Anti-apoptotic Bcl-2 Family Member Mcl-1 Research Articles

Structural transitions in TCTP tumor protein upon binding to the anti-apoptotic protein family member Mcl-1

Translationally Controlled Tumor Protein (TCTP) serves as a pro-survival factor in tumor cells, inhibiting the mitochondrial apoptosis pathway by enhancing the function of anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. TCTP specifically binds to Bcl-xL, preventing Bax-dependent Bcl-xL-induced cytochrome c release, and it reduces Mcl-1 turnover by inhibiting its ubiquitination, thereby decreasing Mcl-1-mediated apoptosis. TCTP harbors a BH3-like motif that forms a β-strand buried in the globular domain of the protein. In contrast, the crystal structure of the TCTP BH3-like peptide in complex with the Bcl-2 family member Bcl-xL reveals an α-helical conformation for the BH3-like motif, suggesting significant structural changes upon complex formation. Employing biochemical and biophysical methods, including limited proteolysis, circular dichroism, NMR, and SAXS, we describe the TCTP complex with the Bcl-2 homolog Mcl-1. Our findings demonstrate that full-length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif, experiencing conformational exchange at the interface on a micro- to milli-second timescale. Concurrently, the TCTP globular domain becomes destabilized, transitioning into a molten-globule state. Furthermore, we establish that the non-canonical residue D16 within the TCTP BH3-like motif reduces stability while enhancing the dynamics of the intermolecular interface. In conclusion, we detail the structural plasticity of TCTP and discuss its implications for partner interactions and future anticancer drug design strategies aimed at targeting TCTP complexes.

ASXL1 Mutations Are Associated with a Response to the Combination of Alvocidib and 5-Azacytidine in Higher-Risk Myelodysplastic Syndromes

Introduction: Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are perspective therapeutic approaches in acute myeloid leukemia (AML) and higher-risk myelodysplastic syndromes (MDS). Inhibitors of BCL-2 family proteins previously showed potent activity against AML and higher-risk MDS. Alvocidib (Alv), a cyclin-dependent kinase 9 inhibitor and indirect transcriptional repressor of the anti-apoptotic BCL-2 family member MCL-1, has previously shown clinical activity in AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). A phase 1b/2 study with Alv and HMAs 5-Azacytidine (5-AZA) or decitabine in higher-risk MDS patients was recently completed (NCT03593915). However, biomarkers of response to 5-AZA + Alv combination are insufficiently characterized. In order to possibly identify biomarkers of response, we performed a comprehensive in vitro assessment of Alv and 5-AZA effects in a cohort of n=45 higher-risk MDS patients. Methods: CD34+ hematopoietic stem and progenitor cells (HSPCs) were isolated from bone marrow (BM) aspirates using positive selection with MACS microbeads. CD34+ cells of aged-matched healthy controls (HC, n=11) were obtained from bone specimen after femur endoprosthesis surgery. HSPCs were expanded for four days in StemSpan SFEM II medium containing StemSpan Myeloid Expansion Supplement (Stem Cell Technologies) and treated with 5-AZA for 48h, Alv for 24h or their sequential combination (5-AZA for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo (CTG) and Annexin-V apoptosis assays. MCL-1 dependency of primary MDS HSPCs was assessed using NOXA-derived T-MS1 peptide. MDS recurrent mutations in BM mononuclear cells were assessed using myeloid NGS panel deep sequencing containing 67 genes. For in vivo tumor transfer experiments, splenic cells from CD45.2+Asxl1Y588X transgenic (Tg) mice were injected intravenously into sublethally irradiated CD45.1+ recipients through tail veins. Engrafted mice were treated with 1 mg/kg 5-AZA intraperitoneally (i.p.) on days 1-3 (qdx) every other week. Alv was administered i.p. at a dose of 2.5 mg/kg on day 5 (qw) in the weeks when 5-AZA was applied. Mice were sacrificed 11 weeks after the transplantation and the percentage of CD45.2+ cells in the BM was assessed using flow cytometric analysis. Results: The combination of 5-AZA+Alv showed an additive cytotoxic effect on MDS cells in CTG cell viability assays (median cell viability was 74%, 74% and 55% for 5-AZA, Alv and combination, respectively, p<0.0001). In annexin-V apoptotic assays, MDS cells were more sensitive to the cytotoxic effect of the combination treatment compared to HC (median percent of apoptotic and dead cells was 37% for MDS vs 26% for HC, p=0.0256). In analogy to in vitro studies, accelerated clearance of xenografted MDS-L cells from the peripheral blood of NSGS mice was observed after 5-AZA+Alv treatment compared to single agents. MCL-1 dependency of primary MDS samples did not significantly correlate with cytotoxic effects of Alv and combination treatment. However, the presence of ASXL1 mutations was significantly associated with higher cytotoxic activity of 5-AZA+Alv combination in univariable (p=0.0044) and multivariable (p=0.042) analyses. Increased sensitivity of BM cells to the cytotoxic effects of 5-AZA+Alv was also observed in Asxl1Y588XTg mice. The combination treatment facilitated clearance of CD45.2+Asxl1Y588X mutant leukemic cells from CD45.1+ recipients compared to 5-AZA monotherapy (p=0.0156). Histological analysis of BM cells after 5-AZA+Alv treatment in CD45.1+ recipients showed decrease in the percentage of immature blasts and improved development of mature BM cells when compared to 5-AZA monotherapy (p=0.0391). Improved cytotoxic effects of Alv and 5-AZA+Alv in ASXL1 mutant compared to ASXL1 wild type MDS samples were associated with higher expression of pro-apoptotic MCL-1 antagonist NOXA (p=0.0017). Conclusions: We provided a pre-clinical rationale for the use of the 5-AZA+Alv combination for higher risks MDS and proposed ASXL1 mutations as potential genetic biomarkers of response in prospective clinical trials.

Dual inhibition of BCL2L1 and MCL1 is highly effective against RET fusion-positive or MET exon 14 skipping mutation-positive lung adenocarcinoma cells

Non-small cell lung carcinomas (NSCLCs), especially lung adenocarcinomas (LUADs), harbor several driver mutations against which highly effective tyrosine kinase inhibitors (TKIs) are available. Although TKIs are generally effective against certain NSCLCs, primary or acquired resistance almost always develops. Driver mutations include RET fusion (∼1–2% of NSCLC cases) and MET exon 14 skipping mutation (METΔex14; ∼3–4%). Surprisingly, the LUAD cell line LC-2/ad with CCDC6-RET fusion thrived independently of RET signaling, and Hs-746T cells harboring METΔex14 plus amplification survived MET silencing. However, these two cell lines were highly sensitive to dual silencing of the representative anti-apoptotic BCL2 family members BCL2L1 and MCL1, undergoing extensive apoptosis in monolayer or 3D on-top culture systems. Moreover, we found that most LUAD cell lines and tissues expressed high levels of BCL2L1 and MCL1 mRNA but extremely low levels of BCL2. Together, these findings suggest that inhibiting BCL2L1 plus MCL1 may represent a new approach to treating LUAD cells irrespective of their driver mutations.

Abstract P209: Collaborative crosstalk between two apoptosis pathways drives synergy of dual inhibition of BCL-2/MDM2 in preclinical models of neuroblastoma

Abstract Introduction: The prognosis of patients with neuroblastoma is poor, and novel therapeutic strategies are needed to improve clinical outcomes. Neuroblastoma often exhibits a low TP53 mutation rate and overexpresses MDM2 and BCL-2 genes. Dual targeting of the MDM2-P53 and BCL-2 apoptotic pathways is an attractive treatment strategy (Vernooij et al, Mol Cancer Ther 2021; Dalton et al, Mol Cancer Ther 2021) and is under clinical development using idasanutlin and venetoclax. However, the mechanisms of action (MOAs) of this combination in neuroblastoma are not fully understood. Alrizomadlin (APG-115) is a potent, orally bioactive MDM2 inhibitor. Lisaftoclax (APG-2575) is a novel BCL-2 selective inhibitor. Both agents have shown clinical activity in solid and hematologic malignancies (Tolcher et al, J Clin Oncol 2021:2506; Ailawadhi et al, J Clin Oncol 2021:7502). We investigated the effects of combined alrizomadlin and lisaftoclax treatment in preclinical models of neuroblastoma and examined novel MOAs associated with this combination. Materials and Methods: Antiproliferative activity was measured by CellTiter-Glo luminescent cell viability assay. Apoptosis was evaluated by annexin V/propidium iodide staining and flow cytometry. Meso-Scale Discovery ELISA and western blot analyses characterized the mechanisms of this combination’s synergistic effects. In animal studies, clinically relevant doses of lisaftoclax were employed. A pulsed high-dose regimen of alrizomadlin was used to achieve best efficacy and manage on-target toxicity. Results: In TP53WT neuroblastoma cell lines IMR-32 and SH-SY5Y, the combination of lisaftoclax and alrizomadlin synergistically inhibited proliferation and induced apoptosis, as evidenced by elevations in caspase-3/7, cleaved PARP, and annexin V+ subpopulations. Tumor growth was synergistically inhibited in 3 distinct subcutaneous mouse xenograft tumor models derived from neuroblastoma cell lines or patient tumors. In the cell lines, alrizomadlin treatment upregulated proapoptotic BCL-2 family member expression (NOXA, BAX, and PUMA) and downregulated antiapoptotic BCL-2 family member MCL-1, likely sensitizing cancer cells to lisaftoclax-induced apoptosis. Treatment of these cells with lisaftoclax and alrizomadlin reduced all 3 major BIM-related complexes, thereby increasing free BIM and triggering cellular apoptosis. Lisaftoclax and alrizomadlin downregulated NMYC and its transcription target MDM2, preventing the development of alrizomadlin resistance mediated by a feedback upregulation of MDM2, and inhibition of the MDM2-P53 apoptotic pathway by alrizomadlin primed cancer cells to BCL-2 inhibition. Conclusions: Alrizomadlin synergizes with lisaftoclax to achieve synthetic lethality through a regulated interaction at multiple molecular nodules, and inhibition of BCL-2 apoptotic pathway by lisaftoclax may prevent cancer cells from developing resistance to MDM2-P53 inhibition. These data warrant clinical development of combined alrizomadlin and lisaftoclax treatment in patients with neuroblastoma. Citation Format: Douglas D. Fang, Qiuqiong Tang, Qixin Wang, Yanhui Kong, Na Li, Feng Zhou, Ran Tao, Dajun Yang, Yifan Zhai. Collaborative crosstalk between two apoptosis pathways drives synergy of dual inhibition of BCL-2/MDM2 in preclinical models of neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P209.

Preclinical Assessment of Alvocidib in Combination with 5-Azacytidine in High-Risk Myelodysplastic Syndromes

Hypomethylating therapy with 5-azacytidine (5-Aza) is a standard-of-care for patients with higher-risk myelodysplastic syndromes (MDS). Response is induced in approximately 50% of 5-Aza treated patients. However, despite robust efficacy in responders, relapse is almost certain. Recently, inhibitors of anti-apoptotic BCL-2 protein family members have shown potent activity against AML and higher-risk MDS in combination with 5-Aza. Alvocidib (Alv), a cyclin-dependent kinase 9 inhibitor and indirect transcriptional repressor of the anti-apoptotic BCL-2 family member MCL-1, has shown anti-leukemic effects in combination with 5-Aza in a phase 1 study of AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). Additionally, Alv has entered a phase 1b/2 study in patients with higher-risk MDS (NCT03593915). In order to possibly identify biomarkers of response, we performed a comprehensive pre-clinical in vitro assessment of Alv combined with 5-Aza using a clinically well-characterized cohort of n=40 MDS (high risk) patients and n=11 healthy controls.CD34+ HSCs were purified from bone marrow (BM) aspirates using positive selection with MACS microbeads. CD34+ cells of healthy controls were obtained from femur head replacement surgery bone specimens. Hematopoietic stem cells (HSCs) were expanded for four days in StemSpan SFEM II medium containing StemSpan Myeloid Expansion Supplement (Stem Cell Technologies) and treated with 5-Aza for 48h, Alv for 24h or their sequential combination (5-Aza for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo and Annexin-V apoptosis assays. MDS recurrent mutations in BM mononuclear cells were assessed using myeloid NGS panel deep sequencing containing 67 genes.The combination of 5-Aza+Alv showed an additive cytotoxic effect on CD34+ MDS cells in CellTiter- Glo cell viability assays (median cell viability = 74%, 73.8% and 55% for 5-Aza, Alv and combination respectively, p<0.0001). In annexin-V apoptotic assay, MDS cells were more sensitive to the cytotoxic effect of the combination treatment compared to healthy CD34+ cells (median % of apoptotic and dead cells = 36.6% for MDS vs 25.6% for healthy group, p=0.0288). Of note, the presence of ASXL1 and ZRSR2 mutations was associated with higher cytotoxic activity of 5-Aza+Alv combination. In particular, ZRSR2 mutations had an independent impact on the cell viability in a multivariable analysis (p=0.035). Overall, we provided pre-clinical support for the use of 5-Aza+Alv combination for higher risks MDS and identified ASXL1 and ZRSR2 mutations as potential genetic biomarkers of response. DisclosuresSchmitt: Affimed GmbH: Research Funding. Jawhar: Celgene: Other: Travel support; Takeda: Honoraria, Other: Travel support; Stemline: Consultancy, Honoraria; Blueprint Medicines: Honoraria; Novartis: Consultancy, Honoraria, Other: Travel support, Speakers Bureau. Foulks: Sumitomo Dainippon Pharma Oncology: Patents & Royalties: WO2021102343A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: CA3103995A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: US11040038B2. Hofmann: Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria. Nowak: Celgene: Honoraria; Takeda: Honoraria; Affimed: Research Funding; Pharmaxis: Current holder of individual stocks in a privately-held company, Research Funding; AbbVie: Other: Investigator on funded clinical trial; Tolero Pharma, Pharmaxis, Apogenix: Research Funding.

Hereditary Chronic Neutrophilic Leukemia in a Four Generation Family without Transformation to Acute Leukemia

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by peripheral blood leukocytosis consisting primarily of segmented neutrophils and band forms, hypercellular bone marrow with granulocytosis, hepatosplenomegaly, and the presence of activating colony-stimulating factor 3 receptor (CSF3R) mutations. Blast transformation occurs frequently in patients with acquired CNL, with a median overall survival of 21 months from diagnosis. Typically, CSF3R mutations in CNL are thought to be somatic; however, we and others have reported rare cases of germline activating CSF3R mutations producing a familial CNL. Here we report the clinical course of a patient with CNL along with definitive evidence of inherited germline transmission of the CSF3R T618I mutation. Spanning four generations, with affected family members of ages 1.6 - 51 years, this is the largest reported pedigree of a family with familial CNL (Figure 1A).The proband is a 49-year-old female referred to our center with a history of lifelong leukocytosis and leukocyte count of 115.1 x 10 9/L with 75% granulocytes and 11% bands, platelet count of 341 x 10 9/L, and hemoglobin of 12.5 g/dL with hematocrit of 38%. The family history was also remarkable for leukocytosis. Prior therapies for the proband included imatinib, splenectomy, and hydroxyurea. Additional testing by our center revealed a T618I CSFR3 mutation, and the absence of mutations in ASXL1 and SETBP1 or a BCR-ABL translocation. Treatment with ruxolitinib resulted in improvement of her leukocyte count to 43.0 x 10 9/L with 73% granulocytes, and reduction in her alkaline phosphatase from 732 IU/L to 296 IU/L. There has been no evidence of gain of any known deleterious somatic mutations that frequently co-occur with somatic T618I CSF3R mutations in CNL in the patient to date.Germline analysis of genomic DNA extracted from cultured mesenchymal stromal cells from the proband and Sanger sequencing demonstrated a heterozygous T618I mutation. Mutational analysis of the proband's family members confirmed a heterozygous CSF3R T618I mutation in all living affected family members, while all unaffected family members tested were homozygous wild type. There has been no evidence of leukemic transformation in any affected family members to date. Mutational analysis was not feasible on the proband's deceased mother and brother with a putative CNL diagnosis due to lack of DNA samples; however, there was no evidence of transformation to acute leukemia in either of the two deceased family members.Because CSF3R can produce anti-apoptotic signaling, we hypothesized that autoactivating T618I mutations could prolong neutrophil survival. Polymorphonuclear cells (PMNs) isolated from the proband and from normal donors were cultured in vitro and apoptosis assessed at 24-hour intervals. Neutrophils expressing the CSF3R T618I had prolonged survival with a >40% decrease in apoptosis after 48 hours in culture (Figure 1B). RNA-seq followed by pathway analysis demonstrated significant decreases in activation of canonical apoptotic pathways in PMNs, including both the extrinsic and mitochondrial dependent pathways. Immunoblotting for candidate anti- and pro-apoptotic proteins revealed increased expression of the anti-apoptotic BCL2 family member MCL1 in T618I-expressing PMNs. Notably, inhibition of MCL1 using S63845 reversed the anti-apoptotic effect induced by ligand-activation of the CSF3R receptor in PMNs (P < 0.001, Figure 1C).In conclusion, we demonstrate hereditary CNL within a large family tree with no observed transformation to acute leukemia in any affected individuals up to age 51, suggesting a potentially more indolent course. Nonetheless, our observations highlight the need for germline testing of patients with CNL to better understand the natural history of CNL. Moreover, our data provide further insight into the pathobiology of CNL and potential novel targets for therapy. [Display omitted] DisclosuresVoorhees: Bristol-Myers Squibb Company.: Other: Data Safety & Monitoring; AbbVie Inc, Bristol-Myers Squibb Company; Consulting Agreement: GlaxoSmithKline, Novartis, Oncopeptides: Other: Advisory Committee.

Synergistic Activity of BH3-Mimetics By Combined Targeting of Anti-Apoptotic Regulators in B-Cell Precursor ALL

In acute lymphoblastic leukemia (ALL), the most frequent malignancy in children and adolescents, deregulated cell death pathways contribute to leukemia development and therapy failure. Apoptosis (programmed cell death) is controlled at the mitochondrial level by different pro- and anti-apoptotic regulators. Molecules of the BCL-2 family are key in regulating intrinsic apoptosis signaling. Pro-apoptotic BH3-only proteins such as BIM and BID activate pro-death proteins like BAX and BAK leading to cell death, while anti-apoptotic BCL-2 family members including BCL-2, MCL-1 and BCL-XL sequester pro-apoptotic molecules, thereby preventing pro-death protein activation and apoptosis induction. Small molecule inhibitors have been developed, which bind to the anti-apoptotic molecules BCL-2, MCL-1 and BCL-XL leading to cell death induction. In particular inhibition of BCL-2, a key regulator of survival in B-cell malignancies including BCP-ALL, by the specific inhibitor venetoclax (VEN) has shown substantial, clinical anti-tumor activity. However, in BCP-ALL heterogeneous sensitivities for VEN have been described suggesting that other BCL-2 family members like MCL-1 and BCL-XL interfere with BCL-2 inhibition thereby counteracting VEN activity leading to poor treatment response.In this study, we compared the effects of inhibition of the anti-apoptotic BCL-2 family members BCL-2 (venetoclax, VEN), MCL-1 (S63845) and BCL-XL (A-1331852), investigated molecular mechanisms and determinants of inhibitor sensitivity, and addressed potential synergistic activity upon simultaneous blockage of BCL-2 together with MCL-1 or BCL-XL in BCP-ALL.First, we investigated the activity of the single inhibitors to induce cell death (positive staining for propidium iodide) in 7 BCP-ALL cell lines and a series of 27 BCP-ALL patient-derived xenograft samples (PDX). Titrating increasing concentrations, we estimated half-maximal effective concentrations (EC 50) for each inhibitor showing heterogenous responses of individual samples to the different inhibitors. Importantly, sensitivities to either inhibitor were not associated with leukemia characteristics including recurrent genetic alterations described in BCP-ALL. We also did not observe similar sensitivities to the inhibitors of individual samples, however some VEN insensitive samples showed sensitivity to MCL-1 and/or BCL-XL-inhibition, suggesting functional substitution of the anti-apoptotic regulators.Next, we investigated anti-apoptotic addictions of BCP-ALL cells to BCL-2 family proteins upon VEN treatment. Analyzing apoptosis signaling after exposure to VEN and BH3-peptides, we identified that ALL cells adapt their anti-apoptotic addiction to MCL-1 and BCL-XL as an escape strategy from VEN-induced cell death. Analyzing protein complexes by co-immunoprecipitation, we found that exposure of ALL cells to VEN rapidly led to reduced BIM/BCL-2 and compensatory increased BIM/MCL-1 complexes. Conversely, S63845 reduced BIM/MCL-1 complexes and led to increased BIM/BCL-2 binding. Importantly, both protein complexes could be effectively disrupted by combination treatment with VEN and S63845, which resulted in release of BIM and promotion of apoptosis signaling.Based on our mechanistic findings, we evaluated BH3-mimetic combinations for cell death induction using multi-dose matrix assays to calculate synergy metrics based on the Bliss independence model. Analyzing combination effects in seven BCP-ALL cell lines and four PDX samples, we identified positive mean synergy scores of VEN with S63845 and A-1331852 in all cases. However, the most synergistic area of the multi-dose-response matrices differed among different leukemias, indicating different extents of addictions and thereby synergies at different drug concentrations.Finally, combined BCL-2 and MCL-1 inhibition was evaluated in vivo in a PDX model of KMT2A-ENL positive pro-B ALL. Combination treatment of VEN with S63845 led to reduced leukemia loads in spleen, bone marrow and CNS as compared to single agent treatment.Taken together, we found heterogeneous responses of BCP-ALL samples to BH3-mimetics antagonizing BCL-2, MCL-1 and BCL-XL. The ability of leukemia cells to adapt their anti-apoptotic dependency from BCL-2 to MCL-1 or BCL-XL can be used as target for combination therapy, demonstrating synergistic activity in PDX samples ex vivo and in vivo. DisclosuresNo relevant conflicts of interest to declare.

Galectin 3 Protein Network Analysis By Reverse Protein Arrays Demonstrates Regulation of MCL-1, PKC Delta and CD74 Associated with Poor Survival in Patients with AML

Galectin 3 (LGALS3) is a glycan binding protein that positively regulates survival signaling by diverse mechanisms. LGALS3 is involved in trafficking of kinases such as RAS and receptors such as CD44 to membranes. LGALS3 gene expression is associated with poor survival in AML (Cheng et al Blood 2013) but its functional role is not clear. We therefore analyzed protein expression of LGALS3 and 230 other proteins by RPPA in 511 AML patients. LGALS3 was significantly elevated in AML blast cells compared to normal CD34+ counterparts and highest in monocytic AML patients. LGALS3 levels were highest in Intermediate and Unfavorable Cytogenetic groups. Pearson correlation of LGALS3 with the other proteins identified a distinct set of 23 proteins positively and 13 proteins negatively correlated with LGALS3 expression levels. LGALS3 positively correlated with the expression of anti-apoptotic BCL2 family member MCL-1. To validate RPPA results, OCI-AML3 cells were transduced with a lentivirus expressing shRNA against LGALS3 or with a control lentivirus expressing shRNA against a non-human gene (i.e. GFP). Suppression of LGALS3 in the cells results in reduced MCL-1 gene and protein expression. As reduction of LGALS3 by shRNA or inhibition of LGALS3 using GCS-100 suppresses phosphorylation of ERK, the mechanism how LGALS3 supports MCL-1 in the AML cells may involve ERK. OCI-AML3 LGALS3 shRNA cells were more sensitive to ABT-737 compared to control cells. RPPA also revealed that LGALS3 was strongly correlated with phosphorylated PKC delta and suppression of LGALS3 by shRNA reduced PKC delta phosphorylation levels implicating LGALS3 as a positive regulator of the kinase. While PKC delta is viewed foremost as a stress kinase, RPPA analysis of phosphorylated (i.e. active) PKC delta in AML patients reveals that elevated levels of active kinase is a prognostic factor associated with poor survival. The pro-survival role of PKC delta in AML may reflect its function as an AKT kinase or as a suppressor of ROS. RPPA also identified a novel positive association of LGALS3 with CD74. OCI-AML3 cells with LGALS3 shRNA did not exhibit altered CD74 expression suggesting that CD74 is upstream of the galectin or possibly that an as yet unidentified protein serves as a common regulator of both LGALS3 and CD74. Stromal cell derived CD74 has been implicated as important in the AML microenvironment as a regulator of IL-8 production (Abdul-Aziz et al Cancer Research 2016) but the role for CD74 in AML blast cells is not clear. CD74 modulates CD44 function so the protein could participate in signaling cascades involving the receptor. Pearson correlation with the other proteins analyzed identified 23 proteins including CD44 and LGALS3 that were positively correlated with CD74 and 8 proteins that were negatively associated with CD74. Though LGALS3 was not positively correlated with CD44, the galectin could potentially modulate CD44 function by influencing its surface expression as LGALS3 is involved in trafficking CD44 to the cell surface. The protein associations of LGALS3 and CD74 were used to assemble protein clusters that were then used for analysis of patient survival. LGALS3 and CD74 were collectively analyzed as part of a group of T-cell regulating proteins analyzed on this array. Within that group 4 different expression patterns (clusters) were defined by hierarchical clustering with LGALS3 being active in 2 of them. The protein cluster identifying active LGALS3 was prognostic for shorter remission duration. AML patients who exhibited both LGALS3 and CD74 active clusters exhibited very poor survival characteristics. For AML patients with both LGALS3 and CD74 active protein cluster, median overall survival was only 24.3 weeks, median remission duration was 17.8 weeks, and no patient survived beyond one year. These data suggest that LGALS3 supports AML cell survival via diverse pathways including positive regulation of MCL-1 and PKC delta and support of a CD74 protein network that identifies a new high risk AML population. It is our hope that strategies targeting LGALS3 and CD74 can be developed to help this new at risk population. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Balancing Properties with Carboxylates: A Lead Optimization Campaign for Selective and Orally Active CDK9 Inhibitors.

Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase involved in the regulation of transcription elongation. An inhibition of CDK9 downregulates a number of short-lived proteins responsible for tumor maintenance and survival, including the antiapoptotic BCL-2 family member MCL-1. As pan-CDK inhibitors under development have faced dosing and toxicity challenges in the clinical setting, we generated selective CDK9 inhibitors that could be amenable to an oral administration. Here, we report the lead optimization of a series of azaindole-based inhibitors. To overcome early challenges with promiscuity and cardiovascular toxicity, carboxylates were introduced into the pharmacophore en route to compounds such as 14 and 16. These CDK9 inhibitors demonstrated a reduced toxicity, adequate pharmacokinetic properties, and a robust in vivo efficacy in mice upon oral dosing.

Breast cancer dependence on MCL-1 is due to its canonical anti-apoptotic function

High levels of the anti-apoptotic BCL-2 family member MCL-1 are frequently found in breast cancer and, appropriately, BH3-mimetic drugs that specifically target MCL-1’s function in apoptosis are in development as anti-cancer therapy. MCL-1 also has reported non-canonical roles that may be relevant in its tumour-promoting effect. Here we investigate the role of MCL-1 in clinically relevant breast cancer models and address whether the canonical role of MCL-1 in apoptosis, which can be targeted using BH3-mimetic drugs, is the major function for MCL-1 in breast cancer. We show that MCL-1 is essential in established tumours with genetic deletion inducing tumour regression and inhibition with the MCL-1-specific BH3-mimetic drug S63845 significantly impeding tumour growth. Importantly, we found that the anti-tumour functions achieved by MCL-1 deletion or inhibition were completely dependent on pro-apoptotic BAX/BAK. Interestingly, we find that MCL-1 is also critical for stem cell activity in human breast cancer cells and high MCL1 expression correlates with stemness markers in tumours. This strongly supports the idea that the key function of MCL-1 in breast cancer is through its anti-apoptotic function. This has important implications for the future use of MCL-1-specific BH3-mimetic drugs in breast cancer treatment.

Molecular mechanisms underlying antitumor activity of camel whey protein against multiple myeloma cells

Treating drug-resistant cancer cells is a clinical challenge and it is also vital to screen for new cancer drugs. Multiple myeloma (MM) is a plasma cell clonal cancer that, despite many experimental therapeutics, remains incurable. In this study, two MM cell line lines U266 and RPMI 8226 were used to determine the impact of camel whey protein (CWP). The CWP IC50 was calculated by MTT examination, while the flow cytometry analysis was used to investigate the chemotaxis responses of MM cells in relation to CXCL12 and the pro-apoptotic effect of CHP. MM cells were treated with CWP and Western blot analysis was used to determine the underlying molecular mechanisms. Dose and time based on the impact of CWP on the cell viability of MM cells with IC50 of 50 μg/ml, without affecting the viability of normal healthy PBMCs. CWP reduced chemotaxis of MM cells significantly from the CXC chemokine ligand 12 (CXCL12). Using Western blot analysis, we found that CWP decreased the activation of AKT, mTOR, PLCβ3, NFαB and ERK, which was mechanistically mediated by CXCL12/CXCR4. In both U266 and RPMI 8226, CWP induced apoptosis by upregulating cytochrome C expression. In addition, CWP mediated the growth arrest of MM cells by robustly decreasing the expression of the anti-apoptotic Bcl-2 family members Bcl-2, Bcl-XL and Mcl-1. Conversely, the expression of pro-apoptotic Bcl-2 family members Bak, Bax and Bim was increased after treatment with CWP. Our data indicates CWP's therapeutic potential for MM cells.

AML-211: Active Galectin 3/CD74 Axis is Associated with Poor Survival Outcome in AML

Context Galectin 3 (LGALS3) participates in diverse survival pathways that support many pro-tumor molecules, especially RAS. However, the role of LGALS3 in acute myeloid leukemia (AML) is not clear. Objective We previously found suppression of LGALS3 in AML cell lines sensitized cells to chemotherapy drugs. Here, we examine protein expression of LGALS3 in 511 AML patients and identify an active LGALS3 protein network. We used reversephase protein analysis for the study. Patients Peripheral blood and bone marrow specimens were collected from 511 patients with newly diagnosed AML. Samples were acquired in accordance with the regulations and protocols (Lab 01-473) approved by the IRB of MDACC. Results LGALS3 was significantly elevated in AML blast cells compared to normal CD34+ counterpart cells and highest in monocytic AML patients. Pearson correlation of LGALS3 with other proteins identified a distinct set of 23 proteins positively and 13 proteins negatively correlated with LGALS3 expression levels. LGALS3 positively correlated with the anti-apoptotic BCL2 family member MCL-1. RPPA also identified novel positive association of LGALS3 with CD74. Pearson correlation with the other proteins analyzed identified 23 proteins, including LGALS3, that were positively correlated with CD74, and 8 proteins that were negatively associated with CD74. The protein associations of LGALS3 and CD74 were used to assemble protein clusters that were used for analysis of patient survival. LGALS3 and CD74 were collectively analyzed as part of a group of T-cell regulating proteins analyzed on this array. Within that group, 4 different expression patterns (clusters) were defined by hierarchical clustering with LGALS3 being active in 2 of them. The protein cluster identifying active LGALS3 was prognostic for shorter remission duration. AML patients who exhibited both LGALS3 and CD74 active clusters exhibited very poor survival characteristics. For AML patients with both LGALS3 and CD74 active protein cluster, median overall survival was only 24.3 weeks, median remission duration was 17.8 weeks, and no patient survived beyond one year. Conclusions These data suggest that LGALS3 supports AML cell survival via diverse pathways, including positive regulation of MCL-1, and support of a CD74 protein network that identifies a new at-risk AML population.

Linking hyperosmotic stress and apoptotic sensitivity.

Cellular responses to hypertonic stress and how these are linked to the induction of or sensitisation to cell death signals are incompletely understood and rarely studied in cancer. Using cell lines derived from head and neck squamous cell carcinoma (HNSCC), Heimer et al. demonstrate that hypertonic environments neutralise the antiapoptotic Bcl-2 family member Mcl-1 by upregulating its antagonist Noxa. Consequently, hypertonically stressed HNSCC cells rely solely on Bcl-xL for survival and succumb to apoptosis when challenged by pharmacological Bcl-xL inhibition. Similar findings were reported in colorectal cancer cells in related manuscripts, suggesting that a common and conserved mechanistic link might exist between hyperosmotic stress and cellular sensitisation to apoptosis.

Boosting the apoptotic response of high-grade serous ovarian cancers with CCNE1-amplification to paclitaxel by targeting APC/C and the pro-survival protein MCL-1.

e18065 Background: Ovarian cancer exhibits the highest mortality rate among gynecological malignancies. Antimitotic agents, such as paclitaxel, are frontline drugs for the treatment of ovarian cancer. They inhibit microtubule dynamics and their efficiency relies on a prolonged mitotic arrest and the strong activation of the spindle assembly checkpoint (SAC). Although ovarian cancers respond well to paclitaxel, the clinical efficacy is limited due to an early onset of drug resistance, which may rely on a compromised mitosis exit associated with weakend intrinsic apoptosis. Accordingly, we aimed at overcoming SAC silencing that occurs rapidly during paclitaxel-induced mitotic arrest. Methods: To do this, we used a specific anaphase-promoting complex/ cyclosome (APC/C) inhibitor to prevent a premature mitotic exit upon paclitaxel treatment. Furthermore, we investigated the role of the anti-apoptotic BCL-2 family member MCL-1 in determining the fate of ovarian cancer cells with CCNE1-amplification that are challenged with clinically relevant dose of paclitaxel. Results: Using time-laps microscopy we demonstrated that APC/C and MCL-1 inhibition under paclitaxel prevents mitotic slippage in ovarian cancer cells and restores death in mitosis (DIM). Consistent with this, the combinatorial treatment reduced the survival of ovarian cancer cells in 2D and 3D cell models. Since a therapeutic ceiling has been reached with taxanes, it is of utmost importance to develop alternative strategies to improve the patient´s survival. Conclusions: Our study provides not only elements to understand the causes of taxane resistance in CCNE1-amplified ovarian cancers but also suggests a new combinatorial strategy that may improve paclitaxel efficacy in this highly lethal gynecological disease.

MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment

Cells experiencing delays in mitotic progression are prone to undergo apoptosis unless they can exit mitosis before proapoptotic factors reach a critical threshold. Microtubule targeting agents (MTAs) arrest cells in mitosis and induce apoptotic cell death engaging the BCL2 network. Degradation of the antiapoptotic BCL2 family member MCL-1 is considered to set the time until onset of apoptosis upon MTA treatment. MCL1 degradation involves its interaction with one of its key binding partners, the proapoptotic BH3-only protein NOXA. Here, we report that the mitochondria-associated E3-ligase MARCH5, best known for its role in mitochondrial quality control and regulation of components of the mitochondrial fission machinery, controls the levels of MCL1/NOXA protein complexes in steady state as well as during mitotic arrest. Inhibition of MARCH5 function sensitizes cancer cells to the proapoptotic effects of MTAs by the accumulation of NOXA and primes cancer cells that may undergo slippage to escape death in mitosis to cell death in the next G1 phase. We propose that inhibition of MARCH5 may be a suitable strategy to sensitize cancer cells to antimitotic drug treatment.

The Combination of CUDC-907 and Gilteritinib Shows Promising Antileukemic Activity in Vitro and In Vivo in Preclinical Models of FLT3-ITD AML

Introduction FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations are found in approximately one quarter of acute myeloid leukemia (AML) cases. Its presence results in constitutive activation of the FLT3 receptor tyrosine kinase and its downstream growth/pro-survival pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT, and confers a poor prognosis. Gilteritinib is a selective inhibitor of FLT3 recently approved by the Food and Drug Administration for treatment of patients with relapsed/refractory AML and a FLT3 mutation. However, gilteritinib exposure induces upregulation of FLT3 - a mechanism of resistance. Previously, we showed that CUDC-907, a dual PI3K/histone deacetylase inhibitor, downregulates FLT3 expression (Li X, et al. Haematologica. 2019; epub ahead of print). We therefore hypothesized that combining CUDC-907 with gilteritinib would abrogate FLT3 upregulation and expression, resulting in synergistic antileukemic activities against FLT3-mutated AML. Methods FLT3-ITD AML cell lines and primary patient samples were treated with gilteritinib or CUDC-907, alone or in combination at clinically achievable concentrations, and subjected to annexin V/propidium iodide staining and flow cytometry analysis to quantify apoptosis. Protein levels of FLT3, Bcl-2 family proteins, and key components of the MAPK/ERK, PI3K/AKT, and JAK/STAT pathways were examined using western blotting. The impact of the observed alterations upon apoptosis were confirmed via overexpression, knockdown, and targeted inhibitor experiments. Real-time RT-PCR was used to determine FLT3 transcript levels. The FLT3-ITD AML cell line MV4-11 was used to generate a xenograft mouse model to assess in vivo efficacy of the two agents. Results CUDC-907 and gilteritinib demonstrated potent synergistic antileukemic effects in FLT3-ITD AML cell lines in vitro and patient samples ex vivo, with combined therapy. CUDC-907 abolished gilteritinib-induced expression of FLT3 in both cell lines and primary patient samples. Gilteritinib treatment reduced p-AKT, p-S6, and p-STAT5 and increased p-ERK, while CUDC-907 reduced p-AKT and p-ERK, and upregulated p-STAT5. The combination of gilteritinib and CUDC-907 decreased not only p-AKT and p-S6, but also p-ERK and p-STAT5. Targeted inhibition of ERK and JAK2/STAT5 signaling by SCH772984 and AZD1480, respectively, confirmed their roles in resistance to gilteritinib and CUDC-907 monotherapies, respectively. Combined gilteritinib and CUDC-907 treatment reduced expression of the anti-apoptotic BCL-2 family member Mcl-1 and increased expression of the pro-apoptotic protein Bim. MCL-1 overexpression and BIM knockdown partially rescued FLT3-ITD AML cells upon drug treatment, confirming their role in the antileukemic activity of combined gilteritinib and CUDC-907. To determine in vivo efficacy of the two agents, NSGS mice were injected with MV4-11 cells. Three days later, the mice were randomized into vehicle control (n=5), 40 mg/kg gilteritinib (oral gavage; n=5), 100 mg/kg CUDC-907 (oral gavage; n=5) or combination (40 mg/kg gilteritinib + 100 mg/kg CUDC-907; n=6) groups. CUDC-907 was given daily for 5 days on, 2 days off, for a total of 4 cycles. Gilteritinib was administered daily for 28 days. Both agents were well tolerated; maximal weight loss was 5.5%, 0.9%, and 6.7% in the CUDC-907, gilteritinib, and combination groups, respectively. Median survival of mice in the vehicle control group was 43 days. Median survival in the CUDC-907 monotherapy and gilteritinib monotherapy arm was 40.5 days and 104 days, respectively. One mouse in the combination therapy arm died on day 138, while the remaining 5 mice in the combination therapy arm continue to survive, as of time of writing (day 168), and are asymptomatic (Figure 1). Conclusion We confirmed that the combination of CUDC-907 plus gilteritinib synergistically induces apoptosis in both FLT3-ITD AML cell lines and primary patient samples, and that gilteritinib-induced FLT3 expression is abolished by CUDC-907. Cooperative inhibition of the PI3K-AKT, JAK-STAT, and RAS-RAF pathways, as well as upregulation of Bim/downregulation of Mcl-1 all appear to contribute to this observed antileukemic synergy. Our cell line-derived xenograft mouse model provides strong evidence of in vivo efficacy and robust grounds for clinical translation of this therapeutic combination. Disclosures No relevant conflicts of interest to declare.

Therapeutic inhibition of Mcl-1 blocks cell survival in estrogen receptor-positive breast cancers.

Cancers often overexpress anti-apoptotic Bcl-2 proteins for cell death evasion, a recognized hallmark of cancer progression. While estrogen receptor (ER)-α+ breast cancers express high levels of three anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1), pharmacological inhibition of Bcl-2 and/or Bcl-xL fails to induce cell death in ERα+ breast cancer cell lines, due to rapid and robust Mcl-1 upregulation. The mechanisms of acute Mcl-1 upregulation in response to Bcl-2/Bcl-xL inhibition remain undefined in in ERα+ breast cancers. We report here that blockade of Bcl-2 or Bcl-xL, alone or together, rapidly induced mTOR signaling in ERα+ breast cancer cells, rapidly increasing cap-dependent Mcl-1 translation. Cells treated with a pharmacological inhibitor of cap-dependent translation, or with the mTORC1 inhibitor RAD001/everolimus, displayed reduced protein levels of Mcl-1 under basal conditions, and failed to upregulate Mcl-1 protein expression following treatment with ABT-263, a pharmacological inhibitor of Bcl-2 and Bcl-xL. Although treatment with ABT-263 alone did not sustain apoptosis in tumor cells in culture or in vivo, ABT-263 plus RAD001 increased apoptosis to a greater extent than either agent used alone. Similarly, combined use of the selective Mcl-1 inhibitor VU661013 with ABT-263 resulted in tumor cell apoptosis and diminished tumor growth in vivo. These findings suggest that rapid Mcl-1 translation drives ABT-263 resistance, but can be combated directly using emerging Mcl-1 inhibitors, or indirectly through existing and approved mTOR inhibitors.

Boosting the apoptotic response of high-grade serous ovarian cancers with CCNE1 amplification to paclitaxel in vitro by targeting APC/C and the pro-survival protein MCL-1.

Ovarian cancer exhibits the highest mortality rate among gynecological malignancies. Antimitotic agents, such as paclitaxel, are frontline drugs for the treatment of ovarian cancer. They inhibit microtubule dynamics and their efficiency relies on a prolonged mitotic arrest and the strong activation of the spindle assembly checkpoint (SAC). Although ovarian cancers respond well to paclitaxel, the clinical efficacy is limited due to an early onset of drug resistance, which may rely on a compromised mitosis exit associated with weakend intrinsic apoptosis. Accordingly, we aimed at overcoming SAC silencing that occurs rapidly during paclitaxel-induced mitotic arrest. To do this, we used a specific anaphase-promoting complex/cyclosome (APC/C) inhibitor to prevent a premature mitotic exit upon paclitaxel treatment. Furthermore, we investigated the role of the antiapoptotic BCL-2 family member MCL-1 in determining the fate of ovarian cancer cells lines with CCNE1 amplification that are challenged with clinically relevant dose of paclitaxel. Using time-laps microscopy, we demonstrated that APC/C and MCL-1 inhibition under paclitaxel prevents mitotic slippage in ovarian cancer cell lines and restores death in mitosis. Consistent with this, the combinatorial treatment reduced the survival of ovarian cancer cells in 2D and 3D cell models. Since a therapeutic ceiling has been reached with taxanes, it is of utmost importance to develop alternative strategies to improve the patient's survival. Thus, our study provides not only elements to understand the causes of taxane resistance in CCNE1-amplified ovarian cancers but also suggests a new combinatorial strategy that may improve paclitaxel-based efficacy in this highly lethal gynecological disease.

Rationally derived drug combinations with the novel Mcl-1 inhibitor EU-5346 in breast cancer.

Recent studies have emphasized a key role for the anti-apoptotic Bcl-2 family member Mcl-1 in conferring tumor cell survival and drug resistance in breast cancer (BC). Mcl-1 inhibitors, such as the BH3-mimetic EU-5346, therefore represent an exciting new class of targeting agents and are a current focus of widespread cancer-drug development efforts. ONCOMINE analysis was utilized to compare expression profiles of Bcl-2 family members across all major BC subgroups. Potential toxicities of EU-5346 were evaluated using iPS-generated cardiomyocytes, blood cells and astrocytes. The anti-BC cell activity of EU-5346-based therapies was evaluated using [3H]-thymidine uptake and spheroid-forming assays as well as immunoblotting and the Chou-Talalay method. Protein level-based activity of EU-5346, the specific anti-Bcl-2 inhibitor ABT-199 and the specific anti-Bcl-xL inhibitor WEHI-539 was verified in Mcl-1Δ/null versus Mcl-1wt/wt MEFs. We previously demonstrated significant anti-tumor activity of EU-5346 in all BC subtypes. Our present results go further and suggest that EU-5346 may induce limited adverse events such as cardiotoxicity, hematotoxicity, and neurotoxicity, frequently observed with other BH3 mimetics. As demonstrated by our mathematical scoring model, the prediction of EU-5643-induced IC50 not only relies on the protein level of Mcl-1 but also on Bak, Bim, and Noxa. Synergistic anti-BC activity of low-dose EU-5346 with the BH3 mimetics ABT-199 or WEHI-539 was observed only in those BC cells expressing Bcl-2 or Bcl-xL, respectively. Similarly, when combined with tamoxifen or trastuzumab, low-dose EU-5346 induced significant anti-BC activity in hormone receptor positive or Her2-positive BC cells, respectively. Finally, EU-5346 in combination with paclitaxel induced synergistic anti-BC activity in both paclitaxel-sensitive and paclitaxel-resistant TNBC cells. These data strongly support the further clinical development of EU-5346 to improve BC patient survival.

Porcine Neonatal Islet-like Cell Clusters Cultured with EGM2 Medium Enhance their Resistance to Apoptosis in Islet Xenotransplantation

Introduction Pig neonatal islet-like cell clusters (NICC) are an attractive source of insulin-producing tissue for potential transplantation treatment of type 1 diabetic patients. However, a considerable loss of NICC after their transplantation due to apoptosis resulted from islet isolation and instant blood mediated inflammatory reaction remains to be overcome. Materials and Methods EGM2 medium was depleted with hydrocortisone and supplemented with 50 mM isobutylmethlxanthine, 10 mM nicotinamide,10 mM glucose. Modified EGM2 medium was used to culture NICC at day 1, the day after isolation and changed every other day. When NICC cultured with modified EGM2 or control medium Ham’s F-10 at day 7, they were collected to do the following assays. AO/EB staining were used to evaluate the viability of NICC. Static assay and oxygen consumption rate analysis were performed to assess the function of NICC. Insulin and glucagon expression was measured by real-time PCR. Serum starvation and Cocl2 treatment was used to simulate the microenvironment post islet transplantation. The viability of NICC was evaluated by FACS and the proposed mechanism was detected by western blotting. Results Compared with Ham’s F-10 medium, culturing NICC with modified EGM2 medium led to increased number and viability of NICC with higher stimulation index, upregulated gene expression of both insulin and glucagon and also higher mitochondria function. Moreover, modified EGM2 medium cultured NICC demonstrated much less apoptotic cells under either serum starvation or CoCl2-induced hypoxia condition than their Ham’s F-10 medium cultured counterparts. The enhanced capability of EGM2 cultured NICC to resist apoptosis was associated with their elevated protein levels of anti-apoptotic Bcl-2 family member Mcl-1. Discussion NICC cultured with EGM2 medium not only improved their viability and mitochondria function but also enhanced their resistance to apoptosis under the hypoxia environment. Conclusion Culturing NICC with modified EGM2 provides a simple and effective approach to enhance their resistance to apoptosis to preserve the initial graft mass for successful islet xenotransplantation. This project was supported by NSFC 81201171 and Hunan Provincial Natural Science Foundation of China 2017JJ3423.