Mcl-1 Inhibitors Research Articles

Abstract 518: The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation

Abstract Richter transformation (RT), characterized by the transformation of chronic lymphocytic leukemia (CLL) to an aggressive large-cell lymphoma, remains a challenge for the therapy. Genetic aberrations, such as Myc activation, TP53 abnormality, NOTCH1 gene mutation or loss of CDKN2A are associated with uncontrolled RT cell proliferation and escaping of apoptosis. Among those changes, gain of Myc activity, found in around 70% of RT, plays a nexus role in the pathogenesis of RT. As a transcription factor, Myc activation has a profound effect on cell survival, proliferation, adhesion and metabolism. Because of its essential role in lymphoma transformation and aggressiveness, Myc is a vital target for therapy toward RT. Fadraciclib (CYC065) is a second-generation CDK inhibitor with selective potency toward CDK2 and CDK9. We have shown previously that by inhibiting CDK9-mediated transcription, fadraciclib reduced expression of the short-lived anti-apoptotic protein Mcl-1 to initiate apoptosis in primary CLL cells. Like Mcl-1, Myc is a quintessential example of an oncogene with rapid turnover in both its mRNA and protein, therefore a promising target of CDK9 inhibition. Thus, we proposed that fadraciclib would target both the survival and proliferation pathways of RT pathogenesis through collaborative inhibition of Mcl-1, Myc and CDK2. First, our data showed that fadraciclib effectively induced apoptosis in the RT cell line HPRT1, as well as the lymphoma cell lines with Myc amplification, including Raji, Ramos and SU-DHL-2. The IC50 for apoptosis was 0.3 µM at 24h in the HPRT1 cells, which is less than the average IC50 for the primary CLL cells (0.8 µM). The IC50 for the lymphoma lines ranged from 0.8 µM to 1.3 µM. Immunoblots showed reduction of RNA Pol2 phosphorylation by fadraciclib, consistent with the inhibition of CDK9. As a result of loss of RNA Pol2 activity and pausing of transcription, there was a clear reduction of both Mcl-1 and Myc mRNA, measured by real-time RT-PCR, that was associated with the reduction of their protein levels. CDK2 was also inhibited by fadraciclib, shown by the reduction of the phosphorylation of both RB and NPM. Second, Fadraciclib inhibited cell proliferation of the HPRT1 cells at an IC50 of 0.25 µM measured by the CellTiter-Glo assay. The IC50 for growth inhibition in the lymphoma lines ranged from 0.5 to 0.7 µM. CFSE tracing demonstrated halting of cell division upon fadraciclib incubation. Cell cycle analysis by Click-iT EdU showed reduction of S-phase population and arresting of cells in both G1 and G2. We are currently working on dissecting the contributions of inhibiting Myc, Mcl-1 and CDK2 individually and together, in apoptosis induction and proliferation blockade. Taken together, these data suggest that fadraciclib is active in RT cells and Myc-dependent lymphoma cell lines and support expansion of its clinical testing in patients with RT. Citation Format: Rong Chen, Yuling Chen, Ping Xiong, William Plunkett. The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 518.

Abstract 4605: AZD4573 in combination with CHOP increases combination benefit in preclinical peripheral T-cell lymphoma models

Abstract Peripheral T-cell Lymphoma (pTCL) makes up about 10% of non-Hodgkin's lymphoma in the United States. Patients with relapsed/refractory pTCL have limited treatment options and poor prognosis. Current standard of care for pTCL revolves around the chemotherapy regimens CHOP/E. Here we examine the efficacy of CDK9 inhibition for the treatment of pTCL. CDK9 regulates transcription through phosphorylation of RNA polymerase II. AZD4573 is a highly potent and selective CDK9 inhibitor; acute inhibition with AZD4573 downregulates short-lived proteins such as MCL-1, BFL-1, and c-MYC, which are often overexpressed in hematologic tumors. It has been shown that the expression pattern of the anti-apoptotic BCL-2 family members in pTCL cell line models closely resembled the expression patterns in pTCL patient samples (Koch et al. 2019). Expression of BCL-2 family members is highly heterogeneous in pTCL, with MCL-1 being the most universally expressed. Using MCL-1 inhibitor AZD5991, we showed statistically significant benefit in survival when combined with CHOP in MCL-1 dependent preclinical pTCL PDX models (Koch et al. 2019). In addition, we have previously reported the importance of the anti-apoptotic protein BFL-1 in cell survival in NHL, including a subset of pTCL (Boiko et al 2021). AZD4573 treatment showed a range of activity across the panel of 18 human pTCL cell lines, in a 6-hour caspase-3/7 activation assay. 14 of the cell lines were sensitive, with EC50 values less than 100 nM, and 13 reached a max caspase activation of over 40%. pTCL subtypes that responded to AZD4573 treatment included ALCL, NK-TCL, and PTCL-NOS and CTCL. Acute AZD4573 treatment resulted in decreased pSer2-RNAP2 and reduced protein levels of c-MYC and MCL-1. To determine what was driving the apoptotic phenotype, we used ribonucleoprotein (RNP) mediated CRISPR knock out (KO) of MYC and MCL1 genes. While pTCL cell lines were dependent on c-MYC and MCL-1 for viability after KO, only MCL-1 KO impaired AZD4573 treatment, suggesting that efficacy is mediated through MCL-1 in ALCL pTCL cell lines assessed. To determine if combining AZD4573 with CHOP improved efficacy, we tested 5 pTCL cell lines in a 6x6 combination dosing matrix and measured viability after 24-72 hours using RealTime-Glo after 24hr exposure to CHOP with AZD4573 added for the last 6hrs. Adding AZD4573 to CHOP in vitro increased the efficacy in pTCL ALCL cell lines with MCL-1 dependency, but not in CTCL cell line with BCL-xL dependency and resistance to AZD4573. CHOP treatment in vitro resulted in a decrease in c-MYC levels but not MCL-1, suggesting that combination benefit may be driven through c-MYC. This data suggests that treatment with AZD4573 as a monotherapy or in combination with CHOP regimen would be an effective therapeutic strategy in pTCL. Citation Format: Danielle Sinead Potter, India Ott, Jamal Saeh, Richard Olsson, Stephen Fawell, Lisa Drew, Justine Roderick. AZD4573 in combination with CHOP increases combination benefit in preclinical peripheral T-cell lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4605.

Abstract 4568: Rational targeting of BCL-xL and/or mTOR enhance the efficacy of KRASG12D inhibitor in pancreatic cancer

Abstract Background: Pancreatic cancer (PaCa) is a highly aggressive malignancy with a very low five-year survival rate secondary to late diagnosis, limited treatment options and resistance to existing therapies. While the emergence of mutant-selective KRASG12D (G12D) inhibitors brings promise for treating PaCa patients carrying these mutations, their effectiveness as single agents is expected to be limited as evident from the clinical efficacy of KRASG12C inhibitors. Experimental Design: First, multiple G12D-mutated PaCa cell lines were treated with a G12D inhibitor (MRTX1133) and subjected to immunoblot analysis for the expression of KRAS effectors and BCL-2 family proteins. Next, a proteolysis-targeting chimera (PROTAC)-based platelet-sparing BCL-xL degrader (DT2216) and/or an mTOR inhibitor (everolimus) were evaluated in combination with MRTX1133 for viability, clonogenicity and apoptosis. Subsequently, mechanistic investigations were carried out using immunoblotting, senescence-associated β-galactosidase staining and qPCR. Finally, therapeutic efficacy and safety of the combinations were evaluated in cell-derived and patient-derived xenograft models. Results: MRTX1133 treatment creates apoptotic vulnerabilities by inducing aberrations in BCL-2 family proteins, with elevated BIM levels and decreased NOXA levels, in G12D-mutated PaCa cells. Furthermore, DT2216 and everolimus were shown to release BIM and stabilize NOXA, respectively, and their combination with MRTX1133 synergistically killed G12D-mutated PaCa cells in vitro and caused significant tumor growth inhibition or regression compared to single agents in vivo. Additionally, MRTX1133 treatment led to induction of senescence in a fraction of PaCa cells. These senescent cells were found to be dependent on BCL-xL and MCL-1 for their survival and were effectively killed with a combination of DT2216 and everolimus through BCL-xL degradation and NOXA-mediated MCL-1 inhibition, respectively. Conclusions: Our findings suggest that the single agent efficacy of MRTX1133 is limited due to apoptosis inhibition and complimentary senescence induction, and therefore its combination with DT2216 and/or everolimus is synergistic, which can potentially overcome resistance, by enhanced apoptosis and clearance of senescent cells. Citation Format: Sajid Khan, Lin Cao, Vignesh Vudhata, Yang Yang, Peiyi Zhang, Guangrong Zheng, Jose G. Trevino, Daohong Zhou. Rational targeting of BCL-xL and/or mTOR enhance the efficacy of KRASG12D inhibitor in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4568.

Development of recombinant protein-based nanoparticle systems for inducing tumor cell apoptosis: In vitro evaluation of their cytotoxic and apoptotic effects on cancer cells

Myeloid leukemia cell differentiation protein (Mcl-1) is a B-cell lymphoma 2 (Bcl-2) family antiapoptotic protein that regulates cellular apoptosis. Mcl-1 expression is important for cancer cell survival and a prospective target in cancer treatment. Bcl-2 homology 3 (BH3) mimetic peptides, such as the Mcl-1-specific MS1 peptide that targets conserved BH3 domains, are highly specific Mcl-1 inhibitors. However, the application of these peptides is limited due to their metabolic instability, degradation by cellular proteases, and low cell permeability. In this study, it was aimed to form nanostructures to increase the cellular uptake and stability of the Mcl-1 inhibitor MS1 peptide. The Bim BH3 mimetic MS1 peptide was produced recombinantly in fusion with mNeonGreen fluorescent protein and polyhistidine (6xHisTag). Self-assembled protein nanoparticles were fabricated from the recombinant MS1-mNeonGreen protein. Furthermore, the MS1-mNeonGreen protein was encapsulated into polymeric nanoparticles. The cytotoxic effects of these nanoparticles on cancer cells [cervical cancer cell line (HeLa), lung cancer cell line (A549) and breast cancer cell line (MDA-MB-231)] and healthy cell line [Human umbilical vein endothelial cells (HUVEC)] was analyzed by MTT test. The apoptotic effect of nanoparticles on these cells was investigated by determining the antiapoptotic and proapoptotic protein levels. As a result, it was revealed that the nanoparticles have a specific cytotoxic effect against the cells and induce apoptosis.

MCL1 inhibition targets Myeloid Derived Suppressors Cells, promotes antitumor immunity and enhances the efficacy of immune checkpoint blockade

Immune checkpoint inhibitors (ICIs) are now the first-line treatment for patients with advanced melanoma. Despite promising clinical results, many patients fail to respond to these therapies. BH3 mimetics, a novel class of small molecule inhibitors that bind and inhibit anti-apoptotic members of the BCL2 family proteins such as BCL2 or MCL1, have been very successful in treating hematologic malignancies. However, there are limited studies on the immunomodulatory role of the BH3 mimetics. Several factors contribute to ICI resistance including myeloid-derived suppressor cells (MDSCs) that exert immunosuppressive effects through direct and indirect inhibition of antitumor immunity. Thus, targeting MDSCs to enhance antitumor immunity has the potential to enhance the efficacy of ICIs. In this study, we show that the MCL1 inhibitor S64315 reduces melanoma tumor growth in an immune cell-dependent manner in mice. Specifically, S64315 enhances antitumor immunity by reducing MDSC frequency and by promoting the activity of CD8+T cells. Additionally, human MDSCs are 10 times more sensitive to S64315 than cutaneous melanoma lines. Further, we found that a higher expression of MCL1 is associated with poor survival for patients treated with anti-PD-1. Finally, combining S64315 and anti-PD-1 significantly slowed tumor growth compared to either agent alone. Together, this proof-of-concept study demonstrates the potential of combining an MCL1 inhibitor with anti-PD-1 in the treatment of melanoma. It justifies the further development of next generation MCL1 inhibitors to improve efficacy of ICIs in treating malignant melanoma.

PBRM1 loss is associated with increased sensitivity to MCL1 and CDK9 inhibition in clear cell renal cancer.

MCL1 is a member of the BCL2 family of apoptosis regulators, which play a critical role in promoting cancer survival and drug resistance. We previously described PRT1419, a potent, MCL1 inhibitor with anti-tumor efficacy in various solid and hematologic malignancies. To identify novel biomarkers that predict sensitivity to MCL1 inhibition, we conducted a gene essentiality analysis using gene dependency data generated from CRISPR/Cas9 cell viability screens. We observed that clear cell renal cancer (ccRCC) cell lines with damaging PBRM1 mutations displayed a strong dependency on MCL1. PBRM1 (BAF180), is a chromatin-targeting subunit of mammalian pBAF complexes. PBRM1 is frequently altered in various cancers particularly ccRCC with ~40% of tumors harboring damaging PBRM1 alterations. We observed potent inhibition of tumor growth and induction of apoptosis by PRT1419 in various preclinical models of PBRM1-mutant ccRCC but not PBRM1-WT. Depletion of PBRM1 in PBRM1-WT ccRCC cell lines induced sensitivity to PRT1419. Mechanistically, PBRM1 depletion coincided with increased expression of pro-apoptotic factors, priming cells for caspase-mediated apoptosis following MCL1 inhibition. Increased MCL1 activity has been described as a resistance mechanism to Sunitinib and Everolimus, two approved agents for ccRCC. PRT1419 synergized with both agents to potently inhibit tumor growth in PBRM1-loss ccRCC. PRT2527, a potent CDK9 inhibitor which depletes MCL1, was similarly efficacious in monotherapy and in combination with Sunitinib in PBRM1-loss cells. Taken together, these findings suggest PBRM1 loss is associated with MCL1i sensitivity in ccRCC and provide rationale for the evaluation of PRT1419 and PRT2527 for the treatment for PBRM1-deficient ccRCC.

Development of Potent Mcl-1 Inhibitors: Structural Investigations on Macrocycles Originating from a DNA-Encoded Chemical Library Screen.

Evasion of apoptosis is critical for the development and growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family, associated with tumor aggressiveness, poor survival, and drug resistance. Development of Mcl-1 inhibitors implies blocking of protein-protein interactions, generally requiring a lengthy optimization process of large, complex molecules. Herein, we describe the use of DNA-encoded chemical library synthesis and screening to directly generate complex, yet conformationally privileged macrocyclic hits that serve as Mcl-1 inhibitors. By applying a conceptual combination of conformational analysis and structure-based design in combination with a robust synthetic platform allowing rapid analoging, we optimized in vitro potency of a lead series into the low nanomolar regime. Additionally, we demonstrate fine-tuning of the physicochemical properties of the macrocyclic compounds, resulting in the identification of lead candidates 57/59 with a balanced profile, which are suitable for future development toward therapeutic use.

Melatonin sensitizes leukemia cells to the MCL1 inhibitors S63845 and A‐1210477 through multiple pathways

AbstractSeveral myeloid cell leukemia sequence 1 protein (MCL1) inhibitors including S64315 have undergone clinical testing for leukemia. Because of the toxicities after MCL1 inhibition, including hematopoietic, hepatic, and cardiac toxicities, there is substantial interest in finding agents that can sensitize leukemia cells to these MCL1 inhibitors. Melatonin is a chronobiotic that promotes chemo‐induced cancer cell death while protecting normal cells from cytotoxic effects. In this study, we found melatonin sensitizes over 10 leukemia cell lines to the MCL1 inhibitors S63845 (S64315 analog) and A‐1210477. Further studies demonstrate that melatonin sensitizes Jurkat cells to S63845 and A‐1210477 independent of melatonin receptors MT1 and MT2, but through multiple mechanisms, including upregulating the death receptor pathway, increasing mitochondrial reactive oxygen species (ROS), inhibiting nuclear factor‐κB (NF‐κB) signaling, and causing cell cycle arrest. First, death receptor pathway inhibition only slightly diminishes the melatonin sensitization of S63845, while inhibiting mitochondrial ROS partially reduces the S63845/melatonin combination‐induced apoptosis and depletion of the mitochondrial pathway totally abolishes it, indicating that both death receptor and mitochondrial apoptosis pathways are involved. Second, transcriptome sequencing analysis found that NF‐κB signaling is downregulated by melatonin that inhibition of NF‐κB signaling by parthenolide also dramatically sensitizes Jurkat cells to S63845. Third, melatonin induces G1 cell cycle arrest and upregulates NOXA while NOXA knockdown diminishes the sensitization to S63845 by melatonin. In addition, a xenograft model suggests that melatonin in combination with S63845 causes shrinkage of leukemic deposit while S63845 or melatonin monotherapy only has limited effects. Thus, our results demonstrate that melatonin efficiently sensitizes various leukemia to the MCL1 inhibitors, potentially allowing the usage of lower doses.

QSAR Analysis and Molecular Docking Studies of Aryl Sulfonamide Derivatives as Mcl-1 Inhibitors and the Influence of Structure and Chirality on the Inhibitory Activity

Background:: Mcl-1 is a kind of antiapoptotic protein and its overexpression is closely related to the occurrence of cancer. Aryl sulfonamide derivatives are expected to become new anticancer agents due to their high inhibitory activity on the Mcl-1 protein. Objective:: The study aimed to establish the QSAR model with good prediction ability and elaborate the influence of structure and chirality on the inhibitory activity. method: Multiple QSAR models were built with different types of descriptors and modeling methods. The molecular docking was performed on compound 45, 25, 26, 24R and 24S. Results:: The comprehensive models including 2D and 3D descriptors demonstrated that nonlinear LSSVM and GP methods gave better results (R2>0.94, RCV2>0.86). The training set had a good predictive power on the test set. The predictive performances of MCCV tests are basically coincident with the results of the single test set. The results of molecular docking showed that the hydrogen bond acceptor at the appropriate position of the substituent on the chiral center can form the hydrogen bond interaction with residue ASN260, which results in the stronger interaction between ligand and protein and higher inhibitory activity. The interaction differences between R and S configuration with Mcl-1 protein are mainly attributed to two residues, HIS224 and ASN260. Two opposite effects lead to the activity of R enantiomer slightly higher than that of S one. The results on chiral compound 24 with ambiguous absolute configuration demonstrated that the steric effect of the substituents on chiral carbon atom is crucial. When there are two substituents with big volume at the same time, high steric effect will prevent the binding of the substituent and the protein, which results in the low inhibitory activity. Conclusion:: The study may provide theoretical guidance on the design and synthesis of novel aryl sulfonamide derivatives with high inhibitory activity

Application of molecular dynamics-based pharmacophore and machine learning approaches to identify novel Mcl1 inhibitors through drug repurposing and mechanics research.

Myeloid cell leukemia 1 (Mcl1), a critical protein that regulates apoptosis, has been considered as a promising target for antitumor drugs. The conventional pharmacophore screening approach has limitations in conformation sampling and data mining. Here, we offered an innovative solution to identify Mcl1 inhibitors with molecular dynamics-refined pharmacophore and machine learning methods. Considering the safety and druggability of FDA-approved drugs, virtual screening of the database was performed to discover Mcl1 inhibitors, and the hit was subsequently validated via TR-FRET, cytotoxicity, and flow cytometry assays. To reveal the binding characteristics shared by the hit and a typical Mcl1 selective inhibitor, we employed quantum mechanics and molecular mechanics (QM/MM) calculations, umbrella sampling, and metadynamics in this work. The combined studies suggested that fluvastatin had promising cell inhibitory potency and was suitable for further investigation. We believe that this research will shed light on the discovery of novel Mcl1 inhibitors that can be used as a supplemental treatment against leukemia and provide a possible method to improve the accuracy of drug repurposing with limited computational resources while balancing the costs of experimentation well.

Dihydroartemisinin Enhances the Therapeutic Efficacy of BH3 Mimetic Inhibitor in Acute Lymphoblastic Leukemia Cells via Inhibition of Mcl-1.

Up-regulation of the anti-apoptotic proteins such as Mcl-1 is associated with the primary and secondary resistance of tumor cells to ABT-737 Bcl-2 inhibitor. The combined treatment of Bcl-2 inhibitors with Mcl-1 inhibitors has been proposed as an attractive therapeutic strategy to overcome this drug resistance. Here, we investigated the effect of dihydroartemisinin on Mcl-1 expression and sensitization of T-ALL cells to ABT-737. The cell growth and survival were tested by the cell proliferation and MTT assays, respectively. The mRNA levels of Bcl-2, Mcl-1, Bax and P21 were examined by qRT-PCR. Apoptosis were detected by Hoechst 33342 staining and caspase-3 activity assay. Our data showed that combination treatment with dihydroartemisinin and ABT-737 caused a significant decrease in the IC50 value and synergistically reduced the cell survival compared with dihydroartemisinin or ABT-737 alone. ABT-737 enhanced the Mcl-1 mRNA expression. Dihydroartemisinin also down-regulated the expression of Bcl-2 and Mcl-1 and enhanced the P21 and Bax expression. Moreover, dihydroartemisinin enhanced the apoptosis induced by ABT-737 in MOLT-4 and MOLT-17 cell lines. In conclusion, dihydroartemisinin demonstrates anti-tumor activities in human ALL cells via inhibition of cell survival and growth. Dihydroartemisinin augments the apoptotic effect of ABT-737 by inhibiting the expression of Mcl-1.

Small molecule Mcl-1 inhibitor for triple negative breast cancer therapy.

Apoptosis is an evolutionarily conserved cell death pathway that plays a crucial role in maintaining tissue homeostasis, orchestrating organismal development, and eliminating damaged cells. Dysregulation of apoptosis can contribute to the pathogenesis of malignant tumors and neurodegenerative diseases. Anticancer drugs typically possess the capacity to induce apoptosis in tumor cells. The Bcl-2 protein family, consisting of 27 members in humans, serves as the key regulator of mitochondrial function. This family can be divided into two functional groups: anti-apoptotic proteins (e.g., Bcl-2, Bcl-xl, Mcl-1) and pro-apoptotic proteins (e.g., Bad, Bax). Mcl-1 exerts its function by binding pro-apoptotic Bcl-2 proteins thereby preventing apoptosis induction. Overexpression of Mcl-1 not only correlates closely with tumorigenesis but also associates significantly with resistance towards targeted therapy and conventional chemotherapy. Effective induction of apoptosis can be achieved through inhibition or interference with Mcl-1. Thus, this mini review discusses existing Mcl-1 inhibitors.

Chemoselective seleno-click amidation in kinetic target-guided synthesis.

Capitalizing on our previous kinetic target-guided synthesis (KTGS) involving the sulfo-click reaction to form N-acylsulfonamide-linked inhibitors in the presence of the protein-protein interaction target Mcl-1, we herein report a seleno-click approach for amide-linked inhibitors of Mcl-1. The seleno-click reaction leverages the enhanced reactivity of selenocarboxylates, enabling the templated amidation with electron-rich azides, thereby expanding the scope of KTGS. The potential of this approach is demonstrated by generating N-benzyl-5-(4-isopropylthiophenol)-2-hydroxyl nicotinamide, a known Mcl-1 inhibitor featuring an amide, via KTGS at 37 °C against Mcl-1. Notably, the seleno-click strategy was also effective at 4 °C, offering a variant for thermally sensitive biological targets.

Statin-induced Mitochondrial Priming Sensitizes Multiple Myeloma Cells to BCL2 and MCL-1 Inhibitors.

BH3 mimetics including venetoclax hold promise for treatment of multiple myeloma but rational combinations are needed to broaden efficacy. This study presents mechanistic and clinical data to support addition of pitavastatin to venetoclax regimens in myeloma. The results open a new avenue for repurposing statins in blood cancer.

Synergistic cytotoxicity of decitabine and YM155 in leukemia cells through upregulation of SLC35F2 and suppression of MCL1 and survivin expression.

The hypomethylation agent decitabine (DAC), in combination with other apoptosis inducers, is considered a potential modality for cancer treatment. We investigated the mechanism underlying the combined cytotoxicity of DAC and YM155 in acute myeloid leukemia (AML) cells because of increasing evidence that YM155 induces apoptosis in cancer cells. Co-administration of DAC and YM155 resulted in synergistic cytotoxicity in AML U937 cells, which was characterized by the induction of apoptosis, NOXA-dependent degradation of MCL1 and survivin, and depolarization of mitochondria. Restoration of MCL1 or survivin expression attenuated DAC/YM155-induced U937 cell death. DAC initiated AKT and p38 MAPK phosphorylation in a Ca2+/ROS-dependent manner, thereby promoting autophagy-mediated degradation of β-TrCP mRNA, leading to increased Sp1 expression. DAC-induced Sp1 expression associated with Ten-eleven-translocation (TET) dioxygenases and p300 was used to upregulate the expression of SLC35F2. Simultaneously, the activation of p38 MAPK induced by DAC, promoted CREB-mediated NOXA expression, resulting in survivin and MCL1 degradation. The synergistic cytotoxicity of DAC and YM155 in U937 cells was dependent on elevated SLC35F2 expression. Additionally, YM155 facilitated DAC-induced degradation of MCL1 and survivin. A similar mechanism explained DAC/YM155-mediated cytotoxicity in AML HL-60 cells. Our data demonstrated that the synergistic cytotoxicity of DAC and YM155 in AML cell lines U937 and HL-60 is dependent on AKT- and p38 MAPK-mediated upregulation of SLC35F2 and p38 MAPK-mediated degradation of survivin and MCL1. This indicates that a treatment regimen that amalgamates YM155 and DAC may be beneficial for AML.

Abstract LB_A01: Rapidly cleared MCL1 inhibitor TTX-810 showing robust efficacy in preclinical tumor models with no effects on cardiomyocytes in vitro and in vivo

Abstract MCL1, an anti-apoptotic protein frequently amplified in cancer, confers resistance to standard of care, thus highlighting its importance as a therapeutic target. However, the involvement of MCL1 in cardiomyocyte function raises concerns for safely targeting the protein in cancer patients. MCL1 inhibitors currently in clinical trials exhibit shortcomings, either due to insufficient potency or extended half-lives that heighten the risk of cardiotoxicity. This study systematically explores MCL1 inhibitor-induced cell dynamics in cancer cells and healthy cardiomyocytes. TTX-810, a potent, selective, and rapidly-cleared MCL1 inhibitor, was used to identify a treatment regimen that induces efficacy, but spares healthy cardiomyocytes. TTX-810 demonstrates potent and reversible binding to the BH3-binding groove of human MCL1 (KD = 0.3 nM). It inhibits the interaction of MCL1 with the pro-apoptotic effector protein BAK or BH3-only protein BIM in cells in a dose- and time-dependent manner, leading to activation of the intrinsic apoptotic cascade and cancer cell death. At suprapharmacologic concentrations and extended exposure durations TTX-810 does induce apoptosis in human iPSC-derived cardiomyocytes, aligning with the recognized mechanism of MCL1 inhibitor-related cardiotoxicity. In contrast, short-term TTX-810 exposure induces efficient cancer cell killing in vitro, and has no impact on cell viability, apoptosis induction, or troponin I release in human iPSC-derived cardiomyocytes, even at high concentration. Similarly, TTX-810 induces strong monotherapy efficacy with once weekly in vivo dosing in an aggressive AML cancer model that is resistant to current standard of care agents like Venetoclax or Azacytidine. Repeated once weekly dosing in dogs over 4-weeks had no impact on troponin I levels in blood, electrocardiogram parameters (PR, QRS, QT and calculated QTcV intervals), heart rate, nor cardiac tissue morphology. In conclusion, this study identifies an optimal TTX-810 treatment regimen that effectively targets cancer cells, while preserving healthy cardiomyocytes. The study also demonstrates that a minimum exposure time is sufficient for efficacy, and this effect is even more pronounced when combined with standard-of-care agents. Moreover, limiting the exposure time expands the TTX-810 therapeutic window with regard to cardiotoxicity, which was validated in GLP toxicology studies in dogs and positions TTX-810 for future clinical development. Citation Format: Ulrike Rauh, Virendar Kaushik, Sabine Pilari, Georgios Kosmidis, Michael Serrano-Wu, Guo Wei, Weiqun Zhang, Stefan Kaulfuss, Kai Thede, Guillaume Poncet-Montange, Douglas Daniels, Christopher T Lemke, Brian Hubbard, Todd R. Golub. Rapidly cleared MCL1 inhibitor TTX-810 showing robust efficacy in preclinical tumor models with no effects on cardiomyocytes in vitro and in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A01.

Exploring the Mechanisms of Venetoclax Resistance Via Drug Screening and Genome-Wide CRISPR Screening

Introduction: Combining Venetoclax, a selective BCL-2 inhibitor, with hypomethylating agents has revolutionized frontline acute myeloid leukemia (AML) treatment. Despite its success, the occurrence of resistance remains a significant concern, limiting the range of available treatment alternatives. Little is known about the mechanism by which the cells escape the treatment. Recent studies have observed MCL1 upregulation in AML cells to promote resistance, and the rationale to use an MCL1 inhibitor became attractive. Although MCL1 inhibitors work exceptionally well in vitro their clinical trials were unsuccessful due to their toxicity. Here, we aim to identify other genes responsible for acquiring resistance and tackle the problem through screening for clinically approved drugs to rapidly benefit the patients with our study. Methods: First, we generated a model of the venetoclax resistance from 3 AML cell lines: MOLM-13, HL-60 and MV4-11. Resistance was achieved through chronic administration of the compound, inhibiting cell viability to 80-90% in multiple rounds by gradually increasing concentrations until the cells developed resistance. We have then characterized these Venetoclax-resistant (VeR) cells using quantitative RT-PCR and RNA sequencing to reveal the mechanisms behind their resistance. VeR cell lines were subjected to drug screening with a library of 859 FDA and EMA-approved compounds with various clinical indications. Cell viability in response to the drug library was assessed after 72h by Cell-Titer Glo and evaluated against the wild-type counterparts. Top-performing compounds were validated in dose-response curves, and synergy scores were identified from combinational treatments. Simultaneously, we performed genome-wide CRISPR/Cas9 knockout screening using Brunello CRISPR knockout library on both MOLM-13 wild-type (WT) and VeR cell lines to identify genes that maintain the resistance to venetoclax and genes that may potentially synergize with targeting BCL-2. Results: Results from the CRISPR screen pointed out multiple genes whose loss of function may contribute to venetoclax resistance in MOLM-13 cells, among which BAX, NOXA, ELAVL1 and TP53 ranked as top hits. On the contrary, we also identified several genes whose loss of function sensitized the cells to venetoclax (e.g., MCL1, OPA1, CDK2, MCAT), suggesting them to be promising therapeutic targets. Among these hits, CDK2 correlates with our drug screening data indicating Flavopiridol as an effective compound in treating VeR cell lines. Moreover, we confirmed the elevated MCL1 gene expression for the MOLM-13 VeR cell line through RNAseq (and qPCR) and detected an altered expression of genes mainly in metabolic pathways and PI3K-Akt signaling pathway via differential gene expression analysis. Our drug screening results revealed overall high efficacy of DNA-damaging agents, proteasome and HDAC inhibitors for both WT and VeR cell lines. We also included an MCL1 inhibitor as a positive control in our drug library and confirmed a synergistic effect when combined with venetoclax. Conclusion: Integrating a range of screening approaches may lead to discovering previously unknown therapeutic targets for venetoclax-resistant AML. Our screens confirmed previously described involvement of the apoptotic pathway genes in venetoclax resistance as well as identified some novel promising targets. Our ongoing efforts involve thorough validations of the identified hits from CRISPR and drug screening to enhance the reliability and translatability of the results. This project was partly supported by grants MUNI/A/1330/2021, MUNI/A/1419/2021 OPRDE (No.CZ.02.2.69/0.0/0.0/19_073/0016943) and project NICR (EU program EXCELES, No. LX22NPO5102).

Long-Acting E. coli-Derived Asparaginase Potentiates the Anti-Leukemic Effect of BCL2 Inhibition, but Not MCL1 Inhibition, in Preclinical Models of Acute Myeloid Leukemia

Background: Asparaginases are enzymes that deplete circulating asparagine and glutamine from plasma and can therefore be used therapeutically for cancers that are sensitive to amino acid depletion. Commercially available asparaginases are isolated fromeither E. coli or Erwinia chrysanthemi (also called crisantaspase), the latter having a higher glutaminase activity. Our previous studies have shown that pegcrisantaspase (PegC), a long-acting pegylated crisantaspase, synergizes with the BCL2 inhibitor, Venetoclax (Ven), to kill several AML cell lines and patient-derived primary AML cells with complex karyotype in vitro and in vivo in a xenograft mouse model. Whether long-acting E. coli-derived asparaginases will have similar anti-AML activity as a single agent or in combination with BCL-2 inhibition, and the potential of asparaginases to synergize with inhibitors of MCL1, a key resistance factor of BCL2 inhibition, has yet to be determined. Methods: Using a panel of 4 human AML cell lines (MOLM14, MV411, MonoMac6, HL60) as well as primary AML patient samples, weestablished the anti-leukemic activity of the BCL2 inhibitor S55746 and the MCL1 inhibitor S63845 alone and in combination with the long-acting E. coli asparaginase, calaspargase pegol-mknl (CalPegA). To determine the ability of S55746 or S63845 with or without CalPegA to inhibit mRNA translation, an established mechanism of Ven-PegC anti-leukemic activity, we measured the dissociation of the translation initiation factor eIF4E from 4EBP1, a negative regulator of cap-dependent mRNA translation, which then allows the formation of translation initiation complexes. The in vivo anti-leukemic efficacy of S55746 and S63845 alone and in combination with CalPegA was tested using an orthotopic AML xenograft mouse model with luciferase-expressing MV411 cells. Results: We found that CalPegA treatment resulted in a dose-dependent inhibition of AML cell proliferation, with IC 50 values ranging from 0.78 to 2.8 IU/mL. Single agent S55746 and S63845 treatment inhibited AML cell proliferation with IC 50 values ranging from 0.01 to 6.7 µM and 0.0001 to 0.08 µM, respectively. In 3 of the human AML cell lines (MOLM-14, MV4-11, and MonoMac6), the addition of a small dose (IC 20) of CalPegA potentiated the anti-leukemic effect of S55746, reducing the IC 50 by approximately 1- 4-fold, and by 10-fold in HL60 cells (Figure 1). This potentiation was also observed in 3 out of 4 primary AML patient samples tested, with the IC 50 of S55746 reduced by 4.5, 18, and 156-fold by the addition of CalPegA. The activity of S63845 was not substantially impacted by the addition of CalPegA in either AML cell lines (Figure 1) or primary samples. Immunoprecipitation of 4EBP1 revealed that treatment with a combination of either S63845 or S55746 with CalPegA increased the binding of eIF4E to 4EBP1 compared to single agent treatments, suggesting an inhibition of translational complex formation. In a MV411 tumor-bearing model, treatment withS55746-CalPegA combination reduced tumor burden over time significantly more than either single agent. S63845 treatment was highly effective at inhibiting leukemia growth over time and was not improved by the addition of CalPegA, as tumor burden in the S63845 and S63845 + CalPegA-treated groups was not significantly different over the course of the study (Figure 2). Conclusions: We report that the E. coli asparaginase, CalPegA, enhances the anti-leukemic effect of the BCL2 inhibitor, S55746, but does not impact the activity of the MCL1 inhibitor, S63845, in AML cell lines, patient derived primary AML samples, and in an AML xenograft mouse model. Ongoing studies are further investigating the anti-leukemic mechanism of S55476/S63845 + CalPegA and examining the efficacy and pharmacodynamic/pharmacokinetic effects of these agents in a patient derived xenograft model of AML.

Identification of vulnerabilities for targeting BCL-2 family members in T-Cell Acute Lymphoblastic Leukemia

T-Cell acute lymphoblastic leukemia (T-ALL) is a disease caused by the malignant transformation of T-cell lineage progenitors. With the use of intensive chemotherapies survival rates have improved, but outcomes particularly of relapsed patients remain poor. In addition, currently used intensive chemotherapies are associated with high rates of treatment-related morbidity and mortality, emphasizing the need to develop new and improved therapies. The intrinsic apoptosis pathway, one of the key pathways controlling cell death, is dysregulated in many cancers and contributes to leukemogenesis and treatment failure. The main steps of this pathway are controlled by proteins of the B-cell lymphoma (BCL-2) family at the outer mitochondrial membrane. Hence, targeting this pathway by BH3-mimetics has emerged as an effective new treatment strategy in different cancers. Venetoclax is a selective BCL-2 inhibitor and is successfully used in CLL and AML, but heterogeneous sensitivity to venetoclax has been described in ALL and inhibitors of other BCL-2 family members including BCL-XL-selective A-1331852 and MCL-1 selective AZD5991 have been developed. Moreover, a dual inhibitor of the anti-apoptotic BCL-2 family members BCL-2 and BCL-XL (AZD4320) with its dendrimer conjugate (AZD0466) has recently shown anti-tumor activity in hematologic cancer models with manageable toxicity. The aims of this study are to evaluate susceptibilities of T-ALL to inhibitors of BCL-2 family proteins, to identify markers of response, to elucidate mechanisms of action and to assess combination effects. First, we analyzed the anti-leukemia activities of inhibitors targeting BCL-2 (venetoclax), BCL-XL (A-1331852) and MCL-1 (AZD5991) and of the dual BCL-2/BCL-XL inhibitor AZD4320 in T-ALL cell lines (N=6) and a series of patient-derived xenograft (PDX) samples (N=8). Inhibition of MCL-1 alone was not effective in the cell lines tested (EC50 >1 µM), but heterogeneous sensitivity was found in PDX samples (EC50 <1µM in 3/8 samples). In cell lines, sensitivity for BCL-2 inhibition was exclusively found in a cell line with an early T-cell precursor ALL phenotype (Loucy). Similar to cell lines, we found insensitivity for BCL-2 inhibition in all PDX samples tested (N=8). For inhibition of BCL-XL, we found heterogeneous sensitivities with 4/6 cell lines and 5/8 PDX samples showing an EC 50 <1 µM. Interestingly, BH3-profiling confirmed BCL-2 dependence in Loucy and BCL-XL dependence in the other cell lines. Importantly, dual inhibition of BCL-2 and BCL-XL showed clear responses in cell lines and PDX samples (median EC 50s 796.1 nM and 721.6 nM). Notably, sensitivity was associated with priming of the BH3-peptide BAD (r s=0.89, p=0.03), providing a marker of response. We next sought to elucidate the molecular mechanisms by which different BH3-mimetics induce cell death in T-ALL. Analyzing protein interactions of BCL-2 family members by co-immunoprecipitation studies, we found that AZD4230 acts on-target by releasing BIM from BCL-2 and BCL-XL, but with compensatory increased protein complexes of BIM and MCL-1. Accordingly, combined inhibition of BCL-2 and BCL-XL with inhibition of MCL-1 leads to release of BIM from all three anti-apoptotic proteins enabling downstream apoptosis signaling. These findings suggest that combined inhibition of BCL-2 and BCL-XL together with MCL-1 may provide synergistic benefits. To further validate these data, we applied dynamic BH3-profiling and determined dependencies of T-ALL samples on BCL-2 family members in response to BCL-2/BCL-XL inhibition. Here, we found that the MCL-1 dependence strongly increases upon BCL-2/BCL-XL inhibition, suggesting MCL-1 as a target for combined inhibition. Based on these results, we performed dose-response matrix analyses to test the effects of combined BCL-2/BCL-XL and MCL-1 inhibition. Most importantly, combined inhibition showed synergism in all cell lines and PDX samples tested (Bliss scores >10), including leukemias being insensitive to single compounds. Taken together, we found vulnerabilities of T-ALL to BCL-2 family inhibition, particularly to dual BCL-2/BCL-XL inhibition by AZD4320, and we demonstrated on-target activities. Using BH3-profiling, we identified BAD-priming as a marker of response for AZD4320 and MCL-1 dependence as a resistance mechanism that can be targeted by combination treatment, suggesting further clinical evaluation.

Drug Resistance Can be Overcome in Multiple Myeloma and Acute Myeloid Leukemia By Targeting Mcl-1 with the Small Chemical KS18

Drug resistance and patient recurrence are still key clinical difficulties in multiple myeloma (MM) treatment, despite the tremendous improvements provided by bortezomib therapy. In many cancers, Mcl-1, a member of the Bcl-2 protein family, helps keep cells alive by preventing their natural death process, apoptosis. The anti-cancer therapeutic promise of Mcl-1 inhibitors is highlighted by the fact that Mcl-1 overexpression increases carcinogenesis and medication resistance. Here, we provide KS18, a powerful pyoluteorin derivative with excellent effectiveness towards Mcl-1-dependent cancer cells, to address this unmet therapeutic need. Based on our results, we conclude that KS18 causes apoptosis in cancer cells through activating the mitochondrial outer membrane permeabilization (MOMP) pathway, with the participation of Bak and Bax, in particular in MM and acute myeloid leukemia (AML). With IC 50 values below 1.5µM, KS18 demonstrated efficacy against bortezomib-resistant MM cells, outperforming other Mcl-1 inhibitors in clinical trials such as S63845, VU661013, and AZD5991. Total apoptosis in MM patient samples treated with KS18 was an impressive 88.8 percent. After four weeks of treatment with a single tolerable dose each week, a model of bortezomib-resistant MM showed considerable tumor shrinkage, demonstrating the powerful anti-tumor efficacy of KS18. According to these results, KS18 may be able to overcome resistance by concentrating on Mcl-1. These encouraging findings have prompted the development of many models of drug-resistant MM and AML for in vivo evaluation of this potential chemical.