib treatment. On the other hand, we found that alisertib treatment causes activation of caspase-2, which promotes apoptosis by cleaving Bid into truncated Bid, a suppressor of both Bcl-xL and Mcl-1. Together, these results define the Bcl-2 protein network critically involved in AURK inhibitor-induced apoptosis and suggest that BH3-mimetics targeting Bcl-xL may help overcome resistance to AURK inhibitors in cancer cells.

1-Sulfonylated 1,2,3,4-tetrahydroquinoline-6-carboxylic acids as simple, readily-accessible MCL-1 inhibitors.

MCL-1 is a member of the BCL-2 family of proteins that regulates the mitochondrial pathway of apoptosis. Overexpression of MCL-1 is associated with the development and progression of a range of human cancers, and is also responsible for the onset of resistance to conventional chemotherapies. Although several MCL-1 inhibitors have now advanced to clinical trials, recent suspensions and terminations reveal the urgency with which new inhibitor chemotypes must be discovered. Building on our previous studies of a chiral, isomeric lead, we report the discovery of a new chemotype to inhibit MCL-1: 1-sulfonylated 1,2,3,4-tetrahydroquinoline-6-carboxylic acid. The nature of the sulfonyl moiety contributed significantly to the resulting inhibitory ability. For example, transforming a phenylsulfonyl group into a 4-chloro-3,5-dimethylphenoxy)phenyl)sulfonyl moiety elicited more than a 73-fold enhancement in inhibiton of MCL-1, possibly through targeting the p2 pocket in the BH3-binding groove, and so it is anticipated that further structure-activity studies here will lead to continued improvements in binding. It should be underscored that this class of MCL-1 inhibitors is readily accessible in four simple steps, is achiral and offers many avenues for optimization, all factors that are welcomed in the search for safe and effective inhibitors of this driver of cancer cell survival.

Inhibition of Mcl-1 Synergistically Enhances the Antileukemic Activity of Gilteritinib and MRX-2843 in Preclinical Models of FLT3-Mutated Acute Myeloid Leukemia.

FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (FLT3-ITD) mutations occur in about 25% of all acute myeloid leukemia (AML) patients and confer a poor prognosis. FLT3 inhibitors have been developed to treat patients with FLT3-mutated AML and have shown promise, though the acquisition of resistance occurs, highlighting the need for combination therapies to prolong the response to FLT3 inhibitors. In this study, we investigated the selective Mcl-1 inhibitor AZD5991 in combination with the FLT3 inhibitors gilteritinib and MRX-2843. The combinations synergistically induce apoptosis in AML cell lines and primary patient samples. The FLT3 inhibitors downregulate c-Myc transcripts through the suppression of the MEK/ERK and JAK2/STAT5 pathways, resulting in the decrease in c-Myc protein. This suppression of c-Myc plays an important role in the antileukemic activity of AZD5991. Interestingly, the suppression of c-Myc enhances AZD5991-inudced cytochrome c release and the subsequent induction of apoptosis. AZD5991 enhances the antileukemic activity of the FLT3 inhibitors gilteritinib and MRX-2843 against FLT3-mutated AML in vitro, warranting further development.

miR-340-5p Mediates Cardiomyocyte Oxidative Stress in Diabetes-Induced Cardiac Dysfunction by Targeting Mcl-1.

Diabetic cardiomyopathy (DCM) is initially characterized by early diastolic dysfunction, left ventricular remodeling, hypertrophy, and myocardial fibrosis, and it is eventually characterized by clinical heart failure. MicroRNAs (miRNAs), endogenous small noncoding RNAs, play significant roles in diabetes mellitus (DM). However, it is still largely unknown about the mechanism that links miRNAs and the development of DCM. Here, we aimed to elucidate the mechanism underlying the potential role of microRNA-340-5p in DCM in db/db mouse, which is a commonly used model of type 2 DM and diabetic complications that lead to heart failure. We first demonstrated that miR-340-5p expression was dramatically increased in heart tissues of mice and cardiomyocytes under diabetic conditions. Overexpression of miR-340-5p exacerbated DCM, which was reflected by extensive myocardial fibrosis and more serious dysfunction in db/db mice as represented by increased apoptotic cardiomyocytes, elevated ROS production, and impaired mitochondrial function. Inhibition of miR-340-5p by a tough decoy (TUD) vector was beneficial for preventing ROS production and apoptosis, thus rescuing diabetic cardiomyopathy. We identified myeloid cell leukemia 1 (Mcl-1) as a major target gene for miR-340-5p and showed that the inhibition of Mcl-1 was responsible for increased functional loss of mitochondria, oxidative stress, and cardiomyocyte apoptosis, thereby caused cardiac dysfunction in diabetic mice. In conclusion, our results showed that miR-340-5p plays a crucial role in the development of DCM and can be targeted for therapeutic intervention.

Inhibition of MCL1 induces apoptosis in anaplastic large cell lymphoma and in primary effusion lymphoma.

Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignancies and contributes to tumorigenesis by inhibiting the apoptotic machinery of the cells. Antagonizing BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening of cell lines applying the LL-100 panel. Anaplastic large cell lymphoma (ALCL) and primary effusion lymphoma (PEL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in cell lines from both malignancies, suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/mTOR inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, offering an alternative to avoid thrombocytopenia which is associated with the use of BCLXL inhibitors.

Cotargeting of Bcl-2 and Mcl-1 shows promising antileukemic activity against AML cells including those with acquired cytarabine resistance.

Acute myeloid leukemia (AML) remains a clinical challenge. Venetoclax is an effective Bcl-2 selective inhibitor approved by the U.S. Food and Drug Administration (FDA) for treatment of AML in patients who are 75 years and older or who have comorbidities. However, resistance to venetoclax limits its clinical efficacy. Mcl-1 has been identified as one determinant of resistance to venetoclax treatment. In this study, we investigate the Mcl-1 inhibitor S63845 in combination with venetoclax in AML cells. We found that S63845 synergizes with venetoclax in AML cell lines and primary patient samples. Bak/Bax double knockdown and treatment with the pan-caspase inhibitor Z-VAD-FMK revealed that the combination induces intrinsic apoptosis in AML cells. Inhibition of Mcl-1 using another Mcl-1 selective inhibitor, AZD5991, also synergistically enhanced apoptosis induced by venetoclax in a caspase-dependent manner. Importantly, S63845 in combination with venetoclax can effectively combat AML cells with acquired resistance to the standard chemotherapy drug cytarabine. In light of these facts, the combined inhibition of Mcl-1 and Bcl-2 shows promise against AML cells, including relapse/refractory AML.

Synergism of a novel MCL­1 downregulator, acriflavine, with navitoclax (ABT­263) in triple­negative breast cancer, lung adenocarcinoma and glioblastoma multiforme.

Myeloid cell leukemia sequence 1 (MCL­1), an anti­apoptotic B­cell lymphoma 2 (BCL­2) family molecule frequently amplified in various human cancer cells, is known to be critical for cancer cell survival. MCL­1 has been recognized as a target molecule for cancer treatment. While various agents have emerged as potential MCL­1 blockers, the present study presented acriflavine (ACF) as a novel MCL­1 inhibitor in triple­negative breast cancer (TNBC). Further evaluation of its treatment potential on lung adenocarcinoma and glioblastoma multiforme (GBM) was also investigated. The anticancer effect of ACF on TNBC cells was demonstrated when MDA­MB­231 and HS578T cells were treated with ACF. ACF significantly induced typical intrinsic apoptosis in TNBCs in a dose­ and time­dependent manner via MCL­1 downregulation. MCL­1 downregulation by ACF treatment was revealed at each phase of protein expression. Initially, transcriptional regulation via reverse transcription­quantitative PCR was validated. Then, post­translational regulation was explained by utilizing an inhibitor against protein biosynthesis and proteasome. Lastly, immunoprecipitation of ubiquitinated MCL­1 confirmed the post­translational downregulation of MCL­1. In addition, the synergistic treatment efficacy of ACF with the well­known MCL­1 inhibitor ABT­263 against the TNBC cells was explored [combination index (CI)<1]. Conjointly, the anticancer effect of ACF was assessed in GBM (U87, U251 and U343), and lung cancer (A549 and NCI­H69) cell lines as well, using immunoblotting, cytotoxicity assay and FACS. The effect of the combination treatment using ACF and ABT­263 was estimated in GBM (U87, U343 and U251), and non­small cell lung cancer (A549) cells likewise. The present study suggested a novel MCL­1 inhibitory function of ACF and the synergistic antitumor effect with ABT­263.

Drug Repurposing: Deferasirox Inhibits the Anti-Apoptotic Activity of Mcl-1.

INTRODUCTION: With the aim of repositioning commercially available drugs for the inhibition of the anti-apoptotic myeloid cell leukemia protein, Mcl-1, implied in various cancers, five molecules, highlighted from a published theoretical screening, were selected to experimentally validate their affinity toward Mcl-1. RESULTS: A detailed NMR study revealed that only two of the five tested drugs, Torsemide and Deferasirox, interacted with Mcl-1. NMR data analysis allowed the complete characterization of the binding mode of both drugs to Mcl-1, including the estimation of their affinity for Mcl-1. Biological assays evidenced that the biological activity of Torsemide was lower as compared to the Deferasirox, which was able to efficiently and selectively inhibit the anti-apoptotic activity of Mcl-1. Finally, docking and molecular dynamics led to a 3D model for the Deferasirox:Mcl-1 complex and revealed the positioning of the drug in the Mcl-1 P2/P3 pockets as well as almost all synthetic Mcl-1 inhibitors. Interestingly, contrary to known synthetic Mcl-1 inhibitors which interact through Arg263, Deferasirox, establishes a salt bridge with Lys234. CONCLUSION: Deferasirox could be a potential candidate for drug repositioning as Mcl-1 inhibitor.

Co-Delivery of Paclitaxel and shMCL-1 by Folic Acid-Modified Nonviral Vector to Overcome Cancer Chemotherapy Resistance.

Acquired chemoresistance presents a major clinical impediment, which is an urgent problem to be solved. Interestingly, myeloma cell leukemia-1 (MCL-1) and folate receptor expression levels are higher in chemotherapy-resistant patients than in pretreatment patients. In this study, a multifunctional folic acid (FA)-targeting core-shell structure is presented for simultaneous delivery of shMCL-1 and paclitaxel (PTX). The transfection efficiency of shMCL-1 with the FA-targeting delivery system is higher than with a nontargeting delivery system in Skov3 and A2780T cells. The FA-targeting system significantly inhibits cell growth, blocks cell cycles, and promotes apoptosis of cancer cells in vitro. The mechanisms involved in inhibiting growth are related to Bcl-2/Bax and cdc2/Cyclin B1 pathways. An analysis of RNA sequencing suggests that shMCL-1 reverses chemoresistance through regulating genes such as regulator of chromosome condensation 2 (RCC2). The synergetic effect of shMCL-1 and PTX effectively inhibits tumor growth in both PTX-resistant and normal cancer models by inducing tumor apoptosis, inhibiting proliferation, and limiting tumor angiogenesis. The study results indicate that a FA-targeting delivery system combining shMCL-1 with PTX can simultaneously target tumor sites and restore the sensitivity of chemotherapy-resistant cancer to PTX. These findings have important implications for patients with normal or PTX-resistant cancer.

CD157 signaling promotes survival of acute myeloid leukemia cells and modulates sensitivity to cytarabine through regulation of anti-apoptotic Mcl-1.

CD157/BST-1 (a member of the ADP-ribosyl cyclase family) is expressed at variable levels in 97% of patients with acute myeloid leukemia (AML), and is currently under investigation as a target for antibody-based immunotherapy. We used peripheral blood and bone marrow samples from patients with AML to analyse the impact of CD157-directed antibodies in AML survival and in response to cytarabine (AraC) ex vivo. The study was extended to the U937, THP1 and OCI-AML3 AML cell lines of which we engineered CD157-low versions by shRNA knockdown. CD157-targeting antibodies enhanced survival, decreased apoptosis and reduced AraC toxicity in AML blasts and cell lines. CD157 signaling activated the PI3K/AKT/mTOR and MAPK/ERK pathways and increased expression of Mcl-1 and Bcl-XL anti-apoptotic proteins, while decreasing expression of Bax pro-apoptotic protein, thus preventing Caspase-3 activation. The primary CD157-mediated anti-apoptotic mechanism was Bak sequestration by Mcl-1. Indeed, the Mcl-1-specific inhibitor S63845 restored apoptosis by disrupting the interaction of Mcl-1 with Bim and Bak and significantly increased AraC toxicity in CD157-high but not in CD157-low AML cells. This study provides a new role for CD157 in AML cell survival, and indicates a potential role of CD157 as a predictive marker of response to therapies exploiting Mcl-1 pharmacological inhibition.

Combination of tyrosine kinase inhibitors and the MCL1 inhibitor S63845 exerts synergistic antitumorigenic effects on CML cells.

Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors.

Design, synthesis, stereochemical determination, molecular docking study, in silico pre-ADMET prediction and anti-proliferative activities of indole-pyrimidine derivatives as Mcl-1 inhibitors.

In this study, fourteen novel indole-pyrimidine hybrids were designed and synthesized. Their chemical structures were confirmed using different spectroscopic techniques (1H NMR, 13C NMR, IR and mass). Their (E) stereochemical configuration was determined theoretically (MM2 property) and experimentally using 2D NMR technique (NOESY experiment). The prepared compounds were subjected to preliminary biological studies as Mcl-1 inhibitors. Most of the compounds exhibited good abilities for targeting Mcl-1 protein, especially, 7d, 7e, 7i and 7k (Ki = 11.19-15.21 nM). These derivatives were further evaluated against Bcl-XL and Bcl-2 proteins. Some compounds were found to have dual Mcl-1/Bcl-XL such as 7i, or Bcl-XL/Bcl-2 inhibitory activity as 7d. The most potent derivatives as Mcl-1 inhibitors were chosen as representative examples for determination of in-vitro anti-proliferative activity against PC-3, K-562 and MDA-MB-231 cell lines. They possessed excellent to good anti-proliferative activities. All of the synthesized compounds were docked into Mcl-1 active site. Drug-likeness properties and in silico pre-ADMET characters were also predicted.

In vivo inducible reverse genetics in patients' tumors to identify individual therapeutic targets.

High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients' leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future.